Venison oligopeptide solution as well as preparation method and application thereof

Abstract

The invention provides a venison oligopeptide solution as well as a preparation method and application thereof, and belongs to the technical field of health food development. The preparation method of the venison oligopeptide solution comprises the following steps: taking venison as a raw material, performing degreasing treatment by using a degreasing enzyme solution, and removing an enzyme hydrolysis product; inoculating the degreased venison with a fermentation inoculant, carrying out solid state fermentation treatment and liquid state fermentation treatment, and filtering after fermentation is completed; carrying out enzymolysis treatment on the fermented venison by using a protease solution, taking supernate after enzymolysis, carrying out enzyme deactivation, and filtering to intercept a 5kDa component, so as to obtain a venison oligopeptide solution; the venison oligopeptide solution disclosed by the invention is high in polypeptide yield, the antioxidant activity and the content of essential amino acids are obviously improved, the venison oligopeptide solution oral liquid is further developed, and the oral liquid is rich in nutrition, free of fishy smell of venison, pleasant in flavor and unique in taste.

Description

Technical Field

[0001] The present invention belongs to the technical field of health food development, and particularly relates to a deer meat oligopeptide solution, a preparation method thereof, and an application thereof. Background Art

[0002] Deer meat is a kind of meat with extremely high nutritional value. It is rich in various nutrients such as protein, fat, minerals, and vitamins, and has many benefits for human health. For example, the protein content in deer meat is relatively high, and the amino acid composition is relatively reasonable, which is easy to be absorbed and utilized by the human body; most of the fat in deer meat is unsaturated fatty acids, which helps to reduce cholesterol and prevent cardiovascular diseases; in addition, deer meat also contains rich minerals such as iron and zinc, as well as vitamin B group, etc., which can enhance human immunity and promote metabolism.

[0003] However, in traditional eating methods, deer meat has some limitations. On the one hand, the fishy smell of deer meat is relatively strong and the taste is relatively rough, which makes many people have a low acceptance of it; on the other hand, the processing difficulty of deer meat is relatively large. Traditional cooking methods are difficult to completely remove its fishy smell, and at the same time, it may also cause the loss of nutrients.

[0004] In order to give full play to the nutritional value of deer meat and meet people's needs for healthy and delicious foods, it is particularly important to develop deep processing technologies for deer meat. As a kind of small molecule protein, oligopeptide has the advantages of small molecular weight, easy absorption, high biological activity, etc., and has received extensive attention in the field of health food in recent years. By preparing deer meat into oligopeptides, the utilization rate of its nutritional value can be improved, and at the same time, its taste and flavor can also be improved.

[0005] At present, although there have been some studies on the preparation methods of deer meat oligopeptides, there are still some deficiencies. For example, the polypeptide yield of some preparation methods is relatively low, resulting in waste of resources; there are also some methods in which the antioxidant activity and essential amino acid content of the prepared deer meat oligopeptides are not high, affecting the exertion of their health care functions. Therefore, it is urgent to develop an efficient preparation method for deer meat oligopeptides to improve the polypeptide yield, enhance the antioxidant activity, enrich the essential amino acid content, and apply it to the development of functional oral liquids, so as to provide consumers with a food with rich nutrition, good taste, and specific health care functions. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a preparation method for a deer meat oligopeptide solution, with a high polypeptide yield, significantly improved antioxidant activity and essential amino acid content, and further develop an oral liquid of the deer meat oligopeptide solution. This oral liquid is rich in nutrition, has no fishy smell of deer meat, and has a pleasant flavor and unique taste.

[0007] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0008] A preparation method of a deer meat oligopeptide solution, comprising the following steps: using deer meat as a raw material, performing defatting treatment with a defatting enzyme solution and removing the enzyme hydrolysis products; inoculating the defatted deer meat with a fermentation inoculum for solid-state fermentation treatment and liquid fermentation treatment, and filtering after fermentation; subjecting the fermented deer meat to enzymatic hydrolysis treatment with a protease solution, taking the supernatant after enzymatic hydrolysis and inactivating the enzyme, and intercepting the 5 kDa component by filtration to obtain the deer meat oligopeptide solution;

[0009] The defatting enzyme solution comprises alkaline lipase and lipase;

[0010] The fermentation inoculum comprises Staphylococcus carnosus GDMCC No.1.955 and Lactobacillus brevis CGMCC No.31284;

[0011] The protease solution comprises trypsin, papain and neutral protease.

