Antibody titer-improving agent using lactic acid bacteria

Abstract

The present invention addresses the problem of providing, as an antibody titer-improving agent (e.g., an adjuvant) that continues the effect of vaccination by vaccines or the like, a safe, simple and economical antibody titer-improving agent (e.g., an adjuvant) that can be easily ingested by all ages, and a method for producing the same. The present invention relates to a method for increasing antibody titer and a method for enhancing the effect of a vaccine using Lactobacillus bulgaricus OLL1073R-1 or a culture thereof, and an antibody titer increasing agent, such as an adjuvant, comprising Lactobacillus bulgaricus OLL1073R-1 or a culture thereof.

Description

[0001] This case is a divisional application of Chinese invention patent application filed on August 26, 2014, with application number CN201480047180.7 and invention title "Antibody Titer Enhancer Using Lactic Acid Bacteria". Technical Field

[0002] This invention relates to antibody titer enhancers using lactic acid bacteria, such as adjuvants. Background Technology

[0003] It is known that respiratory infections such as the common cold or influenza, or gastrointestinal infections such as norovirus, can be fatal to the elderly. Furthermore, in recent years, pregnant women have been identified as having a risk of congenital diseases in their offspring due to rubella infection. Not only pregnant women themselves, but also those living with them are urged to take precautions against rubella. Vaccination is useful as a preventative measure against various infections in the elderly, pregnant women, and children, as well as against influenza and bacterial infections, and as a countermeasure against severe illness.

[0004] Furthermore, young people, such as students, generally have strong immune systems but are less aware of infectious disease prevention measures such as vaccination. However, it is known that young people can also experience a decline in immunity due to factors such as stress, making them more susceptible to infections. Therefore, strengthening immunity is necessary for all age groups. However, vaccination alone may not be sufficient to fully achieve its intended effect, as antibody titers may not be adequately increased and the duration of the effect may be short. Consequently, research has been conducted on various vaccine adjuvants.

[0005] Vaccines are a general term for antigens used to induce active immunization in humans or animals. They include live vaccines using attenuated live pathogens, inactivated or dead bacterial vaccines using killed viruses or pathogenic bacteria, component vaccines using these constituent parts, and toxoids obtained by inactivating toxins that are bacterial products. In addition, there are vaccines used in cancer treatment that use antigens (cancer antigens) that are not expressed in normal cells but are specifically expressed in cancer cells.

[0006] An infection prevention agent containing a component with a molecular weight of 1,000 to 200,000 or less among water-soluble components obtained by fermenting the underground stems of plants in the Dioscoreaceae family (Patent Document 1) is proposed.

[0007] In addition, to enhance immunity, an immune adjuvant that is administered nasally together with an immunogenic substance has been proposed, which contains a culture composition obtained from a culture of Aureobasidium sp. microorganisms as the active ingredient (Patent Document 2).

[0008] In addition, it is proposed that each 100g of food contains at least 10 6A food containing Lactobacillus rhamnosus GG strain and at least 0.5 g of β-glucan isolated from a natural source, and it is proposed to use it as an adjuvant to enhance the immune response to a vaccine (Patent Document 3).

[0009] In addition, a report stated that in healthy elderly individuals aged 70 and over, daily oral administration of a yogurt beverage containing Lactobacillus casei DN-114001, starting four weeks before influenza vaccination, significantly increased antibody titers against the vaccine strain compared to oral administration of a non-fermented milk beverage as an ineffective control (Non-Patent Literature 1).

[0010] In addition, there are reports that oral administration of Lactobacillus fermentum CECT5716 cells resulted in an increase in influenza-specific IgA (Non-Patent Literature 2).

[0011] In addition, there are reports of oral administration of Bifidobacterium animalis subsp. lactis (BB-12) (R) The bacterial cells of *Lactobacillus paracasei* and *Lactobacillus paracasei* subsp. *paracasei* (L. casei 431) (R) A yogurt beverage containing IgG, IgG1, and IgG3, which are specific to influenza, showed an increase (Non-Patent Literature 3).

[0012] In addition, it has been reported that acidic polysaccharides produced by Lactobacillus delbrueckiis sp. bulgaricus OLL1073R-1 and fermented milk made with Lactobacillus delbrueckiis sp. bulgaricus OLL1073R-1 as the starting strain have NK cell activation effects (Patent Document 4).

[0013] Existing technical documents

[0014] Patent documents

[0015] Patent Document 1: Japanese Patent Application Publication No. 04-235921

[0016] Patent Document 2: International Publication No. 2012 / 014978

[0017] Patent Document 3: Japanese Patent Publication No. 2008-529535

[0018] Patent Document 4: Japanese Patent No. 5177728

[0019] Non-patent literature

[0020] Non-patent literature 1: Boge T, Remigy M, Vaudaine S, Tanguy J, Bourdet-Sicard R, Vander Werf S. “A probiotic fermented dairy drink improves antibody response to influenza vaccination in the elderly in two randomised control trials” Vaccine 2009; 27: 5677-5684.

[0021] Non-patent literature 2: Olivares M, Diaz-Ropero MP, Sierra S, Lara-Villoslada F, Fonolla J, Navas M, Rodriguez JM, Xaus J. “Oral intake of Lactobacillus fermentum CECT5716 enhances the effects of influenza vaccination” Nutrition 2007; 23: 254-260

[0022] Non-patent literature 3: Rizzardini et al. “Evaluation of the immune benefits of two probiotic strain Bifdobacterium animalis ssp. lactis, BB-12 and Lactobacillus paracasei ssp. paracasei, L. casei 431 in an influenza vaccination model: a randomized, double-blind, placebo-controled study” Br. J. Nutr. 107: 876-884. 2012.

[0023] Non-patent literature 4: Minna K. Salminen, Hilpi Rautelinf, Soile Tynkkynen, Tuiia Poussa, Maiia Saxelinf, Ville Valtonen, and Asko Jarvinen. "Lactobacillus Bacteremia, Clinical Significance, and Patient Outcome, with Special Focus on Probiotic L.Rhamnosus GG" Clinical Infectious Diseases 2004:38(1January):62-69

[0024] Non-patent document 5: Merja Rautio, Hannele Jousimies-Somer, Heikki Kauma, IlmoPietarinen, Maija Saxelin, Soile Tynkkynen, and Markku Koskela. "Liver AbscessDue to a Lactobacillus rhamnosus Strain Indistinguishable from L.rhamnosusStrain GG" Clinical Infectious Diseases 1999;28:1159-60

[0025] Summary of the invention

[0026] The technical problem that the invention aims to solve

[0027] Although various studies and discussions have been conducted, none of them can be considered safe and easy adjuvants, and they may not be practical enough.

[0028] The invention described in Patent Document 1 requires lengthy cultivation and filtration processes, making its manufacturing method complex and its impact on antibody titers unknown. Furthermore, the invention described in Patent Document 2 administers a culture of *Brachystomata* microorganisms directly via nasal or oral administration during vaccination, facilitating adjuvant uptake. Additionally, the invention described in Patent Document 3 uses *Lactobacillus rhamnosus* GG strain, which, through β-glucan, increases the proliferation rate of *Lactobacillus rhamnosus* GG strain, suggesting its potential benefit as a vaccine adjuvant; however, its adjuvant effect is not confirmed, and no probiotic suitable as a starting material for fermented milk is provided. Furthermore, cases of sepsis and liver abscesses have been reported with *Lactobacillus rhamnosus* GG strain (Non-Patent Documents 4 and 5).

