JP6329125B2 - Stress relieving agent - Google Patents

Description

  The present invention relates to a stress relieving agent containing Lactobacillus gasseri as an active ingredient. The present invention also relates to a stress relieving agent further comprising α-lactalbumin (αLA) as an active ingredient.

  In recent years, there has been a problem of chronic (accumulation) stress due to a mild stress load over a long period of time or a short-term stress not being sufficiently resolved. Even if these chronic stresses are accumulated at a low intensity, they have an effect on the mind and body that is higher than that of a high-intensity, short-term stress, and death due to overwork caused by chronic stress has become a social problem.

  Stress is defined as a biological response to a stimulus that impairs homeostasis. The cause of stress is called a stressor. From the types of external stimuli, physical stressors (cold, noise, radiation, etc.), chemical stressors (oxygen, drugs, etc.), biological stressors (inflammation, infection), psychological stressors (Anger, anxiety, etc.)

  There are two main types of systems that transmit commands from the brain to the body to deal with stressors. One is the hypothalamic-sympathetic-adrenal-medullary axis (SAM axis), known as an emergency response to stress. The other is the hypothalamic-pituitary-adrenal cortex (Hypothalmo-Pituitary-Adrenal axis, HPA axis), which is known as a nonspecific stress response to any external demand.

  The HPA axis, which is a non-specific stress response, first secretes corticotropin-releasing hormone (CRH) from the hypothalamus. CRH secretes Adrenocortico tropic hormone (ACTH) from the pituitary gland. ACTH promotes secretion of glucocorticoids such as cortisol (mainly corticosterone in rats) from the adrenal cortex. This glucocorticoid is generally called a stress hormone.

  Many of the actions of glucocorticoids are involved in physical responses to stress (activation of energy metabolism, promotion of cardiac function) and play an important role in maintaining homeostasis. On the other hand, glucocorticoids are also known to adversely affect the living body, such as reducing the proliferation ability and function of immune cells. For example, persistent secretion of glucocorticoids due to chronic stress causes secondary symptoms such as decreased ability to defend against infection (decreased immune function), emotional disorders such as depression and anxiety, and sleep disorders such as insomnia. .

In recent years, “brain-intestinal correlation” has attracted attention. In the brain-intestinal correlation, various signals generated in the intestine are transmitted to the central nervous system via the nervous system, common information transmitters and receptors such as hormones and cytokines, and affect the information processing process. (Non-patent Document 1). In other words, it is conceivable that stress sensitivity is reduced and stress response is reduced by stimulation from the intestine by intestinal bacteria or probiotics.
The intestine is governed by the brain by autonomic nerves, and when the brain recognizes stress, the information is transmitted to the intestine by information transmission substances such as autonomic nerves, endocrine hormones, and cytokines, causing abnormal peristalsis. On the other hand, when symptoms such as constipation or diarrhea occur in the intestine, it is transmitted to the brain as stress. This bidirectional information transmission mechanism between the brain and the intestine is called “brain-intestinal correlation” (Non-patent Document 1). In recent years, in this brain-intestine correlation, it is becoming clear that intestinal bacteria influence signal transmission from the intestine to the brain. From this, it is considered that stress sensitivity is attenuated and stress response is reduced by stimulation from the intestine with probiotics such as lactic acid bacteria.

  For example, Non-Patent Document 2 discloses that when Lactobacillus johnsonii La1 is administered to rats, the activity of parasympathetic nerves governing the functions of the stomach and gastrointestinal tract is enhanced. Further, in Patent Document 1, Lactobacillus paracatheis, subspices, paracasei lactic acid bacteria FERM-19169 suppresses excessive production of cytokines such as TNF-α, IFN-γ, and IL-6, thereby effectively preventing pathogenic infection. It is disclosed to suppress stress by protecting and suppressing excessive inflammatory reactions and minimizing wear due to diseases and the like. Furthermore, Patent Document 2 describes the effect that acid milk obtained by fermenting animal milk with Lactobacillus lactic acid bacteria including Lactobacillus helveticus ATCC8205 has an effect of preventing and reducing mental and physical symptoms caused by stress. It is disclosed to have.

  However, Non-Patent Document 2 is an effort for acute stress such as gastrointestinal disorders and inflammatory reactions, and Patent Document 1 is examining psychological and physical symptoms but has confirmed the effect on temporary stress. It is unclear whether it is effective enough for chronic stress, which is a problem in modern society.

  The inventors have previously found that Lactobacillus gasseri OLL2809, a probiotic lactic acid bacterium, has an effect of stimulating the immune response of the living body, such as strongly inducing IL-12 from mouse splenocytes. . This lactic acid bacterium acts on the immune response mechanism of the host and is effective in reducing immediate allergic disease (Patent Document 3), delayed hypersensitivity reaction (Patent Document 4), and endometriosis (Patent Document 5). It is clear that there is. IL-12 has the effect of inducing type I helper T cells (Th1) and enhancing the cytotoxic activity of NK cells (Patent Document 3). NK cells play an important role in innate immune mechanisms such as protection from viral infection and elimination of tumor cells.

