I Khoa hpc Ky thuit vi Cdng nghi
D i k kien nuoi thich hop cho sinh tririrng cua vi khua probiotie Lactobacillus acidophilus \ TCC 1225/
G quy mo phong thi nghiem va thiet bj len men JU
Dao Thi Lirong-. H i Thi H j n g . Dirong \ an H Q P r ; . , n . , ™ , . » . „ c ™ . w » . . . ™ - . a » * . « y " . " K ' " " " ' " "
Nf.„ mr, ha, - 1 ;oM nsas .hu>;-n phan h.C-n IM 1 :Ka. . f a ; -han phan bien 1.1 .AA. , .
n.)c ,inh pr„bi,„ic ,r„hB d i i n ki«n ,„ vilra cQi, chung vi k h u i n ^ ; ; - * - ; ' " / f i r h i l d i g c'hiu . r u n g ^ 5 , 12257 p h a n l i p li, .on, Icn men da diryc d a n h jia du-a - " " ' ^ f ? ' ^ ; ; ^ ™ ' ^ ^ , . . (,5„g hoc . i n h trirffng c h o san iruirng rujit nif. phong. Trong nghien cm, na>, chung \ ^ C C - - - ^ du^V 8 ___. j ,^1 n g h i f m , sinh , u i , sinh khiSi cao or quv n,6 phong .hi n g h | t n , v a .rong .h.c. b, len men 3U L r.^^ ^^^q^ J_ ^ _^^^,_^ ^ ^ ^ ^ ^ ^ ^, ^^^^^^^_
t r o n g , „ i . sh,h k h i i Ao . ^ , n , . p h . n g .hi nghijn, - a ,™ng t^i. N ^ e n men ^ ^ ' ^ r ' ^ - ; ; ^ , ; — ; „ ; , „ , „ , , ,
^::i::r^:s:r:=':n:^?:;r,s^..n3n^
mo conR tiRhiC'P cho vi khoan probiotie nav.
Tir khda: L acidophilus \ T C C 12257, Mfi khu5n. quv mo phong thi nghiem va 30 1 p r o b i o t i e , sinh k h 6 i . f hi sd phdn h'Ui: 2.8
flat vftn fli
Ng.iv nu>, v.ii iro cua eae san pham l^r nhien trong v lee lliiie il.iv sire khoe va bao \ e con ngirai chong lai sir gia lUng cua b^nh t^l ng-'iv cang nhan diroc sir quan tam cua colli: d6ng, Trong do, cac san pham sir dung vi sinh vat song (probiotie) nhu cae dugc pham dinh dirong ngay cang gia tang tren the gioi [ 1 ]. Probiolic la cac \ i ^inh v at song ma khi du-gc diing dii Itrgng se mang Iai lai ich sire khoe cho v.ii chu [2]. Cac vi sinh vat probiotie thupc cac nhom khac nhau diroc sir dung duoi dang cac chiing dem le hoac ket hgp Tuy nhien. vi khuan ihugc nhom Laciohacillus va Bifidobaiicriii la cac nhom dien hinh dugc sir dung trong nhicu san pham probiotie cho nguoi va dgng vat. Hau het cac san pham probiotie thucmg mai bao gom cac chung Laciiil\ii.illus khac nhau nhu L acidophilus. L casei. L.
planioniin \ a I m lui oci lo. lai. lis Cac nghien ciru da chimg minh hieu qua eua hoat tinh probiolic v a len ich sire khoe khi
^u dyng Laiiohanllus Vkn jo luimg khoang lO'-lO" CFU ngay [y], Dieu nay d m tren ihue le lii mgt MI luting lon vi sinh vat sc bi ngimg hoai dong trong qua trinh di qua du<Tng lieu hoa ICFITI CO a\it cio va bi anh huimg boi muoi mat Oe >un pham probiotie biru Kb. can co mm so liarng Inn te bao cho moi lieu Do \ ay. cac nghien eiru \ 0 dong hoi, ^mh
tmong va nuoi ciy cac chiing probiotie tao sinh khoi tc bao cao dang dirac cac nganh cong nghiep len men san xuat probiotie va giong cay ngay cang quan tam [4],
Trong s6 cac loai Lactobacillus, cac chiing thugc loai L. acidophilus dugc str dung lam probiotie nhieu nhat, L.
