KR102542030B1 - Makgeolli-derived Lacticaseibacillus paracasei subsp. paracasei DCF0429 and functional postbiotics using the same - Google Patents

Abstract

The present invention relates to rice wine-derived Lacticaseibacillus paracasei subspecies paracasei ( Lacticaseibacillus paracasei subsp. paracasei ) DCF0429 (KCTC15225BP) and functional postbiotics using the same. Jay ( Lacticaseibacillus paracasei subsp. paracasei ) DCF0429 (KCTC15225BP) strain exhibits high antioxidant activity, and postbiotics made by fermenting and killing the strain in soybean embryo medium are also excellent. It can exhibit antioxidant activity, so it has high utilization as a material for various compositions.

Description

Makgeolli-derived Lacticaseibacillus paracasei subspecies Paracasei DCF0429 and functional postbiotics using the same {Makgeolli-derived Lacticaseibacillus paracasei subsp. paracasei DCF0429 and functional postbiotics using the same}

The present invention relates to Makgeolli-derived Lacticaseibacillus paracasei subspecies Paracasei ( Lacticaseibacillus paracasei subsp. paracasei ) DCF0429 (KCTC15225BP) and functional postbiotics using the same, and more specifically, to rice wine isolated from traditional fermented food It relates to Lacticaseibacillus paracasei subsp. paracasei DCF0429 and antioxidant functional postbiotics using the same.

Recently, as interest in health has increased, studies on various health functional foods have been actively conducted. Probiotics refers to living bacteria that are beneficial to health, and mainly include lactic acid bacteria and lactic acid bacteria. It has been expanded from prebiotics, which are nutrients of probiotics, and synbiotics, which refer to probiotics and prebiotics together, to postbiotics, which are metabolites of probiotics, and recently, postbiotics, not probiotics. We are paying attention to the fact that biotics contribute to supporting intestinal health.

Postbiotics is a concept that includes substances produced by probiotics metabolizing or fermenting nutrients, and the dead bodies of bacteria that remain after lactic acid bacteria die. that contributes to intestinal health. Accordingly, there is a high need for new postbiotics, and in particular, their utilization can be increased by having functional properties that are good for health.

On the other hand, postbiotics can be called postbiotics only when beneficial effects are exhibited even when probiotics are killed, not simply postbiotics that are killed by probiotics. Therefore, the method of killing postbiotics is important because the presence and degree of effect are determined according to the method of killing postbiotics and can further enhance health benefits. Therefore, it is also important to develop a germination method in which the efficacy of postbiotics can be demonstrated well.

Republic of Korea Patent Publication No. 10-2011-0124029 (published date: 2011.11.16) relates to a culture solution secreted from rice wine-derived lactic acid bacteria having an antibacterial activity function, and the culture solution secreted from rice wine-derived lactic acid bacteria has antibacterial activity against pathogenic bacteria It has been described as acting as a natural antibacterial agent. Republic of Korea Patent Publication No. 10-2022-0034453 (published date: 2022.03.18) relates to a method for producing postbiotics using kimchi lactic acid bacteria, and uses superfood juice as raw material to mix and ferment with lactic acid bacteria culture medium, and lactic acid bacteria A method for preparing postbiotics, which is a metabolite, is described.

In order to discover new functional postbiotics, the present invention isolates Lacticaseibacillus paracasei subsp. paracasei DCF0429 (KCTC15225BP) from makgeolli, a traditional fermented food, and extracts it from soybean embryo medium. It is intended to provide postbiotics that exhibit excellent antioxidant functionality by fermentation.

the present invention Provided is a Lacticaseibacillus paracasei subsp. paracasei DCF0429 (KCTC15225BP) strain or a dried product thereof isolated from makgeolli.

Meanwhile, in the present invention, the Lacticaseibacillus paracasei subspecies paracasei DCF0429 (KCTC15225BP) may preferably have antioxidant activity.

In addition, the present invention is inoculated with Lacticaseibacillus paracasei subsp. paracasei DCF0429 (KCTC15225BP) in a medium containing glucose in soybean embryo extract and cultured, or a dried product thereof. It provides postbiotics of Lacticaseibacillus paracasei subsp. paracasei ) DCF0429 (KCTC15225BP), characterized in that.

