JP2009102292A - Stress relieving agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new use of lactobacillus FERM P-19169. <P>SOLUTION: Provided is a stress relieving agent containing lactobacillus FERM P-19169 as an active component and based on cytokine production regulating action, phylactic action, fattening action or diarrhea suppressing action. Further provided are a cytokine production regulating agent, a phylactic agent, a fattening agent, and a diarrhea suppressing agent containing lactobacillus FERM P-19169 as an active component. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a stress suppressant, and more particularly to a stress suppressor based on a cytokine production regulating action, an infection defense action, a fattening action or a diarrhea inhibiting action. The present invention further relates to cytokine production regulators, infection protection agents, fattening agents, and diarrhea inhibitors.

  Lactic acid bacteria are a general term for bacteria that produce lactic acid from saccharides by fermentation, and representative examples include Lactobacillus, Bifidobacterium, Pediococcus, and Lactococcus. ), Enterococcus, Streptococcus, and Leuconostoc are known. These lactic acid bacteria are used not only for the production of fermented foods but also for their use as probiotics that improve the bacterial flora in the gastrointestinal tract and have beneficial effects on the host.

  In recent years, for example, lactic acid bacteria FERM P-19169 has been found as a lactic acid bacterium that can grow well in residues produced as a by-product in alcoholic fermentation such as moromi concentrate, and its immunopotentiating action against the pathogenic bacterium has attracted attention (Patent Document 1). ). However, in Patent Document 1, although the superiority of lactic acid bacteria administration based on the mortality rate of mice has been clarified, since the action effect in human livestock or the like has not been demonstrated, there remains a problem for practical use.

  The immune system is equipped with various biological defense mechanisms for protecting the host from tissue damage, pathogenic microorganisms such as viruses and bacteria, or oxidative stress. The immune system produces various cytokines and chemokines in response to external stimuli such as pathogenic microorganisms, causing responses such as phagocytic mobilization, phagocytosis, lymphocyte activation, and NK cell activation. . Such cytokines include interleukin-1 (IL-1), antitumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), Examples include interleukin-12 (IL-12), interleukin-15 (IL-15), and interleukin-18 (IL-18) interferon-γ (IFN-γ).

  In the defense response against pathogenic microorganisms, there are innate immunity that works within several hours after infection and adaptive immunity that works from several days after infection. Innate immunity is a non-antigen-specific reaction and involves monocytes, macrophages, neutrophils, natural killer (NK) cells, and the like. Adaptive immunity is an antigen-specific reaction induced by T lymphocytes and B lymphocytes, and is persistent. In response to innate immunity, particularly inducible innate immunity, stimulation of the products of pathogenic microorganisms results in IL-1, TNF-α, IL-6, IL-8, IL-12, IL-15, IL-18. Various cytokines and chemokines including IFN-γ are produced. This has been shown to cause local ecological defense reactions such as phagocytic mobilization, phagocytosis, lymphocyte activation, and NK cell activation.

  Among these cytokines, overproduction of cytokines called inflammatory cytokines such as IL-1, TNF-α, IL-6, IL-18, IFN-γ, inflammation, fever, ulcers called acute phase responses, It is also known to cause edema, allergy, arthritis and the like, and to cause harm such as systemic shock, disseminated intravascular coagulation syndrome (Non-patent Document 1). In addition, it has been clarified that these inflammatory cytokines are produced by various stresses (Non-patent Document 2). In mice, it is also known that production of IFN-γ is increased by restraint stress (Non-patent Document 3).

  However, there have been no reports so far on administration studies on humans and livestock using lactic acid bacteria as the main component and investigating the cytokine production inhibitory effect.

JP 2005-21156 A Janaway, C.I. A. Jr, translated by Takehiko Kajikawa, "Immunobiology" (Original 5th edition), 2003 Maes, M .; Et al., “In humans, serum polyunsaturated fatty acid levels predict the response of prokineticity to psychological stress”, Biol. Psychiatry. Vol. 47, pp910-920 (2000) Yoshimi Hitomi et al., “Molecular mechanism of atopic dermatitis exacerbation by restraint stress”, Abstracts of the 144th Annual Meeting of the Japan Veterinary Medical Society, pp53 (2007)

  Lactic acid bacteria FERM P-19169 has low added value and is useful for the effective use of distillation residue after alcohol fermentation that has been treated as waste, so that its application is expected to expand. Then, an object of this invention is to provide the new use of lactic acid bacteria FERM P-19169.

  As a result of repeated studies on novel uses of lactic acid bacteria FERM P-19169, the present inventors have demonstrated that lactic acid bacteria FERM P-19169 exert cytokine production regulating action, infection defense action, fattening action and diarrhea inhibiting action, based on these. It was found that the stress load can be suppressed. So far, no stress suppressor has been found that monitors stress fluctuation and has demonstrated a clear and useful stress suppression effect from the monitoring result. The present inventors administered lactic acid bacteria FERM P-19169 in many individuals and measured inflammatory cytokines over time, thereby clearly monitoring the fluctuation of stress for each group. As effects, the present inventors have found useful cytokine production regulating action, infection defense action, fattening effect, and diarrhea suppressing effect. For example, by administering lactic acid bacteria FERM P-19169 to piglets, excessive production of inflammatory cytokines such as TNF-α and IL-6 is suppressed in the weaning period under stress, and E. coli lipopolysaccharide (LPS) in a piglet that is mock-infected, while exhibiting an infection-protecting function, it suppresses a rapid increase in inflammatory cytokines such as TNF-α, IFN-γ, and IL-6, thereby causing an excessive inflammatory reaction Found to suppress. The present inventors have also found fattening action and diarrhea inhibitory action on piglets by lactic acid bacteria FERM P-19169. The stress-suppressing agent of the present invention further shows a lowering effect on the livestock death rate due to stress.

