Actinomycete preparation and use thereof

Abstract

The present invention relates to an actinomycete preparation and the use thereof. The actinomycete preparation is a product obtained by culturing actinomycetes with insect frass. Upon storage for a period of five months, six months, or one year or longer, the content of the actinomycetes in the actinomycete preparation is 1×1011 CFU / g or more.

Description

An actinomycete preparation and its application Technical Field

[0001] This invention relates to the field of microorganisms, and particularly to the field of microbial fertilizers. Background Technology

[0002] Soil pollution and degradation pose serious environmental challenges globally. Under conditions of high-density farming and no fallow periods, soil organic matter declines, soil ecosystems become fragile, and soil-borne diseases become more severe. Therefore, while ensuring food production, seeking effective ways to replenish soil organic matter and restore soil ecological balance has become a primary requirement for sustainable agricultural development.

[0003] Replenishing organic matter is crucial for maintaining soil health and the balance of the microbial community. It can improve soil structure, promote microbial activity, enhance soil fertility, and help reduce the occurrence of soil-borne diseases. Actinomycetes, as an important class of soil microorganisms, can decompose organic matter, releasing nutrients from the soil and thus increasing soil organic matter content and improving soil fertility. They also have functions such as synthesizing secondary metabolites and inhibiting pathogenic microorganisms, thereby promoting plant growth, improving plant resistance, and maintaining soil ecological balance and plant health.

[0004] However, the production and preparation of high-content actinomycete preparations is currently a technical challenge. Existing products have low spore content. For example, the number of spores in actinomycete fertilizers ranges from several thousand to hundreds of millions per gram of preparation. Furthermore, the variety and quantity of existing products are far lower than those of easily produced Bacillus products.

[0005] In addition, the growth rate of actinomycetes is slow and the yield is low when producing actinomycetes on common culture media. They are also easily affected by external contamination during the production process, which limits the production and application of such products. Summary of the Invention

[0006] One aspect of the present invention provides an actinomycete preparation, which is the product of culturing actinomycetes with insect excrement. That is, the actinomycete preparation includes actinomycetes and insect excrement used to culture the actinomycetes. It should be noted that, in practice, the insect excrement in the actinomycete preparation is the insect excrement obtained after the actinomycetes have grown using it as a culture medium.

[0007] In one specific embodiment, the insect excrement is the excrement of the white-spotted scarab beetle and / or the excrement of the silkworm.

[0008] In one specific embodiment, the insect excrement is the excrement of white-spotted flower beetle larvae and / or silkworm larvae.

[0009] In one specific embodiment, the content of the actinomycetes in the actinomycete preparation is 1×10⁻⁶. 11CFU / g or higher. For example, this content is the content after storage for five months, six months, or one year or more.

[0010] In one specific embodiment, the content of the actinomycetes in the actinomycete preparation is 6.5 × 10⁻⁶. 11 CFU / g or higher. For example, this content is the content after storage for five months, six months, or one year or more.

[0011] In one specific embodiment, the content of the actinomycetes in the actinomycete preparation is 2 × 10⁻⁶. 12 CFU / g or higher. For example, this content is the content after storage for three months, five months, six months, or one year or more.

[0012] In one specific embodiment, the content of the actinomycetes in the actinomycete preparation is 3 × 10⁻⁶. 12 CFU / g or higher. For example, this content is the content after two, three, five months or more of storage.

[0013] In one specific embodiment, the content of the actinomycetes in the actinomycete preparation is 4 × 10⁻⁶. 12 CFU / g or higher. For example, this content refers to the content after storage for one month, two months, three months, five months or more.

[0014] In one specific embodiment, the content of the actinomycetes in the actinomycete preparation is 4.8 × 10⁻⁶. 12 CFU / g or higher. For example, this content refers to the content after storage for one month, two months, three months or more.

