JP7357340B2 - Miso for inducing human β-defensin 2 - Google Patents

Description

The present invention relates to miso for inducing human β-defensin 2 and a method for producing the same.

Yokote miso, which has been produced in the Yokote region of Akita Prefecture for some time, is a multi-koji miso with a high ratio of rice malt to soybean ingredients, making it a slightly sweet and flavorful miso. During the Edo period, rice was more expensive than soybeans, so Yokote miso, which contains a high proportion of koji, was said to be a luxurious miso.
In addition, in recent years, the health functions of miso have been attracting attention, and there are reports that continuous intake of miso can suppress the occurrence of cancer, improve high blood pressure, and prevent diabetes. being done. In this way, various knowledge is being obtained about miso in general, but there is not enough knowledge regarding the health functions specific to Yokote miso.

On the other hand, it has long been known that antibacterial substances, especially antibacterial peptides, exist in plants, insects, amphibians, etc. as an innate biological defense mechanism. These are called innate immunity, and it has been elucidated that they play a role in local defense against infection. Antibacterial peptides in humans are collectively called defensins. Defensins have antibacterial activity against bacteria, fungi, and the like, and are known to be involved in various biological defense mechanisms in vivo.
β-defensin is known as a defensin expressed in mucosal epithelium of the skin, lungs, trachea, kidney, reproductive organs, etc. Currently, six types of human β-defensin (human β-defensin 1, human β-defensin 2, human β-defensin 3, human β-defensin 4, human β-defensin 5, and human β-defensin 6) have been isolated and structures determined. . In particular, human β-defensin 2 has the following characteristics: it is particularly strongly expressed in the mucous tissues and skin of the oral cavity, lungs, trachea, eyes, nose, digestive organs, etc., and its expression is induced by bacterial infection and inflammatory cytokine stimulation. It has been elucidated that there is a close relationship with respiratory tract infections and inflammation such as pneumonia.
In recent years, it has been reported that β-defensins are involved not only in local infection defense but also in tissue repair of epithelial tissues and acquired immunity by causing cells such as dendritic cells, T lymphocytes, and monocytes to migrate. ing. It has also been reported that it induces tumor immunity and exhibits antitumor effects, and that it has a growth-inhibiting effect on cancer cells.
If we can promote the production of human β-defensins, which control infection defense, in vivo, we can not only strengthen the direct antibacterial effect, but also further strengthen the body's defense mechanism by mobilizing immune cells at the site of infection. it is conceivable that.

Although the relationship between food and the induction of human β-defensin 2 has been studied so far (Patent Document 1), the relationship between miso and the induction of human β-defensin 2 has never been reported.

Japanese Patent Application Publication No. 2005-270117

The purpose of the present invention is to obtain knowledge regarding the health functions of multi-koji miso and to provide new uses for multi-koji miso.

As a result of various studies to achieve the above object, the present inventors surprisingly found that the multi-koji miso of the present invention highly induces human β-defensin 2 in human cell lines. reached. That is, the present invention is as follows.

1. A composition for inducing human β-defensin 2, comprising miso in which the ratio of soybean and rice malt as raw materials is 2 parts by mass or more of rice malt to 1 part by mass of soybeans.
2. A food composition for inducing human β-defensin 2, comprising miso in which the ratio of soybeans and rice malt as raw materials is 2 parts by mass or more of rice malt to 1 part by mass of soybeans.
3. Miso for inducing human β-defensin 2, in which the ratio of soybeans and rice malt as raw materials is 2 parts by mass or more of rice malt to 1 part by mass of soybeans.
4. A method for producing miso for inducing human β-defensin 2, in which the ratio of soybeans and rice malt as raw materials is 2 parts by mass or more of rice malt to 1 part by mass of soybeans, and after mixing both, the two are fermented and aged at room temperature for 7 to 15 months. .
5. The composition for inducing human β-defensin 2 according to 1 above, wherein the rice malt is rice koji using Aspergillus Aspergillus No. 3 B strain (Akita Konno Shoten Co., Ltd., bacterial number: AOK38), or the composition for inducing human β-defensin 2 according to 2 above. the food composition for inducing human β-defensin 2, or the miso for inducing human β-defensin 2 described in 3 above, or the method for producing the miso for inducing human β-defensin 2 described in 4 above.

