TWI458438B - Fermentation and culture methods, plant fermentation extracts, plant fermentation extract powders, and plant fermentation extract complexes - Google Patents

Description

Fermentation and culture method, plant fermentation extract, plant fermentation extract powder, and plant fermentation extract complex

The present invention relates to the addition of mammals (specifically livestock, pets, etc.), birds (specifically, raising chickens, bird watching, etc.), biogenics, reptiles, fish (specifically aquaculture fish, pet fish). , etc., invertebrates and pharmaceuticals, animal medicines, pharmaceutical products, cosmetics, foods, functional foods, feeds and baths, etc., can also be obtained by fermentation and culture methods for safe and immunogenic substances, plant fermentation extraction a method for producing a substance, a plant fermentation extract containing an immunostimulating substance obtained by fermentation and a culture method, an immunostimulating substance obtained by fermenting the plant extract, a powder, and a plant fermentation extract complex prepared by the plant fermentation extract .

For mammals containing humans (specifically livestock, pets, etc.), birds (specifically for raising chickens, bird watching, etc.), biogenics, reptiles, fish (specifically aquaculture fish, pet fish, etc.), invertebrates, Establishing a disease prevention and treatment system with infection control technology is an important issue. In order to achieve this, it is required to use a method that does not require the use of chemicals, environmental pollution, no resistance to bacteria, and does not accumulate in the human body. In the above-mentioned problem, the present inventors have found that a plant-derived immunostimulating substance such as a wheat water extract can safely achieve disease prevention and treatment effects (Patent Document 1 and Patent Document 2). Further, in order to achieve the above object, it has been found that a low molecular weight lipopolysaccharide obtained from a rice pathogenic bacterium (Pantoea agglomerans) can be used (Non-Patent Document 2). On the other hand, it has been known from recent studies that various substances other than lipopolysaccharide can exhibit an immunostimulating effect, and natural material materials containing these plural immunostimulating substances are attracting attention.

However, fermentation techniques using microorganisms are not only used in the food field, but are also widely used in other fields. For example, the production of wines based on wine, the manufacture of soy sauce or miso, the manufacture of fermented dairy products such as cheese, the manufacture of pharmaceuticals, and the like. The range of microorganisms used in these fermentations is extremely wide, and it is mainly composed of koji (fungi) yeast, lactic acid bacteria, etc., but there is almost no report of the use of gram-negative bacteria. In general, fermentation is a phenomenon in which an organic substance is decomposed by a microorganism, and is broadly produced by a useful substance of a microorganism (Non-Patent Document 3). As a fermentation technique using a microorganism, a representative wine is mentioned. The wine is a fermentation technique using a wine yeast attached to the grape skin, and the product is an alcohol. Further, in the fermentation technique using microorganisms, those using the Gram-negative bacteria include methane fermentation using methanogens, acetic acid fermentation using acetic acid bacteria, and ethanol from the rhizome of agave using Zymomonas mobilis. Fermentation (made of tequila), etc., but the use of edible plants as raw materials, and the use of microorganisms characterized by special symbiosis with the plants for fermentation culture is unknown, and the immunostimulating substances as fermentation products have not attracted attention.

On the other hand, when fermenting by microorganisms, it is necessary to have nutritional conditions that satisfy the fermentation substrate at the time of general microbial growth. In other words, as a carbon source, it is necessary to use a nutrient which is sufficient as a microorganism such as a monosaccharide such as glucose or fructose. Therefore, a fruit such as a grape which originally contains a large amount of fructose can be used as a fermentation substrate without any processing steps, and others must be subjected to a pre-fermentation stage of microorganisms by heating or enzyme treatment. For example, the aforementioned Zymomonas mobilis is a microorganism used in the manufacture of tequila, and the polysaccharide obtained from the rhizome of agave of a non-edible plant is decomposed by fermenting the fermentable monosaccharide. A wine obtained by fermentation of the microorganism to obtain a fermentation product. Therefore, when fermentation is carried out using a general microorganism, a polysaccharide such as starch is not a suitable fermentation substrate. For example, it has been reported that rice pathogenic bacteria (Pantoea agglomerans) cannot decompose starch (Non-Patent Document 4).

The present inventors have established that an aqueous extract of wheat flour contains an active ingredient capable of activating immunity (Non-Patent Document 5). Further, the cereal (wheat, rice), seaweed (kelp bud, kelp, yam, seaweed, etc.), and beans (soybean, red bean) already contain an active ingredient (Non-Patent Document 6). As a biological activity, it has preventive effects such as diseases of humans or mice (diabetes, hyperlipidemia, atopic dermatitis, cancer), and thus can effectively prevent infection of fish, crustaceans, and birds (Patent Document 1 Non-patent document 1). However, in order to achieve the above-mentioned effects, the wheat flour water extract must ingest a large amount of wheat flour.

On the other hand, the rice bacterium (Pantoe agglomerans) is a bacterium that is symbiotic with wheat, and is useful for cultivating wheat by supplying phosphorus and nitrogen to wheat (Non-Patent Document 7). In addition, it is known in Europe that Pantoea agglomerans is not only attached to wheat, but also to the epidermis of fruits such as pears or apples, and when the bacteria adheres, it can prevent rot caused by mold, so the bacteria are used for development. A fungicide which is good for the natural environment without toxicity has been carried out (Non-Patent Document 8). Moreover, the so-called symbiosis is a phenomenon in which the living creatures of different species are generally living together. At this time, the actions or physiology of each other are closely related. Therefore, not only the concept of living in the same place, but also the meaning of the life of the symbiotic. And the necessity, the continuity of the relationship, the spatial positional relationship of the symbiotics, etc., the symbiosis can be divided into various types and differences. Generally, the symbiotic life is divided into the interests of the symbiotic, and the symbiosis is divided into There are three major categories of symbiosis, symbiosis, and parasitism (Non-Patent Document 9). In the case of the rice phytopathogenic bacteria (Pantoe agglomerans), it is known that it can be isolated from any type of wheat in any place (Non-Patent Document 5) or separated from fruits (Non-Patent Documents 10 and 11). The rice pathogenic bacteria (Pantoea agglomerans) can produce antibiotics (Non-Patent Documents 12 and 13), and plants are protected by fungi or other bacteria, and phosphorus and nitrogen are fixed (Non-Patent Document 7). Therefore, the rice pathogenic bacteria (Pantoea agglomerans) often lie on plants and serve as a beneficial role for plants. Sometimes this is not called "symbiosis" and is called "parasitic". Further, the present inventors have known that rice pathogenic bacteria contain an active ingredient capable of activating immunity. In addition, the low molecular weight lipopolysaccharide obtained from the bacterium has preventive effects against various diseases of humans or mice (diabetes, hyperlipidemia, atopic dermatitis, cancer), and infection prevention for fish, crustaceans, and birds. Effective (Patent Document 3, Non-Patent Document 2).

