Composition for inhibiting reduction of acquired immune function due to anti-influenza drug, and method for producing same

Abstract

The composition for suppressing a decrease in acquired immune function according to the present invention contains, as an active ingredient, a lactic acid bacterium product derived from a lactic acid bacterium belonging to the genus Lactobacillus. The composition for suppressing a decrease in acquired immune function suppresses a decrease in acquired immune function caused by the use of an anti-influenza drug. In addition, the lactic acid bacteria belonging to the genus Lactobacillus are preferably bulgaricus. The lactic acid bacteria of the genus Lactobacillus are, for example, German Lactobacillus subsp. Bulgaricus. In addition, the production method according to the present invention is a method for producing a composition for suppressing a decrease in acquired immune function, which is for suppressing a decrease in acquired immune function due to the use of an anti-influenza drug, by supplying a milk raw material to a lactic acid bacterium belonging to the genus Lactobacillus.

Description

[0001] This application is a divisional application of patent application No. 201780042693.2, filed on August 15, 2017, entitled "A composition for inhibiting acquired immune function reduction caused by anti-influenza drugs and a method for manufacturing the same". Technical Field

[0002] This invention relates to a composition for inhibiting acquired immune dysfunction caused by the use of anti-influenza drugs and a method for manufacturing the same. Background Technology

[0003] Influenza is caused by infection with the influenza virus. It is highly contagious, infecting approximately 10 million people annually in Japan. Furthermore, due to its epidemic nature, once an outbreak begins, it can spread rapidly to many people. Elderly individuals are particularly prone to severe illness and are at risk of developing complications such as pneumonia, which can lead to death.

[0004] Currently, in the treatment of influenza, antiviral drugs such as oseltamivir (OSV) are commonly used. It is believed that administering these drugs within 48 hours of symptom onset can inhibit the replication of the influenza virus and shorten the duration of illness. However, administering antiviral drugs reduces the amount of antigens in the body, potentially leading to a decrease in acquired immunity. When acquired immunity is reduced, the production of specific antibodies used to eliminate the virus and prevent reinfection decreases. Therefore, compared to those who do not receive OSV, influenza patients who receive OSV are at a higher risk of reinfection in the following season, which is a concern.

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: Japanese Patent Publication No. 2010-518151

[0008] Patent Document 2: Japanese Patent Application Publication No. 2012-72113

[0009] Patent Document 3: International Publication No. 2012 / 133827 Summary of the Invention

[0010] The technical problem that the invention aims to solve

[0011] In recent years, certain lactic acid bacteria have been confirmed to be effective in the prevention or treatment of influenza infection. For example, Patent Document 1 discloses the use of strains of *Lactobacillus casei* for the manufacture of an orally administered composition for enhancing immunity against influenza after vaccination. Furthermore, Patent Document 2 discloses an influenza prophylactic and / or therapeutic agent containing lactic acid bacteria belonging to *Lactobacillus acidophilus* as an active ingredient. Moreover, Patent Document 3 discloses an anti-influenza virus composition using *Lactobacillus paracasei* as an active ingredient.

[0012] However, none of the aforementioned patent documents 1-3 reported on the suppression of the reduction in immune function against viral infection after administration of anti-influenza drugs.

[0013] Therefore, the object of the present invention is to provide a composition capable of inhibiting the reduction of acquired immune function caused by administration of anti-influenza drugs and a method thereof.

[0014] Technical means to solve technical problems

[0015] In view of the above-mentioned problems, the inventors of this application conducted in-depth research and found that, compared with the case of administering anti-influenza drugs alone, the simultaneous administration of lactic acid bacteria products (lactic acid bacteria products) derived from Lactobacillus and anti-influenza drugs can suppress the reduction of immune function against influenza viruses, thereby completing the present invention.

[0016] According to one aspect of the present invention, a composition for inhibiting acquired immunodeficiency contains a lactic acid bacteria product derived from *Lactobacillus* as an active ingredient, inhibiting acquired immunodeficiency caused by the use of anti-influenza drugs. That is, in the composition for inhibiting acquired immunodeficiency according to one aspect of the present invention, the lactic acid bacteria product derived from *Lactobacillus* is used as the composition itself or as a component of the composition. Furthermore, the term "composition" as used herein includes preparations such as pharmaceuticals, supplements, and food additives, food products (excluding animal and plant products themselves), and food product compositions (including processed food products), as well as substances that animals (including humans) can ingest.

[0017] In the above-mentioned composition for inhibiting the decline of acquired immune function, the above-mentioned lactobacillus species is preferably classified as Bulgarian (bulgaricus).

[0018] In the above-mentioned composition for inhibiting acquired immune function reduction, the lactobacillus is preferably Lactobacillus delbrueckii subsp. bulgaricus.

[0019] The composition described above for inhibiting the decline of acquired immune function is preferably fermented milk.

[0020] The above-mentioned compositions for inhibiting acquired immune dysfunction can also have an inhibitory effect on influenza virus infection.

