KR20120060800A - Fermented Soymilk with High Fructionality and Sensory properties and the Method thereof - Google Patents

Abstract

PURPOSE: Fermented soy milk having excellent functionalities, and a producing method thereof are provided to remove the unique unpleasant smell from soybeans, and to provide the oxygen-resistance and the recognition capability improving function of a purple sweet potato extract. CONSTITUTION: A producing method of fermented soy milk comprises the following steps: soaking soybeans in water, and grinding the soybeans with hot water; heat processing the ground soybeans for extracting, and filtering to obtain soy milk; heat-processing the soy milk at 121 deg C for 5-60 minutes; adding 0.5-2wt% of saccharide into the soy milk, and injecting bacillus subtilis into the soy milk for fermenting; and adding a purple sweet potato extract into the fermented soy milk. The fermentation process is performed at 30-45 deg C until the pH of the soy milk changes to 4.5-6.5.

Description

Fermented Soy Milk with Excellent Functionality and Functionality and Method of Manufacturing the Same {Fermented Soymilk with High Fructionality and Sensory properties and the Method

The present invention relates to a method for producing fermented soy milk excellent in functionality and functionality at the same time and a fermented soy milk produced thereby. The present invention also relates to an antioxidant, memory improvement, cognitive improvement and the composition for preventing or treating dementia diseases and pharmaceutical compositions comprising the extract of fermented soy milk as an active ingredient.

Soybeans have been used in many forms of food and nutrition in Korea, such as tofu, soybean paste, and soy sauce for a long time. It is a high-quality protein source that contains essential amino acids, and is almost as nutritious as milk or eggs. . Soybeans contain about 40% of the protein in all soybeans, with essential amino acids balanced, and other food proteins contain lysine that is likely to be lacking. The fat content is about 18%, mostly unsaturated fatty acids, and more than 50% is the finest linolenic acid. In addition, vitamin E, which helps the action of linolenic acid, is enough to remove cholesterol caused by excessive intake of animal fat. In addition, it is rich in vitamin B, which helps to recover from fatigue, and it is also rich in calcium and iron. In addition, isoflavon in soybean is known to have a strong anti-cancer effect against hormone-related cancers such as breast cancer and prostate cancer, and has been shown to reduce women's menopausal symptoms. Lecithin and saponin in soybeans are effective in preventing various adult diseases by lowering cholesterol levels in the body.In addition, soybeans contain various ingredients that prevent various cancers and adult diseases. It turns out.

Soybeans play an important role as a nutritional source of protein and fat, but they are hard to absorb and digest. In addition, physiologically inhibited substances such as carbohydrates and trypsin inhibitors that are poorly digested may cause problems with absorption of digestion and cause side effects such as diarrhea.

Soybeans include soy milk and fermented soy milk fermented with soy milk. Conventional soy milk is an antitrypsin such as soy odor and soyin present in soybean oil and the smell of fish. Many problems caused digestive problems.

Fermented soybean milk is fermented using soybeans as a raw material, which can be used in food. When only soybeans are consumed, only about 30% is absorbed and the remaining 70% is excreted, but when fermented soybeans More than 90% of nutrients can be absorbed into the body, which is excellent in terms of absorption of nutrients. Fermented soymilk contains enzymes such as amylase, protease, lipase and thrombolytic enzymes produced by microorganisms as well as soybean nutrients. Many enzymes contain various biologically active substances such as peptides, amino acids, oligosaccharides, fatty acids, active isoflavones, phytosterols, lecithins, and saponins, which are produced by decomposition of these enzymes.

      Recently fermented soy milk produced in Korea is mostly produced by using lactic acid bacteria to produce fermented milk, which contains abundant nutrients and various biologically active substances. Soybean odor is the same problem as soymilk, and it is resistant to drinking due to its coarse texture, and its taste also disappears due to fermentation so that it does not meet consumer's preference. There was a problem that the product condensation or an unpleasant taste occurs.

Soymilk drinks currently on the market are formulated with relatively high amounts of flavoring ingredients and flavorful flavors to ensure that the taste of sweeteners, cocoa, fruit juices, etc. is clearly recognized in order to enhance the unpleasant taste and aroma. Added is common. Thus, the compounding quantity of the soymilk component mix | blended in a soymilk beverage will fall by mix | blending various components in comparatively large quantity. Therefore, although the main purpose for the consumer to select soy milk is to consume an active ingredient such as protein or isoflavones derived from soybeans, such a soymilk beverage is formulated with a relatively large amount of the above formulated ingredients to increase palatability. This has caused a problem that the soymilk content in the soymilk beverage is lowered. In addition, commercially available soymilk beverages are relatively sweet and have a very fragrant flavor to mask the fishy taste and smell of soymilk. There was a problem that lack of goodness.

In the present invention, the unpleasant odor of soybean is eliminated and the sweet potato extract has a different taste, and the method of producing fermented soybean milk with excellent functionality and functionality, which has excellent anti-oxidation, memory and cognitive ability, prevention of dementia disease, and improved ability. To provide a fermented soy milk prepared by.

The present invention provides an antioxidant comprising the extract of fermented soy milk as an active ingredient, improving memory, improving cognitive ability or preventing or treating dementia diseases. Further, the antioxidant comprising the extract of fermented soy milk as an active ingredient, It provides a pharmaceutical composition for improving memory, improving cognitive ability and preventing or treating dementia diseases.

In order to solve the above problems of the present invention, the present invention,

(1) pretreating soymilk;

(2) inoculating Bacillus subtilis bacteria with soymilk for fermentation; And

(3) adding a purple sweet potato extract;

It provides a method of producing fermented soy milk excellent in functionality and functionality, including.

The soy milk is

(a) soybean called water;

(b) grinding by adding 7 to 11 times hot water of soybean weight; And

(c) extracting the soy crushed material by heat treatment and then filtration;

It is preferable that it is obtained by the manufacturing method of soymilk containing.

The pretreatment of step (1) is characterized in that the heat treatment for 121 to 10 minutes to 20 minutes.

0.5 to 2% by weight of sugar is preferably added to the soy milk before inoculating Bacillus subtilis bacteria to the soymilk of step (2).

In step (2), Bacillus subtilis 2829-PNU 015 is preferred.

