A simple solid high-density fermentation method of streptomyces
By optimizing the solid-state high-density fermentation method using wheat bran and scarab beetle sand as the fermentation substrate, the problems of low fermentation yield and complex operation of Streptomyces cellulose were solved, achieving efficient propagation and simplified operation, which is convenient for industrial application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
- Filing Date
- 2025-06-09
- Publication Date
- 2026-06-16
AI Technical Summary
Existing fermentation technologies for Streptomyces cellulose suffer from low fermentation yields, complex fermentation components, and cumbersome operation, making it difficult to achieve efficient propagation and industrial application.
Wheat bran and scarab beetle sand were used as fermentation substrates. The initial moisture content and inoculum amount were adjusted, the constant temperature culture conditions were controlled, and the fermentation parameters were optimized to achieve high-density fermentation with a spore content of 10¹³ CFU/g.
It significantly increased the spore content of Streptomyces cellulose to 2.48×10¹³ CFU/g, simplified the operation process, and facilitated industrial production.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial application technology, specifically relating to a high-density fermentation technology for actinomycetes, and more specifically to a simple solid-state high-density fermentation method for Streptomyces cellulosae. Background Technology
[0002] Actinomycetes are a group of prokaryotes widely distributed in soil, air, and water, primarily growing as mycelia and reproducing by spores. They play a variety of important roles in nature and have wide applications in industry and agriculture. The main functions of actinomycetes include antibiotic production, organic matter degradation, nitrogen fixation, enzyme production, vitamin production, plant growth promotion, and immune regulation. Actinomycetes are a major resource microorganism for antibiotic production, accounting for approximately 70% of antibiotics. They can decompose complex organic matter such as cellulose, lignin, and paraffin, playing a vital role in the natural material cycle. Some actinomycetes, such as *Actinomyces frankincense*, can form symbiotic nodules with plants to fix nitrogen, increasing soil fertility. Actinomycetes can produce various enzyme preparations, such as proteases, amylases, and cellulases, for industrial production. They can also produce vitamins such as vitamin B12. Actinomycetes play an important role in steroid transformation and can be used in the pharmaceutical industry. They can be used in wastewater treatment, helping to purify the environment. Actinomycetes can promote plant root growth, increasing plant yield and stress resistance. Secondary metabolites produced by actinomycetes have antibacterial, antifungal, and antitumor effects. Probiotics produced by actinomycetes can regulate intestinal microecological balance and immune responses.
[0003] The industrial application of actinomycetes is inseparable from fermentation technology. The three main optimization directions for industrial fermentation include strains, culture media, and process parameters. Once the strains are selected, the culture media and process parameters become bottlenecks in the industrialization of microorganisms, especially for live microbial agents such as microbial fertilizers and feed additives. This is because, for live microbial agents, the content of effective bacteria directly determines the quality and value of the product. Currently, the most commonly used fermentation processes include solid-state fermentation and liquid-state fermentation.
[0004] Our research team previously screened and isolated an actinomycete strain that can effectively inhibit harmful fungi such as *Streptomyces cellulosae*. After reviewing the literature, we found that there is currently limited research on the fermentation technology of this fungus, with reported fermentation yields of approximately 10... 9 CFU / g. Furthermore, related studies have shown that its fermentation components are quite complex, with ingredients such as corn flour and potassium dihydrogen phosphate added to its formula, making the process rather cumbersome. Summary of the Invention
[0005] The purpose of this invention is to provide a method for high-density solid-state fermentation of actinomycetes. This method can efficiently propagate *Streptomyces cellulosae*, achieving an actinomycete spore content of up to 10⁻⁶. 13 The CFU / g level is significantly higher than the levels reported in existing literature.
[0006] The actinomycete is *Streptomyces cellulosae*, strain number P8-2, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 32350.
[0007] The nucleotide sequence of the 16S rRNA gene of the *Streptomyces celluloseis* contains the DNA molecule shown in SEQ ID NO.1 of the sequence listing.
[0008] The nucleotide sequence of the 16S rRNA gene of *Streptomyces cellulose* was molecularly identified, and a phylogenetic tree was constructed using the NJ method as follows: Figure 1 .
[0009] The morphological characteristics of *Streptomyces cellulosae* P8-2 are as follows: after 7 days of culture on Gao's No. 1 medium, it produces abundant hyphae; the colonies are white, relatively dry, and produce lemon-yellow soluble pigment; the aerial hyphae are light gray, and the hyphal tips produce chain-like spore chains; the spores are round or ovoid (see...). Figure 2 and Figure 3 ).
