Process for the preparation of a fermentation broth containing beta-1,3 / alpha-1,3-glucan

Abstract

This invention pertains to microbial fermentation, specifically a method for preparing a fermentation broth containing β-1,3 / α-1,3-glucan. The method involves inoculating *Rhizobium spp.* into a sterilized fermentation medium containing carbon sources, nitrogen sources, and essential nutrients, followed by aerated fermentation. The carbon source in the fermentation medium is sucrose, and the nitrogen source is soy protein isolate. The aeration rate is controlled in stages, and nitrogen sources and hydrogen peroxide are supplemented during fermentation. This invention solves the problem of low fermentation yield in existing technologies, offering advantages such as high fermentation broth viscosity, a fermentation yield of 25 g / L to 30 g / L, and good water solubility of the prepared product.

Description

Technical Field

[0001] This invention pertains to microbial fermentation, and specifically refers to a method for preparing fermentation broth containing β-1,3 / α-1,3-glucan. Background Technology

[0002] In April 2021, the National Health Commission of China announced that β-1,3 / α-1,3-glucan was listed as a new food ingredient, which specifies the molecular formula of β-1,3 / α-1,3-glucan as (C6H2O). 10 O5) n , n≥615; its structural formula is as follows:

[0003] β-1,3 / α-1,3-glucan is a product made primarily from sucrose, produced by *Rhizobium pusa* (…). Rhizobium pusense It is made through processes such as fermentation, alcohol precipitation, filtration, separation, drying, and pulverization.

[0004] The announcement also specified quality requirements for β-1,3 / α-1,3-glucan, including its properties, content, protein content, and ash content, and clarified the methods for determining its content.

[0005] Existing research indicates that β-1,3 / α-1,3-glucan can promote the proliferation and differentiation of T cells and B cells, systematically enhancing the host's immune response through the synergistic effect of multiple immunomodulatory pathways. This substance can enhance the phagocytic capacity of macrophages, improving their efficiency in clearing harmful substances such as bacteria and viruses; simultaneously, it can promote the secretion of cytokines, including inflammatory factors such as TNF-α, IL-1α, IL-1β, and IL-6, as well as various chemokines. β-1,3 / α-1,3-glucan also possesses multiple physiological functions, including regulating blood sugar, lowering cholesterol, anti-tumor activity, anti-infection, and antioxidant activity. These functions give it broad application prospects in functional foods, health products, and pharmaceuticals.

[0006] The literature with publication number CN106434495A discloses a method for preparing β-1,3-glucan fermentation broth from *Rhizobium praecox*. Rhizobium pusenseCGMCC No. 12954 was inoculated into a sterilized culture medium containing carbon, nitrogen, and necessary nutrients, and fermented with aeration and stirring. Fermentation ended when the viscosity of the culture medium stopped increasing or the fermentation period was ≤48 hours. The existing technology has the following problems: First, the fermentation yield is low, only 5.0–5.5 g / L; this yield level is far from meeting the needs of industrial production, seriously restricting the market promotion and application of β-1,3 / α-1,3-glucan. Second, the fermentation process is difficult to control. Because β-1,3 / α-1,3-glucan is a high-molecular-weight polysaccharide, the viscosity of the fermentation broth increases sharply during fermentation. Studies have shown that fermentation broth viscosity mainly affects product yield through three pathways: product-driven, cell-driven, and low viscosity-dependent. When the product concentration exceeds 15 g / L, mass transfer limitation dominates, and the yield growth rate slows down or even decreases, exhibiting a "bell-shaped curve" characteristic. High-viscosity fermentation broth not only affects the mass transfer efficiency of nutrients and oxygen, but also increases the power consumption of stirring, posing many technical challenges to industrial production. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing fermentation broth containing β-1,3 / α-1,3-glucan. By optimizing the culture medium and fermentation conditions, the method is more conducive to the growth of the strain and the generation of secondary metabolites. The yield of this method is significantly better than that of the prior art.

[0008] The overall technical concept of this invention is: The method for preparing fermentation broth containing β-1,3 / α-1,3-glucan involves inoculating a microbial strain into a sterilized fermentation medium containing a carbon source, nitrogen source, and necessary nutrients, followed by aerated fermentation. The microbial strain is named using the Latin name... Rhizobium pusense The *Prunella vulgaris* species with accession number CGMCC No. 12954 contains: A. Sucrose is used as the carbon source and soy protein isolate is used as the nitrogen source in the fermentation medium; B. During aeration fermentation, the ventilation volume is controlled in stages, including: 0–20 hours: ventilation volume is 0.5 vvm–0.8 vvm; 21–50 hours: ventilation volume is 0.9 vvm–1.1 vvm; From 51 hours until canister discharge: ventilation rate is 1.3vvm to 1.5vvm; The fermentation cycle shall not exceed 70 hours; C. After 28–32 hours of fermentation, add a nitrogen source at a ratio of 0.08%–0.09% of the fermentation liquid volume, using ammonia water and / or ammonium nitrate solution as the nitrogen source; after 50–60 hours of fermentation, add hydrogen peroxide with a volume percentage concentration of 15%–20% at a ratio of 0.04%–0.06% of the fermentation liquid volume.

