Rhizobium massa and application thereof in preparation of selenium-rich beta-1, 3 / alpha-1, 3-glucan fermentation liquor

Abstract

The invention belongs to microbial fermentation, and particularly relates to rhizobium massa and application of rhizobium massa in preparation of selenium-rich beta-1, 3 / alpha-1, 3-glucan fermentation liquor. The preparation method comprises the following steps: inoculating rhizobium pustatae of which the Latin name is Rhizobium push and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.34553 into a sterile fermentation culture medium containing inorganic selenium, and carrying out aerated fermentation, thereby obtaining the selenium-enriched rhizobium pustatae. The method solves the problems that in the prior art, a fermentation strain is low in tolerance to selenium element, the capacity of converting inorganic selenium into organic selenium is weak, and the like, and has the advantages that the product yield and the fermentation liquor viscosity are high, and the selenium content in the selenium-rich beta-1, 3 / alpha-1, 3-glucan product is high.

Description

Technical Field

[0001] The present invention relates to microbial fermentation, in particular to Rhizobium pusa and its application in preparing selenium-enriched beta-1,3 / alpha-1,3-glucan fermentation liquid. Background Art

[0002] As a new type of bioactive polysaccharide, β-1,3 / α-1,3-glucan has a unique structure that supports immune regulation, anti-allergy and other functions, and has shown broad application potential in the fields of food, medicine and cosmetics. On April 25, 2021, it was approved as a new food raw material by the National Health Commission. The announcement clearly stated that β-1,3 / α-1,3-glucan is made from sucrose as the main raw material by Rhizobium pusense fermentation, alcohol precipitation, filtration, separation, drying, and crushing.

[0003] Selenium, as an essential trace element for the human body, has multiple functions. It can enhance the immune system's ability to recognize viruses, foreign bodies, and internal lesions, improve the phagocytic and bactericidal abilities of immune system cells, and increase the levels of immune system cell proliferation and antibody synthesis, thereby enhancing the body's immune function. In 1973, the World Health Organization listed selenium as an essential trace element for the human body. Selenium content in soil varies significantly in different regions. Selenium deficiency is closely related to Keshan disease and may lead to osteomalacia in children. The Chinese Nutrition Society recommends a dietary reference intake of 60 μg / d for adults in my country. Both insufficient and excessive selenium intake can have adverse effects on the human body. Natural selenium-rich foods cannot meet this demand, so it is necessary to find suitable selenium-rich products to meet the human body's selenium needs.

[0004] Selenium-rich foods can be categorized as naturally selenium-rich foods and artificially converted selenium-rich foods based on the pathways and methods of selenium enrichment. Naturally selenium-rich foods, such as selenium-enriched tea and rice, are primarily produced through the biotransformation of crops in soils rich in inorganic selenium to form organic selenium-rich raw materials. Artificial conversion primarily involves the use of plants, animals, and microorganisms as agents to convert inorganic selenium into organic selenium, which is then processed and produced into selenium-rich foods, such as selenium-enriched yeast. Naturally selenium-rich foods are significantly affected by the soil environment, and selenium levels are often unstable. Therefore, research is increasingly focused on the use of artificial conversion. Therefore, considering the role of β-1,3 / α-1,3-glucan and selenium in providing immunity, the research and development of a selenium-enriched β-1,3 / α-1,3-glucan is of great significance.

[0005] The patent documents retrieved by the applicant include:

[0006] The applicant, in patent document 201611113511.X, discloses a Rhizobium pusense strain and a method for producing β-1,3 glucan fermentation broth. The preparation process comprises inoculating Rhizobium pusense, Latin name Rhizobium pusense, with deposit number CGMCC No. 12954, into a sterilized culture broth containing a carbon source, a nitrogen source, and essential nutrients, performing aeration and agitation fermentation, and terminating the fermentation when the culture broth viscosity stops increasing or the fermentation period is ≤48 hours. The fermentation strain has a low tolerance to selenium and is weak in converting inorganic selenium into organic selenium.

[0007] The applicant has not found any domestic application that is identical or similar to this application. Summary of the Invention

[0008] One of the purposes of the present invention is to provide a strain with the Latin name Rhizobium pusense and the preservation number CGMCC No. 34553. The strain has good tolerance to selenium and a strong ability to convert inorganic selenium into organic selenium. The yield of selenium-rich β-1,3 / α-1,3-glucan in the fermentation broth prepared is significantly improved compared with the strain before acclimation.

[0009] A second objective of the present invention is to provide a method for using Rhizobium pusarum in the preparation of selenium-enriched β-1,3 / α-1,3-glucan fermentation broth. Sucrose is used as a carbon source in the fermentation broth; pea hydrolyzate, pea protein powder, potassium nitrate, sodium nitrate, and ammonia water are used as nitrogen sources. Inorganic selenium is added to the fermentation medium, and a fed-batch fermentation process is employed. This method is more conducive to bacterial growth, metabolism, and product synthesis. The yield of selenium-enriched β-1,3 / α-1,3-glucan prepared using this method is significantly superior to that of existing technologies, and the resulting β-1,3 / α-1,3-glucan has a high selenium content.

[0010] The overall technical concept of the present invention is:

[0011] Rhizobium pusense, whose Latin name is Rhizobium pusense, and its preservation number is CGMCC No.34553.

[0012] The applicant of the present invention, whose Latin name is Rhizobium pusense and whose deposit number is CGMCC No. 34553, has submitted it for deposit at the General Microbiology Center of China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on May 15, 2025. The depository is referred to as CGMCC.

[0013] The bacterial strain of the present invention is obtained by domesticating and purifying Rhizobium pusense, whose Latin name is Rhizobium pusense and whose preservation number is CGMCC No. 12954.

[0014] The morphological, physiological and biochemical characteristics of this strain are as follows:

[0015] White colonies, round, with a diameter of 0.03mm to 0.05mm, raised and sticky.

[0016] Rhizobium pusha and its application in preparing selenium-enriched β-1,3 / α-1,3-glucan fermentation broth.

[0017] The specific technical concept of the present invention also includes:

[0018] The invention relates to Rhizobium pusense and its application in preparing selenium-enriched β-1,3 / α-1,3-glucan fermentation broth. The invention relates to Rhizobium pusense, whose Latin name is Rhizobium pusense and whose preservation number is CGMCC No. 34553, being inoculated into a sterile fermentation medium containing inorganic selenium and the selenium is obtained after aeration and fermentation.

