JP2003012534A - Enzyme activity inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an enzyme activity inhibitor which is effective for controlling intestinal bacteria, has an effect for inhibiting the activities of enzymes originating from intestinal microorganisms, and consequently has possibility as a health food which may control the outbreak of large intestinal cancer, or as a raw material for the health food, and further as an agent for preventing the breakout of the large intestinal cancer. SOLUTION: This enzyme activity inhibitor is characterized by symbiotically culturing a plurality of lactic acid bacteria to ferment soybean milk, sterilizing the obtained soybean milk fermentation solution, adding alcohol to the sterilized solution, leaving the mixture for a half year or more to extract with the alcohol, and using the obtained extract containing a specific ingredient in an increased amount as the active ingredient of the enzyme activity inhibitor.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a health food which is effective in the control of intestinal bacteria, has the effect of suppressing the enzyme activity derived from intestinal microorganisms, and thus has the potential to suppress the development of colon cancer. The present invention also relates to an enzyme activity inhibitor which has the potential as a raw material for preventing the development of colon cancer.

[0002]

2. Description of the Related Art Microorganisms living in the human intestine are several hundred billion or more per 1 g of large intestine content, and there are 100 kinds of microorganisms, and they continue to proliferate while maintaining a survival or antagonistic relationship with each other. , Some of the intestinal bacteria are beneficial microbes for humans, and some enter the tissues in the body to cause damage or form harmful components, so the state of intestinal microbes is Lactobacillus beverages represented by yogurt have been used for a long time as a substance that greatly affects health and improves the condition.

Furthermore, recently, substances which are presumed to be produced or secreted by lactic acid bacteria (hereinafter referred to as "lactic acid bacterium fermentation liquid extract").
Say. ) Has been found to improve the state of intestinal microorganisms and has been developed and marketed by the applicants.

The lactic acid bacterium fermentation liquid extract has been identified to some extent, and nucleic acids, amino acids, vitamins and the like are listed as components, but the details thereof can be said to be in an unknown state.

[0005] Such a lactic acid bacterium fermentation liquid extract,
The lactic acid bacterium fermentation liquid extract improves the state of intestinal microorganisms,
Its function is to assist in the synthesis of hormones, the synthesis of vitamins and enzymes, the metabolism of cholesterol, the regulation of blood glucose levels, the regulation of blood pressure, etc.

Regarding the efficacy of the lactic acid bacterium fermentation liquid extract, in addition to improving the state of the intestinal microorganisms, various efficacy such as hepatitis, chronic arthritis, atopic dermatitis, angina pectoris, and various cancers Although the efficacy such as cancer action is mentioned, the fact is that few of them have a concrete pharmacological effect.

On the other hand, in Japan, colon cancer, which is one of the diseases in which the number of patients has rapidly increased after World War II, is caused by the action of enzymes derived from intestinal microorganisms in the food ingested, such as ammonia, amines, sulfides, It is considered to be due to the involvement of indole, nitroso compound, N-hydroxy compound and the like.

[0008]

DISCLOSURE OF THE INVENTION The inventors of the present invention tried to improve various commercially available lactic acid bacterium fermentation liquid extracts, and considered from the physiological activity known for the lactic acid bacterium fermentation liquid extract, and improved lactic acid bacterium fermentation liquid extract. , Speculated that there is an effect of suppressing the enzyme activity derived from intestinal microorganisms, conducted animal experiments, prepared by a specific manufacturing method,
Further, they have found that an extract contained in a lactic acid bacterium fermentation liquid having a specific composition has an excellent effect, and completed the present invention.

[0009]

[Means for Solving the Problems] That is, the invention according to claim 1 of the present invention is characterized by comprising a soymilk fermentation liquid obtained by fermenting soymilk by co-cultivation of a plurality of lactic acid bacteria. It is an inhibitor.

The invention according to claim 2 of the present invention is characterized by comprising an extract obtained by alcohol extraction of a soybean milk fermentation liquid obtained by fermenting soybean milk by co-cultivation of a plurality of lactic acid bacteria. It is an enzyme activity inhibitor.

