JP7515755B2 - Equol-containing food composition and method for producing same - Google Patents

Description

The present invention relates to a food composition containing equol and an organic acid. In particular, the present invention relates to a food composition containing equol and an organic acid, which has an improved flavor, particularly an improved aroma. The present invention also relates to a method for producing the food composition.

Equol-containing compositions are often used primarily as foods, and their flavor, particularly their aroma, is an important factor.
Although it has been known to add flavoring agents to food compositions containing equol to improve their flavor (Patent Document 1) and to add pH adjusters to control the pH (Patent Document 2), the use of pH adjusters to improve flavor was not known.

JP 2015-139424 A Patent No. 5769419

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a food composition containing equol and an organic acid, which has an improved flavor, particularly an improved aroma.
Another object of the present invention is to provide a method for producing a food composition containing equol and an organic acid, which has an improved flavor, particularly an improved aroma, in addition to or other than the above-mentioned object.

In order to solve the above problems, the present inventors have discovered the following invention.
<1> A food composition containing equol and an organic acid.
<2> In the above item <1>, the content of the organic acid is 1.5 to 29% by weight, preferably 1.7 to 25% by weight, and more preferably 2.0 to 22% by weight, based on 100% by weight of the food composition.

<3> In the above item <1> or <2>, the organic acid may be at least one selected from the group consisting of carbonic acid, hydrogen carbonate, citric acid, lactic acid, phosphoric acid, gluconic acid, acetic acid, malic acid, orotic acid, and ammonium salts, alkali metal salts, alkaline earth metal salts, and chelates thereof.
In particular, the organic acid is preferably at least one selected from the group consisting of citric acid, lactic acid, acetic acid, malic acid, orotic acid, phosphoric acid, and gluconic acid, more preferably at least one selected from the group consisting of citric acid, lactic acid, phosphoric acid, gluconic acid, acetic acid, and malic acid, and most preferably at least one selected from the group consisting of citric acid, lactic acid, phosphoric acid, and acetic acid.

<4> In any one of the above items <1> to <3>, the equol may be produced by utilizing a culture process of anaerobic microorganisms.
<5> In the above item <4>, the anaerobic microorganism may be a microorganism belonging to the genus Asaccharobacter or Adlercreutzia.
<6> In either of the above items <4> or <5>, the pH of the culture solution at the end of the culture step is adjusted to 3.0 to 5.5, preferably 3.5 to 5.0, and more preferably 4.0 to 5.0, with the organic acid.

<7> A method for obtaining an equol-containing food composition, comprising the step of culturing at least one equol raw material selected from the group consisting of daidzein glycoside, daidzein, and dihydrodaidzein with an anaerobic microorganism,
A method for producing an equol-containing food composition, characterized in that in the culture process, at least one organic acid selected from the group consisting of carbonic acid, bicarbonate, citric acid, lactic acid, phosphoric acid, gluconic acid, acetic acid, malic acid and orotic acid, as well as their ammonium salts, alkali metal salts, alkaline earth metal salts and chelates are added.
The organic acid is preferably at least one selected from the group consisting of citric acid, lactic acid, acetic acid, malic acid, orotic acid, phosphoric acid, and gluconic acid, more preferably at least one selected from the group consisting of citric acid, lactic acid, phosphoric acid, gluconic acid, acetic acid, and malic acid, and most preferably at least one selected from the group consisting of citric acid, lactic acid, phosphoric acid, and acetic acid.

<8> In the above item <7>, when the equol-containing food composition is taken as 100% by weight, the content of the organic acid is 1.5 to 29% by weight, preferably 1.7 to 25% by weight, and more preferably 2.0 to 22% by weight.
<9> In the above item <7> or <8>, the anaerobic microorganism may be a microorganism belonging to the genus Asaccharobacter or Adlercreutzia.
<10> In any one of the above items <7> to <9>, the pH of the culture solution at the end of the culture step is adjusted to 3.0 to 5.5, preferably 3.5 to 5.0, and more preferably 4.0 to 5.0, with the organic acid.

