Actinomycete culture medium and application thereof
By using water extracts and alkali extracts prepared from insect excrement as components of the actinomycete culture medium, the problems of slow spore growth, low spore production, and susceptibility to contamination in conventional culture media were solved. This resulted in faster spore growth, higher spore production, and reduced susceptibility to contamination, thereby improving culture efficiency and production efficiency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- RUIMAI DEXING (BEIJING) BIOTECHNOLOGY CO LTD
- Filing Date
- 2024-11-14
- Publication Date
- 2026-05-22
AI Technical Summary
Conventional actinomycete culture media result in slow spore growth, low spore production, and susceptibility to contamination, leading to time-consuming and inefficient pure culture of actinomycetes.
Insect excrement was used as the main component of the actinomycete culture medium. Water extracts and alkali extracts were prepared by water extraction or alkali extraction methods. These were combined with agar and other components to form the actinomycete culture medium. The composition of the culture medium was optimized to improve the spore growth rate and sporulation yield, and to reduce contamination by other microorganisms.
It improves the growth rate and sporulation of actinomycete spores, reduces contamination by other microorganisms, simplifies the preparation of culture media, reduces raw material costs, and improves the pure culture and production efficiency of actinomycetes.
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Figure CN122071682A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbiology, and more specifically to an actinomycete culture medium and its application. Background Technology
[0002] Actinomycetes are crucial environmental microorganisms, widely distributed in soil, water bodies, and plant surfaces. They are among the most common soil microorganisms and play a significant role in the function of soil ecosystems. Actinomycetes perform multiple functions in soil ecosystems. First, they play a key role in organic matter decomposition and carbon cycling. They can decompose complex organic matter, converting it into nutrients that plants can absorb and utilize, thus improving soil fertility. Second, actinomycetes can synthesize and release a large number of secondary metabolites, such as antibiotics, antioxidants, and enzymes, which are important for plant growth and the regulation of soil microbial communities. Furthermore, actinomycetes colonize the rhizosphere of plants through antagonism and competition with other microorganisms, directly or indirectly promoting plant growth and health.
[0003] Conventional actinomycete media, such as ISP3 medium, while widely used for the pure culture of actinomycetes, have some drawbacks and limitations. First, spore production in conventional media is slower; actinomycete spores require a longer culture time on conventional media compared to other sporulating bacteria to grow and produce sporulation, leading to the time-consuming and inefficient nature of pure actinomycete culture. Second, the sporulation yield in conventional media is relatively low, limiting the scale and efficiency of actinomycete inoculant production. Furthermore, conventional actinomycete media are susceptible to contamination by other bacteria and fungi during culture, thus affecting the growth and purification of actinomycetes.
[0004] In summary, conventional actinomycete culture media have drawbacks such as slow spore growth, low spore production, and susceptibility to contamination. Therefore, it is necessary to develop and optimize actinomycete culture media that promote rapid spore growth, high spore production, and are less susceptible to contamination, in order to improve the pure culture and production efficiency of actinomycetes. Summary of the Invention
[0005] One aspect of the invention provides the use of insect excrement as a culture medium for actinomycetes, wherein the insect is at least one insect from the family Cetoniidae and / or the family Bombycidae.
[0006] In one specific embodiment, the insect is the white-spotted flower beetle ( Protaetia ( Liocola ) brevitarsis ) and / or silkworms ( Bombyx mori ).
[0007] In one specific embodiment, the actinomycetes are at least one of actinomycetes from feces, soil, or plant rhizosphere soil.
[0008] 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.
[0009] 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.
[0010] In one specific embodiment, the insect excrement is the excrement produced by the larvae of the insect.
[0011] The second invention provides an actinomycete culture medium comprising an aqueous extract and / or an alkaline extract of the insect excrement used in any of the applications described in the first invention.
[0012] In one specific embodiment, the extraction step of the water extract includes: 1) Based on dried feces with a moisture content of less than 3%, mix feces and water at a mass ratio of 1:1 to 1:10, adjust the temperature to 40 degrees Celsius to 100 degrees Celsius, and treat for 1 hour to 16 hours to obtain water treatment solution; 2) The water treatment liquid is subjected to solid-liquid separation to obtain an aqueous extract containing the water extract.
