TWI805237B - 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 comprises Lactobacillus paracaseiGKS6, Bifidobacterium lactisGKK2, Lactobacillus reuteriGKR1, Lactobacillus rhamnosusGKLC1, Lactobacillus plantarumGK4, Lactobacillus plantarumGKD7, Pediococcus acidilacticiGKA4, Bifidobacterium longumGKL7, Bifidobacterium bifidumGKB2, Bifidobacterium breveGKV1, Lactobacillus brevisGKJOY, or the combination thereof.

Description

Use of lactic acid bacteria for preparing antiviral composition

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

Influenza is a respiratory illness 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; Type B and Type C Type B only infects humans, but only slightly mutates. Type B generally causes regional epidemics, while Type C is less likely to cause symptoms in humans. Type A influenza 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 types (H1-H17) ) and 9 species (N1-N9). According to the pairing of these two antigens, it is subdivided into different subtypes, such as H1N1, H3N2 and H2N2.

The novel influenza H1N1 virus has become a serious problem as it causes pandemics in various parts of the world. In early 2009, the new influenza H1N1 virus first appeared in Mexico and caused human infection cases, then triggered a pandemic, and caused more than 600,000 confirmed cases that year, including more than 10,000 deaths. Today's H1N1 influenza The virus has also evolved to become one of the seasonal influenza strains worldwide. The new H1N1 influenza is a type A influenza virus, which is transmitted by droplet and contact. The incubation period is estimated to be 1 to 4 days, and the infectious period is from the day before the onset to 7 days after the onset. Children's cases are usually contagious 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. In some cases, symptoms of diarrhea and vomiting may appear. So far, the epidemiology of the new H1N1 influenza shows that the severity of the disease is still mild, and the death rate is still around 1/1000 to 4. In some high-risk groups, the probability of complications or death will be 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 people, etc.

Vaccination is currently the main means of prevention and treatment against influenza, but unfortunately, the vaccine is still unable to adequately deal with the mutation of the virus. Seasonal influenza virus antigens are easy to accumulate and mutate, so they will continue to evolve in a short period of time and produce new virus strains. Therefore, the World Health Organization predicts and recommends the influenza vaccine combination that should be used in the influenza season every year. However, due to the irregular mutation of the virus, it is difficult to predict accurately. In the past few years, the results of predicting the mutation of the virus have been inaccurate. Ultimately, the vaccines administered do not provide enough protection for the body to be fully effective in preventing a pandemic.

Furthermore, vaccination has certain restrictions. Vaccines need to be stored in a specific temperature range, and their storage period is limited. Factors such as the technical personnel responsible for vaccination and the people who are vaccinated need to be considered. 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. People who are allergic to "egg" protein or other ingredients in the vaccine, 4. Suffering from acute disease, severe chronic disease, acute onset of chronic disease, cold or fever, 5. In the past 14 days, those who have received other vaccines, 6. Those who have taken anti-immune, anti-viral or anti-inflammatory drugs, 7. Patients with autoimmune diseases or virus infections, etc.

In addition, some vaccinators have adverse reactions after vaccination. During the pandemic of the new influenza virus H1N1 in 2009, there were more than 15,000 reported cases of adverse events after people from many countries were vaccinated, and nearly 5% of them would cause patients to be hospitalized, cause permanent disability, prolong the length of 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 be vaccinated. Therefore, 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, they prevent the invasion of pathogenic bacteria by attaching to the host's intestinal tract, which has positive benefits for the host. At present, the research on lactic acid bacteria has become more and more extensive. The literature points out that its efficacy includes improving irritable bowel syndrome, anti-inflammation, improving atopic dermatitis and anti-allergic functions. There are also related reports on the prevention or treatment of common diseases. In addition, many studies have also pointed out that whether lactic acid bacteria are live bacteria or heat-killed bacteria, they all have certain specific health care purposes. Most of the antiviral research on lactic acid bacteria uses the intake of live lactic acid bacteria to enhance the self-immunity of the eater and resist viral infection, which cannot truly represent the effectiveness of drugs in resisting viral infection when the virus invades the body. Accordingly, the present invention provides a use of a composition of lactic acid bacteria, which has the effect of effectively resisting virus-infected cells.

