Phallus impudicus strain Qian P907 and application thereof

Abstract

The invention belongs to the field of edible mushrooms, and particularly discloses a Phallus dongsun strain Qian P907 which is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the Phallus dongsun strain is CGMCC No.40101. The invention further discloses a preparation method of the Phallus dongsun strain Qian P907. The invention also discloses application of the strain in food, beverage, health care product and medicine preparation. The phallus impudicus strain Qian P907 has the advantages of being good in mushroom type, large in sporocarp shape, high in fresh mushroom piece weight, short in cultivation period, high in total sugar content and crude protein content and the like, and is high in commodity value, nutritional value and economic benefit.

Description

Technical Field

[0001] The invention belongs to the field of edible fungi, and in particular relates to a iris strychni strain and also relates to the use of the strain. Background Art

[0002] Dongsun (Phallus dongsun, also known as white ghost pen, bamboo mushroom, bamboo mushroom, no skirt sun, etc.) belongs to the genus Phallus of the family Phallaceae and is a famous medicinal and edible fungus in my country. Dongsun is mainly distributed in Guizhou, Sichuan, Yunnan, Anhui, Guangdong and other places. Dongsun has a crisp taste, delicious taste, rich nutritional value, and is rich in various amino acids, proteins, vitamins, polysaccharides, etc. In addition, Dongsun is also a traditional folk medicine in Asia. It has the effects of promoting blood circulation and relieving pain, inhibiting bacteria, eliminating edema and treating rheumatic joint pain. The active ingredients it contains also have the characteristics of immunomodulation and promoting wound healing. With the improvement of people's living standards and the gradual recognition of the importance of the nutritional value and medicinal value of Dongsun, the demand for Dongsun has greatly increased, but the existing Dongsun production is difficult to meet the demand.

[0003] At present, the cultivation of winter lily in my country mainly has the following problems: (1) There are few cultivated varieties. (2) The mycelium growth rate is low, the production cycle is long, and the traits are unstable. At present, the production cycle of the main cultivated winter lily strains is as long as 9 to 12 months, which requires a lot of labor costs, increases the impact of climate and environment and the risk of pests and diseases, and leads to unstable yield and quality of winter lily. Therefore, in order to meet market demand and reduce costs, breeding new winter lily strains with short cultivation cycles and high yields is the best way to solve the current problems in winter lily production.

[0004] There are abundant wild edible mushroom resources in the primeval forests of southern my country, such as Guizhou, Sichuan, and Yunnan, including wild winter clover resources. The development and domestication of wild winter clover strains can not only enrich people's dining tables and meet people's needs for a high level of life, but also use its medicinal and edible properties to develop higher value-added foods, beverages, health products and medicines, which can increase mushroom farmers' income and export earnings while improving people's health and well-being. Summary of the invention

[0005] The invention aims to provide a winter strychnos nux vomica strain with short cultivation period and high yield.

[0006] Another object of the present invention is to provide uses of the above-mentioned Ilex strychnifolia strain.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The invention provides a Phallus dongsun strain Guizhou P907, which is preserved in the General Microbiological Center of China Microbiological Culture Collection Administration, and its preservation number is CGMCC No.40101.

[0009] The present invention also provides application of the strain Guizhou P907 in food or health products.

[0010] The present invention also provides a food or a health product, wherein the food or the health product contains the strain Guizhou P907 or an extract of Guizhou P907.

[0011] The invention also provides application of the strain Guizhou P907 in beverages.

[0012] The invention also provides a beverage, wherein the beverage contains the strain Guizhou P907 or an extract of Guizhou P907.

[0013] The present invention also provides the use of the strain Guizhou P907 in preparing medicines for improving human immunity.

[0014] The present invention also provides the use of the strain Guizhou P907 in preparing medicines for promoting blood circulation, relieving pain, inhibiting bacteria, eliminating edema or treating rheumatic joint pain.

