Use of bacillus tequilensis gcb-3 in relieving heavy metal antimony poisoning in grass carp

Abstract

The application discloses a use of Bacillus tequilensis GCB-3 in relieving grass carp heavy metal antimony poisoning, wherein the Bacillus tequilensis GCB-3 is preserved in the China General Microbiological Culture Collection Center on January 16, 2024, and the preservation number is CGMCC No. 29629. The Bacillus tequilensis GCB-3 can improve the digestive enzyme activity in the intestinal tract of the grass carp, reduce the accumulation of heavy metal antimony in the intestinal tract and the liver, enhance the structure and function of the intestinal mucosal barrier, and reduce the morphological damage and dysfunction of the liver.

Description

Technical Field

[0001] This invention relates to one use of Bacillus tekirae GCB-3, belonging to the field of new biomaterials technology, and particularly to the use of Bacillus tekirae GCB-3 in alleviating antimony poisoning in grass carp. Background Technology

[0002] Heavy metals are environmental pollutants originating from nature or anthropogenic sources. They are toxic, non-degradable, and bioaccumulative. Once introduced into aquatic environments, they cause excessive concentrations of heavy metals, leading to disease and death in fish and other aquatic organisms. Through the food chain, they pose potential health risks to humans and have long been a key concern for the healthy development of aquaculture. Heavy metals generally refer to metals with a specific gravity greater than 5, comprising approximately 50 types, with common examples including mercury, antimony, chromium, lead, cadmium, manganese, zinc, and nickel. Heavy metals are a widespread pollutant in aquatic waters and are common chemical toxins causing acute poisoning and death in fish. Heavy metals can cause varying degrees of damage to the circulatory, reproductive, nervous, and endocrine systems of fish, as well as the activity of enzymes involved in metabolism. Fish suffering from acute heavy metal poisoning typically have grayish-white gills and secrete large amounts of mucus, forming flocculent deposits that obstruct the gills, causing respiratory distress. Poisoned fish often float on the surface of the water. With the rapid development of urban industrialization, frequent human activities and pollutant emissions have led to an increasingly severe impact of heavy metals on the aquatic environment and aquatic organisms.

[0003] Antimony, as an emerging global environmental pollutant, is not an essential element for life, but rather a potentially toxic trace element and a carcinogenic metalloid. Elemental antimony is more toxic than antimony compounds, and inorganic antimony is more toxic than organic antimony. The trivalent antimony compound Sb(III) is more than 10 times more toxic than Sb(V). In unpolluted natural water bodies in my country, the average concentration of antimony does not exceed 1 μg / L. The "Surface Water Environmental Quality Standard (GB 3838—2002)" stipulates that the maximum allowable concentration of antimony in centralized drinking water surface water sources is 5 μg / L. However, due to industrial development and human activities, the antimony content in some water bodies is constantly increasing. Antimony in soil, water, and the atmosphere can enter the human or animal body through various routes, including respiration, digestion, and skin contact. Currently, the toxic effects of antimony on animals have attracted significant attention. Antimony can have toxic effects on animals, causing damage to different organs through its influence on the nervous system, oxidative stress, genetics, and metabolism. This can manifest as growth retardation, increased deformity rates, gut microbiota dysbiosis, metabolic abnormalities, and altered gene expression.

[0004] In recent years, research on using probiotics to protect the body from heavy metal toxicity has received widespread attention. Probiotics, as a class of live microorganisms that can produce beneficial effects on host health, have been widely used in aquaculture. Some types of probiotics, due to the high content of teichoic acid in their cell walls, can adsorb heavy metals from the environment through biosorption, thus achieving a pollution removal effect. Bacillus, a commonly used probiotic in aquaculture, has been proven to adsorb and remove heavy metals from polluted environments in vitro. In vivo experiments have confirmed that Bacillus coagulans can alleviate the toxic damage caused by cadmium exposure.