INDUSTRIAL APPLICABILITY The present invention can provide a food composition containing equol and an organic acid, which has an improved flavor, particularly an improved aroma.
Furthermore, the present invention can provide a method for producing a food composition containing equol and an organic acid, which has an improved flavor, particularly an improved aroma, in addition to or other than the above effects.

The present invention provides a food composition comprising equol and an organic acid. The present invention particularly provides a food composition comprising equol and an organic acid, which has an improved flavor, particularly an improved aroma.
The present invention also provides a method for producing the food composition.

<Food composition containing equol and organic acid>
The present invention provides a food composition comprising equol and an organic acid. The present invention particularly provides a food composition comprising equol and an organic acid, which has an improved flavor, particularly an improved aroma.
In the present application, the term "flavor" refers to aroma, and in the case of edible products, taste, etc. In the present invention, the "aroma" in particular can be improved.
The food composition of the present invention comprises an organic acid.
The organic acid may be at least one selected from the group consisting of carbonic acid, hydrogen carbonate, citric acid, lactic acid, phosphoric acid, gluconic acid, acetic acid, malic acid, and orotic acid, as well as their ammonium salts, alkali metal salts, alkaline earth metal salts, and chelates. In particular, at least one selected from the group consisting of citric acid, lactic acid, acetic acid, malic acid, orotic acid, phosphoric acid, and gluconic acid is preferred, at least one selected from the group consisting of citric acid, lactic acid, phosphoric acid, gluconic acid, acetic acid, and malic acid is more preferred, and at least one selected from the group consisting of citric acid, lactic acid, phosphoric acid, and acetic acid is most preferred.

The organic acid contains, when the food composition is taken as 100% by weight,
It is preferably 1.5 to 29% by weight, more preferably 1.7 to 25% by weight, and even more preferably 2.0 to 22% by weight.

Equol is preferably produced using an anaerobic microbial cultivation process.
The anaerobic microorganism may be a microorganism belonging to the genus Asaccharobacter or Adlercreutzia.
In addition, the pH of the final culture solution in the culture step is adjusted with the organic acid to 3.0 to 5.5, preferably 3.5 to 5.0, and more preferably 4.0 to 5.0.
The culture step will be described in detail in the method for producing the food composition of the present invention.

The composition of the present invention may contain ingredients other than equol or organic acids, so long as they do not impair the flavor.
Examples of the above-mentioned components include, but are not limited to, amino acids, sugars, vitamins, minerals, isoflavones, dietary fiber, excipients, and the like.

<Method of producing equol-containing food composition>
According to the present invention, the above-mentioned food composition, particularly a food composition having improved flavor, particularly aroma, can be obtained, for example, by the following production method.
The present invention also provides the production method.
The production method includes a step of culturing at least one equol raw material selected from the group consisting of daidzein glycoside, daidzein, and dihydrodaidzein with an anaerobic microorganism, thereby obtaining an equol-containing food composition,
The method is characterized in that the organic acid is added in the culture step.

The method of the present invention comprises the step of culturing at least one equol raw material selected from the group consisting of daidzein glycoside, daidzein and dihydrodaidzein with an anaerobic microorganism.
The conditions for the culture step are not particularly limited as long as equol can be produced. For example, conventionally known conditions can be used, but the conditions are not limited to these.

<<Equol raw material>>
The equol raw material used in the production method of the present invention may be in any form so long as it can be literally used as a raw material for equol.
The equol raw material may be at least one selected from the group consisting of daidzein glycoside, daidzein, and dihydrodaidzein, and may be in any form, such as daidzein glycoside itself, daidzein itself, or dihydrodaidzein itself, or may be a substance containing them, such as soybeans, processed soybeans, soybean hypocotyls, processed soybean hypocotyls, such as soybean hypocotyl extracts, and specifically commercially available isoflavones.