[0019] Furthermore, the following process steps are included:

[0020] A. inoculating Rhizobium pusense (Latin name: Rhizobium pusense, deposit number: CGMCC No. 34553) into a sterile fermentation medium containing a carbon source, a nitrogen source, inorganic selenium, and necessary nutrients, and performing aeration fermentation;

[0021] B. When the viscosity of the fermentation liquid no longer increases or the fermentation is completed after 60 to 70 hours, the selenium-rich β-1,3 / α-1,3-glucan fermentation liquid is obtained.

[0022] To facilitate bacterial growth and product synthesis, the carbon source of the sterile fermentation medium in step A is sucrose; the nitrogen source is selected from pea hydrolyzate, pea protein powder, potassium nitrate, sodium nitrate, ammonia water, or a combination thereof.

[0023] Furthermore, the fermentation medium in step A consists of the following components:

[0024] Sucrose 30g / L~50g / L; sodium dihydrogen phosphate 0.5g / L~1g / L; sodium selenite 1g / L~1.5g / L, the remainder is pea hydrolyzate, pH=7.0.

[0025] Furthermore, the pea hydrolyzate is prepared by the following method:

[0026] a. Crushing and soaking: crush the peas into 4 to 5 petals according to the specification, soak the crushed peas in water for 6 to 8 hours at a concentration of 40% to 50%, and use dilute hydrochloric acid or phosphoric acid to adjust the pH of the mixture during the soaking process to 2.0 to 3.0;

[0027] b. Heating: heating the soaked mixture to 90°C to 100°C and maintaining for 60 to 90 minutes;

[0028] c, solid-liquid separation: use a centrifuge to carry out high-speed centrifugation and solid-liquid separation of the mixture obtained after step b is heated, leaving the supernatant; be diluted with water to a pea concentration of 0.4% to 0.5%, and adjust the clear liquid pH to 6.5 to 7.0 with 10% sodium hydroxide.

[0029] The selenium-enriched β-1,3 / α-1,3-glucan fermentation broth obtained by the method of the present invention has a viscosity of 2500 cp to 3000 cp and a yield of 21 g / L to 28 g / L. The selenium content of the prepared selenium-enriched β-1,3 / α-1,3-glucan product is 10 mg / kg to 20 mg / kg.

[0030] In order to improve the yield, meet the growth of the bacteria and be conducive to the generation of the final product, the nutrients in the fermentation medium are enriched, which is more conducive to the growth and metabolism of the bacteria. The preferred technical solution is that step B includes the following process steps:

[0031] Fermentation 0 to fermentation end: culture temperature 30℃~34℃, inoculation amount 10%~12%, ventilation volume 0.6vvm~0.8vvm;

[0032] Fermentation for 20-30 hours: Add nitrogen source at a concentration of 2g / L-3g / L. The nitrogen source can be selected from one or more of sodium nitrate, potassium nitrate, and ammonia.

[0033] End of fermentation: When the viscosity of the fermentation liquid no longer increases or the fermentation ends after 60 to 70 hours.

[0034] To facilitate industrial production and meet the economic efficiency of industrial production, a preferred embodiment and a preferred technical implementation method are as follows: Step A is to expand the culture of Rhizobium pusense, whose Latin name is Rhizobium pusense and whose deposit number is CGMCC No. 34553, and then inoculate the seed liquid into the sterile fermentation medium in the fermentation tank at an inoculation rate of 10% to 12% by volume of the seed liquid:fermentation medium.

[0035] The expanded culture comprises:

[0036] (1) A single colony of Rhizobium pusense, whose Latin name is Rhizobium pusense and whose deposit number is CGMCC No. 34553, is inoculated on the surface of a shake flask seed culture medium to prepare a shake flask seed;

[0037] (2) The shake flask seeds are expanded and cultured to prepare seed liquid;

[0038] (3) The prepared seed liquid is inoculated into the sterile fermentation medium in the fermentation tank for fermentation according to an inoculation amount of 10% to 12% by volume of the seed liquid: fermentation medium.

[0039] The single colony culture medium of Rhizobium pusa in step (1) of the expanded culture process is composed of the following components:

[0040] Sucrose 20g / L~30g / L; potassium nitrate 2g / L~3g / L; sodium dihydrogen phosphate 0.5g / L~1g / L; pea protein powder 1g / L~2g / L; sodium selenite 1g / L~1.5g / L, agar 15g / L, pH=7.0.

[0041] The process conditions for preparing shake flask seeds in step (1) of the expanded culture process are as follows: a single colony of Rhizobium pusense with a Latin name of Rhizobium pusense and a preservation number of CGMCC No. 34553 is selected and inoculated into a sterilized shake flask seed culture medium, and the shake flask seed culture medium is filled in a 500 ml triangular flask with a capacity of 100 to 150 ml, and shake flask seeds are prepared by shaking culture at a shaking speed of 200 to 220 rpm and a culture temperature of 30°C to 34°C. The culture is carried out until the bacteria are in the logarithmic growth phase. In practice, OD 600 The measured value is controlled, that is, OD 600 ≈0.6~0.8;

[0042] The shake flask seed culture medium consists of the following components:

[0043] Sucrose 20g / L-30g / L; potassium nitrate 2g / L-3g / L; sodium dihydrogen phosphate 0.5g / L-1g / L; pea protein powder 1g / L-2g / L; sodium selenite 1g / L-1.5g / L, the balance is sterile water; pH = 7.0.

[0044] The expanded culture in step (2) of the expanded culture process includes the preparation of primary seeds and secondary seeds in sequence, wherein the primary seed culture solution is composed of the following components:

[0045] Sucrose 20g / L-30g / L; potassium nitrate 2g / L-3g / L; sodium dihydrogen phosphate 0.5g / L-1g / L; pea protein powder 1g / L-2g / L; sodium selenite 1g / L-1.5g / L, the balance is sterile water, pH = 7.0;

[0046] Inoculation volume: shake flask seeds: first-level seed culture solution volume ratio = 5% to 8%, shake flask seeds are inoculated with sterilized first-level seed culture solution and cultured to make first-level seeds. The process conditions for the preparation of first-level seeds are culture temperature 30℃~34℃, ventilation volume 0.6vvm~0.8vvm, culture until the bacteria are in the logarithmic growth phase, and OD 600 The measured value is controlled, that is, OD 600 ≈0.6~0.8.

[0047] The secondary seed culture solution is composed of the following components:

[0048] Sucrose 20g / L-30g / L; potassium nitrate 2g / L-3g / L; sodium dihydrogen phosphate 0.5g / L-1g / L; pea protein powder 1g / L-2g / L; sodium selenite 1g / L-1.5g / L, the balance is sterile water; pH = 7.0;

[0049] The inoculation amount is the volume ratio of primary seed to secondary seed culture solution = 8% to 10%. The primary seed is inoculated into the sterilized secondary seed culture solution and cultured to make the secondary seed. The process conditions in the preparation of the secondary seed are culture temperature 30℃ to 34℃, ventilation volume 0.6vvm to 0.8vvm, and culture until the bacteria are in the logarithmic growth phase. In practice, OD 600 The measured value is controlled, that is, OD 600 ≈0.6~0.8.