[0005] Grass carp is the most farmed fish species in my country and the second most produced freshwater fish in the world. Heavy metal pollution in grass carp and its health risks to consumers urgently require attention. However, due to water pollution and the development of intensive farming methods, grass carp growth is hindered, diseases occur frequently, causing significant economic losses to fishermen and seriously threatening the sustainable development of my country's grass carp farming industry. Summary of the Invention

[0006] The purpose of this invention is to provide the use of Bacillus tekirae GCB-3 in alleviating antimony poisoning in grass carp. This Bacillus tekirae GCB-3 can increase the activity of digestive enzymes in the intestines of grass carp, reduce the accumulation of antimony in the intestines and liver, enhance the structure and function of the intestinal mucosal barrier, and alleviate liver morphological damage and functional impairment.

[0007] The objective of this invention and the technical problem it solves are achieved by the following technical solution. Based on the use of *Bacillus tekirii* GCB-3 in alleviating antimony poisoning in grass carp, as proposed in this invention, *Bacillus tekirii* GCB-3 was deposited on January 16, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 29629.

[0008] Furthermore, the Bacillus tekirae GCB-3 is added to fish feed. The specific preparation method is as follows: Bacillus tekirae liquid and 2% sterile sodium alginate solution are mixed at a volume ratio of 1:2 and sprayed onto the fish feed.

[0009] Furthermore, the Bacillus tekirae GCB-3 is added to the fish diet, and the content of Bacillus tekirae GCB-3, based on the number of viable bacteria, is 1×10⁻⁶. 7 CFU / g-1×10 9 CFU / g.

[0010] Furthermore, the Bacillus tekirae GCB-3 is added to the fish diet, and the content of Bacillus tekirae GCB-3, based on the number of live bacteria, is 1×10⁻⁶.8 CFU / g.

[0011] Furthermore, the fish feed formula contains the following ingredients:

[0012] Fish meal 7%; casein 25%; fish oil 5%; wheat bran 16%; microcrystalline cellulose 11%; wheat starch 30%; calcium dihydrogen phosphate 2%; vitamin and mineral premix 1%; lysine 0.5%; methionine 1%; choline chloride 1.5%.

[0013] Furthermore, the Bacillus tektii GCB-3 is used in grass carp farming to improve the activity of digestive enzymes in the grass carp's intestines; and / or to maintain a good balance of probiotic flora and promote intestinal health.

[0014] Furthermore, the Bacillus tekiria GCB-3 is used in grass carp farming to reduce the accumulation of antimony in the intestines and liver.

[0015] Furthermore, the Bacillus tekiria GCB-3 is used in grass carp farming. Adding GCB-3 to the feed promotes the relative expression of ZO-1, Claudin-b, and MLCK in the grass carp intestine, or promotes the expression and distribution of intestinal tight junction proteins, reduces intestinal permeability, enhances the structure and function of the intestinal mucosal barrier, and / or reduces the content of intestinal endotoxins, enhances intestinal immunity and anti-inflammatory ability, thereby protecting it from the invasion and damage of heavy metal antimony.

[0016] Furthermore, the Bacillus tekiria GCB-3 is used in grass carp farming to reduce liver morphological damage and functional impairment.

[0017] Furthermore, the Bacillus tekiria GCB-3 is used in grass carp farming to regulate the levels of AST, ALT, and TBIL in grass carp serum, upregulate serum albumin levels, or reduce hepatocyte nuclear translocation, reduce cell vacuolation and lipid droplets, improve hemocyte infiltration, and / or activate the Nrf2-Keap1-ARE signaling pathway, reduce oxidative stress, enhance antioxidant capacity, improve liver function, and alleviate endoplasmic reticulum stress, autophagy, and apoptosis.

[0018] By employing the above technical solution, the present invention has the following advantages:

[0019] 1) Stable intestinal colonization: GCB-3 colonizes stably in the intestines of grass carp, which helps maintain a good balance of probiotic flora and promotes intestinal health.

[0020] 2) Improves intestinal function and morphology: GCB-3 can improve intestinal digestive capacity, promote intestinal growth, reduce intestinal permeability, reduce intestinal endotoxin (LPS) content, and enhance intestinal immunity and anti-inflammatory capacity.

