KHOA HOC CdNG N G H l
TOI UU HOA MOT SO DIEU KIEIV SAY PHUIXI TAO
C H £ P H A M B A C T E R I O C I I M D A I M E B O T CO IMGUdiV GOC TU
V I KHUAIM Lactobacillus acldaphllus
NguySn Tliiiy Huong', Nguyin Tlii N^in Phuong'
T6MTAT
Nghidn ciiu nhdm xde dinh cac dilu kifen say phun t6i uu de tao che phdm bacteriocin dang bot ti!r vi khuan Lactobacdlus acidophilus. Cac thi nghidm dupe tiln htoh dl khao sdt anh hudng ciia cac ylu td: Nhiet do khdng khi sdy, luu luong nhdp li^u va ty 1& b6 sung maltodextrln truoc khi s ^ . Thi nghiem tdi uu duoe tiln hdnh dua trfin phuong phdp dap img bl m^t, ciiu tnie co t&ra, s6 lugmg thi nghi$m Id 13 thi nghi6m (tam l§p 1^ 5 Idn) vol hai ylu to anh huong chinh la nhiet d^ say vd ty 16 b6 sung maltodextrin. Ket qua thu duoe nhu sau: nhiet dO khdng khi s^y thich hgp nhdt Id ISSiG^C, nong do chdt khS cua dich thich hpp nhdt dl say Id 15,1 Bx, luu lupng nhap lieu Id 4,67 ml/ phiit. Ven cac dilu kifin say phun nhu trgn da thu dugc che pham bacteriocin c6 hoat tinh 202,208 Ul/ml. Bacteriocin thu dupe Id loai chiu nhi^t. Hoat tinh bacteriocin dupe bao todn trong qud trinh sdy phun.
TCr khda: Bacteriocin, s ^ phun, tii uu h6a, Lactobacillus aadophdus.
1. DATViiNII^
Bacteriocin Id nhiing hpp chat co ban chdt protein do vi khudn sinh tdng hpp vd co kha ndng uc chi sij phdt triln ciia vi khuan khdc co hen he gan voi gidng san xudt Bactenocin dupe tun th^y trong hdu h i t cdc loai vi khuin [6].
Hien nay cdc bacteriocin sdn sinh bdi lactic axit bacteria (LAB) duoe quan tdm nhilu nhat vi LAB dupe coi la vi khuan an todn khi dupe su dung trong cdc thuc p h ^ len men. Cde bacteriocin san sinh boi LAB CO nhiing ling dung quan trpng trong bao quan thuc pham, bacteriocin tu vi khuin lactic da vd dang dupe nghien ciiu nhieu nhdt tren t h i gidi [3].
Viec thuong mai hda cdc san pham bacteriocin Id rdt can thilt, d l cd the dua vdo san xuat cdng nghiOp va ling dung rOng rdi thi phai tao ra dupe san p h ^ dang bdt Hiln tai b Viet Nam cde nghien cuu mdi ehi dimg lai d viec phdn lap gidng cd boat tinh, thu nhdn dich bacteriocin, xac dinh hoat tinh chii chua tdi uu hda phan tao ehl pham d l dua vdo san xuat vd thuong mai. O hudng nghien ciiu nay, da nghiln ciiu tdi im hod qud trinh lln men thu nhdn bacteriocm tu chung L. acidophilus vk buoc dau khao sat dp bin hojit tinh ciia bacteriocm thu dupe [1,2].
Say phun Id mot ky thudt thudng dupe diing d l san xuat edng nghiep cdc ehl pham sinh hpc cdng
nhu cdc sdn pham thue ph&n. San p h ^ thu dupe cd tinh chdt cam quan tdt thdi gian bao quan Idu, boat tinh bin vung Id nhung uu diem Idii sir dung phuong phdp say phun. S^y phun cung Id edeh tdi uu nhdt d l tiln din san xuat chi pham quy md edng nghiep.