[0012] Preferably, the defatting enzyme solution is prepared by mixing alkaline lipase and lipase in a mass ratio of 1:(1.5 - 2), and then adding water in a solid-liquid ratio of 1:(1 - 2).

[0013] Preferably, the defatting treatment comprises: spraying the defatting enzyme solution to moisten the surface of the deer meat, and performing enzymatic hydrolysis at 50 - 60 °C for 25 - 35 min.

[0014] Preferably, the fermentation inoculum is prepared by activation culture, acclimation culture, expansion culture and mixing, including: activation culture: using MRS medium as the activation medium, inoculating the strain in a mass ratio of 2% - 4%, and culturing at 28 - 33 °C for 20 - 28 h; acclimation culture: preparing acclimation media with MRS and deer meat pulp in mass gradients of 10:0, 8:2, 6:4, 4:6, 2:8 and 0:10 respectively, inoculating the activated strain in a mass ratio of 2% - 4%, and culturing at 28 - 33 °C for 20 - 28 h; expansion culture: using deer meat pulp as the expansion medium, inoculating the acclimated strain in a mass ratio of 2% - 4%, and culturing at 28 - 33 °C until the viable count ≥ 1×10 8 CFU / g; mixing the expanded Staphylococcus carnosus GDMCC No.1.955 and Lactobacillus brevis CGMCC No.31284 in a mass ratio of 1:(1 - 1.5).

[0015] Preferably, the solid-state fermentation treatment comprises: inoculating the fermentation inoculum at 1% - 3% of the deer meat mass, and fermenting at 28 - 33 °C for 16 - 24 h; the liquid fermentation treatment comprises: adding water at 0.8 - 1 L / kg based on the mass of the solid-state fermentation treatment product, and fermenting at 35 - 38 °C for 46 - 52 h.

[0016] Preferably, the protease solution is prepared by mixing trypsin, papain and neutral protease in a mass ratio of (0.8 - 1):(1.5 - 2):(1 - 1.5).

[0017] Preferably, the enzymatic hydrolysis treatment of the protease solution includes: the addition amount of the protease solution is 1500 - 2000 U / g, and it is enzymatically hydrolyzed at 35 - 38 °C for 8 - 10 h.

[0018] The present invention also provides a deer meat oligopeptide solution prepared by the above preparation method.

[0019] The present invention also provides the application of the deer meat oligopeptide solution in the preparation of a functional oral liquid.

[0020] The present invention also provides a functional oral liquid, which includes the following components by mass fraction: 70% - 75% of the deer meat oligopeptide solution, 0.04% - 0.06% of turmeric extract, 0.06% - 1% of perilla leaf juice, 4.4% - 6.8% of flavoring agent, 0.1% - 0.2% of stabilizer, and the balance is water.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides a preparation method of a deer meat oligopeptide solution. Through degreasing treatment with a degreasing enzyme, combined treatment of solid-state fermentation and liquid fermentation with a fermentation inoculum, and enzymatic hydrolysis treatment with a protease and retention of the 5 kDa fraction, the yield of polypeptides is effectively improved, and the antioxidant activity and the content of essential amino acids are significantly increased. Using alkaline lipase and lipase to remove the fat on the surface of deer meat makes better use of deer meat protein and avoids negative impacts on the subsequent combined extraction of polypeptides by bacteria and enzymes. The fermentation method combining solid-state fermentation and liquid fermentation enables the large-scale reproduction of strains and the rapid metabolism of microorganisms, increasing the polypeptide content. The deer meat oligopeptide prepared by compound protease has higher antioxidant activity, is more easily absorbed by the human body, has a high content of essential amino acids for the human body, and is rich in health care function value.

[0023] The present invention further develops a functional oral liquid of deer meat oligopeptide solution. On the basis of the deer meat oligopeptide solution, the addition of perilla leaf juice and turmeric extract makes the oral liquid have a unique flavor, good taste, rich nutritional value, no fishy smell of deer meat, convenient to take, and meets the nutritional value requirements of modern people.

