JP7267020B2 - Fermented milk with inhibitory effect on blood sugar level elevation - Google Patents

Description

IPOD FERM BP-10741 NPMD NITE BP-01697 NPMD NITE BP-01814 NPMD NITE BP-01815

TECHNICAL FIELD The present invention relates to fermented milk having an effect of suppressing elevation of blood sugar level.

According to the National Health and Nutrition Survey conducted in Japan in 2007, approximately 8.9 million people are strongly suspected of having diabetes, and 13.2 million people cannot rule out the possibility of diabetes. In other words, there are 22.1 million people with diabetes and potential diabetes patients in Japan, which is a very large number. Diabetes is mainly classified into type 1 diabetes and type 2 diabetes, but many diabetes patients have type 2 diabetes. In type 2 diabetes, insulin function is not sufficiently exerted due to a decrease in insulin secretion or development of insulin resistance due to environmental factors such as lifestyle or genetic factors, resulting in an increase in blood sugar level. Increased blood sugar promotes insulin secretion from pancreatic β-cells, but this gradually exhausts pancreatic β-cells and reduces their ability to secrete insulin, exacerbating the condition. In addition, long-term elevated blood sugar damages blood vessels and other cells and tissues, causing various complications such as arteriosclerosis, retinopathy, neuropathy, and kidney disease. Furthermore, diabetes is also known to be a risk factor for osteoporosis (Non-Patent Document 1). Since type 2 diabetes is a disease that progresses unnoticed with few subjective symptoms, it is very important to prevent it through daily lifestyle habits such as diet.

Diabetes is associated not only with high fasting blood sugar levels, but also with rapid rises in postprandial blood sugar levels. Academically, postprandial hyperglycemia is known to be a risk factor for cardiovascular disease (Non-Patent Document 2). Currently, postprandial hyperglycemia is considered to be a more important risk factor for diabetes and its complications than high fasting blood sugar levels, and it is effective in suppressing rapid postprandial rises in blood sugar levels. A method is needed. α-Glucosidase inhibitors such as acarbose can inhibit postprandial blood glucose elevation by inhibiting glycolysis and delaying sugar absorption, and are used as therapeutic agents for postprandial hyperglycemia. Also, indigestible dextrin is used to suppress postprandial elevation of blood sugar level by suppressing sugar absorption. However, even if the postprandial rise in blood sugar level can be suppressed, this alone does not necessarily lead to the suppression of glucose intolerance underlying diabetes and the prevention of diabetes-related complications. On the other hand, control of blood sugar level requires long-term intake of a blood sugar level elevation inhibitor, and a food composition having a good blood sugar level elevation inhibitory effect is desired.

Patent Document 1 discloses a diabetic complication preventive, ameliorating, and therapeutic agent containing, as active ingredients, a culture or cell of Lactobacillus gasseri and a culture or cell of Bifidobacterium longum. However, Patent Document 1 discloses that colonization of bacteria in the intestinal tract prevents the development of insulin resistance and improves diabetic nephropathy. It needs to be established.

Patent Document 2 discloses a preventive/therapeutic agent for diabetes containing, as an active ingredient, Lactobacillus fermentum NM316 strain that produces polysaccharides. Patent Document 2 discloses that the NM316 strain, which has high intestinal survival, reduces intestinal low-molecular-weight sugars by converting them into indigestible or indigestible macromolecular substances (polysaccharides), thereby preventing obesity and diabetes. can be prevented or treated. However, Patent Document 2 does not describe the preventive effect of diabetic complications.

Patent Document 3 discloses a metabolic syndrome preventive/improvement agent, a blood glucose level elevation inhibitor, a blood glucose level reducing agent, etc., containing Lactobacillus salivarius as an active ingredient. However, Patent Document 3 does not describe the preventive effect of diabetic complications. Furthermore, Patent Document 3 discloses that the effects of lactic acid bacteria and bifidobacteria on sugar and lipid metabolism vary greatly depending on genera and species.

Non-Patent Document 3 reports that ingestion of Caspian Sea yogurt produced using Lactococcus lactis subsp. cremoris FC strain suppresses elevation of blood sugar level. However, Non-Patent Document 3 does not describe whether or not fermented milk produced by microorganisms other than the FC strain has an inhibitory effect on blood sugar level elevation.

Japanese Patent Application Laid-Open No. 2003-252770 Japanese Patent Application Laid-Open No. 2005-40123 JP 2013-147457 A

Ni Y., and Fan D., Medicine (Baltimore). 2017 Dec;96(51):e8811. doi: 10.1097/MD.0000000000008811. Tominaga et al., Diabetes Care. (1999) 22: 920-924 Mori Hideki et al., Geriatrics (2012) p.141-152

An object of the present invention is to provide a food composition having an effect of suppressing an increase in blood sugar level and an effect of suppressing a decrease in bone density.

As a result of extensive studies to solve the above problems, the present inventors have found that fermented milk produced using a predetermined lactic acid bacterium and exopolysaccharide (EPS) derived from the lactic acid bacterium contained in the fermented milk at a high concentration ) has the effect of suppressing an increase in blood sugar level and is also effective in suppressing a decrease in bone density, leading to the completion of the present invention.

That is, the present invention includes the following.
[1] Blood sugar containing 30 mg/kg or more exopolysaccharide (EPS) using Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus Fermented milk for suppressing price increases.
[2] The fermented milk according to [1] above, which is for suppressing an increase in blood sugar level and a decrease in bone density.
[3] The fermented milk of [1] or [2] above, for subjects suffering from diabetes or at risk of developing it.
[4] The above [1], wherein the Lactobacillus delbrueckii subspecies bulgaricus is Lactobacillus delbrueckii subsp. bulgaricus OLL1247 strain (accession number NITE BP-01814) ] The fermented milk according to any one of [3].
[5] The fermented milk according to any one of [1] to [4] above, wherein the Streptococcus thermophilus is Streptococcus thermophilus strain OLS3078 (accession number NITE BP-01697).
[6] A food for suppressing elevation of blood sugar level, comprising the fermented milk according to any one of [1] to [5] above.
[7] A feed for suppressing elevation of blood sugar level, comprising the fermented milk according to any one of [1] to [5] above.
[8] The food according to [6] above, which is for suppressing an increase in blood sugar level and a decrease in bone density.
[9] The feed according to [7] above, which is for suppressing an increase in blood sugar level and a decrease in bone density.
[10] The food according to [6] or [8] above for subjects suffering from diabetes or at risk of developing diabetes.
[11] The feed of [7] or [9] above for subjects suffering from or at risk of developing diabetes.