[0021] 3) Promotes liver health: GCB-3 reduces oxidative stress, enhances antioxidant capacity, improves liver function, and alleviates endoplasmic reticulum stress, autophagy, and apoptosis by activating the Nrf2-Keap1-ARE signaling pathway.

[0022] 4) Reduce antimony poisoning-induced damage: Through the gut-liver axis pathway, GCB-3 can reduce the physical damage caused by antimony poisoning, providing an effective solution to the problems caused by antimony poisoning in grass carp farming. Attached Figure Description

[0023] Figure 1 (a)-1(c) show the effect of Bacillus tekiria GCB-3 on the digestive enzyme activity of grass carp poisoned by antimony.

[0024] Figure 2 (a)-2(c) show the effects of Bacillus tekiria var. tekiria GCB-3 on the concentration of antimony in the intestines, liver, gills and kidneys of grass carp poisoned by antimony.

[0025] Figure 3 (a)-3(f) show the effect of Bacillus tekirae GCB-3 on the expression level of barrier genes in grass carp poisoned by antimony.

[0026] Figure 4 (a)-4(c) show the effects of Bacillus tekiria var. tekiria GCB-3 on the intestinal morphology and structure of grass carp poisoned by antimony.

[0027] Figure 5 (a)-5(f) show the effects of Bacillus tekirae GCB-3 on liver tissue damage in grass carp poisoned by antimony.

[0028] Figure 6 (a)-6(c) show the effects of Bacillus tekiria var. tekiria GCB-3 on the morphology of grass carp liver tissue after antimony poisoning. Detailed Implementation

[0029] This invention isolates a strain of Bacillus amyloliquefaciens, Bacillus tekirae, GCB-3, from the intestines of grass carp, which has significant probiotic properties. In vitro and in vivo tests have demonstrated that this strain is safe and reliable.

[0030] This invention demonstrates that Bacillus tekirae can effectively alleviate antimony poisoning in grass carp by feeding them a diet supplemented with Bacillus tekirae for 28 days. The results were obtained by analyzing indicators such as the grass carp's growth performance, feed utilization rate, digestive enzyme activity, intestinal morphology and function, liver morphology and function, and antimony concentration in various tissues.

[0031] Experiment 1: Bacillus tergentii GCB-3

[0032] A strain of *Bacillus tekirae* (GCB-3) was screened from the intestines of grass carp and deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 16, 2024, with accession number CGMCC No. 29629. It not only possesses the common safety and enzyme-producing capabilities of Bacillus strains, but also exhibits acetic acid production. Acetic acid plays an indispensable role in carbohydrate metabolism in aquatic animals.

[0033] Experiment 2: Effects of Bacillus tergentii GCB-3 on antimony poisoning in grass carp

[0034] Effects of Bacillus tekirii GCB-3 on growth performance and feed utilization of grass carp poisoned by antimony poisoning

[0035] Healthy grass carp were randomly divided into 3 groups, with 3 replicates per group and 30 fish per tank. The groups were as follows: I) Control (CG): Fish were fed a basal diet and antimony-free water; II) Antimony (Sb): Fish were fed a basal diet and exposed to a 5 mg / L potassium antimony tartrate solution (potassium antimony tartrate, purity 99.5% (KSbC4H4O7)); III) Antimony + Bacillus tegmentatus (SbBT): Fish were fed a diet supplemented with Bacillus tegmentatus (1×10⁻⁶). 8 (CFU / g) and exposed to 5 mg / L potassium antimony tartrate solution. The aquaculture trial used a basal diet formulation, as shown in Table 1. Preparation of the Bacillus tekirae diet: Bacillus tekirae liquid was mixed with 2% sterile sodium alginate solution at a ratio of 1:2 (volume ratio) and sprayed onto the basal feed to achieve a Bacillus tekirae concentration of 1×10⁻⁶ CFU / g. 8 CFU / g. The prepared feed should be stored in a 4℃ refrigerator for later use to ensure the stability of the viable Bacillus tekiria strain in the feed.

[0036] After a week of adaptation and stabilization, the experiment officially began, with a rearing period of 28 days. During the rearing experiment, the fish were fed three times a day (08:30, 11:30, and 17:30), with the daily feed amount set at 3% of their total body weight. All water in the experimental treatment group tanks was replaced daily with fresh water containing the same dose of antimony.

