Liquid culture method for improving biomass of ganoderma lingzhi mycelium from hainan tiger milk and application thereof

Abstract

The application discloses a liquid culture method for improving the biomass of Hainan tiger milk Ganoderma lingzhi and application thereof, and relates to the field of liquid culture of tiger milk Ganoderma lingzhi. The liquid culture method of the tiger milk Ganoderma lingzhi comprises the following steps: inoculating activated Hainan tiger milk Ganoderma lingzhi strains into a liquid culture medium for culture to obtain a strain liquid; and inoculating the strain liquid into a Hainan tiger milk Ganoderma lingzhi liquid culture medium for culture to obtain a liquid culture product of the Hainan tiger milk Ganoderma lingzhi. The Hainan tiger milk Ganoderma lingzhi liquid culture medium contains the following raw materials: glucose 25-35.0 g / L, yeast powder 1.5-2.5 g / L, potassium dihydrogen phosphate 2.0-3.0 g / L, and magnesium sulfate 0.5-1.5 g / L. The method can improve the mycelium yield and the content of polyphenol active substances in the liquid culture of the Hainan tiger milk Ganoderma lingzhi, reduce the production cost, meet the market demand, and lay a foundation for large-scale production of the Hainan tiger milk Ganoderma lingzhi and obtaining more mycelium and active ingredients.

Description

Technical Field

[0001] This invention relates to the field of fungal liquid culture technology, specifically to a liquid culture method for increasing the biomass of Ganoderma lucidum mycelium in Hainan and its application. Background Technology

[0002] Hainan Tiger Milk Ganoderma ( Lignosus hainanensi *Ganoderma lucidum* (also known as Hainan Tiger Milk Ganoderma) is an edible and medicinal fungus currently found only in Hainan, China. Rich in various nutrients and active ingredients, such as proteins, polysaccharides, and polyphenols, it possesses antioxidant and anti-cancer properties. Clinically, it is frequently used to treat fever, whooping cough, asthma, cancer, and gastric ulcers, among other diseases, demonstrating its extremely high medicinal value. With the development of modern science and technology, more and more researchers are beginning to study this rare fungus with unique medicinal value, providing scientific evidence for its worth and further promoting its application in various fields.

[0003] With the increasing demand for Hainan Tiger Milk Ganoderma, the harvesting of wild Hainan Tiger Milk Ganoderma is gradually failing to meet public demand. Therefore, researchers are shifting their focus from wild collection to artificial cultivation. Traditional solid-state culture systems have long mycelial growth cycles, with sclerotia maturing in approximately 180 days, resulting in low efficiency for large-scale production. Liquid culture technology, as a highly efficient fungal culture method, offers advantages such as rapid growth and simple cultivation, making large-scale production of Hainan Tiger Milk Ganoderma possible. As a new species within the genus *Ganoderma*, Hainan Tiger Milk Ganoderma possesses unique growth characteristics. Currently, liquid culture methods for Hainan Tiger Milk Ganoderma are still in the exploratory stage, and no publicly available mature technologies have been 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 shortcomings of the prior art, the present invention aims to provide a liquid culture method for increasing the biomass of Ganoderma lucidum mycelium in Hainan.

[0005] The technical solution of this invention is as follows:

[0006] This invention provides a liquid culture method for increasing the biomass of Ganoderma lucidum mycelium in Hainan, comprising the following steps:

[0007] A. The activated Hainan tiger milk Ganoderma lucidum strain was inoculated into a liquid culture medium and cultured to obtain the strain solution;

[0008] B. The obtained inoculum solution was inoculated into Hainan Tiger Milk Ganoderma liquid culture medium and cultured to obtain Hainan Tiger Milk Ganoderma liquid culture product;

[0009] The Hainan tiger milk Ganoderma lucidum liquid culture medium contains the following raw materials: glucose 25-35.0 g / L, yeast powder 1.5-2.5 g / L, potassium dihydrogen phosphate 2.0-3.0 g / L, magnesium sulfate 0.5-1.5 g / L, and the pH is natural.

[0010] Further, the Hainan tiger milk Ganoderma lucidum liquid culture medium contains the following raw materials: glucose 35.0 g / L, yeast powder 1.5 g / L, potassium dihydrogen phosphate 2.5 g / L, magnesium sulfate 0.5 g / L, and the pH is natural.

[0011] Further, the culture time of step B is 6-8 d.

[0012] Further, the culture time of step B is 7 d.

[0013] Further, in step B, the inoculation amount of the strain liquid is 15-25% (v / v).

[0014] Further, in step B, the inoculation amount of the strain liquid is 20% (v / v).

[0015] Further, in step B, the liquid loading amount of the Hainan tiger milk Ganoderma lucidum liquid culture medium is 200-350 mL.

