New oyster mushroom strain and cultivation method and application thereof

Abstract

The invention provides a novel oyster mushroom strain and a cultivation method and application thereof, and relates to the technical field of edible mushrooms. The name of the new pleurotus ostreatus strain is Nanji pleurotus ostreatus No.1, the new pleurotus ostreatus strain is preserved in China Center for Type Culture Collection (CCTCC) on May 12, 2025, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M20251015. The new pleurotus ostreatus strain provided by the invention has the characteristics of high content of crude protein, crude polysaccharide, crude ash and crude dietary fiber, has rich nutritional value, enriches the variety of pleurotus ostreatus, and has great application potential.

Description

Technical Field

[0001] This invention relates to the field of edible fungi technology, and in particular to a new strain of oyster mushroom, its cultivation method, and its application. Background Technology

[0002] Oyster mushroom ( Oyster mushroom (Pleurotus ostreatus) is a widely cultivated edible fungus belonging to the phylum Basidiomycota, class Agaricales, order Agaricales, family Pleurotaceae, and genus Pleurotus. Its fruiting bodies grow in clusters or overlapping layers, with the caps exhibiting imbricate clusters, fan-shaped, shell-shaped, or irregularly funnel-shaped growth. The caps are fleshy, thick, and soft. The color of the cap surface varies depending on light intensity; stronger light results in a darker color, while weaker light results in a lighter color. The gills are pale yellow, varying in length, some extending from the cap edge to the stipe, others only a short section at the cap edge, resembling fan ribs. The stipe is lateral or eccentric, white, and solid; the mycelium is white, robust, and strong; the flesh is white, slightly thick, and soft. Due to its rich nutritional value, short cultivation cycle, and strong environmental adaptability, oyster mushroom has become an important economic crop globally.

[0003] According to relevant records, oyster mushroom fruiting bodies are rich in high-quality protein (20%-30% dry weight), dietary fiber, β-glucan and other polysaccharide active substances, as well as B vitamins, vitamin D precursor (ergosterol), and minerals such as potassium and selenium. Its extracts possess physiological functions such as immunomodulation (by activating macrophages), antioxidant (significant free radical scavenging ability), and lipid-lowering (inhibiting cholesterol synthase activity), and are widely used in the development of functional foods, dietary supplements, and biopharmaceutical raw materials. Currently, the optimal temperature for cultivating oyster mushroom fruiting bodies is mostly between 15-25℃. To further enrich high-quality oyster mushroom germplasm resources and obtain oyster mushroom varieties with better stress resistance, it is necessary to focus on the collection, isolation, domestication, and research and development of new oyster mushroom varieties. Summary of the Invention

[0004] Based on the above, this invention aims to provide a new strain of oyster mushroom, its cultivation method, and its application. The new oyster mushroom strain discovered in this application—Nangong Oyster Mushroom No. 1—is derived from wild oyster mushrooms grown outdoors in Nanjing at low temperatures of 1-5℃ during winter. This strain exhibits strong resistance to low temperatures.

[0005] The technical solution adopted by this invention to achieve its technical objectives is as follows:

[0006] This invention provides a new strain of oyster mushroom, named as follows: The specimen of *Pleurotus ostreatus* No. 1 was deposited at the China Center for Type Culture Collection (CCTCC) on May 12, 2025, at Wuhan University, Wuhan, China (299 Bayi Road, Wuchang District, Wuhan, Hubei Province), with accession number CCTCC NO: M20251015.

[0007] The application further provides a pleurotus ostreatus cultivation species, which is obtained by culturing the pleurotus ostreatus new strain.

[0008] Preferably, the pleurotus ostreatus cultivation species is prepared by the following method.