The invention according to claim 3 of the present invention is to sterilize a soybean milk fermentation liquid obtained by fermenting soybean milk by co-cultivation of a plurality of lactic acid bacteria, add alcohol to the soybean milk fermentation liquid, and apply a period of half a year or more. It is an enzyme activity inhibitor characterized by comprising an extract obtained by performing extraction with alcohol.

The invention according to claim 4 of the present invention comprises an extract obtained by alcohol extraction of a soybean milk fermentation liquid obtained by fermenting soybean milk by co-cultivation of a plurality of lactic acid bacteria, An enzyme activity inhibitor characterized in that the extract contains a total of 20% or more of components having a retention time of 10 minutes or more as measured by liquid chromatography measured under the following conditions. Liquid chromatograph measurement conditions Column: Asahipak GS = 220H Mobile phase: 100 mM sodium phosphate buffer flow rate: 1.0 / min Column temperature: Temperature 40 ° C

The invention according to claim 5 of the present invention comprises an extract obtained by alcohol extraction of a soybean milk fermentation liquid obtained by fermenting soybean milk by co-cultivation of a plurality of lactic acid bacteria, The enzyme activity inhibitor is characterized in that the extract contains 25% or more in total of components having a retention time of 7 minutes or more as measured by liquid chromatography under the following conditions. Liquid chromatograph measurement conditions Column: Asahipak GS = 220H Mobile phase: 100 mM sodium phosphate buffer flow rate: 1.0 / min Column temperature: Temperature 40 ° C

The invention according to claim 6 of the present invention comprises an extract obtained by alcohol extraction of a soybean milk fermentation liquid obtained by fermenting soybean milk by co-cultivation of a plurality of lactic acid bacteria, The retention time of the extract measured by liquid chromatography under the following conditions is 25% or more for components of 7 minutes or longer and 20% for components of 10 minutes or longer.
It is an enzyme activity inhibitor characterized by containing the above. Liquid chromatograph measurement conditions Column: Asahipak GS = 220H Mobile phase: 100 mM sodium phosphate buffer flow rate: 1.0 / min Column temperature: Temperature 40 ° C

Further, the invention according to claim 7 of the present invention comprises a soybean milk fermentation liquid obtained by fermenting soybean milk by co-cultivation of a plurality of lactic acid bacteria, and a nitroreductase activity and an azo enzyme. It is an activity inhibitor.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The enzyme activity inhibitor of the present invention comprises soybean milk fermented liquid as an active ingredient, and the soybean milk fermented liquid is obtained by fermenting soybean milk by co-cultivation of a plurality of lactic acid bacteria. Fermented soy milk, preferably 100 hours to 3
Soymilk fermented liquid itself obtained by culturing for about 00 hours, specifically, those containing various contaminants other than soymilk fermented liquid extract, or solid or liquid obtained by separating individual liquids, soymilk fermented liquid extract However, in order to effectively utilize the soymilk fermented liquid extract, it is preferable to use an extract extracted with an organic solvent as an active ingredient, and more preferably soymilk by co-cultivation of a plurality of lactic acid bacteria. The soybean milk fermented liquid obtained by fermentation is subjected to alcohol extraction to obtain an extract.

A substance that is presumed to be produced or secreted by lactic acid bacteria (hereinafter referred to as a soybean milk fermented extract) present in a soybean milk fermentation liquid obtained by fermentation by co-cultivation does not exert an excellent effect on human health. It is known and there are many commercial products containing them.

The soybean milk fermentation liquor extract in the present invention is obtained from the soybean milk fermentation liquor obtained by fermenting soybean milk by co-cultivation with a plurality of lactic acid bacteria, particularly obtained by alcohol extraction from the soybean milk fermentation liquor. The conditions are as follows: 1) As the alcohol, since it is a food product, it is desirable that it is ethanol, and soybean milk fermentation liquor is added in an approximately equal amount for extraction. 2) The soybean milk fermented liquid used for extraction is sterilized, and as the sterilization method, a method such as heat sterilization or microbial cell destruction sterilization is adopted. The conditions are as follows.

The above-mentioned extraction can be carried out under heating, but it is preferably carried out at room temperature in order to avoid deterioration due to heat, and in a cool dark place in order to avoid the influence of light for a long period of time. Is preferred.

