THE TOLERANCE OF BACILLUS SUBTILIS AND BACILLUS PUMILUS ORIGINATED FROM BOVINE COLUSTRUM TOWARDS ACIDITY AND BILE SALT AS A PROBIOTICS CANDIDATE.
THE TOLERANCE OF Bacillus subtilis AND Bacillus pumilus ORIGINATED FROM BOVINE COLOSTRUM TOWARD
ACICITY AND BILE SALT AS A PROBIOTICS CANDIDATE
1
2
2
Khusnul Khotimah , Ratu Safitri , Nia Rossiana , Anisa Syara
1
2
2
Agriculture and Farms Faculty, Muhammadiyah Malang University, Malang
Biology Department, Mathematics and Natural Science Faculty, Padjadjaran University, Jatinangor
Kj
Introduction
Bo i e olost u is the p e- ilk luid p odu ed the a
a gla ds of atle ate i th B a da o et al.,
. Colost u also o tai p o ioi a te ia F a dso ,
. P o ioi s a e i oo ga is s that a suppo t the ala e of i olo a i
the digesi e t a t. P o ioi a te ia has the ha a te isi s a e tole a e to a idit , tole a e to ile salt, also p odu i g a i i o ial o pou ds. Ba illus is a G a posii e, spo es a e esista t to high te pe atu es. Ba illus is a le to g o at a idit , is a le to
g o at , % ile salt o e t aio s, so e Ba illus p odu e e t a ellula e z es su h as p otease, lipase, a lase, a d ellulase hi h a help the digesi e s ste Chou a d Wei e
; Spi osa et al.,
; Ba osa et al.,
. Ba illus su ilis, Ba illus
lausii, Ba illus pu ilus, a d Ba illus oagula s i luded i o
e ial p o ioi p odu ts Sa de s,
. I this stud e e pe ted spe ies of Ba illus o igi ated f o
o i e olost u that ha e ha a te of a idit a d ile salts tole a e so it a e used as
a pote iall p o ioi a te ia. This stud a e a asis fo the used of p o ioi s f o
o i e olost u to e de eloped i to a o
e ial p o ioi p odu ts.
Materials
Ba te ial ultu e of the ge us Ba illus isolates o tai ed f o
o i e olost u , Al ohol
% , N CH COOH, ile salt O oid L-
, API
CHB Bio e ieu ,
ediu
Nut ie t Aga O oid , ph siologi al NaCl.
Method
The stud o sisted of t o phases, the des ipi e ethod fo the i st phase is the ide ii aio of Ba illus o igi ated of o i e olost u usi g API CHB. The e pe i e tal ethod used i the se o d phase, a el the ha a te izaio of the
Ba illus p o ioi a didates ith o pletel a do ized desig CRD ith fa to s o sisi g of t o tests a e tole a e of a idit test, a d tole a e of ile salt test. Tole a e of a idit test efe s to Ha dia i gsih
ith
fa to ial t eate t i st fa to is the t pe of a didate p o ioi a te ia Ba illus B hi h o sists of t o le els, Ba illus su ilis
a d Ba illus pu ilus
. The se o d t eat e t fa to is the le el of a idit P hi h o sists of le els of pH p , pH p a d
pH p . Tole a e of ile salt test efe s to Bu i et al,
ith
fa to ial t eat e t i st fa to is the t pe of a didate p o ioi a te ia Ba illus B hi h o sists of t o le els, Ba illus a didates A p o ioi
a d Ba illus p o ioi a didate B
. The se o d t eat e t fa to is the o e t aio of ile salts E hi h o sists of t o ile salt o e t aio le el of . % e , a d ile salt o e t aio of . % e . Pa a ete s easu ed at tole a e of a idit test is the u e of
olo ies of CFU/ l to dete i e the highest tole a e to the a idit . Pa a ete s easu ed i tole a e of ile salt test is the u e of olo ies of CFU/ l e e g o o solid ediu o tai i g ile salts . % a d . %. The data o tai ed ill the
Result and Discussion
Ide ii aio ge us Ba illus a te ia f o
o i e olost u
usi g API
CHB has fou d Ba illus su ilis o tai ed ith a u a
. % Figu e
a d Ba illus pu ilus ith a u a
. % Figu e
.
