TWI757735B - Use of lactic acid bacteria for manufacturing an antiviral composition - Google Patents

Abstract

The present invention provides a use of lactic acid bacteria and the active substance thereof for manufacturing an antiviral composition, wherein the lactic acid bacteria comprisesLactobacillus paracasei GKS6,Bifidobacterium lactis GKK2,Lactobacillus reuteri GKR1,Lactobacillus rhamnosus GKLC1,Lactobacillus plantarum GK4,Lactobacillus plantarum GKD7,Pediococcus acidilactici GKA4,Bifidobacterium longum GKL7,Bifidobacterium bifidum GKB2,Bifidobacterium breve GKV1,Lactobacillus brevis GKJOY, or the combination thereof.

Description

Use of lactic acid bacteria for the preparation of antiviral compositions

The present disclosure relates to the use of a lactic acid bacteria and/or its active substances; more specifically, the lactic acid bacteria and/or its active substances are used to prepare a composition for preventing or treating influenza virus infection.

Influenza is a respiratory disease caused by a ribonucleic acid (RNA) virus. Known influenza viruses are divided into three types: type A, type B and type C. Among them, type A can infect humans, pigs, horses, birds, etc., and is more likely to mutate and cause seasonal pandemics; types B and C Type B only infects people, but only slightly mutates. Type B generally causes regional epidemics, while type C is less likely to cause symptoms in humans. Influenza A virus can be based on two glycoproteins on the surface of the virus as antigen types, namely haemagglutinin (H) and neuraminidase (N), which contain 17 species (H1-H17 ) and 9 species (N1-N9). According to the pairing of these two antigens, it is further subdivided into different subtypes, such as H1N1, H3N2 and H2N2.

The new influenza H1N1 virus has caused pandemics all over the world, and has evolved into a serious problem. At the beginning of 2009, after the new influenza H1N1 virus first appeared in Mexico and caused human infection, it immediately caused a pandemic and caused more than 600,000 confirmed cases in that year, including more than 10,000 deaths. Today, H1N1 influenza The virus has also evolved into one of the global seasonal flu strains. The new H1N1 influenza is a type A influenza virus, which is transmitted by droplets and contact The incubation period is estimated to be 1 to 4 days, and the infectious period is from one day before the onset to 7 days after the onset. Children's cases are usually contagious for longer than adult cases and are also the main source of influenza transmission. Symptoms of the new H1N1 flu include fever, cough, sore throat, body aches, headache, chills and fatigue, and in some cases diarrhea and vomiting. Up to now, the epidemiology of the new H1N1 influenza shows that the severity of the disease is still mild, and the mortality rate is still about 1 to 4 in 1,000. In some high-risk groups, the risk of complications or death is higher, including children under five years old, pregnant women, elderly people over 65 years old, people with underlying diseases (such as HIV, diabetes, cardiovascular disease, chronic lung disease), Immunosuppressed patients, obese patients, etc.

Vaccination is currently the main means of prevention and treatment against influenza, but unfortunately, vaccines are still unable to adequately respond to virus mutation. Seasonal influenza virus antigens are easy to accumulate and mutate, so they will continue to evolve in a short period of time, and new virus strains will be produced. Therefore, the World Health Organization forecasts the recommended influenza vaccine combinations for the influenza season every year. However, due to the irregular variation of the virus, it is difficult to predict accurately. In the past few years, the results of the predicted virus mutation have been inaccurate, resulting in many times that the virus strains circulating in the current season do not match the predicted influenza vaccine. Ultimately, the vaccines administered do not provide enough protection to avoid a pandemic.

Furthermore, there are certain restrictions on vaccination. Vaccines need to be stored in a specific temperature range, have a limited shelf life, and also need to be considered by the technicians responsible for vaccination, as well as the people who are vaccinated. In addition, there are some specific groups that are not suitable for vaccination, including: 1. Infants (less than six months old), 2. Pregnant women (within the first three months of pregnancy), 3. Proteins or vaccines against "eggs" Those who are allergic to other ingredients, 4. Those who suffer from acute disease, severe chronic disease, acute onset of chronic disease, cold or fever, 5. Those who have been vaccinated with other vaccines in the past 14 days, 6. Those who have taken anti-immune, anti-viral or anti-inflammatory drugs, 7. Those with autoimmune diseases or viral infections, etc.

