KR102457478B1 - Broccoli Sprouts Fermentation Composition with Enhanced Sulforaphane and Quercetin using Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP) of novel lactic acid bacteria and Manufacturing Method thereof - Google Patents
Broccoli Sprouts Fermentation Composition with Enhanced Sulforaphane and Quercetin using Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP) of novel lactic acid bacteria and Manufacturing Method thereof Download PDFInfo
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- KR102457478B1 KR102457478B1 KR1020190176836A KR20190176836A KR102457478B1 KR 102457478 B1 KR102457478 B1 KR 102457478B1 KR 1020190176836 A KR1020190176836 A KR 1020190176836A KR 20190176836 A KR20190176836 A KR 20190176836A KR 102457478 B1 KR102457478 B1 KR 102457478B1
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Abstract
본 발명은 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)를 이용하여 설포라판 및 쿼세틴이 증강된 새싹브로콜리 유산균 발효물 및 이의 제조방법에 관한 것으로서, 보다 상세하게는, 새싹브로콜리 추출물에 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)를 접종한 접종 조성액을 최적의 발효조건 하에서 발효시켜 설포라판 및 쿼세틴이 증강된 새싹브로콜리 유산균 발효물 및 이의 제조방법에 관한 것이다.
본 발명에 따른 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)를 이용하여 설포라판 및 쿼세틴이 증강된 새싹브로콜리 유산균 발효물의 제조방법은 새싹브로콜리 추출물과 부원료 농축액을 혼합한 혼합물을 가열살균하여 가열 살균액을 제조하는 제1단계;와 상기 가열 살균액에 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)을 접종하여 접종 조성액을 제조하는 제2단계;와 상기 접종 조성액을 발효하는 제3단계;를 포함한다.The present invention is a novel lactic acid bacterium Pediococcus acidilactici DU. LAB. K-1 (KCTC 13963BP) to sulforaphane and quercetin-enhanced sprout broccoli lactic acid fermented product and a method for producing the same, and more particularly, a novel lactic acid bacterium Pediococcus acidilactici DU in sprout broccoli extract. LAB. It relates to a fermented sprouted broccoli lactic acid bacterium with enhanced sulforaphane and quercetin by fermenting the inoculum composition inoculated with K-1 (KCTC 13963BP) under optimal fermentation conditions and a method for producing the same.
A novel lactic acid bacterium Pediococcus acidilactici DU according to the present invention. LAB. The method for producing a fermented sprout broccoli lactic acid bacteria enhanced with sulforaphane and quercetin by using K-1 (KCTC 13963BP) is a first step of preparing a heat sterilizing solution by heat sterilizing a mixture of a sprout broccoli extract and an auxiliary raw material concentrate; and A novel lactic acid bacterium Pediococcus acidilactici DU. LAB. A second step of preparing an inoculation composition by inoculating K-1 (KCTC 13963BP); and a third step of fermenting the inoculation composition.
Description
본 발명은 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)를 이용하여 설포라판 및 쿼세틴이 증강된 새싹브로콜리 유산균 발효물 및 이의 제조방법에 관한 것으로서, 보다 상세하게는, 새싹브로콜리 추출물에 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)를 접종한 접종 조성액을 최적의 발효조건 하에서 발효시켜 설포라판 및 쿼세틴이 증강된 새싹브로콜리 유산균 발효물 및 이의 제조방법에 관한 것이다. The present invention is a novel lactic acid bacterium Pediococcus acidilactici DU. LAB. K-1 (KCTC 13963BP) to sulforaphane and quercetin-enhanced sprout broccoli lactic acid fermented product and a method for producing the same, and more particularly, a novel lactic acid bacterium Pediococcus acidilactici DU in sprout broccoli extract. LAB. It relates to a fermented sprouted broccoli lactic acid bacterium with enhanced sulforaphane and quercetin by fermenting the inoculum composition inoculated with K-1 (KCTC 13963BP) under optimal fermentation conditions and a method for producing the same.
브로콜리(Brasica oleracea var. italica Plenck)는 십자화과에 속하는 채소로 잎과 줄기는 모두 버려지고 작은 꽃봉오리가 다발로 이루어진 꽃만을 식용하며, 한 줄기에서 꽃이 4∼5개 정도 맺히고, 잎은 10∼15장 이상 나와 매우 무성하게 된다. Broccoli ( Brasica oleracea var. italica Plenck) is a vegetable belonging to the cruciferous family. All leaves and stems are discarded and only flowers made up of small flower buds are eaten. With more than 15 chapters, it becomes very lush.
브로콜리는 항산화 물질로 알려진 아스코빅산(ascorbic acid), 베타카로틴(β-carotene), 루틴(rutin), 셀레늄(selenium), 쿼세틴(quercetin) 및 글루타치온(glutathione) 등이 다량 함유되어 있으며, 암세포증식억제 및 해독 효소의 유도 효과가 크다고 알려져 많은 연구가 진행되고 있다. Broccoli contains a large amount of ascorbic acid, beta-carotene, rutin, selenium, quercetin and glutathione, known as antioxidants, and inhibits the growth of cancer cells. and detoxification enzymes are known to have a large inducing effect, and many studies are being conducted.
특히, 브로콜리에는 신체의 자연 치유력을 증강 시켜 암 발행의 위험성을 줄여주는 물질인 설포라판(sulforaphane, S-methylsulfinylbutylisothiocyanate)이 함유되어 있다고 알려져 있는데, 설포라판은 최근 연구에서 발암물질로 전처리한 생쥐의 유선에서 종양발생을 억제하고, 전립선암의 예방에도 유효한 것으로 보고되었다. 또한, in vitro 실험에서 설포라판이 암 예방 효과뿐만 아니라 헬리코박터에 대한 강력한 살균 효과가 있음이 보고되었다. In particular, broccoli is known to contain sulforaphane (S-methylsulfinylbutylisothiocyanate), a substance that reduces the risk of cancer by enhancing the body's natural healing ability. It has been reported to be effective in inhibiting the occurrence and preventing prostate cancer. In addition, it has been reported that sulforaphane has a strong bactericidal effect against Helicobacter as well as a cancer-preventing effect in an in vitro experiment.
종래 새싹브로콜리와 관련한 특허문헌으로, 한국등록특허 제10-1666021호는 회전식 새싹 재배 장치를 이용하여 생산된 브로콜리 새싹을 설포라판 농축액에 침지시킨 후 원적외선 볶음 장치에서 볶아 그 영양성분을 향상시킨 브로콜리 새싹을 이용한 기능성 차와 그의 제조방법을 제시하고 있으며, 한국등록특허 제10-1965391호는 브 콜리 종자에 포졸란 혼합액을 공급하면서 발아 및 새싹을 수득하고, 수득된 브로콜리 새싹을 감압 건조하여 설포라판이 증가된 브로콜리 새싹 추출물을 제시하고 있다.As a patent document related to conventional sprouted broccoli, Korean Patent Registration No. 10-1666021 discloses that broccoli sprouts produced using a rotary sprout cultivation device are immersed in a sulforaphane concentrate and then roasted in a far-infrared roasting device with improved nutritional content. It presents a functional tea and a manufacturing method thereof, and Korean Patent No. 10-1965391 provides germination and sprouts while supplying a pozzolan mixture to broccoli seeds, and drying the obtained broccoli sprouts under reduced pressure to obtain broccoli with increased sulforaphane. Sprout extract is presented.
또한, 한국공개특허 제10-2019-0097741호는 용암해수가 함유된 용수하에서 브로콜리 종자를 발아시키고 발아된 브로콜리 종자에 상기 용암해수가 함유된 용수를 공급하여 제조되는 성장률 및 설포라판 함량이 증가된 브로콜리 새싹 및 이의 생산방법을 제시하고 있으며, 한국등록특허 제10-1807367호는 글루코스 및 프룩토스 중 어느 하나의 당과 브로콜릭 새싹을 혼합한 당 혼합물에 에탄올을 첨가한 후 분획한 브로콜리 새싹 추출물을 유효성분으로 함유하는 항산화 조성물 및 그의 제조방법을 제시하고 있다.In addition, Korea Patent Publication No. 10-2019-0097741 discloses broccoli with increased growth rate and sulforaphane content produced by germinating broccoli seeds under spring water containing lava seawater and supplying water containing lava seawater to the germinated broccoli seeds. Sprouts and their production methods are presented, and Korean Patent No. 10-1807367 is effective in using a broccoli sprout extract fractionated after adding ethanol to a sugar mixture in which any one of glucose and fructose and broccoli sprouts are mixed. An antioxidant composition containing as a component and a method for preparing the same are provided.
본 발명자는 새싹브로콜리에 포함된 유효성분인 설포라판 및 쿼세틴을 증강시키기 위한 연구의 일환으로 새싹브로콜리 추출물에 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)를 접종한 접종 조성액을 최적의 발효조건 하에서 발효시킨 새싹브로콜리 유산균 발효물을 개발하였으며, 상기 새싹브로콜리 유산균 발효물로부터 설포라판 및 쿼세틴이 증강되었음을 확인하여 본 발명에 이르게 되었다. The present inventors developed a novel lactic acid bacterium Pediococcus acidilactici DU in sprout broccoli extract as part of a study to enhance the active ingredients sulforaphane and quercetin contained in sprout broccoli. LAB. K-1 (KCTC 13963BP) inoculated inoculum composition was fermented under optimal fermentation conditions to develop a fermented sprout broccoli lactic acid bacteria, and from the sprout broccoli lactic acid bacteria fermented sulforaphane and quercetin were confirmed to be enhanced, leading to the present invention.
상기와 같은 문제점을 해결하기 위한 본 발명의 목적은 새싹브로콜리 추출물에 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)를 접종한 접종 조성액을 최적의 발효조건 하에서 발효시켜 설포라판 및 쿼세틴이 증강된 새싹브로콜리 유산균 발효물 및 이의 제조방법을 제공하는 것이다.An object of the present invention for solving the above problems is a novel lactic acid bacterium Pediococcus acidilactici DU in sprout broccoli extract. LAB. It is to provide a fermented sprouted broccoli lactic acid bacterium with enhanced sulforaphane and quercetin by fermenting the inoculum composition inoculated with K-1 (KCTC 13963BP) under optimal fermentation conditions and a method for preparing the same.
