High-fat feed added with fermented kelp and application of high-fat feed in phoxinus lagowskii cultivation

Abstract

The invention discloses a high-fat feed added with fermented kelp and application of the high-fat feed in phoxinus lagowskii cultivation, and relates to the technical field of application of high-fat feeds. Comprising the following preparation raw materials: casein, fish meal, peeled soybean meal, fish oil, corn oil, wheat bran, flour, fermented kelp residues, wheat starch, monocalcium phosphate, a compound premix, lysine, methionine and choline chloride. According to the high-fat feed added with the fermented kelp and the application of the high-fat feed in phoxinus lagowskii cultivation, the nutritional characteristics of the kelp are improved through a fermentation means, resource utilization is promoted, meanwhile, the source of aquaculture feed raw materials is expanded, and practical support is provided for reasonable utilization of the fermented kelp in the aquaculture industry; through combined analysis of transcriptomics and metabonomics, the possible mechanism of fermenting kelp to alleviate the lipid metabolism disorder of phoxinus lagowskii under the condition of high-fat feed is explored, the resource utilization of traditional Chinese medicine residues is realized by applying a probiotic fermentation technology, and the disease prevention and control technology of phoxinus lagowskii is improved.

Description

Technical Field

[0001] The invention relates to the technical field of application of high-fat feed, in particular to a high-fat feed added with fermented kelp and application thereof in breeding of mullet. Background Art

[0002] Feed is the basis for the rapid development of intensive aquaculture, and its cost accounts for 40-60% of the total aquaculture cost; protein is the most expensive component in aquatic feed. Due to the global shortage of fish meal and the continuous increase in the price of protein raw materials, in order to reduce the protein content and feed cost in feed, aquaculture production often increases the level of feed fat and reduces the protein content and feed cost through the protein saving effect of fat.

[0003] Lipids are one of the three major nutrients for fish and play an important role in the metabolism of fish. However, excessive intake of fat can lead to lipid metabolism disorders in fish, which in turn damages liver health and induces tissue inflammation and oxidative stress. The liver is an important metabolic and immune organ in fish. Its damage or lesions will lead to metabolic disorders in the fish and cause growth decline; therefore, the aquaculture industry urgently needs relevant technologies to alleviate the negative effects of high-fat feed on fish.

[0004] It has been confirmed in many studies on aquatic animals that kelp, as a common Chinese herbal medicine, has positive effects such as anti-inflammatory, antioxidant, lipid-lowering, and maintaining liver health. However, the current industrial utilization rate of kelp is only about 30%, and the remaining kelp residue still has a large number of components, such as protein, cellulose, and minerals, which have not been fully developed and utilized. Not only does it cause a large amount of waste of kelp natural biological resources, but also a large amount of waste residue rich in organic matter and nutrients will be discharged during the production process, damaging the water environment. Studies have shown that the probiotic fermentation process can promote the release of effective active substances in kelp, improve the absorption and utilization rate of active substances in kelp by aquatic animals, and contribute to the healthy breeding of aquatic animals. At present, kelp is widely added directly to livestock and poultry and aquatic feed, but there are still few cases of adding probiotic fermented kelp to feed. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a high-fat feed added with fermented kelp and application thereof in breeding of mullet, thereby solving the problems raised in the above-mentioned background technology.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: A high-fat feed added with fermented kelp, comprising the following preparation raw materials:

[0007] Casein, fish meal, dehulled soybean meal, fish oil, corn oil, wheat bran, flour, fermented kelp residue, wheat starch, monocalcium phosphate, compound premix, lysine, methionine, choline chloride;

[0008] The specific proportions by percentage are as follows:

[0009] Casein 4-6%, fish meal 12-18%, peeled soybean meal 32-38%, fish oil 1-4%, corn oil 0-5%, wheat bran 8-12%, flour 18-22%, fermented kelp residue 0-6%, wheat starch 1-10%, monocalcium phosphate 1-5%, compound premix 0.1-4%, lysine 0.1-2%, methionine 0.1-2%, choline chloride 0.1-4%.

