Research ArticleGenetic Heterogeneity of Alicyclobacillus Strains Revealed byRFLP Analysis of vdc Region and rpoB Gene

Agnieszka Dekowska Jolanta Niezgoda and Barbara SokoBowska

Prof Waclaw Dabrowski Institute of Agricultural and Food Biotechnology 36 Rakowiecka Street 02-532 Warsaw Poland

Correspondence should be addressed to Agnieszka Dekowska agnieszkadekowskaibprspl

Received 3 August 2018 Accepted 27 September 2018 Published 1 November 2018

Guest Editor Marta Laranjo

Copyright copy 2018 Agnieszka Dekowska et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

PCR-RFLP targeting of the 16S rDNA and rpoB genes as well as the vdc region was applied to identify and differentiate betweenthe spoilage and non-spoilage Alicyclobacillus species Eight reference strains and 75 strains isolated from spoiled juices juiceconcentrates drinks its intermediates and fresh apples were subject to study Hin6I restriction patterns of the 16S rDNA geneenabled distinguishing between all the species analyzed while the rpoB gene and vdc gene cluster analysis also revealed that therewere two major types among the A acidoterrestris isolates one similar to the reference strain A acidoterrestris DSM 2498 and theother similar to the reference strain A acidoterrestris ATCC 49025 Heterogeneity was also observed among the A acidocaldariusisolates RFLP analysis of the 16S rDNA and rpoB genes as well as vdc region can be used successfully in the identification andresearch of intraspecies heterogeneity of the Alicyclobacillus species

1 Introduction

The contamination of fruit juices by Alicyclobacillus hasrecently become one of the most important issues in thejuice and beverage industry These acidophilic thermophilicand spore-forming bacteria are very hard to eliminate fromcontaminated drinks

Alicyclobacillus are Gram positive aerobic soil bornebacteria that are able to grow within a range from pH 20to 60 and at temperatures from 20 to 70∘C [1ndash8] The twomain factors which prevent fruit products from spoilage withmost other bacteria which are thermal treatment and lowpH values are insufficient to eliminate Alicyclobacillus Thespores survive under typical pasteurization conditions andare able to germinate and grow in an acidic environment[9 10]Thermal treatment may even impel germination of thespores [11ndash13]

Despite being nonpathogenic [14] some Alicyclobacillusspecies may cause the spoilage of juices and juice-containingproducts such as nectars and beverages Alicyclobacillusspecies have been found all across the world and theirpresence has been detected on fruit surfaces [15] in juicesproduced from several fruits citrus apple banana berryand stone fruits [1 8 10 16ndash24] in canned tomatoes [25]

and in drinks for example ice tea and isotonic drinks [1726] The spoilage mainly manifests itself as the formationof a medical antiseptic off-odour from compounds pro-duced by the bacteria The main compound associated withspoilage is guaiacol produced from vanillin and vanillicacid [27ndash29] but halophenols 26-dibromophenol and 26-dichlorophenol have also been reported as spoilage agentsAmong the 22 currently known Alicyclobacillus species fivehave been proven to produce an off-odour A acidoterrestrisA acidiphilus A pomorum A cycloheptanicus and Aherbarius [1 9 14 30ndash37]

The classic method for isolating and characterizing Alicy-clobacillus devised by IFU (Internationale Fruchtsaft Union)which is commonly used in the juice and beverage industrytakes about 15 days If present in the tested sample guaiacolcan be detected using the peroxidase method [27 38] bysensory tests ([9 10 39] Siegmund and Pollinger-Zierler2006) or instrumental methods for example HPLC or gaschromatography [28 35 40 41]

The identification of the Alicyclobacillus species basedon their ability to assimilate erythritol with acid production[19 42] mainly allows differentiating between two species Aacidoterrestris and A acidocaldarius

HindawiBioMed Research InternationalVolume 2018 Article ID 9608756 12 pageshttpsdoiorg10115520189608756

2 BioMed Research International

Since the classic microbiological Alicyclobacillus detect-ingmethods are time consuming alternative approaches havebeen adopted like flow cytometry ([30] Pieper et al 2006)Fourier transform infrared spectroscopy [43ndash45] and geneticmethods Except for RAPD-PCR (Yamazaki et al 1997 [46ndash49]) most of the genetic methods used in the studies onAlicyclobacillus target the rDNA operon These include 16SrDNA and ITS region sequencing 16S rDNA RFLP Real-Time PCR and LAMP-PCR of the 16S rDNA fragment ([150] Durak et al 2002 [6 7 51ndash54])

Guaiacol the main spoilage agent is produced by nonox-idative decarboxylation of vanillic acidThe ability to produceguaiacol is associated with presence of vdc gene clusterconsisting of three genes vdcB vdcC and vdcD Chow et al[55] described vdc gene cluster in Streptomyces sp Detectionof the vdc genes of A acidoterrestris using RT-PCR wasdescribed by Niwa and Kawamoto [38] The vdc regionsequence was published byMatsubara [56] To date there areno applications of vdc region analysis for anymicroorganismRpoB gene encoding the 120573 subunit of bacterial RNA poly-merase is one of the single-copy housekeeping genes and iswidely used in studies on bacterial taxonomy These studiesinclude PCR-RFLP analyses of rpoB gene fragments howeverso far there are no reports on using rpoB gene analysis inresearch on of Alicyclobacillus

Our study focuses on the use of rpoB gene vdc region and16SrDNA gene as molecular markers for the identificationand differentiation of Alicyclobacillus

2 Materials and Methods

21 Sample Acquisition and Bacterial Strains Seventy-fivestrains analyzed in this study were isolated from concentratedapple juice (47) concentrated cherry juice (4) fresh apples(3) concentrated strawberry juice (2) concentrated blackcurrant juice (2) tomato juice (2) orange juice (3) cloudy(1) and clear (1) apple juice apple beverage (1) concentratedbeetroot juice (1) concentrated raspberry juice (1) concen-trated orange juice (1) orange beverage (1) banana nectar(1) cherry puree (1) and intermediates used in beverageproduction (3) All the strains were isolated according to themethod described in IFU no 12 September 2004March 2007

Reference strains were obtained from the Leibniz Insti-tute DSMZ ndash German Collection of Microorganisms andCell Cultures (Alicyclobacillus acidiphilus DSM 14558 Ali-cyclobacillus acidocaldarius DSM 446 Alicyclobacillus aci-doterrestrisDSM 2498Alicyclobacillus herbariusDSM 13609Alicyclobacillus hesperidum DSM 12489) and from theAmerican Type Culture Collection (ATCC) (Alicyclobacillusacidoterrestris ATCC 49025 Geobacillus stearothermophilusATCC 7953Bacillus subtilisATCC 6655) Also Alicyclobacil-lus acidocaldarius A1 from our own library collection wasused as a reference strain after biochemical and 16S rDNAsequencing confirmation

22 Biochemical Tests Thestrainswere checked for erythritolutilization and guaiacol production The ability of erythritol

utilization was tested by plating the cultures on agar con-taining 1 erythritol and bromophenol blue as an indicator[42] The ability to produce guaiacol was tested using theperoxidase method [27]

23 DNA Isolation Selected Alicyclobacillus strains werecultured in BAT medium (pH 40plusmn02) at 45∘C for 2ndash3 daysBacillus subtilis was cultured in TSB medium (pH 71plusmn02)at 30∘C for 2 days and Geobacillus stearothermophilus wascultured in TSB medium at 45∘C for 2 days

Bacterial chromosomal DNA was purified using aGenomic Mini Kit (AampA Biotechnology) following themanufacturerrsquos instructions

24 Primer Designing and Amplification All PCR reactionswere performed using a Pequstar 2x Gradient thermocycler(Pequlab)

241 16S rDNA Amplification A fragment of the 16S rDNAgene was amplified using universal primers similar to thatused byWang et al 2010 [57]The primer sequences were 8F(51015840ndash AGAGTTTGATCCTGGCTCAG) E coli positions 8-27and 1512R shortened by 1 nucleotide at 31015840 end (51015840 ndash ACGGC-TACCTTGTTACGACT) E coli positions 1512ndash1493The sizeof the amplification product was 1495 bp

PCR reactions were performed in a total volume of 50120583lcontaining 5 ng of the template DNA 50 pM of each of theprimers and 25120583l of the DreamTaqGreen PCRMasterMix(Thermo Scientific) PCRwas performed under the followingconditions initial denaturation at 94∘C for 2min 40 cycles ofdenaturation at 94∘C for 30 sec annealing at 51∘C for 35 secelongation 72∘C for 1min 40 sec and the final elongation at72∘C for 2min

242 vdc Region Amplification The vdc gene cluster wasamplified using primers vdc fr (51015840 ndash CTGTTGGCTCAA-TGGCGGCTGAGCGAT) vdc rev (51015840 ndash TTATCAGCG-GTTTATCCGCGGTGGAACAGTC) vdc1 fr (51015840 ndash AAC-GACGCAGGTGTGGAAAC) vdc1 rev (51015840 ndash AGCGTG-GGCAAGTTGTCATGTG) vdc K (51015840 ndash TTGGCAACG-GAGAAGTGGGAG) and vdc S (51015840 ndash AATCACGCG-CTGATGATGGG) The 1586 fragment of vdc region con-taining fragments of vdcB and vdcC genes was used asa template for PCR-RFLP and was amplified using theprimers Bur 5 (51015840 GCCGACGTGATGCTCAARGAG-CGCA) and Bur 6 (51015840 GTSGCRTCGAGAATCATCTTG-TG) The primers were designed based on a comparisonof the raw genome sequences derived from Alicyclobacil-lus acidoterrestris ATCC 49025 ([58] GenBank numberAURB010001131) and Alicyclobacillus herbarius DSM 13609(GenBank number AUMH010000321) the sequence pub-lished by Matsubara (GenBank number BD1877781) andsequences obtained from several Alicyclobacillus acidoter-restris strains analyzed in this study Sequence alignmentswere performed using the Serial Cloner program The posi-tions and directions of the vdc primers are described inTable 1and shown in Figure 1

BioMed Research International 3

vdc fr (1 - 27) vdc1 fr(314 - 333)

bur5(560 - 584)

vdc B (245 - 838)

vdcS (1044 - 1063)

vdc C (831 - 2252)vdc region (1 - 2523)

bur6 (2124 - 2146)

vdcK (2202 - 2222) vdc1 rev (2287 - 2308)

vdc rev (2493 - 2523)

vdc D (2290 - 2517)

Figure 1 Vdc region positions of genetic elements and primers

Table 1 The positions and directions of vdc primers

Primer name Primer position (bp) Primer directionvdc fr 1-27 forwardvdc rev 2493-2523 reversevdc1 fr 314-333 forwardvdc1 rev 2287-2308 reversevdc K 2202-2222 forwardvdc S 1044-1063 forwardBur5 560-584 forwardBur6 2124-2146 reverse

The 2523 bp vdc fr ndash vdc rev amplification productcontained the whole vdc region The size of the Bur5-Bur6 amplification product was 1586 bp PCR reactions wereperformed in a total volume of 50120583l containing 25 ng of thetemplate DNA 5 pM of each of the primers and 25120583l ofthe DreamTaq Green PCRMaster Mix (Thermo Scientific)PCR was performed under the following conditions initialdenaturation at 94∘C for 2min 30 cycles of denaturation at94∘C for 30 sec annealing at 59∘C for 50 sec elongation 72∘Cfor 1min 40 sec and the final elongation at 72∘C for 5min

243 rpoB Gene Amplification A fragment of rpoB genewas amplified using Gru3ndashGru6 primers The primerswere designed based on a comparison of the rpoB genesequences of Alicyclobacillus acidocaldarius Bacillus subtilisand Geobacillus stearothermophilus as well as sequencesderived fromA acidoterrestris 49025A hesperidumURH17-3-68 and A herbarius DSM 13609 raw genomic sequencesGru5 and Gru6 primers are nondegenerate versions of theGru3 and Gru4 primers respectively The primer sequenceswere Gru3 (CGYGACGTDCACTAYTCBCACTA) Gru4 (51015840ndash GCCCANACYTCCATCTCRCCRAA) Gru5 (51015840 ndash CGC-GACGTACACTATTCGCACTA) and Gru6 (51015840 ndash GCC-CAAACCTCCATCTCACCAAA) The size of the amplifi-cation product was 1735bp PCR reactions were performedin a total volume of 50 120583l containing 30 ng of the templateDNA 40 pM of each of the primers and 25120583l of theDreamTaq Green PCR Master Mix (Thermo Scientific)PCR was performed under the following conditions initialdenaturation at 94∘C for 2min 35 cycles of denaturation at94∘C for 30 sec annealing at 57∘C (for the Bur5 and Bur6primer pairs) or at 59∘C (for the Bur3 and Bur4 primers)

for 35 sec elongation 72∘C for 1min 45 sec and the finalelongation at 72∘C for 5min

25 PCR-RFLP 5-15120583l of the PCR products were digestedwith BsuRI Hin6I and HphI (Thermo Scientific) in 20 120583lvolumes The samples were incubated at 37∘C for 1-2 hoursand the enzymes were then inactivated with ten-minuteincubation at 80∘C The digests were analyzed on 25ndash3agarose gel

26 DNA Sequencing DNA samples were sequencedusing 8F and shortened 1512R primers for the 16SrDNA gene vdc fr vdc rev vdc1 fr vdc1 rev Bur5Bur6 and two additional primers to fill the gaps vdcS(51015840 ndash AATCACGCGCTGATGATGGG) and vdcK (51015840 ndashTTGGCAACGGAGAAGTGGGAG) for the vdc region andGru3 ndash Gru6 for the rpoB gene

The contig assemblies sequence alignments and phylo-genetic analysis were performed using Serial Cloner software(SerialBasic) and Clustal Omega The sequences were com-pared to theGenBank sequences database using BLAST tools

3 Results

31 RFLP Analysis of 16S rDNA Fragment The 1495 bpfragment of the 16S rDNA gene amplified using 8F andshortened 1512R primers was digested by BsuRI Hin6I andHphI

While BsuRI and HphI digestions did not allow todistinguish between all the species analyzed the patternsobtained by Hin6I digestion were species specific (Figure 2)

All analyzed isolates with their features and RFLP profilesare described in Table 2

32 RFLP Analysis of the vdc Region Fragment The fragmentof vdc region was amplified using Bur5 and Bur6 primersThe product was a single band of 1586 bp and was observedonly for guaiacol producing strains A acidoterrestris Aacidophilus and A herbarius The PCR product was digestedby BsuRI (Figure 3) Hin6I (Figure 4) and HphI (Figure 5)

BsuRI and Hin6I RFLP patterns enabled distinguishingbetween all the species analyzed and divided the A acidoter-restris group into two clusters Type I pattern was identical tothe pattern given by A acidoterrestris DSM 2498 and type IIpattern was identical to A acidoterrestris ATCC 49025 The

4 BioMed Research International

Table2Listof

isolatesa

ndtheirR

FLPprofi

lesAAT

Aacid

oterrestris

AAC

Aacid

ocaldariu

sBA

Brevibacillusa

griBG

bacillus

ginsengihum

i

IsolateSource

Guaiacolprodu

ction

Erythrito

lutilization

RFLP

analysis

16S

Hin61

vdc

BsuR

Ivdc

Hin61

vdc

Hph

IrpoB

BsuR

IrpoB

Hin61

rpoB

Hph

I1

concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

2concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

3interm

ediateforb

everagep

rodu

ction

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

4interm

ediateforb

everagep

rodu

ction

nono

AAC

--

-AAC

(I)

AAC

(I)

AAC

(I)

5concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

6concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

7concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

8concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

9orange

beverage

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

10concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

11concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

12concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

13concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

14concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

15concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

16concentrated

applejuice

yes

nontypical

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

17banana

nectar

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

18orange

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

19concentrated

orange

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

20concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

21orange

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

22concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

23concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

24concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

25fre

shapples

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

26concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

27concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

28concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

29concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

30concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

31concentrated

blackcurrantjuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

32concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

33concentrated

applejuice

yes

nontypical

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

34fre

shapples

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(IA)

AAT

(I)

AAT

(I)

AAT

(I)

BioMed Research International 5

Table2Con

tinued

IsolateSource

Guaiacolprodu

ction

Erythrito

lutilization

RFLP

analysis

16S

Hin61

vdc

BsuR

Ivdc

Hin61

vdc

Hph

IrpoB

BsuR

IrpoB

Hin61

rpoB

Hph

I35

fresh

apples

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

36concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

37concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

38interm

ediateforb

everagep

rodu

ction

nono

AAC

--

-AAC

(I)

AAC

(I)

AAC

(IA)

39concentrated

applejuice

yes

-BG

--

-BG

BGBG

40concentrated

applejuice

yes

-BG

--

-BG

BGBG

41concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

42concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(IIA)

43concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

44spoiledappleb

everage

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

45concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

46spoiledapplejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

47concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

48concentrated

straw

berryjuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

49concentrated

cherry

juice

nono

AAC

--

-AAC

(IA)

AAC

(I)

AAC

(IA)

50tomatojuicefrom

fresh

tomatoes

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

51concentrated

beetroot

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

52concentrated

cherry

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

53concentrated

cherry

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

54concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(IIA)

AAT

(II)

AAT

(II)

55concentrated

cherry

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

56concentrated

raspberryjuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

57concentrated

applejuice

yes

nontypical

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

58orange

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

59concentrated

applejuice

nono

ntypical

AAC

--

-AAC

(IA)

AAC

(I)

AAC

(I)

60concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

61concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

62tomatojuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

63concentrated

applejuice

yes

-BA

--

-BA

BABA

64concentrated

applejuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

65cherry

puree

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

66concentrated

blackcurrantjuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

67concentrated

straw

berryjuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

68clou

dyapplejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

69concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

70concentrated

applejuice

nono

AAC

--

-AAC

(IA)

AAC

(I)

AAC

(I)

71concentrated

applejuice

nono

AAC

--

-AAC

(IA)

AAC

(I)

AAC

(I)

72concentrated

applejuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

73concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

74concentrated

applejuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

75concentrated

applejuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

6 BioMed Research International

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

bp

1000

500

200

Figure 2 Restriction patterns of a 1495 bp fragment of the 16S rDNA gene digested with Hin6I Lanes 6 7 9 14 and 18 A acidoterrestris 23 4 8 10 11 12 13 15 16 and 17 A acidocaldarius 5 Brevibacillus agri 19 A acidoterrestris DSM 2498 20 A acidoterrestris ATCC49025 21A acidiphilus 22 A hesperidum 23 A herbarius 24 A acidocaldarius 25 B subtilis 26 G stearothermophilus lane 1 molecular marker

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 3 Restriction patterns of a 1586 bp fragment of the vdc region digested with BsuRI Lanes 1ndash3 5 7ndash11 13 and 14 A acidoterrestristype I 4 6 12 and 15 A acidoterrestris type II 16 A acidoterrestris DSM 2498 17 A acidoterrestris ATCC49025 18 A acidiphilus 19 Aherbarius 20 molecular marker

HphI patterns confirm this rule with the exception of onestrain which represented the type I pattern in the BsuRI andHin6I analysis

33 RFLP Analysis of the rpoB Gene Fragment The 1735 bpfragment of the rpoB gene was amplified using Gru3 andGru4 or Gru5 and Gru6 primers Gru3 and Gru4 primerswere degenerated versions of Gru5 and Gru6 (respectively)and were used for amplification of isolates other thanA acidoterrestris A acidoterrestris isolates were amplifiedeither with Gru3ndashGru4 or Gru5ndashGru6 primers and theGru5ndashGru6 primer pair was chosen due to the better effi-ciency of the reaction Considering the individual isolates theRFLP patterns were identical for both pairs of primers

Thepatterns obtained by all of the nucleases used (Figures6ndash8) enabled distinguishing between the species analyzedWith exception of two strains the BsuRI patterns dividedthe A acidoterrestris group into two clusters analogicalto the clusters revealed by the vdc region analysis Thepattern (IIA) is shown by two exceptional strains mostresembling the type II pattern of A acidoterrestris andthose strains were classified as type II in other analyses Thereference strain ATCC 49025 shows this type of patternThe A acidocaldarius group was represented by three typesof patterns two of them resembling each other A acido-caldarius DSM 446 belongs to the cluster II of A acido-caldarius group while A acidocaldarius A1 was assigned tocluster I

BioMed Research International 7

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 4 Restriction patterns of a 1586 bp fragment of the vdc region digested with Hin6I Lanes 1ndash3 5 7ndash11 13 and 14 A acidoterrestristype I 4 6 12 and 15A acidoterrestris type II 16A acidoterrestrisDSM 2498 17A acidoterrestris ATCC49025 18 A acidiphilusDSM 1455819 A herbarius DSM 13609 lane 20 molecular marker

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 5 Restriction patterns of a 1586 bp fragment of the vdc region digested with HphI Lanes 2ndash4 6 8ndash12 14 and 15 A acidoterrestristype I 5 7 13 and 16 A acidoterrestris type II 17 A acidoterrestris DSM 2498 18 A acidoterrestris ATCC49025 19 A acidiphilus 20 Aherbarius lane 1 molecular marker

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Figure 6 Restriction patterns of a 1735 bp fragment of the rpoB gene digested with BsuRI Lanes 1ndash4 6 and 8ndash10 A acidoterrestris type I 57 21 22 and 24-26 A acidoterrestris type II 23 A acidoterrestris type IIA 27 A acidocaldarius type II 28 A acidocaldarius type IA 11 Aacidoterrestris DSM 2498 12 A acidoterrestris ATCC49025 13 A acidiphilus 14 A hesperidum 15 A herbarius 16 A acidocaldariusA1 17B subtilis 18 G stearothermophilus lane 19 molecular marker

8 BioMed Research International

Figure 7 Restriction patterns of a 1735 bp fragment of rpoB gene digested with Hin6I Lanes 3 and 5 A acidoterrestris type I 1 2 4 and 6Aacidoterrestris type II 17A acidocaldarius type I 18A acidocaldarius type II 8A acidoterrestrisDSM2498 9A acidoterrestrisATCC4902510A acidiphilus 11 A hesperidum 12A herbarius 13A acidocaldariusA1 14 B subtilis 15G stearothermophilus lanes 7 and 16 molecularmarker

Figure 8 Restriction patterns of a 1735 bp fragment of rpoB gene digested with HphI Lanes 1-4 6 8-11 13 25 and 27 A acidoterrestris I5 7 12 26 and 28 A acidoterrestris II 24 A acidoterrestris IIA 29 A acidocaldarius IA 30 A acidocaldarius type II 15 A acidoterrestrisDSM 2498 16 A acidoterrestris ATCC49025 17 A acidiphilus 18 A hesperidum 19 A herbarius 20 A acidocaldariusA1 21 B subtilis 22G stearothermophilus lanes 14 and 23 molecular marker

DSM_2498 00003231 -00003234 -00025641 0ATCC_49025 00022455 00005656 -00009252 -00001833 051 053 -000013

Figure 9 Phylogenetic tree of partial 16S rRNA sequence of 11 Aacidoterrestris isolatesThe tree was constructed by neighbor joiningmethod using Clustal Omega software

For Hin6I there were two types of patterns for the Aacidoterrestris and A acidocaldarius A acidoterrestris DSM2498 belongs to the cluster I while A acidoterrestris ATCC49025 belongs to the cluster II A acidocaldarius DSM 446represents cluster II of the A acidocaldarius group while AacidocaldariusA1 represents cluster I

HphI endonuclease produces two types of patterns forA acidoterrestris analogically as before and three types ofpatterns for A acidocaldarius One sample of A acidoter-restris cluster II has a slightly different pattern and has been

classified as type II in other analyses Two of the threepatterns of A acidocaldarius differ with only one bandA acidocaldarius DSM 446 represents cluster II of the Aacidocaldarius group while A acidocaldarius A1 representscluster I

34 DNA Sequencing DNA sequencing was performed forselected strains Sequencing of the 16S rDNA gene wasperformed to confirm the proper identification of the isolatesand to identify the non-Alicyclobacillus isolates The 16SrDNA sequence of strains 31 and 34 classified as type Ishowed the greatest similarity toA acidoterrestrisDSM 2498also classified as type I The sequences of strains 33 51 52 5355 and 56 classified as type II showed the greatest similaritytoA acidoterrestrisATCC49025 also classified as type IIThesequence of strain 41 classified as type II showed the greatestsimilarity to A acidoterrestris DSM 3922

Figure 9 shows the phylogenetic tree constructed from the16S rDNA sequences of analyzed strains of A acidoterrestrisThe sequence analysis indicates that A acidoterrestris DSM2498 and two other type I strains are closely related and itshows greater variation within class II strains The isolatesselected for sequencing represent both groups and different

BioMed Research International 9

sources which they were isolated from concentrated applejuice (isolates 33 and 41) fresh apples (isolate 34) concen-trated blackcurrant juice (isolate 31) concentrated raspberryjuice (isolate 56) concentrated beetroot juice (isolate 51)and concentrated cherry juice (isolates 52 53 and 55) Thesequence analysis shows no apparent correlation betweendiversity of the 16S rDNA sequence and the sources of theisolates

Fivewhole vdc gene clusterswere sequenced Twoof themwere isolated from the reference strains A acidoterrestrisDSM 2498 and A acidoterrestris ATCC 49025 2416 bpfragments of the vdc regions were aligned The vdc regionsequence of strains 15 and 34 classified as type I showed999 identity with the vdc sequence of A acidoterrestrisDSM 2498 the sequence of strain 41 classified as typeII showed 993 identity with the vdc sequence A aci-doterrestris ATCC 49025 Vdc sequences of A acidoterrestrisDSM 2498 and A acidoterrestris DSM 49025 showed 945identity For the last pair protein sequences of the geneproducts showed 985 positives and 965 identities forVdcB 994 and 989 for VdcC and 100 and 974 forVdcD

RpoB gene sequencing was performed to confirm thatboth primer pairs Gru3ndashGru4 and Gru5ndashGru6 enabledto amplify the correct DNA fragment and that degenera-tion of the primers did not affect the sequence specificityalthough the degenerated primers produced significantlylower amount of PCR product All of the sequences obtainedshowed greatest similarity to the appropriate rpoB genes

The GenBank accession numbers are KX371237-KX371249 for 16S rDNA sequences KX453673-KX453677for full vdc region sequences KX453678 and KX453679 forpartial vdc sequences of A herbarius and A acidiphilusrespectively and KX453680- KX453682 for partial rpoBsequences

4 Discussion

Among more than one thousand samples of concentratedapple juice tested in our laboratory between 2004 and 201267 was contaminated with Alicyclobacillus sp and 31 ofthe isolates were identified as A acidoterrestris [59] Thestatistics show that Alicyclobacillus spoilage is still a majorconcern in the fruit processing industry

In this study 75 guaiacol producing and non-guaiacolproducing strains were isolated from various fruit juicesconcentrated fruit juices and fresh apples Additionally8 reference strains were used The isolates and referencestrains were examined using classic methods and PCR-RFLP focusing on 16S rDNA and rpoB gene fragments aswell as the vdc region fragment For selected strains DNAsequencing of the 16S rDNA gene was performed Sixty ofthe isolates analyzed were identified as A acidoterrestris12 as A acidocaldarius one as Brevibacillus agri and twoas Bacillus ginsengihumi Four of the isolates which gaveambiguous results during classic identification (nontypicalcolour on an erythritol medium or lack of growth at 65∘Cconnected with lack of guaiacol production) were identified

by genetic methods as A acidoterrestris or A acidocaldariusA acidoterrestris isolates were grouped in two major clusters27 of the isolates belonged to the cluster I and 33 to thecluster II Also A acidocaldarius isolates were grouped intotwo clusters but showed more intracluster diversity

These results support the observations made by Osopaleat al [60] and Durak et al [23] who also reported two geneticclusters among analyzed A acidoterrestris samples by RAPDanalysisand by 16S rDNA sequencing respectively

Most of the strains analyzed in this study were isolatedfrom concentrated apple juice as this is the main subject ofthe Alicyclobacillus focused screening made or outsourced bypolish fruit industry however it is one of the main subjectsof similar screenings performed by fruit industry worldwideA acidoterrestris representing both clusters have been foundin concentrated apple juice For other sources three Aacidoterrestris cluster II isolates have been found in orangejuices two cluster I in concentrated black currant juice andthree cluster II in concentrated cherry juice however theseare only single observations and further research is needed toestablish ifA acidoterrestris strains representing both clustersare found in other juices

Genetic methods are broadly used in the detectioncharacterization and differentiation of microorganisms Theapplication of PCR-RFLP in the characterization of Ali-cyclobacillus and other thermoacidophilic bacteria isolatedfrom the apple juice processing environment has beendescribed by Chen et al [52] although only the 16S rDNAgene was the subject of this study and the Hin6I enzymewas not used RpoB gene has never been subjected to PCR-RFLP or any other genetic analysis of Alicyclobacillus sofar although the value of this gene in taxonomy has beenconfirmed by many studies on other microorganisms Todate there are no reports on the use of the vdc gene cluster intaxonomic studies of microorganisms although its sequencemay be proven valuable for research both on the vectors anddiversity and evolution of the element itself Hin6I restrictionpatterns for 16S rDNAwere sufficient to differentiate betweenall the species analyzed but provided no closer data onintraspecies diversity The diversity was revealed both by therpoB gene and vdc region RFLP analyses

Vdc gene cluster of Streptomyces sp described by Chowet al [55] consists of three genes vdcB (06kb) vdcC (14 kb)and vdcD (02 kb) transcribed as single policistronic mRNAmolecule All three genes were essential to produce guaiacolfrom vanillic acid Niwa and Kawamoto [38] describedanalogical gene cluster in A acidoterrestris consisting ofORF1 (597 bp) ORF2 (1425 bp) and ORF3 (321 bp) Threerespective open reading frameswere found in all five analyzedvdc sequences

Alicyclobacillus acidoterrestris RFLP patterns of boththe rpoB gene and vdc region showed consistently thatthere were two major types among the isolates one similarto the reference strain A acidoterrestris DSM 2498 andthe other similar to the reference strain A acidoterrestrisATCC 49025 Obtaining such consistent data concerningtwo genetic elements of distant function reveals that there isdeeper intraspecies genetic diversity in the A acidoterrestrisspecies This division may be considered when examining

10 BioMed Research International

other features of Alicyclobacillus such as susceptibility totemperature or high hydrostatic pressure in order to establishif there are any differences between the groups

Both the rpoB gene and vdc region seem to be single-copy genetic elements and showed no signs of intragenomicheterogeneity which often makes the RFLP comparison ofrDNA genes harder to perform

The sequence analysis of the 16S rDNA fragment ofselected isolates shows no apparent correlation betweendiversity of the 16S rDNA sequence and the sources whichthe strains were isolated from

In conclusion the application of PCR-RFLP has beenproven to be a fast and reliable method for Alicyclobacillusidentification and differentiation The method is also techni-cally less difficult than most of other molecular techniquesTwo major groups of A acidoterrestris have been identifiedThe primers designed for this study could be useful in furtherresearch on Alicyclobacillus

Data Availability

The DNA sequences obtained during this study have beendeposited in GenBank and the accession numbers areincluded within the article

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

Our research project was fully sponsored by Polish Ministryof Science and Higher Education with funds for statutoryactivity

References

[1] K Yamazaki H Teouka and H Shinano ldquoIsolation and identi-fication of alicyclobacillus acidoterrestris from acidic beveragesrdquoBioscience Biotechnology and Biochemistry vol 60 no 3 pp543ndash545 1996

[2] G DARLAND and T D BROCK ldquoBacillus acidocaldar-ius spnov an Acidophilic Thermophilic Spore-forming Bac-teriumrdquo Journal of General Microbiology vol 67 no 1 pp 9ndash151971

[3] G Deinhard P Blanz K Poralla and E Altan ldquoBacillusacidoterrestris sp nov a new thermotolerant acidophile isolatedfrom different soilsrdquo Systematic and Applied Microbiology vol10 no 1 pp 47ndash53 1987

[4] G Deinhard J Saar W Krischke and K Poralla ldquoBacilluscycloheptanicus sp nov a new thermoacidophile containing 120596-cycloheptane fatty acidsrdquo Systematic and Applied Microbiologyvol 10 no 1 pp 68ndash73 1987

[5] L Albuquerque F A Rainey A P Chung et al ldquoAlicyclobacillushesperidum sp nov and a related genomic species from solfa-taric soils of Sao Miguel in the Azoresrdquo International Journalof Systematic and Evolutionary Microbiology vol 50 no 2 pp451ndash457 2000

[6] K Goto H Matsubara K Mochida et al ldquoAlicyclobacillusherbarius sp nov a novel bacterium containing120596-cycloheptane

fatty acids isolated from herbal teardquo International Journal ofSystematic and EvolutionaryMicrobiology vol 52 no 1 pp 109ndash113 2002

[7] K Goto Y Tanimoto T Tamura et al ldquoIdentificationof thermoacidophilic bacteria and a new Alicyclobacillusgenomic species isolated from acidic environments in JapanrdquoExtremophiles vol 6 no 4 pp 333ndash340 2002

[8] H Matsubara K Goto T Matsumura et al ldquoAlicyclobacillusacidiphilus sp nov a novel thermo-acidophilic 120596-alicyclicfatty acid-containing bacterium isolated from acidic beveragesrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 52 no 5 pp 1681ndash1685 2002

[9] R V Orr R L Shewfelt C J Huang S Tefera and L RBeuchat ldquoDetection of guaiacol produced by Alicyclobacillusacidoterrestris in apple juice by sensory and chromatographicanalyses and comparison with spore and vegetative cell popu-lationsrdquo Journal of Food Protection vol 63 no 11 pp 1517ndash15222000

[10] G L Pettipher M E Osmundson and J MMurphy ldquoMethodsfor the detection and enumeration of Alicyclobacillus acidoter-restris and investigation of growth and production of taint infruit juice and fruit juice-containing drinksrdquo Letters in AppliedMicrobiology vol 24 no 3 pp 185ndash189 1997

[11] A C N F Spinelli A S Sant1015840Ana S Rodrigues Jr andP R Massaguer ldquoInfluence of different filling cooling andstorage conditions on the growth of Alicyclobacillus acidoter-restris CRA7152 in orange juicerdquo Applied and EnvironmentalMicrobiology vol 75 no 23 pp 7409ndash7416 2009

[12] MCMaldonado C Belfiore andA RNavarro ldquoTemperaturesoluble solids and pH effect on Alicyclobacillus acidoterrestrisviability in lemon juice concentraterdquo Journal of IndustrialMicrobiology and Biotechnology vol 35 no 2 pp 141ndash144 2008

[13] M N U Eiroa V C A Junqueira and F L Schmidt ldquoAlicy-clobacillus in orange juice Occurrence and heat resistance ofsporesrdquo Journal of Food Protection vol 62 no 8 pp 883ndash8861999

