Feed and additive for improving content of highly unsaturated fatty acid of largemouth bass and application of feed and additive

Abstract

The invention belongs to the technical field of feeds, and particularly relates to a feed for increasing the content of highly unsaturated fatty acids of largemouth bass, an additive and application of the feed and the additive. By adding the caffeic acid phenethyl ester into the feed, the nutritional value of the feed can be remarkably improved, and breeding feeding experiments prove that the caffeic acid phenethyl ester can activate the activity of Hnf4alpha, promote the expression of highly unsaturated fatty acid synthesis key genes and improve the highly unsaturated fatty acid level of muscular tissues of the micropterus salmoides.

Description

Technical Field

[0001] The invention belongs to the technical field of feed, and more specifically relates to feed for increasing the content of highly unsaturated fatty acids in largemouth bass, an additive and application thereof. Background Art

[0002] Highly unsaturated fatty acids (HAFAs), also known as long-chain polyunsaturated fatty acids (LCPUs), are essential fatty acids for the human body. They play an irreplaceable role in human metabolism and can prevent and treat a variety of diseases. As the primary source of HAFAs for humans, the increasing production of fish aquaculture highlights their importance in meeting human HAFA needs. Largemouth bass, a premium freshwater aquaculture species, is a top choice for consumers and fish farmers due to its delicate texture and rapid growth. Carnivorous fish, such as largemouth bass, have a relatively low capacity to synthesize HAFAs, and therefore require the addition of HA-rich fish oil to their feed to maintain growth and development. However, the recent shortage and high cost of fish oil have severely hampered the healthy and sustainable development of the aquaculture industry. To reduce aquaculture costs, the inclusion of plant-based ingredients in feed has gradually increased, accompanied by a reduction in the amount of fish meal and fish oil. This has led to a significant decrease in HAFA levels in largemouth bass, severely compromising their nutritional quality. In order to improve the nutritional quality of aquatic products and reduce the dependence of aquaculture on fish oil, improving the ability of fish to synthesize endogenous highly unsaturated fatty acids is considered to be an effective solution.

[0003] Current strategies for increasing endogenous highly unsaturated fatty acid synthesis in fish primarily include feed fatty acid ratios, functional feed additives, and gene editing. Feed fatty acid ratios are complex, requiring the precise matching of multiple fat sources. Feed nutritional composition is significantly influenced by the raw materials, making it difficult to control. Furthermore, their applicability is limited across different fish species and within different age ranges within the same species. Therefore, developing a feed additive that enhances the ability of farmed fish to synthesize endogenous highly unsaturated fatty acids, thereby producing high-quality fish products, is a critical challenge for the future aquaculture and feed industries. Summary of the Invention

[0004] The present invention aims to provide a feed, an additive and application thereof for increasing the highly unsaturated fatty acid content of largemouth bass.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The invention provides a feed additive for increasing the content of highly unsaturated fatty acids in largemouth bass. The effective component of the additive is caffeic acid phenethyl ester.

[0007] The present invention adds a certain level of caffeic acid phenethyl ester to feed, activating Hnf4α, a key regulator of highly unsaturated fatty acid synthesis, to promote endogenous highly unsaturated fatty acid synthesis, thereby increasing the level of highly unsaturated fatty acids in largemouth bass and improving their nutritional quality. The present invention provides a functional active substance and method for increasing the level of endogenous highly unsaturated fatty acid synthesis in largemouth bass, enabling easy feed preparation without any ethical or safety concerns.

[0008] The present invention also provides a feed for increasing the highly unsaturated fatty acid content of largemouth bass, which is prepared by mixing a basic feed with the above-mentioned feed additive, wherein the addition amount of the feed additive is 0.1-0.2% of the mass fraction of the basic feed.

[0009] Furthermore, the basic feed ingredients, calculated by mass, include: 40 to 50 parts of fish meal, 4 to 6 parts of fermented black soldier fly slurry, 6 to 8 parts of enzymatically hydrolyzed chicken meal, 5 to 7 parts of chicken liver powder, 5 to 7 parts of enzymatically hydrolyzed soybean meal, 7 to 8 parts of fermented soybean meal, 5 to 7 parts of cottonseed meal, 9 to 11 parts of cassava starch, 1 to 3 parts of soybean oil, 1 to 2 parts of linseed oil, 1 to 2 parts of calcium dihydrogen phosphate, 0.1 to 1 part of choline chloride, 0.5 to 1.5 parts of California sea bass premix, and 0.5 to 1.5 parts of zeolite.

