JP6792781B2 - Fermented seasoning with high antihypertensive peptide content - Google Patents

Description

The present invention relates to a fermented seasoning containing a high amount of the antihypertensive peptides Gly-Tyr and Ser-Tyr, and a method for producing the same.

In recent years, various physiological functions of peptides have been clarified. For example, Gly-Tyr and Ser-Tyr are known to have angiotensin converting enzyme inhibitory action and blood pressure lowering action (Non-Patent Document 1). Therefore, efficient ingestion of Gly-Tyr and Ser-Tyr (hereinafter, may be referred to as antihypertensive peptide Gly-Tyr and antihypertensive peptide Ser-Tyr) is effective in improving hypertension and arteriosclerosis. It can be said that.

On the other hand, soy sauce is a seasoning that has been used for a long time, and the enzyme produced by Jiuqu decomposes soybeans and wheat to produce a rich flavor. This umami is produced by the action of protease on the raw material protein to solubilize it as a peptide, and the action of peptidase on these peptides to decompose them into amino acids such as glutamic acid. So far, as an attempt to contain the antihypertensive peptide Gly-Tyr and the antihypertensive peptide Ser-Tyr in soy sauce, the activities of leucine aminopeptidase-I and leucine aminopeptidase-II in the soy sauce juice have been controlled. A method of suppressing the decomposition of solubilized peptides (Patent Document 1) and a method of combining the use of low protease soy sauce koji with warm digestion of the soy sauce koji have been reported (Patent Document 2). However, it is desired to develop a fermented seasoning having a high blood pressure lowering ability by increasing the antihypertensive peptide Gly-Tyr and the antihypertensive peptide Ser-Tyr produced and accumulated in soy sauce moromi by a simpler method.

Japanese Patent No. 5626748 Japanese Patent No. 5628833

K. Suetsuna, J. et al. Nutr. Biochem. , 1998, (9), p. 415-419

An object of the present invention is to provide a fermented seasoning containing more antihypertensive peptides Gly-Tyr and Ser-Tyr than conventional products and having a higher blood pressure lowering effect.

As a result of diligent research to solve the above problems, the present inventors have obtained soybeans having β-conglycinin of 50 parts by weight or more when the total of β-conglycinin and glycinin in the raw material soybean is 100 parts by weight. The fermented seasoning obtained by mixing with soy sauce koji and adding it to salt solution to prepare moromi and fermenting the moromi at 25 ° C to 60 ° C is surprisingly better than when ordinary soybeans are used as raw materials. Also found that the contents of the antihypertensive peptides Gly-Tyr and Ser-Tyr were significantly increased, leading to the present invention.

That is, the present invention includes the following inventions.
(1) Fermentation characterized in that the content of β-conglycinin with respect to the total amount of β-conglycinin and glycinein in the raw material soybean is 50 w / w% or more, and Gly-Tyr and Ser-Tyr are contained. seasoning.
(2) The fermented seasoning according to (1), wherein the raw material soybean is a hanging material.
(3) The fermented seasoning according to (1) or (2), wherein the raw material soybean contains 11S globulin-deficient or reduced soybean.
(4) The fermented seasoning according to any one of (1) to (3), wherein the raw material soybean contains a single or a plurality of varieties of soybean.
(5) The fermented seasoning according to any one of (1) to (4), which contains 70 μg / ml or more of Gly-Tyr and 100 μg / ml or more of Ser-Tyr per 1 w / w% of total nitrogen concentration.
(6) The fermented seasoning according to any one of (1) to (5), which is a fermented seasoning for lowering blood pressure.
(7) Fermented seasoning containing Gly-Tyr and Ser-Tyr, which comprises a step of mixing raw soybeans, a culture of aspergillus and salt solution to prepare various flavors, and a step of fermenting the various flavors at 25 to 60 ° C. The method for producing a fermented seasoning, wherein the content of β-conglycinin with respect to the total amount of β-conglycinin and glycinin in the raw material soybean is 50 w / w% or more.

According to the present invention, there is provided a fermented seasoning in which the antihypertensive peptide Gly-Tyr and the antihypertensive peptide Ser-Tyr are significantly increased as compared with the conventional product.

FIG. 1 shows the SDS-PAGE analysis results of β-conglycinin and glycinin contained in ordinary soybean and soybean containing high β-conglycinin.

Hereinafter, the present invention will be described in detail.
The fermented seasoning of the present invention has a β-conglycinin content of 50 w / w% or more with respect to the total amount of β-conglycinin and glycine in the raw soybean, and contains Gly-Tyr and Ser-Tyr. It is a feature.