[0024] Biological material preservation description

[0025] The biological material involved in the present invention is Levilactobacillus brevis X4-4, taxonomically named Levilactobacillus brevis. It is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (CGMCC). The address of the preservation unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation number is CGMCC No. 31284, and the preservation date is July 12, 2024. Staphylococcus carnosus GDMCC No. 1.955 was purchased from the Guangdong Microbial Culture Collection Center (GDMCC), and the purchase website is https: / / gdmcc.net / # / resourceDetail?detailId=%22868%22. Detailed implementation manners

[0026] The present invention provides a method for preparing a deer meat oligopeptide solution, which includes degreasing treatment with a degreasing enzyme, two-stage treatment of solid-state fermentation + liquid-state fermentation with a fermentation inoculum, enzymatic hydrolysis treatment with a protease, and filtering and retaining the 5 kDa fraction to obtain the deer meat oligopeptide solution. The specific steps are as follows:

[0027] In the present invention, it is preferred to pretreat the deer meat. After removing broken bones, congestion, injury spots, lymph nodes, dirt, and foreign impurities from the raw deer meat, it is minced through a meat grinder with a 6-12 mm orifice plate, and more preferably minced through a 10 mm orifice plate, which is beneficial for subsequent enzymatic hydrolysis and fermentation treatments.

[0028] In the present invention, a degreasing enzyme solution is used to degrease the deer meat and remove the enzymatic hydrolysis products. Among them, the degreasing enzyme solution includes alkaline lipase and lipase. It is preferred that the degreasing enzyme solution is prepared by mixing alkaline lipase and lipase in a mass ratio of 1:1.5 - 2, and then adding water according to a solid-liquid ratio of 1:1 - 2. More preferably, alkaline lipase and lipase are mixed in a mass ratio of 1:2, and then water is added according to a solid-liquid ratio of 1:1. It is preferred that the degreasing treatment includes spraying the degreasing enzyme solution to moisten the surface of the deer meat, and enzymatic hydrolysis at 50 - 60 °C for 25 - 35 min, and more preferably enzymatic hydrolysis at 55 °C for 30 min. It is preferred that removing the enzymatic hydrolysis products includes shaking and washing with a 0.4% - 0.6% sodium carbonate solution by mass concentration for 8 - 12 min to remove the enzymatic hydrolysis products, more preferably shaking and washing with a 0.5% sodium carbonate solution for 10 min, and even more preferably shaking and washing with pure water for 8 - 12 min and then air-drying naturally. The present invention uses alkaline lipase and lipase to remove the fat on the surface of the deer meat, which can better utilize the deer meat protein and avoid negative impacts on the subsequent combined extraction of polypeptides by bacteria and enzymes.

[0029] In the present invention, the defatted venison is inoculated with a fermentation inoculum for solid-state fermentation treatment and liquid fermentation treatment, and after fermentation is completed, it is filtered. Among them, the fermentation inoculum includes Staphylococcus carnosus GDMCC No. 1.955 and Lactobacillus brevis CGMCC No. 31284. Preferably, the fermentation inoculum is prepared by activation culture, acclimation culture, scale-up culture and mixing, including: (1) Activation culture: Using MRS medium as the activation medium, inoculating the strain at a mass ratio of 2%-4%, and further preferably the inoculation amount is 3%, culturing at 28-33 °C for 20-28 h, further preferably culturing at 30 °C for 24 h, and more preferably performing activation culture 2 times; (2) Acclimation culture: Preparing acclimation media with MRS and venison pulp at mass gradients of 10:0, 8:2, 6:4, 4:6, 2:8 and 0:10 respectively, inoculating the activated strain at a mass ratio of 2%-4%, and further preferably the inoculation amount is 3%, culturing at 28-33 °C for 20-28 h, further preferably culturing at 30 °C for 24 h, and more preferably acclimating once for each gradient medium; (3) Scale-up culture: Using venison pulp as the scale-up medium, inoculating the acclimated strain at a mass ratio of 2%-4%, and further preferably the inoculation amount is 3%, culturing at 28-33 °C until the viable cell count ≥ 1×10 8 CFU / g, and more preferably performing scale-up culture 2 times; (4) Mixing: Mixing the scale-up cultured Staphylococcus carnosus GDMCC No. 1.955 and Lactobacillus brevis CGMCC No. 31284 at a mass ratio of 1:1-1.5. Preferably, the preparation method of the venison pulp for acclimation culture and scale-up culture is: adding 1 L / kg of pure water to the venison after the above-mentioned pretreatment and defatting treatment, and stirring evenly with a pulper to obtain venison pulp.

[0030] In the present invention, preferably, the solid-state fermentation treatment includes: inoculating the fermentation inoculum at 1%-3% of the mass of the venison, and further preferably the inoculation amount is 2%, fermenting at 28-33 °C for 16-24 h, and further preferably fermenting at 30 °C for 20 h. Preferably, the liquid fermentation treatment includes: based on the mass of the solid-state fermentation treatment product, adding 0.8-1 L / kg of water, and further preferably adding 0.9 L / kg of water, fermenting at 35-38 °C for 46-52 h, and further preferably fermenting at 37 °C for 48 h. Preferably, it is filtered through a 180-220 mesh sieve, and further preferably 200 mesh. The present invention adopts a fermentation method combining solid-state fermentation and liquid fermentation. Solid-state fermentation enables the massive reproduction of strains, and during liquid fermentation, the microorganisms that have already reproduced massively can quickly metabolize, increasing the polypeptide content.