According to the present invention, it is possible to provide fermented milk or the like that can be used as a blood sugar level rise inhibitor that is effective in suppressing a decrease in bone density in addition to suppressing blood sugar level elevation, and that can be ingested for a long period of time.

FIG. 1 is a diagram showing the effect of high-EPS fermented milk in suppressing the increase in blood sugar level. Filled circles represent water, filled triangles represent high EPS fermented milk, and open triangles represent IDex. The presence of a statistically significant difference (by Fisher's LSD method) between blood glucose levels at each time point is indicated by different letters of the alphabet. Blood glucose levels were shown as mean±standard error. FIG. 2 is a graph showing the effect of high-EPS fermented milk and low-EPS fermented milk on the blood sugar level elevation. FIG. 2A shows changes in blood glucose levels after sucrose loading, and FIG. 2B shows the area under the curve (AUC) of blood glucose levels after sucrose loading. In FIG. 2A, black triangles represent high-EPS fermented milk, and white circles represent low-EPS fermented milk. Blood glucose levels were shown as mean±standard error. * indicates significant difference p<0.05 by Student's T-test. FIG. 3 is a diagram showing the suppressive effect of high EPS fermented milk on the portal vein blood glucose level. A black triangle indicates high EPS fermented milk, and an open circle indicates low EPS fermented milk. Blood glucose levels were shown as mean±standard error. # indicates a significant difference p<0.1 by Student's T-test, * indicates a significant difference p<0.05 by Student's T-test. FIG. 4 shows the overnight fasting blood glucose level (fasting blood glucose level; at autopsy) after long-term intake of the test diet. The presence of statistically significant differences (by the Tukey-Kramer method) between blood glucose levels in each group are indicated by different letters of the alphabet. Blood glucose levels were shown as mean±standard error. FIG. 5 is a diagram showing the water intake of mice 7 weeks after the start of test feed intake. The presence of statistically significant differences (by the Tukey-Kramer method) between water intakes in each group are represented by different letters of the alphabet. Water intake was shown as mean±standard error. FIG. 6 is a diagram showing the correlation between fasting blood sugar level and blood ucOC concentration after long-term ingestion of the test feed. Only the measured values of the control group, the skim milk powder group, the low EPS fermented milk group and the high EPS fermented milk group were used. FIG. 7 is a diagram showing bone density in II diabetes model mice after long-term ingestion of the test feed. FIG. 7A shows the bone density of the lumbar spine 7 weeks after the start of test feed intake, and FIG. 7B shows the bone density of the femur excised at the time of dissection. The presence of a statistically significant difference (by the Tukey-Kramer method) between total bone densities in each group is represented by different letters of the alphabet. Total bone density was shown as mean±standard error.

The present invention will be described in detail below.
The present invention relates to extracellular polysaccharides obtained by fermentation of milk using Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus as starters; It is based on the knowledge that fermented milk containing EPS) preferably at a high concentration has an effect of suppressing an increase in blood sugar level. The present invention is also based on the finding that long-term ingestion of fermented milk containing such EPS, preferably at a high concentration, improves sugar metabolism and suppresses bone mineral density loss. The present invention provides a blood glucose-raising exopolysaccharide (EPS) containing exopolysaccharide (EPS) prepared using Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. A fermented milk for suppressing and/or suppressing a decrease in bone density is provided.

In the context of the present invention, the term "inhibition" encompasses inhibition and reduction, and refers to, for example, inhibiting an increase in blood glucose level or a decrease in bone density or reducing the increase/decrease thereof (the amount of increase/decrease). .

In the present invention, fermented milk (typically yogurt) refers to milk fermented using microorganisms such as lactic acid bacteria and yeast. Milk used for fermentation (raw material milk) may be any milk such as raw milk, pasteurized milk, concentrated milk, adjusted milk, low-fat milk, non-fat milk, skim milk powder, skim concentrated milk, or any combination thereof. . The milk may be from any mammal such as cows, goats, sheep, horses, camels, buffaloes, etc. It may be milk from cows (milk), for example. The milk may be fat-containing or fat-free. The milk may comprise at least skim milk powder, may be skim milk powder, or may be skim milk powder and raw milk (eg, cow's milk). Milk may be diluted or dissolved in water and used for fermentation. In the production of fermented milk according to the present invention, only water, milk, and the above microorganisms may be used as raw materials. Additional ingredients, including other dairy products (e.g., cream, whey, whey protein, soy milk, soy protein, etc.) may, but need not, be used in the production of the fermented milk according to the present invention. good too. The fermented milk according to the present invention may contain any food additives (for example, sweeteners, flavors, stabilizers, etc.). Alternatively, the fermented milk according to the present invention is produced without adding sugars such as monosaccharides, disaccharides, oligosaccharides (3 to 10 sugars) and polysaccharides other than those contained in milk or dairy products as raw materials. It can be anything.

In the production of the fermented milk according to the invention, the lactic acid bacteria Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus are used as starters for the fermentation.

Examples of Lactobacillus delbrueckii subspecies bulgaricus include, but are not limited to, strains useful for EPS production, such as Lactobacillus delbrueckii subspecies bulgaricus OLL1247 strain and OLL1073R -1 strain is mentioned.

Examples of Streptococcus thermophilus include, but are not limited to, strains useful for EPS production include Streptococcus thermophilus strain OLS3078 and strain OLS3618.

