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

Abstract

The invention belongs to the technical field of microbial fermentation manufacturing, and discloses a ganoderma lucidum fermented turnip hypoxia-tolerant functional base material, a culture medium, a preparation method of the ganoderma lucidum fermented turnip hypoxia-tolerant functional base material and a method for producing the ganoderma lucidum fermented turnip hypoxia-tolerant functional base material through fermentation of the culture medium. The method sequentially comprises the processes of slant strain culture, liquid seed culture and fermentation culture, the fermentation culture is liquid fermentation, and the culture conditions are as follows: the inoculum size is 4-15%, the fermentation temperature is 25-35 DEG C, and the fermentation time is 3-7 days. According to the method, the base material with the hypoxia-resistant function is produced through a liquid fermentation method, the raw material turnip is utilized to a large extent to prepare the base material with the hypoxia-resistant function, related raw materials are provided for subsequent development of hypoxia-resistant health-care food, subsequent processing after fermentation is simple, the production efficiency is high, energy consumption is low, product separation is easy, the production cost is low, and the method is suitable for industrial production. Wide application prospects are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation and manufacturing, in particular to a functional base material and culture medium for fermenting turnips with ganoderma lucidum to resist hypoxia and a preparation method thereof. Background Art

[0002] Hypoxia is widely present in human life and work, especially in plateau environments, high altitudes or during strenuous exercise. When normal human tissue cells cannot obtain the oxygen necessary for metabolic activities, hypoxia will occur, referred to as hypoxia (Hypoxia, Anoxia, Oxygen deficiency). This is a series of symptoms caused by insufficient oxygen intake to supply the body's needs, resulting in a decrease in alveolar oxygen partial pressure and blood oxygen saturation, and tissue cells cannot obtain the required oxygen from the blood for normal oxidative metabolism. These symptoms generally include: rapid heartbeat, dry mouth, purple lips, dizziness, headache, and further nausea, vomiting, loss of appetite, abdominal distension, diarrhea, palpitations, shortness of breath, and even edema, as well as general fatigue, insomnia, coma, etc. Therefore, military, sports, aerospace, underwater operations, tourism and other fields are paying more and more attention to the study of hypoxia.

[0003] At present, the varieties with definite anti-altitude hypoxia effects and relatively wide application, such as Rhodiola rosea, ginseng, and salvia miltiorrhiza, are mostly limited by unclear pharmacological mechanisms or almost exhausted drug resources. Therefore, it is urgent and promising to further explore low-toxic and high-efficiency anti-altitude hypoxia foods and health products, conduct efficacy evaluation, material basis and mechanism research, and vigorously develop new raw materials with hypoxia resistance to ensure the development of ordinary foods and health foods.

[0004] There are few reports on improving the body's hypoxia tolerance through food nutrition measures, especially on preparing hypoxia-resistant functional base materials by microbial fermentation methods to develop health foods. Compared with drug methods, the preparation of hypoxia-resistant functional base materials by microbial fermentation methods has many advantages. It not only has a wide and stable source of raw materials, but also can degrade macromolecular substances such as sugars and lipids into small molecules to improve bioavailability, and can produce a variety of active substances to improve product flavor. At the same time, high-yield strains can be obtained through microbial culture screening, fermentation conditions are easy to control, product quality is guaranteed, and it is easy to large-scale industrial production. Therefore, the production of hypoxia-resistant functional base materials has the advantages of safety, wide source of raw materials, low production cost, and simple production process.

[0005] Ganoderma lucidum is also known as Lingzhi grass and Lingxiancao. Ganoderma is a famous medicinal fungus that has been widely used as a Chinese herbal medicine and functional food in my country and other Asian countries for more than 2,000 years. In recent decades, the bioactive compounds in the fruiting bodies of Ganoderma lucidum have attracted the attention of scholars at home and abroad. These active substances related to pharmacological effects include polysaccharides, triterpenes, sterols, proteins and peptides. Domestic and foreign researchers have confirmed that Ganoderma lucidum polysaccharides are the most important active substances. Ganoderma lucidum polysaccharides have multiple physiological functions, including antioxidant, antibacterial, anti-cancer, anti-aging, anti-inflammatory, heart protection and immune regulation.

