Orange yellow lactarius gaf-m-la1 strain and its separation and expansion method

Abstract

The application discloses an orange-yellow lactarius GAF-M-LA1 strain and a separation and expansion method thereof, and belongs to the technical field of strain separation and expansion. The orange-yellow lactarius GAF-M-LA1 strain has been preserved in the China General Microbiological Culture Collection Center, and the preservation date is June 10, 2022, and the preservation number is CGMCC NO. 40197. A liquid expansion culture medium is prepared by mixing a plant rootlet mixed juice obtained through fermentation and an orange-yellow lactarius crude enzyme liquid, and is used for expansion of the orange-yellow lactarius GAF-M-LA1 strain. In the liquid expansion culture medium, the strain can be continuously expanded, and the mycelium grows rapidly and has a high expansion growth rate. The separation and expansion method disclosed by the application solves the problems of slow mycelium growth and difficult expansion caused by low mycelium purity after separation of the orange-yellow lactarius strain.

Description

Technical Field

[0001] This invention relates to the technical field of bacterial strain isolation and propagation, and in particular to a method for isolating and propagating the orange-yellow lactus strain GAF-M-LA1. Background Technology

[0002] Lactarius deliciosus is a common mycorrhizal edible fungus, mainly distributed in southwestern my country. Its fruiting bodies are delicious, highly nutritious, and rich in various bioactive substances, offering benefits such as boosting immunity and anti-cancer properties, making it popular among consumers. Marketable Lactarius deliciosus primarily relies on manual harvesting from wild habitats, resulting in unstable yields and limited supply. Furthermore, over-harvesting and habitat destruction have led to a sharp decline in wild Lactarius deliciosus populations. Domestic and international research on Lactarius deliciosus mainly focuses on genetic classification and nutritional composition analysis of the fruiting bodies, with limited research on its mycelial tissue culture media and efficient strain propagation. With rising living standards and a large market demand for wild edible fungi, the domestication and cultivation of lactus fungi has become a research hotspot in the edible fungi industry.

[0003] In the acquisition of mycorrhizae and the artificial domestication and cultivation of fruiting bodies of *Lactarius deliciosus*, the low viability of the strains themselves leads to low success rates in strain isolation, slow growth during propagation, and high degeneration rates in subcultures, resulting in low mycorrhizal synthesis rates, thus making domestication and cultivation difficult. *Lactarius deliciosus* var. *orange* is highly susceptible to contamination by other microorganisms in commonly used fungal isolation media, leading to isolation failure. Furthermore, its growth rate during propagation is slow, and strain viability declines with each propagation cycle. To date, there are no reports or studies on methods for isolating and propagating high-quality, efficient *Lactarius deliciosus* var. *orange* strains, making it difficult to efficiently obtain high-quality strains for artificial domestication and cultivation. Therefore, there is an urgent need to find a method for efficiently isolating high-purity *Lactarius deliciosus* var. *orange* strains, and for obtaining *Lactarius deliciosus* mycelia that can be propagated multiple times and grow rapidly. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a *Lactarius citrinum* strain GAF-M-LA1 and its isolation and propagation method, so as to solve the problem of slow mycelial growth and difficulty in propagation caused by low mycelial purity after isolation of *Lactarius citrinum* strain.

[0005] The present invention solves the above-mentioned technical problems through the following technical means:

[0006] The purpose of this invention is to provide a *Lactarius akahatsu* strain GAF-M-LA1, which is a fungus belonging to the genus *Lactarius*. Its preservation information is as follows: the depositary institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is June 10, 2022; the accession number is CGMCCNO.40197; and the suggested classification name is *Lactarius akahatsu*, as shown in Sequence 1, “ITS sequence of *Lactarius akahatsu* strain GAF-M-LA1”.

[0007]

[0008] Another objective of this invention is to provide a method for isolating and propagating the Lactobacillus orange-yellow strain GAF-M-LA1, the specific method of which is as follows:

[0009] S1. Isolation of *Lactarius deliciosus* strain GAF-M-LA1: Healthy, disease-free, fresh, and tender *Lactarius deliciosus* fruiting bodies were treated with 60-75% alcohol. Under aseptic conditions, the middle part of the cap was cut into strips of approximately 0.5 × 0.2 cm to obtain *Lactarius deliciosus* flesh tissue. Under aseptic conditions, the flesh tissue was inoculated onto an isolation medium and cultured in a biochemical incubator at a temperature of 24±1℃ and a humidity of 60%-80% in the dark for 15-20 days. After two generations of isolation and culture, a high-purity, vigorous *Lactarius deliciosus* strain GAF-M-LA1 was obtained.

[0010] Sequence analysis of the isolated Lactarius citrinum GAF-M-LA1 strain yielded a full-length sequence of 624 bp. This Lactarius citrinum strain contains the ITS sequence shown in PatentIn version 3.5 NO.1. The ITS sequence was identified as Lactarius citrinum using Nucleotide BLAST from https: / / www.ncbi.nlm.nih.gov / , with the sequence number ON248533.

[0011] S2. Propagation of Lactobacillus orange-yellow strain GAF-M-LA1: The Lactobacillus orange-yellow spawn was inoculated into liquid propagation medium and placed in a biochemical incubator for 40-60 days in the dark at a temperature of 24±1℃ and a humidity of 60%-80% to obtain the propagated Lactobacillus orange-yellow strain GAF-M-LA1.

