TW202218676A - Use of lactic acid bacteria for increasing resistance against enterovirus 71 - Google Patents

Abstract

The present invention relates to a use of lactic acid bacteria for preparing composition of increasing resistance against enterovirus 71 (EV71), in which the lactic acid bacteria are selected from the group consisting of Lactobacillus plantarumGKM3, GK4, GKD7, GKK1, L. paracaseiGKS6, L. fermentumGKF3, L. caseiGKC1, Bifidobacterium lactisGKK2, L. reuteriGKR1, L. rhamnosusGKLC1, GKY7, Pediococcus acidilacticiGKA4, P. pentosaceusGKP4, B. longumGKL7, B. bifidumGKB2, B. breveGKV1, L. brevisGKJOY, GKL9, B. animalsGKBA and any combination thereof. The composition includes a dead bacterial culture of lactic acid bacteria as an active ingredient, thereby reducing a probability of the EV71 infecting animal cells in vitro, as well as reducing a cytotoxicity of the EV71 in vitro.

Description

Use of Lactic Acid Bacteria to Improve Resistance to Enterovirus 71

The present invention relates to the use of probiotics for preparing a composition for improving resistance to enterovirus, in particular to the use of lactic acid bacteria for preparing a composition for improving resistance to enterovirus type 71.

Enterovirus (EV) in the Picornaviridae family is an icosahedral RNA virus. EV is a general term for a group of virus strains that live in the intestines. However, EVs rarely cause gastrointestinal-related symptoms, but cause symptoms such as influenza-like (such as: fever, sore throat, headache, and nausea, etc.), herpes pharyngeal Angina and/or hand, foot and mouth disease, and some EVs can even cause cardiopulmonary or neurological complications.

EVs can be divided into four categories: coxsackie A virus (CAV) and B (CBV), echovirus, poliovirus, and enterovirus types 68 to 71 (EV68 to EV71), among which EV71 is not only easy to cause hand, foot and mouth disease, but also may cause serious central nervous system diseases such as encephalitis. Children under the age of 5 are at high risk of severe EV71, and severe cases may die. However, there are currently no vaccines and specific drugs for EV71, and only a few drugs can relieve pain caused by fever and ulcers. Therefore, it is necessary to develop medical compositions for preventing and/or treating EV71.

In addition to vaccines and drugs, lactic acid bacteria are also one of the targets currently being developed to prevent and/or treat EV71. Lactic acid bacteria can prevent the invasion of pathogens by attaching to the intestinal tract of the host. In addition, lactic acid bacteria also have the effect of improving physiological functions, such as: improving bowel irritability, anti-inflammatory, improving atopic dermatitis and anti-allergic functions. Current studies have found that some live lactic acid bacteria can also be used to prevent or treat EV71. However, the activity and effective dose of lactic acid bacteria are not easy to control, and the safety of lactic acid bacteria cannot be guaranteed 100%. Although studies have confirmed that the heat-killed bacteria of lactic acid bacteria have the effect of improving physiological functions, there is currently no research to confirm whether the heat-killed bacteria of lactic acid bacteria is not. Also has anti-EV71 effect.

In view of this, it is urgent to provide the use of lactic acid strains for improving resistance to EV71 to solve the above problems.

Therefore, one aspect of the present invention is the use of lactic acid bacteria for preparing a composition for improving resistance to enterovirus 71 (EV71).