  That is, the present invention provides a stress inhibitor based on lactic acid bacteria FERM P-19169 as an active ingredient and based on a cytokine production regulating action, an infection defense action, a fattening action or a diarrhea inhibiting action. According to the stress suppressant of the present invention, when stress due to weaning or environmental change is applied, the stress load can be suppressed through cytokine production regulating action, infection defense action, fattening action or diarrhea inhibiting action.

  The present invention also provides a cytokine production regulator, an infection protective agent, a fattening agent, and a diarrhea suppressant, comprising lactic acid bacteria FERM P-19169 as an active ingredient.

  Lactic acid bacteria FERM P-19169 has a function of suppressing excessive production of cytokines when various stresses are applied. In addition, the cytokine production inhibitory effect here is due to a mild interaction between lactic acid bacteria and the intestinal tract immune system, and does not show rapid immune suppression or pathological reaction. The present invention is the first invention in which an administration test to a living body was actually conducted and the cytokine production inhibitory effect by lactic acid bacteria was verified. In addition, by administering lactic acid bacteria FERM P-19169, while suppressing non-specific biological defense reaction of the host and suppressing excessive cytokine production, it protects against pathogenic infections and minimizes wear due to diseases, etc. It becomes possible to suppress to. Moreover, the weight increase rate of domestic animals, such as a pig, in the fattening period can be improved, and the growth promotion effect is exhibited, thereby shortening the average shipping date of livestock. Furthermore, the incidence of livestock diarrhea and the rate of death (total of accident rate and dredging rate) can be reduced.

  According to the present invention, a novel use of lactic acid bacteria FERM P-19169 is provided. Specifically, a stress suppressor based on lactic acid bacteria FERM P-19169 as an active ingredient and based on cytokine production regulating action, infection defense action, fattening action or diarrhea inhibiting action is provided. According to the present invention, there are further provided a cytokine production regulator, an infection protective agent, a fattening agent and a diarrhea suppressant containing lactic acid bacteria FERM P-19169 as an active ingredient.

  Hereinafter, preferred embodiments of the present invention will be described in detail.

  The stress suppressor of the present invention uses lactic acid bacteria FERM P-19169 as an active ingredient, and suppresses stress based on cytokine production regulating action, infection defense action, fattening action or diarrhea inhibiting action.

  Here, stress means a non-specific reaction of a living body caused by a stressor that is an external stimulus that causes stress, and the stressor includes physical stressors such as temperature, pressure, osmotic pressure, ultraviolet rays, radiation, sound, and electromagnetic waves. Chemical stressors such as drugs, toxic substances, starvation, oxidation, oxygen deficiency, biological stressors such as invasion of bacteria and viruses, and mental stressors such as mental pain and mental tension.

  Stress suppression means suppression of non-specific reactions of the living body caused by the stressor. As non-specific reactions of the living body, elevation of cortisol, C-reactive protein and inflammatory cytokines in the living body is observed. Symptoms include decreased appetite, indigestion, insomnia, and poor physical condition. Especially in the case of livestock, it appears as calming, loose stool, diarrhea, poor growth, poor fattening, etc. As a result, in the presence of a stressor, resistance to external stimuli is reduced, and infections and the like are likely to occur. On the other hand, it can be expected to alleviate these symptoms by suppressing stress.

  As one form of stress suppression in the present invention, for example, TNF-α, IFN-γ, IL, which occurs when a physical or mental stressor such as weaning or environmental change, or a biological stressor such as pathogenic infection is received. The form which suppresses stress by suppressing excessive production of cytokines, such as −6, and effectively protecting against pathogenic infection, suppressing excessive inflammatory reaction, and minimizing wear due to diseases and the like. This form corresponds to stress suppression based on cytokine production regulating action and infection defense action. Another form of stress suppression in the present invention is a form in which resistance to physical, chemical, biological or mental stressors is improved by promoting fattening of livestock, thereby suppressing stress. In yet another form, by reducing the incidence of diarrhea and the death rate of livestock and the like, healthy growth of livestock and the like is promoted, and as a result, resistance to stressors is improved.

  Lactic acid bacteria FERM P-19169 belongs to Lactobacillus paracasei subsp. Paracasei and is deposited as FERM P-19169 at the National Institute of Advanced Industrial Science and Technology / Patent biological depository It is.

  The lactic acid bacterium FERM P-19169 is a standard strain of L. lactobacillus paracatheis subspices paracathesia. paracasei subsp. Paracasei NBRC 15889 shows similar assimilation patterns for many carbohydrates, but some carbohydrates differ in fermentability. That is, in the sugar fermentability test using the API50CH (manufactured by Biomerieux) system, NBRC 15889 is slightly positive when the sugar substrate is sorbose and inulin, and is positive when the sugar substrate is sorbitol and gluconate. FERM P-19169 shows negative when the sugar substrate is sorbose, sorbitol and inulin, and shows a slight positive only when the sugar substrate is gluconate. Lactic acid bacteria FERM P-19169 can be identified by examining the gene sequence by sugar fermentability test using API50CH (manufactured by Biomerieux) system or 16S rDNA gene analysis using extracted genomic DNA.

