Liquid culture method for improving biomass of ganoderma niger mycelium in Hainan and application of liquid culture method

Abstract

The invention discloses a liquid culture method for improving biomass of Hainan ganoderma niger mycelium and application of the liquid culture method, and relates to the field of ganoderma niger liquid culture. The liquid culture method of the polygonum multiflorum comprises the following steps: inoculating an activated Hainan polygonum multiflorum strain into a liquid culture medium, and culturing to obtain a strain liquid; inoculating the strain liquid into a Hainan polygonum multiflorum liquid culture medium for culturing to obtain a Hainan polygonum multiflorum liquid culture product; the Hainan polygonum multiflorum liquid culture medium is prepared from the following raw materials: 25 to 35.0 g / L of glucose, 1.5 to 2.5 g / L of yeast powder, 2.0 to 3.0 g / L of monopotassium phosphate and 0.5 to 1.5 g / L of magnesium sulfate. The method can improve the mycelium yield and polyphenol active substance content of liquid culture of the Hainan polygonum multiflorum, reduce the production cost, meet the market demand, and lay a foundation for large-scale production of the Hainan polygonum multiflorum and acquisition of more mycelia and active components.

Description

Technical Field

[0001] The invention relates to the technical field of fungal liquid culture, and in particular to a liquid culture method for improving the mycelial biomass of Hainan tiger milk ganoderma and an application thereof. Background Art

[0002] Hainan Tiger Milk Ganoderma Lucidum ( Lignosus hainanensi BK Cui is an edible and medicinal fungus currently found only in Hainan, China. Rich in a variety of nutrients and active ingredients, such as proteins, polysaccharides, and polyphenols, it possesses antioxidant and anti-cancer properties. Clinically, it is commonly used to treat a variety of conditions, including fever, whooping cough, asthma, cancer, and gastric ulcers, and possesses exceptionally high medicinal value. With the advancement of modern science and technology, an increasing number of researchers have begun studying Hainan Tiger Milk Ganoderma, a rare fungus with unique medicinal properties. This research has provided scientific evidence for its value and further promoted its application in various fields.

[0003] With the increasing demand for Hainan Tiger Milk Ganoderma, collecting wild Hainan Tiger Milk Ganoderma is increasingly unable to meet public demand. Consequently, relevant researchers have gradually shifted their focus from wild collection to artificial cultivation. Traditional solid culture systems have a long spawning cycle, requiring approximately 180 days for sclerotia to mature, resulting in low efficiency for large-scale production. Liquid culture technology, as an efficient fungal culture method, offers advantages such as rapid growth and simple cultivation, making it possible to mass-produce Hainan Tiger Milk Ganoderma. As a new species in the genus, Hainan Tiger Milk Ganoderma possesses unique growth characteristics. To date, liquid culture methods for Hainan Tiger Milk Ganoderma are still in the exploratory stage, with no publicly available mature technologies reported. Therefore, developing a liquid culture method that can significantly increase the mycelial biomass and polyphenol content of Hainan Tiger Milk Ganoderma is of great significance for fully utilizing its medicinal value and promoting the development of related industries. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention aims to provide a liquid culture method for increasing the mycelial biomass of Hainan Tiger Milk Ganoderma.

[0005] The technical solutions of the present invention are as follows: The present invention provides a liquid culture method for increasing the biomass of Hainan tiger milk ganoderma mycelium, comprising the following steps: A. inoculating the activated Hainan Tiger Milk Ganoderma lucidum strain into a liquid culture medium for cultivation to obtain a strain liquid; B. inoculating the obtained bacterial liquid into a Hainan Tiger Milk Ganoderma lucidum liquid culture medium for culturing to obtain a Hainan Tiger Milk Ganoderma lucidum liquid culture product; The Hainan tiger milk ganoderma lucidum liquid culture medium contains the following raw materials: 25-35.0 g / L of glucose, 1.5-2.5 g / L of yeast powder, 2.0-3.0 g / L of potassium dihydrogen phosphate, and 0.5-1.5 g / L of magnesium sulfate, with a natural pH value.

