Application of protease-producing yeast in broccoli fermented feed

Abstract

The invention discloses an application of protease-producing yeast in broccoli fermented feed. The protease-producing yeast is used in the broccoli fermented feed to ferment and decompose broccoli protein, so that the broccoli protein is decomposed into broccoli polypeptide in an alkaline-acidic environment; wherein the protease-producing yeast comprises Saccharomycopsis fibuligera Ytj-2 and Saccharomyces cerevisiae 99-1, the Saccharomycopsis fibuligera Ytj-2 is classified and named as Saccharomycopsis fibuligera Ytj-2, is deposited in China Center for Type Culture Collection (CCTCC), and has a deposit number of CCTCC NO: M 20221570; the Saccharomyces cerevisiae 99-1 is classified and named as Saccharomyces cerevisiae 99-1, is deposited in China Center for Type Culture Collection (CCTCC), and has a deposit number of CCTCC NO: M 20231104; and the broccoli polypeptide comprises polypeptide fragments of 4 lengths. The method can stably obtain broccoli fermented feed that fully contains broccoli polypeptides, and can continue to ferment when the environment changes, thereby stably obtaining a variety of broccoli polypeptides.

Description

Technical Field

[0001] The invention relates to the technical field of broccoli extracts, and in particular to application of protease-producing yeast in broccoli fermented feed. Background Art

[0002] With the current intensive and large-scale development of animal husbandry, my country's pigs generally have problems such as poor immunity and disease resistance, high morbidity and mortality. The reasons are: first, unscientific feed management, harsh environment, long-distance transportation, and transfers cause stress that reduces the animal's immunity to disease; second, the prevalence of infectious diseases, especially immunosuppressive diseases; third, the abuse of chemical drugs to prevent and treat diseases, which not only causes drug residues and food safety problems, but also causes chronic poisoning of the body and damage to immune tissues and organs; fourth, the abuse of antibiotics and antiviral drugs causes bacterial resistance; fifth, mycotoxin poisoning causes damage to tissues and organs, especially the liver and kidneys, especially the immune organs, resulting in a decrease in the animal's disease resistance and multiple diseases.

[0003] Broccoli (Brassica oleracea L.var.botrytis L), also known as cauliflower, is an annual or biennial plant of the genus Brassica in the family Cruciferae. It is known as the "crown of vegetables" and is rich in biologically active substances, including anti-tumor active substances such as sulforaphane and polysaccharides, and various antioxidant active substances such as polyphenols and flavonoids. It also has high protein and amino acid content, and has very high edible and medicinal health value.

[0004] Foreign studies have shown that many substances extracted from broccoli have the function of improving immunity. Many peptides have become clinical drugs with significant efficacy because of their ability to strengthen human immunity, such as insulin, growth hormone, somatostatin, cerebrolysin peptide vaccine, etc., which have played a role in dealing with modern viruses that previous drugs could not achieve.

[0005] Improving immunity has become an issue that is increasingly valued in the current animal husbandry industry, but there are few literature reports on the isolation of broccoli peptides to enhance the immunity of livestock. For common livestock such as pigs, horses, cattle and sheep, being able to steadily improve breeding levels and provide better quality feed has become a top priority.

[0006] However, during the fermentation process of broccoli, the fermentation environment needs to be kept stable. Ordinary alkaline proteases will lower the pH value of the fermentation environment during the fermentation process until it is unable to ferment for a long time without control. Therefore, the pH value of the internal environment needs to be stabilized during the fermentation process. However, the fermentation progress of each fermentation is difficult to strictly control, and staff are required to conduct multiple tests and adjustments. This makes it difficult to unify and standardize the quality of the feed, which is not conducive to the development of the industry. Summary of the invention

[0007] Based on this, the present invention proposes a Bacillus subtilis, a Bacillus subtilis fermentation product and the application of the two for treating or preventing acute or chronic gastric ulcers in pigs, effectively reducing the probability of occurrence and the degree of deterioration of ulcers.

[0008] The present invention provides an application of protease-producing yeast in broccoli fermented feed, wherein the protease-producing yeast is used in the broccoli fermented feed to ferment and decompose broccoli protein, so that the broccoli protein is decomposed into broccoli polypeptides in an alkaline-acidic environment;

[0009] The protease-producing yeast includes Saccharomycopsis fibuligera Ytj-2 and Saccharomycopsis cerevisiae 99-1. The classification name of Saccharomycopsis fibuligera Ytj-2 is Saccharomycopsis fibuligera Ytj-2, which is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 20221570 and a deposit date of October 14, 2022.

