A protease and its application in preparing pet gastrointestinal health food

Abstract

The present invention relates to a protease and application thereof in preparing gastrointestinal health food for pets. Specifically, the present invention improves the digestion efficiency of protein (especially fish meal) by evolving the protease derived from Bacillus subtilis. The protease is used in preparing pet food, which can significantly reduce the incidence of intestinal diseases and is beneficial to the intestinal health of pets. The protease of the present invention has good application prospects in the field of pet food.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a protease and an application thereof in preparing gastrointestinal health food for pets. Background Art

[0002] As pet owners pay more and more attention to their pets, the eating habits of pets are gradually changing, and high-protein diets for pets are becoming more common. As a result, the odor of pet feces continues to rise. The odor of pet feces caused by high-protein diets has affected the health of pets and the pet-raising experience of pet owners, and the existing pet food formula components have failed to effectively solve the problem of pet feces odor.

[0003] Enzyme preparations are biological products with catalytic functions after purification and processing, and are mainly used to catalyze various chemical reactions in the production process. It has the characteristics of high catalytic efficiency, high specificity, mild action conditions, reduced energy consumption, and reduced chemical pollution. It is widely used in food, textiles, feed, detergents, papermaking, leather, medicine, energy development, environmental protection and other fields.

[0004] In the pet industry, the application of enzyme preparations has also gradually gained attention. First, enzyme preparations can improve the utilization rate of feed by pets, especially the utilization rate of proteins and minerals, thereby facilitating the nutritional absorption of pets. Second, enzyme preparations can also degrade anti-nutritional factors in pet feed and reduce the burden on the pet's intestines. In addition, enzyme preparations can eliminate the anti-nutritional effects of soluble non-starch polysaccharides, reduce the viscosity of intestinal chyme, and change the environment for the growth of harmful microorganisms, thereby significantly improving the health of pets' intestines.

[0005] In terms of pet intestinal health, the addition of enzyme preparations can improve the digestibility of feed for pet dogs and cats, reduce the damage to the kidneys caused by high-protein diets, and reduce the chance of intestinal inflammation. At the same time, certain ingredients in enzyme preparations can promote the proliferation of beneficial bacteria in the pet's intestines, help regulate the balance of intestinal flora, enhance the intestinal resistance to harmful bacteria, and prevent pets from experiencing diarrhea and other discomfort symptoms.

[0006] The present application provides a protease with high-efficiency degradation ability. Using it in pet food can improve the intestinal health of pets and has broad application prospects. Summary of the invention

[0007] In one aspect, the present invention provides a protease for improving protein digestibility, wherein the amino acid sequence of the protease is compared with that of SEQ ID No.2, and the amino acid at position 110 corresponding to SEQ ID No.2 is mutated to R.

[0008] The 110th amino acid is the amino acid site of SEQ ID No. 2 from the N-terminus.

[0009] Compared with the protease shown in SEQ ID No. 2, the above-mentioned protease has significantly improved protein digestion efficiency, especially the degradation efficiency of fish meal, which can be increased by nearly 1 times.

[0010] On the other hand, the present invention also provides a DNA encoding the above-mentioned protease, a recombinant expression vector comprising the DNA, and a host cell comprising the recombinant expression vector.

[0011] In one embodiment, the host cell is selected from Escherichia coli, Bacillus subtilis, Bacillus licheniformis or yeast.

[0012] On the other hand, the present invention also provides a method for preparing the above-mentioned protease, which comprises the step of fermenting the protease using the above-mentioned host cell; optionally, it also comprises the step of purifying the protease.

[0013] On the other hand, the present invention also provides the use of the above-mentioned protease in protein degradation.

[0014] In one embodiment, the protein is derived from livestock and poultry, for example, chicken, duck, cattle, sheep or pig.

[0015] In one embodiment, the protein is derived from fish, such as fish meal.

[0016] In another aspect, the present invention further provides a pet food or pet feed comprising the above-mentioned protease.

[0017] In one embodiment, the pet food or pet feed further comprises one or any combination of amylase, lipase, fish meal, cassava flour, sweet potato flour, pumpkin flour, miscanthus, pork, duck, beet pulp, chicken fat, and manno-oligosaccharides.

[0018] In one embodiment, the content of the protease in the pet food or pet feed is 0.05%-0.2%, preferably, 0.1%-0.15%, more preferably, 0.1%.

[0019] In one embodiment, the content of the amylase is 0.05%-0.2%, preferably, 0.1%.

[0020] In one embodiment, the content of the lipase is 0.01%-0.1%, preferably, 0.05%.

[0021] In one embodiment, the content of fish meal is 20%-50%, preferably, 45%.

