JP5740072B2 - Stress relieving agent using β-1,3-1,6-D-glucan - Google Patents

Description

  The present invention relates to a stress relieving agent, a parasympathetic nerve stimulator and / or a sympathetic nerve inhibitor, and a food or drink composition used for stress relaxation, or for sympathetic nerve suppression and / or parasympathetic nerve stimulation (or stress relaxation). Related to things.

  In today's diversified social environment, an increasing number of people are constantly exposed to stress and complain of physical and mental fatigue. The same mechanism occurs in animals, and stress load such as forced restraint on mice has been reported to increase corticosterone in blood and decrease immunity (especially decrease natural killer (NK) activity). . (Non-patent documents 1, 2, 3)

  The word “stress” was originally proposed by Canadian physiologist H. Selye, and when a living body receives a strong stimulus such as strong heat or pressure from the outside, or when it is injured, the living body undergoes certain modulation. It has been found to cause certain conditions such as adrenal hypertrophy, spleen and thymus atrophy, gastric / duodenal bleeding and ulcers. Such a state is defined as a stress state. This suggests that stress causes hypertension of the cerebral hypothalamus, pituitary gland, endocrine system controlled by the adrenal system, and autonomic nervous system. In addition, American physiologist WB Cannon has also developed a sympathetic nervous system for adrenaline to maintain homeostasis when the body is exposed to strong external physical or mental stimuli. It was discovered that a systemic reaction due to secretion is induced. Specifically, it has been found that the cat is excited to enlarge the pupil, increase the respiratory rate / pulse, increase the blood pressure, and decrease the gastrointestinal function.

Recently, responses in the endocrine system, autonomic nervous system, and immune system have become clear as responses of the body to stress. In response to stress on the living body, hormones such as adrenaline, noradrenaline, and glucocorticoid (also called glucocorticoid) are secreted in response thereto. Adrenaline, noradrenaline, catecholamine and dopamine are secreted from the adrenal medulla, and glucocorticoids, cortisol and corticosterone are secreted from the adrenal cortex. The mechanism is explained as follows. The brain that senses stress sends its discomfort signal to the hypothalamus, which in turn acts on the pituitary gland, from which adrenocorticotropic hormone (ACTH) is secreted, and by that stimulation, glucocorticoids and the like are secreted from the adrenal cortex. , Blood glucose concentration increases. At the same time, the brain that feels the stressor is activated by acting on the autonomic nerve from the hypothalamus. At that time, the sympathetic nerve among the autonomic nerves is stimulated, and adrenaline and the like are secreted from the adrenal medulla, which increases blood sugar and induces an increase in blood pressure. These are the responses of the living body when stress is applied.
If excessive stress continues to be applied, the above-mentioned state will persist, leading to various diseases by inducing delayed healing of the damaged site, muscle atrophy, gastric mucosal injury, blood vessel injury, thymus atrophy, and the like. In particular, damage to the immune system is serious, and it is necessary to reduce the number of lymphocytes and decrease the activity of natural killer cells (NK cells), resulting in exposure to viruses and bacterial infections.
On the other hand, autonomic nerves are also distributed in organs responsible for immunity (thymus, lymph nodes, bone marrow, spleen, etc.), and the influence is thought to be great. In addition, the autonomic nervous system regulates leukocytes, NK cells, and lymphocytes. For example, the activity of NK cells is reduced by adrenaline secreted when sympathetic nerve is dominant. Glucocorticoids are known to weaken lymphocytes and reduce macrophage activity. Conversely, when parasympathetic dominance occurs, the activity of lymphocytes increases. Thus, it is known that the immune system is lowered by stress, which causes many diseases. However, a food material having both an anti-stress effect and an immunostimulatory effect is not known.
In recent years, the number of people with addictive stress is increasing due to factors such as changes in eating habits, lack of exercise, excessive stress, and aging. An increase in the number of stressed persons is announced by the Ministry of Health, Labor and Welfare in 2002. It is clear from

  Against this background, food materials having a function to relieve stress are being actively developed.

  It has been reported that β-1,3-1,6-D-glucan can be prepared from the genus Aureobasidium sp. (Patent Document 1), and has been reported to have intestinal immunity activation activity. (Patent Document 2). However, not limited to its origin, it has not been reported that β-1,3-1,6-D-glucan has a stress relieving action.

JP 2006-104439 A JP 2006-137719 A Biology of stress, Kimiko Murobushi, published by Ohmsha, April 10, 2005, pages 2-55 "Simple Immunology Revised 3rd Edition" Izumi Nakajima, Toshitada Takahashi, Yasunobu Yoshikai Nanedo Published February 20, 2006, pp. 73-82 “Stress and Immunity” by Keiko Hoshi, Blue Backs Kodansha, published May 20, 1993, pages 5-84

  An object of the present invention is to provide a safe stress relieving agent made of a natural material and a food or drink composition having a stress relieving effect.

  Furthermore, it aims at providing the food-drinks composition which consists of a natural material which has the activation effect of the immunity reduced by stress.

  Furthermore, the present invention also provides a safe parasympathetic nerve stimulator and / or sympathetic nerve inhibitor made of a natural material, and a food and drink composition that can stimulate the parasympathetic nerve and / or suppress the sympathetic nerve. And

  The present inventors have maintained the health of β-1,3-1,6-D-glucan (hereinafter referred to as β-glucan in this specification) produced by a microorganism belonging to the genus Aureobasidium sp.・ As a result of repeated studies on effective use for enhancement, the β-glucan whose viscosity has been reduced by alkali treatment has an excellent effect on stress relaxation, and also suppresses and / or stimulates the autonomic nervous system. Have been found to show anti-stress effects. They also found the ability to recover the reduced immune activity when stressed.

  The present invention has been completed based on the above findings, and provides the following stress relieving agents and the like.

The present invention has the following properties (1) to (3):
(1) Derived from microorganisms belonging to the genus Aureobasidium sp.
(2) The ¹ H NMR spectrum of a solution using 1N sodium hydroxide heavy aqueous solution as a solvent has two signals of about 4.7 ppm and about 4.5 ppm.
(3) The viscosity of the aqueous solution at 30 ° C., pH 5.0, concentration 0.5 (w / v%) is 200 cP (mPa · s) or less.
There is provided a stress relieving agent comprising β-1,3-1,6-D-glucan having the formula:

The present invention also provides the following properties (1) to (3):
(1) Derived from microorganisms belonging to the genus Aureobasidium sp.
(2) The ¹ H NMR spectrum of a solution using 1N sodium hydroxide heavy aqueous solution as a solvent has two signals of about 4.7 ppm and about 4.5 ppm.
(3) The viscosity of the aqueous solution at 30 ° C., pH 5.0, concentration 0.5 (w / v%) is 200 cP (mPa · s) or less.
There is provided a parasympathetic nerve stimulator and / or a sympathetic nerve inhibitor, comprising β-1,3-1,6-D-glucan having

The present invention further includes the following properties (1) to (3):
(1) Derived from microorganisms belonging to the genus Aureobasidium sp.
(2) The ¹ H NMR spectrum of a solution using 1N sodium hydroxide heavy aqueous solution as a solvent has two signals of about 4.7 ppm and about 4.5 ppm.
(3) The viscosity of the aqueous solution at 30 ° C., pH 5.0, concentration 0.5 (w / v%) is 200 cP (mPa · s) or less.
There is provided a food and beverage composition comprising β-1,3-1,6-D-glucan having the formula:

  The stress relieving agent of the present invention is effective for prevention, improvement, and / or treatment of symptoms caused by stress.

  In addition, the stress relieving agent of the present invention enhances or stimulates the autonomic nervous system, particularly the parasympathetic nervous system, and / or suppresses the sympathetic nervous system, suggesting an association with stress relaxation.

  Furthermore, since the aqueous solution of β-1,3-1,6-D-glucan in the present invention has a low viscosity, it is useful in that it can be easily ingested and sterilized.