[0029] Furthermore, in Non-Patent Literature 1, the yogurt beverage containing *Lactobacillus casei* DN-114001, which demonstrates an adjuvant effect on influenza vaccines, also requires *Lactobacillus bulgaricus* and *Streptococcus thermophilus* during its fermentation. Additionally, Non-Patent Literatures 2 and 3, which respectively demonstrate the adjuvant effect on influenza vaccines using *Lactobacillus fermentum* CECT5716 and *Bifidobacterium animalis* subsp. *lactobacterium* (BB-12), also demonstrate an adjuvant effect on influenza vaccines. (R) ) and Lactobacillus paracasei subsp. paracasei (L. casei 431) (R) It is unclear whether it can be used as a starting material for fermented milk, etc.

[0030] Furthermore, the invention described in Patent Document 4 is an acidic polysaccharide produced by Lactobacillus bulgaricus OLL1073R-1 and a fermented milk made using Lactobacillus bulgaricus OLL1073R-1 as the starting strain, but the effect explored therein is the activation of NK cells, and the effect on antibody titer is unknown.

[0031] Although various adjuvants have been proposed as described above, there is still no adjuvant that is safe, easy to manufacture, readily available for all ages, and that provides sustained effects by enhancing the rise in antibody titers based on vaccination.

[0032] Therefore, the objective of this invention is to provide a safe, simple, and economical method for the production of an antibody titer enhancer, such as an adjuvant, that can be easily taken by people of all ages, based on the sustained effectiveness of vaccination such as vaccines.

[0033] Technical solutions adopted to solve technical problems

[0034] In order to solve the above-mentioned problems, the inventors conducted repeated and careful research and discovered that Lactobacillus bulgaricus OLL1073R-1, which can be used as the starting strain for fermented milk, has the effect of improving antibody titer, such as adjuvant effect. Further research was conducted and the present invention was completed.

[0035] That is, the present invention relates to the following. [1]

[0037] Antibody titer enhancer containing Lactobacillus bulgaricus OLL1073R-1 and / or its cultures. [2]

[0039] The antibody titer enhancer described in [1] is an adjuvant. [3]

[0041] The antibody titer enhancer described in [2] is a vaccine adjuvant. [4]

[0043] The antibody titer enhancer described in [3] is an adjuvant for infectious disease vaccines. [5]

[0045] As described in [4], the antibody titer enhancer is an infectious disease such as influenza. [6]

[0047] The antibody titer enhancer described in any of [1] to [5], wherein the culture is a composition obtained by culturing Lactobacillus bulgaricus OLL1073R-1 and Streptococcus thermophilus. [7]

[0049] The antibody titer enhancer described in any of [1] to [6], wherein one package contains 9 × 10⁻⁶ Lactobacillus bulgaricus OLL1073R-1 lactic acid bacteria. 7 ~10 12 CFU. [8]

[0051] The antibody titer enhancer described in any of [1] to [7], wherein each package contains an average of 10 to 1000 g or 10 to 1000 ml. [9]

[0053] Products obtained by packaging the antibody titer enhancer described in any one of [1] to [8].

[10]

[0055] Methods for increasing antibody titer that do not include medical procedures, including subject ingestion or administration of an antibody titer enhancer as described in any of [1] to [8].

[11]

[0057] The method described in

[10] involves an intake of 10–1000 g or 10–1000 ml of antibody titer enhancer per day for more than 3 weeks.

[12]

[0059] The method described in

[11] involves continuous intake of an antibody titer enhancer starting at least one week prior to the date of vaccination.

[0060] This invention also relates to the following.

[0061] [I]

[0062] An adjuvant containing Lactobacillus bulgaricus OLL1073R-1 and / or its culture.

[0063] [II]

[0064] The adjuvant as described in [I] is an adjuvant for influenza vaccines.

[0065] [III]

[0066] The adjuvant as described in [I] or [II], wherein the culture is a culture obtained using Lactobacillus bulgaricus OLL1073R-1 and Streptococcus thermophilus.

[0067] [IV]

[0068] The adjuvant as described in any of [I] to [III], wherein one package contains 9 × 10⁻⁶ lactic acid bacteria containing Lactobacillus bulgaricus OLL1073R-1. 7 CFU.

[0069] [V]

[0070] The adjuvant as described in any of [I] to [VI], wherein each package contains an average of 10 to 1000 g or 10 to 1000 ml.

[0071] [VI]

[0072] Methods for enhancing vaccine efficacy that do not include medical procedures, wherein the adjuvant is ingested by the subject or caused to ingest by the subject, as described in any one of [I] to [V].

[0073] [VII]

[0074] The method described in [VI] involves an intake of 10–1000 g or 10–1000 ml of culture per day for more than 3 weeks.

[0075] [VIII]

[0076] Methods that increase antibody titers, excluding medical procedures, include the intake or administration of Lactobacillus bulgaricus OLL1073R-1 and / or its culture to a subject.

[0077] [IX]

[0078] The method described in [VIII] is wherein the culture is a culture obtained using Lactobacillus bulgaricus OLL1073R-1 and Streptococcus thermophilus.

[0079] [X]

[0080] The method described in [VIII] or [IX], wherein the antibody titer is the influenza antibody titer.

[0081] [XI]

[0082] The method described in any of [VIII] to [X], wherein the intake of 10 to 1000 g or 10 to 1000 ml of culture per day is sustained for more than 3 weeks.

[0083] The effects of the invention

[0084] In this invention, *Lactobacillus bulgaricus* OLL1073R-1 and / or its cultures can be used not only for the prevention and treatment of diseases in medicine, but also as a specific health food and a general food for maintaining and enhancing health. In particular, it can increase antibody titers, maintain high titers, and enhance vaccine efficacy. Furthermore, *Lactobacillus bulgaricus* OLL1073R-1 is a strain that can be used as a starting material for fermented milk; therefore, this strain can also be used to provide safe and easily manufactured antibody titer enhancers, such as adjuvants, that can be readily ingested by people of all ages.

[0085] Brief description of the attached diagram

[0086] Figure 1-1 This is a graph showing the changes in antibody titers against H1N1 influenza A.

[0087] Figure 1-2 This is a graph showing the changes in antibody titers against influenza A (H3N2).

[0088] Figure 1-3 This is a graph showing the changes in antibody titers in influenza B.

[0089] Figure 2-1 This is a graph showing the changes in the seroconversion rate of antibodies against H1N1 influenza.

[0090] Figure 2-2 This is a graph showing the changes in the seroconversion rate of antibodies against influenza A (H3N2).

[0091] Figure 2-3This is a graph showing the changes in the seroconversion rate of influenza B antibodies.

[0092] Figure 3-1 This is a graph showing the changes in antibody retention rate for H1N1 influenza A.

[0093] Figure 3-2 This is a graph showing the changes in antibody retention rate for influenza A (H3N2). Figure 3-3 This is a graph showing the changes in antibody retention rate in influenza B.

[0094] Figure 4-1 This is a graph showing the IgG1 antibody titer against the OVA antigen one week after the second ovalbumin (OVA) immunization of mice (day 14).

