Solid-state fermentation method of penicillium oxalicum, penicillium oxalicum complex microbial inoculant and application thereof

Abstract

The invention discloses a solid-state fermentation method of penicillium oxalicum, a penicillium oxalicum complex microbial inoculant and application of the penicillium oxalicum complex microbial inoculant in promoting colonization of the penicillium oxalicum in soil and improving available phosphorus in the soil. The solid-state fermentation method comprises the following steps: mixing a penicillium oxalicum spore suspension and a fermentation substrate for fermentation at 30 + / -0.5 DEG C; the fermentation substrate comprises waste flammulina velutipes residues and wheat bran in a mass ratio of 1: 1. The Penicillium oxalicum complex microbial inoculant comprises the following components in parts by mass: 10-25 parts of a Penicillium oxalicum solid-state fermentation product obtained by a solid-state fermentation method, 20-25 parts of diatomite, 20-30 parts of zeolite powder, 5-10 parts of potassium fulvate and 10-15 parts of humic acid.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a solid-state fermentation method for Penicillium oxalate, a compound inoculum of Penicillium oxalate, and its application. Background Technology

[0002] Penicillium oxalate, as a microbial fertilizer production strain, has the functions of regulating plant growth, improving soil, and remediating heavy metal pollution.

[0003] The applicant's previous research findings, "Screening, Identification and Phosphorus Solubilization Effect of a Strain of Penicillium oxalate" (China Soil and Fertilizer, 2025(3): 233-240), used a highly efficient phosphorus-solubilizing strain isolated from soil as the research object. Experiments were conducted using a poorly soluble phosphorus source medium and soil pots to explore the dissolution effect of the phosphorus-solubilizing strain on the poorly soluble phosphorus source and its effect on increasing available phosphorus in the soil. The results showed that a highly efficient phosphorus-solubilizing strain NP, screened from soil, could produce a clear phosphorus-solubilizing zone on an inorganic phosphorus medium with calcium phosphate as the phosphorus source. The strain was identified as *Penicillium oxalate*. Penicillium oxalicum The strain can utilize ammonium and nitrate nitrogen as nitrogen sources in a culture solution of insoluble phosphorus. After being cultured at 30℃ for 7 days, it can dissolve insoluble phosphorus and increase the concentration of soluble phosphorus. When applied to potted azaleas, the strain can increase the available phosphorus in the soil by 63% after 20 days, an increase of 316 mg / kg. In waste potting soil, it can increase the available phosphorus by 236% after 20 days, an increase of 144 mg / kg.

[0004] Although applying Penicillium oxalate to the soil can increase the available phosphorus in the soil, applying the inoculant alone has problems such as rapid inoculant loss and insufficient colonization.

[0005] In addition, solid-state fermentation of *Penicillium oxalate* typically uses yeast powder, wheat bran, soybean meal, rice bran, etc., as substrates. For example, patent specification CN115851455A discloses a solid-state fermentation method for *Penicillium oxalate*, with the following inoculation and fermentation conditions: using 12.5g of corn flour and 12.5g of wheat bran as the solid substrate for fermentation, adding 10% soluble starch and 10% yeast extract by weight, with a loading amount of 25g, an inoculation amount of 60%, an initial moisture content of 70%, a culture temperature of 33℃, and a culture time of 9 days. Although the materials used in existing solid-state fermentation substrates are slightly cheaper than conventional carbon and nitrogen sources, some costs still need to be incurred. Summary of the Invention

[0006] To address the aforementioned technical problems and shortcomings in the field, this invention provides a solid-state fermentation method for Penicillium oxalate, a compound inoculant for Penicillium oxalate, and their applications.

[0007] The specific technical solution of this invention is as follows: In a first aspect, the present invention provides a solid-state fermentation method for Penicillium oxalate, comprising: mixing a Penicillium oxalate spore suspension and a fermentation substrate and fermenting at 30±0.5℃; The fermentation substrate consists of waste enoki mushroom residue and wheat bran in a 1:1 mass ratio.