On the other hand, in the postbiotics of the present invention, the medium preferably contains 2-4% of soybean germ extract and 2-4% of glucose relative to the total medium.

Meanwhile, in the postbiotics of the present invention, the postbiotics may preferably have antioxidant activity.

The Lacticaseibacillus paracasei subsp. paracasei DCF0429 (KCTC15225BP) strain derived from makgeolli of the present invention exhibits high antioxidant activity and is made by fermenting and killing the strain in a soybean embryo medium. ticks are good too It can exhibit antioxidant activity, so it has high utilization as a material for various compositions.

Figure 1 is a graph of the results of comparative evaluation of the antioxidative ability of 41 strains derived from domestic traditional food through DPPH assay.
Figure 2 is a graph of the results of comparative evaluation of the antioxidative ability of 41 strains derived from domestic traditional food strains of bacteria-excluded cultures through DPPH assay.
Figure 3 is a graph of the results of comparative evaluation of the antioxidative ability of the bacterial lysate of 41 strains derived from domestic traditional food through DPPH assay.
Figure 4 is a process for producing soybean embryo postbiotics, shown in the order of strain culture, dead cells, and freeze-drying from the left.
Figure 5 is a graph of the results of comparative evaluation of the antioxidant capacity of soybean embryo postbiotics using the selected primary strain through DPPH assay.
Figure 6 is a graph of the results of comparative evaluation of the antioxidant capacity of soybean embryo postbiotics using the selected primary strain through TAC assay.
Figure 7 is a graph showing the results of comparative evaluation of the antioxidant activity of the Lacticaseibacillus paracasei subsp. paracasei DCF0429 (KCTC15225BP) compared to the standard strain through DPPH assay.
Figure 8 is a DPPH assay to evaluate the antioxidant activity of each stage of production (before fermentation, after fermentation, after killing cells) of soybean embryo postbiotics using the rice wine-derived Lacticase bacillus paracasei subspecies Paracasei DCF0429 of the present invention. This is the resulting graph.

Postbiotics is a concept that includes substances produced by metabolizing or fermenting nutrients by probiotics and even the dead bodies of bacteria that remain after lactic acid bacteria die. I'm paying attention to ticks. In particular, as it is important for postbiotics to exhibit beneficial effects even when probiotics are killed with postbiotics, it is important to develop a germination method in which the efficacy of postbiotics can be exhibited well.

Accordingly, the present invention provides a Lacticaseibacillus paracasei subsp. paracasei DCF0429 (KCTC15225BP) strain or a dried product thereof isolated from makgeolli. At this time, the strain may have antioxidant activity. In the present invention, in order to select strains with excellent functionality (antioxidant activity), several lactic acid bacteria were isolated and identified among various traditional fermented foods (cabbage kimchi, dongchimi, young radish kimchi, bachelor kimchi, radish radish kimchi, Andong sikhye, makgeolli, and ome technology). Among them, 4 strains with excellent antioxidant activity were selected first. Thereafter, the present invention tested the antioxidant ability of postbiotics prepared after inoculating and culturing the primary selected strains in a medium using soybean embryo extract. Paracasei subspecies Paracasei ( Lacticaseibacillus paracasei subsp. paracasei ) DCF0429 could be selected.

Accordingly, the Lacticasei Bacillus paracasei subspecies paracasei DCF0429 (KCTC15225BP) strain of the present invention was deposited at the Korea Research Institute of Bioscience and Biotechnology (KCTC) on December 06, 2022, and the deposit number Granted KCTC15225BP. The strain is Lacticaseibacillus paracasei subsp. paracasei DCF0429 (KCTC15225BP) and has a DNA sequence encoding 16s rRNA (SEQ ID NO: 1).