상기 과제를 해결하기 위한 본 발명의 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)를 이용하여 설포라판 및 쿼세틴이 증강된 새싹브로콜리 유산균 발효물의 제조방법은 새싹브로콜리 추출물과 부원료 농축액을 혼합한 혼합물을 가열살균하여 가열 살균액을 제조하는 제1단계;와 상기 가열 살균액에 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)을 접종하여 접종 조성액을 제조하는 제2단계;와 상기 접종 조성액을 발효하는 제3단계;를 포함한다.A novel lactic acid bacterium Pediococcus acidilactici DU of the present invention for solving the above problems. LAB. The method for producing a fermented sprout broccoli lactic acid bacteria enhanced with sulforaphane and quercetin by using K-1 (KCTC 13963BP) is a first step of preparing a heat sterilizing solution by heat sterilizing a mixture of a sprout broccoli extract and an auxiliary raw material concentrate; and A novel lactic acid bacterium Pediococcus acidilactici DU. LAB. A second step of preparing an inoculation composition by inoculating K-1 (KCTC 13963BP); and a third step of fermenting the inoculation composition.
상기 제1단계의 새싹브로콜리 추출물은 분쇄액, 농축액, 건조물, 분말 및 이들의 조합 중 어느 하나를 포함한다.The sprout broccoli extract of the first step includes any one of a pulverized liquid, a concentrate, a dry matter, a powder, and a combination thereof.
상기 제1단계의 부원료 농축액은 바나나 농축액, 복숭아농축액, 망고농축액, 딸기농축액, 토마토농축액 및 당근농축액 중 적어도 어느 하나를 포함하며, 1 내지 90 °Brix를 갖는 것을 특징으로 한다.The auxiliary raw material concentrate of the first step includes at least one of banana concentrate, peach concentrate, mango concentrate, strawberry concentrate, tomato concentrate, and carrot concentrate, and it is characterized in that it has 1 to 90 ° Brix.
상기 제1단계는 새싹브로콜리 추출물 100 부피부에 대하여 부원료 농축액 0.1 내지 20 부피부를 첨가하는 것을 특징으로 한다.The first step is characterized by adding 0.1 to 20 parts by volume of the auxiliary raw material concentrate based on 100 parts by volume of the sprout broccoli extract.
상기 부원료 농축액은 갈락토올리고당, 말토올리고당, 프락토올리고당, 이소말토올리고당 및 이들의 조합 중 어느 하나의 당을 더 포함하는 것을 특징으로 한다.The auxiliary raw material concentrate further comprises any one of galactooligosaccharide, maltooligosaccharide, fructooligosaccharide, isomaltooligosaccharide, and combinations thereof.
상기 제2단계에서는 가열 살균액 100중량부에 대하여 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)을 0.1 내지 50중량부 접종하는 것을 특징으로 한다.In the second step, the novel lactic acid bacterium Pediococcus acidilactici DU. LAB. It is characterized by inoculating 0.1 to 50 parts by weight of K-1 (KCTC 13963BP).
상기 제3단계에서는 접종 조성액을 20 내지 50℃에서 6 내지 96시간 발효하는 것을 특징으로 한다.In the third step, the inoculation composition It is characterized in that it is fermented for 6 to 96 hours at 20 to 50 ℃.
상술한 바와 같이, 본 발명에 따른 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)를 이용하여 설포라판 및 쿼세틴이 증강된 새싹브로콜리 유산균 발효물 및 이의 제조방법에 의하면, 균주의 활성도와 베타글로코시데이즈(β-glucosidase) 활성이 높은 분리 동정된 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)을 새싹브로콜리 추출물에 첨가 및 최적의 발효조건 하에서 발효시켜 새싹브로콜리 내 배당체 성분인 루틴(rutin)을 장내 흡수율이 높은 비배당체인 쿼세틴(quercetin)로 효율적으로 전환 또는 부분적 전환하고, 새싹브로콜리 유효성분인 설포라판(sulforaphane)의 함량을 높인 효과가 있다. As described above, the novel lactic acid bacterium Pediococcus acidilactici DU according to the present invention. LAB. According to the fermented sprouted broccoli lactic acid bacteria enhanced with sulforaphane and quercetin by using K-1 (KCTC 13963BP) and a manufacturing method thereof, the activity of the strain and beta-glucosidase activity are high, isolated and identified novel Lactobacillus Pediococcus acidilactici DU. LAB. K-1 (KCTC 13963BP) is added to sprout broccoli extract and fermented under optimal fermentation conditions to efficiently convert or partially convert rutin, a glycoside component in sprout broccoli, into quercetin, a non-glycoside with high intestinal absorption rate. It has the effect of increasing the content of sulforaphane, an active ingredient in sprout broccoli.
도 1은 본 발명에 따른 신규한 유산균으로 발효되어 설포라판(sulforaphane)과 쿼세틴(quercetin)의 함량이 증강된 새싹브로콜리 유산균 발효물 제조단계를 개략적으로 나타낸 흐름도.
도 2는 각종 발효식품으로부터 분리된 유산균.
도 3은 분리 유산균의 베타-글루코시데이즈(β-glucosidase)활성을 보이는 에스큘린(esculin) 고체배지 상 그림.
도 4는 최종 선별 균주 Pediococcus acidilactici DU. LAB. K-1의 뉴클레오타이드 염기서열.
도 5는 최종 선별 균주 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)의 16S rRNA의 계통수 분석.
도 6은 발효시간, 브로콜리 농도 및 발효 부원료 농도에 따른 새싹브로콜리 발효물의 유산균수(12.2-13.2-14.2 Log10 CFU/mL)에 대한 반응표면도.
도 7은 발효시간, 브로콜리 농도 및 발효 부원료 농도에 따른 새싹브로콜리 발효물의 유산함량(12.0-12.5-13.0 mg/mL)에 대한 반응표면도.
도 8은 발효시간, 브로콜리 농도 및 발효 부원료 농도에 따른 새싹브로콜리 발효물의 sulforaphane 증가함량(11-12-13 ㎍/mL)에 대한 반응표면도.
도 9는 발효시간, 브로콜리 농도 및 발효 부원료 농도에 따른 새싹브로콜리 발효물의 rutin 감소함량(8-10-12 ㎍/mL)에 대한 반응표면도.
도 10은 발효시간, 브로콜리 농도 및 발효 부원료 농도에 따른 새싹브로콜리 발효물의 quercetin 증가함량(1.0-1.1-1.2 ㎍/mL)에 대한 반응표면도.
도 11은 발효시간과 브로콜리 농도 및 발효 부원료 농도에 따른 새싹브로콜리 발효물의 유산균수, lactic acid 함량, sulforaphane 증가함량, lutin 감소함량 및 quercetin 증가함량의 발효조건 최적화를 위한 수퍼임포징된(superimposed) 반응표면.
도 12는 새싹브로콜리 유효성분 설포라판(sulforaphan)의 HPLC분석 크로마토 그램. (A); 설포라판(sulforaphan) 표준물질 크로마토 그램, (B); 발효전 새싹브로콜리 추출물의 설포라판 크로마토 그램, (C); 새싹브로콜리 발효물의 설포라판 크로마토 그램
도 13은 새싹브로콜리 유효성분 루틴(rutin)과 쿼세틴(quercetin)의 HPLC분석 크로마토 그램. (A); 루틴(rutin)과 쿼세틴(quercetin) 표준물질 크로마토 그램, (B); 발효전 새싹브로콜리 추출물의 루틴(rutin)과 쿼세틴(quercetin) 크로마토 그램, (C); 새싹브로콜리 발효물의 루틴(rutin)과 쿼세틴(quercetin) 크로마토 그램1 is a flowchart schematically showing the production step of fermented sprout broccoli lactic acid bacteria in which the content of sulforaphane and quercetin is enhanced by being fermented with novel lactic acid bacteria according to the present invention.
Figure 2 is lactic acid bacteria isolated from various fermented foods.
Figure 3 is a beta-glucosidase ( β -glucosidase) of the isolated lactic acid bacteria showing the activity of esculin (esculin) solid medium phase picture.
4 shows the final selection strain Pediococcus acidilactici DU. LAB. The nucleotide sequence of K-1.
Figure 5 shows the final selection strain Pediococcus acidilactici DU. LAB. Phylogenetic analysis of 16S rRNA of K-1 (KCTC 13963BP).
6 is a response surface diagram for the number of lactic acid bacteria (12.2-13.2-14.2 Log10 CFU/mL) of sprouted broccoli fermented products according to fermentation time, broccoli concentration and fermentation auxiliary material concentration.
7 is a response surface diagram for the lactic acid content (12.0-12.5-13.0 mg/mL) of sprouted broccoli fermented products according to fermentation time, broccoli concentration and fermentation auxiliary material concentration.
8 is a response surface diagram for the increased sulforaphane content (11-12-13 μg/mL) of sprouted broccoli fermented products according to fermentation time, broccoli concentration and fermentation auxiliary material concentration.
9 is a response surface diagram of the rutin reduction content (8-10-12 μg/mL) of sprouted broccoli fermented products according to fermentation time, broccoli concentration and fermentation auxiliary material concentration.
10 is a response surface diagram for the increased content of quercetin (1.0-1.1-1.2 μg/mL) in sprouted broccoli fermented products according to fermentation time, broccoli concentration and fermentation auxiliary material concentration.
11 is a superimposed reaction for optimizing fermentation conditions of the number of lactic acid bacteria, lactic acid content, sulforaphane increase, lutin decrease, and quercetin increase content of sprouted broccoli fermented product according to fermentation time, broccoli concentration and fermentation auxiliary material concentration. surface.
Figure 12 is a chromatogram of HPLC analysis of sprout broccoli active ingredient sulforaphan. (A); sulforaphan standard chromatogram, (B); Sulforaphane chromatogram of sprouted broccoli extract before fermentation, (C); Sulforaphane Chromatogram of Sprout Broccoli Fermented Product
13 is an HPLC analysis chromatogram of sprout broccoli active ingredients rutin and quercetin. (A); Chromatograms of rutin and quercetin standards, (B); Chromatograms of rutin and quercetin of sprouted broccoli extract before fermentation, (C); Chromatograms of Rutin and Quercetin in Sprout Broccoli Fermented Product
본 발명의 구체적 특징 및 이점들은 이하에서 첨부도면을 참조하여 상세히 설명한다. 이에 앞서 본 발명에 관련된 기능 및 그 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 구체적인 설명을 생략하기로 한다.Specific features and advantages of the present invention will be described in detail below with reference to the accompanying drawings. Prior to this, if it is determined that the detailed description of the function and its configuration related to the present invention may unnecessarily obscure the gist of the present invention, the detailed description will be omitted.
