Preparation method and application of microbial fermentation straw polysaccharide

Abstract

The invention discloses a method for preparing microbial fermentation straw polysaccharide, which comprises the following steps: (1) treating straw by a bacterium-enzyme synergistic method: mixing soybean meal, straw powder and NaOH, curing, performing enzymolysis on the cured straw powder by using a straw complex enzyme preparation purchased in the market, performing high-pressure sterilization on the enzymolysis product, and drying for later use; (2) fermentation: adding the straw powder subjected to enzymolysis into an LB culture medium, and adding bacillus cereus and bacillus tequilensis for synergistic fermentation; and (3) extracting the fermented straw polysaccharide. According to the method, the straw is used as a substrate, and polysaccharide is produced through fermentation, so that agricultural and sideline product waste straw is effectively utilized, and a new thought is provided for effective utilization of straw resources. Meanwhile, the sustainable straw fermentation polysaccharide resources are used for improving the growth and health of the grass carps, and improving the quality and efficiency of fishery and increasing the yield of farmers.

Description

Technical Field

[0001] The present invention relates to the preparation and application of microbial fermentation polysaccharide, and in particular to a preparation method and application of microbial fermentation straw polysaccharide. Background Art

[0002] Polysaccharides are widely present in living organisms. They are a class of naturally occurring high-molecular-weight polymers composed of aldoses or ketoses linked by glycosidic bonds. They are important biomacromolecules in living organisms and essential for maintaining the normal functioning of life. Plant polysaccharides, also known as plant polysaccharides, are polysaccharides with a degree of polymerization exceeding 10 produced by plant cell metabolism. Many plant polysaccharides exert antioxidant and immunomodulatory effects in organisms. Their low cost and minimal toxicity have made them a key area of ​​research in antioxidant and immunomodulatory research. Plant polysaccharide extraction methods include single extraction and synergistic extraction. Single extraction methods primarily include water extraction and alcohol precipitation, microwave-assisted extraction, ultrasound-assisted extraction, and enzyme extraction. Natural plant polysaccharide extraction technology has evolved from simple cold maceration and decoction methods to today's ultrahigh pressure extraction, ultrafiltration membrane technology, semi-bionic technology, multiphase interaction systems, and supercritical fluid extraction.

[0003] Most new technologies overcome the damage to the polysaccharide structure caused by high temperatures during the extraction process, fully protecting the target product. While increasing yields, these new technologies also place increasing demands on purity. Ultrahigh pressure technology is suitable for the extraction of natural polysaccharide products that require high low-temperature stability, ultrafiltration membrane technology physically extracts and separates polysaccharide products, and supercritical liquid extraction technology achieves precise separation through gradient extraction. The safety and economics of traditional polysaccharide extraction technology cannot be ignored in mass production. Therefore, traditional and modern technologies are now complementing each other, and composite extraction methods are increasingly used in practical applications to achieve the goals of short-term, efficient, and high-purity extraction. Among them, the bacterial-enzyme synergistic fermentation method is low-cost, simple to process, environmentally friendly, and has been extensively researched.

[0004] Microbial fermentation technology generally involves utilizing the metabolic activity of microorganisms, through enzyme treatment or the inherent functions of the microorganisms, to convert difficult-to-use nutrients or anti-nutrients in unconventional feeds into small molecule nutrients required by the animal body. In recent years, there has been considerable research on the application of corn processing by-products. However, due to factors such as their high crude fiber content and anti-nutrient content, these by-products cannot effectively replace other feed ingredients when used in feed. Microbial fermentation of corn processing by-products can enhance their nutritional value and palatability, allowing them to be more effectively used as feed ingredients, thereby improving the production performance of livestock and poultry.

[0005] Jilin Province has abundant straw resources and vast aquaculture areas. How to combine these two advantages and develop related sustainable and healthy development technologies is the main background of this study. Summary of the Invention

[0006] The purpose of the present invention is to overcome the defects of the existing technology and provide a new method for producing straw polysaccharides by microbial fermentation. The method utilizes agricultural and sideline waste straw to produce polysaccharides through a bacterial and enzyme synergistic fermentation method, thereby effectively utilizing straw resources and using sustainably obtained straw fermentation polysaccharide resources to improve the growth and health of grass carp.

