Application of enzymolysis casein in promoting proliferation of porcine endocrine cells

Abstract

The invention discloses an application of enzymatic casein in promotion of porcine endocrine cell proliferation, belongs to the technical field of animal nutrition and feed, and provides an application of enzymatic casein in promotion of porcine endocrine cell proliferation, which does not aim at diagnosis and treatment of diseases. The invention provides a method for promoting pig endocrine cell proliferation by using casein enzymolysis. The method comprises the following steps: adaptively feeding pigs with daily ration with the crude protein content of 13% for 7 days; mixing the daily ration with the crude protein content of 13% with the enzymolysis casein according to the mass ratio of 95.5: 4.5 to obtain the daily ration containing the enzymolysis casein, and feeding twice a day for 28 days. According to the invention, a certain proportion of casein hydrolysate is added into the feed to regulate and control microbe composition of small intestinal mucosa of growing pigs, so that proliferation of small intestinal endocrine cells is further regulated, the secretion level of gastrointestinal hormone is increased, the gastrointestinal transport function of animals is finally enhanced, the absorption of nutrient substances is accelerated, and the growth performance of the pigs is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of animal nutrition and feed, and particularly relates to the application of enzymatic casein in promoting the proliferation of pig endocrine cells. Background Art

[0002] In modern pig farming, improving the growth rate, feed conversion rate and meat quality of pigs has always been the focus of research. As a key system for regulating animal growth, development, metabolism and immune function, the normal operation of the endocrine system is essential for improving the production performance of pigs. In recent years, studies have shown that by adding specific enzymatic products to feed, it can effectively promote animal intestinal health, improve the absorption and utilization rate of nutrients, and thus affect the differentiation and function of endocrine cells. As a high-quality protein source, casein produces peptides and amino acids that are easier to absorb after enzymatic hydrolysis.

[0003] Casein is the most abundant protein in cow's milk. It contains amino acids necessary for animal growth, development and reproduction and has high nutritional value. For pig feed, its nutritional value depends largely on the content of its active nutrients, especially amino acids and other nutrients that provide energy. However, casein tends to form large clots in the stomach, which slows down gastrointestinal transit. Moderate hydrolysis of casein by protease can improve protein utilization. In addition, the form of protein in the diet also affects its absorption and metabolism, and affects gastric emptying rate and the flow of nutrients in the small intestine, which in turn affects pig growth performance. Enzymatic hydrolysis is a common method for preparing hydrolyzed casein. It can not only be mass-produced at a moderate cost, but also the quality of these products is high. Studies have shown that the secretion of gastrointestinal hormones is affected by the form of protein delivery (peptide or protein binding). Further experiments showed that the increase in serum growth hormone-releasing peptide (ghrelin) may be caused by increasing the secretion capacity of gastric ghrelin. Since the host's hormones are mainly derived from endocrine cells, and there are a large number of endocrine cells in the small intestine, serum ghrelin may also be derived from endocrine cells in the small intestine. Ghrelin-secreting cells (X / A-like endocrine cells) in the small intestine are "open" and are more susceptible to nutrients or intestinal microorganisms in the intestinal cavity. In the small intestinal mucosa, the intestinal microbial flora is the closest to the intestinal endocrine. However, the specific mechanism by which intestinal microorganisms affect the differentiation of endocrine cells remains unclear. Intestinal stem cells mainly differentiate into absorptive and secretory cells, and Notch signaling plays a key role in determining the fate of intestinal cells. However, the interaction between microorganisms and Notch signaling on the differentiation mechanism of intestinal endocrine cells is still not fully understood. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes the use of enzymatic casein in promoting the proliferation of porcine endocrine cells. By adding enzymatic casein to the pig diet, the proliferation of endocrine cells of growing pigs is promoted, the secretion of ghrelin by X / A-like endocrine cells is increased, the gastrointestinal transmission thereof is accelerated, and the daily weight gain of pigs is increased.

[0005] To achieve the above-mentioned purpose, the present invention provides the use of enzymatic casein in promoting the proliferation of porcine endocrine cells, which is not aimed at the diagnosis and treatment of diseases.

[0006] Preferably, the porcine endocrine cells are porcine X / A-like endocrine cells.

[0007] Preferably, the enzymatic casein is added to the pig diet to promote the proliferation of pig X / A-like endocrine cells.

[0008] The present invention also provides a method for promoting the proliferation of pig endocrine cells by using the enzymatic casein, comprising the following steps:

[0009] (1) Pigs were adaptively fed a diet containing 13% crude protein for 7 days and were allowed to eat freely during this period;

[0010] (2) The crude protein content of the diet in step (1) is 13% and enzymatic casein is mixed in a mass ratio of 95.5:4.5 to obtain a diet containing enzymatic casein. The diet containing enzymatic casein is fed twice a day, and the feeding amount each time is calculated as 85% of the mass of the free feed intake, and the feeding is continued for 28 days.

[0011] The present invention also provides a preparation for promoting the proliferation of pig X / A-like endocrine cells, wherein the preparation comprises the enzymatic casein.

[0012] The present invention also provides the use of the enzymatic casein in promoting the secretion of pig ghrelin. The enzymatic casein is added to the pig diet for feeding, so as to promote the proliferation of pig X / A-like endocrine cells and increase the ghrelin secretion of the X / A-like endocrine cells.