[0095] Figure 4-2 This is a graph showing the IgG2a antibody titer against the OVA antigen one week after immunization of mice (day 14).

[0096] Figure 4-3 This is a graph showing the IgG2b antibody titer against the OVA antigen one week after immunization of mice (day 14).

[0097] Figure 5-1 This is a graph showing the IgG1 antibody titer against the OVA antigen 2 weeks after mice were immunized with OVA (day 21).

[0098] Figure 5-2 This is a graph showing the IgG2a antibody titer against the OVA antigen two weeks after mice were immunized with OVA (day 21).

[0099] Figure 5-3 This is a graph showing the IgG2b antibody titer against the OVA antigen two weeks after mice were immunized with OVA (day 21).

[0100] Figure 5-4 This is a graph showing the total IgE antibody titer two weeks after mice were immunized with OVA (day 21).

[0101] Figure 6 This is a schematic diagram showing the timeline for measuring influenza-specific antibody titers by blood collection and administration using the HI method at the following dates in Example 1: the start date of intake of the drinking form of yogurt R-1 and the null control beverage (Day 1), 1 week after influenza vaccination (Day 29), 5 weeks after influenza vaccination (Day 57), approximately 8 weeks after influenza vaccination (Day 76), and approximately 10 weeks after influenza vaccination (Day 89, the end date of intake of the drinking form of yogurt R-1 and the null control beverage).

[0102] Figure 7This demonstrates that in Example 2, Group 1 (unfermented milk intake group (ineffective control group)) was given 0.4 ml / animal orally via gastric tube once a day, while Group 2 (yogurt R-1 intake group (R-1 intake group)) was given 0.4 ml / animal orally via gastric tube once a day, containing yogurt R-1 (yogurt obtained by adding the starting bacteria of yogurt R-1 to unfermented milk and fermenting it). After 3 weeks (day 0), both Group 1 (unfermented milk intake group) and Group 2 (yogurt R-1 intake group) received 10 mg / animal (250 μl physiological saline solution) of ovalbumin (OVA) as a model antigen via intraperitoneal administration. Next, immunization was performed again the following week (day 7). One week after the second immunization (day 14), partial blood sampling was performed. Two weeks later (day 21), full blood sampling was performed from the axillary artery under isoflurane anesthesia.

[0103] Methods of implementing the invention

[0104] The "Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1" in this specification was domestically deposited on February 22, 1999, at the Patent Biological Collection Center of the National Institute of Advanced Industrial Science and Technology (IPOD, AIST) (No. 305-8566, Japan, 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Taiwan) under the Budapest Treaty, and was transferred to international collection on November 29, 2006, with the accession number FERM BP-10741.

[0105] As recorded in Budapest Notification No. 282 (http: / / www.wipo.int / treaties / en / notifications / budapest / treat y_budapest_282.html), the Patent Microbial Collection Center of the National Institute of Advanced Industrial Science and Technology (IPOD, NITE) inherited the patent microbial collection business from the Patent Biological Collection Center of the National Institute of Advanced Industrial Science and Technology (IPOD, AIST), and is therefore currently deposited at the Patent Microbial Collection Center of the National Institute of Advanced Industrial Science and Technology (IPOD, NITE) (Room 120, 2-5-8 Kazusa-Kamazu, Kisarazu City, Chiba Prefecture) (Collection No. FERM BP-10741).

[0106] Lactobacillus bulgaricus OLL1073R-1 and / or its cultures can increase antibody titers and maintain high titers.

[0107] In this invention, "culture" refers to a composition obtained by culturing *Lactobacillus bulgaricus* OLL1073R-1, including the culture itself obtained by culturing the bacterial cells, the culture obtained by separating and removing the bacterial cells through centrifugation or other methods, a concentrated culture, a diluted culture, and solids obtained by removing water from the culture, etc., all states of the composition obtained by processing the culture. In this specification, "culture" includes fermentation using *Lactobacillus bulgaricus* OLL1073R-1, and "culture" is the fermentation product of *Lactobacillus bulgaricus* OLL1073R-1, i.e., *Lactobacillus bulgaricus*.

[0108] The culture of *Lactobacillus bulgaricus* OLL1073R-1 can be carried out using conventional methods. For example, the cells can be cultured in a medium containing fresh milk (unpasteurized milk), pasteurized milk, whole milk concentrate, whole milk powder, skim milk, skim milk concentrate, skim milk powder, milk protein concentrate (MPC), whey, whey powder, desalted whey, desalted whey powder, whey protein concentrate (WPC), whey protein isolate (WPI), α-lactalbumin (α-La), β-lactalbumin (β-Lg), casein, sodium caseinate, calcium caseinate, cream, butter, sugars (including lactose), minerals, vitamins, and / or yeast extract, anaerobically at 35–45°C for 2–24 hours. A typical culture medium is one in which yeast extract is added to the product obtained by treating whey with protease, and the pH is adjusted to approximately 7.

[0109] In one embodiment of the present invention, the culture temperature of Lactobacillus bulgaricus OLL1073R-1 is preferably in the range of 30–45°C. More particularly, it is preferably in the range of 32–44°C, and even more preferably in the range of 34–43°C.

[0110] In one embodiment of the present invention, the culture time of Lactobacillus bulgaricus OLL1073R-1 is preferably in the range of 1 to 24 hours. In particular, it is more preferably in the range of 2 to 12 hours, and even more preferably in the range of 3 to 8 hours.

[0111] In one embodiment of the present invention, the culture temperature and the culture time can be any combination. From the viewpoints of manufacturing efficiency, the number of Lactobacillus bulgaricus OLL1073R-1 cells, the acidity of the culture (fermentation product), and flavor, it is preferable to culture (ferment) at 30-45°C for 1-24 hours, more preferably at 32-44°C for 2-12 hours, and even more preferably at 34-43°C for 3-8 hours.

[0112] In one embodiment of the invention, from the viewpoint of production efficiency, the culture is preferably a culture obtained using *Lactobacillus bulgaricus* OLL1073R-1 and *Streptococcus thermophilus*, for example, a composition obtained by culturing *Lactobacillus bulgaricus* OLL1073R-1 and *Streptococcus thermophilus*. In this invention, any strain of *Streptococcus thermophilus* can be used, but *Streptococcus thermophilus* OLS3059 is preferred.

[0113] The "Streptococcus thermophilus OLS3059" in this specification was domestically deposited on February 29, 1996, at the Patent Biology Collection Center of the National Institute of Advanced Industrial Science and Technology (IPOD, AIST) (Japan 305-8566, Higashi 1-1-1 Chuo 6, Tsukuba City, Ibaraki Prefecture) under accession number FERM P-15487 (accession date: February 29, 1996). It was transferred to international collection under the Budapest Treaty on November 29, 2006, and was granted accession number FERM BP-10740.

[0114] As recorded in Budapest Notification No. 282 (http: / / www.wipo.int / treaties / en / notifications / budapest / treaty_budapest_282.html), the Patent Microbial Collection Center of the National Institute of Advanced Industrial Science and Technology (IPOD, NITE) inherited the patent microbial collection business from the Patent Biological Collection Center of the National Institute of Advanced Industrial Science and Technology (IPOD, AIST), and is therefore currently deposited at the Patent Microbial Collection Center of the National Institute of Advanced Industrial Science and Technology (IPOD, NITE) (Room 120, 2-5-8 Kazusa-Kamazu, Kisarazu City, Chiba Prefecture) (Collection No. FERM BP-10740).

