A culture medium and culture method for increasing the viable count of rhizobium huautlense HWHS1

Abstract

The application relates to the technical field of microbial fermentation, and particularly relates to a culture medium for increasing the viable cell count of goat bean new rhizobium HWHS1 and a culture method. The culture medium includes, in 1L, basic components, i.e. glucose, sucrose, yeast powder, calcium chloride, magnesium sulfate heptahydrate, dipotassium hydrogen phosphate and sodium chloride; and active promoting components, i.e. dithiothreitol, trace element solution, ferrous sulfate, disodium ketoglutarate and citric acid. The active promoting components are sterilized separately by filtering sterilization, and are added into the basic components before fermentation. The application shows that DTT has a significant growth promoting effect on rhizobium, and the mixed carbon source system of glucose and sucrose and other basic nutritional components in the application can make the viable cell count break through 12 billion / mL stably, and the highest viable cell count can be more than 20 billion / mL, thereby completely solving the problem of low viable cell count in the prior art.

Description

Technical Field

[0001] This application relates to the technical field of microbial fermentation, and in particular to a culture medium and culture method for increasing the viable count of *Neorhizobium huskum* HWHS1 in goat's bean. Background Technology

[0002] *Neorhizobium clavatum* is a type of beneficial bacteria that can live in symbiosis with peanut roots, converting atmospheric nitrogen into usable nitrogen fertilizer. It is of significant value in reducing chemical fertilizer use, increasing peanut yield, and improving soil. However, the industrial production of *Neorhizobium clavatum* inoculum currently faces several challenging problems:

[0003] The production cycle is too long. *Neorhizobium oryzae*, a slow-growing bacterium, exhibits extremely slow growth and metabolism. Conventional liquid fermentation culture often requires 10-12 days, resulting in extremely low production efficiency. The long cultivation time also significantly increases the risk of contamination by other microorganisms, frequently leading to the scrapping of entire batches. Simultaneously, the effective viable cell count is too low. At the end of fermentation using traditional methods, the viable cell count in the culture medium is typically only a few hundred million CFU / mL, far below the requirements for highly active rhizobium agents in agricultural field applications, severely limiting its widespread adoption in farmland. Furthermore, the culture medium formulation is too complex. Traditional rhizobium culture media often require the addition of dozens of chemical raw materials, resulting in high procurement and preparation costs. Moreover, the quality of raw materials varies greatly between batches, easily leading to inconsistent bacterial counts and unstable quality in different batches. More importantly, technological bottlenecks are difficult to overcome. Existing improvement methods have very limited effectiveness: simply adding some conventional growth promoters can make the bacteria grow slightly faster, but the increase in the final bacterial count is negligible; merely changing the ratio of carbon and nitrogen sources also has very limited room for increasing the bacterial count, and it is still impossible to fundamentally solve the problem of low viable bacterial count.

[0004] Therefore, developing a new Rhizobium culture method for goat bean that uses simple raw materials, has a short culture cycle, and can stably obtain ultra-high viable bacterial counts is an urgent technical problem to be solved in this field. Summary of the Invention

[0005] To address the problems of long culture cycles, low viable cell counts, complex culture media, and unstable effects in the existing technologies, this application provides a culture medium and culture method for increasing the viable cell count of *Neorhizium anisopliae* HWHS1 in goat's bean.

[0006] In a first aspect, this application provides a culture medium for increasing the viable count of *Neorhizium anisopliae* HWHS1 in goat's bean, using the following technical solution:

[0007] A culture medium for increasing the viable count of *Neorhizobium huskum* HWHS1 in goat's bean, per 1 L, comprises: basic components: 6-8 g glucose, 12-16 g sucrose, 3-5 g yeast extract, 0.15-0.25 g calcium chloride, 0.3-0.5 g magnesium sulfate heptahydrate, 0.5-1 g dipotassium hydrogen phosphate, and 0.05-0.15 g sodium chloride; and activity-promoting components: 0.2-1.5 g dithiothreitol, 4-6 mL trace element solution, 0.0006-0.001 g ferrous sulfate, 0.05-0.1 g disodium ketoglutarate, and 0.1-0.3 g citric acid. The activity-promoting components are sterilized separately by filtration and added to the basic components before fermentation.

