Ganoderma lucidum fermented turnip hypoxia-resistant functional base material, culture medium and preparation method thereof

Abstract

The invention belongs to the field of microbial fermentation manufacturing technology, discloses a kind of Ganoderma lucidum fermented turnip hypoxia-resistant functional base material, culture medium and preparation method thereof, utilize culture medium fermentation to produce the method for hypoxia-resistant functional base material, including using Ganoderma lucidum fermented culture medium, obtaining the step of hypoxia-resistant functional base material;The process of slant strain culture, liquid seed culture and fermentation culture is successively included, and the fermentation culture is liquid fermentation, and culture conditions are: inoculum size is 4 15%, fermentation temperature is 25 35 DEG C, and fermentation time is 3 7 days. The present invention produces hypoxia-resistant functional base material by liquid fermentation method, utilizes raw material turnip to a large extent, makes hypoxia-resistant functional base material, provides relevant raw materials for subsequent research and development with hypoxia-resistant health food, and the subsequent processing of fermentation termination is simple, production efficiency is high, energy consumption is low, product separation is easier, production cost is low, and has broad application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation and manufacturing, in particular to a ganoderma lucidum fermented turnip hypoxia-resistant functional base material, a culture medium and a preparation method thereof. Background Art

[0002] Hypoxia is a widespread condition in human life and work, particularly in high altitude environments, at high altitudes, or during strenuous exercise. Hypoxia (also known as hypoxia, anoxia, or oxygen deficiency) occurs when normal human tissue cells lack the oxygen they need for metabolic activity. This condition is caused by insufficient oxygen intake, resulting in a decrease in alveolar oxygen partial pressure and blood oxygen saturation. This leads to a series of symptoms, resulting in a lack of oxygen from the blood for normal oxidative metabolism. These symptoms typically include: rapid heartbeat, dry mouth, bluish lips, dizziness, and headaches. These symptoms may further develop, including nausea, vomiting, loss of appetite, abdominal distension, diarrhea, palpitations, shortness of breath, and even edema, leading to general fatigue, insomnia, and coma. Consequently, research on hypoxia is gaining increasing attention in various fields, including military, sports, aerospace, underwater operations, and tourism.

[0003] Currently, proven anti-altitude hypoxia treatments, such as Rhodiola rosea, ginseng, and salvia miltiorrhiza, are relatively widely used, but are often limited by unclear pharmacological mechanisms or near-exhaustion of drug resources. Therefore, further exploring low-toxicity, high-efficiency anti-altitude hypoxia foods and health supplements, conducting efficacy evaluation, research on their material basis, and mechanisms of action, and vigorously developing new hypoxia-resistant raw materials to support the development of both conventional and health-related foods is both urgent and promising.

[0004] Few studies have reported on improving the body's hypoxia tolerance through dietary nutrition, particularly using microbial fermentation to prepare hypoxia-tolerant functional base materials for the development of health foods. Compared to pharmaceutical approaches, microbial fermentation offers numerous advantages. These include a broad and stable source of raw materials, the ability to degrade macromolecules such as carbohydrates and lipids into smaller molecules, improving bioavailability, and the production of a variety of active substances, enhancing product flavor. Furthermore, high-yield strains can be obtained through microbial culture and screening, and fermentation conditions are easily controlled, ensuring product quality and facilitating large-scale industrial production. Therefore, the production of hypoxia-tolerant functional base materials offers advantages such as safety, a broad source of raw materials, low production costs, and a simple process.

[0005] Ganoderma lucidum, also known as Lingzhicao and Lingxiancao, is a renowned medicinal fungus that has been widely used in my country and other Asian countries as a traditional Chinese medicine and functional food for over 2,000 years. In recent decades, the bioactive compounds in Ganoderma fruiting bodies have garnered considerable attention from scholars both domestically and internationally. These pharmacologically relevant active substances include polysaccharides, triterpenes, sterols, proteins, and peptides. Researchers both domestically and internationally have confirmed that Ganoderma polysaccharides are the primary active substance, exhibiting a variety of physiological functions, including antioxidant, antibacterial, anticancer, anti-aging, anti-inflammatory, cardioprotective, and immune-regulating properties.

[0006] Turnip (Brassica rapa L.), also known as Chamagu, radish root, and turnip, belongs to the genus Brassica in the family Cruciferae. Its tuberous roots are used as medicine, are nutritious, and can be eaten for a long time. It is known as the "holy fruit of longevity" and is a traditional plant used as medicine, food, and feed. Tibetan medical texts such as "Ganlu Bencao Mingjing," "Four Medical Classics," and "New Tibetan Medicine" record that turnip has the benefits of clearing heat and detoxifying, nourishing, enhancing immunity, resisting mutations, protecting against radiation, combating fatigue, delaying aging, and combating hypoxia. Turnip is an essential food for plateau regions and for yaks entering high-altitude areas. It is also sold fresh as a hypoxia-fighting food by Tibetans at some tourist attractions in Tibet. Studies have reported that turnip contains a variety of physiologically active ingredients, including flavonoids, triterpenes, polysaccharides, saponins, and alkaloids. It has the potential to lower blood lipids, lower blood sugar, and have antibacterial properties.