Under the circumstances, the inventors of the present invention have established a method for producing a plant fermentation extract using rice pathogenic bacteria as a method for producing a safe and inexpensive immunostimulating substance. In other words, (1) the main component of the protein contained in the culture solution is replaced by a plant-derived medium, the rice pathogenic bacteria are cultured at a low cost, and the plant components are fermented, and (2) the plant contains a large amount of plants. Raw materials for rice pathogenic bacteria or fermentation products, using this to develop human-containing mammals (specifically livestock, pets, etc.), birds (specifically for raising chickens, bird watching, etc.), biogenic, reptilian, and fish (specifically Aquaculture fish, pet fish, etc., invertebrates and pharmaceuticals, animal medicines, pharmaceutical products, cosmetics, functional foods, food, feed and bath agents have attracted attention. However, it does not mean that a microorganism which is symbiotic to a plant is a plant component such as a raw material derived from a edible plant, and can be utilized as a fermentation substrate. For example, wheat flour is a complex organic substance such as starch which is present in wheat granules, but the rice pathogenic bacterium of wheat symbiotic microorganism is not directly in contact with the outer skin, so whether the rice pathogenic bacterium can be used for fermentation of wheat flour is not a single It can be understood from the relationship between microorganisms and wheat symbiosis. In fact, it is unknown that rice pathogenic bacteria can be used to chemically wheat flour, and there is no relevant report. Conversely, based on the well-known facts to date, rice pathogenic bacteria cannot utilize wheat starch as a fermentation substrate.

Therefore, the saccharide contained in the plant is kept in a state of starch, which is particularly remarkable in the case of edible plants, particularly cereals. Generally, microorganisms do not have high starch utilization functions. For this known part of the universal Gram-negative bacteria fermentable starch. For example, Erwinia is known to be a starch. This fermentation technique is a microorganism in which a large amount of culture is carried out by adding a suitable medium in the case of fermenting starch, which is an amylase activity which is utilized by a microorganism, but a form in which starch is directly used for culture and fermented at the same time is unimaginable. The known technique is a fermentation for the purpose of effectively utilizing the amylase activity of the microorganism, but it is not conceivable that the starch can be used as a matrix to proliferate the microorganism. On the other hand, in the examples of the present invention, since the starch is used as the sole carbon source, the fermentation product is produced in addition to the proliferation of the microorganisms. The present embodiment is not merely a simple fermentation, and the disclosed fermentation culture is also quite large with the known technology. The difference.

On the other hand, on the contrary, some microorganisms maintain the function of decomposing starch, and do not mean that the microorganism proliferates directly with starch as a substrate. When the growth of microorganisms during culture is also for the purpose, the amount of microorganisms added at the beginning of the culture is extremely small, and at this time, even if the microorganism has a small amount of amylase activity, the activity is too weak to sufficiently decompose the matrix, and the microorganism cannot be proliferated. . In fact, most microorganisms cannot proliferate with starch as the sole source of carbon.

However, when rice pathogenic bacteria are used for fermentation and culture in a medium containing wheat flour as a main component, or a plant fermented extract rich in immunostimulating substances can be produced at a low cost (hereinafter, rice pathogenic bacteria are used to contain wheat) The plant fermentation extract obtained by fermentation and culture in a medium containing powder as a main component is called a wheat fermentation extract), and can be provided, for example, in humans, in the field of livestock and aquaculture, and in a safe and safe way to prevent infection. , animal drugs, pharmaceutical products, cosmetics, food, functional foods, feed and bath agents. In the above background, the present inventors have found that rice pathogenic bacteria can be grown under wheat flour as a substrate, and various repeated experiments are carried out to complete the present invention.

The plant fermentation extract provided by the present invention is a general term for a liquid composition obtained by fermentation and culture, a liquid component obtained by solid-liquid separation, and a liquid component obtained by extraction after solid-liquid separation. . That is, the plant fermentation extract is a culture liquid obtained by the fermentation and culture method of the present invention, and all the extracts which can be prepared by using all or a part of the culture liquid using the same. As a matter of course, the plant fermentation extract can be dried and used as a plant fermentation extract powder, or the plant fermentation extract powder can be dissolved in a suitable solution of any concentration, for example, a phosphate buffer containing physiological saline.

Patent Document 1: JP-A-3-218466

Patent Document 2: Japanese Patent Publication No. 8-198902

Patent Document 3: WO 00/57719

Patent Document 4: Japanese Laid-Open Patent Publication No. Hei 6-78756

Patent Document 5: Japanese Patent Publication No. 4-187640

Patent Document 6: Japanese Patent Publication No. 4-49240

Patent Document 7: Japanese Patent Publication No. 4-99481

Patent Document 8: Japanese Patent Publication No. 5-155778

Non-Patent Document 1: Inagawa Yuki Other 8 people, 〝Biotherapy〞, 1991, Vol. 5, No. 4, p617-621

Non-Patent Document 2: Soma, G-I. The other one, 〝Tumor Necrosis Factor: Molecular and Cellular Biology and Clinical Revenvance〞, 1993, p203-220

Non-Patent Document 3: Yamada Toshio Other 6th, 〝Biology Book 3rd Edition, 1983, p1021

Non-Patent Document 4: Gavini, F. Other 6th, 〝Int. J. Syst. Bacteriol.〞, 1989, Vol. 39, p337-345

Non-Patent Document 5: Nishizawa, T. Other 7th, 〝Chem. Pharm. Bull.〞, 1992, Vol. 40, No. 2, p479-483

Non-Patent Document 6: Inagawa, H. Other 8th, 〝Chem. Pharm. Bull.〞, 1992, Vol. 40, No. 4, p994-997

Non-Patent Document 7: Neilson, A. H. 〝 J. Appl. Bacteriol. 〞, 1979, Vol. 46, No. 3, p483-491

Non-Patent Document 8: Nunes, C. Other 3, 〝Int. J. Food Microbiol.〞, 2001, Vol. 70, 1, 2 merger number, p53-61

Non-Patent Document 9: Other 6 of Yamada's Chang Xiong, 3rd Edition of the Biology Book, 1983, p287-288

Non-Patent Document 10: Nunes, C. Other 4, 〝J. Appl. Microbiol.〞, 2002, Vol. 92, No. 2, p247-255

Non-Patent Document 11: Asis, C. A. Jr. 1 other, 〝Lett. Appl. Microbiol. 〞 2004, Vol. 38, No. 1, p19-23

Non-Patent Document 12: Vanneste, J. L. Other 3, 〝J. Bacteriol. 〞 1992, Vol. 174, No. 9, p2785-2796

Non-Patent Document 13: Kearns, L. P. 1 other, 〝Appl Environ Microbiol.〞 1998, Vol. 64, No. 5, p1837-1844

As described above, the immunostimulating substance is often a constituent component or a product of a microorganism contained in the plant itself or symbiotic with the plant. Therefore, in order to obtain an ingestible and safe immunostimulating substance derived from a natural product, the component can be extracted from the edible plant itself (for example, limulus-positive glycolipid, Patent Document 1) or efficiently cultured in a symbiotic microorganism of the edible plant to obtain a constituent component. Or a product (for example, a low molecular weight lipopolysaccharide, Patent Document 2). However, the content of the immunostimulating substance contained in the edible plant is small, and if it is desired to ingest a very large amount of food by the immune activating effect after eating, or if it is not easy to maintain an appropriate amount of the immune activating substance, the effect cannot be achieved. . Moreover, it is necessary to use high cost and lack of practicality when using plant extract as a food or a medicament.

On the other hand, microorganisms that are symbiotic with plants, such as wheat symbiotic bacteria, Pantoea agglomerans contain a low molecular weight lipopolysaccharide component constituting an active ingredient for immunization. However, to date, for the extraction of low molecular weight lipopolysaccharide, it is necessary to use the protein contained in the culture solution as the main component for the animal, for example, it is necessary to use the high-priced culture medium such as NZ amine or trypsin or casein amino acid for the rice pathogenic bacteria. Cultivation. Therefore, it is difficult to provide a highly versatile immunostimulating substance at a low price, and it is unavoidable to simultaneously mix unknown harmful substances from other animals of BSE.

