Application of corn feed containing human lysozyme

Abstract

The invention relates to application of a corn feed containing human lysozyme, the corn feed is used for preparing products for preventing and / or treating animal intestinal diseases, and the amino acid sequence of the human lysozyme is shown as SEQ ID NO.1. The plant corn feed containing the human lysozyme is found for the first time, and can improve the intestinal barrier structure, the mitochondrial function and the immune function of animals suffering from intestinal diseases; the method has the advantages of prolonging the length of the intestinal tract of the animal with the intestinal disease, improving the microvillus state of the intestinal tract of the animal with the intestinal disease to enable the intestinal tract to be regular, improving the desmosome connection state of the intestinal tract of the animal with the intestinal disease to enable the intestinal tract of the animal with the intestinal disease to be orderly and clearly arranged, improving the SOD activity of the intestinal tract of the animal with the intestinal disease and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to application of corn feed containing human-derived lysozyme. Background Art

[0002] Currently, methods for improving animal intestinal health primarily include antibiotics and probiotics. When animals develop intestinal bacterial infections, antibiotics can effectively inhibit further bacterial growth. However, the use of antibiotics often leads to excessive antibiotic residues in animals, posing a food safety risk. Probiotics are also a common solution for animals experiencing intestinal dysfunction. However, the higher cost and slower onset of action of probiotics significantly limit their use.

[0003] Lysozyme has numerous health benefits, including antibacterial and immune-boosting properties, making it a promising alternative to overused antibiotics. Compared to other forms of lysozyme, human lysozyme exhibits stronger antibacterial activity. With the increasing demand for antibiotic alternatives in agriculture, human lysozyme, due to its natural, safe, and highly effective properties, has become a research hotspot in the feed additive field. Compared to the other two, human lysozyme offers advantages: greater safety than antibiotics and lower cost than probiotics, making it a key research area.

[0004] CN108850640A discloses the use of a Pichia yeast fermentation product expressing human lysozyme in a broiler feed additive. The weight percentage of the Pichia yeast fermentation product expressing human lysozyme is 0.1% to 10%. Adding lysozyme to the feed can prevent mold, extend the storage period of the feed, reduce the chance of ingesting moldy feed, and prevent animal infection from the source.

[0005] In the livestock industry, plant-based feeds are an important source of feed, with corn accounting for 60% of pig and poultry feed, 20%-50% of ruminant feed, and 10%-30% of aquatic feed. Genetic engineering techniques are also widely used in corn breeding. Genetically modified corn with high accumulation of human lysozyme may be an effective method for increasing lysozyme production.

[0006] However, no studies have yet shown that human lysozyme transgenic plants can be used as feed to improve animal intestinal health. Summary of the Invention

[0007] The purpose of the present invention is to overcome the above limitations, thereby providing an application of corn feed containing human lysozyme.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] The technical solution of the present invention is to provide a corn feed containing human lysozyme for use in preparing products for preventing and / or treating animal intestinal diseases. The amino acid sequence of the human lysozyme is shown in SEQ ID NO.1.

[0010] SEQ ID NO.1:

[0011] MRVLLVALALLALAASATSKVFERCELARTLKRLGMDGYRGMSLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGVHDEL

[0012] In some specific embodiments, the specific activity of human lysozyme in the corn feed is (0.5-2)×10 8 U / kg

[0013] More preferably, the corn feed may be human lysozyme transgenic corn kernels or human lysozyme transgenic corn kernel powder.

[0014] In some embodiments, the intestinal disease includes colitis and digestive tract bacterial infection.

[0015] In some embodiments, the product is a product for preventing the shortening of the intestine of an animal and / or for lengthening the length of the intestine of an animal suffering from an intestinal disease, the intestinal length being shortened due to the intestinal disease.

[0016] In some specific embodiments, the product is a product for preventing intestinal microvilli disorder in animals and / or improving the state of intestinal microvilli in animals suffering from intestinal diseases, so that the intestinal microvilli appear regular.

[0017] In some specific embodiments, the product is a product for preventing the disorder of the intestinal desmosome connection structure of animals and / or improving the intestinal desmosome connection state of animals suffering from intestinal diseases, so that the intestinal desmosome connection structure is arranged in an orderly and clear manner.

[0018] In some specific embodiments, the product is a product for preventing impaired mitochondrial function in the intestines of animals and / or improving mitochondrial function in the intestines of animals suffering from intestinal diseases.

[0019] In some specific embodiments, the product is a product for preventing the reduction of SOD activity in the intestine of an animal and / or increasing SOD activity in the intestine of an animal suffering from an intestinal disease.

[0020] In some specific embodiments, the product is a product for preventing intestinal immune dysfunction in animals and / or improving intestinal immune function in animals suffering from intestinal diseases.

