KR20210086178A - Method for producing fermented peanut sprout using novel Pediococcus pentosaceus strain and composition for improving constipation containing peanut sprout fermentation produced by the same method - Google Patents

Abstract

The present invention relates to a method for manufacturing a peanut sprout fermented product using a novel Pediococcus pentosaceus strain, and a composition for alleviating constipation containing the peanut sprout fermented product manufactured by the method. By using the peanut sprout fermented product, it is possible to facilitate bowel movements and reduces the number of feces remaining in the large intestine, and thus the present invention can be very usefully used for developing health functional food or pharmaceuticals for improving intestinal functions and alleviating constipation.

Description

Method for producing fermented peanut sprout using novel Pediococcus pentosaceus strain and composition for improving constipation including fermented sprouted peanut prepared by the method {Method for producing fermented peanut sprout using novel Pediococcus pentosaceus strain and composition for improving constipation containing peanut sprout fermentation produced by the same method}

The present invention relates to a method for producing a sprouted peanut fermented product using a novel Pediococcus pentosaceus strain, and a composition for improving constipation comprising the sprouted peanut fermented product prepared by the method.

Constipation is a representative disease caused by malnutrition due to westernized diet and unbalanced nutrition. It is a digestive disease that usually occurs when the bowel movements are small, it is difficult to pass dry and hard stools, or when the discharge of stools is rarer than 2-3 times a week. As a type, it is experienced frequently enough to appeal to 5-20% of the total population, but the contents expressed by each patient are very diverse and it is not easy to define objectively because it is a single symptom group. More than 90% of constipation patients are chronic constipation that occurs idiopathically without a cause, and can be divided into constipation that occurs secondary to causes that cause constipation, such as organic intestinal lesions, metabolic diseases, and taking certain drugs. Chronic constipation has been reported to be associated with reduced quality of life and risk of colon and rectal cancer. The important thing in the treatment of constipation is diet, and sufficient dietary fiber intake is the most suitable and primarily usable for the prevention and treatment of constipation. It is a plant-derived component that is not digested by intestinal enzymes, and absorbs water in the large intestine. This softens the stool and increases its volume. In addition, it helps the growth of beneficial bacteria in the large intestine to enlarge the stool, and some components are fermented by beneficial bacteria in the large intestine, and their metabolites also help relieve constipation. In addition, there are reports that people who are physically active are less constipated. However, in the case of patients who do not have easy diet and exercise, drugs are used secondarily. In the case of bulking and osmotic relievers, abdominal distension and diarrhea may accompany, and long-term use of stimulant drugs may cause various complications. have. Considering the side effects of these therapeutic drugs, the search and research for foods or natural materials that are effective in the prevention and treatment of constipation should be more active.

Peanut ( Arachis hypogagea L.) is an annual herbaceous plant belonging to Leguminosae and is a high-calorie food containing a large amount of fat and protein. However, when sprouted peanuts are sprouted, they contain vitamin C, niacinamide, and pyridoxine. and resveratrol, which is a physiologically active substance effective for antioxidant, is reported to increase 90 times or more with an increase in the content of thiamin. Resveratrol is a physiologically active polyphenol contained in red grapes, peanuts, and cucumber. Germinated vegetables sprouted from seeds and germinated for about a week are smaller in size compared to general vegetables, but contain resveratrol and more nutrients than mature vegetables. Studies have reported that it is significantly higher than that of As such, sprouted peanuts are rich in nutrients and physiologically active ingredients and are expected to have a wide range of uses as functional food materials. Research on phytosterol has been active for several years, and domestic and foreign researchers are mainly focusing on sprouted peanuts and peanuts in Korea. Research on resveratrol content by region and clinical trials are being conducted, but in reality, food is limited to yogurt with sprouted peanuts added. Overseas research also compares resveratrol content of various food raw materials, anti-aging and anti-cancer effects. Research and development of processed products using sprouted peanuts are insignificant, with most research on antioxidant activity such as anti-inflammatory and anti-inflammatory.

Recently, as the efficacy of sprouted peanuts has been known, various studies have been conducted on the characteristics of peanut sprouts, anti-obesity, and anti-inflammatory. The physiological activities of sprouted peanuts include anti-inflammatory and antioxidant effects by inhibiting the expression of inducible nitric oxide synthase (iNOS) or cyclooxygenase-2 (COX-2), known as inflammatory regulators of macrophages. In addition, abundant caffeic acid effectively protects cells from oxidative damage, and has the effect of effective cell protection against activation of Nrf2 (Nuclear Respiratory Factor 2) and UV-B (Ultraviolet-B) induced oxidative stress. In addition, a protective effect by glutamate is shown in nerve cells, and the sprouted peanut extract lowers the expression of peroxisome proliferator-activated receptor (PPAR-γ), which regulates the expression of adiponectin, and MMP-2, an important regulator of adipocyte differentiation. By reducing (matrix metalloproteinase-2) and MMP-9 (matrix metallopeptidase 9), it has an anti-obesity effect that inhibits adipocyte proliferation and differentiation. In addition, according to a recent study, sprouted peanut extract relieves symptoms of erectile dysfunction by inhibiting PDE (phosphodiesterase) activity, relieves oxidative stress in cells in the testicle, and increases hormone synthesis such as testosterone, thereby lowering hormones in addition to prostatic hyperplasia. It is expected to show an effect on the symptoms of male menopause caused by this.

Lactobacillus is one of the most beneficial microorganisms available to humans. As a non-pathogenic bacteria, it can perform intestinal and bacteriostatic actions while inhabiting the human intestine. It also ferments sugars to produce a large amount of lactic acid and works well even under low pH and anaerobic conditions. It has the characteristics of growing and requiring various nutrients. When vegetables are fermented by lactic acid bacteria, their unique flavor and taste are improved, and the sensory properties are improved. Vitamin C and various physiologically active substances are well preserved by organic acids, and vitamin B ₁₂ and vitamin K, which were hardly present in the initial materials, are synthesized. It is known to have advantages such as becoming a hygienic food by inhibiting the growth and proliferation of putrefactive and pathogenic bacteria. Plant lactic acid bacteria are lactic acid bacteria that live in fermented foods such as kimchi and soy sauce and plant foods such as fruits, and are distinguished from animal lactic acid bacteria contained in milk or cheese. According to the data of the Japanese Journal of Probiotics Food Science, there are about 200 kinds of plant lactic acid bacteria, more than 10 kinds of animal lactic acid bacteria, and the most important feature is strong viability. In particular, it is known that plant lactic acid bacteria have a survival rate of more than 90% in artificial gastric juice (pH 2.5), but animal lactic acid bacteria that reproduce in a nutritionally balanced environment are only about 20-30%. It is known that the intake of lactic acid bacteria through plant lactic acid bacteria is more effective than that of animal lactic acid bacteria because Asians, who have been following a vegetarian diet, have longer intestines than Westerners. Research on plant lactic acid bacteria mainly consists of research and invention on pharmaceutical effects, such as exploring the characteristics of lactic acid bacteria, as well as research and invention on characteristics according to lactic acid bacteria culture method and fermentation period.

Meanwhile, Korean Patent Application Laid-Open No. 2015-0139004 discloses 'a method for multiple fermentation of sprouted peanuts and a composition comprising multiple fermented sprouts of peanuts', and Korean Patent Application Publication No. 2015-0093053 discloses 'content of resveratrol in germinated peanuts' It discloses a method for increasing the ', the germinated peanut extract prepared through it, and its use'. However, there has been no disclosure about 'a method for producing a sprouted peanut fermented product using a novel Pediococcus pentosaceus strain and a composition for improving constipation including a sprouted peanut fermented product prepared by the method' of the present invention.

The present invention has been derived from the above needs, the present inventors Pediococcus pentosaceus isolated from fermented food ( Pediococcus pentosaceus ) It was confirmed that the DU.La.P-11 strain had excellent β-glucosidase activity and excellent growth ability in a medium containing sprouted peanut extract. In fermenting the sprouted peanut extract using the DU.La.P-11 strain, by optimizing the concentration of the sprouted peanut extract, the fermentation time, and the concentration conditions of the mixed fruit and vegetable concentrate as an auxiliary material, the fermented sprouted peanut with significantly improved resveratrol content The present invention was completed by confirming that the fermented sprouted peanuts or a beverage containing the same was fed to an animal model inducing constipation by confirming that the effect of improving constipation was excellent.

In order to solve the above problems, the present invention comprises the steps of (1) preparing a concentrate of sprouted peanuts; (2) mixing and sterilizing the mixed fruit and vegetable concentrate with the sprouted peanut concentrate prepared in step (1); and (3) inoculating and fermenting the Pediococcus pentosaceus strain in the mixture of the sprouted peanut concentrate and the mixed concentrate of fruits and vegetables sterilized in the step (2); resveratrol) provides a method for producing fermented sprouted peanuts with enhanced content.

