KR20180019374A - Manufacturing technique for high content of specific ginsenoside compound K using bio-conversion technique - Google Patents

Abstract

The present invention relates to a manufacturing method of increasing the yield of a compound K in a simple manner by increasing a conversion rate to a ginsenoside compound K, and more specifically, includes a method of manufacturing fermented ginseng or red ginseng capable of improving a content of ginsenoside compound K by a two-step bioconversion technique by manufacturing a first culture by using lactic acid bacteria or edible enzymes and then manufacturing a second culture by using the edible enzymes. Accordingly, the present invention has an effect of stably performing an effective conversion mechanism so as to manufacture a high content of a ginsenoside compound K, by going through a process for each step of converting lactic acid bacteria or enzymes from Rb1 to Rd and again from Rd to the compound K through sequential reaction being each used in a step where lactic acid bacteria or enzymes are separated.

Description

[0001] The present invention relates to a method for producing a high-content ginsenoside compound K using a biotransformation technique,

The present invention relates to a method for producing fermented ginseng or fermented red ginseng having an increased content of ginsenoside K, more specifically, fermented ginseng or fermented ginseng having an increased content by increasing the conversion to ginsenoside K by a simple method And a method for producing red ginseng.

Saponin is known to be an effective ingredient of ginseng and red ginseng. Saponin of ginseng and red ginseng is a kind of glycosides, and has a unique chemical structure different from that of saponin found in other plants, and its effect is also greatly different. Saponin of ginseng and red ginseng is called 'ginsenoside' to distinguish it from ginseng saponin.

Recently, according to the development of analytical technology, 30 kinds of ginsenoside chemical structures have been revealed so far. This is much more than 14 kinds of Western ginseng and 15 kinds of Chinese ginseng. Based on glycosides with a dammarane skeleton, ginsenosides are classified into diols (PPD) and triols (PPT), depending on the number of sugars attached to the R1, R2, and R3 positions in the chemical structure Side.

When high pressure is applied to these ginsenosides, enzymes are added or heated during the process, a part of the sugar is removed or new double bonds are formed, creating a unique ginsenoside. Accordingly, various types of ginsenoside-containing products are on the market depending on processing methods of ginseng.

In order for ginsenoside to be absorbed into the body, the diol-based ginsenoside is 20-O-β-D-glucopyranoside 20 (O) -protopanaxadiol 20- glucopyranoside, FGM 1, hereinafter referred to as 'compound K') and the triol ginsenoside was decomposed into 20 (S) -protopanaxatriol (20 (S) -protopanaxatriol, FGM 4) It is absorbed and absorbed, but only a small amount is converted by heat, acid, pressure, digestive enzymes, etc., and is ultimately absorbed into the body.

Recent studies have shown that intestinal microbes are needed when ginseng saponin is converted into such a final product. In order for saponin taken orally to be absorbed to be absorbed, it must be degraded by intestinal microorganisms. Privotella oris is a typical microorganism that saponin metabolizes. In addition, various beneficial bacteria such as lactic acid bacteria participate in saponin metabolism.

In a study that estimated the percentage of people in the general population, 30% of normal people had no bacteria capable of metabolizing saponin, and even though the metabolic capacity was different among the remaining 70% of bacteria, the saponins of the diol and triol were normal It turns out that only a handful of people can do the dialogue. Especially, when continuous chemotherapy or medicines are used, the intestinal flora is not stable and the saponin metabolic rate is further lowered.

In this respect, the fermentation of fermented red ginseng and fermented red ginseng, which can be effective for everyone, by converting the fermented ginseng into a form capable of absorbing the active ingredient of ginseng, regardless of the metabolic function of the individual, It is very desirable.

Korean Patent No. 10-1409761 (2014.06.13), as a prior art for improving the yield of the ginsenoside compound K among the saponin components of ginseng, discloses a method of converting three kinds of enzymes into a compound K However, since three kinds of enzymes, which are food raw materials, are used, inevitable cost increase of the raw material containing the compound K is expected and the conversion rate of the compound K is too low because the conversion is stopped in Rd and F2 during the conversion. Korean Patent Laid-Open No. 10-2016-0076851 (Jan. 26, 2016) discloses a method of converting an enzyme and a Cytolase PCL5 into a compound K. However, for the reasons related to the above-mentioned conversion process, Low.

