HK1096119B - Fermentation and culture method, fermented plant extract, fermented plant extract powder and composition containing the fermented plant extract - Google Patents

Description

Fermentation and culture method, fermented plant extract powder, and composition containing fermented plant extract

Technical Field

The present invention relates to a method for fermentation and culture for obtaining a safe immunopotentiator which can be added to drugs, veterinary drugs, quasi drugs, cosmetics, foods, functional foods, feeds and bath agents related to mammals including humans (specifically domestic animals, pet animals and the like), birds (specifically raised chickens, pet birds and the like), amphibians, reptiles, fish (specifically water-cultured fish, pet fish and the like) and invertebrates, a method for producing a plant fermentation extract, a plant fermentation extract containing an immunopotentiator obtained by the fermentation and culture method, a powder containing an immunopotentiator obtained from the plant fermentation extract, and a plant fermentation extract composition containing the plant fermentation extract.

Background

It is urgent to establish disease prevention and treatment methods including infection prevention techniques for mammals including humans (specifically domestic animals, pet animals, etc.), birds (specifically farmed chickens, pet birds, etc.), amphibians, reptiles, fish (specifically water-farmed fish, pet fish, etc.), and invertebrates. Furthermore, there is an urgent need for a method which can not only achieve the above objects but also eliminate the use of chemicals, environmental pollution, generation of resistant bacteria and accumulation in the human body. The present inventors have found that, in view of the above problems, a plant-derived immunopotentiator such as an aqueous extract of wheat can safely achieve disease prevention and treatment effects (patent document 1, non-patent document 1). Also in order to achieve the above object, the present inventors have found that a low molecular weight lipopolysaccharide obtained from Pantoea agglomerans (Pantoea agglomerans) which is a symbiotic bacterium of wheat can be used (non-patent document 2). Meanwhile, recent studies have shown that various substances other than lipopolysaccharide exhibit an immunopotentiating effect, and these natural materials containing immunopotentiators have attracted attention.

Microbial fermentation technology is widely used not only in the food field but also in a wide range of fields. Fermentation is widely used in the production of wines including wine, soy sauce and bean paste, fermented milk products such as cheese and pharmaceutical production. There are many microorganisms used for fermentation, and rice malt (fungi) yeast and lactic acid bacteria are representative, but the use of gram-negative bacteria is rarely reported. In general, fermentation is a phenomenon in which an organic substance is decomposed by a microorganism, and is broadly intended to mean the production of a useful substance by a microorganism (non-patent document 3). Typical fermentation using microorganisms includes wine brewing. The wine brewing is a fermentation technology of wine yeast attached to grape skin, and the product is alcohol. Among the techniques using microbial fermentation, fermentation using gram-negative bacteria, methane fermentation using methane bacteria, acetic acid fermentation using acetic acid bacteria, and fermentation using Zymomonas mobilis (Zymomonas mobilis) to obtain ethanol from agave tubers (agave wine fermentation) have been known, but little is known about the use of edible plants as a raw material and fermentation culture using microorganisms characterized by having a symbiotic relationship with the plants, and immunopotentiators have never been paid attention as fermentation products. Still further, fermentation and culture methods for the purpose of producing immunopotentiators have never been attracting attention.

On the one hand, when fermentation is carried out using microorganisms, it is usual for the nutrient conditions of the fermentation substrate to be such that the growth of the microorganism is satisfied. That is, it is necessary to have substances available for the microorganism as nutrients, i.e., containing sufficient monosaccharides such as glucose and fructose as a carbon source. Thus, fruits like grapes that are rich in fructose can be used as fermentation substrates without any processing. In other cases, however, microbial fermentation requires pretreatment such as: heating and enzyme treatment. For example, Zymomonas mobilis as taught above is a microorganism used in the fermentation of tequila. In this case, polysaccharides obtained from tubers of the inedible plant agave are decomposed into fermentable monosaccharides by heating, and these monosaccharides are subsequently fermented by microorganisms to obtain alcohol as a fermentation product. Therefore, polysaccharides such as starch are not suitable as fermentation substrates when fermentation culture is performed with typical microorganisms. For example, it has been reported that Pantoea agglomerans cannot decompose starch (non-patent document 4).

We have demonstrated that an aqueous extract of wheat flour contains an active ingredient for enhancing immunity (non-patent document 5). We have also demonstrated that edible grains (wheat, rice), seaweeds (brown algae, kelp, sargassum fusiforme (brown algae) and laver), and beans (soybean and red bean) contain this active ingredient (non-patent document 6). This biological activity has been found to have a preventive effect on diseases (diabetes, hyperlipidemia, atopic dermatitis, cancer) in humans and mice, and also to be effective in preventing infections of fish, crustaceans and chickens (patent document 1, non-patent document 1). However, in order to obtain the above effect by an aqueous extract of wheat flour, a large amount of wheat flour must be ingested.

On the other hand, pantoea agglomerans is a bacterium living symbiotically with wheat, and since this bacterium supplies phosphorus and nitrogen to wheat, it is considered to be useful for wheat breeding (non-patent document 7). Meanwhile, Pantoea agglomerans deposits not only on wheat but also on the epidermis of pears and apples. It has been demonstrated in europe that when such bacteria are deposited, rot disease caused by fungi can be prevented, and development of a nontoxic, environmentally friendly fungicide using such bacteria is under way (non-patent document 8). Symbiosis is the phenomenon defined as "survival of heterogeneous organisms together. In this case, it is usually meant that a close relationship, either behaviorally or physiologically, is often maintained. Therefore, this does not merely mean the concept of living under the same living environment. Symbiotic phenomena are classified and divided into different categories according to the life meaning and importance of symbiotic partners, the sustainability of relationships and the spatial positions of symbiotic partners. Generally, symbiosis is mainly classified according to the presence or absence of benefit/badness from symbiotic subjects: mutualistic symbiosis, partial symbiosis and parasitism. Three types "(non-patent document 9). It is known that Pantoea agglomerans can be isolated from any wheat in any region (non-patent document 5) and also can be isolated from fruits (non-patent documents 10 and 11). It has been reported that pantoea agglomerans protects plants from fungi and other bacteria by producing antibiotics (non-patent documents 12, 13) and performs phosphorus fixation and nitrogen fixation (non-patent document 7). Therefore, Pantoea agglomerans has been considered to be present in plants and to play a plant-benefiting role. Thus, its survival mode is considered to be "symbiotic" rather than "parasitic". In addition, we have demonstrated that an active ingredient that enhances immunity is present in pantoea agglomerans. We have also found that low molecular weight lipopolysaccharides obtained from such bacteria have a preventive effect on diseases (diabetes, hyperlipidemia, atopic dermatitis, cancer) in humans and mice, and also have a preventive effect on infections of fish, crustaceans and chickens (patent document 3, non-patent document 2).

Under such circumstances, we have conceived that a method for producing a fermented plant extract by using Enterobacter agglomerans is established as a method for producing an immunopotentiator safely and inexpensively. That is, we focused on (1) culturing pantoea agglomerans at low cost using a medium containing a main protein component contained in a culture solution derived from a plant and simultaneously fermenting the plant component and (2) preparing a material rich in a plant or a fermentation product containing pantoea agglomerans, thereby paying attention to the development of drugs, drugs for animals, quasi drugs, cosmetics, functional foods, feeds, and bath agents for mammals including humans (specifically, domestic animals, pet animals, etc.), birds (specifically, raised chickens, pet birds, etc.), amphibians, reptiles, fish (specifically, aquaculture fish, pet fish, etc.), and invertebrates. This does not mean, however, that the microorganisms living symbiotically with the plants are able to use plant constituents, such as raw materials derived from edible plants, directly as fermentation substrates. For example, wheat flour is a complex organic matter of starch and similar components present in wheat flour, but isolated microorganisms living symbiotically with wheat, i.e., pantoea agglomerans, do not directly contact the wheat bran. Thus, it could not be demonstrated whether Pantoea agglomerans could be fermented and cultured with wheat flour by the symbiotic relationship of bacteria and wheat. In fact, it is not known at all, nor reported that Enterobacter agglomerans is able to assimilate wheat flour. On the contrary, according to the known facts, it has been described that Enterobacter agglomerans cannot use wheat starch as a fermentation substrate.