In addition, the present invention provides a sprouted peanut fermented product with enhanced resveratrol content prepared by the above method.

In addition, the present invention provides a health functional food composition for preventing or improving constipation comprising the sprouted peanut fermented product as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating constipation comprising the sprouted peanut fermented product as an active ingredient.

When the fermented sprouted peanut of the present invention is used, bowel activity is smooth and the number of feces remaining in the large intestine is reduced, so it can be very usefully used in the development of health functional foods or pharmaceuticals for improving intestinal function and constipation.

1 is a photograph of culturing 11 kinds of lactic acid bacteria isolated from fermented foods by smearing them on MRS agar medium to which bromocresol purple (BCP) is added.
Figure 2 shows the nucleotide sequence of 16S rRNA of Pediococcus pentosaceus DU.La.P-11 strain isolated in the present invention.
Figure 3 shows a phylogenetic diagram of the Pediococcus pentosaceus DU.La.P-11 strain isolated in the present invention.
4 is a four-dimensional response surface analysis results for the number of lactic acid bacteria in sprouted peanut fermented products according to fermentation time, the concentration of sprouted peanut extract, and the concentration of the auxiliary material (fruit and vegetable mixed concentrate).
5 is a four-dimensional response surface analysis result for the lactic acid content of sprouted peanut fermented products according to fermentation time, the concentration of sprouted peanut extract, and the concentration of the auxiliary material (fruit and vegetable mixed concentrate).
6 is a four-dimensional response surface analysis result for the resveratrol content of the sprouted peanut fermented product according to the fermentation time, the concentration of the sprouted peanut extract, and the concentration of the auxiliary material (fruit and vegetable mixed concentrate).
7 is a 4D superimposing for optimal fermentation conditions for the number of lactic acid bacteria, lactic acid content and resveratrol content of sprouted peanut fermented product according to fermentation time, the concentration of sprouted peanut extract and the concentration of auxiliary raw materials (fruit and vegetable mixed concentrate) This is the result of the response surface analysis.
8 is a resveratrol chromatogram result before and after fermentation when a sprouted peanut extract was fermented under optimal fermentation conditions using a Pediococcus pentosaceus DU.La.P-11 strain. (A): standard chromatogram, (B): sprouted peanut extract before fermentation, (C): fermented sprouted peanut.
9 is a graph showing the measurement of the number of stools when the fermented sprouted peanut of the present invention and a beverage containing the same are eaten in an animal model of constipation induced by loperamide administration. Normal; Normal group, Control; Constipation-induced group by administration of loperamide, PC; Loperamide administration and commercial laxative diet group, S1; Loperamide administration and sprouted peanut fermented food group, S2; Loperamide administration and beverage diet containing sprouted peanut fermented product.
10 is a graph showing the weight of feces when the fermented peanut sprouts of the present invention and a beverage containing the same were eaten in an animal model of constipation induced by loperamide administration. Normal; Normal group, Control; Constipation-induced group by administration of loperamide, PC; Loperamide administration and commercial laxative diet group, S1; Loperamide administration and sprouted peanut fermented food group, S2; Loperamide administration and beverage diet containing sprouted peanut fermented product.
11 is a graph showing the measurement of the water content of the stool when the fermented sprouted peanut of the present invention and a beverage containing the same were eaten in an animal model of constipation induced by loperamide administration. Normal; Normal group, Control; Constipation-induced group by administration of loperamide, PC; Loperamide administration and commercial laxative diet group, S1; Loperamide administration and sprouted peanut fermented food group, S2; Loperamide administration and beverage diet containing sprouted peanut fermented product.
12 is a photograph showing the size and shape of the stool remaining in the large intestine when the fermented sprouted peanut of the present invention and a beverage containing the same were fed to an animal model of constipation induced by loperamide administration. Normal; Normal group, Control; Constipation-induced group by administration of loperamide, PC; Loperamide administration and commercial laxative diet group, S1; Loperamide administration and sprouted peanut fermented food group, S2; Loperamide administration and beverage diet containing sprouted peanut fermented product.

In order to achieve the object of the present invention, the present invention

(1) preparing a concentrate of sprouted peanuts;

(2) mixing and sterilizing the mixed fruit and vegetable concentrate with the sprouted peanut concentrate prepared in step (1); and

(3) Inoculating and fermenting a Pediococcus pentosaceus strain in the mixture of the sprouted peanut concentrate and the mixed vegetable concentrate sterilized in the step (2); ) provides a method for producing fermented sprouted peanuts with enhanced content.

In the method for producing a sprouted peanut fermented product of the present invention, the Pediococcus pentosaceus ( Pediococcus pentosaceus ) strain is Pediococcus pentosaceus ( Pediococcus pentosaceus ) As a DU.La.P-11 strain, it was deposited with the Korea Research Institute of Bioscience and Biotechnology on December 20, 2018 (Accession No.: KCTC13781BP). The deposited specific strain is a strain having high beta-glucosidase activity, excellent growth ability in a medium to which sprouted peanut extract is added, and when producing a sprouted peanut fermented product using the strain, specific functionality It is possible to prepare a sprouted peanut fermented product with an enhanced content of resveratrol, a component.

In addition, in the method for producing fermented sprouted peanuts of the present invention, the mixed concentrate is one selected from the group consisting of banana concentrate, peach concentrate, mango concentrate, strawberry concentrate, fig concentrate, tomato concentrate, carrot concentrate, cabbage concentrate, and broccoli concentrate. It may include the above, but is not limited thereto.

In addition, the mixed concentrate of vegetables and galacto-oligosaccharide (galacto-oligosaccharide), malto-oligosaccharide (malto-oligosaccharide), fructo-oligosaccharide (fructo-oligosaccharide) and isomaltooligosaccharide (isomaltooligosaccharide) by adding one or more selected from the group consisting of can be manufactured, but is not limited thereto.

The method for producing a sprouted peanut fermented product of the present invention is specifically

(1) preparing the sprouted peanut concentrate of 1 to 30 Brix by concentrating the extract extracted by adding 5 to 20 times the weight of the sprouted peanut to the sprouted peanut under reduced pressure;

(2) mixing and sterilizing the sprouted peanut concentrate prepared in step (1) by 1 to 20% (v/v) of the mixed fruit and vegetable concentrate of 1 to 20 Brix relative to the weight of the sprouted peanut concentrate; and

(3) Pediococcus pentosaceus ( Pediococcus pentosaceus ) DU.La.P-11 strain having an accession number KCTC13781BP in a mixture of sprouted peanut concentrate and mixed vegetable concentrate sterilized in step (2) above the weight of the mixture 0.1-20% (v/v) inoculation and fermentation at 30-45° C. for 50-60 hours; may include,

more specifically

(1) preparing a sprouted peanut concentrate of 8-15 Brix by concentrating the extract extracted by adding 7 to 15 times the weight of the sprouted peanut to the sprouted peanut under reduced pressure;

(2) mixing and sterilizing the sprouted peanut concentrate prepared in step (1) with 5 to 18% (v/v) of the mixed fruit and vegetable concentrate of 3 to 10 Brix relative to the weight of the sprouted peanut concentrate; and

(3) Pediococcus pentosaceus ( Pediococcus pentosaceus ) DU.La.P-11 strain having an accession number KCTC13781BP in a mixture of sprouted peanut concentrate and mixed vegetable concentrate sterilized in step (2) above the weight of the mixture 1 to 10% (v / v) inoculation and fermenting at 35 to 40 ° C for 52 to 58 hours; may include,

more specifically

(1) preparing the sprouted peanut concentrate of 9-11 Brix by concentrating the extract extracted by adding 7 to 15 times the weight of the sprouted peanut to the sprouted peanut under reduced pressure;

(2) mixing and sterilizing the sprouted peanut concentrate prepared in step (1) by 5 to 18% (v/v) of the mixed fruit and vegetable concentrate of 4 to 6 Brix relative to the weight of the sprouted peanut concentrate; and

(3) Pediococcus pentosaceus ( Pediococcus pentosaceus ) DU.La.P-11 strain having an accession number KCTC13781BP in a mixture of sprouted peanut concentrate and mixed vegetable concentrate sterilized in step (2) above the weight of the mixture 1 to 10% (v / v) inoculation and fermenting at 35 to 40 ° C. for 53 to 57 hours; may include,

even more specifically

(1) preparing the sprouted peanut concentrate of 10 Brix by concentrating the extract extracted by adding 10 times the weight of the sprouted peanut to the sprouted peanut under reduced pressure;

(2) mixing and sterilizing the sprouted peanut concentrate prepared in step (1) by 8.5% (v/v) of the mixed fruit and vegetable concentrate of 5 Brix based on the weight of the sprouted peanut concentrate; and

(3) Pediococcus pentosaceus ( Pediococcus pentosaceus ) DU.La.P-11 strain having an accession number KCTC13781BP in a mixture of sprouted peanut concentrate and mixed vegetable concentrate sterilized in step (2) above the weight of the mixture 2% (v/v) inoculation and fermentation at 37° C. for 55 hours; may include.