Tolerance of Acidity
Based o the the a al sis of a ia e ANOVA esults sho ed that the alue of F ou t > F ta le, hi h ea s Ba illus su ilis a d Ba illus pu ilus has i te a io to pH. Be ause the ANOVA esults a e eal the it ould e follo ed
Du a 's
Muliple Ra ge Test DMRT at sig ii a e le el % Ta le . Based o the Ta le the g o th of B. su ilis a d B. pu ilus i lue ed the pH the highe the pH, the o e olo ies g o . Ba illus su ilis a d Ba illus pu ilus at pH has the est ia ilit . It a e see f o the highest ate i the t eat e t of B. su ilis pH
ith a populaio of .
CFU/ l a d ith a populaio of B. pu ilus .
CFU/ l. I DMRT esults a e see that pH is opi u pH fo the g o th of the
a te ia, this is i a o da e ith the atu e of hi h is Ba illus eut ophils e ui es a opi u pH is . - . . At pH B. su ilis a d B. pu ilus a sill g o
ell. Ba illus su ilis is o e tole a t of a id to pH o pa ed ith Ba illus pu ilus ith a
populaio of .
CFU/ l a d .
CFU/ l. Whe eas at pH B. su ilis a d B. pu ilus a ot g o . Ba te ial Tole a e to a idit aused
the a ilit to ai tai a toplas i pH o e alkali e tha the e t a ellula pH. Highl a idi
o diio s at pH has a e st o g i lue e of B. su ilis a d B. pu ilus. A id i hi iio of a te ial ell g o th o u s th ough the i lue e of de atu aio of e z es that e ist o the ell su fa e, lipopol sa ha ide a d oute e
a e is da aged a d a de ease i
toplas i pH th ough i eased e
a e pe ea ilit Us iai,
. Ba ilus pu ilus a d Ba illus su ilis ased o the esults of tests of tole a e to a id a g o i a id to pH , so B. su ilis a d B. pu ilus has o e of
the e ui e e ts as a a didate p o ioi has tole a e of a idit .
Tolerance of Bile Salts
Based o the a al sis of a ia e ANOVA esult ho ed that the alue of F ou t > F ta le, hi h ea s Ba illus su ilis a d Ba illus pu ilus has i te a io to ile salt o e t aio of . % a d . %. Be ause the ANOVA esults a e eal the it
ould e follo ed Du a 's Muliple Ra ge Test DMRT at sig ii a e le el % Ta le . Based o Ta le sho s that the t eat e t of . % gi es the lo est alue of a te ial ia ilit , he eas . % t eat e t ga e the highest alues of a te ial
ia ilit . It is sho
f o the highest ate i the t eat e t of ile salt o e t aio of . % ith a populaio of .
CFU/ l i Ba illus pu ilus. Ta o k
, states that p o ioi a te ia should e a le to a hie e i the digesi e t a t
ai tai i g a populaio of
CFU/ l a d a p o ide health e eits. Media NA + . % ile salt is a ediu
hose o diio ese les the digesi e t a t o tai i g ile as su fa e-a i e o pou ds that a pe et ate a d ea t ith the toplas i e
a e Hill,
. The highe the o e t aio of ile salts the fe e a te ia g o i g populaio . It a e see that the u e of olo ies of Ba illus su ilis a d Ba illus pu ilus i lue ed the o e t aio of ile salts. Good esista e to ile salts o u s e ause of the ole of pol sa ha ides as a o situe t of the ell all of G a -posii e a te ia. Ba illus su ilis a d Ba illus pu ilus has esista e to ile salts ith a ii al o e t aio . A o di g )a aglia et al.
,
. % ile salt o e t aio is high e ough o e t aio s to sele t st ai s that a e esista t to ile salts, a d all the i o es that su i ed ate g o i
ediu plus . % ile salts, othe ise e esista t to ile salts . This is o e of the e ui e e ts
of a de la ed p o ioi a te ia.
