Probiotic for Aquaculture. Inge Knap Director, Research & Development Chr. Hansen, Denmark

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1 Probiotic for Aquaculture Improving Health and Performance Inge Knap Director, Research & Development Chr. Hansen, Denmark

2 The research for probiotic for aquatic animals is increasing The research for probiotic for aquatic animals is increasing with the demand for environment friendly aquaculture. Although application of probiotic in aquaculture seems to be relatively recent the interest in environment friendly treatments is increasing rapidly (Wang et al 2008)

3 Aquatic animals are quite different from land animals for which most probiotic concepts have been developed. Man and terrestrial livestock undergo embryonic development within an amnion, whereas the larval forms of most fish and shellfish h are released in the external environment at an early ontogenetic stage. The larvae are highly exposed to gastrointestinal microbiota-associated disorders, because they start feeding even though the digestive tract is not fully developed and though the immune system is still incomplete (Gatesoupe 1999).

4 The digestive tract of hydrobionts is an open system constantly in contact with the surrounding environment water. Compared to water the digestive tract is an ecosystem far richer in nutrient and therefore more favourable for the growth of the majority of bacteria This leads to both probiotic opportunities as well as pathogen challenge

5 Introduction Overview Probiotic i for Aquaculture Improving Health and Performance Key criteria for use of probiotics in aquaculture What is a Probiotic? What kind of Probiotics are used? Improving the nutritional utilization Improving the nutritional utilization Mode of action - Improving the nutritional utilization Disease control M d f ti Di t l Mode of action Disease control Bioremediation New Probiotic

6 Key criteria for use of probiotics in aquaculture Improving the nutritional utilization Increased weight Faster growth Lower feed conversion Better protein and fat efficiency ration Disease control Bioremediation

7 What is a Probiotic? The word probiotic was introduced by R.P. Parker in 1974 and his original definition was Organisms and substances which contribute to intestinal microbial balance R. Fuller revised the definition in 1989; Live microbial fed supplements which beneficially affects the host by improving its intestinal microbial balance

8 What kind of Probiotics are used? Main used probiotics Lactic Acid Bacteria (LAB) Bacillus sp Yeast Other aquaculture related Probiotics but less common used; Pseudoalteromonas sp. Pseudomonas sp. and Vibrio fluvialis

Lactic acid bacteria (LAB) Definition of LAB Gram positive organisms Miroaerophilic Ferment hexose sugars to primarily lactic acid Lactococcus, Enterococcus, Pedicoccus, Streptococcus, Leuconostoc

9 Lactic acid bacteria (LAB) Definition of LAB Gram positive organisms Miroaerophilic Ferment hexose sugars to primarily lactic acid Lactococcus, Enterococcus, Pedicoccus, Streptococcus, Leuconostoc and Lactobacillus LAB play a prominent role in the world food supply Perform the main bio-conversations in fermented diary products, meats and vegetables. Critical for the production of silage, wine, coffee, sourdough, yoghurt and other fermented food products LAB naturally associated to mucosal surfaces of animals and LAB s belong to the normal microbiota of the gastrointestinal tract in healthy fish All LAB are GRAS or has QPS status

at present of nitrate/nitrite it can grow anaerobic Bacillus as probiotic is in form of Bacillus

Thermo-tolerant, survives pelletization <95 C Storage stable min.

