Cochu LettApplMicrobiol 2008

ORIGINAL ARTICLE

Maple sap as a rich medium to grow probiotic lactobacilliand to produce lactic acidA. Cochu, D. Fourmier, A. Halasz and J. Hawari

Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada

Introduction

There has been an increased interest in the development

of nutraceuticals and functional foods, specifically in pro-

biotics. However, there has been an increase in the

demand for nondairy-based probiotic products. In a col-

loquium of the American Academy for Microbiology held

in Baltimore in November 2005 (report available at:

http://www.asm.org/Academy/index.asp?bid=2093) and in

a recent review (Sleator and Hill 2008), it was reported

that the use of probiotics could benefit human and ani-

mal health, specifically in the prevention or in the treat-

ment of irritable bowel syndrome, bladder and colon

cancer, urogenital infections, Clostridium difficile infec-

tions, atopic eczema, asthma and diarrhoea caused by

Rotavirus in children. Most probiotics belong to the lactic

acid bacteria (LAB) family, with many species of Lacto-

bacillus, including Lactobacillus acidophilus, Lactobacillus

casei and Lactobacillus plantarum.

In addition to the interest in lactobacilli themselves, lac-

tic acid, which is the primary metabolite of LAB, is widely

used in pharmaceutical, chemical, cosmetic, textile and

food industries. Lactic acid is also used as an acidulant, a

preservative agent, and as precursor for the production of

base chemicals or biodegradable polymers such as polylac-

tic acid (PLA; Vickroy 1985; Kharas et al. 1994). The

worldwide demand for lactic acid is increasing constantly,

and is estimated roughly to range between 130 000 and

150 000 metric tonnes per year (Mirasol 1999). Presently,

lactic acid is commercially produced by fermentation of

sugar cane (Patil et al. 2006; Timbuntam et al. 2006), corn

sugars (Mercier et al. 1992), beet molasses (Kotzamanidis

et al. 2002) and whey (Wee et al. 2006). Most of these agri-

cultural feedstocks first require a pretreatment such as

extraction or hydrolysis of their sugar content, to allow

bacterial fermentation to take place.

Forests in Eastern Canada can be considered as large

reservoirs of maple sap, which contains between 10 and

Keywords

lactic acid, lactobacilli, maple sap, probiotics,

trisaccharides.

Correspondence

Jalal Hawari, Biotechnology Research Institute,

National Research Council of Canada, 6100

Royalmount Avenue, Montreal, Quebec, H4P

2R2, Canada.

E-mail: [email protected]

2008 ⁄ 0471: received 18 March 2008,

revised 18 July 2008 and accepted: 21 July

2008

doi:10.1111/j.1472-765X.2008.02451.x

Abstract

Aims: To demonstrate the feasibility of growing lactobacilli and producing lac-

tic acid using maple sap as a sugar source and to show the importance of oli-

gosaccharides in the processes.

Methods and Results: Two maple sap samples (Cetta and Pinnacle) and puri-

fied sucrose were used as carbon sources in the preparation of three culture

media. Compared with the sucrose-based medium, both maple sap-based

media produced increased viable counts in two strains out of five by a factor

of four to seven. Maple sap-based media also enhanced lactic acid production

in three strains. Cetta sap was found to be more efficient than Pinnacle sap in

stimulating lactic acid production and, was also found to be richer in various

oligosaccharides. The amendment of the Pinnacle-based medium with trisac-

charides significantly stimulated Lactobacillus acidophilus AC-10 to grow and

produce lactic acid.

Conclusions: Maple sap, particularly if rich in oligosaccharides, represents a

good carbon source for the growth of lactobacilli and the production of lactic

acid.

Significance and Impact of the Study: This study provides a proof-of-concept,

using maple sap as a substrate for lactic acid production and for the develop-

ment of a nondairy probiotic drink.

Letters in Applied Microbiology ISSN 0266-8254

500 Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 500–507

ª 2008 The Authors

30 g l)1 of sucrose with trace amounts of glucose and

fructose. Previous studies revealed that it could be directly

used as a raw material without any pretreatment for the

production of exopolysaccharides by Enterobacter agglom-

erans (Morin et al. 1995) or for the production of poly-

hydroxybutyrate (PHB) by fermentation with Alcaligenes

latus (Yezza et al. 2007). The aim of this study was to

determine the suitability of maple sap for the production

of: (i) a high density of viable probiotic lactobacilli, and

(ii) lactic acid, a versatile chemical with potential indus-

trial and biotechnological applications.