(Ingredient soybean)
Soybean storage proteins are classified into 2S, 7S, 11S and 15S globulin by sedimentation coefficient (S) and glycinin, α-, β- and γ-conglycinin by immunological nomenclature. Of these, 7S globulin (β-conglycinin) and 11S globulin (glycinin) make up the majority of soybean storage proteins, and 7S globulin is composed of α, α', and β subunits, while 11S globulin is composed of A1aB2, A1bB1b, A2B1a, A5A4B3 and A3B4 subunits obtained by polymerizing an acidic polypeptide (A) and a basic polypeptide (B) with a disulfide bond (SS bond). The raw material soybean used in the fermented seasoning of the present invention refers to soybean having a β-conglycinin content higher than that of glycinin, and specifically, β-conglycinin with respect to the total amount of β-conglycinin and glycinin in the raw material soybean. The content of soybean may be 50 w / w% or more, but 70 w / w% or more is preferable, 80 w / w% or more is more preferable, 90 w / w% or more is further preferable, and 95 w / w% or more is most preferable. 11S globulin-deficient soybean having an amount of β-conglycinin of 50 w / w% or more based on the total amount of β-conglycinin and glycinin used as the raw material soybean of the present invention (hereinafter referred to as “β-conglycinin high content soybean”) Or reduced soybeans, for example, soybeans in which β-conglycinin (7S globulin) content is increased by deleting part or all of glycinin (11S globulin) such as "Nanahomare" and "Kanto 133", fertilization, etc. Examples thereof include soybeans having a relatively increased β-conglycinin (7S globulin) content under the cultivation conditions of, but are not limited thereto.

As the above soybean varieties, a single variety may be used, or a plurality of varieties may be blended and used. Further, as long as the content of β-conglycinin in the raw material soybean is within the above range, general soybean may be mixed with the soybean containing high β-conglycinin. Furthermore, by mixing separated and purified β-conglycinin (for example, “Lipov” manufactured by Fuji Oil Co., Ltd.) with the raw material soybean, the content of β-conglycinin in the raw material soybean with respect to the total amount of β-conglycinin and glycinin is increased. You may let me.

The raw material soybean can be used as it is, or after being crushed and crushed, and then appropriately subjected to protein denaturation treatment. As the protein denaturation treatment, pressure steaming and swelling treatment, which are widely performed industrially, are preferable.

In the production of the fermented seasoning of the present invention, the above-mentioned raw material soybean is preferably used as a hanging material to be mixed with the jiuqu at the time of preparing various flavors, but it can also be used as a raw material for making koji.

(Jiuqu culture)
As the aspergillus culture, "soy sauce koji" widely and generally used in soy sauce brewing can be used. Specifically, it is obtained by heat-modifying soybeans, defatted soybeans, wheat gluten, etc., and heat-modifying wheat (wheat, barley, bare wheat, hato wheat), rice, etc. It can be obtained by inoculating a solid medium containing a starchy raw material with seed aspergillus and culturing (jiuqu) at 25 to 45 ° C. for 12 to 240 hours. The heat denaturation of wheat is preferably performed by stir-fry crushing, and the heat denaturation of rice is preferably performed by steaming or cooking rice. Here, when soybean is used as a protein raw material, ordinary soybean may be used, or the above-mentioned β-conglycinin-rich soybean may be used. Further, as the aspergillus culture, "liquid cultured aspergillus" used for producing fermented seasonings can also be used. Specifically, it can be obtained by inoculating the seed aspergillus in a liquid medium containing 1 to 5 w / v% of the above protein raw material and starchy raw material and culturing (jiuqu) at 25 to 45 ° C. for 24 to 120 hours. it can. Here, Aspergillus sojae, Aspergillus oryzae, and the like are used as the seed aspergillus.

The blending ratio of the protein raw material and the starch raw material (protein raw material: starch raw material) is not particularly limited, but is, for example, the blending ratio used in dark soy sauce and light soy sauce from 30:70 to 70:30. Is preferable, and 40:60 to 60:40 is more preferable. The water content of the mixture of the protein raw material and the starchy raw material is preferably 35 to 50 w / w%, more preferably 40 to 45 w / w%.

(Preparation of moromi)
Next, the aspergillus culture, soybeans high in β-conglycinin as a hanging material, and saline solution are mixed (prepared) to prepare moromi mash. The conditions for the preparation were a mixture of soybeans high in β-conglycinin and a culture of Jiuqu at a ratio of 5:95 to 95: 5, preferably 10:90 to 80:20, and a weight ratio of 30 to 500%. Preferably 100-300% saline is added. The concentration of the salt solution is preferably 0.1 to 20 w / v%, more preferably 6 to 18 w / v%, as the salt concentration of the moromi juice.

