Lipoteichoic acid from bacillus subtilis and use thereof for maintaining the intestinal health of bullfrogs

Abstract

The present application relates to a kind of bacillus subtilis lipoteichoic acid and its extraction method and application, the extraction method of bacillus subtilis lipoteichoic acid includes: preparation chromatographic column, obtain packed chromatographic column standby;Bacillus subtilis bacterial liquid is added to n-butanol, after stirring uniformly, centrifugal, take middle lower layer water phase, freeze-drying is obtained by vacuum freeze-drying machine with lipoteichoic acid crude extract;The lipoteichoic acid crude extract is dissolved in the equilibrium buffer solution, after centrifugal processing, take supernatant, the supernatant is loaded into the packed chromatographic column, after the supernatant is all entered into the packed chromatographic column, eluent is added to the packed chromatographic column by peristaltic pump, filtrate is collected, and the bacillus subtilis lipoteichoic acid obtained after freeze-drying of the filtrate is obtained.The bacillus subtilis lipoteichoic acid is added to high soybean meal protein feed, can maintain the intestinal health of feeding high soybean meal protein feed bullfrog, improve the feeding effect, with excellent application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for breeding bullfrogs, and in particular to a lipoteichoic acid from Bacillus subtilis and its application. BACKGROUND

[0002] Bullfrogs (Lithobates catesbeianus) have become an important aquaculture species in China due to their fast growth rate, delicious meat, rich nutrition, and strong environmental adaptability. Currently, in the process of bullfrog breeding, plant protein sources have become substitutes for fish meal in order to reduce the amount of fish meal in feed. Soybean meal has become the main plant protein source for replacing fish meal in aquatic animal feed due to its stable supply, low price, high protein content, and relatively balanced essential amino acids. However, it contains various anti-nutritional factors, which can cause intestinal flora imbalance in bullfrogs, leading to soybean meal enteritis in bullfrogs, reducing their growth performance, and thus reducing economic benefits.

[0003] Probiotics can effectively improve the intestinal health of farmed animals and the utilization of soybean meal. Postbiotics also have similar functions to live bacteria. The addition of Bacillus subtilis to bullfrog feed can improve the breeding performance of bullfrogs. However, the high temperature and high pressure conditions during the processing of puffed feed pose a great challenge to the survival of Bacillus subtilis. Lipoteichoic acid is the main component of the cell wall of Bacillus subtilis, which plays an important role in many aspects, including maintaining bacterial structure, participating in bacterial adhesion, promoting host intestinal health, and host-bacterial interactions.

[0004] There are existing technologies for extracting lipoteichoic acid from lactobacillus, such as patent application CN115068510A, which discloses a method for extracting lactobacillus lipoteichoic acid and its anti-inflammatory activity application, including the following steps: (1) extracting lactobacillus by trichloroacetic acid method, Triton X-114 method or n-butanol method to obtain lactobacillus lipoteichoic acid crude body; (2) purifying the lactobacillus lipoteichoic acid crude body by hydrophobic chromatography and anion exchange chromatography to obtain lactobacillus lipoteichoic acid product.

[0005] Lactobacillus lipoteichoic acid stimulates the immune system by regulating the release of related cytokines at the cellular level to exert its anti-inflammatory effect, but lacks data support from in vivo experiments. Due to the lack of in-depth understanding of its specific anti-inflammatory mechanism in the body, it cannot provide support for its application in bullfrog breeding.

[0006] There are also methods for extracting lipoteichoic acid from Clostridium butyricum, for example, patent application CN104161776A discloses a lipoteichoic acid from Clostridium butyricum and its use for regulating the immune response of livestock and poultry, and a TX-114 solution with a mass percentage of 1.5-2.5% is prepared for standby; then 8-16mL of Clostridium butyricum liquid cultured for 45-50h is centrifuged to obtain the wet weight of the bacteria; the TX-114 solution is added and centrifuged to obtain the crude extract of lipoteichoic acid; then the lipoteichoic acid is obtained by column chromatography on a DEAE-cellulose column.