[0037] Measurement of growth indicators:

[0038] The following growth indicators are calculated: weight gain (WGR), specific growth rate (SGR), feed conversion ratio (FCR), condition factor (CF), liver-to-body ratio (HSI), visceral-to-body ratio (VSI), and intestinal index (IBI):

[0039] Weight gain rate (WGR, %) = 100 × (final average weight - initial average weight) / initial average weight;

[0040] Specific growth rate (SGR, % / d) = 100 × (ln final average weight - ln initial average weight) / number of days in the experiment;

[0041] Feed conversion ratio (FCR) = Feed intake / (Final gross weight - Initial gross weight);

[0042] Conditionness (CF, %) = 100 × body weight / body length 3;

[0043] Liver-to-body weight ratio (HSI, %) = 100 × liver and pancreas weight / body weight;

[0044] Visceral mass ratio (VSI, %) = 100 × visceral mass weight / body weight;

[0045] Intestinal body index (IBI, %) = 100 × intestinal weight / body weight.

[0046] Table 1. Experimental feed formulation and nutrient composition (dry matter %)

[0047]

[0048] Compound premix (containing vitamins and minerals): Vitamin A (IU / kg) 3600; Vitamin D3 (IU / kg) 1200; Vitamin E (mg / kg) 20; Vitamin K3 (mg / kg) 5; Vitamin B1 (mg / kg) 5; Vitamin B2 (mg / kg) 7; Vitamin B6 (mg / kg) 6; Vitamin B12 (ug / kg) 20; Calcium pantothenate (mg / kg) 20; Niacin (mg / kg) 30; Folic acid (mg / kg) 1.7; Biotin (mg / kg) 0.05; Vitamin C phosphate (mg / kg) 171.4; Inositol (mg / kg) 90; Mg (Mg / kg) 150; Iron (mg / kg) 120; Zn (mg / kg) 60; Mn (mg / kg) 30; Cu (mg / kg) 4; Co (mg / kg) 0.5; Selenium (mg / kg) 0.1; I (mg / kg).

[0049] Table 2. Effects of Bacillus tekiria on growth performance and feed utilization of grass carp poisoned by antimony poisoning.

[0050]

[0051] In Table 2, compared with the CG group, the grass carp in the Sb group showed significant linear and quadratic decreasing trends in FBW, WGR, SGR, FCR, and PER (P<0.05). However, compared with the Sb group, these indicators in the SbBT group all improved. Meanwhile, the HSI, VSI, ISI, and CF groups showed no significant linear or quadratic trends (P>0.05).

[0052] Heavy metals accumulate in fish, disrupting their normal physiological functions and inhibiting growth and development. These heavy metals may inhibit enzyme activity, interfere with metabolic processes, and reduce appetite and feed intake, thus affecting growth rate and weight gain. The results of this study indicate that antimony exposure does indeed lead to stunted growth and reduced feed utilization efficiency in grass carp.

[0053] Effects of Bacillus tekirii GCB-3 on the digestive enzyme activity of grass carp poisoned by antimony.

[0054] Preparation of crude enzyme solution:

[0055] The intestines were cut into small pieces and weighed (accurate to 0.01g). Nine times the tissue mass of physiological saline was added, and the mixture was homogenized using a tissue homogenizer and centrifuged (4℃, 4000r / min, 10min). The supernatant was collected and placed into a new centrifuge tube and stored in a 4℃ refrigerator. The analysis was completed after 24 hours.

[0056] Enzyme activity assay:

[0057] The digestive enzymes measured in this experiment included proteases, lipases, and amylases, all of which were measured using relevant kits from the Nanjing Jiancheng Biotechnology Research Institute.

[0058] like Figure 1 As shown, compared with the CG group, the levels of the three digestive enzymes in the Sb group showed a significant secondary decreasing trend (P<0.05), while the digestive enzyme levels in the SbBT group did not change significantly compared with the CG group (P>0.05). Supplementing the feed with GCB-3 can effectively increase the activity of digestive enzymes in the intestines of grass carp, enabling more efficient decomposition and absorption of nutrients. This helps improve the digestive function of grass carp, increase feed utilization efficiency, and promote growth and development.