[0022] In one embodiment, the content of cassava flour or sweet potato flour is 10%-20%, preferably, 15%.

[0023] In one embodiment, the content of pumpkin powder, Miscanthus sinensis, pork or duck meat is 5%-10%, preferably 5%.

[0024] In one embodiment, the content of the beet pulp is 1%-5%, preferably 3%.

[0025] In one embodiment, the content of chicken fat is 1%-2%, preferably, 1.5%.

[0026] In one embodiment, the content of manno-oligosaccharide is 0.1%-0.5%, preferably, 0.25%.

[0027] The above contents are all weight percentages.

[0028] On the other hand, the present invention also provides the use of the above-mentioned protease in the preparation of pet food or pet feed.

[0029] On the other hand, the present invention also provides the use of the above-mentioned protease in preparing food or feed that is beneficial to the gastrointestinal health of pets.

[0030] In the present invention, being beneficial to the intestinal health of pets includes reducing the incidence of gastrointestinal diseases of pets and reducing the odor of pet feces.

[0031] In one embodiment, the pet is selected from cats and dogs, preferably, cats.

[0032] The present invention improves the digestion efficiency of protein (fish meal) by evolving the protease derived from Bacillus subtilis; using the protease in preparing pet food can significantly reduce the incidence of intestinal diseases and is beneficial to the intestinal health of pets. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 .Results of hydrolysis efficiency of fish meal by different proteases.

[0034] Figure 2 .The results of the incidence of gastrointestinal diseases in adult cats fed pet feeds containing different proteases.

[0035] Figure 3 .Results of fecal ammonia content in adult cats fed pet feed containing different proteases. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

[0037] In order to make the experimental contents and technical solutions of the present invention more clearly described, the present invention is described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0038] Example 1. Acquisition and optimization of protease from Bacillus subtilis

[0039] According to the literature (US6489108B1), the applicant obtained a protease YqjN from Bacillus subtilis, whose nucleic acid sequence is shown in SEQ ID No.1 and whose amino acid sequence is shown in SEQ ID No.2; it has been reported that the protease can be used in pet feed.

[0040] SEQ ID No.1

[0041]

[0042] SEQ ID No.2

[0043] mnwqtkdellalltslvqyesitgskgevalaeylyfilkdkpyfqkhpddvtphpmddgrsfltalvkkknvkktvllllshfdvvdiedygefkhmackpaellssflekkellpervrrdaesgdwlfgrgtmd mkaglciqlsmleramnghfegnlllitvpdeevnsrgmieavpalkemekkhditltaclnaepmfekfpgdqqqyfytgsigkvlagffckgiethvgepfsglnanlmvseinrllelnadycekvdgevtppp vnlmqkdlkeaysvqtphtavtlfnvlsmkrsaselhqmllktaeqaaeeimsnvrkktqdfqqfepfqpierdvtvltfdelvsrakkragiseteralnyafanrgelgdrdfstkivselaslckedaplivlf yspplypavsskddqlirntadqikhyaaerygialrevqyfpglsdlsylqlekqevdaytsnmplfnrgyslpsgknqalyvpvlnvgpagkdphkwterlympysfevlpdllsftisalkqsesagqllrek

[0044] The applicant analyzed the key amino acids that may affect the activity of YqjN through bioinformatics, including the 40th amino acid (K) and the 110th amino acid (E) of SEQ ID No. 2, and mutated the 40th amino acid K and the 110th amino acid to A and R, respectively, using conventional techniques in the art to obtain mutant proteases K40A, K40R, E110A and E110R.

[0045] Specifically, the variant of YqjN protein was generated by PCR-based site-directed mutagenesis. The specific method is to divide the DNA sequence of YqjN protein into two parts with the mutation site as the center, design two pairs of primers to amplify the two parts of DNA sequence respectively, and introduce the sequence to be mutated on the primers. Finally, the two fragments are loaded into the pcDNA3.3-eGFP vector by Gibson cloning. Fragment amplification kit: TransStart FastPfu DNA Polymerase (containing 2.5mM dNTPs), the specific experimental process is detailed in the instructions. Gel recovery kit: Gel DNA Extraction MiniKit, please refer to the instruction manual for the specific experimental process. Kit used for vector construction: pEASY-Basic Seamless Cloning and Assembly Kit (CU201-03), please refer to the instruction manual for the specific experimental process.

[0046] The protease sequence obtained in this embodiment is shown in the following table:

[0047]

[0048]

[0049] In other embodiments, those skilled in the art may also obtain the protease sequence by other operation methods in the art, for example, obtaining the protease sequence by de novo synthesis.

