KR102431762B1 - Chaga extract enhanced active ingredients and preparation m ethod of the same including two step extraction process with high-temperature pressurization - Google Patents

Abstract

The present invention provides an active ingredient-enhanced chaga mushroom extract using a high-temperature pressurization process and a two-step extraction method, and a method for producing the same, comprising: (a) a first-stage high-temperature/pressure pretreatment process of high-pressure treatment of chaga mushroom pieces at high temperature; (b) a grinding process of pulverizing the chaga mushrooms obtained in the pretreatment process (a) using a food processor; (c) a primary extraction process of hot water extraction of the chaga powder obtained in the grinding process (b); (d) a first concentration drying step of filtering the hot water extract obtained in the first extraction step (c), then concentrating and freeze-drying; (e) a second extraction process of extracting the chaga mushroom gourd (sludge) obtained in the first concentration and drying process (d) with a two-step organic solvent; (f) a second concentration drying step of filtering the organic solvent extract obtained in the second extraction step (e), then concentrating and freeze-drying; (g) a tertiary concentration step of further concentrating the first and second concentrated dried products obtained in the first concentration and drying step (d) and (f) the second concentration and drying step; (h) characterized in that it consists of a final drying process of freeze-drying the tertiary concentrate obtained in the tertiary concentration process (g), and therefore, after high-temperature pressurization of the woody part of Chaga mushroom, two-step extraction according to hot water and solvent extraction conditions By using the method, there is an effect of obtaining a chaga mushroom extract with enhanced content of polyphenol, flavonoid, beta-glucan, betulin and betulinic acid as functional indicator active ingredients, and provides extract materials with confirmed antioxidant activity and safety of active ingredient content It is a very useful invention in the processed food industry because it has an excellent effect to provide various chaga mushroom products as a novel health functional food material with excellent flavor made from liquid, powder, and slurry as well as having an excellent effect.

Description

Chaga extract enhanced active ingredients and preparation m ethod of the same including two step extraction process with high-temperature pressurization}

The present invention relates to a chaga mushroom extract containing a high concentration of active ingredients contained in chaga, and a method for producing the same, and more particularly, to a chaga mushroom wood part after one-step high-temperature pressurization treatment, and then heating the intercellular and intracellular active ingredients with hot water and The most preferred method for preparing the chaga mushroom extract, which is enhanced in polyphenol, flavonoid, beta glucan, betulin and betulinic acid content as an active ingredient serving as a functional food index by applying a two-step extraction method including organic solvent extraction conditions It relates to a method for producing a novel chaga mushroom extract including a two-step solvent extraction method after high-temperature and pressure treatment.

Natural materials or natural resources have been used as simple processing for thousands of years, but as the importance of natural product research has recently risen, it has become a global trend to preserve and study the natural resources of one's own country. Functional substances derived from natural products are more effective in recovering from various diseases or injuries than chemical synthetic products, and have low toxicity in the body and show high preference among consumers without special side effects (refer to Non-Patent Document 1)

Accordingly, research on the search for naturally-derived functional substances derived from herbal medicines and natural products is continuously continuing.

Chaga ( Inonotus obliquus ) belongs to the Hymenochaetaceae of Basidiomycetes and, unlike other mushrooms, penetrates into the trunk of a birch tree in the form of a parasitic strain, grows, accumulates nutrients in the tree, penetrates the bark, and gradually penetrates the bark. It is a mushroom that protrudes to the outside and grows on the surface of a tree in the form of a hard lump (see Non-Patent Document 2).

It is known that chaga mushroom has been used for cancer, gastritis, ulcer and tuberculosis treatment in folklore since the 16th century. , antioxidant and anticancer effects, effects on immune regulation, suppression of weight gain, and improvement of fat metabolism (refer to Non-Patent Documents 3 to 8) have been reported, and optimal physiologically active substances such as immunoactive polysaccharides according to extraction conditions Research on extraction conditions was also conducted (see Non-Patent Document 9).

Among the various functional substances of chaga mushroom, it contains a large amount of polyphenols and flavonoids and acts as a strong antioxidant (see Non-Patent Document 10). Antioxidants are substances that remove harmful free radicals, and have been recognized for their excellent preventive effects on various diseases including aging and lifestyle-related diseases, and thus the development of antioxidants derived from various natural products is actively underway. Free radicals that cause aging and diseases of the human body are formed through biological reactions during normal metabolic processes in the human body, and are known to cause harmful toxicity in cells and tissues to cause diseases (see Non-Patent Document 11).

In addition, various dietary fibers exist in chaga, and the most noteworthy material among them is β-glucan. It has been reported that β-glucan has an anticancer effect on various cancers by confirming that it enhances cellular immunity by restoring the activity of natural killer cells (see Non-Patent Documents 12 and 13).

Betulin is widely used as a starting material for organic synthesis of betulinic acid, betulonic acid, or their derivatives, effective as a therapeutic agent. Scientific facts have been published in many literatures. Through a number of papers, the scientific discovery that betulinic acid has antitumor activity on melanoma (eg, MEL-2, MEL-2 and MEL-4) (see Non-Patent Documents 14 and 15), H9 lymphocytes (lymphocytic) cells have an anti-HIV activity (activity) was announced the results of the study. And as a result of using betulin mixed with acyclovir, it was announced that it had an antiviral effect on herpes simplex viruses and the like (see Non-Patent Document 16).

As a prior art for the extraction method of chaga, it is a general method such as simple hot water extraction, and the chaga is eluted in hot water 5 to 40 times the volume at 30 to 100° C. for 5 to 10 minutes, or 1.2 to 2.5 for 8 hours. A method of extracting while maintaining a pressure of about kg/cm 3 is known.

However, natural products beneficial to the human body, such as mushrooms, contain various polar and non-polar active ingredients, and these active ingredients have different solubility and volatility due to differences in physical and chemical properties. It is considered to be a very important factor for effective dissolution of active ingredients. Regardless of the physical and chemical properties of the active ingredients of Chaga, the extraction and processing method for ingesting as many active ingredients as possible is very important.