Hereinafter, the present invention will be described in detail.
(I) Stress Relieving Agent The stress relieving agent of the present invention contains β-1,3-1,6-D-glucan derived from a microorganism belonging to the genus Aureobasidium sp. This β-1,3-1,6-D-glucan has two signals of about 4.7 ppm and about 4.5 ppm in ¹ H NMR spectrum of a solution using 1N sodium hydroxide heavy aqueous solution as a solvent, The viscosity of the aqueous solution at 30 ° C., pH 5.0, and concentration 0.5 (w / v%) is preferably 200 cP (mPa · s) or less, more preferably 100 cP (mPa · s) or less, and even more preferably 50 cP (mPa).・ S) The following. The lower limit of the viscosity is usually about 10 cP (mPa · s). Since it is well known that NMR measurement values change due to subtle changes in conditions and are accompanied by errors, “about 4.7 ppm” and “about 4.5 ppm” are measured values within the normally expected range. A numerical value including a fluctuation range (for example, ± 0.2).

Β-1,3-1,6-D-glucan produced by Aureobasidium microorganisms β-1,3-1,6-D-glucan produced by Aureobasidium microorganisms is secreted outside the cells. Therefore, it is preferable in that it can be easily recovered and is water-soluble. The microorganism belonging to the genus Aureobasidium can produce from a high molecular weight glucan having a molecular weight of 1 million or more to a low molecular weight glucan having a molecular weight of about tens of thousands according to the culture conditions.

  Among them, those produced by Aureobasidium pullulans are preferable, and Aureobasidium pullulans GM-NH-1A1 strain or GM-NH-1A2 strain (incorporated administrative agency National Institute of Industrial Science and Technology patent biological deposit center) Those produced by FERM P-19285 and FERM P-19286, respectively) are preferred. The GM-NH-1A1 and GM-NH-1A2 strains are mutant strains of the Aureobasidium sp. K-1 strain. The Aureobasidium genus K-1 strain is known to produce two types of β-1,3-1,6-D-glucan having a molecular weight of 2 million or more and about 1 million.

  In addition, β-1,3-1,6-D-glucan produced by Aureobasidium bacteria usually has a sulfur-containing group, whereas β-glucan produced by K-1 strain has a sulfoacetic acid group. (Arg. Biol. Chem., 47, 1167-1172 (1983)), science and industry, 64, 131-135 (1990)). The β-1,3-1,6-D-glucan produced by the GM-NH-1A1 strain and the GM-NH-1A2 strain is also considered to have a sulfoacetic acid group. Among the microorganisms belonging to the genus Aureobasidium, there are also species and strains that produce β-1,3-1,6-D-glucan containing phosphorus-containing groups such as phosphate groups, malate groups, and the like.

  The GM-NH-1A1 and GM-NH-1A2 strains have a high molecular weight β-glucan (fine particle glucan) having an apparent main peak of 500 to 2.5 million and an apparent peak of 2 to 2 as shown in the Examples. It is a strain that produces both 300,000 low molecular weight β-glucan. The fine particle glucan has a primary particle diameter of about 0.05 to 2 μm.

  The solubility of β-1,3-1,6-D-glucan depends on pH and temperature. When β-2,3-1,6-D-glucan is prepared to prepare a 2 mg / ml aqueous solution under the conditions of pH 3.5 and temperature 25 ° C., 50% by weight or more thereof has a primary particle size of 0.05 to 2 μm. Fine particles are formed, and the remainder is dissolved in water. In the present invention, the particle diameter is a value measured by a laser diffraction scattering method.

  When β-1,3-1,6-D-glucan is contained in the preparation as an aqueous solution, an emulsifier such as lecithin or a stabilizer such as cyclic dextrin is added to the aqueous solution to form fine particles. Can be further stabilized.

  When β-1,3-1,6-D-glucan is derived from Aureobasidium pullulans, the binding ratio of β-1,3 bond / β-1,6 bond is 1-1. About 5 and especially about 1.1 to 1.4.

Β-1,3-1,6-D-glucan contained in the stress relieving agent of the present invention β-1,3-1,6-D-glucan contained in the stress relieving agent of the present invention is in an aqueous solution. Has a lower viscosity than that of natural β-1,3-1,6-D-glucan produced by microorganisms belonging to the genus Aureobasidium. This low viscosity β-1,3-1,6-D-glucan has a viscosity at 30 ° C. of a 0.5% (w / v) aqueous solution (pH 5.0), preferably 200 cP (mPa · s) or less. More preferably, it is 100 cP (mPa * s) or less, More preferably, it is 50 cP (mPa * s) or less, More preferably, it is 10 cP or less. In the present invention, the viscosity is a value measured with a BM type rotational viscometer.

This low-viscosity glucan may have the same primary structure as natural β-1,3-1,6-D-glucan produced by an aureobasidium microorganism. Specifically, the ¹ H NMR spectrum of a solution using 1N sodium hydroxide heavy aqueous solution as a solvent has two signals of about 4.7 ppm and about 4.5 ppm. Since it is well known that NMR measurement values change due to subtle changes in conditions and are accompanied by errors, “about 4.7 ppm” and “about 4.5 ppm” are measured values within the normally expected range. A numerical value including a fluctuation range (for example, ± 0.2).

When this glucan is derived from Aureobasidium pullulans (for example, GM-NH-1A1 strain), the obtained β-1,3-1,6-D-glucan is converted into exo-type β-1,3- When hydrolysis is performed with glucanase (Citalase M, manufactured by KAI Kasei Co., Ltd.), release of glucose and gentiobiose can be confirmed as degradation products. From this and the integration ratio of NMR, β-1,3-1,6-D-glucan derived from Aureobasidium pullulans has β-1,6 bonds to the main chain of β-1,3 bonds. A structure in which glucose is branched into one molecule side chain, and the degree of side chain branching of 1,6-bond to 1,3-bond main chain is about 50 to 100%, particularly 50
It is estimated to be ~ 90%.

  The β-1,3-1,6-D-glucan contained in the stress relieving agent of the present invention has a metal ion concentration of 0.4 g per 1 g of the solid content of β-1,3-1,6-D-glucan. Or less, more preferably 0.2 g or less, and even more preferably 0.1 g or less. When β-1,3-1,6-D-glucan is contained in an aqueous solution in the preparation, the metal ion concentration is preferably 120 mg or less, more preferably 50 mg or less, per 100 ml of the aqueous solution. More preferably, it is 20 mg or less.

  The metal ions referred to here include alkali metal ions, alkaline earth metal ions, Group 3 to Group 5 metal ions, transition metal ions, and the like. Include alkali-derived potassium ions and sodium ions used in the production of low-viscosity β-1,3-1,6-D-glucan. The metal ion concentration can be adjusted by ultrafiltration or dialysis. When the metal ion concentration is in the above range, β-1,3-1,6-D-glucan is hardly gelled, aggregated, or precipitated when stored in an aqueous solution or when heat-sterilized in an aqueous solution. . Further, even in a solid preparation, aggregation or the like hardly occurs when redissolved.

  The stress relieving agent of the present invention may contain low-viscosity β-1,3-1,6-D-glucan in a solid state, or a liquid such as an aqueous solution or a fluid state.

Method for producing β-1,3-1,6-D- glucan belonging to the genus Aureobasidium β-1,3-1,6-D-glucan is used, for example, as an organic solvent in the culture supernatant of microorganisms that produce it. Can be obtained as a precipitate.

  Various methods for culturing microorganisms belonging to the genus Aureobasidium to produce β-1,3-1,6-D-glucan have been reported. Examples of carbon sources that can be used include carbohydrates such as sucrose, glucose and fructose, and organic nutrient sources such as peptone and yeast extract.

  Examples of the nitrogen source include inorganic nitrogen sources such as ammonium sulfate, sodium nitrate, and potassium nitrate. In some cases, inorganic salts such as sodium chloride, potassium chloride, phosphate, magnesium salt, calcium salt, and trace metal salts such as iron, copper, manganese and vitamins are used as appropriate to increase the production of β-glucan. It is also an effective method to add a kind or the like.

  Aureobasidium microorganisms are reported to produce high concentrations of β-1,3-1,6-D-glucan when cultured in a medium in which ascorbic acid is added to a tourpec medium containing sucrose as a carbon source. (Arg. Biol. Chem., 47, 1167-1172 (1983)); Science and Industry, 64, 131-135 (1990); JP-A-7-51082). However, the medium is not particularly limited as long as microorganisms grow and produce β-1,3-1,6-D-glucan. If necessary, organic nutrient sources such as yeast extract and peptone may be added.