[0013] In one specific embodiment, the step of crushing the feces is included in or before step 1).
[0014] In one specific embodiment, the extraction step of the alkaline extract includes: I) Based on dried feces with a moisture content of less than 3%, mix feces and water at a mass ratio of 1:1 to 1:10, adjust the pH value to 8 to 12, and treat at a temperature of 30 degrees Celsius to 100 degrees Celsius for 20 to 60 minutes to obtain an alkaline treatment solution. II) Adjust the pH of the alkaline treatment solution to 6 to 8, and allow it to settle at 1 to 50 degrees Celsius for 5 minutes to 48 hours to obtain a crude supernatant; III) The crude supernatant is further subjected to solid-liquid separation to obtain an alkaline extract containing the alkaline extract.
[0015] In one specific embodiment, the step of pulverizing the feces is included in or before step I).
[0016] In one specific implementation, in step 1), the temperature is 40 to 80 degrees Celsius and the processing time is 2 to 8 hours.
[0017] In one specific embodiment, in step II), the pH of the alkaline treatment solution is adjusted to 6.5 to 7.5, and the solution is allowed to settle at 15°C to 40°C for 2 to 24 hours to obtain a crude supernatant.
[0018] In one specific embodiment, step 3) is included after step 2): concentrating the aqueous extract to obtain a concentrated aqueous extract containing the aqueous extract, or drying the aqueous extract to obtain a dry powder of the aqueous extract.
[0019] In one specific embodiment, step IV is included after step III): concentrating the alkaline extract to obtain a concentrated alkaline extract containing the alkaline extract, or drying the alkaline extract to obtain alkaline extract powder.
[0020] In one specific embodiment, in step I), based on dried feces with a moisture content of less than 3%, the feces and water are mixed at a mass ratio of 1:3 to 1:6, the pH value is adjusted to 9 to 11, the temperature is 50 degrees Celsius to 100 degrees Celsius, and the treatment is carried out for 30 to 40 minutes to obtain an alkaline treatment solution.
[0021] In one specific embodiment, in step I), based on dried feces with a moisture content of less than 3%, the feces and water are mixed at a mass ratio of 1:3 to 1:6, the pH value is adjusted to 9 to 11, the temperature is 60 degrees Celsius to 90 degrees Celsius, and the treatment is carried out for 30 to 40 minutes to obtain an alkaline treatment solution.
[0022] In one specific embodiment, in step I), based on dried feces with a moisture content of less than 3%, the feces and water are mixed at a mass ratio of 1:3 to 1:6, the pH value is adjusted to 9 to 11, the temperature is 70 degrees Celsius to 90 degrees Celsius, and the treatment is carried out for 30 to 40 minutes to obtain an alkaline treatment solution.
[0023] In one specific embodiment, in step III), solid-liquid separation is achieved using at least one of a sedimentation centrifuge, an inclined plate sedimentation unit, and an inclined tube sedimentation unit to obtain an alkaline extract containing the alkaline extract.
[0024] In one specific embodiment, the actinomycete culture medium further includes water.
[0025] In one specific embodiment, the dry powder of the water extract and / or alkali extract is 0.125% to 10% by mass of water, which is 100% by mass.
[0026] In one specific embodiment, the dry powder of the water extract and / or alkali extract is 0.25% to 5% by mass of water, which is 100% by mass.
[0027] In one specific embodiment, the dry powder of the water extract and / or alkali extract is 0.25% to 1% by mass of water, which is 100% by mass.
[0028] In one specific embodiment, the dry powder of the water extract and / or alkali extract is 0.5% to 1% by mass of water, which is 100%.
[0029] In one specific embodiment, the actinomycete culture medium further includes agar.
[0030] In one specific embodiment, the agar is 1% to 5% by mass, with water accounting for 100%.
[0031] In one specific embodiment, the agar is 1.5% by mass, with water accounting for 100%.
[0032] The third invention provides the use of the actinomycete culture medium according to any one of the second invention in culturing and / or isolating actinomycetes.