[0021] Furthermore, according to another aspect of the present invention, a method for manufacturing a composition for inhibiting acquired immune function reduction caused by the use of anti-influenza drugs is prepared by supplying milk raw materials to Lactobacillus lactic acid bacteria.

[0022] Beneficial effects

[0023] As described above, the composition of the present invention for inhibiting acquired immunodeficiency contains a lactic acid bacteria product derived from *Lactobacillus* as its active ingredient. Compared to administering an anti-influenza drug alone, by simultaneously administering such a composition for inhibiting acquired immunodeficiency and an anti-influenza drug, the amount of specific antibodies against the influenza virus can be increased. Therefore, the composition for inhibiting acquired immunodeficiency according to the present invention can inhibit acquired immunodeficiency caused by the use of anti-influenza drugs. Furthermore, according to the manufacturing method of the present invention, a composition for inhibiting acquired immunodeficiency caused by the use of anti-influenza drugs can be manufactured. Attached Figure Description

[0024] Figure 1 This is a graph showing the results of Experiment 1 according to this embodiment (the amount of IgA in the lung lavage fluid).

[0025] Figure 2 This is a graph showing the results of Experiment 1 (the amount of IgG in serum) according to this embodiment.

[0026] Figure 3 This is a diagram showing the results of Experiment 2 according to this embodiment. Detailed Implementation

[0027] The present invention will now be described in more detail. However, the present invention is not limited thereto.

[0028] (1) Compositions for inhibiting acquired immune dysfunction

[0029] The composition according to the present invention for inhibiting acquired immunodeficiency contains lactic acid bacteria products derived from *Lactobacillus* as its active ingredient. Here, lactic acid bacteria products include lactic acid bacteria ferments, lactic acid bacteria cultures, lactic acid bacteria metabolites, etc. Lactic acid bacteria ferments refer to the results (including cultures and products) obtained after lactic acid fermentation using lactic acid bacteria. Furthermore, lactic acid bacteria cultures are the results (including cultures and products) obtained by culturing lactic acid bacteria in the presence of a culture medium suitable for lactic acid bacteria cultivation. Lactic acid bacteria metabolites are the results (including products) obtained through the metabolic processes of lactic acid bacteria. Additionally, lactic acid bacteria ferments and lactic acid bacteria cultures sometimes refer to the same substance, and in such cases, they can be used interchangeably.

[0030] Lactic acid bacteria products may or may not contain lactic acid bacteria themselves (including live and dead bacteria). In addition, from the perspective of probiotics, it is preferable to use lactic acid bacteria fermentation products containing live bacteria.

[0031] Furthermore, the term "lactic acid bacteria" in this article refers to a general term for lactic acid-producing microorganisms that utilize glucose as a nutrient source and have a sugar absorption and utilization rate of over 50%. Physiologically, they possess the following characteristics: Gram-positive cocci or bacilli, non-motile, non-spore-forming, and catalase-negative. Lactic acid bacteria have been consumed worldwide since ancient times through fermented milk and other methods, making them extremely safe microorganisms. Lactic acid bacteria are classified into several genera.

[0032] Furthermore, the composition according to the invention for inhibiting acquired immunodeficiency contains a lactic acid bacteria product derived from Lactobacillus species as an active ingredient. That is, the composition according to the invention for inhibiting acquired immunodeficiency contains at least one of the following as an active ingredient: a fermentation product derived from Lactobacillus, a culture of Lactobacillus, and a metabolite of Lactobacillus.

[0033] Examples of lactic acid bacteria in the genus *Lactobacillus* include *Lactobacillus bulgaricus*, *Lactobacillus casei*, *Lactobacillus acidophilus*, and *Lactobacillus plantarum*. Among these *Lactobacillus* species, *Lactobacillus bulgaricus* (also known as Bulgarian lactobacillus) is preferred in this invention. Furthermore, *Lactobacillus delbrueckii* subsp. *bulgaricus* is more preferably used among *Lactobacillus* species.

[0034] In addition, more specifically, Lactobacillus deutschlandiae includes Lactobacillus deutschlandiae OLL1073R-1 (accession number: FERM BP-10741) (hereinafter referred to as "Lactobacillus deutschlandiae R-1 strain"), etc.

[0035] Among various Lactobacillus species, the composition according to the present invention for inhibiting acquired immunodeficiency more preferably contains a lactic acid bacteria product derived from "Bulgaria bulgaricus R-1 strain" as an active ingredient. Bulgaria bulgaricus R-1 strain was deposited in Japan on February 22, 1999 (the deposit date) at the Patent Biological Collection Center of the National Institute of Advanced Industrial Science and Technology (IPOD, AIST) (1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan, accession number FERM P-17227); and on November 29, 2006, it was transferred to international deposit under the Treaty of Budapest, obtaining accession number IPOD FERM BP-10741. In addition, since the Patent Biological Collection Center of the Technical Base for Product Evaluation (IPOD, NITE) inherited the patent microbial collection business from the Patent Biological Collection Center of the National Institute of Advanced Industrial Science and Technology (AIST), the L. bulgaricus R-1 strain is currently deposited at the Patent Biological Collection Center of the Technical Base for Product Evaluation (IPOD, NITE) (Room 122, 2-5-8 Kazusa-ashi, Kisarazu City, Chiba Prefecture, Japan) (Collection No.: FERM BP-10741).

