Feed for improving quality of eriocheir sinensis and preparation method thereof

Abstract

The invention discloses a feed for improving the quality of eriocheir sinensis and a preparation method of the feed, and belongs to the technical field of crab feeds. The feed comprises the following raw materials in parts by weight: 30-40 parts of fish meal, 5-15 parts of chicken meal, 8-12 parts of soybean meal, 4-6 parts of peanut meal, 2-5 parts of egg white powder, 1-5 parts of pig protein powder, 1-5 parts of sesame powder, 15-20 parts of starch, 5-8 parts of soybean oil, 0.5-1 part of 50% choline chloride, 0.5-1.5 parts of monocalcium phosphate, 0.5-1 part of vitamin mineral premix and 1-3 parts of zeolite powder. The crab feed obtained by the invention can obviously improve the quality of the eriocheir sinensis.

Description

Technical Field

[0001] This invention belongs to the field of crab feed technology, specifically relating to a feed for improving the quality of Chinese mitten crab and its preparation method. Background Technology

[0002] The Chinese mitten crab (Eriocheir sinensis) is a special economic aquatic animal farmed in my country. During its cultivation, it is generally fed natural feeds such as corn, sweet potatoes, snails, and chilled fish. To ensure the taste and flavor of the product, fishermen prefer to feed it primarily with chilled fish. However, chilled fish come from wild marine or lake fishery resources, and long-term overfishing can impact ecosystem diversity and lead to the loss of some ecosystem services and functions. While chilled fish can promote the growth of the Chinese mitten crab, its source is unstable and it incurs high costs.

[0003] Currently, although there are aquaculture feeds for Chinese mitten crabs, their fattening effect is poor or they need to be supplemented with frozen fish for farming, which makes it difficult to fundamentally solve the problem.

[0004] Therefore, there is an urgent need for a feed to improve the quality of Chinese mitten crabs. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a feed for improving the quality of Chinese mitten crab and a method for preparing the same. The feed prepared by this invention can improve the quality of both male and female crabs.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a feed for improving the quality of Chinese mitten crabs, comprising the following raw materials in parts by weight:

[0008] Fish meal 30-40 parts, chicken meal 5-15 parts, soybean meal 8-12 parts, peanut meal 4-6 parts, egg white powder 2-5 parts, pork protein powder 1-5 parts, sesame powder 1-5 parts, starch 15-20 parts, soybean oil 5-8 parts, 50% choline chloride 0.5-1 part, calcium dihydrogen phosphate 0.5-1.5 parts, vitamin and mineral premix 0.5-1 part, zeolite powder 1-3 parts.

[0009] Preferably, the raw material composition is 36 parts fish meal, 10 parts chicken meal, 9.10 parts soybean meal, 5 parts peanut meal, 4 parts egg white powder, 3 parts pork protein powder, 5 parts sesame powder, 17 parts starch, 6.50 parts soybean oil, 0.70 parts 50% choline chloride, 1 part calcium dihydrogen phosphate, 0.70 parts vitamin and mineral premix, and 2 parts zeolite powder.

[0010] Preferably, the vitamin and mineral premix comprises 30-60 parts of vitamin B6, 20-50 parts of vitamin B2, 100-160 parts of vitamin E, 600-1000 parts of vitamin C ester, 70-100 parts of calcium dihydrogen phosphate, 130-180 parts of zinc methionine, and 120-250 parts of inositol.

[0011] The present invention provides a method for preparing the above-mentioned feed, comprising the following steps: weighing the raw materials according to the stated weight proportions, then crushing all the feed raw materials separately, passing them through a 60-mesh sieve, mixing them thoroughly and evenly, and making them into pellet feed.

[0012] Preferably, the particle size of the pelleted feed is 1-5 mm.

[0013] It contains at least the following beneficial technical effects:

[0014] The crab feed obtained by this invention can significantly improve the quality of Chinese mitten crab. Attached Figure Description

[0015] Figure 1 The hepatopancreatic index and gonadal index of crabs in each group were calculated.

[0016] Figure 2 The metabolic indicators for each group of crabs are shown.

[0017] Figure 3 The amino acid changes in each group of crabs are shown.

[0018] Figure 4 The changes in fatty acids in each group of crabs.

[0019] Figure 5 This is a transcriptome analysis diagram.