[0033] In one specific embodiment, the use of the actinomycete culture medium according to any one of the second invention in culturing and / or isolating at least one of actinomycetes from insect excrement, soil, or plant rhizosphere.
[0034] The beneficial effects of the present invention are as follows: The actinomycete culture medium provided by the present invention has the characteristics of an actinomycete-specific culture medium. Compared with other culture media, it is not easily contaminated by bacteria and fungi other than actinomycetes. In particular, the sporulation of actinomycetes from insect excrement and soil is high and the sporulation rate is fast. It also has the advantages of low price and easy availability of raw materials and simple method of preparing culture medium. Attached Figure Description
[0035] Figure 1 It displays the names of actinomycetes and species from different habitats.
[0036] Figure 2 The results of spore counting of actinomycetes cultured from different insect excrement particles are shown (10 10 CFU / g).
[0037] Figure 3 The extract yields under different extraction times and temperatures are shown.
[0038] Figure 4 The carbon spectrum integral data (%) of WSFC insect excrement water extract under different extraction times and temperatures are shown.
[0039] Figure 5The carbon spectrum integral data (%) of WSFC insect excrement alkaline extract prepared by various extraction methods according to invention patent CN201611143186.1 are shown.
[0040] Figure 6 The study showed the growth and sporulation of actinomycetes from different sources on insect excrement water extract and alkaline extract media.
[0041] Figure 7 The tested Bacillus species are shown.
[0042] Figure 8 The growth of the tested Bacillus species on different culture media is shown.
[0043] Figure 9 The pathogenic fungi of the tested plant were shown.
[0044] Figure 10 The growth of different pathogenic fungi on WSFC insect excrement water extract medium was compared with that on other media.
[0045] Figure 11 The study compared the sporulation of actinomycetes cultured on conventional actinomycete culture media with those cultured on WSFC insect excrement water extract and alkaline extract media.
[0046] Figure 12 The effects of different concentrations of WSFC insect excrement extract on the culture medium for culturing actinomycetes were demonstrated.
[0047] Figure 13 The study demonstrated the effectiveness of WSFC insect excrement water extract and alkaline extract culture medium in isolating actinomycetes. Detailed Implementation
[0048] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.
[0049] Unless otherwise specified, all reagents used in the embodiments of this invention are commercially available.
[0050] Gao's No. 1 (GS) medium: soluble starch 20 g / L, potassium nitrate 1 g / L, dipotassium hydrogen phosphate 0.5 g / L, magnesium sulfate heptahydrate 0.5 g / L, chloride sodium 0.5 g / L, ferrous sulfate heptahydrate 0.01 g / L, agar 20 g / L, water, pH=7.2.
[0051] ISP3 medium: 20.0 g / L oat flour, 0.001 g / L ferric sulfate heptahydrate, 0.001 g / L manganese chloride, 0.001 g / L zinc sulfate, 20 g / L agar, water, pH=7.2.
[0052] Humic acid medium (HV): Humic acid 0.1 g / L, calcium carbonate 0.02 g / L, disodium hydrogen phosphate 0.5 g / L, magnesium sulfate heptahydrate 0.5 g / L, potassium chloride 1.7 g / L, ferrous sulfate heptahydrate 0.01 g / L, riboflavin 0.0005 g / L, thiamine 0.0005 g / L, vitamin B6 0.0005 g / L, niacin 0.0005 g / L, inositol 0.0005 g / L, pantothenic acid 0.0005 g / L, biotin 0.00025 g / L, para-aminobenzoic acid 0.0005 g / L, agar 20 g / L, water, pH=7.2.
[0053] Straw culture medium: 20 g / L straw powder, 20 g / L agar, water, pH=7.2.
[0054] Mineral humic acid (HA) culture medium: mineral humic acid 0.1 g / L, agar 20 g / L, water, pH=7.2.
[0055] Alkaline lignin medium: 5 g / L alkaline lignin, 1 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 2 g / L ammonium sulfate, 0.1 g / L ferrous sulfate, 20 g / L agar, water, pH=7.2.
