KR100919070B1 - Process for the preparation of fermentation product of esculent roots using microorganism having fermentation activity of saponin-containing esculent roots - Google Patents

Abstract

The present invention (a) the genus Bacillus strains, preferably Bacillus subtilis ( Bacillus ) in a root vegetable containing saponin subtilis ) or Bacillus licheniformis , more preferably Bacillus licheniformis KCCM10885P, followed by fermentation; And (b) separating the fermented root vegetables from the fermentation mixture obtained in step (a), or concentrating or drying the fermented broth obtained from the fermentation mixture obtained. To provide.

Bacillus strain, preferably Bacillus subtilis subtilis ) or Bacillus rickeniformis ( Bacillus) licheniformis ), more preferably Bacillus rickeniformis ( Bacillus) licheniformis ) KCCM10885P has excellent saponin-degrading activity and enables the preparation of root vegetable fermentation containing high concentration of ginsenoside Rh1 and compound K having excellent pharmacological activity. In addition, the obtained root vegetable fermented product has excellent pharmacological activity such as excellent antioxidant activity, anticancer activity, anti-inflammatory activity, and anti-allergic activity. In particular, when fermenting root vegetables such as ginseng using the strain, it is possible to greatly reduce the residual pesticides that may be contained in the raw material.

Description

Process for the preparation of fermentation product of esculent roots using microorganism having fermentation activity of saponin-containing esculent roots}

The present invention relates to a root vegetables fermentation production process of water using a strain having a root vegetables fermentation activity, which contains a saponin, more particularly Bacillus strain having a root vegetables fermentation activity, which contains a saponin, preferably from Bacillus subtilis (Bacillus subtilis ) or Bacillus rickeniformis ( Bacillus) licheniformis ), more preferably Bacillus rickeniformis ( Bacillus) licheniformis ) relates to a method for producing root vegetable fermented products.

Root vegetables containing saponin are, for example, ginseng, deodeok, bellflower, yellow rice, etc., these root vegetables are perennial, many of the pesticides when cultivated easy to infect red bacteria, rot bacteria. Therefore, the residual pesticides of the root vegetables are a problem. In addition, these root vegetables are fragile and difficult to store.

Ginseng is about 11 paper known in the earth as a perennial belonging to the genus sukgeuncho haksang ohgagwa classified ginseng plant, as a representative species (1), ginseng (Panax ginseng CA Meyer is native to the Far East of Asia (Korea, Northern Manchuria, and parts of Russia) and is very effective. (2) Panax quinquefolium L.) grows and grows in the United States and Canada, and (3) Panax notoginseng FHChen grows wild or grows in southwestern Guangxi province from southeastern Yunnan Province of China, and (4) Panax ginseng japonicus CA Meyer) is known to spread to Japan, southwest China and Nepal. Ginseng is not only stored as a commodity in new agricultural plants, but has been used as a valuable medicine since ancient times. Commercial ginseng products are divided into ginseng, which is grown and harvested, white ginseng dried at room temperature, or red ginseng produced by heating the ginseng at 98-100 ° C. In addition, attempts have been made to use ginseng in tissue culture. Many pharmacological experiments so far have revealed that ginseng enhances the nonspecific resistance of the body to stress and has an acidic action. In addition, it has been shown to improve hypertension, enhance insulin action, lower blood sugar in alloxan diabetic mice, synthesize liver liver RNA, protein synthesis, promote sugar and lipid metabolism in rats, etc. (和 효과)百科 圖鑑, Vol. 1, pp. 1-8, Nambatsune Hikki, Hoi-ku-sa, 1980)

Bellflower (P lanticodon grandiflorum A.DC.) is cultivated nationwide in Korea, and is a glycoside of playcodigenin as the main ingredient of placoidin A, C, D, D ₂ and two monoacetates, plaqueco Dean D ₃ , deapio-platycodin D ₃ , and the like. Platicodine D is the main component. In oriental medicine, it is used for dentition, lung fever, tonsillitis and diarrhea. It is grown or wild all over the country.

Deodeok (Codonopsis lanceolata) is also called ginseng, white ginseng, and is used in oriental medicine to remove dentition, expectoration and waste heat. Containing components include beta-D-xylpyranosyl- (1-> 3) -beta-D-glucuronopyranosyl echinocystinic acid (Beta-D-xylopyranosyl- (1-> 3) ) -beta-D-glucuronopyranosyl echinocystic acid), 3-O- [beta-D-xylopyranosyl (1-> 3)-(6'-O-methyl) -beta-D-glucuronopyranosyl ] -30, 16a-dihydroxyoliane-12-ene-28-oic acid 28-O- [PD-xylpyranosyl (1-> 4) -alpha-L-ramnopyranosyl (1-> 2) -alpha-L-arabinopyranosyl] ester (3-O- [beta-D-xylopyranosyl (1-> 3)-(6'-O-methyl) -beta-D-glucuronopyranosyl] -30, 16a -dihydroxyolean-12-ene-28-oic acid 28-O- [PD-xylopyranosyl (1-> 4) -alpha-L-rhamnpyranosyl (1-> 2) -alpha-L-arabinopyranosyl] ester), 3 Beta, 16 alpha-dihydroxyoliane--12-ene-28-ioic acid 28-O- [beta-D-xylpyranosyl (1-> 3) -beta-D-xylpyranosyl (1 -> 4) -alpha-L-ramnopyranosyl (1-> 2) -alpha-L-arabinopyranosyl] ester (3beta, 16alpha-dihydroxyolean-12-ene-28-oic acid 28-O- [beta-D-xylopyranosyl (1- -> 3) -beta-D-xylopyranosyl (1-> 4) -alpha-L-rhamnpyranosyl (1-> 2) -alpha-L-arabinopyranosyl] ester) saponin. It is grown or wild all over the country.

Astragalus membranaceus is used for the purpose of nourishing tonic in oriental medicine, and contains isoflavones, saponins, polysaccharides, etc.A representative saponins include astragalaloside I, II, III, IV, etc. It contains. It is grown or wild all over the country.

On the other hand, as a composition containing conventional ginseng, a composition added with ginseng components by adding a ginseng fragrance component to the existing fermented milk or lactic acid bacteria beverage or ginseng powder or the like has been known.

