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ng dng v mi trng Vi sinh vt aem.asm.org1. doi: 10,1128 /
AEM.71.12.8383 8389,2005 Appl. Mi
trng. Microbiol. - Thng 12 nm 2005 vol. 71 khng c. 12
8.3838389
Trong Vitro Xc nh tnh Prebiotic oligosaccharides ngun gc t sn
xut Orange Juice By-Sn phm Sui1. 2. 3. 4. 5. 6.
K. Manderson 1 , M. Pinart 1 , KM Tuohy 1 , Chng ti Grace 1 , AT
Hotchkiss 2 , W. Widmer 3 ,
7. MP Yadhav 2 , 8. GR Gibson 1 v 9. RA Rastall 1 , *
+ Tc gi chi nhanh Trng Khoa hc Sinh hc Thc phm, i hc Reading, PO
Box 226, Whiteknights, c RG6 6AP, Vng Quc Anh 2. 2 ng khu vc Trung
tm Nghin cu, Dch v Nghin cu Nng nghip, B Nng nghip M, Wyndmoor,
Pennsylvania 19038 3. 3 Citrus v Phng th nghim Subtropical Sn phm,
dch v nghin cu nng nghip, B Nng nghip M, 600 Avenue S, NW, Winter
Haven, Florida 338811.1
Mc tip theo
TM TTTi sn ln men ca oligosaccharides c ngun gc t pectin v cam c
nh gi trong nn vn ha pha trn ca vi khun phn. V cam phn
oligosaccharide c glucose trong rhamnogalacturonan v
oligosaccharides pectic xylogalacturonan. Hai mi bn ting ng h, nhit
v pH c kim sot, khng c khuy k kh phn l nn vn ha c s dng xc nh hiu
qu m cc oligosaccharides bt ngun t cc sn phm cam c trn cc thnh phn
ca h vi sinh vt phn. Cc hiu ng c nh gi thng qua lai ti ch hunh
quang xc nh nhng thay i trong cc qun th vi khun, ln men sn phm cui
cng c phn tch bng sc k lng hiu nng cao nh gi nng acid bo chui ngn,
v sau , mt ch s prebiotic (PI) c xc nh. Oligosaccharides pectic
(POS) c th tng cc s bifidobacterial v Eubacterium rectale , mc d kt
qu l mt ch s tin sinh thp hn t qu trnh trao i cht
fructo-oligosaccharide.Orange sut phn chiu vn duy tr s tng trng ca
hu ht cc qun th vi khun v a ra mt PI tng t nh tinh bt ha tan. Qu
trnh ln men ca POS dn n s gia tng s rectale Eubacterium v ng thi
tng sn xut butyrate. Trong kt lun, nghin cu ny ch ra rng POS c th c
mt tc dng c li trn h vi phn, tuy nhin, mt hiu ng c in tin sinh khng
c tm thy. S gia tng dn s Eubacterium rectale c tm thy, v mc
butyrate tng ln, l li ch tim nng cho cc my ch. Khi nim v sc khe ng
rut iu ch thng qua ch n ung khng phi l mi v ngy tr li vo u ca th k
20. Tuy nhin, n ch l gn y
rng l do c bn m thanh khoa hc c xut v iu tra. Ba cng c iu ch vi
xut hin ( 40 ): vic b sung cc vi sinh vt sng ngoi sinh thc phm (tc
l, cc ch phm sinh hc), kch thch chn lc ca s tng trng v hot ng ca vi
sinh vt bn a mang li li ch cho ng rut (tc l, prebiotics), v s kt hp
ca c hai phng php tip cn (tc l, synbiotics). Tt c ba phng php tip
cn c gng tng s lng vi khun c xem l tch cc cho sc khe tiu ha ca con
ngi, thng l lactobacilli v bifidobacteria. Prebiotics c nh ngha l
"thnh phn thc phm khng tiu ha c chn lc kch thch mt s hn ch ca vi
khun i trng, ci thin sc khe ch nh" ( 12 ). Bifidobacteria v
lactobacilli c gi trc tip c ch s tng trng ca vi khun gy bnh, chng
hn nh mt s loi clostridia (v d, Clostridium difficile , C.
