KR102668553B1 - Lactobacillus pentosus KK18 with biolofical conversion activity of saponin, a method of preparation for composition of fermented sprout ginseng having increased content of ginsenoside Re using the same, fermented red ginseng extracts using thereby and food composition containing the same - Google Patents

Abstract

The present invention relates to a Lactobacillus pentosus KK18 strain having bioconversion ability for saponin, a method for producing a fermented ginseng composition with an increased content of ginsenoside Re using the same, a fermented ginseng sprout composition according to the same, and a food composition containing the same. It's about.
The Lactobacillus pentosus strain of the present invention is food-safe and has an excellent ability to bioconvert saponins contained in ginseng sprouts into ginsenosides Rg2, Re, and F2, which exist only in very trace amounts in ginseng sprouts. . In particular, it makes it possible to produce a fermented ginseng sprout composition with a significantly increased content of ginsenoside Re.
In addition, according to the method for producing a fermented ginseng sprout composition of the present invention, a composition with a significantly increased content of ginsenoside Re can be obtained.
The fermented ginseng sprout composition with increased content of ginsenoside Re is useful for the production of a food composition for improving learning and memory, and enhancing cognitive function.

Description

Lactobacillus pentosus KK18 strain having bioconversion ability for saponin, method for producing a fermented ginseng sprout composition with increased content of ginsenoside Re using the same, fermented ginseng sprout composition resulting therefrom, and food composition containing the same {Lactobacillus pentosus KK18 with biolofical conversion activity of saponin, a method of preparation for composition of fermented sprout ginseng having increased content of ginsenoside Re using the same, fermented red ginseng extracts using thereby and food composition containing the same}

The present invention relates to a Lactobacillus pentosus KK18 strain having bioconversion ability for saponin, a method for producing a fermented ginseng composition with an increased content of ginsenoside Re using the same, a fermented ginseng sprout composition according to the same, and a food composition containing the same. It's about.

The roots of ginseng are mainly used as herbal medicine or as an ingredient in health functional foods. In the case of ginseng sprouts, it is possible to use the entire part for food without discarding any parts, and it can be consumed raw like ssam vegetables or geotjeori, so it is attracting a lot of attention. there is.

The above-mentioned ginseng sprout is a type of ginseng that is usually cultivated for 1 to 2 years. The leaves and stems contain about 7 to 8 times more saponin than the roots. Due to hydroponic cultivation, year-round production, and shortened cultivation period, it is not conventionally grown. It has the advantage of being cheaper than the root ginseng used. However, compared to root ginseng, ginseng sprouts have the disadvantage of containing some components of ginsenosides, especially ginsenoside Re, in very low amounts.

In this regard, it has recently been reported that when ginsenoside Re is ingested in animals, it is effective in improving learning, memory and imagery functions (Tradit Chin Drug Res Clin Pharm, 2007: 18, Pharmacol Biochem Behav, 2012: 101).

In addition, in Patent Document 1, consumption of ginseng fruit extract with increased ginsenoside Re content improves learning function and increases biological markers of indications related to memory formation, improves antioxidant function, and, based on the cholinergic hypothesis, improves the It discloses that it helps improve learning and memory, and enhances cognitive function by inhibiting acetylcholinesterase, an enzyme responsible for the breakdown of acetylcholine.

Additionally, Patent Document 2 discloses a method of increasing the functionality of ginseng sprouts by fermenting them with a mixed strain of Lactobacillus plantarum and Lactobacillus brevis.

The present inventors were studying a method to increase the content of ginsenoside components, especially ginsenoside Re, in ginseng sprouts in order to improve the disadvantage of the relatively low content of effective bioactive ingredients in ginseng sprouts, which can be purchased at low cost. Among them, it was discovered that the ginsenoside Re content could be increased by fermenting ginseng sprouts with Lactobacillus pentosus KK18 strain isolated from kimchi, and the present invention was completed.

KR 10-2027909 B KR 10-2019-0046245 A

The problem to be solved by the present invention is to provide Lactobacillus pentosus KK18 strain (Accession number: KCCM12762P) having bioconversion ability for saponin.

Another object of the present invention is to provide a method for producing a fermented ginseng sprout composition with increased content of ginsenoside R3 using the above strain.

Another object of the present invention is to provide a fermented ginseng sprout composition prepared according to the above method and having an increased content of ginsenoside Re compared to before fermentation.

Another object of the present invention is to provide a food composition containing the fermented ginseng sprout composition.

In order to achieve the above object, the present invention provides Lactobacillus pentosus KK18 strain (Accession number: KCCM12762P), which has bioconversion ability for saponin.

In addition, the present invention includes the steps of (1) grinding ginseng sprout whole plant to produce ginseng sprout whole plant powder; (2) inoculating Lactobacillus pentosus KK18 strain (Accession number: KCCM12762P) into purified water and homogenizing to prepare a strain suspension; and (3) mixing 25 to 40 parts by weight of the strain suspension with respect to 100 parts by weight of the ginseng sprout whole plant powder and then fermenting it. Providing a method for producing a fermented ginseng composition with an increased content of ginsenoside Re, comprising: do.

According to one embodiment of the present invention, the purified water in step (2) may be adjusted to pH 4.8 to 6.4 using an organic acid.

According to one embodiment of the present invention, the strain in step (2) can be inoculated at 0.5 to 2% by weight based on purified water.

According to one embodiment of the present invention, the fermentation in step (3) may be performed at 32 to 42 ° C. for 60 to 72 hours.

In addition, the present invention provides a fermented ginseng sprout composition prepared according to the above method and having an increased content of ginsenoside Re compared to before fermentation.

According to one embodiment of the present invention, the fermented ginseng sprout composition may have a ginsenoside Re content of 0.15 mg/g or more.

According to one embodiment of the present invention, the fermented ginseng sprout composition may have a ginsenoside Rg2 content of 0.15 mg/g or more and a ginsenoside F2 content of 0.10 mg/g or more.