  The culture of lactic acid bacteria FERM P-19169 is not particularly limited as long as it is carried out in general lactic acid bacteria culture media and conditions. For example, a natural medium, a synthetic medium or a semi-synthetic medium can be used. As the medium, any medium containing a nitrogen source and a carbon source may be used, and various inorganic substances may be added. The pH of the medium is preferably 4.0 to 8.0, more preferably 5.0 to 7.0. The culture temperature is preferably 10.0 to 45.0 ° C, more preferably 20.0 to 40.0 ° C. The culture time is about 24 to 96 hours, and aeration or anaerobic culture may be performed. Specifically, for example, static culture can be performed at 37 ° C. using MRS medium (Difco MRS broth). Alternatively, 1.5% agar can be added to the MRS medium and cultured at 37 ° C. using a gas pack anaerobic culture system (Mitsubishi Gas Chemical Co., Ltd.). When using freeze-dried bacteria, restore the bacteria by adding physiological saline or MRS medium, smear on MRS agar plate, incubate at 37.0 ° C, then subculture in liquid medium or agar medium Can be cultured.

  Lactic acid bacteria FERM P-19169 has temperature resistance and alcohol resistance. Further, in addition to a general culture medium for lactic acid bacteria, it can grow well in a 2.5 to 3.0-fold concentrated liquid (moromi concentrated liquid) of alcohol fermentation / distillation residue. By using the moromi concentrate, the lactic acid bacteria FERM P-19169 can be cultured, and such residual liquid can also be used effectively. As such a moromi concentrate, for example, a concentrated concentrate of the residual liquid obtained by subjecting molasses after sugar cane squeezed juice to alcohol fermentation and distillation is preferably used. Moreover, it is preferable to use the 2-8 times diluting water dilution liquid (dilution rate shall be volume ratio) of a mash concentrate, and a 2 times distilled water dilution liquid is especially preferable.

  The lactic acid bacterium FERM P-19169 contained in the stress suppressant of the present invention may be a living bacterium, a bacterium that has been inactivated by heat treatment, UV, formalin treatment, or the like, and a living bacterium is particularly preferable. Lactic acid bacteria FERM P-19169 may be present together with the fermentation product of lactic acid bacteria FERM P-19169. That is, the stress inhibitor of the present invention may contain a fermentation product produced by lactic acid bacteria FERM P-19169.

  The stress-suppressing agent of the present invention can be used as a topical agent such as a liquid, capsule, tablet, granule, powder, pill, fine granule, or troche tablet, or rectal agent. Moreover, in order to reduce the influence by a gastric acid, it is also preferable to use an enteric solvent. The stress suppressant of the present invention may consist only of lactic acid bacteria, and is a pharmaceutically acceptable additive or carrier such as an excipient, a bulking agent, a binder, a wetting agent, a disintegrant, a surface. Activators, lubricants, dispersants, buffers, preservatives, solubilizers, flavoring agents, stabilizers and the like may be included, and if necessary, other lactic acid bacteria may be included.

The stress suppressant of the present invention can be produced, for example, as follows. First, lactic acid bacteria FERM P-19169 are grown and cultured in an appropriate medium, and then the cultured lactic acid bacteria are recovered by natural sedimentation or centrifugation. Subsequently, the collected lactic acid bacteria are diluted and adjusted with the culture supernatant so as to be 1 × 10 ¹⁰ to 2 × 10 ¹¹ CFU / g. In addition to using it as a liquid as it is, after adding a protective agent such as skim milk or lactose, it is processed into a powder or tablet by freeze drying, spray drying, spray granulation, or tableting. Specifically, it is preferable to mix with an equal amount of 20% skim milk liquid and process into a powder form by spray granulation. This is stored at 4 ° C. The stress suppressor produced by such a method shows a viable cell count of 5 × 10 ⁸ CFU / g or more, and is stored at 4 ° C. for 360 days, and the viable cell count of 1 × 10 ⁸ CFU / g or more. Can be maintained.

  Since the stress suppressant of the present invention has a stress suppressive action based on a cytokine production regulating action, an infection protective action, a fattening action, or a diarrhea suppressive action, it can be used for treatment requiring stress reduction or disease prevention. For example, it can be used when subjected to physical, chemical, biological and mental stressors represented by environmental changes, bacterial invasion, and mental labor.

The dose of the stress suppressant of the present invention is preferably determined appropriately according to the administration subject, individual symptoms, age, body weight, and the like. For example, in the case of pigs, the daily dose is preferably 1.0 × 10 ^{7 to} 5.0 × 10 ⁹ CFU / head. When the stress suppressant of the present invention is administered to pigs, it can suppress stress, can protect against pathogenesis without producing excessive inflammatory cytokines even when infected with pathogen, and also has fattening effect and diarrhea suppression The effect can also be expected.

  By containing the stress suppressant of the present invention, a feed additive for livestock is provided. The feed additive for livestock may consist only of a stress suppressor containing lactic acid bacteria FERM P-19169 as an active ingredient, and other general feed additives such as minerals, vitamins, amino acids, dietary fiber, etc. May be included. This livestock feed additive can be a solid, liquid or semi-solid such as a gel. From the viewpoint of storage stability, solid or semi-solid is preferable.