[0012] Furthermore, the separation culture medium in step S1 comprises the following raw materials in parts by weight: 1.0-4.0 parts glucose, 0.3-0.6 parts dipotassium hydrogen phosphate, 0.1-0.3 parts ammonium chloride, 0.05-0.2 parts magnesium sulfate heptahydrate, 0.02-0.06 parts vitamin B1, 1.0-3.0 parts 1wt% sodium chloride solution, 3-6 parts 1wt% calcium chloride solution, 0.5-2.0 parts 1wt% ferric chloride solution, and 15-25 parts agar.

[0013] Furthermore, the liquid culture medium in step S2 includes the following raw materials: fresh potato juice, mixed juice of young plant roots, crude enzyme solution of Lactobacillus angustifolia, and nutrients;

[0014] The mass ratio of the fresh potato juice, the mixed juice of young plant roots, the crude enzyme solution of orange-yellow lactus mushroom, and the nutrients is (10-20):(5-15):(2-4):(0.5-1.5);

[0015] The nutrients comprise the following raw materials by weight: 1-3 parts glucose, 0.3-0.6 parts malt extract powder, 0.3-0.6 parts dipotassium hydrogen phosphate, 0.3-0.5 parts ammonium chloride, 0.1-0.2 parts magnesium sulfate heptahydrate, 0.03-0.06 parts vitamin B1, 0.03-0.06 parts vitamin B6, 2-3 parts 1 wt% sodium chloride solution, 3-6 parts 1 wt% calcium chloride solution, and 1-2 parts 1 wt% ferric chloride.

[0016] Furthermore, the present invention also provides a method for preparing a liquid expansion culture medium, the specific steps of which are as follows:

[0017] After mixing fresh potato juice and plant root mixture evenly, add crude enzyme solution of Lactobacillus citrinum and sonicate at 40-80 kHz to evenly distribute the crude enzyme solution. Stir at 25-28℃ for 2-4 hours, then centrifuge at 4000-6000 r / min to remove the precipitate and take the supernatant. Mix with nutrients and filter aseptically to obtain liquid culture medium.

[0018] Furthermore, the method for preparing the mixed juice of plant young roots is as follows:

[0019] Young roots of Pinus massoniana, Miscanthus sinensis, and Spiraea japonica were separately crushed in a pulverizer to obtain a mixed pulverized material of plant young roots with a particle size of 400-600μm. The material was then poured into an anaerobic fermenter and fermented and liquefied at 25-35℃ for 5-8 days. After centrifugation at 4000-6000r / min for 3-5 minutes, the mixture was aseptically filtered to obtain the mixed juice of plant young roots.

[0020] Furthermore, the preparation method of the crude enzyme solution from the orange-yellow lactus mushroom is as follows:

[0021] Mature, healthy, disease-free Lactarius orange-yellow mushrooms were pulverized in a grinder to obtain 400-600μm Lactarius orange-yellow mushroom pulverized material. Sterile water was added and stirred for 10-20 minutes. Then, the mixture was centrifuged at 4000-6000 r / min. The supernatant was then sterilely filtered to obtain crude enzyme solution of Lactarius orange-yellow mushrooms.

[0022] Furthermore, in step S2, the liquid culture medium for the expansion of the *Lactarius orange-yellow* GAF-M-LA1 strain is replaced with a solid culture medium. The expansion method includes the following steps:

[0023] Under aseptic conditions, the orange-yellow lactus strain GAF-M-LA1 was cut into fan-shaped blocks in a cross pattern along the direction of mycelial growth. The fan-shaped blocks were placed in a solid expansion medium and cultured in a biochemical incubator in the dark for 40-60 days to obtain the expanded orange-yellow lactus strain GAF-M-LA1.

[0024] Furthermore, the solid culture medium in step S2 comprises the following raw materials in parts by weight: 30-50 parts fresh potato juice, 10-30 parts glucose, 0.5-1.5 parts yeast powder, 0.3-0.6 parts malt extract, 1-3 parts peptone, 0.3-0.6 parts dipotassium hydrogen phosphate, 0.1-0.2 parts magnesium sulfate heptahydrate, 0.03-0.06 parts vitamin B1, 2-3 parts 1wt% sodium chloride solution, 3-6 parts 1wt% calcium chloride solution, 1-2 parts 1wt% ferric citrate, and 15-25 parts agar.

[0025] This invention involves mixing and stirring a mixture of plant root extracts and crude enzyme solution from *Lactarius deliciosus* (orange-yellow lactis), then collecting the supernatant. This supernatant is then mixed with nutrients to prepare a liquid culture medium. Compared to other liquid culture media, the plant root extracts in the liquid culture medium prepared by this invention provide organic matter and minerals for the growth of *Lactarius deliciosus*, while the crude enzyme solution decomposes these organic matter and minerals into forms that the mycelium can directly utilize, thus promoting the absorption of nutrients by the mycelium.

