Patent Application
20240182915
In accordance with 37 CFR § 1.831, the present specification makes reference to a Sequence Listing submitted electronically as an .xml file named “PKPA2202KRPR1USA.xml”. The .xml file was generated on Jan. 12, 2024, and is 13000 bytes in size. The entire contents of the Sequence Listing are hereby incorporated by reference.
The present invention relates to mutant Saccharomyces cerevisiae KCTC14983BP, KCTC14984BP and KCTC14985BP. Additionally, the present invention relates to an aldehyde dehydrogenase encoded by a gene having more than 98% homology to the gene of SEQ ID NO: 1. Specifically, the present invention relates to an aldehyde dehydrogenase encoded by the gene of SEQ ID NO: 1 which is characterized in that it contains SEQ ID NO: 2.
In addition, the present invention relates to a food composition that suppresses physiological discomfort in the human body caused by various aldehydes derived from endogenous alcohol compounds. Specifically, it relates to a food or pharmaceutical composition that suppresses auto-brewery symptoms caused by endogenous acetaldehyde.
In addition, the present invention relates to a food composition and pharmaceutical composition for suppressing oxidative stress, containing a lysate of any one or a mixture thereof selected from the group consisting of KCTC13925BP, KCTC 14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP, and KCTC14985BP.
The human body experiences oxidative stress due to various causes such as drinking, smoking, taking medicine, strenuous exercise, and physical and mental stress. Accordingly, various types of endogenous aldehydes are produced in cells of human body.
Additionally, reactive oxygen species (ROS) are generated during the energy production process of mitochondria in cells. Various types of endogenous aldehydes are also produced by lipid peroxidation (LPO) in cell membranes by the reactive oxygen species (ROS).
The endogenous aldehydes produced in this way are very reactive. They easily react with surrounding proteins and modify DNA. As a result, the protein may deteriorate, and its unique function may be reduced or completely lost.
The endogenous aldehydes produced in this way are very reactive. They easily react with surrounding proteins and modify DNA. As a result, the protein may deteriorate, and its unique function may be reduced or completely lost.
As a result, the production and accumulation of endogenous aldehydes causes acceleration of cellular aging or is a root cause of cancer, diabetes, cardiovascular disease, and neurodegenerative diseases.
Various alcohols administered by drinking, taking medicines, etc. are the biggest cause of increasing endogenous aldehydes in the human body. For example, when drinking, 80%-90% of the alcohol undergoes a two-stage enzymatic metabolic decomposition.
In the first stage, alcohol is converted to acetaldehyde (Ach), a toxic metabolic intermediate, by alcohol dehydrogenase (ADH). In the second step, acetaldehyde (Ach) is detoxified into acetic acid by the action of aldehyde dehydrogenase (ALDH) [
During this in vivo alcohol metabolism process, excess acetaldehyde (Ach) generated due to genetic deficiency or excessive drinking directly modifies surrounding proteins or DNA. As a result, it acts as a toxic substance that can cause cancer. The present invention relates to an aldehyde dehydrogenase enzyme that detoxifies endogenous acetaldehyde to prevent it from acting as a toxic substance.
Meanwhile, even in people who do not drink alcohol at all, ethanol can be produced in their bodies by intestinal microorganisms. This endogenous ethanol is also converted to acetaldehyde by ethanol decomposition enzyme. The production of acetaldehyde causes physiological discomfort like to those seen in hangovers caused by drinking, resulting in auto-brewery syndrome (ABS) (Malik F, et al. “Case report and literature review of “auto-brewery syndrome: probably an underdiagnosed medical condition” BMJ Open Gastro 2019).
Auto-brewery syndrome is caused by endogenous ethanol fermentation. The so-called “Gut Fermentation Syndrome” occurs, which shows symptoms of an alcohol hangover even in case that the person did not drink alcohol at all.
According to data from the U.S. National Library of Medicine and Makati Med Hospital, intestinal fermentation syndrome is a disease that occurs when ethanol is spontaneously produced within the body by fungi or bacteria in the gastrointestinal, oral, and urinary systems. Even though a person did not drink alcohol, the action of yeast inside the human body produces endogenous alcohol, showing symptoms of an alcohol hangover. It is caused by alcoholic fermentation of various strains of yeast and various bacteria in the human intestine.
Yeast in the intestines produces carbon dioxide and ethanol during the digestion of food, so even if patients with auto-brew syndrome do not consume alcohol, they experience hangover symptoms due to acetaldehyde produced during the decomposition of alcohol in body.
Although the production of endogenous ethanol is part of the normal digestive process, intestinal fermentation symptoms are caused by yeast or bacteria producing alcohol in the human body. This is mainly found in people with diseases such as diabetes, obesity, and Crohn's disease, but can also occur in healthy people. People with liver dysfunction, such as chronic intestinal obstruction, gastrointestinal paralysis, non-alcoholic fatty liver disease, or non-alcoholic hepatitis, may also experience auto-brewery symptom.
Patients with auto-brewing syndrome may show various symptoms such as vomiting, belching, chronic fatigue syndrom, auto-brewery syndrome, dizziness, disorientation, fainting, irritable bowel symptoms, runny nose, cough, and sinusitis. Chronic fatigue syndrome can lead to health problems such as anxiety, depression, and decreased productivity.
Meanwhile, an increase in acetaldehyde in the human body directly or indirectly causes lipid peroxidation (LPO). This additionally promotes the production of various endogenous harmful aldehydes such as malondialdehyde (MDA) and nonenal (4-Hydroxy nonenal, 4-HNE).
Like acetaldehyde, which is classified as a primary carcinogen by the International Agency for Research on Cancer (IARC) and the World Health Organization (WHO), nonenal and malondialdehyde also modify proteins and cells, and cause cancer. 4-HNE was found to be a biomarker involved in the development of Alzheimer's disease (AD), cataracts, atherosclerosis, diabetes, and cancer.
Malondialdehyde binds to deoxyadenosine or deoxyguanosine in DNA and permanently modifies DNA. In other words, this is a substance that causes cancer. The National Cancer Institute (NCI Thesaurus, NCIt) classifies malondialdehyde as a strong endogenous mutagenic substance and uses it as a biomarker for cardiovascular disease and fatigue.
The human body maintains various life activities by generating energy from mitochondria in cells. In this way, reactive oxygen species (ROS) are inevitably generated during mitochondrial energy production or conversion. This causes a lipid peroxidation (LPO) reaction that destroys the lipid membrane. Through this lipid peroxidation reaction, aldehyde substances such as nonenal (4-HNE), malondialdehyde (MDA), and acetaldehyde (Ach) are produced and accumulated in cells.
Modified proteins such as malondialdehyde-acetaldehyde adducts (MAA) and malondialdehyde-lysine adducts (M-lys) are formed through a chain reaction between endogenous aldehydes and proteins. They are accumulated in cells and increase oxidative stress in the human body [
The increase in oxidative stress disrupts the smooth energy metabolism process in mitochondria, further increasing aldehyde substances such as methylglyoxal (MG) and advanced glycation end products (AGEs) in cells. As a result, intracellular accumulation of aldehyde substances is accelerated.
In this way, when the reactive aldehydes such as HNE and MDA produced by lipid peroxidation (LPO) due to increased free radicals and oxidative stress, or aldehyde substances such as glyceraldehyde-3-phosphate (GA3P), an intermediate in glycolysis, are excessively produced and accumulated in cells, cytotoxicity occurs.
Intracellular accumulation of free radicals or reactive aldehydes weakens intracellular antioxidant defense systems such as glutathione. Disruption of energy metabolism and accumulation of unfolded proteins (UP) ultimately cause an increase in endoplasmic reticulum stress (ER stress). As a result, cytotoxicity occurs, which accelerates aging and triggers various diseases.
Meanwhile, monoamine substances that perform various physiological functions, such as Dopamine (DA), serotonin (5-HT), norepinephrine (NE), epinephrine (Adr), gamma-Aminobutyric acid (GABA), and histamine, exist in the human body.
By the systematic action of monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), catechol-O-methyl transferase (COMT), and alcohol dehydrogenase (ADH), The amine (—NH2) group of monoamine substances is converted to an aldehyde (—CHO) group. The aldehyde group thus converted is finally converted to an acid (—CO2H) group to complete the in vivo metabolism of monoamine substances [
Dopamine (DA) is the raw material for epinephrine (Adr), which regulates the autonomic nervous system. Dopamine is a representative neurotransmitter involved in the development of Parkinson's disease (PD).
L-Dopa, produced from L-phenylalanine, is converted to dopamine (DA) in the dopaminergic neurons of the substantia nigra compacta (SNpc), a specific part of the brain, by the action of enzymes.
Dopamine (DA) thus secreted is metabolized through non-enzymatic reaction where it is converted into neuro-melanin or quinone through autooxidation by radicals. In addition, dopamine may be metabolized by the action of monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), and catechol methyltransferase (COMT) enzymes [
As explained above, the dopamine (DA) metabolic process is divided into two metabolic pathways depending on the order of enzymes acting. In the metabolic pathway where monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), and catechol methyltransferase (COMT) act sequentially, dopamine is converted sequentially to 3,4-dihydroxyphenylacetaldehyde (DOPAL), 3,4-Dihydroxyphenylacetic acid (DOPAC), and homo vanillic acid (HVA).
