KR20210048446A - Method for preparing fermented rice bran by using weissella koreensis and fermented rice bran prepared thereby - Google Patents

Abstract

The present invention relates to a method for preparing fermented rice bran and a fermented rice bran prepared by the method for preparing fermented rice bran. More specifically, the strain used for the preparation of the fermented rice bran of the present invention is a Weissella koreensis DB1 (KCTC 13653BP) strain isolated from kimchi, has producing ability capable of producing citrulline and ornithine from arginine, can grow even in a high-concentration rice bran mixture, and has excellent fermentation efficiency, thereby producing high concentrations of citrulline and ornithine during the fermentation of rice bran, and preparing fermented rice bran with enhanced functionality due to citrulline and ornithine contained at a high concentration. In this aspect, the method for preparing fermented rice bran of the present invention comprises a step of adding a strain of Weissella koreensis DB1 (KCTC 13653BP) isolated from kimchi and providing a citrulline- and ornithine-producing ability. In addition, the fermented rice bran prepared by the method for preparing the fermented rice bran contains high content of citrulline and ornithine in the fermented rice bran, and has excellent functionality. In this aspect, the fermented rice bran may be utilized as a functional food, and the functional food may be a food composition or a health functional food for preventing obesity, containing the fermented rice bran as an active ingredient.

Description

Manufacturing method of fermented rice bran using lactic acid bacteria isolated from kimchi and fermented rice bran produced therefrom {METHOD FOR PREPARING FERMENTED RICE BRAN BY USING WEISSELLA KOREENSIS AND FERMENTED RICE BRAN PREPARED THEREBY}

The present invention relates to a fermented rice bran production method using lactic acid bacteria and a fermented rice bran produced by the above production method.

The prevalence of various lifestyle-related diseases and chronic degenerative diseases, such as overnutrition, environmental pollution, lack of exercise, and increased stress due to the improvement of the economic level, has emerged as a major problem in our society. Among them, obesity is the disease that has attracted the most attention recently. The obesity generally causes an increase in the quantity and number of adipocytes in the body because the energy intake cannot be balanced with the energy expenditure consumed by physical activity, etc. It refers to a state in which adipose tissue is accumulated excessively, and refers to a disease caused by the loss of energy balance in the body due to genetic factors, environmental factors, mental factors, or lifestyle habits such as diet and lack of exercise.

According to the National Health and Nutrition Survey, the prevalence of obesity in Korea is showing an increasing trend, and the Ministry of Health and Welfare reported that 38% of the Korean population consumes more than the recommended intake standard, and that the risk of cardiovascular disease is also increasing. Due to this change in diet, the body is exposed to various risk factors. In particular, foods containing a large amount of saturated fatty acids increase blood lipid levels, causing hypercholesterolemia, and further reduce the incidence of diseases of the circulatory system such as hyperlipidemia and myocardial infarction. Increase.

In relation to the obesity, studies on adipogensis are being conducted. The localization refers to a process in which fat cells are differentiated from adipocytes to accumulate fat, and the localization is known to cause metabolic diseases such as obesity, diabetes, fatty liver and coronary heart disease.

In the adipocytes, lipid metabolism occurs through lipolysis and lipolysis, and when the adipocytes are synthesized, the predipocytes are differentiated into mature adipocytes through proliferation and differentiation processes. The droplets appear in the cytoplasm, and over time they grow and merge into one or several large lipid droplets.

Obesity treatment mechanisms for relieving obesity and maintaining an appropriate weight include controlling appetite, obstructing digestion and absorption of fat, increasing energy consumption, and regulating lipid metabolism. Currently, a commonly used obesity treatment is a drug that inhibits the activity of serotonin and lipolytic enzymes, inhibits the hydrolysis of cholesterol and triglyceride, and helps to excrete unoxidized fat into the stool. Orlistat (XenicalR).

However, sertonin increases blood pressure and should be used with caution in patients with cardiovascular disease, and Olistat has been reported to induce digestive disorders, fat stool, bowel incontinence, and obstruction of fat-soluble vitamin absorption. In some cases, it has been reported to have significant side effects.

Therefore, by suppressing the growth and differentiation of mast cells from natural products that are expected to relatively minimize the possibility of safety problems, the anti-obesity effect against obesity caused by excessive adipose tissue or abnormal fat metabolism is high. There is a demand for the development of substances for the treatment, prevention or improvement of obesity, which is a weak side effect.

On the other hand, rice, which has a long history as a food resource for mankind, is composed of rice husk layer, rice husk layer, snow and endosperm. The rice is obtained in the form of white rice from which the rice hull layer has been removed by grinding the brown rice and brown rice with the rice hull layer removed according to the degree of milling, and is obtained in the form of white rice from which rice bran is removed, such as rice buds, peel, seed skin, and algae layer, and the outer shell of the rice remaining except for the brown rice and white rice. By-products such as rice hull layer, rice hull layer, rice hull layer, and salagi are called rice bran.

Rice bran, previously treated as a by-product, has been reported to contain about 95% of the physiologically active substances contained in rice such as gamma aminobutyric acid (GABA), lecithin, octacosanol, orizanol, tocopherol, and ferulic acid. Since many physiologically active substances exist in the rice bran, it is known that it has various physiological effects such as anti-obesity activity, anti-cholesterol activity, anti-hypertensive activity, hangover relief activity, heavy metal detoxification activity and anti-constipation activity. Is mostly used for livestock feed.

In this regard, the prior patents disclosed are mainly related to the effect of promoting digestion or improving flavor through fermentation of lactic acid bacteria, but prior research on improving the content of active substances for enhancing specific functionality from the active ingredients contained in rice bran has been extremely limited. There are limited aspects. Therefore, there is a growing need for research that is differentiated from the existing methods so that it is possible to improve the profits of farmers and improve the environment through the use of nature through the use of rice bran that is limitedly used or discarded as feed, even though it has various ingredients. have.

10-1733967B 10-1618953B 10-1523139B 10-1357663B 10-1177245B 10-0880382B 10-2018-0133372A 10-2017-0086196A 10-2012-0021788A

In order to solve the above problem, the present inventors have developed lactic acid bacteria, which are microorganisms (GRAS) that are universally secured to improve the functionality of rice bran, in particular, among lactic acid bacteria derived from kimchi, which is a report of lactic acid bacteria having various functions as a fermented food unique to Korea. An object of the present invention is to provide a suitable manufacturing method for manufacturing fermented rice bran, that is, a method for producing fermented rice bran using lactic acid bacteria, by selecting the lactic acid bacteria suitable for fermented rice bran production.

In addition, it is an object of the present invention to provide a fermented rice bran produced by the method for producing fermented rice bran and a functional food using the same.

The technical problems to be achieved by the present invention are not limited to the technical problems mentioned above, and other technical problems that are not mentioned will be clearly understood by those of ordinary skill in the art from the description of the present invention. .

In order to achieve the above object, the present invention provides, in one aspect, a microorganism capable of effectively producing lactic acid bacteria optimal for rice bran fermentation, specifically, citrulline and ornithine, which are substances having an anti-obesity effect from rice bran do.

More specifically, the present invention provides a Weissella koreensis DB1 (Weissella koreensis DB1, KCTC 13653BP) strain isolated from kimchi and capable of producing citrulline and ornithine.

The Weissella koreensis DB1 (KCTC 13653BP) strain may preferably have an optimal growth pH of 5.0 in a medium to which arginine is added.

The present invention, in order to achieve the above object, in another aspect, by using the Weissella koreensis DB1 (Weissella koreensis DB1, KCTC 13653BP) strain, the rice bran fermentation product with enhanced functionality, specifically, citrulline (C ₆ H ₁₃ N ₃ 0 ₃ ) And ornithine (Ornithine, C ₅ H ₁₂ N ₂ 0 ₂ ) It provides a method of preparing a rice bran fermented product with increased content.

More specifically, in the method for producing fermented rice bran, the present invention comprises the step of fermenting by adding a strain of Weissella koreensis DB1 (Weissella koreensis DB1, KCTC 13653BP) isolated from the kimchi and having citrulline and ornithine production capacity. It provides a method for producing fermented rice bran, characterized in that it comprises.

In addition, the present invention is a fermentation raw material production step of mixing rice bran, arginine and water to prepare a mixture; Gelatinized rice bran manufacturing step of preparing a gelatinized rice bran mixture by heating the mixture to gelatinize the rice bran; A strain addition step of adding a Weissella koreensis DB1 (KCTC 13653BP) strain isolated from the kimchi and having citrulline and ornithine producing ability to the gelatinized rice bran mixture; And a fermentation step of fermenting the gelatinized rice bran mixture to which the strain is added to prepare a rice bran fermentation product.

In the fermentation raw material manufacturing step, rice bran is added to the mixture at a concentration of 10% (w/v) to 80% (w/v), and arginine is added to 0.1% (w/v) to 5% (w/v). It can be carried out by adding at a concentration of v). More specifically, in the step of preparing the fermentation raw material, rice bran is added at a concentration of 10% (w/v) to 80% (w/v) based on the total volume of the mixture prepared by mixing water, rice bran and arginine, It can be carried out by adding arginine at a concentration of 0.1% (w/v) to 2% (w/v).

The step of preparing the gelatinized rice bran may be performed by heating the mixture under a temperature condition of 100°C to 140°C and a pressure condition of 0.9 to 1.5 atm for 10 to 20 minutes to gelatinize the rice bran.

The fermentation step may be carried out by fermenting under the condition that the initial pH of the gelatinized rice bran mixture to which the strain is added is pH 6.0 to pH 6.5 and the fermentation temperature is 25°C to 30°C.

In addition, the method for manufacturing the fermented rice bran may further include a step of drying the fermented rice bran of drying the fermented rice bran.

The drying step may be performed by a hot air drying method for 1 hour to 40 hours at a temperature condition of 30°C to 60°C, or a freeze drying method for 20 hours to 100 hours at a temperature condition of -50°C to -80°C.

In order to achieve the above object, the present invention provides, in another aspect, a rice bran fermented product prepared by the method for producing a rice bran fermented product.

The fermented rice bran may preferably have a citrulline content of 2,500 mg/kg to 3,500 mg/kg and an ornithine content of 5,500 mg/kg to 10,500 mg/kg in the fermented rice bran.

In order to achieve the above object, the present invention provides, in another aspect, a food composition for preventing obesity comprising the fermented rice bran as an active ingredient.

The food composition for preventing obesity may preferably be a health functional food.

As described above, rice bran fermented with the Weissella koreensis DB1 (KCTC 13653BP) strain according to the present invention significantly improves the content of citrulline and ornithine in the rice bran, so that the rice bran itself has various functions. In addition, since the anti-obesity activity can be further improved, it not only significantly improves the anti-obesity activity compared to existing rice bran or rice bran fermentation products, but also the lactic acid bacteria, which are strains used in the fermentation process, are GRAS microorganisms, so safety is also secured. Rice bran, whose use is limited as feed or waste, can be manufactured as a high value-added food, contributing to the improvement of profits not only for rice cultivation farmers but also for related industries, so the economic effect is very large, and safety is secured as a food for a long time. Since rice bran and lactic acid bacteria, which are GRAS microorganisms, are used, it can be used for various purposes as a health functional food material, and the industrial effect will be very great.

