KR102705962B1 - Prebiotics composition containing oligosaccharide isolated from soybean fermented food as active ingredient and method for manufacturing same - Google Patents

Abstract

The present invention relates to a prebiotic composition comprising an oligosaccharide isolated from fermented soybean food as an effective ingredient and a method for producing the same. The composition comprises an oligosaccharide isolated from soy sauce or soybean paste as an effective ingredient, and when injected into the body, the composition is not digested by digestive enzymes, increases beneficial intestinal bacteria, and inhibits the growth of harmful bacteria. In addition, the composition exhibits an antibacterial effect, and can lower intestinal inflammatory activity.

Description

{Prebiotics composition containing oligosaccharide isolated from soybean fermented food as active ingredient and method for manufacturing same}

The present invention relates to a prebiotic composition comprising an oligosaccharide isolated from a fermented soybean food as an effective ingredient and a method for producing the same, and more specifically, to a prebiotic composition comprising a novel oligosaccharide isolated from fermented soybean foods such as soy sauce, doenjang, and cheonggukjang and a method for producing the same.

The human intestines are home to about 100 trillion intestinal microorganisms, and they form a complex ecosystem that maintains a certain balance. These intestinal microorganisms help break down and absorb indigestible food, and the enzymes and metabolites they produce are involved in the metabolism of various substances in the human body, and affect the absorption of nutrients and drugs, and the production of toxic substances. They also train the immune system to protect humans from pathogens and diseases.

These intestinal flora include beneficial and harmful bacteria, and health is regulated by their balance. In order to maintain health, it is desirable to maintain intestinal flora with many beneficial bacteria and few harmful bacteria. The flora can change depending on the state of the human body, such as food taken in from the outside, stress, and hormone secretion. If the balance of intestinal microorganism distribution is disrupted due to eating habits and environmental conditions, harmful bacteria can invade and multiply, resulting in bacterial diseases and indigestion, which can have various adverse effects on the body.

To prevent this imbalance, the concept of probiotics emerged as a microbial preparation that can easily help maintain health. Probiotics were defined by the WHO in 2001 as living microorganisms that have a beneficial effect on the host when consumed in appropriate amounts, and have been used to this day. Studies on the activity of probiotics have shown that they have various physiological activities such as antibacterial activity, anticancer effect, enhancing nutrient absorption of the host, strengthening immunity, and scavenging active oxygen.

In other words, probiotics play an important role in maintaining sufficient intestinal function and immunity, and there are many different types of probiotics. Since each species of beneficial bacteria has different effects, complex strain products containing various types of bacteria are being developed rather than products containing a single strain.

Meanwhile, as the concept of probiotics emerged, research began on food for lactic acid bacteria to help them settle in the intestines and help them grow. Food for lactic acid bacteria is collectively called prebiotics. In other words, prebiotics are a type of food for beneficial bacteria to settle and survive in the intestines. If sufficient food is supplied to beneficial bacteria, one will multiply to 20 billion in one day.

Gibson & Roberfroid (1995) first defined prebiotics in their study as non-digestible food ingredients that benefit the host by selectively stimulating the growth and activity of one or a limited number of bacteria in the large intestine and thereby improving health. Commonly known prebiotics include galactooligosaccharides, fructooligosaccharides, maltooligosaccharides, and isomaltooligosaccharides. They are industrially produced using biotechnology, and are applied to various foods due to their unique physicochemical properties. In addition, they act as substrates for lactic acid bacteria, helping the activity of lactic acid bacteria and thus benefiting health.

Therefore, dietary control including prebiotics is a useful method to promote the growth of beneficial bacteria in the intestines and suppress harmful bacteria, and it is known that natural material extracts, breast milk, milk, microorganisms, and seaweed are used.

However, although prebiotics according to conventional technology are generally known to be important in terms of human health and disease prevention, their effects on intestinal flora differ depending on their type, and information on their specific structure and role is insufficient. Therefore, considering the importance of intestinal flora for the general public who want to lead a healthy life and for people with diseases, it is necessary to develop probiotics that promote the growth of beneficial microorganisms in the intestines, and in particular, it is necessary to develop and manufacture probiotics derived from traditional fermented foods whose safety has been proven through long-term consumption.

KR 10-1395875 B1

The purpose of the present invention is to provide a prebiotic composition containing an oligosaccharide isolated from fermented soybean food as an effective ingredient and a method for producing the same.

Another object of the present invention is to provide a probiotic composition containing an oligosaccharide isolated from soy sauce or soybean paste as an effective ingredient, which, when injected into the body, is not digested by digestive enzymes and increases intestinal beneficial bacteria, and a method for producing the same.

Another object of the present invention is to provide a probiotic composition that exhibits an antibacterial effect that suppresses the growth of harmful bacteria in the intestines when injected into the body, thereby reducing intestinal inflammatory activity, and a method for producing the same.

To achieve the above purpose, an oligosaccharide isolated from a fermented soybean food according to one embodiment of the present invention may be included as an effective ingredient.

The above oligosaccharides promote the growth of strains having beneficial effects in the intestines, and these probiotic microorganisms are Lactobacillus , Lactococcus , Enterococcus , Streptococcus , Bifidobacterium , Coprobacillus , Roseburia, Coprococcus , Allobaculum , Peptococcaceae , and Christensenellaceae . Preferably, the above intestinal beneficial bacteria are strains of Bifidobacterium and Lactobacillus.

The above oligosaccharides inhibit the growth of harmful intestinal bacteria, and these microorganisms include strains of the genus Pseudomonas aeruginosa , the genus Vibrio , the genus Staphylococcus , the genus Clostridium perfringens , the genus Eubacterium , the genus Bacteroides , and sulfate reducers .