[0013] The present invention also provides the use of the enzymatic casein in improving the daily weight gain of pigs. The enzymatic casein is added to the daily diet of pigs for feeding, which promotes the proliferation of pig X / A-like endocrine cells, increases the secretion of ghrelin by X / A-like endocrine cells, and improves the daily weight gain of pigs.

[0014] The invention also provides a feed for increasing the daily weight gain of pigs, wherein the feed comprises the enzymatic casein.

[0015] Preferably, the mass content of enzymatically hydrolyzed casein in the feed is 4.5%.

[0016] The invention also provides the use of the enzymatic casein in increasing the abundance of Lactobacillus bacteria in the pig intestine.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects:

[0018] The present invention regulates the composition of small intestinal mucosal microorganisms of growing pigs by adding a certain proportion of enzymatic casein to feed, further regulates the proliferation of small intestinal endocrine cells, increases the secretion level of gastrointestinal hormones, and ultimately promotes the enhancement of animal gastrointestinal transmission function, accelerates the absorption of nutrients, and improves the growth performance of pigs. Taking growing pigs as the research object, the effect of adding enzymatic casein to the diet on the composition of pig small intestinal mucosal microorganisms and the differentiation and function of endocrine cells is explored, and it is proved that the addition of enzymatic casein to the diet increases the relative abundance and number of lactobacilli in the small intestinal mucosa, increases the number of X / A-like endocrine cells in the small intestine of growing pigs, and promotes the increase of ghrelin levels in serum. The addition of enzymatic casein to the diet enhances the ability of pig small intestinal stem cells to differentiate into secretory cells, and lactobacilli promote the expression of key transcription factors (NGN3) of endocrine cells, and increases the number of X / A-like endocrine cells in the small intestine of pigs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The statistical data of the effects of adding casein or hydrolyzed casein to the diet on the growth performance and serum ghrelin of growing pigs, where Casein is the casein group, Hydrolyzed Casein is the hydrolyzed casein group, a is the result of initial body weight, b is the result of final weight, c is the result of average daily weight gain, and d is the result of serum ghrelin level;

[0021] Figure 2 The statistical data of the effects of dietary casein or enzymatic casein on the differentiation of small intestinal endocrine cells in growing pigs, where Casein is the casein group, Hydrolyzed Casein is the enzymatic casein group, the scale is 200 μm, a is the mRNA level of endocrine cell marker genes in the duodenum, b is the mRNA level of endocrine cell marker genes in the jejunum, c is the mRNA level of endocrine cell marker genes in the ileum, d is the number of X / A-like endocrine cells in the small intestinal villi, and e is the result of immunohistochemical staining of X / A-like endocrine cells in the small intestine;

[0022] Figure 3 The statistical results of the effects of casein or hydrolyzed casein in the diet on the differentiation of small intestinal epithelial cells in growing pigs, where Casein is the casein group, Hydrolyzed Casein is the hydrolyzed casein group, a is the mRNA level of duodenal epithelial cell marker genes, b is the mRNA level of jejunal epithelial cell marker genes, and c is the mRNA level of ileal epithelial cell marker genes.

[0023] Figure 4 The statistical data of the effects of dietary casein or enzymatic casein on the expression of transcription factors in small intestinal endocrine cells of growing pigs, where Casein is the casein group, Hydrolyzed Casein is the enzymatic casein group, a is the mRNA level of transcription factors in duodenal endocrine cells, b is the mRNA level of transcription factors in jejunal endocrine cells, c is the mRNA level of transcription factors in ileal endocrine cells, d is the fluorescence image of jejunal NGN3 protein expression, e is the statistical image of jejunal NGN3 protein expression, f is the fluorescence image of ileal NGN3 protein expression, and g is the statistical image of ileal NGN3 protein expression;

[0024] Figure 5 The statistical data show the effects of adding casein or hydrolyzed casein to the diet on the bacterial genus level in the small intestinal mucosa of growing pigs, where Casein is the casein group, Hydrolyzed Casein is the hydrolyzed casein group, a is the bacterial genus level composition result of the relative abundance of more than 1% in the duodenal mucosa, b is the bacterial genus level result of significant changes in the bacterial genus level in the duodenal mucosa, c is the bacterial genus level composition result of the relative abundance of more than 1% in the jejunal mucosa, d is the bacterial genus level result of significant changes in the bacterial genus level in the jejunal mucosa, e is the bacterial genus level composition result of the relative abundance of more than 1% in the ileal mucosa, and f is the bacterial genus level result of significant changes in the bacterial genus level in the ileal mucosa;

[0025] Figure 6 The statistical data show the effect of dietary casein or enzymatic casein addition on the OTU level of Lactobacillus in the small intestinal mucosa of growing pigs. Casein is the casein group, Hydrolyzed Casein is the enzymatic casein group, a is the relative abundance of Lactobacillus amylovora, b is the relative abundance of Lactobacillus mucosa, and c is the relative abundance of Lactobacillus reuteri.