[0020] Figure 6 This is a plot of transcription-metabolism association analysis.

[0021] Figure 7 This represents the changes in various indicators under feed substitution.

[0022] Figure 8 Differences in gonadal lipids in male crabs raised using different fattening methods. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0029] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0030] Example 1

[0031] This invention provides a feed for improving the quality of Chinese mitten crabs, the preparation steps of which are as follows:

[0032] Weigh out the following ingredients according to weight: 36 parts fish meal, 10 parts chicken meal, 9.10 parts soybean meal, 5 parts peanut meal, 4 parts egg white powder, 3 parts pork protein powder, 5 parts sesame powder, 17 parts starch, 6.50 parts soybean oil, 0.70 parts 50% choline chloride, 1 part calcium dihydrogen phosphate, 0.70 parts vitamin and mineral premix, and 2 parts zeolite powder. Then, grind all feed ingredients separately, pass them through a 60-mesh sieve, mix them thoroughly and evenly, and make feed with a particle size of 3mm.

[0033] Example 2

[0034] This invention provides a feed for improving the quality of Chinese mitten crabs, comprising the following raw materials in parts by weight:

[0035] 30 parts fish meal, 5 parts chicken meal, 8 parts soybean meal, 4 parts peanut meal, 2 parts egg white powder, 1 part pork protein powder, 1 part sesame powder, 15 parts starch, 5 parts soybean oil, 0.5 parts 50% choline chloride, 0.5 parts calcium dihydrogen phosphate, 0.5 parts vitamin and mineral premix, and 1 part zeolite powder; then grind all feed ingredients separately, pass them through a 60-mesh sieve, mix them thoroughly and evenly, and make feed with a particle size of 1mm.

[0036] Example 3

[0037] This invention provides a feed for improving the quality of Chinese mitten crabs, comprising the following raw materials in parts by weight:

[0038] 40 parts fish meal, 15 parts chicken meal, 12 parts soybean meal, 6 parts peanut meal, 5 parts egg white powder, 5 parts pork protein powder, 5 parts sesame powder, 20 parts starch, 8 parts soybean oil, 1 part 50% choline chloride, 1.5 parts calcium dihydrogen phosphate, 1 part vitamin and mineral premix, and 3 parts zeolite powder; then, all feed ingredients are crushed separately, passed through a 60-mesh sieve, and thoroughly mixed to make feed with a particle size of 5mm.

[0039] Experimental Example 1

[0040] 1. Laboratory animals and breeding conditions

[0041] Samples were collected after five molts and before market launch. A fattening experiment was conducted on female crabs after their final physiological molt to explore the effects of hepatopancreatic metabolism and its influence on gonadal development before and after sexual maturity in crabs under a frozen fish fattening model. The fattening experiment was conducted in outdoor ponds at a stocking density of 600 crabs / mu (approximately 600 crabs / acre). The initial average weight of female crabs was 103.65±5.23g. The rearing period was 10 weeks. During this period, crabs were fed twice daily at 3-4% of their body weight using the feed prepared in Example 1, at 9:00 and 17:00. The water temperature was 23-28℃, dissolved oxygen >5mg / L, and ammonia nitrogen <0.2mg / L.

[0042] 2. Sample Collection

[0043] Samples were collected after five molts and before market launch. Twenty-one female crabs were selected for each molt. Crabs after the first four molts were designated F1, F2, F3, and F4, respectively. After the fifth molt, they were designated as immature (FU). Before market launch, they were designated as mature (FM). Before collection, the selected crabs were weighed, anesthetized with an ice bath, and dissected. The hepatopancreas and gonads of each crab were weighed, and the tissue samples were rapidly cooled with liquid nitrogen and stored at -80℃. The hepatopancreas and gonads of each group were labeled FUH (female unmature hepatopancreas), FUG (female unmature gonad), FMH (female mature hepatopancreas), and FMG (female mature gonad), respectively.

[0044] 3. Growth index measurement

[0045] Calculate the hepatopancreatic index and gonadal index of each group of crabs after sampling:

[0046] Hepatopancreas index (HSI, %) = Hepatopancreas weight (g) / Body weight (g) × 100% Gonadal index (GSI, %) = Gonadal weight (g) / Body weight (g) × 100%

[0047] The test results are shown below. Figure 1 Where A is body weight (FBW); B is hepatopancreatic index (HSI); and C is gonadal index (GSI).

