Composition for enhancing glycometabolism of micropterus salmoides by targeting IRE1 gene as well as preparation method and application of composition

Abstract

The invention relates to the technical field of aquaculture, in particular to a composition for enhancing glycometabolism of micropterus salmoides by targeting an I RE1 gene, a preparation method and application. The composition can inhibit the expression of the I RE1 gene, improve the endoplasmic reticulum function of the micropterus salmoides fed with the high-sugar feed and improve the sugar metabolism capability of fish bodies.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, and in particular to a composition, a preparation method and application for enhancing sugar metabolism of largemouth bass by targeting IRE1 gene. Background Art

[0002] As an important part of my country's agriculture, aquaculture plays an important role in ensuring food security and increasing farmers' income. With the increase of environmental awareness, how to reduce the impact of aquaculture on the environment and achieve green farming has become an important issue. Sugar is an important source of energy for organisms. Using sugar in feed can reduce dependence on animal protein, improve feed utilization, and significantly reduce feed costs. In addition, sugar mainly comes from plants, which can be continuously regenerated through agricultural planting. Therefore, using sugar in feed can improve the economic benefits and environmental sustainability of farming.

[0003] The sugar metabolism mechanism of fish is significantly different from that of humans and terrestrial animals. Excessive addition of sugars in feed can lead to low disease resistance, slow growth, and high mortality in fish, which makes the utilization rate of sugars in feed by fish relatively low. Therefore, the development of regulators to improve sugar metabolism in fish has significant practical value in production.

[0004] Endoplasmic reticulum stress is considered to be an important mechanism for the occurrence and development of diabetes. Hyperglycemia can cause misfolded and unfolded protein aggregation in the endoplasmic reticulum of pancreatic islet cells and Ca2+ metabolism disorders, triggering an endoplasmic reticulum stress response, leading to islet cell apoptosis, thereby reducing insulin synthesis and secretion, causing the occurrence of diabetes. Inositol-requiring enzyme 1 (IRE1) is a key endoplasmic reticulum stress sensor and signal sensor. Hyperglycemia can activate it and cause endoplasmic reticulum stress, exacerbating diabetic symptoms. At present, a small number of Western medicines have been developed for IRE1 to improve the function of the endoplasmic reticulum and enhance the body's glucose metabolism. However, metabolic regulators developed around IRE1 have not yet been seen in fish. Summary of the invention

[0005] In order to solve the problems in the above-mentioned background technology, the first object of the present invention is to provide a composition for targeting the I RE1 gene to enhance the sugar metabolism of largemouth bass, which can inhibit the expression of the I RE1 gene, improve the endoplasmic reticulum function of largemouth bass fed with high-sugar feed, and improve the sugar metabolism ability of fish.

[0006] To achieve the above purpose, the solution adopted by the present invention to solve the technical problem is: a composition for targeting I RE1 gene to enhance sugar metabolism of largemouth bass, the composition comprises the following raw materials in parts by weight: 1 to 15 parts of allium flavonoids; 3 to 16 parts of claviculinamide; 4 to 31 parts of cucurbitacin B;

[0007] The preparation method of the allium flavonoids comprises the following steps:

[0008] S1, washing the shallot with distilled water, drying it at 50-75°C, and then crushing it, taking 10g of the crushed shallot and adding it to 10-50mL of Mandel salt solution;

[0009] S2, mixing Bacillus amyloliquefaciens and cellulase uniformly to obtain a bacterial enzyme mixture, wherein the mass ratio of Bacillus amyloliquefaciens to cellulase is (1-10):(1-7);

[0010] S3, mixing the bacterial enzyme mixture with the Mandel salt solution prepared in S1, the mass ratio of the bacterial enzyme mixture to the Mandel salt solution is 1g:2mL; obtaining a culture solution, adjusting the pH of the culture solution to 7.0-9.0, and then placing the culture solution in a constant temperature incubator at 35°C-55°C for 24-48h to obtain a bacterial enzyme fermentation solution;

[0011] S4, centrifuging the bacterial enzyme fermentation liquid at a speed of 1000-4000 r / min for 15-60 min, taking the supernatant, and rotary evaporating the supernatant at 85-105° C. to obtain onion flavonoids.