[0050] In order to verify the selenium content in the selenium-enriched β-1,3 / α-1,3-glucan fermentation broth prepared by the present invention, the applicant conducted an experiment using a known method or a determination method reported in existing literature. The specific method is as follows:

[0051] 1. Determination of total selenium content in selenium-enriched β-1,3 / α-1,3 glucan

[0052] 1. Draw a selenium standard curve: Accurately pipette 1 ml of selenium standard solution (selenium content 1000 μg / ml) into an Erlenmeyer flask. Add 5 ml of nitric acid, add glass beads, cover with a small funnel, and chill overnight. Then, slowly digest at low temperature until yellow-brown smoke ceases. Remove and cool. Add 2 ml of 30% hydrogen peroxide, heat briefly, then cool. Use a 100 ml volumetric flask to make a selenium standard solution (10 μg / L). Pipette 0, 0.5 ml, 1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml, and 5.0 ml of the selenium standard solution into Erlenmeyer flasks, respectively. Add 1.0 ml of 5% EDTA-2Na solution and 4 ml of 10 g / L o-phenylenediamine. Add distilled water to 15 ml. Adjust the pH to 2.0 with 5 mol / L NaOH solution. Incubate in the dark for 60 minutes. Add 10 ml of toluene, seal with plastic wrap, and shake on a shaker (120 rpm for 5 minutes). Transfer the mixture to a colorimetric tube and let it stand for 5 minutes. Take the upper organic phase to be measured, using toluene as the reference solution and measure the absorbance at a wavelength of 334 nm. A selenium standard curve was plotted with selenium concentration (x) as the abscissa and absorbance at a wavelength of 334 nm (y) as the ordinate. The regression equation for the selenium standard curve was: y = 10.3337x + 4.2499, with a correlation coefficient R² = 0.9998.

[0053] 2. Determination of Total Selenium Content: Weigh 0.1000 g of powdered selenium-enriched β-1,3 / α-1,3 glucan and add 5 ml of nitric acid. Digest the sample as described above. Bring the digestion solution to volume in a 50 ml volumetric flask. Pipette 5.0 ml into a conical flask and proceed as described above. Substitute the measured absorbance into the standard curve regression equation to determine the selenium content.

[0054] 2. The determination of fermentation yield and fermentation liquid viscosity are both well-known methods, and the applicant will not elaborate on the specific steps.

[0055] The substantial features and significant technical advancements of the present invention are:

[0056] 1. The present invention discloses a Rhizobium pusense strain, whose Latin name is Rhizobium pusense and whose deposit number is CGMCC No. 34553. This strain has good tolerance to selenium and is highly capable of converting inorganic selenium into organic selenium. It can use sucrose as a carbon source, making it easy to produce industrially and at low cost.

[0057] 2. The selenium-rich β-1,3 / α-1,3 glucan fermentation broth prepared by the method of the present invention has a high fermentation yield and viscosity. The fermentation broth viscosity is 2500cp~3000cp, and the yield is 21g / L~28g / L. Compared with the β-1,3 / α-1,3 glucan fermentation broth prepared by the starting strain Rhizobium pusense CGMCC No.12954 before acclimation, the viscosity is 1550cp~1630cp and the yield is 5.0g / L~5.5g / L, which has a significant improvement effect.

[0058] 3. During the fermentation process, nitrogen source is added to stabilize the fermentation control and promote the growth and metabolism of the bacteria.

[0059] 4. The present invention can produce selenium-rich β-1,3 / α-1,3 glucan fermentation broth through an aeration fermentation process. After purification, the selenium content in the selenium-rich β-1,3 / α-1,3 glucan product is 10 to 20 mg / kg. Since β-1,3 / α-1,3-glucan has been identified as a food raw material, and the production process and selenium content in the fermentation product are controllable during industrial fermentation, the present invention provides a new approach for the development of selenium-rich foods, which has strong market competitiveness and broad application prospects.

[0060] The Latin name of the present invention is Rhizobium pusense, and the deposit number is CGMCC No. 34553. The applicant submitted it for deposit at the General Microbiology Center of China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on May 15, 2025. The depository is referred to as CGMCC. DETAILED DESCRIPTION

[0061] The present invention is further described below with reference to the embodiments, but is not intended to limit the present invention. The scope of protection of the present invention is subject to the contents of the claims, and any replacement of equivalent technical means made according to the description does not depart from the scope of protection of the present invention.

[0062] Example 1

[0063] The application of Rhizobium pusa in preparing selenium-enriched β-1,3 / α-1,3-glucan fermentation broth includes the following process steps:

[0064] A. inoculating Rhizobium pusense (Latin name: Rhizobium pusense, deposit number: CGMCC No. 34553) into a sterile fermentation medium containing a carbon source, a nitrogen source, inorganic selenium, and necessary nutrients, and performing aeration fermentation;

[0065] B. When the viscosity of the fermentation liquid no longer increases or the fermentation is completed after 60 hours, a selenium-rich β-1,3 / α-1,3-glucan fermentation liquid is obtained.

[0066] In step A, the carbon source of the sterile fermentation medium is sucrose; the nitrogen source is selected from pea hydrolyzate, pea protein powder, potassium nitrate, sodium nitrate, ammonia water, or a combination thereof.

[0067] The fermentation medium in step A consists of the following components:

[0068] Sucrose 30 g / L; sodium dihydrogen phosphate 0.5 g / L; sodium selenite 1 g / L; the remainder is pea hydrolyzate; pH = 7.0.

[0069] The pea hydrolyzate is prepared by the following method:

[0070] a. Crushing and soaking: crush the peas into 4 to 5 petals according to the specification, soak the crushed peas in water for 6 hours at a concentration of 40%, and use dilute hydrochloric acid or phosphoric acid to adjust the pH of the mixture during the soaking process to 2.0;

[0071] b. Heating: Heat the soaked mixture to 90°C and maintain for 60 minutes;

[0072] c, solid-liquid separation: use a centrifuge to carry out high-speed centrifugation and solid-liquid separation after the mixture obtained after step b is heated up, leaving the supernatant; be diluted with water to a pea concentration of 0.4%, and adjust the clear liquid pH=6.5-7.0 with 10% concentration of sodium hydroxide.

[0073] Step B comprises the following process steps:

[0074] From fermentation 0 to the end of fermentation: culture temperature 30°C, inoculation volume 10%, ventilation volume 0.6 vvm;

[0075] Fermentation for 20-30 hours: Add nitrogen source at a concentration of 2g / L. The nitrogen source can be selected from one or more of sodium nitrate, potassium nitrate, and ammonia water.