[0008] Preferably, in the solid-state fermentation method, the Penicillium oxalate culture medium has the preservation number CCTCC NO: M2024724.

[0009] Preferably, in the solid-state fermentation method, the concentration of the Penicillium oxalate spore suspension is 10. 5 -10 6 per mL.

[0010] Preferably, in the solid-state fermentation method, the water content of the fermentation substrate is 44wt%±1wt%.

[0011] Preferably, in the solid-state fermentation method, the mass percentage of the Penicillium oxalate spore suspension is 5% ± 1%, based on the mass of the fermentation substrate.

[0012] Preferably, in the solid-state fermentation method, the fermentation is static fermentation.

[0013] Preferably, in the solid-state fermentation method, the fermentation time is 5-7 days.

[0014] Secondly, the present invention provides a solid fermentation product of Penicillium oxalate, which is obtained by the solid fermentation method described in the first aspect.

[0015] Thirdly, the present invention provides a Penicillium oxalate compound inoculant, comprising, by weight: 10-25 parts of the Penicillium oxalate solid fermentation product described in the second aspect, 20-25 parts of diatomaceous earth, 20-30 parts of zeolite powder, 5-10 parts of potassium humate, and 10-15 parts of humic acid.

[0016] The solid fermentation product of Penicillium oxalate described in the second aspect, when combined with diatomaceous earth, zeolite powder, potassium humate, and humic acid in the above proportions, has the effect of increasing available phosphorus and promoting the colonization of Penicillium oxalate in soils such as flower pots, which can produce a synergistic effect.

[0017] Fourthly, the present invention provides the application of the solid fermentation product of Penicillium oxalate described in the second aspect or the compound inoculant of Penicillium oxalate described in the third aspect for promoting the colonization of Penicillium oxalate in the soil and increasing the available phosphorus in the soil.

[0018] In the fourth aspect of the application, the Penicillium oxalate solid fermentation product or the Penicillium oxalate compound inoculant is preferably applied to the soil together with organic fertilizer.

[0019] Compared with the prior art, the beneficial effects of this invention are as follows: This invention uses waste enoki mushroom substrate as one of the fermentation matrix components, and mixes it with wheat bran in a specific ratio. This not only reduces fermentation costs and reuses waste materials, reducing environmental pollution, but also significantly increases the number of spores in the resulting fermented product.

[0020] This invention combines Penicillium oxalate solid fermentation product with diatomaceous earth, zeolite powder, potassium humate, and humic acid in a specific ratio, and applies them together with organic fertilizer. This combination can synergistically increase available phosphorus in the soil and promote the colonization of Penicillium oxalate. Specifically: diatomaceous earth provides aeration and drainage; zeolite powder can slowly release soil nutrients and improve soil structure; humic acid provides carbon and energy for beneficial soil microorganisms, accelerating the decomposition of organic matter and nutrient conversion; potassium humate enhances the growth-promoting ability and stress resistance of plants; and organic fertilizer provides comprehensive nutrition to the soil. Attached Figure Description

[0021] Figure 1 This is a photograph of the morphology of strain HM-NP-22 on the culture medium in a specific implementation method.

[0022] Figure 2 This is a microscopic photograph of the HM-NP-22 strain on the culture medium in a specific implementation method.

[0023] Figure 3 This is a diagram showing the results of available phosphorus in the soil under specific implementation methods. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0025] Unless otherwise specified, the operating methods in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0026] Example 1: Isolation, identification, and strain preservation of Penicillium oxalate: I. Separation: Isolation medium: 10 g glucose, 0.3 g MgSO4·7H2O, 5 g calcium phosphate, 0.5 g (NH4)2SO4, 0.3 g NaCl, 0.3 g KCl, 0.03 g MnSO4, 0.01 g FeSO4·7H2O, 0.4 g yeast extract, 20 g agar, 1 L water, natural pH, sterilized at 121℃ for 15 min.