In the production of fermented milk according to the present invention, although not limited to the following, a combination of Lactobacillus delbrueckii subspecies bulgaricus OLL1247 strain and Streptococcus thermophilus OLS3078 strain, It is particularly preferred to use a combination of the Lactobacillus bulgaricus OLL1247 strain and the Streptococcus thermophilus OLS3618 strain, or a combination of the Lactobacillus delbrueckii subspecies OLL1073R-1 strain and the Streptococcus thermophilus OLS3078 strain.

Lactobacillus delbrueckii subsp. bulgaricus strain OLL1247 was registered on March 6, 2014 with the National Institute of Technology and Evaluation Patent Microorganisms Depository Center (NPMD) (Chiba Prefecture, Japan). It has been internationally deposited under the Budapest Treaty under the accession number NITE BP-01814 at Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City.

Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 strain was deposited on February 22, 1999 (original deposit date) at the National Institute of Technology and Evaluation Patent Organism Depository ( NITE-IPOD) (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba, Japan) under the accession number FERM BP-10741 under the Budapest Treaty. On November 29, 2006, this deposited strain was transferred from the domestic deposit (original deposit) to the international deposit under the Budapest Treaty.

Streptococcus thermophilus strain OLS3078 was deposited on August 23, 2013 (original deposit date) at the National Institute of Technology and Evaluation Patent Microorganism Depositary Center (NPMD) (2 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan). -5-8 Room 122) under the Budapest Treaty under the accession number NITE BP-01697.

Streptococcus thermophilus strain OLS3618 was deposited on March 6, 2014 (original deposit date) at National Institute of Technology and Evaluation Patent Microorganism Depositary Center (NPMD) (2 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan). -5-8 Room 122) under the Budapest Treaty under the accession number NITE BP-01815.

The fermented milk according to the present invention contains the lactic acid bacteria Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. In one embodiment, the fermented milk according to the present invention contains Lactobacillus delbrueckii subsp. :1 to 1:1.4, more preferably 1.1:1 to 1:1.3. In one embodiment, the fermented milk according to the invention comprises a total lactic acid bacteria count of 10-100 x ¹⁰⁷ cfu/ml, such as 20-70 x ¹⁰⁷ cfu/ml, although the invention is not limited to this range. .

The fermented milk according to the present invention may be treated before being used for suppressing an increase in blood sugar level or before being blended with other ingredients. The fermented milk according to the present invention may be dried, powdered, granulated, diluted, concentrated, etc., for example. In a preferred embodiment, the fermented milk according to the present invention can be dried by freeze drying, warm air drying, heat drying, or the like. The fermented milk according to the present invention may or may not be sterilized. Sterilization can be performed by a conventional method, for example, radiation sterilization (γ-ray sterilization, electron beam sterilization, ultraviolet sterilization, etc.), heat sterilization (dry heat sterilization, steam sterilization, autoclave sterilization, boiling sterilization, etc.), chemical sterilization. (ethylene oxide gas sterilization, propylene oxide gas sterilization, etc.), plasma sterilization, or the like. Although the lactic acid bacteria in the sterilized fermented milk are dead cells, the total number of lactic acid bacteria and the bacterial count ratio contained in the fermented milk according to the present invention are calculated based on the measured values of viable bacteria immediately before sterilization. good. The Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus in the fermented milk according to the present invention may be viable or dead when used.

The fermented milk according to the present invention contains polysaccharides (exopolysaccharides; EPS) produced during fermentation of milk by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus and released outside the cells. The fermented milk according to the present invention has an EPS of 30 mg/kg or more, preferably 30 mg/kg to 1 g/kg, more preferably 30 mg/kg to 500 mg/kg, even more preferably 30 mg/kg to 100 mg/kg, particularly preferably may comprise 30 mg/kg to 70 mg/kg, such as 35 mg/kg to 65 mg/kg.

The amount of EPS in fermented milk can be measured by a conventional method, but in the present invention, although not limited to the following, it may be measured according to the methods disclosed in JP-A-2018-38356, WO2014/084340, and the like. Specifically, trichloroacetic acid is added to a 10 g sample (high EPS fermented milk) to precipitate proteins, the supernatant is collected to remove protein, and then ethanol is added in an amount twice that of the supernatant. The amount of EPS in the present invention can be measured by leaving overnight under refrigeration, then obtaining a precipitate by centrifugation, adding purified water to the precipitate, and subjecting it to HPLC using a gel filtration column. As an HPLC analyzer, for example, Alliance 2695 Separations Module (Waters) can be used.

The fermented milk according to the present invention can be produced by adding Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus as starters to a fermented milk raw material mixture containing milk and culturing the mixture. Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus may be added to fermented milk raw material mixtures containing milk (e.g. yogurt bases), including, but not limited to, 0.1 to 10% by weight or 1 to 10% by weight. It may be added in an amount of 5% by weight. In a preferred embodiment, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus are added to the fermented milk raw material mixture (eg yogurt base) at an equal count ratio (eg 1.1:1 to 1:1.1). should be added. As an example of a method for producing fermented milk, fermented milk raw materials (including milk) other than the starter are mixed, the resulting mixture is heated to 90 to 98 ° C., for example, 95 ° C. and sterilized, then the starter (lactobacillus・ Add 0.1 to 10% by weight or 1 to 5% by weight, such as 3% by weight, of Delbruchy subsp. A method of fermenting for 5 hours, and after completion of fermentation, stirring and cooling under ice cooling for smoothing, thereby preparing an EPS-containing fermented milk.

The fermented milk according to the present invention and the extracellular polysaccharide (EPS) contained in it at high concentration have an effect of suppressing elevation of blood sugar level. Therefore, the fermented milk according to the present invention can be used for suppressing an increase in blood sugar level of subjects.