[0006] Turnip (Brassica rapa L.), also known as Chamagu, genus yanjing, and manjing, belongs to the genus Brassica of the family Cruciferae. Its tubers are used as medicine, are rich in nutrients, and can be eaten for a long time. It enjoys the reputation of "the holy fruit of longevity" and is a traditional plant for medicine, food, and feed. According to Tibetan medical classics such as "Ganlu Bencao Mingjing", "Four Medical Classics", and "New Tibetan Medicine", turnip has the effects of clearing heat and detoxification, nourishing, enhancing immunity, anti-mutation, anti-radiation, anti-fatigue, delaying aging, and anti-hypoxia. Turnip is a must-have food for plateau areas and yaks entering high-altitude areas. It is also an anti-hypoxia food sold in the form of fresh products by Tibetans in some tourist attractions in Tibetan areas. Studies have reported that turnip contains a variety of physiologically active ingredients such as flavonoids, triterpenes, polysaccharides, saponins, and alkaloids. It has the effects of lowering blood lipids, lowering blood sugar, and antibacterial.

[0007] At present, there are many kinds of raw materials and products with hypoxia resistance at home and abroad. Although these have their own characteristics and priorities for hypoxia resistance and health care, their comprehensive therapeutic effects are not strong, the production process is complicated, and they are not perfect. Chinese invention patent CN111329907A "A composition of genkwa with anti-hypoxia function and its preparation method and application" uses the original juice and extract of traditional Tibetan medicinal and edible plants as the main components, and uses genkwa with bracken and sea buckthorn to obtain good anti-hypoxia effect. The hypoxia resistance time of the composition at normal pressure is 32.93±5.67min, and the hypoxia time of acute cerebral ischemia is 18.46±1.29s. It uses relatively more composite ingredients, and the materials are only preliminarily processed, so the amount of materials used is large. CN110477237A "A genus arborvitae beverage with anti-hypoxia effect and its preparation method and application" discloses an invented genus arborvitae beverage, which uses genus arborvitae, wolfberry, black barley and snow pear as main raw materials, supplemented with niacin and vitamin B 12 , fructose syrup and other auxiliary materials to produce a new genus arborvitae beverage that has the function of improving hypoxia tolerance. In recent years, the number of hypoxia tolerance products developed using genus arborvitae as raw material has gradually increased, but most of them are in the form of beverages, candies, oral liquids, etc. The utilization of raw materials and the convenience of carrying products can be improved.

[0008] In recent years, the number of products with hypoxia resistance developed from genkwa root has gradually increased, but the existing technology still has the following defects:

[0009] 1. Turnip is used in a single form, 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 turnip. Adding an appropriate amount of plant raw materials to the fermentation medium can effectively promote the growth of medicinal fungi and increase the content of functional ingredients in a relatively short period of time.

[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 it 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 directly used as a product or added as an auxiliary material 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 turnip with ganoderma lucidum to resist hypoxia and a preparation method thereof.

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

[0015] A culture medium for producing hypoxia-resistant functional base material by fermenting ganoderma lucidum, wherein 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 powder, ammonium sulfate and ammonium nitrate.

[0018] Furthermore, the formula of the culture medium is: 0.1-0.8% turnip powder, 2-5% glucose, 0.3-0.5% yeast extract powder, 0.1-0.3% MgSO 4 7H 2 O, 0.1-0.3% KH 2 PO 4 and 0.01% Vb 1 , and the rest is water; the above percentages are all mass concentration percentages.

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

[0020] The method comprises the processes of slant culture, liquid seed culture and fermentation culture in sequence, wherein the fermentation culture is liquid fermentation, and the culture conditions are: inoculation amount is 4-15%, fermentation temperature is 25-35°C, and fermentation time is 3-7 days;

[0021] The liquid seed culture medium includes 2-5% glucose, 0.3-0.5% yeast extract powder, 0.1-0.3% MgSO 4 7H 2 O, 0.1-0.3% KH 2 PO 4 and 0.01% Vb 1 , the solvent is water;

[0022] The fermentation medium includes 2-5% glucose, 0.3-0.5% yeast extract, 0.1-0.3% MgSO 4 7H 2 O, 0.1-0.3% KH 2 PO 4 , 0.01% Vb 1 and 0.1-0.8% turnip powder, the solvent is water;

