Wintergrain strain qianp907 and application thereof

Abstract

The application belongs to the field of edible fungi, and specifically discloses a winter truffle (Phallus dongsun) strain Qian P907, which is preserved in the China General Microbiological Culture Collection Center and has a preservation number of CGMCC No. 40101. The application also discloses the use of the strain in food, beverage, health products and medicine preparation. The winter truffle strain Qian P907 has the advantages of good mushroom type, large fruiting body, high single weight of fresh mushroom, short cultivation period, high total sugar content and high crude protein content, and has high commodity value, high nutritional value and high economic benefit.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of edible fungi, and particularly relates to a winter truffle strain. BACKGROUND

[0002] Winter truffle (Phallus dongsun, also known as white ghost pen, bamboo fungus, bamboo fungus, and skirtless truffle) belongs to the family Phallaceae and the genus Phallus, and is a famous medicinal and edible fungus in China. Winter truffle is mainly distributed in Guizhou, Sichuan, Yunnan, Anhui, Guangdong and other places. Winter truffle has a crisp taste and a delicious taste, and is rich in nutrients such as various amino acids, proteins, vitamins, polysaccharides and the like. In addition, winter truffle is also a traditional folk medicine in Asia, and has the effects of activating blood and relieving pain, inhibiting bacteria, reducing edema and treating rheumatic joint pain. The active ingredients contained in winter truffle also have the characteristics of immune regulation and promoting wound healing. With the improvement of people's living standards and the gradual understanding of the importance of the nutritional and medicinal values of winter truffle, the demand for winter truffle has greatly increased, and the existing production of winter truffle is difficult to meet the demand.

[0003] At present, the main problems in the cultivation of winter truffle in China are as follows: (1) there are few cultivated varieties; (2) the mycelial growth rate is low, the production cycle is long, and the traits are unstable. At present, the main cultivated winter truffle strain has a production cycle of 9-12 months, which requires a large amount of labor cost, increases the influence of climate and environment and the risk of diseases and pests, and leads to unstable yield and quality of winter truffle. Therefore, in order to meet the market demand and reduce the cost, breeding a new winter truffle strain with short cultivation cycle and high yield is the best way to solve the existing problems in winter truffle production.

[0004] The primitive forests in southern regions such as Guizhou, Sichuan and Yunnan in China are rich in wild edible fungus resources, including wild winter truffle resources. The development and domestication of wild winter truffle strains not only can enrich people's table and meet the needs of people's high-level life, but also can develop food, beverage, health care products and medicine with higher added value by taking advantage of the characteristics of the fungus being both medicinal and edible, so as to improve people's health level and increase the income of mushroom farmers and export earnings. SUMMARY

[0005] The present application aims to provide a winter truffle strain with short cultivation cycle and high yield.

[0006] Another object of the present application is to provide the use of the winter truffle strain.

[0007] The object of the present application is achieved by the following technical solutions:

[0008] The present application provides a winter truffle (Phallus dongsun) strain Qian P907, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.40101.

[0009] The application also provides application of the strain QianP907 in food or health products.

[0010] The application also provides a food or health product containing the strain QianP907 or extract of QianP907.

[0011] The application also provides application of the strain QianP907 in beverages.

[0012] The application also provides a beverage containing the strain QianP907 or extract of QianP907.

[0013] The application also provides application of the strain QianP907 in preparation of a medicine for improving human immunity.

[0014] The application also provides application of the strain QianP907 in preparation of a medicine for promoting blood circulation, dispelling pain, inhibiting bacteria, removing edema or treating rheumatic arthralgia.

[0015] The application also provides a genetically engineered Fritillaria strain, wherein the starting strain of the genetically engineered Fritillaria strain is the strain QianP907.

[0016] The application also provides a cultivation method of the Fritillaria strain QianP907, comprising the following steps.