[0056] Insect droppings: White Spotted Flower Chafer (WSFC) larvae droppings, Silkworm (SW) larvae droppings, Yellow Meal Worm (YMW) larvae droppings, Rhinoceros Beetle (RB) larvae droppings.
[0057] Test strains: 1 strain of Streptomyces microcyticus isolated from the excrement of WSFC larvae fed with wheat straw, 3 strains of actinomycetes isolated from the excrement of WSFC larvae fed with corn straw, 6 strains of non-excremental actinomycetes from the laboratory of Northeast Agricultural University, 6 strains of pathogenic fungi, 21 standard strains of Bacillus from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, and 2 strains of pathogenic fungi.
[0058] See Actinomycetes from different habitats and their species names. Figure 1 .
[0059] Example 1: Comparison of solid-state fermentation culture of actinomycetes using different insect excrement particles.
[0060] Granular insect excrement from different sources, including WSFC larval excrement, SM larval excrement, RB larval excrement, and YMW larval excrement, were dried to a moisture content of less than 3% and then sterilized by steaming at 121 degrees Celsius for 20 minutes.
[0061] Will Figure 1 The actinomycete spores were prepared into 1×10⁻⁶ spores using sterile water. 8 CFU / g spore suspension was added to different insect excrements at a rate of 0.5 ml per gram of excrement, and incubated at 28°C for 3 days. White spores were produced, resulting in various excrement-spore mixtures. During the incubation process, it was observed that YMW excrement was prone to fungal contamination, indicating that it is not suitable for actinomycete fermentation. RB excrement particles were larger and more prone to breakage. SW and WSFC excrement particles were of moderate size and had greater structural strength, making them less prone to breakage. Consequently, the fermentation body had better aeration, resulting in more prominent white actinomycete spores (i.e., higher spore production).
[0062] The spore count in the insect frass-spore mixture after 3 days of culture was determined by colony forming units (CFUs). This involved serially diluting each insect frass-spore mixture with sterile water, inoculating the appropriate dilution onto HA agar plates using the spread plating method, and incubating at 28°C for 3 days. The number of colonies on the plates was then directly counted to calculate the spore count per gram of insect frass-spore mixture. The results are shown below. Figure 2 . Figure 2 The results showed that for the same actinomycete strain, WSFC insect excrement produced more spores than the other three types, followed by SM insect excrement. Therefore, both WSFC and SM insect excrement are suitable as fermentation media for actinomycetes, especially for those derived from feces, soil, or plant rhizosphere soil.
[0063] Example 2: Preparation and structural composition analysis of WSFC insect excrement water extract.
[0064] Extraction of soluble components from insect excrement: 100 g of dried WSFC insect excrement (moisture content below 3%) was added to a 1 L Erlenmeyer flask, and the volume was adjusted to 1 L with water. Extraction was carried out at a specific temperature for a certain time. Insoluble matter was removed by centrifugation to obtain an aqueous extract. The aqueous extract was then spray-dried to obtain a water-extracted powder. The water-extracted powder was weighed, and the extraction rate was calculated. The extraction rate was calculated as: extraction rate = mass of water-extracted powder / mass of insect excrement. The extraction parameters and extraction rate results are shown below. Figure 3 .
[0065] The results showed that water extracts could be obtained by water immersion at different extraction temperatures and times. When the extraction rate was low, the water extract was light brown; when the extraction rate was high, the water extract was dark brown. The extraction rate increased with increasing temperature and extraction time, but the rate of increase slowed down. The powders obtained by spray drying under different extraction conditions were similar in color, all being light brown powders.
[0066] Using solid 13CDD-MAS NMR analysis was performed on the composition of the water-extracted powder under different extraction conditions. The carbon integral results for each type are shown in the figure. Figure 4 The results showed that the carbon spectra of the water-extracted powders obtained under different extraction conditions were similar, and the content of each type of carbon did not differ significantly, indicating that the carbon components of different structural types tended to be consistent, and thus indicating that the composition of the water-extracted powders obtained under different extraction conditions was basically consistent.