  In addition, the inventors have clarified that αLA has an action of inhibiting the activity of cyclooxygenase-2 to suppress the production of prostaglandins from arachidonic acid and also to suppress the production of IL-6. Yes. From these effects, it has been clarified that αLA reduces menstrual pain caused by inflammation and contributes to improving QOL of healthy women at reproductive age (Patent Document 6). Prostaglandins are one of the inflammatory mediators and exert various inflammatory symptoms such as pain.

  However, the above-mentioned Lactobacillus gasseri OLL2809 and αLA are effective in reducing the immune function caused by the influence of glucocorticoids due to chronic stress (chronic stress), psychological stress such as anxiety and fatigue, etc. There is no mention of effects under stressed conditions.

JP 2010-96751 A Japanese Patent Laid-Open No. 10-045610 International Publication WO2006 / 093022 Pamphlet International Publication WO2012 / 086150 Pamphlet International Publication No. WO2012 / 073924 Pamphlet International Publication WO2008 / 140041 Pamphlet

Aziz Q, Thompson DG., Gastroenterol, 114: 559-578 (1998). Tanida M, Yamano T, Maeda K, Okumura N, Fukushima Y, Nagai K., Neurosci Lett, 389: 109-114 (2005).

  Thus, known stress suppressors are known for stimulating the immune system and improving gastrointestinal disorders, but are effective against various stresses, particularly those associated with chronic stress. It is not known at all how it works. Therefore, the present invention regulates physical and mental health by suppressing the excessive response of the HPA axis and decline in immune function due to chronic stress, and also reducing psychological stress represented by fatigue, anxiety and tension. It is an object of the present invention to provide a stress relieving agent that has no side effects as a medicine, can be used regularly, and can be easily taken as a food or drink.

  The present invention, which has not been known to Lactobacillus gasseri, suppresses the excessive response of the HPA axis caused by chronic stress and the decline of immune function, and reduces psychological stress such as anxiety and tension And found useful as a stress reducing agent. In addition, by using αLA in combination with Lactobacillus gasseri, we found that it reduces daily fatigue, which is one of the symptoms of chronic stress, and found that it is useful as a stress-reducing agent. It was.

The basic features and various aspects of the present invention are listed below.
[1] A stress reducing agent comprising Lactobacillus gasseri as an active ingredient.
[2] The agent according to [1], wherein the stress is chronic stress.
[3] The agent according to [1] or [2], further comprising α-lactalbumin (αLA) as an active ingredient.
[4] Lactobacillus gasseri having a stress reducing effect.
[5] The Lactobacillus gasseri according to [4], which has an action of suppressing an increase in blood stress hormone.
[6] The Lactobacillus gasseri according to [4] or [5], which has an action of suppressing an increase in blood corticosteroids and / or blood glucocorticoids.
[7] A composition containing 1 × 10 ⁷ or more of Lactobacillus gasseri and 40% by weight or more of αLA.
[8] The agent according to any one of [1] to [3], wherein Lactobacillus gasseri is the Lactobacillus gasseri OLL2809 strain deposited under the deposit number NITE BP-72.
[9] Lactobacillus gasseri according to any one of [4] to [6], deposited under the deposit number NITE BP-72.
[10] The composition according to [7], wherein the Lactobacillus gasseri is Lactobacillus gasseri OLL2809 strain deposited under accession number NITE BP-72.
[11] A stress reducing method using Lactobacillus gasseri.
[12] The method according to [11], wherein the stress is chronic stress.
[13] The method according to [11] or [12], further using αLA.
[14] The method according to any one of [11] to [13], wherein the Lactobacillus gasseri is Lactobacillus gasseri OLL2809 strain deposited under accession number NITE BP-72.

  Until now, the present inventors have repeatedly examined the effects of Lactobacillus gasseri or αLA. As a result, the present inventors conducted a submerged stress load test in rats administered with Lactobacillus gasseri OLL2809, and administration of Lactobacillus gasseri OLL2809 slowed the increase in stress hormones such as corticosterone in the blood. Thus, it was found that the decrease in NK activity due to stress load was suppressed. In addition, Lactobacillus gasseri OLL2809 is able to suppress NK activity decline due to intense exercise and reduce psychological stress such as anxiety and tension through a test with athletes who are doing intense exercise on a daily basis. I found it.

  Furthermore, the present inventor has found that daily fatigue can be alleviated by the combined use of OLL2809 and αLA through a test using athletic athletes who perform intense exercise on a daily basis. This effect was objectively verified by a significant reduction in serum derivatives of reactive oxygen metabolites (dROMs) and transforming growth factor (TGF) -β1 levels. The above-described effects have been found to be useful for adjusting physical condition so that, for example, athletes can exhibit excellent athletic performance.

