HK1171961B - Prophylactic composition for influenza infection - Google Patents

Description

Composition for preventing influenza infection

Technical Field

The present invention relates to compositions comprising a propionic acid bacterial culture. Alternatively, the present invention relates to a composition for preventing influenza infection or a method for preventing influenza infection.

Background

Influenza is a highly contagious infection in which the influenza virus is the causative agent. Influenza viruses are negative-strand single-stranded RNA viruses belonging to the Orthomyxoviridae family (Orthomyxoviridae family). Orthomyxoviridae consist of a single genus of influenza virus (one genus and one family). There are three subtypes of influenza a to c viruses, based on differences in their nucleoprotein antigenicity. Influenza a viruses are capable of infecting a wide variety of organisms, such as mammals and birds, as opposed to type b and type c, which infect humans alone as hosts. Therefore, it can easily form mutant viruses having different pathogenicity and antigenicity by undergoing gene hybridization in birds and mammals.

For example, swine influenza is a recent global epidemic that is developed by influenza a subtype H1N1 infection. Swine influenza virus is thought to infect humans, resulting in pandemics. In addition, highly virulent avian influenza virus, which is expected to become a pandemic, can cause a pandemic when it acquires infectivity in humans (Sugawara, t.

Therapeutic agents for inhibiting influenza virus are now commercially available. It is said that when a commercially available influenza therapeutic agent is appropriately administered at an early stage after infection, a sufficient therapeutic effect can be expected. However, influenza remains a dangerous infection, especially in high risk groups including the elderly, children, and people with chronic illnesses. In addition, the emergence of viruses resistant to therapeutic agents has been reported.

Even in the present days with developed therapeutic agents, it is important to vaccinate influenza vaccines against influenza exacerbation. Immunization with influenza vaccines induces neutralizing antibodies against influenza virus, which can prevent virus growth after infection and prevent spread of infection (C, Avendano et al and Zhu Q et al). When the virus has similar antigenicity to that of a virus administered as a vaccine, the effect of preventing exacerbation is said to be high. It is reported that the rate of induction in the elderly is low (17% to 53%) when compared to the rate of induction of neutralizing antibodies by vaccination in healthy adults (70% to 90%) (Goodwin K, etc.). The immune system in the elderly is weakened and may lead to exacerbations and death when the infection occurs. In view of this, it is particularly important to increase the induction rate of neutralizing antibodies in the elderly.

Documents of the prior art

[ patent document ]

[ patent document 1] WO 2001/028547

[ patent document 2] WO 2004/085364

[ patent document 3] WO 2007/023935

[ patent document 4] WO 2005/094850

[ non-patent document 1] Sugawara T. et al, Kansenso-shi (the Journal of infection),2008,82:427-

[ non-patent document 2] World Health Organization: department of communicative Disease Surveillance and Response Global Influenza Programmed,2005.

[ non-patent document 3] C, Avendano et al, J Parenter Enteral Nutr.2004;28:348-54.

[ non-patent document 4] Zhu Q, et al, Biochem Biophys Res Commun.2005;329:87-94.

[ non-patent document 5] Goodwin K, et al, vaccine.2006Feb20;24(8):1159-69.

Disclosure of Invention

[ problem to be solved by the invention ]

It is an object of the present invention to provide a composition comprising a propionic acid bacteria culture. Specifically, the present invention aims to provide a composition having a preventive effect against influenza virus infection. It is another object of the present invention to provide a composition having an intestinal function-regulating effect.

[ means for solving the problems ]

The prophylactic effect of influenza vaccines against viral infection is mainly dependent on the induction of virus neutralizing antibodies. Therefore, stimulating the induction of neutralizing antibodies after vaccination is considered to be effective in enhancing the prophylactic effect of the vaccine. Alternatively, virus-neutralizing antibodies induced as a normal immune response in a patient during a viral infection are important biological defense mechanisms for the patient. The present inventors have conducted extensive studies on components capable of enhancing the induction of virus-neutralizing antibodies caused by influenza vaccines and viral infections. As a result, the present inventors revealed that virus-neutralizing antibody induction at the time of influenza vaccination is enhanced in animals administered with a specific microorganism culture, and thus completed the present invention. In particular, the present invention relates to the following compositions and their uses:

[1] a composition for preventing influenza infection comprising a culture of propionic acid bacteria.

[2] The composition of [1], wherein the propionic acid bacterium is propionibacterium freudenreichii.

[3] The composition of [1], additionally comprising a milk fermentation component and/or an oligosaccharide.

[4] [3] the composition according to, wherein the milk fermentation component is milk or a mixture thereof produced by fermenting milk using a lactic acid bacterium belonging to the genus Lactobacillus and/or a lactic acid bacterium belonging to the genus Streptococcus.

[5] [3] the composition of wherein the milk fermentation component is an unripe cheese.

[6] [3] the composition according to any one of the preceding claims, wherein at least one of the saccharides constituting the oligosaccharide is galactose.

[7] The composition of [3], wherein the composition comprises a propionic acid bacteria culture and a milk fermentation component, and both have been sterilized.

[8] The composition of [1], for enteral administration to an animal receiving influenza vaccination.

[9] A composition of [3], comprising the following nutrients:

a propionic acid bacterial culture;

a milk fermentation component;

an oligosaccharide;

a protein;

a saccharide;

a lipid; and

dietary fiber.

[10] The composition of [9], additionally comprising at least one nutrient selected from the group consisting of: vitamins, minerals, organic acids and organic bases.

[11] An agent for promoting induction of influenza virus neutralizing antibodies in an animal vaccinated with an influenza vaccine comprising a propionic acid bacteria culture.

[12] A method of preventing influenza infection comprising the steps of:

(1) administering a propionic acid bacteria culture to an animal; and

(2) animals were vaccinated with influenza vaccine.

[13] The method for preventing influenza infection described in [12], wherein the step (1) is performed at least once before the step (2), after the step (2), or simultaneously with the step (2).

[14] The method for the prevention of influenza infection described in [12], wherein the propionic acid bacteria culture is administered together with a milk fermentation component and/or an oligosaccharide in step (1).

[15] A composition comprising the following components:

(a) an oligosaccharide;

(b) a milk fermentation component; and

(c) a propionic acid bacterial culture.

[16] [15] the composition according to any one of the preceding claims, wherein at least one of the saccharides constituting the oligosaccharide is galactose.

[17] The composition of [15], which is a composition for modulating intestinal function.

[ Effect of the invention ]

The composition of the present invention enhances the effect of influenza vaccines in inducing neutralizing antibodies. The composition of the present invention can be prepared by mixing components and the like which have been administered to the human body as liquid food. Therefore, the high safety of the composition of the present invention has been secured. Accordingly, the composition of the present invention may be administered not only simultaneously at the time of inoculation but also continuously around the time of inoculation. For continuous administration of the composition of the present invention, a sustained action of promoting the induction of neutralizing antibodies can be expected.

Adjuvants are often incorporated into vaccines to enhance the immune response against viral vaccines, such as influenza virus. Needless to say, a high level of safety is required for adjuvants incorporated into vaccines. Therefore, the development of new adjuvants requires careful safety checks. In contrast, since all the individual components constituting the composition of the present invention have been consumed as food, it proves that there is no risk even when they are continuously administered enterally to the human body. In addition, the compositions of the present invention may be administered enterally in combination with known adjuvants. That is, the present invention is significant not only in terms of safety but also in providing a new option as a means for enhancing an immune response against a vaccine.

In a preferred embodiment, the composition of the invention is provided in combination with components already widely consumed as liquid food or foodstuff. Therefore, continuous consumption of the composition of the present invention as a liquid diet enables the production of a state in which the ability to induce neutralizing antibodies against influenza virus is continuously increased. Influenza virus infection is a serious problem for people at high risk of exacerbation of influenza, such as the elderly or hospitalized patients. However, if the composition of the invention is administered prophylactically as a liquid diet, the ability of the entire population to prevent infection can be improved.

Brief Description of Drawings

Figure 1 shows the experimental protocol (the time points of vaccination and antibody measurement, and the time points of intestinal flora analysis during administration of each diet).

Figure 2 presents a graph showing the effect of administration of milk fermentation components and propionic acid bacteria cultures on neutralizing antibody titers after vaccination. In the figure, the vertical axis shows the log of antibody titers (log10), while the horizontal axis shows the elapsed time (weeks) after vaccination. Statistical significance between the control and test groups was analyzed by the Mann-Whitney U test, while statistical significance between weeks 2 and 6 after vaccination was analyzed by the Wilcoxon signed rank sum test. The symbol indicates a significant difference between week 6 and week 2 in neutralizing antibody titers associated with infection (p < 0.05). A: neutralizing antibody titers against H1N 1; b: neutralizing antibody titers against H3N 2; and C: neutralizing antibody titers against the B1 antigen.

Fig. 3 presents a graph showing the variation of stool scores during the examination. In the figure, the vertical axis shows the Bristol stool grading (1-7), while the horizontal axis shows the administration period (weeks) for each diet. Statistical significance between the control and test groups was analyzed by the Mann-Whitney U test. Statistically significant differences between before and after weekly examinations were analyzed by Wilcoxon signed rank sum test. In addition, the symbol indicates a significant difference (p <0.05) between the control group and the test group, and the symbol "a" indicates that there is a significant difference (p <0.05) compared to when grouped (-4 weeks).

Figure 4 presents a graph showing the effect of milk fermentation components and propionic acid bacteria culture on blood cytokine concentration upon influenza vaccination. On the vertical axis of the graph, A, B and C indicate IL-7 concentration (pg/mL), IL-17 concentration (pg/mL), and TGF-. beta.1 concentration (ng/mL), respectively. The horizontal axis shows the elapsed time (weeks) at the time of blood collection when the time defining the group was-4 weeks. Statistical significance between the two groups, control and test groups, was analyzed by Student's t test (even distribution) or Welch test (uneven distribution). Statistical significance before and during the examination was analyzed by the corresponding t-test for each week.

Modes for carrying out the invention

The present invention provides a composition for preventing influenza infection or a prophylactic agent for influenza infection, comprising a propionic acid bacterium culture.

The prophylactic or preventative composition of the present invention comprises a culture of propionic acid bacteria. "propionic acid bacterium" refers to a gram-positive anaerobic bacterium belonging to the genus Propionibacterium, which is a microorganism that anaerobically produces propionic acid from sugars. Specifically, the following cultures of microorganisms may be added to the composition of the present invention.

Propionibacterium freudenreichii (Propionibacterium freudenreichii);

propionibacterium thoenii (p.thoenii));

propionibacterium (p. acidipronici));

propionibacterium jensenii (p. jensenii)); and the like.

These propionic acid bacteria are microorganisms used for cheese production. In addition, the following microorganisms are also referred to as propionic acid bacteria.

Propionibacterium avidum (p. avidum));

propionibacterium acnes (p.acnes));

propionibacterium lymphadenitum (p. lymphophilium)); or

Propionibacterium granulosum (p. granulosa).

Methods for isolating these microorganisms from nature or fermented milk are known. Propionic acid bacteria and the like used in the production of Swiss cheese (Swiss cheese) can be used. The propionic acid bacterium culture in the present invention refers to a material in which propionic acid bacteria such as those described above are cultured under suitable culture conditions. Methods for culturing propionic acid bacteria are known. In the case of culturing propionic acid bacteria, the conditions described in WO 03/016544A1 and the like can be employed. For example, as a medium for culturing propionic acid bacteria, a composition obtained by adding a beer yeast extract or the like to powdered skim milk or a substance produced by treating powdered skim milk with proteolysis is known. Cultures of propionic acid bacteria can be obtained by inoculating propionibacterium freudenreichii into a suitable medium and culturing them under conditions that allow the propionic acid bacteria to proliferate.