In the metabolic pathway where catechol methyl transferase (COMT), monoamine oxidase (MAO), and aldehyde dehydrogenase (ALDH) act sequentially, dopamine is sequentially converted to 3-methoxytyramine (3-MT), 3-methoxy-4-hydroxy phenylacetaldehyde (MOPAL) and homo vanillic acid (HVA). Through these two metabolic pathways, dopamine is ultimately converted into homo vanillic acid (HVA).
Dopamine is also metabolized into norepinephrine (NE) and epinephrine (Adr), which are hormones used to regulate the autonomic nervous system. In this metabolic process, dopamine is metabolized in the order of 3,4-Dihydroxyphenylglycolaldehyde (DOPEGAL), 3,4-Dihydroxymandelic acid (DOMA) and 4-Hydroxy-3-methoxymandelic acid.
In addition, dopamine is converted sequentially into 3-methoxynorepinephrine (methoxy NE), 4-Hydroxy-3-methoxyphenyl glycolaldehyde (MOPEGAL) and 3-methoxy-4-hydroxymandelic acid by the sequential actions of catechol methyltransferase (COMT), MAO, and aldehyde dehydrogenase (ALDH).
As described above, through the sequential enzymatic actions of monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), and catechol methyl transferase (COMT), the amine (—NH2) functional group of dopamine is converted to aldehyde (—CHO) and finally converted to acid (—CO2H).
Meanwhile, serotonin (5-HT) produced from tryptophan (L-Tryptophan) is closely related to mental health such as learning and sleep. Serotonin is also known as a representative neurotransmitter. Serotonin is metabolized sequentially to 5-Hydroxyindole acetaldehyde (5-HIAL) and 5-Hydroxyindole acetic acid (5-HIAA) through the sequential enzymatic actions of monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), and catechol methyltransferase (COMT).
Serotonin is converted to melatonin by the action of acetyl transferase (N-Acetyl transferase) and catechol methyl transfer enzyme (COMT). Melatonin is changed to 5-methoxyindole-3-acetaldehyde (5-MIAL) by the action of monoamine oxidase (MAO). 5-MIAL thus produced is metabolized into acid by the action of aldehyde dehydrogenase (ALDH) [
Also, 5-Hydroxyindoleacetaldehyde (5-HIAL), produced during serotonin (5-HT) metabolism, has been reported to cause modification of alpha-synuclein (α-Syn) and production of alpha-synuclein oligomers. (Jinsmaa et al. 2015).
When drinking alcohol, normal metabolism of serotonin is disturbed because serotonin binds to alcohol-metabolizing enzymes. As a result, serotonin aldehyde (5-HIAL) is converted to 5-hydroxytryptopol (5-HTOL) and accumulates in the cranial nerves (Shibata et al. 2014).
In the process of metabolizing alcohol into acetic acid, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are rapidly consumed. A deficiency of aldehyde dehydrogenase (ALDH), which should be used in the metabolism of serotonin, may occur.
Due to this lack of aldehyde dehydrogenase (ALDH), serotonin aldehyde (5-HIAL), a metabolite of serotonin (5-HT), accumulates without being decomposed normally, resulting in a toxic effect that deforms proteins in brain nerve cells.
To alleviate the toxic effects of 5-HIAL, an abnormal metabolic pathway is activated to rapidly convert 5-HIAL into 5-hydroxy tryptophol (5-HTOL).
After the decomposition of alcohol in the body is completed, the deficiency of aldehyde dehydrogenase is resolved and the normal metabolic pathway of serotonin is restored, 5-hydroxytryptopol (5-HTOL) is converted back into serotonin aldehyde (5-HIAL) and metabolized through normal metabolism of serotonin.
Due to the toxic effects during the alcohol decomposition process, oxidative stress has already increased, and the function of alcohol dehydrogenase (ADH) has been significantly reduced. Even after alcohol is metabolized in the body, 5-hydroxytryptopol (5-HTOL) accumulates in the human body without being converted into serotonin aldehyde (5-HIAL).
With alcohol consumption, 5-hydroxytryptopol (5-HTOL) abnormally increases in brain nerve cells due to disturbance of the serotonin metabolic pathway due to lack of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) (Shibata et al. 2014).
It had been found that, due to this abnormality in serotonin metabolism, 5-hydroxytryptopol (5-HTOL) increases in the liver, ileum, and spleen, weakening the function of the relevant organs and causing disease. It is becoming.
In conclusion, serotonin aldehyde (5-HIAL) itself is a toxin that causes Parkinson's disease. Additionally, serotonin aldehyde (5-HIAL) distorts the serotonin metabolic pathway and increases modified proteins, causing various diseases such as abnormalities in liver function.
Meanwhile, GABA is converted to succinic semialdehyde (SSA) by monoamine oxidase (MAO), which may act as a toxic aldehyde to nerve cell [
Succinic semialdehyde (SSA), an endogenous aldehyde, is a representative cytotoxic substance that accumulates when the function of aldehyde dehydrogenase (ALDH) is weakened. This is a toxic substance that induces the production and accumulation of gamma hydroxybutyric acid (GHB), which weakens physical functions such as liver function, speaking, and walking. (Buzzi, Andrea et al. 2006).
Meanwhile, histamine is a substance involved in allergic reactions and inflammation. Histamine is one of the substances secreted by the human body for defense against external stimuli (stress). Histamine is a substance secreted in the antigen-antibody response of immune cells such as basophils and mast cells, causing uncomfortable symptoms such as bronchoconstriction (allergic asthma), capillary dilatation, runny nose, and edema.
Histamine is also metabolized by an organized enzyme system involving monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), and histamine-N-methyl transferase (HNMT). Histamine is converted to imidazole acetaldehyde, imidazole acetic acid, and finally imidazole acetic acid riboside [
Histamine is also converted into N-Methyl histamine, N-Methylimidazole acetaldehyde, and N-Methylimidazole acetic acid, sequentially by the action of the enzyme system of histamine-N-methyltransferase (HNMT), MAO and aldehyde dehydrogenase.
In the metabolic process of histamine, the amine group (—NH2) is metabolized to aldehyde (—CHO) and acid (—CO2H) through a detoxification process by monoamine oxidase (MAO), aldehyde dehydrogenase (ALDH), etc. [
Imidazole acetaldehyde, which may be produced when histamine metabolism is abnormal, causes various diseases through a more powerful toxic effect than histamine. The human body is designed to quickly detoxify secreted histamine using an enzyme system to minimize damage to the body.
When an excessively large amount of histamine is secreted at once or when histamine metabolism is abnormal due to a weakened function of aldehyde dehydrogenase (ALDH), the human body reacts excessively to antigens, causing discomfort and shows abnormal symptoms. These phenomena are called histamine intolerance (HIT) or allergic symptom.
Imidazole acetaldehyde or N-methylimidazole acetaldehyde produced from histamine by the action of monoamine oxidase (MAO), should be converted to acid through the action of aldehyde dehydrogenase (ALDH).
When the function of aldehyde dehydrogenase (ALDH) is weakened, aldehyde metabolites originated from histamine have a toxic effect in cell and cause allergic symptoms or histamine hypersensitivity (HIT). In severe cases, Imidazole acetaldehyde, a monoamine aldehyde produced from histamine, disrupts the immune system, and increases the toxic effect of histamine. It may cause chronic itching, psoriasis, atopy, and asthma.
As discussed above, various monoamines such as dopamine (DA), serotonin (5-HT), norepinephrine (NE), epinephrine (Adr), gamma-Aminobutyric acid (GABA) and histamine which act as hormone or neurotransmitter (monoaminergic neurotransmitter) in the human body, may be converted to 3,4-dihydroxyphenylacetaldehyde(DOPAL), 3-methoxy-4-hydroxyphenyl acetaldehyde (MOPAL), 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL), 3-methoxy-4-hydroxyphenylglycolaldehyde(MOPEGAL), 5-Hydroxyindoleacetaldehyde (5-HIAL), 5-methoxyindole-3-acetaldehyde (5-MIAL), Imidazole acetaldehyde and N-Methylimidazole acetaldehyde, by the action of monoamine oxidase.
When aldehyde dehydrogenases in human body do not function normally, various aldehydes produced in the body act as toxic substances to the human body. As a result, various pathological phenomena occur in the human body and aging is accelerated.
There is an urgent need for the development of food or pharmaceutical compositions that may quickly oxidizes and detoxifies various endogenous aldehydes, suppressing various problems caused by accumulation of endogenous aldehydes in the body.
Despite various studies as listed above, a food composition or pharmaceutical composition suppressing an increase of oxidative stress, auto-brewery symptom, chronic itching, psoriasis, atopy, and asthma caused by accumulation of endogenous aldehydes in the body through the detoxification of the various endogenous aldehydes, has not yet been developed.