[0010] Optionally, the specific proportion of the high-fat feed in percentage is as follows:

[0011] Casein 5%, fish meal 15%, peeled soybean meal 35%, fish oil 2%, corn oil 4%, wheat bran 10%, flour 18%, fermented kelp residue 4%, wheat starch 1%, monocalcium phosphate 2%, compound premix 1%, lysine 0.5%, methionine 1%, choline chloride 1.5%.

[0012] Optionally, the specific proportion of the high-fat feed in percentage is as follows:

[0013] Casein 5%, fish meal 15%, peeled soybean meal 35%, fish oil 2%, corn oil 4%, wheat bran 10%, flour 18%, fermented kelp residue 0%, wheat starch 5%, monocalcium phosphate 2%, compound premix 1%, lysine 0.5%, methionine 1%, choline chloride 1.5%.

[0014] Optionally, the specific proportion of the high-fat feed in percentage is as follows:

[0015] Casein 5%, fish meal 15%, peeled soybean meal 35%, fish oil 2%, corn oil 4%, wheat bran 10%, flour 18%, fermented kelp residue 1%, wheat starch 4%, monocalcium phosphate 2%, compound premix 1%, lysine 0.5%, methionine 1%, choline chloride 1.5%.

[0016] Optionally, the specific proportion of the high-fat feed in percentage is as follows:

[0017] Casein 5%, fish meal 15%, peeled soybean meal 35%, fish oil 2%, corn oil 4%, wheat bran 10%, flour 18%, fermented kelp residue 2%, wheat starch 3%, monocalcium phosphate 2%, compound premix 1%, lysine 0.5%, methionine 1%, choline chloride 1.5%.

[0018] An application of a high-fat feed containing fermented kelp in the breeding of mullet, comprising the high-fat feed prepared by fermenting kelp with probiotics, and the specific steps are as follows:

[0019] Step 1: Pre-treat the kelp;

[0020] Step 2: Weigh the bacteria, brown sugar and water in a ratio of 1:10:200, sterilize the water with high pressure, take it out, and let it cool to 40°C to dissolve the brown sugar;

[0021] Step 3: activating the finished starter containing Bacillus subtilis, Bacillus licheniformis, Candida utilis, Enterococcus faecalis and Lactobacillus in a brown sugar solution for 30 minutes to complete the preparation of the fermentation liquid;

[0022] Step 4: Mix the successfully activated fermentation liquid and the pre-treated kelp powder evenly for 4-5 days of primary fermentation;

[0023] Step 5: Add corn flour, bran and glucose again, and inoculate 5% of the total bacterial solution in the bait;

[0024] Step 6: Add water and mix until the water content of the bait is 40% to 45%, and the bait can be rubbed into a ball by hand and falls apart when gently kneaded. Then seal it in a transparent anaerobic fermentation bag and ferment it in a 37°C constant temperature incubator for 3 to 5 days. Turn it over once a day. When a special sour aroma is found, the fermentation is judged to be complete. After the fermented kelp additive is made, it is dried and stored in a dry place for later use.

[0025] Step 7: using fish oil and corn oil as fat sources, fish meal and soybean meal as protein sources, flour and wheat starch as sugar sources, and adding 4.0% fermented kelp by mass percentage;

[0026] Step 8: crush the raw materials, pass through a 60-mesh sieve, weigh and mix according to the formula, make 1.5 mm particles, dry them in the sun and store them at -4°C;

[0027] Step nine: obtain the finished high-fat feed.

[0028] Optionally, in the step 1, the kelp is washed, placed in a 65° C. oven for drying and crushed, and passed through a 60-mesh sieve to obtain kelp powder.

[0029] Optionally, the total bacterial solution concentration in step five is 107 CFU / g, and the mass ratio of LSG1-1 Bacillus licheniformis to LSG2-1 Bacillus subtilis is 5:1.