[0007] Currently, there are many types of raw materials and products with hypoxia resistance effects both at home and abroad. Although these have their own characteristics and priorities for hypoxia resistance and health care, their comprehensive therapeutic effects are weak, the production process is complicated, and they are not yet perfect. Chinese invention patent CN111329907A, "A composition of Daphne dahurica with anti-hypoxia function, its preparation method and application", uses the juice and extract of traditional Tibetan medicinal and edible plants as the main components, and achieves good anti-hypoxia effects by combining Daphne dahurica with Potentilla anserine and Seabuckthorn. The hypoxia resistance time of the composition at normal pressure is 32.93±5.67min, and the hypoxia time for acute cerebral ischemia is 18.46±1.29s. It uses relatively many composite ingredients, and the materials are only preliminarily processed, so the material consumption is large. CN110477237A "A genus arborvitae beverage with anti-hypoxia effect and its preparation method and application" discloses an invention of genus arborvitae beverage, which uses genus arborvitae, wolfberry, black barley and snow pear as main raw materials, supplemented with niacin and vitamin B 12 A new genus arborvitae beverage was prepared with auxiliary materials such as fructose syrup, which has the function of improving hypoxia tolerance. In recent years, the number of hypoxia-resistant products developed with genus arborvitae has gradually increased, but most of them are in the form of beverages, candies, oral liquids, etc. The utilization rate of raw materials and the convenience of carrying products can be improved.

[0008] In recent years, the development of hypoxia-resistant products using genkwa root has gradually increased, but the existing technology still has the following defects:

[0009] 1. Turnip is used in a single form, and is mostly made into products by compounding medicinal and edible plants, drugs or compounds, and the preparation form is too simple.

[0010] 2. It is rare to use fungal fermentation technology to prepare hypoxia-resistant functional products containing turnips. Adding an appropriate amount of plant raw materials to the fermentation medium can effectively promote the growth of medicinal fungi in a relatively short period of time and promote the increase of the content of functional ingredients.

[0011] 3. The preparation process is complicated and the portability can be improved.

[0012] The preparation process of the present invention is simple and clear, and can be obtained by fermentation, concentration, drying and crushing. The prepared hypoxia-resistant functional base material is easy to carry and transport, and has various forms of use. It can be used directly as a product or as an auxiliary material added to other products. Summary of the Invention

[0013] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a functional base material and culture medium for fermenting turnips with Ganoderma lucidum to tolerate hypoxia and a preparation method thereof.

[0014] The technical solution adopted by the present invention to solve its technical problem is:

[0015] The invention discloses a culture medium for producing an oxygen-deficiency-resistant functional base material by fermenting ganoderma lucidum. The culture medium uses powder of turnip roots as a main raw material.

[0016] Furthermore, the culture medium also includes a carbon source, which is one or a combination of sucrose, maltose, glucose, and fructose.

[0017] Furthermore, the culture medium also includes a nitrogen source, which is one or a combination of peptone, yeast extract, ammonium sulfate and ammonium nitrate.

[0018] Furthermore, the culture medium comprises: 0.1-0.8% turnip powder, 2-5% glucose, 0.3-0.5% yeast extract, 0.1-0.3% MgSO47H2O, 0.1-0.3% KH2PO4 and 0.01% Vb1, with the remainder being water; the above percentages are all mass concentration percentages.

[0019] A method for producing an oxygen-resistant functional base material by fermenting the culture medium as described above, comprising the steps of fermenting the culture medium with Ganoderma lucidum to obtain the oxygen-resistant functional base material;

[0020] The process includes slant culture, liquid seed culture and fermentation culture in sequence. The fermentation culture is liquid fermentation. The culture conditions are: inoculation amount of 4-15%, fermentation temperature of 25-35°C, and fermentation time of 3-7 days.

[0021] The liquid seed culture medium comprises 2-5% glucose, 0.3-0.5% yeast extract powder, 0.1-0.3% MgSO47H2O, 0.1-0.3% KH2PO4 and 0.01% Vb1, and the solvent is water;

[0022] The fermentation medium comprises 2-5% glucose, 0.3-0.5% yeast extract powder, 0.1-0.3% MgSO47H2O, 0.1-0.3% KH2PO4, 0.01% Vb1 and 0.1-0.8% turnip powder, and the solvent is water;

[0023] The above percentages are all mass concentration percentages.

