JP6793380B2 - Evaluation method, screening method and manufacturing method of substances that suppress the rise in blood glucose level due to sucrose intake - Google Patents

Description

The present invention relates to a method for evaluating whether or not a substance suppresses an increase in blood glucose level due to sucrose intake, a method for screening the substance, and a method for producing the substance.

An increase in blood glucose level due to excessive sugar intake leads to the onset or worsening of diseases such as diabetes. In fact, the number of patients with type II diabetes as a lifestyle-related disease is increasing worldwide, which has become a social problem. Therefore, suppressing excessive sugar intake is important for a healthy life.

Sucrose is a major sugar contained in foods and is a typical sweetener added to various foods. Sucrose, at least in humans, is broken down into glucose and fructose by α-glycosidases in the intestinal tract, which are absorbed from the intestinal tract, leading to elevated blood glucose levels.
Acarbose and voglibose, which are inhibitors of α-glycosidase, have an effect of inhibiting an increase in postprandial blood glucose level in humans, and are used as antidiabetic agents in humans.

The blood glucose level after eating sucrose is regulated by the action of various organs throughout the body at each stage of decomposition and absorption from the intestinal tract, distribution and metabolism to various organs, and excretion to the outside of the body. Therefore, in order to evaluate an active substance that suppresses an increase in blood glucose level due to sucrose feeding, an experiment using an individual is necessary.
However, a method for searching for a substance exhibiting the above activity has not been established in consideration of such pharmacokinetics.
Conventionally, mammals such as mice and rats have been used to evaluate substances exhibiting a hypoglycemic effect, but it is not only cost effective but also animal protection to use a large number of mammals for experiments. Therefore, a simple method using individual animals has not been established.

So far, the present inventors have reported that the hypoglycemic effect of human insulin, which is an antidiabetic drug, can be evaluated by using a silkworm that has become hyperglycemic by feeding a high glucose diet for a short time (Patent Document 1). 2, Non-Patent Document 1).
However, in the systems reported so far, experiments have been conducted by feeding silkworms with glucose-added diets, and whether or not sucrose, which is generally present in foods, increases the glucose concentration in silkworm body fluids. No consideration was given.

In recent years, the demand for blood glucose elevation inhibitors has been increasing more and more, and conventional evaluation methods, screening methods, and methods for producing blood glucose elevation inhibitors using them are not sufficient, and further improvement is possible. It was desired.

Japanese Unexamined Patent Publication No. 2009-058500 International Publication No. 2010/004916

Matsumoto Y, Sumiya E, Sugita T, Sekimizu K. An Invertebrate Hyperglycemic Model for the Identification of Anti-Diabetic Drugs. PLoS ONE, 6 (3), 2011

The present invention has been made in view of the above background technique, and the subject is whether or not the test substance is a substance that suppresses an increase in human blood glucose level due to sucrose intake by exerting α-glycosidase inhibitory activity. Is to provide a way to evaluate.

As a result of diligent studies to solve the above problems, the present inventors have found that feeding silkworms with sucrose-added diet increases the glucose concentration in body fluids. Furthermore, it was found for the first time that the addition of acarbose and voglibose, which are known as α-glycosidase inhibitors, to the silkworm diet suppresses the increase in glucose concentration in body fluids caused by feeding the diet containing sucrose. That is, it was found for the first time that silkworms can be used to evaluate substances that suppress the increase in human blood glucose level due to sucrose intake by exerting α-glycosidase inhibitory activity.

In addition, as a result of evaluating 18 types of lactic acid bacteria using the above evaluation means, the present inventors suppressed the increase in human blood glucose level due to sucrose intake by demonstrating α-glycosidase inhibitory activity of 5 types of lactic acid bacteria. I found a new thing to do.

Furthermore, the present inventors have identified a lactic acid bacterium that suppresses hyperglycemia due to feeding of sucrose by using the above evaluation method. Yogurt produced using the lactic acid bacterium strain showed an effect of suppressing an increase in blood glucose level in a human sucrose tolerance test. Further, as a result of evaluation using the above evaluation method, the present invention has been made based on the fact that the lactic acid bacterium has an effect of suppressing postprandial hyperglycemia and reducing the onset of diabetes.

That is, the present invention is a method for evaluating whether or not a test substance is a substance that suppresses an increase in human blood glucose level due to sucrose intake by exerting α-glycosidase inhibitory activity, and is at least the following steps ( a), step (b) and step (c),
(A) Step of ingesting sucrose to a test animal,
(B) A step of administering the test substance at the same time as the step (a) or before or after the step (a).
(C) A step of measuring the concentration of sugar in the body fluid of a test animal to which the above test substance has been administered.
It provides a method characterized by having.

Further, the present invention is a method for screening a substance that suppresses an increase in human blood glucose level due to sucrose intake by exerting α-glycosidase inhibitory activity from among test substances, and at least the following step (a), Step (b), step (c) and step (d),
(A) Step of ingesting sucrose to a test animal,
(B) A step of administering the test substance at the same time as the step (a) or before or after the step (a).
(C) A step of measuring the concentration of sugar in the body fluid of a test animal to which the above test substance has been administered.
(D) A step of selecting a substance that reduces the concentration of sugar in the body fluid of the test animal from the above test substances.
It provides a method characterized by having.

The present invention also provides a method for producing a substance that suppresses an increase in human blood glucose level due to sucrose intake by exerting an α-glycosidase inhibitory activity, and is characterized by using the above method. It is a thing.

According to the present invention, "α" is provided by a simple and easy means of solving the above-mentioned problems and the above-mentioned problems, ingesting sucrose and a test substance in a test animal, and measuring the concentration of sugar in the body fluid of the test animal. -Easy, inexpensive, efficient, accurate and appropriate evaluation of whether or not it is a substance that suppresses the rise in human blood glucose level due to sucrose intake by exerting glycosidase inhibitory activity, screening of the substance, etc. Can be done.
A substance having an inhibitory effect on α-glycosidase is expected to suppress an increase in postprandial blood glucose due to excessive feeding of sucrose. Therefore, according to the present invention, α- (in the animal intestine) using a test animal was used. By inhibiting glycosidase activity, it is possible to evaluate, screen, and manufacture "drugs and lactic acid bacteria" that have the effect of suppressing an increase in human blood glucose level.

Using the evaluation method, screening method, etc. of the present invention, a substance that has not been known as "a substance that suppresses an increase in human blood glucose level" has been newly found. For example, "some lactic acid bacteria such as lactic acid bacteria # L1-1 strain" were newly found.
In addition, probiotic research related to blood glucose regulation can be conducted by using the evaluation method of the present invention.

In addition, the Enterococcus faecalis # Ef-1 (Enterococcus faecalis 0831-07) strain obtained in this example suppresses postprandial hyperglycemia due to feeding of human sucrose, and increases the blood glucose level due to sucrose (sucrose) intake. It was found that it is a functional lactic acid bacterium that suppresses sucrose. Therefore, the substance obtained by the evaluation method, the screening method, etc. of the present invention can be used as a food having a blood glucose lowering effect.