[0059] Effects of Bacillus tekirii GCB-3 on antimony concentrations in the intestines, liver, gills, and kidneys of grass carp poisoned by antimony poisoning

[0060] Determination of antimony content:

[0061] After freeze-drying and grinding the sample, 0.1000 g of the sample was accurately weighed and placed in a high-pressure sealed digestion container made of polytetrafluoroethylene (PTFE). Then, 5 mL of nitric acid was added to the container, and the container was sealed and left overnight (10 hours). Next, 1 mL of hydrogen peroxide was added, and the container was tightly sealed with a stainless steel shell and heated in an oven at 140°C for 6 hours. After natural cooling, the inner container was removed, and the sample was evaporated to a thick consistency at 120°C on an electric heating plate. Subsequently, 1 mL of the sample was transferred and diluted to 10 mL with 5% hydrochloric acid containing 5% thiourea and 5% ascorbic acid. The total antimony content was determined using a dual-channel atomic fluorescence spectrophotometer (carrier gas: 5% hydrochloric acid; reducing agent: 2% potassium borohydride + 0.5% potassium hydroxide; carrier gas flow rate: 300 mL / min; shielding gas flow rate: 800 mL / min).

[0062] Antimony accumulation is as follows Figure 2 As shown, the accumulation levels of antimony in grass carp organs were as follows: intestine > liver > kidney > gills. Compared with the Sb group, the SbBT group showed a significant reduction in antimony accumulation in the intestine and liver of grass carp, exhibiting significant linear and quadratic trends.

[0063] Bioaccumulation is one of the main effects of heavy metal pollution on aquatic animals. Aquatic animals can be exposed to heavy metal pollutants in various ways, with water being the primary exposure medium. In polluted water, the higher the concentration of heavy metals, the greater the risk to fish. Different heavy metals have different toxicities and accumulate in organisms to varying degrees. Furthermore, different types of heavy metals have different effects on fish; lead and mercury have a greater impact on the nervous system, while cadmium and chromium have a greater impact on the liver and kidneys. This study found that grass carp accumulated the highest levels of antimony in their intestinal and liver tissues, which may be related to the high tissue affinity of antimony in grass carp tissues. Antimony ions form stable complexes with biomolecules such as proteins in grass carp, leading to preferential accumulation of antimony in tissues such as the intestine and liver. The intestine is the site of direct contact with antimony, while the liver is an important metabolic and detoxification organ, resulting in relatively high antimony accumulation in these two tissues. Moreover, the addition of GCB-3 to the feed reduced antimony accumulation in the intestine and liver, suggesting that GCB-3 may play a role in reducing the accumulation of the heavy metal antimony.

[0064] Effects of Bacillus tekirii GCB-3 on intestinal morphology and barrier gene expression in grass carp poisoned by antimony.

[0065] Histological analysis:

[0066] Following standard sample processing procedures, including sectioning, washing, dehydration, and cleaning, the intestinal sample was embedded in a paraffin block. Subsequently, the sample was cut into 5 μm thick sections, stained with hematoxylin and eosin, mounted on glass slides, and then photographed and measured using an Olympus optical microscope (IX71, Japan).

[0067] Extraction of total mRNA:

[0068] Total mRNA was extracted from the hepatopancreas and intestines (foregut, midgut, and hindgut) of *Sinonovacula roxburghii* using TRIzol reagent (Takara). Dosage and purity (OD260 / OD280) were determined using a NanoDrop 2000 spectrophotometer. mRNA integrity and quality were assessed by 1.0% agarose gel electrophoresis, and the mRNA was stored at -80°C for a short period.

[0069] Primer design and synthesis:

[0070] The gene primers used in this experiment were synthesized by Sangon Biotech Co., Ltd.