It is a graph which shows the stress hormone (ACTH, corticosterone) density | concentration in the blood after a rat water immersion stress load test. Results are expressed as mean + standard deviation. White, gray, and black bars represent the normal group, the stress group, and the Lactobacillus gasseri OLL2809 group, respectively. Evaluation was performed on the 14th day of the test. a and b represent significant differences between different signs (One-way ANOVA, Fisher's PLSD test, P <0.05). It is a graph which shows the oxidative stress marker (8-OHdG) density | concentration in the blood after a rat immersion stress load test. Results are expressed as mean + standard deviation. White, gray, and black bars represent the normal group, the stress group, and the Lactobacillus gasseri OLL2809 group, respectively. Evaluation was performed on the 14th day of the test. a and b indicate that there is a significant difference between different signs (One-way ANOVA, Fisher's PLSD test, P <0.05). It is a graph which shows spleen NK activity after a rat water stress stress test. Results are expressed as mean + standard deviation. White, gray, and black bars represent the normal group, the stress group, and the Lactobacillus gasseri OLL2809 group, respectively. Evaluation was performed on the 14th day of the test. a and b represent significant differences between different signs (One-way ANOVA, Fisher's PLSD test, P <0.05). It is a graph which shows the effect of Lactobacillus gasseri OLL2809 and (alpha) LA in Profiles of Mood States (POMS) which evaluates a mood and an emotional state. The black dots in the graph indicate the results of each subject, and the bars in the graph indicate the average value. The evaluation was performed before the test substance intake and after the intake for 4 weeks. #, *: P <0.1, 0.05. There is a significant difference when compared before and after 4 weeks of test substance intake (Two-way rm ANOVA, Student's paired t test (Bonferroni correction)). It is a graph which shows the effect of Lactobacillus gasseri OLL2809 and (alpha) LA with respect to the feeling of fatigue before and behind exercise load using the visual analog scale (Visual analogue scale (VAS) method. Results are expressed as mean ± standard deviation. White, gray, and black bars represent placebo, OLL2809 only, and OLL2809 + αLA, respectively. The evaluation was performed before and after exercise before taking the test substance and before and after exercise after taking for 4 weeks (Student's paired t test). It is a graph which shows the effect of Lactobacillus gasseri OLL2809 and (alpha) LA with respect to the NK cytotoxic activity fall by 1-hour bicycle ergometer exercise | movement. Results are expressed as mean ± standard deviation. White and black bars represent before and after exercise, respectively. *: P <0.05, significant difference when comparing before and after exercise (Two-way rm ANOVA, Student's paired t test (Bonferroni correction)). n.s .: No significant difference.

  In the present invention, stress refers to a stressor, such as a physical stressor (coldness, noise, radiation, etc.), a chemical stressor (oxygen, a drug, etc.), a biological stressor (inflammation, infection), a psychological stressor (anger, anxiety, etc.). This refers to the phenomenon of biological tension caused by such factors.

  The stress reducing agent of the present invention can be used to relieve stress. The stress relieving agent of the present invention has no side effects as a medicine, can be used regularly, and can be easily ingested as a food or drink.

  The stress relieving agent of the present invention can suppress, for example, a decrease in immune function due to stress. In particular, the stress relieving agent of the present invention has a reducing effect on acute stress and chronic stress by the stressor described above, but can be preferably used in the case of chronic stress in which stress persists. When the stress reducing agent of the present invention is used in the case of chronic stress, for example, NK activity caused by glucocorticoids such as cortisol, corticosterone, and cortisone that are secreted continuously in response to chronic stress. The decrease can be suppressed, and psychological fatigue is also reduced.

  The stress relieving agent of the present invention can improve or prevent secondary symptoms during chronic stress, such as a decrease in immune function, an increase in psychological stress, a delay in wound healing, or a metabolic disorder.

  The stress relieving agent of the present invention can alleviate poor physical condition caused by stress. The stress reducing agent of the present invention can reduce, for example, mental stress (psychological stress) such as anxiety, tension, and fatigue, daily fatigue (chronic fatigue), or chronic fatigue syndrome. Furthermore, the stress reducing agent of the present invention suppresses an increase in stress hormone caused by stress stimulation such as intense exercise, reduces an increase in oxidative stress markers, suppresses a decrease in immune function due to stress load, and improves physical condition. Is to prepare. Therefore, the stress reducing agent of the present invention can also be used for the purpose of maintaining homeostasis (homeostasis), conditioning and physical condition adjustment. As an example, the stress relieving agent of the present invention provides an enhanced immune response and reduced physical fatigue in order for athletes to exhibit superior athletic performance or to prevent or reduce overtraining, It can be used as a sports conditioning agent.

  The stress relieving agent of the present invention contains Lactobacillus gasseri, or Lactobacillus gasseri and αLA as active ingredients. The stress reducing agent of the present invention contains Lactobacillus gasseri as lactic acid bacteria. Any Lactobacillus gasseri can be used as long as it has a stress-reducing effect and a chronic fatigue-reducing effect.

  In the present invention, Lactobacillus gasseri that can be particularly preferably used is Lactobacillus gasseri OLL2809, which was independently isolated from human adult feces. The strain was founded on February 1, 2005 (original deposit date) at the Japan Institute for Product Evaluation Technology Patent Microorganism Depositary Center (Kazusa-Kamazu 2-5-8, Kisarazu City, Chiba Prefecture 292-0818, Japan) As NITE P-72, on January 18, 2006, it was transferred from the original deposit to a deposit under the Budapest Treaty, and the deposit number was deposited as NITE BP-72.