acidophilus la nhom vi khuan khong gay benh va c6 mat trong he vi sinh val duong rupt. d u g c sir dung pho bien lam gi6ng khoi dpng len men sua vai cac hoat tinh co loi n h a kha nang can bang vi sinh vat ducmg tieu hoa, C h u n g cung dugc sir dung trong cac thuc pham len men va Iam probiotie irong cac thuoc va thuc pham bo sung [5], Cac hgp chat protein nhu bacteriocin hoac luong tu bactenocin do L acidophilus san xuat phan lan dugc biel den va d u g c tim thay CO hoat tinh khang khuan manh doi vai cac vi khuan co quan he hg hang gan va cac vi sinh val gay hai khong mong muon Chiing rat hiru ich trong cac ITnh vuc bao quan thirc pham hoac irng dung trong duoc pham, cham soc sire khoe.
Mgl so ehung L acidophilus c6 the tao ra cac cbSl canh iranh va ngan ngira \ i khuan gay benh tir \ ice bam dinh vao cac thu the tren cac te bao bieu mo ciia be mat ruol [61
Nghien cim nay dugc Ihuc hien nham m •. ;'eu phattrifen mo hinh ban cong nghiep san xual smh khui / acidophilus Lhocae irng dung tn lieu sinh h o t \ a prohi ,; dune cac
RHSoM«€\'^Vini'
Khoa hpc Ky thu$t va Cdng nghf i
Culture condition for the growth ofL. acidophilus Vice 12257 in lab and 30 litre bioreactor scale
Th) L u o n g Dao*, Thi H a n g H a , \ ' a n H o p D u o n g ln\litule of Mi<.robiolog\ and Biotechnology.
I Ietnam National Cim ersity. Hanoi Received 7 January 2020: accepted 9 March 2020
moi trucmg va ihong so khac nhau a ca hai dieu ki^-n: nuoi trong phong thi nghidm va thiel bj len men .*() 1. lam lien de cho san \ u a t 6 quy mo cong nghiep.
i i r ttftfng va phimng phap (7 sinh vdt
- Chung L. acidophilus VTCC 12257 dugc luu gi& t?!
Bao lang Giong chuan Viet Nam (VTCC), Vi^n Vi sinh vgt V a Cong nghe sinh hgc, Dai hpc Quoc gia Ha Npi, Abstract:
Probiotie properties in vitro of the L. acidophilus T12.2 ( \ T C C 12257), were isolated from fermented s h r i m p were evaluated based on biological c h a r a c t e r i s t i c s a n d tolerance u n d e r simulated intestinal conditions. In this study, the growth kinetics of the V T C C 12257 strain was studied for high biomass production in a lab a n d 30 litre bioreaetor scale. In case of a l a b . the highest biomass was obtained u n d e r the c u l t u r e conditions as SPY m e d i u m , at 37"C, pH 6.5, inoculum size of 3 % in 32 h; Ihc best sources of carbon and nitrogen were found to be glucose and mixture of peptone a n d yeast e \ l r n e t ; eell d r y weight reached 0.59 g/I. D u r i n g 30 litre b i o r e a c t o r cultivation, the m a x i m a l cell mass obtained at tho agitaUoii speed of 50 r p m , and 16-24 h of i n c u b a t i o n . was 0.72 g/l in pH-eontrolIed culture a n d 0.64 g/l in the uncontrolled p H . This is considered as Ihe lirst step for eell mass production of this probiotie b a c t e r i u m in the industrial scale.
Keywords: biomass, lab and 30 Hire b i o r e a c t o r scale, L.
acidophilus \ TCC 12257, probiotie.
Classiticalion number: 2,8
Vi smh viil kiem dinh: Salmonella eiilerica VTCC-B-04S1)
Mdi trirong
- Moi trucmg MRSA (g/l): pepton-10,0: cao nam men- 5.0; cao thii-10,0; glucoza-20,0; K,HPO,-2.0; CaCO,-5.0.
CH C 0 0 N a - 5 . ( ) , triamoni citrat-2^0: MgSO,.7H,O-0.5S.
MnSO,.4H,O-0,2S. tvvcen 80-1 ml: thach-15.0: nuoc c5l viradu-l l;'pH 6,5±0,2
- MOI irucmg NA (g/l); cao thjt-3,0, pepton-5,0; NaCl- 5.0: pH 7.0±0.2.
