KR20230007957A - Probiotics fermentation products of momordica charantia with enhanced anti-diabetic, anti-dementia and anti-oxidant activity - Google Patents

Abstract

The present invention relates to a probiotic fermented bitter gourd with enhanced anti-diabetic, anti-dementia, and antioxidant functions and a preparation method thereof, and more specifically, provides the preparation method of the fermented bitter gourd with enhanced anti-dementia activities which comprises the step of: preparing a mixture of a bitter gourd extract with a sugar content of 3 to 15 brix by adding a sugar component; and inoculating the prepared mixture with a probiotic strain and fermenting the same and the fermented bitter gourd prepared thereby. The fermented bitter gourd has enhanced anti-dementia, anti-diabetic, and antioxidant activities and can be used as pharmaceutical compositions and health functional food compositions for preventing, treating, or alleviating cognitive dysfunction or diabetes.

Description

Probiotic Bitter gourd fermented product with enhanced anti-diabetic, anti-dementia and antioxidant functions

The present invention relates to a probiotic fermented bitter gourd extract with enhanced antidiabetic, anti-dementia and antioxidant functions, a pharmaceutical composition using the same, and a method for preparing the same.

Bitter melon ( Momordica charantia ) is an annual vine that belongs to the gourd family with large leaves and a kidney-like round shape. It is an edible fruit grown mainly in tropical Asia, Mediterranean Africa, etc., and is known as bitter melon because its taste is very bitter. Polypeptide-p, a phytonutrient contained in bitter gourd, is a kind of peptide known as plant insulin, and acts similarly to insulin in the body. Charantin, a major component of fruits and seeds of bitter gourd, acts on β-cells that promote secretion of insulin, and plant sterol glycosides that give bitterness to bitter gourd are also known to have a hypoglycemic function.

Diabetes mellitus refers to a series of metabolic diseases characterized by hyperglycemia, and in particular, type 2 diabetes is characterized by insulin resistance and reduced insulin secretion. As of 2018, the prevalence of diabetes in Korea is estimated to be 13.8%, 4.94 million people aged 30 years or older, and 27.6%, 2.044 million people aged 65 years or older, showing a consistently high prevalence rate. Diabetes is still the main cause of death in Koreans. It is a disease with serious medical and socioeconomic burdens due to serious complications. As for the drug therapy for type 1 diabetes, multiple insulin injection therapy or insulin pump treatment is used, and type 2 diabetes patients are treated with biguanides, DPP-4 inhibitors, SGLT2 inhibitors, thiazolidinedione, and sulfonylureas. , alpha-glucosidase inhibitors, etc. are being used.

Generation of reactive oxygen species and increase in oxidative stress are known to be causes of decreased insulin secretion, and consequently, β-cell damage is related to complications such as arteriosclerosis or neuropathy in diabetic patients. In addition, reactive oxygen species are known to induce oxidative stress in nerve cells, activate an apoptosis pathway, and consequently induce synaptic damage, thereby affecting the pathogenesis of dementia.

Dementia refers to an acquired multiple disorder that interferes with daily life due to deterioration of functions such as memory, language ability, orientation, judgment and performance ability due to degenerative brain disease or cerebrovascular disease. The main features of Alzheimer's disease, the most common degenerative brain disease that causes dementia, are reduced cholinergic neurons, increased butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) activity, and and accumulation of β-amyloid protein. Increased AChE activity decomposes acetylcholine, reduces its concentration, and causes damage to nerve cells through a series of reactions, resulting in memory deficits. In addition, it has been reported that inhibition of BChE, which is mainly located in glial cells of the brain, is related to cognitive improvement in Alzheimer's patients, and both AChE and BChE can be seen as important factors in Alzheimer's disease.

Various drugs based on the mechanism of dementia are being developed, but due to problems such as low bioavailability and hepatotoxicity, there is a constant need to search for various substances derived from natural products that can replace them.

Republic of Korea Patent Registration No. 10-1074914 (2011.10.18. Notice)

An object of the present invention is to provide a fermented product of a natural product fermented using a probiotic strain and having enhanced anti-dementia, anti-diabetic and antioxidant activities, a pharmaceutical composition using the same, and a method for producing the same.

In order to achieve the above object, the present invention is Leuconostoc mesenteroides MKSR (Leuconostoc mesenteroides MKSR) deposited as KCTC13842BP, Leuconostoc mesenteroides MKJW (Leuconostoc mesenteroides MKJW deposited as KCTC14459BP) in a mixture containing bitter gourd extract and sugar ) and Lactobacillus plantarum MKHA15 deposited as KCTC13928BP, characterized in that inoculated with one or two or more strains selected from the group consisting of, anti-dementia, anti-diabetic or antioxidant fermented bitter gourd provides

In addition, the present invention provides a pharmaceutical composition for preventing or treating cognitive dysfunction and a health functional food composition for preventing or improving containing the fermented product of bitter melon as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating diabetes and a health functional food composition for preventing or improving diabetes containing the fermented product of bitter gourd as an active ingredient.

