Saccharomyces cerevisiae m-5 strain having bioconversion ability for saponin, method for producing fermented ginseng fruit product using the same with increased total and rare ginsenoside content, fermented ginseng fruit product produced using the same, and functional health food composition containing the same

Abstract

To provide a Saccharomyces cerevisiae M-5 strain having bioconversion ability for saponin.SOLUTION: The present invention relates to a Saccharomyces cerevisiae M-5 strain and provides a Saccharomyces cerevisiae M-5 strain (Accession No.: KCCM 13375 P) having bioconversion ability for saponin, a method for producing a fermented ginseng fruit product using the same with increased contents of total ginsenosides and rare ginsenosides, a fermented ginseng fruit product produced by the same, and a functional health food composition containing the same.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a Saccharomyces cerevisiae M-5 strain having bioconversion ability for saponin, a method for producing a fermented ginseng fruit product using the same with increased contents of total ginsenosides and rare ginsenosides, a fermented ginseng fruit product produced using the same, and a functional health food composition containing the same. [Background technology]

[0002] Korean ginseng (Panax ginseng CA Meyer) is the most important medicinal plant in East Asia, including Russia, Europe, Korea, Japan and China, and its roots have been used extensively for medicinal purposes (Yang et al., 2021). The most important pharmacologically active components in Korean ginseng are ginsenosides, which are present in all parts of ginseng. Ginsenosides provide anti-inflammatory, anti-cancer and antioxidant effects, improve blood circulation and memory, protect neurotransmission, hematopoietic, anti-stress and anti-diabetic effects in the human body (Bahukhandi et al., 2021; Chung et al., 2016; Lee et al., 2019).

[0003] According to the structure of ginsenoside aglycone, ginsenosides can be classified into three types: protopanaxadiol-type ginsenosides, protopanaxatriol-type ginsenosides, and oleanane-type ginsenosides. Ginsenoside derivatives are compounds that have a structure in which sugars such as glucose, rhamnose, xylose, and arabinose are ester-bonded to the alcoholic OH groups of R1, R2, and R3 of the non-sugar part (protopanaxadiol or protopanaxatriol), which is a triterpene. The main ginsenosides in ginseng currently known include about 13 common ginsenosides and about 11 rare ginsenosides that are known to be converted from these.

[0004] Korean ginseng can be used as it is as fresh ginseng, dried and used in the form of white ginseng, or steamed and used in the form of red ginseng.

[0005] In recent years, much research has been actively conducted to further increase the content of ginseng saponins, which are only present in these processed ginseng.

[0006] On the other hand, ginseng leaves and fruits contain more ginsenosides than the roots, which are mainly consumed (Chung et al., 2016; Yang et al., 2021). Ginseng fruits have about three times higher ginsenoside content than the roots, and contain more ginsenosides Re and Rd (Ko et al., 2008). In addition, ginseng leaves and fruits contain more polyphenolic substances than the roots and show excellent antioxidant activity (Kim and Ko, 2020).

[0007] The inventors conducted extensive research to develop a ginseng fruit fermented product with increased total ginsenoside and rare ginsenoside contents using ginseng fruit, which contains a large amount of ginsenoside and polyphenol substances. They selected a bacterial strain with excellent ginsenoside bioconversion ability, and confirmed that the ginseng fruit fermented product fermented using the selected bacterial strain has a significantly increased content of total ginsenosides and rare ginsenosides, an excellent ginsenoside index, and a significantly increased intestinal absorption rate of active ingredients such as polyphenols and ginsenosides contained in ginseng fruit, thereby completing the present invention. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Korean Patent Application Publication No. 10-2021-0103055 [Patent Document 2] Korean Patent Application Publication No. 10-2023-0029021 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a Saccharomyces cerevisiae M-5 strain having biotransformation ability towards saponin.

[0010] Another object of the present invention is to provide a method for producing a fermented ginseng fruit product having increased contents of total ginsenosides and rare ginsenosides.

[0011] Yet another object of the present invention is to provide a fermented ginsenoside product produced by the above method, which has an excellent ginsenoside index and contains rare ginsenosides that are not present in the ginseng fruit crushed liquid before fermentation.

[0012] It is still another object of the present invention to provide a functional health food composition containing the fermented ginseng fruit product as an active ingredient. [Means for solving the problem]

[0013] To achieve the above object, the present invention provides a Saccharomyces cerevisiae M-5 strain (Accession No.: KCCM 13375P) having biotransformation ability for saponin.

[0014] According to one embodiment of the present invention, the strain may be isolated from candied ginseng.

[0015] According to one embodiment of the present invention, the strain may use glucose, galactose, α-methyl-d-glucoside, maltose, sucrose, trehalose, melezitose, or raffinose as a carbon source.

[0016] In order to achieve the above-mentioned other objects, the present invention provides a method for producing a fermented ginseng fruit product having increased contents of total ginsenosides and rare ginsenosides, comprising the steps of inoculating a crushed ginseng fruit liquid with Saccharomyces cerevisiae M-5 strain (Accession No.: KCCM 13375P) and then fermenting the liquid.

[0017] According to one embodiment of the present invention, the crushed ginseng fruit liquid may have a pH adjusted to 5-7.

[0018] According to one embodiment of the present invention, the fermentation may be carried out at 25 to 35° C. for 12 hours to 10 days.

[0019] According to one embodiment of the present invention, the fermented ginseng fruit product may have a total ginsenoside content increased by at least 50% compared to that of the crushed ginseng fruit liquid before fermentation.

[0020] According to one embodiment of the present invention, the fermented ginseng fruit product may contain ginsenosides Rg3, Rh4, Rk1 and Rg5 that are not present in the crushed ginseng fruit liquid before fermentation.