[0067] Example 3: Preparation and structural composition analysis of WSFC insect excrement alkaline extract.
[0068] WSFC insect excrement alkali dry powder was prepared according to the extraction methods of various embodiments of invention patent CN201611143186.1, and then subjected to solid-state processing. 13 CDD-MAS NMR analysis, carbon spectrum integrated data are shown below. Figure 5 .in, Figure 5 The sample number extracted in the article is consistent with the invention patent CN201611143186.1, and the alkali mentioned in Table 3 of Example 7 is the type of alkali in step (3).
[0069] The results showed that the carbon components of different structural types in the various alkaline extracts of WSFC insect excrement were basically the same, and the content of each type of carbon element did not differ significantly, indicating that the composition of the alkaline extracts obtained under different extraction conditions was basically the same. However, the structural composition of the WSFC insect excrement alkaline extract was different from that of the WSFC insect excrement aqueous extract, and the content of each type of carbon element varied considerably.
[0070] Example 4: Culture of actinomycetes from different habitats using WSFC insect excrement water extract and alkali extract.
[0071] WSFC insect excrement aqueous extract solid culture medium: Weigh 10g of WSFC insect excrement aqueous 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 insect excrement aqueous extract solid culture medium. Before use, pour 20ml of the 1% WSFC insect excrement aqueous extract solid culture medium into a 90mm diameter petri dish.
[0072] WSFC insect dung extract solid culture medium: Weigh 10g of WSFC insect dung extract powder, add 15g of agar powder, add water, adjust the pH to 7.0, and finally bring the volume to 1L. Sterilize by steaming at 121°C for 20 min to prepare 1% WSFC insect dung extract solid culture medium. Before use, pour 20ml of 1% WSFC insect dung water extract solid culture medium into a 90mm diameter petri dish.
[0073] To test the growth effects of actinomycetes from different habitats and different genera on insect frass culture medium, ten actinomycete strains isolated from different habitats were selected for growth testing. Specific species names are listed below. Figure 1 As shown. The prepared spores of each strain were diluted with sterile water to form 1×10⁻⁶ spores. 8 A CFU / ml suspension was spread at 200 μL per 9 cm diameter petri dish and incubated at 28°C for 3 days. Culture media blocks were then punched out using a 1 cm diameter punch and placed in 50 ml centrifuge tubes. The spores were resuspended in 40 ml of 0.1% Tween solution and cultured on HA plates to determine the CFU count. Results are shown below. Figure 6 .
[0074] Figure 6 The results showed that both the aqueous and alkaline extract media produced very large numbers of spores, and the production of actinomycetes from fecal, soil, and plant rhizosphere soil sources was relatively high; actinomycetes on plant surfaces or within plants could also grow and produce spores, although the number of spores produced was relatively small. These culture results are consistent with... Figure 2 The culture results were basically consistent, that is, the actinomycetes have a certain selectivity for the insect excrement culture medium of the present invention. The growth of actinomycetes from different habitats on the WSFC insect excrement water extract culture medium was different. Among them, the growth of actinomycetes from feces, soil and plant rhizosphere soil was relatively better on the insect excrement culture medium.
[0075] Example 5: Culture of common bacteria and fungi other than actinomycetes in WSFC insect excrement water extract and alkaline extract culture medium.
[0076] Will as Figure 7 Bacillus was prepared into 1×10 8 CFU / ml suspensions were spread onto 1% WSFC insect excrement water extract solid medium, 1% WSFC insect excrement alkaline extract solid medium, and LB medium, respectively. 200 μL was spread on each 9 cm diameter petri dish. The dishes were incubated at 28°C for 3 days. Culture media blocks were 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. Results are shown below. Figure 8 The results showed that Bacillus growth was very weak on WSFC insect feces extract medium, indicating that neither WSFC water extract medium nor WSFC alkaline extract medium is suitable for the growth of highly resistant Bacillus, and therefore has good selectivity for actinomycetes.
