2. Selection of Probiotics The main objective of using probiotics is to re-establish a favourable association between beneficial and harmful microorganisms that make up the microbiota of the intestine of fish. A well-known probiotic should have few specific characteristics in order to exert a positive impacts.
2.1 Characteristics of Probiotic A high-quality probiotic should have the following mentioned characteristics: ? Should be a strain which is capable of exerting a beneficial effect on the host animal, e.g. increased growth or resistance to disease. ? Should not have any side effect; should neither be pathogenic nor toxic, not only with regard to the host species but also with regard to aquatic animals in general and human consumers. ? Should be viable under normal storage conditions and able to survive during industrial process. ? Should be capable of surviving and metabolizing in the gut environment, e.g. resistant to bile and low pH due to organic acids enrichment. ? Possess high ability to multiply in the intestine. ? Possess strong adhesive ability with the gut of the fish. ? Should have strong antagonistic activity against pathogenic microorganisms.
3. Modes of Application of Probiotics in Aquaculture Probiotics can be applied through feeding, injection or immersion or directly by adding in the water. 3.1 Application in Feed Usually, probiotics are used by adding directly in the feed ingredients or by spraying in the prepared feed. In aquaculture, commonly used probiotics are Lactobacillus sp., Bacillus sp. or Saccharomyces cerevisiae. According to the guidelines of Food and Agriculture Organization (FAO) and (World Health Organization (WHO), probiotics should have the capability of surviving while passing through the gut as well as resisting the gastric juices and bile. Additionally, probiotics should have ability to flourish and settle in the gut, which should be safe and effective for the host species.
3.2 Application through Immersion or Injection Probiotics also can be applied by injecting or immersion. A previous study also demonstrated the effect of administration of a well-known probiotic Micrococcus luteus by intra peritoneal injection to Oreochromis niloticus and observed only 25% mortality while 90% mortality with Pseudomonas.
3.3 Direct Application to Culture Water Probiotic application directly in ponds and tanks water also exhibited beneficial effect on fish health by modulating microbial composition and water qualities of water and sediments. Probiotic bacteria, particularly Bacillus spp. and some other species including Aerobacter sp., Nitrobacter sp. and Saccharomyces cerevisiae (yeast), played a remarkable task in the water quality improvement. As a previous study reported that the Gram-positive bacteria such as Bacillus spp. are able to change the organic matter into CO2 efficiently, on the other hand the Gram-negative bacteria are capable to alter a larger amount of organic substance into bacterial biomass or slim. Besides, some probiotic strains are able to provide algicidal activity against some microalgal species, specifically the red tide plankton. The application of nitrifying bacteria into the culture system also effectively removed the excess of ammonia and nitrite from the water which are considered as a foremost water quality problem.
4.1 Effect of Probiotics on Intestinal Ecosystem The gastro-intestinal (GI) tract of fish has been recognized as significant harbours of a multiple microbial community, particularly two discrete groups, such as autochthonous (indigenous) and allochthonous (exogenous). Autochthonous microorganisms have significant effects in the improvement of GI tract of fish, including development and maturation of the intestine and immune system and resistance to infectious pathogenic microbiota. These microorganisms are also able to obstruct the colonization of some other bacteria using several mechanisms such as competition for space and food, secreting antimicrobial compounds as well as being receptors at mucosal surfaces. In that way, probiotic species provide the most favourable positive impacts on the host body by modulating its intestinal microbial balance. Few studies have been carried out to assess the beneficial impacts of probiotics administration on the intestinal autochthonous microflora of fish. As previous studies only have emphasised on the cultivation based techniques in order to know about intestinal microbial population, which distinctly only permit the study of culturable bacteria, while a large number of non-culturable bacteria are lack behind, which may account a enormous effect in the intestine of fish.
4.2 Effect of Probiotics on Digestive Enzymes The digestive organs of an animal have an immense influenced on the composition of food and are accountable for immediate changes in the activities of the digestive enzymes, which is simultaneously related with fish health as well as growth. Even, the effective utilization of feed in fish also seriously influenced by the existence of various digestive fluids and enzymes. The digestion of carbohydrates, fat and protein are mainly dependable on various types of enzymes such as amylase, lipase and protease respectively. There is evidence of increasing feed utilization and growth of Nile tilapia after feeding probiotic diets by increasing the digestion of starch, fat and protein. Increased digestive enzyme activities including amylase, lipase and protease were also noted in Labeo rohita fed with a combination of three probiotics as Bacillus subtilis, Lactococcus lactis and Saccharomyces cerevisiae. Bacteria and their secreted enzymes have played a significant influence on the digestion process of fish by increasing their total digestive enzyme activities in the intestine. The enzymes that are secreted by the probiotic bacteria are recognized as exogenous enzymes. These exogenous enzymes can help to induce the production of endogenous enzymes that are able to endure a broad range of pH compared to those endogenous enzymes, which ultimately able to make delay the digestion phase, in that way they assured the higest utilization of digested nutrients. Nevertheless, it is complicated to differentiate between endo enzymes secreted by fish and exo enzymes produced by the probiotics. There is also evidenced that probionts secreted exoenzymes are capale of increasing the digestive utilization of feed. Probiotics not only have shown a positive effect on the digestive systems of fish, but also in the absorption of digested food components.