[0015] In one specific embodiment, the content of the actinomycetes in the actinomycete preparation is 6 × 10⁻⁶. 12 CFU / g or higher. For example, this content is the content at the end of fermentation or after storage for one, two, three months or more.

[0016] In one specific embodiment, the content of the actinomycetes in the actinomycete preparation is 8 × 10⁻⁶. 12 CFU / g or higher. For example, this content is the content at the end of fermentation or after storage for one, two, three months or more.

[0017] In one specific embodiment, the actinomycetes are at least one of actinomycetes from feces, soil, or plant rhizosphere soil.

[0018] In one specific embodiment, the fecal-derived actinomycetes are at least one of the following: actinomycetes derived from insect larvae feces, actinomycetes derived from mammal feces, and actinomycetes derived from poultry feces.

[0019] In one specific embodiment, the fecal-derived actinomycetes are at least one of the following: actinomycetes derived from the feces of white-spotted flower beetle larvae, actinomycetes derived from the feces of silkworm larvae, actinomycetes derived from cow dung, actinomycetes derived from sheep dung, actinomycetes derived from chicken dung, actinomycetes derived from duck dung, and actinomycetes derived from goose dung.

[0020] The second aspect of the present invention provides a method for preparing an actinomycete preparation as described in any one of the first aspects of the present invention, comprising the following steps:

[0021] 1) Sterilize the insect excrement to obtain sterilized insect excrement;

[0022] 2) Sterilize the water to obtain sterilized water;

[0023] 3) Actinomycete spores are mixed with sterilized water as seeds and inoculated into the insect excrement. After cultivation, the actinomycete preparation is obtained.

[0024] In one specific embodiment, the inoculum amount of the actinomycete spores is 6 × 10⁻⁶. 10 CFU / g of dry insect excrement or higher.

[0025] In one specific embodiment, the inoculum amount of the actinomycete spores is 1.8 × 10⁻⁶. 11 Up to 5.4×10 11 CFU / g dried insect feces.

[0026] In one specific embodiment, the actinomycetes are first cultured in a petri dish using a solid culture medium, and then spores are collected. The spores are then prepared into an aqueous suspension and inoculated into sterilized insect excrement, and cultured to obtain actinomycete seeds. The actinomycete seeds are mixed with sterilized water to obtain a seed suspension. The seed suspension is then inoculated again into fresh, sterilized insect excrement and cultured to obtain the actinomycete preparation. The inoculation amount of the actinomycete seeds is 3 wt% to 9 wt%, with the mass of the re-inoculated insect excrement representing 100%.

[0027] In one specific embodiment, the mass ratio of the insect excrement to the sterilized water is 5:(2 to 3).

[0028] In one specific implementation, the culture temperature is 28 to 30 degrees Celsius, and the culture time is 5 to 7 days.

[0029] In one specific embodiment, in step 1), the insect excrement is sterilized by steam for 15 to 30 minutes; in step 2), the water is sterilized by ultraviolet light for 30 to 1 hour.

[0030] In one specific embodiment, the actinomycetes are at least one of actinomycetes from feces, soil, or plant rhizosphere soil.

[0031] In one specific embodiment, the fecal-derived actinomycetes are at least one of the following: actinomycetes derived from insect larvae feces, actinomycetes derived from mammal feces, and actinomycetes derived from poultry feces.

[0032] In one specific embodiment, the fecal-derived actinomycetes are at least one of the following: actinomycetes derived from the feces of white-spotted flower beetle larvae, actinomycetes derived from the feces of silkworm larvae, actinomycetes derived from cow dung, actinomycetes derived from sheep dung, actinomycetes derived from chicken dung, actinomycetes derived from duck dung, and actinomycetes derived from goose dung. Beneficial effects

[0033] This invention solves the problems of poor aeration, slow sporulation, low spore yield, and poor stability in existing culture media. The actinomycete culture medium provided by this invention possesses the characteristics of a specific actinomycete culture medium. Compared to other culture media, it is less susceptible to contamination by bacteria and fungi other than actinomycetes, especially those derived from insect excrement and soil, resulting in higher sporulation yields and faster sporulation rates. Furthermore, the CFU content becomes more stable with prolonged storage. It also boasts advantages such as inexpensive and readily available raw materials and a simple method for preparing the culture medium. In addition, the insect excrement, a minor component in the actinomycete preparation of this invention, can be used as a fertilizer or a growth-promoting and stress-resistance component for plants. Attached Figure Description

[0034] Figure 1 shows the results of spore counts (CFU / g) of actinomycetes cultured from different insect excrement pellets.