Most of the reported or commercialized methods for producing chaga extracts reported so far are limited to the simple hot water extraction method or the organic solvent extraction method (domestic patent registration No. 10-1662185; see Patent Document 1), and other useful examples on a commercial scale for extracting active ingredients is not known. In particular, chaga is a mushroom having a hard woody tissue grown while parasitic on birch trees for a long time, and the literature on its pretreatment conditions has not been reported at all so far.

Patent No. 1662185 (Method for producing chaga extract containing high concentration of β-glucan) Registered Patent Publication,

Jung SJ, et al. 2004. Screening for antioxidant activity of plant medicinal extracts. J Korean Soc Appl Biol Chem. 47 135-140. Kahlos K. 1994. XII. Inonotus obliquus (Chaga Fungus): In vitro culture and the production of inotodiol, sterols, and other secondary metabolites. Biotechnology in Agriculture and Forestry 26: 179-198. Huang NL. 2002. A mysterioys medicinal fungus in Fussia Inonotys obliquus. Chinese J Edible Mushroom 21: 7-8. Song HY, Lee DJ, Lee BE. 2011. Studies on the anti-pulmonary tuberculosis of Inonotus obliquus. J. Mushroom Sci. Prod. 9: 190-193. Kim PR, Ko SK, Pyo MY. 2010. Effects of hot water extract of chaga mushroom on the proliferation and cytokines production of mouse splenocytes in vitro. Yakhak Hoeji. 54: 187-191. Qi Y, Zhao X, Lim YI, Park KY. 2013. Antioxidant and anticancer effects of edible and medicinal mushrooms. J. Korean Soc. Food Sci. Nutr. 42: 655-662). An CS, Jin HL, Jeon YH, Bak JP, Kim JD, Yoon JH, Lim BO. 2010. Immunoregulatory effects of water extracts of Inonotus obliquus in carbon tetrachloride-induced liver damage animal model. Korean J.Medicinal Crop Sci.18:1-8. Kim BB, Kim MS, Hyun CK. 2016. Suppression of adiposity and improvement of fat metabolism in high-fat diet-induced obese mice treated with an Inonotus obliquus extract. Kor. J. Pharmacogn. 47: 172-178. Kim JC, Yi HC, Lee KU, Hwang KT, Yoo GC. 2015. Optimization of the extraction of bioactive compounds from chaga mushroom (Inonotus obliquus) by the response surface methodology. Kor. J. Food Sci. Technol. 47: 233-239. Cui Y, Kim DS, Park KC. 2005. Antioxidant effect of Inonotus obliquus. Journal of Ethnopharmacology, 96: 79-85. Devy C, Gautier R. 1990. New perspectives on the biochemistry of superoxide anion and the efficiency of superoxide dismutase. Biochem. Pharmacol. 39: 399-405. Tabata K, Ito W, Kojima T, Kawabata S, Misaki A. 1981. Ultrasonic degradation of schizophyllan, an an-titumor polysaccharide produced by Schizophyllum commune fries. Carbohydrate Research. 89(1):121-135. Yamamoto T, Yamashita T, Tsubura E. 1981. Inhibition of pulmonary metastasis of Lewis lung carcinoma by a glucan, Schizophyllan. Invasion Metastasis. 1(1):71-84. Pisha, E. et al., (1995) J. M. Nature Medicine, 1, 1046-1051. Fujioka, T. et al. (1994) J. Nat. Prod. 57, 243-247. Yunhao Gong et al. Antiviral Research 64 (2004) 127-130.

Therefore, an object of the present invention is to devise a device with high utilization under high temperature and high pressure conditions as a first step in order to set the pressure treatment conditions as pretreatment conditions for the xylem xylem, then set the high temperature and pressure conditions, and then set the high temperature and pressure conditions as the second step. To provide a novel Chaga mushroom extract by setting and confirming two-step extraction conditions with enhanced contents of polyphenol, flavonoid, beta-glucan, betulin, and betulinic acid, which are effective active ingredients as functional indicators of food according to solvent extraction conditions. have.

Another object of the present invention is to provide a novel and advanced extract material for which the safety of active ingredient content, such as antioxidants, produced using the above method has been confirmed, and at the same time, a formulation with excellent flavor made from liquid, powder and slurry of various chaga mushroom products.

The above object of the present invention is to provide a method for preparing a chaga extract fortified with active ingredients using a two-stage method of one-step high-temperature and pressure pretreatment and solvent extraction, comprising the steps of: (a) pre-treating the cut pieces of chaga mushroom with high-temperature and pressure; (b) pulverizing the chaga mushroom pretreated under high temperature and pressure in step (a); (c) extracting hot water after mixing water with the pulverized chaga powder of step (b); (d) filtering the hot water extract extracted from the hot water in step (c) and then first concentrating to prepare a chaga mushroom concentrate; (e) extracting after mixing alcohol with the chaga gourd remaining after the filtration of step (d); (f) filtering the alcohol extract extracted in step (e) and then concentrating the second step to prepare a chaga mushroom gourd concentrate; (g) mixing the chaga mushroom concentrate prepared by the primary concentration of step (d) with the chaga mushroom gourd concentrate prepared by the secondary concentration of the step (f) and concentrating tertiary to prepare a concentrate; and (h) freeze-drying the concentrate prepared by the third concentration in step (g).

In the high temperature and pressure pretreatment step (a), the size of the chaga mushroom pieces is preferably 3 to 4 × 3 to 4 × 3 to 4 (cm) in width × length × height, and the pressure is 3.5 to 4.5 bar. It was preferable to add If the size of the piece is larger than the size and the pressure is lower than the pressure of 3.5 bar, the chaga mushroom pieces are not well pressed, and when the size of the piece is larger and the pressure is higher than 4.5 bar, the extraction yield of the mushroom is lower than the pressure low was not desirable.

The powder of chaga obtained in the grinding treatment process (b) is preferably 35 to 45 mesh, and if the fineness is less than 35 mesh, it is not preferable because it does not mix well with water, and if it is larger than 45 mesh, compared to the grinding time The yield was low, which was not preferable.