Qud trinh say phun phu thude vdo nhilu ylu td:
nhilt dd, tdc do bom nhap lieu, ndng dd chat khO ban dau... Nghiln ciiu nay nhdm tdi uu hda mot sd dilu kiln say thich hpp dl thu dupe ehl pham cd hoat tinh tdt nhdt
2. NGUYHVUEUVJtPHUOWIIUPNGHeiCUU 2.1. Nguyin hOu
Dich bacteriocin thu dupe tir qud trinh len men chiing vi khuan Lactobacillus acidophilus L13:
Chung Lactobacillus acidophillus -> lln men (mt MRS bd sung 3,56 g/lit cao nam men, pH 6,2, nhiet dp 32,21°C -^ Dich tho -^ Tua sunlat amdn 70% -> Ly tdm thu tua -^ Hda tan vdo dim —* Loc mdng (membrane) phdn doan 50 kD —* Dich Bacteriocin dirpe dem di say phun [1,2].
Thilt hi say phun: Stt dung thilt hi sdy phun Labplant SD-06AG, Id mdt thiet hi quy md phdng thi nghiem, c6 ca cau phun suong dang vdi phun dp luc khi nen, tdc dO bom nhap heu tii 280 ml/gid din 2115 ml/gid, tdc dO bay hoi tdi da 1,5 lit/gid va ed dp luc khi nen tdi da 7 bar.
' Dai hpc BSch khoa TP HCM
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2.2. Xde dinh hoait t i n h bacteriocin
Xde d m h b o a t tinh bacteriocin (AU/ml) theo p h u o n g phdp p h a loang hai lan liln t i l p (Schilliner vd Rest). Xde dinh dO p h a lodng cao n h a t eho thay vdng vO k h u a n vd xde dinh h o a t tinh bacteriocin (AU/ml) c u a dich len m e n :
A U / m i = D F , x ( l / V b , « ^ AU: don vi boat tinh.
DFi: do p h a loang cao nhat cd vdng lic c h i . D i c h bacteriocin h i r d c k h i sdy p h u n d u p e p h a l o a n g 2 lan Uln t i l p b d n g d e m p h d t p h d t p H 6.
Cde b u d e t i l n h d n h xde dinh hogt tinh;
+ Dd 25 ml m d i t r u d n g N A vdo dia petri v6 t n i n g . D l t h a c h d6ng.
+ T r a i 20 jil dich nudi cdy q u a d e m c h u n g ehi thi.
+ D u e 16 ed d u d n g k i n h 5 n u n t r e n thach. D u e 5 16 x u n g q u a n h dia, each t h d n h dia k h o a n g 1 em, 1 Id c h m h gifta d l ddi chiing.
+ Bom 20 |il dich bacteriocin vdo 16 thach.
+ t l dia d 3 7 ° C , 12-16 gid.
+ K i l m t r a s u tao t h d n h vdng vo k h u l n . D o d u o n g Idnh v d n g vo k h u ^ .
-t- Xde djnh dO p h a loang Idn n h a t cd x u a t hien v d n g k h d n g k h u d n .
Xde dinh n d n g dO chat k h d b d n g c h i l t q u a n g k e . Xde dinh do dm b a n g cdn say a m .
2.3. Phuong p h ^ tdi uu hda
Phuong phap quy hoach thyc nghilm dupe sii dung Id phuong phap ddp ling b l mdt - phuong dn cdu tnie cd tdm (ISM-CCD).
Trudc khi tdi uu hda dich dupe khao sat su dnh hudng ciia ty 1| bd sung maltodextrin, nhiOt dO khong khi sdy, t i e dO bom nhap lieu den boat tinh vd hieu susit thu hdi cua ehl phdm bacteriocin.
Dich bacteriocin dupe bd sung maltodextrin thdnh dung dich lan lupt eo ndng dp chat khd 8,5,10, 12,5,15,17,5, 20 va 22,5 Bx. Sau do sdy phun va'ldem' tra ho?it tinh. D l khao sdt dnh huong eua nhiet dO khong khi say, tiln hdnh sdy phun d cdc nhilt dO 110, 120, 130, 140, 150, 160, 170, 180°C. Anh hudng eua tdc dd bom nhap lieu tdi qua trinh say phun dupe xde dinh bang cdch say phun dich bacteriocin vdi toe
do bom nhdp heu 4,67, 5,35, 6,03, 6,72, 7,40, 8,08 ml/phdt
Chi ph&n sau say dupe xde dinh hogt tinh dl xde dinh gid tri tdm eua cac ylu td anh hudng.