The above oligosaccharides are a combination of monosaccharides of hexose (hex), N-acetylhexosamine (hexNac), deoxyhexose (deoxyhex), pentose (pen), and hexuronic acid (hexA).

The above oligosaccharides can be selected from the group consisting of 474.159, 502.153, 504.169, 515.185, 529.201, 545.196, 586.222, 606.201, 636.211, 648.211, 666.222, 677.238, 707.248, 722.248, 839.291, 864.322, and 869.301, as analyzed by high performance liquid chromatography mass spectrometry (HPLC Q-TOF MS) measurement.

A functional food composition for preventing or improving an intestinal disease according to another embodiment of the present invention may include the probiotics composition.

A pharmaceutical composition for preventing or improving an intestinal disease according to another embodiment of the present invention may include the probiotic composition.

According to another embodiment of the present invention, a method for producing a prebiotics composition comprising an oligosaccharide isolated from a fermented soybean food as an active ingredient may include the steps of: 1) adding water to a fermented soybean food selected from the group consisting of soy sauce, soybean paste, and mixtures thereof to produce a soybean fermentation mixture; 2) vortexing and sonicating the soybean fermentation mixture; 3) mixing methanol and chloroform with the soybean fermentation mixture of step 2 to produce an extraction mixture; 4) vortexing and sonicating the extraction mixture; 5) centrifuging the extraction mixture of step 4); and 6) subjecting the supernatant separated by the centrifugation to solid phase extraction (SPE).

The present invention comprises an oligosaccharide separated from soy sauce or soybean paste as an effective ingredient, and when injected into the body, it is not digested by digestive enzymes and can increase intestinal beneficial bacteria. In addition, it exhibits an antibacterial effect that inhibits the growth of harmful microorganisms, and can reduce intestinal inflammatory activity.

Figure 1 shows the HPLC Q-TOF MS profile measurement results for oligosaccharides extracted from soybean paste according to one embodiment of the present invention.
Figure 2 shows the results of HPLC Q-TOF MS profile measurement for oligosaccharides extracted from soy sauce according to one embodiment of the present invention.
Figure 3 is an experiment to evaluate the inhibitory activity of a prebiotic composition on NO production according to one embodiment of the present invention.
Figure 4 is an experiment to evaluate the inhibitory activity of a prebiotic composition on ROS production according to one embodiment of the present invention.

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 is not limited to the embodiments described herein.

Almost all Korean food is seasoned and flavored with soy sauce, doenjang, and gochujang, so the taste of the sauce is a fundamental factor that determines the taste of the food.

There are no records of when doenjang first began to be eaten in Korea, so it is not known for sure. However, based on the record in China's "Wei Zhi (魏志)" that "Goguryeo was good at making jangyang (藏釀)," it can be assumed that even before the Three Kingdoms period, people had been making and eating a thick mixture of doenjang and soy sauce, and that it was during the Three Kingdoms period that the technology to separate soy sauce and soy sauce was developed. Here, jangyang is interpreted to refer to all fermented processed foods, such as brewing alcohol and making jangdam (Korean soy sauce).

Also, in the early and mid-Joseon Dynasty, written in 《Guhwangchalyo (救荒撮要)》 and 《Jeongbosanrimgyeongje (增補山林經濟)》, there are sections on making soy sauce (造醬法) and 《Jangjepumjo (醬諸品條)》, respectively, which present detailed methods for making good soy sauce, which allows us to easily guess how important soy sauce was to the Korean diet. The beginning of the section on making soy sauce is as follows: "Soy sauce is the best of all flavors. If the soy sauce in your home is not good, even if you have good vegetables and meat, it cannot be considered good food. Even if people in the countryside do not have easy access to meat, if they have various soy sauces that taste good, they do not have to worry about side dishes. First, pay attention to making soy sauce, and it is the right thing to do to obtain good soy sauce by letting it age for a long time." In order to make good soy sauce like this, we have long taken many precautions, and this custom has been passed down to most households even today, when many factory-produced soy sauces are on the market, although the amount has been greatly reduced.

Types of doenjang include traditional doenjang made by soaking soy sauce, straining the broth, and adding edible ingredients; improved doenjang made by adding koji to meju; and eclectic doenjang that is a compromise between the two methods. In addition, there are seasonal specialty soybean pastes, such as dambukjang and makjang made in the spring, jipjang and saenghwangjang made in the summer, cheongtaejang and patjang made in the fall, and cheonggukjang made in the winter.

The traditional method, although slightly different depending on the region, uses soybeans as the main ingredient and allows microorganisms to settle and multiply in a natural state. First, the soybeans are steamed and ground, and the meju is formed into a certain shape such as a wooden needle or sphere, dried for 2-3 days, bound with rice straw, and naturally fermented for about 1-2 months. The meju is then washed and soaked in salt water to age.

The improved method is for mass production of soybean paste. It is a method of inoculating starchy raw materials such as rice, wheat, and barley with Aspergillus oryzae and Bacillus subtilis, mixing the fermented soybeans with steamed soybeans and salt, and maturing the resulting paste.

A compromise method is to make soybean paste with the remaining soy sauce (traditional method), which loses a lot of the meju's ingredients to the soy sauce and reduces its flavor and nutrients, so it is used to make delicious soy sauce and soybean paste. This method involves mixing coarsely ground meju in salt water until thick, fermenting it, mixing it with the remaining meju from the soy sauce, kneading it until sticky, and sealing it.

There are two main ways to make soy sauce: traditional and improved.