[0026] Figure 7 The statistical data show the effect of adding casein or hydrolyzed casein to the diet on the number of lactobacilli in the small intestinal mucosa of growing pigs, where Casein is the casein group, Hydrolyzed Casein is the hydrolyzed casein group, a is the number of lactobacilli, b is the number of amylovora, c is the number of mucosal lactobacilli, and d is the number of Lactobacillus reuteri.

[0027] Figure 8The correlation analysis statistics of serum ghrelin, endocrine cell marker genes and microbial flora, a is the correlation analysis result between serum ghrelin level and genus level flora, b is the correlation analysis result between serum ghrelin level and species level flora, c is the correlation analysis result between endocrine cell marker genes and genus level, d is the correlation analysis result between endocrine cell marker genes and species level;

[0028] Fig. 9 The data are statistically analyzed for the effects of in vitro biomimetic digestion of casein or enzymatic casein products on the proliferation of Lactobacillus amylovorus, where Casein is the casein group, Hydrolyzed Casein is the enzymatic casein group, a is the growth curve of Lactobacillus amylovorus, and b is the number of Lactobacillus amylovorus. DETAILED DESCRIPTION

[0029] 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] The enzymatic casein used in the present invention was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0035] Example 1

[0036] 1 Materials and methods

[0037] 1.1 Animals and experimental design

[0038] This experiment was conducted at an animal testing base. A total of 16 (Duroc×Landrace×Yorkshire) crossbred castrated pigs (19.09±0.61kg) were selected and fed individually in metabolic cages (1.6m high; 3.0m long; 2.5m wide). They were fed a basal diet with a crude protein content of 13% for 7 days before the experiment and were allowed to eat freely. After a 7-day adaptation period, they were randomly divided into two groups, with 8 replicates in each group and one pig in each replicate. The experimental group used a basal diet with a crude protein content of 13%, supplemented with 4.5% casein or 4.5% enzymatic casein. Except for the crude protein level, other nutritional levels met the nutritional standards of the National Research Council of the United States. The dietary composition and nutritional levels of the two experimental groups are shown in Tables 1 and 2. During the experiment, all pigs were kept in an air-conditioned metabolic laboratory. In order to eliminate the experimental differences caused by feed intake in the experiment, equal feeding (each feeding amount was 85% of the free feed intake) was adopted to maintain equal energy and nitrogen intake. Feed was provided twice a day (8:00 AM and 17:00 PM) and the experimental period was 28 days.

[0039] Table 1 Composition of daily diet feed

[0040]

[0041]

[0042] Note: Premix 1 Each kilogram of feed contains 8000IU of vitamin A, 2400IU of vitamin D, 20mg of vitamin E, 15mg of pantothenic acid, 5mg of vitamin B6, 3mg of folic acid, 0.03mg of vitamin B12, 40mg of ascorbic acid, 120mg of iron, 25mg of copper, 20mg of manganese, 120mg of zinc, 0.5mg of iodine, and 0.3mg of selenium.

[0043] Table 2 Nutritional levels of dietary feed

[0044]

[0045] 1.2 Tissue sample collection

[0046] The formal experiment lasted 28 days. On the 29th day of the experiment, all experimental animals were slaughtered and sampled. The abdominal cavity was opened and the small intestine of the pig was immediately taken out and divided into the duodenum, jejunum and ileum. Subsequently, part of the mucosa of the duodenum, jejunum and ileum was scraped off, and part of the remaining tissue was placed in 4% paraformaldehyde, and the other part was stored at -80℃ until further analysis.

[0047] 1.3 Measurement indicators and methods

[0048] 1.3.1 Immunohistochemistry

[0049] In the process of preparing small intestinal tissue (duodenum, jejunum and ileum), the samples need to be fixed in 4% paraformaldehyde first and embedded in paraffin after 48 hours. Next, the embedded samples are sliced ​​using a paraffin slicer, and the thickness of each slice is 4μm. Before proceeding to the next step, the paraffin sections need to be dewaxed, and the sections are placed in xylene (15min, 3 times) in sequence, and then immersed in anhydrous ethanol (5min, 2 times), 85% alcohol (5min, 1 time), 75% alcohol (5min, 1 time), and distilled water (5min). After washing with PBS, the sections are antigen-recovered using sodium citrate antigen retrieval buffer (pH 6), and heated in a microwave oven (medium heat for 7min, stop heat for 7min, medium-low heat for 7min). During use, prevent the sections from drying. After cooling, wash them 3 times with PBS (pH 7.4) on a decolorizing shaker for 5min each time. The sections were soaked in 3% hydrogen peroxide solution and incubated for 20 minutes at room temperature in the dark to block endogenous peroxidase. The sections were then blocked with 5% goat serum at 37°C for 30 minutes. After the blocking solution was removed, the sections were dripped with the primary antibody Ghrelin (1:8000, Abcam, USA) diluted with PBS and placed in a humidified box for overnight incubation at 4°C. The sections were dried and incubated with the secondary antibody (HRP-labeled) of the corresponding species to the primary antibody at room temperature for 50 minutes, then stained with freshly prepared DAB, and then counterstained with hematoxylin for 3 minutes. Finally, the sections were placed in 75% alcohol (5 minutes, once), 85% alcohol (5 minutes, once), anhydrous ethanol (5 minutes, twice), and xylene (5 minutes, once) in order to dehydrate and seal the sections. Finally, the positive cells were counted under a microscope for microscopic examination.