perfringens ) v gy bnh Enterobacteriaceae , thng qua vic sn xut cc
axit bo chui ngn, lm gim pH i trng, sn xut cc hp cht khng khun, v /
hoc cnh tranh cho nn tng trng v cc trang web kt dnh ( 13 , 23 , 27
). Tnh cht bifidogenic ca mt lot cc prebiotics c xc nhn trong c th
trong cc nghin cu thc n ca con ngi ( 11 , 37 ). Nhng prebiotics bao
gm inulin, fructo-oligosaccharides (FOS), galacto-oligosaccharides,
v lactulose ( 42 , 43 , 25 , 44 ). Mc ch ca cng vic ny l nh gi cc
tnh cht tin sinh hc cc oligosaccharides chit xut t v cc sn phm sn
xut nc cam. Citrus d lng cn li sau khi khai thc nc tri cy i din cho
khong mt na khi lng tri cy m t, v khong mt na d lng ny l v ( 2 ).
Hn 2.000.000 tn bt vin bt cy c mi c sn xut hng nm cho thc n gia sc
t d lng ch bin cy c mi, m l mt sn phm gi tr thp. Pectin, mt sn phm
gi tr cao hn, c sn xut t v cam qut, nhng nhu cu th trng cho pectin
l thp so vi pectin tim nng c th c sn xut t cc ngun cung cp trn ton
th gii ca d lng ch bin cy c mi. Oligosaccharides pectic (POS) c sn
xut t pectin thng mi trong mt l phn ng mng t bo enzyme ( 30 ) v sau
nh gi tnh cht tin sinh ( 29 ).Cc oligosaccharides pectic ny c mt
tim nng thp tin sinh so vi FOS, mc d h c chn lc ln men hn l cc
pectin cha m ( 29 ). Oligosaccharides pectic colonocytes cng c bo v
chng li verocytotoxins Escherichia coli ( 32 ) v kch thch apoptosis
trong t bo ung th tuyn ngi i trng ( 31 ). y, chng ti nhm mc ch tm
thy mt cch chi ph-hiu qu hn sn xut oligosaccharides prebiotic
pectic trc tip t v cam sut phn chiu (OA).Fructo-oligosaccharides,
cc prebiotics rng ri nht nghin cu v cc
nh lnh o hin ti th trng, c chn nh mt iu khin tch cc cho nghin cu
qu trnh ln men phn trong ng nghim, trong khi tinh bt, mt
carbohydrate nonprebiotic, c s dng nh mt iu khin khng chuyn. trc Mc
sau
TI LIU V PHNG PHP xc nh tnh chn lc ca qu trnh ln men ca vt liu
kim tra so vi FOS v tinh bt, kim sot pH, khuy ng nn vn ha hng lot
tim slurries con ngi phn c s dng. Vi khun c thc hin bng cch s dng
hunh quang lai ti ch vi cc u d oligonucleotide nhm mc tiu c th 16S
rRNA khu vc ca cc vi sinh vt th nghim. Axit bo chui ngn (SCFA), l
sn phm cui cng ca qu trnh ln men vi khun, c phn tch bng sc k lng
hiu nng cao (HPLC). Vt liu. Mi ho cht u c mua t BDH (Poole, Dorset,
Vng Quc Anh), ngoi tr cho MgSO 4 7H 2 O v l -cysteine HCl, thu c t
Sigma (Poole, Dorset, Vng Quc Anh). Fructo-oligosaccharides
(Raftilose P95) thu c t Orafti (Tienen, B), v tinh bt l t Sigma
(Poole, Dorset, Vng Quc Anh).Ti co Hamlin cam sut phn chiu (phn
trng ca v cam) c ly t mt b x l nc tri cy h cam qut Florida thng mi
v ng lnh ngay lp tc n -20 C cho n khi s dng. Khai thc
oligosaccharides pectic. Orange sut phn chiu c s dng trch xut cc
oligosaccharides pectic. POS sau c iu tr bng cch lc nano loi b
nitrat vt qu ( 14 ). Orange sut phn chiu c pha trn vi mt trng lng
bng nhau ca nc kh Ion ha v sau lm t xay bng cch s dng mt b x l thc
phm m hnh Comitrol 1700 (Urschel phng th nghim, Valparaiso, IN) dn
tt vi mt phn b kch thc ht trung bnh di 0,5 mm.Cc mt t bn sut phn
chiu (9 kg) c cha 4,5 sut phn chiu kg c thm vo mt chic thng cha 57
lt dung dch HNO 3 (pH 1.5) cung cp cho mt nng 1 g peel/12.5 ml HNO
3 , v c iu chnh pH n 1,5 tp trung HNO 3 . B nui cng c sau c khuy
trong 30 pht, v bn c bm vo mt thanh trng (MicroThermics HTSL
Lab-25, Raleigh, NC) cu hnh vi 7,75 mm, bn trong c ng knh ng trao i
nhit (nng khi lng 400 ml, 400 ml gi, lm mt khi lng 400 ml). Thanh
trng c iu chnh n mt nhit nng 120 C, p lc tr li ca 310 n 380 kPa (
ngn chn si), v t l lu lng t 1.200 ml / pht vi nc trc khi bm bn. Sau
khi chuyn i bn, nhit u vo hi nc c tng ln mt cht cho bn nhit 120 C.