[0048] 4. Metabolic index measurement

[0049] Before starting the experiment, approximately 0.2 g of tissue was accurately weighed and added to 0.9% physiological saline at a weight (g):volume (mL) ratio of 1:9. The mixture was mechanically homogenized and centrifuged at 2500 rpm for 10 minutes. The supernatant was used as the test solution. The total protein (TP), glucose (GLU), total cholesterol (TC), total triglycerides (TG), and α-amylase activity (AMS) of the collected hepatopancreatic and gonadal tissues of river crabs were measured. All reagent kits used were purchased from Nanjing Jiancheng Bioengineering Institute, and the assay methods were performed according to the kit instructions.

[0050] TP: Coomassie Brilliant Blue method (Bradford, 1976); GLU: Glucose oxidase method, microplate method; TC: COD-PAP enzyme method, spectrophotometer method; TG: GPO-PAP enzyme method, microplate method; AMS: Starch-iodine colorimetric method.

[0051] The test results are shown below. Figure 2 Among them, glucose is glucose; TP is total protein; TG is triglycerides; and TC is cholesterol.

[0052] 5. Amino acid determination

[0053] Nine crabs were selected from each group to collect hepatopancreas and gonad tissues. Three crabs were randomly selected for average mixing. Three independent samples from each group were measured. The amino acid composition and content of the hepatopancreas and gonad of the Chinese mitten crab were determined using the external standard method. The specific operation steps are as follows:

[0054] (1) Accurately weigh 0.6g of tissue and slowly add 8mL of HCl to ensure that the sample is completely wetted by HCl; (2) Maintain vacuum for 10min, seal with an alcohol burner, and hydrolyze at 110℃ for 24h; (3) Transfer all the hydrolyzed tissue to a volumetric flask with deionized water, and filter to a final volume; (4) Take 1mL of the filtrate and evaporate it to dryness in a vacuum desiccator with NaOH (vacuum drying below 50℃); (5) Add 1mL of 0.05% hydrochloric acid to dissolve the tissue, transfer the dissolved liquid to a centrifuge tube, centrifuge at 10000r / min for 10min, and take the supernatant for analysis using a liquid chromatograph (Anjie Company).

[0055] The test results are shown below. Figure 3 Where A represents the expression of key genes in fatty acid metabolism; BC represents the fatty acid content; D represents the expression of key genes in amino acid metabolism; and E represents the amino acid content.

[0056] Table 1 shows the changes in free amino acids and hydrolyzed amino acids in the hepatopancreas of female crabs before and after maturity.

[0057] Table 1

[0058]

[0059] 6. Fatty acid determination

[0060] Nine crabs were selected from each group to collect hepatopancreas and gonadal tissues. Three crabs were randomly selected for average mixing. After mixing, three independent samples from each group were measured. The percentage of each fatty acid in the total fatty acid content of the hepatopancreas was calculated using the area normalization method, which was taken as the final result (Teshima et al., 1986). The specific operation steps are as follows:

[0061] (1) Take a clean 2mL centrifuge tube, add 1.3mL of 2,6-di-tert-butyl-4-methylphenol (BHT) reagent, add steel balls (cleaned with anhydrous ethanol and sterilized by flame), and homogenize and break up the tissue together with the accurately weighed tissue; (2) Transfer the broken tissue into a clean glass tube, add 2.7mL of BHT reagent, and place at 85℃ for 1.5h; (3) At low temperature, add 1mL of ddH2O and 1mL of n-hexane, and centrifuge at 1000r / min for 10min; (4) Take the upper layer of n-hexane containing fatty acids, blow dry with helium, add 60μL of n-hexane to dissolve, and analyze with gas chromatography-mass spectrometry (Aglilent 7890B-5977A).

[0062] Table 2 shows the changes in fatty acid content in the hepatopancreas of female crabs before and after sexual maturity.

[0063] Table 2

[0064]

[0065]

[0066] 7. Transcriptome analysis

[0067] Nine hepatopancreatic tissue samples were selected from each group for high-throughput sequencing. Within each group, three crabs (0.1g tissue per crab) were randomly mixed, and transcriptome sequencing was performed using three biological replicates. After library quality control, de novo high-throughput sequencing was performed using the Illumina Novaseq 6000 platform. A paired-end (PE) sequencing strategy was employed.

