Probiotics and Its utilization in Aquaculture

PREPARED & PRESENTED

BY:

BHUKYA BHASKAR &

KALYAN,

Introduction

• The World total aquaculture production in 2014 was 73 783 725 tonnes (totalInland 47 102 391t & total marine 26 681 334 t).

• In the decade 2005–2014, fish culture production grew at 5.8 % /yr, down fromthe 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 amodification of the original word “probiotika.”

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 toincrease growth of hydrobionts.

• Recent documented evidence that probiotics can improve the digestibility ofnutrients, increase tolerance to stress, & encourage reproduction.

• FAO has designated use of probiotics as a major means for quality improvementof aquatic environment.

• Goal: make aquaculture products more acceptable to consumers

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” .

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.

Flow chart for selection of probiotics

Source

Isolation of Strain

In Vitro Assessment

In Vivo Assessment

Probiotic Commercialization

Government Agency Approval

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

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

Characteristics of good probiotics(Fuller):

(i) It should be a strain, capable of exerting abeneficial effect on the host animal e.g. increasedgrowth or resistance to disease.

(ii) non-pathogenic and non-toxic.

(iii) present as viable cells preferable in largenumbers.

(iv) capable of surviving & metabolizing in the gutenvironment e.g. resistance to low pH & organicacid.

(v) stable & capable of remaining viable for periodsunder storage and field conditions.

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)

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

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)

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?)

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)

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 immunomodulatorsaltering antitumor responses.

c). Antagonism effect of probiotics : Lactobacillus effective by producing certainweak organic acids as lactic acid & formic acids.

as acidolin, acidophylin, lactobacillin, lactocidin effective against many pathogenicbacteria as Escherichia, Salmonella, Streptococcus, Shigella, Proteus Klebsiella,Pseudomonas, Bacillus and Vibrio thus helps to fight disease.

Lactobacillus balgaricus are potent toxin neutralizer.

Cont…

• d). Antimicrobioses for host benefits:

• Intestinal microbiota of higher animals contributes to resistance against invadingpathogens.

• P. pentosaceous 43200 most promising strain for further research on bacteriocinsmediated protection against C. botulinum hazardous spores.

• e). Probiotic feeding:

• Probiotics once colonized in the gastrointestinal (GI) tract can effect the flora in 3ways as 1). By providing preformed metabolites.

• 2). By continuous provision of viable cells which do not colonize but whichsurvive & metabolize insitu.

• 3). By provision of viable cells which colonize the gut & produce usefulmetabolites insitu.

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

Mode of action of probiotics

Colonization resistance

Suppression of endogenous

pathogens eg: antibiotic

associated diarrhoeaprobiotics

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

Modes of action of probiotics

• Vibrio alginolyticus can be inoculated into shrimp culture with an aim to suppressthe pathogenic vibrio harvey, V. parahaemolyticus, V. splendidus.

• Isolated sp of Carbonobacterium piscicon & C. divergens are producedbacteriocins against L. monocytogens by producing Piscicocin v1 & diversin v41.

• Addition of LAB to rotifers fed to turbot larvae Scopthalmus maximus was foundto 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 virulentstrain of Vibrio anguillarium.

• The effect of flora on digestion in cold water fish is probably negligible as aminimum levels of 107 CFU/gm of intestinal content seem necessary forsignificant bacterial enzyme activity.

Probiotics in Aquaculture

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.

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

Some of the probiotics supplemented products used in Aquaculture

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

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

Aquatic sp used for probiotic application

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

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

Vibrio alginolyticus Salmonids

Probiotics role in Pathogen inhibition in host

Epinephelus coioides

Litopenaeus vannamei

Oncorhynchus mykiss

Anguilla anguilla

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

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

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

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

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).

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

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)

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 scallopsand have shown some inhibition to Vibrio sp. and Aeromonas hydrophila

• Bacillus sp. and Lactobacillus sp. shown to depurate oysters (Crassostreavirginica) against V. vulnificus (Williams et al., 2001)

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 epithelialsurface 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 produceEnzymes as proteases, lipases in bivalves)

• 5). Enhancement of immune response: (probiotics can stimulate non specificimmune system eg: Clostridium butyricum in rainbow trout enhanced phagocytic{leucocytes} activity & resistance to vibriosis.

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 assource 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. Moraxellashowed Antiviral capacity high specificity for poliovirus.

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.

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.

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.

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.

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

Special thanks to

Dr. Yusufzai sir

associate professor,

Head of dept. of Aquaculture

Gratitude To My Beloved Parents Sacrifices, India