According to the above aspect of the present invention, a use of lactic acid bacteria for preparing a composition for improving resistance to EV71 is proposed. The above-mentioned lactic acid bacteria can be selected, for example, from the Lactobacillus plantarum ( Lactobacillus plantarum ) GKM3 with the accession number BCRC 910787, the Lactobacillus paracasei ( L. paracasei ) GKS6 with the accession number BCRC 910788, and the fermented lactobacillus ( L. fermentum ) GKF3, accession number BCRC 910825 L. casei ( L. casei ) GKC1, accession BCRC 910826 Bifidobacterium lactis ( Bifidobacterium lactis ) GKK2, accession BCRC 910827 Lactobacillus lorde ( L. reuteri ) GKR1, L. rhamnosus GKLC1 with accession number BCRC 910828, Lactobacillus germinalis GK4 with accession number BCRC 910858, Lactobacillus germinum GKD7 with accession number BCRC 910877, accession number BCRC 910876 Pediococcus acidilactici ( Pediococcus acidilactici ) GKA4, P. pentosaceus ( P. pentosaceus ) GKP4, accession number BCRC 91087, Lactobacillus germarum GKK1, accession number BCRC 910919, Bifidobacterium longum (BCRC 910988 ) longum ) GKL7, Bifidobacterium breve ( B. bifidum ) GKB2 with accession number BCRC 910986, Bifidobacterium breve ( B. breve ) GKV1 with accession number BCRC 910989, Lactobacillus breve with accession number BCRC 910920 ( L. brevis ) GKJOY, B. animals GKBA with accession number BCRC 910917, L. rhamnosus GKY7 with accession number BCRC 910918, Lactobacillus breve with accession number BCRC 910859 A group consisting of GKL9 and any combination of the above. The above-mentioned Bifidobacterium longum GKL7 and Bifidobacterium bifidum GKB2 were deposited at the Biological Resource Center (BCRC) of the Food Industry Development Research Institute, No. 331, Food Road, Hsinchu, Taiwan on April 10, 2020, while Bifidobacterium breve GKV1 is a Deposited in BCRC on April 13, 2020. The above-mentioned composition may contain a dead bacterial culture of lactic acid bacteria as an active ingredient.

According to the above-mentioned embodiment of the present invention, the above-mentioned composition is used for reducing the probability of EV71 infecting animal cells in vitro or reducing the cytotoxicity of the EV71 in vitro.

According to the above-mentioned embodiment of the present invention, the above-mentioned dead bacteria culture may comprise the dead bacteria cells of the above-mentioned lactic acid bacteria and a fermented liquid.

According to the above embodiments of the present invention, the effective dose of the dead bacteria culture to animal cells may be, for example, less than or equal to 5 volume percent (vol.%).

According to the above-mentioned embodiments of the present invention, the above-mentioned Lactobacillus germinum GKM3, Lactobacillus lordosis GKR1, Lactobacillus rhamnosus GKLC1, Lactobacillus germinum GK4, Lactobacillus germinum GKD7, Pediococcus pentosaceus GKP4, Lactobacillus germinum GKK1, Lactobacillus Effective dose of dead cultures of Bifidobacterium GKL7, Bifidobacterium bifidum GKB2, Bifidobacterium breve GKV1, Lactobacillus breve GKJOY, Bifidobacterium animalis GKBA, Lactobacillus rhamnosus GKY7 and Lactobacillus breve GKL9 to animal cells It can be, for example, less than or equal to 1.25 vol.%.

According to the above-mentioned embodiment of the present invention, the above-mentioned Lactobacillus germinum GKM3, Lactobacillus rhamnosus GKLC1, Lactobacillus germinum GK4, Pediococcus pentosaceus GKP4, Lactobacillus germinum GKK1, Bifidobacterium longum GKL7, Bifidobacterium bifidum GKB2, The effective dose of dead bacteria cultures of Bifidobacterium breve GKV1, Lactobacillus breve GKJOY, Bifidobacterium animalis GKBA, Lactobacillus rhamnosus GKY7 and Lactobacillus breve GKL9 to animal cells is less than or equal to 0.3 vol.%.

According to the above-mentioned embodiments of the present invention, the above-mentioned composition can be, for example, an oral composition.

According to the above-mentioned embodiments of the present invention, the above-mentioned composition can be, for example, a food composition or a pharmaceutical composition.

According to the above-mentioned embodiments of the present invention, the above-mentioned composition may further comprise food or pharmaceutically acceptable carriers, excipients, diluents, adjuvants and/or additives.

According to the above-mentioned embodiments of the present invention, the above-mentioned composition can be, for example, dairy products, fermented dairy products, soy milk, soy milk products, baby food, health food or dietary supplements.

Using the composition of the present invention containing the above-mentioned dead bacteria culture of lactic acid bacteria, not only can the resistance of cells to EV71 be effectively improved in a safer way, but also the composition can be manufactured in a dosage form that is easier to control.