[0068] The analysis results are shown below. Figure 4 A represents differential gene statistics; B represents differential gene clustering analysis; C represents differential gene KEGG signaling pathway enrichment; and D represents key gene expression levels.

[0069] 8. Metabolomics analysis

[0070] Metabolomics analysis was performed on six hepatopancreatic samples from Chinese mitten crabs in each group using high-performance liquid chromatography-tandem high-resolution mass spectrometry in both positive and negative ion modes. The mass spectrometry data were then interpreted using bioinformatics analysis.

[0071] 9. Transcription-Metabolic Association Analysis

[0072] Based on transcriptomic and metabolomic sequencing results of the hepatopancreas of female crabs before and after sexual maturity, differentially expressed mRNAs and metabolites were retrieved. These differentially expressed mRNAs and metabolites were associated with enriched KEGG signaling pathways to identify differentially expressed genes and metabolites that showed significant changes in the same biological process (KEGG pathway), thus pinpointing key genes. Cluster analysis, interaction regulatory network construction, and GO and KEGG enrichment analyses were then performed on the screened key genes and metabolites to reveal the regulatory mechanisms of hepatopancreas metabolism in female crabs before and after sexual maturity from a multi-omics perspective.

[0073] The analysis results are shown below. Figure 5 In this table, A represents the statistical analysis of differentially expressed metabolites; B represents the enrichment analysis of positive differentially expressed metabolites; C represents the enrichment analysis of negative differentially expressed metabolites; D represents the enrichment of KEGG signaling pathway in positive differentially expressed metabolites; E represents the enrichment of KEGG signaling pathway in negative differentially expressed metabolites; and F represents the expression level of key differentially expressed metabolites.

[0074] 10. Validation of key gene expression

[0075] To verify the reliability of the transcriptome and metabolome results, this invention validates the key genes obtained from the transcriptome-metabolome association analysis using real-time quantitative PCR, with the conserved gene EF-1α as an internal reference gene.

[0076] 11. Changes in indicators when using feed to replace chilled fish

[0077] Changes in growth performance, fatty acid and amino acid metabolism of hepatopancreas when using feed to replace fresh fish, see [link to relevant documentation]. Figure 6 A. Growth and hepatopancreatic index; B. Hepatopancreatic protein, fat, and ash content; C. Fatty acid content; D. Expression of key genes in fatty acid metabolism; E. Content of free, hydrolyzed essential, and flavor-enhancing amino acids; F. Expression of key genes in amino acid metabolism.

[0078] Changes in hepatopancreatic metabolism, nutrient deposition, and flavor yield when feed is used to replace fresh fish, see [link to relevant documentation]. Figure 7 A represents the expression of key genes related to liver and pancreatic metabolism and development; B represents the changes in the expression of key liver and pancreatic metabolites.

[0079] Example 2

[0080] male crab

[0081] 1. Laboratory animals and breeding conditions

[0082] Male crabs after their last physiological molt were selected for a fattening experiment to explore changes in gonadal development in crabs under a frozen fish fattening model. The fattening experiment was conducted in an outdoor aquaculture pond at a stocking density of 600 crabs / mu (approximately 600 crabs / acre). The initial average weight of the male crabs was 153.65±12.23g. The rearing period was 6 weeks. During this period, the crabs were fed twice daily (9:00 and 17:00) at a rate of 3-4% of their body weight. The water temperature was maintained at 23-28℃, dissolved oxygen was >5mg / L, and ammonia nitrogen was <0.2mg / L.

[0083] 2. Sample Collection

[0084] After the breeding period ended, gonad samples were collected from each group of male crabs (21 crabs in each group) for experimental analysis.

