With the ban of nutritional ionophore antibiotic usage in EU countries, probiotics have become crucial in animal nutrition because they can enhance immune function, nutrient absorption, and growth. Widely used feed-additive strains include Lactobacillus, Bacillus subtilis, and Saccharomyces cerevisiae. Clostridium butyricum is highly adaptable to short-term high temperatures during feed pelleting and to the environment of the animal's digestive tract because of its protective structure, which consists of an outer spore wall, spore coat, cortex, and core [1]. It can also produce cellulase, amylase, butyric acid, lactic acid, and other beneficial metabolites and has been developed as a natural green probiotic strain that combines the advantages of probiotics such as Bacillus subtilis and Lactobacillus [2]. Butyric acid, which is produced by C. butyricum, as a specific nutrient and energy component of enterocytes, can enhance colonic barrier function [3], nutrient absorption, and growth [4]. Propionic acid can reduce pro-inflammatory factor expression, thereby inhibiting the intestinal inflammation cascade [5]. Acetic acid not only inhibits the invasion and colonization of pathogens but can also be used for milk fat synthesis in the mammary glands of dairy cows [6]. Cellulase, amylase, and other digestive enzymes produced by C. butyricum serve as key enzymes for the absorption of nutrients from feed in animals. They convert polysaccharides into monosaccharides or oligosaccharides, thereby enhancing the absorption of carbohydrates and promoting animal metabolism and growth [7]. C. butyricum is a potential microbial feed additive, but the enzyme activities of cellulase and amylase produced by C. butyricum are lower than those of industrial enzyme-producing microorganisms, which limits its probiotic efficacy. Recently, various biotechnological methods have been used to improve strain performance. Among them, heavy ion beam irradiation, a rapid and efficient mutagenesis method, has been applied to numerous biological mutagenesis breeding programs, especially in the field of microorganisms, and has achieved remarkable effects [8], [9].

In this study, for the first time, a coupled Logistic and Markov chain model was used to screen C. butyricum strains through mutagenesis using carbon ion beam irradiation, and a mutant strain, C. butyricum FZM 240, with optimal enzyme production was obtained. The probiotic properties of this strain were investigated using acid content, high temperature, and simulated gastrointestinal tolerance experiments. C. butyricum FZM 240 was subjected to whole genome sequencing, resequencing, qRT-PCR validation, and homology modeling of the mutant gene-encoded protein, which revealed the influence of carbon ion beam irradiation on its physiological and biochemical properties in vivo and in vitro and its autoregulatory healing in response to changes in the external environment. The potential of this strain as a high-quality probiotic was explored. These findings provide strong theoretical and technical guidance for the application of Clostridium spp. as feed additives.