KR101720051B1 - Pharmaceutical composition comprising fermented Aralia cordata Thunb for preventing or treating arthritis - Google Patents

Abstract

The present invention discloses a composition for preventing or treating arthritis, a food composition, and a method for producing the same, which comprises a fermented product of a fermented product obtained by two stages fermentation with multiple lactic acid bacteria. The fermented product of the present invention is remarkably strengthened by minerals, antioxidants and amino acids as compared with the case of fermentation due to complex fermentation, and is not toxic to cells and inhibits inflammatory reaction through inhibition of factors related to inflammation, And the like.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating arthritis,

The present invention is in the field of functional natural products, and specifically relates to the use of fermented fermented products.

As an aging society, inflammatory diseases, including arthritis, are becoming socially irrespective of gender. Inflammatory diseases caused by such inflammatory reactions include gastritis, colitis, arthritis, nephritis, hepatitis, arteriosclerosis, cancer or degenerative diseases. To date, effective medicines and therapies for the prevention and treatment of arthritic diseases have not been developed. Arthritis refers to inflammatory changes in joints caused by various causes such as aging, mechanical damage, immune disorders, and degenerative arthritis and rheumatoid arthritis.

Degenerative arthritis is called osteoarthritis or abdominal arthritis. Major causes are aging, excessive use of joints, obesity, and genetics. According to the National Statistical Office data, degenerative arthritis is present in 80% of the people aged 55 years or older in Korea.

Rheumatoid arthritis is a type of autoimmune disease, the cause of which is not yet known, but genetic factors, infection, hormone abnormalities are estimated to be. In Korea, the number of people suffering from rheumatoid arthritis is about 670,000, which accounts for 1.4% of the total population (rheumatoid arthritis data), usually occurring in the 20s to 40s, .

As the number of arthritis patients increases, a variety of methods have been tried to treat various drugs, cartilage regeneration, artificial joints, and recently, stem cell treatment. Acetaminophen is a non-steroidal analgesic anti-inflammatory drug. It has a short half-life, so it does not cause any serious problems in analgesic effect during short-term administration. However, it does not cause serious problems in analgesic and anti-inflammatory treatment of chronic diseases such as arthritis. Is required. For this reason, studies on various natural extracts which do not have side effects are currently in progress, and demand for functional foods using natural extracts is steadily increasing.

Korean Patent No. 1388597 discloses a composition containing an extract of a dentifrice root, which comprises a dye of a dentifrice root as an active ingredient, and a therapeutic agent for preventing and treating osteoarthritis.

Korean Patent Registration No. 1179966 discloses a composition containing a mixture of natural extracts containing white rice, and a pharmaceutical composition for preventing and treating arthritis-related diseases comprising a mixture of natural extracts having antiinflammatory activity.

Therefore, it is inevitable to develop a preventive or therapeutic agent for arthritis, which has safer and more excellent effects derived from natural materials. The market demand for development of excellent materials having a new action mechanism from various medicinal plants and agricultural life resources is very high high.

The present invention provides an extract, a composition and a health functional food which can be effectively used for the treatment or prevention of arthritis using natural substances.

In one embodiment, the present invention provides an inhibitory reaction inhibition example comprising a fermented fermented product obtained from a lactic acid bacterium of Lactobacillus rhamnosus GG, L.GG and Lactobacillus plantarum (Lp ) The present invention provides a pharmaceutical composition for preventing or treating arthritis. By the fermentation of the complex lactic acid bacteria of the present invention, useful ingredient content, inflammatory reaction inhibitory activity and viable cell count were maximized compared with no fermentation and monofilament fermentation.

The Lactobacillus lambsosus and Lactobacillus flutarium, which are the multiple lactic acid bacteria used for the fermentation of the composition according to the present invention, are respectively in the range of 1.4 x 10 ⁶ CFU / ml to 1.2 x ^{10 7} CFU / ml, and 0.7 x 10 ⁴ CFU / ml to 0.4 x 10 ⁵ CFU / ml, wherein the Lactobacillus laminocus and the Lactobacillus flutarium are contained in a weight ratio of 1: 0.1 to 0.9, particularly 1: 0.4, based on dry cell weight, As the amount increased, the cell growth was increased.

The composition according to the present invention contains 2 to 6% (w / v) of chewing gum. When the coagulant content is increased, the growth of the cells is increased. The rate of growth of the microorganism was slowed.

In another embodiment, the present invention also provides a composition for preventing or improving arthritis, comprising a fermented product fermented with a complex lactic acid bacterium of Lactobacillus rhamnosus GG, L.GG and Lactobacillus plantarum (Lp ) And the composition may be provided as a health supplement, functional food, beverage or food additive.

As mentioned earlier, the compositions according to the present invention were fortified with antioxidant components and minerals by complex fermentation.

In another embodiment, the present invention also provides a method of producing a virulent fermented product, comprising: inoculating and culturing the virgin bacterium with Lactobacillus rhamnosus GG, L.GG ; And then inoculating and culturing the first culture with a second inoculation with Lactobacillus plantarum (Lp ). When L.GG and Lp were independently inoculated as described above, the final dry cell mass and SOD activity were increased 1.27 times and 1.23 times, respectively, as compared with the fermentation broth inoculated simultaneously.

In the method according to the present invention, the first incubation condition is incubated for 6 days at 37 DEG C with a stirring speed of 100 rpm, and the second incubation condition is carried out for 4 days at 33 DEG C and a stirring speed of 100 rpm. And the lactobacillus lambsosus and the lactobacillus plantarum are each inoculated at a concentration of 1.4 x 10 ⁶ CFU / ml to 1.2 x ^{10 7} CFU / ml, and 0.7 x 10 ⁴ CFU / ml to 0.4 x 10 ⁵ CFU / ml, respectively do.