[0017] (1) mother strain culture: inoculate the strain QianP907 mother strain into a mother strain culture medium, and then place in a 25℃ constant temperature incubator for dark culture for 20-25 days, so that the mycelium fills the culture medium, and the QianP907 mother strain is obtained; wherein the mother strain culture medium comprises the following components: 200g of potato, 20g of glucose, 20g of agar, 0.3g of potassium dihydrogen phosphate, 0.15g of magnesium sulfate, 10mg of vitamin B1, and 1000mL of water; after the mother strain culture medium is prepared, it is sterilized at 121℃ for 30min, and then poured into a 9cm diameter plate;

[0018] (2) original strain culture: the plate filled with the QianP907 mother strain obtained in step (1) is cut into blocks in a cross shape, and then inoculated into an original strain culture medium, 1 plate is inoculated into 1 bottle, and the mycelium fills the strain bottle after 25℃ dark culture for 50 days, and the QianP907 original strain is obtained; wherein the original strain culture medium comprises the following components: 89% of mixed wood chips (fine), 10% of wheat bran, and 1% of lime; the water content is 70%; when prepared, the components are mixed according to the proportions, and then water is added according to the proportion, and then loaded into a 500mL glass strain bottle, sterilized at 121℃ for 1h, and then cooled to room temperature in a clean bench after sterilization.

[0019] (3) Cultivation of the cultivated species: the original strain of Qian P907 obtained in step (2) is transferred to culture medium, about 15 g per bag, and cultured at 25℃ in the dark for 50 days. When the mycelium grows to fill the bag, the cultivated species of Qian P907 is obtained. The culture medium comprises 80% of mixed wood chips, 18% of wheat bran, 1% of lime and 1% of gypsum, and has a water content of 65%. The components are mixed in proportion, and water is added in proportion. The prepared culture medium is placed in a polypropylene plastic bag (16 cm x 35 cm x 0.005 cm), and then the opening is tied with a polypropylene rope. The bag is sterilized at 121℃ for 2 hours, and then cooled to room temperature. The mixed wood chips are coarse wood chips, which need to be pre-wetted to prevent contamination caused by incomplete sterilization;

[0020] (4) Land preparation and ridge building: the cultivation of Winter Chanterelle is selected as a mode of under-forest cultivation. The cultivation site is selected as a forest with high canopy density. The soil is required to be fertile and loose, and the relative air humidity is 70% to 85%. It is best to have a certain inclination to facilitate drainage. When digging the ditch, the width is 50 cm, the depth is 15 to 20 cm, and the length of the ditch is not limited. The bottom of the ditch is leveled, and 3 to 4 cm thick soil is laid to retain water. The width between two ditches is 20 to 25 cm.

[0021] (5) Paving: the cultivation of Qian P907 adopts a cultivation method of one layer of wood, one layer of fungus, and one layer of wood. Fresh broad-leaved trees such as Castanopsis fargesii and Cyclobalanopsis glauca are selected, which are cut into wood blocks with a thickness of 2 to 5 cm and a length of 5 to 10 cm. Then the wood is evenly paved 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 cultivated species of Qian P907 obtained in step (3) is broken into small pieces with a diameter of 3 to 5 cm, which are scattered on the bottom layer of wood. The distance between the fungus and the fungus is 3 to 4 cm. Then a layer of thin bamboo leaves is covered, and then a layer of wood is added.

[0023] (7) Covering soil: clean and loose soil without obvious growth of miscellaneous bacteria is selected. The thickness of the covering soil is 3 to 5 cm. In dry areas, the thickness of the covering soil is slightly thicker, i.e. 5 to 8 cm. The covering soil is in the shape of a tortoise back, and then a layer of pine needles with a thickness of about 2 cm is added to retain moisture.

[0024] (8) Management of fruiting: the temperature for mycelial growth is 16 to 24℃. The temperature for the formation of fruiting bodies of Qian P907 is generally 5 to 12℃, and the fruiting can occur. The humidity requirements are different at different growth stages of Qian P907. During the mycelial growth stage, the humidity is preferably 70% to 85%. During the bud differentiation stage, the relative air humidity is required to be more than 80%. During the fruiting body growth stage, the soil humidity is preferably 80% to 85%, and the relative air humidity is more than 80%. The cultivation environment of Winter Chanterelle requires semi-shade and semi-sunlight, and direct sunlight should be avoided to prevent damage to the buds.

