Education

Probiotics- a new frontier in aquaculture [Fish probiotics]

  • In Print edition September,2020
  • Dr. Partha P. Biswas

The aquaculture has gathered momentum as an industry for over past few decades. The basic paradigm is increased production at almost any cost. This boom in the industry has resulted in social, economic and ecological damage. Outbreaks of viral, bacterial, protozoan and fungal infections experienced devastating economic losses worldwide in nineties of last century. It was mainly due to poor environmental conditions of farms, unbalanced nutrition, generation of toxins, and genetic factors.The need for increased disease resistance, growth of fin & shell fishes, and feed efficiency has brought about the use of probiotics in aquaculture practices.Probiotics have opened a new era in aquaculture. Probiotics reduce the use of chemical additives and medicines especially antibiotics. The overuse of antibiotics can create antibiotic-resistant bacteria. There has been heightened research in developing new dietary supplementation strategies in which various health and growth promoting compounds as probiotics, prebiotics, synbiotics, phytobiotics and other functional dietary supplements have been evaluated.

A prebiotic is a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or the activity of one or a limited number of bacteria in the colon. A number of prebiotics commonly used in aquaculture are fructooligosaccharides (FOS), galactooligosaccharides (GOS), inulin, arabinoxylo-oligosaccharides (AXOS), short-chain fructooligosaccharides (scFOS), and mannan-oligosaccharides (MOS). Synbiotics are nutritional supplements that combine probiotics and prebiotics, enhancing their beneficial effects. Dietary incorporation of MOS in a single or a combined inclusion with Enterococcus faecium ( a probiotic strain )enhances the rainbow trout growth and activates its immune system. Phytobiotics are plant-derived, natural compounds embedded into diets which enhanced animal productivity. Leaves of Peppermint, Mentha piperita is used as appetite, digestion stimulant, antiseptic for fish

Probiotic is a relatively new term which is used to name microorganisms that are associated with the beneficial effects for the host. Generally, probiotics are live and/or dead microbial feed supplements or water additives in the form of mono, multiple strains or in combination with prebiotics or other immunostimulants, which are administered to improve the rearing water quality, to enhance the physiological and immune responses of aquatic animals and to reduce the use of chemicals and antibiotics in aquaculture. While a wide range of micro‐organisms is employed in aquaculture, in which both Gram‐positive (Bacillus, Enterococcus, Lactobacillus, Lactococcus, Microbacterium, Micrococcus, Streptococcus, and Streptomyces) and Gram‐negative bacteria (Aeromonas, Enterobacter, Pseudoalteromonas, Pseudomonas, Rhodopseudomonas, and Vibrio ) are applied effectively. Gram-negative bacteria commonly known to be pathogenic to fish.But some Gram-negative bacteria such as Pseudomonas I-2 have been reported to inhibit V. hervey and V. fluvialis in shrimp culture. Other nonbacteria candidates such as microalgae (Dunaliella spp., Isochrysis galbana , Phaedactylum tricornutum and Tetraselmis suecica) and yeasts (Saccharomyces cerevisiae ) are explored commonly as probiotics for use in aquaculture

PROBIOTICS TO BE USED IN CORRECT DOSE

Overdosage administrations of probiotics can induce immune‐suppression. A dietary supplement with Lactococcus lactis at 108 CFU g−1 improves the growth rate. The application of B. subtilis and B. licheniformis in diets at 109 CFU g−1 improved FCR. Appropriate probiotic levels depend on the probiont species, fish species and their physiological status, rearing conditions and the specific goal of the applications. Inoculum concentration was converted to colony forming units (CFU) per mL from a standard curve previously devised for each strain. Colony-forming unit (CFU) is a unit used in microbiology to estimate the number of viable bacteria or fungal cells in a sample. Some of the automated systems such as the systems from MATLAB allow the cells to be counted without having to stain them.