[0012] Preferably, the composition comprises the following raw materials in parts by weight: 4 to 12 parts of allium flavonoids; 5 to 13 parts of clavicinamide; and 8 to 28 parts of cucurbitacin B.

[0013] Preferably, the composition comprises the following raw materials in parts by weight: 8 to 10 parts of allium flavonoids; 7 to 10 parts of clavicinamide; and 15 to 20 parts of cucurbitacin B.

[0014] Preferably, the mass ratio of Bacillus amyloliquefaciens to cellulase in the bacterial enzyme mixed solution in step S2 is (2.5-3):(1-2.5).

[0015] The second object of the present invention is to provide a method for preparing a composition for enhancing sugar metabolism of largemouth bass by targeting I RE1 gene, which has low preparation cost and stable product performance.

[0016] The method comprises the following steps: crushing androchelidon flavonoids, chrysanthemum amide and cucurbitacin B respectively, the temperature during the crushing process is less than 40 DEG C, and the crushing is carried out to 80-150 meshes.

[0017] Preferably, allium flavonoids, chrysanthemum amide and cucurbitacin B are all crushed to 120-150 mesh.

[0018] The third object of the present invention is to provide a feed for enhancing sugar metabolism of largemouth bass, comprising the above composition.

[0019] Preferably, the mass percentage of the composition in the feed is 0.2-1.2.

[0020] The fourth object of the present invention is to provide a feed for use in preparing a product for enhancing sugar metabolism of largemouth bass.

[0021] The fifth object of the present invention is to provide a composition for use in preparing a product for inhibiting IRE1 gene expression.

[0022] The composition is added into other products to significantly reduce product costs and improve the economic benefits of largemouth bass breeding.

[0023] In summary, the beneficial effects of the present invention are:

[0024] The present application combines enzyme-bacteria combined fermentation technology to produce androstenone, and develops an endoplasmic reticulum composition that enhances sugar metabolism in largemouth bass based on the pharmacological synergistic relationship between androstenone, chrysanthemum amide and cucurbitacin B.

[0025] The composition can select IRE1 as the target and inhibit the expression of the gene, thereby improving the sugar metabolism ability of the fish and increasing the feed utilization rate of the fish.

[0026] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the following specifically cites a preferred embodiment and describes it in detail with the accompanying drawings as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The effect of the composition on IRE1 gene in hepatocytes of largemouth bass;

[0028] Figure 2 The effect of the composition on Calreticulin gene in hepatocytes of largemouth bass;

[0029] Figure 3 The effect of the composition on GRP78 gene in hepatocytes of largemouth bass;

[0030] Figure 4 This is the effect of the composition on the endoplasmic reticulum ratio of hepatocytes of largemouth bass. DETAILED DESCRIPTION

[0031] In order to make the content of the present invention more clearly understood, the present invention is further described below based on specific embodiments in conjunction with the accompanying drawings.

[0032] The preparation method of the Mandel salt solution of this embodiment is:

[0033] S1, prepare constant element solution: add 2.0g K2HPO4, 1.4g (NH4)2SO4, 0.3g MgSO4·7H2O, 0.3g CaCl2 into 600mL double distilled water (water distilled at least twice), and stir until dissolved;

[0034] S2, prepare trace element solution: add 5.0 mg FeSO4·7H2O, 1.6 mg MnSO4·H2O, 1.4 mg ZnSO4·7H2O, and 2.0 mg CoCl2 into 200 mL of double distilled water and stir until dissolved;

[0035] S3, the major element solution and the trace element solution are mixed evenly, the pH is adjusted to 5.5-6.0 with HCl, and then the volume is fixed to 1000 mL with double distilled water to obtain a Mandel salt solution.