For example, Korean Patent Publication No. 10-2001-0000579 (Korean Patent Application No. 10-2000-058997) discloses an acid-glycosylated ginseng composition which is glycosylated by drying under grinding and mixing hydrochloric acid, oxalic acid and sulfuric acid under pressure. Korean Patent Registration No. 192,678 (Korean Patent Application No. 10-1996-0017670) discloses a ginseng composition containing a large amount of Rg3 and Rg5 by treating ginseng at 120-180 ° C. In addition, Korean Patent Registration No. 164,266 (Korean Patent Application No. 10-1996-004217) discloses a manufacturing method that can mass-produce metabolites centered on compound K by metabolizing saponins of ginseng using intestinal bacteria. have. Korean Patent Registration No. 12,545 (Korean Patent Application No. 10-1980-004291) separates the intrinsic fragrance component of ginseng by the conventional method, or enzymatically decomposes ginseng residue which is 0.2-0.8% of organic nitrogen. When the product content is more than 3% suitable for fermentation of lactic acid bacteria, it is adjusted to pH3.8-4.8 with organic acid, and then insoluble polymer protein and fiber are removed and the lactic acid bacteria are cultured in the eluate, followed by the addition of the fragrance component of ginseng. It has been disclosed a method for producing lactic acid bacteria ginseng beverage. In addition, Korean Patent Publication No. 10-1990-0004275 (Korean Patent Application No. 10-1988-012502) discloses a method for preparing an active lactic acid bacteria beverage using ginseng, and Korean Patent Publication No. 10-1998-0000073 (Korea) Patent application No. 10-1996-23750) discloses a fermented milk composition containing ginseng or ginseng and a method for producing the same.

In addition, as a method for obtaining physiologically active saponins using an enzyme, Korean Patent Registration No. 689,745 (Korean Patent Application No. 10-2005-0043187) has a small amount in ginseng by reacting alpha-galactosidase with diol-based ginsenosides. A method of producing compound K in high yield, which is not present in ginsenoside F2 or ginseng present.

In addition, Korean Patent Registration No. 497,895 (Korean Patent Application No. 10-2002-0077081) discloses lactic acid bacteria fermentation products of ginseng, ginseng yogurt containing them, and lactic acid bacteria strains used therein, and Korean Patent Registration No. 472,964 (Korean Patent Application No. 10-2002-0046668) discloses fermented ginseng containing saponin degradation products and a method of manufacturing the same, and Korean Patent Registration No. 618,171 (Korean Patent Application No. 10-2003-0027406) discloses ginseng saponin degradation products. It discloses a fermented red ginseng containing and a method for producing the same.

The conventional technique described above is a technique for producing fermented ginseng by fermenting ginseng to an active ingredient. However, these methods have the disadvantage of using an extract or a slice of ginseng.

In addition, Korean Patent Publication No. 10-2005-0044978 (Korean Patent Application No. 10-2003-0078887) discloses a method of inoculating mushrooms and the like to decompose the residual pesticides of ginseng. However, the above prior art uses a mushroom cultivation method to remove the residual pesticides of ginseng, but the mushroom grows, but the ginseng can not be separated. Therefore, there is a drawback that you must eat mushrooms together. In addition, after cultivating shiitake mushrooms on oak trees, the active ingredients of ginseng will be lost by mushroom cultivation, just as all the nutrients of oak leaves the mushrooms. Therefore, removing pesticides in this way is not a good way to use ginseng.

In addition, Korean Patent Registration No. 425,377 (Korean Patent Application No. 10-2001-0068678) adds water to the water or herbal extracts of herbal medicines to make a water mixture, and then adds cooking oil to the water mixture, stirs, stands or centrifuges. A method for removing fat-soluble residual pesticides contained in a product prepared by extracting herbal medicines, characterized in that the oil-soluble harmful substances such as pesticides contained in the water mixture of the herbal extracts are separated to the cooking oil layer, and then the food oil layer is separated and removed. Has been disclosed. However, the method can be used after the ginseng extract is not suitable for removing the pesticides of the ginseng fuselage.

The present inventors conducted various studies to develop a method for producing a root vegetable fermented product which can produce root vegetable fermented products having excellent pharmacological activity, and also can reduce the content of residual pesticides contained in the root vegetable. As a result, Bacillus strains, preferably Bacillus subtilis ( Bacillus subtilis ) or Bacillus rickeniformis ( Bacillus) licheniformis ), more preferably, when fermenting root vegetables such as ginseng using strains newly isolated from rice bran or rice bran, it was found that not only had saponin degrading activity but also the resulting root vegetable fermentation had excellent pharmacological activity. . In particular, when fermenting root vegetables such as ginseng using the above strains, it was found that can significantly reduce the residual pesticides that may be contained in the raw material.

Therefore, an object of the present invention is to provide a method for producing a root vegetable fermented product using a Bacillus strain having a root vegetable fermentation activity containing saponin.

According to an aspect of the present invention, (a) a Bacillus strain root vegetables containing saponin, preferably from Bacillus subtilis (Bacillus subtilis ) or Bacillus rickeniformis ( Bacillus) licheniformis ), more preferably Bacillus licheniformis KCCM10885P is added to fermentation; And (b) separating the fermented root vegetables from the fermentation mixture obtained in step (a), or concentrating or drying the fermented broth obtained from the fermentation mixture obtained. This is provided. Root vegetables containing the saponin include ginseng, deodeok, bellflower, or astragalus.

In the method according to the present invention, step (a) is a Bacillus strain in the culture medium obtained by dispersing the root vegetables containing saponin in an aqueous medium (aqueous medium), preferably from Bacillus subtilis (Bacillus subtilis ) or Bacillus licheniformis , more preferably Bacillus licheniformis KCCM10885P. In addition, step (a) may be carried out by pickling saponin-containing root vegetables with salt, followed by fermentation by adding the Bacillus strain.

In addition, the Bacillus strain is a Bacillus strain, preferably Bacillus subtilis ( Bacillus subtilis ) or Bacillus licheniformis , more preferably in the form of rice hulls or rice bran containing Bacillus licheniformis KCCM10885P. In addition, the rice husk or rice bran can be preferably used for 10-30 minutes at 100-120 ℃. The fermentation is preferably carried out for 3 to 30 days, preferably 7 to 10 days.