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

[0024] Further, the following steps are included:

[0025] (I) Preparation of turnip powder

[0026] (1) Preparation of turnip powder: Wash and cut turnip into strips, 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, MgS0 4 7H 2 O1.0g, KH 2 PO 4 1.0g, Vitamin B 1 0.1g, agar 20.0g, water to 1L;

[0028] (3) Seed culture medium preparation: 30.0 g glucose, 5.0 g yeast extract powder, MgS0 4 7H 2 O 1.0g, KH 2 PO 4 1.0g, Vitamin B 1 0.1g, pH 6.0, dilute to 1L with water;

[0029] (4) Preparation of fermentation medium: 0.1-0.8% turnip powder, 2-5% glucose, 0.3-0.5% yeast extract powder, 0.1-0.3% MgSO 4 7H 2 O, 0.1-0.3% KH 2 PO 4 , 0.01% Vb 1 , the rest is water; the above percentages are all mass concentration percentages;

[0030] (II) 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 slope, 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 mycelium 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 Erlenmeyer 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 are inoculated into the fermentation medium at an inoculation rate of 4-15%, and cultured at 28°C and 160rpm for 3-7 days;

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

[0038] After the fermentation is terminated, the fermented material is taken out.

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

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

[0041] Use a rotary evaporator to concentrate the fermented product, control the temperature below 60°C, and terminate the concentration process when the dry matter concentration of the fermented 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 use 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, utilizes turnip as a raw material to a large extent, and makes the base material with hypoxia-resistant function, thereby providing relevant raw materials for the subsequent development of hypoxia-resistant health food. 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 invention has broad application prospects.

[0052] 2. The present invention provides a microbial fermentation technology for preparing anoxic functional base material and a production method thereof, which increases the added value of turnip raw materials and has a significant anti-oxic effect, 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 in the metabolism of microorganisms are utilized to degrade cellulose and the like in plant matrix cells to promote the dissolution of active ingredients.

[0054] 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 hypoxia-resistant functional base materials of the present invention has simple process, low requirements on production equipment, saves energy, has low cost, is easy for large-scale industrial production, 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; the liquid fermentation medium thereof comprises 2-5% glucose, 0.3-0.5% yeast extract powder, 0.1-0.3% MgSO 4 7H 2 O, 0.1-0.3% KH2 PO 4 , 0.01%Vb 1 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 normal pressure hypoxia tolerance test and sodium nitrite poisoning survival test, and it was found that compared with the blank, the normal pressure hypoxia tolerance time of the functional base material was increased by 31.19%, which was a very significant difference; and in the sodium nitrite poisoning survival experiment of mice, compared with the blank group, the survival time of the mice in the hypoxia-resistant functional base material was significantly prolonged, increased by 140.46%. The functional base material provided by the present invention can improve the tolerance of the body in an oxygen-deficient environment, 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; it increases the added value of turnip and expands its application.

[0059] 5. The present invention selects an optimized fermentation medium to increase the growth rate and product yield of microorganisms. Modern fermentation engineering technology is used to add turnip powder to the fermentation medium to promote fungal growth, significantly increase the content of ingredients with good efficacy, and promote the dissolution of active ingredients. The powder product of the hypoxia-resistant functional base material is formed by concentration, vacuum freeze drying, crushing and other operations, which is convenient for subsequent use, storage and transportation. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is a normal pressure hypoxia resistance time diagram of the hypoxia resistance functional base material in the present invention; wherein, compared with the blank control group, * P<0.05, ** P < 0.01;

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

[0062] Figure 3 This is a biomass determination diagram 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 in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

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

[0066] A culture medium for producing hypoxia-resistant functional base material by fermenting ganoderma lucidum, wherein 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 powder, ammonium sulfate and ammonium nitrate.

[0069] Preferably, the formula of the culture medium is: 0.1-0.8% turnip powder, 2-5% glucose, 0.3-0.5% yeast extract powder, 0.1-0.3% MgSO 4 7H 2 O, 0.1-0.3% KH 2 PO 4 and 0.01% Vb 1 , and the rest is water; the above percentages are all mass concentration percentages.

