Multifunctional biological protein feed raw material capable of replacing part of soybean meal and application of multifunctional biological protein feed raw material

Abstract

The invention relates to the technical field of biological feed, in particular to a multifunctional biological protein feed raw material capable of replacing part of soybean meal and application of the multifunctional biological protein feed raw material. A large number of experimental studies find that the pichia pastoris protein hydrolysate is used for replacing part of soybean meal in animal daily ration, so that the growth of animals can be effectively promoted, various effects can be exerted, the growth performance of the animals and the apparent digestibility of nutrients in the daily ration can be remarkably improved, and the antioxidant capacity and the immune level of the animals can be remarkably improved; and the Pichia pastoris protein hydrolysate is beneficial to maintaining the integrity of intestinal barriers, improving the micro-ecological environment of the intestinal tracts and the like, so that the Pichia pastoris protein hydrolysate is a biological protein feed raw material which can replace part of soybean meal and has multiple functions.

Description

Technical Field

[0001] The present invention relates to the technical field of biological feeds, and particularly relates to a multifunctional biological protein feed raw material that can replace part of soybean meal and its application. Background Art

[0002] Yeast hydrolysate is a product obtained by externally adding enzyme hydrolysis catalysis or autolysis to yeast, followed by drying or concentration. It is rich in various amino acids, small peptides, glutathione, polysaccharides, nucleotides, vitamins, and trace elements, and is a high-quality and safe protein source. In production, yeast hydrolysate is often used to replace protein raw materials such as fish meal and plasma protein powder. Currently, Saccharomyces cerevisiae and Saccharomyces pastorianus are the most widely used.

[0003] Through existing research, it has been found that the existing yeast hydrolysate has limited effects on growing-finishing pigs. For example, Yi Xianguo et al. have shown that the hydrolysate of Saccharomyces pastorianus can only improve the growth performance, feed efficiency, and the content of serum urea nitrogen in growing pigs, but has no significant effect on the content of serum total protein, creatinine, and carcass grade in growing-finishing pigs. The patent application with the publication number CN119391557A discloses a Pichia pastoris albumin feed additive, and the Pichia pastoris albumin feed is obtained by high-density fermentation of GS115 Pichia pastoris expressing recombinant human albumin. When it is used in pig production, although it can replace part of the soybean meal protein in the diet, it will not have a positive impact on pig growth performance and nutrient digestibility, etc.

[0004] Yeast protein hydrolysate is a green and high-quality resource, and China is rich in yeast strain resources. Exploring yeast protein hydrolysate products that can be effectively used in the field of animal feeds is of great significance for the healthy breeding of animals, especially for the breeding of growing-finishing pigs. Summary of the Invention

[0005] The main purpose of the present invention is to provide a multifunctional biological protein feed raw material that can replace part of soybean meal and its application. Through a large number of experimental studies, the present invention has found that replacing part of the soybean meal in the animal diet with Pichia pastoris protein hydrolysate can effectively promote animal growth and exert various functions, such as significantly improving the growth performance and apparent digestibility of dietary nutrients of animals, significantly enhancing the antioxidant capacity and immune level of animals, helping to maintain the integrity of the intestinal barrier, and improving the intestinal microecological environment, etc. It can be seen that Pichia pastoris protein hydrolysate is a biological protein feed raw material that can replace part of soybean meal and has multiple functions.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, there is provided the application of Pichia pastoris protein hydrolysate in the preparation of a product for promoting animal growth.

[0008] The application includes any one or more of the following (1)-(8):

[0009] (1) Improving growth performance;

[0010] (2) Improving the apparent digestibility of dietary nutrients;

[0011] (3) Reducing blood ammonia levels;

[0012] (4) Improving serum albumin, serum iron and / or creatinine levels;

[0013] (5) Reducing the body's inflammation level and improving the body's immunity;

[0014] (6) Improving antioxidant capacity;

[0015] (7) Reducing intestinal mucosal permeability and enhancing intestinal mucosal barrier function;

[0016] (8) Improving the enrichment of intestinal probiotics.

[0017] In the second aspect of the present invention, there is provided a multifunctional biological protein feed raw material that can replace part of soybean meal. The main component of the multifunctional biological protein feed raw material is Pichia pastoris protein hydrolyzate.

[0018] In the third aspect of the present invention, there is provided a method for promoting the growth of growing-finishing pigs, which is to use Pichia pastoris protein hydrolyzate to replace part of soybean meal in the diet of growing-finishing pigs.

[0019] In the fourth aspect of the present invention, there is provided a feed for promoting the growth of growing-finishing pigs. Calculated by weight, the feed includes the following components:

[0020] 70-75 parts of corn, 15-20 parts of soybean meal, 1-5 parts of Pichia pastoris protein hydrolyzate, 3-5 parts of wheat bran, 1-1.5 parts of soybean oil, 1-1.5 parts of fine stone powder, 1-1.5 parts of calcium hydrogen phosphate, 0.1-0.5 parts of table salt, 0.6-0.65 parts of L-lysine sulfate, 0.03-0.05 parts of DL-methionine, 0.03-0.05 parts of L-threonine, 0.01-0.05 parts of L-tryptophan, 0.3-0.5 parts of premix, 0.03-0.05 parts of phytase, 0.05-0.1 parts of choline chloride, 0.01-0.03 parts of antioxidant.

[0021] Compared with the prior art, the present invention has the following technical advantages:

[0022] The present invention for the first time discovers that the protein hydrolyzate of Pichia pastoris has a significant promoting effect on animal growth. It can improve animal growth performance and the apparent digestibility of dietary nutrients, significantly enhance the antioxidant capacity and immune level, help maintain the integrity of the intestinal barrier, and improve the intestinal microecological environment. Compared with existing yeast hydrolyzate products, the Pichia pastoris protein hydrolyzate described in the present invention has a better comprehensive effect on animal growth, especially for growing-finishing pigs. The present invention confirms that the Pichia pastoris protein hydrolyzate is a multifunctional biological protein feed raw material that can replace part of soybean meal.

