HANGOVER RELIEVER CONTAINING GLUTATHIONE AND ALDEHYDE DEHYDROGENASE

Abstract

A hangover relieving composition including a dry powder, lysate or extract of yeast that produces glutathione and acetaldehyde dehydrogenase. Further, embodiments relate to a hangover relieving composition containing the dry powder, lysate or extract of Saccharomyces cerevisiae Kwon P-1 KCTC13925BP and Saccharomyces cerevisiae Kwon P-2 KCTC14122BP and Saccharomyces cerevisiae Kwon P-3 KCTC14123BP yeast that simultaneously produce glutathione and acetaldehyde dehydrogenase.

Claims

1-5. (canceled)

6. A hangover relieving composition comprising: glutathione and aldehyde dehydrogenase.

7. The hangover relieving composition of claim 6, wherein the glutathione and aldehyde dehydrogenase are derived from any one selected from a group consisting of Saccharomyces cerevisiae yeast, Saccharomyces cerevisiae Kwon P-1 KCTC13925BP, Saccharomyces cerevisiae Kwon P-2 KCTC14122BP, and Saccharomyces cerevisiae Kwon P-3 KCTC14123BP, or a mixture thereof.

8. A Saccharomyces cerevisiae strain mass culture method, comprising: (a) culturing the Saccharomyces cerevisiae strain in a liquid medium, and (b) culturing the Saccharomyces cerevisiae strain further cultured in the (a) step in a solid medium again.

9. The Saccharomyces cerevisiae strain mass culture method of claim 8, wherein the solid medium is any one selected from a group consisting of rice, barley, wheat, corn, and soybeans, or a mixture thereof.

10. The Saccharomyces cerevisiae strain mass culture method of claim 8, wherein the Saccharomyces cerevisiae strain is selected from a group consisting of Saccharomyces cerevisiae Kwon P-1 (KCTC13925BP), Saccharomyces cerevisiae Kwon P-2 (KCTC14122BP), and Saccharomyces cerevisiae Kwon P-3 KCTC14123BP.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG. 1 is a graph showing changes in blood acetaldehyde content of experimental animals according to administration of a composition of the present invention.

[0047] FIG. 2 is a graph showing changes in blood acetaldehyde content of experimental animals according to administration of the composition of the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0048] Hereinafter, the configuration and effects of the present invention will be described in more detail through the following examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples. Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. The following examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

[Example 1] Preparation of Yeast Lysate Containing Glutathione and ALDH

Example 1-1: Saccharomyces cerevisiae Yeast Fermentation Process Containing Glutathione and ALDH

[0049] The ALDH-containing Saccharomyces cerevisiae yeast seed was fermented and cultured for 24 hours in an incubator at 160 rpm and 30° C. using YPD medium (yeast extract, peptone, and glucose containing medium) in a 200 mL flask, the culture was carried out for 72 hours through a 5 L fermenter (Marado-05D-PS, CNS, Korea). After completion of the culture, the yeast was centrifuged using a high-speed centrifuge (Supra R22, Hanil, Korea).

Example 1-2: Preparation of Yeast Lysate Containing Glutathione and ALDH

[0050] The centrifuged ALDH-containing yeast was frozen in a cryogenic freezer (CLN-52U, Nihon freezer, Japan) for 2 days, and then freeze-dried for 2 days in a freeze dryer (FDU-7006, Operon, Korea). After dissolving 3 g of lyophilized yeast powder in 50 mL phosphate-buffered saline (PBS) containing a protease inhibitor (A32955, Thermo fisher, USA), 10 g of 0.5 mm glass beads for cell disruption (11079105, Biospec) were put into the bead homogenizer (Mixer Mill MM400, Retsch, Germany) for 2 minutes a total of 3 times to disrupt the yeast. After centrifugation using a high-speed centrifuge (Supra R22, Hanil, Korea), only the supernatant was separated and freeze-dried for 2 days with a freeze dryer (FDU-7006, Operon, Korea).

