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

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

[0003] Ginsenosides can be classified into three types based on the structure of the ginsenoside aglycone: protopanaxadiol-type ginsenosides, protopanaxatriol-type ginsenosides, and oleanane-type ginsenosides. Ginsenoside derivatives are compounds in which sugars such as glucose, rhamnose, xylose, and arabinose are ester-linked to the alcoholic OH groups (R1, R2, and R3) of the non-sugar triterpene moiety (protopanaxadiol or protopanaxatriol). The major ginsenosides currently known in ginseng include approximately 13 common ginsenosides and approximately 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 found in these processed ginseng.

[0006] On the other hand, ginseng leaves and fruits contain more ginsenosides than the roots, which are primarily consumed (Chung et al., 2016; Yang et al., 2021). Ginseng fruits have approximately three times the ginsenoside content of the roots, and contain high amounts of ginsenosides Re and Rd (Ko et al., 2008). Furthermore, ginseng leaves and fruits contain higher amounts of polyphenols than the roots, and exhibit superior antioxidant activity (Kim and Ko, 2020).

[0007] The present inventors have conducted extensive research to develop a ginseng fruit fermented product with increased total ginsenoside and rare ginsenoside contents using ginseng fruit, which is rich in ginsenosides and polyphenolic 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 not only has significantly increased total ginsenoside and rare ginsenoside contents and an excellent ginsenoside index, but also significantly increases the 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 for 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 ginseng fruit 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] A further object of the present invention is 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 number: 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 an increased content of total ginsenosides and rare ginsenosides, which includes a step 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 ginseng fruit crushed 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 that is increased by at least 50% compared to the ginseng fruit crushed 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, which 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 ginseng fruit product 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 content of the fermented ginseng fruit may be at least 10 μg / mg, the content of ginsenoside Rg2 may be at least 12 μg / mg, the content of ginsenoside Rc may be at least 8 μg / mg, the content of ginsenoside Rb2 may be at least 9 μg / mg, and the content of ginsenoside Rd may be at least 12 μg / mg.

[0025] In addition, in order to achieve yet another of the above-mentioned objects, the present invention provides a fermented ginseng fruit product produced by the above-mentioned method, which has a total content of ginsenosides Rb1, Rg1 and Rg3 of at least 10 μg / mg, and which contains 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 as an active ingredient. [Effects of the Invention]

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

[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 excellent ginsenoside index, and contains rare ginsenosides, making it extremely useful as a functional health food or food ingredient. [Brief explanation of the drawings]

[0029] [Figure 1] These are photographs showing the color change observed after inoculating and culturing strains on esculin-containing MRS agar medium (Fig. 1A) and PDA agar medium (Fig. 1B) to select strains having β-glucosidase activity. [Figure 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. [Figure 3] 1 is a flowchart briefly illustrating a method for producing a fermented ginseng fruit product according to one embodiment of the present invention. [Figure 4] 1 is a graph showing changes in absorbance and pH over time of a fermented ginseng fruit product according to an embodiment of the present invention. [Figure 5]1 is a graph showing changes in pH, reducing sugar, protein and 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 time in a fermented ginseng fruit product according to an example of the present invention. [Figure 7A] 1 is a graph showing the intestinal absorption rate of total polyphenols over time 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, compared with the sample before fermentation. DETAILED DESCRIPTION OF THE INVENTION

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

[0031] In this 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 fermented product. The ginseng fruit fermented product of the present invention in which these indexes exceed 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] As used herein, "rare ginsenoside" refers to a ginsenoside that is contained in trace amounts, almost none, or none at all in raw ginseng or ginseng fruit that has not undergone any additional processing. According to the production method of the present invention, ginsenosides that are mainly contained in raw ginseng fruit crushed liquid that has not undergone any additional processing are converted to rare ginsenosides 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, and more preferably ginsenosides Rg3, Rh4, Rk1, and Rg5.

[0033] One aspect of the present invention relates to a Saccharomyces cerevisiae M-5 strain (accession number: KCCM 13375P) that has the ability to biotransform saponin.

