Genetically engineered probiotic composition for expressing ADH and ALDH and application thereof in hangover alleviating and liver protection

Abstract

The invention discloses a genetically engineered probiotic composition for expressing ADH (adenosine dihydrogenase) and ALDH (aldehyde dehydrogenase) and application of the genetically engineered probiotic composition in hangover alleviating and liver protection. The genetically engineered probiotic composition (such as EcN.1917-ADH and EcN.1917-ALDH) is constructed by introducing genes of alcohol dehydrogenase (ADH, NCBI (National Center for Biotechnology Information) Gene ID: 855349) and acetaldehyde dehydrogenase (ALDH, NCBI Gene ID: 855206) of saccharomyces cerevisiae into probiotics (such as escherichia coli). The engineering bacterium shows remarkable ethanol, acetaldehyde and endurance capacity in an in-vitro experiment, and the ethanol degradation rate is remarkably higher than that of a wild type strain; a mouse drunkenness model verifies that drunkenness and death caused by drinking can be reduced through pretreatment, drunkenness tolerance time is remarkably prolonged, and movement coordination disorder caused by alcohol can be improved. The strain can effectively promote alcohol metabolism and relieve alcoholic liver injury, and a new candidate strain is provided for development of a hangover-alleviating and liver-protecting viable bacterial preparation.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a genetically engineered probiotic composition expressing ADH and ALDH and its application in hangover relief and liver protection. Background Technology

[0002] Alcohol, a common beverage at social gatherings, holds a significant position in the global consumer market due to its unique taste and cultural significance. However, excessive consumption can cause serious harm to human health. According to the World Health Organization (WHO), approximately 3 million people worldwide die each year from alcohol-related illnesses.

[0003] After alcohol enters the body, it is primarily metabolized by the liver, with alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) being key metabolic enzymes. Ethanol is first oxidized by ADH to acetaldehyde (a highly toxic substance), then further metabolized by ALDH to acetic acid, and finally broken down into carbon dioxide and water and excreted from the body. The accumulation of acetaldehyde is a key factor in the development of alcoholic liver disease (ALD). Acetaldehyde has strong hepatotoxicity, leading to oxidative stress, mitochondrial dysfunction, inflammatory responses, and hepatocyte apoptosis. Long-term alcohol abuse can induce fatty liver, alcoholic hepatitis, liver fibrosis, and even cirrhosis and liver cancer.

[0004] Currently, hangover remedies and liver protection products on the market mainly include chemical drugs, plant extracts, and probiotic preparations. The first two have drawbacks, namely, increasing the burden on the liver and kidneys and having limited bioavailability. Probiotics have attracted attention due to their potential to regulate gut microbiota and enhance liver detoxification function; however, the activity of alcohol-degrading enzymes in natural strains is limited, making it difficult to efficiently break down alcohol. In recent years, the development of synthetic biology has provided new ideas for the engineered modification of probiotics. Hangover remedies and liver protection strategies based on engineered probiotics have the advantages of precision, high efficiency, and fewer side effects, and are expected to become an emerging solution for alleviating alcoholic liver damage.

[0005] This invention constructs transgenic engineered probiotics (EcN.1917-ADH, EcN.1917-ALDH) capable of producing ADH and ALDH. Pretreatment with these transgenic probiotics can significantly prolong the duration of intoxication and reduce the mortality rate caused by intoxication.

[0006] Therefore, this invention provides a genetically engineered probiotic composition expressing ADH and ALDH and its application in hangover relief and liver protection, providing new candidate strains for the development of live bacteria preparations for hangover relief and laying the foundation for constructing a highly efficient alcohol metabolism probiotic system. Summary of the Invention

[0007] This invention relates to a genetically engineered probiotic composition expressing ADH and ALDH and its application in alcohol detoxification and liver protection. By increasing the levels of alcohol dehydrogenase and aldehyde dehydrogenase in the body, it promotes alcohol metabolism, reduces alcoholic liver damage, and alleviates the occurrence of alcoholic liver disease (ALD). This invention provides a novel transgenic engineered bacterium, whose significant effects on promoting alcohol metabolism, reducing alcoholic liver damage, and prolonging the duration of intoxication have been verified through in vitro and in vivo experiments.