[0009] The main advantages of using sucrose as the carbon source in this invention are: firstly, sucrose can be directly added to the fermentation system without pretreatment; *Rhizobium pusa* can secrete sucrase to rapidly hydrolyze and utilize it, simplifying the process, shortening preparation time, and eliminating the need for enzyme preparations and pretreatment, thus reducing energy consumption and operating costs; secondly, sucrose has a low molecular weight and contributes very little to the viscosity of the fermentation broth, avoiding viscosity abnormalities and ensuring uniform dissolved oxygen and stable fermentation; and thirdly, *Rhizobium pusa* has a specific utilization advantage for sucrose, slowly releasing glucose and fructose after hydrolysis, avoiding the substitution of sucrose with glucose and fructose. The process has several advantages: First, it inhibits the production of polysaccharide precursors, promotes the synthesis of polysaccharide precursors, and increases glucan yield. Second, sucrose has high purity and few impurities, ensuring stable glucan content and reducing the generation of impurities such as heteropolysaccharides. Third, sucrose is widely available, has a stable price, is convenient to store and transport, and has a high carbon source conversion rate, reducing raw material costs for industrial fermentation and meeting the needs of large-scale production. Fourth, sucrose metabolism can precisely regulate the glycosidic bond ratio of glucan, resulting in more prominent water solubility and immunomodulatory functions, a more uniform molecular weight distribution, and superior physicochemical properties, thus broadening its application scenarios.

[0010] The main advantages of using soy protein isolate as the nitrogen source in this invention are twofold: First, soy protein isolate has a much higher nitrogen content than soybean flour, and after purification to remove impurities such as cellulose and starch, it has high purity and can be directly utilized by Rhizobium pumilus, avoiding interference from impurities, significantly improving nitrogen source conversion rate, and reducing nitrogen waste. Second, soy protein isolate contains no excess impurities and will not increase the viscosity of the fermentation broth, maintaining dissolved oxygen transfer efficiency and avoiding localized nutrient imbalances and metabolic abnormalities caused by impurities in soybean flour. Simultaneously, its high purity reduces the nutritional basis for the growth of other microorganisms, lowers the risk of contamination, and ensures a stable fermentation process. Third, soy protein isolate can be slowly degraded into amino acids, continuously providing a nitrogen source for cell growth and polysaccharide precursor synthesis, avoiding metabolic inhibition caused by uneven nitrogen release from soybean flour, and avoiding the problem of insufficient nitrogen content in soybean oil; its purity can reduce the generation of impurities such as heteropolysaccharides and pigments, and synergistically with sucrose to further improve the yield and purity of glucan; fourth, it can be directly added to the fermentation system, simplifying the process; moreover, soy protein isolate is stable in storage, not easily deteriorated, and has a wide range of sources, making it more cost-effective than the scheme of using soybean flour and soybean oil together, reducing the overall cost of large-scale industrial fermentation.

[0011] The key to controlling and gradually increasing ventilation volume in stages is to adapt to the dissolved oxygen requirements of each fermentation stage. Low ventilation in the early stage meets the needs of cell growth, increased ventilation in the middle stage ensures polysaccharide synthesis, and increased ventilation in the later stage overcomes the viscosity of the fermentation broth and maintains stable dissolved oxygen. It also regulates the carbon and nitrogen metabolism balance to achieve a dynamic balance between cell growth and polysaccharide synthesis, stabilizes the fermentation environment, discharges metabolic waste gas, reduces concentration differences and contamination by other microorganisms, and optimizes the polysaccharide structure to make the molecular weight distribution uniform and improve functional properties.

[0012] The main functions of supplementing nitrogen source during fermentation: Firstly, during fermentation, microbial metabolism causes pH fluctuations in the fermentation broth, affecting the activity of *Rhizobium pusa* and polysaccharide synthesis. Ammonia can quickly neutralize the acidity of the fermentation broth and fine-tune the pH to a suitable range. Ammonium nitrate, after dissociation, can help stabilize the pH. Together, they prevent sudden pH changes. Simultaneously, ammonium nitrate, as an inorganic nitrogen source, supplements nitrogen nutrition, maintains a stable fermentation environment, and ensures normal microbial metabolism. Secondly, both work synergistically with soybean protein isolate to meet the nitrogen requirements of microorganisms at different growth stages. They also regulate the nitrogen release rate, preventing nitrogen excess or deficiency, improving the overall nitrogen conversion rate, further promoting polysaccharide precursor synthesis, and contributing to increased β-1,3 / α-1,3-glucan production. Thirdly, they are adjustable... The fermentation broth's osmotic pressure is controlled, preventing cell dehydration or rupture; it also reduces the growth of unwanted microorganisms during fermentation, and works synergistically with soy protein isolate and sucrose to further enhance fermentation stability, reduce the production of impurities such as heteropolysaccharides, and ensure the purity and structural uniformity of β-1,3 / α-1,3-glucan; fourthly, it is widely available, inexpensive, convenient to replenish, and requires no complex pretreatment, allowing for direct addition as needed; compared to other nitrogen source supplements, both have the functions of nitrogen source replenishment and pH adjustment, offering higher cost-effectiveness and reducing the overall cost of industrial fermentation, meeting the needs of large-scale production. Supplementation should follow the principle of "appropriate amount and timely" to avoid affecting β-1,3 / α-1,3-glucan synthesis.