[0021] According to one embodiment of the present invention, the rare ginsenoside may be one or more selected from ginsenosides Rg3, Rh4, Rk1, Rk3, Rg5 and Rg6.

[0022] According to one embodiment of the present invention, the fermented ginsenoside of the ginseng fruit may have a total content of rare ginsenosides Rg3, Rh4, Rk1, Rk3, Rg5 and Rg6 of at least 10 μg / mg.

[0023] According to one embodiment of the present invention, the fermented ginseng fruit product may have a ginsenoside Re content of at least 65 μg / mg.

[0024] According to one embodiment of the present invention, the ginsenoside Rb1, Rg1 and Rg3 in the fermented ginsenoside extract may have a total content of at least 10 μg / mg, a ginsenoside Rg2 content of at least 12 μg / mg, a ginsenoside Rc content of at least 8 μg / mg, a ginsenoside Rb2 content of at least 9 μg / mg, and a ginsenoside Rd content of at least 12 μg / mg.

[0025] In order to achieve yet another of the above-mentioned objectives, the present invention provides a fermented ginseng fruit product produced by the above-mentioned method, having a sum of ginsenosides Rb1, Rg1 and Rg3 of at least 10 μg / mg, and containing ginsenosides Rg3, Rh4, Rk1 and Rg5 that are not present in the ginseng fruit crushed liquid before fermentation.

[0026] In order to achieve the above-mentioned further object, the present invention provides a functional health food composition containing the fermented ginseng fruit product as an active ingredient. Effect of the Invention

[0027] The Saccharomyces cerevisiae M-5 strain (Accession No.: KCCM 13375P) of the present invention has excellent ginsenoside bioconversion ability. When the strain is inoculated into a crushed ginseng fruit liquid and fermented, the content of ginsenosides Re, Rg2, Rb1, Rc, Rb2, Rd and the content of total ginsenosides are further increased, and a fermented ginseng fruit product containing rare ginsenosides such as ginsenosides Rg3, Rh4, Rk1 and Rg5 that are not present before fermentation and having a further increased intestinal absorption rate of active ingredients can be obtained.

[0028] The fermented ginseng fruit product of the present invention has an increased intestinal absorption rate of polyphenols and ginsenosides compared to before fermentation, has an excellent ginsenoside index, and contains rare ginsenosides, so is extremely useful as a functional health food or food ingredient. [Brief description of the drawings]

[0029] [Figure 1] These are photographs showing the color changes observed after inoculating and culturing strains onto esculin-containing MRS agar medium (Figure 1A) and PDA agar medium (Figure 1B) in order to select strains having β-glucosidase activity. [Diagram 2] 1 is a phylogenetic tree shown by analyzing the 26S rRNA gene sequence of the Saccharomyces cerevisiae M-5 strain of the present invention. [Diagram 3] 1 is a flow chart briefly illustrating a method for producing a fermented ginseng fruit product according to one embodiment of the present invention. [Figure 4] 2 is a graph showing changes in absorbance and pH over fermentation time of a fermented ginseng fruit product according to an embodiment of the present invention. [Diagram 5]1 is a graph showing changes in pH, reducing sugar, protein, polyphenol contents, and viable cell count over the fermentation time of a fermented ginseng fruit product according to an embodiment of the present invention. [Figure 6] 1 is a graph showing changes in ABTS and DPPH scavenging activity over the fermentation time of a fermented ginseng fruit product according to an embodiment of the present invention. [Figure 7A] 1 is a graph showing the intestinal absorption rate over time of total polyphenols in a fermented ginseng fruit product according to an embodiment of the present invention, compared with a sample before fermentation. [Figure 7B] 1 is a graph showing the intestinal absorption rate of each polyphenol in a fermented ginseng fruit product according to one embodiment of the present invention 240 minutes after intestinal permeation, in comparison with a sample before fermentation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The present invention will be described in detail below.

[0031] In the present specification, the "ginsenoside index" can be expressed as (1) the content of rare ginsenosides (the sum of ginsenosides Rg3, Rh4, Rk1, Rk3, Rg5 and Rg6) and / or (2) the sum of Rg1, Rb1 and Rg3 (Rg1+Rb1+Rg3), which may be an index indicating the production efficiency of rare ginsenosides and / or the quality of the ginseng fruit fermentation product. The ginseng fruit fermentation product of the present invention having these indexes exceeding a certain level has a large sum of Rg1, Rb1 and Rg3 and a high content of rare ginsenosides, and therefore has very excellent quality.

[0032] In this specification, "rare ginsenoside" means ginsenoside contained in trace amount, almost non-existent, or non-existent in raw ginseng or ginseng fruit without separate processing. According to the manufacturing method of the present invention, ginsenoside mainly contained in raw ginseng fruit crushed liquid without separate processing is converted into rare ginsenoside by fermentation using Saccharomyces cerevisiae M-5 strain. The rare ginsenoside of the present invention may be one or more ginsenosides selected from ginsenosides Rg3, Rh4, Rk1, Rk3, Rg5 and Rg6, preferably ginsenosides Rg3, Rh4, Rk1, Rk3, Rg5 and Rg6, more preferably ginsenosides Rg3, Rh4, Rk1 and Rg5.

[0033] One aspect of the present invention relates to a Saccharomyces cerevisiae M-5 strain (Accession No.: KCCM 13375P) having biotransformation ability for saponin.