[0115] In this instruction manual, "antibody titer" refers to an indicator of the amount of antibody produced in response to an antigen. It can typically be measured by methods such as hemagglutination inhibition test (HI method), enzyme-linked immunosorbent assay (ELISA method), enzyme immunoassay (EIA method), and latex turbidimetric immunoassay (LTI method).

[0116] Examples of antibody titers include antibody titers against pathogens of infectious diseases caused by viruses or bacteria, such as influenza, measles, mumps, rubella, chickenpox, diphtheria, tetanus, and pertussis, or antibody titers against cancer antigens such as prostate cancer, but these are not limited to these examples. Examples of antibody titers include antibody titers against pathogens of diseases that can be prevented and treated by vaccines, and antibody titers against viruses that cause viral diseases. Examples of antibody titers against pathogens of infectious diseases for which vaccination is recommended, such as influenza, measles, and rubella, are particularly preferred.

[0117] In this specification, "antibody" refers to a glycoprotein molecule produced by B cells in lymphocytes that recognizes and binds to antigens. Furthermore, antibodies are classified into several categories (types) based on differences in their constant region structure. For mammals, based on differences in their constant region structure, they are classified into five categories of immunoglobulins: IgG, IgA, IgM, IgD, and IgE, and each category is further classified into subtypes. In this specification, "antibody" refers to the collective term for the aforementioned categories and subtypes.

[0118] The effector function of an antibody is governed by its Fc region and is largely dependent on the antibody class. Complement activation is limited to IgM and IgG antibodies; the ability to bind the variable region of an antibody to cellular degradation is specifically termed CDC (complement-dependent cytotoxicity). Furthermore, the Fc regions of IgG, IgE, and IgA antibodies bind to their respective specific Fc receptors, activating cells with Fc receptors and playing a role in the intercellular transport of antibodies. In particular, IgG antibodies activate their effector cells—T cells, NK cells, neutrophils, and macrophages—through Fc receptors, killing target cells bound by the antibody's variable region; this is known as ADCC (antibody-dependent cell-mediated cytotoxicity).

[0119] In one embodiment of the present invention, "antibody titer" is IgG antibody titer.

[0120] This invention provides a method for increasing antibody titer, comprising ingesting or induced ingestion of Lactobacillus bulgaricus OLL1073R-1 and / or its culture. Here, the method for increasing antibody titer includes both medical and non-medical procedures performed by a physician.

[0121] In this instruction manual, "ingestion" refers to the process of obtaining nutrients into the body, which may include methods such as food, inhalation, or tube feeding. Typical examples include oral intake and enteral intake, but are not limited to these.

[0122] In one embodiment of the invention, oral intake, particularly through diet, is preferred.

[0123] In this specification, "object" refers to the object to which the antibody titer enhancer, such as an adjuvant, of the present invention is applicable, and examples include mammals, such as humans, preferably humans.

[0124] In this specification, "cause intake" refers to the act of directly or indirectly inducing an object to take in something with a specific visual and / or auditory purpose or effect. Examples include the act of a manufacturer making a proposal, suggestion, or instruction to a salesperson or consumer with a specific purpose or effect, or a salesperson making a proposal to a consumer with a specific purpose or effect, inducing the object to take in something.

[0125] In addition, the present invention also provides an antibody titer enhancer comprising Lactobacillus bulgaricus OLL1073R-1 and / or its culture.

[0126] In this specification, "antibody titer enhancer" refers to a substance that induces antibody production, increases antibody titer, and maintains high titer, as well as compositions containing such substances.

[0127] In this specification, antibody titer enhancers include Lactobacillus bulgaricus OLL1073R-1 itself, cultures of Lactobacillus bulgaricus OLL1073R-1 itself, and compositions comprising them.

[0128] In this instruction manual, antibody titer enhancers may take any form, as long as they are suitable for intake. Examples include pharmaceuticals, quasi-drugs, health functional foods, specific health foods, nutritional functional foods, general foods, health supplements, health foods, nutritional supplements, enteral nutrition agents, oral cosmetics, and animal feed, but they are not limited to these.

[0129] In one embodiment of the present invention, from a preference point of view, the antibody titer enhancer may also be in the form of fermented milk.

[0130] In a preferred embodiment of the present invention, from the viewpoint of manufacturing efficiency, the antibody titer enhancer is a type of yogurt such as plain yogurt, hard yogurt, soft yogurt, or drinkable yogurt.

[0131] In one embodiment of the present invention, from the viewpoint of preservation, it may be in the form of granules, tablets, compressed sugar, capsules, etc.

[0132] In one embodiment of the present invention, when Lactobacillus bulgaricus OLL1073R-1 and / or its culture is in liquid form, from the viewpoint of ease of intake and antibody titer enhancement, it is ideal to consume 5 to 1000 ml per day, preferably 50 to 500 ml, and more preferably 100 to 200 ml.

[0133] In one embodiment of the present invention, when Lactobacillus bulgaricus OLL1073R-1 and / or its culture is in solid or semi-solid form, from the viewpoint of ease of intake and antibody titer enhancement, it is ideal to consume 5 to 1000g per day, preferably 50 to 500g, and more preferably 100 to 200g.

[0134] In one embodiment of the present invention, when Lactobacillus bulgaricus OLL1073R-1 and / or its culture are in dried form, from the viewpoint of ease of intake and antibody titer enhancement, it is ideal to consume 0.1 to 50 g per day, preferably 0.5 to 10 g, and more preferably 1 to 5 g.

[0135] In one embodiment of the present invention, an antibody titer enhancer that provides a stable antibody titer enhancement effect can be provided by packaging an amount suitable for a single intake.

[0136] In one embodiment of the present invention, the antibody titer enhancer can be packaged in an amount suitable for the above-mentioned daily intake.

[0137] Therefore, in one embodiment of the present invention, the antibody titer enhancer may be an antibody titer enhancer comprising the above-mentioned amount of Lactobacillus bulgaricus OLL1073R-1 and / or its culture.

[0138] The present invention also provides articles obtained by packaging antibody titer enhancers. The packaged articles can be made into articles that individually package a quantity suitable for a single intake, articles that package a quantity suitable for several days, such as a week's intake, or articles comprising multiple individually packaged articles, etc.

[0139] In one embodiment of the present invention, the antibody titer enhancer is contained in one package containing 9 × 10 7 CFU or higher, preferably 9×10 8 CFU or higher, preferably 9×10 9 Lactobacillus bulgaricus OLL1073R-1 lactic acid bacteria with a CFU or higher.

[0140] Alternatively, in one embodiment of the invention, the antibody titer enhancer is contained in one package containing 9 × 10 7 ~10 12 CFU, preferably 9×10 8 ~10 12 CFU, or better yet, 9×10 9 ~10 11 Lactobacillus bulgaricus OLL1073R-1 (CFU)

[0141] In this instruction manual, "single package" includes any form, including common packaging forms such as containers with caps, bottles with caps, individual bags, pouches, tubes, blister packs, ampoules, etc. It also includes packaging forms that can be ingested along with the packaging, such as capsules, rice paper, and other edible films.