[0013] The specific technical concept of this invention also includes: To facilitate better cell growth, metabolism, and synthesis of secondary metabolites, a preferred technical implementation is that the fermentation medium comprises the following components by mass percentage: Sucrose 5.0%–7.0%; soy protein isolate 0.16%–0.32%; sodium dihydrogen phosphate 0.15%–0.25%; sodium nitrate 0.18%–0.34%; magnesium sulfate 0.01%–0.02%; ferrous sulfate 0.001%–0.0015%; zinc chloride 0.00005%–0.0001%; ​​defoamer 0.03%–0.05%; pH=6.8–7.5.

[0014] The preferred technique for supplementing nitrogen sources during fermentation is to use ammonia water with a concentration of not less than 18% and ammonium nitrate solution with a mass percentage of 30% to 40%.

[0015] Furthermore, after the ammonium nitrate solution of characteristic C is prepared, it is sterilized at high temperature and then added after cooling to 28℃~30℃.

[0016] The main function of scale-up culture in microbial polysaccharide production is to provide sufficient, highly active, and uniform seed cells for large-scale fermentation, shorten the fermentation cycle, reduce costs, screen for high-quality strains, inhibit contamination by other microorganisms, stabilize the fermentation process, ensure stable polysaccharide yield and quality, and enhance the product's application value. The preferred technical means to achieve this is as follows: During fermentation production, *Rhizobium spp.* (…) Rhizobium pusense After being expanded, CGMCC No. 12954 was inoculated into the fermentation medium at an inoculation rate of 10% to 20% of the volume percentage of seed culture to fermentation medium.

[0017] Furthermore, the aforementioned scale-up culture process includes: (1) The Latin name is Rhizobium pusense The *Rhizobium praecox* strain with accession number CGMCC No. 12954 was inoculated into sterilized primary seed culture medium to prepare primary seed fermentation broth. (2) The primary seed fermentation broth is expanded and cultured to produce a secondary seed fermentation broth; (3) The prepared secondary seed fermentation broth is inoculated into the sterilized fermentation medium, and after aeration and fermentation, a fermentation broth containing β-1,3 / α-1,3-glucan is prepared.

[0018] The process conditions for preparing the primary seed fermentation broth in step (1) of the extended culture process are: Inoculation amount: 0.5%; temperature: 28℃~34℃; ventilation rate: 0.3vvm~0.5vvm; when the viscosity of the primary seed fermentation broth is 50mpa.s~100mpa.s, transfer to the sterilized secondary seed culture medium. The primary seed culture medium comprises the following components by weight percentage: Sucrose 1.0%–2.0%; soy protein isolate 0.16%–0.32%; sodium dihydrogen phosphate 0.15%–0.25%; sodium nitrate 0.18%–0.34%; magnesium sulfate 0.01%–0.02%; ferrous sulfate 0.001%–0.0015%; zinc chloride 0.00005%–0.0001%; ​​defoamer 0.03%–0.05%; pH=6.8–7.5.

[0019] The process conditions for preparing the secondary seed fermentation broth in step (2) of the expanded culture process are as follows: Inoculation amount: 10%–20%; temperature: 28℃–34℃; ventilation rate: 0.3 vvm–0.5 vvm; when the viscosity of the secondary seed fermentation broth is 100 mPa·s–200 mPa·s, transfer the inoculum to the sterilized fermentation medium. The secondary seed culture medium comprises the following components by weight percentage: Sucrose 1.0%–2.0%; soy protein isolate 0.16%–0.32%; sodium dihydrogen phosphate 0.15%–0.25%; sodium nitrate 0.18%–0.34%; magnesium sulfate 0.01%–0.02%; ferrous sulfate 0.001%–0.0015%; zinc chloride 0.00005%–0.0001%; ​​defoamer 0.03%–0.05%; pH=6.8–7.5.

[0020] The viscosity of the primary and secondary seed fermentation broths was measured using a Brookfield viscometer with a No. 4 rotor at a speed of 60 rpm.

[0021] To verify the technical effects of the present invention, the applicant conducted the following experiments: I. Determination of Fermentation Broth Viscosity Based on the fact that β-1,3 / α-1,3-glucan is water-soluble and can produce high viscosity at very low concentrations, the following method is used to determine the viscosity of fermentation broth in the absence of national standards.

[0022] 1. Instruments (1) Brinell viscometer: Measurement error ±5% (2) Rotor model: No. 4.

[0023] 2. Measurement method: Take 500ml of fermentation broth and place it in a beaker. Use a No. 4 rotor at a speed of 60 rpm to measure and read the viscosity value.

[0024] 3. Evaluation results: The viscosity of the fermentation broth was 2500–2700 mPa·s.

[0025] II. Detection of Fermentation Yield There is currently no national standard for yield testing. The industry generally uses the following methods for determination: 1. Instruments (1) Incubator.

[0026] (2) Analytical balance: accurate to 1 mg.

[0027] 2. Detection Method Pour the fermentation broth into a small beaker of a certain volume, stir with a glass rod to remove air bubbles, smooth the surface with the glass rod, and clean the outside of the beaker of fermentation broth. Extract the fermentation broth with 90%–95% ethanol, allow it to settle, squeeze dry with filter cloth, tear it into small pieces, and transfer it completely into a petri dish. Then, place the dish in a 105°C oven and dry until constant weight (about 4 hours). Remove and weigh.