This disclosure provides the use of lactic acid bacteria and/or their active substances, which are used to prepare an anti-influenza virus infection composition, wherein the lactic acid bacteria are: Lactobacillus paracasei ( Lactobacillus paracasei ) GKS6 is deposited in the food industry development foundation with BCRC 910788 Bifidobacterium lactis GKK2 is deposited in BCRC 910826 at the Biological Resource Conservation and Research Center of the Institute of Food Industry Development; Lactobacillus reuteri GKR1 is BCRC 910827 is deposited in the biological resource preservation and research center of the Food Industry Development Institute of the Foundation; Bacillus plantarum ( Lactobacillus plantarum ) GK4 is deposited with BCRC 910858 in the Bioresource Preservation and Research Center of the Food Industry Development Institute of the Foundation; and Research Center; Pediococcus acidilactici GKA4 is deposited in the Bioresources Preservation and Research Center of the Food Industry Development Institute of the Foundation with BCRC 910876; Bifidobacterium longum ( Bifidobacterium longum ) GKL7 is deposited with the Food Industry Foundation with BCRC 910988 Bifidobacterium bifidum ( Bifidobacterium bifidum ) GKB2 is deposited with BCRC 910986 in the biological resource preservation and research center of the Food Industry Development Research Institute; Bifidobacterium breve ( Bifidobacterium breve ) GKV1 Deposited with BCRC 910989 at the Biological Resource Preservation and Research Center of the Food Industry Development Institute of the Foundation; Lactobacillus brevis GKJOY is deposited with the Biological Resource Preservation and Research Center of the Food Industry Development Institute with BCRC 910920; or combination.

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

Preferably, the composition is administered to the subject with an effective amount of 0.2 to 1 g/60 kg 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, the prevention of influenza virus infection refers to subjects administered the composition before influenza virus infection are infected with influenza virus after influenza virus infection, compared to subjects who were not administered the composition before influenza virus infection. The proportion of infected cells is low.

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

Preferably, the active substances of the lactic acid bacteria are prepared by the following method:

(a) taking the lactic acid bacteria and inoculating them in a solid-state medium for solid-state culture to form colonies;

(b) inoculating the colony cultivated in step (a) into the liquid medium for liquid culture to obtain a bacterial liquid of the liquid medium containing bacteria.

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

Unless otherwise specified in this disclosure, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs.

"Antivirus or antiviral" as used herein refers to the treatment or prevention of viral infections. Wherein, "prevention (prevention or preventing)" refers to preventing an individual from the occurrence of a disease, discomfort, or harmful symptom. In a feasible aspect, the "prevention (prevention or preventing)" mentioned herein refers to preventing the patient from being infected with influenza virus or having symptoms of influenza virus infection before the patient is not infected with influenza virus or does not show symptoms of influenza virus infection. symptom. As used herein, "treatment" or "treating" refers to the process of eliminating, terminating, or reducing a disease, disorder, or deleterious symptom in a subject. In one feasible aspect, the "treatment" (treatment or treating) herein refers to alleviating the symptoms of influenza virus infection in the patient when the patient is infected with influenza virus or has symptoms of influenza virus infection.

The "effective amount" mentioned herein refers to an amount, which is sufficient to produce the effect of preventing or treating influenza virus.

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

The "active substance" mentioned in this article refers to the substance selected from the lactic acid bacteria cells used in this disclosure after being processed through 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 solid culture medium containing bacteria and a bacterial liquid of a liquid culture medium containing bacteria.

