KR20220099855A - Composition for anti-inflammation comprising Aronia vinegar - Google Patents

Abstract

The present invention relates to a composition for anti-inflammation comprising aronia vinegar, and more specifically, to a composition for anti-inflammation comprising aronia vinegar, especially, to a food composition for prevention, alleviation, and treatment of inflammation, which is produced by mixing an alcohol extract of the aronia pulverized and a pear concentrate followed by alcoholic fermentation, acetic acid fermentation and centrifugation. The aronia vinegar according to the present invention inhibits NO activity and IL-6 production in macrophages to be useful as a pharmaceutical and food composition for prevention, alleviation, and treatment of the inflammation. Moreover, the aronia vinegar according to the present invention is not cytotoxic and can be safely used as a material derived from natural aronia.

Description

Composition for anti-inflammation comprising Aronia vinegar

The present invention relates to an anti-inflammatory composition comprising aronia vinegar, and more particularly, it contains aronia vinegar prepared by mixing an alcohol extract of pulverized aronia and a pear concentrate, followed by alcohol fermentation and acetic acid fermentation, and centrifugation. It relates to an anti-inflammatory composition, particularly a food composition for the prevention and/or improvement of inflammation.

Inflammation is a defense mechanism occurring in living tissue 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, leading to 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, are cells that regulate inflammatory responses and immune functions through phagocytosis and play a very important role in maintaining homeostasis. Macrophages are activated by stimulation of LPS (lipoplolysaccharide), a component of the outer membrane of Gram-negative bacteria, and play a pivotal role in body defense in the early stage of infection, but macrophages activated by excessive LPS stimulation are induced by an increase in inflammatory cytokines. Among the pro-inflammatory cytokines secreted by LPS from macrophages, IL-6 differentiates B cells into plasma cells and promotes antibody production, converting the acute inflammatory response to the chronic phase (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 the inflammatory response in activated macrophages (Kim JH. et.al. , The Korean Society of Food Preservation, 24:1149-1157, 2018).

In addition, in the inflammatory process in the body, inflammatory factors such as excessive NO (nitric oxide) and PEG2 (prostaglandin E2) are generated by inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). NO is a highly reactive substance and is produced from L-arginine by NOS (NO synthase), and produces a large amount of NO when stimulated by external stimuli or pro-inflammatory cytokines. It has been reported that excessively produced NO promotes inflammatory reactions such as vascular permeability and edema. Another major inflammatory mediator, COX, is an enzyme that promotes the transformation of arachidonic acid into prostaglandins after liberation of arachidonic acid from phospholipids in the cell membrane. It is expressed in large amounts in cells such as macrophages and monocytes by stimulation. The resulting prostaglandins are involved in tumor formation by inhibiting tumor cell death and inducing angiogenesis. Therefore, as a composition for inflammatory diseases and prevention, iNOS, an NO-generating enzyme for the regulation of macrophage-mediated inflammatory response, COX-2, an enzyme that is a step in prostaglandin biosynthesis, and inflammatory cytokines, and their major signaling molecules MAPKs and NF- A substance capable of regulating the expression of κB is attracting attention as a composition for preventing and improving inflammation. In particular, research on a composition for preventing and improving inflammation caused by natural products is being actively conducted (Kim SY. et. al., J. Korean Orient. Med. Ophthalmol. Otolaryngol. Dermatol., 26:54-64, 2013).

Accordingly, the present inventors have completed the present invention by confirming that aronia vinegar has a very excellent anti-inflammatory effect while conducting various studies on aronia (Aronia melanocarpa).

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 comprising aronia vinegar as an active ingredient.

In 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 obtained by mixing the alcohol extract and pear concentrate of the crushed aronia fruit, aging it after alcoholic fermentation, acetic acid fermentation, and centrifugation.

Specifically, the step of preparing the aronia vinegar aronia crushed product; adding a pear concentrate to the aronia lysate; extracting alcohol from the aronia lysate to which the pear concentrate is added; preparing an aronia wine by alcoholic fermentation of the alcohol extract of the aronia crushed product; acetic acid fermentation of the Aronia wine; and centrifuging the acetic acid fermented product to obtain an aronia vinegar supernatant.

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

First, it is a step of preparing an aronia crushed product.

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

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

The pear concentrate refers to a concentrated extract of pear, and the extract of pear may be extracted using water or an organic solvent, but is preferably a water extract of pear, more preferably a hot water extract of 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 crushed aronia. When the content of the pear concentrate is less than 1.5% by weight, the alcohol content of the aronia wine produced afterward does not reach the target level (ie, 10% or more), and when the content of the pear concentrate is more than 2.5% by weight, the content In comparison, a large increase in the alcohol content of aronia wines produced afterward cannot be expected.

