Probiotics for Aquaculture

Transcription

1 Probiotics for Aquaculture Seppo Salminen Functional Foods Forum University of Turku Finland

2 Acquaculture Fish farming Carp and related fish are the most farmed fish (mainly in Asia) at 18,5 million metric tons annually Oysters 4,6 Clams 4,1 Mixed freshwater fish 37 3,7 Shrimp, Prawns 2,5 Salmon, trout 2 Marine capture levelling lli ( million metric tons) Aquaculture on the rise ( million metric tons)

3 Acquaculture Fish farming, shrimps, prawns, clams, oysters Growth importance Fish exposed to a variety of microbes Environmentalist concerns Water W t pollution Disruption of ecosystems Concentration of toxins in edible flesh Window for probiotics?

4 Probiotics Probiotics: live microorganisms that, when administered in adequate amounts, confer a health benefit on the host' Joint FAO/WHO Working Group Report, 2001 Host can be any host Health targets include: Intestinal challenges including gastroenteritis, inflammatory diseases, counteracting antibiotic side-effects In human use usually lactic acid bacteria (LAB) or bifidobacteria Efficacy, safety

5 Probiotics in Fish and Aquaculture

6 SCREENING -using all isolates -- target t groups identification SCREENING TARGET EVALUATION Defining microbiota deviations - target groups SELECTING PROBIOTICS FOR AQUACULTURE SELE ECTION Choose most promising specific strains / species SELECTION CRITERIA Focus on adhesion, competitive exclusion, competition for nutrients, ti t microbiota for weight gain, binding of harmful substances from diet Aquaculture: microbiota irregularity identification as basis for selecting & characterizing probiotics SSMENT ASSE Biomarker impact evaluation Characterization of properties Choose most promising specific strains / species/ combinations for intervention studies Technology considerations Application to end use Focus on strains, target properties and end use combinations New Pro obiotics/ aqu uaculture

7 Selection of Human Probiotics TARGETS DEFINED FOR INTERVENTION STUDIES SPECIFIC PROBIOTIC FOR INTERVENTION TARGET SUBJECTS INTESTINAL MICRO -BIOTA ABERRANCY ASSESSMENT FAECAL MICROBIOTA ASSESSMENT CHARACTERISATION ABERRANT SUBJECTS MICRO- BIOTAABERRANCY ASSESSMENT HEALTHYSUBJECTS NORMAL MICROBIOTA CHARACTERISATION DEFINING TARGET ABERRANCY (ASSESSMENT OF REQUIRED INTERVENTION AND SITE) SPECIFIC PROBIOTIC IDENTIFICATION (ASSESSMENT OF PROBIOTIC PROPERTIES AND ACTIONS)

8 Requirements for fish probiotics Adhesion to fish mucus surfaces Adhesion in different surface areas of importance Adhesion considered species-specific? Competitive exclusion of pathogens Specific fish pathogens, competition for nutrients Balancing intestinal microbiota/barrier Identifying i healthy fish microbiota, strengthening th immune system Right microbiota for weight gain? Benefits for growth

9 Requirements for fish probiotics Specific microbiota promotes weight gain Gordon and coworkers Feed efficiency influenced Characterise the type of microbiota to promote weight gain and feed efficiency Balancing intestinal microbiota/barrier Identifying healthy microbiota, strengthening barrier and resistance to change Modulate microbiota for weight gain

10 Requirements for fish probiotics Should one consider species-specific specific properties? Can human probiotics be used? Studies on competitive exclusion on fish pathogens? Growth and survival of fish?

11 Species-specificity of Probiotics Lactic acid bacteria adhesion in various hosts (%) Adhesion Human Dog Possum Emu Ostrich Rainbow tro 0 LGG La1 LcS Lbulg Ef M74 Ef SF68 Bb12 UK1A SK2A

12 Adhesion of Fish Pathogens Mucus from different sites (%) Adhesion 4,5 4,0 3,5 3,0 25 2,5 2,0 1,5 1,0 0,5 0,0 Oesophagus Stomach Intestine Gills Surface Mucus samples A. salmonicida V. anguilarum Fl.psychrophilum

13 Adhesion of Probiotics on Fish Mucus Fish mucus from different sites 35,0 30,0 Ad dhesion (%) 25,0 20,0 15,0 10,0 5,0 0,0 Oesophagus Stomach Intestine Gills Surface Mucus sample Lactobacillus casei Shirota Lactobacillus rhamnosus LC 705 Bifidobacterium lactis Bb12 Lactobacillus bulgaricus Enterococcus faeciuta Lactobacillus rhamnosus GG Lactobacillus johnsonii La1

14 Impact of Probiotic Culture Supernatants Clearing zones by probiotic culture supernatants 9 10 ) Clear ing zone (mm 1 0 MRS (ph 5,59) MRS (ph 7,1) L. johnsonii (ph 3,78) L. johnsonii (ph 7,65) Bb. Lactis (ph 3,79) Bb. Lactis (ph 7,31) L. GG (ph 3,77) L. GG (ph 7,05) E. faecium (ph 4,33) E. faecium (ph 6,71) L. bulgaricus (ph 4,3) L. bulgaricus (ph 7,25) Supernatants and ph A.salmonicida V. anguilarum Nikoskelainen et al 2003

15 Probiotic Stability in Water Viability of Lactobacillus GG in water Water sample control 10 9 CFU/ml CFU/ml At feeding > min after feeding > min after feeding > min after feeding > hours after Nikoskelainen et al 2005

16 Probiotic Stability in Water Probiotics intended to fish larvae in sea water Survival of Lactobacillus brevis, Lactobacillus casei and Pediococcus acidilactici good Vazquez et al. 2003