[0024] Further, the following steps are included:

[0025] (1) Preparation of turnip powder

[0026] (1) Preparation of turnip powder: Wash and shred turnips, dry them, crush them in a wall-breaking machine, pass them through an 80-mesh sieve, and take them out for later use;

[0027] (2) Preparation of plate culture medium: PDA culture medium: 200.0 g of potato, 20.0 g of glucose, 1.0 g of MgSO4·7H2O, 1.0 g of KH2PO4, 0.1 g of vitamin B1, 20.0 g of agar, and dilute to 1 L with water;

[0028] (3) Seed culture medium preparation: 30.0 g glucose, 5.0 g yeast extract powder, 1.0 g MgSO4·7H2O, 1.0 g KH2PO4, 0.1 g vitamin B1, pH 6.0, dilute to 1 L with water;

[0029] (4) Fermentation medium preparation: 0.1-0.8% turnip powder, 2-5% glucose, 0.3-0.5% yeast extract, 0.1-0.3% MgSO47H2O, 0.1-0.3% KH2PO4, 0.01% Vb1, and the remainder water; the above percentages are all mass concentration percentages;

[0030] (2) The production process of preparing hypoxia-resistant functional base material by liquid culture method may specifically include the following steps:

[0031] (1) Plate culture

[0032] Pick out the mycelial block from the slant, transfer the mycelial block with culture medium to the middle of the plate culture medium, and culture at 28℃ for 6 days;

[0033] (2) Liquid seed culture

[0034] Use a 1 cm diameter puncher to take out the mycelial block from the plate cultured in step (1) and inoculate it into a 250 mL Erlenmeyer flask filled with seed culture medium, with the liquid volume being 40% of the volume of the flask, and culture at 28°C and 160 rpm to obtain liquid seeds;

[0035] (3) 250mL Erlenmeyer flask shake culture

[0036] The cultured liquid seeds were inoculated into the fermentation medium at an inoculum rate of 4-15%, and cultured at 28°C and 160 rpm for 3-7 days;

[0037] (4) Obtaining hypoxia-resistant functional base material

[0038] After terminating the fermentation, taking out the fermented material is obtained.

[0039] Furthermore, the method also includes the step of further processing the fermentation product to form a powder product.

[0040] Furthermore, the fermented product is further processed to form a powder product, as follows:

[0041] Use a rotary evaporator to concentrate the fermentation product, control the temperature below 60°C, and terminate the concentration process when the dry matter concentration of the fermentation liquid reaches below 40%;

[0042] ① Concentration

[0043] Use a rotary evaporator to concentrate the fermentation product and control the temperature below 60°C;

[0044] ② Drying

[0045] Use vacuum freeze drying technology to remove water from the fermentation;

[0046] ③ Crushing

[0047] The dried product is crushed and sieved to obtain a powdery product to prepare an oxygen deficiency resistant functional base material.

[0048] The hypoxia-resistant functional base material obtained by fermentation production according to the above method has the fermentation products of fermentation liquid and solids.

[0049] The application of the hypoxia-resistant functional base material as described above in the preparation of hypoxia-resistant products.

[0050] The advantages and positive effects achieved by the present invention are:

[0051] 1. The present invention produces a base material with hypoxia-resistant function by a liquid fermentation method, and largely utilizes the raw material turnip to prepare the hypoxia-resistant functional base material, providing relevant raw materials for the subsequent development of hypoxia-resistant health foods. The subsequent processing after the fermentation is simple, the production efficiency is high, the energy consumption is low, the product separation is relatively easy, the production cost is low, and the application prospect is broad.

[0052] 2. The present invention provides a microbial fermentation technology for preparing an oxygen-deficient functional base material and a production method thereof, which increases the added value of turnip raw materials and has significant oxygen-deficient efficacy, as follows:

[0053] The present invention utilizes Ganoderma lucidum to ferment turnip to promote the precipitation of functional active ingredients and efficiently extract nutrients. Enzymes produced during microbial metabolism are used to degrade cellulose and other substances in plant matrix cells, promoting the dissolution of active ingredients.

[0054] To enhance the biotransformation ability, adding turnip to the fermentation medium can not only significantly promote the growth of Ganoderma lucidum, but also increase its biomass and polysaccharide production, thereby enhancing the biotransformation effect.

[0055] At the same time, compared with the turnip raw material, the hypoxia resistance effect is significantly improved after being treated by the method of the present invention.

[0056] The culture medium for fermentation has simple ingredients, a wide range of raw material sources, low cost and is easy to prepare.

[0057] 3. The method for producing the hypoxia-resistant functional base material of the present invention has a simple process, low requirements on production equipment, saves energy, is low in cost, is easy to be industrialized on a large scale, and has broad industrial application prospects.