<<Anaerobic microorganisms>>
The equol-utilizing microorganism used in the production method of the present invention is not particularly limited as long as it is an anaerobic microorganism that produces equol from the above-mentioned equol raw material.
Anaerobic microorganisms, for example, can produce equol at temperatures around 37° C. (eg, 30 to 42° C.).
The equol assimilation ability can be confirmed by quantifying daidzein, dihydrodaidzein, equol, etc. in the culture. These quantifications can be performed by those skilled in the art based on the descriptions in, for example, WO2012/033150, JP 2012-135217, JP 2012-135218, JP 2012-135219, etc. An example of these quantification methods is shown below.

For example, ethyl acetate is added to the culture solution, which is then vigorously stirred and centrifuged to remove the ethyl acetate layer. If necessary, the same procedure can be performed several times on the same culture solution, and the ethyl acetate layers can be combined to obtain an equol extract. This extract is concentrated and dried under reduced pressure in an evaporator, and dissolved in methanol. This is then filtered using a membrane such as a polytetrafluoroethylene (PTFE) membrane, and the insoluble matter is removed to obtain a high-performance liquid chromatography measurement sample. Examples of high-performance liquid chromatography conditions include, but are not limited to, the following.

[High performance liquid chromatography conditions]
Column: Phenomenox Luna 5uC18, 2.0mm x 150mm (Shimadzu GLC)
Mobile phase: water/methanol [55:45, v/v]
Flow rate: 0.2 mL / min
Column temperature: 40°C
Detection: UV 280 nm
Retention times: dihydrodaidzein 13.8 min, daidzein 19.6 min, glycitein 22.5 min, equol 25.6 min, genistein 35.0 min

Anaerobic microorganisms may include, but are not limited to, microorganisms classified into the following genera:
Genus Adlercreutzia Genus Asaccharobacter Genus Bacteroides Genus Bifidobacterium Genus Clostridium Genus Eggerthella
Genus Enterococcus Genus Enterorhabdus Genus Eubacterium Genus Finegoldia Genus Lactobacillus Genus Lactococcus Genus Paraeggerthella
Genus Pediococcus Genus Sharpea Genus Slackia Genus Streptococcus Genus Veillonella

Specific examples of anaerobic microorganisms include, but are not limited to, the following microorganisms:
Assaccharobacter celatus
Adlercreutzia equolifaciens
Bacteroides ovatus
Bifidobacterium breve
Bifidobacterium longum
Clostridium sp.
Eggerthella sp.
Enterococcus faecalis
Enterococcus faecium
Enterorhabdus mucosicola
Eubacterium sp.
Finegoldia magna
Lactobacillus fermentum
Lactobacillus mucosae
Lactobacillus paracasei
Lactobacillus plantarum
Lactobacillus rhamnosus
Lactobacillus sp.
Lactococcus garvieae
Paraeggerthella sp.
Pediococcus pentosaceus
Sharpea azabuensis
Slackia equolifaciens
Slackia isoflavoniconvertens
Slackia sp.
Streptococcus constellatus
Streptococcus intermedius
Veillonella sp.

Among the above-mentioned microorganisms, any of the microorganisms described below or related microorganisms having similar species properties to these microorganisms can be given as more preferred anaerobic microorganisms.
- Microorganisms belonging to the genus Asaccharobacter or Adlercreutzia.
In particular, it is preferable that:
・Asaccharobacter celatus DSM 18785 strain ・Adlercreutzia equolifaciens DSM 19450 strain

The above anaerobic microorganisms can be obtained from the depository institutions indicated by their accession numbers. Each accession number indicates that the anaerobic microorganism has been deposited at the following depository institutions:
FERM International Patent Organism Depositary (IPOD)
http://unit.aist.go.jp/pod/ci/index.html
DSM German Collection of Microorganisms and Cell Cultures (DSMZ)
http://www.dsmz.de/
KCCM Korean Culture Center of Microorganisms

