Lactic acid bacteria product and its hangover alleviating use

Abstract

The present invention provides a lactic acid bacteria product, which contains live or dead lactic acid bacteria and / or cell-free extracts thereof. The present invention also provides the use of the product for preparing a product suitable for a subject with increased blood alcohol content, which product is capable of at least one of the following effects on the subject: 1) increasing the subject's alcohol tolerance time; 2) shortening the subject's alcohol time; and 3) reducing the subject's blood alcohol concentration. The present inventors have found that a lactic acid bacteria product, especially a product prepared from ethanol-induced lactic acid bacteria, can greatly increase the alcohol tolerance time, shorten the alcohol time, and reduce the blood alcohol concentration of a drunk person. After ethanol induction, the ethanol dehydrogenase activity of the lactic acid bacteria is greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to a lactic acid bacteria product and its use for alcoholism. BACKGROUND

[0002] The wine culture is an important part of Chinese traditional culture, and has become an indispensable tool in people's social activities. Ethanol is the main component of drinking wine, which can cause nausea and vomiting, memory loss, and lack of concentration when drinking a large amount of alcohol in a short time. Long-term drinking can cause gastric ulcers, alcoholic liver, cirrhosis, and even liver cancer. Ethanol is mainly absorbed into tissues and organs through the oral cavity, esophagus, stomach, and intestinal mucosa. The concentration of ethanol in the blood reaches a peak within 30-60 minutes. The liver is an important place for ethanol metabolism, which is mainly metabolized by three enzyme systems in the body: first, the ethanol dehydrogenase system in the cytoplasm; second, the microsomal ethanol oxidation system; and third, the peroxidase system. The liver has limited capacity to metabolize ethanol, and after acute excessive drinking, a large amount of toxic substances such as acetaldehyde accumulate, causing liver microcirculation disorders, mitochondrial damage, and eventually leading to alcoholic liver damage.

[0003] Ethanol dehydrogenase is a zinc-containing enzyme that exists in large quantities in the liver of humans and animals, plants, and microbial cells, and has a wide range of substrate specificity. Ethanol dehydrogenase uses nicotinamide adenine dinucleotide as a coenzyme to catalyze the reversible reaction between primary alcohols and aldehydes.

[0004] Lactic acid bacteria are a general term for bacteria that can produce a large amount of lactic acid by utilizing fermentable carbohydrates. Probiotics represented by lactic acid bacteria are essential and have important physiological functions for the human body. They are an important part of the human intestinal flora and play an important role in human health, such as improving intestinal health, enhancing the host immune system, and preventing aging. SUMMARY

[0005] In a first aspect, the present application provides a lactic acid bacteria product, which contains lactic acid bacteria in a living or dead form and / or cell-free extracts (products other than the cell wall membrane) of the lactic acid bacteria. Optionally, the lactic acid bacteria are pre-treated by induction, which includes adding ethanol to the culture medium (the implementation forms include directly using culture medium containing ethanol).

[0006] Optionally, the lactic acid bacteria include at least one of Lactococcus lactis, Pediococcus acidilactici, Lactobacillus plantarum, and Streptococcus lactis.

[0007] Optionally, the total amount of ethanol added is 0.5-2.0% of the volume of the culture medium, such as 0.5-1.5%, such as 0.5-1%, such as 0.5-0.75%, such as 0.5-0.6%.

[0008] Optionally, in the Lactococcus lactis or Pediococcus acidilactici induced treatment operation, the total amount of ethanol added is 0.5% of the volume of the culture medium.

[0009] Optionally, in the Lactobacillus plantarum induced treatment operation, the total amount of ethanol added is 0.75% of the volume of the culture medium.

[0010] Optionally, in the Streptococcus lactis induced treatment operation, the total amount of ethanol added is 0.6% of the volume of the culture medium.

