Preparation and application of hangover remedies

Abstract

This invention relates to the field of biological products, and particularly to the preparation and application of hangover relief products. This invention provides an alcohol dehydrogenase mutant, an aldehyde dehydrogenase mutant, an expression vector, a combination of recombinant yeast strains, and yeast single-cell oral capsules. By cloning the alcohol dehydrogenase and aldehyde dehydrogenase genes into the *Saccharomyces cerevisiae* expression plasmid pYX212, and then transforming the *Saccharomyces cerevisiae* into the *Saccharomyces cerevisiae*, engineered yeast is obtained. Utilizing the protective effect of yeast cells, oral administration is achieved, effectively reducing the destruction of recombinant enzymes by gastric juice. The yeast single-cell oral capsules can accelerate alcohol metabolism after daily drinking and promote hepatocyte regeneration; they are convenient and quick to take and have significant effects.

Description

Technical Field

[0001] This invention relates to the field of biological products, and in particular to the preparation and application of hangover relief products. Background Technology

[0002] China boasts a long history of alcohol culture and a diverse range of alcoholic beverages, with drinking becoming an integral part of daily life. Traditional theories suggest that moderate drinking can improve blood circulation, dispel cold and wind, and promote health. However, with rising incomes in my country, alcohol consumption has increased significantly. Modern medical research has demonstrated that excessive drinking is a major health hazard, primarily manifesting as functional damage to vital organs and severe mental disorders. Some individuals lack aldehyde dehydrogenase, resulting in slower alcohol metabolism. Most hangover remedies on the market are purely stimulant; they induce a hormonal effect in the body, leading to acetaldehyde accumulation and alleviating central nervous system depression, thus achieving a sobering effect. While these products have some effect, individual factors significantly influence their effectiveness, resulting in generally limited results. Summary of the Invention

[0003] In view of this, the present invention provides the preparation and application of a hangover relief product, by introducing the alcohol dehydrogenase and acetaldehyde dehydrogenase genes into brewer's yeast respectively, and utilizing the protective effect of yeast cells to effectively reduce the damage of gastric juice to the recombinant enzymes.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides mutants, including alcohol dehydrogenase mutants and / or acetaldehyde dehydrogenase mutants;

[0006] The alcohol dehydrogenase mutant includes an alcohol dehydrogenase with glycine at position 231 mutated to lysine.

[0007] The acetaldehyde dehydrogenase mutants include those with glycine at position 230 mutated to tyrosine and / or alanine at position 231 mutated to serine.

[0008] In some specific embodiments of the present invention, the above-mentioned mutants include:

[0009] The alcohol dehydrogenase mutant has the following characteristics:

[0010] (1) An amino acid sequence as shown in SEQ ID No. 2; or

[0011] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (1), and whose function is the same as or similar to that of (1); or

[0012] (3) An amino acid sequence that is at least 70% homologous to the amino acid sequence shown in (1) or (2).

[0013] and / or

[0014] The acetaldehyde dehydrogenase mutant has the following characteristics:

[0015] (4) An amino acid sequence as shown in SEQ ID No. 4; or

[0016] (5) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (4), and whose function is the same as or similar to that of (4); or

[0017] (6) An amino acid sequence that has at least 70% homology with the amino acid sequence shown in (4) or (5);

[0018] The number of items is between 2 and 100.

[0019] The present invention also provides expression vectors, including expression vector 1 and / or expression vector 2;

[0020] The expression vector 1 includes a gene encoding the alcohol dehydrogenase mutant described above, and an acceptable gene element;

[0021] The expression vector 2 includes a gene encoding the acetaldehyde dehydrogenase mutant described above, as well as an acceptable gene element.

[0022] In some specific embodiments of the present invention, the expression vector described above further includes a plasmid backbone, wherein the plasmid backbone includes pYX212.

[0023] The present invention also provides a combination of recombinant strains, including recombinant strain 1 and recombinant strain 2;

[0024] The recombinant strain 1 includes expression vector 1 in the above-mentioned expression vector;

[0025] The recombinant strain 2 includes expression vector 2 from the above-mentioned expression vectors.

[0026] In some specific embodiments of the present invention, the strains in the above-mentioned recombinant strain combination include Saccharomyces cerevisiae.

[0027] The present invention also provides compositions comprising (I) and (II);

[0028] The (I) includes the above-mentioned combination of recombinant strains;

[0029] The (II) includes one or more of the following: dried pig liver extract powder, vitamin D3, vitamin E, starch, or talc.

[0030] In some specific embodiments of the present invention, the above composition comprises, by weight, per 400 g:

[0031] The above-mentioned recombinant bacterial strain combination is 130-170 g;

[0032] 15-38 g of dried pig liver extract powder;

[0033] Vitamin D3 30~65 mg;

[0034] Vitamin E 45~80 mg;

[0035] Talc 1~3% (w / w);

[0036] Starch balance.

[0037] The present invention also provides the use of the above-described recombinant strain combination or the above-described composition in the following aspects:

[0038] (a) Enhance cell viability; and / or

[0039] (b) Improve total antioxidant capacity; and / or

[0040] (c) Reduce total superoxide dismutase activity; and / or

[0041] (d) Reduce malondialdehyde content; and / or

[0042] (e) Promote alcohol metabolism; and / or

[0043] (f) Improve acute alcoholic liver injury.

[0044] In some specific embodiments of the present invention, the cells described in the above applications include L-02 cells.

[0045] The present invention also provides microbial preparations, including the above-described recombinant strain combination or the above-described composition, and acceptable excipients or adjuvants.

