KR20210035026A - 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 relates to a Chaga mushroom extract with enhanced active ingredients by using a high-temperature pressurization process and a two-step extraction process, and a method of manufacturing the Chaga mushroom extract, capable of providing products of various Chaga mushroom formulations with excellent flavor. According to the present invention, the method of manufacturing the Chaga mushroom extract with enhanced active ingredients by using the high-temperature pressurization process and the two-step extraction process includes: a first-stage high-temperature/pressurization pretreatment process; a grinding process; a primary extraction process; a primary concentration/drying process; a secondary extraction process; a secondary concentration/drying process; a tertiary concentration process; and a drying process.

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 an extract of chaga mushroom containing an active ingredient contained in chaga at a high concentration and a method for producing the same, and more particularly, after the first step high-temperature pressurization of the woody part of chaga mushroom, the intercellular and intracellular active ingredients are hot-watered and Chaga extract with enhanced content of polyphenol, flavonoid, beta glucan, betulin and betulinic acid as an active ingredient serving as a functional food index by applying a two-step extraction method including organic solvent extraction conditions, and the most preferable method for preparing the same It relates to a method for preparing a novel chaga extract including a two-stage solvent extraction method after high-temperature pressurization treatment.

Natural materials or natural materials have been used as simple processing for thousands of years, but as the importance of natural products research has recently emerged, it is becoming a global trend to preserve and study natural materials of the country. Functional substances derived from natural products are more effective in recovering various diseases or injuries than chemical synthetic products, and are less toxic in the body, showing high preference to consumers without any special side effects (see Non-Patent Document 1).

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

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

Chaga is known to have been used in the private sector for the treatment of cancer, gastritis, ulcers and tuberculosis since the 16th century, and the effects of chaga on antituberculosis activity, splenocyte proliferation ability and cytokine production ability through related studies , Antioxidant and anti-cancer effect, effect on immune regulation, suppression of weight gain and improvement of fat metabolism (see Non-Patent Document 3 to Non-Patent Document 8), etc. are reported, and physiologically active substances such as immune-active polysaccharides according to extraction conditions are optimal. A study on the search for 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 powerful antioxidant (see Non-Patent Document 10). Antioxidants are substances that remove harmful free radicals, and have been recognized for their excellent preventive effects against various diseases including aging and lifestyle diseases, and the development of antioxidants derived from various natural products is actively progressing. Free radicals that cause aging and disease in the human body are formed through biological reactions during normal metabolic processes in the human body, and are known to cause diseases by causing harmful toxicity in cells and tissues (see Non-Patent Document 11).

In addition, various dietary fibers exist in chaga mushrooms, and among them, the most notable material is β-glucan. It has been reported that β-glucan exhibits anticancer effects against 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 used for organic synthesis of betulinic acid, betulonic acid, or their derivatives, which are effective as therapeutic agents. Scientific facts have been published in many literatures. The scientific discovery that betulinic acid has antitumor activity in melanomas (e.g., MEL-2, MEL-2 and MEL-4) through a number of papers (see Non-Patent Documents 14 and 15) shows that betulinic acid is Studies have shown that H9 lymphocytes (lymphocytic) cells have anti-HIV activity. In addition, as a result of using betulin mixed with acyclovir, it was announced that it had an antiviral effect on herpes simplex viruses (see Non-Patent Document 16).

Conventional techniques for the extraction method of chaga mushrooms are general methods such as simple hot water extraction, and the chaga mushrooms are eluted for 5 to 10 minutes in 5 to 40 times the volume of hot water at 30 to 100°C, 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 that are beneficial to the human body such as mushrooms contain various polar and non-polar active ingredients, and these active ingredients have different solubility or 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 mushrooms, the extraction processing method to consume as many active ingredients as possible is very important.

Most of the methods for producing chaga mushroom extract reported or commercialized so far are limited to simple hot water extraction methods or organic solvent extraction methods (Korean Patent Registration No. 10-1662185; refer to Patent Document 1), and are useful cases on a commercial scale for extracting other active ingredients. Is not known. In particular, chaga is a mushroom having a hard woody structure grown while parasitic on a birch for a long time, and no literature on its pretreatment conditions has been reported so far.

Patent No. 1662185 (Method for producing chaga extract containing β-glucan at a high concentration) 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.