It is a graph which shows the result of having measured the total sugar amount (A) and glucose concentration (B) in the body fluid of silkworm by ingestion of sucrose. It is a graph which shows the measurement result of α-glycosidase activity in vitro. (A) It is a graph which shows the result of having measured the dose dependence of α-glycosidase activity in the cell disruption fraction of the intestinal tract of silkworm. (B) It is a graph which shows the result of having measured the α-glycosidase activity of the cell disruption fraction (Intestine), the intestinal content (Lumen), and the body fluid (Hemolymph) of the silkworm. (C) It is a graph which shows the α-glycosidase activity inhibitory effect of the silkworm intestinal tract by acarbose. (D) It is a graph which shows the α-glycosidase activity inhibitory effect of the silkworm intestinal tract by voglibose. It is a graph which shows the result of having measured the total sugar amount and glucose concentration in the body fluid of the silkworm which fed the sucrose-containing diet by the addition of acarbose or voglibose. (A) Total sugar amount when acarbose is added, (B) Glucose concentration when acarbose is added, (C) Total sugar amount when voglibose is added, (D) Glucose concentration when voglibose is added It is a graph which shows the result of having measured the glucose concentration in the body fluid of silkworm by feeding sucrose or glucose-containing diet by addition (presence or absence) of acarbose (A) or voglibose (B). It is a graph which shows the result of having measured the glucose concentration in the body fluid of the silkworm by the feeding of the sucrose-containing feed by the addition of each lactic acid bacterium. (A) It is a graph which shows the result of having measured the dose dependence between the glucose concentration in the body fluid of silkworm, and the lactic acid bacterium # L1-1 strain by feeding the sucrose-containing diet. (B) It is a graph which shows the result of having measured the glucose concentration in the body fluid of silkworm by feeding sucrose or glucose-containing feed by addition (presence or absence) of lactic acid bacterium # L1-1 strain. It is a graph which shows the inhibitory effect of the addition of lactic acid bacteria on the increase of glucose concentration in the body fluid of silkworm by feeding a sucrose-containing diet. It is a graph which shows the inhibitory effect of the addition of a lactic acid bacterium (# Ef-1 strain) on the increase of glucose concentration in the body fluid of silkworm by feeding a sucrose-containing diet. It is a graph which shows the inhibitory effect of the addition of lactic acid bacterium (# Ef-1 strain) on the increase of glucose concentration in the body fluid of silkworm by feeding sucrose or glucose-containing diet. It is a graph which shows the inhibitory effect of the addition of the heat-treated bacterium of a lactic acid bacterium (# Ef-1 strain) on the increase of the glucose concentration in the body fluid of the silkworm by feeding a sucrose-containing diet. It is a graph which shows the inhibitory effect of the increase in a human glucose concentration in a sucrose load test by feeding yogurt prepared with lactic acid bacteria (# Ef-1 strain). It is a graph which shows the inhibitory effect of the increase of the glucose concentration in the body fluid of the silkworm by the feeding of the sucrose-containing feed (a) or the glucose-containing feed (b) by the addition of killed lactic acid bacteria (# Ef-1 strain). (A) It is a figure which shows the experimental scheme of the sugar transfer evaluation system in the silkworm intestinal tract in vitro. (B) It is a graph which shows the inhibitory effect of acarbose on the transport of sucrose from the excised intestinal tract to the outside of the intestinal tract. It is a graph which shows the inhibitory effect of sucrose transport from the excised intestinal tract to the outside of the intestinal tract by addition of lactic acid bacteria (# Ef-1 strain). (A) It is a graph which shows the inhibitory effect of the addition of lactic acid bacterium (# Ef-1 strain) on the increase in glucose concentration in the extraintestinal fluid of silkworm. (B) It is a graph which shows the inhibitory effect of the increase in glucose concentration in the extraintestinal fluid of silkworm by the amount of lactic acid bacteria (# Ef-1 strain) added. (C) It is a graph which shows the inhibitory effect on the increase in glucose concentration in the extraintestinal fluid of silkworm by the addition of the heat-treated bacterial cells of lactic acid bacteria (# Ef-1 strain). (D) It is a graph which shows the inhibitory effect of the increase in glucose concentration in the extraintestinal fluid of silkworm by the addition amount of the heat-treated bacterial cells of lactic acid bacteria (# Ef-1 strain). It is a graph which shows the inhibitory effect of glucose transport by the addition of viable lactic acid bacterium (# Ef-1 strain). (A) It is a graph which shows the inhibitory effect of the addition of a viable lactic acid bacterium (# Ef-1 strain) on the increase in glucose concentration in the extraintestinal fluid of silkworm. (B) It is a graph which shows the inhibitory effect of the addition of the heat-treated bacterial cells of lactic acid bacteria (# Ef-1 strain) on the increase in glucose concentration in the extraintestinal fluid of silkworm. (A) It is a graph which shows the α-glycosidase activity inhibitory effect of the silkworm intestinal tract by addition of lactic acid bacterium (# Ef-1 strain). (B) It is a graph which shows the α-glycosidase activity inhibitory effect of a rat intestinal tract by addition of a lactic acid bacterium (# Ef-1 strain). It is a schematic diagram which shows the postprandial hyperglycemia suppressing effect by a lactic acid bacterium (# Ef-1 strain).

Hereinafter, the present invention will be described, but the present invention is not limited to the following specific aspects, and can be arbitrarily modified within the scope of the technical idea.

[Method of evaluating whether or not it is a substance that suppresses the rise in human blood glucose level due to sucrose intake]
The evaluation method of the present invention is a method for evaluating whether or not the test substance is a substance that suppresses an increase in human blood glucose level due to sucrose intake by exerting α-glycosidase inhibitory activity, and is at least the following steps. (A), step (b) and step (c),
(A) Step of ingesting sucrose to a test animal,
(B) A step of administering the test substance at the same time as the step (a) or before or after the step (a).
(C) A step of measuring the concentration of sugar in the body fluid of a test animal to which the above test substance has been administered.
It is a method characterized by having.

In the present invention, "a substance that suppresses an increase in human blood glucose level due to sucrose intake by exerting α-glycosidase inhibitory activity" means "in humans, sucrose is glucose and fructose by inhibiting α-glycosidase activity." It refers to a substance that inhibits the decomposition of sucrose and suppresses the rise in human blood glucose level due to sucrose intake.
The method of the present invention can evaluate whether or not the test substance has α-glycosidase inhibitory activity.

The evaluation method may further include other steps, if necessary. Hereinafter, steps (a), (b), and (c) will be described in order.

<Step (a)>
Step (a) is a step of ingesting sucrose to the test animal.
Sucrose (sucrose) is one of the disaccharides and is hydrolyzed into glucose (dextrose) and fructose (fructose) by enzymes and the like.

Examples of the test animal include mammals such as mice and rats; insects such as silkworms and flies; and the like. In the following points, it is preferable to use silkworm as a test animal.
(1) The silkworm itself is easily available.
(2) A method for breeding silkworms has already been established, which is more convenient for breeding.
(3) Properties similar to the internal organs and organs of mammals such as humans have been known to some extent in previous studies.
(4) The genetic lineage has been established, and genetic homogeneity can be maintained.
(5) Since it is relatively large, moves slowly, and is substantially hairless, it is easy to administer a drug, such as being able to be injected quantitatively.
(6) It has a fat body and body fluid (blood), and it is possible to take them out and quantify the substances contained therein.
(7) Compared to mice, rats, etc., it is cheaper, a large number of individuals can be bred in a narrow space, and there are few ethical problems, so that screening evaluation is easy.
(8) Evaluation can be performed even when the amount of the test substance is small.
(9) It is easy to arrange individuals in the same state, such as by aligning their ages.
(10) It is possible to collect body fluids and analyze components such as sugars, lipids, and enzymes.

In order to evaluate the therapeutic effect of medicines and the effectiveness of foods, it is necessary to evaluate using individual animals, but silkworms do not require a large breeding space and can breed a large number of individuals at low cost. Also, "a method for evaluating whether or not a substance suppresses an increase in human blood glucose level or a method for screening the substance" is also preferable as an in vivo evaluation model.

From the viewpoint of animal protection, experiments using mammals should be conducted in accordance with the 3Rs, which are the international principles: Replacement (development of alternative methods), Reduction (reduction of animal numbers), and Refinement (reduction of animal pain). Must be done (Russell et al., 1959). The use of silkworms as test animals is consistent with the idea of developing alternative methods within the 3Rs.
That is, if silkworm-based evaluation methods, screening methods, manufacturing methods, etc. are used in the exploration stage prior to preclinical studies in food and drug development, the number of mammals that must be sacrificed can be reduced, resulting in cost and cost. Can solve problems from the perspective of animal protection.

The silkworm is preferably a large-sized silkworm from the viewpoints of ease of ingestion of sucrose, ease of administration of a test substance, ease of collecting blood (body fluid) and fat pad, and the like. Here, the "large silkworm" is a silkworm having a body length of 1 cm or more, preferably 1.5 cm or more and 15 cm or less, and particularly preferably 2 cm or more and 5 cm or less. In addition, 4th to 5th instar silkworms are preferable, and 5th instar larvae are particularly preferable.

The method of ingesting sucrose is not particularly limited and can be appropriately selected depending on the purpose. Specific examples include injection into body fluids; oral ingestion by addition to feed (feed); injection into the intestine; etc., which is convenient and can be used in human clinical practice. Therefore, oral ingestion by injection into the intestinal tract or addition to feed (feed) is preferable.

The amount of intake is not particularly limited and can be appropriately selected depending on the feed to be fed, the intake method and the like. In the present invention, the content ratio of sucrose when sucrose is added to the feed (feed) is preferably 2% by mass to 30% by mass, preferably 5% by mass, based on the total amount of the feed (feed) and sucrose. ~ 25% by mass is more preferable, and 8% by mass to 12% by mass is particularly preferable.
If the content ratio or intake is too low, the effect of the test substance may not be clearly confirmed, while if it is too high, the test animal may be injured other than the increase in blood glucose level due to sucrose.
The intake of sucrose is preferably 0.002 g to 0.2 g, more preferably 0.004 g to 0.15 g, and particularly 0.01 g to 0.1 g, per ingestion during the following intake time. preferable.

The ingestion period is not particularly limited as long as the test substance can be evaluated, but is preferably 1 minute to 3 days, more preferably 5 minutes to 1 day, and particularly preferably 10 minutes to 2 hours. For convenience, it is particularly preferable to include the entire amount in one feeding.
If the ingestion period is too short, it may not be possible to ingest a sufficient amount and the effect of the test substance may not be clearly confirmed.

When silkworms are used as test animals, artificial feeds (for example, Silkmate 2S (manufactured by Nosan Corporation), etc.) are preferable as the feeds. Mulberry leaves, which feed silkworms, have been reported to contain several α-glycosidase inhibitory compounds (Konno K et al., Proc. Natl. Acad. Soc. USA, 2006). When leaves are used as food, it is considered that the effect of the α-glycosidase inhibitory compound contained in the mulberry leaf affects the effect of the test substance.
In particular, when silkworm is used as a test animal, it is preferable to mix sucrose in a ratio of 5% by mass to 25% by mass with respect to the total amount of sugar and sucrose contained in the artificial feed, preferably 8% by mass. It is particularly preferable to contain it in a proportion of% to 20% by mass.

<Step (b)>
The step (b) is a step of administering the test substance to the test animal at the same time as the step (a) or before or after the step (a).