[0046] The capsules of this invention have the following effects:

[0047] 1. This process involves cloning the alcohol dehydrogenase and acetaldehyde dehydrogenase genes into the Saccharomyces cerevisiae expression plasmid pYX212, and then transforming them into Saccharomyces cerevisiae to obtain engineered yeast. The recombinant enzymes are expressed in the yeast cells, and the recombinant enzymes are produced. The recombinant enzymes can be orally administered by utilizing the protective effect of yeast cells, i.e., preparing yeast single-cell oral capsules, which effectively reduces the destruction of recombinant enzymes by gastric juice.

[0048] 2. Preparing seed cultures of two recombinant brewing yeasts separately, and then simultaneously introducing them into a fermenter in equal proportions for mixed fermentation simplifies the process and saves costs.

[0049] 3. Taking advantage of the edibility of brewing yeast, the yeast cells can be collected directly after fermentation, eliminating the need for purification of recombinant enzymes and enabling oral administration, which is a simple process.

[0050] 4. A combination of recombinant enzyme, dried pig liver extract powder, vitamin D3, and vitamin E yields a yeast single-cell oral capsule that can accelerate alcohol metabolism after daily drinking and promote liver cell regeneration. It is convenient and quick to take and has significant effects. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0052] Figure 1 Showing the pre-mutant structure of alcohol dehydrogenase;

[0053] Figure 2 Showing the structure of alcohol dehydrogenase after mutation;

[0054] Figure 3 Grayscale analysis of the expression difference of the alcohol dehydrogenase gene before and after optimization; the left side shows the electrophoresis image of the target protein; the right side shows the grayscale analysis of the target protein.

[0055] Figure 4 Showing the pre-mutant structure of acetaldehyde dehydrogenase;

[0056] Figure 5 Showing the structure of acetaldehyde dehydrogenase after mutation;

[0057] Figure 6 The image shows a grayscale analysis of the expression differences of the acetaldehyde dehydrogenase gene before and after optimization; the left side shows the electrophoresis image of the target protein; the right side shows the grayscale analysis of the target protein. Detailed Implementation

[0058] This invention discloses the preparation and application of wine products. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0059] This invention aims to utilize genetic engineering techniques to provide a single-cell oral capsule of recombinant Saccharomyces cerevisiae, which degrades alcohol at its source. The genes for alcohol dehydrogenase and aldehyde dehydrogenase are introduced into Saccharomyces cerevisiae for fermentation and expression of the recombinant enzymes. Saccharomyces cerevisiae itself is edible; therefore, the ethanol and aldehyde dehydrogenase, or other related substances that aid in alcohol metabolism produced by the engineered Saccharomyces cerevisiae, can be used to accelerate the metabolism of alcohol after daily drinking. Currently, no company is actively promoting this project for the hangover relief market; if successfully developed, it will undoubtedly be welcomed by the market.

[0060] [Basic Concept]

[0061] This invention mainly provides a method for preparing yeast single-cell oral capsules and their applications.

[0062] The technical problem to be solved by this invention can be achieved through the following technical solutions.

[0063] Preparation method of yeast single-cell oral capsules:

[0064] Ethanol dehydrogenase and aldehyde dehydrogenase genes were introduced into *Saccharomyces cerevisiae* using genetic engineering techniques. After the strain was constructed, fermentation conditions were explored, progressing from laboratory shake flask fermentation to 10 L small-scale fermentation, then to 100 L pilot-scale fermentation, and finally to fermentation in a 5-ton fermenter. The fermentation broth was centrifuged, washed, and then freeze-dried to obtain the freeze-dried recombinant enzyme for alcohol detoxification. This alcohol-detoxifying enzyme was then compounded with one or more of the following: liver extract, turmeric, N-acetylcysteine, vitamin D3, vitamin E, xylitol, and citric acid, to produce yeast single-cell oral capsules.

[0065] [Information on gene sequence mutants]

[0066] Ethanol dehydrogenase:

[0067] Premutation sequence of human alcohol dehydrogenase ADH1B2 (NM_001286650.2) (codons optimized according to the codon preference of Saccharomyces cerevisiae):

[0068] ATGGTCGCCGTAGGAATTTGCCACACGGATGATCACGTTGTGTCAGGGAACCTGGTTACTCCACTTCCTGTCATCTTGGGCCACGAGGCGGCTGGAATTGTGGAAAGTGTAGGTGAGGGAGTAACGACCGTAAAACCCGGAGACAAAGTGATCCCCTTGTTCACACCGCAATGTGGCAAATGTAGAGTTTGCAAGAACCCCGAAAGTAACTATTGTCTGAAGAATGATTTGGGCAATCCTCGTGGCACACTGCAAGATGGAACCAGGAGATTCACATGCCGTGGGAAACCGATCCACCATTTCTTAGGTACCAGTACATTCTCCCAATATACCGTCGTGGATGAAAACGCGGTTGCCAAGATAGATGCAGCCAGCCCGCTGGAAAAGGTGTGTTTAATCGGTTGCGGATTCAGCACTGGCTACGGGTCAGCCGTCAATGTAGCAAAAGTTACCCCCGGTTCTACCTGCGCGGTATTCGGGCTAGGCGGCGTTGGGTTAAGTGCCGTAATGGGTTGCAAAGCTGCTGGCGCGGCGAGAATCATTGCAGTGGACATTAACAAAGACAAGTTCGCAAAGGCAAAAGAACTGGGTGCTACCGAATGCATCAACCCTCAGGACTACAAAAAACCGATCCAAGAAGTACTTAAAGAGATGACGGATGGTGGGGTTGACTTCTCATTTGAGGTGATT GGACGTTTGGATACAATGATGGCCAGTTTATTGTGTTGCCACGAGGCATGTGGCACCAGCGTTATTGTTGGCGTTCCCCCGGCGAGTCAGAATCTGTCTATCAATCCAATGCTACTACTAACAGGTAGGACCTGGAAGGGAGCAGTTTATGGGGGGTTTAAG AGCAAAGAGGGCATCCCCAAACTGGTGGCGGACTTTATGGCCAAAAAGTTTTCTTTAGACGCCTTGATAACACATGTCTTACCCTTCGAAAAGATAAACGAAGGTTTTGATTTACTACACTCAGGAAAATCCATACGTACCGTATTGACCTTCTGA (SEQ ID No.1)