[0034] In the following example, the 26S rRNA base sequence of the M-5 strain, which is a strain derived from candied ginseng stored in the headquarters (BTC Corporation) research institute, was analyzed for identification and classification of the microorganism, and it was confirmed that it has the nucleic acid sequence of SEQ ID NO: 1. Analysis of the nucleic acid sequence of the strain confirmed that it belongs to Saccharomyces cerevisiae, and the nucleic acid sequence of the strain was requested to Macrogen to examine the homology, and it was confirmed that it is 99.9% identical to the standard strains of Saccharomyces cerevisiae DSM70449 and Saccharomyces cerevisiae RC231. In the nucleic acid sequence of SEQ ID NO: 1, "n" means a base of "a", "g", "c", "t or u", or "unknown or other".

[0035] Therefore, the microorganism of the present invention having the 26S rRNA base sequence of SEQ ID NO: 1 was named Saccharomyces cerevisiae M-5 strain and deposited at the Korea Microorganism Collection on August 1, 2023 (accession number: KCCM 13375P).

[0036] The Saccharomyces cerevisiae M-5 strain of the present invention is a novel Saccharomyces cerevisiae strain derived from candied ginseng.

[0037] The strains use glucose, galactose, α-methyl-d-glucoside, maltose, sucrose, trehalose, melezitose and / or raffinose as carbon sources.

[0038] The Saccharomyces cerevisiae M-5 strain of the present invention has the biotransformation ability for saponin, in particular, converts saponin into ginsenoside, and converts high molecular weight ginsenoside into ginsenoside, especially rare ginsenoside, with increased intestinal absorption rate in the human body, and is therefore very useful for producing fermented ginseng fruit products.

[0039] In addition, the Saccharomyces cerevisiae M-5 strain of the present invention converts ginseng fruit saponin into one rare ginsenoside selected from ginsenosides Rg3, Rh4, Rk1, Rk3, Rg5 and Rg6, and / or ginsenosides Rg2, Rc, Rb2 and Rd, making it possible to produce a ginseng fruit fermentation product with an increased content of a specific ginsenoside, and is therefore very useful for producing health functional food materials.

[0040] Another aspect of the present invention relates to a method for producing a fermented ginseng fruit product having increased contents of total ginsenosides and rare ginsenosides, comprising a step of inoculating a crushed ginseng fruit liquid with Saccharomyces cerevisiae M-5 strain (Accession Number: KCCM 13375P) and then fermenting the liquid.

[0041] Major ginsenosides, which account for over 90% of ginsenosides, have a very low bioabsorption rate due to their high molecular weight. Therefore, in order to increase the efficacy of ginsenosides, a process is required to convert the major ginsenosides into minor ginsenosides, which are relatively well absorbed and have better efficacy. In other words, a conversion process that removes glucose is required for the major ginsenosides to effectively demonstrate physiological activity in the body.

[0042] Therefore, the inventors selected a bacterial strain with excellent bioconversion ability for saponin, and used the selected bacterial strain to convert the high molecular weight ginsenosides in the ginseng fruit crushed liquid into low molecular weight ginsenosides, thereby producing a ginseng fruit fermentation product with increased contents of total ginsenosides and rare ginsenosides and increased intestinal absorption rate of active ingredients such as polyphenols and ginsenosides.

[0043] In the manufacturing method of the present invention, the ginseng fruit may be ginseng fruit extract, ginseng fruit concentrate, ginseng fruit extract, ginseng fruit powder, ginseng fruit concentrate powder, or ginseng fruit crushed liquid, preferably ginseng fruit crushed liquid, ginseng fruit powder, ginseng fruit concentrate, or ginseng fruit concentrate powder, and more preferably ginseng fruit crushed liquid in terms of increasing total ginsenosides and rare ginsenosides.

[0044] In the production method of the present invention, the crushed ginseng fruit liquid may have a crude saponin content of 60 to 100 μg / mg, preferably 70 to 90 μg / mg, based on the solid content.

[0045] The crushed ginseng fruit liquid of the present invention may have a solid content of 2 to 20% by weight, preferably 2 to 10% by weight.

[0046] In the present specification, the ginseng fruit crushed liquid includes not only a crushed liquid obtained by crushing ginseng fruit, but also a processed product of the crushed ginseng fruit liquid. For example, the crushed ginseng fruit liquid may be produced as a powder or a concentrated liquid by a further process such as vacuum distillation, freeze-drying, or spray drying.

[0047] In addition, in the above-mentioned ginseng fruit crushed liquid, it is preferable to add sugar to the ginseng fruit crushed liquid and then adjust the pH to 5 to 7, preferably 5.5 to 6.5, from the viewpoint of increasing the conversion rate of rare ginsenosides to high molecular weight ginsenosides during fermentation by the Saccharomyces cerevisiae M-5 strain of the present invention. In addition, the above-mentioned ginseng fruit crushed liquid may be the above-mentioned pH-adjusted ginseng fruit crushed liquid to which sugar has been added.

[0048] If the pH of the crushed ginseng fruit liquid is outside the above range, the bioconversion rate of high molecular weight ginsenosides, especially the conversion rate to rare ginsenosides, will not reach the expected value.

[0049] In the production method of the present invention, before inoculating the Saccharomyces cerevisiae M-5 strain into the ginseng fruit crushed liquid, a step of sterilization by heating at 80 to 130°C, preferably 80 to 100°C, for 10 to 40 minutes, preferably 20 to 40 minutes, may be carried out.

[0050] The Saccharomyces cerevisiae M-5 strain can be inoculated in the form of a pre-cultured culture solution, specifically, at 2 to 20% by weight, preferably 4 to 10% by weight, based on the ginseng fruit crushed solution.

[0051] The strain concentration of the Saccharomyces cerevisiae M-5 strain preculture solution was 1.0 × 10 6 ~5.0×10 8 cfu / mL, preferably 1.0×10 7 ~5.0×10 8cfu / mL, and even more preferably 1.0×10 7 ~1.0×10 8 It may be cfu / mL.