In addition, the present invention comprises the steps of preparing a mixture of bitter melon extract having a sugar content of 3 to 15 Brix by adding a sugar component; And the prepared mixture was deposited as KCTC13842BP Leuconostoc mesenteroides MKSR (Leuconostoc mesenteroides MKSR), KCTC14459BP deposited Leuconostoc mesenteroides MKJW (Leuconostoc mesenteroides MKJW) and KCTC13928BP deposited Lactobacillus plantarum MKHA15 (Lactobacillus plantarum MKHA15), inoculating and fermenting one or more strains selected from the group consisting of; provides a method for producing fermented bitter melon with enhanced anti-dementia, anti-diabetic or antioxidant activity.

The method for producing fermented bitter gourd fruit according to the present invention provides fermentation conditions such as probiotic strains, sugars, and various minerals, and fermentation conditions such as pH, which can enhance various functionalities of fermented bitter gourd bitter gourd. According to this, it is possible to prepare a fermented product of bitter gourd with enhanced anti-dementia, anti-diabetic and antioxidant activities.

Since the fermented bitter melon with enhanced functionality has excellent anti-dementia activity, it can be used as a pharmaceutical composition and health functional food composition for the prevention, treatment, or improvement of cognitive dysfunction or diabetes.

1 shows a bitter gourd extraction and fermentation process according to an embodiment of the present invention.
Figure 2 shows the results of chromatography for each experimental group. (G, glucose, M, maltose, F, fructose, S, sucrose, O, oligosaccharides, 1B-17B, before fermentation, 1A-17A, after fermentation)
Figure 3 shows the residuals for the effects of bitter gourd extract (X1), sucrose (X2) and maltose (X3) on alpha-glucosidase inhibitory activity as a normalized figure.
Figure 4 shows the results of a response surface model graph for the interaction effect of variables on alpha-glucosidase inhibitory activity. (A: bitter gourd extract (°brix), B: sucrose (mmol))
Figure 5 shows the results of a response surface model graph for the interaction effect of variables on alpha-glucosidase inhibitory activity. (A: bitter gourd extract (°brix), C: maltose (mmol))
Figure 6 shows the results of a response surface model graph for the interaction effect of variables on alpha glucosidase inhibitory activity. (B: sucrose (mmol), C: maltose (mmol))

Hereinafter, the present invention will be described in detail.

Recently, fermentation has been used as a major technology for enhancing the physiological activity of natural products. Accordingly, the present inventors confirmed excellent anti-dementia, anti-diabetic and antioxidant activities in fermented bitter gourd fermented with probiotic strains under specific fermentation conditions. , completed the present invention.

The present invention provides a method for preparing a fermented bitter gourd extract with enhanced anti-dementia, anti-diabetic and antioxidant activities.

In the present specification, " momordica charantia ", as described above, is an annual vine belonging to the Cucurbitaceae family, and is known as "bitter melon" because it tastes very bitter, and in several countries in East Asia and the Caribbean Traditionally, it has been reported to be widely used for medicinal purposes.

In order to achieve the object of the present invention, the present invention is Leuconostoc mesenteroides MKSR (Leuconostoc mesenteroides MKSR) deposited as KCTC13842BP and Leuconostoc mesenteroids MKJW (Leuconostoc mesenteroides MKJW deposited as KCTC14459BP) in a mixture containing bitter gourd extract and sugar Leuconostoc mesenteroides MKJW) and Lactobacillus plantarum MKHA15 deposited as KCTC13928BP, characterized by inoculation with one or two or more strains selected from Bitter gourd is provided.

The sugar may be sucrose, maltose or a mixture thereof, but may preferably be a mixture of sucrose and maltose.

The fermented bitter gourd extract may consist of 4-8 °brix of bitter gourd extract, 350-450 mmol of sucrose, and 100-150 mmol of maltose, and the optimal fermentation conditions obtained as a result of response surface analysis are 5.015 °brix fermented bitter gourd extract, 393.97 mmol of sucrose, and maltose. determined to be 143.386 mmol.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cognitive dysfunction containing fermented bitter melon with enhanced anti-dementia, anti-diabetic and antioxidant activity as an active ingredient.