[0011] Optionally, the ethanol is added to the culture medium in one or multiple times (e.g. 3 times). When added in multiple times: on one hand, the amount of ethanol added each time can be increased successively. As an example of adding ethanol in 3 times, when the total amount of ethanol to be added is 0.5%, 1.0%, 1.5% or 2.0% of the volume of the culture medium, the ratio of the amount of ethanol added successively is 1:3:6; when the total amount of ethanol to be added is 0.6% of the volume of the culture medium, the ratio of the amount of ethanol added successively is 1:2:3; when the total amount of ethanol to be added is 0.75% of the volume of the culture medium, the ratio of the amount of ethanol added successively is 3:5:7. On the other hand, the operation of adding ethanol in adjacent two times can be separated by 0.5-2h, the Lactococcus lactis or Streptococcus lactis can be separated by 2h, the Pediococcus acidilactici can be separated by 1.5h, and the Lactobacillus plantarum can be separated by 0.5h.

[0012] Optionally, the lactic acid bacteria product is made using lactic acid bacteria in the logarithmic phase, and the total culture time required from inoculation can be 12-24h, such as 16h.

[0013] Optionally, the Lactococcus lactis is Lactococcus lactis subsp. Lactis, such as the strain with the accession number CICC 20405.

[0014] Optionally, the Streptococcus lactis is the strain with the accession number CICC 6242.

[0015] Optionally, the Pediococcus acidilactici is the strain with the accession number CICC 20720.

[0016] Optionally, the Lactobacillus plantarum is the strain with the accession number CICC 21790.

[0017] Optionally, the culture medium is MRS (de Man, Rogosa, Sharpe) medium, the culture medium can be liquid, solid or semi-solid medium, and the components thereof can contain sucrose, yeast extract, phosphate, sodium ion, potassium ion, sulfate and magnesium ion, or contain sucrose, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate and magnesium sulfate, such as the following components per liter of culture medium:

[0018]

[0019] Optionally, the culture temperature of the lactic acid bacteria before being made into the lactic acid bacteria product is 30-35°C, such as 31-34°C.

[0020] Optionally, the preparation of the cell-free extract comprises: ultrasonic disruption of the bacteria (the system can be phosphate buffer), centrifugation to collect the supernatant, the centrifugation speed of the supernatant can be 10000-12000 rpm, and the time can be 10-15 min; the centrifugation is carried out at low temperature, such as 2-6°C, and specifically 4°C; and optionally, the supernatant is freeze-dried (which can be carried out under vacuum).

[0021] Optionally, the lactic acid bacteria product is a dry powder, such as a freeze-dried powder.

[0022] In a second aspect, the present application provides a method for preparing a lactic acid bacteria product containing lactic acid bacteria in a living or dead form and / or a cell-free extract thereof, and optionally, the method comprises a pre-induction treatment of the lactic acid bacteria, and the induction treatment comprises adding ethanol to the culture medium (the implementation forms include: directly using a culture medium containing ethanol).

[0023] Optionally, the lactic acid bacteria comprises at least one of Lactococcus lactis, Pediococcus acidilactici, Lactobacillus plantarum and Streptococcus lactis.

[0024] Optionally, the total amount of ethanol added is 0.5-2.0% of the volume of the culture medium, such as 0.5-1.5%, such as 0.5-1%, such as 0.5-0.75%, such as 0.5-0.6%.

[0025] Optionally, in the induction treatment operation of the Lactococcus lactis or Pediococcus acidilactici, the total amount of ethanol added is 0.5% of the volume of the culture medium.

[0026] Optionally, in the induction treatment operation of the Lactobacillus plantarum, the total amount of ethanol added is 0.75% of the volume of the culture medium.

[0027] Optionally, in the induction treatment operation of the Streptococcus lactis, the total amount of ethanol added is 0.6% of the volume of the culture medium.

[0028] Optionally, the ethanol is added to the culture medium once or in multiple times (e.g. 3 times), and when added in multiple times: on one hand, the amount of ethanol added each time can be increased successively, as an example of adding ethanol in 3 times, if the total amount of ethanol to be added is 0.5%, 1.0%, 1.5% or 2.0% of the volume of the culture medium, the ratio of the amount of ethanol added successively is 1:3:6; if the total amount of ethanol to be added is 0.6% of the volume of the culture medium, the ratio of the amount of ethanol added successively is 1:2:3; if the total amount of ethanol to be added is 0.75% of the volume of the culture medium, the ratio of the amount of ethanol added successively is 3:5:7; on the other hand, the operation of adding ethanol in adjacent two times can be spaced 0.5-2h, the Lactococcus lactis or Streptococcus lactis can be spaced 2h, the Pediococcus acidilactici can be spaced 1.5h, and the Lactobacillus plantarum can be spaced 0.5h.