[0076] End of fermentation: Fermentation is completed when the viscosity of the fermentation liquid no longer increases or after 60 hours of fermentation.

[0077] The step A is to expand the culture of Rhizobium pusense, whose Latin name is Rhizobium pusense and whose deposit number is CGMCC No. 34553, and then inoculate the seed liquid into the sterile fermentation medium in the fermentation tank according to an inoculation rate of 10% by volume of the seed liquid: fermentation medium.

[0078] The expanded culture comprises:

[0079] (1) A single colony of Rhizobium pusense, whose Latin name is Rhizobium pusense and whose deposit number is CGMCC No. 34553, is inoculated on the surface of a shake flask seed culture medium to prepare a shake flask seed;

[0080] (2) The shake flask seeds are expanded and cultured to prepare seed liquid;

[0081] (3) The prepared seed liquid was inoculated into the sterile fermentation medium in the fermentation tank for fermentation according to an inoculation amount of 10% by volume of the seed liquid: fermentation medium.

[0082] The single colony culture medium of Rhizobium pusa in step (1) of the expanded culture process is composed of the following components:

[0083] Sucrose 20 g / L; potassium nitrate 2 g / L; sodium dihydrogen phosphate 0.5 g / L; pea protein powder 1 g / L; sodium selenite 1 g / L; agar 15 g / L; pH = 7.0.

[0084] The process conditions for preparing shake flask seeds in step (1) of the expanded culture process are as follows: a single colony of Rhizobium pusense, whose Latin name is Rhizobium pusense and whose deposit number is CGMCC No. 34553, is picked up and inoculated into a sterilized shake flask seed culture medium, and the shake flask seed culture medium is filled in a 500ml triangular flask with a volume of 100ml, and shake flask seeds are prepared by shaking culture, the shaker speed is 200 rpm, the culture temperature is 30°C, and the culture is carried out until the bacteria are in the logarithmic growth phase. In practice, OD 600 The measured value is controlled, that is, OD 600 ≈0.6~0.8;

[0085] The shake flask seed culture medium consists of the following components:

[0086] Sucrose 20 g / L; potassium nitrate 2 g / L; sodium dihydrogen phosphate 0.5 g / L; pea protein powder 1 g / LL; sodium selenite 1 g / L; the balance is sterile water; pH = 7.0.

[0087] The expanded culture in step (2) of the expanded culture process includes the preparation of primary seeds and secondary seeds in sequence, wherein the primary and secondary seed culture solutions are composed of the following components:

[0088] Sucrose 20 g / L; potassium nitrate 2 g / L; sodium dihydrogen phosphate 0.5 g / L; pea protein powder 1 g / L; sodium selenite 1 g / L; the balance is sterile water; pH = 7.0;

[0089] The inoculation amount is the volume ratio of shake flask seeds to primary seed culture solution = 5%. The shake flask seeds are inoculated with the sterilized primary seed culture solution and cultured to make primary seeds. The process conditions in the preparation of primary seeds are culture temperature 30℃, ventilation volume 0.6vvm, and culture until the bacteria are in the logarithmic growth phase. In practice, OD 600 The measured value is controlled, that is, OD 600 ≈0.6~0.8.

[0090] The inoculation volume is 8% of the first-level seed and the second-level seed culture solution. The first-level seed is inoculated into the sterilized second-level seed culture solution and cultured to make the second-level seed. The process conditions in the preparation of the second-level seed are culture temperature 30 ° C, ventilation volume 0.6 vvm, and culture until the bacteria are in the logarithmic growth phase. In practice, OD 600 The measured value is controlled, that is, OD 600 ≈0.6~0.8.

[0091] Example 2

[0092] The difference between this embodiment and embodiment 1 is that:

[0093] The fermentation medium in step A consists of the following components:

[0094] Sucrose 50 g / L; sodium dihydrogen phosphate 1 g / L; sodium selenite 1.5 g / L; the remainder is pea hydrolyzate; pH = 7.0.

[0095] The pea hydrolyzate is prepared by the following method:

[0096] a. Crushing and soaking: crush the peas into 4 to 5 petals according to the specification, soak the crushed peas in water for 8 hours at a concentration of 50%, and use dilute hydrochloric acid or phosphoric acid to adjust the pH of the mixture during the soaking process to 3.0;

[0097] b. Heating: Heat the soaked mixture to 100°C and maintain for 90 minutes;

[0098] C, solid-liquid separation: use centrifuge to carry out high speed centrifugation and solid-liquid separation after the mixture obtained after step b is heated up, leave supernatant; be diluted with water to pea concentration 0.5%, adjust clear liquid pH=7.0 with 10% concentration of sodium hydroxide.

[0099] Step B comprises the following process steps:

[0100] From fermentation 0 to the end of fermentation: culture temperature 34°C, inoculum size 12%, ventilation rate 0.8 vvm;

[0101] Fermentation for 20-30 hours: Add nitrogen source at a concentration of 3g / L. The nitrogen source can be selected from one or more of sodium nitrate, potassium nitrate, and ammonia water.

[0102] End of fermentation: Fermentation is completed when the viscosity of the fermentation liquid no longer increases or after 70 hours of fermentation.

[0103] The step A is to expand the culture of Rhizobium pusense, whose Latin name is Rhizobium pusense and whose deposit number is CGMCC No. 34553, and then inoculate the seed liquid into the sterile fermentation medium in the fermentation tank according to an inoculation rate of 12% by volume of the seed liquid: fermentation medium.

[0104] The expanded culture comprises:

[0105] (1) A single colony of Rhizobium pusa (Latin name: Rhizobiumpusense, deposit number: CGMCC No. 34553) was inoculated on the surface of a shake flask seed culture medium to prepare a shake flask seed;

[0106] (2) The shake flask seeds are expanded and cultured to prepare seed liquid;

[0107] (3) The prepared seed liquid was inoculated into the sterile fermentation medium in the fermentation tank according to an inoculation amount of 12% by volume of the seed liquid: fermentation medium for fermentation.

[0108] The single colony culture medium of Rhizobium pusa in step (1) of the expanded culture process is composed of the following components:

[0109] Sucrose 30 g / L; potassium nitrate 3 g / L; sodium dihydrogen phosphate 1 g / L; pea protein powder 2 g / L; sodium selenite 1.5 g / L; agar 15 g / L; pH = 7.0.