[0052] If the inoculation amount of the strain is less than the lower limit, the fermentation rate may be slowed, whereas if it exceeds the upper limit, the fermentation may proceed excessively, resulting in a decrease in economic efficiency.

[0053] In one specific example, the production method of the present invention involves inoculating the Saccharomyces cerevisiae M-5 strain into the ginseng fruit crushed liquid, followed by fermentation at 10 to 70°C, preferably 20 to 60°C, more preferably 20 to 40°C, even more preferably 25 to 35°C, and particularly preferably 28 to 32°C for 12 hours to 10 days, preferably 16 hours to 6 days, even more preferably 20 hours to 3 days, and particularly preferably 24 hours to 36 hours.

[0054] If the fermentation temperature is below the lower limit, the fermentation period may be prolonged, which may lead to contamination by various bacteria, and if it exceeds the upper limit, the growth of the strain may stop. Also, if the fermentation time is below the lower limit, the fermentation may be insufficient, resulting in a low ginsenoside conversion rate, and if it exceeds the upper limit, the ginsenosides produced by fermentation may begin to decompose, resulting in a decrease in content.

[0055] The above fermentation temperature and time conditions are the optimal conditions for converting ginsenosides in the crushed ginseng fruit liquid into rare ginsenosides.

[0056] The fermented ginseng fruit product can then be heated at 85-95° C. for 10-30 minutes to inactivate the bacterial strain.

[0057] The ginsenoside content of the ginseng fruit fermented product of the present invention produced in this manner is increased by at least 50%, preferably at least 70%, more preferably at least 75%, and even more preferably at least 80% compared to that of the ginseng fruit crushed liquid before fermentation.

[0058] Furthermore, the fermented ginseng fruit product of the present invention contains ginsenosides Rg3, Rh4, Rk1 and Rg5, which are not present in the crushed ginseng fruit liquid before fermentation.

[0059] The sum of ginsenosides Rg3, Rh4, Rk1, Rk3, Rg5 and Rg6, which are rare ginsenosides contained in the fermented ginseng fruit of the present invention, may be at least 10 μg / mg, preferably 10-20 μg / mg, more preferably 13-20 μg / mg, and even more preferably 14-18 μg / mg. The rare ginsenosides contained in the fermented ginseng fruit are increased by about 15-40 times, more specifically about 20-40 times, even more specifically about 25-35 times, and particularly specifically about 28-32 times, compared to before fermentation with the M-5 strain of the present invention.

[0060] Furthermore, the ginsenoside Re content of the fermented ginseng fruit product of the present invention may be at least 70 μg / mg, preferably 70 to 85 μg / mg, more preferably 70 to 80 μg / mg, and even more preferably 75 to 80 μg / mg.

[0061] The total amount of ginsenosides Rb1, Rg1 and Rg3 contained in the fermented ginseng fruit product of the present invention is at least 10 μg / mg, preferably 15 μg / mg or more, more preferably 15 to 25 μg / mg, even more preferably 16 to 22 μg / mg, and particularly preferably 18 to 20 μg / mg. The content of ginsenoside Rg2 is at least 12 μg / mg, preferably 12 to 15 μg / mg, and more preferably 13 to 15 μg / mg. The content of ginsenoside Rc is at least 8 μg / mg, preferably 10 μg / mg or more, and even more preferably 15 to 20 μg / mg. The content of ginsenoside Rb2 is at least 9 μg / mg, preferably 10 μg / mg or more, more preferably 10 to 20 μg / mg, and even more preferably 15 to 20 μg / mg, and the content of ginsenoside Rd is at least 12 μg / mg, preferably 15 μg / mg or more, more preferably 15 to 30 μg / mg, and even more preferably 20 to 30 μg / mg, and especially preferably 25 to 30 μg / mg.

[0062] The greatest feature of the present invention is that by fermenting crushed ginseng fruit liquid with the Saccharomyces cerevisiae M-5 strain of the present invention, the total ginsenoside content is increased, and the ratio of high molecular weight ginsenosides converted into rare ginsenosides such as ginsenosides Rg3, Rh4, Rk1, Rk3, Rg5 and Rg6 is increased, thereby further increasing the intestinal absorption rate of active ingredients such as polyphenols and ginsenosides contained in ginseng fruit.

[0063] Yet another aspect of the present invention relates to a ginseng fruit fermentation product produced by the above-mentioned method, characterized in that the sum of ginsenosides Rb1, Rg1 and Rg3 is at least 15 μg / mg, and that it contains ginsenosides Rg3, Rh4, Rk1 and Rg5 that are not present in the ginseng fruit crushed liquid before fermentation.

[0064] Regarding the content of active ingredients in the fermented ginseng fruit, the "Standards and Specifications for Health Functional Foods" (Notification of the Ministry of Food and Drug Safety No. 2016-143, 2016.12.21.) does not mention ginseng fruit, but states in the section on ginseng item and index component content that the standard value is "0.8-34 mg / g of ginsenoside Rg1 and Rb1 combined," and in the section on ginseng item and index component content that the standard value is "2.5-34 mg / g of ginsenoside Rg1, Rb1, and Rg3 combined." In addition, in the section on "Requirements for final products" of the document, it states that the product should functionally meet the requirements of "improving immunity, improving fatigue, improving blood flow / memory by inhibiting platelet aggregation, antioxidant, and helping menopausal women's health." Furthermore, the detailed items in the document state that in order to help "boost immunity and alleviate fatigue," the content of the index components (Rg1+Rb1+Rg3) must be 3-80mg / g, and in order to help "improve blood flow / memory by inhibiting platelet aggregation, and promote antioxidant properties," the total amount of the index components must be 2.4-80mg / g.

