KHOA HOC CdNG N G H l TOI UU HOA MOT SO DIEU KIEIV SAY PHUIXI TAO C H £ P H A M BACTERIOCIIM D A I M E B O T CO IMGUdiV GOC V I KHUAIM Lactobacillus TU 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 khdng sdy, luu luong nhdp li^u va ty 1& b6 sung maltodextrln truoc 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^ 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 s^y thich hgp nhdt Id ISSiG^C, nong 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 qud trinh sdy phun TCr khda: Bacteriocin, s ^ phun, tii uu h6a, Lactobacillus aadophdus DATViiNII^ Bacteriocin Id nhiing hpp chat co ban chdt protein 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 hdu hit cdc loai vi khuin [6] 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 dl tiln din san xuat chi pham quy md edng nghiep Qud trinh say phun phu thude vdo nhilu ylu td: nhilt dd, tdc bom nhap lieu, ndng dd chat khO ban Hien cdc bacteriocin sdn sinh bdi lactic axit dau Nghiln ciiu nhdm tdi uu hda mot sd dilu bacteria (LAB) duoe quan tdm nhilu nhat vi LAB kiln say thich hpp dl thu dupe ehl pham cd hoat dupe coi la vi khuan an todn dupe su dung tinh tdt nhdt cdc thuc p h ^ len men Cde bacteriocin san sinh boi NGUYHVUEUVJtPHUOWIIUPNGHeiCUU LAB CO nhiing ling dung quan trpng bao quan thuc pham, bacteriocin tu vi khuin lactic da vd dang 2.1 Nguyin hOu dupe nghien ciiu nhieu nhdt tren thi gidi [3] Dich bacteriocin thu dupe tir qud trinh len men Viec thuong mai hda cdc san pham bacteriocin Id chiing vi khuan Lactobacillus acidophilus L13: rdt can thilt, dl cd the dua vdo san xuat cdng nghiOp Chung Lactobacillus acidophillus -> lln men (mt va ling dung rOng rdi thi phai tao dupe san p h ^ MRS bd sung 3,56 g/lit cao nam men, pH 6,2, nhiet dang bdt Hiln tai b Viet Nam cde nghien cuu mdi dp 32,21°C -^ Dich tho -^ Tua sunlat amdn 70% -> Ly ehi dimg lai d viec phdn lap gidng cd boat tinh, thu tdm thu tua -^ Hda tan vdo dim —* Loc mdng nhdn dich bacteriocin, xac dinh hoat tinh chii chua (membrane) phdn doan 50 kD —* Dich Bacteriocin tdi uu hda phan tao ehl pham dl dua vdo san xuat vd dirpe dem di say phun [1,2] thuong mai O hudng nghien ciiu nay, da nghiln ciiu Thilt hi say phun: Stt dung thilt hi sdy phun tdi im hod qud trinh lln men thu nhdn bacteriocm tu Labplant SD-06AG, Id mdt thiet hi quy md phdng thi chung L acidophilus vk buoc dau khao sat dp bin nghiem, c6 ca cau phun suong dang vdi phun dp luc hojit tinh ciia bacteriocm thu dupe [1,2] nen, tdc dO bom nhap heu tii 280 ml/gid din Say phun Id mot ky thudt thudng dupe diing dl san xuat edng nghiep cdc ehl pham sinh hpc cdng 2115 ml/gid, tdc dO bay hoi tdi da 1,5 lit/gid va ed dp luc nen tdi da bar ' Dai hpc BSch khoa TP HCM NONG NGHIEP VA PHAT TRIEN N N G T H N - KY - THANG 9/2015 75 KHOA HOC CdNG NGHfi 2.2 Xde dinh hoait tinh bacteriocin Xde d m h boat 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 pha lodng cao n h a t eho thay vdng vO k h u a n vd xde dinh hoat tinh bacteriocin (AU/ml) cua dich len men: AU/mi = D F , x ( l / V b , « ^ AU: don vi boat tinh DFi: pha loang cao nhat cd vdng lic c h i Dich bacteriocin hirdc k h i sdy p h u n dupe p h a loang lan Uln t i l p bdng d e m phdt phdt pH Cde b u d e tiln h d n h xde dinh hogt tinh; + Dd 25 ml mdi t r u d n g NA vdo dia petri v6 tning D l thach d6ng + Trai 20 jil dich nudi cdy qua d e m chung ehi thi + D u e 16 ed d u d n g kinh n u n tren thach D u e 16 x u n g q u a n h dia, each thdnh dia k h o a n g em, Id c h m h gifta d l ddi chiing + Bom 20 |il dich bacteriocin vdo 16 thach + t l dia d37°C, 12-16 gid + K i l m tra s u tao thdnh vdng vo k h u l n Do d u o n g Idnh vdng vo k h u ^ -t- Xde djnh dO p h a loang Idn n h a t cd xuat hien v d n g k h d n g khudn Xde dinh n d n g dO chat khd b d n g c h i l t quang k e Xde dinh dm b a n g cdn say am 2.3 