[0069] The sequence following the mutation of glycine at position 231 to lysine (mutating the original glycine codon GGA to the lysine codon AAA):

[0070] MVAVGICHTDDHVVSGNLVTPLPVILGHEAAGIVESVGEGVTTVKPGDKVIPLFTPQCGKCRVCKNPESNYCLKNDLGNPRGTLQDGTRRFTCRGKPIHHFLGTSTFSQYTVVDE NAVAKIDAASPLEKVCLIGCGFSTGYGSAVNVAKVTPGSTCAVFGLGGVGLSAVMGCKAAGAARIIAVDINKDKFAKAKELGATECINPQDYKKPIQEVLKEMTDGGVDFSFEVI K RLDTMMASLLCCHEACGTSVIVGVPPASQNLSINPMLLLTGRTWKGAVYGGFKSKEGIPKLVADFMAKKFSLDALITHVLPFEKINEGFDLLHSGKSIRTVLTF (SEQ ID No. 2)

[0071] After site-directed mutagenesis, the alcohol dehydrogenase binding site has a stronger binding capacity. Figure 1 , Figure 2 These are the structures of alcohol dehydrogenase before and after the mutation, respectively. Figure 3 The grayscale analysis of the expression difference of the alcohol dehydrogenase gene before and after optimization is shown in Table 1. In this table, 1 represents the ADH1B2 mutant and 2 represents the original ADH1B2 sequence.

[0072] Table 1

[0073]

[0074] aldehyde dehydrogenase:

[0075] The ALDH2 gene sequence before mutation (codons optimized according to the codon preference of Saccharomyces cerevisiae):

[0076] ATGTCCGCTGCCGCCACGCAAGCAGTGCCTGCTCCAAATCAACAGCCTGAAGTCTTTTGCAATCAAATTTTCATTAATAATGAATGGCACGACGCTGTCAGTCGTAAAACTTTTCCCACCGTGAATCCTAGTACAGGGGAGGTAATTTGTCAAGTTGCGGAGGGCGATAAA GAAGATGTCGACAAGGCGGTTAAGGCTGCCAGAGCCGCCTTTCAACTTGGCAGCCCTTGGAGGCGTATGGACGCATCACATAGGGGCAGACTTTTGAACCGTTTGGCTGATCTGATCGAAAGAGATAGAACTTATTTGGCAGCTTTGGAAACTCTGGATAATGGGAAACCCT ACGTCATAAGTTACTTAGTGGACTTAGACATGGTTTTGAAATGCCTGAGATACTATGCAGGATGGGCTGACAAGTATCATGGTAAGACCATACCAATCGATGGGGATTTTTCAGTTACACAAGACATGAACCGGTTGGGGTTTGTGGGCAAATAATACCGTGGAATTTTCC GCTGTTGATGCAGGCTTGGAAGCTAGGACCCGCATTAGCCACCGGGAACGTTGTTGTTATGAAGGTGGCAGAACAGACGCCTCTTACAGCGTTGTATGTTGCAAACCTAATCAAGGAAGCCGGCTTTCCCCCTGGCGTGGTTAACATCGTGCCGGGTTTTGGACCGACTGCC GGGGCGGCTATAGCGTCTCACGAGGATGTCGATAAGGTCGCGTTTACTGGCTCTACCGAAATTGGACGTGTAATACAAGTAGCTGCGGGGTCCTCCAATCTGAAGCGTGTGACTCTGGAACTGGGCGGCAAGTCTCCGAATATTATAATGAGCGATGCAGACATGGATTGGGCAGTGGAGCAGGCCCACTTTGCCCTTTTCTTTAATCAAGGTCAATGTTGTTGTGCCGGGTCACGTACCTTCGTCCAGGAGGACATTTATGACGAGTTTGTGGAGAGATCAGTGGCACGTGCAAAGTCAAGAGTGGTTGGTAACCCCTTTGATTCAAAGACCGAGCAGGGTCCGCAAGTTGACGAAACACAGTTCAAGAAGATACTTGGTTACATAAATACGGGCAAACAAGAAGGGGCAAAGTTGTTATGTGGTGGAGGGATCGCTGCGGATAGGGGCTATTTTATTCAACCGACAGTTTTTGGAGATGTCCAAGACGGTATGACTATAGCAAAGGAGGAGATATTCGGGCCAGTCATGCAGATCCTAAAGTTTAAGACGATCGAGGAAGTTGTCGGGCGTGCGAATAACAGCACTTACGGTCTGGCCGCCGCAGTTTTTACGAAGGATTTGGATAAAGCGAATTATCTGTCCCAAGCCCTTCAAGCTGGCACAGTGTGGGTTAACTGTTACGATGTTTTCGGAGCTCAAAGCCCTTTCGGTGGGTATAAAATGAGCGGGAGCGGCAGGGAATTAGGCGAGTACGGGCTTCAGGCATACACTGAAGTTAAAACAGTTACTGTAAAGGTTCCACAAAAAAATTCATAA (SEQ ID No.3)

[0077] The sequence after site-directed mutagenesis of glycine at position 230 to tyrosine and alanine at position 231 to serine (mutating the original GGGGC to TACTCG) is:

[0078] MSAAATQAVPAPNQQPEVFCNQIFINNEWHDAVSRKTFPTVNPSTGEVICQVAEGDKEDVDKAVKAARAAFQLGSPWRRMDASHRGRLLNRLADLIERDRTYLAALETLDNGKP YVISYLVDLDMVLKCLRYYAGWADKYHGKTIPIDGDFFSYTRHEPVGVCGQIIPWNFPLLMQAWKLGPALATGNVVVMKVAEQTPLTALYVANLIKEAGFPPGVVNIVPGFGPTA YS AIASHEDVDKVAFTGSTEIGRVIQVAAGSSNLKRVTLELGGKSPNIIMSDADMDWAVEQAHFALFFNQGQCCCAGSRTFVQEDIYDEFVERSVARAKSRVVGNPFDSKTEQGPQVDETQFKKILGYINTGKQEGAKL LCGGGIAADRGYFIQPTVFGDVQDGMTIAKEEIFGPVMQILKFKTIEEVVGRANNSTYGLAAAVFTKDLDKANYLSQALQAGTVWVNCYDVFGAQSPFGGYKMSGSGRELGEYGLQAYTEVKTVTVKVPQKNS (SEQ ID No.4)

[0079] After site-directed mutagenesis, the structure of acetaldehyde dehydrogenase is stable, but mutations at key binding sites enhance the catalytic activity of acetaldehyde dehydrogenase. Figure 4 , Figure 5 These are the structures of acetaldehyde dehydrogenase before and after the mutation, respectively. Figure 6 The grayscale analysis of the expression difference of the acetaldehyde dehydrogenase gene before and after optimization is shown in Table 2. In the table, 1 represents the ADH1B2 mutant and 2 represents the original ADH1B2 sequence.

[0080] Table 2

[0081]

[0082]

Compound Yeast Single-Cell Oral Capsules

[0083] It is a compound formula consisting of recombinant brewer's yeast, dried pig liver extract powder, vitamin D3, and vitamin E, as detailed below:

[0084] 130-170 g of recombinant brewing yeast;

[0085] 15-38 g of dried pig liver extract powder;

[0086] Vitamin D3 30~65 mg;

[0087] Vitamin E 45~80 mg;

[0088] Talc 1~3% (w / w);

[0089] Add starch to a total of 400 g;

[0090] Make 1000 capsules, each weighing 0.4 g.

[0091] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0092] The present invention will be further illustrated below with reference to the embodiments:

[0093] Example 1

[0094] 1. Preparation of recombinant brewer's yeast

[0095] 1.1 Preparation of electroporation plasmids

[0096] 1) Take 50 μL of JM109 glycerol bacteria containing pYX212-ADH1B2 and JM109 glycerol bacteria containing pYX212-ALDH2 and inoculate them into 4 tubes of 5 mL LB liquid medium resistant to Amp and culture overnight.

[0097] 2) Extract the pYX212-ADH1B and pYX212-ALDH2 recombinant plasmids according to the kit instructions, determine their concentrations, and store them at -20℃ for electroporation.

[0098] 1.2 Preparation of competent cells of Saccharomyces cerevisiae

[0099] 1) Take W303 glycerol bacteria and incubate them upside down in an incubator at 28℃~30℃ for 2~3 days;

[0100] 2) Pick a single colony and inoculate it into 5 mL of antibiotic-free YPD medium, incubate overnight at 32°C and 200 rpm;

[0101] 3) Inoculate 1 mL of the bacterial culture from step 2 into an Erlenmeyer flask containing 100 mL of YPD liquid medium and incubate overnight until OD reaches 100%. 600 =1.3~1.5;

[0102] 4) Take 10 mL of bacterial solution, centrifuge at 5000 rpm and 4℃ for 5 min, and then resuspend the particles in 3 mL of pre-cooled sterile water.

[0103] 5) Centrifuge as in step 4, and resuspend the bacterial cells in 750 μL of pre-cooled sterile water.

[0104] 6) Centrifuge as in step 4, and resuspend the bacterial cells in 200 μL of pre-cooled 1M sorbitol.

[0105] 7) Centrifuge as in step 4, resuspend the cells in 100 μL of pre-cooled 1M sorbitol, keep the cells on ice for at least 4 h, and use on the same day.

[0106] 1.3 Electroporation of brewing yeast

[0107] 1) Preparation of the electroporation cup: Before electroporation, soak the electroporation cup in 80% ethanol for 20 min. Align the electrode slit of the electroporation cup with the UV lamp in the laminar flow hood, with the cup lid facing upwards, sterilize by UV irradiation, and then air dry using the laminar flow hood. Thoroughly mix 80 μL of the prepared competent cells with the plasmid and transfer them to a 2 mm ice-cold electroporation reaction cup;

[0108] 2) Incubate the electroporation cup with the cells on ice for 5 minutes;

[0109] 3) Dry the electric rotary cup and use the electric rotary instrument. Conditions: voltage 2 kV, resistance 200 Ω, capacitance 25 μF, and electric shock time 2~25 msec.

[0110] 4) Immediately add 1 mL of 1M pre-cooled sorbitol to the electroporation cup and mix well. Transfer the mixture from the electroporation cup to a sterile 1.5 mL test tube and incubate at 30°C for 5 h.

[0111] 5) Centrifuge quickly, discard 900 μL of supernatant, and take 100 μL to spread on SC-URA plates.

[0112] 6) Incubate upside down in a 30°C incubator until colonies grow.

[0113] 1.4 Yeast genome extraction

[0114] Select colonies with good growth and inoculate them into 5 mL of SC-URA liquid medium. Incubate at 30°C and 180 rpm for 6–8 h. Extract yeast plasmid DNA using a yeast plasmid extraction kit. The specific method is as follows:

[0115] 1) Take 5 mL of yeast culture, centrifuge at 8000 rpm for 1 min, and aspirate the supernatant.