[0016] Further, the culture condition of step B is that the shaking culture temperature is 25℃, and the rotation speed is 160 rpm.

[0017] Further, the culture condition of step A is that the culture temperature is 25℃, the rotation speed is 160 rpm, and the shaking culture is 6 d.

[0018] Further, the present application finds a culture medium which can effectively improve the content of Hainan tiger milk Ganoderma lucidum polyphenols, and the Hainan tiger milk Ganoderma lucidum liquid culture medium contains plant oil and amino acid, preferably, the plant oil is cottonseed oil, and the amino acid is tyrosine.

[0019] Further, the Hainan tiger milk Ganoderma lucidum liquid culture medium contains the following raw materials: glucose 30.0 g / L, yeast powder 1.0 g / L, potassium dihydrogen phosphate 2.5 g / L, magnesium sulfate 0.5 g / L, 5 g / L cottonseed oil, and 0.5 g / L tyrosine, and the pH is natural.

[0020] On the other hand, the present application relates to the application of the liquid culture method in the production of Hainan tiger milk Ganoderma lucidum polyphenols.

[0021] Compared with the prior art, the beneficial results of the present application are that:

[0022] This invention provides the optimal culture medium composition and culture conditions for liquid culture of Ganoderma lucidum (Tiger Milk Ganoderma) from Hainan, which can increase the biomass and phenolic content of Ganoderma lucidum. It provides a reference for large-scale production of Ganoderma lucidum from Hainan and the acquisition of mycelium and physiologically active substances.

[0023] The liquid culture medium provided by this invention has readily available components, a simple culture method, and low cost. It can obtain a large amount of Hainan Tiger Milk Ganoderma culture in a short time, providing a large amount of raw materials for the production of Hainan Tiger Milk Ganoderma related products and the research of active substances. Detailed Implementation

[0024] To better understand the technical content of this invention, the invention will be further described below with reference to specific embodiments.

[0025] Hainan Tiger Milk Ganoderma ( Lignosus hainanensis ) strain, classified and named Lignosus hainanensi sFCATAS 11829, collected from Wuzhishan, Hainan Province, is deposited in the Herbarium of the Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences (FCATAS), with accession number FCATAS 11829. Simultaneously, this strain is also deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025951 and deposit date of April 30, 2025.

[0026] Example 1: Screening for the best carbon source

[0027] Liquid culture medium: Weigh 20.0g glucose, 2.0g peptone, 3.0g potassium dihydrogen phosphate, and 1.5g magnesium sulfate, and add water to a final volume of 1000mL. Sterilize using standard methods to obtain the liquid culture medium.

[0028] Basic culture medium: Weigh 20.0g glucose, 2.0g peptone, 3.0g potassium dihydrogen phosphate, and 1.5g magnesium sulfate, and add water to a final volume of 1000mL. Each bottle contains 200mL. Sterilize using standard methods to prepare the basic culture medium.

[0029] Carbon source optimized culture medium: glucose, sucrose, maltose, fructose and lactose were selected as different carbon sources, and the addition amount was 20.0 g / L. The other conditions were the same as those of the basal culture medium.

[0030] Cultivation methods:

[0031] Activation of Ganoderma lucidum strain from Hainan: The strain preserved at 4℃ was inoculated into PDA medium and cultured in a constant temperature incubator at 25℃ to obtain activated Ganoderma lucidum strain;

[0032] Liquid culture: the strain liquid was inoculated into the carbon source optimized medium at an inoculation amount of 10% (v / v) and cultured at a temperature of 25°C and a rotation speed of 160 r / min, with 3 groups of parallel settings, and oscillation cultured for 7d;

[0033] Liquid culture: the strain liquid was inoculated into the carbon source optimized medium at an inoculation amount of 10% (v / v) and cultured at a temperature of 25°C and a rotation speed of 160 r / min, with 3 groups of parallel settings, and oscillation cultured for 7d;

[0034] Mycelium weighing: after the culture was completed, the mycelium was obtained by suction filtration and placed in a 60°C oven for drying until the weight was constant, and the weight of the mycelium was measured; the results are shown in Table 1.

[0035] Table 1 Influence of different carbon sources on the growth of Hainan tiger milk Ganoderma lucidum mycelium

[0036]

[0037] The experimental results show that glucose, sucrose, maltose, fructose and lactose can all make the mycelium of tiger milk Ganoderma lucidum grow, especially when glucose is added as the carbon source, the dry weight of the mycelium is the highest, reaching 3.41 g / L, which shows that glucose has obvious growth advantage for the mycelium growth of Hainan tiger milk Ganoderma lucidum, so glucose is selected as the carbon source for the liquid culture of Hainan tiger milk Ganoderma lucidum.