[0036] The lactic acid bacteria product contained in the composition for inhibiting acquired immunodeficiency of the present invention is preferably a lactic acid bacteria ferment. This lactic acid bacteria ferment includes lactic acid bacteria ferment and its processed products, such as: culture filtrate or culture supernatant obtained by sterilizing a culture (lactic acid bacteria ferment) using filtration / centrifugation or membrane separation; concentrate, paste, dilution, or dried product (freezing, heating, reduced pressure, etc.) obtained by concentrating the culture filtrate / culture supernatant or lactic acid bacteria ferment using an evaporator or the like. Furthermore, when preparing the processed product, one or a combination of the aforementioned processing steps such as filtration, centrifugation, membrane separation, precipitation, concentration, pasteurization, dilution, and drying can be performed. Moreover, examples of culture media for lactic acid bacteria cultures include, for instance, skim milk powder culture medium and MRS culture medium.

[0037] More specifically, lactic acid bacteria products contained in the composition of the present invention for inhibiting acquired immune dysfunction can be exemplified by lactic acid bacteria ferments obtained by fermenting various substrates using Bacillus bulgaricus R-1 strain.

[0038] The substrate used in fermentation can be anything that can create an environment conducive to the growth or proliferation of L. bulgaricus R-1, resulting in fermentation. This substrate can be, for example, human and animal milk, as well as food materials such as vegetables, fruits, beans, and grains, or it can be a culture medium or raw milk used for the growth or proliferation of microorganisms. The substrate is preferably a food material that can be consumed as food after fermentation. Specifically, it can be a culture medium and raw milk containing raw milk (unsterilized milk), sterilized milk, whole milk concentrate, whole milk powder, skim milk powder, skim milk concentrate, milk protein concentrate (MPC), whey, whey powder, desalted whey, desalted whey powder, whey protein concentrate (WPC), whey protein isolate (WPI), α-lactalbumin, β-lactoglobulin, casein, sodium caseinate, calcium caseinate, cream, butter, soy milk, etc. Among these food materials, it can be a culture medium and raw milk containing sugars (including lactose), minerals, vitamins, yeast extract, etc.

[0039] Furthermore, it is known that *Bulgaria bulgaricus* strain R-1 produces extracellular polysaccharides (EPS) as metabolites. Therefore, the compositions of the present invention for suppressing acquired immunodeficiency may also contain extracellular polysaccharides produced by *Bulgaria bulgaricus* strain R-1. In the present invention, for example, the lower limit of daily intake of extracellular polysaccharides is 500 μg, preferably 1.0 mg, more preferably 2.0 mg. The upper limit is not particularly limited, but is, for example, 8.0 mg.

[0040] In addition, during fermentation, various other fermentation bacteria, such as lactic acid bacteria other than Bacillus bulgaricus R-1 and / or Bacillus natto, yeast, etc., can be used. Specifically, thermophilic bacteria (Streptococcus thermophillus) used as starter cultures in yogurt making and Bacillus natto used in natto fermentation can be used.

[0041] Lactic acid bacteria products are particularly preferred to be fermented milk products or milk cultures of lactic acid bacteria. Examples of fermented milk products or milk cultures include, for instance, fermented milk. Here, "fermented milk" refers to a substance made by fermenting milk. "Fermented milk" includes, but is not limited to, "fermented milk," "lactic acid bacteria beverages," "milk beverages," and "natural cheeses" as defined by, for example, the Ministry Ordinance (Milk and Dairy Products Ordinance) which relates to specifications for the composition of milk and dairy products. For example, fermented milk as defined by the Ministry Ordinance (Milk and Dairy Products Ordinance) refers to milk such as raw milk, cow's milk, special cow's milk, raw goat's milk, sterilized goat's milk, raw sheep's milk, ingredient-adjusted milk, low-fat milk, and processed milk, or milk containing an equal or higher amount of non-fat milk solids, which are fermented by lactic acid bacteria or yeast to produce a solid (hard), paste (soft), or liquid (beverage type) substance or a frozen product of these substances, but is not limited to these.

[0042] Regarding the fermented milk of the present invention, the concentration range of the nonfat milk solids is preferably 4.0% to 12.0%, more preferably 6.0% to 10.0%, and even more preferably 7.0% to 9.0%. Furthermore, the concentration of the milk fat is preferably 0.2% to 4.0%, more preferably 0.3% to 3.0%, and even more preferably 0.4% to 2.0%.

[0043] Yogurt is a typical example of fermented milk. Yogurt includes plain yogurt, set yogurt, soft yogurt, and beverage yogurt, among others.

