Saccharomyces cerevisiae and application thereof in preparation of superoxide dismutase

Abstract

The invention belongs to the technical field of microorganisms and enzyme products thereof, and particularly relates to saccharomyces cerevisiae and application thereof in preparation of superoxide dismutase. The preservation number of the saccharomyces cerevisiae is CCTCC (China Center For Type Culture Collection) NO: M 20242101. The Saccharomyces cerevisiae SOD-1 strain provided by the invention has the characteristics of high yield of superoxide dismutase (SOD), high activity of the produced SOD and good thermal stability, and provides a reliable thallus raw material for production of the SOD. Moreover, SOD prepared through fermentation of the strain has the advantages of being short in growth cycle, low in culture cost, simple in culture condition, simple in post-treatment purification process, high in production efficiency and the like, a feasible way is provided for large-scale production of SOD through biological fermentation, the industrial practicability is high, and the market prospect is wide. In addition, the obtained SOD is high in activity, good in thermal stability and excellent in oxidation resistance, and has a good application prospect in the field of animal feeding as a feed additive.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms and enzyme products thereof, in particular to brewer's yeast and application thereof in preparing superoxide dismutase. Background Art

[0002] Superoxide dismutase (SOD) is widely present in the biological world and is an important component of the antioxidant system in organisms. It can catalyze the dismutation reaction of superoxide anions to generate hydrogen peroxide and oxygen, thereby removing oxygen free radicals in the body and reducing the impact of oxidative stress on the body. It has multiple physiological functions such as anti-aging, improving the body's resistance to various diseases, and enhancing the body's ability to adapt to the external environment. It has broad application prospects in the fields of medicine, food, cosmetics, and agriculture. According to the different metal auxiliary groups contained in SOD, it is usually divided into three types: Cu / Zn-SOD, Mn-SOD, and Fe-SOD.

[0003] At present, there are four main ways to obtain SOD: 1) Extraction from animal blood; 2) Extraction from plants; 3) Production by microbial fermentation; 4) Exogenous expression of SOD by genetic engineering. Among the above methods, animal blood extraction is easily affected by factors such as the source of raw materials, safety of blood products and unstable quality, and has been banned. Plant extraction has problems such as complex process, high cost and low yield. Although genetic engineering can prepare SOD in large quantities, it also has obvious limitations. For example, the biological activity and yield of the enzyme are greatly affected by the expression method, and there are many problems such as low expression level, protein misfolding affecting activity, and complex purification methods. The use of microorganisms such as yeast or bacterial fermentation has the advantages of wide strain sources, fast reproduction, short culture time and low culture cost, and can achieve large-scale production. It has good application potential in the field of SOD preparation.

[0004] The key to producing SOD by microbial fermentation is the selection of excellent strains. However, from the currently reported microorganisms, the SOD production and / or enzyme activity need to be improved, which is not conducive to industrial production. Therefore, obtaining high-yield SOD strains has become the focus of current research. Summary of the invention

[0005] In view of the lack of excellent SOD-producing microorganisms in the prior art, the present invention provides a brewer's yeast. Based on the excellent characteristics of the brewer's yeast strain in high SOD production and high activity of the produced SOD, the present invention further provides the use of the brewer's yeast strain in the preparation of SOD and a preparation method, as well as the use of the produced SOD in improving animal production performance. The present invention is specifically implemented by the following technical solutions:

[0006] The first aspect of the present invention provides a brewer's yeast, with a deposit number of CCTCC NO:M 20242101.

[0007] The second aspect of the present invention provides the use of the above-mentioned Saccharomyces cerevisiae in the preparation of superoxide dismutase.

[0008] The third aspect of the present invention provides a method for preparing superoxide dismutase, comprising the following steps: activating the brewer's yeast as described above and inoculating it into a fermentation medium containing copper ions and zinc ions, culturing for a period of time, collecting the culture solution and centrifuging to obtain bacterial cells, resuspending the bacterial cells and then breaking the bacteria, then placing the cell lysis solution at 70°C for 30-60 minutes, collecting the supernatant, and obtaining an enzyme solution containing superoxide dismutase.

[0009] Furthermore, the fermentation medium includes 0.01-1 mmol / L of copper ions and 0.01-1 mmol / L of zinc ions.

[0010] Furthermore, the fermentation medium comprises the following components in weight percentage: 1% yeast extract powder, 2% peptone, 2% glucose, 0.0025‰ copper sulfate pentahydrate and 0.0028‰ zinc sulfate heptahydrate.

[0011] Furthermore, the culture conditions include: the pH of the fermentation liquid is 5.0, the culture temperature is 28° C., the rotation speed is 200 rpm / min, and the dissolved oxygen content is 40%-60%.

[0012] A fourth aspect of the present invention provides a superoxide dismutase prepared by the above-mentioned method for preparing superoxide dismutase using Saccharomyces cerevisiae.

[0013] The fifth aspect of the present invention provides the use of the superoxide dismutase as described above in improving animal production performance.

[0014] Furthermore, the application method comprises the following steps: mixing the superoxide dismutase dry powder preparation with the animal's basic feed and then feeding the mixture to the animal, wherein the addition amount of the superoxide dismutase dry powder preparation is 0.1-2‰ of the total mass of the basic feed.

