A kind of plant lactobacillus and application thereof

Abstract

The invention discloses a plant lactobacillus and its application. The plant lactobacillus is isolated from the intestinal tract of a healthy human body and can 7 CFU / mL), has strong alcohol sobering ability, and tolerates a certain concentration of bile salt and acid environment in the human intestine; the strain has significant synergistic degradation ability of ethanol and acetaldehyde, can quickly decompose ethanol, shorten the drunkenness time of KM male mice, which is shortened by more than 52% compared with the model group, and the drunkenness tolerance time is extended by more than 212%. At the same time, it enhances the activity of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in the mouse liver, effectively reduces the acetaldehyde content in the mouse blood, can be used to prepare bacterial agents for preventing and / or relieving drunkenness, and provides a new candidate strain for the preparation of live bacteria preparations for sobering up.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, in particular to a plant lactobacillus and also to the application of the plant lactobacillus. Background Art

[0002] Wine culture is an important part of Chinese traditional culture and is prevalent all over the world. With the rapid development of social science and technology and the continuous improvement of people's living standards, people's requirements for quality of life are increasing day by day, and health awareness is gradually increasing. The market demand for hangover products is also increasing, and the requirements for the quality of hangover medicines are also getting higher and higher.

[0003] Excessive drinking is one of the most important causes of morbidity and mortality worldwide. Improper drinking can easily lead to alcohol poisoning and affect liver function. Alcohol is oxidized by alcohol dehydrogenase into toxic and carcinogenic acetaldehyde, increasing the risk of liver disease (including liver cancer). Long-term drinking can also cause brain dysfunction and impaired consciousness.

[0004] Among the existing hangover products, most of them use natural medicinal plants as development materials. For example, the hangover product disclosed in CN202310406106.0 is composed of perilla leaves, cassia seeds, Hovenia dulcis, Imperata root, wolfberry and ginseng, etc.; the hangover product disclosed in CN202310368665.7 is composed of kudzu root extract powder, roxburghii extract powder, bamboo leaf extract powder, wolfberry extract powder, passion fruit extract powder and β-nicotinamide mononucleotide, etc. The raw materials of these hangover preparations are inconvenient to obtain and the ingredients are complex, which affects the research, development and promotion of hangover products in my country.

[0005] In recent years, microecological preparations have gradually become an important type of hangover products. The key to solving alcohol poisoning is that acetaldehyde dehydrogenase decomposes acetaldehyde into acetic acid. Among the currently disclosed hangover probiotics, for example, CN202310085055.6 discloses a probiotic composition for hangover and liver protection, including Lactobacillus johnsonii LJ-G55, Bifidobacterium adolescentis BQ-G66, etc.; CN202211345077.3 discloses a strain of Lactobacillus paracasei SMN-LBK for the preparation of alcoholic fermented beverages or hangover products. CN115322932 B discloses a strain of Lactobacillus plantarum for hangover relief. Animal experiments have shown that taking a bacterial solution with a concentration of 10 one hour before drinking high-concentration liquor can effectively relieve alcohol poisoning. 10CFU / mL, which has the effect of preventing drunkenness, but the dosage of live bacteria used is large, which is not conducive to the later strain production, storage and processing, and human consumption. As a food safety strain, Lactobacillus plantarum is used as the starting point in the existing invention of probiotic strains for alcohol sobering, but there are few studies on enhancing the host's ability to sober up based on the principle of drunkenness. The present invention provides a Lactobacillus plantarum isolated from the intestine of a healthy human body, which has a significant ability to decompose ethanol and acetaldehyde, and is used for the development of hangover relief preparations suitable for human consumption. Summary of the invention

[0006] In view of this, one of the objects of the present invention is to provide a Lactobacillus plantarum; a second object of the present invention is to provide the use of the Lactobacillus plantarum as a hangover product in degrading ethanol or acetaldehyde under acidic environment and / or bile salt conditions.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] 1. A plant lactobacillus, which is classified and named as Lactiplantibacillus plantarum ZX028 and is preserved in China Center for Type Culture Collection with the accession number of CCTCC M 20232705.

[0009] 2. The use of the plant lactobacillus as a hangover sobering product in degrading ethanol or acetaldehyde in an acid environment and / or bile salt conditions.

[0010] Preferably, the acid environment is at a pH of 2.5-6.

[0011] Preferably, the bile salt condition is not higher than 1.0 g / L.

[0012] 3. Application of the plant lactobacillus in the preparation of a product for prolonging tolerance to drunkenness.