In addition, the present invention is inoculated with Lacticaseibacillus paracasei subsp. paracasei DCF0429 (KCTC15225BP) in a medium containing glucose in soybean embryo extract and cultured, or a dried product thereof. It provides postbiotics of Lacticaseibacillus paracasei subsp. paracasei ) DCF0429 (KCTC15225BP), characterized in that. At this time, the medium preferably contains 2-4% of soybean germ extract and 2-4% of glucose compared to the total medium. This is because when the concentration of soybean germ extract increases, the number of viable cells increases, but there is no significant difference at concentrations above the above range. manufactured. In addition, as the concentration of glucose increases, the number of viable cells increases, but as it is confirmed that there is no significant difference at concentrations above the above range, the functionality of postbiotics is best demonstrated and economical efficiency is considered. did As described above, these postbiotics exhibit excellent antioxidant activity by fermenting and using Lacticaseibacillus paracasei subsp. paracasei DCF0429 of the present invention compared to other traditional fermented food-derived strains. It could be prepared as postbiotics.

On the other hand, the soybean embryo extract of the present invention is not limited to any method known in the art, but preferably separating the embryos from soybeans; adding hot water 2 to 13 times (most preferably, 9 times) the weight of the embryo to the separated embryo, grinding and filtering to separate insoluble components; After the separation, drying the recovered soy germ milk (SGM); By culturing the strain of the present invention in a medium using the soybean embryo extract prepared as described above, the excellent antioxidant efficacy of postbiotics can be excellently exhibited even after dead cells.

On the other hand, the postbiotics of the present invention are preferably used in a dried state, and are not limited to any drying method known in the art, but more preferably freeze-drying.

On the other hand, according to the experiment below, the present invention selected strains with excellent antioxidant activity from various traditional fermented foods, and among them, excellent antioxidant activity even when produced with postbiotics that were cultured in a medium using soybean embryo extract and then incorporated into dead cells. Lacticasei Bacillus paracasei subspecies paracasei ( Lacticaseibacillus paracasei subsp. paracasei ) DCF0429 was isolated and identified. As it is a plant-derived lactic acid bacteria, it is safe and exhibits excellent antioxidant ability not only when it is a probiotic but also when it is a dead cell postbiotic, so it can be used as a material for various compositions.

Hereinafter, the contents of the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited only to the following examples, and includes modifications of equivalent technical ideas.

[Example 1: Isolation, identification and evaluation of antioxidant activity of primary selected strains from traditional fermented foods]

In this example, primary selected strains were isolated and identified from traditional fermented foods, and their antioxidant capacity was evaluated.

1) Isolation and identification of lactic acid bacteria from traditional fermented foods

Cabbage kimchi, dongchimi, young radish kimchi, bachelor kimchi, radish kimchi, Andong sikhye, makgeolli, and ome technology were used as traditional fermented foods, and the culture conditions were MRS broth 0.055 g/ml + Agar powder 0.015 g/ml as selective media. ml + Potassium sorbate 0.003 g/ml + L-cysteine monohydrate 0.1 mg/ml + Bromocresol purple 0.05 mg/ml was used and cultured at 37°C for 24 hours.

The gene used for identification was the 16S rRNA gene, and the strains identified were a total of 41 strains, Latilactobacillus sakei subspecies Sakei ( Latilactobacillus sakei subsp. sakei ) (8 strains), Latilactobacillus curvatus ( Latilactobacillus curvatus ) (2 strains), Levilactobacillus brevis ( Levilactobacillus brevis ) (2 strains), Lacticase Bacillus paracasei ( Lacticaseibacillus paracasei ) (4 strains), Lacticase Bacillus paracasei subspecies paracasei ( Lacticaseibacillus paracasei subsp. paracasei ) (strain 1), Lactiplantibacillus plantarum (2 strains), Limosilactobacillus fermentum ( Limosilactobacillus fermentum ) (Strain 1), Leuconostoc citreum ( Leuconostoc citreum ) (3 strains), Leuconostoc mesenteroides ( Leuconostoc mesenteroides ) (8 strains), Weissella cibaria (4 strains), Pediococcus acidilactici (1 strain), Pediococcus pentosaceus ( Pediococcus pentosaceus ) (1 strain), Lactococcus lactis subspecies cremoris ( Lactococcus lactis subsp. cremoris ) (1 strain), Leuconostoc Pseudomesenteroides ( Leuconostoc pseudomesenteroides ) (3 strains), and detailed information is shown in Tables 1 and 2 below.

