TAP CHI SINH HQC. 2013, 35(1): 67-73
N G H I E N ClTU T H U N H A N X V L O O L I G O S A C C H A R I D E (XOS) T t r C A M G A O B A N G C O N G N G H f E N Z Y M E
Tran Thi Nhung', Pham Thj Thu Phuong', Nguyen Thuy HudngS Nguyen Thj Mai Phuang'*
'Vifn Cdng nghf sinh hgc, Vifn Han lam KH & CN Vift Nam, *phuong_nguyen_99@ yahoo.com
^Vifn Cdng nghifp thyc phim
TOM T A T : Cdm g^o Id phy phim cua qud trinh xay xdt gao vd rIt gidu hydratcarbon, djc bifl Id xylan.
Vi thi. ngudn nguySn lifu ndy da va dang dugc sir dyng de sdn xult chit xa hda tan Xylooligosaccharidc (XOS). Mgt s6 vi khuin phd bien trong rugt kit nhu Bifidobacteria vd Lactobacillus co till sir dunjj XOS nhu nguon ca chdt. Thj trudng cho XOS dang ngdy cdng hip dan do nhung lgi the ve cac tinh chit sinh hgc vd cdng nghf so vdi cdc oligosaccharide pho bien khac nhu fructooligosaccharide (FOS) hay galactooligosaccharide (GOS). XOS co thi san xult tir cam gao sit dyng cong nghf hoa hgc hoac cong nghe enzyme. Thiiy phdn cdm gao sir dyng P-1.4-xylanase Id phuong phdp thudng dugc lya chgn de san xult XOS tir cam gao. Hien t^i, Viet Nam vdn dang thilu mgt cong nghf san xult XOS tir cam gao co dg s^ch cao va an todn thyc phdm. Bdi bdo nay trinh bay nhiing ket qud nghien ciru mdi ve thu nhan XOS tir cdm gao sir dung cdng nghf da enzyme than thifn vdi moi trudng. Chi phdm XOS co dg sach tdi 81,4%
da dugc thu nhan blng each thuy phan cdm gao d3 tien xir ly vdi Ultrafio L xylanasc 0,6% ciia hang Novozyme d pH 7,0 tai 50°C trong dem phosphate buffer 100 niM trong 15 gia Day la mgt cong nghe thich hgp dl san xult XOS tir cam gao d Viet Nam.
Tir khoa: Bifidobacteria, Lactobacillus, cam gao, xylanasc, xylooligosaccharidc (XOS).
MO DAU
Vift Nam Id mdt trong nhung nude xuat khiu gao Idn nhIt thi gidi. Theo Bd Ndng nghifp vd Phdt trien ndng thdn, lugng gao xuat khIu nam 2011 dat khoang 7,5 trifu tin. Vi thi lugng cdm gao, phu phdm ciia qua trinh xay xdt gao cung rIt Idn. Cam gao cd gid tri dinh dudng cao vdi thdnh phIn hydratcacbon chiem tir 38,7- 44,3%, trong dd, chit ylu la xylan nen cung la mdt ngudn nguyen lieu tdt cho sdn xuat chdt xa hda tan xylooligosaccharidc (XOS).
XOS la cdc olygomer chira tir 2-7 gdc dudng xylose. XOS cd thi dugc sir dung bdi ca vi khudn Bifidobacteria vd mgt sd Lactobacillus, la nhthig vi khudn phd biln trong rugt kit ciia ngudi [4, 6, 8, 11, 14]. XOS cd nhilu uu vift hon cdc oligosaccharide khde nhu fmctooligosaccharide (FOS) hay galactooligosaccharide (GOS) d cd khia caiih lgi ich sire khde vd cdc dac tinh lien quan den cdng nghf [1]. Vi thi, thj trudng thuong mai cho san phim ndy Id rIt trien vgng [1, 3, 7,9].
Hifn nay, cd hai hudng cdng nghf chinh de thu nhan XOS tir cdm gao la sir dyng cdng nghf hda hgc ho^c cdng nghf enzyme [2, 10, 15].
Hudng thu nhan XOS bang cdng nghf enzym^
dugc quan tdm nhieu do tinh chat than thif n mdi trudng vd dg an todn sdn phdm ciia nd. Qud trinh thiiy phdn cdm gao tao XOS blng enzyme dugc thyc hifn bdi endo P-1-4 xylanase.