[0009] 1) Making a mother strain: collecting a tissue-separated strain of Nanguang Pleurotus ostreatus No. 1, inoculating the strain aseptically on a PDA plate, and placing the PDA plate in a constant-temperature culture at 24 DEG C for 9-10 days until the mycelium covers the plate to obtain the mother strain, wherein the culture medium in the PDA plate is 20% of potato, 2% of glucose, 2.5% of agar, 0.15% of potassium dihydrogen phosphate, 0.15% of magnesium sulfate heptahydrate, and the rest is water;

[0010] 2) Making a primary strain: inoculating the mother strain obtained in step 1) aseptically in a primary strain bottle, and placing the primary strain bottle in a constant-temperature culture at 22-25 DEG C until the mycelium covers the primary strain bottle, wherein the primary strain material in the primary strain bottle is 78% of wood chips, 20% of wheat bran, 1% of sucrose, and 1% of gypsum powder, and the water content is 55-60%;

[0011] 3) Making a cultivation species: inoculating the primary strain obtained in step 2) aseptically in a cultivation bag, and placing the cultivation bag in a culture at 22-25 DEG C until the mycelium covers the cultivation bag to obtain the cultivation species, wherein the cultivation material in the cultivation bag is 93% of cottonseed hulls, 4-6% of wheat bran, 0.8-1.2% of gypsum, 0.8-1.2% of lime, and the water content is 58-62%.

[0012] The application further provides a pleurotus ostreatus fruiting body, which is obtained by culturing the pleurotus ostreatus new strain.

[0013] The application further provides a cultivation method of the pleurotus ostreatus new strain, which is realized based on the above pleurotus ostreatus cultivation species, and the cultivation method comprises the following steps: loading cultivation material into a cultivation bag, sealing, sterilizing at 121 DEG C for 3-4 hours, inoculating the cultivation species after the temperature of the cultivation material decreases to 25-30 DEG C, transferring to indoor culture at 22-25 DEG C, adjusting the relative humidity of air to below 70%, and culturing in the dark, until the mycelium covers the entire cultivation bag, unsealing, controlling the temperature at 15-25 DEG C, and maintaining the relative humidity of air at 88-92% until the fruiting body grows.

[0014] The application further provides a pleurotus ostreatus mycelium, which is obtained by liquid fermentation of the pleurotus ostreatus new strain.

[0015] The application further provides a liquid fermentation method of the new Pleurotus ostreatus strain, comprising the following steps: loading a liquid fermentation medium into a first-stage shake flask, sealing, sterilizing at 121 DEG C for 20-30 minutes, inoculating with a mother strain when the temperature of the flask decreases to 25 DEG C, and culturing at 25 DEG C and 120 rpm for 7-10 days until the wet weight of mycelium per unit volume no longer increases, namely first-stage seed liquid, transferring the first-stage seed liquid into a second-stage shake flask at 10% V / V (volume ratio) in a sterile manner, culturing at 25 DEG C and 120 rpm for 5-7 days until the wet weight of mycelium per unit volume no longer obviously increases, namely second-stage seed liquid, transferring the second-stage seed liquid into a third-stage shake flask at 10% V / V in a sterile manner, culturing at 25 DEG C and 120 rpm for 5-7 days or inoculating into a fermentation tank in a sterile manner, culturing at 25 DEG C, 1.2 VVM of ventilation volume and 150 rpm of stirring speed for 5-7 days, and obtaining the liquid fermentation mycelium of the Nan Gong Pleurotus ostreatus No. 1; the raw material composition of the liquid fermentation medium is 3% of glucose, 0.5% of Angel yeast autolysis powder, 0.15% of potassium dihydrogen phosphate and 0.15% of magnesium sulfate heptahydrate, and the rest is water.

[0016] Preferably, the mother strain is prepared by the following method: collecting the tissue-separated strain Nan Gong Pleurotus ostreatus No. 1, inoculating the strain into a PDA plate in a sterile manner, culturing at 24 DEG C for 9 days until the mycelium covers the plate, and obtaining the mother strain; the culture medium in the PDA plate comprises 20% of potato, 2% of glucose, 2.5% of agar, 0.15% of potassium dihydrogen phosphate and 0.15% of magnesium sulfate heptahydrate, and the rest is water.

[0017] The application further provides application of the new Pleurotus ostreatus strain as a parent in hybrid breeding.

[0018] The application further provides application of the new Pleurotus ostreatus strain in food processing.