Accordingly, an object of the present invention is to design a device with high utilization under high temperature and high pressure conditions as a first step in order to set the pressurization condition as a pretreatment condition for the woody part of chaga mushrooms, and then set the high temperature and pressurized conditions, and the hot water and organic It is used to provide a novel chaga extract by setting and confirming the two-stage extraction conditions in which the 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, are enhanced. have.

Another object of the present invention is to provide a novel and advanced extract material that has confirmed the safety of active ingredient content, such as antioxidants prepared by the above method, and at the same time, a formulation having excellent flavor made of liquid, powder, and slurry. It is intended to provide a variety of chaga products.

The above object of the present invention is a method for producing chaga extract with enhanced active ingredients using a two-stage method of pretreatment with high temperature pressurization and solvent extraction in one step: (a) Step 1 of high-pressure treatment of chaga mushroom pieces at high temperature. A pretreatment process; (b) a pulverization treatment step of pulverizing the chaga mushroom obtained in the high temperature/pressure pretreatment step (a) using a food processor; (c) a primary extraction step of extracting the chaga powder obtained in the pulverizing treatment step (b) with hot water; (d) a first concentrated drying step of filtering the hot water extract obtained in the first extraction step (c), then concentrating and lyophilizing it; (e) a second extraction step of extracting the chaga mushroom gourd (sludge) obtained in the dry concentration step (d) with a two-step organic solvent; (f) the second extraction step (e) a second concentration drying step in which the solvent extract obtained in the extraction is filtered, concentrated, and freeze-dried; (g) a third concentration step of further concentrating the first and second concentrated dried products obtained in the first concentrated drying step (d) and (f) concentrated drying; (h) the third concentration step (g) consists of a drying step of drying the obtained third concentrate.

In the high temperature/pressure pretreatment process (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 desirable to add. When the size of the pieces is larger than the size and the pressure is lower than 3.5 bar pressure, the chaga mushroom pieces are not well pressed, and when the size of the pieces is smaller than that and a pressure higher than 4.5 bar, the extraction yield of the mushrooms is higher than that of the pressurizing pressure. It was not desirable because it was low.

The powder of chaga obtained in the pulverization treatment step (b) is preferably 35 to 45 mesh, and if the powder degree is less than 35 mesh, it is not well mixed with water and is not preferable, and if it is greater than 45 mesh, compared to the crushing time. The yield was low, which was not desirable.

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

Here, when 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 not preferable, and when the temperature is higher than 130°C or the extraction time is longer than 4.5 hours, the energy consumption and extraction time are compared. The extraction yield of the active ingredient was low, which was not preferable.

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

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

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

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

The second and third concentration steps of the steps (f) and (g) are the same as those of the first concentration step, and the drying step of the step (h) is preferably 85 to 95% drying.

The present invention introduces a novel extraction method including a two-stage extraction method of performing a first-stage high-temperature pressurization pretreatment on the woody part of chaga mushrooms, and then extracting the first step of hot water and a second-stage solvent extraction as an active ingredient that becomes a functional index of food. It has the effect of obtaining chaga extract with enhanced contents of polyphenol, flavonoid, beta glucan, betulin and betulinic acid, and can provide an extract food material that has been confirmed to be safe in terms of antioxidant activity and active ingredient content. There is an excellent effect that can provide products of various chaga formulations with excellent flavor made of various materials such as liquid, powder, and slurry.

1 is a diagram schematically illustrating a first-stage chaga mushroom high temperature/pressurized pretreatment apparatus used in the present invention;
Figure 2 is a picture of the comparison of the solubility of the final extracted powder product according to the present invention and the extracts of company N and company B
Figure 3 is a flow chart of the execution of the method for producing a chaga mushroom extract enhanced active ingredients using a two-step extraction method after the high-temperature pressurization of the present invention.
Figure 4 is a photograph showing a public announcement material prepared from 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/pressurization conditions

1. Pre-treatment device design

1 is a schematic diagram of a high-temperature and pressurizing device for pretreatment of chaga mushrooms according to the present invention, and the present invention is effective from a piece of chaga mushroom cut into 3 to 4 × 3 to 4 × 3 to 4 (cm) horizontal × vertical × height It was designed, designed, and manufactured to produce chaga extract with enhanced powder.

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, and 7 is Leg pads, 8 for leg support pipes, 9 for I bolts, and 10 for door hinges.

