Biological function of Clostridium butyricum and its application in aquaculture

（1. Henan Jin Bai He Biotechnology Co. , Ltd., Anyang 455000, Henan China）

Abstract: As one of the probiotics, Clostridium butyricum has a variety of physiological functions such as high heat resistance, acid and alkali resistance, and bile salt resistance. It has been widely used in food and medicine. Compared with other probiotics, the application of Clostridium butyricum as a feed additive in aquaculture is still in its infancy. This article introduces the physiological characteristics of Clostridium butyricum and its mechanism of action in terms of nutrient production, regulation of intestinal microecology, improvement of immunity, repair of intestinal barrier, anti-stress, etc., and summarizes the role of Clostridium butyricum in aquaculture The effect of the application is to provide a reference for the healthy development of the Clostridium butyricum industry and its application in the green production of aquaculture.

Keywords: Clostridium butyricum; mechanism; application

Clostridium butyricum , Clostridium, which is one of the normal bacteria found in the gut of soil, humans and animals. It is a microecological preparation with various biological functions such as regulating intestinal microecological balance, promoting growth and development, improving immunity and antioxidant, and has high development and application value.In 1976, Clostridium butyricum. was used in Japan for feed additives.

1.  Morphology and biological properties of Clostridium butyricum

Clostridium butyricum is a strictly anaerobic Gram-positive bacterium, periflagellum, movement; slightly full oval or round, smooth, shiny, cream, in nutritional lack of growth blocked, the end of the body will form elliptical spores^[1]. As the intestinal flora of human and animal intestines, Clostridium butyricum has many excellent characteristics such as high heat resistance, gastric acid resistance and bile salt resistance. Studies have shown that Clostridium butyricum still has a high survival rate after heat treatment with 100℃, 5 min, 90℃, and 10min, with a growth temperature from 16℃ to 44℃ and pH 1.0.Clostridium butyricum also produces metabolites such as organic acids, enzymes, and vitamins^[2], It has good application prospects in the field of feeding probiotics.

2.  Biological functions of Clostridium butyricum

2.1 Generation of nutrients

In the animal intestinal Clostridium butyricum can produce a variety of enzymes that can help the body digestion, such as cellulase, glycosidase, amylase and protease, under the action of these digestive enzymes, polysaccharides into oligosaccharides, contribute to the body digestion and absorption, produce some oligosaccharides also provide nutrients for other probiotics, conducive to the growth and reproduction of probiotics.In the intestinal tract, Clostridium butyricum can synthesize B vitamins and vitamin K and other substances, but also can promote the absorption of vitamin E, enhance the disease resistance of the animal body, and has an important health care role.Clostridium butyricum has high levels of B vitamin synthesis relative to other species, especially nicotinate synthesis is 3 to 6 times that of other species.Overall, Clostridium butyricum produces a rich vitamin species and plays an important role in promoting the comprehensive absorption of host intestinal nutrients.

2.2 Inhibition of the pathogen proliferation

2.2.1 Secreting of antimicrobial substances

Clostridium butyricum as probiotics can maintain or restore the host intestinal dominant flora in the body, and inhibit the growth of common intestinal pathogenic bacteria such as E. coli, Salmonella, Clostridium perfringens, and H. pylori bacteria through the secretion of short-chain fatty acids, antimicrobial peptides and Clostridium butyricum.Short-chain fatty acids are generally produced by food fibers or glycans in the intestine after fermentation by anaerobic bacteria. The metabolites of Clostridium butyricum mainly include butyrate, lactic acid, acetate and propionic acid.The secretion of organic acids in the intestine can reduce the pH value of the intestinal lumen and limit the growth of harmful pathogenic bacteria^[3], Adjust the microecological balance, reduce enterotoxins, and reduce the diarrhea rate.

Clostridium butyricum is a bacteriocin that inhibits and kills certain Clostridium bacteria, which has a certain bactericidal effect and is antagonistic to the colonization of harmful bacteria.In 1975, Clarke from Aberystwyth University, Wales was first isolated from the Clostridium butyricum NCIB7423 strain and named Clostridium butyricum 7423^[4], Konisky studies found that Clostridium butyricum is a hydrophobic bacteriocin, cells treated with Clostridium butyricum, whose synthesis of DNA, RNA and protein synthesis is inhibited, with strong bacteriostatic capacity.At present, it is widely believed that bacteriocin sterilization acts mainly by destroying the cell membrane and forming permeable pores in the membrane, leading to K + outflow, cell membrane depolarization and ATP leakage, and cell death because of losing lysis. Clostridium butyricum can promote lipophosphowall acid on the cell wall to inhibit the adhesion effect of E. coli in intestinal epithelial cells^[5-6]。

Antimicrobial peptides (AMPs), bioactive small-molecule peptides encoded by ribosomes, are important effectors of the host's innate immune defense system and are used to resist infection by external pathogens.Natural biological antimicrobial peptides have a wide antibacterial spectrum, especially on multiple resistant bacteria, and good thermal stability and water-soluble, almost non-toxic to normal cells in higher animals.And all the antimicrobial peptides have antiviral activity.Mycopeptides can directly chemotaxis ize neutrophils, monocytes, monocytes, mast cells and helper T cells during acute inflammatory reactions, induce gene expression of neutrophils, monocyte chemokines or cytokines, mobilize innate immune cells to the infection site; stimulate mast cells to release histamine, and subsequently increase vascular permeability; increase the expression of the adhesion molecule —— integrin and promote non-regulatory phagocytosis.