(Fermentation and aging of various flavors)
Next, the prepared mash is fermented and aged. Fermentation and aging of moromi are carried out by placing the moromi in a container capable of controlling the temperature and holding the moromi at 25 to 60 ° C., preferably 30 to 55 ° C. for 6 to 720 hours, preferably 12 to 240 hours. During this time, various tastes may be appropriately stirred with a stirring blade, compressed air, or the like.

The above-mentioned fermented and aged moromi mash is in the form of soy sauce mash, and can be used as it is as the fermented seasoning of the present invention. You can also get fermented seasonings.

In addition, whether to inoculate the above-mentioned various flavors after fermentation and aging with soy sauce lactic acid bacteria to sufficiently propagate the lactic acid bacteria and perform lactic acid fermentation, or to inoculate the above-mentioned various flavors with soy sauce yeast to sufficiently propagate the yeast and perform alcoholic fermentation. Alternatively, the lactic acid fermentation and the alcoholic fermentation are carried out in this order to further improve the flavor, which may be used as the fermentation seasoning of the present invention.

Lactic acid fermentation is preferably carried out until 0.5 w / v% or more of lactic acid is produced in the mash. Examples of lactic acid bacteria that can be used for lactic acid fermentation include salt-tolerant soy sauce lactic acid bacteria such as Tetragenococcus halophilus. In the lactic acid fermentation, lactic acid bacteria are added so that the moromi is 1 × 10 ³ to 1 × 10 ⁷ pieces / g moromi, and then the moromi temperature is 20 to 35 ° C, preferably 25 to 30 ° C for 3 days. As mentioned above, it is preferably fermented for 5 to 10 days. In order to make the fermentation vigorous, stirring and aeration may be performed as appropriate.

The various tastes at the start of lactic acid fermentation are usually pH 5.8 to 6.2, but gradually dissolve (hydrolyze), elution of raw materials and lactic acid fermentation are started, and the pH drops to 4.6 to 5.3. , Suitable for yeast growth.

As the yeast used for alcohol fermentation in the present invention, salt-tolerant soybean yeast that can grow vigorously even in various tastes having a high salt concentration of 10 to 20 w / v% is preferable, and for example, Zygosaccharomyces rouxii, Zygosaccharomyces rouxii, Zygosaccharomyces rouxii, etc. Yeasts of the genus Zygosaccharomyces such as Bailli (Zygosaccharomyces bailli), Candida such as Candida versatilus, Candida etchellsii, Candida tropicalis Examples include yeast of the genus.

It is preferable to add the yeast previously cultivated to the moromi so that the number of yeasts is 1 × 10 ³ to 1 × 10 ⁷ per 1 g of the moromi. In addition, yeast that has been dropped into the air or naturally mixed from soy sauce moromi without artificially adding yeast that has been cultivated in advance, or salt-resistant soy sauce yeast that lives in fermenters or the like in which soy sauce moromi is charged (wild). Type yeast) may be used.

In the alcoholic fermentation, after adding yeast, the temperature of the mash is 20 to 35 ° C., and the alcohol content is 0.5 to 4 w / v%, preferably about 3 to 4 w / v%, for a sufficient time, for example. This is done by holding for about 30 to 90 days. The ethyl alcohol concentration in the moromi can be adjusted by the culture time. Further, fermentation and aging of yeast are preferably carried out under aerobic conditions such as intermittent or continuous aeration culture, stirring culture, and aeration stirring culture.

After alcoholic fermentation, the moromi mash is allowed to stand still, and fermentation with yeast and lactic acid bacteria is continued. Then, the moromi is usually aged for several weeks to several months regardless of the degree of aging.

(Squeezing / burning)
The above-mentioned moromi mash after fermentation and aging is squeezed and filtered by a conventional method, then subjected to a heat treatment called "burning", mashed (clarified), filled in a container, and commercialized. Press filtration is a thick-fried method that wraps various flavors in a filter cloth made of synthetic fibers such as nylon and pressurizes them, or a filter that puts various flavors in a filter plate and a filter cloth stretched on a press plate and pressurizes with compressed air or the like. Conventionally known methods such as a press method can be used. The burning may be carried out under the heating conditions used in known soy sauce brewing, for example, at 80 to 85 ° C. for 20 to 60 minutes, or at 110 to 120 ° C. for 5 to 20 seconds. As the method of sedimentation (clarification treatment), conventionally known methods such as membrane treatment, diatomaceous earth filtration, centrifugation, agglutination method, and sedimentation method can be used.