[0142] In this specification, "packaging" includes packaging in any of the packaging forms described above.

[0143] In one embodiment of the present invention, the purpose of the product can be clearly defined by recording the product's use, efficacy, function, types of active ingredients, types of functional ingredients, methods of intake, etc., on a single package or a package containing multiple single packages, and / or by using additional information, such as an instruction manual, supplementary text, etc., and / or by separately publishing information such as brochures.

[0144] In one embodiment of the present invention, it is preferable that the antibody titer enhancer of the present invention includes a description of its use, efficacy, function, types of active ingredients, types of functional ingredients, and methods of administration. The "description" can adopt descriptions suitable for pharmaceuticals, quasi-pharmaceuticals, health functional foods, specific health foods, nutritional functional foods, general foods, health supplements, health foods, nutritional supplements, enteral nutrition agents, oral cosmetics, and animal feed.

[0145] In this application specification, "representation" includes all representations used to inform the intended recipient of the above description, as long as they allow the reader to conceive of or infer the content of the representation. This includes all representations, regardless of their purpose, content, object, or medium. For example, examples include representing the above description on the packaging or container of the product, displaying or publishing the above description in advertisements, price lists, or transaction documents related to the product, or providing information containing this content via electromagnetic (Internet, etc.) methods.

[0146] In one embodiment of the present invention, when the antibody titer enhancer is packaged as a food or beverage, it is preferable to manufacture a food or beverage that is labeled as intended for enhancing antibody titer, such as a food or beverage labeled "for enhancing antibody titer" that has an antibody titer-enhancing effect, a food or beverage labeled "for enhancing antibody titer" that contains Lactobacillus bulgaricus OLL1073R-1, or a food or beverage labeled "for improving immunity" that contains Lactobacillus bulgaricus OLL1073R-1, etc.

[0147] The text used for the above-mentioned notation is not limited to phrases such as "for increasing antibody titer" or "for improving immunity." Any other text that indicates an effect of increasing antibody titer is also included within the scope of this invention. Such text may, for example, be a notation for various purposes that makes the user aware of the effect of increasing antibody titer.

[0148] On the other hand, the content of the label is preferably a label that has been recognized by administrative agencies or the like (for example, a label that has been recognized based on various systems established by administrative agencies and is based on such recognition). It is also preferable to attach such label content to promotional materials and other documents at the point of sale, such as packaging, containers, catalogs, brochures, and POP displays.

[0149] Furthermore, examples can be given for labels such as pharmaceuticals, quasi-pharmaceuticals, health functional foods, foods for specific health uses, nutritional functional foods, general foods, health supplements, health foods, nutritional supplements, enteral nutrition products, oral cosmetics, and animal feed. In particular, examples can be given for labels concerning functionality that are recognized under the systems of various countries, such as labels recognized by the Consumer Affairs Agency of Japan, and labels recognized under the Foods for Specific Health Uses system and similar systems. Examples can be given for labels indicating that the product is a Food for Specific Health Uses, labels indicating that the product is a conditional Food for Specific Health Uses, labels indicating that the product affects the structure and function of the body, and labels indicating a reduction in the risk of disease. Specifically, typical examples can be given for labels indicating the product as a Food for Specific Health Uses as defined by the Health Promotion Law (especially labels indicating health uses) and similar labels.

[0150] In one embodiment of the present invention, in order to improve the antibody titer enhancement effect, the intake of Lactobacillus bulgaricus OLL1073R-1 and / or its cultures and antibody titer enhancers containing them is preferably sustained for more than 3 weeks, more preferably for more than 5 weeks, and more preferably for more than 8 weeks.

[0151] Lactobacillus bulgaricus OLL1073R-1 and / or its cultures can be safely ingested, therefore there is no specific upper limit to the duration of ingestion, and it can be sustained indefinitely. From the perspective of obtaining sufficiently effective antibody titers to enhance efficacy, a baseline of approximately 12 weeks can also be used.

[0152] In one embodiment of the present invention, the antibody titer enhancer is an adjuvant.

[0153] In this specification, "adjuvant" typically refers to an immune adjuvant, which is a component or composition containing such a component that enhances the production of antibodies in response to an administered antigen during immune induction.

[0154] In this specification, "adjuvant" includes Lactobacillus bulgaricus OLL1073R-1 itself, cultures of Lactobacillus bulgaricus OLL1073R-1 itself, and compositions containing them.

[0155] In this invention, the adjuvant can take any form, such as pharmaceuticals, quasi-drugs, health functional foods, specific health foods, nutritional functional foods, general foods, health supplements, health foods, nutritional supplements, enteral nutrition agents, oral cosmetics, feed, etc., but is not limited to these.

[0156] In one embodiment of the present invention, from a preference point of view, the adjuvant may be taken directly in the form of fermented milk.

[0157] In a preferred embodiment of the present invention, from the viewpoint of manufacturing efficiency, the adjuvant is a type of yogurt such as plain yogurt, hard yogurt, soft yogurt, or drinkable yogurt.

[0158] In one embodiment of the present invention, from the viewpoint of preservation, it may be in the form of tablets, compressed sugar, capsules, etc.

[0159] The effectiveness of a vaccine can be enhanced by ingesting or consuming the antibody titer enhancer of the present invention, such as an adjuvant.

[0160] In this invention, "vaccine" refers to a pharmaceutical composition used for the prevention and treatment of infectious diseases. Furthermore, the vaccine may also contain cancer antigens, and includes vaccines used as cancer vaccines in cancer treatment. A vaccine may contain one or more pathogens that eliminate or attenuate pathogenicity, cancer antigens, and one or more common adjuvants, carriers, or other additives.

[0161] Specific examples of vaccines include influenza vaccines, measles vaccines, mumps vaccines, rubella vaccines, MR vaccines, MMR vaccines, varicella vaccines, MMRV vaccines, diphtheria vaccines, tetanus vaccines, pertussis vaccines, DTP vaccines, inactivated poliovirus vaccines, hepatitis B virus vaccines, meningococcal conjugate vaccines, RS virus vaccines, human papillomavirus vaccines, cancer vaccines, and many more, not limited to these.

[0162] In this instruction manual, "enhanced vaccine efficacy" refers to an increase in the antibody titer specific to the vaccine strain.

[0163] In this instruction manual, "methods to enhance vaccine efficacy" include both medical and non-medical procedures performed by physicians.

[0164] The antibody titer enhancer of the present invention, such as an adjuvant, may be in the form of a mixture with the vaccine or in a separate form different from the vaccine. In one embodiment of the present invention, because it has advantages such as long-term uptake and compatibility with various types and forms of vaccines, it is preferable to adopt a separate form different from the vaccine.

[0165] The antibody titer enhancer of the present invention, such as an adjuvant, can also be taken simultaneously with vaccination or before or after vaccination.

[0166] In one embodiment of the present invention, in order to improve the antibody titer enhancement effect, such as the adjuvant effect, the antibody titer enhancer, such as the adjuvant, is ingested for at least 4 weeks, preferably at least 6 weeks, and more preferably at least 8 weeks before and / or after vaccination.

[0167] In one embodiment of the present invention, in order to improve the antibody titer enhancement effect, such as the adjuvant effect, the antibody titer enhancer, such as the adjuvant, is ingested at least one week, preferably at least two weeks, and more preferably at least three weeks before the date of vaccination.