[0015] The present invention also provides a genetically engineered winter clover strain, the starting strain of the genetically engineered winter clover strain is the strain Guizhou P907.

[0016] The present invention also provides a cultivation method of the above-mentioned winter iris strain Guizhou P907, comprising the following steps:

[0017] (1) Stock culture: The stock culture of strain Guizhou P907 is inoculated onto the stock culture medium, and then placed in a 25°C constant temperature incubator for dark culture for 20 to 25 days, until the mycelium covers the culture medium, thereby obtaining the Guizhou P907 stock culture medium; wherein the components of the stock culture medium are: 200 g potato, 20 g glucose, 20 g agar, 0.3 g potassium dihydrogen phosphate, 0.15 g magnesium sulfate, and vitamin B12. 1 10 mg, 1000 mL of water; after the mother culture medium is prepared, sterilize it by high pressure at 121°C for 30 min and pour it into a 9 cm diameter plate;

[0018] (2) Stock culture: The plate covered with the mother strain of Guizhou P907 obtained in step (1) is streaked with a tic-tac-toe method to obtain a bacterial block, which is then transferred to the stock culture medium, one plate is transferred to one bottle, and cultured at 25° C. in the dark for 50 days until the mycelium fills the strain bottle, thereby obtaining the Guizhou P907 stock; wherein the components of the stock culture medium are: 89% miscellaneous sawdust (fine), 10% wheat bran, 1% lime; and 70% water content; when preparing, first take each component according to a proportion, mix them evenly, then add water according to a proportion, and then put them into a 500 mL glass strain bottle, sterilize them at 121° C. under high pressure for 1 h, and after sterilization, place them in a clean bench to cool to room temperature;

[0019] (3) Cultivation of cultivars: transferring the original Guizhou P907 obtained in step (2) to a culture medium, transferring about 15 g to one bag, culturing at 25° C. in the dark for 50 days, until the mycelium fills the bag, thereby obtaining the Guizhou P907 cultivar; wherein the culture medium comprises: 80% sawdust, 18% wheat bran, 1% lime, and 1% gypsum; and a water content of 65%; during preparation, the components are mixed evenly according to a certain proportion, and then water is added according to a certain proportion; the prepared culture medium is put into a polypropylene plastic bag (16 cm×35 cm×0.005 cm), and then tied with a polypropylene rope, and sterilized at 121° C. under high pressure for 2 h, and then cooled to room temperature after the sterilization is completed; wherein the sawdust is coarse sawdust and needs to be pre-wetted in advance to prevent contamination caused by incomplete sterilization;

[0020] (4) Land preparation and bed building: Winter iris cultivation should be carried out in forests with high canopy density. The soil should be fertile and loose, and the relative humidity should be 70% to 85%. It is best to have a certain slope to facilitate drainage. When digging furrows, the width should be 50 cm, the depth should be 15 to 20 cm, and the length should be unlimited. The bottom of the furrow should be leveled, and 3 to 4 cm thick loose soil should be laid to retain water. The width between the two furrows should be 20 to 25 cm.

[0021] (5) Paving: The cultivation method of Guizhou P907 is to use a layer of wood, a layer of fungus, and then a layer of wood. The wood is fresh broad-leaved trees, such as chestnut trees and cycads. They are cut into wood blocks 2 to 5 cm thick and 5 to 10 cm long. The wood is then evenly spread on the bottom of the ditch and compacted to cover the soil in the ditch.

[0022] (6) Sowing: The sowing time is from March to May. The Guizhou P907 cultivar obtained in step (3) is broken into small pieces with a diameter of 3 to 5 cm, and sown on the bottom wood block with a spacing of 3 to 4 cm between the strains. Then, a thin layer of bamboo leaves is covered, and then a layer of wood is added;

[0023] (7) Soil covering: Choose clean, loose soil without obvious bacteria growth. The thickness of the soil covering is 3 to 5 cm. In places prone to drought, the soil covering is slightly thicker, 5 to 8 cm. The soil covering should be in the shape of a turtle's back. Then add a layer of pine needles about 2 cm thick to moisturize the soil.