[0035] Figure 2 shows the growth of the tested Bacillus strains on different culture media.

[0036] Figure 3 shows the growth of pathogenic fungi on insect excrement extract culture medium.

[0037] Figure 4 shows the effect of different insect excrement sterilization methods on the number of microorganisms in insect excrement.

[0038] Figure 5 shows the impact of different water sterilization methods on the number of microorganisms in water.

[0039] Figure 6 shows the effect of inoculum size on the number of spores in the actinomycete preparation.

[0040] Figure 7 shows the effect of moisture content on the number of spores in the actinomycete preparation.

[0041] Figure 8 shows a comparison of the average spore number at which X15 reaches its highest value on different solid particles.

[0042] Figure 9 shows the time it takes for X15 to reach its maximum value on different solid particles.

[0043] Figure 10 shows a comparison of the average number of spores on day 20 after X15 growth on different solid particles.

[0044] Figure 11 shows the change in spore number after the actinomycete preparation has been stored for a long time. Detailed Implementation

[0045] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0046] Unless otherwise specified, all reagents used in the embodiments of this invention are commercially available.

[0047] LB (Luria-Bertani) medium: water, 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH=7.2.

[0048] PDA (Potato Dextrose Agar) medium: water, potato 200 g / L, glucose 20 g / L, agar 20 g / L, pH=7.2.

[0049] Straw powder culture medium: water, corn straw powder 20 g / L, agar 20 g / L, pH=7.2.

[0050] Insect droppings: White Spotted Flower Chafer (WSFC) larvae droppings, Silkworm (SW) larvae droppings, Yellow Meal Worm (YMW) larvae droppings, and Rhinoceros Beetle (RB) larvae droppings.

[0051] Granular culture media: soybean meal medium, corn flour medium and cow dung medium.

[0052] The tested actinomycete strains included: seven strains isolated from the excrement of white-spotted flower beetle larvae fed with wheat straw, namely six strains of *Streptomyces albidoflavus* (X11, X13, X14, X15, X16, X17) and one strain of *Nocardiopsis alba* (X12); three strains isolated from the excrement of *WSFC* larvae fed with corn straw, namely *Streptomyces albidoflavus* 43, *Cellulosimicrobium funkei* 4, and *Streptomyces albidoflavus* 23; and six non-excrement-derived actinomycetes, including *Microbacterium* pseudo-drug-resistant *Microbacterium* from bovine dung. Streptomyces pseudoresistens) 22185, soil-derived Streptomyces p. 1069, cotton rhizosphere soil-derived Streptomyces p. H33, cotton rhizosphere Streptomyces p. H22, tomato endophytic Streptomyces p. 424 and tomato endophytic Streptomyces p. 383.

[0053] The tested Bacillus strains included 21 standard Bacillus strains: Bacillus atrophaeus B01, B05, B13 and B07, Bacillus tequilensis B21 and K01, Bacillus vallismortis B36 and S01, Bacillus halodurans B18, B25 and B34, Bacillus mojavensis B02, Bacillus subtilis B19, B23, B29, B30 and B33, and Bacillus amyloliquefaciens B16, B27, B37 and B39.

