Edible mushroom semi-solid spawn production method and application thereof in liquid spawn preparation

Abstract

The application discloses a kind of edible fungus semi-solid strain production method and its application in liquid strain preparation.The method is: low-temperature preserved edible fungus strain is activated in PDA solid culture medium, and active strain is obtained;Active strain is shaken flask culture in PDB liquid culture medium, and seed liquid is obtained;Seed liquid is shaken flask fermentation culture in fermentation culture medium, and fermentation culture is obtained;Fermentation culture is magnetically high-speed stirred, to break the mycelium therein, and the fermentation culture after mycelium fragmentation treatment is obtained;Sterilized corn flour solution is added to the fermentation culture after mycelium fragmentation treatment, and after mixing, it is placed, and shaken bed culture, and mycelium microspherization culture is obtained;Sterilized miscellaneous sawdust powder is added to the mycelium microspherization culture, and semi-solid fermentation is obtained, and the edible fungus semi-solid strain for liquid strain preparation is obtained.The method is low in requirement for equipment, place and technical personnel, and the obtained edible fungus semi-solid strain can be used for rapidly preparing edible fungus liquid strain.

Description

Technical Field

[0001] This invention belongs to the field of edible fungi and bioengineering, and aims to provide a method for producing semi-solid edible fungi strains and its application in the preparation of liquid strains. Background Technology

[0002] Edible fungi are a food with extremely high nutritional and medicinal value, providing people with abundant nutrition and promoting human health. Currently, edible fungi are mainly produced through artificial cultivation. Most countries in the world have edible fungi cultivation and related industries, making edible fungi cultivation an important agricultural sector globally. The Chinese people have a long tradition of collecting and consuming edible fungi, providing a solid foundation and impetus for the development of the edible fungi industry. After more than 40 years of development, my country's edible fungi cultivation technology has continuously improved, and yields and cultivation areas have steadily increased, making China a major edible fungi producer, accounting for more than 70% of the world's total output. It is a veritable major producer and consumer of edible fungi. Currently, edible fungi have become an important agricultural industry in my country, ranking fifth in total agricultural output value after grains, oils, fruits, and vegetables, with broad development prospects and further promoting my country's agricultural development.

[0003] my country's edible mushroom production is shifting from a family-run, seasonal, and decentralized model to a factory-scale, year-round production model, with increasing production concentration. Factory cultivation has gradually become the main method of edible mushroom production. Factory cultivation of edible mushrooms generally utilizes both solid and liquid spawn for inoculation, and currently both types are used simultaneously. However, solid spawn inoculation has disadvantages such as low inoculation efficiency, slow mycelial germination, slow growth rate, and poor fruiting synchronization, affecting production efficiency and economic benefits. In recent years, the use of liquid spawn in factory cultivation of edible mushrooms has gained increasing popularity and is becoming a future development trend. Currently, most commercially available mushrooms, such as enoki mushrooms, king oyster mushrooms, and shiitake mushrooms, use liquid spawn in their factory cultivation.

[0004] The preparation of liquid spawn for edible fungi is quite complex, typically involving steps such as spawn activation, primary shake-flask culture, secondary shake-flask culture, and liquid spawn fermentation. Spawn activation is particularly time-consuming, usually requiring 5-7 days. Furthermore, the mycelium needs to readjust to the new culture conditions after being transferred to the liquid culture medium, resulting in extremely slow mycelial growth during the primary shake-flask culture stage, generally taking 4-7 days. This leads to an excessively long preparation cycle. In addition, the slow mycelial growth and prolonged culture time during spawn activation and primary shake-flask culture stages increase the risk of contamination, potentially disrupting production plans and impacting output. To promote the industrialized cultivation of edible fungi, it is crucial to develop a novel inoculum with advantages such as high purity, easy preservation, long shelf life, and rapid growth. This inoculum can replace traditional agar master cultures for the immediate and rapid preparation of liquid spawn, eliminating the need for frequent transfer activation operations and shortening shake-flask culture time. Summary of the Invention

[0005] The purpose of this invention is to provide a method for producing semi-solid edible fungi spawn and its application in the preparation of liquid spawn.

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

[0007] A method for producing semi-solid edible fungi spawn that can be used for liquid spawn preparation includes the following steps:

[0008] S1: Microbial activation

[0009] The edible fungi strains preserved at low temperature were inoculated onto PDA solid medium slant for activation culture to obtain activated strains;

[0010] S2: Seed liquid preparation

[0011] The activated bacterial strain was inoculated into PDB liquid medium and cultured in shake flasks to obtain the seed culture.