[0008] This application employs a mixed carbon source system of glucose and sucrose. Glucose, a monosaccharide, can be directly and rapidly absorbed and utilized by rhizobia, propelling the bacteria into a rapid logarithmic growth phase, thereby significantly increasing the total bacterial count per unit volume and acting as a "rapid burst." Sucrose, a disaccharide, requires hydrolysis into monosaccharides by extracellular enzymes before it can be utilized. Its utilization rate is slower, but it can continuously provide nutrition in the later stages of fermentation, preventing premature decline in bacterial count due to the early depletion of glucose alone, thus acting as a "long-term maintenance" agent. The combination of the two can extend the duration of the high-activity plateau phase of the bacteria, providing a wider operating window for industrial post-processing (such as freeze-drying and centrifugal collection).

[0009] Dithiothreitol (DTT) is a potent reducing agent, conventionally known only for protecting proteins from oxidation. This application introduces it for the first time into a rhizobium fermentation system. Its mechanism of action lies in the fact that rhizobia generate a large number of reactive oxygen species (ROS) during rapid reproduction. These ROS attack cell membranes and key enzyme proteins (especially nitrogen-fixing enzymes), leading to decreased cellular metabolic activity. DTT effectively protects the active conformation of these key enzyme proteins by maintaining intracellular redox balance, thereby significantly improving the metabolic activity and cell division rate of single bacteria, resulting in a far greater enrichment effect than conventional growth promoters (such as B vitamins and amino acids). This discovery falls under the category of "new uses of known substances" inventions, exhibiting remarkable non-obviousness.

[0010] Since both DTT and disodium ketoglutarate are heat-sensitive substances, they will completely decompose and become ineffective if sterilized at a conventional high temperature of 121°C. This application specifies that these two core active ingredients must be sterilized by filtration and added only after the basic components have been sterilized at high temperature and cooled. This "separate sterilization and separate addition" process design ensures that the active ingredients in the formula can exert their true effects 100%, which is a key guarantee for the successful scale-up of laboratory results to industrial production. Without this process restriction, the formula will be completely ineffective in large-scale fermentation.

[0011] Boric acid and sodium molybdate in the trace element solution are essential micronutrients for the synthesis of nitrogenase in rhizobia; ferrous sulfate provides iron, which participates in electron transport in the cellular respiratory chain; disodium ketoglutarate is a key intermediate in the tricarboxylic acid cycle, directly providing metabolic energy to the bacteria and accelerating ATP production; citric acid chelates metal ions, reduces precipitation, and promotes the absorption of trace elements, while also serving as a weak acid carbon source that can be slowly utilized by the bacteria. These components work together to provide the optimal microenvironment for the bacteria.

[0012] Optionally, the amount of dithiothreitol added is 0.5-1 g / L.

[0013] Optionally, the pH value of the culture medium is 6.3-6.8; the pH value refers to the pH value after the basic components are sterilized and before the activity-promoting components are added.

[0014] The reason for setting this pH range is that the optimal growth environment for rhizobia is slightly acidic (around pH 6.5), and the pH usually drops by 0.3-0.5 after high-temperature sterilization due to the caramelization of glucose and changes in the phosphate buffer system. Therefore, it is necessary to precisely adjust the pH back to this range after sterilization to ensure that the bacteria are immediately in the optimal acid-base environment after inoculation.

[0015] Optionally, the weight ratio of carbon source to nitrogen source in the culture medium is (4.5-5.5):1.

[0016] Optionally, the trace element solution comprises 0.003-0.005 g / L of boric acid and 0.003-0.005 g / L of sodium molybdate.

