CN113755335B

CN113755335B - Screening method and application of strain capable of alleviating hangover

Info

Publication number: CN113755335B
Application number: CN202110968686.3A
Authority: CN
Inventors: 殷世清; 朱永真; 张甲庆; 徐靖洋; 张凤龙; 李艳琪
Original assignee: Jiangsu Hantide Biomedical Co ltd
Current assignee: Jiangsu Hantide Biomedical Co ltd
Priority date: 2021-08-23
Filing date: 2021-08-23
Publication date: 2022-10-18
Anticipated expiration: 2041-08-23
Also published as: CN113755335A

Abstract

The invention discloses a screening method and application of a strain capable of alleviating hangover, which comprises the following steps: step one, sterilizing a screening culture medium, adding sterile acetaldehyde into the screening culture medium after the sterilization treatment is finished, inoculating pickled vegetables or yoghourt products in an aseptic manner, carrying out anaerobic culture, detecting the acetaldehyde concentration in a liquid phase, and carrying out single colony plate separation on microbial flora with reduced acetaldehyde concentration and survival thallus; wherein the concentration of the sterile acetaldehyde in the screening culture medium is 0.1-0.5%, and the plate is provided with a nutrient-rich culture medium. In the present invention, high throughput screening is performed with the goal of being able to tolerate acetaldehyde and degrade acetaldehyde. Because the final product of acetaldehyde is acetic acid, the invention takes fermentation products of pickles, yoghourt products and the like as initial targets, strains which are tolerant to acetaldehyde are screened, the obtained strains are detected for the capacity of degrading acetaldehyde, and the strains with stronger capacity of degrading acetaldehyde are selected as target strains.

Description

Screening method and application of strain capable of alleviating hangover

Technical Field

The invention relates to the technical field of bioengineering fermentation, in particular to a screening method and application of a strain capable of alleviating hangover.

Background

More than 250 million people die globally every year and are related to alcoholism, and the number is always on the rise, so that the alcoholism seriously harms the physical health of people and even possibly causes a series of social problems.

After drinking, a small part of alcohol can be directly discharged out of the body along with respiration, sweat or urine, and the rest of a large amount of alcohol needs to enter the liver for oxidative decomposition, alcohol is converted into acetaldehyde by alcohol dehydrogenase, then the acetaldehyde is oxidized into acetic acid by liver acetaldehyde dehydrogenase, and finally the acetic acid and the acetic acid are changed into water and carbon dioxide to be discharged out of the body, so that the process causes serious burden on the liver and easily causes liver injury. Acetaldehyde is the most toxic substance causing alcoholic liver injury and can cause hepatocyte injury, inflammation, and extracellular matrix production and fibrosis formation. At present, most of the common anti-alcoholic medicines, health-care products and foods at home and abroad are prepared from traditional Chinese medicinal materials, food materials and the like, and are mainly diuretic chemical synthetic medicines, liver-protecting and stomach-nourishing traditional Chinese medicines, protein peptide medicines and the like, but the drug effect is slow and the anti-alcoholic effect is poor.

Disclosure of Invention

The invention aims to provide a screening method and application of a strain capable of alleviating hangover, and aims to solve the technical problems of slow response and poor hangover alleviating effect of a diuretic chemical drug synthesis technology.

In order to achieve the purpose, the invention provides the following technical scheme:

according to one aspect of the invention, the screening method of the strain capable of alleviating hangover comprises the following steps:

step one, sterilizing a screening culture medium, adding sterile acetaldehyde into the screening culture medium after the sterilization treatment is finished, performing anaerobic culture after a natural fermentation product is inoculated in a sterile manner, detecting the acetaldehyde concentration in a liquid phase, and performing single colony plate separation on a microbial flora with reduced acetaldehyde concentration and survival thallus;

wherein the concentration of the sterile acetaldehyde in the screening culture medium is 0.1-0.5%, and a nutrient-rich culture medium is arranged on the flat plate;

step two, selecting the single bacterial colony on the flat plate, performing pure culture by using a screening culture medium again, crushing the single bacterial colony, detecting the activity of acetaldehyde dehydrogenase in fermentation liquor, and selecting a bacterial strain with higher activity to continue the pure culture;

and step three, centrifuging the strain with higher activity to obtain pure bacteria, then using PBS for resuspension, preparing a bacteria suspension, crushing the bacteria again, measuring the activity of intracellular acetaldehyde dehydrogenase, and selecting the strain with higher activity as a target strain.