The cognitive dysfunction may be selected from the group consisting of dementia, Alzheimer's dementia, vascular dementia, learning disability, amnesia, agnosia, aphasia, apraxia, delirium, and mild cognitive impairment, but is not limited thereto.

The pharmaceutical composition according to the present invention can be prepared according to conventional methods in the pharmaceutical field. The pharmaceutical composition may be formulated with an appropriate pharmaceutically acceptable carrier according to the formulation, and, if necessary, further include excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, and the like. can

In the present specification, "pharmaceutically acceptable" means that it is not toxic to cells or humans exposed to the pharmaceutical composition, and the appropriate carrier does not inhibit the activity and characteristics of the fermented bitter gourd extract according to the present invention. , It may be selected differently depending on the dosage form and formulation.

The pharmaceutical composition can be applied in any dosage form, and more specifically, it can be formulated and used in parenteral dosage forms such as oral dosage forms, external preparations, suppositories and sterile injection solutions according to conventional methods.

Among the oral dosage forms, the solid dosage form is in the form of tablets, pills, powders, granules, capsules, etc., and contains at least one excipient such as starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc. It can be prepared by mixing, and in addition to simple excipients, lubricants such as magnesium stearate and talc may also be included. In addition, in the case of a capsule formulation, a liquid carrier such as fatty oil may be further included in addition to the above-mentioned materials.

Among the oral formulations, liquid formulations include suspensions, solutions for internal use, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is.

The parenteral formulation may include a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized formulation, and a suppository. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, Tween 61, cacao butter, laurin fat, glycerogeratin, and the like may be used. It is not limited thereto, and all suitable agents known in the art may be used.

In addition, calcium or vitamin D3 may be further added to the pharmaceutical composition according to the present invention to enhance therapeutic efficacy.

The pharmaceutical composition according to the present invention can be administered in a pharmaceutically effective amount.

In the present specification, "pharmaceutically effective amount" means an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects.

The effective dosage level of the pharmaceutical composition depends on the purpose of use, the patient's age, sex, weight and health condition, disease type, severity, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment Duration, combination, or factors including drugs used concurrently and other factors well known in the medical arts may be determined differently. For example, although not constant, generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg, may be administered once or several times a day. The dosage is not intended to limit the scope of the present invention in any way.

The pharmaceutical composition may be administered to any animal that may develop cognitive dysfunction or diabetes, and the animal may include, for example, humans and primates as well as livestock such as cattle, pigs, horses, and dogs. .

The pharmaceutical composition may be administered by an appropriate administration route according to the formulation form, and may be administered through various oral or parenteral routes as long as it can reach the target tissue. The method of administration is not particularly limited, and may be administered by conventional methods such as oral, rectal or intravenous, intramuscular, subcutaneous, intrabronchial inhalation, intrauterine dural or intracerebroventricular injection, for example. .

The pharmaceutical composition according to the present invention may be used alone for the prevention or treatment of cognitive dysfunction or diabetes, or may be used in combination with surgery or other drug treatment.

In addition, the present invention provides a health functional food composition for preventing or improving cognitive dysfunction, containing fermented bitter gourd charantia extract having enhanced anti-dementia, anti-diabetic and antioxidant activity as an active ingredient.

In the health functional food composition according to the present invention, the health functional food may be made into a powder, granule, tablet, capsule, syrup or beverage, etc., and there is no limit to the form that the health functional food can take, and the conventional It can include all foods of meaning. For example, beverages and various drinks, fruits and their processed foods (canned fruit, jam, etc.), fish, meat and their processed foods (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.) ), etc., and may include all functional foods in a conventional sense. In addition, food used as feed for animals may also be included.

The health functional food composition may be prepared by further including food chemically acceptable food additives and other appropriate auxiliary components commonly used in the art. The suitability as a food additive can be determined according to the standards and standards for the item in accordance with the general rules of the Food Additive Code and general test methods approved by the Ministry of Food and Drug Safety, unless otherwise specified. Examples of the items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations; and the like.

The other auxiliary ingredients include, for example, flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, colorants, pectic acid, alginic acid, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents, etc. may additionally contain. In particular, as the natural carbohydrate, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol may be used. As the sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame may be used.

The health functional food composition according to the present invention, unlike general drugs, has the advantage of using food as a raw material and has no side effects that may occur when taking drugs for a long time, and is excellent in portability, making it an adjuvant for preventing or improving cognitive dysfunction can be consumed as

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of diabetes containing, as an active ingredient, the fermented product of bitter melon with enhanced anti-dementia, anti-diabetic and antioxidant activities.

In addition, the present invention provides a health functional food composition for preventing or improving diabetes, containing fermented bitter melon with enhanced anti-dementia, anti-diabetic and antioxidant activities as an active ingredient.