[0058] 4. The main raw materials of the culture medium provided by the present invention include turnip root powder; its liquid fermentation medium is composed of 2-5% glucose, 0.3-0.5% yeast extract powder, 0.1-0.3% MgSO47H2O, 0.1-0.3% KH2PO4, 0.01% Vb1 and 0.1-0.8% turnip powder, and the fermentation method can be liquid fermentation. The functional base material prepared after fermentation was subjected to a normal pressure hypoxia resistance test and a sodium nitrite poisoning survival test. It was found that compared with the blank, the normal pressure hypoxia resistance time of the functional base material was increased by 31.19%, which was a very significant difference; and in the sodium nitrite poisoning survival test of mice, the survival time of mice with the hypoxia-resistant functional base material was significantly prolonged compared with the blank group, increasing by 140.46%. The functional base material provided by the present invention can improve the body's tolerance to hypoxia, which is beneficial to alleviate a series of hypoxia symptoms such as altitude sickness. The hypoxia-resistant functional base material of the present invention has the characteristics of simple preparation process and low cost, improves the added value of turnip, and expands its application.

[0059] 5. The present invention selects an optimized fermentation medium to increase microbial growth rate and product yield. Leveraging modern fermentation engineering technology, turnip powder is added to the fermentation medium to promote fungal growth, significantly increase the content of highly effective ingredients, and promote the dissolution of active ingredients. Concentration, vacuum freeze-drying, and pulverization are then used to create a powder product with an oxygen-deficiency-resistant functional base, making it easier to use, store, and transport. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is a graph showing the hypoxia-resistant time of the hypoxia-resistant functional base material under normal pressure; compared with the blank control group, * P<0.05, ** P < 0.01;

[0061] Figure 2 This is a graph showing the survival time of sodium nitrite poisoning in the hypoxia-resistant functional base material of the present invention; compared with the blank control group, * P<0.05, ** P < 0.01;

[0062] Figure 3 This is a diagram of biomass determination in the present invention;

[0063] Figure 4 This is a diagram for determining the polysaccharide content in the present invention. DETAILED DESCRIPTION

[0064] The present invention will be further described below with reference to the following examples. The following examples are descriptive rather than restrictive, and the scope of protection of the present invention cannot be limited by the following examples.

[0065] The various experimental operations involved in the specific embodiments are all routine techniques in the field. For parts not specifically annotated in this document, ordinary technicians in this field can refer to various commonly used reference books, scientific literature or related instructions, manuals, etc. before the filing date of this invention to implement them.

[0066] The invention discloses a culture medium for producing an oxygen-deficiency-resistant functional base material by fermenting ganoderma lucidum. The culture medium uses powder of turnip roots as a main raw material.

[0067] Preferably, the culture medium further comprises a carbon source, which is one or a combination of sucrose, maltose, glucose and fructose.

[0068] Preferably, the culture medium further comprises a nitrogen source, which is one or a combination of peptone, yeast extract, ammonium sulfate and ammonium nitrate.

[0069] Preferably, the culture medium comprises: 0.1-0.8% turnip powder, 2-5% glucose, 0.3-0.5% yeast extract, 0.1-0.3% MgSO47H2O, 0.1-0.3% KH2PO4 and 0.01% Vb1, with the remainder being water; the above percentages are all mass concentration percentages.

[0070] A method for producing an oxygen-resistant functional base material by fermenting the culture medium as described above, comprising the steps of fermenting the culture medium with Ganoderma lucidum to obtain the oxygen-resistant functional base material;

[0071] The process includes slant culture, liquid seed culture and fermentation culture in sequence. The fermentation culture is liquid fermentation. The culture conditions are: inoculation amount of 4-15%, fermentation temperature of 25-35°C, and fermentation time of 3-7 days.

[0072] The liquid seed culture medium comprises 2-5% glucose, 0.3-0.5% yeast extract powder, 0.1-0.3% MgSO47H2O, 0.1-0.3% KH2PO4 and 0.01% Vb1, and the solvent is water;

[0073] The fermentation medium comprises 2-5% glucose, 0.3-0.5% yeast extract powder, 0.1-0.3% MgSO47H2O, 0.1-0.3% KH2PO4, 0.01% Vb1 and 0.1-0.8% turnip powder, and the solvent is water;

[0074] The above percentages are all mass concentration percentages.

[0075] Preferably, the method comprises the following steps:

[0076] (1) Preparation of turnip powder

[0077] (1) Preparation of turnip powder: Wash and shred turnips, dry them, crush them in a wall-breaking machine, pass them through an 80-mesh sieve, and take them out for later use;

[0078] (2) Preparation of plate culture medium: PDA culture medium: 200.0 g of potato, 20.0 g of glucose, 1.0 g of MgSO4·7H2O, 1.0 g of KH2PO4, 0.1 g of vitamin B1, 20.0 g of agar, and dilute to 1 L with water;

[0079] (3) Seed culture medium preparation: 30.0 g glucose, 5.0 g yeast extract powder, 1.0 g MgSO4·7H2O, 1.0 g KH2PO4, 0.1 g vitamin B1, pH 6.0, dilute to 1 L with water;