[0029] Optionally, the lactic acid bacteria product is made using lactic acid bacteria in logarithmic phase, and the total culture time required from inoculation can be 12-24h, such as 16h.

[0030] Optionally, the Lactococcus lactis is Lactococcus lactis subsp. Lactis, such as the strain with accession number CICC 20405.

[0031] Optionally, the Streptococcus lactis is the strain with accession number CICC 6242.

[0032] Optionally, the Pediococcus acidilactici is the strain with accession number CICC 20720.

[0033] Optionally, the Lactobacillus plantarum is the strain with accession number CICC 21790.

[0034] Optionally, the culture medium is MRS (de Man, Rogosa, Sharpe) medium, and the culture medium can be liquid, solid or semi-solid medium, and the components can contain sucrose, yeast extract, phosphate, sodium ion, potassium ion, sulfate and magnesium ion, or contain sucrose, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate and magnesium sulfate, and the components per liter of culture medium are as follows:

[0035]

[0036] Optionally, the culture temperature of the lactic acid bacteria before being made into the lactic acid bacteria product is 30-35°C, preferably 31-34°C.

[0037] Optionally, the preparation of the cell-free extract comprises: ultrasonic disruption of the bacteria (the system can be phosphate buffer), centrifugation to collect the supernatant, the centrifugation speed of the supernatant can be 10000-12000 rpm, and the time can be 10-15 min; the centrifugation is performed at low temperature, such as 2-6°C, and specifically 4°C; and the supernatant can be freeze-dried (which can be performed under vacuum).

[0038] Optionally, the lactic acid bacteria product is a dry powder, such as a freeze-dried powder.

[0039] In a third aspect, the present application provides a method for improving the activity of alcohol dehydrogenase of lactic acid bacteria, which comprises inducing lactic acid bacteria with ethanol to obtain any of the lactic acid bacteria products according to the first aspect of the present application. The method for ethanol induction can use any of the methods according to the second aspect of the present application.

[0040] In a fourth aspect, the present application provides the use of any of the lactic acid bacteria products according to the first aspect of the present application, which is for preparing a product suitable for a subject (human or mouse or other mammal) with increased blood alcohol content, which product can at least produce any of the following effects on the subject:

[0041] 1) increasing the drunk tolerance time of the subject;

[0042] 2) shortening the drunk time of the subject;

[0043] 3) reducing the ethanol concentration in the blood of the subject.

[0044] The increased blood alcohol content refers to a higher blood alcohol content than the normal level, which is usually caused by acute or excessive drinking, and the subject usually shows drunk or even alcohol poisoning.

[0045] The present application has the following beneficial effects:

[0046] The present inventors have found that the lactic acid bacteria product, especially the product prepared after ethanol induction treatment of lactic acid bacteria, can greatly increase the drunk tolerance time of the drunk, shorten the drunk time, and reduce the ethanol concentration in the blood. After ethanol induction treatment, the activity of alcohol dehydrogenase of lactic acid bacteria is greatly improved. DETAILED DESCRIPTION

[0047] The present application is specifically illustrated below.

[0048] In vitro experiment:

[0049] The MRS medium used in the following experiments is as follows:

[0050]

[0051] The determination of ethanol dehydrogenase activity is detected by the improved Valle & Hoch method: 1.5 ml of sodium pyrophosphate buffer with pH 8.8, 0.5 ml of 11.5% (v / v) ethanol, 0.1 ml of 0.25 U / ml ethanol dehydrogenase are added into the measuring tube, 0.1 ml of water solution of the freeze-dried powder of lactic acid bacteria (or 0.1 ml of distilled water for the blank tube) is then added, mixed well and placed in a 37°C water bath for 5 min. After incubation, 1.0 ml of 27 mM β-nicotinamide adenine dinucleotide is added, mixed well and the change of absorbance value is determined under ultraviolet light. The effect of intracellular substance of lactic acid bacteria on ethanol dehydrogenase activity (activation rate) is calculated. The results are shown in the tables. In the tables, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001 and ns represents no significance compared with the 0% blank group.

