Protective agent for prolonging storage time of liquid culture of mesorhizobium huakuii hwhs1 at room temperature and application thereof

Abstract

The application relates to the technical field of microbial fermentation, and particularly relates to a protective agent for prolonging the normal-temperature storage time of goat bean new rhizobium HWHS1 bacterial liquid and application thereof. The protective agent comprises 1,4-piperazine ethanesulfonic acid disodium salt, morpholine ethanesulfonic acid sodium salt, polyvinyl pyrrolidone K30 and sodium ascorbate. Through multi-component synergistic compounding and process optimization, a normal-temperature long-acting preservation system suitable for the goat bean new rhizobium HWHS1 is constructed, without the need of complex processes such as cold chain and freeze-drying, so that the normal-temperature shelf life and live bacteria retention rate of the liquid bacterial agent are significantly improved.

Description

Technical Field

[0001] This application relates to the technical field of microbial fermentation, and in particular to a preservative for extending the shelf life of *Neorhizobium huskum* HWHS1 bacterial culture at room temperature and its application. Background Technology

[0002] Neorhizobium moniliforme is a core functional microorganism that specifically symbiotically fixes nitrogen with peanuts. It can convert inert nitrogen in the air into combined nitrogen that peanuts can directly absorb and utilize, significantly reducing the application of chemical nitrogen fertilizers, improving the rhizosphere soil microecology, and increasing peanut yield and quality. It has extremely high application value in the fields of ecological agriculture, green planting, and reducing fertilizer use while increasing efficiency.

[0003] Most rhizobium products currently available on the market are sold in the form of freeze-dried powder, solid granules, or peat adsorbents, with liquid products being rare. The main reason is that rhizobium microorganisms are inherently delicate – they are accustomed to living in the roots of plants and have weak resistance and poor survival ability when separated from their host environment. Conventional liquid rhizobium inoculants without any added preservatives are prone to stress-induced apoptosis under normal temperature storage and transportation conditions, resulting in a rapid decline in the effective viable cell count. Large-scale cell death occurs after one month of natural storage at room temperature, and after three months of storage, the effective viable cell count is only 200-500 million / mL, far below the effective viable cell standard for agricultural applications, and cannot meet the requirements for large-scale field application.

[0004] Current technologies for preserving Rhizobium respiration rely heavily on stringent low-temperature, anaerobic, and dry storage conditions. Mainstream preservation methods include low-temperature cold chain refrigeration, vacuum freeze-drying, and adsorption preservation using solid media such as peat. All of these technologies have significant drawbacks: low-temperature refrigeration requires a complete cold chain, resulting in high production, storage, and logistics costs, and poor applicability in remote areas; vacuum freeze-drying involves complex processes, high energy consumption, significant mechanical loss of bacterial cells, and requires rehydration and activation before use, making it cumbersome and time-consuming; solid adsorbents have low effective viable cell loads, slow release, and delayed nitrogen fixation.

[0005] Currently, the industry lacks a dedicated liquid composite protection system adapted to the characteristics of the *Rhizobium hygrophorum* strain HWHS1, which cannot achieve long-term stable survival at room temperature, severely restricting the industrial production and large-scale application of highly active liquid rhizobium inoculants.

[0006] Therefore, developing a protective agent that can stably preserve the liquid inoculum of *Neorhizium anisopliae* HWHS1 at room temperature for a long period of time has significant practical implications and industrial value. Summary of the Invention

[0007] This application provides a preservative for extending the shelf life of *Neorhizobium goatii* HWHS1 bacterial culture at room temperature and its application. Through multi-component synergistic compounding and process optimization, this application constructs a long-lasting, room-temperature preservation system adapted to *Neorhizobium goatii* HWHS1, eliminating the need for complex processes such as cold chain and freeze-drying, and significantly improving the room-temperature shelf life and viable cell retention rate of the liquid bacterial agent.

