Production method of Bacillus coagulans spore powder

Abstract

This invention discloses a method for producing Bacillus coagulans spore powder, providing a high-concentration, high-quality spore powder production method for a heat-resistant, multifunctional, tea tree phosphate-solubilizing biocontrol Bacillus coagulans strain. This method can ferment and produce high-viable-spore, highly stress-resistant mature spore fermentation broth, high-viable-spore, centrifuged concentrated spore powder, and high-viable-spore, high-quality spore powder of strain PPX-13 (CGMCC No. 7.608). Preservation tests show that the PPX-13 high-viable-spore, high-quality spore powder product has a shelf life of one year when stored at low cost in a dark, room-temperature environment. Based on the established industrial-scale production process, it can be preliminarily estimated that each ton of high-viable-spore, highly stress-resistant mature spore fermentation broth can produce 8.7 kg of high-viable-spore, centrifuged concentrated spore powder and 392.81 kg of PPX-13 high-concentration, high-quality spore powder product, enabling large-scale industrial fermentation production of Bacillus coagulans spore powder.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and more specifically, to a method for producing Bacillus coagulans spore powder. Background Technology

[0002] my country has been a major tea-producing and tea-growing country since ancient times, producing related tea products. Fujian Province has long been the nation's largest producer of tea, tea varieties, and related products. Furthermore, Ningde City in eastern Fujian is the largest tea-producing city in Fujian and even the entire country, accounting for approximately one-third of Fujian's tea garden or farmland area and tea production. In recent years, due to increased demand for tea, tea garden areas have expanded continuously. Coupled with the long-standing practice of cultivating only a single tea variety in some tea-growing regions, this large-scale, monoculture cultivation method makes tea trees highly susceptible to insect and microbial diseases. Furthermore, extensive management of tea gardens or farmland and deteriorating growing environments have exacerbated tea tree diseases. On the other hand, it has also been found that soil acidification and mineralization in Chinese tea gardens are severe, with insufficient available phosphorus content.

[0003] Anthracnose is a very serious, frequent, and widespread disease affecting tea trees in my country, causing significant losses to tea cultivation and the tea industry every year. Anthracnose is a microbial disease, primarily caused by fungal pathogens. Therefore, the control of fungal pathogens in tea trees is crucial for the sustainable development of tea cultivation and tea enterprises, and has significant economic and market benefits. Currently, the main method for controlling plant fungal diseases is the application of chemical pesticides, combined with other agricultural measures for integrated pest management. However, this has led to the year-round use of chemical pesticides in tea gardens and farmland, resulting in increasingly severe pesticide residues and environmental pollution problems, while also contributing to increased pesticide resistance in pathogens. Therefore, there is a growing demand for more environmentally friendly and effective methods to inhibit various tea tree microbial pathogens. In recent years, with the deepening research on antagonistic microorganisms, the application of antagonistic microbial agents in plant disease control has received increasing public attention.

[0004] Based on current research, the following five agricultural, fermentation industry, environmental, food, and economic issues exist in the development and application of biological control of tea tree fungal diseases and related microbial microecological preparations, bio-fertilizers, and bio-pesticides: (1) the huge demand gap for fertilizers and pesticides after the ban on chemical fertilizers or pesticides; (2) the demand for a large number of high-efficiency, low-cost, long-shelf-life microecological preparations, bio-fertilizers, and bio-pesticides in rural construction; (3) the research and development of bioengineering and fermentation processes for high-efficiency, low-cost fermentation engineering technology for non-grain waste with huge business opportunities and output value; (4) the environmental protection and sustainable management issues of resource recycling and reuse of the huge amount of non-grain waste produced in my country; and (5) the issue of green organic agriculture or food industry with very high economic unit price and output value without the use of chemical fertilizers or pesticides.

[0005] To address the aforementioned bottlenecks and problems, it is imperative to immediately isolate and develop heat-resistant and multifunctional tea tree phosphate-solubilizing biocontrol microorganisms. These microorganisms possess high temperature resistance and multifunctionality, allowing for the microbial conversion of large quantities of non-grain agricultural waste into microbial preparations, biofertilizers, or biopesticides. They also exhibit mesophilic antagonistic activity against several anthracnose fungal pathogens affecting tea trees and can simultaneously enhance and improve soil fertility and the functional microbial community and distribution structure. Furthermore, the future application of these microorganisms in the development and industrial production of efficient, safe, low-cost, and long-shelf-life tea tree microbial microecological preparations, biofertilizers, or biopesticides presents enormous business opportunities and application potential in the organic tea industry. Summary of the Invention

[0006] The purpose of this invention is to provide a method for producing Bacillus coagulans spore powder.

[0007] In previous research, the inventors isolated and developed four highly efficient, safe, and easy-to-manufacture multifunctional tea tree phosphorus-solubilizing microorganisms from the rhizosphere soil of white tea or camellia oleifera from two test tea gardens in eastern Fujian (Pinpinxiang Guanyang White Tea Garden and Wanheng Luyuan Yangzhong Camellia oleifera Garden). Based on their diverse phosphorus-solubilizing activities (dissolving calcium phosphate, hydroxyapatite, aluminum phosphate, iron phosphate, and phosphate rock), phytase activity, and diverse enzyme activities (amylase, cellulose), the inventors further developed these microorganisms. The study compared the activity of two tea tree or one camellia anthracnose fungal isolates (among others: *Colletotrichum gloeosporioides* isolate N425 and ZN81, and *Colletotrichum gloeosporioides* isolate DH9) against mesothermal antagonism. Further, a thermotolerant, multifunctional tea tree phosphate-solubilizing biocontrol *Bacillus coagulans* isolate PPX-13 was selected and classified as *Bacillus coagulans*. Bacillus coagulansThe strain is now deposited at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, accession number CGMCC No. 7.608, deposit date September 8, 2025.

[0008] The isolated PPX-13 was then prepared as a liquid microecological agent and applied to the rhizosphere soil surface of tea trees in the Pinpinxiang Guanyang white tea garden as part of a field trial. The results showed that this bacterium reduced soil acidification by 7.75%, improved soil fertility (increasing organic matter by 9.95%, ammonium nitrogen by 8.88%, nitrate nitrogen by 14.92%, available phosphorus by 29.89%, and available potassium by 27.79%), increased total microbial community by 8.85%, and increased the proportions of actinomycetes, cellulose-decomposing, pectin-decomposing, chitin-decomposing, phosphorus-solubilizing, and nitrogen-fixing microbial communities and their respective percentages in the total microbial community structure by 9.21 and 8.98, 11.84 and 9.19, 12.39 and 9.43, 14.45 and 12.35, 29.85 and 24.71, and 13.15 and 10.59%, respectively. The percentage of mold and its proportion in the total microbial community decreased by 21.23% and 27.74%, respectively; the yield of first-spring tea buds increased by 25.69%. It is recommended to apply the treatment starting in mid-spring when the weather warms up, once every 3 weeks until early autumn, then once every 4-6 weeks until early spring of the following year. The application amount should be approximately 10g of soil covering a 30cm × 30cm square area around the base of the tea tree and a depth of 30cm from the surface. 4 -10 5 cell cm -3 Therefore, the heat-resistant, multifunctional tea tree phosphate-solubilizing Bacillus coagulans isolate PPX-13 has the activity of reducing soil acidification in the rhizosphere of tea trees, improving fertility, improving the microbial ecological environment, increasing the proportion of actinomycetes and functional microbial communities in the total microbial community, inhibiting the proportion of fungal communities in the total microbial community, and increasing the yield of spring tea. It has great potential for the development and industrial production of tea tree microorganisms or microbial biological control microecological preparations, biofertilizers or biopesticides.

[0009] Therefore, this invention will use the heat-resistant, multifunctional tea tree phosphate-solubilizing and bio-acid-inhibiting Bacillus PPX-13 isolate as the test and production target strain for the development of related microecological preparations, bio-fertilizers, or bio-pesticides for green tea planting and organic tea industry. It will also prioritize the development of its high viable spore count and long shelf-life spore powder as the primary product development target. This invention will elaborate on the basic physiology and growth and transspore culture conditions of the heat-resistant, multifunctional tea tree phosphate-solubilizing and bio-acid-inhibiting Bacillus PPX-13 isolate; the formulation of the basic fermentation transspore culture medium and its adjustment to the optimal growth and transspore culture medium formulation; the setting of basic fermentation conditions and their optimization and improvement to the optimal growth and transspore fermentation conditions; the formulation of the production mode and process for high-concentration, long-shelf-life concentrated spore powder and its industrial fermentation and subsequent processing and excipient preparation; and the product development, preservation, and quality assurance tests of the high viable spore count and long-shelf-life spore powder, as well as the formulation of its future commercial specifications. Finally, referring to the industrial mass production systems and equipment commonly used in current microbial fermentation plants, including fermentation, centrifugal concentration of spores and cells, preparation and excipient formation of concentrated spore and cell solids re-vibration suspension, vacuum heating drying and powder preparation, and dilution of concentrated spore powder into spore powder products with high viable spore counts and long shelf life at room temperature, we will establish a standard process and model for the industrial mass production of ton-scale fermentation, transspore fermentation broth, and subsequent processing and excipient preparation. This will enable future microbial fermentation plants to actually mass-produce the high-temperature resistant, multifunctional tea tree phosphate-solubilizing and anticoagulant Bacillus PPX-13 spore powder product, thereby gradually achieving the goal of banning chemical fertilizers and pesticides and the practice of green organic agriculture and sustainable management.

[0010] To achieve the objective of this invention, in a first aspect, this invention provides a method for preparing Bacillus coagulans spore fermentation broth, comprising the following steps: (1) Microaerobic fermentation culture stage: Bacillus coagulans is cultured under conditions of no feeding and no aeration and no additional pressurization in order to achieve the proliferation of vegetative somatic cells; (2) Aerobic transspore stage: When the pH value of the fermentation broth in step (1) stops decreasing and begins to rise, switch to the fermentation culture conditions of feeding, aeration and pressurization to induce the formation of spores by the vegetative cells.

[0011] Preferably, the process switches from step (1) to step (2) after fermentation has been going on for 16-20 hours.

[0012] The common fermentation conditions for steps (1) and (2) are: 20-50 ℃ (preferably 40 ℃), initial pH of the culture medium 5.5-8.0 (preferably pH 6.5), and 100-150 rpm (preferably 150 rpm). The fermentation parameters can be adjusted appropriately according to the fermentation system and equipment used in fermentation production.

[0013] Preferably, the feeding and aeration rate in step (2) is 0.5-1 vvm (preferably 0.5 vvm), and the pressurization pressure is 0-0.1 MPa (preferably 0.1 MPa). The appropriate fermentation parameters can be adjusted according to the fermentation system and equipment during fermentation production.

[0014] Furthermore, the fermentation medium used in steps (1) and (2) comprises: glucose, ammonium chloride, sodium chloride, yeast extract, dipotassium hydrogen phosphate and magnesium sulfate.

[0015] Preferably, the fermentation medium used is: 1 g / L glucose, 10 g / L ammonium chloride, 5 g / L sodium chloride, 10 g / L industrial-grade yeast extract, 2 g / L dipotassium hydrogen phosphate and 0.1 g / L magnesium sulfate.

[0016] Secondly, the present invention provides a method for producing Bacillus coagulans spore powder, the method comprising: (a) Producing spore fermentation broth using the above method; (b) The spore fermentation broth is post-treated to obtain Bacillus coagulans spore powder.

[0017] Further, the post-processing of step (b) includes: centrifuging to collect the cells and spores, mixing with the excipient, and drying until the moisture content of the powder is less than 10% (w / w).

[0018] Preferably, the excipient is silicon dioxide.

[0019] Preferably, the Bacillus coagulans strain is PPX-13, with the preservation number CGMCC No. 7.608.

[0020] Thirdly, the present invention provides Bacillus coagulans spore powder prepared according to the method, wherein the effective number of surviving spores in the powder after being treated in a water bath at 100 °C for 10 minutes is greater than or equal to 2.0 × 10⁻⁶. 9 Effective live spores / g.