[0023] Most existing yeast hydrolyzates are prepared from Saccharomyces cerevisiae or Saccharomyces pastorianus, while the protein hydrolyzate described in the present invention is prepared from Pichia pastoris. On the one hand, compared with other yeasts, Pichia pastoris has the advantages of fast growth and reproduction rate, easy cultivation, low cultivation cost, and suitability for high-density fermentation. The production cost of the protein hydrolyzate described in the present invention is lower. On the other hand, currently, Pichia pastoris is widely used as an expression system for the production of foreign proteins. The application described in the present invention further expands the application field of Pichia pastoris and provides a new direction for the selection of high-quality biological feed protein raw materials. Brief Description of the Drawings

[0024] Figure 1 It is a quadratic regression analysis diagram of the effect of the Pichia pastoris protein hydrolyzate described in the present invention on the growth performance of growing-finishing pigs;

[0025] Figure 2 It is a diagram of the effect of the Pichia pastoris protein hydrolyzate described in the present invention on the α-diversity of intestinal microorganisms in growing-finishing pigs;

[0026] Figure 3 It is the effect of the Pichia pastoris protein hydrolyzate described in the present invention on the β-diversity of intestinal microorganisms in growing-finishing pigs;

[0027] Figure 4 It is the effect of the Pichia pastoris protein hydrolyzate described in the present invention on the composition of intestinal microorganisms at the phylum level;

[0028] Figure 5 It is the effect of the Pichia pastoris protein hydrolyzate described in the present invention on the composition of intestinal microorganisms at the genus level;

[0029] Figure 6 It is the main intestinal microorganisms with significant differences at the genus level;

[0030] Figure 7 It is the LEfSe analysis.

[0031] In the drawings, A - control group, B - treatment group 1, C - treatment group 2, D - treatment group 3, E - treatment group 4. Detailed Embodiments

[0032] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention pertains.

[0033] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0034] To solve the problems described in the background art section, a first aspect of the present invention provides the application of Pichia pastoris protein hydrolysate in the preparation of products for promoting animal growth.

[0035] As a preferred embodiment, the application includes any one or more of the following (1)-(8):

[0036] (1) Improving growth performance;

[0037] (2) Improving the apparent digestibility of dietary nutrients;

[0038] (3) Reducing blood ammonia levels;

[0039] (4) Increasing serum albumin, serum iron, and / or creatinine levels;

[0040] (5) Reducing the body's inflammation level and enhancing the body's immunity;

[0041] (6) Improving antioxidant capacity;

[0042] (7) Reducing intestinal mucosal permeability and enhancing intestinal mucosal barrier function;

[0043] (8) Increasing the enrichment of intestinal probiotics.

[0044] As a preferred embodiment, the improvement of antioxidant capacity includes increasing the activities of superoxide dismutase and glutathione peroxidase, increasing the total antioxidant capacity, and reducing the malondialdehyde content; the reduction of intestinal mucosal permeability and the enhancement of intestinal mucosal barrier function include reducing the activity of diamine oxidase and reducing the content of D-lactic acid.

[0045] As a preferred embodiment, the animals include livestock and poultry.

[0046] Further, the livestock include but are not limited to pigs, cows, sheep, etc. Further still, the pigs are growing and fattening pigs; the poultry include but are not limited to chickens, ducks, geese, etc.

[0047] The second aspect of the present invention provides a multifunctional biological protein feed raw material that can replace part of the soybean meal, and the main component of the multifunctional biological protein feed raw material is Pichia pastoris protein hydrolyzate.

[0048] As a preferred embodiment, the multifunctional biological protein feed raw material consists of Pichia pastoris protein hydrolyzate.

[0049] As a preferred embodiment, the biological protein feed raw material includes any one or several of the following functions (1)-(8):

[0050] (1) Improve growth performance;

[0051] (2) Improve the apparent digestibility of dietary nutrients;

[0052] (3) Reduce blood ammonia levels;

[0053] (4) Increase serum albumin, serum iron and / or creatinine levels;

[0054] (5) Reduce the body's inflammation level and improve the body's immunity;

[0055] (6) Improve antioxidant capacity;

[0056] (7) Reduce intestinal mucosal permeability and enhance intestinal mucosal barrier function;

[0057] (8) Increase the enrichment of intestinal probiotics.

[0058] The third aspect of the present invention provides a method for promoting the growth of growing and fattening pigs, and the method is to use Pichia pastoris protein hydrolyzate to replace part of the soybean meal in the diet of growing and fattening pigs.

[0059] As a preferred embodiment, the proportion of using Pichia pastoris protein hydrolyzate to replace soybean meal is 5%-30%.

[0060] The fourth aspect of the present invention provides a feed for promoting the growth of growing and fattening pigs. Calculated by weight, the feed includes the following components:

[0061] 70 - 75 parts of corn, 15 - 20 parts of soybean meal, 1 - 5 parts of protein hydrolysate of Pichia pastoris, 3 - 5 parts of wheat bran, 1 - 1.5 parts of soybean oil, 1 - 1.5 parts of fine stone powder, 1 - 1.5 parts of calcium hydrogen phosphate, 0.1 - 0.5 parts of table salt, 0.6 - 0.65 parts of L-lysine sulfate, 0.03 - 0.05 parts of DL-methionine, 0.03 - 0.05 parts of L-threonine, 0.01 - 0.05 parts of L-tryptophan, 0.3 - 0.5 parts of premix, 0.03 - 0.05 parts of phytase, 0.05 - 0.1 parts of choline chloride, 0.01 - 0.03 parts of antioxidant.

[0062] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0063] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0064] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0065] In the following examples, unless otherwise specified, the percentage (%) refers to the weight percentage relative to the composition.