[0008] In a first aspect, this application provides a preservative for extending the shelf life of *Neorhizium anisopliae* HWHS1 bacterial culture at room temperature, employing the following technical solution:

[0009] A preservative for extending the shelf life of *Neorhizobium huskum* HWHS1 bacterial culture at room temperature, the preservative comprising disodium 1,4-piperazine ethanesulfonate, sodium morpholine ethanesulfonate, polyvinylpyrrolidone K30, and sodium ascorbate.

[0010] This application combines disodium 1,4-piperazine ethanesulfonate with sodium morpholine ethanesulfonate (as a zwitterionic buffer system to stabilize the osmotic pressure and pH environment of the fermentation broth), polyvinylpyrrolidone K30 (PVP K30, as a polymeric protective agent, can form a flexible protective layer on the cell surface to prevent mechanical damage to the cells and leakage of intracellular substances), and sodium ascorbate (as a potent antioxidant to scavenge extracellular free radicals). These four components form a triple synergistic protection system of "osmotic protection + physical encapsulation + antioxidant," significantly extending the shelf life of the bacterial culture at room temperature.

[0011] Optionally, the concentration of the 1,4-piperazine ethanesulfonate disodium is 0.1-0.3 g / L.

[0012] Optionally, the concentration of the 1,4-piperazine ethanesulfonate disodium is 0.15-0.25 g / L.

[0013] Optionally, the concentration of the sodium morpholine ethanesulfonate is 1-3 g / L.

[0014] Optionally, the concentration of the sodium morpholine ethanesulfonate is 1.5-2.5 g / L.

[0015] Optionally, the concentration of the polyvinylpyrrolidone K30 is 1-3 g / L.

[0016] Optionally, the concentration of the polyvinylpyrrolidone K30 is 1.5-2.5 g / L.

[0017] Optionally, the concentration of the sodium ascorbate is 0.1-0.5 g / L.

[0018] Optionally, the concentration of the sodium ascorbate is 0.2-0.4 g / L.

[0019] Experimental analysis revealed that when the concentrations of disodium 1,4-piperazine ethanesulfonate, sodium morpholine ethanesulfonate, polyvinylpyrrolidone K30, and sodium ascorbate were below the lower limit, the protective efficacy was insufficient; when the concentrations were above the upper limit, not only did the cost increase, but the excessive osmotic pressure or excessive component concentrations may also have a slight inhibitory effect on the bacteria. Therefore, considering both cost and optimal effect, the above-mentioned preferred range was limited.

[0020] Secondly, this application provides the application of the above-mentioned protective agent in the preparation of highly stable liquid inoculum of *Neorhizium anisopliae* HWHS1.

[0021] The application of this protectant in the preparation of highly stable liquid bacterial agents solves the industry pain point that existing rhizobium products (such as freeze-dried powder and peat agents) cannot be stored for a long time at room temperature in liquid form, and significantly reduces the cost of cold chain transportation and storage.

[0022] Thirdly, this application provides a highly stable liquid bacterial agent of *Neorhizium anisopliae* HWHS1. The liquid bacterial agent comprises live *Neorhizium anisopliae* HWHS1 and the aforementioned protective agent. After being stored at room temperature (15-30°C) for 3 months, the effective viable count of the liquid bacterial agent is not less than 3 billion / mL; after being stored for 6 months, the effective viable count is not less than 2 billion / mL.

[0023] The liquid bacterial agent prepared based on the above-mentioned protectant has an excellent shelf life at room temperature. It can meet the agricultural application standard of "effective live bacteria count of not less than 2 billion / mL for 3 months" without the need for additional protective carriers, and has extremely high commercialization value.

[0024] Fourthly, this application provides a method for room temperature preservation of *Neorhizium anisopliae* HWHS1 bacterial suspension, employing the following technical solution:

[0025] A method for room temperature preservation of *Neorhizobium goatii* HWHS1 bacterial suspension, wherein the above-mentioned protective agent is applied to the preparation process of liquid *Neorhizobium goatii* HWHS1 bacterial suspension.

