阅读说明：本技术 一种调节细胞因子、增强免疫功能的复合菌剂及其应用 (Complex microbial inoculant for regulating cell factors and enhancing immune function and application thereof ) 是由 焦顺昌 张文东 吴志勇 魏东 于 2020-08-17 设计创作，主要内容包括：本发明公开了一种调节细胞因子、增强免疫功能的复合菌剂,该复合菌剂包括一株丛毛红曲霉(Monascus pilosus)YL-1以及必要的辅剂,所述复合菌剂的使用方法：(1)丛毛红曲霉(Monascus pilosus)YL-1的种子液制备：(2)用上述复合菌剂进行发酵的方法。所述步骤(2)如下：(1)配置专用培养基,步骤如下：3％葡萄糖,2％木糖,0.5％菊粉,2％鲜沙棘果浆、2％刺梨果浆、1％覆盆子果浆、1％铁皮石斛果浆等；(2)好氧发酵：(3)获得上清液。(The invention discloses a compound microbial inoculum for regulating cell factors and enhancing immune function, which comprises Monascus pilosus (Monascus pilosus) YL-1 and necessary auxiliary agents, and a using method of the compound microbial inoculum comprises the following steps: (1) preparation of Monascus pilosus (Monascus pilosus) YL-1 seed solution: (2) a method for fermenting by using the composite microbial inoculum. The step (2) is as follows: (1) preparing a special culture medium, comprising the following steps: 3% of glucose, 2% of xylose, 0.5% of inulin, 2% of fresh sea-buckthorn fruit pulp, 2% of roxburgh rose fruit pulp, 1% of raspberry fruit pulp, 1% of dendrobium officinale fruit pulp and the like; (2) aerobic fermentation: (3) the supernatant was obtained.)

1. A compound bacterial agent for regulating cell factors, enhancing immunity and treating viral diseases is characterized in that:

comprises Monascus pilosus (Monascus pilosus) YL-1 and necessary auxiliary agents.

2. The use method of the complex microbial inoculum of claim 1 is characterized by comprising the following steps:

(1) preparation of Monascus pilosus (Monascus pilosus) YL-1 seed solution:

(2) a method for fermenting by using the composite microbial inoculum.

3. The method for using the complex microbial inoculum of claim 1, which is characterized in that:

the step (1) is as follows: weighing preserved Monascus pilosus (Monascus pilosus) YL-1, inoculating into solid culture medium, culturing for 24 hr, selecting single colony with good growth in plate, inoculating into liquid culture medium, activating, culturing for 24 hr, and quantifying to 5 × 10⁷(CFU/ml) Monascus pilosus seed liquid,liquid culture conditions: at 30 ℃, 200 r/min;

the solid culture medium comprises the following components in percentage by weight: 1% Yeast Extract, 2% Peptone (Peptone), 2% dextrose (glucose), 2% agar powder, and the balance water.

Liquid culture medium, according to the weight ratio: 1% Yeast Extract, 2% Peptone (Peptone), 2% dextrose (glucose), and the balance water.

4. Use according to claim 2 or 3, characterized in that:

the step (2) is as follows:

(1) preparing a special culture medium, comprising the following steps: according to the weight ratio, 3% of glucose, 2% of xylose, 0.5% of inulin, 2% of fresh sea buckthorn fruit pulp, 2% of roxburgh rose fruit pulp, 1% of raspberry fruit pulp, 1% of dendrobium officinale fruit pulp, 1% of peptone, 0.05% of magnesium sulfate heptahydrate, 3% of ground calcium carbonate, a proper amount of tween 80 and the balance of water; heating to 95 deg.C with steam, sterilizing for 30s, sealing, and cooling to room temperature;

(2) aerobic fermentation:

inoculating activated Monascus pilosus (Monascus pilosus) YL-1 seed liquid according to the inoculation amount of 2% (v/v), and performing aerobic fermentation for 36h at the fermentation temperature of 30 ℃ and the rotation speed of 150 r/min;

(3) heating to 95 deg.C with steam, sterilizing for 1min, centrifuging at 1000r/min for 10min to obtain supernatant.