[0031] In the present invention, the fermented venison is subjected to enzymatic hydrolysis using a protease solution, wherein the protease solution comprises trypsin, papain, and neutral protease. Preferably, the protease solution is prepared by mixing trypsin, papain, and neutral protease in a mass ratio of 0.8 - 1:1.5 - 2:1 - 1.5, and more preferably in a mass ratio of 1:2:1. Preferably, the enzymatic hydrolysis treatment with the protease solution includes: the addition amount of the protease solution is 1500 - 2000 U / g, more preferably 1800 U / g, enzymatic hydrolysis is carried out at 35 - 38 °C for 8 - 10 h, more preferably enzymatic hydrolysis is carried out at 37 °C for 9 h, and constant temperature oscillation is maintained during the enzymatic hydrolysis process. The deer meat oligopeptides prepared by the present invention using composite protease have higher antioxidant activity, are more easily absorbed by the human body, have a high content of essential amino acids for the human body, and are rich in health care function value.

[0032] In the present invention, after enzymatic hydrolysis by protease, the supernatant of the enzymatic hydrolysate is taken and inactivated, and the 5 kDa fraction is retained by filtration to obtain a deer meat oligopeptide solution. Preferably, the enzymatic hydrolysate is centrifuged at 4 °C at a rotation speed of 9000 - 12000 r / min for 15 ± 5 min, more preferably at a rotation speed of 10000 r / min, and the supernatant is taken as deer meat polypeptide. Preferably, inactivation treatment is carried out at a temperature of 90 - 95 °C for 18 - 25 min, more preferably for 20 min. Preferably, after inactivation, pre-filtration is carried out through a microporous membrane, and then the 5 kDa fraction is retained by using a polyethersulfone ultrafiltration membrane to obtain a deer meat oligopeptide solution.

[0033] The present invention also provides a deer meat oligopeptide solution prepared by the above preparation method.

[0034] The present invention also provides the application of the deer meat oligopeptide solution in the preparation of a functional oral liquid.

[0035] The present invention also provides a functional oral liquid, comprising the following components in mass fractions: 70% - 75% of deer meat oligopeptide solution, 0.04% - 0.06% of turmeric extract, 0.06% - 1% of perilla leaf juice, 4.4% - 6.8% of flavoring agent, 0.1% - 0.2% of stabilizer, and the balance is water; preferably, 70% of deer meat oligopeptide solution, 0.05% of turmeric extract, 0.08% of perilla leaf juice, 5.6% of flavoring agent, 0.1% of stabilizer, and the balance is water. More preferably, the flavoring agent includes a sweetening agent and an acidifying agent, 4% - 6% of sweetening agent, selected from one or more of erythritol, xylitol, and stevioside, 0.4% - 0.6% of acidifying agent, selected from one or two of citric acid and malic acid. More preferably, the stabilizer is selected from one or two of disodium ethylenediaminetetraacetate and ε-polylysine.

[0036] In the present invention, the preferred method for preparing turmeric extract is as follows: Fresh turmeric is removed of impurities, cleaned, cut into thin slices with a thickness of 3-5 mm, boiled with water at a material-liquid ratio of 1:50, then placed in a water bath at 85 °C for heat preservation and extraction for 30 min, and then filtered twice by a circulating water vacuum pump. The clarified filtrate is the turmeric extract.

[0037] In the present invention, the preferred method for preparing perilla leaf juice is as follows: Select perilla leaves with fresh leaf surfaces and dark green or dark purple colors, clean them with clear water, add 1 L / kg of water, grind them into a homogeneous slurry with a juicer, filter through a 200-mesh sieve, and sterilize at 121 °C for 15 min to obtain perilla leaf juice. The functional oral liquid prepared by adding perilla leaf juice and turmeric extract in the present invention has a unique flavor, good taste, rich nutritional value, no muttony smell of venison, is convenient to take, and meets the nutritional value requirements of modern people.

[0038] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] Example 1

[0040] A method for preparing an oral liquid of venison oligopeptide solution comprises the following steps:

[0041] (1) Pretreatment of venison: After removing broken bones, congestion, injury spots, lymph nodes, dirt and foreign impurities from the raw venison, it is minced by a meat grinder with a 10-mm orifice plate.