Additionally, the present invention provides a food composition containing the fermented ginseng sprout composition.

The Lactobacillus pentosus strain of the present invention is food-safe and bioconverts saponin contained in ginseng sprouts into ginsenosides Rb1, Rb2, Rg2, Re, and F2, which exist only in very trace amounts in ginseng sprouts. The ability to command is outstanding. In particular, it makes it possible to produce a fermented ginseng sprout composition with a significantly increased content of ginsenoside Re.

In addition, according to the method for producing a fermented ginseng sprout composition of the present invention, a composition with an increased content of ginsenoside Re can be obtained. The fermented ginseng sprout composition with increased content of ginsenoside Re is useful for the production of a food composition for improving learning and memory, and enhancing cognitive function.

Figure 1 is a photograph showing the color change observed after strains were inoculated and cultured on MRS agar medium containing esculin for selection of strains with β-glucosidase activity.
Figure 2 shows the results of TLC analysis of the fermented red ginseng extract after enzyme treatment and inoculation with 13 strains confirmed to have β-glucosidase activity.
Figure 3a shows the results of HPLC analysis of the fermented red ginseng extract after inoculating the G69, CK46, and KK18 strains, respectively, and Figure 3b shows the results of red ginseng fermentation after inoculating the red ginseng extract with the G69, CK46, and KK18 strains, respectively. This is a graph showing the results of TLC analysis of the extract and the Rg3 content according to fermentation period.
Figure 4a shows the results of HPLC analysis of the fermented red ginseng extract after enzyme treatment and inoculation with G69, CK46, and KK18 strains, respectively. Figure 4b shows the results of HPLC analysis of the red ginseng extract after enzyme treatment, and G69, CK46 This is a graph showing the results of TLC analysis of fermented red ginseng extracts fermented after inoculation with and KK18 strains, respectively, and the content of compound K according to the fermentation period.
Figure 5 is a graph showing the DPPH radical scavenging ability, ABTS radical scavenging ability, and FRAP reducing power of strains KK18, G69, and CK46.
Figure 6 is a photograph showing the color change of the API ZYM kit reacted with KK18, G69, and CK46 strains to confirm the enzyme activity of each strain.
Figure 7 is a photograph showing growth inhibition of KK18, G69, and CK46 strains by antibiotic treatment to confirm the antibiotic resistance of each strain.
Figure 8 shows the results of genome analysis of the finally selected Lactobacillus pentosus KK18 strain of the present invention.
Figure 9 is a chromatogram showing the results of analyzing the ginsenoside content of a fermented ginseng sprout composition prepared according to an embodiment of the present invention using HPLC.

Hereinafter, the present invention will be described in detail.

According to one embodiment of the present invention, the present invention provides Lactobacillus pentosus KK18 strain (Accession number: KCCM12762P) having bioconversion ability for saponin.

Lactobacillus pentosus KK18 strain (Accession number: KCCM12762P) of the present invention is a new Lactobacillus pentosus strain derived from kimchi. Although the Lactobacillus pentosus KK18 strain in the present invention was isolated and identified from kimchi, the acquisition route is not limited to this.

The KK18 strain isolated from kimchi in the following examples was found to have the base sequence of SEQ ID NO: 1 as a result of 16S rRNA base sequence analysis for microorganism identification and classification. The base sequence was confirmed for homology through the blast search program provided by NCBI GenBank (http://www.ncbi.nin.gov), and was found to be Lactobacillus pentosus 124-2 strain. It was confirmed that it was 99% identical to (see Figure 1).

Accordingly, the microorganism of the present invention having the 16S rRNA base sequence of SEQ ID NO: 1 was named Lactobacillus pentosus KK18 strain, and was deposited with the Korea Cultivation Society (KCCM) on July 8, 2020 (Accession No. KCCM12762P).

The Lactobacillus pentosus KK18 strain of the present invention has very high β-glucosidase activity, has acid resistance and bile resistance, shows antioxidant activity, and has antibiotic resistance, so it is very useful industrially.

The Lactobacillus pentosus KK18 strain of the present invention converts ginseng sprout saponins into specific ginsenosides, Rb1, Rb2, Rg2, Re, and F2, making it possible to produce a fermented ginseng composition with increased content of specific ginsenosides. , It is very useful in the production of health functional food materials.

According to another embodiment of the present invention, the present invention includes the steps of (1) grinding ginseng sprout whole plant to produce ginseng sprout whole plant powder; (2) inoculating Lactobacillus pentosus KK18 strain (Accession number: KCCM12762P) into purified water and homogenizing to prepare a strain suspension; and (3) mixing 25 to 40 parts by weight of the strain suspension with respect to 100 parts by weight of the ginseng sprout whole plant powder and then fermenting it. Providing a method for producing a fermented ginseng composition with an increased content of ginsenoside Re, comprising: do.

First, in step (1), ginseng sprout whole plant is pulverized to prepare ginseng sprout whole plant powder.

In the present invention, "sprout ginseng" refers to fresh ginseng grown among seedlings of ginseng (young ginseng grown for about a year after sowing) that is less than 24 months old and grown within 90 days, preferably for 30 to 40 days, by controlling the light intensity, temperature and humidity. refers to The saponin content of the ginseng sprout is higher than that of existing fresh ginseng, and the saponin content in the leaves is 6 to 8 times higher than in the roots. You can consume not only the roots of ginseng sprouts, but also the stems and leaves. However, the ginseng sprout has a significantly low content of ginsenosides Rb1, Rb2, Rg2, Re, and F2 among saponins.

In the present invention, it is preferable to use ginseng sprout whole plant, and more preferably, ginseng sprout whole plant grown from ginseng seedlings within 40 days can be used.

In the present invention, “starter” refers to the entire ginseng sprout, including the roots, stems, and leaves.