The number of Bacillus subtilis bacteria inoculated in step (2) is preferably 6 to 9 logCFU / ml.

Soymilk inoculated with Bacillus subtilis bacteria in step (2) is fermented to reach pH 4.5 to 6.5 at 30 to 45.

The content of the purple sweet potato extract in step (3) is 1 to 10% by weight of the total fermented soymilk content.

The concentration of the purple sweet potato extract is preferably 60 to 80 ^o Brix.

The present invention provides fermented soy milk for antioxidant, memory improvement, cognitive ability improvement and prevention or improvement of dementia disease produced by the manufacturing method.

The present invention provides a composition for preventing or treating dementia diseases, including antioxidant, memory improvement, cognitive improvement and dementia comprising the extract of the fermented soy milk as an active ingredient.

The extract is preferably extracted using water, alcohol or a mixed solvent thereof.

The present invention provides a pharmaceutical composition for antioxidant, memory improvement, cognitive improvement and prevention or treatment of dementia diseases comprising the extract of the fermented soy milk as an active ingredient.

The present invention removes the discomfort of soybeans, improves digestion upon ingestion, adds a different taste, improves organoleptic properties, and improves fermented soy milk with excellent functionality such as antioxidant, memory and cognitive ability, and prevention and improvement of dementia diseases. It provides, and provides a composition for the prevention or treatment of antioxidant, memory improvement, cognitive improvement or dementia disease comprising the fermented soy milk as an active ingredient.

The composition comprising the fermented soybean milk or its extract as an active ingredient has no side effects and is stable, and thus industrial value of the present invention will be great because it can be used in various fields such as food and medicine.

1 is a graph showing the DPPH scavenging effect of fortified wormwood, garlic, cornus, purple sweet potato.
Figure 2 is a graph showing the OH scavenging effect of fortified mugwort, garlic, cornus, purple sweet potato.
Figure 3 shows the sensory evaluation of fermented soybean milk added with fortified mugwort, garlic, cornus, purple sweet potato.
Figure 4 shows the sensory evaluation when 1%, 5%, 10% of purple sweet potato hot water extract is added.
5 shows the results of experiments on the object recognition ability of Alzheimer's mice.
6 shows the effects of memory damage protection as a result of T-maze experiments in Alzheimer's-induced mice.
Figure 7 graphically shows the learning progress of the mouse for three days in the Morris Water Maze experiment.
FIG. 8 is a graph comparing the time the mouse visits the shelters when hiding the hides on the last day in the Morris Water Maze experiment and when exposed without hiding.
FIG. 9 is a graph measuring the time the mouse stays in the zone where the evacuation zone is located when the evacuation zone is removed in the Morris Water Maze experiment.

Hereinafter, the present invention will be described in detail.

The present inventors attempted to produce a soymilk product excellent in functionality, which is not only excellent in functionality such as taste and aroma by fermenting soymilk rich in high quality protein, but also conventional soymilk. For the production of such soy milk, fermented bacteria used for plant fermentation were selected, not animal lactobacillus used for milk fermentation, which is generally used for making fermented soy milk. Bacillus subtilis bacteria were screened as strains capable of functioning. Among the Bacillus subtilis bacteria, fermented soy milk fermented with new strains Bacillus subtilis 2805-PNU 014, Bacillus subtilis 2829-PNU 015 and Bacillus subtilis 2825-PNU 016 was most functional and functional. Through this fermentation process, it was confirmed that some of the odors generated during the fermentation process by Bacillus and soybean milk itself and taste of soy milk did not disappear completely, and the sour taste formed by fermentation was somewhat insufficient to appeal to consumers. Among the various plant groups, purple sweet potato was selected while trying to find an additive food which can be used in harmony with the taste of the fermented soy milk to remove the unpleasant taste and the functionality and save the functionality. When the addition of soy milk is greatly reduced in the discomfort of digestion, color, taste, etc. palatability, excellent memory, cognitive ability and dementia treatment effect was confirmed and completed the present invention.

Fermented soybean milk excellent in functionality and functionality of the present invention is characterized in that it comprises the following steps.

(1) pretreatment of soymilk,

(2) fermenting Bacillus subtilis bacteria by inoculating soymilk and

(3) adding the purple sweet potato extract.

In step (1), the soy milk usable is not particularly limited in its kind. Commercially available ones may be purchased or may be subjected to a manufacturing process of soymilk commonly used in the art, but preferably (a) soybean is called water, and (b) hot water of 7 to 11 times the weight of soybean is added. It may be obtained by the method of producing soymilk comprising the step of crushing and (c) heat-extracting and extracting the soy crushed product.

In this case, the soybeans may be selected and called in water, and the temperature called water is not particularly limited, but may be preferably called at 90 to 150 ° C. The soybeans can be added 7 to 11 times, preferably 8 to 10 times, water of the soybean weight, and the temperature of the water is hot water, preferably hot water of 90 to 120 ° C, more preferably 95 ° C. By adding hot water, grinding, the quality of soymilk can be extracted and high yield of effective ingredient can be extracted. The soy crushed product may be extracted by heat treatment and then filtered to obtain soy milk.

Through the pretreatment in step (1) can be improved sterilization, deodorization and absorption of soy milk. The pretreatment step is not particularly limited, but may be performed by heat treatment, and preferably, may be heat treated at 121 to 10 minutes to 20 minutes.

If the heating time is shorter than the above, sterilization of various germs is not sufficiently performed, and soybean milk can grow undesired bacteria such as decayed bacteria or contaminants when fermented into Bacillus subtilis bacteria. have. In addition, the fishy smell of soybean is made of various substances including hexanol, and the effect of deodorization can be obtained through the addition of hot water to the high temperature, and the protease inhibitor or phytate Soymilk after the heat treatment can increase the digestibility because it can destroy the digestive substances contained in soybeans such as.

The Bacillus subtilis bacteria of step (2) are not particularly limited in kind, but are preferably Bacillus subtilis 2805-PNU 014, Bacillus subtilis 2829-PNU 015 and Bacillus subtilis. It may be selected from the one consisting of Tilis 2825-PNU 016, more preferably Bacillus subtilis 2829-PNU 015.