Materials and methods

Maple sap

The maple saps were collected during the Spring of 2007,

immediately frozen and sterilized by filtration through a

0Æ22-lm filtration unit (Millipore) before use. Maple sap

designated Cetta was provided by Mr Djamel Rabia of the

‘Centre d’Experimentation et de Transfert Technologique

en Acericulture’ (Cetta, Pohenegamook, Quebec, Canada).

Pinnacle maple sap was kindly provided by a personal

contact in Baldwin Mills (Quebec, Canada). Both sap

samples were harvested using tubular conduits collecting

system in the middle of March.

Chemical analysis of maple saps

All chemicals were from Sigma-Aldrich. Sucrose, glucose

and fructose were analysed using high-performance liquid

chromatography (HPLC; Waters Chromatography divi-

sion, Milford, MA, USA) equipped with an injector

model 717, a model 600 pump and a 2414 refractive

index detector. The separation was made with an ICsep

ICE-ION-300 column (Transgenomic, San Jose, CA,

USA) of 300 mm · 7Æ8 mm i.d. and an ion guard GC-

801 column (Transgenomics). The mobile phase consisted

of a solution of 0Æ035 N sulfuric acid (pH 4) flowing at

0Æ4 ml min)1. Analyses of total organic carbon (EPA

Method 415Æ1 modified 1999), metals (EPA Method 6020,

1994) and total nitrogen (ASTM D5291, 2007) in the sap

samples were conducted by Maxxam Analytique Inc.

(Montreal, Quebec, Canada). Oligosaccharides with vari-

ous degree of polymerization (d.p.) such as raffinose,

stachyose, maltotriose and 1-kestose were analysed by

liquid chromatography (LC)-mass spectrometry (MS)

using a Bruker micrOTOF-Q mass spectrometer attached

to a Hewlett-Packard 1200 series HPLC system (Bruker

Daltonics, Milton, Canada). The samples were injected

into a 5-lm pore size LC-NH2 column (4Æ6 mm

i.d. · 250 mm; Supelco, Bellefonte, PA, USA) at 35�C.

The solvent system was composed of a mixture of

CH3CN (70% v ⁄ v) ⁄ H2O (30% v ⁄ v) at a flow rate of

1 ml min)1. For mass analysis, positive electrospray ioni-

zation mode was used producing sodium adduct ions

[M+Na]+. The mass range was scanned from 100 to

800 m ⁄ z. In order to better characterize the trisaccharides

found in Cetta sap, the LC-MS method was modified by

eluting the sap through an LC-NH2 column using a sol-

vent mixture composed of acetonitrile (82%) and water

(18%) at a flow rate 1Æ5 ml min)1. As described before,

trisaccharides were detected as their sodium adduct mass

[M+Na]+ using electrospray mass spectrometry in the

positive ionization mode.

Media

One litre of a sucrose-based medium was prepared by mix-

ing the following autoclaved solutions: 50 ml of a 400 g l)1

stock solution of veggietones pea (Oxoid), 25 ml of a

200 g l)1 stock solution of yeast extract (Difco), 10 ml of

a 200 g l)1 stock solution of K2HPO4 (Sigma), 10 ml of a

5 g l)1 stock solution of MnSO4 (Sigma), 10 ml of a

20 g l)1 stock of MgSO4 (Sigma) and 895 ml of a filtered-

sterilized sucrose solution at 22 g l)1 (purified sucrose; EM

Science, Gibbston, NJ), providing 20 g l)1 of sucrose in the

final mixture. The maple sap-based media were prepared

similarly, using 895 ml of either maple sap Cetta or Pinna-

cle instead of the 22 g l)1 sucrose solution. As determined

by HPLC, the final concentration of sucrose in the two

maple sap-based media, Cetta and Pinnacle, were 19Æ00 and

16Æ47 g l)1, respectively. For some experiments, the Pinna-

cle-based medium was also amended with trisaccharides

such as raffinose and maltotriose, each one added at a final

concentration of 1 g l)1.