According to the present invention, the ginseng sprout whole plant may be a powder obtained by pulverizing ginseng sprout whole plant. Fermentation in the form of ginseng sprout whole plant powder expands the reaction surface area of enzymes produced by microorganisms, effectively increasing the active ingredient. Because you can do it.

The ginseng sprout whole plant powder includes the steps of (a) steaming the ginseng sprout whole plant at 95 to 100° C. for 0.5 to 1 hour; (b) drying the steamed ginseng sprout for 40 to 45 minutes at 65 to 70° C. to adjust the moisture content to 25 to 34%; and (c) pulverizing the dried ginseng sprout whole plant.

Next, in step (2) , Lactobacillus pentosus KK18 strain (Accession number: KCCM12762P) is inoculated into purified water and homogenized to prepare a strain suspension.

In the present invention, the purified water is adjusted to pH 4.8 to 6.4, preferably pH 5.2 to 6.0, and more preferably pH 5.4 to 5.8 using an organic acid to produce a specific ginseno during fermentation of the Lactobacillus pentosus KK18 strain of the present invention. This is desirable because it increases the conversion rate of the side.

If the pH of the strain suspension for fermentation of the Lactobacillus pentosus KK18 strain is outside the above range, the expected ginsenoside bioconversion rate will not be achieved.

The Lactobacillus pentosus KK18 strain can be inoculated at 0.5 to 2% by weight, preferably 1.0 to 1.5% by weight, based on the purified water.

If the strain inoculation amount is less than the lower limit, the fermentation speed may be delayed, and if it exceeds the upper limit, abnormal fermentation may proceed and economic efficiency is reduced.

Next, in step (3), 25 to 40 parts by weight, preferably 30 to 35 parts by weight, of the strain suspension is mixed with 100 parts by weight of the ginseng sprout whole plant powder and then fermented.

If the mixing amount of the strain suspension for the ginseng sprout whole plant powder is less than the lower limit, fermentation may not proceed properly, and if it exceeds the upper limit, abnormal fermentation may proceed due to excessive moisture content.

In the present invention, the fermentation can be performed at 32 to 42 ° C. for 60 to 72 hours.

If the fermentation temperature is less than the lower limit, the fermentation period may be prolonged, which may result in bacterial contamination, and if it exceeds the upper limit, the growth of the strain may be stopped. In addition, if the fermentation time is less than the lower limit, fermentation is not sufficient and the ginsenoside conversion rate may be low, and if it exceeds the upper limit, the ginsenosides produced by fermentation begin to decompose and the content decreases. can do.

In addition, according to another embodiment of the present invention, the present invention provides a fermented ginseng sprout composition prepared according to the above method and having an increased content of ginsenoside Re compared to before fermentation.

The contents of “Ginsenoside Re” and “Method for producing fermented ginseng sprout composition” are the same as described above.

The fermented ginseng sprout composition may contain 0.15 mg/g or more of ginsenoside Re, preferably 0.15 to 0.25 mg/g, more preferably 0.16 to 0.20 mg/g.

In addition, the fermented ginseng sprout composition may contain 0.15 mg/g or more of ginsenoside Rg2, preferably 0.15 to 0.25 mg/g, more preferably 0.16 to 0.22 mg/g. In addition, the fermented ginseng sprout composition may contain 0.10 mg/g or more of ginsenoside F2, preferably 0.10 to 0.20 mg/g, more preferably 0.12 to 0.18 mg/g.

The fermented ginseng sprout composition of the present invention is manufactured using ginseng sprouts, which are very inexpensive compared to root ginseng, and the content of ginsenoside Re, which exists only in very trace amounts in ginseng sprouts, is more than 0.15 mg/g, thereby improving learning and memory. , and is useful for the production of food compositions for enhancing cognitive function.

In addition, according to another embodiment of the present invention, the present invention provides a food composition containing the fermented ginseng sprout composition as an active ingredient.

Since the food composition contains a fermented ginseng sprout composition with a ginsenoside Re content of 0.15 mg/g or more as an active ingredient, it can be used to improve learning and memory, and to enhance cognitive function.

The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food, food additives, and feed, and is used for use in humans or animals including livestock. It is intended for consumption.

Food compositions of this type can be prepared in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruits and their processed foods (e.g. canned fruit, bottled foods, jam, mamalade, etc.), fish, meat and their processed foods (e.g. ham, sausages, etc.) corned beef, etc.), bread and noodles (e.g. udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine , vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauce, etc.), etc. can be produced by adding the fermented ginseng composition. In addition, nutritional supplements are not limited to this, but can be prepared by adding the fermented ginseng composition to capsules, tablets, pills, etc. In addition, the health functional food is not limited to this, but for example, the fermented ginseng sprout composition is prepared in the form of tea, juice, and drinks and consumed by liquefying, granulating, encapsulating, and powdering so that it can be consumed (health beverage). can do. In addition, in order to use the fermented ginseng sprout composition in the form of a food additive, it can be prepared in powder form, or extracted and then prepared in the form of a concentrate. In addition, it can be prepared in the form of a composition by mixing the fermented ginseng sprout composition with known active ingredients that are known to have the effect of improving learning and memory, and enhancing cognitive function.

When the food composition of the present invention is used as a health drink composition, the health drink composition may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional drinks. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; polysaccharides such as dextrins and cyclodextrins; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as thaumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the composition of the present invention.

The food composition preferably contains 0.01 to 100% by weight, preferably 0.1 to 50% by weight, of ginseng sprout fermentation composition based on the total weight of the entire food composition.

In addition to the above, the health food of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, It may contain glycerin, alcohol, or carbonating agent. These ingredients can be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail through examples, but the present invention is not limited to the following examples.

<Example>

Example 1: Isolation and identification of strains

1-1: Isolation of strains

Various types of regional kimchi and salted fish from each region were collected and used as kimchi (salted fish) samples for strain isolation. The kimchi and salted fish obtained by region are shown in Table 1 below.