[0024] (8) Management of mushroom production: The optimum temperature for mycelium growth is 16-24°C, and the fruiting temperature for Guizhou P907 fruiting body is generally 5-12°C. Guizhou P907 has different requirements for humidity at different growth stages. During the mycelium growth stage, the humidity is 70%-85%, during the bud differentiation stage, the relative humidity is above 80%, during the fruiting body growth stage, the soil humidity is 80%-85%, and the relative humidity is above 80%. The cultivation environment of winter mulberry requires semi-shade and semi-sun, and direct sunlight should be avoided to damage the mushroom buds.

[0025] (9) Harvesting: The fruiting bodies of Guizhou P907 are generally formed from October to December, and the entire growth cycle from sowing to harvesting is 7 to 8 months. Winter fungus is tender and easy to break, so be especially careful when harvesting. First, use a knife to cut off the bottom mycelium, then gently turn and pull it up. Do not pull it directly to avoid damaging the mycelium and affecting the secondary fruiting.

[0026] The advantages and beneficial technical effects of the present invention are as follows: (1) The stipes of the winter clover strain Guizhou P907 of the present invention are white, cylindrical and hollow, with good mushroom shape and high commercial value. (2) Large flower shape. The stipes of Guizhou P907 are 22 to 28 cm long and 4 to 7 cm in diameter. The average weight of fresh mushrooms is 63.10 g, which is significantly higher than the fresh mushroom weight of 25 to 40 g / piece of other existing winter clover strains. (3) Short cultivation cycle. The period from sowing to the beginning of mushroom harvesting of the winter clover strain of the present invention is 7 to 8 months, while the cultivation period of other existing winter clover strains is 9 to 12 months. The cultivation period of the strain of the present invention is 2 to 4 months shorter than that of other existing winter clover strains. (4) High yield. The existing fresh yield of winter clover is 1.0 to 1.5 kg / m2, while the average fresh yield of the strain Guizhou P907 of the present invention is 2.20 kg / m 2 , which is significantly higher than the fresh product yield of other existing winter iris strains. (5) High nutritional value. The total sugar content of the strain Guizhou P907 of the present invention is 26.75%, which is significantly higher than the total sugar content of other existing winter iris strains of 5-20%; the crude protein content of Guizhou P907 is 17.21%, which is significantly higher than the crude protein content of other existing winter iris strains of 8-16%.

[0027] Biological preservation: The Phallus dongsun strain Guizhou P907 of the present invention is a wild Phallus dongsun strain collected by the inventor in Dafang County, Bijie City, Guizhou Province in 2018, and was artificially domesticated and cultivated; Guizhou P907 was deposited in the General Microbiology Center of the China Culture Collection Administration on February 17, 2022, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and its preservation number is CGMCC NO.40101. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 .Photo of the fruiting body of the winter strychnos nux vomica strain Guizhou P907 of the present invention.

[0029] Figure 2 . Phylogenetic tree diagram of the wintersweetia strain Guizhou P907 of the present invention. DETAILED DESCRIPTION

[0030] The present invention is further described below by way of examples, but the present invention is not limited thereto.