[0006] Furthermore, the Hainan tiger milk ganoderma liquid culture medium contains the following raw materials: 35.0 g / L glucose, 1.5 g / L yeast powder, 2.5 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, and natural pH.

[0007] Furthermore, the culturing time in step B is 6 to 8 days.

[0008] Furthermore, the culturing time in step B is 7 days.

[0009] Furthermore, in step B, the inoculation amount of the bacterial liquid is 15-25% (v / v).

[0010] Furthermore, in step B, the inoculation volume of the bacterial liquid is 20% (v / v).

[0011] Furthermore, in step B, the volume of the Hainan Tiger Milk Ganoderma lucidum liquid culture medium is 200-350 mL.

[0012] Furthermore, the culture conditions in step B are a shaking culture temperature of 25° C. and a rotation speed of 160 rpm.

[0013] Furthermore, the culture conditions of step A are as follows: culture temperature 25° C., rotation speed 160 rpm, and shaking culture for 6 days.

[0014] Furthermore, the present invention has discovered a culture medium that can effectively increase the content of polyphenols in Hainan Tiger Milk Ganoderma Lucidum. The Hainan Tiger Milk Ganoderma Lucidum liquid culture medium contains vegetable oil and amino acids. Preferably, the vegetable oil is cottonseed oil, and the amino acid is tyrosine.

[0015] Furthermore, the Hainan tiger milk ganoderma liquid culture medium contains the following raw materials: 30.0 g / L glucose, 1.0 g / L yeast powder, 2.5 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 5 g / L cottonseed oil, 0.5 g / L tyrosine, and natural pH.

[0016] On the other hand, the present invention relates to the application of the liquid culture method in producing polyphenol substances of Hainan tiger milk ganoderma.

[0017] Compared with the prior art, the present invention has the following advantages: The present invention provides optimal culture medium components and culture conditions for the liquid culture of Hainan tiger milk ganoderma, which can increase the biomass and phenolic content of Hainan tiger milk ganoderma, and provide a reference for large-scale production of Hainan tiger milk ganoderma and obtaining mycelium and physiologically active substances.

[0018] The liquid culture medium provided by the invention has easily available components, a simple culture method and low cost, and can obtain a large amount of Hainan tiger milk ganoderma culture in a short time, thereby providing a large amount of raw materials for the production of Hainan tiger milk ganoderma related products and the research of active substances. DETAILED DESCRIPTION

[0019] In order to better understand the technical content of the present invention, the present invention is further described below in conjunction with specific embodiments.

[0020] Hainan Tiger Milk Ganoderma Lucidum ( Lignosus hainanensis ) strains, classified and named Lignosus hainanensi sFCATAS 11829 was collected from Wuzhishan, Hainan Province, and deposited in the Mycological Collection of the Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences (FCATAS) under the accession number FCATAS 11829. This strain has also been deposited with the China Center for Type Culture Collection, Wuhan University, Wuhan, China, under the accession number CCTCC NO: M 2025951, on April 30, 2025.

[0021] Example 1 Optimal carbon source screening Liquid culture medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Sterilize using conventional methods to prepare liquid culture medium. Basal medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Each bottle contains 200 mL of liquid. Sterilize using conventional methods to prepare the basal medium. Carbon source optimized medium: Glucose, sucrose, maltose, fructose, and lactose were selected as different carbon sources, and the addition amount was 20.0 g / L. Other conditions were the same as those of the basal medium. Cultivation method: Activation of Hainan Tiger Milk Ganoderma lucidum strains: inoculate the strains stored at 4°C into PDA culture medium and culture in a constant temperature incubator at 25°C to obtain activated Tiger Milk Ganoderma lucidum strains; Liquid culture: Take 5 pieces of uniform-sized bacterial cakes and inoculate them into liquid culture medium. Cultivate at 25°C and 160 rpm with shaking for 6 days. Liquid culture: Inoculate the bacterial culture liquid at a 10% (v / v) inoculum into the carbon source optimized medium, culture at 25°C and 160 rpm, set up three parallel groups, and culture with shaking for 7 days; Mycelium weighing: After the culture was completed, the mycelium was taken out by filtration, placed in a 60°C oven to dry to a constant weight, and the mycelium was weighed; the results are shown in Table 1.