[0080] (4) Fermentation medium preparation: 0.1-0.8% turnip powder, 2-5% glucose, 0.3-0.5% yeast extract, 0.1-0.3% MgSO47H2O, 0.1-0.3% KH2PO4, 0.01% Vb1, and the remainder water; the above percentages are all mass concentration percentages;

[0081] (2) The production process of preparing hypoxia-resistant functional base material by liquid culture method may specifically include the following steps:

[0082] (1) Plate culture

[0083] Pick out the mycelial block from the slant, transfer the mycelial block with culture medium to the middle of the plate culture medium, and culture at 28℃ for 6 days;

[0084] (2) Liquid seed culture

[0085] Use a 1 cm diameter puncher to take out the mycelial block from the plate cultured in step (1) and inoculate it into a 250 mL Erlenmeyer flask filled with seed culture medium, with the liquid volume being 40% of the volume of the flask, and culture at 28°C and 160 rpm to obtain liquid seeds;

[0086] (3) 250mL Erlenmeyer flask shake culture

[0087] The cultured liquid seeds were inoculated into the fermentation medium at an inoculum rate of 4-15%, and cultured at 28°C and 160 rpm for 3-7 days;

[0088] (4) Obtaining hypoxia-resistant functional base material

[0089] After terminating the fermentation, taking out the fermented material is obtained.

[0090] Preferably, the method further comprises the step of further processing the fermentation product to form a powder product.

[0091] Preferably, the step of further processing the fermented product to form a powder product is as follows:

[0092] Use a rotary evaporator to concentrate the fermentation product, control the temperature below 60°C, and terminate the concentration process when the dry matter concentration of the fermentation liquid reaches below 40%;

[0093] ① Concentration

[0094] Use a rotary evaporator to concentrate the fermentation product and control the temperature below 60°C;

[0095] ② Drying

[0096] Use vacuum freeze drying technology to remove water from the fermentation;

[0097] ③ Crushing

[0098] The dried product is crushed and sieved to obtain a powdery product to prepare an oxygen deficiency resistant functional base material.

[0099] The hypoxia-resistant functional base material obtained by fermentation production according to the above method has the fermentation products of fermentation liquid and solids.

[0100] The application of the hypoxia-resistant functional base material as described above in the preparation of hypoxia-resistant products.

[0101] Specifically, the relevant preparation and detection are as follows:

[0102] Example 1

[0103] Preparation of hypoxia-resistant functional base material:

[0104] 1. Preparation of Turnip Powder

[0105] (1) Preparation of turnip powder: 3 kg of turnips were purchased from Xinjiang, washed, shredded, and dried, crushed in a wall-breaking machine, passed through an 80-mesh sieve, and taken out for later use.

[0106] (2) Preparation of plate culture medium: PDA medium: 200.0 g potato, 20.0 g glucose, 1.0 g MgSO4·7H2O, 1.0 g KH2PO4, 0.1 g vitamin B1, 20.0 g agar, and dilute to 1 L with water;

[0107] (3) Seed culture medium preparation: glucose 30.0 g, yeast extract 5.0 g, MgSO4·7H2O 1.0 g, KH2PO4 1.0 g, vitamin B1 0.1 g, pH 6.0, dilute to 1 L with water;

[0108] (4) Preparation of fermentation medium: 0.1-0.8% turnip powder, 2-5% glucose, 0.3-0.5% yeast extract, 0.1-0.3% MgSO47H2O, 0.1-0.3% KH2PO4, 0.01% Vb1, and the remainder is water; the above percentages are all mass concentration percentages.

[0109] 2. The production process of preparing hypoxia-resistant functional base material by liquid culture method may specifically include the following steps:

[0110] (1) Plate culture

[0111] The mycelial mass of Ganoderma lucidum was picked out from the slant, and the mycelial mass with the culture medium was transferred to the middle of the plate culture medium in Example 1 and cultured at 28° C. for 6 days.

[0112] (2) Liquid seed culture

[0113] Use a 1 cm diameter puncher to take out the mycelial block from the plate cultured in step (1) and inoculate it into a 250 mL Erlenmeyer flask containing seed culture medium (the liquid volume is 40% of the volume of the Erlenmeyer flask), and culture it at 28°C and 160 rpm to obtain liquid seeds.

[0114] (3) 250mL Erlenmeyer flask shake culture

[0115] The cultured liquid seeds were inoculated into 250 mL Erlenmeyer flasks filled with fermentation medium (the liquid volume was 40% of the volume of the flask) at an inoculum rate of 4-15% (volume ratio), and cultured at 28° C. and 160 rpm for 3-7 days.

[0116] (4) Obtaining hypoxia-resistant functional base material

[0117] After the fermentation is terminated, the fermented material is taken out.

[0118] (5) Post-treatment of hypoxia-resistant functional base materials

[0119] The fermentation product was concentrated by rotary evaporation, and the temperature was controlled below 60°C. The concentration process was terminated when the dry matter concentration of the fermentation liquid reached below 40%.