[0050] 1.3.2 Real-time fluorescence quantitative PCR

[0051] Total RNA was extracted from duodenum, jejunum, and ileum tissues using TRIZOL reagent (TaKaRa Bio, Otsu, Japan). RNA quality and concentration were analyzed using a UV spectrophotometer (NanoDrop-2000; Thermo Fisher Scientific). The OD of RNA samples was 40.3447 W / m2 and 2.3447 W / m2. 260：280 The value was between 1.8 and 2.1, and the RNA quality was considered qualified. After unifying the concentration to 500 ng / μL, the total RNA was reverse transcribed into cDNA using the PrimeScript RT Master Mix kit (TaKaRa Biotechnology Co., Ltd., Otsu, Japan). Real-time fluorescence quantitative PCR was performed using SYBR Premix Ex Taq dye (TaKaRa Biotechnology Co., Ltd., Otsu, Japan) to detect the expression of related genes. The primers were synthesized by Qingke Biotechnology Co., Ltd. (Nanjing, China), and the detailed sequences are shown in Table 3.

[0052] Table 3 List of target genes and qPCE primers

[0053]

[0054]

[0055] 1.3.3 DNA extraction from small intestinal mucosa

[0056] First, total DNA from the small intestinal mucosa (duodenum, jejunum, and ileum) was extracted to quantify lactobacilli. The mucosal tissue was ground with liquid nitrogen and extracted according to the instructions of the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Hilden, Germany). The specific method is as follows: 1. Before the test, the sample to be tested was placed in a 2 mL centrifuge tube and weighed, and then the centrifuge tube was placed on ice; 2. A volume of 1 mL of InhibitEX Buffer was added to each centrifuge tube with sample. Continue to shake for 1 min until the sample is completely homogenized; 3. Heat the suspension at 70 ° C for 5 min and vortex for 15 s; 4. Centrifuge the sample at 14000 rpm at room temperature for 1 min until a precipitate is precipitated; 5. Transfer 25 μL of Proteinase K to a new 2 mL centrifuge tube; 6. Transfer 600 μL of supernatant from step 5 to a 2 mL centrifuge tube containing Proteinase K; 7. Add 600 μL of Buffer AL and vortex for 15 s; 8. Incubate at 70 ° C for 10 min. 9. Add 600 μL of ethanol (96-100%) to the lysate and vortex to mix. 10. Be careful when transferring the lysate to the QIAamp spin column. After transferring 600 μL of lysate to the spin column, close the lid and centrifuge for 1 minute. Continue to the next step and place the QIAamp spin column in a 2 mL collection tube and discard the tube containing the filtrate. 11. Carefully open the QIAamp spin column before proceeding to the next step. First, add 500 μL of Buffer AW1 to the spin column and centrifuge for 1 minute. Then, transfer the QIAamp spin column to a new 2 mL collection tube and discard the old collection tube. 12. Next, add 500 μL of Buffer AW2 to the spin column and centrifuge for 3 minutes. 13. Place the QIAamp spin column in a new 2 mL centrifuge tube and discard the old collection tube with the filtrate. Centrifuge for 3 minutes; 14. Move the treated QIAamp spin column to a new 1.5 mL centrifuge tube. Next, take 50 μL of BufferATE and transfer it to the QIAamp membrane. Leave it at room temperature for 1 minute, then centrifuge for 1 minute to elute the DNA. Finally, use a NanoDrop-2000 spectrophotometer (Thermo Fisher Scientific) to test the quality and concentration of the DNA.

[0057] 1.3.4 High-throughput sequencing and bioinformatics analysis

[0058] The bacterial 16S rRNA gene V3-V4 region was amplified using bacterial primers 341F (5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.47)) and 806R (5'-TTACCGCGGCTGCTGGCAC-3' (SEQ ID NO.48)). The amplicon library was paired-end sequenced on the Illumina MiSeq platform. All 16S rRNA genes were analyzed using QIIME v1.9.1. After quality inspection, the remaining sequences were clustered into operational taxonomic units (OTUs) based on a 97% similarity cutoff using the UCLUST method. The 16S rRNA gene sequences were sorted and assigned values ​​compared with the SILVA 123 database. To explore the effects of dietary casein or enzymatic casein on the small intestinal mucosal microflora of growing pigs.

