utilization of the desirable probiotic species in Aquaculutre best alternatives in replacing utilization of Antibiotics

1. Probiotics and Its utilization in Aquaculture
PREPARED & PRESENTED
BY:
BHUKYA BHASKAR &
KALYAN,

2. Introduction
• The World total aquaculture production in 2014 was 73 783 725 tonnes (total
Inland 47 102 391t & total marine 26 681 334 t).
• In the decade 2005–2014, fish culture production grew at 5.8 % /yr, down from
the 7.2 % /yr achieved in last decade (1995–2004).
• term “probiotic” comes from Greek pro and bios means “prolife”.
• The global market for probiotic ingredients, supplements & foods, reached US
$15,900 million in 2008 & projected to rise up to US $19,600 million in 2013,
growth rate of 4.3% /yr.
• Elie Metchnikoff (1905) 1st to describe the positive role played by some bacteria
& to replace harmful microbes by beneficial microbes”.
• The term probiotic was introduced until 1965 by Lilly and Stillwell as a
modification of the original word “probiotika.”

3. Cont…
• Kozasa made the 1st empirical application of probiotics in aquaculture
• Sperti modified the concept of “tissue extracts that stimulate microbial growth.”
• Use of bacteria as a food source & as a biological control of fish disease was 1st
proposed by Yasuda & Taga (1980).
• The 1st application of probiotics occurred in 1986, to test their ability to
increase growth of hydrobionts.
• Recent documented evidence that probiotics can improve the digestibility of
nutrients, increase tolerance to stress, & encourage reproduction.
• FAO has designated use of probiotics as a major means for quality improvement
of aquatic environment.
• Goal: make aquaculture products more acceptable to consumers

4. Definition of Probiotic
• Probiotics 1st used to describe a microbial feed/food supplement was by Parker in 1974.
Probiotics as “organisms and substances that contribute to intestinal microbial balance.”
• Fuller defined as “live microbial food supplement that benefits the host (human or
animal) by improving the microbial balance of the body” & said that it would be
effective in a range of extreme temperatures and salinity variations.
• In 1998, Guarner & Schaafsma assumed that probiotics are live microorganisms
which, when consumed in adequate amounts, confer health benefits to the host.
• Gatesoupe in 1999, Probiotics as “microbial cells administered in a certain way, which
reaches the gastrointestinal tract & remain alive with the aim of improving health” .

5. Principle & Attributes of of probiotic work
• Principle of probiotic work: based on the competitive exclusion (ecological
process it can beneficially manipulate the microbial composition of gut of host &
environment)
• A microbe that protects its host & prevents disease. Probiotics counter the
decimation of helpful intestinal bacteria by antibiotics.
• Methods commonly used for microencapsulation of probiotics are the emulsion,
extrusion, spray drying & adhesion to starch
• Attributes of probiotics: a). Viability b). Colonization c). Stabilization.

6. Flow chart for selection of probiotics
Source
Isolation of Strain
In Vitro Assessment
In Vivo Assessment
Probiotic Commercialization
Government Agency Approval

7. Culture, Isolation & Identification of probiotic bacteria from host samples
CULTURE & ISOLATION OF BACTERIA: tissue from intestine, kidney, liver,
skin or muscles. incubated in BOD at 30+ or -10 for 24 hr. pure colonies will be
isolated (OIE,2006). Pure culture stored at 200c.
B). Secondary tests:. Nitrate reduction test, Malate utilization test, Urea broth test,
Vogus-proskauer (VP) test, Methyl red test, Indole production test & Simmon’s
citrate utilization test,
Identification of probiotic bacteria:
A) primary test : Motility test, catalase test & oxidase test,
C). Tertiary test for the confirmation of bacteria:
in vitro antagonistic test a). Well diffusion agar assay (WDAA), b). Broth co-culture
assay, c). Cross-streaking method

8. Comparative internal organs observation for
probiotics action in fish & shrimp & oyster

9. Characteristics of good probiotics(Fuller):
(i) It should be a strain, capable of exerting a
beneficial effect on the host animal e.g. increased
growth or resistance to disease.
(ii) non-pathogenic and non-toxic.
(iii) present as viable cells preferable in large
numbers.
(iv) capable of surviving & metabolizing in the gut
environment e.g. resistance to low pH & organic
acid.
(v) stable & capable of remaining viable for periods
under storage and field conditions.