According to the present invention, multi-koji miso can be provided with a new use of inducing human β-defensin 2. For example, by ingesting the multi-koji miso of the present invention, human β-defensin 2 can be induced in the digestive organs such as the large intestine to prevent food poisoning, and the induced human β-defensin 2 can act on immunocompetent cells. It has shown the possibility of exerting antitumor effects.

An example of the method for producing miso of the present invention was shown. The human β-defensin 2 (hBD2) inducing activity of the miso of the present invention was shown. The hBD2-inducing activity of miso aged for half a year was shown. The hBD2-inducing activity of 1.5-year aged miso was shown. The hBD2-inducing activity of miso produced using conventionally used Aspergillus oryzae was demonstrated. A diagram comparing the hBD2-inducing activity of miso aged for six months, soybean:rice malt 1:1 (1x) and 1:2.75 (3x). The hBD2-inducing activity of Aspergillus Aspergillus No. 3B strain and rice malt itself was demonstrated.

The present invention will be explained in detail below.
The soybean raw material used in the present invention is not limited to soybeans used for miso. For example, soybean varieties that are mainly eaten as edamame can also be used. There are no particular restrictions on the rice used to produce rice koji. Aspergillus oryzae or Aspergius soyer may be used to produce rice koji, or both may be used together, but miso with the ability to induce human β-defensin 2 is It is a type of koji mold that can be produced. Koji mold No. 3 B strain (Akita Konno Shoten Co., Ltd. bacterial number: AOK38) sold by Konno Shoten Co., Ltd. is such a koji mold, and it is more preferable to use this koji mold. However, since no induction activity was observed only with Aspergillus Aspergillus No. 3B strain and the rice koji produced using it, it is not the koji mold itself or the rice koji itself that is effective, but rather the metabolism during the production of miso with this koji mold. The product appears to be effective in inducing activity.
In addition, the Aspergillus No. 3 B strain also includes improved strains and mutant strains of the No. 3 B strain that have similar effects.
Furthermore, as a matter of course, when rice malt is produced using Aspergillus Aspergillus No. 3 B strain, the rice malt will contain Aspergillus Aspergillus No. 3 B strain.
The present invention is an invention of a miso that induces human β-defensin 2, which has not been known so far, and the miso of the present invention is not limited to the miso produced using Aspergillus No. 3 B strain.
In miso production, after producing steamed soybeans and rice koji, the steamed soybeans, rice koji, salt, and water are mixed together (hereinafter also referred to as ``shikomi''), but the ratio of soybeans and rice koji before steaming is based on the weight ratio. The ratio of soybean:malt rice = 1:2 or more is preferred, and the ratio of soybean:malt rice = 1:2.5 to 3.5 is more preferred.
At the time of preparation, the proportion of steamed soybeans (the weight is roughly doubled by steaming) is 30-40% by weight, the proportion of rice malt is 40-50% by weight, and the proportion of salt is 8-12% by weight. The proportion of water is preferably 5 to 15% by weight.
When preparing miso, salt-tolerant lactic acid bacteria or salt-tolerant yeast may be added to give sourness and flavor to miso.

The miso of the present invention is a miso for inducing human β-defensin 2, and this is because the miso highly induces human β-defensin 2 at the mRNA level in human cell lines. More specifically, the miso solution containing dissolved miso had a lower mRNA level in Caco-2 cells, which is a cell line for epithelial cells derived from human colon cancer, than the control PBS (phosphate buffered saline). This is to highly induce human β-defensin 2. Caco-2 cells are cells derived from the large intestine, and the miso of the present invention highly induces human β-defensin 2 in the human gastrointestinal tract and may be effective in preventing food poisoning.
Of course, the expression of human β-defensin 2 may be increased at the protein level in Caco-2 cells, or the expression of human β-defensin 2 may be increased at the mRNA level or protein level in other human cell lines. Good too. Furthermore, when administered to experimental animals, the expression of human β-defensin 2 may be increased at the mRNA level and protein level in cells of the gastrointestinal tract.

