Efficacy of Single and Multi-Strain Probiotics on In Vitro Strain Compatibility, Pathogen Inhibition, Biofilm Formation Capability, and Stress Tolerance

SIMPLE SUMMARY: Probiotics are known to be a supplementary strategy which are one of the alternatives to prophylactic treatments such as chemical additives or antibiotics due to a higher demand for environmentally friendly tools to address disease outbreaks in aquaculture. The usage of multi-strain probiotic (MSP) has more benefits and increased potential on a host as opposed to the usage of a single-strain probiotic. The combination of few probiotic strains will help to maximize the benefit spectrum. In vitro screening steps to select a suitable probiotic strain as a candidate for an MSP is highly essential. The aim of this study is to screen and select probiotic strains that were isolated from various aquatic host to produce MSPs. From this study, two potential MSPs were selected based on criteria including the ability of the MSPs to inhibit common aquatic pathogens (Streptococcus agalactiae, Aeromonas hydrophila, Vibrio harveyi, Vibrio parahaemolyticus). These MSPs also showed beneficial characteristics that showed its potential to be applied on an aquatic host. ABSTRACT: Compatibility of each strain in a multi-strain probiotic (MSP), along with its properties, becomes a strong base for its formulation. In this study, single-strain probiotics (SSPs) and multi-strain probiotics (MSPs) were evaluated in vitro for strain compatibility, microbial antagonism, biofilm formation capacity, and stress tolerance. Bacillus amyloliquefaciens L11, Enterococcus hirae LAB3, and Lysinibacillus fusiformis SPS11 were chosen as MSP1 candidates because they showed much stronger antagonism to Aeromonas hydrophila and Streptococcus agalactiae than a single probiotic. MSP 2 candidates were Lysinibacillus fusiformis strains SPS11, A1, and Lysinibacillus sphaericus strain NAS32 because the inhibition zone produced by MSP 2 against Vibrio harveyi and Vibrio parahaemolyticus was much higher than that produced by its constituent SSPs. MSP1 in the co-culture assay reduced (p < 0.05) A. hydrophila count from 9.89 ± 0.1 CFU mL(−1) to 2.14 ± 0.2 CFU mL(−1). The biofilm formation of both MSPs were significantly higher (p < 0.05) than its constituent SSPs and the pathogens. The SSPs in both MSPs generally showed resistance to high temperatures (80, 90, and 100 °C) and a wide range of pH (2 to 9). This in vitro assessment study demonstrates that MSP1 and 2 have the potential to be further explored as multi-strain probiotics on selected aquatic species.