[0054] Eight plant pathogenic fungal strains were tested: *Sclerotium rolfsii* Sr (causing white mold of peanut), *Fusarium asiaticum* Fa (causing Fusarium asiaticum of wheat), *Fusarium oxysporum* Fo (causing damping-off of rice), *Fusarium verticillioide* Fv (causing root rot of maize), *Colletotrichum orbiculare* Co (causing anthracnose of cucumber), *Stagonosporopsis cucurbitacearum* Sc (causing vine blight of pumpkin), *Rhizoctonia solani* Rs (causing sheath blight of rice), and *Corynespora cassiicola* Cc (causing leaf spot of cucumber). Example 1: Comparison of Actinomycetes Cultured by Solid-State Fermentation of Different Insect Excrement Pellets

[0055] Granular insect excrement from different sources, including excrement from white-spotted flower beetle larvae, excrement from two-pronged rhinoceros beetle larvae, and excrement from yellow mealworm larvae, was dried to a moisture content of less than 3% and then sterilized by steaming at 121 degrees Celsius for 20 minutes.

[0056] Actinomycete spores were prepared into 1×10⁻⁶ solutions using sterile water. 8 CFU / g spore suspension was added to different insect excrement at a rate of 0.5 ml per gram of insect excrement and cultured at 28 degrees Celsius for 3 days to produce white spores, thus obtaining the insect excrement-spore mixtures.

[0057] During the cultivation process, it was found that when using mealworm excrement for cultivation, fungal contamination was likely to occur, indicating that it was not suitable for use as an actinomycete fermentation material. The excrement of the two-forked rhinoceros beetle was relatively large and more prone to breakage. The excrement of the white-spotted flower beetle and silkworm excrement were of moderate size, with greater structural strength and less prone to breakage. As a result, the fermentation body had better air permeability, and the white spores of the actinomycetes produced were more obvious (i.e., more spores were produced).

[0058] The spore count in the insect excrement-spore mixture after 3 days of culture was determined using colony forming units (CFUs). This involved serially diluting each insect excrement-spore mixture with sterile water, and then inoculating the appropriate dilution onto mineral humic acid (HA) agar plates using the spread plating method. After 3 days of incubation at 28°C, the number of colonies on the plates was directly counted to calculate the spore count per gram of insect excrement-spore mixture. The results are shown in Figure 1. Figure 1 shows that for the same actinomycete strain, the excrement from the white-spotted beetle produced more spores than other insect excrement strains, followed by silkworm excrement. Therefore, both white-spotted beetle excrement and silkworm excrement are suitable as fermentation media for actinomycetes, especially for those derived from feces, soil, or plant rhizosphere soil. Example 2: Culturing bacteria and fungi other than actinomycetes in insect excrement extract culture medium

[0059] Extraction of soluble insect excrement: Take 100 g of dried white star flower beetle larvae excrement (moisture content below 3%) and add it to a 1L conical flask. Make up the volume to 1L with water and extract at 80 degrees Celsius for 2 hours. Centrifuge to remove insoluble matter and obtain the extract. Spray dry the extract to obtain the extract powder.

[0060] Solid medium containing 1% WSFC (White-spotted Flower Beetle) larval excrement extract: Weigh 10g of dried WSFC larval excrement extract powder, add 15g of agar powder, and bring the volume to 1L. Sterilize by steaming at 121°C for 20 min to prepare 1% WSFC larval excrement extract solid medium. Before use, pour 20ml of the 1% WSFC larval excrement extract solid medium into a 90mm diameter petri dish.

[0061] Bacillus was prepared into 1×10 8 CFU / ml suspensions were spread onto 1% WSFC insect feces extract solid medium, 200 μL per 9 cm diameter petri dish. The dishes were incubated at 28°C for 3 days. Medium blocks were then punched out using a 1 cm diameter punch and placed in 50 ml centrifuge tubes. The resulting spores were resuspended in 40 ml of 0.1% Tween aqueous solution and cultured on LB agar plates to determine the CFU count. The results are shown in Figure 2. The results indicate that Bacillus growth on WSFC insect feces extract medium is very weak, suggesting that WSFC insect feces extract medium is unsuitable for the growth of highly resistant Bacillus, but exhibits good selectivity for actinomycetes.