[0044] Furthermore, when implementing the composition of the present invention for suppressing acquired immunodeficiency as a fermented milk, it is preferable to prepare a single-serving amount in individual packaging. This improves usability because the required amount of the active ingredient can be taken appropriately and conveniently. Here, "single-serving packaging" includes all forms. Examples of packaging forms include containers with lids, bottles with caps, pouches, packets, tubes, etc. In the present invention, the purpose of each individual package or a package containing multiple individual packages can be clearly defined by: descriptions of the product's use, efficacy, method of intake, etc.; and / or by packaging with added descriptions; and / or by disclosing additional descriptions in booklets, etc.

[0045] Furthermore, the composition of the present invention for inhibiting acquired immune dysfunction can also be implemented as a food or beverage in forms other than fermented milk. Specific examples of such food or beverages include, for example, cheese, soft drinks, chewing gum, gummies, jelly, biscuits, etc. However, the form of the food or beverage is not particularly limited.

[0046] Next, the physiological activity of the composition according to the present invention for inhibiting acquired immunodeficiency will be described. The composition of the present invention for inhibiting acquired immunodeficiency inhibits the acquired immunodeficiency caused by the use of anti-influenza drugs.

[0047] Anti-influenza drugs, such as oseltamivir (OSV), zanamivir, peramivir, and lanimivir octanoate hydrate, are known examples. They all inhibit viral replication by inhibiting neuraminidase. It is known that administering such anti-influenza drugs during influenza infection can inhibit viral replication and shorten the duration of illness; however, on the other hand, it can lead to a decrease in acquired immunity against influenza virus infection. Therefore, compared to those who do not receive anti-influenza drugs, those who do receive them are more susceptible to reinfection with influenza in the following season. This is because administering anti-influenza drugs reduces the production of specific antibodies that prevent reinfection. Examples of such specific antibodies include IgA antibodies. It is known that when IgA antibodies are produced in the human respiratory tract and nasal cavity, they act directly on the influenza virus, preventing infection of the respiratory mucosal epithelium.

[0048] In this specification, the decrease in immune function against influenza virus infection caused by administration of anti-influenza drugs, as described above, is referred to as "acquired immunodeficiency." The composition of the present invention for inhibiting acquired immunodeficiency can suppress the "acquired immunodeficiency" caused by the use of anti-influenza drugs. That is, by ingesting the composition of the present invention for inhibiting acquired immunodeficiency, an organism can increase the production of influenza virus-specific antibodies (e.g., IgA antibodies, IgG antibodies, etc.) in the organism. Furthermore, even when anti-influenza drugs are used, by ingesting the composition of the present invention for inhibiting acquired immunodeficiency, the decrease in the production of influenza virus-specific antibodies in the organism can be suppressed.

[0049] Therefore, by simultaneously administering the composition of the present invention for inhibiting acquired immune dysfunction with, for example, an anti-influenza drug, the decrease in immune function against influenza virus infection can be suppressed. Thus, the likelihood of influenza patients using anti-influenza drugs being reinfected with influenza in the following season can be reduced.

[0050] Furthermore, as shown in the following examples, the L. bulgaricus R-1 strain also has the function of inhibiting influenza virus infection. That is, the composition of the present invention for inhibiting acquired immunodeficiency preferably also has an inhibitory effect against influenza virus infection. Thus, by regularly (e.g., daily) taking the composition of the present invention for inhibiting acquired immunodeficiency, influenza infection can also be prevented simultaneously.

[0051] As described above, in order to enhance resistance to influenza viruses and suppress the decline in acquired immune function, it is preferable to regularly (preferably daily) consume the composition of the present invention for suppressing the decline in acquired immune function before infection with influenza. For easy daily consumption of the composition of the present invention for suppressing the decline in acquired immune function, the composition of the present invention for suppressing the decline in acquired immune function is preferably in the form of fermented milk (e.g., yogurt). Yogurt is widely consumed due to its delicious taste and its cosmetic and health benefits. By making the composition of the present invention for suppressing the decline in acquired immune function into the form of yogurt, its required daily intake can be easily achieved.

[0052] Furthermore, as a suitable single intake amount for the composition of the present invention for inhibiting acquired immune function reduction, for example, in the case of fermented milk (e.g., beverage type) with a nonfat milk solids content of 8.0% by weight, it is preferably 50 mL to 200 mL per intake, more preferably 80 mL to 150 mL per intake, and even more preferably 100 mL to 120 mL per intake. Alternatively, for example, in the case of fermented milk (e.g., hard or soft type) with a nonfat milk solids content of 8.0% by weight, it is preferably 50 g to 200 g per intake, more preferably 80 g to 150 g per intake, and even more preferably 100 g to 120 g per intake. In addition, the intake frequency is preferably 0.5 to 5 times per day, more preferably 1 to 3 times per day, and even more preferably 1 to 2 times per day.

[0053] Furthermore, the composition according to the present invention for inhibiting acquired immune dysfunction has the physiological activity of inhibiting acquired immune dysfunction caused by the use of anti-influenza drugs. Therefore, it can be used as an effective ingredient in food and beverages (excluding plants and animals themselves), functional foods, functional beverages, pharmaceuticals, etc. That is, food and beverages (excluding plants and animals themselves), functional foods, functional beverages, and pharmaceuticals containing the composition of the present invention for inhibiting acquired immune dysfunction as an effective ingredient are also included within the scope of the present invention.

