Feed for relieving growth inhibition and glucose metabolism disorder of culter alburnus induced by high-starch feed and preparation method of feed

Abstract

The invention relates to a feed for relieving growth inhibition and glucose metabolism disorder of culter alburnus induced by a high-starch feed and a preparation method thereof, belongs to the field of fish feeds, and particularly relates to the feed for relieving growth inhibition and glucose metabolism disorder of culter alburnus induced by the high-starch feed. The invention aims to solve the technical problems of extremely low tolerance of culter alburnus to high-starch feed, reduced weight gain rate, metabolic disorder and increased insulin resistance index at present. According to the invention, 2% of poly-beta-hydroxybutyrate is added into the feed containing 25% of corn starch, so that the growth performance of culter alburnus can be remarkably improved, and the utilization capacity of glucose is improved and insulin resistance and glucose metabolism disorder induced by high-starch feed are relieved by regulating mechanisms such as AKT / GSK3 beta pathway. The feed disclosed by the invention has the advantages of simple formula components, easily available raw materials, safety and harmlessness, is favorable for developing a novel green additive, and promotes healthy culture of culter alburnus.

Description

Technical Field

[0001] This invention belongs to the field of fish feed, specifically relating to a feed that reduces growth inhibition and sugar metabolism disorders in Culter alburnus induced by high starch feed. Background Technology

[0002] With the rapid development of aquaculture, the shortage of protein resources has become increasingly prominent. Utilizing carbohydrates to partially replace feed protein has become a key strategy for reducing aquaculture costs and nitrogen and phosphorus emissions. However, fish (especially carnivorous fish) generally suffer from poor glucose tolerance and low insulin sensitivity, leading to metabolic disorders, insulin resistance, and growth inhibition when fed high-starch diets. This severely restricts the application of carbohydrates in feed. The Culter alburnus (Basilewsky), a high-value carnivorous fish in my country, exhibits extremely low tolerance to high-starch diets, often displaying decreased weight gain, metabolic disorders, and elevated insulin resistance.

[0003] Poly(β-hydroxybutyrate) (PHB) is a microbially synthesized polyester that degrades into β-hydroxybutyrate (β-HB) in the animal gut. Recent studies have shown that PHB not only improves gut health and alleviates oxidative stress, but its degradation product, β-HB, can also act as a signaling molecule, enhancing glucose utilization by regulating the insulin signaling pathway. However, whether PHB can alleviate metabolic disorders induced by high-starch diets in fish by regulating the insulin signaling pathway remains unclear.

[0004] Therefore, developing PHB-based functional feeds is crucial for overcoming the bottleneck in sugar utilization in Culter alburnus and promoting healthy Culter alburnus farming. Summary of the Invention

[0005] The present invention aims to solve the technical problems of the extremely low tolerance of Culter alburnus to high starch feed, resulting in decreased weight gain, metabolic disorders and increased insulin resistance index, and to provide a feed that reduces growth inhibition and glucose metabolism disorders induced by high starch feed in Culter alburnus.

[0006] The feed of the present invention for mitigating growth inhibition and sugar metabolism disorders induced by high starch feed in Culter alburnus consists of, by mass fraction, 40%–41% fishmeal, 11%–12.5% ​​soybean meal, 11.77%–12% corn gluten meal, 12.5%–25% corn starch, 4.5%–4.73% fish oil, 0.3%–0.5% mineral and vitamin premix, 2%–3% choline chloride, 1%–1.5% calcium dihydrogen phosphate, and 1%–2% poly(β-hydroxybutyrate).

[0007] All the above-mentioned raw materials were purchased commercially and are currently available.

[0008] The above-mentioned method for preparing feed that reduces growth inhibition and sugar metabolism disorder induced by high starch feed in Culter alburnus is as follows: after crushing all the above raw materials, sieve them, mix them thoroughly, make them into granules, dry them in a constant temperature drying oven at 45℃ for 24 hours, seal them in a sealed bag, and store them at -20℃ for later use.