In the present invention, when compared with the soymilk inoculated with the Bacillus subtilis (Bacillus subtilis) bacteria, the antioxidant capacity of the inoculated bacteria is good, Bacillus subtilis 2805-PNU 014, Bacillus among Bacillus subtilis bacteria Subtilis 2829-PNU 015 and Bacillus subtilis 2825-PNU 016 have excellent antioxidant capacity. Generally, Bacillus produces strong ammonia odor during fermentation, whereas Bacillus subtilis 2805-PNU 014, Bacillus subtilis 2829-PNU 015 and Bacillus subtilis 2825-PNU 016 have strong ammonia and ammonia odor. It was not unpleasant and the protease titer was also high. Among them, Bacillus subtilis 2829-PNU 015 is not only the most effective DPPH and OH scavenging effect, but also not so unpleasant that the fermentation odor masks the flavor of soy milk during fermentation. In addition to a reduction in fishy and fishy taste, absorption can be improved to a degree suitable for adults, children or patients with poor digestion upon ingestion.

Before inoculation of the Bacillus subtilis, a temperature suitable for growth of Bacillus subtilis may be selected, and as an example of the present invention, Bacillus subtilis 2805-PNU 014, Bacillus subtilis 2829-PNU 015, or Bacillus subtilis 2825 In the case of -PNU 016, the soymilk pre-treated in step (1) may be cooled to 30 to 45 ° C, preferably 37 to 40 ° C, which is an optimum growth temperature of the Bacillus subtilis.

It may also contain 0.5 to 2%, preferably 1%, of sugar in soymilk prior to inoculation of Bacillus subtilis bacteria. At this time, the sugar may be appropriate glucose. The concentration of the sugar can help the smooth progress of the fermentation and shorten the fermentation time by supplying the appropriate nutrients to fermentation occurs without compromising the taste of fermented soy milk.

When fermentation of the present invention can be inoculated with 1% by weight Bacillus subtilis bacteria of the whole fermented soy milk, the number of bacteria at this time may be 6 to 9 logCFU / ml, preferably 7 to 8 logCFU / ml.

The fermentation temperature is not particularly limited but may be preferably fermented at 30 to 45 ℃, preferably 37 to 40 ℃ 5 hours to 24 hours, the fermentation time is pH 4.5 to 6.5, preferably pH 5.3 It is preferable to determine by the time of the time of becoming -5.7. The solubility of soy protein in soymilk at pH4.5 or below is markedly reduced, which is undesirable, while acid is formed sour and curd is formed, resulting in a significant decrease in functionality, whereas the range of the present invention is acid and curd. Due to the low production of aroma, taste, swallowing texture is not only the best, but also the taste and smell of fish is greatly reduced, the pH range is the most functional.

In addition, when the fermented soybean milk is refrigerated after the end of fermentation, the number of Bacillus subtilis bacteria for 6 to 15 days, preferably 7 to 10 days is preferably maintained at 6 logCFU / ml or more. The maintenance of the number of fermented soybeans during this period provides the sour taste to enhance the taste of fermented soybean milk and lowers the pH to prevent the growth of decayed bacteria for a long time, as well as to maintain the body's balance and health in vivo. It is important to have a probiotics effect.

As described above, when the soymilk is fermented with Bacillus subtilis bacterium, the absorption rate of digestion of soymilk is improved and the fishy taste and smell are reduced compared to the conventional soymilk. Functionality is improved, and various physiologically active substances are produced by Bacillus subtilis bacteria during fermentation process, but functionality is improved, but the taste and smell of conventional soy milk does not disappear completely. The smell is somewhat stronger.

Extraction method of the purple sweet potato extract of the step (3) may be extracted by a method generally used in the art, preferably heat extraction.

The selection of the purple sweet potato as the fermented soy milk additive is not only considering the most preferred taste, appearance, aroma, texture and overall preference when mixed with fermented soy milk, but also synergistic effect by mixing with fermented soy milk, resulting in high antioxidant power, memory and Foods that improve cognition and prevent and improve dementia are selected.

The purple sweet potato heat extract may be added to 1 to 10%, preferably 4 to 6% by weight, most preferably 5% of the total of the fermented soy milk, wherein the concentration of the extract is 60 to 80 ° Brix, preferably Preferably from 65 to 75 ° Brix, most preferably 70 ° Brix. That is, especially when the concentration of the purple sweet potato heat extract is 70 ° Brix and the content is 5% may be the point where the functionality is optimally maximized.

The concentration and content of the extract is lower than the above concentration and content, and the taste and smell of soy milk may not be completely concealed, and the taste may not be balanced because the sour taste is stronger than sweetness. Too dark purple can make the appetite less sweet, and the sweet and strong taste of soy milk can be lost.

The order of addition of the purple sweet potato extract is not limited to the order of the steps of the production process, and may be added at any stage of preparing soymilk, fermenting soymilk or finishing the fermentation, preferably soymilk. It is preferred to add after the fermentation of the.

When the purple sweet potato extract is added during the manufacturing or fermentation of soy milk, in order to remove the fishy taste and aroma of the soy milk, the amount of the purple sweet potato extract has the same effect as that of the extract after the fermentation step. As the amount of soy milk is reduced, the content of active ingredients such as soy protein and isoflavones is reduced, and the sweetness of purple sweet potato masks the unique and protein taste of soy milk itself, and the color of fermented soy milk is purple. It may be appropriate to add the purple sweet potato extract after the fermentation of soy milk, because it becomes too strong to visually reduce appetite.

The addition of the purple sweet potato extract of step (3) offsets the sour taste due to the fermentation process of step (2) and in harmony with the taste of the purple sweet potato to feel the sweet and proteiny taste, the soy milk of the soy milk that remained somewhat after fermentation It can greatly offset taste and smell.

The present invention provides fermented soy milk for antioxidant, memory improvement, cognitive improvement and prevention and treatment of dementia disease prepared according to the above production method.