The amount of feed additives used for livestock can be determined appropriately according to the type of livestock (pigs, cows, cotton sheep, goats, chickens, duck, geese, rabbits, dogs, cats, etc.) The dosage is preferably such that the amount of lactic acid bacteria FERM P-19169 is 1.0 × 10 ^{7 to} 5.0 × 10 ⁹ CFU / head. Moreover, it is preferable to increase / decrease suitably according to the weaning period or fattening period of a pig, or by an environmental change. When the feed additive for livestock of the present invention is added to the feed for pigs and the pigs are bred, the stress of the pigs can be suppressed, and pathogen protection can be achieved without infecting the pathogens and producing excessive inflammatory cytokines. Can also be expected fattening effect and diarrhea suppression effect.

  By containing the stress suppressant of the present invention, functional foods and foods for specified health use can also be provided. Food forms include yogurt, drink yogurt, juice, coffee, sports drinks, milk, soy milk, alcoholic beverages, puddings, jelly, cakes, frozen confectionery and other dairy products such as cheese and butter. Can be mentioned. The amount of lactic acid bacteria FERM P-19169 contained in such a food may be an amount that can suppress stress.

  When the food of the present invention is ingested for a certain period of time, for example, when it receives various stressors such as mental labor, physical fatigue and environmental changes, it can suppress excessive production of inflammatory cytokines even when pathogenic infection occurs, thereby causing pathogenic infection Can be effectively prevented, and an excessive inflammatory reaction can be suppressed, stress can be suppressed, and a healthy body resistant to stress can be obtained.

  By using lactic acid bacteria FERM P-19169 as an active ingredient, a cytokine production regulator, an infection protective agent, a fattening agent, and a diarrhea inhibitor are provided.

Cytokine production regulators can suppress excessive production of inflammatory cytokines and minimize wear due to diseases, etc., so apply to diseases related to excessive secretion of inflammatory cytokines, such as stress. Can do. The dose of the cytokine production regulator is preferably appropriately determined according to individual symptoms, age, weight, and the like. For example, in the case of pigs, the daily dose is preferably 1.0 × 10 ^{7 to} 5.0 × 10 ⁹ CFU / head.

Infectious agents can effectively protect against pathogenic infections and suppress excessive production of inflammatory cytokines, so that diseases such as fever and diarrhea caused by infectious diseases such as microorganisms, viruses, parasites, etc. Can be applied to. It is preferable that the dose of the infection protective agent is appropriately determined according to individual symptoms, age, weight, and the like. For example, in the case of pigs, the daily dose is preferably 1.0 × 10 ^{7 to} 5.0 × 10 ⁹ CFU / head.

Fertilizers suppress overproduction of inflammatory cytokines, effectively protect against pathogenic infections, and exert fattening effects, especially pigs, cows, cotton sheep, goats, chickens, duck, geese, rabbits, dogs It can be applied to breeding cats. It is preferable to appropriately determine the dosage of the fattening agent as necessary. For example, in the case of pigs, the daily dose is preferably 1.0 × 10 ^{7 to} 5.0 × 10 ⁹ CFU / head.

Antidiarrheal agents suppress overproduction of inflammatory cytokines, effectively protect against pathogenic infections, and exert diarrhea-suppressing effects, so that pigs, cows, cotton sheep, goats, chickens, duck, geese, moths, etc. It can be applied to breeding dogs, cats, etc. The dosage of the diarrhea suppressant is preferably determined appropriately as necessary. For example, in the case of pigs, the daily dose is preferably 1.0 × 10 ^{7 to} 5.0 × 10 ⁹ CFU / head.

  EXAMPLES Hereinafter, although the preferable Example of this invention is described in detail, this invention is not limited to these Examples.

(Production Example 1) Production of Lactic Acid Bacteria FERM P-19169 Powdered Product Lactobacillus paracasei FERM P-19169 was grown and cultured in a mash (distilled water dilution) medium, and the cultured lactic acid bacteria were collected by centrifugation. Subsequently, the collected lactic acid bacteria were diluted with the culture supernatant so as to be 1.0 × 10 ^{10 to} 1.0 × 10 ¹¹ CFU / g. This was mixed with an equal amount of 20% skim milk solution and processed into a powdery formulation by spray granulation. The obtained lactic acid bacterium FERM P-19169 powder preparation contained 3.0 × 10 ^{8 to} 3.0 × 10 ⁹ CFU / g of lactic acid bacterium FERM P-19169 as viable bacteria. When the above preparation was stored at 4 ° C. until the test and stored at 4 ° C. for 360 days, the viable cell count of 1.0 × 10 ^{8 to} 3.0 × 10 ⁸ CFU / g or more was maintained.

(Example 1) Cytokine production regulating action by lactic acid bacteria FERM P-19169 Lactic acid bacteria FERM P-19169 was administered to suckling pigs, and the cytokine production regulating action in the weaning period by lactic acid bacteria FERM P-19169 was tested.

  In Examples 1 to 3, a piglet born by one delivery from the same mother pig was taken as one litter, and only piglets in which one abdomen was 8 or more were adopted as test pigs. One group was divided into three abdomen, and the piglets were divided into a non-administration group (n = 33), a forced administration group (n = 27), and a free intake group (n = 29), and tests were sequentially performed for each parturition. In addition, the breed of piglet in each group is W (n = 10) or WLD (n = 21) in the non-administration group, and W (n = 4) or WLD (n = 22) in the forced administration group. In the free intake group, they were W type (n = 12) or LD type (n = 16). The W type is a large Yorkshire type, the L type is a land race type, the D type is a Duroc type, the LD type is a binary hybrid, and the WLD type is a ternary hybrid.