Bacillus subtilis
Figure . Result Idenii aion Ba illus su ilis
Bacillus pumilus
Figure . Result Idenii aion Ba illus pumilus
Ta le . Dun an's Muliple Range Test Toleran e Ba illus su ilis and Ba illus pumilus to pH
Bacteria CFU/ml
pH
Bacillus subtilis
Bacillus pumilus
nd C
a
nd C
a
2
5
4
55,12 x 10 B
16,40 x 10 B
a
b
35,38 x 10 A
19,36 x 10 A
a
b
u
pH B. pu ilus
pH B. pu ilus
pH B. pu ilus
Bacillus subtilis and Bacillus pumilus population growth on
pH , , and agar plates medium
Dife e t lo e ase lete s ead i to the ho izo tal di e io i di ates sig ii a tl dife e t p> .
i ated
pH B. su ilis
8
Des ipio : Dife e t apital lete s ead i to the e i al di e io i di ates sig ii a tl dife e t p> .
d: ot dete
pH B. su ilis
5
8
6
pH B. su ilis
e of olo ies CFU/ l
Ta le . Dun an's Muliple Range Test Toleran e Ba illus su ilis and
Ba illus su ilis
Ba illus pumilus to Bile Salt
Bacteria CFU/ml
Bile salt
(%)
Bacillus subtilis
Bacillus pumilus
8
0,3
, % ile salts
8
6,692 x 10 A
8,745 x 10 A
b
a
8
Ba illus pu ilus
8
3,506 x 10 B
0,5
, % ile salts
5,570 x 10 B
b
, % ile salts
a
Des ipio : Dife e t apital lete s ead i to the e i al di e io i di ates sig ii a tl dife e t p> .
, % ile salts
Bacillus subtilis and Bacillus pumilus population growth on
bile salts 0, % and 0, % agar plates medium
Dife e t lo e ase lete s ead i to the ho izo tal di e io i di ates sig ii a tl dife e t p> .
Conclusion
Based o the test esults of the p o ioi p ope ies of Ba illus su ilis a d Ba illus pu ilus i esista e to lo pH a d ile salts, pe fo ed a al sis of a ia e to dete i e the pote ial oth as a a didate p o ioi
Ba illus pu ilus eet t o e ui e e ts as a a didate p o ioi that is tole a t of lo pH at pH a d pH a d tole a t to ile salts at a o e t aio of . % a d . %.
Reference
Ba osa, T.M., Se a, C.R., La Ragio e, R.M., Wood a d, M.J., a d He i ues, A.O.,
B a da o P, Rassu SPG, La zu A.
. Feedi g Dai
Bu i , N., Cha tha hu , S., Ho gpata ake e, T.
Chou L.). a d Wei e B.
F a dso , R.D.
Hill, M. J.
s. Dala
Puli e G. B i i R Edito J Dai
. S ee i g of La i A id Ba te ia F o
. Role of Gut Ba te ia i Hu a To i olog a d Pha
S i
-
-
.
a olog . Ta lo , Ne Yo k.
. Staisi al P o edu es fo Ag i ultu al Resea h. UI P ess, Jaka ta.
e s as Hu a P o ioi s: Ba illus, Sporola to a illus, a d Brevi a illus. Co p ehe si e Re ie I Food S ie e a d Food Safet Vol. ,
Spi osa, M.R., B a i i, T., Ri a, E., De Feli e, M., Mo elli, L., Pozzi, G., a d Oggio i, M.R.,
o k, G. W.
st ai s of L.a idophilus. J Dai
a d Fisiolog . th Ediio . Gadjah Mada U i e sit P ess, Yog aka ta.
Sa de s M. E, L. Mo elli, a d T.A. To pki s. Spo efo
Ta
e tal Mi o iolog
. A f esh look at the i tesi al
Us iai S, B oto W, da Sei a to H.
. O the fate of i gested Ba illus spo es. Resea h i Mi o iolog
:
-
ik olo a. Ho izo S i ii P ess.
. Cha a te isi s of o 's
)a aglia, A.G., G Ko iu i sk , P. Pe ez a d G. De A to i.
ilk u d usi g p o ioi
:
-
.
Sheep Nut iio Walli g Food : ABI . Pu lishi g.
. Isolaio a d Resista e Test of Se e al Isolates of La to a illus i Lo pH. LIPI , Bogo
Go ez, A.A. da Go ez, K.A.
oile gast oi tesi al t a t. Applied a d E i o
Gast oi tesi al T a s of Ma i e Fish Fo Thei Pote ial Use As P o ioi s. So gkla aka i J. S i. Te h ol.
. Isolaio a d ha a te izaio of a id a d ile tole a t isolates f o
. A i al A ato
Ha di i gsih, R.
La
. S ee i g fo Ba illus isolates i the
a te ia sta te . Jou al of A i al a d Vete i a
. Isolaio a d Cha a te izaio of Biido a te iu
st ai s fo p o ioi fo
S ie es,
:
ulaio . J. Food P ote t.