[0027] Diluent: Tween 80 0.5 g, NaCl 9 g, water 1 L, natural pH, sterilized at 121℃ for 15 min. After cooling, add the corresponding antibiotics at concentrations of 100 µg / mL chloramphenicol, 100 µg / mL kanamycin, and 100 µg / mL streptomycin.

[0028] Soil samples were serially diluted to 10 using a diluent. -4 10 -5 10 -6 10 -7 The samples were spread on the above-mentioned isolation medium, with 3 parallel plates for each gradient. They were incubated upside down at 30°C for 3 days. Strains that could produce obvious transparent phosphate-solubilizing zones on the medium were selected for further pure culture.

[0029] II. Identification: (1) Morphological identification: Observe the morphology of spores and hyphae under a microscope, referring to the Flora of Chinese Fungi (Volume 35) - Penicillium and related genera, Bergey's Manual of Identification, and the Handbook of Fungal Identification. For example... Figure 1 As shown, strain HM-NP-22, after culturing on the medium for 5 days, produced transparent phosphate-solubilizing zones with visible yellowish-brown bacterial patches on the surface. The outer edge of the colony extended outwards in a fluffy manner. With increasing culturing time, brownish-green spores gradually appeared at the edge of the bacterial patches. Figure 2 As shown, oval, light green spores can be observed under a microscope.

[0030] (2) Identification of ITS rDNA: The isolated strains were inoculated into PDA medium and cultured at 28℃ and 160 rpm for 2 days. A small amount of mycelium was scraped off and repeatedly frozen and thawed at -20℃ and 60℃ three times to rupture the mycelium. The PCR reaction system included: ITS1 - 0.6 μL, ITS4 - 0.6 μL, template - a small amount of mycelium, dNTP Mix - 4 μL, 2×KOD buffer - 10 μL, KOD FX - 0.4 μL, dd H2O - 4.4 μL; Amplification reaction conditions: Pre-denaturation: 94℃, 5 min; 31 cycles: 98℃, 10 s; 56℃, 45 s; 68℃, 2 min; Extension: 68℃, 5 min. The ITS1 sequence was 5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID NO: 1), and the ITS4 sequence was 5-TCCTCCGCTTATATGC-3' (SEQ ID NO: 2). The successfully amplified PCR product was sent to Qingke Biotechnology Co., Ltd. for sequencing. After verification, the sequencing result (SEQ ID NO: 3) was compared with the existing sequence in the GenBank database on NCBI using BLAST analysis. The comparison result showed that HM-NP-22 is a Penicillium oxalate strain.

[0031] (3) Physiological and biochemical identification: Detection was performed using physiological and biochemical identification tubes. During the sugar metabolism process, glucose first forms pyruvate through glycolysis and enters different metabolic pathways. The MR test result was positive, and the VP test result was negative, indicating that the metabolic pathway of *Penicillium oxalicum* HM-NP-22 is dominated by the mixed acid pathway, producing a large amount of acidic end products such as lactic acid and acetic acid. Fructose, xylose, mannitol, glucose, arabinose, and maltose were positive, indicating that HM-NP-22 can utilize the above sugars for fermentation. Sucrose, hydrogen sulfide, and gelatin were negative, indicating that HM-NP-22 cannot utilize sucrose, cannot produce cysteine ​​reductase, and therefore cannot form hydrogen sulfide or produce gelatinase to liquefy gelatin. Lysine decarboxylase was negative, and nitrate reduction was negative, indicating that HM-NP-22 does not produce lysine decarboxylase and cannot decarboxylate lysine to produce basic amines. The HM-NP-22 strain lacks nitrate reductase and cannot decarboxylate nitrate (NO3). - ) is reduced to nitrite (NO2) - ) or other products (such as N2, NH3).

[0032] Table 1 shows the physiological and biochemical characteristics of Penicillium oxalate HM-NP-22.