  The test period was 36 ± 2 days in the early feeding period (22 days) and late feeding period (14 ± 2 days). The piglets of each group were raised in the delivery pig house until 4 weeks of age (28 days), and only the sows were moved after weaning. Breeding was carried out with breast milk + milk substitute in the early stage of suckling, and breast milk + artificial milk in the late stage of suckling. As substitute milk and artificial milk, mower up birthday milk (manufactured by ITOCHU FOOD CO., LTD.) And mower up ETSUKE (manufactured by ITOCHU FUJI YUDEN Co., Ltd.) were used, respectively, and no other lactic acid bacteria were added.

The lactic acid bacteria FERM P-19169 preparation obtained in Production Example 1 was used as a living bacterium in both the forced administration group and the free intake group as 1.0 × 10 ⁹ CFU / head / day at the early feeding stage, and 3.0 × at the later feeding stage. The dose was 10 ⁹ CFU / head / day. For the forced administration group, the lactic acid bacteria preparation was diluted in warm water and orally administered at 3 mL once a day in the early feeding period and 9 mL once a day in the later feeding period. The free intake group was divided into two mornings and evenings and mixed with artificial milk.

  During the study period, weighed piglets weekly and recorded clinical symptoms as needed. During the 36 ± 2 days of the breeding period, 2 accidental pigs appeared in the non-administration group, 1 in the forced administration group, and 1 in the free intake group, and these were excluded from the study. The accidental death means the death of piglets and fattening pigs due to crushing death, frailty death, cold death, disease death, etc. by the mother pig. The change in body weight for each group is shown in FIG. From FIG. 1, among the non-administration group (n = 31), the forced administration group (n = 26), and the free intake group (n = 28), the body weight gain of the free intake group was the largest, but a significant difference was observed. There wasn't.

  Cytokine production was measured by sandwich ELISA using piglet plasma samples. For the measurement of TNF-α, a Sine TNF-α ELISA Kit (manufactured by Biosource) using an immobilized anti-TNF-α monoclonal antibody and a biotin-labeled anti-TNF-α monoclonal antibody was used. For the measurement of IFN-γ, a Sine Interferon-gammma (swIFN-γ) ELISA Kit (manufactured by Biosource) using an immobilized anti-IFN-γ monoclonal antibody and a biotin-labeled anti-IFN-γ monoclonal antibody was used. For measurement of IL-6, Quantikine (trademark) and Porcine IL-6 Immunoassay (manufactured by R & D systems) using an immobilized anti-IL-6 polyclonal antibody and a horseradish peroxidase-labeled anti-IL-6 polyclonal antibody were used. . Then, it detected and measured with the microplate reader (made by Tecan). Blood was collected from the jugular vein 2 days before, 2 days, and 9 days after weaning of the piglets. The collected blood was heparinized to obtain 10 mL of heparinized blood. Heparinized blood was divided into three 3 mL portions, and two of them were added to Carsensen et al. (Carstensen, L. et al. (2005). Determination of tumor necrosis factor-alpha responses in piglets in piglets. blood stimulation assay Vet. Immuno. Immunol., Vol. Added. After incubation at 39 ° C. for 2 hours or 20 hours, centrifugation was performed at 10,000 rpm for 10 minutes, and the obtained supernatant was used as a plasma sample with LPS stimulation (LPS 2 hours or LPS 20 hours). The remaining one was centrifuged immediately without adding LPS, and the resulting supernatant was used as a sample without LPS stimulation (LPS 0 hour). All samples were stored at -80 ° C.

  In the analysis of cytokines, first, a preliminary test was performed using two males and two females in each group in order to examine the effect of LPS stimulation and the effect of LPS treatment time. As a result, in the case of TNF-α, the LPS 2 hours showed a higher value than the LPS 20 hours. Also, most of LPS 0 time was below the detection limit. In the case of IL-6, it was hardly detected at LPS 0 time, but it was nearly 10 times higher at LPS 20 hours than LPS 2 hours. In the case of IFN-γ, most were below the detection limit regardless of the presence or absence of LPS stimulation. From the above, it was found that these cytokines were not produced without LPS stimulation. Therefore, in this test, TNF-α was analyzed using a sample of LPS for 2 hours, and IL-6 was analyzed using a sample of LPS for 20 hours, but IFN-γ was not analyzed.

  Among the data of about 300 samples obtained for each cytokine, samples that failed in ELISA and samples that showed clearly higher values for both TNF-α and IL-6 were excluded from the analysis. Data analysis was performed by comparing two groups with two independent t-tests. The Welch method was applied when the variance of data could not be regarded as equal variance by F test. Statistical significance was considered significant when p <0.05.

  The analysis results of the non-administration group (n = 26), the forced administration group (n = 23), and the free intake group (n = 25) are shown in FIG. FIG. 2 shows that TNF-α was significantly lower than that in the non-administered group after 2 days of weaning in the forced administration group and 2 days and 9 days after weaning in the free intake group. On the other hand, 9 days after weaning, the value in the forced administration group was higher than that in the non-administration group. Moreover, IL-6 showed a significantly lower value in the forced administration group and the free intake group than in the non-administered group on any test day, and in particular, the free intake group showed a higher significant difference.

  In the non-administration group, TNF-α showed an overall high value throughout the test period. This is thought to be because the piglet received a strong weaning stressor. On the other hand, administration of lactic acid bacteria FERM P-19169 significantly reduced TNF-α, suggesting that administration of lactic acid bacteria reduced stress due to weaning stressors.