- Moi inrcmg dich thi (g/l): MTI (MRS); peplon-10.0;
cao nam men-5.0. cao thn-lCO; glucoza-20.0; K,HPO^-2.0;
CaCO,-5,0. CH,COONa-5,0; triamoni citrat-2.0:
MgSO,.7H,O-0,58: MnSO_, 4H,O-0,28: tvveen 80-1 ml;
nuoc cat vira dii-l I: pH 6,5±0,2. MT2: tryptone-30,4; cao nam men-8.92. glucose-15,0; pH 6,8, AfT3: glucose-13,4;
cao thit-7.2. sodium pyruvate-3,4, K , H P 0 j - 2 ; sodium acetate-5: ammonium cilrate-2; pH 6,5. MT4: glucose-20;
cao nam men-6: KH,POj-0,25; sodium acelate-5;
triammonium citrate-2; MgSO^.7H,O-0,05; pH 6,5. \fT5:
cao nam men-3: peptone-5; cao maIl-3; glucose-10; sodium sulphile-2.92; basic fushsin-0,47; pH 6,9. AfT6: peptone-6;
cao nam men-6; K.H,P0j-l,5; ammonium citrate-!; tween 80-1: sodium acetale-1; citric acid-0,5; MgSO^.7H,O-0,4;
MnSO^-0.05. glucose-30; pH 6,8. AfT7 (SPY): peptone-25;
glucose-25: cao nam men-25, pH 6,5. \TT8-. lactose-17,7;
cao nam men-18,6; CaCl,-0,9, pH 6,5.
Phirffng phdp
Xac dinh khd ndng smh Inrang: xac dinh sinh khoi: vi khuan dugc nuoi tmh trong ong nghiem nut xoay chira 20 mt dich len men trong phong thi nghiem hoac trong thiet bi len men 301 chira 20 1 moi truang. Mat dp te bao dugc do 6 O D ^ va 1 OD^yy tuang duong voi 0,3 g/I [7],
Hogi linh khdng khudn: sir dung phuang phap khuech tan tren thach theo mo ta ciia Jamaly va cs (2011) [8],
Phirong phdp xac dinh ham lirong glucose: sir dyng
59
I Khoa hpc Ky ihuit va Cdng nghi
ihuiK ihir l ) \ s theo phuong phap cua Miller 11959) [9j / ' / / mih innmg duoc kiem ira tren (hiet b| do pH itico huong dan cua nha san vuat
\gliicn cim dicu kii^-n nudi
- I ya chgn dicu kien nuoi thich hc<y trong phong ih^
nghivm chung vi khuan dui'.. nuoi tinh trong ong nghiem nui \o.iv i2l) ml) trim v jo^i moi tniiTng ( M T I A H M Thu nghiem smh iruong o cac nhiel J o ^•13C nhau (211, 25. .-M .•'5, 4(1, -Js \y 5(j(^-|_ pU if,^,^ ^[,,1 j ^ , ;, jjVn y_ ,^ 1^. j^i^snj;
eay tir 0,2 den l(»"o; cae nguon cjcKm thay ihe la I'niciosc.
galaclosc. glucose, laclosc. maltose, nianmlol, saccharose Unh bpt: nguon mio ihay the la cao mall, cao nam men, cao ihjt. casein, pepton. N.iNO \ , i \ < i . i \ l l i.SO^va ure Thoi gi.in nuoi lien Hic tu H Jen 4S gio J."i Juo.. thu nghiem cho kh.i nang lao smh klioi. pH sau nuoi va ham lu^mg glucose
- L\ra chgn dicu kivn nuoi ihich h\Tp tren ihiet hi len men 30 I: VI khuan duge nuoi irong ihicl bi len men Mi I chua 21' I moi tnrinig. ciiy ''"., giimg. nhiet J o nuin o ' " t . pH ban dau dugc dieu ehinh Ji-n (',5 sau kin khu trung pH kiem soiil i> cung inoi gij iri trung qua innh nuoi dugc dieu chmh bang 2.^\l \ . , l ) | | va 2,5M HCI Trong qua trinh nui^i su dtinu clial chong lao bgi vo trung Khoi luitng le bao duge vac Jinh o cac toe dg khuav HI, 3ti, 5{). 10(1 va 15(1 \ ph .\ai dpih pH sau nuoi. ham liti,Tng glucose \a khoi lu^mg te bao theo thin gian nuoi .S, 1(>. 24, .V.4ll va4v gio duoi dieu kivn pM duge kiem soai \ a kliong kicm soat
Cac Ihi nghivm dugc lien h.inh ' lan, ki-t qua la imng binh cua .^ thu nghidm dgc lap
Ml qua vA diao ly$n
\XK Unh probiotie trong dieu kien in \ tiro ciia chung v i khu.iii \ " r c c 1225" ( T 1 2 2 ) , phan lap lu lom len men o dao Phil Quoc, da dugc danh gia d\ra vao cac dac diem sinh hgc V a kha nftng chong chiu irong dieu kii-n rugl mo phong.
