CROSS-REFERENCE TO RELATED APPLICATION

This application is a 371 of international application of PCT application serial no. PCT/CN2017/102122, filed on Sep. 18, 2017, which claims the priority benefit of China application no. 201710571073.X, filed on Jul. 13, 2017. The entirety of each of the above mentioned patent applications is hereby incorporated by reference herein and made a part of this specification.

BACKGROUND

Technical Field

The disclosure relates to a microbial fermentation composition and use thereof, in particular to an anti-cancer enzymatic composition subjected to microbial fermentation and transformation and use thereof.

Description of Related Art

Due to the stress from life and work as well as lack of exercise, nowadays people are in a sub-healthy state with low immunity, feel drowsy and get colds easily, and often experience gastrointestinal dysfunction. As the body is in the state of low immunity for a long time, the risk of cancer will increase.

Ginseng(Panax ginsengC.A. Meyer) is the root of perennial herb belonging to the genusPanaxof the family Araliaceae, and is known as “king of herbs” as a traditional and precious Chinese medicine.Ginsengis widely used in nourishing health and medical treatment in China, Korea, Japan and even Asian countries. Modern pharmacological studies show thatginsenghas pharmacological activities in the treatment and prevention of, for example, immunomodulation, anti-cancer, anti-ischemia, diabetes, atherosclerosis and anti-inflammatory.

Medicinal fungi refer to a class of fungi that can treat diseases and have medicinal value, that is, their mycelium, fruit bodies, sclerotia, or spores can produce amino acids, proteins, vitamins, polysaccharides, glycosides, alkaloids, and other substances, so they are fungi that can improve health or have the function of preventing, inhibiting or treating diseases. Some of these fungi not only can improve health but also can be eaten, and they are edible fungi as medicine and food. China has abundant resources for medicinal and edible fungi. According to rough estimation, about 2,500 types of such fungi have been recorded worldwide, and nearly 1,000 of them have been reported in China. Studies on the antitumor activity of edible and medicinal fungi mainly focus on the study of polysaccharides. Polysaccharides, proteins, nucleic acids, and lipids account for the four major components of life. In the past, people did not know much about the activity of polysaccharides, and thought that polysaccharides were only nutrients. After the 1960s, it was discovered that polysaccharides have multiple effects: it can control cell division, regulate cell growth and aging, can be used as an immune promoter, and has a therapeutic effect on tumors, cardiovascular diseases and hepatitis, so it has gained more attention since then. Polysaccharides has antitumor activity mainly because of the dextran with the main chain containing β-(1-3) or β-(1-4) and the side chain of β-(1-6). Studies show that the anti-tumor effect of dual-use fungal polysaccharides is performed through host mediating, which can activate T lymphocytes and improve organism's immune function. For example, Yunzhi polysaccharide can activate B cells, lentinan can restore T lymphocytes with decreased immune function, and Schizophyllan polysaccharide uses host reticuloendothelial cells to improve the host's non-specific defense function. These polysaccharides work on organisms with abnormal immune function. In vitro tests show that polysaccharides have no direct cytotoxic effect on the cells themselves.

SUMMARY

In order to solve the problems of low body immunity and reduced collective function, the present disclosure provides a microbial fermentation composition that can improve immunity and has anti-cancer effect.

The microbial fermentation composition of the present disclosure is prepared by selecting from the medicinal and edible fungi, includingAgaricus blazei, Lentinus edodes, Flammulina velutipesandginsengthat can significantly increase energy, after being subjected to extraction and enzymolysis, trace elements such as selenium, zinc and molybdenum are added, and fermentation and transformation are carried out by using probiotics.

The weight g/volume mL percentage of the medicinal and edible fungi is: 1-3% ofAgaricus blazei,2-4% ofLentinus edodes,1-4% ofFlammulina velutipes; the weight g/volume mL percentage of theginsengis 1-3%; the amount of the trace elements is: sodium selenite in an amount of 30-200 μg/100 mL, zinc sulfate heptahydrate 20-85 mg/100 mL, sodium molybdate dihydrate 50-150 μg/100 mL.

The probiotics areBifidobacteriumandLactobacillus.