[0038] Example 2 Screening of the best nitrogen source

[0039] Liquid culture medium: 20.0 g of glucose, 2.0 g of peptone, 3.0 g of potassium dihydrogen phosphate and 1.5 g of magnesium sulfate were weighed, and water was added to make up to 1000 mL. Sterilized by conventional method to prepare the liquid culture medium;

[0040] Basic medium: 20.0 g of glucose, 2.0 g of peptone, 3.0 g of potassium dihydrogen phosphate and 1.5 g of magnesium sulfate were weighed, and water was added to make up to 1000 mL. The amount of liquid in each bottle was 200 mL. Sterilized by conventional method to prepare the basic medium;

[0041] Nitrogen source optimized 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, and the rest of the conditions were the same as the basic medium;

[0042] Culture method:

[0043] Activation of Hainan tiger milk Ganoderma lucidum strain: the strain preserved at 4°C was inoculated into PDA medium and cultured in a 25°C constant temperature incubator to obtain the activated strain of Hainan tiger milk Ganoderma lucidum;

[0044] Liquid strain culture: 5 pieces of uniform and consistent size of the fungus cake were taken and inoculated into the liquid culture medium, and cultured at a temperature of 25°C and a rotation speed of 160 r / min, and oscillation cultured for 6d;

[0045] Liquid culture: inoculate the strain liquid in nitrogen source optimization medium at 10% (v / v) inoculation amount, cultivate at temperature 25℃, rotation speed 160r / min, set 3 groups of parallel, shake culture for 7d;

[0046] Mycelium weighing: after the end of the culture, take out the mycelium, place it in a 60℃ oven to dry to constant weight, weigh the mycelium weight; the results are shown in Table 2.

[0047] Table 2 Influence of different nitrogen sources on the growth of Hainan tiger milk Ganoderma lucidum mycelium

[0048]

[0049] The results show that peptone, yeast powder, urea, beef extract, malt extract powder can make Hainan tiger milk Ganoderma lucidum mycelium grow, especially when adding yeast powder as carbon source, the dry weight of mycelium is the highest, reaching 8.15g / L, which shows that yeast powder has obvious growth advantage for Hainan tiger milk Ganoderma lucidum mycelium growth, so yeast powder is selected as the nitrogen source for liquid culture of Hainan tiger milk Ganoderma lucidum.

[0050] Example 3 Screening of optimal carbon source concentration

[0051] Liquid medium: weigh 20.0g glucose, 2.0g peptone, 3.0g potassium dihydrogen phosphate, 1.5g magnesium sulfate, add water to constant volume to 1000mL. Sterilize using conventional method to prepare the liquid medium;

[0052] Basic medium: weigh 20.0g glucose, 2.0g peptone, 3.0g potassium dihydrogen phosphate, 1.5g magnesium sulfate, add water to constant volume to 1000mL. The liquid volume per bottle is 200mL. Sterilize using conventional method to prepare the basic medium;

[0053] Carbon source concentration optimization medium: select glucose as carbon source, set 15.0g / L, 20.0g / L, 25.0g / L, 30.0g / L, 35.0g / L different carbon source concentrations, and the rest of the conditions are the same as the basic medium;

[0054] Culture method:

[0055] Activation of Hainan tiger milk Ganoderma lucidum strain: inoculate the strain preserved at 4℃ in PDA medium, cultivate in a 25℃ constant temperature incubator to obtain activated Hainan tiger milk Ganoderma lucidum strain;

[0056] Liquid strain culture: take 5 pieces of uniform and consistent size of fungus cake, inoculate in liquid culture medium, cultivate at temperature 25℃, rotation speed 160r / min, shake culture for 6d;

[0057] Liquid culture: inoculate the strain liquid with 10% (v / v) inoculation amount in the carbon source concentration optimized medium, cultivate at 25℃ and 160r / min, set 3 groups in parallel, and shake culture for 7d;

[0058] Mycelium weighing: after the culture, the mycelium was obtained by suction filtration, placed in a 60℃ oven to dry to constant weight, and weighed;

[0059] Table 3 Influence of different carbon source concentrations on the mycelium growth of Hainan Ganoderma lucidum

[0060]

[0061] The experimental results show that the 30.0g / L glucose addition has a good promoting effect on the liquid culture of Hainan Ganoderma lucidum. With the increase of glucose addition, the mycelium dry weight shows a trend of first increasing and then decreasing. When the glucose content is 25.0-35.0g / L, the mycelium dry weight is higher, and when the glucose content is 30.0g / L, the mycelium dry weight reaches the maximum of 5.29g / L.