10. Safety aspects of probiotics
a). Intestinal effects; as relieve effects as promote recovery from diarrhea (rotavirus,
traveler’s). Produce lactase, lactose tolerance & absorption. Relieve constipation, treat
colitis.
b). Immune system effects; as enhance specific & nonspecific response. gastrointestinal
immunity. Reduce chance of infection from pathogens (salmonella, shigella), Inhibit
pathogen growth & translocation
c). Other effects; reduce risk of certain cancer (colon, bladder). Detoxify carcinogens.
Suppress tumors. Lower serum cholesterol. Reduce blood pressure in hypertensives. Treat
food allergies. Synthesize (folic acids, niacin, riboflavin, vit B6 & B12). Rise nutrient
bioavailability. Improve urogenital health. Optimize effects of vaccines (eg: rotavirus
vaccines, typhoid fever vaccines)

11. Aquaculture authority banned antibiotics & drugs residual substance level
1. Chloramphenicol 11. Dimetridazole
2. Nitrofurans ( Furaltadone, Furazolidone,
Furylfuramide, Nifruratel, Nifuroxime,
Nifuraprazine, Nitrofratoin, Nitrofurazone)
12. Metronidazole
3. Neomycin 13. Ronidazole
4. Nalidixic acid 14. Ipronidazole
5. Sulphamethoxzole 15. Other nitroimidazoles
6. Aristolochia spp. & preparation there of 16. Clenbuterole
7. Chloroform 17. Diethylstilbestrol (DES)
8. Chlorpromazine 18. Sulfonamide drugs (except sulfodimethoxine,
sulfabromomethazine & sulfaethoxy pyridazine)
9. Colchicine 19. Fluroquinolones
10. Dapsone 20. Glycopeptides

12. Screening of Putative:
In-vitro antagonism testing of probiotics against pathogens
• Candidates are exposed to pathogen in liquid or solid medium screened for
production of inhibitory compounds (bacteriocins), Siderophores, or on
competition for nutrients must be undertaken with extreme caution: not all media
are suitable
• probionts are finicky as to on which medium they produce inhibitory compounds
(e.g., zobell marine agar vs. TSB)

13. In-vivo Evaluations
• Involves introducing candidate species to host cultures and monitoring growth,
survival, physiochemical parameters
• means of addition: addition to artificial diet, addition to culture water, bathing,
addition via live food
• experimental (allochthonous) infection of host via immersion
• needs long-term evaluation (is the pathogenic effect one of suppression or delay?)

14. Evaluation of Pathogenicity
• Probiotics must not be pathogenic to the host organism - this must be confirmed
prior to acceptance
• host must be challenged under stressed and non-stressed conditions
• usually accomplished by adding probiotic to the culture water
• proper way to do this under monoxenic conditions (only the probiont present) also
look at interaction with other food organisms found simultaneously in culture
(e.g., algae)

15. Function of probiotics:
a). Growth promotion:
(LAB) act on complex carbohydrates (starch, cellulose & synthesize many B –
complex & vitamin K. increase appetite & good health in host. infection control &
from raised digestibility of nutrients.
b). Immunostimulation/ Immunomodulation:
Eg; Lactobacillus casei YIT9018 (BPL) shown to act as immunomodulators
altering antitumor responses.
c). Antagonism effect of probiotics : Lactobacillus effective by producing certain
weak organic acids as lactic acid & formic acids.
as acidolin, acidophylin, lactobacillin, lactocidin effective against many pathogenic
bacteria as Escherichia, Salmonella, Streptococcus, Shigella, Proteus Klebsiella,
Pseudomonas, Bacillus and Vibrio thus helps to fight disease.
 Lactobacillus balgaricus are potent toxin neutralizer.