[0062] Eight pathogenic fungi were inoculated onto 1% WSFC insect frass extract medium, straw powder medium, and PDA medium, respectively, and cultured at 28°C. Growth was recorded, with actinomycetes X15 serving as a control. Figure 3 shows the growth of pathogenic fungi on WSFC insect frass extract medium and other media. Compared to PDA and straw media, the pathogenic fungi showed weaker or almost no growth on WSFC insect frass extract medium. Combined with the results of Example 1, this demonstrates that WSFC insect frass exhibits good selectivity for actinomycetes, especially those isolated from insect frass, animal frass, soil, and plant rhizosphere soil. Example 3: Screening of sterilization methods for insect excrement

[0063] Dried frass of the white-spotted beetle (moisture content below 3%) was placed in 500mL Erlenmeyer flasks at a rate of 200g and sterilized by dry heat at 100°C for 30min. One Erlenmeyer flask constituted one replicate, and three replicates were set up. This sterilization procedure is abbreviated as WSFC-S1.

[0064] Dried frass of the white-spotted flower beetle (moisture content below 3%) was placed in 500mL Erlenmeyer flasks at a rate of 200g and microwaved for 3 minutes. One Erlenmeyer flask constituted one replicate, and three replicates were set up. This sterilization procedure is abbreviated as WSFC-S2.

[0065] Dried frass of the white-spotted beetle (moisture content below 3%) was placed in 500mL Erlenmeyer flasks at a rate of 200g. The flasks were then steam-sterilized at 121°C for 15min and 20min, with one Erlenmeyer flask constituting one replicate, and three replicates were performed. This sterilization procedure is abbreviated as WSFC-S3 (15 min) and WSFC-S4 (20 min).

[0066] Unsterilized dried frass of the white-spotted beetle (moisture content below 3%) was used as a control (CK). Sterilized dried frass of the white-spotted beetle was added to sterilized water at a solid-liquid mass ratio of 1:10 and vortexed for 1 min. 200 μL of the sample was then spread on a solid LB agar plate and incubated at 37°C for 12 h. The experimental results are shown in Figure 4.

[0067] According to the LB culture results in Figure 4, the feces of the white-spotted beetle contain a rich variety of microorganisms. However, after treatment with four sterilization methods, the amount of microorganisms decreased to varying degrees. Among the three sterilization methods, steam sterilization at 121 degrees Celsius was the most effective. Example 4: Screening of water sterilization methods

[0068] In industrial production, if the requirements for water quality are not high, untreated raw water (such as tap water) can be used as production water and treated with ultraviolet sterilization lamps to meet production needs and reduce production costs.

[0069] In this embodiment, 20L of tap water was added to a plastic bucket, and an 11W ultraviolet (UV) sterilization lamp was placed in the water for irradiation at different times: 10 s, 30 s, 3 min, 5 min, 10 min, 30 min, and 1 h, to test the sterilization effect of the UV lamp on microorganisms in the raw water. After sterilization, the water was stirred with a sterile glass rod, and 200 μL of the water sample was spread onto a solid LB agar plate and incubated at 37°C for 12 h. Unsterilized raw water was used as a control (CK). The experimental results are shown in Figure 5.

[0070] Figure 5 shows that the raw water contained few microorganisms; after irradiation with ultraviolet lamps, the growth of microorganisms was significantly inhibited, especially when the irradiation time reached more than 30 minutes, there was almost no microbial growth on the LB plates, indicating that the ultraviolet sterilization effect was significant. Example 5: Effect of inoculum size on the number of spores in actinomycete preparations

[0071] To investigate the effect of inoculum size on the number of spores in actinomycete preparations, this example uses actinomycete X15 as an example. By adjusting the inoculum size, its effect on spore proliferation was observed and analyzed.

[0072] The culture medium was the same as that used in Example 2, consisting of a solid culture medium containing white star flower beetle extract.