[0017] Secondly, this application provides a method for efficiently and stably culturing high-bacterial-count bacterial suspensions of *Neorhizium anisopliae* HWHS1 from goat's bean, employing the following technical solution:

[0018] A method for efficiently and stably culturing high-bacterial-count neorhizobium HWHS1 in goat's bean, the method comprising the following steps:

[0019] (1) Seed liquid preparation: Select a single colony of *Neorhizobium hygrophorum* HWHS1 from goat bean and inoculate it into YMA liquid medium. Culture it in a shaker at 27-29℃ and 180-220 r / min for 6-8 days to obtain seed liquid;

[0020] (2) Primary fermentation: The seed liquid is inoculated into the above culture medium at a volume ratio of 2-4%, and cultured in a fermenter. The ventilation rate is controlled at 0.25-0.35 vvm, the stirring speed is 90-110 r / min, the initial dissolved oxygen is 95-100%, and the culture temperature is 28-29℃.

[0021] (3) Rotary fermentation: When the fermentation broth OD 600When the bacterial culture reaches a concentration of 0.3-0.5 after a 5-fold dilution, it is transferred to another culture tank. The volume ratio of the transferred tanks is controlled at 5-10%. After the transfer, the culture is continued under the same conditions for 4-6 days to obtain a high bacterial count culture.

[0022] This application precisely measures the "OD" through numerous experiments. 600 A 5-fold dilution to 0.3-0.5% is considered the gold standard for transfer. This indicator corresponds to the mid-logarithmic phase (approximately two-thirds of the way through) of bacterial growth. Using a 5-fold dilution instead of directly measuring the stock solution eliminates the nonlinear interference of high-concentration bacterial solution on light scattering, maintaining a good linear relationship between OD value and bacterial concentration, thus accurately determining the growth stage of the bacteria. Transferring at this point avoids both insufficient bacterial count in the early logarithmic phase (too low OD value), which would hinder recovery after transfer, and bacterial aging and decreased activity in the late logarithmic phase (too high OD value), which could lead to no growth or significant autolysis after transfer. This precise quantitative transfer strategy offers significant advantages in process stability compared to traditional methods such as "relying on experience to judge color" or "fixed-time transfer."

[0023] The transfer volume ratio is explicitly limited to 5-10% to ensure the initial bacterial concentration in the new tank is just right. If the transfer volume is less than 5%, the bacteria need a long time to readapt to the new environment and multiply to a sufficient density (extending the lag period), resulting in a longer overall fermentation cycle and making the tank more susceptible to invasion by contaminating bacteria due to the low initial bacterial concentration. If the transfer volume is greater than 10%, it means that a large amount of metabolic byproducts (such as organic acids and ammonia nitrogen) from the primary tank are introduced. Furthermore, due to the large initial bacterial count, the fresh nutrients in the secondary tank will be rapidly depleted, leading to insufficient nutrition in the later stages and an earlier plateau in bacterial count.

[0024] The initial dissolved oxygen level was set at 95-100%, and during fermentation, the dissolved oxygen level was maintained at no less than 30% by adjusting the stirring speed. This ensured that the aerobic bacteria, such as rhizobia, would not experience metabolic obstruction or produce organic acids that inhibit their growth due to lack of oxygen during rapid reproduction. If the dissolved oxygen level fell below 30%, the aerobic respiration of the bacteria would switch to anaerobic fermentation or metabolic stagnation, directly affecting the final accumulation of viable bacteria.

[0025] Optionally, the primary fermentation time is 2.5-3.5 days.

[0026] Optionally, after the culture is completed, the viable count of *Neorhizobium hygrophorum* HWHS1 in the bacterial culture is stabilized at over 12 billion / mL.

[0027] In summary, this application includes at least one of the following beneficial technical effects:

[0028] (1) This application shows that DTT has a significant growth-promoting effect on rhizobia. Combined with the glucose and sucrose mixed carbon source system and other basic nutrients in this application, the number of viable bacteria can stably exceed 12 billion / mL, and can even exceed 20 billion / mL, which completely solves the problem of low viable bacteria count in the prior art.

