Phytobacterium plantarum for reducing virus toxicity and application thereof

Abstract

The invention discloses a plant lactobacillus TSXueLian-3 and application of the plant lactobacillus TSXueLian-3 in reduction of virus toxicity, promotion of proliferation of intestinal probiotics and acceleration of human body rehabilitation. The invention further discloses the plant lactobacillus TSXueLian-3, a fermented product thereof, a preparation method of the plant lactobacillus TSXueLian-3 and a beverage prepared from the fermented product of the plant lactobacillus TSXueLian-3. When the plant lactobacillus TSXueLian-3 provided by the invention is used as a single strain, or is prepared into a composition or a fermented product, the virus toxicity can be reduced, which is specifically reflected as follows: the plant lactobacillus TSXueLian-3 can regulate the balance of intestinal flora, promote the increase of beneficial bacteria such as bifidobacterium, lactobacillus and casein in intestinal tracts, shorten the retention time of viruses in human bodies, and improve the immunity of the human bodies. Virus toxicity can be reduced, and human body rehabilitation can be accelerated; the feed additive contains rich short-chain fatty acids (acetic acid, propionic acid and butyric acid) and bacteriocin, and can reduce virus toxicity.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and particularly to a Lactiplantibacillus plantarum for reducing virus toxicity and its application. Background Art

[0002] More than a hundred enteroviruses (EVs) that can infect humans have been discovered worldwide. Enteroviruses belong to the genus Picornavirus and include poliovirus (PV), Coxsackievirus A / B (CVA, CVB), Echovirus, Norovirus (NoV), Rotavirus, and novel enteroviruses. As the infection time prolongs, these viruses can cause common human infectious diseases such as meningitis, myocarditis, hemorrhagic conjunctivitis, herpangina, upper / lower respiratory tract infections, hand, foot, and mouth disease, conjunctivitis, and acute gastroenteritis, seriously endangering human health.

[0003] Influenza virus is a negative-sense single-stranded RNA virus with a lipid envelope. Its genome contains 8 segments and encodes 10 proteins. It belongs to the Orthomyxoviridae family among respiratory viruses and is the pathogen of human respiratory disease influenza. According to its core protein, influenza virus strains can be classified into 4 genera: A, B, C, and D. Influenza A is more prone to mutation and has strong infectivity compared to other types. According to two proteins on the virus surface, hemagglutinin (HA) and neuraminidase (NA), that is, 18 HAs and 11 NAs, influenza A virus can be classified. These mutants can combine to form various different subtypes or strains, such as H1N1 and H5N1. The mutation rate of influenza B virus is relatively slow and usually does not cause serious diseases like influenza A virus. It is generally common in children, long-term care institutions, and university campuses. After influenza virus infects host respiratory mucosal cells, as the infection time prolongs, it will cause degeneration and necrosis of host cells, inducing local damage to the respiratory tract. If not effectively controlled, the virus will further spread to the lungs and even the blood, causing pneumonia and viremia, and may lead to the death of the host in severe cases, seriously endangering human health.

[0004] During the period of infecting the human body, the virus will not only damage the human immune system but also cause intestinal flora imbalance, exacerbate the condition, and induce various complications. Therefore, accelerating human recovery is beneficial to reducing the toxicity of the virus.

[0005] In addition to directly inhibiting and killing viruses and stimulating the body's immune system itself, probiotics will produce various nutrients such as organic acids (e.g., lactic acid, short-chain fatty acids), mucin, amino acids, bacteriocins, etc. during the in vivo or in vitro fermentation process. These products are affected by factors such as fermentation substrates and bacterial species, resulting in different types and quantities, and significant differences in the produced probiotic effects. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a Lactiplantibacillus plantarum that can reduce the toxicity of viruses. This Lactiplantibacillus plantarum can regulate the balance of the intestinal flora, promote the increase of beneficial bacterial genera such as Bifidobacterium, Lactobacillus, and Casei in the intestine, shorten the residence time of viruses in the human body, facilitate the reduction of virus toxicity, and accelerate the recovery of the human body.

[0007] To solve the above technical problems, the present invention provides the following technical solutions:

[0008] The first aspect of the present invention provides the use of Lactiplantibacillus plantarum TSXueLian-3 in the preparation of a preparation for reducing virus toxicity.