[0007] Clostridium butyricum lipoteichoic acid can regulate the immune function of the intestinal tract of livestock and poultry animals and exert its anti-inflammatory effect. However, bullfrogs are amphibians, and their living environment and body metabolism are quite different from those of livestock and poultry animals, and different strains have different functions, so it is difficult to predict that the effects of lactobacillus lipoteichoic acid and Clostridium butyricum lipoteichoic acid are the same, and therefore, the application of lactobacillus lipoteichoic acid in bullfrog breeding cannot be supported. SUMMARY

[0008] The purpose of the present application is to overcome the problem of frequent intestinal inflammation caused by feeding high-soybean meal content feed in bullfrog breeding, and to provide a Bacillus subtilis lipoteichoic acid and an extraction method thereof.

[0009] Lipoteichoic acid exists in various organisms, and the present application selects Bacillus subtilis as the extraction raw material, because this strain of Bacillus subtilis is a probiotic bacteria native to the intestinal tract of bullfrogs. It has been listed in the "Feed Additive Variety Catalogue (2013)" and can be used as a feed-grade microbial additive. Studies have found that Bacillus subtilis has good tolerance and can colonize in the host's digestive tract, produce various digestive enzymes, consume intestinal oxygen to maintain an anaerobic environment, inhibit the growth of pathogenic bacteria, and have a positive effect on the growth and immunity of the host and the stability of the intestinal flora.

[0010] The difficulty in extracting lipoteichoic acid from Bacillus subtilis lies in maintaining its structural integrity and improving its purity. The present application uses n-butanol-gel chromatography to separate lipoteichoic acid, which can maintain its original structure, improve the purity of the sample, and the extraction yield is greater than 30%.

[0011] The specific scheme is as follows:

[0012] A method for extracting Bacillus subtilis lipoteichoic acid, comprising:

[0013] Preparation of chromatography column: the gel is heated in a water bath, air bubbles are removed, and the treated gel is obtained after cooling to room temperature; the chromatography column is rinsed with the equilibration buffer and added to the specified scale, then the treated gel is poured into the chromatography column and allowed to settle freely, the peristaltic pump is started to add the equilibration buffer to the chromatography column, and when the equilibration buffer is leveled on the surface of the gel, the addition of the equilibration buffer is stopped, and the packed chromatography column is obtained for standby; the equilibration buffer is a mixed solution of n-propanol, ammonium acetate and water;

[0014] Extraction of lipoteichoic acid: the bacterial solution of Bacillus subtilis is added to n-butanol, stirred uniformly, centrifuged, and the middle and lower layer of water phase is taken, and the lipoteichoic acid crude extract is obtained by freeze-drying with a vacuum freeze-drying machine;

[0015] The lipoteichoic acid crude extract is dissolved in the equilibration buffer, and the supernatant is obtained after centrifugal treatment, and the supernatant is loaded into the packed chromatography column, and then the eluent is added to the packed chromatography column through the peristaltic pump, the filtrate is collected, and the Bacillus subtilis lipoteichoic acid is obtained after freeze-drying of the filtrate.

[0016] Further, in the preparation of chromatography column step, the gel is CL-4B gel, preferably, the CL-4B gel is heated in a water bath at 55-65℃ for 10-30min to remove air bubbles, and the treated gel is obtained after cooling to room temperature;

[0017] Preferably, the volume content of n-propanol in the equilibration buffer is 10-20%, the concentration of ammonium acetate is 0.01-0.2M, and the pH of the equilibration buffer is 4.0-5.0;

[0018] Preferably, the size of the chromatography column is (5-15)mm×(20-40cm), preferably 10mm×30cm, the chromatography column is rinsed with the equilibration buffer and added to 0.5-2cm, the gel is poured into the chromatography column and allowed to settle freely to a final height of 5-15cm, then the equilibration buffer is added at a flow rate of 0.05-0.15mL / min using a peristaltic pump, and when the equilibration buffer is leveled on the surface of the gel, the packed chromatography column is obtained for standby.

[0019] Further, in the step of extracting lipoteichoic acid, the volume ratio of the bacterial solution of Bacillus subtilis to n-butanol is 1:1-2;

[0020] Preferably, the centrifugal speed is 5000-10000rpm, and the time is 10-30min;

[0021] Preferably, the eluent is 0.01-0.2M ammonium acetate containing 21-40% n-propanol by volume, and the pH is 4-5.

[0022] Further, the Bacillus subtilis lipoteichoic acid is light brown yellow solid, the extraction yield of the Bacillus subtilis lipoteichoic acid is 33.12±1.58%, n=3.

[0023] Further, the Bacillus subtilis lipoteichoic acid is light brown yellow solid, the infrared spectrum has -NH vibration at 3247 cm -1 , -C=O vibration at 1604 cm -1 , -P=O vibration at 1407 cm -1 , and -C-O vibration at 1098 cm -1 .