[0065] As can be seen from the examples below, the fermented ginsenoside product of the present invention has an excellent ginsenoside composition, since the content of rare ginsenosides such as ginsenosides Rg3, Rh4, Rk1, Rk3, Rg5 and Rg6 is at least 10 μg / mg, and the total of the indicator components (Rg1, Rb1 and Rg3) is at least 10 μg / mg, preferably at least 15 μg / mg.

[0066] Yet another aspect of the present invention relates to a functional health food composition comprising the fermented ginseng fruit as an active ingredient. The fermented ginseng fruit in the functional health food composition is as described above.

[0067] The health functional food composition may be provided in the form of powder, granule, tablet, capsule, syrup or drink, and the health functional food composition may contain other foods or food additives in addition to the active ingredient, and may be used appropriately by a conventional method. The amount of the active ingredient mixed may be appropriately determined depending on the purpose of use, such as prevention, improvement or therapeutic treatment. According to one embodiment, the fermented ginseng extract may be contained in an amount of 0.001 to 80% by weight, preferably 0.001 to 70% by weight, more preferably 0.01 to 60% by weight, even more preferably 0.01 to 30% by weight, and particularly preferably 0.01 to 10% by weight.

[0068] The effective dose of the active ingredient contained in the health food composition may be adjusted depending on various factors including, but not limited to, the type and amount of the active ingredient and other ingredients contained in the composition, the type of dosage form, the age, weight, general health condition, sex and diet of the person taking the composition, the administration time, administration route, intake period, and concurrently used drugs.

[0069] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples. EXAMPLES

[0070] Example 1: Strain isolation and identification.

[0071] Strain isolation.

[0072] A strain with β-glucosidase activity was isolated from candied ginseng (candied ginseng purchased from a local store) using an esculin-containing medium. Specifically, the esculin-containing medium was an MRS or PDA agar medium containing 0.1% esculin and 0.5% ferric ammonium citrate. 9 ml of sterilized water was added to 1 g of candied ginseng, and the diluted solutions were inoculated onto esculin agar medium and incubated at 30°C for 48 hours to observe the change in the color of the medium (Figure 1). Esculin was separated into glucose and esculetin by β-glucosidase, and esculetin reacted with ferric ammonium citrate to form black complexes around the colonies. Therefore, the strains that formed black spots around the colonies cultured on esculin agar medium were judged to have β-glucosidase activity, and among the strains that formed black spots, the strains that formed relatively strong black spots were first selected (Shim et al., 2014).Then, from the selected strains, the strain with the best ginsenoside bioconversion ability was finally selected.

[0073] Strain identification.

[0074] In order to investigate the molecular biological characteristics of the M-5 strain, chromosomal DNA was isolated from the culture medium of each strain, and the chromosomal DNA was subjected to 26S rRNA gene sequence analysis for microbial identification.

[0075] To this end, the selected M-5 strain was inoculated into PDB liquid medium (Difco, Detroit, Michigan, USA), cultured at 30°C and 150 rpm for 48 hours, and then centrifuged to recover the M-5 strain. DNA was extracted from the recovered M-5 strain using a ZR Fungal / Bacterial DNA Miniprep Kit (Zymo Research Corp., CA, USA), and gene fragments were amplified by PCR, and the base sequence was analyzed by Macrogen Co., Ltd. (Seoul, Korea) (Park and Kim, 2011).

[0076] As a result of the base sequence analysis, the M-5 strain was investigated as a strain belonging to Saccharomyces cerevisiae, and was found to have the base sequence of SEQ ID NO: 1. In addition, as a result of analyzing the base sequence by BLAST search, it was found to have 99.9% homology with S. cerevisiae DSM 70449 and S. cerevisiae RC231, and it was confirmed that there is no existing strain having 100% homology with SEQ ID NO: 1. Therefore, the M-5 strain was named S. cerevisiae M-5. Meanwhile, the base sequence of SEQ ID NO: 1 was compared with the 26S rRNA base sequences of strains with high base sequence homology using NCBI BLAST to generate a phylogenetic tree (Figure 2). The phylogenetic tree was analyzed using the neighbor-joining algorithm (Kumar et al., 2004). To measure the reliability of each calculated phylogenetic tree, bootstrap analysis was repeated 1000 times and the robustness was checked using the MEGA 7.0.26 program.

[0077] The isolated M-5 strain was also biochemically identified using an API20C AUX kit to confirm the carbon source utilization. API20C AUX analysis confirmed that the M-5 strain of the present invention uses D-glucose, D-galactose, D-maltose, D-sucrose, D-trehalose, α-methyl-d-glucoside, D-melezitose, and D-raffinose as carbon sources (Table 1). Previous studies have reported that S. cerevisiae isolated from fermented foods metabolizes D-glucose, D-galactose, D-maltose, and D-melezitose (Kang, 2008; Kim et al., 2013b). S. cerevisiae can degrade sucrose and raffinose by invertase (EC 3.2.1.26), and the isolated M-5 strain also showed similar carbon source utilization as S. cerevisiae.

[0078] [Table 1]

[0079] Example 2: Preparation of fermented ginseng fruit product.

[0080] (1) Preparation of M-5 strain preculture medium.

[0081] The isolated M-5 strain was inoculated into PDB liquid medium and pre-cultured at 30°C and 150 rpm for 12 hours. The M-5 strain culture solution (4.3 × 10 7 cfu / mL) were produced.

[0082] (2) Production of crushed ginseng fruit liquid.

