Functional properties of mixed probiotics fermentation broth and its feeding attractant effects

With the rapid development of aquaculture farming industries, high density farming techniques are now widely adopted in China, facilitated by a raft of approaches including oxygenation enhancement, intensive feeding and the frequent addition of antibiotics for disease control. Such intensive industrialized farming methods have improved yields from aquatic farming of animals. As repeatedly noted, they may also lead to the buildup of unused feed, excreta and other biological residues, as well as the accumulation of harmful chemicals such as nitrite, and the application of chemical drugs and antibiotics has led to the proliferation of harmful and resistant bacteria and harmful algae, meanwhile, drug residues also affect the products quality and aquatic animals safety. Probiotics, such as Bacillus, lactic acid bacteria and yeast have been widely used in aquaculture and yielded beneficial effects, mainly in improving water quality, growth performance and reducing the mortality rate by degradation nitrite, inhibiting pathogens, regulating intestinal flora and stimulating the immune system. It is well known that different types of probiotics can express different functions, such as denitrification, enzyme production and antibacterial activity. Mixed strain fermentation, giving multiple functions of fermentation broth, which can reduce production cost, simplify the process and broaden the application range. In this study, we explored the feasibility of multi-functional mixed strain fermentation, 3 strains of probiotics included yeast, lactic acid bacteria and Bacillus with excellent performances (Saccharomyces cerevisiae NJ-02: producing exopolysaccharide, vitamin, nucleotide, etc; Enterococcus faecium SC-01: producing acetic acid, lactic acid and other organic acids, inhibition of pathogens; Bacillus subtilis M7-1: degradation of ammonia nitrogen and nitrite nitrogen, producing extracellular enzymes). After 24 h incubations, the number of Saccharomyces cerevisiae NJ-02, Enterococcus faecium SC-01 and Bacillus subtilis M7-1 viable bacteria counts reached 3.88×108, 2.41×1010, and 5.38×109 CFU/mL, respectively. The results also showed that the biological denitrification and promoted algae (Chlorella vulgaris) growth performance of the mixed fermentation broth were directly related to the number of Bacillus subtilis M7-1 viable bacteria. Further into the process, it was observed that 16 h was the best fermented time for degradation nitrite and algae cultivation. It also showed that the nitrite nitrogen degradation rate reached 89% and the C. vulgaris chlorophyll-a concentration increased by 49.6%. Additionally, mixed strain fermentation broth showed the same enzymatic activity (protease, amylase, cellulase) and antibacterial activity (Vibrio parahaemolyticus) when it was compared to Bacillus subtilis M7-1 and Enterococcus faecium SC-01. Furthermore, the feeding attractant effect was significantly improved than that of the control group and chemical attractant trimethylamine oxide (TMAO) (P<0.05), and was equivalent to that of dimethyl-beta-propiothetin (DMPT) based on the feeding behavior of shrimp. The number of lactic acid bacteria and yeast in shrimp intestinal tract was significantly higher (P<0.05) than that of the control group and the chemical attractant group. In summary, we obtained multi-functional mixed strain fermentation broth such as biological denitrification, promoting algae cultivation, antibacterial activity and biological attractant, this fermentation broth was expected to be used in aquaculture to improve water quality, enhance animal appetite and feed intake and maintain intestinal health. The study provides a microbial resource and technical method for sustainable ecological aquaculture.