[0120] ① Concentration

[0121] The fermentation product was concentrated using a rotary evaporator, and the temperature was controlled below 60°C.

[0122] ② Drying

[0123] The water content of the fermented product was removed using vacuum freeze drying technology.

[0124] ③ Crushing

[0125] The dried product was crushed and sieved to obtain a powdered product, which was used to prepare an oxygen deficiency-resistant functional base material, which was then used for animal experiments.

[0126] Example 2: Animal Experiment Methods

[0127] 2.1. Materials for atmospheric pressure hypoxia resistance experiments

[0128] 250 mL ground-mouth jar, syringe (the needle is oblate to avoid injuring the animal during gavage), stopwatch, vaseline, soda lime (or an equal mass of sodium hydroxide and calcium carbonate, hygroscopic); male mice (CD-1, Henan Sikebes Biotechnology Co., Ltd.), weighing 20 ± 2 g, culture temperature 20 ± 5 °C.

[0129] 48 CD-1 mice were randomly divided into 4 groups, with 12 mice in each group. The drug administration method was as follows:

[0130] The blank control group was gavaged with 0.2 mL of ultrapure water per mouse daily;

[0131] The positive control group (Dazhu Hongjingtian capsule) was gavaged with 0.78 / Kg body weight daily;

[0132] The turnip powder group (the powder refers to the turnip powder prepared in step 1 of Example 1) was gavaged daily at 1 g / kg body weight;

[0133] The hypoxia-resistant functional base material of the present invention (the base material refers to the base material finally prepared in Example 1) was administered orally at a dose of 1 g / kg body weight per day.

[0134] Each mouse was fed 0.2 mL of sample once daily. The mice were gavage-fed for 21 days. One hour after the last feeding, the mice were tested for hypoxia tolerance. The mice were placed in a wide-mouthed bottle pre-filled with 5 g of soda lime. The bottle was sealed with vaseline and airtight. The time until death from hypoxia occurred was measured, using respiratory arrest as an indicator.

[0135] The results of the normobaric hypoxia tolerance experiment in mice are as follows:

[0136] Table 1

[0137] Group n(number) Survival time (min) blank 12 <![CDATA[26.45±0.49 b ]]> Positive 12 <![CDATA[35.12±2.28 a ]]> turnip 12 <![CDATA[29.06±1.03 b ]]> Hypoxia-resistant functional base material 12 <![CDATA[34.7±3.07 a ]]>

[0138] Figure 1 The results showed that the survival time of hypoxic mice in each experimental group increased compared to the blank group. The hypoxia tolerance time of the turnip raw material group was 29.06±1.03 minutes, a 9.87% increase, but the difference was not statistically significant. The hypoxia tolerance time of the hypoxia-resistant functional base was 34.7±3.07 minutes, a 31.19% increase compared to the blank group (P<0.01), indicating that this fermented product has significant hypoxia tolerance. Furthermore, when comparing the turnip powder raw material and the fermented hypoxia-resistant functional base at the same oral dose, the hypoxia-resistant functional base prepared after fermentation showed a significantly improved hypoxia tolerance effect.

[0139] 2.2. Materials for the Sodium Nitrite Poisoning Survival Experiment

[0140] Sodium nitrite, stopwatch, 1 mL syringe; male mice (CD-1, Henan Sikebes Biotechnology Co., Ltd.), weighing 20 ± 2 g, culture temperature 20 ± 5 °C.

[0141] Thirty-six CD-1 mice were randomly divided into three groups, with 12 mice in each group. The drug administration method was as follows:

[0142] The blank control group was gavaged with 0.2 mL of ultrapure water per mouse daily;

[0143] The positive control group (Dazhu Hongjingtian capsule) was gavaged with 0.78 / Kg body weight daily;

[0144] The hypoxia-resistant functional base material of the present invention (the base material refers to the one finally prepared in Example 1) was administered orally at a dose of 1 g / kg body weight daily;

[0145] Since the screening of base materials with hypoxia resistance function selected two tests (sodium nitrite poisoning survival test and normal pressure hypoxia resistance test) as the judgment criteria, the turnip powder group did not test positive in the normal pressure hypoxia resistance test, so the sodium nitrite poisoning test was not performed.

[0146] Each mouse was fed 0.2 mL of sample once daily by gavage for 21 days. Each dose group was continuously orally administered with varying concentrations of the test sample, while the control group received the same volume of solvent. One hour after the last gavage, each group received an intraperitoneal injection of sodium nitrite at a dose of 200-240 mg / kg body weight. Immediately, a timer was placed, and the survival time of the animals was recorded.

[0147] The results of the mouse sodium nitrite poisoning survival experiment are shown in Table 2.