The present invention discloses a composition for preventing or treating arthritis, a food composition, and a method for producing the same, which comprises a fermented product of a fermented product obtained by two stages fermentation with multiple lactic acid bacteria. The fermented product of the present invention is remarkably enhanced in minerals, antioxidants and amino acids as compared with the case of fermentation due to the complex fermentation and the two-stage fermentation, and is not toxic to the cells. Ameliorating an inflammatory reaction involving arthritis, or a disease associated therewith, by inhibiting the inflammatory reaction, and / or alleviating the disease.

FIG. 1 shows a kind of a candidate edible microorganism used in a false fermentation according to an embodiment of the present invention.
FIG. 2 is a graph showing the raw material content ratio according to one embodiment of the present invention. FIG.
FIG. 3 is a process diagram showing an entire production process for producing a fermented complex of lactic acid bacteria according to an embodiment of the present invention.
FIG. 4 shows results of MS-chromatogram qualitative analysis of lactic acid bacteria fermentation untimely MS according to one embodiment of the present invention.
FIG. 5A is a graph showing the total polyphenol and acidity of a miso extract according to an embodiment of the present invention. FIG.
FIG. 5B is a graph showing the total polyphenol and acidity analysis results of an extract of hot water extract according to one embodiment of the present invention. FIG.
FIG. 6A is a graph showing the total amino acid content of a fermented extract obtained by the method according to an embodiment of the present invention. FIG.
FIG. 6B is a graph showing a total amino acid content analysis result of an extract of hot water extract according to one embodiment of the present invention. FIG.
FIG. 7a is an analysis report of major minerals and heavy metal components of a miso fermented extract according to one embodiment of the present invention. FIG.
FIG. 7B is a graph showing analytical results of major minerals and heavy metal components of the hot water extract of crude extract according to one embodiment of the present invention. FIG.
FIG. 8A shows the results of analysis of survival rate of THP-1 cells in the virgin fermented extract according to one embodiment of the present invention.
FIG. 8B shows the results of analysis of survival rate of THP-1 cells in an extract of hot water extract according to one embodiment of the present invention.
FIG. 9A is a graph showing the expression pattern of hTNF-alpha according to one embodiment of the present invention. FIG.
FIG. 9B is a graph showing the hTNF-alpha expression pattern according to one embodiment of the present invention in the treatment with hot water extract.
FIG. 10A is a graph showing hIL-8 expression patterns according to one embodiment of the present invention. FIG.
FIG. 10B is a graph showing the expression pattern of hIL-8 according to one embodiment of the present invention according to the treatment with hot water extract.
FIG. 11A is a graph showing the expression pattern of hMCP-1 according to one embodiment of the present invention in a non-fermented fermented extract treatment. FIG.
FIG. 11B is a graph showing the expression pattern of hMCP-1 according to one embodiment of the present invention according to the treatment with hot water extract.
FIG. 12 is a photograph showing the expression pattern of MMP-9 according to one embodiment of the present invention according to the treatment with hot water extract.

In one embodiment, the present invention relates to a composition for treating or preventing an inflammatory reaction, for example, arthritis, which comprises a viscous fermented product as an active ingredient.

Aralia is a perennial grass belonging to Araliaceae and is a tall grass that grows over 2m in height. It grows strong in the shade of the mountain nation nationwide in our country because it has got the name of the dog in the meaning that it grows strong without moving in the wind. In one embodiment, the term " Aralia cordata Thunb " However, it does not exclude the seeds, leaves, stems and seeds as long as they contain physiological activity.

Herein, the term "fermented product" refers to a final product that has undergone the fermentation process according to the present invention, and may or may not remove the solid content after fermentation. In one embodiment, the solid content after fermentation is sieved to a sieve, such as 100 mesh. Various fermented lactic acid bacteria that produce lactic acid may be used for the fermentation of the present invention.

Fermentation can be carried out in a variety of fermenting bacteria such as lactobacillus such as Lactobacillus rhamnosus GG, L. lactobacillus casei, L. casei and Lactobacillus plantarum (Lp ) Saccharomyces sp. , And Bacillus subtilis .

In one embodiment, Lactobacillus sp. Such as Lactobacillus ramhnosus GG and / or Lactobacillus plantarum may be used. In particular, Lactobacillus rhamnosus GG , L.GG ), and Lactobacillus plantarum (Lp ). The Lactobacillus laminocus and the Lactobacillus flutarium used herein are contained in an amount of 1.4x10 ⁷ CFU / ml to 1.6x10 ⁸ CFU / ml, and 0.8x10 ⁷ CFU / ml to 1.5x10 ⁸ CFU / ml, respectively. The Lactobacillus laminocus and the Lactobacillus plutarium are contained in a weight ratio of 1: 0.1 to 0.9 based on the dry cell weight, and especially in a weight ratio of 1: 0.4.

The chewing gum used in the present invention is a fermentation process in which the active ingredients in the raw material are transferred to various usable components through a fermentation process and in particular, various enzyme components are produced through fermentation, low molecular weight proteins are formed, amino acids are strengthened, And its availability has been maximized.

The solubility of the present invention is in the range of about 2 to 6% (w / v), especially 4% (w / v), of a pharmaceutical composition for inhibiting inflammation, for example for preventing or treating arthritis.

The inflammation of the present invention is characterized by pain, redevelopment, swelling, ultimate impairment of function due to a series of complex interactions between the heat and the cells of the immune system of the infected area. Acute and chronic inflammation can be caused by a variety of factors including arthritis such as osteoarthritis, rheumatoid arthritis, asthma, inflammatory bowel disease, skin inflammatory diseases such as contact dermatitis, atopic dermatitis, dryness, eczema, rose intolerance, Resulting from the excessive biosynthesis of inflammatory mediators involved in a number of inflammatory diseases, such as thermal and radiological burns, e.g., photo-brightening associated with sunburn or chronic skin inflammation.