[0022] Table 1 Effects of different carbon sources on the mycelial growth of Hainan Tiger Milk Ganoderma

[0023] The experimental results showed that glucose, sucrose, maltose, fructose and lactose can all promote the growth of mycelium of Tiger Milk Ganoderma lucidum. Especially when glucose was added as the carbon source, the dry mass of mycelium was the highest, reaching 3.41g / L, indicating that glucose has obvious growth advantages on the growth of mycelium of Hainan Tiger Milk Ganoderma lucidum. Therefore, glucose was selected as the carbon source for liquid culture of Hainan Tiger Milk Ganoderma lucidum.

[0024] Example 2 Optimal nitrogen source screening Liquid culture medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Sterilize using conventional methods to prepare liquid culture medium. Basal medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Each bottle contains 200 mL of liquid. Sterilize using conventional methods to prepare the basal medium. Nitrogen source optimized culture medium: peptone, yeast powder, urea, beef extract, and malt extract powder were selected as different nitrogen sources, and the addition amount was 2.0 g / L. Other conditions were the same as those of the basic culture medium. Cultivation method: Activation of Hainan Tiger Milk Ganoderma lucidum strains: inoculate the strains stored at 4°C into PDA culture medium and culture in a constant temperature incubator at 25°C to obtain Hainan Tiger Milk Ganoderma lucidum activated strains; Liquid culture: Take 5 pieces of uniform-sized bacterial cakes and inoculate them into liquid culture medium. Cultivate at 25°C and 160 rpm with shaking for 6 days. Liquid culture: Inoculate the bacterial culture liquid at a 10% (v / v) inoculum volume into nitrogen-optimized medium, culture at 25°C and 160 rpm, set up three parallel groups, and culture with shaking for 7 days; Mycelium weighing: After the culture was completed, the mycelium was taken out by filtration, placed in a 60°C oven to dry to a constant weight, and the mycelium was weighed; the results are shown in Table 2.

[0025] Table 2 Effects of different nitrogen sources on the mycelial growth of Hainan Tiger Milk Ganoderma

[0026] The results showed that peptone, yeast powder, urea, beef extract and malt extract powder could all promote the growth of Hainan tiger milk Ganoderma mycelium. Especially when yeast powder was added as the carbon source, the dry mass of the mycelium was the highest, reaching 8.15g / L, indicating that yeast powder has a significant growth advantage on the growth of Hainan tiger milk Ganoderma mycelium. Therefore, yeast powder was selected as the nitrogen source for the liquid culture of Hainan tiger milk Ganoderma.

[0027] Example 3 Optimal carbon source concentration screening Liquid culture medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Sterilize using conventional methods to prepare liquid culture medium. Basal medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Each bottle contains 200 mL of liquid. Sterilize using conventional methods to prepare the basal medium. Carbon source concentration optimization culture medium: glucose was selected as the carbon source, and different carbon source concentrations were set at 15.0 g / L, 20.0 g / L, 25.0 g / L, 30.0 g / L, and 35.0 g / L. Other conditions were the same as those of the basal culture medium; Cultivation method: Activation of Hainan Tiger Milk Ganoderma lucidum strains: inoculate the strains stored at 4°C into PDA culture medium and culture in a constant temperature incubator at 25°C to obtain Hainan Tiger Milk Ganoderma lucidum activated strains; Liquid culture: Take 5 pieces of uniform-sized bacterial cakes and inoculate them into liquid culture medium. Cultivate at 25°C and 160 rpm with shaking for 6 days. Liquid culture: Inoculate the bacterial culture liquid at a 10% (v / v) inoculum into the carbon source concentration optimized medium, culture at 25°C and 160 rpm, set up three parallel groups, and culture with shaking for 7 days; Mycelium weighing: After the culture was completed, the mycelium was taken out by filtration and dried in a 60°C oven to a constant weight. The mycelium was weighed; the results are shown in Table 3.

