Tp Khoa hc - Cụng ngh Thy sn S 2/2016 THONG BAO KHOA HOẽC IU KIN THCH HP V KH NNG THY PHN TINH BT V PROTEIN TRONG B SN CA CHNG BACILLUS SUBTILIS C7 SUITABLE FERMENTING CONDITIONS AND HYDROLYSIS OF STARCH AND PROTEIN IN CASSAVA RESIDUE OF BACILLUS SUBTILIS C7 Phm Hng Ngc Thựy1, Nguyn Th Thanh Hi2, Nguyn Minh Trớ3 Ngy nhn bi: 09/10/2014; Ngy phn bin thụng qua: 21/6/2015; Ngy duyt ng: 15/6/2016 TểM TT Chng Bacillus subtilis C7 cú kh nng sinh cỏc enzyme amylase v protease thy phõn tinh bt v protein cú bó sn Bó sn c vi chng Bacillus subtilis C7 mt 106CFU/g hn hp nõng cao hiu qu s dng tinh bt v protein Ch bó sn vi chng Bacillus subtilis C7 thớch hp nh sau: Nhit 36 1C, thi gian 48 gi, t l nc/hn hp l 2/1 (v/w), t l u nnh b sung 10% (w/w) Hiu sut thy phõn tinh bt sau theo ch ụ trờn l 26,42 0,43%; hm lng axit amin t 0,87 0,01% T khúa: Bacillus subtilis C7, bó sn, tinh bt, protein ABSTRACT Bacillus subtilis C7 has ability to synthesize amylase and protease to hydrolyze starch and protein in cassava residue Cassava residue was added Bacillus subtilis C7 at the bacterial density 106CFU/g mixture and fermented in order to enhance the effect of utilizing starch and protein Suitable fermenting conditions of cassava residue with Bacillus subtilis C7 are as follows: Fermenting temperature was 36 1oC, fermenting time was 48 hours, the ratio of water to mixture was 2/1 (v/w), the content of added soybean was 10% (w/w) The hydrolysis productivity of starch after the fermentation was 26.42 0.43% and the content of amino acid was 0.87 0.01% Keywords: Bacillus subtilis C7, cassava residue, starch, protein I T VN Sn c xem l cõy lng thc cú sn lng ln th hai sau cõy lỳa v ang cú xu hng tip tc tng v din tớch v sn lng Hin nay, sn ch yu c dựng lm nguyờn liu cho cỏc nh mỏy ch bin tinh bt; bó sn cụng nghip l ph phm ca quỏ trỡnh sn xut tinh bt, nú chim khong 45% so vi lng sn nguyờn c [6] Thnh phn bó sn cha cht hu c chim 97,2% cht khụ, ú cú tinh bt chim 68%, protein thụ 3,6% [11] Nu khụng s dng ngun bó sn hiu qu s gõy lóng phớ v ụ nhim mụi trng Hin nay, bó sn ó v ang c s dng lm ngun thc n chn nuụi, nhng nhm cung cp thờm cht x l chớnh, cha chỳ ý n khai thỏc hiu qu cỏc cht khỏc cú bó sn nh tinh bt v protein Khoa Cụng ngh thc phm Trng i hc Nha Trang, Vin Cụng ngh sinh hc v Mụi trng Trng i hc Nha Trang, Trng i hc Nha Trang TRNG I HC NHA TRANG 101 Tp Khoa hc - Cụng ngh Thy sn S 2/2016 Chng vi khun Bacillus subtilis C7 c phõn lp v tuyn chn t t cho thy cú kh nng tinh bt v hm lng axit amin sau sinh bn loi enzyme: amylase, protease, cho quỏ trỡnh bó sn vi chng Bacillus phytase, cellulose [5]; hiu qu thy phõn subtilis C7 cellulose v phytase ó c kho sỏt bi 2.2 Phng phỏp phõn tớch cỏc cht Mch Trn Phng Tho v cỏc cng s 2.2.1 Xỏc nh m (2014) [4] Nghiờn cu ny trung vo tỡm c xỏc nh chn thụng s thớch hp m c xỏc nh theo TCVN iu kin thớch hp v ỏnh giỏ kh nng 4326:2001 [7] thy phõn tinh bt v protein ca chng 2.2.2 Xỏc nh hm lng tinh bt Bacillus subtilis C7 vi mc ớch nõng cao hiu qu dựng bó sn lm thc n chn nuụi II I TNG, VT LIU V PHNG PHP NGHIấN CU Hm lng tinh bt 2g mu c xỏc nh thụng qua hm lng ng glucose to thnh t quỏ trỡnh thy phõn tinh bt bng HCl, ú hm lng glucose c xỏc nh bng thuc th DNS (3,5-Axit Dinitrosalicylic) i tng v vt liu nghiờn cu Hm lng tinh bt c tớnh bng cỏch ly 1.1 Chng Bacillus subtilis C7: c phõn hiu s gia hm lng glucose mu lp t mu t (ly mu t t vn, thuc sau thy phõn bng HCl v trc thy phng Vnh Hi, thnh ph Nha Trang) phõn nhõn vi h s 0,9 [3], thớ nghim c Chng ny c lu tr ti Phũng thớ nghim thc hin vi ln lp Vi sinh thuc Trung tõm thớ nghim thc hnh, 2.2.3 Xỏc nh hm lng nit axit amin Trng i hc Nha Trang Hm lng nit axit amin c tớnh bng 1.2 Bó sn: Bó sn c thu nhn t cỏc nh cỏch ly hm lng m formol tr i hm mỏy ch bin tinh bt sn ti Cam Ranh, tnh lng nit NH3 Trong ú hm lng m Khỏnh Hũa v sy khụ n hm lng m t formol c xỏc nh bng phng phỏp 6% tin hnh nghiờn cu chun , hm lng NH3 c xỏc nh theo 1.3 u nnh: u nnh c sy khụ n phng phỏp chng ct lụi cun hi nc [2] hm lng m khong 6%, sau ú xay nh Khi lng mu dựng xỏc nh hm lng v s dng cho nghiờn cu m formol v hm lng nit NH3 l 2g, thớ Phng phỏp nghiờn cu nghim c lp li ln 2.1 B trớ thớ nghim Phng phỏp x lý s liu Bó sn xay nh c b sung thờm bt Kt qu trỡnh by l trung bỡnh cng ca u nnh vi t l ln lt l 0%, 5%, 10%, ln thớ nghim lch chun SD S liu 15%, 20% (w/w), sau ú c thờm nc c x lý bng phn mm MS Excel 2003, vi t l nc/hn hp ln lt l 1/1; 1,5/1; SPSS 16.0 2/1; 2,5/1 (v/w) v c hp vụ trựng nhit 121oC 15 phỳt ri lm ngui Chng III KT QU NGHIấN CU V THO LUN Bacillus subtilis C7 c b sung vi t l nh hng ca thi gian n s thy 10 CFU/g hn hp v cỏc mc nhit phõn tinh bt v protein khỏc 24 1C; 30 1C v 36 1C Bó sn c phi trn vi 5% bt u vi cỏc khong thi gian t 12 n 96 gi nnh (w/w), t l nc/hn hp l 2/1 (v/w) (bc nhy l 12 gi) Hiu sut thy phõn v c hp vụ trựng Sau ú, hn hp c 102 TRNG I HC NHA TRANG Tp Khoa hc - Cụng ngh Thy sn S 2/2016 lm ngui, b sung Bacillus subtilis C7 vi hiu sut thy phõn tinh bt v hm lng t l 10 CFU/g hn hp v c nhit axit amin hn hp chn thi gian phũng thi gian t 12 n 96 gi thớch hp Kt qu c th hin trờn hỡnh Sau tng thi gian , chỳng tụi xỏc nh v hỡnh Hỡnh Hiu sut thy phõn tinh bt theo thi gian Hỡnh Hm lng nit axit amin theo thi gian (Cỏc ch cỏi a, b, c, d th hin s khỏc bit cú ý ngha gia cỏc giỏ tr trung bỡnh (p 0,05)) Khi tng thi gian t gi n 48 gi i vi quỏ trỡnh thy phõn protein, tng thỡ hiu sut thy phõn tinh bt tng C th, thi gian bó sn vi vi khun Bacillus subtilis sau 12 gi hiu sut thy phõn tinh bt C7 t 12 n 36 