In view of the above problems, the present invention provides a fermentation and culture method for obtaining an immunostimulating substance efficiently and inexpensively using a safe raw material, and the plant fermentation extract obtained by the method, the plant fermentation obtained by the plant fermentation extract The extract powder and the plant fermentation extract complex to which the plant fermentation extract powder is added are for the purpose.

The fermentation and culture method of the present invention is characterized in that a raw material derived from an edible plant is fermented by a facultative anaerobic Gram-negative bacterium which is symbiotic in a specific plant, and the facultative anaerobic Gram-negative bacterium is cultured as a bacterium. By.

Further, the starch as a carbon source can be fermented and cultured through a simple process by fermentation of the facultative anaerobic Gram-negative bacteria.

The facultative anaerobic Gram-negative bacteria are preferably facultative anaerobic bacilli.

The facultative anaerobic Gram-negative bacteria is preferably an enterobacteriaceae.

Further, the facultative anaerobic bacterium is preferably of the genus Pantoea, serratia, or enterbacter.

Further, the facultative anaerobic bacterium is a rice pathogenic bacterium (Pantoea agglomerans), and the carbon source may be starch.

Further, the edible plant is preferably cereal, seaweed, or beans, or a mixture thereof.

Further, the raw material of the cereal is wheat flour, rice flour, wheat bran flour, rice bran, or wine cellar, and in particular, the wheat flour contains gluten as a protein source, so that fermentation can be carried out efficiently without using raw materials derived from animals. And training.

Further, the raw material derived from the seaweed is preferably kelp bud powder, cloth crepe or kelp powder.

Further, since the raw material derived from the beans is tofu, it contains a large amount of protein and does not require raw materials derived from animals, and can be efficiently fermented and cultured without using raw materials derived from animals.

Further, the plant fermentation extract of the present invention is characterized by being obtained by the above fermentation and culture method.

Further, the plant fermentation extract powder of the present invention is characterized by being obtained from the above-mentioned plant fermentation extract.

Further, the plant fermentation extract complex of the present invention is characterized in that a plant fermentation extract or a plant fermentation extract powder is added as described above.

Further, the plant fermentation extract complex, wherein the plant fermentation extract complex may be a pharmaceutical, an animal medicine, a pharmaceutical product, a cosmetic, a food, a functional food, a feed, or a body wash.

Further, the plant fermentation extract is preferably a physicochemical property which exhibits macrophage activation energy in the presence of polymyxin B. Further, the aforementioned plant fermentation extract has an immunostimulating activity.

Since the present invention is cultured in a medium which does not contain animal components, it provides a problem that impurities from animal components are not mixed, for example, there is no risk of BSE or unknown harmful substances being mixed, and it is highly safe and inexpensive. Provided in a method for producing a plant fermentation extract, and safely and inexpensively producing an immunostimulating substance comprising a plant fermentation extract or a plant fermentation extract, and the culture liquid, the immunostimulating substance, the extract and the extract powder, and more A pharmaceutical product, an animal medical product, a pharmaceutical external product, a cosmetic, a food, a functional food, a feed, a bathing agent, and the like which are added with the extract or the extract powder are provided.

The raw material from the edible plant is fermented by a facultative anaerobic Gram-negative bacterium that is symbiotic with a specific plant, and the fermentation and culture technique of the facultative anaerobic Gram-negative bacteria can be cultured. It is thought that the routine of fermentation technology up to now cannot be easily imagined.

Further, as an indicator of the immunostimulating effect of the substance, a method of whether or not TNF (TNF-derived activity) is produced by macrophages can be used. And the immunostimulating effect can be quantified by the amount of TNF produced. Among them, limulus-positive plant glycolipids derived from wheat and low-molecular-weight lipopolysaccharides derived from rice pathogenic bacteria are used to review TNF production from macrophages, limulus-positive plant glycolipids from wheat, and low molecular weight lipids from rice-pathogenic bacteria. When the polysaccharide is treated with polymyxin B, TNF is not produced by macrophages, but it is shown by the examples of the present invention that the plant fermentation extract of the present invention produces a large amount of TNF from macrophages when subjected to the above treatment. This is a plant fermentation extract of the present invention obtained as a result of fermentation culture, and has an effect of having an immunostimulating effect different from that of a plant itself which is a raw material thereof and a constituent component of the microorganism itself used for fermentation. .

The best form of implementing the invention

The preferred embodiments of the present invention are described in detail below.

I: Focus on the manufacturing method of plant fermentation extract using rice pathogenic bacteria (Pantoea agglomerans)

In the present invention, the present inventors first discovered that rice pathogenic bacteria can grow starch as a direct carbon source, and invented a method of fermenting using rice pathogenic bacteria and inexpensively producing a wheat fermentation extract rich in an immunostimulating substance as a culture product. As a specific example, it is an effective pharmaceutical product, cosmetics, food, functional food, and feed that can provide an environment-friendly and safe preventable infection in the fields of humans, livestock, and aquaculture.

1: Separation of rice pathogenic bacteria

The wheat flour was suspended in water, and the clear liquid was applied to the L-broth canola medium for cultivation, and microbial colonies appeared. These colonies were identified by microorganisms in a certain way. For example, colonies which are negative for Gram stain, positive for glucose anaerobic metabolism, and negative for oxidase activity are selected, and those having the same properties as standard rice pathogenic bacteria are selected using ID test ‧ EB-20 (Nissui Pharmaceutical Co., Ltd.). The rice pathogenic bacteria which are standard can be obtained by the Institute of Physical Chemistry, Bio-Based Research, Non-Microbial System Preservation Facility (Non-Patent Document 4). In the following description, the percentage indicates the weight unless otherwise specified.

2: Evaluation of immune activating activity

In the form of the present embodiment, as an indicator of the immunostimulating action of the wheat fermentation extract, activation of macrophages can be evaluated by TNF production by macrophages.

3: Low molecular weight lipopolysaccharide from rice pathogenic bacteria

Further, by using fermentation and culture of rice pathogenic bacteria, it is expected to contain a low molecular weight lipopolysaccharide derived from a rice pathogenic bacterium which is one of active ingredients for immunization. The low molecular weight lipopolysaccharide is extremely safe compared to the widely used high molecular weight lipopolysaccharide (hereinafter referred to as lipopolysaccharide), and its biological activity is superior to that of the general lipopolysaccharide. Therefore, the low molecular weight lipopolysaccharide content was determined. The low molecular weight lipopolysaccharide has been described in detail in Patent Document 2. Further, although this example relates to wheat fermented extracts, the present invention does not limit plants to wheat, and the immunostimulating substance is limited to low molecular weight lipopolysaccharides.

Therefore, the rice pathogenic bacteria can be cultured by a known method (Patent Document 2 and Non-Patent Document 8). However, it is known that the main component of the protein contained in the culture solution is derived from an animal, and the cost of the culture medium is relatively high. Moreover, when an animal is administered, for example, a functional food or a functional feed, or is administered transdermally, there is a problem of food safety caused by the incorporation of impurities from the animal such as BSE, and the manufacturing cost is higher, and from the viewpoint of practicality Not a good way. Therefore, the present inventors intend to obtain a natural product having a safe and inexpensive immunostimulating effect, and carry out detailed research results, and the wheat fermentation extract is obtained by performing fermentation and culture methods using rice pathogenic bacteria as shown in the examples. The main component of the protein contained in the culture solution is used in the past for the animal, and the present invention uses the planter. A product of a protein such as casein derived from milk which is decomposed by digestive enzymes is added to the culture liquid. At this time, the original price of the 1 L medium was about 250 yen, but when the wheat flour was replaced, the original price was about 16 yen. Up to now, there has been no fermentation for the purpose of multiplying the fusion activity of plants and their symbiotic microorganisms at a high concentration.