The step (b) may be performed at the same time as the step (a), or may be performed before or after the step (a).
When performed at the same time as step (a), for example, a mixture of sucrose, a test substance and food is administered to the test animal.
When it is carried out after the step (a), for example, it is preferable to administer the test substance between "immediately after the intake of sucrose" and "24 hours after the completion of the intake of sucrose". The period from "immediately after the intake is finished" to "6 hours after the end of the sucrose intake" is more preferable, and "immediately after the sucrose intake is finished" is particularly preferable.
When it is performed before the step (a), for example, it is preferable to ingest sucrose between "immediately after the administration of the test substance" and "after 6 hours have passed after the administration of the test substance". The period from "immediately after the administration of the test substance" to "6 hours after the administration of the test substance" is more preferable, and "immediately after the administration of the test substance is completed" is particularly preferable.

The test substance is not particularly limited, and is selected from animals and plants; microorganisms such as bacteria and yeast or their products; natural products, compounds derived from natural products such as derivatives of natural products; synthetic products; and the like.
More preferably, it is a microorganism or a product thereof, or a compound derived from a natural product, particularly preferably a gram-positive bacterium or a product thereof, and further preferably a lactic acid bacterium or a product thereof.
When the test substance is a microorganism such as a bacterium or yeast, it may be a living microorganism, a dead microorganism, or a processed product of the microorganism.
Examples of dead microorganisms include heat sterilized products, radiation sterilized products, and crushed products of microorganisms.
Examples of the processed product of microorganisms include cultures of microorganisms; concentrates; pastes; spray-dried products, freeze-dried products, vacuum-dried products, dried products such as drum-dried products; liquid products; diluted products; crushed products; sterilization. Processed products; extracts from the cultures; and the like.
The test substance may be one kind or a mixture of the above-mentioned (state) substances.

The method for administering the test substance is not particularly limited and can be appropriately selected depending on the intended purpose. Specific examples thereof include injection into body fluid, oral administration by addition to feed (feed), injection into the intestine, and the like. Oral administration is preferable in terms of simplicity and correspondence with human clinical practice.

The dose of the test substance is not particularly limited and can be appropriately selected depending on the substance to be administered, the administration method and the like. It is also preferable to convert the dose per body weight in humans into the weight of the test animal to be administered. It is also preferable to administer an amount obtained by multiplying the converted value by a predetermined magnification. It is also preferable that the test substance is diluted with physiological saline, water or the like for administration.

The administration period is not particularly limited as long as the decrease in "sugar concentration in body fluid" due to the test substance can be measured, but it is preferable to administer the whole amount at one time or continuously. It is particularly preferable for convenience that the whole amount is contained in one feeding and administered.

<Step (c)>
Step (c) is a step of measuring the concentration of sugar in the body fluid of the test animal to which the test substance is administered.

The above-mentioned "sugar concentration" may be the total sugar concentration contained in the body fluid of the test animal, and is preferably one kind of sugar concentration such as glucose and trehalose, and particularly preferably the glucose concentration. Depending on the method for quantifying the sugar concentration, the total amount of a plurality of types of sugars may be quantified, but the sugar in that case is not limited to one type.
It is particularly preferable that the "sugar concentration" is a "glucose concentration" in terms of excellent evaluation of whether or not the substance suppresses an increase in blood glucose level.

The method for measuring the sugar concentration is not particularly limited, and examples thereof include a phenol sulfuric acid method, anthron sulfuric acid method, carbazole sulfuric acid method and the like for quantification of all sugars; and a glucose oxidase method and the like for quantification of glucose.
There is no particular limitation on the timing of measuring the sugar concentration in the body fluid, from the period immediately after the test substance is administered in step (b) until the effect of the test substance on reducing the sugar concentration disappears. You can select it. Specifically, for example, from the time when the test substance is administered, 1 minute to 1 day is preferable, 20 minutes to 18 hours is more preferable, and 30 minutes to 10 hours is particularly preferable.

As a method for obtaining body fluid from a test animal, for example, the method used in Examples can be used.
The "body fluid" in the present invention includes blood, lymph, tissue fluid, or a mixture thereof. The body fluid to be measured is preferably blood for measuring the blood glucose level.

When measuring the sugar concentration, it is preferable to use a "test animal ingesting sucrose" to which the test substance has not been administered as a control. The test substance is evaluated by how much the sugar concentration was reduced by the administration of the test substance as compared with the control.
For the control of the test substance, for example, it is preferable to administer the same amount of physiological saline.

The number of test animals used in one condition is not particularly limited, but is preferably 1 to 200, more preferably 2 to 40, and particularly preferably 3 to 10. Within this range, correct evaluation is possible both pharmacologically and statistically.

[Method of screening for substances that suppress the increase in human blood glucose level due to sucrose intake]
The screening method of the present invention is a method for screening a substance that suppresses an increase in human blood glucose level due to sucrose intake by exerting α-glycosidase inhibitory activity from among test substances, and is at least the following step (a). , Step (b), Step (c) and Step (d),
(A) Step of ingesting sucrose to a test animal,
(B) A step of administering the test substance at the same time as the step (a) or before or after the step (a).
(C) A step of measuring the concentration of sugar in the body fluid of a test animal to which the above test substance has been administered.
(D) A step of selecting a substance that reduces the concentration of sugar in the body fluid of the test animal from the above test substances.
It is a method characterized by having.

The screening method of the present invention is also a method for screening a substance that is a candidate for the above-mentioned "substance that suppresses an increase in human blood glucose level due to sucrose intake by exerting α-glycosidase inhibitory activity" from among the test substances.
The screening method of the present invention can screen a substance that is a candidate for "a substance having an α-glycosidase inhibitory activity".

The screening method of the present invention may further include other steps if necessary. The steps (a), (b) and (c) are the same as the above evaluation method.

<Step (d)>
The step (d) is a step of selecting a substance that reduces the concentration of sugar in the body fluid of the test animal from the above-mentioned test substances.

The number of test animals used in one condition is not particularly limited, but is preferably 1 to 200, more preferably 2 to 40, and particularly preferably 3 to 10. Within this range, pharmaceutically and statistically correct screening is possible.

According to the screening method of the present invention, "a substance that suppresses the increase in human blood glucose level due to sucrose intake by exerting α-glycosidase inhibitory activity" can be screened in a cost-effective and ethically-free manner. It is possible.

[Method of manufacturing a substance that suppresses the rise in human blood glucose level due to sucrose intake]
The production method of the present invention is characterized by using the above screening method.

The "substance that suppresses the increase in human blood glucose level due to sucrose intake by exerting α-glycosidase inhibitory activity" obtained by the production method of the present invention is used as a drug by mixing with a pharmaceutically acceptable carrier. It may be contained in foods and drinks.
According to the present invention, after screening for a substance that is a candidate for "a substance that suppresses an increase in human blood glucose level due to sucrose intake by demonstrating α-glycosidase inhibitory activity", further by another method using mice, humans, etc. After narrowing down, the narrowed down substance may be used as a drug or may be contained in foods and drinks.

When "a substance that suppresses the increase in human blood glucose level due to ingestion of granules by exerting α-glycosidase inhibitory activity" is used as a drug, the dosage form of the drug is not particularly limited, but for oral administration. Examples of the preparation include tablets, pills, granules, capsules, powders, liquids, suspensions, syrups, sublinguals, etc., and preparations for parenteral administration include injections and transdermal preparations. Examples include absorbents, inhalants, suppositories and the like.

At the time of formulation, it is possible to mix a pharmaceutically acceptable carrier. The type and composition of the carrier can be appropriately determined depending on the administration route and administration method.
As the liquid carrier, for example, water, alcohol, cooking oil and the like can be used. As the solid carrier, for example, amino acids such as lysine, polysaccharides such as cyclodextrin, organic acid salts such as magnesium stearate, cellulose derivatives such as hydroxylpropyl cellulose and the like can be used.

The substances selected in step (d) above include tonicity agents, preservatives, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, excipients, binders, disintegrants, and diluents. Various pharmaceutical additives such as agents, buffers, colorants, and flavoring agents can be blended. In the case of an injection, it is sterilized with an appropriate carrier to obtain a drug.

When "a substance that suppresses the increase in human blood glucose level due to sucrose intake by exerting α-glycosidase inhibitory activity" is used as a food or drink, the type of food or drink is not particularly limited and is appropriately selected according to the purpose. be able to.
Foods and drinks include, for example, confectioneries such as jelly, candy, chocolate, and biscuits; favorite beverages such as green tea, black tea, coffee, and soft drinks; dairy products such as fermented milk, yogurt, and ice cream; vegetable beverages, fruit beverages, etc. Processed vegetables and fruits such as jams; liquid foods such as soups; processed grain products such as breads and noodles; various seasonings; etc.

Further, the above-mentioned food and drink may be produced as, for example, an oral solid preparation such as tablets, granules and capsules, and an oral liquid preparation such as an internal liquid preparation and a syrup preparation.

Enterococcus faecalis (Enterococcus faecalis) # Ef-1 (Enterococcus faecalis 0831-07) is, Chiba Prefecture Kisarazu Kazusa legs bowed in 2-5-8, No. 122 rooms, the National Institute of Technology and Evaluation (N ational Institute of Technology and Evaluation; It is deposited domestically at the Patent Microbial Depositary Center (NPMD) of (hereinafter abbreviated as "NITE"), and is deposited under the deposit number: NITE P-02309 (deposit date: July 26, 2016) , and then international deposit. It is a microorganism that has been changed to NITE BP-02309 (International Deposit Date: May 16, 2017) .