[0010] The classification name of the brewer's yeast 99-1 is Saccharomyces cerevisiae 99-1, which is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 20231104 and a deposit date of June 27, 2023;

[0011] The broccoli polypeptide includes polypeptide fragments of 4 lengths.

[0012] Furthermore, the alkaline-acidic environment includes an environment in which the pH value decreases from 8.1 to 3.1 during the fermentation and decomposition process.

[0013] Furthermore, it also includes:

[0014] The broccoli stem and leaf juice is obtained by pre-treating the fresh stem and leaf of broccoli;

[0015] The broccoli stem and leaf juice is used as a raw material, distilled water is added at a material-liquid mass ratio of 1:15-30, and the protease-producing yeast is added at a temperature of 25-35° C. and an initial pH value of 7.5-8.25, with a 1% inoculation amount, and the enzyme is inactivated after enzymolysis for 10-22 hours under shaking or stirring conditions;

[0016] The mixture was cooled, centrifuged, rotary evaporated, concentrated and freeze-dried to obtain a broccoli polypeptide extract.

[0017] Furthermore, the pretreatment includes: crushing the broccoli stems and leaves into 1-2 cm long fragments with a green feed crusher, beating with a pulper, squeezing with a screw press, filtering, and collecting the squeezed juice to obtain the broccoli stem and leaf juice.

[0018] Furthermore, the method further includes adding a nitrogen source to the broccoli stem and leaf juice for the growth of the protease-producing yeast.

[0019] Furthermore, the nitrogen source includes one or more of peptone, yeast powder, ammonium sulfate, urea and ammonium chloride, and the added amount of the nitrogen source is 10-15 g / L.

[0020] It can be seen from the above technical scheme that the Bacillus subtilis, the Bacillus subtilis fermentation product and the application of the two provided by the present invention have the following beneficial effects:

[0021] The present invention provides a method of fermenting broccoli in broccoli by using two protease-producing yeasts, thereby stably obtaining broccoli fermented feed that sufficiently contains broccoli polypeptides, so that the feed can be effectively used for livestock and animals to enhance their immunity, and can continue to ferment when the environment changes, and can stably obtain a variety of broccoli polypeptides, further capable of stabilizing the quality of the feed, and further promoting the development of the industry. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0023] With the current intensive and large-scale development of animal husbandry, my country's pigs generally have problems such as poor immunity and disease resistance, high morbidity and mortality. The reasons are that unscientific feed management, harsh environment, long-distance transportation, and transfers cause stress that reduces the animal's disease resistance, as well as the frequent occurrence of infectious diseases, the abuse of chemical drugs to prevent and treat diseases, and mycotoxin poisoning, which cause damage to tissues and organs, especially the liver and kidneys, immune organs, resulting in reduced animal resistance and causing diseases.

[0024] Especially for young animals, the digestive system, nervous system and immune system are not fully developed, and they cannot digest nutrients well, which leads to frequent diseases, especially digestive tract diseases such as diarrhea. Therefore, a method is needed to improve the immune and disease resistance of animals from the aspects of feed and diet, and improve the feeding level of livestock.

[0025] Currently, many studies have demonstrated the effects of peptides extracted from soybeans on the immune function of mice. The results show that the peptides can significantly increase the weight of the immune organs of mice and enhance the body's nonspecific immune function. There is also some evidence that soybean peptides can prevent and treat tumors and have a certain inhibitory effect on tumor growth.

[0026] As for broccoli peptides, studies have shown that many substances extracted from broccoli have the function of improving immunity. Many peptides have become clinical drugs with significant efficacy because of their ability to strengthen human immunity, such as insulin, growth hormone, somatostatin, cerebrolysin peptide vaccine, etc., which have played a role in dealing with modern viruses that previous drugs could not achieve.

[0027] However, during the fermentation process of broccoli, the fermentation environment needs to be kept stable. Ordinary alkaline proteases will lower the pH value of the fermentation environment during the fermentation process until it is unable to ferment for a long time without control. Therefore, the pH value of the internal environment needs to be stabilized during the fermentation process. However, the fermentation progress of each fermentation is difficult to strictly control, and staff are required to conduct multiple tests and adjustments. This makes it difficult to unify and standardize the quality of the feed, which is not conducive to the development of the industry.

[0028] Therefore, it is necessary to provide a feed that can enhance animal immunity and a stable method for producing feed, so as to stably improve the immunity and disease resistance of livestock.