[14] I Walls and R Chuyate ldquoIsolation of Alicyclobacillus acidoter-restris from fruit juicesrdquo Journal of AOAC International vol 83no 5 pp 1115ndash1120 2000

[15] M E Parish and R M Goodrich ldquoRecovery of presumptiveAlicyclobacillus strains from orange fruit surfacesrdquo Journal ofFood Protection vol 68 no 10 pp 2196ndash2200 2005

[16] D F Splittstoesser J J Churey and C Y Lee ldquoGrowthcharacteristics of aciduric sporeforming bacilli isolated fromfruit juicesrdquo Journal of Food Protection vol 57 no 12 pp 1080ndash1083 1994

[17] J Baumgart M Husemann and C Schmidt ldquoAlicyclobacil-lus acidoterrestris vorkommen bedeutung und nachweis ingetranken und getrankegrundstoffenrdquo Flussiges Obst vol 64pp 178ndash180 1997

[18] C A Wisse and M E Parish ldquoIsolation and enumeration ofspore-forming thermoacidophilic rod-shaped bacteria fromcitrus processing environmentsrdquo Dairy Food EnvironmentalSanitation vol 18 pp 504ndash509 1998

[19] J Baumgart and S Menje ldquoThe impact of Alicyclobacillusacidoterestris on the quality of juices and soft drinksrdquo FruitProcess vol 10 pp 251ndash254 2000

[20] S Y Eguchi G P Manfio M E Pinhatti E Azuma and SF Variane ldquoAcidotermofilic sporeforming bacteria (ATSB) inorange juices ecology and involvement in the deterioration offruit juices Report of the Research Project Part IIrdquo Fruit Processvol 11 pp 55ndash62 2001

BioMed Research International 11

[21] P AGouws L Gie A Pretorius andNDhansay ldquoIsolation andidentification of Alicyclobacillus acidocaldarius by 16S rDNAfrom mango juice and concentraterdquo International Journal ofFood Science amp Technology vol 40 no 7 pp 789ndash792 2005

[22] K Goto K Mochida Y Kato et al ldquoProposal of six speciesof moderately thermophilic acidophilic endospore-formingbacteria Alicyclobacillus contaminans sp nov Alicyclobacillusfastidiosus sp novAlicyclobacillus kakegawensis sp novAlicy-clobacillus macrosporangiidus sp nov Alicyclobacillus saccharisp nov and Alicyclobacillus shizuokensis sp novrdquo InternationalJournal of Systematic and Evolutionary Microbiology vol 57 no6 pp 1276ndash1285 2007

[23] M Z Durak J J Churey M D Danyluk and R W WoroboldquoIdentification and haplotype distribution of Alicyclobacillusspp from different juices and beveragesrdquo International Journalof Food Microbiology vol 142 no 3 pp 286ndash291 2010

[24] B Sokolowska J Niezgoda A Dekowska et al ldquoIncidence ofAlicyclobacillus spp in Polish apple and dark berry juice con-centrates and the ability of isolated A acidoterrestris strains tospoilage of these juicesrdquo Postępy Nauki i Technologii PrzemysłuRolno-Spozywczego vol 71 no 1 pp 5ndash20 2016

[25] I Walls and R Chuyate ldquoAlicyclobacillus historical perspectiveand preliminary characterization studyrdquo Dairy Food EnvironSanitation18 pp 499ndash503 1998

[26] H-A Duong and N Jensen ldquoSpoilage of iced tea by Alicy-clobacillusrdquo Food Australia vol 52 no 7 p 292 2000

[27] M Niwa and A Kuriyama ldquoA acidoterrestris rapid detectionkitrdquo Fruit Process vol 13 pp 328ndash331 2003

[28] K S Bahceci V Gokmen and J Acar ldquoFormation of guaiacolfrom vanillin by Alicyclobacillus acidoterrestris in apple juice Amodel studyrdquo European Food Research and Technology vol 220no 2 pp 196ndash199 2005

[29] R C Witthuhn E van der Merwe P Venter and M CameronldquoGuaiacol production from ferulic acid vanillin and vanillicacid by Alicyclobacillus acidoterrestrisrdquo International Journal ofFood Microbiology vol 157 no 1 pp 113ndash117 2012

[30] A Borlinghaus and R Engel ldquoAlicyclobacillus incidence in com-mercial apple juice concentrate (AJC) supplies and validationrdquoFruit Process vol 7 pp 262ndash266 1997

[31] D F Splittstoesser C Y Lee and J J Churey ldquoControl ofAlicyclobacillus in the juice industryrdquo Dairy Food EnvironSanitation vol 18 pp 585ndash587 1998

[32] N Jensen ldquoAlicyclobacillus in Australiardquo Food Australia vol 52no 7 pp 282ndash285 2000

[33] IWalls andR Chuyate ldquoSpoilage of fruit juices byAlicyclobacil-lus acidoterrestrisrdquo Food Australia vol 52 no 7 pp 293ndash2952000

[34] N Jensen and F B Whitfield ldquoRole of Alicyclobacillus acidoter-restris in the development of a disinfectant taint in shelf-stablefruit juicerdquo Letters in Applied Microbiology vol 361 pp 9ndash142003

[35] D Gocmen A Elston T Williams M Parish and R LRouseff ldquoIdentification of medicinal off-flavours generated byAlicyclobacillus species in orange juice using GC-olfactometryand GC-MSrdquo Letters in Applied Microbiology vol 40 no 3 pp172ndash177 2005

[36] M Niwa ldquoControl of hazardous bacteria in acidic beveragesby using a guaiacol detection kit (peroxidase methodrdquo FruitProcess vol 15 pp 388ndash392 2005

[37] B Siegmund and B Pollinger-Zierler ldquoGrowth behavior ofoff-flavor-forming microorganisms in apple juicerdquo Journal of

Agricultural and Food Chemistry vol 55 no 16 pp 6692ndash66992007

[38] M Niwa and A Kawamoto ldquoDevelopment of a rapid detectionmethod of A acidoterrestris hazardous bacteria to acidicbeveragerdquo Fruit Process vol 13 pp 102ndash107 2003

[39] T A Eisele and M J Semon ldquoBest estimated aroma andtaste detection threshold for guaiacol in water and apple juicerdquoJournal of Food Science vol 70 no 4 pp S267ndashS269 2005

[40] B Zierler B Siegmund and W Pfannhauser ldquoDeterminationof off-flavour compounds in apple juice caused by microor-ganisms using headspace solid phase microextraction-gaschromatography-mass spectrometryrdquo Analytica Chimica Actavol 520 no 1-2 pp 3ndash11 2004

[41] K S Bahceci and J Acar ldquoModeling the combined effectsof pH temperature and ascorbic acid concentration on theheat resistance of Alicyclobacillus acidoterrestisrdquo InternationalJournal of Food Microbiology vol 120 no 3 pp 266ndash273 2007

[42] J Baumgart ldquoChapter 11 Media for the detection and enu-meration of Alicyclobacillus acidoterrestris and Alicyclobacillusacidocaldarius in foodsrdquo in Progress in Industrial Microbiologyvol 37 pp 161ndash166 Elsevier 2003

[43] M Lin M Al-Holy S-S Chang et al ldquoRapid discriminationof Alicyclobacillus strains in apple juice by Fourier transforminfrared spectroscopyrdquo International Journal of Food Microbiol-ogy vol 105 no 3 pp 369ndash376 2005

[44] J Wang T Yue Y Yuan X Lu J-H Shin and B RascoldquoDiscrimination of alicyclobacillus strains using nitrocellulosemembrane filter and attenuated total reflectance fourier trans-form infrared spectroscopyrdquo Journal of Food Science vol 76 no2 pp 137ndash142 2011

[45] M A Al-Holy M Lin O A Alhaj and M H Abu-GoushldquoDiscrimination between Bacillus and Alicyclobacillus Isolatesin Apple Juice by Fourier Transform Infrared Spectroscopy andMultivariate Analysisrdquo Journal of Food Science vol 80 no 2 ppM399ndashM404 2015

[46] WH Groenewald P A Gouws andR CWitthuhn ldquoIsolationidentification and typification of Alicyclobacillus acidoterrestrisand Alicyclobacillus acidocaldarius strains from orchard soiland the fruit processing environment in South Africardquo FoodMicrobiology vol 26 no 1 pp 71ndash76 2009

[47] J Zhang T Yue and Y Yuan ldquoAlicyclobacillus contaminationin the production line of Kiwi products in Chinardquo PLoS ONEvol 8 no 7 p e67704 2013

[48] I C McKnight M N U Eiroa A S SantrsquoAna and P RMassaguer ldquoAlicyclobacillus acidoterrestris in pasteurized exoticBrazilian fruit juices Isolation genotypic characterization andheat resistancerdquo FoodMicrobiology vol 27 no 8 pp 1016ndash10222010

[49] L Felix-Valenzuela I Guardiola-Avila A Burgara-Estrella MIbarra-Zavala and V Mata-Haro ldquoGenotypic and phenotypicdiversity of Alicyclobacillus acidocaldarius isolatesrdquo Letters inApplied Microbiology vol 61 no 4 pp 367ndash373 2015

[50] J D Wisotzkey P Jurtshuk Jr G E Fox G Deinhard andK Poralla ldquoComparative sequence analyses on the 16S rRNA(rDNA) of Bacillus acidocaldarius Bacillus acidoterrestris andBacillus cycloheptanicus and proposal for creation of a newgenus Alicyclobacillus gen novrdquo International Journal of Sys-tematic Bacteriology vol 42 no 2 pp 263ndash269 1992

[51] C J Connor H Luo B B McSpadden Gardener and HH Wang ldquoDevelopment of a real-time PCR-based systemtargeting the 16S rRNA gene sequence for rapid detection of

12 BioMed Research International

Alicyclobacillus spp in juice productsrdquo International Journal ofFood Microbiology vol 99 no 3 pp 229ndash235 2005

[52] S ChenQ Tang X Zhang et al ldquoIsolation and characterizationof thermo-acidophilic endospore-forming bacteria from theconcentrated apple juice-processing environmentrdquo FoodMicro-biology vol 23 no 5 pp 439ndash445 2006

[53] K Goto A Nishibori Y Wasada K Furuhata M Fukuyamaand M Hara ldquoIdentification of thermo-acidophilic bacteriaisolated from the soil of several Japanese fruit orchardsrdquo Lettersin Applied Microbiology vol 46 no 3 pp 289ndash294 2008

[54] J Chen X Ma Y Yuan and W Zhang ldquoSensitive and rapiddetection ofAlicyclobacillus acidoterrestris using loop-mediatedisothermal amplificationrdquo Journal of the Science of Food andAgriculture vol 91 no 6 pp 1070ndash1074 2011

[55] K T Chow M K Pope and J Davies ldquoCharacterization of avanillic acid non-oxidative decarboxylation gene cluster fromStreptomyces sp D7rdquo Microbiology vol 145 no 9 pp 2393ndash2403 1999

[56] K Matsubara ldquoA method for detecting andor identifying gua-iacol producing micro organismrdquo Patent no JP2003000259A2003

[57] Y Wang T Yue Y Yuan and Z Gao ldquoIsolation and identifi-cation of thermo-acidophilic bacteria from orchards in chinardquoJournal of Food Protection vol 73 pp 390ndash394 2010

[58] M Shemesh R Pasvolsky N Sela S J Green and V andZakinldquoDraft Genome Sequence of Alicyclobacillus acidoterrestrisStrain ATCC 49025rdquo Genome Announcement vol 1 no 5Article ID e00638ndash13 2013

[59] B Sokołowska S Skapska M Fonberg-Broczek et al ldquoFactorsinfluencing the inactivation of Alicyclobacillus acidoterrestrisspores exposed to high hydrostatic pressure in apple juicerdquoHighPressure Research vol 33 no 1 pp 73ndash82 2013

[60] B A Osopale C R Witthuhn J Albertyn and F A Oguntoy-inbo ldquoCulture dependent and independent genomic identifica-tion ofAlicyclobacillus species in contaminated commercial fruitjuicesrdquo Food Microbiology vol 56 pp 21ndash28 2016

Hindawiwwwhindawicom

International Journal of

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Submit your manuscripts atwwwhindawicom

BioMed Research International 3

vdc fr (1 - 27) vdc1 fr(314 - 333)

bur5(560 - 584)

vdc B (245 - 838)

vdcS (1044 - 1063)

vdc C (831 - 2252)vdc region (1 - 2523)

bur6 (2124 - 2146)

vdcK (2202 - 2222) vdc1 rev (2287 - 2308)

vdc rev (2493 - 2523)

vdc D (2290 - 2517)

Figure 1 Vdc region positions of genetic elements and primers

Table 1 The positions and directions of vdc primers

Primer name Primer position (bp) Primer directionvdc fr 1-27 forwardvdc rev 2493-2523 reversevdc1 fr 314-333 forwardvdc1 rev 2287-2308 reversevdc K 2202-2222 forwardvdc S 1044-1063 forwardBur5 560-584 forwardBur6 2124-2146 reverse

The 2523 bp vdc fr ndash vdc rev amplification productcontained the whole vdc region The size of the Bur5-Bur6 amplification product was 1586 bp PCR reactions wereperformed in a total volume of 50120583l containing 25 ng of thetemplate DNA 5 pM of each of the primers and 25120583l ofthe DreamTaq Green PCRMaster Mix (Thermo Scientific)PCR was performed under the following conditions initialdenaturation at 94∘C for 2min 30 cycles of denaturation at94∘C for 30 sec annealing at 59∘C for 50 sec elongation 72∘Cfor 1min 40 sec and the final elongation at 72∘C for 5min

243 rpoB Gene Amplification A fragment of rpoB genewas amplified using Gru3ndashGru6 primers The primerswere designed based on a comparison of the rpoB genesequences of Alicyclobacillus acidocaldarius Bacillus subtilisand Geobacillus stearothermophilus as well as sequencesderived fromA acidoterrestris 49025A hesperidumURH17-3-68 and A herbarius DSM 13609 raw genomic sequencesGru5 and Gru6 primers are nondegenerate versions of theGru3 and Gru4 primers respectively The primer sequenceswere Gru3 (CGYGACGTDCACTAYTCBCACTA) Gru4 (51015840ndash GCCCANACYTCCATCTCRCCRAA) Gru5 (51015840 ndash CGC-GACGTACACTATTCGCACTA) and Gru6 (51015840 ndash GCC-CAAACCTCCATCTCACCAAA) The size of the amplifi-cation product was 1735bp PCR reactions were performedin a total volume of 50 120583l containing 30 ng of the templateDNA 40 pM of each of the primers and 25120583l of theDreamTaq Green PCR Master Mix (Thermo Scientific)PCR was performed under the following conditions initialdenaturation at 94∘C for 2min 35 cycles of denaturation at94∘C for 30 sec annealing at 57∘C (for the Bur5 and Bur6primer pairs) or at 59∘C (for the Bur3 and Bur4 primers)

for 35 sec elongation 72∘C for 1min 45 sec and the finalelongation at 72∘C for 5min

25 PCR-RFLP 5-15120583l of the PCR products were digestedwith BsuRI Hin6I and HphI (Thermo Scientific) in 20 120583lvolumes The samples were incubated at 37∘C for 1-2 hoursand the enzymes were then inactivated with ten-minuteincubation at 80∘C The digests were analyzed on 25ndash3agarose gel

26 DNA Sequencing DNA samples were sequencedusing 8F and shortened 1512R primers for the 16SrDNA gene vdc fr vdc rev vdc1 fr vdc1 rev Bur5Bur6 and two additional primers to fill the gaps vdcS(51015840 ndash AATCACGCGCTGATGATGGG) and vdcK (51015840 ndashTTGGCAACGGAGAAGTGGGAG) for the vdc region andGru3 ndash Gru6 for the rpoB gene

The contig assemblies sequence alignments and phylo-genetic analysis were performed using Serial Cloner software(SerialBasic) and Clustal Omega The sequences were com-pared to theGenBank sequences database using BLAST tools

3 Results

31 RFLP Analysis of 16S rDNA Fragment The 1495 bpfragment of the 16S rDNA gene amplified using 8F andshortened 1512R primers was digested by BsuRI Hin6I andHphI

While BsuRI and HphI digestions did not allow todistinguish between all the species analyzed the patternsobtained by Hin6I digestion were species specific (Figure 2)

All analyzed isolates with their features and RFLP profilesare described in Table 2

32 RFLP Analysis of the vdc Region Fragment The fragmentof vdc region was amplified using Bur5 and Bur6 primersThe product was a single band of 1586 bp and was observedonly for guaiacol producing strains A acidoterrestris Aacidophilus and A herbarius The PCR product was digestedby BsuRI (Figure 3) Hin6I (Figure 4) and HphI (Figure 5)

BsuRI and Hin6I RFLP patterns enabled distinguishingbetween all the species analyzed and divided the A acidoter-restris group into two clusters Type I pattern was identical tothe pattern given by A acidoterrestris DSM 2498 and type IIpattern was identical to A acidoterrestris ATCC 49025 The

4 BioMed Research International

Table2Listof

isolatesa

ndtheirR

FLPprofi

lesAAT

Aacid

oterrestris

AAC

Aacid

ocaldariu

sBA

Brevibacillusa

griBG

bacillus

ginsengihum

i

IsolateSource

Guaiacolprodu

ction

Erythrito

lutilization

RFLP

analysis

16S

Hin61

vdc

BsuR

Ivdc

Hin61

vdc

Hph

IrpoB

BsuR

IrpoB

Hin61

rpoB

Hph

I1

concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

2concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

3interm

ediateforb

everagep

rodu

ction

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

4interm

ediateforb

everagep

rodu

ction

nono

AAC

--

-AAC

(I)

AAC

(I)

AAC

(I)

5concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

6concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

7concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

8concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

9orange

beverage

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

10concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

11concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

12concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

13concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

14concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

15concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

16concentrated

applejuice

yes

nontypical

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

17banana

nectar

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

18orange

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

19concentrated

orange

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

20concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

21orange

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

22concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

23concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

24concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

25fre

shapples

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

26concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

27concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

28concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

29concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

30concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

31concentrated

blackcurrantjuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

32concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

33concentrated

applejuice

yes

nontypical

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

34fre

shapples

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(IA)

AAT

(I)

AAT

(I)

AAT

(I)

BioMed Research International 5

Table2Con

tinued

IsolateSource

Guaiacolprodu

ction

Erythrito

lutilization

RFLP

analysis

16S

Hin61

vdc

BsuR

Ivdc

Hin61

vdc

Hph

IrpoB

BsuR

IrpoB

Hin61

rpoB

Hph

I35

fresh

apples

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

36concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

37concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

38interm

ediateforb

everagep

rodu

ction

nono

AAC

--

-AAC

(I)

AAC

(I)

AAC

(IA)

39concentrated

applejuice

yes

-BG

--

-BG

BGBG

40concentrated

applejuice

yes

-BG

--

-BG

BGBG

41concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

42concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(IIA)

43concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

44spoiledappleb

everage

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

45concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

46spoiledapplejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

47concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

48concentrated

straw

berryjuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

49concentrated

cherry

juice

nono

AAC

--

-AAC

(IA)

AAC

(I)

AAC

(IA)

50tomatojuicefrom

fresh

tomatoes

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

51concentrated

beetroot

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

52concentrated

cherry

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

53concentrated

cherry

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

54concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(IIA)

AAT

(II)

AAT

(II)

55concentrated

cherry

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

56concentrated

raspberryjuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

57concentrated

applejuice

yes

nontypical

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

58orange

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

59concentrated

applejuice

nono

ntypical

AAC

--

-AAC

(IA)

AAC

(I)

AAC

(I)

60concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

61concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

62tomatojuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

63concentrated

applejuice

yes

-BA

--

-BA

BABA

64concentrated

applejuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

65cherry

puree

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

66concentrated

blackcurrantjuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

67concentrated

straw

berryjuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

68clou

dyapplejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

69concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

70concentrated

applejuice

nono

AAC

--

-AAC

(IA)

AAC

(I)

AAC

(I)

71concentrated

applejuice

nono

AAC

--

-AAC

(IA)

AAC

(I)

AAC

(I)

72concentrated

applejuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

73concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

74concentrated

applejuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

75concentrated

applejuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

6 BioMed Research International

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

bp

1000

500

200

Figure 2 Restriction patterns of a 1495 bp fragment of the 16S rDNA gene digested with Hin6I Lanes 6 7 9 14 and 18 A acidoterrestris 23 4 8 10 11 12 13 15 16 and 17 A acidocaldarius 5 Brevibacillus agri 19 A acidoterrestris DSM 2498 20 A acidoterrestris ATCC49025 21A acidiphilus 22 A hesperidum 23 A herbarius 24 A acidocaldarius 25 B subtilis 26 G stearothermophilus lane 1 molecular marker

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 3 Restriction patterns of a 1586 bp fragment of the vdc region digested with BsuRI Lanes 1ndash3 5 7ndash11 13 and 14 A acidoterrestristype I 4 6 12 and 15 A acidoterrestris type II 16 A acidoterrestris DSM 2498 17 A acidoterrestris ATCC49025 18 A acidiphilus 19 Aherbarius 20 molecular marker

HphI patterns confirm this rule with the exception of onestrain which represented the type I pattern in the BsuRI andHin6I analysis

33 RFLP Analysis of the rpoB Gene Fragment The 1735 bpfragment of the rpoB gene was amplified using Gru3 andGru4 or Gru5 and Gru6 primers Gru3 and Gru4 primerswere degenerated versions of Gru5 and Gru6 (respectively)and were used for amplification of isolates other thanA acidoterrestris A acidoterrestris isolates were amplifiedeither with Gru3ndashGru4 or Gru5ndashGru6 primers and theGru5ndashGru6 primer pair was chosen due to the better effi-ciency of the reaction Considering the individual isolates theRFLP patterns were identical for both pairs of primers

Thepatterns obtained by all of the nucleases used (Figures6ndash8) enabled distinguishing between the species analyzedWith exception of two strains the BsuRI patterns dividedthe A acidoterrestris group into two clusters analogicalto the clusters revealed by the vdc region analysis Thepattern (IIA) is shown by two exceptional strains mostresembling the type II pattern of A acidoterrestris andthose strains were classified as type II in other analyses Thereference strain ATCC 49025 shows this type of patternThe A acidocaldarius group was represented by three typesof patterns two of them resembling each other A acido-caldarius DSM 446 belongs to the cluster II of A acido-caldarius group while A acidocaldarius A1 was assigned tocluster I

BioMed Research International 7

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 4 Restriction patterns of a 1586 bp fragment of the vdc region digested with Hin6I Lanes 1ndash3 5 7ndash11 13 and 14 A acidoterrestristype I 4 6 12 and 15A acidoterrestris type II 16A acidoterrestrisDSM 2498 17A acidoterrestris ATCC49025 18 A acidiphilusDSM 1455819 A herbarius DSM 13609 lane 20 molecular marker

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 5 Restriction patterns of a 1586 bp fragment of the vdc region digested with HphI Lanes 2ndash4 6 8ndash12 14 and 15 A acidoterrestristype I 5 7 13 and 16 A acidoterrestris type II 17 A acidoterrestris DSM 2498 18 A acidoterrestris ATCC49025 19 A acidiphilus 20 Aherbarius lane 1 molecular marker

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Figure 6 Restriction patterns of a 1735 bp fragment of the rpoB gene digested with BsuRI Lanes 1ndash4 6 and 8ndash10 A acidoterrestris type I 57 21 22 and 24-26 A acidoterrestris type II 23 A acidoterrestris type IIA 27 A acidocaldarius type II 28 A acidocaldarius type IA 11 Aacidoterrestris DSM 2498 12 A acidoterrestris ATCC49025 13 A acidiphilus 14 A hesperidum 15 A herbarius 16 A acidocaldariusA1 17B subtilis 18 G stearothermophilus lane 19 molecular marker

8 BioMed Research International

Figure 7 Restriction patterns of a 1735 bp fragment of rpoB gene digested with Hin6I Lanes 3 and 5 A acidoterrestris type I 1 2 4 and 6Aacidoterrestris type II 17A acidocaldarius type I 18A acidocaldarius type II 8A acidoterrestrisDSM2498 9A acidoterrestrisATCC4902510A acidiphilus 11 A hesperidum 12A herbarius 13A acidocaldariusA1 14 B subtilis 15G stearothermophilus lanes 7 and 16 molecularmarker

Figure 8 Restriction patterns of a 1735 bp fragment of rpoB gene digested with HphI Lanes 1-4 6 8-11 13 25 and 27 A acidoterrestris I5 7 12 26 and 28 A acidoterrestris II 24 A acidoterrestris IIA 29 A acidocaldarius IA 30 A acidocaldarius type II 15 A acidoterrestrisDSM 2498 16 A acidoterrestris ATCC49025 17 A acidiphilus 18 A hesperidum 19 A herbarius 20 A acidocaldariusA1 21 B subtilis 22G stearothermophilus lanes 14 and 23 molecular marker

DSM_2498 00003231 -00003234 -00025641 0ATCC_49025 00022455 00005656 -00009252 -00001833 051 053 -000013

Figure 9 Phylogenetic tree of partial 16S rRNA sequence of 11 Aacidoterrestris isolatesThe tree was constructed by neighbor joiningmethod using Clustal Omega software

For Hin6I there were two types of patterns for the Aacidoterrestris and A acidocaldarius A acidoterrestris DSM2498 belongs to the cluster I while A acidoterrestris ATCC49025 belongs to the cluster II A acidocaldarius DSM 446represents cluster II of the A acidocaldarius group while AacidocaldariusA1 represents cluster I

HphI endonuclease produces two types of patterns forA acidoterrestris analogically as before and three types ofpatterns for A acidocaldarius One sample of A acidoter-restris cluster II has a slightly different pattern and has been

classified as type II in other analyses Two of the threepatterns of A acidocaldarius differ with only one bandA acidocaldarius DSM 446 represents cluster II of the Aacidocaldarius group while A acidocaldarius A1 representscluster I

34 DNA Sequencing DNA sequencing was performed forselected strains Sequencing of the 16S rDNA gene wasperformed to confirm the proper identification of the isolatesand to identify the non-Alicyclobacillus isolates The 16SrDNA sequence of strains 31 and 34 classified as type Ishowed the greatest similarity toA acidoterrestrisDSM 2498also classified as type I The sequences of strains 33 51 52 5355 and 56 classified as type II showed the greatest similaritytoA acidoterrestrisATCC49025 also classified as type IIThesequence of strain 41 classified as type II showed the greatestsimilarity to A acidoterrestris DSM 3922

Figure 9 shows the phylogenetic tree constructed from the16S rDNA sequences of analyzed strains of A acidoterrestrisThe sequence analysis indicates that A acidoterrestris DSM2498 and two other type I strains are closely related and itshows greater variation within class II strains The isolatesselected for sequencing represent both groups and different

BioMed Research International 9

sources which they were isolated from concentrated applejuice (isolates 33 and 41) fresh apples (isolate 34) concen-trated blackcurrant juice (isolate 31) concentrated raspberryjuice (isolate 56) concentrated beetroot juice (isolate 51)and concentrated cherry juice (isolates 52 53 and 55) Thesequence analysis shows no apparent correlation betweendiversity of the 16S rDNA sequence and the sources of theisolates

Fivewhole vdc gene clusterswere sequenced Twoof themwere isolated from the reference strains A acidoterrestrisDSM 2498 and A acidoterrestris ATCC 49025 2416 bpfragments of the vdc regions were aligned The vdc regionsequence of strains 15 and 34 classified as type I showed999 identity with the vdc sequence of A acidoterrestrisDSM 2498 the sequence of strain 41 classified as typeII showed 993 identity with the vdc sequence A aci-doterrestris ATCC 49025 Vdc sequences of A acidoterrestrisDSM 2498 and A acidoterrestris DSM 49025 showed 945identity For the last pair protein sequences of the geneproducts showed 985 positives and 965 identities forVdcB 994 and 989 for VdcC and 100 and 974 forVdcD

RpoB gene sequencing was performed to confirm thatboth primer pairs Gru3ndashGru4 and Gru5ndashGru6 enabledto amplify the correct DNA fragment and that degenera-tion of the primers did not affect the sequence specificityalthough the degenerated primers produced significantlylower amount of PCR product All of the sequences obtainedshowed greatest similarity to the appropriate rpoB genes

The GenBank accession numbers are KX371237-KX371249 for 16S rDNA sequences KX453673-KX453677for full vdc region sequences KX453678 and KX453679 forpartial vdc sequences of A herbarius and A acidiphilusrespectively and KX453680- KX453682 for partial rpoBsequences

4 Discussion

Among more than one thousand samples of concentratedapple juice tested in our laboratory between 2004 and 201267 was contaminated with Alicyclobacillus sp and 31 ofthe isolates were identified as A acidoterrestris [59] Thestatistics show that Alicyclobacillus spoilage is still a majorconcern in the fruit processing industry

In this study 75 guaiacol producing and non-guaiacolproducing strains were isolated from various fruit juicesconcentrated fruit juices and fresh apples Additionally8 reference strains were used The isolates and referencestrains were examined using classic methods and PCR-RFLP focusing on 16S rDNA and rpoB gene fragments aswell as the vdc region fragment For selected strains DNAsequencing of the 16S rDNA gene was performed Sixty ofthe isolates analyzed were identified as A acidoterrestris12 as A acidocaldarius one as Brevibacillus agri and twoas Bacillus ginsengihumi Four of the isolates which gaveambiguous results during classic identification (nontypicalcolour on an erythritol medium or lack of growth at 65∘Cconnected with lack of guaiacol production) were identified

by genetic methods as A acidoterrestris or A acidocaldariusA acidoterrestris isolates were grouped in two major clusters27 of the isolates belonged to the cluster I and 33 to thecluster II Also A acidocaldarius isolates were grouped intotwo clusters but showed more intracluster diversity

These results support the observations made by Osopaleat al [60] and Durak et al [23] who also reported two geneticclusters among analyzed A acidoterrestris samples by RAPDanalysisand by 16S rDNA sequencing respectively

Most of the strains analyzed in this study were isolatedfrom concentrated apple juice as this is the main subject ofthe Alicyclobacillus focused screening made or outsourced bypolish fruit industry however it is one of the main subjectsof similar screenings performed by fruit industry worldwideA acidoterrestris representing both clusters have been foundin concentrated apple juice For other sources three Aacidoterrestris cluster II isolates have been found in orangejuices two cluster I in concentrated black currant juice andthree cluster II in concentrated cherry juice however theseare only single observations and further research is needed toestablish ifA acidoterrestris strains representing both clustersare found in other juices

Genetic methods are broadly used in the detectioncharacterization and differentiation of microorganisms Theapplication of PCR-RFLP in the characterization of Ali-cyclobacillus and other thermoacidophilic bacteria isolatedfrom the apple juice processing environment has beendescribed by Chen et al [52] although only the 16S rDNAgene was the subject of this study and the Hin6I enzymewas not used RpoB gene has never been subjected to PCR-RFLP or any other genetic analysis of Alicyclobacillus sofar although the value of this gene in taxonomy has beenconfirmed by many studies on other microorganisms Todate there are no reports on the use of the vdc gene cluster intaxonomic studies of microorganisms although its sequencemay be proven valuable for research both on the vectors anddiversity and evolution of the element itself Hin6I restrictionpatterns for 16S rDNAwere sufficient to differentiate betweenall the species analyzed but provided no closer data onintraspecies diversity The diversity was revealed both by therpoB gene and vdc region RFLP analyses

Vdc gene cluster of Streptomyces sp described by Chowet al [55] consists of three genes vdcB (06kb) vdcC (14 kb)and vdcD (02 kb) transcribed as single policistronic mRNAmolecule All three genes were essential to produce guaiacolfrom vanillic acid Niwa and Kawamoto [38] describedanalogical gene cluster in A acidoterrestris consisting ofORF1 (597 bp) ORF2 (1425 bp) and ORF3 (321 bp) Threerespective open reading frameswere found in all five analyzedvdc sequences

Alicyclobacillus acidoterrestris RFLP patterns of boththe rpoB gene and vdc region showed consistently thatthere were two major types among the isolates one similarto the reference strain A acidoterrestris DSM 2498 andthe other similar to the reference strain A acidoterrestrisATCC 49025 Obtaining such consistent data concerningtwo genetic elements of distant function reveals that there isdeeper intraspecies genetic diversity in the A acidoterrestrisspecies This division may be considered when examining

10 BioMed Research International

other features of Alicyclobacillus such as susceptibility totemperature or high hydrostatic pressure in order to establishif there are any differences between the groups

Both the rpoB gene and vdc region seem to be single-copy genetic elements and showed no signs of intragenomicheterogeneity which often makes the RFLP comparison ofrDNA genes harder to perform

The sequence analysis of the 16S rDNA fragment ofselected isolates shows no apparent correlation betweendiversity of the 16S rDNA sequence and the sources whichthe strains were isolated from

In conclusion the application of PCR-RFLP has beenproven to be a fast and reliable method for Alicyclobacillusidentification and differentiation The method is also techni-cally less difficult than most of other molecular techniquesTwo major groups of A acidoterrestris have been identifiedThe primers designed for this study could be useful in furtherresearch on Alicyclobacillus

Data Availability

The DNA sequences obtained during this study have beendeposited in GenBank and the accession numbers areincluded within the article

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

Our research project was fully sponsored by Polish Ministryof Science and Higher Education with funds for statutoryactivity

References

[1] K Yamazaki H Teouka and H Shinano ldquoIsolation and identi-fication of alicyclobacillus acidoterrestris from acidic beveragesrdquoBioscience Biotechnology and Biochemistry vol 60 no 3 pp543ndash545 1996

[2] G DARLAND and T D BROCK ldquoBacillus acidocaldar-ius spnov an Acidophilic Thermophilic Spore-forming Bac-teriumrdquo Journal of General Microbiology vol 67 no 1 pp 9ndash151971

[3] G Deinhard P Blanz K Poralla and E Altan ldquoBacillusacidoterrestris sp nov a new thermotolerant acidophile isolatedfrom different soilsrdquo Systematic and Applied Microbiology vol10 no 1 pp 47ndash53 1987

[4] G Deinhard J Saar W Krischke and K Poralla ldquoBacilluscycloheptanicus sp nov a new thermoacidophile containing 120596-cycloheptane fatty acidsrdquo Systematic and Applied Microbiologyvol 10 no 1 pp 68ndash73 1987

[5] L Albuquerque F A Rainey A P Chung et al ldquoAlicyclobacillushesperidum sp nov and a related genomic species from solfa-taric soils of Sao Miguel in the Azoresrdquo International Journalof Systematic and Evolutionary Microbiology vol 50 no 2 pp451ndash457 2000

[6] K Goto H Matsubara K Mochida et al ldquoAlicyclobacillusherbarius sp nov a novel bacterium containing120596-cycloheptane

fatty acids isolated from herbal teardquo International Journal ofSystematic and EvolutionaryMicrobiology vol 52 no 1 pp 109ndash113 2002