A blood glucose level is an indicator of a subject's ability to metabolize glucose. In sugar metabolism, first, orally ingested sugar is decomposed into glucose by enzymes in the digestive tract and then absorbed into the body. The absorbed glucose is transported through the bloodstream and taken into muscle cells, fat cells, etc. by the action of insulin. Absorbed glucose is also taken up by the liver and converted to glycogen for storage. The liver plays an important role in maintaining glucose metabolism homeostasis, producing glucose from glycogen during fasting and reducing glucose production during feeding, thereby predominately increasing glucose uptake and thus controlling blood glucose levels. is kept stable. Insulin secretion from pancreatic β-cells increases instantaneously when blood glucose levels begin to rise due to ingestion. Insulin promotes synthesis of glycogen from glucose in the liver, suppresses gluconeogenesis, and quickly lowers postprandial blood glucose levels. When glucose tolerance (sugar metabolism regulation function) declines due to aging, bad lifestyle habits, disease, etc., postprandial blood glucose levels tend to rise rapidly and become difficult to drop (postprandial hyperglycemia). It damages blood vessels and other tissues in the body, impairs the function of pancreatic β-cells, and increases the risk of developing cardiovascular disease. As glucose tolerance progresses further, insulin secretion (basal secretion) during periods other than after meals also decreases, resulting in insufficient glucose uptake into muscles and suppression of gluconeogenesis in the liver, resulting in fasting blood sugar levels and Occasional blood sugar levels also rise, increasing tissue damage, leading to the onset and exacerbation of diabetes and its complications.

In the present invention, blood glucose level can be measured by a conventional method. The blood whose blood glucose level is measured may be, for example, venous blood, arterial blood or capillary blood. The blood glucose level may be a plasma blood glucose level, a serum blood glucose level, for example, a venous plasma blood glucose level. However, when evaluating changes in blood sugar levels, it is necessary to compare blood derived from the same blood sampling site and blood sugar levels derived from the same blood treatment method. Measurement of blood glucose level is not limited to the following, but adopts the FAD-GDH enzymatic electrode method, GOD enzymatic electrode method, GOD enzymatic colorimetric method, GOD-POD colorimetric method, and coulometry method for the collected blood sample. Enzyme electrode method, quinoprotein glucose dehydrogenase enzymatic colorimetric method, quinoprotein glucose dehydrogenase enzymatic electrode method, etc. can be used. Such blood glucose measurement can be performed using a commercially available blood glucose meter. Alternatively, the blood glucose level may be measured by a blood glucose level measurement method that does not rely on blood sampling, such as a sensor fixed on the body surface or inside the body, or a laser (for example, the mid-infrared laser method by Yamakawa et al.). Blood glucose meters have also been developed to perform various blood glucose measurement methods.

In a preferred embodiment of the present invention, suppression of blood sugar level elevation includes suppression of postprandial blood sugar level elevation. The fermented milk according to the present invention and the EPS contained therein can suppress (typically alleviate) the postprandial increase in blood sugar level. The effect of suppressing postprandial blood glucose elevation is evaluated based on the area under the blood concentration-time curve (AUC) of blood glucose level after the start of oral carbohydrate intake (after glucose load). can do. Specifically, after measuring the fasting blood sugar level of the subject, the fermented milk or EPS according to the present invention is orally ingested by the subject at the same time as carbohydrate (typically sucrose), and the blood sugar level is measured over time. Then, the total increase in blood glucose level at each time point from the fasting blood glucose level is calculated as the area under the blood concentration-time curve (average value). The area under the blood concentration-time curve of blood glucose level is preferably calculated based on the measured values from the start of oral intake to 120 minutes later. As a negative control, the test was performed in the same procedure except that water instead of the fermented milk or EPS according to the present invention was orally ingested at the same time as carbohydrate (typically sucrose). - Calculate the area under the time curve. The oral intake (oral dose) of the fermented milk or EPS according to the present invention in this evaluation test may be an amount corresponding to 200 to 300 μg EPS/kg body weight. Subjects who orally ingested the fermented milk or EPS of the present invention compared with the area under the blood concentration-time curve of blood glucose levels in negative control subjects (who did not orally ingest the fermented milk or EPS of the present invention) If the area under the blood concentration-time curve of blood glucose level in 2000 is statistically significantly decreased, it can be determined that the fermented milk or EPS according to the present invention has an effect of suppressing the postprandial increase in blood glucose level. Carbohydrate (typically sucrose) loadings for subjects in these assays can range from 300 mg to 3 g/kg body weight, typically 40 to 80 g for humans. It is considered that the effect of suppressing the postprandial increase in blood sugar level that the fermented milk and EPS according to the present invention have is due to the suppression of sugar absorption.

In the present invention, suppression of blood glucose level elevation may include suppression of fasting blood glucose level elevation. Fasting blood glucose level refers to the blood glucose level measured after fasting for 10 hours or more in humans. In a preferred embodiment, ingestion (preferably long-term ingestion) of the fermented milk or EPS according to the present invention suppresses an increase in fasting blood glucose level compared to the case of having an equivalent dietary habit except for not ingesting it. can do. Long-term intake of fermented milk or EPS according to the present invention is not limited to the following, but is preferably 4 weeks or longer, more preferably 6 weeks or longer, and still more preferably 8 weeks or longer, for example, 4 weeks to 1 year or more. It can be done over the above.

High blood sugar conditions, such as those found in diabetes, lead to dehydration, thirst, and increased water intake. In a preferred embodiment, the intake (preferably long-term intake) of the fermented milk according to the present invention and the EPS contained therein is compared with a subject having a similar dietary habit except for not ingesting it in a subject with hyperglycemia. can reduce dehydration and reduce fluid intake.

The effect of suppressing the increase in fasting blood sugar level and the effect of reducing dehydration symptoms as described above are that the fermented milk and EPS according to the present invention improve the sugar metabolism of the subject or suppress the deterioration of the sugar metabolism (prevention or delay).

Furthermore, the fermented milk according to the present invention and the EPS contained therein can suppress a decrease in bone density. In a preferred embodiment, long-term intake of the fermented milk according to the present invention or the EPS contained therein suppresses a decrease in bone density as compared with the case of having an equivalent dietary habit except for not taking the fermented milk for a long period of time. can. The period of long-term intake of the fermented milk or EPS according to the present invention is the same as above.

