T Phn p, Thy s Sinh hc: 26 (2013): 205-212 205 NG VI SINH V I TI Nguy 1 n Th Yn Minh 2 1 Vi Sinh hi hc C 2 H Sinh hi hc C Thông tin chung:  05/02/2013 20/06/2013 Title: Application of microorganisms and enzyme protease to improve the quality of traditional fermented soy-sauce Từ khóa: Aspergillus oryzae, enzyme  Pediococcus halophillus, Zygosaccharomyces rouxii Keywords: Aspergillus oryzae, koji, enzyme protease, Pediococcus halophillus, soy-sauce, Zygosaccharomyces rouxii ABSTRACT The objectives of the study were to produce soy sauce with high quality, free of toxic 3-MCPD (3-monochloropropane-1,2-diol), and to reduce production time, based on these several experiments were carried out. Results of research showed that inoculating pure starter culture of Aspergillus oryzae 10 6 spores/g dry weight (gdw) has shorten the koji incubation time to 52 hours in comparison to 4-5 days of the traditional process. Supplementing of 2% commercializing enzyme into hydrolytic step at 50 o C, 36 hours before adding salt (NaCl), the efficiency of amino acid hydrolysis obtained 44,8% which was more higher than that of the control 8,8%. Aroma of soy sauce was improved by inoculating with the bacteria Pediococcus halophillus 10 4 cells/g and the yeast Zygosaccharomyces rouxii 10 2 cells/g of mixture together in the period of brine fermentation. T  -              Aspergillus oryzae   6  -    50 o    Pediococcus halophillus  4  Zygosaccharomyces rouxii 10 2   1     c ph c s dng ph bin                       ng do cha nhi c s d  bin thc ph       c s d    i trong sn xut       c t   T Phn p, Thy s Sinh hc: 26 (2013): 205-212 206 3-MCPD (3-monochloropropane-1,2-  n sc kh m c     i gian sn xut ng        d thc h nhin xut    n th         m th    i gian sn xut   chp nhn. c ng d sinh hc  ci tin xung    m to ra sn phm  vi thi gian sn xut ng     u ci tin   sn xu    ng   dng vi sinh v   2  2.1 m        c phm, Vin tri Sinh hc, i hc C 2.1.1 u           ng.  Mc ging Aspergillus oryzae n gc t   ng ca Hoa k ATCC       tn tr ging ti Vin  Sinh h  Chng ging vi khun Pediococcus halophillus  m men Zygosacharomyces rouxii t       c phm, Vin  Sinh h  Ch phm enzyme protease (mua ti Vin Sinh hc Nhi   H  Minh). 2.1.2 t   ng: MRSA (Man Rogosa Sharpe Agar); PGA (Potato  glucose  agar)   t: NaHPO 4 .2H 2 O, NaH 2 PO 4. H 2 O, K 2 HPO 4 , SDS (Sodium Dodecyl Sulfat), OPA (Ortho-phthaldialdehyde), DTT (Dithiotreitol), Ethanol, CBB (Coomasie brilliant blue), casein, bromoresol green, methyl red, bromophenol blue, formol, TCA (Trichloroacetic acid), acid boric, acid acetic, acid citric, acid sulfuric, aceton, Na 2 CO 3, MgO, K 2 SO 4 , CuSO 4 , NaOH, (NH 4 ) 2 SO 4  2.2  2.2.1 Khng ca t lm mn ho enzyme protease koji   l mc ging (3 m: 10 4 , 10 5 , 10 6 /g chi gian  koji (3 m: 42, 52, 68 gim lp li 3 l 27 (9 x 3)  m. Ch t  ci ti         2.2.2 Khng ch phm enzyme n kh  protein  ng enzyme b sung (6 m:     i gian th: 12, 24, 36, 48 gi nghim lp li 3 l  m. Ch  kh  formol. 