Korean Patent No. 10-1616104 (Apr. 21, 201) Korean Patent No. 10-1409761 (June 19, 2014) Korean Patent Publication No. 10-2016-0076851 (Jul. 2016) Korean Patent No. 10-0877489 (December 30, 2008) Korean Patent No. 10-0884252 (2009.02.11) Korean Patent No. 10-1346627 (December 24, 2013)

It is an object of the present invention to provide a two-step biotransformation technique and apply it to a conversion mechanism to produce a ginsenoside K, thereby eliminating the stopping of conversion in other products present in the conversion process, Fermented red ginseng or fermented red ginseng containing the senocide compound K is produced.

In order to accomplish the above object, the present invention provides a method for producing Lactobacillus sp. Or pectinase, pectin lyase, polygalacturonase, endoarabinase, cellulase, beta-glucosidase, hemicellulase, A first enzyme, which is at least one selected from hemicellulase, protease, naringinase, pectin esterase and pectin depolymerase, is added to ginseng or red ginseng Producing a first culture; (b) heat treating the first culture; (c) culturing the first culture heat-treated in step (b) above with pectinase, arabinase, cellulase, hemicellulase, protease, Adding a second enzyme in combination to produce a second culture; And (d) heat-treating the second culture. The present invention also provides a method for producing fermented ginseng or fermented red ginseng in which the content of ginsenoside K is increased.

The step (a) may be a step of transforming ginsenoside Rb1 into Rd by culturing the lactobacillus bacteria or the first enzyme added to ginseng or red ginseng.

The Lactobacillus strain of step (a) is selected from the group consisting of Lactobacillus sakei, Lactobacillus alimentarius, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus casei, Wherein the composition is selected from the group consisting of Lactobacillus gasseri, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus bulgaricus and Lactobacillus helveticus. According to one embodiment of the present invention, the lactic acid bacteria may be lactobacillus saccharides.

The first enzyme of step (a) is selected from the group consisting of edible enzymes such as Cytolase PCL5 (Cytolase PCL5, manufacturer: DSM, pectinase, pectin lyase, polygalacturonase and endoarabinase, enzyme producing microorganism: Aspergillus niger, Sumizyme Aspergillus niger), Cellulase KN (Manufacturer: ShinNippon, Type of enzyme: cellulase, hemicellulase, pretease and naringinase, enzyme producing microorganism Aspergillus niger, Rapidase C80Max (manufactured by YC International, Inc.), Aspergillus niger, Crystalzyme APXL (manufacturer: DSM, enzyme type: pectin esterase, pectin depolymerase, cellulase and hemicellulase, enzyme producing microorganism: Aspergillus niger) Aspergillus niger, Rohament CL, Sumizyme SPC, and Rapidase PRESS were used as the enzyme types: pectinase, enzyme producing microorganism: Aspergillus niger, The enzyme may be a compound at least one selected from the group. Korean Patent Laid-Open No. 10-2013-0142707, Lohament CL, Sumizyme SPC, and Rapidays PRESS are disclosed in Korean Patent Laid-Open Publication No. 10-2013-0142707, 2016-0076851, but the conversion rate from ginsenoside Rd to compound K was not high. According to one embodiment of the present invention, the complex enzymes can be used for the conversion to ginsenoside Rd. According to another embodiment of the present invention, the first enzyme may be pectinase, pectin lyase, polygalacturonase and endo arabinase. According to a specific embodiment, the complex enzyme may be cytoResize PCL5.

The addition of the lactobacillus bacteria or the first enzyme to the ginseng or red ginseng in step (a) can be carried out by mixing the lactobacillus germ or the first enzyme with the culture solution containing ginseng or red ginseng. The term "culture" in this specification can be prepared by reacting the mixture at an appropriate temperature after the mixing for an appropriate time. Preferably, the culture can be prepared by reacting for 48 to 72 hours.

In step (b), the conversion to ginsenoside Rd can be stopped by inactivating the Lactobacillus bacteria or the first enzyme of the first culture for efficient conversion of ginsenoside. According to an embodiment of the present invention, the deactivation is preferably performed through heat treatment of the first culture.

The step (c) may be a step of culturing the second enzyme in the first culture to convert the ginsenoside Rd into the compound K. According to an embodiment of the present invention, the second enzyme may be Pyr-Flo (manufacturer: Connell bros, enzyme type: pectinase, arabinase, cellulase, hemicellulase and protease, enzyme producing microorganism: Aspergillus niger).