The fermented milk according to the present invention and the EPS contained therein are suitable for oral intake by subjects suffering from diabetes or at risk of developing diabetes. Alternatively, the fermented milk according to the present invention and the EPS contained therein are suitable for oral intake by subjects suffering from osteoporosis or at risk of developing osteoporosis. In general, subjects with diabetes or at risk of developing diabetes are at risk of developing osteoporosis.

Diabetes can be any type of diabetes, such as type 1 diabetes, type 2 diabetes, gestational diabetes, but preferably type 2 diabetes. Currently, in the diagnosis of type 2 diabetes in humans, the fasting blood sugar level is 126 mg/dl or higher, the blood sugar level is 200 mg/dl or higher after 2 hours of 75 g oral glucose load, the casual blood sugar level is 200 mg/dl or higher, and the HbAlc (hemoglobin A1c) level is Of the 6.5% or more, at least one human subject is determined to be "diabetic type" with high suspicion of diabetes. Also, human subjects with both fasting blood glucose levels of less than 110 mg/dl and blood glucose levels of less than 140 mg/dl after a 2 hour 75 g oral glucose load have been determined to be "normal." Human subjects who do not belong to either the "diabetic type" or the "normal type" are judged to be "borderline type" and fall under the prediabetic group (impaired glucose tolerance). Random blood glucose levels refer to blood glucose levels measured without regard to meal times. The 2-hour postprandial blood glucose level after 75 g of oral glucose load is the blood glucose level (postprandial blood glucose level) measured 2 hours after oral intake of 75 g of glucose. A subject suffering from diabetes can be a human who has been determined to be "diabetic" according to the diagnostic criteria for type 2 diabetes, but is not limited to this as long as they are diagnosed with diabetes. A subject suffering from diabetes may be a non-human mammal in a state of hyperglycemia corresponding to a "diabetic form." On the other hand, a subject at risk of developing diabetes can be a human defined as "borderline" according to the diagnostic criteria for type 2 diabetes, or a corresponding non-human mammal in a relatively hyperglycemic state. Alternatively, a subject at risk of developing diabetes may be a subject with obesity, depression, hypertension, or the like. Subjects at risk of developing diabetes are considered to be more likely to develop diabetes due to poor lifestyle habits (overeating, lack of exercise, smoking, mental stress, lack of sleep, etc.) or genetic predisposition (family history of diabetes, etc.). It may be a subject to be

Subjects suffering from osteoporosis are subjects diagnosed with osteoporosis based on bone mineral density (e.g., young adult average YAM of 70% or less), measured values of bone metabolism markers, etc., and a history of fragility fractures. Obtained, but not limited to, as long as osteoporosis is diagnosed. A subject at risk of developing osteoporosis may be a subject having a disease likely to cause osteoporosis, such as diabetes, hypertension, dyslipidemia, chronic kidney disease, hyperthyroidism, chronic obstructive pulmonary disease. Alternatively, a subject at risk of developing osteoporosis may be a subject using a drug having a bone mass-lowering effect such as a steroid drug or methotrexate, or may be a perimenopausal or postmenopausal subject.

In the present invention, subjects include primates such as humans, monkeys and chimpanzees; rodents such as mice, rats and guinea pigs; domestic animals such as horses, cattle, sheep, goats, pigs, camels and donkeys; dogs, cats and rabbits. It may be any mammal, such as a companion animal such as.

The present invention also provides a method for suppressing elevation of blood sugar level, which comprises administering the fermented milk of the present invention to a subject. According to the method of the present invention, the risk of elevation of blood sugar level can be reduced. Moreover, in the method of the present invention, postprandial elevation of blood sugar level can be suppressed. In the method of the present invention, an increase in fasting blood glucose level can also be suppressed. The method of the present invention can also improve sugar metabolism or suppress a decline in sugar metabolism.

The present invention also provides a method for suppressing a decrease in bone density, comprising administering the fermented milk according to the present invention to a subject. According to the method of the present invention, the risk of lowering bone density can be reduced. In a preferred embodiment, there is also provided a method for suppressing an increase in blood sugar level and a decrease in bone density, comprising administering the fermented milk according to the present invention to a subject.

The present invention also provides a method for treating or preventing diabetes, which comprises administering the fermented milk of the present invention to a subject. The present invention also provides a method for treating or preventing osteoporosis, which comprises administering the fermented milk of the present invention to a subject. The present invention also provides a method for treating or preventing diabetes and osteoporosis, which comprises administering the fermented milk according to the present invention to a subject. In the present invention, "treatment" of diabetes refers to control of blood sugar level so that blood sugar level is maintained in a normal or near-normal range. "Prevention" of diabetes refers to preventing the onset of diabetes, more specifically, preventing abnormal hyperglycemia so as not to meet the diagnostic criteria for "diabetic type" of human type 2 diabetes or a corresponding hyperglycemic state. It refers to preventing or reducing blood sugar level elevation and its exacerbation.

The fermented milk according to the present invention can also be used as an active ingredient of a composition for suppressing an increase in blood sugar level and/or a decrease in bone density, and can also be used as an effective ingredient of a composition for treating or preventing diabetes and/or osteoporosis. You may use it as an ingredient. These compositions may contain pharmaceutically acceptable additives such as carriers, binders, excipients, lubricants, disintegrants, wetting agents, stabilizers, buffers, flavoring agents, preservatives, coloring agents. agents and the like.

Administration of the fermented milk according to the present invention to a subject is not limited, but is preferably administration to the gastrointestinal tract, such as oral administration, nasal administration, or enteral administration.

The dosage (ingestion amount) of the fermented milk according to the present invention is not particularly limited, but the daily dosage is equivalent to EPS 30 μg/kg body weight or more, for example, EPS 50 μg to 1 mg/kg body weight. good.

The present invention also provides a food for suppressing an increase in blood sugar level, which contains the fermented milk according to the present invention. The present invention also provides a feed containing the fermented milk of the present invention for suppressing elevation of blood sugar level. These foods and feeds may be used for suppressing elevation of blood sugar level and suppression of decrease in bone density. The food and feed according to the invention may be for subjects suffering from or at risk of developing diabetes. Their uses and targets are as described above.