17 Aeromonas Challenge in Trout 60,0 50,0 control 40,00 L rhamnosus ,0 20,0 L rhamnosus ,0 0, Time (days) Nikoskelainen et al 2003

18 Changes in Microbiota 10 8 Pre-Challenge 10 7 Challenge Post-Challenge CFU/ /g Vibrio Coliforms Pseudomonas Aeromonas Lactobacillus Lactic acid bacteria Anaerobic Aerobic

19 Changes in Microbiota LAB 10 9 group 10 8 Pre-Challenge Challenge 10 7 Post-Challenge CFU/g Vibrio Coliforms Pseudomonas Aeromonas Lactobacillus Lacticc acid bacteria Anaerobic Aerobic

20 Changes in Microbiota 10 8 Pre-Challenge 10 7 Challenge Post-Challenge LAB group CFU/g Vibrio Coliforms Pseudomonas Aeromonas Lactobacillus Lacticc acid bacteria Anaerobic Aerobic

21 Lactococcus garvieae and Streptococcus iniae Challenge in Trout Challenge results in rapid mortality Several experimental probiotics protect and enhance survival Brunt & Austin 2005

22 Lactococcus garvieae and Streptococcus iniae Challenge in Trout Slightly milder protection provided by inactivated probiotic strains Brunt & Austin 2005

23 Prevention of Pathogen Adhesion Competitive exclusion Inhibition Listeria monocytogenes 4x 3x 2x 4a 3d 3c 3b 3a 2f 2e 2d 2c 2b 2a 4x 3x 2x 4a 3d 3c 3b 3a 2f 2e 2d 2c 2b 2a 1x PJS LC705 Bb99 LGG Displacement % inhibition 1x PJS LC705 Bb99 LGG % inhibition 4x 3x 2x 4a 3d 3c 3b 3a 2f 2e 2d 2c 2b 2a All combinations showed better abilities than single strains 1x PJS LC705 Bb99 LGG % displacement

24 Pathogen exclusion Combination of probiotic can increase pathogen exclusion LGG-LC705-PJS-Bb99 LGG-LC705-PJS-Bb12 E. coli E. coli L.monocytogenes L.monocytogenes S. Typhimurium S. Typhimurium C. difficile St. aureus C.histolyticum B. vulgatus C. difficile St. aureus C.histolyticum B. vulgatus % inhibition of pathogen adhesion % inhibition of pathogen adhesion Inhibition analysis Displacement analysis Competitive exclusion analysis Inhibition analysis Displacement analysis Competitive exclusion analysis Collado et al., 2006

25 Probiotics & Removal of Toxins Several probiotics effective in binding toxins in human subjects, mice and chicken Reduce absorption or metabolism of several toxins or heavy metals Adherence reduced after binding toxins or metals

26 Toxin Removal Removal by Lactobacillus rhamnosus strain GG AFB1 AFM1 OA Trp-P-1 PhIP Folic acid Viable Heat Treated Acid Treated Caffeine Vitamin B % Removed Trp-P-1 > AFB 1, PhIP > OA, AFM 1 > Folic acid, Caffeine, Vitamin B 12

27 Heavy Metal Removal Comparison of strains etal bound % m GG LC705 PJS Bbi E8/99 MIX Cd Pb contact time 60 min

28 Cyanotoxin Removal MC-LR binding 25 % MC-L LR bound MC-LR 0 GG LC705 PJS Bbi E8/99 MIX

29 Toxin Removal From Diet Gastrointestinal tract of humans or animals

30 Probiotics applied in aquaculture (after Irianto & Austin, 2002) Probiotic identity Source Used on Method of application Gram-positive bacteria Bacillus sp. S11 Penaeus monodon Penaeus monodon Premix with feed Bacillus sp. Commercial product Penaeids Water Bacillus sp. Water Added to water Lactobacillus lactis AR21 Mass culture Brachionus plicatilis Feed additive Mixed culture, mostly Bacillus spp. Commercial product Brachionus plicatilis Mixed in water Gram-negative bacteria Vibrio alginolyticus Beach sand Penaeids, salmomids Feed, bath for 10 min Yeast Saccharomyces cerevisiae, S. exiguous, Phaffia rhodozoma Commercial product Litopenaeus vannamei Premix with feed Microalgae Tetraselmis suecica Commercial product Penaeids, Salmo salar Feed

31 List of microorganism authorized as probiotics ot in feedingstuffs s under Council Directive 70/524/EEC Probiotics Bacillus cereus var. toyoi Bacillus licheniformis Bacillus subtilis Enterococcus faecium Lactobacillus casei Lactobacillus farciminis Lactobacillus plantarum Lactobacillus rhamnosus Pediococcus acidilactici Saccharomyces cerevisiae Streptococcus infantarius

32 Other Uses of Probiotic LAB Fermentation processes: Both fish feeds and fish products Traditional way to preserve food and feed Preservation based on acidity, production of antimicrobial substances, competition against food pathogens Fish fermentation: rare and regional - until now

33 Probiotic fish products? Currently few or no applications Attempts to use LAB as preservative Fermentation may be unwanted as it leads to changes in aroma and taste Potential health benefits of LAB used for fish products currently unexplored

34 Probiotic considerations Metabolic activity (growth) of LAB to be avoided (unless fermented product) LAB can be useful in aquaculture Reduction of disease risk in fish, prevention of spoilage to deliver health benefits to humans Inactivated LAB or substances produced by LAB?

35 Concluding Remarks Opportunity for new probiotic uses in growing aquaculture Opportunities for fish products Challenges: demonstration of health benefit(s) and growth improvement in fish, shrimps and humans