[0059] 1.3.5 Lactobacillus quantification

[0060] The absolute quantitative method was used for the quantification of lactobacilli in bacteria. The specific steps for preparing the lactobacillus plasmid standard are as follows: 1) PCR amplification, DNA samples extracted from the mucosa of the duodenum, jejunum and ileum were taken as templates, and PCR amplification was performed using lactobacillus primers (Table 4) with a 50 μL system (25 μL DNA polymerase, 1 μL upstream and downstream primers, 2 μL DNA template, and 21 μL deionized water); 2) PCR product purification, the PCR product of step 1) was subjected to agarose gel electrophoresis, and the band was cut with a clean surgical blade under the UV light of the gel cutter, placed in a sterile 1.5 mL EP tube, and weighed. The purification process used the Sangon DNA purification kit, and the specific experimental steps were carried out according to the instructions; 3) Connection, after obtaining the purified PCR product in the previous step, the rapid DNA connection kit (Sangon Biotechnology, Shanghai) was used for plasmid connection, and the object of connection was pMD-19T plasmid (TakaraBio, Shiga, Japan). The system is as follows: 6 μL of purified PCR product; 1 μL of pMD-19T plasmid; 1 μL of ligation buffer (10); 1 μL of PEG 4000 (50%); 1 μL of T4 DNA ligase. Mix well and incubate overnight at 4°C; 4) Transformation, place 100 μL of competent cells DH5α and 10 μL of ligation solution from the previous step on ice for 30 minutes. Next, place the centrifuge tube in a 42°C water bath for 90 seconds, then immediately place it in an ice bath for 10 minutes; add 1 mL of LB medium without antibiotics to the above bacterial solution, place it in a 37°C constant temperature incubator and culture it for 1 hour; then centrifuge at 4500 rpm for 5 minutes, discard the supernatant, add 40 μL of X-gal solution and 8 μL of IPTG, and mix gently. Spread the above bacterial solution on the LB medium containing ampicillin; culture it in a 37°C incubator overnight. Then, three white colonies were selected from the culture plate, added to LB medium containing ampicillin, and cultured at 37°C for 3 h. The obtained bacterial solution was used for PCR amplification to detect whether there was a target band. Qualified bacterial solution was used for expansion culture, and plasmids were extracted using a plasmid extraction kit (Shanghai Biotechnology Co., Ltd.). Finally, the concentration of plasmid DNA was determined, and the plasmid solution was diluted 10 times in a gradient as a standard curve for lactobacilli.

[0061] Each reaction included 10.0 μL SYBR Green mix, 0.4 μL each of forward and reverse primers, 6.8 μL RNasefree ddH2O, 0.4 μL ROX Reference DyeⅡ, and 2.0 μL DNA. The PCR cycle conditions were as follows: (1) pre-denaturation: 95°C for 30 s; (2) amplification and quantification: 40 cycles, 95°C for 5 s, 60°C for 30 s; (3) fusion: 95°C for 15 s, 60°C for 1 min, and then a melting curve program was performed, the sample was heated from 60°C to 95°C at a heating rate of 0.1°C / s, the fluorescence signal was collected, the CT value was counted, and the bacterial copy number (copy / ng DNA sample) was calculated based on the standard curve. The primers used for bacterial quantification are shown in Table 4.

[0062] Table 4 Lactobacillus primer sequences

[0063]

[0064] 1.3.6 Immunoblotting analysis

[0065] The expression of NGN3 and NOTCH2 proteins in small intestinal tissues was determined by Western blot. The main steps of the experiment were as follows: 1) Grind the jejunal and ileal tissues into powder in liquid nitrogen, weigh 0.3 g, shake and mix in RIPA buffer (Biyuntian, Shanghai, China) containing protease inhibitors, and centrifuge (12000 rpm; 4°C) to collect the supernatant. According to the instructions of the BCA protein assay kit (Biyuntian, Shanghai, China), the protein concentration of the sample was determined and diluted to the same concentration. The diluted protein sample was mixed with 5× loading buffer, heated at 99°C on a metal bath for 10 min, and then cooled on ice for 5 min; 2) SDS-PAGE electrophoresis was performed using FuturePAGETM 4-20% precast gel (ACE, Nanjing), with a voltage of 80 V for the concentrated gel and 120 V for the separation gel; 3) Transfer to membrane, soak the PVDF membrane in methanol for 1 min, and then transfer it to the equilibration solution. After electrophoresis, transfer the membrane using a membrane transfer instrument (GenScript, China); 4) Blocking and antibody incubation. After transfer, place the PVDF membrane in TBST buffer (containing 5% skim milk powder) and incubate on a shaker at room temperature for 1 to 2 hours. After washing the PVDF membrane three times with TBST, place it in a primary antibody working solution of appropriate concentration and incubate it in a refrigerator at 4°C overnight. After taking out the membrane, wash it three times with TBST buffer, each time for 5 minutes. Finally, incubate it in the secondary antibody working solution at room temperature for 1 hour, and then wash the PVDF membrane three times with TBST buffer, each time for 5 minutes. The primary antibodies used included NGN3 (1:1000, Abclonal, China), NOTCH2 (1:1000, Abclonal, China) and β-actin (1:3000, Biosun, China), and the secondary antibody was HRPGoat anti-Rabbit IgG (H+L) (1:5000, Abclonal, China); 5) Imaging detection: absorb the excess liquid with absorbent paper, add BeyoECLPlus (Biyuntian, Shanghai, China) working solution on the membrane, leave it for 2 minutes, and finally use chemiluminescence imager for detection; 6) Quantitative analysis: Use Image J software (NIH, USA) to analyze the gray value of the band, and calculate the relative protein expression of the target protein based on the gray value of the internal reference protein.