Further, the nutrient-rich medium comprises the following components in concentration:

5-10g/L of glucose, 5-10g/L of lactose, 10-20g/L of peptone, 3-5g/L of yeast powder, 2-4g/L of sodium chloride, 0.5-1.5g/L of anhydrous sodium acetate, 0.1-0.5g/L of vitamin C, 0.04g/L of bromocresol purple and 20g/L of agar.

Furthermore, the preparation method of the nutrient-rich culture medium comprises the following steps: blending according to a certain proportion, then adding a certain amount of purified water, uniformly mixing, adjusting the pH value, and then carrying out sterilization treatment;

preferably, the amount of the added purified water is 1L, and the pH value is adjusted to be neutral;

the sterilization treatment at least satisfies: the sterilization time is 20min, and the sterilization temperature is 115 ℃.

Further, in the first step and the second step, the culture temperature of the sterile acetaldehyde in the screening culture medium is 30 +/-1 ℃, and the sterile acetaldehyde is cultured for 24 hours in an anaerobic environment;

preferably, the culture temperature of the sterile acetaldehyde in a screening culture medium is 30 ℃, and the pH value range of the screening culture medium is 7.0 +/-1.

Furthermore, the screening culture medium is prepared by heating and dissolving an M17 culture medium in purified water, and sterilizing the culture medium;

preferably, the screening medium is prepared by dissolving 42.3g of M17 medium in 1L of purified water under heating;

preferably, the preparation conditions at least satisfy: autoclaving for 15min at 115 deg.C.

Further, in step one, the sterile acetaldehyde is prepared by the following method:

using a sterile filter, 40% acetaldehyde was filtered into a sterile container, and 1L of the selection medium contained 2.5ml of sterile 40% acetaldehyde by volume, calculated as a final concentration of 0.1%.

Further, in the second step, when the single colony on the picking plate is subjected to pure culture, the culture temperature is kept at 30 ℃, and shaking anaerobic culture is performed for 24 hours;

the fermentation liquor is directly crushed by an ultrahigh pressure continuous flow cell crusher, the activity of acetaldehyde dehydrogenase is detected by using an acetaldehyde dehydrogenase detection kit, and the relative activity of the acetaldehyde dehydrogenase is detected according to the increased value of the absorbance of the acetaldehyde dehydrogenase at 340 nm.

Further, in the third step, washing the obtained pure bacteria for 2 times by using sterilized normal saline, then using a sterilized PBS solution with the same volume for suspending, then using an ultrahigh pressure continuous flow cell disruption instrument to break the bacterial suspension, then using an acetaldehyde dehydrogenase detection kit to detect the activity of acetaldehyde dehydrogenase, and detecting the relative activity of the acetaldehyde dehydrogenase according to the increase value of the absorbance of the acetaldehyde dehydrogenase at 340 nm;

preferably, the pure thallus is obtained by centrifugation at 6000g for 5 min.

Furthermore, the target bacterial strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms by the patentee, the preservation number is CGMCC NO.22892, the preservation date is 2021, 7 and 13 days, the preservation unit address is No. 3 of No. 1 Siro-Su-Lu of the sunward area in Beijing, and the preservation classification is named Lactococcus lactis. .

According to another aspect of the present invention, there is provided a use of a strain comprising the steps of:

step one, mixing pure cow milk with a stabilizer and a sweetener and then sterilizing;

step two, inoculating the bacterial suspension of the target bacterial strain into the sterilized mixture, and standing for 8-12h at the constant temperature of 30 ℃;

step three, after the product obtained in the step two is coagulated, placing the product in a refrigerator for standing for 4-8 hours, and performing after-ripening;

and step four, preparing the antialcoholic yogurt.