Nh , bn
sau c un nng n 120 C trong vng 20 giy, c t chc nhit 20 giy, v
sau lm lnh n 14 C trong vng 20 giy. Nhit th trong lp rp ng gi t hn
4 C. Bn c bm qua mt hon thin tuabin Pony (Mecat M, Orlando, FL), n
v lc c hc lin tc vi mt mn hnh mt li 53 micron, trong d lng v khng
ha tan c tch ra t cc phng tin khai thc. D lng v khng ha tan c ra
sch bng cch s dng 1 lng nc v kch ng trong 30 pht.X nc c thng qua mt
ln na thng qua vic hon thin turbo, ni cht lng c thu thp v d lng
khng ha tan c loi b. Isopropyl alcohol (IPA) sau c thm vo chit xut
cht lng a nng ln n 70% IPA, v gii php c lu tr trong 12 h ti 5 C kt
ta pectin. Gii php ny sau c bm qua b lc turbo tch kt ta v kt ta
khng tan trong cn c ra sch hai ln na vi 2 phn trn mt n v trng lng
ca IPA 70% v mt ln vi 100% IPA. Cc phn khng tan trong cn sau sy kh
trong l chn khng. Ni trn mt ha tan trong ru c trung ha vi dung dch
NaOH pH gia 6.5 v 7.5, v IPA bc hi 35 C di p lc gim bng cch s dng
Buchi (Brinkman, Westbury, NY) thit b bay hi quay 20 lt. Ni trn mt
dung dch nc c ly tm 9639 x g loi b mt kt ta my hnh thnh sau khi iu
chnh pH. Ni trn mt c vo mt C 18 ct chit pha rn (Altex, Harbor City,
CA) tch cc cc t cc phn phn on khng cc. Cc phn cc c cha POS c ng kh,
v cc phn khng cc c eluted vi ethanol. Lc nano oligosaccharides
pectic. ngi thu thu phn cc, diafiltration lin tc c thc hin bng cch
s dng 1.000 phn t trng lng ct DS-5-DL mng (Osmonics Desal). Cc phn
cc (60 g) c ha tan trong H 2 O (2 lt) cung cp cho hm lng ng tng cng
3%. Gii php ny c lu hnh trong h thng lc trong 10 pht trc khi nng
thc n ban u c xc nh. Hai song song cc t bo lc p sut cao c s dng, vi
tng din tch mng 81 cm 2 . u vo dng chy ca nc trong bn cha thc n chn
nui c kt hp vi tc dng chy trn ngp nh vy m khi lng khng thay i. p
sut v nhit c thit lp ti 20 10 6 Pa v 60 C, tng ng. Thm nhp vo cc
php o thng lng v retentate v thm nhp vo mu c thc hin mi h 2. Th
nghim c hon thnh sau 6 lt tch ly trn ngp thu c. Retentate c cha v
cam POS c ng kh. Trc v sau khi lc nano, ni dung nitrat ca mu c xc
nh bng sc k ion bng cch s dng mt Dionex AS14A ct, mt ct bo v
AG14A,
v mM 3,5 Na 2 CO 3 -1,0 mM NaHCO3 eluent tc 1.2 dng chy ml /
pht. Hiu sut cao sc k trao i anion (HPAEC) Orange v POS c tch ra hn
100 min 25 500 oxalat kali mM phi tuyn, pH 6, gradient bng cch s
dng mt ct PA1 CarboPac (4 250 mm; Dionex Corp) v mt CarboPac PA bo
v ct trn mt h thng Dionex 4500i ( 18 , 19 ). Polygalacturonic axit
thy phn trong mt ni hp 40 pht 125 C trong dung dch axt nitric. Tiu