[0083] In addition, ginseng fruit purchased from a ginseng farm (Goesan, Chungbuk, Korea) was crushed in a mixer. Glucose was added to 1000 ml of the crushed ginseng fruit liquid to a volumetric concentration of 1%, and the pH was adjusted to 6.0 and the liquid was sterilized at 90°C for 30 minutes.

[0084] (3) Fermentation stage.

[0085] The sterilized ginseng fruit crushed liquid (solid content 4 wt%) was inoculated with the M-5 strain culture liquid at 4 wt% and cultured at 30° C. for 24 hours to obtain a ginseng fruit fermentation product (6.2×10 7 cfu / mL) were produced.

[0086] <Test example>.

[0087] The experimental results were expressed as the mean ± standard deviation of three repeated measurements. Statistical significance of the experimental groups was confirmed at the p<0.05 level using one-way ANOVA followed by Tukey's multiple range test using the Statistical Package for the Social Science software (SPSS12, SPSS Inc. Chicago, IL, USA).

[0088] Test Example 1: Analysis of changes in components during fermentation.

[0089] The fermented ginseng fruit product of Example 2 was analyzed for changes in pH, reducing sugar, protein, and polyphenol content over the fermentation period.

[0090] The pH of the fermented ginseng fruit was measured using a pH meter (Orion Star A211, Thermo Scientific, Waltham, MA, USA) (Figure 4). Dry weight was calculated by measuring the weight of the sample before and after freeze-drying. Total sugar and reducing sugar were measured by the phenol-sulfuric acid method (DuBois et al., 1956) and the 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959), respectively, with glucose used as the standard. Changes in protein content were measured using the bicinchoninic acid (BCA) method (Smith et al., 1985), with bovine serum albumin used as the standard. Total polyphenol content was measured by measuring the absorbance at 750 nm using the Folin-Ciocalteu reagent (Singleton et al., 1999), and gallic acid was used as a standard substance (Figure 5).

[0091] As shown in FIG. 4 and FIG. 5, the pH of the fermented ginseng fruit product of the present invention gradually decreased as fermentation proceeded, but gradually increased from the fourth day of fermentation, and the pH was 5.97 on the seventh day of fermentation, which was similar to the initial pH of 6.0. The number of live bacteria showed a tendency to increase rapidly on the first day of fermentation and gradually increase from the second day. The content of reducing sugars gradually decreased from the initial 163.4 μg / mg to 22.7 μg / mg on the seventh day of fermentation as the fermentation time increased. This is because the carbon source was used for the growth of the microorganisms as the fermentation proceeded. In addition, the protein content increased only on the first day of fermentation and decreased from the second day. The polyphenol content was similar to the protein content, and increased only on the first day of fermentation and decreased from the second day.

[0092] Test Example 2: Antioxidant activity during fermentation - radical scavenging ability.

[0093] The antioxidant activity of the fermented ginseng fruit product of Example 2 was analyzed based on the DPPH radical scavenging ability and ABTS radical scavenging ability.

[0094] Radical scavenging activity using 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical was measured by slightly modifying the method of Quang et al. (2003). 100 μl of DPPH solution (0.2 mM) was added to 100 μl of the fermented ginseng fruit product of Example 2 and mixed well. The mixture was left in a dark place at room temperature (30° C.) for 30 minutes, and the absorbance was measured at 520 nm.

[0095] The radical scavenging ability using ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) radical was measured by the method of Re et al. (1999). ABTS and potassium persulfate were mixed to final concentrations of 7.4 mM and 2.6 mM, respectively, and left in the dark at 37°C for 24 hours to form ABTS radical. 200 μl of ABTS radical solution was added to 10 μl of the fermented ginseng fruit product of Example 2 and mixed well, and the absorbance was measured at 414 nm. The radical scavenging ability was measured as the IC value of the sample concentration required to reduce radicals by 50%. 50 The values ​​are expressed as values ​​(Figure 6).

[0096] As shown in FIG. 6, the IC showing the DPPH and ABTS radical scavenging ability of the fermented ginseng fruit product of Example 2 50 The value decreased sharply on the first day of fermentation, and then showed a tendency to increase from the second day of fermentation onwards. Such a result is consistent with the change in polyphenol content during fermentation.

[0097] Test Example 3: Ginsenoside content analysis.

[0098] The change in ginsenoside content in the fermented ginseng fruit product of Example 2 was analyzed.

[0099] To analyze the ginsenoside content of the fermented ginseng fruit, the fermented ginseng fruit was subjected to alcohol extraction. Specifically, 4 times the weight of alcohol was added to the fermented ginseng fruit of Example 2, and the mixture was refluxed at 90°C for 2 hours twice to extract the alcohol. The alcohol extract was collected and used for analysis.

[0100] The ginsenoside contents were analyzed by the existing experimental method (Lee et al., 2009) using an HPLC system (Agilent, Waldbronn, Germany) equipped with a UV detector, and are shown in Table 2 below. The column used was a Cadenza CD-C18 (75 x 4.6 mm, 3 μm, Imtakt Corporation, Kyoto, Japan) treated with a Sep-Pak C18 plus cartridge. The analysis was performed under the following analytical conditions: UV wavelength 203 nm; flow rate 1.2 ml / min; injection volume 5 μl; and column temperature 40°C. The analysis was performed using solvent A (10% acetonitrile) and solvent B (90% acetonitrile). Ginsenosides Rg2 and Rg3 each have S-type and R-type optical isomers in their chemical structures, so the respective contents were calculated as the sum of S-type and R-type.

[0101] [Table 2]

[0102] In the above table, each value is shown as the mean ± SD, and *, **, and *** indicate significant differences at p<0.05, p<0.01, and p<0.001, respectively, when compared with the value on day 0 by Tukey test.