[0168] In one embodiment of the present invention, in order to improve the antibody titer enhancement effect, such as the adjuvant effect, the antibody titer enhancer, such as the adjuvant, is continuously ingested for more than 3 weeks from the date of vaccination, preferably more than 3 weeks, and more preferably more than 5 weeks.

[0169] In one embodiment of the present invention, when the antibody titer enhancer, such as an adjuvant, is in liquid form, from the viewpoint of ease of intake and antibody titer enhancement effect, such as adjuvant effect, it is ideal to take 10 to 1000 ml of the antibody titer enhancer, such as an adjuvant, daily, preferably 50 to 500 ml, and more preferably 100 to 200 ml.

[0170] In one embodiment of the present invention, when the antibody titer enhancer, such as an adjuvant, is in solid or semi-solid form, from the viewpoint of ease of intake and antibody titer enhancement effect, such as adjuvant effect, it is ideal to take 10 to 1000g, preferably 50 to 500g, and more preferably 100 to 200g of the antibody titer enhancer, such as an adjuvant, daily.

[0171] In one embodiment of the present invention, when the antibody titer enhancer, such as an adjuvant, is in dried form, from the viewpoint of ease of intake and antibody titer enhancement effect, such as adjuvant effect, it is ideal to take 0.1 to 50 g of the antibody titer enhancer, such as an adjuvant, daily, preferably 0.5 to 10 g, and more preferably 1 to 5 g.

[0172] In one embodiment of the invention, the antibody titer enhancer, such as an adjuvant, is provided in a packaged form with an amount suitable for a single intake, which can more accurately achieve the effects of the invention.

[0173] Therefore, in one embodiment of the present invention, the antibody titer enhancer, such as an adjuvant, can be packaged in an amount suitable for the above-mentioned daily intake.

[0174] In one embodiment of the present invention, an antibody titer enhancer, such as an adjuvant, is contained in a single package containing 9 × 10 7 CFU or higher, preferably 9×10 8 CFU or higher, preferably 9×10 9 Lactobacillus bulgaricus OLL1073R-1 lactic acid bacteria with a CFU or higher.

[0175] Alternatively, in one embodiment of the invention, the antibody titer enhancer, such as an adjuvant, is contained in a single package containing 9 × 10 7 ~10 12 CFU, preferably 9×10 8 ~10 12 CFU, or better yet, 9×10 9 ~10 11 Lactobacillus bulgaricus OLL1073R-1 (CFU)

[0176] In this invention, in order to improve the antibody titer enhancement effect, such as the adjuvant effect, the intake of the antibody titer enhancer, such as the adjuvant, is ideally sustained for more than 3 weeks, preferably more than 5 weeks, and more preferably more than 8 weeks, regardless of whether it is before or after vaccination.

[0177] Regardless of whether vaccination has been conducted, there is no particular upper limit to the duration of intake of the antibody titer enhancer, such as the adjuvant, of the present invention for safe consumption; it can be sustained indefinitely. From the viewpoint of obtaining a sufficiently effective antibody titer enhancer effect, such as the adjuvant effect, a period of approximately 12 weeks or less can also be used as a benchmark.

[0178] One embodiment of this invention includes food and beverage products, fermented foods, and fermented beverages containing Lactobacillus bulgaricus OLL1073R-1 or its cultures. Examples of fermented foods and beverages include yogurt, cheese, Korean kimchi, Western-style kimchi, German sauerkraut, Indian yogurt, and other lactic acid fermented foods and beverages.

[0179] In one embodiment of the invention, from a preferred viewpoint, the culture of Lactobacillus bulgaricus OLL1073R-1 is preferably fermented milk itself.

[0180] In one embodiment of the invention, from the viewpoint of production efficiency and preference, it is preferable to use fermented milk obtained from Lactobacillus bulgaricus OLL1073R-1 and Streptococcus thermophilus, for example, fermented milk obtained by using Lactobacillus bulgaricus OLL1073R-1 and Streptococcus thermophilus as the starting strain as the culture medium.

[0181] In this specification, "fermented milk" refers to products obtained by fermenting milk, including "fermented milk," "lactic acid bacteria beverages," "milk beverages," and "natural cheeses" as defined by the Ministry Ordinance concerning the composition standards of milk and dairy products (Milk Ordinance). For example, fermented milk refers to "fermented milk" as defined by the Milk Ordinance, which is a product obtained by fermenting raw milk, cow's milk, special cow's milk, raw goat's milk, pasteurized goat's milk, raw sheep's milk, ingredient-adjusted milk, low-fat milk, non-fat milk, and processed milk, or milk containing an equal or greater amount of non-fat milk solids, through lactic acid bacteria or yeast, to form a solid (hard), paste (soft), or liquid (drinking) form, or by freezing them.

[0182] A typical example of fermented milk is yogurt. The international standard defined by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) also stipulates that "a product referred to as yogurt is made from milk and dairy products such as skim milk powder through lactic acid fermentation by two bacteria, Streptococcus thermophilus and Lactobacillus bulgaricus, and the final product contains a large amount of these two bacteria." In this specification, "yogurt" includes the yogurt defined by the FAO / WHO.

[0183] In one embodiment of the present invention, the fermented milk is preferably made using Lactobacillus bulgaricus as the starting bacteria, especially plain yogurt, hard yogurt (set yogurt), soft yogurt, drinking yogurt, and other yogurts.

[0184] In one embodiment of the present invention, from the viewpoint of production efficiency and preference, fermented milk is preferably fermented milk made from Lactobacillus bulgaricus OLL1073R-1 and Streptococcus thermophilus as the starting bacteria.

[0185] The present invention will now be described in more detail based on the embodiments, but the present invention is not limited to these embodiments and various modifications can be made without departing from the technical concept of the present invention. Example

[0186] [Manufacturing Example 1] Manufacturing of Lactobacillus bulgaricus OLL1073R-1 culture and null control

[0187] Lactobacillus bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059 were added as starting bacteria to mixture A, which contains dairy products, sugar, and raw water. The mixture was fermented at 43°C for 4 hours. Then, mixture B, which contains glucose fructose syrup, sugar, pectin, flavoring, stevia, and raw water, was added to produce a drinking-type yogurt (hereinafter referred to as "drinking-type yogurt R-1").

[0188] Mix the same raw materials as those in Mixture A and Mixture B above in the same proportions, add lactic acid to produce a yogurt beverage (hereinafter referred to as the "invalid control beverage").

[0189] The drinking and null control beverages of Yogurt R-1 were filled into capped plastic bottles at 112 ml each.

[0190] [Example 1]

[0191] <Intake Plan>

[0192] A randomized, double-blind, null control trial was conducted on 40 male college students (mean age: 19.4 years (18-25 years)).

[0193] Subjects were divided into two groups: one group consumed one bottle (112 ml) / day of the beverage form of yogurt R-1 prepared in Example 1 (R-1 intake group), while the other group consumed one bottle (112 ml) / day of the null control beverage prepared in Example 1 (null control group). These intake periods were 8 weeks before and 2 weeks after the winter break. Three weeks after the start of the trial, participants received a combination of influenza vaccines (H1N1, H3N2, and influenza B).