[0009] The second aspect of the present invention provides the use of Lactiplantibacillus plantarum TSXueLian-3 in the preparation of a preparation for promoting the proliferation of intestinal probiotics. Among them, the probiotics include, but are not limited to, intestinal beneficial bacteria such as Bifidobacterium, Lactobacillus, and Casei, and for example, may include at least one of Bifidobacterium longum subsp. longum, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus paracasei, and Lactobacillus rhamnosus.

[0010] The third aspect of the present invention provides the use of Lactiplantibacillus plantarum TSXueLian-3 in the preparation of a preparation for relieving influenza symptoms.

[0011] The Lactiplantibacillus plantarum TSXueLian-3 (Lactiplantibacillus plantarum TSXueLian-3) used in the present invention has been described in a Chinese patent application with the publication number CN119351249A (publication date: January 24, 2025) and the title "A Lactiplantibacillus plantarum derived from Saussurea involucrata with strong affinity and improved intestinal constipation and its application". This strain was deposited with the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on September 20, 2024, and the deposit number is GDMCC No. 65165.

[0012] The inventors found during the experiment that compared with other types of Lactiplantibacillus plantarum, the Lactiplantibacillus plantarum TSXueLian-3 provided by the present invention can better regulate the balance of the intestinal flora and promote the increase of beneficial bacterial genera such as Bifidobacterium (such as Bifidobacterium longum subsp. longum, Bifidobacterium bifidum, etc.), Lactobacillus (such as Lactobacillus acidophilus, etc.), and Casei (such as Lactobacillus paracasei, Lactobacillus rhamnosus, etc.) in the intestine; and can shorten the residence time of viruses in the human body, improve the inhibition rate of viruses, thereby reducing virus toxicity and relieving adverse reactions such as high fever, headache, fatigue, and body aches caused by influenza.

[0013] Furthermore, the Lactiplantibacillus plantarum TSXueLian-3 can be in various forms, including but not limited to at least one of bacterial powder, bacterial liquid, and fermentate.

[0014] Furthermore, the preparation includes but not limited to drugs, functional foods, foods, and health foods.

[0015] The fourth aspect of the present invention provides a fermentate, which is obtained by inoculating Lactiplantibacillus plantarum TSXueLian-3 in a composition and then fermenting; wherein, the composition includes the following components in parts by weight: 3-8 parts of cranberry, 10-15 parts of orange, 5-10 parts of coconut meat, 15-25 parts of kiwifruit, 1-5 parts of blueberry, 5-10 parts of blackcurrant, 1-10 parts of anhydrous glucose, and 40-50 parts of water.

[0016] Cranberries contain a variety of functional ingredients beneficial to the human body. Every 100g of cranberries contains 34.3mg of anthocyanins, 0.77mg of proanthocyanidins, 4mg of flavonols, 4.37mg of phenolic acids, 12mg of ellagic acid, 224mg of resveratrol, and 1.05mg of lignans. These anthocyanins, polyphenols, and flavonoids can inhibit the growth of cancer cells and accelerate the apoptosis of cancer cells.

[0017] Oranges are rich in sugar, citric acid, amino acids, dietary fiber, minerals, and vitamin C, which can promote the growth of Lactiplantibacillus plantarum, Lactobacillus, and Bifidobacterium in the intestine, accelerate intestinal peristalsis, promote the excretion of metabolic wastes and harmful substances in the human body, and reduce their toxic effects on the human body.

[0018] Coconut meat is rich in crude fiber, protein, lipids (such as lauric acid and medium-chain fatty acid glycerides), vitamin E, and minerals. Among them, crude fiber and protein can reduce the absorption of cholesterol in the body and accelerate excretion, which is beneficial to reducing blood lipids; lipids and vitamin E have a series of health care functions such as antioxidant, antibacterial, improving human immunity, preventing arteriosclerosis, and preventing obesity. "Compendium of Materia Medica" records that coconut meat has the effects of "tonifying qi and treating wind".

[0019] The pulp of kiwifruit contains rich nutrients and functional factors that maintain human health, can effectively inhibit the synthesis of nitrosamines in the body, and the blocking rate can be as high as 98%. It has a positive preventive effect on the occurrence of various cancers such as breast cancer, lymphoma, and skin cancer, and enjoys the reputation of "the best fruit for anti-cancer".

