WO2023281985A1 - Composition for increasing muscle mass - Google Patents

Abstract

[Problem] Provided is a composition which can be easily ingested and which can increase muscle mass. [Solution] This composition for increasing muscle mass contains (A) guar gum degradation products with an average molecular weight of 1.8 × 10³ to 2.0 × 10⁵ and with at least 70 mass% thereof being within this range, and (B) a guar bean protein, the viscosity of a 1 mass% water solution of the composition, measured at 25°C and 60 rpm using a B-type viscometer, is less than or equal to 50 mPa*s, the guar gum degradation products are contained in endosperm derived from guar, and the composition is obtained by using microbe-derived β-mannanase to hydrolyze and reduce the molecular weight of galactomannan polysaccharide that has a galactose and mannose content ratio (galactose:mannose) in the range 1:1.5-1:2.1, wherein the dietary fiber content, specified by enzymatic-HPLC method, is at least 70 mass%, the content of oligosaccharides of the guar gum degradation products is less than or equal to 15 mass%; of the amino acid compositions in the contained proteins, (glutamic acid + glutamine + aspartic acid + asparagine) ≥ 100 g/100g, (cystine + tyrosine + serine + threonine) ≥ 40mg / 100g, all amino acids ≥ 150mg/100g, and the ratio (A)/(B) is less than or equal 1000.

Description

Composition for increasing muscle mass

The present invention relates to a composition for increasing muscle mass.

Peroxisome proliferator activated receptor γ coactivator-1 (hereinafter referred to as “PGC-1”) is a molecule identified as a transcriptional coactivator that binds to the transcription factor PPARγ. It is known to be involved in enhancement of mitochondrial biosynthesis and maintenance of functions, and to regulate gene expression related to energy production. Since PGC-1 is thought to play a useful role in the transformation of skeletal muscle by exercise training, research and development of substances capable of promoting biosynthesis in the body are being conducted (Patent Document 1). ).
On the other hand, valine, leucine, and isoleucine are known as branched-chain amino acids having branched chains in their side chains. Branched-chain amino acids are essential amino acids that cannot be produced in the body, are endurance amino acids, and are known to act as energy in muscles during exercise (Patent Document 2).
If it is possible to effectively increase PGC-1 and branched chain amino acids, it will effectively act on muscle metabolism, muscle mass increasing action, muscle mass decrease suppressing action, anti-sarcopenic action, anti-frailty action, and anti-metabolic effect based on metabolism improvement. Since it has a syndrome effect, an anti-fatigue effect, and an anti-aging effect, it can be useful not only for athletes, but also for the elderly, the sick, domestic animals and pets.

Republished publication 2018-207741 Japanese Patent Publication No. 2020-528402

However, Patent Document 1 uses a special strain, Enterococcus faecium R30 strain, and lacks versatility. In Patent Document 2, a plurality of substances containing branched-chain amino acids are to be ingested, and a large amount of substances must be ingested, imposing a burden on the ingester.
The present invention has been made to solve the above problems, and an object of the present invention is to provide a composition or the like that can be easily ingested and that can increase muscle mass.

As a result of intensive studies to solve the above-mentioned problems, it was found that a composition containing a specific galactomannan hydrolyzate and guar bean protein has an effect of increasing muscle mass, and the present invention was basically completed. rice field.
Thus, the composition for increasing muscle mass according to the present invention comprises (A) a guar gum decomposition product having an average molecular weight of 1.8×10 ³ to 2.0×10 ⁵ and containing 70% by mass or more of those within the average molecular weight range, and (B) guar bean protein, and the viscosity of a 1% by mass aqueous solution is 50 mPa s or less when measured using a Brookfield viscometer at 25 ° C. and 60 rpm, wherein the guar gum decomposition product is derived from guar Galactomannan polysaccharide contained in the endosperm and having a content ratio of galactose and mannose (galactose:mannose) in the range of 1:1.5 to 1:2.1 is hydrolyzed using microbial-derived β-mannanase to reduce the molecular weight. It has a dietary fiber content of at least 70% by mass and a content of oligosaccharides of guar gum degradation product of 15% by mass or less as defined by the enzyme-HPLC method, and among the amino acids in the protein containing ( glutamic acid + glutamine + aspartic acid + asparagine) ≥ 100 mg/100 g, (cystine + tyrosine + serine + threonine) ≥ 40 mg/100 g, total amino acids ≥ 150 mg/100 g, and (A)/(B) above is 1000 or less.
In the composition, the ratio of (A)/(B) is 25-1000, preferably 100-600, more preferably 200-500. This range provides a sufficient muscle mass-enhancing effect. At this time, the composition preferably has an effect of increasing the expression level of PGC-1 and promoting the production of branched-chain amino acids.