Hereinafter, the contents of the present invention will be described in detail by way of examples. However, the microorganisms described in the examples of the present invention are not limited to rice pathogenic bacteria, and wheat or raw materials used as edible plants are not limited to wheat flour, and may be used in large amounts. Other edible plants that immunize the active material, the raw materials obtained through the general steps, for example, can be applied to kelp buds, cereals (the raw materials from cereals contain wheat flour, rice flour, wheat bran flour, rice bran, or wine cellar, etc.), seaweed (from seaweed The raw material contains kelp bud powder, and cloth crepe or kelp powder), beans (the raw materials from the beans contain bean curd), and the like. These plants are known to contain proteins and sugars and are suitable for fermentation and culture using rice pathogenic bacteria. Further, it is known that these plants are generally symbiotic with a common bacterium, for example, serratia or enter bacteria (Non-Patent Document 4), and microorganisms used for fermentation can of course be used. These plants are symbiotic facultative anaerobic Gram-negative bacteria.

II: The focus of comprehensive invention

(1) The wheat fermentation extract of wheat, the commensal bacteria of the commensal bacteria, and the substance having the immunostimulating action in which the fermentation products are combined is a novel, but the present invention is not limited thereto.

(2) The production of plant fermentation extracts using rice pathogenic bacteria of Gram-negative bacteria is a new technique. However, the invention is not limited thereto.

III: Specific manufacturing method of wheat fermentation extract

(1) Rice pathogenic bacteria are isolated from wheat flour according to a general method (Non-Patent Document 1). Moreover, the strain can be stored in 50% glycerol or the like after isolation and identification only once.

(2) Preparing 0.05 to 5% of salt, 0.005 to 1 mol of phosphate buffer, or mixed salt solution (0.5 to 10% of second sodium phosphate, 0.05 to 5% of potassium phosphate, 0.05 to 0.05) 5% sodium chloride, 0.05 to 5% ammonium chloride).

(3) The wheat flour is suspended in water to a concentration of 0.05 to 10%.

(4) A 0.2 to 3 molar magnesium chloride solution was prepared.

(5) A 0.2 to 3 molar potassium chloride solution was prepared.

(6) 2 to 5 The sterilization operation of the autoclave can be carried out according to the requirements.

(7) 2 to 5 are mixed in an appropriate amount, and after adding water, a suspension containing 0.1 to 5% of wheat flour is obtained. The pH becomes neutral depending on the desired addition of an alkaline solution or an acidic solution.

(8) In the case of 7, 10 to 50,000 units of amylase may be added per 1 L of the medium, and the mixture may be incubated at 10 to 80 ° C for 1 to 24 hours to digest part of the wheat starch.

(9) The steps of 7 to 8 are added to the rice pathogenic bacteria isolated in 1.

(10) 9 Fermentation is carried out at 1 to 40 °C. It can be placed or shaken depending on the situation. Further, it is also possible to carry out stirring for several hours.

(11) Fermenting 10 for 6 hours to one week. The aqueous solution of the wheat flour under fermentation is colored yellow.

An appropriate alkali solution may be added during the fermentation of (12)11 to make the pH neutral, or a wheat flour suspension or an inorganic salt may be added.

(13) After the completion of the fermentation, the solid content of the precipitate is recovered by centrifugation (1000 to 5000 rpm, 10 to 60 minutes). The precipitate is used as a fermented wheat flour and can be used directly as a feed or as a raw material mixed with feed.

(14) Production of Wheat Fermentation Extraction is practicable, and 13 is suspended in water or a salt buffer or the like, and this is heat-treated at 80 to 140 ° C for 10 minutes to 6 hours. The solid component can be removed by centrifugation or filtration. The removed precipitate was again added with water or a buffer for several repeated heat extractions.

(15) A simple purification step can be carried out depending on the use of the 14 wheat fermentation extracts produced. In other words, a salt such as sodium chloride is added to the extract of 14 to have a final concentration of 0.05 to 1 mol/L, and then a solvent such as ethanol is added in an amount of 1 to 3 times the amount of the extract to precipitate. This can be recovered using a centrifugal separator or the like. This precipitate can be washed again with a solvent such as ethanol. This can be dried to make a powder.

A. Example of a method for producing a wheat fermentation extract Example 1 Growth test in rice medium of rice pathogenic bacteria

To determine whether wheat flour is often propagated in the rice commensal bacteria of the symbiotic bacteria when wheat flour is used as a carbon source, the growth of rice pathogenic bacteria in wheat flour solid medium is investigated.

(1) An M9 agar medium containing 0.5% wheat flour as a carbon source was prepared.

(2) One colony of the rice pathogenic bacteria was taken out in 1 ml of PBS via LB agar medium. This was further diluted with PBS from 10 to 10,000 times, and each 0.1 ml was inoculated into 1 M9 agar medium.

(3) After 6 days of culture at 37 ° C, the appearance of colonies was observed. As a result, about 300 colonies were observed in a 10,000-fold diluted 0.1 ml seeded petri dish.

From this, it was confirmed that rice pathogenic bacteria can utilize wheat as a carbon source.

Example 2 Manufacture of wheat fermentation extract

(1) 0.5 g of wheat flour was suspended by adding 5 ml of distilled water, and 0.1 ml of a clarified liquid was added to L-broth agar medium, and cultured overnight at 37 °C.

(2) The yellow colonies were separated, and the bacteria were identified by a general method, and the rice pathogenic bacteria were isolated, suspended in a 50% glycerin solution, and stored in a freezer. A portion of this preservation solution was applied to LB agar medium, and placed at 37 ° C to form an independent colony of rice pathogenic bacteria.

(3) Add 2 g of triangular sodium phosphate ‧7 crystal water, 15 g of first potassium phosphate, 2.5 g of sodium chloride, 5 g of ammonium chloride, and add pure water to make a full amount 1L (inorganic salt mixed solution); take 13.1 g of magnesium chloride ‧ 2 crystal water, add pure water to make the whole amount into 100 mL (calcium chloride solution); put 4L of pure water into a 5L triangular conical flask ( Pure water). The above solution and pure water were placed in an autoclave (TOMY BS-325, 120 ° C, 20 minutes) for sterilization.

(4) Put 24 g of wheat flour (Nisshin powder) in a 1 L triangular conical flask, and add pure water to make the whole amount 600 ml. After the same autoclave sterilization, 3 mg of α-amylase (SIGMA, Bacillus, 1500-3000 units of enzyme activity in 1 mg protein) was added, and heated in a water bath at 65 ° C for 4 to 12 hours (wheat starch amylase). Treatment solution).

(5) Each of the adjusted solutions as shown in Table 1 was placed in a sterilized 3 L Sakaguchi Erlenmeyer flask as a wheat flour medium.

(6) Modulation of inoculum. In a 10 ml wheat flour medium prepared in the same composition as above, one rice bacterial colony isolated from wheat flour was placed in 2, and slowly stirred at 37 ° C for 1 night (12 to 15 hours). The fermentation is carried out to prepare an inoculum for fermentation of wheat flour.

(7) A total amount of 6 is added to 5 and stirred at 37 ° C while fermenting for 20 to 30 hours. The pH of the fermentation broth was measured, and the pH was adjusted to 7 after the addition of ammonia water. Under this sterility, 150 ml of a mixture of wheat starch amylase treatment solution and inorganic salt mixed solution of 37.5 ml was added, and the same fermentation was carried out for 20 to 30 hours. The same operation was repeated for one time and the total fermentation time was 65 to 80 hours.