Alternatively, as ｃA method of culturing propionic acid bacteriｃA to ｃA high concentration, there is ｃA method of culturing propionic acid bacteriｃA in ｃA medium produced by adding minerals and monosaccharides to Whey Protein Concentrate (WPC) or an enzymatic degradation product thereof (japanese patent application laid-open (JP- ｃA) H10-304871 (unexamined, published japanese patent application)). For example, a culture obtainable by culturing propionibacterium freudenreichii in a medium containing whey protein concentrate is a preferred propionibacterium bacteria culture in the present invention. Alternatively, as a highly efficient culture method for culturing propionic acid bacteria, there is a method of culturing bifidobacteria and propionic acid bacteria in different fermentors while circulating a culture solution (JP-A (Kokai) H08-66178). The propionic acid bacteria cultures obtainable by these culturing methods may be mixed into the composition of the present invention.

For example, propionic acid bacteria can be cultured at high density by adding processed whey as a main component of the culture medium. The following ingredients may be shown as examples of processed whey:

whey powder; and

a product of whey or whey powder treated with a protease.

In addition to whey protein, a mixture of minerals and monosaccharides may be added to the medium. In order to reduce the sugar concentration in the medium, whey protein concentrate (which may be hereinafter referred to as "WPC") may be added as a whey protein source. WPC can be obtained by treating whey by dialysis to reduce lactose content. Whey protein concentrates whey protein isolates (which may be referred to hereinafter as "WPI") may be made by further separating the protein components in high purity. The medium composition can be used to culture propionic acid bacteria by adding these components to the medium and adding appropriate amounts of sugar and deficient minerals separately.

Whey is the water-soluble component remaining after removal of, for example, fat, casein, fat-soluble vitamins, etc. from cow's milk. Whey is commonly referred to as cheese whey and curd whey (or sweet whey), which may be obtained as a by-product in the production of natural cheese or rennet curd; or casein whey or quark whey (alternatively referred to as acid whey), which is obtained when producing acid casein or quark from skim milk. The main components of whey are proteins (β -lactoglobulin, α -lactalbumin, etc.), lactose, water-soluble vitamins and salts (mineral components), and their respective characteristics have been explained in studies as a milk component rather than as a whey component.

Examples of "whey-related products" include concentrated whey produced by concentrating whey; whey powder produced by drying whey; a whey protein concentrate (hereinafter also referred to as "WPC") produced by concentrating the main protein of whey using an Ultrafiltration (UF) method or the like and then performing a drying treatment; defatted WPC (low fat, high protein) produced by removing fat from whey using Microfiltration (MF) process or centrifugation, followed by concentration using UF process and drying treatment; a protein isolate (hereinafter also referred to as "WPI") produced by subjecting the main protein of whey to selective fractionation treatment by an ion exchange resin method or a gel filtration method, followed by drying treatment; desalted whey produced by desalting treatment using a Nanofiltration (NF) method and an electrodialysis method, followed by drying treatment; and mineral-concentrated whey produced by subjecting a mineral component derived from whey to a precipitation treatment and then to a concentration treatment by centrifugation or the like. Wherein WPC containing from 15 to 80% (by solid content) dry weight of milk protein is defined as protein concentrated whey powder and classified as a dairy product (concentrated whey, whey powder, WPC and whey protein concentrated powder, as long as it has undergone the production steps specified in the provincial treaty on milk, regardless of the presence or absence of a desalting step) on 3, 30 days of 1998 according to a partial revision of the provincial ordinance on milk.

Whey Protein Concentrate (WPC) is a material obtained by concentrating the main protein of whey using an Ultrafiltration (UF) method or the like, followed by drying treatment. Generally, it is a generic term for materials that are whey proteins at about 25% or more of the solids content. It can be obtained by reducing lactose and salts in whey to increase the relative whey protein content to about 25% to 80% of the solid content. Specifically, WPCs containing milk proteins at 15% to 80% of the dry weight are defined as protein concentrated whey powder according to the province directive on milk.

The standard method for producing Whey Protein Concentrate (WPC) is as follows:

(1) a step of performing membrane separation on whey and then concentrating; or

(2) The whey is subjected to membrane separation, followed by concentration and drying steps.

Conventional apparatuses and methods can be used for the concentration treatment, and for example, a method of heating under reduced pressure, a method using a vacuum evaporator, a vacuum tank, a rising film tube concentrator, a falling film tube concentrator, a plate type concentrator, or the like can be used. In addition, general equipment and methods can also be used for the drying treatment, and for example, spray drying, drum drying, freeze drying, vacuum (reduced pressure) drying can be used.

Whey Protein Isolate (WPI) is a material that can be obtained by concentrating the main proteins of whey by an ion exchange resin method or an electrodialysis method, followed by drying. Generally, WPI is a generic term for materials in which about 85% to 95% of the solids content is whey protein. It can be obtained by reducing lactose, salts, etc. in whey to increase the relative whey protein content to about 90% (85% to 95%) of the solid content.

The standard method for producing Whey Protein Isolate (WPI) is as follows:

(1) a step of subjecting whey to membrane separation, ion exchange resin treatment or electrodialysis treatment, and then concentrating; or

(2) The whey is subjected to membrane separation, ion exchange resin treatment or electrodialysis treatment, followed by concentration and drying steps.

Conventional apparatuses and methods can be used for the concentration treatment, and for example, a method of heating under reduced pressure, a method using a vacuum evaporator, a vacuum tank, a rising film tube concentrator, a falling film tube concentrator, a plate type concentrator, or the like can be used. In addition, common equipment and methods can also be used for the drying treatment, and for example, spray drying, drum drying, freeze drying, vacuum (reduced pressure) drying can be used.

In the present invention, the following composition can be exemplified as a preferable medium composition for culturing propionic acid bacteria. The values shown below are all weight ratios (w/w%). Hereinafter, when the composition is expressed in percentage, weight ratio (w/w%) will be used unless otherwise stated.

Protein content: 1% to 5%, preferably 1.5% to 4.0%

Content of saccharides: 1% to 4%, preferably 1.5% to 3.0%

To obtain such a content, the amount of whey powder, whey protein or protease-treated product thereof added is adjusted. In addition, monosaccharides obtained by subjecting lactose to lactase treatment or glucose, other than lactose, are preferred sugars.

In order to reduce the cost of the culture medium and to obtain a culture suitable for consumption as a food, lactase treated whey mineral solution can be used as a source of sugars and minerals. In particular, WPC may be used as a protein source, while whey minerals may be used as a source of carbohydrates and minerals. By using an optimal ratio mixture of WPC and whey minerals as the components of the medium, propionic acid bacteria can be cultured at a higher concentration than when whey powder is used as the component in the medium. A detailed method for preparing a medium for culturing propionic acid bacteria will be described below.

WPC (bovine) after being reduced is treated with protease to degrade proteins. The protease is an endo-and exo-protease derived from aspergillus oryzae (aspergillus), and is used in an amount of 3% of the amount of the protein to be degraded. The reaction was carried out at 50 ℃ and pH7.0 and stirring was continued for three to five hours until no decrease in pH was observed. Whey minerals are treated with lactase to degrade lactose. The amount of lactase used is 2% to 8% of the amount of carbohydrate to be degraded, the reaction is carried out at 50 ℃ to 60 ℃ (55 ℃ being preferred) and pH5 to 6, and stirring is continued until the protein is completely degraded.

Next, the two solutions are mixed, preferably so that the protein concentration becomes 1% to 5% (preferably 1.5% to 4.0%) and the saccharide concentration becomes 1% to 4% (preferably 1.5% to 3.0%) as a final culture concentration. Finally, components such as yeast extract, sodium sulfate, asparagine, and the like, which are conventionally used in culturing propionic acid bacteria, are added to the medium, and then the pH is adjusted to 5 to 8 (preferably 5.5 to 7.5) to complete the medium preparation. The step of culturing the propionic acid bacterium will be carried out accordingly as described below.

Specifically, the medium temperature was set at 20 ℃ to 40 ℃, and a starter (starter) was inoculated so that the number of viable cells immediately after the start of culture was 10⁷-10⁸cfu/mL. The cells were then cultured for 3 to 4 days. The pH was maintained between 5.5 and 7.5 using aqueous potassium carbonate solution. Glucose may be additionally supplemented during the cultivation. The amount of propionic acid bacteria in the culture obtained in this way amounts to about five times the amount in the conventional culture.

The culture conditions as described above are preferable, particularly for culturing propionic acid bacteria for cheese. In addition to propionibacterium freudenreichii, propionibacterium jensenii, propionibacterium terellenii, and the like may be used as the propionic acid bacteria for cheese. More specifically, a culture capable of producing the following bacterial strains as propionic acid bacteria can be used in the present invention.

Propionibacterium freudenreichii ATCC 6207

Propionibacterium freudenreichii ATCC 8262

Propionibacterium freudenreichii IFO 12424

Propionibacterium freudenreichii IFO 12426

Propionibacterium freudenreichii IFO 12391

Propionibacterium freudenreichii ET-3(FERM BP-8115)

These propionic acid bacteria may be cultured alone or by mixing a plurality of bacterial strains. Alternatively, the plurality of microorganisms may be cultured independently, and then the obtained cultures may be mixed. The culture obtained in this way can be provided directly as such for feeding and drinking. In addition, it may be powdered or subjected to a liquefaction process to produce a functional ingredient, which is then processed into an easily handled form. That is, the culture obtained by culturing the above propionic acid bacterium may be mixed with the composition of the present invention directly or after processing.

Furthermore, the person skilled in the art is able to suitably adjust the medium composition and the culture conditions to further optimize these known methods. For example, various amino acids and salts thereof may be added as nitrogen sources in addition to casein, WPC, and the like, and the ability to proliferate propionic acid bacteria and their preventive effects against influenza infection can be improved. Not only the media composition, but also the culture conditions can be adjusted for optimization. The culture conditions include oxygen concentration of culture atmosphere, temperature, pressure, and the like.

The fraction of the propionic acid bacterium culture of the present invention may be used as long as it retains its preventive effect against influenza virus infection. Thus, the propionic acid bacteria culture includes, for example, the propionic acid bacteria culture itself, culture supernatant, bacterial cells, an extract thereof, a dry powder thereof, or a dilution thereof. Here, "the propionic acid bacterium culture itself" means that the propionic acid bacterium and the medium components are in a mixed state. The dispersion state of the propionic acid bacterium in the medium components may be any state. That is, the propionic acid bacteria may be dispersed or precipitated in the medium components. On the other hand, the "culture supernatant" generally refers to a condition in which the bacterial cells of the propionic acid bacteria are removed from the "propionic acid bacteria culture itself" by filtration, centrifugation, or the like. Furthermore, "bacterial cell" refers to a propionic acid bacterium isolated from the "propionic acid bacterium culture itself". Incomplete separation of the media components and the propionic acid bacterial cells is generally acceptable. Thus, for example, contamination of propionic acid bacterial cells isolated from the culture with media components is acceptable.

For example, when the effect of promoting the induction of neutralizing antibodies after influenza vaccination of a certain fraction of the propionic acid bacterium culture is, for example, 30% or more, preferably 50% or more, or more preferably 70% or more as compared with the same propionic acid bacterium culture (before fractionation), it can be said that the preventive effect of the culture against influenza virus infection is maintained.

The propionic acid bacteria culture can be sterilized after culturing and mixed with the composition of the present invention. Alternatively, the composition that has been mixed with the milk fermentation component may be sterilized. For example, as specified in the provincial directive on cow's milk, cow's milk is generally subjected to the following heat sterilization:

keeping pasteurization at low temperature;

keeping pasteurization at high temperature;

high-temperature short-time pasteurization; or

Ultra high temperature transient pasteurization (ultra high-temperature flash pasteurization).

