Characterization of proteases of

Bacillus subtilis strain 38 isolated from

traditionally fermented soybean in Northern Thailand
Panuwan Chantawannakul
a,
*, Anchalee Oncharoen
a
, Khanungkan Klanbut
a
,
Ekachai Chukeatirote
b
and Saisamorn Lumyong
a
a
Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 Thailand.
b
Department of Biotechnology, School of Science, Mae Fah Luang University,
Chiang Rai 57100 Thailand.
* Corresponding author, E-mail: panuwan@yahoo.com
Received 12 Dec 2001
Accepted 27 Feb 2002
ABSTRACT E i gh ty-two baci llu s strai n s were i solated from T h ai ferm en ted soybean s thua nao , of wh i ch
th i rty-n i n e were i d en ti fi ed as Bacillus subtilis. C ru d e p ro tei n s fro m th ese B. subtilis strai n s were
i n vesti gated for th ei r p roteolyti c acti vi ty. Wh en tested on sk i m m i lk agar, th e cru d e p rotei n s of B.
subtilis strai n 38 exh i bi ted th e h i gh est p roteolyti c acti vi ty a clear zon e wi th an average area of 480
m m
2
) . O p ti m al p H an d tem p eratu re of th e p roteases from B. subtilis strai n 38 were fou n d to be at 6.5
an d 47

C , resp ecti vely. T h e p roteases were u n stable an d rap i d ly d ecreased i n acti vi ty wh en h eated at
60