[0034] In the following example, the 26S rRNA base sequence of the M-5 strain, a strain derived from candied ginseng stored at the headquarters (BTC Corporation) research institute, was analyzed for microbial identification and classification, and it was confirmed to have 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. The nucleic acid sequence of the strain was submitted to Macrogen for homology testing, and it was confirmed to be 99.9% identical to the reference strains of Saccharomyces cerevisiae DSM70449 and Saccharomyces cerevisiae RC231. In the nucleic acid sequence of SEQ ID NO: 1, "n" represents a base that is "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 strain uses glucose, galactose, α-methyl-d-glucoside, maltose, sucrose, trehalose, melezitose, and / or raffinose as a carbon source.

[0038] The Saccharomyces cerevisiae M-5 strain of the present invention has the ability to biotransform saponins, particularly to convert saponins into ginsenosides and high molecular weight ginsenosides into ginsenosides, especially rare ginsenosides, with increased intestinal absorption rates in the human body, making it very useful for producing fermented ginseng fruit products.

[0039] In addition, the Saccharomyces cerevisiae M-5 strain of the present invention converts ginseng fruit saponins 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 fermented ginseng fruit products with increased contents of specific ginsenosides, 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 an increased content of total ginsenosides and rare ginsenosides, which includes a step of inoculating a crushed ginseng fruit liquid with Saccharomyces cerevisiae M-5 strain (Accession No.: 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, to enhance the efficacy of ginsenosides, a process is required to convert them into minor ginsenosides, which are relatively well absorbed and have better efficacy. In other words, a conversion process that removes glucose is required for major ginsenosides to effectively demonstrate their physiological activity in the body.

[0042] Therefore, the inventors selected a bacterial strain with excellent biotransformation ability for saponins, 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 fermented product with increased contents of total ginsenosides and rare ginsenosides and increased intestinal absorption rates 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 pulverized 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 this specification, the ginseng fruit crushed liquid includes not only the crushed liquid obtained by crushing ginseng fruit, but also processed products of the crushed ginseng fruit liquid. For example, the crushed ginseng fruit liquid may be produced as a powder or a concentrated liquid by further processes such as vacuum distillation, freeze-drying, or spray-drying.

[0047] In addition, it is preferable to add sugars to the crushed ginseng fruit 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 with the Saccharomyces cerevisiae M-5 strain of the present invention. Furthermore, the crushed ginseng fruit liquid may be the pH-adjusted crushed ginseng fruit liquid to which sugars have been added.

[0048] If the pH of the ginseng fruit crushed 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 sterilizing the liquid 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, more preferably 1.0 x 10 7 ~1.0×10 8 It may be cfu / mL.

[0052] If the amount of the strain to be inoculated is less than the lower limit, the fermentation rate may be slowed, whereas if the amount is more than 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 ginseng fruit crushed liquid with the Saccharomyces cerevisiae M-5 strain, 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 with various bacteria, and if the fermentation temperature is above 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 the fermentation time is above the upper limit, the ginsenosides produced by fermentation may begin to decompose, resulting in a decrease in the content.

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

[0056] Then, the fermented ginseng fruit product can be heated at 85 to 95°C for 10 to 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 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] Furthermore, the sum of ginsenosides Rg3, Rh4, Rk1, Rk3, Rg5 and Rg6, which are rare ginsenosides contained in the ginseng fruit fermented product of the present invention, may be at least 10 μg / mg, preferably 10 to 20 μg / mg, more preferably 13 to 20 μg / mg, and even more preferably 14 to 18 μg / mg. The amount of rare ginsenosides contained in the ginseng fruit fermented product is increased by about 15 to 40 times, more specifically about 20 to 40 times, even more specifically about 25 to 35 times, and particularly specifically about 28 to 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] Furthermore, the total content 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; and the content of ginsenoside Rc is at least 8 μg / mg, preferably 10 μg / mg or more, and particularly preferably 18 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 may be at least 12 μg / mg, preferably 15 μg / mg or more, more preferably 15 to 30 μg / mg, 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 method, characterized in that the sum of ginsenosides Rb1, Rg1 and Rg3 is at least 15 μg / mg, and the product contains ginsenosides Rg3, Rh4, Rk1 and Rg5, which 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" (Ministry of Food and Drug Safety Notification No. 2016-143, December 21, 2016) does not mention ginseng fruit, but states in the section on ginseng item and index component content that the standard value is "0.8 to 34 mg / g of ginsenosides Rg1 and Rb1 combined," and in the section on ginseng item and index component content that the standard value is "2.5 to 34 mg / g of ginsenosides Rg1, Rb1, and Rg3 combined." The document also states in the section on "Requirements for Final Products" that the final product should functionally meet the following requirements: "Immunity enhancement, fatigue relief, blood flow / memory improvement by inhibiting platelet aggregation, antioxidant properties, and ability to support the health of menopausal women." 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 to 80 mg / g, and in order to help "improve blood flow / memory by inhibiting platelet aggregation, and as an antioxidant," the total amount of index components must be 2.4 to 80 mg / g.

