The intestine of an animal is an organ that communicates with the outside world. The traditional concept believed that it only has the function of digestion and absorption, and did not participate in pathological changes outside the organ. With the deepening of research, everyone realized that the intestinal tract also has a barrier function to prevent harmful substances such as toxins, anti-nutritional factors and bacteria in the intestinal cavity from invading the body. The intestine is not only an important nutrient digestion and absorption organ in the body, but also an important immune and endocrine organ in the body. As the site most frequently exposed to microorganisms in the external environment, the intestine is not only the main site for pathogenic microorganisms and toxins to invade the body, but also the first important barrier to prevent intestinal pathogenic microorganisms from infecting the body [33–36]. The complete intestinal barrier includes four parts:physical barrier, chemical barrier, biological barrier and immune barrier. Each has different molecular regulation mechanisms and biological functions, and is organically combined through their respective signal pathways to jointly defend against foreign antigens from invading the body [37–39].
Digestive enzymes are the main element that constitutes a chemical barrier. The growth of the body is positively related to the digestion and absorption of nutrients. The intestine is the main place where the body absorbs nutrients. Various digestive enzymes in the intestine play an important role in the digestion of nutrients. Therefore, increasing the activity of digestive enzymes is an effective way to improve the digestion of nutrients [40, 41]. Sogarrd proposed for the first time that probiotics can improve animal digestive enzyme activity . At present, a large number of studies have shown that probiotics can significantly increase the activities of protease, amylase and lipase in aquatic animals, and increase the utilization rate of feed [43–45]. Wang et al.  added Bacillus and photosynthetic bacteria to Cyprinus carpio fine feed and found that the intestinal protease, lipase and amylase activities in the experimental group were significantly increased (p < 0.05). Wu et al.  fed Bacillus subtilis Ch9 to grass carp, the intestinal protease and amylase activity of the experimental group was significantly higher than the control group after two weeks (p < 0.05). Suzer et al. found that the protease, lipase and amylase activities of Sparus aurata after ingesting probiotic eutrophic biological diet were higher than the control group. Consistent with the results of previous studies, this study found that adding B. amyloliquefaciens LSG2-8 to the diet can significantly increase the activity of amylase, lipase and protease in the intestinal tract of R. lagowskii. At present, there are different mechanisms for probiotics to improve the activity of digestive enzymes in the intestines of animals. The generally accepted concept is that probiotics produce extracellular enzymes while exerting their effects in the intestinal tract, which may be one of the reasons for the results of this experiment. In addition to the enzymes contained in the animal body itself, probiotics can produce digestive enzymes such as proteases, lipases and amylases during the metabolism process, thereby increasing the host’s intestinal digestive enzyme activity, assisting the body in digesting organic substances, and increasing the conversion rate of food. In addition, when the probiotics enter the digestive tract of aquatic animals with the bait, they can sprout and grow into metabolically active cells in the digestive tract, adjust the structure of the intestinal microbial community, improve the microecological environment, and optimize the physiological activities of the intestinal tract, thereby indirectly promote the body's synthesis and secretion of digestive enzymes.
Non-specific immunoenzyme activity and cytokines are the main indicators to measure the body’s immune barrier function. Important lysosomal enzymes such as alkaline phosphatase, acid phosphatase and lysozyme play an important role in the non-specific immune response. Alkaline phosphatase is a regulatory enzyme that participates in many important functions in all organisms. It can destroy the surface molecular composition of harmful substances, accelerate the degradation of harmful substances, and prevent the reproduction of germs . Acid phosphatase is an enzyme that catalyzes the hydrolysis of phosphate monoesters under acidic conditions. It is mainly involved in the metabolism of phosphate esters and also has the functions of regulating metabolism, energy conversion and signal transduction. As a marker enzyme of lysosomes, studies have found that acid phosphatase is closely related to the normal performance of lysosomal physiological functions . Lysozyme plays an important role in the forefront defense mechanism of fish against infectious pathogens. When pathogens invade, they can stimulate the increase in the concentration of lysozyme in the fish. Lysozyme causes the rupture of pathogenic cells by enzymatically hydrolyzing mucopolysaccharides in the cell wall of pathogens, especially for Gram-positive bacteria or certain specific bacterial cells . In this experiment, we found that adding B. amyloliquefaciens LSG2-8 to the diet at a concentration of 1.0 × 108 CFU/g can significantly increase the activity of LZM, AKP and ACP in the intestinal tract of R. lagowskii (p < 0.05). Consistent with the results of this study, Yang et al.  reported that Bacillus velezensis JW can significantly increase the activity of AKP and ACP in Carassius auratus (p < 0.05). Li et al.  found that Lactobacillus plantarum can significantly increase the activities of AKP and ACP in sea cucumbers (p < 0.05), but has no significant effect on lysozyme (p > 0.05). This difference may be related to environmental factors such as species differences, feed formulations, and the types of probiotics used. At the same time, a large number of studies have confirmed that adding a certain amount of probiotics to the material can improve the immunity of farmed fish [54, 55]. Probiotics can improve fish immunity may be due to the fact that the added probiotics reduce the ammonia nitrogen content in the water body and create a relatively good living environment for fish. Furthermore, probiotics can also produce antibacterial active substances, which can resist the colonization and invasion of pathogenic bacteria and improve the immunity of fish.