In one specific implementation, when 1% by weight of the pear concentrate was used based on 100% by weight of the aronia lysate, the alcohol content of the prepared aronia wine was less than 10% compared to the case where 2% by weight was used, and the yeast metabolite was 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 the aronia lysate to which the pear concentrate was added by varying the concentration of alcohol was extracted with alcohol, the reducing sugar, which is a sugar used for fermentation, was 20% or more when the concentration of alcohol was 20% or more. In light of the previous research results that the alcohol content by yeast fermentation corresponds to 1/2 of the reducing sugar, the reducing sugar content of the alcohol extract must be 20% or more so that the alcohol content of the Aronia wine produced thereafter is at the target level (that is, 10 % or more), it is preferred that the concentration of alcohol is at least 18% or more, preferably 20% or more. However, when the concentration of alcohol is 70%, the content of anthocyanin in the extract is the best compared to the concentration of other alcohol, 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, the extract prepared by extracting the aronia lysate to which the pear concentrate is added may be used as an alcohol extract of aronia lysate or an aronia alcohol extract for convenience.

Next, it is a step of producing an aronia wine by alcoholic fermentation of the alcohol extract of the aronia crushed product.

The alcoholic fermentation is at least one strain selected from the group consisting of Saccharomyces cerevisiae and Saccharomyces boulardii, preferably Saccharomyces cerevisiae and Caromyces Buladai mixed strain, more preferably Saccharomyces cerevisiae refers to the inoculation of the alcohol extract of the aronia lysate and shaking culture. In the case of alcohol fermentation with the Saccharomyces cerevisiae strain, an aronia wine with a high alcohol content can be produced, and when alcohol fermentation with the Caromyces bulladai strain, an aronia wine with a high beta-glucan content can be prepared. can In particular, the Caromyces bulladai strain is a new strain that the applicant newly discovered and deposited as KCTC 14036BP on November 18, 2019 at the Korea Research Institute of Bioscience and Biotechnology.

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 carried out for 70 to 74 hours, preferably 72 hours, it was possible to prepare Aaronia wine having a desired level of alcohol content compared to the time less than that.

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

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 the subsequent acetic acid fermentation. As one specific example, the filtration may be made in the order of a primary nonwoven 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 in oak barrels, preferably for 2 months, before subsequent acetic acid fermentation. The aging may be performed before or after the filtration, preferably after filtration. Due to this aging, pH, acidity, sugar content, etc. of Aronia wine are almost unchanged, but the taste and aroma of Aronia wine have an effect suitable for taste.

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

The acetic acid fermentation refers to 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 after inoculation of acetic acid bacteria.

In addition, 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 carried out by such air injection, and thus it is possible to prepare acetic acid fermentation products having a desired level of pH, acidity, sugar content, and the like.

The acetic acid bacteria is an Acetobacter sp. strain, preferably Acetobacter aceti, based on the total volume % of the Aronia wine 0.8 to 1.2 volume %, preferably 0.9 to 1.1 volume %, most preferably Inoculate at a content of 1% by volume.

When acetic acid fermentation is performed under the above conditions, the pH and acidity of the fermented product, the alcohol content, and the like are contained enough to suit the taste and aroma of the aronia vinegar beverage.

Next, centrifuging the acetic acid fermented product to obtain a supernatant of aronia vinegar.

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

In the present specification, the supernatant obtained by centrifugation of the acetic acid fermented product is called aronia vinegar, and the precipitate by centrifugal separation of the acetic acid fermented product is called aronia vinegar by-product or aronia fermentation by-product.

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

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

In one specific aspect, the present invention provides a pharmaceutical composition for preventing and/or treating inflammation comprising 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 addition to aronia vinegar as an active ingredient, in particular, substances derived from natural materials.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable” means exhibiting non-toxic properties 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 buffers, preservatives, softening agents, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants, and the like.

In addition, the pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. can Furthermore, it can be used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel, or external preparation for skin in the form of a gel. 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, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.

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) or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, and freeze-dried preparations. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

On the other hand, 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 to an individual by an appropriate method, and the administration route of the composition may be administered through any general route as long as it can reach a target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, may be administered intrarectally, but is not limited thereto.

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

The term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level is determined by the sex, age, and weight of the patient. , health condition, disease type, severity, drug activity, drug sensitivity, administration method, administration time, administration route, and excretion rate, treatment period, factors including drugs used in combination or concurrently, and other medical fields. It can be readily determined by one of ordinary skill in the art according to known factors. For administration, the recommended dosage may be administered once a day, or divided into several administrations.

As another specific aspect, the present invention provides a food composition for preventing and / or improving inflammation comprising 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 suitably determined according to the purpose of use (prevention, health or therapeutic treatment), and may further include a food pharmaceutically acceptable food supplement additive. Since the composition of the present invention contains 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 significant limitation on the mixing amount.

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

As used herein, the term “functional food” refers to food manufactured and processed using raw materials or ingredients useful for the human body according to Health Functional Food Act No. 6727, and “functionality” refers to the structure of the human body. And it means ingestion for the purpose of obtaining useful effects for health purposes such as regulating nutrients for function or physiological action.

In addition, the term "health functional food" as used in the present invention refers to a food manufactured and processed by extracting, concentrating, refining, mixing, etc., a specific ingredient as a raw material or a specific ingredient contained in a food raw material for the purpose of health supplementation, It refers to a food designed and processed to sufficiently exert biological control functions, such as biological defense, regulation of biological rhythm, prevention and recovery of disease, etc. with respect to the living body by the above ingredients. It can perform functions related to recovery, etc.