In step (d), the conversion of the ginsenoside K to the ginsenoside K can be stopped by inactivating the second enzyme of the second culture for efficient conversion of the ginsenoside. The inactivation of the second enzyme is preferably carried out through a heat treatment of the second culture.

The ginseng or red ginseng may be an extract obtained by extracting a polar organic solvent with a solvent. According to an embodiment of the present invention, the polar organic solvent may be a 10-70% concentration alcohol. Preferably, the concentration of the alcohol may be 20-50%. More preferably, the concentration of the alcohol may be 40-50%. When the content of extract ginsenoside Rb1 is less than 40%, the content of extract ginsenoside Rb1 is insufficient, and when the content exceeds 50%, there is no statistical significance in comparison with the case of using a lower concentration of the alcohol.

The heat treatment of the first and second cultures may include applying heat at 100-150 占 폚 for 5 minutes to 30 minutes. If the heat treatment is carried out at a temperature lower than 100 ° C., the conversion of ginsenoside becomes very low. If the heat treatment is carried out at a temperature higher than 150 ° C., ginsenosides and other useful substances are destroyed.

The heat treatment may be performed at atmospheric pressure, but may be performed at a pressure higher than atmospheric pressure according to an embodiment of the present invention. According to another embodiment of the present invention, the heat treatment may be performed at a pressure of 1.1-3.0 atmospheres, and according to a particular embodiment, at a pressure of 1.2-1.8 atm.

The heat treatment can be performed for 1-90 minutes. According to one embodiment of the present invention, 5-40 minutes may be performed, and in another embodiment, 5-25 minutes. According to certain embodiments, it may be carried out for 10-20 minutes.

The content of the ginsenoside K may be from 1.00 mg / g to 100.00 mg / g. Preferably, the content of the ginsenoside K is 10.00 mg / g to 50.00 mg / g. More preferably, the content of the ginsenoside K may be from 15.00 mg / g to 30.00 mg / g. This means that ginsenoside K, which is not present in ginseng or red ginseng without the treatment of steps (a) to (d) of the present invention, was produced by using biotransformation technology according to the method of the present invention.

According to one embodiment of the present invention, the ginseng or red ginseng according to the present invention is applied to ginseng, fresh ginseng, ginseng, white ginseng, and red ginseng processed in various forms. Lt; / RTI >

According to the present invention, an effective conversion mechanism can be stably performed by sequentially performing the step of converting Rb1 to Rd and then Rd to a compound K through a sequential reaction using lactic acid bacteria or an enzyme in separate steps, respectively The effect of producing a high content of ginsenoside compound K is obtained.

1 is a result of confirming the ginsenoside component of a first culture prepared through a biotransformation process using Lactobacillus saccharum (A) or Saito Reyes PCL5 (B).
FIG. 2 shows the results of confirming the ginsenoside content of the second culture prepared through the biotransformation process using Pyr-Flo.
FIG. 3 shows the results of confirming the ginsenoside components produced by the bioconversion process using only lactic acid bacteria.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Identification of extraction yield

The extracts were prepared by mixing 300 g of each of 10, 20, 30, 40, 50, 60 and 70% ethanol in 15 g each of white rice ginseng and preparing the same three experimental groups at 80 ° C for 72 hours. Then, the extract was concentrated under reduced pressure to 35 brix, and the content of ginsenoside Rb1 was measured by HPLC (solvent: water) (Table 1).

spirits 10% 20% 30% 40% 50% Rb1 15.91 mg / g 21.18 mg / g 22.86 mg / g 24.03 mg / g 24.24 mg / g

The content of extracted ginsenoside Rb1 in the 20-50% alcohol to 10% alcohol was 133%, 144%, 151%, and 152%, respectively, compared to 40% and 50% (P < 0.05). Therefore, it was confirmed that it is the most economical to conduct the extraction under the condition of selecting 40% alcohol as a solvent at 80 ° C, and the conditions for enhancing the content of ginsenoside Rb1 were established.

Selection of lactic acid bacteria

The lactic acid bacteria used in the present invention were Lactobacillus sakei purchased from KCTC (Korean Collection for Type Cultures). Lactobacillus agar (BD, USA) was used as the culture medium of the lactic acid bacteria, and the culture condition was set at 37 캜 (anaerobic).

