Phallus impudicus strain Qian P216 and application thereof

Abstract

The invention belongs to the field of edible mushrooms, and particularly discloses a phallus impudicus strain Qian P216, and the preservation number of the phallus impudicus strain Qian P216 is CGMCC (China General Microbiological Culture Collection Center) No.40102. The invention also discloses application of the strain in food, beverage, health care product and medicine preparation. The Qian P216 strain disclosed by the invention is pure white in stipe, cylindrical and hollow in stipe, relatively good in mushroom shape and high in commodity value; secondly, the Qian P216 sporocarp is large in flower shape, and the average single weight of fresh mushrooms is 58.27 g / piece; besides, the Qian P216 is short in cultivation period, and the cultivation period is only 7-8 months and is shortened by 2-4 months compared with the cultivation period of other phallus impudicus strains; the Qian P216 also has the advantages of high yield, high total sugar content, high crude protein content and the like.

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 production is difficult to meet the demand. At present, the market price of Dongsun is 280-450 yuan / jin, which is much higher than the price of other edible fungi. Dongsun has become an important way for local farmers to get rich.

[0003] The main problems of winter lily cultivation in my country are as follows: (1) There are few cultivated varieties. (2) The mycelium grows slowly, the production cycle is long, and the traits are unstable. At present, the production cycle of the main winter lily strains is generally 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 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 P216, which is preserved in the General Microbiological Center of China Microbiological Culture Collection Administration, and its preservation number is CGMCC No.40102.

[0009] The present invention also provides application of the strain Guizhou P216 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 P216 or an extract of Guizhou P216.

[0011] The invention also provides application of the strain Guizhou P216 in beverages.

[0012] The invention also provides a beverage, wherein the beverage contains the strain Guizhou P216 or an extract of Guizhou P216.

[0013] The present invention also provides the use of the strain Guizhou P216 in preparing medicines for improving human immunity.

[0014] The present invention also provides the use of the strain Guizhou P216 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 P216.

[0016] The present invention also provides a cultivation method of the above-mentioned winter iris strain Guizhou P216, comprising the following steps:

[0017] (1) Mother culture: The mother culture of strain Guizhou P216 is transferred to the mother culture medium, and then placed in a 25°C constant temperature incubator for dark culture for 20 to 25 days, until the mycelium fills the culture medium, and the Guizhou P216 mother culture is obtained; wherein the mother culture medium comprises: 200 g potato, 20 g glucose, 20 g agar, 0.3 g potassium dihydrogen phosphate, 0.15 g magnesium sulfate, and vitamin B 1 10 mg, 1000 mL of water; after preparation, sterilize at 121°C for 30 min and pour into a 9 cm diameter plate;

[0018] (2) stock culture: the culture medium plate covered with the mother strain of Guizhou P216 obtained in step (1) is streaked with bacterial blocks by the tic-tac-toe method and transferred to the stock culture medium, with one plate transferred to one bottle, and then cultured at 25° C. in the dark for about 50 days until the mycelium fills the strain bottle, thereby obtaining the Guizhou P216 stock; wherein the components of the stock culture medium are: 89% miscellaneous sawdust (fine), 10% wheat bran, 1% lime; and 70% water content; during preparation, the components are mixed evenly according to the proportion, and then water is added according to the proportion; finally, the mixture is placed in a 500 mL glass strain bottle, and sterilized under high pressure at 121° C. for 1 h. After sterilization, the mixture is placed in a clean bench and cooled to room temperature;

[0019] (3) Cultivation of cultivars: transferring the original Guizhou P216 obtained in step (2) to a culture medium, transferring about 15 g to one bag, and culturing at 25°C in the dark for about 50 days until the bag is full of fungi, thereby obtaining the Guizhou P216 cultivar; wherein the composition of the culture medium is: 80% miscellaneous sawdust, 18% wheat bran, 1% lime, and 1% gypsum; and the water content is 65%; during preparation, the components are mixed evenly according to a proportion, and then water is added according to a proportion; after the prepared culture medium is placed in a polypropylene plastic bag (16 cm×35 cm×0.005 cm), the bag is tied with a polypropylene rope, and sterilized at 121°C for 2 h, and then cooled to room temperature after the sterilization is completed; wherein the miscellaneous sawdust is coarse sawdust and needs to be pre-wetted in advance to prevent contamination caused by incomplete sterilization;