[0010] The physiological and biochemical characteristics of Streptomyces cellulosae P8-2 are as follows: In the nine tested culture media, the colonies of strain P8-2 on ISP-1, ISP-4, and ISP-6 media were grayish-white to gray, while on ISP-2, ISP-3, PDA, and Gao's No. 1 media, the colonies were yellow, lemon yellow, orange, and lemon yellow. It could hardly grow on ISP-5 and ISP-7 media. P8-2 produced hyphae and spores on all culture media on which it could grow. P8-2 produces soluble pigments on ISP-2, ISP-3, PDA, and Gao's No. 1 media. The Streptomyces cellulosae strain P8-2 can utilize glucose, maltose, galactose, sucrose, trehalose, raffinose, mannitol, mannose, and xylose; it cannot utilize lactose, fructose, arabinose, sorbitol, or inositol; it can utilize glycine, tyrosine, tryptophan, threonine, asparagine, and urea, but cannot utilize histidine or asparagine.
[0011] The Streptomyces cellulose CGMCC No. 32350 or the composition containing Streptomyces cellulose CGMCC No. 32350 has at least one of the following properties:
[0012] 1) Inhibit pathogens;
[0013] 2) Preparation of pathogen inhibitors;
[0014] 3) Suppress diseases;
[0015] 4) Prepare disease inhibitors.
[0016] The pathogenic fungus is at least one of the following: Trichoderma, Fusarium, more specifically: Trichoderma koningii, T. harzianum, Fusarium equiseti;
[0017] The disease is a fungal disease of edible fungi, specifically at least one of Trichoderma and Fusarium diseases; the edible fungi include one or more of shiitake mushrooms, oyster mushrooms, wood ear mushrooms, button mushrooms, morels, and king oyster mushrooms.
[0018] On the one hand, the present invention provides a fermentation substrate for high-density solid-state fermentation of actinomycetes.
[0019] The fermentation substrate provided by this invention includes wheat bran and scarab beetle sand.
[0020] The mass ratio of wheat bran to scarab beetle sand can be 1-2:1; specifically, it can be 2:1.
[0021] The actinomycete may specifically be Streptomyces cellulosae P8-2.
[0022] On the other hand, the present invention provides a method for high-density solid-state fermentation of actinomycetes.
[0023] The method for high-density solid-state fermentation of actinomycetes provided by this invention includes the following steps:
[0024] Wheat bran and scarab beetle sand were mixed to obtain a fermentation substrate. The initial moisture content of the fermentation substrate was adjusted, and an actinomycete spore suspension was inoculated. The substrate was then cultured at a constant temperature to obtain high-density spores.
[0025] In the method described, the mass ratio of wheat bran to scarab beetle sand can be 1-2:1; specifically, it can be 2:1.
[0026] The initial moisture content of the fermentation substrate is 50%-70%;
[0027] The actinomycete may specifically be Streptomyces cellulosae P8-2;
[0028] Inoculate with 1% of 1×10 7 -1×10 8 A spore suspension of cfu / mL;
[0029] The temperature for the constant temperature culture can be 28℃;
[0030] The constant temperature incubation period can be 4-8 days, specifically 8 days.
[0031] The number of P8-2 spores in the solid fermentation substrate was measured to be 2.48 × 10⁻⁶. 13 cfu / g.
[0032] This invention investigated the effects of fermentation substrate formulation, ratio, moisture content, spore inoculation amount, fermentation temperature, and fermentation time on the spore count of actinomycete P8-2. Experimental results showed that under optimal conditions, after 8 days of fermentation, the number of P8-2 spores in the solid fermentation substrate was 2.48 × 10⁻⁶. 13 The cfu / g concentration is significantly higher than that of existing fermentation methods. Furthermore, the fermentation substrate of this invention is made from a specific ratio of wheat bran and scarab beetle sand, making the raw materials readily available, the operation simple, and easy for industrial-scale production. Attached Figure Description
[0033] Figure 1 The nucleotide sequence of the 16S rRNA gene of Streptomyces cellulose was molecularly identified, and a phylogenetic tree was constructed using the NJ method.
[0034] Figure 2 The colony morphology of Streptomyces cellulosae P8-2 after 7 days of culture on Gao's No. 1 medium.
[0035] Figure 3 Microscopic images of Streptomyces cellulosae P8-2 cells and sporulation morphology after culturing on Gao's No. 1 medium for 7 days.