[7] K Goto Y Tanimoto T Tamura et al ldquoIdentificationof thermoacidophilic bacteria and a new Alicyclobacillusgenomic species isolated from acidic environments in JapanrdquoExtremophiles vol 6 no 4 pp 333ndash340 2002

[8] H Matsubara K Goto T Matsumura et al ldquoAlicyclobacillusacidiphilus sp nov a novel thermo-acidophilic 120596-alicyclicfatty acid-containing bacterium isolated from acidic beveragesrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 52 no 5 pp 1681ndash1685 2002

[9] R V Orr R L Shewfelt C J Huang S Tefera and L RBeuchat ldquoDetection of guaiacol produced by Alicyclobacillusacidoterrestris in apple juice by sensory and chromatographicanalyses and comparison with spore and vegetative cell popu-lationsrdquo Journal of Food Protection vol 63 no 11 pp 1517ndash15222000

[10] G L Pettipher M E Osmundson and J MMurphy ldquoMethodsfor the detection and enumeration of Alicyclobacillus acidoter-restris and investigation of growth and production of taint infruit juice and fruit juice-containing drinksrdquo Letters in AppliedMicrobiology vol 24 no 3 pp 185ndash189 1997

[11] A C N F Spinelli A S Sant1015840Ana S Rodrigues Jr andP R Massaguer ldquoInfluence of different filling cooling andstorage conditions on the growth of Alicyclobacillus acidoter-restris CRA7152 in orange juicerdquo Applied and EnvironmentalMicrobiology vol 75 no 23 pp 7409ndash7416 2009

[12] MCMaldonado C Belfiore andA RNavarro ldquoTemperaturesoluble solids and pH effect on Alicyclobacillus acidoterrestrisviability in lemon juice concentraterdquo Journal of IndustrialMicrobiology and Biotechnology vol 35 no 2 pp 141ndash144 2008

[13] M N U Eiroa V C A Junqueira and F L Schmidt ldquoAlicy-clobacillus in orange juice Occurrence and heat resistance ofsporesrdquo Journal of Food Protection vol 62 no 8 pp 883ndash8861999

[14] I Walls and R Chuyate ldquoIsolation of Alicyclobacillus acidoter-restris from fruit juicesrdquo Journal of AOAC International vol 83no 5 pp 1115ndash1120 2000

[15] M E Parish and R M Goodrich ldquoRecovery of presumptiveAlicyclobacillus strains from orange fruit surfacesrdquo Journal ofFood Protection vol 68 no 10 pp 2196ndash2200 2005

[16] D F Splittstoesser J J Churey and C Y Lee ldquoGrowthcharacteristics of aciduric sporeforming bacilli isolated fromfruit juicesrdquo Journal of Food Protection vol 57 no 12 pp 1080ndash1083 1994

[17] J Baumgart M Husemann and C Schmidt ldquoAlicyclobacil-lus acidoterrestris vorkommen bedeutung und nachweis ingetranken und getrankegrundstoffenrdquo Flussiges Obst vol 64pp 178ndash180 1997

[18] C A Wisse and M E Parish ldquoIsolation and enumeration ofspore-forming thermoacidophilic rod-shaped bacteria fromcitrus processing environmentsrdquo Dairy Food EnvironmentalSanitation vol 18 pp 504ndash509 1998

[19] J Baumgart and S Menje ldquoThe impact of Alicyclobacillusacidoterestris on the quality of juices and soft drinksrdquo FruitProcess vol 10 pp 251ndash254 2000

[20] S Y Eguchi G P Manfio M E Pinhatti E Azuma and SF Variane ldquoAcidotermofilic sporeforming bacteria (ATSB) inorange juices ecology and involvement in the deterioration offruit juices Report of the Research Project Part IIrdquo Fruit Processvol 11 pp 55ndash62 2001

BioMed Research International 11

[21] P AGouws L Gie A Pretorius andNDhansay ldquoIsolation andidentification of Alicyclobacillus acidocaldarius by 16S rDNAfrom mango juice and concentraterdquo International Journal ofFood Science amp Technology vol 40 no 7 pp 789ndash792 2005

[22] K Goto K Mochida Y Kato et al ldquoProposal of six speciesof moderately thermophilic acidophilic endospore-formingbacteria Alicyclobacillus contaminans sp nov Alicyclobacillusfastidiosus sp novAlicyclobacillus kakegawensis sp novAlicy-clobacillus macrosporangiidus sp nov Alicyclobacillus saccharisp nov and Alicyclobacillus shizuokensis sp novrdquo InternationalJournal of Systematic and Evolutionary Microbiology vol 57 no6 pp 1276ndash1285 2007

[23] M Z Durak J J Churey M D Danyluk and R W WoroboldquoIdentification and haplotype distribution of Alicyclobacillusspp from different juices and beveragesrdquo International Journalof Food Microbiology vol 142 no 3 pp 286ndash291 2010

[24] B Sokolowska J Niezgoda A Dekowska et al ldquoIncidence ofAlicyclobacillus spp in Polish apple and dark berry juice con-centrates and the ability of isolated A acidoterrestris strains tospoilage of these juicesrdquo Postępy Nauki i Technologii PrzemysłuRolno-Spozywczego vol 71 no 1 pp 5ndash20 2016

[25] I Walls and R Chuyate ldquoAlicyclobacillus historical perspectiveand preliminary characterization studyrdquo Dairy Food EnvironSanitation18 pp 499ndash503 1998

[26] H-A Duong and N Jensen ldquoSpoilage of iced tea by Alicy-clobacillusrdquo Food Australia vol 52 no 7 p 292 2000

[27] M Niwa and A Kuriyama ldquoA acidoterrestris rapid detectionkitrdquo Fruit Process vol 13 pp 328ndash331 2003

[28] K S Bahceci V Gokmen and J Acar ldquoFormation of guaiacolfrom vanillin by Alicyclobacillus acidoterrestris in apple juice Amodel studyrdquo European Food Research and Technology vol 220no 2 pp 196ndash199 2005

[29] R C Witthuhn E van der Merwe P Venter and M CameronldquoGuaiacol production from ferulic acid vanillin and vanillicacid by Alicyclobacillus acidoterrestrisrdquo International Journal ofFood Microbiology vol 157 no 1 pp 113ndash117 2012

[30] A Borlinghaus and R Engel ldquoAlicyclobacillus incidence in com-mercial apple juice concentrate (AJC) supplies and validationrdquoFruit Process vol 7 pp 262ndash266 1997

[31] D F Splittstoesser C Y Lee and J J Churey ldquoControl ofAlicyclobacillus in the juice industryrdquo Dairy Food EnvironSanitation vol 18 pp 585ndash587 1998

[32] N Jensen ldquoAlicyclobacillus in Australiardquo Food Australia vol 52no 7 pp 282ndash285 2000

[33] IWalls andR Chuyate ldquoSpoilage of fruit juices byAlicyclobacil-lus acidoterrestrisrdquo Food Australia vol 52 no 7 pp 293ndash2952000

[34] N Jensen and F B Whitfield ldquoRole of Alicyclobacillus acidoter-restris in the development of a disinfectant taint in shelf-stablefruit juicerdquo Letters in Applied Microbiology vol 361 pp 9ndash142003

[35] D Gocmen A Elston T Williams M Parish and R LRouseff ldquoIdentification of medicinal off-flavours generated byAlicyclobacillus species in orange juice using GC-olfactometryand GC-MSrdquo Letters in Applied Microbiology vol 40 no 3 pp172ndash177 2005

[36] M Niwa ldquoControl of hazardous bacteria in acidic beveragesby using a guaiacol detection kit (peroxidase methodrdquo FruitProcess vol 15 pp 388ndash392 2005

[37] B Siegmund and B Pollinger-Zierler ldquoGrowth behavior ofoff-flavor-forming microorganisms in apple juicerdquo Journal of

Agricultural and Food Chemistry vol 55 no 16 pp 6692ndash66992007

[38] M Niwa and A Kawamoto ldquoDevelopment of a rapid detectionmethod of A acidoterrestris hazardous bacteria to acidicbeveragerdquo Fruit Process vol 13 pp 102ndash107 2003

[39] T A Eisele and M J Semon ldquoBest estimated aroma andtaste detection threshold for guaiacol in water and apple juicerdquoJournal of Food Science vol 70 no 4 pp S267ndashS269 2005

[40] B Zierler B Siegmund and W Pfannhauser ldquoDeterminationof off-flavour compounds in apple juice caused by microor-ganisms using headspace solid phase microextraction-gaschromatography-mass spectrometryrdquo Analytica Chimica Actavol 520 no 1-2 pp 3ndash11 2004

[41] K S Bahceci and J Acar ldquoModeling the combined effectsof pH temperature and ascorbic acid concentration on theheat resistance of Alicyclobacillus acidoterrestisrdquo InternationalJournal of Food Microbiology vol 120 no 3 pp 266ndash273 2007

[42] J Baumgart ldquoChapter 11 Media for the detection and enu-meration of Alicyclobacillus acidoterrestris and Alicyclobacillusacidocaldarius in foodsrdquo in Progress in Industrial Microbiologyvol 37 pp 161ndash166 Elsevier 2003

[43] M Lin M Al-Holy S-S Chang et al ldquoRapid discriminationof Alicyclobacillus strains in apple juice by Fourier transforminfrared spectroscopyrdquo International Journal of Food Microbiol-ogy vol 105 no 3 pp 369ndash376 2005

[44] J Wang T Yue Y Yuan X Lu J-H Shin and B RascoldquoDiscrimination of alicyclobacillus strains using nitrocellulosemembrane filter and attenuated total reflectance fourier trans-form infrared spectroscopyrdquo Journal of Food Science vol 76 no2 pp 137ndash142 2011

[45] M A Al-Holy M Lin O A Alhaj and M H Abu-GoushldquoDiscrimination between Bacillus and Alicyclobacillus Isolatesin Apple Juice by Fourier Transform Infrared Spectroscopy andMultivariate Analysisrdquo Journal of Food Science vol 80 no 2 ppM399ndashM404 2015

[46] WH Groenewald P A Gouws andR CWitthuhn ldquoIsolationidentification and typification of Alicyclobacillus acidoterrestrisand Alicyclobacillus acidocaldarius strains from orchard soiland the fruit processing environment in South Africardquo FoodMicrobiology vol 26 no 1 pp 71ndash76 2009

[47] J Zhang T Yue and Y Yuan ldquoAlicyclobacillus contaminationin the production line of Kiwi products in Chinardquo PLoS ONEvol 8 no 7 p e67704 2013

[48] I C McKnight M N U Eiroa A S SantrsquoAna and P RMassaguer ldquoAlicyclobacillus acidoterrestris in pasteurized exoticBrazilian fruit juices Isolation genotypic characterization andheat resistancerdquo FoodMicrobiology vol 27 no 8 pp 1016ndash10222010

[49] L Felix-Valenzuela I Guardiola-Avila A Burgara-Estrella MIbarra-Zavala and V Mata-Haro ldquoGenotypic and phenotypicdiversity of Alicyclobacillus acidocaldarius isolatesrdquo Letters inApplied Microbiology vol 61 no 4 pp 367ndash373 2015

[50] J D Wisotzkey P Jurtshuk Jr G E Fox G Deinhard andK Poralla ldquoComparative sequence analyses on the 16S rRNA(rDNA) of Bacillus acidocaldarius Bacillus acidoterrestris andBacillus cycloheptanicus and proposal for creation of a newgenus Alicyclobacillus gen novrdquo International Journal of Sys-tematic Bacteriology vol 42 no 2 pp 263ndash269 1992

[51] C J Connor H Luo B B McSpadden Gardener and HH Wang ldquoDevelopment of a real-time PCR-based systemtargeting the 16S rRNA gene sequence for rapid detection of

12 BioMed Research International

Alicyclobacillus spp in juice productsrdquo International Journal ofFood Microbiology vol 99 no 3 pp 229ndash235 2005

[52] S ChenQ Tang X Zhang et al ldquoIsolation and characterizationof thermo-acidophilic endospore-forming bacteria from theconcentrated apple juice-processing environmentrdquo FoodMicro-biology vol 23 no 5 pp 439ndash445 2006

[53] K Goto A Nishibori Y Wasada K Furuhata M Fukuyamaand M Hara ldquoIdentification of thermo-acidophilic bacteriaisolated from the soil of several Japanese fruit orchardsrdquo Lettersin Applied Microbiology vol 46 no 3 pp 289ndash294 2008

[54] J Chen X Ma Y Yuan and W Zhang ldquoSensitive and rapiddetection ofAlicyclobacillus acidoterrestris using loop-mediatedisothermal amplificationrdquo Journal of the Science of Food andAgriculture vol 91 no 6 pp 1070ndash1074 2011

[55] K T Chow M K Pope and J Davies ldquoCharacterization of avanillic acid non-oxidative decarboxylation gene cluster fromStreptomyces sp D7rdquo Microbiology vol 145 no 9 pp 2393ndash2403 1999

[56] K Matsubara ldquoA method for detecting andor identifying gua-iacol producing micro organismrdquo Patent no JP2003000259A2003

[57] Y Wang T Yue Y Yuan and Z Gao ldquoIsolation and identifi-cation of thermo-acidophilic bacteria from orchards in chinardquoJournal of Food Protection vol 73 pp 390ndash394 2010

[58] M Shemesh R Pasvolsky N Sela S J Green and V andZakinldquoDraft Genome Sequence of Alicyclobacillus acidoterrestrisStrain ATCC 49025rdquo Genome Announcement vol 1 no 5Article ID e00638ndash13 2013

[59] B Sokołowska S Skapska M Fonberg-Broczek et al ldquoFactorsinfluencing the inactivation of Alicyclobacillus acidoterrestrisspores exposed to high hydrostatic pressure in apple juicerdquoHighPressure Research vol 33 no 1 pp 73ndash82 2013

[60] B A Osopale C R Witthuhn J Albertyn and F A Oguntoy-inbo ldquoCulture dependent and independent genomic identifica-tion ofAlicyclobacillus species in contaminated commercial fruitjuicesrdquo Food Microbiology vol 56 pp 21ndash28 2016

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Submit your manuscripts atwwwhindawicom

4 BioMed Research International

Table2Listof

isolatesa

ndtheirR

FLPprofi

lesAAT

Aacid

oterrestris

AAC

Aacid

ocaldariu

sBA

Brevibacillusa

griBG

bacillus

ginsengihum

i

IsolateSource

Guaiacolprodu

ction

Erythrito

lutilization

RFLP

analysis

16S

Hin61

vdc

BsuR

Ivdc

Hin61

vdc

Hph

IrpoB

BsuR

IrpoB

Hin61

rpoB

Hph

I1

concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

2concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

3interm

ediateforb

everagep

rodu

ction

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

4interm

ediateforb

everagep

rodu

ction

nono

AAC

--

-AAC

(I)

AAC

(I)

AAC

(I)

5concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

6concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

7concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

8concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

9orange

beverage

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

10concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

11concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

12concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

13concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

14concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

15concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

16concentrated

applejuice

yes

nontypical

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

17banana

nectar

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

18orange

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

19concentrated

orange

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

20concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

21orange

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

22concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

23concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

24concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

25fre

shapples

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

26concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

27concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

28concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

29concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

30concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

31concentrated

blackcurrantjuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

32concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

33concentrated

applejuice

yes

nontypical

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

34fre

shapples

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(IA)

AAT

(I)

AAT

(I)

AAT

(I)

BioMed Research International 5

Table2Con

tinued

IsolateSource

Guaiacolprodu

ction

Erythrito

lutilization

RFLP

analysis

16S

Hin61

vdc

BsuR

Ivdc

Hin61

vdc

Hph

IrpoB

BsuR

IrpoB

Hin61

rpoB

Hph

I35

fresh

apples

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

36concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

37concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

38interm

ediateforb

everagep

rodu

ction

nono

AAC

--

-AAC

(I)

AAC

(I)

AAC

(IA)

39concentrated

applejuice

yes

-BG

--

-BG

BGBG

40concentrated

applejuice

yes

-BG

--

-BG

BGBG

41concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

42concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(IIA)

43concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

44spoiledappleb

everage

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

45concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

46spoiledapplejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

47concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

48concentrated

straw

berryjuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

49concentrated

cherry

juice

nono

AAC

--

-AAC

(IA)

AAC

(I)

AAC

(IA)

50tomatojuicefrom

fresh

tomatoes

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

51concentrated

beetroot

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

52concentrated

cherry

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

53concentrated

cherry

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

54concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(IIA)

AAT

(II)

AAT

(II)

55concentrated

cherry

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

56concentrated

raspberryjuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

57concentrated

applejuice

yes

nontypical

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

58orange

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

59concentrated

applejuice

nono

ntypical

AAC

--

-AAC

(IA)

AAC

(I)

AAC

(I)

60concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

61concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

62tomatojuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

63concentrated

applejuice

yes

-BA

--

-BA

BABA

64concentrated

applejuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

65cherry

puree

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

66concentrated

blackcurrantjuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

67concentrated

straw

berryjuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

68clou

dyapplejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

69concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

70concentrated

applejuice

nono

AAC

--

-AAC

(IA)

AAC

(I)

AAC

(I)

71concentrated

applejuice

nono

AAC

--

-AAC

(IA)

AAC

(I)

AAC

(I)

72concentrated

applejuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

73concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

74concentrated

applejuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

75concentrated

applejuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

6 BioMed Research International

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

bp

1000

500

200

Figure 2 Restriction patterns of a 1495 bp fragment of the 16S rDNA gene digested with Hin6I Lanes 6 7 9 14 and 18 A acidoterrestris 23 4 8 10 11 12 13 15 16 and 17 A acidocaldarius 5 Brevibacillus agri 19 A acidoterrestris DSM 2498 20 A acidoterrestris ATCC49025 21A acidiphilus 22 A hesperidum 23 A herbarius 24 A acidocaldarius 25 B subtilis 26 G stearothermophilus lane 1 molecular marker

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 3 Restriction patterns of a 1586 bp fragment of the vdc region digested with BsuRI Lanes 1ndash3 5 7ndash11 13 and 14 A acidoterrestristype I 4 6 12 and 15 A acidoterrestris type II 16 A acidoterrestris DSM 2498 17 A acidoterrestris ATCC49025 18 A acidiphilus 19 Aherbarius 20 molecular marker

HphI patterns confirm this rule with the exception of onestrain which represented the type I pattern in the BsuRI andHin6I analysis

33 RFLP Analysis of the rpoB Gene Fragment The 1735 bpfragment of the rpoB gene was amplified using Gru3 andGru4 or Gru5 and Gru6 primers Gru3 and Gru4 primerswere degenerated versions of Gru5 and Gru6 (respectively)and were used for amplification of isolates other thanA acidoterrestris A acidoterrestris isolates were amplifiedeither with Gru3ndashGru4 or Gru5ndashGru6 primers and theGru5ndashGru6 primer pair was chosen due to the better effi-ciency of the reaction Considering the individual isolates theRFLP patterns were identical for both pairs of primers

Thepatterns obtained by all of the nucleases used (Figures6ndash8) enabled distinguishing between the species analyzedWith exception of two strains the BsuRI patterns dividedthe A acidoterrestris group into two clusters analogicalto the clusters revealed by the vdc region analysis Thepattern (IIA) is shown by two exceptional strains mostresembling the type II pattern of A acidoterrestris andthose strains were classified as type II in other analyses Thereference strain ATCC 49025 shows this type of patternThe A acidocaldarius group was represented by three typesof patterns two of them resembling each other A acido-caldarius DSM 446 belongs to the cluster II of A acido-caldarius group while A acidocaldarius A1 was assigned tocluster I

BioMed Research International 7

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 4 Restriction patterns of a 1586 bp fragment of the vdc region digested with Hin6I Lanes 1ndash3 5 7ndash11 13 and 14 A acidoterrestristype I 4 6 12 and 15A acidoterrestris type II 16A acidoterrestrisDSM 2498 17A acidoterrestris ATCC49025 18 A acidiphilusDSM 1455819 A herbarius DSM 13609 lane 20 molecular marker

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 5 Restriction patterns of a 1586 bp fragment of the vdc region digested with HphI Lanes 2ndash4 6 8ndash12 14 and 15 A acidoterrestristype I 5 7 13 and 16 A acidoterrestris type II 17 A acidoterrestris DSM 2498 18 A acidoterrestris ATCC49025 19 A acidiphilus 20 Aherbarius lane 1 molecular marker

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Figure 6 Restriction patterns of a 1735 bp fragment of the rpoB gene digested with BsuRI Lanes 1ndash4 6 and 8ndash10 A acidoterrestris type I 57 21 22 and 24-26 A acidoterrestris type II 23 A acidoterrestris type IIA 27 A acidocaldarius type II 28 A acidocaldarius type IA 11 Aacidoterrestris DSM 2498 12 A acidoterrestris ATCC49025 13 A acidiphilus 14 A hesperidum 15 A herbarius 16 A acidocaldariusA1 17B subtilis 18 G stearothermophilus lane 19 molecular marker

8 BioMed Research International

Figure 7 Restriction patterns of a 1735 bp fragment of rpoB gene digested with Hin6I Lanes 3 and 5 A acidoterrestris type I 1 2 4 and 6Aacidoterrestris type II 17A acidocaldarius type I 18A acidocaldarius type II 8A acidoterrestrisDSM2498 9A acidoterrestrisATCC4902510A acidiphilus 11 A hesperidum 12A herbarius 13A acidocaldariusA1 14 B subtilis 15G stearothermophilus lanes 7 and 16 molecularmarker

Figure 8 Restriction patterns of a 1735 bp fragment of rpoB gene digested with HphI Lanes 1-4 6 8-11 13 25 and 27 A acidoterrestris I5 7 12 26 and 28 A acidoterrestris II 24 A acidoterrestris IIA 29 A acidocaldarius IA 30 A acidocaldarius type II 15 A acidoterrestrisDSM 2498 16 A acidoterrestris ATCC49025 17 A acidiphilus 18 A hesperidum 19 A herbarius 20 A acidocaldariusA1 21 B subtilis 22G stearothermophilus lanes 14 and 23 molecular marker

DSM_2498 00003231 -00003234 -00025641 0ATCC_49025 00022455 00005656 -00009252 -00001833 051 053 -000013

Figure 9 Phylogenetic tree of partial 16S rRNA sequence of 11 Aacidoterrestris isolatesThe tree was constructed by neighbor joiningmethod using Clustal Omega software

For Hin6I there were two types of patterns for the Aacidoterrestris and A acidocaldarius A acidoterrestris DSM2498 belongs to the cluster I while A acidoterrestris ATCC49025 belongs to the cluster II A acidocaldarius DSM 446represents cluster II of the A acidocaldarius group while AacidocaldariusA1 represents cluster I

HphI endonuclease produces two types of patterns forA acidoterrestris analogically as before and three types ofpatterns for A acidocaldarius One sample of A acidoter-restris cluster II has a slightly different pattern and has been

classified as type II in other analyses Two of the threepatterns of A acidocaldarius differ with only one bandA acidocaldarius DSM 446 represents cluster II of the Aacidocaldarius group while A acidocaldarius A1 representscluster I

34 DNA Sequencing DNA sequencing was performed forselected strains Sequencing of the 16S rDNA gene wasperformed to confirm the proper identification of the isolatesand to identify the non-Alicyclobacillus isolates The 16SrDNA sequence of strains 31 and 34 classified as type Ishowed the greatest similarity toA acidoterrestrisDSM 2498also classified as type I The sequences of strains 33 51 52 5355 and 56 classified as type II showed the greatest similaritytoA acidoterrestrisATCC49025 also classified as type IIThesequence of strain 41 classified as type II showed the greatestsimilarity to A acidoterrestris DSM 3922

Figure 9 shows the phylogenetic tree constructed from the16S rDNA sequences of analyzed strains of A acidoterrestrisThe sequence analysis indicates that A acidoterrestris DSM2498 and two other type I strains are closely related and itshows greater variation within class II strains The isolatesselected for sequencing represent both groups and different

BioMed Research International 9

sources which they were isolated from concentrated applejuice (isolates 33 and 41) fresh apples (isolate 34) concen-trated blackcurrant juice (isolate 31) concentrated raspberryjuice (isolate 56) concentrated beetroot juice (isolate 51)and concentrated cherry juice (isolates 52 53 and 55) Thesequence analysis shows no apparent correlation betweendiversity of the 16S rDNA sequence and the sources of theisolates

Fivewhole vdc gene clusterswere sequenced Twoof themwere isolated from the reference strains A acidoterrestrisDSM 2498 and A acidoterrestris ATCC 49025 2416 bpfragments of the vdc regions were aligned The vdc regionsequence of strains 15 and 34 classified as type I showed999 identity with the vdc sequence of A acidoterrestrisDSM 2498 the sequence of strain 41 classified as typeII showed 993 identity with the vdc sequence A aci-doterrestris ATCC 49025 Vdc sequences of A acidoterrestrisDSM 2498 and A acidoterrestris DSM 49025 showed 945identity For the last pair protein sequences of the geneproducts showed 985 positives and 965 identities forVdcB 994 and 989 for VdcC and 100 and 974 forVdcD

RpoB gene sequencing was performed to confirm thatboth primer pairs Gru3ndashGru4 and Gru5ndashGru6 enabledto amplify the correct DNA fragment and that degenera-tion of the primers did not affect the sequence specificityalthough the degenerated primers produced significantlylower amount of PCR product All of the sequences obtainedshowed greatest similarity to the appropriate rpoB genes

The GenBank accession numbers are KX371237-KX371249 for 16S rDNA sequences KX453673-KX453677for full vdc region sequences KX453678 and KX453679 forpartial vdc sequences of A herbarius and A acidiphilusrespectively and KX453680- KX453682 for partial rpoBsequences

4 Discussion

Among more than one thousand samples of concentratedapple juice tested in our laboratory between 2004 and 201267 was contaminated with Alicyclobacillus sp and 31 ofthe isolates were identified as A acidoterrestris [59] Thestatistics show that Alicyclobacillus spoilage is still a majorconcern in the fruit processing industry

In this study 75 guaiacol producing and non-guaiacolproducing strains were isolated from various fruit juicesconcentrated fruit juices and fresh apples Additionally8 reference strains were used The isolates and referencestrains were examined using classic methods and PCR-RFLP focusing on 16S rDNA and rpoB gene fragments aswell as the vdc region fragment For selected strains DNAsequencing of the 16S rDNA gene was performed Sixty ofthe isolates analyzed were identified as A acidoterrestris12 as A acidocaldarius one as Brevibacillus agri and twoas Bacillus ginsengihumi Four of the isolates which gaveambiguous results during classic identification (nontypicalcolour on an erythritol medium or lack of growth at 65∘Cconnected with lack of guaiacol production) were identified

by genetic methods as A acidoterrestris or A acidocaldariusA acidoterrestris isolates were grouped in two major clusters27 of the isolates belonged to the cluster I and 33 to thecluster II Also A acidocaldarius isolates were grouped intotwo clusters but showed more intracluster diversity

These results support the observations made by Osopaleat al [60] and Durak et al [23] who also reported two geneticclusters among analyzed A acidoterrestris samples by RAPDanalysisand by 16S rDNA sequencing respectively

Most of the strains analyzed in this study were isolatedfrom concentrated apple juice as this is the main subject ofthe Alicyclobacillus focused screening made or outsourced bypolish fruit industry however it is one of the main subjectsof similar screenings performed by fruit industry worldwideA acidoterrestris representing both clusters have been foundin concentrated apple juice For other sources three Aacidoterrestris cluster II isolates have been found in orangejuices two cluster I in concentrated black currant juice andthree cluster II in concentrated cherry juice however theseare only single observations and further research is needed toestablish ifA acidoterrestris strains representing both clustersare found in other juices

Genetic methods are broadly used in the detectioncharacterization and differentiation of microorganisms Theapplication of PCR-RFLP in the characterization of Ali-cyclobacillus and other thermoacidophilic bacteria isolatedfrom the apple juice processing environment has beendescribed by Chen et al [52] although only the 16S rDNAgene was the subject of this study and the Hin6I enzymewas not used RpoB gene has never been subjected to PCR-RFLP or any other genetic analysis of Alicyclobacillus sofar although the value of this gene in taxonomy has beenconfirmed by many studies on other microorganisms Todate there are no reports on the use of the vdc gene cluster intaxonomic studies of microorganisms although its sequencemay be proven valuable for research both on the vectors anddiversity and evolution of the element itself Hin6I restrictionpatterns for 16S rDNAwere sufficient to differentiate betweenall the species analyzed but provided no closer data onintraspecies diversity The diversity was revealed both by therpoB gene and vdc region RFLP analyses

Vdc gene cluster of Streptomyces sp described by Chowet al [55] consists of three genes vdcB (06kb) vdcC (14 kb)and vdcD (02 kb) transcribed as single policistronic mRNAmolecule All three genes were essential to produce guaiacolfrom vanillic acid Niwa and Kawamoto [38] describedanalogical gene cluster in A acidoterrestris consisting ofORF1 (597 bp) ORF2 (1425 bp) and ORF3 (321 bp) Threerespective open reading frameswere found in all five analyzedvdc sequences

Alicyclobacillus acidoterrestris RFLP patterns of boththe rpoB gene and vdc region showed consistently thatthere were two major types among the isolates one similarto the reference strain A acidoterrestris DSM 2498 andthe other similar to the reference strain A acidoterrestrisATCC 49025 Obtaining such consistent data concerningtwo genetic elements of distant function reveals that there isdeeper intraspecies genetic diversity in the A acidoterrestrisspecies This division may be considered when examining

10 BioMed Research International

other features of Alicyclobacillus such as susceptibility totemperature or high hydrostatic pressure in order to establishif there are any differences between the groups

Both the rpoB gene and vdc region seem to be single-copy genetic elements and showed no signs of intragenomicheterogeneity which often makes the RFLP comparison ofrDNA genes harder to perform

The sequence analysis of the 16S rDNA fragment ofselected isolates shows no apparent correlation betweendiversity of the 16S rDNA sequence and the sources whichthe strains were isolated from

In conclusion the application of PCR-RFLP has beenproven to be a fast and reliable method for Alicyclobacillusidentification and differentiation The method is also techni-cally less difficult than most of other molecular techniquesTwo major groups of A acidoterrestris have been identifiedThe primers designed for this study could be useful in furtherresearch on Alicyclobacillus

Data Availability

The DNA sequences obtained during this study have beendeposited in GenBank and the accession numbers areincluded within the article

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

Our research project was fully sponsored by Polish Ministryof Science and Higher Education with funds for statutoryactivity

References

[1] K Yamazaki H Teouka and H Shinano ldquoIsolation and identi-fication of alicyclobacillus acidoterrestris from acidic beveragesrdquoBioscience Biotechnology and Biochemistry vol 60 no 3 pp543ndash545 1996

[2] G DARLAND and T D BROCK ldquoBacillus acidocaldar-ius spnov an Acidophilic Thermophilic Spore-forming Bac-teriumrdquo Journal of General Microbiology vol 67 no 1 pp 9ndash151971

[3] G Deinhard P Blanz K Poralla and E Altan ldquoBacillusacidoterrestris sp nov a new thermotolerant acidophile isolatedfrom different soilsrdquo Systematic and Applied Microbiology vol10 no 1 pp 47ndash53 1987

[4] G Deinhard J Saar W Krischke and K Poralla ldquoBacilluscycloheptanicus sp nov a new thermoacidophile containing 120596-cycloheptane fatty acidsrdquo Systematic and Applied Microbiologyvol 10 no 1 pp 68ndash73 1987

[5] L Albuquerque F A Rainey A P Chung et al ldquoAlicyclobacillushesperidum sp nov and a related genomic species from solfa-taric soils of Sao Miguel in the Azoresrdquo International Journalof Systematic and Evolutionary Microbiology vol 50 no 2 pp451ndash457 2000

[6] K Goto H Matsubara K Mochida et al ldquoAlicyclobacillusherbarius sp nov a novel bacterium containing120596-cycloheptane

fatty acids isolated from herbal teardquo International Journal ofSystematic and EvolutionaryMicrobiology vol 52 no 1 pp 109ndash113 2002

[7] K Goto Y Tanimoto T Tamura et al ldquoIdentificationof thermoacidophilic bacteria and a new Alicyclobacillusgenomic species isolated from acidic environments in JapanrdquoExtremophiles vol 6 no 4 pp 333ndash340 2002

[8] H Matsubara K Goto T Matsumura et al ldquoAlicyclobacillusacidiphilus sp nov a novel thermo-acidophilic 120596-alicyclicfatty acid-containing bacterium isolated from acidic beveragesrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 52 no 5 pp 1681ndash1685 2002

[9] R V Orr R L Shewfelt C J Huang S Tefera and L RBeuchat ldquoDetection of guaiacol produced by Alicyclobacillusacidoterrestris in apple juice by sensory and chromatographicanalyses and comparison with spore and vegetative cell popu-lationsrdquo Journal of Food Protection vol 63 no 11 pp 1517ndash15222000

[10] G L Pettipher M E Osmundson and J MMurphy ldquoMethodsfor the detection and enumeration of Alicyclobacillus acidoter-restris and investigation of growth and production of taint infruit juice and fruit juice-containing drinksrdquo Letters in AppliedMicrobiology vol 24 no 3 pp 185ndash189 1997

[11] A C N F Spinelli A S Sant1015840Ana S Rodrigues Jr andP R Massaguer ldquoInfluence of different filling cooling andstorage conditions on the growth of Alicyclobacillus acidoter-restris CRA7152 in orange juicerdquo Applied and EnvironmentalMicrobiology vol 75 no 23 pp 7409ndash7416 2009

[12] MCMaldonado C Belfiore andA RNavarro ldquoTemperaturesoluble solids and pH effect on Alicyclobacillus acidoterrestrisviability in lemon juice concentraterdquo Journal of IndustrialMicrobiology and Biotechnology vol 35 no 2 pp 141ndash144 2008

[13] M N U Eiroa V C A Junqueira and F L Schmidt ldquoAlicy-clobacillus in orange juice Occurrence and heat resistance ofsporesrdquo Journal of Food Protection vol 62 no 8 pp 883ndash8861999

[14] I Walls and R Chuyate ldquoIsolation of Alicyclobacillus acidoter-restris from fruit juicesrdquo Journal of AOAC International vol 83no 5 pp 1115ndash1120 2000

[15] M E Parish and R M Goodrich ldquoRecovery of presumptiveAlicyclobacillus strains from orange fruit surfacesrdquo Journal ofFood Protection vol 68 no 10 pp 2196ndash2200 2005

[16] D F Splittstoesser J J Churey and C Y Lee ldquoGrowthcharacteristics of aciduric sporeforming bacilli isolated fromfruit juicesrdquo Journal of Food Protection vol 57 no 12 pp 1080ndash1083 1994