[0054] Furthermore, the composition of the present invention for inhibiting acquired immune dysfunction can also be implemented as a food product (excluding animal and plant products themselves), functional food, functional beverage, pharmaceutical, etc. That is, according to another aspect of the present invention, the food product, functional food, functional beverage, and pharmaceutical contain any of the above-mentioned lactic acid bacteria products derived from *Lactobacillus* as an active ingredient. Furthermore, it inhibits the acquired immune dysfunction caused by the use of anti-influenza drugs.

[0055] Furthermore, when implementing the composition of the present invention for inhibiting acquired immune dysfunction as a food or beverage, fermented milk is preferred from the viewpoints of production efficiency, ease of consumption, and palatability. In another embodiment of the present invention, fermented milk is yogurt obtained by adding lactobacillus lactic acid bacteria to milk raw materials and fermenting (culturing) the lactic acid bacteria.

[0056] Furthermore, in addition to the composition for suppressing acquired immune function reduction, the food and beverage products of the present invention may also contain known additives that can be found in foods (e.g., functional foods). Examples of such additives include water, sugars, sugar alcohols, starch and processed starch, dietary fiber, milk, processed milk, soy milk, fruit juice, vegetable juice, fruits and vegetables and their processed products, proteins, peptides, amino acids, animal and plant biopharmaceutical extracts, naturally derived polymers (collagen, hyaluronic acid, chondroitin, etc.), vitamins, minerals, thickeners, emulsifiers, preservatives, colorants, and flavorings.

[0057] Furthermore, when the composition of the present invention for inhibiting acquired immunodeficiency is used in a pharmaceutical product, in addition to the lactic acid bacteria product, it may also contain known additives that can be contained in the pharmaceutical product. Examples of such additives include excipients, disintegrants, binders, fluidizing agents, flavoring agents, fragrances, coloring agents, sweeteners, solvents, oils, thickeners, surfactants, gelling agents, stabilizers, preservatives, buffers, suspending agents, and thickeners.

[0058] (2) A method for manufacturing a composition for inhibiting acquired immune dysfunction

[0059] Next, a method for manufacturing the composition for inhibiting acquired immunodeficiency according to the present invention will be described. The method for manufacturing the composition for inhibiting acquired immunodeficiency according to the present invention includes a step of supplying milk raw materials to *Lactobacillus* lactic acid bacteria. Regarding the *Lactobacillus* lactic acid bacteria used, the *Lactobacillus* lactic acid bacteria described in (1) above can be used.

[0060] Examples of dairy raw materials include: animal milk such as cow's milk and its processed products (e.g., skim milk, whole milk powder, condensed milk, casein, whey, fresh cream, compound cream, butter, buttermilk powder, cheese, etc.); and plant-based milk such as soy milk derived from soybeans. Furthermore, dairy raw materials may or may not undergo sterilization. In addition, various additives may be added to dairy raw materials used in the manufacture of compositions for suppressing acquired immunodeficiency.

[0061] By supplying milk raw materials to lactic acid bacteria of the genus Lactobacillus and fermenting or culturing the lactic acid bacteria of the genus Lactobacillus, a lactic acid bacteria product as the main component can be produced. The composition for suppressing the reduction of acquired immune function produced by the production method according to the present invention can be obtained as fermented milk. In this case, the production method according to the present invention can also be said to be a method of supplying milk raw materials to lactic acid bacteria of the genus Lactobacillus and producing fermented milk having the function of suppressing the reduction of acquired immunity.

[0062] Among the raw materials used in the production of this fermented milk, not only the above-mentioned milk raw materials can be contained, but also various other components can be contained. Therefore, as the raw materials used in the production of fermented milk, for example, raw materials called fermented milk raw material mixtures can be cited. A fermented milk raw material mixture refers to a mixture containing raw milk and other components. This fermented milk raw material mixture can be obtained, for example, by heating and dissolving raw materials commonly used in the production of fermented milk such as milk raw materials, water, and other optional components (such as sugar, saccharides, sweeteners, acidulants, minerals, vitamins, flavors, etc.) and mixing them. Raw milk, sterilized milk, skim milk, whole milk powder, skim milk powder, whole milk concentrate, skim milk concentrate, buttermilk, butter, cream, cheese, etc. can also be contained in the milk raw materials. Moreover, whey protein concentrate (WPC), whey protein isolate (WPI), α-lactalbumin (α-La), β-lactoglobulin (β-Lg), etc. can also be contained in the milk raw materials.

[0063] Similar to the conventional method, fermented milk is produced through processes such as the preparation process of the raw material mixture, the (heating) sterilization process of the raw material mixture, the cooling process of the raw material mixture, the addition process of the starter, the fermentation process, and the cooling process of the fermented milk. In the preparation process of the raw material mixture, the raw materials are mixed (prepared). In addition, for the above processes, the usual conditions used in the production of fermented milk can be appropriately adopted. Moreover, it is preferably carried out in the order of the (heating) sterilization process of the raw material mixture, the cooling process of the raw material mixture, the addition process of the starter, the fermentation process, and the cooling process of the fermented milk.