The basic object of the present invention is to provide mutant Saccharomyces cerevisiae KCTC14983BP, mutant Saccharomyces cerevisiae KCTC14984BP and mutant Saccharomyces cerevisiae KCTC14985BP which can produce aldehyde dehydrogenase.
In addition, the primary object of the present invention is to provide food composition containing aldehyde dehydrogenase encoded by a gene with more than 98% homology to the gene of SEQ ID NO: 1 including SEQ ID NO: 2 which promotes decomposition of endogenous aldehyde produced by oxidation of alcohol or an endogenous amine compound.
Another object of the present invention is to provide a food composition and pharmaceutical composition for reducing oxidative stress of human body, which comprises aldehyde dehydrogenase contained in lysate of any one or a mixture thereof selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP KCTC14983BP, KCTC14984BP and KCTC14985BP.
Still yet another object of the present invention is to provide a food composition and a pharmaceutical composition for the prevention of auto-brewery symptom which comprises aldehyde dehydrogenase contained in lysate of any one or a mixture thereof selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP KCTC14983BP, KCTC14984BP and KCTC14985BP.
Still yet another object of the present invention is to provide a food composition and a pharmaceutical composition for the prevention of symptoms of various pathologies caused by endogenous aldehydes, which comprises aldehyde dehydrogenase contained in lysate of any one or a mixture thereof selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP KCTC14983BP, KCTC14984BP and KCTC14985BP.
The objects of the present invention as described above can be accomplished by providing a food composition and a pharmaceutical composition containing aldehyde dehydrogenase that can rapidly decompose endogenous aldehydes contained in lysate of any one or a mixture thereof (hereinafter abbreviated as KARC) selected from the group consisting of KCTC13925BP, KCTC14122BP, KCTC14123BP KCTC14983BP, KCTC14984BP and KCTC14985BP.
Another object of the present invention can also be achieved by providing a food composition or a pharmaceutical composition for the prevention of auto-brewery symptom, containing lysate of any one or a mixture thereof selected from the group consisting of Saccharomyces Cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP.
KARC, a dried powder of lysate of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP, KCTC14983BP, KCTC14984BP and KCTC14985BP, which comprises aldehyde dehydrogenase encoded by SEQ ID NO: 1.
The food composition and the pharmaceutical composition of the present invention shows the effect of suppressing oxidative stress in the human body and suppressing auto-brewery symptoms.
Ethanol or ethanol derivatives (2-subustituted ethanol, R—CH2CH2—OH) are in vivo reversibly converted to acetaldehydes derivatives (R—CH2—CHO) by alcohol dehydrogenase (ADH). Acetaldehydes derivatives, highly toxic substances are irreversibly converted to relatively non-toxic Acetic acid derivatives (R—CH2—CO2H).
Endogenous monoamines (R—C2H4—NH2) are irreversibly converted to highly toxic acetaldehydes (R—CH2—CHO) by monoamine oxidase (MAO) and to acetaldehydes by aldehyde dehydrogenase, ultimately detoxified to acetic acids (R—CH2—CO2H) as same as alcohol metabolism.
In
By monoamine oxidase, dopamine is also converted via norepinephrine (NE) into DOPANAL as like dopegal and mopegal, a known toxic substance, which is finally decomposed to be acid compound by aldehyde dehydrogenase.
In addition, dopamine metabolism does not proceed well for various reasons, such as a decrease on ALDH, to increase DOPANOL (dopamine inducing alcohol) such as DOPOL via DOPANAL in vivo. The reason is that DOPANAL is not converted to less toxic acid compounds.
It is known. Due to the toxicity of DOPANAL (dopamine inducing aldehyde), it is temporarily converted into DOPANOL (dopamine inducing alcohol), a relatively less toxic alcohol and stored. When dopamine metabolism returns to its original state, DOPET, a representative DOPANOL, stored in the body, is metabolized and decomposed into acid through the activation of alcohol dehydrogenase and aldehyde dehydrogenase, which are alcohol metabolism enzymes.
Despite the existence of various dopamine enzymatic metabolism pathways, when enzymatic dopamine metabolism is not progressive well, dopamine is metabolized through a non-enzymatic reaction in which it is spontaneously converted to quinone derivatives by reactive oxygen species (ROS) and then changed to neuro-melanin. In this case, it is also known to cause various diseases due to destruction of homeostasis by rapid changes in melamine distribution.
The monoamine, neurotransmitter as like dopamine (DA), serotonin (5-HT), GABA, and histamine have a common structural structure of two carbon chains and one amine (R—CH2—CH2—NH2). It is oxidized by the monoamine oxidase (MAO) enzyme and converted into endogenous aldehydes (—CHO) such as DOPAL, 5-HIAL, SSA, 4-Imidazole acetaldehyde, and 1-Methylimidazole acetaldehyde, thereby binding and denaturing surrounding proteins, In result the accumulation of denatured proteins within the endoplasmic reticulum acts as a cytotoxic agent to induce cell death. [
In animal experiments in which blood malondialdehyde [
When Parkinson's disease was induced in animals using rotenone, dopamine secretion decreased. Dopamine breakdown metabolism was abnormally suppressed, resulting in a sharp decrease in the production of DOPAC and HVA, and an increase in DOPET, an abnormal metabolite. In the KARC administration group of the present invention, DA, DOPAC, and HVA increased and DOPET, an abnormal metabolite of dopamine, decreased. It is assumed that the KARC of the present invention restores normal dopamine secretion and in vivo dopamine decomposition metabolism.
In tests confirming the reduction of endogenous blood acetaldehyde in the human body [
[
In [
When acetaldehyde was treated with KARC for 1 hour, acetaldehyde, a known for representative endogenous aldehyde and carcinogen, was oxidized 100% not only at 30° C. but also at 37° C. [
Glyoxal, a representative aldehyde produced during energy metabolism in vivo, was reduced by 20.4% by 1 hour at 30° C. and 25.3% by 3 hours by KARC treatment. It also decreased by 23.8% at 1 hour and 23.8% at 3 hours at 37° C. [
In [
When treated with KARC, trans-cinnamaldehyde was reduced by 35.9% in 1 hour and 97.4% in 3 hours at 30° C., and converted to 82.4% in 1 hour and 99.6% in 3 hours at 37° C. [
When treated with KARC, benzaldehyde decreased by 12.2% in 1 hour and 32.0% in 3 hours at 30° C., and converted to 57.4% in 1 hour and 97.1% in 3 hours at 37° C. [
In [
DOPAC was increased at 6 minutes. Therefore, it was confirmed that KARC oxidizes DOPAL and converts it into DOPAC.
Hereinafter, the method for producing dry powder of KARC of the present invention, the lysate of Saccharomyces cerevisiae KCTC13925BP, KCTC14122BP, KCTC14123BP KCTC14983BP, KCTC14984BP, KCTC14985BP, will be described in more detail.
These examples are for illustrative purposes only of compositions that can achieve the purpose of the present invention, and therefore, the scope of the present invention is not limited only to the compositions described in the following examples.
In the present invention, each suspension of makgeolli (traditional Korean wines) was prepared by mixing various types of makgeolli with a 0.9% NaCl solution. The makgeolli suspension was stirred at 200 rpm for 1 hour. The supernatant containing the yeast wild strain was diluted with YPD yeast extract peptone dextrose broth) medium. The diluted solution was prepared to be 10−6 times the original solution.
The diluted solution was smeared on YPD agar medium. The agar medium was statically cultured at 30° C. under aerobic conditions for one week. Saccharomyces cerevisiae was primary screened based on morphological characteristics of colonies, growth characteristics at YM medium and microscopic observation.
The ALDH activity and glutathione content of screened Saccharomyces cerevisiae were measured. Parent strain was selected based on ALDH activity and glutathione production.
Acetaldehyde reacted with Dinitrophenylhydrazine (DNPH) to form acetaldehyde-hydrazone (Ach-DNPH) compound. Ach-DNPH compounds were detected at 360 nm by HPLC equipped with a C18 column. The amount of aldehyde reduced by the decomposition reaction by aldehyde dehydrogenase ALDH) was quantified through the amount of the detected Ach-DNPH compound.
The enzyme reaction was carried out at 30° C. by adding 10 ul of the yeast lysate to 990 ul reaction mixture [50 mM potassium phosphate buffer (pH 8.0), 1.5 mM acetaldehyde and 3 mM NADP+]. After the enzyme reaction was completed, 50 ul of 10 mM DNPH was added to induce the formation of Ach-DNPH. Ach-DNPH formation proceeded at 22° C. for 1 hour.
Ach-DNPH formation was terminated by addition of 3M sodium acetate (pH 9). The Ach-DNPH compound formed was separated by adding twice volume of acetonitrile. The separated Ach-DNPH compound (in ACN) was analyzed by injection into HPLC.
The concentration of the Ach-DNPH compound was analyzed at a wavelength of 360 nm by setting HPLC under the condition of developing a mobile phase (acetonitrile, water) on a C18 column at a rate of 1 ml/min. The area value of the chromatogram obtained as a result of HPLC was converted using the material standard curve of aldehyde-DNPH (Sigma-Aldrich) to quantify the concentration of the Ach-DNPH compound. The reduced concentration of Ach-DNPH per minute, 1 mM, was calculated as 1 unit of ALDH. The activity of ALDH was standardized as Unit/mg-protein.