[0036] The chrysanthemum amide of the present embodiment is an amide organic compound extracted from the leaves of the chrysanthemum cultivar; cucurbitacin B is a tetracyclic triterpenoid compound present in the pedicel of melon.

[0037] 1. Preparation of Compositions 1 to 8

[0038] Preparation method of composition 1

[0039] S1, washing the male onion with distilled water, then drying it at 50°C, then crushing the dried male onion until it can pass through an 80-mesh sieve, taking 10 g of the crushed male onion and adding it to 10 mL of Mandel salt solution;

[0040] S2, mixing Bacillus amyloliquefaciens and cellulase evenly to obtain a bacterial enzyme mixture, wherein the mass ratio of Bacillus amyloliquefaciens to cellulase is 1:1;

[0041] S3, mixing the bacterial enzyme mixture and the Mandel salt solution prepared in S1, the mass of the bacterial enzyme mixture is 1 g, the volume of the Mandel salt solution is 2.5 mL, to obtain a culture solution, and then adjusting the pH of the culture solution to 7.0, and then placing the culture solution in a constant temperature incubator at 35° C. and culturing for 24 hours to obtain a bacterial enzyme fermentation solution;

[0042] S4, centrifuging the fermentation liquid at a speed of 1000 r / min for 15 min, taking the supernatant, and rotary evaporating the supernatant at 85° C. to obtain onion flavonoids.

[0043] S5, crush the onion flavonoids, chrysanthemum amide and cucurbitacin B respectively until they can pass through an 80-mesh sieve, and the temperature during the crushing process is less than 40°C. Then, take 1g of onion flavonoids powder, 3g of chrysanthemum amide powder and 31g of cucurbitacin B powder, mix them thoroughly and evenly to obtain 35g of composition one.

[0044] Preparation method of composition 2

[0045] S1, washing the male onion with distilled water, then drying it at 55°C, then crushing the dried male onion until it can pass through an 80-mesh sieve, taking 10g of the crushed male onion and adding it to 15mL of Mandel salt solution;

[0046] S2, mixing Bacillus amyloliquefaciens and cellulase evenly to obtain a bacterial enzyme mixture, wherein the mass ratio of Bacillus amyloliquefaciens to cellulase is 1:2;

[0047] S3, uniformly mixing the bacterial enzyme mixture with the Mandel salt solution prepared in S1, wherein the mass of the bacterial enzyme mixture is 1 g, and the volume of the Mandel salt solution is 3 mL, to obtain a culture solution, and then adjusting the pH of the culture solution to 7.2, and then placing the culture solution in a constant temperature incubator at 40° C. and culturing for 24 hours to obtain a bacterial enzyme fermentation solution;

[0048] S4, centrifuging the fermentation liquid at a speed of 1200 r / min for 20 min, taking the supernatant, and rotary evaporating the supernatant at 85° C. to obtain onion flavonoids.

[0049] S5, crush the onion flavonoids, chrysanthemum amide and cucurbitacin B respectively until they can pass through a 120-mesh sieve, and the temperature during the crushing process is less than 40°C. Then, take 3g of onion flavonoids powder, 5g of chrysanthemum amide powder and 27g of cucurbitacin B powder, mix them thoroughly and evenly to obtain 35g of composition 2.

[0050] Preparation method of composition 3

[0051] S1, washing the male onion with distilled water, then drying it at 60°C, then crushing the dried male onion until it can pass through an 80-mesh sieve, taking 10 g of the crushed male onion and adding it to 20 mL of Mandel salt solution;

[0052] S2, mixing Bacillus amyloliquefaciens and cellulase evenly to obtain a bacterial enzyme mixture, wherein the mass ratio of Bacillus amyloliquefaciens to cellulase is 3:2;

[0053] S3, uniformly mixing the bacterial enzyme mixture with the Mandel salt solution prepared in S1, wherein the mass of the bacterial enzyme mixture is 2 g, and the volume of the Mandel salt solution is 3 mL, to obtain a culture solution, and then adjusting the pH of the culture solution to 7.5, and then placing the culture solution in a constant temperature incubator at 45° C. and culturing for 24 hours to obtain a bacterial enzyme fermentation solution;

[0054] S4, centrifuging the fermentation liquid at a speed of 1500 r / min for 30 min, taking the supernatant, and rotary evaporating the supernatant at 90° C. to obtain onion flavonoids.