In addition, in the primary hot water extraction process of step (c), it was most preferable to mix the mixture of chaga powder and water in a ratio of 1:20 (w/v) and then extract at 95°C to 130°C for 3.5 to 4.5 hours. .

Here, if the temperature is lower than 95°C or the extraction time is less than 3.5 hours, the extraction of the active ingredient is reduced, which is undesirable, and if the temperature is higher than 130°C or the extraction time is longer than 4.5 hours, compared to the energy consumption and extraction time It was not preferable because the extraction yield of the active ingredient was low.

In the first concentration and drying step of step (d), the hot water extract obtained in step (c) was centrifuged with a centrifuge, filtered with a filter paper, and concentrated with a rotary concentrator.

Here, the centrifugation is preferably centrifugation for 10 to 20 minutes per 4,500Xg to 5,500Xg of the extract.

In addition, in the secondary extraction process of step (e), it was most preferable to extract chaga mushroom gourd (sludge) for 7.5 to 8.5 hours using 45 to 55% alcohol.

Here, less than 45% of alcohol or extraction for less than 7.5 hours is not preferable because the extraction efficiency of the active ingredient is low, and extraction of more than 55% of alcohol or more than 8.5 hours is not preferable because the extraction yield is low compared to the extraction time.

delete

The present invention is an active ingredient serving as a functional index of food by introducing a novel extraction method including a two-step extraction method of performing a first-step high-temperature pressurization of the wood part of Chaga mushroom and then extracting it with a first-step hot water extraction and a second-step solvent extraction. It has the effect of obtaining a chaga mushroom extract with enhanced polyphenol, flavonoid, beta-glucan, betulin and betulinic acid content, and can provide an extract food material that has been confirmed to be safe in terms of antioxidant activity and active ingredient content. , liquid, powder and slurry, etc., have an excellent effect of providing a variety of chaga mushroom formulations with excellent flavor.

1 is a schematic diagram of a first-step high temperature and pressure pretreatment device for chaga mushroom used in the present invention;
Figure 2 is a comparison photo of the solubility of the final extract powder product according to the present invention, N company, B company extract,
3 is an execution flowchart of a method for producing a chaga mushroom extract enhanced with active ingredients using a two-step extraction method after high-temperature pressurization of the present invention.
Figure 4 is a photograph showing the test material prepared with the Russian chaga extract according to the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Example 1. Comparison of active ingredient content according to high temperature and pressure treatment conditions

1. Pretreatment device design

1 is a schematic diagram of the high temperature and pressure device for pre-treatment of chaga according to the present invention, and the present invention is effective from pieces of chaga that are cut into 3 to 4 × 3 to 4 × 3 to 4 (cm) in width × length × height. It was designed, designed and manufactured to produce powder-enhanced chaga extract.

1 is a schematic diagram of a chaga mushroom pretreatment apparatus according to the present invention, where 1 is an inner body, 2 is a side cover, 3 is an open cover, 4 is a cover, 5 is a glass window, 6 is a leg pipe, 7 is a Reference numeral 8 denotes a leg pad, 8 denotes a leg support pipe, 9 denotes an I-bolt, and 10 denotes a door hinge.

The chaga pre-treatment pressurization apparatus shown in FIG. 1 is composed of 10 trays, and increases the internal pressure through water vapor. The air supply unit that supplies water vapor to the inside is located at the bottom, and it automatically adjusts the pressure set through the panel and maintains the internal pressure. A safety device of up to 6 bar is installed at the top and is designed to drop the pressure below the allowable pressure when the internal pressure rises more. It is a remarkable feature of the device of the present invention that steam does not directly touch the chaga. However, when using a high-temperature/pressurization device similar to the device of the present invention, the device may be used if there is no difference in the extraction yield and active ingredient of the final chaga mushroom.

2. Pre-processing condition setting

Chaga pieces of the above-described size were set under various pressure pretreatment conditions (2 bar, 3 bar, 4 bar and 5 bar) and treated for each time (0.5 h and 1 h), and the results are shown in Table 1.

After grinding the chaga mushroom pretreated under high temperature and pressure using a food processor, a particle size of 35 to 45 mesh powder is mixed with water at a mixing ratio of 1:20 (w/v), and then at 100℃, 4 It was extracted under time conditions.

[Preparation of experimental material 1]

After hot water extraction was finished, centrifugation was performed at 5,000Xg for 15 minutes (Mega21R, Hanil Corp., Seoul, Korea), and then filtered using Whatman #1 filter paper (Whatman Co., Maidstone, England). The filtrate obtained above is concentrated using a rotary concentrator (N-1000VW, Eyela Co., Tokyo, Japan) and then freeze-dried (NB-504, Ilshin Co., Dongducheon, Korea), stored at room temperature, and used as a test material did.

2-1. Analysis of polyphenols and flavonoids

The content of phenolic compounds in the hot water extract obtained from chaga mushrooms obtained under high temperature and pressure pretreatment conditions than in the untreated test group was measured to be the highest at 4 bar, and it is shown from Table 1 below that the content of phenolic compounds is rather reduced under the pressure of 5 bar. Confirmed.

On the other hand, according to the treatment time, the phenolic compound content increased with time up to 1 hour of the treatment time, but it was confirmed that the difference was insignificant.