:
-
.
Suppl. .
a te ia. It is k o
that Ba illus su ilis a d
ACICITY AND BILE SALT AS A PROBIOTICS CANDIDATE
1
2
2
Khusnul Khotimah , Ratu Safitri , Nia Rossiana , Anisa Syara
1
2
2
Agriculture and Farms Faculty, Muhammadiyah Malang University, Malang
Biology Department, Mathematics and Natural Science Faculty, Padjadjaran University, Jatinangor
Kj
Introduction
Bo i e olost u is the p e- ilk luid p odu ed the a
a gla ds of atle ate i th B a da o et al.,
. Colost u also o tai p o ioi a te ia F a dso ,
. P o ioi s a e i oo ga is s that a suppo t the ala e of i olo a i
the digesi e t a t. P o ioi a te ia has the ha a te isi s a e tole a e to a idit , tole a e to ile salt, also p odu i g a i i o ial o pou ds. Ba illus is a G a posii e, spo es a e esista t to high te pe atu es. Ba illus is a le to g o at a idit , is a le to
g o at , % ile salt o e t aio s, so e Ba illus p odu e e t a ellula e z es su h as p otease, lipase, a lase, a d ellulase hi h a help the digesi e s ste Chou a d Wei e
; Spi osa et al.,
; Ba osa et al.,
. Ba illus su ilis, Ba illus
lausii, Ba illus pu ilus, a d Ba illus oagula s i luded i o
e ial p o ioi p odu ts Sa de s,
. I this stud e e pe ted spe ies of Ba illus o igi ated f o
o i e olost u that ha e ha a te of a idit a d ile salts tole a e so it a e used as
a pote iall p o ioi a te ia. This stud a e a asis fo the used of p o ioi s f o
o i e olost u to e de eloped i to a o
e ial p o ioi p odu ts.
Materials
Ba te ial ultu e of the ge us Ba illus isolates o tai ed f o
o i e olost u , Al ohol
% , N CH COOH, ile salt O oid L-
, API
CHB Bio e ieu ,
ediu
Nut ie t Aga O oid , ph siologi al NaCl.
Method
The stud o sisted of t o phases, the des ipi e ethod fo the i st phase is the ide ii aio of Ba illus o igi ated of o i e olost u usi g API CHB. The e pe i e tal ethod used i the se o d phase, a el the ha a te izaio of the
Ba illus p o ioi a didates ith o pletel a do ized desig CRD ith fa to s o sisi g of t o tests a e tole a e of a idit test, a d tole a e of ile salt test. Tole a e of a idit test efe s to Ha dia i gsih
ith
fa to ial t eate t i st fa to is the t pe of a didate p o ioi a te ia Ba illus B hi h o sists of t o le els, Ba illus su ilis
a d Ba illus pu ilus
. The se o d t eat e t fa to is the le el of a idit P hi h o sists of le els of pH p , pH p a d
pH p . Tole a e of ile salt test efe s to Bu i et al,
ith
fa to ial t eat e t i st fa to is the t pe of a didate p o ioi a te ia Ba illus B hi h o sists of t o le els, Ba illus a didates A p o ioi
a d Ba illus p o ioi a didate B
. The se o d t eat e t fa to is the o e t aio of ile salts E hi h o sists of t o ile salt o e t aio le el of . % e , a d ile salt o e t aio of . % e . Pa a ete s easu ed at tole a e of a idit test is the u e of
olo ies of CFU/ l to dete i e the highest tole a e to the a idit . Pa a ete s easu ed i tole a e of ile salt test is the u e of olo ies of CFU/ l e e g o o solid ediu o tai i g ile salts . % a d . %. The data o tai ed ill the
Result and Discussion
Ide ii aio ge us Ba illus a te ia f o
o i e olost u
usi g API
CHB has fou d Ba illus su ilis o tai ed ith a u a
. % Figu e
a d Ba illus pu ilus ith a u a
. % Figu e
.