In the present invention, anaerobic microorganisms capable of producing equol are cultured under conditions suitable for the production of equol. In the present invention, conditions suitable for the production of equol refer to conditions under which the survival and activity of anaerobic microorganisms capable of producing equol are maintained. More specifically, gas phase conditions (anaerobic conditions) in which anaerobic microorganisms can survive are maintained, and nutrients are provided to support the activity and growth of the anaerobic microorganisms. Various medium compositions suitable for the survival of anaerobic microorganisms are known. Therefore, for the anaerobic microorganisms capable of producing equol shown above, a person skilled in the art can select an appropriate medium composition. For example, BHI medium manufactured by Difco and the medium used in the examples can be used, but are not limited to these.

Water-soluble organic substances can be added to the medium used in the present invention as a carbon source. Examples of water-soluble organic substances include, but are not limited to, the following compounds:
Sugars such as sorbose, fructose, and glucose;
Alcohols such as methanol;
Organic acids such as valeric acid, butyric acid, propionic acid, acetic acid, formic acid, etc., or their salts.

The concentration of organic matter added to the medium as a carbon source can be adjusted as appropriate to efficiently grow anaerobic microorganisms in the medium. In general, excess or deficiency can be avoided by selecting an addition amount in the range of 0.1 to 10 wt/vol%.

In addition to the carbon sources, a nitrogen source is added to the medium. In the present invention, various nitrogen compounds that can be used in normal fermentation can be used as the nitrogen source. Preferred inorganic nitrogen sources are ammonium salts and nitrate salts. More preferred inorganic nitrogen sources are ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, potassium nitrate, and sodium nitrate.
On the other hand, preferred organic nitrogen sources are amino acids, yeast extract, peptones (e.g., polypeptone N, etc.), meat extract, liver extract, digested serum powder, etc. More preferred organic nitrogen sources are arginine, cysteine, citrulline, lysine, yeast extract, and peptones (e.g., polypeptone N, etc.).

In addition to the carbon and nitrogen sources, other organic or inorganic substances suitable for the production of equol can also be added to the medium. For example, the growth and activity of anaerobic microorganisms can be enhanced by adding cofactors such as vitamins or inorganic compounds such as various salts to the medium. For example, the following are examples of inorganic compounds, vitamins, and microbial growth cofactors derived from plants and animals:

Inorganic compounds Vitamins Potassium dihydrogen phosphate Biotin Magnesium sulfate Folic acid Manganese sulfate Pyridoxine Sodium chloride Thiamine Cobalt chloride Riboflavin Calcium chloride Nicotinic acid Zinc sulfate Pantothenic acid Copper sulfate Vitamin B12
Alum, Thiooctic acid, Sodium molybdate, p-aminobenzoic acid, Potassium chloride, Boric acid, Nickel chloride, Sodium tungstate, Sodium selenate, Ammonium ferrous sulfate

A conventional method can be used to produce a culture solution by adding these inorganic compounds, vitamins, or growth cofactors. The medium can be liquid, semi-solid, or solid. In the present invention, the preferred form of the medium is a liquid medium.
The medium of the present invention can contain dextrins. By culturing anaerobic microorganisms in a medium containing dextrins, a liquid containing equol and dextrins can be prepared without contacting the culture with dextrins after the culture.
Dextrins can be added to the medium before or during the culture of the microorganism.

In the present invention, anaerobic microorganisms can be cultured according to known microbial culture methods. For industrial production, a continuous fermentation system can be used that can continuously supply the medium and substrate gas and has a mechanism for recovering the culture.

When using anaerobic microorganisms in the production of equol-containing food compositions, it is advisable to prevent oxygen from entering the continuous culture system. A normally used culture tank can be used as the incubator. Culture tanks that can also be used to produce equol are commercially available. An anaerobic atmosphere can be created by replacing the oxygen that enters the culture tank with an inert gas such as nitrogen.