In the manufacturing method of the present invention, step (b) may be carried out by separating the fermented root vegetables from the fermentation mixture (fermentation mixture) obtained in step (a), further comprising the step of drying after separation of the fermented root vegetables The fermented root vegetables may be steamed at a temperature of 95 to 110 ° C. and dried at a temperature of about 55 to 65 ° C. In addition, if necessary, the fermented root vegetables are steamed at a temperature of 95 to 110 ° C. and dried at a temperature of about 55 to 65 ° C., and then 0.3 to 0.5 l of ethanol (for example, 70% ethanol) per 100 g of dry matter. The method may further include extracting.

Further, step (b) may be carried out by concentrating or drying the fermentation broth separated from the fermentation mixture obtained in step (a), which fermentation broth is separated from the fermentation mixture obtained in step (a). It may further comprise a washing solution of fermented root vegetables.

The drying of step (b) is preferably carried out by lyophilization.

Bacillus strain, preferably Bacillus subtilis subtilis ) or Bacillus rickeniformis ( Bacillus) licheniformis ), more preferably Bacillus rickeniformis ( Bacillus) newly isolated according to the present invention. licheniformis ) KCCM10885P has excellent saponin-degrading activity. In addition, the root vegetable fermentation product obtained by fermenting root vegetables such as ginseng using a Bacillus strain contains high concentrations of ginsenoside Rh1 and compound K having excellent pharmacological activity, and have excellent antioxidant activity, anticancer activity, anti-inflammatory activity, and Pharmacological activity such as anti-allergic activity. In particular, when fermenting root vegetables such as ginseng using a Bacillus strain, it can greatly reduce the residual pesticides that may be contained in the raw material.

As used herein, the term "root vegetable containing saponin" refers to a root plant containing saponin, and includes, for example, ginseng, deodeok, bellflower, astragalus and the like. In addition, the "root vegetable" can be used as it is without the above-mentioned rhizome plant (that is, not subjected to a separate cutting, cutting, etc.), and includes all that is dried by grinding or cutting according to a conventional method. Preferably, those that do not undergo separate cutting, cutting, etc. may be used.

The present inventors searched a variety of microorganisms having fermentation activity of root vegetables from rice husks and rice bran around the country. In other words, the rice husk and rice bran was treated for 30 minutes at 100 ℃ and suspended in TSB (tryptic soy broth), the supernatant was transplanted to TSA (tryptic soy agar) medium, and then incubated at 37 ℃ for 24 hours to grow colonies Ginseng was added to effectively ferment ginseng to search for strains that produce high concentrations of ginsenoside Rh1 (ginsenoside Rh1) and compound K (compund K).

As a result of the above search, Bacillus strains, in particular Bacillus subtilis or Bacillus rickeniformis ( Bacillus licheniformis ) was found to have the fermentation activity of root vegetables including ginseng. It is surprising that the Bacillus strain has a high fermentation activity against ginseng, which is distinguished from the conventional method for producing ginseng fermented products (ie, fermented ginseng, etc.) using lactic acid bacteria or enteric bacteria.

The present inventors selected about 20 strains having high fermentation activity, and among them, the highest saponin-degrading activity, as well as the smallest off-flavor, were selected as KH-07 strains.

The KH-07 strain was Gram positive bacilli by Gram staining. GDNA of KH-07 strain was isolated using Wizard genomic DNA purification kit (Promega, USA), primer [forward: 5'-TCA CCA AGG CRA CGA TGC G-3 '(SEQ ID NO: 2), reverse: 5' PCR [5 min at 94 ° C. using CGT ATT CAC CGC GGC ATG-3 ′ (SEQ ID NO: 3)]; Thereafter, 30 cycles of 30 seconds (denaturation at 94 ° C., 40 seconds (annealing) at 54 ° C., and 90 seconds at 72 ° C.] were obtained to obtain a PCR product of 1100 bp, which was obtained using a T-easy vector (Promega). JM 109 (Promega) was transformed after ligation. Plasmids were again isolated from the positive clones and sequenced using T7 and SP6 primers.

GenBank The sequence of 16S rDNA of KH-07 strain obtained from the homology search Blast system is shown in SEQ ID NO: 1. Sequence analysis, KH-07 strain is Bacillus subtilis (Bacillus subtilis ) and Bacillus licheniformis , and when compared directly to the two strains, the KH-07 strain showed more similar characteristics to Bacillus licheniformis but was not identical (Table below). 1).

% Homology with KH-07 strains Search (High Homorology Analysis) Align (full method) direct order homology Bacillus subtilis subtilis ) - 98 - Bacillus rickenformis licheniformis ) ATCC 14580 99 99 91

Bacillus known in the bacteriological properties of the newly separated KH-07 strain in accordance with the present invention subtilis strain (Bacillus subtilis ) KCTC1325 and Bacillus rickeniformis ( Bacillus) licheniformis ) compared with KCTC1026 is shown in Table 2 below.

Bacillus subtilis KCTC1325 Bacillus licheniformis KCTC1026 Bacillus licheniformis KH-07 Shape Rod Rod Rod Gram Positive Positive Positive Voges-Prokauer test + + + Indole - - - Methyl red + + + Nitrate reduction - - - Simmon's citrate + + + Oxidase + + + Urease + + + Catalase + + + Casein usability + + + Gelatin Usability + + + Starch Availability + + + D-Glucose Availability + + + L-Arabinose Availability + + + D-Xylose Availability + + + Amygdalin degradation - - + Aerobic growth + + + Anaerobic growth - + +

The KH-07 strain is determined to be a novel strain from the above bacteriological properties, and in particular, as shown in the following examples, it has not only excellent saponin-degrading activity as compared to conventional strains, but also has excellent pharmacological activity of ginsenoside Rh1 ( ginsenoside Rhl) and compound K (compund K) are produced at high concentrations.

Based on the above results, the present inventors deposited the newly isolated KH-07 strain on the Korea Microbiological Preservation Theta (KCCM) on October 17, 2007, and received an accession number of KCCM 10885P.

The present invention includes a method for producing root vegetable fermented products using Bacillus strains. That is, the present invention is (a) the genus Bacillus strains, preferably Bacillus subtilis ( Bacillus ) in the root vegetables containing saponin subtilis ) or Bacillus licheniformis , more preferably Bacillus licheniformis KCCM10885P, followed by fermentation; And (b) separating the fermented root vegetables from the fermentation mixture obtained in step (a), or concentrating or drying the fermented broth obtained from the fermentation mixture obtained. It includes. Root vegetables containing the saponin preferably include ginseng, deodeok, bellflower, or sulfur.