[0013] 4. Application of the plant lactobacillus in preparing a product for shortening the time of drunkenness.

[0014] 5. Application of the plant lactobacillus in improving the enzymatic activities of alcohol dehydrogenase and acetaldehyde dehydrogenase.

[0015] The beneficial effects of the present invention are as follows: the present invention provides a strain of Lactiplantibacillus plantarum ZX028 isolated from the intestine of a healthy human body, which can 7CFU / mL), has strong alcohol sobering ability, and tolerates a certain concentration of bile salt and acid environment in the human intestine; the strain has significant synergistic degradation ability of ethanol and acetaldehyde, can quickly decompose ethanol, shorten the drunkenness time of KM male mice, which is shortened by more than 52% compared with the model group, and the drunkenness tolerance time is extended by more than 212%. At the same time, it enhances the activity of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in the mouse liver, effectively reduces the acetaldehyde content in the mouse blood, can be used to prepare bacterial agents for preventing and / or relieving drunkenness, and provides a new candidate strain for the preparation of live bacteria preparations for sobering up. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0017] Figure 1 The colony morphology of ZX028 on MRS agar medium;

[0018] Figure 2 This is the Gram staining morphology of ZX028 (100×);

[0019] Figure 3 This is the phylogenetic tree of ZX028;

[0020] Figure 4 is the tolerance of ZX028 to different acids;

[0021] Figure 5 is the growth inhibition rate of ZX028 by different concentrations of ethanol;

[0022] Figure 6 is the growth inhibition rate of ZX028 at different concentrations of acetaldehyde.

[0023] Strain preservation

[0024] In the present invention, Lactiplantibacillus plantarum ZX028 is isolated from the intestine of a healthy human body; it is sent to the China Center for Type Culture Collection for preservation, with a preservation number of CCTCC M 20232705, a preservation address of Wuhan University, Wuhan City, Hubei Province, China, a preservation date of December 28, 2023, and is classified and named Lactiplantibacillus plantarum. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0026] Example 1

[0027] The invention discloses a hangover-relieving lactobacillus plantarum ZX028 (Lactiplantibacillus plantarum ZX028), with a preservation number of CCTCC M 20232705.

[0028] 1) Morphological observation of Lactobacillus plantarum ZX028

[0029] The isolated and purified Lactobacillus plantarum ZX028 was activated and streaked onto MRS agar medium containing calcium carbonate, and incubated upside down in a 37°C incubator for 48 h. The colony morphology of the strain was observed. The results are shown in the attached figure. Figure 1 The results showed that the strain grew well on MRS agar medium, with medium-sized colonies, milky white, upwardly convex, moist, neat colony edges, and easy to pick. Gram staining was positive, as shown in Figure 2 shown.

[0030] 2) Molecular biological identification of Lactobacillus plantarum ZX028

[0031] The purified strain was sent to BGI for whole genome sequencing, and the genome sequence has been uploaded to NCBI with the submission number SUB14144161. The sequencing results were compared with the House-keeping Gene database by BLAST. The 19 strains closest to the species level were selected based on 31 housekeeping genes such as dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplB, rplC and rplD. The phylogenetic tree was constructed by using the NJ (Neighbor-Joining) method using MEGA 6.0 software. After alignment, it was found to have the highest homology with Lactobacillus plantarum ( Figure 3 ).

[0032] 3) Gastric acid tolerance test of Lactobacillus plantarum ZX028

[0033] The tolerance of plant lactobacillus ZX028 of the present invention to gastric acid was investigated under the condition of simulating gastric acid. The plant lactobacillus ZX028 strain was inoculated into MRS liquid culture medium at an inoculum size of 2%, and activated and cultured at 37°C for 12h, and activated 3 times continuously. The activated plant lactobacillus ZX028 bacterial solution was inoculated into MRS liquid culture medium at an inoculum size of 2%, and cultured at 37°C for 12h in a constant temperature incubator. The cultivated plant lactobacillus ZX028 bacterial solution was centrifuged at 5000rpm for 10min to collect the bacterial bodies, and the bacterial bodies were resuspended with sterile physiological saline, mixed, and the bacterial suspension was added into sterile physiological saline with a pH value of 2.5, 3, 3.5, 4 and 6 (natural pH of MRS liquid culture medium) at an addition amount of 10%, and the sterile physiological saline with pH 6 was used as a control, and incubated in a constant temperature incubator at 37°C for 4h. The incubated bacterial solution was taken out, diluted 10 times and added with sterile physiological saline, and the number of lactic acid bacteria was detected after vortexing and mixing. The number of live lactic acid bacteria detected was counted and the survival rate was calculated, strain survival rate = test group / control group × 100%.