[0021] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention provides a method for producing high-concentration, high-quality spore powder of a heat-resistant, multifunctional tea tree phosphate-solubilizing and biocontrolling Bacillus coagulans. This method can ferment and produce a high-viable-spore, highly stress-resistant mature spore fermentation broth of the tested heat-resistant, multifunctional tea tree phosphate-solubilizing and biocontrolling Bacillus coagulans isolate PPX-13 (total viable spore count ≥ 2 × 10⁻⁶ at 100 °C). 9 Viable spores / mL and translocation rate ≥ 95%), high viable mature spore count centrifuged concentrated spore powder (total effective viable spore count ≥ 8 × 10⁻⁶ at 100℃). 10Effective live spores / g and moisture <10%) and high-quality spore powder with high live spore count (total effective live spore count ≥ 2 × 10⁻⁶ at 100℃). 9 With effective live spores / g and moisture content <10%, this invention names it PPX-13 high-concentration, high-quality spore powder. Preservation tests show that the PPX-13 high-concentration, high-quality spore powder product specification is a total effective live spore count ≥ 1 × 10⁻⁶ at 100℃. 9 With effective live spores / g and moisture content <10%, it can be stored at low cost in a dark, room-temperature environment with a shelf life of 1 year. Based on the established industrial-scale production process, it can be pre-assessed that each ton of fermentation broth with high live spore count and high stress resistance can produce 8.7 kg of centrifuged concentrated spore powder with high live mature spore count and 392.81 kg of high-concentration, high-quality PPX-13 spore powder product. Attached Figure Description

[0022] Figure 1 In a preferred embodiment of the present invention, the changes and development of microbial cell and spore morphology in the fermentation broth produced by the high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulans isolate PPX-13 in a 7 L small fermenter with 5 L of high viable spore count and high stress resistance mature spore fermentation broth were studied during the pilot-scale production process. This was followed by continuous 10-fold dilution. Bacillus racemilaticus medium The identification and counting of viable spores of isolate PPX-13 were performed using the plate counting method (BR medium) (40 ℃, culture for 5 days). (a) Microscopic examination of vegetative cells (1,000 × oil immersion) of the fermentation broth produced by PPX-13 during the first stage of fermentation transsporulation (microaerobic vegetative cell proliferation culture stage, fermentation hours 16-20); (b) Microscopic examination (1,000 × oil immersion) of the fermentation broth produced by PPX-13 during the second stage of fermentation transsporulation (aerobic culture, starvation and hyperaerobic stress transsporulation culture stage, fermentation hours 16-20 to 96); (c) Colonies of mature viable spores growing in the transsporulation broth of PPX-13 after being treated in a 100 °C water bath for 10 minutes, detected by the BR plate count method with serial 10-fold dilutions; (d) The phosphate-solubilizing transparent zone (indicated by the yellow arrow) of colonies growing from mature viable spores in transspore fermentation broth treated at 100 °C for 10 minutes, detected by the BR plate count method with serial 10-fold dilutions of isolate PPX-13.

[0023] Figure 2In a preferred embodiment of this invention, the high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulant isolate PPX-13 was fermented in a 7 L small fermenter to produce 5 L of high-viable-spore, highly stress-resistant mature spore fermentation broth, and subsequently processed with SiO2 to prepare a high-viable-spore, highly stress-resistant mature spore powder product (named PPX-13 high-concentration, high-quality spore powder, with a total viable spore count resistant to 100 °C of 2.13 ± 0.16 × 10⁻⁶). 9 Effective live spores / g, moisture content 2.90±0.33% (w / w)).

[0024] Figure 3 This is a pre-set mode and flowchart of the industrial mass production process of high-concentration, high-quality spore powder of high-temperature resistant, multifunctional tea tree phosphate-solubilizing and biocontrolling Bacillus coagulans isolate PPX-13, which is a preferred embodiment of the present invention. Detailed Implementation

[0025] This invention aims to overcome the deficiencies in current organic tea industry, green tea tree cultivation, and the lack of suitable multifunctional tea anthracnose biocontrol microecological agents, biofertilizers (especially biophosphate fertilizers), and biopesticides for use in tea gardens in eastern Fujian. It utilizes a heat-resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulans isolate PPX-13 (CGMCC No. 7.608), which can inhibit the fungal pathogens causing anthracnose in several tea trees or Camellia oleifera, and enhance the fertility of the rhizosphere soil, as well as the presence of actinomycetes and functional microbial communities and their proportion in the total microbial community structure. This isolate is intended as a test production strain for various tea anthracnose biocontrol microecological agents, biofertilizers, and biopesticides. Furthermore, it provides a spore fermentation broth (≥ 1 × 10⁻⁶) that allows PPX-13 to undergo fermentation and effective transspore formation (transformation rate ≥ 90%) using a fermenter production system and auxiliary equipment, thereby producing a high-viable mature spore count. 9 The fermentation production mode is based on live spores (mL); and this spore fermentation broth is used as a basis for efficient, time-saving and low-cost processing to produce spore powder with high live spore count and long shelf life at room temperature (1 × 10⁻⁶). 9 -2 × 10 9 Effective live spores / g, moisture content <10%, w / w).

[0026] The present invention adopts the following technical solution: This invention provides a high-concentration, highly stress-resistant, long-shelf-life centrifuged concentrated spore powder of *Bacillus porphyrinus* isolate PPX-13, a thermophilic and multifunctional biocontrol agent for tea tree anthracnose, and a biocontrol agent for tea tree anthracnose. It also outlines the industrial fermentation and subsequent processing methods for its production, the development of high-concentration, high-quality spore powder of PPX-13, and the formulation of future commercial specifications. Specifically, it provides a production process and technology for industrially fermenting and mass-producing spore powder of PPX-13 containing high viable spore counts, high stress resistance, and a long shelf life at room temperature.

[0027] This invention provides a method for producing high-concentration, high-quality spore powder of Bacillus subtilis, a high-temperature resistant, multifunctional, phosphorus-soluble, bio-anti-coagulant fungus from tea trees.

[0028] The present invention also provides the production of fermentation broth containing high viable spore count and high stress resistance mature spores of the heat-resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulans isolate PPX-13, centrifuged concentrated spore powder with high viable spore count, and high-quality spore powder with high viable spore count.

[0029] The present invention also provides a fermentation transspore mode containing the fermentation broth for producing mature spores with high viable spore count and high stress resistance.

[0030] The present invention also provides a fermentation transspore culture medium formula containing the fermentation broth for producing high viable spore count and high stress resistance mature spores (each liter of culture medium contains: 1 g glucose, 10 g ammonium chloride, 5 g sodium chloride, 10 g industrial-grade yeast extract, 2 g dipotassium hydrogen phosphate, and 0.1 g magnesium sulfate).

[0031] The present invention also provides fermentation transspore conditions for a fermentation transspore production mode containing fermentation broth with high viable spore count and high stress resistance to mature spores: (1) Stage 1: from 0 to 16-20 hours (when the pH of the fermentation broth no longer decreases and shows an upward trend), inoculum volume 5% (v / v), 40 ℃, 150 rpm, 0 vvm, 0 MPa, pH not controlled; (2) Stage 2 (from 16-20 hours to 120 hours), 40 ℃, 150 rpm, 0.5 vvm, 0.1 MPa, pH not controlled.

[0032] 1. Study on the basic physiology, growth and transspore culture conditions of PPX-13, a high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulant isolate. To design and formulate the fermentation conditions and production process for producing bacterial cells or spores from the heat-resistant, multifunctional tea tree phosphate-solubilizing and anticoagulant Bacillus isolate PPX-13 using a fermenter, fermentation system, and auxiliary equipment, the basic physiology, growth, and transspore culture conditions of this strain were investigated. The materials and methods are as follows: (1) The optimal temperature range for growth and phosphorus-solubilizing activity of isolate PPX-13, and the determination of its optimal temperature for growth and phosphorus-solubilizing activity. Bacillus racemilaticus The plate assay was performed using medium (BR medium) (after 5 days of incubation). The incubation temperatures were 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 ℃. On the 2nd and 5th days of incubation, the colony diameter (I) and the diameter of the phosphate-solubilizing transparent zone (II) were measured, and the phosphate-solubilizing activity parameter (II / I) was calculated. (2) The initial pH range of the liquid culture medium suitable for the growth of the test strains and the initial pH of the liquid culture medium for optimal growth and transspore formation were tested using the Luria-Bertan medium (LB medium) method. The experimental method is as follows: (a) Inoculate 1 mL of PPX-13 isolate at -80 ℃ with glycerol culture medium in 100 mL of MRS liquid medium and culture at 150 rpm and 40 ℃ for 48 hours, which is recorded as PPX-13 culture medium; (b) Inoculate 5 mL of PPX-13 culture medium in 100 mL of LB liquid medium (contained in a 250 mL shake flask, inoculation amount 5% (v / v)), and then culture in a shake flask at 150 rpm and 40 ℃ for 3 days. Take 10 mL of culture medium every day to detect the total number of viable PPX-13 isolates and the total number of viable spores of PPX-13 isolates at 80 ℃. The viable count of total PPX-13 isolates was determined using the BR plate count method with consecutive 10-fold dilutions (incubated at 40 °C for 5 days), counting the number of colonies with a clear phosphate-solubilizing zone per mL of culture medium. The viable spore count of total PPX-13 isolates at 80 °C was determined using the BR plate count method with consecutive 10-fold dilutions (incubated at 40 °C for 5 days), counting the number of colonies with a clear phosphate-solubilizing zone per mL of culture medium treated at 80 °C for 10 minutes.

[0033] The results are shown in Tables 1-3, indicating the basic physiological and cultural characteristics of the heat-resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulans isolate PPX-13, including growth, phosphate solubilization, transsporination, and transsporination rate: (1) The optimal culture temperature for growth and phosphorus-solubilizing activity is between 20 ℃ and 50 ℃, with the optimum temperature being 40 ℃ (Table 1). Growth and phosphorus-solubilizing activity are significantly impaired at temperatures below 15 ℃ or above 55 ℃. P<0.05) inhibition (Table 1); (2) Based on the optimal growth and phosphorus solubility culture temperature of 40 °C, it was found that after 24 hours of culture in LB liquid medium, the isolate PPX-13 could only grow vegetative cells and no transspores occurred. It was not until 48 hours of culture that vegetative cells successfully transspored into live spores that could withstand 80 °C (Table 2).

[0034] (3) In addition, the initial pH of the liquid culture medium suitable for growth and transsporulation is between 5.5-8.0 and 6.0-7.5, respectively, while the initial pH of the LB liquid culture medium optimal for both growth and transsporulation is 6.5. (Under this initial pH of LB liquid culture medium, the highest total viable count, total number of viable spores resistant to 80 ℃, and transsporulation rate can reach 9.59±1.12 × 10⁻⁶, respectively.) 8 CFU / mL (after 48 hours of incubation), 2.49 ± 0.28 × 10⁻⁶ 7 Live spore CFU / mL (72-hour culture) and 69.90±7.71% (72-hour culture) (Tables 2 and 3). When the initial pH of LB liquid medium was below 5.5 or above 9.0, it significantly reduced the growth, transspore size, and transspore rate of isolate PPX-13. P <0.05) was inhibited, indicating that pH has a great influence on its growth and transspore activity (Tables 2 and 3).