[0055] S5, crush the onion flavonoids, chrysanthemum amide and cucurbitacin B respectively until they can pass through a 120-mesh sieve, and the temperature during the crushing process is less than 40°C. Then, take 5g of onion flavonoids powder, 8g of chrysanthemum amide powder and 22g of cucurbitacin B powder, mix them thoroughly and evenly to obtain 35g of composition 3.

[0056] Preparation method of composition 4

[0057] S1, washing the male onion with distilled water, then drying it at 65°C, then crushing the dried male onion until it can pass through a 100-mesh sieve, taking 10 g of the crushed male onion and adding it to 25 mL of Mandel salt solution;

[0058] S2, mixing Bacillus amyloliquefaciens and cellulase evenly to obtain a bacterial enzyme mixture, wherein the mass ratio of Bacillus amyloliquefaciens to cellulase is 3:5;

[0059] S3, uniformly mixing the bacterial enzyme mixture with the Mandel salt solution prepared in S1, wherein the mass of the bacterial enzyme mixture is 2 g, and the volume of the Mandel salt solution is 3 mL, to obtain a culture solution, and then adjusting the pH of the culture solution to 8.0, and then placing the culture solution in a constant temperature incubator at 45° C. and culturing for 36 hours to obtain a bacterial enzyme fermentation solution;

[0060] S4, centrifuging the bacterial enzyme fermentation liquid at a speed of 2000r / min for 30min, taking the supernatant, and rotary evaporating the supernatant at 95°C to obtain onion flavonoids.

[0061] S5, crush the onion flavonoids, chrysanthemum amide and cucurbitacin B respectively until they can pass through a 120-mesh sieve, and the temperature during the crushing process is less than 40°C. Then, take 8g of onion flavonoids powder, 10g of chrysanthemum amide powder and 17g of cucurbitacin B powder, mix them thoroughly and evenly to obtain 35g of composition 4.

[0062] Preparation method of composition 5

[0063] S1, washing the male onion with distilled water, then drying it at 65°C, then crushing the dried male onion until it can pass through a 120-mesh sieve, taking 10 g of the crushed male onion and adding it to 30 mL of Mandel salt solution;

[0064] S2, mixing Bacillus amyloliquefaciens and cellulase evenly to obtain a bacterial enzyme mixture, wherein the mass ratio of Bacillus amyloliquefaciens to cellulase is 7:5;

[0065] S3, uniformly mixing the bacterial enzyme mixture with the Mandel salt solution prepared in S1, wherein the mass of the bacterial enzyme mixture is 2 g, and the volume of the Mandel salt solution is 3.5 mL, to obtain a culture solution, and then adjusting the pH of the culture solution to 8.5, and then placing the culture solution in a constant temperature incubator at 50° C. and culturing for 36 hours to obtain a bacterial enzyme fermentation solution;

[0066] S4, centrifuging the fermentation liquid at a speed of 2500 r / min for 30 min, taking the supernatant, and rotary evaporating the supernatant at 95° C. to obtain onion flavonoids.

[0067] S5, crush the onion flavonoids, chrysanthemum amide and cucurbitacin B respectively until they can pass through a 120-mesh sieve, and the temperature during the crushing process is less than 40°C. Then, take 10g of the onion flavonoids powder, 12g of the chrysanthemum amide powder and 13g of the cucurbitacin B powder, mix them thoroughly and evenly to obtain 35g of composition 5.