[0116] 2) Add 220 μL of sorbitol buffer to the yeast cells to fully suspend the cells, add 10 μL of yeast cell-breaking enzyme and 5 μL of thiol reducing agent, mix thoroughly, treat at 30℃ for 1 h, centrifuge at 8000 rpm for 1 min, remove the supernatant, add 100 μL of YP1 to the precipitate, and fully suspend the precipitate.

[0117] 3) Add 150 μLYP2 to the EP tube and gently invert it 12-15 times to fully lyse the bacteria.

[0118] 4) Add 200 μL LYP3 to the EP tube, immediately and gently invert it 6-8 times to mix thoroughly, centrifuge at 8000 rpm for 10 min, and transfer the supernatant to another EP tube.

[0119] 5) Add the supernatant from step 4 into the adsorption column, let it stand at room temperature for 2 min, centrifuge at 8000 rpm for 1 min, and discard the waste liquid.

[0120] 6) Add 600 μL of washing solution to the adsorption column, centrifuge at 8000 rpm for 1 min, discard the waste liquid, and repeat twice.

[0121] 7) Centrifuge at 8000 rpm for 2 min, leave the adsorption column open at room temperature for 5 min, place in a new EP tube, add 50 μL of elution buffer preheated at 65℃, let stand at room temperature for 2 min, centrifuge at 8000 rpm for 1 min, and repeat twice.

[0122] 8) Determine the plasmid concentration and store at -20℃.

[0123] 2. Expression of recombinant proteins

[0124] 2.1 Shake flask culture of yeast

[0125] 1) Select three positive strains and inoculate them into 5 mL of SC-URA medium for overnight culture.

[0126] 2) Centrifuge the bacterial culture from step 1 at 2000 rpm for 3 min, discard 4 mL of supernatant and resuspend the bacterial cells.

[0127] 3) Take the bacterial culture from step 2 and inoculate it into an Erlenmeyer flask containing 100 mL of SC-URA medium. Incubate at 30°C and 300 rpm. Use the same conditions to incubate Saccharomyces cerevisiae that has not been transfected with the recombinant plasmid as a negative control.

[0128] 4) At each time point shown below, transfer 1 mL of expression culture to a 1.5 mL microcentrifuge tube. Centrifuge at maximum speed for 2–3 min in a desktop microcentrifuge at room temperature. Time points (hours): 0, 24 (1 day), 48 (2 days), 72 (3 days), 96 (4 days), and then every 24 h. Centrifuge the collected samples at 8000 rpm for 1 min at room temperature, wash with 1 mL PBS, resuspend in 300 µL PBS, and store at -20 °C.

[0129] 2.2 Yeast cell disruption of ALDH2-PYX212 and ADH1B2-pYX212

[0130] The expression vector used was an intracellular expression vector, which required cell disruption to collect proteins and measure enzyme activity. Therefore, the cell disruption conditions for yeast were explored, and the method of repeated freeze-thaw cycles and ultrasonic disruption was selected based on laboratory conditions to disrupt the yeast cell walls.

[0131] 1) Take 1 mL of expression culture from 96 h (3 groups in total), place the sample at -80℃, remove it after 2 h, and place it in a metal bath at 100℃ for 5 min, repeat 3 times. Set the sonication power to 70%, sonicate for 3 s, pause for 5 s, and sonicate for a total of 1 min; add 5 µL of 100 mM PMSF.

[0132] 2) Centrifuge at 8000 rpm for 2 min, and prepare samples from the supernatant precipitate.

[0133] 3. Fermentation

[0134] 3.1 Seed culture

[0135] 1) YPD medium: Dissolve 10 g yeast extract, 20 g peptone, and 20 g glucose in 900 mL of water, bring the volume to 1 L, and autoclave at 115℃ for 30 min.

[0136] 2) Inoculate recombinant Saccharomyces cerevisiae containing the genes for alcohol dehydrogenase and acetaldehyde dehydrogenase into YPD medium at a ratio of 1:50.

[0137] 3) Incubate at 30℃ and 500 rpm for 24 h on a shaker.

[0138] 3.2 50 L Fermentation

[0139] 1) Modified FM2 medium: 42.9 g KH2PO4, 5 g (NH4)2SO4, 1 g CaSO4·2H2O, 14.3 g K2SO4, 11.7 g MgSO4·7H2O, 40 g glycerol, 10 g yeast extract, bring to a final volume of 1 L. Prepare 20 L of medium according to the above formula.

[0140] 2) Feeding: Prepare 10 L of 50% glucose solution and autoclave at 115℃ for 30 min.

[0141] 3) Fermentation process: The culture medium was autoclaved at 121℃ for 20 min, cooled to 30℃, and 80 mL of PTM (2 g CuSO4·5H2O, 0.08 g NaI, 3 g MnSO4·H2O, 0.2 g Na2MoO4·2H2O, 0.02 g H3BO3, 0.5 g CaSO4·2H2O, 0.5 g CoCl2, 7 g ZnCl2, 22 g FeSO4·7H2O, 0.2 g biotin, 1 mL H2SO4, and the volume was adjusted to 1 L). 1 L each of recombinant Saccharomyces cerevisiae containing the genes for alcohol dehydrogenase and acetaldehyde dehydrogenase were inoculated. The fermentation was carried out at 30℃, pH 5.0 (adjusted with 28% ammonia), air flow rate 1 vvm, tank pressure 0.06~0.08 MPa, 100 rpm~150 rpm, and dissolved oxygen controlled above 40%. When dissolved oxygen rapidly rises to above 60%, glucose solution feeding is initiated at a feeding rate of 3 mL / min for 60 h.