  Conditions for aerobic culture of microorganisms belonging to the genus Aureobasidium in the above medium are about 10 to 45 ° C, preferably about 20 to 35 ° C, about 3 to 7, preferably about 3.5 to 5. PH conditions and the like.

  In order to effectively control the culture pH, it is also possible to control the pH of the culture solution with an alkali or an acid. Further, an antifoaming agent may be appropriately added for defoaming the culture solution. The culture time is usually about 1 to 10 days, preferably about 1 to 4 days, whereby β-glucan can be produced. The culture time may be determined while measuring the production amount of β-glucan.

  When a microorganism belonging to the genus Aureobasidium is cultured under aeration and agitation for about 4 to 6 days under the above conditions, 0.1-β-glucan polysaccharide mainly composed of β-1,3-1,6-D-glucan is contained in the culture solution. % To several% (w / v), and the viscosity of the culture solution is from several hundred cP ([mPa · s]) to several thousand cP at 30 ° C. by a BM type rotational viscometer (manufactured by Toki Sangyo Co., Ltd.). It has a very high viscosity ([mPa · s]). By adding, for example, an organic solvent to the supernatant obtained by centrifuging this culture, β-1,3-1,6-D-glucan can be obtained as a precipitate.

Method for producing low-viscosity β-1,3-1,6-D-glucan The above-mentioned culture solution containing β-1,3-1,6-D-glucan having a high viscosity is stirred at room temperature. When alkali is added, the viscosity rapidly decreases.

  The alkali is not particularly limited as long as it is water-soluble and can be used as a pharmaceutical or food additive. For example, an alkali carbonate aqueous solution such as a calcium carbonate aqueous solution, a sodium carbonate aqueous solution, a potassium carbonate aqueous solution, or an ammonium carbonate aqueous solution; an alkali hydroxide aqueous solution such as a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, or a calcium hydroxide aqueous solution; or an ammonia aqueous solution is used. it can. The alkali may be added so that the pH of the culture solution is 12 or more, preferably 13 or more. For example, when using sodium hydroxide to increase the pH of the culture solution, the final concentration of sodium hydroxide is preferably 0.5% (w / v) or higher, more preferably 1.25% (w / v) or higher. Add so that. When alkali is added to the culture solution and stirred well, the viscosity of the culture solution decreases instantaneously.

  Next, insoluble substances such as bacterial cells are separated from the culture solution after the alkali treatment. Since the viscosity of the culture solution is low, a method of allowing the cells to settle naturally (decant method), centrifuging, filtering the whole volume using filter paper or filter cloth, filter press, and membrane filtration (limitation of MF membrane, etc.) Insoluble substances and glucan can be easily separated by a method such as external filtration. In the case of total filtration with filter paper or filter cloth, it is one means to use a filter aid such as celite. Industrially, removal of bacterial cells by a filter press is preferred.

  Next, the solution containing glucan is neutralized by adding an acid. Neutralization may be performed before removal of insoluble matter. The acid is not particularly limited as long as it can be used as a pharmaceutical or food additive. For example, hydrochloric acid, phosphoric acid, sulfuric acid, citric acid, malic acid and the like can be used. The amount of acid used may be such that the solution or culture solution becomes neutral (pH 5-8). That is, neutralization does not necessarily need to be adjusted to pH 7.

  β-1,3-1,6-D-glucan obtained by neutralization after alkali treatment at pH 12 or higher has a viscosity of usually 200 cP at 30 ° C., pH 5.0, and concentration 0.5 (w / v%). Hereinafter, in some cases, it is 50 cP or less. The viscosity varies depending on the production method or purification method.

  The low viscosity β-1,3-1,6-D-glucan treated with alkali does not increase in viscosity even when neutralized. Furthermore, at room temperature (15 to 35 ° C.), even if the liquid is acidified so that the pH is less than 4, the viscosity does not increase.

  In addition, instead of removing the cells after neutralizing and neutralizing the culture supernatant, alkali treatment and neutralization can be performed after removing the cells from the culture supernatant.

  In order to remove soluble contaminants (for example, salts) having a lower molecular weight than glucan from the resulting aqueous solution of glucan, ultrafiltration may be performed, for example.

  Moreover, after carrying out alkali treatment and sterilization, it is possible to perform ultrafiltration under alkaline conditions without neutralization, and thereby purified β-1,3-excellent in transparency, thermal stability and long-term storage stability. 1,6-D-glucan is obtained. The alkaline condition is pH 10 or more, preferably 12 or more, and the upper limit of pH is usually about 13.5.

  The β-1,3-1,6-D-glucan contained in the aqueous solution thus obtained is once dried from the aqueous solution, both when it is dried and used as a solid formulation. It can be deposited. Although the precipitation method of β-1,3-1,6-D-glucan is not particularly limited, for example, ethanol or the like is added to an aqueous solution that is concentrated by ultrafiltration or the like to have a glucan concentration of 1 w / w% or more. Β-1,3-1,6-D-glucan can be precipitated by adding alcohol at an equal volume or more, preferably 2 times or more in volume ratio to the aqueous solution.

  By reducing the viscosity of β-1,3-1,6-D-glucan, concentration by ultrafiltration can be easily performed, so that the amount of alcohol used for alcohol precipitation can be reduced.

  When preparing a solid preparation, the low-viscosity β-1,3-1,6-D-glucan aqueous solution may be directly dried, and the precipitated β-1,3-1,6-D-glucan is dried. May be. Drying can be performed by a known method such as spray drying or freeze drying.

(I) Formulation In the stress relieving agent of the present invention, β-1,3-1,6-D-glucan can be made into an appropriate formulation together with a pharmaceutically acceptable carrier as necessary. Such carriers include excipients, binders, disintegrants, lubricants, moisturizers and the like. Further, a conventional additive such as an antioxidant may be included.

  The form of the preparation is not particularly limited and may be any form such as a tablet, pill, capsule, powder, granule, syrup and the like. When using low-viscosity β-1,3-1,6-D-glucan that has been subjected to alkali treatment, a high-concentration aqueous solution can be prepared. The amount can include an effective amount of β-1,3-1,6-D-glucan.

  As the excipient, known ones can be widely used, for example, various sugars such as lactose, sucrose and glucose; various starches such as potato starch, wheat starch and corn starch; and various celluloses such as crystalline cellulose. Various inorganic salts such as anhydrous calcium hydrogen phosphate and calcium carbonate.

  As the binder, known ones can be used, and examples thereof include crystalline cellulose, pullulan, gum arabic, sodium alginate, polyvinyl pyrrolidone, macrogol and the like.

  As the disintegrant, known ones can be widely used, and examples thereof include carboxymethyl cellulose, carboxymethyl cellulose calcium, hydroxypropyl cellulose, hydroxypropyl starch, starch, sodium alginate and the like.

  As the lubricant, known ones can be widely used, and examples thereof include magnesium stearate, talc, and hardened oil.

  As the wetting agent, known ones can be widely used, and examples thereof include coconut oil, olive oil, sesame oil, peanut oil, soybean phospholipid, glycerin, sorbitol and the like.

  The amount of β-1,3-1,6-D-glucan contained in the preparation may vary depending on the age, body weight, symptoms, administration method, etc. of the subject or patient. In such a case, the daily intake may be contained in an amount of about 1-1000 mg, preferably about 10-500 mg, more preferably about 10-200 mg, and even more preferably about 25-100 mg. If it is in the above intake range, a sufficient stress relieving effect can be obtained, and side effects and toxicity such as diarrhea will not appear.

  In the case of a preparation to be administered once a day, it is sufficient that the necessary amount per day is included in one preparation. For example, in the case of a preparation to be administered three times a day, one third of the necessary amount per day. As long as it is contained in the preparation.

  In the case of solid preparations such as tablets, pills, capsules, powders, granules, β-1,3-1,6-D-glucan is contained in the preparation in an amount of about 0.1 to 100% by weight, It is preferable that about 1 to 50 weight% is contained.

  In the case of a liquid or fluid preparation such as a syrup, β-1,3-1,6-D-glucan is about 0.01 to 2% by weight, particularly 0.05 to 0.5% by weight. It is preferable that it is included. A part of the glucan in the liquid or fluid preparation may not be dissolved.