Types of Kimchi Intake area Gyeonggi-do cabbage kimchi Anyang Jeollanam-do mustard kimchi Yeo su Godeulppaegi, Jeollanam-do Yeo su Chungcheongnam-do cabbage kimchi Cheonan Jeollabuk-do green onion kimchi jeonbuk Jeolla-do cabbage kimchi jeonbuk Chungcheongnam-do mustard kimchi Cheonan Chungcheongnam-do Squid Jeotgal Cheonan Chungcheongnam-do Octopus Jeotgal Cheonan Chungcheongnam-do Kkottogi Jeotgal Cheonan Jeolla-do squid salted fish jeonbuk Gangwon-do Octopus Jeotgal Sokcho

The kimchi or salted fish sample was collected, 10 g of the kimchi or salted fish sample was mixed with 90 mL of MRS broth, homogenized for 3 minutes, and cultured at 37°C for 24 hours. The culture was inoculated into MRS agar medium and then smeared with a glass rod. Thereafter, the plate was cultured in a constant temperature incubator at 37°C for 24 hours. Each colony generated was inoculated onto an MRS agar plate, and cultured at 37°C for 24 hours to isolate lactic acid bacteria colonies. About 1,200 strains were isolated, including the strains isolated in this way and the kimchi-derived strains held in the laboratory of Sunmoon University.

1-2: Selection of strains with β-glucosidase activity

Esculin containing 0.3 g/L and 0.2 g/L ferric ammonium citrate in MRS agar (deMan, Rogosa and Sharpe agar) for selection of strains with β-glucosidase activity. -MRS agar (Esculin-MRS agar) medium was used. The strain was inoculated onto the MRS agar medium containing esculin and cultured at 37°C for 48 hours to observe color changes within the medium (Figure 1). Esculin is separated into glucose and esculin by β-glucosidase, and esculin reacts with ferric ammonium citrate to form a black complex around the colony. Therefore, strains that formed black spots around colonies cultured on esculin agar medium were judged to have β-glucosidase activity and were selected. As a result, among the approximately 1,200 kimchi-derived strains isolated above, 136 strains that produced black spots were obtained, and among them, 29 strains with the darkest black spots were selected.

The β-glucosidase activity test was again performed on the 29 strains selected above, and the results are shown in Table 2 below.

No. Strains β-glucosidase activity No. Strains β-glucosidase activity One CK27 + 16 G69 ++ 2 CK31 + 17 G73 + 3 CK45 ++ 18 G77 + 4 CK46 + 19 G80 + 5 KK18 ++ 20 G86 + 6 NJ15 + 21 G88 ++ 7 NJ22 + 22 G92 + 8 G1 + 23 G93 + 9 G16 + 24 G95 + 10 G17 ++ 25 G117 + 11 G20 + 26 G120 + 12 G33 ++ 27 G126 + 13 G42 + 28 G144 + 14 G63 + 29 SY40 + 15 G64 + black zone + : normal, ++ : strong

Looking at Table 2 above, it can be seen that six strains, CK45, KK18, G17, G33, G69, and G88, show strong black spots.

1-3: Measurement of β-glucosidase enzyme activity

Among the 29 strains that formed black spots on the Esculin MRS agar medium, 6 strains formed relatively strong black spots (CK45, KK18, G17, G33, G69, and G88), and the next strongest black spots were β-Glucosidase activity was measured for 7 strains (G16, G42, G64, NJ22, CK31, CK46, SY40).

To measure β-glucosidase activity, the strain was inoculated 12 hours prior to commercial MRS medium supplemented with 1% carboxymethyl cellulose (CMC), incubated at 37°C for 24 hours, and then incubated at 4°C for 3000 °C. Cells were removed by centrifugation at rpm for 15 minutes, and 0.3 ml of the supernatant was mixed with 0.7 ml of 5 mM ρ-nitrophenyl-β-D-glucopynanoside (ρ-NPG) solution and reacted at 37°C for 10 minutes. The reaction was stopped by adding 1 ml of 0.5 M Na ₂ CO ₃ solution, the absorbance of the produced p-nitrophenol was measured at 450 nm, and the concentration was converted using the calibration curve of ρ-NP as shown in the table below. It is shown in 3.

Strains OD ₄₅₀ β-glucosidase activity (mU/mL) NJ22 2.445 30.34 G16 2.430 30.16 G17 2.404 29.85 G33 2.710 33.53 G42 2.652 32.83 G64 2.255 28.05 G69 2.397 29.76 G88 2.661 32.94 CK31 2.447 30.37 CK45 2.348 29.17 CK46 2.376 29.51 SY40 2.377 29.52 KK18 2.450 30.40

Looking at Table 3 above, it can be seen that the 13 selected strains exhibited high β-glucosidase activity in the range of 28.05 to 33.53 mU/mL.

16s rRNA gene sequence analysis was performed on the 13 strains selected above. As a result of identifying the above strains through base sequence analysis, it was confirmed that all of them belonged to Lactobacillus pentosus or Lactobacillus plantarum. The results of identifying the strains are summarized in Table 4 below.

No Lactobacillus pentosus Lactobacillus Plantarum One NJ22 G16 2 G33 G17 3 G42 G64 4 G88 G69 5 CK31 CK46 6 CK45 SY40 7 KK18

1-4: Selection of strains with excellent ginsenoside bioconversion ability

Among the 13 strains confirmed to have β-glucosidase activity, we attempted to select strains with excellent ginsenoside bioconversion ability.

For this purpose, the red ginseng extract (5% solid content) was treated with enzyme, then 1% (w/w) of the strain was added and cultured with shaking at 37°C, and TLC analysis was performed to determine F2 and compound K (CK) bands. We attempted to select strains with clear ginsenoside bioconversion ability by reducing Rb1.