[0028] Solid content = (number of grams of sample weighed / volume) × 100%.

[0029] 3. Test Results The fermentation yield using the method of this invention is 25 g / L to 30 g / L.

[0030] III. Water Solubility Determination Based on the water solubility of β-1,3 / α-1,3-glucan, which allows solutions to produce high viscosity at very low concentrations, the following method is used to determine the viscosity of a 1% aqueous solution of powder products in the absence of current national standards.

[0031] 1. Instruments (1) Brinell viscometer: measurement error ±5%.

[0032] (2) High-speed mixer.

[0033] (3) Analytical balance: accurate to 1 mg.

[0034] (4) Rotor model: No. 3, No. 4.

[0035] 2. Measurement conditions: Weigh 3.0g of the sample to be tested and add it to 300ml of purified water. Stir for 15 minutes using a high-speed stirrer at 8000 rpm. Then, measure the viscosity using rotor #3 at 60 rpm. If the viscosity value exceeds the detection range of rotor #3, use rotor #4 at 60 rpm. Read the viscosity value.

[0036] 3. Test results: 500~700mpa.s.

[0037] The essential features and significant technical advancements of this invention are as follows: 1. The main functions of using sucrose as a carbon source in this invention are: first, to simplify the fermentation process and reduce operational difficulty and cost; second, to optimize the fermentation environment and improve fermentation stability; third, to adapt to the metabolism of microorganisms and improve the yield and purity of glucan; fourth, to ensure that the raw materials are readily available and cost-effective, making them suitable for industrial production; and fifth, to optimize the product structure and function and enhance its application value.

[0038] 2. The main functions of using soybean protein isolate as the nitrogen source in this invention are: firstly, it has a high and pure nitrogen content, which improves the nitrogen source utilization rate; secondly, it optimizes fermentation stability and reduces contamination by miscellaneous bacteria; thirdly, it adapts to the metabolism of microorganisms and synergistically improves the yield and purity of glucan; and fourthly, it simplifies the process and is suitable for industrialization, reducing overall costs.

[0039] 3. The main functions of the segmented and gradually increasing ventilation volume in this invention during fermentation are: first, to adapt to the dissolved oxygen requirements of different stages of fermentation and ensure normal metabolism; second, to regulate the carbon and nitrogen metabolism balance and promote the synthesis of β-1,3 / α-1,3-glucan; third, to stabilize the fermentation system environment and reduce contamination by miscellaneous bacteria and abnormal metabolism; and fourth, to optimize the structure and function of β-1,3 / α-1,3-glucan and improve the quality of β-1,3 / α-1,3-glucan products.

[0040] 4. The main functions of this invention in supplementing nitrogen sources during fermentation are: first, to precisely regulate the pH of the fermentation broth and stabilize the metabolic environment; second, to synergistically improve the utilization rate of soybean protein isolate; third, to stabilize the fermentation system and optimize the synthesis conditions of β-1,3 / α-1,3-glucan; and fourth, to adapt to industrial production and reduce adjustment costs.

[0041] 5. The main functions of hydrogen peroxide supplementation during fermentation in this invention are: first, to supplement dissolved oxygen in the fermentation broth, which can quickly alleviate the problem of oxygen transfer difficulties caused by the increase in viscosity of the fermentation broth in the later stage of fermentation, providing sufficient oxygen for microbial metabolism and polysaccharide synthesis, and significantly increasing the yield of β-1,3 / α-1,3-glucan; second, to regulate polysaccharide structure and production efficiency; and third, to inhibit the decline in β-1,3 / α-1,3-glucan yield or abnormal quality caused by contamination by miscellaneous bacteria.

[0042] 6. The method of this invention prepares fermentation broth containing β-1,3 / α-1,3-glucan with high fermentation yield and high viscosity. The fermentation yield reaches 25 g / L to 30 g / L, which is nearly 5 times higher than the prior art. The viscosity of the fermentation broth can reach 2000 to 2500 mPa·s. The prepared β-1,3 / α-1,3-glucan has good water solubility, and the viscosity of a 1% aqueous solution can reach 500 to 700 mPa·s. After purification, the product obtained from the fermentation broth containing β-1,3 / α-1,3-glucan meets the quality requirements for β-1,3 / α-1,3-glucan in the "Announcement on 6 New Foods including β-1,3 / α-1,3-glucan and dihydroquercetin" (No. 5 of 2021) issued by the National Health Commission. Detailed Implementation

[0043] The present invention will be further described below with reference to embodiments, but this is not intended to limit the present invention. The scope of protection of the present invention shall be determined by the contents of the claims. Any equivalent technical means substitution made in accordance with the specification shall not depart from the scope of protection of the present invention. Example 1

[0044] The method for preparing fermentation broth containing β-1,3 / α-1,3-glucan involves inoculating a microbial strain into a sterilized fermentation medium containing a carbon source, nitrogen source, and necessary nutrients, followed by aerated fermentation. The microbial strain is named using the Latin name... Rhizobium pusense The *Prunella vulgaris* species with accession number CGMCC No. 12954 contains: A. Sucrose is used as the carbon source and soy protein isolate is used as the nitrogen source in the fermentation medium; B. During aeration fermentation, the ventilation volume is controlled in stages, including: 0–20 hours: ventilation volume is 0.8 vvm; 21–50 hours: Ventilation volume is 1.1 vvm; 51 hours until tank discharge: ventilation rate is 1.5vvm; The fermentation cycle shall not exceed 70 hours; C. After 28-32 hours of fermentation, add nitrogen source at a ratio of 0.09% of the fermentation liquid volume, using ammonia water as the nitrogen source; after 50-60 hours of fermentation, add hydrogen peroxide with a volume percentage concentration of 20% at a ratio of 0.06% of the fermentation liquid volume.