The inflammatory response for which the composition of the present application is effective is arthritis, which includes, but is not limited to, osteoarthritis, rheumatoid arthritis, degenerative arthritis, gouty arthritis, infectious arthritis or Lupus arthritis.

The arthritis of the present invention is a disease name collectively referring to inflammatory changes in joints caused by certain causes such as bacteria or trauma. Arthritis is divided into acute and chronic. Acute arthritis is classified as follows. ① Juvenile arthritis: It is usually caused by trauma and is unknown. It usually occurs only in one joint. ② Long-term fibrosis (arthritis) Arthritis: It occurs during acute rheumatoid arthritis, and there is a turbid effusion in the joints. Fibrosis of the stomach (虚膜) and inflammation of the left side of the severe motion disorder is left. ③ Purulent (arthritic) arthritis: open joints (open wound) or gonorrhea, typhoid, scarlet fever, septicemia (septicemia), such as the plague is common. Infants at 1 to 2 months of age cause dislocations that can not be treated because of severe bone injuries. In adults, periosteal osteomyelitis is a common cause of the eruption of the farmer and the pus into the joints, which is called secondary arthritis arthritis. Chronic arthritis is classified as follows. ① Specificity Inflammation: There is gouty arthritis due to metabolic disorder of uric acid in tuberculosis, phagocytosis or men after middle age. ② Multiple arthritis: It is caused by chronic arthritis rheumatism. It may occur in rapid-onset arthritis (multiple sclerosis), multiple cases of tuberculosis, syphilis or gonorrhea, and it may be one of sepsis. It also includes arthritis, which is called Still's disease. ③ Degenerative osteoarthritis: It is caused by aging or trauma of bones or joints. ④ Hemophilia (hemophilic) arthritis: Hemophilia is due to bleeding within the joint.

Degenerative arthritis, also known as osteoarthritis, occurs predominantly in middle-aged or elderly people. It is a local arthritis caused by a change in articular cartilage caused by aging. Such arthritis results in an increase of inflammatory cytokines and nitric oxide.

Rheumatoid arthritis is a chronic inflammatory disease of unknown origin characterized by multiple arthritis. Initially, the synovial membrane surrounding the joint is inflamed, but the cartilage and bone are gradually inflated to cause destruction and deformation of the joint, and joints as well as joints, anemia, dry syndrome, subcutaneous nodule, pulmonary fibrosis, It is a disease that can invade the whole body including vasculitis and skin ulcer.

In one embodiment of the present invention, it has been confirmed that arthritis is treated and prevented by confirming the inhibitory effects of cytokines and chemokines associated with the inflammatory reaction including arthritis.

As used herein, the term "treatment" refers to any action that improves or alleviates the inflammatory response, for example, arthritis or other conditions associated therewith by administration of the fermented product or compositions comprising it. Those skilled in the art will be able to ascertain, by reference to the data provided by the Korean Medical Association, the precise criteria of the disease for which the composition of the present invention is effective, .

As used herein, the term "prophylactic" refers to any action that inhibits or slows the onset of an inflammatory reaction, such as arthritis, including arthritis, or any other disease resulting therefrom upon administration of the fermented product or composition comprising it do. It will be apparent to those skilled in the art that compositions of the present invention that are effective for inflammation-related diseases can prevent early symptoms of inflammation-related diseases, or such diseases when taken before they appear.

The pharmaceutical composition comprising the coarse fermented product of the present invention may be administered simultaneously or sequentially, and these mixtures may be administered alone or in combination with other pharmaceutically active ingredients for the treatment of arthritis symptoms.

The composition containing the fermented fermented product of the present invention may further contain excipients such as suitable carriers, diluents, preservatives, stabilizers, wetting agents, emulsifiers, solubilizers, sweeteners, coloring agents, osmotic pressure regulators and antioxidants commonly used in the production of pharmaceutical compositions can do. Specific examples include lactose, dextrose, sucrose, sorbitol, mannitol xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone Water, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium, stearate, mineral oil and the like.

The method for administering the pharmaceutical composition for preventing or treating arthritis, including the coarse fermented product of the present invention, can be easily selected according to the formulation, and can be administered to mammals such as livestock, human, and the like in various routes. For example, it may be formulated in the form of powders, tablets, pills, granules, dragees, hard or soft capsules, liquids, emulsions, suspensions, syrups, elixirs, external preparations, suppositories, sterilized injection solutions, Orally or parenterally. In particular, oral administration is preferred.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid formulations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used diluents, various excipients such as wetting agents, sweeteners, have.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used.

Further, the pharmaceutical compositions comprising the coarse fermented product of the present invention can be prepared using methods known in the art or as described in Remington's Pharmaceutical Sciences (recent edition), Mack Publishing Company, Easton PA And the like.

The dosage of the pharmaceutical composition containing the fermented product of the present invention may vary depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, The effective dose of water is typically about 1 to 100 g / day, especially about 10 to 50 g / day, more preferably about 30 g / day for an adult (60 kg). It will be apparent to those skilled in the art that doses may be additive or subtracted, as the dosage can vary depending on various conditions, and thus the dose is not intended to limit the scope of the invention in any way.

The number of administrations can be administered once or several times a day within a desired range, and the administration period is not particularly limited. In addition, the composition containing the extract of the present invention may be added to an arbitrary food and be routinely ingested as such, in addition to oral administration.

The pharmaceutical composition containing the fermented product of the present invention not only provides an excellent therapeutic effect against arthritic diseases, but also has no toxicity and side effects caused by drugs, and can be used safely even when taken for a long time. Accordingly, the fermented fermented product of the present invention can be used as a main ingredient, a food ingredient, and a food additive.

Therefore, from this point of view, the present invention can be provided as a food composition containing the above-mentioned coarse fermented product, for example, a health supplement, a functional food, a food additive and the like.