As a result of the above experiment, various compounds present in chaga were converted into polyphenolic compounds by high temperature/pressure pretreatment, or the physical structure was changed to facilitate extraction of polyphenolic compounds due to high-temperature and high-pressure treatment, resulting in phenolic compound content was judged to increase. The most preferable high-temperature and pressure treatment conditions were 100° C. and 4 bar for 1 hour.

mg/g processing time 2 bar 3 bar 4 bar 5 bar
polyphenols
untreated
(158.6 ± 5.7)
0.5 hours
159.5±5.2
245.0±6.8
348.2±10.4
318.02±11.2
1 hours
160.5±8.3
258.6±7.3
356.2±9.5
316.72±8.3
flavonoids
untreated
(35.97 ± 2.10)
0.5 hours
36.5±1.2
40.6±8.5
50.8±3.2
42.3±8.2
1 hours
36.5±1.3
36.5±7.3
57.3±4.11
52.6±9.3

2-2. Antioxidant (DPPH, ABTS)

Methods for measuring the antioxidant effect of chaga include radical scavenging activity using DPPH (α,α-diphenyl-β-picrylhydrazy l), ABTS[2,2'-azinobis-(3-ethyl-benzoth iazoline-6-sulfonic) acid)] cation (ABTS·+) scavenging ability is a representative measurement. The results of measuring the radical scavenging ability using DPPH after preparing the lyophilized extract powder in a 1% aqueous solution are shown in Table 2 below.

From Table 2, it can be seen from Table 2 that the degree of antioxidant increased according to pressure to 65.7±1.2%, 75.9±1.5%, 81.4±0.7%, and 80.3±0.2% when treated for 0.5 hours, and it was confirmed that it was increased by about 124% compared to the control.

As a result of measuring the cationic radical scavenging ability using ABTS, both untreated and treated groups showed more than 96% of cationic radical scavenging ability.

% processing time 2 bar 3 bar 4 bar 5 bar
DPPH assay
untreated
(65.1 ± 0.4)
0.5 hours
65.7±1.2
75.9±1.5
81.4±0.7
80.3±0.2
1 hours
67.4±0.7
79.3±0.9
85.5±1.0
84.3±0.5
ABTS assay
untreated
(96.4 ± 0.1)
0.5 hours
96.8±0.2
98.6±0.5
97.8±8.5
96.8±3.2
1 hours
97.5±0.4
98.3±0.4
98.4±0.1
97.5±0.7

2-3. Beta Glucan

As a result of measuring the beta-glucan content using the lyophilized extract powder according to the present invention, as shown in Table 3 below, the beta-glucan content was measured to be higher in the high-temperature/pressure pre-treatment test group than in the untreated test group, and as the pre-treatment pressure increased, the beta-glucan content was measured. It was confirmed that the glucan content was increased. At 4 and 5 bar, the 1 hour pretreatment test group had the highest beta-glucan content of 4.2 (w/w, %).

(w/w %) processing time 2 bar 3 bar 4 bar 5 bar
Beta glucan content
untreated
(2.1 ± 0.4)
0.5 hours
2.2±0.2
3.4±0.4
3.9±0.6
4.1±0.7
1 hours
2.3±0.7
3.5±0.9
4.2±0.5
4.2±0.9

When considering the above examples, it is determined that 0.5 to 1 hour treatment at 4 bar is the most suitable pretreatment condition for chaga mushrooms in consideration of the phenolic compound content, antioxidant index, beta glucan content, and economic aspects for industrialization.

Example 2. 2-step extraction method condition setting - non-polar active ingredient extraction condition setting

Known active ingredients of chaga mushroom include phenolic compounds, β-glucan, tricephenolic acid, horomadogen, polyphenol, oxyphenol carboxylic acid, finocin, chaga acid (60%), vanilla acid, paraoxyhyangic acid It contains various polar and non-polar physiologically active substances such as , pterin, sterol, lignin, betulin, and betulinic acid. Secondary organic solvent extraction was performed using alcohol.

1. Alcohol concentration and time setting

The freeze-dried primary extraction sludge is mixed with an organic solvent (50, 70, and 90% alcohol) at a ratio of 1:20 (w/v) at room temperature for 4, 8, and 12 hours and then extracted at 5,000Xg for 15 minutes. After centrifugation (Mega21R, Hanil Corp., Seoul, Korea), it was filtered using Whatman #1 filter paper (Whatman Co., Maidst one, England). The filtrate was concentrated using a rotary concentrator (N-1000VW, Eyela Co., Tokyo, Japan), then freeze-dried (NB-504, Ilshin Co., Dongducheon, Korea), stored at room temperature, and used in the experiment.

1-1. Changes in polyphenol and flavonoid content

The phenolic compound of Chaga mushroom is a representative compound that exerts an antioxidant effect. From Table 4 below, according to the alcohol content of 30, 50 and 70%, the polyphenol content increased as the alcohol concentration and extraction time increased. It was confirmed that the flavonoid content, a representative component of polyphenol, also increased as the alcohol concentration and extraction time increased.

As a result of the experiment, the higher the alcohol content, the lower the solubility and extraction yield. Therefore, as a result, when comparing solubility, polyphenol and flavonoid contents, an extraction time of 8 hours at a 50% alcohol concentration was determined to be the most economical, and the optimum extraction conditions were set.

% processing time alcohol 30% alcohol 50% Alcohol 70%


polyphenols
4 hours
170.6±20.4
222.4±15.8
250.3±14.5
8 hours
200.8±12.6
392.4±20.8
430.4±8.6
12 hours
230.5±10.5
412.4±15.6
465.3±4.6


flavonoids
4 hours
22.8±2.0
52.8±8.5
68.8±5.2
8 hours
35.2±3.2
72.7±5.3
98.4±0.1
12 hours
42.8±5.4
85.8±5.1
92.2±9.6

1-2. Measurement of Antioxidant Degree As a method to measure the antioxidant effect of chaga mushrooms, radical scavenging using DPPH (α,α-diphenyl-β-pi crylhydrazy l), ABTS[2,2'-azinobis-(3-ethyl) -benzothiazo line-6-sulfonic acid)] cation (ABTS +) scavenging activity measurement is a representative example. After preparing the freeze-dried extract powder as a 1% aqueous solution, the radical scavenging ability using DPPH was measured. As shown in Table 5 below, it was confirmed that the radical scavenging ability increased as the alcohol concentration and treatment time increased.

As a result of measuring the cationic radical scavenging ability using ABTS, as shown in Table 5, according to the present invention, it showed a cationic radical scavenging ability of 90% or more in all experimental sections.