[0020] Blueberries are rich in polyphenols, such as ellagic acid, anthocyanins, etc. Among them, ellagic acid is an antioxidant that can combine with harmful free radicals in the human body and has a strong anti-cancer effect, which is beneficial to resisting lung cancer and esophageal cancer; anthocyanins can inhibit the activity of enzymes that cause rapid proliferation of cancer cells.

[0021] Blackcurrants contain various amino acids, antioxidants, many minerals and vitamins that are beneficial to human health, and have functions such as improving the body's immunity and health-care effects of reducing enzyme activity to protect the liver, reducing blood lipids, reducing blood pressure, and anti-aging.

[0022] The inventors found that by mixing fresh fruits such as cranberries, oranges, coconut meat, kiwifruit, blueberries, and blackcurrants in a certain proportion and then adding Lactiplantibacillus plantarum TSXueLian-3 for fermentation, the obtained fermented product is rich in various probiotic factors such as vitamins, amino acids, polyphenols, flavonoids, short-chain fatty acids, and bacteriocins, which is beneficial to increasing the inhibition rate of the virus, thereby being able to reduce the virus toxicity and relieve adverse reactions such as high fever, headache, fatigue, and body aches caused by influenza, and has a significant effect on accelerating the recovery of the human body.

[0023] Preferably, the composition includes the following components by weight: 3 parts of cranberries, 10 parts of oranges, 10 parts of coconut meat, 15 parts of kiwifruit, 5 parts of blueberries, 6 parts of blackcurrants, 10 parts of monohydrate glucose, and 41 parts of water.

[0024] The fifth aspect of the present invention provides a method for preparing the fermented product, including the following steps:

[0025] S1. Take the formulated amounts of cranberries, oranges, coconuts, kiwifruit, blueberries, and blackcurrants, pre-treat them and place them in a wall breaker, add monohydrate glucose and water, and then perform wall-breaking treatment; then adjust the pH to 6.2, sterilize to obtain a liquid material;

[0026] S2. Add the Lactiplantibacillus plantarum TSXueLian-3 bacterial liquid to the liquid material, and after fermentation, obtain the fermented product.

[0027] Further, in step S1, the pre-treatment is as follows: First, wash the cranberries, oranges, coconuts, kiwifruit, blueberries, and blackcurrants with clean water and drain the water; then peel and seed the oranges and take the pulp; cut open the coconut, remove the water, and take the coconut meat; peel the kiwifruit and take the pulp.

[0028] Further, in step S1, the sterilization preferably uses high-temperature sterilization, the sterilization temperature is preferably 121 °C, and the sterilization time is preferably 20 min.

[0029] Further, in step S2, the preparation method of the bacterial liquid is: Take out the freeze-dried and preserved Lactiplantibacillus plantarum TSXueLian-3, activate it 3 times with MRS solid medium and MRS liquid medium, and then adjust the bacterial liquid concentration to 1×10 10 CFU / mL. The inoculation amount of the bacterial liquid is preferably 5 parts.

[0030] Further, in step S2, the fermentation temperature is preferably 37 °C, and the fermentation time is preferably 10 h.

[0031] Furthermore, the fermented product can be further prepared into food, preferably into a beverage.

[0032] The beverage of the present invention creatively selects fresh fruits as the main raw material, adds TSXueLian-3 for fermentation. The obtained beverage not only has a good flavor and high popularity among people, but also has the effect of reducing the toxicity of the virus, has a significant effect on accelerating the recovery of the human body, and can maintain human health when consumed daily.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] 1. The Lactiplantibacillus plantarum TSXueLian-3 provided by the present invention can regulate the balance of the intestinal flora, promote the increase of beneficial bacteria genera such as Bifidobacterium, Lactobacillus, and Lactobacillus casei in the intestine, shorten the residence time of the virus in the human body, is beneficial to reducing the virus toxicity, and accelerating the recovery of the human body.