[0012] S3: Shake-flask fermentation

[0013] The seed culture was inoculated into a fermentation medium and then subjected to shake-flask fermentation to obtain the fermentation culture.

[0014] S4: Mycelial fragmentation treatment

[0015] The fermentation culture was subjected to high-speed magnetic stirring to break the mycelia in the fermentation culture, resulting in a fermentation culture with fragmented mycelia.

[0016] S5: Mycelial Microsphere Culture

[0017] Sterile corn flour solution was added to the fermentation culture after mycelial fragmentation treatment, mixed well, and allowed to stand. The culture was then cultured on a shaker to obtain mycelial microsphere culture.

[0018] S6: Semi-solid fermentation

[0019] Sterilized hardwood powder was added to the mycelial microsphere culture and semi-solid fermentation was carried out to obtain a semi-solid edible fungus strain that can be used for liquid strain preparation.

[0020] The PDA solid culture medium has the following formula: 200 g / L potato, 20 g / L glucose, 20 g / L agar, and the remainder is water; the PDB liquid culture medium has the following formula: 200 g / L potato, 20 g / L glucose, and the remainder is water; the fermentation culture medium has the following formula: 10 g / L glucose, 2 g / L soybean meal, 2 g / L corn flour, 0.5 g / L potassium dihydrogen phosphate, and the remainder is water.

[0021] The above step S1 specifically involves inoculating the edible fungus mother culture stored at 4℃ into a PDA solid culture medium slant, activating it at 22~25℃ for 7~15 days to obtain activated strains.

[0022] The above step S2 is specifically as follows: cut the activated strain into pieces, select 20-30 pieces and inoculate them into a 250mL Erlenmeyer flask containing 80mL of PDB liquid medium, and culture them at 25-28℃ and 150rev / min for 4-7 days. Then, transfer 10% of the inoculum to a 250mL Erlenmeyer flask containing 80mL of PDB liquid medium and culture it at 25-28℃ and 150rev / min for 2-4 days to obtain the seed culture.

[0023] Specifically, step S3 above involves inoculating 10 mL of seed culture into a 250 mL Erlenmeyer flask containing 60 mL of fermentation medium, and fermenting at 25 °C and 150 rev / min for 3 days to obtain the fermentation culture.

[0024] Specifically, step S4 involves adding a sterilized magnetic rotor to the fermentation culture and magnetically stirring at 1000-1200 rev / min for 20-30 minutes. The shearing force generated by the rotation of the magnetic rotor breaks the mycelia in the fermentation culture, resulting in a fermentation culture with fragmented mycelia.

[0025] Specifically, step S5 involves adding sterilized corn flour solution to the fermentation culture after mycelial fragmentation treatment. The volume ratio of the mycelial fragmentation fermentation culture to the corn flour solution is 20:1, and the mass fraction of the corn flour solution is 2%. After mixing, the mixture is allowed to stand for 30-60 minutes, and then cultured in a shaker at 25-28℃ and 120 rev / min for 1 day to allow the mycelia to aggregate and grow into micromycelial balls, thus obtaining a mycelial microsphere culture.

[0026] Specifically, step S6 involves adding sterilized hardwood powder to the mycelial microsphere culture, with a volume-to-mass ratio of 4 mL / 1 g. The mixture is then fermented in a 23°C constant temperature incubator in a semi-solid state for 1-2 days to obtain a semi-solid edible fungus spawn suitable for liquid spawn preparation, which is then stored at 4°C.

[0027] A semi-solid edible fungus strain prepared by the above-described production method.

[0028] The above-mentioned semi-solid edible fungi strain is used in the preparation of liquid edible fungi strains.

[0029] The beneficial effects of this invention are as follows:

[0030] 1) In the semi-solid edible fungi strain production method of the present invention, the process from shake-flask fermentation to semi-solid fermentation is completed in the same container, which can reduce the risk of contamination and ensure the purity of the strain;

[0031] 2) The semi-solid edible fungi spawn production method of the present invention does not require expensive equipment or high-cleanliness environments, and has low requirements for technical personnel;

[0032] 3) The semi-solid edible fungi strains prepared by this invention have a fast mycelial germination rate;

[0033] 4) The semi-solid edible fungi strains obtained by this invention are easy to preserve;

[0034] 5) The semi-solid edible fungi strains produced by this invention have a long shelf life;

[0035] 6) The semi-solid edible fungi strains prepared by this invention are convenient to use and can be used to prepare liquid edible fungi strains instantly and quickly, eliminating the complicated strain activation operation and shortening the shake flask culture time, thus reducing the potential risk of contamination. Attached Figure Description

[0036] Figure 1 Microsphere culture of Pleurotus eryngii mycelium.