Sugars contained in plants are often retained in the form of starch, which is remarkable in edible plants, particularly edible grains. Generally, microorganisms do not have a function of highly assimilating starch. In this regard, a proportion of facultative gram-negative bacteria are known to be capable of fermenting starch. For example: erwinia is known to assimilate starch. However, in this fermentation as described, when fermenting starch, it is desirable to utilize the amylase activity of the microorganism by adding a microorganism which can be cultured in a large amount in another optimal medium, and it has never been thought to carry out the culture itself and the fermentation using starch in combination. In the conventional art, it is considered as fermentation with the aim of effectively utilizing amylase activity of a microorganism only, and there is no plan for culturing a microorganism with starch as a substrate. In one aspect, in the present example, the production of a fermentation product other than the cultivation of a microorganism using starch as a sole carbon source is disclosed, and the present invention is significantly different from the conventional art in that the present example is not only fermentation but fermentation and cultivation.

On the other hand, if a particular microorganism retains the function of decomposing starch, this does not directly indicate that the microorganism is capable of growing using starch as a substrate. In the case of culturing, also in the case of aiming at the growth of microorganisms, the number of microorganisms added at the beginning of culturing is extremely small. In this case, even if the microorganism has amylase activity slightly, the activity is too weak to sufficiently decompose the substrate, and growth of the microorganism cannot be achieved. In fact, it is believed that many microorganisms cannot grow using starch as the sole carbon source.

However, if fermentation and culture are carried out using pantoea agglomerans in a medium whose main component is wheat flour, a fermented plant extract rich in immunopotentiators (hereinafter, a fermented plant extract produced by fermentation and culture using pantoea agglomerans in a medium whose main component is wheat flour is referred to as a fermented wheat extract) is produced at low cost, and as a specific example, it should be possible to provide an environmentally friendly, safe, drug effective for preventing infection, an animal drug, a quasi drug, a cosmetic, a food, a functional food, a feed, and a bathing agent for animal husbandry in the aquaculture field. The present invention has been completed by conducting a large number of experiments in response to the above-mentioned finding that Pantoea agglomerans grows on wheat flour as a substrate.

The fermented plant extract of the present invention is a general term and includes a culture solution itself obtained by fermentation and culture, a liquid fraction obtained by solid/liquid separation of the culture solution, a liquid fraction obtained by extraction of the solid fraction obtained by solid/liquid separation, and the like. That is, the plant fermentation extract includes the culture solution itself obtained by the fermentation and cultivation method according to the present invention, and all extracts that may be prepared using all or a part of the culture solution. It goes without saying that the fermented plant extract can be utilized by drying into fermented plant extract powder or dissolving the fermented plant extract powder in a phosphate buffer solution containing physiological saline in an appropriate solution to an arbitrary concentration.

[ patent document 1] Japanese unexamined patent application publication No. H3-218466

[ patent document 2] Japanese unexamined patent application publication No. H8-198902

[ patent document 3] WO 00/57719

[ patent document 4] Japanese unexamined patent application publication No. H6-78756

[ patent document 5] Japanese unexamined patent application publication No. H4-187640

[ patent document 6] Japanese unexamined patent application publication No. H4-49240

[ patent document 7] Japanese unexamined patent application publication No. H4-99481

[ patent document 8] Japanese unexamined patent application publication No. H5-155778

[ non-patent document 1] Inagawa, H.et al., Biotherapy 5(4), p617-621, 1991

[ non-patent document 2] Soma g.et al, "" Tumor necrosis Factor: molecular and Cellular Biology and Clinical Relevance "p 203-220, 1993

[ non-patent document 3] Yamada T.et al, "Seibutsugaku Jiten" 3 rd., p1021, 1983

[ non-patent document 4] Gavini, F.et al., int.J.Sys t.bacteriol., 39, p337-345, 1989

[ non-patent document 5] Nishizawa T.et al, chem.pharm.Bull., 40(2), p479-483, 1992

[ non-patent document 6] Inagawa H.et al., chem.pharm.Bull., 40(4), p994-997, 1992

[ Nonpatent document 7] Neilson A.H., J.Appl.Bacteriol., 46(3), p483-491, 1979

[ non-patent document 8] Nunes C.et al, int.J.food Microbiol., 70(1-2), p53-61, 2001

[ non-patent document 9] Yamada T.et al, "Seibutsugaku Jiten" 3 rd., p287-288, 1983

[ non-patent document 10] Nunes C.et al, J.Appl.Microbiol., 92(2), p247-255, 2002

[ non-patent document 11] Asis C.A.Jr.et al., Lett.appl.Microbial., 38(1), p19-23, 2004

[ non-patent document 12] Vannester J.L.et., J.bacteriol., 174(9), p2785-2796, 1992

[ non-patent document 13] Kearns L.P.et al, appl.environ. Microbiol., 64(5), p1837-1844, 1988

Disclosure of Invention

[ problems to be solved by the invention ]

As mentioned above, the immunopotentiator is mostly contained in the plant itself and in the components or products of microorganisms living in symbiosis with the plant. Therefore, in order to obtain an immunopotentiator derived from a natural product which is safe to ingest, it is useful to extract a component from the edible plant itself (for example, limulus-positive glycolipid, patent document 1) or to culture a microorganism living symbiotically with the edible plant efficiently to obtain a component or a product thereof (for example, low molecular weight lipopolysaccharide. patent document 2). However, the content of the immunopotentiator in the edible plant is very low, and it is necessary to ingest an extremely large amount of food in order to achieve the immunopotentiation effect by ingestion, and it is not easy to maintain an appropriate amount of the immunopotentiator ingested. Thus, this effect cannot be expected. In addition, when an immunopotentiator is extracted from a plant and used as a food or a medicine, high cost is required and the practicability is poor.

On the one hand, when attention is paid to microorganisms living symbiotically with plants, the bacteria symbiotic with wheat, pantoea agglomerans, contain low molecular weight lipopolysaccharide effective as an effective ingredient for immunostimulation. However, hitherto, in order to extract low molecular weight lipopolysaccharides, pantoea agglomerans has to be cultured in an expensive medium in which main proteins such as NZ amine, tryptone or casamino acid contained are derived from animals. Therefore, it is difficult to provide an immunopotentiator that can be widely used at low cost. At the same time, it cannot be denied that unknown pests such as those derived from BSE animals may be mixed in.

In view of the above problems, the present invention aims to provide a fermentation and culture method for obtaining an immunopotentiator inexpensively and efficiently using safe materials; fermented plant extracts obtained by the method, fermented plant extract powders obtained from fermented plant extracts, and fermented plant extract compositions containing the fermented plant extract powders.

[ means for solving problems ]

The fermentation and cultivation method of the present invention is characterized in that facultative anaerobic gram-negative bacteria exclusively living symbiotically with plants are used for fermentation of materials derived from edible plants while culturing the facultative anaerobic gram-negative bacteria.

Fermentation and cultivation can be carried out in a simple manner by fermenting starch as a carbon source with facultative anaerobic gram-negative bacteria.

It is desirable that the facultative anaerobic gram-negative bacteria are facultative anaerobic bacilli.

Facultative anaerobes belong to the family Enterobacteriaceae (Enterobacteriaceae).

The facultative anaerobacter belongs to the genus Pantoea (Pantoea), Serratia (Serratia) or Enterobacter (Enterobacter).

By using facultative anaerobe pantoea agglomerans, starch can be used as a carbon source.

Desirably, the edible plant is a food grain, seaweed, legume or mixture thereof.

It is also desirable that the raw material derived from the edible grain is wheat flour, rice flour, wheat bran flour, rice bran or distiller's grains. In particular, since wheat flour contains gluten as a protein source, it can be efficiently fermented and cultured even without using raw materials derived from animals.

The raw material derived from seaweed is preferably brown algae powder, Undaria pinnatifida (sporophyll of Undaria pinnatifida) powder or kelp powder.

When the raw material derived from beans is bean curd refuse, it is rich in protein. Thus, it is possible to efficiently ferment and culture even without using raw materials derived from animals.

The fermented plant extract of the invention is characterized by being obtained by a fermentation and culture method.

The fermented plant extract powder of the invention is characterized by being obtained by a fermentation and cultivation method.

The fermented plant extract composition of the invention is characterized by combining a fermented plant extract or a fermented plant extract powder.

The fermented plant extract composition can be used as medicine, animal medicine, quasi-medicine, cosmetic, food, functional food, feed or bathing agent.

It is desired that the plant fermentation extract has the following physicochemical properties.

The fermented plant extract showed macrophage activating ability even in the presence of polymyxin B. The fermented plant extract has immunity enhancing effect. The facultative anaerobic gram-negative bacteria may be bacilli belonging to the genus Pantoea, and the edible plants may be edible grains, seaweed or beans, or a mixture thereof. The facultative anaerobic gram-negative bacteria may be pantoea agglomerans, and the edible plant is an edible grain, seaweed or legume, or a mixture thereof. The raw material derived from the edible grain may be wheat flour, rice flour, wheat bran powder, rice bran or distiller's grains. The raw material derived from the seaweed may be brown seaweed powder, Undaria pinnatifida powder or kelp powder.