5. A fermentation supernatant obtainable by the process of any one of claims 2 to 4.

Technical Field

The invention relates to a compound microbial inoculum for regulating cell factors, enhancing immunologic function and treating viral diseases and application thereof, belonging to the technical field of microbial treatment.

Background

Enhancing immunity refers to enhancing autoimmunity by some means. Immunity is the body's own defense mechanism, and is the body's ability to recognize and destroy any foreign body (virus, bacteria, etc.) that invades from the outside, to treat aged, damaged, dead, denatured self cells, and to recognize and treat mutant cells and virus-infected cells in the body. Immunity refers to the ability of the body to resist external attack and maintain the stability of the environment in the body. The air is filled with various microorganisms: bacteria, viruses, mycoplasma, chlamydia, fungi, and the like. In the case of insufficient immunity, they can be pathogens of early shifts. Although the human body can produce corresponding antibodies to different pathogens to prevent the re-infection, the antibodies have specificity and time limitation, for example, streptococcus antibodies can only protect the body from the re-invasion of streptococcus in a short period of time and cannot prevent the infection of other viruses. The fact that a person with low immunity cannot resist cold virus attacks at all is the true reason for his frequent colds.

There are two types of immunization, cellular and humoral. In cellular immunity, probiotics activate macrophages, which engulf and kill many pathogenic microorganisms while inducing them to release two weapons- -tumor necrosis factor (TNF- α) and interleukin 6 (IL-6). Tumor necrosis factor, as its name implies, is an active factor that has a killing effect on cancer cells, causing bleeding and necrosis of tumors. Interleukins are also important immunologically active factors, which participate in inflammatory reactions and regulate immune functions of the body. Both the extracellular products of the probiotic bacteria and the cell wall peptidoglycan have certain immunological activities. Intact cells (or intact peptidoglycans) can exert immunoadjuvant activity. However, it has been found that macrophage activation can be achieved by the disrupted bacterial cell fluid and even by the genomic DNA of Bifidobacterium. This is why ordinary yoghurt without live bacteria also has a certain immune function. Probiotics can also activate T cells and NK cells (Natural Killer cells), both of which can act as Cell killers, blocking invading cells. Still other studies have shown that certain probiotics promote splenocyte proliferation, increase spleen weight, and enhance immune function. And other cytokines such as interleukin-1, interleukin-10 (IL-1, IL-10) or interferon.

The invention aims to develop a compound microbial inoculum for regulating cell factors and enhancing immune function and application thereof, and further optimizes a culture medium through the synergistic action of a specific microbial inoculum so as to generate a culture with the function of improving immunity.

Disclosure of Invention

The invention aims to develop a compound microbial inoculum for regulating cell factors and enhancing immune function and application thereof, and further optimizes a culture medium through the synergistic action of a specific microbial inoculum so as to generate a culture with the function of improving immunity. The complex microbial inoculum and the fermentation culture thereof are beneficial to the auxiliary treatment and prevention of viral diseases, and can also be directly applied to food fermentation.

Yanli et al, brewed in China, vol.39, No. 1 of 2020, screening and identification of high esterifying ability strains in Tokyo Daqu, disclose a Monascus pilosus (Monascus pilosus) YL-1 strain, which has a known function of high yield of esters and can improve the aroma and flavor of Daqu. The strain is obtained from conventional food, is safe, and can be used for food fermentation. The applicant promises to release to the public within 20 years.

In research and development, the applicant finds that the fermentation liquor of Monascus pilosus does not have the function of remarkably improving the immunity in general, but the fermentation liquor of Monascus pilosus YL-1 has the function of remarkably improving the immunity.

The research and development team further optimizes the fermentation medium of the strain so as to further improve the efficacy of enhancing immunity. The main screening direction of the project is to screen from traditional health care plants, so that the safety is ensured, the fermentation of strains can be stimulated, the fermentation residues can be directly eaten, and the health care effect can be improved.

Through screening, various natural herbaceous plant fruits have the effect of improving the efficacy of the bacterial strain, but the effect is not very obvious, but 4 fruits can greatly improve the health-care effect of the bacterial strain when combined and compounded for use, and have obvious synergistic effects, namely: fructus Hippophae, fructus Rosae Normalis, Rubi fructus, and herba Dendrobii.