[0028] Table 3 Effects of different carbon source concentrations on the mycelial growth of Ganoderma lucidum

[0029] The experimental results showed that the addition of 30.0 g / L of glucose significantly promoted the liquid culture of Hainan Tiger Milk Ganoderma. With increasing glucose addition, the dry mass of mycelium initially increased and then decreased. Mycelial dry mass was highest when the glucose content was between 25.0 and 35.0 g / L, reaching a maximum of 5.29 g / L at 30.0 g / L.

[0030] Example 4 Optimal nitrogen source concentration screening Liquid culture medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Sterilize using conventional methods to prepare liquid culture medium. Basal medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Each bottle contains 200 mL of liquid. Sterilize using conventional methods to prepare the basal medium. Nitrogen source concentration optimization culture medium: yeast powder was selected as the nitrogen source, and different nitrogen source concentrations were set at 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, and 3.0 g / L. Other conditions were the same as those of the basal culture medium; Cultivation method: Activation of Hainan Tiger Milk Ganoderma lucidum strains: The strains stored at 4°C were inoculated into PDA culture medium and cultured in a constant temperature incubator at 25°C to obtain activated Tiger Milk Ganoderma lucidum strains; Liquid culture: Take 5 pieces of uniform-sized bacterial cakes and inoculate them into liquid culture medium. Cultivate at 25°C and 160 rpm with shaking for 6 days. Liquid culture: Inoculate the bacterial culture liquid at a 10% (v / v) inoculum into a nitrogen source concentration-optimized medium, culture at 25°C and a rotation speed of 160 rpm in three replicates, and culture with shaking for 7 days. Mycelium weighing: After the culture was completed, the mycelium was taken out by filtration, placed in a 60°C oven to dry to a constant weight, and the mycelium was weighed; the results are shown in Table 4.

[0031] Table 4 Effects of different nitrogen source concentrations on the mycelial growth of Hainan Tiger Milk Ganoderma

[0032] The experimental results showed that the addition of 1.5 g / L yeast powder significantly promoted the liquid culture of Hainan Tiger Milk Ganoderma lucidum. With increasing yeast powder addition, the dry mass of mycelium initially increased and then decreased. Mycelial dry mass was highest when the yeast powder content was between 1.5 and 2.5 g / L, reaching a maximum of 10.89 g / L at 1.5 g / L.

[0033] Example 5 Optimal potassium dihydrogen phosphate concentration screening Liquid culture medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Sterilize using conventional methods to prepare liquid culture medium. Basal medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Each bottle contains 200 mL of liquid. Sterilize using conventional methods to prepare the basal medium. Potassium dihydrogen phosphate concentration-optimized culture medium: different potassium dihydrogen phosphate concentrations were set at 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, and 3.0 g / L, and the other conditions were the same as those of the basal culture medium; Cultivation method: Activation of Hainan Tiger Milk Ganoderma lucidum strains: The strains stored at 4°C were inoculated into PDA culture medium and cultured in a constant temperature incubator at 25°C to obtain activated Tiger Milk Ganoderma lucidum strains; Liquid culture: Take 5 pieces of uniform-sized bacterial cakes and inoculate them into liquid culture medium. Cultivate at 25°C and 160 rpm with shaking for 6 days. Liquid culture: Inoculate the seed solution at a 10% (v / v) inoculum into a potassium dihydrogen phosphate concentration-optimized medium, culture at 25°C and 160 rpm in three replicates, and shake culture for 7 days. Mycelium weighing: After the culture was completed, the mycelium was taken out by filtration, placed in a 60°C oven to dry to a constant weight, and the mycelium was weighed; the results are shown in Table 5.