It is clear that the blood glucose level of glucose does not rise unless sucrose is broken down into glucose and fructose, whether in the human or silkworm. When sucrose is ingested by silkworms, blood glucose levels such as glucose rise, and α-glycosidase inhibitory activators in humans suppress the rise in silkworms. Therefore, silkworms have a mechanism similar to that of humans. From this, it is clear that the evaluation method / screening method using the "test animal such as silkworm" of the present invention is useful.

On top of that, as shown in FIGS. 5 and 7, many "substances that suppress the increase in blood glucose level of silkworms (humans) due to sucrose intake by exerting α-glycosidase inhibitory activity" However, it was found by the evaluation method and the screening method of the present invention. Moreover, in the lactic acid bacterium # Ef-1 (lactic acid bacterium 0831-07) discovered by using the method of the present invention, the α-glycosidase inhibitory activity was actually confirmed in Example 5 in humans.

Therefore, the "method for producing a substance that suppresses an increase in human blood glucose level due to sucrose ingestion by exerting α-glycosidase inhibitory activity" of the present invention is clear because the extension of the substance is clear.

[Action / Principle]
Although the action / principle of screening for "a substance that suppresses the increase in human blood glucose level due to sucrose intake by exerting α-glycosidase inhibitory activity" by the evaluation method or the like of the present invention is not clear, the following can be considered. ..
For the first time, it was confirmed by the present invention that the blood glucose level rises when sucrose is ingested by silkworms. It has already been reported that when glucose is ingested by silkworms, the blood glucose level rises, but if sucrose is not decomposed into glucose and fructose in the body, the blood glucose level such as glucose does not rise. That is, the mechanism of increasing the blood glucose level by ingesting glucose and the mechanism of increasing the blood glucose level by ingesting sucrose are different, or the latter mechanism of increasing the blood glucose level is accompanied by yet another mechanism of the former mechanism of increasing the blood glucose level.
Therefore, it is considered that the above-mentioned effects are exhibited in silkworms because they may not be completely the same as the mechanism of increasing blood glucose due to human sucrose intake, but at least similar mechanisms exist.

Hereinafter, the present invention will be described in more detail based on Examples and Test Examples, but the present invention is not limited to the specific scope of the following Examples and the like.

<Types of silkworms, breeding conditions, injection conditions>
Fertilized silkworm eggs (hybrid fu, yo x Tsukuba, ne) were purchased from Ehime Sericulture Co., Ltd. The hatched larvae were fed with artificial feed Silkmate 2S (manufactured by Nosan Corporation) at room temperature and raised to the 5th instar larvae.
As the breeding container, a square No. 2 petri dish (Eiken equipment) was used for the eggs to the second instar larvae, and a disposable plastic food pack (Food pack FD Odaka, manufactured by Chuo Kagaku Co., Ltd.) was used for the rest.
The breeding temperature was 27 ° C. Unless otherwise stated, larvae of the 5th instar and 1st day fasted after the 4th instar sleep were used in the experiment.

The sugar-added feed was prepared by mixing artificial feed Silk Mate 2S (manufactured by Nippon Agricultural Products Co., Ltd.) with D-sucrose or D-glucose so as to have a mass ratio of 10% by mass. In addition, the lactic acid bacterium-added bait was mixed with the bait by measuring the wet weight (g) of the pellets obtained by centrifuging the cultured lactic acid bacteria.

<Culture of lactic acid bacteria>
The fungus grew on MRS medium containing 0.5% calcium carbonate. Gram-positive bacteria with a zona pellucida formed around the colony were designated as "lactic acid bacteria". Species of the isolated lactic acid bacteria were identified by a sequence of genes encoding rRNA. Lactic acid bacteria were inoculated into MRS medium and then cultured under static conditions for 1 to 3 days.

<Reagent>
Acarbose was purchased from LKT Laboratories, Inc. Voglibose was distributed by Takeda Pharmaceutical Company Limited.

<Method of quantifying blood glucose level>
The total amount of sugar in the silkworm body fluid was quantified by the phenol sulfate method (Hodge et al).
Body fluid (20 μL) was collected from a cut made with scissors on the first proleg, mixed with 9 times the amount of 0.6N perchloric acid, centrifuged at 3,000 rpm for 10 minutes, and the supernatant. Was used as a body fluid extract (hemolymph extract).
100 μL of body solution extract diluted to an appropriate concentration with distilled water and 100 μL of 5% (w / v) phenol aqueous solution are mixed, 500 μL of concentrated sulfuric acid is added, the mixture is vigorously stirred, and the mixture is allowed to stand at room temperature for 20 minutes and then allowed to stand at 490 nm. The absorbance at was measured. An aqueous glucose solution was used as a standard sugar solution. The glucose concentration in the silkworm body fluid was quantified by a glucometer (Accu-Chek, Roche).

<Measurement of α-glucosidase activity>
Measurements of α-glycosidase activity were performed according to the report by Watanabe et al. (Watanabe S et al., Insect Biochemistry and Molecular Biology, 2013). The higher the concentration of "pNP", the higher the α-glycosidase activity.

<Statistical analysis>
Numerical data was presented as mean ± standard error of the mean (SEM). Significant differences were evaluated using Student's t-test.

<< Lactic acid bacteria glucose metabolism test >>
The glucose metabolism ability of the lactic acid bacterium strain 0831-07 was measured using an API 50 CH kit (Sysmex Corporation). Colonies of the lactic acid bacterium 0831-07 strain were suspended in Suspense Medium (Sysmex Corporation) and prepared to have McFarland turbidity 2. 150 μL of the prepared bacterial solution sample was added to an API plate (Sysmex Corporation), and the cells were cultured at 30 ° C. for 48 hours. After culturing, the metabolic capacity for various sugars was determined by observing the reacted colors.

<< Manufacturing of yogurt >>
0.1 g of cells of the lactic acid bacterium 0831-07 strain grown on MRS agar medium was scraped off with Aese and suspended in 5 mL of 0.9% NaCl. 5 mL of the suspension of the fungus was added to 1 L of milk (Meiji delicious milk, carton box) and stirred. The cells were cultured at 43 ° C. for 20 hours. After confirming gelation, it was stored at 4 ° C. The storage period was 3 weeks or less.

<< Clinical trials using humans >>
A glucose tolerance test was conducted on the subjects using a 50% (w / v) sucrose aqueous solution before and after ingestion of yogurt, and transition data of each blood glucose level was collected. Ten subjects were divided into two groups and randomly performed in an open-label, two-group, two-stage crossover method. The criteria for selecting the subjects are as follows.

<<< Selection Criteria for Healthy Persons in Human Clinical Trials >>>
Those who meet all of the following criteria were targeted.
(1) Persons aged 20 to 60 years old at the time of consent acquisition (2) Healthy adult men and women (3) Prior to participating in the test, receive sufficient explanation about the test product and test, and the subject's free will Person who obtained document consent

<<< Exclusion Criteria >>
Those who violate the following will not be included in the test.
(1) Those who have been diagnosed with diabetes by a doctor and are currently being treated (2) Those who are allergic to dairy products (3) Those who are taking medicines or health foods that may affect blood sugar (4) Those who have a chronic disease and regularly use medicines (5) Those who have had their stomach or intestines removed (6) Others who are responsible for the study (sharing) have judged that they are not suitable as subjects for the study. Person

<<< Restrictions >>>
(1) Supper on the day before the test will be finished from 19:00 to 23:00, and after that, fast until the end of the test. Drinking water (water only) is allowed.
(2) Excessive alcohol intake on the day before the test is prohibited.
(3) Drinking water is prohibited from 1 hour before the glucose load until the end of the test.
(4) On the day of the inspection, smoking is prohibited from waking up to the end of the inspection.
(5) Exercise is prohibited during the inspection. Rest in the day room or on the bed.

A sucrose tolerance test with or without yogurt was carried out at intervals of 2 days or more. The time of sucrose loading in each subject was set to be the same. It was collected from the fingertips of the subject's hand using a puncture device. The blood glucose level of the subject was measured 15 minutes before the sucrose load. The yogurt was ingested 10 minutes before the sucrose load. 200 mL was ingested within 2 minutes. Then, 150 mL of a 50% (w / v) sucrose aqueous solution was drunk (the whole amount was ingested within 1 minute). Blood glucose levels were measured at 15, 30, 45, 60, 90 and 120 minutes after sucrose loading. The blood glucose level was measured using a simple blood glucose meter (Accucheck Aviva (Roche)). The clinical trial was approved by the Ethics Review Committee of Osaki Hospital Tokyo Heart Center.

<< Statistical analysis >>
Numerical data was presented as mean ± standard error of the mean (SEM). Significant differences were evaluated using Student's t-test.

Example 1
[Increase in glucose concentration in silkworm body fluid by feeding sucrose]
So far, the present inventors have found that the total sugar content and glucose concentration in the body fluid of silkworms are increased by feeding silkworms with a diet containing glucose (Non-Patent Document 1).
On the other hand, among the sugars in foods, sucrose is a particularly important cause of obesity and diabetes. Further, in mammals, it is already known that sucrose in food is decomposed into glucose and fructose by α-glycosidase in the intestinal tract, and each sugar is absorbed.
Therefore, it was examined whether the total sugar content and glucose concentration in the body fluid of the silkworm were increased by feeding the silkworm with a diet containing sucrose.