Compared with the prior art, the invention has the beneficial effects that:

in the present invention, high throughput screening is performed with the goal of being resistant to acetaldehyde and able to degrade acetaldehyde. Because the final product of acetaldehyde is acetic acid, the invention takes natural fermentation products such as pickles, yoghourt products and the like as initial targets, strains which are tolerant to acetaldehyde are screened, the obtained strains are detected for the capacity of degrading acetaldehyde, and the strains with stronger capacity of degrading acetaldehyde are selected as target strains.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

step one, sterilizing a screening culture medium, adding sterile acetaldehyde into the screening culture medium after the sterilization treatment is finished, performing anaerobic culture after a natural fermentation product is inoculated in the sterile culture medium in an aseptic manner, detecting the concentration of the acetaldehyde in a liquid phase manner, and performing single colony plate separation on a microbial flora with reduced acetaldehyde concentration and survival thalli;

step two, selecting the single colony on the flat plate, performing pure culture by using a screening culture medium again, crushing the single colony, detecting the activity of acetaldehyde dehydrogenase in fermentation liquor, and selecting a strain with higher activity to continue the pure culture;

The principle utilized by the scheme is as follows: the metabolism of ethanol in human body is completed by alcohol dehydrogenase, and the alcohol dehydrogenase is a rate-limiting enzyme (the enzyme with the slowest catalytic reaction speed in the whole metabolic pathway) and greatly influences the total speed of the whole metabolic pathway, so that the supplement of additional acetaldehyde dehydrogenase for human body is an effective method for removing the harm of ethanol.

The invention carries out high-throughput screening and aims to resist acetaldehyde and degrade acetaldehyde. Because the final product of acetaldehyde is acetic acid, the invention takes natural fermentation products such as pickles, yoghourt products and the like as initial targets, strains which are tolerant to acetaldehyde are screened, the obtained strains are subjected to detection of acetaldehyde degrading capability, and strains with stronger degrading capability are selected as target strains.

In order to facilitate understanding of the technical solution of the present invention, the following examples are further illustrated.

The first embodiment is as follows:

a method for screening strains capable of alleviating hangover comprises the following steps:

wherein the sterile acetaldehyde is prepared by the following method:

filtering 40% acetaldehyde using a sterile filter into a sterile container, 1L of the screening medium containing 2.5ml of sterile 40% acetaldehyde by volume, calculated at a final concentration of 0.1%;

wherein the culture temperature of the sterile acetaldehyde in a screening culture medium is 30 ℃, the sterile acetaldehyde is cultured for 24 hours in an anaerobic environment, and the pH value range of the screening culture medium is 7.0 +/-1;

the screening medium is prepared by heating and dissolving 42.3g of M17 medium in 1L of purified water, and then sterilizing, wherein the preparation conditions at least meet the following conditions: sterilizing under high pressure for 15min at 115 deg.C;

wherein the concentration of the sterile acetaldehyde in the screening culture medium is 0.1-0.5%, the flat plate is provided with a nutrient-rich culture medium, and the nutrient-rich culture medium comprises the following components in concentration:

5-10g/L of glucose, 5-10g/L of lactose, 10-20g/L of peptone, 3-5g/L of yeast powder, 2-4g/L of sodium chloride, 0.5-1.5g/L of anhydrous sodium acetate, 0.1-0.5g/L of vitamin C, 0.04g/L of bromocresol purple and 20g/L of agar;

the preparation method of the nutrient-rich culture medium comprises the following steps: blending according to a proportion, then adding 1L of purified water, mixing uniformly, adjusting the pH value to be neutral, and then carrying out sterilization treatment;

the sterilization treatment at least satisfies: sterilizing for 20min at 115 deg.C;