gi thỡ hm lng axit amin l 12,29 0,34%, nu tng thi gian lờn 48 tng v t cc i thi gian t 24 - 36 gi thỡ hiu sut thy phõn tinh bt tng lờn gi Nhng nu tip tc tng thi gian lờn 48 gp ụi Tuy nhiờn tip tc tng thi gian thỡ gi thỡ hm lng nit axit amin gim 8% v hiu sut thy phõn tinh bt thay i khụng tip tc gim hn 40% n 84 gi Hin ỏng k (p > 0,05) iu ny cú th gii thớch tng ny cú th c gii thớch l kộo nh sau: T 12 gi ti 48 gi hiu sut thy di thi gian thy phõn thỡ mt phn axit amin phõn tinh bt tng dn õy l giai on sinh tng hp mnh m cỏc enzyme thy phõn c cht quỏ trỡnh sinh trng ca Bacillus subtilis C7 giai on ny hot amylase c vi khun Bacillus subtilis C7 s dng v mt phn axit amin tip tc b phõn hy to thnh cỏc sn phm cp thp nờn hm lng axit amin gim Nh vy, thi gian thớch hp cao, quỏ trỡnh thy phõn din mnh Sau ú thy phõn tinh bt v protein l 48 gi tip tc tng thi gian lờn thỡ hot amylase nh hng ca nhit n s thy gim nờn hiu sut thy phõn tinh bt khụng phõn tinh bt v protein tng lờn na Kt qu nghiờn cu ca Bó sn c phi trn vi 5% bt u Gaewchingduang v cng s (2010) cng nnh (w/w), t l nc/hn hp l 2/1 (v/w) cho thy lng ng kh thu hi ln nht Hn hp c hp vụ trựng v lm ngui, sau tin hnh bó sn vi a - amylase thng ú chng Bacillus subtilis C7 c b sung vi mi iu kin nhit 55 C thi t l 106CFU/g hn hp v cỏc nhit 24 gian 48 gi [9] Do ú, thi gian bó sn vi 1oC, 30 1oC, 36 1oC, thi gian c xỏc Bacillus subtilis C7 thớch hp l 48 gi quỏ nh t kt qu thớ nghim trờn Kt qu c trỡnh thy phõn tinh bt t hiu sut cao th hin trờn hỡnh v hỡnh o TRNG I HC NHA TRANG 103 Tp Khoa hc - Cụng ngh Thy sn S 2/2016 Hỡnh nh hng ca nhit n hiu sut Hỡnh nh hng ca nhit n hm thy phõn tinh bt lng nit axit amin (Cỏc ch cỏi a, b, c th hin s khỏc bit cú ý ngha gia cỏc giỏ tr trung bỡnh (p 0,05)) Ba mc nhit l 24 1oC, 30 1oC v 36 1oC c chn nghiờn cu s nh hng ca nhit n s thy phõn tinh bt v protein vỡ kt qu nghiờn cu s c ng dng thy phõn bó sn dựng chn nuụi heo tht, vic la chn ba mc nhit ny cú th ỏp dng vo thc t sn xut Vit Nam iu kin thi tit thay i gia cỏc nm; cỏc a phng trng sn v sn xut tinh bt Khi tng nhit t 24 1oC n 36 1oC thỡ hiu sut thy phõn tinh bt tng Nghiờn cu ca Suman v Ramesh (2010) trờn B subtilis KCPSS-12SS cho thy 35oC l nhit ti u chng ny sinh tng hp -amylase cc i [12] Ngoi ra, Kokab v cng s (2003) nuụi cy rn chng B subtilis trờn mụi trng v chui c bm nh cng cho kt qu tng t [10] Hm lng nit axit amin tng cao nht nhit 36 1C Kt qu ny tng t nh kt qu ca Ahaotu v cng s (2011) nghiờn cu thy phõn protein sn nhit 36 1C thỡ hiu sut thy phõn t hiu qu cao [8] T nhng kt qu trờn cho thy thy phõn bó sn nhit 36 1C thỡ hiu qu thy phõn tinh bt v hm lng axit amin cao hn nhit 24 1C v 30 1C Vỡ vy, chỳng tụi chn nhit bó sn vi chng Bacillus subtilis C7 l 36 1C nh hng ca t l nc n s thy phõn tinh bt v