[0029] (2) This application constructs a mixed carbon source system of "rapid-acting + long-acting": glucose provides energy in the early stage quickly, and sucrose is released slowly to maintain nutrition in the later stage. The two work together to avoid the problem of nutritional imbalance in single carbon source and prolong the plateau period of high activity of bacteria.

[0030] (3) By precisely positioning the timing of the logarithmic transfer tank, this application significantly shortens the total fermentation cycle from the traditional 10-12 days to about 8 days, greatly improving production efficiency and reducing the risk of contamination.

[0031] (4) This application simplifies the culture medium formula to no more than 10 raw materials, which significantly reduces raw material costs and batch-to-batch quality fluctuations. 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] This application provides a culture medium for increasing the viable count of *Neorhizobium huskum* HWHS1 in goat's bean. The culture medium, per 1L, comprises: basic components: 6-8g glucose, 12-16g sucrose, 3-5g yeast extract, 0.15-0.25g calcium chloride, 0.3-0.5g magnesium sulfate heptahydrate, 0.5-1g dipotassium hydrogen phosphate, and 0.05-0.15g sodium chloride; and activity-promoting components: 0.2-1.5g dithiothreitol, 4-6mL trace element solution, 0.0006-0.001g ferrous sulfate, 0.05-0.1g disodium ketoglutarate, and 0.1-0.3g citric acid. The activity-promoting components are sterilized separately by filtration and added to the basic components before fermentation.

[0037] This application also provides a method for efficiently and stably culturing a high-bacterial-count culture of *Neorhizium anisopliae* HWHS1 from goat's bean. The method includes the following steps:

[0038] (1) Seed liquid preparation: Select a single colony of *Neorhizobium hygrophorum* HWHS1 from goat bean and inoculate it into YMA liquid medium. Culture it in a shaker at 27-29℃ and 180-220 r / min for 6-8 days to obtain seed liquid;

[0039] (2) Primary fermentation: The seed liquid is inoculated into the above culture medium at a volume ratio of 2-4%, and cultured in a fermenter. The ventilation rate is controlled at 0.25-0.35 vvm, the stirring speed is 90-110 r / min, the initial dissolved oxygen is 95-100%, and the culture temperature is 28-29℃.

[0040] (3) Rotary fermentation: When the fermentation broth OD 600 When the bacterial culture reaches a concentration of 0.3-0.5 after a 5-fold dilution, it is transferred to another culture tank. The volume ratio of the transferred tanks is controlled at 5-10%. After the transfer, the culture is continued under the same conditions for 4-6 days to obtain a high bacterial count culture.

[0041] In this application, *Neorhizobium galegae* HWHS1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29326, deposited on December 25, 2023, at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] In this application, unless otherwise specified, all basic components are autoclaved at 121°C for 20 minutes; all activity-enhancing components are sterilized by filtration through a 0.22μm filter membrane, and are added aseptically after the basic components have been sterilized and cooled to room temperature.

[0047] The present application will be further described in detail below with reference to the embodiments and test results.

[0048] Example 1

[0049] This embodiment provides a culture medium for increasing the viable count of *Neorhizobium hullerii* HWHS1 in goat's bean.

[0050] 1L of the culture medium contains: basic components—7g glucose, 14g sucrose, 4g yeast extract, 0.2g calcium chloride, 0.4g magnesium sulfate heptahydrate, 0.8g dipotassium hydrogen phosphate, and 0.1g sodium chloride; and activity-enhancing components—0.8g dithiothreitol, 5mL trace element solution (containing 0.004g / L boric acid and 0.004g / L sodium molybdate), 0.0008g ferrous sulfate, 0.07g disodium ketoglutarate, and 0.2g citric acid. After sterilization of the basic components and before adding the activity-enhancing components, the pH is adjusted to 6.5.