6 Vift Nam, vifc nghien cim san xult cdc oligosaccharide tir cam gao gan nhu chua dugc nghien cim. Cd the ndi ring mac dii cam gao la ngudn nguyen lieu gidu xylan nhung den nay chimg ta vdn chua cd cdng nghf phii hgp vd hifu qua de thu dugc XOS cd chat lugng cao.
Bdi bdo ndy trinh bdy cdc ket qud nghien cim mdi ciia chimg tdi vl thu nhan XOS tir cam gao sir dyng cdng nghf enzyme thdn thifn vdi mdi trudng.
VAT LI$U VA PHU^ONG PHAP NGHIEN CUU Cdm gao dugc thu mua tir cdc nhd may vd CO sd xay xdt gao cd uy tin ciia Hd Ngi. Cdm gao sau dd dugc xir ly Idm gidu xylan blng cdc enzyme protease vd a-amylase.
Enzyme Ultraflo L (xylanase) mua tir hang Novozyme (Dan Mach). Cdc hda chit cdn lai dlu dat mire tinh sach phdn tich
Djnh lugrng xylose vdXOS
Tran Thi Nhung, Pham Thi Thu Phuong, Nguyen Thuy Huong, Nguyen Thi Mai Phuong Dya vdo phuong phdp quang phd dl djnh
lugng xylose tdng sd cd trong mlu nghien ciiu sau khi xir ly vdi axit HCl, tir dd tinh dugc hdm lugng XOS dya vdo chdt chudn (Wako), XOS dugc xir ly vdi HCl 1,3 M trong I gid d 100"C de thiiy phan hoan toan xylan thanh xylose. Sau khi trung hda vdi NaOH 1,3 M, mlu nghien ciru dugc ly tdm vd thu djch ndi. Hdm lugng xylose trong dung djch dugc xdc djnh bang Kit D-xylose (Megazyme).
Sdc ky ldp mdng djnh tinh dudng XOS XOS dugc djnh tinli tren bdn sic ky ldp mdng (TLC) silicagel 60 F254 (Merck 1.05554;
20 X 20 cm) sir dyng hf dung mdi phdn tdch n- butanol:axit acetic: HiO vdi ty If 3:1:1. Ban sic ky dugc hifn mdu blng aniline trong hdn hgp axit phthaiic vd n-butanoi bdo hda. Cdc vach dudng hien mdu nau sau khi chay sic ky khodng 90 phut va phun thudc hien mdu.
Xdc dinh hogt tinh xylanase (Endo-l,4-f3- xylanase-EC 3.2.1.8)
Hoat tinh enzyme dugc xdc djnh dya tren vifc do san pham xylose tao thanh khi thiiy phdn 1% xylan oat-spelts (Sigma) trong dem phosphate natri pH 7,0 d 50"C trong 30 phiit.
Mdt don vj hoat tinh enzyme la sd pmol xylose dugc giai phdng trong dieu kien phan irng.
Xylose tao thanh dugc djnh lugng blng Kit do D-xylose. Hoat tinh rieng ciia che pham enzyme la sd don vi enzyme/ing protein chi phdm.
Xdc dinh protein
Ham lugng protein trong mdu nghien cim dugc xac djnh thdng qua phan img mdu vdi thudc thir Bradford sir dyng albumin huylt thanh bd (BSA) lam chit chuln.
KET QUA VA THAO LUAN
Nghien cuu dieu kifn toi uu cho thiiy phan cam gao ciia xylanasc
Hifn nay, tren thj trudng dang thuong m^i mdt sd san pham enzyme xylanase cdng nghifp khde nhau cd ngudn gdc vi sinh vat dl phyc vy cho cdng nghifp chi biln thyc phim nhu sdn
xult bia, bdnh mi hay cho chdn nudi. Vi dy, cdc chi phdm Ultraflo L (Novozyme, Dan Mach), Porzyme (Canada) hay cua mgt sd cdng ty hda chit Trung Qudc. Ket qua phdn tich hoat dO cua 6 mlu enzyme cdng nghifp gdm Porzyme (dang bOt, Canada), Pentopan (d^ng bgt, Trung Qudc), Trichoderma (dang bgt, Trung qudc), Ultraflo L (dang djch, Novozyme) vd Aspergillus (dang bgt, Trung qudc) cho thIy ring hoat dg rieng ciia Ultraflo L (Novozyme) Id cao nhIt, dat 12,68 U/mg protein, cao hon khoang 10 lln so vdi enzyme Aspergillus vd cao hon nhilu lln so vdi cdc enzyme cdn l^i (sd lifu khdng trinh bdy d ddy). Trong s6 cdc enzyme cdng nghifp cd hoat tinh thiiy phdn malt d l sdn xult bia, Ultraflo L cd hdm lugng xylanase cao vd khdng chira beta-glucanase. Vi the, chiing tdi dd sir dyng Ultraflo L Id ngudn xylanase cho qud trinh sail xult XOS tir cdm gao. Dc thu nhan dugc XOS cd hifu suit cao, chung tdi dd tiln hdnh nghien cim cdc dilu kien tdi thich cho qud trinh thiiy phan ndy.