  If it is the said range, (beta) -1,3-1,6-D-glucan of the quantity which a stress reduction effect is fully acquired and a side effect and toxicity do not appear is contained in the dosage amount which is easy to ingest. . In the case of a syrup, a syrup having a viscosity that is easy to drink can be obtained within the above range.

Further, the stress relieving agent of the present invention contains components and additives usually contained in the stress relieving agent as long as the stress relieving effect by β-1,3-1,6-D-glucan is not impaired. Also good.
Subject of Administration The stress relieving agent of the present invention can be suitably administered to mammals including humans exposed to stress. This includes patients who have other diseases besides healthy humans except under stress. Furthermore, since β-1,3-1,6-D-glucan is a safe natural component, a healthy person who is prone to stress can be ingested in a timely or regular manner.

  The stress relieving agent of the present invention has an action of preventing and / or improving physical and mental disorders caused by stress. Here, “prevention” includes not only completely preventing the onset of a disorder caused by stress but also suppressing the degree thereof. “Improvement” includes not only complete recovery from such a malfunction, but also mitigation of the malfunction.

  The stress in the present invention is a concept including stimuli and situations that are mentally and physically burdensome.

  The stress relieving agent of the present invention prevents and / or improves symptoms caused by stress such as depression, anxiety disorder, gastric / duodenal ulcer, irritable bowel syndrome, bronchial asthma, hypertension, autonomic dysfunction, and more. Specifically, post-traumatic stress disorder, stress gastritis, stress ulcer, irritable bowel syndrome, stress asthma, stress hair loss, stress mental disorder, depression, psychosomatic disorder, panic disorder, stress insomnia, Prevent and / or improve stress hypertension, stress headache, stress amenorrhea, stress constipation, stress overeating or anorexia, and stress sexual dysfunction.

(II) Parasympathetic nerve stimulant and / or sympathetic nerve inhibitor (stress relieving agent)
Β-1,3-1,6-D-glucan derived from a microorganism belonging to the genus Aureobasidium sp. Described above suppresses adrenal sympathetic nerve activity, increases gastric parasympathetic (vagus) nerve, spleen Since it suppresses sympathetic nerve activity, it can be used as a sympathetic nerve inhibitor and / or a parasympathetic nerve stimulator. In addition, stress can be relieved to suppress sympathetic nerves and / or stimulate parasympathetic nerves, and can also be used as a stress relieving agent.

As described above, this β-1,3-1,6-D-glucan has two ¹ H NMR spectra of about 4.7 ppm and about 4.5 ppm in a solution using 1N sodium hydroxide heavy aqueous solution as a solvent. The viscosity of the aqueous solution at 30 ° C., pH 5.0 and concentration 0.5 (w / v%) is preferably 200 cP (mPa · s) or less, more preferably 100 cP (mPa · s) or less, It is preferably 50 cP (mPa · s) or less, more preferably 10 cP or less.

  Subjects to be administered include humans suffering from various diseases adversely affected by autonomic nervous system abnormalities, and preferably include humans who feel stress. Moreover, since the said glucan is a natural safe component, a healthy person can always take.

(III) Food / Beverage Composition The food / beverage composition of the present invention contains the β-1,3-1,6-D-glucan described above. This food / beverage product composition contains β-1,3-1,6-D-glucan, and thus has an action of relieving stress and an action of suppressing sympathetic nerve and / or stimulating parasympathetic nerve and relieving stress. Therefore, it can be suitably used as a health food, a functional food, a health functional food such as a nutrition functional food or a food for specified health use. Here, the health food in the present invention generally includes foods generally sold as “healthy”, or foods other than pharmaceuticals that consumers ingest in anticipation of positive effects as good for health. Includes supplements. Moreover, the functional food in the present invention includes food designed so as to express the biological regulation function sufficiently efficiently.

  Therefore, the food / beverage product composition of the present invention is used to relieve stress, or to suppress sympathetic nerves and / or stimulate parasympathetic nerves, or to relieve stress. Can be attached.

  The kind of food / beverage products contained in the food / beverage product composition of the present invention is not particularly limited. As long as β-1,3-1,6-D-glucan can be added, various drinks such as energy drinks, juices, teas, soups, cookies, strawberries, gums, jelly, agar, Any food or drink such as pudding, gummy, chocolate, and processed starch food can be used. Bread, noodles such as udon, dairy products such as yogurt and cheese, processed foods such as dressing and mayonnaise, swallowing supplements, and the like are also suitable. What is necessary is just to mix | blend in the suitable step of a manufacturing process according to the characteristic and objective of each food / beverage products.

  In the food and beverage composition of the present invention, the daily intake is preferably about 1 to 1000 mg, preferably about 10 to 500 mg, more preferably about 10 to 200 mg, and still more preferably about 25 to 100 mg. It is sufficient if β-1,3-1,6-glucan is contained. In particular, in the case of giving to a patient having a disease caused by refractory stress, the amount of β-1,3-1, the daily intake is about 1 to 1000 mg, particularly about 10 to 500 mg. What is necessary is just to contain 6-glucan.

  Since β-1,3-1,6-D-glucan is non-toxic to the human body, there is no particular limitation on the ratio of addition, but considering the characteristics, taste, economy, etc. of each food and drink In the case of a solid, semi-solid or gel food, the amount added is usually about 0.01 to 5% by weight, preferably about 0.01% to 2% by weight, based on the total amount of the composition. Semi-solid foods such as yogurt are also included in solid foods in that fluidity is not required for eating. If it is said range, (beta) -1,3-1,6-glucan of the daily intake effective in stress relaxation will be contained in the amount of foods which can be ingested reasonably. Moreover, if it is the said content ratio of (beta) -1,3-1,6-D-glucan, the solubility of glucan is favorable and a viscosity is low and it is easy to be absorbed.

  For the same reason, when β-1,3-1,6-glucan is contained in a liquid, fluid, or semi-fluid beverage composition, its content is 0. About 01-5 weight% is preferable and about 0.01-2 weight% is more preferable. If it is said range, (beta) -1,3-1,6-glucan of the daily intake effective for stress relaxation will be contained in the food amount which can be ingested reasonably. Moreover, if the content ratio of β-1,3-1,6-D-glucan is within the above range, there is no possibility of causing gelation or an increase in viscosity even by heat treatment such as sterilization. In addition, when the glucan concentration in a drink composition is high, a part may be contained without melt | dissolving.

  The food / beverage composition of the present invention may contain auxiliary components commonly used in the food field within a range not impairing the effects of the present invention. Examples of such auxiliary components include fructooligosaccharides, dairy oligosaccharides, soybean oligosaccharides, oligosaccharides such as isomaltose; lactic acid bacteria such as Bifidobacterium, Lactobacillus, Enterococcus; Agaricus, Maitake, Shiitake, Meshima Cobb, Mushrooms such as Chaga, Hanabiratake, or extracts thereof; Cyclodextrins such as α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin and linear and indigestible dextrins; Citric acid, malic acid, hyaluronic acid Organic acids such as: amino acids such as tryptophan, methionine, theanine, GABA (γ-aminobutyric acid), β-carotenes such as β-carotene, lutein, astaxanthin, fucoxanthin, vitamin A, vitamin C, vitamin E Na Tamines; minerals such as zinc, iron, magnesium, selenium, chromium, copper, manganese, molybdenum, iodine; lactoferrin; royal jelly; propolis; catechins; turmeric; trehalose; Aloe; psyllium; champignon; black vinegar; and various fragrances.

  In particular, β-1,3-1,6-D-glucan of about 0.01 to 5% by weight (particularly 0.01 to 2% by weight), lactic acid bacteria (especially, sterilized lactic acid bacteria powder), oligosaccharides, and / or amino acids. The food-drinks composition containing about 0.01 to 2 weight% of each is preferable. The food / beverage composition in this case may be any solid / semi-solid / gel / liquid / fluid / semi-fluid food / beverage composition.

  In addition, the food and drink composition of the present invention is not mainly composed of general food and drink, and β-1,3-1,6-glucan together with excipients or carriers, etc. as tablets, pills, capsules, For example, it may be a solid so-called supplement preparation (nutritional supplement preparation) formed into a powder, granule or the like. As the excipient, those exemplified in the item of the preparation can be used. In this case, the content of β-1,3-1,6-glucan is preferably about 10 to 80% by weight, more preferably about 10 to 50% by weight, based on the entire composition.