[0037] Figure 2 : King oyster mushroom semi-solid culture.

[0038] Figure 3 Microsphere culture of Pleurotus ostreatus mycelium.

[0039] Figure 4 : Semi-solid mycelium of Pleurotus ostreatus. Detailed Implementation

[0040] The following embodiments are used to further describe the present invention, but these embodiments are merely normative and do not constitute any limitation on the scope of the present invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the present invention, but such modifications and substitutions are still within the protection scope of the present invention.

[0041] In the following examples, the formula of the PDA solid culture medium used was: 200 g / L potato, 20 g / L glucose, 20 g / L agar, and the remainder was water; autoclaved at 121°C for 20 min.

[0042] In the following examples, the formulation of the PDB liquid culture medium used was: 200 g / L potato, 20 g / L glucose, and the remainder water; autoclaved at 121°C for 20 min.

[0043] In the following examples, the fermentation medium was formulated as follows: 10 g / L glucose, 2 g / L soybean meal, 2 g / L corn flour, 0.5 g / L potassium dihydrogen phosphate, and the remainder was water; autoclaved at 121°C for 20 min.

[0044] In the following examples, the corn flour used has a particle size of 60 mesh.

[0045] In the following examples, the hardwood sawdust powder used is obtained by drying, crushing, and sieving hardwood sawdust, and the particle size of the hardwood sawdust powder is 20 mesh.

[0046] Example 1: Preparation of semi-solid mycelium from king oyster mushroom and its application in liquid mycelium production

[0047] A method for preparing a semi-solid mycelium of Pleurotus ostreatus, comprising the following steps:

[0048] S1: Microbial activation

[0049] King oyster mushroom mother culture stored at 4℃ was inoculated into PDA solid medium slant and activated in a constant temperature incubator at 25℃ for 9 days until the mycelium covered the slant, thus obtaining activated strain.

[0050] S2: Seed liquid preparation

[0051] Under aseptic conditions, the activated bacterial culture obtained in step S1 was cut into 0.3cm*0.3cm*0.3cm pieces. 30 pieces were picked and inoculated into 250mL Erlenmeyer flasks containing 80mL PDB liquid medium. The flasks were shaken at 25℃ and 150rev / min for 5 days. Then, the inoculum was transferred to 250mL Erlenmeyer flasks containing 80mL PDB liquid medium at a 10% (v / v) inoculation rate. The flasks were shaken at 25℃ and 150rev / min for 3 days to obtain the seed culture.

[0052] S3: Shake-flask fermentation

[0053] Take 10 mL of the seed culture obtained in step S2 and inoculate it into a 250 mL Erlenmeyer flask containing 60 mL of fermentation medium. Ferment and culture at 25 °C and 150 rev / min for 4 days to obtain the fermentation culture.

[0054] S4: Mycelial fragmentation treatment

[0055] Add a sterilized magnetic rotor (4.5 cm in length) to the fermentation culture obtained in step S3, place it on a magnetic stirrer, and stir magnetically at 1000 rev / min for 30 min. The shear force generated by the rotation of the magnetic rotor will break the hyphae in the fermentation culture to a length of 1-5 mm, thus obtaining a fermentation culture with fragmented hyphae.

[0056] S5: Mycelial Microsphere Culture

[0057] Add a sterilized 2% corn flour solution (by mass fraction) to the fermentation culture obtained in step S4 after mycelial fragmentation treatment. The volume ratio of the fermentation culture after mycelial fragmentation treatment to the corn flour solution is 20:1. After mixing, let it stand for 30 minutes, and then incubate it in a shaker at 25℃ and 120 rev / min for 1 day to allow the mycelia to aggregate and grow into micromycelial balls, thus obtaining a mycelial microsphere culture. In this mycelial microsphere culture, more than 90% of the micromycelial balls have a diameter of less than 0.5 mm. Figure 1 ).

[0058] S6: Semi-solid fermentation

[0059] Add sterile hardwood powder to the mycelial microsphere culture obtained in step S5. The volume-to-mass ratio of the mycelial microsphere culture to the hardwood powder is 4 mL / 1 g. Incubate in a 23℃ constant temperature incubator for 2 days in a semi-solid state to allow the mycelia to adsorb and grow into the substrate, thus obtaining a semi-solid spawn of *Pleurotus eryngii*. Figure 2 Store at 4℃.