10 Bacillus sp. Definition of Bacillus Gram positive organisms Spore formers Bacillus is facultative aerobe but at present of nitrate/nitrite it can grow anaerobic Bacillus as probiotic is in form of Bacillus spores Bacillus spores are ph stable stable at ph 2-3 Trypsin stable Survive stomach transit Thermo-tolerant, survives pelletization <95 C Storage stable min. 24 months when kept dry below 25 C In Feed stable >4 month stability in standard feed formulations Bacillus spores germinate in the intestine, attach to mucus and multiply Bacillus is found in soil and marine sediments

Yeast Yeast are unicellular fungi Dominate fungal diversity in the oceans

β-glucans β-glucans is used as immuno-stimulants Yeast is used in the

Yeast are used in ruminant feeding to reduce oxygen level in the rumen

11 Yeast Yeast are unicellular fungi Dominate fungal diversity in the oceans Large cells times larger than bacteria Cell wall structure comprise β-glucans β-glucans is used as immuno-stimulants Yeast is used in the backing industry and for producing alcoholic beverages. Yeast are used in ruminant feeding to reduce oxygen level in the rumen Natural occurrence in healthy fish Debarryomyces hansenii is a salt loving yeast as Sodium is not toxic

12 Improving the nutritional utilization Improving the nutritional utilization Increased weight Faster growth Lower feed conversion Better protein and fat efficiency ration These effects of using probiotic is well documented within poultry, pigs, ruminant, Few studies in aquaculture

13 Evaluation of Bacillus subtilish as a probiotic to Indian major Carp Labeo rohita (Kunar et al 2006) Trial design: 12 cm long 15 ± 2g L. rohita 100 liter tank with 70 l chlorine free tap water, 27 ± 1 C, Hardness 82mg/l, ph 7.4, dissolved oxygen 6.8 mg/l 2 weeks acclimatization followed with 15 days of treatment. 2 tanks pr. treatment Treatment: T1= control, T2= 5E8 CFU, T3= 1E9 CFU, T4= 5E9 CFU of Bacillus subtilish Results: Treatment T1 Control T2 5E8 CFU T3 1E9 CFU T4 5E9 CFU WG % 30.8 a ± b ± b ± c ±0.8 Superscripts are significant different (P< 0.005

14 Improving the nutritional utilization Other studies reported improved performance of using probiotics: Bacillus sp. improved length and weight of rainbow trout (Raida et al 2003) Growth and feed efficiency improved using the yeast S. cerevisiae ii in Israeli carp (Gatesoupe 2007) The yeast Debaryomyces hansenii improved growth of larval from European sea bass (Gatesoupe 2007) Enterococcus faecium improve growth and feed efficiency of Israeli carp (Ringø 2004)

15 Mode of action - Improving the nutritional utilization Metabolites Bacillus is know to produce proteases, lipases and several carbohydrolases improving nutrient absorption (Ochoa-Solona and Olmos-Soto 2006) Gastrointestinal bacteria in hydrobionts take part in the decomposition of nutrients providing enzymes, amino acids and vitamins (Wang et al 2008) Faster maturation of the intestine Higher brush border membrane enzymes activity after treatment with the yeast S. boulardii to rainbow trout fry suggesting an earlier maturation of the digestive system (Waché et al 2006) Reduce stress Lowering cortisol level Cortison is a hormon directly involved in the stress responses influencing metabolic and osmoregulatory pathways. Lb. delbrueckii showed such effect in of sea bass larvae (Silvi et al 2008) Ensure healthy microbiota in the intestine Significantly higher bacteria counts feeding Bacillus subtilish and Bacillus licheniformis to Nile tilapia fry (Shelby et al 2006) Lb. delbrueckii showed good capability to colonize the gut of sea bass larvae, therefore modifying the gut microbiota and exerting threefold higher effects on the survival of the treated sea bass. (Silvi et al 2008)

16 Disease control Diseases caused by Vibrio spp, Yersinia spp. and Aeromonas spp. are commonly implicated in episodes of mortality in aquaculture Faced with disease problems the common response has been to turn to antimicrobial drugs Vaccination has been a success reducing the use of antibiotic in salmon production (95% reduction), but it is not always possible to use vaccine as a solution eg. shrimps have a non-specific immune response and vaccination can only provide short-term protection against pathogens (Hong et al 2005) Inhibition of pathogens via use of probiotics is an alternative solution. Most probiotic research in aquaculture deals with disease controls