[0026] Optionally, the room temperature protection method specifically involves adding the protectant to the *Neorhizobium huskum* HWHS1 bacterial culture in a stepwise manner. Specifically, before sterilizing the fermentation medium, 1,4-piperazine ethanesulfonate disodium salt, morpholine ethanesulfonate sodium salt, and polyvinylpyrrolidone K30 are added to the fermentation medium and sterilized at high temperature and pressure along with the fermentation medium. After fermentation, the filtered and sterilized sodium ascorbate mother liquor is added to the fermented bacterial culture.

[0027] The stepwise addition process is adopted because sodium ascorbate is easily oxidized and decomposed under high temperature and high pressure (121℃) sterilization conditions, thus losing its antioxidant activity. Therefore, the heat-resistant disodium 1,4-piperazine ethanesulfonate, sodium morpholine ethanesulfonate, and PVPK30 are sterilized together with the culture medium, while sodium ascorbate is added after fermentation by filtration sterilization. This ensures that the activity of each component is not destroyed and that the sterility of the final product is guaranteed.

[0028] In summary, this application includes at least one of the following beneficial technical effects:

[0029] (1) The liquid bacterial agent product of this application can achieve long-term preservation at room temperature of 15-30℃. After 6 months of preservation, the number of effective live bacteria is still not less than 2 billion / mL, which completely changes the predicament of traditional Rhizobium liquid bacterial agents dying on a large scale after 1 month of room temperature storage.

[0030] (2) The liquid bacterial agent product of this application has a simple formula, readily available raw materials and low cost. Its preparation method only requires the use of the conventional process of "heat-resistant components are sterilized with the culture medium and heat-sensitive components are added by step-by-step filtration". It does not rely on expensive large-scale freeze-drying equipment or the whole cold chain, which greatly reduces the production and storage costs of enterprises.

[0031] (3) The protective agent of this application can comprehensively and stably protect the vitality of HWHS1 cells of goat bean neorhizobium in a static state at room temperature through the synergistic effect of multiple components. The prepared liquid bacterial agent product can be directly applied to the field without rehydration and activation, making it extremely convenient to use and suitable for large-scale agricultural promotion. Detailed Implementation

[0032] Before describing the embodiments of this application in detail, it should be understood that the terminology used herein is for the purpose of describing a particular embodiment only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term pertains.

[0033] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0034] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and such ranges or values ​​should be understood to include values ​​close to such ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0035] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0036] In this application, "room temperature storage" refers to a natural storage and transportation environment of 15-30℃. All tests in this application were conducted under standard constant temperature conditions of 25±2℃.

[0037] In this application, *Neorhizobium galegae* HWHS1, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29326, was deposited on December 15, 2023, at the Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing. *Neorhizobium galegae* HWHS1 is a nitrogen-fixing strain with high adaptability to peanut hosts and high nitrogen-fixing efficiency, demonstrating promising prospects for industrial application.

[0038] The screening process for the strain is as follows: Plump, firm root nodules are selected and cut in half with a dissecting knife. If the inside of the nodule is pink or wine-red, proceed to the next test; otherwise, select new nodules. Qualified nodules are first soaked in disinfectant water for 20-30 seconds, then in alcohol for 20-30 seconds, and finally rinsed with sterile water to remove the disinfectant and alcohol. The treated nodules are inoculated into YMA medium with the cut side facing down and cultured upside down. After the rhizobia appear, they are streaked for purification until a single colony is formed. The purified strain is examined under a microscope. Based on the microscopic results, the strain is preliminarily identified as a rhizobium.

