THE TOLERANCE OF BACILLUS SUBTILIS AND BACILLUS PUMILUS ORIGINATED FROM BOVINE COLUSTRUM TOWARDS ACIDITY AND BILE SALT AS A PROBIOTICS CANDIDATE.

(1)

THE TOLERANCE

OF

Bacillus

subtilis

AND

Bacillus pumilus

ORIGINATED FROM BOVINE COLOSTRUM TOWARD

ACICITY AND BILE SALT AS A PROBIOTICS CANDIDATE

Khusnul Khotimah

1

, Ratu Safitri

2

, Nia Rossiana

2

, Anisa Syara

2

1 Agriculture and Farms Faculty, Muhammadiyah Malang University, Malang

2 _{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.

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 eat-e t i st fa to is theat-e t peat-e of a didateat-e p o ioi a teat-e 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

Materials

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 il-it . 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

to-plas 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 e-sista 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.

Figure . Result Idenii aion

Ba illus su ilis

Figure . Result Idenii aion

Ba illus pumilus

Bacillus subtilis

Bacillus pumilus

pH

Bacteria CFU/ml

Bacillus subtilis

Bacillus pumilus

2

nd C

a

4

55,12 x 10

5

B

16,40 x 10

5

B

a

b

6

35,38 x 10

8

A

19,36 x 10

8

A

a

b

Bile salt

(%)

Bacteria CFU/ml

Bacillus subtilis

Bacillus pumilus

0,3

6,692 x 10

8

A

8,745 x 10

8

A

b

a

0,5

3,506 x 10

8

B

5,570 x 10

8

B

b

a

Ta le . Dun an's Muliple Range Test Toleran e

Ba illus su ilis

and

Ba illus pumilus

to Bile Salt

Ta le . Dun an's Muliple Range Test Toleran e

Ba illus su ilis

and

Ba illus pumilus

to pH

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 a te ia. It is k o that Ba illus su ilis a d

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., . S ee i g fo Ba illus isolates i the oile gast oi tesi al t a t. Applied a d E i o e tal Mi o iolog : - .

B a da o P, Rassu SPG, La zu A. . Feedi g Dai La s. Dala Puli e G. B i i R Edito J Dai Sheep Nut iio Walli g Food : ABI . Pu lishi g.

Bu i , N., Cha tha hu , S., Ho gpata ake e, T. . S ee i g of La i A id Ba te ia F 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. Suppl. . Chou L.). a d Wei e B. . Isolaio a d ha a te izaio of a id a d ile tole a t isolates f o st ai s of L.a idophilus. J Dai S i -

-F a dso , R.D. . A i al A ato a d Fisiolog . th Ediio . Gadjah Mada U i e sit P ess, Yog aka ta.

Ha di i gsih, R. . Isolaio a d Resista e Test of Se e al Isolates of La to a illus i Lo pH. LIPI , Bogo . Hill, M. J. . Role of Gut Ba te ia i Hu a To i olog a d Pha a olog . Ta lo , Ne Yo k.

Go ez, A.A. da Go ez, K.A. . Staisi al P o edu es fo Ag i ultu al Resea h. UI P ess, Jaka ta.

Sa de s M. E, L. Mo elli, a d T.A. To pki s. Spo efo 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 the fate of i gested Ba illus spo es. Resea h i Mi o iolog : - .

Ta o k, G. W. . A f esh look at the i tesi al ik olo a. Ho izo S i ii P ess.

Us iai S, B oto W, da Sei a to H. . Cha a te isi s of o 's ilk u d usi g p o ioi a te ia sta te . Jou al of A i al a d Vete i a S ie es, :

-)a aglia, A.G., G Ko iu i sk , P. Pe ez a d G. De A to i. . Isolaio a d Cha a te izaio of Biido a te iu st ai s fo p o ioi fo ulaio . J. Food P ote t. :

-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> .

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> . d: ot dete i ated u e of olo ies CFU/ l

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> .

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> .

pH B. su ilis _pHB. su ilis pH B. su ilis

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

Bacillus subtilis and Bacillus pumilus population growth on

bile salts 0, % and 0, % agar plates medium

, % ile salts _{, % ile salts}

, % ile salts

Ba illus su ilis