[0077] Will as Figure 9 Eight pathogenic fungi were inoculated onto 1% WSFC insect excrement aqueous extract medium, 1% WSFC insect excrement alkaline extract medium, straw powder medium, and PDA medium, respectively, and cultured at 28°C. Growth was recorded. Compared with PDA and straw media, the pathogenic fungi showed weaker or almost no growth on WSFC insect excrement aqueous extract medium and WSFC insect excrement alkaline extract medium. Figure 10 This describes the growth of pathogenic fungi on media such as WSFC insect excrement aqueous extract. Combined with the results of Example 4, it demonstrates that WSFC insect excrement aqueous extract and WSFC insect excrement alkaline extract media exhibit good selectivity for actinomycetes, especially those isolated from insect excrement, animal excrement, soil, and plant rhizosphere soil.
[0078] Example 6: Comparison of sporulation of actinomycetes cultured on conventional actinomycete culture medium with WSFC insect excrement water extract and alkali extract culture medium.
[0079] Six conventional actinomycete culture media—alkaline lignin medium (LA), humic acid medium (HV), Gao's No. 1 medium (GS), oat medium (SP3), mineral humic acid medium (HA), and straw powder medium (Straw)—were compared with 1% WSFC insect excrement aqueous solid medium and 1% WSFC insect excrement alkaline solid medium to compare the sporulation of actinomycete X15. Spores of strain X15 were prepared into 1×10⁻⁶ spores. 8 A CFU / ml suspension was spread in 200 μL portions per petri dish and incubated at 28°C for 14 days. Culture media 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 solution and cultured on HA plates to determine the CFU count. The spore counts obtained from different media at different time points were compared. Results are shown in [Figure Number]. Figure 11 .
[0080] The results showed that the spore production rate of WSFC insect excrement aqueous extract and WSFC insect excrement alkaline extract was better than other media, and the spore number remained relatively stable over a long period of culture, with no significant difference between the two media. The alkaline lignin medium (LA), humic acid medium (HV), and mineral humic acid medium (HA) produced relatively fewer spores (on day 7), with a certain decrease in the later stage, but the spore number in the later stage was higher than that of Gao's No. 1 medium (GS), oat medium (SP3), and straw powder medium (Straw). The spore production rate and total amount of Gao's No. 1 medium (GS), oat medium (SP3), and straw powder medium (Straw) were lower than those of WSFC insect excrement aqueous extract and alkaline extract media, but higher than those of alkaline lignin medium (LA), humic acid medium (HV), and mineral humic acid medium (HA) on day 7, but the spore number decreased rapidly in the later stage.
[0081] The above data indicate that when WSFC insect excrement water extract medium and WSFC insect excrement alkaline extract medium are used to culture actinomycetes, the spore production rate, total amount, and stability are superior to other mediums, and the differences between the two are not significant.
[0082] Example 7: Effects of different concentrations of WSFC insect excrement water extract and alkaline extract culture medium on the culture of actinomycetes.
[0083] Different concentrations of WSFC insect excrement water extract and alkaline extract were prepared. Actinomycetes X15 were streaked onto the culture medium, incubated at 28°C for 24 hours, and the results were observed. (See attached table). Figure 12 Actinomycetes can grow on WSFC insect excrement water extract and alkaline extract media of various concentrations. Among them, the WSFC insect excrement water extract and alkaline extract media with a content of 0.5% to 1% have better culture effect. Actinomycetes can also grow on lower concentrations of 0.125% and 0.25%, but the yield is low.
[0084] Example 8: Comparison of the effects of conventional actinomycete culture medium and WSFC insect excrement water extract and / or alkaline extract culture medium on the isolation of actinomycetes.
[0085] Using rhizosphere soil from healthy peanut plants as the isolation source, actinomycetes were isolated using ISP3, GS, and 1% WSFC insect excrement water extract medium and 1% WSFC insect excrement alkaline extract medium, respectively. The isolation results... Figure 13 As shown in the figure, compared with the nutrient-rich ISP3 and GS media, WSFC insect excrement aqueous extract and alkaline media showed better results in isolating actinomycetes. Specifically, WSFC insect excrement aqueous extract and alkaline extract media exhibited fewer non-actinomycete colonies and smaller colonies, resulting in less interference with actinomycete production and favoring the growth of slower-growing actinomycetes. This resulted in better actinomycete diversity, easier identification and selection of actinomycete clones, and a greater chance of obtaining more diverse actinomycete resources. Identification and calculations revealed that among the identifiable colonies, the proportion of actinomycete colonies obtained from WSFC insect excrement aqueous extract media reached 90%, from WSFC insect excrement alkaline extract media reached 83%, while the proportion obtained from Gao's No. 1 medium (GS) was 45%, and from oat medium (SP3) it was less than 10%.