[0051] The preparation method of the above culture medium is as follows:

[0052] (1) Preparation of basic components: Weigh each component according to the ratio, add it to about 800mL of purified water, place it on a magnetic stirrer and stir until completely dissolved, then add purified water to make up to 1L.

[0053] (2) High temperature sterilization: Dispense the prepared basic culture medium into fermenters or Erlenmeyer flasks and autoclave at 121°C for 20 min.

[0054] (3) Cooling and pH adjustment: After sterilization, allow the basal culture medium to cool naturally to room temperature. Under aseptic conditions, adjust the pH to 6.5 using a 1 mol / L sodium hydroxide solution or hydrochloric acid solution that has been sterilized by filtration through a 0.22 μm filter membrane.

[0055] (4) Pretreatment of active promoting components: Under the aseptic environment of a laminar flow hood, each active promoting component is dissolved in a small amount of sterile water. After mixing evenly, the mixture is filtered and sterilized using a 0.22μm microporous membrane, and the sterile filtrate is collected in a sterile container.

[0056] (5) Aseptic mixing: The aseptic filtrate of the active promoting component obtained in step (4) is slowly added to the basic culture medium whose pH has been adjusted in step (3) under aseptic conditions. While adding, gently shake or stir to make the active component evenly dispersed in the culture medium, thus obtaining the culture medium that increases the viable number of HWHS1 neorhizobium of goat bean.

[0057] Comparative Examples 1-3

[0058] The above comparative examples each provide a culture medium for increasing the viable count of *Neorhizium anisopliae* HWHS1 in goat's bean. The differences from Example 1 are: a different nitrogen source, with yeast powder replaced in equal amounts with tryptone, glutamic acid, or corn flour, as shown in Table 1; the remaining components and preparation methods are consistent with Example 1.

[0059] Table 1. Different nitrogen source schemes and culture results

[0060]

[0061] Example 2

[0062] In this embodiment, the culture medium provided in Example 1 and Comparative Examples 1-3 was used to culture *Neorhizobium goatii* HWHS1.

[0063] The specific method is as follows:

[0064] (1) Seed culture preparation: Select a single colony of *Neorhizobium hupehensis* HWHS1 and inoculate it into YMA liquid medium (formulation: 1L of mannitol 10g, yeast extract 0.8g, dipotassium hydrogen phosphate 0.25g, potassium dihydrogen phosphate 0.25g, magnesium sulfate 0.2g, sodium chloride 0.1g), and culture it in a shaker at 28℃ and 200r / min for 7 days to obtain the seed culture.

[0065] (2) Initial concentration adjustment: Using sterile water as a control, the OD of the seed solution was measured. 600 To ensure that the initial OD values ​​of each group are similar, the high-concentration seed solution is diluted.

[0066] (3) Inoculation culture: Take 3 mL of seed liquid and inoculate it into different culture media as described above, shake well and then place it in a shaker at 28℃ and 200 r / min for culture;

[0067] (4) Viable bacteria count: Samples were taken every 24 hours, and the viable bacteria count was determined by plate counting method.

[0068] The test results are shown in Table 1.

[0069] As shown in Table 1, the number of viable bacteria in the yeast powder group was significantly better than that of other nitrogen sources, thus yeast powder was determined to be the optimal nitrogen source.

[0070] Comparative Examples 4-8

[0071] The above comparative examples each provide a culture medium for increasing the viable count of *Neorhizium anisopliae* HWHS1 in goat's bean. The difference between these and Example 1 lies in the carbon source, as detailed in Table 2; the remaining components and preparation methods are consistent with Example 1.

[0072] Table 2. Different carbon source schemes and cultivation results

[0073]

[0074] Example 3

[0075] In this embodiment, the culture medium provided in Example 1 and Comparative Examples 4-8 was used to culture *Neorhizobium goatii* HWHS1. The specific method was the same as in Example 2, and the detection results are shown in Table 2.