1 is a map showing the producing areas of kimchi obtained in various regions of Korea to secure kimchi lactic acid bacteria according to an embodiment of the present invention.
2 is a result of confirming the ornithine production ability of lactic acid bacteria isolated from kimchi obtained in various regions according to an embodiment of the present invention, the lower part of the photo shows the strain number, and the upper part of the photo The abbreviation indicates the strain name, and Arginine and Ornithine indicate the positions of arginine and ornithine.
3 is a diagram showing the nucleotide sequence of 16S rRNA of Weisella coriensis DB1 strain according to an embodiment of the present invention.
Figure 4 is a graph showing the growth activity according to the temperature of the Weissella coriensis DB1 strain according to an embodiment of the present invention, the horizontal axis of the graph represents the incubation time (hr), the vertical axis of the graph is the degree of microbial growth (Absorbance, A ₆₀₀ ), and the number at the top of the graph indicates the culture temperature.
Figure 5 is a graph showing the growth activity according to the pH of the Weissella coriensis DB1 strain according to an embodiment of the present invention, the horizontal axis of the graph represents the pH condition of the culture medium, the vertical axis of the graph is the degree of microbial growth It represents (log CFU/mL).
6 is a graph showing the growth activity according to pH in a medium to which 1% (w/v) arginine of Weissella coriensis DB1 strain according to an embodiment of the present invention is added, and the horizontal axis of the graph is of the culture medium. It represents the pH condition, and the vertical axis of the graph represents the degree of microbial growth (absorbance, A ₆₀₀ ). In the graph, Control refers to a control group in which 1% (w/v) arginine is added to the MRS medium and pH is not separately adjusted.
7 is a photograph confirming the hemolysis reaction in order to confirm the safety of the Weisella coriensis DB1 strain according to an embodiment of the present invention.
Figure 8 is a result of confirming whether or not bioamine is produced in order to confirm the safety of the Weisella coriensis DB1 strain according to an embodiment of the present invention, and Figure 8a is a positive control Enterococcus faecalis strain (Enterococcus faecalis ATCC 29212). ), and the decarboxylase ability check experiment (Decarboxylase plate method, Bover-Cid) was performed, ATCC 29212 means Enterococcus faecalis strain (Enterococcus faecalis ATCC 29212), DB1 is Weissella coriensis DB1 Means a strain, Figure 8b is a graph confirming the production of bioamine by analyzing the culture medium of Weisella coriensis DB1 strain by HPLC, Standard means the result of HPLC performance of the bioamine standard solution, DB1 is Weisella coriensis DB1 It means the result of HPLC performance of the strain culture solution.
FIG. 9 is a graph showing the growth activity of Weicella coriensis DB1 strain according to pH in a rice bran mixture in which 1% (w/v) arginine and 20% (w/v) rice bran are mixed according to an embodiment of the present invention. As, the horizontal axis of the graph represents the pH condition of the culture medium, the vertical axis of the graph represents the degree of microbial growth (log CFU/mL), purple (24 h) represents the result of culturing for 24 hours, and blue (48 h ) Represents the result of incubation for 48 hours.
FIG. 10 shows the growth activity of Weisella coriensis DB1 strain according to fermentation temperature in a rice bran mixture in which 1% (w/v) arginine and 20% (w/v) rice bran are mixed according to an embodiment of the present invention. As a graph, the horizontal axis of the graph represents the culture temperature condition, the vertical axis of the graph represents the degree of microbial growth (log CFU/mL), purple (24 h) represents the result of culturing for 24 hours, and blue (48 h) Represents the result of incubation for 48 hours.
Figure 11 is a photograph showing the result of the final rice bran fermentation according to the drying method according to an embodiment of the present invention, Figure 11a is a photograph taken of the rice bran fermentation dried by the freeze drying method, Figure 11b is a rice bran fermentation dried by the hot air drying method This is a picture of water.
12 is a photograph showing the results of observation by staining adipocytes of epididymis tissue with Hematoxylin and Eosin (H&E) according to an embodiment of the present invention, where ND means a normal diet group, and HFCD is a high fat high cholesterol diet group. Means, and HFCD-RB refers to a high fat high cholesterol diet group with rice bran added, and HFCD-FRB refers to a high fat high cholesterol diet group with rice bran fermentation hot air dried product.
13 is a graph showing the results of measuring the size of adipocytes by staining adipocytes of epididymis tissue with Hematoxylin and Eosin (H&E) according to an embodiment of the present invention, and the vertical axis of the graph is an average size of adipocytes. (Adipocyte size), the horizontal axis of the graph refers to each experimental group, ND refers to the normal diet group, HFCD refers to the high fat high cholesterol diet group, and HFCD-RB refers to the high fat high cholesterol supplemented with rice bran It means a diet group, and HFCD-FRB means a high fat high cholesterol diet group added with hot air dried rice fermented rice.
FIG. 14 is a photograph showing the results of confirming the adherence of liver tissue by staining liver tissue with Oil-Red O solution according to an embodiment of the present invention, where ND means a normal diet group, and HFCD is high fat high cholesterol. It means a diet group, HFCD-RB means a high fat high cholesterol diet group with rice bran added, and HFCD-FRB means a high fat high cholesterol diet group with rice bran fermentation hot air dried product.

Throughout this specification, a number of citations are referenced and the citations are indicated. The disclosure contents of the cited documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present invention relates to the production and utilization of fermented rice bran with improved anti-obesity activity, that is, rice bran fermented product with increased content of citrulline and ornithine.

Specifically, the present invention relates to a microorganism capable of effectively producing lactic acid bacteria that are optimal for fermentation of rice bran, specifically citrulline and ornithine, which are substances having an anti-obesity effect from rice bran.

More specifically, the present invention relates to a Weissella koreensis DB1 (Weissella koreensis DB1, KCTC 13653BP) strain isolated from kimchi and having citrulline and ornithine-producing ability.

The Weissella koreensis DB1 (KCTC 13653BP) strain may preferably have an optimal growth pH of 5.0 in a medium to which arginine is added.

In addition, the present invention is a rice bran fermentation product with enhanced functionality using the Weissella koreensis DB1 (KCTC 13653BP) strain, specifically, rice bran fermentation with increased citrulline and ornithine content. It relates to a method of making water.

More specifically, the present invention comprises the step of adding a Weissella koreensis DB1 (Weissella koreensis DB1, KCTC 13653BP) strain isolated from the kimchi and having citrulline and ornithine production ability in the method for producing fermented rice bran in the present invention. It relates to a method for producing fermented rice bran, characterized in that.

In addition, the present invention is a fermentation raw material production step of mixing rice bran, arginine and water to prepare a mixture; Gelatinized rice bran manufacturing step of preparing a gelatinized rice bran mixture by heating the mixture to gelatinize the rice bran; A strain addition step of adding a Weissella koreensis DB1 (KCTC 13653BP) strain isolated from the kimchi and having citrulline and ornithine producing ability to the gelatinized rice bran mixture; And a fermentation step of fermenting the gelatinized rice bran mixture to which the strain is added to prepare a rice bran fermentation product.

In the fermentation raw material manufacturing step, rice bran is added to the mixture at a concentration of 10% (w/v) to 80% (w/v), and arginine is added to 0.1% (w/v) to 5% (w/v). It can be carried out by adding at a concentration of v). More specifically, in the step of preparing the fermentation raw material, rice bran is added at a concentration of 10% (w/v) to 80% (w/v) based on the total volume of the mixture prepared by mixing water, rice bran and arginine, It can be carried out by adding arginine at a concentration of 0.1% (w/v) to 5% (w/v).

The step of preparing the gelatinized rice bran may be performed by heating the mixture under a temperature condition of 100°C to 140°C and a pressure condition of 0.9 to 1.5 atm for 10 to 20 minutes to gelatinize the rice bran.

The fermentation step may be carried out by fermenting under the condition that the initial pH of the gelatinized rice bran mixture to which the strain is added is pH 6.0 to pH 6.5 and the fermentation temperature is 25°C to 30°C.

In addition, the method for manufacturing the fermented rice bran may further include a step of drying the fermented rice bran of drying the fermented rice bran.

The drying step may be performed by a hot air drying method for 1 hour to 40 hours at a temperature condition of 30°C to 60°C, or a freeze drying method for 20 hours to 100 hours at a temperature condition of -50°C to -80°C.

In addition, the present invention relates to a rice bran fermented product produced by the method for producing the rice bran fermented product.

The fermented rice bran may preferably have a citrulline content of 2,500 mg/kg to 3,500 mg/kg and an ornithine content of 5,500 mg/kg to 10,500 mg/kg in the fermented rice bran.

In addition, the present invention relates to a food composition for preventing obesity comprising the fermented rice bran as an active ingredient.

The food composition for preventing obesity may preferably be a health functional food.

Unless otherwise specified in the specification, a culture means that a specific microorganism is cultured in a culture medium or a culture medium. The formulation of the culture or the concentrate of the culture is not limited, and for example, the formulation may be liquid or solid.

Unless otherwise specified in the specification, a culture medium is a special purpose to contain nutrients required by a culture target, that is, a microorganism or a tissue of an animal or plant to be cultured in order to cultivate a specific microorganism or tissue of an animal or plant. A material for may be added and mixed. The medium is a concept including all of a natural medium, a synthetic medium or a selective medium, and a concept including a solid medium or a liquid medium depending on the properties.

Unless otherwise specified in the specification, the culture medium means inoculating and culturing the strain in a liquid medium. In addition, the concentrated solution of the culture solution means that the culture solution is concentrated, and the dried product of the culture solution means that the culture solution has been drained of water.

Unless otherwise specified in the specification, a fermentation starter is any one selected from the group consisting of microorganisms including lactic acid bacteria or bacteria involved in fermentation, a culture thereof, a concentrate of the culture, and a dried product of the culture. It means a formulation or composition containing. The fermentation starter is added to the production of fermented foods and the like and is used to provide microorganisms that can grow in fermented foods or as dominant species.

In the case of manufacturing fermented food using the fermentation starter, the quality of the fermented food can be regulated by microorganisms included in the fermentation starter, or the speed or stage of fermentation can be controlled, and fermentation that achieves a specific purpose You can make food.

Unless otherwise specified in the specification, a seed is a microorganism used for fermentation, and refers to a microorganism inoculated into a substrate or food for fermentation, and the seed composition includes as an active ingredient one or more inoculum required to initiate fermentation. Means composition.

Unless otherwise specified in the specification, gelatinization refers to an increase in viscosity and water solubility due to changes in physicochemical properties or structure due to expansion by heat and moisture in the case of heating starch or the like in water or treating with a solvent such as an alkali solution. It refers to a process that changes toward having properties such as an increase in volume, or a phenomenon in which the whole becomes a semi-transparent, almost uniform colloidal material, and is also called gelatinization.

Unless otherwise specified in the specification, food refers to a natural product or processed product containing one or more nutrients, and preferably refers to a state that can be eaten directly through a certain degree of processing, and the Examples are fruits, vegetables, dried or cut products of fruits or vegetables, fruit juice, vegetable juice, mixed juices or chips, noodles, processed livestock food, processed seafood, dairy processed food, fermented food, bean food, grain food , Microbial fermented food, bakery, seasonings, processed meats, acidic beverages, licorice, herbs, and the like. In the present invention, it means including fermented foods, functional foods and processed foods, but is not limited thereto.

Unless otherwise specified in the specification, fermented food refers to food made by adding one or more microorganisms such as lactic acid bacteria or enzymes and using the fermentation action of the microorganisms, and in detail, fermented food seeds are added to the food substrate. It refers to food produced by aging and aging. The fermented food includes all fermented foods such as liquor, bread, kimchi, salted fish, miso, soy sauce, cheese, butter, and yogurt.

The inventors of the present invention selected to improve the anti-obesity effect of rice bran by focusing on the anti-obesity effect of rice bran, facilitating bowel movement, that is, the function of anti-constipation effect, while conducting various studies to increase the utilization of rice bran, and can be used in food. A method of increasing the content of citrulline and ornithine was studied to improve the anti-obesity effect using lactic acid bacteria, which are GRAS microorganisms, and various microorganisms isolated from kimchi collected in all regions of Korea were studied. As a result of performing, the novel Weissella koreensis DB1 strain effectively produced citrulline and ornithine in an arginine-added rice bran mixture prepared by adding arginine and rice bran to water. It was confirmed that the content could be increased, and further research was conducted to confirm the optimum culture pH and optimum culture temperature in the arginine-added rice bran mixture medium of Weicella coriensis DB1 strain. By checking the drying conditions, the present invention was completed based on this.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a microorganism capable of effectively producing lactic acid bacteria that are optimal for fermentation of rice bran, specifically citrulline and ornithine, which are substances having an anti-obesity effect from rice bran.

The microorganism is specifically isolated from kimchi, and may be a Weissella koreensis DB1 (Weissella koreensis DB1, KCTC 13653BP) strain having citrulline and ornithine-producing ability.

* The DB1 strain was isolated and identified by the present inventors and is the first reported strain, and is a lactic acid bacterium isolated from kimchi, and was identified as a Gram-positive monobacterial. The DB1 strain is D-Glucose, D-Fructose, D-Mannose, L-Arabinose, Ribose, D- It was found to have glucose metabolism for xylose (D-Xylose) and N-acetyl glucosamine (N-Acetyl glucosamine). In addition, the 16S rRNA nucleotide sequence was identified as Weisella coriensis with 99.86% homology with Weisella ^{coriensis S-5623 T.} The Weisella coriensis DB1 strain was deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on October 2, 2018, and was given the accession number KCTC 13653BP.

The Weisella coriensis DB1 strain can be grown in a conventional lactic acid bacteria medium, for example, MRS liquid medium or MRS solid medium, or the MRS liquid medium or MRS solid medium can be cultured in a medium to which arginine is added.

The Weisella coriensis DB1 strain can produce citrulline and ornithine, which are substances having excellent anti-obesity activity from arginine or rice bran, as well as citrulline production ability and ornithine production compared to other strains and other strains of the genus Weisella. The ability is remarkably excellent. In addition, the growth activity of the Weisella coriensis DB1 strain is optimal in the pH range of pH 6.0 to pH 8.0, whereas it is optimal at pH 5.0 in the medium to which arginine is added, and pH 6.0 to pH 6.5 in the rice bran mixture to which arginine is added. It was found to be optimal in the pH range of, and as a result of the hemolytic test, antibiotic resistance, and biogenic amine production ability, the safety was confirmed to be very excellent.

Specifically, the Weisella coriensis DB1 strain has an optimum pH of growth in the MRS medium of pH 6.0 to pH 8.0, whereas the MRS medium containing arginine, specifically arginine is 1% (w/w) based on the total volume of the medium. ) In the added MRS medium, the optimum pH is pH 5.0, and arginine is added to the rice bran mixture, specifically, arginine is added by 1% (w/v) based on the total volume of the mixture, and rice bran is 20% based on the total volume of the mixture. (w/v) It may be a strain having an optimum pH of pH 6.0 to pH 6.5 grown in the added rice bran mixture.