The present invention also provides a sprouted peanut fermented product with enhanced resveratrol content prepared by the above method.

In order to further improve the taste and flavor of the sprouted peanut fermented product of the present invention, apple, persimmon, blueberry, blackberry, blackcurrant, acai berry, bokbunja, pomegranate, grape, pear, peach, banana, strawberry, tomato, carrot, One or more fruit extracts selected from plum, plum, cherry, actinidia, mulberry, melon, kiwi, melon, tangerine, orange, grapefruit, tangerine, melon, cherry, melon, black superberry, mango, lemon and pineapple can be added However, it is not limited thereto.

The present invention also provides a health functional food composition for preventing or improving constipation comprising the sprouted peanut fermented product as an active ingredient.

The health functional food composition may be prepared in any one formulation selected from powders, granules, pills, tablets, capsules, candy, syrups and beverages, or may be prepared by adding other foods or ingredients of foods, according to a conventional method. can be appropriately prepared.

Examples of foods to which the active ingredient of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, It may be in any one form selected from tea, drinks, alcoholic beverages, and vitamin complexes, and includes all health functional foods in a conventional sense.

In one embodiment of the present invention, when the health functional food composition of the present invention is prepared in a beverage formulation, in addition to the sprouted peanut fermented product, fructooligosaccharide, polydextrose, citric acid, vitamin C (vitamin C), enzymatically modified stevia, may further include one or more selected from the group consisting of brown rice concentrate and grape concentrate, preferably 1 to 5 parts by weight based on 100 parts by weight of fermented sprouted peanuts parts fructooligosaccharide, 1-5 parts by weight polydextrose, 0.01-1 parts by weight citric acid, 0.01-1 parts by weight vitamin C, 0.01-0.05 parts by weight enzyme-treated stevia ( enzymatically modified stevia), 1 to 5 parts by weight of brown rice concentrate, and 1 to 10 parts by weight of grape concentrate, but is not limited thereto.

The health functional food includes various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, It may contain a pH adjuster, a stabilizer, a preservative, glycerin, alcohol, a carbonation agent used in carbonated beverages, and the like. In addition, it may contain the pulp for the production of natural fruit juices and vegetable beverages. These components may be used independently or in combination.

The health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatine and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used.

The present invention also provides a pharmaceutical composition for preventing or treating constipation comprising the sprouted peanut fermented product as an active ingredient.

The composition of the present invention may further include suitable carriers, excipients and diluents conventionally used in the preparation of pharmaceutical compositions.

The pharmaceutical dosage form of the sprouted peanut fermented product of the present invention may be used in the form of a pharmaceutically acceptable salt thereof, and may be used alone or in combination with other pharmaceutically active compounds as well as in an appropriate group.

The composition comprising the sprouted peanut fermented product according to the present invention is a powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. oral dosage forms, external preparations, suppositories, and sterile injection solutions, respectively, according to a conventional method. It can be formulated and used in the form of Carriers, excipients and diluents that may be included in the composition comprising the sprouted peanut fermented product include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium Various compounds or mixtures including phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can In the case of formulation, it is prepared using diluents or excipients, such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the sprouted peanut fermented product, for example, starch, calcium carbonate, oligosaccharide , sucrose or lactose, gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The preferred dosage of the sprouted peanut fermented product of the present invention varies depending on the patient's condition and weight, the degree of disease, drug form, administration route and period, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the sprouted peanut fermented product of the present invention is preferably administered at 00001 to 100 mg/kg per day, preferably at 0001 to 100 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.

The sprouted peanut fermented product of the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

Hereinafter, an embodiment of the present invention will be described in detail. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Experimental materials and methods

One. Lactobacillus selection

1) Isolation of lactic acid bacteria

Fermented food samples used to isolate lactic acid bacteria were collected by collecting dozens of kimchi and pickled vegetables from households and restaurants and used as lactic acid bacteria separation sample solutions. After diluting ^{each sample solution with sterile distilled water to 10 -1} ~ 10 ^{-5, 0.004} % Bromocresol purple (BCP; Bromocresol purple) was added to a commercially available MRS agar medium by aliquoting 100 μl and plated, and the characteristic colonies of lactic acid bacteria were isolated from the independent colonies that appeared after culturing at 37° C. for 24 hours. The pure isolated lactic acid bacteria were inoculated on MRS agar sloping medium, incubated at 37°C for 24 hours, and then stored at 4°C in refrigerator.

2) Beta-glucosidase (β-Glucosidase) active strain selection

For the selection of strains having beta-glucosidase activity, inoculate the strain on esculin agar medium (esculin 0.1%, peptone 1.8%, ferric citrate 0.1%, agar 2%) containing esculin to the medium. The color change was observed. Esculin is separated into glucose and esculetin by beta-glucosidase, and esculetin reacts with ferric ammonium citrate to form a black complex around the colonies. ) is formed. Therefore, strains forming black spots around colonies cultured on esculin agar medium were judged as strains having beta-glucosidase activity and selected.

3) Beta-glucosidase (β-Glucosidase) activity measurement

Beta-glucosidase activity was measured by inoculating a strain pre-cultured for 12 hours in a commercial MRS liquid medium supplemented with 1% carboxymethyl cellulose (CMC; carboxymethyl cellulose), culturing at 37°C for 24 hours, and then incubating the culture medium at 4°C for 6,000. The cells were removed by centrifugation at rpm for 15 minutes, and 0.5 ml of the supernatant was mixed with 1 ml of a 5 mM ρ-NPG (ρ-nitrophenyl-β-D-glucopynanoside) solution and reacted at 37° C. for 30 minutes. The reaction solution was stopped by adding 1ml of 1M sodium carbonate (Na ₂ CO ₃ ) solution, and the absorbance of the generated para-nitrophenol (ρ-NP; p-nitrophenol) was measured at 405 nm, and then the calibration of para-nitrophenol. Concentrations were converted using the curve.

4) Bacterial growth test according to the concentration of sprouted peanut extract of isolated lactic acid bacteria

The isolated strains were streaked on MRS agar medium to which sprouted peanut extracts were added by concentration (5, 10, 15, 20, 50, and 100% (v/w)) and cultured at 37°C for 24 hours to determine the degree of bacterial growth. observed.

5) Review of Fermentation of Sprout Peanut Extract

In order to investigate the fermentability of the isolated strain on sprouted peanuts, the lactic acid bacteria selected using the extract extracted by adding 10% (w/v) of sprouted peanuts were pre-cultured in MRS liquid medium for 12 hours and then 2% (v/v) v) After inoculation, fermentation was performed by varying the fermentation time.

6) Base sequence analysis and phylogenetic classification of the final selected strain

After sequencing analysis of the 16S rRNA gene base sequence of the finally selected strain, the similarity (%) between the isolated strain and the standard strain on the database was confirmed using the NCBI database.

2. Sprout Peanut Extraction Treatment Conditions

The sprouted peanut raw material used in the present invention was purchased in July of 2018, with a length of around 10 cm, cut to a predetermined size, and extracted at 68° C. for 7 hours by adding 10 times of water. These extracts were concentrated under reduced pressure, and the final concentration was 15 Brix, and the concentration was adjusted as a raw material for lactic acid fermentation.

3. Sprout Peanut Fermentation Conditions

1) Experimental plan for fermentation optimization

To optimize the fermentation conditions of sprouted peanuts, repose surface methodology (RSM) was used. As fermentation conditions, Central Composite Design (CCD) was used. Fermentation conditions of sprouted peanuts were three independent variables: fermentation time (X ₁ : 24, 36, 48, 60, 72 hr), The concentration (X ₂ : 4, 6, 8, 10, 12 Brix) and the concentration (X ₃ : 2, 4, 6, 8, 10Brix) of the mixed vegetable and vegetable concentrate (60 Brix) as an auxiliary material were set, and the dependent variable was after fermentation. The number of lactic acid bacteria (Log10 CFU/mL), lactic acid content and resveratrol content were measured. In addition, the effect of fermentation conditions on the dependent variables of the sprouted peanut extract was analyzed by 4D response surface analysis using the Mathematica program based on the predicted model equation.