[0065] As can be seen from the examples below, the ginseng fruit fermented product of the present invention has an excellent ginsenoside composition, since it contains rare ginsenosides such as ginsenosides Rg3, Rh4, Rk1, Rk3, Rg5 and Rg6 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 containing 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, granules, tablets, capsules, syrup, or beverage. In addition to the active ingredient, other foods or food additives may also be used in the health functional food composition, and the composition may be used appropriately by a conventional method. The amount of the active ingredient may be appropriately determined depending on the intended use, such as preventive, ameliorative, or therapeutic treatment. In 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 content 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 user, 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. [Example]

[0070] Example 1: Isolation and identification of strains.

[0071] Strain isolation.

[0072] Strains with β-glucosidase activity were isolated from candied ginseng (candied ginseng purchased from a local store) using esculin-containing medium. Specifically, the esculin-containing medium was MRS or PDA agar containing 0.1% esculin and 0.5% ferric ammonium citrate. 1 g of candied ginseng was added to 9 ml of sterile water, and serial 10-fold dilutions were inoculated onto esculin agar medium. The medium was incubated at 30°C for 48 hours, and the color change of the medium was observed (Figure 1). Esculin was separated into glucose and esculetin by β-glucosidase, and esculetin reacted with ferric ammonium citrate to form black spots (black complexes) around the colonies. Therefore, strains that formed black spots around colonies cultured on esculin agar medium were determined to have β-glucosidase activity, and strains that formed relatively strong black spots were first selected from the strains that formed black spots (Shim et al., 2014).Then, from the selected strains, the strain with the best ginsenoside bioconversion ability was finally selected.

[0073] Strain identification.

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

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

[0076] Based on the nucleotide sequence analysis, the M-5 strain was identified as a strain belonging to Saccharomyces cerevisiae and was found to have the nucleotide sequence set forth in SEQ ID NO: 1. Furthermore, a BLAST search of the nucleotide sequence revealed 99.9% identity with S. cerevisiae DSM 70449 and S. cerevisiae RC231, confirming that no strains with 100% identity to SEQ ID NO: 1 exist. Therefore, the M-5 strain was designated S. cerevisiae M-5. The nucleotide sequence set forth in SEQ ID NO: 1 was compared with the 26S rRNA sequences of strains with high nucleotide sequence homology using NCBI's 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 confirmed using the MEGA 7.0.26 program.

[0077] The isolated M-5 strain was also biochemically identified using an API20C AUX kit to confirm carbon source utilization. API20C AUX analysis confirmed that the M-5 strain of the present invention utilizes 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 metabolize D-glucose, D-galactose, D-maltose, and D-melezitose (Kang, 2008; Kim et al., 2013b). S. cerevisiae can degrade sucrose and raffinose using an invertase (EC 3.2.1.26), and the isolated M-5 strain exhibited similar carbon source utilization as S. cerevisiae.

[0078] [Table 1]

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

[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 berries purchased from a ginseng farm (Goesan, Chungbuk, South Korea) were crushed in a mixer. Glucose was added to 1000 ml of the crushed ginseng berries to a concentration of 1% by volume, and the pH was adjusted to 6.0 and 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 solution at 4 wt% and cultured at 30°C for 24 hours to obtain a ginseng fruit fermented product (6.2 × 10 7 cfu / mL) were produced.

[0086] <Test example>.