  As a medium for culturing Lactobacillus gasseri which is an active ingredient of the stress relieving agent of the present invention, a medium usually used for a medium of lactic acid bacteria is used. That is, any medium can be used as long as it contains a nitrogen source, an inorganic substance and other nutrients in addition to the main carbon source. As the carbon source, lactose, glucose, sucrose, fructose, starch hydrolyzate, waste molasses and the like can be used according to the assimilation ability of the bacteria used. As the nitrogen source, organic nitrogen-containing substances such as casein hydrolyzate, whey protein hydrolyzate, and soy protein hydrolyzate can be used. In addition, meat extract, fish extract, yeast extract and the like are used as growth promoters.

  Cultivation of Lactobacillus gasseri, which is an active ingredient of the stress reducing agent of the present invention, is desirably performed under anaerobic conditions, but may be performed under microaerobic conditions such as liquid stationary culture that is usually used. For anaerobic culture, a known method such as a method of culturing under a nitrogen gas layer can be applied, but other methods may be used. The culture temperature is generally preferably 30 to 40 ° C., but may be other temperature conditions as long as Lactobacillus gasseri grows. The pH of the medium during culture is preferably maintained at 4.0 to 7.0, but may be other pH conditions as long as Lactobacillus gasseri grows. It can also be cultured under batch culture conditions. The culture time is usually preferably 10 to 24 hours, but may be other culture time as long as Lactobacillus gasseri can grow.

  The Lactobacillus gasseri, which is an active ingredient of the stress reducing agent of the present invention, may be a live cell or a dead cell, and may be a wet cell or a dry cell. In the present specification, “Lactobacillus gasseri” means both Lactobacillus gasseri strains and cells.

Lactobacillus gasseri which is an active ingredient of the stress reducing agent of the present invention may be a “processed product”. In the present invention, the “processed product” of Lactobacillus gasseri is not limited, but, for example, a lactobacillus gasseri suspension (suspension) after completion of the culture, a culture in which the culture solution is used as it is. (Including bacterial cells, culture supernatant and medium components), fermented products (fermented raw milk, skim milk powder or soy milk, etc.), heat-treated products, sterilized or sterilized products by heat, radiation, etc. Products, crushed materials by ultrasonic waves, concentrates, pasted products, dried products (spray dried products, freeze dried products, vacuum dried products, drum dried products, etc.), liquid products, diluted products, and the like. The cell concentration of Lactobacillus gasseri, which is an active ingredient of the stress relieving agent of the present invention, is not particularly limited, but it is 1 × 10 ⁶ cells / g or more in the concentrate and 1 × 10 ⁷ cells / g or more in the dried product. Is preferred.

  The stress relieving agent of the present invention contains αLA as an active ingredient in addition to Lactobacillus gasseri. The αLA of the present invention is not particularly limited in origin, but is preferably derived from a human and / or non-human mammal (such as bovine, sheep, goat, horse, etc.), more preferably human or bovine αLA. The amino acid sequences and base sequences of these αLA are already known and are registered in databases such as EMBL, DDBJ, NCBI. For example, bovine αLA is registered as ACCESSION No. J05147 with DDBJ.

  αLA is derived from the milk of mammals such as humans, cows, sheep, and goats, by ammonium sulfate precipitation ("Latest Revised Dairy Technology Handbook" Dairy Technology Promotion Society, pages 122-125, 1975), ultrafiltration 268879), ion exchange method (Japanese Patent No. 2916047) and the like. The content of αLA in milk varies somewhat depending on the production area, feed, etc., but domestic milk is usually 1.2 g / kg. If milk is used as a raw material for production, a large amount of αLA can be prepared. Moreover, based on an amino acid sequence and a base sequence, you may synthesize | combine chemically or you may manufacture by the genetic engineering method well-known to those skilled in the art. More simply, commercially available αLA (for example, manufactured by Davisco) can be used.

  The stress relieving agent of the present invention may be a drug or food for humans or animals, or may be a material or preparation used by blending with the drug or food. Here, the food has the concept of relieving chronic stress, recovering from physical / psychological fatigue, maintaining homeostasis (homeostasis), conditioning and physical condition, and displays that effect when necessary. Functional foods, foods for the sick, and foods for specified health use.

  As an administration form when the stress relieving agent of the present invention is a pharmaceutical, a dosage form can be selected according to its purpose, administration route, etc., for example, tablets, coated tablets, pills, capsules, granules, Examples include powders, liquids, suspensions, emulsions, syrups, injections, suppositories, soaking agents, decoction, tinctures, etc., but used as parenteral agents such as oral preparations and nutrients by tube feeding It is preferable to do this.

  These various preparations are prepared according to conventional methods as necessary with respect to the active ingredient, such as fillers, extenders, excipients, binders, humectants, disintegrants, surfactants, lubricants, coloring agents, flavoring agents. The pharmaceutical composition can be formulated using known adjuvants that can be usually used in the pharmaceutical preparation technical field, such as solubilizing agents, suspension agents, and coating agents. Moreover, you may contain a coloring agent, a preservative, a fragrance | flavor, a flavoring agent, a sweetening agent, etc. and other pharmaceuticals in this pharmaceutical formulation.

  The stress relieving agent of the present invention is a compound or microorganism that is orally administered alone or mixed with other components that can be usually used in pharmaceuticals and foods, or has other immune response enhancing effects and physical fatigue reducing effects. Is effective in enhancing the immune response and reducing physical fatigue in humans and animals. For example, anxiolytics (eg chlordiazepoxide, oxazolam or diazepam), hypnotics (eg nitrazepam, estazolam or haloxazolam) or vitamins (eg vitamin A, vitamin B1, vitamin B2, vitamin B6, niacinamide, pantothenic acid) , Vitamin B12, vitamin E, biotin, vitamin C, etc.) and the like.