[0036] Figure 4 The effect of fermentation substrate components on sporulation yield was investigated. Among them, 1 was corn cob: wheat bran = 1:1; 2 was corn cob: soybean flour = 1:1; 3 was sawdust: wheat bran = 1:1; 4 was sawdust: soybean flour = 1:1; 5 was scarab beetle sand: soybean flour = 1:1; and 6 was scarab beetle sand: wheat bran = 1:1.
[0037] Figure 5The effect of the ratio of fermentation substrate (wheat bran and scarab beetle sand) on sporulation yield was investigated. The ratios 1:1, 1:1, 1:3, 2:1, and 3:1 represent the mass ratios of wheat bran to scarab beetle sand, respectively.
[0038] Figure 6 The effect of initial substrate moisture content on sporulation rate.
[0039] Figure 7 The effect of inoculum size on sporulation.
[0040] Figure 8 The effect of temperature on sporulation.
[0041] Figure 9 The effect of fermentation time on sporulation yield. Detailed Implementation
[0042] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0044] The scarab beetle sand used in this invention was purchased from the Institute of Biotechnology, Beijing Academy of Agricultural and Forestry Sciences.
[0045] Its main components are shown in Table 1:
[0046] Table 1. Main components of scarab beetle sand
[0047]
[0048] Example 1: Isolation and strain identification of Streptomyces cellulosae P8-2
[0049] 1. Sample collection
[0050] Collected from *Stropharia macrocarpa* specimens in Fangshan District, Beijing.
[0051] 2. Isolation, screening, and antagonistic screening of strains
[0052] The strain was isolated from the cultivation material. 5 grams of fermented edible fungi material (70% poplar sawdust mixed with 30% corn cob, composted outdoors for 10 days, maintaining a moisture content of 55%-60%, and fermentation temperature of 55-60℃) was dissolved in 10 ml of sterile water and shaken at 180 rpm for 10 minutes. The strain was then isolated using conventional serial dilution plating. It was cultured on four different culture media (LB, NA, PDA, and Gao's No. 1) at 28℃. Actinomycete colonies with significant morphological differences and suitable for pure culture were selected, purified, and preserved on Gao's No. 1 medium. Antagonistic bacteria were initially screened and repeatedly screened using the plate confrontation method with *Trichoderma koningii* as the target pathogen. Finally, a strain of actinomycete with strong antibacterial activity was obtained and named P8-2.
[0053] 3. Identification of strains
[0054] Morphological, physiological and biochemical characteristics, and some conserved sequences of strain P8-2 were analyzed. Physiological, biochemical, and environmental tolerance characteristics were determined according to the methods described in "Principles and Methods of Soil Microbiology Research" (Lin Xiangui, Principles and Methods of Soil Microbiology Research [M], Higher Education Press, 2010).
[0055] The results showed that the spores of strain P8-2 were non-spiral, spherical, and oval with smooth surfaces under a 400x optical microscope. The aerial mycelia were light gray to dark gray; on Gaussian agar, the colonies were bright lemon or yellowish-lemon color with pigment penetration; on potato agar, the aerial mycelia were dark gray. The substrate mycelia were abundant and lemon-colored; the strain exhibited rapid gelatin liquefaction, strong milk coagulation and peptone formation, strong starch hydrolysis, and good growth on cellulose. It was negative for tyrosinase and H2S. The physiological and biochemical characteristics of *Streptomyces cellulosae* P8-2 were Gram-positive. It utilized glucose, galactose, sucrose, mannose, inulin, and mannitol; but not arabinose, fructose, lactose, rhamnose, inositol, sorbitol, or sodium acetate. The 16S rDNA sequence of strain P8-2 is shown in SEQ ID No. 1.
[0056] SEQ ID No. 1:
[0057] GGGGGGTGCTTACACATGCAGTCGAACGATGAACCACTTCGGTGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATACTGATCGCCTTGGGCATCCTTGGTGATCGAAAGCTCCGGCGGTGCAGG ATGAGCCCGCGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGA GGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCGAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCGCGTCGGTTGTGA AAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGA
[0058] Example 2: Screening of Fermentation Substrate and Screening of Fermentation Substrate Ratio
[0059] Streptomyces requires a suitable substrate environment to produce large amounts of sporulations. In this study, the following materials were selected as fermentation substrates: corn cob and wheat bran, corn cob and soybean flour, sawdust and wheat bran, sawdust and soybean flour, scarab beetle sand and soybean flour, and scarab beetle sand and wheat bran were mixed at a mass ratio of 1:1. The total weight of the substrate was set at 20g, and 10mL of deionized water was added to adjust the initial moisture content to 50%. After thorough mixing, the mixture was poured into 500mL Erlenmeyer flasks and sterilized at 121℃ for 40min. Subsequently, a 1% (v / v) concentration of 1×10⁻⁶ was inoculated. 7A CFU / g suspension of P8-2 spores was prepared, with three replicates for each treatment group. After incubation at 28°C for 7 days, the spore count of strain P8-2 was determined. The results are as follows: Figure 4 As shown. By Figure 4 It can be seen that the fermentation substrate prepared by mixing scarab beetle sand and wheat bran at a mass ratio of 1:1 had the highest sporulation rate, reaching 6.7 × 10⁻⁶. 9 cfu / g.