[0034] Table 5 Effects of different potassium dihydrogen phosphate concentrations on the mycelial growth of Hainan tiger milk ganoderma

[0035] The experimental results showed that the addition of 2.0 g / L of potassium dihydrogen phosphate significantly promoted the liquid culture of Hainan Tiger Milk Ganoderma. With increasing potassium dihydrogen phosphate addition, the dry mass of mycelium initially increased and then decreased. Mycelial dry mass was highest when the potassium dihydrogen phosphate content was between 2.0 and 3.0 g / L, reaching a maximum of 4.12 g / L at 2.0 g / L.

[0036] Example 6 Optimal magnesium sulfate concentration screening Liquid culture medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Sterilize using conventional methods to prepare liquid culture medium. Basal medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Each bottle contains 200 mL of liquid. Sterilize using conventional methods to prepare the basal medium. Magnesium sulfate concentration optimized culture medium: set different magnesium sulfate concentrations of 0.5g / L, 1.0g / L, 1.5g / L, 2.0g / L, and 2.5g / L, and the other conditions were the same as the basal culture medium; Cultivation method: Activation of Hainan Tiger Milk Ganoderma lucidum strains: The strains stored at 4°C were inoculated into PDA culture medium and cultured in a constant temperature incubator at 25°C to obtain activated Tiger Milk Ganoderma lucidum strains; Liquid culture: Take 5 pieces of uniform-sized bacterial cakes and inoculate them into liquid culture medium. Cultivate at 25°C and 160 rpm with shaking for 6 days. Liquid culture: Inoculate the bacterial culture liquid at a 10% (v / v) inoculum into a medium optimized for magnesium sulfate concentration. Cultivate at 25°C and 160 rpm in three replicates with shaking for 7 days. Mycelium weighing: After the culture was completed, the mycelium was taken out by filtration and dried in a 60°C oven to a constant weight. The mycelium was weighed; the results are shown in Table 6.

[0037] Table 6 Effects of different magnesium sulfate concentrations on the mycelial growth of Hainan tiger milk ganoderma

[0038] The experimental results showed that the addition of 1.0 g / L of magnesium sulfate significantly promoted the liquid culture of Hainan Tiger Milk Ganoderma. With increasing magnesium sulfate addition, the dry mass of mycelium initially increased and then decreased. Mycelial dry mass was highest when the magnesium sulfate content was between 0.5 and 1.5 g / L, reaching a maximum of 6.95 g / L at 1.0 g / L.

[0039] Example 7 Optimal culture time screening Liquid culture medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Sterilize using conventional methods to prepare liquid culture medium. Basal medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Each bottle contains 200 mL of liquid. Sterilize using conventional methods to prepare the basal medium. Optimization of culture time: Based on the basal culture medium, different culture times of 4d, 5d, 6d, 7d, 8d, 9d, 10d, and 11d were set; Cultivation method: Activation of Hainan Tiger Milk Ganoderma lucidum strains: The strains stored at 4°C were inoculated into PDA culture medium and cultured in a constant temperature incubator at 25°C to obtain activated Tiger Milk Ganoderma lucidum strains; Liquid culture: Take 5 pieces of uniform-sized bacterial cakes and inoculate them into liquid culture medium. Cultivate at 25°C and 160 rpm with shaking for 6 days. Liquid culture: Inoculate the seed solution into the basal medium at a 10% (v / v) inoculum volume, culture at 25°C and a rotation speed of 160 r / min, set up three parallel groups, and culture with shaking; Mycelium weighing: After the culture was completed, the mycelium was taken out by filtration and dried in a 60°C oven to a constant weight. The mycelium was weighed; the results are shown in Table 7.

[0040] Table 7 Effects of different culture times on the mycelial growth of Hainan Tiger Milk Ganoderma

[0041] The experimental results showed that 7 days was the optimal culture time for Hainan Tiger Milk Ganoderma lucidum in liquid culture. With increasing culture days, the dry mass of mycelium initially increased, then decreased slightly and gradually leveled off. The dry mass was highest between 6 and 8 days, reaching a maximum of 3.41 g / L at 7 days.