[0087] Experimental results were expressed as the mean ± standard deviation of triplicate measurements. Statistical significance of the experimental groups was confirmed at a 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 changes in pH, reducing sugar, protein, and polyphenol content of the fermented ginseng fruit product of Example 2 over the fermentation period were analyzed.

[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 using 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 absorbance at 750 nm using the Folin-Ciocalteu reagent (Singleton et al., 1999), and gallic acid was used as a standard (Figure 5).

[0091] As shown in Figures 4 and 5, the pH of the fermented ginseng fruit product of the present invention gradually decreased as fermentation progressed, but gradually increased from the fourth day onward. By the seventh day of fermentation, the pH was 5.97, similar to the initial pH of 6.0. The viable cell count increased rapidly on the first day of fermentation and then gradually increased from the second day onward. The reducing sugar content gradually decreased with increasing fermentation time, from an initial value of 163.4 μg / mg to 22.7 μg / mg by the seventh day of fermentation. This is due to the use of carbon sources for microbial growth as fermentation progresses. Furthermore, the protein content increased only on the first day of fermentation and decreased from the second day onward. The polyphenol content, similar to the protein content, increased only on the first day of fermentation and decreased from the second day onward.

[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-picrylhydrazyl (DPPH) radicals was measured using a slightly modified method by Quang et al. (2003). 100 μl of 0.2 mM DPPH solution was added to 100 μl of the fermented ginseng fruit product of Example 2 and mixed thoroughly. The mixture was then left in the dark at room temperature (30°C) for 30 minutes, after which the absorbance was measured at 520 nm.

[0095] The radical scavenging activity 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 then the absorbance was measured at 414 nm. The radical scavenging activity was measured as the IC value of the sample concentration required to reduce radicals by 50%. 50 It is expressed as a value (Figure 6).

[0096] As shown in FIG. 6, the IC scavenging ability of the fermented ginseng fruit product of Example 2 was 50 The values ​​decreased sharply on the first day of fermentation, but then showed a tendency to increase from the second day onwards, which is consistent with the change in polyphenol content during fermentation.

[0097] Test Example 3: Analysis of ginsenoside content.

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

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

[0100] The ginsenoside content was analyzed using an HPLC system (Agilent, Waldbronn, Germany) equipped with a UV detector according to a conventional experimental method (Lee et al., 2009). The results are shown in Table 2. The column used was a Cadenza CD-C18 (75 × 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. Analysis was performed using solvent A (10% acetonitrile) and solvent B (90% acetonitrile). Ginsenosides Rg2 and Rg3 each have optical isomers, S-type and R-type, respectively, and their respective contents were calculated as the sum of the S-type and R-type.

[0101] [Table 2]

[0102] In the above table, each value is shown as mean ± SD, and *, **, and *** indicate significant differences of p<0.05, p<0.01, and p<0.001, respectively, when compared with the value on day 0 by Tukey's 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 ginsenoside fruit of the present invention, the content of protopanaxatriol-type ginsenoside Re was 62.86 μg / mg before fermentation, significantly higher than the contents of other ginsenosides, and increased to 76.60 μg / mg by the seventh day of fermentation. The total content of ginsenoside Rg2, a metabolite of ginsenoside Re, increased from 4.72 μg / mg before fermentation to 13.68 μg / mg by the seventh day of fermentation. In addition, ginsenoside Rg1 increased slightly from 4.5 μg / mg before fermentation to 6.25 μg / mg by the seventh day of fermentation. Among the protopanaxadiol-type ginsenosides, ginsenosides Rb1 and Rb2, which are the major ginsenosides, increased from 2.10 μg / mg to 6.62 μg / mg and from 5.02 μg / mg to 16.10 μg / mg, respectively, by the seventh day of fermentation compared to the first day of fermentation. The concentrations of their metabolites, ginsenosides Rd, Rc, and Rg3, also increased from 7.58 μg / mg to 26.17 μg / mg, 4.74 μg / mg to 13.77 μg / mg, and 0 μg / mg to 5.22 μg / mg, respectively, on the seventh day of fermentation compared to the first day. The rare ginsenosides Rh4, Rg3, Rk1, and Rg5 were not detected before fermentation, but increased to 0.78, 3.38, 1.84, 0.69, and 1.49 μg / mg, respectively, on the seventh day of fermentation. The sum of ginsenosides Rb1, Rg1, and Rg3 also significantly increased from 4.51 μg / mg before fermentation to 18.09 μg / mg on the seventh day of fermentation.