C . Vari ou s p rotease i n h i bi tors were tri ed , on ly 1 , 1 0-p h en an th roli n e d ecreased th e en zym e acti vi ty
i n d i cati n g th e p resen ce of m etallop roteases. I n ad d i ti on , we attem p ted to p rod u ce ferm en ted soybean s
u si n g th e B. subtilisstrai n 38 as a starter cu ltu re.
K E YWO R D S: Thua nao, ferm en ted soybean , B. subtilis, p rotease.
ScienceAsia 28 (2002) : 241-245
INTRODUCTION
I n N orth ern T h ai lan d , ferm en ted soybean s, th e
so -called thua nao , h ave b een p ro d u ced an d
con su m ed locally for several d ecad es. C on ven ti on al
p rod u cti on of thua nao i s as follows: soybean s are
wash ed , so ak ed o vern i gh t, co o k ed b y b o i li n g fo r
abou t 3 4 h , gen tly sm ash ed an d wrap p ed i n si d e
ban an a leaves. T h e ferm en tati on gen erally p roceed s
fo r 2 3 d ays at am b i en t tem p eratu re.
1 , 2
A lter-
n ati vely, th e soybean p rod u cts are allowed to ferm en t
ou td oors exp osed to su n li gh t, resu lti n g i n a d ri ed
form of thua nao th at can be k ep t for several m on th s.
I ts u se i s versati le, an d fresh thua nao, for exam p le
can be con su m ed by steam i n g or roasti n g wh i le th e
d ri ed p rod u cts are i m p ortan t i n gred i en ts i n a vari ety
of local d i sh es.
Si m i lar ferm en ted soybean p rod u cts h ave been
d escri bed i n several cou n tri es, i e kinema i n I n d i a,
3-5
schuidouchi i n C h i n a,
6
an d natto i n Jap an .
7
O th er
related p rod u cts i e ugba an d iru m ad e wi th legu m e
seed s i n stead of soybean s h ave also been rep orted
i n Western A fri ca.
8 , 9
A m o n g th ese, o n ly a few
ferm en ted so yb ean p ro d u cts h ave b een stu d i ed
system ati cally an d m an u factu red i n d u stri ally. T h e
b est-ch aracteri zed ferm en ted so yb ean p ro d u ct i s
p ro b ab ly nattoth e Jap an ese styled ferm en ted
soybean s. Natto i s com m erci ally p rep ared u si n g a
p u re starter cu ltu re of B. subtilis n atto strai n .
7
I t h as
b een esti m ated th at th e co n su m p ti o n o f natto i n
Jap an alon e exceed s 50,000 ton s an n u ally.
Several Bacillus sp eci es h ave been fou n d to be
stron gly associ ated wi th th ese ferm en ted soybean
p rod u cts. F or exam p le, O gbad u et al
1 0
i d en ti fi ed a
variety of Bacillusspecies ie B. subtilis, B. laterosporus,
B. pumilus, B. brevis, B. macerans, B. licheniformis,
B. polymyxa, an d B. coagulans fro m N i geri an
ferm en ted soybean food s. I t sh ou ld also be n oted
th at oth er bacteri al sp eci es su ch as lacti c aci d bacteri a
or Enterococcus sp eci es m ay exi st.
4
H owever, d u e to
th e i n i ti al b o i li n g o f b ean s i n p rep arati o n , th e
ex i sten ce o f n o n -Bacillus sp eci es i s li k ely to b e a
con tam i n ati on d u ri n g an d /or after th e ferm en tati on
process. B. subtilis, a G ram -positive, en dosporeform in g
bacteri a , h as u su ally been fou n d as th e p red om i n an t
m i croorgan i sm i n th ese ferm en ted soybean food s.
T h e abi li ty of B. subtilisto grow over a wi d e p H ran ge
wi th an acti ve growth between p H 5.5 8.5) an d to
p rod u ce several en zym es i e p roteases an d oth er
u sefu l bi ologi cal com p ou n d s
1 1
seem s a li k ely reason
for i ts su p eri ori ty i n th e soybean ferm en tati on . I n
ad d i ti on , th e ben efi ci al effect of B. subtilis ferm en ta-
ti on , p arti cu larly on natto, h as been well- establi sh ed
i n clu d i n g th e p resen ce of gen i stei n an an ti -tu m or
242 ScienceAsia 28 (2002)
agen t) an d so m e p ro teo lyti c en zym es th at can
d egrad e stap h ylococcal en terotoxi n .
1 2-1 6
I n con trast, thua nao p rod u cti on i s sti ll p racti ced
locally wi th li ttle i n form ati on regard i n g m i crobi al
p op u lati on an d ferm en tati on p rocess. O u r ai m was
to exam i n e th e m i croorgan i sm s resp on si ble for su ch
ferm en tati on . I n th i s stu d y, several B. subtilis i solates
were screen ed from thua nao an d i n vesti gated for
th ei r p roteolyti c acti vi ty. I t was fou n d th at B. subtilis
strai n 38 sh owed th e h i gh est p rotease acti vi ty. Som e
fu n d am en tal ch aracteri sti cs o f th e p ro teases were
also determ in ed. I n addition , we attem pted to develop
a lab o rato ry scale p ro cess fo r m ak i n g ferm en ted
soybean s u si n g B. subtilis strai n 38 as a p u re starter
cu ltu re.
MATERIALS AND METHODS
Isolation and classification of spore-forming
bacteria
T h ai ferm en ted soybean p rod u cts thua nao were
collected from si x p rovi n ces i n N orth ern T h ai lan d :
C h i an g M ai , C h i an g R ai , L am p an g, P h rae, P ayao,
an d M ae H o n gso n . A b o u t 0 . 1 g o f sam p le was
tran sferred to 3.0 m l steri le d i sti lled water, sh ak en
vi gorou sly an d h eld at 75

C for 20 m i n to d estroy
all vegetati ve m i crobi al cells. T h e su sp en si on was
th en p lated ou t on n u tri en t agar 1 % p ep ton e, 0.5%
b eef ex tract, 0 . 5 % N aC l, an d 1 . 5 % ag ar) an d
i n cu bated at 37

C for 24 h . B acteri al colon i es were
i solated an d ch aracteri zed by th ei r m orp h ologi cal
an d p h ysi ologi cal p rop erti es.
1 1 ,1 7
T h ese i n clu d ed cell
sh ap e, G ram stai n i n g, p resen ce of sp ore, an d growth
con d i ti on s aerobi c or an aerobi c) . F u rth er classi -
fi cati on was p erform ed u si n g bi och em i cal tests as
d escri bed by N orri s et al.
1 7
Preparation of crude enzyme
I solated bacteri a were cu lti vated i n 50 m l n u tri en t
broth wi th vi gorou s sh ak i n g 1 50 rp m ) at 37

C for
24 h . T h e cu ltu re was th en cen tri fu ged at 1 0,000
rp m for 5 m i n at 4