[ Effect of the invention ]

According to the present invention, since the culture can be performed in a medium containing no animal-derived component, there is no contamination with impurities derived from animal components. Therefore, unknown harmful substances, for example originating from BSE pollution, are unlikely and can provide: a method for producing a fermented plant extract with high safety and at low cost which can be used for various intended uses and a fermented plant extract or fermented plant extract powder containing an immunopotentiator are provided safely and inexpensively; and can provide a culture solution; immunopotentiators and extracts and extract powders; and drugs, animal drugs, quasi drugs, cosmetics, foods, functional foods, feeds and bathing agents containing the extract or extract powder.

This is a fact that has not been thought of nor is it easily speculated from the discovery of conventional fermentation techniques, namely fermentation of edible plant-derived material with facultative anaerobic gram-negative bacteria that live symbiotically exclusively with plants, while culturing facultative anaerobic gram-negative bacteria, achieved in a simple manner

Whether a particular substance produces TNF from macrophages (TNF-inducing activity) may be used as an indicator that an immunopotentiating effect is exhibited. Furthermore, the amount of TNF produced can be used to quantify the immunopotentiating effect. Therefore, production of TNF by macrophages was detected using limulus-positive plant glycolipids derived from wheat flour and low-molecular-weight lipopolysaccharide derived from Pantoea agglomerans. TNF production in macrophages was terminated by treating limulus-positive plant glycolipids derived from wheat flour and low-molecular-weight lipopolysaccharides derived from Pantoea agglomerans with polymyxin B. However, many examples show that TNF is produced from macrophages even when the plant fermentation extract of the invention is treated with polymyxin B. This indicates that the fermented extract of a plant obtained by fermentation and culture has an immunopotentiating effect different from the immunopotentiating effect produced by the components in the plant itself used as a raw material and the microorganism itself used for fermentation.

Drawings

FIG. 1 is a graph showing the results of inhibition of koi herpes by feeds containing fermented wheat extracts.

Detailed Description

Embodiments suitable for the present invention are described in detail below.

I. The basic characteristics of the method for producing fermented plant extracts by using Pantoea agglomerans

In the present invention, we have for the first time found that Pantoea agglomerans can grow directly using starch as a carbon source, and invented a method of inexpensively producing a fermented extract of wheat rich in an immunopotentiator as a fermentation product and a culture product using Pantoea agglomerans. Therefore, the preparation can provide environment-friendly and safe quasi-drugs, cosmetics, foods, functional foods and feeds for effectively preventing human infection and in the livestock industry and the water industry.

1: isolation of Pantoea agglomerans

Wheat flour was suspended in water, and the supernatant was applied to an L broth agar medium, followed by culture to develop microbial colonies. Among these colonies, microorganisms were identified by standard methods. For example, those colonies having the same properties as those of standard Enterobacter agglomerans were selected by screening for gram-stain-negative, glucose anaerobic metabolic reaction-positive and oxidase activity-negative colonies and using the ID test/EB-20 (Nisshinoki pharmaceutical manufacturing). Standard pantoea agglomerans can be obtained from the institute of physical chemistry, center for biological resources (non-patent document 4). In the following description, percentages are weight values unless otherwise specified.

2: evaluation of immunopotentiating Activity

In this embodiment, the ability to activate macrophages is assessed by their production of TNF as an indicator of the immunopotentiation effect exhibited by fermented wheat extracts.

3: low molecular weight lipopolysaccharide derived from Pantoea agglomerans

It is desired to contain a low-molecular-weight lipopolysaccharide derived from pantoea agglomerans as one of the immunopotentiating active ingredients obtained by fermentation and culture of pantoea agglomerans. The low molecular weight lipopolysaccharide has more remarkable safety and higher bioactivity than the commonly used high molecular weight lipopolysaccharide (typical lipopolysaccharide). Thus, the content of low molecular weight lipopolysaccharide was determined. Patent document 2 describes low molecular weight lipopolysaccharides in detail. This example relates to fermented extracts of wheat, but the present invention does not imply that the plant is limited to wheat and that the immunopotentiator is limited to low molecular weight lipopolysaccharides.

Pantoea agglomerans can be cultured by a known method (patent document 2, non-patent document 8), but the major components of proteins contained in the medium are derived from animals, and the medium cost is high. Further, when functional foods and functional feeds are administered to animals or used transdermally, there is a problem of contamination of impurities derived from animals, such as BSE, in terms of food safety, and in addition, production costs are high and the method is poor in practicability. Thus, as a result of extensive studies to obtain a safe and inexpensive natural product having an immunopotentiating effect, the present inventors completed a method of fermenting and culturing with Enterobacter agglomerans to obtain a fermented extract of wheat as shown in examples. The main components of the protein contained in the culture medium have been derived from animals, but the protein is derived from plants in the present invention. Typically, a product obtained by decomposing a protein such as casein derived from milk with a digestive enzyme is added to the culture solution. In this case, the basic cost per liter of the medium is about 250 yen, but if this can be replaced with wheat flour, the basic cost becomes about 16 yen. Fermentation that highly concentrates and synergistically fuses both plant-derived and symbiotic microorganisms' immune enhancing activity has not been seen.

The contents of the present invention will be described in the following examples, but the present invention is not limited to the use of Pantoea agglomerans as the microorganism described in the present examples, nor to the use of wheat as an edible plant or wheat flour as a raw material. The invention can also be applied to raw materials obtained by typical methods from other immunopotentiator-rich edible plants, such as: brown algae, edible grains (including wheat flour, rice flour, wheat bran powder, rice bran or distillers' grains derived from edible grain raw materials), seaweeds (including brown algae powder, Undaria pinnatifida powder or kelp powder derived from seaweed raw materials), and beans (including bean curd dregs derived from bean raw materials). It is well known that these plants contain proteins and carbohydrates. These plants can be suitably used for fermentation and culture of Pantoea agglomerans. It is known that indigenous bacteria (indigenous bacteria), such as bacteria of the genus Serratia or Enterobacter (Enterobacter), live symbiotically with these plants (non-patent document 4). Of course, microorganisms for fermentation include facultative anaerobic gram-negative bacteria that live symbiotically with these plants.

II: summary of the invention

(1) The fermented wheat extract is novel as a substance having an immunopotentiating effect obtained by fusion of wheat, pantoea agglomerans, a bacterium symbiotic therewith, and a fermentation product produced by combination thereof, but the present invention is not limited thereto.

(2) The production of fermented plant extracts using Pantoea agglomerans, a gram-negative bacterium, is novel, but the present invention is not limited thereto.

III: specific method for producing fermented extract of wheat

(1) Pantoea agglomerans were isolated from wheat flour by a standard method (non-patent document 1). Once isolated and identified, the bacteria can be stored in 50% glycerol.

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

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

(4) Preparing 0.2-3 mol of magnesium chloride solution.

(5) Preparing 0.2-3 mol of calcium chloride solution.

(6) In some cases, the solutions (2) to (5) are sterilized by autoclave or the like.

(7) Mixing the solutions (2) - (5) in a proper dosage, and adding water to obtain a 0.1-5% wheat flour suspension. In some cases, the pH is made neutral by adding an alkaline solution or an acidic solution.

(8) In some cases, the wheat starch can be partially digested by adding 10 to 50000 units of amylase per liter of the medium to (7) and incubating at 10 to 80 ℃ for 1 to 24 hours.

(9) Adding the Enterobacter agglomerans separated in (1) to (7) or (8).

(10) Fermenting (9) at 1-40 ℃. In some cases, the fermentation vessel may be left to stand or shaken. Alternatively, the stirring may be performed several hours apart.

(11) Fermenting (10) for 6 hours to one week. When the fermentation proceeds, the wheat flour solution turns yellow.

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

(13) The fermentation was terminated and the solid component was collected as a precipitate by centrifugation (1000 to 5000rpm, 10 to 60 minutes). The precipitate can be used as a wheat flour fermentation product directly for feed or as a raw material to be mixed with feed.

(14) When producing the wheat fermentation extract, suspending (13) in water or salt buffer solution, and heating at 80-140 ℃ for 10 minutes-6 hours. The solid component can be removed by centrifugation or filtration. Water or salt buffer may be added to the removed solid component and the extraction by heating may be repeated several times.

(15) The fermented wheat extract produced in (14) can be further simply purified depending on the intended use. Adding salt such as sodium chloride to the extract of (14) at a final concentration of 0.05-1 mol/L, and adding solvent such as ethanol in an amount of 1-3 times of the extract, wherein precipitation occurs. The precipitate can be collected by centrifugation. The precipitate may be further washed with a solvent such as ethanol. When it is dried, a powder can be prepared.