% processing time alcohol 30% alcohol 50% Alcohol 70%


DPPH assay
4 hours
54.7±1.2
60.9±4.6
61.0±0.7
8 hours
57.9±0.5
68.3±0.2
71.5±1.0
12 hours
60.7±2.2
78.9±1.5
79.4±1.6


ABTS assay
4 hours
93.8±2.2
95.5±0.7
95.8±0.3
8 hours
95.8±1.2
97.8±0.8
97.1±0.2
12 hours
96.8±0.4
98.3±0.4
98.4±0.8

1-3. Beta Glucan

As a result of measuring the beta-glucan content using the freeze-dried extract powder according to the present invention, as shown in Table 6 below, the beta-glucan content was measured to be higher in the pressurized pretreatment test group than in the untreated test group, and the beta-glucan content increased as the pretreatment pressure increased. was able to confirm that In particular, it was measured the highest as 4.2 (w/w, %) in the 1 hour pressure pre-treatment test group at 4 and 5 bar.

(w/w,%) processing time alcohol 30% alcohol 50% Alcohol 70%


Beta glucan content

4 hours
1.2±0.4
2.5±0.8
2.4±0.1
8 hours
1.9±0.1
2.9±0.6
2.8±0.4
12 hours
1.9±0.9
3.2±0.7
3.3±0.5

1-4. Betulin, betulinic acid

As a result of analyzing the content of betulin and betulinic acid using the lyophilized extract powder by HPLC, it was measured as shown in Table 7 below, and was not detected in the hot water extract powder.

When checking the measurement results from Table 7, the content change according to the alcohol content was higher than the increase in betulin and betulinic acid according to the extraction time. In the case of betulin and betulinic acid, 500mg/100g, 200mg under 50% alcohol It was confirmed that /100g or more was extracted.

% processing time alcohol 30% alcohol 50% Alcohol 70%


Betulin
4 hours
354.4±9.7
505.6±25.4
608.5±21.4
8 hours
362.7±10.4
531.5±10.4
616.9±25.6
12 hours
360.5±9.4
546.4±19.7
626.6±30.4


Betulinic acid
4 hours
130.2±1.4
180.8±5.5
201.5±12.4
8 hours
142.0±2.3
182.5±12.5
210.5±10.6
12 hours
153.5±5.5
207.6±10.4
230.8±5.4

As a result of quality analysis at the same concentration (1% aqueous solution) of the extraction powder for each condition, non-polar active ingredients that were not extracted from hot water were extracted from alcoholic solvents, and as the content of alcohol increased, the content of polyphenols, flavonoids, betulin and betulinic acid increased. However, considering the economical aspect for industrialization due to the low extraction yield, the alcohol concentration was set at 50% for 8 hours.

Example 3. Quality measurement of final extract

1. Active ingredient analysis

As a result of comparing the quality of the final extract powder of the present invention and the active ingredient of other companies' products, it was confirmed that they were significantly higher than those of other companies as shown in Table 8 below, and it was confirmed that betulin and betulinic acid were not detected in other companies' products.

division extract powder N company Company B Total polyphenol (mg/g) 317.9±1.8 189.5±6.5 235.3±22.4 Total flavonoids (mg/g) 47.50±2.92 43.19±6.79 44.44±3.37 Antioxidation (%)_DPPH assay 60.23±1.59 67.41±0.85 67.99±0.64 Antioxidation (%)_ABTS assay 98.42±0.10 56.57±0.74 76.28±1.29 Beta-Glucan (w/w, %) 3.7±0.5 1.3±0.5 2.8±0.8 Betulin (mg/100g) 413.1±15.4 Not detected Not detected Betulinic acid (mg/100g) 175.4±7.0 Not detected Not detected

2. Solubility Comparison

When 0.5 g of a sample of the present invention was dissolved in 500 mL of water, it was confirmed that it was transparent and had a very fast dissolution rate compared to other products as shown in FIG. 2 .

Example 4. Expression of flavor components of chaga mushroom extract

Since sugars or amino acids are widely distributed in food, the Maillard reaction is the most common browning reaction. The Maillard reaction not only affects the browning of food and the generation of flavor, but also plays an important role in the antioxidant and antibacterial properties of food, and has been found to be related to aging, geriatric disease and mutagenic inhibition.

As a result of amino acid analysis of the chaga mushroom sludge powder, it was shown in Table 9 below, and it was confirmed that the extracted sludge was also composed of various amino acids.

Amino acids (mg/g) chaga mushroom sludge



non polar Alanine 0.69 Glycine 0.58 Isoleucine 0.52 Leucine 0.81 Methionine ND Proline 0.82 Valine 0.78
Polar Serine 0.58 Threonine 0.71 Cystein 0.04
Basic Lysine 0.33 Arginine 0.28 Histidine 0.19
Acid Aspartic acid 1.11 Glutamic acid 1.18
Aromatic Phenylalanine 0.26 Tyrosine ND
Total amino acid contents (mg/g)

8.87

The sludge after extraction did not show any flavor, so in order to prepare leaching tea for Chaga mushroom, various types of sugar were first added and mixed. At this time, the direct fire roasting conditions were fixed at 100° C. for 4 minutes.

1. Sensory test according to sugar type

As sugars, glucose (glucose), fructose (fructose), and sucrose (sugar, sucrose) were used at 1% each. The results of sensory evaluation are shown in Table 10 below. For sensory quality evaluation, 10 researchers were asked to recognize the characteristics of chaga, and then sensory evaluation of color, flavor, and overall acceptability was assessed using a 5-point scoring method (5: very good, 4: good, 3: average, 2: not good, 1: Very poor). When sensory and color were checked, Sucrose was found to be the best sugar.

division organoleptic properties Color flavor overall glucose 3 0.5 One fructose (fructose) 2 0.5 One sucrose (sugar, sucrose) 3 4 4

2.Sensory test according to sugar concentration

The results of sensory evaluation by treating sucrose by concentration are shown in Table 11 below. In the sensory evaluation of color, 2, 4, and 6% showed the highest values, and in the sensory evaluation of fragrance, 4% and 6% were high in that order, and 4% concentration was evaluated as high in the overall evaluation. It was judged that the higher the sucrose concentration, the more caramelization proceeded, affecting the flavor.