These sterilization methods or sterilization methods may be applied to the propionic acid bacterium culture of the present invention or to a composition comprising them. The sterilization treatment may be performed in a batch unit, or may be performed continuously. The treatment temperature and treatment time may vary depending on the sterilization method, but preferably, it is selected from the range of 50 ℃ to 200 ℃ and 0.1 second to 1 hour depending on the pasteurization method described above. In pasteurizing the propionic acid bacteria culture, it is desirable to maintain the amount of dissolved oxygen in the solution in a reduced state. Accordingly, it is preferable to maintain a continuous inert gas atmosphere during heat sterilization. Examples of the inert gas include nitrogen gas, argon gas, and carbon dioxide gas. Among them, nitrogen is a preferred inert gas because it is present in a large amount in air. Its cost is relatively low and its security is established. In addition, it does not affect the taste and quality of foods and beverages. The present inventors confirmed that the propionic acid bacterium culture retained its preventive effect on influenza after sterilization.

In the present invention, the preventive effect of the propionic acid bacterium culture against influenza infection is maintained after sterilization. In general, the action of lactic acid bacteria on a host depends on the function of the living bacteria. Thus, it was an unexpected discovery that useful functions were identified in the culture after sterilization. There are many reports that probiotics (probiotics) and prebiotics (prebiotics) improve the intestinal environment and activate the immune system. Generally, probiotics refer to microorganisms that, when introduced into the gut of a host in a viable state, can bring about a beneficial effect to the host. In contrast to probiotics, prebiotics refer to substances that provide a beneficial effect to the host by acting on microorganisms that are originally growing in the gut. For example, several types of lactic acid bacterial strains have been reported to have an effect of promoting the induction of neutralizing antibodies against influenza vaccines, particularly in experiments performed with humans. However, since these lactic acid bacteria are probiotics that function as live bacteria, production and quality control are not easy. In addition, live bacterial formulations are often of limited storage stability. For example, even if a live bacterial preparation is stored at a low temperature after production, it is generally not stored for a long period of time.

In contrast to lactic acid bacteria, the propionic acid bacteria cultures of the present invention are prebiotics. In the sterilized culture (prebiotics), there was no change in quality due to the activity of the microorganisms. Therefore, the preventive effect against influenza infection can be stably maintained in the prebiotics. That is, the production and quality control of the composition of the present invention are easy. In addition, the composition of the present invention as a prebiotic can be preserved at normal temperature for a long period of time. In addition, generally, when the effect of oral administration from live lactic acid bacteria is expected, the influence of gastric acid must be considered. This is because lactic acid bacteria are reduced by gastric acid and cannot deliver a sufficient amount of viable bacteria into the intestine. On the other hand, in the present invention, the preventive effect of propionic acid bacteria against influenza infection is independent of the activity of live bacteria. Therefore, a sufficient prophylactic effect against influenza can also be achieved by oral administration.

The propionic acid bacterial culture can be processed into a powder form or a liquid form after sterilization. For example, after a suitable excipient is added to the culture solution so that the solid content of the culture becomes 30 to 40% by weight, it may be dried and then powdered. Powdered skim milk, whey powder, raw starch, dextrin, etc. can be used as excipient. In addition to the above excipients, WPC, Whey Protein Isolate (WPI) and modified starch may also be used as excipients, if necessary. Methods for drying cultures are also known. For example, the culture solution may be directly spray-dried. Specifically, as the modified starch, in addition to dextrin, soluble starch, British dextrin (British gum), oxidized starch, starch ester, starch ether, and the like can be used. Alternatively, the culture solution may be mixed with a reducing solution of excipients, and may be spray-dried after concentration until its solid content reaches 30-40 wt%. By performing a deoxidation treatment (nitrogen sealing, addition of a deoxidizer, or the like) at the time of filling, the dried culture can be stably stored for a long time. In addition, it can be processed into a double powder formulation (0.2% double powder) for convenient use in food.

Propionic acid bacteria cultures are known to have the effect of promoting the proliferation of lactic acid bacteria. A propionic acid bacterium culture having such an action, or a processed product thereof, is called "Bifidobacterium Growth Stimulator (BGS)". BGS may be used as the propionic acid bacterium culture of the present invention as long as it has an effect of promoting production of neutralizing antibodies against influenza virus in influenza vaccine-inoculated animals.

As the propionic acid bacterium culture of the present invention, a whey fermentation product produced by propionic acid bacteria is preferable. For example, a propionic acid bacteria culture obtained by fermenting propionibacterium freudenreichii ET-3 strain producing a bifidogenic growth stimulant in a 10% whey powder reducing solution may be included as an active ingredient in the composition of the present invention. For example, propionic acid bacterial cultures containing BGS are called "Profec" and are approved as active ingredients for food products for specific health uses. As the composition containing "Profec", B.G.S. powder (trade name; manufactured by Meiji Dairies Corporation), and Onaka Katsuryoku tablet (manufactured by Meiji Dairies Corporation; trade name: "Onaka Katsuryoku" is a registered trademark of Meiji Dairies Corporation) are commercially available. Thus, "b.g.s. powder" or "Onaka Katsuryoku tablet" may be used as the composition of the present invention.

Specifically, BGS included in Profec is 1, 4-dihydroxy-2-naphthoic acid (DHNA) and 2-amino-3-carboxy-1, 4-naphthoquinone (ACNQ). Among them, DHNA is a biosynthetic intermediate of vitamin K2 (menaquinones) in microorganisms. These substances promote proliferation by efficiently re-oxidizing NADH produced during energy metabolism of bifidobacteria. Thus, as the propionic acid bacterium culture, one or both of the following components (i) and (ii) may be used. Specifically, the present invention provides a prophylactic agent for influenza infection, which comprises one or both of:

(i)1, 4-dihydroxy-2-naphthoic acid (DHNA); and

(ii) 2-amino-carboxy-1, 4-naphthoquinone (ACNQ).

When the amount of DHNA contained in a propionic acid bacterium culture is taken as a reference, usually for an adult, the dose range of the prophylactic agent for influenza infection comprising the above-mentioned components (i) and/or (ii) in the present invention is usually 0.01 μ g/kg to 100mg/kg of DHNA contained in propionic acid bacteria. In some cases, a smaller dose may be sufficient, but on the other hand, a larger dose may be necessary. Further, the administration may be divided into 2 to 4 administrations per day. The dose can be set by taking into consideration the state of the patient such as age and weight, administration route, degree of the desired preventive effect, and the like.

Since Profec specifically increases human Bifidobacterium enterochium, it is approved as an active ingredient of foods for specific health uses (N.Yoda: ILSI, No.80,5-13 (2004)). Currently, "b.g.s. powder" and "Onaka Katsuryoku tablet" are commercially available as compositions comprising Profec. Therefore, it is not difficult to obtain. However, it is not known that administration of Profec to influenza vaccinated animals can enhance the induction of neutralizing antibodies. In particular, the invention provides agents that promote the induction of neutralizing antibodies in influenza-vaccinated animals, comprising either or both of:

(i)1, 4-dihydroxy-2-naphthoic acid (DHNA); and

(ii) 2-amino-carboxy-1, 4-naphthoquinone (ACNQ). Alternatively, the present invention relates to a pharmaceutical composition for promoting the induction of neutralizing antibodies in an influenza vaccinated animal, said composition comprising one or both of the above components (i) and (ii).

When 1, 4-dihydroxy-2-naphthoic acid (DHNA) is incorporated into the compositions of the present invention, it is desirable to keep the dissolved oxygen in the composition at a low level. This is because dissolved oxygen can break down DHNA, so that DHNA concentration decreases during storage. Methods for reducing the level of dissolved oxygen in compositions comprising DHNA are known (WO 2004/85364). In particular, when the composition is in a liquid state, dissolved oxygen can be replaced with a gas other than oxygen by bubbling an oxygen-free gas into the liquid composition. The preferred oxygen-free gas is nitrogen. In addition, dissolved oxygen can be kept at a low level by adding a compound having oxidation resistance together with DHNA. For the compound having antioxidant properties, known antioxidants can be used. Specifically, as the oxidizing agent, dithionic acid (hyposulfurous acid), ascorbic acid (vitamin C), erythorbic acid, carotene, tocopherol, and polyphenols having an antioxidant function are known.

Polyphenols derived from natural products and polyphenols derived from synthetic products can be used. For example, polyphenols derived from tea, grapes, lemon, coffee, purple potatoes, soybeans, and the like are known. The extracted juice of fruits and vegetables, seeds, plant leaves, etc., or the extract thereof containing a large amount of these polyphenols may be incorporated as polyphenols into the composition of the present invention. For example, extraction using water or an organic solvent can produce a polyphenol extract. Furthermore, concentrates, purified products and dried products of these natural polyphenol-containing products can also be used as polyphenols.

Depending on the type of antioxidant, the level of dissolved oxygen can be reduced by adding the same or greater amount of antioxidant than is added for conventional antioxidant use. For example, when it is desired that DHNA stability can be obtained by adding ascorbic acid alone to the composition without bubbling an inert gas, ascorbic acid may be added in an amount of 0.01 wt% or more based on the total weight of the solution. The antioxidant may be added to the composition prior to adding DHNA to the composition. Alternatively, it may be added to the composition along with DHNA to prevent its degradation. For example, when ascorbic acid is added as an antioxidant, the amount added may be set at 1 μ g to 2g per 100kcal (or per 100g of the composition), typically 150 μ g to 1.5g, and preferably 1mg to 500mg or so.

The propionic acid bacterial culture may be mixed in a proportion of 0.001% to 20%, usually 0.01% to 15%, and preferably 0.01% to 10% of the total composition. Alternatively, when quarks are added as milk fermentation components, the milk fermentation composition comprised in the total composition of the present invention may be set such that the concentration of the quark protein fraction is from about 0.01% to about 30%, preferably from about 0.1% to about 20%, and more preferably from about 0.5% to about 10% of the total composition.

The compositions of the present invention may be prepared in any dosage form, such as liquid form, paste form or dry solid material. The compositions of the present invention may be formulated into compositions by admixing a carrier or pharmaceutically acceptable carrier suitable for enteral administration to a propionic acid bacteria culture. More specifically, it may be formulated into tablets, capsules, granules, powders, syrups, and the like. Alternatively, it may be provided as a propionic acid bacteria culture dispersed in the milk fermentation component. These various preparations can be formulated according to a conventional method using known adjuvants generally used in the technical field of pharmaceutical preparations, such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspending agents, coating agents, solvents, and isotonic agents, as main agents. In addition, minerals such as calcium may be included in appropriate amounts. In addition, vitamins, minerals, organic acids, saccharides, amino acids, peptides, and the like may be added in appropriate amounts. The organic acid includes fatty acids such as short chain fatty acids.

It is known that a whey fermentation product of propionic acid bacteria is expected to have an excellent intestinal regulatory effect. However, it is unknown that propionic acid bacteria cultures show a preventive effect against influenza infection.

Milk fermentation components and oligosaccharides may be additionally added to the composition of the present invention. In particular, the present invention relates to a composition comprising:

(a) an oligosaccharide;

(b) a milk fermentation component; and

(c) a propionic acid bacterial culture.

Generally, oligosaccharides, also called oligosaccharides, refer to compounds that form glycosidic linkages from 2 to 20 sugars. For example, in the present invention, the following sugars may be used as oligosaccharides:

lactosucrose (lactosucrose);

isomalto-oligosaccharides;

fructooligosaccharide;

galacto-oligosaccharides;

a xylooligosaccharide;

soybean oligosaccharides;

aspergillus niger oligosaccharide;

a gentio-oligosaccharide;

lactose;

sucrose;

maltose; and so on.