[0045] The fermentation medium contains the following components by mass percentage: Sucrose 7.0%; soy protein isolate 0.32%; sodium dihydrogen phosphate 0.25%; sodium nitrate 0.34%; magnesium sulfate 0.02%; ferrous sulfate 0.0015%; zinc chloride 0.0001%; ​​defoamer 0.05%; pH = 6.8–7.5.

[0046] The percentage concentration of ammonia in the water shall not be less than 18%.

[0047] After the ammonium nitrate solution is prepared, it is steam-sterilized at 121°C for 30 minutes, and then added after cooling to 28°C~30°C.

[0048] In fermentation production, *Rhizobium praecox* (…) Rhizobium pusense After being expanded, CGMCC No. 12954 was inoculated into the fermentation medium at an inoculation rate of 10% to 20% of the volume percentage of seed culture to fermentation medium.

[0049] The expanded culture process includes: (1) The Latin name is Rhizobium pusense The *Rhizobium praecox* strain with accession number CGMCC No. 12954 was inoculated into sterilized primary seed culture medium to prepare primary seed fermentation broth. (2) The primary seed fermentation broth is expanded and cultured to produce a secondary seed fermentation broth; (3) The prepared secondary seed fermentation broth is inoculated into the sterilized fermentation medium, and after aeration and fermentation, a fermentation broth containing β-1,3 / α-1,3-glucan is prepared.

[0050] The process conditions for preparing the primary seed fermentation broth in step (1) of the extended culture process are: Inoculation amount: 0.5%; temperature: 34℃; ventilation rate: 0.5 vvm; when the viscosity of the primary seed fermentation broth is 50 mpa.s to 100 mpa.s, transfer it to the sterilized secondary seed culture medium. The primary seed culture medium comprises the following components by weight percentage: Sucrose 2.0%; soy protein isolate 0.32%; sodium dihydrogen phosphate 0.25%; sodium nitrate 0.34%; magnesium sulfate 0.02%; ferrous sulfate 0.0015%; zinc chloride 0.0001%; ​​defoamer 0.05%; pH = 6.8–7.5.

[0051] The process conditions for preparing the secondary seed fermentation broth in step (2) of the expanded culture process are as follows: Inoculation amount 20%; temperature 34℃; ventilation rate 0.5 vvm; when the viscosity of the secondary seed fermentation broth is 100 mpa.s to 200 mpa.s, transfer to the sterilized fermentation medium; The secondary seed culture medium comprises the following components by weight percentage: Sucrose 2.0%; soy protein isolate 0.32%; sodium dihydrogen phosphate 0.25%; sodium nitrate 0.34%; magnesium sulfate 0.02%; ferrous sulfate 0.0015%; zinc chloride 0.0001%; ​​defoamer 0.05%; pH = 6.8–7.5.

[0052] The viscosity of the primary and secondary seed fermentation broths was measured using a rotational viscometer with a No. 4 rotor at a speed of 60 rpm. Example 2

[0053] The difference between this embodiment and Embodiment 1 is that: During aerated fermentation, ventilation is controlled in stages, including: 0–20 hours: Ventilation volume is 0.5 vvm; 21–50 hours: ventilation rate is 0.9 vvm; 51 hours until tank discharge: ventilation rate is 1.3vvm; The fermentation cycle shall not exceed 70 hours; After 28–32 hours of fermentation, add a nitrogen source at a ratio of 0.08% of the fermentation liquid volume, using ammonium nitrate solution as the nitrogen source; after 50–60 hours of fermentation, add hydrogen peroxide with a volume percentage concentration of 15% at a ratio of 0.04% of the fermentation liquid volume.

[0054] The fermentation medium contains the following components by mass percentage: Sucrose 5.0%; soy protein isolate 0.16%; sodium dihydrogen phosphate 0.15%; sodium nitrate 0.18%; magnesium sulfate 0.01%; ferrous sulfate 0.001%; ​​zinc chloride 0.00005%; defoamer 0.03%; pH = 6.8–7.5.

[0055] The mass percentage of ammonium nitrate solution is 30% to 40%.

[0056] In fermentation production, *Rhizobium praecox* (…) Rhizobium pusenseAfter being expanded, CGMCC No. 12954 was inoculated into the fermentation medium at an inoculation rate of 10% of the volume percentage of seed culture to fermentation medium.