[0042] (2) Degreasing treatment: Spray the degreasing enzyme solution until the surface is wetted, keep it at 50-60 °C for 30 min, then wash it with 0.5% sodium carbonate solution by shaking for 10 min to remove the enzyme hydrolysis products, and then wash it with pure water by shaking for 10 min and air-dry naturally. The degreasing enzyme solution is prepared by mixing alkaline lipase and lipase in a mass ratio of 1:2 and adding the same mass of pure water and mixing evenly.

[0043] (3) Solid-state fermentation treatment: Take the defatted and air-dried deer meat, evenly spray the compound fermentation inoculum, with the spraying amount being 2% of the mass of the deer meat, and ferment and culture at a constant temperature of 30 °C for 24 h. The compound fermentation inoculum includes Staphylococcus carnosus GDMCC No. 1.955 and Lactobacillus brevis CGMCC No. 31284, which are prepared through activation culture, domestication culture, scale-up culture and mixing: (1) Activation culture: Use MRS medium as the activation medium, inoculate the strain at a mass ratio of 3%, culture at 30 °C for 24 h, and conduct activation culture twice; (2) Domestication culture: Prepare domestication media with MRS and deer meat pulp at mass gradients of 10:0, 8:2, 6:4, 4:6, 2:8 and 0:10 respectively, inoculate the activated strain at a mass ratio of 3%, culture at 30 °C for 24 h, and domesticate once for each gradient medium; (3) Scale-up culture: Use deer meat pulp as the scale-up medium, inoculate the domesticated strain at a mass ratio of 3%, conduct scale-up culture twice at 30 °C until the viable cell count ≥ 1×10 8 CFU / g; (4) Mixing: Mix the scale-up cultured Staphylococcus carnosus GDMCC No. 1.955 and Lactobacillus brevis CGMCC No. 31284 at a mass ratio of 1:1.5

[0044] (4) Liquid-state fermentation treatment: Add sterile water to the solid-state fermentation product, with the addition amount being 0.9 L / kg based on the mass of the solid-state fermentation product, mix evenly, ferment at 37 °C for 48 h, and then pass through a 200-mesh filter screen.

[0045] (5) Compound protease treatment: Take the fermented deer meat and add compound protease, with the enzymatic hydrolysis temperature being 37 °C and the enzymatic hydrolysis time being 10 h, and maintain constant temperature oscillation during the enzymatic hydrolysis process. The compound protease is prepared by mixing trypsin, papain and neutral protease at a mass ratio of 1:2:1, and the addition amount of the compound protease solution is 1500 - 2000 U / g.

[0046] (6) Centrifugation for enzyme inactivation: Take the enzymatic hydrolysate, centrifuge at 4 °C and 10000 r / min for 15 ± 5 min, take the supernatant as deer meat polypeptide, and conduct enzyme inactivation treatment at a temperature of 95 °C for 20 min.

[0047] (7) Retention of oligopeptides: After the deer meat polypeptide is pre-filtered through a microporous membrane, use a polyethersulfone ultrafiltration membrane to retain the 5 kDa component to obtain a deer meat oligopeptide solution, with the polypeptide content reaching 5.04 mg / ml.

[0048] (8) Blending: Blend 70% of deer meat oligopeptide, 0.05% of turmeric extract, 0.08% of perilla leaf juice, 5% of erythritol, 0.6% of citric acid, 0.1% of disodium ethylenediaminetetraacetate and the remaining amount of water by mass fraction to obtain the deer meat oligopeptide oral liquid.

[0049] Comparative Example 1

[0050] The specific implementation method is the same as that of Example 1, except that degreasing treatment is not carried out.

[0051] Comparative Example 2

[0052] The specific implementation method is the same as that of Example 1, except that only solid-state fermentation is carried out and liquid-state fermentation is not carried out.

[0053] Comparative Example 3

[0054] The specific implementation method is the same as that of Example 1, except that only liquid-state fermentation is carried out and solid-state fermentation is not carried out.

[0055] Comparative Example 4

[0056] The specific implementation method is the same as that of Example 1, except that protein complex enzyme treatment is not carried out.

[0057] Comparative Example 5

[0058] The specific implementation method is the same as that of Example 1, except that oligopeptides are not retained by polyethersulfone ultrafiltration membrane.

[0059] Comparative Example 6

[0060] The specific implementation method is the same as that of Example 1, except that turmeric extract and perilla leaf juice are not added during the preparation process.