Experimental design for optimizing sprouted peanut fermentation conditions parameter -2 -One 0 One 2 X ₁ fermentation time (hour) 24 36 48 60 72 X ₂ Sprout Peanut Extract Concentration (Brix) 4 6 8 10 12 X ₃ Concentration of additives (Brix) 2 4 6 8 10

Central synthesis plan for optimizing fermentation of sprouted peanut extract Experiment number ¹⁾ Fermentation conditions Fermentation time (hour) Sprout Peanut Extract Concentration (Brix) Sub-ingredient Concentration (Brix) One 60(1) 10(1) 8(1) 2 60(1) 10(1) 4(-1) 3 60(1) 6(-1) 8(1) 4 60(1) 6(-1) 4(-1) 5 36(-1) 10(1) 8(1) 6 36(-1) 10(1) 4(-1) 7 36(-1) 6(-1) 8(1) 8 36(-1) 6(-1) 4(-1) 9 48(0) 8(0) 6(0) 10 48(0) 8(0) 6(0) 11 48(0) 8(0) 6(0) 12 48(0) 8(0) 6(0) 13 48(0) 8(0) 6(0) 14 72(2) 8(0) 6(0) 15 24(-2) 8(0) 6(0) 16 48(0) 12(2) 6(0) 17 48(0) 4(-2) 6(0) 18 48(0) 8(0) 10(2) 19 48(0) 8(0) 2(-2)

¹⁾ Experimental condition number by central synthesis plan

2) Fermentation

A new lactic acid bacteria (Pediococcus pentosaceus DU.La.P-11(KCTC13781BP)) spawn that was pre-cultured for 12 hours in MRS liquid medium by using sprouted peanut extract as the basic medium and varying the addition amount of the mixed concentrate, which is an auxiliary raw material, according to the experimental plan. After inoculation with 2% (v/v), the fermentation time was varied and fermented.

3) Measurement of growth, pH and acidity of fermented products

The lactic acid bacteria growth of the fermented product was analyzed by measuring the absorbance at 660 nm with a spectrophotometer (Optizen 2120UV, Mecasys co., Ltd., Korea). The pH was measured using a pH meter (Metter Toledo Group, Switzerland), and the acidity was calculated by converting the consumed 0.1N NaOH solution into lactic acid content (%) by neutralizing titration to pH 8.3 with 0.1N NaOH solution. did.

4) Measurement of lactic acid content

For lactic acid content analysis, the sprouted peanut extract and its fermented product sample were filtered with a 0.45 μm membrane filter paper, and HPLC analysis was performed. Lactic acid content analysis conditions were analyzed using a column Aminex ^ⓡ HPX-87H (7.5×300 mm, 9㎛, BIO-RAD Laboratories, USA) with 5mM sulfuric acid at a flow rate of 0.6mL/min and a detector PDA 214nm. did. All standards were used by Sigma (Sigma-Aldrich Co., USA).

5) Resveratrol content measurement

The resveratrol content of the sprouted peanut extract and its fermented product was analyzed by an HPLC system (Alliance e2695, Waters Co., USA) after the sample solution was filtered with a 0.45 μm membrane filter paper (Table 3). For the standard product, a standard calibration curve was prepared using resveratrol (Sigma-Aldrich Co., USA), and then the content was calculated.

4. Constipation improvement animal experiment

1) Breeding and diet of experimental animals

As experimental animals, 50 4-week-old male Sprague Dawley rats were purchased from Hyochang Science (Daegu, Korea), and they were acclimatized by allowing them to freely consume general feed and water for a week. At this time, the laboratory environment was controlled at a temperature of 22±1°C, relative humidity of 63±5%, and light and dark at intervals of 12 hours. After acclimatization for one week, according to the randomized block design, at the second week, 10 animals per treatment group were divided into 5 groups so that the weight of each group was similar, and 3 animals were separated in stainless cages and reared for 4 weeks.

Loperamide (Sigma, USA) is an antidiarrheal agent for the treatment of diarrhea. When administered continuously for a certain period of time, the secretion of mucus in the large intestine and intestinal motility decrease, resulting in a longer transit time, which is a drug that stops diarrhea. In case of administration to the normal group without thyroid disease, it causes constipation. Therefore, the experimental group consisted of a normal group fed a general diet, a control group in which constipation was induced by loperamide administration while feeding a general diet, and a commercial constipation improving agent (Dulcolax S, Boehringer Ingelheim Korea) administered. A positive control group (PC) in which constipation was induced by loperamide administration, an experimental group (S1) in which constipation was induced by loperamide administration after eating the fermented sprouted peanut of the present invention (Preparation Example 1) (S1), fermented sprouted peanut It was composed of an experimental group (S2) in which constipation was induced with loperamide after eating a beverage containing (Preparation Example 2). This animal experiment was approved by the Animal Experimentation Ethics Committee of Daegu University and was carried out according to its regulations.

The duration of the animal experiment was 5 weeks, and the experiment was conducted for 4 weeks after acclimatization to the environment for 1 week. AIN-76A general diet was used for the pellet-type solid feed supplied to the rat diet. The normal group and the control group were fed only a general diet, the S1 experimental group lyophilized the drink containing the sprouted peanut fermented product of Preparation Example 1, and the S2 experimental group lyophilized the drink containing the sprouted peanut fermented product of Preparation Example 2, respectively, and administered 500mg/kg per day. It was administered orally once at a certain time. Among the experimental groups, except for the normal group, the control group, the positive control group, and the S1 and S2 experimental groups were induced by loperamide administration.

2) Treatment of experimental animals

After the dietary intake period for 4 weeks (28 days) was over, the normal group, who did not induce constipation for 5 days from the 29th to the 33rd day, received only 0.9% physiological saline per oral (po), and the control group induced constipation. For the positive control group, S1 and S2 experimental groups, loperamide (5 mg/kg) was dissolved in 0.9% physiological saline and administered orally twice a day (9 am and 9 pm) for 5 days. After measuring the intestinal transit time on the 33rd day of the experiment, on the 34th day, all experimental animals of the experimental group were sacrificed by inhalation anesthesia with ether.

3) Measurement of weight and dietary efficiency

Body weight was measured every week starting from the first feeding day of the experimental diet, and body weight was measured every day during the constipation induction period. The experimental diet was supplied at a fixed time by 100 g once a day, and the amount of food intake was measured by measuring the remaining amount after 24 hours, and the amount of food intake was calculated with the difference.

4) Measurement of the number of stools, stool weight and stool moisture content

The measured values of the number, weight, and water content of stool were divided into two periods before and after induction of constipation. After the fasting plate was installed in the cage of each experimental animal, a total of 4 times were collected on the day of the start of the experiment and every week thereafter. The moisture content of the feces was determined by drying the feces in a dry oven at 70° C. for 24 hours, measuring the weight, and using the difference between the feces weight and the feces weight after drying.

5) Measurement of the number of residual variables in the intestinal tract and observation of the shape

The number of stools in the intestine was measured on the third day after administration of loperamide by taking the entire site from the cecum to the rectum and fixing it with 10% formaldehyde to measure the number of stools remaining in the large intestine. was confirmed as

6) Measurement of serum lipid content

For serum analysis, rats were anesthetized and laparotized on the day of the end of the experiment, blood was collected from the inferior vena cava, and collected in a sterile tube treated with heparin for use in the experiment. The collected blood was centrifuged at 3,000 rpm and 4°C for 15 minutes, and serum was separated and stored at -70°C until analysis. The content of triglycerides in blood is determined by the kit reagent for measuring triglycerides (AM 157S-K, Asan Pharm. Co., Seoul, Korea), and the content of total cholesterol is measured by the kit reagent for measuring total cholesterol (AM 202-K, Asan Pharm. Co.). , Seoul, Korea), and HDL-cholesterol content was measured with a kit reagent for measuring HDL cholesterol (AM 203-K, Asan Pharm. Co., Seoul, Korea). The blood LDL-cholesterol concentration was calculated using the Friedewald formula (LDL cholesterol = (total cholesterol - HDL cholesterol) - (triglycerides / 5)).

7) Liver function enzyme measurement

Blood GOT (Glutamic oxaloacetic transaminase) and GPT (Glutamic pyruvic transaminase) were measured using Reitman-Frankel's method (Reitman, S. et al, 1957, American Journal of Clinical Pathology 28, 56.63.). It was measured using a kit reagent (Asan Pharm. Co., Seoul, Korea).

8) Statistical processing

All experimental results of animal experiments were expressed as mean values and ± standard deviations. Statistical analysis system (SAS version 9.2, SAS Institute Inc., USA) was used for one-way ANOVA analysis and then Duncan's multiple at the level of P < 0.05. Significance was verified by the range test.

Preparation Example 1. Preparation of sprouted peanut fermented product

The sprouted peanut extract was concentrated under reduced pressure at 68° C. for 7 hours by adding 10 times water to the sprouted peanuts cut to a certain size to obtain a sprouted peanut concentrate having a final concentration of 10 Brix. 8.5% of 5 Brix mixed fruit and vegetable concentrate (banana concentrate, peach concentrate, mango concentrate, strawberry concentrate, fig concentrate, tomato concentrate, carrot concentrate, cabbage concentrate and broccoli concentrate) was added to the obtained sprouted peanut concentrate and mixed, followed by mixing at 85°C Sterilized for 30 minutes in a Pediococcus pentosaceus in a mixture of sterilized sprouted peanut concentrate and mixed vegetable concentrate DU.La.P-11 strain (KCTC13781BP) culture solution (10 ⁷ CFU/mL) was inoculated with 2% (v/v) and then fermented at 37° C. for 55 hours to prepare sprouted peanut ferment. Thereafter, sterilization was performed, followed by filtration, and used in the following examples.