In addition, some vaccinated patients experienced adverse reactions after vaccination. During the 2009 novel influenza virus H1N1 pandemic, more than 15,000 adverse events were reported after vaccination by people in many countries, of which nearly 5% resulted in hospitalization, permanent disability, prolonged hospitalization, Serious adverse events such as fetal congenital malformation, life-threatening or death occurred. Adverse reactions after vaccination have reduced the willingness of the general public to receive vaccination, so it is extremely necessary to find effective substances to prevent or treat the infection of the new type of influenza H1N1.

Lactic acid bacteria are a type of probiotics, which generally refer to bacteria that use carbohydrates to ferment and produce large amounts of lactic acid. Lactic acid bacteria are native health-care bacteria. Functionally, by attaching to the intestinal tract of the host, they prevent the invasion of pathogenic bacteria and have positive benefits for the host. At present, the research on lactic acid bacteria has become more and more extensive, and the literature points out that its efficacy includes improving the functions of irritable bowel syndrome, anti-inflammatory, improving atopic dermatitis and anti-allergy, and other related reports on the prevention or treatment of common diseases. In addition, many studies have also pointed out that lactic acid bacteria, whether they are live bacteria or heat-killed bacteria, have some specific health care purposes. In the research of lactic acid bacteria antiviral, the use of ingestion of live lactic acid bacteria to enhance the consumer's own immunity and resist viral infection does not really represent the effectiveness of drugs against viral infection when the virus invades the body. Accordingly, the present invention provides the use of a composition of lactic acid bacteria, which has the effect of effectively resisting virus-infected cells.

The present disclosure provides the use of lactic acid bacteria and/or active substances thereof, which are used for preparing a composition for anti-influenza virus infection, wherein the lactic acid bacteria are: Lactobacillus paracasei GKS6 is deposited in the food industry development consortium with BCRC 910788 Bioresources Conservation and Research Center of the Institute; Bifidobacterium lactis GKK2 is deposited with BCRC 910826 in the Bioresources Conservation and Research Center of the Food Industry Development Institute; Lactobacillus reuteri GKR1 is registered with BCRC 910827 is deposited in the Biological Resource Conservation and Research Center of the Food Industry Development Institute of the Consortium; Lactobacillus rhamnosus GKLC1 is deposited in the Biological Resources Conservation and Research Center of the Food Industry Development Institute of the Consortium as BCRC 910828; endosperm Lactobacillus plantarum GK4 is deposited with BCRC 910858 in the Biological Resource Conservation and Research Center of the Food Industry Development Research Institute; Lactobacillus plantarum GKD7 is deposited with BCRC 910877 in the Biological Resource Conservation of the Food Industry Development Research Institute and research center; Pediococcus acidilactici GKA4 is deposited with BCRC 910876 in the Biological Resource Conservation and Research Center of the Institute of Food Industry Development; Bifidobacterium longum GKL7 is deposited with BCRC 910988 in the Food Industry Consortium Bioresources Conservation and Research Center of the Institute of Development; Bifidobacterium bifidum GKB2 is deposited with BCRC 910986 in the Bioresources Conservation and Research Center of the Food Industry Development Institute; Bifidobacterium breve GKV1 Deposited with BCRC 910989 at the Biological Resource Conservation and Research Center of the Food Industry Development Research Institute; Lactobacillus brevis GKJOY is deposited with BCRC 910920 at the Biological Resources Conservation and Research Center of the Food Industry Development Research Institute; or combination.

Preferably, the active substance is the bacterial liquid of the lactic acid bacteria.

Preferably, wherein the composition is administered to the subject in an effective amount ranging from 0.2 to 1 g/60kg body weight/day.

Preferably, the influenza virus is influenza A virus, influenza B virus or influenza C virus.