2.2.2 Biological antagonism

Clostridium butyricum has a high adhesion effect on the intestinal tract. It can adhere to intestinal epithelial cells to compete with colonization sites, produce competitive inhibition of adhesion to pathogenic bacteria, reduce the opportunity of adhesion and colonization of intestinal mucosa, reduce the proliferation of pathogenic bacteria to a certain extent, and promote the growth of beneficial bacteria^[7]. Clostridium butyricum colonizes the intestinal mucosa for a long time, competes with other microorganisms, limits the survival and reproduction of pathogenic bacteria, and prevents the shifting of "treating bacteria with bacteria".The "occupancy competition" of C. butyrate is essential for the balance of intestinal microbiota, the stability of intestinal bacterial enzyme activity and intestinal permeability, and it was found to play an important role in the adhesion capacity of Clostridium butyricum.

2.3 Promote probiotic growth

The digestive enzymes produced by Clostridium butyricum convert polysaccharides into oligosaccharides conducive to the utilization of probiotics, providing essential growth substances for intestinal beneficial microorganisms, being conducive to the growth and reproduction of probiotics, and improving intestinal health^[7]. In addition, there is cross-breeding in the ecological relationship of microorganisms, a phenomenon that the survival or growth of a microorganism is dependent on another microorganism that lives near it to provide growth factors and nutrient substrates.Studies proved that there is a cross trophic relationship between lactic acid bacteria and butyric acid bacteria, resistant starch and oligo fructose after fermentation to produce lactic acid, and lactic acid as the substrate, lactic acid into butyrate acid, and a lot of butyrate acid reduces the pH in the colon, provides a more suitable living environment for lactic acid bacteria, such as with bifidobacteria, Lactobacillus acidophilus and streptococcus fecal intestinal beneficial bacteria, three bacteria, live bacteria increased by about 24%, 43% and 7%, respectively^[8], It was also found that it showed a strong inhibition effect on intestinal pathogens and corrupt bacteria^[9-10]。

2.4 Enhance the immunity

Clostridium butyricum has immune modulatory ability, act as a stimulating factor, not only can promote the body's immune organ development, activate lymphoid tissue, stimulate the intestinal mucosa immune activity factor to induce secreted immunoglobulin A (SIg A), immunoglobulin M (Ig M), and on the blood white blood cells and cytokines involved in immune regulation, improve the immune function.During regulating inflammatory responses, Clostridium butyricum regulates the body's immune pathways by regulating specific receptor gene expression.TOLL-like receptors in intestinal epithelial cells can recognize and be activated bacterial products. Clostridium butyricum can activate TLR2, regulate the independent pathway of myeloid differentiation factor 88 (MyD88), activate NF-B signaling, regulate downstream gene expression, the secretion of inflammatory factors such as TNF-and IL-8, resist the infringement of pathogenic microorganisms and thus inhibit the inflammatory response^[11]. However, the MyD88-dependent pathway, activating NF-B, mainly mediates the production of inflammatory cytokines, leading to the excessive secretion of proinflammatory factors and destroying the body's autoimmune function.

When the intestinal pathogenic bacteria increase greatly, they often induce the abnormal apoptosis of the intestinal epithelial cells, so that the pathogenic bacteria are easy to enter the deep intestinal tissue and undergo bacterial displacement.Enteropathogenic Escherichia coli (EHEC) is a major class of intestinal infectious pathogens that can cause intestinal disease by inducing apoptosis and necrosis pathways^[12]. Clostridium butyricum inhibited the gene expression of EHEC-induced apoptotic factors, while also inhibiting protein viability^[13]Blocking apoptotic signaling affects the apoptotic process, effectively protects the integrity of the intestinal epithelium, and enhances the body's resistance to pathogenic organisms, thus improving the body's immunity.

