A pleurotus cultivation material fermentation inoculant and application

Abstract

The application discloses a kind of shiitake mushroom culture medium fermentation inoculant, belong to shiitake mushroom fermentation culture medium preparation technical field, the application is screened by the screening test of strain, and fermentation temperature raising agent and ammonia assimilator are screened, in the fermentation process of culture medium, while adding the two inoculants, can effectively promote fermentation temperature, inhibit the growth of miscellaneous bacteria, shorten fermentation period, improve shiitake mushroom bag inoculation quality.

Description

Technical Field

[0001] This invention relates to the field of oyster mushroom culture medium fermentation technology, and more specifically to a microbial agent for oyster mushroom culture medium fermentation and its application. Background Technology

[0002] Oyster mushrooms are the three major cultivated edible fungi in my country, with an annual output of 6.865 million tons, mainly distributed in the Huang-Huai-Hai Plain region, including Henan and Shandong provinces, accounting for 55.2% of the national total. Fermented substrate cultivation of oyster mushrooms does not require high-temperature sterilization or aseptic inoculation. Compared with cooked substrate cultivation, it is cost-effective, energy-saving, and environmentally friendly, and has been listed as a key technology by both agricultural and environmental protection departments, with a usage rate exceeding 70% in the Huang-Huai-Hai Plain. This method involves pre-wetting raw materials such as corn cobs and cottonseed hulls, then piling them in the open air at varying heights, formulas, and heating times. After heating, the piles are turned over every day, and after a fermentation process of about 15 days, inoculation is carried out using an open inoculation method.

[0003] Currently, the standardization and mechanization levels of oyster mushroom cultivation substrate fermentation preparation processes are low, fermentation temperatures are unstable, it is difficult to raise the temperature in winter, ammonia residues are high, oyster mushroom spawn colonization is slow, and there is a lack of simple and effective fermentation quality testing methods, resulting in high contamination rates after oyster mushroom inoculation, inability to raise the temperature and ferment normally in winter, and poor mycelial growth.

[0004] Therefore, how to provide a microbial agent for fermenting oyster mushroom culture medium, addressing the problems of difficulty in heating and high ammonia residue, is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a microbial agent for fermentation of oyster mushroom culture medium, which reduces the contamination rate, shortens the fermentation cycle, and improves the quality of oyster mushroom mycelium.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A fermentation agent for oyster mushroom culture medium includes: a fermentation warming agent and an ammonia assimilation agent. The fermentation warming agent is *Brevibaciiius potassium*, which was deposited on April 20, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27167, and classified as *Brevibaciiius potassium*. The ammonia assimilation agents are *Raoultella planicola* and *Enterobacter tumefaciens*. *Raoultella planicola* was deposited on August 22, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 25564 and classification name *Enterobacter tumefaciens*. *Enterobacter tumefaciens* was also deposited on August 22, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 25563 and classification name *Enterobacter tumefaciens*.

[0008] As an inventive concept with the same technical solution described above, the present invention also seeks protection for the application of the fermentation agent in the preparation of oyster mushroom fermentation culture medium.

[0009] As an inventive concept with the same technical solution as above, this invention also claims protection for a method for preparing oyster mushroom fermentation culture medium, the process of which includes: pre-wetting corn cobs and adding lime, stirring, adding auxiliary materials after 1 day, stirring again, when the average outside temperature is higher than 15℃, naturally raising the temperature to 70℃, turning the compost twice every 24 hours, running the aeration device fan for 1 hour every 2 hours, and the fermentation cycle is 7-8 days; when the average outside temperature is lower than 15℃, adding fermentation warming agent and ammonia assimilation agent, running the aeration device continuously for 12 hours and stopping for 12 hours every day, raising the temperature to above 70℃ after 3 days, then turning the compost once every 24 hours, and completing the fermentation in 10 days.

[0010] Preferably, the auxiliary materials include: bran urea and potassium dihydrogen phosphate, and the corn cob particle size is 0.5-2.5cm, 0.5-1.5cm in winter and 1.0-2.5cm in summer.