In the present invention, "food" refers to food for humans, and may be beverages or other foods. Beverages include, but are not limited to, lactic acid beverages, milk beverages (coffee milk, fruit milk, etc.), tea beverages (green tea, black tea, oolong tea, etc.), fruit and vegetable beverages (oranges, apples, grapes, etc.). (fruit juices such as tomato, carrots, etc.), alcoholic beverages (beer, low-malt beer, wine, etc.), carbonated beverages, soft drinks, and water-based beverages. Foods other than beverages include, but are not limited to, processed foods such as confectionery, instant foods, and seasonings. Existing reference books can be referred to for manufacturing methods of various foods.

The food in the present invention may be a functional food. In the present invention, "functional food" means a food that has a certain level of functionality for living organisms, and includes, for example, food for specified health use (including conditional FOSHU [food for specified health use]) and food with nutrient function claims in Japan. Foods with health claims, foods with functional claims, foods for special dietary uses, dietary supplements, health supplements, supplements (for example, various dosage forms such as tablets, coated tablets, dragees, capsules and liquids) and beauty foods (for example diet food) and other so-called health foods in general. The functional food of the present invention also includes health foods to which health claims based on food standards of Codex (FAO/WHO Joint Food Standards Committee) are applied.

The functional food of the present invention may be food for special uses such as food for sick people, powdered milk for pregnant and nursing women, powdered milk for infants, food for the elderly, and food for nursing care.

The functional food according to the present invention may be solid preparations such as tablets, granules, powders, pills, capsules, etc., liquid preparations such as liquid preparations, suspensions, syrups, gels, pastes, etc. Alternatively, it may be in the form of a normal food (for example, beverage, yogurt, confectionery, etc.).

In the present invention, "feed" refers to food given to non-human mammals. The feed according to the invention may be for the subjects mentioned above.

The food or feed according to the present invention may contain any food component or feed component in addition to the fermented milk according to the present invention. The food and feed according to the present invention can be produced by a conventional method, and the fermented milk according to the present invention can be blended with other food ingredients or feed ingredients in any production process. The fermented milk according to the invention may be treated as described above before being incorporated into other food or feed ingredients.

EXAMPLES The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1] Preparation of high-EPS fermented milk High-EPS fermented milk having an increased EPS content compared to common fermented milk was prepared as follows. Lactobacillus delbrueckii subsp. bulgaricus OLL1247 strain (accession number NITE BP-01814) and Streptococcus thermophilus OLS3078 strain (accession number NITE BP-01697) were used as starters. board. A yogurt base prepared by mixing ingredients other than the starter at the compounding ratio shown in Table 1 was heated to 95°C and sterilized, and then 3% by weight of the starter (including OLL1247 strain and OLS3078 strain with the same number of bacteria) was added. , and fermented at 43°C for 3-5 hours. After completion of fermentation, the yoghurt was smoothed by stirring and cooling under ice cooling, thereby preparing skim milk powder-based high-EPS fermented milk and low-EPS fermented milk.

Table 2 shows the properties of the high-EPS fermented milk thus obtained.

The EPS (extracellular polysaccharide) content of the obtained high-EPS fermented milk was measured according to the following procedure. Deproteinization was performed by adding trichloroacetic acid to a 10 g sample (high EPS fermented milk) to precipitate proteins, and collecting the supernatant. Then, ethanol was added in an amount twice the volume of the supernatant, left overnight under refrigeration, and then centrifuged to obtain a precipitate. Purified water was added to the precipitate, and the precipitate was subjected to HPLC using a gel filtration column to determine the amount of EPS. Alliance 2695 Separations Module (Waters) was used as the HPLC analyzer.

[Example 2] Inhibitory effect of high-EPS fermented milk on increase in blood sugar level Using the high-EPS fermented milk prepared in Example 1, the effect of the high-EPS fermented milk on the change in blood sugar level was examined by a sugar tolerance test as follows.

C57BL/6 mice (male, 9 weeks old; normal mice) were fasted overnight, their body weight and blood glucose level were measured, and the mice were divided into the following 3 groups so that their body weights and blood glucose levels were uniform. .
- Water group (negative control) (n=8)
- High EPS fermented milk group (n=6)
- Indigestible dextrin (IDex) group (positive control) (n=6)

Grouped mice were given water (water group), high EPS fermented milk (high EPS fermented milk group), or indigestible dextrin (IDex; Matsutani Chemical Industry Co., Ltd.) solution (IDex group) in an amount of 5 mL/kg body weight. was orally administered simultaneously with 5 mL/kg body weight of sucrose solution (2 g of sucrose/kg body weight). Said dose of high EPS fermented milk contains 276 μg/kg body weight of EPS. Said dosage of resistant dextrin solution contains 600 mg/kg body weight of resistant dextrin. Before administration and 30, 60, 90, 120 and 180 minutes after administration, tail vein blood (whole body blood) was collected from the mice by puncture, and blood glucose level (blood glucose concentration) was measured. Blood glucose levels were measured using a Breeze 2 sensor (Bayer Yakuhin).

The results are shown in FIG. Blood glucose levels were highest 30 minutes after sucrose load in all of the water group, high EPS fermented milk group, and IDex group. However, the blood glucose levels of the high-EPS fermented milk group and the IDex group after 30 minutes of sucrose loading were statistically significantly lower than those of the water group, and the peak blood glucose level was lowered (Fig. 1). From this, it was shown that the high-EPS fermented milk has the effect of suppressing elevation of blood sugar, similar to indigestible dextrin, which is known to have the effect of suppressing elevation of blood sugar.

In addition, the high EPS fermented milk group to which 276 μg/kg of EPS was administered and the IDex group to which 600 mg/kg of indigestible dextrin was administered showed the same level of blood glucose elevation inhibitory effect (Fig. 1). From this, it was shown that the high-EPS fermented milk and the polysaccharide EPS contained therein have a blood glucose elevation suppressing effect at a much lower dose than the indigestible dextrin.