[0110] The process conditions for preparing shake flask seeds in step (1) of the expanded culture process are as follows: a single colony of Rhizobium pusaensis with the Latin name Rhizobiumpusense and the preservation number CGMCC No. 34553 is selected and inoculated into a sterilized shake flask seed culture medium, and the shake flask seed culture medium is filled in a 500 ml triangular flask with a capacity of 150 ml, and shake flask seeds are prepared by shaking culture, the shaking table speed is 220 rpm, the culture temperature is 34°C, and the culture is carried out until the bacteria are in the logarithmic growth phase. In practice, OD 600 The measured value is controlled, that is, OD 600 ≈0.8;

[0111] The shake flask seed culture medium consists of the following components:

[0112] Sucrose 30 g / L; potassium nitrate 3 g / L; sodium dihydrogen phosphate 1 g / L; pea protein powder 2 g / L; sodium selenite 1.5 g / L; the balance is sterile water; pH = 7.0.

[0113] The expanded culture in step (2) of the expanded culture process includes the preparation of primary seeds and secondary seeds in sequence, wherein the primary and secondary seed culture solutions are composed of the following components:

[0114] Sucrose 30 g / L; potassium nitrate 3 g / L; sodium dihydrogen phosphate 1 g / L; pea protein powder 2 g / L; sodium selenite 1.5 g / L; the balance is sterile water; pH = 7.0;

[0115] The inoculation amount is the volume ratio of shake flask seeds to primary seed culture solution = 8%. The shake flask seeds are inoculated with the sterilized primary seed culture solution and cultured to make primary seeds. The process conditions in the preparation of primary seeds are culture temperature 34 ° C, ventilation volume 0.8vvm, and culture until the bacteria are in the logarithmic growth phase. In practice, OD 600 The measured value is controlled, that is, OD 600 ≈0.6~0.8.

[0116] The inoculation volume is 10% of the first-level seed and the second-level seed culture solution. The first-level seed is inoculated into the sterilized second-level seed culture solution and cultured to make the second-level seed. The process conditions in the preparation of the second-level seed are culture temperature 34 ° C, ventilation volume 0.8vvm, and culture until the bacteria are in the logarithmic growth phase. In practice, OD 600 The measured value is controlled, that is, OD 600 ≈0.6~0.8.

[0117] The rest of the content is the same as that of Example 1.

[0118] Example 3

[0119] The difference between this embodiment and embodiment 1 is that:

[0120] The fermentation medium in step A consists of the following components:

[0121] Sucrose 40 g / L; sodium dihydrogen phosphate 0.75 g / L; sodium selenite 1.3 g / L; the remainder is pea hydrolyzate; pH = 7.0.

[0122] The pea hydrolyzate is prepared by the following method:

[0123] a. Crushing and soaking: crush the peas into 4-5 petals according to the specification, soak the crushed peas in water for 7 hours at a concentration of 45%, and use dilute hydrochloric acid or phosphoric acid to adjust the pH of the mixture during the soaking process to 2.5;

[0124] b. Heating: Heat the soaked mixture to 95°C and maintain for 75 minutes;

[0125] c, solid-liquid separation: use centrifuge to carry out high speed centrifugation and solid-liquid separation after the mixture obtained after step b is heated up, leave supernatant; be diluted with water to pea concentration 0.45%, adjust the clear liquid pH=6.8 with 10% concentration of sodium hydroxide.

[0126] Step B comprises the following process steps:

[0127] From fermentation 0 to the end of fermentation: culture temperature 32°C, inoculum size 11%, ventilation rate 0.7 vvm;

[0128] Fermentation for 20-30 hours: Add nitrogen source at a concentration of 2.5 g / L. The nitrogen source can be selected from one or more of sodium nitrate, potassium nitrate, and ammonia.

[0129] End of fermentation: When the viscosity of the fermentation liquid no longer increases or the fermentation ends after 60 to 70 hours.

[0130] The step A is to expand the culture of Rhizobium pusense, whose Latin name is Rhizobium pusense and whose deposit number is CGMCC No. 34553, and then inoculate the seed liquid into the sterile fermentation medium in the fermenter according to the inoculation rate of 11% of the volume percentage of the seed liquid to the fermentation medium.

[0131] The expanded culture comprises:

[0132] (1) A single colony of Rhizobium pusa (Latin name: Rhizobiumpusense, deposit number: CGMCC No. 34553) was inoculated on the surface of a shake flask seed culture medium to prepare a shake flask seed;

[0133] (2) The shake flask seeds are expanded and cultured to prepare seed liquid;

[0134] (3) The prepared seed liquid was inoculated into the sterile fermentation medium in the fermentation tank for fermentation according to an inoculation amount of 11% of the volume percentage of the seed liquid to the fermentation medium.

[0135] The single colony culture medium of Rhizobium pusa in step (1) of the expanded culture process is composed of the following components:

[0136] Sucrose 25 g / L; potassium nitrate 2.5 g / L; sodium dihydrogen phosphate 0.8 g / L; pea protein powder 1.5 g / L; sodium selenite 1.3 g / L; agar 15 g / L, pH = 7.0.

[0137] The process conditions for preparing shake flask seeds in step (1) of the expanded culture process are as follows: a single colony of Rhizobium pusaensis with the Latin name Rhizobiumpusense and the preservation number CGMCC No. 34553 is selected and inoculated into a sterilized shake flask seed culture medium, and the shake flask seed culture medium is filled in a 500 ml triangular flask with a volume of 130 ml, and shake flask seeds are prepared by shaking culture, the shaking table speed is 210 rpm, the culture temperature is 32°C, and the culture is carried out until the bacteria are in the logarithmic growth phase. In practice, OD 600 The measured value is controlled, that is, OD 600 ≈0.6~0.8;

[0138] The shake flask seed culture medium consists of the following components:

[0139] Sucrose 25 g / L; potassium nitrate 2.5 g / L; sodium dihydrogen phosphate 0.75 g / L; pea protein powder 1.5 g / L; sodium selenite 1.3 g / L, the balance is sterile water; pH = 7.0.

[0140] The expanded culture in step (2) of the expanded culture process includes the preparation of primary seeds and secondary seeds in sequence, wherein the primary and secondary seed culture solutions are composed of the following components:

[0141] Sucrose 25g / L; potassium nitrate 2.5g / L; sodium dihydrogen phosphate 0.75g / L; pea protein powder 1.5g / L; sodium selenite 1.3g / L, the balance is sterile water; pH = 7.0;

[0142] Inoculation volume: shake flask seeds: first-level seed culture solution volume ratio = 5% to 8%, shake flask seeds are inoculated with sterilized first-level seed culture solution and cultured to make first-level seeds. The process conditions for the preparation of first-level seeds are culture temperature 30℃~34℃, ventilation volume 0.6vvm~0.8vvm, culture until the bacteria are in the logarithmic growth phase, and OD 600 The measured value is controlled, that is, OD 600 ≈0.6~0.8.