The chaga mushroom pretreatment pressurizing device shown in FIG. 1 consists of 10 trays, and increases the internal pressure through steam. The air supply that supplies water vapor to the inside is located at the bottom and automatically adjusts the pressure set through the panel and maintains the internal pressure. A safety device of up to 6 bar is installed on the top, and it is designed to drop the pressure below the allowable pressure when the internal pressure rises more than that. It is a remarkable feature of the device of the present invention that steam does not directly come into contact with the chaga mushrooms. However, in the case of using a high-temperature/pressurizing apparatus similar to the apparatus of the present invention, the apparatus may be used as long as there is no difference in the extraction yield and active ingredients of the final chaga mushroom.

2. Pre-processing condition setting

Chaga mushroom pieces of the above-described size were set to 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 crushing the chaga mushrooms pretreated with high temperature and pressure using a food processor, mix the powder with a particle size of 35 ∼ 45 mesh in a ratio of 1:20 (w/v) with water, and then at 100℃, 4 Extracted under time conditions.

[Experimental material preparation 1]

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

2-1. Analysis of polyphenols and polaronoid components

The content of phenolic compounds was highest at 4bar in the hot water extract obtained from chaga mushrooms obtained under high temperature and pressurized pretreatment conditions compared to the untreated test group, and in the case of 5bar pressurization conditions, the content of phenolic compounds decreased from Table 1 below. Confirmed.

On the other hand, depending on the treatment time, the phenolic compound content increased up to 1 hour for 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 to polyphenol 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. It was judged to increase. The most preferable high temperature/pressurization treatment conditions were 1 hour at 100°C and 4 bar.

mg/g Processing time 2bar 3bar 4bar 5bar
Polyphenol
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
Polaronoid
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.Antioxidation (DPPH, ABTS)

To measure the antioxidant effect of chaga mushrooms, radical scavenging activity using DPPH (α,α-diphenyl-β-picrylhydrazy l), ABTS[2,2'-azinobis-(3-ethyl-benzoth iazoline-6-sulfo nic) acid)] Cations (ABTSㆍ+) scavenging ability measurement is typical. The freeze-dried extract powder was prepared as a 1% aqueous solution, and then the radical scavenging ability was measured using DPPH, and the results are shown in Table 2 below.

From Table 2, it was confirmed that the antioxidant level increased with pressure to 65.7±1.2%, 75.9±1.5%, 81.4±0.7%, and 80.3±0.2% at 0.5 hour treatment, and about 124% increase compared to the control.

As a result of measuring the cationic radical scavenging ability using ABTS, it was confirmed that the cation radical scavenging activity was excellent in the case of the chaga extract powder, showing more than 96% of the cationic radical scavenging ability in both the untreated and treated groups.

% Processing time 2bar 3bar 4bar 5bar
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. Betaglucan

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 higher than that of the untreated test group in the high-temperature and pressurized pretreatment test group, and as the pretreatment pressure increased, the beta-glucan content was measured. It was confirmed that the glucan content was increased. The 1 hour pressurized pretreatment test at 4 and 5 bar was the highest with a beta glucan content of 4.2 (w/w, %).

(w/w %) Processing time 2bar 3bar 4bar 5bar
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

Taking the above examples together, in consideration of the phenolic compound content, antioxidant index, beta glucan content, and economical aspects for industrialization, treatment at 4 bar for 0.5 to 1 hour is considered to be the most suitable pretreatment condition for chaga mushrooms.

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

Known active ingredients of chaga mushroom include phenolic compounds, β-glucan, trichephenolic acid, horomadogen, polyphenol, oxyphenolcarboxylic acid, hinocin, chaga acid (60%), vanilla acid, and paraoxyacid. , Pterine, sterol, lignin, betulin, betulinic acid, and other polar and non-polar physiologically active substances. Second organic solvent extraction was performed using alcohol.

1. Set the alcohol concentration and time

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

1-1. Changes in polyphenol and flavonoid content

The phenolic compounds of chaga mushrooms are representative compounds that exhibit antioxidant effects, and in the case of the polyphenol content according to the alcohol content 30, 50 and 70% from Table 4 below, the polyphenol content also increased as the alcohol concentration and extraction time increased. It was confirmed that the flavonoid content, which is 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 were measured. Therefore, as a result, when comparing the solubility, polyphenol and flavonoid contents, an extraction time of 50% alcohol concentration of 8 hours was judged to be the most economical, and the optimum extraction conditions were set.

% Processing time 30% alcohol 50% alcohol 70% alcohol


Polyphenol
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 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-sulfo nic acid)] Cations (ABTS +) scavenging ability measurement is typical. As a result of measuring the radical scavenging ability using DPPH after preparing the freeze-dried extract powder as a 1% aqueous solution, it was confirmed that the radical scavenging ability increased as the alcohol concentration and treatment time increased as shown in Table 5 below.