16. Cont…
• d). Antimicrobioses for host benefits:
• Intestinal microbiota of higher animals contributes to resistance against invading
pathogens.
• P. pentosaceous 43200 most promising strain for further research on bacteriocins
mediated protection against C. botulinum hazardous spores.
• e). Probiotic feeding:
• Probiotics once colonized in the gastrointestinal (GI) tract can effect the flora in 3
ways as 1). By providing preformed metabolites.
• 2). By continuous provision of viable cells which do not colonize but which
survive & metabolize insitu.
• 3). By provision of viable cells which colonize the gut & produce useful
metabolites insitu.

17. Modes of action of probiotics
1). Production of inhibitory compounds/ substances
2). Competion for chemicals or available energy
3). Competion for iron & production of Siderophores
4). Competion for Adhesion sites
5). Enhancement of immune response
6). Improvement of water quality
7). Interaction with phytoplankton
8). Production of enzymes
9). Synthesis of vitamins & absorption of minerals
10). Improvement of stress tolerance
11). Antimicrobial effects
12). Digestive simulation effect
13). Cholesterol – lowering effect

18. Mode of action of probiotics
Colonization resistance
Suppression of endogenous
pathogens eg: antibiotic
associated diarrhoea
probiotics
Normalized intestinal
microbiota
Control of irritable
bowel syndrome
Metabolic effects
Lower level of toxigenic /
mutagenic reaction gut
Reduction risk for colon
cancer
Supply of SCFA & vit
(eg; folate ) colonic
Suppression of exogenous
pathogens eg: travelers
diarrhoea
Bile salts deconjugation
& secretion
immunomodulation
Strengthened innate
immunity
Improved lactose
toleranceLactose hydrolysis
Lower serum cholesterol
Alleviate food allergy
symptoms in infants
Balance immune
response

19. Modes of action of probiotics

20. • Vibrio alginolyticus can be inoculated into shrimp culture with an aim to suppress
the pathogenic vibrio harvey, V. parahaemolyticus, V. splendidus.
• Isolated sp of Carbonobacterium piscicon & C. divergens are produced
bacteriocins against L. monocytogens by producing Piscicocin v1 & diversin v41.
• Addition of LAB to rotifers fed to turbot larvae Scopthalmus maximus was found
to improve growth & increase resistance against pathogenic vibrio.
• Dry feed with LAB Carnobacterium divergens isolated from Atlantic cod (G.
marhua) intestines improved disease resistance of cod fry exposed to virulent
strain of Vibrio anguillarium.
• The effect of flora on digestion in cold water fish is probably negligible as a
minimum levels of 107 CFU/gm of intestinal content seem necessary for
significant bacterial enzyme activity.

21. Probiotics in Aquaculture

22. Cont…
• The effect of flora on digestion in cold water fish is probably negligible as a
minimum levels of 107 CFU/gm of intestinal content seem necessary for
significant bacterial enzyme activity.
• Flavobacterium inhibited growth of Palva lutheri from inoculum of 103 CFU/ml.
• Biocontrol of Aeromonas hydrophila by the Lactococcus lactis suppress protease
production by A. hydrophila which contributes to its virulence in fresh water
fishes as cyprinus carpio & tilapia mossambica by immersion technique proved
successful as antibodies of Lactococcus lactis could be observed in fishes even
after infection by Aeromonas.

24. Few examples of probiotic products used by shrimp farmers
in India
Probiotic brand name Active ingredients/Organisms (as per label Cost/ kg Type of
probiotic
Pro-B Active A Culture/viable spores comprising of Cellulomonas spp., Bacillus spp.,
Nitrasomonas spp. Nitrobacter spp., and Yeast, Xanthophyllomyces spp.,
Enzymes (protease, cellulase, Pectinase, Hemicellulase)
2300/- Water
Avant-Bact MOS (Mannan oligo saccharides), Pediococcus acidilactici MA 18/5 m 1090/- feed
Avant-proW Bacillus subtilis Rosell-179 and Pediococcus acidilactici MA 18/5m 2790/- Water
Black solve Bacillus spp., Nitrasomonas spp., Nitrobacter spp., Rhodococcus spp.,
Cellulomonas spp., Pseudomonas spp., Lactobacillus spp., Aerobacter,
Aspergillus niger, Aspergillus oryzae
729/- water
Bio cult - 120 Soil
Prob solve Bacillus spp., Nitrasomonas spp., Nitrobacter spp., Rhodococcus spp.,
Cellulomonas spp., Pseudomonas spp., Lactobacillus spp., Aerobacter,
Aspergillus niger, Aspergillus oryzae
1458/- feed
Pond solve Nitrasomonas spp., Nitrobacter spp., Rhodobacter, Enzymes (protease,
lipase, amylase) and surfactants
1458/- water
Micro -PS Rhodococcus, Rhodobacter, Nitrasomonas, Nitrobacter and Thiobacillus 165/ lit Water