When this extraction is performed at room temperature, the extraction speed is expected to be slow, but it takes a long period of half a year or more, preferably one year or more to obtain an excellent extract from the soybean milk fermentation broth. It is preferable to carry out the extraction, and by taking such a long time, an excellent soybean milk fermentation liquid extract can be obtained.

Soymilk fermented liquid obtained by co-fermentation with a plurality of lactic acid bacteria is sterilized and alcohol is added,
When the extract is extracted over a period of half a year or more, as will be apparent from the examples described below, the components having a retention time of 10 minutes or longer by liquid chromatography measured under specific conditions are preferably 20% or more in total. The product is a soybean milk fermentation liquor extract which is present in an amount of 25% or more, and the kinds of its components are also increased.

The components are defined in more detail. Soymilk fermented liquor extract or liquid chromatograph in which a total of 25% or more, preferably 30% or more of components having a retention time of 7 minutes or more by liquid chromatography are present. Soymilk fermented liquid extract with a total retention time of 7 minutes or more of 25% or more and 10 minutes or more of total component of 20% or more, preferably 30% or more and 25% or more of soybean milk fermented liquid extract, respectively. is there.

The soybean milk fermentation liquor or its extract of the present invention is
It can be taken orally without any problem, and 0.001 to 0.05 g / kg body weight
It is recommended to be ingested on a daily basis, and since there are no side effects observed for these, it is possible to ingest more to suppress enzyme activity.

[0024]

[Function] Soymilk fermented liquid obtained by fermenting soymilk by co-cultivation with a plurality of lactic acid bacteria, especially the soymilk fermented liquid extract having the above-mentioned constitution, the function originally possessed by the soymilk fermented liquid extract, namely, intestine In addition to stabilizing the regulating action of internal bacteria, it exerts an enzyme activity suppressing effect, that is,
It suppresses the generation of ammonia, amines, sulfides, indoles, nitroso compounds and N-hydroxy compounds produced by the action of enzymes derived from intestinal microorganisms.

This action is exerted by the soybean milk fermentation liquid itself, particularly by the soybean milk fermentation liquid extract having the above-mentioned constitution. However, even if the soybean milk fermentation liquid extract is diluted, minerals and vitamins are added. The soymilk fermentation liquor extract is also produced as a health food with enzyme activity suppression,
Further, as a raw material thereof, it further exerts an action of suppressing enzyme activity, and thus may exert an action as a preventive agent for the development of colon cancer.

[0026]

EXAMPLES The enzyme activity inhibitor of the present invention will be described below in more detail with reference to Examples. Example 1 <Preparation of Fermented Liquid Extract> Prepared are lactic acid bacteria and yeast in combination of 4 types and 4 groups shown in Table 1 below. In the table, A,
B or 1, 2, 3, 4 means that the strains are different.

[0027]

[Table 1]

Lactic acid bacteria of each group were separately cultured for 48 hours while gradually raising the temperature from 20 ° C to 40 ° C using soymilk as a culture medium. After culturing for 48 hours, it was transferred to one container and further cultivated for 96 hours while gradually raising the temperature from 20 ° C to 40 ° C. The soybean milk fermentation broth obtained by the completion of the culture was sterilized by heating, then an equal amount of alcohol was added, and the mixture was stored in a cool dark place for 1 year for extraction. The soybean milk fermentation liquor after extraction was filtered and solid-liquid separated to obtain a slightly yellow transparent soybean milk fermentation liquor extract I.

The fermented liquor extract I thus obtained was analyzed by liquid chromatography under the following conditions. The results are shown in Table 2 and FIG. Liquid chromatograph measurement conditions Column: Asahipak GS = 220H Mobile phase: 100 mM sodium phosphate buffer flow rate: 1.0 / min Column temperature: Temperature 40 ° C. Detector: Ultraviolet spectrophotometer Detection wavelength: 210 nm

[0030]

[Table 2]

Furthermore, the soybean milk fermented liquid extract II which is colorless and transparent was prepared in the same manner as above except that alcohol extraction was carried out all day and night.
Got The soybean milk fermentation liquor extract II obtained is analyzed by liquid chromatography under the above conditions, and the results are shown in Table 3 and FIG.