The wheat flour fermentation solution of (8) 7 was centrifuged (Hitachi, high-speed cooling centrifuge SCR-20B, 5000 rpm, 20 minutes, 4 ° C), and the precipitate was recovered.

Phosphate buffer was added to the precipitate of (9) 8 to make it a suspension of 100 ml in total. 33 ml was taken out and transferred to a 50 ml centrifuge tube, and subjected to hot extraction in a boiling water bath for 30 minutes. After the end of the heating, the mixture was cooled to room temperature, and the liquid was centrifuged (Hitachi, high-speed cooling centrifuge SCR-20B, 10000 rpm, 20 minutes, 20 ° C). After centrifugation, 82 ml of the pale yellow clear liquid was decanted and recovered into another container.

8.9 ml of a 5 molar sodium chloride solution was added to 80 ml of the clear solution of (10) 9, and 178 ml of ethanol was added thereto to make it cloudy. After leaving this in a freezer (-90 ° C) for one night, the liquid was centrifuged (Hitachi, high-speed cooling centrifuge SCR-20B, 10000 rpm, 20 minutes, 4 ° C), and the clear liquid was removed to obtain a precipitate. After the precipitate was suspended by adding 10 ml of 70% ethanol, the liquid was centrifuged (Hitachi, high-speed cooling centrifuge SCR-20B, 1000 rpm, 20 minutes, 20 ° C), and the precipitate was washed. The precipitate was air-dried and dissolved in distilled water to obtain 11 ml of a wheat fermentation extract solution.

(11) Determination of dry weight: 0.3 ml was weighed beforehand and transferred to 1.5 ml of plastic test tube. After freezing, freeze-drying was carried out in a freeze dryer to obtain 7.45 mg. Therefore, the dry weight of the wheat fermentation extract of 10 is 24.8 mg in a 1 ml solution and 273 mg in a total amount of 11 ml.

(12) The wheat fermentation extracts were independently produced eight times in the same manner, and the protein quality of each sample was determined by using Bradford protein concentration measurement method to quantify protein BSA as a standard protein. As a comparison object, purified limulus-positive glycolipid (Patent Document 1) and low-molecular lipopolysaccharide (Patent Document 2) were used. The measurement results are shown in Table 2. The value of the wheat fermentation extract of Tables 2 to 5 and 7 is the content of mg in 1 g of the obtained wheat fermentation extract obtained by the above-mentioned 10.

(13) Measurement of sugar content: Glucose was measured by phenol sulfuric acid method as a standard sugar. The measurement results are shown in Table 3.

(14) Measurement of nucleic acid content: The 100-fold diluted sample was measured for absorbance at 210 to 340 nm. The maximum absorbance of 50 μg was calculated by subtracting the absorbance at 260 nm absorbance to 320 nm and the absorbance at DNA 1OD. The measurement results are shown in Table 4.

(15) Determination of the content of the limulus active substance measured by limulus: the mass of the limulus active substance is a tooxycolor system using the biochemical industry, and the biochemical industry Et-1 is used as a standard limulus active substance. The measurement results are shown in Table 5.

(16) Iodine-starch reaction: dilute 200 times with water using iodine test reagent 1N (12.7 g of iodine and 25 g of potassium iodide added with 10 ml of water, carefully mixed, and added with water to make it 100 ml) 5 μl was added to 0.1 ml of the wheat fermentation extract previously dissolved at a concentration of 1 mg/ml, and carefully stirred. The wheat fermentation extract is immediately converted from lavender to rich purple (positive). Limulus-positive glycolipids and low-molecular-weight lipopolysaccharides did not have the same color development (negative) when operated under the same conditions. The above results are summarized in Table 6.

According to the above results, the wheat fermentation extract and limulus-positive glycolipid, low-molecular-weight lipopolysaccharide in protein content, Tang content, nucleic acid content (limulus-positive glycolipid has no data removed), limulus active substance content, iodine-starch reaction The dissimilarity is shown in all items, and the present invention is a novel substance. The above results are simplified and summarized as shown in Table 7. That is, the plant fermentation extract of the present example is novel in that it differs from the limulus-positive glycolipid low molecular weight lipopolysaccharide by exhibiting the following physicochemical properties. Containing 5 to 15% protein, 20 to 45% sugar, 10 to 35% nucleic acid, and 10 to 40% limulus positive substance, positive in iodine-starch reaction, in the presence of polymyxin B Shows macrophage activation energy.

Too small: much less than the numerical range (mean ± standard deviation) of the wheat fermentation extract.

Too small: much more than the numerical range (mean ± standard deviation) of the wheat fermentation extract.

Example 3 Immune activation of wheat fermentation extract

THP-1 (1×10 ⁶ cells/250 μl: RPMI 1640 medium containing 10% bovine embryo serum) was used as a human myeloid leukemia cell line in a 48-well culture dish for 30 minutes. Pre-culture. The final concentration of each sample was added to 250 μl (final amount 500 μl) at 1 to 10000 ng/ml. A group containing polymyxin B (12.5 μg/ml) in the sample was prepared. After 4 hours of culture, the culture supernatant and cells were recovered. The TNF activity of the clarified liquid was determined using the L-929 cell disorder test. The results are shown in Table 8. Wheat fermentation extract can also produce TNF from macrophages in the presence of polymyxin B, but low molecular weight lipopolysaccharide and limulus positive glycolipids do not produce TNF from macrophages in the presence of polymyxin B. It is therefore known that wheat fermentation extracts have different biological activities compared to low molecular weight lipopolysaccharides or limulus positive glycolipids.

B. Application examples of wheat fermentation extracts for feed Example 4

Feeding chicken feed with wheat fermentation extract (burst suppression effect of large-scale trial of broiler feeding)

A feed containing 430 μg/kg of the wheat fermentation extract produced in Example 2 was produced. The chickens provided are a group of about 5,500 to 6,000 brailer commercial chickens. The control group was a feed that did not contain wheat fermentation extract. For the 3 weeks old after hatching, the feed containing the wheat fermentation extract was administered daily to 7 weeks of age. The number of deaths was measured daily. Dispose of chickens that do not meet the benchmark. The results are shown in Table 9. The removal rate of the test group (feed containing wheat fermentation extract) was 1.9% lower, and that of the control group was 3.3%. The growth rate was 98.1% in the test group and 96.7% in the control group, which showed an increase of 1.4%. A significant difference between the actual number of exits and the number of removals between the test group and the control group was observed, and it was found that there was a significant difference of P < 0.0001 under the χ ² test. From this, it can be seen that the broiler feeding of the feed containing the wheat fermentation extract shows an infection control effect.

Example 5 Feeding of farmed fish with wheat fermented extract (infection control effect of wild fish in squid)

The infection-preventing effect of the feed which was placed in the wheat fermented extract produced in Example 2 was carried out by feeding a group of about 5,200 squid in the field test. The results are shown in Table 10. Death due to streptococcus reached 4.8% when not administered to the control group. Mortality of wheat fermented extracts in the group of wheat fermented extracts (test group) and non-administered group (control group) at 100 μg/kg/day (body weight 1 kg per day) (P<0.00001) There is a significant reduction.

Example 6 Wheat Fermentation Extract (for infection control effect of Koi herpesvirus disease)

(1) Squid: Black squid weighing 70 g. The test was a group of 20.

(2) Adjustment of koi herpes virus: 10 ml of Hanks' buffered saline solution (HBSS) was added to the squid 1 g of the koi herpes virus infection, homogenized, and a 0.45 micron filter was filtered, and the filtrate was used as a virus solution. .

(3) Koi herpes virus infection: The above filtrate was injected into the abdominal cavity of the squid at 600 μl/100 g body weight.