Micro-organisms and growth conditions

Lactobacillus acidophilus R0240 and Lactobacillus helveticus

R0052 were kindly provided by Dr Thomas Tompkins

(Institut Rosell-Lallemand Inc., Montreal, Quebec, Can-

ada) and Lactobacillus rhamnosus (designated strain AC-3)

was isolated from a fresh, commercial white cheese.

Two other lactobacilli, designated as L. casei AC-8 and

L. acidophilus AC-10 were isolated from a concentrated

nondairy-based commercial probiotic product. The iden-

tity of L. rhamnosus AC-3, L. casei AC-8 and L. acidophilus

AC-10 was confirmed by comparing their 16S rDNA gene

sequence, with the 16S rDNA sequences in the NCBI data-

base (data not shown; Altschul et al. 1997).

All bacterial strains used in this study were started

from a glycerol stock and streaked on De Man Rogosa

Sharpe (MRS) agar (De Man et al. 1960). MRS plates

were incubated at 37�C under anaerobic conditions (Gas

Pack anaerobic jar system, BBL). One CFU was used to

A. Cochu et al. Growing lactobacilli in maple sap

ª 2008 The Authors

Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 500–507 501

inoculate precultures in 10 ml of MRS broth for 16 h.

Two per cent (v ⁄ v) of the MRS precultures were used to

inoculate 10 ml of a maple sap (Cetta and Pinnacle) or a

sucrose-based medium, which was incubated overnight at

37�C in 15 ml conical tubes (Falcon; BD Biosciences,

Franklin Lakes, NJ) under static conditions. Subsequently,

20-ml cultures were started in either maple sap or in

sucrose-based media by adding 2% (v ⁄ v) of the corre-

sponding maple sap or sucrose preculture in 20-ml serum

bottles followed by incubation for 16 h at 37�C under sta-

tic conditions. At T0 of growth, initial bacterial popula-

tion was c. 7 log CFU ml)1 for all bacteria, except for L.

acidophilus R0240, which was c. 6 log CFU ml)1.

Analysis of culture media

Aliquots of culture medium were collected and analysed

at T0 and after 16 h of incubation as follows: (i) 0Æ1 ml

was used to determine the viable cell count (CFU), which

was carried out by diluting the samples in 0Æ1% (wt ⁄ v)

peptone water, and spread plating in duplicate on soft

MRS agar plates, which were incubated at 37�C for 48 h

under anaerobic conditions (Gas Pack anaerobic jar sys-

tem, BBL); (ii) 1 ml was used to determine A600 nm; (iii)

3 ml was centrifuged at 12 000 g for 15 min, for pH anal-

ysis and analysis of lactic acid and residual sugars by

HPLC using similar conditions as those described before.

Results

Composition of maple sap samples

Table 1 describes the composition of both Cetta and Pin-

nacle maple sap samples. As determined by HPLC, both

Cetta and Pinnacle sap contained 21Æ0–18Æ2 g l)1 of

sucrose, respectively, and low concentrations of glucose

(0Æ1–0Æ15 g l)1) and fructose (0Æ02–0Æ08 g l)1). Cetta and

Pinnacle saps did not markedly differ in their total

organic carbon and total nitrogen concentrations. How-

ever, the two sap samples differed in their nitrate, sulfate

and sodium content. For example, the concentration of

nitrate and sulfate were 5- and 20-fold higher in Cetta

sap, but in Pinnacle the concentration of sodium was

50-fold lower (data not shown). Calcium, magnesium and

manganese were also present in higher amounts in Cetta

sap than in Pinnacle sap; however, the potassium contents

were slightly similar in both saps (data not shown).

Characterization of oligosaccharides in maple sap

LC-MS analysis of Cetta and Pinnacle saps revealed the

presence of oligosaccharides with d.p. ranging from 3 to

5 (Fig. 1). The area of the peaks corresponding to oligo-

saccharides with a d.p. of 4 and 5 were found to be

greater in Cetta than in Pinnacle sap, but those corre-

sponding to trisaccharides (d.p. 3) were found to be c. 11

times greater in Cetta than in Pinnacle sap (5Æ6 · 107 and

4Æ8 · 106, respectively) (Fig. 1).