As used herein, the term "food" means a natural or processed product containing one or more nutrients, preferably a state of being ready to be eaten through a certain degree of processing, It is intended to include food, food additives, functional foods and beverages as a meaning.

As used herein, the term "functional food" refers to a food group that has been imparted with added value to function or express the function of the food by physical, biochemical, or biotechnological techniques, , And the body control function of disease prevention and recovery is designed to be fully expressed in the living body, and in particular, includes "health functional food".

As used herein, the term "health supplement food" is a food prepared by using raw materials or ingredients having useful functions in the human body, and is useful for health and other purposes such as controlling nutrients and physiological actions on the structure and function of the human body For the purpose of health assisting to obtain the effect, it refers to a food which is made by processing a specific ingredient as a raw material or by extracting, concentrating, refining, mixing, etc. ingredients contained in a food raw material.

In particular, the functional food or the health supplement food according to the present invention refers to a food having an effect of improving symptoms of arthritis or helping alleviation or alleviation of arthritis as compared with before consumption of the food, If you have a normal knowledge, you can refer to the data provided by the Korean Medical Association, and you will know the precise standards of these diseases and judge the degree of improvement.

The functional food and the health supplement food may further include a food-acceptable food supplementary additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of a functional food.

Foods to which the fermented fermented product of the present invention can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, and functional foods. In addition, in the present invention, the food may contain special nutritional foods (e.g., crude oil, spirits, infant food, etc.), meat products, fish products, tofu, jelly, noodles (Such as soy sauce, soybean paste, hot pepper paste, mixed sauce), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese), other processed foods, kimchi, pickled foods But are not limited to, fruits, vegetables, beverages, fermented beverages, etc.), natural seasonings (e.g., ramen soup, etc.). The food, beverage or food additive may be prepared by a conventional production method.

Drinks in the present application mean a general term for drinking or enjoying a taste, and are intended to include functional beverages. The above-mentioned beverage is not particularly limited as long as it contains the fermented fermented product of the present invention as an active ingredient as an essential ingredient at the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient . Examples of such natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrins, cyclodextrins and the like, and Xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably 5 to 12 g per 100 ml of the composition of the present invention. May be further contained.

In the present invention, the functional beverage is used to control the bio-defense rhythm of the beverage group or beverage composition to which the added value is imparted so that the function of the beverage functions for a specific purpose using physical, biochemical, biotechnological techniques, Means a drink which is processed by designing the body so as to sufficiently express the body control function with respect to the living body, and preferably means a drink for improving arthritis.

The above-mentioned functional beverage is not particularly limited to the other ingredients other than the nutritive fermented product of the present invention as an essential ingredient at the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of such natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrins, cyclodextrins and the like, and xylitol , Sorbitol, and erythritol. Natural flavors (tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably 5 to 12 g per 100 mL of the composition of the present invention.

In addition to the above, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, , Organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. These components can be used independently or in combination. The proportion of such additives is not so critical, but may be selected in the range of 0.001 to 20 parts by weight per 100 parts by weight of the fermentation product of the present invention.

In a food intended for the improvement of arthritis, the fermented fermented product of the present invention contains about 0.01 to 90% by weight, about 0.1 to 80% by weight, about 1 to 70%, about 1 to 50% About 1% to about 40%, about 1% to about 30%, about 1% to about 20%, and the like. For example, the beverage composition may comprise 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 mL.

In another aspect, the present invention is also directed to a method of making a chewy extract fermentation product for use in the compositions herein.

The method according to the present invention comprises the step of inoculating and culturing the virulent strain with Lactobacillus rhamnosus GG, L.GG ; And then inoculating the culture with Lactobacillus plantarum (Lp ) and culturing the culture in a second culture. In one embodiment, the first incubating step is incubated for 6 days at 37 DEG C, 100 rpm, and the second incubating step is performed for 4 days at 33 DEG C, 100 rpm. When the Lactobacillus lambosus and Lactobacillus plantarum reached 1.4 x 10 ⁶ to 1.2 x ^{10 7} CFU, respectively, L. p was incubated at 37 ° C for 48 hours and then 0.7 x 10 ⁴ and when the reached 0.4x10 ^5, is inoculated with 1.4x10 ⁶ CFU / ml to about 1.2x10 ⁷ CFU / ml, and 0.7x10 ⁴ CFU / ml to 0.4x10 ⁵ CFU / ml.

The merit used in the method according to the present invention is as mentioned above. The makeup can also be used in dried or unprocessed form, or it can be pretreated in the same way as cutting or grinding.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example 1: Selection of microorganisms suitable for the production of coarse fermented product

Example 1-1 Preparation of Excessive Hot Water Extract

The extracts were mixed with 4% (w / v) of dried chewing gum and water, extracted first for 24 hours at 80 ℃ and then for 15 minutes at 121 ℃. Secondary extraction was used.

Example 1-2 Selection of optimal fermentation microorganisms

Lactobacillus rhamnosus GG (L.GG ) isolated from commercially available dairy products and Lactobacillus casei, L. casei ( Lactobacillus casei, L. casei ) distributed from the College of Agriculture, Kyungpook National University were used as candidates for the edible microorganism 3 strains of lactic acid bacteria of Lactobacillus plantarum (Lp ), 2 yeast strains of Saccharomyces sp. 1 and 2 isolated from makgeolli, and 2 strains of Bacillus subtilis 1 The strains most suitable for producing the fermented fungus were selected from a total of six strains (Fig. 1).

In particular, to select the most suitable edible microorganisms for the fermentation, the six candidate microorganisms were cultured under the same fermentation conditions for 6 days in a liquid medium (12 g of dry miscible product and 300 ml of distilled water) The final strain was selected based on its dry weight and antioxidant activity. SOD activity, a typical antioxidant enzyme, was measured using an SOD assay kit. The test method was performed according to the Food Code. After the reaction, the absorbance at 560 nm was measured using a spectrophotometer, and the SOD activity was calculated as follows.