In this disclosure, in order to resist influenza virus, a composition comprising lactic acid bacteria and/or its active substances has been developed. In a preferred implementation, the above-mentioned lactic acid bacteria include: Lactobacillus paracasei ( Lactobacillus paracasei ) GKS6 is deposited in the Biological Resource Preservation and Research Center of the Food Industry Development Institute of the Foundation with BCRC 910788; Bifidobacterium lactis ( Bifidobacterium lactis ) GKK2 is deposited in the Biological Resource Preservation and Research Center of the Food Industry Development Institute of the Foundation with BCRC 910826; ; Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) GKLC1 is deposited in the Bioresources Preservation and Research Center of the Food Industry Development Research Institute of the Foundation with BCRC 910828; Lactobacillus plantarum ( Lactobacillus plantarum ) GK4 is deposited with the Food Industry Development Research Foundation of the Foundation with BCRC 910858 The Biological Resources Preservation and Research Center of the Institute; Lactobacillus plantarum GKD7 is deposited in BCRC 910877 in the Bioresources Preservation and Research Center of the Institute of Food Industry Development; Pediococcus acidilactici ( Pediococcus acidilactici ) GKA4 is deposited in BCRC 910876 Bifidobacterium longum ( Bifidobacterium longum ) GKL7 is deposited with BCRC 910988 in the Biological Resource Preservation and Research Center of the Food Industry Development Institute; Bifidobacterium bifidum ( Bifidobacterium bifidum ) GKB2 is deposited with BCRC 910986 in the Biological Resource Preservation and Research Center of the Food Industry Development Institute of the Foundation; Research center; Lactobacillus brevis ( Lactobacillus brevis ) GKJOY deposits with BCRC 910920 in the Bioresource Preservation and Research Center of the Food Industry Development Research Institute; or a combination thereof.

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

Regarding the culture of bacteria, lactic acid bacteria are inoculated on a solid-state medium for solid-state culture to activate the bacteria 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 lasts for 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 picked up and 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 for MRS liquid culture is 14 to 18 hours, more preferably 16 hours. In a preferred embodiment, the pH value of the liquid culture is between pH 5.0 and 7.0, more preferably between pH 5.5 and 6.5. In a preferred embodiment, the formulation of the liquid medium is shown in Table 1 below.

Table 1 Element Ratio (weight percent) carbon source 0 to 5 % Protein Potion 0.1~5% yeast extract 0.1~5% nitrogen source 0.1~5% ion 0.01~2% water The remaining part

The bacterial solution obtained after liquid cultivation was adjusted to pH 7.0, and then sterilized at 121 °C for 1 minute. After the temperature of the bacterial solution dropped to room temperature, it was prepared with DMEM culture solution at a concentration of 5%, as the test substance for the following experiments .

The composition of lactic acid bacteria and/or its active substances provided by the present disclosure further includes additives. In a preferred embodiment, the additive can be excipient, preservative, diluent, filler, absorption enhancer, sweetener, or a combination thereof. The excipient may be selected from sodium citrate, calcium carbonate, calcium phosphate, sucrose or combinations thereof. The preservatives can prolong the shelf life of the pharmaceutical composition, eg benzyl alcohol, parabens. The diluent may be selected from water, ethanol, propylene glycol, glycerin or combinations thereof. Fillers may be selected from lactose, galactose, high molecular weight polyethylene glycols or combinations thereof. The absorption enhancer may be selected from dimethylsulfoxide (DMSO), laurocapram, propylene glycol, glycerin, polyethylene glycol, or combinations thereof. The sweetener may be selected from Acesulfame K, aspartame, saccharin, sucralose, neotame or combinations thereof. In addition to the above-listed additives, other suitable additives can be selected as required, provided that the medicinal effect of the composition is not affected.

The composition can be developed into various 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 of not affecting the effect of the bacteria or active substances, the composition of the present disclosure can be made 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.

According to the form of the composition of the present disclosure, an appropriate dose of the composition is administered to a subject at a predetermined time point by an applicable route. In a preferred embodiment, the route of administration includes oral or parenteral, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, respiratory (aerosol), rectal, vaginal or topical (including oral and sublingual). In a preferred embodiment, the predetermined time point includes every meal, every day, and every week. In a preferred embodiment, the subject is human or animal. In a preferred embodiment, the mother who is the subject provides the immunoglobulin to the offspring through the placenta or breastfeeding, so the subject is a mother who is pregnant or breastfeeding.