Generally, miso is classified into rice miso, barley miso, and soybean miso, but the miso of the present invention is a rice miso that uses rice as well as soybeans. Of course, a mixed miso mixture of rice miso and other miso may also be used.
Also, there are two types of miso: raw miso, which has living microorganisms such as koji mold (including miso in which alcohol is added to suppress the action of microorganisms), and miso, which has been heat sterilized to kill the koji mold, but either is fine. . Although the inducing activity is measured using sterilized miso samples, the activity is detected, so it is not a functional probiotic food in which live koji mold and other microorganisms act directly on the human body. It can be said to be a biogenic functional food that contains some component that induces human β-defensin 2 in human cells among the metabolic products of Aspergillus oryzae, including bacterial components.
From this point of view, it is also conceivable to take the ingredients of the miso of the present invention as a supplement, for example.
Furthermore, there is not only one component of the miso of the present invention that is effective in inducing human β-defensin 2, but two or more components may have a synergistic effect.

Hereinafter, the method for producing miso of the present invention will be explained.
Miso is manufactured according to conventional methods. First, polished rice is soaked with water and steamed, then seed koji is added to it to propagate and produce rice koji. Next, the raw soybeans are whole soybeans that are soaked in water and then steamed. Then, mix this rice malt, steamed soybeans, salt, and water. After mixing, leave for a certain period of time to ferment and mature. During the fermentation and ripening period, soybean protein is broken down into amino acids, fat into fatty acids and alcohol, and starch from rice malt into glucose, forming the flavor of miso.
For fermentation and aging, so-called natural fermentation, in which the product is left at room temperature, is preferred. The fermentation/ripening period is preferably 7 to 15 months, more preferably 9 to 15 months. The fermentation/ripening period refers to the period from mixing soybeans, rice malt, salt, and water, that is, from brewing until the miso is completed. More specifically, the term "until finished" refers to, for example, when the product is taken out of the brewery where it was placed during the fermentation and aging period for inspection and packaging. In the case of raw miso, fermentation and aging will continue to some extent after the product is shipped, but this period is not included. Miso prepared in the fall will have a slightly longer fermentation and ripening period due to the low temperatures in the winter, while miso prepared in the spring will ferment and ripen quickly due to the high temperatures in the summer, so the fermentation and ripening period will be slightly shorter. Become.

The present invention will be explained below using examples.

Example 1
[Miso production]
Below, we will describe the method for producing miso at Kura 4, which is one of the breweries where the samples were produced. Miso was produced in almost the same way at other breweries.
1. Preparation of rice malt and steamed soybeans (1) As raw materials, 30 kg of soybeans, 75 kg of rice, and Aspergillus No. 3 B strain (Akita Konno Shoten Co., Ltd., bacterial number: AOK38) were used.
(2) Production of rice koji Rice koji was produced according to the flow of 1 in Figure 1. Rice was soaked in water and steamed. After steaming, heat was released, seeds were set, and the rice was taken into a koji room for a while to produce rice koji. The resulting rice koji weighed 82.5 kg.
(3) Production of steamed soybeans Steamed soybeans were produced according to flow 2 in Figure 1. 30 kg of soybeans were soaked in water for 16 hours or more, then steamed in a pressure cooker, cooled, and crushed to produce steamed soybeans. The weight of the steamed soybeans was approximately twice that of before steaming, and the weight of the resulting steamed soybeans was just under 60 kg.
2. 82.5 kg of the rice malt produced above, a little less than 60 kg of steamed soybeans (equivalent to 30 kg of soybeans before steaming), 18 kg of salt, and 20 L of water were mixed (also called ``shikomi''). After mixing, the mixture was left at room temperature to ferment and mature for a certain period of time.