[0066] 1.3.7 In vitro culture of Lactobacillus amylovorus S1

[0067] The digestion steps of casein or enzymatic casein are as follows: 1) casein or enzymatic casein (1.0 g) was mixed in 9 mL 1× PBS buffer (pH 7.4); 2) the pH was adjusted to 10 with 1 mol / L HCl. 2.0, pepsin (400U / mg protein from porcine gastric mucosa, product number P7125, SigmaAldrich) was added at a substrate ratio of 1:31.25 (pepsin 0.032g); 3) continuous shaking culture at 37°C for 2h, and then the pH was adjusted to 7.5 with 1mol / L NaOH on ice to inactivate the enzyme; 4) trypsin (0.02g) (1645U / mg protein, from porcine pancreas, lyophilized powder, type II-S, product number T7409, SigmaAldrich) was added at a substrate ratio of 1:50, and the reaction mixture was kept under the same conditions as above (continuous shaking culture at 37°C for 2h). After trypsin digestion for 2h, the enzyme activity was terminated by heating at 95°C in a water bath for 5min; 5) finally, the digestion product was lyophilized; 6) Lactobacillus amylovorus S1 was added to the culture medium with casein and enzymatic casein digestion products as the sole nitrogen source and cultured for 16 hours; 7) the growth curve was determined by intermittently measuring the absorbance at 600 nm (total 16 hours, once every 2 hours); 8) finally, the bacterial culture DNA was extracted, and the amylovorus Lactobacillus S1 was absolutely quantified to determine its number in the bacterial culture.

[0068] 1.4 Statistical analysis

[0069] All data were statistically analyzed using SPSS25.0 software (SPSS Inc, IL, USA). The high-throughput sequencing data were tested using the non-parametric Mann-Whitney U statistical method to detect the differences between the two treatment groups. The difference was significant when P<0.05 after correction. The correlation between the serum ghrelin content, endocrine cell marker gene expression and bacterial relative abundance was analyzed using Spearman correlation analysis. P<0.05 was considered to be significantly correlated between the two indicators. The remaining data were compared between the two groups using the independent sample T test, and the data were expressed as mean ± standard error. *, P<0.05; **, P<0.01; ***, P<0.001 was significantly different, and 0.05≤P<0.10 was a trend of change.

[0070] 2 Results and analysis

[0071] 2.1 Effects of dietary casein or enzymatic casein hydrolysis on growth performance and serum ghrelin in growing pigs

[0072] Ghrelin is also called gastric growth hormone releasing peptide. Figure 1 Middle a, Figure 1 Middle b, Figure 1 Medium c and Figure 1 As shown in Figure d, under the same feed intake, the addition of enzymatic casein to the diet significantly (P<0.05) increased the final weight and average daily weight gain of growing pigs. Compared with the addition of casein to the diet, the addition of enzymatic casein significantly (P<0.05) increased the content of ghrelin in the serum of growing pigs.

[0073] 2.2 Effects of dietary casein or enzymatic casein hydrolysis on differentiation of small intestinal epithelial cells in growing pigs

[0074] 2.2.1 Effects of dietary casein or enzymatic casein hydrolysis on endocrine cell differentiation in growing pigs

[0075] Ghrelin in serum is mainly derived from the stomach and small intestine. The marker genes of X / A-like endocrine cells and other endocrine cells in the small intestine were detected (such as Figure 2 Middle a, Figure 2 Medium b and Figure 2 c). The results showed that compared with the casein group, the enzymatic casein group significantly increased the mRNA expression of GHRELIN in the whole small intestine (P<0.05). In the duodenum, the mRNA expression of GIP, CCK and SST in the enzymatic casein group increased significantly (P<0.05); in the jejunum, the addition of enzymatic casein to the diet significantly increased the mRNA expression of GIP (P<0.01) and CCK (P<0.05); in the ileum, compared with the casein group, the mRNA expression of CCK (P<0.001) in the enzymatic casein group was extremely significantly increased, and the mRNA expression of SCT showed an upward trend (P=0.062).

[0076] In order to verify whether the increase in serum ghrelin was due to the enhanced secretion capacity of X / A-like endocrine cells or the increase in the number of X / A-like endocrine cells, immunohistochemical staining was performed using ghrelin as a marker to measure the number of X / A-like endocrine cells in the small intestine. The results showed that in the duodenum, jejunum, and ileum, the number of X / A-positive cells in the small intestinal villi in the enzymatic casein group (e.g. Figure 2 Medium D and Figure 2 The above data indicate that the increase of serum ghrelin concentration is caused by the increase of X / A-like endocrine cells in the small intestine caused by enzymatic casein hydrolysis.

[0077] 2.2.2 Effects of dietary casein or enzymatic casein on epithelial cell differentiation in growing pigs

[0078] In order to further study whether enzymatic casein affects the differentiation of other epithelial cells, the expression of marker genes of other epithelial cells was detected. Compared with the casein group, the enzymatic casein group significantly increased the expression of intestinal stem cell marker gene (LGR5) mRNA in the jejunum and ileum (P<0.05), and there was a trend of increasing the expression of intestinal stem cell marker gene in the duodenum (P=0.080). No significant difference was observed in the mRNA expression of tuft cell marker gene (DCLK1) and Paneth cell marker gene (LYZ1) between the treatment groups in the jejunum. However, in the duodenum, the addition of enzymatic casein to the diet tended to increase the mRNA expression of DCLK1 (P=0.090); in the ileum, the addition of enzymatic casein to the diet significantly (P<0.05) increased the mRNA expression of DCLK1 (P=0.056), and there was a trend of increasing the expression of LYZ1 mRNA (P=0.056) (such as Figure 3 Middle a, Figure 3 Medium b and Figure 3 The above results indicate that the addition of enzymatic casein to the diet promotes the development of intestinal stem cells and has a certain promoting effect on the differentiation of secretory cells.