Among oligosaccharides, there are compounds that are easily degraded under acidic conditions. Therefore, when the composition of the present invention or food containing the composition is acidic, it is preferable to use oligosaccharides that are stable under acidic conditions. For example, an oligosaccharide containing galactose as a constituent sugar is preferable as the oligosaccharide to be mixed under acidic conditions. In the present invention, the oligosaccharide containing galactose as a constituent saccharide is a compound in which 2 to 20 saccharides form glycoside linkages, and includes one in which the saccharide constituting the compound contains one or more galactose molecules. More specifically, raffinose family oligosaccharides (soybean oligosaccharides), galactooligosaccharides, and the like can be designated as preferred oligosaccharides. Among them, galacto-oligosaccharides are the preferred oligosaccharides in the present invention.

In the present invention, galacto-oligosaccharide refers to an oligosaccharide in which galactose is a main constituent sugar. Lactose (Gal (. beta.1-4) Glc) is the basic structure but oligosaccharides with a structure in which one to several galactose residues are linked together, disaccharides in which galactose is linked to glucose. beta. -1,3, etc. (transferred disaccharides), Gal- (Gal) n-Glc (n = 1-18), and Gal- (Gal) n-Gal (n = 1-18) may also be included in the case of galactooligosaccharides. In the present invention, the number of galactose residues constituting galactooligosaccharides may be generally 1 to 20, preferably 1 to 10, and more preferably 1 to 8. Further, the proportion of galactose constituting galactooligosaccharide may be expressed, for example, as 10% or more, preferably 30% or more, and more preferably 50% or more of the total number of monosaccharides constituting the galactooligosaccharide. An example of a method for producing galactooligosaccharides is a method of acting beta-galactosidase on lactose, which has a high galactose transfer capacity (Sawairi, Y., "Function of Galacto-oligosaccharides and Application to Foods" FOOD STYLE21,2, pp.76-78 (1998)). For the β -galactosidase, for example, those derived from microorganisms such as Cryptococcus laurentii (Cryptococcus laurentii) and Bacillus circulans (Bacillus circulans) can be used.

Furthermore, in the legislation and standard systems for (standardized) food products (FOSHU) for specific health care uses, oligosaccharides produced from lactose by the action of β -galactosidase (β -D-galactosylgalactohydrolase, e.c.3.2.1.23, derived from cryptococcus yeast) are defined as galactooligosaccharides. Galactooligosaccharides produced in this manner are those in which one or more galactose groups form glycosidic linkages with the galactose residues of lactose, and 4' -galactosyl-lactose (Gal (. beta.1-4) Glc) is said to be the major component (Director of the priority of Food Safety, Pharmaceutical and Food Safety Bureau, Minist of health, No.0701007 by laboratory and Welfare, 1 st 2005, "Regarding of the setting of the codes and the standards accessing the health, and the term").

Galactooligosaccharides are contained in breast milk. It is known to have an effect of increasing bifidobacteria appropriately in the intestinal tract and is a sugar that is difficult to digest and absorb. In addition, galactooligosaccharides are known to be useful for mineral supplementation after gastrectomy surgery (international publication WO 98/15196). Furthermore, although fructooligosaccharides are hydrolyzed under strongly acidic conditions (Nakamura A., "Changes in microorganisms in Sweet-and-Sour-Pork and Cooked FOOD Using Fructo-oligosacchrides (Furukuto-origo in mochita yanimono tyu deno henka)," Annals of Tokyo Kasei Gakuin University,43, pp.49-53(2003)), lacto-oligosaccharides have properties that are not easily attenuated by degradation and the like under acidic conditions or under heating conditions (Sawairi, Y., "Function of Galacto-oligosacchrides and applicationfoods" (gastrointestinal-no to-syokuhyo) "FOYYLE 21,2, pp.76-1998), and are further useful as a composition for modulating the symptoms of gastrointestinal infection, i.e., a composition that can be used to modulate the effects of a gastrointestinal infection, such as one of the symptoms of the present invention, the composition having an intestinal function-regulating effect can be said to have desirable properties as a composition for use in the prevention of influenza infection.

The oligosaccharides are included in the compositions of the present invention at a concentration of about 0.001% to 20.0%, preferably about 0.05% to 11%, and more preferably about 0.1% to 6% of the total composition content. The amount of mixed oligosaccharide can be adjusted depending on the dosage form, symptoms, body weight, etc.

The composition of the invention may comprise, in addition to the propionic acid bacteria culture, one or both of an oligosaccharide and a milk fermentation component. In the present invention, the milk fermentation component refers to a processed product obtained by fermenting animal milk with the activity of a microorganism or an enzyme. In the present invention, the animal milk includes cow's milk, buffalo's milk, goat's milk, sheep's milk, horse's milk, etc. Among them, bovine milk (cow's milk) has an economic advantage in that a large amount of raw milk can be easily obtained. The milk fermentation component may be produced not only from milk collected from living organisms but also from fractions or processed products thereof. The fraction or processed product of milk includes semi-skimmed milk, reconstituted whole milk, reconstituted skimmed milk, reconstituted semi-skimmed milk, whey, casein, powdered skimmed milk, Whey Protein Concentrate (WPC), Whey Protein Isolate (WPI), butter (butter), buttermilk (butter), cream (cream), and the like. These milk-derived processed products are sometimes referred to as raw milk. The raw milk itself may be used, or after mixing with a different raw milk, as a source of milk fermentation components.

In the present invention, the milk fermentation component may be obtained as a culture produced by adding a microorganism for milk fermentation. Fractions of the microbial culture may be used as the milk fermentation components in the present invention as long as they have a preventive effect against influenza infection when mixed with propionic acid bacteria. The microorganisms added to milk for fermentation purposes are commonly referred to as starters. The microorganisms to be used in the milk fermentation are preferably lactic acid bacteria and bifidobacteria. Specifically, for example, the milk fermentation component can be obtained by using lactic acid bacteria or bifidobacteria belonging to the following genera as a starter:

(ii) lactobacillus;

streptococcus genus;

lactococcus (genus Lactococcus);

leuconostoc (genus Leuconostoc);

pediococcus (genus Pediococcus); and so on.

More specifically, it is known that a milk fermentation component is produced by the following microorganisms. Milk fermentation components obtained using these microorganisms are preferable as the milk fermentation component in the present invention.

And (3) lactobacillus: streptococcus lactis (Streptococcus lactis),

streptococcus casei (Streptococcus cremoris),

streptococcus diacetylactis (Streptococcus diacetylactis),

enterococcus faecium (enterococcus faecium),

enterococcus faecalis (enterococcus faecis),

lactobacillus acidophilus (Lactobacillus acidophilus),

lactobacillus brevis (Lactobacillus brevis),

lactobacillus casei (Lactobacillus casei),

lactobacillus helveticus (Lactobacillus helveticus),

lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus delbrueckii subsp.),

lactobacillus delbrueckii subsp.lactis,

lactobacillus gasseri (Lactobacillus gasseri),

lactobacillus mucosae (Lactobacillus mucosae),

lactobacillus murinus (Lactobacillus murinus),

lactobacillus plantarum (Lactobacillus plantarum),

lactobacillus oralis (Lactobacillus oris),

lactobacillus reuteri (Lactobacillus reuteri), and

lactobacillus rhamnosus (Lactobacillus rhamnosus)

Bifidobacteria: bifidobacterium longum (Bifidobacterium longum),

bifidobacterium bifidum (Bifidobacterium bifidum), and

bifidobacterium breve (Bifidobacterium breve)

Methods for isolating these microorganisms from nature or fermented milk are known. Alternatively, the microorganism which has been isolated can be obtained by aliquoting (subdivisions) from a cell bank. In addition, lactic acid bacteria starters for obtaining milk fermentation components are commercially available. Milk fermentation components produced by commercially available lactic acid bacteria starters may also be used in the compositions of the present invention. Various products are on the market according to the differences in pH and physical properties of the resulting fermented milk. The physical properties of the fermented milk refer to hardness (stiff) and smoothness (smoothness). Commercially available lactic acid bacteria starters can be used as the lactic acid bacteria starter for obtaining the milk fermentation component of the present invention as long as they can promote neutralizing antibody induction of influenza vaccine when used together with propionic acid bacteria culture.

To obtain the milk fermentation component of the present invention, the following microorganisms may be inoculated into raw milk as lactic acid bacteria starter:

lactobacillus bulgaricus (l.bulgaricus);

streptococcus thermophilus (Streptococcus thermophilus) (s. thermophilus); and

lactobacillus lactis (l.lactis).

In the usual production of fermented milk, one, two or more species selected from the group consisting of yeast and lactic acid bacteria other than these lactic acid bacteria may be added to the raw milk. However, in the present invention, in any case, it is preferable to use a mixed starter containing lactobacillus bulgaricus (l.bulgaricus) and streptococcus thermophilus (s.thermophiles), which is a standardized yogurt starter according to Codex standards. Further, when an additional microorganism is added, the additional microorganism may be added to such a mixing starter by considering the fermentation temperature and the fermentation conditions for the target fermented milk. The microorganisms to be additionally mixed into the mixing starter include other lactic acid bacteria such as lactobacillus gasseri (l.gasseri) and Bifidobacterium (Bifidobacterium).

In the present invention, the microorganism to be added as a mixing starter to the raw milk may be selected from microorganisms preserved in a cell bank. Examples of ideal bacterial strains that can be used for the mixed starter are shown below:

lactic acid bacteria starter comprising a mixed culture of the following microorganisms:

lactobacillus bulgaricus (L.bulbgaricus JCM 1002T)

Streptococcus thermophilus (S.thermophilus ATCC 19258)

Lactic acid bacteria starter comprising a mixed culture of the following microorganisms:

streptococcus thermophilus OLS3059(FERM BP-10740)

Streptococcus thermophilus OLS3294(NITE P-77)

Lactobacillus delbrueckii subspecies bulgaricus OLL 1073R-1(FERM BP-10741)

Lactobacillus delbrueckii subspecies bulgaricus OLL 1255(NITE BP-76)

Cheese, natural cheese, yogurt, fermented milk, whey fermented products, whey cheese and the like which can be obtained via fermentation by these microorganisms are included in the milk fermented component of the present invention. For example, the milk fermentation component to be incorporated into the composition of the present invention is obtained by reducing moisture (whey) from fermented milk (yogurt) (e.g., japanese patent No.3,179,555). Proteins derived from fermented milk (yoghurt) have a high nutritional value, as these proteins have an amino acid score of 100, and the digestion and absorption properties of these proteins are improved by fermentation.

For example, among these milk-fermented components, cheese is a preferred milk-fermented component in the present invention. For example, milk in liquid form prepared by combining raw milk in liquid form of one, two or more types may be made into curd (curd) by adding an enzyme or an acid after fermentation with lactic acid bacteria. Cheese is a material produced by removing whey from curd. Regardless of solidification or maturation, the material produced by removing whey from curd is called cheese. Cheese is roughly classified into ripe cheese, which has undergone a ripening process after production, and unripe cheese. Generally, lactic acid bacteria or molds proliferate (ferment) during the ripening of cheese, and each cheese develops a certain characteristic taste. In contrast to mature cheese, unripe cheese maintains the state that whey is produced when it is removed from the curd. Non-ripe cheese (fresh cheese) is preferred as the milk fermentation component in the present invention. There are many types of non-ripe cheese (fresh cheese) including tyrannocheese (cottage cheese), quark (quark cheese), stringcheese (stringcheese), newsatchel (neufcatelchesee), butterfat cheese (cream cheese), mozzarella cheese (mozzarella cheese), whey cheese (ricotta cheese) and mascarapace cheese (mascarapace cheese). Of these fresh cheeses, quarks are preferably used in the present invention. Methods for producing quarks are known (e.g., JP-A (Kokai) H06-228013; Cheese and Fermented Milk Foods, Volume I: Origins and Principles, Frank V.Kosikowski and Vikram V.Misstry, F.V.Kosikowski, L.L.C.,1999, pages 147 to 161). Quark is a type of cheese (generic name), and its nutritional composition, etc. have been elucidated (Milk and Dairy Product Technology, Edgar Sprer; AxelMixa, Marcel Dekker Inc.,1998, pages 245-249).