[0073] Actinomycete culture: Inoculate Actinomycetes X15 onto the above culture medium and incubate at 30 degrees Celsius for 5 days until the plate surface is covered with spores.

[0074] Spore suspension preparation: Scrape the spores from the plate and add them to sterile water, adjusting the concentration to 1×10⁻⁶. 8 CFU / mL was used to prepare a spore suspension.

[0075] Seed preparation: The above spore suspension was evenly inoculated into the dried frass of the white-spotted flower beetle larvae at a concentration of 0.5 ml / g of insect frass (sterilized by steam at 121°C for 20 min). After inoculation, the spores were incubated at 30°C for 5 days to allow for sufficient spore growth and reproduction in the frass, resulting in a seed culture containing 6 × 10⁶ spores. 12 Seeds containing CFU / g of dried insect excrement.

[0076] Inoculation amount setting: Based on the mass of dried larval feces of white-spotted flower beetle, 100% of the inoculation amount is used. According to the different inoculation amounts listed in Figure 6, the prepared seeds are suspended in 10mL of sterilized water, and then inoculated into 20g of dried larval feces of white-spotted flower beetle that has been steam-sterilized at 121 degrees Celsius for 20 minutes, and then thoroughly mixed.

[0077] Fermentation culture: The inoculated samples were placed in an incubator at 30 degrees Celsius and cultured statically for 5 days. The number of spores in each white star flower beetle larvae frass treatment group was taken and the number of spores was calculated after 5 days of culture. The data were significantly different at the P<0.05 level after one-way multiple comparison of variance. The results are shown in Figure 6.

[0078] As shown in Figure 6, when the inoculation amount is 1% (6×10⁻⁶), the inoculation rate is 1% (6×10⁻⁶). 10 The CFU / g dry insect excrement content was increased to 3% (1.8×10). 11 At a concentration of CFU / g dry insect excrement, sporulation significantly increased (P<0.05), indicating that increasing the inoculum amount had a significant impact on increasing the number of sporulations. With further increases in inoculum amount to 5% (3×10⁻⁶), sporulation increased further. 11 CFU / g dry insect excrement), 7% (4.2×10 11 CFU / g dry insect excrement) or 9% (5.4×10) 11 Although the amount of spores produced (CFU / g dry insect excrement) is still increasing, the rate of increase is gradually decreasing. Example 6: Effect of moisture content on the number of spores in actinomycete preparations

[0079] To investigate the effect of moisture content on the number of spores in actinomycete preparations, this example uses actinomycete X15 as an example. X15 spores were added to sterile water to prepare 4ml, 6ml, 8ml, 10ml, and 12ml solutions, each containing 1×10⁻⁶ spores. 8 Five groups of CFU spore suspensions were added to 20g of dried larval feces of *Symplocos rubescens* larvae sterilized at 121°C for 20 min, and mixed thoroughly. After static incubation at 30°C for 5 days, each sample was photographed and the colony forming units (CFU) were counted. Data showed significant differences at the P<0.05 level after one-way multiple comparisons of variance (Figure 7).

[0080] As shown in Figure 7, when the moisture content is between 28.57% and 37.50% (with the total mass of water suspension + dried insect excrement as 100%), the number of spores of actinomycetes is relatively large and the growth is most ideal. Example 7: Evaluation of sporulation rate and spore content of actinomycetes on solid particle culture medium

[0081] Six solid granular culture media (fragments of white-spotted flower beetle larvae, feces of yellow powdery beetle larvae, feces of two-forked rhinoceros beetle larvae, soybean meal, corn flour, and cow dung) were steam-sterilized at 121 degrees Celsius for 20 minutes.