[0142] 3.3 Centrifugal Collection

[0143] After the culture is completed, collect the culture medium, which should be about 30 L. Centrifuge at 3000 rpm and 10℃ for 2 min, discard the supernatant, and continue to centrifuge until the culture medium is used up.

[0144] 3.4 Cleaning

[0145] The collected bacterial cells were resuspended in an appropriate amount of ultrapure water, centrifuged at 3000 rpm and 10℃ for 2 min, the supernatant was discarded, and the process was repeated once.

[0146] 3.5 Freeze-drying

[0147] 1) Resuspend the bacterial cells in an appropriate amount of ultrapure water, combine them, pour them into a freeze-drying tray, and freeze at -80℃ for 24 h.

[0148] 2) Pre-freeze dryer, cold well temperature below -40℃.

[0149] 3) Remove the freeze-drying tray and place it into the freeze dryer.

[0150] 4) Freeze-drying process:

[0151] ① -40℃, vacuum degree 5 Pa, 1 h (start vacuum pump);

[0152] ② -35℃, vacuum degree 10 Pa, 1 h;

[0153] ③ -30℃, vacuum degree 10 Pa, 1 h;

[0154] ④ -20℃, vacuum degree 10 Pa, 1 h;

[0155] ⑤ -10℃, vacuum degree 10 Pa, 1 h;

[0156] ⑥ 0℃, vacuum degree 10 Pa, 6 h;

[0157] ⑦ 4℃, vacuum degree 10 Pa, 24 h;

[0158] ⑧16℃, vacuum degree 10 Pa, 30 min (constant temperature range).

[0159] Collect freeze-dried powder.

[0160] Example 2

[0161] The compound yeast single-cell oral capsules are composed of recombinant Saccharomyces cerevisiae freeze-dried powder, pork liver extract powder (Zhejiang Huadi Pharmaceutical Group Co., Ltd. "Animal Liver Protein Peptide" Q / ZHD0015S-2019), vitamin D3, and vitamin E, as detailed below:

[0162] 130 g of recombinant brewer's yeast freeze-dried powder was obtained in Example 1;

[0163] 15 g of dried pig liver extract powder;

[0164] Vitamin D3 30 mg;

[0165] Vitamin E 45 mg;

[0166] 1% talc;

[0167] Starch balance;

[0168] Make 1000 capsules, each weighing 0.4 g.

[0169] Example 3

[0170] The formula for compound yeast single-cell oral capsules is as follows:

[0171] 150 g of recombinant brewer's yeast freeze-dried powder was prepared in Example 1;

[0172] 25 g of dried pig liver extract powder;

[0173] Vitamin D3 48 mg;

[0174] Vitamin E 65 mg;

[0175] 2% talc;

[0176] Starch balance;

[0177] Make 1000 capsules, each weighing 0.4 g.

[0178] Example 4

[0179] The formula for compound yeast single-cell oral capsules is as follows:

[0180] 170 g of recombinant brewer's yeast freeze-dried powder was obtained in Example 1;

[0181] 38 g of dried pig liver extract powder;

[0182] Vitamin D3 65 mg;

[0183] Vitamin E 80 mg;

[0184] 3% talc;

[0185] Starch balance;

[0186] Prepare 1000 capsules, each weighing 0.4 g.

[0187] Comparative Example

[0188] The formula for compound yeast single-cell oral capsules is as follows:

[0189] 150 g of recombinant brewer's yeast freeze-dried powder was prepared in Example 1;

[0190] Vitamin D3 48 mg;

[0191] Vitamin E 65 mg;

[0192] 2% talc;

[0193] Starch balance;

[0194] Make 1000 capsules, each weighing 0.4 g.

[0195] Example 1

[0196] The nutritional components of the capsules prepared in Examples 2-4 and the comparative examples were analyzed. The peptide detection method was performed according to the liver protein peptide assay (enterprise standard number Q / ZHD0029S-2021) of Zhejiang Huadi Pharmaceutical Group Co., Ltd.; the protein detection method was performed according to GB5009.5-2016. The results are shown in Table 3:

[0197] Table 3: Results of Nutritional Component Analysis

[0198]

[0199] Example 2

[0200] The activity of the yeast single-cell oral capsules prepared in Examples 2-4 and comparative examples was determined by the MTT receptor method.

[0201] 1. Reagents

[0202] (1) 0.01 mol / L phosphate buffer (pH 7.3): Take 8.0 g of sodium chloride, 0.2 g of potassium chloride, 1.15 g of disodium hydrogen phosphate and 0.2 g of potassium dihydrogen phosphate, add 1000 mL of ultrapure water to dissolve, sterilize at 121℃ for 20 min and filter to remove bacteria.

[0203] (2) RPMI-1640 medium: Gibco's RPMI-1640 medium (+Gln)

[0204] (3) 10% fetal bovine serum culture medium: Take 45 mL of the above RPMI-1640 culture medium and add 5 mL of the already dispensed Sijiqing fetal bovine serum.

[0205] (4) RPMI-1640 test solution: Gibco's RPMI-1640 medium (-Gln)

[0206] (5) 10% calf serum test solution: Take 45 mL of the above RPMI-1640 test solution and add 5 mL of the already packaged Sijiqing calf serum.

[0207] (6) 0.25% trypsin-EDTA disodium acetate digestion solution

[0208] (7) Thiazol blue (MTT) solution: Dissolve 50 mg of MTT in 10 mL of 0.01 mol / L phosphate buffer, then incubate at 60°C in a water bath to aid dissolution. Filter to remove impurities and store in a cool, dark place. Dosage should not exceed two weeks (wrap in a light-proof bag, black paper, or aluminum foil).