Effective amount For in vitro cell culture experiments, 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/60 kg body weight/day". Through the following calculation formula, the effective dose obtained from the in vitro cell culture experiment can be converted into the effective dose for animals, and the effective dose for animals can be converted into the effective dose for humans according to the metabolic rate. I. In general (Reagan-Shaw et al ., 2008), 1 "μg/mL" unit (based on the effective amount obtained from in vitro cell culture experiments) can be equivalent to 1 "mg/kg body weight/day" unit ( Based on the effective amount obtained from rat model experiments). Based on the fact that the metabolic rate of rats is 6.2 times that of humans, the effective dosage for humans can be further obtained. II. If the effective dose is 500 μg/mL based on in vitro cell culture experiments, the effective dose used in rats can be calculated as 500 mg/kg body weight/day (ie, 0.5 g/kg body weight/day ). Further, considering the aforementioned difference in metabolic rate, the effective dose for human use can be calculated as about 5 g/60 kg 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 calculated to be about 0.5 g/60 kg body weight/day through the above conversion formula.

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

Embodiment one: strain source

The strains of lactic acid bacteria used in the preparation of the test substances in the examples of the present disclosure are deposited in the Biological Resource Preservation and Research Center of the Food Industry Development Institute. The lactic acid bacteria strains and their corresponding registration numbers are shown in Table 2. Table 2 serial number bacteria Registration number 1 Lactobacillus paracasei GKS6 BCRC 910788 2 Bifidobacterium lactis GKK2 BCRC 910826 3 Lactobacillus reuteri GKR1 BCRC 910827 4 Lactobacillus rhamnosus GKLC1 BCRC 910828 5 Lactobacillus plantarum GK4 BCRC 910858 6 Lactobacillus plantarum GKD7 BCRC 910877 7 Pediococcus acidilactici GKA4 BCRC 910876 8 Bifidobacterium longum GKL7 BCRC 910988 9 Bifidobacterium bifidum GKB2 BCRC 910986 10 Bifidobacterium breve GKV1 BCRC 910989 11 Lactobacillus brevis GKJOY BCRC 910920

Embodiment two: strain culture

Lactic acid bacteria were inoculated on MRS agar as a solid medium, and cultured in a solid state at 37°C for 2 days to activate the bacteria and form colony formation. After the colony growth on the MRS agar was completed, a single colony was 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 cultivation was adjusted to pH 7.0, and then sterilized at 121 °C for 1 minute. After the temperature of the bacterial solution dropped to room temperature, it was prepared with DMEM culture solution at a concentration of 5%, as the test substance for the following experiments .

Example 3: Virus cultivation and amplification (Virus amplify)

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

Example 4: Source and cultivation 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 is BCRC 60004. Subculture was carried out when the cells grew to 80% full. First, the old cell culture medium was sucked off, and the residual serum and metabolites were washed away with phosphate-buffered saline (PBS). After adding 1 mL of trypsin-EDTA, placed in Incubate at 37°C for 3 to 5 minutes, gently pat the side of the cell plate to promote the detachment of the cells attached to the bottom of the plate, and confirm that the cells have been excised by microscopic examination, stop the action of trypsin with 0.5 mL fetal bovine serum FBS, and replace with DMEM medium The cells were washed down and collected in a 50 mL centrifuge tube, and centrifuged at 500 x g for 5 minutes at 4 °C. Then the supernatant was sucked off, and the precipitated cells were redissolved with DMEM culture solution and counted, and the cell concentration was adjusted to 3 x 10 ⁵ cells/dish. Finally, the cells were planted in a cell culture dish, and a total of 10 mL of DMEM medium containing 10% fetal bovine serum (FBS) was added, and cultured in an incubator at 37°C and 5% CO ₂ .

Example 5: Prevention (Pre-treatment) test

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

Example 6: Co-cultivation (Co-treatment) test

In the experimental group, MDCK was planted in a 96-well plate, and placed in an incubator containing 37 ℃ and 5% CO ₂ for overnight culture. After the cells were attached, 5% of the whole fermentation liquid of lactic acid bacteria was mixed with the H1N1 solution of moi = 0.05 After co-cultivating the mixed solution for 1 hour, add the mixed solution to MDCK (overnight culture) 96-well plate containing 1×10 ⁴ cells/well and continue to act for 1 hour in the incubator, remove the original culture solution and add 2% FBS The fresh DMEM culture solution 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. In the negative control group, the virus and lactic acid bacteria liquid were not added to the process of the experimental group. Compared with the process of the experimental group, the positive control group added virus but no lactic acid bacteria solution.