[0082] Actinomycete X15 was prepared to contain 1×10⁻⁶ mmol / L in 10 ml. 8CFU spore suspensions were evenly inoculated into 300g of each of the above-mentioned solid particle culture media and fermented at 30°C. Samples were taken on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, and 20 after inoculation, with three replicates for each treatment. The number of spores in the culture media was counted, and the average number of spores reaching the maximum value of X15 on different solid particles was calculated (Figure 8). The time it took for X15 to reach the maximum value on different solid particles was calculated (Figure 9). The average number of spores reaching the maximum value of X15 on different solid particles was calculated (Figure 10).

[0083] As shown in Figure 8, the average spore count of actinomycetes cultured from the feces of the white-spotted beetle can reach up to 8.4 × 10⁻⁶. 12 The CUF / g was significantly higher than that of other culture media (p < 0.05), indicating that the actinomycetes produced a large number of spores on the spore pellets of the white star flower beetle.

[0084] As shown in Figure 9, X15 reached the maximum number of spores first after growing on the feces of the white-spotted beetle for 7 days, earlier than all other solid particles, indicating that the actinomycetes on the feces of the white-spotted beetle have a fast growth rate and spore production rate.

[0085] As shown in Figure 10, X15 still has a high number of spores on the feces of the white-spotted beetle, which is significantly higher than other culture media (p < 0.05), which is beneficial to prolonging the stable survival period of the spores.

[0086] In summary, the excrement of the white-spotted beetle exhibits excellent performance in all the above measurements, such as rapid sporulation, high spore content, and relatively stable spore number. Therefore, the excrement of the white-spotted beetle can be used as a culture medium for the rapid and stable growth and sporulation of actinomycetes, and actinomycete preparations can be prepared based on it. Example 8: Production of Actinomycete Preparations and Stability of Actinomycete Spores in Actinomycete Preparations

[0087] Environmental preparation: Use ultraviolet lamps to disinfect the sterile room to ensure a sterile environment. Set the temperature to 30 degrees Celsius and maintain the humidity at 90%.

[0088] Material sterilization: Dry frass of white-spotted flower beetle larvae (moisture content below 3%) was sterilized in a steam sterilizer at 121 degrees Celsius for 20 minutes; tap water was sterilized by ultraviolet light irradiation for 30 minutes.

[0089] Spore suspension preparation: Actinomycetes were inoculated onto alkaline lignin medium and cultured at 30°C for 5 days. After the spores had fully colonized, they were added to sterile water to adjust the spore content, preparing a spore concentration of 1×10⁻⁶. 8 A spore suspension of CFU / mL.

[0090] First-grade seed preparation: Add 20g of sterilized dried larvae of the white-spotted flower beetle to a 9cm petri dish, and add 10mL of actinomycete X15 spore suspension. Incubate at 30 degrees Celsius for 5 days.

[0091] Secondary seed preparation: Add 200g of sterilized dried frass of the white-spotted flower beetle larvae to a 1L beaker, along with 100mL of sterile water and 6g of primary seeds (3% inoculum). Incubate at 30°C for 5 days. Multiple 1L beakers can be used to prepare a large quantity of secondary seeds, with a spore content of 7.3 × 10⁻⁶. 12 CFU / g.

[0092] Shallow tray fermentation: Add 3 kg of sterilized dried larvae of the white-spotted flower beetle to each sterilized shallow tray. Add an inoculum solution made by mixing 1.5 L of sterilized water with 90 g of secondary seeds to the tray, stir thoroughly, and then place in a sterile room for 30 minutes.

[0093] Incubate at 7 degrees Celsius for 7 days. After fermentation, directly package the bacteria into 3kg bags, preparing a total of 50 bags of actinomycete preparation.

[0094] The above-mentioned actinomycete preparation was placed under ambient temperature conditions, left open, and samples were taken on the day of packaging. Then, samples were taken every month to test for actinomycete CFU. The results are shown in Figure 11. Although the CFU level decreased to some extent over time, the rate of decrease slowed down with increasing time. The relationship between storage time x (number of months) and CFU level y (10...) is significant.