C . T h e su p ern atan t was collected
an d u sed as th e en zym e solu ti on .
Determination of proteolytic activity
I n i ti ally, p rotease acti vi ty was tested u si n g sk i m
m i lk agar 1 % sk i m m i lk , 0.02% sod i u m azi d e, an d
2.0% agar . F or th i s, 5 l of cru d e p rotei n s p rod u ced
from i solated bacteri a were sp otted on sk i m m i lk
agar p lates. T h e p lates were th en i n cu bated at 37

C
for 1 8 h . P roteolyti c acti vi ty of th e cru d e p rotei n s
was d etected b y o b servi n g th e p resen ce o f clear
zon es. Accord i n g to C oop er,
1 8
acti vi ty of bi ologi cal
su b stan ces i e, an ti b i o ti cs an d en zym es) can b e
exp ressed i n term s of th e squ are of th e d i am eter of
th e clear zon e.
After i n i ti al screen i n g for p roteolyti c acti vi ty, B.
subtilis strai n 3 8 ex h i b i ted h i gh est acti vi ty o f
p roteases an d was u sed th rou gh ou t th e exp eri m en t.
D eterm i n ati on of th e en zym e acti vi ty was p erform ed
u si n g th e azocasei n m eth od .
1 9
20 l of th e cru d e
en zym es were i n cu bated at 37

C i n a m i xtu re 400
l) con tai n i n g 2% azocasei n 230 l) , an d 0.2 M N -
tri s[h yd roxym eth yl]m eth yl-2-am i n oeth an esu lfon i c
aci d T E S) b u ffer p H 7 . 0 1 5 0 l) fo r 2 h . To
term i n ate th e reacti on , 1 .2 m l of 1 0% tri ch loroaceti c
aci d T C A) was ad d ed . All sam p les were allowed to
stan d for 1 5 m i n an d th e su p ern atan t ca 1 .4 m l)
was co llected after cen tri fu gati o n 1 0 , 0 0 0 rp m ; 5
m i n ) . An equ i valen t volu m e of 1 M N aO H was th en
ad d ed an d th e solu ti on was m i xed th orou gh ly p ri or
to m easu ri n g th e absorban ce at 440 n m .
Influence of protease inhibitors
I n h i bi tors, k n own to affect d i fferen t classes of
p ro teases,
2 0 , 2 1
th at were u sed were fo llo ws: 3 , 4 -
d i ch loroi socou m ari n D C I ) , p h en ylm eth ylsu lfon yl-
flu o ri d e P M SF ) , P ep stati n A, E -6 4 tran s-ep o x y-
su cci n yl-leu cylam i d o 4-gu an i d i n o) bu tan e) , an d 1 ,
1 0 -p h en an th ro li n e. T o d eterm i n e wh eth er th e
acti vi ty of p roteases cou ld be affected , each i n h i bi tor
was ad d ed to th e cru d e en zym es an d i n cu bated at
4
o
C for 20 m i n . P rotease acti vi ty was th en m easu red
u si n g th e azocasei n m eth od .
Fermentation of soybean (thua nao) using B. subtilis
strain 38 as inoculant
Soybean s ap p rox. 1 50 g for each exp eri m en t)
were wash ed th orou gh ly an d soak ed i n water for 1 6
h at room tem p eratu re. After d ecan ti n g th e water,
soak ed soybean s were steri li zed by au toclavi n g at
1 21

C for 1 5 m i n . T h ree m l-of sp ore su sp en si on
ca 7 x 1 0
1 0
sp ores/m l) of th e B. subtilis strai n 38
was ad d ed to th e steri li zed soybean s. T h e ferm en ta-
ti on was th en carri ed ou t at 37 C an d 45

C for 72 h
i n th e trad i ti on al T h ai -style ferm en tati on
1 ,2
wh ereas,
i n th e natto-style ferm en tati o n , th e ferm en ted
soybean s were k ep t i n a refri gerator 6 8