List of isolates 1 Strains No. Species separation circle DCF0741 Latilactobacillus sakei subsp. sakei Dongchimi DCF0720 Latilactobacillus sakei subsp. sakei Young Radish Kimchi DCF0723 Latilactobacillus sakei subsp. sakei Ome Technique DCF0731 Latilactobacillus sakei subsp. sakei Kimchi DCF0724 Latilactobacillus sakei subsp. sakei Dongchimi DCF0725 Latilactobacillus sakei subsp. sakei bachelor kimchi DCF0728 Latilactobacillus sakei subsp. sakei Radish kimchi DCF0734 Latilactobacillus sakei subsp. sakei Makgeolli DCF0620 Latilactobacillus curvatus Kimchi DCF0621 Latilactobacillus curvatus Dongchimi DCF1009 Levilactobacillus brevis Dongchimi DCF1010 Levilactobacillus brevis Makgeolli DCF0412 Lacticaseibacillus paracasei Makgeolli DCF0420 Lacticaseibacillus paracasei Makgeolli DCF0422 Lacticaseibacillus paracasei Makgeolli DCF0428 Lacticaseibacillus paracasei Makgeolli DCF0429 Lacticaseibacillus paracasei subsp. paracasei Makgeolli DCF0512 Lactiplantibacillus plantarum Makgeolli DCF0514 Lactiplantibacillus plantarum Makgeolli DCF1401 Limosilactobacillus　fermentum Makgeolli DCF0802 Leuconostoc citreum Makgeolli DCF0803 Leuconostoc citreum Young Radish Kimchi DCF0808 Leuconostoc citreum Radish kimchi DCF0921 Leuconostoc mesenteroides Dongchimi DCF0924 Leuconostoc mesenteroides Dongchimi DCF0910 Leuconostoc mesenteroides Dongchimi DCF0912 Leuconostoc mesenteroides Young Radish Kimchi DCF0913 Leuconostoc mesenteroides Kimchi DCF0915 Leuconostoc mesenteroides Dongchimi DCF0918 Leuconostoc mesenteroides Makgeolli DCF0920 Leuconostoc mesenteroides Makgeolli

List of isolates 2 Strains No. Species separation circle DCF1302 Weissella cibaria Andong Sikhye DCF1304 Weissella cibaria Radish kimchi DCF1305 Weissella cibaria Andong Sikhye DCF1307 Weissella cibaria Radish kimchi DCF1116 Pediococcus acidilactici Ome Technique DCF1204 Pediococcus acidilactici Makgeolli DCF1701 Lactococcus lactis subsp. cremoris Makgeolli DCF1801 Leuconostoc pseudomesenteroides Andong Sikhye DCF1802 Leuconostoc pseudomesenteroides Dongchimi DCF1803 Leuconostoc pseudomesenteroides Radish kimchi

2) Evaluation of antioxidant activity of isolated strains

In order to confirm the difference in antioxidant activity of the 41 strains by property (cell, bacteria-excluded culture medium, bacterial lysate), it was evaluated through DPPH assay. Samples for each property were prepared as follows: ① Bacterial sample: Each isolate was inoculated into MRS broth and cultured at 37°C to an OD ₆₀₀ of 1.0. It was centrifuged with a 1 ml centrifuge (4,000 g for 10 min) of the culture medium, and the cell pellet was resuspended in 1 mL of PBS and used as a sample. ② Bacterial exclusion culture solution sample: Each isolate was inoculated into MRS broth and cultured at 37°C to an OD ₆₀₀ of 1.0. The culture medium was centrifuged with 1 ml centrifuge (4,000 g for 10 min), and the supernatant was used as a sample. ③ Bacterial lysate sample: Each isolate was inoculated into MRS broth and cultured at 37°C to an OD ₆₀₀ of 1.0. Centrifuged with 1 ml centrifuge (4,000 g for 10 min) of the culture medium, resuspended the cell pellet in 1 mL of PBS, and disrupted the cells using a cell homogenizer to use as a sample did The DPPH assay was measured using the DPPH Antioxidant Assay Kit (BIOMAX Co.), each isolate was inoculated into MRS broth, cultured at 37°C to an OD ₆₀₀ of 1.0, and 20 μl of the prepared sample was mixed with 100 μl of Assay buffer and After adding 100 μl of DPPH working solution and reacting at room temperature for 30 minutes by blocking light, absorbance was measured at 517 nm.