[0019] The application has the following beneficial effects:

[0020] The new Pleurotus ostreatus strain is separated from the trees under the building of the State Biochemical Engineering Technology Research Center of Nanjing University of Technology, and is derived from wild Pleurotus ostreatus grown under the condition of 1-5 DEG C low temperature in winter outdoors in Nanjing; the strain shows strong low-temperature resistance, is identified as a new Pleurotus ostreatus strain by morphology and molecular biology, and is named Nan Gong Pleurotus ostreatus No. 1. The application provides a cultivation method and a liquid fermentation method of the Nan Gong Pleurotus ostreatus No. 1, comprising inoculating the cultivation seed of the Nan Gong Pleurotus ostreatus No. 1 into a cultivation bag for cultivation and management and harvesting, and inoculating the liquid seed into a fermentation medium for liquid fermentation. The application further provides quality detection of the strain Nan Gong Pleurotus ostreatus No. 1 and corresponding application. The strain has the characteristics of high contents of crude protein, crude polysaccharide, crude ash and crude dietary fiber, has rich nutritional value, enriches the variety of Pleurotus ostreatus, and has great application potential. Attached Figure Description

[0021] Figure 1 This is a morphological diagram of the wild fruiting body of *Pleurotus ostreatus* No. 1 of this invention.

[0022] Figure 2 This is the phylogenetic tree of the Nan Gong Pleurotus ostreatus No. 1 of the present invention.

[0023] Figure 3 The results of sequence alignment of ITS1 and ITS4 of the present invention, Nan Gong Ping Gu No. 1.

[0024] Figure 4 This is a diagram of the mycelium of the Nan Gong Pleurotus ostreatus No. 1 of this invention on PDA medium.

[0025] Figure 5 This is a diagram showing the mycelial morphology of the Nan Gong Pleurotus ostreatus No. 1 in a liquid culture medium. Detailed Implementation

[0026] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features or steps.

[0027] Unless otherwise stated, any feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0028] The novel oyster mushroom strain of this invention was isolated from the roots of trees downstairs at the National Biochemical Engineering Technology Research Center of Nanjing University of Technology, and originated from wild oyster mushrooms that grow outdoors in Nanjing at low temperatures of 1-5℃ during winter.

[0029] like Figure 1 As shown, the wild-type fruiting bodies of this new strain have the following morphological characteristics:

[0030] The fruiting body is an irregular funnel shape, yellowish-brown, about 8-10 cm long and 5.5-8 cm wide. The gills are pale yellow, varying in length and about 2-4 mm thick, and are easily broken. The stipe is pale yellow, thick and tough, about 2-3 cm in diameter and about 4-6 cm long, with short white hairs on it.

[0031] A phylogenetic tree of the strains of this invention was constructed based on the ITS gene sequence, such as... Figure 2As shown in the phylogenetic tree, the yeast species is used as an outgroup to root, and contains typical species of multiple key groups such as Tricholomataceae, Cortinariaceae and Tricholomataceae. Among them, the Pleurotus species including Pleurotus ostreatus and Pleurotus dryinus form a highly supported single evolutionary branch on the phylogenetic tree, indicating that they have a common ancestor. In addition, the analysis result of the ITS1 molecular sequence (GenBank accession number: AY450345.1) determined by molecular biology is . Combined with the alignment results of the ITS1 and ITS4 molecular sequences ( Figure 3 ), the similarity between the strain Pleurotus 1 of the application and the existing Pleurotus species is as high as 99%, but there are still differences, and the environmental temperature for the growth of the wild fruiting body is significantly lower than that of the existing Pleurotus species, which is quite different from the growth conditions of the Pleurotus recorded in the existing literature, so it is a new species of Pleurotus.

[0032] The original strain is obtained by culturing the tissue separation of the fruiting body in the PDA medium, and is named Pleurotus 1, which has been preserved in the China Center for Type Culture Collection (CCTCC) on May 12, 2025, the address of which is Wuhan University, Wuhan, Hubei Province, China (Wuchang District, Wuhan City, Hubei Province, China), and the preservation number is CCTCC NO: M20251015.