(4) Feed preparation containing wheat fermentation extract: The purchased feed was prepared by mixing the feed of 0, 5, 10, 20 mg/kg in the wheat fermentation extract produced in Example 2.

(5) Method of feeding the bait: feeding the bait once a day for each feed having a weight of 1% by weight. This is equivalent to 0, 50, 100, 200 μg/kg body weight/day, respectively, when converted to the amount of wheat fermentation extract.

(6) Experiment: After one week of feeding the feed containing the wheat fermentation extract, the virus was infested, and the feed containing the wheat fermentation extract was fed for 10 days. The survival rate of squid was observed 10 days after the virus infection operation. The result is shown in Figure 1.

The carp fed without the added wheat fermentation extract died on the sixth day. On the other hand, in the test group fed with the feed containing wheat fermentation extract, the survival rate was significantly increased on the 10th day of infection (the Kaplan-Meier method, the risk rate under the Logrank test was 0.01% or less). In particular, the group fed at 100 μg/kg body weight/day had a 65% survival rate.

C. Manufacture of hand lotion in wheat fermented extract

As the ointment of the fat-soluble base material 1 described in Table 11, about 10% of the wheat fermentation extract produced in Example 2 was added and mixed to form an ointment.

Example 8 Manufacture of moisturizing cream with wheat fermented extract 1. Formulated with moisturizing cream for wheat fermented extract

The ingredients used are shown in Table 12. The group A was dissolved by heating at 70 ° C, and the group B dissolved in 1/4 amount of pure water was heated and dissolved at 70 ° C, dissolved in 1/4 amount of pure water, and dissolved at 70 ° C by heating. Group C, after homogenization and thorough mixing, cooled to 40 ° C. After adding the D group and adjusting the pH to 6.8, the remaining pure water and the appropriate amount of the wheat fermentation extract prepared in Example 2 were added and thoroughly mixed to obtain an emulsion. And the wheat fermentation extract was previously dissolved in pure water to 5 mg/ml, and 0.1 ml was added to 100 g of the emulsion.

2. The effect of moisturizing cream with wheat fermentation extract

This cream was used by a survey of 43 men and women. The result is 18 for the moisturizing effect, 18 for the less effective answer, 2 for the unresponsive answer, and 5 for the unanswered one (a standard symbol check: P< 0.0001). For the improvement effect of dry skin, there are 6 respondents who have an effective effect, 13 who have a slightly effective answer, 0 who have no effect answer, and 24 who have no answer (a specimen symbol check: P< 0.0001). It is considered to be 0 in the case of deterioration of skin condition after use. In addition, four people with mild ectopic symptoms were investigated with this cream, and for the improvement of atopic dermatitis, the number of respondents who thought it was effective was 3, and the answer with a slight effect was 1 Name (a specimen symbol check: P <0.125). Other respondents who believe that the treatment of acne has early recovery is one. In addition, this cream allowed 9 men to use after shaving and conducted a questionnaire survey. Eight people thought that the pain after shaving was reduced, the effect of roughness was prevented, and the scratch was cured early (a specimen symbol check: P) <0.01). In addition, when two patients with symptoms of fifty shoulders were applied to the shoulders and tried to reduce the pain, the effect was considered to be one.

Moreover, when the cream is used in a fire-injured patient, the fire-injured patient with the same degree of skin on both hands is applied by one hand with the cream containing the wheat fermentation extract prepared in Example 2, and the other hand is the wheat-free fermentation extract. After the application of the cream, the hand coated with the cream of the wheat fermented extract obviously has early recovery. When this was applied to 10 fire-injured patients, there was a significant early recovery of the fire wounds of the cream containing the wheat fermentation extract of any one of the films (Fisher direct probabilistic test: P < 0.0001). From the above, it is known that the wheat fermentation extract has a therapeutic effect on fire injury.

Example 9 Manufacture of lotion for wheat fermented extract 1. Put a lotion prescription for wheat fermented extract

The ingredients used are shown in Table 13. The wheat fermentation extract was a wheat fermentation extract solution prepared in Example 2 in which a concentration of 5 mg/ml was previously dissolved in pure water, and 0.1 ml was added for 100 g of the lotion.

2. Effect of putting on the lotion of wheat fermentation extract

Five women were asked to use the lotion to conduct a questionnaire survey. As a result, it was considered to be 5 in the case of moisturizing, and it was considered to be 2 in general. No skin discomfort occurred.

Example 10 Manufacture of body wash containing wheat fermentation extract

A wheat fermentation extract is added for the purpose of improving the function of the living body to prepare a body wash. The basic components of the body wash are shown in Table 14.

The bathing agent in which the wheat fermentation extract was placed was prepared by adding 110 μg of the wheat fermentation extract produced in Example 2. A questionnaire survey was conducted in a general bathtub (160 to 200 liters) used for bathing in a concealed control group with a mixture of extracts and unexposed extracts (1) (2) the degree of difficulty in cooling the water, (3) the effect of fatigue recovery, (4) the degree of easy sleep, (5) the degree of shoulder pain reduction, (6) the treatment effect of muscle pain, (7) the therapeutic effect of neuralgia, (8) ) Low back pain treatment effect, (9) Debilitating physique treatment effect, (10) Hong Kong foot improvement effect, (11) Skin roughness improvement effect, (12) Heterotopic dermatitis treatment effect). As a result, compared with the control group, it was found that there were more than 7% improvement (1) degree of body heat preservation (10%), (2) degree of difficulty in cooling water (7.9%), and (6) treatment effect of muscle pain (13%). ), (8) low back pain treatment effect (16%), (9) debilitating physique treatment effect (10%), (11) skin roughness improvement effect (7.3%), etc. (Mantel-Haenszel test: P < 0.04). From the above results, it was found that when the wheat fermentation extract was used as a body wash, the pain-relieving effect and the body heat retention were indeed improved.

Example 11 Manufacture of candy into wheat fermented extract

(1) The wheat fermentation extract prepared in Example 2 was added in a ratio of 5:5:5:1 in fine sugar, leeches, and water of the raw material, and mixed, and heated and boiled to 120 to 160 °C.

(2) The obtained one is cooled on a cooling iron plate, and then stretched in a rod shape to form about 1 g of granular sugar.

Place an appropriate amount of this granulated sugar in 20 ml of water and dissolve it by heating. As the active ingredient of the wheat fermentation extract in the solution, the amount of lipopolysaccharide was determined to be 4.6 μg/g. The sugar was given to 6 male and female patients who had a sore throat due to a cold. A questionnaire survey was then conducted directly on the improvement of sore throat. For the improvement of sore throat, 6 people felt that there was a reduction (a specimen symbol check: P < 0.03).

Example 12 Alcohol decomposition function of wheat fermentation extract enhances food manufacturing

In the case of the commercially available product of the alcohol decomposition-enhancing food-enhancing food, the wheat-fermented extract produced in the second embodiment was examined for the effectiveness of the new effect of throat soreness.

Purchased goods: trademark "drinking interest"

The composition is shown in Table 15.

The current "drinking" contains Gynostemma pentaphyllum extract and green tea extract, but the lipopolysaccharide which is one of the active ingredients of the plant extract is only about 0.002 μg in one pack. Therefore, when an appropriate amount of wheat fermentation extract having a high content of lipopolysaccharide is added, a new function can be expected. It is preferable to add 1 to 30 μg of 1 g of lipopolysaccharide, which is one of the active ingredients of the wheat fermentation extract, (5 to 150 μg as a wheat fermentation extract), thereby producing a product containing 50 μg of wheat fermentation extract in one package. . In the manufacturing step of drinking alcohol, 2.5 mg of the wheat fermentation extract prepared in Example 2 was added per 100 g of the product. As a result, a new product containing 50 μg of the wheat fermentation extract per 2 g of the product was produced.