As a result of measuring the cationic radical scavenging ability using ABTS, as shown in Table 5, according to the present invention, a cationic radical scavenging ability of 90% or more was shown in all experimental sections, and a high radical scavenging ability was confirmed even in the alcohol extract using hot water extracted sludge.

% Processing time 30% alcohol 50% alcohol 70% alcohol


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.Betaglucan

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 higher than that of the untreated test group, and the beta-glucan content increased as the pretreatment pressure increased. I was able to confirm it. In particular, the highest measurement was 4.2 (w/w, %) in the 1 hour pressurized pretreatment test at 4 and 5 bar.

(w/w,%) Processing time 30% alcohol 50% alcohol 70% alcohol


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 by HPLC using the lyophilized extract powder, 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 of betulin and betulinic acid according to the extraction time.In the case of betulin and betulinic acid, 500mg/100g, 200mg under the condition of 50% or more of the alcohol. It was confirmed that more than /100g was extracted.

% Processing time 30% alcohol 50% alcohol 70% alcohol


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 the quality analysis of the extracted powder by conditions at the same concentration (1% aqueous solution), non-polar active ingredients that were not extracted from hot water were extracted from the alcohol solvent, and the content of polyphenol, flavonoid, betulin and betulinic acid increased as the content of alcohol increased However, considering the economical aspect for industrialization due to the low extraction yield, the alcohol concentration was set from 50% to 8 hours.

Example 3. Quality measurement of final extract

1. Active ingredient analysis

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

division Extract powder Company N Company B Total polyphenol(mg/g) 317.9±1.8 189.5±6.5 235.3±22.4 Total flavonoid(mg/g) 47.50±2.92 43.19±6.79 44.44±3.37 Antioxidant degree (%)_DPPH assay 60.23±1.59 67.41±0.85 67.99±0.64 Antioxidant degree (%)_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 product of the present invention was dissolved in 500 mL of water, it was confirmed that it was transparent and the dissolution rate was very fast 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 foods, the Maillard reaction is the most predominant browning reaction. Maillard reaction not only affects the browning and flavor formation of foods, but also plays an important role in the antioxidant and antibacterial properties of foods, and has been found to be related to aging, adult diseases and mutagenic inhibitory effects.

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

Amino acids (mg/g) Chaga 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

Since flavor does not appear in the extracted sludge, first, various kinds of sugars were added and mixed in order to prepare chaga leached tea. At this time, the direct-fired stir-fry conditions were fixed at 100°C for 4 minutes.

1. Sensory test according to the type of sugar

As the type of sugar, glucose (glucose), fructose (fructose) and sucrose (sugar, sucrose) were each used at 1%. The results of sensory evaluation are shown in Table 10 below. In the sensory quality evaluation, 10 researchers recognized the characteristics of chaga, and then the sensory evaluation for color, flavor and overall acceptability was scored by 5 points (5: very good, 4: good, 3: moderate, 2: not good, 1: Not very good). When checking the sensory and color, the sugar was found to be the best sucrose.

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 for color, the highest in 2, 4, 6%, in the sensory evaluation for fragrance, 4%, 6% in order, and in the overall evaluation, the 4% concentration was evaluated high. The higher the concentration of sucrose, the more caramelization proceeded, and it was judged to have an effect on the fragrance.

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) according to the storage period of the chaga extract is , Antioxidant activity) and microbiological quality stability were analyzed.

2-1. Sample preparation and storage conditions

Chaga mushrooms from Siberia in Russia were purchased, and the surface was rinsed cleanly and used in the experiment.

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

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

Under the condition where the light was blocked, the distribution temperature of the product at room temperature was set to 35℃ and 25℃ and 45℃ for the experimental zones based on the expiration date set storage temperature acceleration test conditions, and stored in an incubator at each temperature. After freezing, in order to check the quality stability of the thawed state, -20℃ was additionally stored.

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

The chaga extract stored in each condition was taken out at intervals of 2 months and the experiment was conducted (FIG. 4).

2-2.Measurement of physicochemical properties

A certain amount of chaga extract stored in a 20 ml glass bottle was taken at each storage temperature 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 chaga extract according to 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 on the final storage day, the pH was 5.45~5.46. It was thought that the storage temperature during the storage period did not significantly affect the pH. Also, the initial storage value showed little difference from the value measured at the 8th month of storage.

term
(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. Measurement of antioxidant level

To measure the antioxidant level, the sample was diluted with 0.1 Brix and used in the experiment to obtain a significant absorbance value.