[0035] In summary, the above results indicate that LB medium can culture the vegetative somatic cells of isolate PPX-13 and transsporinate them into 80℃-resistant spores, with the optimal culture temperature and initial pH of the medium being 40℃ and 6.5, respectively. However, under these conditions, the highest total viable cell count, total number of 80℃-resistant viable spores, and transsporination rate of PPX-13 isolate in LB liquid medium were only 9.59 ± 1.12 × 10⁻⁶. 8 CFU / mL, 2.49±0.28 × 10 7 The viable spore count was 69.90±7.71% per mL. However, in currently available commercially available bacterial spore powders, both domestically and globally, the total viable spore count is ≥ 1 × 10⁻⁶. 9 The viable spore count / g indicates that the above fermentation conditions are insufficient for the industrial-scale production of PPX-13 spore powder with high viable spore counts (Table 2). Currently, for the industrial-scale fermentation production of spore powder from sporulating bacteria, to reduce production and processing costs and extend shelf life at room temperature, the number of viable mature spores with strong environmental resistance per mL of transspore fermentation broth must be ≥ 1 × 10⁻⁶. 9 Only by meeting the minimum fermentation production quality standards can subsequent product quality be effectively and cost-effectively maintained at ≥ 1 × 10⁻⁶. 9Processing and preparation of live spores / g spore powder. Therefore, this invention selects LB medium as the basal medium and adjusts the nitrogen source replacement and concentration, as well as add K2HPO4 and magnesium ions (i.e., various modified LB media) on the fermentation production of vegetative somatic cells and subsequent transspore formation of mature spores from isolate PPX-13. Using an inoculum size of 5% (v / v), a culture temperature of 40 ℃, a stirring speed of 150 rpm, and an initial pH of 6.5 in the liquid medium as the basal fermentation conditions, a small-scale laboratory 2 L fermenter was used to investigate the effects of different modified LB medium formulations, aeration rates, pH control, and oxygen stress on the vegetative somatic cell production and subsequent transspore formation of mature spores from isolate PPX-13. Furthermore, a target for PPX-13 fermentation transspore production ≥ 1 × 10⁻⁶ g of mature spores was established. 9 A suitable and low-cost fermentation transspore medium and fermentation production mode for fermentation broth with live spores / mL of mature spores.

[0036] 2. Formulation of fermentation transsporation medium for PPX-13 basic isolate and adjustment of optimal growth and transsporation medium formulation. The formulation of the fermentation transsporation medium for the basic isolate PPX-13 is shown in Table 4. The primary consideration was to use low-cost and readily available fermentation raw materials or reagents that could be applied to the industrial-scale production or preparation of PPX-13-related microbial fermentation or biotechnology products. Based on LB medium, modified LB medium-1 to LB medium-4 formulations were developed and adjusted as follows: (1) Modified LB medium-1 replaces the expensive peptone in LB medium with the same amount (1%, w / v) of inexpensive inorganic nitrogen source ammonium sulfate, and replaces the expensive yeast extract (5%, w / v) in LB medium with twice the amount (1%, w / v) of inexpensive commercially available industrial-grade yeast extract. Since the moisture content of commercially available industrial-grade yeast extract that can be purchased in large quantities is about 50% (w / w), the amount of yeast extract is increased by 2 times to achieve a more comprehensive balance of nutritional requirements for growth and transsporulation; (2) Modified LB medium-2 is based on modified LB medium-1, but with the addition of 0.2% (w / v) dipotassium hydrogen phosphate and 0.01% (w / w) magnesium sulfate. The reason for adding this phosphorus source and magnesium trace element is based on the fermentation transsporin medium formula of Bacillus coagulans BCRC11592 published in the paper (Study on the stress resistance of a strain of Bacillus coagulans and optimization of its sporulation conditions) by Yu Yue et al. [J]. Feed Industry, 2023, 34(7): 43-47; (3) Modified LB medium-3 is based on modified LB medium-2, replacing the ammonium sulfate in LB medium-2 with ammonium chloride, an inexpensive inorganic nitrogen source with a higher nitrogen content than ammonium sulfate, at the same content (1%, w / v). (4) Modified LB medium-4 is based on modified LB medium-3, but with the addition of 0.01% (v / v) commercially available polyether defoamer to solve the problem of foaming of fermentation liquid caused by aeration during fermentation. At the same time, it is also explored whether the defoamer will affect the growth of isolated PPX-13 cells and the transspore and development of mature spores with high stress resistance.

[0037] 3. Establishment of fermentation and transspore conditions for the basic isolate PPX-13 and optimization and improvement of suitable high-density growth and high-concentration, stress-resistant mature spore transspore fermentation production conditions. The fermentation conditions for testing the sporulation effect of the heat-resistant, multifunctional tea tree phosphate-solubilizing anticoagulant Bacillus isolate PPX-13 are shown in Table 5. The fermentation parameters were designed and implemented based on the fundamental safe operation settings and usage limits of commonly used and cost-effective industrial-scale fermenters and their auxiliary equipment and systems for producing bacterial cells and live spores at the ton-scale. In conjunction with previous studies on the optimal growth temperature and initial pH of the culture medium for isolate PPX-13 (as described in "1. Study on the basic physiology, growth, and transsporulation culture conditions of the heat-resistant, multifunctional tea tree phosphate-solubilizing anticoagulant Bacillus isolate PPX-13"), the following seven fermentation and transsporulation conditions were set, as detailed below: (1) Fermentation transspore conditions-1 is the aerobic fermentation transspore mode that is commonly used in the current industrial fermentation industry for the production of aerobic Bacillus cells and live spores. (2) Fermentation transsporulation conditions-2 are based on fermentation transsporulation conditions-1, but with the addition of controlling the pH to be constant at 6.5 during fermentation. This is because the initial pH of the medium in which the isolate PPX-13 exhibits optimal growth and transsporulation activity is 6.5. Therefore, the pH is controlled to be constant at 6.5 during fermentation (Tables 2 and 3). Furthermore, 40% (w / v) high-concentration calcium carbonate is used to neutralize the acid produced by the isolate PPX-13 during fermentation. This is because the isolate PPX-13 exhibits optimal growth and phosphorus-solubilizing activity when cultured at 40 °C. Therefore, calcium carbonate is used to neutralize the organic acids produced by the isolate PPX-13 during fermentation. (3) Fermentation transspore condition-3 is based on fermentation transspore condition-2, but the organic acids produced by the isolate PPX-13 during fermentation are neutralized with 1 N NaOH solution to explore the effects of adding different alkaline solutions and neutralizing organic acids on the fermentation growth and transspore formation of the isolate PPX-13. (4) Fermentation transsporation condition-4 is based on fermentation transsporation condition-1, but the tank pressure is set to 0.1 MPa instead of 0 MPa in fermentation transsporation condition-1, in order to explore the effects of tank pressure and increased dissolved oxygen on the growth and transsporation of vegetative cells of isolate PPX-13. (5) Fermentation transsporation condition-5 is also based on fermentation transsporation condition-1, but the aeration rate is reduced to 0 vvm to explore the effect of microaerobic fermentation without aeration on the growth and transsporation of vegetative cells of isolate PPX-13. (6) Fermentation transsporation conditions-6 Based on fermentation transsporation conditions-1 and 5, a two-stage fermentation transsporation fermentation mode was innovatively established. (I) The first stage is approximately between 0 and 48 hours of fermentation, which is a microaerobic fermentation culture with no aeration (0 vvm) for high-density vegetative somatic cell proliferation fermentation culture of isolate PPX-13. After 48 hours of fermentation, the second stage begins. (II) The second stage is approximately between 49 hours of fermentation and 120 hours after tank closure, which is an aerobic fermentation culture stage with an aeration rate of 0.5 vvm and a tank pressure of 0.1 MPa. This also innovatively establishes a transsporation fermentation culture mode that promotes transsporation fermentation under starvation and high-oxygen stress. (7) Fermentation Transsporation Conditions-7 are based on Fermentation Transsporation Conditions-6, and innovatively improve the two-stage fermentation transsporation fermentation mode. (I) The first stage is about 0 to 16-20 hours of fermentation. It is a microaerobic fermentation culture with no aeration (0 vvm) for high-density vegetative somatic cell proliferation fermentation culture of isolate PPX-13. It can be stopped when the pH of the fermentation broth no longer decreases and shows an upward trend. This indicates that isolate PPX-13 has almost exhausted all carbon sources and has begun to metabolize nitrogenous substances to produce alkaline metabolites, which causes the pH to rise. It is about to enter the end of the rapid growth period or the stable growth period of no longer increasing vegetative somatic cells, and immediately enters the second stage. (II) The second stage is about 16-20 hours of fermentation to 120 hours after tank collection. It is an aerobic fermentation culture stage with an aeration rate of 0.5 vvm and a tank pressure of 0.1 MPa to increase the dissolved oxygen of the fermentation broth. This is an innovative transsporation fermentation culture mode established by the present invention to promote transsporation fermentation culture with starvation and high oxygen stress. During the second stage, the fermentation broth was taken periodically, and the morphology of the PPX-13 isolate was observed under an oil immersion microscope at 1,000x magnification. The total number of cells and the number of relatively mature individual spores that had detached from the vegetative cells were counted until the majority of the microscopic field of view consisted of "relatively mature individual spores that had detached from the vegetative cells", and the number of these individual spores accounted for ≥ 90% of the total number of cells. Only when these two replicate results appeared at different stages of the overall fermentation process could the fermentation be terminated and the fermentation tank collected (Table 5).

[0038] Therefore, this invention uses LB medium or four modified LB mediums individually combined with seven different transspore fermentation conditions to compare and explore the effects of fermentation growth of isolate PPX-13, transspore formation of live spores, and transspore rate.

[0039] 4. Fermentation and transsporination tests of the heat-resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulans isolate PPX-13 under different test media and fermentation conditions. In the current industrial-scale fermentation production of spore powder from sporogenic bacteria, the minimum fermentation production quality of the fermentation broth containing the number of viable spores is ≥ 1 × 10⁻⁶. 9 A high concentration of live spores per mL is essential for the subsequent processing of the spore powder and for the industrial production of high-concentration live spore powder products. Therefore, in this experiment, if the highest total viable count per mL of transspore fermentation broth is <1 × 10⁻⁶, the desired outcome is achieved. 9 CFU or maximum total viable spore count to withstand 80℃ <1×10⁻⁶ 9 Live spores indicate that the tested culture medium and the fermentation conditions used in conjunction with it are unsuitable for industrial-scale production of transspore fermentation broth for isolate PPX-13, and are therefore considered a failed industrial-scale transspore fermentation test of isolate PPX-13.

[0040] Therefore, in this invention, LB medium and four self-developed modified LB mediums were used as test media (Table 4). Seven self-designed fermentation transsporulation conditions (set with reference to the industrial-grade fermentation equipment, systems, and fermentation parameters used in industrial-scale fermentation production of spore powders of general spore-producing bacteria, and the basic microbial physiological characteristics of isolate PPX-13) were used as test fermentation conditions (Table 5). A 7 L small fermenter (7 L glass fermenter (Biotech-70JG-7000, Shanghai Baoxing Bio-Equipment Engineering Co., Ltd.)) was used to test the fermentation transsporulation of 2 L of isolate PPX-13. The total viable cell count and the total number of viable spores resistant to 80 °C in the transsporulation broth were used as indicators to determine the fermentation transsporulation efficiency. The detection methods for the total viable cell count and the total number of viable spores resistant to 80 °C were as described above in "1. Study on the basic physiology, growth, and transsporulation culture conditions of the high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus PPX-13 isolate".