The preparation method of the microbial fermentation composition is as follows:

A. Extraction of fermentation substrate: Crush the driedginseng, Agaricus blazei, Lentinus edodes, Flammulina velutipes, sieve them through a 65-80 mesh sieve, add drinking water that is 30 times the material weight and soak it in the water for 1 hour, heat and boil for 1.5 hours, and reduce the temperature to 90° C. Adjust and maintain the pH value between 5.5-7.0, add high temperature amylase, add an amount of 20 enzyme activity units×material weight/enzyme activity, maintain the temperature between 80-90° C., and stir the mixture evenly. End the amylase enzymolysis when the material solution and iodine solution no longer turn blue, reduce the temperature of the material solution to 50° C., adjust the pH of the material solution to 5.0-6.0, add pectin lyase in an amount of 0.1% volume of the material solution by volume/volume ratio, maintain the temperature at 45-50° C., carry out enzymolysis for 2 hours, end the enzymolysis and boil for 10 minutes. Inactivate the enzyme, filter, and take the supernatant, concentrate under reduced pressure until the concentration ofAgaricus blazei, Lentinus edodes, Flammulina velutipesandginsengreach the concentration to obtain fermentation substrate extract.

B. Ingredients and sterilization: add sodium selenite, zinc sulfate, sodium molybdate to the fermentation substrate extract, add 0.5% of glucose, 100 mg/100 mL of potassium dihydrogen phosphate, dipotassium hydrogen phosphate trihydrate 100 mg/100 mL, adjust pH value of the material solution, after being autoclaved, a fermentation substrate for subsequent microbial fermentation is obtained.

C. Inoculation and fermentation: Aseptically inoculate two cultures of pre-culturedBifidobacteriumgenus into the sterile fermentation substrate obtained in step B, facultative anaerobic culture for 2 to 6 hours at 38±1° C., re-inoculate at least one culture of pre-culturedLactobacillusand continue fermentation. When the pH of the fermentation broth drops to 4.1, reduce the temperature to 25° C. and maintained for another 24 hours, and end the fermentation.

D. Inactivation: Inactivate the fermentation broth that is completely fermented at 70° C. or 115° C. to obtain the microbial fermentation composition.

In the present disclosure, preferably, the weight g/volume mL percentage of the medicinal and edible fungi is: 2% ofAgaricus blazei,2% ofLentinus edodes, and 1% ofFlammulina velutipes; the weight g/volume mL percentage of theginsengis 2%.

In the present disclosure, preferably, the trace elements and the amount of the same are: sodium selenite in an amount of 70 μg/100 mL, zinc sulfate heptahydrate 50 mg/100 mL, sodium molybdate dihydrate 70 μg/100 mL.

Preferably, in step A of the preparation method, the process parameters in the enzymolysis process are: adjusting the pH to 6.0, adding a high-temperature amylase, maintaining the temperature at 85° C., and stirring uniformly; adjusting the pH of the material solution to 6.0, adding pectin lyase, maintaining the temperature at 45° C., hydrolyzing for 2 hours and ending the enzymolysis.

Preferably, in step B of the preparation method, the pH of the adjusted material solution is 7.0±0.1, and the autoclaving is performed at a temperature of 121° C. for 30 minutes.

Preferably, theBifidobacteriumgenus described in step C of the preparation method refers toBifidobacterium bifidus, Bifidobacterium breve, Bifidobacterium adolescentis, andBifidobacterium infantis; the inoculation volume ratio of each strain is 0.6%, and the culture time is 6 hours. TheLactobacillusisLactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus reuteriorLactobacillus plantarum; the inoculation volume ratio of each strain is 0.5%, and the fermentation temperature is 38±1° C.

Preferably, the inactivation temperature described in step D of the preparation method is 115° C., and the time is 10 minutes.

Preferably, the fermentation substrate extract described in step B of the preparation method is further added with sucrose with a weight mg/volume mL ratio of 3.0%, sucralose in an amount of 30 mg/100 mL and ferrous sulfate heptahydrate in an amount of 5.0 mg/100 mL.

Preferably, in step C of the preparation method, after the fermentation is completed, the oligoxylose syrup sterilized in advance at 115° C. for 40 minutes is added under sterile conditions, and the added amount is 1.5% by weight mg/volume mL ratio.

Another purpose of the present disclosure is to provide the use of the microbial fermentation composition in the preparation of a medicament or a health product for improving the immunity of the organism, preventing and treating tumors.