[Example 3] Evaluation of effect of suppressing increase in blood glucose level of fermented milk with different EPS content For comparison with high EPS fermented milk, low EPS fermented milk was prepared in the same procedure as in Example 1 except for the starter, and shown in Table 1. Prepared according to the compounding ratio. Lactobacillus delbrueckii subsp. bulgaricus; L. bulgaricus OLL1150 strain and Streptococcus thermophilus (S. thermophilus) OLS3078 strain (accession number NITE BP -01697) was used.
The high EPS fermented milk prepared in Example 1 was used.
Table 3 shows a comparison of the properties of low-EPS fermented milk and high-EPS fermented milk.

C57BL/6 mice (male, 10 weeks old) were fasted overnight, then weighed and measured for blood sugar level, and divided into the following two groups so that the body weight and blood sugar levels were equal.
- Low EPS fermented milk group (n=15)
- High EPS fermented milk group (n=15)

Grouped mice were given 5 mL/kg body weight of low EPS fermented milk (low EPS fermented milk group) or high EPS fermented milk (high EPS fermented milk group), and 5 mL/kg body weight of sucrose solution (sucrose 2 g/kg body weight). Said dose of low EPS fermented milk contains 19 μg/kg body weight of EPS and said dose of high EPS fermented milk contains 276 μg/kg of EPS. Before administration and 30, 60, 90, and 120 minutes after administration, tail vein blood (systemic blood) was collected from the mice, and blood glucose levels were measured. Blood collection and blood glucose level measurement were performed in the same manner as in Example 2.

The results are shown in FIG. The high-EPS fermented milk group had statistically significantly lower blood glucose levels 30 minutes and 90 minutes after sucrose loading compared to the low-EPS fermented milk group (Fig. 2A). In addition, the AUC (Area Under the blood concentration-time curve) of blood glucose levels in the high-EPS fermented milk group was also significantly lower than that in the low-EPS fermented milk group (Fig. 2B). . From these results, it was shown that the inhibitory action of the high-EPS fermented milk on blood sugar level elevation was due to EPS.

[Example 4] Inhibitory effect of high-EPS fermented milk on portal vein blood sugar level during sucrose loading In this example, high-EPS fermented milk was administered together with sucrose, Concentration) was measured and compared with the control to examine the effect of high EPS fermented milk on sucrose absorption. It is known that the portal vein blood glucose level directly reflects the amount of glucose absorbed from the intestine.

In order to obtain portal vein blood without affecting the blood glucose level by treatment such as anesthesia, mice with a catheter indwelled in the portal vein (C57BL/6 mouse with indwelling portal vein catheter, male, 11 weeks old) were prepared.

Two days after the portal vein catheterization surgery, the mice were fasted overnight and grouped into the following two groups.
- Low EPS fermented milk group
- High EPS fermented milk group

Grouped mice were given 5 mL/kg body weight of low EPS fermented milk (low EPS fermented milk group) or high EPS fermented milk (high EPS fermented milk group) and 5 mL/kg body weight of sucrose solution (2 g of sucrose). /kg body weight). The low-EPS fermented milk and the high-EPS fermented milk are the same as those used in Example 3. Said dose of low EPS fermented milk contains 19 μg/kg body weight of EPS and said dose of high EPS fermented milk contains 276 μg/kg of EPS. Before administration of the low-EPS fermented milk or high-EPS fermented milk and 15, 30, 60, and 90 minutes after the administration, portal vein blood was collected from the mice through the portal vein catheter, and the portal vein blood glucose level was measured. The measurement of the portal vein blood glucose level was performed in the same manner as the blood glucose level measurement method of Example 2.

The results are shown in FIG. Compared to the low-EPS fermented milk administration group, the high-EPS fermented-milk administration group had a lower portal vein blood glucose level (portal vein blood glucose concentration) 15 minutes after sucrose loading, and Pulse blood glucose levels were also low (Fig. 3).

It was also considered possible that the decrease in systemic blood glucose level observed in Examples 2 and 3 was due to the influence of hormones such as insulin rather than the high EPS fermented milk. However, in this example, the administration of high-EPS fermented milk showed a decrease in portal blood glucose level, indicating that high-EPS fermented milk has an inhibitory effect on blood sugar elevation, and that high-EPS fermented milk has an inhibitory effect on blood glucose elevation. was due to suppression of sucrose absorption.

[Example 5] Effect of long-term intake of high-EPS fermented milk in type II diabetes model mice
(i) Preparation of high-EPS fermented milk and low-EPS fermented milk based on skim milk powder For the preparation of high-EPS fermented milk based on skim milk powder, Lactobacillus delbrueckii subsp. bulgaricus) strain OLL1247 (accession number NITE BP-01814) and Streptococcus thermophilus strain OLS3078 (accession number NITE BP-01697).

Lactobacillus delbrueckii subsp. bulgaricus strain OLL1150 and Streptococcus thermophilus strain OLS3078 (accession number NITE BP-01697) was used.

A yoghurt base prepared by mixing raw materials other than the starter at the compounding ratio shown in Table 4 was heated to 95°C and sterilized. , and fermented at 43° C. for 3-5 hours. After completion of fermentation, the yoghurt was smoothed by stirring and cooling under ice cooling, thereby preparing skim milk powder-based high-EPS fermented milk and low-EPS fermented milk.

Table 5 shows the characteristics of the obtained high-EPS fermented milk and low-EPS fermented milk based on powdered skim milk.

Both the low-EPS fermented milk and the high-EPS fermented milk based on skimmed milk powder were freeze-dried, subjected to electron beam sterilization at 15 kGy, and added to feed in the form of freeze-dried powder in the amounts shown in Table 6 below.

(ii) Long-term ingestion test using type II diabetes model mice
Type II diabetes model mice, db/db mice (male, 5 weeks old, n = 40) and healthy +m/+m mice (n = 8) were obtained from Clea Japan, Inc., and fed with standard purified feed AIN-93M. (CLEA Japan; Table 6) for 1 week to acclimatize the mice, and then used for the evaluation test.

