Probiotics applications in food and agricultural field

3. PROBIOTIC: AN ALTERNATE BIOAGENt IN FOODAND
AGRICULTURAL INDUSTRY
Krutika Patil
Division of Microbiology
ICAR-IARI

4. •The human microbiome
is the collection of all the
microbes found in or on
our bodies.
•Their number is ten
times the human cells.
•The gut microflora is a
main constituent of
intestine’s defence
system
HUMAN MICROBIOME

5. Good and bad microflora

6. • The term Probiotic was coined in 1965 by Lilly and Stillwell.
• The term derived from Latin preposition ‘pro’ and Greek
adjective ‘bios’meaning “FOR LIFE”.
• Organism and substances that have a beneficial effect on the
host animal by contributing to its intestinal microbial balance-
Parker (1974)
• Live microbial feed supplement which beneficially affects the
host animal by improving its intestinal microbial balance-
Fuller (1989)
• Live organisms which, when administered in adequate
amount, confer a health benefit on the host –FAO/WHO
(2002)
PROBIOTICS

7. •Concept of probiotic
•Modify the gut flora and to replace harmful
microbes by useful microbes
•Intestinal autointoxication
•‘Bulgarian Bacillus’
Elie Metkinkoff (1907)
•First isolated Bifidobacterium
from breast fed infants
•Bacillus bifidus communis
•Clinical benefit-treating diarrhea
Henry Tissier (1906)
HISTORY

8. •Outbreak of Shigellosis
•Isolated strain of Escherichia
coli from faeces of a soldier
•E.coli Nessle 1917
•Yakult-a fermented milk product
•Lactobacillus casei shirota
Alfred Nissle (1917)
Dr.Minoru Shirota (1930)

9. What are the Properties of microorganisms to be called as Probiotic?
• Non-pathogenic and non-toxic
• Able to survive the passage through the digestive system
• Able to attach to the intestinal epithelia and colonise
• Able to maintain good viability
• Capable of exerting a beneficial effect on the host
• Stability of desired characteristics during processing, storage
and transportation

10. Probiotic microorganism
Lactobacillus spp.,
• L. acidophilus
• L. casei
• L. rhamnosus
• L. delbrueckii subsp. bulgaricus
• L. paracasei
• L. reuteri
• L. plantarum
• L. gasseri

11. Bifidobacterium spp.,
• B. bifidum
• B. essencis
• B. infantis
• B. longum
• B. lactis
• B. breve
• B. animalis

12. Other microorganisms used as Probiotic:-
• Bacillus subtilis, Bacillus cereus
• Enterococcus faecalis
• Saccharomyces boulardii, S. cerevisiae
• Propionibacterium freudenreichii
• Leuconostoc mesenteroides
• Streptococcus salivarius
Bacillus spp.,
Enterococcus spp.,
Saccharomyces spp.,

13. importaNCE OF Probiotics
• Antibiotic and other drug intake
• Microbial infections
• Unhealthy diet
• Stress
• Age
• Colonic therapies for detoxification

14. MODE OF ACTION as bioagent
a) Competitive
exclusion of
pathogenic
Microorganisms
b) Production of
antimicrobial
substances.
c) Competition for
nutrients &
growth factors.
d) Increase adhesion
to intestinal mucosa.
e) Enhanced
epithelial barrier
function.
f) Enhanced IgA
secretion (Immune
stimulation)

15. PROBIOTIC: IN FOODINDUSTRY

16. PROBIOTICSAGAINSTFOODPATHOGENS
Probiotic strain Pathogen
Lactobacillus rhamnosus GG Listeria monocytogenes
Lactobacillus paracasei PM8 Enterococcus faecalis
Enterococcus durans LAB18s Salmonella enterica subsp. enterica
serovar Typhimurium, Salmonella enterica
subsp. enterica serovar Enteritidis
Lactobacillus acidophilus La-5 Escherichia coli O157:H7
Lactobacillus plantarum DGK-17 Pseudomonas aeruginosa, Klebsiella
pneumoniae
Bifidobacterium longum ATCC15707 Staphylococcus aureus