본 발명은 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)를 이용하여 설포라판 및 쿼세틴이 증강된 새싹브로콜리 유산균 발효물 및 이의 제조방법에 관한 것으로서, 보다 상세하게는, 새싹브로콜리 추출물에 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)를 접종한 접종 조성액을 최적의 발효조건 하에서 발효시켜 설포라판 및 쿼세틴이 증강된 새싹브로콜리 유산균 발효물 및 이의 제조방법에 관한 것이다. The present invention is a novel lactic acid bacterium Pediococcus acidilactici DU. LAB. K-1 (KCTC 13963BP) to sulforaphane and quercetin-enhanced sprout broccoli lactic acid fermented product and a method for producing the same, and more particularly, a novel lactic acid bacterium Pediococcus acidilactici DU in sprout broccoli extract. LAB. It relates to a fermented sprouted broccoli lactic acid bacterium with enhanced sulforaphane and quercetin by fermenting the inoculum composition inoculated with K-1 (KCTC 13963BP) under optimal fermentation conditions and a method for producing the same.
도 1은 본 발명에 따른 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)를 이용하여 설포라판 및 쿼세틴이 증강된 새싹브로콜리 유산균 발효물의 제조방법을 보여주는 순서도이다. 1 is a novel lactic acid bacterium Pediococcus acidilactici DU according to the present invention. LAB. It is a flowchart showing a method for producing a fermented sprouted broccoli lactic acid bacteria enhanced with sulforaphane and quercetin using K-1 (KCTC 13963BP).
본 발명에 따른 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)를 이용하여 설포라판 및 쿼세틴이 증강된 새싹브로콜리 유산균 발효물의 제조방법은 새싹브로콜리 추출물과 부원료 농축액을 혼합한 혼합물을 가열살균하여 가열 살균액을 제조하는 제1단계와 상기 가열 살균액에 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)을 접종하여 접종 조성액을 제조하는 제2단계와 상기 접종 조성액을 발효하는 제3단계를 포함한다.A novel lactic acid bacterium Pediococcus acidilactici DU according to the present invention. LAB. The method for producing a fermented sprout broccoli lactic acid bacteria enhanced with sulforaphane and quercetin using K-1 (KCTC 13963BP) is a first step of preparing a heat sterilizing solution by heat sterilizing a mixture of a sprout broccoli extract and an auxiliary raw material concentrate, and the heating A novel lactic acid bacterium Pediococcus acidilactici DU. LAB. A second step of preparing an inoculation composition by inoculating K-1 (KCTC 13963BP) and a third step of fermenting the inoculation composition.
상기 제1단계에서는 새싹브로콜리 추출물과 부원료 농축액을 혼합한 혼합물을 살균온도 60 내지 135℃ 에서 1 내지 60분간 가열살균하여 가열 살균액을 제조한다In the first step A mixture of the sprout broccoli extract and the auxiliary raw material concentrate is sterilized by heating at a sterilization temperature of 60 to 135° C. for 1 to 60 minutes to prepare a heat sterilizing solution.
상기 새싹브로콜리 추출물은 새싹브로콜리의 분쇄액, 농축액, 건조물, 분말 및 이들의 조합 중 어느 하나를 포함하며, 바람직하게는, 분쇄 새싹브로콜리에 5 내지 10배수의 증류수를 가하여 50 내지 80℃에서 6 내지 12시간 추출 후 원심분리된 상등액을 10 내지 15 °Brix를 갖도록 감압농축한 것을 사용할 수 있다. The sprout broccoli extract includes any one of a crushed solution, a concentrate, a dry product, a powder, and a combination thereof of sprout broccoli, and preferably, 5 to 10 times distilled water is added to the crushed sprout broccoli to 6 to 80° C. After 12 hours of extraction, the centrifuged supernatant may be concentrated under reduced pressure to have 10 to 15 ° Brix.
상기 부원료 농축액은 바나나 농축액, 복숭아농축액, 망고농축액, 딸기농축액, 토마토농축액 및 당근농축액 중 적어도 어느 하나를 포함하며, 이에 한정하는 것은 아니다. The auxiliary raw material concentrate includes at least one of a banana concentrate, a peach concentrate, a mango concentrate, a strawberry concentrate, a tomato concentrate, and a carrot concentrate, but is not limited thereto.
이때, 상기 부원료 농축액은 1 내지 90 °Brix를 갖는 것을 사용할 수 있으며, 바람직하게는 50 내지 80 °Brix를 사용하며, 더욱 바람직하게는, 60°Brix 인 것을 사용할 수 있다.At this time, as the auxiliary raw material concentrate, one having 1 to 90 °Brix may be used, preferably 50 to 80 °Brix may be used, and more preferably, one having 60 °Brix may be used.
상기 제1단계는 새싹브로콜리 추출물 100 부피부에 대하여 부원료 농축액 0.1 내지 20 부피부를 첨가할 수 있으며, 바람직하게는 5 내지 18 부피부를 첨가할 수 있으며, 더욱 바람직하게는 10 부피부를 첨가할 수 있다.In the first step, 0.1 to 20 parts by volume of the auxiliary raw material concentrate may be added with respect to 100 parts by volume of the sprout broccoli extract, preferably 5 to 18 parts by volume, and more preferably 10 parts by volume may be added. can
또한, 상기 부원료 농축액은 갈락토올리고당, 말토올리고당, 프락토올리고당, 이소말토올리고당 및 이들의 조합 중 어느 하나의 당을 더 포함할 수 있다.In addition, the auxiliary raw material concentrate may further include any one of galactooligosaccharides, maltooligosaccharides, fructooligosaccharides, isomaltooligosaccharides, and combinations thereof.
제2단계에서는 상기 가열 살균액에 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)을 접종하여 접종 조성액을 제조한다.In the second step A novel lactic acid bacterium Pediococcus acidilactici DU. LAB. Inoculate K-1 (KCTC 13963BP) to prepare an inoculation composition.
신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)은 김치 수십 종 및 침채류 등의 발효식품으로부터 분리한 것으로, 베타글루코시데이스 활성을 가지며, MRS broth 배지에서 6 내지 24시간 전배양된 전배양액을 사용한다.A novel lactic acid bacterium Pediococcus acidilactici DU. LAB. K-1 (KCTC 13963BP) is isolated from fermented foods such as dozens of kimchi and pickled vegetables, has beta-glucosidase activity, and uses a pre-culture solution pre-cultured for 6 to 24 hours in MRS broth medium.
상기 제2단계에서는 가열 살균액 100중량부에 대하여 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)을 0.1 내지 50중량부 접종하며, 바람직하게는 1 내지 10 중량부를 첨가할 수 있으며, 더욱 바람직하게는 2중량부를 첨가할 수 있다.In the second step, the novel lactic acid bacterium Pediococcus acidilactici DU. LAB. 0.1 to 50 parts by weight of K-1 (KCTC 13963BP) is inoculated, preferably 1 to 10 parts by weight may be added, and more preferably 2 parts by weight may be added.
상기 제3단계에서는 접종 조성액을 발효하는데 접종 조성액을 20 내지 50℃에서 6 내지 96시간 발효하며, 바람직하게는 30 내지 45℃에서 40 내지 70시간 발효할 수 있으며, 더욱 바람직하게는 37℃에서 55시간 동안 발효할 수 있다. In the third step, To ferment the inoculation composition, the inoculation composition is It may be fermented at 20 to 50° C. for 6 to 96 hours, preferably at 30 to 45° C. for 40 to 70 hours, and more preferably at 37° C. for 55 hours.
본 발명에 따른 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)를 이용하여 설포라판 및 쿼세틴이 증강된 새싹브로콜리 유산균 발효물의 제조방법은 상기 3단계 수행 후, 새싹브로콜리 유산균 발효물을 살균온도 60 내지 135℃ 에서 1 내지 60분간 가열살균처리하는 4단계와 10 마이크로 필터 여과기를 통과시켜 여과하는 제5단계를 더 포함할 수 있다. A novel lactic acid bacterium Pediococcus acidilactici DU according to the present invention. LAB. The method for producing fermented sprout broccoli lactic acid bacteria enhanced with sulforaphane and quercetin using K-1 (KCTC 13963BP) is after performing the above three steps, the sprout broccoli lactic acid bacteria fermented product is heat sterilized at a sterilization temperature of 60 to 135° C. for 1 to 60 minutes. It may further include a fourth step and a fifth step of filtering through a 10 micro filter filter.
본 발명에 따른 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)를 이용하여 설포라판 및 쿼세틴이 증강된 새싹브로콜리 유산균 발효물은상술된 제조방법에 의해 제조되며, 이에 관한 상세한 설명은 생략하도록 한다.A novel lactic acid bacterium Pediococcus acidilactici DU according to the present invention. LAB. Sulphoraphane and quercetin-enhanced sprout broccoli lactic acid bacteria fermentation using K-1 (KCTC 13963BP) is prepared by the above-described manufacturing method, and detailed description thereof will be omitted.
이하, 본 발명에 따른 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)를 이용하여 설포라판 및 쿼세틴이 증강된 새싹브로콜리 유산균 발효 음료의 제조방법(이하, 새싹브로콜리 유산균 발효 음료의 제조방법)을 설명하도록 한다.Hereinafter, a novel lactic acid bacterium Pediococcus acidilactici DU according to the present invention. LAB. Using K-1 (KCTC 13963BP), a method for producing a fermented sprouted broccoli lactic acid bacterium drink enhanced with sulforaphane and quercetin (hereinafter, a method for preparing a sprout broccoli lactic acid bacterium fermented beverage) will be described.
본 발명에 따른 새싹브로콜리 유산균 발효 음료의 제조방법은 새싹브로콜리 유산균 발효물을 포함하는 것으로서, 상술된 새싹브로콜리 유산균 발효물의 제조방법의 또한, 상기 제4단계를 거친 가열살균처리된 발효물 또는 상기 제5단계를 거친 여과처리된 여과물 100 중량부에 기호도 향상을 위하여 과실추출물 0.1 내지 99.9 중량부를 첨가하는 6단계를 더 포함할 수 있다. The method for producing a sprout broccoli lactic acid bacteria fermented beverage according to the present invention comprises a sprout broccoli lactic acid bacteria fermented product. A sixth step of adding 0.1 to 99.9 parts by weight of the fruit extract to 100 parts by weight of the filtered filtrate that has undergone five steps may be further included in order to improve the preference.