chun oligosaccharide c c lp bi d bi HPLC ( 17 , 20 , 21 ) hoc thu c
t Sigma. Xt nghim carbohydrate. Cc ni dung carbohydrate tng s POS c
xc nh theo phng php axit phenolsulfuric ( 6 ). Hm lng acid
galacturonic c xc nh bng mt phng php so mu ( 8 ), vi mt s sa i ( 48
). Phn tch tinh bt c thc hin bng cch s dng cc Megazyme (Bray,
Ireland) kit phn tch tng tinh bt. B ti liu ny s dng chu nhit
amylase v amyloglucosidase cho thy phn tinh bt v ng glucose oxidase
/ peroxidase pht hin glucose. Monosaccharide phn tch. Methanolysis
POS c thc hin bng cch thm 100 acetate methyl ml v 400 ml 1,5 M HCl
trong methanol mu 1-mg (sy kh 50 C l chn khng) v un nng 85 C trong
17 h. Sau khi cc mu c lm lnh ti nhit phng, 5 git n butanol c b sung
v cc mu bc hi n kh theo mt dng kh nit. thy phn cc glycosides methyl
hnh thnh bi methanolysis, 0,5 ml 2 M trifluoroacetic axit c b sung,
v cc mu c gia nhit 121 C trong 1 gi. Cc mu c lm lnh ti nhit phng v
bc hi n kh, v 0,5 ml methanol c thm vo v bc hi n kh ba ln.Nc c thm
vo mi mu, v h c lc (0,25 micromet) trc khi n tim. Inositol c thm vo
mi mu trc khi methanolysis nh mt tiu chun ni b. Thy phn c phn tch
ni dung monosaccharide trung lp bng cch pht hin amperometric
HPAEC-xung (PAD) bng cch s dng mt Dionex ICS2500 h thng bao gm mt
CarboPac PA20 ct v ct bo v, 50 EG eluent my pht in m sn xut KOH 10
isocratic mM in thoi di ng giai on, lin tc ti sinh ct anion ci by,
mt bc s 50 my bm hot ng mc 0,5 ml / min, mt ED50 in my d s dng cc
dng sng tim nng tng gp bn ln, v mt mu t ng AS50 vi mt ngn nhit (30
C ct nng). Ni dung monosaccharide c tnh axit c xc nh vi mt h thng
Dionex500 DX, trong bao gm mt my bm gradient GP50 (0,5 ml / pht),
mt ct CarboPac PA20 ct v bo v, mt my d ED40 in (in cc lm vic vng v
in cc tham chiu pH), mt LC25 sc k l (30 C), kh nn PC10
iu khin (postcolumn Ngoi ra 500 mM NaOH), v mt mu t ng AS3500.
Giai on di ng bao gm ca mM isocratic 10 CH 3 COONa, 1 mM NaOH
eluant cho 10 pht, v sau mt gradient tuyn tnh ca 100 n 130 mM CH 3
COONa trong 100 mM NaOH sau 20 min. Phn l nn vn ha qu trnh ln men.
Bn tu ln men thy tinh 280 ml c lp y vi 180 ml ca mt mi trng v trng
prereduced c cha 2 g / nc peptone lt, 2 g / lt chit xut t nm men,
0,1 g / lt NaCl, 0,04 g / lt K 2 HPO 4 , 0,04 g / lt KH 2 PO 4 ,
0,01 g / lt MgSO 4 7H 2 O, 0,01 g / lt CaCl 2 2H 2O, 2 g / lt NaHCO
3 , 0,5 g / lt l -cysteine HCl, 0,5 g / lt mt mui, 4 ml resazurin
0,05 g / lt, 10 ml vitamin K 1 , 2 ml Tween 80, v 10 ml hemin. Mi
trng c iu chnh pH 7.0 v lin tc sparged vi oxy nit (15 ml / pht).