[0103] In the above table, "A" means the sum of ginsenosides Rg3, Rh4, Rk1, Rk3, Rg5 and Rg6 (Rg3+Rh4+Rk1+Rk3+Rg5+Rg6).

[0104] Among the ginsenosides contained in the fermented ginseng fruit product of the present invention, the content of protopanaxatriol ginsenoside Re was 62.86μg / mg before fermentation, which was significantly higher than the contents of other ginsenosides, and increased to 76.60μg / mg on the 7th day of fermentation. The total content of ginsenoside Rg2, a metabolite of ginsenoside Re, increased from 4.72μg / mg before fermentation to 13.68μg / mg on the 7th day of fermentation. In addition, ginsenoside Rg1 increased slightly from 4.5μg / mg before fermentation to 6.25μg / mg on the 7th day of fermentation. Among the protopanaxadiol ginsenosides, ginsenosides Rb1 and Rb2, which are the major ginsenosides, increased from 2.10μg / mg to 6.62μg / mg and from 5.02μg / mg to 16.10μg / mg on the 7th day of fermentation compared to the first day of fermentation, respectively. The metabolites of ginsenosides Rd, Rc and Rg3 also increased from 7.58μg / mg to 26.17μg / mg, 4.74μg / mg to 13.77μg / mg and 0μg / mg to 5.22μg / mg on the 7th day of fermentation compared to the first day of fermentation, respectively. Rare ginsenosides Rh4, Rg3, Rk1 and Rg5 were not detected before fermentation. However, they increased to 0.78, 3.38, 1.84, 0.69 and 1.49μg / mg on the 7th day of fermentation, respectively. The sum of ginsenosides Rb1, Rg1 and Rg3 also increased significantly from 4.51μg / mg before fermentation to 18.09μg / mg on the 7th day of fermentation.

[0105] On the other hand, from the above results, it was found that as the fermentation of the ginseng fruit powder by the yeast isolated according to the present invention progresses, low molecular weight ginsenosides are produced through the bioconversion reaction of high molecular weight ginsenosides, and at the same time, the pectinase secreted breaks down the pectin present in the ginseng fruit cell walls, thereby increasing the extraction efficiency of ginsenosides present in the ginseng fruit, and promoting the decomposition of ginsenoside precursors such as malonyl ginsenoside Rb1 component, thereby increasing the total ginsenoside content.

[0106] Test Example 4: Systemic Absorption Rate Analysis - Intestinal Absorption Rate.

[0107] The intestinal permeability (intestinal absorption rate) of polyphenols contained in the fermented ginseng fruit product of Example 2 was compared before and after fermentation.

[0108] The intestinal absorption rate of fermented ginseng fruit was measured using the intestinal sac of mice according to a conventional method (Shim et al., 2014). Specifically, 8-week-old male Sprague Dawley rats were fasted for 24 hours and then administered CO 2 After sacrifice under anesthesia, the small intestine was quickly removed and washed, and 10 cm of the beginning of the jejunum was collected. The intestine was turned inside out using a glass rod, one end was tied with surgical thread, 1 μl of 0.1% glucose-containing KHB (Krebs-Henseleit bicarbonate) buffer (pH 7.4) was placed in the serosal fluid (inner compartment), and the other end was tied to form an intestinal pouch. The intestinal pouch was placed in a mucosal fluid (outer compartment) containing 1 μl of the fermented ginseng fruit product of Example 2 and 20 μl of KHB buffer. The animal experiment was approved by the Animal Experiment Research Committee of Korea University (KUIACUC-2020-0062).

[0109] During the experiment, the temperature was maintained at 37 °C using a shaking water bath and 5% CO 2 and 95% O 2 The ginseng fruit was continuously fed with fermented ginseng. Samples infiltrated into the intestinal sac were collected at 30, 60, 120, and 240 minutes, and the change in total polyphenol content was measured using Folin-Ciocalteu reagent. After 240 minutes of intestinal permeation, the polyphenol content of the ginseng fruit infiltrated into the intestinal sac before and after fermentation was measured using HPLC (Figures 7A and 7B). Specifically, an existing method (Han et al., 2021) was modified and analyzed using an Agilent HPLC system (Agilent, Waldbronn, Germany) equipped with a UV detector.

[0110] As a reference, a YMC triart C18 column (250 × 4.6 mm, 5 μm, Sungnam, Korea) was used, and the mobile phases were 0.2% formic acid in water (solvent A) and 0.2% formic acid in acetonitrile (solvent B). The gradient conditions were as follows: 5% B from 0 to 2 min, 5% B to 25% B from 2 to 10 min, 25% B to 40% B from 10 to 30 min, 40% B to 50% B from 30 to 40 min, 50% B to 60% B from 40 to 50 min, 60% B to 5% B from 55 to 60 min, and 5% B from 60 to 65 min. The flow rate was 0.8 min / ml, and the injection volume was 20 μl. The detector wavelengths were measured at 260 nm for gallic acid, 292 nm for 3,4-dihydroxybenzoic acid, rutin, and ρ-coumaric acid, and 310 nm for chlorogenic acid, caffeic acid, and trans-ferulic acid.

[0111] As shown in FIG. 7A, the polyphenol content in the sample permeated into the intestinal pouch was found to increase over time. It was confirmed that the intestinal permeability of polyphenols in the fermented ginseng fruit product of Example 2 was significantly higher than that before fermentation. In addition, after 240 minutes of intestinal permeation, the polyphenol content passing through the intestinal pouch was found to have increased rapidly. It was confirmed that after 240 minutes of intestinal permeation, the polyphenol content such as gallic acid, dihydroxybenzoic acid, rutin, caffeic acid, coumaric acid, and ferulic acid in the fermented ginseng fruit product of Example 2 permeated into the intestinal pouch was significantly higher than that before fermentation. It was also confirmed that the total polyphenol content of the fermented ginseng fruit sample before fermentation permeated into the intestinal pouch was 0.37 μg / mg, whereas the total polyphenol content of the fermented ginseng fruit product of Example 2 was 0.46 μg / mg, which was significantly increased.