[0030] The present invention provides a high-fat feed added with fermented kelp and application thereof in breeding of mullet, which has the following beneficial effects:

[0031] The high-fat feed supplemented with fermented kelp and its application in the breeding of Rockfish improves the nutritional characteristics of kelp by fermentation, and the effects of adding different levels of fermented kelp to the high-fat feed on the growth performance, lipid metabolism, immune function and antioxidant capacity of Rockfish, while promoting resource utilization, also expands the source of aquaculture feed raw materials; provides practical support for the rational use of fermented kelp in aquaculture; uses transcriptomics and metabolomics combined analysis to explore the possible mechanism by which fermented kelp alleviates lipid metabolism disorders in Rockfish under high-fat feed conditions, and provides a reference for the application of fermented kelp as a lipid-lowering factor in high-fat feed for fish; uses probiotic fermentation technology to realize the resource utilization of traditional Chinese medicine residues and improve disease prevention and control technology for Rockfish.

[0032] Research on fermented kelp as a Chinese medicinal additive has shown that it is beneficial to improving feed utilization, improving lipid metabolism and immune function of the red snapper. It can not only avoid environmental pollution caused by medicinal residues, but also make it a new feed resource, providing another way for the comprehensive utilization of Chinese medicinal residues. Using kelp residues to make fermented feed can make full use of a variety of unused nutrients and active ingredients, effectively achieve waste utilization and turn waste into treasure, promote the green and healthy development of aquaculture, and provide a theoretical basis and experimental data for the future development of efficient lipid-lowering factors that can be used in high-fat feeds for aquatic animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a schematic diagram of the experimental process of the invention. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0035] Example 1: A high-fat feed (high-fat HF) supplemented with fermented kelp, comprising the following preparation materials:

[0036] Casein, fish meal, dehulled soybean meal, fish oil, corn oil, wheat bran, flour, fermented kelp residue, wheat starch, monocalcium phosphate, compound premix, lysine, methionine, choline chloride;

[0037] The specific proportions by percentage are as follows:

[0038] Casein 5%, fish meal 15%, peeled soybean meal 35%, fish oil 2%, corn oil 4%, wheat bran 10%, flour 18%, fermented kelp residue 0%, wheat starch 5%, monocalcium phosphate 2%, compound premix 1%, lysine 0.5%, methionine 1%, choline chloride 1.5%.

[0039] Example 2: A high-fat feed (HF+1%) with fermented kelp added, comprising the following raw materials:

[0040] Casein, fish meal, dehulled soybean meal, fish oil, corn oil, wheat bran, flour, fermented kelp residue, wheat starch, monocalcium phosphate, compound premix, lysine, methionine, choline chloride;

[0041] The specific proportions by percentage are as follows:

[0042] Casein 5%, fish meal 15%, peeled soybean meal 35%, fish oil 2%, corn oil 4%, wheat bran 10%, flour 18%, fermented kelp residue 1%, wheat starch 4%, monocalcium phosphate 2%, compound premix 1%, lysine 0.5%, methionine 1%, choline chloride 1.5%.

[0043] Example 3: A high-fat feed (HF+2%) supplemented with fermented kelp, comprising the following raw materials:

[0044] Casein, fish meal, dehulled soybean meal, fish oil, corn oil, wheat bran, flour, fermented kelp residue, wheat starch, monocalcium phosphate, compound premix, lysine, methionine, choline chloride;

[0045] The specific proportions by percentage are as follows:

[0046] Casein 5%, fish meal 15%, peeled soybean meal 35%, fish oil 2%, corn oil 4%, wheat bran 10%, flour 18%, fermented kelp residue 2%, wheat starch 3%, monocalcium phosphate 2%, compound premix 1%, lysine 0.5%, methionine 1%, choline chloride 1.5%.

[0047] Example 4: A high-fat feed (HF+4%) supplemented with fermented kelp, comprising the following raw materials:

[0048] Casein, fish meal, dehulled soybean meal, fish oil, corn oil, wheat bran, flour, fermented kelp residue, wheat starch, monocalcium phosphate, compound premix, lysine, methionine, choline chloride;

[0049] The specific proportions by percentage are as follows:

[0050] Casein 5%, fish meal 15%, peeled soybean meal 35%, fish oil 2%, corn oil 4%, wheat bran 10%, flour 18%, fermented kelp residue 4%, wheat starch 1%, monocalcium phosphate 2%, compound premix 1%, lysine 0.5%, methionine 1%, choline chloride 1.5%.