wherein when the single colony on the plate is picked for pure culture, the culture temperature is kept at 30 ℃, and the shake anaerobic culture is carried out for 24 hours;

directly crushing the fermentation liquor by using an ultrahigh pressure continuous flow cell crusher, detecting the activity of acetaldehyde dehydrogenase by using an acetaldehyde dehydrogenase detection kit, and detecting the relative activity of the acetaldehyde dehydrogenase according to the increased value of the absorbance of the acetaldehyde dehydrogenase at 340 nm;

centrifuging the strain with higher activity to obtain pure thalli, then using PBS for resuspension, preparing a strain suspension, crushing the thalli again, measuring the activity of intracellular acetaldehyde dehydrogenase, and selecting the strain with higher activity as a target strain;

washing the obtained pure bacteria for 2 times by using sterilized normal saline, then using sterilized PBS solution with the same volume of suspended weight, then using an ultrahigh pressure continuous flow cell crusher to crush the bacteria suspension, then using an acetaldehyde dehydrogenase detection kit to detect the activity of acetaldehyde dehydrogenase, and measuring the relative activity of the acetaldehyde dehydrogenase according to the increase value of absorbance at 340 nm;

the pure thalli is obtained by centrifugation for 5min at 6000 g;

the target strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms by the patentees, the preservation number is CGMCC NO.22892, the preservation date is 2021 year 7 month 13 day, the preservation unit address is No. 3 of Xilu No. 1 of the North West Chen of the Korean district in Beijing, and the preservation classification is named Lactococcus lactis. .

Examples two-fifty:

following the procedure of example one, except that the strains screened for single colony plate isolation were different, as shown in Table 1:

bacterial strains	Relative enzyme activity	Bacterial strains	Relative enzyme activity	Bacterial strains	Relative enzyme activity
						Example 2	1	Example 19	35	Example 36	5
Example 3	5	Example 20	21	Example 37	2
						Example 4	3	Example 21	4	Example 38	22
Example 5	45	Example-22	33	Example 39	15
						Example 6	12	Example 23	21	Example 40	6
Example 7	2	Example 24	15	Example-41	7
						Example-8	13	Example-25	2	Example-42	46
Example-9	3	Example 26	4	Example 43	54
						Example 10	23	Example 27	17	Example 44	11
Example 11	3	Example 28	21	Example-45	3
						Example-12	11	Example 29	1	Example 46	17
Example-13	4	Example 30	16	Example 47	1
						Example 14	5	Example-31	21	Example-48	57
Example 15	9	Example 32	33	Example 49	12
						Example-16	11	Example 33	21	Example 50	23
Example 17	3	Example 34	1
						Example 18	11	Example 35	8

As can be seen from Table 1, the enzyme activities of the fermented liquid were relatively high in the fifth example, the tenth example, the nineteenth example, the twentieth example, the twenty-two example, the twenty-three example, the twenty-eight example, the thirty-first example, the thirty-two example, the thirty-three example, the thirty-eight example, the forty-two example, the forty-three example, the forty-eight example and the fifty example.

After pure culture is carried out on the strains with higher activity by using a screening culture medium again, the relative enzyme activity is measured, and the relative enzyme activity is shown in a table 2:

bacterial strains	Relative enzyme activity	Strain of bacillus	Relative enzyme activity	Strain of bacillus	Relative enzyme activity
						Example-5	32	Example 23	10	Example 38	13
Example 10	16	Example-28	9	Example 42	43
						Example 19	21	Example-31	13	Example 43	46
Example 20	24	Example 32	26	Example-48	53
						Example-22	15	Example 33	15	Example 50	11

As can be seen from Table 2, the strains of example forty-eight are preferred as the target strains in comparison with the enzyme activities.