[0079] 2.2.3 Effects of dietary casein or enzymatic casein hydrolysis on the expression of transcription factors in small intestinal endocrine cells of growing pigs

[0080] Dietary addition of enzymatic casein significantly increased the expression of endocrine cell subtype marker genes, and endocrine cell differentiation is controlled by many transcription factors. Figure 4 As shown, in the duodenum, compared with the casein group, the mRNA expressions of HES1 (P<0.05) and NEUROD1 (P=0.056) in the casein hydrolyzed group were increased, and the mRNA expression of MATH1 was significantly decreased (P<0.001) ( Figure 4 In the jejunum, casein hydrolysis significantly increased the mRNA and protein expression of NGN3 (P<0.05), and the mRNA expression of HES1 (P=0.057), MATH1 (P=0.095) and ARX (P=0.083) all showed an upward trend (e.g. Figure 4 Middle b, Figure 4 Medium D and Figure 4 The results of ileum showed that the addition of casein to the diet significantly (P<0.05) increased the mRNA expression of MATH1 and significantly (P<0.05) increased the mRNA and protein expression of NGN3, but had no effect on other transcription factors in the ileum (such as Figure 4 Middle c, Figure 4 Medium f and Figure 4 (g) These results suggest that NGN3 is a transcription factor susceptible to casein degradation.

[0081] 2.3 Effects of dietary casein or enzymatic casein hydrolysis on small intestinal microbiota of growing pigs

[0082] In the duodenal mucosal flora, there are 10 genera with a relative abundance greater than 1%, including Lactobacillus, Halomonas, Mitsuokella, Arcobacter, Helicobacter pylori, Prevotella 9, Megasphaera, Campylobacter, Succinivibrio and unidentified Veillonellaceae (e.g. Figure 5 Compared with the casein group, enzymatic casein had a tendency to increase the relative abundance of Lactobacillus (P=0.083) and decrease the relative abundance of Megasphaera (P=0.065), and significantly decreased the relative abundance of Campylobacter (P<0.05) and Succinivibrio (P<0.01) ( Figure 5 (b)

[0083] In the jejunal mucosal flora, there are 13 genera with a relative abundance greater than 1%, including Lactobacillus, Mycoplasma, Halomonas, Helicobacter pylori, unidentified_Chloroplast, Streptococcus, Macrosphaeria, Shigella, Veillonella, Mitsuoka, Sphingomonas, Enterobacter, and Pelagibacterium ( Figure 5 c). Compared with the casein group, enzymatic casein significantly increased the relative abundance of Lactobacillus, Veillonella, and Mitsuoka (P<0.05), and tended to increase Sphingomonas (P=0.083). In addition, enzymatic casein also significantly reduced the relative abundance of Mycoplasma and Streptococcus (P<0.05) in the jejunal mucosa. Figure 5 (d)

[0084] In the ileal mucosal flora, there were 17 genera with a relative abundance greater than 1%, including Lactobacillus, Macrosphaera, Mycoplasma, Campylobacter, Veillonella, Clostridium sensu stricto 1, Halomonas, Terrisporobacter, Helicobacter pylori, Sarcina, Mitsuoka, Romboutsia, Streptococcus, Actinobacillus, Shigella, Arcobacter, and Sphingomonas. Figure 5 Compared with the casein diet, the enzymatic casein diet significantly increased the relative abundance of Lactobacillus and Streptococcus (P<0.01), and significantly decreased the relative abundance of Octacocin (P<0.05). Figure 5 (f)

[0085] At the OTU level, since the genus Lactobacillus showed differences in the three intestinal tracts, the focus was on analyzing the OTUs related to Lactobacillus, mainly including Lactobacillus amylovorus (OTU1), Lactobacillus mucosae (OTU9), and Lactobacillus reuteri (OTU17). Figure 6 Middle a, Figure 6 Medium b and Figure 6 As shown in middle c, in the jejunum, enzymatic casein significantly increased the relative abundance of Lactobacillus amylovorus and Lactobacillus mucosae (P<0.05) compared with the casein diet. In the duodenum, enzymatic casein significantly increased the relative abundance of Lactobacillus reuteri (P<0.01).