[0011] As can be seen from the above technical solution, this invention obtains a variety of microbial agents through screening in the existing oyster mushroom fermentation process. Among them, through a series of experiments, three strains were screened: one thermogenic strain and two ammonia-assimilating strains. The addition of the thermogenic strain can rapidly raise the temperature of the fermentation material at low fermentation temperatures, promote fermentation, and shorten the fermentation cycle. The ammonia-assimilating strain can effectively remove residual ammonia in the fermentation culture material, avoiding the inhibition of oyster mushroom growth. Therefore, this invention, by adding thermogenic agents and ammonia-assimilating agents during the fermentation process of oyster mushroom fermentation culture material, shortens the fermentation cycle, reduces the contamination rate, and improves the quality of oyster mushroom mycelium. Attached Figure Description

[0012] Figure 1 The attached figure shows the antibacterial effect of the fermentation culture medium of the present invention at different fermentation stages;

[0013] Figure 2 The attached figure shows the temperature after the addition of the temperature-increasing agent according to the present invention;

[0014] Figure 3 The attached figure shows the fermentation temperatures of corn cobs of different sizes under low-temperature conditions. Detailed Implementation

[0015] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1: Screening of thermotropic strains

[0017] When the outside temperature is below 5℃ and the substrate temperature is below 15℃ in winter, the temperature of the pre-wetted substrate rises slowly, rarely reaching 70℃ in about a week, thus affecting fermentation efficiency. Existing EM (Effective Microorganisms) warming agents consist of more than 10 microbial communities, including photosynthetic bacteria, yeast, and actinomycetes, resulting in a complex composition. The abundant microbial communities consume a large amount of nutrients in the oyster mushroom substrate. HM (Hyperbial Microorganisms) warming agents are mainly used for the rapid degradation of straw, achieving rapid degradation in a short period. Oyster mushroom substrate fermentation should first satisfy the function of inhibiting mold growth, and secondly, ensure sufficient nutrient supply for oyster mushroom growth and development to guarantee yield; neither of these is suitable for oyster mushroom substrate fermentation. To improve the rapid temperature rise capability of the substrate in winter, a single culture medium-inducing fermentation strain was isolated from the fermentation substrate. Fermentation substrate was collected on days 2, 4, 6, and 8, and labeled PA, PB, PC, and PD, respectively. Weigh 5g of oyster mushroom fermentation material at four different fermentation stages (PA, PB, PC, and PD), dissolve in 45mL of sterile water, place on a shaker, set the temperature to 28℃ and the rotation speed to 180r / min, shake for 30min, remove, let stand at room temperature for 10min, collect the supernatant and serially dilute to 10. -8 Select dilution 10 -6 10 -7 10 -8 The strains were sequentially streaked onto nutrient agar medium. During the PC and PD stages, the selected strains were cultured in a dark room at 45℃ until single colonies appeared. The culture was then streaked again until single colonies appeared. Single colonies were picked and preserved as slant culture. The logarithmic growth phase bacterial culture was mixed with 50% glycerol and stored at -80℃ for long-term preservation. In the previous study, "Isolation and Identification of Functional Bacteria at Different Fermentation Stages of Pleurotus ostreatus Substrate," 11 strains with strong inhibitory effects on Trichoderma and promoting effects on Pleurotus ostreatus growth were screened. Eight strains (PC13, PC18, PC19, PC30; PD6, PD7, PD8, PD9) from the PC and PD stages (numbered 1-8) were selected for thermoregulatory function screening experiments.

[0018] Experimental formulation:

[0019] The mixture consists of 89% corn cobs, 10% wheat bran, and 1% urea, with 5% lime added according to the ratio of substrate to lime. The substrate-to-water ratio is 1:2.5.

[0020] Test methods

[0021] The experiment was conducted during the coldest season, from December 2021 to January 2022. 1000 catties of culture medium was weighed according to the formula, mixed thoroughly, and 0.5% of fermentation agents 1-8 were added respectively, with no agent added serving as the control (CK). The mixture was piled to a height of 70 cm and a width of 1-1.5 m. The heating time and peak temperature were measured. The results showed that strain 7 had a significantly higher heating effect, with the peak temperature being 7°C higher than CK and lasting for 2 days longer. Therefore, strain 7 was selected as the fermentation heating strain. Strain 7 is *Brevibaciiius borstelensis*, deposited on April 20, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27167, and classified as *Brevibaciiius borstelensis* (see Table 1).