[17] J Baumgart M Husemann and C Schmidt ldquoAlicyclobacil-lus acidoterrestris vorkommen bedeutung und nachweis ingetranken und getrankegrundstoffenrdquo Flussiges Obst vol 64pp 178ndash180 1997

[18] C A Wisse and M E Parish ldquoIsolation and enumeration ofspore-forming thermoacidophilic rod-shaped bacteria fromcitrus processing environmentsrdquo Dairy Food EnvironmentalSanitation vol 18 pp 504ndash509 1998

[19] J Baumgart and S Menje ldquoThe impact of Alicyclobacillusacidoterestris on the quality of juices and soft drinksrdquo FruitProcess vol 10 pp 251ndash254 2000

[20] S Y Eguchi G P Manfio M E Pinhatti E Azuma and SF Variane ldquoAcidotermofilic sporeforming bacteria (ATSB) inorange juices ecology and involvement in the deterioration offruit juices Report of the Research Project Part IIrdquo Fruit Processvol 11 pp 55ndash62 2001

BioMed Research International 11

[21] P AGouws L Gie A Pretorius andNDhansay ldquoIsolation andidentification of Alicyclobacillus acidocaldarius by 16S rDNAfrom mango juice and concentraterdquo International Journal ofFood Science amp Technology vol 40 no 7 pp 789ndash792 2005

[22] K Goto K Mochida Y Kato et al ldquoProposal of six speciesof moderately thermophilic acidophilic endospore-formingbacteria Alicyclobacillus contaminans sp nov Alicyclobacillusfastidiosus sp novAlicyclobacillus kakegawensis sp novAlicy-clobacillus macrosporangiidus sp nov Alicyclobacillus saccharisp nov and Alicyclobacillus shizuokensis sp novrdquo InternationalJournal of Systematic and Evolutionary Microbiology vol 57 no6 pp 1276ndash1285 2007

[23] M Z Durak J J Churey M D Danyluk and R W WoroboldquoIdentification and haplotype distribution of Alicyclobacillusspp from different juices and beveragesrdquo International Journalof Food Microbiology vol 142 no 3 pp 286ndash291 2010

[24] B Sokolowska J Niezgoda A Dekowska et al ldquoIncidence ofAlicyclobacillus spp in Polish apple and dark berry juice con-centrates and the ability of isolated A acidoterrestris strains tospoilage of these juicesrdquo Postępy Nauki i Technologii PrzemysłuRolno-Spozywczego vol 71 no 1 pp 5ndash20 2016

[25] I Walls and R Chuyate ldquoAlicyclobacillus historical perspectiveand preliminary characterization studyrdquo Dairy Food EnvironSanitation18 pp 499ndash503 1998

[26] H-A Duong and N Jensen ldquoSpoilage of iced tea by Alicy-clobacillusrdquo Food Australia vol 52 no 7 p 292 2000

[27] M Niwa and A Kuriyama ldquoA acidoterrestris rapid detectionkitrdquo Fruit Process vol 13 pp 328ndash331 2003

[28] K S Bahceci V Gokmen and J Acar ldquoFormation of guaiacolfrom vanillin by Alicyclobacillus acidoterrestris in apple juice Amodel studyrdquo European Food Research and Technology vol 220no 2 pp 196ndash199 2005

[29] R C Witthuhn E van der Merwe P Venter and M CameronldquoGuaiacol production from ferulic acid vanillin and vanillicacid by Alicyclobacillus acidoterrestrisrdquo International Journal ofFood Microbiology vol 157 no 1 pp 113ndash117 2012

[30] A Borlinghaus and R Engel ldquoAlicyclobacillus incidence in com-mercial apple juice concentrate (AJC) supplies and validationrdquoFruit Process vol 7 pp 262ndash266 1997

[31] D F Splittstoesser C Y Lee and J J Churey ldquoControl ofAlicyclobacillus in the juice industryrdquo Dairy Food EnvironSanitation vol 18 pp 585ndash587 1998

[32] N Jensen ldquoAlicyclobacillus in Australiardquo Food Australia vol 52no 7 pp 282ndash285 2000

[33] IWalls andR Chuyate ldquoSpoilage of fruit juices byAlicyclobacil-lus acidoterrestrisrdquo Food Australia vol 52 no 7 pp 293ndash2952000

[34] N Jensen and F B Whitfield ldquoRole of Alicyclobacillus acidoter-restris in the development of a disinfectant taint in shelf-stablefruit juicerdquo Letters in Applied Microbiology vol 361 pp 9ndash142003

[35] D Gocmen A Elston T Williams M Parish and R LRouseff ldquoIdentification of medicinal off-flavours generated byAlicyclobacillus species in orange juice using GC-olfactometryand GC-MSrdquo Letters in Applied Microbiology vol 40 no 3 pp172ndash177 2005

[36] M Niwa ldquoControl of hazardous bacteria in acidic beveragesby using a guaiacol detection kit (peroxidase methodrdquo FruitProcess vol 15 pp 388ndash392 2005

[37] B Siegmund and B Pollinger-Zierler ldquoGrowth behavior ofoff-flavor-forming microorganisms in apple juicerdquo Journal of

Agricultural and Food Chemistry vol 55 no 16 pp 6692ndash66992007

[38] M Niwa and A Kawamoto ldquoDevelopment of a rapid detectionmethod of A acidoterrestris hazardous bacteria to acidicbeveragerdquo Fruit Process vol 13 pp 102ndash107 2003

[39] T A Eisele and M J Semon ldquoBest estimated aroma andtaste detection threshold for guaiacol in water and apple juicerdquoJournal of Food Science vol 70 no 4 pp S267ndashS269 2005

[40] B Zierler B Siegmund and W Pfannhauser ldquoDeterminationof off-flavour compounds in apple juice caused by microor-ganisms using headspace solid phase microextraction-gaschromatography-mass spectrometryrdquo Analytica Chimica Actavol 520 no 1-2 pp 3ndash11 2004

[41] K S Bahceci and J Acar ldquoModeling the combined effectsof pH temperature and ascorbic acid concentration on theheat resistance of Alicyclobacillus acidoterrestisrdquo InternationalJournal of Food Microbiology vol 120 no 3 pp 266ndash273 2007

[42] J Baumgart ldquoChapter 11 Media for the detection and enu-meration of Alicyclobacillus acidoterrestris and Alicyclobacillusacidocaldarius in foodsrdquo in Progress in Industrial Microbiologyvol 37 pp 161ndash166 Elsevier 2003

[43] M Lin M Al-Holy S-S Chang et al ldquoRapid discriminationof Alicyclobacillus strains in apple juice by Fourier transforminfrared spectroscopyrdquo International Journal of Food Microbiol-ogy vol 105 no 3 pp 369ndash376 2005

[44] J Wang T Yue Y Yuan X Lu J-H Shin and B RascoldquoDiscrimination of alicyclobacillus strains using nitrocellulosemembrane filter and attenuated total reflectance fourier trans-form infrared spectroscopyrdquo Journal of Food Science vol 76 no2 pp 137ndash142 2011

[45] M A Al-Holy M Lin O A Alhaj and M H Abu-GoushldquoDiscrimination between Bacillus and Alicyclobacillus Isolatesin Apple Juice by Fourier Transform Infrared Spectroscopy andMultivariate Analysisrdquo Journal of Food Science vol 80 no 2 ppM399ndashM404 2015

[46] WH Groenewald P A Gouws andR CWitthuhn ldquoIsolationidentification and typification of Alicyclobacillus acidoterrestrisand Alicyclobacillus acidocaldarius strains from orchard soiland the fruit processing environment in South Africardquo FoodMicrobiology vol 26 no 1 pp 71ndash76 2009

[47] J Zhang T Yue and Y Yuan ldquoAlicyclobacillus contaminationin the production line of Kiwi products in Chinardquo PLoS ONEvol 8 no 7 p e67704 2013

[48] I C McKnight M N U Eiroa A S SantrsquoAna and P RMassaguer ldquoAlicyclobacillus acidoterrestris in pasteurized exoticBrazilian fruit juices Isolation genotypic characterization andheat resistancerdquo FoodMicrobiology vol 27 no 8 pp 1016ndash10222010

[49] L Felix-Valenzuela I Guardiola-Avila A Burgara-Estrella MIbarra-Zavala and V Mata-Haro ldquoGenotypic and phenotypicdiversity of Alicyclobacillus acidocaldarius isolatesrdquo Letters inApplied Microbiology vol 61 no 4 pp 367ndash373 2015

[50] J D Wisotzkey P Jurtshuk Jr G E Fox G Deinhard andK Poralla ldquoComparative sequence analyses on the 16S rRNA(rDNA) of Bacillus acidocaldarius Bacillus acidoterrestris andBacillus cycloheptanicus and proposal for creation of a newgenus Alicyclobacillus gen novrdquo International Journal of Sys-tematic Bacteriology vol 42 no 2 pp 263ndash269 1992

[51] C J Connor H Luo B B McSpadden Gardener and HH Wang ldquoDevelopment of a real-time PCR-based systemtargeting the 16S rRNA gene sequence for rapid detection of

12 BioMed Research International

Alicyclobacillus spp in juice productsrdquo International Journal ofFood Microbiology vol 99 no 3 pp 229ndash235 2005

[52] S ChenQ Tang X Zhang et al ldquoIsolation and characterizationof thermo-acidophilic endospore-forming bacteria from theconcentrated apple juice-processing environmentrdquo FoodMicro-biology vol 23 no 5 pp 439ndash445 2006

[53] K Goto A Nishibori Y Wasada K Furuhata M Fukuyamaand M Hara ldquoIdentification of thermo-acidophilic bacteriaisolated from the soil of several Japanese fruit orchardsrdquo Lettersin Applied Microbiology vol 46 no 3 pp 289ndash294 2008

[54] J Chen X Ma Y Yuan and W Zhang ldquoSensitive and rapiddetection ofAlicyclobacillus acidoterrestris using loop-mediatedisothermal amplificationrdquo Journal of the Science of Food andAgriculture vol 91 no 6 pp 1070ndash1074 2011

[55] K T Chow M K Pope and J Davies ldquoCharacterization of avanillic acid non-oxidative decarboxylation gene cluster fromStreptomyces sp D7rdquo Microbiology vol 145 no 9 pp 2393ndash2403 1999

[56] K Matsubara ldquoA method for detecting andor identifying gua-iacol producing micro organismrdquo Patent no JP2003000259A2003

[57] Y Wang T Yue Y Yuan and Z Gao ldquoIsolation and identifi-cation of thermo-acidophilic bacteria from orchards in chinardquoJournal of Food Protection vol 73 pp 390ndash394 2010

[58] M Shemesh R Pasvolsky N Sela S J Green and V andZakinldquoDraft Genome Sequence of Alicyclobacillus acidoterrestrisStrain ATCC 49025rdquo Genome Announcement vol 1 no 5Article ID e00638ndash13 2013

[59] B Sokołowska S Skapska M Fonberg-Broczek et al ldquoFactorsinfluencing the inactivation of Alicyclobacillus acidoterrestrisspores exposed to high hydrostatic pressure in apple juicerdquoHighPressure Research vol 33 no 1 pp 73ndash82 2013

[60] B A Osopale C R Witthuhn J Albertyn and F A Oguntoy-inbo ldquoCulture dependent and independent genomic identifica-tion ofAlicyclobacillus species in contaminated commercial fruitjuicesrdquo Food Microbiology vol 56 pp 21ndash28 2016

Hindawiwwwhindawicom

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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

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Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

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Nucleic AcidsJournal of

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Submit your manuscripts atwwwhindawicom

BioMed Research International 5

Table2Con

tinued

IsolateSource

Guaiacolprodu

ction

Erythrito

lutilization

RFLP

analysis

16S

Hin61

vdc

BsuR

Ivdc

Hin61

vdc

Hph

IrpoB

BsuR

IrpoB

Hin61

rpoB

Hph

I35

fresh

apples

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

36concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

37concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

38interm

ediateforb

everagep

rodu

ction

nono

AAC

--

-AAC

(I)

AAC

(I)

AAC

(IA)

39concentrated

applejuice

yes

-BG

--

-BG

BGBG

40concentrated

applejuice

yes

-BG

--

-BG

BGBG

41concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

42concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(IIA)

43concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

44spoiledappleb

everage

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

45concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

46spoiledapplejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

47concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

48concentrated

straw

berryjuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

49concentrated

cherry

juice

nono

AAC

--

-AAC

(IA)

AAC

(I)

AAC

(IA)

50tomatojuicefrom

fresh

tomatoes

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

51concentrated

beetroot

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

52concentrated

cherry

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

53concentrated

cherry

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

54concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(IIA)

AAT

(II)

AAT

(II)

55concentrated

cherry

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

56concentrated

raspberryjuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

57concentrated

applejuice

yes

nontypical

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

58orange

juice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

59concentrated

applejuice

nono

ntypical

AAC

--

-AAC

(IA)

AAC

(I)

AAC

(I)

60concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

61concentrated

applejuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

62tomatojuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

63concentrated

applejuice

yes

-BA

--

-BA

BABA

64concentrated

applejuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

65cherry

puree

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

66concentrated

blackcurrantjuice

yes

yes

AAT

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

AAT

(I)

67concentrated

straw

berryjuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

68clou

dyapplejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

69concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

70concentrated

applejuice

nono

AAC

--

-AAC

(IA)

AAC

(I)

AAC

(I)

71concentrated

applejuice

nono

AAC

--

-AAC

(IA)

AAC

(I)

AAC

(I)

72concentrated

applejuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

73concentrated

applejuice

yes

yes

AAT

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

AAT

(II)

74concentrated

applejuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

75concentrated

applejuice

nono

AAC

--

-AAC

(II)

AAC

(II)

AAC

(II)

6 BioMed Research International

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

bp

1000

500

200

Figure 2 Restriction patterns of a 1495 bp fragment of the 16S rDNA gene digested with Hin6I Lanes 6 7 9 14 and 18 A acidoterrestris 23 4 8 10 11 12 13 15 16 and 17 A acidocaldarius 5 Brevibacillus agri 19 A acidoterrestris DSM 2498 20 A acidoterrestris ATCC49025 21A acidiphilus 22 A hesperidum 23 A herbarius 24 A acidocaldarius 25 B subtilis 26 G stearothermophilus lane 1 molecular marker

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 3 Restriction patterns of a 1586 bp fragment of the vdc region digested with BsuRI Lanes 1ndash3 5 7ndash11 13 and 14 A acidoterrestristype I 4 6 12 and 15 A acidoterrestris type II 16 A acidoterrestris DSM 2498 17 A acidoterrestris ATCC49025 18 A acidiphilus 19 Aherbarius 20 molecular marker

HphI patterns confirm this rule with the exception of onestrain which represented the type I pattern in the BsuRI andHin6I analysis

33 RFLP Analysis of the rpoB Gene Fragment The 1735 bpfragment of the rpoB gene was amplified using Gru3 andGru4 or Gru5 and Gru6 primers Gru3 and Gru4 primerswere degenerated versions of Gru5 and Gru6 (respectively)and were used for amplification of isolates other thanA acidoterrestris A acidoterrestris isolates were amplifiedeither with Gru3ndashGru4 or Gru5ndashGru6 primers and theGru5ndashGru6 primer pair was chosen due to the better effi-ciency of the reaction Considering the individual isolates theRFLP patterns were identical for both pairs of primers

Thepatterns obtained by all of the nucleases used (Figures6ndash8) enabled distinguishing between the species analyzedWith exception of two strains the BsuRI patterns dividedthe A acidoterrestris group into two clusters analogicalto the clusters revealed by the vdc region analysis Thepattern (IIA) is shown by two exceptional strains mostresembling the type II pattern of A acidoterrestris andthose strains were classified as type II in other analyses Thereference strain ATCC 49025 shows this type of patternThe A acidocaldarius group was represented by three typesof patterns two of them resembling each other A acido-caldarius DSM 446 belongs to the cluster II of A acido-caldarius group while A acidocaldarius A1 was assigned tocluster I

BioMed Research International 7

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 4 Restriction patterns of a 1586 bp fragment of the vdc region digested with Hin6I Lanes 1ndash3 5 7ndash11 13 and 14 A acidoterrestristype I 4 6 12 and 15A acidoterrestris type II 16A acidoterrestrisDSM 2498 17A acidoterrestris ATCC49025 18 A acidiphilusDSM 1455819 A herbarius DSM 13609 lane 20 molecular marker

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 5 Restriction patterns of a 1586 bp fragment of the vdc region digested with HphI Lanes 2ndash4 6 8ndash12 14 and 15 A acidoterrestristype I 5 7 13 and 16 A acidoterrestris type II 17 A acidoterrestris DSM 2498 18 A acidoterrestris ATCC49025 19 A acidiphilus 20 Aherbarius lane 1 molecular marker

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Figure 6 Restriction patterns of a 1735 bp fragment of the rpoB gene digested with BsuRI Lanes 1ndash4 6 and 8ndash10 A acidoterrestris type I 57 21 22 and 24-26 A acidoterrestris type II 23 A acidoterrestris type IIA 27 A acidocaldarius type II 28 A acidocaldarius type IA 11 Aacidoterrestris DSM 2498 12 A acidoterrestris ATCC49025 13 A acidiphilus 14 A hesperidum 15 A herbarius 16 A acidocaldariusA1 17B subtilis 18 G stearothermophilus lane 19 molecular marker

8 BioMed Research International

Figure 7 Restriction patterns of a 1735 bp fragment of rpoB gene digested with Hin6I Lanes 3 and 5 A acidoterrestris type I 1 2 4 and 6Aacidoterrestris type II 17A acidocaldarius type I 18A acidocaldarius type II 8A acidoterrestrisDSM2498 9A acidoterrestrisATCC4902510A acidiphilus 11 A hesperidum 12A herbarius 13A acidocaldariusA1 14 B subtilis 15G stearothermophilus lanes 7 and 16 molecularmarker

Figure 8 Restriction patterns of a 1735 bp fragment of rpoB gene digested with HphI Lanes 1-4 6 8-11 13 25 and 27 A acidoterrestris I5 7 12 26 and 28 A acidoterrestris II 24 A acidoterrestris IIA 29 A acidocaldarius IA 30 A acidocaldarius type II 15 A acidoterrestrisDSM 2498 16 A acidoterrestris ATCC49025 17 A acidiphilus 18 A hesperidum 19 A herbarius 20 A acidocaldariusA1 21 B subtilis 22G stearothermophilus lanes 14 and 23 molecular marker

DSM_2498 00003231 -00003234 -00025641 0ATCC_49025 00022455 00005656 -00009252 -00001833 051 053 -000013

Figure 9 Phylogenetic tree of partial 16S rRNA sequence of 11 Aacidoterrestris isolatesThe tree was constructed by neighbor joiningmethod using Clustal Omega software

For Hin6I there were two types of patterns for the Aacidoterrestris and A acidocaldarius A acidoterrestris DSM2498 belongs to the cluster I while A acidoterrestris ATCC49025 belongs to the cluster II A acidocaldarius DSM 446represents cluster II of the A acidocaldarius group while AacidocaldariusA1 represents cluster I

HphI endonuclease produces two types of patterns forA acidoterrestris analogically as before and three types ofpatterns for A acidocaldarius One sample of A acidoter-restris cluster II has a slightly different pattern and has been

classified as type II in other analyses Two of the threepatterns of A acidocaldarius differ with only one bandA acidocaldarius DSM 446 represents cluster II of the Aacidocaldarius group while A acidocaldarius A1 representscluster I

34 DNA Sequencing DNA sequencing was performed forselected strains Sequencing of the 16S rDNA gene wasperformed to confirm the proper identification of the isolatesand to identify the non-Alicyclobacillus isolates The 16SrDNA sequence of strains 31 and 34 classified as type Ishowed the greatest similarity toA acidoterrestrisDSM 2498also classified as type I The sequences of strains 33 51 52 5355 and 56 classified as type II showed the greatest similaritytoA acidoterrestrisATCC49025 also classified as type IIThesequence of strain 41 classified as type II showed the greatestsimilarity to A acidoterrestris DSM 3922

Figure 9 shows the phylogenetic tree constructed from the16S rDNA sequences of analyzed strains of A acidoterrestrisThe sequence analysis indicates that A acidoterrestris DSM2498 and two other type I strains are closely related and itshows greater variation within class II strains The isolatesselected for sequencing represent both groups and different

BioMed Research International 9

sources which they were isolated from concentrated applejuice (isolates 33 and 41) fresh apples (isolate 34) concen-trated blackcurrant juice (isolate 31) concentrated raspberryjuice (isolate 56) concentrated beetroot juice (isolate 51)and concentrated cherry juice (isolates 52 53 and 55) Thesequence analysis shows no apparent correlation betweendiversity of the 16S rDNA sequence and the sources of theisolates

Fivewhole vdc gene clusterswere sequenced Twoof themwere isolated from the reference strains A acidoterrestrisDSM 2498 and A acidoterrestris ATCC 49025 2416 bpfragments of the vdc regions were aligned The vdc regionsequence of strains 15 and 34 classified as type I showed999 identity with the vdc sequence of A acidoterrestrisDSM 2498 the sequence of strain 41 classified as typeII showed 993 identity with the vdc sequence A aci-doterrestris ATCC 49025 Vdc sequences of A acidoterrestrisDSM 2498 and A acidoterrestris DSM 49025 showed 945identity For the last pair protein sequences of the geneproducts showed 985 positives and 965 identities forVdcB 994 and 989 for VdcC and 100 and 974 forVdcD

RpoB gene sequencing was performed to confirm thatboth primer pairs Gru3ndashGru4 and Gru5ndashGru6 enabledto amplify the correct DNA fragment and that degenera-tion of the primers did not affect the sequence specificityalthough the degenerated primers produced significantlylower amount of PCR product All of the sequences obtainedshowed greatest similarity to the appropriate rpoB genes

The GenBank accession numbers are KX371237-KX371249 for 16S rDNA sequences KX453673-KX453677for full vdc region sequences KX453678 and KX453679 forpartial vdc sequences of A herbarius and A acidiphilusrespectively and KX453680- KX453682 for partial rpoBsequences

4 Discussion

Among more than one thousand samples of concentratedapple juice tested in our laboratory between 2004 and 201267 was contaminated with Alicyclobacillus sp and 31 ofthe isolates were identified as A acidoterrestris [59] Thestatistics show that Alicyclobacillus spoilage is still a majorconcern in the fruit processing industry

In this study 75 guaiacol producing and non-guaiacolproducing strains were isolated from various fruit juicesconcentrated fruit juices and fresh apples Additionally8 reference strains were used The isolates and referencestrains were examined using classic methods and PCR-RFLP focusing on 16S rDNA and rpoB gene fragments aswell as the vdc region fragment For selected strains DNAsequencing of the 16S rDNA gene was performed Sixty ofthe isolates analyzed were identified as A acidoterrestris12 as A acidocaldarius one as Brevibacillus agri and twoas Bacillus ginsengihumi Four of the isolates which gaveambiguous results during classic identification (nontypicalcolour on an erythritol medium or lack of growth at 65∘Cconnected with lack of guaiacol production) were identified

by genetic methods as A acidoterrestris or A acidocaldariusA acidoterrestris isolates were grouped in two major clusters27 of the isolates belonged to the cluster I and 33 to thecluster II Also A acidocaldarius isolates were grouped intotwo clusters but showed more intracluster diversity

These results support the observations made by Osopaleat al [60] and Durak et al [23] who also reported two geneticclusters among analyzed A acidoterrestris samples by RAPDanalysisand by 16S rDNA sequencing respectively

Most of the strains analyzed in this study were isolatedfrom concentrated apple juice as this is the main subject ofthe Alicyclobacillus focused screening made or outsourced bypolish fruit industry however it is one of the main subjectsof similar screenings performed by fruit industry worldwideA acidoterrestris representing both clusters have been foundin concentrated apple juice For other sources three Aacidoterrestris cluster II isolates have been found in orangejuices two cluster I in concentrated black currant juice andthree cluster II in concentrated cherry juice however theseare only single observations and further research is needed toestablish ifA acidoterrestris strains representing both clustersare found in other juices

Genetic methods are broadly used in the detectioncharacterization and differentiation of microorganisms Theapplication of PCR-RFLP in the characterization of Ali-cyclobacillus and other thermoacidophilic bacteria isolatedfrom the apple juice processing environment has beendescribed by Chen et al [52] although only the 16S rDNAgene was the subject of this study and the Hin6I enzymewas not used RpoB gene has never been subjected to PCR-RFLP or any other genetic analysis of Alicyclobacillus sofar although the value of this gene in taxonomy has beenconfirmed by many studies on other microorganisms Todate there are no reports on the use of the vdc gene cluster intaxonomic studies of microorganisms although its sequencemay be proven valuable for research both on the vectors anddiversity and evolution of the element itself Hin6I restrictionpatterns for 16S rDNAwere sufficient to differentiate betweenall the species analyzed but provided no closer data onintraspecies diversity The diversity was revealed both by therpoB gene and vdc region RFLP analyses

Vdc gene cluster of Streptomyces sp described by Chowet al [55] consists of three genes vdcB (06kb) vdcC (14 kb)and vdcD (02 kb) transcribed as single policistronic mRNAmolecule All three genes were essential to produce guaiacolfrom vanillic acid Niwa and Kawamoto [38] describedanalogical gene cluster in A acidoterrestris consisting ofORF1 (597 bp) ORF2 (1425 bp) and ORF3 (321 bp) Threerespective open reading frameswere found in all five analyzedvdc sequences

Alicyclobacillus acidoterrestris RFLP patterns of boththe rpoB gene and vdc region showed consistently thatthere were two major types among the isolates one similarto the reference strain A acidoterrestris DSM 2498 andthe other similar to the reference strain A acidoterrestrisATCC 49025 Obtaining such consistent data concerningtwo genetic elements of distant function reveals that there isdeeper intraspecies genetic diversity in the A acidoterrestrisspecies This division may be considered when examining

10 BioMed Research International

other features of Alicyclobacillus such as susceptibility totemperature or high hydrostatic pressure in order to establishif there are any differences between the groups

Both the rpoB gene and vdc region seem to be single-copy genetic elements and showed no signs of intragenomicheterogeneity which often makes the RFLP comparison ofrDNA genes harder to perform

The sequence analysis of the 16S rDNA fragment ofselected isolates shows no apparent correlation betweendiversity of the 16S rDNA sequence and the sources whichthe strains were isolated from

In conclusion the application of PCR-RFLP has beenproven to be a fast and reliable method for Alicyclobacillusidentification and differentiation The method is also techni-cally less difficult than most of other molecular techniquesTwo major groups of A acidoterrestris have been identifiedThe primers designed for this study could be useful in furtherresearch on Alicyclobacillus

Data Availability

The DNA sequences obtained during this study have beendeposited in GenBank and the accession numbers areincluded within the article

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

Our research project was fully sponsored by Polish Ministryof Science and Higher Education with funds for statutoryactivity

References

[1] K Yamazaki H Teouka and H Shinano ldquoIsolation and identi-fication of alicyclobacillus acidoterrestris from acidic beveragesrdquoBioscience Biotechnology and Biochemistry vol 60 no 3 pp543ndash545 1996

[2] G DARLAND and T D BROCK ldquoBacillus acidocaldar-ius spnov an Acidophilic Thermophilic Spore-forming Bac-teriumrdquo Journal of General Microbiology vol 67 no 1 pp 9ndash151971

[3] G Deinhard P Blanz K Poralla and E Altan ldquoBacillusacidoterrestris sp nov a new thermotolerant acidophile isolatedfrom different soilsrdquo Systematic and Applied Microbiology vol10 no 1 pp 47ndash53 1987

[4] G Deinhard J Saar W Krischke and K Poralla ldquoBacilluscycloheptanicus sp nov a new thermoacidophile containing 120596-cycloheptane fatty acidsrdquo Systematic and Applied Microbiologyvol 10 no 1 pp 68ndash73 1987

[5] L Albuquerque F A Rainey A P Chung et al ldquoAlicyclobacillushesperidum sp nov and a related genomic species from solfa-taric soils of Sao Miguel in the Azoresrdquo International Journalof Systematic and Evolutionary Microbiology vol 50 no 2 pp451ndash457 2000

[6] K Goto H Matsubara K Mochida et al ldquoAlicyclobacillusherbarius sp nov a novel bacterium containing120596-cycloheptane

fatty acids isolated from herbal teardquo International Journal ofSystematic and EvolutionaryMicrobiology vol 52 no 1 pp 109ndash113 2002

[7] K Goto Y Tanimoto T Tamura et al ldquoIdentificationof thermoacidophilic bacteria and a new Alicyclobacillusgenomic species isolated from acidic environments in JapanrdquoExtremophiles vol 6 no 4 pp 333ndash340 2002

[8] H Matsubara K Goto T Matsumura et al ldquoAlicyclobacillusacidiphilus sp nov a novel thermo-acidophilic 120596-alicyclicfatty acid-containing bacterium isolated from acidic beveragesrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 52 no 5 pp 1681ndash1685 2002

[9] R V Orr R L Shewfelt C J Huang S Tefera and L RBeuchat ldquoDetection of guaiacol produced by Alicyclobacillusacidoterrestris in apple juice by sensory and chromatographicanalyses and comparison with spore and vegetative cell popu-lationsrdquo Journal of Food Protection vol 63 no 11 pp 1517ndash15222000

[10] G L Pettipher M E Osmundson and J MMurphy ldquoMethodsfor the detection and enumeration of Alicyclobacillus acidoter-restris and investigation of growth and production of taint infruit juice and fruit juice-containing drinksrdquo Letters in AppliedMicrobiology vol 24 no 3 pp 185ndash189 1997

[11] A C N F Spinelli A S Sant1015840Ana S Rodrigues Jr andP R Massaguer ldquoInfluence of different filling cooling andstorage conditions on the growth of Alicyclobacillus acidoter-restris CRA7152 in orange juicerdquo Applied and EnvironmentalMicrobiology vol 75 no 23 pp 7409ndash7416 2009

[12] MCMaldonado C Belfiore andA RNavarro ldquoTemperaturesoluble solids and pH effect on Alicyclobacillus acidoterrestrisviability in lemon juice concentraterdquo Journal of IndustrialMicrobiology and Biotechnology vol 35 no 2 pp 141ndash144 2008

[13] M N U Eiroa V C A Junqueira and F L Schmidt ldquoAlicy-clobacillus in orange juice Occurrence and heat resistance ofsporesrdquo Journal of Food Protection vol 62 no 8 pp 883ndash8861999

[14] I Walls and R Chuyate ldquoIsolation of Alicyclobacillus acidoter-restris from fruit juicesrdquo Journal of AOAC International vol 83no 5 pp 1115ndash1120 2000

[15] M E Parish and R M Goodrich ldquoRecovery of presumptiveAlicyclobacillus strains from orange fruit surfacesrdquo Journal ofFood Protection vol 68 no 10 pp 2196ndash2200 2005

[16] D F Splittstoesser J J Churey and C Y Lee ldquoGrowthcharacteristics of aciduric sporeforming bacilli isolated fromfruit juicesrdquo Journal of Food Protection vol 57 no 12 pp 1080ndash1083 1994

[17] J Baumgart M Husemann and C Schmidt ldquoAlicyclobacil-lus acidoterrestris vorkommen bedeutung und nachweis ingetranken und getrankegrundstoffenrdquo Flussiges Obst vol 64pp 178ndash180 1997

[18] C A Wisse and M E Parish ldquoIsolation and enumeration ofspore-forming thermoacidophilic rod-shaped bacteria fromcitrus processing environmentsrdquo Dairy Food EnvironmentalSanitation vol 18 pp 504ndash509 1998

[19] J Baumgart and S Menje ldquoThe impact of Alicyclobacillusacidoterestris on the quality of juices and soft drinksrdquo FruitProcess vol 10 pp 251ndash254 2000

[20] S Y Eguchi G P Manfio M E Pinhatti E Azuma and SF Variane ldquoAcidotermofilic sporeforming bacteria (ATSB) inorange juices ecology and involvement in the deterioration offruit juices Report of the Research Project Part IIrdquo Fruit Processvol 11 pp 55ndash62 2001

BioMed Research International 11

[21] P AGouws L Gie A Pretorius andNDhansay ldquoIsolation andidentification of Alicyclobacillus acidocaldarius by 16S rDNAfrom mango juice and concentraterdquo International Journal ofFood Science amp Technology vol 40 no 7 pp 789ndash792 2005

[22] K Goto K Mochida Y Kato et al ldquoProposal of six speciesof moderately thermophilic acidophilic endospore-formingbacteria Alicyclobacillus contaminans sp nov Alicyclobacillusfastidiosus sp novAlicyclobacillus kakegawensis sp novAlicy-clobacillus macrosporangiidus sp nov Alicyclobacillus saccharisp nov and Alicyclobacillus shizuokensis sp novrdquo InternationalJournal of Systematic and Evolutionary Microbiology vol 57 no6 pp 1276ndash1285 2007

[23] M Z Durak J J Churey M D Danyluk and R W WoroboldquoIdentification and haplotype distribution of Alicyclobacillusspp from different juices and beveragesrdquo International Journalof Food Microbiology vol 142 no 3 pp 286ndash291 2010

[24] B Sokolowska J Niezgoda A Dekowska et al ldquoIncidence ofAlicyclobacillus spp in Polish apple and dark berry juice con-centrates and the ability of isolated A acidoterrestris strains tospoilage of these juicesrdquo Postępy Nauki i Technologii PrzemysłuRolno-Spozywczego vol 71 no 1 pp 5ndash20 2016

[25] I Walls and R Chuyate ldquoAlicyclobacillus historical perspectiveand preliminary characterization studyrdquo Dairy Food EnvironSanitation18 pp 499ndash503 1998

[26] H-A Duong and N Jensen ldquoSpoilage of iced tea by Alicy-clobacillusrdquo Food Australia vol 52 no 7 p 292 2000

[27] M Niwa and A Kuriyama ldquoA acidoterrestris rapid detectionkitrdquo Fruit Process vol 13 pp 328ndash331 2003

[28] K S Bahceci V Gokmen and J Acar ldquoFormation of guaiacolfrom vanillin by Alicyclobacillus acidoterrestris in apple juice Amodel studyrdquo European Food Research and Technology vol 220no 2 pp 196ndash199 2005

[29] R C Witthuhn E van der Merwe P Venter and M CameronldquoGuaiacol production from ferulic acid vanillin and vanillicacid by Alicyclobacillus acidoterrestrisrdquo International Journal ofFood Microbiology vol 157 no 1 pp 113ndash117 2012

[30] A Borlinghaus and R Engel ldquoAlicyclobacillus incidence in com-mercial apple juice concentrate (AJC) supplies and validationrdquoFruit Process vol 7 pp 262ndash266 1997

[31] D F Splittstoesser C Y Lee and J J Churey ldquoControl ofAlicyclobacillus in the juice industryrdquo Dairy Food EnvironSanitation vol 18 pp 585ndash587 1998