[0071] Real-time quantitative PCR (qRT-PCR) reaction system and conditions:

[0072] Real-time quantitative PCR was performed using the TB Green chimeric fluorescence assay. It was conducted on the StepOnePlus™ Real-Time PCR system (Thermo). This was based on Takara's One Step TB... PrimeScript TM The RT-PCR Kit II was followed according to the instructions, with a reaction volume of 20 μL. All operations were performed on an electronically controlled thermostat to prevent RNA degradation. The qRT-PCR reaction conditions were: reverse transcription 42℃ for 5 min, 95℃ for 10 s; PCR 95℃ for 5 s, 60℃ for 30 s, 40 cycles; 95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s. After the qRT-PCR reaction, β-actin was used as a housekeeping gene, and the 2-ΔΔCT method was used for analysis to calculate the relative expression level of the target gene mRNA in different tissues.

[0073] like Figure 3 As shown, compared with the CG group, the relative expression levels of Occudin, ZO-1, and Claudin-c in the intestine of grass carp in the Sb group were significantly decreased, while the relative expression levels of Occudin, ZO-1, and Claudin-c in the intestine of grass carp were significantly upregulated compared with the Sb group. These gene changes showed significant linear and quadratic relationships (P<0.05). Compared with the CG group, the relative expression levels of ZO-1 and Claudin-b in the intestine of grass carp in the Sb group were significantly decreased (P<0.05). However, compared with the Sb group, the expression levels in the SbBT group were significantly upregulated, showing a significant quadratic trend (P<0.05). Compared with the CG and SbBT groups, the relative expression level of MLCK in the intestine of grass carp in the Sb group showed a significant quadratic decreasing trend (P<0.05).

[0074] Intestinal morphology observation results showed that, compared with the CG group ( Figure 4 Compared to the Sb group, the Sb group showed reduced goblet cells, inflammatory cell infiltration, severe villus fusion, separation of the upper villus from the lamina propria, increased lamina propria width, and reduced muscle layer. In contrast, the SbBT group showed partial recovery of goblet cells, reduced inflammatory cell infiltration, significantly reduced villus fusion, reduced lamina propria width, no improvement in separation of the upper villus from the lamina propria, and no significant increase in muscle layer width.

[0075] The gut is one of the most important organs in fish, and maintaining gut health is crucial for ensuring normal growth and overall health. The morphology and structure of the gut play a significant role in fish gut health. Factors such as inflammatory cell infiltration, villus fusion, villus separation from the lamina propria, and increased lamina propria width may be caused by fish exposure to toxins or harmful substances. Furthermore, the reduction of goblet cells and muscle layer may be related to malnutrition in fish. These phenomena indicate that the fish gut undergoes an inflammatory response, suggesting environmental pollution or dietary problems. Based on the findings of this study, we can conclude that antimony exposure induces an inflammatory response, leading to damage to intestinal epithelial cells, disruption of tight junctions, and increased intestinal permeability. This makes it easier for lipopolysaccharide (LPS) to penetrate the intestinal barrier and enter the bloodstream, resulting in elevated serum LPS levels. Adding GCB-3 to the feed promotes the expression and distribution of tight junction proteins, reduces intestinal permeability, and enhances the structure and function of the intestinal mucosal barrier, thereby protecting it from the invasion and damage of the heavy metal antimony.

[0076] Effects of Bacillus tekirii GCB-3 on the morphology and damage of grass carp liver tissue in antimony poisoning

[0077] Histological analysis:

[0078] Following standard sample processing procedures, including sectioning, washing, dehydration, and cleaning, the liver sample was embedded in a paraffin block. Subsequently, the sample was cut into 5 μm thick sections, stained with hematoxylin and eosin, mounted on glass slides, and then photographed and measured using an Olympus optical microscope (IX71, Japan).

[0079] Enzyme activity assay:

[0080] This experiment measured blood ammonia, AST, ALT, TBIL, and TBA using relevant reagent kits from Nanjing Jiancheng Biotechnology Research Institute.

[0081] like Figure 5As shown, compared with the CG group, the serum AST, ALT, and TBIL levels of grass carp in the Sb group were significantly increased (P<0.05). However, compared with the Sb group, all indicators in the SbBT group were significantly decreased, and this trend showed a significant linear and quadratic relationship (P<0.05). Compared with the CG group, the serum ammonia and TBA levels of grass carp in the Sb group were significantly increased, and this trend showed a significant linear and quadratic relationship (P<0.05). Compared with the CG and SbBT groups, the serum albumin level of grass carp in the Sb group showed a significant quadratic decreasing trend (P<0.05).