[0064] As the medium for culturing lactic acid bacteria, a commonly used medium can be used. That is, any medium can be used as long as it contains a nitrogen source, inorganic substances, and other nutrients in appropriate amounts in addition to the main carbon source. As the carbon source, depending on the nutritional source utilization characteristics (assimilability) of the bacteria used, lactose, glucose, sucrose, fructose, starch hydrolysis products, molasses, etc. can be used. As the nitrogen source, organic nitrogen-containing substances such as casein hydrolysate, whey protein hydrolysate, α-lactalbumin, β-lactoglobulin, glycomacropeptide, and soy protein hydrolysate can be used. In addition, as a growth promoter, meat extract, fish extract, yeast extract, etc. can be used.

[0065] Lactic acid bacteria are preferably cultured under anaerobic conditions, typically under micro-aerobic conditions such as liquid static culture. Furthermore, for anaerobic culture methods, known methods such as culture under a carbon gas phase can be used, or other methods can also be employed. The culture temperature is generally preferably in the range of 30°C to 47°C, more preferably in the range of 35°C to 46°C, and even more preferably in the range of 37°C to 45°C. The pH of the culture medium for lactic acid bacteria culture is preferably maintained in the range of 6 to 7, but other pH ranges are acceptable as long as they are suitable for bacterial growth. The culture time for lactic acid bacteria is generally preferably in the range of 1 hour to 48 hours, more preferably in the range of 8 hours to 36 hours, and even more preferably in the range of 10 hours to 24 hours.

[0066] Typically, fermented milk has a nonfat milk solids content of 8% or more and a lactic acid bacteria or yeast count of 10. 6 10 or more per mL 11 Within the range of less than 1 / mL.

[0067] The manufacturing method of the present invention described above can be used to manufacture a composition for inhibiting acquired immune function reduction caused by the use of anti-influenza drugs. Furthermore, the composition for inhibiting acquired immune function reduction described in (1) above is an example of a composition for inhibiting acquired immune function reduction manufactured by the manufacturing method of the present invention.

[0068] The embodiments disclosed herein should be considered illustrative rather than restrictive in all respects. The scope of the invention is defined not by the foregoing description but by the scope of the claims, and is intended to include all equivalents of the scope of the claims and all modifications within that scope.

[0069] Example

[0070] The present invention will now be illustrated with examples to provide a more detailed description. Furthermore, the examples shown below are merely illustrative and do not limit the scope of the invention.

[0071] (Experiment 1)

[0072] In Experiment 1, the effects of ingesting yogurt made with the Bacillus bulgaricus strain R-1 (hereinafter referred to as "R-1 yogurt") on the amount of anti-influenza-specific antibodies (IgA and IgG antibodies) were investigated. Specifically, the levels of antibodies produced after mild influenza virus infection were investigated between mice pre-treated with R-1 yogurt and mice not treated with R-1 yogurt.

[0073] (1-1) Manufacturing of R-1 Yogurt

[0074] Yogurt is made by adding Lactobacillus bulgaricus subsp. bulgaricus OLL1073R-1 (accession number: FERM BP-10741) (hereinafter referred to as "Bulgarian strain R-1") and thermophilic bacteria as starter cultures to a mixture containing raw milk, skim milk powder, cream, sugar, and stevia.

[0075] (1-2) Mice were given R-1 yogurt, etc.

[0076] Six-week-old female BALB / c mice (SLC Corporation, Japan) were used as experimental subjects. The mice were divided into four groups during the experiment. Nine or ten mice were used in each group.

[0077] MC: Control group (comparative control group to R1 group) given ultrapure water (an alternative to R-1 yogurt) and 0.5% methylcellulose solution.

[0078] R1: Groups given R-1 yogurt and 0.5% methylcellulose solution

[0079] OSV: Groups administered oseltamivir in ultrapure water and dissolved in 0.5% methylcellulose solution.

[0080] OSV+R1: The group given R-1 yogurt and oseltamivir dissolved in 0.5% methylcellulose solution.

[0081] As described above, ultrapure water was used as a comparative control for R-1 yogurt in both the MC and OSV groups. Furthermore, a 0.5% methylcellulose solution (0.5 w / v methylcellulose 400) (Wako Pure Chemicals) was used as a comparative control for OSV in both the MC and R1 groups.

[0082] In the four groups mentioned above, mice in the MC and OSV groups were orally administered ultrapure water (an alternative to R-1 yogurt) for 21 days (3 weeks) before influenza virus infection. The single dose was 0.4 mL. Furthermore, the administration was once daily and continued for 14 days after viral infection.

[0083] In the four groups mentioned above, mice in the R1 group and the OSV+R1 group were orally administered R-1 yogurt for 21 days (3 weeks) before influenza virus infection. The single dose of R-1 yogurt was 0.4 mL. In addition, it was administered once a day and continued for 14 days after viral infection.