[0020] (4) Land preparation and ridge building: The cultivation of Guizhou P216 is carried out in the forest understory mode. The cultivation site is selected in a forest with a high canopy density. The soil should be fertile and loose, and the relative air humidity should be 70% to 85%. It is best to have a certain slope for easy 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 P216 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 P216 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 the growth of mycelium of Guizhou P216 is 16-24°C, and the temperature for the formation of fruiting bodies of Guizhou P216 is generally 5-12°C for mushroom production; Guizhou P216 has different requirements for humidity at different growth stages. During the mycelium growth stage, the humidity is 70%-85%, the relative humidity of the air is above 80% during the bud differentiation stage, and the soil humidity is 80%-85% during the fruiting body growth stage, and the relative humidity of the air is 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;

[0025] (9) Harvesting: The fruiting body of Guizhou P216 is 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 P216 of the present invention are white, cylindrical and hollow, with good mushroom shape and high commercial value. (2) Large mushrooms. The stipes of Guizhou P216 are 20 to 28 cm long and 3 to 6 cm in diameter. The average weight of fresh mushrooms is 58.27 g / piece, 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 cultivation cycle of the Guizhou P216 strain of the present invention from sowing to the beginning of mushroom harvesting is 7 to 8 months, while the cultivation cycle of other existing winter clover strains is generally 9 to 12 months. The cultivation cycle of the strain of the present invention is 2 to 4 months shorter than that of other existing winter clover strains; it not only saves labor costs, but also reduces the impact of climate and environment and the harm of pests and diseases, ensuring the stability of fresh mushroom yield and quality. (4) High yield. The fresh product yield of other existing winter clover strains is generally 1.0 to 1.5 kg / m 2 The average fresh product yield of the strain Guizhou P216 of the present invention was 2.33 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 fresh mushroom of the strain Guizhou P216 of the present invention is 28.81%, which is significantly higher than the total sugar content of other existing winter iris strains of 5-20%; the crude protein content of Guizhou P216 is 17.86%, which is higher than the crude protein content of other existing winter iris strains of 8-16%.

[0027] Biological preservation: The Phallus dongsun strain Guizhou P216 of the present invention is a wild strain collected by the inventor in Dafang County, Bijie City, Guizhou Province in 2018, and was artificially domesticated and cultivated; Guizhou P216 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.40102. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 .Photo of the fruiting body of the winter strychnos nux vomica strain Guizhou P216 of the present invention.

[0029] Figure 2 . Phylogenetic tree diagram of the wintersweetia strain Guizhou P216 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. Unless otherwise specified, the chemical reagents used in the following examples can be purchased from the market.

[0031] Example 1 Collection, separation and domestication process of strain Guizhou P216 of the present invention

[0032] In October 2018, the inventors collected a number of wild Phallus dongsun fruiting bodies in the wild in Yangchang Town, Dafang County, Bijie City, Guizhou Province, and obtained mycelium (strain) through 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 traits were tissue separated every year. All were in 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, vitamin B 1 10mg, 1000mL of water. After preparation, sterilize at 121℃ for 30min, pour into a 9cm diameter plate for cultivation, and select strains with strong mycelium growth, fast growth rate and uniform colony, and then use them as mother strains for the next year's cultivation. In 2021, a strain with good mushroom shape, high yield and short production cycle was selected, and the strain was named Guizhou P216. The fruiting body cap of this strain is 2-7cm high and 2-7cm in diameter. The surface of the cap is grid-like, with dark green and sticky spores attached to the grid. The spores are elliptical to oblong, smooth, colorless and transparent; the stipe is white spongy, hollow, 20-28cm long and 3-6cm in diameter; the trophyte is membranous, grayish white, 4-8cm high and 5-12cm in diameter.