  Since the stress relieving agent according to the present invention has no side effects and can be used regularly, it can be used not only as a medicine but also as a food and drink. Specifically, as foods and drinks, milk, soft drinks, milk drinks, fermented milk, yogurt, cheese, ice cream, bread, biscuits, crackers, confectionery, tablet confectionery, pizza crust, prepared milk powder, liquid food, supplements, beauty Foods, health foods, foods for the sick, foods such as infant milk powder, foods such as infant milk powder, nutritional foods, sports foods, etc. It is also possible to provide the stress reducing agent according to the present invention in any of the above states, for example, solid (powder, granule, etc.), paste, gel, jelly, liquid or suspension.

  Such food can be produced in accordance with a conventional method for ordinary food and beverage compositions such as using Lactobacillus gasseri of the present invention, αLA as it is, or mixing with other foods or food ingredients.

  Examples of other food ingredients include, but are not limited to, water, proteins, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, and flavors.

Here, examples of the protein include whole milk powder, skim milk powder, partially skim milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β -Animal and vegetable proteins such as lactoglobulin, lactoferrin, soy protein, chicken egg protein, meat protein, and hydrolysates thereof.

  Examples of the saccharide include saccharides, processed starch (in addition to dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like.

  Examples of lipids include lard, fish oil, etc., fractionated oils thereof, hydrogenated oils, animal oils such as transesterified oils, palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, Examples include vegetable oils such as hydrogenated oils and transesterified oils.

  Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. And folic acid.

  Examples of the minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey mineral.

  Examples of the organic acid include malic acid, citric acid, lactic acid, and tartaric acid.

  Two or more of these components can be used in combination, and a synthetic product and / or a food containing a large amount thereof may be used.

The intake amount of the stress relieving agent of the present invention varies depending on the sex, age, health condition, etc. of the person who takes it. 1 × 10 ⁷ or more per day, preferably 1 × 10 ⁸ or more, more preferably 1 × 10 ⁹ or more, particularly preferably 1 × 10 ¹⁰ or more, more preferably 1 × 10 ¹¹ The above is mentioned. Further, αLA as an active ingredient is 100 mg or more per day, preferably 250 mg or more, more preferably 500 mg or more, and particularly preferably 1000 mg or more. The upper limit of intake of these stress relieving agents is not particularly limited because there are Lactobacillus gasseri and αLA, which are active ingredients, as general food ingredients. For example, Lactobacillus gasseri OLL2809 cells include 1 × 10 ¹⁵ cells per day, and αLA includes 2000 mg / kg per day. Moreover, the frequency | count of ingestion may be once per day, or may be multiple times. In the present specification, the term “intake” includes “administration”.

  The stress relieving agent of the present invention can be administered to a patient in need of treatment with the composition of the present invention before meals, after meals, between meals and / or before going to bed at one time or divided. The dosage can also be determined individually depending on the age, weight and purpose of administration of the patient being administered.

In addition, Lactobacillus gasseri, or Lactobacillus gasseri and αLA of the present invention are administered to, eaten or fed to mammals, a method for adjusting physical condition, reduction of chronic stress or Provided are a method for alleviating, an increase in blood stress hormones caused by chronic stress, and a method for alleviating or reducing psychological stress due to chronic stress.
The effective amount of the Lactobacillus gasseri, or Lactobacillus gasseri and αLA means the same amount as the intake of the stress relieving agent Lactobacillus gasseri or Lactobacillus gasseri and αLA.

  The stress to which the method of the present invention is applied includes any biological tension caused by a stressor that is physiologically harmful to animals. The stress relieving agent of the present invention is particularly useful for a stress response via the HPA axis.

  Symptoms caused by stress include reduced infection protection, delayed wound healing, decreased immune function, metabolic disorders, cardiovascular disorders (eg chest pain, palpitation, arrhythmia, etc.), digestive disorders (Eg, loss of appetite, stomach pain, nausea, stomach ulcer, constipation, diarrhea), shoulder stiffness, headache, dizziness, floating feeling, eye strain, general fatigue, fatigue, psychological stress such as tension and anxiety, depression, etc. Is mentioned. The stress relieving agent of the present invention is particularly useful for psychological stresses such as decreased immune function, decreased ability to prevent infection, general malaise, fatigue, tension and anxiety.

  The mitigation or alleviation method of the present invention relieves the stress, suppresses the increase in glucocorticoid, suppresses the decrease in immune function associated with the stress, and relieves psychological stress and fatigue such as anxiety and tension. , Maintain homeostasis in the body and adjust daily physical condition.

  In addition, all prior art documents cited in the present specification are incorporated herein by reference.

  EXAMPLES Hereinafter, although this invention is demonstrated further more concretely based on an Example, this invention is not limited to a following example.