[0010] The present invention also provides application of the feed additive or the feed in increasing the content of highly unsaturated fatty acids in largemouth bass.

[0011] Furthermore, increasing the highly unsaturated fatty acid content of largemouth bass includes increasing the content of docosahexaenoic acid in largemouth bass meat.

[0012] Furthermore, increasing the highly unsaturated fatty acid content of largemouth bass includes increasing the content of eicosapentaenoic acid in largemouth bass meat.

[0013] Furthermore, increasing the highly unsaturated fatty acid content of largemouth bass includes increasing the content of arachidonic acid in largemouth bass meat.

[0014] Furthermore, increasing the highly unsaturated fatty acid content of largemouth bass includes increasing the content of eicosatrienoic acid in largemouth bass meat.

[0015] The present invention has the following beneficial effects:

[0016] The present invention significantly increases the levels of arachidonic acid (C20:4n-6), eicosapentaenoic acid (C20:5n-3), docosahexaenoic acid (C22:6n-3) and eicosatrienoic acid (C20:3n-3) in the highly unsaturated fatty acids in the muscle tissue of largemouth bass by promoting the synthesis level of endogenous highly unsaturated fatty acids, thereby improving the nutritional value of largemouth bass. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a molecular simulation of the interaction conformation between caffeic acid phenethyl ester and Hnf4α.

[0018] Figure 2 The expression levels of genes related to highly unsaturated fatty acid synthesis in the liver tissue of largemouth seabass in different feed groups are shown in the figure, where ** indicates P < 0.01 and * indicates P < 0.05.

[0019] Figure 3 This is a ChIP-qPCR analysis of the enrichment level of Hnf4α in the promoters of genes related to highly unsaturated fatty acid synthesis in the liver tissue of largemouth bass. A is the statistical diagram of the enrichment level of the amplified fads2a gene promoter region, B is the statistical diagram of the enrichment level of the amplified fads2b gene promoter region, and C is the statistical diagram of the enrichment level of the amplified elovl5 gene promoter region. DETAILED DESCRIPTION

[0020] The present invention is described in detail below with reference to specific examples, but these examples should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0021] Example 1

[0022] 1. Molecular docking technology was used to virtually screen small molecule compounds that interact with Hnf4α.

[0023] 1. Molecular docking simulation: Based on the sequence of largemouth bass Hnf4α protein, AlphaFold2 was used to perform protein modeling, and Maestro 13.0 was used to optimize the hydrogen atom orientation of the protein model and minimize the energy under the OPLS4 force field. Then, based on the active pocket structure of human and mouse Hnf4α, the protein model was simulated by 2021-4 Active pocket prediction of Hnf4α in largemouth bass was performed. Considering the economic benefits and easy availability of small molecule compounds derived from natural compounds, the Taoshu natural compound library (L6020) was selected for virtual screening. Structure-based virtual screening was performed using The vsw (Virtual Screening Workflow) module in the 2021-4 software was completed, and the docking used three Glide algorithms with different accuracy levels: HTVS, SP, and XP. The three algorithms gradually improved in accuracy. Finally, the binding free energy of the output conformation was calculated. According to the result score, caffeic acid phenethyl ester was screened to have the best binding activity with the largemouth bass Hnf4α protein.

[0024] 2. Experimental results: Figure 1 As shown, caffeic acid phenethyl ester effectively binds to the active pocket of the largemouth bass Hnf4α protein, with multiple intermolecular interactions, including hydrogen bonding with amino acids G236, S180, L218, and R225. Furthermore, hydrophobic interactions with A222, L219, V347, V250, I344, V177, L235, N343, and V253 further strengthen the binding. These numerous interactions demonstrate that caffeic acid phenethyl ester binds strongly to the Hnf4α protein, potentially modulating its activity.