The traditional method is made at home. Traditionally, soybeans were boiled in meju around December and naturally fermented. Then, the meju was soaked in salt water around February to April of the following year and aged for 1 to 2 months. After that, the meju was removed and soy sauce was boiled to adjust the taste and moisture. Since various types of bacteria and molds multiply in the traditional meju and hydrolyze the soybeans, substances with an unpleasant taste and smell were created, and soy sauce often did not taste very good.

However, recently, due to the advancement of microbiology, the cultivation of yeast isolated from yellow grains, which has a strong ability to hydrolyze proteins and starches, and the process of inoculating boiled soybeans with the yeast made from this and fermenting them to make meju, which is then dried, have been commercialized and sold on the market. If you make soy sauce with this improved meju using the same method as the traditional method, you get a high-quality soy sauce with a pure sweetness. Improved soy sauce has a good flavor because it contains amino acids, maltose, glucose, etc. produced by yellow grains, as well as alcohol and lactic acid produced by yeast and lactic acid bacteria.

The salt concentration of soy sauce is usually 18 to 20%, and the brown color of soy sauce is due to melanin and melanoidin, which are breakdown products of amino acids. Some commercially available soy sauces are colored with caramel (starch syrup). The smell of soy sauce is a mixture of alcohol, ketone, aldehyde, volatile acid, ester, and phenol, and the unique taste of soy sauce is due to β-methyl mercaptopropyl alcohol.

In addition to the methods mentioned above, there are various manufacturing methods in each region of our country, and the microorganisms present in doenjang and ganjang differ depending on the environment of each region, which results in unique regional characteristics. Since ancient times, it has been said that good soy sauce is necessary to bring out the flavor of food, so soy sauce and doenjang have been important seasonings in Korean food. There are also various types of fermented soybean foods in China and Japan. In China, doubanjang and doushi are representative fermented soybean foods, and in Japan, natto and miso are representative fermented soybean foods. It is known that Korean doenjang and ganjang have characteristic flavors and aromas because different microorganisms are involved in the fermentation.

Doenjang and soy sauce made by fermenting soybeans are known to be excellent in preventing aging and cancer, and are also known to have the effect of strengthening liver function because they contain eight essential amino acids. In addition, they are known to be excellent in promoting digestion and preventing constipation, and specifically, while the digestion and absorption rate of raw soybeans is about 55%, that of fermented soybean paste is as high as 85%. In addition, carbohydrates such as dietary fiber present in raw soybeans change into various forms through fermentation, making them easier to digest and promoting intestinal peristalsis compared to when regular soybeans are consumed. The present invention is a prebiotics composition comprising an oligosaccharide isolated from a fermented soybean food as an effective ingredient, and is characterized by comprising an oligosaccharide isolated from soy sauce or soybean paste.

As explained above, fermented soybean paste and soy sauce are traditional fermented foods widely consumed in Korea and have historical value from a food and cultural perspective. Soybean paste and soy sauce are used as important sources of nutrients and flavorings for Koreans and are popular worldwide. The ingredients of soybean paste and soy sauce are closely related to their quality and nutritional benefits.

Among the components contained in soybeans, dietary fiber, raffinose, and stachyose are known to be indigestible by human digestive enzymes. These carbohydrates can be hydrolyzed or transformed during the fermentation process, which may provide additional physiological effects on intestinal microflora.

In general, the prebiotic properties of oligosaccharides are largely dependent on their structural conformation, including the degree of polymerization between monosaccharides constituting the oligosaccharides and the monosaccharide composition. However, due to structural complexity, studies have not been conducted well, but recent advances in mass spectrometry have made it possible to profile various oligosaccharides in fermented foods.

Accordingly, the present invention separated and confirmed oligosaccharides in soybean paste or soy sauce, and confirmed excellent prebiotic effects and anti-inflammatory effects by the separated oligosaccharides.

More specifically, the oligosaccharide of the present invention promotes the growth of strains having a beneficial effect in the intestines, and these probiotic microorganisms are Lactobacillus , Lactococcus , Enterococcus , Streptococcus , Bifidobacterium , Coprobacillus , Roseburia, Coprococcus , Allobaculum , Peptococcaceae , and Christensenellaceae .

Lactobacillus , Bifidobacterium , Coprobacillus , Roseburia , Coprococcus , Allobaculum, Peptococcaceae, and Christensenellaceae, known as beneficial intestinal microorganisms, feed on the oligosaccharides isolated from the soybean paste or soy sauce, thereby inhibiting the growth of harmful bacteria in the intestines and facilitating the growth of beneficial bacteria.

The above oligosaccharides can be selected from the group consisting of 474.159, 502.153, 504.169, 515.185, 529.201, 545.196, 586.222, 606.201, 636.211, 648.211, 666.222, 677.238, 707.248, 722.248, 839.291, 864.322, and 869.301, as analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS) measurement.

The monosaccharide composition of the oligosaccharide separated from the soybean paste or soy sauce of the present invention was measured by gas chromatography mass spectrometry using an Agilent 6890A GC, a DB-1 fused silica capillary column with a 30 m × 0.25 μm inner diameter and a 0.25 μm film thickness, nitrogen (flow rate 2.5 mL/min, 17 psi) as a carrier gas, pulse split mode (split ratio 2:1), injector and FID temperatures of 280°C, and GC temperature programming, increasing the temperature from 120°C to 200°C (1.5°C/min), maintaining it at 200°C for 5 minutes, and then running it at 250°C for 2 minutes.

The above prebiotic composition comprises an oligosaccharide and a strain isolated from soy sauce or soybean paste, and the strain may be selected from the group consisting of Lactobacillus , Bifidobacterium , Coprobacillus , Roseburia , Coprococcus , Allobaculum , Peptococcaceae , Christensenellaceae , and mixtures thereof.