protein Bó sn c phi trn 5% u nnh (w/w), t l nc/h hp c nghiờn cu ln lt l 1/1; 1,5/1; 2/1; 2,5/1 (v/w) Sau ú hn hp c hp vụ trựng, lm ngui v b sung chng Bacillus subtilis C7 vi t l 106CFU/g hn hp Sau 48 gi nhit 36 1oC, tin hnh xỏc nh hiu sut thy phõn tinh bt v hm lng axit amin cỏc mu cú t l nc b sung khỏc Kt qu c th hin trờn hỡnh v hỡnh Hỡnh nh hng ca t l nc/hn hp Hỡnh nh hng ca t l nc/hn hp n hiu sut thy phõn tinh bt n hm lng nit axit amin (Cỏc ch cỏi a, b, c th hin s khỏc bit cú ý ngha gia cỏc giỏ tr trung bỡnh (p 0,05)) 104 TRNG I HC NHA TRANG Tp Khoa hc - Cụng ngh Thy sn Khi tng t l nc/hn hp t 1/1 (v/w) lờn 2/1 (v/w) thỡ hiu sut thy phõn tinh bt tng Hiu sut thy phõn tinh bt t cao nht t l nc/hn hp l 2/1 (v/w) v thp nht t l nc/hn hp l 1/1 (v/w) Nu tng t l nc/hn hp lờn 2,5/1 (v/w) thỡ hiu sut thy phõn tinh bt thay i khụng ỏng k (p > 0,05) iu ny cú th gii thớch nh sau: Nu m tng cao quỏ 55 - 70% s lm gim thoỏng khớ ca mụi trng, cũn thp hn s kỡm hóm sinh trng v phỏt trin ca vi khun cng nh s sinh tng hp to enzyme amylase Trong iu kin vụ trựng tt (trong t m phũng thớ nghim) hot lc amylase cao nht thu c m mụi trng 65-68% [1] Do ú, bó sn vi Bacillus subtilis C7 nờn chn t l nc/hn hp l 2/1 (v/w) s thớch hp cho quỏ trỡnh thy phõn tinh bt i vi quỏ trỡnh thy phõn protein, tng t l nc/hn hp t 1/1 (v/w) n 2,5/1 (v/w) S 2/2016 thỡ hm lng nit axit amin tng C th, t l 1/1 (v/w) hm lng nit axit amin l 0,61 0,01%; tng t l lờn 2/1 (v/w) thỡ hm lng nit axit amin l 0,81 0,01% Khi t l nc/hn hp tng n 2,5/1 (v/w) thỡ hm lng nit axit amin thay i khụng ỏng k (p > 0,05) T nhng kt qu trờn cho thy t l nc/ hn hp l 2/1 (v/w) l thớch hp nht nh hng ca t l u nnh n s thy phõn tinh bt v protein Bó sn c b sung thờm bt u nnh vi t l bt u nnh ln lt l 0%, 5%, 10%, 15% v 20% (w/w) Sau ú cho thờm nc vi t l nc/hn hp l 2/1 (v/w) v hp vụ trựng Hn hp c ngui v b sung chng Bacillus subtilis C7 vi mt 106CFU/g ri tin hnh nhit 36 1oC 48 gi Hiu sut thy phõn tinh bt v hm lng axit amin c th hin trờn hỡnh v hỡnh Hỡnh nh hng ca t l u nnh n hiu Hỡnh nh hng ca t l u nnh n hm sut thy phõn tinh bt lng nit axit amin (Cỏc ch cỏi a, b, c, d th hin s khỏc bit cú ý ngha gia cỏc giỏ tr trung bỡnh (p 0,05)) Hiu sut thy phõn tinh bt t cao nht b sung 10% u nnh, thp nht khụng b sung u nnh Khi tip tc tng t l u nnh b sung lờn 15% v 20% thỡ hiu sut thy phõn tinh bt li gim C th, hiu sut thy phõn tinh bt mu khụng b sung u nnh l 15,27 0,22%, b sung 10% u nnh thỡ hiu sut thy phõn tinh bt tng lờn, t 26,42 0,43% Hiu sut thy phõn tinh bt t l u nnh 15% v 20% gim so vi b sung 10% u nnh, ln lt l 24,29 0,19 % v 24,75 0,45% Hm lng nit axit amin tng t l u nnh 5% v 10%, gim dn t l u nnh b sung tng hn 10% C th, 5% hm lng nit axit amin 0,83 0,03%, nu tng t l u nnh lờn 10% thỡ hm lng