[0025] Effects of caffeic acid phenethyl ester supplementation on the synthesis of highly unsaturated fatty acids in largemouth bass

[0026] 1. Experimental Feed: The feed formula (by weight) is shown in Table 1. Feed ingredients, including fish meal, fermented black soldier fly slurry, enzymatically hydrolyzed chicken meal, chicken liver meal, enzymatically hydrolyzed soybean meal, fermented soybean meal, cottonseed meal, cassava starch, soybean oil, linseed oil, calcium dihydrogen phosphate, choline chloride, California sea bass premix, and zeolite powder, were purchased from Zhejiang Hanbei Biotechnology Co., Ltd. All ingredients were ground and passed through a 60-mesh sieve, weighed according to the appropriate proportions, and 14% water was added. The feed was mechanically mixed, pelletized by a pelletizer, and dried before use.

[0027] 2. Culture Management: 180 largemouth bass weighing 30 g were randomly placed in six culture tanks, 30 per tank, with three replicates per group. The culture period was 56 days. During this period, the fish were fed twice daily to satiation. The water temperature was maintained at 26°C–29°C, dissolved oxygen >7 mg / L, pH 6.8–7.3, ammonia nitrogen <0.3 mg / L, and nitrite <0.15 mg / L.

[0028] Table 1: Feed formula settings

[0029] Element control group Experimental group fishmeal 45 servings 45 servings Fermented black soldier fly paste 5 servings 5 servings Enzymatic chicken powder 6.95 servings 6.95 servings Chicken liver powder 6 servings 6 servings Enzymatically hydrolyzed soybean meal 6 servings 6 servings Fermented soybean meal 7.5 servings 7.5 servings cottonseed meal 6 servings 6 servings Tapioca starch 10.05 servings 10.05 servings soybean oil 2 servings 2 servings flaxseed oil 1.5 servings 1.5 servings Calcium dihydrogen phosphate 1.5 servings 1.5 servings Choline chloride 0.5 serving 0.5 serving California bass premix 1 serving 1 serving Zeolite powder 1 serving 0.9 parts Caffeic acid phenethyl ester 0 copies 0.1 part total 100 copies 100 copies

[0030] 3. Sample Collection and Index Measurement: After the completion of the breeding experiment, the experimental fish were fasted for 24 hours. The fatty acid composition of dorsal muscle tissue was determined from 9 randomly selected experimental fish in each group. Muscle tissues from 3 fish were pooled into one sample, with 3 replicates per group. Total RNA was extracted from the liver tissues of 3 randomly selected experimental fish in each group to measure the mRNA expression levels of the highly unsaturated fatty acid synthesis-related genes fads2a, fads2b, and elovl5. ChIP-qPCR was also performed to determine the enrichment of Hnf4α protein in the promoter regions of the highly unsaturated fatty acid synthesis-related genes fads2a, fads2b, and elovl5. Muscle tissues from 3 fish were pooled into one sample, with 3 replicates per group.

[0031] Fatty acid composition analysis: Total lipids from largemouth bass liver were extracted using the chloroform / methanol method and methyl-esterified with boron trifluoride. Fatty acid composition was determined by GC-MS. A 1 μL sample was injected; the injection temperature was 260°C, the detection temperature was 270°C, and the column temperature program was: initial temperature 150°C, hold for 5 minutes, then increase to 230°C at a rate of 5°C / min, hold for 20 minutes. Hydrogen and air flow rates were 30 and 40 mL / min, respectively, with nitrogen as the carrier gas at a flow rate of 1.5 mL / min. Chromatographic peaks were calibrated with fatty acid standards, and fatty acid content and composition were calculated.

[0032] Gene expression analysis: Liver RNA was extracted and reverse transcribed into cDNA, and then real-time fluorescence quantitative PCR was performed with β-actin and 18sRNA as double internal references. -ΔΔCT Methods The mRNA expression levels of highly unsaturated fatty acid synthesis-related genes fads2a, fads2b and elovl5 in the liver tissue of largemouth bass were quantitatively determined.

[0033] ChIP-qPCR: Liver tissue from largemouth bass was collected and ChIP-qPCR analysis was performed according to the Sonication ChIP Kit (ABclonal; RK20258) instructions. Protein-DNA complexes were fixed with 1% formaldehyde cross-linking for 10 minutes at room temperature, and cross-linking was terminated by the addition of 50 mM glycine. After washing with PBS, nuclei were extracted with nuclear lysis buffer. Chromatin was fragmented by sonication, and then a specific anti-Hnf4α antibody (ABclonal; A20865) and protein A / G magnetic beads were added. A control group was treated with IgG antibody. The cells were incubated overnight at 4°C. After magnetic separation, the complexes were washed with high-salt buffer, LiCl buffer, and TE buffer, and then eluted. Cross-links were reversed by adding 2 μL of 20 mg / mL proteinase K to the DNA purification eluate at 65°C overnight. DNA was then recovered by phenol-chloroform extraction and column purification. The recovered DNA and input DNA were used as templates to amplify the promoter regions of the fads2a, fads2b, and elovl5 genes.