Example 7: MTS detection

Seed 1×10 ⁴ cells/well of MDCK in a 96-well plate and culture in a 5% CO ₂ incubator at 37°C for 24 hours. Then add samples with different concentrations to act for 48 hours, and then use MTS to detect the cell viability of the samples. The ratio of culture solution to MTS reagent is 5:1. After adding MTS reagent to the cell solution, react in the incubator in the dark for 1 hour, and then use an enzyme immunoassay reader (ELISA reader) to measure the absorbance of the sample at 490 nm Values were used to calculate cell viability.

Test statistics

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

test results

In the prevention test and the co-cultivation test, the experimental group administered with virus and lactic acid bacteria solution, the positive control group administered with virus but not administered with lactic acid bacteria solution, and the negative control group administered with neither virus nor lactic acid bacteria solution The cell viability was used as a benchmark (100%), and the cell viability is shown in Figures 1 to 11. In the prevention test and co-cultivation test, the experimental group administered with virus and lactic acid bacteria solution, compared with the positive control group administered with virus but not administered with lactic acid bacteria solution, the rate of increase in cell survival rate is shown in Table 3: Table 3 serial number bacteria preventive test co-culture test 1 GKS6 13.31% (p<0.01) 23.35% (p<0.05) 2 GKK2 12.56% (p<0.05) 15.43% (p<0.01) 3 GKR1 13.19% (p<0.05) 13.70% (p<0.001) 4 GKLC1 13.61% (p<0.05) 26.16% (p<0.01) 5 GK4 15.82% (p<0.05) 24.54% (p<0.01) 6 GKD7 8.26% (p<0.01) 16.90% (p<0.05) 7 GKA4 11.33% (p<0.001) 24.70% (p<0.001) 8 GKL7 14.89% (p<0.05) 25.08% (p<0.05) 9 GKB2 11.99% (p<0.05) 27.96% (p<0.01) 10 GKV1 12.54% (p<0.05) 25.82% (p<0.05) 11 GKJOY 8.99% (p<0.01) 27.35% (p<0.01)

From Figure 1 to Figure 11, relative to the cell survival rate of the negative control group that was not administered with virus or lactic acid bacteria liquid as a benchmark (100%), the positive control that was administered with virus but not administered with lactic acid bacteria liquid The cell survival rate of each bacterial species in the group was between 20% and 40%, about 30%. Compared with the negative control group, the survival rate of the cells in the positive control group decreased significantly, indicating that the administered virus was toxic to the cells, causing a large number of cells to lose activity or even die.

From Table 3, compared with the cell survival rate of the positive control group in the prevention test, the cell survival rate of the experimental group increased between 5% and 20%. Compared with the positive control group, more cells remained active in the experimental group, which showed that pre-cultivating cells with lactic acid bacteria in the experimental group could help reduce the proportion of cells infected with the virus later, or slow down the degree of virus infection later, and achieve anti-virus Effect.

Also from Table 3, compared with the cell survival rate of the positive control group in the co-culture experiment, the cell survival rate of the experimental group was increased between 10% and 30%. Compared with the positive control group, more cells remained active in the experimental group, which showed that the experimental group co-cultivated the virus and lactic acid bacteria in advance, and then infected or administered the cells at the same time, which helped to reduce the infectivity of the virus and achieve antiviral effects. .

Embodiment eight: composition preparation

If the lactic acid bacteria disclosed in the present disclosure are used as food for antiviral purposes, the following compositions 1 to 2 are used as illustrative examples.

Composition 1: Take the bacterial powder of GKS6 (20 wt%), fully mix it with benzyl alcohol (8 wt%) as a preservative, and glycerin (7 wt%) as a diluent, and dissolve in pure water (65 wt% ), stored at 4°C for later use. The aforementioned wt% refers to the ratio of each component to the total weight of the composition.

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

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

If the lactic acid bacteria disclosed in the present disclosure are used in liquid dosage form as a medical product for antiviral purposes, the following compositions 3 to 4 are used as illustrative examples.