As a result, as shown in FIG. 1, Latilactobacillus curvatus DCF0620 strain identified from cabbage kimchi among 41 cell samples showed the highest antioxidant capacity at 27.47%. In addition, as shown in Figure 2, Leuconostoc mesenteroides DCF0913 strain identified from cabbage kimchi among 41 bacteria-excluded culture samples showed the highest antioxidant capacity at 37.29%. In addition, as shown in FIG. 3, Lacticaseibacillus paracasei subspecies paracasei identified in makgeolli among 41 bacterial lysate samples ( Lacticaseibacillus paracasei subsp. paracasei ) DCF0429 strain showed the highest antioxidant capacity at 26.27%. However, since they are metabolites inside lactic acid bacteria when manufacturing postbiotics, four strains ( Lacticaseibacillus paracasei subsp. paracasei DCF0429, Lactiplantibacillus plantarum DCF0512, Leuconostoc citreum DCF0803, Leuconostoc citreum DCF0808) with excellent antioxidant activity in bacterial lysates were selected as the primary strains. was selected.

[Example 2: Preparation of soybean embryo medium and selection of final strains through evaluation of antioxidant activity of the primary selected strains in the medium]

In this Example, a soybean embryo medium was prepared, and the final strain was selected through the evaluation of the antioxidant activity of the primary selected strain to be selected in Example 1 in the medium.

1) Preparation of soybean embryo medium

Embryos were isolated from soybeans, and insoluble components were separated by grinding and filtering by adding hot water at a level of 9 times the weight of the embryos. The recovered soy germ milk (SGM) was lyophilized to prepare a powdered soy germ extract. As a result of previous research, as the concentration of soybean germ extract increased, the number of viable cells increased, but as it was verified that there was no significant difference at a concentration of 3% or more, it was judged that it would be economically efficient to prepare a medium at a concentration of 3%. Therefore, a soybean embryo medium was prepared by mixing 3% glucose with a soybean germ extract concentration of 3%.

2) Selection of final strains through evaluation of antioxidant activity of primary selected strains in soybean embryo medium

The soybean embryo medium prepared above was inoculated with the four primary selected strains selected in Example 1 and cultured for 24 hours. The method for preparing these postbiotics is as follows: Inoculation of each isolated strain into MRS broth And, after culturing at 37 ℃ to OD ₆₀₀ 1.0, it was inoculated into soybean embryo medium prepared by 1% (v / v) and cultured at 37 ℃ for 3 hours. The cultured samples were killed under conditions of 121° C. for 15 minutes. The killed samples were lyophilized and used to prepare soybean embryo postbiotics powder. Figure 4 shows the process for preparing soybean embryo postbiotics (from the left, strain culture → dead cells → freeze-drying). For the prepared soybean embryo postbiotics, it was verified that no viable cells were present and all cells were killed through a viable cell count test.

In order to analyze the functional changes of the four soybean embryo postbiotics, the antioxidant capacity was evaluated by DPPH assay and TAC assay. DPPH assay was measured using DPPH Antioxidant Assay Kit (BIOMAX, BO-DPH-200), and each soybean embryo postbiotics powder was dissolved in distilled water at 100 mg/L (w/v) to prepare a sample, 100 μl of Assay buffer and 100 μl of DPPH working solution were added to 20 μl of the prepared sample, and the mixture was reacted for 30 minutes at room temperature by blocking light, and the absorbance was measured at 517 nm. The TAC assay was measured using the Total Antioxidant Capacity Assay Kit (BIOMAX, BO-TAC-200), and each soybean embryo postbiotics powder was dissolved in distilled water at 100 mg/L (w/v) to prepare a sample. , After adding 100 μl of Copper Reagent and 100 μl of Reaction buffer to 100 μl of the prepared sample, reacting at room temperature for 30 minutes by blocking light, the absorbance was measured at 450 nm.