Preferably, the influenza virus is H1N1.

Preferably, the anti-influenza virus refers to the prevention or treatment of influenza virus infection.

Preferably, wherein the prevention of influenza virus infection refers to that a subject who was administered the composition before influenza virus infection was infected with influenza virus after influenza virus infection, relative to subjects who were not administered the composition before influenza virus infection. The proportion of infected cells is lower.

Preferably, wherein the treatment of influenza virus infection refers to that the subject who is administered the composition after influenza virus infection is infected with influenza virus compared to subjects who are not administered the composition after influenza virus infection. The proportion of infected cells is lower.

Preferably, wherein the active substance of these lactic acid bacteria is prepared by the following method: (a) get the thalline of this lactic acid bacteria and inoculate it in solid medium for solid-state culture to form colonies; (b) the colony (colony) cultivated in step (a) ) is inoculated into a liquid medium for liquid culture to obtain a bacterial liquid containing a liquid medium of bacterial cells.

Preferably, wherein the composition comprises an additive selected from the group consisting of excipients, preservatives, diluents, fillers, absorption enhancers, sweeteners or combinations thereof.

Preferably, the composition is a medicine, feed, beverage, nutritional supplement, dairy product, food or health food.

Preferably, the composition is in the form of powder, lozenge, suppository, microcapsule, ampule, liquid spray or suppository.

Figure 1 shows the survival rate of Lactobacillus paracasei GKS6 on H1N1-infected MDCK cells in the prevention test (Pre-treatment) and the co-culture test (Co-treatment) (P<0.01 for the prevention test, P<0.01 for the co-culture test) <0.05).

Figure 2 shows the survival rate of Bifidobacterium lactis GKK2 against H1N1-infected MDCK cells in the prevention test and the co-culture test (P<0.05 for the prevention test, P<0.01 for the co-culture test).

Figure 3 shows the survival rate of Lactobacillus reuteri GKR1 on H1N1-infected MDCK cells in the prevention test and the co-culture test (P<0.05 for the prevention test, P<0.01 for the co-culture test).

Figure 4 shows the survival rate of Lactobacillus rhamnosus GKLC1 on H1N1-infected MDCK cells in the prevention test and the co-culture test (P<0.05 for the prevention test and P<0.01 for the co-culture test).

Figure 5 shows the survival rate of Lactobacillus plantarum GK4 against H1N1-infected MDCK cells in the prevention test and the co-culture test (P<0.05 for Pre-treatment, P<0.01 for the co-culture test).

Figure 6 shows the survival rate of Lactobacillus plantarum GKD7 on H1N1-infected MDCK cells in the prevention test and co-culture test (Pre-treatment P<0.01, co-culture test P<0.05).

Figure 7 shows the survival rate of Pediococcus acidilactici GKA4 on H1N1-infected MDCK cells in the prevention test and the co-culture test (P<0.01 for the prevention test, P<0.01 for the co-culture test).

Figure 8 shows the survival rate of Bifidobacterium longum GKL7 on H1N1-infected MDCK cells in the prevention test and the co-culture test (P<0.05 for the prevention test and P<0.05 for the co-culture test).

Figure 9 shows the survival rate of Bifidobacterium bifidum GKB2 on H1N1-infected MDCK cells in the prevention test and the co-culture test (P<0.05 for the prevention test, P<0.01 for the co-culture test).

Figure 10 shows the survival rate of Bifidobacterium breve GKV1 against H1N1-infected MDCK cells in the prevention test and the co-culture test (P<0.05 for the prevention test and P<0.05 for the co-culture test).

Figure 11 shows the survival rate of Lactobacillus brevis GKJOY on H1N1-infected MDCK cells in the prevention test and the co-culture test (P<0.01 for the prevention test, P<0.05 for the co-culture test).

All technical and scientific terms used in the present disclosure have the same meanings as commonly understood by those of ordinary skill in the technical field to which the present invention belongs, unless otherwise specified in the present disclosure.