Depending on the shape of the culture tank, an agitator or similar device can be used to thoroughly agitate the culture medium. By agitating the culture in the culture tank, the opportunities for the medium components and substrate gas to come into contact with the anaerobic microorganisms can be increased, optimizing the efficiency of equol production. The substrate gas can also be supplied as nanobubbles.

In the present invention, equol may be produced in a closed system such as a bottle or test tube sealed with a rubber stopper without ventilation, but it may also be produced in a gas phase consisting of one or more gases including hydrogen. When produced in a gas phase, there is no particular limitation on the hydrogen concentration.

When the method of the present invention is carried out in the gas phase, the combination of gases constituting the gas phase is not particularly limited, and in addition to hydrogen, one or more gases selected from carbon dioxide, nitrogen, etc. can be used as constituent components. In order to efficiently recover equol, it is preferable that the amount of mixed gas constituting the gas phase aerated into the culture tank is 0.01 to 2.0 V/V/M gas volume/liquid volume/min.

In the present invention, anaerobic microorganisms can be cultured at normal pressure, but when pressurized, the pressurization conditions are not particularly limited as long as they allow the microorganisms to grow. Preferred pressurization conditions include, but are not limited to, a range of 0.02 to 0.2 MPa.

To increase the amount of equol recovered, the temperature of the culture tank is not particularly limited, but is preferably 30°C to 40°C, and more preferably 33°C to 38°C.
The fermentation time can be appropriately set depending on the amount of equol produced, the amount of isoflavones remaining, etc. Examples of the fermentation time include, but are not limited to, 8 to 120 hours, preferably 12 to 72 hours, and particularly preferably 16 to 60 hours.

An organic acid can be added to the culture solution at the end of the culture process to improve flavor. There are no restrictions on the amount added as long as the flavor improvement effect is achieved, but for example, the pH can be used as an indicator. When adding the organic acid, it is recommended to adjust the pH of the culture solution to 3.0 to 5.5, preferably 3.5 to 5.0, and more preferably 4.0 to 5.0 with the organic acid.

The fermentation culture obtained by the culture process of the present invention can be used in a solid form by heat drying, spray drying, or freeze drying, as necessary. Heat drying or spray drying can be performed, for example, using a spray dryer. Freeze drying can be performed using a freeze dryer. The fermentation culture that has been heat dried, spray dried, or freeze dried can be subjected to a powdering process as necessary.

The food composition containing equol obtained by the culture process of the present invention can be provided as an ingredient for food and beverages (including supplements), etc.

When the equol-containing food composition of the present invention is provided as food or drink, it can be used as a general food, a food for specified health uses, a nutritional supplement, a functional food, a food for the sick, a food additive, etc. Examples of the form of food that contains the equol-containing food composition of the present invention include soft drinks, milk, pudding, jelly, candy, gum, gummy candy, yogurt, chocolate, soup, cookies, snacks, ice cream, popsicles, bread, cake, cream puffs, ham, meat sauce, curry, stew, cheese, butter, dressing, supplements, beverages with specified health benefits, and nutritional drinks.

The equol-containing food composition of the present invention can be made into an equol-containing composition by using water, proteins, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juice, flavors, etc. as main components. Examples of proteins include animal and vegetable proteins such as whole milk powder, skim milk powder, partially skim milk powder, casein, soy protein, egg protein, meat protein, etc., as well as their hydrolysates, butter, etc. Examples of carbohydrates include sugars, processed starches (textlin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fibers, etc. Examples of lipids include animal fats and oils such as lard, fish oil, fractionated oils thereof, hydrogenated oils, and interesterified oils, and vegetable fats and oils such as palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, hydrogenated oils, and interesterified oils. Examples of vitamins include vitamin A, carotenes, B vitamins, vitamin C, D vitamins, vitamin E, K vitamins, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline, folic acid, etc., and examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, whey minerals, etc. Two or more of these components can be used in combination, and synthetic products and/or foods containing large amounts of these may also be used.