         SOD (%) = (1- (A-B) / C) 100

A: Measure absorbance after adding sample to xanthine oxidase reagent

B: Absorbance measurement after addition of blank buffer instead of xanthine oxidase reagent

C: Absorbance measurement after addition of distilled water to xanthine oxidase reagent

As a result, the dry weight of the final lactic acid bacteria was lower than that of yeast or bacillus in the same conditions as the following [Table 1]. However, not only the flavor after fermentation was excellent but also the activity of SOD related to antioxidant activity was higher than yeast or bacillus , And three kinds of lactic acid bacteria were firstly extracted as candidate microorganisms for non - fermentation.

[Table 1]

In order to select the most suitable microorganism for the fermentation of the first lactic acid bacteria, we tried to ferment lactic acid bacteria with L. p, another lactic acid bacteria of kimchi lactic acid bacteria, and the number of live bacteria, pH, The analysis was compared.

As shown in the following Table 2, pH was largely lowered during fermentation with lactic acid bacteria, and when the fermented lactic acid bacteria L. p and lactic acid bacteria L. GG were mixed, the flavor including flavor and aroma, And the two strains were finally selected and used for the combined fermentation.

[Table 2]

The total polyphenol content and antioxidative SOD activity of the fermented broth prepared by the first - selected lactic acid bacteria were compared with those of other lactic acid bacteria. The total polyphenol content was determined by colorimetrically modifying the Folin-Denis method. The total polyphenol content of the extracts was expressed as mg gallic acid per g after the standard curves were generated using gallic acid (Sigma, USA).

As shown in [Table 3], total polyphenol content and SOD activity were increased when combined fermentation of Lactobacillus plantarum and other dairy lactic acid bacteria compared to the antioxidant activity of each lactic acid bacteria. The total polyphenol content and the SOD activity of L. plantarum and L. GG were 1.2 times higher and 1.4 times higher than those of non - fermented hot - water extracts, respectively, .

[Table 3]

Example 2 Optimization of Fermentation Process of Lactic Acid Bacterium Unsaturated

In order to obtain the best raw material costs, 2%, 4%, 6% and 8% (w / v) of differentiated raw materials, which were dried in 100 ml of distilled water, were separately added to prepare sterilized media. Example The selected two kinds of lactic acid bacteria according to the first in a MRS broth OXOID's lactic acid bacteria L. GG-only medium is 37 ℃, 48 hours after incubation when reached ^{1.4x10 6 ~ 1.2x10 7 CFU, L.} p is 33 ℃ After 48 hours of incubation, when the concentration reached 0.7x104 to 0.4x105 CFU, 5 ml of each culture was inoculated on the previously prepared culture broth and incubated at 37 ° C and 100 rpm for 7 days. The turbidity of the culture was measured by absorbance method (A600 nm) Respectively.

As a result, as shown in FIG. 2, the growth rate of the cell was increased with the increase of the simpleactivity until the raw material content ratio was 2 to 6% (w / v), but the growth rate of the cell was slowed at 8% Respectively. In the case of medium supplemented with 4% (w / v) of dry loquat, the highest absorbance value was obtained after 6 days of culture, and even if no more raw materials were supplied, Was determined to be 4% (w / v) as a dry standard.

In order to determine the optimum lactic acid bacterial inoculation amount, the culture conditions including the coenzyme content were the same and the mixing ratio of L. p and L. GG was 1: 1, and the inoculation ratio of the combined lactic acid bacteria was 2 to 10% (v / v) 2%, and incubated for 6 days. Then, the cell dry weight was measured. As shown in Table 4, when the inoculation ratio of lactic acid bacteria inoculated with 2% of L. p and L. GG was 4% (v / v), the cell growth was increased as the seed amount increased, , And the optimal inoculum size was determined to be inoculated with 2% (v / v) of each of the two lactic acid bacteria cultured in the MRS medium.

The inoculated amount of Lactobacillus laminocus and Lactobacillus plutarum was in the range of 1: 0.1 to 0.9 g / g, based on dry cell weight, after drying the same volume of culture medium at 65 DEG C for 18 hours, ), And specifically may be included at a ratio of 1: 0.4 (g / g).

[Table 4]

In order to maximize the antioxidant activity of the fermented lactic acid fermentation broth, the growth characteristics and antioxidant activities of the fermented broth were investigated. In method 1, two L. p and L. GG lactobacillus strains were cultured in MRS medium and then inoculated with 2% (v / v), respectively, in the challenge medium, whereas in method 2, , L. gG was inoculated with 2% (v / v) of L. gG, which had better cell growth, and cultured at 37 ° C and 100 rpm for 6 days. When the pH of the culture medium was slightly lowered, % (v / v), and incubated for 4 days at 33 ° C and 100 rpm for 10 days in total.

The results of the analysis of the final dry cell mass and antioxidative SOD activity of the lactic acid fermentation broth prepared according to Methods 1 and 2 are shown in Table 5 below. When Lp was independently inoculated, which was able to grow vigorously in weak acidity, LGG with excellent early growth and SOD activity were 1.27 times and 1.23 times, respectively, as compared with the fermentation broth inoculated with two kinds of lactic acid bacteria simultaneously And the final fermentation method was determined based on this.

[Table 5]

To determine the optimum agitation speed, the agitation speed of the culture apparatus was increased by 50 rpm in the range of 0 to 300 rpm, and the dry cell weight and total polyphenol content obtained after 10 days of culture were measured. As the agitation speed increased, the cell growth was slightly increased, and after 100 rpm, the cell growth was not significantly affected. Also, the total polyphenol content was the highest at 100 rpm, and the optimum agitation speed was determined as 100 rpm [Table 6].