Concentration (%, w/w) organoleptic properties Color flavor overall 2 3 2 2 4 3 4 4 6 3 3 2 8 2 One 2 10 2 One One

As a result of the experiment, the optimal roasting conditions for flavor improvement were set to 4% sucrose, 100℃, 4 minutes, and direct fire roasting conditions.

[Experimental material preparation and test method 2]

In accordance with the expansion of the functional food and RTD (Ready to Drink) beverage market, in the present invention, the physicochemical (polyphenol, beta-glucan, betulin and betulinic acid , antioxidant activity) and microbiological quality stability were analyzed.

2-1. Sample preparation and storage conditions

We purchased chaga from Siberia, Russia, and rinsed the surface cleanly and used it in the experiment.

After pretreatment through a steamer, the first hot water extraction and the second solvent extraction of the filtration foil were mixed, and then concentrated with 10Brix and used for the analysis experiment.

The chaga mushroom extract was sterilized at 95°C or higher for 10 minutes and sealed in 10 ml each in a 20 ml glass bottle. In order to measure the quality change under the conditions of direct sunlight, it was stored in a well-lit window.

In the condition where the light is blocked, based on the shelf life setting storage temperature accelerated test conditions, 35℃, the distribution temperature of the products distributed at room temperature, and 25℃ and 45℃ as the experimental zone were set and stored in the incubator at each temperature. After freezing, -20℃ was additionally set and stored to check the quality stability of the thawed state.

In addition, in order to confirm the stability of the precipitation according to the change in pH, the pH was adjusted to 4 and 8 and stored at 35 °C, and the storage stability with respect to pH was investigated.

The chaga extract stored in each condition was taken out every 2 months and the experiment was carried out (FIG. 4).

2-2. Measurement of physicochemical properties

A certain amount of the chaga extract stored in a 20ml glass bottle at each storage temperature was taken and measured using a pH meter (STAR-A111, Orion) and a refractometer (PR-101, ATAGO Co., Tokyo, Japan).

Table 12 shows the changes in physicochemical properties of the chaga extract according to the storage temperature for each storage period. The initial pH of storage was 5.30, the highest during storage was 5.46, the lowest was 5.38, and the pH was 5.45~5.46 on the last day of storage. The sugar content also showed little difference between the initial value of storage and the value measured at the 8th month of storage.

period
(month) temperature -20℃ 25 ℃ 35 ℃ 45 ℃ pH 0 5.30 2 5.38 5.42 5.42 5.41 4 5.41 5.44 5.43 5.46 6 5.41 5.43 5.42 5.41 8 5.45 5.46 5.45 5.46 °Brix 0 10.0 2 10.0 10.0 10.1 10.0 4 9.9 9.9 10 9.9 6 10.0 10.0 10.0 10.0 8 10.0 10.1 10.1 10.0

2-3. Antioxidant level measurement

For the measurement of antioxidant, the sample was diluted with 0.1 Brix to obtain a significant absorbance value and used in the experiment.

The DPPH method is a method of measuring the antioxidant effect by electron donating action according to the degree of discoloration of dark purple by reduction by antioxidant substances such as tocopherol, ascorbate, flavonoid compounds, sulfur-containing amino acids such as glutation, maillard-type browning substances, and peptides. way.

As a result of measuring the radical scavenging ability using DPPH after diluting the chaga extract according to the storage temperature for each storage period to 0.1 Brix, it was found that all of -20℃, 25℃, 35℃, and 45℃ had an electron donating effect of more than 70%. It was confirmed that the antioxidant effect was maintained even when the storage period was increased.

As a result of measuring the cation radical scavenging ability using ABTS, the degree of antioxidant decreased as the temperature increased, but it showed a cationic radical scavenging ability of 70% or more in all experimental sections, indicating that the antioxidant activity was high even with an increase in the storage period (Table 13). .

period
(month) temperature -20℃ 25 ℃ 35 ℃ 45 ℃ Antioxidant degree (%)
DPPH assay 0 70.53 ± 0.31 2 73.23 ± 0.07 73.15 ± 0.29 72.85 ± 0.32 72.82 ± 0.13 4 71.72 ± 0.38 71.28 ± 0.23 71.28 ± 0.40 71.54 ± 0.12 6 71.61 ± 0.03 71.44 ± 0.08 71.41 ± 0.10 71.14 ± 0.10 8 71.49 ± 0.60 71.42 ± 0.57 71.38 ± 0.55 71.03 ± 0.41 Antioxidant degree (%)
ABTS assay 0 81.44 ± 0.30 2 77.60 ± 1.53 74.68 ± 1.29 73.97 ± 1.99 74.17 ± 1.00 4 80.86 ± 0.15 77.97 ± 0.37 77.24 ± 0.34 76.96 ± 0.22 6 80.44 ± 0.27 76.69 ± 0.28 76.27 ± 0.23 75.75 ± 0.55 8 80.38 ± 0.31 76.24 ± 0.16 75.61 ± 0.16 75.02 ± 0.16

3-1. Free radical scavenging effect using DPPH (a,a-diphenyl-β-picrylhydrazyl)

In order to confirm the free radical scavenging effect that causes oxidative stress, the electron-donating ability or radical-scavenging activity of the chaga water extract for the DPPH radical, which is a free radical, was evaluated by Blois MS (1958)'s modification method. (Kim JH et al. 2002). That is, in a container that blocks light, 1 mL of 0.15 mM DPPH solution dissolved in 99.9% methanol and 0.25 mL of each extraction sample solution were added, mixed well, and absorbance was measured at 517 nm after 30 minutes for comparison.

3-2.ABTS[2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid)] cation (ABTS·+) scavenging effect

According to the method of Leong and Shui (19. Leong LP, Shui G. 2002. An investigation of antioxidant capacity of fruits in Singapore markets. Food Chem 76:69-75), the antioxidative activity of chaga extract was measured for the scavenging ability of ABTS cations. was tested.