[0015] Furthermore, the preparation method of the superoxide dismutase dry powder preparation comprises: mixing an enzyme solution containing superoxide dismutase with corn starch, and drying to prepare the superoxide dismutase dry powder preparation, wherein the specific activity of the superoxide dismutase dry powder preparation is 1-3 million U / g.

[0016] Furthermore, the animal includes laying hens or sows, and the animal production performance includes late laying hen production performance or sow antioxidant performance.

[0017] The advantages and positive effects of the present invention are:

[0018] 1. The Saccharomyces cerevisiae SOD-1 strain provided by the present invention has the characteristics of high superoxide dismutase (SOD) production, high SOD activity and good thermal stability, and provides a reliable bacterial raw material for SOD production. Moreover, the fermentation preparation of SOD by the aforementioned strain has the advantages of short growth cycle, low culture cost, simple culture conditions, simple post-treatment purification process and high production efficiency, which provides a feasible way for the large-scale production of SOD by biological fermentation, high industrial practicability and broad market prospects.

[0019] 2. The SOD separated by fermenting Saccharomyces cerevisiae SOD-1 has high activity, good thermal stability and excellent antioxidant performance. It has good application prospects in the field of animal breeding as a feed additive, is beneficial to improving the antioxidant capacity of breeding animals and alleviating the inflammatory response of the liver, reducing the body's oxidative stress damage, and improving the production performance of animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 This is the electrophoresis diagram of the purification of superoxide dismutase produced by fermentation of Saccharomyces cerevisiae SOD-1 in the embodiment of the present invention;

[0022] Figure 2 The enzyme activity curve of superoxide dismutase produced by fermentation of Saccharomyces cerevisiae SOD-1 in the embodiment of the present invention at 70°C for different time periods;

[0023] Figure 3 This is a line graph of egg production rate of late-stage laying hens fed with SOD dry powder preparations at different times according to an embodiment of the present invention;

[0024] Figure 4 This is a bar graph of egg quality of late laying hens fed with SOD dry powder preparation for 6 weeks according to the embodiment of the present invention, which includes eggshell strength, egg white height, yolk color, Haugh value and egg fat content from left to right;

[0025] Figure 5 This is a bar graph of serum antioxidant levels of late-stage laying hens fed with SOD dry powder preparations for 6 weeks according to the present invention, which includes superoxide dismutase, glutathione peroxidase, catalase, total antioxidant capacity and malondialdehyde from left to right.

[0026] Figure 6This is a tissue section diagram of liver fat distribution of late-stage laying hens fed with SOD dry powder preparation for 6 weeks according to the embodiment of the present invention;

[0027] Figure 7 This is a bar graph of serum lipid content of late-stage laying hens fed with SOD dry powder preparations for 6 weeks according to an embodiment of the present invention, which includes total cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol from left to right;

[0028] Figure 8 This is a bar graph of liver lipid content of late-stage laying hens fed with SOD dry powder preparations for 6 weeks according to an embodiment of the present invention, which includes total cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol from left to right;

[0029] Fig. 9 This is a bar graph of the expression of genes related to liver fat synthesis in late-stage laying hens fed with SOD dry powder preparations for 6 weeks according to the embodiment of the present invention, including fatty acid synthase, peroxisome proliferator-activated receptor γ and transmembrane protein 68 from left to right;

[0030] Fig.10 This is a tissue section diagram of the liver morphology of late-stage laying hens fed with SOD dry powder preparation for 6 weeks according to the embodiment of the present invention;

[0031] Fig.11 This is a bar graph of the expression levels of genes related to liver inflammatory factors after late-stage laying hens were fed the SOD dry powder preparation for 6 weeks according to the example of the present invention, including interleukin-1β, interleukin-6 and interleukin-10 from left to right. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the examples. The equipment and reagents used in each embodiment and test example can be obtained from commercial sources unless otherwise specified. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] Based on the information contained in this application, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined process, properties or components, because these embodiments and other descriptions are only for illustrating specific aspects of the present invention. In fact, various changes that the technicians in this field or related fields can obviously make to the embodiments of the present invention are all covered within the scope of the appended claims.

[0034] In order to better understand the present invention rather than limit the scope of the present invention, all the numerals and other numerical values ​​used in this application indicating dosage, percentage, etc. should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as being obtained according to the reported significant figures and by conventional rounding methods. In addition, the meaning of the terms "include", "comprise", "contain", "have" and the like is non-restrictive, and other steps and other ingredients that do not affect the results can be added.

[0035] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0036] An embodiment of the present invention provides a brewer's yeast, the preservation number is CCTCC NO: M 20242101, the preservation date is September 27, 2024, the strain was deposited in the China Type Culture Collection on September 27, 2024, the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province (Wuhan University), the culture name is brewer's yeast SOD-1, and the taxonomic name is Saccharomyces cerevisiae.

[0037] The invention domesticates saccharomyces cerevisiae by adding high concentrations of copper and zinc ions, induces the saccharomyces cerevisiae to express superoxide dismutase (SOD), and uses the SOD enzyme activity of the saccharomyces cerevisiae fermentation liquid at different domestication times as an index to separate and obtain the saccharomyces cerevisiae SOD-1 strain of the invention. The enzyme activity of saccharomyces cerevisiae SOD-1 fermented for 48 hours is measured, and the SOD activity of the fermentation culture liquid can reach 193U / mL, and the SOD yield calculated by the wet weight of the saccharomyces cerevisiae can reach 2412.5U / g; it can be seen that the strain of the invention has the characteristics of high SOD production, high SOD activity and good stability, and provides a reliable saccharomyces raw material for the large-scale production of SOD. In addition, the production of SOD by fermentation using the strain of the present invention has the advantage of simple separation and purification of the target enzyme. Specifically, the fermentation liquid is centrifuged to collect the bacteria, the bacteria are broken and then subjected to heat denaturation treatment at high temperature (e.g., 70°C) for a period of time (e.g., 30-60 minutes), and then the supernatant is collected by centrifugation to separate and purify the SOD enzyme solution with a purity greater than 85%, which is conducive to the large-scale production of SOD and has good market potential in the industry of preparing SOD by biological fermentation.

[0038] Another embodiment of the present invention provides the use of the Saccharomyces cerevisiae SOD-1 as described above in the preparation of superoxide dismutase.

[0039] The SOD produced by the brewer's yeast SOD-1 fermentation of the present invention has the advantages of high yield and good activity, and the brewer's yeast has a short growth cycle, low culture cost, simple culture conditions, and is easy to realize industrial production, providing a feasible approach for large-scale preparation of SOD by biological fermentation.

[0040] Based on the same inventive concept as above, the present invention also provides a method for preparing superoxide dismutase by fermenting Saccharomyces cerevisiae SOD-1, comprising the following steps:

[0041] The activated Saccharomyces cerevisiae SOD-1 is inoculated into a fermentation medium containing copper ions and zinc ions and cultured for a period of time, the culture solution is collected and centrifuged to obtain bacterial cells, the bacterial cells are resuspended and then broken into bacteria, and then the cell lysis solution is placed at 70° C. for 30-60 minutes, and the supernatant is collected to obtain an enzyme solution containing superoxide dismutase.

[0042] Since the brewer's yeast SOD-1 of the present invention has a high yield of SOD, and SOD has good thermal stability, the enzyme activity loss is within 6% when kept in a 70°C water bath for 1 hour, and more than 80% of the enzyme activity can still be retained when kept in a 2-hour heat bath. Therefore, the present invention can remove foreign proteins by simple high-temperature heat denaturation treatment, and finally obtain SOD with a purity greater than 85% by centrifugation of the denatured precipitated foreign proteins. In short, the fermentation production of SOD using the brewer's yeast SOD-1 of the present invention has the advantages of simple separation and purification, high enzyme yield and high production efficiency, and has high industrial applicability.

[0043] The method for activating brewer's yeast adopts conventional methods in the art, such as inoculating it in LB medium or YPD medium commonly used for yeast and culturing it for a period of time. The culture time can be adaptively adjusted based on the bacterial concentration, which will not be repeated here.

[0044] Optionally, the fermentation medium comprises copper ions at a molar concentration of 0.01-1 mmol / L and zinc ions at a molar concentration of 0.01-1 mmol / L.

[0045] In a preferred embodiment, the fermentation medium comprises the following components in weight percentage: 1% yeast extract powder, 2% peptone, 2% glucose, 0.0025‰ copper sulfate pentahydrate and 0.0028‰ zinc sulfate heptahydrate.

[0046] The fermentation culture conditions adopt conventional yeast culture conditions, such as fermentation liquid pH 5.0, culture at 28°C, 200rpm / min and dissolved oxygen 40%-60%. The fermentation culture time is adaptively adjusted according to the bacterial yield and enzyme activity index. Generally speaking, a culture time of 48h can obtain high content and high activity SOD.

[0047] Another embodiment of the present invention provides a superoxide dismutase, which is prepared by the above-mentioned method for preparing superoxide dismutase by fermenting Saccharomyces cerevisiae SOD-1.

[0048] The SOD separated by fermenting Saccharomyces cerevisiae SOD-1 has the characteristics of high activity and good thermal stability, excellent antioxidant performance, and has good application prospects in the field of animal breeding as a feed additive, is beneficial to improving the antioxidant capacity of breeding animals and alleviating the inflammatory response of the liver, reducing the oxidative stress damage of the body, and improving the production performance of animals.

[0049] Based on this, another embodiment of the present invention provides a superoxide dismutase dry powder preparation, comprising the superoxide dismutase as described above and acceptable excipients.

[0050] The SOD enzyme solution obtained by fermenting Saccharomyces cerevisiae SOD-1 is high in specific activity and concentration. By adding acceptable auxiliary materials to disperse SOD, it is beneficial to dry the SOD enzyme solution, obtain dry powder with good dispersibility, and then directly mix it with basic feed in the future. The advantages of the superoxide dismutase dry powder preparation over the prior art are basically the same as the advantages of the superoxide dismutase over the prior art as described above, and will not be repeated here.

[0051] Acceptable excipients refer to components that do not interfere with the efficacy of the biological activity of the active ingredient - SOD and have no obvious toxicity to the body (including human or animal body) at the concentration at which they are applied, including any one or a combination of at least two of solvents, dispersants, diluents, fillers, wetting agents, binders, disintegrants, lubricants, suspending agents, excipients, flavoring agents, protective agents, etc. It is well known in the art to use the above components in enzyme preparations.

[0052] Optionally, the acceptable auxiliary material is corn starch.

[0053] Optionally, the SOD specific activity of the superoxide dismutase dry powder preparation is 1-3 million U / g. Specifically, it can be 100,000 U / g. After corn starch and SOD enzyme solution are mixed, they are dried to obtain the dry powder preparation of the present invention.

[0054] The SOD specific activity of the dry powder preparation is adjusted by controlling the amount of corn starch added. For example, if the enzyme solution activity is 100,000 U / mL, 1g of starch is added and then dried.

[0055] The embodiments of the present invention also provide the use of the superoxide dismutase or superoxide dismutase dry powder preparation as described above in improving the production performance of animals.

[0056] Optionally, the animal includes laying hens or sows, and the animal production performance includes late laying hen production performance or sow antioxidant performance.

[0057] The superoxide dismutase of the present invention can improve the production performance of late-stage laying hens, which generally refers to the late stage of laying eggs, 60 weeks of age and later. Specifically, the superoxide dismutase of the present invention includes: 1) improving the egg production rate of late-stage laying hens, and maintaining the egg production rate of late-stage laying hens at about 95%; 2) improving the quality of eggs, and improving the eggshell strength, egg white height, yolk color, Haugh value and fat content of eggs; 3) enhancing serum antioxidant capacity, improving the levels of SOD, glutathione peroxidase, catalase and total antioxidant capacity in serum, and reducing the level of malondialdehyde in serum; 4) promoting liver fatty acid metabolism, reducing the accumulation of fat in the liver, reducing lipid levels, including triglycerides, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, inhibiting the synthesis and expression of fatty acid synthase, peroxisome proliferator-activated receptor γ, and transmembrane protein 68 genes; 5) inhibiting the occurrence of liver inflammation, reducing the infiltration of inflammatory cells in the liver, and inhibiting the synthesis and expression of interleukin-1β, interleukin-6 and interleukin-10 genes.

[0058] The superoxide dismutase of the present invention can improve the antioxidant performance of sows, specifically including: 1) improving serum antioxidant capacity, improving the superoxide dismutase, glutathione peroxidase, catalase activities and glutathione content and total antioxidant capacity level in serum; 2) reducing serum oxidative damage level, reducing the malondialdehyde, hydrogen peroxide and tumor necrosis factor-α content in serum.

[0059] In practical applications, the superoxide dismutase of the present invention or its dry powder preparation is directly mixed with the basic feed of the animal and then fed to the animal. Since the specific activity of pure SOD after drying is very high, it is preferably prepared as a dry powder preparation and mixed with the basic feed, and the addition amount of the dry powder preparation is generally 0.1-2‰ of the total mass of the basic feed. The addition amount is slightly different according to the difference in feeding animals, and those skilled in the art can adjust it according to feeding needs. For example, when raising laying hens, the addition amount can preferably be 0.2‰, and when raising large animals such as sows, the addition amount can be increased, preferably 1‰.

[0060] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as the conditions described in the Molecular Cloning Laboratory Guide (Fourth Edition) published by Cold Spring Harbor Laboratory, or usually under the conditions recommended by the manufacturer.

[0061] The superoxide dismutase (SOD) activity detection in the present invention is carried out in accordance with the national standard of the People's Republic of China "GB / T41906-2022 Superoxide dismutase activity detection method". The enzyme activity is defined as: at 25°C, the amount of enzyme that inhibits the self-oxidation rate of pyrogallol by 50% per minute in 1 mL of reaction solution is one superoxide dismutase activity unit (U).

[0062] Example 1: Domestication, Isolation and Identification of Saccharomyces cerevisiae SOD-1 with High SOD Production

[0063] Saccharomyces cerevisiae, also known as baker's yeast or budding yeast, is commonly used to make bread, steamed buns and brew wine. As a high-quality functional and nutritional feed, Saccharomyces cerevisiae is approved to be directly added to animal feed, and it can express Cu / Zn-SOD itself. Cu / Zn-SOD exerts catalytic activity through the copper and zinc ions chelated thereto. The lack of these two metal ions will cause it to lose its enzyme activity. Therefore, it can be coerced into producing SOD with high enzyme activity by a high concentration of copper and zinc ions in the culture environment. The original Saccharomyces cerevisiae strain used for screening in the present invention is provided by Hubei Huada Ruier Technology Co., Ltd. After directed domestication in 0-1mM copper ion and 0-1mM zinc ion culture solution, Saccharomyces cerevisiae SOD-1 with high SOD production is isolated. Specifically comprising the following steps:

[0064] (1) Activation of Saccharomyces cerevisiae: The Saccharomyces cerevisiae seed solution was inoculated into YPD medium (pH 5.0) and activated for 24 h at 28°C and 200 rpm / min. YPD medium includes the following components: 1% yeast extract powder, 2% peptone and 2% glucose, sterilized at 115°C for 30 min, and 2% glucose was sterilized separately and then cooled and mixed.

[0065] (2) Induction of Saccharomyces cerevisiae: The activated culture solution was inoculated into YPD medium (30 mL) containing different concentrations of copper ions and zinc ions, and cultured at 28°C, pH 5.0, and 200 rpm / min for 24 h, 48 h, and 72 h, respectively. The culture solution was then collected and centrifuged at 12000 g for 5 min to collect the bacteria, and 5 mL of resuspension buffer was added to the collected bacteria, and the SOD activity of the cell lysis solution was sampled and measured. The resuspension buffer for the bacteria was a phosphate buffer, which included the following components: 0.059% sodium dihydrogen phosphate dihydrate and 0.58% disodium hydrogen phosphate dodecahydrate.

[0066] The results of SOD activity detection of bacterial cell disruption fluid are shown in Table 1. It can be seen that the grain wine yeast cultivated for 48 hours at a concentration of 0.1 mM / L copper and zinc ions has the highest enzyme activity, and the SOD enzyme activity is increased by 58.73% compared with the original strain.

[0067] Table 1 Results of superoxide dismutase activity assay under different copper and zinc ion concentrations

[0068]

[0069] (3) Screening of brewer's yeast: The group with the highest enzyme activity improvement, i.e., the culture solution cultured at 0.1 mM / L copper and zinc ion concentrations for 48 h, was selected and diluted in multiple proportions and spread on solid YPD medium containing 0.1 mM / L copper and zinc ions, respectively. The culture was placed in a 28°C incubator and incubated upside down for 2-4 days. Single colonies with typical yeast colony characteristics were randomly selected and inoculated into YPD medium containing 0.1 mM / L copper and zinc ions, and cultured at 28°C and 200 rpm for 48 h to obtain candidate yeast strains.

[0070] (4) Identification of Saccharomyces cerevisiae: The screened Saccharomyces cerevisiae was preliminarily identified by morphology. Its colonies were milky white and shiny, flat and with neat edges, and most of them were round or oval. At the same time, the screened Saccharomyces cerevisiae was sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for sequencing identification. The sequencing primers were universal primers ITS1: 5'-TCCGTAGGTGAACCTGCGG-3'; ITS4: 5'-TCCTCCGCTTATTGATATGC-3'. The ITS sequence determination results are shown in SEQ ID NO.1. Blast comparison analysis was performed in the NCBI database. The similarity and homology between the screened strain and the Saccharomyces cerevisiae isolate JGF21 (GenBank: MZ089538.1) was 97.1%, and it could be identified as Saccharomyces cerevisiae, and named Saccharomyces cerevisiae SOD-1.

[0071] The ITS gene of Saccharomyces cerevisiae SOD-1 is as follows:

[0072] (See SEQ ID NO.1).

[0073] The above-mentioned Saccharomyces cerevisiae SOD-1 was deposited in the China Center for Type Culture Collection (CCTCC) on September 27, 2024, with the deposit number CCTCC M 20242101. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province (Wuhan University), and the culture classification is named Saccharomyces cerevisiae.

[0074] Example 2 Study on the characteristics of SOD produced by fermentation of Saccharomyces cerevisiae SOD-1

[0075] The production of SOD by fermentation of Saccharomyces cerevisiae SOD-1 includes the following steps:

[0076] (1) Activation of Saccharomyces cerevisiae SOD-1 strains: Saccharomyces cerevisiae SOD-1 was inoculated into YPD medium and activated at 28°C and 200 rpm / min for 24 h.

[0077] (2) Fermentation of Saccharomyces cerevisiae SOD-1: The activated Saccharomyces cerevisiae SOD-1 seed solution was inoculated into the fermentation medium (pH 5.0) at a 3% inoculum amount, and the dissolved oxygen content was controlled at 40%-60%. The fermentation culture was carried out at 28°C and 200 rpm / min for 48 hours. The fermentation medium included the following components: 1% yeast extract powder, 2% peptone, 2% glucose, 0.0025‰ copper sulfate pentahydrate and 0.0028‰ zinc sulfate heptahydrate, sterilized at 115°C for 30 minutes, and 2% glucose was sterilized separately and then cooled and mixed.

[0078] (3) Saccharomyces cerevisiae SOD-1 cells and fragmentation: After the fermentation culture was completed, the culture medium was collected and concentrated using a disc centrifuge, and the cells were resuspended in a resuspension buffer at a weight-to-volume ratio of 1:5, that is, 5 mL of resuspension buffer was added per gram of wet weight of the cells, and then the cells were fragmented using a high-pressure homogenizer at a pressure of 1500 bar. This was repeated 5 times to obtain a cell fragmentation liquid containing SOD.

[0079] (4) Heat the cell suspension containing SOD at 70°C for 30 min and centrifuge to obtain the supernatant containing SOD, which is the SOD enzyme solution. Mix the supernatant with loading buffer and perform SDS-PAGE. The electrophoresis pattern is as follows: Figure 1 As shown, lane 1 is a molecular marker, and lanes 2-5 are repeated spotting of SOD enzyme solution. As can be seen from the figure, the molecular weight of the SOD of the present invention is 16kDa, and the purity of SOD in the enzyme solution is relatively high, reaching more than 85%.

[0080] By measuring the SOD protein yield and enzyme activity of different batches of fermentation broth, it is calculated that 50-80 g of wet bacteria can be obtained per liter (L) of fermentation broth of the brewer's yeast SOD-1 of the present invention, the SOD activity of the bacterial cell crushing liquid is 2000-2500 U / g wet bacteria, and the enzyme amount that can be purified is 30 mg-50 mg.

[0081] SOD temperature tolerance: The SOD enzyme solution obtained in step (4) was divided into 10 tubes with a volume of 5 mL, and kept in a 70°C water bath for 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 hours, respectively, and the enzyme activity at different denaturation times was measured. Figure 2As shown. Taking the enzyme solution without heat treatment as 100%, the relative enzyme activities at different heating times are 94.86%, 81.20%, 68.29%, 57.43%, 45.40%, 36.05%, 34.56%, 28.61%, 25.77%, and 19.79%, respectively. It can be seen that the SOD produced by fermenting Saccharomyces cerevisiae SOD-1 of the present invention has good thermal stability. The enzyme activity is basically not affected when kept in a 70°C water bath for less than 1 hour. When kept in a 70°C water bath for 2 hours, the enzyme activity can still be retained by more than 80%.

[0082] Since the SOD enzyme of the present invention has good thermal stability, the cell disruption solution can be subjected to a high-temperature treatment at 70°C for 30 minutes. In this step, most of the proteins in the cell contents are precipitated due to thermal denaturation, while the SOD enzyme of the present invention is not affected. Therefore, after a one-step heat treatment, a SOD enzyme with high purity can be obtained by centrifugation, which greatly simplifies the preparation process.

[0083] Example 3 Study on improving egg-laying performance of late-stage laying hens by using feed additives containing SOD

[0084] Under normal circumstances, the body's oxidation level and antioxidant level are in a balanced state. However, with age and the need for production, the balance between the two is broken, resulting in a weakened ability of the liver and ovaries to cope with oxidative stress, thus affecting their function. In addition, the liver's lipid metabolism is active, and lipid peroxidation will further lead to oxidative stress. SOD can scavenge oxygen free radicals and reduce their oxidative stress effects on the body. Therefore, adding exogenous SOD can improve the antioxidant capacity of poultry and livestock, which is beneficial to their production and recovery.

[0085] This example aims to explore the effect of SOD enzyme produced by Saccharomyces cerevisiae SOD-1 on the egg-laying performance of late laying hens. The SOD enzyme solution prepared above was mixed with corn starch and fully dried in a 60°C oven, and the specific activity of SOD in the dry powder was adjusted to 100,000 U / g. The dry powder preparation was used as a feed additive in an amount of 0.2‰, that is, 0.2g of the aforementioned feed additive was added per kg of basic feed. The basic feed during the experiment was CP Group's 324 laying hen compound feed (article number: CP 324), and its composition and nutritional level are shown in Table 2.

[0086] Table 2 Composition of basic feed for laying hens

[0087]

[0088] Grouping and feeding of laying hens: The laying hens were of Hailan brown laying hens, 58 weeks old, and the experimental site was Hongteng laying hens breeding farm in Gong'an County, Jingzhou City, Hubei Province. The laying hens were randomly divided into a control group (NC) and a SOD experimental group (SOD), with 6 replicates in each group and 10 chickens in each replicate. The control group (NC) was fed with a basic feed, and the SOD experimental group (SOD) was fed with 0.2‰ of the SOD dry powder of the present invention on the basis of the control group (NC). During the experiment, the laying hens were free to eat and drink, and the other conditions were carried out according to the standards of the breeding farm. The entire experimental period was 6 weeks, i.e. 42 days.

[0089] Sample collection: During the entire test period, eggs were collected and counted at 12 noon every day. On the last day of the sixth week of the test period, i.e., day 42, all eggs were collected, and one laying hen was randomly selected in each replicate for venous blood sampling using a coagulation tube and then killed. The liver was separated and cleaned in saline, and a portion was placed in 4% paraformaldehyde for fixation, and a portion was placed in a 2 mL cryotube and frozen in liquid nitrogen for testing.

[0090] Data analysis: The data were statistically analyzed in Microsoft Excel 2019, and the differences between the groups were analyzed by Ttest using GraphPad Prism 8. The results were expressed as mean ± standard error (mean ± SE). P < 0.05 indicated a significant difference, where * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0091] 3.1. Egg production statistics

[0092] Egg production rate (%) = (number of eggs laid / number of laying hens) × 100%. The changes in egg production rate during the experiment are as follows: Figure 3 As shown in the figure, in the initial stage, the egg production rates of the control group (NC) and the SOD experimental group (SOD) were both around 90%. As the age of the laying hens increased, the egg production rate of the control group (NC) showed a downward trend, while the egg production rate of the SOD experimental group (SOD) gradually maintained at around 95%, indicating that feeding SOD in the late laying period of laying hens can greatly improve the egg production rate of laying hens.

[0093] 3.2. Egg quality testing

[0094] The eggshell strength was tested using an eggshell strength tester (Guangzhou Runhu Instrument Co., Ltd., Guangzhou, China), the albumen height, yolk color, and Haugh value were tested using a multifunctional egg quality analyzer (Japan Robotmation Co., Ltd., Nanjing, China), and the egg fat content was tested in accordance with the National Standard of the People’s Republic of China GB 5009.6-2016 “Determination of Fat in Foods”. The test steps and calculation formulas were strictly followed in accordance with the methods specified in this standard.

[0095] After the experiment, the quality of eggs changed as follows: Figure 4 As shown in the data, there were significant differences between the control group (NC) and the SOD experimental group (SOD) in eggshell strength, egg white height, yolk color, Haugh value, egg fat content, etc., and the control group (NC) was significantly lower than the SOD experimental group (SOD), indicating that feeding SOD to laying hens during egg laying can significantly improve the quality of eggs.

[0096] 3.3 Serum antioxidant level detection

[0097] The detection kits for superoxide dismutase (catalog number: A001-1-2), glutathione peroxidase (catalog number: A005-1-2), catalase (catalog number: A007-1-1), total antioxidant capacity (catalog number: A015-2-1), and malondialdehyde (catalog number: A003-1-2) in serum were purchased from Nanjing Jiancheng Bioengineering Institute, and the detection steps and calculation formulas were carried out according to the kit instructions.

[0098] After the experiment, the serum biochemical indicators of laying hens were as follows Figure 5 As shown in the data, there were significant differences between the control group (NC) and the SOD experimental group (SOD) in superoxide dismutase, glutathione peroxidase, catalase, total antioxidant capacity, etc., and the control group (NC) was significantly lower than the SOD experimental group (SOD), while the malondialdehyde level in the control group (NC) was significantly higher than that in the SOD experimental group (SOD), indicating that feeding SOD during laying period can significantly improve the antioxidant capacity of laying hens.

[0099] 3.4. Liver lipid distribution and liver and serum lipid content detection

[0100] The lipid distribution in the liver of laying hens was analyzed by frozen sections of liver tissue, oil red O staining, and microscopic examination. The results were as follows: Figure 6 As shown, Oil Red O staining can stain the lipid droplets in the liver into orange to bright red. It can be seen from the figure that the lipid droplet content of the control group (NC) is significantly higher than that of the SOD experimental group (SOD), indicating that feeding SOD to late laying hens can significantly reduce the accumulation of fat in the liver of laying hens, and has the effect of treating or preventing fatty liver.

[0101] The detection steps and calculation formulas of lipid content in liver and serum were carried out according to the instructions of the corresponding kits. The detection kits for triglyceride (item number: A110-1-1), total cholesterol (item number: A111-1-1), low-density lipoprotein cholesterol (item number: A113-1-1), and high-density lipoprotein cholesterol (item number: A112-1-1) were purchased from Nanjing Jiancheng Bioengineering Institute.

[0102] The results of serum and liver lipid content tests were as follows: Figure 7-8 As shown. It can be seen that the control group (NC) was significantly lower than the SOD experimental group (SOD) in terms of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). This shows that feeding SOD to late-stage laying hens can significantly increase lipid transfer in the liver and transfer lipids to eggs through the liver-blood-egg pathway, thereby reducing lipid accumulation in the liver and improving egg quality.

[0103] 3.5. Detection of relative expression of genes related to lipid synthesis

[0104] Total RNA was extracted from the liver using the SPARKeasy tissue / cell RNA rapid extraction kit (purchased from Shandong Sikejie Biotechnology Co., Ltd., catalog number: AC0202-B), and then reverse transcribed into cDNA using the SPARKscriptⅡAll-in-oneRTSuperMixfor qPCR (purchased from Shandong Sikejie Biotechnology Co., Ltd., catalog number: AG0305-B) reverse transcription kit. The expression levels of genes were detected using real-time quantitative detection (RT-qPCR). Fat synthesis-related genes included fatty acid synthase (FASN), peroxisome proliferator-activated receptor γ (PPARG), and transmembrane protein 68 (TMEM68). 2×SYBRGreenqPCRMix (purchased from Shandong Sikejie Biotechnology Co., Ltd., catalog number: AH0104-B) reagent was used for pretreatment before loading, and the results were detected on a CFX96Touch fluorescent quantitative PCR instrument (Bio-Rad, USA). -△△Ct Methods The relative expression level of each gene was calculated and normalized by the internal reference gene β-actin.

[0105] The relative expression of lipid synthesis related genes is as follows Fig. 9 As shown in the figure, the expression levels of genes such as fatty acid synthase, peroxisome proliferator-activated receptor γ, and transmembrane protein 68 in the control group (NC) were significantly lower than those in the SOD experimental group (SOD), indicating that feeding SOD to late laying hens can significantly inhibit lipid synthesis in the liver, thereby inhibiting the accumulation of lipids in the liver.

[0106] 3.6. Observation of liver tissue morphology and detection of inflammatory factor expression

[0107] Paraffin-embedded liver tissue sections were taken for HE staining, and the liver morphology was analyzed under a microscope. Fig.10 The liver cells in the field of view have clear structures, round nuclei, rich cytoplasm, and no necrosis or degeneration. However, focal infiltration of inflammatory cells can be seen in the liver tissue of the control group (NC), while only a small amount of inflammatory cells infiltrate the liver tissue of the SOD experimental group (SOD), as shown by the red arrows.

[0108] The expression of genes related to liver inflammatory factors Fig.11 As shown in the figure, the expression levels of inflammatory factors such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10) in the control group (NC) were significantly higher than those in the SOD experimental group (SOD), indicating that feeding SOD to late laying hens can significantly inhibit the inflammatory response of the liver and achieve the effect of protecting the liver.

[0109] The primer sequences used in the above examples are shown in Table 3.

[0110] Table 3 Primer sequences used for RT-qPCR

[0111]

[0112]

[0113] Based on the above data, it can be seen that feeding SOD to late-stage laying hens can regulate lipid metabolism in the liver and promote lipid transfer, while improving the body's antioxidant capacity, alleviating the fatty liver phenomenon in late-stage laying hens, and at the same time slowing down the inflammatory response of the liver. Multiple factors work together to promote the increase in their egg production rate.

[0114] Example 4 Study on improving the antioxidant performance of sows by feed additives containing SOD

[0115] This example aims to explore the effect of SOD enzyme produced by Saccharomyces cerevisiae SOD-1 as a feed additive on the antioxidant capacity of farrowing sows. The preparation method of the feed additive comprises: mixing the above-prepared SOD enzyme solution with corn starch and fully drying it in a 60°C oven, and adjusting the specific activity of SOD in the dry powder to 100,000 U / g. The amount of feed additive added is 1‰ of the basic feed. The composition and nutritional level of the basic feed are shown in Table 4.

[0116] Table 4 Composition of basic feed for sows

[0117]

[0118] Note: 1) Premix for pregnancy provides Cu5mg, I0.15mg, Fe83mg, Mn20mg, Zn128mg, VA13400IU, VD per kg of feed. 3 3000IU, VE22.4mg and VK 3 3mg. The lactation premix provides Cu20mg, I0.12mg, Fe80mg, Mn44mg, Zn88mg, VA10000IU, VD per kg of feed. 3 1400IU, VE4IU, VK 31.28mg. 2) Nutritional levels are calculated values.

[0119] Sow grouping and feeding: The sow breed is "Landrace × Large White" dual-purpose sows. The sows are divided into a control group (NC) and a SOD experimental group (SOD) according to the body condition score, with 10 replicates in each group and 3 pigs in each replicate. The experiment started on the 86th day of sow gestation and ended on the 21st day of lactation after delivery. The control group (NC) was fed with a basic feed, and the SOD experimental group (SOD) was fed with 1‰ of the SOD dry powder described in the present invention on the basis of the control group (NC).

[0120] Sample collection: Six sows with similar body conditions and the same farrowing date were selected from each group for blood sampling from the anterior vena cava. The blood was centrifuged to separate the serum after static sedimentation, stored on dry ice and transported back to the laboratory at -80℃ for testing.

[0121] The relevant kits of Nanjing Jiancheng Bioengineering Research Institute were used to detect the antioxidant indexes of sow serum, including catalase, superoxide dismutase, glutathione peroxidase activity, glutathione (item number: A006-2-1) content and total antioxidant capacity, and the serum oxidative damage indexes, including malondialdehyde, hydrogen peroxide (item number: A064-1-1) and tumor necrosis factor-α (item number: H052-1-2) content, were also measured. The relevant results are shown in Table 5-6.

[0122] Table 5 Effect of SOD dry powder preparation on antioxidant indexes of sows

[0123]

[0124] Table 6 Effects of SOD dry powder preparation on sow serum oxidative damage indicators

[0125]

[0126] Compared with the control group, the antioxidant activities of serum T-AOC, GSH-Px, GSH, CAT, and SOD in the SOD experimental group were significantly increased (P<0.05), and the activities of serum T-AOC, GSH-Px, GSH, CAT, and SOD increased by 144.86%, 107.5%, 13.75%, 32.85%, and 113.44%, respectively. 2 O 2 , MDA and TNF-α levels were significantly decreased (P<0.05). 2 O 2 , MDA and TNF-α contents decreased by 256.30%, 133.70% and 158.90% respectively. The results showed that adding SOD to the diet could alleviate the oxidative stress of sows and enhance their antioxidant capacity.

[0127] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A brewer's yeast, It is characterized in that The deposit number is CCTCC NO:M 20242101.

2. Use of the saccharomyces cerevisiae as claimed in claim 1 in the preparation of superoxide dismutase.

3. A method for preparing superoxide dismutase, It is characterized in that The following steps are involved: The activated brewer's yeast as claimed in claim 1 is inoculated into a fermentation medium containing copper ions and zinc ions, cultured for a period of time, the culture solution is collected and centrifuged to obtain bacterial cells, the bacterial cells are resuspended and then broken, and then the cell disruption solution is placed at 70° C. for 30-60 minutes, and the supernatant is collected to obtain an enzyme solution containing superoxide dismutase.

4. The method for preparing superoxide dismutase according to claim 3, It is characterized in that The fermentation medium comprises 0.01-1 mmol / L of copper ions and 0.01-1 mmol / L of zinc ions.

5. The method for preparing superoxide dismutase according to claim 4, It is characterized in that The fermentation medium comprises the following components in weight percentage: 1% yeast extract powder, 2% peptone, 2% glucose, 0.0025‰ copper sulfate pentahydrate and 0.0028‰ zinc sulfate heptahydrate.

6. A superoxide dismutase, It is characterized in that The superoxide dismutase is prepared by the method for preparing superoxide dismutase according to any one of claims 3 to 5.

7. Use of the superoxide dismutase as claimed in claim 6 in improving animal production performance.

8. Use of the superoxide dismutase according to claim 7 in improving animal production performance, It is characterized in that The application method comprises the following steps: The superoxide dismutase dry powder preparation is mixed with the basic feed of the animal and then fed to the animal. The addition amount of the superoxide dismutase dry powder preparation is 0.1-2‰ of the total mass of the basic feed.

9. Use of the superoxide dismutase according to claim 8 in improving animal production performance, It is characterized in that The preparation method of the superoxide dismutase dry powder preparation comprises: The enzyme solution containing superoxide dismutase is mixed with corn starch and then dried to prepare the superoxide dismutase dry powder preparation, and the specific activity of the superoxide dismutase dry powder preparation is 1-3 million U / g.

10. Use of the superoxide dismutase according to claim 7 in improving animal production performance, It is characterized in that The animal includes laying hens or sows, and the animal production performance includes late laying hen production performance or sow antioxidant performance.

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