[0033] Example 2 Classification and Identification of the Strain Guizhou P216 of the Present Invention

[0034] (1) Morphological identification of the strain Guizhou P216 of the present invention

[0035] The buds of the strain Guizhou P216 of the present invention grow singly or in clusters, the fruiting body has no sterile skirt, the cap is bell-shaped, the top is flat after maturity, and there are perforations. The surface of the cap is grid-like, and dark green, sticky sporophytes are attached to the grid. The spores are elliptical to oblong, smooth, colorless and transparent; the stipe is white, spongy, hollow and has a grid; the stipe is grayish white and has mycelial cords. According to the description and photos of the morphological characteristics of winter sun on page 478 of "Chinese Edible Fungi Cultivation" (Zhang Jinxia et al., China Agricultural Press, 2020), the strain Guizhou P216 of the present invention is consistent with the morphological characteristics of winter sun, indicating that the strain Guizhou P216 of the present invention belongs to the genus Phallus dongsun.

[0036] (2) Molecular biological classification and identification of the strain Guizhou P216 of the present invention

[0037] The genomic DNA of Guizhou P216 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 primer sequences for ITS-PCR amplification are as follows:

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

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

[0040] The ITS-PCR amplification system (20 μL) is as follows: 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 reaction conditions of the ITS-PCR amplification are: pre-denaturation 94℃ 4min; 94℃ 30s, 56℃ 30s, 72℃ 1min, 35 cycles; 72℃ extension 10min. As a result, a DNA molecule fragment with a size of 503bp was amplified, and the DNA molecule fragment was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0041] Results The nucleotide sequence of the obtained DNA molecule fragment is shown in SEQ ID NO: 1. The sequence was compared with the NCBI database by BLAST online. The results showed that the strain sequence had the highest similarity with the ITS sequence of Phallus dongsun, with a sequence similarity of up to 96%. A phylogenetic tree was constructed. Results (see Figure 2 ) The strain Guizhou P216 of the present invention belongs to the genus Phallus dongsun, and the strain is different from any existing Phallus dongsun strain and is a new Phallus dongsun strain.

[0042] Example 3 Cultivation test of the winter safflower strain Guizhou P216 of the present invention

[0043] Proceed as follows:

[0044] (1) Mother culture: Inoculate the mother culture of Guizhou P216 (the Guizhou P216 strain has been deposited in the General Microbiology Center of the China Microbiological Culture Management Committee, and its deposit number is: CGMCC No: 40102) onto the mother culture medium, place it in a 25°C constant temperature incubator and culture it in the dark for 20 days. The mycelium will cover the culture medium, and the Guizhou P216 mother culture will be obtained. The mother culture medium comprises: 200g potato, 20g glucose, 20g agar, 0.3g potassium dihydrogen phosphate, 0.15g magnesium sulfate, 10mg vitamin B1, and 1000mL water. After preparation, sterilize it at 121°C for 30min and pour it into a 9cm diameter plate.

[0045] (2) Original seed culture: Use the tic-tac-toe method to streak the bacterial blocks from the Guizhou P216 mother plate covered with culture medium obtained in step (1) and transfer them to the original seed culture medium, transfer one plate to one bottle, and then culture at 25°C in the dark for about 50 days until the mycelium fills the culture bottle, thus obtaining the Guizhou P216 original seed. The original seed culture medium comprises the following components: 89% miscellaneous sawdust (fine), 10% wheat bran, and 1% lime; the water content is about 70%; take each component in proportion, mix them evenly, and then add water in proportion; put the prepared original seed culture medium into a 500mL glass culture bottle, sterilize it under high pressure at 121°C for 1h, and place it in a clean bench to cool to room temperature after sterilization.