[0026] The interaction between crude enzyme solution of *Lactarius deliciosus* and a mixture of plant root juices was used to prepare a liquid culture medium for the propagation of *Lactarius deliciosus* strains. The resulting strains exhibited high growth rates, strong propagation capabilities, and rapid growth. However, directly using plant pulverization to prepare the liquid culture medium resulted in insufficient utilization of the crude enzyme solution due to the large molecular weight of organic matter and the fixed inorganic minerals within the plant tissues. Consequently, the *Lactarius deliciosus* mycelia could not fully utilize the organic matter and inorganic minerals, leading to low growth rates, poor propagation capabilities, and slow growth rates. Therefore, it is necessary to anaerobically ferment and liquefy the pulverized plant mixture to reduce the molecular weight of organic matter in the plant root juice before culturing *Lactarius deliciosus* mycelia. Furthermore, the crude enzyme solution needs to be present with young fruiting bodies at a suitable temperature to allow the utilization of organic matter and minerals in the plant root juice. The *Lactarius deliciosus* strain GAF-M-LA1 obtained using this method exhibited rapid mycelial growth, low degeneration rate, continuous propagation capability, and high propagation growth rate. Using liquid culture medium for expansion provides an effective way to efficiently obtain high-quality strains of Lactobacillus orange-yellow for artificial domestication and cultivation, which is of great significance for the development and application of this type of edible fungus.

[0027] Beneficial effects:

[0028] 1. This invention successfully isolated the orange-yellow lactus strain GAF-M-LA1. The strain obtained by expanding the orange-yellow lactus strain GAF-M-LA1 in liquid or solid expansion medium can be used for continuous expansion and the mycelium grows rapidly.

[0029] 2. After anaerobic fermentation, the mixed juice of young plant roots has a low molecular weight of organic matter. When it reacts with the crude enzyme solution of Lactobacillus orange-yellow, it can be directly utilized by the tender fruiting bodies and mycelia, promoting the absorption of nutrients. This results in rapid mycelial separation, fast growth, and strong expansion capacity of the strain cultured in liquid culture medium.

[0030] 3. The *Lactarius orange-yellow* strain GAF-M-LA1 obtained under the isolated and propagated culture conditions exhibited vigorous mycelial activity, strong competitive ability, high success rate of continuous propagation, and low degeneration rate. This provides an effective way to efficiently obtain high-quality *Lactarius orange-yellow* strains for artificial domestication and cultivation, and is of great significance for the development and application of this type of edible fungus.

[0031] Instruction manual illustrations

[0032] Figure 1 Example 4: Mycelial growth of Lactobacillus orange-yellow strain GAF-M-LA1 after isolation;

[0033] Figure 2 Example 4: Growth of Lactobacillus orange-yellow (GAF-M-LA1) strain after liquid propagation;

[0034] Figure 3 Example 7: Growth of Lactobacillus orange-yellow (GAF-M-LA1) strain after solid culture. Detailed Implementation

[0035] The following will be described in conjunction with the embodiments and appendices. Figure 1-3 The present invention will be described in detail below:

[0036] Before preparing the culture medium, all the raw materials should be prepared. Therefore, it is necessary to prepare fresh potato juice, mixed juice of young plant roots and crude enzyme solution of orange-yellow lactus mushroom.

[0037] Preparation of fresh potato juice:

[0038] Wash and peel 1 kg of fresh potatoes, crush them with 5 times the amount of water, extract the juice, and filter it through 4 layers of gauze to obtain fresh potato juice.

[0039] Preparation of mixed juice from young plant roots:

[0040] Weigh out 4g of young roots of Pinus massoniana, 1.5g of young roots of Dictamnus dasycarpus and 2.5g of young roots of Spiraea japonica, mix them evenly and pour them into a grinder to grind them to 400-600μm. The resulting plant mixture was then poured into an anaerobic fermentation tank and fermented and liquefied at 25℃ for 6 days. After centrifugation at 5000r / min for 4 minutes, the supernatant was collected to obtain the mixed juice of young plant roots.

[0041] Preparation of crude enzyme solution from Lactobacillus angustifolia:

[0042] 4g of mature Lactarius orange-yellow mushroom cells that are healthy and free from pests and diseases are poured into a grinder and ground into 400-600μm fine particles. 0.15L of sterile water is added and stirred for 15min. The mixture is then centrifuged at 5000r / min to obtain the supernatant, which is used to prepare crude enzyme solution of Lactarius orange-yellow mushroom.

[0043] Example 1: Culture medium preparation

[0044] Preparation of isolation culture medium 1:

[0045] Weigh or measure the raw materials shown in Table 1 and add them to 1L of distilled water. Dissolve them by microwave heating and autoclave at 121℃. After cooling to room temperature, pour them into a petri dish to prepare the separation culture medium.

[0046] Table 1

[0047]

[0048] Preparation of solid expansion culture medium 1:

[0049] Weigh or measure the raw materials shown in Table 2 and add them to 1L of distilled water. Dissolve them by microwave heating and autoclave at 121℃. After cooling to room temperature, pour them into a petri dish to prepare the separation culture medium.

[0050] Table 2

[0051]

[0052] Preparation of liquid expansion culture medium 1:

[0053] Weigh or measure the raw materials shown in Table 3 and prepare the liquid expansion culture medium according to the following steps:

[0054] After the fresh potato juice and the mixed juice of young plant roots were evenly mixed, the crude enzyme solution of orange-yellow lactus mushroom was added and sonicated at 60 kHz. After stirring at 27 ℃ for 3 h, the precipitate was removed by centrifugation at 5000 r / min and the supernatant was collected. Nutrients were added to the supernatant and mixed thoroughly. After autoclaving at 121 ℃, the liquid culture medium was obtained.