[0008] In a first aspect, the present invention provides a genetically engineered probiotic composition expressing ADH and ALDH, wherein the genetically engineered bacteria promote the alcohol metabolism process, alleviate alcoholic liver damage, and reduce the occurrence of alcoholic liver disease (ALD) by expressing ADH and ALDH.

[0009] Furthermore, the genetically engineered probiotics include, but are not limited to: Escherichia coli, Clostridium butyricum, lactic acid bacteria, Bacillus subtilis, yeast, etc.

[0010] Furthermore, the recombinant plasmid vectors include, but are not limited to, pET28a(+), pET28b, pET32a, pMTL82151, pMTL007, etc.

[0011] Furthermore, the genetically engineered strains ADH (NCBI Gene ID: 855349) and ALDH (NCBI Gene ID: 855206) genes are derived from Saccharomyces cerevisiae S288C.

[0012] In a second aspect, the present invention provides the application of the genetically engineered probiotic composition according to any one of the first aspects, the application comprising:

[0013] It is used to reduce the occurrence of alcoholic liver disease, including but not limited to alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis, and alcoholic cirrhosis.

[0014] It is used to enhance the tolerance of probiotics to ethanol and acetaldehyde in the body and promote the metabolism of alcohol.

[0015] It was used to prolong the time of intoxication and reduce the mortality of mice due to intoxication.

[0016] This invention provides a genetically engineered probiotic composition with highly efficient alcohol metabolism. This engineered bacterium can significantly improve alcohol metabolism efficiency by specifically expressing ADH and ALDH. Its construction process overcomes the limitations of traditional hangover remedies in terms of enzyme activity and stability. In vitro and in vivo experiments verified that this transgenic engineered bacterium exhibits excellent ethanol and acetaldehyde metabolic activity in a simulated in vivo environment, providing a novel technical pathway for the development of hangover remedies and liver-protecting biological agents. Its main advantages and beneficial effects include:

[0017] Enhanced tolerance to ethanol and acetaldehyde: By significantly enhancing the tolerance of probiotics to ethanol and acetaldehyde, the genetically engineered bacteria improve the survival rate of probiotics in ethanol and acetaldehyde environments, ensuring that the genetically engineered bacteria can fully promote the alcohol metabolism process.

[0018] Increased ethanol and acetaldehyde degradation capacity: Transgenic engineered bacteria can significantly enhance the ethanol and acetaldehyde degradation rate of probiotics, promote alcohol metabolism, and reduce alcohol damage to organs such as the liver and intestines.

[0019] Prolonging the duration of intoxication: In in vivo experiments, the transgenic engineered bacteria significantly prolonged the duration of intoxication in mice after drinking alcohol, reducing mortality and intoxication rates caused by alcohol consumption. Simultaneously, it improved coordination in mice after drinking alcohol. Attached Figure Description

[0020] Figure 1 Colony morphology of EcN.1917-ADH and EcN.1917-ALDH on LB agar medium;

[0021] Figure 2 Agarose gel electrophoresis image of recombinant plasmids expressing ADH and ALDH;

[0022] Figure 3 The growth inhibition rate of EcN.1917-ADH by different concentrations of ethanol;

[0023] Figure 4 Growth inhibition rate of EcN1917-ALDH at different concentrations of acetaldehyde;

[0024] Figure 5 The ability of genetically engineered bacteria to degrade ethanol

[0025] Figure 6 Evaluation of the in vivo alcohol detoxification effect of transgenic engineered bacteria Detailed Implementation

[0026] This invention relates to a genetically engineered probiotic composition expressing ADH and ALDH and its application in hangover relief and liver protection. Specifically, it describes the preparation of the engineered bacteria and their effects on alcohol tolerance in vitro and in vivo, degradation of ethanol and acetaldehyde, prolonging the duration of intoxication, and improving alcoholic liver damage. The technical solutions in the embodiments of this invention are clearly and completely described below. Obviously, the described examples are only a part of the embodiments of this invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0027] Implementation Example 1: Preparation of Transgenic Engineered Bacteria

[0028] First, the ADH (NCBI Gene ID: 855349) and ALDH (NCBI Gene ID: 855206) genes from *Saccharomyces cerevisiae* S288C were inserted into recombinant plasmid vectors including, but not limited to, pET28a(+), pET28b, pET32a, pMTL82151, and pMTL007 to construct recombinant plasmids. These recombinant plasmids were then transformed into competent bacteria, including but not limited to *Escherichia coli*, *Clostridium butyricum*, lactic acid bacteria, *Bacillus subtilis*, and yeast. The colony morphology of the strains was observed, and the results are shown in the attached figure. Figure 1 As shown, the strain grows well on LB agar medium, and the colonies are milky white, smooth, moist, and round with neat edges.