The proportion of the equol-containing food composition in these foods can be appropriately set depending on the type of food, the equol content, the age and sex of the person taking it, the expected effect, etc. An example is a ratio of 0.01 to 100 g, preferably 0.1 to 10 g, and more preferably 0.5 to 5 g per 100 g of food, but is not limited to these. The daily intake of a food containing an equol-containing food composition will vary depending on the equol content in the composition, the age and weight of the person taking it, the number of times it is taken, etc., but an example is an amount of the composition equivalent to 0.01 to 10 g per day for an adult.
The present invention will be described based on examples, but the scope of the present invention is not limited to the following examples.

Example 1
(Preparation of pre-culture medium)
1 L of Anaerobe Basal Broth (ABB) medium (Thermo Scientific) was dispensed into a 2 L fermentation tank, which was then sterilized by replacing the gas with nitrogen gas.
(Preparation of main culture medium)
Daidzein was added to the ABB medium to a concentration of 600 mg/L, and 1 L of the mixture was dispensed into a 2 L fermentation tank, after which the tank was sterilized by replacing the gas with nitrogen gas.
(Preculture)
After inoculating the preculture medium with Asaccharobacter celatus DSM 18785 strain, the gas was replaced with nitrogen gas and the medium was cultured at 37° C. for 1 day.

(Main culture)
Each of the above precultured strains was inoculated into the main culture medium, and the gas was replaced with a hydrogen/nitrogen mixed gas, followed by culturing at 37° C. for 5 days.
(Acidification step)
The pH of the liquid obtained after the completion of the main culture was lowered to 5.0 by adding citric acid, lactic acid, phosphoric acid, gluconic acid, acetic acid, malic acid, and orotic acid, and then sterilized by heating.
(freeze drying)
After completion of the main culture, the liquid was sterilized using a filter with a pore size of 0.2 μm and then freeze-dried to obtain Powder 1.

Example 2
Powder 2 was obtained in the same manner as in Example 1, except that Adlercreutzia equolifaciens DSM 19450 strain was used instead of Asaccharobacter celatus DSM 18785 strain in Example 1.

Comparative Example 1
Powder C1 was obtained in the same manner as in Example 1, except that 10% hydrochloric acid was used instead of the various organic acids in Example 1.
Comparative Example 2
Powder C2 was obtained in the same manner as in Example 2, except that 10% hydrochloric acid was used instead of the various organic acids in Example 2.

(Aroma evaluation)
The resulting powders 1, 2, C1, and C2 were evaluated for fragrance. Four men and women in their 30s to 50s were selected to evaluate the fragrance through a sensory test, and the results were rated AAA to A according to the following criteria.

AAA: Good fragrance;
AA: There is a smell from the culture medium, but it is not bothersome;
A: It has a scent derived from the culture medium.

The results of the sensory test are shown in Tables 1 and 2. In the tables, "AC" represents the Asaccharobacter celatus DSM 18785 strain, and "AE" represents the Adlercreutzia equolifaciens DSM 19450 strain.
As is clear from Tables 1 and 2, it was confirmed that powders 1 and 2 of Examples 1 and 2, i.e., powders obtained by using an organic acid as a pH adjuster, have a reduced characteristic odor derived from the medium components compared to powders C1 and C2 of Comparative Examples 1 and 2, i.e., powders obtained by using hydrochloric acid as a pH adjuster. The content of each organic acid in the powder is shown in Table 3.

Claims (1)

A food composition comprising lactic acid or gluconic acid, and equol, wherein the content of said lactic acid or gluconic acid is 1.5 to 29% by weight when the food composition is taken as 100% by weight, and said food composition is a jelly or a soft drink .