[0055] Table 3

[0056] 15g 10g 3g 1g

[0057] Weigh out 2.0g of glucose, 0.5g of malt extract, 0.4g of dipotassium hydrogen phosphate, 0.4g of ammonium chloride, 0.15g of magnesium sulfate heptahydrate, 0.05g of vitamin B1, 0.05g of vitamin B6, 2.5g of 1wt% sodium chloride solution, 4g of 1wt% calcium chloride solution, and 1.5g of 1wt% ferric chloride solution. Mix them to obtain the nutrients. Finally, weigh out 1g of the nutrients for Table 3.

[0058] Example 2: Preparation of Culture Medium II

[0059] Preparation of isolation culture medium II:

[0060] Weigh or measure the raw materials shown in Table 4 and add them to 1L of distilled water. Dissolve them by microwave heating and autoclave at 121℃. After cooling to room temperature, pour them into a petri dish to prepare the separation culture medium.

[0061] Table 4

[0062]

[0063]

[0064] Solid expansion culture medium two:

[0065] Weigh or measure the raw materials shown in Table 5 and add them to 1L of distilled water. Dissolve them by microwave heating and autoclave at 121℃. After cooling to room temperature, pour them into a petri dish to prepare the separation culture medium.

[0066] Table 5

[0067]

[0068] Preparation of liquid expansion culture medium II:

[0069] Weigh or measure the raw materials shown in Table 6 and prepare the liquid expansion culture medium according to the following steps:

[0070] After mixing the prepared fresh potato juice and the mixed juice of plant young roots evenly, the prepared crude enzyme solution of orange-yellow lactus mushroom was added and sonicated at 40 kHz. After stirring at 28 ℃ for 2 h, the precipitate was removed by centrifugation at 6000 r / min and the supernatant was collected. Nutrients were added to the supernatant and mixed thoroughly. After autoclaving at 121 ℃, the liquid culture medium was obtained.

[0071] Table 6

[0072] 10g 5g 2g 0.5g

[0073] Weigh out the following ingredients according to their mass: 3.0g glucose, 0.6g malt extract, 0.3g dipotassium hydrogen phosphate, 0.3g ammonium chloride, 0.2g magnesium sulfate heptahydrate, 0.03g vitamin B1, 0.03g vitamin B6, 3g 1wt% sodium chloride solution, 6g 1wt% calcium chloride solution, and 1g 1wt% ferric chloride solution. Mix them to obtain the nutrients. Weigh out 0.5g of the nutrients for Table 6.

[0074] Example 3: Culture medium preparation

[0075] Preparation of isolation culture medium, part three:

[0076] Weigh or measure the raw materials shown in Table 7 and add them to 1L of distilled water. Dissolve them by microwave heating and autoclave at 121℃. After cooling to room temperature, pour them into a petri dish to prepare the separation culture medium.

[0077] Table 7

[0078]

[0079] Preparation of solid expansion culture medium, part three:

[0080] Weigh or measure the raw materials shown in Table 8 and add them to 1L of distilled water. Dissolve them by microwave heating and autoclave at 121℃. After cooling to room temperature, pour them into a petri dish to prepare the separation culture medium.

[0081] Table 8

[0082]

[0083] Preparation of liquid expansion culture medium, part three:

[0084] Weigh or measure the raw materials shown in Table 9 and prepare the liquid expansion culture medium according to the following steps:

[0085] After mixing the prepared fresh potato juice and the mixed juice of plant young roots evenly, the prepared crude enzyme solution of orange-yellow lactus mushroom was added and sonicated at 80 kHz. After stirring at 25 ℃ for 4 h, the precipitate was removed by centrifugation at 4000 r / min and the supernatant was collected. Nutrients were added to the supernatant and mixed thoroughly. After autoclaving at 121 ℃, the liquid culture medium was obtained.

[0086] Table 9

[0087] 20g 15g 4g 1.5g

[0088] Weigh out the following ingredients according to their mass: 1.0g glucose, 0.3g malt extract, 0.6g dipotassium hydrogen phosphate, 0.5g ammonium chloride, 0.1g magnesium sulfate heptahydrate, 0.06g vitamin B1, 0.06g vitamin B6, 2g 1wt% sodium chloride solution, 3g 1wt% calcium chloride solution, and 2g 1wt% ferric chloride solution. Mix them to obtain the nutrients. Weigh out 1.5g of the nutrients for Table 9.

[0089] Example 4: Propagation method of Lactobacillus orange-yellow strain (using liquid propagation medium)

[0090] In this embodiment, the isolation medium 1 and liquid expansion medium 1 prepared in Example 1 were used to isolate and expand the Lactobacillus orange-yellow strain GAF-M-LA1. The specific steps are as follows:

[0091] S1. Isolation of *Lactarius deliciosus* strain GAF-M-LA1: Healthy, disease-free, fresh, and tender *Lactarius deliciosus* fruiting bodies were treated with 70% alcohol. Under aseptic conditions, the middle part of the cap was cut into strips of approximately 0.5 × 0.2 cm to obtain *Lactarius deliciosus* flesh tissue. Under aseptic conditions, the flesh tissue was inoculated onto isolation medium 1 and cultured in a biochemical incubator at a temperature of 24±1℃ and a humidity of 70% for 18 days in the dark. After two generations of isolation and culture, a high-purity, vigorous *Lactarius deliciosus* strain GAF-M-LA1 was obtained.

