KR102557702B1 - Composition for anti-inflammation comprising Aronia vinegar - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition containing aronia vinegar, and more specifically, contains aronia vinegar prepared by mixing alcohol extract and pear concentrate of pulverized aronia, followed by alcoholic fermentation and acetic acid fermentation, followed by centrifugation. It relates to an anti-inflammatory composition, particularly a food composition for preventing and/or improving inflammation. Aronia vinegar according to the present invention inhibits NO activity in macrophages and inhibits the production of IL-6, so it is useful as a pharmaceutical and food composition for preventing, improving, and treating inflammation. Moreover, the aronia vinegar according to the present invention is non-cytotoxic and can be safely used because it is a material derived from natural aronia.

Description

Composition for anti-inflammation comprising Aronia vinegar

The present invention relates to an anti-inflammatory composition containing aronia vinegar, and more specifically, contains aronia vinegar prepared by mixing alcohol extract and pear concentrate of pulverized aronia, followed by alcoholic fermentation and acetic acid fermentation, followed by centrifugation. It relates to an anti-inflammatory composition, particularly a food composition for preventing and/or improving inflammation.

Inflammation is a defense mechanism that occurs in biological tissues to respond to external stimuli such as physical and chemical stimuli or tissue damage and bacterial infection. However, the continuous inflammatory response rather increases vascular permeability by vasoactive substances such as histamine, prostaglandins, and leukotriene, which induces chronic inflammation or promotes mucosal damage, resulting in functional disorders such as pain, edema, and fever. It causes a variety of diseases, including inflammatory diseases. Macrophages, one of the cells involved in the inflammatory response in the body, play a very important role in maintaining homeostasis as cells that regulate inflammatory responses and immune functions through phagocytosis. Macrophages are activated by stimulation of LPS (lipopolysaccharide), a component of the outer membrane of Gram-negative bacteria, and play a pivotal role in biological defense in the early stage of infection. However, macrophages activated by excessive LPS stimulation are induced to increase inflammatory cytokines. Among the pro-inflammatory cytokines secreted by LPS in macrophages, IL-6 converts an acute inflammatory response into a chronic phase by differentiating B cells into plasma cells and promoting antibody production (Kang BK. et al. al., Microbiol. Biotechnol. Lett., 44:236-245, 2016). In addition, TNF-α is a cytokine involved in systemic inflammation, and excessive production induces fever and apoptosis, and IL-1β acts as a major mediator of inflammatory responses in activated macrophages (Kim JH. et.al. , The Korean Society of Food Preservation, 24:1149-1157, 2018).

In the inflammatory process in the body, inflammatory factors such as excessive nitric oxide (NO) and prostaglandin E2 (PEG2) are produced by inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). NO is a highly reactive substance and is produced from L-arginine by NO synthase (NOS), and when stimulated by external stimuli or pro-inflammatory cytokines, a large amount of NO is produced. It has been reported that overproduced NO promotes inflammatory responses such as vascular permeability and edema. COX, another major inflammatory mediator, is an enzyme that promotes the conversion of arachidonic acid to prostaglandin after the release of arachidonic acid from phospholipids in cell membranes. It is expressed in large amounts in cells such as macrophages and monocytes by stimulation. The resulting prostaglandin suppresses tumor apoptosis and induces angiogenesis to be involved in tumor formation. Therefore, as a composition for preventing inflammatory diseases and macrophage-mediated inflammatory responses, NO-generating enzyme iNOS, prostaglandin biosynthesis step enzyme COX-2, and expression control of inflammatory cytokines and their major signaling molecules, MAPKs and NF- Substances capable of regulating the expression of κB are attracting attention as compositions for preventing and improving inflammation. In particular, studies on compositions for preventing and improving inflammation by natural products are being actively conducted (Kim SY. et. al., J. Korean Orient. Med. Ophthalmol. Otolaryngol. Dermatol., 26:54-64, 2013).

Accordingly, while the present inventors were conducting various studies on Aronia (Aronia melanocarpa), it was confirmed that Aronia vinegar had a very excellent anti-inflammatory effect and completed the present invention.

One object of the present invention is to provide an anti-inflammatory composition comprising aronia vinegar as an active ingredient.

Another object of the present invention is to provide a food composition for preventing and / or improving inflammation containing aronia vinegar as an active ingredient.

As one aspect, the present invention provides an anti-inflammatory composition comprising aronia vinegar as an active ingredient.

In the present invention, the aronia vinegar refers to the supernatant by mixing the alcohol extract and pear concentrate of the aronia fruit lysate, aging it after alcohol fermentation, fermenting it with acetic acid, and centrifuging.

Specifically, the aronia vinegar comprising the steps of preparing aronia lysate; Adding pear concentrate to the aronia lysate; Alcohol extraction of the aronia lysate to which the pear concentrate is added; Preparing Aronia wine by alcoholic fermentation of the alcohol extract of the Aaronia lysate; Acetic acid fermentation of the Aronia wine; And centrifuging the acetic acid fermentation product to obtain a supernatant of aronia vinegar; includes.

Hereinafter, the manufacturing method of the aronia vinegar will be described in detail.

First, it is a step of preparing aronia lysate.

In the present invention, the aronia refers to aronia fruit, and the aronia lye refers to grinding the aronia fruit by a conventional method such as a mixer or grinder.

Next, it is a step of adding pear concentrate to the aronia lysate.

The pear concentrate refers to a concentrated pear extract. The pear extract may be extracted using water or an organic solvent, but is preferably a water extract of the pear, more preferably a hot water extract of the pear.

The pear concentrate is added in an amount of 1.5 to 2.5% by weight, preferably 1.8 to 2.2% by weight, more preferably 2% by weight based on 100% by weight of the aronia lysate. If the content of the pear concentrate is less than 1.5% by weight, the alcohol content of the aronia wine produced thereafter does not reach the target level (ie, 10% or more), and if the content of the pear concentrate exceeds 2.5% by weight, the content In comparison, a large increase in the alcohol content of Aronia wine produced later cannot be expected.

In one specific embodiment, when 1% by weight of the pear concentrate was used based on 100% by weight of aronia lysate, the alcohol content of the prepared aronia wine was less than 10% compared to when 2% by weight was used, and yeast metabolites It was confirmed that the content of one, beta-glucan, was also significantly reduced.

Next, it is a step of extracting alcohol from the aronia lysate to which the pear concentrate is added.

The alcohol extraction refers to extraction with alcohol having an alcohol concentration of 20 to 80%, preferably 50 to 80%, more preferably 60 to 80%, and most preferably 70%.

In one specific implementation, when alcohol was extracted from aronia lysate to which pear concentrate was added at different concentrations of alcohol, reducing sugar, a sugar used in fermentation, was 20% or more when the concentration of alcohol was 20% or more. In view of the previous research results that the alcohol content by yeast fermentation corresponds to 1/2 of the reducing sugar level, the reducing sugar content of the alcohol extract must be 20% or more so that the alcohol content of the aronia wine produced thereafter reaches the target level (i.e., 10%). % or more), it is preferable that the concentration of alcohol is at least 18% or more, preferably 20% or more. However, when the alcohol concentration is 70%, the anthocyanin content in the extract is the highest compared to other alcohol concentrations, so 70% alcohol is most preferably used.

The alcohol extraction may be performed at 80 to 100°C, preferably 90 to 100°C for 2 to 4 hours, preferably 2.5 to 3.5 hours, and most preferably 3 hours.

In the present specification, an extract prepared by extracting alcohol from the aronia lysate to which the pear concentrate is added may be used as an alcohol extract of the aronia lysate or an aronia lysate extract for convenience.

Next, it is a step of producing Aronia wine by alcoholic fermentation of the alcohol extract of the aronia lysate.

The alcoholic fermentation is carried out using one or more strains selected from the group consisting of Saccharomyces cerevisiae and Saccharomyces boulardii, preferably Saccharomyces cerevisiae and Caromyces It refers to a mixture of Buladai strains, more preferably Saccharomyces cerevisiae, inoculated into the alcohol extract of the aronia lysate and cultured with shaking. Aronia wine with high alcohol content can be produced when alcoholic fermentation is performed with the Saccharomyces cerevisiae strain, and Aronia wine with high beta-glucan content can be produced when alcoholic fermentation is performed with the Karomyces bulla strain. can In particular, the Karomyces Bulladi strain is a new strain deposited as KCTC 14036BP on November 18, 2019 with the Korea Research Institute of Bioscience and Biotechnology as a newly discovered strain by the present applicant.

The shaking culture is performed for 70 to 74 hours at room temperature at 100 to 130 rpm.

In one specific implementation, when the alcoholic fermentation was performed for 70 to 74 hours, preferably 72 hours, it was possible to produce Aronia wine with a target level of alcohol content compared to less time.

During the alcohol fermentation, the inoculation of the fermentation strain is 0.08 to 0.12% by volume, preferably 0.09 to 0.11% by volume, and most preferably 0.1% by volume based on the total volume% of the alcohol extract of the aronia lysate.

In the present specification, the fermented product by alcoholic fermentation of the alcohol extract of the aronia lysate may be collectively referred to as aronia wine for convenience.

On the other hand, the prepared aronia wine may be filtered before subsequent acetic acid fermentation. As one specific example, the filtration may be performed in the order of a primary non-woven fabric filter and a secondary paper filter. After this filtration, acetic acid fermentation can be performed smoothly.

In addition, the prepared aronia wine may be aged for 1 to 3 months, preferably 2 months, in oak barrels before subsequent acetic acid fermentation. The aging may be performed before or after the filtration, preferably after filtration. By this aging, the pH, acidity, sugar content, etc. of Aronia wine are almost unchanged, but the taste and aroma of Aronia wine have an effect suitable for the taste.

Next, it is a step of acetic acid fermentation in the aronia wine.

The acetic acid fermentation refers to inoculation of acetic acid bacteria and fermentation at room temperature for 160 to 170 hours, preferably 165 to 170 hours, more preferably 166 to 170 hours, and most preferably 168 hours at room temperature.

In the acetic acid fermentation, it is preferable to inject air for 1 hour per day during the fermentation process. Acetic acid fermentation is effectively progressed by such air injection, and a fermented acetic acid product having a desired level of pH, acidity, sugar content, etc. can be produced.

The acetobacter is a strain of the genus Acetobacter, preferably Acetobacter aceti, and is 0.8 to 1.2% by volume, preferably 0.9 to 1.1% by volume, most preferably 0.9 to 1.1% by volume, based on the total volume% of Aronia wine. It is inoculated with an amount of 1% by volume.

In the case of acetic acid fermentation under the above-mentioned conditions, the pH, acidity, and alcohol content of the fermented product are contained enough to suit the taste and aroma of the aronia vinegar beverage.

Next, it is a step of centrifuging the acetic acid fermentation product to obtain a supernatant of aronia vinegar.

The supernatant by centrifugation is aronia vinegar, but the precipitate by centrifugation is a by-product of aronia vinegar and can be dried and powdered by a method such as hot air drying and used as a material such as food.

In this specification, the supernatant obtained by centrifugation of the acetic acid fermentation is referred to as aronia vinegar, and the precipitate obtained by centrifugation of the acetic acid fermentation is referred to as aronia vinegar by-product or aronia fermentation by-product.

Aronia vinegar prepared by the above method had excellent NO inhibitory activity and inhibited the production of IL-6 among cytokines compared to aronia vinegar by-products. Thus, the aronia vinegar is useful as an anti-inflammatory application.

The anti-inflammatory composition of the present invention can be used as a pharmaceutical composition for preventing and/or treating inflammation, and a food composition for preventing and/or improving inflammation.

As one specific aspect, the present invention provides a pharmaceutical composition for preventing and / or treating inflammation containing aronia vinegar.

Aronia vinegar contained in the pharmaceutical composition of the present invention is as described above.

The pharmaceutical composition of the present invention may further include other substances known to have anti-inflammatory effects, in particular substances derived from natural materials, in addition to aronia vinegar as an active ingredient.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" of the present invention means exhibiting characteristics that are not toxic to cells or humans exposed to the composition. The carrier may be used without limitation as long as it is known in the art such as a buffer, a preservative, a pain reliever, a solubilizer, an isotonic agent, a stabilizer, a base, an excipient, a lubricant, and the like.