[0035] 2. The present invention uses fresh fruits such as cranberries, oranges, coconut meat, kiwifruits, blueberries, and blackcurrants as the main raw materials, and adds Lactiplantibacillus plantarum TSXueLian-3 for fermentation. The obtained beverage not only has a good flavor and high popularity among people, but also is rich in various probiotic factors such as vitamins, amino acids, polyphenols, flavonoids, short-chain fatty acids, and bacteriocins, which is beneficial to increasing the inhibition rate of the virus, reducing the virus toxicity, alleviating adverse reactions such as high fever, headache, fatigue, and body aches caused by influenza, and has a significant effect on accelerating the recovery of the human body. Detailed implementation manners

[0036] The following further illustrates the present invention with specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments given are not intended to limit the present invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention belongs. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0038] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0039] Experimental example 1

[0040] 1. Experimental strains

[0041] (1) Lactobacillus plantarum CICC 25283 (Accession No.: CICC 25283), Lactobacillus plantarum TSXueLian-3, Lactobacillus plantarum GDMCC 1.4958 (Accession No.: GDMCC 1.4958), Lactobacillus plantarum GDMCC 1.4920 (Accession No.: GDMCC 1.4920).

[0042] (2) Commercially available standard strains: Bifidobacterium longum subsp. longum, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus rhamnosus.

[0043] 2. Sample preparation

[0044] (1) Solution A: Take out the freeze-dried and preserved Lactobacillus plantarum, activate it for 3 generations with MRS solid medium and MRS liquid medium, and then adjust the bacterial liquid concentration to 1×10 10 CFU / mL.

[0045] (2) Solution B: Inoculate Solution A into MRS medium at an inoculation amount of 1%, mix evenly, and anaerobically culture in a 37°C constant temperature incubator for 48 h.

[0046] (3) Solution 1: Wash the soybeans, drain them, and then make them into soy milk. The ratio of soy milk, granulated sugar and purified water is 2:1:30. After boiling, cool it to 37°C, inoculate 1% of Solution A, stir evenly, and ferment at 37°C for 10 h.

[0047] (4) Solution 2: Wash the soybeans, drain them, and then make them into soy milk. The ratio of soy milk, granulated sugar and purified water is 2:1:30. After boiling, cool it to 37°C.

[0048] 3. In vitro culture of commercially available standard strains

[0049] (1) Solution C: Take out the freeze-dried and preserved strain, activate it for 3 generations with MRS solid medium and MRS liquid medium, and then adjust the bacterial liquid concentration to 1×10 10 CFU / mL.

[0050] (2) Solution D: Inoculate Solution C into MRS medium at an inoculation amount of 1%, mix evenly, and anaerobically culture in a 37°C constant temperature incubator for 48 h.

[0051] (3) Solution E

[0052] 1) When the commercially available strain is a Bifidobacterium such as Bifidobacterium longum subsp. longum or Bifidobacterium bifidum, the preparation method of Solution E is: Inoculate Solution A at an inoculation amount of 1% and Solution C at an inoculation amount of 1% into MRS medium, mix evenly, and anaerobically culture in a 37°C constant temperature incubator for 48 h.

[0053] 2) When the commercially available strains are from the genus Lactobacillus (such as: Lactobacillus acidophilus), genus Casei (such as: Lactobacillus paracasei, Lactobacillus rhamnosus), the preparation method of solution E is as follows: Inoculate solution A and solution C into MRS medium at an inoculation amount of 0.5% respectively, mix evenly, and anaerobically culture in a constant temperature incubator at 37°C for 48 h.

[0054] 4. Index detection

[0055] (1) When the commercially available strains are from the genus Bifidobacterium such as Bifidobacterium longum subsp. longum, Bifidobacterium bifidum, etc., determine the viable count of Bifidobacterium in solution D and solution E according to the method of GB 4789.35; when the commercially available strains are from the genus Lactobacillus such as Lactobacillus acidophilus, etc., determine the viable count of total lactic acid bacteria in solution B, solution D and solution E according to the method of GB 4789.35; when the commercially available strains are from the genus Casei such as Lactobacillus paracasei, Lactobacillus rhamnosus, etc., determine the viable count of total lactic acid bacteria in solution B, solution D and solution E according to the method of GB 4789.35. The results are shown in Table 1.