The above prebiotic composition not only inhibits the growth of harmful bacteria in the intestines and facilitates the growth of beneficial bacteria, but also reduces intestinal inflammatory activity due to its anti-inflammatory activity. This is an anti-inflammatory effect caused by oligosaccharides contained in soy sauce or soybean paste, and the oligosaccharides are characterized by being delivered to the intestines without being decomposed in the stomach, thereby not only activating the growth of beneficial bacteria in the intestines, but also exhibiting anti-inflammatory activity.

Preferably, the probiotic composition is characterized in that it additionally contains an oligosaccharide isolated from soy sauce or soybean paste and a natural extract. The natural extract is not easily decomposed in the stomach like the oligosaccharide and is delivered to the intestine, so that it can further increase the effect of the oligosaccharide on the proliferation of beneficial bacteria in the intestine, and can also exhibit a synergistic effect of the anti-inflammatory effect.

The above natural extracts are Rumex acetosella L. extract and Rumex spp. extract.

The above baby's milkweed (Rumex acetosella L.) is a perennial herb of the family Rumex in the order Rumex in the order Dicotyledons, and grows on roadsides and vacant lots. It reproduces by spreading out rhizomes, and the stems are erect, 20–50 cm tall, have hair-like projections, have serrated vertical lines, are purple, and taste sour together with the leaves. The leaves that grow from the roots grow in clusters, have long petioles, are lanceolate, have sharp tips, and have ear-like projections that spread out to the left and right at the base. The leaves on the stem grow alternately, have the same shape as the leaves that grow from the roots, but are smaller. The flowers are dioecious, bloom in May–June, are reddish green, and form a panicle at the end of the stem. The male flowers are 3 mm in diameter, and have six sepals and stamens each. The female flowers are smaller than the male flowers, have six sepals, one pistil, a three-lobed style and a finely divided stigma. The fruit is an achene, oval, 2 mm long, brown, and has three serrated lines.

The root of the above-mentioned Rumex acetosella L. is called Sanmo (酸模), and the leaf is called Sanmoyeop (酸模葉), and is used for medicinal purposes. The root contains tannin, chrysophanein, and hyperin, and has the effects of clearing away heat, diuresis, relieving heat, and killing insects, and can treat fever, impotence, urination, aging, hemoptysis, malignant boils, and flatulence.

The leaves of the above Rumex acetosella L. contain vitexin, hyperin, violaxanthin, and 63 mg% of vitamin C, and 17.3-36.7% of tannin, and the stems and leaves contain calcium oxalate and tartaric acid. When applied externally, it has the effect of relieving boils and boils and can treat boils.

The above-mentioned Allium senescens, like the commonly known chives, is a perennial monocotyledonous plant belonging to the lily family, and is mainly distributed around Ulleungdo and the northern region. The bulb is egg-shaped and oval, and has a thin membranous outer skin and no fibers. In addition, Allium senescens has thick leaves and is fleshy, so it contains a lot of moisture and essential oils. In oriental medicine, it is classified as a plant with a warm nature, and it is known that the private sector has used Allium senescens for detoxification and indigestion since ancient times, as well as for food. Recently, it has been revealed that it contains a large amount of polyphenol, which is known as an antioxidant, and therefore it is expected to have excellent effects in preventing aging, which is a major concern of modern people.

The prebiotic composition of the present invention is characterized in that it contains 1 to 5 parts by weight of a baby's breath extract and 1 to 5 parts by weight of a chive extract per 100 parts by weight of an oligosaccharide isolated from soy sauce or soybean paste, and when used in a mixture within the above range, it is possible to exhibit an effect of proliferating intestinal beneficial bacteria and an anti-inflammatory effect.

In addition, the present invention provides a health functional food composition for preventing or improving intestinal disease, which comprises the prebiotic composition as an effective ingredient.

In a health functional food composition according to one embodiment of the present invention, the intestinal disease may be any one selected from the group consisting of functional dyspepsia, ulcerative dyspepsia, non-ulcer dyspepsia, gastroesophageal reflux disease, reflux esophagitis, atony, pseudo-intestinal obstruction, gastric motility paralysis, constipation, irritable bowel syndrome, irritable colitis, diabetic gastrointestinal motility disorder, gastrointestinal motility disorder due to chemotherapy, intestinal obstruction due to gastrointestinal motility disorder, gastrointestinal motility disorder due to myotonic dystrophy, and non-cardiac chest pain, but is not limited thereto.

The health functional food composition of the present invention can be used in various ways, such as in drugs, foods, and beverages for preventing oxidation. The functional food of the present invention can be used in, for example, various foods, candies, chocolates, beverages, gums, teas, vitamin complexes, health supplements, etc., and can be used in the form of powders, granules, tablets, capsules, or beverages.

The prebiotics composition containing the oligosaccharide isolated from the fermented soybean food of the present invention as an effective ingredient can be added to food or beverage for the purpose of increasing intestinal beneficial bacteria. At this time, the amount of the prebiotics composition in the food or beverage can generally be added in the amount of 0.01 to 50 wt%, preferably 0.1 to 20 wt%, of the total food weight for the health functional food composition of the present invention, and in the amount of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml for the health beverage composition.

The health beverage composition of the present invention has no particular restrictions on the liquid component except that it contains the probiotics composition as an essential component in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional components like a conventional beverage. Examples of the above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as polysaccharides such as maltose and sucrose, conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the above-mentioned natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of these additives is not so important, but is typically selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The composition including the pharmaceutically acceptable carrier may be in various oral or parenteral dosage forms. When formulating, it may be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid preparations may be prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

In addition, the pharmaceutical composition of the present invention may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.