[0066] In the following examples, the protein hydrolysate of Pichia pastoris is a commercially available product, and the manufacturer is Ningxia Future Biotechnology Co., Ltd.; the main components and contents of the protein hydrolysate of Pichia pastoris are: crude protein 50.49%, crude fiber 0.07%, crude fat 3.19%, calcium 0.03%, phosphorus 1.73%.

[0067] Example 1

[0068] A method for promoting the growth of growing and finishing pigs: adding 1.25% of the protein hydrolysate of Pichia pastoris to the diet of growing and finishing pigs, which replaces 5.95% of the soybean meal in the diet.

[0069] A feed for promoting the growth of growing and finishing pigs, calculated by weight, the feed includes the following components:

[0070] 70.03 parts of corn, 19.77 parts of soybean meal, 1.25 parts of protein hydrolysate of Pichia pastoris, 3.98 parts of wheat bran, 1.00 parts of soybean oil, 1.39 parts of fine stone powder, 1.09 parts of calcium hydrogen phosphate, 0.30 parts of table salt, 0.60 parts of L-lysine sulfate, 0.05 parts of DL-methionine, 0.04 parts of L-threonine, 0.02 parts of L-tryptophan, 0.34 parts of premix, 0.04 parts of phytase, 0.08 parts of choline chloride, 0.01 parts of antioxidant.

[0071] Example 2

[0072] A method for promoting the growth of growing-finishing pigs: Add 2.5% of Pichia pastoris protein hydrolyzate to the diet of growing-finishing pigs, which replaces 12.03% of soybean meal in the diet.

[0073] A feed for promoting the growth of growing-finishing pigs, calculated by weight, the feed comprises the following components:

[0074] 70.28 parts of corn, 18.27 parts of soybean meal, 2.50 parts of Pichia pastoris protein hydrolyzate, 3.98 parts of wheat bran, 1.00 part of soybean oil, 1.39 parts of fine stone powder, 1.09 parts of calcium hydrogen phosphate, 0.30 part of salt, 0.60 part of L-lysine sulfate, 0.05 part of DL-methionine, 0.04 part of L-threonine, 0.03 part of L-tryptophan, 0.34 part of premix, 0.04 part of phytase, 0.08 part of choline chloride, 0.01 part of antioxidant.

[0075] Example 3

[0076] A method for promoting the growth of growing-finishing pigs: Add 3.75% of Pichia pastoris protein hydrolyzate to the diet of growing-finishing pigs, which replaces 18.32% of soybean meal in the diet.

[0077] A feed for promoting the growth of growing-finishing pigs, calculated by weight, the feed comprises the following components:

[0078] 70.46 parts of corn, 16.72 parts of soybean meal, 3.75 parts of Pichia pastoris protein hydrolyzate, 3.98 parts of wheat bran, 1.00 part of soybean oil, 1.39 parts of fine stone powder, 1.19 parts of calcium hydrogen phosphate, 0.30 part of salt, 0.61 part of L-lysine sulfate, 0.05 part of DL-methionine, 0.04 part of L-threonine, 0.04 part of L-tryptophan, 0.34 part of premix, 0.04 part of phytase, 0.08 part of choline chloride, 0.01 part of antioxidant.

[0079] Example 4

[0080] A method for promoting the growth of growing-finishing pigs: Add 5% of Pichia pastoris protein hydrolyzate to the diet of growing-finishing pigs, which replaces 24.73% of soybean meal in the diet.

[0081] A feed for promoting the growth of growing-finishing pigs, calculated by weight, the feed comprises the following components:

[0082] 70.69 parts of corn, 15.22 parts of soybean meal, 5.00 parts of protein hydrolysate of Pichia pastoris, 3.98 parts of wheat bran, 1.00 part of soybean oil, 1.39 parts of fine stone powder, 1.19 parts of calcium hydrogen phosphate, 0.30 part of table salt, 0.62 part of L-lysine sulfate, 0.05 part of DL-methionine, 0.05 part of L-threonine, 0.04 part of L-tryptophan, 0.34 part of premix, 0.04 part of phytase, 0.08 part of choline chloride, 0.01 part of antioxidant.

[0083] Example 5

[0084] A method for promoting the growth of growing-finishing pigs: Add 2.86% of protein hydrolysate of Pichia pastoris to the diet of growing-finishing pigs, which replaces 13.77% of soybean meal in the diet.

[0085] A feed for promoting the growth of growing-finishing pigs, calculated by weight, the feed comprises the following components:

[0086] 70.28 parts of corn, 17.91 parts of soybean meal, 2.86 parts of protein hydrolysate of Pichia pastoris, 3.98 parts of wheat bran, 1.00 part of soybean oil, 1.39 parts of fine stone powder, 1.09 parts of calcium hydrogen phosphate, 0.30 part of table salt, 0.60 part of L-lysine sulfate, 0.05 part of DL-methionine, 0.04 part of L-threonine, 0.03 part of L-tryptophan, 0.34 part of premix, 0.04 part of phytase, 0.08 part of choline chloride, 0.01 part of antioxidant.

[0087] Example 6

[0088] A method for promoting the growth of growing-finishing pigs: Add 2.79% of protein hydrolysate of Pichia pastoris to the diet of growing-finishing pigs, which replaces 13.43% of soybean meal in the diet.

[0089] A feed for promoting the growth of growing-finishing pigs, calculated by weight, the feed comprises the following components:

[0090] 70.28 parts of corn, 17.98 parts of soybean meal, 2.79 parts of protein hydrolysate of Pichia pastoris, 3.98 parts of wheat bran, 1.00 part of soybean oil, 1.39 parts of fine stone powder, 1.09 parts of calcium hydrogen phosphate, 0.30 part of table salt, 0.60 part of L-lysine sulfate, 0.05 part of DL-methionine, 0.04 part of L-threonine, 0.03 part of L-tryptophan, 0.34 part of premix, 0.04 part of phytase, 0.08 part of choline chloride, 0.01 part of antioxidant.