[Example 1]
Examination of Lactobacillus gasseri OLL2809 to reduce stress and improve immune suppression [Experimental method]
After acclimating 6 weeks old male Sprague Dawlye rats (SPF) by individual breeding, the stress load was applied to the `` normal group '' without stress load, `` stress group '' with stress load, and Lactobacillus gasseri OLL2809. Divided into 3 groups of “OLL2809 group” (Day 1). All groups were reared normally in the same manner as during the acclimatization period, and then were reared for 5 days (Day 9 to Day 13) in cages filled with water up to a height of 1.5 cm from the bottom in the stress group and OLL2809 group from Day 9. From Day 1 to Day 14, OLL2809 strain at 40 mg / kg body weight / day was forcibly administered intragastrically using a gastric tube. Only the solvent water was administered to the normal group and the stress group in the same manner. Rats were dissected on Day 14 and NK activity of splenocytes and blood stress hormones were measured.
For the NK activity of splenocytes, a method using flow cytometry with YAC-1 cells as target cells was used. 1 × 10 ⁶ rat spleen effector cells and 2.5 × 10 ⁴ cells of Dio (3,3'-dioctadecyloxacarbocyanine perchlorate (Dio; Sigma-Aldrich, St. Louis, MO)) labeled YAC -1 target cells were mixed in a 96-well round bottom plate and cultured for 4 hours in a 37 ° C CO ₂ incubator. 15 minutes before the end of the culture, 20 μL of 0.5 mg / mL propidium iodide (Sigma-Aldrich) was added to stain dead cells. YAC-1 target cell death was analyzed using FACSCalibur flow cytometry and CellQuest software (BD Biosciences, Flanklin Lakes, NJ), and the death rate was defined as NK activity.
Three levels of stress hormones in the blood were measured: ACTH (adrenocorticotropic hormone), corticosterone, and 8-OHdG. These were measured using a kit. ACTH; ACTH (Rat) EIA KIT (Phoenix Pharmaceuticals, Burlingame, CA), Corticosterone; Corticosterone EIA Kit (Enzo Life Science, Farmingdale, NY), 8-OHdG; High-sensitivity 8-OHdG Check, (Nikken Seil, Shizuoka).

[Experimental result]
As a result of measuring ACTH and corticosterone, stress response mediators of the HPA axis, a significant increase was observed in the stress group compared to the normal group (Fig. 1). On the other hand, in the OLL2809 group, these values increased compared with the normal group due to stress load, but were not significant. In addition, 8-OHdG, an oxidative stress marker, showed similar changes (Fig. 2).
When NK spleen activity was measured, a significant decrease was observed in the stress group compared to the normal group (Fig. 3). On the other hand, in the OLL2809 group compared with the stress group, NK activity was significantly enhanced and recovered to the same level as in the normal group. This change was the opposite of corticosterone. From these results, it was clarified that ingestion of OLL2809 strain has the effect of maintaining high NK activity that is reduced by stress.
Therefore, these results revealed that administration of OLL2809 strain reduced the stress response via the HPA axis.

[Example 2] Examination of condition improvement effect in athletes of Lactobacillus gasseri OLL2809 and αLA (1) Subjects Healthy university students who satisfy the following criteria were enrolled in clinical trials as subjects (n = 44): (i ) A healthy Japanese man who is 30 years old or younger at the time of obtaining consent, and (ii) Exercise that requires endurance on a daily basis (more than 5 days a week).
Furthermore, subjects who did not violate the following exclusion criteria were included as subjects. Exclusion (cancellation / dropout) criteria: (i) Subjects who declined to continue the study due to free will; (ii) Those who developed allergic diseases such as hay fever during the study period; (iii) Continued the study due to the onset of some disease (Iv) Those who are found to be not in compliance with the instructions of the investigator or investigator; (v) The number of ingestions during the test substance intake period Those who have reached 10% of the scheduled number.
In carrying out this case, written consent was obtained from all test subjects who received an explanation of the study content from the investigator. If the subject was under 20 years of age, the consent of the person and their guardians was obtained. This study was conducted in compliance with the Declaration of Helsinki, and was conducted with consideration given to the safety of subjects. This test was conducted with the approval of the Ethics Review Committee of the Meiji Research Headquarters.

(2) Test method A randomized placebo-controlled double-blind method (parallel comparison test among three groups) was performed. After a 1-week pre-observation period, placebo and Lactobacillus gasseri OLL2809 only group (hereinafter referred to as OLL2809 group), and Lactobacillus gasseri OLL2809 plus αLA group (hereinafter referred to as OLL2809 + αLA group) ) 3 groups. Each subject received a placebo or tablet containing the test article for 4 weeks. The test was performed before and after ingestion of the test substance, and exercise load, questionnaire survey, and blood test were performed as described below. The blood test after exercise was performed approximately 1 hour after the completion of 1 hour of exercise.

[Exercise load]
Using an electromagnetic brake bicycle ergometer, subjects were allowed to perform a one-hour bicycle ergometer exercise at an intensity that maintained 70% of the predicted maximum heart rate reserve throughout the exercise (70% HRreserve). 70% HRreserve was calculated by the following Carbonen equation:
70% HRreserve = [(220-age)-resting heart rate] x 0.7 + resting heart rate.
Heart rate was monitored every 5 minutes throughout the exercise test.