[32] N Jensen ldquoAlicyclobacillus in Australiardquo Food Australia vol 52no 7 pp 282ndash285 2000

[33] IWalls andR Chuyate ldquoSpoilage of fruit juices byAlicyclobacil-lus acidoterrestrisrdquo Food Australia vol 52 no 7 pp 293ndash2952000

[34] N Jensen and F B Whitfield ldquoRole of Alicyclobacillus acidoter-restris in the development of a disinfectant taint in shelf-stablefruit juicerdquo Letters in Applied Microbiology vol 361 pp 9ndash142003

[35] D Gocmen A Elston T Williams M Parish and R LRouseff ldquoIdentification of medicinal off-flavours generated byAlicyclobacillus species in orange juice using GC-olfactometryand GC-MSrdquo Letters in Applied Microbiology vol 40 no 3 pp172ndash177 2005

[36] M Niwa ldquoControl of hazardous bacteria in acidic beveragesby using a guaiacol detection kit (peroxidase methodrdquo FruitProcess vol 15 pp 388ndash392 2005

[37] B Siegmund and B Pollinger-Zierler ldquoGrowth behavior ofoff-flavor-forming microorganisms in apple juicerdquo Journal of

Agricultural and Food Chemistry vol 55 no 16 pp 6692ndash66992007

[38] M Niwa and A Kawamoto ldquoDevelopment of a rapid detectionmethod of A acidoterrestris hazardous bacteria to acidicbeveragerdquo Fruit Process vol 13 pp 102ndash107 2003

[39] T A Eisele and M J Semon ldquoBest estimated aroma andtaste detection threshold for guaiacol in water and apple juicerdquoJournal of Food Science vol 70 no 4 pp S267ndashS269 2005

[40] B Zierler B Siegmund and W Pfannhauser ldquoDeterminationof off-flavour compounds in apple juice caused by microor-ganisms using headspace solid phase microextraction-gaschromatography-mass spectrometryrdquo Analytica Chimica Actavol 520 no 1-2 pp 3ndash11 2004

[41] K S Bahceci and J Acar ldquoModeling the combined effectsof pH temperature and ascorbic acid concentration on theheat resistance of Alicyclobacillus acidoterrestisrdquo InternationalJournal of Food Microbiology vol 120 no 3 pp 266ndash273 2007

[42] J Baumgart ldquoChapter 11 Media for the detection and enu-meration of Alicyclobacillus acidoterrestris and Alicyclobacillusacidocaldarius in foodsrdquo in Progress in Industrial Microbiologyvol 37 pp 161ndash166 Elsevier 2003

[43] M Lin M Al-Holy S-S Chang et al ldquoRapid discriminationof Alicyclobacillus strains in apple juice by Fourier transforminfrared spectroscopyrdquo International Journal of Food Microbiol-ogy vol 105 no 3 pp 369ndash376 2005

[44] J Wang T Yue Y Yuan X Lu J-H Shin and B RascoldquoDiscrimination of alicyclobacillus strains using nitrocellulosemembrane filter and attenuated total reflectance fourier trans-form infrared spectroscopyrdquo Journal of Food Science vol 76 no2 pp 137ndash142 2011

[45] M A Al-Holy M Lin O A Alhaj and M H Abu-GoushldquoDiscrimination between Bacillus and Alicyclobacillus Isolatesin Apple Juice by Fourier Transform Infrared Spectroscopy andMultivariate Analysisrdquo Journal of Food Science vol 80 no 2 ppM399ndashM404 2015

[46] WH Groenewald P A Gouws andR CWitthuhn ldquoIsolationidentification and typification of Alicyclobacillus acidoterrestrisand Alicyclobacillus acidocaldarius strains from orchard soiland the fruit processing environment in South Africardquo FoodMicrobiology vol 26 no 1 pp 71ndash76 2009

[47] J Zhang T Yue and Y Yuan ldquoAlicyclobacillus contaminationin the production line of Kiwi products in Chinardquo PLoS ONEvol 8 no 7 p e67704 2013

[48] I C McKnight M N U Eiroa A S SantrsquoAna and P RMassaguer ldquoAlicyclobacillus acidoterrestris in pasteurized exoticBrazilian fruit juices Isolation genotypic characterization andheat resistancerdquo FoodMicrobiology vol 27 no 8 pp 1016ndash10222010

[49] L Felix-Valenzuela I Guardiola-Avila A Burgara-Estrella MIbarra-Zavala and V Mata-Haro ldquoGenotypic and phenotypicdiversity of Alicyclobacillus acidocaldarius isolatesrdquo Letters inApplied Microbiology vol 61 no 4 pp 367ndash373 2015

[50] J D Wisotzkey P Jurtshuk Jr G E Fox G Deinhard andK Poralla ldquoComparative sequence analyses on the 16S rRNA(rDNA) of Bacillus acidocaldarius Bacillus acidoterrestris andBacillus cycloheptanicus and proposal for creation of a newgenus Alicyclobacillus gen novrdquo International Journal of Sys-tematic Bacteriology vol 42 no 2 pp 263ndash269 1992

[51] C J Connor H Luo B B McSpadden Gardener and HH Wang ldquoDevelopment of a real-time PCR-based systemtargeting the 16S rRNA gene sequence for rapid detection of

12 BioMed Research International

Alicyclobacillus spp in juice productsrdquo International Journal ofFood Microbiology vol 99 no 3 pp 229ndash235 2005

[52] S ChenQ Tang X Zhang et al ldquoIsolation and characterizationof thermo-acidophilic endospore-forming bacteria from theconcentrated apple juice-processing environmentrdquo FoodMicro-biology vol 23 no 5 pp 439ndash445 2006

[53] K Goto A Nishibori Y Wasada K Furuhata M Fukuyamaand M Hara ldquoIdentification of thermo-acidophilic bacteriaisolated from the soil of several Japanese fruit orchardsrdquo Lettersin Applied Microbiology vol 46 no 3 pp 289ndash294 2008

[54] J Chen X Ma Y Yuan and W Zhang ldquoSensitive and rapiddetection ofAlicyclobacillus acidoterrestris using loop-mediatedisothermal amplificationrdquo Journal of the Science of Food andAgriculture vol 91 no 6 pp 1070ndash1074 2011

[55] K T Chow M K Pope and J Davies ldquoCharacterization of avanillic acid non-oxidative decarboxylation gene cluster fromStreptomyces sp D7rdquo Microbiology vol 145 no 9 pp 2393ndash2403 1999

[56] K Matsubara ldquoA method for detecting andor identifying gua-iacol producing micro organismrdquo Patent no JP2003000259A2003

[57] Y Wang T Yue Y Yuan and Z Gao ldquoIsolation and identifi-cation of thermo-acidophilic bacteria from orchards in chinardquoJournal of Food Protection vol 73 pp 390ndash394 2010

[58] M Shemesh R Pasvolsky N Sela S J Green and V andZakinldquoDraft Genome Sequence of Alicyclobacillus acidoterrestrisStrain ATCC 49025rdquo Genome Announcement vol 1 no 5Article ID e00638ndash13 2013

[59] B Sokołowska S Skapska M Fonberg-Broczek et al ldquoFactorsinfluencing the inactivation of Alicyclobacillus acidoterrestrisspores exposed to high hydrostatic pressure in apple juicerdquoHighPressure Research vol 33 no 1 pp 73ndash82 2013

[60] B A Osopale C R Witthuhn J Albertyn and F A Oguntoy-inbo ldquoCulture dependent and independent genomic identifica-tion ofAlicyclobacillus species in contaminated commercial fruitjuicesrdquo Food Microbiology vol 56 pp 21ndash28 2016

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6 BioMed Research International

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

bp

1000

500

200

Figure 2 Restriction patterns of a 1495 bp fragment of the 16S rDNA gene digested with Hin6I Lanes 6 7 9 14 and 18 A acidoterrestris 23 4 8 10 11 12 13 15 16 and 17 A acidocaldarius 5 Brevibacillus agri 19 A acidoterrestris DSM 2498 20 A acidoterrestris ATCC49025 21A acidiphilus 22 A hesperidum 23 A herbarius 24 A acidocaldarius 25 B subtilis 26 G stearothermophilus lane 1 molecular marker

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 3 Restriction patterns of a 1586 bp fragment of the vdc region digested with BsuRI Lanes 1ndash3 5 7ndash11 13 and 14 A acidoterrestristype I 4 6 12 and 15 A acidoterrestris type II 16 A acidoterrestris DSM 2498 17 A acidoterrestris ATCC49025 18 A acidiphilus 19 Aherbarius 20 molecular marker

HphI patterns confirm this rule with the exception of onestrain which represented the type I pattern in the BsuRI andHin6I analysis

33 RFLP Analysis of the rpoB Gene Fragment The 1735 bpfragment of the rpoB gene was amplified using Gru3 andGru4 or Gru5 and Gru6 primers Gru3 and Gru4 primerswere degenerated versions of Gru5 and Gru6 (respectively)and were used for amplification of isolates other thanA acidoterrestris A acidoterrestris isolates were amplifiedeither with Gru3ndashGru4 or Gru5ndashGru6 primers and theGru5ndashGru6 primer pair was chosen due to the better effi-ciency of the reaction Considering the individual isolates theRFLP patterns were identical for both pairs of primers

Thepatterns obtained by all of the nucleases used (Figures6ndash8) enabled distinguishing between the species analyzedWith exception of two strains the BsuRI patterns dividedthe A acidoterrestris group into two clusters analogicalto the clusters revealed by the vdc region analysis Thepattern (IIA) is shown by two exceptional strains mostresembling the type II pattern of A acidoterrestris andthose strains were classified as type II in other analyses Thereference strain ATCC 49025 shows this type of patternThe A acidocaldarius group was represented by three typesof patterns two of them resembling each other A acido-caldarius DSM 446 belongs to the cluster II of A acido-caldarius group while A acidocaldarius A1 was assigned tocluster I

BioMed Research International 7

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 4 Restriction patterns of a 1586 bp fragment of the vdc region digested with Hin6I Lanes 1ndash3 5 7ndash11 13 and 14 A acidoterrestristype I 4 6 12 and 15A acidoterrestris type II 16A acidoterrestrisDSM 2498 17A acidoterrestris ATCC49025 18 A acidiphilusDSM 1455819 A herbarius DSM 13609 lane 20 molecular marker

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 5 Restriction patterns of a 1586 bp fragment of the vdc region digested with HphI Lanes 2ndash4 6 8ndash12 14 and 15 A acidoterrestristype I 5 7 13 and 16 A acidoterrestris type II 17 A acidoterrestris DSM 2498 18 A acidoterrestris ATCC49025 19 A acidiphilus 20 Aherbarius lane 1 molecular marker

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Figure 6 Restriction patterns of a 1735 bp fragment of the rpoB gene digested with BsuRI Lanes 1ndash4 6 and 8ndash10 A acidoterrestris type I 57 21 22 and 24-26 A acidoterrestris type II 23 A acidoterrestris type IIA 27 A acidocaldarius type II 28 A acidocaldarius type IA 11 Aacidoterrestris DSM 2498 12 A acidoterrestris ATCC49025 13 A acidiphilus 14 A hesperidum 15 A herbarius 16 A acidocaldariusA1 17B subtilis 18 G stearothermophilus lane 19 molecular marker

8 BioMed Research International

Figure 7 Restriction patterns of a 1735 bp fragment of rpoB gene digested with Hin6I Lanes 3 and 5 A acidoterrestris type I 1 2 4 and 6Aacidoterrestris type II 17A acidocaldarius type I 18A acidocaldarius type II 8A acidoterrestrisDSM2498 9A acidoterrestrisATCC4902510A acidiphilus 11 A hesperidum 12A herbarius 13A acidocaldariusA1 14 B subtilis 15G stearothermophilus lanes 7 and 16 molecularmarker

Figure 8 Restriction patterns of a 1735 bp fragment of rpoB gene digested with HphI Lanes 1-4 6 8-11 13 25 and 27 A acidoterrestris I5 7 12 26 and 28 A acidoterrestris II 24 A acidoterrestris IIA 29 A acidocaldarius IA 30 A acidocaldarius type II 15 A acidoterrestrisDSM 2498 16 A acidoterrestris ATCC49025 17 A acidiphilus 18 A hesperidum 19 A herbarius 20 A acidocaldariusA1 21 B subtilis 22G stearothermophilus lanes 14 and 23 molecular marker

DSM_2498 00003231 -00003234 -00025641 0ATCC_49025 00022455 00005656 -00009252 -00001833 051 053 -000013

Figure 9 Phylogenetic tree of partial 16S rRNA sequence of 11 Aacidoterrestris isolatesThe tree was constructed by neighbor joiningmethod using Clustal Omega software

For Hin6I there were two types of patterns for the Aacidoterrestris and A acidocaldarius A acidoterrestris DSM2498 belongs to the cluster I while A acidoterrestris ATCC49025 belongs to the cluster II A acidocaldarius DSM 446represents cluster II of the A acidocaldarius group while AacidocaldariusA1 represents cluster I

HphI endonuclease produces two types of patterns forA acidoterrestris analogically as before and three types ofpatterns for A acidocaldarius One sample of A acidoter-restris cluster II has a slightly different pattern and has been

classified as type II in other analyses Two of the threepatterns of A acidocaldarius differ with only one bandA acidocaldarius DSM 446 represents cluster II of the Aacidocaldarius group while A acidocaldarius A1 representscluster I

34 DNA Sequencing DNA sequencing was performed forselected strains Sequencing of the 16S rDNA gene wasperformed to confirm the proper identification of the isolatesand to identify the non-Alicyclobacillus isolates The 16SrDNA sequence of strains 31 and 34 classified as type Ishowed the greatest similarity toA acidoterrestrisDSM 2498also classified as type I The sequences of strains 33 51 52 5355 and 56 classified as type II showed the greatest similaritytoA acidoterrestrisATCC49025 also classified as type IIThesequence of strain 41 classified as type II showed the greatestsimilarity to A acidoterrestris DSM 3922

Figure 9 shows the phylogenetic tree constructed from the16S rDNA sequences of analyzed strains of A acidoterrestrisThe sequence analysis indicates that A acidoterrestris DSM2498 and two other type I strains are closely related and itshows greater variation within class II strains The isolatesselected for sequencing represent both groups and different

BioMed Research International 9

sources which they were isolated from concentrated applejuice (isolates 33 and 41) fresh apples (isolate 34) concen-trated blackcurrant juice (isolate 31) concentrated raspberryjuice (isolate 56) concentrated beetroot juice (isolate 51)and concentrated cherry juice (isolates 52 53 and 55) Thesequence analysis shows no apparent correlation betweendiversity of the 16S rDNA sequence and the sources of theisolates

Fivewhole vdc gene clusterswere sequenced Twoof themwere isolated from the reference strains A acidoterrestrisDSM 2498 and A acidoterrestris ATCC 49025 2416 bpfragments of the vdc regions were aligned The vdc regionsequence of strains 15 and 34 classified as type I showed999 identity with the vdc sequence of A acidoterrestrisDSM 2498 the sequence of strain 41 classified as typeII showed 993 identity with the vdc sequence A aci-doterrestris ATCC 49025 Vdc sequences of A acidoterrestrisDSM 2498 and A acidoterrestris DSM 49025 showed 945identity For the last pair protein sequences of the geneproducts showed 985 positives and 965 identities forVdcB 994 and 989 for VdcC and 100 and 974 forVdcD

RpoB gene sequencing was performed to confirm thatboth primer pairs Gru3ndashGru4 and Gru5ndashGru6 enabledto amplify the correct DNA fragment and that degenera-tion of the primers did not affect the sequence specificityalthough the degenerated primers produced significantlylower amount of PCR product All of the sequences obtainedshowed greatest similarity to the appropriate rpoB genes

The GenBank accession numbers are KX371237-KX371249 for 16S rDNA sequences KX453673-KX453677for full vdc region sequences KX453678 and KX453679 forpartial vdc sequences of A herbarius and A acidiphilusrespectively and KX453680- KX453682 for partial rpoBsequences

4 Discussion

Among more than one thousand samples of concentratedapple juice tested in our laboratory between 2004 and 201267 was contaminated with Alicyclobacillus sp and 31 ofthe isolates were identified as A acidoterrestris [59] Thestatistics show that Alicyclobacillus spoilage is still a majorconcern in the fruit processing industry

In this study 75 guaiacol producing and non-guaiacolproducing strains were isolated from various fruit juicesconcentrated fruit juices and fresh apples Additionally8 reference strains were used The isolates and referencestrains were examined using classic methods and PCR-RFLP focusing on 16S rDNA and rpoB gene fragments aswell as the vdc region fragment For selected strains DNAsequencing of the 16S rDNA gene was performed Sixty ofthe isolates analyzed were identified as A acidoterrestris12 as A acidocaldarius one as Brevibacillus agri and twoas Bacillus ginsengihumi Four of the isolates which gaveambiguous results during classic identification (nontypicalcolour on an erythritol medium or lack of growth at 65∘Cconnected with lack of guaiacol production) were identified

by genetic methods as A acidoterrestris or A acidocaldariusA acidoterrestris isolates were grouped in two major clusters27 of the isolates belonged to the cluster I and 33 to thecluster II Also A acidocaldarius isolates were grouped intotwo clusters but showed more intracluster diversity

These results support the observations made by Osopaleat al [60] and Durak et al [23] who also reported two geneticclusters among analyzed A acidoterrestris samples by RAPDanalysisand by 16S rDNA sequencing respectively

Most of the strains analyzed in this study were isolatedfrom concentrated apple juice as this is the main subject ofthe Alicyclobacillus focused screening made or outsourced bypolish fruit industry however it is one of the main subjectsof similar screenings performed by fruit industry worldwideA acidoterrestris representing both clusters have been foundin concentrated apple juice For other sources three Aacidoterrestris cluster II isolates have been found in orangejuices two cluster I in concentrated black currant juice andthree cluster II in concentrated cherry juice however theseare only single observations and further research is needed toestablish ifA acidoterrestris strains representing both clustersare found in other juices

Genetic methods are broadly used in the detectioncharacterization and differentiation of microorganisms Theapplication of PCR-RFLP in the characterization of Ali-cyclobacillus and other thermoacidophilic bacteria isolatedfrom the apple juice processing environment has beendescribed by Chen et al [52] although only the 16S rDNAgene was the subject of this study and the Hin6I enzymewas not used RpoB gene has never been subjected to PCR-RFLP or any other genetic analysis of Alicyclobacillus sofar although the value of this gene in taxonomy has beenconfirmed by many studies on other microorganisms Todate there are no reports on the use of the vdc gene cluster intaxonomic studies of microorganisms although its sequencemay be proven valuable for research both on the vectors anddiversity and evolution of the element itself Hin6I restrictionpatterns for 16S rDNAwere sufficient to differentiate betweenall the species analyzed but provided no closer data onintraspecies diversity The diversity was revealed both by therpoB gene and vdc region RFLP analyses

Vdc gene cluster of Streptomyces sp described by Chowet al [55] consists of three genes vdcB (06kb) vdcC (14 kb)and vdcD (02 kb) transcribed as single policistronic mRNAmolecule All three genes were essential to produce guaiacolfrom vanillic acid Niwa and Kawamoto [38] describedanalogical gene cluster in A acidoterrestris consisting ofORF1 (597 bp) ORF2 (1425 bp) and ORF3 (321 bp) Threerespective open reading frameswere found in all five analyzedvdc sequences

Alicyclobacillus acidoterrestris RFLP patterns of boththe rpoB gene and vdc region showed consistently thatthere were two major types among the isolates one similarto the reference strain A acidoterrestris DSM 2498 andthe other similar to the reference strain A acidoterrestrisATCC 49025 Obtaining such consistent data concerningtwo genetic elements of distant function reveals that there isdeeper intraspecies genetic diversity in the A acidoterrestrisspecies This division may be considered when examining

10 BioMed Research International

other features of Alicyclobacillus such as susceptibility totemperature or high hydrostatic pressure in order to establishif there are any differences between the groups

Both the rpoB gene and vdc region seem to be single-copy genetic elements and showed no signs of intragenomicheterogeneity which often makes the RFLP comparison ofrDNA genes harder to perform

The sequence analysis of the 16S rDNA fragment ofselected isolates shows no apparent correlation betweendiversity of the 16S rDNA sequence and the sources whichthe strains were isolated from

In conclusion the application of PCR-RFLP has beenproven to be a fast and reliable method for Alicyclobacillusidentification and differentiation The method is also techni-cally less difficult than most of other molecular techniquesTwo major groups of A acidoterrestris have been identifiedThe primers designed for this study could be useful in furtherresearch on Alicyclobacillus

Data Availability

The DNA sequences obtained during this study have beendeposited in GenBank and the accession numbers areincluded within the article

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

Our research project was fully sponsored by Polish Ministryof Science and Higher Education with funds for statutoryactivity

References

[1] K Yamazaki H Teouka and H Shinano ldquoIsolation and identi-fication of alicyclobacillus acidoterrestris from acidic beveragesrdquoBioscience Biotechnology and Biochemistry vol 60 no 3 pp543ndash545 1996

[2] G DARLAND and T D BROCK ldquoBacillus acidocaldar-ius spnov an Acidophilic Thermophilic Spore-forming Bac-teriumrdquo Journal of General Microbiology vol 67 no 1 pp 9ndash151971

[3] G Deinhard P Blanz K Poralla and E Altan ldquoBacillusacidoterrestris sp nov a new thermotolerant acidophile isolatedfrom different soilsrdquo Systematic and Applied Microbiology vol10 no 1 pp 47ndash53 1987

[4] G Deinhard J Saar W Krischke and K Poralla ldquoBacilluscycloheptanicus sp nov a new thermoacidophile containing 120596-cycloheptane fatty acidsrdquo Systematic and Applied Microbiologyvol 10 no 1 pp 68ndash73 1987

[5] L Albuquerque F A Rainey A P Chung et al ldquoAlicyclobacillushesperidum sp nov and a related genomic species from solfa-taric soils of Sao Miguel in the Azoresrdquo International Journalof Systematic and Evolutionary Microbiology vol 50 no 2 pp451ndash457 2000

[6] K Goto H Matsubara K Mochida et al ldquoAlicyclobacillusherbarius sp nov a novel bacterium containing120596-cycloheptane

fatty acids isolated from herbal teardquo International Journal ofSystematic and EvolutionaryMicrobiology vol 52 no 1 pp 109ndash113 2002

[7] K Goto Y Tanimoto T Tamura et al ldquoIdentificationof thermoacidophilic bacteria and a new Alicyclobacillusgenomic species isolated from acidic environments in JapanrdquoExtremophiles vol 6 no 4 pp 333ndash340 2002

[8] H Matsubara K Goto T Matsumura et al ldquoAlicyclobacillusacidiphilus sp nov a novel thermo-acidophilic 120596-alicyclicfatty acid-containing bacterium isolated from acidic beveragesrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 52 no 5 pp 1681ndash1685 2002

[9] R V Orr R L Shewfelt C J Huang S Tefera and L RBeuchat ldquoDetection of guaiacol produced by Alicyclobacillusacidoterrestris in apple juice by sensory and chromatographicanalyses and comparison with spore and vegetative cell popu-lationsrdquo Journal of Food Protection vol 63 no 11 pp 1517ndash15222000

[10] G L Pettipher M E Osmundson and J MMurphy ldquoMethodsfor the detection and enumeration of Alicyclobacillus acidoter-restris and investigation of growth and production of taint infruit juice and fruit juice-containing drinksrdquo Letters in AppliedMicrobiology vol 24 no 3 pp 185ndash189 1997

[11] A C N F Spinelli A S Sant1015840Ana S Rodrigues Jr andP R Massaguer ldquoInfluence of different filling cooling andstorage conditions on the growth of Alicyclobacillus acidoter-restris CRA7152 in orange juicerdquo Applied and EnvironmentalMicrobiology vol 75 no 23 pp 7409ndash7416 2009

[12] MCMaldonado C Belfiore andA RNavarro ldquoTemperaturesoluble solids and pH effect on Alicyclobacillus acidoterrestrisviability in lemon juice concentraterdquo Journal of IndustrialMicrobiology and Biotechnology vol 35 no 2 pp 141ndash144 2008

[13] M N U Eiroa V C A Junqueira and F L Schmidt ldquoAlicy-clobacillus in orange juice Occurrence and heat resistance ofsporesrdquo Journal of Food Protection vol 62 no 8 pp 883ndash8861999

[14] I Walls and R Chuyate ldquoIsolation of Alicyclobacillus acidoter-restris from fruit juicesrdquo Journal of AOAC International vol 83no 5 pp 1115ndash1120 2000

[15] M E Parish and R M Goodrich ldquoRecovery of presumptiveAlicyclobacillus strains from orange fruit surfacesrdquo Journal ofFood Protection vol 68 no 10 pp 2196ndash2200 2005

[16] D F Splittstoesser J J Churey and C Y Lee ldquoGrowthcharacteristics of aciduric sporeforming bacilli isolated fromfruit juicesrdquo Journal of Food Protection vol 57 no 12 pp 1080ndash1083 1994

[17] J Baumgart M Husemann and C Schmidt ldquoAlicyclobacil-lus acidoterrestris vorkommen bedeutung und nachweis ingetranken und getrankegrundstoffenrdquo Flussiges Obst vol 64pp 178ndash180 1997

[18] C A Wisse and M E Parish ldquoIsolation and enumeration ofspore-forming thermoacidophilic rod-shaped bacteria fromcitrus processing environmentsrdquo Dairy Food EnvironmentalSanitation vol 18 pp 504ndash509 1998

[19] J Baumgart and S Menje ldquoThe impact of Alicyclobacillusacidoterestris on the quality of juices and soft drinksrdquo FruitProcess vol 10 pp 251ndash254 2000

[20] S Y Eguchi G P Manfio M E Pinhatti E Azuma and SF Variane ldquoAcidotermofilic sporeforming bacteria (ATSB) inorange juices ecology and involvement in the deterioration offruit juices Report of the Research Project Part IIrdquo Fruit Processvol 11 pp 55ndash62 2001

BioMed Research International 11

[21] P AGouws L Gie A Pretorius andNDhansay ldquoIsolation andidentification of Alicyclobacillus acidocaldarius by 16S rDNAfrom mango juice and concentraterdquo International Journal ofFood Science amp Technology vol 40 no 7 pp 789ndash792 2005

[22] K Goto K Mochida Y Kato et al ldquoProposal of six speciesof moderately thermophilic acidophilic endospore-formingbacteria Alicyclobacillus contaminans sp nov Alicyclobacillusfastidiosus sp novAlicyclobacillus kakegawensis sp novAlicy-clobacillus macrosporangiidus sp nov Alicyclobacillus saccharisp nov and Alicyclobacillus shizuokensis sp novrdquo InternationalJournal of Systematic and Evolutionary Microbiology vol 57 no6 pp 1276ndash1285 2007

[23] M Z Durak J J Churey M D Danyluk and R W WoroboldquoIdentification and haplotype distribution of Alicyclobacillusspp from different juices and beveragesrdquo International Journalof Food Microbiology vol 142 no 3 pp 286ndash291 2010

[24] B Sokolowska J Niezgoda A Dekowska et al ldquoIncidence ofAlicyclobacillus spp in Polish apple and dark berry juice con-centrates and the ability of isolated A acidoterrestris strains tospoilage of these juicesrdquo Postępy Nauki i Technologii PrzemysłuRolno-Spozywczego vol 71 no 1 pp 5ndash20 2016

[25] I Walls and R Chuyate ldquoAlicyclobacillus historical perspectiveand preliminary characterization studyrdquo Dairy Food EnvironSanitation18 pp 499ndash503 1998

[26] H-A Duong and N Jensen ldquoSpoilage of iced tea by Alicy-clobacillusrdquo Food Australia vol 52 no 7 p 292 2000

[27] M Niwa and A Kuriyama ldquoA acidoterrestris rapid detectionkitrdquo Fruit Process vol 13 pp 328ndash331 2003

[28] K S Bahceci V Gokmen and J Acar ldquoFormation of guaiacolfrom vanillin by Alicyclobacillus acidoterrestris in apple juice Amodel studyrdquo European Food Research and Technology vol 220no 2 pp 196ndash199 2005

[29] R C Witthuhn E van der Merwe P Venter and M CameronldquoGuaiacol production from ferulic acid vanillin and vanillicacid by Alicyclobacillus acidoterrestrisrdquo International Journal ofFood Microbiology vol 157 no 1 pp 113ndash117 2012

[30] A Borlinghaus and R Engel ldquoAlicyclobacillus incidence in com-mercial apple juice concentrate (AJC) supplies and validationrdquoFruit Process vol 7 pp 262ndash266 1997

[31] D F Splittstoesser C Y Lee and J J Churey ldquoControl ofAlicyclobacillus in the juice industryrdquo Dairy Food EnvironSanitation vol 18 pp 585ndash587 1998

[32] N Jensen ldquoAlicyclobacillus in Australiardquo Food Australia vol 52no 7 pp 282ndash285 2000

[33] IWalls andR Chuyate ldquoSpoilage of fruit juices byAlicyclobacil-lus acidoterrestrisrdquo Food Australia vol 52 no 7 pp 293ndash2952000

[34] N Jensen and F B Whitfield ldquoRole of Alicyclobacillus acidoter-restris in the development of a disinfectant taint in shelf-stablefruit juicerdquo Letters in Applied Microbiology vol 361 pp 9ndash142003

[35] D Gocmen A Elston T Williams M Parish and R LRouseff ldquoIdentification of medicinal off-flavours generated byAlicyclobacillus species in orange juice using GC-olfactometryand GC-MSrdquo Letters in Applied Microbiology vol 40 no 3 pp172ndash177 2005

[36] M Niwa ldquoControl of hazardous bacteria in acidic beveragesby using a guaiacol detection kit (peroxidase methodrdquo FruitProcess vol 15 pp 388ndash392 2005

[37] B Siegmund and B Pollinger-Zierler ldquoGrowth behavior ofoff-flavor-forming microorganisms in apple juicerdquo Journal of

Agricultural and Food Chemistry vol 55 no 16 pp 6692ndash66992007

[38] M Niwa and A Kawamoto ldquoDevelopment of a rapid detectionmethod of A acidoterrestris hazardous bacteria to acidicbeveragerdquo Fruit Process vol 13 pp 102ndash107 2003

[39] T A Eisele and M J Semon ldquoBest estimated aroma andtaste detection threshold for guaiacol in water and apple juicerdquoJournal of Food Science vol 70 no 4 pp S267ndashS269 2005

[40] B Zierler B Siegmund and W Pfannhauser ldquoDeterminationof off-flavour compounds in apple juice caused by microor-ganisms using headspace solid phase microextraction-gaschromatography-mass spectrometryrdquo Analytica Chimica Actavol 520 no 1-2 pp 3ndash11 2004

[41] K S Bahceci and J Acar ldquoModeling the combined effectsof pH temperature and ascorbic acid concentration on theheat resistance of Alicyclobacillus acidoterrestisrdquo InternationalJournal of Food Microbiology vol 120 no 3 pp 266ndash273 2007

[42] J Baumgart ldquoChapter 11 Media for the detection and enu-meration of Alicyclobacillus acidoterrestris and Alicyclobacillusacidocaldarius in foodsrdquo in Progress in Industrial Microbiologyvol 37 pp 161ndash166 Elsevier 2003

[43] M Lin M Al-Holy S-S Chang et al ldquoRapid discriminationof Alicyclobacillus strains in apple juice by Fourier transforminfrared spectroscopyrdquo International Journal of Food Microbiol-ogy vol 105 no 3 pp 369ndash376 2005

[44] J Wang T Yue Y Yuan X Lu J-H Shin and B RascoldquoDiscrimination of alicyclobacillus strains using nitrocellulosemembrane filter and attenuated total reflectance fourier trans-form infrared spectroscopyrdquo Journal of Food Science vol 76 no2 pp 137ndash142 2011

[45] M A Al-Holy M Lin O A Alhaj and M H Abu-GoushldquoDiscrimination between Bacillus and Alicyclobacillus Isolatesin Apple Juice by Fourier Transform Infrared Spectroscopy andMultivariate Analysisrdquo Journal of Food Science vol 80 no 2 ppM399ndashM404 2015

[46] WH Groenewald P A Gouws andR CWitthuhn ldquoIsolationidentification and typification of Alicyclobacillus acidoterrestrisand Alicyclobacillus acidocaldarius strains from orchard soiland the fruit processing environment in South Africardquo FoodMicrobiology vol 26 no 1 pp 71ndash76 2009

[47] J Zhang T Yue and Y Yuan ldquoAlicyclobacillus contaminationin the production line of Kiwi products in Chinardquo PLoS ONEvol 8 no 7 p e67704 2013

[48] I C McKnight M N U Eiroa A S SantrsquoAna and P RMassaguer ldquoAlicyclobacillus acidoterrestris in pasteurized exoticBrazilian fruit juices Isolation genotypic characterization andheat resistancerdquo FoodMicrobiology vol 27 no 8 pp 1016ndash10222010

[49] L Felix-Valenzuela I Guardiola-Avila A Burgara-Estrella MIbarra-Zavala and V Mata-Haro ldquoGenotypic and phenotypicdiversity of Alicyclobacillus acidocaldarius isolatesrdquo Letters inApplied Microbiology vol 61 no 4 pp 367ndash373 2015

[50] J D Wisotzkey P Jurtshuk Jr G E Fox G Deinhard andK Poralla ldquoComparative sequence analyses on the 16S rRNA(rDNA) of Bacillus acidocaldarius Bacillus acidoterrestris andBacillus cycloheptanicus and proposal for creation of a newgenus Alicyclobacillus gen novrdquo International Journal of Sys-tematic Bacteriology vol 42 no 2 pp 263ndash269 1992

[51] C J Connor H Luo B B McSpadden Gardener and HH Wang ldquoDevelopment of a real-time PCR-based systemtargeting the 16S rRNA gene sequence for rapid detection of

12 BioMed Research International

Alicyclobacillus spp in juice productsrdquo International Journal ofFood Microbiology vol 99 no 3 pp 229ndash235 2005

[52] S ChenQ Tang X Zhang et al ldquoIsolation and characterizationof thermo-acidophilic endospore-forming bacteria from theconcentrated apple juice-processing environmentrdquo FoodMicro-biology vol 23 no 5 pp 439ndash445 2006

[53] K Goto A Nishibori Y Wasada K Furuhata M Fukuyamaand M Hara ldquoIdentification of thermo-acidophilic bacteriaisolated from the soil of several Japanese fruit orchardsrdquo Lettersin Applied Microbiology vol 46 no 3 pp 289ndash294 2008

[54] J Chen X Ma Y Yuan and W Zhang ldquoSensitive and rapiddetection ofAlicyclobacillus acidoterrestris using loop-mediatedisothermal amplificationrdquo Journal of the Science of Food andAgriculture vol 91 no 6 pp 1070ndash1074 2011