25. Some of the probiotics supplemented products used in Aquaculture

26. Production of inhibitory compounds by probiotics
• Release of chemicals having a bactericidal or bacteriostatic effect ultimate result:
competitive edge for nutrients/energy
• production sites: in host intestine, on its surface, or in culture medium
• products: antibiotics, bacteriocins, Siderophores, lysozymes, proteases, hydrogen
peroxide, organic acids (pH change)
• Lactobacillus sp. produces bacteriocins (toxins) marine bacteria produce bacteriolytic
enzymes against V. parahaemolyticus
• Alteromonas sp. produces monastatin, shown to be inhibitory against Aeromonas
hydrophila inhibitory effects have been shown by probiotics against aquaculture
pathogens

27. Different applications of probiotics in aquaculture
probiotics as growth promoter
Identity of the probiotic Applied to aquatic species
Bacillus sp. S11 Penaeus monodon
Bacillus sp. Catfish
Carnobacterium divergens Gadus morhua
Alteromonas CA2 Crassostrea gigas
Lactobacillus helveticus Scophthalmus maximus
Lactobacillus lactis AR21 Brachionus plicatilis
Streptococcus thermophilus Scophthalmus maximus
Streptomyces Xiphophorus helleri
L. casei Poeciliopsis gracilis
Bacillus NL 110, Vibrio NE 17 Macrobrachium rosenbergii
Bacillus coagulans Cyprinus carpio koi

28. Aquatic sp used for probiotic
application

29. Commercially used probiotics organisms in aquaculture
Lactobacillus species Bifidobacterium species Streptococcus species
L. Acidophilus B. bifidum S. thermophilus
L. Paracasei B. breve
L. Casei B. lactis
L. Plantarum B. longun
L. fermentum
L. reuteri
L. Gasseri
L. rhamnous
L. Johnsonii
L. Salivarius
L. Lact

30. Probiotics role in Pathogen inhibition
Probiotics sp Host cultured sp Probiotics sp Host cultured sp
Bacillus sp. Penaeids V. fluvialis Oncorhynchus mykiss
Enterococcus faecium SF 68 Anguilla anguilla Tetraselmis suecica Salmo salar
L. rhamnosus ATCC53103 Oncorhynchus mykiss Carnobacterium sp. Hg4-
03
Hepialus
gonggaensis larvae
Micrococcus luteus A1-6 Oncorhynchus mykiss Lactobacillus acidophilus Clarias gariepinus
Pseudomonas fluorescens Oncorhynchus mykiss Bacillus spp., Enterococcu
ssp.
Farfantepenaeus
brasiliensis
P. fluorescens AH2 Oncorhynchus mykiss Lactococcus lactis Epinephelus coioides
Pseudomonassp. Oncorhynchus mykiss
Roseobacter sp. BS. 107 Scallop larvae
Saccharomyces cerevisiae,
S. exiguous, Phaffia
rhodozyma
Litopenaeus vannamei

31. Probiotics role in Pathogen inhibition in host
Epinephelus coioides
Litopenaeus vannamei
Oncorhynchus mykiss
Anguilla anguilla

32. Nutrient digestibility
Nutrient digestibility
L. helveticus Scophthalmus maximus
Bacillus NL
110, Vibrio NE 17
Macrobrachium
rosenbergii
Carnobacterium sp. Hg4-
03
Hepialus
gonggaensis larvae
Lactobacillus acidophilus Clarias gariepinus
Shewanella
putrefaciens Pdp11
Solea senegalensis Solea senegalensis
Shewanella putrefaciens