Example 2
[Measurement of human β-defensin 2 (hBD2) inducing activity when multi-koji miso is applied to Caco-2 cells]
1. Experimental example 1 <Measurement of hBD2-inducing activity of multi-koji miso>
(1) The raw materials were soybeans: rice koji = 1:2.75, the koji mold was koji mold No. 3 B strain, and the fermentation and aging period was 1 year. Multi-koji miso was produced in the same manner as in Example 1 and used as a sample. did. PBS was used as a control.
(2) The expression intensity of hBD2 mRNA was measured by the following method.
RNA extraction was performed using the RNeasy Mini kit (Qiagen) (I), and cDNA synthesis was performed using the Super Script III First-Strand kit (Invitrogen) (II). Further, PCR after cDNA synthesis was performed using primers corresponding to each target gene (FASMAC, sequence list shown in Table 1), and amplification was performed using KOD-Multi&Epi (Toyobo) (III).

(I) Extraction of RNA a. Add 1 mg/ml of each sample dissolved in 1× PBS to Caco-2 cells (cell count was done the day before to give 1.6×106 cells/dish) and incubate at 37°C for 3 hours. Co-incubated for hours. The sample solution used was one that had been sterilized at 100°C for 5 minutes.
B. Cells were lysed with 600 μl of Buffer RLT Lysis, followed by 10 strokes with a 21G syringe to disrupt the cells.
C. 600 μl of 70% ethanol prepared in advance was added and pipetted 10 times with a blue tip to form an ethanol precipitate. 600 μl of this was placed in a spin column and centrifuged at 10,000 rpm for 15 seconds. The filtrate was removed, and 600 μl of the remaining solution was again added to the same spin column and centrifuged in the same manner. The work after adding ethanol was done quickly.
D. 700 μl of Buffer RW1 was added to the spin column, centrifuged at 10,000 rpm for 15 seconds, and the filtrate was removed.
Ho. 500 μl of Buffer RPE was added to the spin column, centrifuged at 10,000 rpm for 15 seconds, and the filtrate was removed. Thereafter, 500 μl of Buffer RPE was added again, centrifuged at 10,000 rpm for 2 minutes, and the filtrate was removed.
F. Prepare a new tube without a lid, set the column, and centrifuge at 15,000 rpm for 1 minute without adding anything.
to. The column was set in a new 1.5 ml tube, 30 μl of RNase-free water was added, and centrifuged at 10,000 rpm for 1 minute. The obtained samples were immediately stored on ice.
(II) Synthesis of cDNA (a) The concentration of the RNA sample obtained in the RNA extraction in (I) was measured using Nanodrop and Q5000.
(b) Prepared as shown in Table 2 using Super Script III First-Strand kit.

(c) The prepared solution was incubated at 65°C for 5 min, 4°C for 1 min, and 25°C at ∞.
(d) During the incubation, the cDNA Synthesis Mix shown in Table 3 was prepared and stored on ice.

(e) The prepared cDNA Synthesis Mix was added to the incubated sample and pipetted. Thereafter, PCR was immediately performed under the PCR conditions shown in Table 4.

(f) After completion of PCR, the sample was transferred to ice, 1 μl of RNase H was added, and the sample was incubated at 37°C for 20 minutes.
to. The prepared cDNA was stored at -20°C.
(III) Amplification of target gene Super Script III First-Strand kit (Invitrogen) was used to amplify the target gene.
(b) To amplify the target DNA, it was prepared as shown in Table 5 and PCR was performed. PCR conditions were as shown in Tables 6 and 7. In this experiment, the β-actin gene was also amplified as an internal control.