Tilp theo xdy dung ma trdn thyc nghiOm vd tiln hanh thi nghilm tdi uu. D l khao sat viing tdi uu, sii dimg k l ho^ich bdc hai tdm xoay cho 3 ylu td, mdi ylu td tiln hdnh tai 5 miic: -a, -1, 0, +1, +a (vdi a = 1,68), gia tri tgi 0 la gid tn tam. Sd Idn thi nghiem dupe xac dinh nhu sau: N = 2'+ 2*3+ 3 = 17. Tinh toan he sd hdi quy vdi ham muc tilu Id hoat tmh bacteriocin.
Phuong trinh hdi quy cd dang Y = b,, + biXj + b2X2 + baXs + buXii + b22X22 + bsjXas + bjaXi)^ + b23XaA3 + b 13X1X3
T r o n g dd b j , b2, b3 Id cdc h i s d bdc 1; b u , ^^2, b ^ Id cde h i s d bdc 2; bi2, b23, bjs Id c a c h e s d t u o n g tac c u a tiing c a p y l u t d ; Xi, X^, X3, Xn, X22, X33, X12, X23, Xi3 Id cdc b i l n dOe Idp.
Sd h e u thi n g h i e m s a u d d d u g e x i i ly b d n g p y n m i m vi tinh d l c h p n r a k i t q u a tdi u u .
Sau k h i cd k i t q u d tdi u u h i tinh todn d u doan, t i l n hdnh t h i n g h i e m k i l m t r a lai d l d u a r a k e t lu^n tdi u u sau ciing.
8. KfrquANGiraicuu
3.1. H o ^ t t i n h ban d d u ciia dich t r u d c Idii s ^ Dich bacteriocin t n i d c k h i s a y d u p e xde dmh b o a t tinh t h e o p h u o n g p h d p p h a l o a n g h a i Idn lien tiep. H o a t tinh t h u d u p e t r e n c h i t h i Bacillus sp. la 204.800 A U / m l , h i e Id d dO p h a lodng 2 ^ vdn con b o a t tinh k h d n g Bacillus s p .
H o ^ t tinh t h u d u p e trOn e h i t h i ^-CoAthdp hon, chi dat 800 A U / m l (do p h a l o a n g c a o n h d t la 2 ^ .
Khao sdt ha^t t i n h t e n SabnoneBa s p . c u n g cao b a n g Bacillus s p . D i l u ndy c h i i n g td, dich bacteriocin d a n g n g h i e n eiiu thd h i | n h o a t t i n h k h d n g khudn ddi vdi chiing vi k h u f o G r a m d u o n g e a o b a n vi khuan G r a m d m . T r o n g c d c c d n g b d t r u d e day v^
bacteriocin c u a vi k h u i n G r a m d u o n g , R. W . Jack va c p n g sir (1995) c i m g d a e h i r a r ^ h o a t tinh ciia bacteriocin do G r a m d u o n g t a o r a c d k h a n a n g lic c h i m a n h ddi vdi c d c c h u n g G r a m d u o n g [4].
3.2. Khdo s d t c d c y l u t d dnh h u 6 n g d i n qui trinh s f y phun
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3.2.1. Anh hudng cua him lugng maltodextrin vio dich tivdc khi sdy
Hdm luong maltodextrin bd sung vdo dich trude khi sdy ed anh hudng den hieu sudt thu hdi cOa ehl pham, bd sung maltodextrin cdng nhilu thi lupng bOt thu dupe sau sdy cang nhilu, tuy nhien, hi|u suat thu hdi chi phdm lai khOng eao. Ham lugng bacteriocm cd trong mdu Id khOng thay ddi nen ciu din ndng dO chat khd 15 Bx Id hoat tinh bdt dau giam dan. Kit qua boat tinh thi hiln trong bang 1.
