thesis on probiotic, part 2, abadin

8/17/2019 Thesis on Probiotic, Part 2, Abadin

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1

CHAPTER 3

RESULTS AND DISCUSSION

3.1. Isolation of Lactic Acid Bacteria

15 samples of buffalo milk taken from healthy buffalos were used as isolation

source. Lactic acid bacteria were isolated from various mediums at 37 °C under

anaerobic conditions. Some ram neative bacteria were detected on !"# aar but no

one on $%S aar. &lso lots of catalase positive bacteria and yeast were observed.

!he reason could be the contamination from buffalo's breast skin. (rom appro)imately

*++ isolates, -+ isolates remained at the end of the isolation, purification after the loss of

unstable isolates durin purification and subculturin steps. &ll of the isolates were ram

positive catalase neative rods and cocci. t was understood that the isolates from buffalo

milk were so sensitive to the subculturin. &lso some of them were lost because of bein

intolerant to /0+°C and also /*+°C cryopreservation.

3.. Pro!iotic Pro"erties

3..1. Resistance to Lo# "H

ein resistant to low p2 is one of the maor selection criteria for probiotic

strains 4uwehand, et al. 1666, ak8r *++39. Since, to reach the small intestine they

have to pass throuh from the stressful conditions of stomach 4Chou and :eimer 1666,

ak8r *++39. &lthouh in the stomach, p2 can be as low as 1.+, in most in vitro assays

p2 3.+ has been preferred. ;ue to the fact that a sinificant decrease in the viability of

strains is often observed at p2 *.+ and below 4"rasad, et al. 16609.

(or selection the strains resistant to low p2, "S p2/adusted to 3.+ was used.

!he time that takes durin the diestion in the stomach is 3 hours. So all the isolates

were detected whether they were resistant to p2 3.+ durin 3 hours. &fter the

e)amination of all the isolates, the isolates that survive in p2 3.+ were taken to the ne)t

step. &ccordin to this e)periment only three isolates were resistant to low p2. !wo of


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them are bacilli and the other is cocci. =;-*+ values

10

9

8

7

6 17

5 83

4 95

3

2

1

0

0 1 2 3

time(hour)

(iure 3.*. Survival in p2 3.+ > cfu values

&s it is seen from the raphics &S17 bacilli is very stable in p2 3.+ which means

that this isolate is able to survive in this p2 value. &S03 bacilli is also stable durin 3

O D ( 6 2 0 )

L o g c f u / m l


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hours. &S65 is able to tolerate p2 3.+ but it is more sensitive to low p2 than &S17 and

&S03.

:hen the two methods were compared, both of them ave the same results for

&S17, nearly the same for &S03, but to decide the survival in p2 3.+ cfu values seem

more reliable for &S65.

3... Tolerance a$ainst Bile

!he strains, resistant to low p2, were screened for their ability to tolerate the

bile salt. &lthouh the bile concentration of the human astro intestinal tract varies, the

mean intestinal bile concentration is believed to be +.3? w@v and the stayin time is

suested to be A h 4"rasad, et al. 16609.

Strains were detected in +.3? durin A hours. !he cfu values and =;-*+ were

observed. &ccordin to the results all of the isolates are resistant to +.3? bile salt. &S17

and &S03 are more tolerant than &S65. &ll of the isolates are also able to row in +.3?

bile salt as they survive.

0.08

0.07

0.06

0.0517

0.04 83

950.03

0.02

0.01

0

0 1 2 3 4

time(hour)

(iure 3.3. !olerance aainst +.3? bile > =;-*+ values

O D ( 6 2 0 )


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9

8

7

617

583

495

3

2

1

0

0 1 2 3 4

time(hour)

(iure 3.A. !olerance aainst +.3? bile > cfu values

3..3. Anti%icro!ial Acti&it'

!he selected strains were e)amined accordin to their antimicrobial activity. (or

this purpose, strains were detected aainst the indicator microoranisms Salmonella

thyphimurium CC$ 5AA5, Escherichia coli =157B27 C!C 1*6+++ and Escherichia

coli %%L /3++0. !he diameter of inhibition Dones 4!able 3.19 showed that all of the

isolates have antibacterial effec t on the indicator microoranisms. !he tests were

applied two times and the averaes of diameters of Dones were iven.