[0039] The identification process of the strain is as follows: The purified strain obtained above was sent to a third-party company for sequencing to obtain its 16S rRNA (as shown in SEQ ID NO 1). Multiple reference strain sequences were obtained from the NCBI (GenBank) database. The 16S rRNA of this strain and the reference strains were analyzed using the software BioEdit and MEGA11. A phylogenetic tree of this strain and the reference strains was constructed, thus identifying this strain as *Neorhizobium galegae* HWHS1.

[0040] In this application, the general fermentation basal medium formula (per liter) is as follows: glucose 6-8g, sucrose 12-16g, yeast extract 3-5g, calcium chloride 0.1-0.3g, magnesium sulfate heptahydrate 0.3-0.5g, dipotassium hydrogen phosphate 0.5-1g, ammonium chloride 0.5-2g, monosodium glutamate 0.1-0.2g, citric acid 0.1-0.3g, ferrous sulfate 0.005-0.01g, sodium molybdate 0.01-0.03g, sodium carboxymethyl cellulose 0.1-0.3g, and 0.2mL (0.02%) of bufotoxin. This medium is a conventional fermentation medium for rhizobia and can stably support the growth and reproduction of the bacteria.

[0041] In summary, a formulation for a highly stable *Neorhizium anisopliae* HWHS1 bacterial culture containing the aforementioned protectants, based on a conventional fermentation medium, includes the following protectants at the following concentrations: 0.1-0.3 g / L disodium 1,4-piperazine ethanesulfonate, 1-3 g / L sodium morpholine ethanesulfonate, 1-3 g / L polyvinylpyrrolidone K30, and 0.1-0.5 g / L sodium ascorbate.

[0042] In this application, the protective agents are uniformly added in a stepwise manner: except for sodium ascorbate, the other protective agent components (disodium 1,4-piperazine ethanesulfonate, sodium morpholine ethanesulfonate, and polyvinylpyrrolidone K30) are added before the culture medium is sterilized and simultaneously sterilized at 121°C and 0.1MPa for 20 minutes; sodium ascorbate is prepared separately as a sterile stock solution, filtered through a 0.22μm sterile aqueous filter membrane for sterilization, and aseptically added after the culture medium has cooled to room temperature and the bacterial fermentation is completed.

[0043] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0044] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0045] The present application will be further described in detail below with reference to the embodiments and test results.

[0046] Example 1

[0047] This embodiment provides a preservative for extending the shelf life of *Neorhizium anisopliae* HWHS1 bacterial culture at room temperature. The specific formulation is shown in Table 1.

[0048] The aforementioned protective agents were added to the *Neorhizobium huskum* HWHS1 bacterial culture in a stepwise manner. Specifically, before sterilizing the fermentation medium, disodium 1,4-piperazine ethanesulfonate, sodium morpholine ethanesulfonate, and polyvinylpyrrolidone K30 were added to the fermentation medium, which was then sterilized at high temperature and pressure along with the fermentation medium. After fermentation, the filtered and sterilized sodium ascorbate mother liquor was added to the fermented bacterial culture.

[0049] The general fermentation basal culture medium formula (per liter) is as follows: glucose 7g, sucrose 14g, yeast powder 4g, calcium chloride 0.2g, magnesium sulfate heptahydrate 0.4g, dipotassium hydrogen phosphate 0.8g, ammonium chloride 1g, monosodium glutamate 0.15g, citric acid 0.2g, ferrous sulfate 0.008g, sodium molybdate 0.02g, sodium carboxymethyl cellulose 0.2g, and bubbly agent 0.2mL.

[0050] Example 2-17

[0051] The above embodiments provide a preservative for extending the shelf life of *Neorhizium anisopliae* HWHS1 bacterial culture at room temperature. The specific formulations are shown in Table 1, and all other aspects remain consistent with Example 1.

[0052] The differences between Examples 1-17 are as follows:

[0053] The difference between Examples 1-5 is that the concentration of disodium 1,4-piperazine ethanesulfonate is different.