[0009] The above-prepared feed is used for feeding Culter alburnus as follows: feed twice a day during the feeding period, at 7:00 AM and 6:00 PM, with a feeding amount of 3% to 5% of the fish's body weight. Change the water once every 2 days, replacing 1 / 4 to 1 / 3 of the water each time.

[0010] This invention, by adding 2% polyβ-hydroxybutyrate to a feed containing 25% corn starch, significantly improves the growth performance of Culter alburnus (a type of culter fish). It also improves glucose utilization and alleviates insulin resistance and glucose metabolism disorders induced by high-starch feed by regulating mechanisms such as the AKT / GSK3β pathway. The feed formulation of this invention has simple components, readily available raw materials, and is safe and harmless, contributing to the development of novel green additives and promoting healthy Culter alburnus farming.

[0011] The present invention has the following beneficial effects:

[0012] (1) By adding 2% by weight of polyβ-hydroxybutyrate to the high starch feed of Culter alburnus, this invention significantly alleviates the problems of decreased growth performance and decreased feed utilization induced by high starch feed in Culter alburnus, and effectively improves the liver damage problem induced by high starch feed in Culter alburnus.

[0013] (2) The addition of poly-β-hydroxybutyrate to feed in this invention helps regulate the blood glucose level of Culter alburnus, promotes glycolysis and glycogen synthesis, improves glucose tolerance, alleviates insulin resistance, and improves insulin sensitivity.

[0014] (3) Poly-β-hydroxybutyrate can effectively alleviate the inhibition of the AKT / GSK3β pathway in the liver of Culter alburnus caused by high starch diet, increase the phosphorylation level of AKT and GSK3β, promote the absorption and utilization of glucose by Culter alburnus, and optimize the glucose metabolism pathway in Culter alburnus.

[0015] In summary, the method of adding poly-β-hydroxybutyrate to the high-starch feed for Culter alburnus provided by this invention has multiple beneficial effects, such as promoting growth, improving liver health, regulating blood sugar, and reducing insulin resistance, providing strong support for the healthy breeding of Culter alburnus. Attached Figure Description

[0016] Figure 1 A graph showing the effects of different feeds on the growth performance and feed efficiency of Culter alburnus.

[0017] Figure 2A graph showing the effects of different feeds on serum biochemical parameters of Culter alburnus;

[0018] Figure 3 A graph showing the effect of different feeds on the insulin resistance index of Culter alburnus.

[0019] Figure 4 A graph showing the effect of different feeds on glucose tolerance in Culter alburnus;

[0020] Figure 5 A graph showing the effect of different feeds on liver glycogen content in Culter alba;

[0021] Figure 6 A graph showing the effects of different diets on the activity of enzymes related to liver glucose metabolism in Culter albopictus.

[0022] Figure 7 A graph showing the effects of different diets on the levels of AKT and GSK3β proteins in the liver of Culter alburnus. Detailed Implementation

[0023] Specific Implementation Method 1: This implementation method is a feed for reducing growth inhibition and sugar metabolism disorders induced by high starch feed in Culter alburnus. It consists of 40%~41% fish meal, 11%~12.5% ​​soybean meal, 11.77%~12% corn gluten meal, 12.5%~25% corn starch, 4.5%~4.73% fish oil, 0.3%~0.5% mineral and vitamin premix, 2%~3% choline chloride, 1%~1.5% calcium dihydrogen phosphate, and 1%~2% poly(β-hydroxybutyrate).

[0024] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the fish meal contains 65 wt% crude protein and 8.4 wt% crude fat. Everything else is the same as in Specific Implementation Method One.

[0025] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method One or Two in that the soybean meal contains 44.9 wt% crude protein and 1.5 wt% crude fat. Everything else is the same as in Specific Implementation Method One or Two.

[0026] Specific Implementation Method Four: This implementation method differs from Specific Implementation Methods One to Three in that the corn gluten powder contains 63.5 wt% crude protein and 5.4 wt% crude fat. Everything else is the same as in Specific Implementation Methods One to Three.