In addition, the pharmaceutical composition of the present invention is formulated according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions. can Furthermore, it may be used in the form of ointments, lotions, sprays, patches, creams, powders, suspensions, gels, or skin external preparations in the form of gels. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the aronia vinegar, for example, starch, calcium carbonate, sucrose It is prepared by mixing sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions for oral 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. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, and lyophilized formulations. 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.

Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" of the present invention means introducing a predetermined substance into a subject by an appropriate method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, but not limited thereto.

The term "individual" refers to all animals such as rats, mice, and livestock, including humans. Preferably, it may be a mammal including a human.

The term "pharmaceutically effective amount" means an amount that is sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is determined by the patient's sex, age, and weight , health condition, type of disease, severity, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration, and excretion rate, duration of treatment, factors including drugs used in combination or concomitantly, and other medical fields well known. It can be easily determined by a person skilled in the art according to known factors. Administration may be administered once a day at the recommended dose, or may be administered in several divided doses.

As another specific aspect, the present invention provides a food composition for preventing and / or improving inflammation containing aronia vinegar.

When the composition of the present invention is used as a food additive, the aronia vinegar may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment), and may further include food additives acceptable in food science. Since the composition of the present invention uses a compound derived from a fraction of a natural extract as an active ingredient, there is no problem in terms of stability, so there is no great limitation on the mixing amount.

The food composition of the present invention may include all food in a conventional sense, and may be used interchangeably with terms known in the art, such as functional food and health functional food.

The term "functional food" of the present invention refers to food manufactured and processed using raw materials or ingredients having useful functionality for the human body in accordance with the Act on Health Functional Foods No. 6727, and "functional" refers to the structure of the human body. And it refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients for functions or physiological functions.

In addition, the term "health functional food" of the present invention refers to food manufactured and processed by using specific ingredients as raw materials or by extracting, concentrating, refining, mixing, etc. specific ingredients contained in food ingredients for the purpose of health supplementation. It refers to food designed and processed by the above ingredients to sufficiently exert bioregulatory functions such as biodefense, regulation of biorhythm, prevention and recovery of disease, etc. It can perform functions related to recovery, etc.

There are no restrictions on the types of foods in which the composition of the present invention can be used. In addition, the composition containing the aronia vinegar of the present invention as an active ingredient can be prepared by mixing suitable other auxiliary ingredients that may be contained in food and known additives according to the selection of those skilled in the art. Examples of food that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and There are vitamin complexes, etc., and it can be prepared by adding aronia vinegar according to the present invention to jellies and juices prepared as a main component.

In addition, foods that can be applied to the present invention include, for example, special nutritional foods (e.g., formula milk, infant, baby food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), health supplement food , seasonings (eg soy sauce, soybean paste, gochujang, mixed paste, etc.), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various types of kimchi, pickled vegetables, etc.) ), beverages (eg fruit, vegetable drinks, soy milk, fermented beverages, etc.), and natural seasonings (eg, ramen soup, etc.).

When the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweeteners, flavoring agents, or natural carbohydrates as additional components, like conventional beverages. In addition to the above, the health functional food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, and glycerin. , alcohol, a carbonating agent used in carbonated beverages, and the like. In addition, it may contain fruit flesh for the manufacture of natural fruit juice, fruit juice beverages and vegetable beverages.

Aronia vinegar according to the present invention inhibits NO activity in macrophages and inhibits the production of IL-6, so it is useful as a pharmaceutical and food composition for preventing, improving, and treating inflammation. Moreover, since the aronia vinegar according to the present invention is non-cytotoxic and is a material derived from natural aronia, it can be safely used in animals including humans.

1 is an optimal process flow chart for mass production of aronia vinegar according to an embodiment of the present invention.
Figure 2 is a picture confirming the cytotoxicity of aronia vinegar and aronia vinegar by-products according to an embodiment of the present invention.
Figure 3 is a picture confirming the NO inhibitory activity of aronia vinegar and aronia vinegar by-products according to an embodiment of the present invention.
Figure 4 is a picture confirming the production of cytokines of aronia vinegar and aronia vinegar by-products according to an embodiment of the present invention.

Hereinafter, examples and the like will be described in detail to aid understanding of the present invention. However, the embodiments according to the present invention can be modified in many different forms, and the scope of the present invention should not be construed as being limited to the following examples. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Example 1: Optimal yeast strain experiment for producing fermented aronia

Aronia fruits purchased directly from aronia cultivating farms were washed and crushed with a blender (Mufti Quick 7-MQ736, BRAUN, Germany). 1 L of the aronia lysate was used as a medium for this experiment, and the medium was sterilized at 121 ° C for 15 minutes and then allowed to cool for 2 hours. Since then, Saccharomyces cerevisiae (KCTC NO. 27756), known as yeast used for fermentation of beer or bread, and Saccharomyces boulardii srcnm 001, a new strain discovered by the present applicant , Kazachstnia servazzii, known as an acid-resistant yeast found in kombucha tea leaves, and 4 species of Galactomyces geotrichum, whose metabolites are used as cosmetics, were added at 0.1% of the volume of the medium. Inoculated and incubated at 120 rpm at 27 ° C for 44 to 46 hours. After cultivation, the fermentation characteristics of the aronia lysate by four types of yeast fermentation were analyzed as follows.

Experiment method

1) Measurement of pH, titratable acidity and amino acid degree

The pH of the fermentation product was diluted by adding 9 mL of distilled water to 1 mL of the sample, and then measured using a pH meter (pH-meter P-71, HORIBA, Kyoto, Japan). The titratable acidity was obtained by titrating after mixing 1 mL of sample and 1 mL of NaOH, dropping a 1% Phenolphthalein solution reagent, and checking the color change section. The amino acid level was tested by adding 9 mL of distilled water to 1 mL of sample, titrating with 0.1 N NaOH until reaching pH 8.3, and converting the amount used into tartaric acid content (%, v/v).

2) Measurement of reducing sugar content

The reducing sugar content of the fermentation product was measured according to the dinitro salicylic acid (DNS) method. 3 mL of DNS reagent was added to 1 mL of a diluted sample solution of the supernatant obtained by centrifugation of the fermented product (1,500 xg, 20 min), and the color was developed at 100 ° C. for 5 minutes. After cooling to room temperature in the dark for 50 minutes, absorbance was measured at 550 nm. The reducing sugar content was calculated after preparing a standard curve using glucose as the standard material.

3) Measurement of total sugar content

The total sugar content of the fermented product was measured using the phenol-sulfuric acid method. After 2-fold dilution of the fermented product, 1 mL of the mixture was weighed, and then 1 mL of phenol solution was added and mixed well. Thereafter, 5 mL of concentrated sulfuric acid was added and reacted by exotherm as strongly as possible. After adding sulfuric acid, the mixture was allowed to stand at room temperature for 40 minutes, and absorbance was measured at 470 nm. The total sugar content was calculated after preparing a standard curve using glucose as the standard material.

4) Measurement of °Brix (Soluble sugar content) content

The Brix content of the fermented product was measured using a sugar meter (Pocket refractometer PAL-2, ATAGO, Kyoto, Japan). After quantifying 500 ul of the fermented product, the average value was calculated by repeating the measurement three times. When measuring, the experiment was conducted after adjusting the zero point using distilled water.

5) Measurement of viable cell count

The number of viable cells in the fermented product was taken by taking 1 mL of the fermented product, diluted 10 ⁴ , 10 ⁵ , and 10 ⁶ stepwise with sterile physiological saline by a 10-fold dilution method, smeared 20 μL on the medium, and then placed in a 30 ° C incubator (Shaking incubator, Daewon Science, Co., Ltd.). Jaecheon, Korea), colony colonies formed by culturing for 24 hours were counted and expressed as colony forming unit (CFU/mL).

6) Measurement of beta glucan content

Beta-glucan content was analyzed using Megazyme Kit (Mushroom and Yeast β-glucan Assay Procedure K-YBGL). The measured values of total glucan and α-glucan measured at absorbance 510nm are available on the website of www.megazyme.com along with the absorbance value of the reaction solution obtained by reacting glucose solution (1mg/mL) with the GOPOD reagent. Calculated by referring to the calculation formula for the content of Calc content (%, w / w). Finally, beta-glucan was calculated by subtracting the alpha-glucan content from the total glucan content. In Equation 1 below, the experiment was conducted in accordance with the Ministry of Food and Drug Safety health functional food regulations.

[Equation 1]

Total glucan content (mg/g) = C X (a X b )/S X 0.9

Alpha glucan content (mg/g) = C X (a X b )/S X 0.9

Beta glucan content (mg/g) = total glucan content - alpha glucan

C: Concentration of test solution (ug/mL)

a: Total amount of test solution (mL)

b: dilution factor

S: sample weight (mg)

0.9: beta glucan conversion factor (160/180)

7) DO, BOD measurement

The amount of dissolved oxygen and biochemical oxygen demand of the fermentation product were measured using (DO-31P, TOADKK Co., Ltd., Tokyo, Japan). 15 mL of the fermented product was quantified and repeated three times to calculate the average value. However, the amount of dissolved oxygen on the 0th day of fermentation was converted to 100% and the biological oxygen demand was converted to 0%, and the experiment was conducted.

8) Alcohol content measurement

The alcohol content of the fermented product was measured according to the liquor analysis regulations of the National Tax Service. After taking 100mL of the sample in a measuring cylinder, it was transferred to a 500mL Erlenmeyer flask. The measuring cylinder into which the sample was placed was washed three times with 10 mL of distilled water, and the solution was combined with an Erlenmeyer flask. An Erlenmeyer flask was connected to one side of the cooler, and a 100mL measuring cylinder was connected to the other side with a water pipe. Heat was applied to the sample using a Soxhlet heater. When the distillate reached 70mL, distillation was stopped, and distilled water was replenished, and the measuring cylinder was used up to the 100mL mark. After shaking well and measuring with a spirit meter, temperature correction was performed by the alcohol frequency conversion table.

9) Statistical processing

The experimental results were expressed as mean values and standard deviations (mean±SD), and statistical processing was performed by one-way ANOVA using SPSS (statist ictal package for social science, 24, SPSS Inc. Chicago, IL, USA), followed by Duncan's Significance was verified at the P<0.05 level by a multiple range test.

Experiment result

Table 1 shows the pH change of alcohol fermentation for four strains of Aronia yeast. pH showed a tendency to decrease slightly with yeast fermentation time, but it was found that fermentation proceeded normally without abnormal fermentation according to yeast fermentation time. S. cerevisiae showed a slight decrease from 4.24±0.01 at the beginning of fermentation to 3.64±0.02 at 46 hours of fermentation.

pH of fermented aronia lysate using four strains of yeast Yeast pH Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 4.24±0.01 4.04±0.02 3.88±0.01 3.84±0.02 3.70±0.01 3.65±0.02 3.65±0.01 3.64±0.02 S. boulardii 4.23±0.02 4.02±0.01 3.87±0.02 3.80±0.00 3.60±0.01 3.45±0.02 3.45±0.02 3.41±0.01 K. servazzii 4.26±0.00 4.11±0.02 4.01±0.02 3.95±0.01 3.88±0.01 3.74±0.02 3.74±0.02 3.72±0.01 G. trichum 4.28±0.01 4.15±0.02 4.04±0.01 4.02±0.02 3.92±0.01 3.84±0.02 3.84±0.01 3.80±0.01

Table 2 shows the acidity change of alcoholic fermentation for four strains of Aronia yeast. In the case of acidity, there was a tendency to increase slightly with yeast fermentation time, but it was found that it did not affect yeast fermentation. The S. cerevisiae strain showed a slight increase from 0.65±0.02 at the beginning of fermentation to 0.77±0.01 at 46 hours of fermentation.