[0105] On the other hand, from the above results, it was found that as the ginseng fruit powder is fermented by the yeast isolated according to the present invention, 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 malonylginsenoside 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 ginseng fruit fermentation product was measured using rat intestinal sacs according to a previously published method (Shim et al., 2014). Specifically, 8-week-old male Sprague-Dawley rats were fasted for 24 hours and then sacrificed under CO2 anesthesia. The small intestine was rapidly removed and washed, and the initial 10 cm of the jejunum was collected. The intestine was turned inside out using a glass rod, and one end was tied with surgical suture. 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 sac. The intestinal sac was placed in the mucosal fluid (outer compartment) containing 1 μl of the ginseng fruit fermentation product of Example 2 and 20 μl of KHB buffer. The animal experiments were approved by the Animal Experimentation and 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% CO₂ and 95% O₂ were continuously supplied. Samples were collected at 30, 60, 120, and 240 minutes after infiltration into the intestinal sac, and changes in total polyphenol content were measured using the Folin-Ciocalteu reagent. After 240 minutes of intestinal permeation, the polyphenol content of the ginseng berries infiltrated into the intestinal sac before and after fermentation was measured using HPLC (Figures 7A and 7B). Specifically, a modified version of a previous method (Han et al., 2021) was used for analysis 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. 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 Figure 7A, the polyphenol content in the sample permeated into the intestinal sac increased 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. Furthermore, after 240 minutes of intestinal permeation, a rapid increase in the content of polyphenols passing through the intestinal sac was observed. After 240 minutes of intestinal permeation, it was confirmed that the content of polyphenols 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 sac was significantly higher than that before fermentation. Furthermore, it was confirmed that the total polyphenol content of the fermented ginseng fruit sample permeated into the intestinal sac was 0.37 μg / mg, while the total polyphenol content of the fermented ginseng fruit product of Example 2 was 0.46 μg / mg, a significant increase.

[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 sac were measured using HPLC (Table 3).

[0113] [Table 3]

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

[0115] Table 3 confirms that the content of all ginsenosides in the ginseng fruit fermented product of Example 2 that had permeated the intestinal sac 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 approximately 40% to 5.95 μg / mg after fermentation with the M-5 strain of the present invention. In addition, the intestinal permeation rates of ginsenosides Rh1, Rg2, and Rb1 contained in ginseng fruit were found to be more than two-fold higher when fermented with the M-5 strain of the present invention compared to before fermentation.

[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 biotransform the saponin of the present invention, has significantly better intestinal permeability (absorption rate) of polyphenols and ginsenosides than non-fermented ginseng fruit crushed liquid, and has a higher content of total ginsenosides and rare ginsenosides, thereby showing excellent ginsenoside indicators.

[0117] Although the present invention has been described above with reference to the preferred embodiment, various modifications and variations can be made without departing from the spirit and scope of the invention, and the appended claims include all such modifications and variations that fall within the spirit and scope of the invention.

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

Claims

1. Saccharomyces cerevisiae M-5 strain (accession number: KCCM 13375P) has the ability to biotransform saponins.

2. The strain according to claim 1, wherein the strain is isolated from candied ginseng.

3. 2. The strain of claim 1, wherein the strain 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 with increased contents of total ginsenosides and rare ginsenosides, comprising the steps of inoculating a crushed ginseng fruit solution with Saccharomyces cerevisiae M-5 strain (Accession No.: KCCM 13375P) and then fermenting the solution.

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

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

7. A method for producing a ginseng fruit fermented product with increased contents of total ginsenosides and rare ginsenosides, as described in claim 4, characterized in that the total ginsenoside content of the ginseng fruit fermented product is increased by at least 50% compared to the ginseng fruit crushed liquid before fermentation.

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

11. The method for producing a ginseng fruit fermented 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 is at least 65 μg / mg.

12. The fermented ginseng fruit product 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 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.

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