There is no limitation on the kind of food in which the composition of the present invention can be used. In addition, the composition comprising aronia vinegar of the present invention as an active ingredient can be prepared by mixing other suitable auxiliary ingredients that may be contained in food and known additives according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There are vitamin complexes, and the like, and it can be prepared by adding the 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 (eg, formula milk, infant food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (eg, ramen, noodles, etc.), health supplements , Seasoned foods (eg soy sauce, soybean paste, red pepper paste, mixed soy sauce, etc.), sauces, sweets (eg snacks), dairy products (eg fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickles, etc.) ), beverages (eg, fruit, vegetable beverages, 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 ingredients, as in a conventional beverage. In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin , alcohol, a carbonation agent used in carbonated beverages, and the like. In addition, it may contain the pulp for the production of natural fruit juice, fruit juice beverage and vegetable beverage.

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 the prevention, improvement, and treatment of 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 for animals including humans.

1 is an optimal process flow chart for mass production of aronia vinegar according to an embodiment of the present invention.
2 is a diagram confirming the cytotoxicity of aronia vinegar and aronia vinegar by-products according to an embodiment of the present invention.
3 is a diagram confirming the NO inhibitory activity of aronia vinegar and aronia vinegar by-products according to an embodiment of the present invention.
4 is a diagram confirming the production amount of cytokines in 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 help the understanding of the present invention. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

Example 1: Optimal yeast strain experiment for production of aronia fermented products

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

Experimental method

1) Measurement of pH, titrated acidity and amino acid level

The pH of the fermented 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 titrated acidity was obtained by mixing 1 mL of sample and 1 mL of NaOH, adding 1% Phenolphthalein solution reagent, checking the section where the color changes, and then titrating to obtain the value. Amino acid level was tested by adding 9 mL of distilled water to 1 mL of sample, then titrating it with 0.1 N NaOH until pH 8.3 was reached, and converting the consumed amount into tartaric acid content (%, v/v).

2) Measurement of reducing sugar content

The reducing sugar content of the fermented 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 obtained by centrifuging the fermented product (1,500 x g, 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 a 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 diluting the fermented product 2 times, 1 mL was quantified, and then 1 mL of phenol solution was added and mixed well. Then, 5 mL of concentrated sulfuric acid was added and the reaction was exothermic as strongly as possible. After adding sulfuric acid, the absorbance was measured at 470 nm after standing at room temperature for 40 minutes. Total sugar content was calculated after preparing a standard curve using glucose as a standard substance.

4) °Brix (Soluble sugar content) content measurement

Brix content of the fermented product was measured using a saccharometer (Pocket refractometer PAL-2, ATAGO, Kyoto, Japan). After measuring 500 ul of the fermented product, the average value was calculated by repeating measurement three times. During the measurement, the experiment was performed after setting the zero point using distilled water.

5) Measurement of viable cell count

For the number of viable cells in the fermented product, take 1 mL of the fermented product, dilute it stepwise by 10 ⁴ , 10 ⁵ , 10 ⁶ times with sterile physiological saline, and spread 20 μL on the medium. In Jaecheon, Korea), colonies formed by culturing for 24 hours were counted and expressed as colony forming unit (CFU/mL).

6) Beta glucan content measurement

Beta-glucan content was analyzed using Megazyme Kit (Mushroom and Yeast β-glucan Assay Procedure K-YBGL). Total glucan and α-glucan measured at absorbance 510 nm are the absorbance values of the reaction solution obtained by reacting glucose solution (1 mg/mL) with GOPOD reagent together with Mega on www.megazyme.com homepage. -Calc It was calculated as the content (%, w/w) value referring to the content calculation formula. Finally, all beta-glucans were calculated by subtracting the alpha-glucan content from the glucan content. Equation 1 below was tested in accordance with the Ministry of Food and Drug Safety's Health Functional Food Standard.

[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 coefficient (160/180)

7) DO, BOD measurement

The amount of dissolved oxygen and biochemical oxygen demand of the fermented product were measured using (DO-31P, TOADKK, Tokyo, Japan). 15 mL of the fermented product was measured and repeated 3 times to calculate the average value. However, the dissolved oxygen amount of the 0th day of fermentation was converted into 100% and the biological oxygen demand was converted to 0%, and the experiment was carried out.

8) Determination of alcohol content

The alcohol content of the fermented product was measured in accordance with the National Tax Service's Alcohol Analysis Regulations. After taking 100mL of the sample in a measuring cylinder, it was transferred to a 500mL Erlenmeyer flask. After washing the measuring cylinder containing the sample 3 times with 10mL of distilled water, the solution was added to the Erlenmeyer flask. An Erlenmeyer flask was connected to one side of the cooler, and a 100 mL measuring cylinder was connected by hand to the other side. Heat was applied to the sample using a Soxhlet heater. When the distillate reached 70 mL, distillation was stopped, distilled water was added, and the volume was adjusted to the 100 mL scale of the measuring cylinder. After shaking well and measuring with an alcohol meter, temperature correction was performed according to the alcohol content conversion table.