[0025] (9) Harvesting: The fruiting body formation time of Qian P907 is generally from October to December, and the whole growth cycle from sowing to harvesting is 7-8 months. The winter truffle is tender and easy to break, so special care should be taken during harvesting. First, cut off the bottom of the mycelium with a small knife, then gently turn and pull it up, and do not pull it directly to avoid damaging the mycelium and affecting the second mushroom.

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

[0027] Biological preservation: The winter truffle (Phallus dongsun) strain Qian P907 of the present application is a wild winter truffle strain collected by the present inventor in Dafang County, Bijie City, Guizhou Province in 2018, and is artificially domesticated and cultivated; Qian P907 was preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Haidian District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, on February 17, 2022, and its preservation number is CGMCC NO. 40101. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 . The fruiting body photo of the winter truffle strain Qian P907 of the present application.

[0029] Figure 2 . The phylogenetic tree of the winter truffle strain Qian P907 of the present application. DETAILED DESCRIPTION

[0030] The present application will be further described by examples, but does not constitute any limitation on the present application.

[0031] Example 1 Collection, isolation and domestication process of the strain Qian P907 of the present application

[0032] In October 2018, the inventors collected multiple wild winter truffle fruiting bodies in the Dafang County of Bijie City, Guizhou Province, and obtained mycelium (strain) through fruiting body tissue separation. Through morphological characteristics, ITS-PCR detection, phylogenetic tree construction, and gene fragment size and sequence, it was identified as Phallus dongsun. The strain obtained by tissue separation was cultivated from 2019 to 2021, and the fruiting bodies with good commercial traits were subjected to tissue separation every year. The mycelium with robust growth, fast growth rate, and uniform colony was selected as the mother strain for cultivation in the next year. In 2021, a strain with good mushroom type, high yield, and short production cycle was selected, which was named as Qian P907. The fruiting body of the strain has a cap height of 2-7 cm, a cap diameter of 2-7 cm, a cap color of dark green, a surface grid, a dark green and sticky spore body attached in the grid, oval to long oval spores, smooth and colorless transparent, a white sponge and hollow stem with a length of 22-28 cm and a diameter of 4-7 cm, and a film-like volva with a grayish white color, a height of 4-8 cm, and a diameter of 5-12 cm.

[0033] Example 2: Classification and identification of the strain Qian P907 of the present application

[0034] (1) Morphological identification of Qian P907

[0035] The Qian P907 bud is single or clustered, the fruiting body is sterile skirt, the cap is bell-shaped, the top is flat after maturation, and there are perforations. The cap surface is grid-shaped, and a dark green and sticky spore body is attached in the grid. The spores are oval to long oval, smooth, and colorless transparent. The stem is white and sponge-like, hollow, and has a grid. The volva is grayish white, and has a cord. According to the corresponding morphological characteristics and photos of winter truffle in the 478th page of Chinese Edible Fungus Cultivation (Zhang Jinxia, Cai Weiming, Huang Chenyang, et al., China Agriculture Press, 2020), the morphological characteristics of Qian P907 are consistent with those of winter truffle, indicating that Qian P907 belongs to the Phallus dongsun species.

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

[0037] The genomic DNA of the QianP907 strain is extracted by using a new plant genomic DNA extraction kit CW0531 (Beijing Kangwei Century Company), and the ITS-PCR amplification is carried out by using universal primers ITS4 and ITS5 to obtain an amplification 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 as follows: 1 μL of 20-50 ng / μL DNA template, 2 μL of 10×Buffer (with Mg 2+ ), 0.5 μL of 2.5 mM dNTP, 0.2 μL of 5 U / μl DNA polymerase, 0.5 μL of 0.2 μM primer, and adding double-distilled water to 20 μL. The ITS-PCR amplification reaction conditions are as follows: pre-denaturation at 94℃ for 4 min; 94℃ for 30 s, 56℃ for 30 s, 72℃ for 1 min, 35 cycles; and extension at 72℃ for 10 min.