As used herein, "antivirus or antiviral" refers to the treatment or prevention of viral infection. Wherein "prevention (prevention or preventing)" refers to the protection of an individual from the occurrence of a disease, discomfort, or detrimental symptom. In a possible aspect, "prevention (preventing)" as used herein refers to preventing the patient from contracting influenza virus or having the risk of contracting influenza virus when the patient is not infected with influenza virus or has no symptoms of influenza virus infection. symptom. "Treatment or treating" as used herein refers to the process of eliminating, ending, or reducing the symptoms of a disease, discomfort, or detriment in an individual. In a feasible aspect, this paper The "treatment" or "treating" refers to alleviating the symptoms of influenza virus infection in a patient when the patient is infected with influenza virus or has symptoms of influenza virus infection.

As used herein, an "effective amount" refers to an amount to be used which is sufficient to effect the prevention or treatment of influenza virus.

The term "cell" as used herein refers to the structure of the lactic acid bacteria used in the present disclosure in various culture stages. In a specific aspect, bacterial system refers to the complete or partial structure of lactic acid bacteria. In a specific aspect, the bacterial system refers to a colony of lactic acid bacteria groups formed by the division and reproduction of a single bacterium. In a specific aspect, the cultivation stage refers to solid state cultivation, liquid cultivation, or liquid scale-up inoculated in a fermenter.

The "active substance" mentioned herein refers to the substance screened out after the lactic acid bacteria cells used in the present disclosure are processed by specific experimental steps, or the mixture of the cells and the culture medium or culture solution. In a specific aspect, the active substance refers to a bacterial liquid containing a solid medium containing bacterial cells and a liquid medium containing bacterial cells.

The present disclosure develops a composition comprising lactic acid bacteria and/or active substances thereof in order to resist influenza virus. In a preferred embodiment, the above-mentioned lactic acid bacteria comprise: Lactobacillus paracasei ( Lactobacillus paracasei ) GKS6 is deposited in the Biological Resource Conservation and Research Center of the Food Industry Development Institute of the consortium with BCRC 910788; Bifidobacterium lactis ( Bifidobacterium lactis ) ) GKK2 is deposited with BCRC 910826 at the Biological Resource Conservation and Research Center of the Food Industry Development Research Institute; Lactobacillus reuteri GKR1 is deposited with BCRC 910827 at the Biological Resources Conservation and Research Center of the Food Industry Development Research Institute. ; Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) GKLC1 is deposited with BCRC 910828 in the Biological Resource Conservation and Research Center of the Food Industry Development Institute; Lactobacillus plantarum GK4 is deposited with BCRC 910858 in the Food Industry Development Research Consortium Bioresources Conservation and Research Center of the Institute; Lactobacillus plantarum GKD7 is deposited with BCRC 910877 in the Biological Resources Conservation and Research Center of the Food Industry Development Research Institute; Pediococcus acidilactici GKA4 is deposited with BCRC 910876 Bioresources Conservation and Research Center of the Food Industry Development Institute of the Consortium; Bifidobacterium longum GKL7 is deposited in the Bioresources Conservation and Research Center of the Food Industry Development Institute of the Consortium with BCRC 910988; Bifidobacterium longum ( Bifidobacterium bifidum ) GKB2 is deposited with BCRC 910986 in the Biological Resource Conservation and Research Center of the Food Industry Development Research Institute; Bifidobacterium breve GKV1 is deposited with BCRC 910989 in the Biological Resources Conservation and Research Center of the Food Industry Development Institute Research Center; Lactobacillus brevis GKJOY is deposited with BCRC 910920 at the Biological Resource Conservation and Research Center of the Food Industry Development Research Institute; or a combination thereof.

However, the strains described in the present disclosure are not limited to being obtained through this channel, and those with ordinary knowledge in the technical field can also obtain the strains of these lactic acid bacteria from other microbial strain preservation units.