9) Statistical processing

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

Experiment result

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

pH of fermented aronia lysate using 4 yeast strains 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 changes in acidity of alcoholic fermentation for four strains of aronia yeast. In the case of acidity, there was a tendency to slightly increase according to the yeast fermentation time, but it was found that this did not affect the yeast fermentation. S. cerevisiae strain showed a slight increase from 0.65±0.02 at the start of fermentation to 0.77±0.01 for 46 hours of fermentation.

Acidity (%) of fermented aronia lysate using 4 yeast strains 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

Table 3 shows the changes in the amino acid level of alcohol fermentation for the four strains of Aronia yeast. In the case of amino acids, there was a tendency to slightly increase according to the yeast fermentation time, but it was found that this did not affect the yeast fermentation. S. cerevisiae strain showed a slight increase from 0.68±0.02 at the start of fermentation to 0.86±0.01 for 46 hours of fermentation.

Amino acid degree (%) 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 alcoholic fermentation for four strains of aronia yeast. In the case of total sugar, it decreased according to the yeast fermentation time, which showed a decreasing trend according to the sugar metabolism process of the yeast fermentation strain. The S. cerevisiae strain showed a high decrease due to glucose metabolism after the S. boulardii strain from 8.52±1.42 at the start of fermentation to 0.98±0.32 for 46 hours of fermentation.

Total sugar content (%) of fermented aronia lysate using 4 yeast strains 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 reducing sugar changes in alcoholic fermentation for the four strains of Aronia yeast. In the case of reducing sugar, it decreased according to the yeast fermentation time, which showed the same decreasing trend as the total sugar according to the sugar metabolism of the yeast fermentation strain. S. cerevisiae strain showed a tendency to decrease from 5.52±0.66 at the start of fermentation to 0.55±0.23 for 46 hours of fermentation, which is the same as the total sugar due to glucose metabolism after S. boulardii strain.

Reducing sugar content (%) of fermented aronia lysate using 4 yeast strains 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 change in the number of viable cells in alcoholic fermentation for the four strains of aronia yeast. In the case of the number of viable cells, when 0 hours of yeast fermentation was set as 0, the S. cerevisiae strain decreased from 5.52±0.66 at the start of fermentation to 0.55±0.23 for 46 hours after S. boulardii strain, which was the same as the total sugar due to glucose metabolism. showed a tendency to

Number of viable cells (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 changes in the alcohol content of alcohol fermentation for the four strains of aronia yeast. In the case of alcohol content, when 0 hours of yeast fermentation was set as 0, it was confirmed that the S. cerevisiae strain made the highest alcohol content by sugar metabolism from 0 to 46 hours of fermentation at 3.10±0.10.

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

Beta-glucan (%) of fermented Aronia lysate using 4 yeast strains Yeast Beta glucan 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, as a result of measuring the component change by yeast strain when the same fermentation conditions were given to select the strain most suitable for alcohol production among the four yeast strains, the S. cerevisiae strain had the highest alcohol content production ability. 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 excellent in alcohol production, and S. boulardii, which is excellent in metabolites.

Example 2: Establishment of manufacturing conditions for aronia extract

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

In Example 1, it was determined that 10% of alcohol could not be produced by simple crushing of aronia, so that the crushed aronia was extracted with hot water according to the content of each alcohol concentration. proceeded. Total sugar and reducing sugar content were the same as in Example 1 above. The results 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 alcohol 5% alcohol 10% Alcohol 20% Alcohol 30% alcohol 40% 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

Total sugar (Reducing sugar content) (%) of hot water extract of aronia lysate or extract by alcohol concentration Time (number of times) Condition water 100 alcohol 5% alcohol 10% Alcohol 20% Alcohol 30% alcohol 40% 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 in the present invention is 10% alcohol. When 10% of 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. Therefore, when calculating the fermentation time, it is confirmed that when extracting 10% alcohol and 20% of aronia crushed alcohol, the reducing 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 the content of each alcohol concentration in aronia lysate

To measure the total anthocyanin content, an extract was prepared by the same method as in 2-1 above for each alcohol concentration of the aronia lysate, and ethanol containing 1% HCl was added thereto, followed by sonication for 15 minutes to extract anthocyanins, and then 10 at 4000 rpm. The supernatant obtained by centrifugation for minutes was used for the experiment. After diluting the sample 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, measured at 510 and 700 nm absorbance, and then the following formula 2 was used to measure the total anthocyanin content. For the standard material, the anthocyanin lambda max value was applied when measuring after confirming the highest 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: Dilutiion factor

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

V = final sample volume

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

mg/gram)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. in the fermented product of 20% alcohol extract of aronia lysate

In Example 2, since it was confirmed that the reducing sugar content of the medium extracted with 20% alcohol from the aronia lysate was 28%, the pH, acidity, amino acid, total sugar, Reducing sugar, number of viable cells and alcohol content were measured. The results are shown in Tables 12 to 18 below. On the other hand, the 20% alcohol extract of aronia crushed product used in this experiment was concentrated after extraction of alcohol, and then the experiment was carried out, which was to blow alcohol to make 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

Aronia lysate using 4 types of yeast (20% alcohol extraction) Acidity (%) of fermented product 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

Aronia lysate using 4 types of yeast (20% alcohol extraction) Amino acid content (%) 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

Aronia lysate using 4 types of yeast (20% alcohol extraction) Total 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 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 (%) in fermentation 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) Number of viable cells in fermented product (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

Aronia lysate using 4 types of yeast (20% alcohol extraction) Alcohol content (%) of fermented product 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 production capacity of the aronia fermented product increased when the 20% alcohol extract was used, but it was confirmed that the alcohol content was less than 10%, which is one object of the present invention. . Therefore, in the next experiment, we tried to use sweetened pear concentrate without using sweetened sugar. In addition, in the subsequent experiment, 70% of alcohol was used in consideration of the anthocyanin content of Example 2-2.