Acidity (%) of fermented aronia lysate using four strains of yeast Yeast Acidity Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0.65±0.02 0.68±0.01 0.72±0.02 0.74±0.01 0.74±0.01 0.80±0.00 0.80±0.02 0.77±0.01 S. boulardii 0.70±0.01 0.72±0.01 0.74±0.02 0.78±0.03 0.80±0.02 0.82±0.02 0.82±0.01 0.81±0.02 K. servazzii 0.62±0.01 0.64±0.02 0.66±0.03 0.68±0.02 0.70±0.01 0.72±0.00 0.72±0.20 0.71±0.03 G. trichum 0.64±0.01 0.66±0.02 0.66±0.02 0.68±0.01 0.68±0.01 0.70±0.02 0.70±0.00 0.68±0.02

Changes in the amino acid degree of alcohol fermentation for four strains of Aronia yeast are shown in Table 3. In the case of amino acids, there was a tendency to increase slightly with yeast fermentation time, but this did not appear to affect yeast fermentation. The S. cerevisiae strain showed a slight increase from 0.68±0.02 at the beginning of fermentation to 0.86±0.01 at 46 hours of fermentation.

Amino acid content of fermented aronia lysate using 4 yeast strains (%) Yeast Amino acidity Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0.68±0.02 0.70±0.01 0.74±0.02 0.76±0.01 0.78±0.02 0.82±0.01 0.84±0.00 0.86±0.01 S. boulardii 0.72±0.01 0.74±0.02 0.76±0.01 0.81±0.02 0.82±0.01 0.84±0.02 0.86±0.02 0.88±0.01 K. servazzii 0.70±0.01 0.71±0.02 0.72±0.01 0.74±0.03 0.76±0.2 0.76±0.02 0.78±0.03 0.80±0.02 G. trichum 0.71±0.02 0.72±0.02 0.74±0.01 0.76±0.01 0.78±0.01 0.80±0.02 0.80±0.02 0.82±0.00

Table 4 shows the total sugar changes in alcohol fermentation for four strains of Aronia yeast. In the case of total sugar, it decreased according to the yeast fermentation time, which was shown to decrease according to the sugar metabolism process of the yeast fermentation strain. The S. cerevisiae strain showed a tendency to decrease from 8.52±1.42 at the start of fermentation to 0.98±0.32 after 46 hours of fermentation, following S. boulardii strain, by sugar metabolism.

Total sugar content (%) of fermented aronia lysate using 4 strains of yeast Yeast Total sugar content Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 8.52±1.42 8.21±0.77 7.14±0.84 5.21±0.71 3.51±0.62 1.71±0.51 1.02±0.24 0.98±0.32 S. boulardii 8.44±1.21 7.74±1.03 6.58±1.33 4.98±0.24 2.91±0.12 1.42±0.25 0.88±0.26 0.42±0.11 K. servazzii 8.52±0.88 8.11±0.74 7.71±1.01 6.44±1.14 5.87±1.03 4.32±0.58 3.31±0.41 2.98±0.33 G. trichum 8.42±0.65 8.11±0.66 7.88±0.74 7.02±0.51 6.78±0.26 6.05±0.31 4.02±0.42 3.88±0.22

Table 5 shows the changes in reducing sugars in alcohol fermentation for four strains of Aronia yeast. In the case of reducing sugar, it decreased according to yeast fermentation time, which showed the same decreasing trend as total sugar according to the sugar metabolism process of yeast fermentation strain. The S. cerevisiae strain showed a tendency to decrease from 5.52±0.66 at the beginning of fermentation to 0.55±0.23 after 46 hours of fermentation, the same as the total sugar by the sugar metabolism process next to the S. boulardii strain.

Reducing sugar content (%) of fermented aronia lysate using 4 strains of yeast Yeast Reducing sugar content Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 5.52±0.66 5.01±0.74 4.14±0.61 3.25±0.51 2.44±0.55 1.25±0.32 0.87±0.21 0.55±0.23 S. boulardii 5.54±0.31 5.02±0.42 4.22±0.77 3.01±0.11 2.25±0.18 1.14±0.19 0.74±0.08 0.48±0.03 K. servazzii 5.53±0.36 5.02±0.31 4.66±0.21 4.03±0.18 3.87±0.17 3.66±0.15 3.14±0.14 2.74±0.13 G. trichum 5.54±0.32 5.02±0.25 4.76±0.28 4.33±0.31 3.57±0.42 3.36±0.14 2.98±0.12 2.66±0.08

Table 6 shows the changes in the number of viable cells in alcohol fermentation for four strains of Aronia yeast. In the case of the viable cell count, when 0 hour of yeast fermentation was set as 0, the S. cerevisiae strain decreased from 5.52±0.66 at the beginning of fermentation to 0.55±0.23 at 46 hours of fermentation, following S. boulardii strain, as high as total sugar by glycolysis. showed a tendency to

Viable cell count (10 ⁶ CFU/mL) (%) of fermented aronia lysate using 4 yeast strains Yeast Viable cell count Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0±.0.00 4.40±1.12 10.23±2.12 18.40±2.14 35.50±1.98 58.20±1.74 88.40±2.14 80.10±3.51 S. boulardii 0±0.00 10.21±1.23 18.21±2.54 33.30±2.99 45.20±2.55 71.40±3.54 98.10±2.14 100.01±5.87 K. servazzii 0±0.00 2.20±1.54 5.50±1.84 8.80±1.41 14.20±3.01 21.20±1.44 44.40±1.98 54.50±2.14 G. trichum 0±0.00 1.42±1.68 2.41±1.99 3.60±0.88 8.80±0.77 11.40±1.44 13.20±0.98 21.30±1.87

Table 7 shows the alcohol content change of alcohol fermentation for four strains of Aronia yeast. In the case of alcohol content, when 0 hour of yeast fermentation was set as 0, it was confirmed that the S. cerevisiae strain made the highest alcohol content by sugar metabolism, with 3.10 ± 0.10 from 0 fermentation start to 46 hours fermentation.

Alcohol content (%) of fermented aronia lysate using 4 yeast strains Yeast Alcohol content Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0.00±.0.00 0.60±0.1 1.10±0.10 1.30±0.10 1.80±0.20 2.20±0.10 02.40±0.2 3.10±0.10 S. boulardii 0.00±0.00 0.50±0.2 1.00±0.10 1.10±0.20 1.40±.0.20 1.70±0.20 1.90±0.10 2.70±0.20 K. servazzii 0.00±0.00 0.40±0.10 0.80±0.20 1.00±0.10 1.20±0.10 1.40±0.10 1.50±0.20 1.60±0.10 G. trichum 0.00±0.00 0.00±0.00 0.10±0.00 0.40±0.10 0.80±0.10 1.10±0.20 1.20±0.10 1.40±0.20

Table 8 shows changes in beta-glucan content of alcohol fermentation for four strains of Aronia yeast. In the case of beta-glucan content, when 0 hour of yeast fermentation was set as 0, the S. cerevisiae strain was 2.7±0.1 from fermentation start 0 to fermentation 46 hours, the highest after S. boulardii strain 3.5±0.2 among the metabolites of the yeast fermentation process. confirmed to make.

Beta glucan (%) of fermented aronia lysate using 4 yeast strains Yeast Beta-glucans Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0±0.0 1.1±0.1 1.3±0.1 1.5±0.1 1.7±0.2 1.9±0.1 2.4±0.1 2.7±0.1 S. boulardii 0±0.0 1.4±0.2 1.6±0.2 2±0.1 2.4±0.1 3±0.1 3.1±0.2 3.5±0.2 K. servazzii 0±0.0 0.4±0.1 0.7±0.1 1.1±0.2 1.4±0.1 1.7±0.2 2.1±0.1 2.3±0.1 G. trichum 0±0.0 0±.0.0 0.4±0.0 0.4±0.1 0.7±0.1 0.7±0.1 1±0.1 1.4±0.2

As can be seen from the above and experimental results, in order to select the strain most suitable for producing alcohol among the four strains of yeast, when the same fermentation conditions were given, the change in the components of each yeast strain was measured, and as a result, the S. cerevisiae strain had the highest alcohol content producing ability. It was judged to be In addition, when comparing the results of alcohol and yeast metabolites in the selection of strains, it was decided to select S. cerevisiae, which is a general baker's yeast and has excellent alcohol production, and S. boulardii, which has excellent metabolites.

Example 2: Establishment of Manufacturing Conditions for Aronia Extract

2-1. Investigation of total sugar and reducing sugar content according to alcohol concentration of aronia lysate

In Example 1, it was determined that 10% of alcohol could not be produced by simple crushing of aronia, and hot water extraction of the crushed aronia according to the content of alcohol concentration was carried out and total sugar and reducing sugar were compared to find the optimal extraction conditions. proceeded. Total sugar and reducing sugar contents were the same as in Example 1. The results for this are shown in Tables 9 and 10.

Reducing sugar content (%) of hot water extract of aronia lysate or extract by alcohol concentration Time (number of times) condition water 100 5% alcohol 10% alcohol 20% alcohol 30% alcohol 40% alcohol One 8.52 9.12 10.04 24.15 27.41 27.55 2 8.44 9.05 10.12 24.44 27.33 27.44 3 8.48 9.08 10.22 24.24 27.21 27.57 4 8.52 9.04 9.89 24.33 27.011 27.60 5 8.47 9.11 10.01 24.57 27.34 27.51 Average 8.48 9.08 10.06 24.34 27.25 27.53 STDEV 0.034 0.035 0.123 0.165 0.015 0.061

Reducing sugar content (%) of hot water extract of aronia lysate or extract by alcohol concentration Time (number of times) condition water 100 5% alcohol 10% alcohol 20% alcohol 30% alcohol 40% alcohol One 9.55 10.89 13.51 28.14 30.11 30.99 2 9.58 11.02 13.66 28.12 30.14 31.01 3 9.66 11.04 13.64 28.33 30.21 30.99 4 9.79 10.92 13.72 28.01 30.09 31.11 5 9.89 10.94 13.77 28.14 30.04 31.14 Average 9.69 10.96 13.66 28.14 30.11 31.05 STDEV 0.128 0.058 0.087 0.103 0.056 0.064

The target value in the present invention is 10% alcohol. When 10% alcohol was used, total sugar was 13.66±0.087%. When referring to the results of other studies, it is mentioned that the alcohol content is about 1/2 of the total sugar. Accordingly, when calculating in consideration of the fermentation time, it is confirmed that when extracting 10% alcohol and 20% alcohol from aronia lysate, reducing sugar, the sugar used for fermentation, is 28%, and the alcohol content is about 14%. Therefore, it is judged that when extracting with 20% or more of alcohol, a large amount of alcohol can be made during fermentation.

2-2. Investigation of anthocyanin content according to alcohol concentration of aronia lysate

In order to measure the total anthocyanin content, extracts were prepared by the same method as in 2-1 for each alcohol concentration of the aronia lysate, and 1% HCl was added to the extract, and then anthocyanins were extracted by sonication for 15 minutes, followed by 10 at 4000 rpm. The supernatant obtained by centrifugation was used for the experiment. After diluting the sample according to the experimental concentration, 0.025 M Potassium Chloride buffer (pH 1.0) and 0.4M Sodium acetate buffer (pH 4.5) were mixed, reacted for 15 minutes, and measured at 510 and 700 nm absorbance, followed by the following equation 2 was used to determine the total anthocyanin content. For the standard material, the value of anthocyanin lambdamax was applied when measuring after confirming the maximum absorbance.

[Equation 2]

Total anthocyanin content (mg/gram) = (A × MW × DF × 1000) / ε × V

A (absorbance) = (A510 nm-A700 nm)pH 1.0 - (A510 nm-A700 nm)pH 4.5;

MW (cyanidine-3-glucoside molecular weight) = 322.17

DF: dilution factor

ε(cyanidin chloride molar absorbance) = 24,600 M-1cm-1,

V = final volume of sample

As can be seen in Table 11 below, as a result of comparing the measurement of anthocyanin content of 10%-100% alcohol extract, the anthocyanin content was highest in the 70% alcohol extract in the first experiment.