[0046] (3) Cultivation of cultivars: Transfer the original Guizhou P216 obtained in step (2) to the culture medium, transfer about 15 g to one bag, and culture at 25°C in the dark for about 50 days until the bag is full of fungi, thus obtaining the Guizhou P216 cultivar. The culture medium comprises: 80% miscellaneous sawdust, 18% wheat bran, 1% lime, and 1% gypsum; the water content is 65%; when preparing, take each component in proportion and mix them evenly, and then add water in proportion; then put the prepared culture medium into a polypropylene plastic bag (16cm×35cm×0.005cm) and tie it with a polypropylene rope, sterilize it under high pressure at 121°C for 2 hours, 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 ridge construction: The cultivation of Guizhou P216 selects the under-forest cultivation mode, and the cultivation site selects a forest with a high density. The soil should be fertile and loose in texture, and the relative air humidity should be 70% to 85%. It is best to have a certain slope to facilitate drainage. When digging ditches, the width should be 50 cm, the depth should be 15 to 20 cm, and the length should be unlimited. The bottom of the ditch should be leveled, and 3 to 4 cm thick loose soil should be laid to retain water. The width of the two ditches should be 20 to 25 cm.

[0048] (5) Paving: The cultivation method of Guizhou P216 is to place a layer of wood, a layer of fungus and then a 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 April 1 to 10. Break the Guizhou P216 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 about 2 cm thick to moisturize the soil.

[0051] (8) Management of mushroom production: Temperature management is the key in the cultivation of winter mulberry. Qian P216 is a low-temperature variety. The mycelium growth temperature is preferably 16-24°C. The fruiting temperature of Qian P216 is generally guaranteed to be 5-12°C for mushroom production. Qian P216 has different requirements for humidity at different growth stages. In the mycelium growth stage, the humidity is preferably 70%-85%. In the bud differentiation stage, the relative humidity of the air is required to be above 80%. In the fruiting stage, the soil humidity is preferably 80%-85%, and the relative humidity of the air is above 80%. The cultivation environment of winter mulberry requires half shade and half sun, and direct sunlight should be avoided to damage the mushroom buds.

[0052] (9) Harvesting: The fruiting body of Guizhou P216 is formed from November 1 to November 30, and the entire growth cycle from sowing to harvesting is about 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) The fruiting bodies of Guizhou P216 were measured after harvest and the morphology of the fruiting bodies of Guizhou P216 was described; the weight of each plant was weighed and the average fresh weight was calculated. 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 fruiting body stipe of the winter clover strain Guizhou P216 of the present invention is white, cylindrical and hollow, with a good mushroom shape and high commercial value. Secondly, the fruiting body is large, with a stipe length of 20 to 28 cm and a diameter of 3 to 6 cm. The average weight of fresh mushrooms is 58.27 g, which is higher than the fresh mushroom weight of 25 to 40 g / piece of other existing winter clover strains. Guizhou P216 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 P216 has a high yield. After cultivation and yield measurement, the average fresh product yield is 2.33 kg / m 2 , significantly higher than other existing winter iris strains 1.0 ~ 1.5 kg / m 2 The fresh product yield is high. After testing, the total sugar content of Guizhou P216 is 28.81%, which is significantly higher than the total sugar content of other existing winter clover strains of 5-20%; Sixth, the crude protein content of Guizhou P216 is 17.86%, which is higher than the crude protein content of other existing winter clover strains of 8-16%.

[0055] The above results show that the winter mulberry strain Guizhou P216 of the present invention has the advantages of good mushroom shape, large fruiting body shape, high fresh mushroom unit weight, short cultivation period, high yield, and high total sugar content and crude protein content. In short, it has high nutritional value, high functional product development and utilization value, high added value, and brings high economic benefits to mushroom farmers.

Claims

1. A Phallus dongsun strain Guizhou P216, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.40102.

2. Use of the strain Guizhou P216 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 P216 or an extract of Guizhou P216 according to claim 1.

4. Use of the strain Guizhou P216 according to claim 1 in beverages.

5. A beverage, characterized in that The beverage contains the strain Guizhou P216 or an extract of Guizhou P216 according to claim 1.

6. Use of the strain Guizhou P216 according to claim 1 in preparing drugs for improving human immunity.

7. Use of the strain Guizhou P216 according to claim 1 in the preparation of drugs for promoting blood circulation and relieving pain, inhibiting bacteria, reducing edema or treating rheumatic joint pain.

8. A genetically engineered winter safflower strain, characterized in that: The starting strain of the genetically engineered Ilex sanguinea strain is the strain Guizhou P216 described in claim 1.

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