Two bacterial strains (SH13 and SH24) were isolated from Litopenaeus vannamei and showed probiotic potential. They demonstrated high acid and bile tolerance as well as antagonistic activity against common fish and human pathogens. SH13 and SH24 both showed over 90% antioxidant activity. These strains could be useful probiotics in aquaculture and also warrant further research for human applications.

1. Isolation and Characterization of Bacillus Species
with Probiotic Efficiency Against Common Aquatic
Pathogens in Litopenaeus vannamei
Under the Guidance Of
Dr. C. Amutha
Assistant Professor
Dept. of Animal Behaviour and Physiology
School of Biological Sciences
Madurai Kamaraj University
Madurai-625021
Presented by
Allapichai Riyaskhan B
21MIB02

2. INTRODUCTION
• The marine ecosystem has a higher diversity of living organisms compared
to the land that provides numerous to human societies.
• Arthropoda is the largest phylum of all living organisms and has members
with characteristic deselection and segmented bodies class.
• Crustacean is the largest and the most economically important group of
Marine arthropods among them crabs, shrimps and prawns are some of the
most attractive species and give attention relation to their use as health
foods.
• Crustacean is mostly aquatic with some species adapted to terrestrial
environments.

3. Litopenaeus vannamei (White Shrimp)
• Shrimp aquaculture is a thriving and globally recognized industry. Shrimp
export accounts for 17% of all marine foods exported globally.
• The Pacific white shrimp Lithogenous vannamei the representative species
of crustaceans is the major cultured shrimp species with the highest yield in
the world.
• The white leg shrimp Lithogenous vannamei commercially important
shrimp species which accounts for 80percentage global production.
• Antibiotic usage is widespread in freshwater shrimp farms, brackish-water
shrimp farms, and shrimp hatcheries. The present Indian requirements for
antibiotics in fish and prawn feed are mostly based on coastal aquaculture
recommendations.

4. PROBIOTICS
• The term probiotic comes from Greek meaning pro-life
• In fact, terms such as friendly bacteria, friendly or healthy are commonly
used to describe probiotics.
• The use of probiotics in aquaculture is new and differs from its use in
mammals and other terrestrial animals such as cattle, pigs, and poultry.
• Probiotics or beneficial microorganisms are also beginning to play a role in
aquaculture. This involves the use of live bacteria or yeast in the diet or
culture water to ensure that the gut is initially colonized with beneficial
organisms or to improve digestion.

5. OBJECTIVES
1. Isolation and identification of probiotic bacteria from White shrimp
(Litopenaeus vannamei).
2. To screen the functional properties (Acid and Bile tolerance) of probiotic
strains.
3. To identify bacterial genius and the antagonistic activity against Fish and
Human pathogens.
4. To determine the antioxidant activity of the identified strains using DPPH
assay.

6. METHODOLOGY
Shrimp Samples
•Dissection and
culture of
Hepatopancreas
Isolation of
Bacteria
•Pure culture
Acid and Bile
tolerance
Identification of
Bacterial strain
•Morphological
and Biochemical
characterization
Probiotic
Characterization
• Antioxidant
property
• Antagonistic
activity

7. RESULTS
Isolation of Bacteria
Gram Staining
Gram positive (SH13) Gram positive (SH24)

8. BIO CHEMICALANALYSIS
CITRATE POSITIVE Oxidase positive -blue color CATALASE POSITIVE COAGULASE POSITIVE

9. After probiotic characterization (2%
Bile salt Tolerance, 2 pH Acid
Tolerance Antibiotics Susceptibility
Test and Antagonistic Activity 2
Bacterial stains had probiotic
nature.

10. Optical Density for 2% Bile Salt Tolerance Test
Stain
Name
Incubation Time
0
Time
2
Hour
s
4
Hour
s
6
Hour
s
8
Hours
24
Hours
SH 1 1.414 1.343 1.35 1.387 1.494 1.337
SH 2 1.538 1.074 1.428 1.214 1.511 1.469
SH 3 1.175 1.386 1.462 1.469 1.269 1.547
SH 4 1.591 1.691 1.628 1.603 1.463 1.587
SH 5 1.607 1.683 1.688 1.645 1.586 1.582
SH 6 1.627 1.248 1.222 1.155 1.598 1.526
SH 7 1.389 1.558 1.531 1.524 1.34 1.53
SH 8 1.324 1.55 1.562 1.557 1.073 1.517
SH 9 1.587 1.124 1.104 0.957 1.489 1.125
SH 10 1.477 1.315 1.319 1.225 1.445 1.316
SH 13 1.22
8
1.484 1.53 1.611 1.602 1.775
SH 24 1.25
2
1.369 1.411 1.485 1.679 1.837
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
SH 1 SH 2 SH 3 SH 4 SH 5 SH 6 SH 7 SH 8 SH 9 SH 10 SH 13 SH 24
OPTICAL DENSITY FOR 2 %BILESALT TOLERANCE TEST
Optical Density for 2% Bile Salt Tolerance Test Incubation Time 0 Time
Optical Density for 2% Bile Salt Tolerance Test Incubation Time 2 Hours
Optical Density for 2% Bile Salt Tolerance Test Incubation Time 4 Hours
Optical Density for 2% Bile Salt Tolerance Test Incubation Time 6 Hours
Optical Density for 2% Bile Salt Tolerance Test Incubation Time 8 Hours
From the 31 samples, it is found that SH
13 & SH 24 SH13 and SH24 good survival
and sustainable against 2% bile salt
tolerance test.