Regarding the bacterial culture, the lactic acid bacterial cells are inoculated on a solid medium to perform solid-state culture to activate the bacterial cells and form colony formation. In a preferred embodiment, the temperature of the solid-state culture is 27 to 42°C, more preferably 32 to 37°C. In a preferred embodiment, the solid-state culture time is 1 to 4 days, more preferably 2 to 3 days. In a preferred embodiment, the solid medium is MRS agar. After the colony growth on the solid medium is completed, a single colony is inoculated into a test tube containing MRS liquid medium, and activated by liquid culture. In a preferred embodiment, the temperature of MRS liquid culture is 30 to 55°C, more preferably 32 to 45°C. In a preferred embodiment, the time of MRS liquid culture is 14 to 18 hours, more preferably 16 hours. Time. In a preferred embodiment, the pH value of the liquid culture is pH 5.0 to 7.0, more preferably pH 5.5 to 6.5. In a preferred embodiment, the formulation of the liquid culture medium is shown in Table 1 below.

The bacterial solution obtained after liquid culture is adjusted to pH 7.0, and then sterilized at 121°C for 1 minute. After the bacterial solution temperature is lowered to room temperature, the concentration of DMEM culture solution is configured to be 5%, as the test substance of the following experiments. .

The composition of lactic acid bacteria and/or active substances thereof provided by the present disclosure further comprises additives. In a preferred embodiment, the additive may be an excipient, a preservative, a diluent, a filler, an absorption enhancer, a sweetener, or a combination thereof. The excipient may be selected from sodium citrate, calcium carbonate, calcium phosphate, sucrose, or a combination thereof. The preservatives extend the shelf life of pharmaceutical compositions, eg, benzyl alcohol, parabens. The diluent may be selected from water, ethanol, propylene glycol, glycerol, or a combination thereof. The filler can be selected from lactose, galactose, high molecular weight polyethylene glycols or combinations thereof. Absorption enhancers may be selected from dimethylsulfoxide (DMSO), lauroketone, propylene glycol, glycerol, polyethylene glycol, or combinations thereof. The sweetener may be selected from Acesulfame K, aspartame, saccharin, sucralose/sucralose, neotame (neotame) or a combination thereof. In addition to the additives listed above, other suitable additives can be selected according to the requirements without affecting the medicinal effect of the composition.

The composition can be developed into different commercial products in the field of medicine. In a preferred embodiment, the composition is a medicine, feed, beverage, nutritional supplement, dairy product, food or health food.

Under the premise that the composition of the present disclosure does not affect the effect of the bacteria or active substances, the composition can be prepared into any pharmaceutical form according to the needs of the recipient. In a preferred embodiment, the composition is in the form of powder, lozenge, suppository, microcapsule, ampoule/ampule, liquid spray or suppository.

The composition of the present disclosure is administered to a subject at a suitable dose at a predetermined time point and by a suitable route according to the form of the composition. In a preferred embodiment, the route of administration comprises oral or parenteral, and also includes intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, respiratory (aerosol), rectal, vaginal or topical (including oral and sublingual). In a preferred embodiment, the predetermined time points include every meal, every day, and every week. In a preferred embodiment, the subject is a human or an animal.

Effective amounts are used in in vitro cell culture experiments, and the total volume of cell culture medium used in each culture is defined as "μg/mL". In animal experiments, the effective dose is defined as "g/60kg body weight/day". Through the following calculation formula, the effective dose obtained from in vitro cell culture experiments can be converted into an effective dose for animals, and the effective dose for animals can be converted into an effective dose for humans according to the metabolic rate.

I. In general (Reagan-Shaw et al. , 2008), 1 "μg/mL" unit (effective amount based on in vitro cell culture experiments) can be equivalent to 1 "mg/kg body weight/day" unit ( The effective dose based on rat model experiments). Based on the known metabolic rate of rats is 6.2 times that of humans, the effective dose for humans can be further obtained.

II. If the effective amount is 500 μg/mL based on in vitro cell culture experiments, the effective amount used in rats can be calculated as 500 mg/kg body weight/day (ie, 0.5 g/kg body weight/day). Further, taking into account the aforementioned differences in metabolic rates, an effective amount for human use can be calculated to be about 5g/60kg body weight/day.