Bdng 1. Anh huong ciia ndng dO cMt Wid din hogt tinh c h i phim bacteriocin sau Idii s ^ N6ngdQ chat kho
cua dich (Bx) 8,5 10 12,5
15 17,5 20 22,5
Hoat tinh ch^ pham sau khi s^y (AU/ml)
195.984,7 209.814,4 211.658,3 219.956,1 209.583,9 203.130,0 191.374,8 Kit qua nay cung khd tuong ddng vdi mOt sd nghiln cuu v l say phun bacteriocin, Radoslaw DembczyAski vd cdc cpng su (2008) khi khao sdt sdy phun diverein dugc ttiu nhdn tii Camobacterium divergens da xac dinh dugc ndng dp maltodextrin thich hpp d l sdy phun nen Idn hon 10% [5].
3.2.2. Anh hirdng cua nhidt dp sdy
Nhiet do Id yeu td quan trpng nhdt trong qud trinh sdy. Nhiet do khdng ehi dnh hudng din hilu sudt ciia qua trinh say md cdn anh hudng din chat lupng ciia san phdm thu dupe sau say. Bactenocin cd ban chat Id protein nIn dupe xIp vdo nhdm nh^y cam vdi n h i l t nhilt dp qua cao se 1dm biln tinh, anh hudng din hoat tinh cua chi phdm, tuy nhiln nhiet do say neu qud thap se lam cho dp dm cua san pham cao gdy nen hien tugng hdm dinh tren binh thu mdu, gay khd khan cho qud trinh thu mdu ddng thdi anh hudng din thoi gian bao quan ciia che pham. Bang kit qua cho thdy hoat tinh ciia ehl pham tdng khi tdng nhiet dd sdy, hieu sudt thu hdi cung tang, tuy nhien chi tdng den ISO'C, khi nhiOt dp say eao hon 150°C thi hoat tinh bdt ddu giam ddn, hilu suat thu hdi cd giam nhimg khdng dang ke. Nhu vdy dya vdo boat tinh ta thdy nhi|t dd thich hop nhat d l say phun thu ehl phdm bacteriocm Id 150°C.
Bang 2.
Nhiet dp ec)
110 120
Anh huong cua n h i ^ do khdng khi s^y d^n loat tinh d^a ch^ phim hacteriocin
Hoat tinh ch^
phdm sau khi say (AU/ml) 171.782,68 172.935,16 130 1 172.704,66 140 1 174.087,63
Nhi?t dp CO
150 160 170 180
Hoat tinh ch^
p h ^ sau khi siv (AU/ml) 195.062,68 181.693,97 179.389,02 171.552,19 Kit qua nay hodn todn phii hpp vdi cac nghien cuu trudc dd vl bacteriocin ciia cde chung Lactobacillus acidophilus. S. Oh va cdng su trong nghiln ciiu ndm 2000 dd thu dO nhay ciia bacteriocin Lactobacillus acidophilus tao ra vdi nhilt dd va thu dupe k i t qua boat tinh bacteriocin vdn cdn sau khi xu 15^20 phiit d l 2 r C [6].
3.2.3. Anh hudng cua tdc dd bam nhap Udu Tdc do boTO nhap hlu cd dnh hudng Idn d i n luu liigng dong nhap h l u , nang suat tiiilt bi va ca nhiet dd khdng khi dau ra. Tdc dp bom nhdp ueu tang ddng nghia vdi thdi gian luu ciia vdt Ulu say trong budng say giam, lugng hoi nude thoat ra bi dich qua it hon 1dm dd am tang, phdn hat dm dinh lai trong buong sdy tang ddn din hilu suat thu hdi san phdm sau qua trinh St^ phun giam.
Tuy nhien, bang k i t qua eho thdy ddi vdi e h l pham bacteriocm, tdc dO bom trong khoang khao sdt 4,67 ml/phut din 8,08 ml/phiit khOng gay bien dOng nhilu din hogt tinh e h l phdm sau sdy. Tdc dd bom cdng tdng ihi thdi gian luu cua nguyin hlu trong budng sdy giam vi vdy nen dp am cua chi pham se tdng ddn, dd ^ anh hudng din thdi gian bao quan eua ehl pham. "Vi vdy, chiing tdi chon tdc dp bom xHiw lilu cho qud trinh say phun Id 4,67 ml/phiit.