A food or drink according to another invention is characterized by containing the muscle mass-increasing composition. Furthermore, it should have PGC-1 expression level increasing action, branched chain amino acid production promoting action, muscle mass decrease suppressing action, anti-sarcopenia action, anti-frailty action, anti-metabolic syndrome action, anti-fatigue action and anti-aging action. is preferred.
Further, a pharmaceutical product according to another invention is characterized by containing the muscle mass-increasing composition. Furthermore, it should have PGC-1 expression level increasing action, branched chain amino acid production promoting action, muscle mass decrease suppressing action, anti-sarcopenia action, anti-frailty action, anti-metabolic syndrome action, anti-fatigue action and anti-aging action. is preferred.
The food and drink in the present invention includes food and beverages, such as nutritional supplements, health foods, foods for specified health uses, foods with function claims, foods for diet therapy, general health foods, supplements, tea beverages, and coffee. Beverages, juices, soft drinks, drinks, cooked rice, bread, noodles, dairy products, processed eggs, processed marine and livestock foods, confectionery, oils and fats and processed foods, seasonings, side dishes and the like. Pharmaceuticals include pharmaceuticals or quasi-drugs, and are preferably oral formulations or enteral formulations, and can be in the form of liquids, tablets, granules, pills, syrups, and the like.

The feed in the present invention refers to food to be fed to organisms other than humans, and its form is not particularly limited. The organisms to which the feed can be applied are not particularly limited, but examples include farmed animals and pet animals. Farmed animals include livestock such as horses, cattle, pigs, sheep, goats, camels, and llamas, experimental animals such as mice, rats, guinea pigs, and rabbits, and poultry such as chickens, ducks, turkeys, and ostriches. be. Pet animals include, for example, dogs and cats.

According to the present invention, it is possible to provide a composition or the like that can be easily ingested and that can promote an increase in muscle mass.

Fig. 10 is a graph showing the measurement results of muscle weights in a high-fat diet group and a PHGG group in a PHGG intake test using lifestyle-related disease model mice. 2 is a graph showing LDL-C measurement results in a high-fat diet group and a PHGG group. 1 is a graph showing the results of examining free amino acids in a control group (group C) and a PHGG group (group P) in a PHGG intake test to pigs. Results for (A) free leucine, (B) free isoleucine, (C) free valine, and (D) free branched-chain amino acids are shown, respectively. In the graph, "*" indicates significant at 5% risk (p<0.05), and "**" indicates significant at 1% risk (p<0.01) (same in FIG. 4). Fig. 2 is a graph showing the results of examining free amino acids in a control group (group C) and a PHGG group (group P). Results for (A) free methionine, (B) free threonine, and (C) free essential amino acids are shown, respectively.

Next, embodiments of the present invention will be described with reference to the drawings. The technical scope of the present invention is not limited by these embodiments, and can be implemented in various forms without changing the gist of the invention.
Guar gum is a water-soluble natural polysaccharide obtained from the endosperm (more precisely, the cotyledons) of guar beans, and has two molecules of mannose linked in a linear chain and one molecule of galactose as a side chain. It is a polysaccharide and has an average molecular weight of about 2.0×10 ⁵ to 3.0×10 ⁵ . Guar gum is known to have physiological effects such as blood sugar level elevation inhibitory effect, cholesterol lowering effect, and bowel movement improving effect. In the present invention, the guar gum hydrolyzate is made from beans derived from the annual leguminous plant guar (scientific name: Cyanopsis tetragoloba), which is used for food in India, Pakistan, etc., and the galactomannan polysaccharide contained in the endosperm is hydrolyzed. and water-soluble dietary fiber obtained by reducing the molecular weight. The method for hydrolyzing guar gum is not particularly limited and may be an enzymatic decomposition method, an acid decomposition method, or the like.