[0091] Test Example

[0092] 1.1 Test animals and test design

[0093] A total of 180 healthy "Landrace×Large White" crossbred pigs with similar parity, age, and body weight were selected for the experiment. The initial average body weight of the experimental pigs was 31.48±0.91 kg. The experimental pigs were randomly divided into 5 treatment groups, namely the control group (basal diet group), treatment group 1 (with 1.25% addition of Pichia pastoris protein hydrolysate), treatment group 2 (with 2.5% addition of Pichia pastoris protein hydrolysate), treatment group 3 (with 3.75% addition of Pichia pastoris protein hydrolysate), and treatment group 4 (with 5% addition of Pichia pastoris protein hydrolysate). There were 6 replicates in each treatment, and each replicate had 6 pigs (4 boars and 2 sows).

[0094] The Pichia pastoris protein hydrolysate used in this experiment is a commercially available product produced by Ningxia Future Biotechnology Co., Ltd. The main components and contents of the Pichia pastoris protein hydrolysate are as follows: crude protein 50.49%, crude fiber 0.07%, crude fat 3.19%, calcium 0.03%, and phosphorus 1.73%.

[0095] 1.2 Experimental diets

[0096] The experimental diets were formulated as powder according to the NRC (2012) swine nutritional requirement standards. The diet compositions and nutritional levels of each group are shown in Table 1. Diet samples of the control group and the experimental groups were collected, pulverized, passed through a 40-mesh sieve, and stored at -20°C.

[0097] Table 1 Diet compositions and nutritional levels

[0098]

[0099] Note: 1 The premix provided per kilogram of diet: VA 6500 IU, VD3 2400 IU, VE 20 mg, VK3 2.4 mg, VB1 2.4 mg, VB2 6.6 mg, VB6 3 mg, VB 12 0.025 mg, niacin 25 mg, pantothenic acid 13 mg, biotin 0.2 mg, Cu 25 mg, Fe 150 mg, Zn 80 mg, Mn 50 mg, I 0.6 mg, Se 0.3 mg.

[0100] 2 Except for the digestible energy of pigs, other nutritional levels are measured values.

[0101] 1.3 Feeding and management

[0102] The experimental base adopts strict biosafety protection measures to ensure the all-in-all-out of animals and the complete isolation of the breeding personnel. The temperature in the experimental pig house is controlled at 20 - 28 °C, with natural light and normal ventilation. During the entire experiment, the experimental diet is in powder form, and the experimental pigs are allowed to eat and drink freely. They are immunized according to the original pig farm immunization program, and the feces are cleaned daily.

[0103] The pre-feeding period of the experiment is 5 days. In the first 2 days of the pre-feeding period, the basic diet is fed, and on the 1st, 3rd, and 5th days of the pre-feeding period, the basic diet is gradually replaced with 10%, 50%, and 100% of the experimental diet respectively. The formal experimental period is 76 days, during which the health status (diarrhea or other diseases, death, etc.) of the experimental pigs is observed and recorded daily.

[0104] 1.4 Determination indexes and methods

[0105] 1.4.1 Growth performance

[0106] The body weights of the pigs are recorded at the beginning and end of the experiment. Feed is withheld for 12 hours the night before weighing, and the pigs are weighed on an empty stomach in groups of replicates. During the experiment, the daily feed intake is recorded; after the experiment, the feed intakes of each replicate group are counted, and the average daily gain, average daily feed intake, and feed-to-gain ratio are calculated, etc.

[0107] 1.4.2 Apparent digestibility of dietary nutrients

[0108] The acid-insoluble ash method of endogenous indicator is used to determine the apparent total tract digestibility (ATTD) of dietary nutrients. From day 70 to 72 of the experiment, fresh feces are collected regularly. About 400 g of feces are collected from each replicate group every day, 200 g of which is added with 10 mL of 4 mol / L HCl for treatment and stored in a refrigerator at 4 °C. After the experiment, the fecal samples of each replicate group every 3 days are mixed in the same proportion, then dried in an oven at 65 °C, conditioned for 24 hours, and pulverized for standby.

[0109] The content of dry matter (DM) in the diet is determined by the direct drying method (GB / T 6435). The crude protein (CP), ether extract (EE), crude fiber (CF), calcium, total phosphorus, amino acids, and crude ash in the diet and fecal samples are determined by national standard methods (GB / T 6432, GB / T 6433, GB / T 6434, GB / T 6436, GB / T 6437, GB / T 6438). The acid-insoluble ash in the diet and fecal samples is determined with reference to national standard GB / T 23742, and the gross energy (GE) in the diet and fecal samples is determined by an oxygen bomb calorimeter (Parr 6400).

[0110] The calculation formula for the ATTD of dietary nutrients is as follows:

[0111]

[0112] Wherein, AIAd is the content of acid-insoluble ash in the diet; Nf is the content of nutrients in the fecal sample; AIAf is the content of acid-insoluble ash in the fecal sample; Nd is the content of nutrients in the diet.

[0113] 1.4.3 Blood routine, biochemical indexes and immune indexes

[0114] On the 76th day of the experiment, under fasting conditions in the morning, 1 boar was randomly selected from each replicate group of each treatment group, and 3 tubes of blood were collected from the anterior vena cava (1 tube with anticoagulant and 2 tubes without anticoagulant), which were used for the determination of blood routine, serum biochemistry, serum immune indexes and serum antioxidant indexes respectively. The anticoagulated blood was used to detect blood routine indexes such as white blood cell count, red blood cell count, platelet count, hemoglobin content, hematocrit, lymphocyte count, monocyte count, neutrophil count, eosinophil count and basophil count by an automatic blood cell analyzer (Mindray BC-5180CRP); for the blood samples without anticoagulant, serum was prepared after low-temperature centrifugation, and serum biochemical indexes (including alanine aminotransferase activity, aspartate aminotransferase activity, alkaline phosphatase activity, total protein, glucose and urea nitrogen, etc.) were detected by a Roche automatic biochemical analyzer (Cobas c311, Roche); for another blood sample without anticoagulant, serum was prepared after low-temperature centrifugation, and the contents of serum interleukin (IL-2, IL-4, IL-6 and IL-10), tumor necrosis factor (TNF-α) and interferon (IFN-γ), etc. were determined by ELISA method using an enzyme-linked immunosorbent analyzer (Thermo Fisher Scientific Thermo-MK3). At the same time, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC) and malondialdehyde (MDA), D-lactic acid (D-Lactic Acid), DAO and immunoglobulins (IgG, IgM and IgA) were detected. The specific operation steps were carried out strictly according to the kit instructions.