To better characterize the d.p. 3 oligosaccharides shown

in Fig. 1, a modified LC-MS method was employed to

improve separation of overlapping signals to enhance

their detection. Figure 2a represents a typical LC-MS

chromatogram of trisaccharides in Cetta maple sap. The

peak at 25Æ2 min exhibits a sodium adduct mass

[M+Na]+ at 527 Da with a retention time (r.t.) similar to

that of 1-kestose (b-d-Fruf-(2 fi 1)b-d-Fruf(2 fi 1)a-

d-Glup). The minor LC-MS peak was observed with a

r.t. at 30Æ1 min and a [M+Na]+ at 527 Da and matched

the two trisaccharides, raffinose (O-a-galactopyrano-

syl(1 fi 6)a-d-glucopyranosyl-b-d-fructofuranoside) and

maltotriose (O-a-dd-glucopyranosyl-(1 fi 4)-O-a-d-

glucopyranosyl-(1 fi 4)-d-glucose), with each showing

the same r.t. at 30Æ1 min and [M+Na]+ at 527 Da. The

identities of these sugars and the major compound

eluting at 21Æ5 min will be discussed latter.

Growing lactobacilli in sucrose- and maple sap-based

media

The growth of L. acidophilus R0240, L. helveticus R0052

and L. acidophilus AC-10 resulted in a drop in pH (from

7Æ2 to below 4Æ5, not shown), and significant sucrose con-

sumption (1Æ3–6Æ0 g l)1; Table 2). Whichever medium

was used, with the exception of L. acidophilus R0240 that

had low final bacterial counts, the viable counts of all

other strains reached c. 9 log CFU ml)1 after 16 h of fer-

mentation at 37�C (Fig. 3a). Extending the fermentation

time from 16 to 24 h did not result in an increased viable

count (data not shown). While L. rhamnosus AC-3,

L. acidophilus R0240 and L. casei AC-8 grew similarly in

the sucrose- and maple-based culture media, L. helveticus

R0052 and L. acidophilus AC-10 grew four to seven times

more in the maple sap-based media (Fig. 3a) and also

produced more lactic acid (1Æ89–4Æ94 g l)1) than the

other strains (Fig. 3b). No other volatile organic acids

Table 1 Composition (in g l)1) of the maple sap samples used

Sap component

Cetta maple sap

(Pohenegamook,

Quebec, Canada)

Pinnacle maple sap

(Balwin Mills,

Quebec, Canada)

Fructose 0Æ080 0Æ026

Glucose 0Æ145 0Æ098

Sucrose 21Æ001 18Æ201

Total organic carbon 10Æ300 9Æ210

Total nitrogen <0Æ100 <0Æ100

Growing lactobacilli in maple sap A. Cochu et al.


ª 2008 The Authors

such as acetic acid were detected in the cultures. For all

cultures, except for L. casei AC-8, Cetta maple sap led to

higher lactic acid production than the Pinnacle maple

sap. It is interesting to note that L. helveticus R0052 and

L. acidophilus AC-10 both consumed increased amounts

of sucrose when grown in maple sap-based media

(Table 2). On the other hand, Table 2 shows that the

three other strains (AC-3, R0240 and AC-8) generally

consumed comparable amounts of sucrose in sucrose-

based and in Pinnacle-based media, but much less in

Cetta-based medium. To understand what factors in

maple sap could contribute to increased growth, sucrose

consumption and lactic acid production, complementary

experiments of bacterial growth in maple sap-based media

were performed.