III. According to the following test results, the effective dose based on the cell test is 50 μg/mL, and the effective dose for humans is about 0.5g/60kg body weight/day through the above conversion formula.

In a preferred embodiment, the effective amount of the lactic acid bacteria cells or active substances contained in the composition is 0.05g to 5g/60kg body weight/day, more preferably 0.1g to 2g/60kg body weight/day, More preferably, it is 0.2g to 1g/60kg body weight/day.

Example 1: Source of bacteria

The strains of lactic acid bacteria used in the preparation of the test substances in the embodiments of the present disclosure are deposited in the Biological Resource Preservation and Research Center of the Institute of Food Industry Development. The lactic acid bacteria strains and their corresponding deposit numbers are listed in Table 2 below.

Embodiment 2: bacterial culture

The lactic acid bacteria cells were inoculated on MRS agar as a solid medium, and the bacteria cells were activated by solid-state culture at 37° C. for 2 days, and colony formation was formed. After the colony growth on the MRS agar was completed, a single colony was picked up and inoculated into a test tube containing MRS liquid medium, and activated by liquid culture at 37°C for 16 hours. The formulation of the liquid medium is shown in Table 1 above. The bacterial solution obtained after liquid culture is adjusted to pH 7.0, and then sterilized at 121°C for 1 minute. After the bacterial solution temperature is lowered to room temperature, the concentration of DMEM culture solution is configured to be 5%, as the test substance of the following experiments. .

Example 3: Virus cultivation and amplification (Virus amplify)

Influenza A virus (H1N1) was injected into the allantoic membrane of embryonated eggs of specific pathogen free (SPF) chickens at a dose of about 100 half of the tissue culture infective dose (50% tissue culture infective dose, TCID50) in 100 μL . Next, seal the hole of the egg allantoic membrane with wax, put it in an incubator for 1 to 2 days to allow the virus to multiply, and then put it at 4°C to wait for coagulation. Finally, the liquid in the allantoic membrane of the virus-injected chicken embryo egg was drawn out and stored at -80°C to be used as a virus solution for experiments.

Example 4: Source and culture of cell lines

Canine kidney epithelial cells (Madin-Darby Canine Kidney cells, MDCK) were purchased from the Bioresource Collection and Research Center (BCRC) of the Food Industry Research Institute, and the cell line number was BCRC 60004. When the cells grow to eighth full, the subculture is carried out. First, the old cell culture medium is removed, and the residual serum and metabolites are washed with phosphate-buffered saline PBS. After adding 1 mL of trypsin typsin-EDTA, it is placed in 37 Act at ℃ for 3 to 5 minutes, gently tap the side of the cell plate to promote the detachment of the cells attached to the bottom of the plate. After confirming that the cells have been excised by microscopic examination, the action of trypsin is stopped with 0.5 mL of fetal bovine serum FBS, and the cells are washed with DMEM medium. Cells were plated and collected in 50 mL centrifuge tubes and centrifuged at 500 x g for 5 min at 4°C. Then, the supernatant was removed by suction, and the precipitated cells were re-thawed with DMEM medium and counted, and the cell concentration was adjusted to 3×10 ⁵ cells/dish. Finally, the cells were seeded in a cell culture dish, and a total of 10 mL of DMEM medium containing 10% Fetal Bovine Serum (FBS) was added, and the cells were cultured in an incubator at 37°C and 5% CO ₂ .

Example 5: Pre-treatment test

In the experimental group, 1×10 ⁴ cells/well of MDCK were seeded in a 96-well dish and cultured overnight in an incubator containing 37°C and 5% CO ₂ . After the MDCK was attached to the 96-well plate, a 5% concentration of lactic acid bacteria solution was added and acted in an incubator for 1 hour. Then the culture medium was aspirated, and H1N1 solution with a multiplicity of infection (MOI) moi=0.05 was added to the cells for 1 hour in the incubator. The culture medium was removed and fresh DMEM medium containing 2% FBS was added. The cells were cultured for 48 hours in a 37°C, 5% CO ₂ incubator, and then analyzed for cell viability. Compared with the process of the experimental group, the negative control group did not add virus and lactic acid bacteria liquid. Compared with the experimental group, the positive control group added virus but no lactic acid bacteria solution.