17. Livestock
Poultry Aquaculture
PROBIOTIC: IN AGRICULTURE

18. PROBIOTICS AGAINST FARMANIMALPATHOGENS
Application Pathogen Probiotic strain
Poultry Campylobacter jejuni Lactobacillus salivarius SMXD51,
L. gasseri SBT2055, L. paracasei J.R
+ L. rhamnosus 15b
Salmonella enterica subsp.
enterica serovar Enteritidis
Bacillus cereus var. toyoi
Clostridium perfringens Lactobacillus johnsonii F19185
Pig Escherichia coli Enterococcus faecium NCIMB 11181
Clostridium perfringens Lactobacillus fermentum I5007
goat Clostridium spp. Lactobacillus plantarum PCA 236
Shigella & Salmonella Lactobacillus reuteri DDL 19
Cattle E. coli O157:H7 Lactobacillus acidophilus strain NP51

19. Continued….
Application Pathogen Probiotic strain
Aquaculture Edwardsiella tarda Enterococcus faecium SF68
Aeromonas salmonicida ssp.
salmonicida
Lactococcus lactis ssp. lactis
CLFP 100
Vibrio harveyi Streptococcus phocae PI8o

20. CASE STUDY -1
OBJECTIVE
• To decipher the effect of oral intubation of Bacillus subtilis on
Aeromonas hydrophila-induced intestinal mucosal barrier function
damage and inflammation in grass carp
Weiguang Kong, Can Huang, Ying Tang, Ding Zhang, Zhixin Wu & Xiaoxuan Chen
(Kong et al., (2017) Scientific Reports 7: 1588 DOI:10.1038/s41598-017-01336-9)

21. MATERIALS & METHODS
1.Specimen: 2 year old Grass carp(Ctenopharyngodon idella)
2.Bacterial Strains: B. subtilis Ch9 and A. hydrophila
3.Infection experiment: 100L plastic tank with aerated tap water
a) control group
b) A. hydrophila group
c) B. subtilis + A. hydrophila
4.Sampling: 6 hpi, 12 hpi, 24 hpi, 48 hpi, 72 hpi, 96 hpi, and
120 hpi (hpi - hours post infection)
5. Detection of intestinal permeability:
a. serum D-lactic acid: fish D-lactic ELISA assay kit
b. Evans Blue(EB)
6. Histological assessment: 3D digital slice scanner

22. RESULT
Effect of A. hydrophila on the concentration of intestinal EB and
serum D-lactic acid after oral intubation with B. subtilis

23. Histological changes of the intestines 48 h after oral application
of A. hydrophila
Groups Intestinal villus Number per
villus
Number per
mm2
Length (μm) Width (μm) Goblet cells Inflammatory
cells
Control 367.62 ±
23.70
110.32 ±
7.80
34.3 ± 6.22 1357.05 ±
271.88
A. hydrophila 296.49 ±
19.62
171.75 ±
16.03
40.1 ± 7.43 4014.06 ±
872.12
B. subtilis + A.
hydrophila
375.71 ±
14.26
108.87 ±
7.02
63.6 ± 14.10 1922.84 ±
373.25
Effect of A. hydrophila on the intestinal length, width of villi, No.of villus of goblet,
inflammatory cells and after orally intubation with B. subtilis
A) Control
B) A.
hydrophila
C) B. subtilis +
A.
hydrophila

24. CONCLUSION
• The amount of EB and serum level of D-lactic acid
concentration was markedly higher in the A.hydrophila group
compared to control and protective group 48 hpi and 72 hpi
with A. hydrophila. This indicates that protective group having
B. subtilis could largely prevent the increase in intestinal
mucosal permeability caused by A.hydrophila.
• Disruption of intestinal barrier function is often accompanied
by intestinal inflammation. Administration of the probiotic B.
subtilis Ch9 prior to oral intubation could prevent functional
damage to intestinal mucosal barrier and reduced inflammation
induced by A. hydrophila in grass carp.