CHARACTERISTICS OF GOOD PROBIOTICS

(i) It should be a strain, capable of exerting a beneficial effect on the host animal like increased growth or resistance to disease etc.
(ii) Non-pathogenic and non-toxic.
(iii) Present as viable cells preferable in large numbers.
(iv) Capable of surviving in the gut environment e.g. resistance to low pH & organic acid etc.
(v) Stable & capable of remaining viable for periods under storage and field conditions.
This is to be remembered that results of using probiotics can be affected by improper management methods and product quality.
• Incorrect application methods.
• Incorrect claims of probiotics, which can’t be fulfilled (e. g. for white spot syndrome).
• Too low bacterial concentrations (colony forming units should be above 108/g or CFU/ml ).
• Poor bacterial stability during production and storage (shelf life).
High levels of viable organisms and stability during production and storage are important criteria for the selection of suitable strains. The safety of strains are carefully assessed, as well, and care is taken that transmission of antibiotic resistance or virulent plasmids must not take place. Of further great importance are the survival and growth of beneficial bacteria in culture conditions, and their ability to colonize the gut of the aquatic animals. So considering these factors one must not compromise with low price probiotics & products of unfamiliar companies. Farmers should stick only to products of those companies who have good R & D wing.

MODES OF ACTION OF PROBIOTICS

1) Production of inhibitory compounds/substances.
2) Competition 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) It is required at optimum dose as overdosage administrations of probiotics can induce immune‐suppression(application of B. subtilis and B. licheniformis in diets at 109 CFU g−1 improved FCR, specific growth rate, weight gain and protein efficiency ratio ).
14) Prolonged administrations of probiotics can induce immune‐suppression.

SOURCE OF PROBIOTICS

In the last three decades, several probiotic microorganisms have been identified, characterized and applied in aquaculture.These beneficial microorganisms can be of host or non-host origin. It is highlighted that host-associated microorganisms offer a great prospect as a source of probiotics with diverse biochemical features.Bacteria obtained from intestine of aquatic as well as terrestrial animals are commonly used as probiotics in aquaculture. Several bacterial species such as Vibrio and Pseudomonas spp. Are isolated from marine fishes are being proposed as probiotics. Probiotics from terrestrial environment have been documented conferring numerous benefits to the cultured animals.

SINGLE AND COMBINATION

Probiotics can be applied singly or in combination Most studies on probiotics have focused on the use of single cultures, and it is largely speculative whether two or even multiple combinations of probiotic strains would be beneficial. Probiotics based on a single strain are less effective than those based on mixed strains.Multistrain and multispecies probiotics enhanced protection against pathogenic infection. A mixture of B. subtilis and Lactobacillus acidophilus enhance bacteriocidal activity. Positive effects of multistrain probiotics on the survival and growth of rohu (Labeo rohita ) was seen at hatchling and fry stages, but not at later stages.

APPLICATION METHODS

Probiotics administration varies from direct oral/water routine or feed additives, in which the former is considered the most practical method for prawn probiotics.The administration via feeding (dry feed) definitely has limitations during early larval stages due to immature digestive tracts of fish in that stage of development Commonly, probiotics can be added directly into culture water. Bacillus subtilis has been widely administered as an appropriate probiotic agent to control the water quality.The mode of action of bacterial inocula is claimed to be the enhancement of natural processes such as organic matter degradation, nitrification, ammonia removal, denitrification, sulfide oxidation, and degradation of toxic pollutants.Pond waters are usually treated with the inoculum at 5mg l-1,initially, 2.5 mg l-1 after week 1, and 0.5 mg l-1 for the next two weeks.Extracellular enzymes are only the first step in the degradation process.

DIFFERENT STRAINS OF PROBIOTIC USED IN AQUACULTURE

Probiotics that currently used in aquaculture industry include a wide range of taxa – from Lactobacillus, Bifidobacterium, Pediococcus, Streptococcus and Carnobacterium spp. to Bacillus, Flavobacterium, Cytophaga, Pseudomonas, Alteromonas, Aeromonas, Enterococcus, Nitrosomonas, Nitrobacter, and Vibrio spp., yeast (Saccharomyces, Debaryomyces) and etc. Most common probiotics belong to Lactic acid bacteria, genus Vibrio, Bacillus, Pseudomonas and Roseobacter.Vibrio alginolyncus when introduced in larval rearing tanks caused a reduction in the incidence and severity of luminous vibriosis caused by Vibrio harveyi and improvement in growth of shrimp larvae. The population level of lactic acid bacteria associated with the gastro intestinal tract of the fish is affected by physiological, nutritional and environmental factors related to age, food habits, season, salinity and stress. Though they are rarely present in juvenile fish reared on artificial feed but may dominate in the intestinal flora if they are supplemented in the feed. To mention but few, strains of probiotic use in aquaculture are: Lactococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus delbrueckii.The advantage of using Bacillus strains as a probiotic in feed is their ability to survive the pelletization process.