[Evaluation item]
[I] POMS
The POMS questionnaire was completed twice, before the observation period and before exercise 4 weeks after ingestion. Each score was summed to score six subscales: TA (tension-anxiety), D (depression-depression), AH (anger-hostility), V (liveness), F (fatigue), and C (confused). According to the POMS manual, these total raw scores were converted into T-scores for parametric statistical analysis (Yokoyama K, Araki S. POMS Japanese manual. Kaneko Syobo, Tokyo, 1994 (In Japanese).).
[Ii] Fatigue VAS
Fatigue VAS was used to measure subject fatigue. The evaluation was performed four times before and after exercise load before the start of intake, and before and after exercise load 4 weeks after intake. VAS was rated on a straight line of 100mm, with the right side "no fatigue" and the left side "the strongest fatigue ever experienced". The subject was marked on the straight line at a position where he felt he best represented his current perception of his condition. Next, the VAS fatigue score was determined by measuring the distance (mm) from the left end of the straight line to the mark.
[Iii] NK activity In two observation periods before and after ingestion, blood was collected 4 times in total, 2 times before and after exercise, and NK activity was measured by BML.
[Iv] Blood and blood biochemistry tests Blood tests (red blood cell count, white blood cell count, platelet count, hemoglobin) and blood biochemistry tests (blood glucose level, total protein, alanine aminotransferase (ALT), asparagine aminotransferase (AST), lactic acid Dehydrogenase (LDH), total bilirubin, γ-transpeptidase, alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine, uric acid, total cholesterol, triglycerides, high density lipoprotein (HDL)) 4 weeks later, before exercise. These were mainly evaluated as safety indicators.
TGF-β1 and dROMs were evaluated by collecting blood 4 times in total, 2 times before and after exercise, in 2 observation periods before and after intake.

(3) Manufacture of test object The test object was manufactured in Meiji Co., Ltd.
Lactobacillus gasseri OLL2809 freeze-dried powder is inoculated with 1% pre-culture of Lactobacillus gasseri OLL2809 in a whey and lactose-based medium (whey decomposition medium), 37 ° C, lower limit pH 4.5 (6 mol / L NaOH automatically adjusted), and cultured for 18 hours under nitrogen aeration. Thereafter, the culture solution was concentrated 20 times and heat-treated at 85 ° C. for 60 minutes. After cooling, it was freeze-dried, and the dried product was pulverized to obtain a freeze-dried Lactobacillus gasseri OLL2809 powder.
OLL2809 tablets taken by the OLL2809 group produced 1 mg of Lactobacillus gasseri OLL2809 lyophilized powder and 500 mg tablets per tablet containing excipients and the like. OLL2809 + αLA tablets taken by the OLL2809 + αLA group produced 16.7 mg of Lactobacillus gasseri OLL2809 freeze-dried powder, 150 mg of αLA, 500 mg tablets per tablet containing excipients and the like. The placebo tablets ingested by the placebo group were prepared by blending dextrin instead of Lactobacillus gasseri OLL2809 lyophilized powder and αLA, and adding excipients to give 500 mg per tablet. These made every tablet indistinguishable in appearance, color and taste.

(4) Test substance intake In each group, each tablet was ingested continuously 2 tablets once a day, 3 times a day (morning, noon, evening) for 4 weeks. That is, the daily intake of OLL2809 group is 100 mg (5 × 10 ¹⁰ cells) of L. gasseri OLL2809 freeze-dried powder, and the intake of OLL2809 + αLA group is 100 mg of Lactobacillus gasseri OLL2809 freeze-dried powder. (5 × 10 ¹⁰ cells) and αLA are 900 mg.

(5) Statistical analysis Blind results were reviewed prior to key opening, and handling of dropped cases was determined based on blind data. This evaluation was performed on all subjects who completed the study.
The results are shown as mean ± standard deviation. One-way analysis of variance (One-way ANOVA) was used for subject background factors. Two-way repeated-measures (rm) ANOVA (fatigue) was conducted using two factors: the test factor and the observation period factor before and after intake. Sensitivity survey and NK activity were the difference between before and after exercise in each observation period). When significant, post hoc analysis was performed, and comparison before and after intake within the group was performed by Student's paired t test (Bonferroni correction). P <0.05 was judged as significant.

(6) Results [Subject background factors]
Forty-four people (14 people in the placebo group, 15 people in the OLL2809 group, 15 people in the OLL2809 + αLA group) participated in the study, and all of them completed the study period. There were no cases of withdrawal or dropout due to compliance violations or adverse events. All subjects were finally included in the analysis. Table 1 shows the subjects' background factors recorded before ingestion. There were no significant differences between the three groups in all items.

(Table 1)
Subject background factor

[POMS]
The results of POMS are shown in Table 2 and FIG. In each group, before intake and after 4 weeks, the placebo group showed no significant difference between 44.9 ± 9.9 before intake and 43.0 ± 6.0 after 4 weeks, but the OLL2809 group Showed a significant decrease at 43.6 ± 8.2 before ingestion and 40.6 ± 6.9 after 4 weeks (P = 0.006). Furthermore, in the OLL2809 + αLA group, a tendency to decrease to 42.6 ± 6.1 before ingestion and 39.7 ± 6.3 after 4 weeks was observed (P = 0.095).
In “Fatigue”, there was no significant difference between the placebo group and OLL2809 group before and after ingestion, but only OLL2809 + αLA group tended to decrease to 43.8 ± 6.1 before ingestion and 40.3 ± 5.2 after 4 weeks.
From the above results, OLL2809 reduced psychological stress such as tension and anxiety, and OLL2809 and αLA were found to reduce daily fatigue, which is felt by chronic stress.

