Auricularia plicata strain rich in iron and crude fibers and cultivation method and application thereof

Abstract

The invention discloses an auricularia plicata strain rich in iron and crude fibers and a cultivation method and application thereof, and belongs to the field of edible fungus cultivation. A new Auricularia plicata strain is separated and purified from an Auricularia plicata fruiting body on an anise tree in Dariaotun, love village, Yujiang district, Baijiang city, Guangxi Zhuang Autonomous District, is named as' Guiyun No.5 ', is preserved in Guangdong Microbial Culture Collection Center, and has a preservation number of GDMCC No: 64801. According to the method, a set of auricularia plicata cultivation method is constructed through condition optimization, large-scale cultivation and planting can be achieved through the cultivation method, the planted auricularia plicata is rich in iron and crude fibers, and the method has important significance on deep development of edible mushrooms and preparation of the edible mushrooms into food or medicine rich in iron and crude fibers.

Description

Technical Field

[0001] The invention relates to the field of edible fungus cultivation, in particular to a Auricularia auricula strain and a cultivation method and application thereof. Background Art

[0002] Auricularia delicata is a species of the genus Auricularia in the family Auriculariales. It is an edible and medicinal fungus.

[0003] Auricularia auricula is mainly distributed in tropical and subtropical areas in southwest my country. At present, it is mainly collected in the wild. There are a few literature reports on artificial cultivation of Auricularia auricula, but it has not yet been fully industrialized. Bacterial strains and cultivation techniques are the key technical links for industrialization. The acquisition of Auricularia auricula strains is usually achieved by separation and purification of fruiting bodies and direct application in cultivation production. However, due to the characteristics of Auricularia auricula varieties, their mycelium grows slowly and is prone to degeneration. The shortcomings of existing technologies are that there are few strains that can be cultivated, the strain rejuvenation technology is backward, and the cultivation conditions are in the stage of small-scale experimental research. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of wrinkled fungus rich in iron and crude fiber and a cultivation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention isolates a strain of wrinkled fungus rich in iron and crude fiber, and constructs a set of cultivation methods by optimizing cultivation conditions, which can realize large-scale cultivation and planting.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a wrinkled fungus (Auricularia delicata) strain, named "Guiyun No. 5", the preservation number of the wrinkled fungus strain is GDMCC No: 64801, the preservation time is June 24, 2024, the preservation unit is Guangdong Microbiological Culture Collection Center, and the preservation address is Guangdong Academy of Sciences Institute of Microbiology, 5th Floor, Building 59, Compound 100 Xianlie Middle Road, Guangzhou.

[0007] The present invention also provides application of the Auricularia auricularia strain in preparing food or medicine rich in iron and / or crude fiber.

[0008] The invention also provides a food rich in iron and crude fiber, comprising the Auricularia auricularia auricula strain.

[0009] The present invention also provides a method for cultivating the Auricularia auricularia auricula strain, comprising the following steps:

[0010] (1) collecting the Auricularia auricula separated from tissues, inoculating the Auricularia auricula into a PDA slant culture medium aseptically, and culturing the culture medium in a dark environment at a constant temperature of 25° C. until the mycelium grows all over the slant culture medium, thereby obtaining a production mother culture;

[0011] (2) The production mother seed is aseptically inoculated into a stock seed bag, and placed in a constant temperature dark culture at 25° C. until the mycelium is fully fed with the material, thereby obtaining the stock seed; the culture medium in the stock seed bag comprises the following raw materials in parts by weight: 60 parts of cottonseed hulls, 25 parts of wheat bran, 1.2 parts of superphosphate, 1 part of gypsum, and 50 parts of water;

[0012] (3) The original seed is aseptically inoculated into a production bag, and placed in a dark culture at a constant temperature of 25° C. until the mycelium is fully fed with the material, thereby obtaining the production seed; the culture medium loaded into the production bag comprises the following raw materials in parts by weight: 100 parts of broad-leaved wood chips, 25 parts of bran, 20 parts of sugarcane bagasse, 20 parts of cottonseed hulls, 1.0 parts of superphosphate, 1.5 parts of gypsum and 55 parts of water;

[0013] (4) After the cultivation material is bagged and sterilized, the production species is inoculated, and the inoculated bacterial sticks are piled up in a "well" shape, the temperature is controlled at 25° C., and the cultivation is shaded; the culture medium loaded in the cultivation material includes the following raw materials in parts by weight: 100 parts of miscellaneous wood chips, 15 parts of wheat bran, 0.5 parts of gypsum, 0.5 parts of lime, 12 parts of cottonseed hulls, and 80 parts of water;

[0014] (5) When the mycelium has grown all over the bag, the mycelium is punctured when it is physiologically mature. After puncturing, the mushroom sticks are stacked in a "well" shape for ventilation and cultivation. When primordia are formed at the puncture holes, the ears can be placed on the ground and managed;

[0015] (6) Harvest the wrinkled fungus after it matures.