Preparation Example 2. Preparation of beverage containing sprouted barley fermented product

Beverages containing sprouted barley fermented products Based on 100 parts by weight of the sprouted peanut fermented product of Preparation Example 1, 3 parts by weight of fructooligosaccharide, 3 parts by weight of polydextrose, 0.05 parts by weight of citric acid, 0.05 parts by weight of vitamin C (vitamin C) , 0.02 parts by weight of enzymatically modified stevia, 2 parts by weight of brown rice concentrate and 5 parts by weight of grape concentrate were added and mixed.

Example 1. Isolation and identification of novel lactic acid bacteria

1) Separation of lactic acid bacteria from fermented foods

To isolate lactic acid bacteria from fermented foods, MRS agar medium containing BPB (Bromocresol purple) was used. After diluting several fermented food samples to ^{a concentration of 10 -1} to 10 ^-4 using physiological sterile saline, smearing them on MRS agar medium, culturing at 37°C, observing the size, color, shape, and transparency of the strain. 11 types of lactic acid bacteria were isolated and subcultured to purely isolated single colonies and named them DU.La.P-1~11.

2) Beta-glucosidase (β-Glucosidase) active strain selection

After culturing using esculin agar to select a beta-glucosidase active strain, a strain forming a black complex around a colony was selected as a beta-glucosidase active strain. was considered. Among the isolated strains, all isolated strains except the DU.La.P-1 strain formed black spots around the colonies, indicating that beta-glucosidase activity was present (FIG. 1). In addition, as a result of measuring beta-glucosidase activity against 11 isolated lactic acid bacteria, the beta-glucosidase activity of the DU.La.P-1 strain that did not form black spots on the esculin agar medium was 2.59 mU. /mL was the lowest, and the DU.La.P-11 strain had the highest beta-glucosidase activity of 5.30 mU/mL (Table 4).

Measurement of black spot length and beta-glucosidase activity of isolated lactic acid bacteria isolate Black spot length (mm) Beta-glucosidase activity
(unit: mU/ml) DU.La.P-1 0.00±0.00 2.59±0.06 DU.La.P-2 16.00±1.00 4.70±0.10 DU.La.P-3 11.67±1.53 4.52±0.16 DU.La.P-4 19.33±1.53 4.61±0.04 DU.La.P-5 17.67±0.58 4.77±0.13 DU.La.P-6 13.67±0.58 4.88±0.03 DU.La.P-7 20.33±0.58 5.08±0.09 DU.La.P-8 20.00±2.00 4.87±0.15 DU.La.P-9 22.33±1.53 4.60±0.04 DU.La.P-10 19.33±1.15 4.65±0.05 DU.La.P-11 21.00±1.00 5.30±0.03

3) Growth ability of isolated strains according to the concentration of sprouted peanut extract

As a result of examining the growth ability of lactic acid bacteria isolated on agar medium containing 5, 10, 15, 20, 50 and 100% (v/w) sprouted peanut extract, DU.La.P-1 and DU.La. All isolated lactic acid bacteria, except for the P-3 strain, were found to have distinctly formed colonies in the medium containing sprouted peanut extract (Table 5).

Growth ability of isolated strains on agar medium containing sprouted peanut extract isolate strain Growth ability according to the added concentration (%) of sprouted peanut extract 5 10 15 20 50 100 DU.La.P-1 ++ ++ + + + + DU.La.P-2 +++ +++ +++ +++ +++ +++ DU.La.P-3 ++ ++ ++ ++ ++ ++ DU.La.P-4 +++ +++ +++ +++ +++ +++ DU.La.P-5 +++ +++ +++ +++ +++ +++ DU.La.P-6 +++ +++ +++ +++ +++ +++ DU.La.P-7 +++ +++ +++ +++ +++ +++ DU.La.P-8 +++ +++ +++ +++ +++ +++ DU.La.P-9 +++ +++ +++ +++ +++ +++ DU.La.P-10 +++ +++ +++ +++ +++ +++ DU.La.P-11 +++ +++ +++ +++ +++ +++

+; Weak growth, ++; medium growth, +++; strong fertility.

4) Final strain selection

In order to examine whether the sprouted peanuts were fermented for the isolated lactic acid bacteria, the beta-glucosidase activity was good for three strains (DU.La.P-7, DU.La.P-8 and DU.La.P-). 11) was inoculated into 10 Brix sprouted peanut extract and cultured at 37°C for 48 hours to measure the number of viable cells, pH and acidity to confirm the growth and fermentation ability of the strain. As a result, all three strains were found to be able to grow in sprouted peanuts, and the DU.La.P-7 and DU.La.P-11 strains appeared to proliferate vigorously within 24 hours, and after fermentation The DU.La.P-11 strain showed the largest increase in the number of bacteria. In the case of pH, it appeared that the fermentation time decreased in all strains, and there was no significant difference between the strains. In the case of acidity, it was found that there was no significant difference between the strains, and it was found that the acidity tended to increase a lot during 24 hours of fermentation. Therefore, the DU.La.P-11 strain, which has excellent beta-glucosidase activity and the largest increase in the number of lactic acid bacteria and the decrease in pH, was finally selected as the fermented strain of peanut sprout.

Changes in pH, acidity and number of viable cells of isolated strains on agar medium with sprouted peanut extract added Fermentation time (day) isolate DU. La. P-7 DU. La. P-8 DU. La. P-11 pH 0 5.44±0.00 5.37±0.00 5.32±0.00 One 3.70±0.01 3.68±0.00 3.70±0.00 2 3.67±0.01 3.67±0.00 3.65±0.00 Acidity (%) 0 0.05±0.00 0.05±0.00 0.05±0.01 One 0.22±0.01 0.25±0.01 0.24±0.01 2 0.24±0.00 0.26±0.00 0.25±0.00 number of lactic acid bacteria
(Log10 CFU/ml) 0 10.87±0.01 11.08±0.02 11.12±0.03 One 17.47±0.03 13.54±0.34 17.49±0.01 2 18.12±0.03 17.29.±0.01 18.43±0.01

5) Identification of the final selection strain (DU. La. P-11)

Among the finally isolated strains, DU with the highest beta-glucosidase activity and fermentation power. La. P-11 nucleotide sequence of the 16s rDNA gene of the strains (SEQ ID NOS. 1, 2) analysis by the comparison with NCBI blast DB, Phedi O Rhodococcus genus (Pediococcus sp.) Phedi O Lactococcus pen soil three mouse (Pediococcus belonging to pentosaceus ) and showed 99% homology (Table 7). After aligning the nucleotide sequences using the Bioedit program and the Clustal X program, the close relationship was investigated. As a result, it showed 99% homology with Pediococcus pentosaceus DSM 20336 (FIG. 3). Therefore, the DU.La.P-11 strain was identified as Pediococcus pentosaceus, and this Pediococcus pentosaceus The strain was named DU.La.P-11 and was deposited at the Korea Institute of Biotechnology and Biotechnology Biological Resources Center (KCTC) on December 20, 2018, and was given an accession number KCTC13781BP.

16s rDNA sequencing results of DU.La.P-11 strain standard strain homology Difference Pediococcus pentosaceus DSM 20336 1446/1449 (99%) 2/1449 (0%) Pediococcus stilesii FAIR-E 180 1424/1448 (99%) 0/1448 (0%) Pediococcus acidilactici DSM 20284 1421/1449 (98%) 2/1449 (0%) Pediococcus claussenii ATCC BAA-344 1409/1451 (97%) 4/1451 (0%) Pediococcus argentinicus CRL 776 1408/1450 (97%) 4/1450 (0%) Pediococcus acidilactici NGRI 0510Q 1411/1455 (97%) 16/1455 (1%) Pediococcus claussenii P06 1406/1451 (97%) 3/1451 (0%)

Example 2. Optimization of fermentation conditions of sprouted peanut extract

1) Fermentation characteristics and active ingredient content according to fermentation conditions

Table 8 shows the characteristics of fermented products obtained under 19 fermentation conditions designed by the central synthesis plan with fermentation time, sprouted peanut extract concentration, and auxiliary raw material (fruit and vegetable mixed concentrate) concentration as independent variables to set the optimal fermentation conditions for sprouted peanut extract. same. Response surface analysis was performed using this, and regression equations for the number of lactic acid bacteria, lactic acid content, and resveratrol content values were obtained (Table 9). In addition, the optimal fermentation conditions and fermentation characteristic values for each variable were predicted and shown in Table 10, and the results of their 4-dimensional response surface analysis are shown in FIGS. 5 to 7 .