[0092] The *Lactarius deliciosus* strain GAF-M-LA1 obtained in this embodiment has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 10, 2022, with accession number CGMCC NO.40197, as shown in Sequence 1, “ITS Sequence of *Lactarius deliciosus* strain GAF-M-LA1”:

[0093]

[0094] S2. Propagation of Lactobacillus orange-yellow strain GAF-M-LA1 in liquid culture medium:

[0095] The orange-yellow lactus spawn was inoculated into liquid expansion medium and cultured in a biochemical incubator in the dark for 50 days at a temperature of 24±1℃ and a humidity of 70% to obtain the expanded orange-yellow lactus GAF-M-LA1 strain.

[0096] Example 5:

[0097] In this embodiment, the isolation medium II and liquid expansion medium II prepared in Example 2 were used to isolate and expand the Lactobacillus orange-yellow strain GAF-M-LA1. The specific steps were the same as in Example 4.

[0098] Example 6:

[0099] In this embodiment, the isolation medium III and liquid expansion medium III prepared in Example 3 were used to isolate and expand the Lactobacillus orange-yellow strain GAF-M-LA1. The specific steps were the same as in Example 4.

[0100] Example 7: Propagation method of Lactobacillus orange-yellow strain (propagation on solid culture medium)

[0101] In this embodiment, the isolation medium 1 and solid expansion medium 1 prepared in Example 1 were used to isolate and expand the Lactobacillus orange-yellow strain GAF-M-LA1. The specific steps are as follows:

[0102] S1. Isolation of *Lactarius deliciosus* strain GAF-M-LA1: Healthy, disease-free, fresh, and tender *Lactarius deliciosus* fruiting bodies were treated with 70% alcohol. Under aseptic conditions, the middle part of the cap was cut into strips of approximately 0.5 × 0.2 cm to obtain *Lactarius deliciosus* flesh tissue. Under aseptic conditions, the flesh tissue was inoculated onto isolation medium 1 and cultured in a biochemical incubator at a temperature of 24±1℃ and a humidity of 70% for 18 days in the dark. After two generations of isolation and culture, a high-purity, vigorous *Lactarius deliciosus* strain GAF-M-LA1 was obtained.

[0103] S2. Propagation of *Lactarius orange-yellow* strain GAF-M-LA1 on solid culture medium:

[0104] Under aseptic conditions, the orange-yellow lactus strain GAF-M-LA1 was cut into fan-shaped blocks along the direction of mycelial growth in a cross pattern. The fan-shaped blocks were inoculated onto solid expansion medium 1 and cultured in a biochemical incubator in the dark for 50 days at a temperature of 24±1℃ and a humidity of 70% to obtain the expanded orange-yellow lactus strain GAF-M-LA1.

[0105] Example 8:

[0106] In this embodiment, the isolation medium II and solid expansion medium II prepared in Example 2 were used to isolate and expand the Lactobacillus orange-yellow strain GAF-M-LA1. The specific steps were the same as in Example 7.

[0107] Example 9:

[0108] In this embodiment, the isolation medium III and solid expansion medium III prepared in Example 3 were used to isolate and expand the Lactobacillus orange-yellow strain GAF-M-LA1. The specific steps were the same as in Example 7.

[0109] In order to compare with the expansion methods of Example 4 (expansion in liquid culture medium) and Example 7 (expansion in solid culture medium), the present invention also uses the expansion methods of Comparative Examples 1-9 for expansion.

[0110] The specific preparation methods for Comparative Examples 1-6 are as follows:

[0111] Comparative Example 1:

[0112] This comparative example is compared with Example 4. The isolation steps for the *Lactarius orange-yellow* strain GAF-M-LA1 in step S1 are the same, the only difference being the liquid culture medium used in step S2. The liquid culture medium in Comparative Example 1 is replaced with a common liquid culture medium for mushrooms. All other isolation and expansion steps are the same. The specific preparation method for the common liquid culture medium for mushroom isolation is as follows:

[0113] Weigh out 2.5g of glucose, 0.5g of dipotassium hydrogen phosphate, 0.2g of ammonium chloride, 0.15g of magnesium sulfate heptahydrate, 0.05g of vitamin B1, 2.5g of 1wt% sodium chloride solution, 5g of 1wt% calcium chloride solution, and 1.5g of 1wt% ferric chloride solution, respectively. Dissolve and dilute with 1L of distilled water, and sterilize at 121℃ for 20min to obtain liquid culture medium.

[0114] Comparative Example 2:

[0115] This comparative example is compared with Example 4. The isolation steps for the *Lactarius orange-yellow* GAF-M-LA1 strain are the same in step S1, except that the plant root mixture is not added to the liquid expansion medium in step S2. All other isolation and expansion steps are the same. The specific preparation method of the liquid expansion medium is as follows:

[0116] 15g of fresh potato juice and 3g of crude enzyme solution from Lactobacillus citrinum were weighed and mixed evenly. The mixture was then sonicated at 60kHz to ensure even distribution of the crude enzyme solution. After stirring at 27℃ for 3 hours, the mixture was centrifuged at 5000r / min to remove the precipitate and collect the supernatant. The supernatant was then mixed evenly with 1g of nutrients at 100℃ and aseptically filtered to obtain the liquid culture medium.