TWO FORMS OF AQUACULTURE PROBIOTICS

Aquatic probiotics are marketed in two forms: 1) Dry forms: the dry probiotics that come in packets can be given with feed or applied to water. They have many benefits, such as safety, easy using, longer shelf life and etc. 2) Liquid forms: the hatcheries generally use liquid forms which are live and ready to act. These liquid forms are directly added to hatchery tanks or blended with farm feed. The liquid forms can be applied any time of the day in indoor hatchery tanks, while it should be applied either in the morning or in the evening in outdoor tanks. Liquid forms give positive results in lesser time when compared to the dry and spore form bacteria, though they are lower in density. There are no reports of any harmful effect for probiotics but it is found that the biological oxygen demand level may temporarily be increased on its application; therefore it is advisable to provide subsurface aeration to expedite the establishment of probiotics organisms. A minimum dissolved oxygen level of 3% is recommended during probiotics treatment. The development of suitable probiotics for aquaculture is not a simple task. It requires empirical and fundamental research, full-scale trials as well as the development of appropriate monitoring tools and production under stringent quality control. A performing mixture of probiotic strains can be designed after evaluating the ability of individual strains to grow in low/high salinity under micro-aerophilic or anaerobic conditions, produce various enzymes, and more importantly, produce a range of inhibitory compounds.

BENEFIT OF USING PROBIOTICS IN AQUACULTURE
WATER PROBIOTICS FOR IMPROVEMENT OF WATER QUALITY

Aquaculture probiotics have a very important role to play in the degradation of organic matter thereby significantly reducing the sludge and slime formation. As a result, water quality would improve by reducing the disease (including Vibrio sp., Aeromonas sp. and viruses) incidences, enhancing zooplankton numbers, reducing odours and ultimately enhancing aquacultural production. By speeding up the rate of organic matter breakdown, free amino acids and glucose are also released providing food sources for the beneficial microorganisms. Inorganic forms of nitrogen, such as ammonia, nitrate and nitrite are also reduced. Gram-positive bacteria, especially Bacillus spp., have been able to convert organic substances to carbon dioxide more efficiently. The probiotic bacterium Bacillus licheniformis significantly decreases ammonia levels. A mixture of B. licheniformis, B. subtilis and Bacillus pumilus has been found to increase growth performance and immune defense in larvae.

WATER PROBIOTICS IN BIOFLOC TANK/POND

In conventional fish and crustacean culture systems, the nitrogen compounds transformation is made by autotrophic microorganisms of Nitrospira, Nitrobacter and Nitrosomonas genera. However, an excess of organic matter in form of carbon, inhibit their efficiency by limiting their growth. Contrary, in Biofloc systems, nitrogen compounds transformation is more efficient, because this process is made by facultative heterotrophic bacteria that correspond principally to Bacillus and Pseudomonas genera. These two genders are more efficiently in organic matter presence, which allow increasing their population abundances quickly and oxide-reduction process. Bacillus subtilis and yeast Rhodotorulla sp can be identified in culture medium and showed probiotic characteristic. It is from weeks 4 to 5 it was observed the increasing of heterotrophic bacterial diversity represented by: Sphingomonas paucimobilis, Pseudomonas luteola, Pseudomonas mendocina, Bacillus sp., Micrococcus sp. and Rhodotorula sp. yeast. Authors remark that there could be variations between develop microbial groups with respect culture specie, food and specially carbohydrate source applied. Bacillus amyloliquefaciens probiotic is added to improve the immune system.

GUT PROBIOTICS FOR IMPROVEMENT IN DIGESTION

Bacillus cereus strain E when given with feed causes increased fish growth due to more efficient use of the food. Similar result of using B. subtilis, B. licheniformis, and Enterococcus faecium shows that these probiotics if provided for 10 weeks along with the diet of fish a good growth can be recorded. Probiotic yeast (Saccharomyces cerevisiae strain X2180) causes fish growth and activity and expression of antioxidative key enzymes (catalase, glutathione peroxidase, and superoxide dismutase).In white shrimp Bacillus secretes a wide range of exoenzymes that complement the activities of the fish and increases enzymatic digestion.