Indeed, the two sap samples, Cetta and Pinnacle, varied

in their chemical compositions (Table 1) and we attrib-

uted the variation in lactic acid production (no significant

difference was observed on bacterial growth) between the

two samples as due to cumulative effect of maple sap

components. To demonstrate the effect of oligosaccha-

rides in promoting lactobacilli growth and in increasing

their lactic acid production, we monitored the disappear-

ance of the oligosaccharides initially present in the Cetta

maple-based medium. After 16 h of incubation, LC-MS

analysis showed that L. helveticus R0052 and L. acidophi-

lus AC-10 consumed c. 70% of the d.p. 4 oligosaccharide

appearing at 19Æ2 min and 50% of the major d.p. 3 oligo-

saccharides shown in Fig. 2a (data not shown). Since Pin-

nacle sap contained less oligosaccharides, we thus

amended the Pinnacle sap-based medium with a mixture

of two d.p. 3 oligosaccharides, raffinose (1 g l)1) and

maltotriose (1 g l)1) and used it to grow L. acidophilus

AC-10. Because A600 nm was previously found to perfectly

correlate with CFU (data not shown), this parameter was

used to measure bacterial growth. The lactobacilli grew

approximately three times more in the Pinnacle medium

amended with raffinose and maltotriose, and produced

double the concentration of lactic acid than when grown

in the nonamended Pinnacle medium (Table 3). It should

be noted that the average lactic acid concentration

obtained in the nonamended Pinnacle sap-based medium

presented in Table 3 is different from the average lactic

acid concentration previously shown Fig. 3b (2Æ95 and

1Æ89 g l)1, respectively). Despite the fact that the 20-ml,

16-h culture was done similarly, the incubation times of

the precultures varied between the two experiments

explaining the variation observed.

d.p.

3d.

p. 3

d.p.

4 d.p.

4

d.p.

5

d.p.

3d.

p. 3

d.p.

4

d.p.

5

0 10 20 30 40 50 Time (min)0·00

0·25

0·50

0·75

1·00

1·25

x104

Intens·(a)

(b)

0 10 20 30 40 50 Time (min)0·00

0·25

0·50

0·75

1·00

1·25

x104

Intens·

Figure 1 Liquid-chromatography–mass

spectrometry profile of the oligosaccharides

with degree of polymerization (d.p.) from

3 to 5 in Cetta (a) and Pinnacle (b) maple

saps obtained by ESI-Qq-TOF mass

spectrometer using positive ionization mode.


ª 2008 The Authors


Discussion

In this study, we have investigated the use of maple sap

as a new feedstock for the production of probiotic lacto-

bacilli and lactic acid. The LAB strains were selected

based on their ability to yield high concentrations of via-

ble cells when grown on maple sap-based media (Fig. 3a).

Our results clearly demonstrate that (i) maple sap consti-

tutes a good carbon source; (ii) supported the growth of

L. rhamnosus AC-3, L. helveticus R0052, L. casei AC-8 and

L. acidophilus AC-10 at c. 9 log CFU ml)1 and (iii)

allowed L. helveticus R0052 and L. acidophilus AC-10 (the

best lactic acid producers of the study) to produce up to

5 g l)1 of lactic acid after 16 h of fermentation at 37�C in

Cetta sap-based medium (Fig. 3b). Figure 3 also shows

that L. rhamnosus AC-3 and L. casei AC-8 are good

candidates for production of probiotics because lactic

acid if produced would be in trace amounts. Viable bacte-

rial counts and lactic acid production were correlated

with significant sucrose consumption. These results show

that maple sap was favourable compared with other

sucrose-based feedstocks, such as beet juice and soymilk

0 10 20 30 40 50 Time (min)0·0

0·5

1·0

1·5

x105

0 10 20 30 40 50 Time (min)0·0

0·5

1·0

1·5

x105

0·0

0·5

1·0

1·5

x1051-Kestose spiked maple sap

Raffinose spiked maple sap

Maltotriose spiked maple sap

0 10 20 30 40 50 Time (min)

0 10 20 30 40 50 Time (min)0·0

0·5

1·0

1·5

(a)

(b)

(c)

(d)

x105

Figure 2 Extracted ion chromatograms

([M+Na]+ at m ⁄ z 527) of the trisaccharides

from Cetta maple sap (a), 1-kestose spiked

(b), raffinose spiked (c) and maltotriose spiked

(d) Cetta maple sap.



ª 2008 The Authors

containing 58 and 28 g l)1 of sucrose respectively, each of

which were shown to support the growth of lactobacilli

(8–9 log CFU ml)1) and the production of lactic acid

(2Æ3–5Æ3 g l)1) under similar culture conditions, i.e. batch

cultures conducted without pH control or lactic acid

removal (Garro et al. 1998; Yoon et al. 2005).