[Table 6]

Example 3 Whole production process for producing fermented lactic acid bacteria complex lactic acid bacteria

A fermented fermented product was prepared on the basis of the optimum strain selection and culture conditions according to Examples 1 and 2. FIG. 3 is an overall process diagram of manufacturing a fermented fermented product. In order to maximize the extraction of useful components contained in the fermentation broth, the fermentation broth is subjected to high temperature and high pressure treatment to obtain a fermented fermented product under the fermentation conditions described above, And the total polyphenol contents were investigated. When the fermented broth fermented with complex lactic acid bacteria was sterilized at 121 ℃ for 10 minutes, the total polyphenol content was 48.2 mg / 100g and the total polyphenol content was not changed significantly by sterilization.

In order to remove solids required for formulation and storage stability, the liquid filtrate from the primary filtration body is filtered with a second filter paper (Wattman), and finally, purified using a molecular weight 50,000 daltons UF membrane Respectively. In the case of the liquid fermentation extract obtained after the purification, there was no significant difference from the total polyphenol content before the removal of the solid content. The liquid fermented extract was used as a powdery form by liquid phase or lyophilization, and was used for the analysis of the fecal fermentation product in the refrigerator.

Example 4 Analysis of Physicochemical Properties and Major Components of Exfoliated Fermented Product

To evaluate the physicochemical properties of fermented monoculture fermented with mixed lactic acid bacteria, the fermentation broth was sterilized, cells and solids were removed, purified, and the water solubility, pH and thermal stability were measured using freeze - dried powder and liquid samples. The results are shown in Table 7. In the comparison group, unsweetened hot water extract without fermentation process was used under the same conditions.

[Table 7]

The major minerals of non - fermented fermented extracts were compared and analyzed in the same conditions as non - fermented hot - water extracts. Contents of major physiologically active minerals such as magnesium, copper and zinc were analyzed by atomic absorption spectrophotometer. As shown in Table 8, major minerals contained in the fermented extract showed higher values than hot-water extracts.

[Table 8]

(C ₂₀ H ₃₀ O ₂ , molecular weight 302.45), which is known as an index substance that affects various physiological activities such as arthritis, and arthritis, is investigated in order to investigate the effect of multiple lactic acid fermentation on the content of contaminating acid The extracts of hot water extract (Unsaturated 1) and lactobacillus fermented extract (Unsaturated 2) were qualitatively analyzed as a liquid sample. At this time, the standard substance was used in the analysis of the herbal medicine by the Korea Food and Drug Administration. The analysis was commissioned to Gyeonggi Bio Center to obtain more objective results and analyzed by MS-chromatogram.

The solvent used for the analysis was MeOH (B & J) and the reagent was IPE (Sigma).

[Table 10]

As shown in FIG. 4, in the case of lactic acid bacteria fermentation untreated analysis, the same peak as that of the reference material was found at 23.86 and 23.85 minutes in both the hot water extract and the fermentation untreated sample, which were raw materials, and the MS spectrum , And it was found to be consistent with the theoretical value at the level of 1.4 ppm.

The qualitative analysis of lactic acid bacteria fermentation toxicity revealed that the continental acid was included, and then quantitatively analyzed by MS-chromatogram. For quantitative analysis, the injection volume was 5 μL, the scan range was SIM: m / z 303, mass width = 5, and the other conditions were analyzed in the same manner as the qualitative analysis. As a result, as shown in Table 11, continental acid was not measured in the hot water extract (Unsaturated 1) diluted in the same multiples but in the fermented non-fermented fermentation extract (Unsaturated 2), the concentration of 0.294 ± 0.028 mg / ml of continental acid was quantitatively analyzed , And it was confirmed that the content of continental acid which is an indicator component in the fermentation of complex lactic acid bacteria was increased in the case of virulence.

[Table 11]

The total polyphenol content and acidity of the combined lactic acid bacteria fermented non-fermented extract were analyzed and analyzed by the Food Analysis Research Center of Suwon Women's University. The results are shown in FIGS. 5a and 5b. FIG. 5a shows the total polyphenol content and acidity of L. fermented extract, and the total polyphenol content was 48.63 mg / 100 g in the fermentation broth prepared by fermentation with L. GG and L. p, Polyphenol and acidity analysis report showed 29.34mg / 100g. Therefore, it was confirmed that total polyphenol content related to antioxidation was remarkably increased in the fermented extract of virgin yeast compared to the hot - water extract.

The major amino acid contents of the fructose fermented with the lactic acid bacteria were analyzed and analyzed by the Food Analysis Research Center of Suwon University. FIG. 6A is a test result of the extract of fermented soybean paste, and FIG. 6B is a test report of the extract of hot soybean extract. The total amino acid content was about 132.78 mg / 100g, which was almost 2.5 times higher than 52.36 mg / 100 of the hot water extract. The total amino acid content of the fermented extract was 20% Respectively.

In addition, the basic physiologically active minerals and fermentation broth of fermented lactic acid bacteria were investigated. In consideration of the use of roots, the contents of heavy metals were analyzed and analyzed by the Korea Food and Drug Administration (KFDA) designated Food Hygiene Inspection Agency. The results are shown in Figs. 7A and 7B. In the case of the non-fermented extract of Fig. 7a, the content of potassium, sodium, phosphorus, manganese, magnesium, zinc and copper, which are major minerals that affect the physiological activity, And phosphorus were 7.9 mg / 100g and 114.7 mg / 100g, respectively, compared to 4.04 mg / 100g and 68.5 mg / 100g of hot water extract, respectively.

In addition, according to the test report, heavy metals of tin, lead, cadmium, arsenic and mercury were not detected in the simvastatin extract fermented with complex lactic acid bacteria, confirming the basic safety of the fermented extract fermented with complex lactic acid bacteria I could.