The enzyme used in the enzymatic decomposition method is not particularly limited as long as it is an enzyme that hydrolyzes linear mannose chains, but it is preferable to use β-mannanase derived from bacteria belonging to the genus Aspergillus or Rhizopus. The upper limit of the average molecular weight distribution of the guar gum decomposition product is 2×10 ⁵ or less, preferably 1.0×10 ⁵ or less, more preferably 2.5×10 ⁴ or less. The lower limit of the average molecular weight distribution of the guar gum decomposition products is 1.8×10 ² or more, preferably 3.0×10 ³ or more. If the average molecular weight exceeds 2×10 ⁵ , the viscosity will be too high and it will be difficult to include it in food and drink. ^The method for ^measuring the ^{molecular weight} distribution is not particularly limited. There is a method using a liquid chromatography method, and the like.
The guar gum decomposition product of the present invention contains 70% by mass or more, preferably 80% by mass or more of those having the average molecular weight within the above range.

Galactose is a type of monosaccharide classified as aldohexose, and has a molecular formula of C ₆ H ₁₂ O ₆ and a molecular weight of 180 (both of which are the same as glucose). The configuration is 2nd (2nd from the top in the Fisher projection formula), the -OH at the 5th position is in the same direction, and the 3rd and 4th positions are in the opposite direction, and the 5th position of D-galactose is D-glycerol. Same as aldehyde.
Mannose is a kind of monosaccharide classified as aldohexose, and has a molecular formula of C ₆ H ₁₂ O ₆ and a molecular weight of 180 (both of which are the same as glucose). The steric configuration is such that the —OH at the 2nd and 3rd positions are in the same direction and the 4th and 5th positions are in the opposite direction, and the steric configuration at the 5th position of D-mannose is the same as that of D-glyceraldehyde. Mannose is poorly metabolized in humans and hardly enters the glycolysis system when orally ingested.
The oligosaccharide content was 6 to 15% by mass (15% by mass or less) according to the high-performance liquid chromatograph analysis chart.

Amino acid composition in proteins can be measured by a known method using, for example, HPLC. Asparagine and glutamine are converted to aspartic acid and glutamic acid, respectively, during protein hydrolysis in pretreatment for amino acid analysis, and asparagine and aspartic acid, and glutamic acid and glutamine cannot be distinguished during measurement. Therefore, these are collectively quantified as aspartic acid and glutamic acid.
The muscle mass-increasing composition of the present invention can be orally ingested as it is or mixed with foods, beverages, and the like. The dosage for oral administration is not particularly limited, but is 0.5 g to 70 g (preferably 3 g to 30 g, more preferably 6 g to 18 g) per adult per day.

<Preparation of guar gum degradation product (PHGG)>
Example 1
After adjusting the pH to 4.5 by adding 0.1 N hydrochloric acid to 900 g of water, 0.2 g of β-mannanase derived from bacteria belonging to the genus Aspergillus (manufactured by Novo Nordisk Bioindustry) and 100 g of guar gum powder were added and mixed. The mixture was reacted at 40-45°C for 24 hours. After the reaction, the enzyme was deactivated by heating at 90°C for 15 minutes. The reaction solution was separated by suction filtration, and the clear solution obtained by removing insoluble matter was concentrated under reduced pressure (Yamato evaporator). A solid content of 20% by mass was obtained. This was dried with a spray dryer (manufactured by Okawara Kakoki Co., Ltd.) to obtain 65 g of a guar gum decomposition product as a powder.
A 0.5 (w/v) % concentration aqueous solution was obtained by dissolving the guar gum decomposition product in water. Polyethylene glycol (average molecular weight: 2×10 ² , 2×10 ³ , 2×10 ⁴ and 1×10 ⁵ ) was used as a molecular weight marker, and high performance liquid chromatography (column: YMC-Pack Diol-120, detector : Differential refractometer), the average molecular weight was about 20,000. 85% by mass or more was contained with an average molecular weight of 1.8×10 ³ to 2.0×10 ⁵ .