[0056] Table 1 Viable count of bacterial strain solutions under different culture conditions (×10 10 CFU / mL)

[0057]

[0058]

[0059] Please refer to Table 1. The viable count of Bifidobacterium in solution E of Bifidobacterium longum subsp. longum and Bifidobacterium bifidum is higher than that in solution D in some cases and lower in others, indicating that Lactiplantibacillus plantarum has different effects on the growth of strains from the genus Bifidobacterium. Some strains can play a promoting role, while some strains do not. Among them, the viable count of Bifidobacterium in solution E corresponding to Lactiplantibacillus plantarum TSXueLian-3 is the highest, indicating that Lactiplantibacillus plantarum TSXueLian-3 has the best promoting effect on Bifidobacterium.

[0060] The viable count of total lactic acid bacteria in solution E of Lactobacillus acidophilus, Lactobacillus paracasei, and Lactobacillus rhamnosus is higher than that in solution B and solution D, indicating that when Lactiplantibacillus plantarum is mixed and fermented with the genus Lactobacillus and the genus Casei, the effect is better than single-strain fermentation, with a synergistic effect, and can increase the viable count of total lactic acid bacteria. Among them, the viable count of total lactic acid bacteria in solution E corresponding to Lactiplantibacillus plantarum TSXueLian-3 in the genus Lactobacillus and the genus Casei is the highest, indicating that it has the best effect on promoting the proliferation of lactic acid bacteria and the best effect on promoting the increase of beneficial bacteria in the intestine.

[0061] (2) Human testing

[0062] 1) Recruit 40 adult patients with influenza A (H1N1) virus respiratory tract infection. Inclusion criteria: aged 18 - 50 years old; positive nucleic acid test for influenza A (H1N1) virus; meeting the diagnostic criteria for respiratory tract infection; traditional Chinese medicine dialectical classification: wind-heat. Exclusion criteria: patients with concurrent bacterial infection, congenital heart disease, liver and kidney failure; those who withdraw from this study midway.

[0063] 2) Randomly divide the patients into 5 groups, with 8 patients in each group. The grouping is shown in Table 2. For all groups of patients, in addition to taking medications on time as prescribed by the doctor every day, samples are taken 1 hour after lunch every day for 7 days. During the trial period, the interval between taking medications and consuming samples is ≥2 hours. On the 8th day of the trial, the curative effect is evaluated and adverse reactions and symptom remission time are recorded. The evaluation criteria are as follows: (a) Marked effect: symptoms such as high fever, headache, fatigue, whole body aches, etc. completely disappear; (b) Effective: symptoms such as high fever, headache, fatigue, whole body aches, etc. are significantly improved; (c) Ineffective: not meeting the above criteria. The results are shown in Table 3.

[0064] Table 2 Patient grouping and sample trial situation

[0065]

[0066]

[0067] Table 3 Improvement of patients' symptoms

[0068]

[0069] As can be seen from Table 3, compared with Group A, the total number of patients with marked effect and effective number in Groups B - E has increased, indicating that consuming the product prepared by fermenting Lactobacillus plantarum can promote the recovery of virus-infected patients, and the remission time of adverse reactions such as high fever, headache, fatigue, and whole body aches has also decreased. Among them, the total number of patients with marked effect and effective number in Group C is the largest, and the remission time of adverse reactions such as high fever, headache, fatigue, and whole body aches is the shortest, indicating that consuming the product prepared by fermenting Lactobacillus plantarum TSXueLian - 3 has the best effect in promoting the recovery of virus-infected patients, can shorten the residence time of the virus in the human body, reduce the virus toxicity, and accelerate human recovery.

[0070] Combining the experimental results in Table 1 and Table 3, choosing Lactobacillus plantarum TSXueLian - 3 as the best strain to promote the increase of beneficial bacteria in the intestine can reduce virus toxicity and promote human recovery.

[0071] Example 1

[0072] This example provides a preparation method for a drink to reduce virus toxicity, including the following steps:

[0073] S1. Select fresh, non-moldy, ripe cranberries, oranges, coconuts, kiwis, blueberries, blackcurrants, and qualified monohydrate glucose.

[0074] S2. Wash the cranberries, oranges, coconuts, kiwis, blueberries, and blackcurrants with clean water and drain the water; after peeling and deseeding the oranges, take the pulp; after cutting open the coconut and removing the water, take the coconut meat; after peeling the kiwi, take the pulp; then weigh each component according to Table 4 and set aside.