!able 3.1. ;iameter of inhibition Dones

IsolatesIndicator (icroor$anis%s

Dia%eter of in)i!ition *ones+%%,

No Salmonella

thyphimurium Escherichia coli

Escherichia coli

O1-/H

1. *+ A1 **

03 10 A3 *5

- 15 3- 15

L

o g c f u / m l


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3.3. P)'siolo$ical and Bioc)e%ical C)aracteri*ation

&ll of the isolates were subected to Eram stainin and they were e)amined

under liht microscope. &ll the strains ave blue/ purple color with staininF hence they

all were Eram positive bacteria. &S17 was bacilli with lon and rounded end 4(iure

3.5.a9. &S03 was cocobacilli morpholoy 4(iure 3.5.b9 while &S65 was cocci with

spherical morpholoy.

4a9 4b9

(iure 3.5. $icroscopic imaes +a, &S17 +!, &S03

solates were tested for catalase activity. !hey were all catalase neative 4do not

show catalase activity9.

!o test the as production from lucose test tubes were observed for 5 days.

&S65 showed no as production while as production was observed from &S17 and

&S03. !his indicates that, &S17 and &S03 are heterofermentative cultures whereas

&S65 is homofermentative.

&nother criterion for the identification the isolates was the ability of rowth at

different temperatures. (rom the results of 7 days observation, all of the isolates can

row at A5 °C however they cannot row at 1+ °C and 15 °C.

Erowth at different aCl concentrations was observed. &ll of the isolates have

the ability to row at *? aCl concentration. &S17 and &S03 do not show the ability to

row at -.5? aCl concentration however &S65 can row at this concentration.

&rinine hydrolysis test was another step to follow the identification procedure.

!he isolates which ave the briht orane were accepted that they can produce

ammonia from arinine. !he yellow color indicated neative arinine hydrolysis.


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&ccordin to this test, &S17 can not hydrolyDe arinine while both &S03 and &S65 can

produce ammonia from arinine.

!he most useful test for the determination of strain differences is carbohydrate

fermentation.


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A S -

A S 0 3

A S 1 .

Co

c o c c i

b a c i l l i

b a c i l l i

Shape

/ / / Catalase

/ G G as from lucose

G G / &mmonia from &rinine

G G G *? aCl

G / / -.5? aCl

/ / / Erowth at 1+°C

/ / / Erowth at 15°C

G G G Erowth at A5°C

G G G Elucose

/ / G Hylose

/ G G %ibose

/ / / $eleDitose

/ G G &rabinose

/ / / $annitol

/ G /!rehalose

G G G $elibiose

G G G %affinose

G G G Ealactose

G / / Salicin

G G G $altose

G G G Sucrose

G G / / $annose

G G G (ructose

G G G Lactose

/ / / %hamnose

/ / / Sorbitol

!a

ble3..iochemical!est%esultsofslates

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A+

&ccordin to the biochemical test results &S17 produced as from lucose while

did not produce ammonia from arinine. t tolerated only ?* aCl concentrations and

only rew at A5 °C. !his isolate ave positive results with the carbohydrates, lucose,

)ylose, ribose, arabinose, melibiose, raffinose, alactose, maltose, sucrose, fructose and

lactose. &S03 produced both as from lucose and ammonia from arinine. t was

resistant to ?* salt concentrations and rew at A5 °C. !his isolate ave positive test

results with suars, lucose, ribose, arabinose, trehalose, melibiose, raffinose, alactose,

maltose, sucrose, fructose and lactose. :hen these biochemical test results are compared

with the literature information 4!able 3.39, it seems that &S17 is like to be Lactobacillus

oris, &S03 is like to be Lactobacillus fermentum and &S 65 is like to be Streptococcus

ssp. (or the future e)periments &S17 and &S03 were chosen.