[0148] Table 2

[0149] Group n(number) Survival time (min) blank 12 <![CDATA[19.03±1.13 c ]]> Positive 12 <![CDATA[23.2±0.9 b ]]> Hypoxia-resistant functional base material 12 <![CDATA[45.76±1.26 a ]]>

[0150] Figure 2 The results showed that the survival time of mice in the hypoxia-tolerant functional base was significantly prolonged compared to the blank group, reaching 45.76±0.25 minutes, an increase of 140.46%. This difference was highly significant (P<0.01), indicating that the fermented product has a highly significant hypoxia-tolerant function. In summary, the hypoxia-tolerant functional base tested positive in both the atmospheric hypoxia-tolerant test and the sodium nitrite poisoning survival test, indicating that the functional base has hypoxia-tolerant function.

[0151] Example 3

[0152] Detection of active substance content in hypoxia-resistant functional base materials:

[0153] 3.1 Biomass determination

[0154] 5 mL of the fermentation product obtained from the 250 mL Erlenmeyer flask shake culturing in step (3) of Example 1, prepared by the liquid culture method for preparing anoxic-tolerant functional substrate, was collected and filtered to separate the mycelium from the fermentation broth. The mycelium was rinsed three times with distilled water until clean. The mycelium was dried in a 60°C incubator and weighed to calculate the dry weight of the mycelium using the differential method.

[0155] 3.2 Determination of polysaccharide content

[0156] Preparation of phenol solution: Weigh 6 g of phenol, dissolve it in distilled water, shake well and transfer it completely to a 100 mL volumetric flask, add distilled water to the scale, then transfer it to a brown bottle, mix and shake well, store it in a dark place and use it later (prepare it immediately).

[0157] Accurately weigh 0.15g of glucose standard, dissolve it in distilled water, shake well and transfer it completely to a 1000mL volumetric flask, add distilled water to the scale to make a 0.15g / L glucose standard solution. Respectively draw 0, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, and 0.20mL of 150mg / L glucose standard solution in a 10mL test tube for precise measurement, add 0.20mL of distilled water, and use 0.40mL of distilled water as a blank control. Then add 0.20mL of 6% phenol reagent and 1mL of concentrated sulfuric acid to each test tube, mix well, put it in a fume hood, cool to room temperature, and measure the absorbance at 490nm. The horizontal axis is the glucose concentration, the vertical axis is the absorbance value, and the standard curve is drawn to obtain the regression equation y=2.87891x+0.01052(R 2 =0.99824)

[0158] Determination of polysaccharide content in samples:

[0159] Take 0.20 mL of the sample to be tested into a test tube, measure the absorbance according to the above operation, and calculate the polysaccharide content according to the formula.

[0160] Polysaccharide content (g / L) = (0.9C*5*V1*n) / V2;

[0161] C: concentration of standard glucose solution (mg / L);

[0162] V1:OD 490 The corresponding volume of standard glucose solution (mL);

[0163] V2: volume of sample solution (mL);

[0164] n: sample dilution multiple;

[0165] 0.9: Correction factor.

[0166] Take the fermentation product obtained by shaking the 250 mL Erlenmeyer flask in step (3) of Example 1 in the preparation of hypoxia-resistant functional base by liquid culture method, centrifuge and take the supernatant, and detect the absorbance value at a wavelength of 490 nm according to the method in the standard curve. Substitute the absorbance value into the standard curve to obtain the polysaccharide content in the fermentation broth. Figure 3 and Figure 4As shown, after adding turnip powder to the culture medium, the polysaccharide content of the fermentation liquid increased significantly to 2.05±0.07g / L, the bacteria grew vigorously, the growth of Ganoderma lucidum mycelium increased significantly, and the biomass was 7.91±0.23g / L.

[0167] Example 4

[0168] On the basis of Example 1, the additives in the fermentation medium were changed so that turnip powder was not added to the fermentation medium. Other culture steps were the same to obtain Ganoderma lucidum fermentation product. The biomass and polysaccharide content were determined according to the steps of Example 3. Figure 3 and Figure 4 As shown, when turnip powder was not added to the fermentation medium, the polysaccharide content of the fermentation liquid was 1.45±0.05 g / L, the biomass was 6.54±0.37 g / L, the mycelial growth was slow, and the biomass was not as high as that when turnip powder was added to the fermentation medium.

[0169] Comparing Examples 3 and 4, the fermentation product prepared by adding turnip powder to the culture medium and fermenting with Ganoderma lucidum had higher biomass and polysaccharide yields in the fermentation broth than the fermentation product without turnip powder. Polysaccharide yield increased from 1.45±0.05 g / L to 2.05±0.07 g / L, and biomass increased from 6.54±0.37 g / L to 7.91±0.23 g / L. Adding turnip powder to the culture medium did not inhibit the growth of Ganoderma lucidum. Turnip powder not only ensured the normal growth of Ganoderma lucidum, but also helped increase the fermentation product mass and the secretion of polysaccharides, a secondary metabolite. Therefore, it can be used as a high-quality plant nutrient substrate for Ganoderma lucidum liquid fermentation systems.