Cytokine, as an indispensable mediator that secreted from immune cells with regulating the immune response, repairing damaged tissues and defensing against infection. Interleukin (IL) and transforming growth factor (TGF) are the main cytokines in fish. The interleukins in fish have both pro-inflammatory and anti-inflammatory effects. IL-1β and IL-8 are widely accepted as two important pro-inflammatory factors that commonly used as indicator genes in response to bacterial and viral invasion [56, 57]. IL-10 and TGF-β are pleiotropic anti-inflammatory factors that are effective at relieving inflammation by inhibiting release of inflammatory cytokines . Many experiments have proved that probiotics can regulate and induce the immune factors of the intestinal mucosal cells of animals. Christensen et al.  found 6 strains of Lactobacillus, namely Lactobacillus Reuteri DSM12246, Lactobacillus plantarum Lb1, Lactobacillus fermentum Lb20, Lactobacillus casei CHCC3137 ), Lactobacillus plantarum 299v and Lactobacillus johnsonii La1 can induce the increase of the expression of IL-12 and tumor necrosis factor TNF in the intestinal dendritic cells of mice to varying degrees. In fish probiotics research reports, different probiotics will also regulate the differential expression of some immune factors in the intestine. Previous studies have shown that dietary administration with 1.0 × 109CFU/g L. brevis JCM 1170 and L. acidophilus JCM 1132 significantly increased (p < 0.05) TGF-β gene expression of hybrid tilapia . Reyes-Becerril et al. also through probiotics in vivo experiments that when 1.0 × 106 CFU/g active Debaryomyces hansenii was added to feed golden-head sea bream for 4 weeks, found that the expression of immune genes in the intestine of sea bream (including Hep, IgM, TCR-β, CSF-1R, TNF-α and IL-1β) were down-regulated . In the present study, supplementation of B. amyloliquefaciens LSG2-8 1.0 × 108 CFU/g diets significantly up-regulated the expression of IL-10 mRNA and TGF-β mRNA, and down-regulated the expression of IL-8 mRNA and IL-1-β mRNA in the foregut, midgut and hindgut of R. lagowskii. However, Kim et al. found that two active probiotics, Carnobacterium maltaromaticum B26 and Carnobacterium divergens B33 were isolated from healthy rainbow trout intestines. They all mixed with the intestinal cells of rainbow trout for 6 h and 12 h, except for the complement C3 gene, other intestinal cytokines (such as IL-1β, IL-8, TNF-α and TGF-β) were not found to be up-regulated . The minor differences may be related to the species variation, feed formula, the probiotics species used and other environmental factors.
The intestinal physical barrier plays an important role in resisting the invasion of pathogenic bacteria, maintaining the balance of intestinal flora, and protecting the health of the body. The integrity of the physical barrier includes the integrity of the tight junctions between the intestinal mucosal cells and the integrity of the intestinal mucosal epithelial cells. Zo-1 is an important tight junction protein, which is involved in the maintenance of polarity and material transport in epithelial cells [63, 64]. Claudin-3 is a transmembrane protein, which is essential for tight junctions between intestinal mucosal cells [65, 66]. Probiotics can regulate the permeability of the intestinal mucosal barrier by regulating the expression of tight junction proteins. Previous studies have shown that colitis mice eat compound probiotics, the expression levels of ZO-1, Claudin, Occludin and other genes in the intestine were all up-regulated. The results showed that the composite probiotics can protect the integrity of the intestinal mucosal barrier by reducing the permeability of the intestinal mucosa . The probiotic Lactobacillus brevis SBC8803 and Lactobacillus rhamnosus GG and their supernatants could increase the expression of tight junction proteins in the intestinal tract of mice with alcoholic liver disease and humans [68–70]. Similarly, in this experiment, we found that B. amyloliquefaciens LSG2-8 could significantly up-regulate the expression of ZO-1 mRNA and Claudin-3 mRNA genes in the intestinal tract of R. lagowskii,thereby reducing the permeability of the intestinal tract and improving the integrity of the intestinal mucosal barrier, which was consistent with the results of Jiang et al . The mechanism of probiotics regulating the physical barrier of the intestine may be as follows: probiotics can prevent pathogenic bacteria from being fixed in the intestine, promote mucus secretion in the intestinal mucosa and increase the thickness of the mucosal layer, thereby improving the barrier function of the intestinal physical membrane.