[0024] The application also protects the application of the Bacillus subtilis lipoteichoic acid in bullfrog breeding.

[0025] Further, the application is to add the Bacillus subtilis lipoteichoic acid to the expanded feed with soybean meal content of 50-60 wt% in a proportion of 20-35 mg / kg, for breeding bullfrog.

[0026] Further, the effect for breeding bullfrog is at least one of the following (1)-(11):

[0027] (1) the feed efficiency is 0.97±0.03;

[0028] (2) the protein efficiency is 2.31±0.07;

[0029] (3) the serum C3 and C4 content of bullfrog is improved;

[0030] (4) the number of jejunum villus cup cells of bullfrog is improved;

[0031] (5) the jejunum pH and ileum pH of bullfrog are both reduced;

[0032] (6) the jejunum protease and lipase activity level of bullfrog is improved;

[0033] (7) the muc2, muc3 and fut1 gene expression level of bullfrog is improved;

[0034] (8) the mucosa fold height and muscle layer thickness of bullfrog is improved;

[0035] (9) the zo-1 gene expression level of bullfrog is improved;

[0036] (10) the serum DAO activity and ET content of bullfrog is reduced;

[0037] (11) the jak2, stat3 and mek1 / 2 gene expression level of bullfrog is improved.

[0038] The present application also protects a bullfrog feed comprising the Bacillus subtilis lipoteichoic acid.

[0039] Further, the bullfrog feed is an expanded feed, the crude protein content is 40-42wt%, the crude fat content is 8-9wt%, the total calcium content is 0.4-0.5wt%, and the total phosphorus content is 0.7-0.8wt%; the content of soybean meal in the expanded feed is greater than or equal to 50wt%, the Bacillus subtilis lipoteichoic acid is added into the bullfrog feed in a proportion of 20-35mg / kg, and the preparation method is as follows:

[0040] After the raw materials are crushed and sieved, the crushed raw materials are uniformly mixed according to the proportion, squid paste and distilled water are added, the double-screw extruder is used to prepare the expanded feed, the expanded feed is dried at 55°C for 6h, and the crude product is obtained for standby use;

[0041] The Bacillus subtilis lipoteichoic acid is uniformly mixed with sterile distilled water, uniformly sprayed on the surface of the crude product, uniformly stirred, blown dry at 30-35°C, then soybean oil and fish oil are added, uniformly mixed, and then air-dried in a cool place to obtain the bullfrog feed.

[0042] Beneficial effects:

[0043] In the present application, the Bacillus subtilis (LCBS1) lipoteichoic acid is added into the high-soybean-meal-protein feed, which can improve the growth performance, feed utilization rate and immunity of bullfrogs, promote intestinal cell proliferation, relieve soybean meal enteritis, and make the digestive tract tissue structure more complete and healthy. The Bacillus subtilis (LCBS1) lipoteichoic acid shows good probiotic effect on bullfrogs. BRIEF DESCRIPTION OF DRAWINGS

[0044] In order to more clearly illustrate the technical solutions of the present application, the following will briefly introduce the drawings. Obviously, the drawings described below only relate to some embodiments of the present application, rather than limiting the present application.

[0045] Figure 1 is a lipoteichoic acid extract photo provided by an embodiment of the present application;

[0046] Figure 2 is an infrared spectrum diagram of a lipoteichoic acid sample provided by an embodiment of the present application;

[0047] Figure 3 is a jejunum PAS staining diagram provided by an embodiment of the present application;

[0048] Figure 4 is an expression diagram of jejunum mucin and fucosyltransferase genes provided by an embodiment of the present application;

[0049] Figure 5is a HE staining image of the jejunum provided by one embodiment of the present application;

[0050] Figure 6 is an expression image of the tight junction protein gene provided by one embodiment of the present application;

[0051] Figure 7 is an expression image of the cell proliferation gene provided by one embodiment of the present application.

[0052] Note: Figure 5 The arrow in the middle indicates that the lamina propria is separated from the mucosa, and the triangle indicates that the submucosa is separated from the muscularis. DETAILED DESCRIPTION

[0053] The preferred embodiments of the present application will be described in more detail below. Although the preferred embodiments of the present application are described below, it is understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. If a specific technique or condition is not specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained commercially. In the following examples, unless otherwise specified, "%" means weight percent, and "parts" means weight parts.