Anh hudng ctia pH den kha ndng thuy phdn cdm gao ciia xylanasc
Dung djch cdm gao trong dfm phosphate natri 100 mM, d cdc pH 5,0; 6,0; 7,0 dugc bl sung xylanase vdi ndng dg 0,3% vd tiln hdnh thuy phdn d 50°C trong khoang thdi gian 21 gid.
Hdm lugng dudng xylose sau thiiy phdn dugc kiem tra de ddnh gid khd ndng thiiy phdn ciia enzyme. Kit qua thu dugc cho thdy, sau khi thiiy phdn d pH 7,0 trong 21 gid, hdm lugng dudng xylose d?t gid trj AA340 blng 0,660, tuong irng vdi ham lugng XOS Id 6,8%, cao hon ddng ke so vdi gid trj nay thu dugc khi thiiy phan tai dieu kifn pH 6,0 (5,46%) vd cao hem tdi gan 50% so vdi thiiy phan tai dieu kifn pH 5,0 (4,9%) (bang I). Ket qud ndy cung cd thi ly gidi dugc Id do xylanase trong Ultraflo L Id enzyme cd ngudn gdc tir vi khudn (Bacillus) nen viing pH tdi thich cho hoat dgng ciia nd d gin cac gid trj pH trung tinh, khde vdi cdc xylanase cd ngudn gdc tir nlm (Aspergillus), thudng c6 pH tdi thich cho hoat dgng d vimg axit (pH 5,0) [12].
TAP CHI SINH HQC, 2013, 35(1) 67-73 Bang 1. Anh hudng cua pH din kha ndng thiiy phdn cdm g?io ciia xylanase
Thdi gian (gid) 0 3 21
XOS (%) pH5,0
0,007 4,59 4,90
pH6,0 0,007 5.05 5.46
pH7.0 0,005 5,46 6,80 Bdng 2. Anh hudng ciia nhift dg din kha ndng thiiy phdn cdm g^io cua xylanase
Thdi gian (gid) 0 3 21
XOS (%) 37"C
0,002 1,71 2,93
45 "C 0,004 1,96 3.40
50 "C 0,005 4,03 5,46
55 "C 0,003 4,22 5.00 Anh hudng ciia ciia nhift dg den khd ndng
thiiy phan cdm gao ciia xylanase Dung djch cdm gao trong dfm phosphate natri 100 mM, pH 7,0 dugc bd sung xylanase vdi ndng dd 0,3% va tiln hdnh thuy phdn d cdc nhift dg 37°C; 45°C; 50°C; 55°C trong khodng thdi gian 21 gid. Ham lugng xylose trong sdn phdm thiiy phdn sau dd dugc djnh lugng de ddnh gid hifu qua thuy phdn.
Ket qud thu dugc d bang 2 cho thay, hdm lugng XOS tai nhiet do thiiy phan 50''C sau 21 gid Id cao hon dang ke so vdi d cdc nhift dg khde. Dya tren cdc sd lieu thu dugc cd the thIy, hdm lugng chat nay cao hon gIp 2 lln so vdi thuy phan d nhift do 37°C trong 21 gid (5,46%
so vdi 2,93%) vd cao hon khodng 70% so vdi
Bdng 3. Anh hudng cua ndng do enzyme den kha nang thiiy phdn cdm g^o ciia xylanase thuy phdn d nhift dg 45''C (3,4%). O nhift dg cao hon 50''C, khd nang thiiy phdn ciia enzyme bj gidm di. Cdc sd lifu thu dugc da cho thdy nhift dg 50°C la tdi thich cho xylanase Ultraflo L thuy phdn cdm gao.
Anh hud-ng ciia nong dg enzyme den kha ndng thiiy phan cdm gao ciia xylanase
Dung djch cdm gao trong dem phosphate natri 100 mM, pH 7,0 dugc bd sung xylanase vdi cdc ndng do khde nhau la 0,15%; 0,3%;
0,6%; 0,9%. Qud trinh thiiy phan dugc thuc hifn d 50°C trong 21 gid. Ket qua thiiy phan dugc trinh bay d bang 3. Sd lieu thu dugc cho thay sir dyng xylanase d ndng dg 0,6% la thich hgp nhdt de thiiy phan cam gao thu XOS.