Examples fifty one to fifty six:

the procedure of example one was followed except that the initial pH of the screening media was varied as shown in Table 3:

initial pH value	OD600	Final pH value
			3.0	1.2	2.6
4.0	2.4	3.4
			5.0	4.0	4.1
6.0	7.1	4.9
			7.0	10.6	5.1
8.0	7.5	5.9

As can be seen from Table 3, the OD600 showed the maximum absorbance at an initial pH of 7.0, indicating that the optimum growth pH of the strain was 7.0.

OD600 refers to the absorbance of a certain solution at a wavelength of 600nm, and the absorbance is proportional to the concentration of the light-absorbing substance in the solution, i.e., the concentration of the bacterial suspension is proportional to the absorbance, as can be seen from the definition of the absorbance.

step one, mixing pure cow milk with a stabilizer and a sweetening agent and then sterilizing;

step three, after the product obtained in the step two is curd, placing the curd in a refrigerator for standing for 4 to 8 hours, and performing after-ripening;

and step four, preparing the yoghurt for relieving the effect of alcohol.

The yogurt obtained by the method can be drunk after drinking because the thallus contains abundant acetaldehyde dehydrogenase, and is helpful for relieving discomfort after drinking and alleviating hangover.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which includes the appended claims and their equivalents.

Claims

1. A screening method of a strain capable of alleviating hangover is characterized by comprising the following steps:

step one, sterilizing a screening culture medium, adding sterile acetaldehyde into the screening culture medium after the sterilization treatment is finished, performing anaerobic culture after aseptically inoculating pickled vegetables or yogurt products, detecting the concentration of the acetaldehyde in a liquid phase, and performing single colony plate separation on microbial flora with reduced acetaldehyde concentration and survival thalli;

wherein the sterile acetaldehyde is prepared by the following method: filtering 40% acetaldehyde using a sterile filter into a sterile container, 1L of the screening medium containing 2.5ml of sterile 40% acetaldehyde by volume, calculated at a final concentration of 0.1%;

wherein the screening culture medium is prepared by heating and dissolving an M17 culture medium in purified water and sterilizing the culture medium;

2. The method for screening a strain capable of alleviating hangover according to claim 1, wherein:

the nutrient-rich medium comprises the following components in concentration:

3. The method for screening a strain capable of alleviating hangover according to claim 2, wherein:

the preparation method of the nutrient-rich culture medium comprises the following steps: blending according to a certain proportion, then adding a certain amount of purified water, uniformly mixing, adjusting the pH value, and then carrying out sterilization treatment; adding the purified water in an amount of 1L, and adjusting the pH value to be neutral; the sterilization treatment at least satisfies: the sterilization time is 20min, and the sterilization temperature is 115 ℃.

4. The method for screening a strain capable of neutralizing the effect of alcohol according to claim 1, wherein:

in the first step and the second step, the culture temperature of the sterile acetaldehyde in a screening culture medium is 30 +/-1 ℃, and the sterile acetaldehyde is cultured for 24 hours in an anaerobic environment; the pH value range of the screening culture medium is 7.0 +/-1.

5. The method for screening a strain capable of alleviating hangover according to claim 4, wherein:

the screening culture medium is prepared by dissolving 42.3g of M17 culture medium in 1L of purified water by heating; the preparation conditions are as follows: autoclaving for 15min at 115 deg.C.

6. The method for screening a strain capable of alleviating hangover according to claim 1, wherein:

in the second step, when the single colony on the picking plate is subjected to pure culture, the culture temperature is kept at 30 ℃, and shake anaerobic culture is performed for 24 hours;

7. The method for screening a strain capable of alleviating hangover according to claim 1, wherein:

washing the obtained pure bacteria for 2 times by using sterilized normal saline, then using sterilized PBS solution for equal volume suspension, then using an ultrahigh pressure continuous flow cell crusher to crush the bacteria suspension, then using an acetaldehyde dehydrogenase detection kit to detect the activity of the acetaldehyde dehydrogenase, and detecting the relative activity of the acetaldehyde dehydrogenase according to the increased value of the absorbance of the acetaldehyde dehydrogenase at 340 nm; the pure thallus is obtained by centrifugation for 5min at 6000 g.