nit axit amin tng lờn 0,87 0,01% Nu tip tc tng t l u nnh lờn 20% thỡ hm lng nit axit amin gim cũn 0,84 0,01% Khi tng t l u nnh lờn 15% v 20% thỡ hm lng nit axit amin v hiu sut thy phõn tinh bt cú xu hng gim dn mụi trng cú nhiu thnh phn cht dinh dng nờn TRNG I HC NHA TRANG 105 Tp Khoa hc - Cụng ngh Thy sn vi khun Bacillus subtilis C7 s dng trc tip v lm chm quỏ trỡnh thy phõn tinh bt v protein, mt phn axit amin s c vi khun s dng, mt phn tip tc b phõn hy to NH3 Do ú, chỳng tụi chn t l u nnh b sung bó sn l 10% (w/w) IV KT LUN V KIN NGH Kt lun Ch bó sn vi chng Bacillus subtlis C7 (mt 106CFU/g hn hp) hiu qu S 2/2016 thy phõn tinh bt v protein cao l: Nhit 36 1C, thi gian 48 gi, t l nc/hn hp l 2/1 (v/w), t l u nnh b sung 10% (w/w) Hiu sut thy phõn tinh bt sau theo ch trờn l 26,42 0,43%; hm lng axit amin t 0,87 0,01% Kin ngh ng dng quy trỡnh bó sn vi chng Bacillus subtlis C7 chn nuụi heo tht TI LIU THAM KHO Ting Vit Quỏch nh, 1982 S dng ch phm enzyme cụng nghip thc phm NXB Khoa hc v K thut H Ni Nguyn Th Hin, T Vit Phỳ, Trn Thanh i, 2010 Phõn tớch thc phm NXB Lao ng Nguyn c Lng, Cao Cng, 2003 Thớ nghim cụng ngh sinh hc, Tp 1: Thớ nghim húa sinh hc NXB i hc Quc Gia TP HCM Mch Trn Phng Tho, Phm Hng Ngc Thựy, Nguyn Minh Trớ, 2014 Kh nng thy phõn phytate v cellulose bó sn ca chng Bacillus subtilis C7 Tp Khoa hc - Cụng ngh thy sn S 3/2014, trang 180-184 Nguyn Minh Trớ, Mch Trn Phng Tho, Nguyn Th Thanh Hi, Phm Hng Ngc Thựy, 2013 Tuyn chn chng Bacillus spp sinh enzyme v nhõn sinh trờn mụi trng bó sn Tp Nụng nghip v Phỏt trin Nụng thụn S 223/2013, trang 84-88 Bựi Quang Tun, 2005 bó sn lm thc n d tr cho trõu bũ Tp chn nuụi s 7 TCVN 4326:2001 (ISO 6496:1999) Thc n chn nuụi - Xỏc nh m v hm lng cht bay hi khỏc Ting Anh Ahaotu, I., Oqueke, C C., Owuamanam, C I., Ahaotu, N N , Nwosu, J N., 2011 Protein improvement in Gari by the use of pure cultures of microorganisms involved in the natural fermentation process Pakistan Journal of Biological Sciences, 14: 933-938 Gaewchingduang, S., Pengthemkeerati, P., 2010 Enhancing efciency for reducing sugar from cassava bagasse by pretreatment World Academy of Science, Engineering and Technology, 4: 625-628 10 Kokab, S., Asghar, M., Rehman, K., Asad, M J., Adedyo, O., 2003 Bio-Processing of Banana Peel for -amylase production by Bacillus subtilis International Journal of Agriculture and Biology, 1: 36-39 11 Sriroth, K., Chollakup, R., Chotineeranat, S., Piyachomkwan, K., Oates, C G., 2000 Processing of cassava waste for improved biomass utilization Bioresource Technology, 71: 63-69 12 Suman, S and Ramesh, K 2010 Production of a thermostable extracellular amylase from thermophilic Bacillus species Journal of Pharmaceutical Sciences and Research, 2: 149-154 106 TRNG I HC NHA TRANG