[0016] Preferably, in step (2), the inoculation amount of the production mother seed is 8 g per original seed bag.

[0017] Preferably, in step (3), the inoculation amount of the original seed is 8 g for each production bag.

[0018] Preferably, in step (4), the inoculation amount of the production seed is 10 g per cultivation bag.

[0019] The present invention discloses the following technical effects:

[0020] The invention separates and purifies a new wrinkled fungus strain from fungus fruiting bodies, establishes a cultivation method for the wrinkled fungus after optimizing conditions, detects the yield and nutritional components of the wrinkled fungus after cultivation, and finds that the yield of the wrinkled fungus can reach the standard of the existing fungus cultivation yield, indicating that the wrinkled fungus separated and purified by the invention can be cultivated on a large scale; in addition, it is found in nutritional component analysis that the iron content and crude fiber content of the wrinkled fungus are much higher than those of other varieties of fungus, indicating that the wrinkled fungus can be used for supplementing iron elements and crude fiber, and is of great significance for preparing foods or medicines rich in iron and crude fiber. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 To purify the strain with better growth;

[0023] Figure 2 The fungus grown in the bag after puncturing and the harvested fungus;

[0024] Figure 3 The results of determination of iron content in different fungus species are shown in Figure 2.

[0025] Figure 4 The results of the determination of crude fiber content of different fungus;

[0026] Figure 5 The results of the determination of moisture content of different fungus;

[0027] Figure 6 The results are for the determination of protein content in different fungus;

[0028] Figure 7 The results of determination of ash content of different fungus;

[0029] Figure 8 The results are the determination results of polysaccharide contents in different fungus;

[0030] Fig. 9 These are the results of determining the total sugar content of different fungus. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Example 1 Isolation, purification and identification of strains

[0037] 1. Isolation and purification of strains

[0038] (1) Fruiting body collection: Fruiting bodies were collected from the star anise tree in Nahua Village, Luofu Village, Daleng Township, Youjiang District, Baise City, Guangxi Zhuang Autonomous Region in August 2018.

[0039] (2) Tissue separation: Rinse the debris on the fruiting body with running water and wipe dry. Place in 75% alcohol to sterilize the surface for 5 minutes. Wash the surface with sterilized water on a clean bench for 3 times, dry the water, cut the fruiting body with a blade, tear open the inner layer of the fruiting body, cut into 0.5-1cm squares, and stick the inner layer on the PDA culture dish.

[0040] (3) Strain purification: Incubate in the dark at 25°C for 4 days, remove the contaminated culture dishes, and select the uncontaminated culture dishes with good growth to obtain the strain (see Figure 1). Use an inoculation needle to pick up a 0.5 cm-sized bacterial block and inoculate it on the PDA culture medium for strain purification. After 3 days, pick up a 0.5 cm-sized bacterial block and inoculate it on the culture slant. Culture it in the dark at 25°C for 5 days to obtain the purified test tube strain of Auricularia auricula.

[0041] 2. Strain identification

[0042] The test fruiting bodies and corresponding mycelium were used to extract DNA according to the CTAB method, and PCR amplification and ITS sequence determination were performed. The ITS of the samples was compared with the wrinkled fungus (Auricularia delicata) in Genbank by Blast. Identities = 572 / 575 (99%), Gaps = 3 / 572 (0%). The analysis confirmed that the isolated pure culture was short-haired Auricularia mycelium.

[0043] Biological characteristics: The fruiting body is 5.72cm x 8.21cm in size; the ear base is small and the ear blade is thin; the fruiting body is flaky, mostly clustered, and the edges are shallowly cracked; the surface of the hymenium is obviously wrinkled and reticular. The fruiting body is elastic, gelatinous, translucent, yellow-brown, and leathery, thin, and dark brown when dry. The sterile surface is soft hairy; the cross section has an obvious medullary layer.

[0044] The ITS sequence primers are: ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO: 2), ITS4 (5'-TCCTCCGCT TATTGATATGC-3', SEQ ID NO: 3).

[0045] Amplification reaction system: TaKaRa Ex Taq enzyme 0.25μL, 10×Ex Taq Buffer 3μL, MgCl2 1.5μL, dNTP Mixture 2.5μL, ITS1 / ITS4 0.25μL each, DNA template 1μL, ddH2O supplemented to 25μL.

[0046] The reaction program was: 95°C for 3 min; 95°C for 30 s, 56°C for 45 s, 72°C for 1 min, 35 cycles; 72°C for 4 min.