The probiotic composition of the present invention has been used for food and medicinal purposes since ancient times, and there is no special restriction on the administration dosage, which may vary depending on the degree of absorption in the body, body weight, patient's age, sex, health condition, diet, administration time, administration method, excretion rate, disease severity, etc. In general, the probiotic composition can be administered in an amount of about 10 to 1,000 mg per 1 kg of body weight, and more specifically, about 50 to 500 mg per 1 kg of body weight. A pharmaceutical composition containing the probiotic composition of the present invention as an active ingredient is manufactured in consideration of the effective dosage range, and a unit dosage form formulated in this way can be administered several times at regular intervals using a specialized administration method according to the judgment of a specialist who monitors or observes the administration of the drug and the needs of an individual, if necessary.

According to another embodiment of the present invention, a method for producing a prebiotics composition comprising an oligosaccharide isolated from a fermented soybean food as an active ingredient may include the steps of: 1) adding water to a fermented soybean food selected from the group consisting of soy sauce, soybean paste, and mixtures thereof to produce a soybean fermentation mixture; 2) vortexing and sonicating the soybean fermentation mixture; 3) mixing methanol and chloroform with the soybean fermentation mixture of step 2 to produce an extraction mixture; 4) vortexing and sonicating the extraction mixture; 5) centrifuging the extraction mixture of step 4); and 6) subjecting the supernatant separated by the centrifugation to solid phase extraction (SPE).

More specifically, the step 1) above is a step of preparing a mixture by mixing water into a fermented soybean food, wherein the fermented soybean food is soybean paste or soy sauce. That is, it is a step of preparing a mixture by mixing water into soybean paste or soy sauce.

Thereafter, the mixture is subjected to a shaking and ultrasonic treatment process. More specifically, the mixture is shaken for 5 to 10 minutes, and treated for 5 to 10 minutes using an ultrasonic device at 200 to 300 W. Thereafter, methanol and chloroform are mixed with the mixture, and at this time, methanol and chloroform are mixed at a weight ratio of 1:2.

Afterwards, it was shaken for 5 to 10 minutes and treated for 5 to 10 minutes using an ultrasonic device at 200 to 300 W.

Thereafter, centrifugation was performed at 4°C for 30 minutes, and the supernatant was obtained by centrifugation, and then the oligosaccharides were separated using a solid phase extraction method. The oligosaccharides may be selected from the group consisting of 474.159, 502.153, 504.169, 515.185, 529.201, 545.196, 586.222, 606.201, 636.211, 648.211, 666.222, 677.238, 707.248, 722.248, 839.291, 864.322, and 869.301, as analyzed by liquid chromatography mass spectrometry (HPLC Q-TOF MS) measurement.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only intended to illustrate the present invention, and the content of the present invention is not limited to the following examples.

Manufacturing example 1

0.02 g of soybean paste was mixed with 100 ml of water, and primary extraction was performed using the Folch extraction method. Specifically, 1 ml of the mixture of water and soybean paste was separated, shaken for 5 to 10 minutes, and treated for 5 to 10 minutes at 200 to 300 W using an ultrasonic device. Thereafter, 1.33 ml of methanol and 2.66 ml of chloroform were mixed, shaken for 5 to 10 minutes, and treated for 5 to 10 minutes at 200 to 300 W using an ultrasonic device. Centrifugation was performed at 4°C for 30 minutes, and the supernatant was obtained by centrifugation, after which oligosaccharides were separated using a solid phase extraction method.

The above solid phase extraction method used a carbon 8 cartridge to elute oligosaccharides into water, then used a graphite carbon cartridge to further adsorb the oligosaccharides contained in soybean paste, and then eluted using a 40% acetonitrile solution dissolved in 0.1% TFA to separate the oligosaccharides.

Manufacturing example 2

Oligosaccharides were isolated in the same manner, except that 0.1 ml of soy sauce was used instead of the above-mentioned soybean paste.

Experimental Example 1

Oligosaccharide component analysis

To analyze the oligosaccharides separated in the above Manufacturing Example 1 and Manufacturing Example 1, liquid chromatography mass (HPLC Q-TOF MS) analysis was performed, and the analysis conditions were as follows:

Agilent 6530 HPLC Q-TOF MS System

-Column: Porous graphitized carbon (100 x 2.1 mm I.D., 3 μm particle size)

-Mobile phase A: 0.1% formic acid (v/v) and 3% acetonitrile dissolved in water

-Mobile phase B: 90% acetonitrile (v/v) and 0.1% formic acid dissolved in water

-Flow rate: 1.0 mL/min

-Linear gradient: 0 to 2.5 min, 0% B; 2.5 to 20 min, 0 to 16% B; 20 to 30 min, 16 to 44% B; 30 to 35 min, 44 to 100% B, then 100% B for 10 min; 45 to 45.01 min, 100 to 0% B, then 0% B for 10 min.

- Collision energy: 1.3V/100Da (offset -3.5V)

The results of the above analysis are as shown in Figures 1 and 2.

The above Table 1 and Figure 1 show the analysis results of oligosaccharides contained in soybean paste, and Table 2 and Figure 2 show the analysis results of oligosaccharides contained in soy sauce. According to the analysis in Table 1 and Table 2, when the molecular weights of the oligosaccharides were confirmed with each other, it was confirmed that oligosaccharides having molecular weights (Mw) of 474.159, 502.153, 504.169, 515.185, 529.201, 545.196, 586.222, 606.201, 636.211, 648.211, 666.222, 677.238, 707.248, 722.248, 839.291, 864.322, and 869.301 were contained identically.