[0143] The inoculation amount is the volume ratio of primary seed to secondary seed culture solution = 8% to 10%. The primary seed is inoculated into the sterilized secondary seed culture solution and cultured to make the secondary seed. The process conditions in the preparation of the secondary seed are culture temperature 30℃ to 34℃, ventilation volume 0.6vvm to 0.8vvm, and culture until the bacteria are in the logarithmic growth phase. In practice, OD 600 The measured value is controlled, that is, OD 600 ≈0.6~0.8.

[0144] The rest of the content is the same as that of Example 1.

[0145] Example 4

[0146] The difference between this embodiment and embodiment 1 is that:

[0147] The fermentation medium in step A consists of the following components:

[0148] Sucrose 45 g / L; sodium dihydrogen phosphate 0.9 g / L; sodium selenite 1.4 g / L; the remainder is pea hydrolyzate; pH = 7.0.

[0149] The pea hydrolyzate is prepared by the following method:

[0150] a. Crushing and soaking: crush the peas into 4 to 5 petals according to the specification, soak the crushed peas in water for 7.5 hours at a concentration of 48%, and use dilute hydrochloric acid or phosphoric acid to adjust the pH of the mixture during the soaking process to 2.8;

[0151] b. Heating: Heat the soaked mixture to 98°C and maintain for 80 minutes;

[0152] c, solid-liquid separation: use a centrifuge to carry out high-speed centrifugation and solid-liquid separation of the mixture obtained after step b is heated, leaving the supernatant; be diluted with water to a pea concentration of 0.4% to 0.5%, and adjust the clear liquid pH to 6.5 to 7.0 with 10% sodium hydroxide.

[0153] Step B comprises the following process steps:

[0154] From fermentation 0 to the end of fermentation: culture temperature 33°C, inoculum size 11.5%, ventilation volume 0.75 vvm;

[0155] Fermentation for 20-30 hours: Add nitrogen source at a concentration of 2.8 g / L. The nitrogen source can be selected from one or more of sodium nitrate, potassium nitrate, and ammonia.

[0156] End of fermentation: When the viscosity of the fermentation liquid no longer increases or the fermentation ends after 60 to 70 hours.

[0157] The step A is to expand the culture of Rhizobium pusense, whose Latin name is Rhizobium pusense and whose deposit number is CGMCC No. 34553, and then inoculate the seed liquid into the sterile fermentation medium in the fermenter according to the inoculation rate of 11.5% of the volume percentage of the seed liquid to the fermentation medium.

[0158] The expanded culture comprises:

[0159] (1) A single colony of Rhizobium pusense, whose Latin name is Rhizobium pusense and whose deposit number is CGMCC No. 34553, is inoculated on the surface of a shake flask seed culture medium to prepare a shake flask seed;

[0160] (2) The shake flask seeds are expanded and cultured to prepare seed liquid;

[0161] (3) The prepared seed liquid was inoculated into the sterile fermentation medium in the fermentation tank for fermentation according to an inoculation amount of 11.5% of the volume percentage of the seed liquid to the fermentation medium.

[0162] The single colony culture medium of Rhizobium pusa in step (1) of the expanded culture process is composed of the following components:

[0163] Sucrose 28g / L; potassium nitrate 2.8g / L; sodium dihydrogen phosphate 0.9g / L; pea protein powder 1.8g / L; sodium selenite 1.4g / L, agar 15g / L, pH=7.0.

[0164] The process conditions for preparing shake flask seeds in step (1) of the expanded culture process are as follows: a single colony of Rhizobium pusense, whose Latin name is Rhizobium pusense and whose deposit number is CGMCC No. 34553, is picked up and inoculated into a sterilized shake flask seed culture medium, and the shake flask seed culture medium is filled in a 500 ml triangular flask with a volume of 140 ml. The shake flask seeds are prepared by shaking culture, the shaker speed is 215 rpm, the culture temperature is 33°C, and the culture is carried out until the bacteria are in the logarithmic growth phase. In practice, the OD value is used. 600 The measured value is controlled, that is, OD 600 ≈0.6~0.8;

[0165] The shake flask seed culture medium consists of the following components:

[0166] Sucrose 28 g / L; potassium nitrate 2.8 g / L; sodium dihydrogen phosphate 0.9 g / L; pea protein powder 1.8 g / L; sodium selenite 1.4 g / L, the balance is sterile water; pH = 7.0.

[0167] The expanded culture in step (2) of the expanded culture process includes the preparation of primary seeds and secondary seeds in sequence, wherein the primary and secondary seed culture solutions are composed of the following components:

[0168] Sucrose 28g / L; potassium nitrate 2.8g / L; sodium dihydrogen phosphate 0.9g / L; pea protein powder 1.9g / L; sodium selenite 1.4g / L, the balance is sterile water, pH = 7.0;

[0169] Inoculation volume: shake flask seeds: first-level seed culture solution volume ratio = 7%, shake flask seeds are inoculated with sterilized first-level seed culture solution and cultured to make first-level seeds. The process conditions in the preparation of first-level seeds are culture temperature 33 ° C, ventilation volume 0.75 vvm, culture until the bacteria are in the logarithmic growth phase, and OD is used in practice. 600 The measured value is controlled, that is, OD 600 ≈0.6~0.8.

[0170] The inoculation volume is 9% of the first-level seed and the second-level seed culture solution. The first-level seed is inoculated into the sterilized second-level seed culture solution and cultured to make the second-level seed. The process conditions in the preparation of the second-level seed are culture temperature 33 ° C, ventilation volume 0.75 vvm, and culture until the bacteria are in the logarithmic growth phase. In practice, OD 600 The measured value is controlled, that is, OD 600 ≈0.6~0.8.

[0171] The rest of the content is the same as Example 1.

[0172] Example 5

[0173] The difference between this embodiment and embodiment 1 is that:

[0174] The fermentation medium in step A consists of the following components:

[0175] Sucrose 35 g / L; sodium dihydrogen phosphate 0.6 g / L; sodium selenite 1.1 g / L; the remainder is pea hydrolyzate; pH = 7.0.

[0176] The pea hydrolyzate is prepared by the following method:

[0177] a. Crushing and soaking: crush the peas into 4 to 5 petals according to the specification, soak the crushed peas in water for 6 to 8 hours at a concentration of 42%, and use dilute hydrochloric acid or phosphoric acid to adjust the pH of the mixture during the soaking process to 2.0 to 3.0;

[0178] b. Heating: Heat the soaked mixture to 93°C and hold for 65 minutes;

[0179] c, solid-liquid separation: use a centrifuge to carry out high-speed centrifugation and solid-liquid separation of the mixture obtained after step b is heated; be diluted with water to a pea concentration of 0.42%, and adjust the clear liquid pH to 6.5-7.0 with 10% sodium hydroxide.