Tolerance of Acidity
Based o the the a al sis of a ia e ANOVA esults sho ed that the alue of F ou t > F ta le, hi h ea s Ba illus su ilis a d Ba illus pu ilus has i te a io to pH. Be ause the ANOVA esults a e eal the it ould e follo ed
Du a 's
Muliple Ra ge Test DMRT at sig ii a e le el % Ta le . Based o the Ta le the g o th of B. su ilis a d B. pu ilus i lue ed the pH the highe the pH, the o e olo ies g o . Ba illus su ilis a d Ba illus pu ilus at pH has the est ia ilit . It a e see f o the highest ate i the t eat e t of B. su ilis pH
ith a populaio of .
CFU/ l a d ith a populaio of B. pu ilus .
CFU/ l. I DMRT esults a e see that pH is opi u pH fo the g o th of the
a te ia, this is i a o da e ith the atu e of hi h is Ba illus eut ophils e ui es a opi u pH is . - . . At pH B. su ilis a d B. pu ilus a sill g o
ell. Ba illus su ilis is o e tole a t of a id to pH o pa ed ith Ba illus pu ilus ith a
populaio of .
CFU/ l a d .
CFU/ l. Whe eas at pH B. su ilis a d B. pu ilus a ot g o . Ba te ial Tole a e to a idit aused
the a ilit to ai tai a toplas i pH o e alkali e tha the e t a ellula pH. Highl a idi
o diio s at pH has a e st o g i lue e of B. su ilis a d B. pu ilus. A id i hi iio of a te ial ell g o th o u s th ough the i lue e of de atu aio of e z es that e ist o the ell su fa e, lipopol sa ha ide a d oute e
a e is da aged a d a de ease i
toplas i pH th ough i eased e
a e pe ea ilit Us iai,
. Ba ilus pu ilus a d Ba illus su ilis ased o the esults of tests of tole a e to a id a g o i a id to pH , so B. su ilis a d B. pu ilus has o e of
the e ui e e ts as a a didate p o ioi has tole a e of a idit .
Tolerance of Bile Salts
Based o the a al sis of a ia e ANOVA esult ho ed that the alue of F ou t > F ta le, hi h ea s Ba illus su ilis a d Ba illus pu ilus has i te a io to ile salt o e t aio of . % a d . %. Be ause the ANOVA esults a e eal the it
ould e follo ed Du a 's Muliple Ra ge Test DMRT at sig ii a e le el % Ta le . Based o Ta le sho s that the t eat e t of . % gi es the lo est alue of a te ial ia ilit , he eas . % t eat e t ga e the highest alues of a te ial
ia ilit . It is sho
f o the highest ate i the t eat e t of ile salt o e t aio of . % ith a populaio of .
CFU/ l i Ba illus pu ilus. Ta o k
, states that p o ioi a te ia should e a le to a hie e i the digesi e t a t
ai tai i g a populaio of
CFU/ l a d a p o ide health e eits. Media NA + . % ile salt is a ediu
hose o diio ese les the digesi e t a t o tai i g ile as su fa e-a i e o pou ds that a pe et ate a d ea t ith the toplas i e
a e Hill,
. The highe the o e t aio of ile salts the fe e a te ia g o i g populaio . It a e see that the u e of olo ies of Ba illus su ilis a d Ba illus pu ilus i lue ed the o e t aio of ile salts. Good esista e to ile salts o u s e ause of the ole of pol sa ha ides as a o situe t of the ell all of G a -posii e a te ia. Ba illus su ilis a d Ba illus pu ilus has esista e to ile salts ith a ii al o e t aio . A o di g )a aglia et al.
,
. % ile salt o e t aio is high e ough o e t aio s to sele t st ai s that a e esista t to ile salts, a d all the i o es that su i ed ate g o i
ediu plus . % ile salts, othe ise e esista t to ile salts . This is o e of the e ui e e ts
of a de la ed p o ioi a te ia.
Bacillus subtilis
Figure . Result Idenii aion Ba illus su ilis
Bacillus pumilus
Figure . Result Idenii aion Ba illus pumilus
Ta le . Dun an's Muliple Range Test Toleran e Ba illus su ilis and Ba illus pumilus to pH
Bacteria CFU/ml
pH
Bacillus subtilis
Bacillus pumilus
nd C
a
nd C
a
2
5
4
55,12 x 10 B
16,40 x 10 B
a
b
35,38 x 10 A
19,36 x 10 A
a
b
u
pH B. pu ilus
pH B. pu ilus
pH B. pu ilus
Bacillus subtilis and Bacillus pumilus population growth on
pH , , and agar plates medium
Dife e t lo e ase lete s ead i to the ho izo tal di e io i di ates sig ii a tl dife e t p> .