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

[0071] The method comprises the processes of slant culture, liquid seed culture and fermentation culture in sequence, wherein the fermentation culture is liquid fermentation, and the culture conditions are: inoculation amount is 4-15%, fermentation temperature is 25-35°C, and fermentation time is 3-7 days;

[0072] The liquid seed culture medium includes 2-5% glucose, 0.3-0.5% yeast extract powder, 0.1-0.3% MgSO 4 7H 2 O, 0.1-0.3% KH 2 PO 4 and 0.01% Vb 1 , the solvent is water;

[0073] The fermentation medium includes 2-5% glucose, 0.3-0.5% yeast extract, 0.1-0.3% MgSO 4 7H 2 O, 0.1-0.3% KH 2 PO 4 , 0.01% Vb 1and 0.1-0.8% turnip powder, the solvent is water;

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

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

[0076] (I) Preparation of turnip powder

[0077] (1) Preparation of turnip powder: Wash and cut turnip into strips, 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, MgS0 4 7H 2 O1.0g, KH 2 PO 4 1.0g, Vitamin B 1 0.1g, agar 20.0g, water to 1L;

[0079] (3) Seed culture medium preparation: 30.0 g glucose, 5.0 g yeast extract powder, MgS0 4 7H 2 O 1.0g, KH 2 PO 4 1.0g, Vitamin B 1 0.1g, pH 6.0, dilute to 1L with water;

[0080] (4) Preparation of fermentation medium: 0.1-0.8% turnip powder, 2-5% glucose, 0.3-0.5% yeast extract powder, 0.1-0.3% MgSO 4 7H 2 O, 0.1-0.3% KH 2 PO 4 , 0.01% Vb 1 , the rest is water; the above percentages are all mass concentration percentages;

[0081] (II) 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 slope, 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 mycelium 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 Erlenmeyer 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 are inoculated into the fermentation medium at an inoculation rate of 4-15%, and cultured at 28°C and 160rpm for 3-7 days;

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

[0089] After the fermentation is terminated, the fermented material is taken out.

[0090] Preferably, the method further comprises the step of further processing the fermented 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 fermented product, control the temperature below 60°C, and terminate the concentration process when the dry matter concentration of the fermented 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 use 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 turnip purchased from Xinjiang was washed, shredded, and dried. The turnip was 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 culture medium: 200.0 g potato, 20.0 g glucose, MgS0 4 7H 2 O 1.0g, KH 2 PO 4 1.0g, Vitamin B 1 0.1g, agar 20.0g, water to 1L;

[0107] (3) Seed culture medium preparation: 30.0 g glucose, 5.0 g yeast extract powder, MgS0 4 7H 2 O 1.0g, KH 2 PO 4 1.0g, Vitamin B 1 0.1g, pH 6.0, dilute to 1L with water;

[0108] (4) Preparation of fermentation medium: 0.1-0.8% turnip powder, 2-5% glucose, 0.3-0.5% yeast extract powder, 0.1-0.3% MgSO 4 7H 2 O, 0.1-0.3% KH 2 PO 4 , 0.01% Vb 1 , and the rest 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 mycelium 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 (liquid volume was 40% of the volume of the Erlenmeyer flask) containing fermentation medium 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 fermented product was concentrated by a rotary evaporator, the temperature was controlled below 60°C, and the concentration process was terminated when the dry matter concentration of the fermented liquid reached below 40%.

[0120] ① Concentration

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

[0122] ② Drying

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

[0124] ③ Crushing

[0125] The dried product is crushed and sieved to obtain a powdery product, and an oxygen-deficiency-resistant functional base material is prepared, which is used for animal experiments.

[0126] Example 2: Animal Experiment Methods

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

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

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

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

[0131] The positive control group (Dazhu Hongjingtian capsule) was intragastrically administered 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 intragastrically administered with 1 g / kg body weight daily;

[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 intragastrically administered with 1 g / kg body weight per day.

[0134] Each mouse was fed 0.2 mL of sample each time, once a day. The mice were fed by gavage with a syringe for 21 days. One hour after the last feeding, the mice were tested for their ability to tolerate hypoxia. The mice were placed in a wide-mouthed bottle pre-filled with 5 g of soda lime, the bottle mouth was sealed with vaseline, and the lid was tightly covered to prevent air leakage. The time was immediately counted, and respiratory arrest was used as an indicator to observe the time when the mice died due to hypoxia.