[0115] 1.4.4 Fecal microbiota sequencing

[0116] According to the sampling requirements of the microbiological experiment, fresh feces of the experimental pigs were collected on the morning of the 76th day of the experiment. Fresh feces of 1 pig were collected from each replicate of each group: when it was observed that the pigs began to show defecation reaction, the feces were caught with a sterile plate, and about 2 g of fecal samples at a depth of 3 - 5 cm were collected with a sterile sampling spoon and transferred to a sterile cryopreservation tube, immediately placed in liquid nitrogen, and then transferred to an ultra-low temperature freezer at -80 °C for fecal flora analysis.

[0117] The total microbial DNA of pig fecal samples was extracted using a magnetic bead method DNA extraction kit (Magen, China). After extraction, 2% agarose gel electrophoresis and a UV spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) were used to detect the total amount and quality of the extracted microbial DNA. Specific primers were designed for the V3-V4 hypervariable region of the bacterial 16S rDNA gene with qualified total amount and quality, and PCR amplification was carried out using an ABI type (Applied Biosystems, Foster City, CA, USA) PCR instrument. The specific primer sequences were: 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). Sequencing and data analysis using the Ilumina Novaseq 6000 PE250 (Illumina Inc., San Diego, CA, USA) were completed by Shanghai Yuanxin Biotechnology Co., Ltd. (Shanghai, China). The DNA samples of microorganisms were detected, PCR amplified, purified, library constructed, sequenced, and data analyzed according to the company's standard methods. Software such as Mothur (version v.1.30.1) and Usearch (vsesion 7.0) were used to perform OTU (Operational Taxonomic Units) clustering and bioinformatics statistical analysis on the sequencing sequences at a 97% similarity level.

[0118] 1.5 Statistical analysis

[0119] The experimental data were analyzed by one-way ANOVA using SPSS Statistics 20 software, and Duncan's multiple comparisons were performed for each group. The experimental results were expressed as the mean value of each group and the standard error of the mean (SEM) between groups. A P value less than 0.05 was considered statistically significant, and a P value greater than 0.05 and less than 0.1 indicated a tendency of statistical difference; at the same time, regression curve analysis was used to analyze the linear or quadratic effects between different concentrations of Pichia pastoris protein hydrolysate and evaluation indicators. When the P value of the quadratic effect was less than 0.05, a quadratic regression curve analysis was performed on the addition amount of different concentrations of Pichia pastoris protein hydrolysate and the evaluation indicators.

[0120] 2. Results and analysis

[0121] 2.1 Effects of Pichia pastoris protein hydrolysate on the growth performance of growing-finishing pigs

[0122] The effects of Pichia pastoris protein hydrolysate on the growth performance of finishing pigs are shown in Table 2.

[0123] Table 2 Effects of Pichia pastoris protein hydrolysate on growth performance of growing-finishing pigs

[0124]

[0125] Note: 1 The number of samples in each group n = 6.

[0126] For data in the same row with a, b, c, different letters indicate significant differences between the data (P < 0.05); no letters or the same letters indicate no significant differences. The same applies hereinafter.

[0127] Taking the replicate group as the unit, there were no significant differences in the initial weights of pigs in all experimental groups, and no diarrhea or death occurred in all pigs during the experiment. The addition level of Pichia pastoris protein hydrolysate had linear and quadratic effects on the final weight and average daily gain of finishing pigs (P < 0.05). Compared with the control group, with no significant difference in the initial weight, the final weights of growing-finishing pigs increased by 0.9 kg, 6.1 kg, 4.3 kg, and 1.3 kg respectively, and the average daily gains increased by 0.017 kg, 0.082 kg, 0.056 kg, and 0.015 kg respectively. Among them, the final weight (P < 0.05) and average daily gain (P < 0.01) of the finishing pigs in treatment group 2 with 2.5% Pichia pastoris protein hydrolysate reached a significant level of difference, and the average daily gain of treatment group 3 with 3.75% Pichia pastoris protein hydrolysate also increased significantly (P < 0.05). The average daily feed intakes of the experimental groups with Pichia pastoris protein hydrolysate increased by 0.08 kg, 0.243 kg, 0.132 kg, and 0.106 kg respectively, and the increase in feed intake of treatment group 2 with 2.5% Pichia pastoris protein hydrolysate was the most significant compared with the control group (P < 0.05).

[0128] The quadratic regression analysis of the effect of the addition level of Pichia pastoris protein hydrolysate in the diet on the growth performance of growing-finishing pigs is as Figure 1 shown. The results of the prediction equation show that when the addition amount of Pichia pastoris protein hydrolysate is 2.86%, the final weight of finishing pigs reaches the maximum value of 116.4 kg; when the addition amount of Pichia pastoris protein hydrolysate is 2.79%, the average daily gain reaches the maximum value of 1.118 kg.

[0129] 2.2 Effects of Pichia pastoris protein hydrolysate on apparent digestibility of dietary nutrients in growing-finishing pigs

[0130] The effects of adding Pichia pastoris protein hydrolysate in the diet on the apparent digestibility of dietary nutrients in growing-finishing pigs are shown in Table 3.

[0131] Table 3 Effects of Pichia pastoris protein hydrolysate on apparent digestibility of dietary nutrients in growing-finishing pigs

[0132]

[0133]

[0134] Note: 1 The number of samples in each group n = 6.