The general production process for non-ripened cheese is as follows. First, curd is produced from raw milk. A starter is inoculated to the raw milk and, after cultivation, rennet is further added to produce curd. The raw milk may be pretreated, if necessary, before the curd is produced. For example, to reduce the difference in quality between production batches, the quality may be adjusted by mixing multiple types of milk sources. This process is called normalization. Furthermore, homogenization (homogenization) may be applied to mechanically break down fat globules in milk. Alternatively, the removal of microorganisms mixed in the milk source by centrifugation and heat treatment sterilization may be performed.

The solid component that can be obtained by separating whey from the curd obtained is an unripe cheese (fresh cheese). Methods for separating whey from curd by centrifugation and membrane separation are known. For example, centrifugal separators such as quark separators are used for whey separation. Alternatively, the separation process can be made efficient by cutting or heating the curd beforehand when needed.

More specifically, fresh cheese, which can be obtained from the following sources and procedures, is preferred as the milk fermentation component of the present invention. In the following steps, primarily Lactobacillus bulgaricus and/or Streptococcus thermophilus can be used for the fermentation:

heating and sterilizing skim milk of cattle;

initiating fermentation by inoculating 0.5% to 5% lactic acid bacteria starter;

separating whey from the formed curd when the pH reaches 4.6; and

the curd obtained by separating the whey is cooled to obtain an unripe cheese.

In general terms, the non-ripened cheese that can be so produced may also be referred to as quark. Examples of compositions of non-ripened cheese are as follows:

from 17% to 19% of the total solids content,

11% to 13% protein;

1% or less fat (i.e., 0% to 1%),

from 2% to 8% of saccharides, and

2% or less lactose (i.e., 0% to 2%).

The milk fermentation component can be incorporated in an amount such as a proportion of about 0.01% to 30%, usually about 0.1% to 20%, preferably about 0.5% to 10% of the protein derived from the milk fermentation component in the total composition. Alternatively, 0.01g to 33g, 0.1g to 22g, 0.5g to 11g, preferably 2g to 6g, and more preferably 2.5g to 4.5g may be mixed into 100mL of the composition. Alternatively, the protein derived from the milk fermentation component contained in the total composition of the present invention may be set to about 0.1% to 100%, preferably about 1% to 100%, and more preferably about 30% to 100% of the mass of the protein in the total composition.

Further, the milk fermentation component fermented by adding lactic acid bacteria belonging to the genus lactococcus as a lactic acid bacteria starter to the heat-sterilized skim milk may be the non-mature cheese of the present invention. More specifically, lactic acid bacteria starters incorporating the following microorganisms can be used:

the bacteria of the lactococcus lactis are,

lactococcus casei (Lactococcus cremoris), and

and a microorganism belonging to the genus Leuconostoc.

The curd can be obtained by adding a lactic acid bacteria starter produced by mixing microorganisms such as those described above to heat-sterilized skim milk, followed by culturing. The non-ripened cheese may also be obtained by removing whey from the milk fermentation components. The milk-fermenting ingredient in the present invention also includes an unripe cheese which can be obtained by cutting curd in advance using a cutting tool (cutter) and separating whey while heating.

In addition, the non-ripened cheese of the present invention also includes a material produced by adding rennet to raw milk and then solidifying. Rennet is a raw material (source) containing rennet (EC3.4.23.4) as a main ingredient for producing cheese and the like.

The compositions of the present invention may be administered enterally. Enteral administration refers to the delivery of the compositions of the present invention to the intestinal tract. Thus, enteral administration includes not only oral administration, but also administration by enema (intestinal infusion), and various types of methods using tube feeding administration. Tube feeding is a method of directly administering liquid food or the like to the digestive tract through a tube to a patient who has poor oral ingestion of meals. Depending on the differences in the method of tube placement, there are the following routes of administration:

through the nasal cavity (nasal feeding)

External fistula

Percutaneous gastroscopy gastrostomy (PEG)

Jejunostomy

External fistulae refer to the delivery of tubes into the alimentary canal from outside the abdominal wall, including percutaneous gastroscopy and jejunostomy. In addition, enema administration of the composition perfused from the anus to the rectum is also included in enteral administration. In the case of oral administration, the composition may be in any dosage form. However, for administration by tube or enema administration, it is advantageous to prepare the composition of the present invention in paste, semi-solid or liquid form.

The compositions of the invention are useful for preventing influenza virus infection in an animal. In the present invention, "animal" refers to mammals and birds that are hosts of influenza virus. In addition to humans, cases of influenza virus infection are known to be present in mammals such as pigs and the like. It can be applied to cattle, goats, sheep, horses, buffalos, camels, etc., as well as pets, animals raised in zoos and domestic animals, in addition to pigs. Birds, including poultry such as chickens, and many wild birds also exhibit influenza virus infection. For all these animal species, a prophylactic effect against influenza infection by administration of the composition of the present invention can be expected. When the composition of the present invention is administered to an animal, the composition of the present invention may be administered by mixing into feed.

When the milk fermentation component is incorporated into the composition of the present invention, the dose (intake amount) (solid content) of the milk fermentation component is, for example, about 1mg to 20g, preferably about 10mg to 15g, and more preferably about 50mg to 10g per kg body weight per day. The dosage may be adjusted depending on the dosage form, symptoms, sex, age, body weight, and the like.

For example, when a milk fermentation component is mixed into the composition of the present invention and administered to a human, the dose is usually about 0.05g to 1,500g, preferably about 0.05g to 1,000g, and more preferably about 2.5g to 800g (solid content) per day. This may suitably be administered once to those subjects in need of promotion of neutralising antibody induction by a composition of the invention, or separately and administered before, after or between meals, and/or at bedtime. The dose can be individually and appropriately adjusted depending on the age and weight of the subject to be administered and the purpose of administration. Furthermore, the composition of the present invention may be used as a substitute for a meal or it may be used as a dietary supplement.

The propionic acid bacteria culture constituting the composition of the present invention, and additionally added milk fermentation components or oligosaccharides have been used as nutrients and components of liquid diet. Thus, the composition of the present invention can be produced by mixing a nutritional agent or a liquid diet containing these components. For example, propionic acid bacterial cultures containing BGS are called "Profec" and are approved as active ingredients for food products for specific health uses. Products named "b.g.s. powder" and "Onaka Katsuryoku tablet" include "Profec", which is a whey fermentation product produced by fermentation of propionic acid bacteria. In addition, the product named "fibrin YH" (MeijiDairies Corporation) is a liquid diet containing a milk fermentation component. Therefore, the composition of the present invention can be obtained by mixing these products according to the above mixing ratio.

Furthermore, a kit for preventing influenza infection can be constructed by combining a composition comprising a culture of propionic acid bacteria with a composition comprising a milk fermentation component. More specifically, the present invention relates to a kit for preventing influenza infection, comprising (is compounded of):

(a) a composition comprising a propionic acid bacterial culture; and

(b) a composition comprising a milk fermentation component. Alternatively, the present invention provides a kit for promoting induction of influenza virus neutralizing antibodies in an influenza vaccine-vaccinated animal, wherein the kit comprises the above-described compositions (a) and (b). For example, the kit of the invention may be constituted by combining a nutritional composition comprising a culture of propionic acid bacteria and a nutritional composition comprising a milk fermentation component. These nutritional components are distributed as a liquid diet or nutrient.

The present inventors found that in humans ingesting propionic acid bacteria cultures, the induction of neutralizing antibodies due to influenza vaccination was enhanced. More specifically, the present invention provides compositions comprising a culture of propionic acid bacteria to be administered enterally to an animal receiving influenza vaccination. Alternatively, the present invention provides an agent for promoting influenza virus neutralization antibody induction in an influenza vaccine-vaccinated animal, wherein the agent comprises a propionic acid bacteria culture. Furthermore, in a preferred embodiment, the composition or agent of the invention for promoting the induction of neutralizing antibodies may additionally comprise one or both of a milk fermentation component and an oligosaccharide.

Furthermore, the present invention relates to the use of a propionic acid bacteria culture for the preparation of an agent for promoting the induction of influenza virus neutralizing antibodies in an influenza vaccine vaccinated animal. Alternatively, the invention relates to a propionic acid bacteria culture for promoting neutralizing antibody induction in influenza vaccine vaccinated animals. In addition, the present invention relates to a method for producing an agent that promotes the induction of neutralizing antibodies in an influenza-vaccinated animal, comprising the step of mixing a propionic acid bacteria culture with a pharmaceutically acceptable carrier.

The invention also relates to the use of a propionic acid bacteria culture for the preparation of a prophylactic agent against influenza infection. Alternatively, the invention relates to a propionic acid bacterial culture for use in the prevention of influenza infection.

Alternatively, the present invention provides a pharmaceutical composition for promoting influenza virus neutralization antibody induction in an influenza vaccine vaccinated animal, wherein the composition comprises a propionic acid bacteria culture. Furthermore, the present invention relates to the use of a propionic acid bacteria culture for the preparation of a pharmaceutical composition for promoting the induction of neutralizing antibodies against influenza virus in an animal vaccinated with influenza virus. The pharmaceutical compositions of the present invention comprise a pharmaceutically effective amount of a culture of propionic acid bacteria. Carriers suitable for oral or enteral administration may be incorporated into the pharmaceutical compositions of the invention. The pharmaceutical compositions of the invention may also be provided and administered as a liquid diet for the purpose of inducing neutralizing antibodies in influenza-vaccinated animals.

In the present invention, the influenza vaccine includes a vaccine administered for the purpose of preventing influenza virus infection and exacerbation after infection. Inactivated vaccines put into practical use in japan at present, and nasal vaccines put into practical use in other countries are included in the influenza vaccine of the present invention. Inactivated vaccines are typically administered subcutaneously, intradermally, or intramuscularly. On the other hand, nasal vaccines are live virus vaccines. The purpose of spraying nasal vaccines into the nasal cavity is to induce IgA antibodies in the respiratory mucosa, which have a high preventive effect on viral infections.

Alternatively, the present invention provides a method for preventing influenza infection, the method comprising the steps of:

(1) administering a propionic acid bacteria culture to an animal; and

(2) animals were vaccinated with influenza vaccine.

In the present invention, the prevention of influenza infection specifically includes one or both of prevention of influenza virus infection and prevention of exacerbation after influenza virus infection. Influenza infection is an infectious disease accompanied by a variety of symptoms caused by infection with the pathogenic influenza virus. In the present invention, influenza infection may sometimes be simply referred to as "influenza". Exacerbations of influenza infection include the following conditions:

the symptoms of the infection become severe;

an increase in the type of symptoms of the infection;

an increase in virus-infected tissue or infected cells; and

viral propagation in vivo.

Thus, inhibiting at least one of these conditions means prevention of influenza infection in the present invention. In the present invention, the prevention of influenza infection includes the prevention of influenza virus infection. More specifically, enhancement of induction of virus-neutralizing antibodies is included in the prevention of influenza virus infection. The level of production of neutralizing antibodies induced in vivo by influenza vaccination may decrease over time. Prevention of reduction of neutralizing antibodies helps to prevent influenza infection. Thus, prevention of a reduction in the level of production of neutralizing antibodies is also included in promoting induction of virus neutralizing antibodies.

Neutralizing antibodies help prevent the spread of influenza-infected tissues or influenza-infected cells in vivo. The animal species for which the composition of the present invention or the method of the present invention can be expected to have a preventive effect against influenza infection is a host animal of influenza virus. More specifically, examples include humans, animals including humans, and animals other than humans.