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

We purchased and used 5 kg of 100-year-old aronia products sold by the Aaronia Research Society of Mokgol Co., Ltd. The crushed liquid used for fermentation was primarily used to separate the branches and fruits of aronia. Afterwards, the fruits were washed in tap water. The washed fruits were crushed with a blender (Mufti Quick 7-MQ736, BRAUN, Germany). After hot water extraction at 100°C for 3 hours under the 70% alcohol condition (40g aronia crushed product + 800g alcohol), the aronia crushed product was concentrated at 45°C, 70-80 RPM, 80mbar conditions for 3 hours to blow off the alcohol. Afterwards, this aronia lysate was used as a medium, and the pear concentrate was used as a raw material containing 65.21±3.24% of total sugar and 44.02±4.14% of reducing sugar. After adding 1% or 2% by weight of the total volume to the concentrated aronia crushed product as a pear concentrate, it was sterilized at 121° C. for 15 minutes and then allowed to cool for 2 hours. As in Example 1, 4 types of yeast were inoculated with 0.1% of the medium volume and cultured at 140 rpm at 27° C. for 44 to 46 hours. After culturing, the fermentation characteristics of the alcohol extract of aronia lysate were analyzed in the same manner as in Example 1, and as a result, the results when 1 wt% pear concentrate was mixed in Tables 19 to 26 below, Table 27 To Table 34 shows the results when mixing 2 wt% pear concentrate.

Aronia lysate using 4 types of yeast (70% alcohol extraction) + pH of mixed fermented pear concentrate 1% 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

Acidity (%) of aronia lysate using 4 types of yeast (70% alcohol extraction) + 1% pear concentrate 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

Amino acid content (%) of aronia lysate using 4 types of yeast (70% alcohol extraction) + 1% pear concentrate mixed fermentation Yeast amino acid 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 Total 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) + reducing sugar (%) of mixed fermented pear concentrate 1% 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) + Number of viable cells in 1% mixed fermented 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±1.35 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

Aronia lysate using 4 types of yeast (70% alcohol extraction) + 1% pear concentrate alcohol content (%) 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 aronia lysate using 4 types of yeast (70% alcohol extraction) + 1% pear concentrate mixed fermentation (g/100g) Yeast Beta glucan 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) + pH of 2% pear concentrate mixed fermentation 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

Acidity (%) of aronia lysate using 4 types of yeast (70% alcohol extraction) + 2% pear concentrate 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

Amino acid content (%) of aronia lysate using 4 types of yeast (70% alcohol extraction) + 2% pear concentrate mixed fermentation Yeast amino acid 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) + 2% pear concentrate Total sugar (%) 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

Reducing sugar (%) of aronia lysate using 4 types of yeast (70% alcohol extraction) + 2% pear concentrate mixed fermentation 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) + Number of viable cells in mixed fermented product 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 mixed fermented 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 mixed fermented pear concentrate 2% (g/100g) Yeast Beta glucan 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 than when 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 that the S. boulardii strain was higher than the S. cerevisiae strain.

After that, the next experiment was carried out after supplementing with 2% of the pear concentrate, selecting the S. cerevisiae strain and the S. boulardii strain as the optimal strain.

Example 5: Measurement of alcohol content, etc. of fermented products according to fermentation time

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

After culturing, the fermentation characteristics according to the fermentation time of the alcohol extract of aronia lysate were analyzed in the same manner as in Example 1, and as a result, the results when the fermentation time was 44 hours are shown in Tables 35 to 44 below. Tables 45 to 54 show the results when the fermentation time of 2 wt% was 72 hours.

Aronia lysate (70% alcohol extract solution) + 2% pear concentrate pH of fermented medium (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 (70% alcohol extract solution) + acidity (%) of pear concentrate 2% medium fermentation (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

Amino acid content (%) of aronia lysate (70% alcohol extraction solution) + 2% pear concentrate 2% culture medium (44 hours) Yeast amino acid 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 (70% alcohol extract solution) + 2% pear concentrate 2% total sugar (%) of fermented medium (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 (70% alcohol extract solution) + reducing sugar (%) of pear concentrate 2% medium fermentation (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 (70% alcohol extract solution) + viable cell count (10 ⁶ CFU/mL) of pear concentrate 2% medium fermentation (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 (70% alcohol extract solution) + alcohol content (%) of pear concentrate 2% medium fermentation (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 (70% alcohol extract solution) + beta-glucan content of pear concentrate 2% culture medium (g/100g)) (44 hours) Yeast Beta glucan 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