[0084] (1-3) Influenza virus infection and OSV administration in mice

[0085] Mice in groups (1-2) above were infected with influenza virus nasally at a dose of 0.5 pfu (plaque-forming units) per mouse. The influenza virus used was influenza A virus (IAV) / Puerto Rico / 8 / 1934 (PR8) (H1N1) (hereinafter referred to as PR8).

[0086] Following viral infection, mice in the MC and R1 groups were orally administered 0.5% methylcellulose (OSV-free) as an OSV solvent. The single dose of methylcellulose was 0.1 mL. Furthermore, the administration was twice daily for 14 days.

[0087] Following viral infection, mice in the OSV group and the OSV+R1 group were orally administered oseltamivir (phosphate) (Fnakoshi Co., Ltd.), an antiviral drug for influenza, dissolved in 0.5% methylcellulose. The single dose of oseltamivir (phosphate) was 0.1 mg / 0.1 mL / mouse. The administration was twice daily for 14 days.

[0088] As mentioned above, mice in the OSV group and OSV+R1 group were given anti-influenza drug (OSV), while mice in the MC group and R1 group were not given anti-influenza drug.

[0089] (1-4) Evaluation of anti-influenza specific antibody titer (antibody titer) using ELISA

[0090] The titer of anti-influenza specific antibodies was evaluated using ELISA on day 14 of viral infection. Specifically, the titer of IgA antibodies in mouse lung lavage fluid and the titer of IgG antibodies in mouse serum were evaluated.

[0091] Perform ELISA according to the following steps. Add antigen conditioning solution (PR8 (0.5 μg / mL) BSA (0.1%) / PBS) at 100 μL / well to a 96-well plate to immobilize the antigen (0.05 μg / well). After incubating at 4°C for 12 hours, wash each well three times with washing buffer (50 mM Tris-HCl (pH 8.0), 0.14 M NaCl, 0.05% Tween 20). Add sufficient blocking buffer (50 mM Tris-HCl (pH 8.0), 0.14 M NaCl, 1% BSA) to each well and incubate at 37°C for 2 hours. After washing each well three times with washing buffer, add 100 μL / well of the measurement sample (obtained from lung washing fluid or serum of each group of mice) appropriately diluted with sample buffer (50 mM Tris-HCl (pH 8.0), 0.14 M NaCl, 0.05% Tween 20, 1% BSA). Wash each well five times with washing buffer.

[0092] Sufficient amounts of 10,000-fold diluted HRP-conjugated anti-mouse IgG (manufactured by Bethyl Laboratories, #A90-131P) or 2,000-fold diluted HRP-conjugated anti-mouse IgA (manufactured by Bethyl Laboratories, #A90-103P) were added to each well. Each well was then washed five times with washing buffer. Chromogenic reagent (TMB (3,3',5,5'-tetramethylbenzidine), manufactured by KPL, SureBlue, #52-00-02) was added to 100 μL / well, and after incubation at room temperature for 15 minutes, stop solution (TMB stop solution, manufactured by KPL, #50-85-05) was added to 100 μL / well. The absorbance of each sample was then measured at 450 nm to evaluate the anti-influenza specific antibody titer.

[0093] Figure 1 The results of IgA determination are shown in the figure. Figure 2 The results of IgG measurement are shown. In each graph, the vertical bars represent the standard deviation within each group (MC group, OSV group, R1 group, OSV+R1 group). Additionally, the inter-group measurements are also shown. The marker indicates a significant difference when the risk rate (crisis rate) is less than 5%. The label means that there is a significant difference when the risk rate is less than 1%.

[0094] like Figure 1 As shown, compared with the group given oseltamivir alone (OSV group), the amount of IgA antibodies in lung lavage fluid was significantly increased in the group given R-1 yogurt and oseltamivir (OSV+R1 group). Furthermore, as... Figure 2 As shown, the amount of IgG antibodies in serum was significantly increased in the OSV+R1 group compared with the OSV group.

[0095] Based on the above results, it was confirmed that R-1 yogurt made using Lactobacillus bulgaricus strain R-1 (Lactobacillus bulgaricus subsp. OLL1073R-1) has an inhibitory effect on acquired immune function reduction caused by anti-influenza drugs.

[0096] (Experiment 2)

[0097] In Experiment 2, nasal cleaning fluid was collected from mice in the groups (MC, OSV, R1, and OSV+R1) obtained from Experiments 1 (1-3) above. 50 μL of this nasal cleaning fluid was neutralized with influenza virus PR8 (100 pfu). This solution was then applied to MDCK cells (canine kidney-derived cells), and the number of infected cells was counted after 16 hours to evaluate the influenza virus neutralizing activity of R-1 yogurt.