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!able 3.3. Literature nformation of iochemical !est %esults sourceB 4%oos, et al. *++5, 2ammes and 2ertel 16659

S t r a i n s

a s f r o m l u c o s e

& m m o n i a f r o m & r i n i n e

E r o w t h a t 1 5 ° C

E r o w t h a t A 5 ° C

$ e l e D i t o s e

& r a b i n o s e

! r e h a l o s e

$ e l i b i o s e

% a f f i n o s e

E a l a c t o s e

% h a m n o s e

Lactobacillusoris bacilli / G / / G G G G / G ; d G G G / G G d G G / ;

Lactobacillus fermentum bacilli / G G / G G d G / d / d G G G / G G w G G / ;

SymbolsB GB 6+? or more strains are positive, /B 6+?or more are neative, dB 11/06? of strains are positive, wB weak positive reaction, ;B

no data available

A1

A1


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1+

CHAPTER 2

CONCLUSION AND UTURE PERSPECTI4E

CharacteriDation and determination of probiotic properties of Lactic &cid

acteria isolated from buffalo milk was the aim of this study. !o determine the probiotic

properties different tests were applied such as resistance to low p2 and bile salt and

antimicrobial activity tests. &fter the determination of potential probiotic isolates, these

isolates were characteriDed by phenotypic and enotypic methods. (or the phenotypic

characteriDation, morpholoic e)amination, resistance to different temperatures and salt

concentrations, as production from lucose, ammonia production from arinine,determination of suar fermentaiton profiles were applied. (inally the followin results

were obtainedF

1. Lactic &cid acteria were isolated from buffalo milk.

*. "robiotic properties of isolated bacteria were determined. =nly 3 of them

showed resistance to low p2, tolerance to bile salt, antimicrobial activity

aainst some indicator microoranisms.

3. "henotyic and enotypic identifications were effectively diferentiate the

isolates especially suar fermentation patterns support the enotypiccharacteriDation results. !wo of them was determined that they could be

potential probiotic strains even if some forward tests were applied.

n this study the first step was taken to use the isolates as cultures for probiotic

products. !he main criteria of bein probiotic strains were determined and the selected

isolates were identified. !herefore some future studies should be performed to use these

isolates reliably. t will be beneficial to test the followin characteristicsF

1. Clinical studies for human health.

*. !echnoloical properties 4strain stability, viability in products, bacteriophae

resistance9.

3. &ntibiotic resistance.


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REERENCES

&nelis, $., Sirausa, S., erloco, $., Caputo, L., Settanni, L., &lfonsi, E., &merio,

$., Erandi, &., %ani, &., and Eobbetti, $. *++-. Selection of potential probioticLactobacilli from pi feces to be used as additives in pelleted fedin. Research in

Microbiology. 157B76*/0+1.

rady, L.I., Eallaher, ;.;., usta, (.(. *+++. !he role of probiotic cultures in the

prevention of colon cancer. The Journal of Nutrition. 13+BA1+S/A1AS.

ulut, . *++3. solation and molecular characteriDation of lactic acid bacteria from

cheese. IYTE Thesis of Ms.

ak8r, . *++3. ;etermination of some probiotic properties on Lactobacilli and

ifidobacteria. Anara !ni"ersity Thesis of #h.$.

Cardinal, $.I., $ehrous, I., Lacroi), C., Simard, %.


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Collins, I.K., !hornton, E., Sullivan, E.=. 1660. Selection of probiotic strains for

human applications. International $airy Journal 0BA07/A6+.

;uas, ., $ercenier, &., Lenoir/:inkoop, ., &rnaud, C., ;uas, ., and "ostaire,


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2olDapfel, :.2., 2aberer, "., Snell, I., Schilliner, M., 2uis in't Jeld, I. 1660.

=verview of ut flora and probiotics. International Journal of %oo& Microbiology

A1B05/1+1.