The 5th instar 1st day silkworm is fed a "normal diet" or a "10% by mass sucrose diet" containing 10% by mass of sucrose with respect to the total mass of the diet (10% Sucrose diet). It was. Then, the body fluid of the silkworm was collected over time by the above-mentioned method or the like. The total sugar content and glucose concentration in the body fluid were measured, and a significant difference test was performed using Student's t-test.

The results are shown in FIG. In FIG. 1, the horizontal axis is the elapsed time (unit: time) since feeding the silkworm, and the vertical axis is the total sugar level (A) or glucose concentration (B) (unit: time). In mg / mL), error bars indicate standard error (SEM). Black circles show the results when a normal diet is given, and white circles show the results when a 10% by mass sucrose diet is given. Experiments were performed with n = 5 / group.

As a result of FIG. 1, it was found that the total sugar concentration and the glucose concentration in the body fluids of the silkworms fed the "10% sucrose diet" were all rapidly increased as compared with the silkworms fed the "normal diet". From the above results, it was found that sucrose was decomposed into glucose and fructose by an enzymatic reaction in the intestinal tract of silkworms, and each sugar was absorbed and transferred into the body fluid, thereby increasing the total sugar concentration and the glucose concentration in the body fluid. Do you get it.

Example 2
[Inhibitory effect of acarbose and voglibose on the increase in glucose concentration in silkworm body fluid by feeding sucrose]
It is known that there is a gene encoding α-glycosidase that decomposes sucrose into glucose and fructose in the silkworm genome (Watanabe S et al., Insect Biochemistry and Molecular Biology, 2013).
Therefore, next, it was examined whether or not sucrose is decomposed by α-glycosidase in the intestinal tract in silkworms.
After feeding silkworms on the 1st day of the 5th instar with normal food for 1 day, the intestinal tract, intestinal contents (Lumen), and body fluid (Hemolymph) were collected, and the intestinal tract whose α-glycosidase activity was measured was crushed by sonication. The cell disruption fraction (Intestine) was used. Experiments were performed in groups n = 4-5 / group.

FIG. 2 shows the measurement results of α-glycosidase activity. The higher the concentration (nmol) of "Produced pNP", the higher the α-glycosidase activity.
The horizontal axis of FIG. 2A is the concentration of protein (μg / mL) contained in the cell disruption fraction of the intestinal tract.

FIG. 2A shows the dose-dependent measurement results of α-glycosidase activity in the cell disruption fraction of the intestinal tract. It was confirmed that the cell disruption fraction of the intestinal tract had α-glycosidase activity.

FIG. 2B shows the results of measuring the α-glycosidase activity per 1 μg of each of the intestinal cell disruption fraction (Intestine), intestinal content (Lumen), and body fluid (Hemolymph).
It was confirmed that there is α-glycosidase activity in the silkworm intestinal cell disruption fluid and body fluid. In addition, the specific activity of α-glycosidase activity in the silkworm intestinal cell disruption solution was higher than the specific activity of the intestinal contents.

Next, it was examined whether Acarbose or Voglibose inhibits the α-glycosidase activity of the silkworm intestinal tract.
After feeding silkworms on the 1st day of 5th instar with normal food for 1 day, the intestinal tract was collected, and acarbose or voglibose was added to the cell disruption fraction (Intestine) disrupted by sonication to measure α-glycosidase activity. .. Experiments were performed with n = 5 / group.

The results are shown in FIGS. 2C and 2D. The α-glycosidase activity in the silkworm intestinal cell disruption solution was inhibited by the addition of acarbose (Fig. 2C) and voglibose (Fig. 2D), which are typical α-glycosidase inhibitors.

Next, it was examined whether the increase in sugar concentration in the body fluid of silkworms due to sucrose feeding could be suppressed by the addition of acarbose and voglibose to the diet.
Silkworms on the first day of the fifth instar were fed a 10% by mass sucrose diet or a 10 mass% sucrose diet supplemented with acarbose or voglibose for 1 hour. The body fluid of the silkworm was collected as described above, and the total sugar content and glucose concentration in the body fluid were measured. A significant difference test was performed using Student's t-test.

The results are shown in FIG. FIG. 3A shows the total sugar amount when acarbose is added, FIG. 3B shows the glucose concentration when acarbose is added, FIG. 3C shows the total sugar amount when voglibose is added, and FIG. 3D shows the glucose concentration when voglibose is added. The measurement result is shown.
The horizontal axis in FIG. 3 is the concentration (content) of acarbose or voglibose contained in the bait (unit: mass%). * Indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001, and error bars indicate standard error (SEM). Experiments were performed with n = 5 / group.

As a result of FIG. 3, the total sugar concentration and the glucose concentration in the body fluid of the silkworm that ate the diet containing acarbose having a final content of 8% by mass or bogribose having a final content of 4% by mass in the diet containing sucrose were both compounds. The results were lower than those of silkworms that ate a non-additive diet (acarbose or boglibose).

In mammals, acarbose and boglibose have been reported to suppress the increase in blood glucose level due to the feeding of sucrose, but not the increase in blood glucose level due to the feeding of the monosaccharide glucose (Tschope D et al). ., Cardiovasc Diabetol., 2013). Therefore, in silkworms as well, it was examined whether or not the effects of these α-glycosidase inhibitors differ depending on the sugar contained in the diet.
10% by mass sucrose diet; 10% by mass glucose diet; "10% by mass sucrose diet or 10% by mass glucose diet" for "acarbose (final content 8% by mass)" or "boglibose (final)" for silkworms on the first day of 5th instar The diet was fed with a content of 4% by mass) for 1 hour.
The body fluid of the silkworm was collected in the same manner as described above, and the glucose concentration in the body fluid was measured. A significant difference test was performed using Student's t-test.

The results are shown in FIG. FIG. 4A shows the measurement results of glucose concentration when acarbose was added, and FIG. 4B shows the measurement results when voglibose was added. In the horizontal axis, "+ Sucrose" indicates a 10 mass% sucrose diet, and "+ Glucose" indicates a 10 mass% glucose diet. *** indicates P <0.001, and error bars indicate standard error (SEM). Experiments were performed with n = 5 / group.

As a result of FIG. 4, the glucose concentration in the body fluid of the silkworm, which was increased by adding 10% by mass of sucrose to the diet, was suppressed by adding acarbose having a final content of 8% by mass or bogribose having a final content of 4% by mass to the diet. It was. On the other hand, when 10% by mass glucose was added to the diet, the effects of these compounds (acarbose or voglibose) were not observed.
From the above results, it was found that in silkworms as well as in mammals, acarbose and voglibose bring about an inhibitory effect on blood glucose elevation by an α-glycosidase inhibitory effect.

Example 3
[Effect of lactic acid bacteria on increase in glucose concentration in silkworm body fluid by feeding sucrose]
Acarbose, an α-glycosidase inhibitor, has been reported to be produced by a Gram-positive bacterium called Actinoplanes sp. SE50 / 110 (Schwientek P et al., BMC Genomics, 2012). It has also been reported that the addition of Lactobacillus rhamnosus, a type of lactic acid bacterium, to the diet of mice suppresses the increase in blood glucose concentration caused by sucrose (Honda K et al., J. Clin. Biochem. Nutr. , 2012). Furthermore, it has been reported that the heat-treated bacterial cell fraction of lactic acid bacteria has α-glycosidase inhibitory activity (Panwar H et al., Eur J Nutr, 2014).

Therefore, from the lactic acid bacterium library (Table 1) possessed by the present inventors, lactic acid bacteria that inhibit "increase in glucose concentration in silkworm body fluid due to sucrose feeding" were screened.
Silkworms on the 1st day of the 5th instar were fed a diet containing 10% by mass of sucrose and various lactic acid bacteria (final content 25% by mass, Table 1) for 1 hour. The body fluid of the silkworm was collected in the same manner as described above, and the glucose concentration in the body fluid was measured. A significant difference test was performed using Student's t-test.

The results are shown in FIG. The vertical axis shows the ratio (%) of the blood glucose level when the blood glucose level of the silkworm after feeding the control (10 mass% sucrose diet without lactic acid bacteria) for 1 hour is 100 mass%. * Indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001, and error bars indicate standard error (SEM). Experiments were performed in groups n = 4-15 / group.

As a result of FIG. 5, it was found that for some lactic acid bacteria, addition of them to a 10% by mass sucrose diet suppressed the increase in the blood glucose level of silkworms.

Next, the glucose concentration in the body fluid of the silkworm was measured by feeding the sucrose-containing diet by adding the lactic acid bacterium # L1-1 strain, which was most remarkably suppressed in the increase in blood glucose in FIG.
The silkworms on the first day of the 5th instar were fed with a 10 mass% sucrose diet containing lactic acid bacteria (LAB) # L1-1 (final content 0 to 50 mass%) for 1 hour. It was. The body fluid of the silkworm was collected as described above, and the glucose concentration in the body fluid was measured.

The results are shown in FIG. 6A. In the lactic acid bacterium # L1-1 strain in which the suppression of the increase in blood glucose was most remarkable in FIG. 5, the effect of suppressing the increase in blood glucose depending on the amount of cells added to the diet was observed.

In addition, "difference in inhibitory effect on increase in glucose concentration in silkworm body fluid by feeding sucrose or glucose-containing diet" was verified by adding or not adding lactic acid bacterium # L1-1 strain.
10% by mass sucrose diet; 10% by mass glucose diet; "Lactobacillus # L1-1 strain (final content 25% by mass)" for each of "10% by mass sucrose diet or 10% by mass glucose diet" ) Was added for 1 hour. The body fluid of the silkworm was collected, and the glucose concentration in the body fluid was measured as described above. A significant difference test was performed using Student's t-test.