Phuong p h ^ tdi uu hda Phuong phap quy hoach thyc nghilm dupe sii dung Id phuong phap ddp ling bl mdt - phuong dn cdu tnie cd tdm (ISM-CCD) Trudc tdi uu hda dich dupe khao sat su dnh hudng ciia ty 1| bd sung maltodextrin, nhiOt dO khong sdy, tie 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 sdy phun va'ldem' tra ho?it tinh D l khao sdt dnh huong eua nhiet dO khong 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 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 ylu td, mdi ylu td tiln hdnh tai miic: -a, -1, 0, +1, +a (vdi a = 1,68), gia tri tgi la gid tn tam Sd Idn thi nghiem dupe xac dinh nhu sau: N = 2'+ 2*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 bj, b2, b3 Id cdc h i sd bdc 1; b u , ^^2, b ^ Id cde h i sd bdc 2; bi2, b23, bjs Id cac h e sd tuong tac cua tiing cap y l u td; 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 sau d d d u g e x i i ly bdng p y n m i m vi tinh d l chpn 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 KfrquANGiraicuu 3.1 Ho^t tinh ban ddu 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 boat tinh theo p h u o n g phdp p h a loang hai 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 , hie Id d dO p h a lodng ^ vdn boat tinh k h d n g Bacillus sp Ho^t tinh t h u dupe trOn ehi t h i ^-CoAthdp hon, chi dat 800 A U / m l (do p h a loang cao n h d t la ^ Khao sdt ha^t tinh t e n SabnoneBa sp cung cao b a n g Bacillus sp D i l u ndy chiing td, dich bacteriocin d a n g nghien eiiu thd h i | n h o a t tinh 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 Gram dm T r o n g cdc c d n g b d t r u d e day v^ bacteriocin cua 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 ehi r a r ^ h o a t tinh ciia bacteriocin G r a m d u o n g tao r a cd k h a nang lic c h i m a n h ddi vdi cdc 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 n g d i n qui trinh s f y phun 76 NONG NGHIEP VA PHAT TRIEN N O N G THON - KY - THANG 9/2015 KHOA HOC CdNG NGHE 3.2.1 Anh hudng cua him lugng maltodextrin vio dich tivdc sdy Hdm luong maltodextrin bd sung vdo dich trude 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, nhien, hi|u suat thu hdi chi phdm lai khOng eao Ham lugng bacteriocm cd 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 bang Bdng Anh huong ciia ndng dO cMt Wid din hogt tinh chi 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 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 cung khd tuong ddng vdi mOt sd nghiln cuu v l say phun bacteriocin, Radoslaw DembczyAski vd cdc cpng su (2008) 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 Id yeu td quan trpng nhdt qud trinh sdy Nhiet 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 nhilt nhilt dp qua cao se 1dm biln tinh, anh hudng din hoat tinh cua chi phdm, nhiln nhiet 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 tdng nhiet dd sdy, hieu sudt thu hdi cung tang, nhien chi tdng den ISO'C, 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 Anh huong cua n h i ^ khdng s^y d^n loat tinh d^a ch^ phim hacteriocin Hoat tinh ch^ Hoat tinh ch^ Nhiet Nhi?t p h ^ sau phdm sau dp ec) dp CO siv (AU/ml) say (AU/ml) 195.062,68 110 171.782,68 150 181.693,97 160 120 172.935,16 179.389,02 170 130 172.704,66 171.552,19 180 140 174.087,63 Kit qua hodn todn phii hpp vdi cac nghien cuu trudc dd vl bacteriocin ciia cde chung Lactobacillus acidophilus S Oh va cdng su nghiln ciiu ndm 2000 dd thu dO nhay ciia bacteriocin Lactobacillus acidophilus tao vdi nhilt dd va thu dupe kit qua boat tinh bacteriocin vdn cdn sau xu 15^20 phiit d l r C [6] 3.2.3 Anh hudng cua tdc dd bam nhap Udu Tdc 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 dau Tdc dp bom nhdp ueu tang ddng nghia vdi thdi gian luu ciia vdt Ulu say budng say giam, lugng hoi nude thoat bi dich qua it hon 1dm dd am tang, phdn hat dm dinh