As used herein, the term "root vegetable fermented product" refers to a fermented root vegetable obtained according to the present invention (i.e., fermented root vegetable, for example, fermented ginseng, fermented deodeok, fermented bellflower, fermented coriander, etc.) or fermented obtained according to the present invention. And the remaining fermentation broth, its concentrate, or its concentrated dried product. In addition, the "root vegetable fermentation" means that the fermented root vegetable described above is further processed, for example steamed at a temperature of 95-110 ° C. and dried at a temperature of about 55-65 ° C .; And steamed at a temperature of 95-110 ° C. and dried at a temperature of about 55-65 ° C., followed by extraction with 0.3-0.5 l of ethanol (eg 70% ethanol) per 100 g of dry matter. That is, the root vegetable fermented product includes a fermented ginseng and the like prepared by further processing red ginseng or red ginseng extract.

In the production method of the present invention, step (a) is a Bacillus strain, preferably Bacillus sub, in a culture medium obtained by dispersing root vegetables such as ginseng, deodeok, bellflower, and astragalus in an aqueous medium, for example, water. Tillis ( Bacillus subtilis ) or Bacillus rickeniformis ( Bacillus) licheniformis ), more preferably Bacillus rickeniformis ( Bacillus) licheniformis ) can be performed by fermentation by adding KCCM10885P. The amount of the aqueous medium, such as water, is not significantly limited. For example, water of about 10 times the weight of root vegetables can be used.

Root vegetables such as ginseng, Deodeok, bellflower, Astragalus, it is preferable to perform a pre-treatment process salted before performing the culture. That is, it is preferable to suppress the production of various germs by pickling according to a conventional method with 4-12 g of salt with respect to 100 g of root vegetables.

Said Bacillus strain, preferably Bacillus subtilis or Bacillus rickeniformis ( Bacillus licheniformis ), more preferably Bacillus rickeniformis ( Bacillus) licheniformis ) KCCM10885P can be added as is or in starter form. When the cells are added as it is, the content of the cells can be added, for example, 100,000 to 1 million, but is not significantly limited.

In addition, when the strain of the genus Bacillus in the form of a starter, the strain may be added by inoculating chaff or rice bran. The starter is Bacillus strain, preferably Bacillus subtilis ( Bacillus subtilis ) or Bacillus licheniformis , more preferably Bacillus licheniformis KCCM10885P about 100,000 can be obtained by adding to normal rice husk or rice bran. Preferably, when the starter-type chaff or rice bran is used after treatment at 100 to 120 ° C. for 10 to 30 minutes, the yield of the fermented product may be increased, and an unpleasant odor may be reduced during fermentation.

The fermentation may be performed for 3 to 30 days, preferably 7 to 10 days, but is not limited thereto. For example, Bacillus strains, preferably Bacillus subtilis subtilis ) or Bacillus licheniformis , more preferably Bacillus licheniformis KCCM10885P, when fermented with intact cells for 15 days, preferably about 10 days at room temperature or about 37 The fermentation may be carried out at ℃ ℃, when inoculated into the rice husk or rice bran in the form of a starter fermentation may be carried out for 30 days, preferably about 1 week at room temperature or about 37 ℃.

In addition, the fermentation may be carried out by pickling fermentation. For example, add 4-12 g of salt to 100 g of dried ginseng and mix the pickled ginseng with an auxiliary material (salt and rice husk or rice bran starter (about 1-4 g per 100 g of ginseng)) in an onggi, wooden barrel or plastic barrel. Put the pickled ginseng on the bottom so that there is no gap between them, add the subsidiary ingredients, put the ginseng again, and repeat it so that it does not float in the container, cover it with a heavy stone or wood and press it tightly for 15 days, preferably about This can be done by aging at room temperature for one week.

The production method of the present invention is to separate the fermented root vegetables from the fermentation mixture (fermentation mixture) obtained in step (a), or to concentrate or dry the fermentation broth separated from the fermentation mixture (step (b)). It includes. The concentration may be a conventional method such as concentrated under reduced pressure, the drying may be a method such as reduced pressure drying, freeze drying. Preferably the drying may be carried out by lyophilization.

In the manufacturing method of the present invention, step (b) may be carried out by separating the fermented root vegetables from the fermentation mixture (fermentation mixture) obtained in step (a), further comprising the step of drying after separation of the fermented root vegetables The fermented root vegetables may be steamed at a temperature of 95 to 110 ° C. and dried at a temperature of about 55 to 65 ° C. In addition, if necessary, the fermented root vegetables are steamed at a temperature of 95 to 110 ° C. and dried at a temperature of about 55 to 65 ° C., and then 0.3 to 0.5 l of ethanol (for example, 70% ethanol) per 100 g of dry matter. The method may further include extracting.

Further, step (b) may be carried out by concentrating or drying the fermentation broth separated from the fermentation mixture obtained in step (a), which fermentation broth is separated from the fermentation mixture obtained in step (a). It may further comprise a washing solution of the fermented root vegetables (for example, washing liquid obtained by washing at least one time with about 0.5 to 1 water per 100 g of fermented root vegetables).

The root vegetable fermentation product obtained according to the present invention may be prepared into a pharmaceutical composition together with conventional pharmaceutical additives or as a functional health food (eg, a beverage). For example, 0.1-80% of the root vegetable fermentation product obtained according to the present invention, 5-10% of sugar, 0.05-0.3% citric acid, 0.005-0.02% of caramel, and 0.1-1% of vitamin C are mixed therewith, 94% purified water is mixed to make a syrup. The syrup is sterilized at 85 to 98 ° C. for 20 to 180 seconds, mixed with cooling water at a ratio of 1: 4, and then carbonated gas is infused with 0.5 to 0.82% of carbonated beverage. Can be prepared. In addition, the homogeneous blend of subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%), water (75%) and the root vegetable fermentation product obtained in accordance with the present invention are instantaneous. After sterilization, it may be packaged in a small packaging container such as a glass bottle or a plastic bottle to prepare a health beverage. The composition ratio may be arbitrarily changed according to regional and ethnic preferences such as preference, demand hierarchy, demand country, use purpose, and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the invention, but not to limit the scope of the invention.