The fermented seasoning of the present invention contains 1.2 times or more of the antihypertensive peptides Gly-Tyr and Ser-Tyr as compared with the conventional product. Specifically, the fermented seasoning of the present invention has Gly-Tyr of 70 μg / ml or more, preferably 80 μg / ml or more, and Ser-Tyr of 100 μg / ml or more, preferably 100 μg / ml or more, per 1 w / w% of total nitrogen concentration. It contains 110 μg / ml or more and can be expected to have a more excellent blood pressure lowering effect.

In addition, the fermented seasoning of the present invention contains a significant amount of the above-mentioned antihypertensive peptide, but has almost no bitterness, has a mellow and abundant umami and richness, and has a mild and excellent aroma, and is deliciously ingested as a seasoning. It can be done.

The antihypertensive peptide can also be concentrated or separated and purified from the fermented seasoning. Examples of the method include vacuum concentration, ultrafiltration, dialysis, and various types of chromatography. These are commonly used methods.

The fermented seasoning of the present invention may be used as it is, but it may be used as it is, or it may be made into a paste, solid or powder by freeze-drying, spray-drying, drum-drying or the like by a conventional method, and then various soy sauces. It can also be used as a soy sauce-containing seasoning by adding it to liquid seasonings such as (noodle soup, etc.), sauce (sauce for roasted meat, etc.), ponzu, sauce, dressing, soup, etc. In addition, it can be provided as a food or drink for lowering blood pressure by adding it to foods of marine products and livestock meat products (kamaboko, ham, etc.).

In addition, since the fermented seasoning of the present invention contains a significant amount of antihypertensive peptide, it can be used as a fermented seasoning for lowering blood pressure for the purpose of preventing and / or ameliorating a disease in which the pathological condition is improved by lowering blood pressure. Examples of the above-mentioned diseases include, but are not limited to, hypertension, thrombosis, arteriosclerosis, angina, myocardial infarction, and cerebral infarction. The fermented seasoning for lowering blood pressure of the present invention is used to include foods other than pharmaceuticals that can be ingested for the purpose of maintaining or promoting health, such as health foods, functional foods, health functional foods, or special purpose foods. Health foods include foods provided under the names of dietary supplements, dietary supplements, supplements and the like. Health functional foods are defined by the Food Hygiene Law or the Food Promotion Law, and are based on specified health foods and nutritionally functional foods that can display specific health effects, functions of nutritional components, reduction of disease risk, etc., and scientific evidence. Includes foods with functional claims that can display the contents notified to the Commissioner of the Consumer Affairs Agency. In addition, special-purpose foods include foods for the sick, foods for the elderly, foods for pregnant women, etc. that indicate that they are suitable for a specific target person or a patient having a specific disease.

Hereinafter, the present invention will be described in more detail with reference to Examples, but these Examples do not limit the present invention.

(Reference example) Breeding of soybeans high in β-conglycinin and measurement of β-conglycinin content
(1) Breeding of β-conglycinin-rich soybeans Breeding of β-conglycinin-rich soybeans is described in the literature (Yagasaki, K., N. Kaizuma and K. Kitamura (1996) Inheritance of glycinin subunits and characterization of glycinin molecules lacking the subunits. According to the method described in in soybean (Glycine max (L.) Merr.). Breeding Science 46 (1): 11-15.), The procedure was performed as follows. Backcrossing was performed with the 11S-deficient soybean "Nanahomare" as the gene donor and the normal soybean "Sachiyutaka" as the backcross parent. The obtained BC ₁ F ₁ seeds were cultivated in the field, and BC ₁ F ₂ seeds were harvested. Individuals lacking the three subunits of 11S were selected from the harvested BC ₁ F ₂ seeds by SDS-polyacrylamide gel electrophoresis, and then returned to "Sachiyutaka" for crossing. This operation was repeated twice, and the obtained BC ₃ F ₁ seeds were cultivated in the field to harvest BC ₃ F ₂ seeds. Individuals lacking the three subunits of 11S by SDS-polyacrylamide gel electrophoresis were selected from these BC ₃ F ₂ seeds, and the generation was advanced to BC ₃ F _{3 in} order to achieve genetic immobilization. From the BC ₃ F ₃ generation onward, soybeans lacking 11S and having a high β-conglycinin content were produced by selecting strains with excellent agricultural characteristics.