  In particular, β-1,3-1,6-D-glucan of about 10 to 80% by weight (especially 10 to 50% by weight), lactic acid bacteria (in particular, sterilized lactic acid bacteria powder), oligosaccharides, and / or amino acids 1 to What contains about 10 weight% is preferable.

The food / beverage composition of the present invention is ingested when necessary or daily by a person who is stressed or stressed, a person who feels stress, or a person suffering from a disease caused by abnormalities in the autonomic nervous system. It is suitable for.
EXAMPLES Next, the present invention will be described in detail with reference to examples and test examples, but the present invention is not limited to these examples.

(1) Preparation of low viscosity β-1,3-1,6-glucan
(1-1) Production of β-glucan After placing 100 ml of a liquid medium having the composition shown in Table 1 in a 500 ml shoulder flask and autoclaving at 121 ° C. for 15 minutes, Aureobasidium pullulans GM-NH-1A1 strain (FERM P-19285) is aseptically inoculated from a slant with the same medium composition and inoculated at a rate of 130 rpm with aeration and stirring at 30 ° C. for 24 hours. A seed culture solution was prepared by culturing for a time.

Next, 200 L of medium having the same composition is placed in a 300 L culture apparatus (manufactured by Maruhishi Bioengineer), autoclaved at 121 ° C. for 15 minutes, and 2 L of the seed culture obtained as described above is aseptic. The cells were inoculated and aerated and stirred at 200 rpm, 27 ° C., 40 L / min. The pH of the medium was controlled within the range of pH 4.2 to 4.5 using sodium hydroxide and hydrochloric acid. The turbidity after 96 hours was 23 OD at OD 660 nm, the polysaccharide concentration was 0.5% (w / v), and the substituted sulfoacetic acid content was 0.09%.
<Measurement of polysaccharide concentration>
The polysaccharide concentration was collected by sampling several ml of the culture solution, centrifuging and removing the cells, and then adding ethanol to the supernatant so that the final concentration was 66% (v / v) to precipitate the polysaccharide. Then, it melt | dissolved in ion-exchange water and quantified with the phenol sulfuric acid method.
<Measurement of substituted sulfo content>
Similarly, ethanol was added to the culture supernatant from which the cells had been removed so that the final concentration was 66%, and β-glucan was collected by precipitation. Thereafter, the sample was dissolved again in ion-exchanged water, centrifuged again, sodium chloride was added to the supernatant so that the final concentration was 0.9%, and β-glucan was again collected with 66% ethanol. This β-glucan recovery and purification operation was further repeated twice, and the resulting β-glucan aqueous solution was dialyzed against ion-exchanged water and then freeze-dried to obtain a β-glucan powder.

  This β-glucan powder was combusted and absorbed by the combustion tube and analyzed by ion chromatography. As a result, the S content was 239 mg / kg, and the substituted sulfoacetic acid content calculated from this value was 0.09%.

(1-2) Alkali treatment When the viscosity of the culture solution obtained as described above was measured at 30 ° C. and 12 rpm using a BM type rotational viscometer (manufactured by Tokyo Keiki), 1500 cP ((mPa · s) )Met. The rotor used for the measurement was selected appropriately according to the viscosity.

  When 25% (w / w) sodium hydroxide was added to this culture solution so that the final concentration of sodium hydroxide was 2.4% (w / v) and stirred (pH 13.6), the viscosity decreased instantaneously. did. Subsequently, after neutralizing with 50% (w / v) aqueous citric acid solution to pH 5.0, the viscosity at a concentration of 0.5 (w / v%) was measured. The viscosity at that time (30 ° C.) Was 20 cP ([mPa · s]).

  Next, 1 wt% of KC Flock (manufactured by Nippon Paper Industries Co., Ltd.) was added to the culture broth as a filter aid, and the cells were removed using a Kamata filter press (manufactured by Kamata Kikai). About 230 L). The polysaccharide concentration was 0.5% (w / v), and the recovery rate was almost 100%.

(1-3) Desalination of β-glucan aqueous solution After dilution of the above β-glucan aqueous solution (culture filtrate) to 0.3%, ultrafiltration (UF) membrane (molecular weight cut 50,000, manufactured by Nitto Denko Corporation) Was used, and finally the sodium ion concentration was lowered to 20 mg / 100 ml, and then the pH was adjusted to 3.5 with a 50% (w / v) aqueous citric acid solution.

Subsequently, sterilization was performed by holding at 95 ° C. for 3 minutes using a hot filling heating unit (manufactured by Nisaka Manufacturing Co., Ltd.) to obtain a final β-glucan aqueous solution. The concentration of β-glucan at this time was 0.22% (w / v) as measured by the phenol sulfuric acid method. The total yield from the culture was about 73%.
<Measurement of sulfur content>
Further, the obtained β-glucan aqueous solution was dialyzed with ion-exchanged water and then freeze-dried to obtain β-glucan powder. From the compositional analysis result of this β-glucan, the S content was 330 mg / kg, and the substituted sulfoacetic acid content calculated from this was 0.12%.
<Confirmation of combined state>
Moreover, since the wavelength shift from 480 nm to about 525 nm could be confirmed by the Congo red method, the above-described culture filtrate that had been desalted contained glucan containing β-1,3 bonds. Proven (K. Ogawa, Carbohydrate Research, 67, 527-535 (1978), supervised by Tadayuki Imanaka, Development of the use of microorganisms, 1012-1015, NTS (2002)). The shift difference to the maximum value at that time was Δ0.48 / 500 μg polysaccharide.

  15 ml of the culture filtrate was taken out, 30 ml of ethanol was added, and centrifuged at 4 ° C. and 1000 rpm for 10 minutes to collect the precipitated polysaccharide. Wash with 66% ethanol, centrifuge at 4 ° C, 1000 rpm for 10 minutes, add 2 ml of ion-exchanged water and 1 ml of 1N sodium hydroxide aqueous solution to the precipitated polysaccharide, stir, then heat at 60 ° C for 1 hour to precipitate. Dissolved. Next, after freezing at −80 ° C., vacuum lyophilization was performed overnight, and the dried powder was dissolved in 1 ml of 1N sodium hydroxide heavy aqueous solution and subjected to two-dimensional NMR.

¹ H NMR spectra with a correlation between the two-dimensional ^{NMR (13 C- 1 H COSY NMR} ) 106ppm shown in FIG. In this spectrum, two signals of 4.7 ppm and around 4.5 ppm were obtained.

As a result, it was proved that this β-glucan is β-1,3-1,6-D glucan (supervised by Tadayuki Imanaka, development of microbe utilization, 1012-1015, NTS (2002 )). From the integration ratio of each ¹ H NMR signal, the ratio of β-1,3 bond / β-1,6 bond was found to be 1.15.
<Particle size measurement>
Next, when the particle size of the culture solution was measured using a laser diffraction / scattering type particle size distribution measuring apparatus (LA-920 manufactured by HORIBA), the particles were about 0.3 μm and 100 μm in size. I was seen. Subsequently, when particle size measurement was performed while irradiating with ultrasonic waves, the 100 μm peak disappeared as soon as it was seen, the 0.3 μm peak increased, and finally only 0.3 μm. The particle size distribution of the culture solution when irradiated with ultrasonic waves is shown in FIG.

  The 0.3 μm peak is a peak due to primary particles of β-1,3-1,6-D-glucan, and the 100 to 200 μm peak is a primary particle of β-1,3-1,6-D-glucan. This is considered to be a peak due to aggregated secondary particles.

Further, it was confirmed that the secondary particles disappeared in the same manner even when stirred with a magnetic stirrer and lightly shaken, and easily broken into primary particles. Therefore, the secondary particles are considered to be very loosely aggregated (slowly aggregated state).
<Molecular weight measurement>
Further, Toyopearl HW65 manufactured by Tosoh Corporation (column size 75 cm × φ1 cm, excluded molecular weight 2.5 million (dextran)) was subjected to gel filtration chromatography using 0.1 M sodium hydroxide aqueous solution as an eluent, and dissolved β- When the molecular weight of a solution containing 1,3-1,6-D-glucan and β-1,3-1,6-D glucan primary particles was measured, the dissolved β-1,3-1,6- Two types of low molecular fractions having peaks of 2 to 300,000 derived from D-glucan and apparently high molecular fractions of 500 to 2.5 million derived from primary particles were detected. Shodex pullulan was used as a molecular weight marker.