[0041] The results show that the size of the DNA molecule segment is 544 bp, and the sequencing is carried out by sending to Shengong Bioengineering Co., Ltd. The nucleotide sequence of the DNA molecule segment is shown as SEQ ID NO: 1. The sequence is subjected to BLAST online comparison in the NCBI database, and the results show that the sequence of the strain is most similar to the sequence of Phallus dongsun, with a similarity of 92.42%, and a phylogenetic tree is constructed. The results show that the strain QianP907 belongs to Phallus dongsun, and is different from any existing strain, and is a new Phallus dongsun strain. Figure 2

[0042] Example 3 Cultivation test of the Phallus dongsun strain QianP907 of the application

[0043] The following method is used:

[0044] ​(1)Mother culture: inoculate Qian P907 mother strain (Qian P907 strain is preserved in China General Microbiological Culture Collection Center, and its preservation number is CGMCC No: 40101) to mother culture medium, and place in a 25℃ constant temperature incubator for dark culture for 20-25 days. The mycelium grows on the medium to obtain Qian P907 mother strain. The composition of the mother culture medium is: potato 200g, glucose 20g, agar 20g, potassium dihydrogen phosphate 0.3g, magnesium sulfate 0.15g, vitamin B1 10mg, water 1000mL. The mother culture medium is prepared according to the usual method, then autoclaved at 121℃ for 30min, and poured into a 9cm diameter plate.

[0045] (2)Primary culture: the Qian P907 mother strain with mycelium growing on the medium obtained in step (1) is inoculated into primary culture medium by using a cross-shaped method to transfer the mycelium block to the primary culture medium, 1 plate is transferred to 1 bottle, and then cultured at 25℃ in the dark for 50 days. The mycelium grows on the bottle to obtain Qian P907 primary strain. The composition of the primary culture medium is: mixed wood chips (fine) 89%, wheat bran 10%, lime 1%; water content 70%; when preparing, the components are mixed according to the proportion, then water is added according to the proportion, and then the mixture is loaded into a 500mL glass bottle. The bottle is autoclaved at 121℃ for 1h, and then cooled to room temperature in a clean bench after sterilization.

[0046] (3)Culture of cultivated species: the Qian P907 primary strain obtained in step (2) is transferred to the culture medium, and 15g of the culture medium is transferred to a bag. The bag is cultured at 25℃ in the dark for 50 days, and the mycelium grows on the bag to obtain Qian P907 cultivated species. The composition of the culture medium is: mixed wood chips 80%, wheat bran 18%, lime 1%, and gypsum 1%; water content 65%; when preparing, the components are mixed according to the proportion, and then water is added according to the proportion. The prepared culture medium is loaded into a polypropylene plastic bag (16cm×35cm×0.005cm), and then the opening is tied with a polypropylene rope. The bag is autoclaved at 121℃ for 2h, and then cooled to room temperature after sterilization. The mixed wood chips in the culture medium are coarse wood chips, which need to be pre-wetted to prevent contamination caused by incomplete sterilization.

[0047] (4)Ground preparation and ridge building: the cultivation of Yuxingcao selects a forest understory mode, and the cultivation site selects a forest with high canopy density. The soil is fertile and loose, the relative humidity of air is 70%-85%, and preferably has a certain inclination to facilitate drainage. When digging the ditch and ridge, the width is 50cm, the depth is 15-20cm, and the length of the ditch is unlimited. The bottom of the ditch is leveled, 3-4cm thick soil is laid on the bottom of the ditch for water retention, and the width between the two ditches is 20-25cm.

[0048] (5)Paving: The cultivation of winter truffles uses a cultivation method of one layer of wood, one layer of spawn, and another layer of wood. Fresh broad-leaved trees such as Castanopsis fargesii and Cyclobalanopsis glauca are selected and cut into wood blocks with a thickness of 2-5 cm and a length of 5-10 cm. The wood is then evenly paved on the bottom of the ditch and compacted to cover the soil in the ditch.