In addition, the method for producing fermented bitter gourd extract according to the present invention comprises the steps of preparing a mixture of bitter gourd extract having a sugar content of 3 to 15 brix by adding a sugar component; And the above prepared mixture was deposited as KCTC13842BP, Leuconostoc mesenteroides MKSR ( Leuconostoc mesenteroides MKSR ), Leuconostoc mesenteroides MKJW deposited as KCTC14459BP, and Lactobacillus plantarum MKHA15 deposited as KCTC13928BP plantarum MKHA15), inoculating with one or two or more strains selected from the group consisting of and fermenting; may include.

As used herein, "extract" refers to a substance obtained by extracting a component of a natural substance regardless of the extraction method, extraction solvent, extracted component, or the form of the extract. It may include all materials that can be obtained by processing or processing by the method.

The extract may be extracted according to a method commonly used in the art, for example, hot water extraction, ultrasonic extraction, filtration, reflux extraction, etc., which are performed alone or in combination of two or more methods. It can be.

The bitter gourd extract according to the present invention may be extracted with water, C1 to C4 alcohol, or a mixture thereof as a solvent, preferably extracted with water at 100 to 130 ° C. one or more times, but is not limited thereto.

In the above production method, the step of preparing a mixture of bitter gourd extract may be performed by mixing sugar components with bitter gourd extract so that the sugar content is 3 to 15 brix, preferably 4 to 12 brix, but is limited thereto It is not.

The sugar component is sucrose, maltose, glucose, galactose, lactose, mannose, maltooligosaccharide, fructooligosaccharide, starch ) And may be one or two or more selected from the group consisting of dextrin, but is not limited thereto.

Preferably, the sugar component may consist of 350-450 mmol of sucrose and 100-150 mmol of maltose.

The step of preparing a mixture of the bitter gourd extract is to add yeast extract, peptone, potassium phosphate, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate to the bitter gourd extract ( ferrous sulfate heptahydrate), sodium chloride (sodium chloride), manganese sulfate (manganese sulfate) and calcium chloride dihydrate (calcium chloride dihydrate), one or two or more nutritional supplements selected from the group consisting of may be further added, but is not limited thereto not.

In the above production method, the fermentation step may be performed for 20 to 30 hours at a pH of 6 to 7 and a temperature of 25 to 40 ° C, but is not limited thereto.

In the manufacturing method, the anti-dementia, anti-diabetic and antioxidant activities of the fermented bitter melon extract can be further enhanced by appropriately adjusting the sugar concentration, nutritional supplement and pH range of the mixture of the bitter gourd extract according to the strain.

In addition, the present invention provides a reagent composition for inhibiting acetylcholinesterase (AChE) or butyrylcholinesterase (BuChE) containing fermented bitter melon as an active ingredient.

Corresponding features may be substituted in the foregoing.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Example 1> Preparation of fermented bitter melon

In order to check the growth of bacteria according to Momordica charantia L. extract concentration, nutritional supplements and pH control, first, as shown in Figure 1 below, bitter gourd extract and water were mixed at a weight ratio of 1:15 Next, the first extraction was performed by heating at 121 ° C. for 5 hours, and then the second extraction was performed by adding more water and heating at 105 ° C. for 3 hours to prepare a bitter gourd extract. The extract was prepared by mixing sugar and maltose after adding water to have a sugar content of 4, 8, or 12 brix.

bitter gourd extract concentrations (12, 8, 4 brix), nutritional supplements [yeast extract, 0.5%, peptone, 0.5%, 2% potassium phosphate; YPK] and mineral mixture [magnesium sulfate heptahydrate (0.02%), ferrous sulfate heptahydrate (0.001%), sodium chloride (0.001%), manganese sulfate (0.001%) %), calcium chloride dihydrate (0.013%)] and pH adjustment (near pH 6.5) after sterilization with a high-pressure sterilizer (121 ℃, 20 minutes) 5% (v / v), Leuconostok Mesenteroids MKSR ( Leuconostoc mesenteroides MKSR, KCTC13842BP), Leuconostoc mesenteroides MKJW ( Leuconostoc mesenteroides MKJW, KCTC14459BP), or Lactobacillus plantarum MKHA15 ( Lactobacillus plantarum MKHA15, KCTC13928BP) (about 5.8-6.8 log CFU/ml) Inoculated and fermented.

<Example 2> Preparation of powder formulation of fermented bitter melon

1. Preparation of powdered excipients

Dextrin [glucose equivalent (D.E.): 1~25], fructofiber (Degree of polymerization: 5~25), oligosaccharide, cyclodextrin (alpha, beta, gamma), water-soluble dietary fiber, lactose, dextran, indigestible maltose Dextrin [glucose equivalent (D.E.): 5-25] was prepared.