[0029] Based on this, according to the above situation, the present invention provides an application of protease-producing yeast in broccoli fermented feed, wherein the protease-producing yeast is used in broccoli fermented feed to ferment and decompose broccoli protein, so that the broccoli protein is decomposed into broccoli polypeptides in an alkaline-acidic environment;

[0030] Among them, the protease-producing yeasts include Saccharomycopsis fibuligera Ytj-2 and Saccharomycopsis cerevisiae 99-1. The classification of Saccharomycopsis fibuligera Ytj-2 is named Saccharomycopsis fibuligera Ytj-2, which is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 20221570.

[0031] The classification of Saccharomyces cerevisiae 99-1 is named Saccharomyces cerevisiae 99-1, and it is deposited in China Center for Type Culture Collection (CCTCC) with the deposit number CCTCC NO: M20231104;

[0032] Broccoli peptides include peptide fragments of 4 lengths.

[0033] The present invention provides a method of fermenting broccoli in broccoli by using two protease-producing yeasts, thereby stably obtaining broccoli fermented feed that sufficiently contains broccoli polypeptides, so that the feed can be effectively used for livestock and animals to enhance their immunity, and can continue to ferment when the environment changes, and can stably obtain a variety of broccoli polypeptides, further capable of stabilizing the quality of the feed, and further promoting the development of the industry.

[0034] Furthermore, the alkaline-acidic environment includes an environment in which the pH value decreases from 8.1 to 3.1 during the fermentation and decomposition process.

[0035] According to an embodiment of the present invention, during the process of protease-producing yeast fermenting and decomposing broccoli protein, the protease-producing yeast secretes alkaline protease, neutral protease and acidic protease to enzymatically hydrolyze the broccoli protein to obtain broccoli polypeptide.

[0036] The alkaline protease, neutral protease and acidic protease produced by the capsular yeast Ytj-2 and the cerevisiae 99-1 can stably decompose the broccoli protein in the environment, cut the broccoli protein at different sites in different fermentation stages, and further obtain broccoli peptides with multiple peptide chains.

[0037] Furthermore, it also includes:

[0038] The broccoli stem and leaf juice is obtained by pre-treating the fresh stem and leaf of broccoli;

[0039] Using broccoli stem and leaf juice as raw material, adding distilled water at a material-liquid mass ratio of 1:15-30, adding protease-producing yeast at a temperature of 25-35°C and an initial pH value of 7.5-8.25, with a 1% inoculation amount, performing enzymolysis for 10-22 hours under shaking or stirring conditions, and then inactivating the enzyme;

[0040] The mixture was cooled, centrifuged, rotary evaporated, concentrated and freeze-dried to obtain a broccoli polypeptide extract.

[0041] According to an embodiment of the present invention, the mass ratio of slurry to liquid may be 1:15, 1:20, 1:25, 1:30, etc.

[0042] According to an embodiment of the present invention, the fermentation temperature may be 25°C, 28°C, 30°C, 32°C or 35°C, etc.

[0043] According to an embodiment of the present invention, the initial pH value may be 7.5, 7.7, 8, 8.1 or 8.25, etc.

[0044] According to an embodiment of the present invention, the enzymatic hydrolysis time can be 10 h, 12 h, 15 h, 18 h, 20 h, 22 h, etc.

[0045] According to an embodiment of the present invention, the pretreatment includes: crushing the broccoli stems and leaves into 1-2 cm long fragments with a green fodder crusher, beating with a pulper, squeezing with a screw press, filtering, and collecting the squeezed juice to obtain broccoli stem and leaf juice.

[0046] Broccoli stem and leaf juice contains a variety of elements for the growth of protease-producing yeast, such as sugars and inorganic salts. It is rich in glucose and soluble starch, as well as rich in calcium, magnesium, zinc, copper and other inorganic salts, which can effectively promote the growth and reproduction of protease-producing yeast.

[0047] According to an embodiment of the present invention, a nitrogen source is further added to the broccoli stem and leaf juice for the growth of protease-producing yeast.

[0048] According to an embodiment of the present invention, the nitrogen source includes one or more of peptone, yeast powder, ammonium sulfate, urea and ammonium chloride, and the added amount of the nitrogen source is 10-15 g / L.