After setting the ranges of each independent variable as shown in Table 1 according to the central synthesis programming method, 19 fermentation conditions were set as shown in Table 2 using Design Expert, and sprouted peanut extract was fermented. The number of lactic acid bacteria in the sprouted peanut extract according to the fermentation conditions was in the range of 11.75 to 14.86 Log10 CFU/mL (Table 8), and the regression equation based on this was shown in Table 9. ^{The R 2} value for the number of lactic acid bacteria was 0.8574, which showed high reliability, and the P- value showed a significant level within 1%. On the other hand, the lack of fit test value is a statistic that tests the fit of the model. When the P value is less than 0.05, there is a problem with the model fit, and when it is larger than this, the model is judged to be suitable. As a result of ANOVA, the P- value of the lack of fit test for the model of the number of lactic acid bacteria was 0.0981, indicating that the model was suitable (Table 9). In the effect on fermentation conditions, the number of lactic acid bacteria was found to be affected by the sprouted peanut extract concentration and fermentation time, and was not affected by the concentration of auxiliary materials (Table 10). As a result of the regression analysis predicted by the response surface model according to the fermentation conditions, the normal point was the maximum point, and as a result of calculating the optimal point, the maximum value of the number of lactic acid bacteria was 15.20 log10 CFU/mL, and the fermentation condition at this time was a fermentation time of 61.02 hours. , the sprouted peanut extract concentration was 10.86 Brix and the auxiliary material concentration was 5.81 Brix (Table 11). The four-dimensional response surface for the number of lactic acid bacteria in the fermented product obtained according to the experimental conditions showed the form of a maximum point as shown in FIG. 4, and it was found that the sprouted peanut extract concentration increased and the fermentation time increased.

Experimental value of fermentative properties for setting optimal fermentation conditions of sprouted peanut extract by response surface analysis Experiment number ¹⁾ Fermentation characteristics and active ingredient content Number of lactic acid bacteria (Log10 CFU/ml) Lactic acid content (mg/ml) Resveratrol content (μg/ml) One 14.80 12.89 0.66 2 14.85 13.16 0.80 3 12.12 9.59 0.37 4 12.08 10.43 0.41 5 13.98 11.15 0.62 6 13.84 10.80 0.87 7 12.96 9.53 0.47 8 12.57 8.96 0.46 9 14.03 11.07 0.65 10 14.44 11.20 0.74 11 14.86 11.39 0.73 12 14.74 11.73 0.66 13 14.57 11.33 0.79 14 14.04 11.67 0.75 15 11.75 8.87 0.63 16 13.84 12.19 0.66 17 12.04 9.05 0.31 18 13.41 10.21 0.54 19 13.08 10.89 0.70

¹⁾ Experiment number by central synthesis programming method

Polynomial calculated by RSM program to set optimal fermentation conditions for sprouted peanut extract Number of lactic acid bacteria (Log10 CFU/ml) Lactic acid content (mg/ml) Resveratrol content
(μg/ml) Intercept(β0) Linear -0.576081 -1.056976 -1.239189 β1 0.183941 0.187479 -0.000144 β2 1.216774 0.470498 0.341639 β3 1.219611 0.938421 0.133260 Quadratic β11 -0.002836 -0.001798 -0.000066864 β22 -0.099291 -0.042863 -0.015220 β33 -0.080228 -0.047238 -0.006782 cross product β12 0.016458 0.013385 0.000625 β13 -0.002813 -0.010573 0.000313 β23 -0.010625 0.010938 -0.011250 Lack of fit
t-value 4.10 2.81 2.43 P-value 0.0981 0.164 0.2047 R ² 0.8754 0.9604 0.8730 Significance 0.0039 <.0001 0.0042

Results of regression analysis to establish optimal conditions for fermentation of sprouted peanut extract response variable F-Ratio Fermentation time (hour) Sprout Peanut Extract Concentration
(Brix) Concentration of additives
(Brix) Number of lactic acid bacteria (Log10 CFU/ml) 6.09 ^** 11.60 ^*** 2.29 Lactic acid content (mg/ml) 21.82 ^*** 34.48 ^*** 3.08 ^* Resveratrol content (μg/ml) 0.19 13.84 ^*** 2.77 ^*

*; p < 0.1, **; p < 0.05, ***; p < 0.01.

In addition, the lactic acid content according to the fermentation conditions was found to be in the range of 8.87 to 13.16 mg/mL (Table 8), and the regression formula for the lactic acid content based on this is shown in Table 9, and the R ² value is 0.9604, which is within 1%. significance was confirmed. As a result of ANOVA, the P-value of the test for lack of fit of lactic acid content was 0.1694, indicating that the model was suitable (Table 9). As shown in Table 10, the effect on fermentation conditions was the greatest of fermentation time and the concentration of sprouted peanut extract, followed by the concentration of additives. The normal point predicted by the response surface model according to the fermentation conditions was the maximum point, and the maximum value was 13.65%, and it was confirmed that the fermentation time was 62.63 hours, the sprout peanut extract concentration was 11.08 Brix, and the auxiliary material concentration was 5.26 Brix (Table 11). . The change of the lactic acid content according to the fermentation conditions through the four-dimensional reaction surface is shown in FIG. 5 .

The resveratrol content according to the fermentation conditions was in the range of 0.31 to 0.87 μg/mL (Table 8), and the regression equation based on this is shown in Table 9. The R ² value of the regression equation for this model was 0.8730, 1 Significance within % was recognized. As a result of ANOVA, the P-value of the lack of fit test for the fermentation condition model of resveratrol content was 0.2047, indicating that the model was suitable (Table 9). The resveratrol content was found to be affected the most by the sprouted peanut extract concentration, followed by the concentration of additives, and was not affected by the fermentation time (Table 10). As shown in Table 11, the predicted normal point of the resveratrol content was the maximum, and the maximum value was 0.85 μg/mL, at this time, the fermentation time was 52.19 hours, the sprout peanut extract concentration 10.71 Brix, and the auxiliary material concentration 3.14 Brix It was confirmed. As for the resveratrol content of the fermented product, it was found that the resveratrol content increased as the sprouted peanut extract concentration and the auxiliary material concentration were higher, when viewed from the four-dimensional response surface according to the fermentation conditions (FIG. 6).

Optimal Fermentation Conditions for Sprout Peanuts for Fermentation Characteristics response variable predictive response
shape response value fermentation time
(hour) Fresh Peanut Extract Concentration (Brix) Concentration of additives
(Brix) result number of lactic acid bacteria Ieast 57.12 4.31 5.71 11.29 maximum maximum 61.02 10.86 5.81 15.20 lactic acid content Ieast 27.07 6.50 4.73 8.67 maximum maximum 62.63 11.08 5.26 13.65 Resveratrol content Ieast 49.95 4.06 5.36 0.20 maximum maximum 52.19 10.71 3.14 0.85

2) Prediction and empirical experiment of optimal fermentation conditions of sprouted peanut extract

In order to set the fermentation conditions of the sprouted peanut extract, each reaction surface was superimposing to obtain optimal fermentation conditions that satisfy all of the lactic acid bacteria number, lactic acid content and resveratrol content. It was. The optimal fermentation conditions range for the sprouted peanut extract were 50 to 60 hours of fermentation time, 8 to 12 Brix of the sprouted peanut extract concentration, and 4 to 6 Brix of the auxiliary material (fruit and vegetable concentrate) concentration (Table 12). Therefore, in order to confirm the reliability of the model formula for the predicted results, actual fermentation is performed under arbitrary conditions, that is, fermentation time 55 hours, sprouted peanut extract concentration 10 Brix, and auxiliary material concentration 5 Brix within the predicted optimal condition range. And as a result of measuring the number of lactic acid bacteria, lactic acid content and resveratrol content of the fermented product, it was found to be at a level similar to the predicted values (Table 13).

Prediction of optimal fermentation conditions range for fermentation characteristics and active ingredient content changes of sprouted peanuts by superimposing the reaction surface Fermentation conditions Range of predicted conditions (optimal point) Fermentation time (hour) 50-60 (55) Sprout Peanut Concentration (Brix) 8-12 (10) Sub-ingredient Concentration (Brix) 4-6(5)

Comparison of predicted condition values and measured condition values for the fermentation of sprouted peanuts Predicted condition (A) ¹⁾ Measured condition (B) ^2)* B/A×100(%) Number of lactic acid bacteria (Log10 CFU/ml) 15.04 14.85±1.05 98.73 Lactic acid content (mg/ml) 12.75 12.16±0.98 95.37 Resveratrol content (μg/ml) 0.81 0.75±0.07 88.23

¹⁾ The value calculated from the prediction equation for the response variable.

²⁾ Optimal conditions for independent variables: fermentation time 55 hours, sprouted peanut concentration 10 Brix, auxiliary material concentration 5 Brix

^{*) The} mean value is three replicate standard deviations.