상기 과실추출물은 착즙액, 농축액, 분쇄액, 분말 및 이들의 조합 중 어느 하나의 형태로 첨가될 수 있으며, 사과, 감, 블루베리, 블랙베리, 블랙커런트, 아사이베리, 복분자, 석류, 포도, 배, 복숭아, 바나나, 딸기, 토마토, 당근, 자두, 매실, 앵두, 다래, 오디, 머루, 키위, 참외, 귤, 오렌지, 자몽, 귤, 멜론, 체리, 멜론, 블랙수퍼베리, 망고, 레몬, 파인애플 및 이들의 조합 중 어느 하나로부터 추출되는 추출물을 사용할 수 있으나, 기호도 향상을 위한 것이라면 이에 한정하는 것은 아니다. The fruit extract may be added in the form of any one of juice, concentrate, pulverized liquid, powder, and combinations thereof, and may include apples, persimmons, blueberries, blackberries, blackcurrants, acai berries, bokbunja, pomegranates, grapes, Pear, Peach, Banana, Strawberry, Tomato, Carrot, Plum, Plum, Cherry, Actinidia, Odi, Meruru, Kiwi, Melon, Tangerine, Orange, Grapefruit, Tangerine, Melon, Cherry, Melon, Black Superberry, Mango, Lemon, An extract extracted from any one of pineapple and combinations thereof may be used, but it is not limited thereto if it is intended to improve taste.
본 발명에 따른 새싹브로콜리 유산균 발효 음료는 상술된 새싹브로콜리 유산균 발효 음료의 제조방법에 의해 제조되며, 이에 관한 상세한 설명은 생략하도록 한다.Sprout broccoli lactic acid bacteria fermented beverage according to the present invention is prepared by the method for producing the above-described sprout broccoli lactic acid bacteria fermented beverage, and detailed description thereof will be omitted.
본 발명에서는 새싹브로콜리 유산균 발효물은 장내 흡수율이 높은 비배당체성분인 쿼세틴(quercetin)과 새싹브로콜리 유효성분인 설포라판(sulforaphane)의 함량이 높아 음료뿐만 아니라 건강보조식품, 건강기능식품 등에 다양한 제품 및 제형의 형태로 활용될 수 있다.In the present invention, sprout broccoli lactic acid bacteria fermented product has a high content of quercetin, a non-glycolysate component with high intestinal absorption, and sulforaphane, an active ingredient in sprout broccoli, as well as beverages, as well as various products and formulations such as health supplements and health functional foods. can be used in the form of
이하, 본 발명의 실시예 및 실험예를 들어 상세히 설명한다. 단, 하기의 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예는 한정되는 것은 아니다.Hereinafter, examples and experimental examples of the present invention will be described in detail. However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.
실험재료와 방법Experimental materials and methods
1. 유산균1. Lactobacillus
1-1. 유산균의 분리1-1. Isolation of lactic acid bacteria
유산균 분리에 사용한 발효식품 시료는 가정집 및 음식점에서 담근 김치 수십 종 및 침채류 등을 수집하여 유산균 분리 시료액으로 사용하였으며, 각 시료액을 멸균증류수로 10-1 ∼ 10-5으로 희석한 후 0.004% 브로모크레졸퍼플(BCP)가 첨가된 시판용 MRS 한천배지에 100 μl 씩 분주 도말하고, 37℃에서 24시간 배양 후 나타난 독립된 콜로니 중 균주의 크기, 색, 모양, 투명도 등을 관찰하여 유산균의 특징적인 콜로니를 순수 분리하였다. 순수 분리한 유산균은 MRS 한천사면배지에 접종하여 37℃에서 24시간 배양한 후 4℃ 냉장보관하면서 사용하였다.Fermented food samples used to isolate lactic acid bacteria were collected by collecting dozens of kimchi and pickled vegetables from households and restaurants and used as lactic acid bacteria separation sample solutions. After diluting each sample solution with sterile distilled water to 10 -1 to 10 -5 , 0.004 % Bromocresol Purple (BCP) is added to a commercial MRS agar medium in an aliquot of 100 μl, and the size, color, shape, and transparency of the strains are observed among the independent colonies that appeared after culturing at 37°C for 24 hours. Red colonies were isolated. The pure isolated lactic acid bacteria were inoculated on MRS agar sloping medium, incubated at 37°C for 24 hours, and then used while refrigerated at 4°C.
1-2. 베타글루코시데이스(β-Glucosidase) 활성 균주 선발1-2. Selection of beta-glucosidase (β-Glucosidase) active strains
베타글루코시데이스 활성을 갖는 균주의 선발을 위해 에스쿨린 한천(esculin 0.1%, peptones 1.8%, ferric citrate 0.1%, agar 2%)법을 이용하여 에스쿨린(esculin)이 함유된 에스쿨린 한천배지에 균주를 접종하여 배지 내에서의 색깔 변화를 관찰하였다. 에스쿨린은 베타글루코시데이스에 의하여 글루코스(glucose)와 에스쿨린으로 분리되며 에스쿨린은 페릭 암모니움 시트레이트(ferric ammonium citrate)와 반응하여 콜로니주위에 검은색 반점(black complex)를 형성하게 된다. 따라서 에스쿨린 한천배지에서 배양된 콜로니주위에 검은색 반점를 형성하는 균주를 베타글루코시데이스 활성을 가지는 균주로 판단하여 선별하였다.For the selection of strains with beta-glucosidase activity, esculin agar (esculin 0.1%, peptones 1.8%, ferric citrate 0.1%,
1-3. 베타글루코시데이스(β-Glucosidase) 활성능 측정1-3. Measurement of beta-glucosidase activity
베타글루코시데이스 활성 측정은 1% 카르복시메칠셀루로스(carboxymethyl cellulose, CMC)가 첨가된 시판용 MRS 배지에 12시간 전배양한 균주를 접종하여 37℃에서 24시간 배양 후, 배양액을 4℃에서 6000 rpm으로 15분간 원심분리하여 세포를 제거하고, 상층액 0.5 ml를 취하여 1 ml의 5 mM 파라-니트로페닐-베타-디-글루코피라노사이드(ρ-nitrophenyl-β-D-glucopynanoside, ρ-NPG)용액과 혼합한 후 37℃에서 30분간 반응시켰다. 반응액은 1 ml의 1 M 탄산나트륨(Na2CO3)용액을 첨가하여 반응을 중지시키고, 생성된 파라-니트로페놀(p-nitrophenol, ρ-NP)를 405 nm에서 흡광도를 측정한 후 파라-니트로페놀의 검량곡선을 이용하여 농도를 환산하였다.Beta-glucosidase activity was measured by inoculating a strain pre-cultured for 12 hours in a commercial MRS medium supplemented with 1% carboxymethyl cellulose (CMC), culturing at 37°C for 24 hours, and then incubating the culture medium at 4°C at 6000 rpm. Remove cells by centrifugation for 15 minutes, take 0.5 ml of the supernatant, and 1 ml of 5 mM para-nitrophenyl-beta-di-glucopyranoside (ρ-nitrophenyl-β-D-glucopynanoside, ρ-NPG) After mixing with the solution, it was reacted at 37°C for 30 minutes. The reaction solution was stopped by adding 1 ml of 1 M sodium carbonate (Na 2 CO 3 ) solution, and the resulting para-nitrophenol (p-nitrophenol, ρ-NP) was measured for absorbance at 405 nm and then para- The concentration was converted using the calibration curve of nitrophenol.
1-4. 분리 유산균주의 새싹브로콜리 추출액 농도에 따른 균 생장실험1-4. Bacterial growth test according to the concentration of the sprouted broccoli extract of the isolated lactic acid strain
새싹브로콜리 추출액을 농도별로 (5, 10, 15, 20, 50 및 100%) 첨가한 MRS agar에 분리 균주를 획선 도말하여 37℃에서 24시간 배양 후 균의 생장정도를 관찰하였다.The isolated strains were streaked on MRS agar to which sprouted broccoli extracts were added by concentration (5, 10, 15, 20, 50 and 100%), and the growth degree of the bacteria was observed after culturing at 37° C. for 24 hours.
1-5. 새싹채소 추출액의 유산발효 검토1-5. Lactic acid fermentation review of sprout vegetable extract
분리균주의 새싹브로콜리에 대한 발효능을 조사하기 위해 새싹브로콜리 10%를 첨가하여 추출한 추출물을 기본 배지로 하여 위에서 선별된 유산균 3종을 MRS broth 배지에 12시간 전배양 후 2% 접종 후 발효시간을 달리하여 발효하였다. In order to investigate the fermentation ability of the isolated strain on sprout broccoli, the extract extracted by adding 10% of sprout broccoli was used as a basic medium, and the three types of lactic acid bacteria selected above were pre-cultured in MRS broth medium for 12 hours and then the fermentation time was measured after inoculation with 2%. It was fermented differently.
1-6. 최종선별균주의 염기서열분석과 계통분류 1-6. Base sequencing and phylogenetic classification of last-selected strains
최종 선별된 균주의 16S rRNA 유전자는 시퀀싱(sequencing) 을 통하여 분석하였으며, NCBI 데이터베이스(database)를 이용하여 분리된 균주와 데이터베이스상의 표준균주(type strain)와의 유사성(similarity, %)을 확인하였다.The 16S rRNA gene of the finally selected strain was analyzed through sequencing, and the similarity (%) between the isolated strain and the type strain on the database was confirmed using the NCBI database.
2. 새싹브로콜리 추출물2. Sprout Broccoli Extract
2-1. 새싹브로콜리 추출물의 준비2-1. Preparation of Sprout Broccoli Extract
본 발명에서 사용된 새싹브로콜리 원료는 2018년에 7월에 수확된 것 중 길이가 10 cm 전후의 것을 구입한 후, 일정한 크기로 잘라 10배수의 물을 가하여 67℃에서 7시간 추출하였다. 이들 추출물을 감압농축하여 최종 농도를 15 °Brix로 하여 유산발효를 위한 원료로 사용하였다.Sprout broccoli raw materials used in the present invention were harvested in July of 2018, after purchasing those with a length of about 10 cm, cut to a certain size, added 10 times water, and extracted at 67 ° C. for 7 hours. These extracts were concentrated under reduced pressure and the final concentration was 15 °Brix, which was used as a raw material for lactic acid fermentation.