The ornithine (Ornithine, C ₅ H ₁₂ N ₂ 0 ₂ ) is not an amino acid constituting a protein, but is an amino acid that is usually contained in food, and is included in a large amount, for example, in freshwater bivalve clams or rapeseeds. The ornithine is known to have anti-obesity activity, induction of muscle synthesis, secretion of growth hormone (GH), promotion of basic metabolism, improvement of liver function, and improvement of skin beauty. For example, the ornithine is a food material that increases muscle synthesis by secreting growth hormone and promotes basal metabolism to prevent obesity, and has been widely used as a dietary supplement or medicine in the United States and European countries. It was found to have new functionalities such as skin care effects such as wrinkle improvement. In addition, in Japan, it can be used as a food material in the form of L-ornithine hydrochloride, but in Korea, it is difficult to use because it is not yet registered as a food additive by the KFDA. Therefore, in Korea, ornithine is not added as an additive, but may be used in the form of a fermented food in which the ornithine content is increased by increasing the content of ornithine during the fermentation process.

Until now, studies to increase the content of ornithine in fermented foods by producing ornithine during the fermentation process have been conducted in relation to dairy products such as cheese, a representative fermented food in the West. The research is very limited. Research results published so far are also on lactic acid bacteria capable of producing ornithine, such as Korean Patent No. 096258, Korean Patent Publication No. 2017-0073839, or Korean Patent Publication No. 2012-0021788. Only limited research has been conducted to be used.

The citrulline (Citrulline, C ₆ H ₁₃ N ₃ 0 ₃ ) is an amino acid present in the seeds of the gourd family, and it has been found that it exists in nature as a free amino acid. It is not known to be clear. The citrulline is known to have anti-obesity activity, induction of muscle synthesis, diuretic and blood circulation improvement. For example, the citrulline is known to be effective in hypertension because it has the ability to improve blood flow by dilation of blood vessels, that is, the ability to remove free radicals in the body, and it is known to be effective in kidney disease and constipation, and induces muscle synthesis. , It is known to have the effect of relaxing muscles and improving body functions. Therefore, in western countries such as the United States and Europe, citrulline is consumed in the form of pharmaceuticals or functional foods.

However, in Korea, such as confirming the functionality of citrulline as in Korean Patent No. 1754227 or Korean Patent Publication No. 2017-0033101, or extracting citrulline from watermelon peels as in Korean Patent No. 1038433, etc. It has been investigated that only studies on extracting citrulline are limited, and studies on producing citrulline using lactic acid bacteria derived from fermented strains, especially Korean traditional fermented foods, have not been conducted at all.

In addition, the present invention is a rice bran fermentation product with enhanced functionality using the Weissella koreensis DB1 (KCTC 13653BP) strain, specifically, rice bran fermentation with increased citrulline and ornithine content. It relates to a method of making water.

The method for preparing the fermented rice bran is a step of adding a strain of Weissella koreensis DB1 (Weissella koreensis DB1, KCTC 13653BP), which is separated from the kimchi and has citrulline and ornithine production capacity, in the rice bran ferment production method. , Separated from the kimchi, and fermented by adding the Weissella koreensis DB1 (Weissella koreensis DB1, KCTC 13653BP) strain having citrulline and ornithine producing ability, that is, isolated from the kimchi, and having citrulline and ornithine production ability Weissella koreensis DB1 (Weissella koreensis DB1, KCTC 13653BP) may include the step of fermenting rice bran by adding a strain.

More specifically, the method of preparing the fermented rice bran includes a fermentation raw material manufacturing step of preparing a mixture by washing rice bran, and then mixing rice bran, arginine, and water; Gelatinized rice bran manufacturing step of preparing a gelatinized rice bran mixture by heating the mixture to gelatinize the rice bran; A strain addition step of adding a Weissella koreensis DB1 (KCTC 13653BP) strain isolated from the kimchi and having citrulline and ornithine producing ability to the gelatinized rice bran mixture; And a fermentation step of fermenting the rice bran mixture to which the strain is added to prepare a rice bran fermentation product.

The fermentation refers to a phenomenon in which a complex organic substance is converted into a simple compound by an enzyme action to release free energy, and generally refers to a phenomenon in which a microorganism decomposes an organic substance and accumulates metabolites. The fermentation increases the nutritional value of food by biosynthesizing essential amino acids or improving the digestibility of protein content and fiber, and also improves the bioavailability of micronutrients such as calcium, phosphorus or various vitamins. Let it.

Representative microorganisms used in this fermentation process include lactic acid bacteria, yeast, and acetic acid bacteria. Among them, lactic acid bacteria (LAB), a strain that has secured safety due to long-term fermented food manufacturing, is mainly used. . The lactic acid bacteria is a microorganism (GRAS, Generally Recognized As Safe) that has been widely ingested as food, such as fermented foods over a long period of time around the world, or has been used as a fermentation strain of foods and has been recognized for safety.

In the step of preparing the fermentation raw material, rice bran in the mixture is 10% (w/v) to 80% (w/v) or 12% (w/v) to 70% (w/v), preferably 15% (w /v) to 60% (w/v), more preferably 17% (w/v) to 50% (w/v) or 20% (w/v) to 50% (w/v) or 19% (w/v) to 30% (w/v), and arginine is added at a concentration of 0.1% (w/w) to 5% (w/w), preferably 0.1% (w/v) To 2% (w/v), more preferably 0.2% (w/v) to 1.7% (w/v), even more preferably 0.5% (w/v) to 1.5% (w/v) It can be carried out by a method of adding to. More specifically, in the step of preparing the fermentation raw material, rice bran is added at a concentration of 10% (w/v) to 80% (w/v) based on the total volume of the mixture prepared by mixing water, rice bran and arginine, It can be carried out by adding arginine at a concentration of 0.1% (w/v) to 5% (w/v). For example, the fermentation raw material manufacturing step is 10% (w/v) to 80% (w/v) based on the total volume of the mixture prepared by mixing rice bran, specifically washed and dried rice bran with water, rice bran, and arginine in water. v), and arginine was added at a concentration of 0.1% (w/v) to 5% (w/v) based on the total volume of the mixture prepared by mixing water, rice bran and arginine. After, it can be carried out by a method of stirring so that it can be well mixed.

The step of preparing the gelatinized rice bran is a temperature condition of 80°C to 160°C or 100°C to 140°C, preferably 110°C to 130°C, and 0.7 to 2.0 atm or 0.9 to 1.5 atm, preferably 1.0 atm It can be carried out by a method of gelatinizing the rice bran by heating for 1 minute to 60 minutes, preferably 5 minutes to 40 minutes, more preferably 10 minutes to 20 minutes under pressure conditions of to 1.25 atm. Since the step of preparing the gelatinized rice bran is to apply high temperature heat, not only the rice bran is gelatinized to increase fermentation efficiency, but also the mixture, that is, the rice bran mixture, is sterilized to increase the fermentation efficiency by microorganisms other than the strain of the present invention in the fermentation process. Deterioration or generation of by-products can be controlled. The step of preparing the gelatinized rice bran may further include a process of cooling the gelatinized rice bran mixture by heating to room temperature, for example, 10 ℃ to 25 ℃ or 15 ℃ to 20 ℃ after heating.

The strain addition step may be performed by adding a Weissella koreensis DB1 (KCTC 13653BP) strain isolated from the kimchi to the gelatinized rice bran mixture and having citrulline and ornithine producing ability. More specifically, the step of adding the strain includes 0.1% (v/v) to 10% (v/v) or 0.2% (v/v) to 5.0% based on the volume ratio of the strain suspension or culture solution to the gelatinous rice bran mixture. (v/v) or 0.5% (v/v) to 2.0% (v/v) can be added, and the strain concentration of the strain suspension or culture is 4 log CFU/mL to 9 log CFU/mL or 5 log CFU/mL to 8 log CFU/mL or 6 log CFU/mL to 7 log CFU/mL.

In the fermentation step, the initial pH of the gelatinized rice bran mixture to which the strain is added is pH 5.0 to pH 8.0, preferably pH 5.5 to pH 7.0, most preferably pH 6.0 to pH 6.5, and the fermentation temperature is 20° C. It can be carried out by fermenting at a temperature condition of 35°C, preferably 25°C to 30°C.

The fermentation step may improve fermentation efficiency by performing the fermentation at the optimum growth pH in rice bran fermentation unlike the optimum growth pH in the culture medium to which the strain is added or the arginine of the strain is added. Whereas normal lactic acid bacteria have the same or similar range of growth optimal pH even when the medium composition is different, the Weissella koreensis DB1 (KCTC 13653BP) strain is a lactic acid bacteria or Weissella coriensis in rice bran fermentation. It was confirmed that the strain has optimal growth activity at a pH of a different condition than the optimal growth pH of the strain (Fig. 7), and since it was confirmed that the amount of ornithine production was also optimal at this optimal pH, the characteristics of the Weisella coriensis DB1 strain were utilized. It can significantly improve the efficiency of rice bran fermentation.

In addition, the method for manufacturing the fermented rice bran may further include a step of drying the fermented rice bran of drying the fermented rice bran.

The drying step is a hot air drying method, preferably performed by a hot air drying method for 1 hour to 40 hours at a temperature condition of 30°C to 60°C, or a freeze drying method, preferably at a temperature condition of -50°C to -80°C for 20 hours It can be carried out by a freeze drying method for up to 100 hours.

The hot air drying can be performed for 1 hour to 40 hours, preferably 5 to 30 hours, more preferably 10 to 20 hours at a temperature condition of 30°C to 60°C, preferably 40°C to 50°C. have.

The freeze-drying is from -50°C to -80°C, preferably from -70°C to -80°C for 20 to 100 hours, preferably 40 to 80 hours, more preferably 48 to 72 hours While you can do it.

In addition, the present invention may be a rice bran fermented product manufactured by the method for producing a rice bran fermented product.

Specifically, the fermented rice bran is separated from kimchi, and fermented rice bran fermentation comprising the step of fermenting by adding a strain of Weissella koreensis DB1 (KCTC 13653BP) having citrulline and ornithine producing ability. Rice bran fermented product prepared by a water production method or a fermentation raw material production step of mixing rice bran, arginine and water to prepare a mixture; Gelatinized rice bran manufacturing step of preparing a gelatinized rice bran mixture by heating the mixture to gelatinize the rice bran; A strain addition step of adding a Weissella koreensis DB1 (KCTC 13653BP) strain isolated from the kimchi and having citrulline and ornithine producing ability to the gelatinized rice bran mixture; And it may be a rice bran fermented product prepared by a method for producing a rice bran fermented product comprising a fermentation step of fermenting the gelatinized rice bran mixture to which the strain is added to prepare a rice bran fermented product.

The fermented rice bran has a citrulline content of 2,500 mg/kg or more, an ornithine content of 5,500 mg/kg or more, and preferably has a citrulline content of 2,700 mg/kg or more, based on the total weight of the rice bran fermentation in the rice bran fermentation product. , Ornithine content may be 7,000 mg/kg or more, more preferably citrulline content is 2,800 mg/kg or more, and ornithine content may be 8,000 mg/kg or more.

In addition, the fermented rice bran has a citrulline content of 2,500 mg/kg to 3,500 mg/kg and an ornithine content of 5,500 mg/kg to 10,500 mg/kg based on the total weight of the fermented rice bran in the fermented rice bran, preferably Preferably, the citrulline content is 2,700 mg/kg to 3,200 mg/kg, the ornithine content is 7,000 mg/kg to 9,500 mg/kg, more preferably the citrulline content is 2,800 mg/kg to 2,900 mg/kg, The ornithine content may be 8,000 mg/kg to 8,800 mg/kg.

Since the rice bran fermentation product contains a high content of citrulline and ornithine, in addition to the functionality of the existing rice bran or rice lactic acid bacteria fermentation product, anti-obesity activity, blood circulation improvement ability, liver function improvement ability, diuresis by additional citrulline and ornithine It may be that the function, the ability to improve the body function or improve the skin beauty such as wrinkle improvement is added, or the functionality of the existing rice bran or rice lactic acid bacteria fermentation has been enhanced.

In this dimension, the fermented rice bran of the present invention is a functional food or a raw material of a functional food, preferably a functional food or a raw material of a functional food for preventing or improving obesity, or a functional food or a raw material of a functional food for preventing or improving hyperlipidemia. Can be used as.

In this aspect, the present invention may relate to a food composition for improving or preventing obesity comprising the fermented rice bran produced by the method for producing fermented rice bran as an active ingredient.

The food composition for improving or preventing obesity may preferably be a health functional food.

In another aspect, the present invention may relate to a food composition for improving or preventing hyperlipidemia comprising the fermented rice bran produced by the method for producing fermented rice bran as an active ingredient.

The food composition for improving or preventing hyperlipidemia may preferably be a health functional food.