Example 3. Comparison of useful component content of sprouted peanuts before and after fermentation of lactic acid bacteria

Lactobacillus Pediococcus pentosaceus isolated in the present invention ( Pediococcus pentosaceus) When the sprouted peanut extract was fermented under optimal fermentation conditions using the DU.La.P-11 strain (KCTC13781BP), the results of examining the lactic acid and resveratrol content changes of sprouted peanuts before and after fermentation are shown in Table 14, and resveratrol before and after fermentation A chromatogram of the content analyzed by HPLC is shown in FIG. 8 . In the case of fermentation characteristics, lactic acid was not detected at all in sprouted peanut extract, which is the raw material, but it was found to be 12.16 mg/mL by fermentation in the fermented product prepared using the DU.La.P-11 strain of the present invention. It was found that proper fermentation occurred, and the resveratrol content was increased about 37.5 times in the fermented product compared to the sprouted peanut extract.

Changes in the content of useful ingredients before and after fermentation of sprouted peanut extract content Sprout Peanut Extract (9 Brix) Sprout Peanut Fermented Product Lactic acid (mg/mL) - 12.16±0.17 Resveratrol (μg/mL) 0.02±0.001 0.75±0.03

Example 4. Efficacy of fermented sprouted peanuts

1) Weight change and dietary intake

Table 15 shows the changes in body weight and dietary intake for each group of constipated rats during the experiment period. As for the change in body weight, at the end of the experiment, the group administered orally administered the fermented sprouted peanut (S1) and the beverage containing it (S2) showed a tendency to decrease in weight compared to the normal group (Normal). It was found that the weight decreased significantly compared to the group. On the other hand, it was found that the control group had an increase in body weight compared to the normal group. All the experimental groups induced constipation with loperamide in the dietary intake showed a significant difference and decreased the dietary intake than the normal group. In addition, the control group induced constipation with loperamide and the experimental group (S1 and S2) induced with loperamide after eating fermented sprouted peanut (S1) and a beverage (S2) containing the same were compared. As a result, it was found that the dietary intake was higher in all experimental groups than in the control group, and in particular, it was found that it was significantly increased in the experimental group (S2) containing sprouted peanut fermented product. Dietary efficiency showed a significant difference between the normal group and the control group inducing constipation with loperamide, and the diet efficiency of the control group was high, and compared to the control group, fermented sprouted peanuts (S1) and beverages containing the same (S2) The dietary efficiency of the experimental group (S1 and S2), which induced constipation with loperamide while eating , was significantly lower at the P < 0.05 level.

Weight gain, dietary intake and dietary efficiency of fed rats with experimental diet for 5 weeks military ¹⁾ Weight gain (g) Dietary intake (g/day) Dietary intake efficiency (%) Normal 173.3±10.31 ^ba 24.74±1.72 ^a 0.22±0.01 ^cb Control 181.3±16.20 ^a 23.34±2.21 ^c 0.27±0.02 ^a Positive control (PC) 152.7±24.26 ^c 21.51±1.64 ^c 0.22±0.04 ^cb S1 152.5±19.88 ^c 21.51±1.67 ^c 0.22±0.03 ^cb S2 160.9±18.62 ^bc 21.18±1.88 ^b 0.21±0.03 ^c

¹⁾ Normal: Normal diet.

Control: Normal diet and loperamide administration group.

PC: Commercial laxatives (Dulcolax S tablets) diet and loperamide administration group.

S1: Fermented peanut sprout diet and loperamide administration group.

S2: Beverage diet containing sprouted peanut fermented product and group administered with loperamide.

2) organ weight

Table 16 shows the liver, kidney and spleen weights of the experimental animals. The weight of the liver was highest in the control group induced with loperamide, and the weight of the liver compared to the control group in the fermented sprouted peanut or beverage diet and loperamide administration experimental group (S1 and S2). was low, and showed a tendency to increase compared to the normal group, and there was no significant difference. In the case of kidney weight, the experimental group inducing constipation with loperamide showed a lower tendency than the normal group and the control group, and in particular, the sprouted peanut fermented product experimental group (S1) was significantly lower. The weight of the spleen showed the same trend as the weight of the liver, which was lower in the experimental group than in the control group, and was higher than that in the normal group, and there was no significant difference between the experimental groups.

Comparison of liver, kidney, and spleen weights in mice military ¹⁾ Liver weight (g) Kidney weight (g) Spleen weight (g) Normal 15.75±1.63 ^2)a3) 2.52±0.24 ^ba 0.72±0.12 ^a Control 17.50±2.06 ^a 2.64±0.24 ^a 0.78±0.09 ^a Positive control (PC) 16.92±2.18 ^a 2.46±0.25b ^a 0.74±0.12 ^a S1 16.82±2.63 ^a 2.39±0.27 ^b 0.72±0.09 ^a S2 16.91±1.55 ^a 2.43±0.18 ^ba 0.76±0.10 ^a

3) fat weight

Abdominal fat weight of rats orally administered with sprouted peanut fermented product (S1) and drink containing sprouted peanut fermented product (S2) while inducing constipation showed a tendency to be lower than that of normal group and control group, S1 And in the S2 experimental group, significance was recognized at the level of P < 0.05. The weight of kidney fat also showed the same trend as the weight of abdominal fat, indicating that the experimental group (S1, S2) had a lower weight than the normal group and the control group. The weight of epididymal fat was significantly lower in both the experimental groups inducing constipation than in the control group inducing constipation (Table 17).

Fat weight by region of experimental mice military ¹⁾ Abdominal fat weight (g) Kidney fat weight (g) Epididymal fat weight (g) Normal 3.42±0.68 ^2)ba3) 1.62±0.29 ^a 0.18±0.04 ^cb Control 3.62±0.70 ^a 1.65±0.38 ^a 0.32±0.07 ^a Positive control (PC) 2.61±0.68 ^c 1.15±0.0.23 ^c 0.20±0.02 ^b S1 2.65±0.54 ^c 1.43±0.30 ^ba 0.20±0.03 ^b S2 2.95±0.44 ^c 1.34±0.13 ^bc 0.22±0.03 ^b

4) Number of stools, stool weight, stool moisture content

The number of feces, feces weight, and moisture content of feces per experimental animal were measured by dividing into before and after induction of constipation. As shown in Table 18, there was no significant difference in the number of stools excreted from each group for 4 weeks before induction of constipation between the normal group and the control group, and there was no significant difference between the positive control group (PC) and the sprouted peanut fermentation. The water test group (S1) and the drink test group (S2) containing fermented sprouted peanuts had more stools than the control group, but there was no significant difference. As a result of measuring the number of stools, weight of stools, and water content of stools in the experimental animals induced with constipation after administration of loperamide, the number of stools decreased after inducing constipation, and compared with the normal group that did not induce constipation. There was a significant decrease when Therefore, it was confirmed that constipation was properly induced. All of the experimental groups (S1, S2) that induced constipation, the same as the control group in which the number of stools were reduced, decreased compared to before the induction of constipation. However, compared to the control group, the number of stools was significantly greater (FIG. 9), and the stool weight also showed a similar tendency to the number of stools (FIG. 10). In the case of the water content of the feces, there was a tendency to increase in the fermented sprouted peanut test group (S1) than in the normal group and the control group before induction of constipation, but there was no significant difference. Also, after inducing constipation, the moisture content of the feces was higher than in the normal group. has decreased, but Compared with the control group, all the experimental groups showed a tendency to increase (FIG. 11).

5) Number of residual stools in the intestinal tract

All experimental animals were sacrificed on the last day of the experiment, and as a result of measuring the number of feces remaining in the colon, it was confirmed that the smallest number of residual feces was 0.90±0.57 in the normal group that did not induce constipation, and loperamide It was confirmed that there were 5.00±1.25 of the control groups induced with constipation, and the number was significantly higher. And compared to the control group, S1, a fermented sprouted peanut fermented product, had 3.20±1.14, and S2, a beverage experimental group containing fermented sprouted peanut, had 3.56±1.51, indicating that the number of residual stools in the intestinal tract was significantly lower than that of the control group. The significant difference in the number of residual stools in the intestinal tract of the normal group and the control group confirmed once again the induction of constipation by loperamide, and the sprouted peanut fermented product experimental group also confirmed that the number of residual stools was smaller than that of the control group even though constipation was induced. Thus, it was confirmed that the sprouted peanut fermented product had an effect on smooth bowel movements (Table 19 and FIG. 12).