2-2. 새싹브로콜리 유산발효조건 최적화2-2. Optimization of lactic acid fermentation conditions for sprouted broccoli
새싹브로콜리의 유산발효조건을 최적화하기 위하여 반응표면분석법(repose surface methodology, RSM)을 이용하였다. 발효조건은 중심합성계획법(Central Composite Design, CCD)을 이용하였다. 새싹브로콜리의 발효조건은 표 1, 2와 같이 세 개의 독립변수를 5수준으로 발효시간(X1 : 24, 36, 48, 60, 72 hr) 및 새싹브로콜리 추출물의 농도(X2 : 4, 6, 8, 10, 12 °Brix), 부원료 농축액(60 °Brix) 농도 (X3 : 2, 4, 6, 8, 10 °Brix)로 하였고, 종속변수는 유산발효 후 유산균수(Log10 CFU/mL), pH 감소량, 산도증가량, lactic acid 함량, 유효성분(rutin, quercetin 및 sulforaphan)의 함량을 측정하였다. 또한, 유산발효 조건이 새싹브로콜리 추출물의 종속변수(발효특성 및 유효성분 함량 변화)에 미치는 영향을 예측된 모델식을 바탕으로 Mathematica program을 이용하여 4차원 반응표면분석으로 해석하였다.To optimize the lactic acid fermentation conditions of sprout broccoli, repose surface methodology (RSM) was used. As fermentation conditions, Central Composite Design (CCD) was used. Fermentation conditions of sprout broccoli are as shown in Tables 1 and 2, with three independent variables at 5 levels, fermentation time (X 1 : 24, 36, 48, 60, 72 hr) and concentration of sprout broccoli extract (X 2 : 4, 6). , 8, 10, 12 °Brix), and the concentration of the auxiliary raw material concentrate (60 °Brix) (X 3 : 2, 4, 6, 8, 10 °Brix), and the dependent variable was the number of lactic acid bacteria after lactic acid fermentation (Log10 CFU/mL ), pH decrease, acidity increase, lactic acid content, and active ingredients (rutin, quercetin and sulforaphan) were measured. In addition, the effect of lactic acid fermentation conditions on the dependent variables (fermentation characteristics and active ingredient content change) of the sprout broccoli extract was analyzed by using the Mathematica program based on the predicted model equation and 4D response surface analysis.
1)중심합성계획에 의한 실험조건 번호. 1) Experimental condition number by central synthesis plan.
3. 유산균 발효추출물3. Lactobacillus fermentation extract
3-1. 발효3-1. fermentation
새싹브로콜리 추출물을 기본 배지로 하여 실험계획에 따라 부원료 농축액(60 °Brix)의 첨가량을 달리하여, MRS 액체배지에서 12시간 전배양된 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)을 종균으로 하여 2% 접종 후 발효시간을 달리하여 발효하였다.A novel lactic acid bacterium Pediococcus acidilactici DU was pre-cultured for 12 hours in MRS liquid medium by using sprout broccoli extract as a basic medium and varying the amount of supplementary material concentrate (60 °Brix) according to the experimental plan. LAB. After 2% inoculation with K-1 (KCTC 13963BP) as a seed, fermentation was performed at different fermentation times.
3-2. 발효물의 생육도, pH 및 산도 측정3-2. Measurement of viability, pH and acidity of fermented products
발효물의 유산균 생육도는 분광광도계(Optizen 2120UV, Mecasys co., Ltd., Korea)로 660nm에서 흡광도를 측정하였다. pH는 pH미터(Metter Toledo Group, Switzerland)를 사용하여, 산도는 0.1 N NaOH용액으로 pH 8.3으로 중화 적정하여 소비된 0.1 N NaOH용액을 유산함량(%)으로 환산하였다. The lactic acid bacteria growth of the fermented product was measured by absorbance at 660 nm with a spectrophotometer (Optizen 2120UV, Mecasys co., Ltd., Korea). The pH was adjusted using a pH meter (Metter Toledo Group, Switzerland), and the acidity was neutralized to pH 8.3 with 0.1 N NaOH solution, and the consumed 0.1 N NaOH solution was converted to lactic acid content (%).
3-3. 유산(Lactic acid) 함량 측정 3-3. Determination of lactic acid content
Lactic acid 함량 분석을 위해 추출물 및 발효물 시료를 0.45 ㎛ 막여과지로 여과하여 HPLC로 분석하였다. Lactic acid 함량 분석조건은 컬럼 Aminex®HPX-87H(7.5×300 mm, 9㎛, BIO-RAD Laboratories, USA)를 사용하여 5 mM 황산(sulfuric acid)을 유속 0.6mL/min의 조건으로 검출기 PDA 214 nm에서 분석하였다. 모든 표준물질은 시그마사(Sigma-Aldrich Co., USA) 제품으로 사용하였다.For lactic acid content analysis, extracts and fermented samples were filtered with 0.45 μm membrane filter paper and analyzed by HPLC. Lactic acid content analysis conditions were performed using a column Aminex ® HPX-87H (7.5×300 mm, 9㎛, BIO-RAD Laboratories, USA) with 5 mM sulfuric acid at a flow rate of 0.6 mL/min. Analyzed in nm. All standard materials were used by Sigma (Sigma-Aldrich Co., USA).
3-4. 유효성분 함량 측정3-4. Active ingredient content measurement
유효성분 함량은 새싹브로콜리 추출물 및 발효물 시료 용액을 0.45 ㎛ 막여과지로 여과한 후, HPLC시스템(Alliance e2695, Waters Co., USA)로 분석하였다. 분석조건은 표 3에 나타내었다. 표준품은 설포라판(Sulforaphane), 루틴(Rutin ), 쿼세틴(quercetin) 등(Sigma-Aldrich Co., USA)을 이용하여 표준 검량선을 작성한 후 각각의 함량을 계산하였다.Active ingredient content was analyzed by HPLC system (Alliance e2695, Waters Co., USA) after filtering the sample solution of sprout broccoli extract and fermented product with 0.45 μm membrane filter paper. The analysis conditions are shown in Table 3. For standard products, a standard calibration curve was prepared using Sulforaphane, Rutin, Quercetin, etc. (Sigma-Aldrich Co., USA), and then the content of each was calculated.
실험결과Experiment result
4. 신규한 유산균의 분리 동정4. Isolation and identification of novel lactic acid bacteria
4-1. 발효식품으로부터 유산균 분리4-1. Separation of lactic acid bacteria from fermented foods
발효식품(김치) 시료로부터 11종의 유산균을 분리하였다. 일부 발효식품에서는 숙성에 따라 유산균이 거의 검출되지 않는 시료도 있었다. 발효식품으로부터 분리한 균주는 DU. La. B-1∼7과 DU. LAB. K-1∼3로 명명하였으며, 분리한 균주는 계대배양을 실시하여 단독 colony를 순수분리하였다. 발효식품의 종류와 숙성도에 따른 colony의 분포는 달랐으며 희석배수가 10-3~10-5 일 때 균주의 분리가 가장 용이하였다(도 2). 11 types of lactic acid bacteria were isolated from fermented food (kimchi) samples. In some fermented foods, there were samples in which lactic acid bacteria were hardly detected according to aging. The strain isolated from fermented food was DU. La. B-1 to 7 and DU. LAB. It was named K-1~3, and the isolated strain was subcultured to purely isolate a single colony. The distribution of colonies according to the type of fermented food and the degree of ripening was different, and when the dilution was 10 -3 to 10 -5 , it was easiest to isolate the strain (FIG. 2).
4-2. 베타-글루코시데이즈(β-glucosidase) 활성 균주 선발 4-2. Beta-glucosidase (β-glucosidase) active strain selection
β-glucosidase 활성 균주를 선발하고자 esculin agar법을 사용하였다. 분리 유산균들이 esculin에 의해 black complex의 생성 여부에 따른 결과는 도 3과 같으며, black complex 길이는 표 4와 같다. Esculin agar에서 black complex 생성은 DU. La. B-1균주를 제외한 모든 분리유산균에서 colony 주변에 black coomplex를 형성하여 β-glucosidase 활성이 있는 것으로 나타났으며, DU. LAB. K-3 균주가 black complex 길이가 가장 크게 나타났다(도 3). 또한, 분리된 11종의 유산균에 대한 β-glucosidase활성을 측정한 결과는 표 4와 같이 esculin agar에서 black complex 생성하지 않은 DU. La. B-1균주의 β-glucosidase 활성이 2.74 mU/mL로 가장 낮게 나타났으며, DU. La. B-7균주가 β-glucosidase 활성이 5.85 mU/mL로 가장 높은 활성을 나타내었다. The esculin agar method was used to select β-glucosidase active strains. The results according to whether the isolated lactic acid bacteria produced the black complex by esculin are shown in FIG. 3, and the length of the black complex is shown in Table 4. Generation of the black complex in Esculin agar was performed by DU. La. All isolated lactic acid bacteria except B-1 strain formed a black coomplex around the colony and showed β-glucosidase activity, DU. LAB. The K-3 strain showed the largest black complex length (FIG. 3). In addition, as shown in Table 4, the results of measuring β-glucosidase activity against 11 isolated lactic acid bacteria were DU without black complex formation in esculin agar. La. The β-glucosidase activity of strain B-1 was the lowest at 2.74 mU/mL, and DU. La. Strain B-7 showed the highest activity with β-glucosidase activity of 5.85 mU/mL.
4-3. 분리 균주의 새싹채소 추출액 농도에 따른 균 생장실험4-3. Bacterial growth test according to the concentration of the sprouted vegetable extract of the isolated strain
새싹브로콜리 추출액 단독으로 5, 10, 15, 20, 50, 100% 함유한 고체배지에서 분리 유산균주의 생육능을 검토한 결과는 표 5와 같다. DU. La. B-1과 DU, LAB. K-7균주를 제외한 모든 분리 유산균들은 새싹브로콜리 추출액이 100% 함유된 배지에서 colony가 뚜렷하게 생성된 것을 알 수 있었다. Table 5 shows the results of examining the viability of isolated lactic acid strains in solid medium containing 5, 10, 15, 20, 50, and 100% of sprout broccoli extract alone. DU. La. B-1 and DU, LAB. All isolated lactic acid bacteria except strain K-7 showed clear colony formation in the medium containing 100% of sprout broccoli extract.