[0085] 3. Sample processing and analysis

[0086] 3.1 Measurement of growth indicators

[0087] Calculate the hepatopancreatic index and gonadal index of each group of crabs after sampling:

[0088] Hepatopancreas index (HSI, %) = Hepatopancreas weight (g) / Body weight (g) × 100% Gonadal index (GSI, %) = Gonadal weight (g) / Body weight (g) × 100%

[0089] 3.2 Measurement of metabolic indicators

[0090] Before starting the experiment, approximately 0.2 g of tissue was accurately weighed and added to 0.9% physiological saline at a weight (g):volume (mL) ratio of 1:9. The mixture was mechanically homogenized and centrifuged at 2500 rpm for 10 minutes. The supernatant was used as the test solution. The total protein (TP), glucose (GLU), total cholesterol (TC), total triglycerides (TG), and α-amylase activity (AMS) of the collected hepatopancreatic and gonadal tissues of river crabs were measured. All reagent kits used were purchased from Nanjing Jiancheng Bioengineering Institute, and the assay methods were performed according to the kit instructions.

[0091] TP: Coomassie Brilliant Blue method (Bradford, 1976); GLU: Glucose oxidase method, microplate method; TC: COD-PAP enzyme method, spectrophotometer method; TG: GPO-PAP enzyme method, microplate method; AMS: Starch-iodine colorimetric method.

[0092] 3.3 Transcriptome Analysis

[0093] Nine glandular tissue samples were selected from each group for high-throughput sequencing. Within each group, three crabs (0.1g tissue per crab) were randomly mixed, and transcriptome sequencing was performed using three biological replicates. After library quality control, de novo high-throughput sequencing was performed using the Illumina Novaseq 6000 platform. A paired-end (PE) sequencing strategy was employed.

[0094] 3.3 Metabolomics Analysis

[0095] Metabolomics analysis was performed on six Chinese mitten crab gonad samples in each group using high performance liquid chromatography-tandem high resolution mass spectrometry in both positive and negative ion modes. The mass spectrometry data were then interpreted using bioinformatics analysis.

[0096] 3.4 Data Processing

[0097] Independent samples t-tests were performed on various indicators of Chinese mitten crabs before and after maturity using SPSS 22.0 software. * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01), and *** indicates P<0.001 (the same applies below). Images were created using GraphPad 8.0 software.

[0098] The effects of feed substitution for chilled fish on the gonadal lipidome, see Figure 8 Where A represents changes in gonadal lipids; B represents clustering and VIP analysis of key differential metabolites; C represents ROC analysis of key lipids; D represents lipid classification; E represents expression levels of key differential lipids; F represents KEGG functional clustering of key differential lipids; G represents KEGG topological analysis of key differential lipids; and H represents the synthesis mechanism of lipid PC.

[0099] As can be seen from the above, the quality of both female and male crabs improved after replacing frozen fish with feed. Therefore, this feed can improve the quality of Chinese mitten crab.

[0100] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A feed for improving the quality of Chinese mitten crab, characterized in that, Including the following parts by weight of raw materials: Fish meal 30-40 parts, chicken meal 5-15 parts, soybean meal 8-12 parts, peanut meal 4-6 parts, egg white powder 2-5 parts, pork protein powder 1-5 parts, sesame powder 1-5 parts, starch 15-20 parts, soybean oil 5-8 parts, 50% choline chloride 0.5-1 part, calcium dihydrogen phosphate 0.5-1.5 parts, vitamin and mineral premix 0.5-1 part, zeolite powder 1-3 parts.

2. The feed according to claim 1, characterized in that, The raw material composition is as follows: 36 parts fish meal, 10 parts chicken meal, 9.10 parts soybean meal, 5 parts peanut meal, 4 parts egg white powder, 3 parts pork protein powder, 5 parts sesame powder, 17 parts starch, 6.50 parts soybean oil, 0.70 parts 50% choline chloride, 1 part calcium dihydrogen phosphate, 0.70 parts vitamin and mineral premix, and 2 parts zeolite powder.

3. The feed according to claim 1, characterized in that, The vitamin and mineral premix includes 30-60 parts of vitamin B6, 20-50 parts of vitamin B2, 100-160 parts of vitamin E, 600-1000 parts of vitamin C ester, 70-100 parts of calcium dihydrogen phosphate, 130-180 parts of zinc methionine, and 120-250 parts of inositol.

4. The method for preparing feed according to any one of claims 1-3, characterized in that, The process includes the following steps: weighing the raw materials according to the stated weight proportions, then crushing all feed ingredients separately, passing them through a 60-mesh sieve, mixing them thoroughly and evenly, and making them into pelleted feed.

5. The preparation method according to claim 4, characterized in that, The particle size of pelleted feed is 1-5mm.

Images