[0057] The process conditions for preparing the primary seed fermentation broth in step (1) of the extended culture process are: Inoculation amount: 0.5%; temperature: 28℃; ventilation rate: 0.3 vvm; when the viscosity of the primary seed fermentation broth is 50 mpa.s to 100 mpa.s, transfer it to the sterilized secondary seed culture medium. The primary seed culture medium comprises the following components by weight percentage: Sucrose 1.0%; soy protein isolate 0.16%; sodium dihydrogen phosphate 0.15%; sodium nitrate 0.18%; magnesium sulfate 0.01%; ferrous sulfate 0.001%; ​​zinc chloride 0.00005%; defoamer 0.03%; pH = 6.8–7.5.

[0058] The process conditions for preparing the secondary seed fermentation broth in step (2) of the expanded culture process are as follows: Inoculation amount 10%; temperature 28℃; ventilation rate 0.3 vvm; when the viscosity of the secondary seed fermentation broth is 100 mpa.s to 200 mpa.s, transfer to the sterilized fermentation medium; The secondary seed culture medium comprises the following components by weight percentage: Sucrose 1.0%; soy protein isolate 0.16%; sodium dihydrogen phosphate 0.15%; sodium nitrate 0.18%; magnesium sulfate 0.01%; ferrous sulfate 0.001%; ​​zinc chloride 0.00005%; defoamer 0.03%; pH = 6.8–7.5.

[0059] The rest of the content is the same as in Example 1. Example 3

[0060] The difference between this embodiment and Embodiment 1 is that: During aerated fermentation, ventilation is controlled in stages, including: 0–20 hours: ventilation rate is 0.65 vvm; 21–50 hours: Ventilation volume is 1.0 vvm; 51 hours until tank discharge: ventilation rate is 1.4 vvm; The fermentation cycle shall not exceed 70 hours; After 28–32 hours of fermentation, add a nitrogen source at a ratio of 0.085% of the fermentation liquid volume, using ammonia water and ammonium nitrate solution as the nitrogen source; after 50–60 hours of fermentation, add hydrogen peroxide with a volume percentage concentration of 17.5% at a ratio of 0.05% of the fermentation liquid volume.

[0061] The fermentation medium contains the following components by mass percentage: Sucrose 6.0%; soy protein isolate 0.24%; sodium dihydrogen phosphate 0.2%; sodium nitrate 0.26%; magnesium sulfate 0.015%; ferrous sulfate 0.0013%; zinc chloride 0.00008%; defoamer 0.04%; pH = 6.8–7.5.

[0062] The percentage concentration of ammonia water is not less than 18%, and the mass percentage of ammonium nitrate solution is 35%.

[0063] In fermentation production, *Rhizobium praecox* (…) Rhizobium pusense After being expanded, CGMCC No. 12954 was inoculated into the fermentation medium at an inoculation rate of 15% of the volume percentage of seed culture to fermentation medium.

[0064] The process conditions for preparing the primary seed fermentation broth in step (1) of the extended culture process are: Inoculation amount: 0.5%; temperature: 31℃; ventilation rate: 0.4 vvm; when the viscosity of the primary seed fermentation broth is 50 mpa.s to 100 mpa.s, transfer it to the sterilized secondary seed culture medium. The primary seed culture medium comprises the following components by weight percentage: Sucrose 1.5%; soy protein isolate 0.24%; sodium dihydrogen phosphate 0.2%; sodium nitrate 0.26%; magnesium sulfate 0.015%; ferrous sulfate 0.0013%; zinc chloride 0.00008%; defoamer 0.04%; pH = 6.8–7.5.

[0065] The process conditions for preparing the secondary seed fermentation broth in step (2) of the expanded culture process are as follows: Inoculation amount 15%; temperature 31℃; ventilation rate 0.4 vvm; when the viscosity of the secondary seed fermentation broth is 100 mpa.s to 200 mpa.s, transfer to sterilized fermentation medium; The secondary seed culture medium comprises the following components by weight percentage: Sucrose 1.5%; soy protein isolate 0.24%; sodium dihydrogen phosphate 0.2%; sodium nitrate 0.26%; magnesium sulfate 0.015%; ferrous sulfate 0.0013%; zinc chloride 0.00008%; defoamer 0.04%; pH = 6.8–7.5.

[0066] The rest of the content is the same as in Example 1. Example 4

[0067] The difference between this embodiment and Embodiment 1 is that: During aerated fermentation, ventilation is controlled in stages, including: 0–20 hours: Ventilation volume is 0.6 vvm; 21–50 hours: ventilation rate is 0.95 vvm; 51 hours until tank discharge: ventilation rate is 1.35 vvm; The fermentation cycle shall not exceed 70 hours; After 28–32 hours of fermentation, add a nitrogen source at a ratio of 0.083% of the fermentation liquid volume, using ammonium nitrate solution as the nitrogen source; after 50–60 hours of fermentation, add hydrogen peroxide with a volume percentage concentration of 16% at a ratio of 0.045% of the fermentation liquid volume.

[0068] The fermentation medium contains the following components by mass percentage: Sucrose 5.5%; soy protein isolate 0.2%; sodium dihydrogen phosphate 0.18%; sodium nitrate 0.2%; magnesium sulfate 0.013%; ferrous sulfate 0.0012%; zinc chloride 0.00006%; defoamer 0.035%; pH = 6.8–7.5.

[0069] The percentage concentration of ammonia water is not less than 18%, and the mass percentage of ammonium nitrate solution is 33%.