[0051] The kelp used was from Jilin Huinan Changlong Biochemical Pharmaceutical Co., Ltd. It was the herbal waste left over from the production of Haikun Shenxi Capsules after the extraction of brown algae polysaccharide components.

[0052] The finished fermentation agent selected in this experiment was from Henan Ruiteli Biotechnology Co., Ltd., and LSG1-1 Bacillus licheniformis and LSG2-1 Bacillus subtilis were provided by the Fisheries Laboratory of Jilin Agricultural University.

[0053] Control group: a high-fat diet supplemented with fermented kelp, comprising the following preparation materials:

[0054] Casein, fish meal, dehulled soybean meal, fish oil, corn oil, wheat bran, flour, fermented kelp residue, wheat starch, monocalcium phosphate, compound premix, lysine, methionine, choline chloride;

[0055] The specific proportions by percentage are as follows:

[0056] Casein 5%, fish meal 15%, dehulled soybean meal 35%, fish oil 2%, wheat bran 10%, flour 22%, wheat starch 5%, monocalcium phosphate 2%, compound premix 1%, lysine 0.5%, methionine 1%, choline chloride 1.5%.

[0057] The comparative test is as follows: Figure 1 As shown:

[0058] First, the kelp is pretreated: the kelp is washed, dried in an oven at 65°C and crushed, and passed through a 60-mesh sieve to obtain kelp powder; the weight of the strain, brown sugar and water is weighed in a ratio of 1:10:200, the water is sterilized under high pressure and then taken out, and the brown sugar is dissolved after being dried to 40°C; the finished starter containing strains such as Bacillus subtilis, Bacillus licheniformis, Candida utilis, Enterococcus faecalis and Lactobacillus is activated in a brown sugar solution for 30 minutes, and the fermentation liquid is prepared; the successfully activated fermentation liquid is evenly mixed with the pretreated kelp powder for the primary fermentation of 4-5 days; and carbon sources such as corn flour, bran and glucose are added again, and 5% of the total bacterial liquid of the bait (concentration is 107CF U / g), LSG1-1 Bacillus licheniformis and LSG2-1 Bacillus subtilis mass ratio of 5:1; add water and mix until the water content of the bait is about 40% to 45%, and after reaching the standard of being rubbed into a ball by hand and falling apart by light kneading, seal it in a transparent anaerobic fermentation special bag, and ferment it in a 37°C constant temperature incubator for 3 to 5 days, turn it over once a day, and judge that the fermentation is over when a special sour aroma is found. After making the fermented kelp additive, dry it and store it in a dry place for later use; finally, the effective ingredients in the fermented kelp were determined, and the results showed that the total sugar content was 175.74 (mg / g), the alginate content was 4.56%, the crude protein content was 20.35%, and the crude fiber content was 38.42%;

[0059] With fish oil and corn oil as fat sources, fish meal and soybean meal as protein sources, flour and wheat starch as sugar sources, the experiment was divided into 5 groups, namely, the basic feed ND group (crude fat 4.30%); high-fat feed Control group (crude fat 8.24%); high-fat feed added with different levels of fermented kelp FK-1 group (1.0%, crude fat 8.19%), FK-2 group (2.0%, crude fat 8.21%) and FK-4 group (4.0%, crude fat 8.15%); feed formula and main nutrients are shown in Table 1-2; the raw materials were crushed, sieved through a 60-mesh sieve, weighed according to the formula and mixed to make 1.5mm particles, dried in the sun and stored at -4°C for later use. The specific raw material formula is shown in the following table;

[0060]

[0061]

[0062] Rockfish was purchased from Huinan City, Jilin Province. The experiment was conducted in the aquaculture room of Jilin Agricultural University. After 15 days of pre-feeding, 600 healthy Rockfish with an initial body weight of (4.05±0.01) g / tail were selected and randomly divided into 5 groups (3 replicates in each group, 40 fish in each replicate) and raised for 8 weeks. During this period, they were fed 3 times a day (07:00 in the morning, 12:00 noon and 17:00 in the afternoon). The feed feeding amount, mortality of experimental fish and water quality were recorded every day. The water was changed once every 2 days, and 1 / 4 to 1 / 3 of the water was changed each time to keep the aquaculture water quality fresh. During the aquaculture experiment, natural light was used, oxygen was supplied for 24 hours, the water temperature was 20℃±1℃, the dissolved oxygen was>5mg / L, the ammonia nitrogen was<0.3mg / L, the nitrite was<0.05mg / L, and the pH was 7.5-8.5.