The DPPH method measures the antioxidant effect by electron donating action according to the degree of discoloration of dark purple by reduction by antioxidants such as tocopherol, ascorbate, flavonoid compounds, sulfur-containing amino acids such as glutation, maillard browning substances, and peptide That's the 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, it was found that -20℃, 25℃, 35℃, and 45℃ all 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 cationic radical scavenging ability using ABTS, the degree of antioxidant decreased as the temperature increased, but showed a cationic radical scavenging activity of more than 70% in all experimental sections, indicating that the antioxidant power was high even with an increase in storage period (Table 13) .

term
(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. Effect of free radical scavenging 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 Chaga water extract against DPPH radical, which is a free radical, was modified by Blois MS (1958). (Kim JH et al. 2002). That is, 1 mL of a 0.15 mM DPPH solution dissolved in 99.9% methanol and 0.25 mL of each extracted sample solution were added and mixed well using a light-blocked container, and the absorbance was measured at 517 nm after 30 minutes and compared.

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 measurement of the antioxidant capacity of chaga extract was determined by the scavenging ability of ABTS cations. Was tested.

ABTS reacts with free groups (hydroxyl, peroxyl, alkoxyl, inorganic radicals) to produce relatively stable cations (ABTS·+) that absorb light at 414 nm. The solution was prepared by mixing a 7.4 mM ABTS solution with a 2.6 mM potassium persulphate solution, reacting for 12 to 16 hours in the dark, and diluting the absorbance at 414 nm to about 1.5. 50 μL of a 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 was reduced by the phenolic compound of the extract and colored molybdenum blue (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 a 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 the absorbance at 750 nm after 30 minutes. In addition, the absorbance of each extract was compared with the gallic acid calibration curve used as a standard material to determine the total phenol content (μg gallic acid equivalent/mL). Was used. The experimental measurement was repeated three times and the average value was used.

Polyphenols are very diverse, including tannin, and have recently been reported to have various functionalities. (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 chaga extract was almost the same from 26.79 mg/ml for the first time to 25.90 mg/ml at 45° C. storage after 8 months. Therefore, it was found that the polyphenol content was almost unchanged depending on the storage period and temperature (Table 14).

term
(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

To measure the beta-glucan content, each sample was lyophilized and tested in a powder state.

After calculating 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 by the difference in sugar contents other than α-glucan and glucan. .

After adding 37% hydrochloric acid to the crude β-glucan powder sample, it was hydrolyzed at 100° C. for 2 hours, and then the pH was adjusted using 2N KOH. The hydrolyzed solution was diluted with 200 mM sodium acetate buffer (pH 5.0) and centrifuged. 100 μl of the supernatant was taken, and exo 1,3-β-glucanase and β-glucosidase were added, followed by reaction at 40° C. for 1 hour.

After that, 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 sugar contents other than α-glucan and 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 were added, and a 40° C. water bath (Water bath C-WBE, Chang Shin Science Co. ) For 30 minutes and then centrifuged (1500g, 10 minutes). 100 µl of the centrifuged supernatant was taken, 100 µl of 200 mM sodium acetate buffer (pH 5.0) and 3 µl of a glucose checking reagent were added to react at 40° C. for 20 minutes, and the absorbance was measured at 510 nm.

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

[Equation]

Total Glucan (% w/w) = ΔE × F/W × 90

α-glucan (% w/w) = ΔE × F/W × 9.27

β-glucan = total glucan-α-glucan

In the above equation, 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 lyophilized extract powder, as shown in the table, the beta-glucan content was measured to be 25 mg/g or more, 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).

term
(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 betulin, a standard sample, was dissolved in 20 mL of HPLC-grade ethanol at room temperature to make a mother solution with a concentration of 5.0 g/L, and this mother solution was diluted to obtain a concentration of 4.0 g/L, 3.0 g/L, and 2.0 g/L. L reagent was prepared and analyzed by HPLC. 10 mg of betulinic acid, a standard sample, was dissolved in 20 mL of HPLC-grade ethanol to prepare a 0.5 g/L mother liquor, and this mother liquor was diluted to make 0.4 g/L, 0.3 g/L, and 0.2 g/L reagents. It was analyzed by HPLC (HPLC Column: Nova-Pak C18-ODS reversed phase, 4 μm, 3.9x300mm, eluent: CH3CN:H2O=86:14 v/v). The concentration of betulin contained in an unknown reagent was determined using a calibration curve created by linear regression by plotting the area of the HPLC peak obtained by analyzing each sample as a concentration.