The sea-buckthorn fruit is rich in nutrition, and active substances such as various vitamins, fatty acids, trace elements, linolein, sea-buckthorn flavone, superoxide and the like and various amino acids required by a human body are determined to be contained in the sea-buckthorn fruit. Wherein the content of vitamin C is extremely high, and the content of vitamin C in per 100 g of fruit juice can reach 825-1100 mg, which is 2-3 times of that of kiwi fruit, and the vitamin C is the beauty of the king of vitamin C. 7.5 to 10 percent of sugar and 3 to 5 percent of acid. The sea-buckthorn leaf contains crude protein 15.75%, crude fat 9.48%, crude fiber 14.04% and nitrogen-free extract 54.84%, and the sea-buckthorn leaf can be used for preparing health tea. The seabuckthorn oil contains 206 active substances beneficial to human bodies, wherein 46 bioactive substances comprise a large amount of vitamin E, vitamin A, flavone and the like, has special pharmacological properties of resisting fatigue, enhancing organism vitality, resisting cancer and the like, has the effects of protecting and accelerating the repair of gastric mucosa, increasing the drug property of intestinal bifidobacteria, reducing plasma cholesterol and reducing the content of cholesterol in blood vessel walls, can prevent and treat hyperlipidemia and atherosclerosis, and has the effect of promoting wound healing.

The roxburgh rose mainly contains the following nutrient substances, including: carbohydrate: the total amount of the hydrates in the fruit of Rosa roxburghii Tratt is about 13%, and the main ingredients are fiber, sucrose, glucose, fructose, etc. Wherein the content of cellulose is 4-7%, the content of sucrose is 1.4-3.5%, the content of glucose is 0.1-0.9%, and the content of fructose is 0.6-1.2%; protein and amino acid: protein 0.6-0.7%; the amino acids include 18 kinds of amino acids such as aspartic acid, threonine, valine, methionine, phenylalanine, lysine, histidine and arginine. 8 of them are essential amino acids for human body, 2 are semi-essential amino acids; ③ vitamins and tannins: mainly comprises vitamin C, vitamin P, vitamin B group (vitamin B1, B2, etc.), vitamin E, folic acid, carotene, etc., and tannin, wherein the content of vitamin C is high. The detection of professor Lorentz and Song garden chrysanthemum of nutriologists shows that every 100 g of roxburgh rose pulp contains vitamin C2075-2725 mg, which is 800 times of apple, 400 times of banana, 100 times of tangerine, 22 times of tomato and 10 times of kiwi fruit, and is called as vitamin C King, and the coronas of all fruits and vegetables in the male parent; fourthly, trace elements: the main spring comprises iron (Fe), zinc (Zn), strontium (Sr), selenium (Se), rubidium (Rb), manganese (Mn), copper (Cu), cobalt (Co), nickel (Ni), fluorine (F), aluminum (A1), vanadium (v), silicon (Si) and the like. Wherein Fe, Zn, Se, Mn, Cu, Co, Ni, F, V and Si are essential trace elements for human body; enzyme: mainly including superoxide dismutase (SOD), peroxidase, etc.

The raspberry fruits are edible and are cultivated in Europe for a long time, and most of the cultivars are used as fruits. The raspberry fruit contains abundant nutrient elements such as vitamin A, vitamin C, calcium, potassium, magnesium and the like and a large amount of fiber. Per 100 g Rubi fructus, water content is 87%, protein content is 0.9 g, fiber content is 4.7 g, and heat of 209.3 kilojoules can be provided. The raspberry can effectively relieve cardiovascular diseases such as angina pectoris and the like, but slight diarrhea can be caused sometimes. The raspberry fruit is sour, sweet and delicious, and has the reputation of 'gold fruit'. Raspberry is rich in salicylic acid, phenolic acid, etc. Salicylic acid is called natural aspirin, is widely used for easing pain, relieving heat, resisting blood coagulation and effectively preventing thrombus. If the raspberry is eaten for a long time, the heart can be effectively protected, and cardiovascular and cerebrovascular diseases such as hypertension, vascular wall atherosclerosis, cardiovascular and cerebrovascular embrittlement and rupture and the like can be prevented.