The analysis results are shown in Tables 1 and 2 below.

Observed Mass Theoretical mass Mass error(ppm) Oligosaccharide composition Hex HexNAc Deoxyhex Pen HexA 474.160 474.159 -3.374 3 - - 1 502.155 502.153 -3.385 1 - 1 - 1 504.171 504.169 -2.975 3 - - - 515.187 515.185 -2.717 1 1 - 1 529.201 529.201 -1.266 1 1 1 - 545.198 545.196 -3.797 2 1 - - 586.222 586.222 -0.461 1 2 - - 606.201 606.201 0.049 2 - - 2 636.213 636.211 -2.515 3 - - 1 648.213 648.211 -2.468 1 2 1 666.223 666.222 -1.951 4 - - - 677.240 677.238 -2.614 2 1 - 1 691.256 691.254 -3.183 2 1 1 - 707.250 707.248 -2.644 3 1 - - 722.250 722.248 -3.143 1 - 1 3 732.281 732.280 -1.229 1 2 1 - 839.293 839.291 -3.181 3 1 - 1 864.324 864.322 -1.851 1 2 1 1 869.303 869.301 -2.496 4 1 - - 1031.356 1031.354 -2.133 5 1 - - 1042.372 1042.370 -1.919 3 2 - 1 1188.428 1188.428 0.084 3 2 1 1

Observed Mass Theoretical mass Mass error(ppm) Oligosaccharide composition Hex HexNAc Deoxyhex Pen HexA 444.149 444.148 -2.702 1 - - 2 474.160 474.159 -3.163 2 - - 1 488.176 488.174 -4.711 2 1 - 502.154 502.153 -1.593 1 1 1 504.170 504.169 -2.380 3 - - - 515.186 515.185 -2.523 1 1 - 1 529.201 529.201 -1.323 1 1 1 - 545.198 545.196 -3.852 2 1 - - 586.222 586.222 -0.290 1 2 - - 606.202 606.201 -2.474 2 - - 2 620.217 620.216 -1.612 2 - 1 1 636.212 636.211 -1.729 3 - - 1 648.212 648.211 -1.543 1 2 1 661.246 661.243 -4.991 1 1 1 1 666.224 666.222 -2.852 4 - - - 677.239 677.238 -1.034 2 1 1 707.249 707.248 -1.513 3 1 - - 722.249 722.248 -0.928 1 - 1 3 828.276 828.275 -1.207 5 - - - 839.291 839.291 -0.322 3 1 - 1 864.324 864.322 -1.620 1 2 1 1 869.302 869.301 -1.231 4 1 - - 900.297 900.296 -1.222 3 - - 3 990.329 990.327 -2.020 6 - - 1001.343 1001.344 0.799 4 1 - 1 1031.357 1031.354 -2.618 5 1 - - 1042.372 1042.370 -1.823 3 2 - 1 1152.380 1152.380 0.347 7 - - - 1188.427 1188.428 0.757 3 2 1 1 1193.407 1193.407 0.084 6 1 - -

In addition, to analyze the monosaccharide composition, oligosaccharides were decomposed into monosaccharides by acid treatment and then analyzed. The oligosaccharide sample was mixed with 5 ml of 2 M trifluoroacetic acid, hydrolyzed by reaction at 100 ° C for 2 hours, and dried using nitrogen. After that, 0.3 ml of silylation reagent was mixed, silanized, and dried using nitrogen. The silane reagent was a mixture of HMDS/TMCS/Pyridine in a weight ratio of 2:1:10. The above dried reagent was analyzed by gas chromatography mass spectrometry using an Agilent 6890A GC, a DB-1 fused silica capillary column with a 30 m × 0.25 μm i.d., 0.25 μm film thickness, nitrogen as a carrier gas (flow rate 2.5 mL/min, 17 psi), pulse split mode (split ratio 2:1), injector and FID temperatures of 280°C, and GC temperature programming, ramping from 120°C to 200°C (1.5°C/min), holding at 200°C for 5 minutes, and then running at 250°C for 2 minutes.

As a result, the monosaccharides that make up the oligosaccharides of soy sauce and soybean paste are hexose, glucose, fructose, mannose, and galactose, N-acetylhexosamine is N-acetylglucosamine (GlcNAC), deoxyhexose is fucose and rhamnose, and pentose is xylose and arabinose.

Manufacturing example 3

Preparation of natural extracts

The young shoots of Baby's Breath were washed, dried, and pulverized. After adding distilled water as an extraction solvent in a ratio of 1:10 (w:v), they were completely soaked, and the extraction was repeated three times for 3 hours each while refluxing at 80℃. The extract was filtered through Whatman No. 2 filter paper. The filtrate was concentrated under reduced pressure at 60℃ to prepare a powder sample, which was used in the experiment. Baby's Breath extract (RE) was prepared by hot water extraction, concentration, and filtration.

Preparation of extract of Dumebuchu

The extract of chives (AE) was prepared using the same method as that for preparing the extract of the aforementioned baby swimming pool.

Preparation of probiotic composition

A probiotic composition was prepared by mixing oligosaccharides isolated from soy sauce (SO) or soybean paste (SPO) and natural extracts as follows.