[0032]

[Table 3]

<Sensory test> Twenty-four randomly selected subjects were divided into two groups, one group was designated as A group, soybean milk ferment solution extract II, and the other group was designated as B group, soybean milk ferment solution extract I, and morning and evening. 10 drops (about 0.4c
c) was dripped in an appropriate amount of water, hot water or tea and allowed to drink for 30 days.

During the test drinking period, each of the following items was evaluated for its own condition by the following evaluation method (5-point method). In addition, () is the display in the attached figure. <Evaluation items> 1. Stomach condition (solid line with ◆) 2. Intestinal tone (solid line on black square) 3. Defecation (solid line on ●) 4. Smell of flight (solid line with ×) 5. Fart smell (dotted line on ◇) 6. Urine output (□ is the dotted line) 7. Appetite (dotted circle) 8. Fatigue (dotted line)

<Evaluation method> The state before the start is 3 (reference value)
The following 5 grades are used to score the status of the daily evaluation items. 5: Very good 4: Improved 3: No change 2: Bad 1: Very bad

Evaluation value one week after the start of the test and before the end of the test 1
Tables 4 to 7 below show the results of comparative evaluations by calculating the average of weekly evaluation values, and FIGS. 3 and 4 show the results. Each table is as follows. Table 4: First week of Group A Table 5: Last week of Group A Table 6: First week of Group B Table 7: Last week of Group B

[0037]

[Table 4]

[0038]

[Table 5]

[0039]

[Table 6]

[0040]

[Table 7]

<Enzyme activity inhibition confirmation test> C3H / Hen mice (SPF, 5 weeks old, male, average weight 25 g) purchased from Sankyo Lab Service Co., Ltd. were used in the test.
3 animals per cage, 23 ° C, 55% relative humidity in a constant temperature and humidity small animal breeding apparatus, high protein as a feed,
MR-A1, which is a high-fat feed, and sterilized tap water as drinking water were freely ingested and bred. As a sample, the soybean milk fermentation liquor extract I was diluted with ultrapure water (10
, 100 times, 1000 times), and 0.2 ml / 1 time / day at 0 to 10 days, 25 to 30 days from 9:00 to 11:00 in 40 days of the whole experiment period. It was orally administered. The enzymatic activity was performed on fresh feces excreted in the morning of the measurement day, and the feces collected with tweezers were dispersed in 0.2M Tris-HCl buffer solution (pH 7.9) that had been anaerobically cooled with nitrogen gas. Final concentration 150 mg /
Adjust the volume to ml, add φ0.2 mm glass beads of the same mass as the feces, and stir while aeration with nitrogen gas, then 2
The supernatant obtained by separation at 00 × g for 10 minutes was further anaerobically ventilated with nitrogen gas for measurement.
Enzyme activity is measured by nitroreductase (nitrored)
actase) and azoreductase (azoreduct)
(ase) activity was performed by the method described in the literature (G
oldin et al, 1980; Goldin a
nd Gorbach, 1984).

The measurement results are shown in FIGS. Figure 1
It is a figure showing the nitroreductase activity in the feces of a mouse (solid line) and a control mouse (dotted line) administered with a 0-fold diluted soymilk fermented liquid extract, and the arrow in the figure shows the effect of the soymilk fermented liquid extract And the dotted double-headed arrow indicates the day of administration (same in each figure below). FIG. 6 is a graph showing nitroreductase activity in the feces of a mouse (solid line) and a control mouse (dotted line) administered with a 1000-fold diluted soybean milk fermentation broth extract. FIG. 7 is a diagram showing the azoreductase activity in the feces of a mouse (solid line) and a control mouse (dotted line) administered with a 10-fold diluted soybean milk extract. FIG. 8 is a graph showing the azoreductase activity in the feces of a mouse (solid line) and a control mouse (dotted line) administered with a 1000-fold diluted soybean milk fermentation broth extract.