[0050] Example 2. Expression of protease and verification of fish meal degradation activity

[0051] In this embodiment, Escherichia coli BL21 was used to express and purify the protease obtained in Example 1, and the specific operation was as follows:

[0052] According to the DNA sequences of YqjN, K40A, K40R, E110A and E110R, primers were designed for PCR amplification, and the PCR products were verified by agarose gel electrophoresis, leaving a reaction product with clear bands. The digested PCR products were purified, and the experimental steps were referred to the GV-High-Efficiency DNA Fragments Purification Kit of Beijing Dingguo Changsheng Biotechnology Co., Ltd.; the genes of YqjN, K40A, K40R, E110A and E110R were connected to the plasmid pET28a, respectively, to obtain pET28a plasmids expressing different proteases, which were named pET28a-YqjN, pET28a-K40A, pET28a-K40R, pET28a-E110A and pET28a-E110R, respectively.

[0053] Add 10 μL of purified pET28a plasmid expressing different proteases to 50 μL of E. coli DH5α competent cells, ice bath for 30 minutes; heat shock at 42°C for 90 seconds; ice bath for 2 minutes, add 1 mL of liquid LB, and culture in a shaker at 37°C for 1-1.5 hours; centrifuge at 8000 rpm for 2 minutes, discard the supernatant (leave a little bottom liquid). Spread the remaining solution on an LB plate containing 50 μg / mL kanamycin, and culture it upside down at 37°C overnight; pick a single clone the next day, inoculate it in 5 mL of LB medium containing 50 μg / mL kanamycin, and culture it at 37°C 200 rpm overnight; extract the plasmid.

[0054] Add 2 μL of correctly sequenced pET28a plasmid expressing different proteases to 50 μL of E. coli BL21 competent cells, ice bath for 30 minutes; heat shock at 42°C for 90 seconds; ice bath for 2 minutes, add 1 mL of liquid LB, culture in a shaker at 37°C for 1-1.5 hours; centrifuge at 8000rpm for 2 minutes, and discard the supernatant (leave a little bottom liquid). Spread the remaining solution on an LB plate containing 50 μg / mL kanamycin, spread evenly until dry, and culture inverted at 37°C overnight; pick a single clone the next day, inoculate it in 5 mL of LB medium containing antibiotics, and culture it at 37°C 200rpm overnight to obtain recombinant engineered bacteria containing pET28a plasmids expressing different proteases.

[0055] The fermentation expression and purification of the above-mentioned proteases YqjN, K40A, K40R, E110A and E110R are as follows:

[0056] The recombinant engineered bacteria containing the above-mentioned protease were cultured in 5 mL LB medium (containing 50 μg / mL kanamycin) at 37°C and 200 rpm overnight;

[0057] Transfer the overnight cultured bacterial solution to a 1L Erlenmeyer flask containing 300mL LB medium (containing 50μg / mL kanamycin), and culture at 37°C for about 3h until OD600=0.6-0.8; add IPTG with a final concentration of 0.5mM, and induce the culture at 20°C for 20h; centrifuge at 8000rpm, 4°C for 10min to obtain bacterial precipitate;

[0058] Resuspend the bacteria in NaH2PO4-NaCl buffer at pH 8.0 and place the bacterial solution in a 100 mL centrifuge tube; place the centrifuge tube on ice and disrupt the cells by ultrasound, with the time set to 3s ON, 3s OFF, for a total of 300 times; centrifuge at 11000rpm, 4℃ for 30min; filter the supernatant with a 0.22μm filter, combine the column filler with the filtrate, and prepare for affinity purification.

[0059] The target protein containing 6×His tag was affinity purified using Ni Sepharose 6Fast Flow affinity column from GE Healthcare.

[0060] According to the above operation steps, purified recombinant proteases YqjN, K40R, E110A and E110R were successfully obtained; K40A protease was not purified successfully, which may be related to the fact that amino acid mutations affect protein expression.

[0061] In other embodiments, other expression systems may be used to express and purify the above-mentioned protease, for example, a yeast expression system, a Bacillus subtilis expression system, a Bacillus licheniformis expression system, etc. Other tags may also be used to purify the protein, for example, a GST tag, etc.

[0062] The obtained recombinant proteases YqjN, K40R, E110A and E110R were diluted to 0.1 g / mL for fish meal degradation experiments. Fish meal is often used in pet feed as a high-quality protein. Determining the degradation rate of fish meal can reflect the degradation activity of different proteases, and thus can reflect their digestion efficiency of protein in pet feed.