Example  One. KH -07 Fermentation of Ginseng Using Rice Bran Inoculated with Strain

About 100,000 KH-07 strains were inoculated into 100 g of rice bran and treated for 30 minutes in an oven at 60 ° C., 80 ° C., 100 ° C., and 120 ° C., respectively.

Ginseng (Ginseng) was washed with water and pickled with 5% brine for 3 hours. The raw materials (salt and the rice bran) were mixed on the pottery and placed on the bottom, and the pickled ginseng was placed so as not to float, and then, the raw ingredients were added again, the ginseng was laid again, and this was repeatedly put on the pottery and pressed with a heavy stone. Ginseng radishes were aged at 37 ° C. for about one week so that carbohydrates in the rice bran were fermented and the yeast and beneficial bacteria grew to produce lactic acid and butyric acid. Ginseng fermented product was separated, washed with water, and then dried naturally to obtain a solid ginseng fermented product (ie, fermented ginseng).

Example  2. Bacillus  Fermentation of Ginseng Using Chaff Containing Strains

For rice hulls from around the country, the microorganisms contained in the rice husk were tested and Bacillus strains, in particular Bacillus subtilis ( Bacillus) subtilis ) or Bacillus rickeniformis ( Bacillus) licheniformis ) were selected as the rice hull containing a relatively large amount of strains. The ginseng fermented product was prepared in the same manner as in Example 1, except that the selected chaff was used by treating the oven at 60 ° C., 80 ° C., 100 ° C., and 120 ° C. for 30 minutes.

The results of measuring the degree of fermentation (growth of bacteria) and the unpleasant odor of the ginseng fermented products obtained in Examples 1 and 2 according to the respective treatment conditions are shown in Table 3 below.

Fermentation (growth of bacteria) degree smell 60 ℃ 80 ℃ 100 ℃ 120 ℃ Room temperature 60 ℃ 80 ℃ 100 ℃ 120 ℃ Example 1 - + +++ ++ --- - + +++ Example 2 - + ++ ++ --- - + ++

--- Severe hyphae formation / severe odor

-Mycelium forms / odors

-Slight hyphae formation / slight odor

+ Fermentation / slight smell of lactic acid

++ Proper fermentation / lactic acid smell

+++ Good fermentation / strong lactic acid smell

From the results of Table 3, when fermented using chaff or rice bran as a starter, it can be seen that it is preferable to use for 10 to 30 minutes at 100-120 ℃.

Example  3.

After fermenting the ginseng fermented product (ie, fermented ginseng) after aging in Example 1, the remaining fermentation broth and the washing solution of the ginseng fermented product (ie, fermented ginseng) were combined and filtered, and the obtained solution was concentrated under reduced pressure, and then lyophilized. To obtain ginseng fermented powder.

Example  4.

The ginseng fermentation product (ie, fermented ginseng) obtained in Example 1 was treated under steam at 98 ° C. for 3 hours, treated at 60 ° C. for 48 hours, and then dried to prepare a red ginseng fermented product.

Example  5.

500 ml of water was added to ginseng powder (100 g), and 100,000 KH-07 strains were added, fermented at 37 ° C. for 10 days, and the supernatant was lyophilized to prepare a ginseng fermented product.

Example  6.

500 ml of water was added to 100 g of ginseng powder, and 100,000 strains of Bacillus subtilis KCTC 1325 were added thereto, followed by fermentation at 37 ° C. for 10 days, and the supernatant was lyophilized to prepare a ginseng fermented product.

Example  7.

Ginseng powder (100 g) was added 500 ml of water, and 100,000 Bacillus licheniformis KCTC1026 strains were added, fermented at 37 ° C for 10 days, and the supernatant was lyophilized to prepare a ginseng fermented product.

Example  8

100,000 KH-07 strains were inoculated into 100 g of rice bran and treated in an oven at about 100 ° C. for 30 minutes each.

The bellflower roots were washed with water and rinsed with 5% salt for 3 hours. The raw materials (salt and rice bran) were mixed on the pottery and placed on the bottom, and the pickled bellflower was placed so as not to float. Bellflower radish was fermented at 37 ° C for about one week so that carbohydrates in the rice bran were fermented and the yeast and beneficial bacteria grew to produce lactic acid and butyric acid. The bellflower fermented product was separated, washed with water, and then dried naturally to obtain a solid bellflower fermented product (ie, fermented bellflower).

Example  9

Except for using the deodeok instead of the bellflower root fermented in the same manner as in Example 7 to obtain a solid deodeok fermentation (that is, fermented deodeok).

Example  10

Fermented in the same manner as in Example 7 except for using Astragalus instead of the bellflower root to obtain a solid Astragalus fermentation (ie fermentation Astragalus).

Test Example  1. Saponin Content Analysis

The saponin content of the root vegetable fermentation obtained in Examples 1 to 7 was analyzed. That is, 2 g of each sample was taken (However, Example 3 took 0.5 g of dry powder) and extracted three times with methanol (100 ml). The extract was concentrated under reduced pressure, suspended in 100 ml of water, extracted three times with ether (100 ml) and concentrated under reduced pressure. Then extracted three times with butanol (100 ml) and concentrated under reduced pressure, the obtained concentrate was dissolved in 100 ml of methanol to give 100 mg of the saponin fraction, which was obtained by TLC (developing solvent: CHCl ₃ : MeOH: H ₂ O = 65: 35:10, Coloring reagent: 5% methanolic acid solution, Detector: Shimadzu TLC Scanner CS-9301PC). The results are shown in Table 4 below.

