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 . %.

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