[0007] The purpose of the present invention and the technical problem solved are achieved by adopting the following technical solutions. According to the present invention, a method for producing straw polysaccharides by microbial fermentation comprises the following steps:

[0008] (1) Treating straw by a bacterial enzyme method: soybean meal, straw powder, and NaOH are mixed and matured, and the matured straw powder is enzymatically hydrolyzed using a commercially available straw complex enzyme preparation. The enzymatic hydrolysis product is sterilized under high pressure and then dried for later use;

[0009] (2) Fermentation: Enzymatically hydrolyzed straw powder was added to LB medium, and Bacillus cereus and Bacillus tequila were added for synergistic fermentation;

[0010] (3) Extraction of polysaccharides from fermented straw.

[0011] Furthermore, in the step (1), the straw powder is sieved through 60 mesh; the mass ratio of the soybean meal and straw powder added is 1:5; the NaOH concentration is 1.2-4%, and the mass ratio of the soybean meal straw powder mixture to the NaOH solution is controlled at 1:1-1.5; and the aging is carried out at room temperature for 24-36 hours.

[0012] Furthermore, in step (2),

[0013] The preparation of the culture medium comprises the following steps: weighing tryptone, yeast extract, and sodium chloride according to the proportion of LB culture medium, adding enzymatically hydrolyzed straw powder, adding double-distilled water to the volume of the culture medium, adjusting the pH to 7.2 with NaOH, and sterilizing;

[0014] The amount of enzymatically hydrolyzed straw powder added is 50-60 g straw powder per 1000 mL of culture medium;

[0015] The density of the Bacillus cereus and Bacillus tequila strains is 1.5×10 8 cells / mL, 0.5 mL of each of the two bacterial solutions was added to every 100 mL of culture medium;

[0016] The fermentation conditions are: temperature 30° C., rotation speed 160 rpm, and fermentation time 48 h.

[0017] Furthermore, in step (2),

[0018] The Bacillus cereus is selected from Bacillus cereus LSG-1; the Bacillus tequila is selected from Bacillus tequila GCB-3.

[0019] Furthermore, in step (3), the extraction of the fermented straw polysaccharide comprises the following steps:

[0020] The fermentation broth obtained is collected and filtered, and concentrated by rotary evaporation. The concentrate is subjected to the Savag method to remove free crude protein; a chloroform-n-butanol solution having a volume of 1 / 4 of the crude polysaccharide solution is added to the crude polysaccharide solution, the mixture is shaken thoroughly, and the mixture is centrifuged to separate the aqueous phase from the chloroform phase; a chloroform-n-butanol solution having a volume of 1 / 4 of the aqueous phase is added, and the above process is repeated until no white precipitate is present at the interface between the liquid and the aqueous phase;

[0021] Add 3 times the volume of anhydrous ethanol to the purified straw crude polysaccharide solution, let it stand at 4°C for 12-24 hours to obtain fermented straw polysaccharide solids; centrifuge at 4°C, 5000r / min, discard the supernatant, and take the precipitate for freeze-drying.

[0022] Furthermore, in step (3),

[0023] The chloroform-n-butanol is pre-prepared into a mixed solution with a volume ratio of 4:1;

[0024] The shaking time is 30 min;

[0025] The centrifugation time is 1 min.

[0026] The present invention provides a fermented straw polysaccharide prepared by the above-mentioned method for preparing microbial fermented straw polysaccharide, wherein the monosaccharide composition comprises the following percentage components:

[0027] 15.99% mannose, 7.58% glucosamine, 1.1% ribose, 2.02% rhamnose, 1.65% glucuronic acid, 0.4% galacturonic acid, 2.08% galactosamine, 32.07% glucose, 14.59% galactose, 11.73% xylose, 8.99% arabinose, 1.8% fucose.

[0028] The present invention proposes the use of the fermented straw polysaccharide prepared by the above-mentioned method for preparing the microbial fermented straw polysaccharide as a fish feed immunopotentiator.

[0029] Furthermore, the added amount of the fermented straw polysaccharide is 0.5-4 g / kg feed; preferably 2-4 g / kg; preferably 2-3 g / kg; preferably 2 g / kg.