% Content in saponin fraction ingredient Example White ginseng Red ginseng  One 2 3 4 5 6 7 Ginsenoside Rb1 10.1 8.5 6.5 3.5 9.5 15.0 14.1 14.5 6.4 Ginsenoside Rd 6.5 5.1 5.3 1.1 7.5 3.5 4.5 2.1 1.5 Ginsenoside Rg3 <0.1 <0.1 <0.1 5.7 <0.1 <0.1 <0.1 Not detected 7.8 Ginsenoside Rh1 0.9 1.2 1.5 2.1 1.1 0.2 0.3 Not detected 0.1 Ginsenoside Rh2 0.5 0.3 0.1 1.1 0.1 0.1 0.1 Not detected <0.1 Ginsenoside Rh3 <0.1 <0.1 <0.1 0.3 <0.1 <0.1 <0.1 Not detected <0.1 Compound K 1.9 2.1 2.3 2.0 1.9 1.2 1.2 Not detected Not detected Protopananacidio Diol <0.1 <0.1 <0.1 0.1 0.1 <0.1 <0.1 Not detected <0.1 Proto-Panasak Triol <0.1 0.1 <0.1 0.2 0.1 <0.1 <0.1 Not detected <0.1

From the results of Table 4, when the root vegetable is fermented using the genus Bacillus strains, ginsenoside Rd (ginsenoside Rd), ginsenoside Rh1 (compound K) and compound K (compound K) increased, and after fermentation When prepared in the form of red ginseng, ginsenosides Rh2, Rh3, Compound K, Protopananadiol, and Protopananatriol can be seen to increase compared to red ginseng.

Test Example  2. Evaluation of Fermentation Effect on Pesticide Content in Pesticide-Containing Ginseng

A ginseng fermented product was prepared in the same manner as in Example 1 or 2 except that 2 ppm of Malathion or Chlorpyrifos was used. 100 g of each ginseng fermentation product fermented with ginseng, chaff, and rice bran was added to 500 ml of water, extracted continuously, extracted for 2 hours, and concentrated under reduced pressure to 50 ml. 100 ml of acetonitrile were added thereto, homogenized, the acetonitrile was removed under reduced pressure in a water bath of 40 ° C. or lower, and the resulting aqueous solution was centrifuged at 8,500 rpm for 5 minutes to obtain an extract.

An Amberlite XAD8 resin was suspended in a 20 mm inner column in water, filled to a height of 40 mm, and then flowed into 200 ml of water, followed by 100 ml of a mixed solution of 5% sodium chloride solution: methanol (1: 1). The extract obtained above was added to this column and eluted with 200 ml of methanol. 100 ml of water was added to the eluate, and methanol was removed under reduced pressure in a water bath at 40 ° C or lower. The aqueous solution was transferred to a separatory funnel, and 5 g of sodium chloride and 50 ml of 20% dichloromethane-containing benzene were added thereto, stirred vigorously for 5 minutes, and left to stand. The dichloromethane and benzene layers were taken into a separatory funnel. 50 ml of 20% dichloromethane-containing benzene was added to the aqueous layer, and the above procedure was repeated. The dichloromethane and benzene layers were combined, passed through anhydrous sodium sulfate column, and dehydrated. The column was washed with about 20 ml of benzene, and then 40 Concentrated under reduced pressure to about 5 ml in a water bath below ℃.

A column of 15 mm in diameter was filled with 5 g of a mixture of activated carbon: microcrystalline cellulose powder (1:10) and then about 5 g of anhydrous sodium sulfate in benzene, respectively, until a small amount of benzene remained at the top. Outflow. The concentrate was added to this column and eluted with 150 ml of benzene. The eluate was almost removed under reduced pressure in a water bath of 40 ° C. or lower, and completely removed benzene with nitrogen gas at room temperature. The residue was immediately dissolved in acetone and analyzed by gas chromatography according to the conditions in the food industry. The results are shown in Table 5 below.

Pesticide Content (ppm) Malathion Chlorpyrifos Ginseng 2.1 1.9 Chaff Fermented Ginseng 0.1 0.2 Rice Bran Fermented Ginseng Not detected Not detected

From the results of Table 5, it can be seen that the fermentation of ginseng according to the present invention to prepare a ginseng fermented product can significantly lower the residual pesticides.

Test Example  3: Determination of antioxidant activity

The antioxidant activity was evaluated by measuring the radical scavenging ability of DPPH (1,1-diphenyl-2-picrylhydrazyl) on the root vegetable fermentation products prepared in Examples 1 to 10. White ginseng, red ginseng, Gilgyeong, deodeok and Astragalus were also used as samples for comparison of antioxidant activity.

500 μl of 60 μM DPPH (in EtOH) 500 ul sample was added and reacted at room temperature for 30 minutes, and then absorbance was measured at 520 nm. Ethanol was used instead of DPPH as a blank, and the degree of DPPH radical removal was determined as 100% when water was used instead of the sample as a control. Caffeic acid was used as a control drug. IC ₅₀ (50% inhibitory concentration, μg / ml) for each sample was calculated as shown in Table 6 below.

Processed sample IC ₅₀ μg / ml) Example 1 15 Example 2 45 Example 3 23 Example 4 32 Example 5 65 Example 6 95 Example 7 85 Example 8 35 Example 9 29 Example 10 45 White ginseng 125 Red ginseng 110 Street view 154 Deodeok 145 Astragalus 96

From the results of Table 6, the fermented product obtained by the present invention has a superior antioxidant activity than the extract of the protozoa, especially the newly isolated KH-07 strain according to the present invention is more excellent than the known Bacillus strains It can be seen that it has activity.

Test Example  4. Anticancer activity measurement

The anticancer activity of the root vegetable ferments prepared in Examples 1 to 10 was determined by Carmichael et al., Carmichael, J. et al; Cancer. Res ., 47 , 936-940, 1987). White ginseng, red ginseng, Gilgyeong, deodeok and Astragalus were also used as samples for the comparison of anticancer activity.

Human-derived cancer cell lines (HepG2; Korea Cell Line Bank, Cat. No. 58065) and lung cancer cell lines (A-549; Korea Cell Line Bank, Cat. No. 10185) were 10% FBS and 1% antibiotic-antifungal agents, respectively. Antibiotics-antimycotics, GIBCO, Cat.No. 15240-062) and 2.2 g / L of sodium bicarbonate added RPMI 1640 medium solution (Sigma, R-6504) were cultured as follows (5% CO ₂ ).