[0041] This invention uses five test culture media (Table 4) and seven test fermentation transsporation conditions (Table 5) to conduct fermentation transsporation experiments on 11 batches of isolate PPX-13. During the fermentation transsporation process, the total viable cell count and the total number of viable spores resistant to 80 °C in the produced transsporation broth must be ≥ 1 × 10⁻⁶. 9 CFU / mL and ≥ 1 × 10 9The live spores / mL ratio is the minimum acceptable standard for successful fermentation transspore formation. If any indicator fails to meet the acceptable standard, the process is considered a failure. The results are shown in Table 6, as detailed below. (1) The first batch was carried out using "LB medium (Table 4) + fermentation transsporulation conditions-1 (Table 5)," which is one of the common aerobic transsporulation fermentation modes for aerobic spore-forming bacteria. The highest total viable count and total number of viable spores resistant to 80 °C were found to occur at 48 and 72 hours of fermentation transsporulation, respectively, but these were only 7.48 ± 0.34 × 10⁻⁶. 8 CFU / mL and 4.18±0.44 × 10 6 The number of live spores / mL did not reach the total live bacteria count ≥ 1 × 10⁻⁶ set by this invention. 9 CFU / mL and total viable spore count ≥1×10⁻⁶ at 80℃ 9 The production quality standard for live spores / mL was not met, therefore it was deemed unqualified; furthermore, the total viable count of the fermentation broth at 72 hours after transspore formation was <1 × 10⁻⁶. 9 Therefore, after 72 hours of transspore fermentation, the total number of viable spores resistant to 80°C produced by the fermentation broth will not exceed the total number of viable spores resistant to 80°C ≥ 1 × 10⁻⁶ set in this invention. 9 The production quality standard of live spores / mL was therefore deemed a failure, and all experiments at fermentation transsporation hours 96 and 120 were discontinued. This result also indicates that LB medium and the general aerobic transsporation fermentation mode for aerobic Bacillus sporogenes are not suitable for high-density growth and transsporination of isolate PPX-13. (2) The second batch was carried out using "LB medium (Table 4) + fermentation transsporulation conditions-2 (Table 5)," which is one of the current common aerobic fermentation modes for aerobic acid-producing and spore-forming Bacillus transsporulation using a high concentration of calcium carbonate (40%, w / v) to maintain a constant pH of 6.5. The effects of this mode on the fermentation growth and transsporulation of isolate PPX-13 were investigated. The highest total viable count and the highest number of viable spores resistant to 80 °C both occurred at 48 hours of fermentation transsporulation, but were only 8.73 ± 0.94 × 10⁻⁶, respectively. 8 CFU / mL and <1 × 10 5 The number of live spores / mL was deemed unqualified, indicating that LB medium, constant pH control at 6.5 (using high concentration of calcium carbonate (40%, w / v)), and general aerobic acidogenic spore-forming bacteria transspore fermentation mode are not suitable for high-density growth and transspore formation of isolate PPX-13. (3) The third batch was carried out using "LB medium (Table 4) + fermentation transsporulation conditions-3 (Table 5)," which is one of the common aerobic fermentation modes for aerobic acid-producing and spore-forming Bacillus transsporulation, with pH controlled at a constant 6.5 using 1 N NaOH solution. The effects of this mode on the fermentation growth and transsporulation of isolate PPX-13 were investigated. The highest total viable cell count and the total number of viable spores resistant to 80 °C both occurred at 48 hours of fermentation transsporulation, but were only 6.43 ± 0.81 × 10⁻⁶, respectively. 7 CFU / mL and <1 × 10 5 The number of live spores per mL was deemed insufficient, indicating that LB medium, a constant pH of 6.5 (using 1 N NaOH solution), and the typical aerobic acid-producing and spore-forming Bacillus transsporination fermentation method are not suitable for the high-density growth and transsporination of isolate PPX-13. Furthermore, the highest total viable count was as low as 6.43 ± 0.81 × 10⁻⁶. 7 CFU / mL. This result also indicates that, compared to weakly alkaline calcium carbonate, which has a milder effect on microbial growth and transsporulation activity, the addition of strongly alkaline NaOH has a significant effect. P <0.05) inhibited the fermentation growth and transspore formation of isolate PPX-13; (4) The fourth batch was carried out using "LB medium (Table 4) + fermentation transsporulation conditions-4 (Table 5)," which is one of the common aerobic transsporulation fermentation modes for aerobic spore-forming bacteria. Compared with fermentation transsporulation condition-1, fermentation transsporulation condition-4 increased the tank pressure setting from 0 MPa to 0.1 MPa to increase the dissolved oxygen content in the transsporulation fermentation broth. The results showed that the overall fermentation growth and physiological activity curves of the isolate PPX-13 were very similar to those of the first batch, but the highest total viable count and the total number of viable spores resistant to 80 ℃ were 6.67 ± 0.70 × 10⁻⁶. 8 CFU / mL and 6.37±0.44 × 10 6 The viable spores / mL were all below the production quality standards set in this invention, and therefore were deemed unqualified. This also indicates that LB medium, 0.1 MPa tank pressure, and the general aerobic transsporation fermentation mode for aerobic spore-forming bacteria are not suitable for the high-density growth and transsporination of isolate PPX-13. In addition, compared with the first batch of transsporation fermentation at 0 MPa tank pressure, the 0.1 MPa tank pressure environment reduced the total viable count of isolate PPX-13, but increased its total number of viable spores resistant to 80 °C and the transsporination rate. It can be seen that oxygen is an abiotic stress for isolate PPX-13, inhibiting its growth, but also promoting its transsporination because oxygen is an abiotic stress that inhibits the growth of isolate PPX-13. (5) The fifth batch was conducted using "Modified LB Medium-1 (Table 4) + Fermentation Transsporation Conditions-4 (Table 5)". Based on the experimental results of batches 1-4 above, it was found that (I) the total viable count of isolate PPX-13 fermented on LB medium could not reach ≥1 × 10⁻⁶. 9 CFU / mL, and the peptone and yeast extract in LB medium are expensive fermentation raw materials, which is not conducive to saving and reducing the overall production cost. They must be replaced with cheaper raw materials that are generally available in large quantities on the market. Therefore, this invention replaces the expensive peptone in LB medium with an inorganic nitrogen source with the same content (1%, w / v) and a higher nitrogen content than peptone - ammonium sulfate; and replaces the expensive yeast extract in LB medium (5%, w / v) with twice the content (1%, w / v) of cheap commercially available industrial-grade yeast extract, because the moisture content of commercially available industrial-grade yeast extract that can be purchased in large quantities is about 50% (w / w); (II) Adjusting the pH to a constant 6.5 and increasing dissolved oxygen cannot increase the total viable number of isolate PPX-13, and may even have an inhibitory effect. However, increasing dissolved oxygen is an oxygen stress for isolate PPX-13, which can increase its total number of viable spores resistant to 80 °C and transspore rate. Therefore, this batch used modified LB medium-1 (Table 4) + fermentation transsporulation conditions-4 (Table 5) for the transsporulation experiment of isolate PPX-13 to investigate the effects of nitrogen source adjustment and modification of the medium and dissolved oxygen stress on the fermentation transsporulation of isolate PPX-13. The results indicated that the highest total viable count still appeared at 48 hours of fermentation transsporulation, and had significantly increased ( P The value was increased from <0.05 to 1.01±0.14 × 10⁻⁵. 9 The total viable count of the transspore fermentation broth has reached ≥ 1 × 10⁻⁶ CFU / mL, as specified in this invention. 9 The production quality standard is CFU / mL. Furthermore, the total number of viable spores resistant to 80 °C gradually increases with the fermentation and transspore formation time, reaching 2.84 ± 0.32 × 10⁻⁶ at 72 hours. 7 Live spores / mL, but the total viable bacterial count had already decreased to 3.15 ± 0.39 × 10⁻⁶. 7 Therefore, after 72 hours of transspore fermentation, the total number of viable spores resistant to 80℃ produced by the fermentation broth will not exceed the total number of viable spores resistant to 80℃ set in this invention as ≥ 1 × 10⁻⁶. 9The production quality standard of live spores / mL was therefore deemed a failure, and no further experiments were conducted at fermentation transsporation hours 96 and 120. However, this result also indicates that, compared to peptone, the inorganic nitrogen source ammonium sulfate, with its higher nitrogen content, increases the nitrogen content of the transsporation fermentation broth, directly increasing the growth of vegetative cells of isolate PPX-13 and subsequent transsporation activity. This suggests that increasing the nitrogen content in the fermentation broth enhances the growth and transsporation of isolate PPX-13. (6) The 6th batch was carried out using “Modified LB medium-2 (Table 4) + fermentation transsporulation conditions-4 (Table 5)”. Based on the formulation of fermentation transsporulation medium for Bacillus coagulans BCRC11592 published in the paper by Yu Yue et al. (Study on the stress resistance of a strain of Bacillus coagulans and optimization of its sporulation conditions [J]. Feed Industry, 2023, 34(7): 43-47), Modified LB medium-2 was designed by adding 0.2% (w / v) dipotassium hydrogen phosphate and 0.01% (w / w) magnesium sulfate to supplement the phosphorus and magnesium ions reduced by the replacement of peptone with ammonium sulfate. The results showed that the overall fermentation growth and transsporation physiological activity curves of isolate PPX-13 were very similar to those of batch 5, and the total viable cell count and the total number of viable spores resistant to 80 °C were also slightly higher than those of batch 5. This indicates that the addition of 0.2% (w / v) dipotassium hydrogen phosphate and 0.01% (w / w) magnesium sulfate can improve the growth and transsporation activity of isolate PPX-13. Therefore, these were listed as the basic components and dosage formulations of its fermentation transsporation medium. However, the highest total number of viable spores resistant to 80 °C was only 5.14 ± 0.60 × 10⁻⁶. 7 The viable spores / mL (appearing at 72 hours of fermentation culture) still cannot reach ≥ 1 × 10⁻⁶. 9 The production quality standard for live spores / mL was therefore determined to be a failed and unqualified test for this batch. (7) Batch 7 was conducted using “Modified LB Medium-3 (Table 4) + Fermentation Transsporation Conditions-4 (Table 5)”. Based on the results of batch 5, increasing the nitrogen content in the fermentation broth improved the growth and transsporation of isolate PPX-13. Therefore, Modified LB Medium-3 was based on Modified LB Medium-2, using the same amount (1%, w / v) of inexpensive inorganic nitrogen source—ammonium chloride—which has a higher nitrogen content than ammonium sulfate, to replace the ammonium sulfate in LB Medium-2. This further increased the nitrogen content of the transsporation fermentation broth, and the effect of replacing (1%, w / v) ammonium sulfate with the same amount of ammonium chloride and the increased nitrogen content on the fermentation growth and transsporation of isolate PPX-13 was investigated. The results showed that the overall physiological activity curves of PPX-13's fermentation growth and transsporation were very similar to those of batches 5 and 6, and the total viable cell count and the total number of viable spores resistant to 80 °C were also significantly higher. PThe concentration of nitrogen in the PPX-13 fermentation broth was <0.05%, higher than batches 5 and 6. This indicates that increasing the nitrogen content of the fermentation broth and replacing its nitrogen source from (1%, w / v) ammonium sulfate with the same amount of ammonium chloride significantly improved the growth and transspore activity of isolate PPX-13. Therefore, 1% (w / v) ammonium chloride (with the same amount of ammonium sulfate) was listed as the basic component and dosage formulation of the fermentation transspore medium for isolate PPX-13. However, the highest total number of viable spores resistant to 80℃ was 2.90 ± 0.37 × 10⁻⁶. 8 The viable spores / mL (appearing at 72 hours of fermentation culture) still did not reach ≥ 1 × 10⁻⁶. 9 The production quality standard for live spores / mL was therefore determined to be a failed and unqualified test for this batch. (8) Batch 8 was conducted using “Modified LB Medium-3 (Table 4) + Fermentation Transsporation Conditions-5 (Table 5)”. Based on the results of batch 4, dissolved oxygen in the fermentation broth was an adverse condition for the growth of isolate PPX-13, but this adverse condition also promoted transsporation. Therefore, this batch used fermentation transsporation condition-5 (0 vvm and 0 MPa) to test the fermentation growth and transsporation of isolate PPX-13, exploring the effect of a microaerobic environment on the fermentation growth and transsporation of isolate PPX-13. The results showed that at 48 hours of transsporation fermentation, the total viable count in the fermentation broth could reach as high as 2.50 ± 0.30 × 10⁻⁶. 9 CFU / mL, significantly ( P <0.05) is higher than batch 7, which had ventilation (0.5 vvm) and controlled tank pressure (0.1 MPa), but the highest total number of viable spores resistant to 80℃ is significantly lower. P <0.05) is lower than that of batch 7. Therefore, it can be concluded that isolate PPX-13 is indeed inhibited by oxygen. However, oxygen is also a stress factor that promotes the transsporulation activity of isolate PPX-13. In the absence of dissolved oxygen, the transsporulation activity of isolate PPX-13 is significantly (…). P The concentration of viable cells decreased to <0.05. Additionally, in the 8th batch of fermentation transsporic tests, at 72 hours of fermentation, the total viable cell count was (8.09 ± 0.44 × 10⁻⁵). 7 CFU / mL) and total viable spore count (5.93 ± 0.75 × 10⁻⁶). 7 The number of live spores (mL) was far below the set production quality standard, so the experiment was deemed a failure and all subsequent experiments were terminated.