Example 5 Evaluation of basal cytotoxicity of extractive fermented extract

Cell viability assay (cell viability assay) was performed to evaluate the basal cytotoxicity of virulent fermented extract. The survival rate of the cells was determined by diluting the prepared fermented extract and then measuring the number of cells with a microscope. THP-1 cells associated with the inflammatory reaction were suspended in RPMI 1640 medium containing 10% FBS to a concentration of 2.0 x 10 ⁴ cells / well, and 200 μl of each was inoculated into each well of a 96-well plate. The fermented extract was incubated for 24 hours at an appropriate concentration, and then 50 μl of the fermented extract was removed from each well. The number of cells per unit area was measured using a hemacytometer.

As shown in FIG. 8A, when the concentration of 500 μg / ml of non-fermented fermented extract was treated at 500 μg / ml, the THP-1 cell survival rate However, as shown in FIG. 8B, in the case of the extract of hot water extract, the number of cells was slightly decreased when the concentration was 500 μg / ml. Therefore, compared to the hot - water extract, the complex of fermented lactobacillus showed excellent results in the evaluation of basal cytotoxicity.

Example 6 Evaluation of Safety of Fermentation Combination Using Laboratory Animals

In order to evaluate the stability of the fermented extract, the single oral dose toxicity study of hot - water fermented hot - water extracts was carried out in accordance with the Toxicological Test Standards for Drugs and other Notification No. 2013-121 of Korea Food and Drug Administration. The test substance is an extract of chewing gum fermented with complex lactic acid bacteria.

Experimental animals were SPF / VAF Outbred CrljOri: CD1 [ICR] male mice of the same age (OrientBio, Seungnam, Korea) [ANNEX I, II] [Approval no. Of the Committee]. DHU2014-013; ANNEX III]. Experimental results were analyzed in a total of 10 groups including 5 control per group including medium control. The dosing method and dosage were as follows: Clinical application route / Approximate lethal dose was set at an appropriate stage and sterile distilled water was used as a solvent and administered orally.

2,000, 1,000 and 500 mg / kg of hot-water fermented hot water extract and 2,00 mg / kg of Phellinus linteus extract were administered once orally. In the control group of male and female mice, only oral administration of sterile distilled water (20 ml / kg) was used instead of the test substance.

In order to obtain single-oral toxicity data of mouse of fermented hot-water extract, 2,000 mg / kg of rodent dose limit was set as the highest dose group and the azithromycin was administered according to the Food and Drug Administration Notification No. 2013-121 " 2, 1000 and 500 mg / kg were administered to medium and low dose groups. To compare toxicity, 2,000 mg / kg of maleic anhydrase extract and male and female medium control groups were separately prepared.

The results of this study were as follows: 1) The mortality, clinical symptoms, weight changes, autopsy findings, organ weight, and histopathological changes (14 chronological cases: lung, heart, thymus, kidney, adrenal, spleen, testis / ovary, liver, pancreas, , Epididymal / uterine and subcutaneous lymph nodes).

As a result of animal experiments, the mortality rate was not observed during the 14-day experimental period, and the death related to the administration of non-fermented hot-water extract until the dose limit of 2,000 mg / kg, which was the rodent limiting dose, was not recognized.

Clinical symptoms were not related to clinical symptoms associated with administration of exogenous fermented hot - water extract or hot - water extract.

No significant changes in body weight and body weight were observed during the whole experimental period compared to the same gender media control group in all the non - fermented hot - water extracts or hot -

Changes in the long term weight were not significantly different from those of the same sex-media control group in all the wild-type fermented hot-water extract-treated male and female rats, and no significant change in the organ weights associated with administration of the hot water extract was observed .

The autopsy findings were sporadically observed in all experimental groups including the male and female control groups, with mild or moderate [1 ~ 2 +] pulmonary congestion, thrombus atrophy, spleen enlargement, uterine edema, or sublingual lymph node enlargement. No visual examination findings related to the administration of the extract were observed.

Histopathological examination revealed that the incidental lesions such as mild [1+] pulmonary hemorrhage, decreased thymic cortical lymphocyte count, splenic red watery lymph node enlargement, local inflammatory cell infiltration of the liver, and lymph node enlargement were found in all experimental groups (1/5; 20%) of mild cystic formation in the kidney was observed only in the group treated with 500 mg / kg of fermented hot-water extract of female females. Histopathological changes related to the administration of fermented hot- And no histopathologic changes associated with the administration of exogenous hydrothermal extract were observed.

No deaths were observed up to the dose limit of 2,000 mg / kg, which was the limit dose of rodent administration in this experiment, and the single oral dose lethal dose and the approximate lethal dose of the wild-type fermented hot-water extract were 2,000 mg kg / day. No specific clinical symptoms or target organ was observed. No deaths, clinical symptoms, weight, long-term weight, visual and histopathological changes associated with 2,000 mg / kg of hot water extracts were observed.

As a result of single dose oral toxicity test in mouse of fermented hot water extract of fACR (fACR), the death toll of 2,000 mg / kg, which is the dose limit of rodent administration, was not recognized, and the half lethal dose and the approximate lethal dose were judged to be 2,000 mg / No other toxic symptoms were observed, and it was considered as a relatively safe drug. No deaths, clinical symptoms, weight, long-term weight, visual or histopathological changes associated with 2,000 mg / kg of hot water extracts were also observed.

Example 7 Assessment of Inhibitory Effect of Fermentation Nocturnal Inflammation Response on Arthritis

To investigate the anti-inflammatory effects of the fermented extracts, the THP-1 cell line, which is a human monocyte, was used to investigate the effects of hTNF-alpha and hIL-8 among arthritis inflammation-related cytokines and arthritis pain And on the expression of hMCP-1, a chemokine. Lactic acid fermented non-fermented extracts and extracts of hot-water extracts were dissolved in tertiary distilled water at concentrations of 10, 30, 50, 100, 300 and 500 μg / ml, respectively. Then THP-1 cells were treated with human monocyte THP-1 cells. LPS (lipopolysaccharide) was treated as a model for stimulation. Twenty four hours after the treatment, the culture supernatant was separated and subjected to ELISA to determine the concentration of cytokines and chemokines.