[Example 2] Mass Production of Saccharomyces cerevisiae Strain by Two-Step Fermentation Process

[0051] Saccharomyces cerevisiae KwonP-1 strain (KCTC13925BP) was inoculated into YPD medium containing 2% peptone, 1% yeast extract, and 2% glucose and cultured at 30° C. and fermented at 200 rpm, 1 vvm in a fermentor (Fermentor, Cobiotech) until the OD600 nm value reached 50.

[0052] The recovered cells are mixed with the already sterilized rice fermented powder at a ratio of 10%, the moisture content is adjusted to 60%, and the solid phase is cultured for 2 days at 30° C. and then dried at 50° C. to adjust the final moisture content to 7%, and yeast-fermented rice fermented powder was prepared.

[0053] The fermentation composition of the present invention thus prepared contained a maximum of 600 units/g of ALDH. Considering that the rice fermented powder by the wild-type Saccharomyces cerevisiae yeast strain known to date generally contains about 2 unit/g of ALDH, it was confirmed that the ALDH content of the fermented composition of the present invention was increased by about 300 times.

[0054] Table 1 shows the results of evaluating the acetaldehyde decomposition ability of the compositions (1 to 4) of the present invention prepared by the two-stage fermentation process of the present invention for 5 minutes.

TABLE-US-00001 TABLE 1 Reaction NADPH Suspension Unit/g time (m) (mM) vol. (ml) powder Average One 5 1.732 0.20 693.0 681.4 One 5 1.675 0.20 669.8 2 5 1.722 0.20 688.9 682.2 2 5 1.689 0.20 675.6 3 5 1.770 0.20 707.9 702.1 3 5 1.741 0.20 696.3 4 5 1.639 0.20 655.8 665.7 4 5 1.689 0.20 675.6

[0055] The ALDH coenzyme NAD was added as an enzyme activator to the dry pulverized powder of the fermentation composition of the present invention prepared in this way, and citric acid, magnesium stearate, DL methionine, vitamin C and lactic acid bacteria (Lactobacillus plantium 10 7/g), zinc oxide, and silicon dioxide were added to prepare a hangover reliever of the present invention.

[0056] Through animal testing of the hangover reliever of the present invention, the concentration of acetaldehyde in the blood was measured after alcohol intake, and the hangover reliever of the present invention significantly and rapidly reduced the concentration of acetaldehyde in the blood compared to the conventional hangover reliever.

[0057] In addition, for the human clinical trial of the hangover reliever of the present invention, voluntary clinical trial volunteers were subjected to a genomic test to divide the volunteers into an experimental group, which is an ALDH2 possessing group that is able to decompose aldehydes, and an experimental group, which is genetically deficient in the ability to decompose aldehydes and has a ALDH2*2 mutant gene.

[0058] As a result of the hangover relieving ability test in the human body for 15 hours, significant differences in aldehyde degradation ability were confirmed in both the ALDH2 retention test group and the ALDH2*2 gene mutation test group. The hangover reliever of the present invention was able to efficiently remove acetaldehyde in both experimental groups. In particular, it efficiently removes aldehydes even in the ALDH 2*2 gene mutation experimental group, where it is difficult to degrade aldehydes, it was confirmed that the hangover reliever of the present invention is effective in decomposing aldehydes and relieving hangovers due to the content enhancement of ALDH and Glutathione.

[Example 3] Measurement of Hangover Relief Effect of the Composition of the Present Invention

Example 3-1: Animal Test for Changes in Blood Acetaldehyde Over Time

[0059] Table 2 shows the animal test results on the temporal change of acetaldehyde in blood after ethanol administration.

TABLE-US-00002 TABLE 2 Blood Acetaldehyde (mg/L) 0 hr 1 hr 3 hr 5 hr 8 hr Control group 0 0.03 0.02 0.03 0.02 Ethanol administration 0 0.52 0.49 0.3 0.19 group Ethanol and the 0 0.48 0.33 0.23 0.18 composition administered at 73 mg/kg Ethanol and the 0 0.46 0.24 0.22 0.12 composition 220 mg/kg administered group

[0060] Table 3 shows the results of the animal test of the cumulative amount of aldehydes in the blood (mg/L.Math.hr).