[0042] (9) Batch 9 was conducted using “Modified LB medium-3 (Table 4) + fermentation transsporulation conditions-6 (Table 5)”. Based on the experimental results of batches 7 and 8, it was found that the isolate PPX-13 needed to grow vegetative cells under a microaerobic fermentation environment, and then promote its transsporulation into mature spores under aerobic stress. Therefore, this batch innovatively designed a two-stage fermentation transsporulation mode. The first stage was from 0 to 48 hours of transsporulation fermentation, with a microaerobic environment of no aeration (0 vvm) and tank pressure (0 MPa) to grow a large number of vegetative cells with vigorous microbial activity. The second stage was from 49 to 120 hours of transsporulation fermentation, with aeration turned on (0.5 vvm) and tank pressure controlled (0.1 MPa) to create an oxygen stress environment with high dissolved oxygen. At this time, the nutrients in the fermentation broth were almost exhausted, thus creating a starvation environment for vegetative cells. In this way, both oxygen and starvation stress were created to promote the transsporulation of isolate PPX-13. The results showed that using this fermentation mode did indeed yield the highest total viable count (2.38 ± 0.27 × 10⁻⁶) that met production quality standards at 48 hours after fermentation transsporulation. 9 The result (CFU / mL) meets the predetermined production target (total viable count ≥ 1 × 10⁻⁶). 9 The production quality standard is CFU / mL; although the translocation efficiency and count of total viable spores resistant to 80℃ are significantly improved ( P <0.05) higher than batch 8 without ventilation and tank pressure, but significantly ( P The concentration of viable cells in the fermentation transsporation test was <0.05, lower than that of batch 7, which had aeration (0.5 vvm) and controlled tank pressure (0.1 MPa) from the start of transsporation. This result indicates that microaerobic fermentation transsporation can indeed significantly increase the total viable cell count of isolate PPX-13, but the subsequent aerobic and starvation stresses were initiated too late. This caused the vegetative cells of isolate PPX-13 to become nutrient-depleted, resulting in weakened microbial activity and preventing them from effectively entering the transsporation physiological and metabolic stage to transform into mature spores. Therefore, during the overall fermentation transsporation of isolate PPX-13, it is necessary to select appropriate and easily identifiable physiological and metabolic indicators that are suitable for easy identification and industrial-scale production, during the early stage of fermentation, when the viable cell count and microbial activity are high, to establish an earlier fermentation transsporation time point (<48 hours) to maintain high microbial activity and promote transsporation. In addition, in the fermentation transsporation test of batch 9, at 72 hours of fermentation, the total viable cell count was (7.61±0.84 × 10⁻⁶). 7 CFU / mL) and total viable spore count at 80℃ (2.08 ± 0.26 × 10⁻⁶). 7 The number of live spores (mL) was far below the set production quality standard, therefore the experiment was deemed a failure and all subsequent related experiments were terminated.

[0043] (10) The 10th batch was conducted using “Modified LB medium-3 (Table 4) + fermentation transsporulation conditions-7 (Table 5)”. Based on the experimental results of the 9th batch, it is known that, in accordance with the principle of considering the actual industrial production of viable spore powder from sporogenic bacteria, in the early stage of fermentation transsporulation of isolate PPX-13, it is necessary to find a suitable and easily identifiable physiological metabolic index with a large number of vegetative somatic cells and high microbial activity, and to establish an earlier fermentation transsporulation time point (<48 hours) to maintain the high number of vegetative somatic cells and high microbial activity of isolate PPX-13, thereby promoting its transsporulation. Therefore, based on the fermentation transsporulation results of the 9th batch, it was found that during the 16th-20th hour of fermentation of isolate PPX-13, the pH of the fermentation broth stopped decreasing and began to rise. This fermentation time point indicates that isolate PPX-13 has exhausted all carbon sources in the fermentation broth, thus becoming unable to continue producing organic acid-acidified fermentation broth. It also indicates that the nutrients in the fermentation broth are nearing depletion, and the metabolism of nitrogen sources has begun, producing alkaline metabolites that cause the pH of the fermentation broth to rise. This result also shows that the microbial fermentation curve of isolate PPX-13 is at the end of its rapid growth phase or has reached a stable phase, with its microbial growth and activity beginning to decline. Therefore, this invention selects the unique microbial physiological characteristics of PPX-13's fermentation transspores as the fermentation transspore time point to initiate oxygen and starvation stress. Specifically, during the 16th-20th hour of PPX-13's fermentation transspore process, once the pH of the fermentation broth stops declining and begins to rise, aeration (0.5 vvm) and tank pressure control (0.1 MPa) are immediately initiated to simultaneously create oxygen and starvation stress to promote transspore formation into mature spores. The results indicated that, compared to batches 7 and 9, the total viable cell count, total number of viable spores resistant to 80 °C, and transfection rate were significantly higher at multiple fermentation time points during the overall fermentation and transfection process. P The total viable count and the total number of viable spores resistant to 80 °C both reached the total viable count ≥ 1 × 10⁻⁵ as set in this invention after 72 hours of fermentation (<0.05). 9 CFU / mL and total viable spore count ≥ 1 × 10⁻⁶ at 80 °C 9 The production quality standard of live spores / mL is therefore deemed qualified. In this invention, this fermentation mode - "modified LB medium-3 + fermentation transspore conditions-7" is named "PPX-13 fermentation transspore mode", while modified LB medium-3 (Table 4) and fermentation transspore conditions-7 (Table 5) are named "PPX-13 fermentation transspore medium" and "PPX-13 fermentation transspore conditions" respectively.

[0044] (11) The 11th batch was carried out with "Improved LB Medium - 4 (Table 4) + Fermentation Transconjugation Conditions - 7 (Table 5)". This experiment was based on Improved LB Medium - 3, with an additional 0.01% (v / v) commercially available polyether antifoaming agent added to prepare Improved LB Medium - 4, thereby solving the problem of foaming of the fermentation broth caused by aeration during the transconjugation fermentation of the isolate PPX - 13 using the PPX - 13 fermentation transconjugation mode in industrial fermentation production. At the same time, it was also explored whether the antifoaming agent would affect the growth of the isolate PPX - 13 cells and the transconjugation and development of highly stress - resistant mature spores. The results showed that the physiological activity curves of the overall fermentation growth and transconjugation of the isolate PPX - 13 were very similar to those of the 10th batch, and the total viable cell count, total heat - resistant 80 °C viable spore count, and transconjugation rate measured at each time point during the fermentation transconjugation process were also very close, without significant ( P <0.05) differences. After 72 hours of fermentation, the total viable cell count and the total heat - resistant 80 °C viable spore count both reached the production quality standards set by this invention, namely, the total viable cell count ≥ 1 × 10 9 CFU / mL and the total heat - resistant 80 °C viable spore count ≥ 1× 10 9 viable spores / mL, so it was judged as qualified. From these results, it can be seen that adding 0.01% (v / v) commercially available polyether antifoaming agent does not affect the fermentation growth and transconjugation activity of the isolate PPX - 13, and this antifoaming agent can be applied to the mass production of the industrial high - concentration viable spore fermentation broth of the isolate PPX - 13 to reduce the risk of contamination by miscellaneous bacteria caused by aeration - induced foaming of the fermentation broth and the loss of fermentation equipment. However, in the actual operation of the fermentation transconjugation of the isolate PPX - 13 in a small - scale fermenter, no obvious foaming phenomenon of the fermentation broth was observed during the fermentation production process of the high - concentration viable spore fermentation broth of the isolate PPX - 13 using the PPX - 13 fermentation transconjugation mode. Therefore, considering cost reduction and simplifying the on - site industrial fermentation operation process, the antifoaming agent can be not added during the fermentation production of the high - concentration viable spore fermentation broth of the isolate PPX - 13.

[0045] 5. Development of the PPX - 13 Transconjugation Fermentation Mode and Application of the 2 - L High - Viable - Spore - Count and Highly Stress - Resistant Mature Spore Fermentation Transconjugation Test of the Isolate PPX - 13 Conducted Thereby Based on and synthesizing points 1-4 of the above-mentioned invention, this invention innovatively establishes a fermentation production mode (named PPX-13 fermentation transspore mode) for the fermentation broth of the high-viable spore count fermentation liquid of the high-temperature resistant, multifunctional tea tree phosphate-solubilizing, bio-anti-coagulant Bacillus isolate PPX-13. The main innovation is the establishment of a "two-stage fermentation transspore mode," where the first stage is microaerobic fermentation culture for the proliferation and fermentation of vegetative cells of isolate PPX-13; the second stage is aerobic fermentation culture, using aerobic, starvation, and high-oxygen stress conditions for the transspore fermentation culture of isolate PPX-13. The overall fermentation time is set at 96 hours (Table 7). Because during the fermentation transspore process of isolate PPX-13 using the PPX-13 fermentation transspore mode, its cell morphology changes from long, vegetative cells of the bacterium, then gradually transspores into teliospores carrying vegetative cells, and finally forms individual spores detached from the vegetative cells. Figure 1 (a) and (b). Therefore, in microbial physiology and spore development research, individual spores detached from vegetative cells are more mature transgenic spores, exhibiting higher environmental resistance. They are also the preferred choice for industrial production of high-quality, long-shelf-life spore powders or other related microbial biotechnology products, and represent the necessary spore morphology for the industrial mass production of these products. Therefore, in the overall PPX-13 fermentation transgenic process, after entering the second stage of aerobic, starvation, and hyperaerobic stress fermentation culture, appropriate amounts of fermentation broth were periodically taken for observation of the PPX-13 spore morphology under a 1,000x oil immersion microscope. The total number of cells and the number of mature individual spores detached from vegetative cells were counted until the vast majority of the microscopic field of view consisted of "mature individual spores detached from vegetative cells," and the transgenic rate of these mature individual spores was ≥ 90% of the total number of cells. This result was repeated twice during different fermentation stages throughout the overall fermentation process before the fermentation was considered complete and the fermentation tank was collected (Table 7).

[0046] In a 2 L transspore fermentation experiment using the PPX-13 transspore fermentation mode, the total viable cell count and the total number of viable spores resistant to 80 and 100 °C were measured to assess the microbial activity and effectiveness of the PPX-13 isolate in forming highly resistant mature spores. The experimental methods for determining the total viable cell count and the total number of viable spores resistant to 80 °C are as described in point 1 of the invention above. The total number of viable spores resistant to 100 °C was determined using the BR plate counting method with consecutive 10-fold dilutions (incubated at 40 °C for 5 days), counting the number of colonies per mL of fermentation broth exhibiting a clear phosphate-solubilizing zone after a 10-minute water bath treatment at 100 °C. This method is also established as a quality assurance test for distinguishing, identifying, and calculating colonies grown from vegetative cells, spores, or highly resistant mature spores (those resistant to 100°C) of isolate PPX-13 using a simple BR plate assay. The quality assurance standard is that a single colony growing on a BR plate must show a clear phosphate-solubilizing zone to be distinguishable as isolate PPX-13. Since isolate PPX-13 is a thermophilic, multifunctional phosphate-solubilizing Bacillus, a clear phosphate-solubilizing zone must appear around the colony after 5 days of incubation on a BR plate at 40°C. If this phosphate-solubilizing zone is not present, it is definitely not isolate PPX-13. Instead, it is likely contaminated by environmental bacteria during fermentation or post-processing of isolate PPX-13's vegetative cells, spores, or transspores (those resistant to 100°C). Such contaminants should not be included in the PPX-13 vegetative cells, spores, or highly resistant mature spores (those resistant to 100°C). The number of viable microbial colonies in the PPX-13 isolate (effective viable mature spores at ℃) was calculated, and this test was used to perform a simple quality assurance test to distinguish between isolate PPX-13 and environmental contaminants. Spores that survived a 100℃ boiling water bath for 10 minutes in the PPX-13 spore fermentation broth can be considered transspores with high stress resistance. Figure 1 (c and d). The results are shown in Table 8. A rise in the pH of the fermentation broth was observed at 18.0 hours of transspore fermentation, and a continuous upward trend in pH was confirmed at 18.5 hours. Aeration (0.5 vvm) was immediately initiated, and a tank pressure of 0.1 MPa was established to proceed to the second stage. Subsequently, a continuous upward trend was observed in the pH of the fermentation broth, the number of individual spores, the transspore conversion rate of individual spores, the total number of viable spores resistant to 80 °C, the total number of viable spores resistant to 100 °C (transformed into highly stress-resistant mature spores), and the transspore conversion rate of mature spores. During fermentation from 82 to 120 hours, the total number of cells and individual spores were observed and counted using a 1,000 × oil immersion microscope. Confirmed experimental results showed a transspore conversion rate ≥ 90% with two or more replicates. Therefore, the fermentation was stopped at 120 hours. The fermentation broth product at the time of collection had a total viable cell count and a total number of viable spores (transgenic mature spores) resistant to 100℃ as high as 2.15 ± 0.23 × 10⁻⁶.9 CFU / mL and 2.13±0.23 × 10 9 The number of live spores per mL indicates that this transspore fermentation broth contains high numbers of live spores and is highly resistant to adverse conditions, and also significantly exceeds the total live cell count (≥ 1 × 10⁻⁶) set in this invention. 9 CFU / mL and total viable spore count ≥ 1 × 10⁻⁶ at 80 °C 9 The production quality standard of live spores / mL was therefore deemed qualified, and the translocation rate of mature transspores was ≥ 90% (Table 8). Furthermore, it was found that at fermentation hours 82.0 and 96.0, observation and counting of total cells and individual spores using a 1000× oil immersion microscope yielded definitive results with a translocation rate of individual spores ≥ 90% in two replicates. Moreover, at fermentation hour 96, the total viable cell count and the total number of viable spores (translocated mature spores) resistant to 100℃ reached as high as 2.34 ± 0.30 × 10⁻⁶. 9 CFU / mL and 2.23±0.27 ×10 9 The number of live spores / mL significantly exceeded the production quality standards set by this invention and was deemed qualified. Furthermore, the transgenic mature spore transgenic rate was ≥ 90% (Table 8). Therefore, the fermentation time of the fermentation broth of the whole isolate PPX-13 with high live spore count and high stress resistance to mature spores, carried out in a fermentation tank and its auxiliary systems and equipment, can be shortened to 96 hours, which can further reduce the industrial fermentation cost and the turnover and machine occupation costs of the fermentation tank and its auxiliary systems and equipment.