The active ingredient is preferably separated from the kimchi, and fermenting rice bran fermentation comprising the step of fermenting by adding a Weissella koreensis DB1 (KCTC 13653BP) strain having citrulline and ornithine producing ability. Rice bran fermented product prepared by a water production method or a fermentation raw material production step of mixing rice bran, arginine and water to prepare a mixture; Gelatinized rice bran manufacturing step of preparing a gelatinized rice bran mixture by heating the mixture to gelatinize the rice bran; A strain addition step of adding a Weissella koreensis DB1 (KCTC 13653BP) strain isolated from the kimchi and having citrulline and ornithine producing ability to the gelatinized rice bran mixture; And it may be a rice bran fermented product prepared by a method for producing a rice bran fermented product comprising a fermentation step of fermenting the gelatinized rice bran mixture to which the strain is added to prepare a rice bran fermented product.

The fermented rice bran has a citrulline content of 2,500 mg/kg or more, an ornithine content of 5,500 mg/kg or more, and preferably has a citrulline content of 2,700 mg/kg or more, based on the total weight of the rice bran fermentation in the rice bran fermentation product. , Ornithine content may be 7,000 mg/kg or more, more preferably citrulline content is 2,800 mg/kg or more, and ornithine content may be 8,000 mg/kg or more.

In addition, the fermented rice bran has a citrulline content of 2,500 mg/kg to 3,500 mg/kg and an ornithine content of 5,500 mg/kg to 10,500 mg/kg based on the total weight of the fermented rice bran in the fermented rice bran, preferably Preferably, the citrulline content is 2,700 mg/kg to 3,200 mg/kg, the ornithine content is 7,000 mg/kg to 9,500 mg/kg, more preferably the citrulline content is 2,800 mg/kg to 2,900 mg/kg, The ornithine content may be 8,000 mg/kg to 8,800 mg/kg.

The obesity refers to a state in which adipose tissue is excessively accumulated due to an increase in the quantity and number of adipocytes in the body, and the increase in the quantity and number of these adipocytes causes localization, that is, the differentiation of adipocytes and the accumulation of fat. Occurs by

The hyperlipidemia refers to a condition that can cause a disease due to a high level of fat in the blood or a disease caused by a high content of fat in the blood. The hyperlipidemia is divided into endogenous hyperlipidemia and exogenous hyperlipidemia, and endogenous hyperlipidemia is often caused by abnormal cholesterol metabolism, and exogenous hyperlipidemia is often caused by excessive intake of food. With regard to the cause of the onset of the exogenous hyperlipidemia, it has been reported that an increase in the blood concentration of triglycerides caused by ingestion of food is closely related.

Cholesterol control means that cholesterol levels in the body are controlled in an appropriate range so that the accumulation of cholesterol in plasma and tissues, especially blood vessels, does not occur. A disease in which cholesterol is accumulated above an appropriate level is called hypercholesterolosis, and abnormal cholesterol control functions occur for a long time, and if the hypercholesterolosis is maintained for a certain period of time or longer, it may lead to atherosclerosis.

The food composition for improving or preventing obesity or the food composition for improving or preventing hyperlipidemia may be used to suppress, improve or prevent obesity or hyperlipidemia. The food composition may be a functional food composition, specifically a health functional food composition for preventing or improving the obesity or hyperlipidemia. The health functional food composition may be a health functional beverage.

The food composition for improving or preventing obesity or the food composition for improving or preventing hyperlipidemia is from 0.0001% to 99.999% by weight or 0.001% by weight to the total weight of the rice bran fermented product extract prepared by the method for producing fermented rice bran fermented product. 99.9% by weight or 0.01% by weight to 99% by weight or 0.1% by weight to 90% by weight or 1% by weight to 75% by weight or 3% by weight to 50% by weight may be included.

The food composition for improving or preventing obesity or the food composition for improving or preventing hyperlipidemia, comprising the fermented rice bran produced by the fermented rice bran produced by the method for producing fermented rice bran can be directly applied to animals, including humans. The animal is a group of organisms corresponding to plants, mainly ingesting organic matter as nutrients, and refers to a differentiated digestive organ, excretory organ, and respiratory organ, and may be preferably a mammal, more preferably a human.

The rice bran fermented product prepared by the method for producing fermented rice bran can be used alone in the food composition for improving or preventing obesity or the food composition for improving or preventing hyperlipidemia, and other pharmacologically acceptable carriers, excipients, It may additionally contain a diluent or an accessory ingredient.

The food is a natural product or processed product containing one or more nutrients, and may be in a state that can be eaten directly through a certain processing process, and as a general meaning, health functional foods, beverages, food additives and beverages It is intended to include all additives and the like.

Foods of the present invention include, for example, various foods, beverages, gum, tea, vitamin complexes, and health functional foods. In addition, the food in the present invention includes special nutritional foods (e.g., formula, infant food, etc.), processed meat products, fish meat products, tofu, muk, noodles (e.g., ramen, noodles, etc.), health supplements, seasoning foods (e.g. , Soy sauce, soybean paste, red pepper paste, mixed sauce, etc.), sauces, confectionery (e.g., snacks, etc.), dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.), beverages ( Examples include, but are not limited to, fruit vegetable beverages, fermented beverages, soy milk, etc.), and natural seasonings (eg, ramen soup, etc.). The foods, health functional foods, beverages, food additives, and beverage additives may be prepared by conventional manufacturing methods.

In the present invention, the health functional food refers to a food group or food composition that has added value to act and express the function of the food by using physical, biochemical, and biotechnological techniques, etc. It refers to food that is designed and processed to sufficiently express the weight control function related to recovery, etc. to the living body.

The health functional food may include food additives acceptable for food, and may further include suitable carriers, excipients, and diluents commonly used in the manufacture of health functional foods.

In the present invention, the beverage refers to a generic term for drinking to quench thirst or to enjoy the taste, and is intended to include a health functional beverage. The beverage is an essential component at the indicated ratio, and there is no particular limitation on other components other than containing the fermented rice bran as an active ingredient, and as an ordinary beverage, various flavoring agents or natural carbohydrates can be contained as an additional component. have.

Examples of the above natural carbohydrates include monosaccharides, such as disaccharides such as glucose and fructose, such as maltose, sucrose, and the like, and polysaccharides, such as dextrin, cyclodextrin, and the like, and conventional sugars, and These are sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate may be generally about 1 to 20 g, preferably 5 to 12 g, per 100 mL of the food composition of the present invention. It may further contain pulp for the manufacture of beverages.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring and natural flavoring agents, coloring agents and heavy weight agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid, and It may contain salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like. These components may be used independently or in combination. The proportion of these additives is not so important, but may be selected in the range of 0 to 200,000 parts by weight per 100 parts by weight of the rice bran fermentation of the present invention.

In the present invention, a health-functional beverage refers to a beverage group or beverage composition that has added value to act and express the function of the beverage for a specific purpose by using physical, biochemical, and biotechnological techniques, etc., to control the biological defense rhythm of the beverage composition, and to prevent diseases. It refers to a beverage that has been designed and processed to fully express the function of gymnastics for and recovery.

The health functional beverage is an essential ingredient in the indicated ratio, and there is no particular limitation on other ingredients other than containing the rice bran fermented product of the present invention, and may contain various flavoring agents or natural carbohydrates, etc. as an additional ingredient like a normal beverage. .

Examples of the natural carbohydrates include monosaccharides, such as disaccharides such as glucose and fructose, such as maltose, sucrose, and the like, and polysaccharides, such as dextrin, cyclodextrin, and the like, and xylitol. , Sugar alcohols such as sorbitol and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate may be generally about 1 g to 20 g, preferably 5 g to 12 g per 100 mL of the composition of the present invention.

In addition, the composition of the present invention may further contain flesh for the manufacture of natural fruit juice, fruit juice beverage, and vegetable beverage.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring and natural flavoring agents, coloring agents and heavy weight agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid, and It may contain salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like. These components may be used independently or in combination. The proportion of these additives is not so important, but may be selected in the range of 0 to 200,000 parts by weight per 100 parts by weight of the rice bran fermentation of the present invention.

In the present invention, the health functional beverage refers to a beverage group or a beverage group that has added value to act and express the function of the beverage for a specific purpose by using physical, biochemical, and biotechnological techniques, etc., to control the biological defense rhythm of the beverage composition, and to prevent diseases. It refers to a beverage that is designed and processed to sufficiently express weight control functions related to and recovery to the living body.

The health functional beverage is an essential component in the indicated ratio, except for containing the rice bran fermented product of the present invention, there is no particular limitation on other components, and may contain various flavoring agents or natural carbohydrates, etc. as an additional component, like a normal beverage. .

Examples of the natural carbohydrates may be monosaccharides such as glucose or fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, or sugar alcohols such as xylitol, sorbitol, and erythritol. . The flavoring agent may be a natural flavoring agent such as taumatin, rebaudioside A, or a stevia extract such as glycyrrhizin, or a synthetic flavoring agent such as saccharin or aspartame.

The amount of the natural carbohydrate added may be generally about 1 g to 20 g, preferably 5 g to 12 g per 100 mL of the food composition of the present invention. In addition, the composition of the present invention may further contain flesh for the manufacture of natural fruit juice, fruit juice beverage, and vegetable beverage.

In addition, in a food composition for preventing or improving obesity comprising fermented rice bran as an active ingredient or a food composition for preventing or improving hyperlipidemia comprising fermented rice bran as an active ingredient, the active ingredient is 0.01% by weight to the total weight of the food. It may be included in an amount of 15% by weight, and the beverage composition may be included in an amount of 0.02 g to 5 g, preferably 0.3 g to 1 g, based on 100 mL.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in various different forms, and the examples described herein are for illustrative purposes only, and the present invention is not limited to the following examples.

In this experiment, the same experiment was performed three times, and the data obtained from each experiment was expressed as mean and standard deviation (Mean ± SEM) using the SAS 9.1 version statistical program. In order to verify the significance between groups, it was analyzed through one-way ANOVA, and the post-hoc test was verified by Duncan's multiple range test at P <0.05 level between each experimental group.

<Example> Manufacturing and utilization of fermented rice bran

Example 1. Isolation and identification of strains

Ornithine in advance among lactic acid bacteria isolated from kimchi samples in the optimal ripening period collected from 36 homes, famous houses, restaurants, temples, and companies nationwide in five regions including Seoul, Gyeonggi, Gangwon, Chungcheong, Yeongnam and Honam. The 40 types of kimchi lactic acid bacteria in Table 1, which were confirmed to have productive ability, were isolated and used as an experimental strain.

In order to select the lactic acid bacteria for the production of rice bran fermentation, 1% arginine was added to the MRS liquid medium, and the 40 strains of Table 1 were stationarily cultured at 30° C. for 48 hours, and then each of the strains was cultured. The degree of ornithine production in one culture was analyzed using Thin Layer Chromatography (TLC). Specifically, the culture solution obtained by culturing each strain was centrifuged for 3 minutes at 9,950 xg and 4° C., and then a supernatant was obtained, and 2 μL of the supernatant was dropped on a TLC plate (Merck, Germany), and then acetic acid ( acetic acid, Darjung, Republic of Korea), 1-butanol (1-butanol, Junsei co., Japan) and tertiary distilled water based on volume 3: 12: 5 (acetic acid: 1-butanol: tertiary distilled water, v/v ) Was developed twice in 7 cm increments in the developing solvent. After the development is complete, the TLC plate is dried at room temperature and then immersed in a 0.5% ninhydrin (Alfa Aesar, USA) solution and heated to see whether or not a spot is visible in the same position as compared to the arginine and ornithine standards. And by comparing how dark the corresponding spot (spot), it was confirmed the degree of production of ornithine of each strain. The results of the development of the TLC plate are shown in FIG. 2 below.

As shown in FIG. 2, of the 40 strains, 7 strains, specifically, strain 3 (GH), strain 10 (GL), strain 12 (DB1), strain 20 (CM), 23 Strains No. (IS), No. 38 (CGM1) and No. 40 (HJ) were visually confirmed to have excellent ornithine-producing ability.

Based on the results of FIG. 2, in order to confirm the ornithine production ability of the seven strains, the degree of ornithine production was confirmed by using HPLC through amino acid analysis.

Specifically, the culture solution obtained by culturing the 7 strains was centrifuged at 9,950 xg and 4°C for 3 minutes, and then a supernatant was obtained, and the resulting supernatant was filtered through a 0.2 μm membrane filter (Sartotius, Germany) to prevent impurities. The etc. were removed and used for analysis.

OPA (O-phthalaldehyde)-FMOC (fluorenylmethyl chloroformate) was derivatized and analyzed by HPLC (Ultimate 3000, Thremo Scientific Dionex, USA). The column oven temperature was set to 40°C, and an Inno C18 column (4.6 x 150 mm, 5 μm, Youngjin Biochrom, Korea) was used. The mobile phase was a mixture of 40 mM sodium phosphate (pH 7.0), water: acetonitrile: methanol = 10: 45: 45, A and B, respectively, at a flow rate of 1.5 mL/min. The gradient conditions are: Solution A: Solution B, from 0 to 24 minutes, 95: 5 (v/v, %), 24 to 25 minutes, 45: 55, 23 to 34.5 minutes, 20: 80, 34.5 minutes to 95: 5 It was set up and analyzed. The amino acid content was calculated using a standard curve prepared based on the results of HPLC analysis of standard amino acids.