[0033] Example 1

[0034] This embodiment mainly studies the cultivation of Pleurotus 1, which includes the following aspects:

[0035] I. Preparation of the cultivated species

[0036] The main steps include:

[0037] (1) Making the mother strain: collecting the tissue-separated strain Pleurotus 1, and aseptically inoculating the strain into a PDA plate, and placing it in a constant temperature incubator at 24℃ for 9 days until the mycelium covers the plate (as shown in Figure 4 ), and the mother strain is obtained, wherein the culture medium of the PDA plate is 20% potato, 2% glucose, 2.5% agar, 0.15% potassium dihydrogen phosphate, 0.15% magnesium sulfate heptahydrate, and the rest is water.

[0038] (2) Making the original strain: aseptically inoculating the mother strain into the original strain bottle, and placing it in a constant temperature incubator at 22-25℃ until the mycelium covers the original strain bottle, wherein the raw material composition in the original strain bottle is 78% sawdust, 20% wheat bran, 1% sucrose, 1% gypsum powder, and the water content is 55-60%.

[0039] (3) Preparation of the cultivation species: the original species is aseptically inoculated into a cultivation bag and cultured at 22-25°C until the mycelium fills the cultivation bag, thereby obtaining the cultivation species. The raw material composition of the cultivation material is 93% cottonseed hulls, 5% wheat bran, 1% gypsum, and 1% lime by mass percentage, and the water content is 60%. The cultivation material is loaded into a gusset bag, and a plastic neck ring and a lid are covered, thereby obtaining the cultivation species bag.

[0040] II. Cultivation

[0041] The cultivation material is loaded into a polyethylene gusset bag, sealed with a plastic neck ring and a lid, and sterilized at 121°C for 3-4 hours. After sterilization, when the temperature of the cultivation material decreases to 25-30°C, the cultivation species is inoculated and transferred to a room at 22-25°C. The relative humidity of the air is adjusted to below 70%, and the culture is carried out in the dark. Pay attention to ventilation. When the mycelium fills the entire cultivation bag (30-40 days), remove the plastic neck ring and lid from the bag opening. The temperature is controlled at 15-25°C, and the relative humidity of the air is maintained at about 90% until the fruiting bodies grow out (this process takes about 7 days). The composition of the cultivation material is 93% cottonseed hulls, 5% wheat bran, 1% gypsum, and 1% lime by mass percentage, and the water content is about 60%.

[0042] III. Quality identification of fruiting bodies

[0043] Harvest the first flush and the second flush of the South Engineering Pleurotus ostreatus No. 1 at the same time, and determine the contents of crude protein, crude polysaccharide, crude ash, and crude dietary fiber. Take the average value. The determination of crude protein refers to the "National Food Safety Standard Determination of Protein in Food" (GB 5009.5-2016); the determination of crude polysaccharide content refers to the "Determination of Crude Polysaccharide in Edible Fungi by Spectrophotometry" (NY / T 1676-2023); the determination of crude ash content refers to the "National Food Safety Standard Determination of Ash in Food" GB 5009.4-2016; and the determination of crude dietary fiber content refers to the "National Food Safety Standard Determination of Dietary Fiber in Food" (GB 5009.88-2023). The results are shown in Table 1.

[0044] Table 1. Quality determination of fruiting bodies of the first flush and the second flush of South Engineering Pleurotus ostreatus No. 1

[0045] As can be seen from Table 1, the contents of crude protein, crude polysaccharide, and crude ash of the South Engineering Pleurotus ostreatus No. 1 of the present application are higher than those of common Pleurotus ostreatus varieties on the market (Table 2), and the nutritional value is relatively rich.

[0046] Table 2. Nutrient content in common Pleurotus ostreatus

[0047] Example 2

[0048] The present embodiment mainly studies the liquid fermentation method of Nangong Pleurotus 1, which specifically includes:

[0049] (1) Making mother culture: collect Nangong Pleurotus 1 separated by tissue, inoculate the strain aseptically on a PDA plate, and place it in a constant temperature incubator at 24°C for about 9 days until the mycelium covers the plate, and then obtain the mother culture. The PDA culture medium is 20% potato, 2% glucose, 2.5% agar, 0.15% potassium dihydrogen phosphate, 0.15% magnesium sulfate heptahydrate, and the rest is water.