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

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

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

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

Without further elaboration, it is believed that one skilled in the art to which this invention pertains, based on the foregoing description, can utilize the present invention to the widest extent. Therefore, it can be understood that the above description is only used for illustration and not to limit the rest of the disclosure in any way.

none

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

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

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

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

Figure 6 shows the survival rate of Lactobacillus plantarum ( 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 ( Pediococcus acidilactici ) GKA4 on H1N1-infected MDCK cells in the prevention test and the co-culture test (P<0.01 for the prevention test and P<0.01 for the co-culture test).

Fig. 8 shows the survival rate of Bifidobacterium longum GKL7 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).

Fig. 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 and P<0.01 for the co-culture test).

Fig. 10 shows the survival rate of Bifidobacterium breve GKV1 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 11 shows the survival rate of Lactobacillus brevis GKJOY against H1N1-infected MDCK cells in the prevention test and the co-culture test (P<0.01 for the prevention test and P<0.05 for the co-culture test).

Lactobacillus paracasei GKS6 BCRC 910788;

Bifidobacterium lactis GKK2 BCRC 910826;

Lactobacillus reuteri GKR1 BCRC 910827;

Lactobacillus rhamnosus GKLC1 BCRC 910828;

Lactobacillus plantarum GK4 BCRC 910858;

Lactobacillus plantarum GKD7 BCRC 910877;

Pediococcus acidilactici GKA4 BCRC 910876;

Bifidobacterium longum GKL7 BCRC 910988;

Bifidobacterium bifidum GKB2 BCRC 910986;

Bifidobacterium breve GKV1 BCRC 910989;

Lactobacillus brevis GKJOY BCRC 910920.

Claims (8)

A use of lactic acid bacteria, which is used to prepare an anti-influenza A virus (H1N1) composition, wherein the lactic acid bacteria include: Lactobacillus paracasei ( Lactobacillus paracasei ) GKS6 is deposited in BCRC 910788 in the Institute of Food Industry Development Resource Conservation and Research Center; Bifidobacterium lactis GKK2 is deposited in BCRC 910826 at the Biological Resource Conservation and Research Center of the Food Industry Development Institute of the Foundation; Lactobacillus reuteri GKR1 is deposited in the consortium with BCRC 910827 The Biological Resource Preservation and Research Center of the Food Industry Development Research Institute; Lactobacillus plantarum GK4 is deposited in the Biological Resource Preservation and Research Center of the Food Industry Development Research Institute of the Foundation with BCRC 910858; Lactobacillus plantarum GKD7 Deposited with BCRC 910877 in the Biological Resource Preservation and Research Center of the Food Industry Development Institute of the Foundation; Bifidobacterium longum GKL7 is deposited with BCRC 910988 at the Biological Resource Preservation and Research Center of the Institute of Food Industry Development; Bifidobacterium bifidum GKB2 is deposited with BCRC 910986 at the Institute of Food Industry Development Bifidobacterium breve GKV1 is deposited in BCRC 910989 at the Biological Resource Conservation and Research Center of the Food Industry Development Research Institute; or Lactobacillus brevis GKJOY is deposited in BCRC 910920 Bioresource Preservation and Research Center of Food Industry Development Research Institute of Foundation. The use as described in claim 1, wherein the composition is administered to the subject in an effective amount, and the effective amount is 0.05g to 5g/60kg body weight/day. The use as described in Claim 1, wherein the anti-influenza A virus (H1N1) refers to preventing or treating the influenza A virus (H1N1) infection. The use as described in claim 3, wherein the prevention of influenza type A virus (H1N1) infection refers to the experimenter who has not been given the composition before the infection of influenza type A virus (H1N1), the influenza type A virus (H1N1 ) subjects administered the composition before infection have a higher cell survival rate after infection with influenza A virus (H1N1). The use as described in claim 3, wherein the treatment of type A influenza virus (H1N1) infection refers to the subject who has not been given the composition after infection with type A influenza virus (H1N1), the type A influenza virus (H1N1 ) after infection with the composition, the survival rate of cells after infection with influenza A virus (H1N1) is higher. The use according to claim 1, wherein the composition comprises an additive selected from the following group: excipient, preservative, diluent, filler, absorption enhancer, sweetener or a combination 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 as described in Claim 1, wherein the composition is in the form of powder, lozenge, suppository, microcapsule, ampoule, liquid spray or suppository.

Images