Continuing from the above, the present invention provides a use of lactic acid bacteria for preparing a composition for improving resistance to enterovirus 71 (EV71), wherein the composition may comprise a dead bacteria culture of lactic acid bacteria as an active ingredient .

The "lactic acid bacteria" refers to bacteria that produce a large amount of lactic acid by fermenting carbohydrates. In general, lactic acid bacteria can be isolated from dairy products and/or fermented products, such as strains of Lactobacillus , Bifidobacterium and/or Pediococcus . In one embodiment, the lactic acid bacteria can be a strain deposited at the Biological Resource Center (BCRC) of the Food Industry Development Research Institute, No. 331, Food Road, Hsinchu, Taiwan, and it is, for example, selected from Lactobacillus germinalis (BCRC 910787) Lactobacillus plantarum ) GKM3, accession number BCRC 910788 L. paracasei GKS6, accession number BCRC 910824 Lactobacillus fermentum ( L. paracasei ) GKF3, accession number BCRC 910825 Lactobacillus casei ( L. casei ) GKC1, Bifidobacterium lactis GKK2 with accession number BCRC 910826, L. reuteri GKR1 with accession number BCRC 910827, Lactobacillus rhamnosus ( L. reuteri ) with accession number BCRC 910828 rhamnosus ) GKLC1, Lactobacillus germinum GK4 with accession number BCRC 910858, Lactobacillus germinum GKD7 with accession number BCRC 910877, Pediococcus acidilactici GKA4 with accession number BCRC 910876, pentose with accession number BCRC 910879 Pediococcus ( P. pentosaceus ) GKP4, Lactobacillus germarum GKK1 with accession number BCRC 910919, Bifidobacterium longum ( B. longum ) GKL7 with accession number BCRC 910988, Bifidobacterium bifidum with accession number BCRC 910986 ( B Bifidum ) GKB2, Bifidobacterium breve ( B. breve ) GKV1 under BCRC 910989, Lactobacillus brevis ( L. brevis ) GKJOY under BCRC 910920, Bifidobacterium animalis ( B ) under BCRC 910917 Animals) GKBA, L. rhamnosus GKY7 with accession number BCRC 910918, Lactobacillus breve GKL9 with accession number BCRC 910859 and any combination of the above groups.

It is supplemented that the above-mentioned Lactobacillus germinum GKM3 and Lactobacillus paracasei GKS6 were deposited on July 14, 2017, and Lactobacillus fermentum GKF3, Lactobacillus casei GKC1, Bifidobacterium lactis GKK2, Lactobacillus lorde GKR1 and rhamnosus Lactobacillus saccharobacterium GKLC1 was deposited on February 12, 2018, Lactobacillus germinum GK4 and Lactobacillus breve GKL9 were deposited on November 19, 2018, and Lactobacillus germinum GKD7, Pediococcus lactis GKA4 and Pediococcus pentosaceus GKP4 were deposited On March 6, 2019, Lactobacillus germinum GKK1, Lactobacillus breve GKJOY, Bifidobacterium animalis GKBA and Lactobacillus rhamnosus GKY7 were deposited on July 18, 2019, and Bifidobacterium longum GKL7 and Bifidobacterium bifidum Bifidobacterium GKB2 was deposited on April 10, 2020, and Bifidobacterium breve GKV1 was deposited on April 13, 2020.

The "dead bacteria culture" may include, but is not limited to, dead bacteria cells of lactic acid bacteria and fermented liquid. Specifically, the dead bacteria culture refers to a product obtained by the lactic acid bacteria after the culture step and the sterilization step. The cultivation method is not particularly limited, and can be conventional liquid cultivation to obtain bacterial liquid, wherein the bacterial liquid includes live bacteria and fermented liquid. The method of sterilization is not particularly limited, and can be conventional high-temperature sterilization, for example: treating the above-mentioned bacterial liquid at a high temperature of 121° C. for 1 minute to obtain dead bacterial cells from the living bacteria in the bacterial liquid. Compared with live bacteria of lactic acid bacteria or their lyophilized powder, dead bacteria cultures are safer and easier to produce.

The "resistance to EV71" can be divided into "reduce the probability of infecting animal cells in vitro" and "reduce cytotoxicity in vitro". Specifically, "reduce the probability of infecting animal cells in vitro" means that after exposure to EV71, animal cells in vitro are less susceptible to infection, and "reduce cytotoxicity in vitro" means that even if animal cells are infected with EV71, they are not likely to die.