[0070] In fermentation production, *Rhizobium praecox* (…) Rhizobium pusense After being expanded, CGMCC No. 12954 was inoculated into the fermentation medium at an inoculation rate of 10% to 20% of the volume percentage of seed culture to fermentation medium.

[0071] The process conditions for preparing the primary seed fermentation broth in step (1) of the extended culture process are: Inoculation amount: 0.5%; temperature: 28℃~34℃; ventilation rate: 0.35vvm; when the viscosity of the primary seed fermentation broth is 50mpa.s~100mpa.s, transfer it to the sterilized secondary seed culture medium. The primary seed culture medium comprises the following components by weight percentage: Sucrose 1.3%; soy protein isolate 0.2%; sodium dihydrogen phosphate 0.18%; sodium nitrate 0.22%; magnesium sulfate 0.013%; ferrous sulfate 0.0011%; zinc chloride 0.00006%; defoamer 0.035%; pH = 6.8–7.5.

[0072] The process conditions for preparing the secondary seed fermentation broth in step (2) of the expanded culture process are as follows: Inoculation amount 13%; temperature 29℃; ventilation rate 0.35 vvm; when the viscosity of the secondary seed fermentation broth is 100 mpa.s to 200 mpa.s, transfer to sterilized fermentation medium; The secondary seed culture medium comprises the following components by weight percentage: Sucrose 1.3%; soy protein isolate 0.2%; sodium dihydrogen phosphate 0.18%; sodium nitrate 0.22%; magnesium sulfate 0.013%; ferrous sulfate 0.0011%; zinc chloride 0.00006%; defoamer 0.035%; pH = 6.8–7.5.

[0073] The rest of the content is the same as in Example 1. Example 5

[0074] The difference between this embodiment and Embodiment 1 is that: During aerated fermentation, ventilation is controlled in stages, including: 0–20 hours: ventilation volume is 0.7 vvm; 21–50 hours: ventilation rate is 1.05 vvm; 51 hours until tank discharge: ventilation rate is 1.45 vvm; The fermentation cycle shall not exceed 70 hours; After 28–32 hours of fermentation, add a nitrogen source at a ratio of 0.087% of the fermentation liquid volume, using ammonia water and ammonium nitrate solution as the nitrogen source; after 50–60 hours of fermentation, add hydrogen peroxide with a volume percentage concentration of 19% at a ratio of 0.055% of the fermentation liquid volume.

[0075] The fermentation medium contains the following components by mass percentage: Sucrose 6.5%; soy protein isolate 0.3%; sodium dihydrogen phosphate 0.22%; sodium nitrate 0.3%; magnesium sulfate 0.018%; ferrous sulfate 0.0014%; zinc chloride 0.00009%; defoamer 0.045%; pH = 6.8–7.5.

[0076] The percentage concentration of ammonia water is not less than 18%, and the mass percentage of ammonium nitrate solution is 38%.

[0077] In fermentation production, *Rhizobium praecox* (…) Rhizobium pusense After being expanded, CGMCC No. 12954 was inoculated into the fermentation medium at an inoculation rate of 10% to 20% of the volume percentage of seed culture to fermentation medium.

[0078] The process conditions for preparing the primary seed fermentation broth in step (1) of the extended culture process are: Inoculation amount: 0.5%; temperature: 32℃; ventilation rate: 0.45 vvm; when the viscosity of the primary seed fermentation broth is 50 mpa.s to 100 mpa.s, transfer it to the sterilized secondary seed culture medium. The primary seed culture medium comprises the following components by weight percentage: Sucrose 1.8%; soy protein isolate 0.3%; sodium dihydrogen phosphate 0.22%; sodium nitrate 0.3%; magnesium sulfate 0.018%; ferrous sulfate 0.0014%; zinc chloride 0.00009%; defoamer 0.045%; pH = 6.8–7.5.

[0079] The process conditions for preparing the secondary seed fermentation broth in step (2) of the expanded culture process are as follows: Inoculation amount 18%; temperature 32℃; ventilation rate 0.45 vvm; when the viscosity of the secondary seed fermentation broth is 100 mpa.s to 200 mpa.s, transfer to sterilized fermentation medium; The secondary seed culture medium comprises the following components by weight percentage: Sucrose 1.8%; soy protein isolate 0.3%; sodium dihydrogen phosphate 0.23%; sodium nitrate 0.3%; magnesium sulfate 0.018%; ferrous sulfate 0.0014%; zinc chloride 0.00008%; defoamer 0.045%; pH = 6.8–7.5. The remaining contents are the same as in Example 1.