[0030] Furthermore, the formula of the fish feed, calculated in g / kg addition amount, comprises the following ingredients:

[0031] Fish meal 280g / kg, flour 200g / kg, wheat bran 180g / kg, peeled soybean meal 160g / kg, microcrystalline cellulose 80g / kg, fish oil 20g / kg, corn oil 20g / kg, calcium dihydrogen phosphate 20g / kg, 50% choline chloride 15g / kg, 1% compound premix 10g / kg, methionine 10g / kg, lysine 5g / kg, fermented straw polysaccharide 0.5-4g / kg.

[0032] The fish feed preparation method comprises the following steps:

[0033] The feed ingredients are crushed, sieved, weighed, and mixed. The extracted crude polysaccharides from the straw are added to the base feed. After mixing evenly, oil and water are sprayed on the feed, stirring evenly during the spraying process.

[0034] Spray water until the feed can be grasped into a ball without falling apart, put the raw materials into the machine for mechanical extrusion into strips, and dry in an oven at 50-70℃ for 12-24 hours.

[0035] The feed is put into a pellet mill to make a feed with good palatability and uniform size for use in breeding experiments.

[0036] By means of the above technical solution, the method for producing straw polysaccharides by microbial fermentation and its application of the present invention have at least the following advantages:

[0037] 1) This study uses straw as a matrix and produces polysaccharides through fermentation, thereby effectively utilizing agricultural and sideline waste straw and providing new ideas for the effective utilization of straw resources.

[0038] 2) At the same time, sustainably obtained straw fermentation polysaccharide resources are used to improve the growth and health of grass carp, promote the quality and efficiency of fisheries and increase farmers' income.

[0039] 3) This study provides basic data for straw fermentation polysaccharide technology and its effects as an immunopotentiator on the growth, antioxidant and immune systems of grass carp, and provides a theoretical basis and technical support for the effective utilization of straw resources and healthy farming of grass carp in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 (A)-1(C) are the free radical scavenging abilities of fermented straw polysaccharides in effect test example 1.

[0041] Figure 2 This is the quadratic regression curve of the added amount of fermented straw polysaccharide and weight gain in effect test example 2.

[0042] Figure 3 It is the survival rate of Aeromonas hydrophila challenge in effect test example 2.

[0043] Figure 4 (A)-4(F) are the immune gene expressions in the liver of grass carp in effect test example 2. DETAILED DESCRIPTION

[0044] This study investigated two key issues: microbial fermentation of straw polysaccharides and healthy grass carp farming. First, straw was treated using a bacterial-enzyme co-fermentation method to release nutrients. Fermentation was then used to further reduce anti-nutritional factors in the straw and obtain fermented straw polysaccharides. The polysaccharides extracted from the straw were then fed to grass carp as an immune enhancer for eight weeks. The effects of the fermented straw polysaccharides on the growth and health of the grass carp were evaluated by measuring their growth performance, nutritional metabolism, antioxidant capacity, immunity, and resistance to infection with Aeromonas hydrophila.

[0045] The fermentation strains used in the embodiments of the present invention are: Bacillus cereus LSG-1, which was deposited in the General Microbiology Center of the China Culture Collection Administration Committee on April 20, 2020, with a deposit number of CGMCC No. 19595; Bacillus tequila GCB-3, which was deposited in the General Microbiology Center of the China Culture Collection Administration Committee on January 16, 2024, with a deposit number of CGMCC No. 29629.

[0046] Example Preparation of Straw Polysaccharide by Microbial Fermentation

[0047] Microbial fermentation of straw

[0048] Straw pretreatment: Soybean meal (40 g), sieved (60 mesh) straw powder (200 g), and 1.2% NaOH (50 mL) were mixed evenly and aged at room temperature for 24 h. The aged straw powder was enzymatically hydrolyzed using a commercially available straw complex enzyme preparation. The hydrolyzed product was sterilized by autoclaving and then dried for later use.

[0049] Fermentation strain composition: Bacillus cereus LSG-1 and Bacillus tequila GCB-3 were used for synergistic fermentation, with a strain density of 1.5×10 8 cells / mL, and 0.5 mL of each of the two bacterial solutions was added to every 100 mL of culture medium.