ABTS reacts with free radicals (hydroxyl, peroxyl, alkoxyl, inorganic radicals) to produce a relatively stable cation (ABTS·+) that absorbs at 414 nm. To prepare the solution, a 2.6 mM potassium persulphate solution was mixed with a 7.4 mM ABTS solution and reacted in a dark place for 12 to 16 hours, and then diluted so that the absorbance at 414 nm was about 1.5. 50 μL of the sample was mixed with 1 mL of this dilution, reacted at room temperature for 90 minutes, and absorbance was measured at 414 nm for comparison.

3-3.Measurement of polyphenol content

The total phenol content of the sample was quantitatively analyzed using the principle that the Folin-Ciocalteu reagent is reduced by the phenolic compound of the extract to develop a molybdenum blue color (Choi Y, Him M, Shin J, Park J, Lee J. 2003. The antioxidant activities of the some commercial teas. J Korean Soc Foof Sci Nutr 32: 723-727). 2 mL of 2% Na2CO3 solution was added to 100 μL of each extract sample, left for 3 minutes, and then 100 μL of 50% Folin-Ciocalteu reagent was added to measure absorbance at 750 nm after 30 minutes. And the absorbance of each extract was compared with the gallic acid calibration curve used as a standard to determine the total phenol content (μg gallic acid equivalent/mL). was used. Experimental measurements were repeated three times and the average value was used.

Polyphenols are very diverse, including tannins, and have recently been reported to have various functions. (Lee KW, Kundu KJ, Kim SO, Chun KS, Lee HJ, Surh YJ, CoCoa Polyphenols inhibit phorbol ester-induced superoxide anion formation in cultured HL-60 cells and expression of cyclooxygenase-2 and activation of NF-kappaB and MAPKs in mouse skin in vivo.Journal of Nutrition.2006;136(5):1150-5). (superoxide anion formation in cultured HL-60 cells and expression of cyclooxygenase-2 and activation of NF-kappaB and MAPKs in mouse skin in vivo. Journal of Nutrition. 2006;136(5):1150-5). The polyphenol content in the chaga extract was almost the same from 26.79 mg/ml at the beginning to 25.90 mg/ml at 45°C after 8 months. Therefore, the polyphenol content according to the storage period and temperature showed almost no change (Table 14).

period
(month) temperature -20℃ 25 ℃ 35 ℃ 45 ℃ polyphenol content
(mg/ml) 0 26.79 ± 0.17 2 25.93 ± 0.33 25.46 ± 0.48 24.02 ± 0.96 24.99 ± 0.58 4 25.62 ± 0.80 26.75 ± 0.78 25.08 ± 0.81 26.24 ± 1.13 6 26.79 ± 1.75 25.50 ± 1.57 25.97 ± 0.60 26.93 ± 1.07 8 26.53 ± 1.05 26.37 ± 1.09 27.22 ± 0.34 25.90 ± 1.96

3-4.Measurement of beta-glucan content

For measurement of beta-glucan content, each sample was freeze-dried and tested in a powder state.

After determining the total glucan and non-glucan sugar contents using the Mushroom and Yeast β-glucan kit (Megazyme, Ireland), the β-glucan content (% w/w) was measured as the difference between α-glucan and non-glucan sugar contents. .

After adding 37% hydrochloric acid to the crude β-glucan powder sample and hydrolyzing it at 100° C. for 2 hours, the pH was adjusted using 2N KOH. The hydrolyzate was diluted with 200 mM sodium acetate buffer (pH 5.0) and centrifuged. Take 100 μl of the supernatant, add exo 1,3-β-glucanase and β-glucosidase, and react at 40° C. for 1 hour.

After that, a glucose determination reagent was added and reacted at 40° C. for 20 minutes, and absorbance was measured at 510 nm (UV/VIS Spectrophtometer V-530, JASCO, Japan) to determine the total glucan and sugar content other than glucan. analyzed.

For α-glucan and sugar content other than glucan, 2 ml of 2 M KOH was added to the sample, fixed in a container filled with ice, and stirred with a stirrer (Magnetic Stirrer C-MAG HS7, IKA Labortechnik, Germany) for 20 minutes. 8 ml of 1.2M sodium acetate buffer (pH3.8) was added, and 200 μl of amyloglucosidase and invertase was added thereto, followed by a 40° C. water bath (Water bath C-WBE, Chang Shin Science Co.). ), followed by centrifugation (1500 g, 10 minutes) for 30 minutes. 100 µl of the centrifuged supernatant was taken, 100 µl of 200 mM sodium acetate buffer (pH 5.0) and 3 ml of a glucose confirmation reagent were added, reacted at 40°C for 20 minutes, and absorbance was measured at 510 nm.

The contents of total glucan, α-glucan and β-glucan were calculated by the following equation.

[Equation]

Total glucans (% w/w) = ΔE × F/W × 90

α-glucan (% w/w) = ΔE × F/W × 9.27

β-glucan = total glucan - α-glucan

In the above formula, ΔE = eaction absorbance - blank absorbance

F = 100 / GOPOD reagent absorbance for 100μg of D-glucose standard

W = weight of sample

As a result of measuring the beta-glucan content using the lyophilized extract powder, as shown in the table, the beta-glucan content was measured to be 25 mg/g or more in all treatment groups, and it was confirmed that there was no significant change in the beta-glucan content depending on the storage period and temperature became (Table 15).

period
(month) temperature -20℃ 25 ℃ 35 ℃ 45 ℃ Beta glucan content
(mg/g) 0 25.78 ± 0.05 2 26.67 ± 0.95 25.95 ± 1.00 24.80 ± 0.83 24.69 ± 0.41 4 24.08 ± 0.20 25.14 ± 0.20 25.32 ± 0.20 25.34 ± 0.44 6 26.37 ± 0.33 27.54 ± 0.89 24.11 ± 0.56 22.83 ± 0.63 8 24.91 ± 0.28 24.46 ± 0.25 24.76 ± 0.10 23.65 ± 0.43