[0054] Example 1 Extraction of Lipoteichoic Acid from Bacillus subtilis LCBS1

[0055] Chromatography column packing: CL-4B gel was placed in a water bath at 60°C for 30 min to remove air bubbles, and then cooled to room temperature. A 10 mm x 30 cm chromatography column was rinsed with equilibration buffer (0.1 M ammonium acetate containing 15% n-propanol, pH 4.7) and added to 1 cm. The CL-4B gel was poured into the chromatography column and allowed to settle freely (the final height was about 10 cm). The equilibration buffer was added at a flow rate of 0.1 mL / min using a peristaltic pump. When the equilibration buffer was leveled on the surface of the gel, the sample was added for purification.

[0056] Extraction of lipoteichoic acid: 8 x 10 7Add an equal volume of n-butanol to the solution (CFU / mL), place in a shaker, and stir at 21°C for 1 h (120 rpm). Then centrifuge at 8000 rpm for 20 min, collect the middle and lower aqueous phase, and freeze-dry using a vacuum freeze dryer to obtain crude lipoteichoic acid extract. Dissolve the crude extract in 3 mL of equilibration buffer, centrifuge at 8000 rpm for 20 min, and load the supernatant at a constant flow rate of 0.2 mL / min. After all the sample has entered the gel, add 1 column volume of elution buffer (0.1 M ammonium acetate containing 30% n-propanol, pH 4.7), and continuously elute using a peristaltic pump at a flow rate of 0.8 mL / min, keeping the eluent sufficient. Collect the filtrate. The lipoteichoic acid (LTA) obtained after lyophilizing the filtrate is a pale brownish-yellow solid. Figure 1 As shown.

[0057] The yield of lipoteichoic acid from Bacillus subtilis is calculated using the following formula:

[0058] Yield (%) = (Dry weight of lipoteichoic acid / Dry weight of bacterial cells) × 100%

[0059] The yield of lipoteichoic acid was calculated to be 33.12 ± 1.58% (n = 3).

[0060] Example 2: Extraction of lipoteichoic acid from Bacillus subtilis LCBS1

[0061] Column packing: Incubate CL-4B gel in a water bath at 65°C for 30 minutes to remove air bubbles, then cool to room temperature. Rinse the 10mm × 30cm column with equilibration buffer (0.1M ammonium acetate containing 10% n-propanol, pH = 4-5) to a depth of 1.5cm. Pour CL-4B gel into the column and allow it to settle freely (to a final height of approximately 12cm). Add equilibration buffer using a peristaltic pump at a flow rate of 0.15mL / min. Once the equilibration buffer level is even with the gel surface, add the sample for purification.

[0062] Extraction of lipoteichoic acid: Take 8×10⁻⁶ bacterial suspension of Bacillus subtilis LCBS1. 7 Add an equal volume of n-butanol to the solution (CFU / mL), place in a shaker, and stir at 21°C for 1 h (120 rpm). Then centrifuge at 5000 rpm for 30 min, collect the middle and lower aqueous phase, and freeze-dry using a vacuum freeze dryer to obtain crude lipoteichoic acid extract. Dissolve the crude extract in 3 mL of equilibration buffer, centrifuge at 5000 rpm for 30 min, and load the supernatant at a constant flow rate of 0.2 mL / min. After all the sample has entered the gel, add 1 column volume of elution buffer (0.05 M ammonium acetate containing 25% n-propanol, pH = 4-5). Elute continuously using a peristaltic pump at a flow rate of 0.12 mL / min, ensuring sufficient eluent, and collect the filtrate. The lipoteichoic acid (LTA) obtained after lyophilizing the filtrate is a pale brownish-yellow solid.

[0063] Example 3 Extraction of Lipoteichoic acid from Bacillus subtilis LCBS1

[0064] Chromatography column packing: CL-4B gel was placed in a water bath at 55°C for 30 minutes to remove air bubbles, and then cooled to room temperature. A 10 mm x 30 cm chromatography column was rinsed with equilibration buffer (0.1 M ammonium acetate containing 15% n-propanol, pH 4.7) and added to 2 cm. The CL-4B gel was poured into the chromatography column and allowed to settle freely (the final height was about 15 cm). The equilibration buffer was added at a flow rate of 0.1 mL / min using a peristaltic pump. When the equilibration buffer was leveled on the surface of the gel, the sample was added for purification.