[0135] Compared with the control group, adding 2.5% of Pichia pastoris protein hydrolysate in the diet significantly increased the apparent digestibility of gross energy (P<0.01), dry matter (P<0.01), crude protein (P<0.05), crude fiber (P<0.01) and crude ash (P<0.05) in the diet, and the addition level of Pichia pastoris protein hydrolysate had a quadratic effect on the apparent digestibility of crude fiber in the diet (P<0.05). Adding different percentages of Pichia pastoris protein hydrolysate in the diet all increased the apparent digestibility of dry matter, crude protein, crude fiber, etc. to varying degrees.

[0136] 2.3 Effects of Pichia pastoris protein hydrolysate on blood routine and serum biochemical indexes of growing-finishing pigs

[0137] The effects of adding Pichia pastoris protein hydrolysate in the diet on blood routine indexes of growing-finishing pigs are shown in Table 4.

[0138] Table 4 Effects of Pichia pastoris protein hydrolysate on blood routine indexes of growing-finishing pigs

[0139]

[0140] Note: 1 The number of samples in each group n = 6.

[0141] Compared with the control group, treatment group 4 significantly increased the number of eosinophils, basophils and their corresponding proportions in the blood (P<0.05); treatment group 3 increased the mean corpuscular hemoglobin content (P<0.05) and mean corpuscular hemoglobin concentration (P<0.01); while treatment group 2 had a tendency to increase hemoglobin concentration, mean corpuscular hemoglobin content and mean corpuscular hemoglobin concentration (0.05<P<0.1). In addition, the addition level of Pichia pastoris protein hydrolysate had a linear effect on the number of eosinophils and their proportion, basophils and their proportion, mean corpuscular hemoglobin concentration in the blood (P<0.05), and the addition level of Pichia pastoris protein hydrolysate had a quadratic regression effect on the proportion of eosinophils, basophils and mean corpuscular hemoglobin concentration in the blood (P<0.05).

[0142] The effects of adding Pichia pastoris protein hydrolysate in the diet on serum biochemical indexes of growing-finishing pigs are shown in Table 5.

[0143] Table 5 Effects of Pichia pastoris protein hydrolysate on serum biochemical indexes of growing-finishing pigs

[0144]

[0145] Note: 1 The number of samples in each group n = 6.

[0146] As shown in Table 5, compared with the control group, the experimental groups with different concentration gradients of Pichia pastoris protein hydrolysate added in the diet increased the contents of albumin (ALB) and creatinine (CREA) in serum to a certain extent. The serum creatinine levels in treatment group 1 and treatment group 4 increased significantly (P<0.01), and the serum albumin in treatment group 1, treatment group 2 and treatment group 4 increased significantly (P<0.05), indicating that treatment group 2 with 2.5% Pichia pastoris protein hydrolysate added was more conducive to the synthesis of liver protein on the basis of not significantly affecting muscle metabolism. It is worth noting that compared with the control group, adding different concentrations of Pichia pastoris protein hydrolysate could significantly reduce the blood ammonia (NH3) level of finishing pigs (P<0.001), and the blood ammonia level in treatment group 4 with 5% Pichia pastoris protein hydrolysate added decreased most significantly (P<0.001), indicating that Pichia pastoris protein hydrolysate replacing part of soybean meal may reduce the decomposition of tissue amino acids in the body or improve the digestion and absorption of intestinal proteins, which is more conducive to the efficient utilization of nitrogen. In addition, compared pairwise with the control group, adding 2.5% Pichia pastoris protein hydrolysate in the diet increased the content of serum iron (Iron) in finishing pigs (P<0.05).

[0147] 2.4 Effects of Pichia pastoris protein hydrolysate on serum oxidative stress and intestinal permeability-related indexes of growing-finishing pigs

[0148] The results obtained are shown in Table 6.

[0149] Table 6 Effects of Pichia pastoris protein hydrolysate on serum antioxidant and intestinal permeability-related indexes of growing-finishing pigs

[0150]

[0151] Note: 1 The number of samples in each group n = 6.

[0152] As shown in Table 6, compared with the control group, the activities of superoxide dismutase (SOD) (P = 0.001), glutathione peroxidase (GSH-Px) (P < 0.05), and total antioxidant capacity (T-AOC) (P < 0.01) in the sera of treatment groups 2, 3, and 4 were significantly increased to varying degrees, the content of serum lipid peroxide malondialdehyde (MDA) was decreased (P < 0.01), and treatment group 1 could significantly increase the total antioxidant capacity (T-AOC) of fattening pigs (P < 0.05). Diamine oxidase (DAO) and D-lactic acid in the blood are key indicators reflecting intestinal mucosal integrity and intestinal permeability, and their serum levels are positively correlated with intestinal permeability within a certain range.

[0153] Compared with the control group, adding protein hydrolysate of Pichia pastoris could significantly decrease the activity of DAO (P = 0.001) and the concentration of D-lactic acid (P < 0.01). In addition, the addition levels of protein hydrolysate of Pichia pastoris had significant linear and quadratic effects on SOD, GSH-Px, T-AOC, MDA, DAO, and D-LA in the serum (P < 0.01). The above results indicate that adding protein hydrolysate of Pichia pastoris to the diet can improve the antioxidant capacity of fattening pigs, reduce intestinal mucosal permeability, and enhance intestinal mucosal barrier function.

[0154] 2.5 Effects of protein hydrolysate of Pichia pastoris on serum immune-related indexes of growing and fattening pigs

[0155] The results obtained are shown in Table 7.

[0156] Table 7 Effects of protein hydrolysate of Pichia pastoris on serum immune-related indexes of growing and fattening pigs

[0157]

[0158]

[0159] Note: 1 The number of samples in each group n = 6.