A. Examples relating to methods for producing fermented extracts of wheat

[ example 1]

Growth study of Pantoea agglomerans in wheat flour culture medium

In order to confirm whether or not the growth of Enterobacter agglomerans, which is a bacterium symbiotic with wheat in an indigenous place, was possible using wheat flour as a carbon source, the growth of Enterobacter agglomerans in a wheat flour solid medium was examined.

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

(2) One colony of Enterobacter agglomerans was picked out of LB agar medium and suspended in 1ml of PBS. This was then diluted 10-fold to 10,000-fold and 0.1ml of each aliquot was inoculated into M9 agar medium in (1).

(3) After 6 days of incubation at 37 ℃, the appearance of colonies was observed. As a result, about 300 colonies were observed in a lidded culture dish inoculated with 0.1ml diluted 10000 times.

This confirmed that Pantoea agglomerans can utilize wheat flour as a carbon source.

[ example 2]

Production of fermented wheat extract

(1) To 0.5g of wheat flour was added (5ml) distilled water to suspend, and 0.1ml of the suspension was added to L broth agar medium and cultured overnight at 37 ℃.

(2) Yellow colonies were isolated, bacteria identified by standard methods, pantoea agglomerans isolated, suspended in a 50% glycerol solution and stored in a refrigerator. A part of the stock was applied to LB agar medium, which was left to stand at 37 ℃ to prepare individual colonies of Enterobacter agglomerans.

(3) Into a 2-liter culture flask, 64g of sodium dihydrogenphosphate heptahydrate, 15g of potassium monohydrogenphosphate, 2.5g of sodium chloride, and 5g of ammonium chloride were added, followed by addition of purified water to make a total volume of 1 liter (inorganic salt mixed solution). Purified water was added to 13.1g of magnesium chloride dihydrate to make a total volume of 100ml (magnesium chloride solution). Purified water was added to 11.1g of calcium chloride to make a total volume of 100ml (calcium chloride solution). Purified water (4L) was added to a 5L Erlenmeyer flask (purified water). Both the above solution and purified water were autoclaved (TOMY BS-325, 120 ℃, 20 minutes).

(4) Wheat flour (24g) (manufactured by Nisshinoki flour Co., Ltd.) and purified water were put in a 1L Erlenmeyer flask to make a total volume of 600 ml. After the same autoclaving, 3mg of alpha-amylase (SIGMA, Bacillus, enzyme activity 1500-3000 units/mg protein) was added and heated in a water bath at 65 ℃ for 12 hours (amylase-treated wheat flour solution).

(5) The prepared solutions were placed in a 3L sterilized shake flask in the amounts shown in Table 1 to prepare a wheat flour medium.

[ Table 1]

Material	Dosage of
		Inorganic salt mixed solution	200ml
Purified water	550ml
		Amylase-treated wheat flour solution	200ml
Magnesium chloride solution	2.0ml
		Calcium chloride solution	0.1ml

(6) Preparation of inoculum: a colony of Enterobacter agglomerans isolated from the wheat flour of (2) was added to 10ml of a wheat flour culture medium having the same composition as that of (5) previously prepared, and fermented overnight (12 to 15 hours) at 37 ℃ with gentle stirring to prepare an inoculum for wheat flour fermentation.

(7) Adding the whole amount of (6) into (5), and stirring and fermenting at 37 ℃ for 20-30 hours. The pH of the fermentation broth was adjusted to pH7 by the addition of aqueous ammonia. Under the aseptic condition, 150ml of amylase treated wheat flour solution is added into 37.5ml of inorganic salt mixed solution, and the mixture is fermented for 20-30 hours in the same way. The same operation is repeated for 65-80 hours.

(8) The wheat flour fermentation solution was centrifuged (Hitachi, high speed refrigerated centrifuge, SCR-20B, 5000rpm, 20 minutes, 4 ℃), and the precipitate was collected.

(9) Adding phosphate buffer solution into the precipitate of (8), suspending to obtain a total volume of 100ml, transferring each 33ml of the suspension into a 50ml centrifuge tube, and heating in a boiling water bath for extraction for 30 minutes. After termination of heating, the solution was cooled to room temperature and centrifuged (Hitachi, high speed refrigerated centrifuge, SCR-20B, 10000rpm, 20 min, 20 ℃). After centrifugation, 82ml of the pale yellow supernatant was poured into another container and collected.

(10) To 80ml of the supernatant (9) was added a sodium chloride solution (8.9ml, 5 mol). When 178ml of ethanol was added, white turbidity appeared. It was left to stand overnight in a refrigerator (-90 ℃ C.), and the solution was centrifuged (Hitachi, high speed refrigerated centrifuge, SCR-20B, 10000rpm, 20 minutes, 4 ℃ C.). The supernatant was removed to obtain a precipitate. 10ml of 70% cold ethanol was added to the precipitate, and after suspension, the solution was centrifuged (Hitachi, high speed refrigerated centrifuge, SCR-20B, 10000rpm, 20 minutes, 20 ℃), and the precipitate was washed. Air drying the precipitate and dissolving in distilled water to obtain 11ml of fermented wheat extract.

(11) Measurement of dry weight: 0.3ml was transferred into a weighed 1.5ml plastic tube, frozen, and then lyophilized with a lyophilizer, thereby weighing 7.45 mg. Thus, the dry weight of the fermented extract of medium and small wheat in (10) was 24.8mg per 1ml of the solution and 273mg per 11ml of the total amount.

(12) The wheat fermentation extract was independently produced 8 times in the same manner, and the amount of protein in each sample was measured by the Bradford method using BSA as a standard protein for protein quantification. Purified limulus-positive glycolipids (patent document 1) and low-molecular lipopolysaccharides (patent document 2) were used as controls. The measurement results are shown in table 2. In tables 2 to 5 and 7, the numerical values corresponding to the fermented wheat extracts indicate the mg weight obtained by drying each 1g of the fermented wheat extract obtained in (10) above.

(13) And (3) measuring the sugar content: the sugar content was measured by the phenol-sulfuric acid method using glucose as a standard sugar. The measurement results are shown in table 3.

(14) Measuring the content of nucleic acid: and measuring the absorption of the sample diluted by 100 times at 210-340 nm. The maximum content was calculated from the value obtained by subtracting the absorbance at 320nm from the absorbance at 260nm and the DNA absorbance at 50. mu.g/10D. The measurement results are shown in table 4.

(15) Measuring limulus active substance content by limulus assay: for the measurement, the Toxi-color system was supplied by Biochemical industries, and ET-1 from Biochemical industries was used as a standard limulus active substance. The measurement results are shown in table 5.

(16) Iodine-starch reaction: when in use, the iodine reagent 1N (10 ml of water is added into 12.7g of iodine and 25g of potassium iodide, the mixture is mixed evenly, and water is added to the mixture until the volume is 100ml) is diluted by 200 times with water. Adding it (5 μ L) to 0.1mL of fermented extract of wheat previously dissolved to 1mg/mL, and mixing. In the wheat fermented extract, the solution immediately changed from light purple to dark purple (positive). The same procedure did not result in such a color change (negative) in limulus-positive glycolipids and low molecular weight lipopolysaccharides. The above results are summarized in table 6.

The above results show that it is apparent that the fermented wheat extract is different from limulus-positive glycolipid and low-molecular-weight lipopolysaccharide in protein content, sugar content, nucleic acid content (except limulus-positive glycolipid because there is no data), limulus-positive substance content and iodine-starch reaction, and it is apparent that the present substance is novel. The above results are briefly summarized in table 7. Namely, the fermented plant extract of the present example is novel, and shows the following physicochemical properties different from those of limulus-positive glycolipid and low-molecular-weight lipopolysaccharide. The wheat fermentation extract has a protein content of 5-15%, a sugar content of 20-45%, a nucleic acid content of 10-35%, a limulus positive substance content of 10-40%, an iodine-starch reaction positive and shows macrophage activation ability even in the presence of polymyxin B.