Iacobsen, C.., ielsen, J.%., 2ayford, &.


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$artin, %., =livares, $., $arin, $.L., (ernandeD, L., Haus, I., %odriueD, I.$. *++5.

"robiotic potential of 3 Lactobacilli strains isolated from breast milk. J ,um Lact .

*1419B0/17.

$ombelli, ., Eismondo, $.%. *+++. !he use of probiotics in medical practice.

International Journal of Antimicrobial Agents 1-B531/53-.

$oreira, I. L. S., $ota, %. $., 2orta, $.(., !ei)eira, S. $%., euman


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%oos, S.,

approachin a definition. Am. J. (lin. Nutr. 73B3-1S/3-AS.


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APPENDI5 A

CHE(ICALS OR (ICROBIOLO6ICAL E5PERI(ENTSAND (OLECULAR CHARACTERI7ATION

!able &.1. Chemicals Msed in $icrobioloical


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!able &.1. Chemicals Msed in $icrobioloical


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5-

APPENDI5 B

RECIPES OR CULTURE (EDIA AND BIOCHE(ICAL

TESTS

B.1. (RS Brot)

In$redients $9l

"epton 1+.+

Lab/Lemco meat e)tract 1+.+

#east


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K *2"=A *

Sodium acetate 5.+

!riammonium citrate *.+

$S=A.72*= +.*

$nS=A.A2*= +.+5

&ar 15.+

;eioniDed water 1+++ml

&ll inredients were dissolved in deioniDed water and p2 was adusted to -.3.

$edium was steriliDed by autoclavin at 1*1°C for 15 min.

B.3. TP: Brot)

In$redients $9l

!rypticase peptone 1+.+

"hytone peptone 5.+

Elucose 15.+

#east e)tract *.5

!ween 0+ 1 ml

Cysteine 2CL +.5

K *2"=A *.+

$Cl*-2*= +.5

&ll the inredients were suspended into deioniDed water, and p2 was adusted to

-.5. !hen solution was dispensed to the test tubes and autoclaved at 11+ °C for 3+ min.

B.2. TP: A$ar

In$redients $9l

!rypticase peptone 1+.+

"hytone peptone 5.+

Elucose 15.+

#east e)tract *.5


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!ween 0+ 1 ml

Cysteine 2CL +.5

K *2"=A *.+

$Cl*-2*= +.5

&ar/aar +.5?

&ll the inredients were suspended into deioniDed water, and p2 was adusted

to-.5. !hen solution was autoclaved at 11+°C for 3+ min.

B.-. (odified (RS Brot) for Testin$ t)e 6ro#t) at Different

Te%"erat;res

In$redients $9l

"epton 1+.+


#east


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B.


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$S=A.72*= +.*

$nS=A.A2*= +.+5



$edium was dispensed into test tubes and inverted durham tubes were distributed to

each test tube, and lastly steriliDed by autoclavin at 1*1°C for 15 min.

B.0. (odified (RS for Car!o)'drate 3er%entations

In$redients $9l

"epton 1+.+


#east


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Sodium acetate 5.+

!riammonium citrate *.+

$S=A.72*= +.*

$nS=A.A2*= +.+5

&rinine 1,5



$edium was steriliDed by autoclavin at 1*1°C for 15 min.


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APPENDI5 C

CARBOH:DRATES USED OR CARBOH:DRATE

ER(ENTATION TESTS

Suar solutions prepared at concentration 1+?

1. ;4G9 Hylose

*. ;4/9 %ibose

3. $eleDitose

A. L4G9 &rabinose

5. $annitol

-. ;4G9 !rehalose

7. $elibiose

0. %affinose

6. ;4G9 Ealactose

1+. $altose

11. Sucrose

1*. ;4G9 $annose

13. (ructose

1A. Lactose

15. %hamnose

1-. Sorbitol

17. Elucose

Suar solution prepared at concentration 5?

10. ;4/9 Salicin

thesis on probiotic, part 2, abadin

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