Example 6: Co-treatment test

In the experimental group, MDCK was seeded in a 96-well plate and placed in an incubator containing 37°C and 5% CO ₂ for overnight culture. After the cells were attached, 5% lactic acid bacteria fermentation broth was mixed with H1N1 solution with moi=0.05. In order to co-culture for 1 hour after the mixed solution, add the mixed solution to MDCK (overnight culture) 96-well plate containing 1×10 ⁴ cells/well and continue to act in the incubator for 1 hour, remove the original culture solution and add 2% FBS The fresh DMEM medium was cultured in a 37°C, 5% CO ₂ incubator for 48 hours, and finally the cell viability of MDCK was measured with the MTS detection kit. Compared with the process of the experimental group, the negative control group did not add virus and lactic acid bacteria liquid. Compared with the experimental group, the positive control group added virus but no lactic acid bacteria solution.

Embodiment 7: MTS detection

1×10 ⁴ cells/well of MDCK were seeded in a 96-well dish and cultured in a 5% CO ₂ incubator at 37°C for 24 hours. Then, samples of different concentrations were added for 48 hours, and then the cell viability of the samples was detected by MTS. The ratio of culture solution to MTS reagent is 5:1. After adding MTS reagent to the cell solution, react in the incubator for 1 hour in the dark, and then use an ELISA reader to measure the absorbance of the sample at 490nm. Calculate cell viability.

Test Statistics

The experimental data are expressed as mean and standard deviation (S.D.). The statistical analysis method used Student's t-test to test the difference between the test substance group and the control group, and p<0.05 indicated a significant difference.

test results

In the prevention test and co-cultivation test, the experimental group administered with the virus and the lactic acid bacteria liquid, the positive control group with the virus but not with the lactic acid bacteria liquid, and the negative control group with neither the virus nor the lactic acid bacteria liquid. The cell viability was used as a benchmark (100%), and the cell viability was shown in Figures 1 to 11 . In the prevention test and the co-cultivation test, the experimental group in which the virus and the lactic acid bacteria liquid were administered, compared to the positive control group that was administered the virus but not the lactic acid bacteria liquid, the improvement ratio of the cell survival rate was as follows in Table 3:

1 to 11, with respect to the cell survival rate of the negative control group in which neither virus nor lactic acid bacteria liquid was administered as a benchmark (100%), the positive control in which the virus was administered but the lactic acid bacteria liquid was not administered. The survival rate of cells in each strain ranged from 20% to 40%, about 30%. The cell viability of the positive control group was significantly decreased relative to the negative control group, indicating that the administered virus was toxic to the cells, causing a large number of cells to lose viability or even die.

It can be seen from Table 3 that in the prevention test, the cell survival rate of the experimental group was increased between 5% and 20% compared with the cell survival rate of the positive control group. Compared with the positive control group, the experimental group had more The cells remained active, showing that the experimental group pre-incubated the cells with the lactic acid bacteria liquid, which helped to reduce the proportion of cells infected by the virus later, or slowed down the degree of subsequent virus infection, and achieved the antiviral effect.

Also from Table 3, in the co-culture experiment, compared with the cell survival rate of the positive control group, the cell survival rate of the experimental group increased by 10% to 30%. Compared with the positive control group, the experimental group has more cells that retain activity, which shows that the experimental group co-cultures the virus with the lactic acid bacteria solution in advance, and then infects or administers the cells at the same time, which helps to reduce the infectivity of the virus and achieve the antiviral effect. .

Example 8: Composition preparation

If the lactic acid bacteria of the present disclosure are applied to antiviral purposes as a food, the following aspects of the compositions 1 to 2 are used as illustrative examples.

Composition 1: Take the bacterial powder (20wt%) of GKS6, mix well with benzyl alcohol (8wt%) as a preservative and glycerol (7wt%) as a diluent, and dissolve in pure water (65wt%), store Reserve at 4°C. The aforementioned wt% refers to the proportion of each component in the total weight of the composition.