C ) for 48
h after th e fi rst 24 h ferm en tati on . T h e p H of th e
ferm en ted soybean s was m on i tored every 6 h .
RESULTS AND DISCUSSION
I n th i s stu d y, several sam p les of thua nao were
co llected lo cally fro m d i fferen t so u rces i n si x
p ro vi n ces i n th e N o rth ern p art o f T h ai lan d . To
ScienceAsia 28 (2002) 243
d estroy all vegetati ve cells an d th u s selecti vely i solate
b acteri a i n th e gen u s Bacillus, each sam p le was
resu sp en d ed i n d i sti lled water, i n cu bated at 75

C
fo r 2 0 m i n an d sp read o n n u tri en t agar p lates.
E i gh ty-two b acteri al i so lates were o b tai n ed an d
fu rth er i d en ti fi ed u si n g b i o ch em i cal assays as
su ggested b y N o rri s et al.
1 7
K ey i d en ti fi cati o n o f
Bacillus sp eci es i n clu d ed catalase assay, Vo ges
P ro sk au r V P ) test, gro wth u n d er an aero b i c co n -
d i ti on , an d starch h yd rolysi s. O f all i solated bacteri a,
th i rty-n i n e 4 7 . 5 % ) i so lates were classi fi ed as B.
subtilis yi eld i n g p osi ti ve r eacti on for catalase, VP,
an d starch h yd ro lysi s tests, an d u n ab le to gro w
u n d er an aerobi c con d i ti on ) . T h e B. subtilis sp eci es
i s of p arti cu lar i n terest d u e to i ts p revalen ce i n th ese
ferm en ted food s.
2, 1 0
I n ad d i ti on , u se of B. subilis i n
th e food i n d u stry i s recom m en d ed by th e U S F D A
as on e of th e G R AS G en erally R ecogn i zed As Safe)
organ i sm s.
22, 23
T h e B. subtilis i solates were th en ch aracteri zed
for p rotease p rod u cti on . U si n g sk i m m i lk agar, th e
p roteolyti c acti vi ty cou ld be d etected by th e p resen ce
of a clear zon e. I t was fou n d th at B. subtilis strai n
38 yi eld ed th e h i gh est p rotease acti vi ty wi th a clear
zon e of ap p roxi m ately 480 m m
2
F i g 1 ) . R elati on sh i p
b etween p ro tease p ro d u cti o n an d gro wth o f B.
subtilis strai n 38 was d eterm i n ed . F or th i s, B. subtilis
strai n 3 8 was cu lti vated i n n u tri en t b ro th . C ell
d en si ty as m easu red by absorban ce at 660 n m ) an d
p rotease acti vi ty as m easu red by p resen ce of th e
clear zon e on sk i m m i lk agar were m on i tored every
4 h . T h e resu lts sh o wed th at th e p ro d u cti o n o f
p roteases was d ep en d en t on bacteri al growth F i g
2) . D u ri n g th e baci llu s growth , cell m orp h ology was
also ex am i n ed : all b acteri al cells ap p eared i n
vegetati ve fo rm s d u ri n g th e fi rst 1 2 h , b u t sp o re
fo rm ati o n was o b served b etween 1 6 an d 2 4 h o f
th e i n cu bati on p eri od . T h i s resu lt su ggested th at
p ro d u cti o n o f p ro teases reach ed th e h i gh est level
d u ri n g th e ex p o n en ti al p h ase an d co n ti n u ed
con stan t level wh en sp ores were form ed stati on ary
state) .
T h e effect of som e p h ysi cal factors, su ch as p H
an d tem p eratu re, on th e acti vi ty of th e cru d e en zym e
were i n vesti gated u si n g azocasei n as a su bstrate F i g
3 an d 4) . T h e op ti m u m tem p eratu re an d p H for th e
en zym e acti vi ty were 47

C an d 6.5, resp ecti vely. I n
ad d i ti o n , wh en testi n g th e en zym e stab i li ty was
tested at d i fferen t tem p eratu res 40