[55] K T Chow M K Pope and J Davies ldquoCharacterization of avanillic acid non-oxidative decarboxylation gene cluster fromStreptomyces sp D7rdquo Microbiology vol 145 no 9 pp 2393ndash2403 1999

[56] K Matsubara ldquoA method for detecting andor identifying gua-iacol producing micro organismrdquo Patent no JP2003000259A2003

[57] Y Wang T Yue Y Yuan and Z Gao ldquoIsolation and identifi-cation of thermo-acidophilic bacteria from orchards in chinardquoJournal of Food Protection vol 73 pp 390ndash394 2010

[58] M Shemesh R Pasvolsky N Sela S J Green and V andZakinldquoDraft Genome Sequence of Alicyclobacillus acidoterrestrisStrain ATCC 49025rdquo Genome Announcement vol 1 no 5Article ID e00638ndash13 2013

[59] B Sokołowska S Skapska M Fonberg-Broczek et al ldquoFactorsinfluencing the inactivation of Alicyclobacillus acidoterrestrisspores exposed to high hydrostatic pressure in apple juicerdquoHighPressure Research vol 33 no 1 pp 73ndash82 2013

[60] B A Osopale C R Witthuhn J Albertyn and F A Oguntoy-inbo ldquoCulture dependent and independent genomic identifica-tion ofAlicyclobacillus species in contaminated commercial fruitjuicesrdquo Food Microbiology vol 56 pp 21ndash28 2016

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BioMed Research International 7

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 4 Restriction patterns of a 1586 bp fragment of the vdc region digested with Hin6I Lanes 1ndash3 5 7ndash11 13 and 14 A acidoterrestristype I 4 6 12 and 15A acidoterrestris type II 16A acidoterrestrisDSM 2498 17A acidoterrestris ATCC49025 18 A acidiphilusDSM 1455819 A herbarius DSM 13609 lane 20 molecular marker

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 5 Restriction patterns of a 1586 bp fragment of the vdc region digested with HphI Lanes 2ndash4 6 8ndash12 14 and 15 A acidoterrestristype I 5 7 13 and 16 A acidoterrestris type II 17 A acidoterrestris DSM 2498 18 A acidoterrestris ATCC49025 19 A acidiphilus 20 Aherbarius lane 1 molecular marker

bp

1000

500

200

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Figure 6 Restriction patterns of a 1735 bp fragment of the rpoB gene digested with BsuRI Lanes 1ndash4 6 and 8ndash10 A acidoterrestris type I 57 21 22 and 24-26 A acidoterrestris type II 23 A acidoterrestris type IIA 27 A acidocaldarius type II 28 A acidocaldarius type IA 11 Aacidoterrestris DSM 2498 12 A acidoterrestris ATCC49025 13 A acidiphilus 14 A hesperidum 15 A herbarius 16 A acidocaldariusA1 17B subtilis 18 G stearothermophilus lane 19 molecular marker

8 BioMed Research International

Figure 7 Restriction patterns of a 1735 bp fragment of rpoB gene digested with Hin6I Lanes 3 and 5 A acidoterrestris type I 1 2 4 and 6Aacidoterrestris type II 17A acidocaldarius type I 18A acidocaldarius type II 8A acidoterrestrisDSM2498 9A acidoterrestrisATCC4902510A acidiphilus 11 A hesperidum 12A herbarius 13A acidocaldariusA1 14 B subtilis 15G stearothermophilus lanes 7 and 16 molecularmarker

Figure 8 Restriction patterns of a 1735 bp fragment of rpoB gene digested with HphI Lanes 1-4 6 8-11 13 25 and 27 A acidoterrestris I5 7 12 26 and 28 A acidoterrestris II 24 A acidoterrestris IIA 29 A acidocaldarius IA 30 A acidocaldarius type II 15 A acidoterrestrisDSM 2498 16 A acidoterrestris ATCC49025 17 A acidiphilus 18 A hesperidum 19 A herbarius 20 A acidocaldariusA1 21 B subtilis 22G stearothermophilus lanes 14 and 23 molecular marker

DSM_2498 00003231 -00003234 -00025641 0ATCC_49025 00022455 00005656 -00009252 -00001833 051 053 -000013

Figure 9 Phylogenetic tree of partial 16S rRNA sequence of 11 Aacidoterrestris isolatesThe tree was constructed by neighbor joiningmethod using Clustal Omega software

For Hin6I there were two types of patterns for the Aacidoterrestris and A acidocaldarius A acidoterrestris DSM2498 belongs to the cluster I while A acidoterrestris ATCC49025 belongs to the cluster II A acidocaldarius DSM 446represents cluster II of the A acidocaldarius group while AacidocaldariusA1 represents cluster I

HphI endonuclease produces two types of patterns forA acidoterrestris analogically as before and three types ofpatterns for A acidocaldarius One sample of A acidoter-restris cluster II has a slightly different pattern and has been

classified as type II in other analyses Two of the threepatterns of A acidocaldarius differ with only one bandA acidocaldarius DSM 446 represents cluster II of the Aacidocaldarius group while A acidocaldarius A1 representscluster I

34 DNA Sequencing DNA sequencing was performed forselected strains Sequencing of the 16S rDNA gene wasperformed to confirm the proper identification of the isolatesand to identify the non-Alicyclobacillus isolates The 16SrDNA sequence of strains 31 and 34 classified as type Ishowed the greatest similarity toA acidoterrestrisDSM 2498also classified as type I The sequences of strains 33 51 52 5355 and 56 classified as type II showed the greatest similaritytoA acidoterrestrisATCC49025 also classified as type IIThesequence of strain 41 classified as type II showed the greatestsimilarity to A acidoterrestris DSM 3922

Figure 9 shows the phylogenetic tree constructed from the16S rDNA sequences of analyzed strains of A acidoterrestrisThe sequence analysis indicates that A acidoterrestris DSM2498 and two other type I strains are closely related and itshows greater variation within class II strains The isolatesselected for sequencing represent both groups and different

BioMed Research International 9

sources which they were isolated from concentrated applejuice (isolates 33 and 41) fresh apples (isolate 34) concen-trated blackcurrant juice (isolate 31) concentrated raspberryjuice (isolate 56) concentrated beetroot juice (isolate 51)and concentrated cherry juice (isolates 52 53 and 55) Thesequence analysis shows no apparent correlation betweendiversity of the 16S rDNA sequence and the sources of theisolates

Fivewhole vdc gene clusterswere sequenced Twoof themwere isolated from the reference strains A acidoterrestrisDSM 2498 and A acidoterrestris ATCC 49025 2416 bpfragments of the vdc regions were aligned The vdc regionsequence of strains 15 and 34 classified as type I showed999 identity with the vdc sequence of A acidoterrestrisDSM 2498 the sequence of strain 41 classified as typeII showed 993 identity with the vdc sequence A aci-doterrestris ATCC 49025 Vdc sequences of A acidoterrestrisDSM 2498 and A acidoterrestris DSM 49025 showed 945identity For the last pair protein sequences of the geneproducts showed 985 positives and 965 identities forVdcB 994 and 989 for VdcC and 100 and 974 forVdcD

RpoB gene sequencing was performed to confirm thatboth primer pairs Gru3ndashGru4 and Gru5ndashGru6 enabledto amplify the correct DNA fragment and that degenera-tion of the primers did not affect the sequence specificityalthough the degenerated primers produced significantlylower amount of PCR product All of the sequences obtainedshowed greatest similarity to the appropriate rpoB genes

The GenBank accession numbers are KX371237-KX371249 for 16S rDNA sequences KX453673-KX453677for full vdc region sequences KX453678 and KX453679 forpartial vdc sequences of A herbarius and A acidiphilusrespectively and KX453680- KX453682 for partial rpoBsequences

4 Discussion

Among more than one thousand samples of concentratedapple juice tested in our laboratory between 2004 and 201267 was contaminated with Alicyclobacillus sp and 31 ofthe isolates were identified as A acidoterrestris [59] Thestatistics show that Alicyclobacillus spoilage is still a majorconcern in the fruit processing industry

In this study 75 guaiacol producing and non-guaiacolproducing strains were isolated from various fruit juicesconcentrated fruit juices and fresh apples Additionally8 reference strains were used The isolates and referencestrains were examined using classic methods and PCR-RFLP focusing on 16S rDNA and rpoB gene fragments aswell as the vdc region fragment For selected strains DNAsequencing of the 16S rDNA gene was performed Sixty ofthe isolates analyzed were identified as A acidoterrestris12 as A acidocaldarius one as Brevibacillus agri and twoas Bacillus ginsengihumi Four of the isolates which gaveambiguous results during classic identification (nontypicalcolour on an erythritol medium or lack of growth at 65∘Cconnected with lack of guaiacol production) were identified

by genetic methods as A acidoterrestris or A acidocaldariusA acidoterrestris isolates were grouped in two major clusters27 of the isolates belonged to the cluster I and 33 to thecluster II Also A acidocaldarius isolates were grouped intotwo clusters but showed more intracluster diversity

These results support the observations made by Osopaleat al [60] and Durak et al [23] who also reported two geneticclusters among analyzed A acidoterrestris samples by RAPDanalysisand by 16S rDNA sequencing respectively

Most of the strains analyzed in this study were isolatedfrom concentrated apple juice as this is the main subject ofthe Alicyclobacillus focused screening made or outsourced bypolish fruit industry however it is one of the main subjectsof similar screenings performed by fruit industry worldwideA acidoterrestris representing both clusters have been foundin concentrated apple juice For other sources three Aacidoterrestris cluster II isolates have been found in orangejuices two cluster I in concentrated black currant juice andthree cluster II in concentrated cherry juice however theseare only single observations and further research is needed toestablish ifA acidoterrestris strains representing both clustersare found in other juices

Genetic methods are broadly used in the detectioncharacterization and differentiation of microorganisms Theapplication of PCR-RFLP in the characterization of Ali-cyclobacillus and other thermoacidophilic bacteria isolatedfrom the apple juice processing environment has beendescribed by Chen et al [52] although only the 16S rDNAgene was the subject of this study and the Hin6I enzymewas not used RpoB gene has never been subjected to PCR-RFLP or any other genetic analysis of Alicyclobacillus sofar although the value of this gene in taxonomy has beenconfirmed by many studies on other microorganisms Todate there are no reports on the use of the vdc gene cluster intaxonomic studies of microorganisms although its sequencemay be proven valuable for research both on the vectors anddiversity and evolution of the element itself Hin6I restrictionpatterns for 16S rDNAwere sufficient to differentiate betweenall the species analyzed but provided no closer data onintraspecies diversity The diversity was revealed both by therpoB gene and vdc region RFLP analyses

Vdc gene cluster of Streptomyces sp described by Chowet al [55] consists of three genes vdcB (06kb) vdcC (14 kb)and vdcD (02 kb) transcribed as single policistronic mRNAmolecule All three genes were essential to produce guaiacolfrom vanillic acid Niwa and Kawamoto [38] describedanalogical gene cluster in A acidoterrestris consisting ofORF1 (597 bp) ORF2 (1425 bp) and ORF3 (321 bp) Threerespective open reading frameswere found in all five analyzedvdc sequences

Alicyclobacillus acidoterrestris RFLP patterns of boththe rpoB gene and vdc region showed consistently thatthere were two major types among the isolates one similarto the reference strain A acidoterrestris DSM 2498 andthe other similar to the reference strain A acidoterrestrisATCC 49025 Obtaining such consistent data concerningtwo genetic elements of distant function reveals that there isdeeper intraspecies genetic diversity in the A acidoterrestrisspecies This division may be considered when examining

10 BioMed Research International

other features of Alicyclobacillus such as susceptibility totemperature or high hydrostatic pressure in order to establishif there are any differences between the groups

Both the rpoB gene and vdc region seem to be single-copy genetic elements and showed no signs of intragenomicheterogeneity which often makes the RFLP comparison ofrDNA genes harder to perform

The sequence analysis of the 16S rDNA fragment ofselected isolates shows no apparent correlation betweendiversity of the 16S rDNA sequence and the sources whichthe strains were isolated from

In conclusion the application of PCR-RFLP has beenproven to be a fast and reliable method for Alicyclobacillusidentification and differentiation The method is also techni-cally less difficult than most of other molecular techniquesTwo major groups of A acidoterrestris have been identifiedThe primers designed for this study could be useful in furtherresearch on Alicyclobacillus

Data Availability

The DNA sequences obtained during this study have beendeposited in GenBank and the accession numbers areincluded within the article

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

Our research project was fully sponsored by Polish Ministryof Science and Higher Education with funds for statutoryactivity

References

[1] K Yamazaki H Teouka and H Shinano ldquoIsolation and identi-fication of alicyclobacillus acidoterrestris from acidic beveragesrdquoBioscience Biotechnology and Biochemistry vol 60 no 3 pp543ndash545 1996

[2] G DARLAND and T D BROCK ldquoBacillus acidocaldar-ius spnov an Acidophilic Thermophilic Spore-forming Bac-teriumrdquo Journal of General Microbiology vol 67 no 1 pp 9ndash151971

[3] G Deinhard P Blanz K Poralla and E Altan ldquoBacillusacidoterrestris sp nov a new thermotolerant acidophile isolatedfrom different soilsrdquo Systematic and Applied Microbiology vol10 no 1 pp 47ndash53 1987

[4] G Deinhard J Saar W Krischke and K Poralla ldquoBacilluscycloheptanicus sp nov a new thermoacidophile containing 120596-cycloheptane fatty acidsrdquo Systematic and Applied Microbiologyvol 10 no 1 pp 68ndash73 1987

[5] L Albuquerque F A Rainey A P Chung et al ldquoAlicyclobacillushesperidum sp nov and a related genomic species from solfa-taric soils of Sao Miguel in the Azoresrdquo International Journalof Systematic and Evolutionary Microbiology vol 50 no 2 pp451ndash457 2000

[6] K Goto H Matsubara K Mochida et al ldquoAlicyclobacillusherbarius sp nov a novel bacterium containing120596-cycloheptane

fatty acids isolated from herbal teardquo International Journal ofSystematic and EvolutionaryMicrobiology vol 52 no 1 pp 109ndash113 2002

[7] K Goto Y Tanimoto T Tamura et al ldquoIdentificationof thermoacidophilic bacteria and a new Alicyclobacillusgenomic species isolated from acidic environments in JapanrdquoExtremophiles vol 6 no 4 pp 333ndash340 2002

[8] H Matsubara K Goto T Matsumura et al ldquoAlicyclobacillusacidiphilus sp nov a novel thermo-acidophilic 120596-alicyclicfatty acid-containing bacterium isolated from acidic beveragesrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 52 no 5 pp 1681ndash1685 2002

[9] R V Orr R L Shewfelt C J Huang S Tefera and L RBeuchat ldquoDetection of guaiacol produced by Alicyclobacillusacidoterrestris in apple juice by sensory and chromatographicanalyses and comparison with spore and vegetative cell popu-lationsrdquo Journal of Food Protection vol 63 no 11 pp 1517ndash15222000

[10] G L Pettipher M E Osmundson and J MMurphy ldquoMethodsfor the detection and enumeration of Alicyclobacillus acidoter-restris and investigation of growth and production of taint infruit juice and fruit juice-containing drinksrdquo Letters in AppliedMicrobiology vol 24 no 3 pp 185ndash189 1997

[11] A C N F Spinelli A S Sant1015840Ana S Rodrigues Jr andP R Massaguer ldquoInfluence of different filling cooling andstorage conditions on the growth of Alicyclobacillus acidoter-restris CRA7152 in orange juicerdquo Applied and EnvironmentalMicrobiology vol 75 no 23 pp 7409ndash7416 2009

[12] MCMaldonado C Belfiore andA RNavarro ldquoTemperaturesoluble solids and pH effect on Alicyclobacillus acidoterrestrisviability in lemon juice concentraterdquo Journal of IndustrialMicrobiology and Biotechnology vol 35 no 2 pp 141ndash144 2008

[13] M N U Eiroa V C A Junqueira and F L Schmidt ldquoAlicy-clobacillus in orange juice Occurrence and heat resistance ofsporesrdquo Journal of Food Protection vol 62 no 8 pp 883ndash8861999

[14] I Walls and R Chuyate ldquoIsolation of Alicyclobacillus acidoter-restris from fruit juicesrdquo Journal of AOAC International vol 83no 5 pp 1115ndash1120 2000

[15] M E Parish and R M Goodrich ldquoRecovery of presumptiveAlicyclobacillus strains from orange fruit surfacesrdquo Journal ofFood Protection vol 68 no 10 pp 2196ndash2200 2005

[16] D F Splittstoesser J J Churey and C Y Lee ldquoGrowthcharacteristics of aciduric sporeforming bacilli isolated fromfruit juicesrdquo Journal of Food Protection vol 57 no 12 pp 1080ndash1083 1994

[17] J Baumgart M Husemann and C Schmidt ldquoAlicyclobacil-lus acidoterrestris vorkommen bedeutung und nachweis ingetranken und getrankegrundstoffenrdquo Flussiges Obst vol 64pp 178ndash180 1997

[18] C A Wisse and M E Parish ldquoIsolation and enumeration ofspore-forming thermoacidophilic rod-shaped bacteria fromcitrus processing environmentsrdquo Dairy Food EnvironmentalSanitation vol 18 pp 504ndash509 1998

[19] J Baumgart and S Menje ldquoThe impact of Alicyclobacillusacidoterestris on the quality of juices and soft drinksrdquo FruitProcess vol 10 pp 251ndash254 2000

[20] S Y Eguchi G P Manfio M E Pinhatti E Azuma and SF Variane ldquoAcidotermofilic sporeforming bacteria (ATSB) inorange juices ecology and involvement in the deterioration offruit juices Report of the Research Project Part IIrdquo Fruit Processvol 11 pp 55ndash62 2001

BioMed Research International 11

[21] P AGouws L Gie A Pretorius andNDhansay ldquoIsolation andidentification of Alicyclobacillus acidocaldarius by 16S rDNAfrom mango juice and concentraterdquo International Journal ofFood Science amp Technology vol 40 no 7 pp 789ndash792 2005

[22] K Goto K Mochida Y Kato et al ldquoProposal of six speciesof moderately thermophilic acidophilic endospore-formingbacteria Alicyclobacillus contaminans sp nov Alicyclobacillusfastidiosus sp novAlicyclobacillus kakegawensis sp novAlicy-clobacillus macrosporangiidus sp nov Alicyclobacillus saccharisp nov and Alicyclobacillus shizuokensis sp novrdquo InternationalJournal of Systematic and Evolutionary Microbiology vol 57 no6 pp 1276ndash1285 2007

[23] M Z Durak J J Churey M D Danyluk and R W WoroboldquoIdentification and haplotype distribution of Alicyclobacillusspp from different juices and beveragesrdquo International Journalof Food Microbiology vol 142 no 3 pp 286ndash291 2010

[24] B Sokolowska J Niezgoda A Dekowska et al ldquoIncidence ofAlicyclobacillus spp in Polish apple and dark berry juice con-centrates and the ability of isolated A acidoterrestris strains tospoilage of these juicesrdquo Postępy Nauki i Technologii PrzemysłuRolno-Spozywczego vol 71 no 1 pp 5ndash20 2016

[25] I Walls and R Chuyate ldquoAlicyclobacillus historical perspectiveand preliminary characterization studyrdquo Dairy Food EnvironSanitation18 pp 499ndash503 1998

[26] H-A Duong and N Jensen ldquoSpoilage of iced tea by Alicy-clobacillusrdquo Food Australia vol 52 no 7 p 292 2000

[27] M Niwa and A Kuriyama ldquoA acidoterrestris rapid detectionkitrdquo Fruit Process vol 13 pp 328ndash331 2003

[28] K S Bahceci V Gokmen and J Acar ldquoFormation of guaiacolfrom vanillin by Alicyclobacillus acidoterrestris in apple juice Amodel studyrdquo European Food Research and Technology vol 220no 2 pp 196ndash199 2005

[29] R C Witthuhn E van der Merwe P Venter and M CameronldquoGuaiacol production from ferulic acid vanillin and vanillicacid by Alicyclobacillus acidoterrestrisrdquo International Journal ofFood Microbiology vol 157 no 1 pp 113ndash117 2012

[30] A Borlinghaus and R Engel ldquoAlicyclobacillus incidence in com-mercial apple juice concentrate (AJC) supplies and validationrdquoFruit Process vol 7 pp 262ndash266 1997

[31] D F Splittstoesser C Y Lee and J J Churey ldquoControl ofAlicyclobacillus in the juice industryrdquo Dairy Food EnvironSanitation vol 18 pp 585ndash587 1998

[32] N Jensen ldquoAlicyclobacillus in Australiardquo Food Australia vol 52no 7 pp 282ndash285 2000

[33] IWalls andR Chuyate ldquoSpoilage of fruit juices byAlicyclobacil-lus acidoterrestrisrdquo Food Australia vol 52 no 7 pp 293ndash2952000

[34] N Jensen and F B Whitfield ldquoRole of Alicyclobacillus acidoter-restris in the development of a disinfectant taint in shelf-stablefruit juicerdquo Letters in Applied Microbiology vol 361 pp 9ndash142003

[35] D Gocmen A Elston T Williams M Parish and R LRouseff ldquoIdentification of medicinal off-flavours generated byAlicyclobacillus species in orange juice using GC-olfactometryand GC-MSrdquo Letters in Applied Microbiology vol 40 no 3 pp172ndash177 2005

[36] M Niwa ldquoControl of hazardous bacteria in acidic beveragesby using a guaiacol detection kit (peroxidase methodrdquo FruitProcess vol 15 pp 388ndash392 2005

[37] B Siegmund and B Pollinger-Zierler ldquoGrowth behavior ofoff-flavor-forming microorganisms in apple juicerdquo Journal of

Agricultural and Food Chemistry vol 55 no 16 pp 6692ndash66992007

[38] M Niwa and A Kawamoto ldquoDevelopment of a rapid detectionmethod of A acidoterrestris hazardous bacteria to acidicbeveragerdquo Fruit Process vol 13 pp 102ndash107 2003

[39] T A Eisele and M J Semon ldquoBest estimated aroma andtaste detection threshold for guaiacol in water and apple juicerdquoJournal of Food Science vol 70 no 4 pp S267ndashS269 2005

[40] B Zierler B Siegmund and W Pfannhauser ldquoDeterminationof off-flavour compounds in apple juice caused by microor-ganisms using headspace solid phase microextraction-gaschromatography-mass spectrometryrdquo Analytica Chimica Actavol 520 no 1-2 pp 3ndash11 2004

[41] K S Bahceci and J Acar ldquoModeling the combined effectsof pH temperature and ascorbic acid concentration on theheat resistance of Alicyclobacillus acidoterrestisrdquo InternationalJournal of Food Microbiology vol 120 no 3 pp 266ndash273 2007

[42] J Baumgart ldquoChapter 11 Media for the detection and enu-meration of Alicyclobacillus acidoterrestris and Alicyclobacillusacidocaldarius in foodsrdquo in Progress in Industrial Microbiologyvol 37 pp 161ndash166 Elsevier 2003

[43] M Lin M Al-Holy S-S Chang et al ldquoRapid discriminationof Alicyclobacillus strains in apple juice by Fourier transforminfrared spectroscopyrdquo International Journal of Food Microbiol-ogy vol 105 no 3 pp 369ndash376 2005

[44] J Wang T Yue Y Yuan X Lu J-H Shin and B RascoldquoDiscrimination of alicyclobacillus strains using nitrocellulosemembrane filter and attenuated total reflectance fourier trans-form infrared spectroscopyrdquo Journal of Food Science vol 76 no2 pp 137ndash142 2011

[45] M A Al-Holy M Lin O A Alhaj and M H Abu-GoushldquoDiscrimination between Bacillus and Alicyclobacillus Isolatesin Apple Juice by Fourier Transform Infrared Spectroscopy andMultivariate Analysisrdquo Journal of Food Science vol 80 no 2 ppM399ndashM404 2015

[46] WH Groenewald P A Gouws andR CWitthuhn ldquoIsolationidentification and typification of Alicyclobacillus acidoterrestrisand Alicyclobacillus acidocaldarius strains from orchard soiland the fruit processing environment in South Africardquo FoodMicrobiology vol 26 no 1 pp 71ndash76 2009

[47] J Zhang T Yue and Y Yuan ldquoAlicyclobacillus contaminationin the production line of Kiwi products in Chinardquo PLoS ONEvol 8 no 7 p e67704 2013

[48] I C McKnight M N U Eiroa A S SantrsquoAna and P RMassaguer ldquoAlicyclobacillus acidoterrestris in pasteurized exoticBrazilian fruit juices Isolation genotypic characterization andheat resistancerdquo FoodMicrobiology vol 27 no 8 pp 1016ndash10222010

[49] L Felix-Valenzuela I Guardiola-Avila A Burgara-Estrella MIbarra-Zavala and V Mata-Haro ldquoGenotypic and phenotypicdiversity of Alicyclobacillus acidocaldarius isolatesrdquo Letters inApplied Microbiology vol 61 no 4 pp 367ndash373 2015

[50] J D Wisotzkey P Jurtshuk Jr G E Fox G Deinhard andK Poralla ldquoComparative sequence analyses on the 16S rRNA(rDNA) of Bacillus acidocaldarius Bacillus acidoterrestris andBacillus cycloheptanicus and proposal for creation of a newgenus Alicyclobacillus gen novrdquo International Journal of Sys-tematic Bacteriology vol 42 no 2 pp 263ndash269 1992

[51] C J Connor H Luo B B McSpadden Gardener and HH Wang ldquoDevelopment of a real-time PCR-based systemtargeting the 16S rRNA gene sequence for rapid detection of

12 BioMed Research International

Alicyclobacillus spp in juice productsrdquo International Journal ofFood Microbiology vol 99 no 3 pp 229ndash235 2005

[52] S ChenQ Tang X Zhang et al ldquoIsolation and characterizationof thermo-acidophilic endospore-forming bacteria from theconcentrated apple juice-processing environmentrdquo FoodMicro-biology vol 23 no 5 pp 439ndash445 2006

[53] K Goto A Nishibori Y Wasada K Furuhata M Fukuyamaand M Hara ldquoIdentification of thermo-acidophilic bacteriaisolated from the soil of several Japanese fruit orchardsrdquo Lettersin Applied Microbiology vol 46 no 3 pp 289ndash294 2008

[54] J Chen X Ma Y Yuan and W Zhang ldquoSensitive and rapiddetection ofAlicyclobacillus acidoterrestris using loop-mediatedisothermal amplificationrdquo Journal of the Science of Food andAgriculture vol 91 no 6 pp 1070ndash1074 2011

[55] K T Chow M K Pope and J Davies ldquoCharacterization of avanillic acid non-oxidative decarboxylation gene cluster fromStreptomyces sp D7rdquo Microbiology vol 145 no 9 pp 2393ndash2403 1999

[56] K Matsubara ldquoA method for detecting andor identifying gua-iacol producing micro organismrdquo Patent no JP2003000259A2003

[57] Y Wang T Yue Y Yuan and Z Gao ldquoIsolation and identifi-cation of thermo-acidophilic bacteria from orchards in chinardquoJournal of Food Protection vol 73 pp 390ndash394 2010

[58] M Shemesh R Pasvolsky N Sela S J Green and V andZakinldquoDraft Genome Sequence of Alicyclobacillus acidoterrestrisStrain ATCC 49025rdquo Genome Announcement vol 1 no 5Article ID e00638ndash13 2013

[59] B Sokołowska S Skapska M Fonberg-Broczek et al ldquoFactorsinfluencing the inactivation of Alicyclobacillus acidoterrestrisspores exposed to high hydrostatic pressure in apple juicerdquoHighPressure Research vol 33 no 1 pp 73ndash82 2013

[60] B A Osopale C R Witthuhn J Albertyn and F A Oguntoy-inbo ldquoCulture dependent and independent genomic identifica-tion ofAlicyclobacillus species in contaminated commercial fruitjuicesrdquo Food Microbiology vol 56 pp 21ndash28 2016

Hindawiwwwhindawicom

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GenomicsInternational Journal of

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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

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Submit your manuscripts atwwwhindawicom

8 BioMed Research International

Figure 7 Restriction patterns of a 1735 bp fragment of rpoB gene digested with Hin6I Lanes 3 and 5 A acidoterrestris type I 1 2 4 and 6Aacidoterrestris type II 17A acidocaldarius type I 18A acidocaldarius type II 8A acidoterrestrisDSM2498 9A acidoterrestrisATCC4902510A acidiphilus 11 A hesperidum 12A herbarius 13A acidocaldariusA1 14 B subtilis 15G stearothermophilus lanes 7 and 16 molecularmarker

Figure 8 Restriction patterns of a 1735 bp fragment of rpoB gene digested with HphI Lanes 1-4 6 8-11 13 25 and 27 A acidoterrestris I5 7 12 26 and 28 A acidoterrestris II 24 A acidoterrestris IIA 29 A acidocaldarius IA 30 A acidocaldarius type II 15 A acidoterrestrisDSM 2498 16 A acidoterrestris ATCC49025 17 A acidiphilus 18 A hesperidum 19 A herbarius 20 A acidocaldariusA1 21 B subtilis 22G stearothermophilus lanes 14 and 23 molecular marker

DSM_2498 00003231 -00003234 -00025641 0ATCC_49025 00022455 00005656 -00009252 -00001833 051 053 -000013

Figure 9 Phylogenetic tree of partial 16S rRNA sequence of 11 Aacidoterrestris isolatesThe tree was constructed by neighbor joiningmethod using Clustal Omega software

For Hin6I there were two types of patterns for the Aacidoterrestris and A acidocaldarius A acidoterrestris DSM2498 belongs to the cluster I while A acidoterrestris ATCC49025 belongs to the cluster II A acidocaldarius DSM 446represents cluster II of the A acidocaldarius group while AacidocaldariusA1 represents cluster I

HphI endonuclease produces two types of patterns forA acidoterrestris analogically as before and three types ofpatterns for A acidocaldarius One sample of A acidoter-restris cluster II has a slightly different pattern and has been

classified as type II in other analyses Two of the threepatterns of A acidocaldarius differ with only one bandA acidocaldarius DSM 446 represents cluster II of the Aacidocaldarius group while A acidocaldarius A1 representscluster I

34 DNA Sequencing DNA sequencing was performed forselected strains Sequencing of the 16S rDNA gene wasperformed to confirm the proper identification of the isolatesand to identify the non-Alicyclobacillus isolates The 16SrDNA sequence of strains 31 and 34 classified as type Ishowed the greatest similarity toA acidoterrestrisDSM 2498also classified as type I The sequences of strains 33 51 52 5355 and 56 classified as type II showed the greatest similaritytoA acidoterrestrisATCC49025 also classified as type IIThesequence of strain 41 classified as type II showed the greatestsimilarity to A acidoterrestris DSM 3922

Figure 9 shows the phylogenetic tree constructed from the16S rDNA sequences of analyzed strains of A acidoterrestrisThe sequence analysis indicates that A acidoterrestris DSM2498 and two other type I strains are closely related and itshows greater variation within class II strains The isolatesselected for sequencing represent both groups and different

BioMed Research International 9

sources which they were isolated from concentrated applejuice (isolates 33 and 41) fresh apples (isolate 34) concen-trated blackcurrant juice (isolate 31) concentrated raspberryjuice (isolate 56) concentrated beetroot juice (isolate 51)and concentrated cherry juice (isolates 52 53 and 55) Thesequence analysis shows no apparent correlation betweendiversity of the 16S rDNA sequence and the sources of theisolates

Fivewhole vdc gene clusterswere sequenced Twoof themwere isolated from the reference strains A acidoterrestrisDSM 2498 and A acidoterrestris ATCC 49025 2416 bpfragments of the vdc regions were aligned The vdc regionsequence of strains 15 and 34 classified as type I showed999 identity with the vdc sequence of A acidoterrestrisDSM 2498 the sequence of strain 41 classified as typeII showed 993 identity with the vdc sequence A aci-doterrestris ATCC 49025 Vdc sequences of A acidoterrestrisDSM 2498 and A acidoterrestris DSM 49025 showed 945identity For the last pair protein sequences of the geneproducts showed 985 positives and 965 identities forVdcB 994 and 989 for VdcC and 100 and 974 forVdcD

RpoB gene sequencing was performed to confirm thatboth primer pairs Gru3ndashGru4 and Gru5ndashGru6 enabledto amplify the correct DNA fragment and that degenera-tion of the primers did not affect the sequence specificityalthough the degenerated primers produced significantlylower amount of PCR product All of the sequences obtainedshowed greatest similarity to the appropriate rpoB genes

The GenBank accession numbers are KX371237-KX371249 for 16S rDNA sequences KX453673-KX453677for full vdc region sequences KX453678 and KX453679 forpartial vdc sequences of A herbarius and A acidiphilusrespectively and KX453680- KX453682 for partial rpoBsequences

4 Discussion

Among more than one thousand samples of concentratedapple juice tested in our laboratory between 2004 and 201267 was contaminated with Alicyclobacillus sp and 31 ofthe isolates were identified as A acidoterrestris [59] Thestatistics show that Alicyclobacillus spoilage is still a majorconcern in the fruit processing industry

In this study 75 guaiacol producing and non-guaiacolproducing strains were isolated from various fruit juicesconcentrated fruit juices and fresh apples Additionally8 reference strains were used The isolates and referencestrains were examined using classic methods and PCR-RFLP focusing on 16S rDNA and rpoB gene fragments aswell as the vdc region fragment For selected strains DNAsequencing of the 16S rDNA gene was performed Sixty ofthe isolates analyzed were identified as A acidoterrestris12 as A acidocaldarius one as Brevibacillus agri and twoas Bacillus ginsengihumi Four of the isolates which gaveambiguous results during classic identification (nontypicalcolour on an erythritol medium or lack of growth at 65∘Cconnected with lack of guaiacol production) were identified

by genetic methods as A acidoterrestris or A acidocaldariusA acidoterrestris isolates were grouped in two major clusters27 of the isolates belonged to the cluster I and 33 to thecluster II Also A acidocaldarius isolates were grouped intotwo clusters but showed more intracluster diversity

These results support the observations made by Osopaleat al [60] and Durak et al [23] who also reported two geneticclusters among analyzed A acidoterrestris samples by RAPDanalysisand by 16S rDNA sequencing respectively

Most of the strains analyzed in this study were isolatedfrom concentrated apple juice as this is the main subject ofthe Alicyclobacillus focused screening made or outsourced bypolish fruit industry however it is one of the main subjectsof similar screenings performed by fruit industry worldwideA acidoterrestris representing both clusters have been foundin concentrated apple juice For other sources three Aacidoterrestris cluster II isolates have been found in orangejuices two cluster I in concentrated black currant juice andthree cluster II in concentrated cherry juice however theseare only single observations and further research is needed toestablish ifA acidoterrestris strains representing both clustersare found in other juices