The calculation results of amino acids contained in the culture medium of the seven strains are shown in Table 2 below. The units of each amino acid in Table 2 below are expressed in units of mg (mg/L) based on 1 L of the analysis sample. In addition, N.D in Table 2 means that the corresponding amino acid was not found.

As shown in Table 2, it was confirmed that the DB1 strain had remarkably excellent ornithine-producing ability among the seven strains confirmed to have excellent ornithine-producing ability in HPLC results. Of the seven strains, most strains excluding the DB1 strain were 5,000 mg to 6,500 mg, specifically 5,059.53 mg to 6,520.55 mg, whereas the DB1 strain was 8,373.59 mg, which was found to have better ornithine production ability than other strains. Is 165.5% compared to the GH strain, and 110.1% compared to the HJ strain, showing excellent ornithine-producing ability. From the above results, the DB1 strain was selected and isolated as the strain having the best ornithine-producing ability.

In order to identify the DB1 strain selected as the strain having the best ornithine-producing ability, morphological and biochemical properties were observed. The investigation of the morphological characteristics was performed by observing the morphology of bacteria using a microscope and whether or not Gram was stained, and the results are shown in Table 3 below.

Characteristics Strain (DB1) Gram-stain Positive (+) Cell morphology Monococci

As shown in Table 3, the isolated DB1 strain is a Gram-positive monobacterial bacteria that form round, smooth and ivory-colored flat colonies when cultured at 30°C for 48 hours using MRS plate medium. Confirmed. In addition, as a result of performing a catalase test, it was confirmed as catalase-negative. A biochemical investigation was performed to identify the isolated strain. The biochemical investigation was performed by examining the sugar availability using the API 50CHL system (BioMerieux, France) and then confirming using the strain identification program (http://apiweb.biomerieux.com). The results of the investigation of sugar availability using the API 50CHL system are shown in Table 4 below.

In Table 4,-or + is determined on the basis of sugar metabolism or sugar utilization, and is indicated as-when there is no sugar metabolism for each sugar, and + when there is sugar metabolism.

As shown in Table 4, the DB1 strain is L-Arabinose, Ribose, D-Xylose, D-Glucose, D-Fructose It was confirmed to have glucose metabolism against D-Fructose, D-Mannose and N-Acetyl glucosamine.

As a result of the morphological analysis and biochemical analysis performed through the glucose metabolic analysis, the DB1 strain was estimated to be Weissella koreensis , and to finally identify it, 16S rRNA sequence analysis was performed. Specifically, the 16S rRNA nucleotide sequence of DB1 was determined as shown in FIG. 3, and 16S rRNA nucleotide sequence analysis was performed by comparing it with the nucleotide sequence of other strains registered in GenBank.

The nucleotide sequence of the 16S rRNA of the isolated strain was performed in the following manner. After the chromosomal DNA of the isolate was isolated using a Genomic DNA purification kit (Qiagen, Germany), PCR was performed using primers having the sequence shown in Table 5 below.

Primers Sequence (5′→ 3′) 785F GGATTAGATACCCTGGTA 907R CCGTCAATTCMTTTRAGTTT

In the PCR, after performing a pre-denaturation step at 94° C. for 1 minute, the process of 30 seconds at 94° C., 30 seconds at 55° C. and 1 minute at 72° C. was repeated 30 times, and 10 at 72° C. It proceeded by a method of performing a minute termination step (termination). The amplified PCR product was purified using a multiscreen filter plate (Millipore Co., USA), and a sequencing reaction was performed using the BigDye(R) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). For the DNA sample containing the amplified product, the nucleotide sequence was determined using an ABI Prism Prism 3730xl DNA analyzer (Applied Biosystems), and as shown in FIG. 3 from the result, a total nucleotide sequence of 1,460 bp was confirmed. The nucleotide sequence was compared with the nucleotide sequence registered as a ribosomal RNA gene sequence in GenBank using the BLASTN program, and the homology between the nucleotide sequences was compared and analyzed using the Clustal X program.

* The 16S rRNA base sequence of strain DB1 was determined to represent the way Cellar Corey N-Sys S-5623 ^T strain (Weissella koreensis S-5623 ^T) and 99.86% homology. Therefore, finally, the DB1 strain was identified as Weissella koreensis , and the DB1 strain was named Weissella koreensis DB1.

Hereinafter, the Weissella koreensis DB1 strain was deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on October 2, 2018, and was given the accession number KCTC 13653BP.

Example 2. Confirmation of growth characteristics and functionality of isolated strains

2-1. Confirmation of optimum growth pH and temperature conditions of isolated strains

The optimal growth temperature and growth pH conditions of the Weissella koreensis DB1 strain were confirmed as follows.

First, the Weissella koreensis DB1 strain was inoculated in MRS medium (Difco, USA) at 1% (v/v), about 6.70 log CFU/mL, and the culture temperature was 5℃ at intervals of 5℃. The culture was carried out at a temperature range of to 30°C, and the degree of growth of the strain was confirmed by measuring the absorbance at 600 nm for each time. The results of measuring the growth degree are shown in FIG. 4.

As shown in FIG. 4, it was confirmed that the optimum growth temperature was 30°C. Specifically, in the case of 5°C, the degree of growth reached a plateau after 11 days, and the absorbance (A ₆₀₀ ) measured at the time of 11 days was confirmed to be 1.787 ± 0.012, and in the case of 10°C , After 4 days, the growth level entered a plateau, and the absorbance (A ₆₀₀ ) measured at 4 days was confirmed to be 2.535 ± 0.021, and in the case of 15°C, after 4 days At the time point, the degree of growth reached a plateau, and the absorbance (A ₆₀₀ ) measured at the time of 4 days was confirmed to be 2.955 ± 0.133, and in the case of 25°C, the degree of growth at the time of 20 hours had passed. _{The absorbance (A 600} ) measured at the time of 20 hours elapsed was confirmed to be 3.162 ± 0.157, and in the case of 30°C, the most rapid growth activity was confirmed at the time point 16 hours elapsed, and 16 hours From the time point elapsed, the number of cells gradually increased, but the degree was limited, and the absorbance (A ₆₀₀ ) measured at the time of 16 hours was found to be 3.035 ± 0.174, and the absorbance (A ₆₀₀ ) was 3.000 in the shortest time. It was confirmed that 30°C increased above is the optimum growth temperature.

By varying the pH at 30° C., which was identified as the optimal growth temperature, the optimal growth pH of the Weisella coriensis DB1 strain was confirmed. Specifically, the Weisella coriensis DB1 strain was 1% in MRS medium (Difco, USA) adjusted to pH 4.0, pH 5.0, pH 6.0, pH 7.0 and pH 8.0 using 5 N HCl or 5 N NaOH. (v/v), inoculated at about 6.70 log CFU/mL, cultured for 16 hours while varying the culture pH, and then measured the absorbance at 600 nm to confirm the growth degree of the strain. The results of measuring the growth degree are shown in FIG. 5.

As shown in FIG. 5, it was confirmed that the optimum growth pH was between pH 6 and pH 8. Specifically, from pH 4 to pH 6, the growth activity increases as the pH increases and approaches neutrality, whereas in pH 6 to pH 8, the viable cell number is 8.32 log CFU/mL to 8.84 log CFU/mL. Showing a similar degree, the growth pH was found to be between pH 6 and pH 8.

Based on the above-identified results, the optimal growth conditions for the Weissella coriensis DB1 strain to produce ornithine, that is, the optimal growth pH in the growth conditions to which arginine is added, is from pH 6 to the identified optimal growth pH condition. It was further checked whether it coincided with pH 8.

Specifically, the pH was varied using the MRS medium to which 1% (w/v) arginine was added at 30° C., which was confirmed as the optimum growth temperature, and the optimum growth pH of the Weisella coriensis DB1 strain was confirmed. Specifically, 1% (w/v) arginine adjusted to pH 4.0, pH 5.0, pH 6.0, pH 6.5 and pH 7.0 using 5 N HCl or 5 N NaOH was added to the Weisella coriensis DB1 strain Inoculated with 1% (v/v), about 6.70 log CFU/mL in the MRS medium, cultured for 24 hours with different culture pH, and then measured the absorbance at 600 nm to confirm the growth degree of the strain. The results of measuring the growth degree are shown in FIG. 6.

As shown in FIG. 6, it was confirmed that the optimum growth pH was measured using an existing MRS medium, that is, pH 5, unlike pH 6 to pH 8. Specifically, it was confirmed that the growth activity was low at pH 4, whereas the absorbance (A ₆₀₀ ) at pH 5 was higher than 5.000 (5.08 ± 0.11) and was found to be the best, whereas at pH 6 (A ₆₀₀ 4.52 ± 0.20), the pH The growth activity decreased as it increased and approached neutrality.In the case where the pH was not adjusted with the medium containing 1% (w/v) arginine in the MRS medium (initial pH was pH 7.2 to pH 7.5), the pH was 7 It was found to be lower, and it was confirmed that the initial pH was adjusted to pH 5 in the growing condition to which arginine was added.

2-2. Confirmation of optimal ornithine production conditions of isolated strains

Based on the results confirmed in 2-1, in order to confirm the ornithine production capacity according to the culture conditions of the Weissella koreensis DB1 strain, 1 adjusted the initial pH to pH 5 at 30°C. %(w/v) arginine-added MRS medium and 1% (w/v) arginine-added MRS medium (pH 7.2 to pH 7.5) for 48 hours, 24 hours elapsed The degree of ornithine production at the time point and 48 hours elapsed was confirmed.

The ornithine production ability was confirmed through an amino acid analysis method using HPLC in the same manner as described above.

Specifically, the culture solution obtained by culturing the Weicella coriensis DB1 strain in each of the above conditions was centrifuged for 3 minutes at 9,950 xg and 4°C for 3 minutes, and then a supernatant was obtained, and the obtained supernatant was used as a 0.2 μm membrane filter (Sartorius , Germany) to remove impurities, etc., and used for analysis. OPA (O-phthalaldehyde)-FMOC (fluorenylmethyl chloroformate) was derivatized and analyzed by HPLC (Ultimate 3000, Thremo Scientific Dionex, USA). The column oven temperature was set to 40°C, and an Inno C ₁₈ column (4.6 × 150 mm, 5 μm, Youngjin Biochrom, Korea) was used as the column. The mobile phase was a mixture of 40 mM sodium phosphate (pH 7.0), water: acetonitrile: methanol = 10: 45: 45, A and B, respectively, at a flow rate of 1.5 mL/min. The gradient conditions are: Solution A: Solution B, from 0 to 24 minutes, 95: 5 (v/v, %), 24 to 25 minutes, 45: 55, 23 to 34.5 minutes, 20: 80, 34.5 minutes to 95: 5 It was set up and analyzed. The amino acid content was calculated using a standard curve prepared based on the results of HPLC analysis of standard amino acids.

Table 6 shows the results of measuring the content of each amino acid identified through HPLC after culturing in MRS medium to which 1% (w/v) arginine was added, in which the initial pH, which is the optimum production condition, was adjusted to pH 5. .

As shown in Table 6, the content of ornithine cultured in MRS medium containing 1% (w/v) arginine in which the initial pH was adjusted to pH 5 after 48 hours elapsed was 11,090.80 mg/ It was confirmed as L, and it was confirmed that ornithine production ability was very excellent, and citrulline, which is known to have anti-obesity activity in addition to ornithine, was also confirmed to be 319.33 mg/L, and it was also confirmed that citrulline production ability was also present.

Specifically, when the initial pH was not adjusted, the content of ornithine was confirmed to be 8,373.59 mg/L after 48 hours, and 1% (w/v) arginine was added with the initial pH adjusted to pH 5. It was confirmed that when cultured in the medium for 48 hours (11,090.80 mg/L), the productivity improved by more than 32% to 132.4% compared to the control without adjusting the initial pH.

In addition, even when compared with other strains producing ornithine in the results of previous studies (The Korean Journal of Microbiology, Vol 45, No. 4, p. 339-345), the strain of the present invention has remarkably excellent ornithine producing ability. Was confirmed. For comparison with the previous study results, when cultured for 48 hours without adjusting the initial pH as in the previous study results, the content of ornithine produced in 1 L of the culture medium per hour was compared, and the results are shown in Table 7 below. .

Strain name Ornithine production capacity (mg/L/hr) Weissella koreensis OK1-4 27.01 Weissella koreensis OK1-6 31.41 Weissella koreensis KCCM 41517 29.23 Lactobacillus hilgardii KCCM 40759 21.27 Weissella koreensis DB1 174.45

As shown in Table 7 above, in the case of lactic acid bacteria capable of producing ornithine from existing arginine, the ornithine production ability was confirmed to be about 21 mg/L/hr to 31.5 mg/L/hr, whereas weissella coriensis of the present invention DB1 ( Weissella koreensis DB1) strain was confirmed to have a production capacity of 174.45 mg/L/hr, which is about 5.6 to 8.2 times better than the existing strain. In addition, in the existing studies, lactic acid bacteria capable of producing citrulline were not identified at all. On the other hand, it was confirmed that the Weicella coriensis DB1 strain of the present invention can also produce citrulline.