Furthermore, the prophylactic effect against influenza infection produced by the composition of the present invention is independent of the antigenicity of influenza virus. Therefore, it is effective in preventing infection with various types of influenza viruses. In particular, influenza viruses belonging to type A and subtypes thereof are preferred, and the present invention should be able to prevent influenza viruses infected therewith. More specifically, influenza a viruses in which the host is human, pig, avian, or the like are preferable targets for prevention in the present invention.

For example, the enhancement induced by neutralizing antibodies can be confirmed as follows. That is, the same influenza vaccine was administered to the group to which the composition of the present invention was administered as well as to the group to which the composition of the present invention was not administered. Then, the virus-neutralizing antibody-induced states in the two groups can be compared. At this time, the constitution of each group was divided so that the conditions were the same except for the application of the composition. In other words, it is desirable to assign conditions such as health status, age, physique, and sex ratio so that there is no deviation after the two groups. It is also desirable to make the genetic profile as uniform as possible. Therefore, when a human is used as a subject, a group of the same race is desirable. When confirming the preventive effect in non-human animals, it is attempted to use a genetically identical group.

Under these conditions, the composition administration and vaccination for both groups were performed according to the same schedule. In addition, when the induction of neutralizing antibodies was significantly enhanced in the group using the composition of the present invention, the preventive effect of the composition against influenza infection could be confirmed. Here, the enhancement of the induction of neutralizing antibodies means that, for example, any of the following can be confirmed. In the indices shown below, the neutralizing antibody titer can be evaluated, for example, by comparing the proportion of individuals who exceeded the infection-inhibiting antibody titer between the two groups (incidence).

When the antibody titer of the neutralizing antibody is rapidly increased,

when the production of neutralizing antibodies is continued for a long time, or

When high neutralizing antibody titers are achieved.

Therefore, if the number of individuals in which any of the above effects is confirmed is significantly increased by administration of the composition of the present invention, the preventive effect of the composition against influenza infection can be confirmed. Methods for quantitatively evaluating neutralizing antibody titers are known. For example, the infection inhibitory effect of the antibody can be confirmed by administering infectious influenza virus and cultured cells. Serial dilutions of the antibodies allowed quantitative comparison of infection inhibition.

In the present invention, the propionic acid bacterium culture is administered before or after the time of vaccination or simultaneously with vaccination. Continuous administration of the propionic acid bacterial culture from before to after vaccination is preferred. More specifically, the present invention provides compositions comprising a propionic acid bacterial culture for use as such: the enteral administration is continued around the time of vaccination of the animals that will be vaccinated with the influenza vaccine. For example, the time before and after the vaccination means when the day of vaccination is set to 0 th day, -150 days to +150 days, usually, -60 days to +60 days, or-8 weeks to +8 weeks.

During this period, a pharmaceutically effective amount of the composition of the invention is administered at least once daily. The daily dose may be divided and administered in multiple batches. Alternatively, it may be administered every other day. Further, in the present invention, an effective amount of the propionic acid bacterium culture may be administered in combination with various types of compositions at different mixing ratios.

In fact, the present inventors confirmed that the induction of neutralizing antibodies by the vaccine was promoted in humans who ingested propionic acid bacteria cultures before and after the time of influenza vaccination. Accordingly, the present invention provides a method for promoting or enhancing neutralizing antibody induction in an animal administered an influenza vaccine, wherein the method comprises the steps of:

(1) administering a propionic acid bacteria culture to an animal; and

(2) animals were vaccinated with influenza vaccine.

The composition of the present invention is expected to exert not only the effect of promoting the induction of neutralizing antibodies in vaccination but also the effect of enhancing the induction of neutralizing antibodies even upon viral infection. That is, even in patients infected with influenza virus, the composition of the present invention can suppress the spread of viral infection in patients and alleviate symptoms by increasing the ability of the patients themselves to produce virus-neutralizing antibodies. In particular, when a highly virulent virus is infected, not only the risk of inducing an airway infection but also the risk of inducing systemic symptoms are expected. By the patient taking the composition of the present invention continuously, it is expected that the ability to induce neutralizing antibodies against infectious viruses can be increased, and exacerbation can be prevented.

The composition of the present invention can be used in the form of any medicine or food and drink. For example, the induction of neutralizing antibodies by influenza vaccines can be enhanced by administering the composition directly as a medicament. Alternatively, it can be ingested as a food for special use, such as a food for special health use, or a food having a nutritional function, with the expectation that the preventive effect of the influenza vaccine is enhanced. Further, regardless of forms such as liquid form, paste form, solid form and powder form, the composition can be ingested as a food by adding the composition to various types of foods and drinks. Examples of foods and beverages include cow's milk, soft drinks, fermented milks, yogurt, cheese, bread, cookies, crackers (crackers), pizza crusts, modified powdered milk (modified powdered milk), liquid foods, patient foods, nutritional foods, frozen foods, food compositions, processed foods, and other commercially available foods. When the composition of the present invention is prepared in the form of an acidic drug or food and drink, its pH may be set at pH2.0 to pH6.0, and preferably pH3.0 to pH 5.0.

When the composition of the present invention is continuously administered to an animal, it can be administered as a nutrient, food, beverage or meal. When the composition of the present invention is administered as a nutrient, food, beverage or meal, its nutritional components may be adjusted by mixing additional nutrients in addition to the milk fermentation component and the propionic acid bacteria culture. Water, proteins, sugars, fats, vitamins, minerals, organic acids, short chain fatty acids, organic bases, fruit juices, flavors, and the like may be used as additional nutrients in the present invention. For these nutrients, the following components may be used:

protein: (animal and plant proteins, or degradation products thereof)

Whole milk powder, powdered skim milk, powdered semi-skim milk, casein, whey powder, whey protein concentrate, whey protein isolate, alpha-casein, beta-casein, kappa-casein, beta-lactoglobulin, lactoferrin, soy protein, chicken egg protein, meat protein, and the like;

milk-derived fats and carbohydrates: butter, whey minerals, cream, non-protein nitrogen, sialic acid, phospholipids, various types of milk-derived components such as lactose, and the like.

Peptides and amino acids: peptides and various types of amino acids such as tyrosine phosphopeptides, arginine and lysine.

Saccharides: modified starches (dextrins (maltodextrin, indigestible dextrin, etc.), soluble starches, British starches (British starch), oxidized starches, starch esters, starch ethers, etc.), dietary fibers, etc.;

oils and fats: animal oils and fats such as lard, fish oil, and the like, as well as isolated oils, hydrogenated oils, transesterified oils (transesterified oils); and vegetable oils such as palm oil, safflower oil, corn oil, rapeseed oil and palm oil, as well as isolated oils, hydrogenated oils and transesterified oils;

vitamins: vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, nicotinic acid (niacin), nicotinic acid (nicotinic acid), pantothenic acid, biotin, inositol, choline, folic acid, etc.;

minerals: calcium, phosphorus, potassium, chlorine, magnesium, sodium, copper, iron, manganese, zinc, selenium, chromium, molybdenum, and the like;

organic acid: malic acid, citric acid, lactic acid, tartaric acid, and the like; and

short chain fatty acids: acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, and the like.

Any chemically synthesized substance or natural product derived component may be used as these additional nutrients. Alternatively, a food containing the objective component may be mixed as a raw material. These components may be mixed by combining at least one, two, or more types according to the composition of the intended nutrient. The composition may be in the form of a solid or a liquid. In addition, it can be made in the form of a gel or semi-solid. Thus, the nutritional agent may be administered as a liquid diet.

In fact, as indicated in the examples described below, the compositions of the invention promote the induction of influenza virus neutralizing antibodies in host animals vaccinated with influenza vaccine by ingestion thereof as a liquid diet comprising a culture of propionic acid bacteria. Thus, a composition having a liquid diet composition in which a propionic acid bacteria culture is incorporated is a preferred embodiment of the present invention. That is, the present invention relates to a method for producing a liquid diet that promotes induction of neutralizing antibodies against influenza virus in a subject that has been vaccinated with influenza vaccine, wherein the method comprises a step of mixing a propionic acid bacteria culture into the liquid diet. Alternatively, the present invention provides a method for providing a liquid diet having the ability to promote influenza virus-neutralizing antibody induction in a subject vaccinated with an influenza vaccine, the method comprising the step of mixing a propionic acid bacteria culture into the liquid diet. Furthermore, the present invention provides a prophylactic composition for influenza infection, comprising the following nutrients:

a propionic acid bacterial culture;

a milk fermentation component;

an oligosaccharide;

a protein;

a saccharide;

a lipid; and

dietary fiber.

In the above composition, the propionic acid bacteria culture may be, for example, one or both of: (i)1, 4-dihydroxy-2-naphthoic acid (DHNA), and (ii) 2-amino-3-carboxy-1, 4-naphthoquinone (ACNQ). Furthermore, when the milk fermentation component in the above composition contains protein, protein of a different source may be additionally mixed as protein. For example, a milk component may be blended in addition to the milk fermentation component, and similarly, a saccharide other than an oligosaccharide may be blended as the saccharide in the above composition.

The various components constituting the composition of the present invention may be appropriately adjusted according to various conditions such as the physique, age and sex of the subject to which the composition is administered as a liquid diet. More specifically, compositions such as the following may be shown as a common composition (per 100 mL):

propionic acid bacterial cultures: 1mg to 22g, typically 10mg to 17g, and preferably 10mg to 11 g; or 0.01 μ g to 15mg, usually 0.5 μ g to 10mg, and preferably 0.5 μ g to 0.1mg, in terms of the amount of DHNA;

milk fermentation components: from 0.01g to 33g, usually from 0.1g to 22g, preferably from 0.5g to 11g, in terms of protein amount;

oligosaccharide: 1mg to 20g, typically 50mg to 11g, and preferably 0.1g to 6 g;

protein: 0.01g to 50g, usually 0.1g to 30g, and preferably 0.5g to 15 g;

saccharides: from 0.1g to 40g, usually from 0.5g to 30g, preferably from 1g to 25 g;

lipid: 0.1g to 20g, usually 0.3g to 15g, preferably 0.6g to 10 g; and

dietary fiber: 0g to 15g, usually 0g to 10g, and preferably 0g to 8 g.

In the above composition, when a culture of propionic acid bacteria containing DHNA is mixed, the culture may be mixed in an amount equivalent to DHNA to obtain the above composition. Similarly, the oligosaccharides incorporated into the above-described composition may be incorporated as part of the saccharide. That is, the oligosaccharide constitutes a part of the saccharide in the above composition.

In addition, in the composition of the present invention, at least one nutrient selected from the group consisting of: vitamins, minerals, organic acids or short chain fatty acids, and organic bases. When these components are mixed, the composition may be appropriately adjusted according to various conditions such as the physique, age and sex of the subject to which the liquid diet is administered. More specifically, the following composition can be shown as a general composition (per 100 mL):

vitamins: 0g to 2g, usually 0g to 1.5g, preferably 0mg to 500 mg;

minerals: 0g to 5g, usually 0g to 3g, preferably 0g to 2 g; and

organic acids or short chain fatty acids: 0g to 5g, usually 0g to 3g, preferably 0g to 2 g.

More specifically, the present invention provides a method for producing a liquid diet for enhancing neutralizing antibody induction by influenza vaccine, wherein the method comprises a step of mixing the above nutrients according to the above composition. The liquid diet produced by the present invention can be designated as liquid diet induced by promoting neutralizing antibodies against influenza virus in a subject by administering the liquid diet to the subject vaccinated with influenza before and after the influenza vaccination time.