Total Anthocyanin Content (CE mg/gram) Concentration
(1000μg/mL) filtrate sludge filtrate + sludge 10% 2.095±0.03 0.559±0.02 2.654 20% 1.641±0.02 0.489±0.04 2.130 30% 3.082±0.04 0.332±0.03 3.414 40% 3.728±0.08 0.201±0.05 3.929 50% 2.811±0.02 0.236±0.03 3.047 60% 3.370±0.03 0.288±0.03 3.658 70% 6.286±0.05 0.271±0.06 6.557 80% 5.247±0.11 0.279±0.05 5.527 90% 5.160±0.03 0.253±0.02 5.413 100% 4.287±0.02 0.672±0.04 4.959

Example 3: Measurement of alcohol content, etc. of fermented product of 20% alcohol extract of aronia lysate

In Example 2, it was confirmed that the medium in which 20% alcohol was extracted from the aronia lysate had a reducing sugar content of 28%, so pH, acidity, amino acid level, total sugar, Reducing sugar, viable cell count and alcohol content were measured. The results are shown in Tables 12 to 18 below. On the other hand, the 20% alcohol extract of the aronia lysate used in this experiment was concentrated after alcohol extraction, and then the experiment was conducted, which was to blow alcohol to produce wine for food after yeast fermentation.

pH of fermented aronia lysate (20% alcohol extraction) using 4 types of yeast Yeast pH Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 4.34±0.01 4.24±0.02 4.14±0.01 3.84±0.02 3.70±0.01 3.65±0.02 3.65±0.01 3.64±0.02 S. boulardii 4.33±0.02 4.02±0.01 3.87±0.02 3.80±0.00 3.60±0.01 3.45±0.02 3.45±0.02 3.41±0.01 K. servazzii 4.36±0.00 4.11±0.02 4.01±0.02 3.95±0.01 3.88±0.01 3.74±0.02 3.74±0.02 3.72±0.01 G. trichum 4.29±0.01 4.15±0.02 4.04±0.01 4.02±0.02 3.92±0.01 3.84±0.02 3.84±0.01 3.80±0.01

Acidity (%) of fermented aronia lysate (20% alcohol extraction) using 4 types of yeast Yeast Acidity Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0.75±0.02 0.77±0.01 0.80±0.02 0.84±0.01 0.84±0.01 0.90±0.00 0.92±0.02 0.94±0.01 S. boulardii 0.78±0.01 0.78±0.01 0.80±0.02 0.82±0.03 0.84±0.02 0.82±0.02 0.86±0.01 0.86±0.02 K. servazzii 0.62±0.01 0.64±0.02 0.66±0.03 0.68±0.02 0.70±0.01 0.72±0.00 0.72±0.20 0.71±0.03 G. trichum 0.66±0.01 0.66±0.02 0.66±0.02 0.68±0.01 0.68±0.01 0.70±0.02 0.70±0.00 0.68±0.02

Amino acid content of fermented aronia lysate (20% alcohol extraction) using 4 types of yeast (%) Yeast Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0.68±0.02 0.70±0.01 0.74±0.02 0.76±0.01 0.78±0.02 0.82±0.01 0.84±0.00 0.86±0.01 S. boulardii 0.72±0.01 0.74±0.02 0.76±0.01 0.81±0.02 0.82±0.01 0.84±0.02 0.86±0.02 0.88±0.01 K. servazzii 0.70±0.01 0.71±0.02 0.72±0.01 0.74±0.03 0.76±0.02 0.76±0.02 0.78±0.03 0.80±0.02 G. trichum 0.71±0.02 0.72±0.02 0.74±0.01 0.76±0.01 0.78±0.01 0.80±0.02 0.80±0.02 0.82±0.00

Total sugar (%) of fermented aronia lysate (20% alcohol extraction) using 4 types of yeast Yeast Total sugar content Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 28.14±1.77 20.14±1.85 18.42±1.42 14.42±1.21 12.14±1.01 10.14±1.14 6.14±1.25 2.21±0.47 S. boulardii 28.15±1.54 19.14±1.42 15.21±1.11 13.33±0.58 11.44±0.87 8.81±0.77 4.21±0.25 1.32±0.14 K. servazzii 28.14±0.98 20.15±1.14 18.44±1.32 16.21±1.32 14.28±1.33 11.14±1.14 7.71±0.84 4.25±0.71 G. trichum 28.47±1.02 22.21±0.47 19.22±1.35 17.14±1.41 15.17±1.00 13.66±0.71 8.87±0.62 6.21±0.08

Aronia lysate using 4 types of yeast (20% alcohol extraction) Reducing sugar (%) of fermented product Yeast Reducing sugar content Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 25.14±2,14 15.21±1,84 14.22±1,47 13.14±1,42 10.21±0,98 7.77±0,97 4.35±0,88 1.14±0,14 S. boulardii 25.15±1,84 17.14±1,47 15.31±1,65 12.14±1,32 11.41±1,21 7.01±0.71 3.21±0.54 0.98±0.33 K. servazzii 26.14±1.36 19.14±1.21 16.58±1.24 13.33±0.87 12.55±1.01 10.00±1.07 5.55±0.87 3.14±0.61 G. trichum 26.22±1.41 20.14±1.35 18.01±1.32 15.21±1.24 13.21±0.92 9.59±0.84 7.66±0.71 4.42±0.62

Aronia lysate using 4 types of yeast (20% alcohol extraction) viable cell count (10 ⁶ CFU/mL) (%) Yeast Viable cell count Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0±0.00 10.40±1.51 20.40±1.46 30.50±3.12 35.50±3.25 66.60±4.15 99.80±6.25 118.20±9.87 S. boulardii 0±0.00 13.20±2.51 18.20±1.77 32.20±6.51 45,20±4.55 71,40±6.21 92,40±7.74 93,50±6.87 K. servazzii 0±0.00 2,20±0.81 5,50±0.41 8,70±0.42 13,20±0.33 21,10±1.01 40,20±1.21 51,40±3.54 G. trichum 0±0.00 1.40±0.21 2.30±0.31 3.60±0.25 8.70±0.33 11.20±0.47 13.10±1.01 21.20±1.04

Alcohol content (%) of fermented aronia lysate (20% alcohol extraction) using 4 types of yeast Yeast Alcohol content Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0±0.0 1.4±0.2 3.3±0.1 5.5±0.2 6.1±0.1 8.4±0.3 8.5±0.3 9.5±0.3 S. boulardii 0±0.0 1.2±0.1 2.1±0.1 2.8±0.2 4.8±0.2 5.2±0.2 6.4±0.1 7.0±0.1 K. servazzii 0±0.0 0.4±0.1 1.0±0.2 1.4±0.1 2.0±0.1 2.4±0.2 3.1±0.2 3.4±0.2 G. trichum 0±0.0 0.4±0.1 0.8±0.1 1.5±0.2 1.7±0.1 2.1±0.2 2.5±0.2 3.0±0.2

As can be seen in Tables 12 to 18, it was confirmed that the alcohol-producing ability of the fermented aronia increased when the 20% alcohol extract was used, but it was confirmed that the alcohol content of 10%, which is one purpose of the present invention, was not reached . Therefore, in the next experiment, we tried to use pear concentrate without using supplement sugar, but supplement sugar. Also, in subsequent experiments, 70% alcohol was used in consideration of the anthocyanin content of Example 2-2.

Example 4: Measurement of alcohol content of fermented product according to sugar content

5 kg of 100-year-old aronia products sold by the Mokggol Aronia Research Association were purchased and used, and the lysate used for fermentation was primarily used to separate the branches and fruits of aronia. Afterwards, the fruits were washed in tap water. The washed fruit was crushed with a blender (Mufti Quick 7-MQ736, BRAUN, Germany). Then, after hot water extraction at 100 ° C for 3 hours under the condition of 70% alcohol (40 g of aronia lysate + 800 g of alcohol), the aronia lysate was concentrated for 3 hours under the conditions of 45 ° C, 70 ~ 80 RPM, 80 mbar to blow the alcohol. Afterwards, this aronia lysate was used as a medium, and pear concentrate was used as a raw material with 65.21 ± 3.24% of total sugar and 44.02 ± 4.14% of reducing sugar. After adding 1% by weight or 2% by weight of the total volume to the concentrated aronia lysate as a pear concentrate, sterilization at 121 ° C. for 15 minutes, followed by cooling for 2 hours. As in Example 1, four types of yeast were inoculated at 0.1% of the medium volume and cultured at 140 rpm and 27 ° C for 44 to 46 hours. After cultivation, the fermentation characteristics of the alcohol extract of the aronia lysate were analyzed by the same method as that described in Example 1, and as a result, the results of mixing 1% by weight pear concentrate in Tables 19 to 26 below are shown in Table 27 below. To Table 34 shows the results when 2% by weight pear concentrate was mixed.

Aronia lysate using 4 types of yeast (70% alcohol extraction) + 1% pear concentrate pH of fermented mixture Yeast pH Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 4.26±0.01 4.15±0.02 4.05±0.01 3.75±0.02 3.73±0.01 3.68±0.02 3.65±0.01 3.64±0.02 S. boulardii 4.24±0.02 3.94±0.01 3.78±0.02 3.72±0.00 3.64±0.01 3.48±0.02 3.45±0.02 3.41±0.01 K. servazzii 4.28±0.00 4.00±0.02 3.93±0.02 3.88±0.01 3.90±0.01 3.76±0.02 3.74±0.02 3.72±0.01 G. trichum 4.20±0.01 4.04±0.02 4.01±0.01 3.94±0.02 3.93±0.01 3.87±0.02 3.84±0.01 3.80±0.01

Aronia lysate using 4 types of yeast (70% alcohol extraction) + acidity (%) of fermented mixture of pear concentrate 1% Yeast Acidity Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0.68±0.02 0.70±0.01 0.72±0.02 0.74±0.01 0.76±0.01 0.82±0.00 0.94±0.02 0.94±0.01 S. boulardii 0.70±0.01 0.72±0.01 0.72±0.02 0.74±0.03 0.74±0.02 0.78±0.02 0.84±0.01 0.86±0.02 K. servazzii 0.72±0.01 0.74±0.02 0.76±0.03 0.78±0.02 0.80±0.01 0.82±0.00 0.82±0.20 0.91±0.03 G. trichum 0.76±0.01 0.76±0.02 0.78±0.02 0.78±0.01 0.80±0.01 0.80±0.02 0.82±0.00 0.82±0.02

Aronia lysate using 4 types of yeast (70% alcohol extraction) + Amino acid content of fermented mixture of pear concentrate 1% (%) Yeast Amino acids Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0.68±0.02 0.70±0.01 0.74±0.02 0.76±0.01 0.78±0.02 0.82±0.01 0.84±0.00 0.86±0.01 S. boulardii 0.72±0.01 0.74±0.02 0.76±0.01 0.81±0.02 0.82±0.01 0.84±0.02 0.86±0.02 0.88±0.01 K. servazzii 0.70±0.01 0.71±0.02 0.72±0.01 0.74±0.03 0.76±0.2 0.76±0.02 0.78±0.03 0.80±0.02 G. trichum 0.71±0.02 0.72±0.02 0.74±0.01 0.76±0.01 0.78±0.01 0.80±0.02 0.80±0.02 0.82±0.00

Aronia lysate using 4 types of yeast (70% alcohol extraction) + 1% pear concentrate mixed fermented sugar (%) Yeast Total sugar content Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 28.14±1.77 20.14±1.85 18.42±1.42 14.42±1.21 12.14±1.01 10.14±1.14 6.14±1.25 2.21±0.47 S. boulardii 28.15±1.54 19.14±1.42 15.21±1.11 13.33±0.58 11.44±0.87 8.81±0.77 4.21±0.25 1.32±0.14 K. servazzii 29.22±0.98 20.15±1.14 18.44±1.32 16.21±1.32 14.28±1.33 11.14±1.14 7.71±0.84 4.25±0.71 G. trichum 30.25±1.02 22.21±0.47 19.22±1.35 17.14±1.41 15.17±1.00 13.66±0.71 8.87±0.62 6.21±0.08