Chung VI khuan dugc d|nh danh la /, acidophilus \ T C C 1225" dtra v;io hinh ihai \ a trinh lu - \ D \ r 16S. Chung nav n u n g dav du dac tmh probiotie nhu smh irucmg a . " C.
CO kha nang smh cac en/ynie ngoai bao iP-galactosidase.
cellulase va protease), co kha nang uc che mgt so vi khuan gay bvnh dirong rugl va pho khang rong ii> djch nuoi, chiu muoi mat (ll.5",i). ton tai trong dieu kien khae nghiet cua d^ dav va rupt. co kha ning bam dmh toi vao cac \k bao bieu mo nigi va nhay cam vai mot si^i loai khang sinh ihone dyng Ihu nghtcm. C.\c kci qua da chung mmh L acidophilus N Tt C 1225" IJ mgt chung probiolic dav hira hen [10]
Tmng nghien cuu nav, chung \ TCC 1225" dugc nghien cuu cac dicu kii-n nuoi thieh hgp cho san \uat sinh khoi cao oquy mo phong ihi nghiem v j thiel bi len men 30 1.
,^eh,nS^u.ien nu^i,h,ehh„ tron, phan, M
nghiem L ' •:..,e>,..,L..e..i.,h,heSTCCC:^^^^
„ . h , c n , Hra cho,, n,o, - - ' - - " "^ ^^^^.^^ „„5i a u g c 5,onc. nciio,, caebon. nguon n,Io >,, mm u^
Jjni, cia neng bicl-
. „,^, ,r.r,n,g nu.n d , " .ren cac nglncn enu . m a c d.ay
j„oc .„c i„cn ;o, >, ^i>-n.- '•«-•-•• "r. ,''""f; ""'''r
e,™ Kl,:, nang ,,,o »,nl, kho, ,.i kha nang khang Sanonea ,.. , , , c u a i , « / . / , ' / ' ' " ' " - ^ ' T ' - - ' " ' " • • ' ' '^'"' "' • M^ih kho. ti bao phu thuoc >;,o nh,eu itiu kien. trong do ,hanh phin moi ,ru,Vng co ^a, tro >o cung quan trgng. ICet
^ua tronc hinh I eho thiy. lat ea cac mo, trucmg dirge lim ngh,;'m lieu cb thi ho trg te bao sinh tnrdng va san smh ehat khang khuan. nhimg a mire dg khae nhau, Trong thi nghigm naN. mo, truong MTI (MRS) ^.\ MTV (SPY) eg the tag ra kh.^, lugng li bilo kho cao nhiit trong so nhirng mgi truong nuii, sau:-lg,o(l)..'i7-0..^<)gl)
" Hoar linh klia™ Lhuan -•— Ktioi luijng
MTI VIT: V[T< MT4 MT5 MT6 MT'
Hinh 1. iLFa chon moi trifong nuoi thich hcfp.
Kha nang khang Salmonella enlerica cung thay d6i gitra cac moi truang thir nghiem, kel qua khang khuSn cao nh^t tren 2 moi truang MRS va SPY va khong co su khac biet dang ke giua 2 moi trucmg nay (27 va 28 m m ) , San xuk bacteriocin phu thugc vao loai moi Injcmg nuoi c i y va bi anh hucmg bai ihanh phan moi truang, M R S dirong nhu la moi inrang phii hgp hon so vdi cac moi truang d u g c Ihu nghiem khac, ket qua tuong t y da d u g c quan sat trong cac nghien ciru Iruac day [11]. vi no co Igi cho su phat t n i n ciia vi khuan va mat do te bao cao thucmg co kii cho viec san
\ual bacteriocin, nhung nhijng nam gan day. viec sir dung mgt 50 thuc pham Iam nguyen lieu tho se kinh le JIQ^ p i Heenan va cs (2002) [12] da nuoi vi khuan pri,l-,|,,iit: trong moi trucmg c o b a n CO chiia peptone dau nan' ,- r-.^rn men va glucose monohydrate. Trong thi nghi,,- | | ,
WiNOMGrt, / \ani "'^' *
Khoa hpc Ky thuit va Cong nghi i
va cs (20141 [7], Iga chgn moi iruong SPY co khoi lucmg te bao kho cao nhat (2,84 g/l) trong so nhtmg moi trucmg thir nghidm sau 24 gio nuoi Laciohacillus delbnteckii sp.
butgancus WiCC-B-02, Moi trudng MT7 (SPY) dugc chgn cho cac buac nghien ciru tiep theo trong bao cao nay.
ll
Hinh 2, Li^a chpn nhift dg, pH nuoi va ty 1$ giong cay thich hgp.