[0062] Example 4 Screening of the Optimal Nitrogen Source Concentration

[0063] Liquid medium: weigh 20.0g glucose, 2.0g peptone, 3.0g potassium dihydrogen phosphate, and 1.5g magnesium sulfate, and add water to 1000mL. Sterilize using the conventional method to prepare the liquid medium;

[0064] Basic medium: weigh 20.0g glucose, 2.0g peptone, 3.0g potassium dihydrogen phosphate, and 1.5g magnesium sulfate, and add water to 1000mL. The liquid volume in each bottle is 200mL. After sterilization using the conventional method, the basic medium is prepared;

[0065] Nitrogen source concentration optimized medium: select yeast powder as the nitrogen source, set different nitrogen source concentrations of 1.0g / L, 1.5g / L, 2.0g / L, 2.5g / L, and 3.0g / L, and the rest of the conditions are the same as the basic medium;

[0066] Cultivation method:

[0067] Activation of Hainan Ganoderma lucidum strain: inoculate the strain preserved at 4℃ in PDA medium, and cultivate in a 25℃ constant temperature incubator to obtain the activated strain of Hainan Ganoderma lucidum;

[0068] Liquid strain culture: take 5 uniform and consistent size of fungus cakes, inoculate in the liquid medium, cultivate at 25℃ and 160r / min, and shake culture for 6d;

[0069] Liquid culture: inoculate the strain liquid with 10% (v / v) inoculation amount in nitrogen source concentration optimized medium, cultivate at temperature 25℃, rotation speed 160r / min, set 3 groups in parallel, shake culture for 7d;

[0070] Mycelium weighing: after the end of the culture, take out the mycelium, obtain the mycelium by suction filtration, place it in a 60℃ oven to dry to constant weight, weigh the mycelium weight; the results are shown in Table 4.

[0071] Table 4 Influence of different nitrogen source concentrations on the mycelium growth of Hainan tiger milk Ganoderma lucidum

[0072]

[0073] The experimental results show that the addition amount of 1.5g / L yeast powder has a good promoting effect on the liquid culture of Hainan tiger milk Ganoderma lucidum. With the increase of the addition amount of yeast powder, the dry weight of mycelium shows a trend of first increasing and then decreasing. When the content of yeast powder is 1.5~2.5g / L, the dry weight of mycelium is higher, and when it is 1.5g / L, the dry weight of mycelium reaches the maximum, which is 10.89g / L.

[0074] Example 5 Screening of the Optimal Potassium Dihydrogen Phosphate Concentration

[0075] Liquid culture medium: weigh 20.0g of glucose, 2.0g of peptone, 3.0g of potassium dihydrogen phosphate, and 1.5g of magnesium sulfate, and add water to make up to 1000mL. Sterilize using the conventional method to prepare the liquid culture medium;

[0076] Basic medium: weigh 20.0g of glucose, 2.0g of peptone, 3.0g of potassium dihydrogen phosphate, and 1.5g of magnesium sulfate, and add water to make up to 1000mL. The liquid volume per bottle is 200mL. After sterilization using the conventional method, the basic medium is prepared;

[0077] Potassium dihydrogen phosphate concentration optimized medium: set 1.0g / L, 1.5g / L, 2.0g / L, 2.5g / L, and 3.0g / L different potassium dihydrogen phosphate concentrations, and the rest of the conditions are the same as the basic medium;

[0078] Cultivation method:

[0079] Activation of Hainan tiger milk Ganoderma lucidum strain: inoculate the strain preserved at 4℃ in PDA medium, and cultivate in a 25℃ constant temperature incubator to obtain the activated tiger milk Ganoderma lucidum strain;

[0080] Liquid strain culture: take 5 uniform and consistent size of fungus cakes, inoculate in liquid culture medium, cultivate at temperature 25℃, rotation speed 160r / min, shake culture for 6d;

[0081] Liquid culture: the seed liquid was inoculated in the potassium dihydrogen phosphate concentration optimized medium at an inoculation amount of 10% (v / v) and cultured at a temperature of 25°C and a rotation speed of 160 r / min, 3 groups of parallel were set, and the liquid culture was performed for 7 days;

[0082] Mycelium weighing: after the culture was completed, the mycelium was obtained by suction filtration, placed in a 60°C oven for drying until the weight was constant, and the weight of the mycelium was weighed; the results are shown in Table 5.

[0083] Table 5 Influence of different potassium dihydrogen phosphate concentrations on the mycelium growth of Hainan tiger breast Ganoderma lucidum

[0084]

[0085] The experimental results show that the addition amount of 2.0 g / L of potassium dihydrogen phosphate has a good promoting effect on the liquid culture of Hainan tiger breast Ganoderma lucidum. With the increase of the addition amount of potassium dihydrogen phosphate, the dry weight of the mycelium presents a trend of first increasing and then decreasing. When the content of potassium dihydrogen phosphate is 2.0-3.0 g / L, the dry weight of the mycelium is higher, and when the content of potassium dihydrogen phosphate is 2.0 g / L, the dry weight of the mycelium reaches the maximum of 4.12 g / L.