[0034] The experimental results are as attached Figure 4 As shown: the survival rate of the strain at pH 4 and 3.5 was 95.82% and 88.86% respectively, and the survival rate was less affected by the pH of gastric acid; the survival rate at pH 3.0 and 2.5 was still 69.61% and 58.47%. Generally, after eating, the pH value of gastric juice is about 3.5, indicating that Lactobacillus plantarum ZX028 can tolerate gastric acid with a pH value of >2.5.

[0035] 4) Test on bile salt tolerance of Lactobacillus plantarum ZX028

[0036] The plant lactobacillus ZX028 strain was inoculated into MRS liquid culture medium at an inoculum size of 2%, and activated and cultured at 37°C for 12 hours, and activated three times continuously. The activated ZX028 bacterial solution was inoculated into MRS liquid culture medium at an inoculum size of 2%, and cultured at 37°C for 12 hours in a constant temperature incubator. The cultured bacterial solution was centrifuged at 5000rpm for 10 minutes to collect the bacterial cells, and the bacterial cells were resuspended in sterile physiological saline, mixed, and the bacterial suspension was added to the MRS culture medium with bile salt concentration of 1.0g / L and 0g / L (initial bacterial solution) at an addition amount of 10%, and the bile salt concentration was 0g / L as the control group. Then incubated in a 37°C constant temperature incubator for 4 hours. The incubated bacterial solution was taken out, diluted 10 times and added with sterile physiological saline, and the number of lactic acid bacteria was detected after vortexing and mixing. The number of live lactic acid bacteria detected was counted, and the survival rate was calculated. The calculation formula was as follows: strain survival rate (%) = test group / control group × 100%.

[0037] Table 1. Results of bile salt tolerance test of Lactobacillus plantarum ZX028

[0038]

[0039] The results in Table 1 show that when the bile salt concentration is 1.0 g / L, the survival rate of the strain is 79.82%, and it takes 1 to 4 hours for food to pass through the small intestine. The tolerance time is set to the longest time that food stays in the small intestine (4 hours), indicating that the ZX028 strain can tolerate a certain concentration of bile salt environment in the intestine for a longer period of time.

[0040] In summary, the Lactobacillus plantarum ZX028 of the present invention grows well on MRS agar medium, has good gastric acid and bile salt tolerance, and is suitable for oral administration.

[0041] Example 2

[0042] A composition of this embodiment includes live strains of Lactobacillus plantarum ZX028, wherein the live strains of Lactobacillus plantarum ZX028 are ≥ 2×10 9 CFU / g.

[0043] 1) Test on the tolerance of the composition to ethanol and acetaldehyde

[0044] The strain of Lactobacillus plantarum ZX028 was inoculated into MRS liquid culture medium at an inoculation rate of 2%, and activated and cultured at 37°C for 12 hours, and activated three times continuously; the activated bacterial solution was inoculated into MRS liquid culture medium containing 0%, 1%, 2%, 4%, 6%, 8% (v / v) ethanol and 0%, 0.05%, 0.1%, 0.2%, 0.3% and 0.4% (m / v) acetaldehyde at an inoculation rate of 2%, and cultured at 37°C in a constant temperature incubator for 15 hours, the fermentation liquid was mixed by vortex oscillation, and the OD of the fermentation liquid was measured by ultraviolet spectrophotometer. 600 , to investigate the effects of ethanol and acetaldehyde on the growth of the composite. The results are shown in the attached Figure 5 and 6 As shown. The results show that when the acetaldehyde content of plant lactobacillus ZX028 is 0.05%, 0.10% and 0.20%, the growth rate is 90.87%, 77.20% and 46.15% respectively, and even when the acetaldehyde content is 0.30%, ZX028 still has a survival rate of 6.60%; when the ethanol content of plant lactobacillus ZX028 is 1%, 2%, 4%, and 6%, the growth rate is 95.19%, 83.01%, 66.70% and 33.43% respectively, so ZX028 can tolerate higher concentrations of ethanol and acetaldehyde. Generally, after people drink alcohol, when the ethanol concentration in the human body exceeds 1.27% (about 1000mg / L), the phenomenon of alcohol poisoning may be caused. It can be seen that plant lactobacillus ZX028 is active in the gastrointestinal environment of excessive drinking.