[0112] In addition, after 240 min of intestinal permeation, the ginsenoside contents of the ginseng fruit samples before and after fermentation that had permeated the intestinal pouch were measured using HPLC (Table 3).

[0113] [Table 3]

[0114] In the above table, each value is shown as the mean ± SD, and * and *** indicate significant differences at p<0.05 and p<0.001, respectively, when compared with the value before fermentation by Tukey test.

[0115] From Table 3, it can be seen that the content of all ginsenosides in the fermented ginseng fruit product of Example 2 that had permeated into the intestinal pouch was significantly higher than that of the sample before fermentation. In particular, the intestinal permeation amount of ginsenoside Re, which is contained in large amounts in ginseng fruit, was 4.28 μg / mg before fermentation and increased by about 40% to 5.95 μg / mg after fermentation with the M-5 strain of the present invention. In addition, it was found that the intestinal permeability of ginsenosides Rh1, Rg2 and Rb1 contained in ginseng fruit increased by more than two-fold compared to that before fermentation when fermented with the M-5 strain of the present invention.

[0116] From the above results, it was specifically confirmed that the ginseng fruit fermentation product using the Saccharomyces cerevisiae M-5 strain, which has the ability to bioconvert the saponin of the present invention, has significantly superior intestinal permeability (absorption rate) of polyphenols and ginsenosides compared to non-fermented ginseng fruit crushed liquid, has a higher content of total ginsenosides and rare ginsenosides, and exhibits excellent ginsenoside indicators.

[0117] Although the present invention has been described above as the preferred embodiment, various modifications and variations are possible without departing from the spirit and scope of the invention, and the appended claims include such modifications and variations that fall within the spirit and scope of the invention.

[0118] [Accession number] Depository institution: Korea Center for Culture Collection of Microorganisms (KCCM) Accession number: KCCM 13375P Date of acceptance: 20230801

Claims

1. Saccharomyces cerevisiae M-5 strain (Accession No.: KCCM 13375P) having biotransformation ability for saponin.

2. The strain according to claim 1, characterized in that the strain is isolated from candied ginseng.

3. The strain according to claim 1, which uses glucose, galactose, α-methyl-d-glucoside, maltose, sucrose, trehalose, melezitose or raffinose as a carbon source.

4. A method for producing a fermented ginseng fruit product having increased contents of total ginsenosides and rare ginsenosides, comprising the steps of inoculating a crushed ginseng fruit liquid with Saccharomyces cerevisiae M-5 strain (Accession No.: KCCM 13375P) and then fermenting the liquid.

5. The method for producing a fermented ginseng fruit product having increased contents of total ginsenosides and rare ginsenosides according to claim 4, characterized in that the pH of the crushed ginseng fruit liquid is adjusted to 5 to 7.

6. The method for producing a fermented ginseng fruit product having increased contents of total ginsenosides and rare ginsenosides according to claim 4, wherein the fermentation is carried out at 25-35°C for 12 hours to 10 days.

7. A method for producing a ginsenoside- and rare ginsenoside-enhanced ginsenosides-containing fermented ginseng fruit product as described in claim 4, characterized in that the total ginsenoside content of the fermented ginseng fruit product is at least 50% higher than that of the ginsenosides-containing ...

8. The method for producing a ginseng fruit fermentation product having increased contents of total ginsenosides and rare ginsenosides as described in claim 4, characterized in that the ginseng fruit fermentation product contains ginsenosides Rg3, Rh4, Rk1 and Rg5 that are not present in the ginseng fruit crushed liquid before fermentation.

9. The method for producing a fermented ginsenoside of ginsenosides having increased contents of total ginsenosides and rare ginsenosides, as described in claim 4, characterized in that the rare ginsenosides are one or more selected from ginsenosides Rg3, Rh4, Rk1, Rk3, Rg5 and Rg6.

10. The method for producing a ginseng fruit fermentation product having increased content of total ginsenosides and rare ginsenosides as described in claim 4, characterized in that the sum of rare ginsenosides Rg3, Rh4, Rk1, Rk3, Rg5 and Rg6 is at least 10 μg / mg.

11. The method for producing a ginsenoside Re fermented ginseng fruit product having increased contents of total ginsenosides and rare ginsenosides, as described in claim 4, characterized in that the ginsenoside Re content of the ginsenoside Re fermented ginseng fruit product is at least 65 μg / mg.

12. The fermented ginseng fruit product is the sum of ginsenosides Rb1, Rg1 and Rg3 is at least 10 μg / mg; A content of ginsenoside Rg2 of at least 12 μg / mg; A content of ginsenoside Rc of at least 8 μg / mg; A content of ginsenoside Rb2 of at least 9 μg / mg; and The method for producing the fermented ginseng fruit product with increased contents of total ginsenosides and rare ginsenosides according to claim 4, characterized in that the content of ginsenoside Rd is at least 12 μg / mg.

13. A fermented ginseng fruit product produced by the method of any one of claims 4 to 12, characterized in that the sum of ginsenosides Rb1, Rg1 and Rg3 is at least 10 μg / mg, and the product contains ginsenosides Rg3, Rh4, Rk1 and Rg5 that are not present in the ginseng fruit crushed liquid before fermentation.

14. A functional health food composition comprising the fermented ginseng fruit product of claim 13 as an active ingredient.

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