[0031] Example 1 Collection, separation and domestication process of strain Guizhou P907 of the present invention

[0032] In October 2018, the inventors collected multiple wild Phallus dongsun fruiting bodies in Dafang County, Bijie City, Guizhou Province, and obtained mycelium (strain) by fruiting body tissue separation. After morphological characteristics, ITS-PCR detection, phylogenetic tree construction, and gene fragment size and sequence, it was identified as Phallus dongsun. The strains obtained by tissue separation were cultivated from 2019 to 2021, and the fruiting bodies with good commercial properties were separated every year. They were all cultured on the mother culture medium (the mother culture medium formula is: 200g potato, 20g glucose, 20g agar, 0.3g potassium dihydrogen phosphate, 0.15g magnesium sulfate, 10mg vitamin B1, 1000mL water. After preparation, 121℃ high pressure sterilization for 30min, poured into a 9cm diameter plate), and the strains with strong mycelium growth, fast growth rate and uniform colony were selected, and then used as the mother culture for the next year. In 2021, a strain with good mushroom shape, high yield and short production cycle was selected and named Guizhou P907. The cap of the fruiting body of this strain is 2-7 cm high and 2-7 cm in diameter. The cap is dark green in color and has a grid-like surface. The grid is attached with dark green, sticky spores. The spores are oval to oblong, smooth, colorless and transparent. The stipe is white, spongy, hollow, 22-28 cm long and 4-7 cm in diameter. The trophyte is membranous, grayish white, 4-8 cm high and 5-12 cm in diameter.

[0033] Example 2 Classification and Identification of the Strain Guizhou P907 of the Present Invention:

[0034] (1) Morphological identification of Guizhou P907:

[0035] The buds of Guizhou P907 grow singly or in clusters, and the fruiting bodies have no sterile skirts. The cap is bell-shaped, flat on the top and perforated when mature. The cap surface is grid-like, and dark green, sticky sporophytes are attached to the grid. The spores are oval to oblong, smooth, colorless and transparent; the stipe is white, spongy, hollow and grid-like; the stipe is grayish white with mycelial cords. According to the description and photos of the corresponding morphological characteristics of Dongsun on page 478 of "Chinese Edible Fungi Cultivation" (Zhang Jinxia, ​​Cai Weiming, Huang Chenyang, etc., China Agricultural Press, 2020), Guizhou P907 has the same morphological characteristics as Dongsun, indicating that Guizhou P907 belongs to the species of Phallus dongsun of the genus Phallus.

[0036] (2) Molecular biological classification and identification of the strain Guizhou P907 of the present invention

[0037] The genomic DNA of the Guizhou P907 strain was extracted using a new plant genomic DNA extraction kit CW0531 (Beijing Kangwei Century Company), and ITS-PCR amplification was performed using universal primers ITS4 and ITS5 to obtain the amplified product. The sequences of the ITS4 and ITS5 primers are as follows:

[0038] ITS5: 5'-GGAAGTAAAAGTCGTAACAAGG-3';

[0039] ITS4: 5'-TCCTCCGCTTATTGATATGC-3'.

[0040] The ITS-PCR amplification reaction system (20 μL) is: 1 μL of 20-50 ng / μL DNA template, 10× Buffer (with Mg 2+ ) 2μL, 2.5mM dNTP 0.5μL, 5U / μl DNA polymerase 0.2μL, 0.2μM primers 0.5μL each, add double distilled water to 20μL. The ITS-PCR amplification reaction conditions are: pre-denaturation at 94℃ for 4min; 94℃ for 30s, 56℃ for 30s, 72℃ for 1min, 35 cycles; 72℃ extension for 10min.

[0041] Results A DNA fragment of 544 bp was obtained by amplification and sent to Sangon Biotech Co., Ltd. for sequencing. The nucleotide sequence of the DNA fragment is shown in SEQ ID NO: 1. The sequence was compared online with BLAST in the NCBI database. The results showed that the strain sequence had the highest similarity with that of Phallus dongsun, with a similarity of 92.42%. A phylogenetic tree was constructed. Figure 2 It can be known that the strain Guizhou P907 of the present invention belongs to the genus Phallus dongsun, and is different from any existing strains and is a new Phallus dongsun strain.