The results are shown in FIG. 6B. * Indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001, and error bars indicate standard error (SEM). Experiments were performed with n = 5 / group.

As a result of FIG. 6B, the effect of suppressing the increase in blood glucose by the lactic acid bacterium Lactococcus lactis # L1-1 strain was not observed in the case of the diet containing 10% by mass glucose.
The above results suggest that the suppression of blood glucose elevation by feeding sucrose of the Lactococcus lactis # L1-1 strain is due to the suppression of sugar absorption from the intestinal tract by inhibition of α-glycosidase in the silkworm intestinal tract.

Example 4
<Identification of lactic acid bacteria that suppress the increase in glucose concentration in silkworm body fluids due to sucrose feeding>
Furthermore, in addition to the lactic acid bacteria listed in Table 1, we searched the lactic acid bacteria library (Table 2) owned by the inventors for lactic acid bacteria that significantly inhibit the increase in glucose concentration in the body fluid of silkworms due to sucrose feeding. did.

Silkworms on the 1st day of the 5th instar were fed a diet containing 10% by mass of sucrose diet and various lactic acid bacteria shown in Table 1 (25% of the total diet) for 1 hour. After that, the body fluid of the silkworm was collected, the glucose concentration in the body fluid was measured, and a significant difference test was performed using Student's t-test. The results are shown in FIG.
In FIG. 7, the vertical axis shows the glucose concentration (mg / dL). “Control” on the horizontal axis shows the results when the silkworms were fed a 10% by mass sucrose diet containing no lactic acid bacteria. * Is P <0.05, and error bars indicate standard error (SEM). It was performed at n = 3 per group.

As a result of FIG. 7, the Enterococcus faecalis 0831-07 (Enterococcus faecalis # Ef-1) strain markedly suppressed the increase in the blood glucose level of silkworms due to the feeding of a 10% by mass sucrose diet.
Hereinafter, Enterococcus faecalis 0831-07 may be abbreviated as "lactic acid bacterium 0831-07" or "lactic acid bacterium # Ef-1".

Next, the silkworms on the first day of the fifth instar were fed a diet containing a 10 mass% sucrose diet supplemented with a lactic acid bacterium 0831-07 strain (0 to 50 mass% with respect to the total diet) for 1 hour. After that, the body fluid of the silkworm was collected, and the glucose concentration in the body fluid was measured. The results are shown in FIG.
In FIG. 8, the vertical axis shows the glucose concentration (mg / dL), and the horizontal axis shows the content (mass%) of the lactic acid bacterium 0831-07 strain. ** is P <0.01, and error bars indicate standard error (SEM). It was performed at n = 5 per group.

As a result of FIG. 8, the effect of suppressing the increase in blood glucose by the lactic acid bacterium strain 0831-07 was dependent on the amount of cells added to the diet.

Next, a 10% by mass sucrose diet or "a diet obtained by adding a lactic acid bacterium 0831-07 strain (25% by mass to the total diet) to a 10 mass% glucose diet" was added to the 5th instar 1st day silkworm for 1 hour. Gave. The body fluid of the silkworm was collected and the glucose concentration in the body fluid was measured. A significant difference test was performed using Student's t-test. The results are shown in FIG.
In FIG. 9, the vertical axis shows the glucose concentration (mg / dL). “+ Sucrose” on the horizontal axis indicates a 10 mass% sucrose diet, and “+ Glucose” indicates a 10 mass% glucose diet. *** is P <0.001, and error bars indicate standard error (SEM). It was performed at n = 7 per group.

As a result of FIG. 9, the lactic acid bacterium 0831-07 (lactic acid bacterium # Ef-1) strain remarkably suppressed "the increase in the blood glucose level of silkworms due to the feeding of a 10 mass% sucrose diet".
It should be noted that the inhibitory effect of the lactic acid bacterium 0831-07 strain was also observed on the increase in glucose concentration in the body fluid when glucose was fed.
In addition, the lactic acid bacterium strain 0831-07 was able to grow in a medium containing glucose or fructose.

Next, "lactic acid bacteria 0831-07 strain (25% by mass with respect to the whole diet)" or "lactic acid bacteria 0831-07 strain heat-treated by autoclave treatment" on the 5th instar 1st day silkworm, 10 mass% sucrose diet ( A diet containing 25% by mass) ”was given for 1 hour. Then, the body fluid of the silkworm was collected, and the glucose concentration in the body fluid was measured. A significant difference test was performed using Student's t-test. The results are shown in FIG.
In FIG. 10, the vertical axis shows the glucose concentration (mg / dL). “Control” on the horizontal axis shows the results when the silkworms were fed a 10% by mass sucrose diet containing no lactic acid bacteria. “-” Indicates a lactic acid bacterium 0831-07 (# Ef-1) strain that has not been heat-treated, and “Heat-killed” indicates a lactic acid bacterium 0831-07 strain that has been heat-treated by an autoclave treatment.

As a result of FIG. 10, the heat-treated bacterial cell fraction of the lactic acid bacterium 0831-07 strain suppressed the increase in glucose concentration in the body fluid of the silkworm due to feeding of the sucrose diet, as in the case of the untreated viable bacteria (FIG. 10). ).
Therefore, it was suggested that the inhibitory effect of the lactic acid bacterium 0831-07 strain on the increase in blood glucose by feeding sucrose was due to the inhibition of α-glycosidase in the silkworm intestinal tract by the heat treatment resistant component of the lactic acid bacterium 0831-07 strain.

Example 5
<Inhibitory effect of lactic acid bacterium 0831-07 strain on increase in blood glucose level due to human sucrose feeding>
Next, in order to verify whether the lactic acid bacterium 0831-07 strain suppresses postprandial hyperglycemia in humans, yogurt was produced from milk using the lactic acid bacterium 0831-07 strain.
A sucrose (sucrose) tolerance test was performed on the subjects by a crossover method between when the yogurt was not ingested and when the yogurt was ingested.
Subjects in the yogurt intake group ingested 200 mL of yogurt 10 minutes before sucrose loading. Then, both the subjects in the yogurt non-ingestion group and the yogurt ingestion group drank 150 mL of a 50% (w / v) sucrose aqueous solution. Blood glucose levels were measured at 15, 30, 45, 60, 90 and 120 minutes after sucrose loading. The blood glucose level was measured by collecting a small amount of blood from the fingertip and using a simple blood glucose meter. A significant difference test was performed using Student's t-test. The results are shown in FIG.
In FIG. 11, the vertical axis shows the glucose concentration (mg / dL). The horizontal axis shows the elapsed time (minutes) after ingestion (loading) of sucrose. * Is P <0.05, and error bars indicate standard error (SEM). It was performed at n = 10 per group.

As a result of FIG. 11, the subjects who ate the yogurt produced using the lactic acid bacterium strain 0831-07 had lower blood glucose levels 45 minutes after sucrose loading as compared with the non-feeding group (FIG. 11).
Therefore, it was suggested that the bacterial cell component of the lactic acid bacterium 0831-07 strain has an effect of suppressing an increase in blood glucose level due to ingestion of sucrose (sucrose) in humans.

Example 6
<Inhibitory effect of addition of killed lactic acid bacteria to increase glucose concentration in silkworm body fluid by feeding sucrose-containing food>
Heat-killed bacterial cell fraction of lactic acid bacterium 0831-07 (# Ef-1) strain or autoclaved lactic acid bacterium 0831-07 (# Ef-1) strain on a diet containing 10% by mass sucrose (Heat-killed # Ef-1) Was fed to silkworms on the 1st day of the 5th instar for 1 hour with the addition of sucrose added at 6.3, 12.5, and 25% by mass based on the total diet. The body fluid of the silkworm was collected and the glucose concentration in the body fluid was measured. The results are shown in FIG. 12a.

In FIG. 12a, the vertical axis shows the glucose concentration (mg / dL). “No bacteria” on the horizontal axis shows the results when the silkworm was fed a 10% by mass sucrose diet containing no lactic acid bacterium 0831-07 (# Ef-1) strain. "Viable # Ef-1 content" indicates the content of the unheat-treated lactic acid bacterium 0831-07 strain, and "Heat-killed # Ef-1 content" indicates the content of the heat-treated lactic acid bacterium 0831-07 strain.
*** is P <0.001, ** is p <0.01, and error bars indicate standard error (SEM). It was carried out at n = 11-14 per group.

As a result of FIG. 12a, it was found that the inhibitory effect of the lactic acid bacterium strain 0831-07 on the increase in blood glucose was enhanced depending on the amount of cells added to the diet. In addition, the heat-treated bacterial cell fraction of the lactic acid bacterium 0831-07 strain also suppressed the increase in glucose concentration in the silkworm body fluid due to feeding of the sucrose-containing diet in a dose-dependent manner.

Next, a heat-treated bacterial cell fraction (Heat-killed # Ef-1) of a lactic acid bacterium 0831-07 (# Ef-1) strain or an autoclaved lactic acid bacterium 0831-07 (# Ef-1) strain on a 10 mass% glucose diet. ) Was added so as to be 25% by mass based on the total diet, and the silkworms on the first day of 5th instar were fed for 1 hour. The body fluid of the silkworm was collected, the glucose concentration in the body fluid was measured, and a significant difference test was performed using Student's t-test. The results are shown in FIG. 12b.