[0034] 4. Data Processing and Statistical Analysis: The experimental results were expressed as mean ± standard deviation (mean ± SD). t-test was used to compare the two groups of data. Statistical analysis was performed using IBM SPSS Statistics 22. P < 0.05 was considered significant.

[0035] 5. Experimental results: As shown in Table 2, the addition of caffeic acid phenethyl ester to feed significantly increased the levels of arachidonic acid (C20:4n-6), eicosapentaenoic acid (C20:5n-3), docosahexaenoic acid (C22:6n-3) and eicosatrienoic acid (C20:3n-3) in the highly unsaturated fatty acids in the muscle tissue of largemouth bass, thereby improving the nutritional value of largemouth bass. Figure 2 As shown in the results, the addition of caffeic acid phenethyl ester to the feed significantly activated the expression levels of genes fads2a, fads2b and elovl5 related to the synthesis of highly unsaturated fatty acids in the liver tissue of largemouth bass, indicating that caffeic acid phenethyl ester promoted the synthesis level of endogenous highly unsaturated fatty acids in largemouth bass. Figure 3 As shown in the results, the addition of caffeic acid phenethyl ester to feed significantly increased the enrichment level of Hnf4α in the promoter region of highly unsaturated fatty acid synthesis-related genes fads2a, fads2b and elovl5, suggesting that caffeic acid phenethyl ester activates the transcriptional regulatory activity of Hnf4α to activate the expression of highly unsaturated fatty acid synthesis-related genes fads2a, fads2b and elovl5, thereby improving the endogenous highly unsaturated fatty acid synthesis capacity.

[0036] Table 2 Fatty acid composition of muscle tissue of largemouth bass in different feed groups

[0037]

[0038]

[0039] Note: ** indicates P < 0.01, * indicates P < 0.05.

[0040] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0041] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0042] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A feed additive for increasing the content of highly unsaturated fatty acids in largemouth bass, characterized in that: The active ingredient of the additive is caffeic acid phenethyl ester.

2. A feed for increasing the content of highly unsaturated fatty acids in largemouth bass, characterized in that: The feed additive is mixed with a basic feed and the feed additive according to claim 1, wherein the addition amount of the feed additive is 0.1 to 0.2% of the mass fraction of the basic feed.

3. A feed for increasing the content of highly unsaturated fatty acids in largemouth bass according to claim 2, characterized in that: The basic feed ingredients, calculated by mass, include: 40 to 50 parts of fish meal, 4 to 6 parts of fermented black soldier fly slurry, 6 to 8 parts of enzymatically hydrolyzed chicken meal, 5 to 7 parts of chicken liver powder, 5 to 7 parts of enzymatically hydrolyzed soybean meal, 7 to 8 parts of fermented soybean meal, 5 to 7 parts of cottonseed meal, 9 to 11 parts of cassava starch, 1 to 3 parts of soybean oil, 1 to 2 parts of linseed oil, 1 to 2 parts of calcium dihydrogen phosphate, 0.1 to 1 part of choline chloride, 0.5 to 1.5 parts of California sea bass premix, and 0.5 to 1.5 parts of zeolite.

4. Use of the feed additive according to claim 1 or the feed according to claim 2 in increasing the content of highly unsaturated fatty acids in largemouth bass.

5. The use according to claim 4, characterized in that The method of increasing the highly unsaturated fatty acid content of largemouth bass comprises increasing the content of docosahexaenoic acid in largemouth bass meat.

6. The use according to claim 4, characterized in that The method of increasing the highly unsaturated fatty acid content of largemouth bass comprises increasing the content of eicosapentaenoic acid in largemouth bass meat.

7. The use according to claim 4, characterized in that The method of increasing the highly unsaturated fatty acid content of largemouth bass comprises increasing the arachidonic acid content in largemouth bass meat.

8. The use according to claim 4, characterized in that The method of increasing the highly unsaturated fatty acid content of largemouth bass comprises increasing the content of eicosatrienoic acid in largemouth bass meat.

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