[0033] Table 1 III. Preservation of Microbial Strains: Penicillium oxalate ( Penicillium oxalicum HM-NP-22 was deposited on April 19, 2024, at the China Center for Type Culture Collection (CCTCC, located at Wuhan University, Wuhan, Hubei Province), with accession number CCTCC NO: M 2024724.

[0034] Example 2: Penicillium oxalate is used for solid-state fermentation of waste mushroom residue: I. Preparation of Seed Liquid - Penicillium oxalate Spore Suspension: Using a solid culture medium confluent with *Penicillium oxalate* HM-NP-22, and with visible green spores surrounding the hyphae, 10 mL of physiological saline containing 0.05% Tween was poured onto a plate. Spores and hyphae were gently scraped off with a spatula, and the liquid was collected and shaken to mix, yielding a *Penicillium oxalate* spore suspension. After dilution and plating verification, the spore suspension concentration was found to be 10⁻⁶. 5 -10 6 per mL.

[0035] II. Pretreatment of mushroom residue: Crush the waste enoki mushroom residue and air-dry it for later use. Set up fermentation groups: mushroom residue group, wheat bran group, mushroom residue + wheat bran (mass ratio 1:1) group, and mushroom residue + wheat bran (mass ratio 1:2) group. The moisture content of each group was set at 44 wt%. Use 250 ml Erlenmeyer flasks, with each flask containing 30 g. Treat the above materials in an autoclave for 30 minutes to inactivate any remaining bacteria and fungi through high temperature and high pressure.

[0036] III. Fermentation by Penicillium oxalate: The spore suspension was added to the above fermentation groups at a rate of 5 wt% (based on the mass of the fermentation substrate), and placed in a static fermentation environment at 30°C. The mixture was shaken once at 24 h and 48 h to ensure full contact between the spore suspension and the material. After 7 days, the mixture was diluted, coated, and the spore count was determined. Table 2 shows the viable cell counts obtained from different fermentation formulations.

[0037] Table 2 The results of this embodiment show that when waste mushroom residue and wheat bran are mixed in a 1:1 mass ratio as the fermentation substrate, with a loading of 30g and a moisture content of 44wt%, and inoculated into the solid fermentation medium at a seed inoculation rate of 5wt%, the *Penicillium oxalate* HM-NP-22 strain exhibits the highest sporulation yield, reaching 7.92 × 10⁻⁶. 9 / g. Therefore, it can be seen that Penicillium oxalate in this invention can utilize waste mushroom residue for solid-state fermentation, shortening the fermentation cycle and increasing the number of viable spores.

[0038] Example 3: The compounding of Penicillium oxalate inoculant with diatomaceous earth, zeolite powder, humic acid, potassium fulvate, and organic fertilizer, and its effect on increasing available phosphorus in flower soil: I. Material Preparation: Using the Penicillium oxalate solid fermentation product from Example 2 (mushroom residue + wheat bran (1:1) group), 10 parts by weight of Penicillium oxalate solid fermentation product, 25 parts by weight of diatomaceous earth, 30 parts by weight of zeolite powder, 10 parts by weight of potassium humate and 15 parts by weight of humic acid were mixed to obtain a Penicillium oxalate compound inoculant.

[0039] II. Combination of Penicillium oxalate compound inoculant with organic fertilizer: Choose commercially available organic fertilizer. During the potting process for potted flowers, mix the above-mentioned Penicillium oxalate compound inoculant, organic fertilizer, and flower soil before potting. Follow the soil mixing instructions in Table 3.

[0040] Table 3 III. Results of available phosphorus testing in soil: Twenty-eight days after soil filling, soil samples were taken from the potted plants using a soil sampling drill and sent for testing. The testing methods followed NY / T 1121.7-2014 Soil Testing Part 7: Determination of Available Phosphorus in Soil. Soil pH was determined according to NY / T 1377-2007. The results for available phosphorus in the soil are as follows: Figure 3 As shown.