[0042] Example 8 Optimal inoculum size screening Liquid culture medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Sterilize using conventional methods to prepare liquid culture medium. Basal medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Each bottle contains 200 mL of liquid. Sterilize using conventional methods to prepare the basal medium. Optimization of inoculation volume: Based on the basal culture medium, set different inoculation volumes of 5%, 10%, 15%, 20%, and 25%; Cultivation method: Activation of Hainan Tiger Milk Ganoderma lucidum strains: The strains stored at 4°C were inoculated into PDA culture medium and cultured in a constant temperature incubator at 25°C to obtain activated Tiger Milk Ganoderma lucidum strains; Liquid culture: Take 5 pieces of uniform-sized bacterial cakes and inoculate them into liquid culture medium. Cultivate at 25°C and 160 rpm with shaking for 6 days. Liquid culture: Inoculate the bacterial culture liquid into the basal culture medium at different inoculum sizes, culture at 25°C and 160 rpm, set up three parallel groups, and culture with shaking for 7 days; Mycelium weighing: After the culture was completed, the mycelium was taken out by filtration and dried in a 60°C oven to a constant weight. The mycelium was weighed; the results are shown in Table 8.

[0043] Table 8 Effects of different inoculum amounts on the mycelial growth of Hainan Tiger Milk Ganoderma

[0044] Experimental results showed that 20% (v / v) is the optimal inoculum size for liquid culture of Hainan Tiger Milk Ganoderma. With increasing inoculum size, the dry mass of mycelium initially increases and then decreases. Mycelial dry mass is highest when the inoculum size is between 15% and 25%, reaching a maximum of 4.65 g / L at 20%.

[0045] Example 9 Optimal Liquid Volume Screening Liquid culture medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Sterilize using conventional methods to prepare liquid culture medium. Basal medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Sterilize using conventional methods to prepare the basal medium. Optimization of liquid volume: set different liquid volumes of 150mL, 200mL, 250mL, 300mL, and 350mL, and the other conditions are the same as those of the basal culture medium; Cultivation method: Activation of Hainan Tiger Milk Ganoderma lucidum strains: inoculate the strains stored at 4°C into PDA culture medium and culture in a constant temperature incubator at 25°C to obtain Hainan Tiger Milk Ganoderma lucidum activated strains; Liquid culture: Take 5 pieces of uniform-sized bacterial cakes and inoculate them into liquid culture medium. Cultivate at 25°C and 160 rpm with shaking for 6 days. Liquid culture: Inoculate the seed solution at 10% (v / v) inoculum into basal culture medium with different liquid volumes. Culture at 25°C and 160 rpm in three parallel groups with shaking for 7 days. Mycelium weighing: After the culture was completed, the mycelium was taken out by filtration, placed in a 60°C oven to dry to a constant weight, and the mycelium was weighed; the results are shown in Table 9.

[0046] Table 9 Effects of different liquid volumes on the mycelial growth of Hainan Tiger Milk Ganoderma

[0047] The experimental results showed that 300 mL was the optimal liquid volume for the liquid culture of Hainan Tiger Milk Ganoderma. With increasing liquid volume, the dry mass of mycelium initially increased and then leveled off, reaching a maximum of 3.45 g / L at an inoculum volume of 300 mL.

[0048] Example 10 Orthogonal test Table 10 Orthogonal experimental design table

[0049] Liquid culture medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Sterilize using conventional methods to prepare liquid culture medium. Orthogonal test medium: Prepare medium according to the concentration of each component in the orthogonal test design table. Each bottle contains 300 mL of liquid. Sterilize using conventional methods to prepare 9 sets of orthogonal test medium. Cultivation method: Activation of Hainan Tiger Milk Ganoderma lucidum strains: inoculate the strains stored at 4°C into PDA culture medium and culture in a constant temperature incubator at 25°C to obtain Hainan Tiger Milk Ganoderma lucidum activated strains; Liquid culture: Take 5 pieces of uniform-sized bacterial cakes and inoculate them into liquid culture medium. Cultivate at 25°C and 160 rpm with shaking for 6 days. Liquid culture: Inoculate the seed solution into the orthogonal test medium at a 20% inoculum size, culture at 25°C and a rotation speed of 160 r / min, set up three parallel groups, and culture with shaking for 7 days; Mycelium weighing: After the culture was completed, the mycelium was taken out by filtration, placed in a 60°C oven to dry to a constant weight, and the mycelium was weighed; the results are shown in Table 11.