10% (wt / vol) phn bn t ba nh ti tr khe mnh c chun b bng cch s dng
prereduced 0,1 M phosphate m nc mui sinh l (pH 7.0) v sau trn ln
trong mt stomacher (m hnh 6041, Seward khoa hc, Vng quc Anh) cho
120. Mi tu c tim 20 ml bn v g 2 carbohydrate (POS,
fructo-oligosaccharides, cam sut phn chiu, hoc tinh bt). Mu phn c
ly t mt con c v hai tnh nguyn vin n c tui trung bnh l 24 nm ngi
khng dng thuc khng sinh trong t nht 6 thng trc khi cung cp cc mu v
nhng ngi khng c tin s ri lon tiu ha. Fermentors c duy tr 37 C, v mu
bn sao c g b sau khi 0, 5, 10, v 24 gi m s vi khun v SCFA phn tch
(46 , 29 ). Qu trnh ln men c chy trn ba dp ring bit vi nh ti tr khc
nhau. Thay i hunh quang trong lai ti ch. trong qun th vi khun phn
ngi c nh gi bi hunh quang lai ti ch vi cc u d 16S rRNA. Cc u d 16S
rRNA, c th cho cc lp hc ch yu ca h vi sinh ng rut (Bacteroides,
bifidobacteria,: Clostridium histolyticum , Eubacterium rectale v
lactobacilli / enterococci), c sn xut v c dn nhn nh du hunh quang.
Cc u d s dng Bif164 ( 26 ), Bac303 ( 28 ), Erec482 ( 10 ), His150 (
10 ), v Lab158 ( 16 ). Cc axit nucleic vt 4'6-diamidino-2
phenylindole (DAPI) c s dng m tng s vi khun. Mu qu trnh ln men c
pha long v c nh qua m trong Paraformaldehyde 4% 4 C trong mt t l
1:03 ca mu Paraformaldehyde 4% (vol / vol). Nhng t bo ny sau ra sch
vi nc mui phosphate-m (0,1 M, pH 7,0), resuspended ml 150 phosphate
m nc mui cng vi 150 ethanol ml, v c lu tr -20 C ti thiu l 1 gi.
Treo t bo c sau thm vo hn hp lai bao gm 4,78 g / lt Tris-HCl (30
mM), 79,69 g / lt NaCl (1,36 M), 15 ml 10% sodium dodecyl sulfate
gii php (pH 7.2), v 4 M HCl 15 ml v lc thng qua mt b lc 0,2 micron
(Millipore, Watford, Vng Quc Anh). Lai to c thc hin nhit thch hp
cho cc u d. Sau , hn hp lai chn khng lc
(Millipore, Watford, Vng Quc Anh), v b lc c t trn mt knh hin vi
v kim tra bng cch s dng knh hin vi hunh quang, chng hn cc nhm vi
khun c th c lit k ( 36 ). . Phn tch acid bo chui ngn mt millilliter
mu c g b t cc fermentors vn ha hng lot v ly tm 13.000 g trong 5
pht, v 20 ml c tim vo mt h thng HPLC (LaChrom L7100; Merck, Poole,
Vng Quc Anh) trang b mt my d ch s khc x v mt vi phun t ng. Cc ct c
s dng l mt ion-loi tr Aminex HPX-87H ct (7,8 bng 300 mm; Bio-Rad,
Watford, Vng Quc Anh) duy tr mc 50 C. Eluent l 0,005 mM H 2 SO 4 ti
mt tc dng chy 0,6 ml / pht. D liu c thu thp bng cch s dng JCL6000
phn mm (Jones sc k, Wales, Vng Quc Anh). nh lng ca cc mu c thc hin
bng cch s dng cc ng cong hiu chun axetic, axit propionic, butyric,
lactic nng gia 0,5 v 100 mM, v kt qu c th hin trong mmol / lt ( 36
). Cc th nghim c lp i lp li trong ba ln. Phn tch thng k. Trong trng
hp s khc bit trong phng php iu tr c tm thy, sinh vin hai mu th
nghim t c thc hin gia cc mu. S khc bit c coi l ng k khi P