[0065] Extraction of Lipoteichoic acid: 8 x 10 7 CFU / mL of Bacillus subtilis LCBS1 was added with an equal volume of n-butanol, and placed in a shaker for 1 h (120 rpm) at 21°C. Then, centrifugation was performed at 10,000 rpm for 10 min, and the middle and lower water phases were collected. The crude Lipoteichoic acid extract was obtained by freeze-drying using a vacuum freeze dryer. The crude extract was dissolved in 3 mL of equilibration buffer, centrifuged at 10,000 rpm for 10 min, and the supernatant was loaded at a constant flow rate of 0.2 mL / min. After the sample was completely introduced into the gel, 1 times the column volume of elution buffer (0.12 M ammonium acetate containing 40% n-propanol, pH 4-5) was added. The elution was continued at a flow rate of 0.10 mL / min using a peristaltic pump, and the eluate was maintained while the filtrate was collected. The Lipoteichoic acid (LTA) obtained by freeze-drying the filtrate was a light brown-yellow solid.

[0066] Example 4 Identification of Lipoteichoic acid from Bacillus subtilis

[0067] The sample prepared in Example 1 was analyzed by infrared spectroscopy. 1 mg of the sample prepared in Example 1 was thoroughly mixed with 100 mg of potassium bromide, and then pressed into a tablet. The Fourier transform infrared spectrometer was used to analyze the key functional groups of the sample immediately.

[0068] The infrared spectrum of the sample prepared in Example 1 is shown in FIG. 1. Figure 2 , Figure 2 The -NH vibration is at 3247 cm -1 The -C=O vibration is at 1604 cm -1 The -P=O vibration is at 1407 cm -1 The -C–O vibration is at 1098 cm -1 There are multiple characteristic absorption peaks of Lipoteichoic acid, indicating that the extract contains substances with glycerophosphate structure, amide, and sugar groups, etc. It is proved that the sample prepared in Example 1 is Lipoteichoic acid.

[0069] Example 5 Preparation of expanded feed

[0070] The soybean meal was used as the main protein source, and fish oil and soybean oil were used as the main fat source to prepare high-soybean meal feed with 41.90% crude protein and 8.8% crude fat. The raw material was ground through a 60-mesh screen. The raw materials were mixed according to the ratio, and squid paste and distilled water were added. The mixture was extruded into a diameter of 4.0 mm by a twin-screw extruder (TSE65). The feed was dried at 55°C for 6 h and then stored in a refrigerator at -20°C.

[0071] Table 1 Formulation and nutritional composition of high-soybean meal feed (% dry matter)

[0072]

[0073]

[0074] In this example, the control group (CG) and the lipoteichoic acid group (LTA) were designed for the extruded feed. According to the conversion of yield, the experimental group was added with the same amount of lipoteichoic acid as 8.0 x 10 7 CFU / mL, i.e. 29.07 mg / kg. The control group was not added with lipoteichoic acid.

[0075] Lipoteichoic acid group (LTA): Lipoteichoic acid was mixed with an appropriate amount of sterile distilled water, and then evenly sprayed on the surface of the extruded feed. After stirring, the mixture was blown dry at 30°C. Soybean oil and fish oil were added and mixed evenly, and then air-dried in a cool place. The feed was packed in sealed bags and stored at -20°C for later use.

[0076] Control group (CG): An appropriate amount of sterile distilled water was evenly sprayed on the surface of the extruded feed, and then stirred evenly. The mixture was blown dry at 30°C. The same amount of soybean oil and fish oil as the lipoteichoic acid group (LTA) was added, and then mixed evenly and air-dried in a cool place. The feed was packed in sealed bags and stored at -20°C for later use.

[0077] Example 6 Cultivation experiment

[0078] The experiment was conducted at the aquaculture test site of Jimei University. The bullfrogs were the same batch of tadpoles, which were temporarily raised in aquaculture barrels with a water depth of 5 cm (π x 80 2 x 90 cm). During this period, commercial feed was fed to the bullfrogs to adapt to the aquaculture environment. After 2 weeks of temporary cultivation, 78 healthy young frogs (initial weight: 36.40 ± 0.06 g) were randomly transferred to 6 aquaculture barrels (π x 30 2 x 90 cm) with a water depth of 5 cm. Each group had 3 replicates, with 13 frogs in each replicate. The bullfrogs were fed twice a day at 8:00 and 17:00, and the residual feed was collected and the water was changed 30 min later. The feeding and death of the bullfrogs in each aquaculture barrel were recorded, and the experimental period was 56 days. The water temperature was 29-33°C.