33. Water quality Probiotics role in Aquaculture
Probiotic strain in
Water quality
Succeeded host sp
Bacillus NL 110, Vibrio sp. NE
17
Macrobrachium rosenbergii
Lactobacillus acidophilus Clarias gariepinus
B. coagulans SC8168 Pennaeus vannamei
Bacillus sp., Saccharomyces sp
.
Penaeus monodon
Penaeus monodon Bacillus sp.
Bacillus NL 110, Vibrio sp. NE
17
Macrobrachium rosenbergii
Bacillus sp. Penaeus monodon

35. Probiotics role in stress tolerance Stress tolerance in Aquaculture
Lactobacillus delbrueckii Dicentrarchus labrax
Alteromonas sp. Sparus auratus
B. subtilis, L. acidophilus,
S. cerevisiae
Paralichthys olivaceus
L. casei Poecilopsis gracilis
Pediococcus acidilactici Litopenaeus stylirostris
Shewanella
putrefaciens Pdp11
Mahi mahi
Sparus auratus
Paralichthys olivaceus
Litopenaeus stylirostris
Dicentrarchus labrax

36. Reproduction improvement
probiotics for
Reproduction
improvement
Host sp used for
Reproduction
improvement
Bacillus subtilis Poecilia reticulata,
Xiphophorus maculatus
L. rhamnosus Danio rerio
L. acidophilus, L. casei,
Enterococcus faecium,
Bifidobacterium
thermophilum
Xiphophorus helleri
Poecilia reticulata

37. Recent Findings: finfish
• Digestive tract of finfish contains 108 cells/g (Ringo et al., 1995)
• For cod, Gadus gadus, gut is colonized by similar bacteria as found in eggs
(Hansen and Olafsen, 1999)
• Putative probiotics added as soon as possible after hatching in order to colonize
gut prior to feeding (Ringo and Vadstein, 1998)
• Turbot and dab harbor bacteria capable of suppressing growth of V. anguillarum
(Ollson et al., 1992)
• V. alginolyticus was effective in reducing disease caused by Aeromonas
salmonicida in Atlantic salmon (Austin et al., 1995).

38. Recent Findings: finfish
• Kennedy et al. (1998) showed addition of a Gram-positive probiotic increased
survival, size uniformity, and growth rate of snook, red drum, spotted sea trout and
striped mullet.
• Gram et al. (1999) reported a strain of Pseudomonas fluorescens reduced mortality
of 40 g rainbow trout infected with pathogenic V. anguillarum
• Garcia-de-la-Banda et al. (1992) added Streptococcus lactis and Lactobacillus
bulgaricus to rotifers and Artemia sp. nauplii and recorded 6x higher survival
Nikoskelainen et al. (2003) showed immune enhancement in rainbow trout via
Lactobacillus rhamnosus supplemented in feeds

39. Recent Findings: shrimp
• Broad application in hatcheries, used as food source (e.g., soil bacteria for P. monodon
nauplii)Improved survival (57% vs. 0%) after 13 days against V. anguillarum
• Improved survival of L. vannamei PL’s inoculated with V. alginolyticus (non-pathogenic)
vs. oxytet and control (Garriques and Arivalo, 1995)
• probiotics in Ecuador in 1992, hatchery down-time between batches was reduced from 7
days per month to 21 days per year, production volumes increased by 35% &
antimicrobial use decreased by 94%
• In shrimp hatcheries in New Caledonia, a strain of Pseudoalteromonas piscicida was
found to inhibit the growth of Vibrio sp. (Saulnier et al., 2000)

40. Recent Findings: bivalave molluscs
• Most research has focused on nutritional contributions to mollusc larvae
• most work in vitro wherein autochthonous strains have been isolated from scallops
and have shown some inhibition to Vibrio sp. and Aeromonas hydrophila
• Bacillus sp. and Lactobacillus sp. shown to depurate oysters (Crassostrea
virginica) against V. vulnificus (Williams et al., 2001)