[0086] Example 6 Screening of the Optimal Magnesium Sulfate Concentration

[0087] Liquid culture medium: 20.0 g of glucose, 2.0 g of peptone, 3.0 g of potassium dihydrogen phosphate and 1.5 g of magnesium sulfate were weighed, and water was added to make up to 1000 mL. The liquid culture medium was prepared by sterilization using a conventional method;

[0088] Basic medium: 20.0 g of glucose, 2.0 g of peptone, 3.0 g of potassium dihydrogen phosphate and 1.5 g of magnesium sulfate were weighed, and water was added to make up to 1000 mL. The liquid culture medium was prepared by sterilization using a conventional method;

[0089] Magnesium sulfate concentration optimized medium: 0.5 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L and 2.5 g / L of magnesium sulfate were set, and the rest of the conditions were the same as the basic medium;

[0090] Culture method:

[0091] Activation of Hainan tiger breast Ganoderma lucidum strain: the strain preserved at 4°C was inoculated in PDA medium and cultured in a 25°C constant temperature incubator to obtain the activated strain of tiger breast Ganoderma lucidum;

[0092] Liquid strain culture: 5 pieces of uniform and consistent fungus cakes were taken and inoculated in the liquid culture medium, and cultured at a temperature of 25°C and a rotation speed of 160 r / min, and the liquid culture was performed for 6 days;

[0093] Liquid culture: inoculate the strain liquid with 10% (v / v) inoculation amount in magnesium sulfate concentration optimized medium, cultivate at temperature 25℃, rotation speed 160r / min, set 3 groups in parallel, shake culture for 7d;

[0094] Mycelium weighing: after the end of the culture, take out the mycelium, dry it in a 60℃ oven until the weight is constant, and weigh the mycelium; the results are shown in Table 6.

[0095] Table 6 Influence of different magnesium sulfate concentrations on the growth of Hainan tiger breast Ganoderma lucidum mycelium

[0096]

[0097] The experimental results show that the addition of 1.0g / L magnesium sulfate has a good promoting effect on the liquid culture of Hainan tiger breast Ganoderma lucidum. With the increase of magnesium sulfate addition, the dry weight of mycelium shows a trend of first increasing and then decreasing. When the magnesium sulfate content is 0.5-1.5g / L, the dry weight of mycelium is higher, and when the magnesium sulfate content is 1.0g / L, the dry weight of mycelium reaches the maximum of 6.95g / L.

[0098] Example 7 Screening of optimal culture time

[0099] Liquid medium: weigh 20.0g glucose, 2.0g peptone, 3.0g potassium dihydrogen phosphate and 1.5g magnesium sulfate respectively, and add water to make up to 1000mL. Sterilize using conventional method to prepare the liquid medium;

[0100] Basic medium: weigh 20.0g glucose, 2.0g peptone, 3.0g potassium dihydrogen phosphate and 1.5g magnesium sulfate respectively, and add water to make up to 1000mL. The liquid volume per bottle is 200mL. After sterilization using conventional method, the basic medium is prepared;

[0101] Culture time optimization: based on the basic medium, set different culture times of 4d, 5d, 6d, 7d, 8d, 9d, 10d and 11d;

[0102] Culture method:

[0103] Activation of Hainan tiger breast Ganoderma lucidum strain: inoculate the strain preserved at 4℃ in PDA medium, and cultivate in a 25℃ constant temperature incubator to obtain activated tiger breast Ganoderma lucidum strain;

[0104] Liquid strain culture: take 5 uniform and consistent size of fungus cakes, inoculate in liquid medium, cultivate at temperature 25℃, rotation speed 160r / min, and shake culture for 6d;

[0105] Liquid culture: seed liquid was inoculated in the basic medium at an inoculation amount of 10% (v / v) and cultured at a temperature of 25°C and a rotation speed of 160 r / min, with 3 groups in parallel, and shock culture;

[0106] Mycelium weighing: after the culture was completed, the mycelium was obtained by suction filtration, placed in a 60°C oven for drying until the weight was constant, and weighed;

[0107] Table 7 Influence of different culture times on the growth of Hainan tiger milk Ganoderma lucidum mycelium

[0108]

[0109] The experimental results show that 7d is the best culture time for liquid culture of Hainan tiger milk Ganoderma lucidum. With the increase of culture days, the dry weight of mycelium shows a trend of first increasing, then slightly decreasing and gradually tending to be flat. When the culture days are 6-8d, the dry weight of mycelium is relatively high, and when the culture days are 7d, the dry weight of mycelium reaches the maximum, which is 3.41g / L.