The main active ingredient ofginsengis ginsenosides, and now there are more than 50 kinds of monomeric saponins that have been separated with confirmed structure. Ginsenoside is a glycoside compound composed of aglycon connected with sugar, which belongs to triterpenoid saponin. Ginsenosides are divided into 3 types according to the structure of aglycones: diol-type ginsenosides (PPD), triol-type ginsenosides (PPT), and oleanolic-type ginsenosides. Both PPD and PPT ginsenosides are dammarane-type tetracyclic triterpenoids, which account for the majority of ginsenosides, and are currently considered to be one of the main active ingredients ofginseng. PPD-type saponins include, for example, ginsenosides Rb1, Rd, Rh2, etc., and PPT-type saponins include, for example, ginsenosides Re, Rg1, Rg2, etc., among which the content of ginsenosides Rb1, Rd, Re, etc. are higher, while the content of ginsenosides Rg3, Compound K, Rh2 are very rare and substantially do not exist in artificially cultivatedginseng, so it is a rare ginsenoside.

After oral administration of ginsenosides orginsengextracts, ginsenosides are hydrolyzed to produce deglycosylated metabolites in an acidic gastric juice environment. In the intestine, glycoside hydrolases (such as β-glucosidase, a-rhamnoside, xylosidase, etc.) deglycosylation expressed by the intestinal flora can generate secondary metabolites or aglycones, which are absorbed into the systemic circulation by means of transmembrane transport, etc., and thus exert their efficacy. The differences between various ginsenosides are mainly the differences in the number and type of aglycones, glycosyl groups, and positions of glycosidic bonds. It has been proved that gastrointestinal metabolism position of saponins ingredients in traditional Chinese medicine is an important node that affects the absorption and efficacy of saponins ingredients. The glycoside hydrolase system expressed by the intestinal flora is the key factor in the node that mediates metabolism of saponin ingredients in the intestine.

Flammulina velutipesis beautiful in appearance and delicious, and has been widely used in medicated diets because of its various medicinal effects, also commonly known as edible and medicinal fungus and ornamental fungus. Studies have found that there are abundant anti-tumor active ingredients inFlammulina velutipes, including polysaccharides, flammulin, amycin, amycin protein and so on. Polysaccharide is one of the main active ingredients ofFlammulina velutipes, and related functional evaluations have shown that polysaccharide has the function of improving immunity and anti-tumor effects.Flammulina velutipescontains flammulin, which is a special alkaline protein that can strengthen the defense function of organism's immune system, and has a significant inhibitory effect on cancer cells.

Lentinan can increase the activity of natural killer cells (NK cells) and the production of lymphokine-activated killer cells (LAK cells), which can activate cellular immune function and produce tumor necrosis factor. When combined with chemotherapy drugs, it can significantly improve the anti-tumor effect. Studies have shown that the level of biological activity of polysaccharides is related to its structure, and the introduction of chemical groups often enhances the activity of polysaccharides or makes polysaccharides generate new activities. Therefore, appropriate modification of polysaccharide structure is one of the focuses of the current research on polysaccharide.

Agaricus blazei, also known as Brazilian mushroom, is a precious and rare medicinal and edible fungus that is originated from Brazil and is rich in sugar and protein. Studies have shown thatAgaricus blazeihas the effects of regulating immunity, anti-tumor, anti-virus, anti-inflammatory, anti-fatigue and liver protection.

Trace element selenium, mainly presence in the form of selenium cysteine in protein, is a trace element necessary for human life activities and has a wide range of biological functions. A large number of studies have proven that selenium has anti-cancer and anti-cancer effects, and is a powerful inhibitor of breast cancer, liver cancer, skin cancer, colon cancer and gastric cancer, being capable of inhibiting adenogastric cancer and pregastric cancer of rats induced by carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNC), and preventing 3-methylcholanthrene (3-MCA) from inducing sarcomas of mice, thereby reducing the risk of sarcomas.

The trace element zinc is involved in the synthesis of metallothionein, thereby inhibiting the formation of free radicals. The zinc oxide can inhibit the destruction of single-strand DNA of human skin fibroblasts exposed to ultraviolet A1 radiation. Therefore, Zn may have a negative correlation with cancer.

Molybdenum is an essential micronutrient for animals and humans, which is a component of various enzymes that participates in and affects the metabolism of many substances in the body. Meanwhile, molybdenum has good prevention and treatment effects on pain, liver disease, cardiovascular and cerebrovascular disease as well as endemic diseases such as Keshan disease, dental caries, small cell hypochromic anemia, and regional hair loss.