25. CASE STUDY-2
Natacha C. Gómez, Juan M. P. Ramiro, Beatriz X. V. Quecan and
Bernadette D. G. de Melo Franco
OBJECTIVE: To evaluate the potential probiotic traits of LAB isolated
from different fermented Brazilian products and their inhibition Effect
against Escherichia coli O157:H7, Listeria monocytogenes and S.
typhimurium biofilm formation.
(Gomez et al., (2016) Frontiers in Microbiology 7:863.doi: 10.3389/fmicb.2016.00863)
Received: 22 February 2016
Accepted: 23 May 2016

26. MATERIALS & METHODS
• Bacterial Strains:
Identificati
on code
Probiotic Strains
MBSa1 Lactobacillus sakei
MBSa3 Lactobacillus curvatus
VB69 Lactococcus lactis
VB94 Lactococcus lactis
40 Lactobacillus casei
352 Lactobacillus helveticus
368 Lactococcus lactis
113 Weisella viridescens
Pathogenic strains
Listeria monocytogenes
Escherichia coli O157:H7
Salmonella typhimurium
• Auto-Aggregation and Co-Aggregation Assays
• Biofilm Assay
• Inhibition of Biofilm Formation

27. RESULTS
Auto-aggregation of lactic acid bacteria strains cells re-
suspended in PBS (pH 7.1) evaluated after 24h incubation at
37°C

28. Co-Aggregation aggregation values recorded for lactic acid bacteria
strains with Listeria monocytogenes , Salmonella typhimurium and
Escherichia coli O157:H7 after 24h incubation at 37°C in PBS (pH 7.1)

29. MBSa1
MBSa3
VB69
VB94
40
352
368
113
48 hrs
1.8
1.6
1.2
1
0.8
0.6
0.4
0.2
0
Biofilm Formation Of LAB using the microtiter plate assay

30. A. Listeria
monocytogenes
1. Positive control
2. W. viridescens 113
3. Lactobacillus casei 40
4. Lactobacillus
helveticus 352
5. L. lactis 94
6. L. lactis 69
Quantification of pathogen biofilms in presence of probiotic strains

31. 1
1
1. Positive control B. S. Typhimurium
2. W. viridescens 113 C. E. coli O157:H7
3. Lactobacillus casei 40
4. Lactobacillus
helveticus 352
5. L. lactis 94
6. L. lactis 69

32. CONCLUSION
• Lactobacillus curvatus MBSa3 exhibited the highest co-aggregation (69%
with Listeria monocytogenes and 74.6% with E. coli O157:H7) and in this
case pathogenic biofilms were not detected after three times of incubation
tested, 24, 48,and 72 hours.
• The study showed the potential of probiotic LAB biofilms for the control of
Listeria monocytogenes, S. Typhimurium and E. coli O157:H7 biofilms
formation.

33. Conclusion
• Biocontrol property of some probiotics can be exploited
against certain pathogenic strains of bacteria.
• Due to the potential antimicrobial functions, probiotics can be
effectively used to control the pathogens of poultry, live stock
and aquaculture.
• Probiotics can be used as environment friendly agents for
controlling a number of microbial pathogens.
• They can be used to counteract the proliferation of several
bacteria which form biofilm leading to food contamination.
• Therefore, the probiotics offer great potential in food and
agriculture industry due to an array of applications they
possess.

34. Future DIRECTIONS
• More intensive studies are required on probiotics to enhance
their utility as biocontrol in food and agriculture industry
• The consumers must be made aware on the safety, efficacy,
sensory appeal, brand and marketing of probiotics for greater
acceptability.
• Detailed studies on probiotics in vivo is required to critically
evaluate the interaction between host and probiotic strains.
• Advanced throughput technologies to be conducted to
understand the molecular basis of relationship between
probiotic organisms and their host.