[0027] Specific Implementation Method Five: This implementation method differs from Specific Implementation Method Four in that each kilogram of the mineral and vitamin premix contains the following substances: Vitamin A, 960,000 IU; Vitamin D, 560,000 IU; Vitamin E, 24,000 mg; Vitamin K, 1,620 mg; Nicotinamide, 4,580 mg; Pantothenic Acid, 7,020 mg; Folic Acid, 1,060 mg; Biotin, 40 mg; Inositol, 30,200 mg; Copper, 961 mg; Iron, 40,600 mg; Manganese, 5,520 mg; Cobalt, 64 mg; Iodine, 134 mg; Zinc, 23,200 mg; Selenium, 84 mg. Everything else is the same as in Specific Implementation Method Four.

[0028] Specific Implementation Method Six: This implementation method differs from Specific Implementation Method Five in that the chemical structural formula of the polyβ-hydroxybutyrate is: . Everything else is the same as in Specific Implementation Method 5.

[0029] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Method One in that the feed, by mass fraction, consists of 40% fishmeal, 12.5% ​​soybean meal, 11.77% corn gluten meal, 25% corn starch, 4.73% fish oil, 0.5% mineral and vitamin premix, 2% choline chloride, 1.5% calcium dihydrogen phosphate, and 2% poly(β-hydroxybutyrate). Everything else is the same as in Specific Implementation Method One.

[0030] Specific Implementation Method Eight: This implementation method is the preparation method of feed for reducing growth inhibition and sugar metabolism disorder induced by high starch feed in Culter alburnus according to Specific Implementation Method One. The specific process is as follows: all raw materials are crushed and sieved, thoroughly mixed and made into granules, dried in a constant temperature forced-air drying oven at 45℃ for 24 hours, sealed in a sealed bag, and stored at -20℃ for later use.

[0031] The invention was verified using the following experiments:

[0032] Experiment 1: This experiment is to develop a feed that can alleviate growth inhibition and sugar metabolism disorders induced by high starch feed in Culter alburnus. The feed consists of 40% fish meal, 12.5% ​​soybean meal, 11.77% corn gluten meal, 25% corn starch, 4.73% fish oil, 0.5% mineral and vitamin premix, 2% choline chloride, 1.5% calcium dihydrogen phosphate and 2% poly(β-hydroxybutyrate).

[0033] The fish meal contains 65 wt% crude protein and 8.4 wt% crude fat.

[0034] The soybean meal contains 44.9 wt% crude protein and 1.5 wt% crude fat.

[0035] The corn gluten meal contains 63.5 wt% crude protein and 5.4 wt% crude fat.

[0036] The mineral and vitamin premix mentioned above was purchased from Shenyang Boeing Feed Co., Ltd. Each kilogram of the premix contains the following substances: Vitamin A, 960,000 IU; Vitamin D, 560,000 IU; Vitamin E, 24,000 mg; Vitamin K, 1620 mg; Nicotinamide, 4580 mg; Pantothenic acid, 7020 mg; Folic acid, 1060 mg; Biotin, 40 mg; Inositol, 30200 mg; Copper, 961 mg; Iron, 40600 mg; Manganese, 5520 mg; Cobalt, 64 mg; Iodine, 134 mg; Zinc, 23200 mg; Selenium, 84 mg.

[0037] The poly-β-hydroxybutyrate was purchased from Ningbo Tianan Biomaterials Co., Ltd., China, and its chemical structural formula is as follows: .

[0038] The above feed is prepared as follows: all the above raw materials are crushed and sieved, thoroughly mixed and made into pellets, dried in a constant temperature drying oven at 45℃ for 24 hours, sealed in a sealed bag and stored at -20℃ for later use, and denoted as HCP.

