Probiotics for cultured freshwater fish

Tilapia dominates the aquaculture industry in many tropical and subtropical countries and is one of the most important protein sources from freshwater fish¹. Traditionally, antibiotics and chemicals have been used to treat infectious diseases in fish. As an alternative to the use of antibiotics, probiotics originated from the native gastrointestinal microbiota of fish have been increasingly common within the past two decades². Probiotic organisms include bacteria, yeast, and filamentous fungi often originate from the GIT of fish, and can be applied individually or in mixtures or consortia.

In 2017, more than 150 million tons of fish were produced worldwide, with China being the largest producer country with 4 million tons of total product³. By 2030, it is expected that close to 62% of consumed fish will come from aquaculture and 38% from wild-caught fish⁴. However, one of the main difficulties in the commercial cultivation of aquatic organisms is the appearance of infectious diseases that hamper industry sustainability. Several researchers and producers point to disease as the leading cause of losses in production and economic resources⁵.

Probiotic bacteria give multiple benefits to fish, such as growth promotion, inhibition of pathogen colonisation, and improvement of nutrient digestion, water quality, and stress tolerance, as well as enhancement of reproduction⁶. Yeast is the second group of microorganisms with probiotic potential. Yeast as probiotic supplements in the fish diet offer benefits that include modulation of the digestive microbiota, enhancement of immune responses, contributions to intestinal enzymatic physiology, and enhanced growth performance⁷. The third group of microorganisms with probiotic potential are the filamentous fungi. These fungi stimulate antioxidant response and the immune system, and additionally stimulate the production of various digestive enzymes including amylases, cellulases, β-glucanases, xylanases, proteases, and lipases⁸. Generally, these probiotic microbes are non-fish derived. In contrast, a multi-strain probiotic culture, maintained in continuous culture, was recently developed from Nile tilapia (Oreochromis niloticus) gastrointestinal microbiota. A Cetobacterium sp. was the dominant genus, and the multi-strain culture had in vitro antibacterial activity against fish pathogens such as Streptococcus agalactiae and Aeromonas hydrophila⁹. Results identified three bacteria within the continuous culture with distinct antibacterial activity against these pathogens.

It is essential to define the probiotic dosage for a specific fish and environment in order to avoid economic losses due to overdose or too low a dosage¹⁰. Most of the journal articles sourced suggested the use of probiotic concentrations of approximately 1 × 10⁶ CFU/g of feed showed significant improvement in growth performance, resistance to infection, and immune modulation. Growth performance and health status improved in trout¹¹, and tilapia¹², with a probiotic microbial concentration around 1 × 10⁶ CFU/g of feed. According to Merrifield et al.¹³ that concentration of probiotics is necessary to ensure the colonisation of the probiotic in the intestinal tract of fish.

A very high concentration of probiotics, exceeding what is optimal, could result in wasted energy and nutrients, and, for that reason, it is necessary to test the functional dosage of probiotics in every particular situation¹⁴. Farias et al.¹⁵ showed that a higher dosage than needed could partially suppress probiotic responses. The lower concentration tested (1 × 10⁷ CFU/g) was enough to offer an improvement in growth performance and nutrient utilisation in Oncorhynchus mykiss¹¹ and Salmo salar¹⁰. Bhujel et al.¹⁶ used regression analysis to define the optimal concentration of the two commercial probiotic formulations in Labeo rohita, showing that the effective dosages were higher concentrations than that suggested by the manufacturers.

Another critical factor for probiotic efficacy is the administration period. Researchers generally administrated multi-strain probiotics over periods of approximately 30 days (28–30 days), 45 days (42–49 days) and 60 (56–60 days). Addo et al.¹⁷ found that growth performance was low at 21 days of treatment with a mixture of two Bacillus strains (SB3086 and SB3615). Similarly, Bacillus subtilis, Saccharomyces cerevisiae and Aspergillus oryzae did not show improvement in growth performance over 28 days of treatment¹⁸. However, the administration of Micrococcus sp. and Bacillus sp. enhanced growth performance of Etropus suratensis at day 28 in comparison to 14 days of administration. Other authors considered that more than 45 days are needed to confirm the probiotic potential offered by a mixture of probiotics. Giri et al.¹⁹ showed that after 60 days, growth performance and immune modulation improved. Similarly, growth parameters improved with the administration of Enterococcus faecium and Geotrichum candidum to L. rohita²⁰ for 45 days and 90 days. The administration of a probiotic in Piaractus mesopotamicus showed an increase in survival and biomass production benefits offered by microorganisms administered over a more extended period²¹. Merrifield et al.¹³ demonstrated that a continuous administration enhanced colonisation and probiotic activity in the rainbow trout (O. mykiss) gastrointestinal tract.