[0054] The difference between Examples 3 and 6-9 is that the concentration of sodium morpholine ethanesulfonate is different.

[0055] The difference between Examples 3 and 10-13 is that the concentration of polyvinylpyrrolidone K30 is different.

[0056] The difference between Examples 3 and 14-17 is that the concentration of sodium ascorbate is different.

[0057] Table 1 Formulation of Protective Agent

[0058]

[0059] Comparative Example 1

[0060] This comparative example provides a preservative for extending the shelf life of *Neorhizium anisopliae* HWHS1 bacterial culture at room temperature. It differs from Example 3 in that polyvinylpyrrolidone K30 is not added; the specific formulation is shown in Table 1. All other aspects remain consistent with Example 1.

[0061] Comparative Examples 2-4

[0062] The above comparative examples each provide a preservative for extending the shelf life of *Neorhizium anisopliae* HWHS1 bacterial culture at room temperature. The difference between them and Example 3 is that polyvinylpyrrolidone K30 and sodium ascorbate were not added, and other types of components were added, specifically: Comparative Example 2 also added sucrose, Comparative Example 3 also added Tween 80, and Comparative Example 4 also added xanthan gum. The specific formulations are shown in Table 1, and all other aspects remain consistent with Example 1.

[0063] Comparative Example 5

[0064] This comparative example provides a preservative for extending the shelf life of *Neorhizium anisopliae* HWHS1 bacterial culture at room temperature. It differs from Example 3 in that sodium ascorbate is not added, and the specific formulation is shown in Table 1. All other aspects remain consistent with Example 1.

[0065] Comparative Example 6

[0066] This comparative example provides a preservative for extending the shelf life of *Neorhizium anisopliae* HWHS1 bacterial culture at room temperature. It differs from Example 3 in that sodium ascorbate is not added, and valine is added; the specific formulation is shown in Table 1. All other aspects remain the same as in Example 1.

[0067] Comparative Example 7

[0068] This comparative example provides a preservative for extending the shelf life of *Neorhizium anisopliae* HWHS1 bacterial culture at room temperature. It differs from Example 3 in that it does not contain sodium morpholine ethanesulfonate and sodium ascorbate, but instead contains valine and sodium dodecyl sarcosinate, as shown in Table 1. All other aspects remain consistent with Example 1.

[0069] Performance testing

[0070] The *Neorhizobium goatii* HWHS1 was cultured in fermentation medium containing the protectants added to the above-described examples and comparative examples, stored at room temperature, and the number of viable bacteria was measured in months 0, 1, 2, 3, and 6.

[0071] The effective viable cell count was determined using the plate dilution and plating method. The procedure was as follows: bacterial culture samples were taken at each time point and serially diluted with sterile physiological saline. Appropriate dilutions were then plated onto YMA solid agar plates, with three replicates for each dilution. The plates were incubated at 28°C for 3-5 days. After colonies appeared, the viable cells were counted, and the effective viable cell count per milliliter of sample was calculated based on the dilution factor. Each treatment was performed in triplicate, and the average result was taken.

[0072] The test results are shown in Table 1.

[0073] As shown in Table 1, compared with the comparative examples, Examples 1-17 using the four-component compound protective agent of this application all showed a significant inhibitory trend in viable cell decay during room temperature storage. In particular, Example 3 showed the flattest decay curve of effective viable cell count during room temperature storage for up to 6 months, indicating that the components of this application achieved the best synergistic protective effect under this preferred ratio.

[0074] Based on the trends observed in the comparative examples, PVP K30 and sodium ascorbate are the "core elements" for maintaining the long-term survival of the bacterial culture. Comparative Example 1 (lacking PVP K30) saw its viable cell count drop significantly to single digits (approximately 500 million / mL) by month 6; while Comparative Examples 5 (lacking sodium ascorbate) and 6 (using valine instead of sodium ascorbate) exhibited a more dramatic accelerated decline, with viable cell counts reaching zero by month 3. This indicates that without any of the key components described in this application, the system cannot prevent oxidative stress and lysis / apoptosis of the bacteria at room temperature.