2. Powdering method

First, a concentrate was prepared by concentrating the fermented bitter gourd extract prepared in Example 1 to 5-30%, and then a mixed solution was prepared by adding the prepared excipients to the prepared concentrate. The prepared mixture was adsorbed by heating and stirring at 45 to 70 ° C for 20 to 40 minutes, sterilized at 70 to 90 ° C for 10 to 20 minutes, and then dried.

The drying method may use spray drying (Inlet Temp. 135 ~ 195 ℃, Outlet Temp. 70 ~ 95 ℃), vacuum drying (Room Temp. 45 ~ 85 ℃) or freeze drying method, but among them, the spray drying method is most efficient

<Example 3> Fermentation of bitter gourd extract for optimization experiment

1. Experiment materials and strains used

Bitter gourd and water were mixed, followed by primary extraction by heating at 121° C. for 5 hours, and then secondary extraction by heating at 105° C. for 3 hours by adding more water to prepare a bitter gourd extract. Bitter gourd extract was stored at -20 ℃ until use. The strain used for fermenting bitter gourd extract was Leuconostoc mesenteroides MKJW (KCTC14459BP) isolated from kimchi, and was used in the experiment after culturing in MRS broth for 24 hours. It was inoculated so that the initial number of bacteria was about 7 log CPU/mL and fermented for 24 hours.

2. Fermentation conditions

Response surface methodology (RSM) was performed using the Stat-Ease program (Design-Expert® software, version 12, Stat-Ease, Inc., Minneapolis, MN, USA) to optimize the fermentation conditions of bitter gourd extract. conducted.

independent variable density I middle go X ₁ : Bitter gourd extract (°brix) 5 10 15 X ₂ : sucrose (mmol) 80 240 400 X ₃ : maltose (mmol) 0 125 250

As in Table 1, the factors considered as independent variables (Xn), that is, concentration of bitter gourd extract (X1; 5-15 °brix), sucrose concentration (X2; 80-240 mmol), maltose concentration (X3; 125- 250 mmol), the experimental range was set in three stages.

Run X ₁ X ₂ X ₃ One 10 240 125 2 5 240 250 3 15 80 125 4 15 240 250 5 10 400 250 6 10 240 125 7 10 80 250 8 15 400 125 9 5 240 0 10 15 240 0 11 5 400 125 12 10 240 125 13 5 80 125 14 10 240 125 15 10 400 0 16 10 240 125 17 10 80 0

As shown in Table 2 above, fermentation experiments were conducted by setting 17 groups. As a dependent variable affected by the independent variable, alpha glucosidase inhibitory activity was measured and the value was used in the analysis. In all experimental groups, YPK (0.5% yeast extract, 0.5% peptone, 2% K ₂ HPO ₄ ) and mineral mixture (0.02% MgSO₄, 0.001% FeSO₄, 0.001% NaCl, 0.001% MnSO₄, 0.013% CaCl₂) were included, Fermentation was carried out at 30 ° C for 24 hours.

<Experimental Example 1> pH analysis of fermented bitter gourd extract

After inoculating the strains according to Example 1, pH was measured after fermentation for 24 hours at a temperature of 30 ° C for MKSR and MKJW and 37 ° C for MKHA15 in a shaking incubator at 140 rpm. The control group was analyzed after standing for 24 hours at 140 rpm and 37° C. in a shaking incubator after adding the same amount of 5% (v/v) sterilized 0.1% peptone without inoculating the bacteria.

As a result, as shown in Table 3 below, the lower the concentration of bitter gourd extract, the lower the pH. In the case of MKSR and MKJW, the pH change in the pH-adjusted group was large, and in MKHA15, the pH change was relatively greater when nutrient supplements were added than when the pH was adjusted.

In Table 3, “YH” indicates the addition of nutritional supplements (yeast extract, peptone and potassium phosphate) and mineral mixture to a mixture of sucrose, maltose, and bitter gourd extract (Momordica charantia L.) and pH It refers to the group fermented by adjusting the value.

The "Y" means a group fermented by adding a nutritional supplement and a mineral mixture to a mixture of sucrose, maltose, and bitter gourd extract.

The "H" refers to a group fermented by adjusting the pH value of a mixture of sucrose, maltose, and bitter gourd extract.

The "-" means a group in which only the bitter gourd extract was fermented with each strain. Hereinafter, the same applies.