2.2.3 Khng ca chng ging vi khun Pediococcus halophillusm men Zygosacharomyces rouxii n cht    c b sung vi khu m men: (1) b sung vi khun - n       sung vi khun c kim tra pH d T Phn p, Thy s Sinh hc: 26 (2013): 205-212 207 cho nt s t i khun (3 m: 10 2 , 10 4 , 10 6 t  dt s t m men (3 m : 10 2 , 10 4 , 10 6 t    m lp l       m. Ch      n xui ti n nghim thc tm ng thi 2 nghim thc i ch         m thc sn xu c n thng. Ch o m amin, mt s vi khum men. 2.2.4 n xuc m tra chng sn phm m sn xu i tin v t  nghic hin. -MCPD, aflatoxin,  i m . 3 KT QU O LUN 3.1 ng ca t lm m thn hoa enzyme protease koji    m kh   ng ca t l nm m  i gian  ng n ho         ng 1 cho thy ho koji ti thm 52 gi vc ging ch 6  t cao nht  nghim thc 6 (5,13 U/g mu). V   c gi   6  /gck) nu tip ti gian  n 68 gi (nghim thc 9), hong mc 4,73 U/g m  ng kh   khu  trin ch       din ra mi koji chuy  enzyme protease. Bng 1: Hong protein, hoc hiu ca koji Nghi m thc T hp thi gian (gi l mc g/gck) Ho koji (U/g mu) ng protein (mg/g mu) Hoc hiu (U/g mu) 1 42-10 4 2,55 h 153,40 g 16,62 g 2 42-10 5 3,28 g 166,51 f 19,69 b 3 42-10 6 4,08 f 213,22 e 19,14 d 4 52-10 4 4,09 f 238,70 d 17,13 f 5 52-10 5 4,38 d 250,71 a 17,47 ef 6 52-10 6 5,13 a 242,50 c 21,15 a 7 68-10 4 4,27 e 242,13 c 17,63 e 8 68-10 5 4,88 b 249,30 a 19,57 bc 9 68-10 6 4,73 c 245,70 b 19,25 cd CV(%) 1,35 0,65 1,39 *S liu trong ba 3 ln lp lt c mang ch s   i t l mc ging 10 4  5 i gian  t 42 - 68 gi hot   p t       nm mc ny mn h khun ty chi nghim thc chng t l 10 6 t thi gian   52 gi i t l mc ging ch 3 m 10 4 , 10 5 , 10 6 /gck s cho kt qu i hot        u)  nghim thc 4, (4,38 U/g mu)  nghim thc 5, (5,13 U/g mu)  nghim thc 6, cho thy t l mc ging ch n ho    ng thi  nghim thc 6 vi thi gian    t l mc ging 10 6 /gck cho ho   c hiu cao nht 21,15 (U/g m  ca T Phn p, Thy s Sinh hc: 26 (2013): 205-212 208 Nguyng (2002), thi gian thu nhn   ng 36 - 60 gi  ng Th Thanh Thu (2003) thc 40  48 gi khi m     u chng Aspergillus to enzyme cao nh  mi b      c Phm, 1998).    y t l mc gi  i gian  koji rt   n ho  protease c l nm m   6    i thi gian   ti nghim sau. 3.2 ng ch ph n kh  protein nh ho Kt qu    phm enzyme  Bng 2 c s d  . Bng 2: Ho protease Ch s Ch phm enzyme protease Ho 12,20 ng protein (mg/g) 352,80 Hoc hiu (U/g) 34,58 Kt qu  lia 3 ln lp li c khi ti       c mu  n thy       50 o    sung ch phm enzyme protease. Ch ph c b sung  5 mi m ly mu 12; 24; 36; 48 (gi). Kt qu nh hiu sut thm amin khi b sung ch phm enzyme protease  Bng 3. Bu sut th Nghim thc T hi gian N amin (mgN/gck) Hiu sut (%) 1 0  12 11,53 o 25,74 o 2 0  24 16,41 j 36,63 j 3 0  36 17,78 g 39,68 g 4 0  48 16,71 i 37,29 i 5 1,0  12 12,55 m 28,01 m 6 1,0  24 17,71 g 39,55 g 7 1,0- 36 18,46 d 41,21 d 8 1,0  48 18,84 c 42,07 c 9 1,5  12 12,32 n 27,51 n 10 1,5  24 19,18 b 42,82 b 11 1,5  36 19,33 b 43,15 b 12 1,5  48 17,87 g 39,89 g 13 