Fermented soy milk of the present invention can feel the sweet and proteiny taste in harmony with the taste of purple sweet potato provides a different taste from the conventional fermented soy milk and greatly reduces the fishy taste and smell of soy milk that has been pointed out as a problem of fermented soy milk. In addition, it can be seen that it is excellent in antioxidant, memory improvement, cognition improvement, inhibition of lipid peroxidation, inhibition of NO production and prevention and improvement of dementia disease. Fermented soy milk for the prevention and improvement of Bacillus subtilis, preferably the new strains Bacillus subtilis 2805-PNU 014, Bacillus subtilis 2829-PNU 015 or Bacillus subtilis 2825-PNU 016, more preferably Is fermented by Bacillus subtilis 2829-PNU 015 (Korea Microorganism Conservation Center, Accession No.:KCCM 42923) Generating a variety of bioactive substances by the Bacillus bacterium, and the functionality of the fermented soy milk improved by the addition of purple sweet potato extract, and there are functional can be enhanced also by introducing a unique flavor.

The present invention provides a composition for the prevention or treatment of antioxidant, memory improvement, cognitive improvement and dementia disease comprising the extract of the fermented soy milk as an active ingredient.

The fermented soy milk extract may be extracted according to a general method used in the art, but may be preferably extracted using water, alcohol or a mixed solvent thereof, and more preferably, extracted with methanol.

In a preferred embodiment of the present invention, the prepared fermented soymilk may be lyophilized and powdered and extracted using methanol, and 2 to 20 times the weight of the powdered fermented soymilk, preferably 2 to 15 times the methanol. It can be extracted by adding.

The composition is excellent in anti-oxidation, memory, cognitive ability and prevention and improvement of dementia disease and has a low side effect and stable, can be used as a raw material for food, medicine, cosmetics, etc., and also can be directly taken in the pharmaceutically acceptable range Could be

The present invention provides an antioxidant, memory improvement, cognitive improvement and the prevention or treatment of dementia diseases comprising the extract of the fermented soy milk as an active ingredient.

The pharmaceutical composition of the present invention, the total weight of the composition comprises 0.1 to 90% by weight of the fermented soy milk extract.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The pharmaceutical compositions of the present invention may be prepared in any formulation conventionally prepared in the art (e.g., Remington's Pharmaceutical Science, latest edition; Mack Publishing Company, Easton PA), for example granules, fines, powders, It may be administered as a pharmaceutical composition for oral administration such as hard capsules, soft capsules, syrups, emulsions, suspensions or solutions, and may be intravenous, intramuscular or subcutaneous injections, mucus, suppositories, transdermal absorbents, It is also possible to administer as a pharmaceutical composition for parenteral administration such as a transmucosal absorbent, a nasal drop, an ear drop, an eye drop, an inhalant, a cream, an ointment, a pape. The formulation prepared as a pharmaceutical composition in powder form may be dissolved in use and used as an injection or mucus.

For the preparation of pharmaceutical compositions, additives for the preparation of solids or liquids may be used. The additive for preparation may be either organic or inorganic. In other words, when preparing oral solid preparations, excipients, binders, disintegrating agents, lubricants, coloring agents and the like are added to the main medicine, and then tablets, coating tablets, granules, powders, Preparations in the form of capsules and the like can be prepared. Examples of excipients to be used include lactose, sucrose, white sugar, glucose, corn starch, starch, talc, sorbet, crystalline cellulose, dextrin, kaolin, calcium carbonate, silicon dioxide and the like. Examples of the binder include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, Dextrin, pectin and the like. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. As a coloring agent, if it is normally permitted to add to a pharmaceutical, all can be used. These tablets and granules can be appropriately coated according to sugar, gelatin coating, and other needs. Moreover, preservatives, antioxidants, etc. can be added as needed.

Inert diluents commonly used in the preparation of liquid preparations for oral administration, for example emulsions, syrups, suspensions, solutions, may be used, for example water or vegetable oils. In addition to the inert diluent, this preparation may be formulated with an adjuvant such as a wetting agent, suspending aid, sweetener, fragrance, colorant or preservative. After the liquid formulation is prepared, it may be filled into a capsule of absorbable material such as gelatin. As a solvent or suspending agent used for preparation of a parenteral administration agent, for example, an injection or a suppository, water, propylene glycol, polyethylene glycol, benzyl alcohol, ethyl oleate, lecithin are mentioned, for example. As a base used for manufacture of a suppository, a cacao butter, an emulsified cacao butter, a laurin butter, and a witezol are mentioned, for example. The preparation method of the formulation is not particularly limited, and any method commonly used in the art can be used.

In the case of injectable preparations, for example, diluents such as water, ethyl alcohol, macrogol, propylene glycol, citric acid, acetic acid, phosphoric acid, lactic acid, sodium lactate, sulfuric acid and sodium hydroxide; PH adjusters and buffers such as sodium citrate, sodium acetate and sodium phosphate; Stabilizers, such as sodium pyrosulfite, ethylenediamine tetraacetic acid, thioglycolic acid, and thio lactic acid, etc. can be used. In this case, in order to prepare an isotonic solution, a sufficient amount of salt, glucose, mannitol, or glycerin may be blended in the formulation, or a conventional dissolution aid, analgesic agent, or local anesthetic may be used.

In the case of ointments such as pastes, creams, and gels, bases, stabilizers, wetting agents, preservatives, and the like, which are commonly used, may be blended as necessary, and the components may be mixed and formulated by conventional methods. .

The dosage of the pharmaceutical of the present invention is not particularly limited, but the extract of the present invention may be administered at 0.001 to 1000 mg / kg, preferably 0.1 to 500 mg / kg per day. Administration may be administered once a day or may be divided several times. However, since the dosage of the extract of the present invention is determined in view of various related factors such as the route of administration, the age, sex, weight, severity of the disease, health condition, etc., the dosage does not limit the scope of the present invention in any aspect. It should not be understood to be limiting.

The present invention provides a health food for preventing or treating dementia diseases, including antioxidant, memory improvement, cognitive improvement and dementia comprising the extract of the fermented soy milk as an active ingredient.

Foods to which the composition of the present invention can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, health functional foods, and the like. Other components other than the composition of the present invention have special limitations. It may contain various flavors or natural carbohydrates and the like as an additional ingredient, as in normal beverages.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples. However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

Example 1  : Preparation of Materials

Example 1 -1 Cheonggukjang

Cheonggukjang purchased six representative Cheonggukjang from the Sunchang area, and among them, Cheonggukjang, the most antioxidant among the three Cheonggukjangs selected through sensory evaluation, was used as Cheonggukjang for the isolation of Bacillus bacteria.