41. Benefits of probiotics in aquaculture
• 1). Production of inhibitory compounds: (bacteriocins, sideropheres,
lysozymes, proteases, hydrogen peroxides)
• 2). Competion for adhesion sites (probiotics on gut or external mucous epithelial
surface against pathogens as Vibrio anguillarium, Aeromonas hydrophila)
• 3). Competion for nutrients
• 4). Source of nutrients & enzymatic contribution to digestion: (bacteriodes &
Clostridium sp. Supplying fatty acids & vitamins to host. Probiotics produce
Enzymes as proteases, lipases in bivalves)
• 5). Enhancement of immune response: (probiotics can stimulate non specific
immune system eg: Clostridium butyricum in rainbow trout enhanced phagocytic
{leucocytes} activity & resistance to vibriosis.

42. Benefits of probiotics in aquaculture
• Lactobacillus rhamnosus (strain ATCC 53103) @ 105 cfu g-1 feed, stimulated the
respiratiory burst in rainbow trout (O. mykiss)
• 6). Influence on water quality : (Nitrosomonas { convert ammonia to nitrite},
Nitrobacter {convert nitrite to notrate}, methane reducing bacteria use CO2 as
source of molecular O2)
• 7). Interaction with phytoplankton :
(eg: probiotics algicidal effect microalgae red tide plankton)
• 8). Antiviral activity: (Pseudomonas sp., Vibrio sp., Aeromonas sp., &
coryniferms isolated from salmonid hatchery had antiviral activity against IHNV
{infectous hematopoitic necrosis virus} with > 50% plaque reduction. Moraxella
showed Antiviral capacity high specificity for poliovirus.

43. Limitation of probiotics use
• Probiotics can be used in advance as prevention tools i.e. these should be used
continuously before the outbreaks of diseases.
• Probiotics can prevent disease rather than treatment of disease.
• Probiotics can be established well in static or low water exchange systems (re-
circulatory system).
• Probiotics are effective if applied as soon as the water medium is sterilized before
contamination with other microbes.
• Probiotics can easily be destroyed by any other chemical or drug which generally
interferes with the establishment of useful microbes.

44. Future perspectives of probiotics
• Fermented African foods & beverages as Ogi & kunnu, to modulated= host immunity
is a very promising of research.
• Further screening of already discovered probiotics involves in vitro & in vivo
investigation to evaluate their potential as immunostimulatory agents.
• Genetic engineering of probiotics to make them more efficacious should be pursued.
• Should be focus on the mechanisms of action within system, which stimulate the in vivo
effects.
• Oligonucleotide sequence, which triggers innate immunity system, will go a long way
to help understand the mechanism by which immunostimulatory potentials of probiotics
are mediated.

45. Future perspectives of probiotics
• This investigation reveal which CpG motif within probiotic DNA (CpG DNA)
actually responsible for stimulation of immune system of humans.
• Phytase enzymes can rise the nutritional value of food & feed by liberating
inorganic phosphate from phytate, the major storage form of phosphate in plants.
• Bacillus coagulans is a lactic acid producing & spore forming probiotic bacteria
recombinant tested for Phytase activity on test plates at PH (5, 6, 7, 8, 9, 10) to
obtain any changing in PH optimum of the enzyme in the new host but no Phytase
activity was observed on the plates.

46. conclusion
• FAO should be recommend the sustainable use of Probiotics & their complex
(Biofloc) in replacing the Antibiotics & excess artificial feed in Aquaculture to
discourage aquatic habitat pollution.
• Enhance ecofriendly, sustainable, consumer safety.
• Utilized in multiple aquaculture production process due to its multiple beneficial
action both externally & internally.
• Lack of knowledge on modes of actions is very evident.

47. Cont…
• Reduce pollutants harmful effects by their direct utilization for microbial food
production process which makes them very less harmful form (degradation,
bioremediation, symbiotic, commensals, modification & other beneficial
unknown action of probiotic organisms).
• More information on competitive process between bacteria is required.
• More information on relationship between bacteria & their micro biota required

48. Special thanks to
Dr. Yusufzai sir
associate professor,
Head of dept. of Aquaculture
Gratitude To My Beloved Parents Sacrifices, India