Through modern scientific research, the dendrobium polysaccharide and other effective components in the dendrobium officinale can increase the activity of T cells and B cells in vivo, so that the body immunity is improved. Meanwhile, the dendrobium officinale has good effects of stabilizing blood sugar, reducing blood fat, relieving insomnia, promoting gastrointestinal health, relieving constipation and improving eyesight. Especially young women often eat dendrobium officinale, can effectively improve the annual symptoms, and has obvious effects of maintaining beauty and keeping young and delaying senility. The data are cited in dendrobe-related literature in CNKI (China journal network). Among the Jiudaoxiancao, Dendrobium officinale is the only herb of immortal grass that doesn't stick to the soil. Wild dendrobium officinale grows from stone cracks of cliff cliffs or on ancient trees, and the growth of the dendrobium officinale stops once the dendrobium officinale dips in the ground gas. It is a kind of Mesona chinensis which can absorb sunlight, rain and dew, but does not contain earth gas. Therefore, Xiancao has a figurative meaning. 03. Not all dendrobium stems are dendrobium officinale. The dendrobium candidum is one of more than seventy dendrobium in China with the best drug effect, and the rich polysaccharide and dendrobine are multiple times of those of other dendrobium.

The invention discovers for the first time that the fermentation liquor of Monascus pilosus (Monascus pilosus) YL-1 has the function of improving immunity, and also discovers for the first time that the combined use of the seabuckthorn fruit, the roxburgh rose fruit, the raspberry and the dendrobium officinale can greatly and obviously improve the immunity.

The technical scheme of the invention is as follows:

a compound bacterial agent for regulating cell factors and enhancing immune function is characterized in that:

comprises Monascus pilosus (Monascus pilosus) YL-1 and necessary auxiliary agents.

Preparation of Monascus pilosus (Monascus pilosus) YL-1 seed solution:

weighing preserved Monascus pilosus (Monascus pilosus) YL-1, inoculating into solid culture medium, culturing for 24 hr, selecting single colony with good growth in plate, inoculating into liquid culture medium, activating, culturing for 24 hr, and quantifying to 5 × 10⁷(CFU/ml) Monascus pilosus seed fluid. The culture conditions are as follows: 30 ℃ and 200 r/min.

Solid medium: according to the weight ratio, 1% Yeast Extract (Yeast Extract), 2% Peptone (Peptone), 2% dextrose (glucose), 2% agar powder, and the balance water.

Liquid culture medium: by weight, 1% Yeast Extract (Yeast Extract), 2% Peptone (Peptone), 2% dextrose (glucose), and the balance water.

The method for fermenting by using the composite microbial inoculum comprises the following steps:

(1) preparing a special culture medium, comprising the following steps: according to the weight ratio, 3% of glucose, 2% of xylose, 0.5% of inulin, 2% of fresh sea buckthorn fruit pulp, 2% of roxburgh rose fruit pulp, 1% of raspberry fruit pulp, 1% of dendrobium officinale fruit pulp, 1% of peptone, 0.05% of magnesium sulfate heptahydrate, 3% of ground calcium carbonate, a proper amount of tween 80 and the balance of water; heating to 95 deg.C with steam, sterilizing for 30s, sealing, and cooling to room temperature;

(2) aerobic fermentation:

inoculating activated Monascus pilosus (Monascus pilosus) YL-1 seed liquid according to the inoculation amount of 2%, and performing aerobic fermentation for 36h at the fermentation temperature of 30 ℃ and the rotation speed of 150 r/min.

(3) Heating to 95 deg.C with steam, sterilizing for 1min, centrifuging at 1000r/min for 10min to obtain supernatant.

The invention has the advantages that:

the invention discovers for the first time that the fermentation liquor of Monascus pilosus (Monascus pilosus) YL-1 has the function of improving immunity, and also discovers for the first time that the combined use of the seabuckthorn fruit, the roxburgh rose fruit, the raspberry and the dendrobium officinale can greatly and obviously improve the immunity.

Detailed Description

The following examples of the present invention are described in detail, and are only for the purpose of illustrating the present invention and are not to be construed as limiting the present invention.

Specific examples of the present invention are described below.

The inoculation amount of the invention is v/v.