[0021] In some embodiments, the animal is selected from any one of chicken, duck, goose, pig, and mouse.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention is the first discovery of a plant-based corn feed containing human lysozyme. It can improve the intestinal barrier structure, mitochondrial function, and immune function of animals with intestinal diseases. This is reflected in the following: extending the intestinal length of animals with intestinal diseases, improving the microvilli of the intestines of animals with intestinal diseases to make them regular, improving the desmosome junctions of the intestines of animals with intestinal diseases to make them orderly and clear, and increasing the SOD activity in the intestines of animals with intestinal diseases. In addition, the plant-based corn containing human lysozyme of the present invention does not affect corn yield (100-kernel weight, starch content) or corn quality (protein content). BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 To investigate the accumulation and biological activity of human lysozyme protein in transgenic corn kernels.

[0025] Figure 2 The colon length of mice with DSS-induced colitis was changed after feeding transgenic corn containing human lysozyme.

[0026] Figure 3 Transmission electron microscopy was used to observe the effect of human lysozyme transgenic corn feed on the intestinal barrier structure of colitis mice.

[0027] Figure 4 Analysis of the activity of SOD protein in colitis mice fed with human lysozyme transgenic corn feed.

[0028] Figure 5 GO analysis of transcriptome data of intestinal tissues of colitis mice fed a transgenic corn diet containing human lysozyme. DETAILED DESCRIPTION

[0029] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0030] In the following examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0031] Example 1

[0032] Endosperm proteins were extracted from mature transgenic corn kernels and control (CK) corn kernels from ears expressing human lysozyme (HLZ) protein. The kernels were soaked in water to separate the endosperm from the embryo and pericarp. The endosperm samples were dried to constant weight and pulverized in a mortar and pestle under liquid nitrogen. Total protein was extracted from the powder using extraction buffer (12.5 mM sodium borate, pH 10.0, 1% (w / v) SDS, and 2% (v / v) β-mercaptoethanol). To separate alcohol-soluble proteins from total protein, anhydrous ethanol was added to an aliquot of the sample to a final concentration of 70% (v / v). After incubation at 37°C for 30 minutes, the mixture was centrifuged at 12,000 rpm for 10 minutes. The supernatant contained alcohol-soluble proteins, while insoluble proteins were non-alcohol-soluble proteins.

[0033] HLZ protein transgenic corn is a transgenic corn material that expresses HLZ protein in corn endosperm. Its construction process refers to patent ZL 20221 0501552.5, and the amino acid sequence of the human lysate used is shown in SEQ ID NO.1.

[0034] SEQ ID NO.1:

[0035] MRVLLVALALLALAASATSKVFERCELARTLKRLGMDGYRGMSLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGVHDEL

[0036] SDS-PAGE analysis showed that there was a specific protein band of 10 to 15 kD in the total protein and non-alcohol-soluble protein of the endosperm of the transgenic lines, which was consistent with the size of HLZ (14 kD). Figure 1 a).

[0037] Western blotting of endosperm proteins extracted from mature transgenic and non-transgenic kernels using an HLZ antibody confirmed that the new band observed in SDS-PAGE analysis was HLZ. No band was detected in CK. These results indicate that HLZ is highly accumulated in the maize endosperm, with the highest accumulation in transgenic lines 1 and 4 ( Figure 1 b).

[0038] Lysozyme activity was determined using total protein extracted from transgenic endosperm. Mature kernels of transgenic line 4 were ground into a fine powder using a mortar and pestle under liquid nitrogen. After drying, 20 mg of the powder sample was weighed and the lysozyme protein was isolated from the powder using extraction buffer (normal saline, 1% (v / v) PMSF, 2% (v / v) β-mercaptoethanol). Using a lysozyme assay kit (bjbalb, Beijing, China), 0.2 mL of sample was mixed with 2 mL of bacterial suspension and incubated at 37°C for 15 minutes. The transmittance of the bacterial suspension was measured at a wavelength of 530 nm, and the lysozyme activity was calculated according to the lysozyme activity calculation formula.

[0039] The calculation formula of lysozyme activity is:

[0040]

[0041] C 标准 :Standard concentration 2.5μg / mL;

[0042] N: dilution multiple of the sample before testing.

[0043] like Figure 1 c The experimental results showed that the total protein extracted from the transgenic endosperm had a strong lysozyme activity of about 300 U / mg.

[0044] Example 2

[0045] In this example, an 8-week-old DSS (dextran sulphate sodium)-induced acute colitis mouse model was used to perform functional analysis of corn feed rich in human lysozyme protein.

[0046] Mouse diets were customized using kernels of HLZ transgenic maize line 4 and CK kernels.