[0039] Two feeds with the same nitrogen and fat content as HCP were prepared and compared, designated CK and HC, respectively. The raw material composition of the feeds is shown in Table 1 below:

[0040] Table 1 Feed formulation and nutrient levels (air-dried basis, mass percentage)

[0041]

[0042] The Culter alburnus used in the experiment were provided by the Harbin Academy of Agricultural Sciences. They were temporarily housed for 15 days before the start of the feeding experiment to allow them to acclimatize to the culture environment. 360 healthy Culter alburnus [initial average body weight: (3.30±0.06) g / fish] were selected and divided into 3 groups (each fed one of the 3 feeds listed in Table 1), with 3 replicates per group and 40 fish per replicate, for an 8-week feeding experiment. During the experiment, fish were fed twice daily, at 7:00 AM and 6:00 PM, at a rate of 4% of their body weight. Water was changed every 2 days, with 1 / 4 of the water replaced each time.

[0043] 1.2 Sample Collection

[0044] After an 8-week feeding trial, the fish were fasted for 24 hours and anesthetized with MS-222 (ethyl 3-aminobenzoate, Sigma-Aldrich, USA). The number and weight of Culter alburnus in each replicate were recorded. Eighteen Culter alburnus were randomly selected from each replicate, and blood was collected from their tail veins using a syringe. The samples were allowed to stand at 4°C for 24 hours, then centrifuged at 1000g for 10 minutes at 4°C. The supernatant was collected and stored at -80°C. Liver tissue was collected from 15 Culter alburnus, frozen in liquid nitrogen, and stored at -80°C for later use. Liver tissue was collected from 3 Culter alburnus and stored in 4% paraformaldehyde solution.

[0045] The test data below are expressed as mean ± standard error (SEM). Independent samples t-tests were performed using SPSS software, and p < 0.05 was considered statistically significant.

[0046] 1.3 Glucose Tolerance Test: After an 8-week feeding trial, fish were fasted for 24 hours. Twenty Culter alburnus were randomly selected from each replicate and anesthetized with MS-222 before injection and injected with glucose (1 g / kg body weight). Serum samples were collected from three fish in each replicate before injection, and at 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours after injection to measure glucose levels.

[0047] Depend on Figure 4 It was found that after glucose injection, the serum glucose levels of Culter alburnus in all groups reached their peak at 1 hour and returned to baseline levels at 6 hours. Furthermore, before glucose injection (0 hours), and at 1, 3, 12, and 24 hours after injection, the serum glucose levels of Culter alburnus in the HC group were significantly higher than those in the CK group (P < 0.05); conversely, before glucose injection (0 hours), and at 1, 12, and 24 hours after injection, the serum glucose levels of Culter alburnus in the HCP group were significantly lower than those in the HC group (P < 0.05). This indicates that adding 2% poly(β-hydroxybutyrate) to the feed can effectively improve glucose tolerance in Culter alburnus.

[0048] 1.4 Sample Determination

[0049] 1.4.1 Determination of growth performance, feed utilization, and insulin resistance index

[0050] Calculate growth performance, feed utilization, and insulin resistance index using the following formulas:

[0051] Body mass gain rate (WG, %) = 100% × (FW - IW) / IW;

[0052] Specific growth rate (SGR, %·d) -1 )=100% × (lnFW-lnIW) / t;

[0053] Feed efficiency (FE, %) = 100% × (FW - IW) / I t ;

[0054] The HOMA-IR index is calculated as: FG × FI / 22.8.

[0055] In the formula: FW and IW are the final and initial fish mass, respectively, in grams; I t The dry feed intake is expressed in grams (g); t is the test time, expressed in days (d); FG is fasting blood glucose, expressed in mmol / L; FI is fasting serum insulin, expressed in mU / L.

[0056] like Figure 1 As shown, compared with the CK group, the body mass, weight gain rate, specific growth rate and feed efficiency of the HC group were significantly reduced (P<0.05); adding 2% polyβ-hydroxybutyrate to the feed can significantly alleviate the reduction in body mass, weight gain rate, specific growth rate and feed efficiency induced by high starch feed in the HC group (P<0.05).

[0057] Figure 3 This indicates that the insulin resistance index of Culter alburnus in the HC group was significantly higher than that in the CK group (P<0.05), while the insulin resistance index of Culter alburnus in the HCP group was significantly lower than that in the HC group (P<0.05).