TABLE-US-00003 TABLE 3 Accumulated amount of Decrease acetaldehyde in the blood rate Control group 0.26 Ethanol administration 2.57 group Ethanol and the composition 2 −22% 73 mg/kg administered group Ethanol and the composition 1.27 −51% 220 mg/kg administered group

Example 3-2: Analysis of Ethanol and Isetaldehyde in Blood of Human Clinical Trial Volunteers

[0061] Volunteers for the human clinical trial were selected from 43 healthy adult males between their 20s and 40s who can drink soju based on an average alcohol content of 20 degrees at a time of drinking, and the clinical trial was conducted once a week on Friday at 17:00 for a total of 4 weeks. Clinical trials were conducted through a camp for a total of 15 hours at 8 o'clock the day after admission to the hospital, and a total of 23 patients finally completed the clinical trials due to personal circumstances during the clinical process.

[0062] On the first day of the camp, the amount of change in alcohol concentration and acetaldehyde concentration was measured after drinking 10 glasses of soju, which is a blood alcohol metabolism measurement in each time period, on the second day of the camp, the amount of changes in blood alcohol metabolism was measured after drinking 10 glasses of soju 30 minutes after taking 73 mg/kg of the composition of the present invention, and on the second day of the camp, the amount of change in blood alcohol metabolism was measured after drinking 10 glasses of soju 30 minutes after taking 220 mg/kg of the composition of the present invention.

[0063] In the group taking the composition containing 500 mg/day of the double-fermented dry powder and 1500 mg of fermented rice powder of the present invention, the blood concentration of Acetaldehyde, which is a causative agent of hangovers and a strong carcinogen in the body, was significantly reduced in a dose-dependent manner compared to the alcohol alone group. In addition, the residual amount of blood alcohol was also significantly decreased in a dose-dependent manner with the composition of the present invention.

[0064] Table 4 shows the decrease in blood alcohol concentration of human clinical trial volunteers.

TABLE-US-00004 TABLE 4 Alcohol Alcohol Peak accumulation reduction Decrease Cmax (g .Math. hr/dL) (%) rate (g/L) Alcohol only 30.852  100% 7.583 administration group Ethanol and 29.693 96.2% −3.8% 5.548 composition 73 mg/kg administration group Ethanol and 25.271 85.7% −14.9% 4.18 composition 220 mg/kg administration group

[0065] Table 5 shows the decrease in the residual amount of acetaldehyde in the blood of volunteers in human clinical trials.

TABLE-US-00005 TABLE 5 Residual amount Residue of acetaldehyde reduc- Peak in blood tion Decrease Cmax (mg .Math. hr/dL) (%) rate (mg/dL) Ethanol 13.02  100% 1.65 administration group Ethanol and 9.39 72.1% −27.9% 1.2 composition 73 mg/kg administration group Ethanol and 5.22 68.0% −32.0% 1.3 composition 220 mg/kg administration group

Example 3-3: Test for Confirming Changes in Ethanol and Acetaldehyde According to ALDH Gene Mutation

[0066] For the recruitment of volunteers for the human clinical trial, 43 adult males in their 20s to 40s who can drink soju based on an average alcohol content of 20% at a time of drinking were selected. For a total of 4 weeks, a clinical trial was conducted through a camp for a total of 15 hours at 8 o'clock the day after admission to the hospital, and a total of 23 patients finally completed the clinical trials due to personal circumstances during the clinical process.