Thdi gian (gier)
0 21
XOS(%) Ndng dg enzyme 0
0,008 4,21
0.15%
0,008 5,05
0,3%
0.007 5,06
0,6%
0,005 6,49
0,9%
0,006 5,79 Bdng 4. Anh hudng ciia thdi gian den khd ndng thiiy phan cdm gao ciia xylanase
Thdi gian (gid) 0 5 10 15 21
XOS (%) 0,009
3,06 3,98 5,12 5,32
Tran Thi Nhung. Pham Thi Thu Phuong Nguyen Thuy Huong, Nguyen Tht Mai Phuong Anh hudng ciia thdi gian den khd ndng thiiy
phan cdm g^o cua xylanasc
Dung djch cdm gao trong dfm phosphate natri 100 mM, pi I 7,0 dugc bd sung xylanase vdi ndng dg 0,3% vd tiln hdnh thuy phan a nhift do 50°C trong cdc khoang thdi gian 0; 5;
10; 15 vd 21 gid. Kit qua xdc djnh hdm lugng
XOS trong djch thuy phdn cdm g?io dugc trinh bdy d bdng 4. Sd lifu d bang 4 vd hinh 1 cho thdy, tai thdi dilm thuy phdn 15 gid, hdm lugng XOS d^t cao nhIt (5,32%). Sau thdi dilm ndy, hdm lugng XOS cung tdng nhung khdng ddng kl. Nhu v^y, thdi gian thuy phdn 15 gid la thich hgp d l thu dugc XOS cd hdm lugng cao nhlt.
Xylose Xylobiose -
.Xylolriose .
«» • • • #
Hinh 1. Sac ky dd TLC djch thiiy phdn cdm gao sir dung qua trinh d hinh 5 1. Xylose; 2. XOS; 3. 0 gid; 4 5 gid; 5. 10 gid; 6.
15 gid; 7. 21 gid.
Xylose XOS Ehchihuy Dichthuy phan 1 phan 2 Hinh 2. So sdnh cdc sdn pham thuy phdn
cdm gao theo cdc qud trinh khac nhau
Hinh 3. San pham XOS sau say phun
Er::' '
Hinh 4. Phan tich thdnh phdn dich thiiy phdn cdm gao vdi xylanase blng HPLC. A. Dich thuy phdn cam gao sau khi loai tinh bgt vd protein dugc xir ly vdi xylanase; B. XOS chuln 0,25%.
TAP CHI SINH HQC, 2013,35(1) 67-73
I Cdm gao |
, i ,
Djch cam tho trong dfm phosphate nalti 100 mM, pH = 7,0 (1:7 w/v)
i
I Lam giau xylan bang thdy phan vdi protease vi o-amylas
]
Cim g?o da xii ly Thu hoi djchrira 1 Dfm phosphate natri 100 mM, pH = 6,8(l:7w/v) 1
1
1 B6 sung Ultraflo L. \1 d 50°C trong 15 gio 1
i
1 ^ 1 I
i
1 Say thu san pham 1
Chin nuoi
Hinh 5. So dd qua trinh sdn xudt XOS tir cam gao sir dung cdng nghf da enzyme Kilm tra sdn phim thiiy phan cdm gao vdi
xylanse blng TLC (hinh I) cho thIy, san pham chmh ciia qud trinh thiiy phdn ndy la xylobiose.
Day Id ngudn XOS dd dugc chirng minh Id thich hgp cho cdc vi khuIn Bifidobacteria vd mgt sd Lactobacillus trong rugt kit ddng hda [6, 11].
Nhu vdy, dilu kifn thich hgp cho thiiy phan cdm gao dl thu XOS Id pH 7,0 d nhiet do 5 0 ^ trong thdi gian 15 gid vdi ndng do enzyme 0,6%. San phdm ciia qud trinh thiiy phdn cdm gao vdi dilu kifn lya chgn nay chii ylu la xylobiose, tiep theo la xylose vd mdt sd XOS mach ngln khde nhu xylotriose, xylotetraose.