[0050] (2) Making primary seed liquid: pour the liquid fermentation medium into a primary shake flask, seal the flask with a rubber band and high-temperature-resistant sealing film, and sterilize it at 121°C for 20-30 minutes. When the temperature of the flask drops to 25°C, inoculate the mother culture, and place it in a constant temperature incubator at 25°C and 120 rpm for 7-10 days until the wet weight of mycelium per unit volume no longer increases, which is the primary seed liquid. The formula of the primary seed liquid fermentation medium is 3% glucose, 0.3% Angel yeast autolysis powder, 0.15% potassium dihydrogen phosphate, 0.15% magnesium sulfate heptahydrate, and the rest is water.

[0051] (3) Making secondary seed liquid: inoculate the primary seed liquid aseptically into a secondary shake flask at 10% V / V, and place it in a constant temperature incubator at 25°C and 120 rpm for about 7 days until the wet weight of mycelium per unit volume no longer increases significantly, which is the secondary seed liquid. The formula of the secondary seed liquid fermentation medium is 3% glucose, 0.3% Angel yeast autolysis powder, 0.15% potassium dihydrogen phosphate, 0.15% magnesium sulfate heptahydrate, and the rest is water.

[0052] (4) Making Pleurotus liquid fermentation mycelium: inoculate the secondary seed liquid aseptically into a tertiary shake flask at 10% V / V, and place it in a constant temperature incubator at 25°C and 120 rpm for 5-7 days, or inoculate it aseptically into a fermentation tank, and place it in a constant temperature incubator at 25°C, with aeration rate of 1.2 VVM and stirring speed of 150 rpm for 5-7 days, to obtain the Nangong Pleurotus 1 liquid fermentation mycelium, as shown in Figure 5 The formula of the secondary seed liquid fermentation medium is 3% glucose, 0.5% Angel yeast autolysis powder, 0.15% potassium dihydrogen phosphate, 0.15% magnesium sulfate heptahydrate, and the rest is water.

[0053] The mycelium of the above-mentioned Nangong Pleurotus 1 strain was subjected to quality identification, and the mycelium was dried at 50-60°C after separation, and the contents of crude protein, crude polysaccharide and crude dietary fiber were determined. The average number was taken. The detection method is the same as that of Pleurotus fruiting body. The results are shown in Table 3:

[0054] Table 3. Quality determination of Nangong Pleurotus 1 strain liquid fermentation mycelium

[0055] As shown in Table 3, the contents of crude protein, crude polysaccharide, crude ash and crude dietary fiber in the liquid fermentation mycelium of the P. ostreatus No. 1 are all high, and the liquid fermentation mycelium can be extracted to increase the product added value in food and health products, and expand the application scenarios of the P. ostreatus.

[0056] For example, the strain of the present application can be widely applied in the field of food processing.

[0057] Application 1: The strain of the present application is used for processing P. ostreatus powder, and is added into pet food or livestock feed to improve the nutritional value of animal food.

[0058] Application 2: Used for making dried mushroom food.

[0059] Application 3: Used as an additive for cereal food and bread.

[0060] Meanwhile, since the strain of the present application has good low-temperature stress resistance, it can be used for hybrid breeding with other P. ostreatus varieties to enrich the germplasm resources of P. ostreatus.

[0061] In summary, compared with the existing P. ostreatus strains, the fruiting body of the P. ostreatus No. 1 of the present application has the following characteristics: the fruiting body is yellow-brown and irregular funnel-shaped, the cap is smooth, and the gill is white and has a netted gill. Through quality analysis, it is found that the strain also has the characteristics of high contents of crude protein, crude polysaccharide, crude ash and crude fiber.

[0062] The above-described embodiments only express several embodiments of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which all belong to the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. A novel strain of oyster mushroom, characterized in that, Name is The *Pleurotus ostreatus* No. 1 was deposited at the China Center for Type Culture Collection (CCTCC) on May 12, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20251015.

2. A cultivar of oyster mushroom, characterized in that, It was obtained by culturing the new oyster mushroom strain described in claim 1.