Number of feces in the colon of mice military ¹⁾ Number of stools in colon (count/day) Normal 0.90±0.57 ^c Control 5.00±1.25 ^a Positive control (PC) 2.70±1.42 ^b S1 3.20±1.14 ^b S2 3.56±1.51 ^b

6) Measurement of serum lipid content

To observe the effect of fermented sprouted peanut diet on the blood lipid content of rats, blood was collected and changes in triglyceride and cholesterol in the serum of experimental animals were measured. As a result, as shown in Table 20, in the case of triglycerides, the highest level was 59.52±17.38 mg/dL in the control group induced with loperamide, and the content of triglycerides compared to the control group in both experimental groups (S1, S2). It showed a tendency to appear significantly less, and the content showed a tendency to be less than that of the normal group that did not induce constipation, but there was no significant difference. And in the case of total cholesterol content, the experimental group (S2) of the drink containing sprouted peanut fermented product showed a significantly lower value than the control group that induced constipation. The HDL-cholesterol content was the highest (18.83±3.80 mg/dL) in the normal group that did not induce constipation, and the lowest in the control group induced with loperamide was 12.90±3.91 mg/dL. In the case of the sprouted peanut fermented product test group (S1), the content showed a tendency to decrease compared to the normal group, but showed a higher content than the control group. The measured value of the LDL-cholesterol content was 37.42±9.40 mg/dL in the control group, indicating the highest content, and both the experimental groups (S1, S2) were found to have a lower content than the control group.

Triglyceride, total cholesterol, HDL-cholesterol, LDL-cholesterol (mg/dL) in serum of mice military ¹⁾ neutral lipid total cholesterol HDL-Cholesterol LDL-Cholesterol Normal 50.90±15.22 ^ba 53.71±7.93 ^bac 18.83±3.80 ^{a 24.70} ±11.82 bdc Control 59.52±17.38 ^a 62.23±6.24 ^a 12.90±3.91 ^b 37.42±9.40 ^a Positive control (PC) 43.55±11.61 ^bc 45.87±8.95 ^c 15.17±2.89 ^ba 21.99±9.49 ^dc S1 40.74±7.85 ^bc 53.61±8.16 ^bac 15.23±1.60 ^ba 30.23±7.20 ^bac S2 39.68±11.76 ^bc 51.68±9.44 ^bc 16.40±4.08 ^ba 27.34±10.13 ^bdc

7) Blood GOT and GPT activity

GOT and GPT enzymes are secreted into the blood when cells are destroyed or die, and a certain amount can be detected in the blood as well. Due to a decrease in the energy supply within the cell due to fatty liver, alcohol intake, obesity, etc., K ^- flows out of the cell, and Na ^- , Ca ² - and water flow into the cell, causing the cell to expand and the cell membrane to expand, resulting in GOT and GPT Since the enzyme flows out from within the cytoplasm, its content increases when necrosis and tissue destruction of hepatocytes progress due to hepatotoxicity. Therefore, enzyme activity related to liver function was measured in this experiment. As a result, as shown in Table 21, the blood GOT activity was significantly higher in the control group induced with loperamide as 38.48±6.49 Kramen/mL than in the normal group, and the sprouted peanut ferment (S1) and In the case of the experimental group (S2), which ate the beverage containing the beverage, the content showed a tendency to be higher than that of the normal group, but showed a slightly lower content than the control group. As a result of analyzing the GPT activity, the control group showed the highest content value of 15.64±2.28 Kramen/mL, and the content of the sprouted peanut fermented product (S1) and the experimental group (S2) in which the beverage containing it was fed compared to the control This tended to appear significantly lower, and the content was significantly lower than that of the normal group that did not induce constipation.

Serum GOT and GPT of mice military ¹⁾ GOT ²⁾ activity (Kramen/mL) GPT activity (Kramen/mL) Normal 32.61±4.17 ^b 12.91±1.30 ^b Control 38.48±6.49 ^a 15.64±2.28 ^a Positive control (PC) 34.61±3.89 ^ba 11.81±1.64 ^b S1 34.58±3.58 ^ba 11.87±1.65 ^b S2 32.73±4.97 ^b 8.81±1.10 ^c

Korea Institute of Bioscience and Biotechnology KCTC13781BP 20181220

<110> Fresh Bell Co., Ltd. Industry Academic Cooperation Foundation, Daegu University <120> Method for producing fermented peanut sprout using novel Pediococcus pentosaceus strain and composition for improving constipation containing peanut sprout fermentation produced by the same method <130> PN19514 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1448 <212> DNA <213> Pediococcus pentosaceus <400> 1 gcaagtcgaa cgaacttccg ttaattgatt atgacgtact tgtactgatt gagattttaa 60 cacgaagtga gtggcgaacg ggtgagtaac acgtgggtaa cctgcccaga agtaggggat 120 aacacctgga aacagatgct aataccgtat aacagagaaa accgcatggt tttcttttaa 180 aagatggctc tgctatcact tctggatgga cccgcggcgt attagctagt tggtgaggta 240 aaggctcacc aaggcagtga tacgtagccg acctgagagg gtaatcggcc acatgggac 300 tgagacacgg cccagactcc tacgggaggc agcagtaggg aatcttccac aatggacgca 360 agtctgatgg agcaacgccg cgtgagtgaa gaagggtttc ggctcgtaaa gctctgttgt 420 taaagaagaa cgtgggtaag agtaactgtt tacccagtga cggtatttaa ccagaaagcc 480 acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt atccggattt 540 attgggcgta aagcgagcgc aggcggtctt ttaagtctaa tgtgaaagcc ttcggctcaa 600 ccgaagaagt gcattggaaa ctgggagact tgagtgcaga agaggacagt ggaactccat 660 gtgtagcggt gaaatgcgta gatatatgga agaacaccag tggcgaaggc ggctgtctgg 720 tctgcaactg acgctgaggc tcgaaagcat gggtagcgaa caggattaga taccctggta 780 gtccatgccg taaacgatga tactaagtg ttggagggtt tccgcccttc agtgctgcag 840 ctaacgcatt aagtaatccg cctggggagt acgaccgcaa ggttgaaact caaaagaatt 900 gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagctacg cgaagaacct 960 taccaggtct tgacatcttc tgacagtcta agagattaga ggttcccttc ggggacagaa 1020 tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 1080 acgagcgcaa cccttattac tagttgccag cattaagttg ggcactctag tgagactgcc 1140 ggtgacaaac cggaggaagg tggggacgac gtcaaatcat catgcccctt atgacctggg 1200 ctacacacgt gctacaatgg atggtacaac gagtcgcgag accgcgaggt taagctaatc 1260 tcttaaaacc attctcagtt cggactgtag gctgcaactc gcctacacga agtcggaatc 1320 gctagtaatc gcggatcagc atgccgcggt gaatacgttc ccgggccttg tacacaccgc 1380 ccgtcacacc atgagagttt gtaacaccca aagccggtgg ggtaaccttt taggagctag 1440 ccgtctaa 1448

Claims (7)

(1) preparing a concentrate of sprouted peanuts;
(2) mixing and sterilizing the mixed fruit and vegetable concentrate with the sprouted peanut concentrate prepared in step (1); and
(3) Inoculating and fermenting a Pediococcus pentosaceus strain in a mixture of the sprouted peanut concentrate and the mixed concentrate of fruits and vegetables sterilized in the step (2); ) Method for manufacturing sprouted peanut fermented product with enhanced content The method of claim 1, wherein the Pediococcus pentosaceus ( Pediococcus pentosaceus ) strain is a Pediococcus pentosaceus DU.La.P-11 strain whose accession number is KCTC13781BP. Method for producing sprouted peanut fermented product. The method according to claim 1, wherein the mixed concentrate is banana concentrate, peach concentrate, mango concentrate, strawberry concentrate, fig concentrate, tomato concentrate, carrot concentrate, cabbage concentrate, and broccoli concentrate It comprises at least one selected from the group consisting of concentrate. A method for producing fermented sprouted peanuts with enhanced resveratrol content. According to claim 1,
(1) preparing the sprouted peanut concentrate of 9-11 Brix by concentrating the extract extracted by adding 5 to 20 times the weight of the sprouted peanut to the sprouted peanut under reduced pressure;
(2) mixing and sterilizing the sprouted peanut concentrate prepared in step (1) by 1-20% (v/v) of the mixed concentrate of 4-6 Brix fruits and vegetables based on the weight of the sprouted peanut concentrate; and
(3) Pediococcus pentosaceus ( Pediococcus pentosaceus ) DU.La.P-11 strain having an accession number KCTC13781BP in a mixture of sprouted peanut concentrate and mixed vegetable concentrate sterilized in step (2) above the weight of the mixture 0.1-20% (v/v) inoculated and fermented at 30-45° C. for 53-57 hours; Resveratrol content-enhanced manufacturing method of sprouted peanut fermented product comprising a. The fermented sprouted peanuts with enhanced resveratrol content prepared by the method of any one of claims 1 to 4. A health functional food composition for preventing or improving constipation comprising the fermented sprouted peanut of claim 5 as an active ingredient. A pharmaceutical composition for preventing or treating constipation comprising the fermented sprouted peanut of claim 5 as an active ingredient.

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