C , 50

C , an d
6 0

C ) , wi th th e B. subtilis p ro teases b ei n g m o st
affected at 60

C as th ey exh i bi ted a rap i d d ecrease

i n en zym e acti vi ty. I n cu bati on at 40

C an d 50

C
sh owed sli gh t effect on en zym e acti vi ty as m ore th an
80 an d 50% of p roteolyti c acti vi ty resp ecti vely cou ld
sti ll be d etected after 60 m i n assay F i g 4B ) .
P rotease en zym es of B. subtilis gen erally can be
categori zed i n to two grou p s wi th resp ect to th ei r
o p ti m al p H n eu tral an d alk ali n e p ro teases. T h e
form er exh i bi t op ti m al p H at 7.0, wh ereas th e latter
h ave p H op ti m a between 9 1 1 .
20
T h erefore, th e
cru d e p rotease en zym es p rod u ced from B. subtilis
strai n 38 belon g to th e n eu tral grou p si n ce th ey h ad
o p ti m al p H at 6 . 5 . To fu rth er ch aracteri ze th e
Fig 1. C om p arati ve resu lts of p roteolyti c acti vi ty of B. subtilis
strai n s i solated from thua nao, T h ai ferm en ted soybean s.
T h e p roteolyti c acti vi ty was assayed u si n g sk i m m i lk agar
an d exp ressed as a squ are of th e clear zon e d i am eter.
1 8
Fig 2. R elati on sh i p between cell growth an d p rotease p rod u cti on
of B. subtilisstrai n 38. C ell growth I was m easu red by
ab so rb an ce at 6 6 0 n m an d p ro tease acti vi ty I) was
d eterm i n ed by p resen ce of th e clear zon e on sk i m m i lk
agar.
Fig 3. E ffect of p H on acti vi ty of th e Bacillus subtilis 38 p roteases.
G ood 's bi ologi cal bu ffers M E S, P I P E S, T E S, TAP S, C H E S
Si gm a) ) were u sed to cover a p H ran ge 5.5-1 0 followi n g
th e m an u factu re's recom m en d ati on . T h e cru d e extract
u sed for each bu ffer was 20 l.
244 ScienceAsia 28 (2002)
p ro teases, vari o u s typ es o f i n h i b i to rs were u sed .
O n ly 1 , 1 0-p h en an th roli n e was clearly able to i n h i bi t
th e en zym e acti vi ty T ab le 1 ) i n d i cati n g th at th e
p rotease i s a m etallop rotease s .
F or m an y d ecad es, thua nao h as been p rod u ced
i n a con ven ti on al m an n er. T h e ferm en tati on p rocess
th u s m ai n ly d ep en d s on a m i xtu re of bacteri al sp eci es
alth ou gh , n ot su rp ri si n gly, th e Bacillus sp eci es h ave
p layed a k ey role. T h ere i s also a lack of system ati c
p roced u re i n clu d i n g n o tem p eratu re con trol. H en ce,
th e qu ali ty of th e ferm en ted p rod u cts i s n ot u n i form .
U si n g a su i table B. subtilis sp eci es as a p u re starter
cu ltu re i s a p ro m i si n g ap p ro ach to i m p ro ve th e
ferm en tati on p rocess, wh i ch m ay lead to com m erci al-
scale p rod u cti on . C on sequ en tly, we attem p ted to
u ti li ze B. subtilis strai n 38 i solated i n a laboratory-
scale soybean ferm en tati on . F or th i s, ferm en tati on
of soybean s was carri ed ou t i n both trad i ti on al T h ai
an d natto styles at 3 7

C an d 4 5

C . D u ri n g th e
ferm en tati on p rocess, ch an ges i n p H were m on i tored
every 6 h . G en erally, th e tren d s of p H ch an ge of
each ferm en tati on batch were si m i lar wi th i n i ti al p H
of 6.0 an d grad u ally i n creased to ap p roxi m ately 8.0
after 72 h ferm en tati on F i g 5) . T h e i n creased i n
p H of each batch was p ossi bly d u e to p roteolysi s
an d am m on i a release.
3
Accord i n g to F i g 5, th e p H
ch an ges were m ost d ram ati c at 1 2 h of ferm en tati on .
T h e d i fferen ce d u e to tem p eratu re con d i ti on s 37 C
an d 45

C ) was also clearly observed at th i s p eri od ,
both ferm en ted soybean sam p les obtai n ed by th e
thua nao an d nattom eth od s sh owed h i gh er p H 7.59
an d 7.85) at 45