[0079] The experimental results are as follows:

[0080] (1) Growth performance and feed utilization

[0081] As shown in Table 2, after 56 days of cultivation, the average weight, weight gain rate, feed efficiency and protein efficiency of the LTA group were significantly higher than those of the control group (P <0.05). There was no significant difference in feed intake rate, survival rate, liver body ratio and leg body ratio between the two groups (P >0.05).

[0082] From the above data, it can be concluded that the addition of LTA in high soybean meal feed can promote the growth performance of bullfrog and improve the utilization of feed by bullfrog.

[0083] Table 2 Effect of experimental feed on the growth and biological characteristics of bullfrog

[0084]

[0085] Note: The means with different superscripts in the same row are significantly different (P≤0.05), and the following table is the same.

[0086] (2) Apparent digestibility of feed nutrients by bullfrog

[0087] The apparent digestibility of experimental feed by bullfrog is shown in Table 3. The apparent digestibility of crude protein, calcium and phosphorus in the LTA group was significantly higher than that in the control group (P <0.05). There was no significant difference in the apparent digestibility of dry matter between the two groups (P >0.05), which indicated that LTA could improve the digestibility of high soybean meal feed by bullfrog and promote the utilization of soybean meal protein.

[0088] Table 3 Effect of experimental feed on the apparent digestibility of nutrients in feed by bullfrog (%)

[0089]

[0090] (3) Serum immunity of bullfrog

[0091] As shown in Table 4, the serum C3 and C4 contents of the LTA group were significantly higher than those of the control group (P <0.05). There was no significant difference in serum AKP activity between the two groups (P >0.05). This indicates that LTA can improve the serum immunity of bullfrog.

[0092] Table 4 Effect of experimental feed on the serum immunity of bullfrog

[0093]

[0094] (4) Intestinal chemical barrier of bullfrog

[0095] As Figure 3As shown, the LTA group had a higher number of goblet cells. Quantitative analysis revealed that the number of goblet cells per villus in the LTA group was significantly higher than that in the control group (Table 5) (P<0.05). Furthermore, the jejunal and ileal pH of the LTA group were significantly lower than those of the control group. Conversely, the jejunal protease and lipase activities, as well as the expression levels of muc2, muc3, and fut1 genes, were significantly higher in the LTA group than in the control group (Table 5 and 1). Figure 4 (P<0.05). These results indicate that the addition of LTA to a high soybean meal diet can improve the intestinal chemical barrier function of bullfrogs, promote the secretion of intestinal mucus, and maintain intestinal health.

[0096] Table 5. Effects of experimental diets on the chemical barrier of the jejunum in bullfrogs.

[0097]

[0098] (5) Physical barrier of bullfrog intestine

[0099] like Figure 5 As shown, in the control group, the jejunal mucosal folds of bullfrogs were atrophied, villi were sparse, the muscle layer was narrowed, the lamina propria was separated from the mucosa, and the submucosa was separated from the muscle layer, exhibiting typical characteristics associated with soybean meal-type enteritis. Conversely, the addition of LTA to a high soybean meal diet could reduce jejunal mucosal damage in bullfrogs and alleviate the symptoms of soybean meal-type enteritis.

[0100] Quantitative analysis of tissue sections also revealed (see Table 6) that the mucosal fold height and muscle layer thickness in the LTA group were significantly higher than those in the control group (P < 0.05). Among the intestinal permeability-related indicators, serum DAO activity and ET content in the LTA group were significantly lower than those in the control group (P < 0.05).

[0101] Meanwhile, the expression level of zo-1 in the LTA group was significantly higher than that in the control group (see...). Figure 6 (P < 0.05).

[0102] Therefore, adding LTA to high soybean meal protein feed can alleviate soybean meal-induced intestinal damage and reduce intestinal permeability.

[0103] Table 6. Effects of experimental diets on the jejunal tissue structure of bullfrogs

[0104]

[0105] (6) Cell proliferation genes

[0106] As shown in Figure 7, the expression levels of jak2, stat3, and mek1 / 2 in the LTA group were significantly higher than those in the control group (P<0.05). The expression level of e2f4 in the LTA group showed an upregulation trend, but the difference was not significant (P>0.05).