Example 1 -2 soymilk

It was purchased from Donghwa Food (Yangsan, Korea) for the production of fermented soymilk.

Example 1 -3 Bacillus Fungus

Among the best of the three antioxidant ability Soybean Soybean Soybean selected during a typical six kinds of Doi areas through the sensory evaluation Bacilllus subtilis 2805- PNU 014, Bacillus subtilis 2829- PNU 015, Bacillus subtilis 2825-PNU 016 was isolated and used.

Example 1 -4 Functional material

Cheonho Foods Co., Ltd., which is conducting joint research for antioxidant, cognitive ability improvement and Alzheimer's disease prevention, was supplied with hot water extract of fortified lion wormwood, garlic, cornus milk and purple sweet potato.

Example 2  : Fermented Soymilk  Produce

Six kinds of Cheonggukjang were purchased from Sunchang area, Jeonbuk, and three kinds of Cheonggukjang with the most sensuality were selected through sensory evaluation, and one type of Cheonggukjang with the most effective effect was selected by measuring their antioxidant activity. Three kinds of Bacillus in this Cheonggukjang Bacillus subtilis bacteria were separated from the received pre-sale disclosure to the Korea Culture Center of Microorganisms strains to compare it with the active subtilis fungus, and Bacillus used commercially Bacillus among three bacteria isolated from Cheonggukjang as a result of fermenting soymilk with five kinds of bacteria including subtilis Subtilis 2829 PNU-015 showed the best results in antioxidant activity, so it was used for fermenting soymilk as an optimal fermentation strain to enhance antioxidant activity (Accession No .: KCCM 42923, Deposit Date March 26, 2009) .

To ferment soy milk, add 1% glucose to soy milk, sterilize at 121 for 15 minutes and cool to 37 ° C. Then Bacillus selected as fermented strain subtilis 2829 PNU - 015 bacteria were inoculated with 1%. The fermentation was stopped in a shaking water bath at 37 ° C. for about 8 hours when the pH reached 5.5 ± 0.2. Functional fermented soymilk production is added to the soymilk fermented as described above so that the 70 ° Brix purple sweet potato hot water extract has a final concentration of 5%. The fermented soymilk thus prepared was lyophilized and powdered and extracted three times for 24 hours at room temperature with 10% of 100% methanol, and the filtrate was concentrated under reduced pressure at 36-40 ° C. with a vacuum rotary concentrator to obtain an extract. It was used for the experiment of antioxidant activity.

Example 3  : Bacillus subtilis Antioxidant Effect of Bacteria

Bacillus isolated from Cheonggukjang, the best antioxidant activity described above subtilis 2805 PNU -014 , Bacillus subtilis 2829 PNU -015 , Bacillus subtilis 2825 PNU -016 and Bacillus from Commercial Microbial Conservation Center The antioxidant activity to select the most outstanding among bacteria subtilis bacteria as 1,1-diphenyl-2-picrylhydrazyl ( DPPH) scavenging, OH (hydroxyl radical) scavenging Bacillus The antioxidant activity of the subtilis strains was examined.

Each Bacilllus in Tables 1 and 2 below DPPH scavenging effect and OH scavenging effect of fermented soymilk fermented with subtilis were shown.

Treatment (μg / mL) DPPH radical scavenging activity (%) SM FSM-1 FSM-2 FSM-3 FSM-4 FSM-5 100 23.6 ± 0.3 ^d 25.4 ± 0.3 ^bc 20.0 ± 0.2 ^e 28.1 ± 0.6 ^a 24.6 ± 0.1 ^c 25.7 ± 0.1 ^b 500 50.2 ± 0.4 ^e 59.2 ± 0.5 ^d 61.0 ± 0.4 ^d 78.9 ± 0.2 ^a 69.6 ± 0.4 ^b 65.2 ± 2.1 ^c 1000 68.4 ± 0.7 ^d 74.0 ± 0.7 ^c 77.3 ± 1.0 ^c 98.1 ± 3.1 ^b 104.1 ± 0.6 ^a 97.3 ± 2.2 ^b

SM: Soymilk

FSM-1: Fermented soymilk by Bacillus subtilis 11316 from KCCM

FSM-2: Fermented soymilk by Bacillus subtilis from Vilac

FSM-3: Fermented soymilk by Bacillus subtilis PNU 2805-014

FSM-4: Fermented soymilk by Bacillus subtilis PNU 2829-015

FSM-5: Fermented soymilk by Bacillus subtilis PNU 2825-016

Values are mean ± SD

^ae Means with the different letters are signigicantly different (p <0.05) by Ducan's multiple range test.

Treatment (㎍ / mL) OH radical scavenging activity (%) SM FSM-1 FSM-2 FSM-3 FM-4 FSM-5 100 85.7 ± 0.0 ^d 86.8 ± 0.2 ^cd 82.7 ± 0.2 ^e 89.4 ± 0.2 ^a 88.8 ± 0.4 ^ab 87.7 ± 0.7 ^bc 500 90.0 ± 0.2 ^b 83.3 ± 0.2 ^c 82.1 ± 0.9 ^c 88.0 ± 0.1 ^b 89.8 ± 0.7 ^b 97.1 ± 2.6 ^a 1000 82.6 ± 0.3 ^b 89.5 ± 0.6 ^a 83.8 ± 1.4 ^b 84.6 ± 1.1 ^b 90.8 ± 2.2 ^a 84.5 ± 0.7 ^b

SM: Soymilk

FSM-1: Fermented soymilk by Bacillus subtilis 11316 from KCCM

FSM-2: Fermented soymilk by Bacillus subtilis from Vilac

FSM-3: Fermented soymilk by Bacillus subtilis PNU 2805-014

FSM-4: Fermented soymilk by Bacillus subtilis PNU 2829-015

FSM-5: Fermented soymilk by Bacillus subtilis PNU 2825-016

Values are mean ± SD

^ae Means with the different letters are signigicantly different (p <0.05) by Ducan's multiple range test.