[0117] Comparative Example 3:

[0118] This comparative example is compared with Example 4. The isolation steps for the *Lactarius orange-yellow* GAF-M-LA1 strain in step S1 are the same, except that the crude enzyme solution of *Lactarius orange-yellow* is not added when preparing the liquid expansion culture medium in step S2. All other isolation and expansion steps are the same, and the specific preparation method is as follows:

[0119] Measure 15g of fresh potato juice and 10g of mixed plant root juice, mix them evenly, stir at 27℃ for 3h, centrifuge at 5000r / min to remove the precipitate and take the supernatant, mix the supernatant with 1g of nutrients at 100℃, and filter aseptically to obtain the liquid expansion culture medium.

[0120] Comparative Example 4:

[0121] This comparative example is compared with Example 4, the only difference being that the healthy, disease-free, fresh and tender orange-yellow latex fruiting bodies in step S1 are replaced with robust, disease-free, mature orange-yellow latex fruiting bodies. The other isolation and expansion culture conditions are the same. Step S1 is as follows:

[0122] S1. Isolation of the Orange-Yellow Lactarius GAF-M-LA1 strain: Mature Orange-Yellow Lactarius fruiting bodies with robust growth and free from pests and diseases were treated with 70% alcohol. Under aseptic conditions, the middle part of the cap was cut into strips of about 0.5×0.2cm to obtain Orange-Yellow Lactarius flesh tissue. Under aseptic conditions, the flesh tissue was inoculated onto isolation medium 1 and cultured in a biochemical incubator at a temperature of 24±1℃ and a humidity of 70% for 18 days in the dark. After two generations of isolation culture, a high-purity and vigorous Orange-Yellow Lactarius GAF-M-LA1 strain was obtained.

[0123] Comparative Example 5:

[0124] This comparative example is a comparative example with Example 4. The isolation steps for the *Lactarius orange-yellow* strain GAF-M-LA1 in step S1 are the same, except that after inoculating the strain into the liquid expansion medium in step S2, the temperature for the light-protected culture in step S2 is changed to 28±1℃, while the humidity remains at 70%. The remaining isolation and expansion steps are the same. Step S2 is as follows:

[0125] S2. Propagation of Lactobacillus orange-yellow strain GAF-M-LA1 in liquid culture medium:

[0126] The orange-yellow lactus spawn was inoculated into liquid expansion medium and cultured in a biochemical incubator in the dark for 50 days at a temperature of 28±1℃ and a humidity of 70% to obtain the expanded orange-yellow lactus GAF-M-LA1 strain.

[0127] Comparative Example 6:

[0128] This comparative example is a comparative example with Example 4. The isolation steps for the *Lactarius orange-yellow* strain S1, GAF-M-LA1, are the same, except that after inoculating the strain in step S1 into the liquid expansion medium, the temperature for incubation in the dark is changed to 20±1℃, while the humidity remains at 70%. The remaining isolation and expansion steps are the same. Step S2 is as follows:

[0129] S2. Propagation of Lactobacillus orange-yellow strain GAF-M-LA1 in liquid culture medium:

[0130] The orange-yellow lactus spawn was inoculated into liquid expansion medium and cultured in a biochemical incubator in the dark for 50 days at a temperature of 20±1℃ and a humidity of 70% to obtain the expanded orange-yellow lactus GAF-M-LA1 strain.

[0131] Comparative Example 7:

[0132] This comparative example is compared with Example 7, the only difference being that the healthy, disease-free, fresh and tender orange-yellow latex fruiting bodies in step S1 are replaced with robust, disease-free, mature orange-yellow latex fruiting bodies. The other isolation and expansion culture conditions are the same. Step S1 is as follows:

[0133] S1. Isolation of the Orange-Yellow Lactarius GAF-M-LA1 strain: Mature Orange-Yellow Lactarius fruiting bodies with robust growth and free from pests and diseases were treated with 70% alcohol. Under aseptic conditions, the middle part of the cap was cut into strips of about 0.5×0.2cm to obtain Orange-Yellow Lactarius flesh tissue. Under aseptic conditions, the flesh tissue was inoculated onto isolation medium 1 and cultured in a biochemical incubator at a temperature of 24±1℃ and a humidity of 70% for 18 days in the dark. After two generations of isolation and culture, the vigorous Orange-Yellow Lactarius GAF-M-LA1 strain was obtained.

[0134] Comparative Example 8:

[0135] This comparative example is a comparative example with Example 7, the only difference being that after the bacterial strain in step S2 is inoculated into the solid expansion culture medium, the temperature for the light-protected culture in step S2 is changed to 28±1℃, while the humidity remains at 70%. The remaining isolation and expansion steps are the same. Step S2 is as follows:

[0136] S2. Propagation of *Lactarius orange-yellow* strain GAF-M-LA1 on solid culture medium:

[0137] Under aseptic conditions, the orange-yellow lactus strain GAF-M-LA1 was cut into fan-shaped blocks in a cross pattern along the direction of mycelial growth. The fan-shaped blocks were inoculated onto solid expansion medium 1 and cultured in a biochemical incubator in the dark for 50 days at a temperature of 28±1℃ and a humidity of 70% to obtain the expanded orange-yellow lactus strain GAF-M-LA1.