Aronia lysate (70% alcohol extract solution) + DO (%) of pear concentrate 2% medium fermentation (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 (70% alcohol extract solution) + BOD (%) of pear concentrate 2% medium fermentation (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 (70% alcohol extract solution) + pH of pear concentrate 2% medium fermentation (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 (70% alcohol extract solution) + Pear concentrate 2% acidity (%) of fermented medium (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

Amino acid content (%) of aronia lysate (70% alcohol extract solution) + 2% pear concentrate 2% culture medium (72 hours) Yeast amino acid 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 (70% alcohol extract solution) + 2% pear concentrate 2% total sugar (%) of fermented medium (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 (70% alcohol extract solution) + reducing sugar (%) of pear concentrate 2% medium fermentation (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 (70% alcohol extract solution) + viable cell count (10 ⁶ CFU/mL) of pear concentrate 2% medium fermentation (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% extract 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 (70% alcohol extract solution) + beta-glucan content of pear concentrate 2% fermented medium (g/100g)) (72 hours) Yeast Beta glucan 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

Aronia lysate (Alcohol 70% Extract 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

BOD (%) of alcohol production experiment in fermenter of aronia lysate (70% alcohol extraction solution) + 2% pear concentrate medium (S. cerevisiae, S. boulardii) (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 comparative experiments for 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 as a yeast metabolite, and the alcohol content also decreased the fermentation time. It was confirmed that the increase was increased with the increase of 24 hours. Therefore, for subsequent acetic acid fermentation, it was determined that 72 hours of fermentation was optimal because it was advantageous to have a high alcohol content.

Example 6: Alcohol production and acetic acid fermentation experiment of fermented product

In the same manner as in the experimental method of Example 5, the aronia lysate (70% alcohol extraction solution) and 2% by weight of the pear concentrate were mixed, and then the yeast was cultured for 72 hours. And after the secondary paper filter was put back into the jar fermenter, acetobacter aceti (Acetobacter aceti (ATCC15973)) was inoculated with 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

Acidity (%) of alcohol production and acetic acid production experiments of aronia lysate (70% alcohol extraction solution) + 2% pear concentrate 2% culture 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. Amino acid content (%) of alcohol production experiment and acetic acid production experiment in fermenter of optimal strain of Aronia lysate (70% alcohol extraction solution) + 2% pear concentrate (S. cerevisiae, S. boulardii) (168 hours) ) Yeast Amino accord 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

Aronia lysate (70% alcohol extraction solution) + alcohol content (%) in 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 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 (70% alcohol extraction solution) + Beta-glucan content (g/100g)) of alcohol production experiment and acetic acid production experiment of 2% fermented pear concentrate (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 S. boulardii strain, which produces a lot of metabolite beta-glucan, it was confirmed that the decrease was smaller than that of S. cerevisiae strain due to acetic acid fermentation. Therefore, it is judged that both strains are optimal conditions depending on the use and purpose. If you want to increase the amount of vinegar and acetic acid, acetic acid fermentation after yeast fermentation with S. cerevisiae strain is the optimal condition, and if you want metabolites, it is judged that acetic acid fermentation after yeast fermentation with S. boulardii strain is the optimal condition.

Example 7: Industrial Yeast Fermentation and Oak Barrel Aging Experiment

Experiments were conducted to confirm the suitability of industrial yeast fermentation for mass production. Considering the RPM characteristics of the large alcohol fermenter, a mixture of 70% alcohol extract and 2% pear concentrate of aronia lysate was added by setting it to 5%. The oak barrels are exchanged with purified water on a daily basis and soaked for 5 days. The fermented aronia fermented broth was moved to an oak barrel and aged at 6° C. for 2 months.

Although the industrially fermented aronia fermented broth was not described herein, the results were similar to those of Example 6.

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

As a result, in the case of acidity, it was found to be 1.55~1.57% before ripening, and the acidity of Oak-1 and Oak-2 for 1 month was 1.52% and 1.57%, respectively, and the acidity after 2 months of aging was 1.50% and 1.55%. In the case of acidity (%), it was found to be kept constant until 2 months of aging. In the case of pH, it showed a value of 3.936-4.001 before ripening, and the pH value showed a tendency to slightly increase to 4.014-4.045 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, which was consistent with the % sugar content of industrial large yeast fermentation. It is thought that as the aging continues, the sugar content will decrease.

In the case of alcohol content, as the aging progressed, the alcohol content showed a tendency to increase. In the second month of aging, the alcohol content increased to 10.5% and 11.0% for Oak-1 and Oak-2, respectively, and it is considered that the post-fermentation continued due to the influence of the low temperature environment and oak barrels.

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

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

In order to confirm the optimal conditions for the aronia acetic acid fermentation process, a 5L jar fermentor of Kobiotech was used. Total acidity, pH, sugar content (brix%), alcohol content (%), DO, and BOD were measured according to the fermentation time of the jar fermentor by the following analysis method. Acetic acid fermentation is performed using the aronia yeast (S. cerevisiae) fermentation broth described in Examples, etc., after adding the seed seed, and then correcting the initial acidity to 1.5 to 2.0%, followed by fermentation conditions with an aeration VVM of 0.5, 250 rpm, and 30 ° C. was set to carry out aerobic fermentation for 14 days. The specific process is shown in FIG. 1 .