Experimental Example 1 Experimental example 2 Experimental Example 3 Experimental Example 4 Experimental Example 5 Experimental example 6 Experimental example 7 Experimental example 8 SPO 100 - 100 100 100 - - - SO - 100 - - - 100 100 100 RE - - 0.5 3 10 0.5 3 10 AE - - 0.5 3 10 0.5 3 10

(unit weight)

Experimental example 2

Evaluation of the proliferation of beneficial intestinal bacteria

For the above oligosaccharides, a prebiotics experiment was conducted. The compositions of Experimental Examples 1 to 8 were added at 10 wt%, and 1 mL of the cultured strain was added. After culturing for 24 hours in an incubator, the growth rate of the strain was confirmed. The strains used in the experiment were Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus plantarum, Bifidobacterium longum, Bifidobacterium bifidum, and Bifidobacterium animalis subsp. lactis. A total of 7 lactic acid strains were used. MRS medium (BD, Sparks, USA) was used for culturing Lactobacillus strains, and Bifidobacterium strains were cultured in MRS medium with 0.05% (w/v) L-cysteine hydrochloride (Sigma-Aldrich Co.,) added (MRSC). Each strain was statically cultured at 37°C for 24 hours, and experiments were conducted after passage 3 times.

Experimental Example 1 Experimental example 2 Incubation time Incubation time 0h 24h 0h 24h Lactobacillus rhamnosus ¹ 4.11±0.57 14.24±0.72 3.21±0.57 12.24±0.32 Lactobacillus fermentum ² 2.47±0.12 15.47±0.51 1.27±0.22 13.17±0.41 Lactobacillus casei ³ 1.86±0.56 16.57±0.17 1.45±0.86 15.56±0.27 Lactobacillus plantarum ⁴ 0.58±0.47 13.54±0.37 0.89±0.27 14.34±0.36 Bifidobacterium longum ⁵ 0.89±0.14 11.28±0.36 1.81±0.13 12.29±0.16 Bifidobacterium bifidum ⁶ 2.24±0.11 11.28±0.34 1.14±0.21 13.18±0.31 Bifidobacterium animalis subsp. Lactis ⁷ 1.56±0.81 14.98±0.12 2.56±0.11 12.78±0.46

(×10 ⁷ CFU/mL)

Experimental Example 3 Experimental example 4 Experimental Example 5 Incubation time Incubation time Incubation time 0h 24h 0h 24h 0h 24h 1 4.21±0.57 13.24±0.72 3.11±0.57 16.12±0.21 4.11±0.57 13.44±0.52 2 2.45±0.12 15.17±0.51 3.47±0.12 15.45±0.11 2.47±0.12 15.37±0.21 3 1.84±0.57 15.53±0.27 1.56±0.56 16.57±0.18 1.56±0.56 16.17±0.26 4 0.78±0.57 13.14±0.17 1.58±0.47 15.14±0.37 0.68±0.47 14.14±0.87 5 0.65±0.44 11.38±0.26 1.89±0.14 15.18±0.16 0.49±0.14 11.58±0.46 6 2.14±0.21 10.78±0.14 3.24±0.21 14.28±0.14 2.14±0.11 11.68±0.12 7 1.36±0.82 14.18±0.22 2.54±0.82 15.68±0.19 1.46±0.81 14.18±0.21

(×10 ⁷ CFU/mL)

Experimental example 6 Experimental example 7 Experimental Example 8 Incubation time Incubation time Incubation time 0h 24h 0h 24h 0h 24h 1 2.21±0.47 12.23±0.32 3.41±0.17 16.52±0.20 3.22±0.17 12.24±0.31 2 1.21±0.21 11.17±0.21 4.47±0.22 16.15±0.32 1.47±0.13 12.17±0.31 3 1.41±0.76 15.56±0.17 2.56±0.56 16.57±0.28 1.61±0.66 14.46±0.24 4 0.67±0.25 15.34±0.24 2.58±0.36 16.14±0.57 0.59±0.24 14.44±0.16 5 1.11±0.11 11.38±0.16 2.88±0.24 15.98±0.46 1.51±0.11 13.19±0.15 6 1.34±0.20 12.18±0.31 4.25±0.24 16.28±0.13 1.44±0.31 12.18±0.32 7 2.51±0.21 14.18±0.16 3.53±0.81 15.58±0.29 2.16±0.21 11.68±0.47

(×10 ⁷ CFU/mL)

According to the above experimental results, it was confirmed that when the oligosaccharide composition separated from soy sauce or soybean paste was mixed with intestinal beneficial bacteria, the beneficial bacteria proliferated. In addition, it was confirmed that intestinal beneficial bacteria proliferated in a composition mixed with natural extracts, and in particular, it was confirmed that a significant proliferation effect was exhibited in a composition mixed within the scope of the present invention.

Experimental Example 3

Evaluation of inhibition of growth of harmful intestinal bacteria

The disks through which the compositions of Experimental Examples 1 to 8 were filtered were placed one by one on an MHA (Muller Hinton Agar) medium coated with intestinal harmful bacteria such as bacteria, and cultured at 37°C for 24-48 hours. The antibacterial effect was measured by measuring the diameter of the inhibition zone around the disk. The intestinal harmful bacteria used in the experiment were three types of harmful strains, Salmonella typhi, Escherichia coli O157:H7, and Shigella sonnei. Salmonella typhi, Escherichia coli O157:H7 , and Shigella sonnei were dispensed and applied to each MHA (Muller Hinton Agar) medium. The disks through which the compositions of Experimental Examples 1 to 8 were filtered were pressed onto each MHA medium coated with the three types of harmful strains, and after cultured at 37°C for 24-48 hours, the diameter of the transparent zone around the disk where bacterial growth was inhibited was measured to measure the antibacterial effect.

According to the above experimental results, it was confirmed that each composition exhibited an excellent effect of inhibiting the growth of harmful intestinal bacteria, and in particular, it was confirmed that the most excellent effect of inhibiting the growth of harmful bacteria was exhibited in Experimental Examples 2 and 4.