<Experimental Example 2> Viable cell count analysis of fermented bitter gourd extract

After inoculating the strains according to Example 1, MKSR and MKJW were fermented at 30 ° C. and MKHA15 at 37 ° C. for 24 hours in a shaking incubator at 140 rpm, and then the number of viable cells was measured. The control (control) was analyzed after the addition of the same amount of 5% (v / v) sterilized 0.1% peptone without inoculation of bacteria, and then left in a shaking incubator at 140 rpm and 37 ° C for 24 hours. All values are expressed as mean ± SD (n = 2).

As a result, as shown in Table 4 below, as the concentration of bitter gourd extract increased, the viable cell count decreased, but MKHA15 showed the highest viable cell count at 8 brix. In MKSR and MKJW, when the concentration of bitter gourd extract was 12 brix, the pH control was a bigger growth factor than the addition of nutritional supplements, and in the case of MKHA15, the addition of nutritional supplements was more important than pH control. In addition, when the concentration of bitter melon extract was 4 or 8 brix, the group added with nutritional supplements showed a higher growth rate than the group with adjusted pH.

<Experimental Example 3> Analysis of digestive enzyme inhibitory activity

Alpha-glucosidase inhibitory activity was measured by adding 50μL of 0.02M sodium phosphate buffer (pH 6.8), 2mM pNPG (4-Nitrophenyl α-D-gl ucopyranoside) solution and 50μL of alpha-glucosidase enzyme. After adding 50 μL of a 10-fold dilution of the fermented extract, the mixture was reacted at 37° C. for 40 minutes, 1.5 mL of 0.1 M Na ₂ CO ₃ was added, and the absorbance was measured at 405 nm. Acarbose, an antidiabetic drug, was tested as a positive control. All values are expressed as mean±SD (n=3).

[Equation 1]

Inhibition activity (inhibitionm, %) = {1-( Abs _sample - Abs _{sample blank} )/( Abs _control - Abs _blank ) }*100

Abs _sample : Absorbance of the sample

Abs _{sample blank} : Absorbance of sample blank test

Abs _control : Absorbance of the control group

Abs _blank : Absorbance of blank test

As a result, as shown in Table 5 below, the higher the concentration of bitter gourd extract, the higher the alpha-glucosidase inhibitory activity. There was no significant difference according to the inoculum at the concentration of 12 brix between the fermented product of bitter gourd extract and the control group, but at the concentration of 4 or 8 brix, the group with addition of nutritional supplement and pH control and the group with addition of nutritional supplement showed no significant difference according to fermentation. It can be seen that there is an enemy difference.

* In Table 3, other lowercase letters in the same row and other uppercase letters in the same column indicate significant differences (P<0.05). Hereinafter, the same applies.

<Experimental Example 4> Antioxidant activity analysis

DPPH radical scavenging ability was measured by modifying the method of Lin et al. (2000). In the experiment, 300 μL of the 40-fold dilution of the fermented product of bitter gourd extract and 1.2 mL of 0.2 mM DPPH (2,2-Diphenyl-1-picrylhydrazyl: Sigma Aldrich, StLouis, USA) were mixed and reacted in the dark for 30 minutes, and then the absorbance was measured at 517 nm. It was calculated by substituting into Equation 2 below. Ascorbic acid was tested as a positive control.

[Equation 2]

DPPH radical scavenging activity (%) = {1-( Abs _sample - Abs _{sample blank} )/ Abs _control }*100

Abs _sample : Absorbance of the sample

Abs _{sample blank} : Absorbance of sample blank test

Abs _control : Absorbance of the control group

As a result, as shown in Table 6 below, most of the MKSR except for the fermented bitter gourd extract fermented group at the concentration of 4, 8 or 12 brix and the group in which nutritional supplements were added to the concentration of 12 brix of the bitter gourd extract. and MKJW strains showed higher antioxidant activity, and MKHA showed no significant difference from the control group.

<Experimental Example 5> Analysis of cholinease inhibitory activity

Acetylcholinesterase (acetylcholinesterase, AChE) enzyme inhibitory activity measurement was measured by modifying the method of F. Sharififar et al. (2012). Mix 125μL of a 10-fold dilution of fermented product of bitter gourd extract diluted with 0.2M SPB (pH7.7), 40μL of dithionitrobenzoic acid (DTNB), and 5μL of AChE (SigmaAldrich, StLouis, USA) for 5 minutes After standing, 7.5 μL of acetylthiocholine iodide was added, and the absorbance was measured at 412 nm for 15 minutes, and the absorbance was substituted into Equation 3 below. All values were expressed as mean ± SD (n = 4), and galantamine was used as a positive control.