[0045] 2) Determination of the ability of the composition to degrade acetaldehyde

[0046] The plant lactobacillus ZX028 strain is inoculated into an MRS liquid culture medium at an inoculation rate of 2%, and activated and cultured at 37° C. for 12 hours, and activated three times in succession; the activated bacterial solution is inoculated into MRS liquid culture medium containing 0.5%, 1% and 2% (m / v) acetaldehyde respectively at an inoculation rate of 2%, and statically cultured at 37° C. in a constant temperature incubator for 21 hours, 1 mL of the bacterial solution is centrifuged at 5000 rpm for 10 minutes, and the supernatant is filtered through a 0.22 μm aqueous phase filter membrane, and the acetaldehyde content in the system is determined by a gas chromatograph.

[0047] Table 2. Determination results of acetaldehyde degradation by the composition

[0048]

[0049] The results in Table 2 show that when the acetaldehyde content is 0.10%, the degradation rate of acetaldehyde by Lactobacillus plantarum ZX028 is as high as 80%, indicating that the ZX028 strain can play a role in sobering up in the human intestine.

[0050] In summary, the composition of the present invention comprising Lactobacillus plantarum ZX028 has strong tolerance to ethanol and acetaldehyde, and can degrade acetaldehyde to exert the effect of sobering up, and can be used to prepare functional food or medicine for sobering up.

[0051] Example 3

[0052] A composition of this embodiment includes live strains of Lactobacillus plantarum ZX028, wherein the live strains of Lactobacillus plantarum ZX028 are ≥ 2×10 7 CFU / g.

[0053] The Lactobacillus plantarum ZX028 strain was inoculated into MRS liquid medium at a 2% inoculum volume, and activated and cultured at 37°C for 12 hours, and activated three times in succession. The activated ZX028 bacterial solution was centrifuged at 5000 rpm for 10 min to collect the bacterial cells, and the bacterial cells were resuspended in sterile saline and mixed. The number of viable Lactobacillus plantarum ZX028 cells was adjusted to 2×10 7 CFU / g.

[0054] Animal experiment: 15 healthy KM male mice aged 6 weeks were fed adaptively for 1 week and randomly divided into 3 groups (3-6 mice in each group), namely acute intoxication model group, Lactobacillus plantarum ZX028 group (ZX028) and Myrkl positive group. After fasting for 6 hours, all mice were gavaged with 200 μL sterile saline in the intoxication model group and 200 μL 2×10 7CFU / g of Lactobacillus plantarum ZX028 and the Myrkl positive group were gavaged with a live bacterial suspension dissolved in sterile saline (the gavage amount was converted to mouse weight based on an adult body weight of 60-70 kg). After 30 minutes, 1000 μL or 500 μL of 40-proof liquor was gavaged.

[0055] 1) Effects of the composition on behavioral indicators of drunk mice

[0056] All mice were fed 1000mL of 40-proof liquor; Intoxication index: Keeping the back down for 20s or more is the intoxication index, that is, the time when the righting reflex disappears; Sobering index: Free movement, spirit, and smooth fur are the sobering index, that is, the time when the righting reflex recovers; Intoxication tolerance time = time when the righting reflex disappears - alcohol feeding time; Intoxication time = time when the righting reflex recovers - time when the righting reflex disappears. The test results are shown in Table 3:

[0057] Table 3 Recording and analysis results of behavioral indicators of drunk mice

[0058]

[0059] Note: The same lowercase letters in the same column indicate no significant difference (P>0.05), and adjacent letters indicate significant difference (P<0.05).

[0060] The drunkenness rate of the ZX028 group was 80%, which was better than the model group and the Myrkl group. In addition, by comparing the drunkenness tolerance time and drunkenness time of drunken KM mice in different groups, it was found that the overall drunkenness tolerance time of drunken mice in the ZX028 group was increased by 212.86% and 102.12% compared with the model group and the Myrkl group, respectively, and the drunkenness time was shortened by 52.77% and 26.63% compared with the model group and the Myrkl group, respectively. Obviously, the present invention is superior to the model group and the positive Myrkl group.

[0061] 1) Determination of acetaldehyde content in blood of drunk mice by the composition

[0062] All mice were gavaged with 500 mL of 40% liquor; the eyeballs were removed to collect blood and the mice were killed by cervical dislocation. The blood samples were centrifuged at 2800 r / min for 15 minutes at 4°C, and the supernatant (serum) was collected and frozen at -80°C for analysis of blood acetaldehyde content.