[0076] As shown in Table 2, the glucose + sucrose mixed carbon source group in Example 1 had the highest number of viable bacteria; the glycerol group showed a decline after day 6; the soluble starch group had the lowest number of viable bacteria due to insufficient nutrition caused by easy clumping; the number of bacteria was low when sucrose was used alone as a carbon source, but the effect was significantly improved when combined with glucose, indicating that the two have a synergistic effect.

[0077] Examples 4-7

[0078] The above embodiments provide a culture medium for increasing the viable count of *Neorhizium anisopliae* HWHS1 in goat's bean. The difference between these and Embodiment 1 lies in the amount of DTT added, as shown in Table 3; the remaining operational steps are consistent with Embodiment 1.

[0079] Comparative Examples 9-10

[0080] The above comparative examples each provide a culture medium for increasing the viable count of *Neorhizium anisopliae* HWHS1 in goat's bean. The difference between these and Example 1 lies in the amount of DTT added, as shown in Table 3; all other operating steps remain the same as in Example 1.

[0081] Table 3. Culture results with different DTT addition levels.

[0082]

[0083] Example 8

[0084] In this embodiment, the *Neorhizobium goatii* HWHS1 was cultured using the culture media provided in Examples 1, 4-7, and Comparative Examples 9-10. The specific method was the same as in Example 2, and the detection results are shown in Table 3.

[0085] As shown in Table 3, compared with the blank control without any DTT, the addition of DTT resulted in a significant increase in bacterial count. As the DTT concentration gradually increased from 0 to 0.8 g / L, the viable bacterial count at each stage showed a steady and significant upward trend. However, when the concentration was further increased to 1.5 g / L or even 2.0 g / L, the bacterial count decreased instead of increasing, exhibiting a clear parabolic trend of first increasing and then decreasing. This indicates that an appropriate amount of DTT can greatly activate bacterial activity, but excessive amounts, due to their strong reducing properties, can have a toxic effect on cells. Therefore, this application limits the optimal DTT addition range to 0.2-1.5 g / L, more preferably 0.5-1 g / L.

[0086] Examples 9-13

[0087] The above embodiments provide a culture medium for increasing the viable count of *Neorhizium anisopliae* HWHS1 in goat's bean. The difference between these and Embodiment 1 lies in the carbon-to-nitrogen ratio, as shown in Table 4; all other operating steps remain the same as in Embodiment 1.

[0088] Comparative Examples 11-12

[0089] The above comparative examples each provide a culture medium for increasing the viable count of *Neorhizium anisopliae* HWHS1 in goat's bean. The difference between these and Example 1 lies in the carbon-to-nitrogen ratio, as shown in Table 4; all other operating steps remain the same as in Example 1.

[0090] Table 4. Culture results for different carbon-nitrogen ratios

[0091]

[0092] Example 14

[0093] In this embodiment, the culture medium provided in Examples 1, 9-13, and Comparative Examples 11-12 was used to culture *Neorhizobium goatii* HWHS1. The specific method was the same as in Example 2, and the detection results are shown in Table 4.

[0094] As shown in Table 4, the number of viable bacteria is higher when the carbon-to-nitrogen ratio is in the range of (4.5-5.5):1, with the highest number of viable bacteria at a carbon-to-nitrogen ratio of 5.25:1, which is determined to be the optimal carbon-to-nitrogen ratio. The number of bacteria decreases significantly when the carbon-to-nitrogen ratio is too low (3:1) or too high (7:1).

[0095] Example 15

[0096] This embodiment describes a method for efficiently and stably culturing a high-bacterial-count culture of *Neorhizium anisopliae* HWHS1 from goat's bean. The culture medium provided in Example 1 is used for cultivation.