[0180] Step B comprises the following process steps:

[0181] From fermentation 0 to the end of fermentation: culture temperature 31°C, inoculum size 10.5%, ventilation volume 0.65 vvm;

[0182] Fermentation for 20-30 hours: Add nitrogen source at a concentration of 2.2 g / L. The nitrogen source can be selected from one or more of sodium nitrate, potassium nitrate, and ammonia.

[0183] End of fermentation: When the viscosity of the fermentation liquid no longer increases or the fermentation ends after 60 to 70 hours.

[0184] The step A is to expand the culture of Rhizobium pusense, whose Latin name is Rhizobium pusense and whose deposit number is CGMCC No. 34553, and then inoculate the seed liquid into the sterile fermentation medium in the fermentation tank according to an inoculation rate of 10% to 12% by volume of the seed liquid: fermentation medium.

[0185] The expanded culture comprises:

[0186] (1) A single colony of Rhizobium pusense, whose Latin name is Rhizobium pusense and whose deposit number is CGMCC No. 34553, is inoculated on the surface of a shake flask seed culture medium to prepare a shake flask seed;

[0187] (2) The shake flask seeds are expanded and cultured to prepare seed liquid;

[0188] (3) The prepared seed liquid was inoculated into the sterile fermentation medium in the fermentation tank for fermentation according to an inoculation amount of 10.5% of the volume percentage of the seed liquid to the fermentation medium.

[0189] The single colony culture medium of Rhizobium pusa in step (1) of the expanded culture process is composed of the following components:

[0190] Sucrose 22 g / L; potassium nitrate 2.2 g / L; sodium dihydrogen phosphate 0.6 g / L; pea protein powder 1.2 g / L; sodium selenite 1.2 g / L; agar 15 g / L, pH = 7.0.

[0191] The process conditions for preparing shake flask seeds in step (1) of the expanded culture process are as follows: a single colony of Rhizobium pusense, whose Latin name is Rhizobium pusense and whose deposit number is CGMCC No. 34553, is picked up and inoculated into a sterilized shake flask seed culture medium, and the shake flask seed culture medium is filled in a 500 ml triangular flask with a volume of 110 ml, and shake flask seeds are prepared by shaking culture, the shaker speed is 205 rpm, the culture temperature is 31°C, and the culture is carried out until the bacteria are in the logarithmic growth phase. In practice, OD 600 The measured value is controlled, that is, OD 600 ≈0.6~0.8;

[0192] The shake flask seed culture medium consists of the following components:

[0193] Sucrose 22 g / L; potassium nitrate 2.2 g / L; sodium dihydrogen phosphate 0.6 g / L; pea protein powder 1.2 g / L; sodium selenite 1.2 g / L; the balance is sterile water; pH = 7.0.

[0194] The expanded culture in step (2) of the expanded culture process includes the preparation of primary seeds and secondary seeds in sequence, wherein the primary and secondary seed culture solutions are composed of the following components:

[0195] Sucrose 22g / L; potassium nitrate 2.2g / L; sodium dihydrogen phosphate 0.6g / L; pea protein powder 1.2g / L; sodium selenite 1.2g / L; the balance is sterile water; pH = 7.0;

[0196] Inoculation volume: shake flask seeds: first-level seed culture solution volume ratio = 6%, shake flask seeds are inoculated with sterilized first-level seed culture solution and cultured to make first-level seeds. The process conditions in the preparation of first-level seeds are culture temperature 31 ° C, ventilation volume 0.65vvm, culture until the bacteria are in the logarithmic growth phase, and OD is used in practice. 600 The measured value is controlled, that is, OD 600 ≈0.6~0.8.

[0197] The inoculation volume is 8.5% of the first-level seed and the second-level seed culture solution. The first-level seed is inoculated into the sterilized second-level seed culture solution and cultured to make the second-level seed. The process conditions in the preparation of the second-level seed are culture temperature 31 ° C, ventilation volume 0.65 vvm, culture until the bacteria are in the logarithmic growth phase, and the OD value is used in practice. 600 The measured value is controlled, that is, OD 600 ≈0.6~0.8.

[0198] The rest of the content is the same as Example 1.

[0199] The applicant tested the fermentation yield, viscosity of the fermentation broth, and selenium content in the purified selenium-enriched β-1,3 / α-1,3-glucan in Examples 1 to 5. The relevant results are as follows:

[0200] Table 1 Test results of fermentation yield, fermentation liquid viscosity and selenium content in products in Examples 1 to 5

[0201] Fermentation yield (g / L) Fermentation broth viscosity (cp) Selenium content (mg / kg) Example 1 21 2550 10 Example 2 26 2700 20 Example 3 26 2680 19 Example 4 28 2700 20 Example 5 26 3000 15

Claims

1. Rhizobium pusense, whose Latin name is Rhizobium pusense, and whose deposit number is CGMCC No. 34553.

2. Use of the Rhizobium pushauri according to claim 1 in preparing selenium-enriched β-1,3 / α-1,3-glucan fermentation broth.

3. The use of Rhizobium pusaensis according to claim 2 in the preparation of selenium-enriched β-1,3 / α-1,3-glucan fermentation broth, characterized in that The rhizobium pusense with the Latin name of Rhizobium pusense and the preservation number of CGMCC No.34553 is inoculated into a sterile fermentation medium containing inorganic selenium, and the product is obtained after aeration and fermentation.

4. The use of Rhizobium pusaensis according to claim 3 in preparing selenium-enriched β-1,3 / α-1,3-glucan fermentation broth, characterized in that The process steps include: A. inoculating Rhizobium pusense (Latin name: Rhizobium pusense, deposit number: CGMCC No. 34553) into a sterile fermentation medium containing a carbon source, a nitrogen source, inorganic selenium, and necessary nutrients, and performing aeration fermentation; B. When the viscosity of the fermentation liquid no longer increases or the fermentation is completed after 60 to 70 hours, the selenium-rich β-1,3 / α-1,3-glucan fermentation liquid is obtained.

5. The use of Rhizobium pusaensis according to claim 4 in preparing selenium-enriched β-1,3 / α-1,3-glucan fermentation broth, characterized in that In the step A, the carbon source of the sterile fermentation medium is sucrose; the nitrogen source is selected from pea hydrolyzate, pea protein powder, potassium nitrate, sodium nitrate, ammonia water or a combination thereof.

6. Use of the Rhizobium pusaensis according to claim 4 in preparing selenium-enriched β-1,3 / α-1,3-glucan fermentation broth, characterized in that The viscosity of the selenium-enriched β-1,3 / α-1,3-glucan fermentation broth is 2500cp~3000cp, and the polysaccharide yield is 21g / L~28g / L.