[0049] According to an embodiment of the present invention, in the early stage of fermentation, the protease-producing yeast needs to be supplemented with a nitrogen source during its growth process. In the early stage of fermentation, the decomposition efficiency of broccoli protein by the protease-producing yeast is reduced. Artificially supplementing the nitrogen source in the early stage of fermentation can enable the protease-producing yeast to grow and multiply rapidly, thereby further increasing the level of protease-producing yeast in the fermentation product, thereby increasing its fermentation rate.

[0050] The technical solution of the present invention is described in detail below through preferred embodiments. It should be noted that the specific embodiments below are only used for illustration and are not used to limit the present invention.

[0051] Example 1: Characterization of protease producing yeast.

[0052] (1) Protease activity and protein accumulation efficiency of the two strains

[0053] The 18S rDNA technology was used to sequence the 18S rDNA strains of Ytj-2 and Saccharomyces cerevisiae 99-1 using modern high-throughput microbial culture technology (microplate culture), and the protease activity and protein accumulation efficiency of the two strains were obtained by comparative identification through NCBI. The results are shown in Table 1.

[0054] Table 1. Protease activity and protein accumulation efficiency of two strains

[0055]

[0056] From the results in the above table, it can be seen that the alkaline protease, neutral protease and acidic protease produced by Saccharomyces cerevisiae Ytj-2 and Saccharomyces cerevisiae 99-1 enable them to be used in environments with changing pH values, and have good protease activity and protein accumulation efficiency. When used in the fermentation process of broccoli protein, broccoli polypeptides with various peptide chains can be obtained.

[0057] (2) Effects of different pH values ​​on the growth of the two strains

[0058] The initial pH of the culture medium was adjusted to 3.1, 4.1, 5.1, 6.1, 7.1, 8.1 and 9.1 to study the effects of different initial pH on the growth of Saccharomyces cerevisiae Ytj-2 and Saccharomyces cerevisiae 99-1. The results are shown in Table 2.

[0059] Table 2. Effect of different pH on the growth of two strains

[0060]

[0061]

[0062] As shown in Table 2, the results showed that when the initial pH of the culture medium was between 3 and 8.1, it had no significant effect on the growth of strains MQ-5 and Ytj-2. 600 They were all around 1.65, so the initial pH range of the culture medium was selected as 3-8.1 in subsequent experiments.

[0063] (3) Effect of broccoli stem and leaf juice on the fermentation of two strains

[0064] Broccoli stem and leaf juice was used to conduct a small test in a 10 mL Erlenmeyer flask and a 5 L fermentation tank. Two strains were inoculated to verify the feasibility of fermenting broccoli stem and leaf juice. The results are shown in Table 3.

[0065] Table 3. Verification test results

[0066]

[0067] The results of the verification experiment showed that under the optimal process conditions obtained above, the yeast growth OD 600 The average value was 1.691 with good repeatability, indicating that fermentation with broccoli stem and leaf juice was feasible and could be used in the research and application of yeast microbial protein synthesis.

[0068] Example 2: Investigating the effect of protease-producing yeast fermentation on the types of broccoli polypeptides.

[0069] Broccoli stem and leaf juice was used as raw material, distilled water was added at a material-liquid mass ratio of 1:15, protease-producing yeast was added at a temperature of 30°C and an initial pH of 8, and fermentation was carried out in a 10mL conical flask. With a 1% inoculum amount, under oscillation or stirring conditions, the enzyme was inactivated after enzymolysis for 22 hours; the extract was cooled, centrifuged, rotary evaporated, concentrated and freeze-dried to obtain broccoli polypeptide extract, which was used as the experimental group sample.

[0070] 0.1 g of commercially available alkaline protease (purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd.), 0.1 g of neutral protease (purchased from Shandong Longyuan Bioengineering Co., Ltd.) or 0.1 g of acidic protease (purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd.) were fermented according to the above method, and the pH values ​​were adjusted to 8, 7 and 4, respectively. After fermentation for 22 hours, the broccoli polypeptide content was determined and used as samples of control groups 1-3.

[0071] The above three proteases were compounded and fermented according to the above method. The broccoli polypeptide content was determined under the condition of initial pH value of 8. They were used as 4 groups of samples in the control group to determine whether they could produce the four broccoli polypeptides of the present invention. The results are shown in Table 4.

[0072] Table 4. Protease activity and protein accumulation efficiency of two strains

[0073]

[0074] It can be seen from Table 4 that the method provided by the present invention can effectively obtain 4 types of broccoli proteins, and it can be seen from the protein content in Table 4 that the three proteases that can adapt to different pH values ​​obtained by fermentation with protease-producing yeast have a compounding effect, so that it has better protein decomposition efficiency, while in the control group 4, it is difficult to obtain broccoli polypeptide B by artificially compounding the three proteases, and because the recognition and cleavage sites of the proteases are different and they affect each other to a certain extent, broccoli polypeptide C cannot be obtained after compounding.

