Mixed culture of probiotics on a solid-state medium: An efficient method to produce an affordable probiotic feed additive

The overuse and abuse of antibiotics in animal husbandry is an ongoing problem. While probiotics could be an alternative, their effectiveness, stability and production cost are key factors that need to be addressed first. This study used a mixed culture of Bifidobacterium bifidum, Clostridium butyricum, Bacillus subtilis and Bacillus licheniformis on a simple and inexpensive solid-state medium generated by pouring a liquid MRS medium, which was designed to favor the dense growth of lactobacilli, onto wheat bran at a ratio of 1:2 by weight. Using this method, we achieved a very high number of live bacteria (NLB), at 3.93 × 10 CFU/g, without the need for expensive anaerobic equipment. The mixed culture thus achieved striking results that were up to 10,000 times better than the pure culture and did not require special anaerobic equipment. A real-time PCR analysis demonstrated that the shares of the four strains in the mixed culture probiotics (MCPs) were 2, 17, 46, and 35%, respectively, which indicates that the fermentation product contained an uneven distribution of the four probiotic target bacteria. The MCPs possessed good storage stability at room temperature, and the NLB was greater than 10 CFU/g after 30 days at 25°C, which made it easier to transport and store. They also demonstrated good stability in artificial digestion fluids, with an NLB of over 10 CFU/g after sequential treatments, which enabled them to maintain effectiveness in the animal gastrointestinal tract. Finally, the MCPs were fed to the mealworms (Tenebrio molitor) and raccoon dogs (Nyctereutes procyonoides) in order to test their effectiveness, and the growth rates of both significantly improved with the MCPs feeding. This study thus demonstrated that culturing MCPs on a solid-state medium is an efficient method to affordably produce probiotic feed additives that can improve the performance of very physiologically different animals, such as the mealworm and raccoon dog, which indicates their potential for very wide applicability.