As a result of analyzing the content of betulin and betulinic acid in the chaga extract concentrated with 10 Brix by HPLC, it was measured as shown in the table. Unlike the measurement results using the lyophilized extract powder, betulinic acid was not detected, and it was confirmed that the content of betulin decreased with increasing temperature rather than the change with period (Table 16).

term
(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. Microorganism test

To measure the number of microorganisms contained in the chaga extract, the number of bacteria and the coliform dry film medium were used, and 1 ml of the sample dilution solution was dispensed into each medium, cultured at 35°C for 48 hours using a standard plate method, and colonies were counted. It is 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 extract are shown in the table. General bacteria and coliforms were found to be microbiologically safe as they were not detected in all samples during the storage period. Therefore, the consumption of chaga extract is considered to be food hygiene safe, and it was found to be suitable for the standard value (less than 100 per ml of the number of bacteria, coliform group: negative) in the food industry (Table 17).

term
(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 sedimentation stability of the chaga extract stored at each temperature and the chaga extract according to the pH change, the degree of sedimentation was expressed as O, △, and X.

It was confirmed that precipitation did not occur even when the storage period was prolonged and the temperature increased, so that the chaga extract was in an aqueous state and showed excellent sedimentation safety during storage (Table 18).

term
(month) Temperature pH -20 ℃ 15 ℃ 35 ℃ 45 ℃ 4 8 Presence or absence of precipitation
(O, △, X) 2 × × × × × × 4 × × × × × × 6 × × × × × × 8 × × × × × ×

3-8. Chromaticity measurement

To measure the change in chromaticity of chaga extract in 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), b (+ yellow,-blue) values were measured by repeating three times and expressed as an average value.

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

term
(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: leg pipe
7: leg pad 8: leg support pipe
9: I bolt 10: door hinge

Claims (7)

(a) a one-step high temperature/pressurization pretreatment process of high-pressure treatment of chaga mushroom pieces at high temperature;
(b) a pulverizing step of pulverizing the chaga mushroom obtained in the process (a) high temperature/pressurization using a food processor;
(c) a first extraction step of extracting the chaga powder obtained in the pulverizing step (b) with hot water;
(d) a first concentrated drying step of filtering the hot water extract obtained in the step (c) hot water extraction, then concentrating and lyophilizing it;
(e) a second extraction step of extracting the chaga mushroom gourd (sludge) obtained from the step (d) concentration drying with a second step organic solvent;
(f) a second concentration drying step in which the extract obtained in the step (e) is filtered, concentrated, and freeze-dried;
(g) a third concentration step of further concentrating the first and second concentrated dried products obtained in the above steps (d) and (f) concentrated drying;
(h) The method for producing chaga mushroom extract, characterized in that the effectiveness of the third concentrate obtained in the above step (g) is lyophilized and dried using a two-step extraction method and a high-temperature pressurization treatment. The method according to claim 1, wherein the size of the chaga mushroom pieces in the step (a) is 3 to 4 × 3 to 4 × 3 to 4 (cm) in width × length × height, and the pressure is 3.5 to 4.5 bar. A method for producing chaga mushroom extract characterized by enhanced active ingredients using high-temperature pressurization treatment and a two-step extraction method. The method of claim 1, wherein the powder of chaga obtained in the step (b) is 35 to 45 mesh, wherein the active ingredient is strengthened using a high-temperature pressurization treatment and a two-step extraction method. . The method of claim 1, wherein the first extraction step of step (c) comprises mixing chaga powder and water in a ratio of 1:20 (w/v) and then extracting at 95°C to 130°C for 3.5 to 4.5 hours. A method for producing chaga mushroom extract characterized by enhanced active ingredients using a high-temperature pressurization treatment and a two-step extraction method. The high-temperature pressurization treatment according to claim 1, wherein in the second step of the second organic solvent extraction step of the step (e), the chaga mushroom gourd (sludge) is extracted for 7.5 to 8.5 hours using 45 to 55% alcohol. A method for producing chaga mushroom extract characterized by enhanced active ingredients using a two-step extraction method. An active ingredient-enhanced chaga extract prepared by the method of any one of claims 1 to 5 using a high-temperature pressurization treatment and a two-step extraction method. A food composition containing the chaga extract of claim 6.

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