  The reason why the gavage group increased after 9 days of weaning may be due to stress due to gavage, other stress, or infection with some pathogen. In addition, IL-6 did not increase 2 days after weaning in the non-administered group, and showed a high value on any test day. It is thought that was applied. Considering that the test period was between June 12 and August 17, it was possible that pigs that were vulnerable to heat received a high-temperature stressor.

  The values for the non-administration group were high and the standard deviation was large, whereas the values for the gavage group and the free intake group were significantly lower and the standard deviation was also small. It was also effective against some environmental stresses, suggesting that the health status of the piglets in the test group was stably maintained. In addition, the free intake group was more effective than the gavage group, and the increase in body weight was better than the other groups. Was guessed.

(Example 2) Infection protective action by lactic acid bacteria FERM P-19169 Lactic acid bacteria FERM P-19169 was administered to suckling pigs and tested for infection protective action by lactic acid bacteria FERM P-19169 in the weaning period.

  In the same manner as in Example 1, lactic acid bacteria were administered. The test was conducted using a binary or ternary cross piglet after completion of the 5-week administration test. The non-administration group, the gavage group and the free intake group were selected from 2 abdomen, 6 males and 6 females each, and divided into 2 groups so that the body weights were equal, one with the LPS inoculation group and the other with the LPS non Inoculated area.

  Blood samples before LPS inoculation were performed on 5 week-old piglets for which the lactic acid bacteria administration test was completed. Then, Nakajima et al. (Nakajima, L. et al. (2000). Involvement of apoptosis in the endotoxomic conditions of the liver and kidsneys. P. 62, J. et. , 2.5 mg of LPS (Escherichia coli 055: B5, manufactured by SIGMA) was dissolved in 10 mL of PBS and inoculated into the ear vein so that the dose was 20 μg / kg, and mock infection was performed. Non-inoculated areas were inoculated with PBS only. Thereafter, 1.5, 3, 6, 12, and 24 hours later, blood was collected from the jugular vein, treated with heparin, and then centrifuged in the same manner as in Example 1. The obtained plasma sample was stored at −20 ° C.

  For TNF-α, IL-6, and IFN-γ in plasma, changes over time due to LPS inoculation were measured by ELISA according to the method described in Example 1. In addition, clinical symptoms due to LPS inoculation were also recorded.

  The average weights of the piglets used in the test were 12.4 kg, 9.7 kg, and 11.1 kg in the non-administration group, the forced administration group, and the free intake group, respectively. There were no piglets that showed clinical symptoms during the study period. Approximately 220 samples obtained for each cytokine were analyzed. Among them, samples that showed clearly abnormal values by ELISA were excluded from the analysis. Data analysis was performed by comparing two groups with two independent t-tests. Statistical significance was considered significant when p <0.05.

  The result of the analysis is shown in FIG. From FIG. 3, by inoculation with LPS, TNF-α, IL-6, and IFN-γ in piglet plasma increased rapidly, peaked at 1.5 hours or 3 hours, and then 6 hours later. It was found that the value decreased to the value before the test. TNF-α showed a maximum value at 1.5 hours, and showed a high value of 10 μg / mL or more in the non-administration group and the gavage group, whereas in the free administration group, TNF-α was 1.5 hours. Although an increase was observed, the value was significantly lower than that of the other groups ((A) in FIG. 3).

  IL-6 production also showed a transient increase and decrease after LPS inoculation, but peaked at 1.5 hours in the gavage and free intake groups, but increased further in the non-administered group Then, after showing a peak at 3 hours, it turned to decrease, and at 3 hours, a significant difference was observed between the non-administration group and the free intake group (p <0.05) ((B) of FIG. 3).

  In addition, it was confirmed that IFN-γ was induced by LPS inoculation, and at 3 hours, the forced administration group and the free intake group showed significantly lower values than the non-administration group ((C) of FIG. 3). ).

  In the LPS non-inoculated group inoculated with PBS only, production of these cytokines was hardly observed.

  From the above results, the stimulation of the mock infection by LPS inoculation first produces TNF-α rapidly, and subsequently produces IL-6 and IFN-γ, and the immune response is completed within 6 hours. I understood. These cytokines are known as inflammatory cytokines that protect against infection while causing excessive inflammation such as fever and systemic shock in the body. In this study, lactic acid bacteria FERM P-19169 suppressed the rapid production of these cytokines, suggesting that it regulates the amount of cytokines produced as an infection defense and suppresses excessive inflammation. .

(Example 3) Fattening effect by lactic acid bacteria FERM P-19169 Lactic acid bacteria FERM P-19169 was administered to a piglet in the fattening period, and the fattening action was tested.

  The piglets were divided into a non-administration group (n = 30), a forced administration group (n = 33) and a free intake group (n = 28), and one group was divided into three abdomen. The female breed was W or LW and the male breed was D or W. The piglets were kept in the delivery piggery until 4 weeks (28th), and only the mother pigs were moved after weaning.

The test period was 83 to 97 days before shipment (110 kg) for pigs that reached 30 kg. The fattening period was divided into three stages of 30-50 kg stage (stage I), 50-90 kg stage (stage II), 90-110 kg stage (stage III), and the lactic acid bacteria FERM P-19169 preparation obtained in Production Example 1 As live bacteria, 1.0 × 10 ⁹ CFU / head / day in stage I, 2.0 × 10 ⁹ CFU / head / day in stage II, 3.0 × 10 ⁹ CFU / head / day in stage III, new It was mixed with the feed for testing meat production capacity (TDN 74.5% or more, CP 14.5% or more, manufactured by Suguji Shokai Co., Ltd.) and allowed to ingest freely.