[0047] The amplified ITS sequence (SEQ ID NO: 1) is as follows:

[0048] .

[0049] The strain isolated and identified was Auricularia delicata 2018051504 (abbreviated as: Auricularia delicata 504), named Auricularia delicata "Guiyun No. 5", and was deposited in Guangdong Provincial Microbiological Culture Collection on June 24, 2024. The preservation address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC No: 64801.

[0050] Embodiment 2 Cultivation and application of Auricularia auricula

[0051] (1) Preparation of mother culture: Collect the strain of Auricularia auriculariae "Guiyun No. 5" separated by tissue, aseptically inoculate the strain into the mother culture medium (PDA slant culture medium), place it in a 25°C incubator for constant temperature and dark culture until the mycelium grows all over the slant to obtain the mother culture.

[0052] (2) Preparation of stock seed: The production mother seed is aseptically inoculated into a stock seed bag (the culture medium in the bag includes the following raw materials in parts by weight: 60 parts of cottonseed hulls, 25 parts of wheat bran, 1.2 parts of superphosphate, 1 part of gypsum, and 50 parts of water), with an inoculum amount of 8 g per bag, and placed in a 25°C incubator for constant temperature dark culture until the mycelium is fully fed with the material to obtain the stock seed;

[0053] (3) Preparation of production seeds: The original seeds are aseptically inoculated into a production seed bag (the culture medium in the bag includes the following raw materials by weight: 100 parts of broad-leaved wood chips, 25 parts of bran, 20 parts of bagasse, 20 parts of cottonseed hulls, 1.0 parts of superphosphate, 1.5 parts of gypsum, and 55 parts of water), with an inoculum size of 8 g per bag, and placed in a 25° C. incubator for constant temperature dark culture until the mycelium is fully fed with the material, and the production seeds are obtained;

[0054] (4) Preparation of mushroom bags: Use a 15cm×55cm×0.005cm polyethylene bag with a folded corner. Put the culture material (composed of the following components by weight: 100 parts of sawdust, 15 parts of wheat bran, 0.5 parts of gypsum, 0.5 parts of lime, 12 parts of cottonseed hulls and 80 parts of water) into the bag, and then sterilize it in an autoclave. The temperature is raised to 100°C within 4 hours after entering the pot, and the temperature is kept constant for 15 hours. The fire cannot be stopped during this period. After sterilization, let the temperature in the pot drop naturally to 50-60°C, take out the material rod while it is hot, and move it into a cooling room or inoculation room. When the temperature of the material rod drops below 28°C, inoculate it according to the aseptic operation requirements.

[0055] (5) Inoculation: Make 4 holes in the bag for inoculation, with a hole diameter of 2.5 cm and a hole depth of 2.5 cm; the inoculation amount is 10 g;

[0056] (6) Cultivation: The inoculated mushroom sticks are piled up in a “well” shape, the temperature is controlled at 25°C, and the mycelium is cultured in a darkened state. The relative humidity in the pile is lower than 65%. The mycelium will grow fully in about 50 days. During this period, the pile is turned, spread, and ventilated according to the ambient temperature and mycelium growth.

[0057] (7) Puncture: Puncture when the mycelium fills the bag and reaches physiological maturity. Use the bag-free method to puncture holes. The number of holes is 250-300 per bag. The hole diameter is 0.4 cm and the hole depth is 0.5 cm. After puncturing, the mushroom sticks are stacked in a "well" shape and ventilated for 10 days. When primordia are formed at the hole mouth (see Figure 2 ) can be put to the ground and ears can be pulled out.

[0058] (8) Placement: Place the mushrooms in a greenhouse or outdoors on a sunny day with a temperature of about 25°C. Set up a simple rack with mushroom sticks leaning against it at an angle of about 60 degrees, with a distance of 15 cm between the mushroom sticks.

[0059] (9) Management of mushrooms: Use water mist spray belts or spray pipes to maintain the humidity of mushroom sticks, spray 3 to 5 times a day, each time for 20 to 40 minutes; when the auricles grow to more than 1 cm, the number of spraying can be reduced by 1 to 2 times, and the relative humidity can be maintained above 95%; harvest when the auricularia auricula is mature (see Figure 2 ).

[0060] Under the above cultivation conditions, different fungus species were cultivated at the same time. After the fungus species were harvested when mature, the yields of the different fungus species and the nutritional elements of the fungus species were measured.

[0061] (1) Yield measurement

[0062] After harvesting, the fungus was weighed for wet weight and the data was recorded. After weighing, it was dried. The dried fungus was weighed again and the data was recorded. Finally, the dried fungus was stored in a 4°C refrigerator for subsequent experiments.