[0050] LB medium composition: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride (NaCl), 60 g of enzymatically hydrolyzed straw powder, double-distilled water to 1000 mL, 5 mol / L NaOH (about 0.2 mL) to adjust the pH to 7.2, and sterilize at 121°C for 30 min.

[0051] Fermentation conditions: Use a 300 mL Erlenmeyer flask with a 200 mL fermentation volume, sealed with a breathable membrane, and culture in an air bath shaker. Fermentation conditions: 30°C, 160 rpm, and 48 hours.

[0052] Extraction of polysaccharides from fermented straw

[0053] The fermentation broth was collected and filtered to remove solids. The broth was then concentrated on a rotary evaporator at 50-69°C. Free crude protein was removed from the concentrate using the Savag method. The crude polysaccharide solution was then added to a chloroform-n-butanol solution (pre-prepared to a 4:1 volume ratio) at 1 / 4 its volume. The mixture was dispensed into stoppered test tubes and shaken thoroughly for 30 minutes. The aqueous phase was then centrifuged for 1 minute to separate the chloroform phase. A chloroform-n-butanol solution equivalent to 1 / 4 its volume was added to the aqueous phase. This process was repeated until no white precipitate remained at the interface between the two liquids.

[0054] Add 3 times the volume of anhydrous ethanol to the purified straw crude polysaccharide solution, let it stand at 4°C for 24 hours to obtain fermented straw polysaccharide solids. Centrifuge at 4°C, 5000 r / min, discard the supernatant, and freeze-dry the precipitate.

[0055] The activity analysis and polysaccharide composition of fermented straw polysaccharides are shown in Table 1.

[0056] Table 1 Composition of monosaccharides in fermented straw polysaccharides (liquid phase analysis)

[0057] Element External standard method (mg / mL) Relative percentage (%) Mannose 0.122 15.99 Glucosamine 0.084 7.58 Ribose 0.008 1.10 Rhamnose 0.018 2.02 Glucuronic acid 0.018 1.65 Galacturonic acid 0.003 0.40 Galactosamine 0.020 2.08 glucose 0.301 32.07 Galactose 0.105 14.59 Xylose 0.075 11.73 arabinose 0.051 8.99 Fucose 0.012 1.80

[0058] Application Example Preparation of Fermented Straw Polysaccharide Feed

[0059] Formula of fermented straw polysaccharide feed:

[0060] Table 2 Formula of fermented straw polysaccharide feed

[0061]

[0062]

[0063] Gradient fermented straw polysaccharide feed fermented straw polysaccharide addition amount:

[0064] Table 3 Addition amount of fermented straw polysaccharide

[0065] Grouping Addition amount (g / kg) CK 0 SP05 0.5 SP10 1 SP20 2 SP40 4

[0066] Experimental feed production process:

[0067] The feed ingredients are crushed, sieved, weighed, and mixed. The extracted crude polysaccharides from the straw are then added to the base feed in a gradient (from least to most). After mixing thoroughly, oil and water are sprayed on the feed, stirring evenly during the spraying process. Water is sprayed until the feed can be grasped and does not fall apart. The raw materials are then placed in a machine for mechanical extrusion into strips and dried in an oven at 50-70°C for 12-24 hours. The feed is then placed in a pellet mill to produce a palatable, uniformly sized feed for use in breeding experiments.

[0068] Effect Test Example 1 Determination of Antioxidant Related Indexes of Fermented Straw Polysaccharides

[0069] Experimental methods: DPPH free radical scavenging ability, superoxide anion scavenging rate and hydrogen free radical scavenging ability were all tested using the kits of Shanghai Jianglai Biotechnology Co., Ltd.

[0070] Determination of antioxidant related indicators such as Figure 1 The results are as follows: The results of this experimental study showed that the DPPH free radical scavenging ability of fermented straw polysaccharide reached 74.39% at a fermented straw polysaccharide concentration of 0.6 mg / mL, the superoxide anion (ASA) scavenging rate was 33.42%, and the light free radical scavenging capacity (AHR) was 36.83%. This shows that the fermented straw polysaccharide prepared by this method has strong antioxidant capacity.