[0086] 2.4 Effects of dietary casein or enzymatic casein addition on the number of Lactobacillus in the small intestinal mucosa of growing pigs

[0087] To further confirm the effect of dietary casein or enzymatic casein on the growth of Lactobacillus in the small intestinal mucosa of pigs, real-time fluorescence quantitative PCR was used to determine the number of Lactobacillus in the small intestinal mucosa. The results showed that compared with the casein group, the enzymatic casein group significantly increased the number of total Lactobacillus in the jejunum and ileum (P<0.05). In the duodenum, the number of total Lactobacillus in the enzymatic casein group (P=0.080) tended to increase compared with the casein group (e.g. Figure 7 In the whole small intestinal mucosa, the number of Lactobacillus amylovorus in the casein group was significantly higher than that in the casein group (P<0.05). Figure 7 Compared with the casein group, the number of Lactobacillus mucosae in the jejunum and ileum of the enzymatic casein group was significantly increased (P<0.05). Figure 7 There was no difference in the number of Lactobacillus reuteri between the two treatment groups (Fig. Figure 7 (d)

[0088] 2.5 Correlation analysis between serum ghrelin, endocrine cell marker genes and microbial flora

[0089] The above results show that the addition of enzymatic casein to the diet significantly changed the microbial flora of the small intestinal mucosa, especially the effect on the jejunal mucosal lactobacilli. Therefore, the correlation analysis between serum ghrelin levels, endocrine cell marker genes and genus and species levels of bacteria in the jejunal mucosa of pigs was conducted. Figure 8 As shown in the results, at the genus level, serum ghrelin concentration was significantly positively correlated with Lactobacillus (P<0.001) and Helicobacter (P<0.05), and significantly negatively correlated with Streptococcus (P<0.05); the marker genes of X / A-like endocrine cells were significantly positively correlated with Lactobacillus (P<0.001). At the species level, serum ghrelin levels were significantly positively correlated with Lactobacillus amylovorus; the marker genes of X / A-like endocrine cells were significantly positively correlated with Lactobacillus amylovorus (P<0.001). The above results indicate that Lactobacillus amylovorus may affect the level of serum ghrelin and the expression of related marker genes.

[0090] 2.6 Effects of in vitro biomimetic digestion of casein or enzymatic casein hydrolysis products on the proliferation of Lactobacillus amylovorus

[0091] In order to simulate the changes that casein or enzymatic casein undergoes when entering the small intestine from the stomach, and to explore whether enzymatic casein has an effect on the proliferation of the key lactobacillus (Lactobacillus amylovorus), Lactobacillus amylovorus was cultured in a modified MRS medium, with pepsin-trypsin digested casein or enzymatic casein residues as the only nitrogen source (as shown in Table 5). Fig. 9As shown in Figure 2, after casein or enzymatic casein digestion, the digestion residue of enzymatic casein is more conducive to the growth of Lactobacillus amylovorus, while the digestion residue of casein is not as effective as the digestion residue of enzymatic casein in promoting the growth of Lactobacillus amylovorus (e.g. Fig. 9 qPCR results showed that at 16 h, the number of Lactobacillus amylovorus in the digestion residue of the enzymatic casein group was significantly higher than that of the casein group (P<0.001, Fig. 9 (b)

[0092] Table 5 Single nitrogen source culture medium formula

[0093] Culture medium Casein Enzymatic casein Unit: g / L Casein residue 10.00 - g Enzymatic casein residue - 10.00 g glucose 20.00 20.00 g Dipotassium hydrogen phosphate 2.00 2.00 g Triammonium citrate 2.00 2.00 g Sodium acetate 5.00 5.00 g Magnesium sulfate 0.20 0.20 g Manganese sulfate 0.05 0.05 g Twain 80 1.00 1.00 mL

[0094] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of enzymatic casein in promoting the proliferation of porcine endocrine cells, characterized in that: It is not intended for the diagnosis or treatment of disease.

2. The application according to claim 1, characterized in that: The porcine endocrine cells are porcine X / A-like endocrine cells.

3. The use according to claim 1 or 2, characterized in that: The enzymatic casein is added into the diet of pigs for feeding, so as to promote the proliferation of pig X / A-like endocrine cells.

4. A method for promoting the proliferation of porcine endocrine cells using enzymatic casein as claimed in claim 1, characterized in that: The following steps are involved: (1) Pigs were adaptively fed a diet containing 13% crude protein for 7 days and were allowed to eat freely during this period; (2) The crude protein content of the diet in step (1) is 13% and enzymatic casein is mixed in a mass ratio of 95.5:4.5 to obtain a diet containing enzymatic casein. The diet containing enzymatic casein is fed twice a day, and the feeding amount each time is calculated as 85% of the mass of the free feed intake, and the feeding is continued for 28 days.

5. A preparation for promoting the proliferation of porcine X / A-like endocrine cells, characterized in that: The preparation comprises the enzymatically hydrolyzed casein according to claim 1.

6. The use of enzymatic casein in promoting the secretion of pig ghrelin as claimed in claim 1, characterized in that: The enzymatic casein is added to the daily diet of pigs for feeding, so as to promote the proliferation of pig X / A-like endocrine cells and increase the secretion of ghrelin by X / A-like endocrine cells.

7. The use of enzymatic casein in improving daily weight gain of pigs as claimed in claim 1, characterized in that: The enzymatic casein is added to the daily diet of pigs for feeding, which promotes the proliferation of pig X / A-like endocrine cells, increases the secretion of ghrelin by X / A-like endocrine cells, and improves the daily weight gain of pigs.

8. A feed for increasing the daily weight gain of pigs, characterized in that: The feed comprises the enzymatically hydrolyzed casein according to claim 1.

9. The feed according to claim 8, characterized in that: The mass content of enzymatic casein in the feed is 4.5%.

10. Use of enzymatic casein as claimed in claim 1 in increasing the abundance of Lactobacillus bacteria in the intestinal tract of pigs.

Images