[0022] Table 1

[0023] Maximum temperature (℃) 74 71 68 73 76 72 78 72 71 When the temperature rises (d) 10 9 8 9 5 7 5 7 8 Duration (d) 5 5 6 5 5 4 6 4 4

[0024] Application of strain No. 27 in Example

[0025] From January 5 to January 20, 2022, an experiment was conducted at the oyster mushroom trough fermentation base in Huixian County, Xinxiang City, Henan Province.

[0026] The outside temperature is -5℃ to -5℃.

[0027] Culture medium formula: 88% corn cob, 10% wheat bran, 1% urea, and 1% potassium dihydrogen phosphate.

[0028] Fermentation strain culture: LB liquid culture.

[0029] Two fermentation tanks and outdoor composting were used. Each tank contained 35,000 kg of substrate, which was pre-wetted by spraying for 24 hours. Bran and urea were added, with three tanks: Tank 1 added at 2.5% (No. 7), and Tank 2 added at 5% (No. 7). The compost was turned twice with a turner. Static cultivation was carried out, with a Roots blower providing ventilation for 12 hours daily, followed by a 12-hour static period. The temperature rise and time of the substrate were observed.

[0030] Outdoor fermentation was carried out in three separate piles, each containing 10,000 catties of material, 1 meter high, 5 meters wide, and of unlimited length. After the culture media were piled, they were covered with plastic film. Three days later, 2.5% concentration of strain No. 5 and 2.5% concentration of strain No. 7 were added to piles No. 3 and No. 4, respectively. The inhibition rate of the culture media was then tested.

[0031] The results are shown in Table 2, indicating that the heating time of the added heating element was shortened by 4-8 days compared to the control (CK).

[0032] After all fermentation processes were completed, the culture media showed a 100% inhibition rate, and the contamination rate of the oyster mushroom spawn bags after inoculation was 0%.

[0033] Therefore, we use a 2.5%-5% concentration of No. 7 inoculant as a warming agent for the fermentation of oyster mushroom substrate in winter.

[0034] Table 2

[0035]

[0036] Example 3 Screening of ammonia assimilation agents

[0037] To address the problem of residual ammonia in the culture medium, which inhibits the growth of oyster mushrooms when present in large quantities, an ammonia-assimilating bacterial agent and its application method were developed. Heterotrophic ammonia-assimilating bacteria isolated from sludge, belonging to the genus *Ralstonia*, inhibited oyster mushroom mycelial growth after co-culturing with *oyster mushrooms*, and therefore cannot be used for assimilating residual ammonia in the fermentation medium of *oyster mushrooms*. After primary and secondary screening in media containing 0.025%, 0.05%, and 0.5% NH4Cl, nine bacterial strains with high ammonia assimilation rates were obtained from the *oyster mushroom* fermentation medium. These nine strains exhibited ammonia assimilation rates exceeding 60% for 0.05% NH4Cl and 50% for 0.5% NH4Cl, and did not oxidize ammonia to produce nitrite. The survival rate of these nine strains after culturing at 50℃ for 24 hours was 3%-10%. 16S rRNA gene sequence analysis revealed that 6 strains belonged to the genus *Enterobacter*, possibly *Enterobacter ertabaci*, while the other 3 strains belonged to the genera *Klebsiella* and *Raoultella*, respectively. No antagonism was observed among the 6 *Enterobacter* strains, of which 48... # and 63 # The bacteria exhibited good ammonia assimilation rate and temperature tolerance, making them suitable for preparing ammonia assimilation agents for fermenting oyster mushroom substrate. The ammonia assimilation agents were *Raoultella planicola* and *Enterobacter tumefaciens*. *Raoultella planicola* was deposited on August 22, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 25564 and classification name *Enterobacter tumefaciens*. *Enterobacter tumefaciens* was also deposited on August 22, 2022, at the same CGMCC, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 25563 and classification name *Enterobacter tumefaciens*.