[0042] Example 3 Cultivation test of the winter safflower strain Guizhou P907 of the present invention

[0043] Proceed as follows:

[0044] (1) Stock culture: Inoculate the Guizhou P907 stock culture (the Guizhou P907 strain is deposited in the General Microbiology Center of the China Microbiological Culture Management Committee, and its deposit number is: CGMCC No: 40101) into the stock culture medium, place it in a 25°C constant temperature incubator and culture it in the dark for 20 to 25 days, until the mycelium fills the culture medium, and the Guizhou P907 stock culture is obtained. The stock culture medium comprises: 200g potato, 20g glucose, 20g agar, 0.3g potassium dihydrogen phosphate, 0.15g magnesium sulfate, and vitamin B1 10 mg, 1000 mL of water. The mother culture medium was prepared according to the usual method, then sterilized by high pressure at 121°C for 30 min, and poured into a 9 cm diameter plate.

[0045] (2) Stock culture: The mother strain of Guizhou P907 that is full of culture medium obtained in step (1) is transferred to the stock culture medium by tic-tac-toe method, and one plate is transferred to one bottle, and then cultured at 25°C in the dark for 50 days, and the mycelium is full of the culture bottle, and the Guizhou P907 stock is obtained. The stock culture medium is composed of: 89% miscellaneous sawdust (fine), 10% wheat bran, 1% lime; 70% water content; when preparing, the components are mixed evenly according to the proportion, and then water is added according to the proportion, and then loaded into a 500mL glass culture bottle, and sterilized at 121°C for 1h. After sterilization, it is placed in a clean bench to cool to room temperature.

[0046] (3) Cultivation of cultivars: Transfer the original Guizhou P907 obtained in step (2) to the culture medium, transfer about 15 g to one bag, and culture at 25°C in the dark for 50 days until the bag is full of fungi, and the Guizhou P907 cultivar can be obtained. The composition of the culture medium is: 80% miscellaneous sawdust, 18% wheat bran, 1% lime, 1% gypsum; the water content is 65%; when preparing, mix the components evenly according to the proportion, and then add water according to the proportion; put the prepared culture medium into a polypropylene plastic bag (16cm×35cm×0.005cm), then tie it with a polypropylene rope, sterilize it at 121°C for 2h, and cool it to room temperature after sterilization; the miscellaneous sawdust in the culture medium is coarse sawdust, which needs to be pre-wetted in advance to prevent contamination caused by incomplete sterilization.

[0047] (4) Land preparation and bed building: Winter iris cultivation should be carried out under forests. The cultivation site should be a forest with a high canopy density. The soil should be fertile and loose, and the relative humidity should be 70% to 85%. It is best to have a certain slope for easy drainage. When digging trenches, the width should be 50 cm, the depth should be 15 to 20 cm, and the length should be unlimited. The bottom of the trench should be leveled, and 3 to 4 cm thick loose soil should be laid to retain water. The width between the two trenches should be 20 to 25 cm.

[0048] (5) Material laying: Winter mulberry is cultivated with a layer of wood, a layer of fungus and another layer of wood. The wood is fresh broad-leaved trees, such as chestnut and cycad. The wood is cut into blocks 2 to 5 cm thick and 5 to 10 cm long. The wood is then evenly spread on the bottom of the ditch and compacted to cover the soil in the ditch.

[0049] (6) Sowing: The sowing time is from April 1 to April 10. Break the Guizhou P907 cultivar obtained in step (3) into small pieces with a diameter of 3 to 5 cm and sow them on the bottom wood block with a spacing of 3 to 4 cm between each strain. Then cover them with a thin layer of bamboo leaves and then a layer of wood.

[0050] (7) Soil covering: Choose clean, loose soil without obvious bacterial growth. The thickness of the soil covering should be 3 to 5 cm. In drought-prone areas, the soil covering should be slightly thicker, 5 to 8 cm. The soil covering should be in the shape of a turtle's back. Then, add a layer of pine needles 2 cm thick to moisturize the soil.