(2) Measurement of β-conglycinin amount The β-conglycinin amount was measured as follows. Milcer IFM-620DG (manufactured by Iwatani Corp.) contains soybeans with high β-conglycinin content obtained from one general foreign soybean, four general domestic soybeans (A, B, C, D) and (1). ) Was used for grinding. Ten times the amount of pure water was added to the ground soybean powder, and the mixture was crushed with a polytron homogenizer. Centrifugation at 4,000 G for 15 minutes gave a water-soluble fraction sample of the supernatant. 10 μL of a sample diluted 100-fold with pure water was subjected to SDS-PAGE. SDS-PAGE was performed according to a conventional method. The gel after SDS-PAGE was stained with a Quick-CBB kit (manufactured by Wako Pure Chemical Industries, Ltd.) and imaged with a scanner. Images of the stained gel are shown in FIG. 1, and the bands indicated by arrows are β-conglycinin α and α'subunits, β-conglycinin β subunit, glycinin acidic IIb subunit, and glycinin acidic (acidic), respectively, from the top. ) I and IIa subunits, glycinin basic subunits. The intensity of each band was quantified using the image analysis software ImageJ, and the ratio of the total of the β-conglycinin bands to the total of the β-conglycinin and glycinin bands was calculated as the β-conglycinin ratio. The results are shown in Table 1.

(Example 1, Comparative Example 1) Production of fermented seasoning containing peptide 25-40 by adding spores of Aspergillus oryzae aspergillus oryzae to a solid medium containing equal amounts of heat-modified soybean (general soybean) and wheat. Soy sauce koji was obtained by making koji at ° C for 72 hours. Next, 260 g of heat-modified soybean (soybean containing high β-conglycinin, domestic soybean A), 60 g of soy sauce koji, 50 g of salt, and 200 mL of water were mixed in an Erlenmeyer flask, and stirred at 50 ° C. and 140 rpm for 3 days. I got various tastes. Here, as the soybeans to be mixed with the soy sauce koji, those using β-conglycinin-rich soybeans are referred to as Example 1, and those using domestic soybean A are referred to as Comparative Example 1. After preparing the mash, insoluble solids were removed from the mash by filtration through a filter paper to obtain a clarified solution. Further, the clarified solution was heated at 85 ° C. for 30 minutes to perform enzyme deactivation and sterilization. The mixture was centrifuged at 10,000 g for 10 minutes, and the supernatant was collected to obtain a liquid peptide-containing fermented seasoning.

The components of the obtained peptide-containing fermented seasoning were analyzed. Soluble total nitrogen (TN) was measured according to a conventional method using a combustion type (improved Dumas method) nitrogen measuring device Dumasarm (manufactured by Gerhard Japan Co., Ltd.). The amounts of Gly-Tyr and Ser-Tyr were determined by the addition calibration curve method using an LC-MS / MS system 2695-Quattro Micro API (manufactured by Waters). The results are shown in Table 2.

As shown in Table 2, the fermented seasoning of Example 1 using β-conglycinin-rich soybean is a hypotensive peptide Gly, as compared with the fermented seasoning of Comparative Example 1 using general domestic soybean A. The amounts of -Tyr and Ser-Tyr were significantly higher.

The present invention can be used in the field of manufacturing such as soy sauce or seasonings containing soy sauce.

Claims (6)

The content of β-conglycinin with respect to the total amount of β-conglycinin and glycine in the raw material soybean is 50 w / w% or more, and Gly-Tyr is 70 μg / ml or more per 1 w / w% of total nitrogen concentration, Ser- A fermented seasoning characterized by containing 100 μg / ml or more of Tyr. The raw material soybeans are subjected raw material fermented seasoning according to claim 1. The fermented seasoning according to claim 1 or 2 , wherein the raw material soybean contains 11S globulin-deficient or reduced soybean. The fermented seasoning according to any one of claims 1 to 3 , wherein the raw material soybean contains a single or a plurality of varieties of soybean. The fermented seasoning according to any one of claims 1 to 4 , which is a fermented seasoning for lowering blood pressure. A method for producing a fermented seasoning containing Gly-Tyr and Ser-Tyr, which comprises a step of mixing raw soybeans, a culture of aspergillus and salt solution to prepare various flavors, and a step of fermenting the various flavors at 25 to 60 ° C. A method for producing a fermented seasoning, wherein the content of β-conglycinin with respect to the total amount of β-conglycinin and glycinin in the raw material soybean is 50 w / w% or more.

Images