Aronia lysate using 4 types of yeast (70% alcohol extraction) + 1% pear concentrate mixed fermented sugar (%) Yeast Reducing sugar content Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 25.14±2,14 15.21±1,84 14.22±1,47 13.14±1,42 10.21±0,98 7.77±0,97 4.35±0,88 1.14±0,14 S. boulardii 25.15±1,84 17.14±1,47 15.31±1,65 12.14±1,32 11.41±1,21 7.01±0.71 3.21±0.54 0.98±0.33 K. servazzii 26.14±1.36 19.14±1.21 16.58±1.24 13.33±0.87 12.55±1.01 10.00±1.07 5.55±0.87 3.14±0.61 G. trichum 26.22±1.41 20.14±1.35 18.01±1.32 15.21±1.24 13.21±0.92 9.59±0.84 7.66±0.71 4.42±0.62

Aronia lysate using 4 types of yeast (70% alcohol extraction) + viable cell count of fermented mixture of pear concentrate 1% (10 ⁶ CFU/mL) Yeast Viable cell count Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0±0.00 10.40±1.35 20.40±1.47 30.50±1.14 35.50±1.84 61.60±2.14 92.50±1.82 100.01±2.01 S. boulardii 0±0.00 13.20±1.33 18.20±1.02 31.20±5.14 45.20±3.25 71.40±4.44 91.10±3.69 99.50±5.04 K. servazzii 0±0.00 2.20±0.89 5.50±1.01 8.70±1.14 14.20±1.32 21.20±1.25 44.40±1.47 55.50±2.22 G. trichum 0±0.00 1.40±0.44 2.30±0.39 3.60±0.22 8.70±0.89 11.20±1.01 13.10±0.87 20.50±1.98

Alcohol content (%) of fermented aronia lysate using 4 types of yeast (70% alcohol extraction) + 1% pear concentrate Yeast Alcohol content Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0±0.0 1.4±0.2 3.3±0.1 6.2±0.2 7.8±0.1 9.4±0.2 10.2±0.3 10.4±0.1 S. boulardii 0±0.0 1.2±0.1 2.8±0.2 5.5±0.2 7.4±0.1 8.2±0.2 9.1±0.3 9.9±0.2 K. servazzii 0±0.2 0.4±0.1 0.4±0.2 1.4±0.1 2.1±0.1 2.5±0.2 3.0±0.0 3.1±0.2 G. trichum 0±0.0 0.4±0.1 0.8±0.1 1.5±0.1 2.1±0.2 2.7±0.1 3.5±0.2 4.0±0.1

Yeast metabolite content of fermented aronia lysate using 4 types of yeast (70% alcohol extraction) + 1% pear concentrate (g/100g) Yeast Beta-glucans Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0.0±0.0 1.1±0.1 1.3±0.2 1.8±0.1 2.5±0.3 3.2±0.1 4.4±0.2 5.9±0.1 S. boulardii 0.0±0.0 1.8±0.2 2.0±0.2 2.5±0.1 4.2±0.2 5.8±0.1 6.1±0.2 7.4±0.1 K. servazzii 0.0±0.1 0.1±0.1 0.3±0.1 0.4±0.2 0.9±0.1 1.2±0.3 1.4±0.2 1.5±0.2 G. trichum 0.0±0.0 0.0±0.0 0.5±0.0 0.6±0.0 0.8±0.2 0.9±0.2 1.0±0.1 1.5±0.1

Aronia lysate using 4 types of yeast (70% alcohol extraction) + 2% pear concentrate pH of mixed fermented product Yeast pH Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 4.24±0.01 4.14±0.02 4.04±0.01 3.74±0.02 3.70±0.01 3.65±0.02 3.65±0.01 3.64±0.02 S. boulardii 4.23±0.02 3.92±0.01 3.77±0.02 3.70±0.00 3.60±0.01 3.45±0.02 3.45±0.04 3.41±0.01 K. servazzii 4.26±0.00 4.01±0.02 3.91±0.02 3.85±0.01 3.88±0.01 3.74±0.01 3.74±0.02 3.72±0.01 G. trichum 4.19±0.01 4.05±0.02 4.00±0.01 3.92±0.02 3.92±0.01 3.84±0.02 3.84±0.01 3.80±0.01

Aronia lysate using 4 types of yeast (70% alcohol extraction) + acidity (%) of fermented mixture of pear concentrate 2% Yeast Acidity Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0.65±0.02 0.67±0.01 0.70±0.02 0.74±0.01 0.74±0.01 0.80±0.00 0.92±0.02 0.94±0.01 S. boulardii 0.68±0.01 0.68±0.01 0.70±0.02 0.72±0.03 0.74±0.02 0.78±0.02 0.84±0.01 0.86±0.02 K. servazzii 0.62±0.01 0.64±0.02 0.66±0.03 0.68±0.02 0.70±0.01 0.72±0.00 0.72±0.20 0.71±0.03 G. trichum 0.66±0.01 0.66±0.02 0.66±0.02 0.68±0.01 0.68±0.01 0.70±0.02 0.70±0.00 0.68±0.02

Aronia lysate using 4 types of yeast (70% alcohol extraction) + amino acid level of fermented mixture of pear concentrate 2% (%) Yeast Amino acids Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0.68±0.02 0.70±0.01 0.74±0.02 0.76±0.01 0.78±0.02 0.82±0.01 0.84±0.00 0.86±0.01 S. boulardii 0.72±0.01 0.74±0.02 0.76±0.01 0.81±0.02 0.82±0.01 0.84±0.02 0.86±0.02 0.88±0.01 K. servazzii 0.70±0.01 0.71±0.02 0.72±0.01 0.74±0.03 0.76±0.02 0.76±0.02 0.78±0.03 0.80±0.02 G. trichum 0.71±0.02 0.72±0.02 0.74±0.01 0.76±0.01 0.78±0.01 0.80±0.02 0.80±0.02 0.82±0.00

Aronia lysate using 4 types of yeast (70% alcohol extraction) + total sugar (%) of fermented mixture of pear concentrate 2% Yeast Total sugar content Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 32.24±2.64 25.44±2.01 17.55±1.87 15.11±1.44 10.14±1.46 9.99±1.87 6.14±1.66 2.21±0.87 S. boulardii 31.18±2.41 26.12±1.87 19.21±1.48 13.22±1.54 11.41±1.05 10.02±1.02 7.71±0.87 2.31±0.74 K. servazzii 31.22±2.34 27.44±2.14 20.41±1.87 15.17±1.72 13.21±1.62 10.88±1.52 9.95±1.21 4.25±1.04 G. trichum 31.58±1.89 28.17±1.25 22.24±2.01 17.99±1.47 15.22±1.33 13.66±1.29 10.23±1.07 9.98±0.88

Aronia lysate using 4 types of yeast (70% alcohol extraction) + reducing sugar (%) of fermented mixture of pear concentrate 2% Yeast Reducing sugar content Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 26.14±2,14 16.21±1,84 15.22±1,47 14.14±1,42 10.31±0,98 7.47±0,97 4.35±0,88 1.14±0,14 S. boulardii 26.15±1,84 18.14±1,47 16.31±1,65 13.14±1,32 11.51±1,21 6.91±0.71 3.21±0.54 0.98±0.33 K. servazzii 27.14±1.36 20.14±1.21 17.58±1.24 14.33±0.87 12.65±1.01 9.91±1.07 5.55±0.87 3.14±0.61 G. trichum 27.22±1.41 21.14±1.35 19.01±1.32 16.21±1.24 13.31±0.92 9.39±0.84 7.66±0.71 4.42±0.62

Aronia lysate using 4 types of yeast (70% alcohol extraction) + viable cell count of fermented mixture of 2% pear concentrate (10 ⁶ CFU/mL) Yeast Viable cell count Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0±0.00 10.40±1.04 20.40±1.52 30.50±1.33 35.50±1.27 66.60±1.87 91.20±5.21 100.00±4.44 S. boulardii 0±0.00 13.20±1.21 18.20±1.32 33.30±1.65 45.20±1.82 71.40±2.01 92.40±2.31 99.30±3.12 K. servazzii 0±0.00 2.20±0.07 5.50±0.14 8.80±0.32 14.20±0.54 21.20±0.68 44.40±0.74 52.50±1.47 G. trichum 0±0.00 1.40±0.08 2.40±0.14 3.60±0.21 8.70±0.32 10.40±1.02 12.80±1.23 21.30±1.32

Aronia lysate using 4 types of yeast (70% alcohol extraction) + alcohol content (%) of fermented mixture of pear concentrate 2% Yeast Alcohol content Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0±0.0 1.4±0.1 3.3±0.2 7.1±0.1 8.2±0.1 9.0±0.1 10.1±0.2 12.5±0.1 S. boulardii 0±0.0 1.2±0.2 2.8±0.1 5.5±0.2 7.4±0.1 8.2±0.1 9.1±0.2 10.4±0.2 K. servazzii 0±0.0 0.4±0.1 0.4±0.1 1.4±0.1 2.0±0.2 2.8±0.2 4.0±0.2 4.5±0.3 G. trichum 0±0.0 0.4±0.2 0.8±0.1 1.5±0.2 2.1±0.2 2.7±0.1 3.5±0.2 4.0±0.2

Aronia lysate using 4 types of yeast (70% alcohol extraction) + Beta-glucan content of fermented mixture of 2% pear concentrate (g/ 100g) Yeast Beta-glucans Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0±0.0 1.5±0.1 2.1±0.2 2.8±0.1 4.5±0.3 6.2±0.1 7.4±0.2 7.9±0.1 S. boulardii 0±0.0 2.1±0.2 4.0±0.2 5.5±0.1 6.2±0.2 7.8±0.1 8.1±0.2 9.4±0.1 K. servazzii 0±0.1 0.4±0.1 0.9±0.1 1.4±0.2 1.9±0.1 2.2±0.3 2.4±0.2 2.5±0.2 G. trichum 0±0.0 0±0.0 0.5±0.0 0.6±0.0 0.8±0.2 0.9±0.2 1.4±0.1 1.5±0.1

As can be seen in Tables 19 to 34, it was confirmed that the alcohol content of the fermented product was 10% or more when 2% by weight of the pear concentrate was added compared to the case where 1% by weight of the pear concentrate was added to the 70% alcohol extract. In particular, in the case of the S. cerevisiae strain, the alcohol content was higher than that of the S. boulardii strain, but the content of beta-glucan, one of the yeast metabolites, was confirmed to be higher in the S. boulardii strain than in the S. cerevisiae strain.

Afterwards, the next experiment was carried out after supplementing with 2% pear concentrate and selecting S. cerevisiae and S. boulardii strains as the optimal strains.

Example 5: Measurement of alcohol content of fermented product according to fermentation time

5 kg of 100-year-old aronia products sold by the Moksagol Aronia Research Society were purchased and used, and the lysate used for fermentation was primarily separated from the branches and fruits of aronia. Afterwards, the fruit was washed in tap water. The washed fruit was crushed with a blender (Mufti Quick 7-MQ736, BRAUN, Germany). Then, hot water extraction at 100°C for 3 hours under the condition of 70% alcohol (40g of aronia lysate + 800g of alcohol), which was confirmed through the previous supplement experiment, followed by 3 hours of aronia at 45°C, 70-80 RPM, 80mbar to blow off the alcohol. The lysate was concentrated. Afterwards, this aronia lysate was used as a medium, and pear concentrate was used as a raw material with 65.21 ± 3.24% of total sugar and 44.02 ± 4.14% of reducing sugar. 2% of the total volume was added as pear concentrate to the concentrated aronia lysate. This experiment was conducted with a 5L fermenter (KB -250, KO Biotech Co., Ltd., Incheon, Korea), and the volume of the experiment medium was at least 3.5 L. After preparing the fermented medium, it was sterilized at 121 ° C for 15 minutes and allowed to cool for 3 hours. Thereafter, Saccharomyces cerevisiae (KCTC NO. 27756) and Saccharomyces boulardii srcnm 001 were inoculated at 0.1% of the volume of the medium and cultured at 27° C. for 44 hours or 72 hours at 120 rpm and oxygen aeration rate of VVM 0.2.