[0029] Example 2: Agarose gel electrophoresis images of transgenic engineered bacteria ADH and ALDH

[0030] First, the engineered bacteria were cultured overnight in liquid medium at 37°C and 200 rpm. Then, 5 ml of the extracted plasmid was used for agarose gel electrophoresis. Agarose powder was weighed and dissolved in 1×TAE buffer. The solution was microwaved until completely dissolved, nucleic acid dye was added and mixed thoroughly, then poured into the gel casting tank, and a comb was inserted. The gel was allowed to solidify at room temperature. The DNA sample was then mixed with loading buffer in 1×TAE buffer at 80V for electrophoresis. The results were observed under an imaging system. Figure 2 As shown, lane 1 is the plasmid expressing ADH, and lane 2 is the plasmid expressing ALDH. Bright target bands appear at the positions corresponding to the markers in both lanes.

[0031] Implementation Example 3: Ethanol Tolerance of Engineered Escherichia coli

[0032] Wild-type bacteria and the prepared engineered strains were cultured separately until turbidity was achieved. Different doses of ethanol were added to the culture medium to achieve ethanol concentrations of 0%, 2%, 6%, and 10%, respectively. The medium was then incubated at room temperature with shaking, and the OD value of each medium was recorded every 2 hours. 600 Numerical values ​​were used to compare the growth of different strains at different ethanol concentrations. Results are as follows: Figure 3 As shown, compared to wild-type bacteria that lose their tolerance to ethanol at a concentration of 2%, the engineered bacteria, after transgenic modification, exhibited good activity at a concentration of 10% ethanol, and the number of bacteria in the culture medium increased with the duration of culture. This indicates that the engineered probiotics significantly improve bacterial tolerance to ethanol, increase the survival rate of probiotics in an ethanol environment, and ensure that the transgenic engineered bacteria can fully promote the alcohol metabolism process.

[0033] Implementation Example 4: Acetaldehyde Tolerance of Engineered Escherichia coli

[0034] Wild-type bacteria and the prepared engineered strains were cultured separately until turbidity was achieved. Different doses of acetaldehyde were added to the culture medium to achieve concentrations of 0%, 0.1%, 0.3%, and 0.5%, respectively. The medium was then incubated at room temperature with shaking, and the OD values ​​of each medium were recorded every 2 hours. 600 Numerical values ​​were used to compare the growth of different strains under different acetaldehyde concentrations. Results are as follows: Figure 4 As shown, compared to wild-type bacteria that lose their tolerance to acetaldehyde at a concentration of 0.3%, the genetically engineered bacteria exhibit good activity at a concentration of 0.5% acetaldehyde, and the number of bacteria in the culture medium increases with the duration of culture. This indicates that the engineered probiotics significantly improve bacterial tolerance to acetaldehyde, increase the survival rate of probiotics in an acetaldehyde environment, and ensure that the genetically engineered bacteria can fully promote the alcohol metabolism process.

[0035] Implementation Example 5: Ethanol Degradation Rate of Engineered Escherichia coli

[0036] The engineered bacterial strain was inoculated into LB liquid medium at a 1% inoculum and cultured with shaking at room temperature. 2% ethanol was added to the medium, and the medium was cultured with shaking at room temperature for 6 hours. Samples were taken every two hours, filtered through a 0.22 μm sterile filter, and the ethanol content in the system was determined using gas chromatography. The results are as follows: Figure 5 As shown, compared with wild-type bacteria, engineered bacteria can significantly degrade ethanol in the culture medium, indicating that engineered strains can effectively degrade alcohol.