Xac dinh do sach ciia che phdm XOS sau khi thuy phan
Sdn phim XOS thu dugc sau khi say phun djch thuy phan (hinh 3) dugc xdc djnh dd sach tren mdy phan tich sic ky Idng hifu ndng cao (HPLC). Kit qua thu dugc d hinh 4A vd 4B cho thIy. chi phdm sau khi thuy phdn chira phIn Idn
Id xylobiose. xylotriose vd mdt phdn xylose, phii hgp vdi kit qud phdn tich TLC d tren. Sdn phdm XOS thu dugc cd do sach dat 81,4%.
Todn bg qua trinh thu nhan XOS sir dyng cdng nghf da enzyme dugc tdm tit trong sa dd d hinh 5. Hifu sudt sdn phdm dugc tinh dya tren hdm lugng chit khd thu dugc/trgng lugng nguyen lieu ddu vdo. Hifu sudt thu hdi XOS sau khi thiiy phan cdm gao sir dung qua trinh neu tren dat 13,2% (13,2 gram XOS tren 100 gram cdm gao dd xir ly) vdi dg sach dat 81,4%. Cd the thdy ring, qua trinh thu nhan XOS ciia chiing tdi dua ra d day don gian, thdn thifn vdi mdi trudng vd hifu qua hon cdc qud trinh XOS dd dugc cdng bd trudc ddy vdi cam gao vd Idi ngd [4, 5, 10, 13, 15]. Cy thi Id: i) Khdng sir dyng hda chit de xir ly nguyen lifu lam gidu xylan nen ddm bao an todn thyc phdm; ii) Khdng cd sdn phdm phy thai ra mdi trudng nen khdng phdi xir ly mdi trudng; iii) Cdc enzyme sir dyng d l sdn xult XOS dlu phd biln trong
T^-an Thi Nhung, Pham Thi Thu Phuong, Nguyen Thuy Huong, Nguyen Thi Mai Phuong chl biln thyc phdm vd gid thdnh thIp; iv) San
pham thu dugc cd dg sach tuong dii cao. Chinh nhiing yeu td ndy sg gdp phdn Idm giam gid thdnh sdn phdm XOS thu dugc vd nhd dd cd tinh c^nh tranh cao vdi cdc sdn phdm XOS thuong mpi khde tren thj trudng.
KET LUAN
Dd thu nhdn dugc XOS tir cdm gao d qui md phdng thi nghifm cd dg sach dat 81,4% blng qui trinh thiiy phdn cdm gao dd xir ly loai bd protein vd tinh bgt vdi enzyme xylanase (Ultraflo L, Novozyme) d nhift dg 50''C t^i pH 7,0 vdi ndng do enzyme 0,6% trong thdi gian 15 gid.
L&i dim ffn: Cdng trinh ndy dugc hodn thanh vdi sy hd trg vl kinh phi ciia dl tdi KC.04.TN01/11-
15. Bd Khoa hgc vd Cdng nghe, 2012.
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TAP CHI SINH HQC, 2013,35(1): 67-73
O P T I M I S A T I O N O F RICE BRAN H Y D R O L V Z A T I O N BY X Y L A N A S E F O R X Y L O O L I G O S A C C H A R I D E P R O D U C T I O N
Tran Thi Nhung', Pham Thi Thu Phuong', Nguyen Thuy Huong\ Nguyen Thi Mai Phuong'"
'institute of Biotechnology , VAST
^Food Industries and Research Institute SUMMARY
Rice bran is a subsidy product of rice processing. It is rich in carbohydrate, especially xylan therefore, has being used for production of soluble fiber oligosaccharide including xylooligosaccharides (XOS). XOS has been proven to be fermented by beneficial bacteria Bifidobacteria and Lactobacillus in colon The market for XOS is increasing rapidly due to its advantages in biological and technological properties compared to other common oligosaccharides, such as fmctooligosaccharide (FOS) or galactooligosaccharide (GOS).
XOS can be produced from rice bran by using cither chemical or enzymatic hydrolyzation technologies.
The hydrolyzation using P-l,4-xylanase is commonly used to produce XOS from rice bran. However, an appropriate technology for XOS production from rice bran with high purity and food safe in Vietnam is still badly needed
This paper presents new research results on XOS production from rice bran by using a multienzymatic and environmental friendly technology. A XOS preparation with purity level of 81.4%> has been obtained by hydrolyzing protease and a-amylase pretreated rice bran with 0.6% commercial Ultrafio L xylanase from Novozyme at pH 7.0 and 50°C in 100 mM phosphate buffer for 15 hours. This is an appropriate technology for XOS production from rice bran in Vietnam.
Keywords: Bifidobacteria, Lactobacillus, rice bran, xylanase, xylooligosaccharide (XOS).
Ngdy nhdn bdi: 9-5-2012