[0097] The method specifically includes the following steps:

[0098] (1) Seed culture preparation: Select a single colony of *Neorhizobium hupehensis* HWHS1 and inoculate it into YMA liquid medium (formulation: 1L of mannitol 10g, yeast extract 0.8g, dipotassium hydrogen phosphate 0.25g, potassium dihydrogen phosphate 0.25g, magnesium sulfate 0.2g, sodium chloride 0.1g), and culture it in a shaker at 28℃ and 200r / min for 7 days to obtain the seed culture.

[0099] (2) Primary fermentation: The seed liquid was inoculated into the above culture medium at a volume ratio of 3%, and cultured in a fermenter. The ventilation rate was controlled at 0.3 vvm, the stirring speed was 100 r / min, the initial dissolved oxygen was 100%, and the culture temperature was 28℃. The dissolved oxygen was maintained at no less than 30% throughout the process by adjusting the stirring speed.

[0100] (3) Transfer fermentation: After 3 days of cultivation, the OD of the fermentation broth at this time is 600When the bacterial culture reaches a value of 0.3-0.5 (corresponding to 2 / 3 of the logarithmic growth phase) after a 5-fold dilution, it is transferred to a secondary fermenter at a transfer volume ratio of 10%. After the transfer, it is cultured under the same conditions for another 5 days to obtain a high bacterial culture.

[0101] After the culture was completed, the viable bacterial count was determined by plate counting method and found to be 20.2 billion / mL, which reached the expected high bacterial count level.

[0102] 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.

[0103] 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 culture medium for increasing the viable count of *Neorhizobium huskum* HWHS1 in goat's bean, characterized in that, The culture medium comprises, per 1 L: Basic ingredients: 6-8g glucose, 12-16g sucrose, 3-5g yeast powder, 0.15-0.25g calcium chloride, 0.3-0.5g magnesium sulfate heptahydrate, 0.5-1g dipotassium hydrogen phosphate, 0.05-0.15g sodium chloride; Active ingredients: Dithiothreitol 0.2-1.5g, trace element solution 4-6mL, ferrous sulfate 0.0006-0.001g, disodium ketoglutarate 0.05-0.1g, citric acid 0.1-0.3g; The active ingredient is sterilized separately by filtration and added to the base ingredients before fermentation.

2. The culture medium according to claim 1, characterized in that, The amount of dithiothreitol added is 0.5-1 g / L.

3. The culture medium according to claim 1, characterized in that, The pH value of the culture medium is 6.3-6.8; the pH value refers to the pH value after the basic components are sterilized and before the activity-promoting components are added.

4. The culture medium according to claim 1, characterized in that, The weight ratio of carbon source to nitrogen source in the culture medium is (4.5-5.5):

1.

5. The culture medium according to claim 1, characterized in that, The trace element solution comprises 0.003-0.005 g / L of boric acid and 0.003-0.005 g / L of sodium molybdate.

6. A method for efficiently and stably culturing high-bacterial-count bacterial suspensions of *Neorhizobium huskum* HWHS1 from goat's bean, characterized in that... The method includes the following steps: (1) Primary fermentation: The seed liquid is inoculated into the culture medium described in any one of claims 1-5 at a volume ratio of 2-4%, and cultured in a fermenter, with the ventilation rate controlled at 0.25-0.35 vvm, the stirring speed at 90-110 r / min, the initial dissolved oxygen at 95-100%, and the culture temperature at 28-29℃; (2) Rotary fermentation: When the fermentation broth OD 600 When the bacterial culture reaches a concentration of 0.3-0.5 after a 5-fold dilution, it is transferred to another culture tank. The volume ratio of the transferred tanks is controlled at 5-10%. After the transfer, the culture is continued under the same conditions for 4-6 days to obtain a high bacterial count culture.

7. The method according to claim 6, characterized in that, The primary fermentation time is 2.5-3.5 days.

8. The method according to claim 6, characterized in that, After the culture was completed, the viable count of *Neorhizobium goatii* HWHS1 in the bacterial culture was stable at over 12 billion / mL.

Images