[0052] Example 1a:

[0053] Using MRS medium, the strain of Lactococcus lactis subsp. Lactis with preservation number CICC 20405 is activated twice and then inoculated. The temperature of the incubator is kept at about 32°C. 0.1 ml, 0.3 ml and 0.6 ml of ethanol is added into 200 ml of MRS medium in the experimental group in turn, and the same amount of water is added into the control group in turn. After adding ethanol each time, the treatment of each ethanol gradient is performed for 2 h. The operation of the parallel experiment of three ethanol concentrations is the same as above (the amount of added ethanol is also increased by 1:3:6 in turn, and the control group is also added with water in correspondence). Only the total amount of added ethanol in the medium is different. The ratio of the total volume of added ethanol to MRS medium in the three parallel experiments is 1%, 1.5% and 2.0% respectively.

[0054] After adding the above ethanol, the continuous culture is performed to the logarithmic phase (the total culture time is 16 h). The bacterial cells are centrifuged and washed with 75 mM phosphate buffer. The mass of bacterial cells is matched with the volume of phosphate buffer at 1 g:40 ml. The bacterial cells are collected by centrifugation after ultrasonic disruption in 50 ml of phosphate buffer. The supernatant after ultrasonic disruption is collected by centrifugation at a speed of 12000 rpm for 10 min at a temperature of 4°C. The bacteria are removed by membrane filtration. The obtained supernatant is vacuum freeze-dried. Finally, the freeze-dried powder of Lactococcus lactis is prepared. The effect on ethanol dehydrogenase activity is detected in vitro. The experimental results are shown as follows:

[0055] Table 1a Effect of intracellular substance of Lactococcus lactis under different ethanol concentrations on ethanol dehydrogenase activity

[0056]

[0057] Example 1b:

[0058] Experimental ideas, methods refer to example 1a, the only difference is that ethanol is not added 3 times, but 1 time to the final required concentration (0.5-2%), the results are compared as follows in Table 1b:

[0059] Table 1b the effect of intracellular material of Lactococcus lactis under different ethanol concentration induction on ethanol dehydrogenase activity

[0060]

[0061] Example 2:

[0062] Using MRS medium, the frozen Lactobacillus plantarum (preserved number CICC21790) was activated twice and inoculated, keeping the incubator temperature at about 31℃, 0.1ml, 0.3ml, 0.6ml of ethanol was added to the experimental group 200ml MRS medium to achieve a total ethanol concentration of 0.5%, and the control group was added with the same amount of water, and each ethanol gradient was treated for 0.5h after adding ethanol; another parallel experiment with different ethanol concentrations was operated as above (the amount of ethanol added was also gradually increased, and the control group was also added with water), only the total amount of ethanol added in the medium was different, that is, the ratio of the total volume of ethanol to MRS medium was 0.75%, 1% respectively); after adding the above ethanol, it was continuously cultured to the logarithmic phase (the total culture time was 16h); the bacterial cells were centrifuged and washed with 75mM phosphate buffer, the mass ratio of bacterial cells to phosphate buffer was 1g:30ml; the bacterial cells were collected by centrifugation again; the bacterial cells were resuspended in 50ml phosphate buffer and ultrasonically broken; the supernatant was collected by centrifugation at a speed of 12000rpm for 10min at 4℃; the bacteria were removed by membrane filtration, and the obtained supernatant was vacuum freeze-dried to prepare Lactobacillus plantarum freeze-dried powder, and then the effect on ethanol dehydrogenase activity was detected in vitro, and the experimental results are shown as follows:

[0063] Table 2 the effect of intracellular material of Lactobacillus plantarum under different ethanol concentration induction on ethanol dehydrogenase activity

[0064]

[0065] Example 3:

[0066] Using MRS medium, the frozen Streptococcus lactis (preserved number CICC 6242) was activated twice and inoculated, keeping the incubator temperature at about 33℃, 0.1ml, 0.3ml and 0.6ml of ethanol was added into 200ml MRS medium of the experimental group in turn, and the control group was added with the same amount of water in turn, and each ethanol gradient was treated for 1.5h after adding ethanol each time; another parallel experiment of ethanol concentration was operated as above (the amount of added ethanol was also increased gradually, and the control group was also added with water correspondingly), only the total amount of added ethanol in the medium was different, i.e. the ratio of the total volume of ethanol to MRS medium in the parallel experiment was 0.6% and 0.75% respectively; after adding the above ethanol, it was continuously cultured to the logarithmic phase (the total culture time was 16h); the bacterial cells were centrifuged and washed with 75mM phosphate buffer, the mass of bacterial cells was 1g and the volume of phosphate buffer was 30ml; the bacterial cells were collected by centrifugation again; the bacterial cells were resuspended in 50ml phosphate buffer and ultrasonic broken; the supernatant after ultrasonic breaking was collected by centrifugation at a speed of 12000rpm for 15min at 4℃; the bacteria were removed by membrane filtration, and finally the Streptococcus lactis freeze-dried powder was prepared by vacuum freeze-drying, and then the effect on the activity of alcohol dehydrogenase was detected in vitro, and the experimental results are shown below:

[0067] Table 3 Effect of Streptococcus lactis on alcohol dehydrogenase activity induced by gradient ethanol

[0068]

[0069] Example 4:

[0070] Using MRS medium, the frozen Pediococcus acidilactici (preserved number CICC20720) strain was inoculated after two activations, the temperature of the incubator was kept at about 34℃, 0.1ml, 0.3ml and 0.6ml of ethanol was added into 200ml MRS medium of the experimental group in turn, and the same amount of water was added into the control group; after adding ethanol each time, each ethanol gradient was treated for 1.5h; another parallel experiment of ethanol concentration was operated as above (the amount of added ethanol was also increased gradually, and the control group was also added with water correspondingly), only the total amount of added ethanol in the medium was different, i.e. the ratio of the total volume of ethanol to MRS medium was 0.6% and 0.75% respectively; after adding the above ethanol, the continuous culture was carried out to the logarithmic phase (the total culture time was 16h); the bacterial body was centrifuged, washed with 75mM phosphate buffer, and the mass of bacterial body was mixed with 50ml of phosphate buffer; the bacterial body was collected by centrifugation again; the bacterial body was resuspended in 60ml of phosphate buffer, and was subjected to ultrasonic disruption; the supernatant was collected by centrifugation at a speed of 10000rpm for 20min at 4℃; the bacteria were removed by membrane filtration, the obtained supernatant was vacuum freeze-dried to prepare Pediococcus acidilactici freeze-dried powder, and then the effect on the activity of ethanol dehydrogenase was detected in vitro, and the experimental results are shown in Table 4 below:

[0071]

[0072]

[0073] In vivo experiment:

[0074] 1. Drugs and reagents

[0075] Positive drug: bifendate drop pills.

[0076] Reagent: 56-degree baijiu.

[0077] 2. Experimental animals and methods

[0078] Experimental subjects: mice, provided by the Henan Animal Experiment Center.

[0079] Experimental method: 100 mice were adaptively fed and randomly divided into 5 groups, 20 mice in each group. They were divided into a blank group, a model group, a positive group, a lactococcus group and an ethanol induction group. The behavior index was observed after acute drunkenness on the first day, and continuous gavage was performed for seven days. Two hours after gavage with alcohol on the seventh day, the eyeball was taken for blood. The blood sample was centrifuged at a speed of 6000rpm, a temperature of 4℃ and a time of 5min to prepare serum.

[0080] Blank group: 0.1ml / 10g of normal saline, 10-12ml / kg of normal saline was gavaged after 0.5-1h.

[0081] Model group: 0.1ml / 10g normal saline, 10-12ml / kg 56 degree baijiu was given by gavage after 0.5-1h.

[0082] Positive group: 1.5mg / 10g bifendate drop pill emulsion, 10-12ml / kg 56 degree baijiu was given by gavage after 0.5-1h.

[0083] Lactococcus group: 2.0mg / 10g lactobacillus powder (the lactobacillus powder prepared in the foregoing in-vitro experiment embodiment 1a 0% blank group) aqueous solution, 10-12ml / kg 56 degree baijiu was given by gavage after 0.5-1h.

[0084] Ethanol induction group: 2.0mg / 10g lactobacillus powder (the lactobacillus powder prepared in the foregoing in-vitro experiment embodiment 1a 0.5% ethanol induction group) aqueous solution, 10-12ml / kg 56 degree baijiu was given by gavage after 0.5-1h.