[0071] Effect Test Example 2 Effects of Fermented Straw Polysaccharide Feed on Growth and Immunity of Grass Carp

[0072] Feeding experiment:

[0073] The feeding experiment was conducted in a temperature-controlled, single-circuit aquaculture system at Jilin Agricultural University's aquarium eco-room. Pre-feeding was performed for 15 days before the start of the feeding experiment. During the feeding experiment, the water temperature was maintained at 25 ± 1°C, ammonia nitrogen < 0.3 mg / L, nitrite < 0.05 mg / L, dissolved oxygen > 5 mg / L, and the pH was controlled between 7.5 and 8.5. Four experimental groups with different concentrations were set up as a control group, with three replicates per group. Over an 8-week feeding experiment, the fish were fed at full capacity twice a day (8:00 AM and 5:00 PM) using hand-spreading. The water was changed every two days, with 1 / 4 to 1 / 3 of the water replaced each time, to maintain fresh water quality. The feeding status of each bucket of fish was recorded daily, and their activity was observed.

[0074] Sample collection:

[0075] After the feeding experiment, the fish were deprived of food for 24 hours, and then live dissection and sample collection were performed on ice trays. The group weight of each bucket of fish was weighed, and 8 fish were randomly selected from each bucket. They were anesthetized with 200 mg / L MS-222. After the fish skin was dried, their body length and weight were measured, and blood was collected from the tail vein. After blood collection, the fish were dissected, and the viscera, hepatopancreas, and intestines were removed and weighed for growth index measurement. Five fish were randomly selected from each bucket, and the hepatopancreas and intestines were quickly removed, quickly frozen in liquid nitrogen, and stored in a -80°C freezer for future use in the measurement of growth and immune-related gene expression.

[0076] Effects of fermented straw polysaccharides on the growth of grass carp

[0077] Calculate the weight gain rate, specific growth rate, and feed coefficient according to the following formula:

[0078] Weight gain rate (WGR, %) = (Wt-W0) / W0×100;

[0079] Specific growth rate (SGR, % / d) = (ln Wt - ln W0) / T × 100;

[0080] Feed coefficient (FCE) = F / (Wt-W0);

[0081] Fullness (CF, g / cm 3 )=Wt / L 3 ×100;

[0082] Intestine index (%) = 100 × intestinal weight (g / tail) / body weight (g / tail);

[0083] Intestinal length index (%) = 100 × intestinal length (cm) / body length (cm).

[0084] Where W0 is the total weight of the fish at the beginning of the experiment (g), Wt is the total weight of the fish at the end of the experiment (g), F is the food intake (g), T is the breeding time (d), and L is the body length of the fish at the end (cm).

[0085] Table 4 Effects of gradient addition of fermented straw polysaccharides on grass carp growth

[0086]

[0087] Table 5 Body parameters of grass carp

[0088]

[0089] Tables 4 and 5 show that when the addition of fermented straw polysaccharide to feed is 2-4 g / kg, the addition of fermented straw polysaccharide has a positive effect on the growth of grass carp.

[0090] pass Figure 2 It is preliminarily inferred that when the fermented straw polysaccharide is added at an amount of 2-3g / kg, it has a promoting effect on the growth of grass carp.

[0091] Aeromonas hydrophila challenge experiment

[0092] Experimental procedures: The pathogen was isolated from enteritis grass carp and preserved in our laboratory. Aeromonas hydrophila was cultured in LB liquid medium at 28°C for 12 h. The medium was removed after centrifugation and the drug was resuspended in sterile saline to 1.0×10 8 Grass carp were infected with 0.2 ml of Aeromonas hydrophila per fish via intraperitoneal injection, while the control group received the same dose of saline. The challenge period was 7 days, with the water temperature and pH at 25 ± 1°C and 7.0 ± 0.4, respectively, and dissolved oxygen controlled at 6.0 mg / L. Infection was performed under natural light.

[0093] Figure 3 Different points represent the average survival rate. Each group was repeated three times (3×10=30 fish). The results showed that when 2 g / kg of fermented straw polysaccharide was added to the feed, the survival rate of grass carp reached 73.33%. It was preliminarily inferred that the addition of fermented straw polysaccharide played a protective role against the infection of grass carp by Aeromonas hydrophila.