[0068] Preparation method of composition 6

[0069] S1, washing the male onion with distilled water, then drying it at 70°C, then crushing the dried male onion until it can pass through a 120-mesh sieve, taking 10 g of the crushed male onion and adding it to 30 mL of Mandel salt solution;

[0070] S2, mixing Bacillus amyloliquefaciens and cellulase evenly to obtain a bacterial enzyme mixture, wherein the mass ratio of Bacillus amyloliquefaciens to cellulase is 8:5;

[0071] S3, uniformly mixing the bacterial enzyme mixture with the Mandel salt solution prepared in S1, wherein the mass of the bacterial enzyme mixture is 2 g, and the volume of the Mandel salt solution is 4 mL, to obtain a culture solution, and then adjusting the pH of the culture solution to 9.0, and then placing the culture solution in a constant temperature incubator at 55° C. and culturing for 48 hours to obtain a bacterial enzyme fermentation solution;

[0072] S4, centrifuging the bacterial enzyme fermentation liquid at a speed of 3000 r / min for 35 minutes, taking the supernatant, and rotary evaporating the supernatant at 100° C. to obtain androstenol flavonoids.

[0073] S5, crush the onion flavonoids, chrysanthemum amide and cucurbitacin B separately until they can pass through a 120-mesh sieve. The temperature during the crushing process is less than 40°C. Then, take 12g of onion flavonoids powder, 12g of chrysanthemum amide powder and 11g of cucurbitacin B powder, mix them thoroughly and evenly to obtain 35g of composition 6.

[0074] Preparation method of composition 7

[0075] S1, washing the male onion with distilled water, then drying it at 75°C, then crushing the dried male onion until it can pass through a 150-mesh sieve, taking 10 g of the crushed male onion and adding it to 30 mL of Mandel salt solution;

[0076] S2, mixing Bacillus amyloliquefaciens and cellulase evenly to obtain a bacterial enzyme mixture, wherein the mass ratio of Bacillus amyloliquefaciens to cellulase is 9:5.5;

[0077] S3, uniformly mixing the bacterial enzyme mixture with the Mandel salt solution prepared in S1, wherein the mass of the bacterial enzyme mixture is 2 g, and the volume of the Mandel salt solution is 4.5 mL, to obtain a culture solution, and then adjusting the pH of the culture solution to 9.0, and then placing the culture solution in a constant temperature incubator at 55° C. and culturing for 48 hours to obtain a bacterial enzyme fermentation solution;

[0078] S4, centrifuging the fermentation liquid at a speed of 3500 r / min for 40 min, taking the supernatant, and rotary evaporating the supernatant at 105° C. to obtain onion flavonoids.

[0079] S5, crush the onion flavonoids, chrysanthemum amide and cucurbitacin B separately until they can pass through a 120-mesh sieve, and the temperature during the crushing process is less than 40°C. Then, take 14g of onion flavonoids powder, 15g of chrysanthemum amide powder and 6g of cucurbitacin B powder, mix them thoroughly and evenly to obtain 35g of composition 7.

[0080] Preparation method of composition 8

[0081] S1, washing the male onion with distilled water, then drying it at 75°C, then crushing the dried male onion until it can pass through a 150-mesh sieve, taking 10 g of the crushed male onion and adding it to 50 mL of Mandel salt solution;

[0082] S2, mixing Bacillus amyloliquefaciens and cellulase evenly to obtain a bacterial enzyme mixture, wherein the mass ratio of Bacillus amyloliquefaciens to cellulase is 10:7;

[0083] S3, uniformly mixing the bacterial enzyme mixture with the Mandel salt solution prepared in S1, wherein the mass of the bacterial enzyme mixture is 2 g, and the volume of the Mandel salt solution is 5 mL, to obtain a culture solution, and then adjusting the pH of the culture solution to 9.0, and then placing the culture solution in a constant temperature incubator at 55° C. and culturing for 48 hours to obtain a bacterial enzyme fermentation solution;

[0084] S4, centrifuging the fermentation liquid at a speed of 4000 r / min for 60 min, taking the supernatant, and rotary evaporating the supernatant at 105° C. to obtain onion flavonoids.