Each of the cancer cell lines were treated with 0.25% trypsin to remove the cells from the flask, and the number of cells was adjusted to 3 × 10 ⁴ cells / well, and 180 μl per well was put into a 96 well culture plate at 37 ° C., 5% CO ₂ for 24 hours. Cells were attached by culturing in a conditional saturated CO ₂ incubator. Sterilize each sample to 10 mg / ml by autoclaving and add 20µl per well to make the final concentration of the sample to 1 mg / ml, and then saturate at 37 ℃ and 5% CO _2. The incubator was incubated for 48 hours. After 48 hours, 50 μl of 20 mg / ml MTT reagent (Cig. Cat. No. M-5655, Sigma) was added to each well, followed by further reaction in a CO ₂ incubator for 4 hours, and then 100 μl of DMSO was added to the remaining precipitate. The cytotoxicity was measured by measuring the absorbance at 580 nm with an ELISA analyzer (Bio. TeK Instrument Inc., USA). The results are shown in Table 7 below.

Treatment Sample ED50 (μg / ml) Lung Cancer Cell Line (A-549) Liver Cancer Cell Line (HepG2) Example 1 185 165 Example 2 215 195 Example 3 190 185 Example 4 250 233 Example 5 285 267 Example 6 295 285 Example 7 > 300 252 Example 8 211 245 Example 9 195 215 Example 10 > 300 285 White ginseng > 300 > 200 Red ginseng > 300 > 200 Street view > 300 295 Deodeok > 300 285 Astragalus > 300 > 300

From the results of Table 7, the fermented product obtained by the present invention has a very good anticancer activity, in particular, the newly isolated KH-07 strain according to the present invention has a better anticancer activity than the known strain of the genus Bacillus Can be.

Test Example  5. Inflammatory Cytokine TNF - alpha  And Allergic Cytokine IL - 4 of  Production Inhibition Activity Assessment

The production inhibitory activity of inflammatory cytokine TNF-alpha and allergic cytokine IL-4 was evaluated for the root vegetable fermentation products prepared in Examples 1 to 10. For the comparison of activity, water extracts of white ginseng, red ginseng, Gilgyeong, deodeok and Astragalus were also used as samples.

DBL (Dulbeccos' modified Eagle's medium, Sigma, 22256) containing 10% FBS (fetal bovine serum) and L-glutamine in rat basophil cell line (rat basophil cell line, Cat.No. 22256) (Sigma D-5648) was incubated for 2 hours in a humidified 5% CO ₂ incubator at 37 ° C., and the adherent cells were suspended using trypsin-EDTA solution. Used for this test.

The RBL-2H3 cells were each ⁵ × 10 ⁵ in 24 wells. After dispensing cells / well, mouse monoclonal IgE was inoculated with 0.5 µg / ml for 12 hours, followed by sensitization. The cells were washed with 0.5 ml of siraganian buffer (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl ₂ , 25 mM PIPES, 40 mM NaOH, pH7.2) and then again 0.16 ml of siraguanian buffer (5.6 mM). Glucose, 1 mM CaCl ₂ , 0.1% BSA was added) and then incubated at 37 ° C. for 10 minutes. Each sample was added 0.04 ml each time, and after 20 minutes, the cells were activated for 60 minutes at 37 ° C. with 0.02 ml of antigen (DNP-BSA 1 μg / ml), followed by centrifugation at 2000 rpm for 10 minutes. 0.025 ml of supernatant was transferred to 96 wells and IL-4 (interleukin-4) and TNF-alpha (tumor necrosis factor-alpha) were measured using the Endogen Elisa kit (USA). The results are shown in Table 8 below.

Treatment Sample Treatment concentration (mg / ml) TNF-alpha concentration (pg / ml) IL-4 concentration (pg / ml) Normal - 12 1.2 Allergic Induction - 573 16.4 Example 1 0.01 415 12.5 0.05 387 9.5 Example 2 0.01 452 12.8 0.05 389 10.2 Example 3 0.01 478 13.2 0.05 412 9.1 Example 4 0.01 455 12.1 0.05 412 8.7 Example 5 0.01 478 12.5 0.05 388 9.5 Example 6 0.01 487 12.5 0.05 435 10.2 Example 7 0.01 485 13.2 0.05 425 11.5 Example 8 0.01 467 12.9 0.05 432 11.2 Example 9 0.01 501 11.5 0.05 457 10.5 Example 10 0.01 495 13.5 0.01 451 12.0 Ginseng Extract 0.01 514 14.2 0.05 482 14.1 Red Ginseng Extract 0.01 497 13.2 0.05 465 12.9 Gilyeong extract 0.01 512 13.8 0.05 499 11.5 Deodeok Extract 0.01 525 12.9 0.05 477 11.5 Astragalus Extract 0.01 518 16.2 0.05 465 14.5

From the results in Table 8, it can be seen that the fermented product obtained by the present invention has very good inhibitory activity of inflammatory cytokine TNF-alpha and allergic cytokine IL-4.

Test Example  6. Anti-allergy  Active evaluation

Anti-allergic activity was evaluated for the root vegetable fermentation prepared in Examples 1 to 10. White ginseng, red ginseng, Gilgyeong, deodeok, astragalus, and azelastine (control, Sigma, USA) were also used as samples for comparison of activity.

Assessment of allergic activity was performed using a passive transdermal anaphylaxis experimental animal model. The back of the mouse (ICR-based Korean animal) in which 10 μg of a solution diluted with physiological saline of the mouse against DNP-BSA (Dinitrophenol-human serum albumin (Sigma, A-6661)) was diluted with ether. ), And manually sensitized by injection (50 µl), and after 48 hours, 0.2 ml of saline solution containing 0.2 mg of DNP-HSA and 1.6 mg of evans blue was injected into the tail vein and killed by cervical distal bone 30 minutes later. The amount of Evans blue pigment leaked into was measured. The test sample was administered 1 hour before the Evans blue. A portion of the back of the mouse (1cm ² of the site where IgE was injected) was cut out into a test tube, and 0.7 ml of 1N-KOH was added thereto and incubated overnight at 37 ° C. 4 ml of 0.6N phosphate-acetone mixed solution (5:13) was added to the test tube, followed by shaking with Vortex and filtration to give colorimetric determination at 620 nm. The test results are shown in Table 9 below.

Treatment Sample Dosage (mg / kg) % Inhibition Saline solution 100 0 Example 1 100 54 Example 2 100 51 Example 3 100 55 Example 4 100 48 Example 5 100 49 Example 6 100 47 Example 7 100 48 Example 8 100 54 Example 9 100 45 Example 10 100 42 White ginseng 100 41 Red ginseng 100 38 Street view 100 40 Deodeok 100 25 Astragalus 100 31 Azelastine 100 85

From the results of Table 9, it can be seen that the fermented product obtained by the present invention has very excellent anaphylaxis inhibitory activity.