Yeast cells were harvested by centrifuging 1 ml of Saccharomyces cerevisiae culture medium. A suspension was prepared by adding 1 ml of water to the harvested yeast cells. Glutathione was extracted by stirring the suspension at 1,000 rpm at 85° C. for 2 hours. The suspension was centrifuged to remove yeast cells, and the supernatant was filtered through a 0.22 μm filter to obtain a sample containing glutathione.
The concentration of glutathione in the sample was analyzed by HPLC (Shimazu LC-20AD) equipped with a C18 column. The concentration of glutathione was analyzed at a wavelength of 210 nm under conditions in which the mobile phase (2.02 g/L Sodium 1-heptanesulfonate monohydrate, 6.8 g/L Potassium dihydrogen phosphate, pH 3.0, methanol mixture) was developed at a rate of 1 ml/min. The area value of the chromatogram obtained as a result of HPLC was analyzed using the standard curve of glutathione.
ALDH activity and glutathione content were analyzed for 200 different types of yeast obtained from Korean makgeolli. The 10 types of yeast listed in [Table 1] had higher ALDH activity or glutathione production ability than other yeasts.
The ALDH activity of Yeast #97 was 0.10 Unit/mg-protein, the second highest overall. The glutathione content of Yeast #97 was 0.42%, the highest among all of yeast 97 was selected as the parent strain and a mutation induction procedure was performed.
Identification was performed to confirm the exact species of the wild-type parent strain (Yeast #97, Wild-type yeast). To ensure sufficient yeast cells for DNA extraction, only colonies of a single yeast were plated on YPD agar medium. DNA was extracted using a Genomic DNA prep kit (HiGene™, BIOFACT Co., Ltd., Daejeon, Korea) according to the manufacturer's instructions.
To amplify rRNA gene on ITS region of the yeast, polymerase chain reaction (PCR) was performed on yeast chromosomal DNA using the ITS5 (forward) and ITS4 (reverse) primers. DNA sequencing of PCR result was analyzed.
The DNA sequence of the parent strain was isolated using the Bioedit program. The reverse strand of the PCR result was converted into a paired base sequence through a reverse completion process.
It was confirmed that the sequence of the forward strand matched the paired sequence of the reverse strand by the Cluster X program. The parent strain which was matching the sequence information confirmed through the above experimental process was identified by using the BLAST database provided by the U.S. National Center for Biotechnology Information (NCBI). As a result of identification, it was found that rRNA in the ITS of the parent strain was 100% identical to that of saccharomyces cerevisiae.
The mutation induction process for the wild-type Saccharomyces cerevisiae parent strain was conducted according to the method described in U.S. patent application Ser. No. 17/176,365.
To induce mutations in the yeast parent strain, wild yeast strains that produce both ALDH and glutathione were treated with ethyl methane sulfonate (EMS) or nitrosoguanidine (NGD). Yeast strains in which mutations were induced were exposed to various concentrations of methylglyoxal. A mutant strain with excellent adaptability to methylglyoxal was selected. Selected yeast strains were exposed to various concentrations of lysine. A mutant strain with excellent adaptability to lysine was selected. Thirty mutant strains with excellent adaptability to methylglyoxal and lysine were obtained. Each of the 30 yeasts was evaluated through five characteristics: growth curve, ALDH activity, ADH activity, coenzyme content, and glutathione content.
Saccharomyces cerevisiae is a crab tree positive microorganism and produces ethanol simultaneously with growth under aerobic conditions. Cultivating yeast with high yields requires Saccharomyces cerevisiae with high ethanol tolerance.
YPD media with different ethanol concentrations (no ethanol, 5%, 7%, and 10%) were prepared. Culture medium of Saccharomyces cerevisiae(yeast) adjusted to OD=1 at 660 nm was prepared. Each mixture of the prepared YPD medium and yeast culture medium was diluted at a ratio of 99:1. Finally, YPD media containing yeast with four different concentrations of alcohol were prepared. Each YPD medium mixed with yeast was cultured with shaking at 30 #C and 200 rpm. The growth curve ofthe mutant strain was measured every 3 hours for 48 hours. The growth curve of each mutant strains are evaluated through three characteristics: time (or period) of lag phase, specific growth rate (OD660 nm/hr) of exponential phase, and maximum density (OD660 nm).
The higher concentration of ethanol in YPD medium, the longer the time taken for the lag phase. The maximum density and specific growth rate decreased. As a result of comparing the maximum density of mutant strains at low concentration (ethanol 5%) and high concentration (ethanol 10%), it was found that in the case of nine mutant strains, 50% of growth was even maintained at high concentration compared to growth at low concentration. The growth characteristics of the nine mutant strains that distinguished them from other strains were a short lag phase and a high specific growth rate.
The activity of alcohol dehydrogenase (ADH) was measured by adding 10 μl of yeast lysate to 990 μl of the reaction mixture with the composition of 50 mM potassium phosphate buffer (pH 8.0), 2 mM NAD+ and 1% ethanol. The activity of aldehyde dehydrogenase (ALDH) was measured by adding 10 μl of yeast lysate to 990 μl of the reaction mixture with the composition of 50 mM potassium phosphate buffer (pH 8.0), 3 mM NAD+ and 1.5 mM acetaldehyde. The enzymatic reaction of ADH and ALDH was carried out at 30° C. for 5 minutes, and the concentration of NAD(P)H produced as a result of the enzyme reaction was measured through absorbance at 340 nm.
The enzyme activities of nine mutant strains (K-1 to K-9) selected in the present invention were measured. The ADH activity of the mutant strain was a minimum of 382.69 units/g and a maximum of 975.29 units/g. The ADH activity of the mutant strain increased at least 5.1 times and up to 13.1 times compared to the type strain (reference yeast, Saccharomyces cerevisiae KCTC7296). The ALDH activity of the mutant strain was a minimum of 15.23 unit/g and a maximum of 72.16 unit/g. The ALDH activity of the mutant strain increased by at least 5.3 and up to 24.9 times compared to the enzyme activity of the type-strain.
Six mutant strains (K-1, 4, 6, 7, 8, and 9) showed similar increase rate of enzyme activity of ADH and ALDH compared to the type strain. The enzyme activity of ALDH in the three mutant strains (K-2, 3, and 5) was 18.3, 23.2 and 24.9 times higher, respectively, compared to the type-strain. The enzyme activity of ADH in the three mutant strains (K-2, 3, and 5) was 9.7, 11.6, and 13.1 times higher respectively, compared to the type-strain. The rate of increase in enzyme activity of ALDH for the three mutant strains (K-2, 3, and 5) was twice as high as that of ADH.
The present inventors named three novel mutant strains (K-2, 3, and 5) adapted to increase aldehyde dehydrogenase (ALDH) activity as PicoYP, PicoYP-01, and PicoYP-02, respectively. The three novel mutant strains were deposited at the Korea Research Institute of Bioscience and Biotechnology's Biological Resources Center and were assigned the deposit numbers of KCTC14983BP, KCTC14984BP, and KCTC14985BP, respectively.
NADtotal and NADPtotal in lysates extracted from mutant strains were measured with NADH/NAD+ assay kit and NADPH/NADP+ assay kit, respectively. NAD(P) in the sample was converted to NAD(P)H using NAD(P) cycling buffer and NAD(P) cycling enzyme mix. The chromophoric test reaction was induced with NAD(P) developer measured as absorbance at 450 nm. The chromophoric test reaction was measured as absorbance at 450 nm. The absorbance of the samples was plugged into the equation corresponding to the standard curve, and the NAD(P) total was calculated in the yeast lysate.
The coenzyme content of nine mutant strains (K-1 to K-9) selected in the present invention was measured. The NADtotal of the mutant strains had a minimum of 126 nmole/g and a maximum of 195 nmole/g. The NADtotal of the mutant strain increased at least 7.3 times and up to 10.8 times compared to the type-strain. The NADPtotal content of the mutant strain was a minimum of 2.4 nmole/g and a maximum of 5.8 nmole/g. The NADP total content of the mutant strain increased at least 11.4 times and up to 27.6 times compared to the type-strain.
In the six mutant strains (K-1,4,6,7,8,9), the increase rate of NADPtotal was less than twice the increase rate of NADtotal. The NADPtotal content increase rates of the three novel mutant strains (PicoYP, PicoYP-01, and PicoYP-02) were 25.7, 22.9, and 27.6 times, respectively. The NAD total content increase rates of the three novel mutant strains were 10.8, 9.9, and 11.3 times, respectively. The NADPtotal increase rate of the three novel mutant strains was more than twice the NADtotal increase rate.