From the result roll, the Weissella coriensis DB1 strain of the present invention has not only excellent ornithine production ability, but also citrulline production ability, and thus it was confirmed as an optimal strain for the production of an active ingredient for improving anti-obesity or hyperlipidemia.

2-3. Confirmation of the safety of isolated strains

Based on the results confirmed in Examples 2-1 and 2-2, the safety of the Weissella koreensis DB1 (Weissella koreensis DB1) strain was confirmed.

First, in order for lactic acid bacteria to be utilized as a fermentation strain for food production, a probiotic active agent, or a probiotic microorganism, safety without hemolytic toxicity to the human body is required. In this regard, with respect to whether the Weicella coriensis DB1 strain can induce destruction or degradation of red blood cells, hemolytic properties were examined.

에서 48시간 배양하여 균체주위에 투명환의 생성여부로 용혈성을 판단하였으며, 그 결과를 도 7에 나타내었다.Specifically, the hemolytic confirmation is after sterilization of a blood agar base (Oxoid, UK), after making a plate medium by adding 7% horse blood (Oxoid, UK), and then streaking the Weisella coriensis DB1 strain. , 30

The hemolytic properties were determined by incubation for 48 hours in the cells, and the hemolytic properties were determined by the formation of transparent rings around the cells, and the results are shown in FIG.

As shown in FIG. 7, the Weicella coriensis DB1 does not induce a hemolytic reaction in any case (α-hemolysis and β-hemolysis) in the hemolytic test because a transparent ring caused by destruction of red blood cells around the cells is not generated at all. It was confirmed that it does not. Therefore, it was confirmed that the Weisella coriensis DB1 had no harmful effects on the human body such as hemolytic properties, and it was confirmed that it has safety as a seed of fermented food.

In addition, when lactic acid bacteria ingested as food or medicine changes in the body's immune system due to substances that are produced to grow on their own, safety problems may arise. In the case of lactic acid bacteria, which are related GRAS microorganisms, basic safety is not required. As a result, problems related to antibiotic resistance in the body are receiving more attention due to lactic acid bacteria. In this regard, the Weicella coriensis DB1 strain was tested for antibiotic resistance.

In order to confirm the safety of the Weisella coriensis DB1 strain, the sensitivity to 8 kinds of antibiotics that are mainly used in the past was measured using a broth microdilution method. The eight antibiotics were Ampicillin (AMP), Vacomycin (VAC), Gentamycin (GEN), Kanamycin (KAN), Streptomycin (STR), Erythromycin (ERY), Tetracycline (TET), Chloramphenicol (CHL), and Sigma-Aldrich It was purchased from (USA) and used after dissolving in a prescribed solvent. In addition, the medium for measuring the antibiotic sensitivity is a Muller-Hinton (MH, BBL Muller Hinton Broth, Becton Dickinson and Company, USA) liquid medium with 0.5% dextrose added by adjusting the antibiotics for each concentration. I did.

The antibiotic sensitivity was performed by measuring the minimum inhibitory concentration (MIC) of the antibiotic. Specifically, the minimum growth inhibitory concentration for antibiotics of the strain was performed in the following manner. First, after inoculating 1% (v/v) in MRS liquid medium and incubating for 24 hours at 30° C., the culture medium was centrifuged for 4 minutes at 9,950 xg to recover the cells, and then MH containing the 0.5% Dextrose. It was suspended in a liquid medium (Muller-Hinton broth) and _{adjusted to an OD 600} value of 0.4 to 0.5, and the suspension was diluted 1:10 in MH liquid medium. The suspension of the strain, that is, the diluted culture solution was added to the prepared medium by diluting the antibiotic step by step, and then incubated at 30° C. for 24 hours under anaerobic conditions. Susceptibility was determined by measuring turbidity and absorbance at 600 nm (Ultrospec 2100 pro, Amersham Biosciences, Sweden) to confirm growth. As a control for turbidity and growth, the result of culturing the strain in MH liquid medium to which antibiotics were not added was used. Since there is no break point for Weissella koreensis suggested by the European Food Safety Authority (EFSA, 2012), the sensitivity analysis for the strain is related to antibiotic resistance of Weissella koreensis. Antibiotic susceptibility or resistance was determined by comparison with experimental values confirmed in other studies performed. Table 8 below shows the measurement results and experimental values of Weisella Corrients confirmed in other studies. The unit of Table 8 below is MIC (μg / mL).

As shown in Table 8, the Weissella coriensis DB1 strain, which is the strain of the present invention, showed a lower minimum growth inhibitory concentration compared to the results of a preliminary study on all eight kinds of antibiotics, and was susceptible to all of the eight kinds of antibiotics. Was found to have. From the above results, it was confirmed that the Weisella coriensis DB1 strain exhibits only a standard level of antibiotic resistance, which does not present a risk to human health, and thus does not have antibiotic resistance, that is, it is a safe strain that can be used as a probiotic.

In addition, it was recently confirmed whether bioamines were produced, which is a problem related to fermented foods in Korea as well as in the West. The bio-amine production was compared with the previously reported strain Enterococcus faecalis ATCC 29212 as having bio-amine-producing ability, and the decarboxylase activity confirmation experiment (Decarboxylase plate method, Bover-Cid) and in vivo The degree of amine production was confirmed by an experiment confirming by HPLC.

For the decarboxylase activity confirmation experiment, first, 1 mL of the strain culture solution was added to 9 mL of a liquid medium to which 0.1% of Tyrosine was added as a precursor amino acid, followed by incubation for 24 hours, and then prepared at a concentration of 9.0 log CFU/mL. The prepared culture was carried out by dropping 10 μL on a decarboxylase plate to which 1% Tyrosine was added to check the color change around the colony formed by the corresponding strain. The result of confirming the change in the surrounding color of the formed colony is shown in FIG. 8A.

As shown in FIG. 8A, ATCC 29212 ( Enterococcus faecalis ATCC 29212) on the left has a vivid purple color around the colony, confirming that it has bioamine-producing ability, whereas DB1 (Weissella coriensis DB1 of the present invention) ) Did not change at all around the colony, so it was judged that there was no bioamine-producing ability.

In addition, in order to confirm the bioamine-producing ability of Weissella coriensis DB1, HPLC was performed on the culture medium of the Weissella coriensis DB1 strain, and whether or not bioamine was produced and the amount of bioamine produced were quantitatively verified.

Verification using the HPLC was performed in the following manner. First, in order to verify whether or not bioamine is produced using HPLC, a bioamine standard solution (BA standard solution) was prepared. The bioamine standard solution was prepared using a total of 9 types of bioamine and an internal standard (IS). Specifically, the nine biological amines are tryptamine (tryptamine hydrochloride, TRY, 49.12 mg), putrescine (putrescine dihydrochloride, PUT, 73.2 mg), cadaverine (cadaverine dihydrochloride, CAD, 68.56 mg), and spur midyne (TRY, 49.12 mg). spermidine trihydrochloride, SPD, 70.16 mg), phenylethylamine (2-phenylethylamine hydrochloride, PHE, 52.08 mg), spermine (spermine tetrahydrochloride, SPM, 68.8 mg), histamine (histamine dihydrochloride, HIS, 66.24 mg), tyramine (tyramine hydrochloride) , TYR, 50.72 mg) and agmatine (agmatine sulfate, AGM, 70.16 mg), and the 9 kinds of biological amines and internal standards (1,7-diaminoheptane, IS, 40 mg) were added to each of 0.1 N HCl 40 mL. It was dissolved and prepared at a final concentration of 1 mg/mL. The biological amine standard solution (BA standard solution) was prepared by mixing the biological amine and the internal standard substance (IS) at a concentration of 0.1 mg/mL, respectively.

에서 24시간 전배양한 후, MRS broth와 MRSO broth(상기 MRS broth에 0.1% 오르니틴(Ornithine, 전구체)를 첨가한 배지)에 각각 전배양한 균주를 10%씩 접종하여 24시간 동안 배양하였다. 상기 24시간 동안 배양한 후, 4

및 9,950 x g의 조건으로 15분간 원심분리한 후, 상등액만을 분리하여 균체를 제거하고 배양 상징액을 회수하였다. 상기 회수한 배양 상징액을 0.2 μm membrane filter로 제균한 후, 동결건조하여 10배 농축하였다. 상기 10배 농축한 농축 배양 상징액 2 mL에 5% trichloroacetic acid(TCA)-0.4 M perchloric acid(HClO4) 10 mL을 가하고, 상기 내부표준물질(IS, 1,7-diaminoheptane)를 0.2 mg/mL첨가하여 추출하였다. 상기 생체아민 및 내부표준물질이 포함된 생체아민 표준용액과 상기 추출된 배양 상징 추출액에 대해 우선 유도 체화를 수행한 후, HPLC를 수행하였다. In addition, the method of extracting a biological amine from the culture medium of Weicella coriensis DB1 strain for performing the HPLC is used by modifying the method of Hwang et al. (Journal of Chromatography B, Vol 693, pp. 23-30 (1997)). I did. More specifically, the Weisella coriensis DB1 strain was each 30

After pre-culture in 24 hours, the strains pre-cultured in MRS broth and MRSO broth (a medium in which 0.1% ornithine (precursor) was added to the MRS broth) were each inoculated with 10% of the strains and cultured for 24 hours. After incubation for the above 24 hours, 4

And after centrifuging for 15 minutes under the conditions of 9,950 xg, only the supernatant was separated to remove the cells, and the culture supernatant was recovered. The collected culture supernatant was sterilized with a 0.2 μm membrane filter, and then lyophilized and concentrated 10 times. 10 mL of 5% trichloroacetic acid (TCA)-0.4 M perchloric acid (HClO4) was added to 2 mL of the 10-fold concentrated culture supernatant, and 0.2 mg/mL of the internal standard (IS, 1,7-diaminoheptane) was added. And extracted. The bioamine standard solution containing the bioamine and the internal standard and the extracted culture symbol extract were first subjected to derivatization, followed by HPLC.

에서 40분간 반응시켰다(benzoylation). 상기 각각의 반응액에 포화된 NaCl 용액(saturated NaCl) 2 mL를 가하여 반응을 정지시키고, 디에틸 에테르(diethyl ether) 3 mL를 첨가하여 추출한 다음, 상징액 1.5 mL을 분취하여 질소 가스로 농축하였다. 상기 질소가스로 농축한 상징액을 50% 메탄올 수용액(이동상) 500 μL에 용해하여 HPLC 분석 시료로 사용하였다. 또한, 상기 생체아민 및 내부표준물질을 이용하여 정확한 생체아민 분석을 위해 HPLC 분석 조건을 설정하였다. 상기 HPLC를 수행한 기계에 관한 사항 및 수행조건과 최종 분석조건으로 결정된 HPLC의 분석조건을 하기 표 9 및 표 10에 나타내었다.The derivatization process performed before performing the HPLC and the derivative and process of the extracted concentrated culture symbol extract were performed in the following manner. 1 mL of 2 M NaOH and 10 μL of benzoyl chloride were added to the bioamine standard mixture solution and the extracted concentrated culture supernatant extract, respectively, and 30

It was reacted for 40 minutes at (benzoylation). The reaction was stopped by adding 2 mL of saturated NaCl solution to each of the reaction solutions, followed by extraction with 3 mL of diethyl ether, and 1.5 mL of the supernatant was aliquoted and concentrated with nitrogen gas. The supernatant concentrated with nitrogen gas was dissolved in 500 μL of a 50% methanol aqueous solution (mobile phase) and used as an HPLC analysis sample. In addition, HPLC analysis conditions were set for accurate bioamine analysis using the bioamine and internal standard material. Tables 9 and 10 show the details of the machine that performed the HPLC and the conditions for the HPLC analysis determined as the final analysis conditions.

Instrument Youngin instrument Detector UV-730D (254 nm) Column SunFire C ₁₈ (4.6 x 150 nm, 3.5 μm, Waters) Solvent A: water: methanol (50: 50) / B: water: methanol (10: 90) Oven temperature 40℃ Injection Vol 20 μL

Time(min) Flow rate (mL/min) A(%) B(%) 0 0.4 100 0 15 0.4 0 100 25 0.4 0 100 30 0.4 100 0 55 0.4 100 0

The HPLC results performed under the conditions of Table 9 and Table 10 are shown in FIG. 8B. As shown in FIG. 8B, it was confirmed that Weicella coriensis DB1 did not generate bioamine even in quantitative experimental results.

From the above results, the Weicella Coriansis DB1 of the present invention is not only universally secured as a GRAS microorganism, but also as a result of a hemolytic test, an antibiotic resistance test, and a bioamine-producing ability test, all of which were confirmed negative, and individual safety was also secured. Confirmed.

Example 3. Functional rice bran production using isolated strains

3-1. Confirmation of functional rice bran production conditions using isolated strains

Using the Weissella koreensis DB1 (Weissella koreensis DB1) strain, which has the citrulline and ornithine production ability, and ornithine production ability is remarkably superior to other strains, the optimum conditions are used to prepare the rice bran fermentation product. Confirmed.