[0037] Implementation Example 6: Establishing a Mouse Model of Intoxication

[0038] Forty healthy male Kunming rats aged 6-8 weeks were acclimatized for one week and randomly divided into 5 groups of 8 rats each. After a 6-hour fast with unlimited water intake, all mice were administered different doses of 56° alcohol via gavage. The state of intoxication was observed after gavage. Mice were placed on their backs facing down on a table; the absence of the righting reflex for 30 seconds or more was considered an indicator of intoxication. The time from alcohol administration to the disappearance of the righting reflex was recorded as the intoxication time. The intoxication rate and mortality rate of mice at different alcohol doses were also recorded.

[0039]

[0040] The results showed that the 420uL group, which had the highest intoxication rate and the lowest mortality rate, was selected as the optimal gavage volume.

[0041] Implementation Example 7: Investigating the Intracellular Alcohol Detoxification Effect of Engineered Bacteria

[0042] 1) Loss of righting reflex. Forty healthy male Kunming rats aged 6-8 weeks were acclimatized for one week and randomly divided into four groups of 10 each: acute alcohol intoxication model (NC group), Zbiotics positive control (PC group), EcN.1917 group, and EcN.1917-ADH / ALDH group. After fasting for 6 hours with free access to water, the alcohol intoxication model (NC group) was administered 200 μL of sterile saline by gavage, the Zbiotics positive control (PC group) was administered 100 μL of Zbiotics by gavage, and the EcN.1917 group and EcN.1917-ADH / ALDH group were administered 100 μL of Zbiotics by gavage. 9 EcN.1917 or cfu / mL

[0043] Mice were administered EcN.1917-ADH / ALDH via gavage. Thirty minutes later, 420 μL of 56% ABV liquor was administered. The state of intoxication in mice was observed after gavage, and the time from administration of alcohol to the disappearance of the righting reflex was recorded as the intoxication time. The intoxication rate and mortality rate of each group were recorded.

[0044]

[0045] The pretreated EcN.1917-ADH / ALDH group had a drunkenness rate of 20% and a mortality rate of 0%, significantly lower than the NC and EcN.1917 groups. This indicates that the transgenic engineered bacteria can significantly reduce alcohol-induced drunkenness and mortality.

[0046] 2) Steel cable experiment:

[0047] Forty healthy male Kunming rats aged 6-8 weeks were randomly divided into four groups of 10 each: an acute alcohol intoxication model group (NC group), a non-alcoholic group, and a control group.

[0048] Zbiotics positive control PC group, EcN.1917 group, and EcN.1917-ADH / ALDH group. All mice were fasted for 6 hours but allowed free water. A 50cm long, 0.5-1cm diameter rod (30-50cm off the ground) was fixed at both ends to a support frame. The mice were placed in the center of the steel cable, and the time from placement to fall was recorded within 60 seconds (if a mouse did not fall within 60 seconds, it was counted as 60 seconds). Results are as follows: Figure 6 As shown, EcN.1917-ADH / ALDH treatment can improve the effect of alcohol on motor coordination in mice.

Claims

1. A genetically engineered probiotic composition expressing ADH and ALDH and its application in hangover relief and liver protection, characterized in that... This method includes the following steps: inserting the ADH (NCBI Gene ID: 855349) and ALDH (NCBI Gene ID: 855206) genes from Saccharomyces cerevisiae S288C into plasmid vectors including but not limited to pET28a(+), pET28b, pET32a, pMTL82151, and pMTL007 to construct recombinant plasmids.

2. A genetically engineered probiotic composition expressing ADH and ALDH and its application in hangover relief and liver protection, characterized in that: Recombinant plasmids are transformed into competent bacteria, including but not limited to: Escherichia coli, Clostridium butyricum, lactic acid bacteria, Bacillus subtilis, yeast, etc. Single colonies grown on corresponding resistant solid plates are transgenic engineered bacteria.

3. The application of the genetically engineered probiotic composition as described in claim 2 in the treatment of hangovers and liver protection, characterized in that: Transgenic engineered bacteria significantly enhance the tolerance of probiotics to ethanol and acetaldehyde, improve the survival rate of probiotics in ethanol and acetaldehyde environments, and increase the acetaldehyde degradation rate of probiotics. This effectively prolongs the duration of intoxication in mice after alcohol consumption and reduces the mortality rate and intoxication rate caused by alcohol in mice.

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