11. 0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
SH 1 SH 2 SH 3 SH 4 SH 5 SH 6 SH 7 SH 8 SH 9 SH 10 SH 13 SH 24
OPTICAL DENSITY FOR 2pH ACID TOLERANCE
TEST
Optical Density for 2pH Acid Tolerance Test Incubation Time 0 Time
Optical Density for 2pH Acid Tolerance Test Incubation Time 1 Hour
Optical Density for 2pH Acid Tolerance Test Incubation Time 2 Hours
Optical Density for 2pH Acid Tolerance Test Incubation Time 3 Hours
Optical Density for 2pH Acid Tolerance Test Incubation Time 4 Hours
Optical Density for 2pH Acid Tolerance Test Incubation Time 24 Hours
Optical Density for 2pH Acid Tolerance Test
Stain
Name
Incubation Time
0 Time 1 Hour
2
Hours
3
Hours
4
Hours
24
Hours
SH 1 0.154 0.156 0.156 0.154 0.152 0.151
SH 2 0.156 0.155 0.155 0.151 0.151 0.149
SH 3 0.158 0.159 0.156 0.155 0.154 0.15
SH 4 0.15 0.15 0.15 0.148 0.153 0.154
SH 5 0.157 0.162 0.165 0.164 0.166 0.168
SH 6 0.146 0.147 0.144 0.154 0.147 0.146
SH 7 0.152 0.153 0.152 0.149 0.155 0.155
SH 8 0.152 0.154 0.152 0.15 0.154 0.154
SH 9 0.148 0.148 0.147 0.159 0.149 0.149
SH 10 0.15 0.152 0.152 0.025 0.152 0.151
SH 13 0.14 0.148 0.148 0.172 0.189 0.214
SH 24 0.262 0.3 0.298 0.312 0.318 0.356
From the 31 samples, it is found that SH
13 & SH 24 SH13 and SH24 good survival
and sustainable against 2% acid pH salt
tolerance test

12. ANTIBIOTIC SUSCEPTIBILITY (KIRBY-BAUER) TEST
Antagonistic Activity (Well Diffusion method)
Fish Pathogen Zone of Inhibition of Strains
BSH 1 BSH
2
Enterobacter sp. 12 11
Proteus vulgaris - -
Vibrio cholerae 11 5
Aeromonas hydrophilla 13 6
Edwardseilla tarda 9 2
Staphylococcus aureus 6 -
Bacillus cerus - 6
Human Pathogens
ATCC 25922 10 4
ATCC 2146BAA - 4
MTCC 733 10 5

13. Antioxidant Activity using DPPH
Scavenging rate (%)= [1-(Ab Sample - Ab Blank)/Ab Control] * 100
Blank - 0.046, Control - 0.023
Stain Absorbance Scavenging
rate %
Stain Absorbance Scavenging
rate %
SH 1 0.069 43.25% SH 17 0.133 53.99%
SH 2 0.117 23.80% SH 18 0.15 43.56%
SH 3 0.125 38.90% SH 19 0.158 38.65%
SH 4 0.129 36.44% SH 20 0.144 47.24%
SH 5 0.172 30.06% SH 21 0.136 52.15%
SH 6 0.142 48.47% SH 22 0.141 49.08%
SH 7 0.123 40.12% SH 23 0.153 41.72%
SH 8 0.119 22.58% SH 24 0.161 85.81%
SH 9 0.163 35.58% SH 25 0.138 50.92%
SH 10 0.138 50.92% SH 26 0.173 29.45%
SH 11 0.157 39.26% SH 27 0.141 49.08%
SH 12 0.158 38.65% SH 28 0.173 29.45%
SH 13 0.156 93.25% SH 29 0.118 33.19%
SH 14 0.13 55.83% SH 30 0.149 44.17%
SH 15 0.162 36.20% SH 31 0.167 33.13%
SH 16 0.106 40.55%

14. SUMMARY & CONCLUSION
From the present study, 2 Bacillus sp.
which have high probiotic potentiality against
common pathogens that affect aquatic creatures
were characterized. These two bacterial strains
can be used for future research in aquaculture
and they also showed activity against human
pathogens so they can be also used for human
research also.