[0160] As shown in Table 7, the addition of Pichia pastoris protein hydrolysate to the diet had varying degrees of effects on the levels of anti-inflammatory factors and immunoglobulins in the serum of finishing pigs. Compared with the control group, the treatment 1 group significantly increased the serum anti-inflammatory factor IL-10 (P<0.05), the treatment 2 group significantly increased serum IL-10 (P<0.05), as well as immunoglobulins IgA (P<0.05) and IgM (P<0.01). The treatment 3 group significantly increased the serum anti-inflammatory factors IL-4 (P<0.05) and IFN-γ (P<0.05), as well as immunoglobulins IgG (P<0.05), IgA (P<0.01) and IgM (P<0.001). The treatment 4 group significantly increased the levels of serum IgA (P<0.01) and IgM (P<0.001). In addition, there were both obvious linear relationships (P≤0.001) and quadratic regression effects (P = 0.001) between the changes in the levels of immunoglobulins IgA and IgM and the addition dose of Pichia pastoris protein hydrolysate; there was a quadratic regression relationship (P<0.05) between the change in IL-10 level and the addition dose of Pichia pastoris protein hydrolysate; there was a linear relationship (P<0.05) between the change in IFN-γ level and the addition dose of Pichia pastoris protein hydrolysate. The above measurement results indicate that under normal feeding conditions, adding Pichia pastoris protein hydrolysate to the diet of growing-finishing pigs helps to reduce the body's inflammation level and enhance the body's disease resistance.

[0161] 2.6 Effects of Pichia pastoris protein hydrolysate on the intestinal microbiota composition of growing-finishing pigs

[0162] 2.6.1 Effects of Pichia pastoris protein hydrolysate on the α-diversity and β-diversity of intestinal microbiota

[0163] The Chao and Ace indices measure species abundance, that is, the number of species, and the Shannon and Simpson indices are used to measure species diversity, which are affected by the species abundance and species evenness in the sample community. The results of this experiment show that different levels of Pichia pastoris protein hydrolysate in the diet had significant effects on the α-diversity of the intestinal microbiota of the experimental pigs ( Figure 2 ). As Figure 2 can be seen, compared with the control group, the treatment 1 group and the treatment 3 group reduced the Ace index (P<0.05) and Chao index (P<0.05) of the microbiota, that is, reduced the number of microbial species. The treatment 2 group (2.5%) and the treatment 4 group (5%) had no significant effect on the number of microbial species. Both the Shannon index and the Simpson index are indicators used to estimate microbial diversity. The larger the Simpson index value, the lower the community diversity; the larger the Shannon value, the higher the community diversity. Figure 2The results showed that, compared with the control group, the treatment group 2 (2.5%) significantly reduced the Simpson index (P<0.05), and the Shannon index increased by 4.76%. The above data indicated that adding 2.5% of Pichia pastoris protein hydrolysate to the diet could improve the diversity of intestinal microbiota without affecting the number of intestinal microbial species in growing-finishing pigs, which was beneficial to the stability of the intestinal microecological environment.

[0164] The β-diversity of the microbiota was evaluated using principal coordinate analysis (PCoA) based on unweighted_unifrac ( Figure 3 ). The analysis results showed that there was effective separation between the control group and each experimental group (P<0.01), indicating that adding Pichia pastoris protein hydrolysate had a significant effect on the intestinal microbial function of growing-finishing pigs, and there were obvious differences in the fecal microbial flora among different groups.

[0165] 2.6.2 Effects of Pichia pastoris protein hydrolysate on the microbial composition at the phylum level and genus level of intestinal microbiota

[0166] As Figure 4 known, at the phylum level, the dominant flora included Firmicutes, Bacteroidota, Proteobacteria, Spirochaetota, Desulfobacterota, Actinobacteriota, etc., accounting for more than 96% of the relative abundance at the phylum level.

[0167] At the genus level ( Figure 5) The dominant genera included unclassified Muribaculaceae, Streptococcus, Christensenellaceae_R-7_group, Rikenellaceae_RC9_gut_group, Treponema, Prevotella, Lactobacillus, Prevotellaceae_NK3B31_group, etc. Among them, compared with the control group, the relative abundances of unclassified Clostridia_UCG-014 in treatment group 1 and treatment group 2 were significantly increased (P<0.05), the relative abundances of Lachnospiraceae_XPB1014 in treatment group 1 and treatment group 3 decreased (P<0.05), the relative abundances of unclassified Paludibacteraceae (P<0.05) and Prevotellaceae_UCG-003 (P<0.05) in treatment group 4 were significantly increased, the relative abundance of uncultured Prevotellaceae in treatment group 3 increased (P<0.05), the relative abundance of Bradymonadales in treatment group 1 increased (P<0.05), but the abundance of unclassified Bacteroidales in treatment group 3 decreased (P<0.05)( Figure 6)。Research shows that the abundance of the Clostridia_UCG-014 genus is negatively correlated with colitis, while the abundance of Lachnospiraceae_XPB1014 is positively correlated with body inflammation and blood lipid levels. The changes in their relative abundances are both beneficial to enhancing the intestinal mucosal barrier of the finishing pigs in experimental groups 1, 2, and 3 and reducing the body's inflammation level. The Bradymonadales genus is a facultative predatory bacterium, which is beneficial to the construction of the intestinal microbial community and the recycling of nutrient elements. The increase in the abundance of the Bradymonadales genus in treatment group 1 may have a certain improvement in the stability of the intestinal flora composition or the recycling efficiency of nutrients in finishing pigs. The Prevotellaceae genus is considered to be related to the synthesis of short-chain fatty acids, and the increase in its abundance helps to protect the intestinal mucosal barrier, indicating that treatment groups 3 and 4 may improve the protection of the intestinal mucosa by regulating the synthesis of short-chain fatty acids, and the abundance of the conditional pathogenic bacterium genus Bacteroidales in treatment group 3 decreases compared with the control group. Based on the results of the changes in the abundances of intestinal bacteria at the genus level, it shows that adding different concentrations of Pichia pastoris protein hydrolysate to the diet of growing-finishing pigs can increase the proportion of probiotics to varying degrees, promote the metabolism of nutrients in the intestine, and the addition amounts of 3.75% and 5% are more helpful for enhancing the intestinal mucosal barrier of growing-finishing pigs.