[0038] Example 4 verifies the effects of fermentation warming agent and ammonia assimilation agent.

[0039] The raw materials (88% corn cobs, 10% wheat bran, 1% urea, and 1% potassium dihydrogen phosphate) were pre-wetted and then mixed with lime. When the average outside temperature was below 15°C, a fermentation warming agent and an ammonia assimilation agent were added. The aeration device was run continuously for 12 hours a day, raising the temperature to 75°C in three days. Subsequently, the pile was turned over once every 24 hours, and fermentation was completed in 10 days.

[0040] A properly fermented oyster mushroom substrate needs to simultaneously inhibit mold growth and promote oyster mushroom development. Methods relying solely on color, odor, or ammonia content measurement cannot accurately determine if the substrate possesses both functions. Therefore, accurately detecting the quality of the fermented substrate before bagging directly impacts the contamination rate of the subsequent mushroom bags.

[0041] 500g of fermentation culture material from days 2, 4, 6, 8, and 10 were taken and labeled T1-T5, with the blank serving as the control (CK). The culture material was soaked in water at a ratio of 1:2, and the filtered liquid was replenished to 1000ml. PDA was added to prepare agar plates, which were then inoculated with *Trichoderma* and *Pleurotus ostreatus* strains, respectively. The results showed that the culture material from days 6 and 8 exhibited inhibition rates of 95% and 99% against *Penicillium* and *Trichoderma* mycelia, respectively. Figure 1 The inhibition rate against the two types of mold spores reached 100%. Higher concentrations resulted in stronger antibacterial activity. The extract from the culture medium fermented for 10 days showed the most significant promoting effect on the mycelial growth of *Pleurotus ostreatus*, especially at a volume fraction of 80%, where the mycelial growth rate and biomass increased by 0.53 cm·d compared to the control. -1 The dose was 1.79 mg; there was no significant difference from day 8. See Tables 3 and 4;

[0042] Table 3

[0043]

[0044] Table 4

[0045] <![CDATA[Mycelial growth rate / (cm·d -1 )Growth rate]]> 1.26d 1.27d 1.42c 1.54b 1.58a 1.59a Mycelial growth Status +++ - + + + +

[0046] At the same time, the temperature change during fermentation was measured, see Figure 2 .

[0047] Example 5 describes the preparation of fermentation feed using corn with different particle sizes as raw materials. The process is the same as in Example 1, with particle sizes of 0.5-1.5 cm, 1.5-2.5 cm, and 2.5-5.5 cm, respectively. The changes in fermentation temperature are statistically analyzed. Figure 3 .

[0048] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0049] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A mushroom culture medium fermentation inoculum, characterized by, include: The fermentation warming agent and ammonia assimilation agent are as follows: The fermentation warming agent is *Brevibaciiius borstelensis*, deposited on April 20, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27167 and classified as *Brevibaciiius borstelensis*; the ammonia assimilation agents are *Raoultella phytoregenesis* and *Enterobacter xylophilus*. *Raoultella phytoregenesis* was deposited on August 22, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 25564 and classified as *Raoultella*. planicola; The *Enterobacter tabaci* described was deposited on August 22, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 25563 and classification name *Enterobacter tabaci*.

2. The application of the fermentation agent according to claim 1 in the preparation of oyster mushroom fermentation culture medium.

3. A method for preparing a fermentation medium for Pleurotus ostreatus, characterized by, The process includes: pre-wetting corn cobs, adding lime and stirring; after 1 day, adding auxiliary materials and stirring; when the average outside temperature is below 15℃, adding the fermentation warming agent and ammonia assimilation agent as described in claim 1, running the aeration blower continuously for 12 hours and stopping for 12 hours, raising the temperature to above 70℃ in 3 days, and then turning the pile once every 24 hours, and completing the fermentation in 10 days.

4. The method according to claim 3, wherein the method is characterized by, The auxiliary materials include wheat bran, urea, and potassium dihydrogen phosphate.

5. The method for preparing a fermentation culture medium for oyster mushrooms according to claim 3, characterized in that, The diameter of corn cobs is 0.5-2.5cm, 0.5-1.5cm in winter and 1.0-2.5cm in summer.

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