Analysis method

1) Total acid and pH measurement

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

[Equation 3]

Total Acid (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 saccharometer (ATAGO, Pocket Refractometer, P AL-2) by taking 500 μL of the fermented product.

3) Determination of alcohol content

Alcohol content was measured in accordance with the National Tax Service's Alcohol Analysis Regulations. After taking 100 mL of fermented product in a measuring cylinder, it was transferred to a round flask. After washing the measuring cylinder containing the fermented product with 10 mL of distilled water 3 times or more, the solution was combined. Using a Soxhlet heater, heat was applied to the round flask containing the fermented product for distillation. When the distillate reached 70 mL, distillation was stopped and distilled water was added to make 100 mL. After stirring, the alcohol content was indicated using an alcohol system (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, 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 calculated by converting 100% and biological oxygen demand to 0%.

Analysis

1) Total acid and pH measurement

Acidity and pH changes according to the period of acetic acid fermentation using aronia yeast fermented 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 change in acidity increased by about 0.4% to 2.13% until the 8th day of fermentation, but sharply increased 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 shown from 3.87 in the initial stage to 3.60 after the completion of the acetic acid fermentation.

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 showed a tendency to decrease gradually as the 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 found to be 15.1% on the 4th day of fermentation and 14.5% on the 8th day, and 14.5% on the 14th day after the end of fermentation, the same as on the 8th day of fermentation.

Sugar content change according to aronia acetic acid fermentation time Sugar content Brix (%) change 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) Determination of alcohol content

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 to acetic acid through fermentation, and in general, the alcohol content is reported to be 4-8% as acetic acid fermentation proceeds. The initial alcohol content of the AAF-0.5 sample group was 8.2%, and decreased to 4.0% on the 4th day of fermentation and 1.7% on the 8th day of fermentation. The alcohol content on the 14th day after the end of fermentation was 0.5%.

Alcohol change according to aronia acetic acid fermentation time Alcohol (%) change 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 microorganisms enter the growth cessation and death phase. The aeration amount was set at VVM 0.5, and the dissolved oxygen and biochemical oxygen demand at 0 hours of fermentation were converted to 100% and 0%, respectively. On the 4th day of acetic acid fermentation, the dissolved oxygen content of 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 by 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 time Change in BOD(%) 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 completion of the acetic acid fermentation using the jar fermentor of Example 8-1, industrial acetic acid fermentation was performed for mass production. Considering the RPM characteristics of the remaining large fermenters and the fermenter environment except for the temperature of 30℃ among fermentation conditions, air injection was performed for 1 hour per day. The fermentation broth after acetic acid fermentation was completed was separated from the fermentation broth from the aronia fermentation by-products using an industrial continuous centrifuge. The separated by-products were subjected to hot air drying and powdered.

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 are shown.

1) Total acid and pH measurement

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 days, 4 days, 8 days, 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% until the 8th day of fermentation, and it was found that the acetic acid yield increased by about 46.61% from the initial acidity to 4.20% at the 14th day of fermentation. In the case of pH, it was confirmed that the value decreased as the acetic acid fermentation progressed, and the pH was 3.61 after the completion of the acetic acid fermentation from the initial pH of 4.01.

Change 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 0th day of fermentation was 16.7%, and it continued to decrease 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 tendency of sugar content change in the Lab scale of Example 8-1.

Sugar content change according to Aronia large acetic acid fermentation time Sugar content Brix (%) change 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) Determination of alcohol content

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 to acetic acid through fermentation, and in general, the alcohol content is reported to be 4-8% as acetic acid fermentation proceeds. 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 3.5% on the 8th day of fermentation. The alcohol content also showed a decreasing pattern in the same manner as the change in the alcohol content in the Lab scale of Example 8-1, but the alcohol content at 14 days after the end of fermentation was 1.83%. This is considered to be a difference in air injection due to the shape of the fermenter, unlike the jar fermentor.

Alcohol change according to Aronia large acetic acid fermentation time Alcohol (%) change 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 used in RPMI 1640 medium containing 10% FBS (fetalbovine serum), 1% antibiotics, and 2-ME (2-mercaptoethanol) at 37°C, 4%. Incubated in an incubator controlled in CO2.

The cultured mouse macrophage line (RAW264.7) was dispensed in a 96-well plate at a rate of 5X10 ⁴ cells/100 μl per well, followed by overnight incubation. After 1 hour of LPS treatment at a concentration of 1 μg/ml, the prepared samples were treated by concentration and incubated with CO ₂ 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 FIG. 2 , it was confirmed that there was no cytotoxicity at the concentration used in the experiment ( FIG. 2 ).

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

In macrophages, when an inflammatory response is triggered by external stimuli such as LPS, various pathological responses that secrete NO, produce various substances such as inflammatory cytokines, and regulate the inflammatory response occur. The anti-inflammatory efficacy was investigated by treating the experimental sample in RAW264.7 cells induced by LPS.