The increased lactic acid production and sucrose con-

sumption by L. helveticus R0052 and L. acidophilus AC-10

in Cetta maple sap compared with Pinnacle maple sap led

us to search for variations in their chemical compositions.

The maple sap samples came from two different regions

of Quebec and literature indicates that the location and

Table 2 Sucrose consumption (in g l)1) by various lactobacilli after 16 h of fermentation in purified sucrose-based medium or in maple sap-based

media*

Strains

Sucrose-based

medium

Cetta-based

medium

Pinnacle-based

medium

Lactobacillus rhamnosus AC-3 1Æ05 ± 0Æ13 0Æ00 ± 0Æ04 1Æ01 ± 0Æ16

Lactobacillus acidophilus R0240 2Æ41 ± 0Æ29 1Æ26 ± 0Æ09 2Æ47 ± 0Æ13

Lactobacillus helveticus R0052 3Æ42 ± 0Æ04 5Æ64 ± 0Æ11 4Æ41 ± 0Æ08

Lactobacillus casei AC-8 0Æ92 ± 0Æ11 0Æ48 ± 0Æ09 0Æ95 ± 0Æ24

Lactobacillus acidophilus AC-10 3Æ20 ± 0Æ10 5Æ95 ± 0Æ03 4Æ57 ± 0Æ12

*The initial concentrations of sucrose in sucrose-, Cetta- and Pinnacle-based media were respectively, 20Æ0, 19Æ0 and 16Æ5 g l)1. Glucose and fruc-

tose were totally consumed by all cultures.

7

8

9

10(a)

(b)

L. rhamnosus AC-3

L. acidophilusR0240

L. helveticusR0052

L. casei AC-8 L. acidophilus AC-10

log

CF

U m

l–1

0·00

0·83 0·92 0·75

0·35

1·98

4·94 4·82

0·00

1·20

1·98 1·89

0·00 0·00 0·000

2

4

6

L· rhamnosus AC-3

L· acidophilusR0240

L· helveticusR0052

L· casei AC-8 L· acidophilus AC-10

Lact

ic a

cid

prod

uced

(g

l–1)

Figure 3 Bacterial viable count (a) and lactic acid produced (b) by lactobacilli in sucrose-based (black), Cetta (white) or Pinnacle (grey) sap-based

media after 16 h of fermentation at 37�C. CFU, colony-forming units; L., Lactobacillus. Values indicate averages from two distinct cultures and

bars represent the SE.


ª 2008 The Authors


several other factors that include, the age of maple trees,

period and method of maple harvest, will largely influ-

ence sap composition in terms of sugars, minerals, phe-

nolic compounds, vitamins and organic acids (Morselli

1975; Kuentz et al. 1976; Stuckel and Low 1996). Despite

the fact that some differences between the two saps were

noted in the concentrations of nitrate, sulfate, sodium,

magnesium and manganese, the amendment of sap with

culture medium components such as phosphate, yeast

extract, veggietones pea, manganese and magnesium likely

rendered these differences insignificant. Also because

sucrose was always partially consumed by the lactobacilli

cultures, the difference in sucrose concentration does not

explain the difference in growth and lactic acid produc-

tion observed in the two sap samples, Cetta and Pinnacle.

However, the most significant difference observed

between the two maple sap samples was in their content

of oligosaccharides. Both maple saps revealed the presence

of oligosaccharides with a d.p. ranging from 3 to 5. We

found that Cetta sap that contained a higher content of

oligosaccharides, particularly trisaccharides, showed a

slightly higher bacterial growth and higher yield of lactic

acid than Pinnacle. For example, Cetta sap was found to

contain 1-kestose as one of the two major trisaccharides

as confirmed by comparison with a reference standard

(Fig. 2a,b). The second major trisaccharide was tentatively

identified as neokestose (Fig. 2a) based on previous

assignment made by Haq and Adams (1961) who also

reported neokestose as a major trisaccharide in maple

sap. Whereas the minor LC-MS peak with a r.t. at

30Æ1 min was tentatively identified as raffinose as con-

firmed by Porter et al. (1954) who reported the presence

of this sugar based on detailed structural analysis of the

trisaccharide in maple sap. However, more recently Bazi-

net et al. (2007) suggested the trisaccharide to be a malto-

triose based on HPLC ⁄ refractive index analysis alone.