  In order to separate the water-soluble β-1,3-1,6-D-glucan and fine particles, a β-1,3-1,6-D-glucan solution containing the fine particle fraction and the soluble fraction is used. When filtration was performed with a filter (0.2 μm) manufactured by Advantech, 500 to 2.5 million polymer fractions disappeared. From this, it was found that the polymer fraction corresponds to primary particles of β-1,3-1,6-D-glucan and secondary particles in which primary particles are aggregated. Therefore, the molecular weight of water-soluble β-1,3-1,6-D-glucan is considered to be 2 to 300,000.

(2) Preparation of powdered glucan In (1), β-1,3-1,6-containing fine particle β-1,3-1,6-D-glucan prepared by alkali treatment and bacterial cell removal treatment Ethanol was added to the D-glucan aqueous solution so that the final concentration was 66% (v / v) to precipitate polysaccharide glucan, which was collected by centrifugation. Subsequently, ethanol and water were removed by a freeze-drying method to obtain dry β-1,3-1,6-D-glucan. The yield at that time was 95% or more compared to the total sugar concentration before ethanol precipitation.

  Subsequently, the obtained dried β-1,3-1,6-D-glucan was dissolved and dispersed in water so that the final concentration was 0.3% (w / v), and then the same as described above. Gel chromatography was performed using Toyopearl HW65 (column size: 75 cm × φ1 cm, excluded molecular weight: 2.5 million (dextran)) manufactured by Toyopearl, and the molecular weight was measured. It was found that the molecular weight was composed of two types, a low molecular fraction having a peak of 2 to 300,000 and a high molecular fraction having an apparent appearance of 500 to 2.5 million. Here, a pullulan manufactured by Shodex was used as a molecular weight marker.

  On the other hand, in order to separate water-soluble β-1,3-1,6-D-glucan and fine particles, an aqueous solution of β-1,3-1,6-D-glucan prepared by this method (fine particles and solubilized glucan were mixed). When the product was filtered with a filter (0.2 μm) manufactured by Advantech, 500 to 2.5 million polymer fractions disappeared. Therefore, even if the β-1,3-1,6-D-glucan obtained by this method is dried, if it is redissolved, it is the same as β-1,3-1,6-D-glucan before drying. It was demonstrated to reproduce the physical behavior of

(3) Production of high purity β-1,3-1,6-D-glucan powder (1) 90 L of the culture solution (polysaccharide concentration 0.5% (5 mg / ml)) was added to 50% aqueous citric acid solution After neutralization with 9 kg, the cells were removed through a Kamata filter press 40D-4 pre-coated with 1.8 kg of filter aid (Nippon Paper Chemicals Powder Cellulose KC Flock). The filtrate was concentrated to 9 L with an ultrafiltration spiral element (NTU3150-S4 manufactured by Nitto Denko). While stirring this concentrated solution, 18 L of ethanol was added to obtain a glucan / ethanol / water slurry. The viscosity of the slurry was 22 mPa · s (30 ° C.) with a BM viscometer. The mixture was allowed to stand at room temperature for 3 hours, and about 17 L of the supernatant (ethanol / water) was removed. The viscosity of the remaining slurry was 45 mPa · s (30 ° C.). 10 L of this concentrated slurry was spray-dried using a Sakamoto Giken type spray dryer R-3 to obtain 360 g of β-1,3-1,6-D-glucan powder (recovery rate 80%). As a result of analyzing the NMR spectrum, the purity of the obtained β-1,3-1,6-D-glucan was 90% or more.
In addition, when the obtained β-1,3-1,6-D-glucan powder was dissolved in 1N sodium hydroxide heavy aqueous solution and the NMR spectrum was measured, the 1H NMR spectrum was about 4.7 ppm and about 4.5 ppm. Two signals were obtained. Moreover, the viscosity of the obtained β-1,3-1,6-D-glucan powder having a concentration of 0.5 (w / v%) was 200 cP or less (pH 5.0, 30 ° C.).

(4) Examination of stress relaxation effect
Improvement Effect on Restraint Stress in Mice It was confirmed as follows that low viscosity β-1,3-1,6-glucan suppresses stress induced by restraint.
1) As the substance to be used for reducing the viscosity of the substance , the high-purity β-1,3-1,6-glucan powder obtained in the item (3) above is used in ultrapure water, and the dosage is 25 mg, 50 mg, 100 mg. / Kg prepared was used.

2) Animal used Balb / c mice (male, 8 weeks old) were purchased from Nippon Claire Co., Ltd., pre-bred for 1 week, and then healthy mice were selected and used for experiments. This animal experiment is planned in accordance with the “Ehime University Animal Experiment Guidelines” and “Standards for the Breeding and Storage of Experimental Animals” (No. 6 of the Prime Minister's Notification in March 1980) and approved by the Animal Ethics Committee of Ehime University. Was carried out.

3) Restrained stress-loaded β-glucan was orally administered every morning for 7 days. Forced restraint was performed for 12 hours from 19:00 on the third, fifth, and seventh days of administration to 7:00 the next morning. For forced restraint, a 50 ml plastic tube with 100 or more vent holes was used, and care was taken not to be injured or painful when restrained. In addition to the restraint control group (water is administered orally every morning and enforced restraint), the control group that is not restrained and does not give food and water at night (fast food control) Group).

That is, the first group (normal group) was not restrained, and in addition to normal food and water intake, super sterilized water was orally administered once a day for 7 consecutive days.

Group 2 (Fast Food Control Group) was not restrained, but food and water were not given for 12 hours from 19:00 to 7:00 of the following morning on the 3rd, 5th and 7th days of administration. In addition, ultra-sterilized water was orally administered once a day for 7 consecutive days.

In the third group (restraint control group), forced restraint was performed for 12 hours from 19:00 to 7:00 on the next morning on the third, fifth and seventh days of administration. In addition, ultra-sterilized water was orally administered once a day for 7 consecutive days.

In the fourth group (β-glucan 25 mg / kg administration group), forced restraint was performed for 12 hours from 19:00 on the third day, the fifth day, and the seventh day to 7 o'clock in the next morning. In addition, β-glucan 25 mg / kg was orally administered once per day for 7 consecutive days per mouse individual.

In the fifth group (β-glucan 50 mg / kg administration group), forced restraint was performed for 12 hours from 19:00 on the third, fifth and seventh days of administration to 7:00 on the next morning. In addition, β-glucan 50 mg / kg was orally administered once per day for 7 consecutive days per mouse individual.

In the sixth group (β-glucan 100 mg / kg administration group), forced restraint was performed for 12 hours from 19:00 on the third day, the fifth day, and the seventh day of administration to 7 o'clock the next morning. In addition, β-glucan 100 mg / kg was orally administered once per day for 7 consecutive days per mouse.

On the 7th day (third time), the spleen was aseptically removed under ether anesthesia simultaneously with the restraint removal, and blood was collected from the inferior vena cava.

4) Measurement of blood corticosterone concentration The collected blood was centrifuged to separate plasma, and stored at -20 ° C or lower until measurement. The blood corticosterone concentration was measured using an EIA kit (Diagnostic Systems Laboratories, Inc., TX, USA).

5) Evaluation and measurement of spleen immune function The aseptically removed spleen was homogenized in a culture solution (RPMI 1640 containing 5% fetal bovine serum and antibiotics) and filtered through a nylon mesh to separate spleen cells. A part of the spleen cells was added to a multiwell plate, and concanavalin A (ConA) was added to 10 μg / ml, and cultured in a CO 2 incubator (37 ° C., 5% CO 2). After 48 hours, the culture supernatant was collected and stored at −20 ° C. or lower until measurement. Interleukin 6 (IL-6) and IL-12 in the culture were measured using ELISA kits (R & D Systems, Inc., MN, USA and Pierce Biotechnology, Inc., IL, USA), respectively.

  Lymphocytes were separated from the separated spleen cells using a lymphocyte separation reagent (CEDALANE Laboratories Ltd., Ontario, Canada). NK-sensitive mouse lymphoma cells YAC-1 were fluorescently labeled with BCSCF-AM (DOJINDO Laboratories, Kumamoto, Japan) as target cells. Mouse lymphocytes and YAC-1 cells were adjusted to 100: 1, added to the multiwell plate, and cultured for 2 hours in a CO2 incubator (37 ° C., 5% CO2). The culture supernatant was collected, the fluorescence intensity was measured, and the% of the total fluorescence intensity of the labeled YAC-1 cells was calculated and used as NK activity.