As is clear from these results, the nitroreductase activity and the azoreductase activity in the feces of the mice that were orally administered with the soymilk fermented liquid extract were decreased, and the soymilk fermented liquid extract was derived from intestinal microorganisms. It is clear that the action of the enzyme is suppressed. Therefore, the soymilk fermented liquid extract suppresses the generation of ammonia, amines, sulfides, indoles, nitroso compounds and N-hydroxy compounds produced by the action of enzymes derived from intestinal microorganisms. Theoretically, the results of the reduction of stool odor and fart odor were confirmed.

[0044]

EFFECTS OF THE INVENTION According to the present invention, the regulating action of intestinal bacteria possessed by the conventionally well-known soymilk fermentation liquid extract can be more effectively and stably exhibited. In addition, the soybean milk fermented liquid extract provided by the present invention has the action of suppressing the activity of the enzyme derived from intestinal microorganisms, and of course, itself, as well as diluted, added minerals and vitamins. After that, since the effect does not change, the soybean milk fermented liquid extract is excellent as a health food having an enzyme activity suppressing effect and as a raw material thereof, and is widely used.

[Brief description of drawings]

FIG. 1 is a liquid chromatograph chart of lactic acid bacterium fermentation liquid extract I.

FIG. 2 is a liquid chromatograph chart of lactic acid bacterium fermentation liquid extract II.

FIG. 3 is a diagram showing a sensory test result in Group A.

FIG. 4 is a diagram showing the results of a sensory test in Group B.

FIG. 5 is a graph showing a decrease in nitroreductase activity due to a soymilk fermentation broth extract (10-fold dilution).

FIG. 6 is a diagram showing a decrease in nitroreductase activity due to a soymilk fermentation broth extract (diluted 1000 times).

FIG. 7 is a graph showing a decrease in azoreductase activity due to a soymilk fermentation broth extract (10-fold dilution).

FIG. 8 is a graph showing a decrease in azoreductase activity due to a soymilk fermentation broth extract (diluted 1000 times).

[Explanation of symbols]

None

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 43/00 111 A61P 43/00 111 C12N 9/99 C12N 9/99 F term (reference) 4B018 MD58 MD86 ME08 ME14 MF01 MF13 4C087 AA01 AA02 AA04 BC57 CA10 CA11 MA52 NA14 ZB26 ZC41 4C088 AB61 AC04 CA14 MA52 NA14 ZA66 ZB26

Claims (7)

[Claims] 1. An enzyme activity inhibitor comprising a soybean milk fermentation liquid obtained by fermenting soybean milk by co-culturing a plurality of lactic acid bacteria. 2. An enzyme activity inhibitor, which comprises an extract obtained by alcohol extraction of a soybean milk fermentation liquid obtained by fermenting soybean milk by co-culturing a plurality of lactic acid bacteria. 3. The enzyme activity inhibitor characterized in that the alcohol extraction is carried out by adding alcohol to a sterilized soymilk fermentation liquid and taking a period of half a year or more. 4. The extract according to claim 2, wherein the extract contains a total of 20% or more of components having a retention time of 10 minutes or more as measured by liquid chromatography measured under the following conditions. Enzyme activity inhibitor. Liquid chromatograph measurement conditions Column: Asahipak GS = 220H Mobile phase: 100 mM sodium phosphate buffer flow rate: 1.0 / min Column temperature: Temperature 40 ° C 5. The extract according to claim 2, wherein the extract contains 25% or more in total of components having a retention time of 7 minutes or more as measured by liquid chromatography under the following conditions. Tumor suppressor. Liquid chromatograph measurement conditions Column: Asahipak GS = 220H Mobile phase: 100 mM sodium phosphate buffer flow rate: 1.0 / min Column temperature: Temperature 40 ° C 6. The extract contains a total of 25% or more of components having a retention time of 7 minutes or more and a total of 20% or more of components having a retention time of 7 minutes or more measured under the following conditions. The enzyme activity inhibitor according to claim 2 or 3, characterized in that it is present. Liquid chromatograph measurement conditions Column: Asahipak GS = 220H Mobile phase: 100 mM sodium phosphate buffer flow rate: 1.0 / min Column temperature: Temperature 40 ° C 7. The enzyme activity inhibitor according to claim 1, wherein the enzyme activity is a nitroreductase activity or an azoreductase activity.