Bang 3. Anh hudng c6a t^c dd bom n h | p Hlu din hojit tinh cfla cfal p h ^ bacteriodn Tdc do bom
nhap lieu (ml/phlit) 4,67 5,35 6,03 6,72 7,40 8,08
Hoat tinh che pham sau khi sdy
(AU/ml) 195.293,17 195.984,66 195.523,67 195.754,16 195.523,67 195.754,16
DO i n cua chO ph&n
5,45 5,82 5,59 5,69 7,12 7,46
KHOA HOC CdNG N G H |
3.3. Tdi uu cdc dilu kiln s ^ phun dich bacteriocin
Vi thilt hi say cd tdc dd bom tdi thilu Id 4,67 ml/phlit, kit qua khao sat cho thay tdc dd bom tu 4,67 din 8,08 ml/phiit khdng 1dm biln ddng nhilu boat tinh ciia che pham sau khi say nen chpn tdc dO bom 4,67 ml/phlit la tdc dp ^ i uu cho nghien cuu nay.
Tdm cua 2 yeu td dnh hudng edn lai: nhiet dp 150°C, ty le bd sung maltodextrin: 15Bx. Tiln hanh quy h o ^ h thyc nghiem gdm 13 thi nghiem. Kit qua dupe thi hien trong bang 4.
He sd hdi quy (R^ tinh dupe Id 0,9578. Dilu nay thi hiln r^ng cd 95,78% sd h | u thuc nghiem tuong thich vdi sd hdu tiOn doan theo md hinh. Gia tri R^
0,75 thi hiln md hinh tuong thich vdi thuc nghiem cd nghia Id su tuong quan rdt chdt che giira cdc ylu
td thi nghilm va hoat tinh sinh ra. M
Bang 4. K ^ qui thi nghiOm tOi uu Thi
nghiOm 1 2 3 4 5 6 7 8 9 10 11 12 13
NOng do chltkh6 (BiO 12,5 12,5 17,5 17,5 15 15 11,5 18,5 15 15 15 15 15
NhiOtdO say (°C) 140 160 140 160 136 164 150 150 150 150 150 150 150
Hoat tinh bacteriocm
(Ul/ml) 187,143 192,502 184,932 190,808 188,701 198,567 193,564 186,561 201,640 203,628 202,560 202,560 202,560
Hinh 1. Kit qua tdi mi hda a . B l m d t d ^ u n g _ b. Vdng ddng tdm Hiyc nghilm kilm chting:
Tien hdnh sdy phun dich bacteriocin vdi cac thdng sd: nhiet dd 153,6''C, ndng dp chdt khd 15,1 Bx vd tdc dp bom n h ^ lilu 4,67 ml/phiit Hoat tinh thu dupe dat 202,208 Ul/ml tuong thich 99% so vdi md hinh tdi uu.
Phuomg trinh hdi quy: Y=202584+2587, 39Xi- 1418,46X2-3496,07Xi^-4659,06X2^+86,715IXiX2_
(Vdi F=31,77, p-value=0,001). Nhu vdy, khi tang nhiet do say thi boat tinh chi phdm cung tdng tuy nhien neu tilp tuc tdng 'ihiet dO len cao qud thi boat tinh chi phdm 1^ cd xu hudng giam ddn, cd anh hirdng thuan len hoat tinh cua bacteriocin. Tuong tu nhu vdy, ndng dp chdt khd ciing anh hudng len hoat tinh trong qud trinh sdy, khi tang ndng dd chat khd thi boat tinh ciing tSng len tuy nhien hojit tinh ehi tang din miic nhat dinh, nlu vdn dip tuc tdng hdm luong chat khd cung khong tang dupe hoat tinh bacteriocin cua ehe pham.