As can be seen in Tables 1 and 2 Bacillus Subtilis 2829 PNU- 015 exhibited the highest antioxidant activity among the five Bacillus strains tested with 104.1% DPOH scavenging effect and 90.8% OH scavenging effect. Thus Bacillus, the strain with the highest antioxidant activity subtilis 2829 PNU- 015 was deposited on March 26, 2009 (Korea Microbiological Conservation Center, Accession No.:KCCM 42923).

Example 4  : Selection and addition amount of functional material with excellent antioxidant functionality

Example  4-1 Antioxidant ability  Measure

DPPH scavenging effect and OH scavenging effect of the extracts of fortified lion wormwood, garlic, cornus and purple sweet potato, which are manufactured and used as health functional food, were measured and summarized in FIGS. 1 and 2. Fortified lion wormwood, garlic, cornus and purple sweet potato were supplied from Cheonho Foods (Busan, Korea).

DPPH scavenging activity is measured by Hatano et al. (Hatano T, Edamatsu R, Hiramatsu M, Fujita Y, Yasuhara T, Yoshida T, Okuda T .: Effects of the interaction of tannins with co-existing substances, .Effects of tannins and related polyphenols on superoxide anion radical, and on 1,1-diphenyl-2-picrylhydrazyl radical.Chem.Pharm.Bull. 37: 2016-2021 (1989)).

100 μM of the sample dissolved in ethanol (EtOH) and 100 μM of the 60 μM DPPH solution were mixed in a 96-well plate for 30 minutes, and the absorbance was measured at 540 nm. The free radical scavenging effect was expressed as a percentage (%) compared to the control without the sample.

OH scavenging activity was determined by Chung et al. (Chung SK, Osawa T, Kawajusgu S .: Hydroxyl radical-scavenging effects of spices and scavengers fro brown musard ( Brassica nigra ) .J. Biosci.Biotech.Biochem. 61: 118-123 (1997) ) Was used.

According to the Fenton reaction, 10mM FeSO ₄ 7H ₂ O-EDTA was mixed with 10mM 2-deoxyribose solution and sample solution by concentration, and then 10mM H ₂ O ₂ was added and incubated at 37 ° C for 4 hours. 2.8% trichloroacetic acid (TCA) and 1.0% thiobarbituric acid (TBA) solution were added to the mixture, and the mixture was boiled for 10 minutes, cooled and absorbed at 520 nm.

1 and 2, the purple sweet potato hot water extract exhibits a high scavenging effect of 90% or more in the DPPH scavenging effect, and the OH scavenging ability also shows a scavenging ability of 80% or more. In addition, the functionality was also evaluated by mixing 5% of the hot water extracts of fortified sweet potato mugwort, garlic, cornus and purple sweet potato to prepare fermented soymilk.

Example  4-2 Sensuality  Measure

To measure sensory characteristics, a panel of 10 graduate students (10 women in the Department of Food and Nutrition, Pusan National University) were studied. The evaluation items were five items: appearance, flavor, taste, texture, and overall acceptability, with 1 being the worst and 9 being the best. The average value was obtained and expressed as QDA profile. As shown in FIG. 3, the sensory evaluation of the fermented soymilk added with purple sweet potato hot water extract was significantly higher than that of other functional materials.

In order to determine the concentration of purple sweet potato hot water extract selected according to the above contents, fermented soy milk was added with purple sweet potato hot water extract of 70 ° Brix concentration to the fermented soy milk at 1%, 5% and 10% concentrations. Was carried out. As a result, 5%>10%> 1% addition showed good functionalities. As shown in FIG. 4, most of the items showed similar scores at the 5% and 10% addition levels, but the appearance of color and fragrance was 10 The addition of 5% was higher than the addition of%.

Example 5  : Functional Fermented soy milk  Antioxidative effect

Bacillus by adding 5% of 70 ° Brix purple sweet potato hot water extract to soymilk In order to investigate the antioxidant effect of the functional fermented soybean milk fermented with subtilis bacteria, the DPPH scavenging effect and the OH scavenging effect were tested in the same manner as in Example 3 above, and the results obtained are summarized in Table 3 below.

Treatment (μg / ml) Scavenging activity (%) DPPH OH 100 14.3 ± 0.2 ^d 92.8 ± 0.2 ^d 250 47.8 ± 0.7 ^c 96.2 ± 0.2 ^c 500 70.5 ± 0.3 ^b 97.4 ± 0.2 ^b 1000 131.0 ± 7.5 ^a 102.8 ± 0.4 ^a

Values are mean ± SD.

^ad Means with the different letters are significantly different (p <0.05) by Duncan'smultiplerangetest.

As shown in Table 3, the DPPH and OH scavenging effects of the functional fermented soybean milk were increased in a concentration-dependent manner, confirming the antioxidant functionality of the fermented soymilk added with purple sweet potato hot water extract.

Example 6 : Functional Fermented soy milk  Cognitive Improving Effects in Alzheimer's-induced Mice

Example 6 -1 Experimental Animals and Breeding

Thirty five ICR mice (25-27 g, male) of 5 weeks old were purchased from Orient Bio Co., Ltd. and stabilized with a general diet for 1 week before being used for the experiment. The temperature and humidity of the feeding room were maintained at 20 ± 2 ℃, 50 ± 10%, the contrast was maintained in the light-dark cycle every 12 hours. Forage, solid feeds from Purina (Korea, crude protein 21.1%, crude fat 3.5%, crude fiber 5.0%, crude ash 8.0%, calcium 0.6%, phosphorus 0.6%) were used as drinking water.

The experimental animals were acclimated to the laboratory environment for 1 week, and then the weight of each group was adjusted equally, so that the sweet potato hot water after the normal, control (Amyloid-β _25-35 treatment, control) and Aβ _25-35 treatment The fermented soymilk extract was divided into 100mg / kg / day oral administration group and Aβ treatment, and the purple sweet potato hot water extract added fermented soymilk 200mg / kg / day oral administration group. The normal group was then injected with sterile saline to the crown and the other groups (control, 100 mg / kg / day, 200 mg / kg / day) were injected with Aβ _25-35 .