Animal cell experiments confirmed that before cells were exposed to EV71, or when cells were exposed to EV71, administration of the above-mentioned dead bacteria cultures of lactic acid bacteria to cells could increase the cell survival rate, indicating that the dead bacteria cultures of lactic acid bacteria could reduce EV71 in vitro. Infection probability of animal cells. In another experiment, after the cells were exposed to EV71, the dead bacteria culture of the above-mentioned lactic acid bacteria was administered to the cells, which could increase the cell survival rate, indicating that the dead bacteria culture of lactic acid bacteria could reduce the cytotoxicity of EV71 in vitro.

In one embodiment, the effective dose of dead bacteria culture to animal cells may be, for example, less than or equal to 5 volume percent (vol.%), which is equivalent to the bacterial count of 10 ⁶ CFU to 10 ⁹ CFU per milliliter of dead bacteria culture.

In one embodiment, the above-mentioned Lactobacillus germinum GKM3, Lactobacillus lordosis GKR1, Lactobacillus rhamnosus GKLC1, Lactobacillus germinum GK4, Lactobacillus germinum GKD7, Pediococcus pentosaceus GKP4, Lactobacillus germinum GKK1, Bifidobacterium longum The effective dose of GKL7, Bifidobacterium bifidum GKB2, Bifidobacterium breve GKV1, Lactobacillus breve GKJOY, Bifidobacterium animalis GKBA, Lactobacillus rhamnosus GKY7 and Lactobacillus breve GKL9 may be, for example, less than or equal to 1.25 vol.%.

In one embodiment, Lactobacillus germinum GKM3, Lactobacillus rhamnosus GKLC1, Lactobacillus germinum GK4, Pediococcus pentosaceus GKP4, Lactobacillus germinum GKK1, Bifidobacterium longum GKL7, Bifidobacterium bifidum GKB2, Bifidobacterium breve The effective dose of dead cultures of Bacillus GKV1, Lactobacillus breve GKJOY, Bifidobacterium animalis GKBA, Lactobacillus rhamnosus GKY7 and Lactobacillus breve GKL9 to animal cells may be, for example, less than or equal to 0.3 vol.%.

In one embodiment, the above-mentioned composition can be, for example, an oral composition, and the type of the oral composition is not particularly limited, as long as it contains the above-mentioned dead bacteria culture. In one embodiment, the above-mentioned oral composition is dairy products, fermented dairy products, soy milk, soy milk products, baby food, health food or dietary supplement and the like.

The dosage form of the composition of the present invention is not particularly limited. In one embodiment, the dosage form of the composition is an aqueous solution, suspension, dispersion, emulsion (single-phase or multi-phase dispersion system, single- or multi-chamber liposome), hydrogel, gel, solid lipid nanoparticles, tablet formulations, granules, powders and/or capsules, etc.

In one embodiment, the composition may be, for example, a food composition or a pharmaceutical composition. In one embodiment, the composition may optionally contain food or pharmaceutically acceptable carriers, excipients, diluents, adjuvants and/or additives, such as solvents, emulsifiers, suspending agents, disintegrating agents, Binders, stabilizers, chelating agents, diluents, gelling agents, preservatives, lubricants and/or absorption delaying agents, etc.

Several embodiments are used below to illustrate the application of the present invention, but they are not intended to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make various changes and modifications without departing from the spirit and scope of the present invention. retouch.

Embodiment 1, the making of dead bacteria culture

First, lactic acid bacteria strains No. 1 to No. 19 were obtained from BCRC, and the above lactic acid bacteria were inoculated on Man Rogosa and Sharp (MRS) agar medium, followed by solid-state culture at 37° C. for 2 days to obtain a single colony of each strain. , wherein the strains No.1 to No.19 are: Lactobacillus germarum GKM3, Lactobacillus paracasei GKS6, Lactobacillus fermentum GKF3, Lactobacillus casei GKC1, Bifidobacterium lactis GKK2, Lactobacillus lorde GKR1, Lactobacillus rhamnosus Bacillus GKLC1, Lactobacillus germinum GK4, Lactobacillus germinum GKD7, Pediococcus lactis GKA4, Pediococcus pentosaceus GKP4, Lactobacillus germinum GKK1, Bifidobacterium longum GKL7, Bifidobacterium bifidum GKB2, Bifidobacterium breve GKV1, Lactobacillus brevis Bacillus GKJOY, Bifidobacterium animalis GKBA, Lactobacillus rhamnosus GKY7 and Lactobacillus breve GKL9.