The digestive ability of fish is closely related to its intestinal tissue structure. A complete intestinal tissue structure is the prerequisite for improving the digestive ability of fish, and the study of intestinal morphological characteristics is also the main way to understand whether the physiological condition of fish is normal . The intestine of fish is not as complicated as the intestine of mammals, and has obvious differentiation. It is mainly composed of four parts: mucosal layer, submucosal layer, muscle layer and serosal layer. The increase in the height of the folds on the mucosal layer can expand the absorption surface area of nutrients, which is more conducive to the digestion and absorption of nutrients, and the mucosal layer is the main adsorption site for the flora in the intestine . Therefore, the structural changes of the mucosal layer can be observed to understand the influence of B. amyloliquefaciens LSG2-8 on the intestinal structure of R. lagowskii. The main role of the intestinal muscularis is to promote intestinal peristalsis, which is composed of smooth muscle, so increasing the thickness of the intestinal muscularis can indirectly promote the digestion and absorption of nutrients by fish. The results of this experiment showed that the height of the intestinal fold, the width of the lamina propria and the thickness of the muscle layer of R. lagowskii with B. amyloliquefaciens LSG2-8 were higher than those of the control group. This shows that the addition of B. amyloliquefaciens LSG2-8 can promote the increase of intestinal fold height, lamina propria width and muscle layer thickness of specific cultured subjects, and can improve the structure of intestinal tissue.
Intestinal microbes play an important role in the regulation of animal nutrition metabolism, immunity and diseases. From the sparse curve of the sample, it can be seen that the sequencing results of this experiment basically cover all the microorganisms in the intestine of R. lagowskii, and from the Venn diagram, it can be seen that the proportion of OTU shared between each sample is relatively high, which shows that under the same environmental conditions, the similarity of the microbial flora in the intestinal tract of R. lagowskii fed with different levels of B. amyloliquefaciens LSG2-8 was higher. We also found that feeding different levels of B. amyloliquefaciens LSG2-8 R. lagowskii’s gut microbes are mainly composed of Proteobacteria, Firmicutes, Bacteroides and Actinomycetes. This result is consistent with some research findings. The level composition of the intestinal microbiota of carp and grass carp is similar [74, 75]. Among them, Firmicutes and Bacteroides play an important role in the process of carbohydrate metabolism and nutrient absorption ; Actinomycetes are mostly effective antibiotics, and their most important role is to produce and extract antibiotics. In addition, it can also recover biological materials with complex classification .The normal distribution of beneficial and harmful bacteria in the intestines of fish is in a dynamic balance. When the body is attacked by pathogens, the dynamic balance of intestinal flora will be broken, resulting in a sharp increase in the number of harmful bacteria in the intestine. The number of beneficial bacteria is decreasing, which leads to a decline in the body's immune function and disease outbreaks [78, 79]. Studies have found that the potential pathogens that cause bacterial diseases in aquaculture animals are widely distributed in the intestines of fish [80, 81]. Similarly, in this experiment, we found that there are Aeromonas bacteria in the intestines of R. lagowskii and Flavobacterium, Aeromonas and Flavobacterium are common conditional pathogens. In the prediction of PICRUs, we found that the addition of B. amyloliquefaciens LSG2-8 to feed can reduce the relative abundance of microorganisms related to immune system diseases and metabolism-related diseases in the intestinal tract of R. lagowskii’s stalk. It shows that B. amyloliquefaciens LSG2-8 can maintain the balance of the intestinal flora of R. lagowskii by promoting the survival and reproduction of beneficial bacteria and inhibiting the growth of harmful bacteria. And we speculate that B. amyloliquefaciens LSG2-8 may inhibit the growth of pathogenic bacteria by secreting antibacterial substances and competing with pathogens for living space and nutrients. However, its mechanism of action on intestinal flora still needs further study.