At this time, the red ginseng extract was obtained by adding 10% by weight of red ginseng powder to 70% (w/w) fermented alcohol, extracting at 75 ℃ for 5 hours to obtain an extract of 15 Brix, and then placing it in a constant temperature water bath at 50 ℃ using a rotary concentrator. It was obtained by concentrating to 30 Brix.

In addition, the enzyme treatment was performed by adding Rapidase C80max 5% (w/w) and Pyr-flo 5% (w/w) to the red ginseng extract (solid content 5%) and reacting for 24 hours, followed by Ultimase MFC 5% ( w/w) and reacted for 72 hours.

The results of TLC analysis using the fermented red ginseng extract obtained by enzymatically treating the red ginseng extract as described above and then inoculating and fermenting the strain are shown in Figure 2.

Looking at Figure 2, the F2 and compound K (CK) bands appeared clearly in the G69, CK46, and KK18 strains, and the F2 and compound K (CK) bands appeared most strongly in the KK18 strain.

Accordingly, strains G69, CK46, and KK18, which clearly showed F2 and compound K (CK) bands through the TLC analysis, were selected as strains with excellent ginsenoside bioconversion ability, and the KK18 strain with the strongest bands was preferentially selected. Selected.

1-5: Confirmation of bioconversion ability of selected strains

To confirm the bioconversion ability of the selected KK18 strain, G69, and CK46 strains by reducing Rb1, 5% (w/w) of each of the above strains was inoculated into red ginseng extract (5% solid content) and cultured with shaking at 37°C. Afterwards, TLC analysis and HPLC analysis were performed (Figures 3a and 3b).

Figure 3a shows the results of HPLC analysis of the fermented red ginseng extract after inoculating the G69, CK46, and KK18 strains, respectively, and Figure 3b shows the results of red ginseng fermentation after inoculating the red ginseng extract with the G69, CK46, and KK18 strains, respectively. This is a graph showing the results of TLC analysis of the extract and the Rg3 content according to fermentation period.

Looking at Figure 3, it can be seen that Rb1 decreased and Rg3 content increased due to fermentation of the red rice ginseng extract. At this time, F2 and compound K were not produced by the fermentation.

In addition, the red ginseng extract (5% solid content) was treated with enzymes, and then the above strains were inoculated at 1% (w/w) each, cultured with shaking at 37°C, and then TLC analysis and HPLC analysis were performed (Figures 4a and 4b). ). The enzyme treatment was performed by adding Rapidase C80max 5% (w/w) and Pyr-flo 5% (w/w) to the red ginseng extract (solid content 5%) and reacting for 24 hours, followed by Ultimase MFC 5% (w/w). w) is added and reacted for 72 hours.

Figure 4a shows the results of HPLC analysis of the fermented red ginseng extract after enzyme treatment and inoculation with G69, CK46, and KK18 strains, respectively. Figure 4b shows the results of HPLC analysis of the red ginseng extract after enzyme treatment, and G69, CK46 This is a graph showing the results of TLC analysis of fermented red ginseng extracts fermented after inoculation with and KK18 strains, respectively, and the content of compound K according to the fermentation period.

Looking at Figure 4, as a result of TLC using the fermented product before and after fermentation, the F2 band increased on the 2nd day of fermentation for all three strains, and in particular, the KK18 strain had a compound K (CK) band on the 2nd and 3rd days of fermentation. You can see a clear increase.

In addition, as a result of quantifying the contents of ginsenoside F2 and compound K according to fermentation time by HPLC, the KK18 strain was found to increase the content of compound K by 12.5% by the 3rd day of fermentation. Therefore, strain KK18 was confirmed as a strain having bioconversion activity from ginsenoside F2 to compound K. In addition, the content of Compound K was found to decrease on the 5th day of fermentation after inoculation of the KK18 strain, and the conditions of culturing for 3 days after adding the KK18 strain were optimal for the production of Compound K by simultaneous enzyme conversion and lactic acid bacteria fermentation. It was found that

Meanwhile, the F2 content was found to increase in fermented products inoculated with the G69 strain and the CK46 strain, but the content of compound K hardly increased. Therefore, the above two strains appear to have no or minimal activity in bioconverting ginsenoside F2 to compound K.

1-6: Acid and bile resistance

In order for lactic acid bacteria to have probiotic activity, they must be resistant to gastric juice and bile and survive in order to reach the intestines and exert physiological activity. Therefore, we sought to confirm the acid resistance and bile resistance of the selected strains.

In order to determine the effect of resistance to gastric acid and bile acid, the selection strain was cultured in LB liquid medium at 30°C for 18 hours and then used as the initial inoculation culture for the experiment.

For the acid resistance analysis, 5% of the initial inoculation culture was inoculated into each LB liquid medium adjusted to pH 2.5, incubated with shaking at 30°C for 30 minutes, and the degree of acid resistance was confirmed by measuring the number of viable bacteria. In addition, in the bile resistance analysis, 5% of the initial inoculation culture was inoculated into LB liquid medium containing 0.3% bile (oxgall), cultured with shaking at 30°C for 6 hours, and then bile resistance was confirmed by measuring the number of viable bacteria. The control group for each experiment was defined as the number of viable cells reacting in a medium without pH adjustment and a medium without bile added, and the acid resistance and bile resistance of each strain are shown in Table 5 below as a % ratio to the control group. It was.

strain　 control group acid resistance Bile tolerance acid % bile salt % Lactobacillus plantarum
G69 83.37 88.60 Lactobacillus plantarum
CK46 84.57 91.87 Lactobacillus pentosus
KK18 84.08 88.72

Looking at Table 5, the KK18 strain, G69, and CK46 strains, which are strains selected in the present invention, were found to have a survival rate of more than 80% in gastric acid and bile, respectively, and were confirmed to have resistance to gastric acid and bile. Therefore, it is believed that the above strains are probiotics and that many live bacteria will stably reach the intestines when the strains are ingested.