[0047] 6. Summary and a brief description of the results of each stage in the subsequent experiments and the overall industrial pilot production process of high-concentration, high-quality PPX-13 spore powder. The most crucial core technology for the industrial-scale production of spore powders or other related microbial preparations from spore-producing bacteria with high viable spore counts, high quality, and long shelf life at room temperature, both domestically and internationally, lies in the innovation and establishment of an industrial-scale fermentation transsporination model and process for a transsporination fermentation broth with a high viable spore count and high resistance to environmental stress. Therefore, this invention innovatively establishes an industrial-scale fermentation transsporination model for a high-viable spore count and highly stress-resistant mature spore fermentation broth of the high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulans isolate PPX-13, named the "PPX-13 Fermentation Transsporination Model." This model is applied to the related fermentation production and product development of isolate PPX-13, as well as the formulation of its industrial-scale production process and procedures, as described below: (1) Small-scale industrial fermentation trial production of 5 L high viable spore count and high stress resistance mature spore fermentation broth (in a 7 L fermenter) was carried out in a total of 3 batches. All batches clearly met the production quality assurance standards set by the present invention and were all judged to be qualified production batches. The microbial fermentation growth curves of these 3 batches of trial production showed significant reproducibility and production stability. Therefore, it can be applied to industrial production and reduce production risks. A total of 13,923.4 mL of high viable spore count and high stress resistance mature spore fermentation broth was produced. (2) All 13,923.4 mL of the fermentation broth containing high viable spore counts and high stress resistance of mature spores produced above were collected and further processed (including ultra-high speed centrifugation to extract cells and spore sediment solids, SiO2 exfoliation, drying at 60 ℃, and uniform pulverization) to prepare centrifuged concentrated spore powder containing high viable mature spore counts. A total of 121.58 g was produced, with a yield of 0.87±0.05% (w / v) per unit of fermentation broth containing high viable spore counts and high stress resistance of mature spores, and each gram of spore powder contained 9.03±1.14× 10⁻⁶ spores. 10 It can withstand 100 ℃ and produce viable mature spores with a moisture content of 3.10±0.52% (w / w). (3) Take 50 g of the PPX-13 isolate with high viable mature spore count, centrifuge to concentrate the spore powder, and dilute it appropriately with an appropriate amount of SiO2 to prepare a powder with a concentration ≥ 2 × 10⁻⁶. 9 The PPX-13 high-activity mature spore powder product, named "PPX-13 high-concentration high-quality spore powder" in this invention, yielded a total production of 2,250.01 g, with each gram containing 2.13 ± 0.16 × 10⁻⁶ spores. 9 It can withstand 100 ℃ and produce viable mature spores with a moisture content of 2.90±0.33% (w / w). (4) 1,200 g of high-concentration, high-quality PPX-13 spore powder was subjected to a one-year sealed preservation test under 4°C refrigeration and dark room temperature (i.e., stored in a general sealed biological material and product storage room, without any temperature control equipment or outdoor sunlight, and without any volatile chemicals, but with ventilation and dehumidification equipment and a small amount of indoor white LED light). Each storage environment and condition had 600 g of the tested PPX-13 high-concentration, high-quality spore powder, which was then divided into three groups of 200 g each for triple replication. The results showed that the survival rates of the total effective viable microbial population and the total effective viable spore population resistant to 80°C and 100°C were not significantly different when the tested PPX-13 high-concentration, high-quality spore powder was stored under 4°C refrigeration and dark room temperature. PThe PPX-13 high-concentration, high-quality spore powder exhibits high reproducibility and stability in production and microbial activity, making it suitable for industrial production and reducing production risks. Furthermore, after approximately 180 and 360 days of storage, the survival rates of the total effective viable microbial population and the total effective viable spore population resistant to 80℃ and 100℃, respectively, are ≥92.84% and 84.04%, while the moisture content (w / w) is ≤2.97% and 3.84%, respectively. This also indicates that after 360 days of storage in a dark, room-temperature environment, the total number of mature spores resistant to 100℃ in the PPX-13 high-concentration, high-quality spore powder is ≥1.81 × 10⁻⁶. 9 Effective live spores / g. Therefore, the specification for PPX-13 high-concentration, high-quality spore powder can be formulated as ≥ 1.0 × 10⁻⁶. 9 It can withstand 100 ℃ with effective live mature spores / g and moisture content <10% (w / w). It should be stored in a sealed container at a dark room temperature. The shelf life is 1 year. (5) Develop an industrial mass production model and process for PPX-13 high-concentration, high-quality spore powder products. It is estimated that each ton of PPX-13 isolate with high viable spore count and high stress resistance mature spore fermentation broth can produce approximately 8.7 kg of centrifuged concentrated PPX-13 isolate with high viable mature spore count and approximately 392.81 kg of PPX-13 high-concentration, high-quality spore powder products. The overall production operation time is approximately 13-14 days (if the time required for counting the total number of 100℃ effective viable mature spores and quality assurance work of the produced PPX-13 high-concentration, high-quality spore powder products is considered, the overall operation time is approximately 18-19 days).

[0048] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0049] Example 1: Small-scale production demonstration of 5 L of high viable spore count and high stress resistance mature viable spore fermentation broth of PPX-13, a high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulant isolate, using the PPX-13 fermentation transspore mode. The results are shown in Tables 9-11. The small-scale production of 5 L high viable spore count and high stress-resistant mature spore fermentation broth of PPX-13 isolates in these three batches showed similar trends in pH, total cell (including spore) count, single spore translocation rate, total viable cell count, total number of viable spores resistant to 80 °C, total number of viable spores resistant to 100 °C (translocated mature spores), and mature spore translocation rate. These trends were also similar to those of the 2 L high viable spore count and high stress-resistant mature spore fermentation translocation test of PPX-13 isolates (Table 8). This indicates that the production of high viable spore count and high stress-resistant mature spore fermentation broth of PPX-13 isolates using the PPX-13 translocation fermentation model has a certain degree of stability and reproducibility. Therefore, it can be applied to industrial production and reduce production risks. The total viable cell count and total viable spore count (capable of withstanding 100℃) of the fermentation broth produced in these three batches, which exhibited high viable spore counts and high stress resistance, significantly exceeded the total viable cell count ≥ 1 × 10⁻⁶ set by this invention. 9 CFU / mL and total viable spore count ≥ 1 × 10⁻⁶ at 80 °C 9 The production quality standard of live spores / mL was thus deemed qualified, and the fermentation time only required 96 hours. Therefore, the overall fermentation time can be shortened to 96 hours, further reducing the industrial fermentation cost and the turnaround and occupation costs of the fermenter and its auxiliary equipment and systems. Furthermore, the individual spore translocation rate and the high-stress-resistant mature spore translocation rate of these three batches significantly exceeded 90% at 96 hours of fermentation or at the end of fermentation, indicating that the PPX-13 fermentation translocation mode can effectively promote the translocation of PPX-13 isolates into highly stress-resistant mature spores. This has great potential for the industrial-scale production of high-concentration, high-quality spore powder of PPX-13 isolates or other related microbial biotechnology products. The small-scale pilot production of these three batches of 5 L high-live-spore-count, highly stress-resistant mature live spore fermentation broth yielded a total of 13,923.4 mL, which will be used for the subsequent preparation and pilot production of high-live-spore centrifuged concentrated spore powder.

[0050] Example 2: Small-scale pilot production demonstration of high-concentration, high-quality PPX-13 mycelium powder product post-processing using fermentation broth of high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulans isolate PPX-13 with high viable spore count and high stress resistance. The specific small-scale pilot production model for the post-processing of high-concentration, high-quality PPX-13 mycelial powder products using fermentation broth containing high-viable-spore-count, highly stress-resistant mature PPX-13 isolates is as follows: (1) The fermentation broth of mature spores of PPX-13 isolate with high viable spore count and high resistance to stress was centrifuged at 10,000 rpm and 25 ℃ for 30 minutes. The supernatant was carefully poured off, and the cells and spores were removed as much as possible. (2) The obtained centrifuged solids of cells and spores were added with sterile water at a wet weight to volume ratio of 1 g: 0.5 mL, and then the centrifuged solids were resuspended by vigorous shaking to complete the preparation of concentrated cell and spore suspension. (3) Then, add the excipient (SiO2) of the obtained centrifuged solids wet weight at a weight ratio of 1:1 to the concentrated cell and spore suspension, mix thoroughly and evenly, put it into an iron pan, and dry it at 60 °C overnight until the moisture content is <10% (w / w). The moisture content test is conducted as follows: 5 g of spore powder sample is accurately weighed into three clean and dry empty glass dishes. The weight of each glass dish containing the spore powder sample is recorded. The glass dishes are then placed in an electric constant temperature drying oven and dried overnight at 105 ℃. After drying, the glass dishes containing the spore powder sample are placed in a desiccator to cool to room temperature and then accurately weighed. The weight of the glass dish containing the spore powder sample before drying is subtracted from the weight of the glass dish containing the spore powder sample after drying. The difference is divided by the corresponding weight of the spore powder sample (the result is calculated as a percentage, w / w (%)), which is the moisture content of the spore powder sample. (4) The dried PPX-13 isolate with high active mature spore count centrifuged concentrated spore powder prepared by SiO2 excipient is uniformly mixed using a mixer. The processing method is to uniformly stir at 1,000 rpm for 30 seconds to complete the production of PPX-13 isolate with high active mature spore count centrifuged concentrated spore powder. It is then sealed and stored in a dark, room temperature, dry place. (5) Accurately weigh an appropriate amount of PPX-13 isolate with high viable mature spore count, centrifuge and concentrate the spore powder, treat it in a 100 ℃ water bath for 10 minutes, then dilute it 10 times and spread it on BR plate medium and incubate it at 40 ℃ for 5 days. Count and calculate the number of 100 ℃ effective viable mature spore colonies with obvious phosphorus-soluble transparent zone in each gram of spore powder (that is, the identification and calculation of 100 ℃ effective viable mature spore colonies of PPX-13 isolate with high stress resistance by simple BR plate detection method for quality assurance test); (6) Based on the measured number of viable mature spores at 100 ℃, add an appropriate amount of SiO2 to the PPX-13 isolate with a high number of viable mature spores, centrifuge and concentrate the spore powder, then dilute appropriately and mix thoroughly to ensure that each gram of spore powder contains ≥ 2 × 10⁻⁶ spores. 9 The production and preparation of PPX-13 high-concentration, high-quality spore powder products can be completed by producing viable, mature spores that can withstand 100℃.