상대적 성장도: +; 성장도 약함, ++; 성장도 중간, +++; 성장도 강함Relative Growth: +; Weak growth, ++; Medium growth, +++; strong growth
4-4. 최종균주 선별4-4. Final strain selection
분리한 유산 균주에 대한 새싹브로콜리 발효 여부를 검토하기 위하여 표 4에서 β-glucosidase 활성능이 좋은 3종의 균주(DU. La. B-7. B-8, DU. LAB. K-1)를 배양하여 10 °Brix의 새싹브로콜리 추출액에 접종하여 37℃ incubator에서 48시간 배양하여 생균수, pH, 산도를 측정하여 균주의 생장 및 발효능을 확인하였다(표 6). 그 결과 3종의 균주 모두가 새싹브로콜리에서 생장이 가능한 것으로 나타났으며, 세 균주 모두가 24시간 내에 왕성하게 증식하는 것으로 보였으며, 발효 종료 후 DU. LAB. K-1에서 가장 많은 균수 증가량을 보였다. pH의 경우는 모든 균주에서 발효시간이 경과할수록 감소하는 것으로 나타났으며, DU. LAB. K-1 균주에서 가장 낮은 pH 값이 측정되었다. 산도의 경우는 균주 간에 큰 차이가 없는 것으로 나타났으며, 발효 24시간 동안 산도가 많이 증가하는 경향을 보였으며, 24시간 후 48시간까지는 산도의 변화가 거의 없는 것으로 나타났다. 따라서 새싹브로콜리의 발효를 위해서는 유산균수 증가량과 pH 감소폭이 가장 큰 DU. LAB. K-1 균주를 발효균주로 최종선정하였다.In Table 4, three strains with good β-glucosidase activity (DU. La. B-7. B-8, DU. LAB. K-1) were analyzed to examine whether the isolated lactic acid strain was fermented with sprout broccoli. Incubated and inoculated into sprouted broccoli extract at 10 °Brix, incubated for 48 hours in an incubator at 37 °C, the number of viable cells, pH, and acidity were measured to confirm the growth and fermentation ability of the strain (Table 6). As a result, all three strains were shown to be able to grow in sprout broccoli, and all three strains appeared to proliferate vigorously within 24 hours. LAB. K-1 showed the largest increase in the number of bacteria. In the case of pH, it was shown that the fermentation time decreased in all strains, and DU. LAB. The lowest pH value was measured in the K-1 strain. In the case of acidity, it was found that there was no significant difference between the strains, and the acidity showed a tendency to increase a lot during 24 hours of fermentation, and there was almost no change in acidity until 48 hours after 24 hours. Therefore, for the fermentation of sprout broccoli, DU has the largest increase in the number of lactic acid bacteria and the decrease in pH. LAB. The K-1 strain was finally selected as the fermentation strain.
4-5. 최종 선발균주(DU. LAB. K-1)의 동정4-5. Identification of the final selection strain (DU. LAB. K-1)
최종 분리된 균주 중 베타-글루코시데이즈 활성능과 발효력이 가장 높은 1균주인 DU. LAB. K-1를 16s rDNA 유전자의 염기서열을 분석하여 NCBI blast DB와 비교한 결과(표 7), Pediococcus속에 속하는 Pediococcus acidilactici과 98~99% 상동성을 보였다. NCBI Blast 검색를 통해 Pediococcus속 내에 DU. LAB. K-1균주와 유연관계가 가까운 종들을 찾고 Pediococcus acidilactici DU. LAB. K-1 뉴클레오타이드 염기서열(도 4) 검색을 통하여 동일 균주들의 염기서열을 조사하였다. Bioedit program과 Clustal X program을 이용하여 염기서열을 alignment한 후 근연관계를 조사해 본 결과 도 5와 같으며 Pediococcus acidilactici DSM 20284와 Pediococcus acidilactici NGRI 0510Q와 각각 99%와 98% 상동성을 보였다. 따라서 분리균주 DU. LAB. K-1균주를 Pediococcus acidilactici DU. LAB. K-1으로 명명하였으며 국제미생물 기탁기관인 한국생명공학연구원 생물자원센터(KCTC) 2019년 9월 30일자로 특허기탁 하였으며 기탁번호 KCTC 13963BP를 부여받았다.Among the finally isolated strains, DU, the
5. 새싹브로콜리 추출물의 유산발효조건 최적화5. Optimization of lactic acid fermentation conditions for sprout broccoli extract
5-1. 유산발효조건에 따른 발효적 특성 및 유효성분 함량5-1. Fermentation characteristics and active ingredient content according to lactic acid fermentation conditions
새싹브로콜리 추출물의 최적 유산발효 조건을 설정하기 위해 발효시간, 새싹브로콜리 추출물 농도 및 부원료 농축액 농도를 독립변수로 하여 중심합성계획에 의해 설계된 19구의 발효조건에서 얻어진 발효물의 특성은 표 8과 같다. 각각의 결과를 이용하여 반응표면분석을 실시하고, 유산균수, lactic acid 함량, sulforaphane함량, rutin 및 quercetin 함량값에 대한 회귀식을 얻었다(표 9). 또한 변수별 최적발효조건과 발효 특성값을 예측하여 표 10에 나타냈으며, 이들의 4차원 반응표면을 발효시간, 새싹브로콜리 추출물농도, 및 부원료 농축액 농도를 독립변수로 하여 도 6∼도 9에 나타내었다.Table 8 shows the characteristics of the fermented product obtained in 19 fermentation conditions designed by the central synthesis plan with fermentation time, sprout broccoli extract concentration, and auxiliary raw material concentrate concentration as independent variables to set the optimal lactic acid fermentation conditions for sprout broccoli extract. Response surface analysis was performed using each result, and regression equations were obtained for the number of lactic acid bacteria, lactic acid content, sulforaphane content, and rutin and quercetin content values (Table 9). In addition, the optimal fermentation conditions and fermentation characteristic values for each variable were predicted and shown in Table 10, and their four-dimensional response surface is shown in FIGS. It was.
중심합성계획법에 따라 표 1과 같은 각 독립변수의 범위를 설정한 후, Design Expert를 이용하여 표 2와 같이 19가지의 발효 조건을 설정하고 새싹브로콜리 추출물을 유산발효하였다. 발효조건에 따른 새싹브로콜리 발효물의 유산균 수는 11.41∼14.55 Log10 CFU/mL의 범위로 나타났으며(표 8), 이를 바탕으로 한 회귀식은 표 9와 같다. 유산균수에 대한 R2값은 0.9786으로 높은 신뢰도를 보였으며, P-value는 1% 이내 유의수준을 보였다. 효소처리 조건에 대한 영향에서 환원당 함량의 경우 발효시간 > 새싹브로콜리 추출물 농도 > 부원료 농축액 농도 순으로 세 가지 조건 모두에서 영향을 크게 받는 것으로 나타났다(표 10). 한편 lack of fit(적합결여검정) 값은 model의 적합도를 검정하는 통계량으로 P값이 0.05보다 작을 때 model의 적합성에 문제가 있으며, 이보다 클 때 model이 적합하다고 판단하는 통계값이다. 유산균수 값의 ANOVA 분석을 통한 모형에 대한 적합성을 검증한 적합결여도(lack of fit)의 P-value는 0.0535로 나타나 model이 적합한 것으로 나타났다(표 9). 발효조건에 따라 반응표면모델로 예측된 회귀분석 결과 정상점은 최대점으로 나타났으며, 최적점을 산출한 결과 유산균수의 최댓값은 14.99 Log10 CFU/mL이고 이때의 발효조건은 발효시간 70.85 hr, 새싹브로콜리 추출물 농도 8.64 °Brix 및 부원료 농축액 농도 7.03 °Brix로 나타났다(표 10). 실험조건에 따라 얻은 발효물의 유산균수에 대한 4차원 반응표면은 도 6 같이 최대점의 형태를 나타내었으며, 새싹브로콜리 추출물 농도가 높고, 발효시간이 길수록 증가하는 것으로 나타났다. After setting the ranges of each independent variable as shown in Table 1 according to the central synthesis programming method, 19 fermentation conditions were set as shown in Table 2 using Design Expert, and lactic acid fermentation of sprout broccoli extract was performed. The number of lactic acid bacteria in the sprouted broccoli fermented product according to the fermentation conditions was in the range of 11.41 to 14.55 Log10 CFU/mL (Table 8), and the regression equation based on this was shown in Table 9. The R 2 value for the number of lactic acid bacteria was 0.9786, which showed high reliability, and the P -value showed a significance level within 1%. In the effect on the enzyme treatment conditions, the reducing sugar content was found to be greatly affected by all three conditions in the order of fermentation time > sprout broccoli extract concentration > auxiliary raw material concentration concentration (Table 10). On the other hand, the lack of fit value is a statistic that tests the fitness of the model. When the P value is less than 0.05, there is a problem with the model's fit, and when it is larger than this, the model is judged to be suitable. The P -value of the lack of fit, which verified the fit of the model through ANOVA analysis of the number of lactic acid bacteria, was 0.0535, indicating that the model was suitable (Table 9). As a result of the regression analysis predicted by the response surface model according to the fermentation conditions, the normal point was found to be the maximum point. The sprout broccoli extract concentration was 8.64 °Brix and the concentration of the auxiliary raw material concentrate was 7.03 °Brix (Table 10). The four-dimensional response surface for the number of lactic acid bacteria in the fermented product obtained according to the experimental conditions showed the form of a maximum point as shown in FIG. 6, and it was found that the sprout broccoli extract concentration was high and the fermentation time increased.
1)중심합성계획법에 의한 실험번호 1) Experiment number by central synthesis programming method
*Significant at 10% level; **significant at 5% level; ***significant at 1% level.*Significant at 10% level; **significant at 5% level; ***significant at 1% level.