[0060] The semi-solid spawn of *Pleurotus eryngii* obtained in this embodiment was stored at 4℃ for 15 days. Under aseptic conditions, 0.5g was inoculated into a 250mL Erlenmeyer flask containing 80mL of PDB liquid culture medium. The flask was then shake-cultured at 23℃ and 150rev / min for 4 days to obtain the liquid spawn of *Pleurotus eryngii*. This liquid spawn was then inoculated at a rate of 5% (v / v) into a 10L fermenter with 7L of fermentation medium. The fermentation was carried out at 23℃ and an aeration rate of 1vvm for 4 days to obtain the liquid spawn for fermentation tank. Measurements showed that the number of mycelial balls and mycelial aggregates in this liquid spawn for fermentation tank reached over 420 per milliliter.

[0061] Example 2: Preparation of semi-solid spawn of Pleurotus ostreatus and its application in liquid spawn production

[0062] A method for preparing a semi-solid spawn of Pleurotus ostreatus, comprising the following steps:

[0063] S1: Microbial activation

[0064] The oyster mushroom mother culture stored at 4℃ was inoculated into a PDA solid medium slant and activated in a 25℃ constant temperature incubator for 9 days until the mycelium covered the slant, thus obtaining the activated strain.

[0065] S2: Seed liquid preparation

[0066] Under aseptic conditions, the activated bacterial culture obtained in step S1 was cut into 0.3cm*0.3cm*0.3cm pieces. 30 pieces were picked and inoculated into 250mL Erlenmeyer flasks containing 80mL PDB liquid medium. The flasks were shaken at 25℃ and 150rev / min for 5 days. Then, the inoculum was transferred to 250mL Erlenmeyer flasks containing 80mL PDB liquid medium at a 10% (v / v) inoculation rate. The flasks were shaken at 25℃ and 150rev / min for 3 days to obtain the seed culture.

[0067] S3: Shake-flask fermentation

[0068] Take 10 mL of the seed culture obtained in step S2 and inoculate it into a 250 mL Erlenmeyer flask containing 60 mL of fermentation medium. Ferment and culture at 25 °C and 150 rev / min for 4 days to obtain the fermentation culture.

[0069] S4: Mycelial fragmentation treatment

[0070] Add a sterilized magnetic rotor (4.5 cm in length) to the fermentation culture obtained in step S3, place it on a magnetic stirrer, and stir magnetically at 1200 rev / min for 20 min. The shear force generated by the rotation of the magnetic rotor breaks the hyphae in the fermentation culture to a length of 1~5.0 mm, thus obtaining the fermentation culture after hyphae fragmentation treatment.

[0071] S5: Mycelial Microsphere Culture

[0072] Add a sterilized 2% corn flour solution (by mass fraction) to the fermentation culture obtained in step S4 after mycelial fragmentation treatment. The volume ratio of the mycelial fragmentation fermentation culture to the corn flour solution is 20:1. After mixing, let it stand for 30 minutes, and then incubate it in a shaker at 25℃ and 120 rev / min for 1 day to allow the mycelia to aggregate and grow into micromycelial balls, thus obtaining a mycelial microsphere culture. In this mycelial microsphere culture, more than 96% of the micromycelial balls have a diameter of less than 0.5 mm. Figure 3 ).

[0073] S6: Semi-solid fermentation

[0074] Add sterile hardwood powder to the mycelial microsphere culture obtained in step S5. The volume-to-mass ratio of the mycelial microsphere culture to the hardwood powder is 4 mL / 1 g. Ferment in a constant temperature incubator at 23℃ for 2 days to allow the mycelia to adsorb and grow into the substrate, obtaining a semi-solid spawn of Pleurotus ostreatus. Transfer the spawn to a blue-capped bottle. Figure 4Store at 4℃.

[0075] The semi-solid spawn of *Pleurotus ostreatus* obtained in this embodiment was stored at 4℃ for 30 days. Under aseptic conditions, 4g was inoculated into a 1000mL Erlenmeyer flask containing 300mL of PDB liquid culture medium. The flask was then shake-cultured at 23℃ and 130rev / min for 4 days to obtain the *Pleurotus ostreatus* shake-flask liquid spawn. This liquid spawn was then inoculated at a rate of 4% (v / v) into a 100L fermenter with 70L of fermentation medium. The fermentation was carried out at 23℃ and an aeration rate of 1vvm for 4 days to obtain the *Pleurotus ostreatus* fermenter liquid spawn. Measurements showed that the number of mycelial balls and mycelial aggregates in this *Pleurotus ostreatus* fermenter liquid spawn reached over 250 per milliliter.