Further, when the viscosity of the 1% by mass aqueous solution was measured using a Brookfield viscometer at 25° C. and 60 rpm, it was 8 mPa·s.
When the content ratio of galactose and mannose (galactose:mannose) was measured, it was 1:1.7.
The dietary fiber content was measured by an enzyme-HPLC method and found to be 90% by mass.
The oligosaccharide content was 7.3% by mass.
Further, amino acid analysis of the guar bean protein in this example was performed, and the results were as shown in Table 1. From these results, (glutamic acid + glutamine + aspartic acid + asparagine) / total amino acids, (glutamic acid + glutamine + aspartic acid + asparagine) / (arginine + lysine + histidine), (cystine + tyrosine) / total amino acids, (valine + histidine) + proline + leucine)/total amino acids were 0.47, 5.0, 0.11 and 0.10, respectively.
Further, the ratio of guar gum hydrolyzate to guar bean protein was found to be guar gum hydrolyzate:guar bean protein=279:1.

　なお、アミノ酸分析の特性上、グルタミン酸＋グルタミン（Glu+Gln）はグルタミン酸として、アスパラギン酸+アスパラギン（Asp+Asn）はアスパラギンとして、システイン（Cys）はシスチンとして検出されるため、これを考慮して表中の記載を行った。

Due to the characteristics of amino acid analysis, glutamic acid + glutamine (Glu + Gln) is detected as glutamic acid, aspartic acid + asparagine (Asp + Asn) is detected as asparagine, and cysteine (Cys) is detected as cystine. The description in the table was performed.

Example 2
After adjusting the pH to 3 by adding 0.1 N hydrochloric acid to 900 g of water, 0.15 g of β-mannanase derived from bacteria belonging to the genus Aspergillus (manufactured by Novo Nordisk Bioindustry) and 100 g of guar gum powder were added and mixed. The mixture was reacted at 40-45°C for 24 hours. After the reaction, the enzyme was deactivated by heating at 90°C for 15 minutes. The reaction solution was separated by suction filtration, and the clear solution obtained by removing insoluble matter was concentrated under reduced pressure (Yamato evaporator). A solid content of 20% by mass was obtained. This was dried with a spray dryer (manufactured by Okawara Kakoki Co., Ltd.) to obtain 68 g of guar gum decomposition product as a powder.
When the average molecular weight of the guar gum decomposition product was determined in the same manner as in Example 1, it was about 2.5×10 ⁴ . According to the results of the HPLC chart, 85% by mass or more of those having an average molecular weight of 1.8×10 ³ to 2.0×10 ⁴ were contained.
Also, the viscosity of the 1% by mass aqueous solution was measured using a Brookfield viscometer at 25° C. and 60 rpm, and found to be 10 mPa·s.

When the content ratio of galactose and mannose (galactose:mannose) was measured, it was 1:1.8.
The dietary fiber content was measured by an enzyme-HPLC method and found to be 89% by mass.
The oligosaccharide content was 10% by mass.
In addition, amino acid analysis of the guar bean protein in this example revealed (glutamic acid + glutamine + aspartic acid + asparagine) / total amino acids, (glutamic acid + glutamine + aspartic acid + asparagine) / (arginine + lysine + histidine). , (cystine+tyrosine)/total amino acids, and (valine+histidine+proline+leucine)/total amino acids were 0.56, 22.3, 0.16 and 0.03, respectively.
Further, the ratio of guar gum hydrolyzate to guar bean protein was found to be guar gum hydrolyzate:guar bean protein=418:1.