Example 6 -2 Induced memory damage in experimental animals

A total of 32 ICR mice, 8 of each group, were anesthetized with ether, followed by Laursen and Belknap methods (Laursen SE, Belknap JK .: Intracerebroventricular injections in mice.Some methodological refinements. J. Pharmacol. Methods 16: 355-357 (1986) A) _25-35 was injected into 10μL Hemilton micro-syringe with a 26-gauge needle and 5μL of Aβ _25-35 was injected into the crown of each experimental animal at a depth of 2.2 mm to cause memory damage. At this time, the Normal group was injected with sterile saline.

Example 6 -3 cognitive ability behavior test of experimental animals

 ① Object recognition experiment

A black box without a lid was used to observe the object recognition ability. One day before the experiment, place the same object (A ₁ and A ₂ ) in the box on both sides and allow 10 minutes for the two objects to acclimate. After 24 hours, one of the two objects the previous day was replaced with a new object (A ₁ and B), and the searchability of the old and new objects was numerically compared. The objects A ₁ , A _2, and B used in this experiment were all made of plastic and were similar in size and color. The objects were placed diagonally in a black box and the mouse started from the center of the two objects. At the end of the experiment, the boxes and objects were disinfected with 70% alcohol.

Object recognition tests are known to be useful for measuring visual cognitive memory (Tang YP, Shimiqu E, Dube GR, Rampon C, Kerchner GA, Zhuo M .: Genetic enhancement of learning and memory in mice.Nature 401: 63 -69 (1999)) Presenting the same two objects to the mouse gives a similar number of touches, but after 24 hours, if one of the two objects is replaced with a new object, the mouse usually becomes new because of the habit of being more curious about the new object. The rate of touching the object becomes larger. However, mice with memory impairment are unable to distinguish between new objects and there is no increase in touch rate.

Experimental results of whether or not the sweet potato hot water extract added fermented soybean milk methanol extract dissolved in bottled water orally administered mouse object.

As shown in Figure 5, the control group can be seen that there is almost no difference in the frequency of contact with A ₁ and B 43.57% and 44.67%. However, the group that orally administered the fermented soymilk extract with purple sweet potato at 100mg / kg / day recognized 54.67% of the new objects and 54.74% of the group administered orally at 200mg / kg / day, which is the level recognized by the normal group. It is similar to 59.32%, indicating that the frequency of contact with new objects is high. Based on these results, it can be seen that purple sweet potato hot water extract has a protective effect against visual cognitive memory impairment.

 ② T-maze experiment

According to Montgomery's method (Montgomery KC .: A test of two explanations of spontaneous alternation. J. Comp. Physiol. 45: 287-293 (1952)), T-maze experiments were performed. It is a black T-maze-type maze and consists of a starting box, left arm, and right arm, and a detachable barrier is installed in the right arm passage. One day before the experiment, the gate was blocked by the T-maze's right arm passage and the left arm was left open at the starting point. Move the mouse freely for 10 minutes to allow time for adjustment. After 24 hours, the left arm and the right arm also open along with the number of times the mouse enters the left and right arms for 10 minutes. Passage was considered to have entered the passage by half of the mouse trunk.

Mice are curious about new objects and new spaces. Experimental results of T-maze are shown in FIG. 6.

As shown in FIG. 6, in the case of the control group, the access degrees of the left arm (old route) and the right arm (new route) were 47.85% and 52.15%. However, the oral administration of purple sweet potato fermented soymilk extract at 100mg / kg / day showed 54.57% of the new route and 54.75% of the oral administration at 200mg / kg / day. Results similar to 57.02%. As a result, it can be seen that the fermented soymilk added with purple sweet potato hot water extract has a protective effect against Alzheimer's induced memory damage.

③ Experiment of water water maze

According to Morris's experimental method (Morris R .: Developments of a water-maze procedure for studying spatial learning in the rat.J. Neurosci. Methods 11: 47-60 (1984)) In addition, a circular escape pad was placed about 5 mm below the water surface in the middle of one section. The skim milk was removed in 22 ± 2 ° C. water so that the hides could not be seen by the mouse when swimming. Different shaped stickers were attached to the walls of the tank, which were divided into four parts so that the mouse used the space cues around the tank to find the escape. In addition, during the experiment, all subjects around the tank, such as instruments, shelves, and experimenters, were always conducted at the same location.

In the second stage, the first stage was the learning phase to find the escape in the maze (3 days) and the second stage was the memory test on the 4th day. The experiment began with the mouse being placed in the tank along the tank wall, where the starting point was the central wall of each quadrant, the starting quadrants were randomly selected in no particular order, and the location of the escape zone was not changed during the learning period. . Mice were trained to recognize the space location of the shelters for three days three times a day (done within 12 hours at four hour intervals). If 60 seconds had not been found, the experimenter gently guided the mouse to the shelters to find the location of the shelters. Mice were allowed to rest there for 30 seconds as a reward for the reaction when they climbed the shelters. After learning the mouse for 3 days, three days to measure the memory capacity was measured on the fourth day.

The results of the learning phase in which the mouse visits the shelters within 60 seconds for 3 days are shown in FIG. 7. This result shows how much the mouse is learning.

As shown in FIG. 7, it can be seen that the control group took longer to visit the shelter than the normal group or the group which orally administered the fermented soy milk extract with purple sweet potato. These results indicate that purple sweet potato added fermented soy milk protects memory loss.

First, the evacuation zone was placed in a bath containing skim milk powder and the time for the mouse to visit the evacuation zone was measured. Secondly, the hides were removed from the tank with skim milk powder, and the mice were started to measure the time spent in the area where the hides were originally located. Third, the evacuation zone was placed in the tank without skim milk powder, and the time for the mouse to visit the evacuation zone within 60 seconds was measured. The first and second experimental results are shown in comparison with FIG. 8.

As shown in FIG. 8, it can be seen that the control group takes the longest time to find the hide, regardless of whether the hide is hidden or exposed due to skim milk powder.

The third result is shown in FIG. The time the mouse stays in the place where the shelter was.

As shown in FIG. 9, it can be seen that the control group has the shortest time to stay in the place where the escape was. These results indicate that the control group did not remember the place where the shelter was located, and the groups that orally administered fermented soymilk extract with other purple sweet potato were longer in the area where the shelter was located. It is believed to remember the location of the evacuation zone. It can be seen that the purple sweet potato added fermented soy milk has a protective effect against memory damage.

