Royal University of Phnom Penh

Faculty of Engineering
Topic: Effect of the Bacteriocin-producing Bacillus cereus
Strain HVR22
on the Preservation of Fish Fillets

Presenter: SORN channthy Lecturer : PHOEURK chanrith

Year :3 Department : Biological Engineering
Department : Biological Engineering

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Content
I. Introduction
II. Objectives
III. Material and Methods
IV. Results and Discussion
V. Conclusion

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I. Introduction
 The Bacillus cereus strain HVR22 is an isolate from budu.
 which has antimicrobial activity toward Escherichia coli,
Staphylococcus aureus , Salmonella typhi , Bacillus subtilis and
Listeria monocytogenes bacteria.
 Budu is a traditional fish-fermented
product from West Sumatra.

Budu
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I. Introduction
 The LAB is a fermented bacteria that acts as biopreservative
because it can produce antimicrobial compounds, such as
-lactic acid
-hydrogen peroxide (H2O2),
-diacetyl
-carbon dioxide (CO2)
-and bacteriocins

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I. Introduction
 Bacteriocins are polypeptide compounds that act as
antimicrobial compounds.
 Bacteriocins can be used as a starter culture
 component of the food probiotic and as biopreservative,
extending the shelf life of different food products.
 Bacteriocins can be produced by gram-positive and gram-
negative bacteria.
 Mostly Bacillus spp and LABs because they are “Generally
Recognized as Safe (GRAS)”

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II. Objectives

 Evaluate the effect of partial purification of bacteriocins on

pathogen bacteria and to observe of its effect on the cellular
morphology of Escherichia coli with applications to fish fillets.

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 II. Materials and Methods
2.1 Antagonistic activity of bacteriocins

Budu MRSA meduim centrifuged at 6000

incubated in 37C for 24 h
rpm for 10 min

1 oz of bacteria was
added to 10 mL
incubated in 37C for 24 h E.coli extracted bacteriocin
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 II. Materials and Methods
2.2 Scanning electron microscopy
Remove and resuspension

Poly-L-lysine treated Immersing the chips

Suspension with 30min chamber in OSO4 in cacodylate
silicon wafer chips
3% glutaraldehyde buffer 30 min
in Na-cacodylate

JEM-JEOL-JSM-5310LV type sputter coater Dehydrated in ethanol ( 50,

70, 95(2x) and 100 (2x) ) for
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10 min
 II. Materials and Methods
2.3 Transmission electron microscopy

Washed twice with Fixed in 2.5% glutaraldehyde Post-fixed in 2%

centrifugation osmium tetroxide
1M phosphate buffer and 2% formaldehyde in
phosphate buffer 10 day 45min

Dehydrated in
acetone (30-100%)

Observed with a Post-strained with

tipe JEOL-1010 Embedding in Araldite-
2% uranyl acetate
Durcupan for 72h
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II. Materials and Methods
2.3 Applications of bacteriocins to fresh fish fillets

Soaks 30 min Remove

Store between
-2 and 0 C

Fresh fish fillets 10g Crude bacteriocins Sterile HDPE plastic

 Note: for control no soaks it.

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III. Results and Discussions
3.1 Antagonistic activity of bacteriocins
 The Bacillus cereus strain HVR22 isolates had the highest inhibitory
effect against the Escherichia coli in 14 mm.
 The same effect was also found in Moroccan sardines (Sardina
pilchardus ) fermented fish.
• The bakasang fermentation inhibition zone diameter against
Escherichia coli was 15 mm

Fig. 1: Antimicrobial activity of isolate Bacillus cereus strain HVR22 on

inhibiting the growth of Escherichia coli bacteria

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III. Results and Discussions
3.2 Scanning electron microscopy
 The cell surface was
-wrinkled
-rough
-formed irregular shapes
-a few cells with septa that had not yet split.
 Antibacterial bacteriocins
Fig. 2: Electron micrograph of Escherichia coli bacteria
-disrupted the cell membrane target exposed to antibacterial of the Bacillus cereus strain
HVR22 using Scanning Electronic Microscopy (SEM)
-cause bacteriolysis (lysis of bacterial cells)
-damage to the peptidoglycan layer

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III. Results and Discussions
3.3 Transmission electron microscopy
 A few visible changes, including
-irregular cytoplasm
-Formation of a space between cytoplasm
and cell membrane
-Wrinkled cells (collapse) and cell lysis

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III. Results and Discussions
3.4 Application of bacteriocins to fresh fish fillets

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IV. Conclusions
 Antimicrobial compounds produced by the Bacillus cereus strain
HVR22 can damage the cell membrane and cellular structures
inside Escherichia coli cells.
 Antimicrobial compounds of the Bacillus cereus strain HVR22
can inhibit the growth of Salmonella sp. and Escherichia sp. in
snapper fish fillet until 7 days of storage and can be used as
natural preservatives.

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Quantity per Container:1 Oz

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