[0063] 100uL of serum was taken and derivatized with 100uL of 2,4-DNPH dissolved in 6nmol HCl in a 60°C water bath for 90min. After the derivatization, the sample was cooled to room temperature in an ice water bath, filtered through a 0.22um aqueous filter membrane, and then determined by liquid chromatography. The acetaldehyde content in the sample was analyzed by the external standard method.

[0064] Table 4. Blood acetaldehyde test results of drunk mice

[0065]

[0066] From the blood acetaldehyde detection results of drunken mice in Table 4, we can see that 1 hour after drinking, the blood acetaldehyde levels in the ZX028 group and the positive Myrkl group were reduced by 12.39% and 10.61% respectively compared with the model group. From 1 hour to 2 hours after drinking, the blood acetaldehyde degradation levels of the ZX028 group and the positive Myrkl group were 59.26 mg / L and 26.78 mg / L respectively. The ability of the ZX028 group to degrade blood acetaldehyde was significantly better than that of the positive Myrkl group.

[0067] 2) Effect of the composition on the activities of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in the liver of drunk mice

[0068] In this Example 2), each group of mice was killed by cervical dislocation, and the liver was removed immediately after dissection. A 0.5-1.0 g liver tissue block was cut from each mouse with surgical scissors, placed in a cryopreservation tube, and immediately placed in liquid nitrogen for testing. The enzyme activity levels of mouse liver alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were determined according to the kit method (Nanjing Jiancheng).

[0069] Table 5. ADH and ALDH test results of drunken mice in each group

[0070]

[0071] Note: The same lowercase letters in the same column indicate no significant difference (P>0.05), and adjacent letters indicate significant difference (P<0.05).

[0072] The test results in Table 5 show that compared with the model group and the positive group, the mice fed with 200 μL of live bacteria before being fed with liquor had a 7 CFU / g of Lactobacillus plantarum ZX028 bacterial liquid can increase the activity of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in the liver of mice and enhance the ability of mice to sober up.

[0073] 3) Effect of the composition on body weight and liver index of mice

[0074] Twenty healthy KM male mice aged 5 weeks were fed adaptively for 1 week and randomly divided into 4 groups (5 mice in each group), namely normal group, model group, Lactobacillus plantarum ZX028 group (ZX028) and Myrkl positive group. The model group was gavaged with 200 μL sterile saline, and the ZX028 group was gavaged with 200uL 2×10 7CFU / g of Lactobacillus plantarum ZX028, and the Myrkl positive group were gavaged with a live bacterial suspension dissolved in sterile saline (the gavage amount was converted to mouse body weight based on an adult body weight of 60-70 kg). After 30 minutes, 200 μL of 40-proof liquor was gavaged intragastrically, and this gavage method was continued for one week. The mice in each group were weighed and recorded; the mice were killed by cervical dislocation, and the mice were dissected immediately, the livers were removed, washed with saline, dried, and the total liver weight was weighed and recorded.

[0075] Table 6. Test results of the effect of the composition on the body weight and liver index of mice

[0076]

[0077] Note: The same lowercase letters in the same column indicate no significant difference (P>0.05), adjacent letters indicate significant difference (P<0.05), and non-adjacent letters indicate extremely significant difference (P<0.01).

[0078] Excessive drinking leads to decreased appetite and absorption function of mice, and weight loss; liver index represents the proportion of liver to the whole body weight. Liver damage can cause liver enlargement, dysplasia and cirrhosis in mice, and the corresponding proportion of liver to body weight also increases. Liver index can reflect liver damage to a certain extent.

[0079] As shown in Table 6, compared with the normal group, the body weight of the mice in the model group was significantly reduced (P<0.01), and the liver index was the highest. The ZX028 group could significantly improve the weight changes of mice and reduce the liver index of mice, indicating that the composition can alleviate the weight loss and liver damage caused by drinking to a certain extent.

[0080] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A plant lactobacillus ( Lactiplantibacillus plantarum ), characterized in that: The Lactobacillus plantarum is classified and named as Lactobacillus plantarum ZX028, and is preserved in China Center for Type Culture Collection with a preservation number of CCTCCNO: M 20232705.

2. Use of the plant lactobacillus described in claim 1 in preparing a hangover sobering product.

3. Use of the plant lactobacillus according to claim 1 in preparing a product for prolonging tolerance to drunkenness.

4. Use of the plant lactobacillus according to claim 1 in preparing a product for shortening the time of drunkenness.

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