[0060] The sporulation yield of strain P8-2 in different substrates indicates that substrate type has a significant impact on sporulation capacity. Figure 4 As shown, in a composite substrate of scarab beetle sand and wheat bran, P8-2 exhibited the best sporulation performance, with a sporulation yield of 6.7 × 10⁻⁶. 9 The sporulation rate (cfu / g) showed significant differences compared to other substrates. The lowest sporulation rate (1.5 × 10⁻⁶) was observed in the corn cob and wheat bran substrates. 8 cfu / g. From the cultivation process, obvious spore formation was observed after 4–5 days of cultivation in a substrate of scarab beetle sand and wheat bran, and a uniform spore covering layer was formed after 6–7 days. Therefore, the combination of scarab beetle sand and wheat bran was determined to be the optimal solid-state fermentation substrate.
[0061] To determine the optimal fermentation substrate ratio, fermentation substrates were prepared using wheat bran and scarab beetle sand at mass ratios of 1:1, 1:2, 1:3, 2:1, and 3:1, with a total substrate mass of 20g and an initial moisture content of 50%. After thorough mixing, the substrates were sterilized at 121℃ for 40min, and then inoculated with 1% 1×10⁻⁶ ppm of a certain amount of a certain chemical solution. 7 After preparing the cfu / mL spore suspension, the cells were incubated at 28°C for 7 days. Three replicates were performed at different ratios. Sporulation of P8-2 was determined using the dilution plate count method (results are shown below). Figure 5 (As shown), the optimal ratio of fermentation substrate was selected. (From...) Figure 5 It can be seen that when wheat bran and scarab beetle sand are mixed in a 1:2 ratio in the substrate, the sporulation yield reaches 1.15 × 10⁻⁶. 10 The cfu / g ratio was 2.3-5.7 times higher than other ratios. The final determined solid-state fermentation substrate ratio for P8-2 was wheat bran to scarab beetle sand = 1:2.
[0062] Example 3: The effect of initial moisture content on fermentation
[0063] The effect of initial moisture content on fermentation was investigated: The moisture content of the substrate (wheat bran and scarab beetle sand in a mass ratio of 2:1, with a total substrate mass of 20g) was adjusted by using deionized water, with moisture content gradients of 50%, 60%, 70%, 80%, and 90%. The mixture was thoroughly mixed and poured into 500mL Erlenmeyer flasks, sterilized at 121℃ for 40min, cooled, and then inoculated with 1% 1×10⁻⁶ ppm of a certain solution. 7CFU / mL spore suspension was incubated at 28°C for 7 days in a constant temperature incubator. Different moisture content treatments were set up in triplicate. The sporulation rate of P8-2 was determined by dilution plate counting method to obtain the optimal moisture content.
[0064] Figure 6 The study demonstrated the effect of different moisture contents on the sporulation rate of the strain. Within the moisture content range of 50-70%, sporulation rate showed a positive correlation with moisture content. However, when the moisture content exceeded 70%, sporulation rate decreased sharply. This indicates that suitable substrate moisture content is a key factor in maintaining the sporulation capacity of the strain. Within the moisture content range of 50-70%, sufficient moisture without affecting substrate aeration creates favorable conditions for strain growth and sporulation. However, when the moisture content exceeds 70%, excessive humidity may lead to poor substrate aeration, inhibiting the metabolic activity of the strain and thus causing a decrease in sporulation rate.
[0065] Example 4: Effect of inoculum size on fermentation
[0066] Investigating the effect of inoculum size on fermentation: A well-mixed substrate (wheat bran and scarab beetle sand in a mass ratio of 2:1, total substrate mass of 20g, substrate moisture content of 70%) was placed into a 500mL Erlenmeyer flask, sterilized at 121℃ for 40min, cooled, and then inoculated with 1% 1×10⁻⁶ styrene solution. 6 1×10 7 1×10 8 1×10 9 CFU / mL spore suspension was incubated at 28°C for 7 days in a constant temperature incubator. Different inoculum amounts were treated in triplicate. The sporulation rate of P8-2 was determined by dilution plate counting method.