First, in order to prepare a rice bran fermented product, a fermented strain and rice bran as a fermentation raw material were prepared by the following method.

The fermentation strain was inoculated with Weissella coriensis DB1 strain in MRS medium, cultured at 30°C for 24 hours, and then 1% of the culture medium was inoculated into MRS medium, followed by passage culture in which cultured at 30°C for 24 hours. Times, that is, after performing the first and second times, the second passage culture was centrifuged at 10,000 xg and 4° C. for 15 minutes, and then the supernatant was removed and the cells were recovered. Sterile water was added to the recovered cells and suspended at a concentration of about 6.70 log CFU/mL, and used as a seed for fermentation of rice bran.

The fermentation raw rice bran is 20% (w/v, rice bran weight/volume of distilled water), 45% (w/v, rice bran weight/volume of distilled water) and 50% (w /v, rice bran weight/distilled water volume), and then arginine was added at a concentration of 1% (w/v, arginine weight/distilled water volume).Experimental example (addition of 1% arginine) And Comparative Example (1% arginine not added) was prepared by dividing. Rice bran were gelatinized by heating the prepared rice bran mixture of each of the experimental examples and comparative examples under a temperature condition of 121°C and a pressure condition of 1.1 atm using an autoclave for 15 minutes. The gelatinized rice bran was cooled to room temperature (15° C. to 20° C.) and used as a rice bran fermentation object.

1% (v/v) of the rice bran fermentation seeds of about 6.70 log CFU/mL were inoculated into the rice bran fermentation solution to which arginine, which is the target of rice bran fermentation, and sufficiently mixed using a sterilized glass rod, and then at 30° C. for 48 hours During the fermentation was carried out. The fermented rice bran fermentation product was analyzed for changes in the number of viable bacteria and amino acid content in the fermented product every 24 hours.

The number of viable cells was analyzed by the plate count method, and the change in amino acid content was analyzed by the HPLC analysis performed in Example 2-2 on the fermented product after 24 hours.

The plate count method was performed in the following manner. First, the fermented product was mixed with sterilized water of 4 times the amount of the fermented product based on the volume ratio, and then juiced using a sterilized gauze. A dilution was prepared while diluting the juiced juice by 10 times by a stepwise dilution method (Serial dilution, 10 times dilution). The diluted stepwise dilution was dispensed on an MRS solid medium (MRS plate) and plated, incubated at 30° C. for 48 hours, and the number of colonies was counted every 24 hours and calculated as CFU/mL.

The measured number of viable cells is shown in Table 11 below.

Additive content Viable cell count (log CFU/mL) Rice bran content (w/v) Arginine content (w/v) 24 hours 48 hours 20% 0% 7.33±0.50 7.34±0.40 One% 7.47±0.36 7.48±0.42 45% 0% 7.56±0.34 7.56±0.40 One% 8.13±0.16 7.49±0.39 50% 0% 7.48±0.25 7.31±0.25 One% 8.11±0.19 7.46±0.22

As shown in Table 11, in all of the added rice bran content (20%, 45%, and 50%) based on the lapse of 24 hours, the number of viable cells of the fermentation strain Weisella coriensis DB1 strain was 1% (w /v) It was confirmed that the number of viable cells increased by about 0.1 to 0.6 log CFU/mL with the addition of arginine, and it was confirmed that the number of viable cells was significantly increased by the addition of arginine. In particular, the Weisella coriensis DB1 strain was confirmed to be able to fermentation because it was found to grow effectively even in a condition in which 50% or more of the culture stock solution was solid (50% rice bran and 1% arginine). In the case of general microorganisms, microbial fermentation is limited at high solids concentrations, thus exhibiting a limited effect in the industrialization of microbial fermentation strains where efficiency is important, whereas the Weisella coriensis DB1 strain, which is the strain of the present invention, ferments microorganisms even at high solids concentrations. Was confirmed to be effective, and the industrial effect was expected to be great.

3-2. Confirmation of optimal fermentation conditions for rice bran fermentation

It has the citrulline and ornithine production ability identified as a strain suitable for rice bran fermentation in 3-1, and the ornithine production ability is remarkably superior to other strains. In order to confirm the conditions, the optimum fermentation pH and the optimum fermentation temperature were confirmed.

The optimum fermentation pH and optimum fermentation temperature were confirmed by measuring the number of viable cells by measuring the number of viable cells identified in 3-1 under each fermentation condition. Specifically, sterilized water was added to the recovered cells prepared in Example 3-1 and suspended at a concentration of about 6.70 log CFU/mL, and the rice bran fermentation seeds were 20% (w/v) in tertiary distilled water. , Rice bran weight/volume of distilled water), and then arginine was added at a concentration of 1% (w/v, arginine weight/volume of distilled water), and then a temperature of 121℃ using an autoclave. The rice bran was gelatinized by heating under conditions and a pressure of 1.1 atm for 15 minutes, cooled to room temperature, and then inoculated into the prepared rice bran fermentation target, and fermentation was performed for 48 hours under each condition. In the fermented rice bran fermentation, the number of viable bacteria in the fermentation product was measured every 24 hours by a plate count method as in the method of 3-1. Fermentation was performed with different fermentation conditions, first, fermentation was performed at 30° C. with different pH, and then the optimum pH was checked, and thereafter, the fermentation was performed by varying the temperature at the optimum pH.

After fermentation according to the fermentation conditions, specifically fermentation temperature and fermentation pH conditions, the results of confirming the number of viable cells of the Weisella coriensis DB1 strain are shown in FIGS. 9 and 10, respectively.

As shown in FIG. 9, the optimum fermentation pH was confirmed to be pH 6.0 to pH 6.5. Specifically, after adjusting the pH of the rice bran fermentation object gelatinizing the rice bran to pH 4.0 to pH 7.0 using 5 N HCl or 5 N NaOH, fermentation for 48 hours at each pH, and the number of viable bacteria every 24 hours. , As the pH increased from pH 4.0, the number of viable cells increased as the pH became weaker, and the number of viable cells increased significantly at pH 6.0, and it was confirmed that the number of viable cells was 8.10 log CFU/mL based on 24 hours, and the viable cells similar to pH 6.0 at pH 6.5. While the number was confirmed to be 8.20 log CFU/mL, the number of viable cells was significantly lowered at pH 7.0, and pH 6.0 to pH 6.5 were found to be optimal for fermenting rice bran using the Weisella coriensis DB1 strain.

In addition, as shown in FIG. 10, the optimum fermentation temperature was confirmed to be 25 ℃ to 30 ℃. As a result of varying the temperature at pH 6.5, which is the optimum pH identified in FIG. 10, fermentation did not proceed well when the fermentation temperature was less than 20°C, whereas at 25°C, the number of viable cells was significantly increased and thus, based on 24 hours. The number of viable cells was found to be 7.97 log CFU/mL, and the fermentation efficiency was higher than 25°C at 30°C, and the number of viable cells was confirmed to be 8.10 log CFU/mL, whereas the number of viable cells at 37°C compared to 30°C. Significantly low, the optimum fermentation temperature was found to be 25 ℃ to 30 ℃, more preferably 30 ℃.

According to the results of FIGS. 9 and 10, the optimal fermentation conditions for the production of functional rice bran fermentation products using the Weisella coriensis DB1 strain are a pH condition of pH 6.0 to pH 6.5 and a temperature condition of 25°C to 30°C. Confirmed.

In addition, the optimum fermentation pH was confirmed by analyzing changes in the contents of arginine, ornithine, and citrulline through HPLC analysis performed in 2-2. Specifically, sterilized water was added to the recovered cells prepared in Example 3-1 and suspended at a concentration of about 6.70 log CFU/mL, and the rice bran fermentation seeds were 20% (w/v) in tertiary distilled water. , Rice bran weight/volume of distilled water), and then arginine was added at a concentration of 1% (w/v, arginine weight/volume of distilled water), and then a temperature of 121℃ using an autoclave. After inoculating the rice bran fermentation target prepared by heating the rice bran by heating for 15 minutes under conditions and pressure conditions of 1.1 atm and cooling it to room temperature, the initial pH of the rice bran fermentation target is changed, and fermentation is performed for 48 hours under each condition. Performed. The entire fermented product after 48 hours was dried to obtain a dried rice bran fermented product.

Changes in the contents of arginine, ornithine, and citrulline were analyzed by the HPLC analysis performed in Example 2-2 on the obtained fermented rice bran, and the results are shown in Table 12 below.

No Peak Name Amino acid content (mg/L) pH 5.0 pH 6.0 pH 6.5 pH 7.0 One Arginine 37,694.66 32,724.56 32,899.84 37,437.62 2 Ornithine 5,864.21 8,495.68 8,358.40 6,092.42 3 Citrulline 201.68 1,897.84 1,882.88 51.21 Total 43,760.56 43,118.08 43,141.12 43,581.24

As shown in Table 12, in terms of citrulline production and ornithine production, the optimum pH was found to be from pH 6 to pH 6.5, confirming the same results as measuring the number of viable cells. Therefore, for fermentation of rice bran using the Weisella coriensis DB1 strain, it was confirmed that fermentation at pH 6 to pH 6.5 using a fermented product containing arginine to rice bran was optimal. Corriensis DB1 strain is suitable for rice bran fermentation, not only produces ornithine through rice bran fermentation, but also can produce citrulline and ornithine in a remarkably high yield when arginine is added to the rice bran fermentation. For the rice bran fermentation using the Corianensis DB1 strain, it was found that fermentation at 30° C. and pH 6.0 to pH 6.5 using a fermented product containing arginine to rice bran was optimal.

3-3. Confirmation of drying method of fermented rice bran

In order to improve the ease of storage and use of the prepared rice bran fermented product, it was dried to prepare a rice bran fermented product. In order to confirm a drying method suitable for the preparation of the dried rice bran fermentation, the fermented rice bran prepared in 3-2 was dried by hot air drying and freeze drying, respectively.

The hot air drying was performed by hot air drying the fermented rice bran prepared in 3-2 at 40° C. for 16 hours. The freeze drying was performed at -70°C for 72 hours by a freeze drying method. The rice bran fermented products obtained by drying by the respective drying methods are shown in Figs. 11A (freeze drying) and Fig. 11B.

For the rice bran fermented dried products prepared by each of the above drying methods, the original rice bran (fresh rice bran) was used as a comparative example, and a sensory test was performed. The sensory test was conducted on 10 sensory workers among college students and master's doctorate researchers in the Department of Food Science and Nutrition at Chosun University who had been trained for more than 6 months. Each sample of Sangik was placed on a flat white plastic plate and provided to the sensory personnel in a random order. It was carried out by an appropriate sensory test method.

As a result of the sensory test, the raw rice bran as a comparative example had a rough texture and a characteristic straw-like taste and aroma that can be felt in grains before milling, and it was evaluated that even after chewing and swallowing, a lot of residues remained in the mouth and felt uncomfortable. On the other hand, in the case of the freeze-dried fermented rice bran, the texture is similar to finely ground rice flour or soybean flour, and it seems to dissolve in the mouth because it is soft, and there is almost no residue in the mouth even after swallowing. In addition, the rice bran fermented hot air dried product was evaluated as having a bitter texture than the freeze-dried product dried by the freeze drying method, so that it had a chewy taste, and was moderately soft and combined with acidity and savory taste.

After dissolving the amino acid components contained in the rice bran fermented hot air dried product and the rice bran original product itself, which were dried by the hot air drying method, which was evaluated as having the best acceptability in the above evaluation, each of the high algae was dissolved in tertiary distilled water, and then carried out in Example 2-2. It was analyzed by HPLC analysis, and the results are shown in Table 13 below. The units in Table 13 below represent the content (mg) of the corresponding amino acid per kg of dry matter to be analyzed (mg/kg). In addition, N.D in Table 13 means that the corresponding amino acid was not found.

As shown in Table 13, in the case of citrulline, 2,816.02 mg of citrulline was confirmed in the dry rice fermentation hot air, and it was confirmed that the citrulline content was increased by about 166 times compared to 17.01 mg in the fresh rice bran, and even in the case of ornithine While ornithine was not found in the original rice bran, when the rice bran was fermented by adding the Weissella coriensis DB1 strain of the present invention, the ornithine content of the fermented product 8,744.10 mg was confirmed, and the ornithine content was remarkably increased. Was confirmed.

The total amount of ornithine and citrulline, which are anti-obesity components, was 11,560.12 mg/kg in the final rice bran fermented dried product prepared by the optimal fermented rice bran fermentation conditions and method, and the fermented rice bran fermented product prepared by the method of the present invention has an anti-obesity effect. Was evaluated to be excellent. The anti-obesity effect of the functional rice bran fermentation hot air dried product using the Weissella coriensis DB1 strain, which has the final citrulline and ornithine production ability evaluated to be excellent in the anti-obesity effect, and has remarkably superior ornithine production ability compared to other strains. Animal experiments were performed to confirm.

3-4. Confirmation of anti-obesity effect of fermented rice bran

The anti-obesity effect of the rice bran fermented hot air dried product was confirmed through animal experiments.