Example 6-4 Inhibition of Lipid Peroxidation and NO Production in Experimental Animals

 ① TBARS measurement

After homogenizing the liver, brain and kidney tissues with physiological saline, mix the tissue homogenate with 1% phosphoric acid and 0.67% thiobarbubituric acid (TBA) solution. After boiling this mixture for 45 minutes, it is cooled in ice and 2 ml of 1-butanol is added to the mixture, followed by centrifugation at 3000 rpm for 10 minutes. Supernatants were measured at 535 nm and 520 nm absorbance.

The degree of inhibition of lipid peroxidation in each tissue is shown in Table 4 below.

Normal Control 100mg / kg / day 200mg / kg / day Liver 19.15 ± 1.25 ^ab 28.33 ± 8.14 ^a 19.83 ± 1.89 ^ab 14.17 ± 1.51 ^b Brain 11.70 ± 0.73 ^b 36.90 ± 3.61 ^a 11.81 ± 0.43 ^b 9.56 ± 1.13 ^b Kidney 5.39 ± 0.66 ^b 9.74 ± 0.93 ^a 9.32 ± 0.46 ^a 5.88 ± 0.64 ^b

(Unit: nmole / mg protein)

Normal: Normal group not treated with Amyloid-β _25-35

Control: Amyloid-β _25-35 treatment group

100mg / kg / day: Oral administration of fermented soymilk with purple sweet potato after Amyloid-β _25-35 treatment

200mg / kg / day: Fermented soymilk added with purple sweet potato after Amyloid-β _25-35 treatment 200mg / kg / day oral administration group

Values are mean ± SD

^ab Means with the different letters are significantly different (p <0.05) by Ducan's multiple range test.

As can be seen from Table 4, the liver, brain, and kidneys of the experimental animals were extracted to measure the amount of lipid peroxides produced. As a result, the level of lipid peroxidation of each organ was low in mice orally administered with the extract of fermented soymilk containing purple sweet potato hot water extract. You can see that. From these results, it was confirmed that purple sweet potato added fermented soymilk has a protective effect on lipid peroxidation.

 ② NO production inhibition measurement

After homogenizing the tissue with physiological saline, 150 μL of the tissue supernatant obtained by centrifugation at 3000 rpm for 10 minutes and 130 μL of secondary distilled water are mixed. Then, 5% phosphoric acid solution containing 1% sulfanilic acid and 0.1% N- (1-naphthyl) ethlene-dianide dihydrochloride solution were added in a ratio of 1: 1 (v / v) immediately before use and 20 μL was added. After standing at room temperature for 30 minutes, it was measured at 540 nm absorbance.

The degree of NO production inhibition of each tissue is shown in Table 5 below.

Normal Control 100mg / kg / day 200mg / kg / day Liver 79.51 ± 4.31 ^b 93.23 ± 1.16 ^a 84.25 ± 3.07 ^b 71.73 ± 4.38 ^b Brain 23.49 ± 1.49 ^b 29.95 ± 1.51 ^a 23.18 ± 1.18 ^b 14.81 ± 0.8 ^b Kidney 33.3 ± 2.56 ^ab 35.5 ± 2.05 ^a 29.68 ± 6.59 ^ab 27.17 ± 3.4 ^b

(Unit: μmol / L / mg protein)

Normal: Normal group not treated with Amyloid-β _25-35

Control: Amyloid-β _25-35 treatment group

100mg / kg / day: Oral administration of fermented soymilk with purple sweet potato after Amyloid-β _25-35 treatment

200mg / kg / day: Fermented soymilk added with purple sweet potato after Amyloid-β _25-35 treatment 200mg / kg / day oral administration group

Values are mean ± SD

^ab Means with the different letters are significantly different (p <0.05) by Ducan's multiple range test.

As shown in Table 5, between the experimental animal, the brain, kidneys removed by only one processing the Amyloid-β _25-35 results in production of purple sweet potato extract, fermented soy milk for oral administration the mice were measured for degree of generated NO It is generated less than the control group, and the result is similar to that of the normal group. In the brain, the mice fed oral administration of fermented soymilk extract with purple sweet potato 200mg / kg / day showed less NO production than the normal group. From these results, it can be seen that the purple sweet potato added fermented soybean milk showed a suppression of NO production.

Korea Microorganism Conservation Center (overseas) KCCM42923 20090326

Claims (10)

(1) pretreating soymilk;
(2) inoculating Bacillus subtilis bacteria with soymilk for fermentation; And
(3) adding a purple sweet potato extract;
Containing, a method of producing fermented soy milk for improving memory and cognitive ability. The method of claim 1,
Soy milk in step (1)
(a) soybean called water;
(b) grinding by adding 7 to 11 times hot water of soybean weight; And
(c) extracting the soy crushed material by heat treatment and then filtration;
Fermented soymilk production method characterized in that obtained by the method for producing soymilk comprising a. The method of claim 1,
The pretreatment of step (1) is a fermented soymilk production method, characterized in that the heat treatment for 5 minutes to 60 minutes at 121 ℃. The method of claim 1,
The soymilk of step (2) is a method for producing fermented soymilk, characterized in that 0.5 to 2% by weight of sugar is added before inoculation of Bacillus subtilis bacteria. The method of claim 1,
In step (2), the Bacillus subtilis bacteria production method characterized in that the accession number KCCM 42923. The method of claim 1,
The method of producing fermented soy milk, characterized in that the number of Bacillus subtilis bacteria inoculated in step (2) is 6 to 9 logCFU / ml. The method of claim 1,
The soymilk inoculated with Bacillus subtilis bacteria in step (2) was fermented so as to reach pH 4.5 to 6.5 at 30 to 45 ℃. The method of claim 1,
The content of the purple sweet potato extract in step (3) is 1 to 10% by weight of the total fermented soymilk content manufacturing method of fermented soy milk. The method of claim 1,
The concentration of the purple sweet potato extract in step (3) is 60 to 80 ^o Brix production method, characterized in that the Brix. Fermented soy milk for memory improvement and cognitive ability improvement prepared by the method of any one of claims 1 to 9.

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