[0094] Immune gene expression in the hepatopancreas of grass carp

[0095] Experimental steps: Trizol (Shanghai Shenggong Biotechnology Co., Ltd.) was used to extract hepatopancreatic RNA. Fluorescence quantitative PCR was performed using the One Step TB Green staining method and the One Step TB Green PrimeScript TM RT-PCR Kit II (Takara) was used in the ABI 7500 RT-PCR system.

[0096] The 20 μL reaction system consisted of 10 μL One Step TB Green RT-PCR Buffer 4 (2x), 0.8 μL PrimeScript 1Step Enzyme Mix 2, 0.4 μL ROX reference dye (50x), 2 μL total RNA, 5.2 μL RNaseFree dH2O, and 0.8 μL each of PCR forward and reverse primers.

[0097] All primers were designed by Premier 5.0 software and synthesized by Shanghai Sangon Biotechnology Service Co., Ltd. All target genes were analyzed with β-actin as the internal control. -ΔΔCT The relative gene expression was calculated by the method.

[0098] pass Figure 4 AF analysis showed that the gene expression level of ikkα in the hepatopancreas increased with the increase of fermented straw polysaccharide addition, among which the treatment groups SFP10, SFP20 and SFP40 were significantly higher than that of the CK group (p<0.05); the gene expression level of ikkβ in the hepatopancreas decreased with the increase of fermented straw polysaccharide addition, and the fermented straw polysaccharide addition group was significantly lower than that of the CK group (p<0.05); the gene expression level of ikkγ in the hepatopancreas was significantly increased in the SFP40 group compared with the CK group (p<0.05); the gene expression level of il-6 in the hepatopancreas of the SFP10 and SFP40 groups was significantly higher than that of the CK group (p<0.05); there was no significant difference in the gene expression level of TNF-α in the hepatopancreas among the groups (p>0.05).

[0099] Grass carp enzyme-linked immunosorbent assay

[0100] Experimental Procedure: Weigh an appropriate amount of hepatopancreas and record its exact weight. Add normal saline at a ratio of 1 g tissue specimen to 9 mL of normal saline. Place a 10% tissue homogenate in an ice-water bath and centrifuge at 4000 rpm for 10 minutes at 4°C. Carefully aspirate the supernatant for analysis. Kits were purchased from Changchun Yibo Biotechnology Co., Ltd. and were used to measure immune enzyme activities, including IL-10, IL-1β, complement C3, complement C4, TNF-α, and TGF-β.

[0101] Table 6 ELISA indexes of grass carp hepatopancreas

[0102]

[0103] According to the analysis of enzyme-linked immunosorbent assay indicators of grass carp hepatopancreas in Table 6, the IL-10 concentration in the hepatopancreas of SFP20 and SFP40 groups was significantly reduced (p<0.05); the iL-1β concentration in the hepatopancreas of grass carp increased with the increase of fermented straw polysaccharide content in the feed (p<0.05); the complement C3 content showed a trend of first decreasing and then increasing, and the C3 concentration in the hepatopancreas of SFP05, 10 and 20 groups was significantly lower than that in the CK group and SFP40 group (p<0.05); the complement C4 content in the hepatopancreas of the fermented straw polysaccharide-added group was significantly lower than that in the CK group (p<0.05).

[0104] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for producing straw polysaccharides by microbial fermentation, characterized in that: The following steps are involved: 1) Treating straw by bacterial enzyme method: soybean meal, straw powder and NaOH are mixed and matured, the matured straw powder is enzymolyzed with straw composite enzyme preparation purchased from the market, and the enzymolysis product is sterilized by high pressure and dried for later use; 2) Fermentation: adding enzymatically hydrolyzed straw powder to LB medium, and adding Bacillus cereus and Bacillus tequila for cooperative fermentation; 3) Extraction of polysaccharides from fermented straw.

2. The method for producing straw polysaccharides by microbial fermentation according to claim 1, characterized in that: In the step 1), The straw powder is sieved through 60 mesh; The mass ratio of soybean meal to straw powder added is 1:5; The NaOH concentration is 1.2%-4%, and the mass ratio of the soybean meal straw powder mixture to the NaOH solution is 1:1-1.5; The aging is carried out at room temperature for 24-36 hours.