A single colony was respectively inoculated into 20 ml of MRS culture solution, and liquid culture was performed at 37° C. for 16 hours to obtain a bacterial solution. After the above bacterial liquid was adjusted to pH 7.0, high temperature sterilization was performed at a temperature of 121° C. for 1 minute to obtain a dead bacterial culture. It should be noted that in solid-state culture and liquid culture, strain No.5, strain No.13 to strain No.15 and strain No.17 need to be carried out in an anaerobic environment, while other strains are carried out in an aerobic environment. . At this time, the bacterial count of the bacterial solution was about 10 ⁶ CFU/mL to 10 ⁹ CFU/mL.

Embodiment 2, Vero cells and cell culture steps

African green monkey kidney cells (African green monkey kidney cells) Vero cells (Accession No.: ATCC CCL-81) are highly sensitive to various types of viruses, so Vero cells are widely used in the study of the molecular mechanism of virus infection, vaccines and recombinant proteins research, such as: rabies, mink distemper, and EV71 vaccine development. In this example, Vero cells were obtained from the American Type Culture Collection (ATCC).

In the cell culture step, Vero cells were cultured in a cell culture medium containing ₂ vol% fetal bovine serum (FBS) in RPMI- 1640.

Example 3. Evaluation of the dead bacteria culture of lactic acid bacteria to enhance the resistance of cells to EV71

Pre-treatment, co-treatment and post-treatment of Vero cells with dead bacteria cultures of lactic acid bacteria as prophylactic, co-culture and therapeutic tests to evaluate lactic acid bacteria Effects of dead bacterial cultures on the resistance of Vero cells to EV17.

In the first control group, Vero cells were subjected only to the above cell culture steps for 48 hours. In the second control group, the virus exposure treatment was performed for 1 hour, followed by the cell culture step for 48 hours, wherein the virus exposure treatment was performed by culturing Vero cells with EV71-containing cell culture medium at 37°C, and EV71 virus infection The multiplicity of infection (MOI) was 0.25.

MTS assay using tetrazolium salt 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) to evaluate different groups Cell viability in Vero cells.

In the prophylaxis test, Vero cells were sequentially subjected to pretreatment for 1 hour, virus exposure treatment for 1 hour, and cell culture step for 48 hours, wherein the pretreatment was performed with 5 vol.%, 1.25 vol.% or 0.3125 vol. % of the dead bacterial culture in cell culture medium Vero cells were incubated at 37°C for 1 hour. In the prophylaxis test, cultures of dead bacteria from strain No. 14 to strain No. 19 were used. Cell viability was analyzed by MTS assay, as shown in Figure 1 .

1 is a bar graph showing the cell viability of the prevention test according to an embodiment of the present invention, wherein the vertical axis represents the cell viability with the first control group as 100%, and the horizontal axis represents the groups, from left to right The first control group, the second control group, the strain No. 14 group, the strain No. 15 group, the strain No. 16 group, the strain No. 17 group, the strain No. 18 group and the strain No. 19 group, "- ” indicates that Vero cells are not in contact with EV71, “+” indicates that Vero cells are in contact with EV71, bar 101 indicates the first control group, bar 103 indicates the second control group, and bar 105 indicates that the concentration of dead bacteria culture was 5 vol .%, bar 107 indicates that the concentration of dead bacteria culture is 1.25 vol.%, bar 109 indicates that the concentration of dead bacteria culture is 0.3125 vol.%, "***" indicates that there is a statistically significant difference compared with the second control group. significant difference (p<0.001).