After cultivation, the fermentation characteristics according to the fermentation time of the alcohol extract of the aronia lysate were analyzed by the same method as described in Example 1, and as a result, the results when the fermentation time was 44 hours in Tables 35 to 44 below, Tables 45 to 54 show the results when the 2% by weight fermentation time was 72 hours.

Aronia lysate (alcohol 70% extraction solution) + pear concentrate 2% medium fermentation pH (44 hours) Yeast pH Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 4.24±0.01 4.14±0.02 4.04±0.01 3.74±0.02 3.70±0.01 3.65±0.02 3.65±0.01 3.64±0.02 S. boulardii 4.23±0.02 3.92±0.01 3.77±0.02 3.70±0.00 3.60±0.01 3.45±0.02 3.45±0.02 3.41±0.01

Aronia lysate (alcohol 70% extraction solution) + pear concentrate 2% acidity (%) of fermented medium (44 hours) Yeast Acidity Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0.68±0.02 0.70±0.01 0.74±0.02 0.78±0.01 0.82±0.01 0.84±0.00 0.90±0.02 0.94±0.01 S. boulardii 0.70±0.01 0.69±0.01 0.72±0.02 0.76±0.03 0.77±0.02 0.78±0.02 0.88±0.01 0.90±0.02

Aronia lysate (alcohol 70% extraction solution) + Amino acid content (%) of fermented pear concentrate 2% medium (44 hours) Yeast Amino acids Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0.68±0.02 0.70±0.01 0.74±0.02 0.76±0.01 0.78±0.02 0.82±0.01 0.84±0.00 0.86±0.01 S. boulardii 0.72±0.01 0.74±0.02 0.76±0.01 0.81±0.02 0.82±0.01 0.84±0.02 0.86±0.02 0.88±0.01

Aronia lysate (alcohol 70% extraction solution) + pear concentrate 2% medium fermentation total sugar (%) (44 hours) Yeast Total sugar content Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 32.24±2.64 25.44±2.01 17.55±1.87 15.11±1.44 10.14±1.46 9.99±1.87 3.14±1.66 1.21±0.87 S. boulardii 31.18±2.41 26.12±1.87 19.21±1.48 13.22±1.54 11.41±1.05 10.02±1.02 5.71±0.87 1.91±0.74

Aronia lysate (alcohol 70% extraction solution) + pear concentrate 2% reducing sugar (%) of fermented medium (44 hours) Yeast Reducing sugar content Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 26.14±2,14 16.21±1,84 15.22±1,47 14.14±1,42 10.31±0,98 7.47±0,97 4.35±0,88 1.14±0,14 S. boulardii 26.15±1,84 18.14±1,47 16.31±1,65 13.14±1,32 11.51±1,21 6.91±0.71 3.21±0.54 0.98±0.33

Aronia lysate (alcohol 70% extraction solution) + viable cell count of fermented pear concentrate 2% medium (10 ⁶ CFU/mL) (44 hours) Yeast Viable cell count Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0.0±0.00 10.40±1.04 20.40±1.52 30.50±1.33 35.50±1.27 66.60±1.87 91.20±5.21 100.01±4.44 S. boulardii 0.0±0.00 13.20±1.21 18.20±1.32 33.30±1.65 45.20±1.82 71.40±2.01 92.40±2.31 99.30±3.12

Aronia lysate (alcohol 70% extraction solution) + pear concentrate 2% alcohol content (%) of fermented medium (44 hours) Yeast Alcohol content Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0.0±0.0 1.4±0.1 3.3±0.2 7.1±0.1 8.2±0.1 9.0±0.1 10.1±0.2 13.8±0.1 S. boulardii 0.0±0.0 1.2±0.2 2.8±0.1 5.5±0.2 7.4±0.1 8.2±0.1 9.1±0.2 9.8.±0.2

Aronia lysate (alcohol 70% extraction solution) + pear concentrate 2% medium fermented beta-glucan content (g/ 100g)) (44 hours) Yeast Beta-glucans Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0.0±0.0 1.5±0.1 2.1±0.2 2.8±0.1 4.5±0.3 6.2±0.1 7.4±0.2 8.9±0.1 S. boulardii 0.0±0.0 2.1±0.2 4.0±0.2 5.5±0.1 6.2±0.2 7.8±0.1 8.1±0.2 12.4±0.1

Aaronia lysate (alcohol 70% extraction solution) + pear concentrate 2% DO (%) of fermented medium (44 hours) Yeast DO Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 100.0±0.0 95.2±2.1 90.1±5.4 85.4±4.1 81.2±2.2 80.4±3.1 78.5±1.8 77.2±1.7 S. boulardii 100.0±0.0 96.3±1.8 91.2±1.7 88.2±1.5 82.1±2.2 81.4±2.4 79.3±1.1 76.8±1.2

Aronia lysate (alcohol 70% extract solution) + BOD (%) of fermented pear concentrate 2% medium (44 hours) Yeast BOD Time (h) 0 12 18 24 30 36 42 46 S. cerevisiae 0.0±0.0 3.1±0.1 8.4±0.6 12.1±0.7 17.4±1.3 19.2±0.9 20.4±1.4 21.5±1.8 S. boulardii 0.0±0.0 2.5±0.1 7.7±0.2 11.2±0.1 15.2±1.7 18.2±1.2 19.2±0.8 20.1±1.1

Aronia lysate (alcohol 70% extraction solution) + pear concentrate 2% medium fermentation pH (72 hours) Yeast pH Time (h) 0 12 18 24 30 36 42 46 52 58 64 72 S. cerevisiae 4.23±0.01 4.02±0.02 3.86±0.02 3.81±0.03 3.70±0.01 3.61±0.02 3.62±0.02 3.60±0.03 3.55±0.02 3.51±0.02 3.44±0.02 3.36±0.02 S. boulardii 4.23±0.01 4.05±0.02 3.87±0.03 3.77±0.02 3.63±0.02 3.40±0.01 3.25±0.02 3.12±0.03 3.11±0.02 3.05±0.02 3.04±0.01 3.02±0.01

Aronia lysate (alcohol 70% extraction solution) + pear concentrate 2% medium acidity (%) (72 hours) Yeast Acidity Time (h) 0 12 18 24 30 36 42 46 52 58 64 72 S. cerevisiae 0.67±0.01 0.71±0.02 0.74±0.02 0.76±0.01 0.78±0.02 0.80±0.01 0.82±0.01 0.87±0.02 0.88±0.01 0.90±0.01 0.92±0.02 0.94±0.02 S. boulardii 0.70±0.01 0.72±0.02 0.74±0.01 0.79±0.02 0.81±0.02 0.82±0.03 0.84±0.03 0.85±0.03 0.85±0.02 0.86±0.02 0.87±0.02 0.88±0.02

Aronia lysate (alcohol 70% extraction solution) + pear concentrate 2% amino acid level (%) of fermented medium (72 hours) Yeast Amino acids Time (h) 0 12 18 24 30 36 42 46 52 58 64 72 S. cerevisiae 0.76±0.01 0.76±0.02 0.78±0.01 0.80±0.02 0.82±0.03 0.84±0.02 0.88±0.01 0.92±0.01 0.91±0.02 0.93±0.01 0.94±0.02 0.95±0.01 S. boulardii 0.74±0.02 0.76±0.03 0.80±0.02 0.84±0.01 0.86±0.02 0.86±0.01 0.90±0.01 0.92±0.02 0.94±0.02 0.95±0.03 0.96±0.03 0.99±0.03

Aronia lysate (alcohol 70% extraction solution) + pear concentrate 2% medium fermentation total sugar (%) (72 hours) Yeast Total sugar content Time (h) 0 12 18 24 30 36 42 46 52 58 64 72 S. cerevisiae 32.27±3.21 26.44±3.09 18.65±2.84 15.31±2.14 12.55±1.87 11.47±1.48 9.87±1.09 7.14±0.87 5.21±0.65 3.14±0.56 1.51±0.21 0.87±0.19 S. boulardii 31.19±4.87 26.15±3.21 19.42±1.14 13.33±1.32 12.41±1.14 10.02±2.12 9.25±1.04 8.22±0.87 6.25±0.74 4.21±0.32 2.58±0.27 1.11±0.21

Aronia lysate (alcohol 70% extraction solution) + pear concentrate 2% reducing sugar (%) of fermented medium (72 hours) Yeast Reducing sugar content Time (h) 0 12 18 24 30 36 42 46 52 58 64 72 S. cerevisiae 26.22±2.15 17.21±1.84 15.33±1.42 13.14±2.16 10.33±1.47 7.77±1.36 6.32±1.32 4.58±2.12 3.28±2.16 2.54±1.58 1.05±1.47 0.57±0.08 S. boulardii 25.15±2.65 17.24±1.87 16.27±1.48 14.95±1.52 13.52±1.32 9.58±0.87 7.25±0.74 6.25±0.59 4.33±0.48 3.17±0.43 2.01±0.39 0.98±0.33

Aronia lysate (alcohol 70% extraction solution) + viable cell count of fermented pear concentrate 2% medium (10 ⁶ CFU/mL) (72 hours) Yeast Viable cell count Time (h) 0 12 18 24 30 36 42 46 52 58 64 72 S. cerevisiae 0.00±0.00 10.40±1.04 20.40±1.52 30.50±1.33 35.50±1.27 66.60±1.87 91.20±5.21 100.00±4.44 101.01±5.14 99,40±4.33 85,20±4.21 71,40±3.21 S. boulardii 0.00±0.0 13.20±1.21 18.20±1.32 33.30±1.65 45.20±1.82 71.40±2.01 92.40±2.31 99.30±3.12 102.02±3.69 101,10±4.25 99,50±4.61 98,20±3.51

Aronia lysate (alcohol 70% extraction solution) + pear concentrate 2% alcohol content of fermented medium (%) Yeast Alcohol content Time (h) 0 12 18 24 30 36 42 46 52 58 64 72 S. cerevisiae 0.0±0.0 1.4±0.1 3.3±0.2 7.1±0.1 8.2±0.1 9.0±0.1 9.2±0.1 9.5±0.2 9.8±0.2 10.1±0.2 13.8±0.1 15.2±0.2 S. boulardii 0.0±0.0 1.2±0.2 2.8±0.1 5.5±0.2 7.4±0.1 8.2±0.1 8.4±0.3 8.4±0.2 8.8±0.2 9.1±0.2 9.8.±0.2 12.1±0.2

Aronia lysate (alcohol 70% extract solution) + pear concentrate 2% medium fermented beta-glucan content (g/ 100g)) (72 hours) Yeast Beta-glucans Time (h) 0 12 18 24 30 36 42 46 52 58 64 72 S. cerevisiae 0.0±0.0 1.5±0.1 2.1±0.2 2.8±0.1 4.5±0.3 6.2±0.1 7.4±0.2 7.7±0.1 8.2±0.2 8.8±0.1 9.0±0.3 9.2±0.3 S. boulardii 0.0±0.0 2.1±0.2 4.0±0.2 5.5±0.1 6.2±0.2 7.8±0.1 8.1±0.2 12.4±0.1 12.9±0.4 13.8±0.3 14.2±0.3 14.9±0.3

Aaronia lysate (alcohol 70% extraction solution) + pear concentrate 2% DO (%) of fermented medium (72 hours) Yeast DO Time (h) 0 12 18 24 30 36 42 46 52 58 64 72 S. cerevisiae 100.0±0.0 .95.2±2.1 90.1±5.4 85.4±4.1 81.2±2.2 80.4±3.1 78.5±1.8 77.2±1.7 73.1±1.9 70.5±1.8 69.5±2.1 68.7±2.3 S. boulardii 100.0±0.0 96.3±1.8 91.2±1.7 88.2±1.5 82.1±2.2 81.4±2.4 79.3±1.1 76.8±1.2 74.1±1.5 72.1±1.8 70.9±1.4 69.9±1.5

Aronia lysate (alcohol 70% extract solution) + pear concentrate 2% medium (S. cerevisiae, S. boulardii) BOD (%) of alcohol production experiment in the fermenter of the optimal strain (72 hours) Yeast BOD Time (h) 0 12 18 24 30 36 42 46 52 58 64 72 S. cerevisiae 0±0.0 3.1±0.1 8.4±0.6 12.1±0.7 17.4±1.3 19.2±0.9 20.4±1.4 21.5±1.8 24.1±1.2 27.1±1.3 30.1±1.4 31.4±0.9 S. boulardii 0±0.0 2.5±0.1 7.7±0.2 11.2±0.1 15.2±1.7 18.2±1.2 19.2±0.8 20.1±1.1 22.1±0.8 23.4±0.7 24.4±1.1 25.2±1.2

As can be seen from the above results, as a result of a comparative experiment of 72 hours and 44 hours with 5L fermented, it was confirmed that the 72-hour fermentation time was superior to the 44-hour fermentation time in terms of yeast metabolites, and the alcohol content also increased the fermentation time. It was confirmed that it increased with the increase of 24 hours. Therefore, since a high alcohol content is advantageous for acetic acid fermentation, it was determined that 72 hours of fermentation was optimal.