[0194] <Evaluation Items>

[0195] Blood samples were collected on the first day of intake of the drinking version of Yogurt R-1 and the null control beverage (day 1), one week after influenza vaccination (day 29), five weeks after influenza vaccination (day 57), approximately eight weeks after influenza vaccination (day 76), and approximately ten weeks after influenza vaccination (day 89, the end day of intake of the drinking version of Yogurt R-1 and the null control beverage). The titer of influenza-specific antibodies was determined by the HI method.

[0196] These schedules are shown in Figure 6 .

[0197] As a result, approximately 8 weeks after influenza vaccination (a mixture of influenza A (H1N1), influenza A (H3N2), and influenza B), the R-1 uptake group showed significantly higher antibody titers for influenza A (H3N2) compared to the ineffective control group. Figure 1-2 ).

[0198] At this point, compared with the ineffective control group, the overall antibody titer for H1N1 influenza A in the R-1 intake group showed a low value. Figure 1-1 ).

[0199] In addition, the antibody titer for influenza B was significantly higher in the R-1 intake group compared to the ineffective control group. Figure 1-3 ).

[0200] Here, "antibody titer" has the following meaning.

[0201] <Antibody titer>

[0202] After infection with a virus, antibodies are produced in the serum. Antibody titer is an indicator of the amount of antibodies produced against antigens such as viruses.

[0203] For example, influenza viruses have the property of causing red blood cells in animals to agglutinate. If antibodies are present in the blood, the viral antigens react with the antibodies to form an antigen-antibody reaction, inhibiting the agglutination ability of red blood cells. The method that utilizes this property to determine the amount of antiviral antibodies in the blood is the erythrocyte agglutination inhibition test, which is used to determine the "antibody titer" of viruses such as measles and rubella.

[0204] Specifically, the serum was diluted to a specified dilution factor, and a certain amount of virus was added to allow it to react. A suspension of red blood cells was then added, and the dilution factor at which red blood cell agglutination was inhibited was observed. The final dilution factor at which red blood cell agglutination was completely inhibited was taken as the HI "antibody titer".

[0205] On the other hand, approximately 5 weeks after influenza vaccination (a mixture of influenza A (H1N1), influenza A (H3N2), and influenza B), the R-1 uptake group showed significantly higher seroconversion rates for influenza A (H1N1) and influenza A (H3N2) antibodies compared to the ineffective control group. Figure 2-1 , 2-2 ).

[0206] Furthermore, regarding the seroconversion rate of influenza A (H3N2), in the R-1 intake group, approximately 8 weeks after influenza vaccination, it was higher than the international standard (European Medicines Agency standard) of 40% for vaccine effectiveness, while in the ineffective control group, it did not exceed 40% after influenza vaccination. Figure 2-2 ).

[0207] Regarding the seroconversion rate of influenza B antibodies, the R-1 uptake group consistently maintained a rate above 40% after influenza vaccination, while the ineffective control group showed a rate below 40% approximately 8 weeks after influenza vaccination. Figure 2-3 ).

[0208] Figures 2-1 to 2-3 The dashed line representing 40% indicates the international standard for vaccine effectiveness (the standard of the European Medicines Agency).

[0209] Here, "antibody seroconversion rate" has the following meaning.

[0210] <Antibody seroconversion rate>

[0211] The antibody seroconversion rate is the proportion of subjects whose HI "antibody titer" was less than 10 before vaccination and greater than 40 after vaccination, or whose HI "antibody titer" was greater than 10 before vaccination and increased more than fourfold after vaccination.

[0212] For both influenza A H1N1 and influenza A H3N2, the antibody retention rates, in both the R-1 uptake group and the ineffective control group, were higher than 70% from before influenza vaccination, which is considered an international standard for vaccine effectiveness (the European Medicines Agency standard). Figure 3-1 , Figure 3-2 ).

[0213] Regarding the antibody retention rate for influenza B, in the R-1 uptake group, it was higher than 70%, the standard for influenza vaccine effectiveness, at 1 week and approximately 5 weeks after vaccination, while in the ineffective control group, it did not exceed 70% after influenza vaccination. Figure 3-3 ).

[0214] Figures 3-1 to 3-3 The dashed line indicating 70% represents the international standard for vaccine effectiveness (the standard of the European Medicines Agency).

[0215] Here, "antibody retention rate" has the following meaning.

[0216] <Antibody retention rate>

[0217] The antibody retention rate is the proportion of people whose HI "antibody titer" is more than 40 times higher.

[0218] The novel influenza A (H3N2) and influenza B strains suggest the possibility that the intake of Lactobacillus bulgaricus OLL1073R-1 and / or its cultures may contribute to higher antibody titers in the new influenza vaccines, such as through adjuvant effects.

[0219] The results above show that even for young people with typically strong immune systems, such as students, antibody titers are increased by ingesting Lactobacillus bulgaricus OLL1073R-1 and / or its culture as described in this invention.

[0220] It can increase antibody titer in young people, so it is speculated that it can lead to increased antibody titer in all age groups, including the elderly, pregnant women, and children.

[0221] [Example 2]

[0222] <Animal Experimentation Plan>

[0223] Four-week-old female BALB / c mice (Charles River Co., Ltd., Japan) were allowed free access to MF solid feed (Oriental Yeast Industry Co., Ltd.) and UV-sterilized water for 7 days, and were then acclimatized through routine feeding. Based on average weight and similar deviations, they were divided into groups of 8 and 10 mice. Until the end of the experiment, Group 1 (unfermented milk intake group (ineffective control group)) was given 0.4 ml / mouse once daily via gastric tube to receive unfermented milk (cow's milk, skim milk powder, sugar, and sweetener). Group 2 (yogurt R-1 intake group (R-1 intake group)) was given 0.4 ml / mouse once daily via gastric tube to receive yogurt R-1 (yogurt obtained by adding the starting bacteria of yogurt R-1 to unfermented milk and fermenting it). Three weeks after administration (day 0), both group 1 (unfermented milk intake group) and group 2 (yogurt R-1 intake group) received 10 mg / animal (250 μl saline solution) of ovalbumin (OVA) as a model antigen via intraperitoneal administration. Subsequently, immunization was repeated every other week (day 7). Partial blood collection was performed one week after the second immunization (day 14), and complete blood collection was performed two weeks later (day 21) via axillary artery under isoflurane anesthesia. The collected blood samples were centrifuged, plasma was prepared, and the OVA-specific antibody titer in the plasma was determined by ELISA. The antibody titers of group 1 (unfermented milk intake group) and group 2 (yogurt R-1 intake group) were compared. Statistical analysis was performed using a t-test.

[0224] These schedules are shown in Figure 7 .

[0225] <ELISA assay>

[0226] The ELISA assays used were commercially available ELISA kits from Bethyl Laboratories, Inc.: Mouse IgG1 ELISA Quantitative Kit (catalog number E90-105), Mouse IgG2a ELISA Quantitative Kit (catalog number E90-107), Mouse IgG2b ELISA Quantitative Kit (catalog number E90-109), and Mouse IgE ELISA Quantitative Kit (catalog number E90-115). Except for the initial application of OVA to the ELISA plate, the assay was performed essentially according to the described ELISA kit instructions and standard ELISA methods. The sample plates used were F96 CERT. MAXISORPNUNC-IMMUNO PLATE (NUNC Corporation, catalog number 439454). For IgE antibody titers, the assay was performed according to the described ELISA kit instructions.