7. The use of Rhizobium pushauri according to claim 5 in preparing selenium-enriched β-1,3 / α-1,3-glucan fermentation broth, characterized in that The fermentation medium in step A consists of the following components: Sucrose 30g / L~50g / L; sodium dihydrogen phosphate 0.5g / L~1g / L; sodium selenite 1g / L~1.5g / L, the remainder is pea hydrolyzate, pH=7.

0.

8. Use of Rhizobium pushauri according to claim 7 in preparing selenium-enriched β-1,3 / α-1,3-glucan fermentation broth, characterized in that The pea hydrolyzate is prepared by the following method: a. Crushing and soaking: crush the peas into 4 to 5 petals according to the specification, soak the crushed peas in water for 6 to 8 hours at a concentration of 40% to 50%, and use dilute hydrochloric acid or phosphoric acid to adjust the pH of the mixture during the soaking process to 2.0 to 3.0; b. Heating: heating the soaked mixture to 90°C to 100°C and maintaining for 60 to 90 minutes; c, solid-liquid separation: use a centrifuge to carry out high-speed centrifugation and solid-liquid separation of the mixture obtained after step b is heated, leaving the supernatant; be diluted with water to a pea concentration of 0.4% to 0.5%, and adjust the clear liquid pH to 6.5 to 7.0 with 10% sodium hydroxide.

9. The use of Rhizobium pushauri according to claim 4 in preparing selenium-enriched β-1,3 / α-1,3-glucan fermentation broth, wherein the step B comprises the following process steps: From fermentation hour 0 to the end of fermentation: culture temperature 30℃~34℃, inoculation amount 10%~12%, ventilation volume 0.6vvm~0.8vvm; Fermentation for 20-30 hours: Add nitrogen source at a concentration of 2g / L-3g / L. The nitrogen source can be selected from one or more of sodium nitrate, potassium nitrate, and ammonia. End of fermentation: When the viscosity of the fermentation liquid no longer increases or the fermentation ends after 60 to 70 hours.

10. Use of the Rhizobium pushauri according to claim 5 in preparing selenium-enriched β-1,3 / α-1,3-glucan fermentation broth, characterized in that The step A is to expand the culture of Rhizobium pusense, whose Latin name is Rhizobium pusense and whose deposit number is CGMCC No. 34553, and then inoculate the seed liquid into the sterile fermentation medium in the fermentation tank according to the inoculation amount of 10% to 12% of the volume percentage of the seed liquid to the fermentation medium.

11. Use of Rhizobium pushauri according to claim 10 in preparing selenium-enriched β-1,3 / α-1,3-glucan fermentation broth, characterized in that The expanded culture comprises: (1) A single colony of Rhizobium pusense, whose Latin name is Rhizobium pusense and whose deposit number is CGMCC No. 34553, is inoculated on the surface of a shake flask seed culture medium to prepare a shake flask seed; (2) The shake flask seeds are expanded and cultured to prepare seed liquid; (3) The prepared seed liquid is inoculated into the sterile fermentation medium in the fermentation tank for fermentation according to an inoculation amount of 10% to 12% by volume of the seed liquid: fermentation medium.

12. Use of Rhizobium pushauri according to claim 11 in preparing selenium-enriched β-1,3 / α-1,3-glucan fermentation broth, characterized in that The single colony culture medium of Rhizobium pusa in step (1) of the expanded culture process is composed of the following components: Sucrose 20g / L~30g / L; potassium nitrate 2g / L~3g / L; sodium dihydrogen phosphate 0.5g / L~1g / L; pea protein powder 1g / L~2g / L; sodium selenite 1g / L~1.5g / L, agar 15g / L, pH=7.

0.

13. Use of Rhizobium pushauri according to claim 11 in preparing selenium-enriched β-1,3 / α-1,3-glucan fermentation broth, characterized in that The process conditions for preparing shake flask seeds in step (1) of the expanded culture process are as follows: a single colony of Rhizobium pusense, whose Latin name is Rhizobium pusense and whose deposit number is CGMCC No. 34553, is picked and inoculated into a sterilized shake flask seed culture medium, and the shake flask seed culture medium is filled in a 500 ml triangular flask with a capacity of 100 to 150 ml, and shake flask seeds are prepared by shaking culture at a shaking speed of 200 to 220 rpm and a culture temperature of 30° C. to 34° C. until the bacteria are in the logarithmic growth phase; The shake flask seed culture medium consists of the following components: Sucrose 20g / L-30g / L; potassium nitrate 2g / L-3g / L; sodium dihydrogen phosphate 0.5g / L-1g / L; pea protein powder 1g / L-2g / L; sodium selenite 1g / L-1.5g / L, the balance is sterile water; pH = 7.

0.

14. Use of Rhizobium pushauri according to claim 11 in preparing selenium-enriched β-1,3 / α-1,3-glucan fermentation broth, characterized in that The expanded culture in step (2) of the expanded culture process includes the preparation of primary seeds and secondary seeds in sequence, wherein the primary seed culture solution is composed of the following components: Sucrose 20g / L-30g / L; potassium nitrate 2g / L-3g / L; sodium dihydrogen phosphate 0.5g / L-1g / L; pea protein powder 1g / L-2g / L; sodium selenite 1g / L-1.5g / L, the balance is sterile water, pH = 7.0; The volume ratio of inoculation amount of shake flask seeds: primary seed culture solution is 5% to 8%. The shake flask seeds are inoculated with the sterilized primary seed culture solution and cultured to prepare the primary seeds. The process conditions in the preparation of the primary seeds are culture temperature of 30°C to 34°C, ventilation volume of 0.6vvm to 0.8vvm, and culture until the bacteria are in the logarithmic growth phase.

15. The method for producing β-1,3 glucan fermentation broth using Rhizobium bodhisattva according to claim 14, characterized in that The secondary seed culture solution is composed of the following components: Sucrose 20g / L-30g / L; potassium nitrate 2g / L-3g / L; sodium dihydrogen phosphate 0.5g / L-1g / L; pea protein powder 1g / L-2g / L; sodium selenite 1g / L-1.5g / L, the balance is sterile water; pH = 7.0; The inoculation amount is the volume ratio of the primary seed to the secondary seed culture solution = 8% to 10%. The primary seeds are inoculated into the sterilized secondary seed culture solution and cultured to prepare the secondary seeds. The process conditions in the preparation process of the secondary seeds are culture temperature of 30° C. to 34° C., ventilation volume of 0.6 vvm to 0.8 vvm, and culture until the bacteria are in the logarithmic growth phase.