Example 3
After adjusting the pH to 4 by adding 0.1 N hydrochloric acid to 900 g of water, 0.25 g of β-mannanase derived from bacteria belonging to the genus Aspergillus (manufactured by Novo Nordisk Bioindustry) and 100 g of guar gum powder were added and mixed. The mixture was reacted at 50-55°C for 12 hours. After the reaction, the enzyme was deactivated by heating at 90°C for 15 minutes. The reaction solution was separated by suction filtration, and the clear solution obtained by removing insoluble matter was concentrated under reduced pressure (Yamato evaporator). A solid content of 20% by mass was obtained. This was dried with a spray dryer (manufactured by Okawara Kakoki Co., Ltd.) to obtain 70 g of a guar gum decomposition product as a powder.
When the average molecular weight of the guar gum decomposition product was determined in the same manner as in Example 1, it was about 1.5×10 ⁴ . According to the results of the HPLC chart, 86% by mass or more of those having an average molecular weight of 1.8×10 ³ to 2.0×10 ⁵ were contained.

Further, when the viscosity of the 1% by mass aqueous solution was measured using a Brookfield viscometer at 25° C. and 60 rpm, it was 9 mPa·s.
When the content ratio of galactose and mannose (galactose:mannose) was measured, it was 1:2.0.
The dietary fiber content was measured by an enzyme-HPLC method and found to be 88% by mass.
The oligosaccharide content was 9% by mass.
In addition, amino acid analysis of the guar bean protein in this example revealed (glutamic acid + glutamine + aspartic acid + asparagine) / total amino acids, (glutamic acid + glutamine + aspartic acid + asparagine) / (arginine + lysine + histidine). , (cystine+tyrosine)/total amino acids, and (valine+histidine+proline+leucine)/total amino acids were 0.48, 9.6, 0.17 and 0.05, respectively.
Further, the ratio of guar gum hydrolyzate to guar bean protein was found to be guar gum hydrolyzate:guar bean protein=357:1.

<PHGG intake test using lifestyle-related disease model mice>
1. Test Method (1) Experimental Animals, Test Feeds, and Breeding Method Four-week-old C57BL/6NJcl strain male mice (CLEA Japan, Inc.) were used. After one week of preliminary breeding with the AIN-93G feed, the animals were divided into groups and fed the test feed ad libitum for 12 weeks.
In the control high-fat diet group, approximately 30% of lard was added to the AIN-93G composition so that fat-derived calories were 60%. In the PHGG group, 5% of the dietary fiber cellulose in the composition of the high-fat diet group was completely replaced with PHGG. As PHGG, the one of Example 3 was used.
Body fat percentage and muscle weight were measured by computed tomography (CT) scanning on the 77th day of rearing. An autopsy was performed on the 84th day, and blood was collected and organs were extracted. A block of about 5 mm square was taken from the liver for microarray analysis, immersed in RNAlater (Thermo Fisher Scientific Co., Ltd.) at 4°C overnight, and then stored at -80°C.

(2) Measurement of muscle weight On day 77 of this feeding, body composition was measured using an X-ray CT apparatus Latheta LCT-200 (Hitachi, Ltd.). Under isoflurane anesthesia, the region from the second rib to the caudal tip of the pelvis was measured at intervals of 2 mm, and the muscle weight was calculated using Latheta's attached software.
(3) Contract Analysis of Serum Components Total cholesterol (T-CHO), triglyceride (TG), non-esterified fatty acid (NEFA), LDL cholesterol (low density lipoprotein cholesterol: LDL-C) , HDL cholesterol (high density lipoprotein cholesterol: HDL-C), glucose (GLU), and glycoalbumin (GA) measurements were consigned to the Nagahama Life Science Laboratory.
(4) DNA microarray of liver After immersion in RNAlater, total RNA was extracted from a liver sample stored at -80°C, fragmented and labeled sense strand cDNA was synthesized, and subjected to DNA microarray analysis. After normalizing the obtained data, comparison between the two groups was performed, and genes whose expression varied significantly between the high-fat diet group and the PHGG group were extracted. Furthermore, enrichment analysis was performed to investigate the function of the expression-changed genes.