2.5 Protection of the intestinal mucosal barrier

Buttrate is the most efficient short chain fatty acid in intestinal epithelial cells. The priority of short chain fatty acid is butyrate, propionic acid and acetic acid, which accounts for 70% of colonic energy supply and demand.In addition, butyrate can promote colonic protein synthesis, promote cell proliferation, regeneration and repair of intestinal epithelial tissue cells, and reverse the increase in intestinal mucosal permeability^[14]. Buttrate is the main nutrient for the regeneration of intestinal epithelial tissue cells, which can increase the number of intestinal epithelial cells, increase the villus height and make the shallow crypts, indicating that it improves the intestinal morphological structure and enhances the nutrient absorption ability^[15]。

Buttrate realizes gene regulation by inhibiting the activity of histone deacetylase enzyme: butyrate can increase the expression of galactin-1 expression in colon cancer cells, this lectin binding to galactoside is related to cancer cell differentiation, and participates in cell adhesion to prevent intestinal cancer; butyrate can also upregulate the expression of mucin genes in the mucus layer, secrete mucus, and effectively protect against harmful microbial invasion.Buttrate can also reverse the LPS-induced decreased expression of tight-junction proteins and closed proteins, enhance tight cell-to-cell junctions, reduce cell permeability, and enhance intestinal barrier function.

2.6  Anti-stress

Under normal circumstances, the dynamic balance between the body radicals and the antioxidant defense system, when the animal is subjected to external stress, not only the blood glucose concentration, creatine kinase and lactate dehydrogenase content increased^[16-17]It will also produce excessive free radicals, thus causing damage to the body.It have shown that H produced by Clostridium butyricum metabolism₂And butyrate can enhance the body's antioxidant enzyme activity, such as peroxidase, superoxide dismutase, also prototype coenzyme I and also prototype coenzyme II, to selectively remove the free radicals and reactive oxygen species produced by the body under stress^[18], butyrate can also significantly reduce hydrogen peroxide-induced DNA damage in colocytes, increase GSH content in the colonic mucosa, exert antioxidant effects, and reduce oxidative stress^[19]. Furthermore, Clostridium butyricum can also regulate antioxidase expression by inducing the expression of the organism and Nrf2, a transcription factor, thereby inhibiting oxidative stress in the liver^[20]. Xing Shuai et al took weaned piglets as the research object, designed 4 groups of tests, the control group and test 1 group fed the basic diet, supplemented with C. butyrate 0.05% (number of live bacteria 1.0*10¹⁰cfu·kg^-1), With E. coli 2 weeks after feeding, the serum index showed that 41.20% dehydrogenase and blood glucose increased by 44.60% (P <0.01) and 20% compared with controls, respectively, indicating that Clostridium butyricum enhanced the body's stress resistance^[21]。

3.  Application of Clostridium butyricum in aquaculture

With the beginning of antiban at the feed end, green, environmental protection and residue-free probiotics feed additives have gradually entered people's vision. Clostridium butyricum, because of its unique strain advantages, has become the leader in probiotics feed additives, in South American white shrimp and Japanese marsh shrimp^[22-24]In the study, spraying Clostridium butyricum on the surface of feed can improve the digestive enzyme vitality of shrimp intestines, resist stress resistance and regulate the composition of its intestinal flora.Pan^[25]The antagonism of Clostridium butyricum on the adhesion ability of two fish pathogens, aeromonas hydrophilophilus and V. eelis, showed that Clostridium butyricum could inhibit their colonization in the fish gut.Sumon class^[26]It was found that after feeding a feed containing Clostridium butyricum for 60d, its gastrointestinal weight and growth rate were significantly increased, and its body immunity was enhanced. At the same time, in vitro tests proved that the growth of the pathogenic bacterium Vibrii could be inhibited in the breeding process; Duan Yafei et al^[297]The effect of Clostridium butyricum addition to diet on shrimp was studied, and it was found that Clostridium butyricum improved the growth performance and feed utilization rate of shrimp, promoted its intestinal structure, digestive enzymes and SCFA content, and improved the intestinal immune function under ammonia stress conditions.

He Ruipeng et al^[28]The addition of CBYG01 to the gentian grouper diet showed that the diet was added 1*10¹¹CFU / kg caseate bacteria significantly improved the grouper growth performance and antioxidant capacity compared to the control (P <0.05).Shu Hu et al^[29]It was found that adding 0.5% caseate bacteria to the gem feed significantly increased the final body weight of the gem perch (P <0.05), and significantly improved the intestinal digestive enzyme activity and serum antioxidant capacity (P <0.05).Song Zengfu^[30]Adding Clostridium butyricum to the feed can increase the daily weight and reduce the bait coefficient; increase IgM concentration in plasma, improve intestinal digestive enzymes and serum lysozyme activity; promote the growth of beneficial bacteria and inhibit the growth of E. coli, E. enteri and other pathogenic bacteria.In addition, in the intestinal epithelial cell adhesion test of fish, Clostridium butyricum could significantly inhibit the adhesion of V. eel, reducing the adhesion rate by 7.03% (P <0.05).In the in vitro experiments^[31], Clostridium butyricum has obvious inhibitory effects on the apoptosis induced by Salmonella enterica enteritis and V. parahaemolyticus (P <0.05), indicating that Clostridium butyricum can prevent and treat the infection caused by Salmonella enteritis and V. parahaemolyticus.