In order to drink karaoke, there are 20 adults and men with sore throat, and each of them takes the original "drinking interest", and the other 10 take "drinking interest" containing wheat fermented extract. The effect of alcohol decomposition and the effect of sore throat on the well-known effect are reviewed. Thereafter, a questionnaire survey was conducted directly on the effect of sore throat relief. As a result, it is considered that the "drinking of the fermented wheat extract" has a reduction effect on the sore throat, and 8 of the 10 people have a reduction effect on the "drinking interest rate". There was a significant difference (Fisher direct probability test: P < 0.012).

E. Pharmacodynamic examples of wheat fermentation extracts Example 13 Manufacture of glycerin solution for wheat fermentation extract (therapeutic effect on atopic dermatitis)

For the face, hands, feet, body, neck, wrist, back and other rashes, consciously symptomatic moderate to severe symptoms of refractory atopic dermatitis, 9 males and females (25 to 34 years old), will contain examples The 50 mg/ml 50% glycerin solution of the wheat fermentation extract produced by the two was administered to the patient 2 to 3 times a day, and 2 to 3 ml once. Patients with a conscious state (itching sensation) were classified as mild, moderate, and severe. The patient was re-examined after 2 weeks to 2 months to evaluate the effect. The results were significantly more effective (significant improvement in rash and almost disappeared from the conscious state) of 4 (44%), effective (light improvement in rash and reduction in conscious state) of 4 (44%), unchanged to 1 (11%), the deterioration to 0 (one specimen symbol test: P < 0.03), from the above, the effective rate was 89%.

Example 14 Analgesic effect of wheat fermentation extract

The wheat fermentation extract obtained in Example 2 was dissolved in distilled water, and a probe was used for each mouse to carry out oral administration of 0.2 ml. After 90 minutes, 0.7% acetic acid was intraperitoneally administered, and after 5 minutes, the condition was observed, and the number of body twitches caused after 30 minutes was measured. The results indicate that the necessary amount of each sample in which the distilled water was administered to the control tissue 30% of the body twitch was blocked in Table 6.

The active ingredient of the low molecular weight lipopolysaccharide from the large intestine was 7 as the wheat fermentation extract at 1 hour, indicating that the wheat fermentation extract had an excellent analgesic effect.

Example 15 Inhibition effect of atopic dermatitis on wheat fermentation extract

To investigate the effect of atopic dermatitis on wheat fermentation extracts, a type I allergy model was introduced. For a group of 3 to 4 male BALB/c mice, 1 μg/mouse of anti-nitrobenzene and mouse monoclonal antibodies were administered intravenously. One hour later, the wheat fermentation extract (4 μg/mouse) prepared in Example 2 was intradermally administered intradermally or orally (100 μg/mouse), and after 1 hour, the surface of the ear shell of the fish mouse was coated. 20 μl of cloth containing 0.25% dinitrofluorobenzene in acetone-olive oil mixed solution (4:1) as an antigen. The thickness of the ear shell was measured by a thickness gauge at 1, 2, 24, and 48 hours after coating, and the difference (Δ) from the thickness before coating was used as the degree of edema. The drug administration effect was an early reaction confirmed after the antigen was administered for 1 hour, and a delayed reaction which was induced 24 hours later, and the inhibition rate was determined by the following formula for each inhibition. {Inhibition rate = (1 - edema of △ ear shell after administration of drug / edema of △ ear shell of control) × 100} The results are shown in Table 17. It is known from the table that the wheat fermentation extract can inhibit allergic reactions by intradermal administration or oral administration.

Example 16 Infection prevention effect of wheat fermentation extract

In order to investigate the infection prevention effect of wheat fermentation extract, a model of methicillin-resistant Staphylococcus aureus (MRSA) infection was introduced. For a group of 10 male BALB/c mice (6-8 weeks old), 200 mg/kg cyclophosphamide (CY) was intraperitoneally administered, and after 5 days, the wheat fermentation extraction obtained in Example 2 was carried out. Intradermal administration of the substance. After 3 hours, MRSA (3 × 10 ⁷ colony forming units (CFU)) was administered intravenously, and the number of days of survival was examined. The results are shown in Table 18. It was found from the table that the wheat fermentation extract was statistically significantly inferior to the physiological saline (control group) group (χ ² assay, P < 0.001), indicating an infection prevention effect on MRSA.

Example 17 Cancer metastasis treatment effect of wheat fermentation extract

To investigate the effect of cancer cell transfer treatment of wheat fermentation extracts, a lung metastasis model of MethA cancer cells was introduced. For a group of 10 male BALB/c mice (6-8 weeks old), 1×10 ⁵ cells of MethA cells were administered intravenously, and 12 days later, 4 days in a row, the wheat fermentation extract prepared in Example 2 was subjected to Intradermal administration. After 20 days of cell transplantation, the rats were dissected and the lungs were removed for formalin fixation. The lungs were observed by the naked eye and the number of nodules was determined. The results are shown in Table 19. It is known from the table that the wheat fermentation extract has a statistically significant difference (t-test, P < 0.001) compared with physiological saline (control group), indicating a therapeutic effect on metastasis of lung cancer cells of MethA.

F. Related examples of bean curd fermentation extract Example 18 Manufacture of bean curd fermentation extract

(1) In a 2 liter triangular conical flask, 1.0 L of water, 0.2 g of potassium phosphate, 1.15 g of potassium phosphate, 8 g of salt, and 0.2 g of potassium chloride were placed.

(2) 20 g of dried bean curd residue was added to 1.

(3) Sterilize 2 in an autoclave.

(4) Modulation of inoculum. One colony of rice pathogenic bacteria isolated from wheat flour was placed in 5 ml of the 2% bean curd medium prepared in the previous composition, and slowly stirred at 37 ° C for 1 night (15 hours) to ferment to complete the bean curd residue. Prepare with inoculum.

(5) 4 whole amount was added to 3, and fermentation was carried out for 48 hours while stirring slowly at 37 °C.

The tofu residue fermentation solution of (6) 5 was subjected to heating extraction at 120 ° C for 20 minutes in an autoclave. This was centrifuged (Putian 8800, 2000 rpm, 10 minutes), and the clear liquid was recovered as a fermentation extract of bean curd residue.

(7) Measurement of dry weight: 0.3 ml was preliminarily weighed and transferred to a 1.5 ml plastic test tube, and after freezing, it was 5.97 mg when freeze-dried by a freeze dryer. Therefore, the dry weight of the fermented extract of the bean curd residue of 6 was 19.9 mg per 1 ml of the solution, and the total amount was 19.9 g when it was 1000 ml.

(8) Protein quality of a 10-fold diluted sample was determined by Bradford protein concentration assay using protein quantification BSA as a standard protein. The results are shown in Table 15.

(9) Measurement of nucleic acid content: The 100-fold diluted sample was subjected to absorbance measurement at 210 to 340 nm. The maximum absorbance of 50 μg was calculated by subtracting the absorbance at 260 nm absorbance to 320 nm and the absorbance at DNA 1OD.

(10) Measurement of sugar content: Glucose was measured by phenol sulfuric acid method as a standard fine.

(11) Determination of the content of the limulus active substance measured by limulus: the mass of the limulus active substance is a tooxycolor system using the biochemical industry, and the biochemical industry Et-1 is used as a standard limulus active substance.