As a result, referring to FIG. 9A, it was confirmed that hatching fermented extract had a tendency to inhibit hTNF-alpha production significantly when stimulated with LPS (Lipopolysaccharide), which is the main inflammation inducer, It means the possibility of making. On the other hand, referring to FIG. 9B, it can be confirmed that the hot water extract of unswerved hot water does not have a significant effect on hTNF-alpha production when treated at the same concentration. Therefore, it was concluded that the extract of fermented soybean paste showed better anti - inflammatory effect than that of hot - water extract.

In the case of hIL-8 among the cytokines produced during the inflammatory reaction, referring to FIG. 10A, the hatching fermented extract fermented with Lactobacillus acidophilus showed a tendency that hIL-8 expression was significantly suppressed by treatment with LPS I could confirm. However, referring to FIG. 10B, it was found that the extract of hot water extract had little effect on hIL-8 expression.

We investigated the effect of non-fermented extracts on the expression of hMCP-1, recently reported to be associated with arthritis pain induction in chemokines produced during inflammation. Referring to FIG. 11A, it was confirmed that hatching fermentation extract significantly inhibited hMCP-1 expression when LPS induced inflammation. However, it can be seen from FIG. 11b that the extract of hot water extract had little effect on hMCP-1 expression.

Example 8 Influence of Fermentation Dose on MMP-9 Activity (in vitro)

The activity of MMP-9 and 2 ((Matrix metallopeptidase-9, 2) was measured by using THP-1 cell line to determine the effect of gelatin- MMP-9 and MMP-2 associated with the inflammation reaction in the sodium dodecyl sulfate polyacrylamide gel containing 0.1% gelatin were subjected to electrophoresis and then analyzed by MMP- -9 and MMP-2 is found to gel in the culture conditions for the enzymatic activity by measuring the activity of MMP-9 and MMP-2 secreted by the breakdown level of gelatin, THP-1 cells to 1.0 x 10 ⁵ cells / well were suspended in serum-free RPMI 1640 medium and 100 μl of each was inoculated into each well of a 96-well plate. The extracts of non-fermented and non-fermented hot-water extracts were stimulated with lipopolysaccharide. Separated after hours The gel was electrophoresed on 10% SDS polyacrylamide gel with FOZ buffer solution (4% SDS, 20% glycerol, 0.01% bromo phenol, 0.125 M Tris-Cl) and placed in 2.5% Triton X-100 for 40 minutes After removing SDS, the gel was immersed in digestion buffer (50 mM Tris-HCl, pH 7.6, 0.15 M NaCl, 10 mM CaCl 2, 0.02% NaNO 3) and cultured at 37 ° C for 12 hours.

After the culture, the gel was stained with 0.1% Coomassie Brilliant Blue R-250 and the degradation of the gelatin was confirmed by discoloration. As shown in FIG. 12, when LPS stimulation was applied at the concentration of 300 μg / ml and 500 μg / ml, the tendency to inhibit the production of MMP-9 was confirmed, unlike the hot water extract of the same concentration, May be able to inhibit cartilage tissue destruction, which may help prevent arthritis. However, MMP-2 activity was not significantly affected.

Preparation Example 1. Preparation of liquid preparation

Fermented product 10,000 mg

Sugar 200mg

100 mg of isomerized sugar

Lemon incense quantity

Purified water was added to make a total volume of 1000 ml. The above components were mixed according to a conventional method for producing a liquid agent, and then filled in a brown bottle and sterilized to prepare a liquid agent.

Preparation Example 2. Preparation of health drinks

 Fermented product 10,000 mg

Citric acid 1000mg

Oligosaccharide 100 g

2 g of mesyl concentrate

Taurine 1g

Purified water was added to the entire 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the resulting solution was stirred and heated at 85 DEG C for about 1 hour. The resulting solution was filtered and sterilized in a 2 L sterilized container. The resulting solution was refrigerated, ≪ / RTI >

Preparation Example 3. Preparation of health food

 5000 mg fermented product

Vitamin mixture quantity

Vitamin A acetate 70 mg

Vitamin E 1.0mg

0.13 mg of vitamin B1

0.15 mg of vitamin B2

Vitamin B6 0.5mg

Vitamin B12 0.2mg

Vitamin C 10mg

Biotin 10mg

Nicotinic acid amide 1.7 mg

Folic acid 50mg

Calcium pantothenate 0.5mg

Vitamin E 1.0mg

Mineral mixture quantity

1.75 mg ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Calcium phosphate diphosphate 55 mg

Potassium citrate 90mg

Calcium carbonate 100 mg

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is relatively mixed with a suitable preparation for health food, the compounding ratio may be arbitrarily modified, and the above ingredients may be mixed according to a conventional method of manufacturing health food, The granular formulation of health food was prepared.

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

Claims (10)

A method for producing an intact fermented product having enhanced antioxidant components and minerals,
( Lactobacillus rhamnosus GG, L.GG ) is inoculated to the chewing gum, wherein the chewing gum is contained in an amount of 2 to 6% by weight, and the first culture is carried out at 37 DEG C, 100 rpm for 6 days Cultured, and
Lactobacillus plantarum (Lp ) is inoculated to the first culture, and the second culture is carried out at 33 DEG C and 100 rpm for 4 days, wherein Lactobacillus laminosus and Lactobacillus plantarum are cultured in a dry cell Wherein the composition is inoculated at a ratio of 1: 0.4 on a weight basis.

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