In FIG. 12b, the vertical axis shows the glucose concentration (mg / dL). “No bacteria” on the horizontal axis shows the results when the silkworms were fed a 10 mass% glucose diet containing no lactic acid bacterium 0831-07 (# Ef-1) strain. "Viable" was fed a 10% by mass glucose diet containing an unheat-treated lactic acid bacterium 0831-07 strain, and "Heat-killed" was fed a 10% by mass glucose diet containing a heat-treated lactic acid bacterium 0831-07 strain. The result of the time is shown.
* Is P <0.05, ** is P <0.01, and error bars indicate standard error (SEM). It was performed at n = 6 to 7 per group.

As a result of FIG. 12b, the lactic acid bacterium strain 0831-07, which had not been heat-treated, suppressed an increase in glucose concentration in the silkworm body fluid due to feeding of a glucose-containing diet. On the other hand, the heat-treated cells of the lactic acid bacterium 0831-07 strain were not found to have an activity of suppressing an increase in glucose concentration in the silkworm body fluid after glucose feeding.

The above results show that the viable lactic acid bacterium 0831-07 (# Ef-1) strain suppresses the increase in glucose concentration in the silkworm body fluid after ingestion of sucrose and glucose, and that the lactic acid bacterium 0831-07 (# Ef-1) It was suggested that the heat-treated cells of the strain still have the activity of suppressing the increase in glucose concentration in the body fluid of silkworms after ingestion of sucrose.

Example 7
<Construction of experimental scheme for sugar transfer evaluation system in silkworm in vitro>

Next, we attempted to elucidate the mechanism by which the lactic acid bacterium strain 0831-07 suppresses the increase in blood glucose of silkworms after feeding sucrose. In mammals, sucrose is known to be decomposed into glucose and fructose by α-glycosidase in the intestinal tract and absorbed from the intestinal tract. First, an experimental system was constructed to analyze the process by which sucrose in the intestinal tract of silkworms is decomposed by α-glycosidase and glucose is transferred to the outside of the intestinal tract.
The intestinal tract of the silkworm on the first day of the fifth instar was removed and tied with a thread so that the solution could be put in it. A sucrose solution was added into the intestinal tract of the silkworm and tied with a thread so that the solution did not leak. Then, it was incubated in PBS to quantify the glucose transported out of the intestinal tract.
“Experimental scheme of sugar transfer evaluation system in silkworm in vitro” is shown in FIG. 13a.

Using the above experimental scheme, a sucrose solution in the silkworm intestine or a sample obtained by adding acarbose (40 mg / mL) to the sucrose solution is placed in the silkworm intestine, incubated at 27 ° C., and glucose in the extraintestinal fluid over time. The concentration was measured. The results are shown in FIG. 13b.
In FIG. 13b, the vertical axis shows the glucose concentration (mg / dL). The horizontal axis shows the incubation time (minutes).

As a result of FIG. 13b, it was found that the glucose concentration in the extraintestinal fluid increased in a time-dependent manner (black circles in FIG. 13b). In addition, the increase in glucose concentration in the extraintestinal fluid was suppressed by adding acarbose, which is an α-glycosidase inhibitor (white circle in FIG. 13b). From this result, it was suggested that sucrose is decomposed into glucose and fructose by α-glycosidase in the intestinal tract of silkworms and further transported to the outside of the intestinal tract.

Example 8
<Mechanism analysis of postprandial hyperglycemia suppression after eating sucrose by lactic acid bacterium strain 0831-07>
Using the experimental scheme constructed in Example 7, a sucrose solution or a sample obtained by adding a sucrose solution to a sucrose solution with a lactic acid bacterium 0831-07 strain (250 mg (wet weight) / mL) was placed in the intestinal tract of the silkworm, 27. The silkworm was incubated at ° C, and the glucose concentration of the extraintestinal fluid was measured over time. The results are shown in FIG. 14a.
In FIG. 14a, the vertical axis shows the glucose concentration (mg / dL). The horizontal axis shows the incubation time (minutes).

In addition, a sucrose solution in the intestinal tract of the silkworm, or a sample obtained by adding a lactic acid bacterium strain 0831-07 (31 mg, 63 mg, 125 mg, 250 mg (wet weight) / mL) to the sucrose solution is placed in the intestinal tract of the silkworm and incubated at 27 ° C. , The glucose concentration of the extraintestinal fluid after 60 minutes was measured. The results are shown in FIG. 14b.
In FIG. 14b, the vertical axis shows the glucose concentration (mg / dL). The horizontal axis shows the amount (mg / mL) of the lactic acid bacterium 0831-07 (# Ef-1) strain. * Is P <0.05, ** is p <0.01, and error bars indicate standard error (SEM). It was performed at n = 3 to 5 per group.

As a result of FIGS. 14a and 14b, when a viable lactic acid bacterium strain 0831-07 was added to the sucrose solution in the intestinal tract of silkworm, the increase in glucose concentration in the extraintestinal fluid was suppressed. It was also found that the inhibitory effect was enhanced depending on the amount of cells (Fig. 14b).

Next, a sucrose solution or a heat-treated bacterial cell fraction (Heat-killed # Ef-1) (250 mg (wet weight) / mL) of an autoclaved lactic acid bacterium strain 0831-07 was added to the sucrose solution in the intestinal tract of silkworms. The sample was placed in the intestinal tract of the silkworm, incubated at 27 ° C., and the glucose concentration of the extraintestinal fluid was measured over time. The results are shown in FIG. 14c.
In FIG. 14c, the vertical axis shows the glucose concentration (mg / dL). The horizontal axis shows the incubation time (minutes).

In addition, a heat-treated bacterial cell fraction (Heat-killed # Ef-1) (31 mg, 63 mg, 125 mg, 250 mg (wet weight)) of a sucrose solution or a lactic acid bacterium 0831-07 strain autoclaved in a sucrose solution in the intestinal tract of silkworms / The sample to which mL) was added was placed in the intestinal tract of a silkworm, incubated at 27 ° C., and the glucose concentration of the extraintestinal solution after 60 minutes was measured. The results are shown in FIG. 14d.
In FIG. 14d, the vertical axis shows the glucose concentration (mg / dL). The horizontal axis shows the amount (mg / mL) of the heat-treated bacterial cell fraction (Heat-killed # Ef-1) of the autoclaved lactic acid bacterium 0831-07 strain. ** is p <0.01, and error bars indicate standard error (SEM). It was performed at n = 3 to 5 per group.

As a result of FIGS. 14c and 14d, the increase in glucose concentration in the extraintestinal fluid was suppressed even when the heat-treated bacterial cell fraction of the lactic acid bacterium 0831-07 strain was added. It was also found that the inhibitory effect was enhanced depending on the amount of cells (Fig. 14d).
From these results, it was suggested that the heat resistance factor of the lactic acid bacterium strain 0831-07 inhibits the process of transfer of the molecule (glucose) obtained by decomposing sucrose in the intestinal tract of silkworm to the outside of the intestinal tract.

Next, a glucose solution in the intestinal tract of the silkworm, or a sample obtained by adding a lactic acid bacterium strain 0831-07 (250 mg (wet weight) / mL) to the glucose solution, was placed in the intestinal tract of the silkworm, incubated at 27 ° C., and incubated over time. The glucose concentration of the external solution was measured. The results are shown in FIG. 15a.
In FIG. 15a, the vertical axis shows the glucose concentration (mg / dL). The horizontal axis shows the incubation time (minutes).

As a result of FIG. 15a, even when the glucose solution was sealed in the intestinal tract of the silkworm, permeation of glucose out of the intestinal tract was observed with the passage of time.

In addition, a sample in which a glucose solution or a heat-treated bacterial cell fraction (Heat-killed # Ef-1) (250 mg (wet weight) / mL) of an autoclaved lactic acid bacterium strain 0831-07 was added to the glucose solution in the intestinal tract of the silkworm. Was placed in the intestinal tract of silkworms and incubated at 27 ° C., and the glucose concentration of the extraintestinal fluid was measured over time. The results are shown in FIG. 15b.
In FIG. 15b, the vertical axis shows the glucose concentration (mg / dL). “No bacteria” on the horizontal axis shows the result of a glucose solution containing no lactic acid bacterium 0831-07 (# Ef-1) strain. "Viable" shows the result of the glucose solution to which the unheat-treated lactic acid bacterium 0831-07 strain was added, and "Heat-killed" shows the result of the glucose solution to which the heat-treated lactic acid bacterium 0831-07 strain was added. * Is p <0.05, and error bars indicate standard error (SEM). It was performed at n = 3 to 4 per group.

It was found that when a viable lactic acid bacterium strain 0831-07 was added into the intestinal tract, an increase in glucose concentration in the extraintestinal fluid was suppressed (FIGS. 15a and 15a and b). On the other hand, when the heat-treated bacterial cell fraction of the lactic acid bacterium 0831-07 strain was added, the increase in glucose concentration in the extraintestinal fluid was not suppressed (FIG. 15b).
From these results, it was suggested that the heat-sensitive factor of the lactic acid bacterium strain 0831-07 inhibits glucose transport from the intestinal tract to the outside of the intestinal tract of silkworms.