PROBIOTICS TO INCREASE STRESS TOLERANCE IN FISH

Aquaculture practices demand intensive productions in shorter times, causing stress in crop species. One of the firsts formal reports on this field studied the supplementation of Lactobacillus delbrueckii ssp. in the diet of European sea bass at time intervals of 25 to 59 days.Hormone cortisol was quantified in fish tissue as stress marker, since it is directly involved in the animal’s response to stress. Cortisol levels obtained in the treated fishes were significantly lower than those in the control.Commercial probiotics containing Bacillus subtilis, Lactobacillus acidophilus, Clostridium butyricum, and Saccharomyces cerevisiae showed greater tolerance in the stress test than the control group.

Probiotics on reproductive performance

Probiotic supplementation of B. subtilis isolated from intestine of Cirrhinus mrigala when given to other species of fishes it resulted increase in the gonadosomatic index, fecundity, viability, and production of fry from the female fish.

PROBIOTICS USED FOR INHIBITION OF PATHOGENS

Probiotic microorganisms have the ability to release chemical substances with bactericidal or bacteriostatic effect on pathogenic bacteria that are in the intestine of the host, thus constituting a barrier against the proliferation of opportunistic pathogens. In general, the antibacterial effect is due to one or more of the following factors: production of antibiotics, bacteriocins, siderophores, enzymes (lysozymes, proteases) and/or hydrogen peroxide, as well as alteration of the intestinal pH due to the generation of organic acids. Carnobacterium sp.is used against many fish pathogens.In the case of shrimp, studies have focused on the evaluation of probiotics such as Bacillus cereus, Paenibacillus polymyxa, and Pseudomonas sp.as biocontrol agents against pathogens of various Vibrio species.

PROBIOTICS USED AS GROWTH PROMOTER

Probiotics have been used in aquaculture to increase the growth of cultivated species, in reality it is not known whether these products increase the appetite, or if, by their nature, improve digestibility.The use of probiotics as growth promoters of edible fishes has been reported. Diet of Nile tilapia (Oreochromis niloticus) was amended with a probiotic Streptococcus strain, increasing significantly the content of crude protein and crude lipid in the fish, also increased in weight. When feed was supplemented with Bacillus subtilis and Streptomyces, finding also suggest increase in growth and survivality.

PROBIOTICS IN LARVAL CULTURE

Several bacteria have been used in the larval culture of aquatic organisms either delivered directly into the water freeze-dried, or via live carriers such as Artemia nauplii or rotifers. Vibrio alginolyticus is a frequently tested bacterium with promising results which has characteristics of conferring some degree of protection against disease. But this Vibrio may require some caution since some strains could be pathogenic. It could be a probiotic candidate for shrimp larviculture. Improvement in water quality of tank of rearing larvae has been recorded during the addition of probiotics, especially Bacillus spp. The rationale is that gram-positive Bacillus spp. are generally more efficient in converting organic matters back to CO2.

PROBIOTICS IN IMMUNE RESPONSE ENHANCEMENT

Lactococcus lactis was found to cause increases in immune functions. Resistance of shrimp, Litopenaeus vannamei against Vibrio harveyi and white spot syndrome can be enhanced by administration of of Bacillus . B. subtilis, Tetraselmis chuii( a marine alga), and P. tricornutum, as feed supply singly or in combination, exhibited up-regulating effects on immune parameters in fishes.

CONCLUSION

The application of probiotics in aquaculture shows promise, but needs further comprehensive research. Our country needs to strengthen its research and development efforts, quality control, and introduction of HACCP and ISO standards regarding production of probiotics. Microbial management in both the hatchery system and grow-out system are becoming an integral part of aquaculture practices. Therefore, greater effort in microbial intervention studies is imperative. Future studies are required to exclude the possibility that when probiotic product are accumulated in the animal it will not form a consumer risk. Safety of the workers formulating the product should also be addressed. Impact of the microbial additive on the environment is another issue of future investigation.

About Author:

Dr. Partha P. Biswas
Incharge-Fisheries Training & Culture Unit
Simurali Krishi Kendra
Simurali, Dist.-Nadia, West Bengal
Email :parthapbis2006@yahoo.co.in

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