[0075] S3. Add the cranberries, oranges, coconut meat, kiwis, blueberries, and blackcurrants to the blender in sequence, and then add monohydrate glucose and purified water. After feeding, blend at 36000 r / min for 2 min, adjust the pH to 6.2, sterilize at 121 °C for 20 min, and cool to 37 °C to obtain liquid 1.

[0076] S4. Take out the freeze-dried and preserved Lactiplantibacillus plantarum TSXueLian-3, activate it for 3 generations with MRS solid medium and MRS liquid medium, and then adjust the bacterial liquid concentration to 1×10 10 CFU / mL.

[0077] S5. Take 5 portions of the bacterial liquid and add it to liquid 1, stir evenly, ferment at 37 °C for 10 h, and then refrigerate at 4 °C for 12 h to obtain liquid 2.

[0078] S6. Pass liquid 2 through filters with mesh sizes of 100, 200, 300, 400, and 500 meshes in sequence at 4 °C, collect the filtrate, and the obtained filtrate is the drink.

[0079] Comparative Examples 1-7

[0080] The preparation methods of Comparative Examples 1-7 are the same as those of Example 1, except that: the ratios of the fermentation raw materials are different. The specific ratios of the fermentation raw materials are shown in Table 4.

[0081] Table 4

[0082]

[0083] Experimental Example 2

[0084] 1. Determination of the content of short-chain fatty acids (acetic acid, propionic acid, butyric acid)

[0085] (1) Solution preparation

[0086] Solution A: Take methanol and pass it through a membrane.

[0087] Solution B: Take 100 μL of 2-ethylbutyric acid, place it in a 10 mL volumetric flask, add methanol to the scale, and pass it through a membrane.

[0088] Solution C: Prepare a mixed solution containing 13 μg / mL acetic acid, 5 μg / mL propionic acid, and 5 μg / mL butyric acid, make up the volume with methanol, and filter through a membrane.

[0089] (2) Sample solution treatment

[0090] Take 0.5 g of the drink, add 2 ml of methanol, vortex for 2 min, centrifuge at 5000 r / min for 10 min, aspirate the supernatant, centrifuge at 13000 r / min for 10 min at 4 °C, take 1 ml of the supernatant, add 1 μL of sulfuric acid and 3 μL of the internal standard solution, vortex for 2 min, filter through a membrane, and make 3 parallel samples for each sample.

[0091] (3) Chromatographic conditions: Elite-WAX capillary column (30 m × 0.25 mm × 0.25 μm), programmed temperature conditions: initial column temperature 50 °C, increase the temperature to 180 °C at a rate of 50 °C / min, increase the temperature to 210 °C at a rate of 2 °C / min, increase the temperature to 260 °C at a rate of 3 °C / min, and hold for 3 min. The injection port temperature is 200 °C, the carrier gas is high-purity helium, the flow rate is 1.0 mL / min, the column head pressure is 100 kPa; the injection volume is 2 μL, and the split mode (split ratio is 5:1).

[0092] (4) Mass spectrometry conditions: Ionization mode EI, collision energy 70 eV, ion source temperature 250 °C, ion transfer line temperature 250 °C, solvent delay time 2 min, scanning mode: full scan, 40 - 500 m / z.

[0093] (5) Inject Solution A, Solution B, Solution C, and the treated sample solution, calculate the content of short-chain fatty acids in the sample according to the peak area, and find the average value. The results are shown in Table 5.

[0094] Table 5 Content of short-chain fatty acids (μg / g) in the drinks of Example 1 and Comparative Examples 1 - 7

[0095]

[0096] Short-chain fatty acids, also known as volatile fatty acids, are organic fatty acids composed of 1 - 6 carbon atoms, which can inhibit the proliferation of tumor cells, control the expression of proto-oncogenes, promote the differentiation and apoptosis of tumor cells, and play an anti-tumor role. As can be seen from Table 5, the contents of short-chain fatty acids (acetic acid, propionic acid, butyric acid) in the drinks of different examples and comparative examples are different. Among them, the content of short-chain fatty acids (acetic acid, propionic acid, butyric acid) in the drink of Example 1 is the highest. This shows that different formulations will lead to different fermentation effects of Lactiplantibacillus plantarum TSXueLian-3. The lack of any one of cranberry, orange, coconut, kiwifruit, blueberry, and blackcurrant will cause a significant decrease in the content of short-chain fatty acids such as acetic acid, propionic acid, and butyric acid in the fermentation products.