The intestinal flora is an important “microecological organ” of the human body. As a living system symbiotic with the host, it participates in many physiological processes. The intestinal flora disorder is gradually considered to be an important pathological feature of certain metabolic diseases and mental diseases, and is closely related to the occurrence and development of such diseases. Intestinal flora disorders often aggravate the symptoms of the disease, and the disease process can adversely affect the intestinal flora and exacerbate the degree of intestinal flora. In addition, a variety of external factors can affect the homeostasis of the intestinal flora, such as stress, abuse of antibiotic, etc., which can often cause intestinal flora disorders. Systematic characterization of the composition and function of the intestinal flora is an important means for related research on the intestinal flora. Modern drug metabolism research shows that after many natural medicines are taken orally and enter the body, they come into contact with the microbial flora in the intestine, and bacteria metabolize and transform the drug to transform the precursor substance of the drug into the active ingredient of the drug to exert effect.

It is allegedly reported that lactic acid bacteria have anti-cancer effects, but the mechanism is not very clear. The possible mechanisms are: strengthening the organism's immune system; adjusting the intestinal flora to prevent the formation of carcinogens in the intestine; producing anti-mutagenic substances; degrading or adsorbing carcinogens, etc.

When the organism consumes too much greasy food, the fat increases in the body and metabolism increases as well. The cholate in liver is responsible for lipid metabolism by digesting fats through emulsification. When cholate passes through the intestine of the body and encounter pathogenic bacteria, carcinogens will be generated and cause bowel cancer. However, with the presence of probiotics, pathogenic bacteria will be suppressed, so the presence of cholate does not easily induce carcinogens. Scientists have also confirmed the above fact through experiments. Probiotic drinks can reduce the possibility of genetic damage to the intestinal cells in the body, thereby preventing certain cancers.

Lactobacillusis the main probiotic in the female reproductive tract. Heat-inactivatedLactobacillushas strong adhesion to cervical cancer cell strain HeLa cells. It has been confirmed that improving the immunogenicity of tumor cells and restoring the immune surveillance function of the tumor cells are important strategies for anti-tumor treatment. Studies conducted by Wang Hongyan et al. found that heat-inactivatedLactobacilluscan indeed increase the immunogenicity of HeLa cells of the cervical cancer cell strain and activate the anti-tumor effects of T cells and NK cells, which provides new clues for probiotic-assisted tumor immunotherapy. Studies conducted by Chen Yutang et al. show that oral administration of probiotic for patients with liver cancer and liver cirrhosis during interventional treatment can effectively solve the intestinal flora imbalance of patients, and can reduce the risk of some liver cirrhosis complications in the short term without resulting significant functional impairment to liver.

Compared with the related art, the present disclosure selects probiotics for fermentation to transform inorganic trace elements such as selenium, zinc, molybdenum, etc. into organic trace elements, thereby reduces the accumulation toxicity and mutagenicity of inorganic trace elements, and improving the biological activity of trace elements. The selected medicinal and edible fungi andginsengare rich in polysaccharides. After probiotic fermentation, the molecular weight of the above-mentioned fungi andginsengis significantly reduced, which improves the tumor suppressing effect of fungal polysaccharide.

The formula of the fermentation composition containsginsengextract, and the ginsenosides in the fermentation process are transformed into deglycosylation through metabolism of probiotics to generate secondary metabolites or aglycones, which are absorbed into the systemic circulation through transmembrane transport, etc. and then exert a medicinal effect. The difference between various ginsenosides lies mainly in aglycones. In the fermentation composition, the content of rare ginsenoside Rg3 increased significantly.

In experiments of vitro inhibition of HepGII cells (hepatocellular carcinoma cells) and Hela cells (cervical cancer cells) by the fermentation composition, it is shown that the fermentation composition subjected to probiotics fermentation had a significant inhibitory effect on tumor cell proliferation. As compared with the unfermented composition, the fermented composition has a significantly enhanced inhibitory effect on experimental tumor cells, and the difference is remarkable. In addition, the inhibitory effect of the fermentation composition on the tumor cells used in the test increases with the increase of the adopted concentration. The in vivo inhibitory effect of the fermentation composition on solid tumors of mouse S180 shows that the antitumor effect of the probiotic fermentation composition in high, medium and low doses is significantly improved. Although the related mechanism is not clear, the results of this study prove that no inhibitory effect on tumor cells was observed in the composition without probiotic transformation. Under the in vitro effect of intestinal beneficial flora,ginseng, Agaricus blazei, Lentinus edodes, or other fungal polysaccharides which are subjected to some known or unknown chemical modification or biological transformation or the probiotic bacteria themselves and their metabolites on tumor cells have higher inhibitory activity.