C th an th ei r cou n terp arts at 37

C
7.24 an d 7.01 ) . T h e h i gh er p H valu es at 45

C were
li k ely to be th e resu lt of h i gh er acti vi ty of B. subtilis
proteases. T h erefore, ferm en tation at h igh tem peratu re
4 5

C ) m ay rep resen t a rap i d m ean s fo r so yb ean
ferm en tati on . B y stati sti cal an alysi s SP SS/F ri edm an ) ,
soybean ferm en tati on by B. subtilis 38 at 45 C a
gave m o re sati sfacto ry o d o r an d co lo r o f fi n al
p ro d u cts th an th at at 3 7
o
C p < 0 . 0 1 ) . I t wi ll b e
wo rth wh i le ex am i n e th e n u tri ti o n al v alu es o f
ferm en ted soybean s i n th e fu tu re exp eri m en ts. F rom
th i s stu d y, th e selected strai n m ay be regard ed as a
p o ten ti al starti n g cu ltu re d u e to i ts h i gh ly acti ve
p ro teo lyti c acti vi ty an d h en ce rap i d ferm en tati o n
Table 1. Effect of inhibitors on activity of protease
enzymes obtained from B. subtilis strain 38.
Inhibitors used
a
Specificity Relative
activity
b
None 100
1,10-phenanthroline Metalloprotease 10.39
DCI Serine protease 84.87
E64 Cysteine protease 93.18
PMSF Serine protease 94.07
Pepstatin A Aspatyl protease 100
a
T h e fi n al con cen trati on of th ese i n h i bi tors i n th e reacti on i s
as follows: 1 m M for 1 ,1 0-p h en an th roli n e; 0.1 m M for D C I
an d E 64; 0.5 m M for P M SF, an d 0.5 g/m l for P ep stati n A.
b
T h i s assay was p erfo rm ed u si n g azo casei n m eth o d
1 9
. T h e
relati ve acti vi ty was calcu lated u si n g th e A440n m valu e of
th e con trol reacti on n o i n h i bi tor as 1 00 % en zym e acti vi ty.
T h e cru d e extract u sed i n each treatm en t was 1 5 l.
Fig 4. E ffect of tem p eratu re on p rotease acti vi ty A) an d stabi li ty
B ) . F o r tem p eratu re stab i li ty B ) , th e cru d e en zym e
solu ti on were h eld at 40 C +, 50 C I) , an d 60 C O ) ,
an d at i n tervals su bsam p les 20l, 0-60 m i n ) were rem oved
an d th en assay ed b y th e azo casei n m eth o d at
37 C . M E S bu ffer p H 6.5) was u sed for both exp eri m en ts.
Fig 5. C h an ge i n p H d u ri n g soybean ferm en tati on . F erm en tati on
of soybean s was carri ed ou t i n th e trad i ti on al T h ai thua
nao an d n atto styles; each ferm en tati o n b atch was
p erform ed at 37 C -thua nao, 37 C ;

-thua nao, 45 C ;
O -natto, 37 C ; -natto, 45 C .
ScienceAsia 28 (2002) 245
wou ld be exp ected . T h e n eu tral p roteases p rod u ced
d u ri n g th e ou tgrowth or log p h ase p resu m ably p lay
an i m p ortan t role i n soybean ferm en tati on . T h i s
strai n h o ld s great p ro m i se fo r th e p ro d u ct, an d
provides attractive possibilities for fu rth er optim ization
as an i n d u stri al strai n to i m p rove th e soci o-econ om i c
ben efi ts of trad i ti on al food s.
ACKNOWLEDGEMENTS
We wo u ld li k e to ack n o wled ge th e P ro f D r
N atth B h am arap ravati research fu n d , C h i an g M ai
U n i versi ty, C h i an g M ai , T h ai lan d , fo r fi n an ci al
su p p o rt. T h an k s also to A sso ci ate P ro fesso r D r
Arayar Jati sati en r for h er ad vi ce i n stati sti cal an alysi s.
I n ad d i ti on , we wou ld li k e to exp ress ou r th an k s to
D ep artm en t of B i ology, F acu lty of Sci en ce, C h i an g
M ai U n i versi ty, C h i an g M ai , T h ai lan d for p rovi d i n g
laboratory faci li ti es.
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