The compositions of the invention may be prepared by mixing a propionic acid bacteria culture with a pharmaceutically acceptable carrier. When additional components are further mixed, the above-mentioned saccharides, proteins, fats, etc. are mixed as a carrier or a liquid diet, and the composition is prepared by uniform mixing. In general, the composition may be prepared such that the protein derived from the bacterial culture and milk fermentation components is up to a proportion of about 1 wt% or more, for example, about 30 wt% or more, preferably 70 wt% or more, and more preferably about 100 wt% of the protein in the composition. When the composition of the present invention is prepared as a liquid diet, it is desirable to prepare 0.1 to 3kcal, or preferably 0.7 to 2kcal per 1 mL. In addition, when mixed, at least one or more additional components consisting of vitamins, minerals and dietary fiber may be added. The dietary fiber can be divided into water-soluble dietary fiber and insoluble dietary fiber, and either one of them can be used. Specifically, the following components may be exemplified as the water-soluble dietary fiber:

pectins (protopectin, pectinic acid, and pectic acid);

guar gum hydrolysate;

glucomannan;

galactomannan;

psyllium;

corn fiber;

alginic acid;

an alginic acid hydrolysate;

carrageenan (carrageenin); and

indigestible dextrin.

Examples of insoluble dietary fibers include crystalline cellulose, sugar beet fiber, and wheat bran. Preferably, pectin, guar gum hydrolysate or indigestible dextrin may be used. In addition, in some cases, flavors and other agents may be added.

The various types of components described above may be mixed and then filled into containers and heat sterilized as necessary to produce a liquid diet or enteral nutrient. When the pH of the composition is acidic, the heat treatment may be performed under milder conditions than usual. For example, although the heat sterilization treatment of a neutral liquid diet uses the tank sterilization (recovery) condition at 120 ℃ to 130 ℃ for 20 minutes to 40 minutes or the indirect sterilization condition at 140 ℃ to 145 ℃ for 4 seconds to 10 seconds, the composition of the present invention may be subjected to the tank sterilization at 80 ℃ to 90 ℃ for 15 minutes to 30 minutes or the indirect sterilization at 95 ℃ to 110 ℃ for 20 seconds to 60 seconds. Taste can be improved and heat labile components can be mixed by using mild sterilization conditions. In addition, the composition of the present invention can be made into a gel using agar or gelatin, can be made into granular foods and medicines by spray drying or the like, and can also be made into solid foods and medicines.

The compositions of the invention may be produced by methods known in the art of liquid diet and enteral nutrition. For example, a method of heat-sterilizing a composition in a liquid form in advance and then aseptically filling it into a container (for example, a method combining a UHT sterilization method and an aseptic packaging method), and a method of filling a liquid composition into a container and then heat-sterilizing it together with the container (for example, a tank sterilization method and an autoclave sterilization method) can be used. More specifically, when the composition is used in a liquid form, a homogenized product (homogenized liquid) based on the composition is heat-sterilized again at about 120 to 145 ℃ for 1 to 10 seconds and cooled, if necessary, and then aseptically filled, or aseptically filled into cans or soft packs and tank-sterilized. Furthermore, when the composition is used in the form of a powder, for example, the homogenate is spray-dried or freeze-dried.

Hereinafter, the present invention will be described in more detail, but it should not be construed as being limited to the individual embodiments described below. In the present invention, when the raw materials are tempered (added/mixed), they are tempered while being heated. When the tempering temperature is set at a high temperature such as 90 ℃, the protein is solidified (coagulated), and when the tempering temperature is set at a low temperature such as 2 ℃, the protein is hardly dissolved or dispersed in water or the like. Thus, for the tempering step, the temperature is preferably from 5 ℃ to 85 ℃, more preferably from 15 ℃ to 75 ℃, even more preferably from 25 ℃ to 55 ℃, and particularly preferably from 35 ℃ to 50 ℃. In this case, it is preferable to adopt an appropriate mixing time in consideration of the growth of bacteria (contaminating bacteria and the like) in the mixed solution.

In the present invention, the blended liquid is homogenized after high-temperature sterilization. The reason is that proteins may be denatured and viscosity may be increased (thickened) by high-temperature sterilization (heating), and the thickening degree can be reduced by homogenization after high-temperature sterilization. Here, homogenization after high-temperature sterilization refers to homogenization after high-temperature sterilization before filling into a container or the like to be a product, and the number of times of homogenization is not limited to one, and may be a plurality of times such as two or more times. For example, when the mixed solution is sterilized and secondarily sterilized as it is (as is), homogenization is performed again after the secondary sterilization. Furthermore, the tempering liquid may be homogenized after the sterilization, and when the second sterilization is performed, the homogenization may be performed again in a second round after the second sterilization. Further, the prepared liquid may be homogenized after sterilization, and may be homogenized again for the second time without sterilization. More specifically, in the present invention, it is important to perform homogenization at least once after the preparation liquid is sterilized at high temperature and before the preparation liquid is filled into a container or the like to be a product.

On the other hand, even after homogenizing the blended liquid subjected to high-temperature sterilization (sterilization liquid), it may be sterilized again to such an extent that the sterilization liquid is not caused. For example, the sterilization of the mixed solution may be followed by homogenization, and then, without high-temperature sterilization, it may be sterilized again for a second time. At this time, as the high-temperature sterilization step, for example, a heating history (thermal history) corresponds to a temperature of 100 ℃ to 150 ℃ and a holding time of 1 second to 30 seconds; preferably 115 ℃ to 145 ℃ for 1 second to 20 seconds; more preferably from 120 ℃ to 145 ℃ for from 1 second to 10 seconds; and even more preferably from 125 ℃ to 145 ℃ for from 1 second to 5 seconds. When high temperature sterilization is performed, proteins are denatured and the sterilization solution is easily thickened. In other words, if high-temperature sterilization is not performed, the mixed solution is difficult to thicken after sterilization. Therefore, the effect of reducing the thickening degree by homogenization is particularly effective in the case of high-temperature sterilization.

In addition, when high-temperature sterilization is performed, the pressure (pressurization or depressurization) of the prepared liquid can be adjusted. At this time, normally, for the purpose of preventing the boiling of the preparation, for example, the sterilization pressure is set to about 1kg/cm²To 10kg/cm². That is, in the high-temperature sterilization of the present invention, such pressure may be applied in addition to the temperature (heating). The equipment for high-temperature sterilization comprises plate type heat exchanger and tube type heat exchangerHeat exchangers, steam injection type sterilizers, and joule heating type sterilizers. On the other hand, in the homogenization, if a homogenizer is used, for example, the temperature is set at about 10 to 60 ℃, and the flow rate is set at about 100 to 10,000L/h, and the pressure is set at 10 to 100MPa, preferably 20 to 80MPa, more preferably 30 to 70MPa, and more preferably 20 to 50 MPa. In addition, the conditions of the operations such as high-temperature sterilization and homogenization can be changed, if necessary, and performed by various treatments.

Hereinafter, the present invention will be described with reference to more specific examples, but the present invention is not limited thereto. In the tempering step, warm water at the aforementioned temperature is stirred in a tank, and in consideration of ease of mixing and dispersion, raw materials other than the vitamin mixture (mixed vitamin component) are added, mixed, and stirred to produce a tempering liquid. The order of addition of the raw materials to facilitate mixing and dispersion varies depending on the amount and nature of the raw materials. Thus, depending on the composition, the various types of mixed components may be added at one time (at once) or separately in various orders. Specifically, for example, one method is to add sugars, proteins, oils and fats, and then minerals in that order. Another example is a method of adding a portion of the sugars, proteins other sugars, minerals, then oils and fats in that order. Further, another example is a method of adding oil and fat, protein, sugar, and then minerals in this order. To the sterilized solution, a vitamin mixture (mixed vitamin component), a flavoring agent (perfume), and the like are added and mixed to produce a final sterilized solution. The final sterilized solution was subjected to steam injection type heat sterilization and then homogenized using a homogenizer (homogenization by two-stage pressurization) to produce a composition.

The composition of the present invention can be used as an enteral nutrient having a preventive effect against influenza infection. That is, when the composition of the present invention is mixed into a liquid diet or an enteral nutrient, since the liquid diet or the enteral nutrient itself has a preventive effect against influenza infection, it can be used not only as a food but also for the purpose of preventing influenza infection. It can be used for preparing food with at least one of the following functions, such as specific health food and nutritious functional food:

prophylactic effects against influenza infection;

enhancing neutralizing antibody-induced effects; or

The effect of preventing the reduction of neutralizing antibody titer after vaccination.

In facilities where there are many elderly and hospitalized patients, it can be a preventive means for enhancing infection prevention effect of influenza vaccine. Alternatively, it can be used as an infant food for influenza prevention for infants, or a general nutritional food for influenza infection prevention. Furthermore, in a preferred embodiment, the composition of the present invention may be formulated into a composition having an intestinal regulatory effect. Therefore, it can be used as a nutritional supplement drug or a food for preventing influenza infection, which has an intestinal function regulating effect.

All prior art references cited herein are incorporated by reference into this specification. Hereinafter, the present invention will be described in further detail with reference to examples.

Examples

Example 1 measurement of antibody titer after influenza vaccine inoculation to tube-fed patients:

[ method ]

Patients receiving tube feeding were divided into two groups, test and control groups, and the effect of propionic acid bacteria cultures on neutralizing antibody induction of influenza vaccines was evaluated. The patient background for the control and test groups is summarized in table 1. No significant differences in patient background were shown between groups when analyzed by Student's t-test (homogeneous distribution) or Welch test (heterogeneous distribution).

Patient background for control and test groups

[ Table 1]

Statistical significance was analyzed by Student's t-test (uniform distribution) or Welch test (non-uniform distribution).

The liquid diet administered to each group was as follows:

control group (11 persons): conventionally making into liquid diet;

test group (11 persons; however, 12 persons were subjected to stool scoring and flora analysis): test liquid diet and propionic acid bacterial cultures were applied simultaneously.

A "regular composition liquid diet" is a liquid diet containing milk proteins as proteins, but no milk fermentation components or propionic acid bacterial cultures.

On the other hand, the test liquid diet was a liquid diet containing a milk fermentation fraction (3.8g/100kcal) as a protein. The milk fermentation components included in the test liquid diet were produced as follows: fermenting skim milk with Lactobacillus bulgaricus and Streptococcus thermophilus, and concentrating; adding Mel, vitamins, minerals, edible oil, dietary fiber and dextrin, and sterilizing.

Further, the propionic acid bacterium culture is a Propionibacterium freudenreichii (a propionic acid bacterium) culture. In addition, the propionic acid bacterial culture contains DHNA (1, 4-dihydroxy-2-naphthoic acid), which is a component derived from the propionic acid bacterial culture. The simultaneous administration of the test liquid diet and the propionic acid bacteria culture is considered to be the administration of a new nutritional composition containing the milk fermentation component and the propionic acid bacteria culture. In addition, galacto-oligosaccharides are administered as oligosaccharides.

There was no substantial difference between the conventional composition liquid diet and the test liquid diet, except that the test liquid diet contained a milk fermentation component. At the end of the examples, the composition (average) of these diets is shown (table 6). Each component was administered to both the control group (regular composition liquid diet administration group) and the test group (test liquid diet administration group) so that there was no significant difference based on calories. As a result, the following amounts of milk fermentation components and propionic acid bacteria culture were added to the patients in the test group, respectively:

milk fermentation components: average 33 g/day; and

propionic acid bacterial cultures: on average, 1 g/day (about 13. mu.g/day when converted to DHNA amounts, or 1.6. mu.g DHNA per 100kcal caloric intake).

The date of the start of administration of the above diet in each group was defined as-4 weeks (time of grouping), and after four weeks (set as week 0), the upper arm was inoculated subcutaneously with 0.5mL of influenza vaccine (H1N1 type, H3N2 type, or B type vaccine) (Chemo-series-Therapeutic Research Institute) (fig. 1). The number of bifidobacteria in the faeces was calculated and the intestinal flora was compared 4 weeks before and 4 weeks after vaccination. Blood was collected from all subjects in each group according to the following schedule, and antibody titers against the vaccine and cytokine concentrations in the blood were determined. To determine cytokine concentrations in blood, blood was collected according to the following schedule:

on the day of grouping (week-4);

at the time of influenza vaccination (week 0); and

2 weeks (week 2) and 6 weeks (week 6) after vaccination.