  During the study period, weighed piglets every week and recorded clinical symptoms as needed. The growth status of pigs is shown in Table 1 and (A) of FIG. During the fattening period, 2 out of 4 pigs that caused diarrhea were observed in the non-administration group, and 1 showed 4 symptoms of diarrhea. One animal showed diarrhea and fever symptoms at the same time and was treated with ampicillin and slipilin. However, no pigs showing such symptoms were observed in the administration group. From Table 1, the shipping age is 159.8 days in the non-administration group and 156.3 days in the administration group, and the shipping weight is 111.4 kg in the non-administration group and 113.4 kg in the administration group. there were. Although there was no significant difference, it was found that the administration group was shipped about 3 days (2.2%) earlier, 2 kg (1.8%) heavier, and shipped.

  The average daily weight gain during the fattening period is shown in Table 2 and FIG. From Table 2, the daily average weight gain (weight gain) from 30 kg showed a high tendency in the administration group in both the early fattening period and the late fattening period. In particular, in the late fattening period, the average daily weight gain of the non-administration group was 918.4 g, whereas that of the administration group was 1030.6 g, which was 12.2% compared to the non-administration group. It turned out that it increased a lot.

  From the above results, it was proved that there is a fattening effect by administration of lactic acid bacteria FERM P-19169. In particular, it has been clarified that the weight gain rate in the late fattening period is effective. Lactic acid bacteria themselves do not have high-calorie nutritional components that promote fattening, and the amount of lactic acid bacteria added this time is less than 1% of the feed, so that the stress-suppressing effect of lactic acid bacteria administration is healthy for pigs. It is thought that it contributed to growth.

(Example 4) Lactic acid bacteria administration test to suckling and weaning piglets Lactic acid bacteria preparations reduce the accident rate (accident mortality) of suckling and weaning piglets, suppress diarrhea, and fattening investigated.

  The test was conducted at a medium-scale pig farmer (Chiba Prefecture) about 400 mother pigs. One piglet born from the same mother pig by one delivery was taken as one litter, and only piglets with 7 or more litters born within 6 days were adopted as test pigs. The group composition is as follows: during the sucking period, the non-administration group is divided into 8 stomachs (n = 81), the administration group is 7 stomachs (n = 79), and then the weaning period is divided into 15 groups × 4 rooms by grouping This was performed in the group (n = 60) and the administration group (n = 60). The piglet varieties were all LWD ternary hybrids. Note that the L type indicates a land race type, the W type indicates a large Yorkshire type, and the D type indicates a Duroc type.

  The test period was divided into the late lactation period (27 days from the 14th day to the 40th day) and the weaning period (48 days from the 41st day to the 88th day). Postpartum piglets were housed in the delivery house for each belly, and after weaning (day 28), only the mother pigs were moved. On the 41st day, the animals moved to the weaning house and divided into groups so that the body weights were almost the same in 4 rooms of 15 each. Breed with breast milk until day 13 after birth, breast milk and commercially available artificial milk PH 5 (Nisshin Marubeni Feed Co., Ltd.) from day 14, only artificial milk from day 28, formula feed customer from day 41 The animals were reared by switching to L (Nisshin Marubeni Feed Co., Ltd.). The piglets were always bred with free drinking and constant salary.

  The lactic acid bacteria FERM P-19169 preparation obtained in Production Example 1 was added at 1% by weight to artificial milk during the sucking period, and 0.1% by weight was added to the formulated feed during the weaning period to allow free intake.

  The piglets were weighed three times during delivery, weaning, and moving to the fattening house. Clinical symptoms were recorded at any time. When diarrhea was confirmed during the suckling period, an appropriate amount of Starosan F (manufactured by Kyoritsu Pharmaceutical Co., Ltd.) was sprinkled in the pig bun with the piglet, and the breeding environment was improved by drying the floor and preventing contamination. In addition, piglets that showed some clinical symptoms during the weaning period were treated by injection of antibiotics (ampicillin, kanamycin). Furthermore, blood was collected from 3 animals from each room and 12 animals from each group at the end of the test, and cytokine production was measured in the same manner as in Example 1.

  The accident rate (excluding crushing) for each group during the test period is shown in FIG. During the study period of 75 days, accident rates other than crushing were 3.9% in the non-administration group (n = 75), 1.3% in the administration group (n = 75), and non-administration group in the weaning period It was 1.7% in (n = 60) and 0% in the administration group (n = 60). Compared with the non-administered group, the accident rate during the sucking period was reduced to 1/3 in the administration group, and the tendency was low in the weaning period. Accidental deaths other than crushing are thought to be caused by frailty or disease infection, and the decrease in the accident rate other than crushing in the treatment group suggested that the piglet was growing healthy.

  Table 3 shows the incidence of diarrhea for each belly during the late feeding period. Diarrhea was confirmed only during the suckling period. The incidence of diarrhea that developed diarrhea even once was 75.5% in the non-administration group, compared with 25.3% in the administration group. Decreased to 3.

  The weight change for each group is shown in FIG. In the feeding period, there was no significant difference in weight gain between the non-administered group (n = 75) and the administered group (n = 75), but in the weaning period, the administered group (n = 60) n = 60), a 5.0% weight gain was observed.

  From the above results, it was suggested that administration of the lactic acid bacterium Lactobacillus paracasei FERM P-19169 has the effect of suppressing the occurrence of diarrhea and reducing the accident rate of piglets. In addition, it was speculated that the weight of the administration group increased during the weaning period because the piglet grew healthy due to the effect of suppressing the occurrence of diarrhea.