[0110] Example 8 Screening of the best inoculation amount

[0111] Liquid culture medium: 20.0g of glucose, 2.0g of peptone, 3.0g of potassium dihydrogen phosphate, and 1.5g of magnesium sulfate were weighed, and water was added to make up to 1000mL. The liquid culture medium was prepared by sterilization with a conventional method;

[0112] Basic medium: 20.0g of glucose, 2.0g of peptone, 3.0g of potassium dihydrogen phosphate, and 1.5g of magnesium sulfate were weighed, and water was added to make up to 1000mL. The amount of liquid in each bottle was 200mL. The basic medium was prepared by sterilization with a conventional method;

[0113] Inoculation amount optimization: based on the basic medium, different inoculation amounts of 5%, 10%, 15%, 20%, and 25% were set;

[0114] Culture method:

[0115] Activation of Hainan tiger milk Ganoderma lucidum strain: the strain preserved at 4°C was inoculated in PDA medium and cultured in a 25°C constant temperature incubator to obtain the activated tiger milk Ganoderma strain;

[0116] Liquid strain culture: 5 pieces of uniform and consistent fungus cakes were inoculated in the liquid culture medium and cultured at a temperature of 25°C and a rotation speed of 160 r / min, with shock culture for 6d;

[0117] Liquid culture: the strain liquid was inoculated in the basic medium at different inoculation amounts, cultured at a temperature of 25°C and a rotation speed of 160 r / min, with 3 groups in parallel, and shock culture for 7d;

[0118] Mycelium weighing: After the end of the culture, the mycelium was obtained by suction filtration and placed in a 60°C oven to dry to constant weight. The weight of the mycelium was measured; the results are shown in Table 8.

[0119] Table 8 Effect of different inoculation amounts on the growth of Hainan Tiger Breast Ganoderma lucidum mycelium

[0120]

[0121] The experimental results show that 20% (v / v) is the best inoculation amount for Hainan Tiger Breast Ganoderma lucidum liquid culture. With the increase of the inoculation amount, the dry weight of the mycelium shows a trend of first increasing and then decreasing. When the inoculation amount is 15% to 25%, the dry weight of the mycelium is relatively high, and when the inoculation amount is 20%, the dry weight of the mycelium reaches a maximum of 4.65 g / L.

[0122] Example 9 Screening of the Optimal Liquid Loading Amount

[0123] Liquid medium: 20.0 g of glucose, 2.0 g of peptone, 3.0 g of potassium dihydrogen phosphate, and 1.5 g of magnesium sulfate were weighed, and water was added to make up to 1000 mL. The liquid medium was sterilized using a conventional method;

[0124] Basic medium: 20.0 g of glucose, 2.0 g of peptone, 3.0 g of potassium dihydrogen phosphate, and 1.5 g of magnesium sulfate were weighed, and water was added to make up to 1000 mL. The basic medium was prepared after sterilization using a conventional method;

[0125] Liquid loading amount optimization: 150 mL, 200 mL, 250 mL, 300 mL, and 350 mL different liquid loading amounts were set, and the rest of the conditions were the same as the basic medium;

[0126] Cultivation method:

[0127] Activation of Hainan Tiger Breast Ganoderma lucidum strain: The strain preserved at 4°C was inoculated in PDA medium and cultured in a 25°C constant temperature incubator to obtain activated Hainan Tiger Breast Ganoderma lucidum strain;

[0128] Liquid strain culture: 5 uniform size fungus cakes were inoculated in the liquid medium and cultured at a temperature of 25°C and a rotation speed of 160 r / min for 6 days;

[0129] Liquid culture: The seed liquid was inoculated in the basic medium with different liquid loading amounts at an inoculation amount of 10% (v / v) and cultured at a temperature of 25°C and a rotation speed of 160 r / min for 7 days. Three groups of parallel experiments were set;

[0130] Mycelium weighing: After the end of the culture, the mycelium was obtained by suction filtration and placed in a 60°C oven to dry to constant weight. The weight of the mycelium was measured; the results are shown in Table 9.

[0131] Table 9 Influence of different liquid loading on the growth of Hainan tiger breast Ganoderma lucidum mycelium

[0132]

[0133] The experimental results show that 300 mL is the best liquid loading for liquid culture of Hainan tiger breast Ganoderma lucidum. With the increase of liquid loading, the dry weight of mycelium shows a trend of first increasing and then tending to be flat, and when the inoculum amount is 300 mL, the dry weight of mycelium reaches the maximum of 3.45 g / L.