The glutathione content of the nine mutant strains was measured in the same manner as Example 1-2. The glutathione content of the mutant strains ranged from a minimum of 0.85% to a maximum of 1.05%. The glutathione content of the mutant strain increased at least 2.7 times and up to 3.3 times compared to the type strain. In three novel mutant strains (PicoYP, PicoYP-01, PicoYP-02), the increase rate of ALDH activity and coenzyme content were higher compared to others.
The three novel mutant yeasts (PicoYP, PicoYP-01, PicoYP-02) had similar glutathione production abilities to the existing deposited strains (Kwon P-1, Kwon P-2, Kwon P-3). The three novel mutant yeasts had significantly increased ADH and ALDH enzyme activities and coenzyme contents compared to the existing deposited strains.
It was investigated the carbon source preference for growth of three mutant strains (KwonP-1, KwonP-2, KwonP-3) with high ALDH and glutathione, for which a domestic patent application was filed on Feb. 18, 2020. Various carbon sources used by the reference yeast strain (KCTC7296) for growth were measured. To find the maximum ability of producing ALDH, it was investigated the carbon source preference for growth of three new mutant strains (PicoYP, PicoYP-01, and PicoYP-02).
The characteristic and novelty of carbon source preference of strains was analyzed by API 50 CHL kit (API systems, BIOMERIEUX, SA, France).
Preparing the 15 ml of conical tube included 8 ml of YPD medium. Each of the seven mutant strains was inoculated into the prepared conical tube.
After culturing the inoculated conical tubes at 30° C. and 200 rpm for 24 hours, each of the seven mutant strains was secured and extracted from the stage of exponential growth phase. To eliminate the influence of the carbon source contained in the residual YPD medium, the yeast was washed three times using a centrifuge. A yeast suspension of 2McFarland concentration was prepared using API 50 CHL medium. The prepared yeast suspension was filled into the tube of the strip. The strip onto which the suspension was dispensed was cultured at 30° C. for 24 hours.
API 50 CHL medium used for API testing was purple. When acids were produced through energy metabolism, API 50 CHL medium turns blue, green, and finally yellow. In the end, it was recorded which type of carbon source was used by mutant strains based on the color change as like: Purple x, Blue+, Green++, and Yellow+++.
All of the seven mutant strains tested used 19 kinds of carbon sources for energy production and growth: L-arabinose, ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, N-acetyl-glucosamine, arbutin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, raffinose, gentiobiose.
Rhamnose was used by only three mutant strains: KwonP-1, PicoYP-01, PicoYP-02. Sorbitol was used by four mutant strains: KwonP-1, KwonP-3, PicoYP-01, PicoYP-02. α-methyl-D-mannoside was used by four mutant strains: type strain, KwonP-1, KwonP-2, PicoYP-02. Amygdalin was used by six mutant strains: KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, PicoYP-02. D-turanose was used by four mutant strains: type-strain, KwonP-1, KwonP-3, PicoYP-02. D-tagatose was used by three mutant strains: type-strain, KwonP-3, PicoYP-3. Gluconate was used only by type-strain.
Mannitol and sorbitol, which correspond to alcoholic carbon sources, had a significant effect on yeast growth. The three types of novel mutant strains differed from the other four types of yeast in the type of sugar used for growth. The use of the preferred alcoholic carbon source was slightly different between the three new mutant strains (PicoYP, PicoYP-01 and PicoYP-02) [Table 5].
When KARC is administered orally, in order for the enzyme activity to be maintained in the intestine, the enzyme activity must be passed safely without being destroyed by stomach acid, which secretes powerful proteolytic enzymes such as pepsin.
NaOH solution was added to artificial gastric fluid at pH=1.17 to artificially generate two simulated solutions at pH=3 and pH=5, which resemble the human gastric environment during food digestion. 1 g of KARC was added to 7 ml of artificial gastric fluid and 7 ml of two simulated solutions and mixed at 36.5° C. for 5, 30, 60, and 90 min respectively. NaOH solution was added to reaction mixture to adjust acidity to pH=7, respectively. 10 ml of sample for analysis were taken from the adjusted solution at pH=7, respectively. The activity of ALDH was analyzed from each sample.
Under the condition of pH=1.17, the ALDH activity of the sample decreased by more than 92.88% compared to the control group during 5 minutes of reaction. Under the condition of pH=1.17, the ALDH activity of the sample decreased by an average of 98.89% for 90 minutes. The ALDH activity of the samples decreased by an average of 96.66% over 90 min at pH=3 and 56.83% at pH=5. Ultimately the ALDH activity at pH=3 and 5 remained relatively higher than that at pH=1.17 during the 90-min reaction.
In detail, the ALDH activity of KwonP-1 (KCTC13925BP) at pH=1.17 decreased by 90.94% compared to the control group to 5.57 unit/g when reacted for 5 minutes. The ALDH activity of KwonP-1 decreased by 98.57% to 0.88 unit/g for 90 minutes [
The ALDH activity of KwonP-2 (KCTC14122BP) at pH=1.17 decreased by 91.18% to 5.43 unit/g when reacted for 5 minutes. The ALDH activity of KwonP-2 decreased by 98.81% to 0.73 unit/g for 90 minutes [
The ALDH activity of KwonP-3 (KCTC14123BP) at pH=1.17 decreased by 89.99% to 6.16 unit/g when reacted for 5 minutes. The ALDH activity of KwonP-3 decreased by 97.85% to 1.32 unit/g for 90 minutes [
The ALDH activity of PicoYP (KCTC14983BP) at pH=1.17 decreased by 92.84% to 4.40 unit/g when reacted for 5 minutes. The ALDH activity of PicoYP decreased by 98.33% to 1.03 unit/g for 90 minutes [
The ALDH activity of PicoYP-01 (KCTC14984BP) at pH=1.17 decreased by 95.71% to 2.64 unit/g when reacted for 5 minutes. The ALDH activity of PicoYP-01 decreased by 99.76% to 0.15 unit/g for 90 minutes [
The ALDH activity of PicoYP-02 (KCTC14985BP) at pH=1.17 decreased by 96.66% to 2.05 unit/g when reacted for 5 minutes. The ALDH activity of PicoYP-02 decreased by 99.76% to 0.15 unit/g for 90 minutes [
pH 1.17 is the pH of the raw gastric juice secreted. When you eat food, the pH rises from 3 to 5 when raw gastric fluids and food mix in the stomach, so it is unlikely that a pH of 1.17 will be reached. Nevertheless, ALDH activity in the mutant strain was retained even at pH 1.17, which is an extreme condition.
In the end, the ALDH enzyme activity of the novel mutant strains (PicoYP, PicoYP-01, PicoYP-02) was maintained at 2 unit/g to 5 unit/g even though it decreased from 92% to 97% under strongly acidic conditions of pH=1.17. 2-5 units of enzyme activity remain, which is sufficient to function in the intestines. It even remained higher at pH=3 and pH=5 compared to pH=1.17. This was the reason for reaching the conclusion that new mutant strains (PicoYP, PicoYP-01, PicoYP-02) could be administered orally.
Each was inoculated into YPD medium (2% peptone, 1% yeast extract, 2% glucose) and primary seed culture was performed at 30° C. and 200 rpm for 18 hours. 20 ml of cultured seed was inoculated into 1980 ml of YPD medium and cultured again in 5 L. Cultivation in a 5 L culture tank was carried out at 30° C. and 200 rpm for 48 hours. Growth curve at OD660 nm and enzyme activity were analyzed using 10 ml of sample collected from secondary culture.
The maximum density (OD660 nm) of KwonP-1 (KCTC13925BP) was 134.4. The maximum density of KwonP-1 was 4.35% higher than that of the type-strain (KCTC7296). The growth curve characteristics and specific growth rate (OD660 nm/hr) of KwonP-1 were similar to those of the type-strain. The ALDH activity of KwonP-1 was 33.6 unit/g. The ALDH activity of KwonP-1 was 11.96 times higher than that of the type-strain [
The maximum density (OD660 nm) of KwonP-2 (KCTC14122BP) was 133.8. The maximum density of KwonP-2 was 3.88% higher than that of the type-strain. The growth of KwonP-2 ended earlier than that of the type-strain. The specific growth rate (OD660 nm/hr) of KwonP-2 was 14.8% higher than that of the type-strain. The ALDH activity of KwonP-2 was 31.5 unit/g. The ALDH activity of KwonP-2 was 11.21 times higher than that of the type-strain [
The maximum density (OD660 nm) of KwonP-3 (KCTC14123BP) was 134.1. The maximum density of KwonP-3 was 4.12% higher than that of the type-strain. The growth of KwonP-3 ended earlier than that of the type-strain. The specific growth rate (OD660 nm/hr) of KwonP-3 was 6.08% higher than that of the type-strain. The ALDH activity of KwonP-3 was 29.5 unit/g. The ALDH activity of KwonP-3 was 10.5 times higher than that of the type-strain [
The maximum density (OD660 nm) of PicoYP (KCTC14983BP) was 123.8. The maximum density of PicoYP was 3.88% higher than that of type-strain. The growth curve characteristics of PicoYP were similar to those of type-strain. The specific growth rate (OD660 nm/hr) of PicoYP was 6.22% higher than that of the type-strain. The ALDH activity of PicoYP was 44.2 unit/g. The ALDH activity of PicoYP was 15.73 times higher than that of the type-strain [
The maximum density (OD660 nm) of PicoYP-01 (KCTC14984BP) was 126.9. The maximum density of PicoYP-01 was 1.47% higher than that of the type-strain. The growth curve characteristics of PicoYP-01 were similar to those of type-strain. The specific growth rate (OD660 nm/hr) of PicoYP-01 was 2.14% higher than that of the type-strain. The ALDH activity of PicoYP-01 was 47.1 unit/g. The ALDH activity of PicoYP-01 was 16.76 times higher than that of the type-strain [
The maximum density (OD660 nm) of PicoYP-02 (KCTC14985BP) was 148.1. The maximum density of PicoYP-02 was 14.99% higher than that of the type-strain. The growth curve of PicoYP-02 was located at the top compared to the type-strain. The specific growth rate (OD660 nm/hr) of PicoYP-02 was 9.64% lower than that of the type-strain. The ALDH activity of PicoYP-02 was 52.68 unit/g. The ALDH activity of PicoYP-02 was 18.75 times higher than that of the type-strain [
To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysate, proteases were removed and inhibited. To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysate cell debris was removed. The dried product or lysate of the mutant strain was mixed to prepare the KARC composition.