Nghien cuu ve anh huong ciia cac yeu to vat ly (bao gom nhiet dg va pH) den vi?c san xuat sinh khoi cua chiing VTCC 12257 dugc thyc hien tren moi trucmg MT7 dich the (hinb 2) 6 tat ca cac nhiet dg dugc Ihir nghiem {20, 25, 30. 35, 40. 45 va 50''C), sinh kh6i tang len khi tang nbiijl dg den 40°C, sinh khoi cao nhat dugc quan sat thay a 35-40°C dat 0,575-0,587 g/l. Tang qua 40°C. nhiet do nuoi CO anh hirong xau den sinh truang. Hau het cac thanh vien ciia vi kliuan lactic la mesophilic, tang truang toi iru cr nhiet dg 35-40°C va tat ca cac hoat dgng trao doi chat, bao gom san xuat bacteriocin cung xay ra trong khoang nhiet do nay [13], Kci qua cua Kheirallah va cs(2014) [14] cong bo hoat linh khang khuan toi da ciia cac chat chuyen boa ctia B.
bifidum triing vdi nhiet do toi uu cho su phat trien cita te bao d 37°C. Favaro va cs (2012) [15] cung c6 ket qua tuang ty khi nghien cim tren Enterococcus faecium. Viec giam hoat tinh khang khuan do cac chat chuyen hda dugc tao ra d nhiet dg cao hem va thap ban co the dugc coi la tang tnrdng cham, dan den lain cham qua trinh tong hgp cac hop chat khang khuan [16]. Nhiel do 37''C dugc lya cbgn cho cac nghien ciru liep theo.
Dg pH dugc biet la rat quan trong doi vdi sinh khoi ciing nhu san pham bacteriocin ciia vi khuan lactic, bdi su tong hgp. hap phu ciia bacteriocin vao le bao va sy phan hiiy protein phu thugc vao pH [ 13]. Chiing VTCC 12257 c6 kha nang sinh trudng d cac pH nghien cuu tir 3-9: sinh khoi tang tir pH 3-7 va giam nj pH 8-9; dat gia tri cao nhat d pH 6-7.
Day la chiing co kha nang chiu pH thap tot, sinh khdi dat
gan 50^0 d pH3 so vdi sinh khdi cao nhal d pH 6-7 (hinh 2). pH dugc chgn de nudi vi khuan \TCC 12257 la 6.5. Kei qua iren tucmg ty vdi bao cao ciia Enan va Aiamri t20(lM [17] nudi L. plantarum. sinh khoi va hgp chat khang khuan cao nhat (plantaricin Ugl). khi do pH ban dau dugc kiem soal dmirc 6.5. Cac nghien cuu kliac cung bao cao boat u'nh khang khuan cao nhat dugc tao ra vdi pH ban dau 6.5 bdi Enterococcus faecium trong Cheese Whey bd sung [15] va Laciocaccus laciis trong vang sua [18]. M5t khac. Ghaly va cs (2003) [19] da bao cao rang, viec san .xual a\it lactic ciia Lactobacillus helvelicus dugc lang cudng rat nhieu tu Cheese Wbcy dugc bo sung cao nam men va lactamine dudi pH kiem soat 5,5.
Ty Ie giong cay la mgt trong nhiJng thdng sd quan trgng trong nghien cuu dieu ki^n len men. Trong thi nghidm nay.
ty Ie gidng 0.2-10% (v/v) dugc kiem tra. sinh khoi thap nhal khi cay 0.2% giong; sinh khoi tSng dan khi cay lugng gidng lang den 5% va co xu hudng giam nhe khi cay giong tren 7% (hinh 2). Ty le gidng t6t nhat cua chung VTCC 12257 de tao sinh kh6i la 3-5%. Kheirallah va cs (2014) [14] bao cao ty le giong tot nhal nudi Btfidobaclenum hifiduiu de I90 hgp chit khang khuan la 2%. Khi ly I? giong thap hon hoac cao han t\' le nay. hoat tinh khang khuan giam. Ty le giong thap, te bao B bifidum khong dii de tao cac hgp chat khang khuan, khi tang xy le giong toi uu lam cho cac le bao d trang thai hoan thien va bat dau san xuat cac hgp chat khang khuan de duy tri sy song ciia vi khuan. Viec san xuat toi da c^c hgp chat khang khuan tucmg ung vdi sinh khoi te bao toi da.