1-7: Antioxidant activity

We sought to confirm the antioxidant activity of the selected KK18 strain, and G69 and CK46 strains. For this purpose, DPPH radical scavenging ability, ABTS radical scavenging ability, and FRAP reducing power were measured as follows (FIG. 5).

< DPPH radical scavenging ability >

For the DPPH radical scavenging ability, the supernatant obtained by culturing the selected KK18 strain in LB liquid medium at 30°C for 18 hours and then centrifuging was used as an analysis sample. 0.2 ml of the analysis sample and 0.8 ml of DPPH solution were mixed, reacted in the dark for 30 minutes, and the absorbance of the reactant was measured at 517 nm. In the case of the negative control, the DPPH radical scavenging ability was performed in the same manner as above, replacing each composition with distilled water, and the difference in absorbance was calculated as a percentage (%) by substituting the difference in absorbance into the equation below.

Radical scavenging ability (%) = [1-(absorbance of negative control group ÷ absorbance of experimental group)] × 100

< ABTS radical scavenging ability >

ABTS radical scavenging ability is measured using the principle that blue radicals gradually change color to colorless, that is, gradually fade according to antioxidant activity. This analysis has the advantage of being able to measure in a short time because the decolorization reaction ends quickly within 1 minute. ABTS radical scavenging ability is obtained by first mixing K2S2O8 reagent and methanol in a ratio of 2:1 to form ABTS cation radicals in the dark for 16 hours, then diluting this with methanol and adjusting the final concentration to an absorbance value of 0.8ㅁ0.2 at 732 nm. used.

Specifically, 0.1 ml of each analysis sample was added to 0.9 ml of ABTS reagent, reacted for 1 minute, and absorbance was quickly measured at 732 nm. The negative control was performed in the same manner using distilled water instead of the sample, and the difference in absorbance was calculated as a percentage (%) using the above formula.

< FRAP reducing power >

FRAP (ferric reducing antioxidant power) reducing power corresponds to reduction during the redox reaction, and is expressed as an indicator of antioxidant activity by measuring the absorbance value when reducing Fe ³⁺ ions to Fe ²⁺ ions.

Specifically, 30mM acetate buffer (30mM, pH 3.6), TPTZ reagent (10mM, dissolved in 6N hydrochloric acid) and FeCl ₃ solution (20mM, dissolved in distilled water) were mixed at 100:10:10 (v/v/ The FRAP reagent was prepared by first mixing in the ratio v). The FRAP reagent was pre-reacted in a water bath at 37°C for 15 minutes, and then 0.05 ml of each analysis sample was added to the test tube. 0.85 ml of the pre-reacted FRAP reagent was added thereto, and the mixture was incubated for 15 minutes at 37°C in the same manner as above. It was allowed to react for a minute. After reaction, the absorbance of a portion was measured at 593 nm.

Figure 5 is a graph showing the DPPH radical scavenging ability, ABTS radical scavenging ability, and FRAP reducing power of the selected KK18 strain, and G69 and CK46 strain cultures.

Looking at Figure 5, it can be seen that the G69 strain exhibits the highest DPPH radical scavenging ability (43%) and ABTS radical scavenging ability (30%). The KK18 strain showed relatively high FRAP reducing power and was confirmed to have good antioxidant activity with DPPH radical scavenging ability and ABTS scavenging ability of 30% and 25%, respectively.

1-8: Confirmation of enzyme activity using API ZYM kit

To confirm the enzyme activity of the selected strain, the API ZYM kit (BioMerieux, Marcy-I'Etoile, France), which was designed based on the substrate availability of a total of 19 different enzymes, was used. The selected lactic acid bacteria were smeared on MRS agar medium, the colonies cultured at 37°C for 48 hours were resuspended in 2 mL suspension medium, the turbidity was adjusted to 5-6 with McFarland (BioMerieux), and 60 ㎕ was dispensed into each tube and incubated at 37°C. After culturing for 4 hours, a drop of ZYM A and ZYM B reagent was added to each tube and reacted at room temperature for 5 minutes to determine the activity of each substrate enzyme by color change. It is expressed as a value from 0 to 5 according to the degree of color change, where 0 is a negative reaction, 5 (= 40 nmol) is the maximum intensity response, and 4 to 1 are intermediate values of 30, 20, 10, and 5 nmol, respectively. If it is 2 or more, it is judged positive. In addition, the degree of color change was indicated as “+” when it was 2 to 3, “++” when it was 3 to 4, and “+++” when it was 4 to 5. In general, in order for lactic acid bacteria to be used as probiotics, the enzymes they produce are also very important, so the API ZYM kit was used to check whether the finally selected KK18 strain produced enzymes. As a result, esterase lipase, lipase, leucin arylamidase, valine arylamidase, acid phosphatase, naphthol-AS -Naphol-AS-phosphohydrolase, β-galactosidase, β-glucosidase and N-acetyl-β-glucosaminidase (β- glucosaminidase) activity was confirmed (see Table 6 and Figure 6).

Enzymes L. plantarum G69 L. plantarum CK46 L. pentosus KK18 One Control +++ +++ +++ 2 Alkaline phosphatase - + - 3 Esterase (C4) - - - 4 Esterase Lipase (C8) - + + 5 Lipase(C14) - + + 6 Leucine arylamidase ++ +++ +++ 7 Valine arylamidase - ++ ++ 8 Cystine arylamidase - - - 9 Trypsin - - - 10 α-chymotrypsin - - - 11 Acid phosphatase - + + 12 Naphthol-AS-
phosphohydrolase + + ++ 13 α-galactosidase + + - 14 β-galactosidase +++ +++ ++ 15 β-glucuronidase - - - 16 α-glucosidase + + - 17 β-glucosidase ++ ++ +++ 18 N-acetyl-
β-glucosaminidase +++ ++ + 19 α-mannosidase + - - 20 α-frucosidase - - -

Looking at Table 6, G69, CK46, and KK18 strains all exhibited β-glucosidase activity, and the KK18 strain exhibited higher β-glucosidase activity than the G69 and CK46 strains.