  The results of cytokine analysis at the end of the test are shown in FIG. From FIG. 7, it was recognized that both TNF-α and IL-6 decreased in the administration group. This suggested that, as in Example 1, the stress was reduced by suppressing excessive production of inflammatory cytokines, and the health status of the piglet was stably maintained.

(Example 5) Administration test of lactic acid bacteria to fattening pigs The effect of reducing the death rate of fattening pigs (total of accident rate and drought rate due to poor growth) by administration of lactic acid bacteria preparations was examined.

  The test was conducted at a medium-scale pig farmer (Chiba Prefecture) about 400 mother pigs. As in Example 4, fattening pigs were divided into 14 to 15 heads × 4 rooms by grouping, and were performed in the non-administration group (n = 58) and the administration group (n = 59). The test pigs were weighed before the start of the test to confirm that there was no variation. All pig breeds were LWD ternary hybrids. Note that the L type indicates a land race type, the W type indicates a large Yorkshire type, and the D type indicates a Duroc type.

  The test period was 57 days during the fattening period (from 30 kg to shipment). During the test period, they are raised on a mixed diet that does not contain antibiotics. The first half of fattening (from 30 kg to 70 kg) is Step B (JA East Kumiai Feed Co., Ltd.), and the second half of fattening (from 70 kg to shipment) is IP Kamo C (JA East Japan Kumiai) Feed Co., Ltd.) was used. The fattening pigs were always raised with free drinking and constant feeding. During the test, they were vaccinated against swine erysipelas and Aujeszky's disease.

  The lactic acid bacteria FERM P-19169 preparation obtained in Production Example 1 was added to the mixed feed of the administration group by 0.1% by weight.

  During the test period, clinical symptoms of fattening pigs were recorded at any time, and the number of accidental deaths and the number of migrating heads (pox) due to poor growth were also recorded. Furthermore, pigs that showed any clinical symptoms during the study period were treated with antibiotics and the like. Immediately after the start of the study, swine pleural pneumonia infection was observed in both the administration group and the non-administration group, and antibiotics (fluorocol) were orally administered for 5 days by adding feed to all heads, and if necessary, by injection Antibiotic treatment was performed.

  FIG. 8 shows the result of death rate (total of accident rate and dredging rate) until the end of the test. The accident rate until the end of the study was 5 (8.6%) in the non-administration group, 3 (5.1%) in the administration group, and 2 in the non-administration group (3.5%) ), 1 in the administration group (1.7%). The death rate was 12.1% in the non-administration group and 6.8% in the administration group, and it was found that the death rate in the administration group was reduced by 43.8% compared to the non-administration group. This suggests that Lactobacillus paracasei FERM P-19169 exerts an infection-protecting effect and suppresses overproduction of inflammatory cytokines due to pathogenic infection and the like, and can stably maintain a healthy state.

It is a figure which shows the influence on the weight gain in the weaning period by administration of the lactic acid bacterium FERM P-19169 with respect to the piglet of a lactation period. It is a figure which shows the influence on the production of cytokine in the weaning period by administration of the lactic acid bacterium FERM P-19169 with respect to the piglet of a suckling period. In addition, A is a figure which shows the influence on production of TNF- (alpha), B is a figure which shows the influence on production of IL-6. It is a figure which shows the influence on the production of the cytokine at the time of the mock infection in the weaning period by administration of the lactic acid bacterium FERM P-19169 with respect to the piglet of a lactation period. In addition, A is a figure which shows the influence on production of TNF- (alpha), B is a figure which shows the influence on production of IL-6, C is a figure which shows the influence on production of IFN- (gamma). It is a figure which shows the influence on the fattening state by administration of the lactic acid bacteria FERM P-19169 in the piglet of fattening period. In addition, A is a figure which shows the growth condition during the fattening period, and B is a figure which shows the daily average body weight gain during the fattening period. It is a figure which shows the influence on the accident rate (except for crushing death) by administration of the lactic acid bacteria FERM P-19169 in the piglet of a nursing period and a weaning period. It is a figure which shows the influence on the weight change by administration of the lactic acid bacteria FERM P-19169 in the piglet of a suckling period and a weaning period. It is a figure which shows the influence on the production | generation of TNF- (alpha) and IL-6 at the time of completion | finish of the test by the administration of the lactic acid bacteria FERM P-19169 with respect to the piglet of a nursing period and a weaning period. It is a figure which shows the influence on the death rate by administration of lactic acid bacteria FERM P-19169 in the piglet of fattening period.

Claims (8)

  A stress suppressor comprising lactic acid bacteria FERM P-19169 as an active ingredient and based on cytokine production regulating action, infection defense action, fattening action or diarrhea inhibiting action.   The stress suppressor according to claim 1, wherein the cytokine production regulating action is a TNF-α, IFN-γ or IL-6 production inhibitory action.   A livestock feed additive comprising the stress suppressant according to claim 1 or 2.   A cytokine production regulator comprising lactic acid bacteria FERM P-19169 as an active ingredient.   The cytokine production regulator according to claim 4, which is a production inhibitor of TNF-α, IFN-γ, or IL-6.   An infection protective agent comprising lactic acid bacteria FERM P-19169 as an active ingredient.   A fattening agent containing lactic acid bacteria FERM P-19169 as an active ingredient.   A diarrhea inhibitor comprising lactic acid bacteria FERM P-19169 as an active ingredient.

Images