[0085] S5, crush the onion flavonoids, chrysanthemum amide and cucurbitacin B respectively until they can pass through a 120-mesh sieve, and the temperature during the crushing process is less than 40°C. Then, take 15g of onion flavonoids powder, 16g of chrysanthemum amide powder and 4g of cucurbitacin B powder, mix them thoroughly, and obtain 35g of composition 8.

[0086] 2. Largemouth bass rearing experiment and result analysis

[0087] The components and proportions of the premix used in all the following experiments were the same. Each kilogram of premix consisted of the following minerals and vitamins: CuSO4·5H2O, 2.0 g; FeSO4·7H2O, 25 g; ZnSO4·7H2O, 22 g; MnSO4·4H2O, 7 g; Na2SeO3, 0.04 g; KI, 0.026 g; CoCl2·6H2O, 0.1 g; VA, 900,0 00IU; VD, 200,000IU; VE, 4500mg; VK3, 220mg; VB1, 320mg; VB2, 1090mg; Niacin 2800mg; VB5, 2000mg; VB6, 500mg; VB12, 1.6mg; VC, 5000mg; Pantothenic acid, 1000mg; Folic acid, 165mg; Choline, 60000mg; Inositol, 15000mg.

[0088] Largemouth bass rearing experiment and result analysis

[0089] Example 1 and Comparative Examples 1 to 4 of the present application are all configured with equal fat and equal nitrogen feed (the feed sugar level is 20%), wherein Comparative Example 2 adds 1.0wt% of androstenol flavonoids, Comparative Example 3 adds 1.0wt% of clavicinamide, Comparative Example 4 adds 1.0wt% of cucurbitacin B group, and Example 1 adds 1.0wt% of composition 1. The specific formula composition of the feed of Example 1 and Comparative Examples 1 to 4 is shown in Table 1.

[0090] Table 1

[0091]

[0092] The animals used in the experiment were largemouth bass fry with an initial weight of 30.2±1.01g, which were purchased from a breeding farm in Guangzhou. Experimental method and steps: (1) First, 600 fry of uniform size were randomly placed in 20 cages (specifications: 2.0m×1.0m×1.0m), with 30 fish in each cage; (2) Then, the fry were cultured for 12 weeks using conventional feeding methods, during which the feeding and growth of the fry were observed; (3) After the culture was completed, blood and tissue samples of the largemouth bass were collected for subsequent index analysis. The effects of the feeds of Example 1 and Comparative Examples 1 to 4 on the growth, physiological and biochemical indicators of largemouth bass are shown in Table 2.

[0093] Table 2

[0094]

[0095] The results showed that composition 1 significantly increased the weight gain rate and liver endoplasmic reticulum ratio of largemouth bass fed high-sugar feed, but significantly reduced the feed conversion rate, blood sugar level, IRE1 enzyme activity, and IRE1, GRP78 and Calreticulin gene expression. This shows that composition 1 is superior in promoting fish growth, increasing feed utilization, improving endoplasmic reticulum function and lowering blood sugar.

[0096] Largemouth bass rearing experiment and result analysis 2

[0097] Examples 1 to 4 of the present application and Comparative Example 1 are all configured with equal fat and nitrogen feeds (the sugar level of the feed is 20%), wherein Example 1 adds 1.0wt% of Composition 1, Example 2 adds 1.0wt% of Composition 2, Example 3 adds 1.0wt% of Composition 3, and Example 4 adds 1.0wt% of Composition 4. The specific formula compositions of the feeds of Examples 1 to 4 and Comparative Example 1 are shown in Table 3.

[0098] Table 3

[0099]

[0100] The animals used in the experiment were largemouth bass fry with an initial weight of 10.01±0.21g, which were purchased from a breeding farm in Guangzhou. Experimental method and steps: (1) First, 600 fry of uniform size were randomly placed in 20 cages (specifications: 2.0m×1.0m×1.0m), with 30 fish in each cage; (2) Then, the fry were cultured for 12 weeks using conventional feeding methods, during which the feeding and growth of the fry were observed; (3) After the culture was completed, blood and tissue samples of the largemouth bass were collected for subsequent index analysis. The effects of the feeds of Examples 1 to 4 and Comparative Example 1 on the growth, physiological and biochemical indicators of largemouth bass are shown in Table 4.