유산발효조건에 따른 새싹브로콜리 발효물에 대한 lactic acid 함량은 10.76∼14.05 mg/mL의 범위로 나타났으며(표 8), 이를 바탕으로 한 lactic acid 함량의 회귀식은 표 9와 같고 R2값은 0.8762로 1% 이내의 수준에서 유의성이 확인되었다. ANOVA 분석을 통한 lactic acid 함량의 모형에 대한 적합성을 검증을 위한 적합결여도(lack of fit)의 P-value은 0.2174로 나타나 model이 적합한 것으로 나타났다(표 9). 유산발효조건에 대한 영향은 표 10에서와 같이 새싹브로콜리 추출물 농도에 영향이 가장 크며, 그 다음으로 발효시간, 부원료 농축액 농도 순으로 영향을 받는 것으로 나타났다. 유산발효조건에 따라 반응표면모델로 예측된 정상점은 최대점으로 최대값이 13.99 mg/mL이었고, 이때 발효 시간은 58.51 hr, 새싹브로콜리농도 11.47 °Brix 및 부원료 농축액 농도 6.90 °Brix이었다(표 11). 4차원 반응표면을 통한 유산발효조건에 따른 lactic acid 함량의 변화는 도 7와 같이 새싹브로콜리 추출물 농도가 높을수록 lactic acid 함량이 증가하는 것으로 나타났다.The lactic acid content of the sprouted broccoli fermented product according to the lactic acid fermentation conditions was in the range of 10.76∼14.05 mg/mL (Table 8), and the regression formula for the lactic acid content based on this was shown in Table 9, and the Significance was confirmed at a level within 1% of 0.8762. The P -value of the lack of fit for verifying the suitability of the model of the lactic acid content through ANOVA analysis was 0.2174, indicating that the model was suitable (Table 9). As shown in Table 10, the effect on the lactic acid fermentation conditions was the greatest on the concentration of sprout broccoli extract, followed by the fermentation time and the concentration of the auxiliary material concentrate. The normal point predicted by the response surface model according to the lactic acid fermentation conditions was the maximum point, and the maximum value was 13.99 mg/mL, and the fermentation time was 58.51 hr, the sprout broccoli concentration was 11.47 °Brix, and the concentration of the auxiliary material concentrate was 6.90 °Brix (Table 11). ). The change of the lactic acid content according to the lactic acid fermentation conditions through the four-dimensional reaction surface showed that the lactic acid content increased as the sprout broccoli extract concentration increased, as shown in FIG. 7 .
발효조건에 따른 새싹브로콜리 발효물의 sulforaphane 증가함량은 8.97∼12.34 ㎍/mL의 범위로 나타났으며(표 8), 이를 바탕으로 한 회귀식은 표 9와 같다. Sulforaphane 함량에 대한 R2값은 0.8471로 높은 신뢰도를 보였으며, P-value는 1% 이내 유의수준을 보였다. 한편 sulforaphane함량의 model에 대한 ANOVA 분석을 통한 모형에 대한 적합성을 검증을 위한 적합결여도(lack of fit)의 P-value는 0.2147로 나타나 model이 적합한 것으로 나타났다(표 9). 발효조건에 대한 영향에서 sulforaphane 함량의 경우 새싹브로콜리 추출물 농도 > 발효시간 순으로 영향을 크게 받는 것으로 나타났으며, 부원료 농축액 농도에서는 영향을 받지 않는 것을 알 수 있었다(표 10). 발효조건에 따라 반응표면모델로 예측된 회귀분석 결과 정상점은 안장점으로 나타났으며, 능선분석을 하여 최적점을 산출한 결과 sulforaphane 함량의 최댓값은 13.41 ㎍/mL이고 이때의 발효조건은 발효시간 56.79hr, 새싹브로콜리 추출물 농도 4.48 °Brix 및 부원료 농축액 농도 7.20 °Brix로 나타났다(표 11). 실험조건에 따라 얻은 발효물의 sulforaphane 함량에 대한 4차원 반응표면은 도 8과 같이 안장점의 형태를 나타내었으며, 새싹브로콜리 추출물 농도가 낮고, 발효시간이 길수록 증가하는 것으로 나타났다. The increased content of sulforaphane in sprouted broccoli fermented products according to the fermentation conditions was in the range of 8.97 to 12.34 μg/mL (Table 8), and the regression equation based on this was shown in Table 9. The R 2 value for the sulforaphane content was 0.8471, which showed high reliability, and the P -value showed a significance level within 1%. On the other hand, the P -value of the lack of fit (lack of fit) for verifying the fit of the model through ANOVA analysis of the sulforaphane content model was 0.2147, indicating that the model was suitable (Table 9). In the effect on the fermentation conditions, it was found that the sulforaphane content was significantly affected in the order of sprout broccoli extract concentration > fermentation time, and was not affected by the concentration of the auxiliary material concentrate (Table 10). As a result of the regression analysis predicted by the response surface model according to the fermentation conditions, the normal point was found to be the saddle point. At 56.79 hr, the broccoli sprout extract concentration was 4.48 °Brix and the concentration of the auxiliary material concentrate was 7.20 °Brix (Table 11). The four-dimensional response surface for the sulforaphane content of the fermented product obtained according to the experimental conditions showed the shape of a saddle point as shown in FIG. 8, and it was found that the sprout broccoli extract concentration was low and the fermentation time increased.
발효조건에 따른 새싹브로콜리 발효물의 rutin 감소 함량은 2.89∼12.64 ㎍/mL의 범위로 나타났으며(표 8), 이를 바탕으로 한 회귀식은 표 9와 같으며, 이 모델에 대한 회귀식의 R2 값은 0.9767로 1% 이내의 유의성이 인정되었다. ANOVA 분석을 통한 모형에 대한 적합성을 검증을 위한 적합결여도(lack of fit)의 P-value는 0.0506으로 나타나 model이 적합한 것으로 나타났다(표 9). Rutin 감소 함량은 새싹브로콜리 추출물 농도와 발효시간에 영향을 크게 받는 것으로 나타났다(표 10). 표 11과 같이 rutin 함량의 예측된 정상점은 안장점으로 나타났으며, 능선분석을 하여 최대값을 산출한 결과 rutin 함량의 최댓값이 12.765 ㎍/mL이었고, 이때 발효시간 53.50 시간, 새싹브로콜리 추출물 농도 11.76 °Brix 및 부원료 농축액 농도 6.98 °Brix이었다. 새싹브로콜리 발효물의 rutin 함량은 발효조건에 따른 4차원 반응표면에서 볼 때 새싹브로콜리 추출물 농도가 높을수록 rutin 감소 함량이 증가하는 것으로 나타났다(도 9). 이는 추출몰의 rutin이 발효에 사용된 유산균의 β-glucosidase의 활성에 의하여 배당체인 rutin이 비배당체인 quercetin으로 전환되므로 rutin은 감소되고 quercetin은 증가되는 것을 알 수 있다.The rutin reduction content of sprouted broccoli fermented products according to fermentation conditions was in the range of 2.89-12.64 μg/mL (Table 8), and the regression equation based on this was shown in Table 9, and R 2 of the regression equation for this model The value was 0.9767, and significance within 1% was recognized. The P -value of the lack of fit to verify the fit of the model through ANOVA analysis was 0.0506, indicating that the model was suitable (Table 9). It was found that the reduced Rutin content was significantly affected by the sprout broccoli extract concentration and fermentation time (Table 10). As shown in Table 11, the predicted normal point of the rutin content was the saddle point, and as a result of calculating the maximum value through ridge analysis, the maximum value of the rutin content was 12.765 μg/mL, at this time, the fermentation time 53.50 hours, the sprout broccoli extract concentration The concentration of the 11.76 °Brix and auxiliary material concentrates was 6.98 °Brix. As for the rutin content of the fermented sprout broccoli, it was found that the higher the concentration of the sprout broccoli extract, the higher the rutin reduction content was seen from the four-dimensional response surface according to the fermentation conditions (FIG. 9). It can be seen that rutin is reduced and quercetin is increased because rutin, a glycoside, is converted to quercetin, a non-glycoside, by the activity of β-glucosidase of lactic acid bacteria used for fermentation.
발효조건에 따른 새싹브로콜리 발효물의 quercetin 증가 함량 범위는 0.85∼1.62 ㎍/mL의 범위로 나타났으며(표 8), 이를 바탕으로 한 회귀식은 표 9와 같다. 이 모델에 대한 회귀식의 R2 값은 0.9120로 1% 이내의 유의성이 인정되었으며, Quercetin 증가 함량의 model에 대한 ANOVA 분석을 통한 모형에 대한 적합성을 검증을 위한 적합결여도(lack of fit)의 P-value는 0.0843으로 나타나 model이 적합한 것으로 나타났다(표 9). 새싹브로콜리 농도, 부원료 농축액 농도, 발효시간 순으로 모두에서 영향을 받는 것으로 나타났다(표 10). 유산발효조건에 따라 반응표면모델로 예측된 정상점이 안장점으로 나타났으며, 최적점을 산출한 결과 quercetin 증가 함량의 최대값은 1.65 ㎍/mL이었고, 이때 발효 시간은 37.05 hr, 새싹브로콜리 농도 4.51 °Brix 및 부원료 농축액 농도 5.25 °Brix이었다 (표 11). 4차원 반응표면을 통한 유산발효조건에 따른 quercetin 증가 함량의 변화는 도 10에 나타내었다.The increased content of quercetin in sprouted broccoli fermented products according to fermentation conditions was in the range of 0.85 to 1.62 μg/mL (Table 8), and the regression equation based on this was shown in Table 9. The R 2 value of the regression equation for this model was 0.9120, which was found to be significant within 1%, and the Lack of fit for verifying the fit of the model through ANOVA analysis for the model with increased quercetin content. The P -value was 0.0843, indicating that the model was suitable (Table 9). It was found to be affected by the sprout broccoli concentration, the concentration of the auxiliary material concentrate, and the fermentation time in the order (Table 10). According to the lactic acid fermentation conditions, the normal point predicted by the response surface model was the saddle point, and as a result of calculating the optimal point, the maximum value of the quercetin increase was 1.65 μg/mL, at this time, the fermentation time was 37.05 hr, and the sprout broccoli concentration was 4.51. The concentration of °Brix and additives was 5.25 °Brix (Table 11). The change in the content of quercetin increased according to the lactic acid fermentation conditions through the four-dimensional reaction surface is shown in FIG. 10 .