In addition, the G69, CK46, and KK18 strains all showed no activity against the carcinogenic enzyme β-glucuronidase, confirming their safety against carcinogenesis.

1-9: Antibiotic susceptibility analysis

In the case of lactic acid bacteria, they are widely used as materials for food and medicine, and therefore, confirming antibiotic resistance is a very important factor. Therefore, antibiotic susceptibility analysis was performed on the selected strains of G69, CK46, and KK18.

Antibiotic susceptibility testing was conducted using the disk diffusion method of the National Committee for Clinical Laboratory Standards (NCCLS). The antibiotic disc (BBL, Sensi-disc, Becton Dickinson Co., NJ, USA) used was ampicillin, gentamicin, kanamycin, streptomycin, tetracycline, and ciprofloxacin ( Eight types were used, including Ciprofloxacin, Chloramphenicol, and Doxycycline. The selected strains were cultured in MRS liquid medium, ^plated on Müller Hint agar (Merck, Darmstadf, Germany) at 1.0 The size of the inhibitory ring for each antibiotic was measured, and susceptibility and resistance were determined according to the National Committee for Clinical Laboratory Standards (NCCLS) guidelines (see Figure 7). Data values were measured repeatedly three times and expressed as mean and standard deviation.

Antibiotic L. plantarum G69 L. plantarum CK46 L. pentosus
KK18 L. rhamnosus
GG Ampicillin S S S S Gentamicin R R R R Kanamycin R R R R Streptomycin R R R R Tetracycline S S S S Ciprofloxacin R R R R Chloramphenicol S S S S Doxycycline S S S S

*S; Sensitive, R; Resistance

Looking at Table 7 above, it was confirmed that G69, CK46, and KK18 strains all exhibited the same resistance as the commercial strain Lactobacillus rhamnosus GG strain. That is, it was inhibited by ampicillin, tetracycline, chloramphenicol, and doxycycline, but showed resistance to gentamicin, kanamycin, streptomycin, and ciprofloxacin. Since strains of the Lactobacillus genus are known to have inherent resistance to antibiotics showing resistance, it is believed that the above three strains can be used industrially.

1-10: Molecular biological identification of selected strains

Through Examples 1-1 to 1-9, it was found that β-glucosidase activity was high, ginsenoside bioconversion ability was excellent, and in particular, it had bioconversion activity of ginsenoside F2 to compound K, and was acid-resistant and resistant. Lactobacillus pentosus KK18 strain was finally selected as a strain with excellent bile properties, antioxidant activity, and antibiotic sensitivity.

To investigate the molecular biological characteristics of the finally selected Lactobacillus pentosus KK18 strain, chromosomal DNA was isolated from the strain culture medium, and the entire 16s rRNA gene sequence analysis was performed using the chromosomal DNA. The identified strain was found to belong to Lactobacillus pentosus , and the base sequence (16s rRNA sequence) of the identified strain is shown in SEQ ID NO: 1.

As a result of comparing the base sequence of this strain with other strains registered in GenBank, it was found to be a standard strain. It showed 99% homology with the standard Lactobacillus pentosus 124-2 strain, and it was confirmed that no strain with 100% homology with SEQ ID NO: 1 exists (see FIG. 8). Therefore, the present inventors deposited the Lactobacillus pentosus KK18 strain with the Korean Cultivation Society (KCCM) on July 8, 2020, and received accession number KCCM12762P.

Example 2: Preparation of ginseng sprout fermentation composition

(1) Manufacturing of ginseng sprout powder

Ginseng sprouts were purchased from Daul Sprouts, and whole plants (leaves, stems, and roots) were used. The ginseng whole plant was washed three times with tap water to remove foreign substances, then placed in a steaming device and steamed at 95°C for 1 hour with steam. Afterwards, it was placed in a dryer maintained at 70°C and dried for 45 minutes to adjust the moisture content to 30%, and then pulverized to a size of approximately 1.5 × 1.5 mm using a stamp cutter to prepare ginseng sprout whole plant powder.

(2) Preparation of strain suspension

Organic acid was added to purified water to adjust the pH to 5.1. A strain suspension was prepared by inoculating 1.2% by weight of Lactobacillus pentosus KK18 strain culture pre-cultured in purified water at pH 5.6 and then homogenizing.

(3) Fermentation

35 parts by weight of the strain suspension prepared in (2) above was sprayed onto 100 parts by weight of the ginseng sprout whole plant powder prepared in (1) above, and then fermented at 37°C for 65 hours. The fermented product after the fermentation was dried with hot air at 90°C for 5 hours to prepare a fermented ginseng sprout composition.

Comparative Example 1: Fermentation at pH 3.5

A fermented ginseng sprout composition was prepared in the same manner as in Example 2, except that when preparing the strain suspension in step (2), the pH of the purified water was adjusted to pH 3.5 instead of pH 5.6.

Comparative Example 2: Fermentation at pH 7.0

A fermented ginseng sprout composition was prepared in the same manner as in Example 2, except that when preparing the strain suspension in step (2), the pH of the purified water was adjusted to pH 7.0 instead of pH 5.6.

Comparative Example 3: Fermentation with Lactobacillus Plantarum G69 strain

It was carried out in the same manner as in Example 2, except that when preparing the strain suspension in step (2), Lactobacillus plantarum G69 strain was inoculated instead of Lactobacillus pentosus KK18 strain to prepare a fermented ginseng sprout composition.

Comparative Example 4: Fermentation with Lactobacillus Plantarum CK46 strain

It was carried out in the same manner as in Example 2, except that when preparing the strain suspension in step (2), Lactobacillus plantarum CK46 strain was inoculated instead of Lactobacillus pentosus KK18 strain to prepare a fermented ginseng sprout composition.