Neutralizing antibodies were measured by hemagglutination inhibition assay (HI method), blood cytokines were measured by ELISA method, and the number of bifidobacteria in feces was measured by real-time PCR method. The results of the measurements before and after the grouping time, and at weeks 2 and 5 after vaccination were compared for both groups. At this time, an antibody titer of less than 10 was defined as 5.

Further, with respect to the value of the stool score, the score was recorded every day from the time of grouping (week-4) for each patient according to the Bristol stool classification (table 2), and the average score for that week was analyzed. The Bristol stool grading has seven grades from 1 to 7, with 1 being defined as the hardest stool, 7 being defined as the watery stool, and 3 and 4 being defined as the normal stools.

Statistical analysis was performed as follows. Statistical significance of the expression rate of infection-inhibiting antibodies was analyzed by chi-square test. For neutralizing antibody titers, log10 (antibody titers) was calculated, then analyzed for statistical significance between the two groups by the Mann-Whitney U test, and after vaccination by the Wilcoxon signed rank sum test. With regard to the cytokine concentration and the intestinal flora in the blood, the statistical significance between the two groups was analyzed by Student's t-test (homogeneous distribution) or Welch-test (heterogeneous distribution), and the statistical significance before and during the weekly test was analyzed by the respective t-test. Statistical significance of stool scores between the two groups was analyzed by the Mann-Whitney U test and by the Wilconxon signed rank sum test before and during the test.

[ Table 2] Bristol stool sample

[ results ]

Infection-inhibiting antibody titers were set to 40 or higher (Kojimahara N, et al, vaccine.2006; 24: 5966-9, Scharpe J, et al, Am J Kidney Dis.2009; 54:77-85), and the expression rates of infection-inhibiting antibodies at weeks 2 and 6 after vaccination were determined for subjects whose average antibody titers were lower than 40 at the time of fractionation (week-4) and at the time of vaccination (week 0). The results obtained were analyzed by chi-square test. With respect to the H1N1 and H3N2 antigens, the expression rate of the subjects reaching the infection-suppressing antibody titer was higher in the test group than in the control group for both the week 2 and week 6 time points. Regarding the B-1 antigen, the expression rates in both groups were 9% at week 6, but the test group had a higher expression rate at week 2 than the control group. Furthermore, when significance testing was performed between the two groups, the infection-suppressing antibody titer expression rate for H3N2 antigen was significantly higher in the test group at week 6 (table 3).

Thus, by comparative experiments, it was revealed that the neutralizing antibody induction of influenza vaccine was enhanced by the simultaneous intake of milk fermentation components and propionic acid bacteria culture.

Expression rate of infection-suppressing antibody

[ Table 3]

	H1N1	H3N2	B-1
					n week 2 week 6	n week 2 week 6	n week 2 week 6
Control group	10 30%(3) 10%(1)	10 50%(5) 10%(1)	11 18%(2) 9%(1)
				Test group	9 44%(4) 44%(4)	11 64%(7) 64%(7)	11 27%(3) 9%(1)
P value	0.86 0.24	0.85 0.04	11

() Indicating the number of patients

Comparing the neutralizing antibody titers at weeks 2 and 6 after influenza vaccination, the antibody titers against H1N1 and H3N2 antigens in the control group were significantly reduced compared to week 2 at week 6. On the other hand, in the test group, the neutralizing antibody titer did not change between week 2 and week 6 (fig. 2A and 2B).

Next, when analyzing the intestinal flora before and after the influenza vaccination time, the number of Bifidobacterium enteric (Bifidobacterium) (log10 (bacteria number/g stool)) in the test group increased from 6.39 ± 1.91 to 7.37 ± 2.4, but in the control group from 6.65 ± 2.97 to 6.18 ± 2.80 without any change (table 4).

Variation in the number of bifidobacteria in the intestine (unit: number of bacteria/1 g of feces log)

[ Table 4]

In addition, the stool score values of the test groups were significantly reduced at weeks-1, 0, 1,2, and 6 as compared to the time of grouping (week-4), and an improvement in stool was observed. On the other hand, the fraction values in the control group during the administration were not significantly reduced compared to the start of the administration (fig. 3). In addition, fecal scores were significantly reduced in the test group at weeks 3 and 6 compared to the control group.

Regarding the change in the blood cytokine concentration, no large change in any cytokine could be observed between the test group and the control group. However, within the test group, IL-7 was significantly increased at week 6 compared to before vaccination (week-4). At this time, no change in IL-7 concentration was observed at weeks-4 and 6 in the control group.

[IL-7]

In the test group, the IL-7 concentration was significantly increased at week 6 compared to week-4 (at the time of grouping or at the time of first blood sampling), but showed no significant change in the control group compared to week-4 (fig. 4). Furthermore, no significant difference was observed between the control group and the test group.

[IL-17]

In both the test and control groups, the IL-17 concentration decreased in week 6 compared to week-4 (at the time of grouping or at the time of first blood sampling) (control group: p <0.05, test group: p >0.05) (FIG. 4). No significant difference was observed between the control and test groups.

[TGF-β1]

In both groups, no significant change was observed in blood TGF- β 1 concentration during administration compared to week-4. On the other hand, a significant increase was observed in the test group compared to the control group at week 0 and week 2. However, since blood TGF- β 1 concentration tended to be higher in the test group than the control group at the time of grouping (week-4) (p =0.074), it was considered that there was no difference between the two groups in the effect on blood TGF- β 1 concentration (fig. 4).

As can be seen from the above results, the combined administration of the milk fermentation composition and the propionic acid bacteria culture has a specific enhancing effect on neutralizing antibody induction upon influenza vaccination. Furthermore, the combined administration of the milk fermentation component and the propionic acid bacteria culture showed a clear intestinal function regulating effect.

EXAMPLE 2 method for producing novel liquid diet

In the tempering step, warm water was stirred in a tank in consideration of ease of mixing and dispersion, and raw materials other than the vitamin mixture (mixed vitamin component) were charged in the tank in the order of fat and oil, milk fermentation component protein, sugar, minerals, and propionic acid bacterial culture (table 5). The milk fermentation component was prepared by lactic acid fermentation using the following microorganisms as lactic acid bacteria starter:

streptococcus thermophilus OLS3059(FERM BP-10740); and

lactobacillus delbrueckii subspecies bulgaricus OLL 1073R-1(FERM BP-10741).

The preparation was heat-sterilized by a steam injection method, and then homogenized using a homogenizer (homogenization by two-stage pressurization) to produce a sterilized solution. To the sterilization liquid, a vitamin mixture (mixed vitamin component) and a flavoring agent (perfume) are added and mixed to produce a final sterilization liquid. The final sterilized solution was further heat-sterilized by a steam injection method (two-step sterilization), and then homogenized using a homogenizer (homogenization by two-stage pressurization) to produce a composition. The composition has good quality and taste.

[ Table 5]

The novel liquid diet comprises the following components:

(composition per 100kcal)

The amount of vitamin a is expressed as retinol equivalent.

EXAMPLE 3 method for producing novel liquid diet

A composition was obtained in a similar manner to that described in example 2 except that the milk fermentation component was prepared by lactic acid fermentation using the following microorganisms as lactic acid bacteria starter. The quality and taste of this composition was good as in example 2.

Microorganisms used as starter for lactic acid bacteria:

streptococcus thermophilus OLS3294(NITE P-77); and

lactobacillus delbrueckii subspecies bulgaricus OLL 1255(NITE BP-76).

EXAMPLE 4 method for producing novel liquid diet

A composition was obtained in a similar manner to that described in example 2 except that the milk fermentation component was prepared by lactic acid fermentation using lactic acid bacteria starter (Lactobacillus bulgaricus and Streptococcus thermophilus inoculated for production of "Meiji Bulgaria yogurt" manufactured by Meiji Dairies Corporation). The quality and taste of this composition was good as in example 2.

[ Table 6]

Liquid diet composition for comparison:

(composition per 100kcal)

(*): the amount of vitamin a is expressed as retinol equivalent.

Industrial applicability

The composition containing the propionic acid bacteria culture provided by the present invention can be used as a prophylactic agent for influenza infection by administering it together with a milk fermentation component. The compositions of the invention can enhance induction of influenza virus neutralizing antibodies in animals vaccinated with influenza vaccines. Thus, by administering the composition of the present invention to an animal vaccinated with an influenza vaccine, the effect of the vaccine in preventing infection or exacerbation can be enhanced. The composition of the present invention may also be incorporated into food or nutritional agents such as liquid foods to produce a composition for oral ingestion, which may be expected to produce an effect of preventing influenza. Furthermore, the composition of the present invention may additionally contain milk fermentation components and oligosaccharides. An intestinal function modulating effect by administration of the composition can also be expected.

Claims (18)

1. Use of a propionic acid bacteria culture comprising (a) one or both of 1, 4-dihydroxy-2-naphthoic acid (DHNA) and 2-amino-3-carboxy-1, 4-naphthoquinone (ACNQ) and (b) a milk fermentation component in the preparation of a composition for the prevention of influenza infection.

2. The use of claim 1, wherein the propionic acid bacterium is a gram-positive anaerobic bacterium belonging to the genus propionibacterium, which anaerobically produces propionic acid from sugars.

3. The use of claim 1, wherein the propionic acid bacterium is propionibacterium freudenreichii.

4. The use of claim 1, wherein the composition additionally comprises an oligosaccharide.

5. The use according to any one of claims 1 to 4, wherein the milk fermentation component is milk or a mixture thereof produced by fermenting milk with a lactic acid bacterium belonging to the genus Lactobacillus and/or a lactic acid bacterium belonging to the genus Streptococcus.

6. The use of any one of claims 1-4, wherein the milk fermentation component is an unripe cheese.

7. The use of claim 4, wherein at least one of the sugars constituting the aforementioned oligosaccharides is galactose.

8. The use of claim 1, wherein both the propionic acid bacteria culture and the milk fermentation component have been sterilized.

9. The use of any one of claims 1-4, 7 and 8, wherein the composition is for enteral administration to an animal receiving influenza vaccination.

10. The use of claim 5, wherein the composition is for enteral administration to an animal receiving influenza vaccination.

11. The use of claim 6, wherein the composition is for enteral administration to an animal receiving influenza vaccination.

12. The use of claim 4, wherein the composition comprises the following nutrients:

a propionic acid bacterial culture;

a milk fermentation component;

an oligosaccharide;

a protein;

a saccharide;

a lipid; and

dietary fiber.

13. The use of claim 12, wherein the composition additionally comprises at least one nutrient selected from the group consisting of: vitamins, minerals, organic acids and organic bases.

14. Use of a propionic acid bacteria culture comprising (a) one or both of 1, 4-dihydroxy-2-naphthoic acid (DHNA) and 2-amino-3-carboxy-1, 4-naphthoquinone (ACNQ) and (b) a milk fermentation fraction in the manufacture of an agent for promoting induction of influenza virus neutralizing antibodies in an influenza vaccine vaccinated animal.

15. The use of claim 14, wherein the milk fermentation component is a processed product obtained by fermenting animal milk by the activity of a microorganism or an enzyme.

16. The use of claim 14, wherein the propionic acid bacterium is a gram-positive anaerobic bacterium belonging to the genus propionibacterium, which anaerobically produces propionic acid from sugars.

17. The use of claim 14, wherein the propionic acid bacterium is propionibacterium freudenreichii.

18. Use according to claim 15, wherein the microorganism is a lactic acid bacterium belonging to the genus Lactobacillus and/or a lactic acid bacterium belonging to the genus Streptococcus, or a mixture thereof.