[0101] Table 4

[0102]

[0103] The results showed that composition 4 significantly increased the weight gain rate and liver endoplasmic reticulum ratio of largemouth bass fed high-sugar feed, but significantly reduced the feed conversion rate, blood sugar level, IRE1 enzyme activity, and IRE1, GRP78 and Calreticulin gene expression. This shows that composition 4 is superior in promoting fish growth, increasing feed utilization, improving endoplasmic reticulum function and lowering blood sugar.

[0104] Largemouth bass rearing experiment and result analysis 3

[0105] Examples 4 to 8 of the present application are all configured with equal fat and equal nitrogen feeds (the sugar level of the feed is 20%), wherein Example 5 adds 1.0wt% of Composition 5 as a regulator, Example 6 adds 1.0wt% of Composition 6, Example 7 adds 1.0wt% of Composition 7, and Example 8 adds 1.0wt% of Composition 8. The specific formula compositions of the feeds of Examples 4 to 8 are shown in Table 5.

[0106] Table 5

[0108]

[0109] The animals used in the experiment were largemouth bass fry with an initial weight of 10.01±0.21g, which were purchased from a breeding farm in Guangzhou. Experimental method and steps: (1) First, 600 fry of uniform size were randomly placed in 20 cages (specifications: 2.0m×1.0m×1.0m), with 30 fish in each cage; (2) Then, the fry were cultured for 12 weeks using conventional feeding methods, during which the feeding and growth of the fry were observed; (3) After the culture was completed, blood and tissue samples of the largemouth bass were collected for subsequent index analysis. The effects of the feeds of Examples 4 to 8 on the growth, physiological and biochemical indicators of largemouth bass are shown in Table 6.

[0110] Table 6

[0111]

[0112] The results showed that composition 8 significantly increased the weight gain rate and liver endoplasmic reticulum ratio of largemouth bass fed high-sugar feed, but significantly reduced the feed conversion rate, blood sugar level, IRE1 enzyme activity, and IRE1, GRP78 and Calreticulin gene expression. This shows that composition 8 is superior in promoting fish growth, increasing feed utilization, improving endoplasmic reticulum function and lowering blood sugar.

[0113] The detection and analysis methods used in this application are as follows:

[0114] 1. Determination of growth and body shape indicators

[0115] Weight gain rate (%) = (weight of fish at the end of the experiment - weight of fish at the beginning of the experiment) / weight of fish at the beginning of the experiment × 100;

[0116] Feed coefficient = total amount of feed input (kg) / (weight of fish at the end of the experiment - weight of fish at the beginning of the experiment);

[0117] The experimental data were analyzed using SPSS27.0 (Chicago, USA) statistical software, and one-way analysis of variance (ANOVA) was performed. Before analysis, the Shapiro-Wilk test was used to evaluate the normality of the data, and the Levene variance homogeneity test was used to evaluate the homogeneity of variance. When analyzing the experimental data, different lowercase letters in the same row of data in the table indicate significant differences (P<0.05), and the same lowercase letters indicate no significant differences (P>0.05)

[0118] The reagents, methods and equipment not described in this application are conventional reagents, methods and equipment in this technical field. The aquaculture experiments were carried out at the teaching and research base of the College of Oceanography, South China Agricultural University.