Further confirmation to our assignments of the three tri-

saccharides was obtained by proper spiking of each ana-

lyte separately during HPLC analysis (Fig. 2b–d).

Literature reports indicated that raffinose-like oligosac-

charides could enhance the acidification rate and the pop-

ulation levels of strains of L. acidophilus and

Bifidobacterium lactis (Martınez-Villaluenga et al. 2005).

More recently, it was shown that also fructo-oligosaccha-

rides can enhance the production of various bacteriocins

by LAB (Chen et al. 2007) and the soybean fructo-oligo-

saccharides, inulin and raffinose, are able to enhance the

growth of various probiotic bacteria (Su et al. 2007). In

the present study, when the Pinnacle-based medium was

amended with the two trisaccharides, raffinose (1Æ0 g l)1)

and maltotriose (1Æ0 g l)1), L. acidophilus AC-10 grew

approximately three times faster and the lactic acid yield

increased from 2Æ95 g l)1 to 5Æ95 g l)1.

In conclusion, our results revealed that maple sap can

be considered as a remarkable renewable feedstock for

developing a nondairy drink with probiotic lactobacilli.

Among the strains tested, L. rhamnosus AC-3 and L. casei

AC-8 represented the best choices for the probiotic drink

owing to their high viable cells count and low lactic acid

production. Finally, maple sap-based media may serve as

a convenient substrate for significant lactic acid produc-

tion by L. helveticus R0052 and L. acidophilus AC-10

without pretreatments of maple sap.

Acknowledgements

The authors wish to thank Stephane Deschamps, Chan-

tale Beaulieu, Louise Paquet, Karine Trudel and Alain

Corriveau for their excellent technical assistance. They

also thank Punita Mehta for the revision of the

manuscript.

References

Altschul, S.F., Madden, T.L., Schaffer, A., Zhang, J., Zhang, Z.,

Miller, W. and Lipman, D.J. (1997) Gapped BLAST and

PSI-BLAST: a new generation of protein database search

programs. Nucleic Acids Res 25, 3389–3402.

ASTM D5291 (2007) Standard Test Methods for Instrumental

Determination of Carbon, Hydrogen, and Nitrogen. Ameri-

can Society for Testing and Materials International.

Bazinet, L., Gaudreau, H., Lavigne, D. and Martin, N. (2007)

Partial demineralization of maple sap by electrodialysis:

impact on syrup sensory and physicochemical characteris-

tics. J Sci Food Agricult 87, 1691–1698.

Chen, Y.S., Srionnual, S., Onda, T. and Yanagida, F. (2007)

Effects of prebiotic oligosaccharides and trehalose on

growth and production of bacteriocins by lactic acid bacte-

ria. Lett Appl Microbiol 45, 190–193.

De Man, J.C., Rogosa, M. and Sharpe, M.E. (1960) A medium

for the cultivation of lactobacilli. J Appl Bacteriol 23, 130–

135.

EPA Method 415Æ1 (1999) Total Organic Carbon in Water. US

Environmental Protection Agency.

EPA Method 6020 (1994) Inductively Coupled Plasma – Mass

Spectrometry. US Environmental Protection Agency.

Table 3 Growth of Lactobacillus acidophilus AC-10 as measured by

the A600 nm and production of lactic acid in Pinnacle-based medium

and Pinnacle-based medium amended with raffinose (1 g l)1) and

maltotriose (1 g l)1) after 16 h of incubation at 37�C

Medium A600 nm Lactic acid (g l)1)

Pinnacle sap 0Æ58 ± 0Æ058 2Æ95 ± 0Æ102

Amended Pinnacle sap 1Æ60 ± 0Æ066 5Æ95 ± 0Æ260

Values indicate averages from two distinct cultures.



ª 2008 The Authors

Garro, M.S., Font de Valdez, G., Oliver, G. and Savoy de Gi-

ori, G. (1998) Growth characteristics and fermentation

products of Streptococcus salivarius subs. thermophilus, Lac-

tobacillus casei and L. fermentum in soymilk. Z Lebensm

Unters Forsch A 206, 72–75.