[0051] The results are shown in Tables 12 and 13. Figure 2As shown, the fermentation broth of PPX-13 isolates with high viable spore counts and high stress resistance, produced in 5 L batches using the PPX-13 fermentation transspore model, was further processed into small-scale production, which also produced three batches of PPX-13 isolates with high viable mature spore counts, centrifuged concentrated spore powder. The various indicators, parameters, and yields during the production process were similar to the moisture content, total effective viable microorganisms, and total effective viable spore populations resistant to 80 ℃ and 100 ℃ of the prepared spore powder. This indicates that the subsequent small-scale trial production model is stable, highly reproducible, and has low production risk, and therefore can be used for large-scale industrial mass production (Tables 12 and 13). These three batches produced a total of 121.58 g of centrifuged concentrated spore powder of PPX-13 isolate with high viable mature spore count. Therefore, the yield of centrifuged concentrated spore powder of PPX-13 isolate with high viable mature spore count and high stress resistance per unit unit of fermentation broth was 0.87 ± 0.05% (w / v) (Table 12), and each gram of powder contained 9.03 ± 1.14 × 10⁻⁶ mg / L. 10 The PPX-13 isolate was tested at 100 °C and found to be viable mature spores with a moisture content of 3.10 ± 0.52% (w / w) (Table 13). Subsequently, 50 g of the isolate was centrifuged to concentrate the spore powder, which was then appropriately diluted with SiO2 to prepare a concentration ≥ 2 × 10⁻⁶. 9 The PPX-13 high-concentration, high-quality spore powder product with an effective live spore count / g is named "PPX-13 high-concentration, high-quality spore powder" in this invention. A total of 2,257.50 g was produced. Each unit of PPX-13 high-concentration, high-quality spore powder with a high effective live spore count can be diluted to produce 45.15 units of PPX-13 high-concentration, high-quality spore powder, with a yield of 4,515%. It can also be further estimated that the yield of PPX-13 high-concentration, high-quality spore powder from each unit of PPX-13 high-concentration, high-resistance mature spore fermentation broth with a high effective live spore count is 3,928.05%. The PPX-13 high-concentration, high-quality spore powder produced in this post-processing process underwent quality assurance testing (i.e., the identification and calculation of colonies of PPX-13 high-resistance mature spores grown at 100℃ using a simple BR plate assay) yielded 2.13 ± 0.16 × 10⁻⁶ ppm. 9 Effective viable spores / g at 100℃, moisture 2.90±0.33% (w / w) (Table 14 and Figure 2 The remaining 71.58 g of centrifuged concentrated PPX-13 spore powder with high viable mature spore count was stored at 4 °C for subsequent related experiments and for the production of additional high-concentration, high-quality PPX-13 spore powder.

[0052] Example 3: Demonstration of the long shelf life at room temperature of PPX-13 high-concentration, high-quality spore powder product from the high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulant isolate PPX-13. The long shelf life test of PPX-13 high-concentration, high-quality spore powder at room temperature was conducted using a sealed, room-temperature storage test of general Bacillus spore powder. The specific experimental method is as follows: (1) The tested PPX-13 high-concentration, high-quality spore powder product was prepared in Example 2 above, and it had a concentration of 2.13 ± 0.16 × 10⁻⁶. 9 Effective viable spores / g at 100℃, moisture 2.90±0.33% (w / w) (Table 14 and Figure 2 ); (2) Weigh 1,200 g of the tested PPX-13 high-concentration high-quality spore powder and conduct a one-year sealed preservation test at 4 ℃ refrigeration and dark room temperature. For each preservation environment and condition, 600 g of the same tested PPX-13 high-concentration high-quality spore powder was used, and then divided into 3 groups of 200 g each for triple replication test. (3) On days 30, 90, 180, 270, and 360, 30 g of the high-concentration, high-quality PPX-13 spore powder was precisely weighed and sampled for the detection of total viable microorganisms, total viable spores resistant to 80℃, total viable spores resistant to 100℃, and moisture content. The detection methods were as described in "5. PPX-13 Transspore Fermentation Model Development and Application: 2 L High Viable Spore Count, High Stress Resistance Mature Spore Fermentation Transspore Experiment of PPX-13 Isolate" and Example 2. After sampling the high-concentration, high-quality PPX-13 spore powder sample, it was immediately and completely sealed to prevent the spore powder from absorbing moisture from the air, which would lead to an increase in moisture content, and to prevent contamination by other microorganisms. (4) The survival rate of the total effective viable microbial community and the effective spore survival rate (%) of the total effective viable spore community of the PPX-13 high-concentration high-quality spore powder at each test time point of the preservation test are calculated as follows: the total number of effective viable microorganisms and the total number of effective viable spores resistant to 80℃ and 100℃ measured at the time of preservation test are divided by the total number of effective viable microorganisms and the total number of effective viable spores resistant to 80℃ and 100℃ on the production day (day 0).

[0053] The results are shown in Table 14. The survival rates of the total viable microbial population, the total viable spores resistant to 80℃, and the total viable spores resistant to 100℃ were not significantly different when the high-concentration, high-quality PPX-13 spore powder was stored at 4℃ and in a dark, room-temperature environment. PThe difference was <0.05%, but both decreased slowly with prolonged storage time, which is related to the death of effective microbial vegetative cells and spores; the moisture content, however, increased slowly with prolonged storage time, which is related to the absorption of moisture from the air by the fungal powder, and the moisture content of the fungal powder stored in a dark room temperature was significantly ( P <0.05) Refrigerated at 4 ℃. When the tested high-concentration, high-quality PPX-13 spore powder was refrigerated at 4 ℃ and stored at dark room temperature for approximately 180 and 360 days, respectively, the survival rates of effective microbial vegetative cells or spores were ≥ 92.84% and 84.04%, respectively, while the moisture content (w / w) was ≤ 2.97% and 3.84%, respectively. These results indicate that the mature PPX-13 spores produced using the innovative PPX-13 fermentation transsporulation method of this invention do indeed have high resistance to environmental stress and a high survival rate at dark room temperature. It also indicates that the PPX-13 spore powder or related microbial biotechnology products produced using the PPX-13 fermentation transsporulation method can also have high quality and a long shelf life at dark room temperature. This provides substantial support for the transportation, storage, quality requirements, promotion, distributor marketing, and inventory management of these products in production or sales. Furthermore, it was also shown that after 360 days of storage in a dark, room-temperature environment, the total number of viable spores resistant to 100℃ in PPX-13 high-concentration, high-quality spore powder could be ≥ 1.81 × 10⁻⁶ based on experimental results and conversion estimates. 9 With an effective live spore count / g and a moisture content ≤ 3.84% (w / w), the specifications and quality assurance standards for PPX-13 high-concentration, high-quality spore powder can be formulated as ≥ 1.0 × 10⁻⁶. 9 Live spores / g and moisture content <10% (w / w), store in a sealed container at a dark, room temperature, shelf life is 1 year.

[0054] Example 4: Industrial-scale production model and process of high-concentration, high-quality spore powder product of PPX-13, a high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulant isolate. The industrial-scale production model and flow chart for PPX-13 high-concentration, high-quality spore powder provided by this invention are based on the fermenters, auxiliary equipment and systems, and post-processing equipment commonly used in large-scale (ton-level) microbial fermentation plants both domestically and internationally, and their typical production models and usage parameters for spore powder or microbial biotechnology powder products. Figure 3 As shown, the details are as follows: (1) First, the ton-scale industrial production mode of centrifuged concentrated spore powder of PPX-13 isolate with high number of viable mature spores was carried out. After working sequence 1-4, it is estimated that each ton of fermentation broth of PPX-13 isolate with high number of viable mature spores and high resistance to stress can produce about 8.7 kg of centrifuged concentrated spore powder of PPX-13 isolate with high number of viable mature spores. (2) After work sequence 5 and 6, the PPX-13 isolate with high viable mature spore count produced above is centrifuged and concentrated into spore powder, and then appropriately diluted with an appropriate amount of SiO2 to prepare PPX-13 high concentration and high quality spore powder. It is estimated that each ton of PPX-13 isolate with high viable spore count and high stress resistance mature spore fermentation broth can produce about 392.81 kg of PPX-13 high concentration and high quality spore powder. (3) The overall production operation time is approximately 13-14 days. If the time required for counting and quality assurance of the total number of viable spores at 100℃ in the produced PPX-13 high-concentration high-quality spore powder is taken into account, the overall operation time is approximately 18-19 days. However, it is necessary to make appropriate optimizations based on the actual large-scale production situation (e.g., 100-1,000 L) of the fermentation plant, the actual on-site operation, and the availability of equipment and raw materials for the fermentation plant. A suitable SOP for the actual on-site production of PPX-13 high-concentration high-quality spore powder should be formulated according to local conditions.

[0055] Table 1. Optimal growth and phosphate-solubilizing activity test results of the tested heat-resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulans isolate PPX-13 (based on...). Bacillus racemilaticus (BR medium plate assay, cultured for 5 days)

[0056] A,B,C,D,E,F,G,H,I Different capital letters in the shoulder labels of data in the same column indicate significant differences (Tukey, P <0.05), identical or no letters indicate no significant difference (Tukey, P ≥ 0.05).

[0057] a,b,c,d,e Different lowercase letters in the shoulder labels of peer data indicate significant differences (Tukey, P <0.05), identical or no letters indicate no significant difference (Tukey, P ≥ 0.05).

[0058] Data are expressed as mean ± standard deviation (mean ± SD, n ≥ 3).

[0059] Table 2. Optimal growth and initial culture pH test of the tested heat-resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulans isolate PPX-13 (cultured for 3 days using the Luria-Bertan medium (LB medium) liquid detection method).

[0060] A,B,C,D,E,F,G,H Different capital letters in the shoulder labels of data in the same column indicate significant differences (Tukey, P <0.05), identical or no letters indicate no significant difference (Tukey, P ≥ 0.05).

[0061] Data are expressed as mean ± standard deviation (mean ± SD, n ≥ 3).

[0062] ND: Not detected.

[0063] Table 3. Growth characteristics, culture and transspore conditions of the tested heat-resistant, multifunctional tea tree phosphate-solubilizing Bacillus coagulant isolate PPX-13.

[0064] Each experiment was performed in at least three replicates.

[0065] Table 4. Test culture medium designed and used in this invention for testing the fermentation sporulation effect of the high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulant isolate PPX-13.

[0066] 1 Purchased from Guangdong Huankai Microbial Technology Co., Ltd.

[0067] 2 This industrial-grade yeast extract was purchased from Beijing Hongrun Baoshun Technology Co., Ltd., product serial number: HRBS-Y017C, and is used for microbial fermentation.

[0068] 3 This industrial-grade polyether defoamer is from Yantai Hengxin Chemical Technology Co., Ltd., product serial number: THI-X288A, and is used for microbial fermentation.

[0069] Table 5. Fermentation conditions for testing the sporulation effect of the high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulant isolate PPX-13 designed and tested in this invention.

[0070] 1Preparation of inoculum: Inoculate 1 mL of 1 mL isolate of PPX-13 at -80 ℃ with glycerol culture medium in 100 mL of MRS liquid medium and incubate at 150 rpm and 40 ℃ for 48 hours. This culture is recorded as PPX-13 MRS inoculum.

[0071] Table 6. High-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulant isolate PPX-13 under different test media, fermentation conditions, and 7 L small fermenter 2 L fermentation transsporination test

[0072] Note: The 7 L small fermenter used in this experiment is a 7 L glass fermenter (Biotech-70JG-7000, Shanghai Baoxing Bio-Equipment Engineering Co., Ltd.).

[0073] 1 As shown in Table 4 above.

[0074] 2 As shown in Table 5 above.

[0075] 3 The total viable count was determined by the BR plate count method with 10-fold consecutive dilutions (incubated at 40 °C for 5 days), counting the number of colonies with a clear phosphate-solubilizing zone per mL of fermentation broth.