[0063] Samples were taken on the 28th day (mid-term) and the 56th day (final stage) respectively; the fish were stopped from feeding for 1 day, and the group mass of each bucket of fish was recorded; 8 fish were randomly selected from each bucket, and their body length, weight, visceral mass weight, hepatopancreas weight, intestinal length and intestinal weight were measured; blood was collected from the tail vein with a syringe, and the supernatant was collected after centrifugation; the hepatopancreas and intestine were taken, frozen in liquid nitrogen, and stored at -80℃ for later use; the muscles above the lateral line and below the dorsal fin were taken and stored at -20℃ for later use;

[0064] The growth performance related indicators are calculated by the following formula:

[0065] Weight gain rate (WGR, %) = 100% × (WF-WC) / WC;

[0066] Specific growth rate (SGR, % / d) = 100% × (lnWF-lnWC) / T Feed efficiency (FER) = (WF-WC) / IF × 100%;

[0067] Hepatosomal ratio (HSI, %) = 100% × WH / WF;

[0068] Viscera-to-body ratio (VSI, %) = 100% × WV / WF;

[0069] Fullness (CF, %) = 100% × WF / L3;

[0070] Intestinal body index (IBI, %) = 100% × WI / WF;

[0071] Intestinal length index (ILI, %) = 100% × IL / L;

[0072] The nutritional components of muscle were determined using the national standard method:

[0073] (1) Crude protein - Kjeldahl method (GB5009.5-2016);

[0074] (2) Crude lipid: Soxhlet ether extraction method (GB5009.6-2016);

[0075] (3) Ash: Muffle furnace calcination method (GB5009.4-2016);

[0076] (4) Moisture: 105°C constant temperature drying weight loss method (GB5009.3-2016).

[0077] The amino acid content of the muscle of the Lophatherum rothenbergii was determined by Beijing Kekengou Technology Co., Ltd. The steps are as follows: freeze-dry the muscle of the Lophatherum rothenbergii for 24 hours and then crush it, take 0.1g of the dry sample and add it to an acid hydrolysis tube, add 10mL of hydrochloric acid diluent (1:1), acid hydrolyze at 110℃ for 24 hours, filter, make the volume to 20mL, take 1mL into a deacidification tube, deacidify, add 2mL of amino acid diluent, mix well and filter into a sample bottle, and use a fully automatic amino acid analyzer for determination;

[0078] Sample processing is as follows:

[0079] Trimming: Remove the liver and intestines and trim them to 0.5×0.5 cm 2 The tissue blocks were placed in embedding boxes and numbered;

[0080] Dehydration: After washing the tissue samples with running water for 2 hours, immerse them in 50%, 75%, 85%, 95% (Ⅰ), 95% (Ⅱ), 100% (Ⅰ), and 100% (Ⅱ) ethanol for 1 hour each;

[0081] Transparency: After dehydration, immerse the tissue sample in xylene (I) and xylene (II) for 5 min each; Wax immersion: In a 60°C water bath, immerse the tissue sample in soft wax (I), soft wax (II), hard wax (I), and hard wax (II) for 30 min each;

[0082] Embedding: Finally, pour melted hard wax into the stacked paper box and place the wax-soaked tissue sample with the cross section facing down;

[0083] Sectioning: After the hard wax solidifies and cools, trim the wax block and cut it into 4-7 μm thick slices with a slicer. Expand it in 37-42℃ water, gently pick it up with a slide, absorb the moisture with filter paper, bake it at 37℃, and dry it at 37℃;

[0084] Staining: The sections were placed in xylene (Ⅰ), xylene (Ⅱ), and a mixture of xylene and alcohol (1:1) for 3 min each, and then immersed in absolute alcohol and gradient alcohol until distilled water for 1 min; stained with hematoxylin solution for 4 min, differentiated for 10 s, washed with anti-blue for 4 min, and stained with eosin; then placed in 85%, 95% (Ⅰ), and 95% (Ⅱ) ethanol for dehydration, and finally transparentized with xylene;