[0061] Test Example 1

[0062] The polypeptide content, oligopeptide percentage, ABTS+ scavenging rate, DPPH free radical scavenging rate and essential amino acid score in the deer meat oligopeptide oral liquid of Example 1 and Comparative Examples 1-6 were detected respectively.

[0063] Among them, the polypeptide content was determined by the trichloroacetic acid soluble nitrogen method; the oligopeptide content was determined by high performance liquid chromatography-mass spectrometry (LC-MS), and the percentage of oligopeptides with a relative molecular mass below 5000 in the total polypeptides was calculated; the ABTS+ scavenging rate and DPPH free radical scavenging ability were determined by an antioxidant capacity kit; the essential amino acid score was determined by the EAAI scoring method. The sensory evaluation is shown in Table 1.

[0064] Table 1 Sensory evaluation criteria for deer meat oligopeptide oral liquid

[0065]

[0066]

[0067] Test results:

[0068] As shown in Table 2, the method of combining bacteria and enzymes was used to prepare the deer meat oligopeptide oral liquid, which had a high polypeptide content and a relatively high proportion of oligopeptides. Ultrafiltration membranes were used to retain the oligopeptides, resulting in a slight decrease in the polypeptide content, a significant increase in antioxidant capacity, and an improvement in the essential amino acid score. The amino acid content was close to the requirement of essential amino acids for human protein synthesis, and the degree of amino acid utilization was high. In addition, adding perilla leaf juice and turmeric extract could remove the fishy smell of deer meat, improve the overall flavor and taste, and significantly enhance the sensory evaluation.

[0069] Table 2 Measurement results of various indexes of deer meat oligopeptide oral liquid in Example 1 and Comparative Examples 1-6

[0070]

[0071] Experimental Example 2

[0072] Twenty food professionals were selected to score the deer meat oligopeptide oral liquids prepared in Example 1 and Comparative Examples 1-6 according to the sensory scoring criteria in Table 1. The specific results are shown in Table 3. It can be seen that the deer meat polypeptide oral liquid prepared by the method of Example 1 had uniform color, good stability, a suitable sweet and sour taste, a unique taste, a special flavor of deer meat, and no fishy smell. The deer meat polypeptide oral liquid prepared by the method of Example 1 had obvious advantages in terms of taste, flavor, nutritional value, and sensory performance.

[0073] Table 3 Sensory scores of deer meat oligopeptide oral liquid in Example 1 and Comparative Examples 1-6

[0074]

[0075]

[0076] Example 2

[0077] A preparation method of a deer meat oligopeptide solution oral liquid is as follows:

[0078] (1) Pretreatment of deer meat: After removing broken bones, congestion, injury marks, lymph nodes, dirt, and foreign impurities from the raw deer meat, it was minced through a meat grinder with a 10 mm orifice plate.

[0079] (2) Degreasing treatment: Spray the degreasing enzyme solution until the surface is wetted. After maintaining at 55 °C for 30 min, wash it with 0.5% sodium carbonate solution by shaking for 10 min to remove the enzyme hydrolysis products, and then wash it with pure water by shaking for 10 min and air-dry naturally. The degreasing enzyme solution was prepared by mixing alkaline lipase and lipase in a mass ratio of 1:1.5 and adding water in a solid-liquid ratio of 1:2 and mixing evenly.

[0080] (3) Solid-state fermentation treatment: Take the defatted and air-dried deer meat, evenly spray the compound fermentation inoculum, with the spraying amount being 1% of the mass of the deer meat, and ferment and culture at a constant temperature of 28°C for 24 h. The compound fermentation inoculum includes Staphylococcus carnosus GDMCC No. 1.955 and Lactobacillus brevis CGMCC No. 31284, which are prepared through activation culture, acclimation culture, scale-up culture, and mixing:

[0081] Activation culture: Use MRS medium as the activation medium, inoculate the strain at a mass ratio of 2%, culture at 30°C for 24 h, and perform activation culture 2 times;

[0082] Acclimation culture: Prepare acclimation media with MRS and deer meat pulp at mass gradients of 10:0, 8:2, 6:4, 4:6, 2:8, and 0:10 respectively, inoculate the activated strain at a mass ratio of 2%, culture at 30°C for 24 h, and acclimate once for each gradient medium;

[0083] Scale-up culture: Use deer meat pulp as the scale-up medium, inoculate the acclimated strain at a mass ratio of 2%, perform scale-up culture at 30°C for 2 times until the viable cell count ≥ 1×10 8 CFU / g;

[0084] Mixing: Mix the scale-up cultured Staphylococcus carnosus GDMCC No. 1.955 and Lactobacillus brevis CGMCC No. 31284 at a mass ratio of 1:1.