Example 19 Immunostimulating effect of bean curd extract

THP-1 (1×10 ⁶ cells/250 μl: RPMI 1640 medium containing 10% bovine embryo serum) was used as a human myeloid leukemia cell line in a 48-well culture dish for 30 minutes. Pre-culture. The final concentration of each sample was added to 250 μl (final amount 500 μl) at 100 to 10000 ng/ml. A group containing polymyxin B (12.5 μg/ml) in the sample (to be a group containing only 100 ng/ml of polymyxin B) was prepared. After 4 hours of culture, the culture supernatant and cells were recovered. The TNF activity of the clarified liquid was determined using the L-929 cell disorder test. The results are shown in Table 21. The fermentation extract of bean curd can also produce TNF from macrophages in the presence of polymyxin B, but low molecular weight lipopolysaccharide and limulus positive glycolipid do not produce TNF from macrophages in the presence of polymyxin B. . Therefore, it can be known that the bean curd fermentation extract has a different biological activity than the low molecular weight lipopolysaccharide or the limulus positive glycolipid.

N.D.: not determined

G. Related examples of rice flour fermentation extract Manufacture of rice flour fermentation extract Example 20

(1) In a 2 liter triangular conical flask, 1.0 L of water, 0.2 g of potassium phosphate, 1.15 g of potassium phosphate, 8 g of salt, and 0.2 g of potassium chloride were placed.

(2) Add 20 g of dry rice flour to 1.

(3) Sterilize 2 in an autoclave.

(4) Modulation of inoculum. One colony of rice pathogenic bacteria isolated from wheat flour was placed in 5 ml of the 2% dried medium prepared in the previous composition, and slowly stirred at 37 ° C for 1 night (15 hours) to ferment the rice flour. Preparation of inoculum.

(5) 4 whole amount was added to 3, and fermentation was carried out for 72 hours with gentle stirring at 37 °C.

The rice flour fermentation solution of (6) 5 was subjected to heating extraction at 120 ° C for 20 minutes in an autoclave. This was centrifuged (Putian 8800, 2000 rpm, 10 minutes), and the clear liquid was recovered as a rice flour fermentation extract.

(7) Determination of the content of the limulus active substance measured by limulus: the mass of the limulus active substance is a tooxycolor system using the biochemical industry, and the biochemical industry Et-1 is used as a standard limulus active substance. The content of the limulus active substance in the rice flour fermentation extract was determined to be 1.7 μl/mg.

Example 21 Immune activation of rice flour extract

THP-1 (1×10 ⁶ cells/250 μl: RPMI 1640 medium containing 10% bovine embryo serum) was used as a human myeloid leukemia cell line in a 48-well culture dish for 30 minutes. Pre-culture. The final concentration of each sample was added to 250 μl (final amount 500 μl) at 1 to 10000 ng/ml. A group containing polymyxin B (12.5 μg/ml) in the sample was prepared. After 4 hours of culture, the culture supernatant and cells were recovered. The TNF activity of the clarified liquid was determined using the L-929 cell disorder test. The results are shown in Table 22. Rice flour fermentation extract can also produce TNF from macrophages in the presence of polymyxin B, but low molecular weight lipopolysaccharide and limulus positive glycolipid do not produce TNF from macrophages in the presence of polymyxin B. Therefore, it can be known that the rice flour fermentation extract has a different biological activity than the low molecular weight lipopolysaccharide or the limulus positive glycolipid.

H. Related examples of kelp bud fermentation extract Example 22 Manufacture of kelp buds and fabric fermentation

(1) In a 2 liter triangular conical flask, 1.0 L of water, 0.2 g of potassium phosphate, 1.15 g of potassium phosphate, 8 g of salt, and 0.2 g of potassium chloride were placed.

(2) Add 20 g of kelp buds and cloth glutinous rice flour to 1.

(3) Sterilize 2 in an autoclave.

(4) Modulation of inoculum. One colony of rice pathogenic bacteria isolated from kelp buds and cloth buds was placed in 5 ml of the 2% dried medium prepared in the previous composition, and slowly stirred at 37 ° C for 1 night (15 hours) to ferment. Preparation of kelp buds and fabrics for inoculum.

(5) 4 whole amount was added to 3, and fermentation was carried out for 72 hours with gentle stirring at 37 °C.

The kelp bud and cloth fermentation solution of (6) 5 was heated and extracted in an autoclave at 120 ° C for 20 minutes. This was centrifuged (Putian 8800, 2000 rpm, 10 minutes), and the clear liquid was recovered as a fermentation extract of kelp buds and cloth.

(7) Determination of the content of the limulus active substance measured by limulus: the mass of the limulus active substance is a tooxycolor system using the biochemical industry, and the biochemical industry Et-1 is used as a standard limulus active substance. The content of the limulus active substance in the kelp bud and the fabric extract was determined to be 132 μl/mg.

Example 23 Immune activation of kelp buds and fabric extracts

THP-1 (1×10 ⁶ cells/250 μl: RPMI 1640 medium containing 10% bovine embryo serum) was used as a human myeloid leukemia cell line in a 48-well culture dish for 30 minutes. Pre-culture. The final concentration of each sample was added to 250 μl (final amount 500 μl) at 1 to 10000 ng/ml. A group containing polymyxin B (12.5 μg/ml) in the sample was prepared. After 4 hours of culture, the culture supernatant and cells were recovered. The TNF activity of the clarified liquid was determined using the L-929 cell disorder test. The results are shown in Table 22. The kelp bud and fabric extract can also produce TNF from macrophages in the presence of polymyxin B, but low molecular weight lipopolysaccharide and limulus positive glycolipid in the presence of polymyxin B are not in macrophages. Produce TNF. Therefore, it can be known that the kelp bud and the cloth extract fermentation extract have different biological activities compared to the low molecular weight lipopolysaccharide or the limulus positive glycolipid.

Industrial availability

The invention relates to a plant fermentation extract which can manufacture a safe immune activating substance inexpensively, and the obtained plant fermentation extract can be utilized for a mammal containing humans (specifically, livestock, pets, etc.), birds (specifically, raising chicken, rewarding Birds, etc., biogenic, reptilian, fish (specifically aquaculture fish, pet fish, etc.), invertebrates and pharmaceuticals, animal medicines, pharmaceutical products, cosmetics, food, functional foods, feed and Bathing agents, etc.

Fig. 1 is a graph showing the effect of suppressing the incidence of koi herpes by adding a feed of wheat fermented extract.

Claims (7)

A fermentation and culture method characterized in that a raw material containing a saccharide derived from a food plant and having a main component of a polysaccharide is fermented by a rice pathogenic bacterium (Pantoe agglomerans) alone, and the rice is cultivated separately under the same conditions. Pathogenic bacteria. A plant fermentation extract characterized in that a raw material containing a saccharide derived from a food plant and having a main component of a polysaccharide is fermented by a rice pathogenic bacterium alone, and the rice pathogenic bacterium is cultured separately. A plant fermentation extract powder characterized by being obtained from a plant fermentation extract according to item 2 of the patent application. A plant fermentation extract complex characterized by adding a plant fermentation extract according to item 2 of the patent application or a plant fermentation extract powder according to claim 3 of the patent application. The plant fermentation extract complex according to claim 4, wherein the plant fermentation extract complex is a pharmaceutical, an animal medicine, a pharmaceutical product, a cosmetic, a food, a functional food, a feed, or a bathing agent. . The plant fermentation extract of claim 2, which is a physicochemical property showing the activation energy of macrophages even in the presence of polymyxin B. A plant fermentation extract according to claim 2 of the patent application, which has an immunostimulating activity.