[0085] Sealing: remove excess xylene and seal with neutral gum;

[0086] The preparation of crude enzyme solution is as follows:

[0087] At 4°C, the intestinal samples were thawed, cut into pieces, weighed (accurate to 0.01 g), diluted 10 times with saline, homogenized for 5 min, centrifuged at 5000 r / min and 4°C for 10 min, and the supernatant was taken as the crude enzyme solution, placed in a centrifuge tube, and stored in a refrigerator at 4°C. The test was completed within 24 hours.

[0088] The enzyme activity was determined as follows:

[0089] Kits were used to determine the activities of protease, amylase and lipase in the intestine of Rockfish, as well as intestinal permeability indicators such as T-CHO, TG, HDL-C, LDL-C, AST, ALT activity, D-lactic acid, DAO, ET-1 and 5-HT in the serum; kit method was used to determine the contents of AST, ALT, ALT, ACP, AKP, LZM, IgM, C3, C4 complement and ROS, MDA, SOD, CAT, GPX, GST, GSH in the serum and hepatopancreas of Rockfish after 28 days (mid-term) and 56 days (final stage) of feeding; at the same time, the activities of lipases such as HSL, LPL, ATGL, ACC, FAS and CPT-1 in the hepatopancreas and muscle were determined.

[0090] The preparation of crude enzyme solution is as follows:

[0091] At 4°C, thaw the liver and intestinal tract (foregut, midgut, hindgut) samples, cut them into pieces, weigh them (accurate to 0.01g), dilute them 10 times with physiological saline, homogenize them for 5 minutes, centrifuge them at 5000r / min and 4°C for 10 minutes, take the supernatant as the crude enzyme solution, put it in a centrifuge tube, store it in a refrigerator at 4°C, and complete the test within 24 hours.

[0092] Design and synthesis of primers for intestinal tight junction, liver, spleen, kidney and muscle related gene expression Primers were designed using Premier 5.0 software.

[0093] RNA was extracted using the Trizol method as follows:

[0094] 1) Add 200 μl of chloroform, shake and mix to extract RNA;

[0095] 2) Stand for 5 min, centrifuge at 12000 rpm, 4°C for 15 min;

[0096] 3) Aspirate the supernatant into a new 1.5 ml centrifuge tube;

[0097] 4) Add an equal amount of isopropanol and mix gently to precipitate RNA;

[0098] 5) Let stand for 10 min, centrifuge at 12000 rpm, 4°C for 10 min, and discard the supernatant;

[0099] 6) Add 1 ml of 75% ethanol to wash and neutralize the isopropanol. Gently shake the RNA precipitate to float, centrifuge at 12000 rpm, 4°C for 5 min, and discard the supernatant. Then, aspirate the excess ethanol and dry naturally at room temperature.

[0100] 7) Add 10-50 μL ddH2O and wait for the RNA concentration to be determined. The RNAOD value within the range of 1.8 < 260 / 280 < 2.0 can be used for subsequent experiments;

[0101] Reverse transcription reaction system (20 μL): add 14 μL of ddH2O, 4 μL of Prime_Script_RT_Master_Mix, and 2 μL of RNA in the following order, and follow the conditions of the reverse transcription kit at 37°C for 15 min; 85°C for 5 s to obtain template cDNA;

[0102] 1) Reaction system (20 μL): add ddH2O 7.2 μL, upstream primer 0.4 μL, downstream primer 0.4 μL, SYBR Premix Ex Tap TM II 10 μL, template cDNA in the following order: 2 μL;

[0103] 2) Reaction conditions: pre-denaturation: 95°C for 3 min; denaturation: 95°C for 30 s; annealing: 60°C for 20 s; extension: 72°C for 30 s (40 cycles from denaturation to extension); melting curve: 70°C-95°C, 0.5°C / 6 s.