[0134] Example 10 Orthogonal test

[0135] Table 10 Orthogonal test design table

[0136]

[0137] Liquid medium: 20.0 g of glucose, 2.0 g of peptone, 3.0 g of potassium dihydrogen phosphate, and 1.5 g of magnesium sulfate were weighed, respectively, and water was added to make up to 1000 mL. The conventional method was used for sterilization to prepare the liquid medium;

[0138] Orthogonal test medium: the medium was prepared according to the concentration of each component in the orthogonal test design table. The liquid loading in each bottle was 300 mL. After sterilization using the conventional method, 9 groups of orthogonal test medium were prepared;

[0139] Cultivation method:

[0140] Activation of Hainan tiger breast Ganoderma lucidum strain: the strain preserved at 4℃ was inoculated in PDA medium and cultured in a constant temperature incubator at 25℃ to obtain activated Hainan tiger breast Ganoderma lucidum strain;

[0141] Liquid strain culture: 5 uniform and consistent fungus cakes were taken and inoculated in liquid culture medium, and cultured at a temperature of 25℃ and a speed of 160 r / min for 6 days of shaking culture;

[0142] Liquid culture: the seed liquid was inoculated in the orthogonal test medium at an inoculum amount of 20%, and cultured at a temperature of 25℃ and a speed of 160 r / min for 7 days of shaking culture, with 3 groups in parallel;

[0143] Mycelium weighing: after the culture was completed, the mycelium was taken out, and the mycelium was obtained by suction filtration and placed in a 60℃ oven for drying to constant weight. The weight of the mycelium was weighed; the results are shown in Table 11.

[0144] Table 11 Results of orthogonal test

[0145]

[0146] The analysis of the orthogonal test results shows that the four influencing factors have the following influence degree on the growth of Hainan Tiger Breast Ganoderma lucidum from large to small: B>A>D>C, and the optimal combination of liquid culture of Hainan Tiger Breast Ganoderma lucidum is A2B1C3D1, that is, 2.5 g / L of potassium dihydrogen phosphate, 0.5 g / L of magnesium sulfate, 35 g / L of glucose, and 1.5 g / L of yeast powder.

[0147] Example 11 verification test

[0148] Liquid medium: 20.0 g of glucose, 2.0 g of proteose peptone, 3.0 g of potassium dihydrogen phosphate, and 1.5 g of magnesium sulfate were weighed, and water was added to make up to 1000 mL. The liquid medium was sterilized by using a conventional method;

[0149] Verification test medium: 35.0 g of glucose, 1.5 g of yeast powder, 2.5 g of potassium dihydrogen phosphate, and 0.5 g of magnesium sulfate were weighed, and water was added to make up to 1000 mL. The liquid volume of each bottle was 300 mL. The verification test medium was prepared after sterilization by using a conventional method;

[0150] Culture method:

[0151] Activation of Hainan Tiger Breast Ganoderma lucidum strain: the strain preserved at 4°C was inoculated in PDA medium and cultured in a constant temperature incubator at 25°C to obtain the activated strain of Hainan Tiger Breast Ganoderma lucidum;

[0152] Liquid strain culture: 5 pieces of uniform and consistent fungus cakes were inoculated in the liquid medium and cultured at a temperature of 25°C and a rotation speed of 160 r / min for 6 days;

[0153] Liquid culture: the seed liquid was inoculated in the verification test medium at an inoculation amount of 20%, and cultured at a temperature of 25°C and a rotation speed of 160 r / min for 7 days, with 3 groups in parallel;

[0154] Mycelium weighing: after the culture was completed, the mycelium was obtained by suction filtration and placed in a 60°C oven for drying until the weight was constant, and the weight of the mycelium was weighed; further, the content of polyphenol in the mycelium was detected. The content of polyphenol was determined by spectrophotometry according to the standard of T / AHFIA005-2018 “Determination of total polyphenol content in plant extracts and their products”.

[0155] Results: the dry weight of the mycelium obtained after weighing was 22.04 g / L, which was better than the results of the 9 groups in the orthogonal test. The polyphenol content detection result showed that after 7 days of liquid culture, the polyphenol content in the mycelium was 4.1914 mg / g. This example proves that the optimal combination obtained by the orthogonal test is feasible, and it is a suitable liquid culture method for Hainan Tiger Breast Ganoderma lucidum.

[0156] Example 12 Medium optimization and detection of polyphenolic active ingredients

[0157] During the exploration of the liquid medium formula, it was found that some amino acids and oils could significantly improve the content of polyphenolic active ingredients in mycelium. Some relevant research data are as follows:

[0158] Liquid medium: 20.0 g of glucose, 2.0 g of peptone, 3.0 g of potassium dihydrogen phosphate, and 1.5 g of magnesium sulfate were weighed, and water was added to make up to 1000 mL. Sterilize using the conventional method to prepare the liquid medium;

[0159] Basic medium: 35.0 g of glucose, 1.5 g of yeast powder, 2.5 g of potassium dihydrogen phosphate, and 0.5 g of magnesium sulfate were weighed, and water was added to make up to 1000 mL. The amount of liquid in each bottle is 300 mL. After sterilization using the conventional method, the basic medium is prepared;