The mutant strain and the medium in which it was cultured contained various substances, such as yeast metabolites and proteolytic enzymes secreted by yeast. In order to extract and preserve ALDH, coenzyme, and glutathione present in yeast, it is necessary to sufficiently remove substances outside the yeast fungus, and for this purpose, a washing process was performed. Washing of the mutant strain was carried out by dispensing 40 ml of culture medium into 50 ml conical tubes, centrifuging at 13,000 rpm for 15 minutes, and removing the supernatant.
As a result of centrifugation, residual medium remained inside the pellet produced by the yeast bacteria clumping together. After adding 30 ml of purified water, the pellet was sufficiently loosened by vortex, and the previous process was repeated three times to sufficiently remove the remaining medium.
The ethanol resistance of yeast is known to be up to 13%, and yeast bacteria die when exposed to high concentrations of ethanol. The washed pellet was sufficiently dissolved using 10 ml of 20% ethanol solution to induce the death of yeast bacteria. The pellet dissolved in ethanol was stirred at 100 rpm for 30 minutes to proceed with the yeast death process. When the reaction time was completed, 30 ml purified water was added to lower the ethanol concentration to 5%. The previous washing process was repeated three times to sufficiently remove ethanol.
To preserve ALDH and ADH from the decomposition action of proteases present in yeast cells, 10 ml of 1×PBS was prepared by dissolving 2 tablets of protease inhibitor (Pierce protease inhibitor mini tablets, EDTA-free, Thermo Scientific). The above solution was added to the washed yeast pellet and sufficiently released.
To prepare a lysate of the mutant strain prepared in the present invention, 4 g of glass beads were added and stirred to break the yeast cell wall. To prevent denaturation of the enzyme due to the heat generated during the process of crushing the yeast, vortex for 30 seconds and ice incubation for 30 seconds were repeated six times.
After the yeast cell wall disruption was completed, 10 ml of 100 mM potassium phosphate buffer was added and mixed by vortex for 3-5 seconds. It was centrifuged at 13,000 rpm for 15 minutes to remove cell structures such as yeast cell walls and glass beads. The supernatant was filtered through a 0.2 μm filter (Minisart® Syringe Filter, Sartorius, Goettingen, ermany) to prepare the KARC composition.
To preserve the enzymes (ALDH, ADH) contained in the mutant enzyme lysate, intracellular proteases were removed and inhibited, and cell debris such as cell walls were removed. The KARC composition was prepared with a lysate selected from the 6 mutant strains (KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, PicoYP-02), or a mixture thereof in a free ratio [Table 6].
KARC 1 was manufactured from KwonP-1. The enzyme activity of ADH and ALDH of KARC 1 were 461.4 unit/g and 28.6 unit/g, respectively. In KARC 1, the content of coenzymes of NADtotal and NADPtotal were 176.2 nmole/g and 5.1 nmole/g, respectively. The GSH content of KARC 1 was 0.98 wt %.
KARC 2 was manufactured from KwonP-2. the enzyme activity of ADH and ALDH of KARC 2 were 482.1 unit/g and 29.8 unit/g, respectively. In KARC 2, the content of coenzymes of NADtotal and NADPtotal were 175.4 nmole/g and 5.2 nmole/g, respectively. The GSH content of KARC 2 was 0.96 wt %.
KARC 3 was manufactured from KwonP-3. the enzyme activity of ADH and ALDH of KARC 2 were 477.5 unit/g and 28.1 unit/g, respectively. In KARC 3, the content of coenzymes of NADtotal and NADPtotal were 177.2 nmole/g and 5.1 nmole/g, respectively. The GSH content of KARC 3 was 1.00 wt %.
KARC 4 was manufactured from PicoYP. the enzyme activity of ADH and ALDH of KARC 2 were 586.8 unit/g and 33.8 unit/g, respectively. In KARC 4, the content of coenzymes of NADtotal and NADPtotal were 184.3 nmole/g and 5.7 nmole/g, respectively. The GSH content of KARC 4 was 0.84 wt %.
KARC 5 was manufactured from PicoYP-01. the enzyme activity of ADH and ALDH of KARC 5 were 621.6 unit/g and 38.2 unit/g, respectively. In KARC 5, the content of coenzymes of NADtotal and NADPtotal were 186.9 nmole/g and 5.6 nmole/g, respectively. The GSH content of KARC 5 was 0.84 wt %.
KARC 6 was manufactured from PicoYP-02. the enzyme activity of ADH and ALDH of KARC 5 were 664.1 unit/g and 41.6 unit/g, respectively. In KARC 6, the content of coenzymes of NADtotal and NADPtotal were 195.0 nmole/g and 5.8 nmole/g, respectively. The GSH content of KARC 6 was 0.88 wt %.
KARC was manufactured by freely mixing dry powders and lysates prepared from six deposit strains. The average enzyme activities of ADH and ALDH in the composition of KARC were 547.6 unit/g and 33.1 unit/g, respectively. The average contents of coenzyme NADtotal and coenzyme NADPtotal in the composition of KARC were 180.4 nmole/g and 5.4 nmole/g, respectively. The average content of glutathione in the composition of KARC was 0.84 wt %.
The aldehyde decomposition ability of KARC was kept on during the lysate production process. KARC showed the ability to remove endogenous aldehydes such as HNE, MDA, and 3,4-dihydroxyphenyl acetaldehyde (DOPAL).
It was investigated the differences between both ALD (yeast aldehyde dehydrogenase) of the mutant strains and parent strain. Whole genome sequencing was performed on the parent strain and mutant strains of KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, and PicoYP-02. The mutant strain cells were obtained by culturing pure strains on solid medium. The genome sequence of the mutant strain obtained were analyzed.
Among ALDs (yeast aldehyde dehydrogenases) in the novel mutant strains, ALD2 (SEQ ID NO:3) was found to be condensed with ALD3 (SEQ ID NO:4) on chromosome 13. A non-coding region of 689 nucleotides was located between the ALD2 and ALD3 coding genes
The ALD2 and ALD3 existed continuously in the same genome. ALD2 and ALD3 encoded respective aldehyde dehydrogenases. ALD2 coding gene was almost similar to ALD3, consist of 1,521 nucleotides and 506 amino acids, but had an 8.2% difference in sequence. ALD2 and ALD3 they were identified as separate aldehyde dehydrogenases that differed from each other in 125 base sequences (8.2%).
In the six mutant strains (KwonP-1, KwonP-2, KwonP-3, PicoYP, PicoYP-01, PicoYP-02), there is no stop codon at the end of the ALD2 sequence, so proteins are synthesized continuously. As a result, a new, larger ALDH enzyme is created by linking a part of ALD2 and ALD3[SEQ ID NO: 1].
ALD2[SEQ ID NO. 3] of the type-strain (KCTC7296) consisted of 30 nucleotide sequences (5′-GTTCACATAAATCTCTCTTTGGACAACTAA-3′) coding 9 amino acids (N-VHINLSLDN-C) at the terminal, excluding the stop codon.
ALD2 of the six mutant strains consisted of specific 42 nucleotide sequences (5′-AGATATAGATTATACACATTTAGAAAATTAGCCAAAAGAAAA-3′) coding 14 amino acids (N-RYRLYTFRKLAKRK-C) between 5′-terminal of ALD2 and ALD3, [SEQ ID NO. 2].
There was no stop codon at the end of the sequence in ALD2 coding gene by deleted from the 1492nd nucleotide of ALD2 to 647th nucleotide of non-coding region. Finally, the six deposited mutant strains had new mutated gene consist of total 3,054 bases coding novel ALD. [SEQ ID NO: 1].
For the acetaldehyde and MDA animal experiments, 5-week-old male Sprague Dawley (SD) rats (Rat) were used. The KARC composition was orally administered to rats at 10 units/kg or 20 units/kg, and alcohol (3 g/kg) was orally administered to the rats 30 minutes after KARC injection.