[0209] Preparation of the test solution: Take an appropriate amount of the test sample, dilute it with water to 10 mg / mL, and use RPMI-1640 detection solution to prepare a solution containing 100 μg of peptide per mL.

[0210] The test samples refer to the capsules shown in Examples 2-4 and the comparative examples.

[0211] 2. Measurement

[0212] (1) Place the 1) RPMI-1640 culture medium, 2) 1640 detection solution, and 3) 0.25% trypsin-EDTA disodium acetate digestion solution, which are refrigerated in the refrigerator, into a water bath at 37°C for rewarming.

[0213] (2) After wiping the pipette, pipette tip, 96-well cell culture plate, 0.01 mol / L phosphate buffer (pH 7.3), syringe, disposable 0.22 filter membrane and PBS with 75% ethanol, place them in a clean bench and irradiate with ultraviolet light for 30 min.

[0214] (3) Turn on the CO2 incubator, remove the cell culture flask, and observe cell growth under an inverted microscope. Ideally, the cells should be 80% full at the bottom of the flask (L-02 cells should be cultured in 10% fetal bovine serum medium until the logarithmic growth phase). Discard the culture medium. Wash the cells twice with 0.01 mol / L phosphate buffer (pH 7.3), then digest them with 0.25% trypsin-EDTA-disodium EDTA solution (digestion time approximately 5 minutes, until a mesh-like structure appears at the bottom of the flask). Add 10% fetal bovine serum detection solution to stop the digestion. Transfer the cells to a 15 mL centrifuge tube and centrifuge at 1500 rpm for 3 minutes. Discard the supernatant and dilute with 10% fetal bovine serum detection solution to a concentration of (2.5 × 10⁻⁶) per mL. 4 )~(5×10 4 (The large square in the center of the counting chamber corresponds to sixteen square squares at each of the four corners, with a total of 12-14 cells at each corner.) Spread the cell suspension onto a 96-well cell culture plate, 100 μL per well. Leave 3 wells with 100 μL of 10% fetal bovine serum detection solution as blank controls. Incubate at 37°C in a 5% CO2 saturated water vapor incubator for 3-4 hours to allow the cells to adhere. After 3-4 hours, add 100 μL of the test solution to each well of the sample group. Each batch of test samples is prepared in triplicate. For the cell control group and blank control group, add 100 μL of RPMI-1640 and 100 μL of the detection solution to each well, respectively. Incubate at 37°C in a 5% CO2 saturated vapor incubator for 44 hours. After incubation, remove the culture plate, aspirate the culture medium, and wash each well once with 0.01 mol / L phosphate buffer (pH 7.3). Then add 100 μL of the above phosphate buffer (pH 7.3) and 20 μL of MTT solution to each well and continue incubation for 4 hours. After incubation, discard the culture medium, add 100 μL of dimethyl sulfoxide to each well, and shake gently on a shaker for 10 min. Measure the absorbance (A value) at 570 nm using a microplate reader.

[0215] 3. Calculation:

[0216]

[0217] The results are shown in Table 4:

[0218] Table 4: Cell Viability

[0219]

[0220] *Indicates a highly significant difference compared to the control group, P<0.01;

[0221] #The results showed a significant difference compared to the control group, P<0.05;

[0222] The results showed that the yeast single-cell oral capsules provided in Examples 2-4 could significantly improve cell activity (P<0.05).

[0223] Example 3

[0224] Animal efficacy tests were conducted on Example 3 and the comparative example.

[0225] 1. Grouping and feeding of laboratory animals

[0226] ICR mice were randomly divided into 7 groups: blank control group, alcohol model group, Saccharomyces cerevisiae control group (ADH1B2 / ALDH2-pYX212 without induced expression), experimental group (including high-dose group, medium-dose group, and low-dose group), and comparative group.

[0227] The alcohol group was given 52-degree Hongxing Erguotou (5.8 grams of Hongxing Erguotou per kilogram of body weight) by gavage from Beijing Hongxing Co., Ltd., and an additional 100 µL of physiological saline was administered by gavage.

[0228] The blank control group was given normal saline by gavage, and the gavage volume was the same as that of the alcohol group.

[0229] The control group and the alcohol group were given the same dose of Red Star Erguotou by gavage, and in addition, 100 µL of 1 mg / mL non-active lyophilized powder resuspension (ADH1B2 / ALDH2-pYX212 without induced expression) were given by gavage.

[0230] The experimental group was treated the same as the control group of Saccharomyces cerevisiae. In addition, the experimental group was administered different amounts of the dry powder resuspension of Example 3 by gavage. The high-dose group was 10 mg / mL of the dry powder resuspension of Example 3, the medium-dose group was 1 mg / mL of the dry powder resuspension of Example 3, and the low-dose group was 0.1 mg / mL of the dry powder resuspension of Example 3.

[0231] The control group was treated the same as the Saccharomyces cerevisiae control group, and was additionally administered 1 mg / mL of the control group dry powder suspension by gavage.

[0232] Mice were given free access to sterilized standard rat feed and sterilized drinking water. Weight changes were recorded for each group of mice during the initial cage placement, acclimatization, and treatment phases. During drug administration, the mice's mental state, activity level, diet, coat luster, and any mortality were observed, recorded, and compared among the treatment groups. Details of the experimental groups are shown in Table 5.

[0233] Table 5

[0234]

[0235] 2. Sample Collection and Processing

[0236] After the final gavage treatment, mice in each group were fasted but allowed free access to water. Samples were collected and mice were sacrificed 16 hours later. Serum samples were collected and frozen at -80℃. The left lobe of the liver was removed for tissue sectioning; tissue from the right lobe of the liver was thoroughly homogenized to prepare a 10% liver tissue homogenate.