[ Table 2]

Protein content of fermented extract

Sample (I)	Protein content (mg/g)
		Wheat fermentation extract 1	60
Wheat fermentation extract 2	71
		Wheat fermentation extract 3	90
Wheat fermentation extract 4	105
		Wheat fermentation extract 5	103
Wheat fermentation extract 6	82
		Wheat fermentation extract 7	88
Fermented wheat extract 8	88
		Limulus positive glycolipid	40
Low molecular weight lipopolysaccharide	3.8 or less

[ Table 3]

Sugar content of fermented extract

Sample (I)	Sugar content (mg/g)
		Wheat fermentation extract 1	318
Wheat fermentation extract 2	428
		Wheat fermentation extract 3	313
Wheat fermentation extract 4	232
		Wheat fermentation extract 5	372
Fermentation of wheatExtract 6	324
		Wheat fermentation extract 7	298
Fermented wheat extract 8	329
		Limulus positive glycolipid	133
Low molecular weight lipopolysaccharide	668

[ Table 4]

Nucleic acid content of fermentation extract

Sample (I)	Nucleic acid content (mg/g)
		Wheat fermentation extract 1	102
Wheat fermentation extract 2	102
		Wheat fermentation extract 3	226
Wheat fermentation extract 4	291
		Wheat fermentation extract 5	302
Wheat fermentation extract 6	240
		Wheat fermentation extract 7	218
Fermented wheat extract 8	216
		Limulus positive glycolipid	Not reported
Low molecular weight lipopolysaccharide	2.8

[0178]

[ Table 5]

Limulus active substance content of fermented extract

Sample (I)	Limulus active substance content (mg/g)
		Wheat fermentation extract 1	242
Wheat fermentation extract 2	118
		Wheat fermentation extract 3	125
Wheat fermentation extract 4	458
		Wheat fermentation extract 5	224
Wheat fermentation extract 6	231
		Wheat fermentation extract 7	356
Fermented wheat extract 8	289
		Limulus positive glycolipid	970
Low molecular weight lipopolysaccharide	993

[ Table 6]

Iodine-starch reaction of fermentation extract

Sample (I)	Measurement of
		Wheat fermentation extract 1	Positive for
Wheat fermentation extract 2	Positive for
		Wheat fermentation extract 3	Positive for
Wheat fermentation extract 4	Positive for
		Wheat fermentation extract 5	Positive for
Wheat fermentation extract 6	Positive for
		Wheat fermentation extract 7	Positive for
Fermented wheat extract 8	Positive for
		Limulus positive glycolipid	Negative of
Low molecular weight lipopolysaccharide	Negative of

[ Table 7]

Summary of differences between wheat fermentation extracts and similar products

Sample (I)	Protein content	Sugar content	Nucleic acid content	Content of limulus active substance	Iodine-starch reaction
						Wheat fermentation extract (mean value + -standard deviation)	86±15mg/g	327±57mg/g	212±75mg/g	255±113mg/g	Positive for
Limulus positive glycolipid	Too small	Too small	Not testing	Too much	Negative of
						Low molecular weight lipopolysaccharide	Too small	Too much	Too small	Too much	Negative of

Too small: significantly lower than the range of values (mean. + -. standard deviation) of the wheat fermented extract

Too much: significantly higher than the range of values (mean. + -. standard deviation) of the wheat fermented extract

[ example 3]

Immunity enhancing effect of fermented wheat extract

The acute myelogenous leukemia cell line THP-1 (1X 10) to be used as human macrophage⁶/250. mu.L of RPMI1640 medium containing 10% fetal bovine serum) were placed in 48-well plates and pre-incubated for 30 minutes. Then 250. mu.L of medium (final volume 500. mu.L) was added to give a final concentration of 1-10000ng/mL per sample. A group containing polymyxin B (12.5. mu.g/mL) in the sample was set up. After 4 hours of culture, culture supernatant and cells were collected. The TNF activity of the supernatants was measured by the cytotoxic assay using L-929. The results are shown in Table 8. TNF production by macrophages even in the presence of polymyxin B is possible with fermented wheat extracts, but with low molecular weightsLipopolysaccharide and limulus positive glycolipid, and macrophage can not produce TNF in the presence of polymyxin B. From this point, it is apparent that the fermented wheat extract has biological activities different from those of low molecular weight lipopolysaccharide and limulus positive glycolipid.

[ Table 8]

Production of macrophage TNF by fermented wheat extract and inhibitory action by polymyxin B (TNF-inducing activity of fermented wheat extract)

Sample concentration (ng/ml)	Fermented wheat extract containing polymyxin B	Fermented wheat extract without polymyxin B	Low molecular weight lipopolysaccharide added with polymyxin B	Low molecular weight lipopolysaccharide without polymyxin B	Limulus positive glycolipid containing polymyxin B	Limulus-positive glycolipid without polymyxin B
							0	0	0	0	0	0	0
1	0	0	0	0.64	0	1.2
							10	0	1.2	0	6.3	0	4.2
100	0	8.7	0	10.2	0	14.2
							1000	1.7	28.3	0	6.3	0	26.2
10000	26	50.4	0	3.8	0	13.2

B. Application example of wheat fermentation extract to feed

[ example 4]

Chicken raising feed containing wheat fermentation extract (large-scale research on suppression effect of death rate in broiler raising)

A feed containing 430. mu.g/kg of the fermented wheat extract prepared in example 2 was prepared. Each group uses about 5500-6000 commercial chickens suitable for roasting. The control group was fed with the feed containing no fermented wheat extract. The feed containing the fermented extract of wheat was supplied to the chickens which had hatched for 3 weeks, and was supplied daily for 7 weeks after hatching. The number of dead chickens was counted daily. Chickens that do not meet shipping criteria are discarded. The results are shown in Table 9. The removal rate of the test group (feed containing wheat fermentation extract) was low, 1.9%, and the control group was 3.3%. The feeding rate of the test group was 98.1%, and that of the control group was 96.7%. Thus, an increase in the feeding rate of 1.4% was observed. The number of actually delivered chickens and the number of removed chickens of the test group and the control group are subjected to significance difference test, and the difference is x²Significant differences were observed in the assay with p < 0.0001. From the above, it is seen that the feed for broiler chickens containing the fermented extract of wheat has an infection preventing effect.

[ Table 9]

Function of broiler chicken feeding feed containing wheat fermentation extract

	Test group	Control group
			Number of chicks	5906	5525
Actual number of chickens delivered	5792	5345
			Removing chicken number	114	180
Removal rate	1.9％	3.3％
			Rate of rearing	98.1％	96.7％

[ example 5]

Fish feed containing fermented wheat extract (infection prevention effect in herring open-air test)

To test the infection-preventing effect, approximately 5200 herring/group tested in the open air were fed with the feed containing the fermented extract of wheat produced in example 2. The results are shown in Table 10. The streptococcus mortality rate in the control group not given was 4.8%. In the group (test group) fed 100. mu.g/kg per day (per 1kg body weight and per day), a significant reduction in mortality (p < 0.0001) was observed compared to the non-administered group (control group).

[ Table 10]

Infection prevention of herring by feed containing fermented wheat extract in open-air test

Treatment of	Number of fish raised	Death number of fish	Mortality rate	Significance difference test (x)2Inspection)
					Control group	5201	249	4.79
Test group	5193	101	1.94	(p＜0.0001)

[ example 6]

Fish feed containing fermented wheat extract for preventing koi herpes (koi fishes) infection

(1) Carp: black carp weighing 70g was used. The test was performed on 20 carps/group.

(2) Preparing koi herpesvirus: 10ml of Hanks Balanced Salt Solution (HBSS) was added to 1g of carp gills dying from Koi herpes infection, homogenized, filtered through a 0.45 μm filter and the filtrate was made into virus solution.

(3) Koi herpesvirus infection: the filtrate was injected intraperitoneally (600. mu.L/100 g body weight).

(4) Preparing a feed containing a wheat fermentation extract: the fermented wheat extracts prepared in example 2 were mixed with commercial feeds at the ratios of 0, 5, 10 and 20 mg/kg.

(5) The feeding method comprises the following steps: once a day at 1% feed/body weight each. Corresponding to an amount of fermented wheat extract of 0, 50, 100 or 200. mu.g/kg body weight/day.

(6) Experiment: feeding Cyprinus Carpio with feed containing wheat fermented extract for one week, infecting with virus, and feeding for 10 days. The survival rate of carp within 10 days after virus infection was observed. The results are shown in FIG. 1.

All carp in the group not supplied with the fermented wheat extract at the end of day 6 died. Meanwhile, the survival rate in the group fed with the fermented extract of wheat at the 10 th day after infection showed a significant improvement (Kaplan Meier method, logrank test, risk percentage is 0.01% or less). Specifically, the survival rate of the group to which the fermented extract of wheat was supplied at 100. mu.g/kg body weight/day was 65%.

C. Application example of fermented extract of wheat to cosmetics and bathing agent

[ example 7]

Production of hand cream containing wheat fermentation extract

About 10% of the fermented wheat extract prepared in example 2 was mixed with an ointment of fat-soluble base 1 according to the formulation described in table 11 to obtain an ointment.