After the administration was completed, blood samples were collected from the tail vein at 0, 1, 3, 5, and 8 hours after KARC injection, and after centrifugation, plasma was stored at −80° C. [
7-week-old male Wistar rats (7 weeks old, 250 g, n=8-10) were used. Rotenone solution (2.5 mg rotenone/ml, 20 μl DMSO/ml) was prepared using natural oil (middle chain triglycerides). Mice were intraperitoneal injection administered rotenone solution (2.5 mg/kg) daily for 60 days.
Two administration methods were employed to confirm the Parkinson's disease prevention and treatment effects of KARC. KARC (20 units/kg) was administered orally at the same time as rotenone administration to observe the effect of preventing Parkinson's disease. KARC (20 units/kg) or L-dopa were administered orally at the two weeks after rotenone administration to observe the effect of therapeutic Parkinson's disease. To quantify dopamine, brain tissues were isolated and stored at −80° C. in liquid nitrogen. [
The total acetaldehyde reduction effect by oral administration of KARC was assessed using an Acetaldehyde assay kit (LSBio, Seattle, WA, USA). 20 μl of each sample was dispensed into two wells of a 96 well plate. 80 μl of working reagent (75 μl assay buffer, 8 μl NAD/MTT, 1 μl Enzyme A, 1 μl Enzyme B) was dispensed into one well. In the remaining well, 80 μl of blank working reagent (75 μl assay buffer, 8 μl NAD/MTT, 1 μl Enzyme B) was dispensed. The plate after dispensing was lightly mixed and reacted at room temperature for 30 minutes. When the reaction was completed, the absorbance was measured at 565 nm (520-600 nm).
The concentration of acetaldehyde reached the maximum 1 hour after ethanol administration and showed a tendency to decrease in the KARC composition administration group. In the KARC administration group, acetaldehyde concentration significantly decreased compared to the control group (Vehicle) 1, 3, and 5 hours after ethanol administration. In the KARC high-dose administration group (F), the blood acetaldehyde concentration was 0.356, 0.224, and 0.091 mM, respectively, which decreased by 39.2%, 58.4%, and 72.1% compared to the control group [
Total malondialdehyde content in blood was analyzed using the OxiTec™ TBARS assay kit according to the manufacturer's protocol (ZeptoMetric, Buffalo, NY, USA). 100 μl sample, 100 μl 8.1% SDS solution, and 4 ml color indicator (TBA, 10% NaOH solution, 20% acetic acid) were added to the conical tube, and then reacted in a constant temperature water bath at 95° C. for 60 minutes. After completion of the reaction, the sample was centrifuged at 4° C. and 1,600 rpm for 10 minutes and stabilized at room temperature for 30 minutes. 150 μl of supernatant was transferred to a 96 well plate, and absorbance was measured at 530-540 nm.
In the control group (Vehicle), the concentration of MDA in the blood reached the maximum 3 hours after ethanol administration, whereas in the group administered KARC, it reached the maximum value 1 hour after ethanol administration. The concentration of MDA in the blood decreased, showing a significant difference from the control group 3 and 5 hours after ethanol administration. The blood MDA concentration of the KARC high-dose administration group (F) was 0.232 and 0.137 μM, respectively, a decrease of 80.4% and 86.3% compared to the control group [
These results showed that oral administration of KARC was effective in reducing various endogenous aldehydes such as acetaldehyde and malondialdehyde in the blood.
To measure the effect of reducing dopamine-derived DOPAL by oral administration of KARC, the DOPAL content in rat brain striatonigra samples was measured by HPLC/MMS. After dissolving the sample in trichloroacetic acid (3.0 M/100 ul), isoproterenol (1 nmol/ml, 100 ul) was added and pretreated by centrifugation using a toyopak SP carton (Toso, Tokyo, Japan).
To dissolve the adsorbed amine compound, 0.6M KCl-acetonitrile (1:1, 2 ml) was treated, and DPE reagent was added to the solution to induce fluorescence. The final solution produced as a result of the reaction was injected into HPLC to measure dopamine.
To measure the DOPAC and HVA content in the sample, the sample was dissolved in HClO4 (300 l), and the supernatant was obtained by homogenization and centrifugation (50,000 g, 4° C., 15 minutes). The supernatant was filtered and DOPAC and HVA contents were measured through HPLC/MMS. The content of DOPAL was calculated as ng/g tissue.
To investigate changes in dopamine metabolism in the brain of PD model animals using rotenone, DA, DOPAL, DOPAC, and HVA were measured using HPLC [
As a result of the measurement, the levels of DA, DOPAL, DOPAC, and HVA in the brain of the control group were measured at 1542, 22, 620, and 970 ng/g tissue weight, respectively. In the group where PD was induced using rotenone, the levels of DA, DOPAC and HVA were decreased by 1021, 234, and 102 ng/g tissue weight, respectively compared to the control group. But the level of DOPAL was relatively increased 70 ng/g weight.
In the group administered the reference drug L-Dopa, DA and DOPAL levels in the brain increased 1816 and 96 ng/g tissue weight compared to the control group, respectively, but DOPAC and HVA levels decreased 281 and 126 ng/g tissue weight. Similar results to the rotenone administration group were observed.
On the other hand, in the group administered KARC to the Parkinson's induction model, DA, DOPAL, DOPAC, and HVA in brain tissue were 1290, 21, 510, and 790 ng/g tissue weight, respectively. DA, DOPAC, and HVA increased compared to the rotenone administered group, and DOPAL relatively decreased.
In particular, in KARC pre-administered group for preventive purposes, DA and DOPAL increased 1522 and 18 ng/g tissue weight compared to the control group, respectively, and DOPAC and HVA also increased 590 and 860 ng/g tissue weight, resulting in all levels of DA, DOPAL, DOPAC and HVA were almost consistent with the results of the control group.
It was used the ratio of dopamine turnover index ((DOPAC+HVA)/DA), which is used to indirectly check the amount of DOPAL that remains unmetabolized during DA metabolism because DA is metabolized through DOPAL and DOPAC and is ultimately metabolized into HVA [
As a result of calculating the dopamine conversion index [(DOPAC+HVA)/DA], It was 103.1% in the control group, 100.8% in the KARC pre-administration group, and 95.3% in the KARC post-administration group, which means that dopamine metabolism in vivo was progressive well in three groups.
On the other hand, compared to the three groups, there was a 32.9% decrease in the rotenone group and a 22.4% decrease in the L-dopa group, which means that dopamine metabolic dysfunction was abnormally caused by Parkinson's disease.
This means that DOPAL, a metabolic intermediate, is accumulated in vivo. KARC administration inhibits the accumulation of DOPAL, a neurotoxin, and recovers dopamine metabolism to normal, accelerating to increase DOPAC and HVA, which are relatively less toxic than DOPAL. As a result, KARC has the effect of preventing and treating PD with restoring DA metabolic function.
Reactive oxygen species or oxidative stress increases when drinking alcohol due to excessive acetaldehyde (Ach) produced by alcohol dehydrogenase (ADH). Aldehyde dehydrogenase (ALDH) acts to convert it into acetic acid and excrete it out of the body. In the case of aldehyde dehydrogenase gene mutation or excessive aldehyde caused by excessive alcohol cause peroxidation of fat.
The resulting acetaldehyde and malondialdehyde worsen oxidative stress and interfere with mitochondrial energy metabolism. Endoplasmic reticulum stress is induced through the accumulation of denatured proteins in cells, leading to cell death.
The concentration of blood acetaldehyde was measured over time following alcohol consumption [
At a dose of KARC 20 units/kg administration, the AUC of blood acetaldehyde (Ach) decreased significantly by 55.71% compared to alcohol consumption alone, measuring 5.22±0.99 mg·h/dL (P<0.001). When comparing the KARC 10 units/kg administration group with the KARC 20 units/kg administration group, the blood acetaldehyde (Ach) in the KARC 20 units/kg group decreased significantly (P=0.034). KARC demonstrated dose-dependent reduction in the total amount of blood acetaldehyde (Ach) over time.
The reduction in blood acetaldehyde (Ach) concentration due to KARC administration has a positive impact on reducing oxidative stress and promoting health.
The concentration of blood malondialdehyde (MDA) was measured during the chemotherapy period [
In the control group, the blood MDA concentration ranged from 0.427 μM to 0.885 μM with a substantial variability. In the KARC administration group, the range was significantly reduced, with values ranging from 0.158 μM to 0.269 μM. This not only confirmed the effect of reducing blood MDA concentration but also stabilizing it, as demonstrated in [
Various factors, such as drug intake, stress, and intense physical exercise, lead to an increase in intracellular reactive oxygen species. This triggers lipid peroxidation reactions and oxidative processes in endogenous amines such as dopamine, norepinephrine, serotonin, histamine, and more. Reactive aldehyde compounds, including 4-hydroxynonenal (HNE), malondialdehyde (MDA), acetaldehyde (Ach), and dopamine-induced aldehyde, accumulate within cells, exacerbating oxidative stress.