[0095] 12 The relationship between CFU / g and the function y = 7.3929e follows an exponential function. -0.202x (R) 2 = 0.992). According to this function, the CFU content will tend to stabilize as the storage time increases. According to this function, the CFU content after 12 months of storage is approximately 6.5 × 10⁻⁶. 11 CFU / g. Industrial applicability

[0096] Compared to other culture media, the actinomycete culture medium used in the actinomycete preparation of this invention is less susceptible to contamination by bacteria and fungi other than actinomycetes. In particular, actinomycetes derived from insect excrement and soil exhibit high sporulation rates and stable CFU content with prolonged storage. Furthermore, it offers advantages such as inexpensive and readily available raw materials and simple culture medium preparation methods. In addition, the insect excrement, a minor component in the actinomycete preparation of this invention, can be used as a fertilizer or a growth-promoting and stress-resistance component for plants.

Claims

1. An actinomycete preparation, which is a product of culturing actinomycetes with insect excrement.

2. The preparation of Actinomyces according to claim 1, characterized by The insect excrement is the excrement of the white-spotted scarab beetle and / or the excrement of the silkworm beetle. Preferably, the insect excrement is the excrement of white-spotted flower beetle larvae and / or silkworm larvae.

3. The preparation of Actinomyces according to claim 1, characterized by, The content of the actinomycete in the actinomycete preparation is 1 x 10 11 CFU / g or more; Preferably, the content of said actinomycetes in said actinomycetes preparation is of 6.5 x 10 11 CFU / g or more; Preferably, the content of said actinomycetes in said actinomycetes preparation is 2 x 10 12 CFU / g or more.

4. The preparation of Actinomyces according to claim 1, characterized by, The actinomycetes are at least one of the actinomycetes from feces, soil, or plant rhizosphere soil. Preferably, the fecal-derived actinomycetes are at least one of the following: actinomycetes derived from insect larvae feces, actinomycetes derived from mammal feces, and actinomycetes derived from poultry feces. Preferably, the actinomycetes from feces are at least one of the following: actinomycetes from the feces of white-spotted flower beetle larvae, actinomycetes from the feces of silkworm larvae, actinomycetes from cow dung, actinomycetes from sheep dung, actinomycetes from chicken dung, actinomycetes from duck dung, and actinomycetes from goose dung.

5. A method for preparing the actinomycete preparation according to any one of claims 1 to 4, comprising the following steps: 1) Sterilize the insect excrement to obtain sterilized insect excrement; 2) Sterilize the water to obtain sterilized water; 3) Actinomycete spores are mixed with sterilized water as seeds and inoculated into the insect excrement. After cultivation, the actinomycete preparation is obtained.

6. The method of claim 5, wherein, The inoculation amount of the actinomyces spores is 6 x 10 10 CFU / g dry insect feces or more; Preferably, the inoculum of actinomycete spores is between 1.8 x 10 11 and 5.4 x 10 11 CFU / g of dry insect feces.

7. The method of claim 5, wherein, First, the actinomycetes are cultured in a petri dish using a solid culture medium, and then spores are collected. The spores are prepared into an aqueous suspension and inoculated into sterilized insect excrement, and cultured to obtain actinomycete seeds. The actinomycete seeds are mixed with sterilized water to obtain a seed suspension. The seed suspension is then inoculated again into fresh, sterilized insect excrement and cultured to obtain the actinomycete preparation. The inoculation amount of the actinomycete seeds is 3 wt% to 9 wt%, based on the mass of the re-inoculated insect excrement as 100%.

8. The method of claim 5, wherein, The mass ratio of the insect excrement to the sterilized water is 5:(2 to 3).

9. The method of claim 5, wherein, The incubation temperature is 28 to 30 degrees Celsius, and the incubation time is 5 to 7 days.

10. The method of claim 5, wherein, In step 1), the insect excrement is sterilized by steam for 15 to 30 minutes; In step 2), the water is sterilized by ultraviolet sterilization for 30 to 1 hour.

Images