[0138] Comparative Example 9:

[0139] This comparative example is a comparative example with Example 7, the only difference being that after the bacterial strain in step S1 is inoculated into the solid expansion culture medium, the temperature for incubation in the dark is changed to 20±1℃, while the humidity remains at 70%. The remaining isolation and expansion steps are the same. Step S2 is as follows:

[0140] S2. Propagation of *Lactarius orange-yellow* strain GAF-M-LA1 on solid culture medium:

[0141] Under aseptic conditions, the orange-yellow lactus strain GAF-M-LA1 was cut into fan-shaped blocks in a cross pattern along the direction of mycelial growth. The fan-shaped blocks were inoculated onto solid expansion medium 1 and cultured in a biochemical incubator in the dark for 50 days at a temperature of 20±1℃ and a humidity of 70% to obtain the expanded orange-yellow lactus strain GAF-M-LA1.

[0142] Experiment 1: Growth of Lactobacillus orange-yellow strain GAF-M-LA1 after isolation

[0143] The mycelial color, mycelial density, colony morphology and propagation ability of the *Lactarius cirrhosa* strains isolated in Example 4 and Comparative Example 4 were recorded, and the results are shown in Table 10.

[0144] Table 10. Growth of *Lactarius orange-yellow* strain GAF-M-LA1 after isolation.

[0145]

[0146] In Example 4, the growth of the strain after isolation is as follows: Figure 1 As shown, the mycelium of the Lactobacillus orange-yellow strain GAF-M-LA1 is white, sparse, long, and grows radially.

[0147] Experiment 2: Growth rate of Lactobacillus orange-yellow inoculum GAF-M-LA1 strain in liquid culture medium

[0148] Comparative Example 1, although successfully isolated, failed to expand, so there is no data on the growth rate after expansion. Therefore, only the growth rate of the *Lactarius citrinum* GAF-M-LA1 strain from Examples 4 and Comparative Examples 2-6 after expansion is recorded. Since Examples 4 and Comparative Examples 2-6 both used liquid expansion medium for expansion culture, the liquid expansion growth rate is recorded as shown in Table 11.

[0149] Table 11 Growth rate of Lactobacillus orange-yellow var. GAF-M-LA1 strain in liquid culture medium

[0150]

[0151] The growth of the *Lactarius deliciosus* strain after expansion in the liquid culture medium of Example 4 is as follows: Figure 2As shown, the mycelium grows densely, and pale yellow, fluffy, twisted mycelium can grow in 50 days. The success rate of continuous propagation is high, and the mycelium is vigorous.

[0152] Experiment 3: Growth rate of Lactobacillus orange-yellow inoculum GAF-M-LA1 strain in solid culture medium

[0153] The methods of Example 7 and Comparative Examples 7-9 were used to expand the culture of *Lactarius orange-yellow* using solid culture medium, and the growth rate of the strains under the expansion culture medium conditions was statistically analyzed. Since both Example 7 and Comparative Examples 7-9 used solid culture medium for expansion culture, the solid culture growth rate is recorded as shown in Table 12.

[0154] Table 12. Solid culture growth rate of Lactobacillus orange-yellow strain GAF-M-LA1

[0155]

[0156]

[0157] The growth of the *Lactarius deliciosus* strain after expansion in the liquid culture medium of Example 4 is as follows: Figure 3 As shown, the growth of the *Lactarius deliciosus* strain after solid-state propagation is as follows: Figure 2 As shown, the mycelium grows densely, and pale yellow, fluffy, twisted mycelium can grow in 50 days. The success rate of continuous propagation is high, and the mycelium is vigorous.

[0158] Data Analysis:

[0159] As shown in Table 10, the relevant data results of Example 4 indicate that the *Lactarius deliciosus* strain GAF-M-LA1 was successfully obtained after isolation in Example 4. The mycelium of the strain is white, sparse, long, and grows radially. Figure 1 As shown, the colonies are small and dense, the strain can be continuously propagated, and the mycelium grows rapidly. From the relevant data results of Example 4 in Table 10, it can be seen that after isolating the *Lactarius deliciosus* GAF-M-LA1 strain and propagating it in liquid culture medium, the growth rate of the *Lactarius deliciosus* GAF-M-LA1 strain in liquid culture medium was 94.67%, indicating that the obtained *Lactarius deliciosus* GAF-M-LA1 strain has strong propagation ability and grows rapidly. From the relevant data results in Table 12, it can be seen that after isolating the *Lactarius deliciosus* GAF-M-LA1 strain in Example 7 and propagating it in solid culture medium, the growth rate of the *Lactarius deliciosus* GAF-M-LA1 strain in solid culture medium was 90.67%, indicating that it can be continuously propagated, and the mycelium grows rapidly.

[0160] Comparing the data of Example 4 and Comparative Example 4 in Table 10, it can be seen that Comparative Example 4 used mature orange-yellow latex fruiting bodies that were vigorous and free from pests and diseases for isolation and propagation. After the strain was isolated, the mycelium of the orange-yellow latex GAF-M-LA1 strain was pale yellow, sparse, long and twisted, and the colonies were small and sparse. It could be propagated, but the mycelial growth was slow. This was mainly because the mycelium and spore differentiation of the mature orange-yellow latex fruiting bodies entered the plateau or decline phase.

[0161] In Comparative Example 1, when the liquid expansion medium was replaced with ordinary liquid expansion medium, no new mycelia grew and the liquid expansion of the Lactobacillus orange-yellow strain was not possible. This indicates that ordinary liquid expansion medium cannot be used for the isolation and expansion of Lactobacillus orange-yellow GAF-M-LA1.