[0119] Figure 1-4 The effects of the feed components of Example 1 and Comparative Examples 1 to 4 on the expression of IRE1 gene, Calreticulin gene, GRP78 gene expression and endoplasmic reticulum ratio in hepatocytes of largemouth bass, respectively, are shown in the results. The expression of IRE1 gene in hepatocytes, the expression of glucose-regulated protein 78 (GRP78) gene in endoplasmic reticulum stress-related genes, and the expression of Calreticulin gene in Comparative Example 1 are significantly higher than those in other experimental groups, indicating that the allium flavonoids, champignonamide and cucurbitacin B in the composition can enhance the sugar metabolism capacity of largemouth bass. The expression of IRE1, GRP78 and Calreticulin genes in hepatocytes of Example 1 is significantly lower than that in other experimental groups, while the endoplasmic reticulum ratio in the cells is significantly higher than that in other experimental groups, indicating that the composition can inhibit the endoplasmic reticulum stress of hepatocytes induced by high sugar by downregulating the expression of IRE1 gene, thereby greatly increasing the amount of endoplasmic reticulum.

[0120] The embodiments described above are only preferred implementation modes of the present invention and cannot be used to limit the protection scope of the present invention. Any non-substantial changes and modifications made by those skilled in the art on the basis of the invention shall fall within the protection scope of the present invention.

Claims

1. A composition for targeting the IRE1 gene to enhance sugar metabolism in largemouth bass, characterized in that: The composition comprises the following raw materials in parts by weight: 1 to 15 parts of allium flavonoids; 3 to 16 parts of clavicinamide; 4 to 31 parts of cucurbitacin B; The preparation method of the allium flavonoids comprises the following steps: S1, washing the shallot with distilled water, drying it at 50-75°C, and then crushing it, taking 10g of the crushed shallot and adding it to 10-50mL of Mandel salt solution; S2, mixing Bacillus amyloliquefaciens and cellulase uniformly to obtain a bacterial enzyme mixture, wherein the mass ratio of Bacillus amyloliquefaciens to cellulase is (1-10):(1-7); S3, mixing the bacterial enzyme mixture with the Mandel salt solution prepared in S1, wherein the mass ratio of the bacterial enzyme mixture to the Mandel salt solution is (1-2) g:(2-5) mL; obtaining a culture solution, adjusting the pH of the culture solution to 7.0-9.0, and then placing the culture solution in a constant temperature incubator at 35° C. to 55° C. for 24-48 hours to obtain a bacterial enzyme fermentation solution; S4, centrifuging the bacterial enzyme fermentation liquid at a speed of 1000-4000 r / min for 15-60 min, taking the supernatant, and rotary evaporating the supernatant at 85-105° C. to obtain onion flavonoids.

2. A composition for enhancing sugar metabolism in largemouth bass by targeting IRE1 gene according to claim 1, characterized in that: The composition comprises the following raw materials in parts by weight: 4-12 parts of allium flavonoids; 5-13 parts of clavicinamide; and 8-28 parts of cucurbitacin B.

3. A composition for enhancing sugar metabolism of largemouth bass by targeting IRE1 gene according to claim 2, characterized in that: The composition comprises the following raw materials in parts by weight: 8-10 parts of allium flavonoids; 7-10 parts of clavicinamide; and 15-20 parts of cucurbitacin B.

4. The composition for targeting IRE1 gene to enhance sugar metabolism in largemouth bass according to claim 1, characterized in that: The mass ratio of Bacillus amyloliquefaciens to cellulase in the bacterial enzyme mixed solution in step S2 is (2.5-3):(1-2.5).

5. A method for preparing a composition for enhancing sugar metabolism of largemouth bass by targeting IRE1 gene according to any one of claims 1 to 4, characterized in that: The following steps are involved: The onion flavonoids, chrysanthemum amide and cucurbitacin B are crushed into 120-150 meshes respectively, and the temperature during the crushing process is less than 40°C.

6. A feed for enhancing sugar metabolism of largemouth bass, characterized in that: A composition comprising any one of claims 1 to 4.

7. A feed for enhancing sugar metabolism of largemouth bass according to claim 6, characterized in that: The mass percentage of the composition in the feed is 0.2-1.

2.

8. Use of a feed in preparing a product for enhancing sugar metabolism of largemouth bass, characterized in that: Use the feed as described in claim 6.

9. Use of a composition in preparing a product for inhibiting IRE1 gene expression, characterized in that: A composition as claimed in any one of claims 1 to 4 is used.

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