As shown in Figure 1, the cell viability of the pretreated Vero cells was significantly increased relative to the second control group, wherein when the concentration of dead bacteria culture was 5 vol. The cell survival rates of No. 14 group, strain No. 15 group, strain No. 16 group, strain No. 17 group, strain No. 18 group and strain No. 19 group were increased by 45.39%, 35.90%, 32.82% and 29.39% respectively. %, 28.56% and 31.94%, and the dead bacteria cultures of the above strains can also significantly improve the cell survival rate when the concentrations are 1.25 vol.% and 0.3125 vol.%. This result shows that the dead bacteria cultures of strain No. 14 to strain No. 19 can reduce the probability of EV71 infecting animal cells in vitro.

In the co-culture experiment, the Vero cells were co-treated, wherein the co-treatment was that the Vero cells were first exposed to the virus for 1 hour, and then cultured in the cell culture medium for 48 hours. Liquid cultures contained not only EV71 but also 5 vol.%, 1.25 vol.% or 0.3125 vol.% of dead bacteria cultures. In the co-cultivation experiments, cultures of dead bacteria of strain No. 1, strain No. 5 to strain No. 9 were used. The cell viability of the co-culture assay was analyzed by the MTS assay, as shown in FIG. 2 .

2 is a bar graph showing the cell viability of the co-culture test according to an embodiment of the present invention, wherein the vertical axis represents the cell viability with the first control group as 100%, and the horizontal axis represents the groups, from left to The right is the first control group, the second control group, the strain No. 1 group, the strain No. 5 group, the strain No. 6 group, the strain No. 7 group, the strain No. 8 and the strain No. 9 group, "- ” indicates that Vero cells are not in contact with EV71, “+” indicates that Vero cells are in contact with EV71, bar 201 indicates the first control group, bar 203 indicates the second control group, and bar 205 indicates that the concentration of dead bacteria culture was 5 vol .%, bar 207 indicates that the concentration of dead bacteria culture is 1.25 vol.%, bar 209 indicates that the concentration of dead bacteria culture is 0.3125 vol.%, "**" and "***" indicate the same as the second control group There is a significant difference in comparison, wherein "**" means p<0.01 and "***" means p<0.001.

As shown in Figure 2, compared with the second control group, the cell viability of the co-treated Vero cells was significantly increased, wherein compared with the second control group, the strain No. 1 group, the strain No. 5 group, the strain No. The cell survival rates of group 6, strain No. 7, strain No. 8 and strain No. 9 were increased by 10.63%, 8.78%, 8.37%, 8.58%, 12.06% and 5.88%, respectively. The dead bacteria cultures of strain No. 1 group, strain No. 6 group, strain No. 7 group, strain No. 8 group and strain No. 9 group can also significantly improve the cell survival rate when the concentration is 1.25 vol.% , the cell survival rate of strain No. 1 group, strain No. 7 group and strain No. 8 group was also significantly improved when the concentration was 0.3125 vol.%. This result shows that the dead bacteria cultures of strain No. 1, strain No. 5 to strain No. 9 can reduce the probability of EV71 infecting animals to cells in vitro.

In the treatment trial, virus exposure treatment for 1 hour, post-treatment for 1 hour, and cell culture step for 48 hours were performed sequentially, wherein post-treatment was performed with 5 vol.%, 1.25 vol.% or 0.3125 vol.% Cell cultures of dead bacteria cultures Vero cells were grown at 37°C, and dead bacteria cultures of strain No. 1 to strain No. 15 and strain No. 17 to strain No. 19 were used in the treatment test. The cell viability of the treatment assays was analyzed by the MTS assay, as shown in FIG. 3 .

3 is a bar graph showing cell viability in a treatment test according to an embodiment of the present invention, wherein the vertical axis represents the cell viability with the first control group as 100%, and the horizontal axis represents groups, from left to right They are the first control group, the second control group, the strain No. 1 group to the strain No. 15 group, the strain No. 17 group to the strain No. 19 group, "-" means that the Vero cells are not in contact with EV71, "+" Indicates that Vero cells are in contact with EV71, bar 301 indicates the first control group, bar 303 indicates the second control group, bar 305 indicates that the concentration of dead bacteria culture is 5 vol.%, and bar 307 indicates that the concentration of dead bacteria culture is 5 vol.%. The concentration of 1.25 vol.%, the straight bar 309 indicates that the concentration of dead bacteria culture is 0.3125 vol.%, "**" and "***" indicate significant differences compared with the second control group, where "**" represents p<0.01 and "***" represents p<0.001.