[0097] 2. Evaluation of bacteriocin content

[0098] (1) Experimental virus: CV-A6

[0099] (2) Cell culture

[0100] 1) Cell resuscitation: Take out the cryopreservation tube of human rhabdomyosarcoma (RD) cells from liquid nitrogen, immediately place it in a 37°C water bath and shake rapidly until completely thawed, and centrifuge at 1000 r / min for 5 min at room temperature. Discard the supernatant, add cell culture medium, gently pipette to resuspend, place it in a cell culture incubator at 37°C and 5% CO2, and replace the fresh culture medium after 12 h.

[0101] 2) Cell passage: When the RD cells grow to more than 90% of the bottom area of the cell bottle, discard the culture medium, wash twice with 0.01 M PBS, add 1 mL of 0.25% trypsin, place it in a cell culture incubator at 37°C and 5% CO2 for 1 min. Discard the trypsin, place it in a cell culture incubator at 37°C and 5% CO2, observe under an inverted microscope until the cell gaps are obvious, add the culture medium, pipette the cells until evenly dispersed, adjust the cell density, and place it in a cell culture incubator at 37°C and 5% CO2.

[0102] (3) Acid solution: Containing 0.5 mg / mL lactic acid, 0.5 mg / mL acetic acid, 0.5 mg / mL propionic acid, 0.5 mg / mL butyric acid, and the solvent is purified water.

[0103] (4) Collect RD cells in the logarithmic phase, adjust the cell density to 5×104 / mL with the growth medium, seed into a 96-well plate, 100 μL per well, and culture in a cell culture incubator at 37°C with 5% CO2 for 24 h. Discard the liquid and wash twice with PBS.

[0104] 1) Group A: Add purified water to the 96-well plate, 100 μL per well (MOI = 1), and culture in a cell culture incubator at 37°C with 5% CO 2 for 1 h. Discard the liquid, add the maintenance medium, 100 μL per well, and incubate in a cell culture incubator at 37°C with 5% CO 2 until 48 hpi.

[0105] 2) Group B: Mix purified water and CV-A6 virus solution at a ratio of 30:1, add to the 96-well plate, 100 μL per well (MOI = 1), and culture in a cell culture incubator at 37°C with 5% CO 2 for 1 h. Discard the liquid, add the maintenance medium, 100 μL per well, and incubate in a cell culture incubator at 37°C with 5% CO 2 until 48 hpi.

[0106] 3) Group C: Mix the acid solution and CV-A6 virus solution at a ratio of 30:1, add 100 μL to each well of a 96-well plate (MOI = 1), and incubate in a cell culture incubator at 37°C with 5% CO 2 for 1 h. Discard the liquid, add the maintenance medium, 100 μL to each well, and incubate in a cell culture incubator at 37°C with 5% CO 2 until 48 hpi.

[0107] 4) Sample group: Mix the drink and CV-A6 virus solution at a ratio of 30:1, add 100 μL to each well of a 96-well plate (MOI = 1), and incubate in a cell culture incubator at 37°C with 5% CO 2 for 1 h. Discard the liquid, add the maintenance medium, 100 μL to each well, and incubate in a cell culture incubator at 37°C with 5% CO 2 until 48 hpi. The grouping method is shown in Table 6, and three parallel samples are made for each group.

[0108] Table 6 Types of drinks and grouping in the sample group

[0109]

[0110]

[0111] (5) Observe the cytopathic effect under a microscope. Discard the liquid from the 96-well plate, wash twice with PBS, add 100 μL of maintenance medium and 10 μL of CCK8 to each well, and incubate in a cell culture incubator at 37°C with 5% CO 2 for 1 h. Measure the OD value at 450 nm with an enzyme-labeled instrument, calculate the virus inhibition rate, and find the average value. The results are shown in Table 7. The experimental groups refer to Group B, Group C, and the sample group. According to the virus inhibition situation, evaluate the content of bacteriocin. The better the inhibition effect, the higher the content of bacteriocin.