[0135] The results of the mouse normobaric hypoxia tolerance experiment 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 hypoxia tolerance time of each experimental group under normal pressure was higher than that of the blank group during the experiment. The hypoxia tolerance time of the turnip raw material group under normal pressure was 29.06±1.03min, an increase of 9.87%, but there was no significant difference, and there was no statistical significance; the hypoxia tolerance time of the hypoxia-resistant functional base material under normal pressure was 34.7±3.07min, an increase of 31.19%, which was significantly different from the blank (P<0.01), indicating that the fermented material had significant hypoxia tolerance; at the same time, the turnip powder raw material and the hypoxia-resistant functional base material after fermentation were compared at the same intragastric dose, and the hypoxia-resistant functional base material prepared after fermentation had a significant improvement in hypoxia tolerance.

[0139] 2.2. Materials for the sodium nitrite poisoning survival experiment

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

[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 ultrapure water 0.2 mL / mouse per day;

[0143] The positive control group (Dazhu Hongjingtian capsule) was intragastrically administered 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 intragastrically administered with 1 g / kg body weight per day;

[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 carried out.

[0146] Each mouse was fed 0.2 mL of sample each time, once a day. The mice were fed by gavage with syringes for 21 days. Each dose group was continuously given different concentrations of the test sample orally, and the control group was given the same volume of solvent. One hour after the last gavage, each group of animals was intraperitoneally injected with sodium nitrite at a dose of 200-240 mg / kg BW, and the time was immediately counted to record the survival time of the animals.

[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 each experimental group poisoned by sodium nitrite was prolonged compared with the blank group, which was 45.76±0.25min, an increase of 140.46%, which was significantly different from the blank group (P<0.01), indicating that the fermented product had a significant hypoxia tolerance function. To summarize the above experimental results, the results of the hypoxia tolerance functional base in the normal pressure hypoxia tolerance test and the sodium nitrite poisoning survival test were positive, indicating that the functional base had hypoxia tolerance.

[0151] Example 3

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

[0153] 3.1 Biomass determination

[0154] Take 5 mL of the fermentation product obtained by shaking a 250 mL Erlenmeyer flask in step (3) of Example 1 in preparing anoxic functional substrate by liquid culture, and separate the mycelium and the fermentation liquid by suction filtration. Rinse the mycelium with distilled water three times until it is clean. Dry the mycelium in a 60°C thermostat and weigh it to calculate the dry weight of the mycelium by 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 until the mark, then transfer it to a brown bottle, mix and shake well, store it in a dark place and set aside (prepare it for immediate use).

[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. Pipette 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*V 1 *n) / V 2 ;

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

[0162] V 1 :OD 490 The corresponding volume of standard glucose solution (mL);

[0163] V 2 : Sample solution volume (mL);

[0164] n: sample dilution multiple;

[0165] 0.9: Correction factor.

[0166] Take the fermentation product obtained by culturing in a 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 4 As shown, after adding turnip powder to the culture medium, the polysaccharide content of the fermentation liquid was greatly increased 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. The other culture steps were the same to obtain the Ganoderma lucidum fermentation product, and the biomass and polysaccharide content were determined according to the steps of Example 3. Figure 3 and Figure 4 As shown, when turnip powder is not added to the fermentation medium, the polysaccharide content of the fermentation liquid is 1.45±0.05 g / L, the biomass is 6.54±0.37 g / L, the mycelium growth state is slow, and the biomass is not as high as that when turnip powder is added to the fermentation medium.

[0169] Comparing Examples 3 and 4, the fermented product obtained by adding turnip to the culture medium and fermenting with Ganoderma lucidum has higher biomass and polysaccharide yield in the fermentation broth than the fermented product without addition, and the polysaccharide yield increases from 1.45±0.05g / L to 2.05±0.07g / L, and the biomass increases from 6.54±0.37g / L to 7.91±0.23g / L. The growth of Ganoderma lucidum is not inhibited after adding turnip powder to the culture medium. Turnip can ensure the normal growth of Ganoderma lucidum, and it is also beneficial to increase the amount of fermented product bacteria and the secretion of secondary metabolites polysaccharides, so it can be used as a high-quality plant nutrient matrix for Ganoderma lucidum liquid fermentation system.