= • 6
I''
L
I
, l h '
+-' C^' •.*' C-»"' V* .J-*,s-f>^ "-* '"JZ.
Hinh 3. Lua chgn nguon caebon, nitc thich hgp.
Cac nguon carbon khac nhau da dugc su dung cho sinh trudng cua chiing VTCC 12257 (hinh 3). glucose, maltose va fructose la nguon carbon tot nhat trong so cac nguon dugc Ihir nghiem. irong lugng te bao khd toi da la 0,58-0.59
I Khoa hpc Ky thuit vi Cdng nghi
g I Manmiol v 3 tmh bgi tan cho irong lugng te bao kho thap nh.iiiiMs-n.2l E U. Tronc bao cao cua Daihn va cs l2t IM
|20]. sinli kiidi ii bao cua Laciohacillus kefiranofaciens dat 0.96 g I khi nudi iren glucose, sau do la sucrosc. Iacto>c va manmiol. Tuy nhien. dm vdi L. acidophilus .\\2. glucose la nguon carbon cho mat do le bao thap hon lactose, glycerol manniiol. eUianol va fniciose. 0 L acidophilus \TCC 4962.
manmtol dugc vac djnh la ngudn carbon tot nhat de lang iruong ti- bao va lo^i bo cholesterol [:i]. Cac loai ducmg khac nhau da dugc sir dyng irong nhieu nghien cim de toi uu san xuii baciemxin o cac chung vi khuan khac nhau.
luv nhicn. glucose van Juoc eoi la neu6n carbon toi nhat kich Ihich san \udt bacteriocin. phdn Um cac thue nghiem da chimg minh nang sual bacteriocin cao hen quan den sy hien divn cua glucose trong moi trudng sinh irucmg chit khong phai cac monosacant khac. Glucose dugc coi la nguon carbon chinh cua hSu hi-t cac v i sinh val bdi vi no dugc dong hda \ a chuyen hoa niing lugng te bao nhanh Tac dgng tieu eye ciia linh bg< doi v oi bacteriocin co the do v i khuan da bi hup phy len be m$t cua cac phan tu unh bgi, Iam han che su dyng eiia chimg [13],
Mail hel cae moi UTjdng nudi dugc su dyng cho vi khuan laclic ihuinig dugc b6 sung ngudn nita, la nguon co anh hmrng Km den san vuat sinh khoi Nudi ehung VTCC I22>"
iren moi trudng M 17. ihay tan lugt cac ngudn nita hCm co la cao mall, cao nam men. cao ihit. pepton, tryptone. ure: va eac ngudn vdco: N.iNO , N a \ 0 . (Mlj.'^O. Nguon nito anh hudng nhieu den smii innmg cua chiing VTCC 1225".
sinh khdi cao htm khi ihay bang cac nguon hiiu ca don tir (1.24-0.4" 111 t ac nguon nua vo ccr deu cho ket qua ihap hon dang ke so vdi nguon niia huu co. chi dal 0.03-0.20 g I N.iNt), cdn gay uc che sy stnh tnjdng. Khoi lugng te bao cho moi ngui^n nua theo ihu ly giam dan nhu sau cao nam men > peptone ^ iryptone > cao malt > cao thjl > ure
>(NH I ^O -NaNC^NaNt) (hinh 3) Tuy nhien, nguon nua hon hgp ciia cao nam men va pepion (DC -) cho sinh khin cao nhdt. dat 0.5'^ gl. Do do. hdn hgp ciia cao nam men v a pepton d3 dugc su dyng lam nguon nito cho cac thi nghi?m liOp theo, Cao nam men la nguon nita dugc sii dung phd bi^n nhdt cung cap ham luong phiic hgp v itamin va tien chdt ON \ ben eanh nito huu co cho v i khuan laclic. Lee va cs (2(11 Ot [21 ] thu duge mat dg te bao cao nhdt khi cao nam men Juoc sit dyng lam ngudn nito nuoi L. acidophilus A12.
di?t Eta trj ^.15 Log CFU ml. Mai Jo te bao tuong doi cho mot ngudn nito theo thir ty giam dan nhu sau: cao nam men
> peptone > tryptone cao mail > cao thit. Thi va cs (2003) [22] da chung minh rdng, su ket hgp cua peptone thjl vai cac nguon nua khac nhu cao nam men giup vi khuan phat
Hinh 4 Lya chpn thdi gian nuoi thich hgp d quy mo phong thi nghiem.