Haq, S. and Adams, G.A. (1961) Oligosaccharides from the

sap of sugar maple (Acer saccharum marsh). Can J Chem

39, 1165–1170.

Kharas, G.B., Sanchez-Riera, F. and Severson, D.K. (1994)

Polymers of lactic acid. In Plastics from Microbes: Microbial

Synthesis of Polymers and Polymer Precursors ed. Mobley,

D.P. pp. 93–137. Munich, Germany: Carl Hanser Verlag.

Kotzamanidis, C., Roukas, T. and Skaracis, G. (2002) Optimi-

zation of lactic acid production from beet molasses by Lac-

tobacillus delbrueckii NCIMB 8130. World J Microbiol

Biotechnol 18, 441–448.

Kuentz, A., Simard, R.E., Zee, J.A. and Desmarais, M. (1976)

Comparison of two methods for analysis of minerals in

maple syrups. Can Inst Food Sci Technol J 9, 147–150.

Martınez-Villaluenga, C., Frıas, J., Vidal-Valverde, C. and

Gomez, R. (2005) Raffinose family of oligosaccharides

from lupin seeds as prebiotics: application in dairy prod-

ucts. J Food Protect 68, 1246–1252.

Mercier, P., Yerushalmi, L., Rouleau, D. and Dochain, D.

(1992) Kinetics of lactic acid fermentation on glucose and

corn by Lactobacillus amylophilus. J Chem Technol Biotech-

nol 55, 111–121.

Mirasol, F. (1999) Lactic acid prices falter as competition

toughen. Chem Market Reporter 255, 16.

Morin, A., Heckert, M., Poitras, E., Leblanc, D., Brion, F. and

Moresoli, C. (1995) Exopolysaccharide production on low-

grade maple sap by Enterobacter agglomerans grown in

small-scale bioreactors. J Appl Bacteriol 79, 30–37.

Morselli, M.F. (1975) Nutritional value of maple syrup. Natl

Maple Syrup Digest 14, 12.

Patil, S.S., Kadam, S.R., Patil, S.S., Bastawde, K.B., Khire, J.M.

and Gokhale, D.V. (2006) Production of lactic acid and

fructose from media with cane sugar using mutant of

Lactobacillus delbrueckii NCIM 2365. Lett Appl Microbiol

43, 53–57.

Porter, W.L., Hoban, N. and Willits, C.O. (1954) Contribution

to the carbohydrate chemistry of maple sap and syrup.

Food Res 19, 597–602.

Sleator, R.D. and Hill, C. (2008) New frontiers in probiotic

research. Lett Appl Microbiol 46, 143–147.

Stuckel, J.G. and Low, N.H. (1996) The chemical composition

of 80 pure maple syrup samples produced in North Amer-

ica. Food Res Int 29, 373–379.

Su, P., Henricksson, A. and Mitchell, A. (2007) Prebiotics

enhance survival and prolong the retention period of spe-

cific probiotic inocula in an in vivo murine model. J Appl

Microbiol 103, 2392–2400.

Timbuntam, W., Sriroth, K. and Tokiwa, Y. (2006) Lactic acid

production from sugar-cane juice by a newly isolated Lac-

tobacillus sp. Biotechnol Lett 28, 811–814.

Vickroy, T.B. (1985) Lactic acid. In Comprehensive Biotechnol-

ogy: The Principles, Applications and Regulations of Biotech-

nology in Industry, Agriculture and Medicine ed. Moo-

Young, M. pp. 761–776. New York, USA: Pergamon Press.

Wee, Y.-J., Kim, J.-N. and Ryu, H.-W. (2006) Biotechnological

production of lactic acid and its recent applications. Food

Technol Biotechnol 44, 163–172.

Yezza, A., Halasz, A., Levadoux, W. and Hawari, J. (2007) Pro-

duction of poly-b-hydroxybutyrate (PHB) by Alcaligenes

latus from maple sap. Appl Microbiol Biotechnol 77, 269–

274.

Yoon, K., Woodams, E. and Hang, Y. (2005) Fermentation of

beet juice by beneficial lactic acid bacteria. Lebensm Wiss

U Technol 38, 73–75.


ª 2008 The Authors


Documents

Cochu LettApplMicrobiol 2008