[ Table 11]

Composition (I)	Dosage of
		White petrolatum	250g
Stearyl alcohol	200g
		Propylene glycol	120g
Polyoxyethylene hardened castor oil 60	40g
		Glycerol monostearate	10g
Para-oxybenzoic acid methyl ester	1g
		Propyl p-oxybenzoate	1g
Purified water	Proper amount of

[ example 8]

Preparation method of moisturizing cream containing wheat fermentation extract

1. Formula of moisturizing cream containing wheat fermentation extract

The ingredients used are shown in table 12. The combination A was dissolved by heating at 70 ℃, the combination B was mixed with purified water in an amount of 1: 4 and heated/dissolved at 70 ℃, and the combination C was mixed with purified water in an amount of 1: 4 and heated/dissolved at 70 ℃, and both were added to A. This mixture was thoroughly mixed by a homogenizer and cooled to 40 ℃. The composition D was added thereto, and the pH was adjusted to 6.8. Subsequently, an appropriate amount of the remaining purified water and the fermented wheat extract prepared in example 2 were added and mixed to obtain an emulsion. The fermented extract of wheat was dissolved in purified water at 5mg/mL, and 0.1mL was added to 100g of the emulsion.

[ Table 12]

Composition (I)	w/w％	Combination of
			Squalane	5.0	A
Olive oil	10.0	A
			Jojoba oil	5.0	A
Stearic acid	4.0	A
			Polyoxyethylene sorbitan monostearate (20E.O.)	1.8	A
Methyl polysiloxane	0.3	A
			Sorbitan monostearate	0.5	B
Self-emulsifying glyceryl monostearate	3.0	B
			Para-oxybenzoic acid methyl ester	0.2	B
Propyl p-oxybenzoate	0.2	B
			1, 3-butanediol	5.0	B
Concentrated glycerin	6.0	B
			Carboxyvinyl polymer	0.22	C
Potassium hydroxide	Proper amount of	D
			Wheat fermentation extract (5mg/ml)	0.1
Purified water	Proper amount of
			Total up to	100.00

2. Moisturizing cream containing wheat fermentation extract

This cream was used by 43 men and women and was subjected to questionnaire. As a result, 18 responses did have moisturizing effect, 18 responses had slight moisturizing effect, 2 responses did not have moisturizing effect, and 5 responses did not (one sample sign test: p < 0.0001) for moisturizing effect. For the improvement effect of rough skin, 6 responses were indeed effective, 13 responses were slightly effective, no response was effective, and 24 were not (one sample sign test: p < 0.0001). For skin condition deterioration after use, no human response deteriorated. This cream was used by 4 people with mild atopy and questionnaire was performed. For the ameliorating effect of atopic dermatitis, 3 responded to it with certainty and 1 responded to it with slight effect (one sample sign test: p < 0.125). In addition, 1 person responded to acne and the scar healed rapidly. This cream was used by 9 people after shaving and questionnaires were performed. 8 answers it effectively reduced pain after shaving, prevented dryness and prematurely healed razor wounds (one sample sign test: p < 0.01). In addition, the cream was used for shoulder relief by 2 persons with age-induced shoulder stiffness. 1 answers that it is valid.

In addition, the cream is used by burn patients. In patients with the same degree of skin burn on both hands, cream containing the fermented wheat extract prepared in example 2 was used on one hand, and cream containing no fermented wheat extract prepared in example 2 was used on the other hand. The hands treated with the cream containing the wheat fermented extract healed significantly faster. The cream was used by 10 burn patients including this case. Thus, the wound healing was faster at all sites treated with the skin cream containing the wheat fermented extract than at the sites treated with the skin cream without the wheat fermented extract (Fisher exact probability: p < 0.001). From the above, it is seen that the fermented wheat extract shows an effect of treating burn.

[ example 9]

Production of astringent containing fermented wheat extract

1. Formula of toning lotion containing wheat fermentation extract

The ingredients used are shown in table 13. The fermented extract of wheat produced in example 2 was dissolved in purified water at 5mg/mL, and 0.1mL of the solution was added to 100g of cosmetic water.

[ Table 13]

Composition (I)	％
		Citric acid sodium salt	0.1
Carboxypyrrolidone sodium salt	1.0
		1, 3-butanediol	5.0
POE(30)POP(6)Decyltetradecylether	0.6
		Purified water	Proper amount of
Wheat fermentation extract (5mg/ml)	0.1
		Preservative	Proper amount of
Ethanol	10.0
		Total up to	100.0

2. Cosmetic lotion containing fermented extract of wheat

This lotion was used by 5 women and a questionnaire was performed. As a result, 3 women responded to it with good moisturizing effect, and 2 women responded to it with general moisturizing effect. All of these women had no skin problems.

[ example 10]

Preparation method of bathing agent containing fermented extract of wheat

A bathing agent containing fermented extract of wheat is prepared for improving body function. The basic components of the bathing agent are shown in Table 14.

[ Table 14]

Composition (I)	Content (wt.)
		Sodium sulfate	25.0g
Calcium silicate	0.26g
		Perfume (citrus junos)	0.5g

[0269]

A bathing agent containing the fermented wheat extract was prepared by adding 110. mu.g of the fermented wheat extract prepared in example 2 to the above components. The bathing agents containing the extract and the bathing agents containing no extract were arbitrarily given to 102 subjects who had bathed in (160-. As a result, it was observed that the improvement was 7% or more in (1) the degree of body warmth (10%), (2) the unpleasant feeling of feeling cold after bathing (7.9%), (6) the effect on muscular pain (13%), (8) the effect on lumbar pain (16%), (9) the effect on cold temperature sensitivity (10%) and (11) the improving effect on dry skin (7.3%) compared to the control (Mantel-Haenszel test: p < 0.04). From the above results, it was observed that the bathing agent using the fermented extract of wheat produced an effect of relieving muscular pain and improving body warmth.

D. Application example of wheat fermentation extract to functional food

[ example 11]

Production of candy containing fermented wheat extract

(1) As raw materials, sugar grains, starch syrup, water and the mixture of the fermented wheat extract prepared in example 2 were mixed at a ratio of 5: 1 and heated and cooked at 120-160 ℃.

(2) The resultant of (1) was cooled on a steel plate for cooling, stretched into a rod shape, and die-cast into pellets of about 1g to obtain candies.

The candy is dissolved in 20ml of water by heating. The amount of lipopolysaccharide as an active ingredient of the fermented extract of wheat in this solution was measured to be 4.6. mu.g/g. The candy was ingested by 6 men and women suffering from cold and sore throat. Thereafter, questionnaires on sore throats were conducted. For sore throat, 6 people all felt a reduction in sore throat (one sample sign test: p < 0.03).

[ example 12]

Production of alcoholic decomposition functional food containing fermented extract of wheat

The fermented wheat extract prepared in example 2 was mixed with a commercial product as an alcoholic decomposition functional food, and it was examined whether a new effect of alleviating sore throat was observed.

Commercial product: trademark "Nondeoiki"

The compositions are shown in Table 15.

[ Table 15]

Composition (I)	Ratio of component contents
		Powdered sugar	78.98％
Vitamin C	10.00％
		Toyoriden-P	5.00％
Vitamin B2	0.02％
		Spice (menthol)	0.50％
Gancao vine (Gynostemma pentaphylla) (saponin)	3.50％
		T-flavour Cone 13189B (flavonoid)	2.00％

Today 'noni' contains cranberry (Gynostemma pentaphylla) extract and green tea extract, but only lipopolysaccharide, one of the active ingredients of the plant extract, at about 0.002 μ g/bag. Therefore, it is desirable to obtain new functions by adding a proper amount of lipopolysaccharide-rich wheat fermentation extract. It is desirable to mix 1-30 μ g/2 g/bag of lipopolysaccharide of one of the active ingredients of fermented extract of wheat (such as fermented extract of wheat 5-150 μ g). Thus, first, a product in which 50. mu.g of the fermented extract of wheat is mixed per bag is produced. During the production of "Nondeoiki", 2.5mg of fermented wheat extract was added per 100g of the product. As a result, a new product containing 50. mu.g of the fermented extract of wheat per 2g of the product was produced.

With 20 adults and women of sore throat after drinking alcohol and karaoke as experimental subjects, conventional "non oiki" and "non oiki" containing fermented wheat extract were administered to 10 persons, respectively, and the enhancing effect on the alcohol-decomposing ability of the known effects and the alleviating effect on sore throat were examined. Immediately thereafter, questionnaires were conducted on the effect of alleviating sore throats. As a result, 8 of 10 persons receiving "non oiki" containing the fermented extract of wheat were observed to be relieved, but 2 of 10 persons receiving conventional "non oiki" were relieved. Thus, a statistically significant difference was observed compared to the control (Fisher exact probability: p < 0.012).