[0049] (6)Seeding: The seeding time is from April 1st to April 10th. The Qian P907 cultivated species obtained in step (3) is broken into small pieces with a diameter of 3-5 cm and scattered on the bottom layer of wood, with a spacing of 3-4 cm between the spawn. A thin layer of bamboo leaves is then covered, followed by another layer of wood.

[0050] (7)Covering soil: Clean, loose soil without obvious growth of miscellaneous bacteria is selected, and the covering soil thickness is 3-5 cm. In areas prone to drought, the covering soil thickness is slightly thicker, at 5-8 cm. The covering soil is in the shape of a tortoise back, and then a layer of pine needles with a thickness of 2 cm is added for moisture retention.

[0051] (8)Mushroom management: Temperature management is the key during the cultivation of winter truffles. It belongs to a low-temperature variety, and the mycelium growth temperature is suitable for 16-24℃. The formation temperature of winter truffle fruiting bodies is generally 5-12℃, which can produce mushrooms. The humidity requirements are different at different growth stages. During the mycelium growth stage, the humidity is suitable for 70%-85%, the air relative humidity during the bud differentiation stage is required to be more than 80%, the soil humidity during the fruiting body growth stage is suitable for 80%-85%, and the air relative humidity is more than 80%. Winter truffles do not require high light intensity, and the cultivation environment requires semi-shade and semi-sunlight, which can avoid direct sunlight to cause damage to the mushroom buds.

[0052] (9)Harvesting and evaluation: The formation time of Qian P907 fruiting bodies is from November 1st to November 30th, and the entire growth cycle from seeding to harvesting is 7-8 months. Winter truffles are brittle and easy to break, so special care should be taken during harvesting. First, cut off the bottom mycelium with a small knife, then gently turn and pull it up, do not pull it directly, avoid damaging the mycelium and affecting the second mushroom.

[0053] (10)Measure and describe the morphology of Qian P907 fruiting bodies after harvesting; weigh the weight of single plant and calculate the average fresh weight. Total sugar determination refers to GB / T 15672-2009 "National Standard Determination of Total Sugar Content in Edible Fungi"; protein content determination refers to GB5009.5-2016 "Food Safety National Standard Determination of Protein in Food".

[0054] Results (see Figure 1The stipe of the winter truffle strain Qian P907 is white, the stipe is cylindrical and hollow, the cap shape is good, and the commodity value is high. Secondly, the fruiting body of Qian P907 has a large size, the stipe is 22-28 cm long and 4-7 cm in diameter, the average fresh mushroom single weight is 63.10 g, which is significantly higher than the fresh mushroom single weight of 25-40 g of other existing winter truffle strains. In addition, the cultivation period of Qian P907 is short, the period from sowing to starting to harvest is 7-8 months, which is 2-4 months shorter than the cultivation period of 9-12 months of other existing winter truffle strains; the yield of Qian P907 is high, and the average fresh yield is 2.20 kg / m 2 , which is significantly higher than the fresh yield of 1.0-1.5 kg / m 2 of other existing winter truffle strains. The total sugar content of Qian P907 is 26.75%, which is significantly higher than the total sugar content of 5-20% of other existing winter truffle strains. The crude protein content of Qian P907 is 17.21%, which is significantly higher than the crude protein content of 8-16% of other existing winter truffle strains.

[0055] The above results show that the winter truffle strain Qian P907 has the advantages of good cap shape, large fruiting body size, high fresh mushroom single weight, short cultivation period, high total sugar content and high crude protein content, and in general, the commodity value is high, the nutritional value is high, has high functional product development and utilization value, high added value, and high economic benefit.

Claims

1. A strain of Dictyophora indusiata (Vent.ex Pers.) Fisch. Phallus dongsun strain Qian P907, which is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, and its deposit number is CGMCC No. 40101.

2. The strain QianP907 of claim 1 is applied in food.

3. A food product, characterized by, The food contains the strain QianP907 of claim 1.

4. The strain QianP907 of claim 1 is applied in beverage.

5. A beverage, characterized in that The beverage contains the strain QianP907 of claim 1.

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