Butyrylcholi nesterase (BChE) enzyme inhibitory activity was measured by mixing 125 μL of a 30-fold dilution of fermented product of bitter gourd extract, 40 μL of dithionitrobenzoic acid (DTNB), and 5 μL of Acetyl cholinesterase (AChE) enzyme, leaving it for 5 minutes, then butyryl Choline iodide (butyrylcholine iodide) 7.5μL was added and the absorbance was measured at 412nm for 15 minutes and measured by substituting into the following formula 3. All values were expressed as mean ± SD (n = 2), and galantamine was used as a positive control.

[Equation 3]

AChE and BuChE inhibitory activity = [1 - ( S _sample / S _control )] × 100

S _sample : slope of the sample

S _control : slope of the control group

As a result, as shown in Tables 7 and 8 below, acetylcholinesterase inhibitory activity except for the fermentation group of 4 or 8 brix concentration of bitter gourd extract and the group in which the pH was adjusted to the concentration of 4 brix of bitter gourd extract, in which fermentation was not performed well. and the inhibitory activity of the fermented group was significantly higher than that of the control group. Butyrylcholinesterase inhibitory activity showed a significant difference according to the concentration of bitter gourd extract, but there was no significant difference between the fermented group and the control group.

<Experimental Example 6> Optimization experiment for alpha glucosidase inhibitory activity

In order to perform an optimization experiment, the Goji extract was fermented as in Example 3 above. As shown in Table 2, fermentation was carried out by setting 17 groups, and then each group was chromatographed, residuals were checked to find the optimal conditions for alpha glucosidase inhibitory activity, and the variables for alpha glucosidase inhibitory activity The response surface model graph for the interaction effect was confirmed in 3D.

Run Log CFU/mL pH TSS (°brix) α-glucosidase inhibition 0 h 24h 0 h 24h 0 h 24h One 7.21 9.56 6.56 4.41 26.7 25.2 86.82 2 7.21 9.89 6.31 4.05 25.5 23.9 92.65 3 7.21 9.54 6.56 4.89 25.9 24.1 75.67 4 7.21 9.45 6.46 5.24 34.5 32.9 76.70 5 7.21 9.67 6.38 4.62 35.6 33.8 80.27 6 7.21 9.60 6.46 4.41 26.8 25.3 85.93 7 7.21 9.73 6.50 4.60 24.8 23.0 76.92 8 7.21 8.30 6.35 5.44 36.4 34.7 65.43 9 7.21 9.66 6.63 4.07 17.8 16.6 81.36 10 7.21 9.20 6.48 4.83 27.9 26.2 67.87 11 7.21 10.17 6.54 4.02 27.5 26.1 97.50 12 7.21 9.46 6.52 4.38 26.6 25.0 87.62 13 7.21 9.15 6.64 4.39 15.8 14.3 68.67 14 7.21 9.62 6.49 4.40 26.7 25.2 86.39 15 7.21 9.75 6.50 4.44 28.6 27.4 78.56 16 7.21 9.49 6.48 4.40 26.7 25.3 85.11 17 7.21 9.79 6.61 4.58 17.0 15.5 65.17

As a result, according to Table 9, when MKJW was inoculated with 7.21 Log CFU/mL in all experimental groups, lactic acid bacteria proliferated within the range of 8.3 - 10.2 Log CFU/mL. Alpha glucosidase inhibitory activity ranged from 65.2 to 97.5%. The highest inhibition rate (97.5%) was achieved when fermented with Experimental Group 11 (5°brix of bitter gourd extract, 400 mmol of sucrose, 125 mmol of maltose). When fermented with the addition of sucrose, Leuconostoc megenteroise is distinguished from lactic acid bacteria of the Lactobacillus family by its ability to produce dextran, a complex polysaccharide. In addition, according to FIG. 2, it was confirmed that some leuconostoc megenteroses, including MKJW and MKSR, produced oligosaccharides when fermented by adding sucrose and maltose together. In the case of the experimental groups (Nos. 9, 10, 15, and 17) in which maltose was not added, the alpha-glucosidase inhibitory activity was relatively low even though the number of viable cells was increased. It was confirmed that experimental group 11 (lane 11A) in FIG. 2 produced more oligosaccharides than 9A, 10A, 15A, and 17A.

sum of squares df mean square F-value P-value Quadratic model 1411.60 9 156.84 91.97 <0.0001 X ₁ 371.40 One 371.40 217.78 <0.0001 X ₂ 156.01 One 156.01 91.48 <0.0001 X ₃ 141.01 One 141.01 82.68 <0.0001 x ₁ x ₂ 381.36 One 381.36 223.61 <0.0001 X ₁ X ₃ 1.52 One 1.52 0.8911 0.3766 X ₂ X ₃ 25.20 One 25.20 14.78 0.0063 X ₁ ² 27.87 One 27.87 16.34 0.0049 X ₂ ² 205.44 One 205.44 120.46 <0.0001 X ₃ ² 72.82 One 72.82 42.70 0.0003 Residual 11.94 One 1.71 Lack of Fit 8.38 7 2.79 3.14 0.1492 Pure Error 3.56 4 0.8903 Correlation total 1423.53 16