[0051] (8) Management of mushroom production: Temperature management is the key in the cultivation of winter mulberry. Winter mulberry is a low-temperature variety. The mycelium growth temperature is preferably 16-24°C, and the fruiting temperature of winter mulberry is generally guaranteed to be 5-12°C for mushroom production. Winter mulberry has different requirements for humidity at different growth stages. In the mycelium growth stage, the humidity is preferably 70%-85%, the bud differentiation stage requires the relative humidity of the air to be above 80%, and the fruiting stage requires the soil humidity to be 80%-85%, and the relative humidity of the air to be above 80%. Winter mulberry does not require much light for growth, and the cultivation environment requires half shade and half sun for the best, and direct sunlight should be avoided to damage the mushroom buds.

[0052] (9) Harvesting and evaluation: The fruiting body of Guizhou P907 is formed between November 1 and November 30, and the entire growth cycle from sowing to harvesting is 7 to 8 months. Winter fungus is tender and easy to break, so be especially careful when harvesting. First, cut off the bottom mycelium with a knife, then gently turn and pull it up. Do not pull it directly to avoid damaging the mycelium and affecting the secondary fruiting.

[0053] (10) After harvest, measure and describe the fruiting body morphology of Guizhou P907; weigh the weight of each plant and calculate the average fresh weight. The total sugar content was determined according to GB / T 15672-2009 "National Standard for Determination of Total Sugar Content in Edible Fungi"; the protein content was determined according to GB5009.5-2016 "National Food Safety Standard for Determination of Protein in Food".

[0054] Results (see Figure 1 ) The stipes of the winter clover strain Guizhou P907 of the present invention are white, cylindrical and hollow, with a good mushroom shape and high commercial value. Secondly, the fruiting body of Guizhou P907 is large, with a stipe length of 22 to 28 cm and a diameter of 4 to 7 cm. The average weight of fresh mushrooms is 63.10 g, which is significantly higher than the fresh mushroom weight of 25 to 40 g / piece of other existing winter clover strains. In addition, Guizhou P907 has a short cultivation cycle, from sowing to the beginning of mushroom harvesting, which is 7 to 8 months, which is 2 to 4 months shorter than the 9 to 12-month cultivation cycle of other existing winter clover strains; Guizhou P907 has a high yield. After cultivation and yield measurement, the average fresh product yield is 2.20 kg / m 2 , which is significantly higher than other existing winter safflower by 1.0-1.5kg / m 2 The total sugar content of Guizhou P907 is 26.75%, which is significantly higher than the total sugar content of other existing winter iris strains of 5-20%. The crude protein content of Guizhou P907 is 17.21%, which is significantly higher than the crude protein content of other existing winter iris strains of 8-16%.

[0055] The above results show that the winter mulberry strain Guizhou P907 of the present invention has the advantages of good mushroom shape, large fruiting body, high fresh mushroom unit weight, short cultivation period, high total sugar content and crude protein content. In short, it has high commodity value, high nutritional value, high functional product development and utilization value, high added value and high economic benefits.

Claims

1. A Phallus dongsun strain Guizhou P907, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.40101.

2. Use of the strain Guizhou P907 according to claim 1 in food or health products.

3. A food or health product, characterized in that: The food or health product contains the strain Guizhou P907 or an extract of Guizhou P907 according to claim 1.

4. Use of the strain Guizhou P907 according to claim 1 in beverages.

5. A beverage, characterized in that The beverage contains the strain Guizhou P907 or an extract of Guizhou P907 according to claim 1.

6. Use of the strain Guizhou P907 according to claim 1 in preparing drugs for improving human immunity.

7. Use of the strain Guizhou P907 according to claim 1 in the preparation of drugs for promoting blood circulation, relieving pain, inhibiting bacteria, reducing edema or treating rheumatic joint pain.

8. A genetically engineered winter clover strain, the starting strain of the genetically engineered winter clover strain is the strain Guizhou P907 described in claim 1.

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