Nsihien ciru dgng bgc phat trign ciia te bao, pH thay d6i \a lugng glucose tieu thy trong qua trinh nudi cua L.
acidophilus VTCC 12257 duac tiln hanh trong Ihdi gian 48 Eio lien tuc. Tc^ bao phat trien vdi pha lag ng^n va dat khoi lugne ik bao cao nhdt (0,59 g/l) sau 24 gid nuoi. Khoi lugng t^ bao cao duy tri dSn 40 gid nudi va giam nhe sau dd, dat 0.45 g'l vao 48 gid nudi. DiSu nay cho thSy, sinh trudng dat den plia 6n dinh sau 32 gid cd thg la kk qua cua su tich lu cac san phdm phy hoac gidi ban chat dinh dudng. Trong qua Irinh lang trudng t^ bao, pH moi trudng giam dan ya dai gia tri 3,92 sau 24 gid nudi. Sau thdi gian do, pH van giu nguyen trong khoang 3,8 cho den khi kk thuc qua trinh nuoi. Toe dg lieu thu glucose nhanh han trong 16 gid nuoi ban ddu. Vao cu6i thdi gian nudi, 10,48% lugng glucose da khong dugc sir dung bdi ti bao budc vao pha dn djnh va ban che su dung ngudn caebon (hinh 4). Dudng cong sinh tnrdng te bao ciia L. acidophilus A12 dugc xay dung trong mdi trudng MRS bdi Lee vacs (2010) [21]. Pha lag khoang 2 gid sau do la pha logarit tir 2 den 8 gid. Tang trudng te bao dugc duy tri them 8 gid. So luong tdi da cua te bao dugc quan sat thdy d 16 gid vdi mat do te bao la 8,23 Log CFU/ml [21]. Dgng hpc ciia su tang trudng te bao va san xudt axit laclic cOa L. delbrueckii sp. hulgaricus WICC B-02 dugc nudi irong moi irudng ldi tru hda chua 3 thanh phdn chinh glucose, cao nam men vd peptone dugc bao cao [7]. Te bao budc vao pha lag trong 2 gid nuoi, sau te bao bal dau tang theo cdp sd nhan, dal trgng lugng khd ldi da la 3,14 g/l, giai doan on djnh sau 10 gid. Do pH ciia moi trudng giam ddn
\a ihdp nhal Id 5,28: sau do lang len 5.76 khi ket ihiic qua tnnh nudi Dieu nay cd Ihe dugc quy cho su hinh thanh cac ion amoni ly do tir phan img ciia axit lactic v a a\u amin cua L delbrueckii sp. bulgaricus do gidi thich su gi.i tang dg pH cua mot trudng.
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Tdc dg khudy cd vai trd Idn doi vdi sinh irudng ciia cac V i sinh vdt khi nudi irong cac thiet bi len men. nd vira co lac dyng dao trgn mdi trudng vira giiip phdn lan deu dinh duong uep .\iic vdi be mdt, Idm tang kha nang hap thu dinh dudng cua te bao. Chiing VTCC 12257 duoc nudi tren thiet bj 30 1 chiia 20 1 mdi Irudng MT7. a 37"C. voi 3% gidng, thdi gian 24 gia. Sinh khdi lang khi tang loc do khuay tu 10-50 \ 'ph va cd \u ihe giam nhe khi lang cao hon. Toe do khuay thich hgp cho sy sinh trudng ciia chiing VTCC 12257 Id 50 v /ph vdi sinh kh6i dat 0,62 g/l (hinh 5)
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Hinh 5. Lya chgn che 6g khuay thich hgp.
Hinh 6. Lya chon thdi gian nuoi thich hgp tren thiet bi 30 I.
Anh huong ciia pH duac kiem soat v a khdng dugc kiem soat trong qua trinh len men theo me cua chiing VTCC 12257 tren thiitbj Ien men 30 1, a37-C trong 48 gid vdi pH ban ddu la 6,5 Hinh 6 bieu thi su ihay doi cua sinh khoi. tieu
ihu glucose va diay ddi pH trong dieu ki?n pH dugc kiem soat vd khdng kiem soat. cac mdu dugc lay trong mdi 8 gid.