E. Examples relating to the therapeutic Effect of fermented extracts of wheat

[ example 13]

Preparation of Glycerol solution containing fermented extract of wheat (therapeutic effect on atopic dermatitis)

A50% glycerin solution containing 50. mu.g/ml of the fermented wheat extract prepared in example 2 was administered 2 to 3 times a day in a dose of 2 to 3ml per dose to 9 male and female patients (25 to 34 years) suffering from intractable atopic dermatitis, with skin rash observed on their face, hands, legs, trunk, neck, arms and back, and the subject's symptoms were moderate to severe. The symptoms (pruritus) of the subjects were classified into mild, moderate and severe grades according to patient complaints. After 2 weeks to 2 months from use, the patient is again referred to and the effect is evaluated. As a result, 4 (44%) cases were completely responded (rash was significantly improved and symptoms of the subject almost disappeared), 4 (44%) cases were partially responded (slight improvement in rash and reduction in symptoms of the subject), 1 (11%) case was unchanged, and 0 was deteriorated (one sample sign test: p < 0.03). From the above results, it was determined that the effective rate was 89%.

[ example 14]

Analgesic effect of fermented extract of wheat

The fermented extract of wheat prepared in example 2 was dissolved in distilled water and administered orally to mice in an gavage machine at 0.2ml per mouse. After 90 minutes, the mice were injected with 0.7% acetic acid in the abdominal cavity. After observing the mice for 5 minutes, the number of writings induced within 30 minutes was calculated. Results are shown in table 16 for each sample required to inhibit 30% of the number of twists compared to the distilled water control. The effective activity of the low molecular weight lipopolysaccharide derived from the fermented wheat extract is 1, and the effective activity of the fermented wheat extract is 7, showing that the fermented wheat extract shows excellent analgesic effect.

[ Table 16]

Analgesic effect of fermented wheat extract on pain induced by acetic acid in mice

Treatment of	Amount that results in 30% inhibition	Relative activity
			Distilled water	230±190mg	1
Fermented extract of wheat	33±35mg	7.0

[ example 15]

Inhibitory effect of fermented wheat extract on atopic dermatitis

To examine the effect of wheat fermentation extract on atopic dermatitis, a type I allergy model was introduced. BALB/c male mice (3-4 per group) were injected intravenously with anti-monoclonal antibodies (1. mu.g per mouse). After 1 hour, the fermented extract of wheat prepared in example 2 was injected subcutaneously (in the abdomen) (4. mu.g per mouse) or orally (100. mu.g per mouse). After another 1 hour, 20. mu.L of acetone-olive oil (4: 1) mixed solution containing 0.25% dinitrofluorobenzene was applied to the upper and lower surfaces of the auricle of the mouse as an allergen. Auricle thickness was measured with a thickness gauge after 1, 2, 24 and 48 hours of application. The value (Δ) obtained by subtracting the thickness immediately before application is the edema level. The inhibitory effect of the drug administration on the early-stage reaction observed 1 hour after allergen injection and the delayed reaction caused 24 hours after allergen injection was obtained from the inhibition ratio obtained by the following formula. Inhibition ratio (1-auricle edema Δ after administration/auricle edema Δ of control) × 100. The results are shown in Table 17. The table shows that the fermented wheat extract inhibits allergic reactions by subcutaneous injection and oral administration.

[ Table 17]

Inhibition of allergic reactions by fermented wheat extracts

Administration method of wheat fermentation extract	Dosage (/ mouse)	Inhibition (%) (after 1 hour)	Inhibition (%) (after 24 hours)
				Subcutaneous injection	4μg	81.0	102.1
Is administered orally	100μg	41.3	60.8

[ example 16]

Infection prevention effect of fermented wheat extract

To test the infection prevention effect of the wheat fermented extract, a methicillin-resistant staphylococcus aureus (MRSA) infection model was introduced. To BALB/c male mice (6-8 weeks old) (10 mice per group), cyclophosphamide (CY, 200mg/kg) was intraperitoneally injected, and after 5 days, the fermented wheat extract prepared in example 2 was subcutaneously injected. After 3 hours, MRSA (3X 10) was injected intravenously⁷Individual Colony Forming Units (CFU)), and days of survival were examined. The results are shown in Table 18. The table shows that the fermented wheat extract showed a statistically significant difference in the MRSA infection preventing effect (x) compared to the normal saline group (control)²And (4) checking: p < 0.001).

[ Table 18]

Prevention effect of fermented wheat extract on MRSA infection

Administration of drugs	Survival rate	Rate of risk
			Physiological saline	0/10
Wheat fermentation extract (0.004 mug)	9/10	P＜0.001
			Wheat fermentation extract (0.04 mug)	6/10	P＜0.005

[ example 17]

Therapeutic effect of fermented extract of wheat on metastatic cancer

To examine the therapeutic effect of fermented wheat extracts on metastatic cancer, a pulmonary metastatic model of Meth a cells was introduced. BALB/c male mice (6-8 weeks old) (10 mice per group) were intravenously injected with Meth A cancer cells (1X 10)⁵One cell), 12 days later, 4 consecutive days were subcutaneously injected with the fermented wheat extract prepared in example 2. 20 days after the transplantation of the cells, autopsy was performed, and the lungs were soaked and fixed with formalin. The lungs were visually observed and the number of nodules was counted. The results are shown in Table 19. The table shows that the wheat fermented extract showed a statistically significant difference in the treatment effect of Meth a lung metastatic cancer compared to the saline group (control) (t-test: p < 0.001).

[ Table 19]

Therapeutic effect of wheat fermentation extract on Meth A lung metastatic cancer

Administration of drugs	Knot number (mean. + -. standard deviation)	Rate of risk
			Physiological saline	60±11
Wheat hairYeast extract (40 mug/kg)	33±8	P＜0.001
			Wheat fermentation extract (400 mug/kg)	19±6	P＜0.001

F. Examples relating to fermented extract of bean curd lees

[ example 18]

Production of fermented extract of bean curd lees

(1)1.0L of water, 0.2g of potassium monohydrogen phosphate, 1.15g of sodium dihydrogen phosphate, 8g of sodium chloride and 0.2g of potassium chloride were placed in a 2-liter Erlenmeyer flask.

(2) Dried bean curd refuse (20g) was added to (1).

(3) And (2) sterilizing by using an autoclave.

(4) Preparation of inoculum: a colony of pantoea agglomerans separated from wheat flour was added to 5ml of 2% bean curd lees medium having the same composition as previously prepared, and fermented overnight (15 hours) at 37 ℃ with gentle stirring to prepare an inoculum for bean curd lees fermentation.

(5) The whole amount of (4) was added to (3), and the mixture was fermented at 37 ℃ for 48 hours with gentle stirring.

(6) (5) heating and extracting the fermented solution of bean curd residue in a pressure cooker at 120 deg.C for 20 min. The resultant was centrifuged (Kubota 8800, 2000rpm, 10 minutes), and the supernatant was collected to prepare a fermented extract of bean curd lees.

(7) Measurement of dry weight: 0.3ml was transferred into a weighed 1.5ml plastic tube, frozen and then lyophilized with a lyophilizer to obtain 5.97 mg. Thus, the dry weight of the fermented extract of bean curd lees in (6) is 19.9mg per 1ml of the solution and 19.9g per 1000ml of the total amount.

(8) Protein quantification BSA was used as a standard protein, and the amount of protein in the 10-fold diluted sample was measured by the Bradford method. The results are shown in Table 15.

(9) Measuring the content of nucleic acid: the absorption at 210-340nm of the 100-fold diluted sample was measured. The maximum content was calculated from the value obtained by subtracting the absorbance at 320nm from the absorbance at 260nm and the absorbance of DNA at 50. mu.g/10D.

(10) And (3) measuring the sugar content: the sugar content was measured by the phenol-sulfuric acid method using glucose as a standard sugar. .

(11) Measuring limulus active substance content by limulus assay: for the measurement, the Toxi-color system was supplied by Biochemical industries, Inc., and ET-1 was used as a standard limulus active substance. The measurement results are shown in table 20.