[0063] Specifically, 100 mL of water was taken, the pH was adjusted to 6.0, and then 10 ml of different recombinant proteases with a concentration of 0.1 g / mL and 50 g of fish meal were added. The mixture was hydrolyzed at 37°C for 6 h, and the remaining fish meal was dried to constant weight. The degradation efficiency of the fish meal was calculated. No protease was added to the control group (CK).

[0064] The results are as follows Figure 1 As shown, compared with YqjN, K40R mutation had almost no effect on the hydrolysis efficiency of fish meal, E110A mutation significantly reduced the hydrolysis efficiency of fish meal, and E110R mutation significantly increased the hydrolysis efficiency of protease on fish meal. This shows that E110R protease is more conducive to protein degradation than wild-type protease YqjN; in the following experiments, the inventors further tested the efficiency of E110R protease in pet feed.

[0065] Example 3. Effects of proteases on pet intestinal health

[0066] In this embodiment, proteases YqjN and E110R are selected and prepared with other beneficial ingredients to prepare pet feed, which is then fed to pets to observe the effects on the intestinal health of the pets.

[0067] In this embodiment, Bacillus subtilis WB800N and expression vector pHT01 commonly used in the art are used to express and purify proteases YqjN and E110R, which is common technical knowledge in the art. In other embodiments, other expression vectors, such as pHT43, may also be used; other host bacteria may also be used for expression and purification, such as Bacillus licheniformis.

[0068] Specifically, in this embodiment, protease is used to prepare a feed for feeding cats, and the specific ingredients of the feed are shown in the following table (the amount of each substance is provided on a weight percentage basis):

[0069] Ingredients Content (wt%) Protease (YqjN or E110R) 0.1 Amylase 0.1 Lipase 0.05 Fishmeal 45 Tapioca Flour 15 Sweet potato flour 15 Pumpkin powder 5 Miscanthus 5 pork 5 duck 5 Beet pulp 3 Chicken fat 1.5 Manno-oligosaccharide 0.25

[0070] In the above table, except for protease, other ingredients such as amylase, lipase, and fish meal are all commercially available products.

[0071] In the above table, the activity of protease is 48120 U / g, the activity of commercial amylase is 20000 U / g, and the activity of commercial lipase is 20000 U / g.

[0072] According to the above table, cat food containing protease YqjN or protease E110R was prepared, and cat food without any protease (0.1% fish meal was added to replace protease) was used as control (CK); a cat feeding test was conducted, 10 adult cats were fed with each cat food, and the incidence of gastrointestinal diseases in adult cats and the average content of ammonia in feces were recorded. The data was derived from the test results of 30 adult cats followed up for 480 hours. The results are as follows Figure 2 and Figure 3 shown.

[0073] like Figure 2 As shown, the incidence of gastrointestinal diseases in adult cats was significantly reduced when cat food containing protease YqjN or protease E110R was fed to adult cats compared with cat food without added protease; among them, cat food containing protease E110R can further reduce the incidence of gastrointestinal diseases in adult cats compared with cat food containing protease YqjN.

[0074] like Figure 3 As shown, when adult cats were fed cat food containing protease YqjN or protease E110R, the average content of ammonia in the feces of adult cats was significantly reduced compared with cat food without adding protease; among them, cat food containing protease E110R can further reduce the content of ammonia in the feces of adult cats compared with cat food containing protease YqjN.

[0075] The above experiments show that compared with protease YqjN, protease E110R can significantly improve the digestibility of protein; using it in pet food or pet feed can promote pets' digestion and absorption of protein, which is beneficial to the intestinal health of pets.

[0076] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A protease for improving protein digestibility, characterized in that: Compared with SEQ ID No. 2, the amino acid sequence of the protease is mutated to R at position 110 corresponding to SEQ ID No.

2.

2. A DNA encoding the protease according to claim 1.

3. A recombinant expression vector comprising the DNA of claim 2.

4. A host cell comprising the recombinant expression vector according to claim 3.

5. A method for preparing the protease according to claim 1, comprising the step of fermenting the protease using the host cell according to claim 4.

6. Use of the protease according to claim 1 in protein degradation.

7. A pet food or pet feed, characterized in that: The pet food or pet feed comprises the protease according to claim 1.

8. The pet food or pet feed according to claim 7, characterized in that: The pet food or pet feed further comprises one or any combination of amylase, lipase, fish meal, cassava flour, sweet potato flour, pumpkin flour, miscanthus, pork, duck, beet pulp, chicken fat, and manno-oligosaccharide.

9. Use of the protease according to claim 1 in preparing pet food or pet feed.

10. The use according to claim 9, characterized in that: The pet food or pet feed is beneficial to the gastrointestinal health of pets.

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