[0067] Among them, about 22 people participated in the alcohol metabolism-related genetic test, and obtained consent for the experiment and information use, and a total of 22 people performed three types of genomic test, ADH1B (Alcohol dehydrogenase 1B), ALDH2 (Aldehyde dehydrogenase 2), and CPY2E1 P450, which are involved in alcohol metabolism in vivo, it was confirmed that the blood concentration of Acetaldehyde, a substance that causes hangovers and a strong carcinogen in the body, decreased in a dose-dependent manner in both the ALDH2 non-mutant group and the ALDH2*2 mutant group compared to the alcohol only administration group.

[0068] In the case of the ALDH2*2 gene mutation group, it is known that even a small amount of alcohol shows a very high blood acetaldehyde concentration, the effect of reducing blood aldehydes has never been observed or reported through conventional hangover relieving drinks or conventional hangover relieving foods and drugs. However, in the case of administration of the hangover relieving composition of the present invention, the effect of reducing blood Acetaldehyde in the ALDH2*2 gene mutant group is very remarkable.

[0069] Table 6 shows the alcohol content (g hr/L) of the normal ALDH gene carrying group and the ALDH gene mutant group.

TABLE-US-00006 TABLE 6 ALDH gene Normal ALDH gene mutant group carrying group Ethanol administration 42.55 28.13 group Ethanol and composition 36.13 29.86 73 mg/kg administration group Ethanol and composition 40.87 24.94 220 mg/kg administration group

[0070] Table 7 shows the average blood acetaldehyde content (g hr/L) of the normal ALDH gene carrying group and the ALDH gene mutant group.

TABLE-US-00007 TABLE 7 ALDH gene Normal ALDH gene mutant group carrying group Ethanol administration 10.56 13.47 group Ethanol and composition 8.78 7.57 73 mg/kg administration group Ethanol and composition 8.43 3.92 220 mg/kg administration group

[Example 4] Toxicity Test of the Hangover Relieving Composition of the Present Invention

Example 4-1. Preparation of Laboratory Animals

[0071] As experimental animals, female and male ICR mice (7 weeks old) were received and acclimatized for 7 days. During the acclimatization period, general symptoms were observed, and only healthy animals were used for the test. Feed and water were ingested ad libitum, and group separation was performed so that there were 5 males and 5 females in each group based on the average body weight of about 20 g the day before oral administration.

Example 4-2 Administration of the Hangover Relieving Composition of the Present Invention

[0072] The test substance was prepared by dissolving in physiological saline so that the doses of the test animals were 0, 750, 3000, and 5000 mg/Kg, respectively, based on the content of the yeast lysate containing GSH and ALDH of the present invention. The standard of administration dose complied with the Korea National Toxicology Program (KNTP) toxicity test manual of the Ministry of Food and Drug Safety, and the maximum applied dose 5000 mg/Kg guided by the KNTP manual was applied as the maximum concentration of this experiment. Samples prepared for each group were orally administered once to each test animal, and physiological saline was administered to the normal group (G1).

Example 4-3. Observation and Autopsy

[0073] Symptoms were observed at least once a day from the date of acquisition to the day of autopsy for all animals in the test group, and symptoms were observed for 7 days after oral administration. After the type of symptom observation, an autopsy was performed, and changes in each organ were visually observed at the time of autopsy.

[0074] As a result of a single-dose toxicity test using the yeast lysate containing glutathione and ALDH of the present invention in mice, no mortality was observed for 7 days, and no peculiarities such as weight gain and feed intake, etc. were found at a concentration of up to 5000 mg/kg. Also, no unusual findings were found in the autopsy results after the observation was completed.

[0075] [Accession Number]

[0076] Name of deposit institution: Korea Research Institute of Bioscience and Biotechnology

[0077] Accession number: KCTC13925BP

[0078] Deposit date: 20190822

[0079] Name of deposit institution: Korea Research Institute of Bioscience and Biotechnology

[0080] Accession number: KCTC14122BP

[0081] Deposit date: 20200130

[0082] Name of deposit institution: Korea Research Institute of Bioscience and Biotechnology

[0083] Accession number: KCTC14123BP

[0084] Deposit date: 20200130