[0168] LEfSe analysis (linear discriminant analysis effect size, LEfSe), that is, LDA Effect Size analysis, can achieve comparisons between multiple groups to find species with significant differences in abundance between groups (i.e., Biomarkers). Figure 7It is a bar chart showing the LDA values of significantly different species, used to display the significantly enriched species within each group and their impact levels. According to the LDA score chart, the treatment group 1 was significantly enriched in Bacteroidia_g_unclassified, Bacteroidia_o_unclassified, Bacteroidia_f_unclassified, Eubacterium__hallii_group, and Christensenellaceae_g_uncultured; the treatment group 2 had significantly different enriched abundances of o_WCHB1_41, c_Kiritimatiellae, WCHB1_41_g_norank, WCHB1_41_f_norank, and g_Dorea; the treatment group 3 was significantly enriched in g_Oribacterium, g_Prevotella_7, g_Faecalibacterium, g_Ligilactobacillus, g_GCA_900066575, Lachnospiraceae_g_uncultured, g_Asteroleplasma, g_Erysipelotrichaceae_UCG_006, and g_Senegalimassilia; the treatment group 4 was significantly enriched in f-Paludibacteraceae, g_Paludibacteraceae_g_unclassified, g_Paludibacteraceae_UCG_001, p_251_o5_g_norank, p_251_o5, g_Anaerostipes, f_Oxalobacteraceae, g_Oxalobacter, and g_Erysipelatoclostridium. From Figure 7 it can be seen that adding Pichia pastoris protein hydrolysate to the diet of finishing pigs helps to enrich some probiotics and metabolism-related bacteria. Moreover, with the increase of the addition amount, the more types of intestinal microorganisms are enriched, and the greater the impact, which is beneficial to reducing the colonization of pathogenic microorganisms and improving the intestinal environment.

[0169] From the above results, it can be known that adding Pichia pastoris protein hydrolysate to the diet can improve the growth performance of growing-finishing pigs and the apparent digestibility of some dietary nutrients, significantly improve the antioxidant capacity and immune level of finishing pigs, help maintain the integrity of the intestinal barrier, and improve the intestinal microecological environment. The Pichia pastoris protein hydrolysate described in the present invention is a multifunctional biological protein feed raw material that can replace part of soybean meal.

[0170] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of Pichia pastoris protein hydrolysate in the preparation of products for promoting animal growth.

2. Use of the Pichia pastoris protein hydrolyzate according to claim 1 in the preparation of a product for promoting animal growth, characterized in that, The said use includes any one or more of the following (1)-(8): (1) Improving growth performance; (2) Improving the apparent digestibility of dietary nutrients; (3) Reducing blood ammonia level; (4) Increasing the levels of serum albumin, serum iron and / or creatinine; (5) Reducing the body's inflammation level and enhancing the body's immunity; (6) Improving antioxidant capacity; (7) Reducing intestinal mucosal permeability and enhancing intestinal mucosal barrier function; (8) Increasing the enrichment of intestinal probiotics.

3. Use of the Pichia pastoris protein hydrolyzate according to claim 2 in the preparation of a product for promoting animal growth, characterized in that, The said improvement in antioxidant capacity includes increasing the activities of superoxide dismutase and glutathione peroxidase, increasing the total antioxidant capacity and reducing the content of malondialdehyde; The said reduction of intestinal mucosal permeability and enhancement of intestinal mucosal barrier function include reducing the activity of diamine oxidase and reducing the content of D-lactic acid.

4. Use of the Pichia pastoris protein hydrolyzate according to claim 1 in the preparation of a product for promoting animal growth, characterized in that, The said animals include livestock and poultry.

5. A multifunctional biological protein feed raw material that can replace part of soybean meal, characterized in that, Its main component is Pichia pastoris protein hydrolysate.

6. The multifunctional biological protein feed raw material capable of substituting part of soybean meal according to claim 5, wherein It consists of Pichia pastoris protein hydrolysate.

7. The multifunctional biological protein feed raw material capable of substituting part of soybean meal according to claim 5 or 6, characterized in that The said biological protein feed raw material includes any one or more of the following (1)-(8) functions: (1) Improving growth performance; (2) Improving the apparent digestibility of dietary nutrients; (3) Reducing blood ammonia level; (4) Increasing the levels of serum albumin, serum iron and / or creatinine; (5) Reducing the body's inflammation level and enhancing the body's immunity; (6) Improving antioxidant capacity; (7) Reducing intestinal mucosal permeability and enhancing intestinal mucosal barrier function; (8) Increasing the enrichment of intestinal probiotics.

8. A method for promoting the growth of growing and finishing pigs, characterized in that, Use Pichia pastoris protein hydrolysate to replace part of the soybean meal in the diet of growing-finishing pigs.

9. The method for promoting the growth of growing and fattening pigs according to claim 8, characterized in that, The proportion of using Pichia pastoris protein hydrolysate to replace soybean meal is 5%-30%.

10. A feed for promoting the growth of growing and fattening pigs, characterized in that, By weight, the said feed includes the following components: 70-75 parts of corn, 15-20 parts of soybean meal, 1-5 parts of Pichia pastoris protein hydrolysate, 3-5 parts of wheat bran, 1-1.5 parts of soybean oil, 1-1.5 parts of fine stone powder, 1-1.5 parts of calcium hydrogen phosphate, 0.1-0.5 parts of salt, 0.6-0.65 parts of L-lysine sulfate, 0.03-0.05 parts of DL-methionine, 0.03-0.05 parts of L-threonine, 0.01-0.05 parts of L-tryptophan, 0.3-0.5 parts of premix, 0.03-0.05 parts of phytase, 0.05-0.1 parts of choline chloride, 0.01-0.03 parts of antioxidant.

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