2,0  12 13,56 l 30,27 l 14 2,0  24 19,95 a 44,53 a 15 2,0  36 20,04 a 44,74 a 16 2,0  48 19,91 a 44,46 a 17 2,5  12 14,48 k 32,33 k 18 2,5  24 18,26 ef 40,77 ef 19 2,5  36 18,46 de 41,20 de 20 2,5  48 18,12 f 40,45 f 21 3,0  12 12,62 m 28,18 m 22 3,0  24 17,33 b 38,69 h 23 3,0  36 19,21 b 42,88 b 24 3,0  48 18,91 c 42,22 c CV(%) 0,708 0,697 *S liu trong ba 3 ln lp lt c mang ch s   T Phn p, Thy s Sinh hc: 26 (2013): 205-212 209 Kt qu Bng 3 cho th  m amin   m th   sung enzyme protease cho kt qu      thi mi ch sung enzyme protease. Nghim thc 15 vi  ng enzyme protease b sung 2% sau 36 gi th       u so vi m i ch     ( nghim th                                             c  sung enzyme vi n     phn       cnh tranh gia    t trong vic g    ng ca enzyme. B m th sung enzyme protease cho kt qu  so vi mi ch                  nghim th            m thc      m th     s  u sut thi v protease b   i gian trong sut        (2004) ch ph    t endoprotease ct ni ph cho ra nhin peptide ng ng th   kt hp vi           tip t            yme 1; 1,5; 2,5; 3  2%, nghim thc 15 (36 gi th                               th    th         gi                  nghim tip theo. 3.3 ng chng ging vi khun Pediococcus halophillus m men Zygosachromyces rouxii n ch  n th     t dch th   c chuy      c mui.       sung chng vi khun, nm men chu m t   t       Sn phc cm b sung vi khum men s m quan v  (so vi mi chng) nhm mn ra nghim thc hay sn phm tt nh  ti      nghim sau. Kt qu c    T Phn p, Thy s Sinh hc: 26 (2013): 205-212 210 :  sung vi khun ni ch sung vi khun, nm men, I1: b sung 10 2  2 tbmn/g, I2: 10 2  4 tbmn/g, I3: 10 2  6 tbmn/g, I4:10 4 tbvk/g  2 tbmn/g, I5: 10 4  4 tbmn/g, I6: 10 4  6 tbmn/g, I7: 10 6  2 tbmn/g, I8: 10 6  10 4 tbmn/g, I9: 10 6  6  sung vi khum tra pH khong 5,5 ri b sung ni ch sung vi khun, nm men, II1: 10 2  b sung 10 2 tbmn/g, II2: 10 2 c, sau 10 4 tbmn/g; II3: 10 2 c, sau 10 6 tbmn/g; II4: 10 4 c, sau 10 2 tbmn/g; II5: 10 4 c, sau 10 4 tbmn/g; II6: 10 4 c, sau 10 6 tbmn/g; II7: 10 6 c, sau 10 2 tbmn/g; II8: 10 6 c, sau 10 4 tbmn/g; II9: 10 6 c, sau 10 6 tbmn/g Kt qu        thy nghim thc I4 (b sung vi khut s 10 4 m men 10 2 t  m cao nh so vi m thi. Theo Jansen et al. (2003) vic b     n  Pediococcus halophillus  m men Zygosaccharomyces rouxii     trng trong vic t      sn ph   n chu mn bi        o v chua nh ng th   c bo qun sn ph  nm men s d t  u o     o v    ng thn  git tacid hu n ng v  i acid acetic, vi aci     t     acid amin phn ng v   t  sc t  c t     trn v  c t lo   n trong . T kt qu m thn  ti  n xu   i tin, ng thi nghim thi chng (m sung ch pht  sung ch phm thc sn xun thng. m amin  Bng 4 cho th      theo th       cao nht (9,36 gN/L)  nghim th      9 u so vm thi chng    m: (8,3 gN/L) nghim th    nghim th        nghim thc 4 (truyn thm amin thp nht. T Phn p, Thy s Sinh hc: 26 (2013): 205-212 211 Bng 4: m th Thi gian  m amin (gN/L)  CV (%) NT1 NT2 NT3 NT4 0 5,22 5,22 