[0075] Example 3: Investigating the effect of fermentation products obtained by protease-producing yeast fermentation on the proliferation ability of mouse lymphocytes

[0076] Animals were continuously gavaged with the four fermentation products obtained in Example 2 for one month. One hour after the last gavage, the animals were killed by cervical dislocation, the spleen was aseptically removed, and placed in a small dish containing an appropriate amount of sterile Hank's solution. The spleen was ground to make a single cell suspension, filtered through a 200-mesh screen, washed twice with Hank's solution, and centrifuged for 10 min each time (1000 r / min). The cells were then suspended in 1 mL of RPMI1640 complete culture medium, and the cell concentration was adjusted to 3×106 cells / mL with culture medium. The cell suspension was added to 2 wells of a 24-well plate, 1 mL per well, 75 μL ConA solution (final concentration 7.5 μL / mL) was added to the experimental well, and the other well was used as a control. The plates were cultured in a 37° C., 5% CO2 incubator for 72 h. 4 hours before the end of the culture, 0.7 mL of supernatant was removed from each well, 0.7 mL of RPMI 1640 culture medium without calf serum was added, and 50 μL / well of MTT (5 mg / ml) was added, and the culture was continued for 4 hours. After the culture was completed, acidic isopropanol (96 ml isopropanol + 4 ml 1M HCl) was added to each well, and the mixture was mixed by pipetting, and the light absorbance was measured by an enzyme marker at a wavelength of 570 nm. The proliferation capacity of lymphocytes was expressed by subtracting the OD value of the well without Con A from the OD value of the well with Con A, and the results are shown in Table 5.

[0077] Table 5. Proliferation capacity of mouse lymphocytes

[0078]

[0079] As shown in the results in Table 5, the present invention provides a method of fermenting broccoli with two protease-producing yeasts, which can stably obtain broccoli fermented feed that sufficiently contains broccoli polypeptides, so that the feed can be effectively used for livestock and enhance their immunity.

[0080] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of protease-producing yeast in broccoli fermented feed, characterized in that: Protease-producing yeast is used in broccoli fermented feed to ferment and decompose broccoli protein, so that the broccoli protein is decomposed into broccoli polypeptides in an alkaline-acidic environment; The protease-producing yeasts are Saccharomycopsis fibuligera Ytj-2 and Saccharomycopsis cerevisiae 99-1. The classification name of Saccharomycopsis fibuligera Ytj-2 is Saccharomycopsis fibuligera Ytj-2, which is deposited in China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 20221570. The classification of the brewer's yeast 99-1 is named as Saccharomyces cerevisiae 99-1, and it is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 20231104; The broccoli polypeptide includes polypeptide fragments of 4 lengths.

2. The use according to claim 1, characterized in that: The alkaline-acidic environment includes an environment in which the pH value decreases from 8.1 to 3.1 during the fermentation and decomposition process.

3. The use according to claim 1, characterized in that: Also includes: The broccoli stem and leaf juice is obtained by pre-treating the fresh stem and leaf of broccoli; The broccoli stem and leaf juice is used as a raw material, distilled water is added at a material-liquid mass ratio of 1:15-30, and the protease-producing yeast is added at a temperature of 25-35° C. and an initial pH value of 7.5-8.25, with an inoculum amount of 1%, and the enzyme is inactivated after enzymolysis for 10-22 hours under shaking or stirring conditions; The mixture was cooled, centrifuged, rotary evaporated, concentrated and freeze-dried to obtain a broccoli polypeptide extract.

4. The use according to claim 3, characterized in that: The pretreatment comprises: crushing the broccoli stems and leaves into 1-2 cm long fragments by a green feed crusher, beating with a pulper, squeezing with a screw press, filtering, and collecting the squeezed juice to obtain the broccoli stem and leaf juice.

5. The use according to claim 4, characterized in that: The method also includes adding a nitrogen source into the broccoli stem and leaf juice for the growth of the protease-producing yeast.

6. The use according to claim 5, characterized in that: The nitrogen source includes one or more of peptone, yeast powder, ammonium sulfate, urea and ammonium chloride, and the added amount of the nitrogen source is 10-15 g / L.

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