2. Test results (1) Muscle weight Fig. 1 shows the measurement results of muscle weight. Muscle weight was significantly (p<0.05) higher in the PHGG group than in the high-fat diet group.
(2) Serum Components FIG. 2 shows the measurement results of LDL-C. LDL cholesterol was significantly (p<0.05) lower in the PHGG group than in the high-fat diet group.
(3) DNA microarray analysis of liver Table 2 shows genes that were significantly increased (↑) or decreased (↓) by DNA microarray analysis of liver.

In contrast to the high-fat diet group, the PHGG group showed decreased expression of Cd36, SREBP1, Cyp51, Fdft1, Hmgscs1, and Nsdh1, which are genes involved in fat accumulation and cholesterol biosynthesis. In addition, as a result of enrichment analysis, it was shown that it contributes to suppression of fat accumulation, suppression of cholesterol elevation, and improvement of insulin resistance. PHGG intake significantly increased the expression of PGC1α and AMPK, which regulate mitochondrial biogenesis and energy metabolism.

<PHGG intake test to pigs>
1. Test method (1) Experimental animal, test feed, and breeding method A test was conducted on meat pigs raised on a general farm. The control group was fed a normal diet for meat pigs, and the PHGG group was fed the same diet as the control group with 0.06% PHGG added without interruption from weaning to shipping. As PHGG, the one of Example 1 was used.
Three animals were randomly selected from each group, totaling six animals, and slaughtered on the 160th day of rearing. After 5 days from slaughter, about 30 g of skeletal muscle was collected from the shoulder and analyzed for free amino acids. served to
(2) Measurement of Free Amino Acids Except for tryptophan, amino acid automatic analysis was used, and tryptophan was analyzed using high performance liquid chromatography.

2. Test Results Table 3 shows the average measurement results of free amino acid amounts in the control group and the PHGG measurement group (n=3). FIG. 3 shows the measurement results of leucine, isoleucine, valine and branched chain amino acids.

Compared to the control group (C group), the PHGG group (P group) had significantly more free leucine, free isoleucine, free valine, and free branched-chain amino acids in skeletal muscle, and also significantly more free methionine, free threonine, and free essential amino acids. was common in Branched-chain amino acids are essential amino acids that cannot be produced in the body, are endurance amino acids, and are known to work as energy in muscles during exercise. In addition, PHGG is known to promote muscle synthesis, so we found that PHGG works favorably for muscle building in the elderly and infirm.
As described above, according to the present embodiment, it was possible to provide a composition or the like that can be easily ingested and that can promote an increase in muscle mass.

Claims (4)

(A) a guar gum decomposition product having an average molecular weight of 1.8×10 ³ to 2.0×10 ⁵ and containing 70% by mass or more of those within the average molecular weight range; and (B) guar bean protein; The viscosity of a 1% by mass aqueous solution is 50 mPa s or less when measured at 25 ° C. and 60 rpm using
The guar gum hydrolyzate is a galactomannan polysaccharide contained in guar-derived endosperm and having a content ratio of galactose and mannose (galactose:mannose) in the range of 1:1.5 to 1:2.1 using microbial-derived β-mannanase. It is obtained by decomposing and reducing the molecular weight, and has a dietary fiber content of at least 70% by mass and an oligosaccharide content of guar gum decomposition product of 15% by mass or less as determined by the enzyme-HPLC method, Among the amino acids contained in the protein, (glutamic acid + glutamine + aspartic acid + asparagine) ≥ 100 mg/100 g, (cystine + tyrosine + serine + threonine) ≥ 40 mg/100 g, total amino acids ≥ 150 mg/100 g. , a composition for increasing muscle mass, wherein the ratio of (A)/(B) is 1000 or less. A food or drink containing the muscle mass-increasing composition according to claim 1 . A pharmaceutical product containing the muscle mass-increasing composition according to claim 1 . A feed containing the composition for increasing muscle mass according to claim 1 .

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