[0058] 1.4.2 Enzyme activity assay

[0059] Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), glucose (GLU), triglycerides (TG), and total cholesterol (TC) levels were detected using commercial kits (Nanjing Jiancheng Biological Institute, China); serum β-hydroxybutyrate (β-HB) and insulin (INS) levels were detected using fish ELISA kits (Yancheng Junxing Biotechnology Co., Ltd., China).

[0060] The activities of glucose-6-phosphatase (G6P), hexokinase (HK), phosphofructokinase (PFK), phosphoenolpyruvate carboxylkinase (PEPCK), pyruvate kinase (PK), fructose-1,6-bisphosphate aldolase (FBA), glycogen synthase (GCS), and glycogen phosphorylase a (Gpa) in the liver were detected using commercial kits (Shanghai Enzyme-Link Biotechnology Co., Ltd., China).

[0061] Depend on Figure 2The results showed that the serum AST, ALT, and LLT activities of Culter albus in the HC group were significantly higher than those in the CK group (P < 0.05), while the serum AST and ALT activities of Culter albus in the HCP group were significantly lower than those in the HC group (P < 0.05). This indicates that supplementing the feed with 2% poly-β-hydroxybutyrate can effectively alleviate liver damage induced by high-starch feed in Culter albus. The serum β-hydroxybutyrate content of Culter albus in the HCP group was significantly higher than that in the HC group (P < 0.05). The serum glucose, insulin, triglyceride, and total cholesterol levels of Culter albus in the HC group were significantly higher than those in the CK group (P < 0.05), while the serum glucose, insulin, triglyceride, and total cholesterol levels of Culter albus in the HCP group were significantly lower than those in the HC group (P < 0.05).

[0062] Figure 6 The results showed that, compared with the CK group, the activities of glucose-6-phosphatase, phosphofructokinase, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphate aldolase in the liver of *Culter albopictus* were significantly increased in the HC group (P < 0.05), while the activities of glucose-6-phosphatase, phosphofructokinase, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphate aldolase in the liver of *Culter albopictus* were significantly lower in the HCP group than in the HC group (P < 0.05). Compared with the CK group, the activities of hexokinase, pyruvate kinase, glycogen synthase, and glycogen phosphorylase a in the liver of *Culter albopictus* were significantly decreased in the HC group, while the activities of hexokinase and glycogen synthase in the liver of *Culter albopictus* were significantly higher in the HCP group than in the HC group (P < 0.05), and the activity of glycogen phosphorylase a in the liver of *Culter albopictus* was significantly lower in the HCP group than in the HC group (P < 0.05). This indicates that long-term feeding of high-starch diets leads to impaired glucose utilization and abnormal gluconeogenesis activation in the liver of Culter alburnus, revealing an insulin resistance-like pathological state in the liver. Supplementation with 2% PHB in the feed significantly alleviated these phenomena.

[0063] 1.4.3 PAS staining (e.g.) Figure 5 )

[0064] PAS staining was used to detect glycogen synthesis in the liver. Liver samples were dehydrated sequentially with ethanol, xylene, and paraffin, then embedded in paraffin and sectioned. Alcin blue and periodic acid (Wuhan Servi Biotechnology Co., Ltd., China) were used to stain the sections, and images were acquired and analyzed under a microscope.

[0065] Figure 5 The results, combined with PAS glycogen staining and glycogen content measurement, showed that the liver glycogen content of the HC group was significantly higher than that of the CK group (P<0.05), while the liver glycogen content of the HCP group was significantly higher than that of the HC group (P<0.05).

[0066] 1.4.4 Western blot for protein immunoblotting

[0067] The levels of lipid metabolism proteins in the hepatopancreas of carp were detected by Western blotting: First, an appropriate amount of carp liver sample was taken, lysed with lysis buffer, and centrifuged (12000 rpm, 4℃) for 30 min. The supernatant was collected, and the protein concentration was detected using a BCA protein quantification kit. The supernatant was mixed with 5× loading buffer at a volume ratio of 4:1 and incubated in a 95℃ water bath for 5-10 min to denature the protein. SDS-PAGE gel electrophoresis, Western blotting reaction, and protein band gray value analysis were performed. Antibody information is shown in Table 2.