[0082] Liver morphology observation results showed that, compared with the CG group ( Figure 6 In the Sb group, hepatocyte nuclear translocation, severe vacuolation, hemocyte infiltration, increased hepatic sinusoids, and a significant increase in lipid droplets were observed. Compared with the Sb group, the SbBT group showed reduced hepatocyte nuclear translocation, decreased cell vacuolation and lipid droplets, and significantly improved hemocyte infiltration.

[0083] AST, ALT, blood ammonia, albumin, total bilirubin, and total bile acids are indicators of liver function impairment in fish. By monitoring changes in these liver function indicators, it is possible to assess whether liver damage or disease exists in fish and to determine their liver health status. AST and ALT are mainly found in the liver, and elevated levels in these indicators suggest liver dysfunction.

[0084] On the other hand, albumin is the main plasma protein synthesized by the liver, and a decrease in its synthesis indicates abnormal liver function. When liver function is impaired and the liver cannot effectively convert ammonia into urea and adequately excrete bilirubin and bile, it leads to elevated levels of blood ammonia, TBIL, and TBA.

[0085] The results of this study on these liver function impairment indicators show that exposure to antimony can lead to liver dysfunction. Morphological observations also revealed that the liver of grass carp may have undergone damage to cellular structure and function, inflammatory response, sinusoidal changes, and lipid metabolism disorders. Adding GCB-3 to the diet can effectively alleviate liver morphological damage and functional impairment.

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. Bacillus tergentii GCB-3 ( Bacillus tequilensis The use of GCB-3 in alleviating antimony poisoning in grass carp is characterized by, The Bacillus tekirae GCB-3 was deposited at the China General Microbiological Culture Collection Center on January 16, 2024, with accession number CGMCC No. 29629.

2. The Bacillus tergentii GCB-3 as described in claim 1 ( Bacillus tequilensis The use of GCB-3 in alleviating antimony poisoning in grass carp is characterized by, The Bacillus tekirae GCB-3 is added to fish feed. The specific preparation method is as follows: Bacillus tekirae liquid and 2% sterile sodium alginate solution are mixed at a volume ratio of 1:2 and sprayed onto the fish feed.

3. The Bacillus tergentii GCB-3 as described in claim 1 ( Bacillus tequilensis The use of GCB-3 in alleviating antimony poisoning in grass carp is characterized by, The Bacillus tekirae GCB-3 was added to the fish diet, and the content of Bacillus tekirae GCB-3, based on the number of live bacteria, was 1×10⁻⁶. 7 CFU / g up to 1×10 9 CFU / g.

4. The Bacillus tergentii GCB-3 as described in claim 3 ( Bacillus tequilensis The use of GCB-3 in alleviating antimony poisoning in grass carp is characterized by, The Bacillus tekirae GCB-3 was added to the fish diet, and the content of Bacillus tekirae GCB-3, based on the number of live bacteria, was 1×10⁻⁶. 8 CFU / g.

5. The Bacillus tergentii GCB-3 as described in claim 1 ( Bacillus tequilensis The use of GCB-3 in alleviating antimony poisoning in grass carp is characterized by, The Bacillus tekiria var. tekiria is used in grass carp farming to improve the activity of digestive enzymes in the grass carp's intestines.

6. The Bacillus tergentii GCB-3 as described in claim 1 ( Bacillus tequilensis The use of GCB-3 in alleviating antimony poisoning in grass carp is characterized by, The Bacillus tekiria var. tekiria GCB-3 is used in grass carp farming to reduce the accumulation of the heavy metal antimony in the intestines and liver.

7. The Bacillus tergentii GCB-3 as described in claim 1 ( Bacillus tequilensis The use of GCB-3 in alleviating antimony poisoning in grass carp is characterized by, The Bacillus tekiria var. tekiria is used in grass carp farming to reduce liver morphological damage.

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