[0170] Prior art products with hypoxia tolerance were evaluated based on animal or human hypoxia tolerance tests. The present invention uses two animal tests: a normal pressure hypoxia tolerance test and a sodium nitrite poisoning survival test. This simplified experimental procedure reduces complex operations, making the process more efficient. The results are easy to observe and analyze, reducing reliance on specialized equipment. Furthermore, the use of biotechnology through fermentation simplifies the extraction and purification steps of conventional composite hypoxia tolerance products, improving the utilization rate of the active ingredients in the plant material and conserving resources.

[0171] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A culture medium for producing an oxygen-deficient functional base material by fermenting Ganoderma lucidum, characterized in that: The culture medium uses turnip root powder as a main raw material; The culture medium has a formula of: 0.1-0.8% turnip powder, 2-5% glucose, 0.3-0.5% yeast extract powder, 0.1-0.3% MgSO4·7H2O, 0.1-0.3% KH2PO4 and 0.01% Vb1, with the remainder being water; the above percentages are all mass concentration percentages.

2. A method for producing an oxygen-deficient functional base material by fermentation using the culture medium according to claim 1, characterized in that: The method comprises the steps of fermenting the culture medium with Ganoderma lucidum to obtain an oxygen-deficient functional base material; The process includes slant culture, liquid seed culture and fermentation culture in sequence. The fermentation culture is liquid fermentation, and the culture conditions are: inoculation amount of 4-15%, fermentation temperature of 25-35°C, and fermentation time of 3-7 days; The liquid seed culture medium is: 2-5% glucose, 0.3-0.5% yeast extract powder, 0.1-0.3% MgSO4·7H2O, 0.1-0.3% KH2PO4 and 0.01% Vb1, and the solvent is water; The fermentation medium is: 2-5% glucose, 0.3-0.5% yeast extract powder, 0.1-0.3% MgSO4·7H2O, 0.1-0.3% KH2PO4, 0.01% Vb1 and 0.1-0.8% turnip powder, and the solvent is water; The above percentages are all mass concentration percentages.

3. The method according to claim 2, wherein: The steps include: (1) Preparation of turnip powder (1) Preparation of turnip powder: Wash and shred turnips, dry them, crush them in a wall-breaking machine, pass them through an 80-mesh sieve, and take them out for later use; (2) Preparation of plate culture medium: PDA medium: 200.0 g of potato, 20.0 g of glucose, 1.0 g of MgSO4·7H2O, 1.0 g of KH2PO4, 0.1 g of vitamin B1, 20.0 g of agar, and dilute to 1 L with water; (3) Seed culture medium preparation: 30.0 g glucose, 5.0 g yeast extract powder, 1.0 g MgSO4·7H2O, 1.0 g KH2PO4, 0.1 g vitamin B1, pH 6.0, dilute to 1 L with water; (4) Preparation of fermentation medium: 0.1-0.8% turnip powder, 2-5% glucose, 0.3-0.5% yeast extract, 0.1-0.3% MgSO4·7H2O, 0.1-0.3% KH2PO4, 0.01% Vb1, and the rest is water; the above percentages are all mass concentration percentages; (2) The production process for preparing hypoxia-resistant functional base material by liquid culture method may specifically include the following steps: (1) Plate culture Pick out the mycelial block from the slant, transfer the mycelial block with culture medium to the middle of the plate culture medium, and culture at 28℃ for 6 days; (2) Liquid seed culture Use a 1 cm diameter puncher to take out the mycelial block from the plate cultured in step (1) and inoculate it into a 250 mL Erlenmeyer flask filled with seed culture medium, with the liquid volume being 40% of the volume of the flask, and culture at 28°C and 160 rpm to obtain liquid seeds; (3) 250mL Erlenmeyer flask shake culture The cultured liquid seeds were inoculated into the fermentation medium at an inoculum rate of 4-15%, and cultured at 28°C and 160 rpm for 3-7 days; (4) Obtaining hypoxia-resistant functional base materials After terminating the fermentation, taking out the fermented material is obtained.

4. The method according to claim 2 or 3, characterized in that: The method further comprises the step of further processing the fermented product to form a powder product.

5. The method according to claim 4, characterized in that: The steps for further processing the fermented product to form a powder product are as follows: Use a rotary evaporator to concentrate the fermentation product, control the temperature below 60°C, and terminate the concentration process when the dry matter concentration of the fermentation liquid reaches more than 40%; ① Concentration Use a rotary evaporator to concentrate the fermentation product and control the temperature below 60°C; ② Drying Use vacuum freeze drying technology to remove water from the fermentation; ③ Crushing The dried product is crushed and sieved to obtain a powdery product to prepare an oxygen deficiency resistant functional base material.

6. The hypoxia-resistant functional base material obtained by fermentation production according to the method of any one of claims 2 to 4.

7. Use of the hypoxia-resistant functional base material according to claim 6 in the preparation of hypoxia-resistant products.

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