[0107] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details of the above-described embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0108] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, various possible combinations are not described again by the present application.

[0109] In addition, various different embodiments of the present application can also be combined in any manner, as long as it does not deviate from the idea of the present application, and it should also be considered as disclosed by the present application.

Claims

1. Use of Bacillus subtilis lipoteichoic acid in the preparation of high soybean meal feed for bullfrogs, characterized in that: The feed is an expanded feed, the crude protein content is 40-42wt%, the crude fat content is 8-9wt%, the total calcium content is 0.4-0.5wt%, and the total phosphorus content is 0.7-0.8wt%; the content of soybean meal in the feed is greater than or equal to 50wt%, and the bacillus subtilis lipoteichoic acid is added into the feed in a proportion of 20-35mg / kg, and the effect for breeding bullfrogs is that the number of jejunum villus cup cells of the bullfrogs is increased; the jejunum pH and ileum pH of the bullfrogs are both reduced; the jejunum protease and lipase activity levels of the bullfrogs are increased; the jejunum Muc2, Muc3 and Fut1 gene expression level of the bullfrogs is increased; the mucosal fold height and muscle layer thickness of the bullfrogs are increased; the Zo-1 gene expression level of the bullfrogs is increased; the serum DAO activity and ET content of the bullfrogs are reduced.

2. Use according to claim 1, characterized in that: The extraction method of Bacillus subtilis lipoteichoic acid comprises the following steps: preparing a chromatographic column; heating a gel in a water bath, removing air bubbles, and cooling to room temperature to obtain a treated gel; rinsing the chromatographic column with an equilibration buffer and adding it to a specified scale, then pouring the treated gel into the chromatographic column for free settling, starting a peristaltic pump to add the equilibration buffer to the chromatographic column, and stopping adding the equilibration buffer when the equilibration buffer is leveled on the surface of the gel to obtain a packed chromatographic column for standby use; the equilibration buffer is a mixed solution of n-propanol, ammonium acetate and water; Extracting lipoteichoic acid: adding Bacillus subtilis bacterial solution to n-butanol, stirring uniformly, centrifuging, taking the middle and lower layer of water phase, and freeze-drying with a vacuum freeze dryer to obtain a crude extract of lipoteichoic acid; Dissolving the crude extract of lipoteichoic acid in the equilibration buffer, taking the supernatant after centrifugal treatment, and loading the supernatant into the packed chromatographic column; after the supernatant enters the packed chromatographic column, adding an eluent to the packed chromatographic column through a peristaltic pump, collecting the filtrate, and freeze-drying the filtrate to obtain Bacillus subtilis lipoteichoic acid.

3. Use according to claim 2, characterized in that: In the step of preparing a chromatographic column, the gel is CL-4B gel.

4. Use according to claim 3, characterized in that: The CL-4B gel is heated in a water bath at 55-65 ℃ for 10-30 min to remove air bubbles, and then cooled to room temperature to obtain a treated gel.

5. Use according to claim 3, characterized in that: The volume content of n-propanol in the equilibration buffer is 10-20%, the concentration of ammonium acetate is 0.01-0.2 M, and the pH of the equilibration buffer is 4.0-5.

0.

6. Use according to claim 3, characterized in that: The size of the chromatographic column is 5-15 mm × 20-40 cm, the chromatographic column is rinsed with the equilibration buffer and added to 0.5-2 cm, the gel is poured into the chromatographic column for free settling to a final height of 5-15 cm, then the equilibration buffer is added at a flow rate of 0.05-0.15 mL / min using a peristaltic pump, and the packed chromatographic column is obtained for standby use when the equilibration buffer is leveled on the surface of the gel.

7. Use according to claim 6, characterized in that: The size of the chromatographic column is 10 mm × 30 cm.

8. Use according to claim 2, characterized in that: In the step of extracting lipoteichoic acid, the volume ratio of Bacillus subtilis bacterial solution to n-butanol is 1:1-2.

9. Use according to claim 8, characterized in that: The centrifugal speed is 5000-10000 rpm, and the time is 10-30 min.

10. Use according to claim 8, characterized in that: The eluent is 0.01-0.2 M ammonium acetate containing 21-40% n-propanol by volume, and the pH is 4-5.

11. Use according to claim 1, characterized in that: The Bacillus subtilis lipoteichoic acid is added to an expanded feed with a soybean meal content of 50-60 wt% at a ratio of 20-35 mg / kg for breeding bullfrogs.

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