As shown in Figure 3, the cell viability of the post-treated Vero cells was significantly increased relative to the Vero cells of the second control group, wherein when the concentration of dead bacteria culture was 5 vol.%, compared with the second control group group, the cell survival rate of strain No. 1 group to strain No. 15 group, strain No. 17 group to strain No. 19 group increased by 26.49%, 15.27%, 10.84%, 12.73%, 15.55%, 11.60%, 12.00 respectively %, 7.55%, 15.90%, 7.83%, 11.93%, 7.80%, 15.34%, 11.30%, 5.72%, 8.15%, 7.48% and 16.41%. When the concentration of the dead bacteria culture was 0.3125 vol.% for the strain No. 1 group, the strain No. 9 group, the strain No. 11 group to the strain No. 14 group and the strain No. 19 group, compared with the second control group, The cell survival rate also increased significantly, among which strain No. 1 group, strain No. 11 group to strain No. 14 group and strain No. 19 group also significantly increased cell survival when the concentration of dead bacteria culture was 0.3125 vol.% Rate. This result shows that dead bacteria cultures of strain No. 1 to strain No. 15 and strain No. 17 to strain No. 19 can reduce the cytotoxicity of EV71 in vitro.

The above results show that the dead bacteria culture of lactic acid bacteria can reduce the probability of EV71 infecting animal cells in vitro and reduce the cytotoxicity in vitro, which means that the application of the dead bacteria culture of lactic acid bacteria of the present invention can indeed improve the resistance of animal cells to EV71, thereby Has the potential to reduce an individual's chance of being infected with EV71, reduce the chance of developing EV71 infection, reduce symptoms of EV71 onset, or speed up the recovery of EV71-infected patients.

It can be seen from the above examples that the use of the dead bacteria culture of lactic acid bacteria of the present invention for preparing the composition against EV71 has the advantage of using the dead bacteria culture of at least one strain, which is not only easy to manufacture and dose control, but also improves the use of safety. In addition, the dead bacteria culture of lactic acid bacteria of the present invention not only effectively reduces the probability of EV71 infecting animal cells in vitro, but also effectively reduces the cytotoxicity of EV71 in vitro. The efficacy of EV71's resistance.

It should be understood that although Vero cells are used in the present invention to establish a cell model, however, it should not be used to limit the type of cells to which the dead bacterial culture of the lactic acid bacteria of the present invention is administered. Anyone with ordinary knowledge in the technical field to which the present invention pertains will know that the above-mentioned dead bacteria culture of acid bacteria not only has the effect of enhancing the resistance to EV71 on Vero cells, but also in other cells such as intestinal cells, respiratory system cells and/or nerve cells. Cells also had similar or identical results.

It should be added that, although the present invention takes a specific process, a specific cell line, a specific administration method and/or a specific analysis method as an example, it is described that the dead bacteria culture of the lactic acid bacteria of the present invention is used for the preparation of anti-EV71. The purpose of the composition, but any person with ordinary knowledge in the technical field to which the present invention belongs will know that the present invention is not limited to this, and without departing from the spirit and scope of the present invention, the present invention contains the composition of the dead bacteria culture of lactic acid bacteria. Other processes or other analytical methods may be used.

Although the present invention has been disclosed above with several embodiments, it is not intended to limit the present invention. Anyone with ordinary knowledge in the technical field to which the present invention belongs, without departing from the spirit and scope of the present invention, can make various Therefore, the scope of protection of the present invention should be determined by the scope of the appended patent application.

101, 103, 105, 107, 109, 201, 203, 205, 207, 209, 301, 303, 305, 307, 309: Straight

In order to make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the detailed description of the accompanying drawings is as follows: [ Fig. 1 ] is a bar graph showing the cell viability of the prevention test according to an embodiment of the present invention. [Fig. 2] is a bar graph showing the cell viability of the co-culture test according to an embodiment of the present invention. [Fig. [ Fig. 3 ] is a bar graph showing cell viability in a treatment test according to an embodiment of the present invention.