Claims

1. A method for producing semi-solid edible fungi spawn that can be used for liquid spawn preparation, characterized in that: Includes the following steps: S1: Microbial activation The edible fungi strains preserved at low temperature were inoculated onto PDA solid medium slant for activation culture to obtain activated strains; S2: Seed liquid preparation The activated bacterial strain was inoculated into PDB liquid medium and cultured in shake flasks to obtain the seed culture. S3: Shake-flask fermentation The seed culture was inoculated into a fermentation medium and then subjected to shake-flask fermentation to obtain the fermentation culture. S4: Mycelial fragmentation treatment The fermentation culture was subjected to high-speed magnetic stirring to break the mycelia in the fermentation culture, resulting in a fermentation culture with fragmented mycelia. S5: Mycelial Microsphere Culture Sterile corn flour solution was added to the fermentation culture after mycelial fragmentation treatment, mixed well, and allowed to stand. The culture was then cultured on a shaker to obtain mycelial microsphere culture. S6: Semi-solid fermentation Sterilized hardwood powder was added to the mycelial microsphere culture and semi-solid fermentation was carried out to obtain a semi-solid edible fungus strain that can be used for liquid strain preparation. The PDA solid culture medium has the following formula: 200 g / L potato, 20 g / L glucose, 20 g / L agar, and the remainder is water; the PDB liquid culture medium has the following formula: 200 g / L potato, 20 g / L glucose, and the remainder is water; the fermentation culture medium has the following formula: 10 g / L glucose, 2 g / L soybean meal, 2 g / L corn flour, 0.5 g / L potassium dihydrogen phosphate, and the remainder is water.

2. The production method according to claim 1, characterized in that: Step S1 specifically involves inoculating the edible fungus mother culture stored at 4℃ into a PDA solid culture medium slant and activating it at 22~25℃ for 7~15 days to obtain activated strains.

3. The production method according to claim 1, characterized in that: Step S2 specifically involves: cutting the activated bacterial strain into bacterial blocks, selecting 20-30 bacterial blocks and inoculating them into a 250mL Erlenmeyer flask containing 80mL of PDB liquid culture medium, and culturing them at 25-28℃ and 150rev / min for 4-7 days. Then, at an inoculum volume of 10%, the strain is transferred to a 250mL Erlenmeyer flask containing 80mL of PDB liquid culture medium and cultured at 25-28℃ and 150rev / min for 2-4 days to obtain the seed culture.

4. The production method according to claim 1, characterized in that: Step S3 specifically involves: taking 10 mL of seed culture and inoculating it into a 250 mL Erlenmeyer flask containing 60 mL of fermentation medium, and fermenting it at 25 °C and 150 rev / min for 3 days to obtain the fermentation culture.

5. The production method according to claim 1, characterized in that: Step S4 specifically involves adding a sterilized magnetic rotor to the fermentation culture and magnetically stirring at 1000-1200 rev / min for 20-30 minutes to break the mycelia in the fermentation culture through the shearing force generated by the rotation of the magnetic rotor, thereby obtaining a fermentation culture with fragmented mycelia.

6. The production method according to claim 1, characterized in that: Step S5 specifically involves adding sterilized corn flour solution to the fermentation culture after mycelial fragmentation treatment. The volume ratio of the fermentation culture after mycelial fragmentation treatment to the corn flour solution is 20:1, and the mass fraction of the corn flour solution is 2%. After mixing, the mixture is allowed to stand for 30-60 minutes, and then cultured in a shaker at 25-28℃ and 120 rev / min for 1 day to allow the mycelia to aggregate and grow into micromycelial balls, thus obtaining a mycelial microsphere culture.

7. The production method according to claim 1, characterized in that: Step S6 specifically involves adding sterilized hardwood powder to the mycelial microsphere culture, with a volume-to-mass ratio of 4 mL / 1 g for the mycelial microsphere culture and the hardwood powder. The mixture is then fermented in a 23°C constant temperature incubator in a semi-solid state for 1-2 days to obtain a semi-solid edible fungus strain that can be used for liquid spawn preparation. The strain is then stored at 4°C.

8. A semi-solid edible fungus strain prepared by the production method according to any one of claims 1 to 7.

9. The application of the semi-solid edible fungi strain as described in claim 8 in the preparation of liquid edible fungi strains.

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