[0237] 3. Method for determining total antioxidant capacity

[0238] Total antioxidant capacity was determined according to the instructions of the Total Antioxidant Capacity (T-AOC) Test Kit from Beijing Solarbio Science & Technology Co., Ltd.

[0239] 4. Method for determining total superoxide dismutase activity

[0240] Total superoxide dismutase activity was determined according to the instructions of the Superoxide Dismutase (SOD) Activity Assay Kit from Beijing Solarbio Science & Technology Co., Ltd.

[0241] 5. Method for determining malondialdehyde content

[0242] The malondialdehyde (MDA) content was determined according to the instructions of the MDA content detection kit from Beijing Solarbio Technology Co., Ltd.

[0243] 6. Results and Analysis

[0244] The antioxidant indices of mouse liver tissue were compared as shown in Table 6.

[0245] Table 6

[0246]

[0247] (1) Total antioxidant capacity data show:

[0248] Compared with the blank control group, the alcohol group decreased by 36.3% and the low-dose group decreased by 26.2%, with significant differences (P<0.05); although the other groups all had some decreases, the differences were not significant.

[0249] Compared with the alcohol group, the total antioxidant capacity of the control group, low-dose group, medium-dose group and Saccharomyces cerevisiae control group increased to some extent, but the difference was not significant; compared with the alcohol group, the total antioxidant capacity of the high-dose group increased by 56.1%, which was significant (P<0.05).

[0250] (2) Total superoxide dismutase activity data showed:

[0251] Compared with the blank control group, the total superoxide dismutase activity in the alcohol group increased by 25.1%, which was statistically significant (P<0.05). While the other Saccharomyces cerevisiae treatment groups also showed increases, the differences were not statistically significant. Compared with the alcohol group, the control group showed a decrease of approximately 9.8%, the low-dose and medium-dose groups both decreased by approximately 13.7%, the Saccharomyces cerevisiae control group decreased by 7.8%, with no statistically significant difference; the high-dose group showed a decrease of 22.9%, which was statistically significant (P<0.05).

[0252] (3) The results of the malondialdehyde content determination are as follows:

[0253] Compared with the blank control group, the alcohol content increased by 135.2%, the low-dose group by 98.5%, the medium-dose group by 65.3%, the brewer's yeast control group by 80.2%, and the comparative group by 85.8%, with significant differences (P<0.05); the high-dose group only increased by 23.2%, with no significant difference. Compared with the alcohol group, the low-dose group decreased by 15.6%, with no significant difference; compared with the alcohol group, the high-dose group decreased by 47.6% in malondialdehyde content, with a significant difference (P<0.05).

[0254] 7. Conclusion

[0255] The degree of liver damage can be determined by analyzing and comparing liver antioxidant indicators. The most important indicators are total antioxidant capacity and malondialdehyde (MDA) content, which reflects lipid peroxidation. Superoxide dismutase (SOD) is a component of total antioxidant capacity. Analysis of antioxidant levels showed no significant differences between the comparative group, low-dose group, medium-dose group, and the Saccharomyces cerevisiae control group and the alcohol group. Similarly, no significant differences were found between the high-dose group, medium-dose group, and the Saccharomyces cerevisiae control group and the control group. These results indicate that the high-dose group has a significant protective effect. Furthermore, SOD activity increased in the alcohol group, suggesting that alcohol intake has a certain inducing effect on SOD, inducing SOD expression in the liver and promoting oxidative damage repair. Antioxidant data indicate that the high-dose mixture dry powder resuspension has a significant protective effect against acute alcoholic liver injury in mice.

[0256] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An expression carrier, characterized in that, Including expression vector 1 and expression vector 2; The expression vector 1 includes a gene encoding an alcohol dehydrogenase mutant, and acceptable gene elements; The amino acid sequence of the alcohol dehydrogenase mutant is shown in SEQ ID No. 2; The expression vector 2 includes a gene encoding an acetaldehyde dehydrogenase mutant, as well as acceptable gene elements; The amino acid sequence of the acetaldehyde dehydrogenase mutant is shown in SEQ ID No.

4.

2. A combination of recombinant bacterial strains, characterized in that, Including recombinant strain 1 and recombinant strain 2; The recombinant strain 1 includes expression vector 1 as described in claim 1; The recombinant strain 2 includes expression vector 2 as described in claim 1.

3. The recombinant strain combination as described in claim 2, characterized in that, The strains include Saccharomyces cerevisiae.

4. A composition, characterized in that, Including (I) and (II); The (I) includes the recombinant strain combination as described in claim 2; The (II) includes one or more of the following: dried pig liver extract powder, vitamin D3, vitamin E, starch, or talc.

5. The composition according to claim 4, characterized in that, By weight, each 400 g contains: 130 g to 170 g of the recombinant strain combination as described in claim 2 or 3; Pig liver extract powder, 15 g ~ 38 g; Vitamin D3 30 mg ~ 65 mg; Vitamin E 45 mg~80 mg; Talc 1%~3% (w / w); Starch balance.

6. The use of the recombinant strain combination as described in claim 2 or 3, or the composition as described in claim 4 or 5, in the preparation of a product having the following functions, characterized in that, The function is one or more of the following: (a) Enhance cell viability; and / or (b) Improve total antioxidant capacity; and / or (c) Reduce total superoxide dismutase activity; and / or (d) Reduce malondialdehyde content; and / or (e) Promote alcohol metabolism; and / or (f) Improve acute alcoholic liver injury.

7. A microbial preparation, characterized in that, Includes the recombinant strain combination as described in claim 2 or 3, or the composition as described in claim 4 or 5, and acceptable excipients or adjuvants.

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