In view of the above results, when ginseng, red ginseng, Astragalus, Gilgyeong, Deodeok and the like are fermented with a Bacillus strain, preferably KH-07 strain, or rice bran containing the same, yellow bran, which is isolated according to the present invention. Since the prevention and treatment of diseases such as aging, cancer, and allergies shows enhanced efficacy, it is possible to manufacture medicines or health supplements containing fermented ginseng, red ginseng, Astragalus, Gilgyeong, and deodeok of higher quality. It is expected.

Formulation example  One. Powder  Produce

According to the preparation method of powder in the Pharmacopoeia General Formulation, powder was prepared in the following component content per packet.

Example 1 Dry Powder 50 mg

Lactose ··········· 100 mg

Talc 10 mg

Formulation example  2. Preparation of Tablets

According to the preparation method of tablets in the pharmacopeia formulation, tablets were prepared in the following component contents per tablet.

Example 1 Dry Powder 50 mg

Corn starch ㆍ ..100 mg

Lactose · ・ ・ ・ ・ ・ ・ ・ ・ ・ 100 mg

Magnesium Stearate 2 mg

Formulation example  3. Manufacture of capsule

According to the preparation method of capsules in the pharmacopeia formulation, capsules were prepared in the following component contents per capsule.

Example 1 Dry Powder 50 mg

Corn starch ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ 100 mg

Lactose ····················· 100 mg

Magnesium stearate 2 mg

Formulation example  4. Preparation of Injectables

Injectable drugs were prepared according to the preparation method of injectable drugs in the pharmacopeia of the pharmacopeia according to the following ingredient content per ampule (2 ml).

Example 1 Dry Powder 50 mg

Sterile distilled water for injection ㆍ ············

pH regulator ㆍ ···········

Formulation example  5. Liquid  Produce

According to the preparation method of the liquid formulation in the Pharmacopoeia General Regulations, the liquid formulation was prepared per 100 ml of the liquid formulation.

Example 1 Dry Powder 50 mg

Isosugars 10 g

Mannitol ······· 5 g

Purified water ㆍ ············

<110> KIM, DongHyun <120> Process for the preparation of fermentation product of esculent roots using microorganism having fermentation activity of saponin-containing esculent roots <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 1097 <212> DNA <213> Bacillus licheniformis <400> 1 aggcgacgat gcgtagccga cctgagaggg tgatcggcca cactgggact gagacacggc 60 ccagactcct acgggaggca gcagtaggga atcttccgca atggacgaaa gtctgacgga 120 gcaacgccgc gtgagtgatg aaggttttcg gatcgtaaaa ctctgttgtt agggaagaac 180 aagtaccgtt cgaatagggc ggtaccttga cggtacctaa ccagaaagcc acggctaact 240 acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggaatt attgggcgta 300 aagcgcgcgc aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg 360 tcattggaaa ctggggaact tgagtgcaga agaggagagt ggaattccac gtgtagcggt 420 gaaatgcgta gagatgtgga ggaacaccag tggcgaaggc gactctctgg tctgtaactg 480 acgctgaggc gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg 540 taaacgatga gtgctaagtg ttagagggtt tccgcccttt agtgctgcag caaacgcatt 600 aagcactccg cctggggagt acggtcgcaa gactgaaact caaaggaatt gacgggggcc 660 cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct 720 tgacatcctc tgacaaccct agagataggg cttccccttc gggggcagag tgacaggtgg 780 tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 840 cccttgatct tagttgccag cattcagttg ggcactctaa ggtgactgcc ggtgacaaac 900 cggaggaagg tggggatgac gtcaaatcat catgcccctt atgacctggg ctacacacgt 960 gctacaatgg gcagaacaaa ggcagcgaag ccgcgaggct aagccaatcc cacaaatctg 1020 ttctcagttc ggatcgcagt ctgcaactcg actgcgtgaa gctggaatcg ctagtaatcg 1080 cggatcagca tgccgcg 1097 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 tcaccaaggc racgatgcg 19 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 cgtattcacc gcggcatg 18

Claims (17)

(a) fermentation by adding Bacillus licheniformis KCCM10885P to a root vegetable containing saponin; And (b) separating the fermented root vegetables from the fermentation mixture obtained in step (a), or concentrating or drying the fermented broth obtained from the fermentation mixture obtained. . delete delete delete The method of producing a root vegetable fermented product of claim 1, wherein the root vegetable containing saponin is ginseng, deodeok, bellflower, or astragalus. The method of claim 1, wherein step (a) is carried out by adding a Bacillus strain to the culture medium obtained by dispersing a saponin-containing root vegetable in an aqueous medium. The method of claim 1, wherein step (a) is carried out by pickling saponin-containing root vegetables with salt and then fermenting the strain of genus Bacillus. The method of claim 1, wherein the Bacillus licheniformis KCCM10885P is added in the form of rice bran or rice bran containing Bacillus licheniformis KCCM10885P. 9. The method of claim 8, wherein the rice hull or rice bran is treated for 10 to 30 minutes at 100 to 120 ℃. The method of claim 1, wherein the fermentation of root vegetables is characterized in that the fermentation is carried out for 3 to 30 days. The method of claim 1, wherein step (b) is performed by separating the fermented root vegetables from the fermentation mixture obtained in step (a). 12. The method of claim 11, further comprising the step of drying after separation. The method of claim 11, further comprising the step of steaming the fermented root vegetables at a temperature of 95 to 110 ℃ and dried at a temperature of about 55 to 65 ℃. The method of claim 11, further comprising the step of steaming the fermented root vegetables at a temperature of 95 to 110 ℃ and dried at a temperature of about 55 to 65 ℃, and then extracted with 0.3 to 0.5 l of ethanol per 100 g of the dried Root vegetable fermented product manufacturing method characterized in that. The method of claim 1, wherein step (b) is performed by concentrating or drying the fermentation broth separated from the fermentation mixture obtained in step (a). The method of claim 15, wherein the fermentation broth further comprises a washing solution of fermented root vegetables separated from the fermentation mixture (fermentation mixture) obtained in step (a). The method of claim 1, wherein the drying is performed by lyophilization.