脾臓重量は平均値±標準誤差で示した（n=6）。
＊はP<0.05で拘束ストレスコントロール群との間に有意差あり
One-way ANOVA検定で有意な差異が認められたものについて、さらにFishers Protected LSDによる多重検定群間検定を行った。 6) Results
<Spleen weight>
The spleen weight of the fasting control group was significantly reduced compared to the normal group. Moreover, the spleen weight after repeated restraints was significantly reduced as compared with the normal group and the fasting control group. Β-glucan had no effect on the decrease in spleen weight due to restraint stress. (Table 2)

The spleen weight was expressed as mean ± standard error (n = 6).
* P <0.05 and there is a significant difference from the restraint stress control group
About the one-way ANOVA test by which the significant difference was recognized, the multiple test group test by Fishers Protected LSD was further performed.

<Blood corticosterone concentration>
The blood corticosterone concentration was significantly increased by restraint stress compared to the normal group. Moreover, the tendency which rises also by fasting was recognized. In contrast, in the β-glucan administration group, a tendency to suppress an increase in blood corticosterone concentration was observed, and in the β-glucan 50 mg / kg administration group, compared to the restrained control group and the fasting control group. A significant decrease in blood corticosterone concentration was observed. (Figure 1)

<Cytokine secretion capacity of spleen cells>
IL-12 and IL-6 secreted from spleen cells isolated from the removed spleen were significantly reduced by restraint stress. In contrast, the β-glucan administration group suppressed the decrease due to restraint stress of IL-12 and IL-6, and in the β-glucan 100 mg / kg administration group, the secretion amount of IL-12 was significant compared to the restraint control group Had risen to. However, this reduction-inhibiting effect did not reach the high secretion amount of the normal group and the fasting control group. (FIGS. 2 and 3)

  Concanavalin A stimulation increased IL-12 secretion from spleen cells by about 2-fold and IL-6 secretion by about 31-fold. IL-12 and IL-6 secretion from spleen cells stimulated with concanavalin A was significantly reduced by restraint stress. In contrast, administration of β-glucan (100 mg / kg) significantly suppressed the decrease in IL-12 and IL-6 due to restraint stress. (FIGS. 2 and 3)

NK活性は平均値±標準誤差で示した（n=6）。
エフェクター細胞/ターゲット細胞 ＝ 100 : 1
＊はP<0.05で拘束ストレスコントロール群との間に有意差あり
One-way ANOVA検定で有意な差異が認められたものについて、さらにFishers Protected LSDによる多重検定群間検定を行った。 <NK activity of spleen lymphocytes>
The NK activity of spleen lymphocytes was significantly reduced by restraint stress. Administration of β-glucan (50 mg / kg and 100 mg / kg) significantly suppressed the decrease in NK activity due to restraint stress. (Table 3)

NK activity was expressed as mean ± standard error (n = 6).
Effector cells / target cells = 100: 1
* P <0.05 and there is a significant difference from the restraint stress control group
About the one-way ANOVA test by which the significant difference was recognized, the multiple test group test by Fishers Protected LSD was further performed.

  As a result, it can be seen that the viscosity-reduced glucan has an antagonistic action against restraint stress in mice.

(5) Functional evaluation using electrophysiological measurement method for rat autonomic nervous system <br/> Using rats to study the action of β-glucan on autonomic nervous system (sympathetic and parasympathetic nerves) related to stress The activity of the sympathetic nerve that controls the adrenal gland and spleen and the parasympathetic (vagus) nerve that controls the stomach were measured electrophysiologically.
1) Dissolve the low-viscosity glucan used in the above item (3) by dissolving the high-purity β-1,3-1,6-glucan powder in water heated to 60 ° C and cooling to room temperature. Administered into the duodenum.

2) The animals used were Wistar male rats having a body weight of about 300 g and kept in a constant temperature animal room at 24 ° C. under a light / dark cycle every 12 hours (lighted from 8 o'clock to 20 o'clock) for one week or more. Food (Oriental yeast, MF) and water were fed ad libitum.

3) Electrophysiological measurement of the autonomic nervous system To examine autonomic nervous activity, the abdomen was urinated under urethane (1 g / kg, ip) anesthesia in the middle of the light period after 3 hours fasting, and the adrenal gland and spleen were controlled. The sympathetic nerve and the parasympathetic nerve governing the stomach were picked up with a silver electrode, and the nerve activity was measured by the method described above (see references 1-10). The tube was inserted into the trachea from the start of surgery to the end of measurement to secure the airway, and the body temperature (rat rectal temperature) was maintained at 35.0 ± 0.5 ° C. with a heat retaining device.
○ Reference 1-10
Reference 1: Yamano T. et al. Neurosci. Lett. 313: 78-82, 2001.
Reference 2: Niijima A. et al. Auton. Neurosci .: Basic & Clin. 97: 99-102, 2002.
Reference 3: Nagai K. et al. Exp. Biol. Med. (Maywood) 228: 1138-1145, 2003.
Reference 4: Shen J. et al. Neurosci. 380: 289-294, 2005.
Reference 5: Shen J. et al. Neurosci. 383: 188-193, 2005.
Reference 6: Tanida M. et al. Am. J. Physiol. 288: R447-455, 2005.
Reference 7: Tanida M, et al. Brain Res. 1058: 44-55, 2005.
Reference 8: Tanida M. et al. Neurosci. Lett. 398: 102-106, 2006.
Reference 9: Tanida M. et al. Neurosci. Lett. 389: 109-114, 2005.
Reference 10: Yamano T. et al. Life Sci. (In press) 2006.

  For intraduodenal administration, a polyethylene tube inserted into the duodenum was used, the dose per animal was 1 ml, and the administration rate was 1 ml / min. Regarding the administration effect, 10 ng to 10 mg of β-glucan was dissolved in 1 ml of water and administered into the duodenum, and the activity of the autonomic nerve was measured electrophysiologically to measure the increase or decrease of the activity. As a control experiment, 1 ml of water as a solvent was administered into the duodenum under the same conditions. Data was analyzed by the average value of power generation frequency (pulse / 5 sec) per 5 seconds every 5 minutes, expressed as a percentage with the value before administration as 100%, and expressed as an average value ± standard error. Statistical calculations were performed using ANOVA with repeated measures and Mann-Whitney U-test.

4) Results
<Determination of β-glucan dosage for measurement of adrenal sympathetic nerve activity>
Actual data obtained by measuring 10 ng to 10 mg of β-glucan in the duodenum and measuring adrenal sympathetic nerve activity (ASNA), and average activity every 5 minutes with the pre-dose value as 100% It is expressed as a quantity. Adrenal sympathetic nerve activity decreased at doses ranging from 1 μg (= 1000 ng) to 10 mg, and the most marked decrease by 35 minutes after administration was when 10 μg of β-glucan was administered. Therefore, we examined changes in the activity of the adrenal sympathetic nerve when 10 μg, which was the most effective, was administered into the duodenum.

<Effect of β-glucan intraduodenal administration on adrenal sympathetic nerve activity>
Changes in actual adrenal sympathetic nerve activity were examined when 10 μg of β-glucan and water, which was a solvent used as a control experiment, were administered into the duodenum, respectively. Almost no change was observed in the nerve activity when the control water was administered, but the adrenal sympathetic nerve activity was significantly decreased by intraduodenal administration of 10 μg β-glucan. These data are shown in FIG. 4 as the average activity amount every 5 minutes with the neural activity before administration as 100%.

The adrenal sympathetic nerve activity (ASNA) hardly changes in the water-treated control animals, whereas in the 10 μg β-glucan group, the activity decreases rapidly from 5 to 15 minutes after administration, and then about 30 to 40 Stayed at the% level. When the values of both groups were analyzed by the analysis of variance (ANOVA with repeated measures) as a group from 5 minutes to 60 minutes, a significant difference (p <0.0005; F = 364) was found between the two values. The values of adrenal sympathetic nerve activity before administration were 102.5 ± 2.6 spikes / 5 sec and 132.7 ± 15.7 spikes / 5 sec in the water administration group and β-glucan administration group, respectively. Not recognized (p = 0.4 by Mann-Whitney U-test).