3-5. Betulin, betulinic acid content

Betulin (Sigma: B8795, minimum 98%) and betulinic acid (Sigma: 855057, minimum 90%) were used as standard samples. 100 mg of standard sample betulin was dissolved in 20 mL of HPLC-grade ethanol at room temperature to prepare a mother solution with a concentration of 5.0 g/L. L of reagent was prepared and analyzed by HPLC. The standard sample, 10 mg of betulinic acid, was dissolved in 20 mL of HPLC-grade ethanol to prepare a 0.5 g/L mother solution and analyzed. Analyzed by HPLC [HPLC Column: Nova-Pak C18-ODS reversed phase, 4 μm, 3.9×300 mm, eluent: CH3CN:H2O=86:14 v/v). The concentration of betulin contained in an unknown reagent was determined using a calibration curve made by linear regression by plotting the area of the HPLC peak obtained by analyzing each sample as the concentration.

The content of betulin and betulinic acid in the chaga extract concentrated with 10 Brix was analyzed by HPLC and measured as shown in the table. Contrary to the measurement results using the lyophilized extract powder, betulinic acid was not detected, and it was confirmed that the content of betulinic acid decreased as the temperature increased rather than the change over time (Table 16).

period
(month) temperature -20℃ 25 ℃ 35 ℃ 45 ℃ Betulin
(ppm) 0 11.93 2 11.93 11.73 9.65 8.02 4 12.56 10.28 11.42 9.26 6 10.14 12.43 10.21 7.02 8 10.64 9.41 9.56 8.06 Betulinic acid
(ppm) 0 - 2 - - - - 4 - - - - 6 - - - - 8 - - - -

3-6. Microbial test

To measure the number of microorganisms in the chaga extract, dry film medium for the number of bacteria and coliform group was used, and 1 ml of the sample dilution was dispensed into each medium and cultured at 35°C for 48 hours using the standard plate method, followed by counting colonies. It was expressed as Colony Forming Unit (CFU) per 1 ml of sample.

The changes in microorganisms according to the storage period and temperature of the chaga mushroom extract are shown in the table. Normal bacteria and coliform groups were not detected in all samples during storage, indicating that they were microbiologically safe. Therefore, ingestion of chaga mushroom extract is judged to be food hygienic and suitable for food standards (100 or less per 1 ml of number of bacteria, coliform group: negative) (Table 17).

period
(month) temperature -20℃ 25 ℃ 35 ℃ 45 ℃ common bacteria
(CFU/ml) 2 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 8 0 0 0 0 0 0 0 0 coliform
(CFU/ml) 2 - - - - 4 - - - - 6 - - - - 8 - - - -

3-7. Sedimentation stability test

In order to evaluate the precipitation stability of the chaga extract stored at each temperature and the chaga extract according to the change in pH, the degree of precipitation was shown as O, Δ, and X.

It was confirmed that precipitation did not occur even when the storage period was prolonged and the temperature was increased, and the chaga extract showed excellent sedimentation stability during storage in a water-soluble state (Table 18).

period
(month) temperature pH -20℃ 15 ℃ 35 ℃ 45 ℃ 4 8 Precipitation
(O, △, X) 2 × × × × × × 4 × × × × × × 6 × × × × × × 8 × × × × × ×

3-8. Chromaticity measurement

To measure the chromaticity change of the chaga extract under direct sunlight, take a certain amount of a sample stored in a 20 ml glass bottle near a well-lit window and use a colorimeter (Spectrophotometer CM-3500d, Konica Minolta Co., Ltd., Japan). L (brightness), a (+ red, - green), and b (+ yellow, - blue) values were repeatedly measured three times and expressed as an average value.

The chromaticity change of the chaga extract during storage in direct sunlight did not show significant changes in L, a, and b values, so it was confirmed that the chaga extract was stable to chromaticity change in direct sunlight (Table 19).

period
(month) L (brightness) a (redness) b (yellowness) 0 4.19 ± 0.22 -0.03 ± 0.02 -0.32 ±0.12 2 3.06 ± 0.14 -0.02 ± 0.01 -0.13 ± 0.08 4 4.96 ± 0.96 -0.09 ± 0.07 -0.76 ± 0.63 6 4.19 ± 0.37 -0.07 ± 0.04 -0.70 ± 0.21 8 3.74 ± 0.62 -0.02 ± 0.01 -0.21 ± 0.13

1: inner body 2: side cover
3: open cover 4: cover
5: glass window 6: bridge pipe
7: leg pad 8: leg support pipe
9: I bolt 10: door hinge

Claims (7)

(a) pre-treating the chaga mushroom pieces cut into 3 to 4 × 3 to 4 × 3 to 4 cm in width × length × height at 100° C. and 3.5 to 4.5 bar for 1 hour at high temperature and pressure;
(b) pulverizing the chaga mushroom pretreated under high temperature and pressure in step (a) to 35-45 mesh;
(c) mixing the pulverized chaga powder of step (b) with water in a ratio of 1:20 (w:v), followed by hot water extraction at 95-130° C. for 3.5-4.5 hours;
(d) filtering the hot water extract extracted from the hot water in step (c) and then first concentrating to prepare a chaga mushroom concentrate;
(e) mixing 45-55% alcohol with the remaining chaga mushroom gourd after the filtration of step (d) in a 1:20 (w:v) ratio, followed by extraction for 7.5-8.5 hours;
(f) filtering the alcohol extract extracted in step (e) and then concentrating the second step to prepare a chaga mushroom gourd concentrate;
(g) mixing the chaga mushroom concentrate prepared by the first concentration in step (d) with the chaga mushroom gourd concentrate prepared in the second concentration in step (f), and then concentrating tertiary to prepare a concentrate; and
(h) A method for producing a chaga extract with enhanced content of betulin and betulinic acid, characterized in that it comprises the step of freeze-drying the concentrate prepared by tertiary concentration of step (g). delete delete delete delete delete delete

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