[ Table 20]

Component content of fermented extract of bean curd residue

Composition (I)	(mg/g)
		Protein	112
Candy	537
		Nucleic acids	Not detected
Limulus active substance	10

[ example 19]

Immunity enhancing effect of fermented extract of bean curd lees

The acute myelogenous leukemia cell line THP-1 (1X 10) to be used as human macrophage⁶/250. mu.L of RPMI1640 medium containing 10% fetal calf serum) were placed in 48-well plates and pre-incubated for 30 minutes. Subsequently, 250. mu.L of medium (final volume 500. mu.L) was added to give a final concentration of 100-10000 ng/mL per sample. A sample group containing polymyxin B (12.5. mu.g/mL) was set (a group of 100ng/mL containing no polymyxin B alone). After 4 hours of culture, culture supernatant and cells were collected. The TNF activity of the supernatants was measured by the cytotoxic assay using L-929. The results are shown in Table 21. TNF can be produced by macrophages even in the presence of polymyxin B by using the fermented extract of bean curd refuse, but TNF cannot be produced by macrophages in the presence of polymyxin B by using low molecular weight lipopolysaccharide. From this point, it is apparent that the fermented extract of bean curd refuse has biological activity different from low molecular weight lipopolysaccharide.

[ Table 21]

TNF production by macrophage cell via fermented extract of bean curd lees and inhibitory action by polymyxin B (TNF-inducing activity of fermented extract of bean curd lees)

TNF-inducing activity of fermented extract of bean curd lees and inhibitory effect of polymyxin B

Sample concentration (ng/ml)	Fermented extract of bean curd residue containing polymyxin B	Fermented extract of bean curd residue without polymyxin B	Low molecular weight lipopolysaccharide added with polymyxin B	Low molecular weight lipopolysaccharide without polymyxin B	Limulus positive glycolipid containing polymyxin B	Limulus positive glycolipid without polymyxin B
							0	0	0	0	0	0	0
100	N.D.	0.45	0	0.39	0	3.27
							1000	0.38	4.6	0	0.42	0.5	11.3
10000	11.1	11.1	0	0.28	14.4	25.3

N.D.: is not made

G. Examples relating to fermented extracts of rice flour

[ example 20]

Production of fermented extract of rice flour

(1)1.0L of water, 0.2g of potassium monohydrogen phosphate, 1.15g of sodium dihydrogen phosphate, 8g of sodium chloride and 0.2g of potassium chloride were placed in a 2-liter Erlenmeyer flask.

(2) Dried rice flour (20g) was added to (1).

(3) And (2) sterilizing by using an autoclave.

(4) Preparation of inoculum: an colony of Pantoea agglomerans isolated from wheat flour was added to 5ml of a medium containing 2% of rice flour having the same composition as previously prepared, and fermented overnight (15 hours) at 37 ℃ with gentle stirring to prepare an inoculum for rice flour fermentation.

(5) The whole amount of (4) was added to (3), and the mixture was fermented at 37 ℃ for 72 hours with gentle stirring.

(6) And (5) heating and extracting the rice flour fermentation solution in a pressure cooker at 120 ℃ for 20 minutes. This was centrifuged (Kubota 8800, 2000rpm, 10 minutes) and the supernatant was collected to make a rice flour fermentation extract.

(7) Measuring limulus active substance content by limulus assay: for the measurement, the Toxi-color system was supplied by Biochemical industries, and ET-1 from Biochemical industries was used as a standard limulus active substance. The content of limulus active substance in the rice flour fermented extract was measured to be 1.7. mu.g/mL.

[ example 21]

Immunity enhancing effect of rice flour fermented extract

The acute myelogenous leukemia cell line THP-1 (1X 10) to be used as human macrophage⁶/250. mu.L of RPMI1640 medium containing 10% fetal calf serum) were placed in 48-well plates and pre-incubated for 30 minutes. Subsequently, 250. mu.L of medium (final volume 500L) was added to give a final concentration of 1-10000ng/mL per sample. A sample group containing polymyxin B (12.5. mu.g/mL) was established. After 4 hours of culture, culture supernatant and cells were collected. The TNF activity of the supernatants was measured by the cytotoxic assay using L-929. The results are shown in Table 22. Fermentation of the extract with rice flour allowed macrophages to produce TNF even in the presence of polymyxin B, but with low molecular weight lipopolysaccharides, macrophages were unable to produce TNF in the presence of polymyxin B. From this point, it is apparent that the fermented extract of rice flour has biological activities different from those of low molecular weight lipopolysaccharide and limulus positive glycolipid.

[ Table 22]

TNF production by macrophages via rice flour fermentation extracts and inhibition by polymyxin B (TNF-inducing activity of rice flour extracts)

Sample concentration (ng/ml)	Rice flour fermentation extract added with polymyxin B	Fermented extract of rice flour without polymyxin B	Limulus positive glycolipid containing polymyxin B	Limulus positive glycolipid without polymyxin B
					0	0	0	0	0
1	0	0	0	0.1
					10	0	0	0	2.2
100	0	0.1	0	6.1
					1000	0.1	0.6	0	23.9
10000	0.5	2.4	0	29.3

H. Examples relating to fermented extracts of brown algae

[ example 22]

Preparation method of fermented extract of Undaria pinnatifida (brown seaweed) of brown algae

(1)1.0L of water, 0.2g of potassium monohydrogen phosphate, 1.15g of sodium dihydrogen phosphate, 8g of sodium chloride and 0.2g of potassium chloride were placed in a 2-liter Erlenmeyer flask.

(2) Dried brown algae Undaria pinnatifida (20g) was added to (1).

(3) And (2) sterilizing by using an autoclave.

(4) Preparation of inoculum: an inoculum for fermentation of brown algae wakame was prepared by adding a colony of pantoea agglomerans isolated from wheat flour to 5ml of 2% brown algae wakame medium having the same composition as previously prepared, and fermenting overnight (15 hours) at 37 ℃ with gentle stirring.

(5) The whole amount of (4) was added to (3), and the mixture was fermented at 37 ℃ for 72 hours with gentle stirring.

(6) And (5) heating and extracting the brown algae undaria pinnatifida fermentation solution in a pressure cooker at 120 ℃ for 20 minutes. The extract was centrifuged (Kubota 8800, 2000rpm, 10 minutes), and the supernatant was collected to prepare an extract of fermented Undaria brown algae.

(7) Measuring limulus active substance content by limulus assay: for the measurement, the Toxi-color system was supplied by Biochemical industries, Inc., and ET-1 was used as a standard limulus active substance. The content of limulus active substance in the fermented extract of brown algae Undaria pinnatifida was found to be 132. mu.g/mL.

[ example 23]

Immunity enhancing effect of fermented extract of brown algae Undaria pinnatifida

THP-1 (1X 10) to be used as human macrophage acute myelogenous leukemia cell line⁶/250. mu.L of RPMI1640 medium containing 10% fetal calf serum) were placed in 48-well plates and pre-incubated for 30 minutes. Subsequently, 250. mu.L of medium (final volume 500. mu.L) was added to give a final concentration of 1-10000ng/mL per sample. A sample group containing polymyxin B (12.5. mu.g/mL) was set up. After 4 hours of culture, culture supernatant and cells were collected. The TNF activity of the supernatants was measured by the cytotoxic assay using L-929. The results are shown in Table 23. The fermented extract of brown algae Undaria pinnatifida can produce TNF from macrophages even in the presence of polymyxin B, but low molecular weight lipopolysaccharide macrophages cannot produce TNF in the presence of polymyxin B. From this point, it is apparent that the fermented extract of brown algae Undaria pinnatifida has biological activities different from those of low molecular weight lipopolysaccharide and limulus positive glycolipid.

[ Table 23]

TNF production by macrophages due to Undaria pinnatifida extract and inhibitory effect by polymyxin B (TNF-inducing activity of Undaria pinnatifida extract)

Sample concentration (ng/ml)	Undaria pinnatifida extract containing polymyxin B	Phaeophyta Undaria Pinnatifida extract without polymyxin B	Limulus positive glycolipid containing polymyxin B	Limulus positive glycolipid without polymyxin B
					0	0	0	0	0
1	0	0	0	0.1
					10	0	0	0	2.2
100	0	3.2	0	6.1
					1000	2.4	14.4	0	13.9
10000	18.7	31.8	0	29.3

[0401] [ Industrial applicability]

According to the present invention, a fermented plant extract as a safe immunopotentiator can be produced inexpensively. The fermented plant extract obtained by this method can be used in drugs, drugs for animals, quasi drugs, cosmetics, foods, functional foods, feeds and bath agents for mammals including humans (specifically domestic animals, pet animals, etc.), birds (specifically raised chickens, pet birds, etc.), amphibians, reptiles, fish (specifically water-cultured fish, pet fish, etc.) and invertebrates.

Claims (2)

1. A method of fermentation and culture characterized by using M9 medium containing 0.5% wheat flour or wheat flour treated with amylase, fermenting with Enterobacter agglomerans symbiotically coexisting with wheat, and simultaneously culturing said Enterobacter agglomerans.

2. A method of fermentation and culture characterized by fermenting bean curd refuse with Enterobacter agglomerans living symbiotically with wheat and simultaneously culturing said Enterobacter agglomerans.