5,40 5,13 5,24 j 8,6 1,5 5,77 6,63 6,87 5,63 6,22 h 3,0 5,50 6,67 6,77 5,30 6,05 i 4,5 6,73 6,53 6,57 5,10 6.23 h 6,0 7,50 7,20 7,50 5,61 6,98 g 7,5 7,00 7,20 7,97 6,56 7,19 f 9,0 8,30 8,50 9,36 7,80 8,50 a 10,5 7,10 8,46 9,16 7,63 8,33 b 12,0 7,87 8,20 8,73 7,20 8,00 c 13,5 7,60 8,10 8,50 7,06 7,83 d 15,0 7,37 7,76 8,46 6,93 7,63 e  6,90 c 7,32 b 7,76 a 6,36 d CV (%) 15,0 n thng. S liu trong b ca 3 ln lp lt c mang ch s  (           m d p tc di s bi        trong su             th 15   m th   m thc       men-    m amin cao nht (8,m amin thp nh  nghim th-NT4 (6,93 gN/L), ng th y s  v  - so v     ng enzyme- NT1 (7,37 gN/L). M        m th    thm thi chng (NT1; NT2, NT4). Kt qu Bng 4 cho thy  r m amin cao nht      u so v           c hiu qu ca vic b    n th        m amin cao   i NT1      sung enzyme).  to ra sn ph  dng nghim th   vn xut. 3.4 n xuc m tra chng sn phm T  t     i tin vi 4  (1) to koji; (2) th    c mui;     Kt qu    Bng 5 cho thy sn ph      i ti t nh ca B Y T. Sn phm m t so vn phm th ng (4,0 -11 gN/L)      ng cao v  ng ng thi sn phm      hin din ca 3-  T Phn p, Thy s Sinh hc: 26 (2013): 205-212 212 B        ng ch yu ca sn ph   i ti  n phm th ng Ch  n quy nh  ci tin    CHIN-SU (Nht Ca) Soy sauce 501S- Senpio food company ng (g/L) Theo CBNXS 15,4 8,96 4,0 10 -11   4 8,4 - - 6 -7 NaCl (g/L) 140 - 220 163,8 160 150 155  165 Aflatoxin   - - - 3-MCPD 1mg/kg KPH 1mg/kg 1mg/kg - E. Coli   - - - Coliformes fecaux 10 2 /mL 0/mL - - - S. aureus 3/mL 0/mL - - - Cl. perfringens 10/mL 0/mL - - - Salmonella   - - - 4 KT LUN  chng ging thun Aspergillus oryzae vi mt s 10 6 /g chn thi gian  koji xu    so v    truyn thng kho- t hp b sung 2% ch phm enzyme     n th    50 o C, 24 gi c khi b sung mui cho hiu sut th m amin cao (44,53%). B sung vi khun Pediococcus halophillus vi mt s 10 4 t   m men Zygosaccharomyces rouxii 10 2 t   c mui cho sn phm n  t          -MCPD. U THAM KHO 1. B Y T, 2005. Danh mn v sinh i vc  thc ph theo Quynh s - a B ng B Y t, B Y ti. 2.   hc protein. Vi ngh Sinh hi hc C 3. ng Th ng di hc Quc gia, Tp.H  4.  thum quan. Nxb Khoa h thui. 5.  i Nghip, 2004. Thc ti hc Quc gia, Tp.H  6. p. Nxb i. 7. c Ph vi sinh vi. 8. Nguyc ng, 2002. Vi sinh vt hc i hc Quc gia TP.H  9. Steinkraus. K. H., 2004. Industrialization of Indigenous Fermented Food, 2 nd Edition, CRC Press, USA. 10. Jansen. M, J. H. Veurink, Gert  Jan W. Euverink, L. Dijkhuizen, 2003. Growth of the salt  tolerant yeast Zygosaccharomyces rouxii in microtiter plate: effects of NaCl, pH and temperature on growth and fusel alcohol production from branched  chain amino acids. FEMS Yeast Research. 11. Wood, J. B. Brian, 1994. Technology transfer and Indigenous fermented foods. Food Research International, 27, pp 269  280. .   c phm, Vi n  Sinh h  Ch phm enzyme protease (mua ti Vi n Sinh hc Nhi .  tn tr ging ti Vi n  Sinh h  Chng ging vi khun Pediococcus halophillus  m men Zygosacharomyces