[0170] Products with hypoxia tolerance developed in the prior art use animal experiments or hypoxia tolerance population experiments as indicators to determine hypoxia tolerance. The present invention uses two animal experiments: normal pressure hypoxia tolerance experiment and sodium nitrite poisoning survival experiment as indicators to determine that the product of the present invention has hypoxia tolerance. The experimental steps are simplified, and complex operations are reduced, making the experimental process more efficient; the experimental results are easy to observe and analyze, reducing dependence on professional equipment. In addition, biotechnology is used to simplify the extraction and purification steps of hypoxia tolerance products of general composite compositions through fermentation operations, thereby improving the utilization rate of effective ingredients of plant raw materials and saving resources.

[0171] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate 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 hypoxia-resistant functional base material by fermentation of Ganoderma lucidum, characterized in that: The culture medium uses turnip root powder as a main raw material.

2. The culture medium according to claim 1, characterized in that: The culture medium also includes a carbon source, which is one or a combination of sucrose, maltose, glucose and fructose.

3. The culture medium according to claim 2, characterized in that: The culture medium also includes a nitrogen source, which is one or a combination of peptone, yeast extract powder, ammonium sulfate and ammonium nitrate.

4. The culture medium according to claim 2 or 3, characterized in that: The formula of the culture medium is: 0.1-0.8% turnip powder, 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 rest is water; the above percentages are all mass concentration percentages.

5. A method for producing anoxic-resistant functional base material by fermentation using the culture medium according to any one of claims 1 to 4, characterized in that: The method comprises the steps of fermenting the culture medium with Ganoderma lucidum to obtain an oxygen-deficiency-resistant functional base material; The method comprises the processes of slant culture, liquid seed culture and fermentation culture in sequence, wherein the fermentation culture is liquid fermentation, and the culture conditions are: inoculation amount is 4-15%, fermentation temperature is 25-35°C, and fermentation time is 3-7 days; 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; The fermentation medium includes 2-5% glucose, 0.3-0.5% yeast extract, 0.1-0.3% MgSO47H2O, 0.1-0.3% KH2PO4, 0.01% Vb1 and 0.1-0.8% turnip powder, the solvent is water; The above percentages are all mass concentration percentages.

6. The method according to claim 5, characterized in that: The steps include: (I) Preparation of turnip powder (1) Preparation of turnip powder: Wash and cut turnip into shreds, 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 culture medium: 200.0 g of potato, 20.0 g of glucose, 1.0 g of MgS04·7H2O, 1.0 g of KH2PO4, 0.1 g of vitamin B1, 20.0 g of agar, and dilute to 1 L with water; (3) Preparation of seed culture medium: 30.0 g glucose, 5.0 g yeast extract powder, 1.0 g MgS04·7H2O, 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% MgSO47H2O, 0.1-0.3% KH2PO4, 0.01% Vb1, and the rest is water; the above percentages are all mass concentration percentages; (II) The production process of 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 the 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 mycelium 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 Erlenmeyer flask, and culture at 28°C and 160 rpm to obtain liquid seeds; (3) 250mL Erlenmeyer flask shake culture The cultured liquid seeds are inoculated into the fermentation medium at an inoculation rate of 4-15%, and cultured at 28°C and 160rpm for 3-7 days; (4) Obtaining hypoxia-resistant functional base material After the fermentation is terminated, the fermented material is taken out.

7. The method according to claim 5 or 6, characterized in that: The method further comprises the step of further processing the fermented product to form a powder product.

8. The method according to claim 7, characterized in that: The steps of further processing the fermented product to form a powder product are as follows: Use a rotary evaporator to concentrate the fermented product, control the temperature below 60°C, and terminate the concentration process when the dry matter concentration of the fermented liquid reaches below 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.

9. The hypoxia-resistant functional base material obtained by fermentation production according to any one of claims 5 to 8.

10. Use of the hypoxia-resistant functional base material according to claim 9 in the preparation of hypoxia-resistant products.

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