[0075] Comparative Examples 2-4 used other commonly used preservatives in the industry (such as sucrose, Tween 80, and xanthan gum) to replace the active components of this application, and none of them could withstand 6 months of storage at room temperature. Of particular note is Comparative Example 4, which used the conventional physical colloid xanthan gum. Although it performed reasonably well in the third month, it also rapidly deteriorated below the shelf life threshold later on. This fully demonstrates the irreplaceable and unique role of the specific four-component combination in this application.

[0076] Comparing the test results of Examples 1-17, it can be seen that when the concentration of a specific component is slightly higher or slightly lower than the preferred range (such as in Examples 10 and 14), although the rate of decline of viable bacteria count is slightly faster than in Example 3, the effective viable bacteria count remains stable at over 2 billion / mL in the 6th month. This indicates that the formulation of this application has a wide applicability window and can significantly reduce the risk of dosage error during industrial addition.

[0077] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A preservative for extending the shelf life of *Neorhizobium arvense* HWHS1 bacterial culture at room temperature, characterized in that, The protective agents include disodium 1,4-piperazine ethanesulfonate, sodium morpholine ethanesulfonate, polyvinylpyrrolidone K30, and sodium ascorbate.

2. The protective agent according to claim 1, characterized in that, The concentration of the 1,4-piperazine ethanesulfonate disodium is 0.1-0.3 g / L; Optionally, the concentration of the 1,4-piperazine ethanesulfonate disodium is 0.15-0.25 g / L.

3. The protective agent according to claim 1, characterized in that, The concentration of the sodium morpholine ethanesulfonate is 1-3 g / L; Optionally, the concentration of the sodium morpholine ethanesulfonate is 1.5-2.5 g / L.

4. The protective agent according to claim 1, characterized in that, The concentration of polyvinylpyrrolidone K30 is 1-3 g / L; Optionally, the concentration of the polyvinylpyrrolidone K30 is 1.5-2.5 g / L.

5. The protective agent according to claim 1, characterized in that, The concentration of the sodium ascorbate is 0.1-0.5 g / L; Optionally, the concentration of the sodium ascorbate is 0.2-0.4 g / L.

6. The use of the protective agent according to any one of claims 1-5 in the preparation of a highly stable liquid inoculum of *Neorhizium anisopliae* HWHS1.

7. A highly stable liquid inoculum agent of *Neorhizium anisopliae* HWHS1, characterized in that, The liquid bacterial agent comprises live *Neorhizobium goatii* HWHS1 and the protective agent according to any one of claims 1-5.

8. The liquid bacterial agent according to claim 7, characterized in that, The liquid bacterial agent, after being stored at room temperature (15-30℃) for 3 months, has an effective viable count of no less than 3 billion / mL; after being stored for 6 months, the effective viable count is no less than 2 billion / mL.

9. A method for room temperature preservation of *Neorhizium anisopliae* HWHS1 bacterial suspension, characterized in that, The protective agent described in any one of claims 1-5 is applied in the preparation process of liquid inoculum of *Neorhizium anisopliae* HWHS1.

10. The method for room temperature preservation of *Neorhizium anisopliae* HWHS1 bacterial suspension according to claim 9, characterized in that, The specific method for room temperature protection is as follows: the protective agent is added to the *Neorhizobium huskum* HWHS1 bacterial culture in a stepwise manner. Specifically, before sterilizing the fermentation medium, 1,4-piperazine ethanesulfonate disodium salt, morpholine ethanesulfonate sodium salt, and polyvinylpyrrolidone K30 are added to the fermentation medium and sterilized at high temperature and pressure along with the fermentation medium. After fermentation, the filtered and sterilized sodium ascorbate mother liquor is added to the fermented bacterial culture.

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