First, for the experimental animals, 40 6-week-old male C57BL/6N mice were purchased from a central laboratory animal (Seoul, Korea) and adapted to solid feed at the Chosun University Laboratory Animal Center for 1 week, followed by randomized block design. By dividing 10 animals for each treatment group, 1 dog was separated and reared in a stainless steel cage. The experimental animal center breeding conditions maintained a temperature condition of 18 ± 2 ℃ and a humidity condition of 50 to 55% relative humidity, and the photoperiod (08:00 to 20:00) and the dark cycle were adjusted at 12 hour intervals.

After the 7 days, the experimental animals were subjected to a normal diet group (ND), a high fat high cholesterol diet group (high-fat/high-cholesterol group, HFCD), high fat according to a randomized com-plete block desigh. High cholesterol diet and 5% (w/w, additive weight/total weight of diet) rice bran group (high-fat/high-cholesterol group + 5% (w/w) rice bran group, HFCD-RB) and high fat Divide groups of 10 animals into a high cholesterol diet and 5% (w/w) rice bran fermentation hot air dried product addition group (high-fat/high-cholesterol group + 5% (w/w) fermented rice bran group, HFCD-FRB) Used in the experiment.

During the experiment, dietary intake and body weight were measured once a week at a certain time, and water and feed were ingested without restriction. Among the diets used in the above experiment, the normal diet and the high fat high cholesterol diet were prepared based on the AIN-93 tablet diet (Reeves et al., J Nutr. 123: 1939-1951, 1993), and the normal diet was 10 kcal compared to calories. It was prepared by supplying% fat, and the high fat high cholesterol diet was prepared by adding 35 kcal% fat and 1.25% cholesterol compared to calories.

Experimental animals in each of the experimental groups maintained the diet for 10 weeks. After maintaining the diet for the above 10 weeks, the anti-obesity effect was confirmed by measuring the weight of each part of the liver and adipose tissue of the experimental animal, histopathological examination of the liver tissue, and measurement of the size of adipocytes.

The weight of the liver and adipose tissue of the experimental animal was measured by the following method. First, the experimental animals were sacrificed and the organs were harvested. Specifically, the experimental animal was fed water and fasted for 12 hours, anesthetized with ethyl ether, and the organs of the experimental animal after laparotomy, specifically liver and white adipose tissue (epididymal adipose tissue, mesenteric adipose tissue, etc.) Tissues and adipose tissue around the kidney) were removed, washed with physiological saline, water was removed, and the weight was measured.

The weight of the extracted liver and white adipose tissue was calculated by calculating the relative weight to 100 g of the fasted body weight before autopsy, and the results are shown in Table 14 below. The units in Table 14 below represent the content (g) of fat per 100 g of the experimental animal to be analyzed (g/100 g body wt.). AT in Table 14 below means adipose tissue, and Total white AT (Total white adipose tissue) means the result of summing the measured weights of the adipose tissues of the four extracted tissues. do.

division Normal diet
(ND) High fat
High cholesterol
Diet (HFCD) High fat high cholesterol diet + rice bran
(HFCD-RB) High fat high cholesterol diet + fermented rice bran
(HFCD-FRB) Total white adipose tissue
(Total white AT) 0.87±0.01 1.44±0.04 1.25±0.04 1.08±0.04 Epididymis adipose tissue
(Epididymal
white AT) 0.46±0.02 0.82±0.04 0.69±0.03 0.64±0.02 Mesenteric adipose tissue
(Mesenteric
white AT) 0.22±0.01 0.32±0.02 0.26±0.05 0.24±0.01 Back fat tissue
(Retroperitioneal white AT) 0.09±0.01 0.26±0.02 0.21±0.04 0.21±0.02 Adipose tissue around the kidney
(Perinenal
white AT) 0.02±0.00 0.06±0.02 0.05±0.01 0.04±0.01

According to previous studies, it was confirmed that the abnormal health symptoms induced by obesity are due to an increase in white adipose tissue located in the abdominal cavity rather than the weight gain itself.In order to confirm the anti-obesity effect of the rice bran fermented hot air dry matter, the weight of the white adipose tissue Was measured. Specifically, the weights of epididymal adipose tissue, mesenteric adipose tissue, etc. and adipose tissue around the kidney were measured, and the results are shown in Table 14. As shown in Table 14, the normal formula In the high-fat/high-cholesterol group (HFCD group) compared to this group (normal diet group, ND group), including the weight of total white adipose tissue, epididymal adipose tissue, mesenteric adipose tissue, and back adipose tissue And it was found that the weight of the adipose tissue around the kidney was significantly increased. On the other hand, even in the case of a high-fat, high-cholesterol diet, rice bran group, HFCD- added rice bran group (high-fat/high-cholesterol + 5% (w/w) rice bran group, HFCD-) RB group) significantly reduced the weight of total white adipose tissue, epididymal adipose tissue, mesenteric adipose tissue, back adipose tissue, and peri-renal adipose tissue, compared to the high fat high cholesterol diet group (HFCD group). It was confirmed to be. In particular, in the case of the rice bran fermented rice bran group (HFCD-FRB) in which 5% (w/w) dry rice bran fermentation was ingested together with hot air dried rice bran fermented rice (high-fat/high-cholesterol + 5% (w/w) fermented rice bran group). , It was confirmed that the weight of the adipose tissue was significantly reduced compared to the high-fat high-cholesterol diet group as well as the rice gang source addition group, and it was confirmed that the anti-obesity effect of rice bran was significantly improved through the fermentation process.

By comparing the size of adipocytes in the epididymis tissues of the extracted experimental animals, the anti-obesity effect was confirmed. Specifically, the extracted epididymis tissue was cut to a constant size, fixed with 10% formalin solution for 24 hours, and then excess fixative was removed from flowing water, and the tissue was The moisture in the inside was removed. Thereafter, after removing ethanol from the tissue using xylene, paraffin treatment was performed to fill the tissue space, and then cut to a thickness of 3 μm to prepare an epididymal tissue slide. The epididymis tissue section was attached to a slide, dried, and then stained with hematoxylin and eosin (H&E stain, Hematoxylin and eosin stain), and then adipocytes were observed with a light microscope (TS100, Nikon, Japan). It is shown in Figure 12. In addition, the result of observing the adipocytes, that is, a picture of adipocytes, was analyzed using an image analyzer program (National Institute of Mental Health, USA), and the sizes of adipocytes between the experimental groups were compared, and the results are shown. It is shown in 13.

As shown in FIGS. 12 and 13, in the case of the high fat high cholesterol diet group (HFCD group) compared to the normal diet group (ND group), the size of the epididymal adipocytes was enlarged, and thus it was confirmed that it was observed larger and more clearly. On the other hand, in the case of the ingested rice bran fermentation hot air dried product added group (HFCD-FRB group), it was confirmed that the size of adipocytes was significantly decreased compared to the high fat high cholesterol diet group (HFCD group). It was confirmed that cell hypertrophy was suppressed. In the case of the rice grain added group (HFCD-RB group), the size of adipocytes decreased compared to the high fat high cholesterol diet group (HFCD group), but no significant difference was found.

에서 Cryocut Microtme(Leica CM1800, 독일)를 사용하여 3 μm두께로 잘라 간 조직 절편을 제작하였다. 상기 간 조직 절편을 slide에 부착시켜 말린 다음 Oil-Red O 용액으로 1시간 처리하여 염색한 후, 수세, 주화 및 탈수단계를 차례로 수행한 다음 봉입제로 봉입한 후, 광학 현미경(light microscope)으로 관찰하여 지방세포를 관찰하였으며, 그 결과를 하기 도 14에 나타내었다.In addition, pathological histologic examination was performed on the liver of the extracted experimental animals to confirm the anti-obesity effect. Specifically, after fixing the extracted liver with 4% paraformaldehyde solution, -25

Using a Cryocut Microtme (Leica CM1800, Germany) was cut to a thickness of 3 μm to prepare a liver tissue section. After attaching the liver tissue section to a slide, drying it, treating it with an Oil-Red O solution for 1 hour, dyeing, washing, coining, and dehydrating in order, then encapsulating with an encapsulant, and observed with a light microscope. The adipocytes were observed, and the results are shown in FIG. 14 below.

As shown in FIG. 14, in the case of the high fat high cholesterol diet group (HFCD group) compared to the normal diet group (ND group), it was confirmed that fat was accumulated in the liver tissue. Specifically, in the case of the high-fat high-cholesterol diet group (HFCD group), red-stained fat cells with Oil-Red O solution were identified throughout the liver tissue, and fat accumulation in hepatocytes in the liver tissue was clearly confirmed. On the other hand, in the case of the rice bran fermentation hot air dried product added group (HFCD-FRB group) and the rice bran added group (HFCD-RB group), fat accumulation in liver cells was limited compared to the high fat high cholesterol diet group (HFCD group). In particular, in the case of the rice bran fermented hot air dried product added group (HFCD-FRB group), the color was similar to that of the normal diet group (ND group), whereas the rice bran added group (HFCD-RB group) was found to be slightly purple. , It was found that the anti-obesity effect was more excellent when the rice bran fermented product was added than the rice bran.

The Weisella coriensis DB1 strain is a microorganism with excellent safety suitable for fermentation of rice bran, and can produce citrulline and ornithine by fermenting rice bran, and when produced by the production method of the present invention using the Weisella coriensis DB1 strain, Rice bran fermentation products with remarkably increased citrulline and ornithine, which are not found in the rice bran itself or only limited components, can be prepared, and such high content of anti-obesity components, namely, rice bran fermentation hot air dried products including citrulline and ornithine, Not only has the obesity effect excellent, but it also has excellent preference, so it is expected that it can be used in various purposes for exerting the anti-obesity effect.

Korea Research Institute of Bioscience and Biotechnology KCTC13653BP 20181002

<110> Industry University Cooperation Foundation Chosun University <120> METHOD FOR PREPARING FERMENTED RICE BRAN BY USING WEISSELLA KOREENSIS AND FERMENTED RICE BRAN PREPARED THEREBY <130> CDP20190912 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 1460 <212> RNA <213> Artificial Sequence <220> <223> Weissella koreensis DB1 <400> 1 aatacatgca agtcgaacgc attgtggttg aaatgatatg aagaacttgt tcagatttga 60 ttttcaacat tgcaatgagt ggcgaacggg tgagtaacac gtgggaaacc tacctcttag 120 caggggataa catttggaaa caaatgctaa taccgtataa taattaaaac cgcatggttt 180 tagtttaaaa gatggtcttg ctatcactaa gagatggtcc cgcggtgtat tagttagttg 240 gtgaggtaat ggctcaccaa gacaatgata catagccgag ttgagagact gaccggccac 300 aatgggactg agacacggcc catactccta cgggaggcag cagtagggaa tcttccacaa 360 tggacgaaag tctgatggag caacgccgcg tgtgtgatga agggtttcgg ctcgtaaaac 420 actgttgtaa gagaagaatg acattgagag taactgttca atgtgtgacg gtatcttacc 480 agaaaggaac ggctaaatac gtgccagcag ccgcggtaat acgtatgttc caagcgttat 540 ccggatttat tgggcgtaaa gcgagcgcag acggttattt aagtctgaag tgaaagccct 600 cggctcaacc gaggaattgc tttggaaact ggataacttg agtgcagtag aggaaagtgg 660 aactccatgt gtagcggtga aatgcgtaga tatatggaag aacaccagtg gcgaaggcgg 720 ctttctggac tgtaactgac gttgaggctc gaaagtgtgg gtagcaaaca ggattagata 780 ccctggtagt ccacactgta aacgatgagt gctagttgtt cgagggtttc cgcccttgag 840 tgacgaagct aacgcattaa gcactccgcc tggggagtac gaccgcaagg ttgaaactca 900 aaggaattga cggggacccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960 aagaacctta ccaggtcttg acatcctttg accactccag agatggagct ttcccttcgg 1020 ggacaaagtg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa 1080 gtcccgcaac gagcgcaacc cttattgtta gttgccagca tttagttggg cactctagca 1140 agactgccgg tgacaaaccg gaggaaggcg gggatgacgt caaatcatca tgccccttat 1200 gacctgggct acacacgtgc tacaatggca agtacaacga gtcgccaacc cgcgagggtg 1260 cgcaaatctc ttaaagcttg tctcagttcg gactgtaggc tgcaactcgc ctacacgaag 1320 tcggaatcgc tagtaatcgc ggatcagcac gccgcggtga atacgttccc gggtcttgta 1380 cacaccgccc gtcacaccat gagagtttgt aacacccaaa gtcggtgagg taacctttta 1440 ttaggagcca gccgcctaag 1460

Claims (2)

Weissella koreensis DB1 (Weissella koreensis DB1, KCTC 13653BP) strain isolated from kimchi and having citrulline and ornithine production capacity. The method of claim 1,
The way Cellar Corey N-Sys DB1 (Weissella koreensis DB1, KCTC 13653BP ) strain Arginine (Arginine) is a way Cellar optimum growth pH in the added medium is. Characterized in that the pH 5.0 Corey N-Sys DB1 (Weissella koreensis DB1, KCTC 13653BP ) Strain.

Images