[0080] The applicant tested the fermentation yield and the viscosity and water solubility of the prepared fermentation broth in Examples 1-5, and the relevant results are as follows: Yield (g / L) Fermentation broth viscosity (mPa·s) Water-soluble (mPa·s) Fermentation broth prepared by the method in Example 1 25.1 2460 500 Fermentation broth prepared by the method in Example 2 26.4 2410 600 Fermentation broth prepared by the method in Example 3 28.2 2320 600 Fermentation broth prepared by the method in Example 4 29.1 2280 500 Fermentation broth prepared by the method in Example 5 26.8 2090 700

Claims

1. A method for preparing fermentation broth containing β-1,3 / α-1,3-glucan involves inoculating a microbial strain into a sterilized fermentation medium containing a carbon source, nitrogen source, and necessary nutrients, followed by aerated fermentation. The microbial strain is named using the Latin name... Rhizobium pusense The *Prunella vulgaris* species with accession number CGMCC No. 12954 is characterized by: A. Sucrose is used as the carbon source and soy protein isolate is used as the nitrogen source in the fermentation medium; B. During aeration fermentation, the ventilation volume is controlled in stages, including: 0–20 hours: ventilation volume is 0.5 vvm–0.8 vvm; 21–50 hours: ventilation volume is 0.9 vvm–1.1 vvm; From 51 hours until canister discharge: ventilation rate is 1.3vvm to 1.5vvm; The fermentation cycle shall not exceed 70 hours; C. After 28–32 hours of fermentation, add a nitrogen source at a ratio of 0.08%–0.09% of the fermentation liquid volume, using ammonia water and / or ammonium nitrate solution as the nitrogen source; after 50–60 hours of fermentation, add hydrogen peroxide with a volume percentage concentration of 15%–20% at a ratio of 0.04%–0.06% of the fermentation liquid volume.

2. The method for preparing fermentation broth containing β-1,3 / α-1,3-glucan according to claim 1, characterized in that... The fermentation medium for A contains the following components by mass percentage: Sucrose 5.0%–7.0%; soy protein isolate 0.16%–0.32%; sodium dihydrogen phosphate 0.15%–0.25%; sodium nitrate 0.18%–0.34%; magnesium sulfate 0.01%–0.02%; ferrous sulfate 0.001%–0.0015%; zinc chloride 0.00005%–0.0001%; ​​defoamer 0.03%–0.05%; pH=6.8–7.

5.

3. The method for preparing fermentation broth containing β-1,3 / α-1,3-glucan according to claim 1, characterized in that feature C The percentage concentration of ammonia in the solution is not less than 18%, and the mass percentage of ammonium nitrate solution is 30% to 40%.

4. The method for preparing fermentation broth containing β-1,3 / α-1,3-glucan according to claim 3, characterized in that... After the ammonium nitrate solution of C is prepared, it is sterilized at high temperature, and then added after cooling to 28℃~30℃.

5. The method for preparing fermentation broth containing β-1,3 / α-1,3-glucan according to any one of claims 1 to 4, characterized in that... It is the root nodule bacterium ( Rhizobium pusense After being expanded, CGMCC No. 12954 was inoculated into the fermentation medium at an inoculation rate of 10% to 20% of the volume percentage of seed culture to fermentation medium.

6. The method for preparing fermentation broth containing β-1,3 / α-1,3-glucan according to claim 5, characterized in that... The aforementioned scale-up culture process includes: (1) The Latin name is Rhizobium pusense The *Rhizobium praecox* strain with accession number CGMCC No. 12954 was inoculated into sterilized primary seed culture medium to prepare primary seed fermentation broth. (2) The primary seed fermentation broth is expanded and cultured to produce a secondary seed fermentation broth; (3) The prepared secondary seed fermentation broth is inoculated into the sterilized fermentation medium, and after aeration and fermentation, a fermentation broth containing β-1,3 / α-1,3-glucan is prepared.

7. The method for preparing fermentation broth containing β-1,3 / α-1,3-glucan according to claim 6, characterized in that... The process conditions for preparing the primary seed fermentation broth in step (1) of the expanded culture process are as follows: Inoculation amount: 0.5%; temperature: 28℃~34℃; ventilation rate: 0.3vvm~0.5vvm; when the viscosity of the primary seed fermentation broth is 50mpa.s~100mpa.s, transfer to the sterilized secondary seed culture medium. The primary seed culture medium comprises the following components by weight percentage: Sucrose 1.0%–2.0%; soy protein isolate 0.16%–0.32%; sodium dihydrogen phosphate 0.15%–0.25%; sodium nitrate 0.18%–0.34%; magnesium sulfate 0.01%–0.02%; ferrous sulfate 0.001%–0.0015%; zinc chloride 0.00005%–0.0001%; ​​defoamer 0.03%–0.05%; pH=6.8–7.

5.

8. The method for preparing fermentation broth containing β-1,3 / α-1,3-glucan according to claim 6, characterized in that... The process conditions for preparing the secondary seed fermentation broth in step (2) of the expanded culture process are as follows: Inoculation amount: 10%–20%; temperature: 28℃–34℃; ventilation rate: 0.3 vvm–0.5 vvm; when the viscosity of the secondary seed fermentation broth is 100 mPa·s–200 mPa·s, transfer the inoculum to the sterilized fermentation medium. The secondary seed culture medium comprises the following components by weight percentage: Sucrose 1.0%–2.0%; soy protein isolate 0.16%–0.32%; sodium dihydrogen phosphate 0.15%–0.25%; sodium nitrate 0.18%–0.34%; magnesium sulfate 0.01%–0.02%; ferrous sulfate 0.001%–0.0015%; zinc chloride 0.00005%–0.0001%; ​​defoamer 0.03%–0.05%; pH=6.8–7.

5.

9. The method for preparing fermentation broth containing β-1,3 / α-1,3-glucan according to claim 6, characterized in that... The viscosity of the primary and secondary seed fermentation broths was measured using a Brookfield viscometer with a No. 4 rotor at a speed of 60 rpm.

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