[0104] The growth genes were determined as follows:

[0105] The contents of IGF, ERK, PI3K, S6K and GH were detected by fluorescence quantitative PCR method; 4.4.2.7.6 Determination of immune and antioxidant related genes The contents of ZO-1, Occludin-a, Occludin-b, Claudin-2, Claudin-4, TGF-β, IL-10, IL-1β, TNF-α, NF-κB, IL-8, IL-6, Nrf2 and GSH-PX, CAT, HO-1, Maf, Keap1, GCLC, CuZn-SOD were detected in the Lophatherum bream fed for 28 days, 56 days and 56 days after LPS infection according to the fluorescence quantitative PCR method;

[0106] Acquisition of raw data for transcriptome analysis:

[0107] 1) RNA extraction;

[0108] 2) mRNA purification and shearing;

[0109] 3) Synthesize the first strand of cDNA;

[0110] 4) Synthesis of the second strand of cDNA and purification of the second strand product;

[0111] 5) End repair and 3'-end A addition;

[0112] 6) Connecting joints;

[0113] 7) Purification of ligation products and fragment selection;

[0114] 8) PCR amplification;

[0115] 9) Bioinformatics analysis of PCR product purification data, as follows:

[0116] A. Sequencing data and its quality control;

[0117] B. Transcriptome sequencing data assembly;

[0118] C. Transcriptome sequencing library quality assessment;

[0119] D. Unigene functional annotation;

[0120] E. Gene expression analysis;

[0121] F, differential expression analysis;

[0122] The acquisition of raw data for metabolomics analysis is as follows:

[0123] 1) Metabolite extraction;

[0124] 2) On-machine testing;

[0125] 3) Data processing and data analysis:

[0126] A. Data evaluation;

[0127] B. Sample grouping data analysis;

[0128] Real-time quantitative PCR (qPCR) was used to quantitatively verify some differentially expressed genes obtained by transcriptome analysis, β-actin was used as an internal reference, and the cycle (CyclethresholdCt) was used to compare the relative expression levels of differentially expressed genes in liver samples, and the relative quantitative 2-ΔΔCt method was used for calculation;

[0129] The obtained data were processed and analyzed using the 2-ΔΔCT method; after the obtained experimental data were sorted, the data were subjected to one-way analysis of variance using SPSS (20.0) software. If the experimental data analysis of variance was significant, Duncan's multiple comparisons were further performed, and the experimental data were expressed as (mean ± standard error), and P < 0.05 was considered significant. Discriminant analysis (DA) was used to compare two categories, such as the added group and the control group, and its main purpose was to determine the metabolites with different abundances between the two categories; partial least squares / projection (PLS) and orthogonal PLS (OPLS) were used for potential structure analysis.

[0130] in conclusion:

[0131] 1) The activity of the effective components of kelp residues was improved after fermentation with probiotics;

[0132] 2) Adding fermented kelp residue to high-fat diet can regulate the growth performance of P. loblollyii;

[0133] 3) The addition of fermented kelp residue can alleviate the negative effects of high-fat diet on hepatopancreatic lipid metabolism in L.

[0134] 4) Fermented kelp can improve the immune function and antioxidant capacity of the serum of P. loblollyii, thereby alleviating the immune function damage and oxidative stress caused by high-fat diet;

[0135] 5) Fermented kelp has a significant effect on the lipid metabolism of the hepatopancreas of P. loblolly at the transcriptional and metabolic levels. The key pathways affecting the health of the hepatopancreas may be the fatty acid biosynthesis pathway and the bile acid secretion pathway.

[0136] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A high-fat feed added with fermented kelp, characterized in that: The preparation includes the following raw materials: Casein, fish meal, dehulled soybean meal, fish oil, corn oil, wheat bran, flour, fermented kelp residue, wheat starch, monocalcium phosphate, compound premix, lysine, methionine, choline chloride; The specific proportions by percentage are as follows: Casein 4-6%, fish meal 12-18%, peeled soybean meal 32-38%, fish oil 1-4%, corn oil 0-5%, wheat bran 8-12%, flour 18-22%, fermented kelp residue 0-6%, wheat starch 1-10%, monocalcium phosphate 1-5%, compound premix 0.1-4%, lysine 0.1-2%, methionine 0.1-2%, choline chloride 0.1-4%.