[0098] The result is as follows Figure 3 As shown in the figure. In the graph, each vertical bar represents the standard deviation within each group (MC group, OSV group, R1 group, OSV+R1 group). Additionally, the inter-group... The labeling indicates a significant difference when the risk rate is less than 5%. The marker signifies a significant difference at a risk rate of less than 1%. A significant reduction in infected cell count was observed in the R-1 yogurt administration group (R1 group) compared to the control group (MC group). Furthermore, no significant difference was found between the R-1 yogurt and OSV administration group (OSV+R1 group) and the OSV administration group (OSV group); however, as... Figure 3 As shown, compared with the OSV group, a slight decrease in the number of infected cells was found in the OSV+R1 group.

[0099] Based on the above results, it was confirmed that R-1 yogurt made using Lactobacillus bulgaricus strain R-1 (Lactobacillus bulgaricus subsp. OLL1073R-1) has the effect of enhancing neutralizing activity against influenza virus.

[0100] Collection Number:

[0101] FERM BP-10741.

Claims

1. Use of Lactobacillus lactic acid bacteria or lactic acid bacteria products derived from Lactobacillus lactic acid bacteria in the manufacture of a composition for inhibiting acquired immunodeficiency, said composition inhibiting acquired immunodeficiency caused by the use of an anti-influenza drug having neuraminidase inhibitory activity.

2. The use as described in claim 1, wherein, The lactobacillus species is classified as Bulgarian (Bulgarian).

3. The use as described in claim 1, wherein, The lactobacillus mentioned is Lactobacillus delbrueckii subsp. bulgaricus.

4. The use as described in claim 3, wherein, The Lactobacillus bulgaricus subspecies mentioned is Lactobacillus bulgaricus subspecies OLL1073R-1 with accession number FERMBP-10741.

5. The use as described in any one of claims 1-4, wherein, The lactic acid bacteria products are lactic acid bacteria ferments, lactic acid bacteria cultures, or lactic acid bacteria metabolites.

6. The use as described in claim 5, wherein, The lactic acid bacteria ferment is a lactic acid bacteria ferment containing live bacteria.

7. The use as described in claim 5 or 6, wherein, The lactic acid bacteria product is fermented milk.

8. The use as described in claim 7, wherein, The concentration of nonfat milk solids in the fermented milk is in the range of 4.0% to 12.0%, preferably 6.0% to 10.0%, and more preferably 7.0% to 9.0%. The concentration of milk fat in the fermented milk is 0.2% to 4.0%, preferably 0.3% to 3.0%, and more preferably 0.4% to 2.0%.

9. The use as described in claim 7 or 8, wherein, The fermented milk is yogurt.

10. The use as described in claim 9, wherein, The yogurt may be plain yogurt, hard yogurt, soft yogurt, or beverage yogurt.

11. The use as described in any one of claims 7-10, wherein, The fermented milk is packaged in individual portions suitable for a single serving.

12. The use as described in claim 11, wherein, In the case of beverage-type fermented milk with a nonfat milk solids content of 8.0% by weight, the suitable amount for a single consumption is 50 mL to 200 mL, preferably 80 mL to 150 mL, and more preferably 100 mL to 120 mL; or, In the case of hard or soft fermented milk with a fat-free milk solids content of 8.0% by weight, the suitable amount for a single intake is 50g to 200g, preferably 80g to 150g, and more preferably 100g to 120g.

13. The use as described in any one of claims 1-12, wherein, The anti-influenza drug with neuraminidase inhibitory activity is oseltamivir, zanamivir, peramivir, or lanimivir octanoate hydrate.

14. The use as described in claim 13, wherein, The anti-influenza drug with neuraminidase inhibitory activity is oseltamivir.

15. The use as described in any one of claims 1-14, wherein, The composition also has an inhibitory effect on influenza virus infection.

16. The use as described in any one of claims 1-15, wherein, The composition itself is used as a food product, functional food, functional beverage, or pharmaceutical product; or, the composition is included as an active ingredient in a food product, functional food, functional beverage, or pharmaceutical product.

17. A method for manufacturing a composition for inhibiting acquired immune dysfunction, said composition containing a lactic acid bacteria product derived from Lactobacillus as an active ingredient, inhibiting acquired immune dysfunction caused by the use of an anti-influenza drug with neuraminidase inhibitory activity.

18. The method of claim 17, wherein, The lactobacillus species is classified as Bulgarian.

19. The method of claim 17, wherein, The lactobacillus mentioned is *Lactobacillus deutschlandii* subsp. *bulgaricus*.

20. The method of claim 19, wherein, The Lactobacillus bulgaricus subspecies mentioned is Lactobacillus bulgaricus subspecies OLL1073R-1 with accession number FERMBP-10741.

21. The method according to any one of claims 17-20, wherein, Substrate is supplied to the lactobacillus species.

22. The method according to any one of claims 17-21, wherein, Milk raw materials are supplied to the lactobacillus bacteria.

23. The method according to any one of claims 17-22, wherein, The composition is fermented milk.

24. The method of claim 23, wherein, The fermented milk has a fat-free milk solids content of 8% or more and a lactic acid bacteria count of 10. 6 10 or more per mL 11 Within the range of less than 1 / mL.

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