[0050] Table 11 Orthogonal test results

[0051] Analysis of the orthogonal test results showed that the influence of the four factors on the mycelium growth of Hainan tiger milk Ganoderma lucidum is B>A>D>C from large to small, and the optimal combination for liquid culture of Hainan tiger milk Ganoderma lucidum is A2B1C3D1, that is, 2.5g / L potassium dihydrogen phosphate, 0.5g / L magnesium sulfate, 35g / L glucose, and 1.5g / L yeast powder.

[0052] Example 11 Verification Test Liquid culture medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Sterilize using conventional methods to prepare liquid culture medium. Verification test medium: Weigh 35.0 g glucose, 1.5 g yeast extract, 2.5 g potassium dihydrogen phosphate, and 0.5 g magnesium sulfate, and dilute to 1000 mL with water. Each bottle contains 300 mL of liquid. Sterilize using conventional methods to prepare the verification test medium. Cultivation method: Activation of Hainan Tiger Milk Ganoderma lucidum strains: inoculate the strains stored at 4°C into PDA culture medium and culture in a constant temperature incubator at 25°C to obtain Hainan Tiger Milk Ganoderma lucidum activated strains; Liquid culture: Take 5 pieces of uniform-sized bacterial cakes and inoculate them into liquid culture medium. Cultivate at 25°C and 160 rpm with shaking for 6 days. Liquid culture: Inoculate the seed solution into the validation test medium at a 20% inoculum size, culture at 25°C and 160 rpm, set up three parallel groups, and culture with shaking for 7 days; Mycelium Weighing: After the incubation period, the mycelium was removed and filtered to obtain the mycelium. The mycelium was then dried in a 60°C oven to a constant weight and weighed. Furthermore, the polyphenol content in the mycelium was determined. Polyphenol Content Determination: Polyphenol content was determined spectrophotometrically according to the standard T / AHFIA005-2018, "Determination of Total Polyphenols in Plant Extracts and Their Products."

[0053] Results: The dry weight of the mycelium was 22.04 g / L, which was superior to the nine experiments in the orthogonal experiment table. Polyphenol content testing revealed a polyphenol content of 4.1914 mg / g after seven days of liquid culture. This example demonstrates the feasibility of the optimal combination identified in the orthogonal experiment and demonstrates its suitability for the liquid culture of Hainan Tiger Milk Ganoderma.

[0054] Example 12 Culture medium optimization and detection of polyphenol active ingredients While exploring liquid culture medium formulations, we discovered that certain amino acids and oils can significantly increase the content of polyphenolic active ingredients in mycelium. Some relevant research data are as follows: Liquid culture medium: Weigh 20.0 g glucose, 2.0 g peptone, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate, and dilute to 1000 mL with water. Sterilize using conventional methods to prepare liquid culture medium. Basal medium: Weigh 35.0 g glucose, 1.5 g yeast extract, 2.5 g potassium dihydrogen phosphate, and 0.5 g magnesium sulfate, and dilute to 1000 mL with water. Each bottle contains 300 mL of liquid. Sterilize using conventional methods to prepare the basal medium. Optimize the culture medium: replace the equivalent amount of glucose + yeast powder in the basal medium with 0.5g vegetable oil + 0.5g amino acid, and keep the other conditions the same as the basal medium; that is: A1: 35.0g glucose, 1.5g yeast powder, 2.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.5g cottonseed oil, 0.5g tyrosine A2: 35.0g glucose, 1.5g yeast powder, 2.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.5g soybean oil, 0.5g tyrosine A3: 35.0g glucose, 1.5g yeast powder, 2.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.5g cottonseed oil, 0.5g glycine A4: 35.0g glucose, 1.5g yeast extract, 2.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.5g soybean oil, 0.5g glycine Cultivation method: Activation of Hainan Tiger Milk Ganoderma lucidum strains: inoculate the strains stored at 4°C into PDA culture medium and culture in a constant temperature incubator at 25°C to obtain Hainan Tiger Milk Ganoderma lucidum activated strains; Liquid culture: Take 5 pieces of uniform-sized bacterial cakes and inoculate them into liquid culture medium. Cultivate at 25°C and 160 rpm with shaking for 6 days. Liquid culture: 20% (v / v) seed solution was inoculated into the optimized culture medium, cultured at 25°C and 160 rpm in three parallel groups, and shaken for 7 days. Mycelium Weighing: After the incubation period, the mycelium was removed and filtered to obtain the mycelium. The mycelium was then dried in a 60°C oven to a constant weight and weighed. The polyphenol content in the mycelium was determined. The results are shown in Table 12.