Genetic methods are broadly used in the detectioncharacterization and differentiation of microorganisms Theapplication of PCR-RFLP in the characterization of Ali-cyclobacillus and other thermoacidophilic bacteria isolatedfrom the apple juice processing environment has beendescribed by Chen et al [52] although only the 16S rDNAgene was the subject of this study and the Hin6I enzymewas not used RpoB gene has never been subjected to PCR-RFLP or any other genetic analysis of Alicyclobacillus sofar although the value of this gene in taxonomy has beenconfirmed by many studies on other microorganisms Todate there are no reports on the use of the vdc gene cluster intaxonomic studies of microorganisms although its sequencemay be proven valuable for research both on the vectors anddiversity and evolution of the element itself Hin6I restrictionpatterns for 16S rDNAwere sufficient to differentiate betweenall the species analyzed but provided no closer data onintraspecies diversity The diversity was revealed both by therpoB gene and vdc region RFLP analyses

Vdc gene cluster of Streptomyces sp described by Chowet al [55] consists of three genes vdcB (06kb) vdcC (14 kb)and vdcD (02 kb) transcribed as single policistronic mRNAmolecule All three genes were essential to produce guaiacolfrom vanillic acid Niwa and Kawamoto [38] describedanalogical gene cluster in A acidoterrestris consisting ofORF1 (597 bp) ORF2 (1425 bp) and ORF3 (321 bp) Threerespective open reading frameswere found in all five analyzedvdc sequences

Alicyclobacillus acidoterrestris RFLP patterns of boththe rpoB gene and vdc region showed consistently thatthere were two major types among the isolates one similarto the reference strain A acidoterrestris DSM 2498 andthe other similar to the reference strain A acidoterrestrisATCC 49025 Obtaining such consistent data concerningtwo genetic elements of distant function reveals that there isdeeper intraspecies genetic diversity in the A acidoterrestrisspecies This division may be considered when examining

10 BioMed Research International

other features of Alicyclobacillus such as susceptibility totemperature or high hydrostatic pressure in order to establishif there are any differences between the groups

Both the rpoB gene and vdc region seem to be single-copy genetic elements and showed no signs of intragenomicheterogeneity which often makes the RFLP comparison ofrDNA genes harder to perform

The sequence analysis of the 16S rDNA fragment ofselected isolates shows no apparent correlation betweendiversity of the 16S rDNA sequence and the sources whichthe strains were isolated from

In conclusion the application of PCR-RFLP has beenproven to be a fast and reliable method for Alicyclobacillusidentification and differentiation The method is also techni-cally less difficult than most of other molecular techniquesTwo major groups of A acidoterrestris have been identifiedThe primers designed for this study could be useful in furtherresearch on Alicyclobacillus

Data Availability

The DNA sequences obtained during this study have beendeposited in GenBank and the accession numbers areincluded within the article

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

Our research project was fully sponsored by Polish Ministryof Science and Higher Education with funds for statutoryactivity

References

[1] K Yamazaki H Teouka and H Shinano ldquoIsolation and identi-fication of alicyclobacillus acidoterrestris from acidic beveragesrdquoBioscience Biotechnology and Biochemistry vol 60 no 3 pp543ndash545 1996

[2] G DARLAND and T D BROCK ldquoBacillus acidocaldar-ius spnov an Acidophilic Thermophilic Spore-forming Bac-teriumrdquo Journal of General Microbiology vol 67 no 1 pp 9ndash151971

[3] G Deinhard P Blanz K Poralla and E Altan ldquoBacillusacidoterrestris sp nov a new thermotolerant acidophile isolatedfrom different soilsrdquo Systematic and Applied Microbiology vol10 no 1 pp 47ndash53 1987

[4] G Deinhard J Saar W Krischke and K Poralla ldquoBacilluscycloheptanicus sp nov a new thermoacidophile containing 120596-cycloheptane fatty acidsrdquo Systematic and Applied Microbiologyvol 10 no 1 pp 68ndash73 1987

[5] L Albuquerque F A Rainey A P Chung et al ldquoAlicyclobacillushesperidum sp nov and a related genomic species from solfa-taric soils of Sao Miguel in the Azoresrdquo International Journalof Systematic and Evolutionary Microbiology vol 50 no 2 pp451ndash457 2000

[6] K Goto H Matsubara K Mochida et al ldquoAlicyclobacillusherbarius sp nov a novel bacterium containing120596-cycloheptane

fatty acids isolated from herbal teardquo International Journal ofSystematic and EvolutionaryMicrobiology vol 52 no 1 pp 109ndash113 2002

[7] K Goto Y Tanimoto T Tamura et al ldquoIdentificationof thermoacidophilic bacteria and a new Alicyclobacillusgenomic species isolated from acidic environments in JapanrdquoExtremophiles vol 6 no 4 pp 333ndash340 2002

[8] H Matsubara K Goto T Matsumura et al ldquoAlicyclobacillusacidiphilus sp nov a novel thermo-acidophilic 120596-alicyclicfatty acid-containing bacterium isolated from acidic beveragesrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 52 no 5 pp 1681ndash1685 2002

[9] R V Orr R L Shewfelt C J Huang S Tefera and L RBeuchat ldquoDetection of guaiacol produced by Alicyclobacillusacidoterrestris in apple juice by sensory and chromatographicanalyses and comparison with spore and vegetative cell popu-lationsrdquo Journal of Food Protection vol 63 no 11 pp 1517ndash15222000

[10] G L Pettipher M E Osmundson and J MMurphy ldquoMethodsfor the detection and enumeration of Alicyclobacillus acidoter-restris and investigation of growth and production of taint infruit juice and fruit juice-containing drinksrdquo Letters in AppliedMicrobiology vol 24 no 3 pp 185ndash189 1997

[11] A C N F Spinelli A S Sant1015840Ana S Rodrigues Jr andP R Massaguer ldquoInfluence of different filling cooling andstorage conditions on the growth of Alicyclobacillus acidoter-restris CRA7152 in orange juicerdquo Applied and EnvironmentalMicrobiology vol 75 no 23 pp 7409ndash7416 2009

[12] MCMaldonado C Belfiore andA RNavarro ldquoTemperaturesoluble solids and pH effect on Alicyclobacillus acidoterrestrisviability in lemon juice concentraterdquo Journal of IndustrialMicrobiology and Biotechnology vol 35 no 2 pp 141ndash144 2008

[13] M N U Eiroa V C A Junqueira and F L Schmidt ldquoAlicy-clobacillus in orange juice Occurrence and heat resistance ofsporesrdquo Journal of Food Protection vol 62 no 8 pp 883ndash8861999

[14] I Walls and R Chuyate ldquoIsolation of Alicyclobacillus acidoter-restris from fruit juicesrdquo Journal of AOAC International vol 83no 5 pp 1115ndash1120 2000

[15] M E Parish and R M Goodrich ldquoRecovery of presumptiveAlicyclobacillus strains from orange fruit surfacesrdquo Journal ofFood Protection vol 68 no 10 pp 2196ndash2200 2005

[16] D F Splittstoesser J J Churey and C Y Lee ldquoGrowthcharacteristics of aciduric sporeforming bacilli isolated fromfruit juicesrdquo Journal of Food Protection vol 57 no 12 pp 1080ndash1083 1994

[17] J Baumgart M Husemann and C Schmidt ldquoAlicyclobacil-lus acidoterrestris vorkommen bedeutung und nachweis ingetranken und getrankegrundstoffenrdquo Flussiges Obst vol 64pp 178ndash180 1997

[18] C A Wisse and M E Parish ldquoIsolation and enumeration ofspore-forming thermoacidophilic rod-shaped bacteria fromcitrus processing environmentsrdquo Dairy Food EnvironmentalSanitation vol 18 pp 504ndash509 1998

[19] J Baumgart and S Menje ldquoThe impact of Alicyclobacillusacidoterestris on the quality of juices and soft drinksrdquo FruitProcess vol 10 pp 251ndash254 2000

[20] S Y Eguchi G P Manfio M E Pinhatti E Azuma and SF Variane ldquoAcidotermofilic sporeforming bacteria (ATSB) inorange juices ecology and involvement in the deterioration offruit juices Report of the Research Project Part IIrdquo Fruit Processvol 11 pp 55ndash62 2001

BioMed Research International 11

[21] P AGouws L Gie A Pretorius andNDhansay ldquoIsolation andidentification of Alicyclobacillus acidocaldarius by 16S rDNAfrom mango juice and concentraterdquo International Journal ofFood Science amp Technology vol 40 no 7 pp 789ndash792 2005

[22] K Goto K Mochida Y Kato et al ldquoProposal of six speciesof moderately thermophilic acidophilic endospore-formingbacteria Alicyclobacillus contaminans sp nov Alicyclobacillusfastidiosus sp novAlicyclobacillus kakegawensis sp novAlicy-clobacillus macrosporangiidus sp nov Alicyclobacillus saccharisp nov and Alicyclobacillus shizuokensis sp novrdquo InternationalJournal of Systematic and Evolutionary Microbiology vol 57 no6 pp 1276ndash1285 2007

[23] M Z Durak J J Churey M D Danyluk and R W WoroboldquoIdentification and haplotype distribution of Alicyclobacillusspp from different juices and beveragesrdquo International Journalof Food Microbiology vol 142 no 3 pp 286ndash291 2010

[24] B Sokolowska J Niezgoda A Dekowska et al ldquoIncidence ofAlicyclobacillus spp in Polish apple and dark berry juice con-centrates and the ability of isolated A acidoterrestris strains tospoilage of these juicesrdquo Postępy Nauki i Technologii PrzemysłuRolno-Spozywczego vol 71 no 1 pp 5ndash20 2016

[25] I Walls and R Chuyate ldquoAlicyclobacillus historical perspectiveand preliminary characterization studyrdquo Dairy Food EnvironSanitation18 pp 499ndash503 1998

[26] H-A Duong and N Jensen ldquoSpoilage of iced tea by Alicy-clobacillusrdquo Food Australia vol 52 no 7 p 292 2000

[27] M Niwa and A Kuriyama ldquoA acidoterrestris rapid detectionkitrdquo Fruit Process vol 13 pp 328ndash331 2003

[28] K S Bahceci V Gokmen and J Acar ldquoFormation of guaiacolfrom vanillin by Alicyclobacillus acidoterrestris in apple juice Amodel studyrdquo European Food Research and Technology vol 220no 2 pp 196ndash199 2005

[29] R C Witthuhn E van der Merwe P Venter and M CameronldquoGuaiacol production from ferulic acid vanillin and vanillicacid by Alicyclobacillus acidoterrestrisrdquo International Journal ofFood Microbiology vol 157 no 1 pp 113ndash117 2012

[30] A Borlinghaus and R Engel ldquoAlicyclobacillus incidence in com-mercial apple juice concentrate (AJC) supplies and validationrdquoFruit Process vol 7 pp 262ndash266 1997

[31] D F Splittstoesser C Y Lee and J J Churey ldquoControl ofAlicyclobacillus in the juice industryrdquo Dairy Food EnvironSanitation vol 18 pp 585ndash587 1998

[32] N Jensen ldquoAlicyclobacillus in Australiardquo Food Australia vol 52no 7 pp 282ndash285 2000

[33] IWalls andR Chuyate ldquoSpoilage of fruit juices byAlicyclobacil-lus acidoterrestrisrdquo Food Australia vol 52 no 7 pp 293ndash2952000

[34] N Jensen and F B Whitfield ldquoRole of Alicyclobacillus acidoter-restris in the development of a disinfectant taint in shelf-stablefruit juicerdquo Letters in Applied Microbiology vol 361 pp 9ndash142003

[35] D Gocmen A Elston T Williams M Parish and R LRouseff ldquoIdentification of medicinal off-flavours generated byAlicyclobacillus species in orange juice using GC-olfactometryand GC-MSrdquo Letters in Applied Microbiology vol 40 no 3 pp172ndash177 2005

[36] M Niwa ldquoControl of hazardous bacteria in acidic beveragesby using a guaiacol detection kit (peroxidase methodrdquo FruitProcess vol 15 pp 388ndash392 2005

[37] B Siegmund and B Pollinger-Zierler ldquoGrowth behavior ofoff-flavor-forming microorganisms in apple juicerdquo Journal of

Agricultural and Food Chemistry vol 55 no 16 pp 6692ndash66992007

[38] M Niwa and A Kawamoto ldquoDevelopment of a rapid detectionmethod of A acidoterrestris hazardous bacteria to acidicbeveragerdquo Fruit Process vol 13 pp 102ndash107 2003

[39] T A Eisele and M J Semon ldquoBest estimated aroma andtaste detection threshold for guaiacol in water and apple juicerdquoJournal of Food Science vol 70 no 4 pp S267ndashS269 2005

[40] B Zierler B Siegmund and W Pfannhauser ldquoDeterminationof off-flavour compounds in apple juice caused by microor-ganisms using headspace solid phase microextraction-gaschromatography-mass spectrometryrdquo Analytica Chimica Actavol 520 no 1-2 pp 3ndash11 2004

[41] K S Bahceci and J Acar ldquoModeling the combined effectsof pH temperature and ascorbic acid concentration on theheat resistance of Alicyclobacillus acidoterrestisrdquo InternationalJournal of Food Microbiology vol 120 no 3 pp 266ndash273 2007

[42] J Baumgart ldquoChapter 11 Media for the detection and enu-meration of Alicyclobacillus acidoterrestris and Alicyclobacillusacidocaldarius in foodsrdquo in Progress in Industrial Microbiologyvol 37 pp 161ndash166 Elsevier 2003

[43] M Lin M Al-Holy S-S Chang et al ldquoRapid discriminationof Alicyclobacillus strains in apple juice by Fourier transforminfrared spectroscopyrdquo International Journal of Food Microbiol-ogy vol 105 no 3 pp 369ndash376 2005

[44] J Wang T Yue Y Yuan X Lu J-H Shin and B RascoldquoDiscrimination of alicyclobacillus strains using nitrocellulosemembrane filter and attenuated total reflectance fourier trans-form infrared spectroscopyrdquo Journal of Food Science vol 76 no2 pp 137ndash142 2011

[45] M A Al-Holy M Lin O A Alhaj and M H Abu-GoushldquoDiscrimination between Bacillus and Alicyclobacillus Isolatesin Apple Juice by Fourier Transform Infrared Spectroscopy andMultivariate Analysisrdquo Journal of Food Science vol 80 no 2 ppM399ndashM404 2015

[46] WH Groenewald P A Gouws andR CWitthuhn ldquoIsolationidentification and typification of Alicyclobacillus acidoterrestrisand Alicyclobacillus acidocaldarius strains from orchard soiland the fruit processing environment in South Africardquo FoodMicrobiology vol 26 no 1 pp 71ndash76 2009

[47] J Zhang T Yue and Y Yuan ldquoAlicyclobacillus contaminationin the production line of Kiwi products in Chinardquo PLoS ONEvol 8 no 7 p e67704 2013

[48] I C McKnight M N U Eiroa A S SantrsquoAna and P RMassaguer ldquoAlicyclobacillus acidoterrestris in pasteurized exoticBrazilian fruit juices Isolation genotypic characterization andheat resistancerdquo FoodMicrobiology vol 27 no 8 pp 1016ndash10222010

[49] L Felix-Valenzuela I Guardiola-Avila A Burgara-Estrella MIbarra-Zavala and V Mata-Haro ldquoGenotypic and phenotypicdiversity of Alicyclobacillus acidocaldarius isolatesrdquo Letters inApplied Microbiology vol 61 no 4 pp 367ndash373 2015

[50] J D Wisotzkey P Jurtshuk Jr G E Fox G Deinhard andK Poralla ldquoComparative sequence analyses on the 16S rRNA(rDNA) of Bacillus acidocaldarius Bacillus acidoterrestris andBacillus cycloheptanicus and proposal for creation of a newgenus Alicyclobacillus gen novrdquo International Journal of Sys-tematic Bacteriology vol 42 no 2 pp 263ndash269 1992

[51] C J Connor H Luo B B McSpadden Gardener and HH Wang ldquoDevelopment of a real-time PCR-based systemtargeting the 16S rRNA gene sequence for rapid detection of

12 BioMed Research International

Alicyclobacillus spp in juice productsrdquo International Journal ofFood Microbiology vol 99 no 3 pp 229ndash235 2005

[52] S ChenQ Tang X Zhang et al ldquoIsolation and characterizationof thermo-acidophilic endospore-forming bacteria from theconcentrated apple juice-processing environmentrdquo FoodMicro-biology vol 23 no 5 pp 439ndash445 2006

[53] K Goto A Nishibori Y Wasada K Furuhata M Fukuyamaand M Hara ldquoIdentification of thermo-acidophilic bacteriaisolated from the soil of several Japanese fruit orchardsrdquo Lettersin Applied Microbiology vol 46 no 3 pp 289ndash294 2008

[54] J Chen X Ma Y Yuan and W Zhang ldquoSensitive and rapiddetection ofAlicyclobacillus acidoterrestris using loop-mediatedisothermal amplificationrdquo Journal of the Science of Food andAgriculture vol 91 no 6 pp 1070ndash1074 2011

[55] K T Chow M K Pope and J Davies ldquoCharacterization of avanillic acid non-oxidative decarboxylation gene cluster fromStreptomyces sp D7rdquo Microbiology vol 145 no 9 pp 2393ndash2403 1999

[56] K Matsubara ldquoA method for detecting andor identifying gua-iacol producing micro organismrdquo Patent no JP2003000259A2003

[57] Y Wang T Yue Y Yuan and Z Gao ldquoIsolation and identifi-cation of thermo-acidophilic bacteria from orchards in chinardquoJournal of Food Protection vol 73 pp 390ndash394 2010

[58] M Shemesh R Pasvolsky N Sela S J Green and V andZakinldquoDraft Genome Sequence of Alicyclobacillus acidoterrestrisStrain ATCC 49025rdquo Genome Announcement vol 1 no 5Article ID e00638ndash13 2013

[59] B Sokołowska S Skapska M Fonberg-Broczek et al ldquoFactorsinfluencing the inactivation of Alicyclobacillus acidoterrestrisspores exposed to high hydrostatic pressure in apple juicerdquoHighPressure Research vol 33 no 1 pp 73ndash82 2013

[60] B A Osopale C R Witthuhn J Albertyn and F A Oguntoy-inbo ldquoCulture dependent and independent genomic identifica-tion ofAlicyclobacillus species in contaminated commercial fruitjuicesrdquo Food Microbiology vol 56 pp 21ndash28 2016

Hindawiwwwhindawicom

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Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

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GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

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Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

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Hindawiwwwhindawicom Volume 2018

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Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

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Hindawiwwwhindawicom Volume 2018

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ArchaeaHindawiwwwhindawicom Volume 2018

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Nucleic AcidsJournal of

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Submit your manuscripts atwwwhindawicom

BioMed Research International 9

sources which they were isolated from concentrated applejuice (isolates 33 and 41) fresh apples (isolate 34) concen-trated blackcurrant juice (isolate 31) concentrated raspberryjuice (isolate 56) concentrated beetroot juice (isolate 51)and concentrated cherry juice (isolates 52 53 and 55) Thesequence analysis shows no apparent correlation betweendiversity of the 16S rDNA sequence and the sources of theisolates

Fivewhole vdc gene clusterswere sequenced Twoof themwere isolated from the reference strains A acidoterrestrisDSM 2498 and A acidoterrestris ATCC 49025 2416 bpfragments of the vdc regions were aligned The vdc regionsequence of strains 15 and 34 classified as type I showed999 identity with the vdc sequence of A acidoterrestrisDSM 2498 the sequence of strain 41 classified as typeII showed 993 identity with the vdc sequence A aci-doterrestris ATCC 49025 Vdc sequences of A acidoterrestrisDSM 2498 and A acidoterrestris DSM 49025 showed 945identity For the last pair protein sequences of the geneproducts showed 985 positives and 965 identities forVdcB 994 and 989 for VdcC and 100 and 974 forVdcD

RpoB gene sequencing was performed to confirm thatboth primer pairs Gru3ndashGru4 and Gru5ndashGru6 enabledto amplify the correct DNA fragment and that degenera-tion of the primers did not affect the sequence specificityalthough the degenerated primers produced significantlylower amount of PCR product All of the sequences obtainedshowed greatest similarity to the appropriate rpoB genes

The GenBank accession numbers are KX371237-KX371249 for 16S rDNA sequences KX453673-KX453677for full vdc region sequences KX453678 and KX453679 forpartial vdc sequences of A herbarius and A acidiphilusrespectively and KX453680- KX453682 for partial rpoBsequences

4 Discussion

Among more than one thousand samples of concentratedapple juice tested in our laboratory between 2004 and 201267 was contaminated with Alicyclobacillus sp and 31 ofthe isolates were identified as A acidoterrestris [59] Thestatistics show that Alicyclobacillus spoilage is still a majorconcern in the fruit processing industry

In this study 75 guaiacol producing and non-guaiacolproducing strains were isolated from various fruit juicesconcentrated fruit juices and fresh apples Additionally8 reference strains were used The isolates and referencestrains were examined using classic methods and PCR-RFLP focusing on 16S rDNA and rpoB gene fragments aswell as the vdc region fragment For selected strains DNAsequencing of the 16S rDNA gene was performed Sixty ofthe isolates analyzed were identified as A acidoterrestris12 as A acidocaldarius one as Brevibacillus agri and twoas Bacillus ginsengihumi Four of the isolates which gaveambiguous results during classic identification (nontypicalcolour on an erythritol medium or lack of growth at 65∘Cconnected with lack of guaiacol production) were identified

by genetic methods as A acidoterrestris or A acidocaldariusA acidoterrestris isolates were grouped in two major clusters27 of the isolates belonged to the cluster I and 33 to thecluster II Also A acidocaldarius isolates were grouped intotwo clusters but showed more intracluster diversity

These results support the observations made by Osopaleat al [60] and Durak et al [23] who also reported two geneticclusters among analyzed A acidoterrestris samples by RAPDanalysisand by 16S rDNA sequencing respectively

Most of the strains analyzed in this study were isolatedfrom concentrated apple juice as this is the main subject ofthe Alicyclobacillus focused screening made or outsourced bypolish fruit industry however it is one of the main subjectsof similar screenings performed by fruit industry worldwideA acidoterrestris representing both clusters have been foundin concentrated apple juice For other sources three Aacidoterrestris cluster II isolates have been found in orangejuices two cluster I in concentrated black currant juice andthree cluster II in concentrated cherry juice however theseare only single observations and further research is needed toestablish ifA acidoterrestris strains representing both clustersare found in other juices

Genetic methods are broadly used in the detectioncharacterization and differentiation of microorganisms Theapplication of PCR-RFLP in the characterization of Ali-cyclobacillus and other thermoacidophilic bacteria isolatedfrom the apple juice processing environment has beendescribed by Chen et al [52] although only the 16S rDNAgene was the subject of this study and the Hin6I enzymewas not used RpoB gene has never been subjected to PCR-RFLP or any other genetic analysis of Alicyclobacillus sofar although the value of this gene in taxonomy has beenconfirmed by many studies on other microorganisms Todate there are no reports on the use of the vdc gene cluster intaxonomic studies of microorganisms although its sequencemay be proven valuable for research both on the vectors anddiversity and evolution of the element itself Hin6I restrictionpatterns for 16S rDNAwere sufficient to differentiate betweenall the species analyzed but provided no closer data onintraspecies diversity The diversity was revealed both by therpoB gene and vdc region RFLP analyses

Vdc gene cluster of Streptomyces sp described by Chowet al [55] consists of three genes vdcB (06kb) vdcC (14 kb)and vdcD (02 kb) transcribed as single policistronic mRNAmolecule All three genes were essential to produce guaiacolfrom vanillic acid Niwa and Kawamoto [38] describedanalogical gene cluster in A acidoterrestris consisting ofORF1 (597 bp) ORF2 (1425 bp) and ORF3 (321 bp) Threerespective open reading frameswere found in all five analyzedvdc sequences

Alicyclobacillus acidoterrestris RFLP patterns of boththe rpoB gene and vdc region showed consistently thatthere were two major types among the isolates one similarto the reference strain A acidoterrestris DSM 2498 andthe other similar to the reference strain A acidoterrestrisATCC 49025 Obtaining such consistent data concerningtwo genetic elements of distant function reveals that there isdeeper intraspecies genetic diversity in the A acidoterrestrisspecies This division may be considered when examining

10 BioMed Research International

other features of Alicyclobacillus such as susceptibility totemperature or high hydrostatic pressure in order to establishif there are any differences between the groups

Both the rpoB gene and vdc region seem to be single-copy genetic elements and showed no signs of intragenomicheterogeneity which often makes the RFLP comparison ofrDNA genes harder to perform

The sequence analysis of the 16S rDNA fragment ofselected isolates shows no apparent correlation betweendiversity of the 16S rDNA sequence and the sources whichthe strains were isolated from

In conclusion the application of PCR-RFLP has beenproven to be a fast and reliable method for Alicyclobacillusidentification and differentiation The method is also techni-cally less difficult than most of other molecular techniquesTwo major groups of A acidoterrestris have been identifiedThe primers designed for this study could be useful in furtherresearch on Alicyclobacillus

Data Availability

The DNA sequences obtained during this study have beendeposited in GenBank and the accession numbers areincluded within the article

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

Our research project was fully sponsored by Polish Ministryof Science and Higher Education with funds for statutoryactivity

References

[1] K Yamazaki H Teouka and H Shinano ldquoIsolation and identi-fication of alicyclobacillus acidoterrestris from acidic beveragesrdquoBioscience Biotechnology and Biochemistry vol 60 no 3 pp543ndash545 1996

[2] G DARLAND and T D BROCK ldquoBacillus acidocaldar-ius spnov an Acidophilic Thermophilic Spore-forming Bac-teriumrdquo Journal of General Microbiology vol 67 no 1 pp 9ndash151971

[3] G Deinhard P Blanz K Poralla and E Altan ldquoBacillusacidoterrestris sp nov a new thermotolerant acidophile isolatedfrom different soilsrdquo Systematic and Applied Microbiology vol10 no 1 pp 47ndash53 1987

[4] G Deinhard J Saar W Krischke and K Poralla ldquoBacilluscycloheptanicus sp nov a new thermoacidophile containing 120596-cycloheptane fatty acidsrdquo Systematic and Applied Microbiologyvol 10 no 1 pp 68ndash73 1987

[5] L Albuquerque F A Rainey A P Chung et al ldquoAlicyclobacillushesperidum sp nov and a related genomic species from solfa-taric soils of Sao Miguel in the Azoresrdquo International Journalof Systematic and Evolutionary Microbiology vol 50 no 2 pp451ndash457 2000

[6] K Goto H Matsubara K Mochida et al ldquoAlicyclobacillusherbarius sp nov a novel bacterium containing120596-cycloheptane

fatty acids isolated from herbal teardquo International Journal ofSystematic and EvolutionaryMicrobiology vol 52 no 1 pp 109ndash113 2002

[7] K Goto Y Tanimoto T Tamura et al ldquoIdentificationof thermoacidophilic bacteria and a new Alicyclobacillusgenomic species isolated from acidic environments in JapanrdquoExtremophiles vol 6 no 4 pp 333ndash340 2002

[8] H Matsubara K Goto T Matsumura et al ldquoAlicyclobacillusacidiphilus sp nov a novel thermo-acidophilic 120596-alicyclicfatty acid-containing bacterium isolated from acidic beveragesrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 52 no 5 pp 1681ndash1685 2002

[9] R V Orr R L Shewfelt C J Huang S Tefera and L RBeuchat ldquoDetection of guaiacol produced by Alicyclobacillusacidoterrestris in apple juice by sensory and chromatographicanalyses and comparison with spore and vegetative cell popu-lationsrdquo Journal of Food Protection vol 63 no 11 pp 1517ndash15222000

[10] G L Pettipher M E Osmundson and J MMurphy ldquoMethodsfor the detection and enumeration of Alicyclobacillus acidoter-restris and investigation of growth and production of taint infruit juice and fruit juice-containing drinksrdquo Letters in AppliedMicrobiology vol 24 no 3 pp 185ndash189 1997

[11] A C N F Spinelli A S Sant1015840Ana S Rodrigues Jr andP R Massaguer ldquoInfluence of different filling cooling andstorage conditions on the growth of Alicyclobacillus acidoter-restris CRA7152 in orange juicerdquo Applied and EnvironmentalMicrobiology vol 75 no 23 pp 7409ndash7416 2009

[12] MCMaldonado C Belfiore andA RNavarro ldquoTemperaturesoluble solids and pH effect on Alicyclobacillus acidoterrestrisviability in lemon juice concentraterdquo Journal of IndustrialMicrobiology and Biotechnology vol 35 no 2 pp 141ndash144 2008

[13] M N U Eiroa V C A Junqueira and F L Schmidt ldquoAlicy-clobacillus in orange juice Occurrence and heat resistance ofsporesrdquo Journal of Food Protection vol 62 no 8 pp 883ndash8861999

[14] I Walls and R Chuyate ldquoIsolation of Alicyclobacillus acidoter-restris from fruit juicesrdquo Journal of AOAC International vol 83no 5 pp 1115ndash1120 2000

[15] M E Parish and R M Goodrich ldquoRecovery of presumptiveAlicyclobacillus strains from orange fruit surfacesrdquo Journal ofFood Protection vol 68 no 10 pp 2196ndash2200 2005

[16] D F Splittstoesser J J Churey and C Y Lee ldquoGrowthcharacteristics of aciduric sporeforming bacilli isolated fromfruit juicesrdquo Journal of Food Protection vol 57 no 12 pp 1080ndash1083 1994

[17] J Baumgart M Husemann and C Schmidt ldquoAlicyclobacil-lus acidoterrestris vorkommen bedeutung und nachweis ingetranken und getrankegrundstoffenrdquo Flussiges Obst vol 64pp 178ndash180 1997

[18] C A Wisse and M E Parish ldquoIsolation and enumeration ofspore-forming thermoacidophilic rod-shaped bacteria fromcitrus processing environmentsrdquo Dairy Food EnvironmentalSanitation vol 18 pp 504ndash509 1998

[19] J Baumgart and S Menje ldquoThe impact of Alicyclobacillusacidoterestris on the quality of juices and soft drinksrdquo FruitProcess vol 10 pp 251ndash254 2000

[20] S Y Eguchi G P Manfio M E Pinhatti E Azuma and SF Variane ldquoAcidotermofilic sporeforming bacteria (ATSB) inorange juices ecology and involvement in the deterioration offruit juices Report of the Research Project Part IIrdquo Fruit Processvol 11 pp 55ndash62 2001

BioMed Research International 11

[21] P AGouws L Gie A Pretorius andNDhansay ldquoIsolation andidentification of Alicyclobacillus acidocaldarius by 16S rDNAfrom mango juice and concentraterdquo International Journal ofFood Science amp Technology vol 40 no 7 pp 789ndash792 2005

[22] K Goto K Mochida Y Kato et al ldquoProposal of six speciesof moderately thermophilic acidophilic endospore-formingbacteria Alicyclobacillus contaminans sp nov Alicyclobacillusfastidiosus sp novAlicyclobacillus kakegawensis sp novAlicy-clobacillus macrosporangiidus sp nov Alicyclobacillus saccharisp nov and Alicyclobacillus shizuokensis sp novrdquo InternationalJournal of Systematic and Evolutionary Microbiology vol 57 no6 pp 1276ndash1285 2007

[23] M Z Durak J J Churey M D Danyluk and R W WoroboldquoIdentification and haplotype distribution of Alicyclobacillusspp from different juices and beveragesrdquo International Journalof Food Microbiology vol 142 no 3 pp 286ndash291 2010

[24] B Sokolowska J Niezgoda A Dekowska et al ldquoIncidence ofAlicyclobacillus spp in Polish apple and dark berry juice con-centrates and the ability of isolated A acidoterrestris strains tospoilage of these juicesrdquo Postępy Nauki i Technologii PrzemysłuRolno-Spozywczego vol 71 no 1 pp 5ndash20 2016

[25] I Walls and R Chuyate ldquoAlicyclobacillus historical perspectiveand preliminary characterization studyrdquo Dairy Food EnvironSanitation18 pp 499ndash503 1998

[26] H-A Duong and N Jensen ldquoSpoilage of iced tea by Alicy-clobacillusrdquo Food Australia vol 52 no 7 p 292 2000

[27] M Niwa and A Kuriyama ldquoA acidoterrestris rapid detectionkitrdquo Fruit Process vol 13 pp 328ndash331 2003

[28] K S Bahceci V Gokmen and J Acar ldquoFormation of guaiacolfrom vanillin by Alicyclobacillus acidoterrestris in apple juice Amodel studyrdquo European Food Research and Technology vol 220no 2 pp 196ndash199 2005

[29] R C Witthuhn E van der Merwe P Venter and M CameronldquoGuaiacol production from ferulic acid vanillin and vanillicacid by Alicyclobacillus acidoterrestrisrdquo International Journal ofFood Microbiology vol 157 no 1 pp 113ndash117 2012

[30] A Borlinghaus and R Engel ldquoAlicyclobacillus incidence in com-mercial apple juice concentrate (AJC) supplies and validationrdquoFruit Process vol 7 pp 262ndash266 1997

[31] D F Splittstoesser C Y Lee and J J Churey ldquoControl ofAlicyclobacillus in the juice industryrdquo Dairy Food EnvironSanitation vol 18 pp 585ndash587 1998

[32] N Jensen ldquoAlicyclobacillus in Australiardquo Food Australia vol 52no 7 pp 282ndash285 2000

[33] IWalls andR Chuyate ldquoSpoilage of fruit juices byAlicyclobacil-lus acidoterrestrisrdquo Food Australia vol 52 no 7 pp 293ndash2952000

[34] N Jensen and F B Whitfield ldquoRole of Alicyclobacillus acidoter-restris in the development of a disinfectant taint in shelf-stablefruit juicerdquo Letters in Applied Microbiology vol 361 pp 9ndash142003

[35] D Gocmen A Elston T Williams M Parish and R LRouseff ldquoIdentification of medicinal off-flavours generated byAlicyclobacillus species in orange juice using GC-olfactometryand GC-MSrdquo Letters in Applied Microbiology vol 40 no 3 pp172ndash177 2005

[36] M Niwa ldquoControl of hazardous bacteria in acidic beveragesby using a guaiacol detection kit (peroxidase methodrdquo FruitProcess vol 15 pp 388ndash392 2005

[37] B Siegmund and B Pollinger-Zierler ldquoGrowth behavior ofoff-flavor-forming microorganisms in apple juicerdquo Journal of

Agricultural and Food Chemistry vol 55 no 16 pp 6692ndash66992007

[38] M Niwa and A Kawamoto ldquoDevelopment of a rapid detectionmethod of A acidoterrestris hazardous bacteria to acidicbeveragerdquo Fruit Process vol 13 pp 102ndash107 2003

[39] T A Eisele and M J Semon ldquoBest estimated aroma andtaste detection threshold for guaiacol in water and apple juicerdquoJournal of Food Science vol 70 no 4 pp S267ndashS269 2005