[0076] 4 The total number of viable spores resistant to 80 °C was determined by BR plate counting with 10-fold dilutions (incubated at 40 °C for 5 days), counting the number of colonies per mL of fermentation broth with a clear phosphate-solubilizing zone after 10 minutes of water bath treatment at 80 °C.

[0077] ND: Not detected.

[0078] Each experiment was performed in at least three replicates, and the data are expressed as mean ± standard deviation (mean ± SD, n ≥ 3).

[0079] Table 7. Fermentation production mode settings for high viable spore count and high stress resistance spore fermentation broth of high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulans isolate PPX-13 (named PPX-13 fermentation transspore mode).

[0080] Table 8. Various indicators, parameters, microbial community microscopic observation and counting, and detection of total viable bacteria and viable spores during the fermentation and transsporation experiment of 2 L high viable spore count and high stress resistance spore fermentation broth using the PPX-13 high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulans isolate PPX-13 in a 7 L small fermenter.

[0081] Note 1: When the pH of the spore fermentation broth stops decreasing and begins to rise during fermentation, it immediately enters the second stage.

[0082] Note 2: At fermentation times of 82.0 and 120.0 hours, the total number of cells (including spores) and the number of individual spores were observed and counted using a 1,000 × oil immersion microscope. If the conversion rate of individual spores was ≥ 90% in two or more replicates, the fermentation was completed.

[0083] 1 This pH value is the pH measured in the fermentation tank.

[0084] 2 Spores that have detached from vegetative cells.

[0085] 3 Spores that can survive a 100°C boiling water bath for 10 minutes in the spore fermentation broth can be considered as transgenic mature spores with high stress resistance.

[0086] ND: Not detected.

[0087] Each experiment was performed in at least three replicates, and the data are expressed as mean ± standard deviation (mean ± SD, n ≥ 3).

[0088] Table 9. Various indicators, parameters, microbial community observations and counting, and detection of total viable bacteria and viable spores during the fermentation preparation of the first batch of high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulans isolate PPX-13 using the PPX-13 fermentation transspore mode and a 7 L small fermenter (5 L fermentation volume).

[0089] Note 1: When the pH of the spore fermentation broth stops decreasing and begins to rise during fermentation, it immediately enters the second stage.

[0090] Note 2: At fermentation times of 83.0 and 97.0 hours, the total number of cells and individual spores were observed and counted using a 1,000× oil immersion microscope. Valid test results with two or more replicates showing a single spore translocation rate ≥ 90% were considered valid, and the fermentation tank was then closed.

[0091] 1 This pH value is the pH measured in the fermentation tank.

[0092] 2 Spores that have detached from vegetative cells.

[0093] 3 Spores that can withstand a 100°C boiling water bath for 10 minutes and survive in the spore fermentation broth can be considered as transgenic mature spores with high stress resistance.

[0094] ND: Not detected.

[0095] Each experiment was performed in at least three replicates, and the data are expressed as mean ± standard deviation (mean ± SD, n ≥ 3).

[0096] Table 10. Various indicators, parameters, microbial community observations and counting, and detection of total viable bacteria and viable spores during the fermentation preparation of the second batch of high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulans isolate PPX-13 using the PPX-13 fermentation transspore mode and a 7 L small fermenter (5 L fermentation volume).

[0097] Note 1: When the pH of the spore fermentation broth stops decreasing and begins to rise during fermentation, it immediately enters the second stage.

[0098] Note 2: At fermentation times of 71.0 and 91.5 hours, the total number of cells and individual spores were observed and counted using a 1,000× oil immersion microscope. Valid test results with two or more replicates showing a single spore translocation rate ≥ 90% were considered for successful fermentation and the fermentation tank was closed.

[0099] 1 This pH value is the pH measured in the fermentation tank.

[0100] 2 Spores that have detached from vegetative cells.

[0101] 3 Spores that can withstand a 100°C boiling water bath for 10 minutes and survive in the spore fermentation broth can be considered as transgenic mature spores with high stress resistance.

[0102] ND: Not detected.

[0103] Each experiment was performed in at least three replicates, and the data are expressed as mean ± standard deviation (mean ± SD, n ≥ 3).

[0104] Table 11. Various indicators, parameters, microbial community observations and counting, and detection of total viable bacteria and viable spores during the fermentation preparation of the third batch of high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulans isolate PPX-13 using the PPX-13 fermentation transspore mode and a 5 L fermentation tank (7 L small fermenter).

[0105] Note 1: When the pH of the spore fermentation broth stops decreasing and begins to rise during fermentation, it immediately enters the second stage.

[0106] Note 2: At fermentation times of 82.5 and 91.0 hours, the total number of cells and individual spores were observed and counted using a 1,000× oil immersion microscope. Valid test results with two or more replicates showing a single spore translocation rate ≥ 90% were considered valid, and the fermentation tank was then closed.

[0107] 1 This pH value is the pH measured in the fermentation tank.

[0108] 2 Spores that have detached from vegetative cells.

[0109] 3 Spores that can withstand a 100°C boiling water bath for 10 minutes and survive in the spore fermentation broth can be considered as transgenic mature spores with high stress resistance.

[0110] ND: Not detected.

[0111] Each experiment was performed in at least three replicates, and the data are expressed as mean ± standard deviation (mean ± SD, n ≥ 3).

[0112] Table 12. Production batches of high-activity mature spore count centrifuged concentrated bacterial powder of the high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulant isolate PPX-13, and the detection of various indicators, parameters, and yields during the production process.

[0113] 1 The fermentation broth of this isolate PPX-13, which has a high number of viable spores and is highly resistant to adverse conditions, was produced using a 5-L small-scale pilot production test conducted in a 7 L laboratory fermenter (7 L glass fermenter Biotech-70JG-7000, Shanghai Baoxing Bio-Equipment Engineering Co., Ltd.).

[0114] 2The obtained centrifuged cell and spore solids were mixed with sterile water at a wet weight to volume ratio of 1 g: 0.5 mL. The mixture was then vigorously shaken to resuspend the solids, thus preparing a concentrated cell and spore suspension. Next, an excipient (SiO2) at a wet weight ratio of 1:1 was added to the concentrated cell and spore suspension and thoroughly mixed. The mixture was then placed in an iron pan and dried at 60 °C overnight until the moisture content was <10% (w / w). The dried SiO2-excipiented PPX-13 high-viability mature spore concentrated spore powder was then uniformly mixed using a mixer at 1,000 rpm for 30 seconds. This completed the production of the PPX-13 high-viability mature spore concentrated spore powder. The total effective viable microorganisms and the total effective viable mature spore populations resistant to 80 °C and 100 °C were then detected, and the powder was sealed and stored in a dry place at room temperature.

[0115] Table 13. Detection of moisture, total viable microorganisms, and total viable spore population in three batches of high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulant isolate PPX-13 high viable mature spore count centrifuged concentrated bacterial powder produced by fermentation trial using a 7 L small fermenter with 5 L of high viable spore count and high stress resistance, and subsequent powder preparation trial using SiO2 excipients.

[0116] A Different capital letters in the shoulder labels of data in the same column indicate significant differences (Tukey, P <0.05), identical or no letters indicate no significant difference (Tukey, P ≥ 0.05).

[0117] a Different lowercase letters in the shoulder labels of peer data indicate significant differences (Tukey, P <0.05), identical or no letters indicate no significant difference (Tukey, P ≥ 0.05).

[0118] Each experiment was performed in at least three replicates, and the data are expressed as mean ± standard deviation (mean ± SD, n ≥ 3).

[0119] Table 14 Preservation test and survival rate of high-concentration, high-quality spore powder of Bacillus PPX-13, a high-temperature resistant, multifunctional tea tree phosphate-solubilizing biocontrol agent. Note 1: The number of effective microbial colonies with obvious phosphate-solubilizing transparent zones per gram of the tested high-concentration, high-quality spore powder of the heat-resistant, multifunctional tea tree phosphate-solubilizing biocontrol Bacillus coagulans isolate PPX-13, measured on the production day (day 0) after treatment in a 100 ℃ water bath for 10 minutes, was 2.13 ± 0.16 × 10⁻⁶. 9 CFU g -1 (Its moisture content is 2.90 ± 0.33%, w / w).

[0120] Note 2: The survival rate (%) is calculated as follows: (The number of effective microbial colonies with obvious phosphorus-solubilizing transparent zones per gram of bacterial powder, measured by the BR plate counting method (incubated at 40 ℃ for 5 days) with or without a 10-fold dilution and treated in a water bath at 80 or 100 ℃ for 10 minutes, is divided by the number of effective microbial colonies with obvious phosphorus-solubilizing transparent zones per gram of bacterial powder, measured on the corresponding production day (day 0), with or without a 10-fold dilution and treated in a water bath at 80 or 100 ℃ for 10 minutes) × 100%.

[0121] 1. Stored in a general sealed biological material and product storage room, without any temperature control equipment or outdoor sunlight, and without any volatile chemicals, but with exhaust equipment for ventilation and dehumidification, and a small amount of indoor white LED light for illumination.

[0122] A,B Different capital letters in the shoulder labels of data in the same column indicate significant differences (Tukey, P <0.05), identical or no letters indicate no significant difference (Tukey, P ≥ 0.05).

[0123] a,b,c,d,e Different lowercase letters in the shoulder labels of peer data indicate significant differences (Tukey, P <0.05), identical or no letters indicate no significant difference (Tukey, P ≥ 0.05).

[0124] Each experiment was performed in at least three replicates, and the data are expressed as mean ± standard deviation (mean ± SD, n ≥ 3).

[0125] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing Bacillus coagulans spore fermentation broth, characterized in that, Includes the following steps: (1) Microaerobic fermentation culture stage: Bacillus coagulans is cultured under conditions of no feeding and no additional pressure to achieve the proliferation of vegetative somatic cells; (2) Aerobic transspore stage: When the pH value of the fermentation broth in step (1) stops decreasing and begins to rise, switch to the fermentation culture conditions of feeding, aeration and pressurization to induce the formation of spores by the vegetative cells.

2. The method according to claim 1, characterized in that, When fermentation has been going on for 16-20 hours, switch from step (1) to step (2).

3. The method according to claim 1, characterized in that, The common fermentation conditions for steps (1) and (2) are: 20-50℃, initial pH of the culture medium 5.5-8.0, and 100-150 rpm; preferably 40℃, initial pH of the culture medium 6.5, and 150 rpm. The fermentation parameters can be adjusted appropriately according to the fermentation system and equipment used in fermentation production.

4. The method according to claim 1, characterized in that, The feeding and aeration rate in step (2) is 0.5-1 vvm, and the pressurization pressure is 0-0.1 MPa; preferably 0.5 vvm and 0.1 MPa, and the appropriate fermentation parameters can be adjusted according to the fermentation system and equipment during fermentation production.

5. The method according to claim 1, characterized in that, The fermentation medium used in steps (1) and (2) comprises glucose, ammonium chloride, sodium chloride, yeast extract, dipotassium hydrogen phosphate and magnesium sulfate.

6. The method according to claim 5, characterized in that, The fermentation medium used in steps (1) and (2) is: 1 g / L glucose, 10 g / L ammonium chloride, 5 g / L sodium chloride, 10 g / L industrial-grade yeast extract, 2 g / L dipotassium hydrogen phosphate and 0.1 g / L magnesium sulfate.

7. A method for producing Bacillus coagulans spore powder, characterized in that, The method includes: (a) Producing spore fermentation broth using the method described in any one of claims 1-6; (b) The spore fermentation broth is post-treated to obtain Bacillus coagulans spore powder.

8. The method according to claim 7, characterized in that, The post-processing in step (b) includes: centrifuging to collect the cells and spores, mixing with excipients, and drying until the moisture content of the powder is less than 10% (w / w); Preferably, the excipient is silicon dioxide.

9. The method according to any one of claims 1-8, characterized in that, The Bacillus coagulans strain is PPX-13, with accession number CGMCC No. 7.

608.

10. The Bacillus coagulans spore powder prepared according to the method of claim 9, characterized in that, After being treated in a 100°C water bath for 10 minutes, the bacterial powder had a viable spore count greater than or equal to 2.0 × 10⁻⁶. 9 Effective live spores / g.

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