[0047] The feed formula is as follows:

[0048] Ratio g Energy kcal Casein 130 520 Cystine 3 12 corn starch 187.5 750 maltodextrin 132 528 sucrose 100 400 cellulose 50 soybean oil 0 0 Multiple Minerals 35 Multidimensional 10 40 choline 2.5 Non-GMO / GMO corn flour 350 1750 Human lysozyme activity <![CDATA[1.05×10 8 U / kg]]> total 1000 4000 Mass ratio Energy ratio protein 0.203 0.203 Fat 0.070 0.158 carbohydrate 0.640 0.640

[0049] The groups are as follows:

[0050] ①DSS-induced + HLZ-containing feed group (DSS+HLZ-Feed group); ②DSS-induced + HLZ-free feed group (DSS+CK-Feed group); ③As control: pure water and normal feed group (Vehicle+CK-Feed group).

[0051] Eight-week-old C57BL / 6J mice were randomly divided into groups of six. From the start of the experiment until the end, mice in each group had free access to drinking water and the corresponding HLZ-Feed or CK-Feed. On day 8, the drinking water of the DSS-induced group was replaced with a 3% DSS-containing aqueous solution (ad libitum). On day 12, the 3% DSS-containing solution was replaced with pure water (ad libitum). After four days of feeding, mice were sacrificed for specimen collection.

[0052] After 8 days of DSS induction, the food intake and water intake of mice (DSS+HLZ-Feed group and DSS+CK-Feed group) decreased compared with the control group (Vehicle+CK-Feed group). Macroscopic phenotypic observation showed that the weight of mice began to decrease rapidly from the 11th day, and the fecal trait score and bleeding degree increased significantly ( Figure 2 a).

[0053] ① Stool consistency score refers to the Bristol stool classification system, as follows:

[0054] Type 1: Hard balls (difficult to pass);

[0055] Type II: sausage-shaped, but with a bumpy surface;

[0056] Type 3: sausage-shaped, but with cracks on the surface;

[0057] Type 4: like a sausage or snake, with a smooth surface;

[0058] Type 5: soft block with smooth broken edges (easy to pass);

[0059] Type 6: fluffy masses with rough edges, mushy stools;

[0060] Type 7: Watery, no solid lumps (completely liquid).

[0061] ② The bloody stool scoring standard refers to the DAI scoring standard, which is as follows:

[0062]

[0063] The specific data are as follows:

[0064] Stool consistency score D8 D9 D10 D11 D12 D13 D14 Normal+control feed 0 0 0 0 0 0 0 DSS+control diet 0 0 0 1 1.5 1.5 2.5 DSS+experimental feed 0 0 0 1 1 1 1.5 Bloody stool score D8 D9 D10 D11 D12 D13 D14 Normal+control feed 0 0 0 0 0 0 0 DSS+control diet 0 0 0 2 4 4 4 DSS+experimental feed 0 0 0 2 2 3 4

[0065] These results indicate that the DSS-induced colitis mouse model was successfully established. Figure 2 b and c showed that DSS-induced colitis led to colon shortening (P < 0.001), while the colon length of mice fed HLZ feed (DSS+HLZ-Feed group) was restored (P < 0.05).

[0066] Example 3

[0067] The mouse colon tissue constructed in Example 2 was immersed in paraformaldehyde and post-fixed with osmium tetroxide. The fixed sample was dehydrated to 100% with a gradient of ethanol, then transferred to a propylene oxide solution and slowly embedded in acrylic resin. Thin sections (70 nm) were cut using a diamond knife microtome (Reichert Ultracut E). The sections were placed on a 100-mesh copper grid and stained with uranyl acetate for 30 minutes and lead citrate for 15 minutes. The sections were observed using a transmission electron microscope (Hitachi H7600).

[0068] like Figure 3 Transmission electron microscopy (TEM) observation results of a showed that compared with the Vehicle+CK-Feed group, the intestinal microvilli structure of mice in the DSS+CK-Feed group was severely damaged, and the intestinal microvilli of mice in the DSS+HLZ-Feed group were regular. It can be seen that HLZ feed improved the intestinal microvilli of DSS-induced mice, making them appear regular and villi-like.

[0069] like Figure 3 Transmission electron microscopy (TEM) observation results of b showed that in terms of intestinal barrier integrity and epithelial cell junctions, it was observed that compared with the Vehicle+CK-Feed group, the desmosome junction structure of the DSS+CK-Feed group was disordered and the internal structure was difficult to identify; while the desmosome junction structure of the DSS+HLZ-Feed group was arranged in an orderly and clear manner. It can be seen that HLZ feed also improved the desmosome junction status of DSS-induced colitis mice.

[0070] Example 4

[0071] Superoxide dismutase (SOD) is an important antioxidant enzyme. Increased activity of SOD can enhance the scavenging ability of mitochondria-derived reactive oxygen species (ROS) in cells, thereby achieving functional stability of mitochondria and cells.