Example 6: Experiments on alcohol production and acetic acid fermentation of fermented products

In the same way as in Example 5, after mixing the aronia lysate (70% alcohol extraction solution) and 2% by weight of the pear concentrate, the yeast was cultured for 72 hours, and the yeast cultured aronia fermented product was filtered through a primary non-woven fabric. And after a second paper filter, it was put back into the jar fermenter, and Acetobacter aceti (ATCC15973) was inoculated at 1% of the total volume, followed by acetic acid fermentation for 7 days. The acetic acid fermented product was analyzed in the same manner as in Example 1, and the results are shown in Tables 55 to 59 below.

Yeast pH Time (h) 0 12 24 36 48 60 72 84 96 108 120 132 1441 156 168 S. cerevisiae 3.55±0.02 3.50±0.02 3.40±0.02 3.38±0.03 3.37±0.01 3.35±0.02 3.40±0.02 3.38±0.03 3.35±0.02 3.31±0.02 3.30±0.02 3.30±0.02 3.28±0.02 3.27±0.02 3.26±0.02 S. boulardii 3.58±0.01 3.54±0.02 3.50±0.03 3.40±0.02 3.35±0.02 3.33±0.01 3.30±0.02 3.28±0.03 3.27±0.02 3.26±0.02 3.25±0.01 3.20±0.01 3.18±0.03 3.17±0.01 3.17±0.01

Aronia lysate (alcohol 70% extraction solution) + acidity (%) of alcohol production and acetic acid production experiment of fermented pear concentrate 2% medium (168 hours) Yeast Acidity Time (h) 0 12 24 36 48 60 72 84 96 108 120 132 1441 156 168 S. cerevisiae 0.94±0.02 1.02±0.02 1.14±0.02 1.52±0.01 1.98±0.02 2.14±0.01 2.48±0.01 3.02±0.02 3.48±0.01 3.88±0.01 4.14±0.02 4.87±0.02 5.02±0.02 5.21±0.02 6.08±0.02 S. boulardii 0.88±0.02 0.92±0.02 1.04±0.01 1.32±0.02 1.62±0.02 1.89±0.03 2.14±0.03 2.57±0.03 2.98±0.02 3.08±0.02 3.58±0.02 4.08±0.02 4.49±0.03 4.87±0.01 5.02±0.02

Table 64. Aronia lysate (alcohol 70% extract solution) + pear concentrate 2% medium (S. cerevisiae, S. boulardii) Amino acid content (%) of alcohol production experiment and acetic acid production experiment in the fermenter of the optimal strain (168 hours ) Yeast Amino acod Time (h) 0 12 24 36 48 60 72 84 96 108 120 132 1441 156 168 S. cerevisiae 0.95±0.01 1.04±0.02 1.10±0.02 1.42±0.01 1.78±0.02 2.04±0.01 2.18±0.01 2.89±0.02 3.18±0.01 3.48±0.01 3.88±0.02 4.07±0.02 4,66±0.02 5.01±0.02 5.41±0.02 S. boulardii 0.99±0.03 0.90±0.02 1.02±0.01 1.12±0.02 1.32±0.02 1.79±0.03 2.04±0.03 2.27±0.03 2.68±0.02 2.98±0.02 3.08±0.02 3.28±0.02 3.49±0.03 3.87±0.01 4.02±0.02

Alcohol content (%) of alcohol production experiment and acetic acid production experiment of aronia lysate (alcohol 70% extraction solution) + pear concentrate 2% medium fermentation (168 hours) Acetic acid Alcohol content Time (h) 0 24 72 96 120 168 S. cerevisiae 15.2±0.2 15.1±0.1 8.8±0.2 6.2±0.1 5.1±0.3 3.1±0.2 S. boulardii 12.1±0.2 12.1±0.2 7.8±0.1 5.4±0.2 3.2±0.1 1.9±0.1

Aronia lysate (alcohol 70% extraction solution) + beta-glucan content (g/ 100g) of alcohol production experiment and acetic acid production experiment of fermented pear concentrate 2% medium (168 hours) Acetic acid Alcohol content Time (h) 0 24 72 96 120 168 S. cerevisiae 9.2±0.3 7.1±0.1 5.8±0.2 4.2±0.1 3.1±0.3 1.1±0.2 S. boulardii 14.9±0.3 12.0±0.2 10.8±0.1 8.4±0.2 7.2±0.1 6.9±0.1

As can be seen from the above results, it was confirmed that there was a significant difference in the amount of acetic acid produced depending on the yeast. In addition, in the case of the S. boulardii strain, which produces a large amount of metabolite beta-glucan, it was confirmed that the decrease was smaller than that of the S. cerevisiae strain by acetic acid fermentation. Accordingly, it is determined that both strains are optimal conditions depending on use and purpose. To increase the amount of vinegar and acetic acid, yeast fermentation with S. cerevisiae strain followed by acetic acid fermentation is the optimal condition, and if metabolites are desired, yeast fermentation with S. boulardii strain followed by acetic acid fermentation is considered the optimal condition.

Example 7: Industrial yeast fermentation and oak barrel aging experiments

An experiment was conducted to confirm the suitability of industrial yeast fermentation for mass production. A mixture of 70% alcohol extract and 2% pear concentrate of aronia lysate was added by setting it to 5% in consideration of the RPM characteristics of a large alcohol fermenter. The oak barrel was soaked for 5 days while exchanging purified water on a daily cycle, and after completion, internal disinfection was carried out with sodium sulfite (1g/1L) to suppress the growth of germs. Fermented aronia fermented liquid was moved to an oak barrel and aged at 6 ° C for 2 months.

The industrially fermented aronia fermentation broth was similar to the results of Example 6, although not described herein.

In addition, after moving the industrially fermented aronia fermented liquid into an oak barrel, the acidity, pH, Brix content, and alcohol content before aging, after 1 month of aging, and after 2 months of aging were measured in the same manner as in Example 1. did

As a result, the acidity was 1.55~1.57% before aging, and the acidity of 1 month of aging was 1.52% and 1.57% of Oak-1 and Oak-2, respectively, and the acidity of 2 months of aging was 1.50% and 1.55%. In the case of acidity (%), it was found that it was kept constant until 2 months of aging. In the case of pH, the value was 3.936 to 4.001 before aging, and the pH value slightly increased to 4.014 to 4.045 after 2 months of aging, but there was no significant difference.

In the case of Brix content, before aging, Oak-1 and Oak-2 were 17.0% and 17.5%, respectively, consistent with the % sugar content of industrial large-scale yeast fermentation. It is thought that the longer the aging, the lower the sugar content.

In the case of alcohol content, the alcohol content tended to increase as aging progressed. The alcohol content in the 2nd month of aging increased to 10.5% and 11.0% in Oak-1 and Oak-2, respectively, and it is thought that post-fermentation is continuously progressing due to the influence of low-temperature environment and oak barrel.

Example 8: Establishment of mass production process for optimal acetic acid fermentation of aronia

8-1. Establishment of optimal acetic acid fermentation process for aronia

In order to confirm the optimal conditions of the aronia acetic acid fermentation process, it was carried out using a 5L Jar fermentor from Kobiotech Co., Ltd. Total acidity, pH, sugar content (brix%), alcohol content (%), DO, and BOD were measured according to the fermentation progress time of the Jar fermentor by the following analysis method. Acetic acid fermentation uses the aronia yeast (S. cerevisiae) fermentation liquid described in the above examples, etc., after adding seeds, correcting the initial acidity to 1.5 ~ 2.0%, and then fermenting conditions at aeration rate VVM 0.5, 250 rpm, 30 ℃ was set to proceed with aerobic fermentation for 14 days. The specific process is shown in Figure 1.

analysis method

1) Measurement of total acid and pH

For the total acid content, after centrifuging the sample during the acetic acid fermentation period, 1 mL of the supernatant was taken, neutralized and titrated with 0.1 N NaOH solution using 1% Phenolphthalein as an indicator, and acidity (%) was shown by calculating with an acetic acid conversion factor (Equation see 3). The pH of the fermentation product was measured by taking a 20 mL sample and using a pH meter (SevenEasy, Mettler-Toledo AG, Schwerzenbach, Switzerland).

[Equation 3]

Total Acidity (%) = [(0.1N-NaOH Consumption × 0.006g × F) / Sample volume] (mL) × 100

F: 0.1N-NaOH factor

2) Brix (Soluble solid content) content measurement

Sugar content was measured using a sugar meter (ATAGO, Pocket Refractometer, P AL-2) by taking 500 μL of the fermented product.

3) Alcohol content measurement

Alcohol content was measured according to the National Tax Service's liquor analysis regulations. After taking 100 mL of the fermentation product in a measuring cylinder, it was transferred to a round flask. After washing the measuring cylinder containing the fermented product three or more times with 10 mL of distilled water, the solutions were combined. Heat was applied to the round flask containing the fermented product using a Soxhlet heater to distill it. When the distillate reached 70 mL, distillation was stopped and distilled water was replenished to make 100 mL. After stirring, the alcohol content was indicated using a spirit meter (ATAGO, PAL-2REFRACTOMETER).

4) DO and BOD measurement

The amount of dissolved oxygen and biochemical oxygen demand of the fermentation broth were measured using (DO-31P, TOADKK Co., Ltd., Tokyo, Japan). The amount of dissolved oxygen was measured by quantifying 15 mL of the fermentation broth, and the amount of dissolved oxygen on the 0th day of fermentation was converted into 100% and the biological oxygen demand was calculated as 0%.

Analysis

1) Measurement of total acid and pH

Acidity and pH changes according to the period of acetic acid fermentation using Aronia yeast fermentation product (wine) are shown in Tables 60 and 61. The initial acidity of the AAF-0.5 sample group before the start of acetic acid fermentation was 1.78%. The acidity change increased by about 0.4% to 2.13% until the 8th day of fermentation, but it increased rapidly to 4.14% on the 14th day of fermentation. In the case of pH, it was confirmed that the value decreased as the acetic acid fermentation progressed, and it was found to be 3.60 after the acetic acid fermentation was completed from 3.87 at the beginning.

Changes in total acidity content according to aronia acetic acid fermentation time Acidity (%) content according to acetic acid fermentation time ¹⁾ Time 0 days 4 days 8 days 14 days AAF-0.5 ²⁾ 1.78±0.013) 1.99±0.01 2.13±0.01 4.14±0.01

pH change according to aronia acetic acid fermentation time pH change according to acetic acid fermentation time1) Time 0 days 4 days 8 days 14 days AAF-0.52) 3.87 3.78 3.72 3.60

2) Brix (Soluble solid content) content measurement

Table 62 shows the change in sugar content according to the period of acetic acid fermentation using Aronia wine. The sugar content on day 0 of the initial fermentation was 16.1% and gradually decreased as acetic acid fermentation progressed. It is judged that sugar content acts on acetic acid fermentation as a carbon source necessary for initial fermentation, and it was 15.1% on the 4th day of fermentation and 14.5% on the 8th day, and 14.5% on the 14th day after fermentation was completed, which was the same as the 8th day of fermentation.