According to Table 10 and FIGS. 3 to 6, the suitability of the model for the alpha-glucosidase inhibitory activity of a total of 17 experimental groups using three variables was shown. As a result of statistical model analysis, optimal conditions for high alpha-glucosidase inhibitory activity were determined to be 5.015 °brix of bitter gourd extract, 393.97 mmol of sucrose, and 143.386 mmol of maltose. As a result of response surface analysis, the fermentation results actually tested under optimal fermentation conditions are shown in Table 11 below, and according to Table 12 below, alpha glucosidase inhibitory activity was 98.02%, which was not significantly different from the predicted result (P > 0.05 ).

Log CFU/mL pH TSS (°brix) 0 h 24h 0 h 24h 0 h 24h Optimal condition ¹⁾ 7.19±0.03 9.64±0.06 6.85±0.02 4.15±0.01 25.75±0.05 23.80±0.00

Predicted value Observed value P -value α-glucosidase inhibition activity (%) 98.02 98.02±1.20 0.998

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. Do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC13842BP 20190425 Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC14459BP 20210121 Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC13928BP 20190827

Claims (13)

In a mixture containing bitter gourd extract and sugar, Leuconostoc mesenteroides MKSR deposited as KCTC13842BP, Leuconostoc mesenteroides MKJW deposited as KCTC14459BP, and Lactobacillus plantarum deposited as KCTC13928BP MKHA15 ( Lactobacillus plantarum MKHA15) Characterized in that inoculated with one or two or more strains selected from the group consisting of, anti-dementia, anti-diabetic or antioxidant fermented product of bitter melon. The fermented bitter gourd product according to claim 1, wherein the sugar is sucrose, maltose or a mixture thereof. [Claim 2] The fermented product of bitter melon according to claim 1, characterized in that it consists of 4-8 °brix of bitter gourd extract, 350-450 mmol of sucrose and 100-150 mmol of maltose. A pharmaceutical composition for the prevention or treatment of cognitive dysfunction containing the fermented bitter gourd extract according to claim 1 as an active ingredient. According to claim 4,
The cognitive dysfunction,
A pharmaceutical composition for preventing or treating cognitive dysfunction, characterized in that it is selected from the group consisting of dementia, Alzheimer's dementia, vascular dementia, learning disability, forgetfulness, agnosia, aphasia, apraxia, delirium and mild cognitive impairment. A health functional food composition for preventing or improving cognitive dysfunction containing the fermented bitter gourd extract according to claim 1 as an active ingredient. A pharmaceutical composition for preventing or treating diabetes containing the fermented product of bitter melon according to claim 1 as an active ingredient. A health functional food composition for preventing or improving diabetes containing the fermented product of bitter melon according to claim 1 as an active ingredient. Preparing a mixture of bitter melon extract having a sugar content of 3 to 15 brix by adding a sugar component; and
The prepared mixture was prepared by Leuconostoc mesenteroides MKSR deposited as KCTC13842BP, Leuconostoc mesenteroides MKJW deposited as KCTC14459BP, and Lactobacillus plantarum MKHA15 deposited as KCTC13928BP MKHA15) inoculated with one or two or more strains selected from the group consisting of and fermented; anti-dementia, anti-diabetic or anti-oxidant activity is enhanced, containing a fermented bitter gourd extract manufacturing method. According to claim 9,
The sugar component,
Sucrose, maltose, glucose, galactose, lactose, mannose, m altooligosaccharide, fructooligosaccharide, starch ) and dextrin (dextrin) characterized in that one or more than one selected from the group consisting of, bitter melon fermented product manufacturing method. According to claim 9,
The sugar component is a method for producing fermented bitter gourd, characterized in that consisting of 350-450 mmol of sucrose and 100-150 mmol of maltose. According to claim 9,
To prepare the mixture,
Yeast extract, peptone, potassium phosphate, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, sodium chloride in the bitter gourd extract , Manganese sulfate (manganese sulfate) and calcium chloride dihydrate (calcium chloride dihydrate) characterized in that by further adding one or two or more nutritional supplements selected from the group consisting of, bitter melon fermented product manufacturing method. According to claim 9,
The fermentation step is
Characterized in that it is carried out for 20 to 30 hours at a pH of 6 to 7 and a temperature of 25 to 40 ° C., a method for producing fermented bitter gourd.

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