Study on Bacillus spp. isolated from fermented foods and fish samples for

some probiotic properties.

Artitaya Buatong

Master of Science Thesis in Microbiology

Overview Probiotic, Bacillus spp.

Introduction1.

Goal for the development of health food supplements.

Objective2.

Use for screening functional potential strains.

Methodologies3.

Report of potential strains.

Results4.

Summary of significant result in this study

Conclusions5.

Health functionality

- cholesterol reducing

- antioxidant activity

- antimicrobial activity(Hwang, et al., 2013)

“Live microorganisms which,

when administered in

adequate amounts, confer a

health benefit to the host”

(FAO/WHO, 2002)

Probiotic

List of probiotics

Lactobacillus, Lactococcus,

Bifidobacterium, Leuconostoc,

Pediococcus, Bacillus (Isolauri, Salminen, & Ouwehand, 2004)

1

Antioxidant

- Reduce the amount of free radicals

- Reduce the risk of developing certain

diseases such as cancer, heart disease,

stroke, cataracts, Parkinson's,

Alzheimer's and arthritis

- Prevent direct cell damage caused by

chain reaction that free radicals initiate

- Reduce the signs of aging by preventing

the oxidation of skin cells

- Improves digestion- Relieves asthma and allergy symptoms

4

2

Bacteriocin

Bacteriocins are bacterial ribosomally

synthesized, extracellular peptides or proteins

with an antibacterial activity

Lactococcus lactis: Nisin

Pediococcus acidilactici: Pediocin PA-1

Bacillus subtilis: subtilinNisin

3

endospore

survive extreme

conditions- nutrient deficiency

- Heat

- solvents

- enzymes

Functional properties

- Antioxidant

- Reduce cholesterol

- Antimicrobial activity

Probiotic properties

- survive in gastrointestinal

(GI) tract

- adhere to epithelial cells

Bacillus spp.Gram positive

Rod shape

Obligate aerobes or facultative anaerobes

4

Objective

To investigate some probiotic properties including

antioxidant and antimicrobial activities of Bacillus spp.

isolated from fermented foods and fish intestines

5

Pre-screening of

antioxidant-

producing strains

DPPH free radical

scavenging ability

culture supernatant bacteriocin-like

substances

Preliminary

purification using

NH4SO4

precipitation

Overall experiment

6

57 strains of Bacillus were obtained

from Dr. Tongpim’s research Lab,

Department of Microbiology, Faculty of

science, Khon Kaen University.

They were isolated from fish gut and Thai

traditional fermented foods in the North-

Eastern region of Thailand.

Bacterial cultures

9 7

Pre-screening method for antioxidant-producing strains (Takao et al, 1994)

Nutrient agar (NA)

35 °C, 24 h.sterile filter

paper on

the agar

plate

24 h.

sprayed with

0.2 mM DPPH solution

positive

negative

Pre-screening of antioxidant-producing strains (Takao et al, 1994)

10

8

Pre-screening method

Fig. 1 Color of Bacillus colonies on paper after sprayed with 0.2 mM DPPH solution

14 strains of Bacillus could produce antioxidants

Antioxidant-producing strains

11

9

10

DPPH free radical scavenging ability (Jin et al., 2012; Ding et al., 2017 )

0.2 mM

DPPH solution,

Vortexed Centrifuged

Bacterial

suspension

(OD600 = 1)

1 ml 3 ml

dark, 30 min

Scavenging activity (%) = 1- (Asample / Acontrol) x 100

measured A517

* PBS: control

12

DPPH free radical scavenging abilityS

cav

engin

g a

ctiv

ity (

%)

31.3 30.2

25.8

1.1

30.228.8

19.4

10.2

0.4

30.7

19.9 21.0 20.0

6.8

0

5

10

15

20

25

30

35

40

11

Antimicrobial activity (Huang & Adams, 2004)

culture supernatant preparation

Bacillus culture

18 h

Centrifuge7000 rpm ,4ºC, 10 min

Supernatant

Agar well diffusion

assay

catalase enzyme

(1 mg/ml final concentration)

37°, 30 min

pretreated

supernatant

Adjust to pH 7

Filter-

sterilized

14

12

Antimicrobial activity of the culture supernatant

Inhibition Zone (mm)

Bacterial strain

E.

coli

O1

57

:H7

MR

SA

B. ce

reu

s

S. T

yp

him

uri

um

S. a

ure

us

P.

aer

ug

inosa

L. m

on

ocy

togen

es

L. in

ocu

a

Sh

igel

lasp

.

V.

para

haem

oly

ticu

s

Bacillus siamensis B44v 8 8 14 9 9 25 13.5 14.5 9 0

Bacillus sp. strain TP24 8 9 15 9 8 9 8 10.5 9 0

Bacillus sp. strain CF2 0 0 8 0 0 0 12 8 0 0

13

Inhibition Zone (mm)

Bacterial strain

E. co

li O

157:H

7

MR

SA

B. ce

reu

s

S. T

yp

him

uri

um

S. au

reu

s

P. aer

ugin

osa

L. m

on

ocy

togen

es

L.

inocu

a

Sh

igel

lasp

.

V.

para

haem

oly

ticu

s

Bacillus siamensis B44v 8 8 14 10 8 20 12 14 9 0

Bacillus sp. strain TP24 0 0 9.5 0 0 0 0 0 0 0

Antimicrobial activity of the pretreated culture supernatant

14

Partial purification of bacteriocin-like substances

ammonium sulfate

Precipitation,

20, 40, 60, 80%

saturation

4°C, overnightCentrifuge

Dissolve

protein sedimentin 10 mM PBS pH 7.0

dialysis

1 kDa cut-off membrane

Agar well

diffusion assay

Crude

protein

Culture

supernatant

17

16

17

ผลการคดัเลือกเปอร์เซ็นตแ์อมโมเนียมซลัเฟตในการตกตะกอน bacteriocinInhibition Zone (mm)

Ammonium sulfate

saturation (%)

E.

coli

O1

57

:H7

MR

SA

B.

cere

us

S. T

yp

him

uri

um

S. a

ure

us

P.

aer

ug

inosa

L. m

on

ocy

togen

es

L. in

ocu

a

Sh

igel

lasp

.

V. para

haem

oly

ticu

s

20 10 9 13 1 1 1 21 21 9 0

40 15 16 21 15 14 12 25 25 15 10

60 15 15 2 14 12 11 25 25 15 10

80 14 15 2 14 12 11 25 25 15 10

Antimicrobial activity of the crude bacteriocin

Conclusion

exhibited 25.8% DPPH scavenging activityBacillus

siamensis

B44vProduce bacteriocin against E. Coli O157:H7, MRSA,

B. Cereus, S. Thyphimurium, S. aureus, P. aeruginosa,

L. inocua, L. monocytogenase, Shigella sp.,

V. parahaemolyticus

B. siamensis B44V has a good potential to be used

in developing functional foods. 19

20

ACKNOWLEDGMENTS

The research capability enhancement program through

graduate student scholarship, Faculty of Science,

Khon Kaen University

THANK YOU

FOR YOUR KIND ATTENTION

21

22

Assoc. Prof. Dr. Saowanit Tongpim Assoc. Prof. Dr. Yanee Trongpanich

Dr. Ratchanu MeidongMicrobiology Division,

Faculty of Science, Khon Kaen University

ACKNOWLEDGMENTS