Generally, it is recommended that probiotics be applied in the earlier growth stages. The administration of two types of probiotic mixtures in L. rohita at different stages showed that growth performance and survival improved in hatchlings and fry, but administration only in the advanced fry stage did not affect survival and growth¹⁶. Likewise, Jha et al.²² demonstrated that early administration improved survival and growth of L. rohita hatchlings and fry. In contrast, probiotic administration to advanced fry did not cause any effect. Similarly, Ridha et al.²³ showed that application of two types of probiotic mixtures improved growth parameters at the juvenile stage, more than when administered at the adult stage. However, the recommendation is to evaluate probiotics applications from earlier stages to market size with continuous administration and treatments being withdrawn at different periods¹⁶.

The method of administration has been shown to affect probiotic performance²⁴. The method most commonly used for administering probiotic mixtures is incorporation into the feed (92.8%), followed by direct incorporation into the water (4.8%) and in live food (1.6%). The process for incorporating the probiotic into feed has different stages: mixing the probiotic with feed, adding water, pelletising to the selected size, drying, packaging, labeling, and storing until feeding.

There are a few journal articles in which the authors measured the viability of the bacteria incorporated into the feed. Aly et al.²⁵ centrifuged and washed the probiotics with a buffer, and added a concentration of 1 × 10⁹ CFU/g to feed. The feed was blended in an automatic mixer and pelletised. The pellets were dried in an oven at 45°C and the probiotic viability was measured weekly over five weeks of storage at 4°C and 25°C. Results showed that B. pumilus survived at both temperatures over the five weeks. Meanwhile, Citrobacter freundii and B. firmus survived at 4°C during the five weeks, but at 25°C, they were viable for one or two weeks, respectively. In another study, probiotics were centrifuged, and bacterial pellets were resuspended in nutrient broth²⁶. The probiotic suspension was sprayed at ~2 × 10⁹ CFU/g on feed in plastic trays and air-dried in a microbial cabinet at room temperature (19°C). Viability was measured after three months of storage at 4°C, showing that Exiguobacterium JHEb1, Vibrio JH1, and Enterococcus JHLDc were viable at concentrations of more than 1 × 10⁷ CFU/g of feed over the three months evaluated. Finally, Bacillus amyloliquefaciens 54A and B. pumilus 47B at three different concentrations (1 × 10⁹, 3 × 10⁹ and 5 × 10⁹ CFU/g of feed) were mixed with feed and viability at 4°C was evaluated every week showing that the probiotic level decreased 10% after every three weeks of storage²⁷. From the processing perspective to incorporate the fish probiotics, Bacillus spp. would be more suitable to be incorporated in the fish feed or pellets, since they are spore-forming bacteria, which allow heat resistance during the pellet compression process²⁸. Floating fish feed is more desirable for fish. Therefore, probiotics that survive the preparation process for dry pellet would be more effective as fish feed.

Selection of probiotic strains should consider the potential unexpected transmission of the strains to human, or their genetic characteristics to other bacteria. Therefore, strains identified as probiotics for fish might not necessarily be suitable for commercial probiotics. Strains such as E. faecium and C. freundii are considered as opportunistic pathogenic bacteria in humans^{29, 30}. Therefore, these strains are not recommended for future fish probiotics products.

In summary, probiotic microorganisms are a healthy, sustainable, and environmentally friendly approach in comparison with antibiotics to reduce the loss of aquaculture fish in disease outbreaks. Probiotics offer several benefits, including improved growth, immune modulation, and disease resistance. However, it is important to define the proper dosage, the administration period, fish stage at administration, administration method, and probiotic viability during production and storage in order to offer optimal probiotic efficacy in aquaculture fish. At the same time, it is important to evaluate its safety application on humans, other animals, and the environment.

The authors declare no conflicts of interest.