<Determination of β-glucan dosage for gastric parasympathetic (vagus) nerve activity measurement>
Changes in actual gastric sympathetic nerve activity (GVNA) were measured when β-glucan in an amount of 0.1 μg to 10 μg was administered into the duodenum. Administration of 0.1 μg β-glucan slightly reduced gastric parasympathetic nerve activity, whereas administration of 1 μg and 10 μg β-glucan significantly increased gastric parasympathetic nerve activity. Therefore, the effect of 1 μg β-glucan, which was the most effective of these, on the gastric parasympathetic nerve activity was examined using 3 rats each with water administration.

<Effects of intraduodenal administration of β-glucan on gastric parasympathetic (vagus) nerve activity>
Changes in actual gastric parasympathetic (vagus) nerve activity were examined when 1 μg of β-glucan and water, which was a solvent used as a control experiment, were each administered intraduodenum. FIG. 5 shows the actual measurement data at that time and the average activity amount every 5 minutes with the neural activity before administration as 100%.

Control water administration hardly changed the gastric parasympathetic nerve activity, but 1 μg β-glucan administration into the duodenum was significantly higher than that after water administration (P <0.0005, F = 22.9). Was raised. The value of gastric sympathetic nerve activity before administration was 112.3 ± 28.3 spikes / 5sec and 129.4 ± 41.1 spikes / 5sec in the water administration group and β-glucan administration group, respectively, and there was no statistically significant difference. (In Mann-Whitney U-test, p = 0.4).

<Determination of β-glucan dosage for splenic sympathetic nerve activity>
Changes in splenic sympathetic nerve activity (SSNA) after intraduodenal administration of 0.1 μg to 10 μg β-glucan were measured. Depending on the doses of 0.1 μg, 1 μg, and 10 μg, β-glucan suppressed splenic sympathetic nerve activity. Thus, the effect of intraduodenal administration of 10 μg β-glucan and water on splenic sympathetic nerve activity was examined using three rats.

<Effect of intraduodenal administration of β-glucan on splenic sympathetic nerve activity>
Changes in actual splenic sympathetic nerve activity were examined when 10 μg of β-glucan and water, which was a solvent used as a control experiment, were each administered intraduodenum. The result is shown in FIG.

Administration of control water slightly increased splenic sympathetic nerve activity, but administration of 10 μg β-glucan significantly increased splenic sympathetic nerve activity (P <0.0005, F = 68.9) compared to after water administration. The splenic sympathetic nerve activity before administration was 116.0 ± 17.9 spikes / 5sec and 116.2 ± 16.6 spikes / 5sec in the water administration group and β-glucan administration group, respectively, and no statistically significant difference was observed ( P = 1) at Mann-Whitney U-test.

  These results indicate that 0.1 to 10 μg of β-glucan suppresses the activity of sympathetic nerves governing the adrenal gland and spleen and promotes the activity of gastric parasympathetic nerves.

  The above facts indicate that β-glucan suppresses sympathetic nerve activity governing the adrenal gland and spleen, promotes gastric parasympathetic nerve activity, lowers blood sugar, lowers blood pressure, promotes digestion and absorption in the intestine, promotes eating, It suggests that it has the effect of promoting immunity and improving constipation by causing increased NK activity. Inhibition of splenic sympathetic nerve activity predominates the lymphocyte Th1 (T helper 1) cell line, and is expected to reduce allergic reactions such as hay fever.

(6) Formulation example of food and beverage composition Formulation example 1 (cookie)
Powder β-1,3-1,6-D-glucan 1% by weight
Bactericidal lactic acid bacteria powder 0.2% by weight
Catechin 1% by weight
Cookies

Formulation Example 2 (Supplement)
Powder β-1,3-1,6-D-glucan 10% by weight
Collagen peptide 42% by weight
Hyaluronic acid 0.06% by weight
Bactericidal lactic acid bacteria powder 1% by weight
Vitamin C 10% by weight
Vitamin B2 0.03% by weight
Vitamin B6 0.03% by weight
Excipient (such as starch)

Formulation Example 3 (Supplement)
Powder β-1,3-1,6-D-glucan 1% by weight
Collagen peptide 42% by weight
Hyaluronic acid 0.06% by weight
Vitamin C 10% by weight
Vitamin B2 0.03% by weight
Vitamin B6 0.03% by weight
Niacin 0.15% by weight
Excipient (such as starch)

Formulation Example 4 (Drink)
β-1,3-1,6-D-glucan aqueous solution (0.2 wt% β-glucan aqueous solution) 61.5 wt%
Bactericidal lactic acid bacteria powder 0.03% by weight
Milk oligosaccharide 0.8% by weight
Lactoferrin 0.09% by weight
Sweetener (sucralose) 0.03% by weight
Citric acid 0.22% by weight
Fragrance 0.37% by weight
Water balance

Formulation Example 5 (Drink)
β-1,3-1,6-D-glucan aqueous solution (0.2 wt% β-glucan aqueous solution) 61.5 wt%
Bactericidal lactic acid bacteria powder 0.03% by weight
Theanine 0.8% by weight
GABA 0.09 wt%
Sweetener (sucralose) 0.03% by weight
Citric acid 0.22% by weight
Fragrance 0.37% by weight
Water balance

Formulation Example 6 (Drink)
Powder β-1,3-1,6-D-glucan (derived from genus Aureobasidium) 0.2% by weight
Safflower extract 7%
Ginkgo biloba extract 7%
Ginseng extract 7%
Pomegranate extract 1%
Amakusa extract 3.5%
Cinnamon extract 3.5%
Chen peel extract 3.5%
Turmeric extract 2.1%
Ginger extract 1%
Honey 3%
Water balance

Figure 3 shows the effect of β-glucan on plasma corticosterone concentrations in Balb / c mice exposed to restraint stress. The plasma corticosterone concentration was expressed as mean ± standard error (n = 6). * P <0.05. Significant difference between restraint stress control group and one-way ANOVA test. Significant difference was observed by one-way ANOVA test. Figure 3 shows the effect of β-glucan on IL-12 secretion from the spleen of Balb / c mice exposed to restraint stress. Spleen cells were cultured in RPMI 1640 containing 5% FBS for 48 hours with or without concanavalin A (final conc. 10 μg / ml). * P <0.05. Significant difference between restraint stress control group and one-way ANOVA test. Significant difference was observed by one-way ANOVA test. Figure 6 shows the effect of β-glucan on IL-6 secretion from spleens of Balb / c mice exposed to restraint stress. Spleen cells were cultured in RPMI 1640 containing 5% FBS for 48 hours with or without concanavalin A (final conc. 10 μg / ml). * P <0.05. Significant difference between restraint stress control group and one-way ANOVA test. Significant difference was observed by one-way ANOVA test. The change of the adrenal sympathetic nerve activity (ASNA) by the duodenum administration of Daiso beta-glucan (10ug / ml) is shown. The change of the gastric parasympathetic nerve activity (GVNA) by the duodenum administration of Daiso beta-glucan (1ug / ml) is shown. The change of the splenic sympathetic nerve activity (SSNA) by the duodenum administration of Daiso beta-glucan (10ug / ml) is shown. NMR spectrum is shown. Attribution: ¹ H-NMR peak near 4.5 ppm: 1-position hydrogen (β1 → 6 bond) 4.4729 ppm ¹ H-NMR peak near 4.7 ppm: 1-position hydrogen (β1 → 3 bond) 4 7258ppm The particle size distribution after ultrasonic irradiation is shown.

Claims (1)

The following properties (1) to (3):
(1) derived from Aureobasidium pullulans GM-NH-1A1 strain or Aureobasidium pullulans GM-NH-1A2 strain;
(2) ¹ H NMR spectrum of a solution using 1N sodium hydroxide heavy aqueous solution as a solvent has two signals of about 4.7 ppm and about 4.5 ppm;
(3) The viscosity of the aqueous solution at 30 ° C., pH 5.0, concentration 0.5 (w / v%) is 200 cP (mPa · s) or less.
A blood corticosterone lowering agent in a stress state, which comprises β-1,3-1,6-D-glucan having the formula:

Images