[0072] This example uses a SOD detection kit (S0101S, Beyotime) to detect SOD activity. Sample detection was performed according to the instructions provided by the kit, specifically:

[0073] (1) The mouse colon tissue constructed in Example 2 was treated with physiological saline (0.9% NaCl, containing 0.16 mg / ml sodium heparin) to remove blood and obtain a clean colon tissue sample.

[0074] (2) Take an appropriate amount of treated tissue sample and add 100 μl of SOD sample preparation solution per 10 mg of tissue, homogenize on ice, and measure the protein concentration.

[0075] (3) The detection system was prepared according to the instructions. Each reaction system contained 20 μL of protein sample, 160 μL of WST-8 working solution, and 20 μL of reaction starting working solution.

[0076] (4) The reaction system was incubated at 37°C for 30 minutes and then the absorbance was measured using a microplate reader at 450 nm. Six biological replicates were performed for each data set, and the data were read using a microplate reader.

[0077] like Figure 4 The analysis results showed that the SOD level in the colon tissue of mice in the DSS+CK-Feed group was significantly lower than that in the Vehicle+CK-Feed group, while the SOD level in the colon tissue of mice in the DSS+HLZ-Feed group was significantly higher than that in the DSS+CK-Feed group. It can be seen that HLZ feed improved the SOD level affected by DSS.

[0078] Example 5

[0079] Total RNA (10 μg) was extracted from colon tissue (9767, TAKARA) and then RNA concentration and quality were determined using a 3.0 fluorometer. Total RNA integrity was assessed using an Agilent 2100 Bioanalyzer, and samples with a RIN value higher than 7.0 were used for high-throughput sequencing.

[0080] 1 mg of RNA was used for RNA library construction. RNA-seq libraries were prepared according to Illumina's standard instructions (TruSeqStandard RNA LT Guide), quality assessed using an Agilent 2100 Bioanalyzer, and sequenced on an Illumina HiSeq X10 according to the manufacturer's instructions. Differentially expressed genes were defined as those with a fold change greater than 2 or less than 0.5 and a P < 0.05.

[0081] A total of 935 and 476 differentially expressed genes were identified in the comparisons between the DSS+CK-Feed / Vehicle+CK-Feed and DSS+HLZ-Feed / DSS+CK-Feed groups, respectively.

[0082] Cluster analysis based on the gene ontology (GO, https: / / geneontology.org) signaling pathway showed that Figure 5 As shown in a, compared with the Vehicle+CK-Feed group, DSS induced changes in a large number of immune-related pathways in the colon tissue of mice, such as immunoglobulin complexes, T cell association, and MHCII protein binding. Figure 5As shown in b, immune-related pathways were also enriched in the DSS+HLZ-Feed group compared with the DSS+CK-Feed group, such as immunoglobulin complexes, T cell-related pathways, and MHCII protein binding. These results suggest that DSS induces inflammation mainly by altering colonic immune-related signaling pathways, while HLZ feed may improve acute colitis by restoring DSS-induced immune dysfunction.

[0083] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. Use of corn feed containing human lysozyme in the preparation of a product for preventing and / or treating animal intestinal diseases, wherein the amino acid sequence of the human lysozyme is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The specific activity of human lysozyme in the corn feed is (0.5-2)×10 8 U / kg.

3. The use according to claim 1, characterized in that The intestinal diseases include colitis and digestive tract bacterial infection diseases.

4. The use according to claim 1, characterized in that The product is a product for preventing the shortening of the intestinal tract of animals and / or lengthening the intestinal tract length of animals suffering from intestinal diseases.

5. The use according to claim 1, characterized in that The product is a product for preventing intestinal microvilli disorder in animals and / or improving the state of intestinal microvilli in animals suffering from intestinal diseases, so that the intestinal microvilli appear regular.

6. The use according to claim 1, characterized in that The product is a product for preventing the disorder of the desmosome connection structure in the intestine of animals and / or improving the desmosome connection state in the intestine of animals suffering from intestinal diseases, so that the desmosome connection structure in the intestine is arranged in an orderly and clear manner.

7. The use according to claim 1, characterized in that The product is a product for preventing damage to the intestinal mitochondrial function of animals and / or improving the intestinal mitochondrial function of animals suffering from intestinal diseases.

8. The use according to claim 1, characterized in that The product is a product for preventing the reduction of SOD activity in the intestinal tract of animals and / or increasing SOD activity in the intestinal tract of animals suffering from intestinal diseases.

9. The use according to claim 1, characterized in that The product is a product for preventing intestinal immune dysfunction in animals and / or improving intestinal immune function in animals suffering from intestinal diseases.

10. The use according to claim 1, characterized in that The animal is selected from any one of chicken, duck, goose, pig and mouse.

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