< Test example>

Test example: Ginsenoside content

HPLC was used to analyze the content of ginsenosides contained in the fermented ginseng sprout composition prepared in the above examples and comparative examples.

Specifically, to measure the content of ginsenosides, 5 mg of each 5 mg of ginsenoside standard products (Rb1, Rb2, Rg2, Re, and F2) were precisely measured and then dissolved in 1 ml of methanol. Afterwards, it was diluted 10 times and then again 50 times and 100 times to prepare standard solutions of three concentrations. The prepared standard solution was set as a standard for measuring the content of ginsenoside.

Meanwhile, 10 g of the ginseng sprout fermentation composition prepared according to the above Examples and Comparative Examples was dissolved in 100 ml of 70% methanol, extracted three times by refluxing in a constant temperature water bath at 80°C, and concentrated under reduced pressure. Afterwards, distilled water was added, followed by diethyl ether to remove fat-soluble components, extracted three times with saturated n-butanol, concentrated under reduced pressure, and the ginsenoside content was analyzed using HPLC (Figures 8 and 9 ). Figure 9 is a chromatogram showing the results of analyzing the ginsenoside content of the fermented ginseng sprout composition prepared according to the example using HPLC.

division Ginsenoside content (mg/g) control group Example 2 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Rb1 0.00 0.02 0.00 0.00 0.00 0.00 Rb2 0.00 0.01 0.00 0.00 0.00 0.00 Rg2 0.05 0.19 0.11 0.07 0.05 0.08 Re 0.01 0.28 0.08 0.06 0.04 0.05 F2 0.04 0.14 0.09 0.10 0.09 0.11 Sum 0.10 0.64 0.28 0.23 0.18 0.24

Looking at Table 8, it can be seen that the content of ginsenosides Rg2, Re, and F2 in the fermented ginseng sprout composition according to the example was significantly increased compared to the composition according to the comparative example.

Through these results, it was confirmed that the specific ginsenoside content of the fermented ginseng composition can be effectively increased when using the strain and production method of the present invention.

Although the present invention has been described in terms of the above-mentioned preferred embodiments, various modifications and variations can be made without departing from the gist and scope of the invention. Additionally, the appended claims cover such modifications or variations as fall within the subject matter of the present invention.

<110> Sunmoon University Industry-Academic Collaboration Foundation <120> Lactobacillus pentosus KK18 with biolofical conversion activity of saponin, a method of preparation for composition of fermented sprout ginseng having increased content of ginsenoside Re using the same, fermented red ginseng extracts using thereby and food composition containing the same <130>HPC-9398 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 720 <212> DNA <213> Lactobacillus pentosus <400> 1 gacgaacgct ggcggcgtgc ctaatacatg caagtcgaac gaactctggt attgattggt 60 gcttgcatca tgatttacat ttgagtgagt ggcgaactgg tgagtaacac gtgggaaacc 120 tgcccagaag cgggggataa cacctggaaa cagatgctaa taccgcataa caacttggac 180 cgcatggtcc gagtttgaaa gatggcttcg gctatcactt ttggatggtc ccgcggcgta 240 ttagctagat ggtggggtaa cggctcacca tggcaatgat acgtagccga cctgagaggg 300 taatcggcca cattgggact gagacacggc ccaaactcct acgggaggca gcagtaggga 360 atcttccaca atggacgaaa gtctgatgga gcaacgccgc gtgagtgaag aagggtttcg 420 gctcgtaaaa ctctgttgtt aaagaagaac atatctgaga gtaactgttc aggtattgac 480 ggtatttaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg 540 gcaagcgttg tccggattta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat 600 gtgaaagcct tcggctcaac cgaagaagtg catcggaaac tgggaaactt gagtgcagaa 660 gaggacagtg gaactccatg tgtagcggtg aaatgcgtag atatatggaa gaacaccagt 720 720

Claims (10)

delete (1) Grinding ginseng sprouts to produce ginseng sprouts powder;
(2) Lactobacillus pentosus KK18 strain (Accession number: KCCM12762P) having the 16S rRNA base sequence of SEQ ID NO: 1 was inoculated into purified water adjusted to pH 4.8 to 6.4 using organic acid and homogenized to obtain a strain suspension. manufacturing step; and
(3) Mixing 25 to 40 parts by weight of the strain suspension with respect to 100 parts by weight of the ginseng sprout whole plant powder and then fermenting it. A method of producing a fermented ginseng composition with an increased content of ginsenoside Re. The method of claim 2, wherein the ginseng sprout powder in step (1) is
(a) steaming the ginseng sprout for 0.5 to 1 hour at 95 to 100°C;
(b) drying the steamed ginseng sprout for 40 to 45 minutes at 65 to 70° C. to adjust the moisture content to 25 to 34%; and
(c) pulverizing the dried ginseng sprout whole plant; A method for producing a fermented ginseng composition with an increased content of ginsenoside Re. delete According to paragraph 2,
A method for producing a fermented ginseng sprout composition with an increased content of ginsenoside Re, characterized in that the strain in step (2) is inoculated at 0.5 to 2% by weight based on purified water. According to paragraph 2,
A method for producing a fermented ginseng sprout composition with increased content of ginsenoside Re, characterized in that the fermentation in step (3) is performed at 32 to 42 ° C. for 60 to 72 hours. A fermented ginseng sprout composition prepared according to the method of any one of claims 2, 3, 5, and 6, and having an increased content of ginsenoside Re compared to before fermentation. In clause 7,
The fermented ginseng sprout composition is characterized in that the ginsenoside Re content is 0.15 mg/g or more. In clause 7,
The fermented ginseng sprout composition is characterized in that the ginsenoside Rg2 content is 0.15 mg/g or more and the ginsenoside F2 content is 0.10 mg/g or more. A food composition comprising the fermented ginseng sprout composition of claim 7.