[0085] 3. Evaluation method:

[0086] Drunk / wake-up index:

[0087] Drunk index: keeping back for 30s and above is drunk index, i.e. the time of disappearance of righting reflex.

[0088] Wake-up index: free movement, spirit and smooth hair are wake-up indexes, i.e. the time of recovery of righting reflex.

[0089] Drunk tolerance time = time of disappearance of righting reflex - time of wine pouring

[0090] Drunk time = time of recovery of righting reflex - time of disappearance of righting reflex

[0091] The detection results are shown in Table 5 as follows:

[0092]

[0093] Compared with the model group, ** represents p<0.01, and *** represents p<0.001.

[0094] Obviously, the present application is much better than the model group, and the effects of the ethanol induction group in two aspects are very significant. Further analysis of the advantages of the ethanol induction group in reducing the blood ethanol concentration adopts gas chromatography to detect the ethanol content:

[0095] 0.5mL of the whole blood sample prepared by blood sampling is taken, 0.1mL of tert-butyl alcohol, 1mL of dimethyl sulfoxide and 2g of anhydrous sodium sulfate are added, mixed, centrifuged, and 1μL of supernatant is injected.

[0096] The comparison of the detection results is shown in Table 6.

[0097] Table 6. Effect of intracellular substance of lactic acid bacteria induced by ethanol on ethanol content in acute drunken mice

[0098]

[0099] Table 6. ** means p < 0.01, *** means p < 0.001 compared with the model group.

Claims

1. A lactic acid bacteria product, said product comprising a cell-free extract of lactic acid bacteria, said lactic acid bacteria being previously subjected to an induction treatment, said induction treatment comprising the addition of ethanol to the culture medium, said lactic acid bacteria being Lactococcus lactis with the accession number CICC 20405, said cell-free extract being the product of the bacteria except for the cell wall membrane, said ethanol being added in a total amount of 0.5% of the volume of the culture medium.

2. The lactic acid bacterial product according to claim 1, characterized in that Said ethanol is added to the culture medium at one time or in several times.

3. The lactic acid bacterial product according to claim 2, characterized in that Said ethanol is added to the culture medium in three times.

4. The lactic acid bacterial product according to claim 2 or 3, characterized in that When added in several times, the amount of ethanol added each time is increased.

5. The lactic acid bacterial product according to claim 4, characterized in that The interval between the operation of adding ethanol in two adjacent times is 0.5-2h.

6. The lactic acid bacterial product according to claim 1, characterized in that Said lactic acid bacteria product is made using lactic acid bacteria in logarithmic phase.

7. The lactic acid bacterial product according to claim 1, characterized in that Said culture medium is MRS culture medium.

8. The lactic acid bacterial product according to claim 7, characterized in that Said culture medium is liquid, solid or semi-solid culture medium.

9. The lactic acid bacterial product according to claim 7, characterized in that The components of said culture medium comprise sucrose, yeast extract, phosphate, sodium ion, potassium ion, sulfate and magnesium ion.

10. The lactic acid bacterial product according to claim 7, characterized in that Said culture medium comprises sucrose, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate and magnesium sulfate.

11. The lactic acid bacterial product according to claim 1, characterized in that The preparation of said cell-free extract comprises ultrasonic disruption of the bacteria, and collection of the supernatant after centrifugation.

12. The lactic acid bacterial product according to claim 11, characterized in that The preparation further comprises freeze-drying of the supernatant.

13. The lactic acid bacterial product according to claim 11, characterized in that The rotation speed of said centrifugation is 10000-12000rpm.

14. The lactic acid bacterial product according to claim 11, characterized in that The centrifugation time is 10-15min.

15. The lactic acid bacterial product of claim 1, wherein Said lactic acid bacteria product is dry powder.

16. The lactic acid bacterial product of claim 1, wherein Said lactic acid bacteria product is freeze-dried powder.

17. Use of the lactic acid bacteria product of any of the preceding claims, said use being for the preparation of a product suitable for a subject with increased blood alcohol content, which product is capable of at least one of the following effects on said subject: 1) increasing the time of his drunkenness tolerance; 2) shortening the time of his drunkenness; 3) reducing the ethanol concentration in his blood.

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