17 Evaluation of Bacillus subtilis as a probiotic to Indian major Carp Labeo rohita (Kunar et al 2006) Trial design: 12 cm long 15 ± 2g L. rohita 100 liter tank with 70 l chlorine free tap water, 27 ± 1 C, Hardness 82mg/l, ph 7.4, dissolved oxygen 6.8 mg/l 2 weeks acclimatization followed with 15 days of treatment + challenge with Aeromonas hydrophila. 2 tanks pr. treatment Treatment: T1= control challenge, T2= 5E8 CFU, T3= 1E9 CFU, T4= 5E9 of Bacillus subtilis, CFU, T5= no challenge no treatment Results: Treatment T1 T2 T3 T4 T5 Survival % d ± c ± bc ± ab ± a ±0.00 Superscripts are significant different (P< 0.005)

18 Disease control in shrimp aquaculture with probiotic bacteria (Moriarty 1999) Vibrio harveyi have been implicated as the main bacterial pathogen of shrimps Vibrio sp. got resistant to every antibiotic used in the Philippines Chlorine has been used in hatchery and ponds but it developed multiple resistant bacteria When probiotic Bacillus was used no disease was experienced and indeed survival was very high (80-100%) Pond # 1 Probiotic 2 Probiotic 3 Probiotic 4 Control 5 Control Stocking N/m 2 Days of culture Survival % Control Harvest (tonne/ha)

19 Mode of action disease control Immune stimulation Li et al (2007) improved immune response in white shrimp (Litopenaeus vannamei) 3 types of haemocytes were significantly higher after treatment with Bacillus licheniformis Toxin degradation Bacillus Licheniformis has shown capability to reduce the effect of Clostridia perfringens toxin in chicken (Knap et al 2008) In a shrimp challenge model the mortality of Vibrio harveyi infection was significantly reduced by the presence of probiotic Bacillus in the tank water (Hong et al 2005) Pathogen exclusion One of the first theories for explaining effect of Probiotic and it is well documented in land animal species. Some bacteria have antiviral effect. The exact mechanism is not known. Laboratory studies indicate that extracellular agents from the bacteria inactivate the viruses (Wang et al 2008) Bacterocin production The intestinal micro flora of hydrobionts play an important role in the resistance to infectious diseases by producing antibacterial metabolites (Wang et al 2008) Bacillus is know to produce a variety of bacterocins:

20 Urdaci & Pinchuk (2005) Overview bacterocins in Bacillus

21 Bioremediation Aquaculture generates considerable amount of wastes: Metabolic by-products Residual feed Faecal matter Residues from prophylactic p & therapeutic treatments High concentration of these component generates stress and some could be toxic Bi di ti i t i t lit b f Bioremediation is a way to improve water quality by use of bacteria and enzymes

22 Use of bioremediation Resulting in lower accumulation of slime or organic matters in the bottom of the pond Better penetration of oxygen Bio-control resulting in low pathogen pressure Bacillus spp, Lactobacillus and Phenibacillus together with nitrifying bacteria have been reported to be key in this process Bacillus spp is most often reported Fast multiplication Producing high levels of extracellular enzyme complexes Pathogen exclusion Reduce total ammonium nitrogen

23 New Probiotic Many potential strains easy to isolate from intestine Focus should be Careful identification Preferably GRAS or with QPS status Safety non pathetic Free of transferable antibiotic resistance Scalable and economic production process Storage stable Easy to apply and use for end-user Convincing efficacy documentation Consistent performance

24 Use of probiotic in the aquaculture industry is expanding for the benefit of the animal health and the economy in the aquaculture production.

25 Thank you for your attention

lbs farmed fish per capita consumption lbs farmed fish per capita consumption

Delbert M. Gatlin III Department of Wildlife and Fisheries Sciences and Intercollegiate Faculty of Nutrition Texas A&M University System 1970 1.5 lbs farmed fish per capita consumption 2006 17 lbs farmed