3. The method for producing straw polysaccharide by microbial fermentation according to claim 1, characterized in that: In the step 2), The preparation of the culture medium comprises the following steps: Weigh tryptone, yeast extract, and sodium chloride according to the proportion of LB medium, add enzymatically hydrolyzed straw powder, add double distilled water to the volume of the medium, adjust the pH to 7.2 with NaOH, and sterilize; The amount of the enzymatically hydrolyzed straw powder added is 50-60 g of straw powder per 1000 mL of culture medium; The strain density of Bacillus cereus and Bacillus tequila is 1.5×10 8 cells / mL, 0.5 mL of bacterial solution was added to every 100 mL of culture medium; The fermentation conditions are: temperature 30° C., rotation speed 160 rpm, and fermentation time 48 h.

4. The method for producing straw polysaccharide by microbial fermentation according to claim 1, characterized in that: In the step 2), the Bacillus cereus is selected from Bacillus cereus LSG-1, which was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on April 20, 2020, with a deposit number of CGMCC No.19595; The Bacillus tequila is Bacillus tequila GCB-3, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 16, 2024, with the deposit number CGMCC No.29629.

5. The method for producing straw polysaccharide by microbial fermentation according to claim 1, characterized in that: In the step 3), the extraction of the fermented straw polysaccharide comprises the following steps: The obtained fermentation liquid is collected and filtered, concentrated by rotary evaporation, and the free crude protein is removed from the concentrate by Savag method; a chloroform-n-butanol solution having a volume of 1 / 4 of the crude polysaccharide solution is added to the crude polysaccharide solution, and after sufficient shaking, the solution is centrifuged to separate the aqueous phase from the chloroform phase; a chloroform-n-butanol solution having a volume of 1 / 4 of the aqueous phase is added to the aqueous phase, and the above process is repeated until there is no white precipitate at the interface of the liquid surface; Add 3 times the volume of anhydrous ethanol to the purified crude straw polysaccharide solution, let it stand at 4°C for 12-24 hours to obtain fermented straw polysaccharide solids; centrifuge at 4°C, 5000r / min to discard the supernatant, and take the precipitate for freeze-drying.

6. The method for producing straw polysaccharide by microbial fermentation as claimed in claim 5, characterized in that: In the step 3), The chloroform-n-butanol is pre-prepared into a mixed solution with a volume ratio of 4:1; The shaking time is 30 min; The centrifugation time is 1 min.

7. The fermented straw polysaccharide prepared by the method for producing microbial fermented straw polysaccharide according to any one of claims 1 to 6, characterized in that: Its monosaccharide composition contains the following percentage components: 15.99% mannose, 7.58% glucosamine, 1.1% ribose, 2.02% rhamnose, 1.65% glucuronic acid, 0.4% galacturonic acid, 2.08% galactosamine, 32.07% glucose, 14.59% galactose, 11.73% xylose, 8.99% arabinose, 1.8% fucose.

8. Use of the fermented straw polysaccharide prepared by the method for preparing the microbial fermented straw polysaccharide according to any one of claims 1 to 6 as an immunopotentiator for fish feed.

9. The use of the fermented straw polysaccharide prepared by the method for preparing the microbial fermented straw polysaccharide according to claim 8 as a fish feed immunopotentiator, characterized in that: The added amount of the fermented straw polysaccharide is 0.5-4g / kg feed; preferably 2-4g / kg; preferably 2-3g / kg; preferably 2g / kg.

10. The use of the fermented straw polysaccharide prepared by the method for preparing the microbial fermented straw polysaccharide according to claim 8 as a fish feed immunopotentiator, characterized in that: The formula of the fish feed, calculated by g / kg addition, comprises the following ingredients: Fish meal 280g / kg, flour 200g / kg, wheat bran 180g / kg, peeled soybean meal 160g / kg, microcrystalline cellulose 80g / kg, fish oil 20g / kg, corn oil 20g / kg, calcium dihydrogen phosphate 20g / kg, 50% choline chloride 15g / kg, 1% compound premix 10g / kg, methionine 10g / kg, lysine 5g / kg, fermented straw polysaccharide 0.5-4g / kg.

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