[0063] (2) Determination of iron content

[0064] Refer to GB 5009.90-2016, National Food Safety Standard - Determination of Iron in Food.

[0065] (3) Determination of moisture content

[0066] Refer to GB 5009.3-2016, National Food Safety Standard - Determination of Water Content in Food.

[0067] (4) Protein determination

[0068] Refer to GB 5009.5-2016, National Food Safety Standard - Method for Determination of Protein in Food.

[0069] (5) Determination of crude fiber

[0070] Refer to GB / T 5009.10-2003, Determination of crude fiber in plant foods.

[0071] (6) Determination of ash content

[0072] Refer to GB 5009.4-2016, National Food Safety Standard for Determination of Ash in Food.

[0073] (7) Determination of polysaccharides and total sugars

[0074] Refer to GB / T 15672-2009, determination method of total sugars and polysaccharides in edible fungi.

[0075] The yield determination results of different fungus are shown in Table 1 below.

[0076] Table 1 Determination of yield of different fungus

[0077]

[0078] The above results indicate that the Auricularia auricula isolated and purified by the present invention can reach the current production level of different types of Auricularia auricula.

[0079] The results of the determination of the nutritional content are shown in Figure 3-Figure 9 The results showed that the iron content, crude fiber content, moisture content and protein content of the wrinkled fungus separated and purified by the present invention were significantly different from those of other fungi, especially the iron content was about 1.5-2.3 times that of the other fungi except the short-haired fungus, and the crude fiber content was 1.7-4.1 times that of the other fungi. The ash content, polysaccharide and total sugar content of the wrinkled fungus were lower or had no significant difference compared with other fungi.

[0080] According to the above results, it can be seen that the present invention has isolated a new strain of Auricularia auricula, and it is the first strain of Auricularia auricula found to have a higher iron content and crude fiber content, which is of great significance for applying Auricularia auricula to the preparation of foods or medicines with high iron content or crude fiber content.

[0081] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Auricularia delicata, characterized in that: The deposit number of the Auricularia auricularia auricularia strain is GDMCC No: 64801.

2. Use of the Auricularia auricularia strain as claimed in claim 1 in preparing food rich in iron and / or crude fiber.

3. A food rich in iron and crude fiber, characterized in that: The invention comprises the Auricularia auricularia strain according to claim 1.

4. A method for cultivating the Auricularia auricularia auricula strain according to claim 1, characterized in that: The following steps are involved: (1) collecting the Auricularia auricula separated from tissues, inoculating the Auricularia auricula into a PDA slant culture medium aseptically, and culturing the culture medium in a dark environment at a constant temperature of 25° C. until the mycelium grows all over the slant culture medium, thereby obtaining a production mother culture; (2) The production mother seed is aseptically inoculated into a stock seed bag, and placed in a constant temperature dark culture at 25° C. until the mycelium is fully fed with the material, thereby obtaining the stock seed; the culture medium in the stock seed bag comprises the following raw materials in parts by weight: 60 parts of cottonseed hulls, 25 parts of wheat bran, 1.2 parts of superphosphate, 1 part of gypsum, and 50 parts of water; (3) The original seed is aseptically inoculated into a production bag, and placed in a dark culture at a constant temperature of 25° C. until the mycelium is fully fed with the material, thereby obtaining the production seed; the culture medium loaded into the production bag comprises the following raw materials in parts by weight: 100 parts of broad-leaved wood chips, 25 parts of bran, 20 parts of sugarcane bagasse, 20 parts of cottonseed hulls, 1.0 parts of superphosphate, 1.5 parts of gypsum and 55 parts of water; (4) After the cultivation material is bagged and sterilized, the production species is inoculated, and the inoculated bacterial sticks are piled up in a "well" shape, the temperature is controlled at 25° C., and the cultivation is shaded; the culture medium loaded in the cultivation material includes the following raw materials in parts by weight: 100 parts of sawdust, 15 parts of wheat bran, 0.5 parts of gypsum, 0.5 parts of lime, 12 parts of cottonseed hulls, and 80 parts of water; (5) When the mycelium has grown all over the bag and the mycelium is physiologically mature, the mycelium is punctured and the mycelium sticks are stacked in a "well" shape for ventilation and cultivation; When the primordium is formed at the puncture hole, the ears can be placed on the ground and managed; (6) Harvest the wrinkled fungus after it matures.

5. The cultivation method according to claim 4, characterized in that In step (2), the inoculation amount of the production mother seed is 8 g per original seed bag.

6. The cultivation method according to claim 4, characterized in that In step (3), the inoculation amount of the original seed is 8 g for each production bag.

7. The cultivation method according to claim 4, characterized in that In step (4), the inoculation amount of the production seed is 10 g per cultivation bag.

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