[0055] Table 12 Test results of mycelium yield and polyphenol content of Hainan Tiger Milk Ganoderma

[0056] Results: Mycelial dry weights ranged from 20.16 to 21.94 g / L, and mycelial biomass remained high. This suggests that replacing glucose and yeast extract with vegetable oils and amino acids had minimal impact on mycelial yield, maintaining the highest mycelial yield for Hainan Tiger Milk Ganoderma lucidum. Phenolic active substance content analysis revealed that the addition of cottonseed oil and tyrosine significantly increased polyphenol content, reaching a peak of 168.617 mg / g on the seventh day of culture.

[0057] The above descriptions are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A liquid culture method for increasing the mycelial biomass of Hainan tiger milk ganoderma, characterized in that: The following steps are involved: A. inoculating the activated Hainan Tiger Milk Ganoderma lucidum strain into Hainan Tiger Milk Ganoderma lucidum liquid culture medium for cultivation to obtain a strain liquid; B. inoculating the obtained bacterial liquid into a Hainan Tiger Milk Ganoderma lucidum liquid culture medium for culturing to obtain a Hainan Tiger Milk Ganoderma lucidum liquid culture product; The Hainan Tiger Milk Ganoderma lucidum liquid culture medium contains the following raw materials: 25-35.0 g / L glucose, 1.5-2.5 g / L yeast powder, 2.0-3.0 g / L potassium dihydrogen phosphate, 0.5-1.5 g / L magnesium sulfate, and has a natural pH value. The Hainan tiger milk ganoderma lucidum liquid culture medium also contains cottonseed oil and tyrosine.

2. The liquid culture method for increasing the mycelial biomass of Hainan Tiger Milk Ganoderma according to claim 1, characterized in that: The culture time of step B is 6 to 8 days.

3. The liquid culture method for increasing the mycelial biomass of Hainan Tiger Milk Ganoderma according to claim 1, characterized in that: The incubation time of step B is 7 days.

4. The liquid culture method for increasing the mycelial biomass of Hainan Tiger Milk Ganoderma according to claim 1, characterized in that: In step B, the inoculation volume of the bacterial culture solution is 15-25% (v / v).

5. The liquid culture method for increasing the mycelial biomass of Hainan Tiger Milk Ganoderma according to claim 1, characterized in that: In step B, the inoculation volume of the bacterial liquid is 20% (v / v).

6. The liquid culture method for increasing the mycelial biomass of Hainan Tiger Milk Ganoderma according to claim 1, characterized in that: In step B, the volume of Hainan Tiger Milk Ganoderma lucidum liquid culture medium is 200-350 mL.

7. The liquid culture method for increasing the mycelial biomass of Hainan Tiger Milk Ganoderma according to claim 1, characterized in that: The Hainan Tiger Milk Ganoderma lucidum liquid culture medium contains the following raw materials: 30.0 g / L glucose, 1.0 g / L yeast powder, 2.5 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 5 g / L cottonseed oil, 0.5 g / L tyrosine, and has a natural pH.

8. Use of the liquid culture method according to any one of claims 1 to 7 in producing polyphenols from Hainan tiger milk ganoderma.

Images