After acclimation, the mice were fasted for 6 hours, blood was collected from the tail vein, and fasting blood glucose levels were measured. Mice were grouped into the following groups so that fasting blood glucose levels and body weights were equal. The type of mice in each group and the food to be fed are given in parentheses.

- Normal group (+m/+m mice, AIN-93M diet)
- Control group (db/db mice, high starch diet)
- Skim milk powder group (db/db mice, skim milk diet)
- Low EPS fermented milk group (db/db mice, low EPS fermented milk diet)
- High EPS fermented milk group (db/db mouse, high EPS fermented milk diet)
- Acarbose group (db/db, acarbose diet; positive control)
Mice in each group were fed test diets prepared according to Table 6 ad libitum for 8 weeks.

Regarding the component composition in Table 6, the amounts of components other than acarbose (calculated value) are measured values. Acarbose (trade name: Glucobay ^(R) ) is an α-glucosidase inhibitor and is used as a therapeutic drug for diabetes to suppress postprandial hyperglycemia. A high-starch diet is a high-calorie diet.

At the time of necropsy (after about 14 hours of overnight fasting from the night of the 57th day after the start of intake of the test feed), tail vein blood (whole body blood) was collected by puncture, and the blood sugar level was measured. Blood glucose levels were measured using Niprostat Strip XP3 (Nipro).

FIG. 4 shows the blood glucose level after overnight fasting (fasting blood glucose level) in each group. Fasting blood glucose levels indicate the basal capacity of glucose metabolism.

Also, as shown in FIG. 4, the powdered skim milk group showed higher post-overnight fasting blood glucose levels compared to the control group. In addition, the low-EPS fermented milk group and the high-EPS fermented milk group showed statistically significantly lower blood glucose levels after overnight fasting than the skim milk powder group.

In addition, the water intake of the mice was measured 7 weeks after the initiation of intake of the test diet. The amount of water intake was calculated from the difference in the weight of the water bottle before watering and two days after watering.

The results are shown in FIG. Water intake, which is known to increase with an increase in blood sugar level, was significantly lower in the high-EPS fermented milk group than in the skim milk powder group.

From the above results, it was shown that sugar metabolism was improved in the high-EPS fermented milk group compared to the skim milk powder group.

Furthermore, the blood collected at the time of dissection was measured for blood undercarboxylated osteocalcin (ucOC) concentration using a Mouse Glu-Osteocalcin High Sensitive EIA Kit (Takara Bio Inc.). As shown in Fig. 6, the blood ucOC concentration showed a statistically significant negative correlation with blood glucose level after overnight fasting (fasting blood glucose level) at autopsy, and the lower the blood glucose level, the higher the value tended to be. was in Recently, it has been reported that osteocalcin released from bone (particularly undercarboxylated osteocalcin) plays a role in improving glucose metabolism (Lee et al., 2007, Cell, 130: 456-469). Therefore, it was suggested that ucOC may also be involved in the glucose metabolism improving effect of high-EPS fermented milk.

Diabetic patients are known to be prone to osteoporosis. Therefore, in order to investigate the effects of high-EPS fermented milk on the bones of diabetic model mice, X-ray CT images of the 2nd to 4th lumbar vertebrae of mice and the femurs at the time of dissection were taken 7 weeks after the start of intake of the test diet. Total bone density (BMD) was measured.

The results are shown in FIG. Compared to the healthy group, the control group of type II diabetes model mice showed a marked decrease in bone density (FIGS. 7A and B). Even in the acarbose group, in which acarbose reduces fasting blood glucose levels, the lumbar spine BMD and femoral bone density at the time of autopsy 7 weeks after the start of intake of the test diet were equal to or lower than those of the control group. (FIGS. 7A and B). In other words, it was shown that an α-glucosidase inhibitor (acarbose) can lower blood sugar levels but cannot prevent the onset of osteoporosis associated with diabetes. On the other hand, the high EPS fermented milk group showed high lumbar bone density and femoral bone density (Figs. 7A and B). Therefore, high-EPS fermented milk was shown to be useful not only for suppressing elevation of blood sugar levels in diabetic patients, but also for suppressing bone mineral density loss and preventing osteoporosis. Since the administration of α-glucosidase inhibitors, which lower blood sugar levels, did not suppress the loss of bone density, it is considered that the osteoporosis preventive effect of high-EPS fermented milk is not induced solely by the suppression of blood sugar level elevation.

The fermented milk according to the present invention can be used for suppressing elevation of blood sugar level and/or suppression of decrease in bone density. The fermented milk according to the present invention is also very useful in treating and preventing diabetes and osteoporosis.

Claims (12)

Using Lactobacillus delbrueckii subsp. bulgaricus OLL1247 strain (accession number NITE BP-01814) and Streptococcus thermophilus OLS3078 strain (accession number NITE BP-01697) , A fermented milk containing 30 mg/kg or more of extracellular polysaccharide (EPS) for suppressing elevation of blood sugar level. The fermented milk according to claim 1, which is for suppressing elevation of blood sugar level and suppression of decrease in bone density. 3. Fermented milk according to claim 1 or 2 for subjects suffering from or at risk of developing diabetes. 3. Fermented milk according to claim 2 for subjects with diseases predisposing to osteoporosis. A food for suppressing an increase in blood sugar level, comprising the fermented milk according to any one of claims 1 to 4 . A feed for suppressing elevation of blood sugar level, comprising the fermented milk according to any one of claims 1 to 4 . 6. The food according to claim 5 , which is for suppressing an increase in blood sugar level and a decrease in bone density. The feed according to claim 6 , which is for suppressing blood sugar level elevation and bone density decrease suppression. 8. Food according to claim 5 or 7 for subjects suffering from or at risk of developing diabetes. 9. A feed according to claim 6 or 8 for subjects suffering from or at risk of developing diabetes. 8. Food according to claim 7, for subjects with diseases predisposing to osteoporosis. 9. A feed according to claim 8 for subjects with diseases predisposing to osteoporosis.

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