Experimental example 4

Anti-inflammatory evaluation of probiotic compositions

1) Cytotoxicity evaluation

RAW264.7 cells, a mouse macrophage cell line used in the experiment, were obtained from the Korea Cell Line Bank (KCLB), and DMEM medium containing 10 wt% fetal bovine serum (FBS) and 1 wt% penicillin-streptomycin was used for cell culture. The cells were cultured in a CO ₂ incubator (37°C, 5% CO ₂ ), and the cell toxicity of the extract was measured using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction method.

Cells were seeded at ^5Х104 per well in a 96-well plate and attached for 24 hours. The extracts were diluted to a certain concentration and treated with the cells. After 30 minutes, 1 μg/mL lipopolysaccharide (LPS) was treated and cultured for 24 hours. After the medium containing the sample was removed, serum-free medium and 5 mg/mL MTT solution were added and cultured for 2 more hours at 37°C. DMSO was dispensed, sonicated, stirred for 10 minutes, and then the absorbance was measured at 570 nm to determine the cell viability.

When RAW264.7 cells were treated with 1 ㎍/mL of LPS and Experimental Examples 1 to 8, respectively, and cell viability was evaluated. As a result, oligosaccharides isolated from soybeans or soy sauce were not toxic even at a concentration of 800 ㎍/mL, and RE and AE were not toxic up to 600 ㎍/mL.

2) Evaluation of inhibitory activity on NO production

Mouse macrophage RAW264.7 was dispensed into a 24-well plate at a concentration of 5Х10 ⁵ cells/well and cultured for 24 hours, and then treated with the composition of each experimental example. After culturing for 30 minutes, LPS (1 μg/mL) was treated and cultured for 20 or 24 hours, and the cell supernatant was collected. The collected cell culture supernatant was centrifuged (1000 rpm, 10 min, 4°C), 50 μL was mixed with 50 μL of sulfanilamide solution, blocked from light for 5 minutes and reacted, then mixed with 50 μL of NED solution, reacted at room temperature for 10 minutes, and the absorbance was measured at 540 nm using an ELISA reader (Epoch, Bioteck, Winooski, VT, Germany).

The experimental results are as shown in Fig. 3. As shown in Fig. 3, it was confirmed that each experimental composition exhibited excellent NO production inhibition activity compared to the control group treated only with LPS, and in particular, it was confirmed that the most excellent anti-inflammatory effects were exhibited in Experimental Examples 4 and 7.

3) Effect of inhibiting ROS production

The inhibitory efficacy was measured by inducing intracellular inflammatory response with LPS to induce ROS production using an intracellular ROS assay kit (Cell biolabs, USA). 5Х10 ⁴ cells/well of RAW 264.7 cells were seeded in a 96-well black plate, cultured, and then replaced with serum-free DMEM medium. After culturing the cells again for 24 hours, the samples were treated at various concentrations and cultured for 24 hours at 37°C, 5% CO ₂ conditions. After washing three times with PBS (pH 7.4), 100 μL of 1XDCFHDA was added to the medium, cultured for 1 hour at 37°C, 5% CO ₂ , and then washed three times with PBS again. After adding 100 μL of lysis buffer and mixing, fluorescence was measured at excitation 485 nm and emission 535 nm using a fluorescence microplate reader (Perkin-Elmer Inc., Waltham, MA, USA) to evaluate the ROS production inhibition activity.

The experimental results are as shown in Fig. 4. As shown in Fig. 4, it was confirmed that each experimental composition exhibited excellent ROS production inhibition activity compared to the control group treated only with LPS, and in particular, it was confirmed that the most excellent anti-inflammatory effects were exhibited in Experimental Examples 4 and 7.

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention.

Claims (7)

Contains oligosaccharides isolated from fermented soybean foods, extracts of baby spindle, and extracts of chives as active ingredients
A prebiotic composition for food. In the first paragraph,
The above oligosaccharide increases at least one intestinal microorganism selected from the group consisting of Lactobacillus , Lactococcus , Enterococcus , Streptococcus , Bifidobacterium , Coprobacillus , Roseburia, Coprococcus , Allobaculum , Peptococcaceae , and Christensenellaceae .
A prebiotic composition for food. In the first paragraph,
The above oligosaccharide is a monosaccharide selected from the group consisting of hexose (hex), N-acetylhexosamine (hexNac), deoxyhexose (deoxyhex), pentose (pen), hexuronic acid (hexA), and mixtures thereof.
A prebiotic composition for food. In the first paragraph,
The above oligosaccharide is selected from the group consisting of 474.159, 502.153, 504.169, 515.185, 529.201, 545.196, 586.222, 606.201, 636.211, 648.211, 666.222, 677.238, 707.248, 722.248, 839.291, 864.322, and 869.301, as analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS).
A prebiotic composition for food. A food probiotic composition comprising any one of claims 1 to 4.
A functional food composition for the proliferation of beneficial intestinal bacteria, inhibition of harmful bacteria, and anti-inflammation. delete 1) A step of preparing a soybean fermentation mixture by adding water to a fermented soybean food;
2) A step of vortexing and sonicating the above soybean fermentation mixture;
3) A step of preparing an extraction mixture by mixing methanol and chloroform into the soybean fermentation mixture of step 2);
4) A step of vortexing and sonicating the above extracted mixture;
5) A step of centrifuging the extracted mixture of step 4);
6) A step of separating oligosaccharides by performing solid phase extraction (SPE) on the supernatant separated by the above centrifugation; and
7) A step of mixing the baby swimming extract and the chive extract into the above oligosaccharide.
A method for preparing a prebiotic composition.