Ứng dụng vi sinh vật và enzyme protease để cải tiến chất lượng nước tương lên men truyền thống potx

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.