These aldehydes subsequently react with surrounding proteins and undergo secondary metabolic processes to form stable end products such as Malondialdehyde-acetaldehyde adduct (MAA) and Malondialdehyde lysine adducts (M-lys adducts), known as Advanced Lipid Peroxidation End Products. The accumulation of these products exerts toxic effects on various cells, further intensifying oxidative stress.
This cumulative oxidative stress disrupts mitochondrial energy metabolism within cells and leads to the buildup of aldehyde intermediates in aldehyde-based sugar metabolism, including methylglyoxal (MG) and glyceraldehyde-3-phosphate (GA3P). The chain reaction involving aldehydes results in the accumulation of stable final glycoxidation products known as advanced glycation end products (AGEs), which weaken intracellular antioxidant defense systems like glutathione (GSH). These processes elevate endoplasmic reticulum (ER) stress, leading to increased cellular apoptosis in nerve cells.
The increase in reactive oxygen species and oxidative stress is associated with elevated levels of reactive aldehydes like HNE and MDA, as well as modified proteins such as advanced glycation end products (AGEs) and advanced lipid peroxidation end products (ALEs). This cascade of events is known to involve mutual reinforcement and amplification, leading to heightened endoplasmic reticulum stress (ER stress).
KARC administration effectively regulated malondialdehyde, a marker for active oxygen and oxidative stress, demonstrating the potential for reducing oxidative stress and improving the constancy of endoplasmic reticulum (ER) stress. KARC significantly reduced malondialdehyde concentrations in the bloodstream, illustrating its capability to reduce active oxygen and oxidative stress.
By lowering the levels of acetaldehyde and malondialdehyde in human blood, KARC exhibited its potential to prevent and remedy ER stress through the reduction of active oxygen and oxidative stress. This suggests that by modulating intracellular active oxygen and oxidative stress, KARC inhibits neuronal cell apoptosis, consequently suppressing and preventing Parkinson's disease. This leads to improvements in behavioral and motor functions.
The experimental animals were female and male ICR mice (7 weeks old). The received ICR mice were acclimatized for 7 days. The general symptoms of the adopted mice were observed during the acclimatization period, and only healthy animals were used for short-term administration toxicity tests. Feed and water were consumed ad libitum. Based on the average body weight of about 20 g the day before oral administration, groups were separated into 10 groups, 5 for each group, and 5 for each group.
The test substance was prepared by dissolving it in physiological saline so that the dosage for experimental animals was 0, 750, 3,000, and 5,000 mg/kg, respectively, based on the content of the mutant yeast lysate KARC of the present invention.
The standards for administered dosage were in accordance with the Ministry of Food and Drug Safety's Korea national Toxicology Program (KNTP) toxicity test manual. The maximum application dose of 5,000 mg/kg guided by the KNTP manual was set as the maximum concentration for this experiment. The samples prepared for each group were orally administered once to each test animal. For the normal group (G1), physiological saline was administered.
For animals in all test groups, symptoms of mice were observed at least once a day from the date of acquisition to the date of necropsy. Symptoms were observed for 7 days after oral administration. After observing the rat's symptoms, an autopsy was performed. During the autopsy of the rat, changes in each organ were observed with the naked eye.
A single-dose toxicity test of the ALDH-containing KARC composition of the present invention was conducted using mice. As a result, no cases of mouse death were observed for 7 days at concentrations of the mutant yeast KARC up to 5,000 mg/kg. No unusual features, such as weight gain or changes in feed intake, were found in the mice. No unusual findings were found in the autopsy results conducted after the end of observation.
The present invention confirmed the effect of KARC in reducing exogenous and endogenous aldehydes. As a result of reacting KARC (300 mg/ml) with various aldehydes (1 mM) at 37° C. for 3 hours, 3,4-Dihydroxylphenyl acetaldehyde (DOPAL) decreased by 24.4%, succinic semialdehyde (SSA) decreased by 74.9%, glyoxal decreased by 23.8%, cinnamaldehyde decreased by 99.6%, and benzaldehyde decreased by 97.1%. In the case of acetaldehyde, it decreased by 100.0% even after reacting at 30° C. for 1 hour. [
Potassium chloride (KCl) was dissolved in a 50 mM of pH 7.5 HEPES buffer solution to be 200 mM. For experiments with acetaldehyde, glyoxal, DOPAL, cinnamaldehyde, and benzaldehyde, 935 μl of buffer solution, 15 μl of 100 mM EDTA aqueous solution, 30 μl of 100 mM NADP+ aqueous solution, 10 μl of 100 mM aldehydes in Demineralized water (DW) or acetonitrile solution, and 10 μl of 300 mg/mL KARC were dispensed into microtubes. As a negative control, 935 μl of buffer solution, 15 μl of 100 mM EDTA aqueous solution, 30 μl of 100 mM NADP+ aqueous solution, 10 μl of 100 mM aldehyde in DW or acetonitrile solution, and 10 μl of DW were dispensed into a microtube.
For experiments with SSA, 845 μl of buffer, 15 μl of 100 mM EDTA aqueous solution, 30 μl of 100 mM NADP+ aqueous solution, 10 μl of 10 mM SSA in acetonitrile solution, and 10 μl of 300 mg/mL KARC were dispensed into microtubes. As a negative control, 845 μl of buffer solution, 15 μl of 100 mM EDTA aqueous solution, 30 μl of 100 mM NADP+ aqueous solution, 100 μl of 10 mM SSA in acetonitrile solution, and 10 μl of DW were dispensed into a microtube.
The reactants were shakes at 30° C. or 37° C. for 1 hour or 3 hours using thermo shaker.
For experiments using the representative aliphatic aldehydes: SSA, acetaldehyde, glyoxal, a 500 μl of each reaction was aliquoted into a microtube at the end of the reaction. 470 μl of methanol, 20 μl of 50 mM DNPH in acetonitrile solution, and 10 μl of 6N HCl were additionally dispensed into the microtube containing the reaction solution, and heated at 70° C. for 40 minutes. Alternatively, 480 μl of methanol, 10 μl of 100 mM DHBA in acetonitrile solution, and 10 μl of 6N HCl were added and heated at 70° C. for 40 min. After the heated solution was cooled, 10 μl was quantified and injected into HPLC for analysis.
For experiments with DOPAL, cinnamaldehyde, and benzaldehyde, representative of aromatic aldehydes, 10 μl of the solution reacted with KARC, without heating with DNPH or DHBA, was aliquoted and injected into the HPLC for analysis.
HPLC system (Waters Alliance 2690/2695 HPLC with Waters 2996 PDA detector) was used for analysis. The analytical column was 150 mm×4.6 mm i.d. packed with C18, 5 μm particle size (Shimadzu Scientific Instruments, Kyoto, Japan).
In gradient, it started at 80% of water (lv/v % trifluoroacetic acid) and deployed in reverse phase to 20% after 15 minutes. Absorbance was analyzed at wavelengths of 254 nm, 310 nm, or 360 nm with an ultraviolet detector.
The results were confirmed by the progress of the reaction in which aldehyde was consumed through the reduction of DNPH-aldehyde conjugates or DHBA-aldehyde conjugates in the experimental group compared to the negative control group.
Food and pharmaceutical compositions containing KARC as an active ingredient for suppressing auto-brewing symptoms were prepared. It is possible to prepare food or pharmaceutical compositions of various composition ratios containing KARC powder. As an example, the powder composition according to the present invention has the function of suppressing auto-brewing symptoms and oxidative stress through ingestion of 13 g of the composition twice a day. The weight ratio between the components and phases of the food or pharmaceutical composition containing the powder composition is shown in [Table 7].
In the food and pharmaceutical composition, KARC dry powder, excipients, and natural sweeteners such as fructo-oligosaccharides, enzyme-treated stevia (Stevia), anhydrous citric acid, iso-maltodextrins (Iso-malto), and xylitol, citrus juice powder, and citrus flavor powder were added. Processing and testing of raw materials and final products of food or pharmaceutical compositions were conducted in accordance with the general test methods and the Health Functional Foods Act described in the Korean Food Code. KARC-containing food or pharmaceutical compositions decompose endogenous aldehydes and exhibit the effect of suppressing auto-brewing symptoms and oxidative stress. KARC-containing foods or pharmaceutical compositions can prevent or improve irritating bowel syndromes.
Through the above examples, the mutant yeast composition KARC containing aldehyde dehydrogenase was described in detail: manufacturing methods, pharmacological effects, administration methods, therapeutically effective doses for disease models, short-term administration acute toxicity, and representative examples of food or pharmaceutical compositions. Although the efficacy of KARC has been described in detail through the above examples, these are only examples of the present invention.
A person skilled in the art can easily derive various modifications and other embodiments equivalent to the present invention from the above-described embodiments of the present invention.
Even foods or therapeutic agents containing a modified form of aldehyde dehydrogenase that embodies the technical gist of the present invention described in the patent claims fall within the scope of legal protection of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2022-0165794 | Dec 2022 | KR | national |
| 10-2023-0153007 | Nov 2023 | KR | national |