lai buong sdy tang ddn din hilu suat thu hdi san phdm sau qua trinh St^ phun giam Tuy nhien, bang kit qua eho thdy ddi vdi ehl pham bacteriocm, tdc dO bom khoang khao sdt 4,67 ml/phut din 8,08 ml/phiit khOng gay bien dOng nhilu din hogt tinh ehl phdm sau sdy Tdc dd bom cdng tdng ihi thdi gian luu cua nguyin hlu 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 Anh hudng c6a t^c dd bom nh|p Hlu din hojit tinh cfla cfal p h ^ bacteriodn Hoat tinh che Tdc bom DO i n cua nhap lieu pham sau sdy chO ph&n (AU/ml) (ml/phlit) 195.293,17 5,45 4,67 5,82 195.984,66 5,35 5,59 195.523,67 6,03 195.754,16 5,69 6,72 7,12 195.523,67 7,40 195.754,16 7,46 8,08 SJ/II NONG NGHllP VA PHAT TRIEN NONG TH6N - KY - THANG 9/2015 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 say nen chpn tdc dO bom 4,67 ml/phlit la tdc dp ^ i uu cho nghien cuu He sd hdi quy (R^ tinh dupe Id 0,9578 Dilu 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 M Tdm cua 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 bang Bang K ^ qui thi nghiOm tOi uu Hoat tinh NOng NhiOtdO bacteriocm chltkh6 say (°C) (Ul/ml) (BiO 12,5 140 187,143 192,502 12,5 160 184,932 17,5 140 17,5 160 190,808 15 136 188,701 15 164 198,567 193,564 11,5 150 18,5 150 186,561 15 150 201,640 15 150 203,628 15 150 202,560 15 150 202,560 15 150 202,560 Thi nghiOm 10 11 12 13 Hinh Kit qua tdi mi hda b Vdng ddng tdm a.Blmdtd^ung_ 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, 39Xi1418,46X2-3496,07Xi^-4659,06X2^+86,715IXiX2_ (Vdi F=31,77, p-value=0,001) Nhu vdy, tang nhiet say thi boat tinh chi phdm cung tdng 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 qud trinh sdy, tang ndng dd chat khd thi boat tinh ciing tSng len 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 say thich hpp nhdt la 153,e'C, ndng dd chdt khd cua djch thich hpp nhdt dl sdy la 15,1 Bx vd hoat tinh tdi uu 202931 Ul/ml 78 Hinh H o ^ tinh chi p h ^ sau 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 chi pham thu dupe 202,208 Ul/ml Bacteriocin thu dupe Id lo?i chiu nhiet Hojit tinh bacteriocin dugc bao toan qua trinh sdy phun TAI UEU THAM KHAO Nguyin Dang Khoa, Nguyin TTniy Huong Khao sdt mpt sd ngudn hydrateaebon vd nito hiiu co NONG NGHIEP VA PHAT TRIEN NONG THON - KY - 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, (18), 204-210,2013 R Dembczynski, P Lubbe, W Bialas, A Sip, Nguyen Ddng Khoa, Nguyin Thiiy Huong T Jankowski, W Grajek, R Mareeik, P CypUk Khao sat dp bin 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 H Chen and D G Hoover (2003) University of Life Sciences, Poland, 11 Bacteriocms and their Food ^phcations S Oh, S H Kim, R W Worobo Comprehensive Reviews in Food Science and Food Characterization and Purification of a Bacteriocin Safety, 2(2): 82-100 Produced by a Potential Probiotic Culture, 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 conditionsformaking the powder bacteriocin from Lactobacillus acidophilus The experiments were investigated at the center of thesefactorsfor investigating the influence: temperature of drying air,flowrate 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 times) with two mainfeictorsare 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 usefordrying is 15.1 Bx,flowrate input is 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 N N G NGHIEP V A PHAT TRIEN NONG THON - KY - THANG 9/2015 79 ... cac nghien cuu trudc dd vl bacteriocin ciia cde chung Lactobacillus acidophilus S Oh va cdng su nghiln ciiu ndm 2000 dd thu dO nhay ciia bacteriocin Lactobacillus acidophilus tao vdi nhilt dd... say phun bacteriocin, Radoslaw DembczyAski vd cdc cpng su (2008) khao sdt sdy phun diverein dugc ttiu nhdn tii Camobacterium divergens da xac dinh dugc ndng dp maltodextrin thich hpp d l sdy phun. .. 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