Composition 2: GKS6 of composition 1 was replaced with GKK2 (20 wt%), and the rest of the composition was the same as composition 1.

Under the example that the present disclosure provides the composition 1 and the composition 2, those skilled in the art can understand that other lactic acid bacteria strains other than GKS6 and GKK2 in the present disclosure can also be used to prepare the compositions.

If the lactic acid bacteria of the present disclosure are used in liquid dosage forms as medicinal products for antiviral purposes, the following aspects of compositions 3 to 4 are used as illustrative examples.

Composition 3: Take the bacterial powder (20wt%) of GKS6, mix well with benzyl alcohol (8wt%) as a preservative, glycerol (7wt%) as a diluent, and sucrose (10wt%) as a diluent, and dissolve. In pure water (55wt%), stored at 4 ℃ for later use. The aforementioned wt% refers to the proportion of each component in the total weight of the composition.

Composition 4: GKS6 of composition 3 was replaced with GKK2 (20 wt%), and the rest of the composition was the same as composition 3.

Under the example that the present disclosure provides the composition 1 and the composition 2, those skilled in the art can understand that other lactic acid bacteria strains other than GKS6 and GKK2 in the present disclosure can also be used to prepare the compositions.

The details of each specific example of the claimed invention are described above. Other features of the invention claimed in the present disclosure have been clearly presented by the detailed description of each of the foregoing specific examples and the scope of the claims.

Without further elaboration, it is believed that one of ordinary skill in the art to which this invention pertains can, based on the foregoing description, utilize the present invention to its broadest extent. Therefore, it is to be understood that the above description is for illustrative purposes only, and is not intended to limit the remainder of the disclosure in any way.

【Biological Material Deposit】

Lactobacillus paracasei GKS6 BCRC 910788; Bifidobacterium lactis GKK2 BCRC 910826; Lactobacillus reuteri GKR1 BCRC 910827; Lactobacillus rhamnosus GKLC1 BCRC 910828; Germ Lactobacillus plantarum GK4 BCRC 910858; Lactobacillus plantarum GKD7 BCRC 910877; Pediococcus acidilactici GKA4 BCRC 910876; Bifidobacterium longum GKL7 BCRC 910988; Bifidobacterium bifidum Bifidobacterium bifidum ) GKB2 BCRC 910986; Bifidobacterium breve GKV1 BCRC 910989; Lactobacillus brevis GKJOY BCRC 910920.

Claims (8)

Use of a lactic acid bacteria, which is used to prepare a composition against influenza A virus (H1N1), wherein the lactic acid bacteria comprises: Lactobacillus rhamnosus GKLC1 is deposited in the Food Industry Development Institute of a consortium with BCRC 910828 Biological Resource Conservation and Research Center. The use of claim 1, wherein the composition is administered to the subject in an effective amount ranging from 0.05g to 5g/60kg body weight/day. The use according to claim 1, wherein the anti-influenza A virus (H1N1) refers to the prevention or treatment of the influenza A virus (H1N1) infection. The use as claimed in claim 3, wherein the prevention of influenza A virus (H1N1) infection refers to influenza A virus (H1N1) infection in subjects not administered the composition before infection ) subjects administered the composition prior to infection had higher cell viability following influenza A (H1N1) infection. The use as claimed in claim 3, wherein the treatment of influenza A (H1N1) infection refers to influenza A (H1N1) infection with respect to a subject not administered the composition after infection with influenza A (H1N1) ) post-infection subjects administered the composition had higher cell viability following influenza A (H1N1) infection. The use of claim 1, wherein the composition comprises an additive selected from the group consisting of excipients, preservatives, diluents, fillers, absorption enhancers, sweeteners, or combinations thereof. The use as described in claim 1, wherein the composition is a medicine, feed, beverage, nutritional supplement, dairy product, food or health food. The use according to claim 1, wherein the composition is in the form of powder, lozenge, suppository, microcapsule, ampule, liquid spray or plug.

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