[0085] (4) Liquid-state fermentation treatment: Add sterile water to the solid-state fermentation product, with the addition amount being 0.8 L / kg based on the mass of the solid-state fermentation product, mix evenly, ferment at 35°C for 52 h, and then pass through a 200-mesh filter screen.

[0086] (5) Compound protease treatment: Take the fermented deer meat and add compound protease, with the enzymatic hydrolysis temperature being 35°C and the enzymatic hydrolysis time being 10 h, and maintain constant temperature oscillation during the enzymatic hydrolysis process. The compound protease is prepared by mixing trypsin, papain, and neutral protease at a mass ratio of 0.8:1.5:1, and the addition amount of the compound protease solution is 1500 U / g.

[0087] (6) Centrifugation for enzyme inactivation: Take the enzymatic hydrolysate, centrifuge at 4°C and 10000 r / min for 15 ± 5 min, take the supernatant as deer meat polypeptide, and perform enzyme inactivation treatment at a temperature of 90°C for 20 min.

[0088] (7) Retention of oligopeptides: After the deer meat polypeptide is pre-filtered through a microfiltration membrane, use a polyethersulfone ultrafiltration membrane to retain the 5 kDa component to obtain a deer meat oligopeptide solution, and the polypeptide content reaches 4.85 mg / ml.

[0089] (8) Preparation: Prepare the oral liquid of oligopeptides from venison by mixing 72% of oligopeptides from venison, 0.04% of turmeric extract, 0.06% of perilla leaf juice, 4% of xylitol, 0.4% of malic acid, 0.1% of ε-polylysine and the balance of water according to mass fraction.

[0090] Example 3

[0091] A preparation method of an oral liquid of a solution of oligopeptides from venison comprises the following steps:

[0092] (1) Pretreatment of venison: After removing broken bones, congestion, injury spots, lymph nodes, dirt and foreign impurities from the raw venison, it is minced through a meat grinder with a 10-mm orifice plate.

[0093] (2) Degreasing treatment: Spray the degreasing enzyme solution until the surface is wetted, keep it at 60 °C for 30 min, then wash it with 0.5% sodium carbonate solution by shaking for 10 min to remove the enzyme hydrolysis products, and then wash it with pure water by shaking for 10 min and air-dry it naturally. The degreasing enzyme solution is prepared by mixing alkaline lipase and lipase in a mass ratio of 1:2 and adding water in a solid-liquid ratio of 1:1 and mixing evenly.

[0094] (3) Solid-state fermentation treatment: Take the degreased and air-dried venison, evenly spray the compound fermentation inoculum, and the spraying amount is 3% of the mass of the venison, and ferment and culture it at a constant temperature of 33 °C for 16 h. The compound fermentation inoculum includes Staphylococcus carnosus GDMCC No. 1.955 and Lactobacillus brevis CGMCC No. 31284, which are prepared through activation culture, domestication culture, scale-up culture and mixing:

[0095] Activation culture: Use MRS medium as the activation medium, inoculate the strain according to a mass ratio of 4%, culture it at 30 °C for 24 h, and perform activation culture twice;

[0096] Domestication culture: Prepare domestication media with MRS and venison pulp respectively according to mass gradients of 10:0, 8:2, 6:4, 4:6, 2:8 and 0:10, inoculate the activated strain according to a mass ratio of 4%, culture it at 30 °C for 24 h, and domesticate each gradient medium once;

[0097] Scale-up culture: Use venison pulp as the scale-up medium, inoculate the domesticated strain according to a mass ratio of 4%, perform scale-up culture twice at 30 °C until the viable count ≥ 1×10 8 CFU / g;

[0098] Mixing: Mix the scale-up cultured Staphylococcus carnosus GDMCC No. 1.955 and Lactobacillus brevis CGMCC No. 31284 in a mass ratio of 1:1.2.

[0099] (4) Liquid fermentation treatment: In the solid fermentation product, add sterile water. The addition amount is 1 L / kg based on the mass of the solid fermentation product. Mix evenly, ferment at 38 °C for 46 h, and then filter through a 200-mesh sieve.

[0100] (5) Compound protease treatment: Take the fermented deer meat and add compound protease. The enzymatic hydrolysis temperature is 38 °C, and the enzymatic hydrolysis time is 8 h. Keep constant temperature oscillation during the enzymatic hydrolysis process. The compound protease is made by mixing trypsin, papain, and neutral protease according to the mass ratio of 1:2:1.5. The addition amount of the compound protease solution is 2000 U / g.