Kit qua tdi uu nhu sau: nhiet dd khdng khi say thich hpp nhdt la 153,e'C, ndng dd chdt khd cua djch thich hpp nhdt d l sdy la 15,1 Bx vd hoat tinh tdi uu 202931 Ul/ml.
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Hinh 2. H o ^ tinh c h i p h ^ sau khi s ^ 4.KErLUiiN
Bang phuong phdp tdi uu hda quy hoach thyc nghiem, da xde dinh dilu kiln say phun cho boat tinh bacteriocin tdi uu nhat nhiet dd say 153,6''C, ndng dd chdt kho ban ddu: 15,1 Bx, tdc dp bora nhdp liOu 4,67 ml/phiit. Hoat tinh c h i pham thu dupe 202,208 Ul/ml. Bacteriocin thu dupe Id lo?i chiu nhiet. Hojit tinh bacteriocin dugc bao toan trong qua trinh sdy phun.
TAI UEU THAM KHAO
1. Nguyin Dang Khoa, Nguyin TTniy Huong.
Khao sdt mpt sd ngudn hydrateaebon vd nito hiiu co
NONG NGHIEP VA PHAT TRIEN N O N G THON - KY 2 - THANG 9/2016
KHOA HOC CdNG NGH|
M ndng cao kha ndng sinh tdng hpp bacteriocm cua and Molecular Biology reviews. Vol. 59, no. 2, pp.
chung Lactobaeilhis acidophilus. Tap chi Phdn tich 171-200.
Hda, Ly va Smh hpc, 4 (18), 204-210,2013. 5. R Dembczynski, P. Lubbe, W. Bialas, A. Sip, 2. Nguyen Ddng Khoa, Nguyin Thiiy Huong. T. Jankowski, W. Grajek, R. Mareeik, P. CypUk Khao sat dp b i n boat tinh cua bacteriocm dupe (2008). Production of dry diverein preparation by Lactobacillus acidophilus tdng hpp. Tgp chi Phdn spray drying and lyophihzation. Electronic Joumal of tichHda,LyvdSinhhpc,4(18),204r210,2013. ^''^sh Agricultual universities. Department of Biotechnology and Food Microbiology. PoznaA 3. H. Chen and D. G. Hoover (2003).
Bacteriocms and their Food .^phcations.
Comprehensive Reviews in Food Science and Food Safety, 2(2): 82-100.
University of Life Sciences, Poland, 11.
6. S. Oh, S. H. Kim, R. W. Worobo.
Characterization and Purification of a Bacteriocin Produced by a Potential Probiotic Culture, 4. R. W. Jack, I. R. Taqq, B. Ray (1995). Lactobacillus acidophilus 30SC Joumal of Dauy Bacterioeins of gram-positive bacteria. Microbiology Science. Vol. 83 No. 12 2000.
OPTlMIZA'nON OFTECHNOLOGICAL PARAMETERS IN THE SPRAY DRYING OF BACTERIOCIN FROM iBctobadllus addopbibis
Nguyen TliuyHuong^, Nguyen Tlii Nam Phuong^
'University of Technology VNU-HCM Summary
The stuffy used for determine the spray-dried optimal conditions for making the powder bacteriocin from Lactobacillus acidophilus. The experiments were investigated at the center of these factors for investigating the influence: temperature of drying air, flow rate input and additional maltodextrin before drying. Optimal experiment was investigated based on the response surface mediod, center of structure, the number of laboratory esperiments are 13 (center repeat 5 times) with two main feictors are the drying temperature and the ratio additional maltodextrin. The results were as follows: the best drying temperature is 153.6°C, the best dry concentration use for drying is 15.1 Bx, flow rate input is 5 rpm (4.67 ml/min). The spray drying conditions as above we obtain bacteriocin active preparations 202.208 Ul/ml. Bacteriocin we obtain was heat stable. Bacteriocm active was maintained during spray drying.
Keywords: Bactsnodn, spray drying, optimization, Lactobacillus acidophilus.
Ngudi phdn bi|n: PGS.TS. Le Anh Diic Ngdy nhdn bdi: 15/5/2015 Ngdy tiidng qua phdn biln: 15/6/2015 Ngdy duyet ddng: 22/6/2015