[0160] Optimized medium: 0.5 g of plant oil + 0.5 g of amino acid replaces the same amount of glucose + yeast powder in the basic medium, and the rest of the conditions are the same as the basic medium; that is:

[0161] A1: 35.0 g of glucose, 1.5 g of yeast powder, 2.5 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 0.5 g of cottonseed oil, and 0.5 g of tyrosine

[0162] A2: 35.0 g of glucose, 1.5 g of yeast powder, 2.5 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 0.5 g of soybean oil, and 0.5 g of tyrosine

[0163] A3: 35.0 g of glucose, 1.5 g of yeast powder, 2.5 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 0.5 g of cottonseed oil, and 0.5 g of glycine

[0164] A4: 35.0 g of glucose, 1.5 g of yeast powder, 2.5 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 0.5 g of soybean oil, and 0.5 g of glycine

[0165] Culture method:

[0166] Activation of Hainan Tiger Breast Ganoderma lucidum strain: The strain preserved at 4℃ was inoculated in PDA medium and cultured in a constant temperature incubator at 25℃ to obtain the activated strain of Hainan Tiger Breast Ganoderma lucidum;

[0167] Liquid strain culture: 5 pieces of uniform size of the fungus cake were inoculated in the liquid medium and cultured at a temperature of 25℃ and a speed of 160 r / min for 6 days of shaking culture;

[0168] Liquid culture: the seed liquid was inoculated in the optimized medium at 20% (v / v) inoculation amount, and cultured at 25℃ with a rotation speed of 160 r / min, 3 groups of parallel were set, and the liquid culture was shaken for 7 days;

[0169] Mycelium weighing: after the culture was finished, the mycelium was taken out, and the mycelium was obtained by suction filtration, placed in a 60℃ oven and dried to constant weight, and the weight of the mycelium was weighed; the content of polyphenol in the mycelium was detected. The results are shown in Table 12.

[0170] Table 12 Mycelium yield and polyphenol content detection results of Hainan Tiger Breast Ganoderma lucidum

[0171]

[0172] Results: the dry weight of the mycelium obtained after weighing was 20.16~21.94g / L, the mycelium biomass remained at a high level, indicating that the plant oil and the amino acid replaced part of the glucose and the yeast powder had little effect on the yield of the mycelium, and the yield of the mycelium of Hainan Tiger Breast Ganoderma lucidum still remained at the highest level. From the detection results of the content of the phenolic active substances, it can be seen that the addition of cottonseed oil and tyrosine significantly improved the content of the polyphenolic substances, and the content of the polyphenolic substances reached a peak value of 168.617mg / g on the 7th day of culture.

[0173] The above only describes some embodiments of the present application, and is not used to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application, all belong to the protection scope of the present application.

Claims

1. A liquid culture method for increasing the biomass of Ganoderma lucidum mycelium in Hainan, characterized in that, Includes the following steps: A. The activated Hainan Tiger Milk Ganoderma lucidum strain was inoculated into Hainan Tiger Milk Ganoderma lucidum liquid culture medium and cultured to obtain the strain liquid; B. The obtained inoculum solution was inoculated into Hainan Tiger Milk Ganoderma liquid culture medium for culture to obtain Hainan Tiger Milk Ganoderma liquid culture product; The Hainan Tiger Milk Ganoderma lucidum liquid culture medium contains the following raw materials: glucose 25~35.0g / L, yeast powder 1.5~2.5g / L, potassium dihydrogen phosphate 2.0~3.0g / L, magnesium sulfate 0.5~1.5g / L, and natural pH; The Hainan Tiger Milk Ganoderma liquid culture medium also contains cottonseed oil and tyrosine.

2. The liquid culture method for increasing the biomass of Ganoderma lucidum mycelium in Hainan according to claim 1, characterized in that, The incubation time for step B is 6-8 days.

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

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

5. The liquid culture method for increasing the biomass of Ganoderma lucidum mycelium in Hainan according to claim 1, characterized in that, Step B: The inoculum volume is 20% (v / v).

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

7. The liquid culture method for increasing the biomass of Ganoderma lucidum mycelium in Hainan according to claim 1, characterized in that, The Hainan Tiger Milk Ganoderma lucidum liquid culture medium contains the following raw materials: glucose 35.0 g / L, yeast powder 1.5 g / L, potassium dihydrogen phosphate 2.5 g / L, magnesium sulfate 0.5 g / L, cottonseed oil 0.5 g / L, tyrosine 0.5 g / L, and natural pH.

8. The application of the liquid culture method according to any one of claims 1 to 7 in the production of Ganoderma lucidum polyphenols from Hainan tiger milk.

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