i ated
pH B. su ilis
8
Des ipio : Dife e t apital lete s ead i to the e i al di e io i di ates sig ii a tl dife e t p> .
d: ot dete
pH B. su ilis
5
8
6
pH B. su ilis
e of olo ies CFU/ l
Ta le . Dun an's Muliple Range Test Toleran e Ba illus su ilis and
Ba illus su ilis
Ba illus pumilus to Bile Salt
Bacteria CFU/ml
Bile salt
(%)
Bacillus subtilis
Bacillus pumilus
8
0,3
, % ile salts
8
6,692 x 10 A
8,745 x 10 A
b
a
8
Ba illus pu ilus
8
3,506 x 10 B
0,5
, % ile salts
5,570 x 10 B
b
, % ile salts
a
Des ipio : Dife e t apital lete s ead i to the e i al di e io i di ates sig ii a tl dife e t p> .
, % ile salts
Bacillus subtilis and Bacillus pumilus population growth on
bile salts 0, % and 0, % agar plates medium
Dife e t lo e ase lete s ead i to the ho izo tal di e io i di ates sig ii a tl dife e t p> .
Conclusion
Based o the test esults of the p o ioi p ope ies of Ba illus su ilis a d Ba illus pu ilus i esista e to lo pH a d ile salts, pe fo ed a al sis of a ia e to dete i e the pote ial oth as a a didate p o ioi
Ba illus pu ilus eet t o e ui e e ts as a a didate p o ioi that is tole a t of lo pH at pH a d pH a d tole a t to ile salts at a o e t aio of . % a d . %.
Reference
Ba osa, T.M., Se a, C.R., La Ragio e, R.M., Wood a d, M.J., a d He i ues, A.O.,
B a da o P, Rassu SPG, La zu A.
. Feedi g Dai
Bu i , N., Cha tha hu , S., Ho gpata ake e, T.
Chou L.). a d Wei e B.
F a dso , R.D.
Hill, M. J.
s. Dala
Puli e G. B i i R Edito J Dai
. S ee i g of La i A id Ba te ia F o
. Role of Gut Ba te ia i Hu a To i olog a d Pha
S i
-
-
.
a olog . Ta lo , Ne Yo k.
. Staisi al P o edu es fo Ag i ultu al Resea h. UI P ess, Jaka ta.
e s as Hu a P o ioi s: Ba illus, Sporola to a illus, a d Brevi a illus. Co p ehe si e Re ie I Food S ie e a d Food Safet Vol. ,
Spi osa, M.R., B a i i, T., Ri a, E., De Feli e, M., Mo elli, L., Pozzi, G., a d Oggio i, M.R.,
o k, G. W.
st ai s of L.a idophilus. J Dai
a d Fisiolog . th Ediio . Gadjah Mada U i e sit P ess, Yog aka ta.
Sa de s M. E, L. Mo elli, a d T.A. To pki s. Spo efo
Ta
e tal Mi o iolog
. A f esh look at the i tesi al
Us iai S, B oto W, da Sei a to H.
. O the fate of i gested Ba illus spo es. Resea h i Mi o iolog
:
-
ik olo a. Ho izo S i ii P ess.
. Cha a te isi s of o 's
)a aglia, A.G., G Ko iu i sk , P. Pe ez a d G. De A to i.
ilk u d usi g p o ioi
:
-
.
Sheep Nut iio Walli g Food : ABI . Pu lishi g.
. Isolaio a d Resista e Test of Se e al Isolates of La to a illus i Lo pH. LIPI , Bogo
Go ez, A.A. da Go ez, K.A.
oile gast oi tesi al t a t. Applied a d E i o
Gast oi tesi al T a s of Ma i e Fish Fo Thei Pote ial Use As P o ioi s. So gkla aka i J. S i. Te h ol.
. Isolaio a d ha a te izaio of a id a d ile tole a t isolates f o
. A i al A ato
Ha di i gsih, R.
La
. S ee i g fo Ba illus isolates i the
a te ia sta te . Jou al of A i al a d Vete i a
. Isolaio a d Cha a te izaio of Biido a te iu
st ai s fo p o ioi fo
S ie es,
:
ulaio . J. Food P ote t.
:
-
.
Suppl. .
a te ia. It is k o
that Ba illus su ilis a d