Specifically, the stabilized NO oxide, NO ₂ (Nitrite) was measured using the Griess reaction. First, RAW264.7 cells were seeded in a 96-well plate at a rate of 5X10 ⁴ cells/100 μl per well, followed by overnight incubation. After LPS was treated at a concentration of 1 μg/ml and incubated for 1 hour with CO₂, the prepared samples were treated by concentration and incubated with CO₂ for 24 hours. Then, put the culture supernatant into a 96 well plate, and add 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). After reacting for 10 minutes, absorbance was measured at 550 nm with a microplate reader.

As can be seen in FIG. 3 , aronia vinegar and aronia vinegar by-product exhibited NO production inhibition rates of about 21% and 14%, respectively, and aronia vinegar had a superior NO production inhibition rate compared to aronia vinegar by-products.

9-3. Cytokine production assay

The inflammatory response is induced by an increase in inflammatory cytokines due to an excessive immune response. Representative cytokines include TNF-α, IL-6, IL-1β, and GM-CSF. Among the pro-inflammatory cytokines secreted by LPS from macrophages, IL-6 differentiates B cells into plasma cells and promotes antibody production, converting the acute inflammatory response to a chronic stage. In addition, TNF-α is a cytokine involved in systemic inflammation. In addition to macrophages, TNF-α is also produced by NK cells and CD4 T cells, and excessive production induces fever and apoptosis. IL-1β acts as a major mediator of the inflammatory response by activating caspase-1 in activated macrophages, and, in particular, induces an excessive inflammatory response 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 the inflammatory cytokines induced by LPS in RAW 264.7 cells was confirmed.

Specifically, RAW264.7 cells were seeded in a 24-well plate, followed by overnight incubation. Thereafter, LPS was applied to the wells at a concentration of 1 μg/ml, and samples were treated by concentration and incubated with CO₂ for 24 hours. Then, the cell culture supernatant was harvested. The cytokines IL-6, TNF-α, GM-CSF, and IL-1β contained in the supernatant were measured using the enzyme-linked immunosorbent assay (ELISA). That is, the primary antibody capture antibody was diluted in a coating buffer in a plate-bottom micro-well, dispensed at 100 μl/well, and after overnight at 4°C, washed with a washing solution. The washed micro-wells were blocked with PBS to which 10% FBS was added, and the culture supernatant collected in the experiment was diluted at an appropriate ratio, 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 the enzyme reagent was added. Finally, a substrate was added to develop color and then 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 FIG. 4, aronia vinegar inhibited cytokine production compared to aronia vinegar by-product, and in particular, it was confirmed that there was a significant effect on the amount of IL-6 production.

Claims (4)

As an anti-inflammatory composition comprising aronia vinegar,
The aronia vinegar, preparing an aronia crushed product; adding a pear concentrate to the aronia lysate; extracting alcohol from the aronia lysate to which the pear concentrate is added; preparing an aronia wine by alcoholic fermentation of the alcohol extract of the aronia crushed product; acetic acid fermentation of the Aronia wine; and centrifuging the acetic acid fermented product to obtain an aronia vinegar supernatant. Anti-inflammatory composition, characterized in that prepared from.
The method of claim 1, wherein the aronia vinegar comprises: preparing a crushed aronia; adding a pear concentrate to the crushed aronia in an amount of 2% by weight based on the total weight of the crushed aronia; extracting the aronia lysate to which the pear concentrate is added with a 70% alcohol solvent at 80 to 100° C. for 2 to 4 hours; The alcohol extract of the aronia lysate is at least one strain selected from the group consisting of Saccharomyces cerevisiae and Saccharomyces boulardii at 100 to 130rpm at room temperature for 70 to 74 hours. During alcoholic fermentation to prepare Aronia wine; acetic acid fermentation of the Aronia wine while injecting air for 1 hour per day for 160 to 170 hours; and centrifuging the acetic acid fermented product to obtain an aronia vinegar supernatant. Anti-inflammatory composition, characterized in that prepared from.
As a food composition for preventing and improving inflammation comprising aronia vinegar,
The aronia vinegar, preparing an aronia crushed product; adding a pear concentrate to the aronia lysate; extracting alcohol from the aronia lysate to which the pear concentrate is added; preparing an aronia wine by alcoholic fermentation of the alcohol extract of the aronia crushed product; acetic acid fermentation of the Aronia wine; and centrifuging the acetic acid fermented product to obtain an aronia vinegar supernatant. A food composition for preventing and improving inflammation, characterized in that it is prepared from.
The method of claim 3, wherein the aronia vinegar comprises: preparing a crushed aronia product; adding a pear concentrate to the crushed aronia in an amount of 2% by weight based on the total weight of the crushed aronia; extracting the aronia lysate to which the pear concentrate is added with a 70% alcohol solvent at 80 to 100° C. for 2 to 4 hours; The alcohol extract of the aronia lysate is at least one strain selected from the group consisting of Saccharomyces cerevisiae and Saccharomyces boulardii at 100 to 130rpm at room temperature for 70 to 74 hours. During alcoholic fermentation to prepare Aronia wine; acetic acid fermentation of the Aronia wine while injecting air for 1 hour per day for 160 to 170 hours; and centrifuging the acetic acid fermented product to obtain an aronia vinegar supernatant. A food composition for preventing and improving inflammation, characterized in that it is prepared from.

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