Khdi lugng te bao ihu dugc tu ca hai qua trinh len men tren thiel bi 30 I deu cao hon so v at kei qua thu dugc lir quy md phdng thi nghiem Sinh khoi te bao toi da thu dugc Ian lugt la 0.72 va 0.64 g-'l doi \di qua trinh len men dugc kiem soal va khdng kiem soai pH, tang khoang 16,7 va 6,3"n so vai khoi lugng tdi da thu dugc trong quy md phong thi nghi^'m (0.6 g 1, hinh 4). Dieu nay co the dugc quy cho cac dieu kign nuoi tot han rir thiel bi dugc khuay trgn va phdn phoi cac thanh phan moi trudng cung nhu cac sdn phdm chuyen hda.
Ngoai ra, su gidm ciia sinh khoi te bao trong qua trinh len men khong dugc kiem soal pH co the dugc giai thich dya tren thuc te Id viec san sinh lactic lao ra mgl miii trucmg co linh axil cao. uc che sy phat trien cua te bao.
O dieu kien nuoi pH kicm soat, khoi lugng te bdo d^t tdi da sau 16-24 gia va duy tri den 32 gid. sau do giam nh^. Tdc do lieu thu glucose nhanh Irong 16 gid nuoi. Vao cuoi thdi gian nudi. iugng glucose con lai la 8,9%. Nuoi o dieu kiijn pH khong kiem soat (pH ban dau la 6,5). sinh trudng cua chiing VTCC 12257 cung tang theo cap so nhan, sinh khoi ciing dat tdi da sau 16-24 gid. Tuy nhien. gia Irj nay thdp hon so vdi qua trinh nuoi pl-1 dugc kiem soat va cao han khi nuoi trong dieu ki?n phong thi nghidm. Mdt khac, pH cua moi trudng giam dang ke. dal gid trj khoang 3,96 trong 16 gid dau tien do su hinh ihanh axit. Toe d^ tieu thy glucose cua chiing VTCC 12257 nhanh hon trong 16 gid nuoi ban dau, lugng glucose con Iai Id 10,5%) vdo thdi gian cuoi.
Trong nghien ciru ciia Elsayed va cs (2014) [7], nuoi chiing L. delbrueckii sp bulgaricus tren thiet bi len men 16 1, chira 8 I moi trudng loi uu d che dp pH dugc kiem soat va khdng kiem soat. khoi lugng te bdo toi da 6,59 g/l dat dugc sau 8 gid dudi dieu kien pH dugc kiem sodt va 4,88 g/l sau 12 gid dudi dieu kien pH khong kiem soat. Glucose giam rat nhanh trong 6 gid nuoi dudi dieu kien pH dugc kiem soat va 10 gid trong dieu kien pH khong kiem soat. Khoi lugng te bao loi da cung cao hem trong dieu kien pH dugc kiem soat (7.5 gl) so vdi dieu kien pH khdng dugc kiem soat (5,38 g/l) khi nuoi L. salivarius trong thiet bi len men 16 I [23].
Ket luan
• Chung i acidophilus VTCC 12257 cho sinh khoi cao nhat yen moi trudng MT7 (SPY), nhiet dd 37"C, pH 6,5, ty le giong ca\ 3%, ngudn caebon la glucose, ngudn nilo la hdn hgp cua pepton va cao nam men; thdi gian nudi 24-32 gid trong quy md phdng thi nghiem.
• Khoi Iugng te bao tdi da cao hon irong dieu ki?n pH dugc kiem sodt so vdi dieu kien pH khong dugc kiem soai khi nuoi \TCC 12257 trong thiet bj Ien men 30 I d ldc dg khuay 50 \ ph: ihdi gian Ien men 16-24 gid.
63
I Khoa hpc Ky thuit vi Cdng nghi
I I I CAM ON
I. ac lac gia \in cam on Du an FIRST - Tieu hgp phan 2a cua Nhom hi,Tp tac "Tang cuong ndng luc lam chu cong nghv Icn men >an \uai enzyme, probiotie dap ung nhu cau trong nunc v a ndng cao kha ning tu chu eua \ ien \'i sinh v at V a t i«ng nghC- Mnh hiK. Dai hgc Oude gia Ha Nor da ho tro kinh phi de thyc hien cong trinh nay
TU U E l THAM KHAO
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