[0162] Comparing the data from Example 4 and Comparative Examples 2-3 in Table 11, it can be seen that Comparative Example 2 did not add the mixed juice of young plant roots when preparing the liquid culture medium; Comparative Example 3 did not add the active enzyme of *Lactarius deliciosus*. The success rate of liquid propagation of *Lactarius deliciosus* GAF-M-LA1 strains in both Comparative Examples 2 and 3 was reduced. This indicates that the mixed juice of young plant roots and the active enzyme of *Lactarius deliciosus* interact. The active enzyme converts the organic matter in the mixed juice of young plant roots into organic matter and minerals that are easily absorbed and utilized by *Lactarius deliciosus* mycelia. Therefore, the nutrients in the prepared isolation culture medium are more easily utilized by *Lactarius deliciosus* mycelia, resulting in faster growth and stronger propagation ability of the *Lactarius deliciosus* GAF-M-LA1 strain during isolation and propagation.

[0163] Comparing the data from Example 4 and Comparative Examples 4-6 in Table 11, and Example 4 and Comparative Examples 7-9 in Table 12, it can be seen that the selection of fruiting bodies during the isolation of *Lactarius deliciosus* strains and the temperature of the light-protected culture during expansion both affect the isolation and expansion of *Lactarius deliciosus* strain GAF-M-LA1. In Comparative Examples 4 and 7, replacing the fruiting bodies with robust, disease-free, mature *Lactarius deliciosus* fruiting bodies resulted in a decrease in the growth rate of *Lactarius deliciosus* strain GAF-M-LA1 after expansion. In Comparative Examples 5-6 and 8-9, changing the light-protected culture temperature to 20±1℃ and 28±1℃ respectively, the growth rate of *Lactarius deliciosus* strain GAF-M-LA1 decreased in both cases. This indicates that young fruiting bodies are required for continuous expansion of *Lactarius deliciosus* using the culture medium of this invention. In addition, excessively high or low temperatures during the cultivation process in the dark can affect the activity of Lactobacillus orange-yellow, resulting in insufficient utilization of organic matter in the mixed plant juice, which leads to slow growth and reduced cultivation capacity of the isolated Lactobacillus orange-yellow GAF-M-LA1 strain.

[0164] This invention involves mixing fermented and liquefied plant root juice with crude enzyme solution of Lactobacillus orange-yellow, and then using the mixture to prepare a liquid culture medium for Lactobacillus orange-yellow strain. The strain can be continuously cultured, and the mycelium grows rapidly, with strong culture capacity and high culture growth rate.

[0165] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A method for propagating the *Lactarius orange-yellow* strain GAF-M-LA1, characterized in that, The method includes the following steps: Propagation of Lactobacillus orange-yellow strain GAF-M-LA1: The Lactobacillus orange-yellow strain was inoculated into liquid expansion medium and placed in a biochemical incubator for 40-60 days in the dark at a temperature of 24±1℃ and a humidity of 60%-80%. The liquid culture medium used in the above steps includes the following raw materials: fresh potato juice, mixed juice of young plant roots, crude enzyme solution of Lactobacillus orange-yellow, and nutrients; The mass ratio of the fresh potato juice, the mixed juice of young plant roots, the crude enzyme solution of orange-yellow lactus mushroom, and the nutrients is (10-20):(5-15):(2-4):(0.5-1.5). The nutrients comprise the following raw materials in parts by weight: 1-3 parts glucose, 0.3-0.6 parts malt extract powder, 0.3-0.6 parts dipotassium hydrogen phosphate, 0.3-0.5 parts ammonium chloride, 0.1-0.2 parts magnesium sulfate heptahydrate, 0.03-0.06 parts vitamin B1, 0.03-0.06 parts vitamin B6, 2-3 parts 1wt% sodium chloride solution, 3-6 parts 1wt% calcium chloride solution, and 1-2 parts 1wt% ferric chloride. The Lactobacillus orange-yellow strain GAF-M-LA1 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 10, 2022, with accession number CGMCC NO.40197.

2. The method for propagating the *Lactarius orange-yellow* strain GAF-M-LA1 according to claim 1, characterized in that, The preparation method of the liquid expansion culture medium is as follows: After mixing fresh potato juice and plant root juice evenly, add crude enzyme solution of Lactobacillus citrinum and sonicate at 40-80kHz to evenly distribute the crude enzyme solution. Stir at 25-28℃ for 2-4 hours, centrifuge to remove precipitate, and pour the supernatant into an Erlenmeyer flask. Add nutrients to fully dissolve and filter aseptically to obtain liquid culture medium.

3. The method for propagating the *Lactarius orange-yellow* strain GAF-M-LA1 according to claim 2, characterized in that, The preparation method of the mixed juice of plant young roots is as follows: The young roots of Pinus massoniana, Miscanthus sinensis, and Spiraea japonica were crushed and mixed evenly to obtain a mixed crushed plant root material. This material was then poured into an anaerobic fermentation tank for fermentation and liquefaction. Subsequently, the mixture was centrifuged to obtain a mixed juice of the plant roots.

4. The method for propagating the *Lactarius orange-yellow* strain GAF-M-LA1 according to claim 3, characterized in that, The preparation method of the crude enzyme solution of Lactobacillus orange-yellow includes the following steps: Crush the mature mycelium of healthy, disease-free Lactobacillus orange-yellow and add sterile water. Stir for 10-20 minutes, centrifuge, and separate the supernatant. Filter the supernatant aseptically to obtain crude enzyme solution of Lactobacillus orange-yellow.

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