(b) After completion of PCR, 4 μl of 6×SB was added to each sample, pipetted, and agarose gel electrophoresis was performed. Agarose gels were prepared at a concentration of 1% and prestained with 0.5 μl of ethidium bromide (EtBr). 15 μl of each sample was loaded and electrophoresed at 100 V.
(IV) Analysis The gel after electrophoresis was photographed under ultraviolet light, and the obtained images were analyzed using image analysis software "ImageJ".
(3) Results Figure 2 shows the expression intensity of hBD2 mRNA when multi-koji miso was served. Miso from Kura 5, 4, and 3 showed hBD2-inducing activity. Kura 2 and Kura 6 also tended to have high induction activities.

Example 3
[Study on the relationship between fermentation/ripening period and hBD2 inducing activity]
In order to investigate the influence of the fermentation/ripening period on hBD2 induction activity, we investigated the induction activity when the fermentation/ripening period was 6 months and 1.5 years.1. Experimental Example 2 <Measurement of hBD2-inducing activity of half-year fermented and aged multi-malt miso>
(1) A multi-koji miso prepared in the same manner as in Example 1 was used as a sample, using soybean:rice koji = 1:2.75 as raw materials, No. 3 B strain of koji mold, and a fermentation/ripening period of half a year.
(2) Measurement of hBD2 mRNA expression intensity was performed in the same manner as in Experimental Example 1 (2).
(3) Results Figure 3 shows the expression intensity of hBD2 mRNA when fermented and aged miso was served for half a year. The hBD2-inducing activity of miso from Kura 1 to 6 tended to be slightly higher in Kura 3, 4, and 6, but it could not be confirmed that it was higher than when PBS was provided. Note that E12 is an IL-12-inducing lactic acid bacteria supplement and is a sample unrelated to the results.
2. Experimental Example 3 <Measurement of hBD2 expression activity of 1.5-year fermented and aged multi-malt miso>
(1) The raw materials were soybean: rice koji = 1:2.75, the koji mold was No. 3 B strain, the fermentation and aging period was 1.5 years, and multi-koji miso was produced in the same manner as in Example 1 and used as a sample. The breweries with two samples each use different varieties of soybeans.
For reference, a commercially available miso that did not use Aspergillus Aspergillus No. 3 B strain, had a soybean:rice malt ratio of less than 1:2, and had an unknown fermentation and aging period was also sampled as a general miso.
(2) Measurement of hBD2 mRNA expression intensity was performed in the same manner as in Experimental Example 1 (2).
(3) Results
Figure 4 shows the expression intensity of hBD2 when 1.5-year fermented and aged miso was served. Miso from Kura 6-5 and Kura 2-7 showed slightly weak hBD2-inducing activity, but no inducing activity was confirmed from the other nine samples.
In addition, hBD2 induction activity could not be confirmed from the commercially available sample, so when combined with the results in Experimental Example 1 in which induction activity was confirmed in the multi-koji miso of the present invention, the superiority of the multi-koji miso of the present invention can be confirmed. was suggested.
3. Summary of Results Although there are some variations in the results depending on the brewery, miso that has been fermented and aged for 1 year has or tends to have hBD2-inducing activity (Experiment Example 1, Figure 2), while miso that has been fermented for 6 months or 1.5 years has Except for some, no hBD2-inducing activity was shown. This suggests that a fermentation/ripening period of about one year is more preferable.

Comparative example 4
[Study on the relationship between the type of Aspergillus strain and hBD2-inducing activity]
Comparative experiment example 4
(1) In order to examine the influence of differences in the strains of Aspergillus oryzae, miso was produced using various types of Aspergillus oryzae that have been used in the past, instead of Aspergillus No. 3B strain, and used as samples. Multi-koji miso was produced in the same manner as in Example 1, except for the fermentation and aging period of 1 year, using a soybean:rice koji ratio of 1:2.75 and the difference from Aspergillus No. 3 B strain. .
(2) Measurement of hBD2 mRNA expression intensity was performed in the same manner as in Experimental Example 1 (2).
(3) Results The hBD2-inducing activity of the one-year fermented and aged multi-koji miso produced using conventional koji mold is shown in FIG. The one-year fermented and aged multi-koji miso from any of the breweries did not exhibit hBD2-inducing activity. In Experimental Example 1 (Figure 2) using Aspergillus Aspergillus No. 3 B strain, all one-year fermented and aged multi-koji miso exhibited hBD2-inducing activity or had a tendency to do so. I found it important to use it.