Bifidobacterium longum ( Bifidobacterium longum ) GKL7 , Bifidobacterium longum ( B. bifidum ) GKB2 and B. breve ( B. breve ) GKV1 are deposited in the biological resources of the Institute of Food Industry Development, No. 331, Food Road, Hsinchu, Taiwan Center (BCRC), the deposit numbers are BCRC 910988, BCRC 910986 and BCRC 910989, of which the deposit date of Bifidobacterium longum GKL7 and Bifidobacterium bifidum GKB2 is April 10, 2020, and the deposit date of Bifidobacterium breve GKV1 The date is April 13, 2020.

301, 303, 305, 307, 309: Straight

Claims (7)

A kind of lactic acid bacteria is used for preparing the purposes of the composition that improves the resistance to enterovirus 71 type (Enterovirus 71, EV71), wherein this lactic acid bacteria is selected from the paracasei ( Lactobacillus paracasei ) whose deposit number is BCRC 910788 ) GKS6, L. fermentum GKF3 with accession number BCRC 910824, L. casei GKC1 with accession number BCRC 910825, Bifidobacterium lactis GKK2 with accession number BCRC 910826 , Lactobacillus rhamnosus ( L. rhamnosus ) GKLC1 with accession number BCRC 910828, Pediococcus. acidilactici GKA4 with accession number BCRC 910876, Pediococcus pentosaceus ( P. pentosaceus ) with accession number BCRC 910879 GKP4, Bifidobacterium longum ( B. longum ) GKL7 with accession number BCRC 910988, Bifidobacterium breve ( B. breve ) GKV1 with accession number BCRC 910989, Bifidobacterium animalis ( B. animals ) with accession number BCRC 910917 ) GKBA, Lactobacillus brevis ( L. brevis ) GKL9 whose deposit number is BCRC 910859 and a group formed by any combination of the above, the Bifidobacterium longum GKL7 was deposited at No. 331, Food Road, Hsinchu, Taiwan on April 10, 2020 Consortium Food Industry Development Research Institute Biological Resource Center (BCRC), the Bifidobacterium breve GKV1 was deposited with the BCRC on April 13, 2020, and the composition comprises an effective dose of a thermokilled culture of the lactic acid bacteria As an active ingredient, the heat-killed bacteria culture is obtained by carrying out a liquid culture with Man Rogosa and Sharp (MRS) medium at 37°C, and then performing high temperature sterilization at a temperature of 121°C. The Bifidobacterium lactis GKK2 , this Bifidobacterium longum GKL7, this Bifidobacterium breve GKV1 and this Bifidobacterium animalis GKBA carry out this liquid culture in an anaerobic condition, and this Lactobacillus paracasei GKS6, this Lactobacillus fermentum GKF3, this Lactobacillus casei GKC1, the Lactobacillus rhamnosus GKLC1, the Pediococcus lactis GKA4, the Pediococcus pentosaceus GKP4 and the Lactobacillus breve GKL9 were cultured in an aerobic condition for the liquid culture. Use of the lactic acid bacteria as claimed in claim 1 for preparing a composition for enhancing resistance to EV71, wherein the composition is used for reducing a probability of the EV71 infecting an animal cell in vitro or reducing a cytotoxicity of the EV71 in vitro . The use of the lactic acid bacteria according to claim 1 for preparing a composition for improving resistance to EV71, wherein the heat-killed bacteria culture comprises a dead bacterial cell and a fermented liquid of the lactic acid bacteria. Use of the lactic acid bacteria as claimed in claim 1 for preparing a composition for improving resistance to EV71, wherein the composition is an oral composition. Use of the lactic acid bacteria as claimed in claim 1 for preparing a composition for improving resistance to EV71, wherein the composition is a food composition or a pharmaceutical composition. Use of the lactic acid bacteria as claimed in claim 1 for preparing a composition for improving resistance to EV71, wherein the composition further comprises a food or a pharmaceutically acceptable carrier, an excipient, a diluent, an adjuvant and/or an additive. Use of the lactic acid bacteria according to claim 1 for preparing a composition for improving resistance to EV71, wherein the composition is a dairy product, a fermented dairy product, a soy milk, a soy milk product, a baby food, a health food or a dietary supplement.

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