[0067] Figure 7 This study demonstrates the variation in spore production of strain P8-2 in a solid substrate under different inoculation concentration gradients. At an inoculation concentration of 1%... 6 cfu / mL, 10 7 At a spore concentration of CFU / mL, spore yield showed a significant increasing trend with increasing initial inoculum concentration; when the inoculum concentration reached 10... 7 At cfu / mL, the spore yield reached its maximum value of 1.26 × 10⁻⁶. 12 cfu / g; when the inoculation concentration exceeds 10 8 At cfu / mL, spore production decreased to 5.2 × 10⁻⁶. 11 CFU / g. This indicates that both excessively high and low inoculum concentrations are detrimental to spore germination and reproduction. The optimal inoculum concentration is 1% × 10⁻⁶. 7 When the spore concentration is CFU / mL, it can ensure rapid colonization of the strain and maximize the sporulation rate.
[0068] Example 5: The effect of temperature on fermentation
[0069] Investigating the effect of temperature on fermentation: 1% 10 7 Fermentation substrates (wheat bran and scarab beetle sand in a mass ratio of 2:1, total substrate mass of 20g, substrate moisture content of 70%) inoculated with cfu / mL were placed in constant temperature incubators at 26℃, 28℃, 30℃, and 32℃ and cultured continuously for 7 days. Each temperature treatment was replicated 3 times. The sporulation rate of P8-2 was determined by the dilution plate count method, and the optimal fermentation temperature was screened.
[0070] The changes in spore production of strain P8-2 in a solid fermentation substrate under different culture temperatures showed that the spore production reached its maximum value of 1.24 × 10⁻⁶ at a fermentation temperature of 28℃. 12 cfu / g. Sporulation initially decreased after the temperature exceeded 28℃, then began to increase again. Figure 8 (As shown).
[0071] Example 6: Determination of Fermentation Endpoint
[0072] According to the selected optimal substrate formula, ratio (wheat bran and scarab beetle sand in a mass ratio of 2:1, total substrate mass of 20g), moisture content (70%), and inoculum size (1% 1×10⁻⁶). 7 The spores were inoculated with cfu / mL and cultured in a constant temperature incubator at 28℃. Starting from day 4 of fermentation, the number of P8-2 spores in the solid fermentation substrate was measured for 6 consecutive days.
[0073] Depend on Figure 9 It can be seen that the sporulation rate of P8-2 increased rapidly from day 6, from 2.6 × 10⁻⁶ on day 6. 12 CFU / g rose to 2.48 × 10⁻⁶ on the eighth day. 13 The CFU / g level decreased after day eight because the substrate nutrients were insufficient to sustain the growth of a large number of bacterial strains, leading to autophagy and a decline in bacterial count. Therefore, day eight was chosen as the endpoint of substrate fermentation.
[0074] Example 7: Solid-state fermentation culture of actinomycete P8-2
[0075] Preparation of fermentation substrate: Mix wheat bran and scarab beetle sand at a mass ratio of 2:1, adjust the initial moisture content of the fermentation substrate to 70%, and inoculate with 1% of 1×10 8 A suspension of actinomycete P8-2 spores (cfu / mL) was cultured in a 28℃ incubator for 8 days. The number of P8-2 spores in the solid fermentation substrate was measured to be 2.48 × 10⁻⁶. 13 cfu / g.
[0076] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.
Claims
1. A method for high-density solid-state fermentation of actinomycetes, comprising the following steps: Wheat bran and scarab beetle sand were mixed to obtain a fermentation substrate. The initial moisture content of the fermentation substrate was adjusted, and a suspension of actinomycete spores was inoculated. The substrate was then cultured at a constant temperature to obtain high-density spores. The actinomycetes are Streptomyces cellulose ( Streptomyces cellulosae P8-2, its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 32350; The mass ratio of wheat bran to scarab beetle sand is 1:2; The initial moisture content of the fermentation substrate is 70%; Inoculate with 1% of 1×10 7 A spore suspension of cfu / mL; The temperature for the constant temperature incubation was 28°C. The constant temperature incubation period is 8 days; The number of P8-2 spores in the solid fermentation substrate was measured to be 2.48 × 10⁻⁶. 13 cfu / g.