[0068] Table 2 Antibody Information

[0069]

[0070] Depend on Figure 7 It was found that, compared with the CK group, the levels of p-AKT and p-GSK3β in the liver of Culter alburnus in the HC group were significantly decreased (P < 0.05), while the levels of p-AKT and p-GSK3β in the liver of Culter alburnus in the HCP group were significantly higher than those in the HC group (P < 0.05); the level of GSK3β in the liver of Culter alburnus in the HC group was significantly higher than that in the CK group (P < 0.05), while the level of GSK3β in the liver of Culter alburnus in the HCP group was significantly lower than that in the HC group (P < 0.05).

[0071] In conclusion, supplementing the feed with 2% poly-β-hydroxybutyrate can effectively alleviate growth inhibition, insulin resistance, and hepatic glucose metabolism disorders induced by high-starch feed in Culter alburnus, and increase the phosphorylation levels of hepatic AKT and GSK3β.

Claims

1. A feed that alleviates growth inhibition and glucose metabolism disorders induced by high-starch feed in Culter alburnus, characterized in that... The feed is composed of 40-41% of fish meal, 11-12.5% of soybean meal, 11.77-12% of corn gluten meal, 12.5-25% of corn starch, 4.5-4.73% of fish oil, 0.3-0.5% of mineral and vitamin premix, 2-3% of choline chloride, 1-1.5% of calcium dihydrogen phosphate and 1-2% of poly-beta-hydroxybutyric acid.

2. The feed for alleviating the growth inhibition and sugar metabolism disorder of Procypris percnurus induced by high-starch feed according to claim 1, characterized in that The fish meal contains 65wt% of crude protein and 8.4wt% of crude fat.

3. The feed for alleviating the growth inhibition and sugar metabolism disorder of Procypris percnurus induced by high-starch feed according to claim 1, characterized in that The soybean meal contains 44.9wt% of crude protein and 1.5wt% of crude fat.

4. The feed for alleviating the growth inhibition and sugar metabolism disorder of Procypris percnurus induced by high-starch feed according to claim 1, characterized in that The corn gluten meal contains 63.5wt% of crude protein and 5.4wt% of crude fat.

5. The feed for alleviating the growth inhibition and sugar metabolism disorder of Procypris percnurus induced by high-starch feed according to claim 1, characterized in that The mineral and vitamin premix contains the following per kilogram: vitamin A, 960000IU; vitamin D, 560000IU; vitamin E, 24000mg; vitamin K, 1620mg; nicotinamide, 4580mg; pantothenic acid, 7020mg; folic acid, 1060mg; biotin, 40mg; myo-inositol, 30200mg; copper, 961mg; iron, 40600mg; manganese, 5520mg; cobalt, 64mg; iodine, 134mg; zinc, 23200mg; selenium, 84mg. The chemical structural formula of the poly-beta-hydroxybutyric acid is:

6. The feed for alleviating the growth inhibition and sugar metabolism disorder of Procypris percnurus induced by high-starch feed according to claim 1, characterized in that The feed is composed of 40-41% of fish meal, 11-12.5% of soybean meal, 11.77-12% of corn gluten meal, 12.5-25% of corn starch, 4.5-4.73% of fish oil, 0.3-0.5% of mineral and vitamin premix, 2-3% of choline chloride, 1-1.5% of calcium dihydrogen phosphate and 1-2% of poly-beta-hydroxybutyric acid. 。 7. The feed for alleviating the growth inhibition and sugar metabolism disorder of Procypris percnurus induced by high-starch feed according to claim 1, characterized in that The preparation method comprises the following steps: after all raw materials are crushed and sieved, the mixture is uniformly mixed, granulated, dried in a constant temperature drying oven at 45℃ for 24h, sealed in a bag, and stored at-20℃ for standby use.

8. The method for preparing a feed for alleviating the growth inhibition and sugar metabolism disorder of Procypris percnurus induced by high-starch feed according to claim 1, characterized in that ​

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