[0227] Add 100 μg / ml of OVA dissolved in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6) to the sample plate at 100 μl / well (for IgE-only ELISA assays, add 100 μl / well of the coating antibody solution provided with the ELISA kit to the sample plate). Prepare the coating buffer as described in the ELISA kit instructions. Incubate the sample plate overnight at 4°C. After washing four times with washing buffer (1×TBS, 0.05% Tween-20), add 200 μl / well of blocking solution (1% BSA, 1×TBS) and incubate for 30 minutes at room temperature. After washing four times with washing buffer, add 100 μl / well of standard sample and incubate for 1 hour at room temperature. After washing four times with washing buffer, add 100 μl / well of secondary antibody and incubate the sample plate for 1 hour at room temperature. After washing four times with washing buffer, add substrate solution (BD OptEIA) at 100 μl / well. (R) The TMB substrate reagent kit (BD Bioscience, catalog number 555214) was incubated at room temperature for 15 minutes in the dark. 100 μl of stop solution (0.18 M H₂SO₄) was added per well, and the absorbance (OD) was set to 450 nm for each sample. 1×TBS was prepared by diluting 10×TBS (500 mM Tris, 1.4 M NaCl, pH 8.0). Blocking solution was also used for sample dilution and secondary antibody solution preparation. The results of the above ELISA assays are shown in [image / description]. Figures 4-1 to 4-3 and Figures 5-1 to 5-4 .

[0228] <Calculation of Antibody Titer>

[0229] The antibody titer in plasma was calculated by creating a calibration curve through serial dilution of a pre-prepared high-concentration standard plasma containing OVA-specific antibodies. In this case, the IgG1 antibody titer in the standard plasma was set to 10. 7 Any unit (AU) / ml, IgG2a antibody titer set to 10. 6 It is calculated from AU / ml.

[0230] <Preparation of Standard Plasma>

[0231] The preparation of standard plasma was carried out through the following steps. Five 4-week-old female BALB / c mice (Charles River Co., Ltd., Japan) were allowed free access to MF solid feed (Oriental Yeast Industry Co., Ltd.) and UV-sterilized water for 7 days, and were acclimatized through routine feeding. After acclimatization (day 0), 100 μg / mouse (250 μl Freund's complete adjuvant emulsion) of OVA was administered intraperitoneally. Two weeks after the first immunization (day 14), 100 μg / mouse (250 μl Freund's incomplete adjuvant emulsion) of OVA was administered intraperitoneally. Two weeks after the second immunization (day 28), whole blood was collected from the axillary artery under isoflurane anesthesia. The collected blood was centrifuged to prepare plasma, and the titers of OVA-specific IgG1 and IgG2a antibodies in the plasma were determined by ELISA. Based on the results, plasma with high OVA-specific antibody titers was used as standard plasma.

[0232] One week after OVA immunization (day 14), antibody titers of IgG1 and IgG2b against the OVA antigen did not show significant differences between the ineffective control group and the R-1 uptake group, but a tendency for higher mean titers was observed. Figure 4-1 and Figure 4-3 The antibody titer for IgG2a against the OVA antigen was significantly higher in the R-1 uptake group compared to the ineffective control group. Figure 4-2 ).

[0233] Furthermore, two weeks after OVA immunization (day 21), there was no significant difference in antibody titers of IgG1 against the OVA antigen between the ineffective control group and the R-1 uptake group. However, compared with the ineffective control group, the R-1 uptake group showed a tendency for a higher average antibody titer. Figure 5-1 The antibody titer for IgG2a against the OVA antigen was significantly higher in the R-1 uptake group compared to the ineffective control group. Figure 5-2 The antibody titer for IgG2b against the OVA antigen was significantly higher in the R-1 uptake group compared to the ineffective control group. Figure 5-3 ).

[0234] Furthermore, when comparing antibody titers one week (day 14) and two weeks (day 21) after OVA immunization, the antibody titers of IgG1, IgG2a, and IgG2b remained high in the R-1 intake group, especially the antibody titers of IgG2a and IgG2b, which remained significantly high in the R-1 intake group. Therefore, this demonstrates that antibody production is maintained at a high level through ingestion of Lactobacillus bulgaricus OLL1073R-1 and / or its culture.

[0235] Two weeks after OVA immunization (day 21), no significant difference was observed in total IgE antibody titer between the ineffective control group and the R-1 uptake group. Figure 5-4Based on this result, it can be inferred that the intake of R-1 does not lead to an increase in IgE antibody titer.

[0236] The following results show that ingestion of Lactobacillus bulgaricus OLL1073R-1 and / or its cultures significantly induced antibody production of IgG, particularly IgG2a and IgG2b. Furthermore, antibody production to maintain these antibodies was shown to be at high levels.

[0237] It is hypothesized that the uptake of Lactobacillus bulgaricus OLL1073R-1 and / or its culture increases the production of antibodies against IgG2a and IgG2b, which possess complement activation capabilities and the ability to activate effector cells such as T cells, NK cells, neutrophils, and macrophages, as well as enhancing CDC and ADCC activities. Therefore, it is anticipated that the uptake of this culture will be beneficial for the comprehensive prevention of infectious diseases and the prevention of severe illness. Furthermore, since the uptake of this culture increases the production of various antibodies in mice, it is hypothesized that the production of various antibodies in humans will also increase, potentially contributing to the comprehensive prevention of infectious diseases and the prevention of severe illness.

[0238] Based on the above results, it is hoped that Lactobacillus bulgaricus OLL1073R-1 and / or its cultures can provide safe and simple antibody titer enhancers, such as adjuvants, that can induce antigen-specific immune responses.

Claims

1. Uses of Lactobacillus delbrueckiis sp. bulgaricus OLL1073R-1 and / or its cultures, wherein, The Lactobacillus delbrueckiis sp. bulgaricus OLL1073R-1 and / or its culture are used to manufacture a vaccine adjuvant for increasing antibody titers in the blood; the vaccine adjuvant is an adjuvant for an infectious disease vaccine; the infectious disease is influenza; The influenza strains are selected from the following group: influenza A (H3N2), influenza B, or a combination thereof; Furthermore, the antibody seroconversion rate of the vaccine reaches 40%, where 40% is the international standard threshold for vaccine effectiveness.

2. The use as described in claim 1, wherein, The influenza referred to are influenza A (H3N2) and influenza B.

3. The use as described in claim 1, wherein, The influenza mentioned is influenza A, H3N2.

4. The use as described in claim 1, wherein, The influenza mentioned is influenza B.

5. The use as described in claim 1, wherein, The culture is a combination obtained by culturing Lactobacillus delbrueckiis sp. bulgaricus OLL1073R-1 and Streptococcus thermophilus.

6. The use as described in claim 1, wherein, One package of adjuvant contains 9 × 10⁶ lactic acid bacteria containing Lactobacillus bulgaricus OLL1073R-1. 7 ~10 12 CFU.

7. The use as described in claim 6, wherein, Each package contains an average of 10–1000g or 10–1000ml of adjuvant.

8. The use as described in claim 6, wherein, Each package contains an average of 100–200g or 100–200ml of adjuvant.

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