Claims
1. The application of insect excrement as a culture medium for actinomycetes, among which, The insect is at least one insect from the family Cetoniidae and / or the family Bombycidae.
2. The application according to claim 1, characterized in that, The insect in question is the white-spotted flower beetle ( Protaetia ( Liocola ) brevitarsis ) and / or silkworms ( Bombyx mori ); and / or 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.
3. The application according to claim 1 or 2, characterized in that, The insect excrement is the excrement produced by the larvae of the insect.
4. An actinomycete culture medium comprising an aqueous extract and / or an alkaline extract of the insect excrement used in any one of claims 1 to 3.
5. The actinomycete culture medium according to claim 4, characterized in that, The extraction steps of the aqueous extract include: 1) Based on dried feces with a moisture content of less than 3%, mix feces and water at a mass ratio of 1:1 to 1:10, adjust the temperature to 40 degrees Celsius to 100 degrees Celsius, and treat for 1 hour to 16 hours to obtain water treatment solution; 2) The water treatment solution is subjected to solid-liquid separation to obtain an aqueous extract containing the water extract; Optionally, in or before step 1), a step of pulverizing the feces is also included; or The extraction steps of the alkaline extract include: I) Based on dried feces with a moisture content of less than 3%, mix feces and water at a mass ratio of 1:1 to 1:10, adjust the pH value to 8 to 12, and treat at a temperature of 30 degrees Celsius to 100 degrees Celsius for 20 to 60 minutes to obtain an alkaline treatment solution. II) Adjust the pH of the alkaline treatment solution to 6 to 8, and allow it to settle at 1 to 50 degrees Celsius for 5 minutes to 48 hours to obtain a crude supernatant; III) The crude supernatant is further subjected to solid-liquid separation to obtain an alkaline extract containing the alkaline extract; Optionally, a step of pulverizing the feces may be included in or before step I).
6. The actinomycete culture medium according to claim 5, characterized in that, In step 1), the temperature is 40 to 80 degrees Celsius, and the processing time is 2 to 8 hours; or In step II), the pH of the alkaline treatment solution is adjusted to 6.5 to 7.5, and the solution is allowed to settle at 15°C to 40°C for 2 to 24 hours to obtain a crude supernatant.
7. The actinomycete culture medium according to claim 5, characterized in that, Following step 2), step 3) is further included: concentrating the aqueous extract to obtain a concentrated aqueous extract containing the aqueous extract, or drying the aqueous extract to obtain a dry powder of the aqueous extract; or The process includes step IV after step III): concentrating the alkaline extract to obtain a concentrated alkaline extract containing the alkaline extract, or drying the alkaline extract to obtain an alkaline extract powder.
8. The actinomycete culture medium according to claim 4, characterized in that, The actinomycete culture medium also includes water; Preferably, the dry powder of the water extract and / or alkali extract is 0.125% to 10% based on the mass of water (100%). Preferably, the dry powder of the water extract and / or alkali extract is 0.125% to 5% by mass, based on the mass of water (100%). More preferably, the dry powder of the water extract and / or alkali extract is 0.5% to 1% based on the mass of water (100%).
9. The actinomycete culture medium according to claim 8, characterized in that, The actinomycete culture medium also includes agar; Preferably, the agar content is 1% to 5% by mass, with water accounting for 100% of the total mass.
10. The use of the actinomycete culture medium according to any one of claims 4 to 8 in the cultivation and / or isolation of actinomycetes, particularly in the cultivation and / or isolation of at least one of fecal, soil or plant rhizosphere-derived actinomycetes.