3. The oyster mushroom cultivar according to claim 2, characterized in that, The oyster mushroom spawn was prepared by the following method: 1) Preparation of mother culture: Collect the tissue-isolated spore of *Pleurotus ostreatus* No. 1, aseptically inoculate the spore onto a PDA plate, and incubate at a constant temperature of 24℃ for 9-10 days until the mycelium covers the plate to obtain the mother culture. The culture medium in the PDA plate is composed of 20% potato, 2% glucose, 2.5% agar, 0.15% potassium dihydrogen phosphate, 0.15% magnesium sulfate heptahydrate, and the remainder is water. 2) Preparation of primary culture: The mother culture obtained in step 1) is aseptically inoculated into the primary culture bottle and placed in a constant temperature culture at 22-25℃ until the mycelium fully grows in the primary culture bottle. The primary culture material in the primary culture bottle is composed of 78% sawdust, 20% wheat bran, 1% sucrose, 1% gypsum powder, and has a moisture content of 55-60% by weight percentage. 3) Preparation of spawn: The original spawn obtained in step 2) is aseptically inoculated into a cultivation bag and incubated at 22-25℃ until the mycelium fully grows in the cultivation bag to obtain the spawn. The cultivation material in the cultivation bag is composed of cottonseed hulls 93%, wheat bran 4-6%, gypsum 0.8-1.2%, lime 0.8-1.2%, and has a moisture content of 58-62%.

4. A fruiting body of *Pleurotus ostreatus*, characterized in that, The fruiting bodies of the oyster mushroom are obtained by culturing the new oyster mushroom strain described in claim 1.

5. A cultivation method for a new strain of oyster mushroom, characterized in that, Based on the oyster mushroom cultivation spawn described in claim 3, the cultivation method includes: filling the cultivation material into a cultivation bag, sealing it, sterilizing it at 121℃ for 3-4 hours, and after sterilization, when the temperature of the cultivation material drops to 25-30℃, inoculating it with the cultivation spawn, transferring it to an indoor environment at 22-25℃, adjusting the relative humidity to below 70%, and cultivating it in the dark. When the mycelium has covered the entire cultivation bag, the seal is removed, the temperature is controlled at 15-25℃, and the relative humidity is maintained at 88-92%, until fruiting bodies grow.

6. A type of oyster mushroom mycelium, characterized in that, The oyster mushroom mycelium was obtained by liquid fermentation of the new oyster mushroom strain described in claim 1.

7. The liquid fermentation method for the new strain of Pleurotus ostreatus according to claim 1, characterized in that, include: Liquid fermentation medium is poured into a primary shake flask, sealed, and sterilized at 121°C for 20-30 minutes using moist heat. When the flask temperature drops to 25°C, the mother culture is inoculated and incubated at 25°C and 120 rpm for 7-10 days, until the wet weight of mycelium per unit volume no longer increases (this is the primary seed culture). The primary seed culture is then aseptically transferred at 10% v / v to a secondary shake flask and incubated at 25°C and 120 rpm for 5-7 days, until the wet weight of mycelium per unit volume no longer increases significantly (this is the secondary seed culture). The secondary seed culture is then aseptically transferred at 10% v / v to a tertiary ... The mycelium of *Pleurotus ostreatus* No. 1 was obtained by aseptically inoculating it into a fermenter at 25°C, with an aeration rate of 1.2 VVM and a stirring speed of 150 rpm for 5-7 days. The raw material composition of the liquid fermentation medium is as follows (by mass percentage): glucose 3%, Angel yeast autolysate 0.5%, potassium dihydrogen phosphate 0.15%, magnesium sulfate heptahydrate 0.15%, and the remainder is water.

8. The liquid fermentation method according to claim 7, characterized in that, The mother culture was prepared by the following method: the spawn of *Pleurotus ostreatus* No. 1, isolated from tissue, was aseptically inoculated onto a PDA plate and incubated at a constant temperature of 24°C for 9 days until the mycelium covered the plate. The culture medium in the PDA plate, by mass percentage, consisted of 20% potato, 2% glucose, 2.5% agar, 0.15% potassium dihydrogen phosphate, 0.15% magnesium sulfate heptahydrate, and the remainder was water.

9. The application of the new oyster mushroom strain according to claim 1 as a parent in hybridization breeding.

10. The application of the new oyster mushroom strain according to claim 1 in food processing.

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