(Table 2)
Effects of OLL2809 and αLA on mood profile test (POMS) scores

[Evaluation of fatigue due to exercise load using VAS method]
Fig. 5 shows the result of fatigue caused by exercise load using the VAS method. When the fatigue feeling before exercise was compared before and after ingestion within each group, a significant decrease in the VAS score was observed after 4 weeks in the OLL2809 + αLA group (P = 0.026). This result was consistent with the POMS result. Therefore, when a correlation analysis was performed on the test values before exercise, a significant positive correlation was found between the fatigue feeling of POMS and the VAS method (r = 0.293, n = 88, P = 0.0053). From these results, it was suggested that in the OLL2809 + αLA group, chronic fatigue was reduced by ingesting the test substance for 4 weeks.

[NK cytotoxic activity]
FIG. 6 shows the results of peripheral blood NK cell disorder activity before and after exercise performed before the test substance ingestion and peripheral blood NK activity before and after exercise performed 4 weeks after the ingestion.
Before ingestion, NK activity decreased to -12.4 ± 11.0% in the placebo group, -12.2 ± 12.3% in the OLL2809 group, and -14.5 ± 11.4% in the OLL2809 + αLA group after 1 hour of exercise. On the other hand, the difference between before and after exercise at 4 weeks was -8.8 ± 12.0% in the placebo group, -2.7 ± 14.2% in the OLL2809 group, and -4.3 ± 10.2% in the OLL2809 + αLA group, respectively.
When comparing before and after exercise load in each group, a significant decrease in NK activity was observed in all groups before exercise. On the other hand, after 4 weeks, the placebo group (P = 0.016) showed a significant decrease due to exercise load as before ingestion, whereas the OLL2809 group (P = 0.476) and OLL2809 + αLA group (P = 0.125). ) Showed no significant decrease in NK activity. That is, it has been clarified that the intake of OLL2809 strain reduces the decrease in NK activity due to chronic stress such as exercise load.

[Blood dROMs, TGF-β1]
Table 3 shows the results of dROMs and TGF-β1 in peripheral blood.
dROMs is an index indicating the degree of oxidative damage caused by active oxygen and free radicals, that is, the degree of oxidative stress. When comparing the pre-exercise values of dROMs, there was no significant difference between 303 ± 56 U.CARR before ingestion and 298 ± 80 U.CARR after 4 weeks in the placebo group, but in the OLL2809 group, there was no significant difference. Showed a tendency to decrease to 298 ± 50 U.CARR and 283 ± 27 U.CARR after 4 weeks (P = 0.089). Furthermore, in the OLL2809 + αLA group, it significantly decreased to 317 ± 58 U.CARR before ingestion and 284 ± 53 U.CARR after 4 weeks (P = 0.033). These values were not affected by exercise load.
TGF-β1 is a cytokine that is suggested to be a causative agent of central fatigue. TGF-β1 was compared to pre-exercise only before taking 1.29 ± 0.35, 1.33 ± 0.27, 1.35 ± 0.32 ng / ml in the placebo group, OLL2809 group, OLL2809 + αLA group, 1.23 ± 0.18 after 4 weeks, There was no significant difference between groups with 1.20 ± 0.15, 1.19 ± 0.19 ng / ml. When compared with pre-ingestion within each group, there was no significant change in the placebo group, but there was a tendency to decrease in the OLL2809 group (P = 0.075). Furthermore, a significant decrease was observed in the OLL2809 + αLA group (P = 0.035).

(Table 3)
Effect of test substance intake on peripheral blood TGF-β1 and dROMs

  Lactobacillus gasseri OLL2809 and αLA have been shown to improve the condition in athletes. Lactobacillus gaselli OLL2809 intake has been found to be effective in reducing mental stress and maintaining immunity that declines after strenuous exercise. It was. In addition to Lactobacillus gasseri OLL2809, ingestion of αLA was found to reduce daily fatigue. Based on the above results, taking only Lactobacillus gasseri OLL2809 or αLA in addition to Lactobacillus gasseri OLL2809 reduces the decline in immune function against chronic stress of daily training of athletes At the same time, it became clear that it is useful for adjusting the physical condition (improving the condition) of athletes by demonstrating the effect of relieving psychological aspects (psychological stress) such as fatigue.

  The Lactobacillus gasseri or Lactobacillus gasseri and αLA-provided stress-reducing agent provided based on the present invention alleviate poor physical condition caused by chronic stress when taken orally.

Claims (3)

Lactobacillus gasseri (Lactobacillus gasseri) and α-lactalbumin (αLA) as an active ingredient, a stress relieving agent , wherein the Lactobacillus gasseri is deposited under accession number NITE BP-72 An agent which is a gasseri OLL2809 strain (Lactobacillus gasseri OLL2809) .   The agent according to claim 1, wherein the stress is chronic stress. The agent according to claim 1 or 2, comprising 1 × 10 ⁷ or more of Lactobacillus gasseri and 40% by weight or more of αLA.

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