[0101] (6) Centrifugation for enzyme inactivation: Take the enzymatic hydrolysate and centrifuge it at 4 °C and 10000 r / min for 15 ± 5 min. Take the supernatant as deer meat polypeptide and perform enzyme inactivation treatment at 95 °C for 20 min.

[0102] (7) Retention of oligopeptides: After the deer meat polypeptide is pre-filtered through a microporous membrane, use a polyethersulfone ultrafiltration membrane to retain the 5 kDa component to obtain a deer meat oligopeptide solution, and the polypeptide content reaches 5.12 mg / ml.

[0103] (8) Blending: Blend 75% of deer meat oligopeptide, 0.06% of turmeric extract, 1% of perilla leaf juice, 6% of stevioside, 0.6% of citric acid, 0.1% of ε-polylysine, 0.1% of disodium ethylenediaminetetraacetate, and the balance of water by mass fraction to obtain the deer meat oligopeptide oral liquid.

[0104] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a venison oligopeptide solution, characterized in that: The following steps are involved: Deer meat is used as a raw material, a degreasing enzyme solution is used for defatting and enzymatic hydrolysis products are removed; the defatted deer meat is inoculated with a fermentation agent for solid-state fermentation and liquid-state fermentation, and filtered after the fermentation is completed; the fermented deer meat is enzymatically hydrolyzed with a protease solution, the supernatant is taken after the enzymatic hydrolysis and the enzyme is inactivated, and a 5kDa component is intercepted by filtration to obtain a deer meat oligopeptide solution; The delipidase solution comprises alkaline lipase and lipase; The fermentation bacteria include Staphylococcus carnosus GDMCC No.1.955 and Lactobacillus breve CGMCC No.31284; The protease solution includes trypsin, papain and neutral protease.

2. The preparation method according to claim 1, characterized in that: The delipidase solution is prepared by mixing alkaline lipase and lipase in a mass ratio of 1:(1.5-2), and then adding water in a solid-liquid ratio of 1:(1-2).

3. The preparation method according to claim 1, characterized in that: The degreasing treatment comprises: spraying a degreasing enzyme solution to soak the surface of the venison, and performing enzymolysis at 50-60° C. for 25-35 minutes.

4. The preparation method according to claim 1, characterized in that: The fermentation bacteria agent is prepared by activation culture, domestication culture, expansion culture and mixing, and comprises: Activation culture: Use MRS medium as activation medium, inoculate the strain at a mass ratio of 2%-4%, and culture at 28-33℃ for 20-28h; Acclimation culture: prepare acclimation culture medium with MRS and venison slurry in mass gradients of 10:0, 8:2, 6:4, 4:6, 2:8 and 0:10, inoculate activated bacteria at a mass ratio of 2%-4%, and culture at 28-33°C for 20-28h; Expansion culture: Use venison pulp as expansion culture medium, inoculate domesticated bacteria at a mass ratio of 2%-4%, and culture at 28-33℃ until the number of viable bacteria is ≥1×10 8 CFU / g; The expanded cultured Staphylococcus carnosus GDMCC No.1.955 and Lactobacillus breve CGMCC No.31284 were mixed in a mass ratio of 1:(1-1.5).

5. The preparation method according to claim 1, characterized in that: The solid-state fermentation treatment includes: inoculating fermentation bacteria at 1%-3% of the weight of venison, and fermenting at 28-33°C for 16-24 hours; the liquid fermentation treatment includes: adding water at 0.8-1L / kg based on the weight of the solid-state fermentation product, and fermenting at 35-38°C for 46-52 hours.

6. The preparation method according to claim 1, characterized in that: The protease solution is prepared by mixing trypsin, papain and neutral protease in a mass ratio of (0.8-1):(1.5-2):(1-1.5).

7. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis treatment with the protease solution includes: adding 1500-2000 U / g of the protease solution and performing enzymatic hydrolysis at 35-38° C. for 8-10 hours.

8. The venison oligopeptide solution obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the venison oligopeptide solution according to claim 8 in the preparation of a functional oral liquid.

10. A functional oral liquid, characterized in that: The invention comprises the following components in mass fraction: 70%-75% of the venison oligopeptide solution as claimed in claim 8, 0.04%-0.06% of turmeric extract, 0.06%-1% of perilla leaf juice, 4.4%-6.8% of flavoring agent, 0.1%-0.2% of stabilizer, and the balance is water.

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