Example 1. Preparation of concentrate

The extracts were prepared by adding 20% of 40% alcohol as a solvent to white rice germ, concentrating it under reduced pressure to 50 brix, and removing the solvent to prepare a concentrate.

Example 2. Preparation of first culture using lactic acid bacteria

10 times more purified water than the concentrate prepared according to Example 1 was diluted and Lactobacillus sakei was added. The addition amount was 0.1% (v / v) of the total weight of the diluted red ginseng concentrate. After the addition, the first culture was prepared by culturing at 30 DEG C for 72 hours. After the first culture was completed, it was sterilized by heat treatment at 121 캜 and 1.5 atm for 15 minutes in order to prevent further ginsenoside conversion.

Thereafter, saponin bioconverted by the strain of the present invention was analyzed. The recovered first culture was separated by using butanol as a solvent, and the butanol layer was analyzed by HPLC.

HPLC analysis was performed using a Waters LC system (Waters, USA), columns SUPERCO C18 (25 cm ㅧ 4.6 ㎜, 5 ㎛) and sample loading amount 10 ㎕. The mobile phase was obtained by concentration gradient with 100% water and 100% acetonitrile for 110 minutes. The flow rate was 1.2 ml per minute and was detected at 203 nm (Figure 1A).

Example 3. Preparation of first culture using enzyme

(Cytolase PCL5, manufactured by DSM, enzyme type: pectinase, pectin lyase, polygalacturonase, and endorrabinae), which is an edible enzyme of the same amount in place of Lactobacillus casei, , Enzyme-producing microorganism: Aspergillus niger) (Fig. 1B).

Example 4 Preparation of Second Culture Using Enzyme

Pyr-Flo, an edible enzyme, was added to the first culture prepared in Example 3 at 0.1% (v / v) of the total weight. After the addition, the cells were cultured at 50-60 ° C for 72 hours.

When the preparation of the second culture was completed, the enzyme was sterilized by heat treatment at 121 캜 and 1.5 atm for 15 minutes to prevent further conversion.

The components were then confirmed by HPLC analysis as in Example 2 (FIG. 2). As a result of the measurement, it was confirmed that the content of the ginsenoside compound K was greatly increased as compared with the culture 1. The content of the ginsenoside components converted in the order of Rb1, Rd, and Compound K (CK) according to the method of the present invention was measured by HPLC (Table 2).

Ingredients extract The first culture Second culture Rb1 19.23 mg / g 0.30 mg / g 0.28 mg / g Rd 0.86 mg / g 21.56 mg / g 0.39 mg / g CK 0 mg / g 0 mg / g 20.24 mg / g

Comparative Example. Preparation of culture using only lactic acid bacteria

Bifidobacterium bifidum (Bifidobacterium bifidum, Bifidobacterium agar, BD, USA, culture condition: 100 mg / ml) was prepared in the same manner as in Example 4 except that Lactobacillus sake was added to prepare a first culture. 37 캜, anaerobic) was added to prepare a second culture.

The components were then confirmed by HPLC analysis as in Example 2 (FIG. 3).

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

(a) Lactobacillus; Or Cytolase PCL5, Sumizyme AC, Cellulase KN, Crystalzyme APXL, Rapidase C80Max, Rohament CL, Preparing a first culture by adding a first enzyme selected from the group consisting of Sumizyme SPC (Sumizyme SPC) and Rapidase PRESS (Rapidase Press) to ginseng or red ginseng;
(b) heat treating the first culture;
(c) culturing the first culture heat-treated in step (b) above with pectinase, arabinase, cellulase, hemicellulase, protease, Adding a second enzyme in combination to produce a second culture; And
(d) heat treating said second culture
Wherein the content of the ginsenoside compound (K) is increased.
The method according to claim 1, wherein the second enzyme is Pyr-Flo.
The method according to claim 1, wherein the ginseng or red ginseng is an extract extracted with a polar organic solvent.
4. The method according to claim 3, wherein the polar organic solvent is a solvent.
The method according to claim 1, wherein the heat treatment is a treatment by application of heat at 100-150 占 폚.
6. The method of claim 5, wherein the application of heat is performed for 5 to 40 minutes.
6. The method of claim 5, wherein the application of heat is performed for 5 to 25 minutes.
The method according to claim 1,
Wherein the content of the ginsenoside K is 1.00 mg / g to 100.00 mg / g.

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