Change in sugar content according to aronia acetic acid fermentation time Change in sugar content Brix (%) according to acetic acid fermentation time1) Time 0 days 4 days 8 days 14 days AAF-0.52) 16.1±0.003) 15.1±0.00 14.5±0.00 14.5±0.00

3) Alcohol content measurement

Table 63 shows the alcohol change according to the period of acetic acid fermentation using Aronia wine. The alcohol component is a substance that is converted into acetic acid through fermentation, and in general, as acetic acid fermentation proceeds, the alcohol content is reported to be 4-8%. The initial alcohol content of the AAF-0.5 sample group was 8.2%, decreased to 4.0% on the 4th day of fermentation and 1.7% on the 8th day of fermentation, and the alcohol content on the 14th day of fermentation was 0.5%.

Alcohol change according to aronia acetic acid fermentation time Change in alcohol (%) according to acetic acid fermentation time1) Time 0 days 4 days 8 days 14 days AAF-0.52) 8.2±0.293) 4.0±0.00 1.7±0.29 0.5±0.00

4) DO, BOD measurement

Changes in DO and BOD according to the period of acetic acid fermentation using aronia wine (fermented product) are shown in Tables 64 and 65. Acetic acid bacteria require a lot of oxygen for rapid growth and smooth metabolism in the early growth phase. In addition, the amount of dissolved oxygen increases as the number of viable cells decreases as the growth of microorganisms enters the stage of cessation and death. The amount of aeration was set at VVM 0.5, and the amount of dissolved oxygen and biochemical oxygen demand at 0 hour of fermentation were converted to 100% and 0%, respectively. On the 4th day of acetic acid fermentation, the amount of dissolved oxygen in AAF-0.5 was 73.5%, and the biochemical oxygen demand was 26.5%. In the case of demand, it showed a tendency to increase up to 98.8%.

DO change in aronia acetic acid fermentation time DO (%) change according to acetic acid fermentation time1) Time 0 days 4 days 8 days 14 days AAF-0.52) 100.0 73.5 26.3 1.2

Changes in BOD during aronia acetic acid fermentation BOD (%) change according to acetic acid fermentation time1) Time 0 days 4 days 8 days 14 days AAF-0.52) 0.0 26.5 73.7 98.8

8-2. Establishment of optimal acetic acid fermentation process for aronia

After the acetic acid fermentation using the Jar fermentor of Example 8-1 was completed, industrial acetic acid fermentation for mass production was performed. Among the fermentation conditions, air injection was performed for 1 hour per day in consideration of the RPM characteristics of the large fermentation vats and the fermenter environment, except for the temperature of 30 ° C. The fermented liquid after the acetic acid fermentation was separated from the fermented liquid by-product of the aronia fermentation using an industrial continuous centrifugal separator. The separated by-product was powdered by hot air drying.

Total acidity, pH, sugar content (brix%), alcohol content (%), DO, and BOD according to the fermentation progress time of the prepared aronia fermentation by-product were measured by the analysis method of Example 8-1, and the results are as follows Tables 65 to 68 show.

1) Measurement of total acid and pH

Changes in acidity and pH according to the period of industrial acetic acid fermentation using aronia wine (fermented product) are shown in Tables 66 and 67. Samples were collected and measured at intervals of 0, 4, 8, and 14 days, and the initial acidity of BAF-1 before the start of acetic acid fermentation was 1.79%. The change in acidity increased to 3.08% by the 8th day of fermentation, and by the 14th day of fermentation, the acidity was 4.20%, indicating that the acetic acid yield increased by about 46.61% compared to the initial acidity. In the case of pH, it was confirmed that the value decreased as the acetic acid fermentation proceeded, and the pH was 3.61 after the acetic acid fermentation was completed from the initial pH of 4.01.

Changes in total acidity content according to aronia large acetic acid fermentation time Acidity (%) content according to acetic acid fermentation time1) Time 0 days 4 days 8 days 14 days BAF-12) 1.79±0.003) 1.97±0.00 3.08±0.02 4.20±0.01

pH change according to Aronia large acetic acid fermentation time pH change according to acetic acid fermentation time1) Time 0 days 4 days 8 days 14 days BAF-12) 4.01 3.93 3.75 3.61

2) Brix (Soluble solid content) content measurement

Table 68 shows the change in sugar content according to the period of industrial acetic acid fermentation using Aronia wine. The sugar content on the initial day 0 of fermentation was 16.7%, and it decreased continuously as acetic acid fermentation progressed to 14.8% on the 4th day of fermentation, 13.9% on the 8th day of fermentation, and 13.2% on the 14th day of fermentation. It showed the same pattern as the trend of sugar content change that proceeded on the Lab scale of Example 8-1.

Change in sugar content according to aronia large acetic acid fermentation time Change in sugar content Brix (%) according to acetic acid fermentation time1) Time 0 days 4 days 8 days 14 days BAF-12) 16.7±0.003) 14.8±0.00 13.9±0.00 13.2±0.00

3) Alcohol content measurement

Table 69 shows the alcohol change according to the period of industrial acetic acid fermentation using Aronia wine. The alcohol component is a substance that is converted into acetic acid through fermentation, and in general, as acetic acid fermentation proceeds, the alcohol content is reported to be 4-8%. The initial alcohol content of the BAF-1 sample group was 8.0% and decreased to 6.2% on the 4th day of fermentation and to 3.5% on the 8th day of fermentation. The alcohol content also showed the same decreasing pattern as the alcohol content change on the Lab scale of Example 8-1, but the alcohol content on the 14th day after fermentation was terminated was 1.83%. This is considered to be the difference in air injection due to the shape of the fermentation device, unlike the Jar fermentor.

Alcohol change according to Aronia large acetic acid fermentation time Change in alcohol (%) according to acetic acid fermentation time1) Time 0 days 4 days 8 days 14 days BAF-12) 8.0±0.003) 6.2±0.29 3.5±0.00 1.83±0.29

Example 9: Anti-inflammatory assay of aronia vinegar

9-1. Cytotoxicity assay of aronia vinegar

RAW 264.7 cells, a macrophage cell line used in the experiment, were purchased from the Korea Cell Line Bank and cultured in RPMI 1640 medium containing 10% FBS (fetalbovine serum), 1% antibiotics, and 2-ME (2-mercaptoethanol) at 37°C and 4% It was cultured in an incubator controlled under CO2.

The cultured mouse macrophage cell line (RAW264.7) was dispensed per well at 5X10 ⁴ cells/100 μl in a 96 well plate, followed by overnight. After 1 hour after treatment with LPS at a concentration of 1 μg/ml, the prepared samples were treated by concentration and subjected to CO ₂ incubation for 24 hours. Then, the culture supernatant was removed, treated with CCK-8 reagent, and absorbance was measured at 450 nm using a Microplate reader.

As a result, as can be seen in Figure 2, it was confirmed that there was no cytotoxicity at the concentration used in the experiment (Figure 2).

9-2. Test of NO activity inhibitory effect of aronia vinegar

In macrophages, when an inflammatory response occurs due to external stimuli such as LPS, NO is secreted, various substances such as inflammatory cytokines are produced, and various pathological responses that control the inflammatory response occur. Experimental samples were treated in RAW264.7 cells, in which an inflammatory response was induced by LPS, and anti-inflammatory efficacy was investigated.

Specifically, NO ₂ (Nitrite), a stabilized NO oxide, was measured using the Griess reaction. First, RAW264.7 cells were dispensed in 5X10 ⁴ cells/100 μl per well in a 96 well plate, followed by overnight. After treating LPS at a concentration of 1μg/ml and CO₂ incubation for 1 hour, the prepared samples were treated by concentration and CO₂ incubation was performed for 24 hours. Then, the culture supernatant was placed in a 96 well plate, and the same amount of Griess reagent (0.1% N-1-naphtyl-ethylendiamine in H ₂ O : 1% sulfanilamide in 5% H ₃ PO ₄ = 1 : 1) was added to the mixture. After reacting for 10 minutes, absorbance was measured at 550 nm using a Microplate reader.

As can be seen in Figure 3, aronia vinegar and aronia vinegar by-products showed NO production inhibition rates of about 21% and 14%, respectively, and aronia vinegar was superior to aronia vinegar by-products in NO production inhibition rate.

9-3. Cytokine production test

The inflammatory response is induced by an increase in inflammatory cytokines due to an excessive immune response, and representative cytokines include TNF-α, IL-6, IL-1β, and GM-CSF. Among the pro-inflammatory cytokines secreted by LPS in macrophages, IL-6 differentiates B cells into plasma cells, promotes antibody production, and converts an acute inflammatory response into a chronic phase. In addition, TNF-α is a cytokine involved in systemic inflammation. It is produced not only by macrophages but also by NK cells and CD4 T cells. Excessive production of TNF-α induces fever and apoptosis. IL-1β acts as a major mediator of inflammatory responses by activating caspase-1 in activated macrophages, and in particular induces excessive inflammatory responses by activating Th cells. Excessive secretion of pro-inflammatory cytokines is a factor in various diseases such as sepsis, Alzheimer's disease, cancer, and inflammatory bowel disease.

Accordingly, the effect of aronia vinegar on inflammatory cytokines induced by LPS in RAW 264.7 cells was confirmed.

Specifically, RAW264.7 cells were seeded in a 24-well plate and then overnight. Thereafter, LPS was added to the well at a concentration of 1 μg/ml, and the samples were treated for each concentration, followed by CO₂ incubation for 24 hours. Then, the cell culture supernatant was collected. The cytokines IL-6, TNF-α, GM-CSF, and IL-1β contained in the supernatant were measured using enzyme-linked immunosorbent assay (ELISA). That is, the primary antibody capture antibody was diluted in coating buffer in the plate-bottom micro-well, dispensed at 100 μl/well, overnight at 4° C., and washed with a washing solution. The washed micro-well was blocked with PBS supplemented with 10% FBS, and the culture supernatant collected in the experiment was diluted in an appropriate ratio and then dispensed into each well and reacted at room temperature. Then, after reacting with 100 μl/well of the biotin-attached secondary antibody at room temperature for a certain period of time, 100 μl/well of enzyme reagent was added. Finally, a substrate was added to develop color, and then the color was measured using a microplate reader. The measured concentrations of IL-6, TNF-α, GM-CSF, and IL-1β were converted using a standard curve.

As a result, as can be seen in Figure 4, it was confirmed that aronia vinegar inhibited cytokine production compared to aronia vinegar by-products, and in particular, had a significant effect on IL-6 production.

Claims (4)

delete As an anti-inflammatory composition containing aronia vinegar,
The aronia vinegar is preparing an aronia lysate; Adding pear concentrate to the aronia lysate at 2% by weight based on the total weight of the aronia lysate; Extracting the aronia lysate to which the pear concentrate was added at 80 to 100 ° C. for 2 to 4 hours with 70% alcohol solvent; The alcohol extract of the aronia lysate was alcoholicly fermented at 100 to 130 rpm at room temperature for 70 to 74 hours with a mixed strain of Saccharomyces cerevisiae and Saccharomyces boulardii to obtain Aronia making wine; Fermenting the aronia wine with acetic acid while injecting air for 1 hour per day for 160 to 170 hours; And centrifuging the acetic acid fermentation product to obtain a supernatant of aronia vinegar; anti-inflammatory composition characterized in that produced from. delete As a food composition for preventing and improving inflammation containing aronia vinegar,
The aronia vinegar is preparing an aronia lysate; Adding pear concentrate to the aronia lysate at 2% by weight based on the total weight of the aronia lysate; Extracting the aronia lysate to which the pear concentrate was added at 80 to 100 ° C. for 2 to 4 hours with 70% alcohol solvent; The alcohol extract of the aronia lysate was alcoholicly fermented at 100 to 130 rpm at room temperature for 70 to 74 hours with a mixed strain of Saccharomyces cerevisiae and Saccharomyces boulardii to obtain Aronia making wine; Fermenting the aronia wine with acetic acid while injecting air for 1 hour per day for 160 to 170 hours; And centrifuging the acetic acid fermentation product to obtain a supernatant, which is aronia vinegar. A food composition for preventing and improving inflammation, characterized in that produced from.