As a result, among the four soybean embryo postbiotics, as shown in FIG. 5 (DPPH assay) and FIG. 6 (TAC assay) , Lacticaseibacillus paracasei subspecies paracasei DCF0429 strain is the most excellent. It was confirmed that it exhibits antioxidant activity. Accordingly, among the four types of soybean embryo postbiotics, the strain Lacticaseibacillus paracasei subsp. paracasei DCF0429 strain, which is the best strain in the antioxidant experiment, was selected as the final postbiotics strain.

On the other hand, when the sequence encoding the 16S rRNA of the Lacticaseibacillus paracasei subsp. paracasei DCF0429 strain selected as the final strain was identified in Example 1, using the primers shown in Table 3 below, , Lacticase Bacillus paracasei subspecies paracasei ( Lacticaseibacillus paracasei subsp. paracasei ) DCF0429 strain was isolated, and the sequence encoding its 16S rRNA was described in SEQ ID NO: 3.

strain name gene name Primer name sequence information sequence number Lacticaseibacillus paracasei subsp. paracasei 16S rRNA gene F primer 5'-AGA GTT TGA TCM TGG CTC AG-3' SEQ ID No. 1 R primer 5'-GGT TAC CTT GTT ACG ACT TC-3' SEQ ID No. 2

The inventors of the present invention, on December 06, 2022, to the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) for the final selected strain Lacticaseibacillus paracasei subspecies paracasei DCF0429 (Accession No. : KCTC15225BP) was deposited.

3) Comparison of antioxidant capacity of the selected final strain with the standard strain

The finally selected Lacticase Bacillus paracasei subspecies paracasei ( Lacticaseibacillus paracasei subsp. paracasei ) DCF0429 strain was compared with the antioxidant activity level using the standard strain. As a standard strain , Lacticaseibacillus paracasei subsp. paracasei KACC12361 obtained from the Korean Agricultural Culture Collection (KACC) was used. As a result, as shown in FIG. 7, it was confirmed that the Lacticaseibacillus paracasei subsp. paracasei DCF0429 strain exhibits higher antioxidant activity than the standard strain (KACC12361). Through this, it was found that the finally selected strain has a higher antioxidant capacity than the standard strain, and thus has excellent utility as a postbiotics.

[Example 3: Preparation of postbiotics and evaluation of their antioxidant activity]

In this example, postbiotics were prepared using the Lacticaseibacillus paracasei subsp. paracasei strain DCF0429, which was finally selected in Example 2, and its antioxidant activity was evaluated. The postbiotics preparation method was the same as in Example 2 above.

When preparing postbiotics, changes in antioxidant activity during heat treatment and freeze-drying were confirmed, and this was evaluated by comparing antioxidant activity at the stage before fermentation, immediately after fermentation, and after killing cells. As a result, as shown in FIG. 8, the antioxidant activity was increased through fermentation, and it was confirmed that the antioxidant activity was excellent even after the germination process.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC15225BP 20221206

Claims (5)

Lysate of Lacticaseibacillus paracasei subsp. paracasei DCF0429 (KCTC15225BP) strain isolated from makgeolli.
According to claim 1,
The lysate of the strain of Lacticaseibacillus paracasei subsp. paracasei DCF0429 ( KCTC15225BP ) is characterized in that it has antioxidant activity. .paracasei ) Lysate of DCF0429 (KCTC15225BP) strain.
Paracasei subspecies paracasei ( Lacticaseibacillus paracasei subsp. paracasei ) DCF0429 (KCTC15225BP) is inoculated and cultured in a medium containing soybean embryo extract and glucose, characterized in that it comprises a culture that is killed. Postbiotics of Lacticaseibacillus paracasei subsp. paracasei DCF0429 (KCTC15225BP).
According to claim 3,
The badge is
Post bio of Lacticaseibacillus paracasei subsp. paracasei DCF0429 (KCTC15225BP) characterized by containing 2-4% by weight of soybean embryo extract and 2-4% by weight of glucose relative to the total medium ticks.
According to claim 3,
The postbiotics,
Postbiotics of Lacticaseibacillus paracasei subsp. paracasei DCF0429 (KCTC15225BP), characterized by antioxidant activity.

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