Example 9
<Inhibition of α-glycosidase activity in the intestinal tract of silkworms or rats by lactic acid bacterium strain 0831-07>
Next, it was examined whether the heat-treated fraction of the lactic acid bacterium 0831-07 strain inhibits the α-glycosidase activity of the silkworm intestinal tract. The measurement of α-glycosidase was carried out in the same manner as in Example 2.
The silkworms on the first day of the fifth instar were fed with normal food for one day. The α-glycosidase activity was measured by adding a cell crushed fraction obtained by crushing the intestinal tract of the silkworm by ultrasonic treatment and a heat-treated bacterial cell fraction (Heat-killed # Ef-1) of a lactic acid bacterium strain 0831-07. The results are shown in FIG. 16a.
In FIG. 16a, the vertical axis shows the concentration (nmol) of "Produced pNP". The higher the concentration (nmol) of "Produced pNP", the higher the α-glycosidase activity. The horizontal axis shows the amount (mg / mL) of the heat-treated bacterial cell fraction (Heat-killed # Ef-1) of the lactic acid bacterium 0831-07 strain.

As a result of FIG. 16a, α-glycosidase activity was observed in the cell disruption fraction of the silkworm intestinal tract, and it was found that the heat-treated fraction of the lactic acid bacterium 0831-07 strain inhibited this activity in a dose-dependent manner.

Next, the rat intestinal acetone extract fraction and the heat-treated bacterial cell fraction (Heat-killed # Ef-1) of the lactic acid bacterium 0831-07 strain were added to measure the α-glycosidase activity, and a significant difference was measured using Student's t-test. The test was performed. The results are shown in FIG. 16b.
In FIG. 16b, the vertical axis shows the concentration (nmol) of "Produced pNP". The higher the concentration (nmol) of "Produced pNP", the higher the α-glycosidase activity. The horizontal axis shows the amount (mg / mL) of the heat-treated bacterial cell fraction (Heat-killed # Ef-1) of the lactic acid bacterium 0831-07 strain. *** is p <0.001, and error bars indicate standard error (SEM). It was performed at n = 3 per group.

As a result of FIG. 16b, the heat-treated fraction of the lactic acid bacterium strain 0831-07 also inhibited the α-glycosidase activity in the rat intestinal lysate in a dose-dependent manner. Therefore, the heat-treated fraction of the lactic acid bacterium 0831-07 strain inhibits the α-glycosidase activity in the intestinal tract of silkworms and mammals, and the process in which sucrose in the intestinal tract is decomposed into glucose and fructose and transferred to the outside of the intestinal tract It was suggested that it was inhibited.

<Summary of Examples>
As confirmed by acarbose and voglibose, which are "inhibitors of α-glycosidase in humans," a test substance is a substance that suppresses the increase in human blood glucose level due to sucrose ingestion by using the method of the present invention. It turned out that it can be accurately evaluated whether or not it is.
In addition, since it was found that at least some lactic acid bacteria newly suppress the increase in blood glucose level, a substance (a candidate substance) that suppresses the increase in blood glucose level in humans by using the method of the present invention. Was found to be able to be screened.

Sucrose is a sweetener added to many dishes as well as sweets such as cakes. Foods high in sucrose are high in calories. Currently, the onset of lifestyle-related diseases such as obesity and blood sugar level rise due to excessive calorie intake and the subsequent onset of diabetes is a problem, and it is important to be careful not to raise the blood sugar level on a daily basis. It is said to be very important in controlling the onset. Diet and exercise therapy are considered to be effective for the prevention of lifestyle-related diseases, but it is often difficult to continue them. In the diet, the calorie intake is limited so that the postprandial blood glucose level does not rise.

Therefore, the development of food additives that suppress the rise in blood glucose level is considered to be effective in implementing a continuous diet. Ingestion of "substances such as lactic acid bacteria", which has been shown to have an effect of inhibiting an increase in blood glucose level in vivo by the evaluation method of the present invention, is more effective for the diet of obese and diabetic patients and their reserves. Expected to be targeted.

Further, in this example, a blood glucose elevation inhibition evaluation system using silkworm was used in the strains of the genera Lactococcus, Lactobacillus, Leuconostoc, and Enterococcus. It became clear that there are strains that are judged to be effective. These bacteria are used as lactic acid bacteria in the production of fermented foods.
In addition, among the five strains of Lactococcus lactis, one strain was determined to be effective by the blood glucose elevation inhibition evaluation system using silkworm. This result suggests that there is a difference in the α-glycosidase inhibitory effect among the strains of the same Lactococcus lactis.

Further, in order to obtain a strain that suppresses an increase in blood glucose level, an evaluation test using animals is required, and the evaluation method or screening method of the present invention can be used to quickly and easily screen (select) useful lactic acid bacteria. It turned out.

In addition, from the above example, yogurt prepared from the lactic acid bacterium 0831-07 (lactic acid bacterium # Ef-1) strain found as a result of screening using the silkworm evaluation system suppresses postprandial hyperglycemia due to human sucrose feeding. It was shown to do.
Therefore, the lactic acid bacterium strain 0831-07 obtained in this example is a functional lactic acid bacterium that suppresses an increase in blood glucose level due to ingestion of sucrose (sucrose). Yogurt made with the lactic acid bacteria is expected to be more effective in the diet of obese and diabetic patients and their reserve humans.

In addition, the lactic acid bacterium strain 0831-07 was found to have an activity of inhibiting α-glycosidase in the silkworm intestinal tract and an activity of inhibiting the transport of glucose from the intestinal tract to the outside of the intestinal tract. It was suggested that these activities possessed by the bacterium suppress the increase in blood glucose level of silkworms fed sucrose. FIG. 17 shows a schematic diagram showing the postprandial hyperglycemia suppressing effect of the lactic acid bacterium strain 0831-07.
In FIG. 17, "Lumen" is "luminal", "α-glucosidase" is "α-glycosidase", "Glycolysis" is "sucrose decomposition", and "Heat stable factor (s)" is "heat resistance factor", ""Heat sensitive factor (s)" is "heat sensitive factor", "Transport" is "transport", "Sucrose" is "sucrose", "Glucose" is "glucose", "Fructose" is "fructose", "E. faecalis"(# Ef-1) "indicates" lactic acid bacterium 0831-07 ".

In addition, the heat-treated cells of the lactic acid bacterium 0831-07 strain retained the activity of suppressing postprandial hyperglycemia due to sucrose feeding of silkworms. This fraction inhibited the activity of α-glycosidase, an enzyme that degrades silkworm intestinal sucrose, but not glucose transport in the intestinal tract. Therefore, it was suggested that the effect of the lactic acid bacterium 0831-07 strain on suppressing the increase in blood glucose of silkworms ingesting sucrose was mainly due to the inhibition of α-glycosidase activity (Fig. 17).

In order to evaluate the effectiveness of food, it is necessary to evaluate using individual animals. Mammals such as mice and rats, which have been conventionally used as experimental animals, are expensive to carry out screening using a large number of individuals. On the other hand, silkworms do not require a large breeding space and can breed a large number of individuals at low cost. Furthermore, from the viewpoint of animal protection, experiments using mammals are conducted in accordance with the international principles of the 3Rs, namely Replacement (development of alternative methods), Reduction (reduction of animal numbers), and Refinement (reduction of animal pain). Must be (Russell et al., 1959).
The use of silkworms as alternative animals is consistent with the idea of Relative Replacement in the 3Rs. The use of silkworms in the search for functional foods could reduce the number of mammals sacrificed and solve problems in terms of cost and animal protection.

Not only can a novel "substance that suppresses the increase in human blood glucose level due to sucrose intake by exerting α-glycosidase inhibitory activity" be searched for by the evaluation method of the present invention, but also the evaluation method of the present invention can be used to regulate blood glucose. It can be used for probiotic research related to sucrose, and is widely used in the fields of pharmaceuticals, general foods, health foods, etc.

This application is based on Japanese Patent Application No. 2015-199959, which was filed on October 8, 2015, and Japanese Patent Application No. 2016-159554, which was filed on August 16, 2016. , All the contents of those applications are cited herein and incorporated as disclosure of the specification of the present invention.

Claims (4)

A method for evaluating whether or not a test substance is a substance that suppresses an increase in human blood glucose level due to sucrose intake by exerting α-glycosidase inhibitory activity, and is a method for evaluating at least the following steps (a) and (b). ) And step (c),
(A) Step of feeding silkworms with sucrose,
(B) A step of administering the test substance at the same time as the step (a) or before or after the step (a).
(C) A step of measuring the sugar concentration in the body fluid or intestinal cells of the silkworm to which the test substance has been administered.
A method characterized by having. A method for screening a substance that suppresses an increase in human blood glucose level due to sucrose intake by exerting α-glycosidase inhibitory activity from among the test substances, and is a method of at least the following steps (a), (b), and step. (C) and step (d),
(A) Step of feeding silkworms with sucrose,
(B) A step of administering the test substance at the same time as the step (a) or before or after the step (a).
(C) A step of measuring the sugar concentration in the body fluid or intestinal cells of the silkworm to which the test substance has been administered.
(D) A step of selecting a substance that reduces the concentration of sugar in the body fluid or intestinal cells of silkworms from the above test substances.
A method characterized by having. The method according to claim 1 or 2, wherein the test substance is lactic acid bacteria or a product thereof. A method for producing a substance that suppresses an increase in human blood glucose level due to sucrose intake by exerting α-glycosidase inhibitory activity.
A manufacturing method comprising the method according to claim 2 or 3.