2. The high-fat feed added with fermented kelp according to claim 1, characterized in that: The specific proportion of the high-fat feed in percentage is as follows: Casein 5%, fish meal 15%, peeled soybean meal 35%, fish oil 2%, corn oil 4%, wheat bran 10%, flour 18%, fermented kelp residue 4%, wheat starch 1%, monocalcium phosphate 2%, compound premix 1%, lysine 0.5%, methionine 1%, choline chloride 1.5%.

3. The high-fat feed added with fermented kelp according to claim 1, characterized in that: The specific proportion of the high-fat feed in percentage is as follows: Casein 5%, fish meal 15%, peeled soybean meal 35%, fish oil 2%, corn oil 4%, wheat bran 10%, flour 18%, fermented kelp residue 0%, wheat starch 5%, monocalcium phosphate 2%, compound premix 1%, lysine 0.5%, methionine 1%, choline chloride 1.5%.

4. The high-fat feed added with fermented kelp according to claim 1, characterized in that: The specific proportion of the high-fat feed in percentage is as follows: Casein 5%, fish meal 15%, peeled soybean meal 35%, fish oil 2%, corn oil 4%, wheat bran 10%, flour 18%, fermented kelp residue 1%, wheat starch 4%, monocalcium phosphate 2%, compound premix 1%, lysine 0.5%, methionine 1%, choline chloride 1.5%.

5. The high-fat feed added with fermented kelp according to claim 1, characterized in that: The specific proportion of the high-fat feed in percentage is as follows: Casein 5%, fish meal 15%, peeled soybean meal 35%, fish oil 2%, corn oil 4%, wheat bran 10%, flour 18%, fermented kelp residue 2%, wheat starch 3%, monocalcium phosphate 2%, compound premix 1%, lysine 0.5%, methionine 1%, choline chloride 1.5%.

6. An application of a high-fat feed containing fermented kelp as claimed in any one of claims 1 to 5 in the cultivation of P. rostrata, characterized in that: The method comprises the following steps: Step 1: Pre-treat the kelp; Step 2: Weigh the bacteria, brown sugar and water in a ratio of 1:10:200, sterilize the water with high pressure, take it out, and let it cool to 40°C to dissolve the brown sugar; Step 3: activating the finished starter containing Bacillus subtilis, Bacillus licheniformis, Candida utilis, Enterococcus faecalis and Lactobacillus in a brown sugar solution for 30 minutes to complete the preparation of the fermentation liquid; Step 4: Mix the successfully activated fermentation liquid and the pre-treated kelp powder evenly for 4-5 days of primary fermentation; Step 5: Add corn flour, bran and glucose again, and inoculate 5% of the total bacterial solution in the bait; Step 6: Add water and mix until the water content of the bait is 40% to 45%, and the bait can be rubbed into a ball by hand and falls apart when gently kneaded. Then seal it in a transparent anaerobic fermentation bag and ferment it in a 37°C constant temperature incubator for 3 to 5 days. Turn it over once a day. When a special sour aroma is found, the fermentation is considered complete. After the fermented kelp additive is made, dry it and store it in a dry place for later use. Step 7: using fish oil and corn oil as fat sources, fish meal and soybean meal as protein sources, flour and wheat starch as sugar sources, and adding 4.0% fermented kelp by mass percentage; Step 8: crush the raw materials, pass through a 60-mesh sieve, weigh and mix according to the formula, make 1.5 mm particles, dry them in the sun and store them at -4°C; Step nine: obtain the finished high-fat feed.

7. The use of a high-fat feed added with fermented kelp in the breeding of Psoralea corylifolia according to claim 6, characterized in that: In the step 1, the kelp is washed and placed in a 65° C. oven to dry and crush, and passed through a 60-mesh sieve to obtain kelp powder.

8. The use of a high-fat feed supplemented with fermented kelp in the culture of Pseudosciaena sutchuenensis according to claim 6, characterized in that: The total bacterial solution concentration in step 5 is 107 CFU / g, and the mass ratio of LSG1-1 Bacillus licheniformis to LSG2-1 Bacillus subtilis is 5:1.

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