[0112] Virus inhibition rate % = (OD of experimental group - OD of Group B) / (OD of Group A - OD of Group B) × 100%

[0113] Table 7 Effects of the drinks in Example 1 and Comparative Examples 1 - 7 on the virus

[0114] Group Virus inhibition rate (%) Group A 0 Group B 0 Group C 14.15±1.13 Group D 28.86±1.54 Group E 36.45±1.65 Group F 30.03±3.46 Group G 26.29±1.97 Group H 41.34±5.03 Group I 54.91±3.70 Group J 50.11±3.18 Group K 75.68±5.31

[0115] The proteinaceous metabolites produced by probiotics with antiviral effects are mainly various bacteriocins. Bacteriocins are antimicrobial peptides synthesized ribosomally by many bacteria during the primary growth stage and have antimicrobial, anticancer, and anti-biofilm effects. They are the main substances in probiotic metabolites that exert antiviral effects. As shown in Table 7, the virus inhibition rate in Group C is less than 20%, the virus inhibition rates in Groups D-J are between 25% and 55%, and the virus inhibition rate in Group K is the highest, exceeding 70%. This indicates that a simple acid solution (containing lactic acid, acetic acid, propionic acid, and butyric acid) has a certain inhibitory effect on the virus, but the effect is poor. However, the drink obtained by fermenting various fresh fruits with Lactiplantibacillus plantarum TSXueLian-3 has a better antibacterial effect than the acid solution. This is because in addition to short-chain fatty acids such as acetic acid, propionic acid, and butyric acid, the fermented drink is also rich in bacteriocins, so it can enhance the inhibitory effect on the virus. The fermentation effect of Example 1 is the best, resulting in the highest content of bacteriocins in the drink, so the virus inhibition rate is the highest.

[0116] Combining the experimental results in Table 5 and Table 7, Example 1 was selected as the best formulation for the drink. The obtained formulation can inhibit the growth of the virus, which is beneficial to reducing the toxicity of the virus to the body and reducing the virus toxicity.

[0117] The above-described embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Those skilled in the art of this technology can understand that: without departing from the spirit and scope of the present invention and the appended claims, various substitutions, changes, deletions, additions, and modifications are possible. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. Application of Lactobacillus plantarum TSXueLian-3 in the preparation of preparations for reducing viral toxicity.

2. Application of Lactobacillus plantarum TSXueLian-3 in the preparation of preparations that promote the proliferation of intestinal probiotics.

3. The use according to claim 2, characterized in that: The probiotics include at least one of Bifidobacterium longum subspecies longum, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus paracasei, and Lactobacillus rhamnosus.

4. Application of Lactobacillus plantarum TSXueLian-3 in the preparation of preparations for relieving influenza symptoms.

5. The use according to any one of claims 1 to 4, characterized in that: The plant lactobacillus comprises at least one of bacterial powder, bacterial liquid and fermentation product.

6. The use according to any one of claims 1 to 4, characterized in that: The preparations include medicines, functional foods, foods and health foods.

7. A fermented product, characterized in that The fermented product is obtained by inoculating the plant lactobacillus TSXueLian-3 described in claim 1 into the composition and fermenting it; The composition comprises the following components by weight: 3 to 8 parts of cranberry, 10 to 15 parts of orange, 5 to 10 parts of coconut meat, 15 to 25 parts of kiwi fruit, 1 to 5 parts of blueberry, 5 to 10 parts of blackcurrant, 1 to 10 parts of glucose monohydrate, and 40 to 50 parts of water.

8. A fermentation product according to claim 7, characterized in that: The composition comprises the following components by weight: 3 parts of cranberry, 10 parts of orange, 10 parts of coconut meat, 15 parts of kiwi fruit, 5 parts of blueberry, 6 parts of blackcurrant, 10 parts of glucose monohydrate and 41 parts of water.

9. The method for preparing a fermented product according to claim 7 or 8, characterized in that: The following steps are involved: S1. Take the formula amount of cranberry, orange, coconut, kiwi, blueberry and black currant, place it in a wall breaking machine after pretreatment, add glucose monohydrate and water and then break the wall; then adjust the pH to 6.2, and sterilize to obtain a liquid; S2. Adding the bacterial liquid of Lactobacillus plantarum TSXueLian-3 into the feed liquid, and obtaining the fermented product after fermentation.

10. A beverage, characterized in that: The drink comprises the fermented product according to claim 7 or 8.

Images