Reference example 5
[Study on the relationship between the proportion of rice malt and hBD2 inducing activity]
Reference experiment example 5
(1) In order to investigate the relationship between the ratio of rice koji and hBD2 induction activity, we used samples with soybean:rice koji = 1:1 (hereinafter referred to as 1x) and miso with a ratio of 1:2.75 (hereinafter referred to as 3x). , the induction activities were compared. PBS was used as a control, and 1x and 3x samples from the same stock were paired to compare the hBD2 induction activities of 1x and 3x.
A sample was produced in the same manner as in Experimental Example 1 (2) except for the ratio of rice malt to soybean using the No. 3 B strain of koji mold and the fermentation/ripening period of half a year. There are two sets of samples each for Kura 4 and Kura 2 (Figure 6), but the types of soybeans are different for each set.
(2) Measurement of hBD2 mRNA expression intensity was performed in the same manner as in Experimental Example 1 (2).
(3) Results and discussion Since the fermentation and aging period is half a year, the hBD2-inducing activity of the 3x sample is not clear, so it is only for reference, but in any set, the hBD2-inducing activity of the 3x sample is higher than that of the 1x sample. tended to be large (Figure 6). Note that the sample marked with circle 2 is a sample that is unrelated to the results.

Comparative example 6
[Measurement of hBD2-inducing activity of Aspergillus oryzae and rice malt]
Comparative experiment example 6
(1) In order to confirm whether Aspergillus ore or rice malt itself has hBD2-inducing activity, Aspergillus ore or rice malt was used as a sample.
(2) Measurement of hBD2 mRNA expression intensity was performed in the same manner as in Experimental Example 1 (2).
(3) Results The hBD2 inducing activity when Aspergillus oryzae and rice malt were provided is shown in FIG. Since none of them showed hBD2-inducing activity, it was found that the koji mold or rice malt itself did not have hBD2-inducing activity, but rather the miso made using these had hBD2-inducing activity.

The miso for inducing human β-defensin 2 of the present invention provides a new use for multi-koji miso, can contribute to expanding demand for multi-koji miso, and contributes to the food industry that produces multi-malt miso.

Claims (8)

A composition for inducing human β-defensin 2, comprising miso in which the ratio of soybeans and rice malt as raw materials is 2 parts by mass or more of rice malt to 1 part by mass of soybeans. A food composition for inducing human β-defensin 2, comprising miso in which the ratio of soybeans and rice malt as raw materials is 2 parts by mass or more of rice malt to 1 part by mass of soybeans. Miso for inducing human β-defensin 2, in which the ratio of soybeans and rice malt as raw materials is 2 parts by mass or more of rice malt to 1 part by mass of soybeans. A method for producing miso for inducing human β-defensin 2, in which the ratio of soybeans and rice koji as raw materials is 2 parts by mass or more of rice koji to 1 part by mass of soybeans, and after mixing the two, the two are fermented and aged at room temperature for 7 to 15 months. . The composition for inducing human β-defensin 2 according to claim 1, wherein the rice malt is rice malt using Aspergillus Aspergillus No. 3 B strain (Akita Konno Shoten Co., Ltd., bacterial number: AOK38). The food composition for inducing human β-defensin 2 according to claim 2, wherein the rice malt is rice malt using Aspergillus Aspergillus No. 3 B strain (Akita Konno Shoten Co., Ltd., bacterial number: AOK38). The miso for inducing human β-defensin 2 according to claim 3, wherein the rice malt is rice malt using Aspergillus No. 3 B strain (Akita Konno Shoten Co., Ltd., bacterial number: AOK38). 5. The method for producing miso for inducing human β-defensin 2 according to claim 4, wherein the rice malt is rice malt using Aspergillus No. 3 B strain (Akita Konno Shoten Co., Ltd., bacterial number: AOK38).