[40] B Zierler B Siegmund and W Pfannhauser ldquoDeterminationof off-flavour compounds in apple juice caused by microor-ganisms using headspace solid phase microextraction-gaschromatography-mass spectrometryrdquo Analytica Chimica Actavol 520 no 1-2 pp 3ndash11 2004

[41] K S Bahceci and J Acar ldquoModeling the combined effectsof pH temperature and ascorbic acid concentration on theheat resistance of Alicyclobacillus acidoterrestisrdquo InternationalJournal of Food Microbiology vol 120 no 3 pp 266ndash273 2007

[42] J Baumgart ldquoChapter 11 Media for the detection and enu-meration of Alicyclobacillus acidoterrestris and Alicyclobacillusacidocaldarius in foodsrdquo in Progress in Industrial Microbiologyvol 37 pp 161ndash166 Elsevier 2003

[43] M Lin M Al-Holy S-S Chang et al ldquoRapid discriminationof Alicyclobacillus strains in apple juice by Fourier transforminfrared spectroscopyrdquo International Journal of Food Microbiol-ogy vol 105 no 3 pp 369ndash376 2005

[44] J Wang T Yue Y Yuan X Lu J-H Shin and B RascoldquoDiscrimination of alicyclobacillus strains using nitrocellulosemembrane filter and attenuated total reflectance fourier trans-form infrared spectroscopyrdquo Journal of Food Science vol 76 no2 pp 137ndash142 2011

[45] M A Al-Holy M Lin O A Alhaj and M H Abu-GoushldquoDiscrimination between Bacillus and Alicyclobacillus Isolatesin Apple Juice by Fourier Transform Infrared Spectroscopy andMultivariate Analysisrdquo Journal of Food Science vol 80 no 2 ppM399ndashM404 2015

[46] WH Groenewald P A Gouws andR CWitthuhn ldquoIsolationidentification and typification of Alicyclobacillus acidoterrestrisand Alicyclobacillus acidocaldarius strains from orchard soiland the fruit processing environment in South Africardquo FoodMicrobiology vol 26 no 1 pp 71ndash76 2009

[47] J Zhang T Yue and Y Yuan ldquoAlicyclobacillus contaminationin the production line of Kiwi products in Chinardquo PLoS ONEvol 8 no 7 p e67704 2013

[48] I C McKnight M N U Eiroa A S SantrsquoAna and P RMassaguer ldquoAlicyclobacillus acidoterrestris in pasteurized exoticBrazilian fruit juices Isolation genotypic characterization andheat resistancerdquo FoodMicrobiology vol 27 no 8 pp 1016ndash10222010

[49] L Felix-Valenzuela I Guardiola-Avila A Burgara-Estrella MIbarra-Zavala and V Mata-Haro ldquoGenotypic and phenotypicdiversity of Alicyclobacillus acidocaldarius isolatesrdquo Letters inApplied Microbiology vol 61 no 4 pp 367ndash373 2015

[50] J D Wisotzkey P Jurtshuk Jr G E Fox G Deinhard andK Poralla ldquoComparative sequence analyses on the 16S rRNA(rDNA) of Bacillus acidocaldarius Bacillus acidoterrestris andBacillus cycloheptanicus and proposal for creation of a newgenus Alicyclobacillus gen novrdquo International Journal of Sys-tematic Bacteriology vol 42 no 2 pp 263ndash269 1992

[51] C J Connor H Luo B B McSpadden Gardener and HH Wang ldquoDevelopment of a real-time PCR-based systemtargeting the 16S rRNA gene sequence for rapid detection of

12 BioMed Research International

Alicyclobacillus spp in juice productsrdquo International Journal ofFood Microbiology vol 99 no 3 pp 229ndash235 2005

[52] S ChenQ Tang X Zhang et al ldquoIsolation and characterizationof thermo-acidophilic endospore-forming bacteria from theconcentrated apple juice-processing environmentrdquo FoodMicro-biology vol 23 no 5 pp 439ndash445 2006

[53] K Goto A Nishibori Y Wasada K Furuhata M Fukuyamaand M Hara ldquoIdentification of thermo-acidophilic bacteriaisolated from the soil of several Japanese fruit orchardsrdquo Lettersin Applied Microbiology vol 46 no 3 pp 289ndash294 2008

[54] J Chen X Ma Y Yuan and W Zhang ldquoSensitive and rapiddetection ofAlicyclobacillus acidoterrestris using loop-mediatedisothermal amplificationrdquo Journal of the Science of Food andAgriculture vol 91 no 6 pp 1070ndash1074 2011

[55] K T Chow M K Pope and J Davies ldquoCharacterization of avanillic acid non-oxidative decarboxylation gene cluster fromStreptomyces sp D7rdquo Microbiology vol 145 no 9 pp 2393ndash2403 1999

[56] K Matsubara ldquoA method for detecting andor identifying gua-iacol producing micro organismrdquo Patent no JP2003000259A2003

[57] Y Wang T Yue Y Yuan and Z Gao ldquoIsolation and identifi-cation of thermo-acidophilic bacteria from orchards in chinardquoJournal of Food Protection vol 73 pp 390ndash394 2010

[58] M Shemesh R Pasvolsky N Sela S J Green and V andZakinldquoDraft Genome Sequence of Alicyclobacillus acidoterrestrisStrain ATCC 49025rdquo Genome Announcement vol 1 no 5Article ID e00638ndash13 2013

[59] B Sokołowska S Skapska M Fonberg-Broczek et al ldquoFactorsinfluencing the inactivation of Alicyclobacillus acidoterrestrisspores exposed to high hydrostatic pressure in apple juicerdquoHighPressure Research vol 33 no 1 pp 73ndash82 2013

[60] B A Osopale C R Witthuhn J Albertyn and F A Oguntoy-inbo ldquoCulture dependent and independent genomic identifica-tion ofAlicyclobacillus species in contaminated commercial fruitjuicesrdquo Food Microbiology vol 56 pp 21ndash28 2016

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

10 BioMed Research International

other features of Alicyclobacillus such as susceptibility totemperature or high hydrostatic pressure in order to establishif there are any differences between the groups

Both the rpoB gene and vdc region seem to be single-copy genetic elements and showed no signs of intragenomicheterogeneity which often makes the RFLP comparison ofrDNA genes harder to perform

The sequence analysis of the 16S rDNA fragment ofselected isolates shows no apparent correlation betweendiversity of the 16S rDNA sequence and the sources whichthe strains were isolated from

In conclusion the application of PCR-RFLP has beenproven to be a fast and reliable method for Alicyclobacillusidentification and differentiation The method is also techni-cally less difficult than most of other molecular techniquesTwo major groups of A acidoterrestris have been identifiedThe primers designed for this study could be useful in furtherresearch on Alicyclobacillus

Data Availability

The DNA sequences obtained during this study have beendeposited in GenBank and the accession numbers areincluded within the article

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

Our research project was fully sponsored by Polish Ministryof Science and Higher Education with funds for statutoryactivity

References

[1] K Yamazaki H Teouka and H Shinano ldquoIsolation and identi-fication of alicyclobacillus acidoterrestris from acidic beveragesrdquoBioscience Biotechnology and Biochemistry vol 60 no 3 pp543ndash545 1996

[2] G DARLAND and T D BROCK ldquoBacillus acidocaldar-ius spnov an Acidophilic Thermophilic Spore-forming Bac-teriumrdquo Journal of General Microbiology vol 67 no 1 pp 9ndash151971

[3] G Deinhard P Blanz K Poralla and E Altan ldquoBacillusacidoterrestris sp nov a new thermotolerant acidophile isolatedfrom different soilsrdquo Systematic and Applied Microbiology vol10 no 1 pp 47ndash53 1987

[4] G Deinhard J Saar W Krischke and K Poralla ldquoBacilluscycloheptanicus sp nov a new thermoacidophile containing 120596-cycloheptane fatty acidsrdquo Systematic and Applied Microbiologyvol 10 no 1 pp 68ndash73 1987

[5] L Albuquerque F A Rainey A P Chung et al ldquoAlicyclobacillushesperidum sp nov and a related genomic species from solfa-taric soils of Sao Miguel in the Azoresrdquo International Journalof Systematic and Evolutionary Microbiology vol 50 no 2 pp451ndash457 2000

[6] K Goto H Matsubara K Mochida et al ldquoAlicyclobacillusherbarius sp nov a novel bacterium containing120596-cycloheptane

fatty acids isolated from herbal teardquo International Journal ofSystematic and EvolutionaryMicrobiology vol 52 no 1 pp 109ndash113 2002

[7] K Goto Y Tanimoto T Tamura et al ldquoIdentificationof thermoacidophilic bacteria and a new Alicyclobacillusgenomic species isolated from acidic environments in JapanrdquoExtremophiles vol 6 no 4 pp 333ndash340 2002

[8] H Matsubara K Goto T Matsumura et al ldquoAlicyclobacillusacidiphilus sp nov a novel thermo-acidophilic 120596-alicyclicfatty acid-containing bacterium isolated from acidic beveragesrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 52 no 5 pp 1681ndash1685 2002

[9] R V Orr R L Shewfelt C J Huang S Tefera and L RBeuchat ldquoDetection of guaiacol produced by Alicyclobacillusacidoterrestris in apple juice by sensory and chromatographicanalyses and comparison with spore and vegetative cell popu-lationsrdquo Journal of Food Protection vol 63 no 11 pp 1517ndash15222000

[10] G L Pettipher M E Osmundson and J MMurphy ldquoMethodsfor the detection and enumeration of Alicyclobacillus acidoter-restris and investigation of growth and production of taint infruit juice and fruit juice-containing drinksrdquo Letters in AppliedMicrobiology vol 24 no 3 pp 185ndash189 1997

[11] A C N F Spinelli A S Sant1015840Ana S Rodrigues Jr andP R Massaguer ldquoInfluence of different filling cooling andstorage conditions on the growth of Alicyclobacillus acidoter-restris CRA7152 in orange juicerdquo Applied and EnvironmentalMicrobiology vol 75 no 23 pp 7409ndash7416 2009

[12] MCMaldonado C Belfiore andA RNavarro ldquoTemperaturesoluble solids and pH effect on Alicyclobacillus acidoterrestrisviability in lemon juice concentraterdquo Journal of IndustrialMicrobiology and Biotechnology vol 35 no 2 pp 141ndash144 2008

[13] M N U Eiroa V C A Junqueira and F L Schmidt ldquoAlicy-clobacillus in orange juice Occurrence and heat resistance ofsporesrdquo Journal of Food Protection vol 62 no 8 pp 883ndash8861999

[14] I Walls and R Chuyate ldquoIsolation of Alicyclobacillus acidoter-restris from fruit juicesrdquo Journal of AOAC International vol 83no 5 pp 1115ndash1120 2000

[15] M E Parish and R M Goodrich ldquoRecovery of presumptiveAlicyclobacillus strains from orange fruit surfacesrdquo Journal ofFood Protection vol 68 no 10 pp 2196ndash2200 2005

[16] D F Splittstoesser J J Churey and C Y Lee ldquoGrowthcharacteristics of aciduric sporeforming bacilli isolated fromfruit juicesrdquo Journal of Food Protection vol 57 no 12 pp 1080ndash1083 1994

[17] J Baumgart M Husemann and C Schmidt ldquoAlicyclobacil-lus acidoterrestris vorkommen bedeutung und nachweis ingetranken und getrankegrundstoffenrdquo Flussiges Obst vol 64pp 178ndash180 1997

[18] C A Wisse and M E Parish ldquoIsolation and enumeration ofspore-forming thermoacidophilic rod-shaped bacteria fromcitrus processing environmentsrdquo Dairy Food EnvironmentalSanitation vol 18 pp 504ndash509 1998

[19] J Baumgart and S Menje ldquoThe impact of Alicyclobacillusacidoterestris on the quality of juices and soft drinksrdquo FruitProcess vol 10 pp 251ndash254 2000

[20] S Y Eguchi G P Manfio M E Pinhatti E Azuma and SF Variane ldquoAcidotermofilic sporeforming bacteria (ATSB) inorange juices ecology and involvement in the deterioration offruit juices Report of the Research Project Part IIrdquo Fruit Processvol 11 pp 55ndash62 2001

BioMed Research International 11

[21] P AGouws L Gie A Pretorius andNDhansay ldquoIsolation andidentification of Alicyclobacillus acidocaldarius by 16S rDNAfrom mango juice and concentraterdquo International Journal ofFood Science amp Technology vol 40 no 7 pp 789ndash792 2005

[22] K Goto K Mochida Y Kato et al ldquoProposal of six speciesof moderately thermophilic acidophilic endospore-formingbacteria Alicyclobacillus contaminans sp nov Alicyclobacillusfastidiosus sp novAlicyclobacillus kakegawensis sp novAlicy-clobacillus macrosporangiidus sp nov Alicyclobacillus saccharisp nov and Alicyclobacillus shizuokensis sp novrdquo InternationalJournal of Systematic and Evolutionary Microbiology vol 57 no6 pp 1276ndash1285 2007

[23] M Z Durak J J Churey M D Danyluk and R W WoroboldquoIdentification and haplotype distribution of Alicyclobacillusspp from different juices and beveragesrdquo International Journalof Food Microbiology vol 142 no 3 pp 286ndash291 2010

[24] B Sokolowska J Niezgoda A Dekowska et al ldquoIncidence ofAlicyclobacillus spp in Polish apple and dark berry juice con-centrates and the ability of isolated A acidoterrestris strains tospoilage of these juicesrdquo Postępy Nauki i Technologii PrzemysłuRolno-Spozywczego vol 71 no 1 pp 5ndash20 2016

[25] I Walls and R Chuyate ldquoAlicyclobacillus historical perspectiveand preliminary characterization studyrdquo Dairy Food EnvironSanitation18 pp 499ndash503 1998

[26] H-A Duong and N Jensen ldquoSpoilage of iced tea by Alicy-clobacillusrdquo Food Australia vol 52 no 7 p 292 2000

[27] M Niwa and A Kuriyama ldquoA acidoterrestris rapid detectionkitrdquo Fruit Process vol 13 pp 328ndash331 2003

[28] K S Bahceci V Gokmen and J Acar ldquoFormation of guaiacolfrom vanillin by Alicyclobacillus acidoterrestris in apple juice Amodel studyrdquo European Food Research and Technology vol 220no 2 pp 196ndash199 2005

[29] R C Witthuhn E van der Merwe P Venter and M CameronldquoGuaiacol production from ferulic acid vanillin and vanillicacid by Alicyclobacillus acidoterrestrisrdquo International Journal ofFood Microbiology vol 157 no 1 pp 113ndash117 2012

[30] A Borlinghaus and R Engel ldquoAlicyclobacillus incidence in com-mercial apple juice concentrate (AJC) supplies and validationrdquoFruit Process vol 7 pp 262ndash266 1997

[31] D F Splittstoesser C Y Lee and J J Churey ldquoControl ofAlicyclobacillus in the juice industryrdquo Dairy Food EnvironSanitation vol 18 pp 585ndash587 1998

[32] N Jensen ldquoAlicyclobacillus in Australiardquo Food Australia vol 52no 7 pp 282ndash285 2000

[33] IWalls andR Chuyate ldquoSpoilage of fruit juices byAlicyclobacil-lus acidoterrestrisrdquo Food Australia vol 52 no 7 pp 293ndash2952000

[34] N Jensen and F B Whitfield ldquoRole of Alicyclobacillus acidoter-restris in the development of a disinfectant taint in shelf-stablefruit juicerdquo Letters in Applied Microbiology vol 361 pp 9ndash142003

[35] D Gocmen A Elston T Williams M Parish and R LRouseff ldquoIdentification of medicinal off-flavours generated byAlicyclobacillus species in orange juice using GC-olfactometryand GC-MSrdquo Letters in Applied Microbiology vol 40 no 3 pp172ndash177 2005

[36] M Niwa ldquoControl of hazardous bacteria in acidic beveragesby using a guaiacol detection kit (peroxidase methodrdquo FruitProcess vol 15 pp 388ndash392 2005

[37] B Siegmund and B Pollinger-Zierler ldquoGrowth behavior ofoff-flavor-forming microorganisms in apple juicerdquo Journal of

Agricultural and Food Chemistry vol 55 no 16 pp 6692ndash66992007

[38] M Niwa and A Kawamoto ldquoDevelopment of a rapid detectionmethod of A acidoterrestris hazardous bacteria to acidicbeveragerdquo Fruit Process vol 13 pp 102ndash107 2003

[39] T A Eisele and M J Semon ldquoBest estimated aroma andtaste detection threshold for guaiacol in water and apple juicerdquoJournal of Food Science vol 70 no 4 pp S267ndashS269 2005

[40] B Zierler B Siegmund and W Pfannhauser ldquoDeterminationof off-flavour compounds in apple juice caused by microor-ganisms using headspace solid phase microextraction-gaschromatography-mass spectrometryrdquo Analytica Chimica Actavol 520 no 1-2 pp 3ndash11 2004

[41] K S Bahceci and J Acar ldquoModeling the combined effectsof pH temperature and ascorbic acid concentration on theheat resistance of Alicyclobacillus acidoterrestisrdquo InternationalJournal of Food Microbiology vol 120 no 3 pp 266ndash273 2007

[42] J Baumgart ldquoChapter 11 Media for the detection and enu-meration of Alicyclobacillus acidoterrestris and Alicyclobacillusacidocaldarius in foodsrdquo in Progress in Industrial Microbiologyvol 37 pp 161ndash166 Elsevier 2003

[43] M Lin M Al-Holy S-S Chang et al ldquoRapid discriminationof Alicyclobacillus strains in apple juice by Fourier transforminfrared spectroscopyrdquo International Journal of Food Microbiol-ogy vol 105 no 3 pp 369ndash376 2005

[44] J Wang T Yue Y Yuan X Lu J-H Shin and B RascoldquoDiscrimination of alicyclobacillus strains using nitrocellulosemembrane filter and attenuated total reflectance fourier trans-form infrared spectroscopyrdquo Journal of Food Science vol 76 no2 pp 137ndash142 2011

[45] M A Al-Holy M Lin O A Alhaj and M H Abu-GoushldquoDiscrimination between Bacillus and Alicyclobacillus Isolatesin Apple Juice by Fourier Transform Infrared Spectroscopy andMultivariate Analysisrdquo Journal of Food Science vol 80 no 2 ppM399ndashM404 2015

[46] WH Groenewald P A Gouws andR CWitthuhn ldquoIsolationidentification and typification of Alicyclobacillus acidoterrestrisand Alicyclobacillus acidocaldarius strains from orchard soiland the fruit processing environment in South Africardquo FoodMicrobiology vol 26 no 1 pp 71ndash76 2009

[47] J Zhang T Yue and Y Yuan ldquoAlicyclobacillus contaminationin the production line of Kiwi products in Chinardquo PLoS ONEvol 8 no 7 p e67704 2013

[48] I C McKnight M N U Eiroa A S SantrsquoAna and P RMassaguer ldquoAlicyclobacillus acidoterrestris in pasteurized exoticBrazilian fruit juices Isolation genotypic characterization andheat resistancerdquo FoodMicrobiology vol 27 no 8 pp 1016ndash10222010

[49] L Felix-Valenzuela I Guardiola-Avila A Burgara-Estrella MIbarra-Zavala and V Mata-Haro ldquoGenotypic and phenotypicdiversity of Alicyclobacillus acidocaldarius isolatesrdquo Letters inApplied Microbiology vol 61 no 4 pp 367ndash373 2015

[50] J D Wisotzkey P Jurtshuk Jr G E Fox G Deinhard andK Poralla ldquoComparative sequence analyses on the 16S rRNA(rDNA) of Bacillus acidocaldarius Bacillus acidoterrestris andBacillus cycloheptanicus and proposal for creation of a newgenus Alicyclobacillus gen novrdquo International Journal of Sys-tematic Bacteriology vol 42 no 2 pp 263ndash269 1992

[51] C J Connor H Luo B B McSpadden Gardener and HH Wang ldquoDevelopment of a real-time PCR-based systemtargeting the 16S rRNA gene sequence for rapid detection of

12 BioMed Research International

Alicyclobacillus spp in juice productsrdquo International Journal ofFood Microbiology vol 99 no 3 pp 229ndash235 2005

[52] S ChenQ Tang X Zhang et al ldquoIsolation and characterizationof thermo-acidophilic endospore-forming bacteria from theconcentrated apple juice-processing environmentrdquo FoodMicro-biology vol 23 no 5 pp 439ndash445 2006

[53] K Goto A Nishibori Y Wasada K Furuhata M Fukuyamaand M Hara ldquoIdentification of thermo-acidophilic bacteriaisolated from the soil of several Japanese fruit orchardsrdquo Lettersin Applied Microbiology vol 46 no 3 pp 289ndash294 2008

[54] J Chen X Ma Y Yuan and W Zhang ldquoSensitive and rapiddetection ofAlicyclobacillus acidoterrestris using loop-mediatedisothermal amplificationrdquo Journal of the Science of Food andAgriculture vol 91 no 6 pp 1070ndash1074 2011

[55] K T Chow M K Pope and J Davies ldquoCharacterization of avanillic acid non-oxidative decarboxylation gene cluster fromStreptomyces sp D7rdquo Microbiology vol 145 no 9 pp 2393ndash2403 1999

[56] K Matsubara ldquoA method for detecting andor identifying gua-iacol producing micro organismrdquo Patent no JP2003000259A2003

[57] Y Wang T Yue Y Yuan and Z Gao ldquoIsolation and identifi-cation of thermo-acidophilic bacteria from orchards in chinardquoJournal of Food Protection vol 73 pp 390ndash394 2010

[58] M Shemesh R Pasvolsky N Sela S J Green and V andZakinldquoDraft Genome Sequence of Alicyclobacillus acidoterrestrisStrain ATCC 49025rdquo Genome Announcement vol 1 no 5Article ID e00638ndash13 2013

[59] B Sokołowska S Skapska M Fonberg-Broczek et al ldquoFactorsinfluencing the inactivation of Alicyclobacillus acidoterrestrisspores exposed to high hydrostatic pressure in apple juicerdquoHighPressure Research vol 33 no 1 pp 73ndash82 2013

[60] B A Osopale C R Witthuhn J Albertyn and F A Oguntoy-inbo ldquoCulture dependent and independent genomic identifica-tion ofAlicyclobacillus species in contaminated commercial fruitjuicesrdquo Food Microbiology vol 56 pp 21ndash28 2016

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 11

[21] P AGouws L Gie A Pretorius andNDhansay ldquoIsolation andidentification of Alicyclobacillus acidocaldarius by 16S rDNAfrom mango juice and concentraterdquo International Journal ofFood Science amp Technology vol 40 no 7 pp 789ndash792 2005

[22] K Goto K Mochida Y Kato et al ldquoProposal of six speciesof moderately thermophilic acidophilic endospore-formingbacteria Alicyclobacillus contaminans sp nov Alicyclobacillusfastidiosus sp novAlicyclobacillus kakegawensis sp novAlicy-clobacillus macrosporangiidus sp nov Alicyclobacillus saccharisp nov and Alicyclobacillus shizuokensis sp novrdquo InternationalJournal of Systematic and Evolutionary Microbiology vol 57 no6 pp 1276ndash1285 2007

[23] M Z Durak J J Churey M D Danyluk and R W WoroboldquoIdentification and haplotype distribution of Alicyclobacillusspp from different juices and beveragesrdquo International Journalof Food Microbiology vol 142 no 3 pp 286ndash291 2010

[24] B Sokolowska J Niezgoda A Dekowska et al ldquoIncidence ofAlicyclobacillus spp in Polish apple and dark berry juice con-centrates and the ability of isolated A acidoterrestris strains tospoilage of these juicesrdquo Postępy Nauki i Technologii PrzemysłuRolno-Spozywczego vol 71 no 1 pp 5ndash20 2016

[25] I Walls and R Chuyate ldquoAlicyclobacillus historical perspectiveand preliminary characterization studyrdquo Dairy Food EnvironSanitation18 pp 499ndash503 1998

[26] H-A Duong and N Jensen ldquoSpoilage of iced tea by Alicy-clobacillusrdquo Food Australia vol 52 no 7 p 292 2000

[27] M Niwa and A Kuriyama ldquoA acidoterrestris rapid detectionkitrdquo Fruit Process vol 13 pp 328ndash331 2003

[28] K S Bahceci V Gokmen and J Acar ldquoFormation of guaiacolfrom vanillin by Alicyclobacillus acidoterrestris in apple juice Amodel studyrdquo European Food Research and Technology vol 220no 2 pp 196ndash199 2005

[29] R C Witthuhn E van der Merwe P Venter and M CameronldquoGuaiacol production from ferulic acid vanillin and vanillicacid by Alicyclobacillus acidoterrestrisrdquo International Journal ofFood Microbiology vol 157 no 1 pp 113ndash117 2012

[30] A Borlinghaus and R Engel ldquoAlicyclobacillus incidence in com-mercial apple juice concentrate (AJC) supplies and validationrdquoFruit Process vol 7 pp 262ndash266 1997

[31] D F Splittstoesser C Y Lee and J J Churey ldquoControl ofAlicyclobacillus in the juice industryrdquo Dairy Food EnvironSanitation vol 18 pp 585ndash587 1998

[32] N Jensen ldquoAlicyclobacillus in Australiardquo Food Australia vol 52no 7 pp 282ndash285 2000

[33] IWalls andR Chuyate ldquoSpoilage of fruit juices byAlicyclobacil-lus acidoterrestrisrdquo Food Australia vol 52 no 7 pp 293ndash2952000

[34] N Jensen and F B Whitfield ldquoRole of Alicyclobacillus acidoter-restris in the development of a disinfectant taint in shelf-stablefruit juicerdquo Letters in Applied Microbiology vol 361 pp 9ndash142003

[35] D Gocmen A Elston T Williams M Parish and R LRouseff ldquoIdentification of medicinal off-flavours generated byAlicyclobacillus species in orange juice using GC-olfactometryand GC-MSrdquo Letters in Applied Microbiology vol 40 no 3 pp172ndash177 2005

[36] M Niwa ldquoControl of hazardous bacteria in acidic beveragesby using a guaiacol detection kit (peroxidase methodrdquo FruitProcess vol 15 pp 388ndash392 2005

[37] B Siegmund and B Pollinger-Zierler ldquoGrowth behavior ofoff-flavor-forming microorganisms in apple juicerdquo Journal of

Agricultural and Food Chemistry vol 55 no 16 pp 6692ndash66992007

[38] M Niwa and A Kawamoto ldquoDevelopment of a rapid detectionmethod of A acidoterrestris hazardous bacteria to acidicbeveragerdquo Fruit Process vol 13 pp 102ndash107 2003

[39] T A Eisele and M J Semon ldquoBest estimated aroma andtaste detection threshold for guaiacol in water and apple juicerdquoJournal of Food Science vol 70 no 4 pp S267ndashS269 2005

[40] B Zierler B Siegmund and W Pfannhauser ldquoDeterminationof off-flavour compounds in apple juice caused by microor-ganisms using headspace solid phase microextraction-gaschromatography-mass spectrometryrdquo Analytica Chimica Actavol 520 no 1-2 pp 3ndash11 2004

[41] K S Bahceci and J Acar ldquoModeling the combined effectsof pH temperature and ascorbic acid concentration on theheat resistance of Alicyclobacillus acidoterrestisrdquo InternationalJournal of Food Microbiology vol 120 no 3 pp 266ndash273 2007

[42] J Baumgart ldquoChapter 11 Media for the detection and enu-meration of Alicyclobacillus acidoterrestris and Alicyclobacillusacidocaldarius in foodsrdquo in Progress in Industrial Microbiologyvol 37 pp 161ndash166 Elsevier 2003

[43] M Lin M Al-Holy S-S Chang et al ldquoRapid discriminationof Alicyclobacillus strains in apple juice by Fourier transforminfrared spectroscopyrdquo International Journal of Food Microbiol-ogy vol 105 no 3 pp 369ndash376 2005

[44] J Wang T Yue Y Yuan X Lu J-H Shin and B RascoldquoDiscrimination of alicyclobacillus strains using nitrocellulosemembrane filter and attenuated total reflectance fourier trans-form infrared spectroscopyrdquo Journal of Food Science vol 76 no2 pp 137ndash142 2011

[45] M A Al-Holy M Lin O A Alhaj and M H Abu-GoushldquoDiscrimination between Bacillus and Alicyclobacillus Isolatesin Apple Juice by Fourier Transform Infrared Spectroscopy andMultivariate Analysisrdquo Journal of Food Science vol 80 no 2 ppM399ndashM404 2015

[46] WH Groenewald P A Gouws andR CWitthuhn ldquoIsolationidentification and typification of Alicyclobacillus acidoterrestrisand Alicyclobacillus acidocaldarius strains from orchard soiland the fruit processing environment in South Africardquo FoodMicrobiology vol 26 no 1 pp 71ndash76 2009

[47] J Zhang T Yue and Y Yuan ldquoAlicyclobacillus contaminationin the production line of Kiwi products in Chinardquo PLoS ONEvol 8 no 7 p e67704 2013

[48] I C McKnight M N U Eiroa A S SantrsquoAna and P RMassaguer ldquoAlicyclobacillus acidoterrestris in pasteurized exoticBrazilian fruit juices Isolation genotypic characterization andheat resistancerdquo FoodMicrobiology vol 27 no 8 pp 1016ndash10222010

[49] L Felix-Valenzuela I Guardiola-Avila A Burgara-Estrella MIbarra-Zavala and V Mata-Haro ldquoGenotypic and phenotypicdiversity of Alicyclobacillus acidocaldarius isolatesrdquo Letters inApplied Microbiology vol 61 no 4 pp 367ndash373 2015

[50] J D Wisotzkey P Jurtshuk Jr G E Fox G Deinhard andK Poralla ldquoComparative sequence analyses on the 16S rRNA(rDNA) of Bacillus acidocaldarius Bacillus acidoterrestris andBacillus cycloheptanicus and proposal for creation of a newgenus Alicyclobacillus gen novrdquo International Journal of Sys-tematic Bacteriology vol 42 no 2 pp 263ndash269 1992

[51] C J Connor H Luo B B McSpadden Gardener and HH Wang ldquoDevelopment of a real-time PCR-based systemtargeting the 16S rRNA gene sequence for rapid detection of

12 BioMed Research International

Alicyclobacillus spp in juice productsrdquo International Journal ofFood Microbiology vol 99 no 3 pp 229ndash235 2005

[52] S ChenQ Tang X Zhang et al ldquoIsolation and characterizationof thermo-acidophilic endospore-forming bacteria from theconcentrated apple juice-processing environmentrdquo FoodMicro-biology vol 23 no 5 pp 439ndash445 2006

[53] K Goto A Nishibori Y Wasada K Furuhata M Fukuyamaand M Hara ldquoIdentification of thermo-acidophilic bacteriaisolated from the soil of several Japanese fruit orchardsrdquo Lettersin Applied Microbiology vol 46 no 3 pp 289ndash294 2008

[54] J Chen X Ma Y Yuan and W Zhang ldquoSensitive and rapiddetection ofAlicyclobacillus acidoterrestris using loop-mediatedisothermal amplificationrdquo Journal of the Science of Food andAgriculture vol 91 no 6 pp 1070ndash1074 2011

[55] K T Chow M K Pope and J Davies ldquoCharacterization of avanillic acid non-oxidative decarboxylation gene cluster fromStreptomyces sp D7rdquo Microbiology vol 145 no 9 pp 2393ndash2403 1999

[56] K Matsubara ldquoA method for detecting andor identifying gua-iacol producing micro organismrdquo Patent no JP2003000259A2003

[57] Y Wang T Yue Y Yuan and Z Gao ldquoIsolation and identifi-cation of thermo-acidophilic bacteria from orchards in chinardquoJournal of Food Protection vol 73 pp 390ndash394 2010

[58] M Shemesh R Pasvolsky N Sela S J Green and V andZakinldquoDraft Genome Sequence of Alicyclobacillus acidoterrestrisStrain ATCC 49025rdquo Genome Announcement vol 1 no 5Article ID e00638ndash13 2013

[59] B Sokołowska S Skapska M Fonberg-Broczek et al ldquoFactorsinfluencing the inactivation of Alicyclobacillus acidoterrestrisspores exposed to high hydrostatic pressure in apple juicerdquoHighPressure Research vol 33 no 1 pp 73ndash82 2013

[60] B A Osopale C R Witthuhn J Albertyn and F A Oguntoy-inbo ldquoCulture dependent and independent genomic identifica-tion ofAlicyclobacillus species in contaminated commercial fruitjuicesrdquo Food Microbiology vol 56 pp 21ndash28 2016

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

12 BioMed Research International

Alicyclobacillus spp in juice productsrdquo International Journal ofFood Microbiology vol 99 no 3 pp 229ndash235 2005

[52] S ChenQ Tang X Zhang et al ldquoIsolation and characterizationof thermo-acidophilic endospore-forming bacteria from theconcentrated apple juice-processing environmentrdquo FoodMicro-biology vol 23 no 5 pp 439ndash445 2006

[53] K Goto A Nishibori Y Wasada K Furuhata M Fukuyamaand M Hara ldquoIdentification of thermo-acidophilic bacteriaisolated from the soil of several Japanese fruit orchardsrdquo Lettersin Applied Microbiology vol 46 no 3 pp 289ndash294 2008

[54] J Chen X Ma Y Yuan and W Zhang ldquoSensitive and rapiddetection ofAlicyclobacillus acidoterrestris using loop-mediatedisothermal amplificationrdquo Journal of the Science of Food andAgriculture vol 91 no 6 pp 1070ndash1074 2011

[55] K T Chow M K Pope and J Davies ldquoCharacterization of avanillic acid non-oxidative decarboxylation gene cluster fromStreptomyces sp D7rdquo Microbiology vol 145 no 9 pp 2393ndash2403 1999

[56] K Matsubara ldquoA method for detecting andor identifying gua-iacol producing micro organismrdquo Patent no JP2003000259A2003

[57] Y Wang T Yue Y Yuan and Z Gao ldquoIsolation and identifi-cation of thermo-acidophilic bacteria from orchards in chinardquoJournal of Food Protection vol 73 pp 390ndash394 2010

[58] M Shemesh R Pasvolsky N Sela S J Green and V andZakinldquoDraft Genome Sequence of Alicyclobacillus acidoterrestrisStrain ATCC 49025rdquo Genome Announcement vol 1 no 5Article ID e00638ndash13 2013

[59] B Sokołowska S Skapska M Fonberg-Broczek et al ldquoFactorsinfluencing the inactivation of Alicyclobacillus acidoterrestrisspores exposed to high hydrostatic pressure in apple juicerdquoHighPressure Research vol 33 no 1 pp 73ndash82 2013

[60] B A Osopale C R Witthuhn J Albertyn and F A Oguntoy-inbo ldquoCulture dependent and independent genomic identifica-tion ofAlicyclobacillus species in contaminated commercial fruitjuicesrdquo Food Microbiology vol 56 pp 21ndash28 2016

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom