Selenium-rich enterococcus faecium microbial agent and preparation method thereof

Abstract

The invention discloses a selenium-rich enterococcus faecium microbial inoculum and a preparation method thereof.The selenium-rich enterococcus faecium microbial inoculum is prepared through a biological fermentation technology and contains organic selenium capable of being absorbed by organisms, the content of nano-selenium in the microbial inoculum is 10-120 micrograms / gram, and the number of active colonies of selenium-rich enterococcus faecium is 1 * 10 < 9 >-5 * 10 < 10 > CFU / gram. By adding the nano-selenium, the acid resistance and bile resistance of the enterococcus faecium are improved, and the survival rate of the enterococcus faecium in the digestive tract is increased, so that the bioavailability of the microbial inoculum is improved. Nano-selenium has antioxidant, anti-inflammatory and immunoregulation biological activities and the like, and after the nano-selenium is combined with enterococcus faecium, the overall functionality of the microbial inoculum can be enhanced, and more comprehensive health management is provided for hosts.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a selenium-enriched Enterococcus faecium agent and a preparation method thereof. Background Art

[0002] With the acceleration of modern life and changes in dietary structure, people are paying more and more attention to health issues. Probiotics, as active microorganisms that can have a beneficial effect on the host, have become a hot topic in the research of health foods and medicines. Probiotics have a positive effect on human health through multiple mechanisms such as improving the balance of intestinal microorganisms, enhancing immunity, and inhibiting the growth of harmful bacteria.

[0003] However, traditional probiotic products have some limitations. First, the stability and activity of probiotics are key factors affecting their effectiveness. During production, storage and transportation, probiotics are easily affected by environmental factors, resulting in decreased activity. Secondly, the bioavailability of probiotics is also a problem. When many probiotics pass through the digestive tract, the number of live bacteria reaching the intestine is greatly reduced due to the destruction of gastric acid and bile. In addition, the functionality of probiotics also needs to be further improved to meet the health needs of different populations.

[0004] The development of nanotechnology has provided new ideas for the research and application of probiotics. As a nanomaterial with multiple biological activities such as antioxidant, anti-inflammatory, and immunomodulatory, nano-selenium has been widely used in medicine, food, and cosmetics. Combining nano-selenium with probiotics can not only improve the stability and bioavailability of probiotics, but also enhance the functionality of probiotics, achieving the effect of 1+1>2.

[0005] There are various methods for preparing nano-selenium, including chemical synthesis, physical vapor deposition, and biosynthesis. Among them, biosynthesis is considered to be a promising method for preparing nano-selenium due to its advantages such as environmental friendliness, low cost, and easy large-scale production. However, there are still some problems with the existing biosynthetic nano-selenium technology, such as the difficulty in controlling the particle size, morphology, and dispersibility of nano-selenium, the low efficiency of combining nano-selenium with probiotics, and the biological safety and stability of nano-selenium.

[0006] In view of these problems, the present invention proposes a method for preparing nano-selenium Enterococcus faecium. As a common probiotic, Enterococcus faecium has the advantages of acid resistance, bile resistance, and easy colonization, and is an ideal nano-selenium carrier. By optimizing the culture medium formula, fermentation conditions and freeze-drying process, the present invention successfully prepared highly active and highly stable nano-selenium Enterococcus faecium, providing a new solution for the research and application of probiotics. Summary of the invention

[0007] In order to solve the problems of the prior art, the purpose of the present invention is to provide a selenium-enriched Enterococcus faecium agent and a preparation method thereof.

[0008] In order to achieve the above-mentioned object, the present invention adopts the following technical scheme: The first aspect of the present invention is to provide a selenium-enriched Enterococcus faecium agent. The second aspect of the present invention is to provide a method for preparing a selenium-enriched Enterococcus faecium agent. The third aspect of the present invention is to provide a method for preparing a selenium-enriched Enterococcus faecium freeze-dried powder. The fourth aspect of the present invention is a freeze-dried powder of selenium-enriched Enterococcus faecium.

[0009] The selenium-rich Enterococcus faecium bacterial agent of the present invention is prepared by a biological fermentation process and contains organic selenium that can be absorbed by the body, wherein the content of nano-selenium in the bacterial agent is 10-120 micrograms / gram, and the number of active colonies of the selenium-rich Enterococcus faecium is 1×10 9 Up to 5×10 10 CFU / g.

[0010] Furthermore, the selenium-enriched Enterococcus faecium agent is provided in the form of tablets, freeze-dried powder, capsules, dairy products, stirred yogurt or solidified yogurt.

[0011] The preparation method of the selenium-enriched Enterococcus faecium agent of the present invention comprises the following steps:

[0012] a. Prepare Enterococcus faecium culture medium and adjust the pH to 7.4 using NaOH;

[0013] b. Sterilize the culture medium at 121°C for 20 minutes and cool to room temperature;

[0014] c. Inoculate the thawed Enterococcus faecium seed solution into the culture medium and activate it in a constant temperature incubator at 40°C for 24 hours;

[0015] d. The activated Enterococcus faecium seed solution was inoculated into 1L of culture medium at a 3% inoculum volume, and nano-selenium was added after sterilization by filtration through a 0.22μm filter membrane to a final concentration of 10μg / ml of the nano-selenium solution, and cultured in a constant temperature incubator at 40°C for 24 hours;

[0016] e. The fermented bacteria were collected by centrifugation at 8000 g, 10 min, and 4° C. to obtain a selenium-rich Enterococcus faecalis agent.

[0017] Furthermore, the preparation of the Enterococcus faecium culture medium includes the following components:

[0018]

[0019]

[0020] Further, a. using 20 mM phosphate buffer PBS, 0.9% NaCl, 5% skimmed milk powder, adjust the pH to 7.0 of the sterilization solution to resuspend the bacterial sludge, the ratio is 50 ml solution to resuspend 1 gram of bacterial sludge; b. the resuspended bacterial sludge is freeze-dried, and the freeze-drying procedure includes pre-freezing to -45°C for 1.5 hours, followed by main drying at -45°C for 4 hours, and then gradually heating to 20°C and final drying under a vacuum degree of 0.2 mba.

[0021] Furthermore, in step (d), the method for preparing the nano-selenium solution comprises the following steps:

[0022] (1) Add stabilizer to a beaker;

[0023] (2) slowly adding the aqueous solution containing the reducing agent into the beaker, and maintaining magnetic stirring during the addition process until the stabilizer is completely dissolved;

[0024] (3) slowly adding the sodium selenite aqueous solution to the solution obtained in step (2), during which the solution in step (2) is under magnetic stirring;

[0025] (4) After the sodium selenite aqueous solution is added, the pH is immediately adjusted to a predetermined range using a sodium hydroxide solution, and then the magnetic stirring is continued for 30 minutes to obtain a red nano-selenium solution.

[0026] Further, in step (1), the stabilizer is selected from at least one of epigallocatechin gallate EGCG, chitosan, gum arabic, bovine serum albumin BSA or sodium hyaluronate; the concentration range of the epigallocatechin gallate EGCG is 0.1mM-40mM; the concentration range of the chitosan is 0.01%-0.7%, and the molecular weight range of the chitosan is 100KD-1000KD; the concentration range of the gum arabic is 0.04%-0.4%, and the molecular weight range of the gum arabic is 200KD-2000KD; the concentration range of the bovine serum albumin BSA is 0.4mg / ml-60mg / ml, and the molecular weight range of the bovine serum albumin BSA is 200KD-2000KD; the concentration range of the sodium hyaluronate is 0.02%-0.25%, and the molecular weight range of the sodium hyaluronate is 200KD-2000KD.

[0027] In step (2), the reducing agent is selected from at least one of reduced glutathione GSH or vitamin C (Vc);

[0028] Furthermore, in step (2), the final concentration of the reducing agent in the reaction system is 5mM-50mM glutathione GSH and 5mM-100mM vitamin C Vc; in step (3), the final concentration of sodium selenite in the reaction system is 5mM; in step (4), in the step of adjusting the pH, the pH value ranges from 5.0 to 9.0.

[0029] The present invention provides a method for preparing a selenium-enriched Enterococcus faecium freeze-dried powder, which is characterized by comprising the following steps: a. using 20 mM phosphate buffer (PBS), 0.9% NaCl, 5% skimmed milk powder, and adjusting the pH to 7.0 of a sterilizing solution to resuspend the bacterial mud, the ratio being 50 ml of the solution to resuspend 1 gram of the bacterial mud;

[0030] b. The resuspended bacterial sludge is freeze-dried, and the freeze-drying procedure includes pre-freezing to -45°C for 1.5 hours, followed by main drying at -45°C for 4 hours, and then gradually warming to final drying at 20°C. After freeze-drying, the sludge is collected to obtain selenium-enriched Enterococcus faecium freeze-dried powder; the vacuum degree of main drying and terminal drying is maintained at 0.2mba.

[0031] c. Dilute the lyophilized powder to the desired concentration using skimmed milk powder or other fillers.

[0032] Further, in step (c), 10 g of 5.0 x 10 10 The selenium-enriched Enterococcus faecium mother powder was diluted to a concentration of 1.0x10 cfu / g in 50 g. 10 cfu / g of bacterial powder as an example, using the gradient dilution method, first weigh 10.0 g of 5.0x10 10 cfu / g mother powder, add 2.5g skim milk powder, mix well, and get 12.5g of 4.0x10 10 cfu / g of bacterial powder; then 12.5 g of 4.0x10 10 cfu / g of bacterial powder, add 12.5 g of skimmed milk powder and mix well to obtain 25.0 g of 2.0x10 10 cfu / g of bacterial powder; finally, 25.0 g of 2.0x10 10 cfu / g of bacterial powder was mixed with 25.0 g of skimmed milk powder to obtain 50.0 g of a concentration of 1.0x10 10 cfu / g selenium-enriched Enterococcus faecium freeze-dried powder.

[0033] The selenium-enriched Enterococcus faecium freeze-dried powder of the invention is prepared by a preparation method.

[0034] Beneficial effects: The addition of nano-selenium in the present invention improves the acid and bile resistance of Enterococcus faecium, increases its survival rate in the digestive tract, and thus improves the bioavailability of the bacterial agent. Nano-selenium has biological activities such as antioxidant, anti-inflammatory and immunomodulatory. After combining with Enterococcus faecium, it can enhance the overall functionality of the bacterial agent and provide more comprehensive health management for the host.

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] Highly active bacterial agent: Through specific culture medium formula and fermentation conditions, the selenium-rich Enterococcus faecalis bacterial agent prepared has high activity, ensuring the biological efficacy of the bacterial agent.

[0037] Stable selenium content: By precisely controlling the amount of nano-selenium added, the stable selenium content in the inoculant is ensured, which helps to achieve the expected health benefits.

[0038] Enhanced stability: The optimized freeze-drying process improves the stability of the bacterial agent, allowing it to remain active during storage and transportation, extending the shelf life of the product.

[0039] Environmentally friendly production process: The biosynthesis method is used to prepare nano-selenium, which reduces the environmental pollution that may be caused by the chemical synthesis process and meets the requirements of green production.

[0040] Easy to scale up production: The preparation of the culture medium and the fermentation process are standardized, which makes it easy to scale up in industrial production to meet market demand.

[0041] High safety: The selected Enterococcus faecium CICC 10840 strain is a known safe probiotic strain, combined with the biosynthesis method of nano-selenium to ensure the safety of the product.

[0042] Wide range of applications: The prepared selenium-rich Enterococcus faecalis agent can be widely used in food, health products, medicines and other fields, providing consumers with diversified health choices.

[0043] Scientific quality control: Through live bacteria counting and ICP-MS analysis, the accuracy and consistency of live bacteria count and selenium content in the inoculant are ensured, facilitating scientific quality control. DETAILED DESCRIPTION

[0044] The following will be combined with the test examples of the present invention to clearly and completely describe the technical solutions in the test examples of the present invention. Obviously, the test examples described are only part of the test examples of the present invention, not all of the test examples. Based on the test examples in the present invention, all other test examples obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0045] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0046] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0047] It should be understood that in the various test examples of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the test examples of the present application.

[0048] The terms used in the test examples of this application are only for the purpose of describing specific test examples, and are not intended to limit this application. The singular forms "a", "said" and "the" used in the test examples of this application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0049] The weight of the relevant components mentioned in the test example description of this application can not only refer to the specific content of each component, but also indicate the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the test example description of this application, it is within the scope disclosed in the test example description of this application. Specifically, the mass described in the test example description of this application can be μg, mg, g, kg and other mass units known in the chemical industry.

[0050] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. For example, without departing from the scope of the test examples of this application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0051] Unless otherwise specified, the reagents and materials used in the present invention are prepared by conventional methods or obtained from commercial channels.

[0052] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual points, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0053] The first aspect of the embodiment of the present application provides a nano-selenium-rich Enterococcus faecium agent, which is prepared by a biological fermentation process and contains organic selenium that can be absorbed by the body, wherein the selenium content in the agent is 10-120 μg / g, and the number of active colonies of the nano-selenium Enterococcus faecium is 1×10 9 Up to 5×10 10 CFU / g.

[0054] In some embodiments, the selenium-enriched Enterococcus faecium agent is in the form of a tablet.

[0055] In some embodiments, the selenium-enriched Enterococcus faecium agent is a freeze-dried powder.

[0056] In some embodiments, the selenium-enriched Enterococcus faecium agent is a capsule.

[0057] In some embodiments, the selenium-enriched Enterococcus faecium agent is a dairy product.

[0058] In some embodiments, the selenium-enriched Enterococcus faecium agent is stirred yogurt.

[0059] In some embodiments, the selenium-enriched Enterococcus faecium agent is solidified yogurt.

[0060] The second aspect of the present application embodiment provides a method for preparing a fecal enterococcus agent, comprising the following steps:

[0061] a. Prepare Enterococcus faecium culture medium and adjust the pH to 7.4 using NaOH;

[0062] b. Sterilize the culture medium at 121°C for 20 minutes and cool to room temperature;

[0063] c. Inoculate the thawed Enterococcus faecium seed solution into the culture medium and activate it in a constant temperature incubator at 40°C for 24 hours;

[0064] d. The activated Enterococcus faecium seed solution was inoculated into 1L of culture medium at a 3% inoculum volume, and nano-selenium was added after sterilization by filtration through a 0.22μm filter membrane to a final concentration of 10μg / ml of the nano-selenium solution, and cultured in a constant temperature incubator at 40°C for 24 hours;

[0065] e. The fermented bacteria were collected by centrifugation at 8000 g, 10 min, and 4° C. to obtain a selenium-rich Enterococcus faecalis agent.

[0066] Enterococcus faecium was obtained from China Industrial Microbiological Culture Collection Center, strain number: CICC 10840.

[0067] In some embodiments, the preparation of Enterococcus faecium culture medium comprises the following components:

[0068]

[0069] In some embodiments, in step (d), the method for preparing the nano-selenium solution comprises the following steps:

[0070] (1) Add stabilizer to a beaker;

[0071] (2) slowly adding the aqueous solution containing the reducing agent into the beaker, and maintaining magnetic stirring during the addition process until the stabilizer is completely dissolved;

[0072] (3) slowly adding the sodium selenite aqueous solution to the solution obtained in step (2), during which the solution in step (2) is under magnetic stirring;

[0073] (4) After the sodium selenite aqueous solution is added, the pH is immediately adjusted to a predetermined range using a sodium hydroxide solution, and then the magnetic stirring is continued for 30 minutes to obtain a red nano-selenium solution.

[0074] In some embodiments, in step (1), the stabilizer is epigallocatechin gallate (EGCG).

[0075] In some embodiments, in step (1), the stabilizer is chitosan.

[0076] In some embodiments, in step (1), the stabilizer is gum arabic.

[0077] In some embodiments, in step (1), the stabilizer is bovine serum albumin (BSA).

[0078] In some embodiments, in step (1), the stabilizer is sodium hyaluronate.

[0079] In some embodiments, in step (2), the reducing agent is reduced glutathione GSH.

[0080] In some embodiments, in step (2), the reducing agent is vitamin C (Vc).

[0081] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.1 mM to 40 mM (for EGCG).

[0082] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.01% to 0.7% (for chitosan).

[0083] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.04% to 0.4% (for gum arabic).

[0084] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.4 mg / ml to 60 mg / ml (for BSA).

[0085] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.02% to 0.25% (for sodium hyaluronate).

[0086] In some embodiments, in step (2), the final concentration of the reducing agent in the reaction system is 5 mM-50 mM (GSH).

[0087] In some embodiments, in step (2), the final concentration of the reducing agent in the reaction system is 5 mM-100 mM (Vc).

[0088] In step (3), the final concentration of sodium selenite in the reaction system is 5 mM.

[0089] In some embodiments, in step (1), the molecular weight of the stabilizer is in the range of 100 KD - 1000 KD (for chitosan).

[0090] In some embodiments, in step (1), the molecular weight of the stabilizer ranges from 200 KD to 2000 KD (for gum arabic).

[0091] In some embodiments, in step (1), the molecular weight of the stabilizer ranges from 200 KD to 2000 KD (for sodium hyaluronate).

[0092] In step (4), in the step of adjusting the pH, the pH value ranges from 5.0 to 9.0.

[0093] In some embodiments, the peak value of the particle size distribution of the nano-selenium prepared by the above steps can be precisely controlled between 20nm-60nm, and the particle size is mainly distributed between 10nm-150nm.

[0094] The third aspect of the present invention is to provide a method for preparing a selenium-enriched Enterococcus faecium freeze-dried powder, characterized by comprising the following steps: a. using 20 mM phosphate buffer (PBS), 0.9% NaCl, 5% skimmed milk powder, and adjusting the pH to 7.0 of the sterilization solution to resuspend the bacterial mud, the ratio being 50 ml of the solution to resuspend 1 gram of the bacterial mud;

[0095] b. The resuspended bacterial sludge was freeze-dried. The freeze-drying procedure included pre-freezing to -45°C for 1.5 hours, followed by main drying at -45°C for 4 hours, and then gradually warming to final drying at 20°C. After freeze-drying, the lyophilized powder of selenium-enriched Enterococcus faecium was obtained. The vacuum degree of main drying and terminal drying was maintained at 0.2mba.

[0096] c. Dilute the lyophilized powder to the desired concentration using skimmed milk powder or other fillers.

[0097] In some embodiments, in step (c), 10 g of 5.0 x 10 10 The selenium-enriched Enterococcus faecium mother powder was diluted to a concentration of 1.0x10 cfu / g in 50 g. 10 cfu / g of bacterial powder as an example, using the gradient dilution method, first weigh 10.0 g of 5.0x10 10 cfu / g mother powder, add 2.5g skim milk powder, mix well, and get 12.5g of 4.0x10 10 cfu / g of bacterial powder; then 12.5 g of 4.0x10 10 cfu / g of bacterial powder, add 12.5 g of skimmed milk powder and mix well to obtain 25.0 g of 2.0x10 10 cfu / g of bacterial powder; finally, 25.0 g of 2.0x10 10 cfu / g of bacterial powder was mixed with 25.0 g of skimmed milk powder to obtain 50.0 g of a concentration of 1.0x10 10 cfu / g selenium-enriched Enterococcus faecium freeze-dried powder.

[0098] The fourth aspect of the present invention is to provide a selenium-enriched Enterococcus faecium freeze-dried powder prepared by a preparation method.

[0099] Example 1

[0100] The preparation method of the selenium-enriched Enterococcus faecium agent of the present invention comprises the following steps:

[0101] (1) Fermentation of Nano-Selenium Enterococcus faecium

[0102] Prepare Enterococcus faecium culture medium, prepare 1000mL / bottle, 5 bottles; 200mL / bottle, 1 bottle. Sterilize at 121℃ for 20min, and cool to room temperature. The formula of Enterococcus faecium fermentation medium is shown in Table 1:

[0103] Table 1

[0104]

[0105] Fermentation process: 1 mL of thawed Enterococcus faecalis seed liquid is inoculated into 200 mL of culture medium, and the culture medium is placed in a constant temperature incubator at 40°C for activation for 24 hours; the activated Enterococcus faecalis seed liquid is inoculated into a blue-capped bottle containing 1 L of culture medium at an inoculation rate of 3%, and then nano-selenium that has been sterilized by filtration through a 0.22 μm filter membrane is added to make the final concentration of nano-selenium 10 μg / ml, and the culture medium is placed in a constant temperature incubator at 40°C for 24 hours, and the fermented bacteria are collected by centrifugation.

[0106] Example 2

[0107] The difference between Example 2 and Example 1 is that in the method for preparing the nano-selenium solution, in step (1), the stabilizer is chitosan, the reducing agent is reduced glutathione GSH, the concentration range of chitosan is 0.01%-0.7%, and the molecular weight range of chitosan is 100KD-1000KD.

[0108] In step (2), the final concentration of the reducing agent in the reaction system is 5 mM-100 mM (Vc).

[0109] Example 3

[0110] The difference between Example 3 and Example 1 is that in the preparation method of the nano-selenium solution, in step (1), the stabilizer is gum arabic, the reducing agent is vitamin C (Vc), the concentration range of gum arabic is 0.04%-0.4%, and the molecular weight range of gum arabic is 200KD-2000KD.

[0111] Example 4

[0112] The difference between Example 4 and Example 1 is that in the method for preparing the nano-selenium solution, in step (1), the stabilizer is bovine serum albumin BSA, and the concentration range of the bovine serum albumin BSA is 0.4 mg / ml-60 mg / ml.

[0113] Example 5

[0114] The difference between Example 5 and Example 1 is that in the preparation method of the nano-selenium solution, in step (1), the stabilizer is sodium hyaluronate. The concentration range of sodium hyaluronate is 0.02%-0.25%, and the molecular weight range of sodium hyaluronate is 200KD-2000KD.

[0115] Example 6

[0116] The method for preparing a selenium-enriched Enterococcus faecium freeze-dried powder of the present invention is characterized by comprising the following steps: a. using 20 mM phosphate buffer (PBS), 0.9% NaCl, 5% skimmed milk powder, and adjusting the pH to 7.0 of a sterilizing solution to resuspend the bacterial mud, the ratio being 50 ml of the solution to resuspend 1 gram of the bacterial mud;

[0117] b. The resuspended bacterial sludge was freeze-dried. The freeze-drying procedure included pre-freezing to -45°C for 1.5 hours, followed by main drying at -45°C for 4 hours, and then gradually warming to final drying at 20°C. After freeze-drying, the lyophilized powder of selenium-enriched Enterococcus faecium was obtained. The vacuum degree of main drying and terminal drying was maintained at 0.2mba.

[0118] Taking 10 grams of 5.0x10^10 cfu / g selenium-enriched Enterococcus faecium mother powder diluted to 50 grams of 1.0x10^10 cfu / g bacterial powder as an example, the gradient dilution method is used. First, 10.0 grams of 5.0x10^10 cfu / g mother powder is weighed, 2.5 grams of skim milk powder is added, and mixed to obtain 12.5 grams of 4.0x10^10 cfu / g bacterial powder; then, 10 grams of 5.0x10^10 cfu / g mother powder is weighed, 2.5 grams of skim milk powder is added, and mixed to obtain 12.5 grams of 4.0x10^10 cfu / g bacterial powder; then, 10 grams of 5.0x10^10 cfu / g bacterial powder is added to 10 grams of 5.0x10^10 cfu / g mother powder ... bacterial powder; then, 10 grams of 5.0x10^10 cfu / g bacterial powder is added to 10 grams of 5.0x10^10 cfu / g bacterial powder; then, 10 grams of 5.0x10^10 cfu / g bacterial powder is added to Add 12.5 grams of skimmed milk powder to 2.5 grams of 4.0x10^10 cfu / g bacterial powder and mix well to obtain 25.0 grams of 2.0x10^10 cfu / g bacterial powder; finally, mix 25.0 grams of 2.0x10^10 cfu / g bacterial powder and 25.0 grams of skimmed milk powder to obtain 50.0 grams of selenium-enriched faecium Enterococcus lyophilized powder with a concentration of 1.0x10^10 cfu / g.

[0119] c Take a certain amount of freeze-dried powder, dissolve it to a certain concentration with sterile water, count the live bacteria of nano-selenium Enterococcus faecium, and analyze the selenium content in the freeze-dried powder by inductively coupled plasma mass spectrometry (ICP-MS).

[0120] d. Perform live bacterial count and ICP-MS analysis on the freeze-dried powder to ensure that the live bacterial count reaches ≧5.0x 10 10 cfu / g, selenium content reaches ≧50μg / 1.0x10 10 cfu;

[0121] e. Dilute the above freeze-dried powder with skim milk powder or other fillers to make the content of nano-selenium 10-120 μg / g and the number of active colonies of selenium-rich Enterococcus faecium 1×10 9 Up to 5×10 10 CFU / g, and selenium-enriched Lactobacillus rhamnosus freeze-dried powder was obtained.

[0122] The freeze-drying procedure is shown in Table 2:

[0123] Table 2

[0124]

[0125] The selenium-rich Enterococcus faecium agent prepared by nano-selenium in the present invention has an active colony count of 1×10 9 Up to 5×10 10 CFU / g, the actual measured content of selenium in the bacterial agent (ICP-MS method) is: 10-120 micrograms / g.

[0126] The present invention proposes a method for preparing nano-selenium Enterococcus faecium. As a common probiotic, Enterococcus faecium has the advantages of acid resistance, bile resistance, and easy colonization, and is an ideal nano-selenium carrier. By optimizing the culture medium formula, fermentation conditions and freeze-drying process, the present invention successfully prepared highly active and highly stable nano-selenium Enterococcus faecium, providing a new solution for the research and application of probiotics.

[0127] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above test examples, and the above test examples and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims

1. A selenium-rich Enterococcus faecium agent, characterized in that: The selenium-rich Enterococcus faecium agent is prepared by a biological fermentation process and contains organic selenium that can be absorbed by the body, wherein the content of nano-selenium in the agent is 10-120 μg / g, and the number of active colonies of the selenium-rich Enterococcus faecium is 1×10 9 Up to 5×10 10 CFU / g.

2. The selenium-enriched Enterococcus faecium agent according to claim 1, characterized in that: The selenium-enriched Enterococcus faecium agent is provided in the form of tablets, freeze-dried powder, capsules, dairy products, stirred yogurt or solidified yogurt.

3. The method for preparing the selenium-enriched Enterococcus faecium agent according to claim 1, characterized in that The steps include: a. Prepare Enterococcus faecium culture medium and adjust the pH to 7.4 using NaOH; b. Sterilize the culture medium at 121°C for 20 minutes and cool to room temperature; c. Inoculate the thawed Enterococcus faecium seed solution into the culture medium and activate it in a constant temperature incubator at 40°C for 24 hours; d. The activated Enterococcus faecium seed solution was inoculated into 1L of culture medium at a 3% inoculum volume, and nano-selenium was added after sterilization by filtration through a 0.22μm filter membrane to a final concentration of 10μg / ml of the nano-selenium solution, and cultured in a constant temperature incubator at 40°C for 24 hours; e. The fermented bacteria were collected by centrifugation at 8000 g, 10 min, and 4° C. to obtain a selenium-rich Enterococcus faecalis agent.

4. The method for preparing the selenium-enriched Enterococcus faecium inoculum according to claim 3, characterized in that: The preparation of Enterococcus faecium culture medium includes the following components:

5. The method for preparing the selenium-enriched Enterococcus faecium inoculum according to claim 3, characterized in that: a. Use 20mM phosphate buffer (PBS), 0.9% NaCl, 5% skimmed milk powder, adjust the pH to 7.0 of the sterilization solution to resuspend the bacterial sludge, the ratio is 50ml solution to resuspend 1 gram of bacterial sludge; b. The resuspended bacterial sludge is freeze-dried, and the freeze-drying procedure includes pre-freezing to -45℃ for 1.5 hours, followed by main drying at -45℃ for 4 hours, and then gradually heating to 20℃ and final drying under 0.2mba vacuum.

6. The method for preparing the selenium-enriched Enterococcus faecium inoculum according to claim 3, characterized in that: In step (d), the method for preparing the nano-selenium solution comprises the following steps: (1) Add stabilizer to a beaker; (2) slowly adding the aqueous solution containing the reducing agent into the beaker, and maintaining magnetic stirring during the addition process until the stabilizer is completely dissolved; (3) slowly adding the sodium selenite aqueous solution to the solution obtained in step (2), during which the solution in step (2) is under magnetic stirring; (4) After the sodium selenite aqueous solution is added, the pH is immediately adjusted to a predetermined range using a sodium hydroxide solution, and then the magnetic stirring is continued for 30 minutes to obtain a red nano-selenium solution.

7. The method for preparing nano-selenium of the selenium-enriched Enterococcus faecium inoculant according to claim 6, characterized in that: In step (1), the stabilizer is selected from at least one of epigallocatechin gallate EGCG, chitosan, gum arabic, bovine serum albumin BSA or sodium hyaluronate; the concentration range of epigallocatechin gallate EGCG is 0.1mM-40mM; the concentration range of chitosan is 0.01%-0.7%, and the molecular weight range of chitosan is 100KD-1000KD; the concentration range of gum arabic is 0.04%-0.4%, and the molecular weight range of gum arabic is 200KD-2000KD; the concentration range of bovine serum albumin BSA is 0.4mg / ml-60mg / ml, and the molecular weight range of bovine serum albumin BSA is 200KD-2000KD; the concentration range of sodium hyaluronate is 0.02%-0.25%, and the molecular weight range of sodium hyaluronate is 200KD-2000KD; In step (2), the reducing agent is selected from at least one of reduced glutathione GSH or vitamin C (Vc); in step (2), the final concentration of the reducing agent in the reaction system is 5mM-50mM glutathione GSH and 5mM-100mM vitamin C Vc; In step (3), the final concentration of sodium selenite in the reaction system is 5 mM; in step (4), in the step of adjusting the pH, the pH value ranges from 5.0 to 9.

0.

8. A method for preparing the selenium-enriched Enterococcus faecium freeze-dried powder according to claim 2, characterized in that The method comprises the following steps: a. using 20 mM phosphate buffer PB, 0.9% NaCl, 5% skimmed milk powder, and adjusting the pH to 7.0 of the sterilization solution to resuspend the bacterial sludge, with the ratio of 50 ml of solution to resuspend 1 gram of bacterial sludge; b. The resuspended bacterial sludge is freeze-dried, and the freeze-drying procedure includes pre-freezing to -45°C for 1.5 hours, followed by main drying at -45°C for 4 hours, and then gradually warming to final drying at 20°C. After freeze-drying, the sludge is collected to obtain selenium-enriched Enterococcus faecium freeze-dried powder; the vacuum degree of main drying and terminal drying is maintained at 0.2mba. c. Dilute the lyophilized powder to the desired concentration using skimmed milk powder or other fillers.

9. The method for preparing the selenium-enriched Enterococcus faecium freeze-dried powder according to claim 8, characterized in that: In step (d), 10 g of 5.0 x 10 10 The selenium-enriched Enterococcus faecium mother powder was diluted to a concentration of 1.0x10 cfu / g in 50 g. 10 cfu / g of bacterial powder as an example, using the gradient dilution method, first weigh 10.0 g of 5.0x10 10 cfu / g mother powder, add 2.5g skim milk powder, mix well, and get 12.5g of 4.0x10 10 cfu / g of bacterial powder; then 12.5 g of 4.0x10 10 cfu / g of bacterial powder, add 12.5 g of skimmed milk powder and mix well to obtain 25.0 g of 2.0x10 10 cfu / g of bacterial powder; finally, 25.0 g of 2.0x10 10 cfu / g of bacterial powder was mixed with 25.0 g of skimmed milk powder to obtain 50.0 g of a concentration of 1.0x10 10 cfu / g selenium-enriched Enterococcus faecium freeze-dried powder.

10. A selenium-enriched Enterococcus faecium freeze-dried powder, prepared by the preparation method according to claim 9.

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