[0041] The results of this embodiment show that, compared with the control group, applying organic fertilizer, Penicillium oxalate solid-state fermentation product, and Penicillium oxalate compound inoculant alone can increase soil available phosphorus by 38.8%, 12.2%, and 24.4%, respectively; applying organic fertilizer in combination with Penicillium oxalate solid-state fermentation product can increase soil available phosphorus by 58.8%; and applying organic fertilizer in combination with Penicillium oxalate compound inoculant can increase soil available phosphorus by 68.8%. These results indicate that organic fertilizer, Penicillium oxalate solid-state fermentation product, and Penicillium oxalate compound inoculant can all increase soil available phosphorus; and applying organic fertilizer in combination with Penicillium oxalate solid-state fermentation product and Penicillium oxalate compound inoculant can synergistically increase soil available phosphorus.

[0042] Example 4: Colonization of Penicillium oxalate in soil: Soil samples from potted flowers taken by a soil sampling drill after 28 and 56 days of soil loading in Example 3 were diluted and coated according to Example 1. The number of spore colonizations is shown in Table 4.

[0043] Table 4 The results of this embodiment show that *Penicillium oxalate* can colonize in soil, with a colonized spore count of 7.2 × 10⁻⁶ after 28 days. 6 The number of colonizing spores per g, when combined with diatomaceous earth, zeolite powder, humic acid, and potassium humate, is 2.4 × 10⁶. 7 The number of spores / g increased by 3.3 times; the number of colonized spores after Penicillium oxalate was combined with organic fertilizer was 4.3 × 10⁶. 6 The number of spores / g increased by 5.9 times. After being applied in combination with organic fertilizer, the number of colonized spores reached 7.5 × 10⁶. 7 The number of spores / g increased 1.7 times compared to the organic fertilizer + Penicillium oxalate inoculant group, and 3.1 times compared to the single compound inoculant group. This demonstrates that Penicillium oxalate inoculant can colonize the soil, and its combined application with diatomaceous earth, zeolite powder, humic acid, and potassium fulvate organic fertilizer effectively increases the number of colonized spores in the soil.

[0044] Furthermore, it should be understood that after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A solid-state fermentation method for Penicillium oxalate, characterized in that, include: Mix the Penicillium oxalate spore suspension with the fermentation substrate and ferment at 30±0.5℃; The fermentation substrate consists of waste enoki mushroom residue and wheat bran in a 1:1 mass ratio.

2. The solid-state fermentation method according to claim 1, characterized in that, The preservation number of the Penicillium oxalate strain is CCTCC NO: M 2024724.

3. The solid-state fermentation method according to claim 1, characterized in that, The concentration of the Penicillium oxalate spore suspension was 10. 5 -10 6 per mL.

4. The solid-state fermentation method according to claim 1, characterized in that, The water content of the fermentation substrate is 44wt%±1wt%.

5. The solid-state fermentation method according to claim 1, characterized in that, Based on the mass of the fermentation substrate, the mass percentage of the Penicillium oxalate spore suspension is 5% ± 1%.

6. The solid-state fermentation method according to claim 1, characterized in that, The fermentation is a static fermentation; The fermentation time is 5-7 days.

7. A solid-state fermentation product of Penicillium oxalate, characterized in that, Obtained by the solid-state fermentation method according to any one of claims 1-6.

8. A compound inoculant for Penicillium oxalate, characterized in that, The product comprises, by weight parts: 10-25 parts of the Penicillium oxalate solid fermentation product as described in claim 7, 20-25 parts of diatomaceous earth, 20-30 parts of zeolite powder, 5-10 parts of potassium humate, and 10-15 parts of humic acid.

9. The application of the solid fermentation product of Penicillium oxalate according to claim 7 or the compound inoculant of Penicillium oxalate according to claim 8 for promoting the colonization of Penicillium oxalate in the soil and increasing the available phosphorus in the soil.

10. The application according to claim 9, characterized in that, The solid fermentation product of Penicillium oxalate or the compound inoculant of Penicillium oxalate is applied to the soil together with organic fertilizer.

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