5-2. 새싹브로콜리 추출물의 최적 유산발효조건 예측 및 실증실험5-2. Prediction and empirical experiment of optimal lactic acid fermentation conditions of sprout broccoli extract
새싹브로콜리 추출물의 발효조건을 설정하기 위하여 유산균 수, lactic acid 함량, sulforaphane 증가 함량, rutin 감소함량 및 quercetin 증가 함량의 결과에 대하여 4차원 반응표면을 superimposing(겹침)하여 도 11의 겹쳐진 부분으로써 최적 발효조건을 나타내었다(표 12). 즉, 새싹브로콜리 추출물의 최적 발효조건 범위는 발효시간 50~60 시간, 새싹브로콜리 추출물 농도 8~12 °Brix 및 부원료 농축액 농도 4~7 °Brix로 나타났다. 따라서 이와 같은 예측결과에 대한 모델식의 신뢰성을 확인하기 위하여 예측된 최적 조건의 범위 내에서 임의의 조건 즉, 발효시간 55시간, 새싹브로콜리 추출물 농도 10 °Brix 및 부원료 농축액 농도 6 °Brix을 대입하여 실제 유산발효를 실시하여 발효 특성을 분석하였다(표 13). In order to set the fermentation conditions of the sprout broccoli extract, the 4D response surface was superimposing (overlapping) the results of the number of lactic acid bacteria, the lactic acid content, the sulforaphane increase content, the rutin decrease content, and the quercetin increase content. The conditions are shown (Table 12). In other words, the optimal fermentation conditions range for sprout broccoli extract was 50 to 60 hours for fermentation time, 8 to 12 °Brix for sprout broccoli extract, and 4 to 7 °Brix for the concentration of auxiliary material concentrate. Therefore, in order to confirm the reliability of the model formula for such prediction results, arbitrary conditions, that is, fermentation time 55 hours, sprout
1)반응변수에 대한 예측식으로 부터 계산된 값. 1) The value calculated from the prediction equation for the response variable.
2)독립변수의 최적조건 : 발효시간 55 hr, 새싹 브로콜리 농도 9 °Brix, 부원료 농도 6 °Brix 2) Optimal conditions for independent variables: fermentation time 55 hr, sprout broccoli concentration 9 °Brix,
*) 평균값은 3반복 표준편차. *) The mean value is three replicate standard deviations.
6. 유산균 발효 전후 새싹브로콜리의 유효성분 함량 비교6. Comparison of active ingredient content of sprout broccoli before and after fermentation of lactic acid bacteria
본 발명이 추구한 새싹브로콜리 추출물을 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)에 의하여 최적발효 조건으로 발효되었을 때 발효 전후의 새싹브로콜리의 유효성분 변화를 조사한 결과는 표 14와 같다. 또한, 이들을 HPLC로 분석한 크로마토그램은 도 12와 같다. 발효 특성상 lactic acid의 경우 원료인 새싹브로콜리 추출물은 전혀 검출되지 않았으나 발효물은 유산발효에 의하여 13.43 mg/mL로 나타나 최적조건에서 유산균 발효가 적절한 발효가 일어났음을 알 수 있다. 유효성분의 경우도 새싹브로콜리 원료내 함량보다 대부분 그 함량이 변화 된 것을 볼 수 있었다. sulforaphan함량의 경우, 새싹브로콜리 추출물 원료대비 약 341%가 증가하였다. 루틴(rutin)의 경우 새싹브로콜리 추출물 원료 대비 발효물은 약 60.4%가 감소하였다. 또한, quercetin 함량의 경우 새싹브로콜리 추출물 대비 발효물이 약 49.1%가 증가하였다. 이는 새싹 브로콜리 원료의 배당체인 rutin성분이 유산균 발효에 의하여 β-glucosidase의 작용으로 비배당체인 quercetin성분으로 생물전환된 결과로 나타나, 본 발명의 발효처리가 효과적인 처리인 것으로 보인다. A novel lactic acid bacterium Pediococcus acidilactici DU. LAB. Table 14 shows the changes in the active ingredients of sprout broccoli before and after fermentation when fermented under optimal fermentation conditions by K-1 (KCTC 13963BP). In addition, chromatograms analyzed by HPLC are shown in FIG. 12 . In the case of lactic acid, the raw material, sprout broccoli extract, was not detected at all in the case of fermentation characteristics, but the fermented product was 13.43 mg/mL by lactic acid fermentation, indicating that fermentation of lactic acid bacteria was appropriate under optimal conditions. In the case of the active ingredient, it was found that most of the content was changed compared to the content in the raw material of sprout broccoli. In the case of sulforaphan content, about 341% increased compared to the raw material of sprout broccoli extract. In the case of rutin, the fermented product decreased by about 60.4% compared to the raw material of sprout broccoli extract. In addition, in the case of the quercetin content, the fermented product increased by about 49.1% compared to the sprout broccoli extract. This is the result of bioconversion of rutin component, a glycoside of sprouted broccoli raw material, into a non-glycoside quercetin component by the action of β-glucosidase by fermentation of lactic acid bacteria, suggesting that the fermentation treatment of the present invention is an effective treatment.
이상과 같이 본 발명은 첨부된 도면을 참조하여 바람직한 실시예를 중심으로 설명하였지만 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 본 발명의 특허청구범위에 기재된 기술적 사상 및 영역으로부터 벗어나지 않는 범위 내에서 본 발명을 다양하게 수정 또는 변형하여 실시할 수 있다. 따라서 본 발명의 범주는 이러한 많은 변형의 예들을 포함하도록 기술된 청구범위에 의해서 해석되어야 한다.As described above, the present invention has been mainly described with reference to the accompanying drawings, but those of ordinary skill in the art to which the present invention pertains within the scope not departing from the technical spirit and scope described in the claims of the present invention Various modifications or variations of the present invention can be practiced. Accordingly, the scope of the present invention should be construed by the appended claims to include examples of many such modifications.
기탁기관명 : 한국생명공학연구원Name of deposit institution: Korea Research Institute of Bioscience and Biotechnology
수탁번호 : KCTC13963BPAccession number: KCTC13963BP
수탁일자 : 20190930Deposit date: 20190930
<110> Industry Academic Cooperation Foundation, Daegu University Fresh Bell Co., Ltd. <120> Broccoli Sprouts Fermentation Composition with Enhanced Sulforaphane and Quercetin using Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP) of novel lactic acid bacteria and Manufacturing Method thereof <130> PX19-336 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1431 <212> DNA <213> Artificial Sequence <220> <223> DU.LAB.K-1 <400> 1 gaacttccgt taattgatta tgaggtgctt gcactgaatg agattttaac acgaagtgag 60 tggcggacgg gtgagtaaca cgtgggtaac ctgcccagaa gcaggggata acacctggaa 120 acagatgcta ataccgtata acagagaaaa ccgcctggtt ttcttttaaa agatggctct 180 gctatcactt ctggatggac ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca 240 aggcgatgat gcgtagccga cctgagaggg taatcggcca cattgggact gagacacggc 300 ccagactcct acgggaggca gcagtaggga atcttccaca atggacgcaa gtctgatgga 360 gcaacgccgc gtgagtgaag aagggtttcg gctcgtaaag ctctgttgtt aaagaagaac 420 gtgggtgaga gtaactgttc acccagtgac ggtatttaac cagaaagcca cggctaacta 480 cgtgccagca gccgcggtaa tacgtaggtg gcaagcgtta tccggattta ttgggcgtaa 540 agcgagcgca ggcggtcttt taagtctaat gtgaaagcct tcggctcaac cgaagaagtg 600 cattggaaac tgggagactt gagtgcagaa gaggacagtg gaactccatg tgtagcggtg 660 aaatgcgtag atatatggaa gaacaccagt ggcgaaggcg gctgtctggt ctgtaactga 720 cgctgaggct cgaaagcatg ggtagcgaac aggattagat accctggtag tccatgccgt 780 aaacgatgat tactaagtgt tggagggttt ccgcccttca gtgctgcagc taacgcatta 840 agtaatccgc ctggggagta cgaccgcaag gttgaaactc aaaagaattg acgggggccc 900 gcacaagcgg tggagcatgt ggtttaattc gaagctacgc gaagaacctt accaggtctt 960 gacatcttct gccaacctaa gagattaggc gttcccttcg gggacagaat gacaggtggt 1020 gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080 ccttattact agttgccagc attcagttgg gcactctagt gagactgccg gtgacaaacc 1140 ggaggaaggt ggggacgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg 1200 ctacaatgga tggtacaacg agtcgcgaaa ccgcgaggtt tagctaatct cttaaaacca 1260 ttctcagttc ggactgtagg ctgcaactcg cctacacgaa gtcggaatcg ctagtaatcg 1320 cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1380 tgagagtttg taacacccaa agccggtggg gtaaccttta ggagctagcc g 1431 <110> Industry Academic Cooperation Foundation, Daegu University Fresh Bell Co., Ltd. <120> Broccoli Sprouts Fermentation Composition with Enhanced Sulforaphane and Quercetin using Pediococcus acidilactici DU. LAB. K-1 (KCTC 13963BP) of novel lactic acid bacteria and Manufacturing Method <130> PX19-336 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1431 <212> DNA <213> Artificial Sequence <220> <223> DU.LAB.K-1 <400> 1 gaacttccgt taattgatta tgaggtgctt gcactgaatg agattttaac acgaagtgag 60 tggcggacgg gtgagtaaca cgtgggtaac ctgcccagaa gcaggggata acacctggaa 120 acagatgcta ataccgtata acagagaaaa ccgcctggtt ttcttttaaa agatggctct 180 gctatcactt ctggatggac ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca 240 aggcgatgat gcgtagccga cctgagaggg taatcggcca cattgggact gagacacggc 300 ccagactcct acgggaggca gcagtaggga atcttccaca atggacgcaa gtctgatgga 360 gcaacgccgc gtgagtgaag aagggtttcg gctcgtaaag ctctgttgtt aaagaagaac 420 gtgggtgaga gtaactgttc acccagtgac ggtatttaac cagaaagcca cggctaacta 480 cgtgccagca gccgcggtaa tacgtaggtg gcaagcgtta tccggattta ttgggcgtaa 540 agcgagcgca ggcggtcttt taagtctaat gtgaaagcct tcggctcaac cgaagaagtg 600 cattggaaac tgggagactt gagtgcagaa gaggacagtg gaactccatg tgtagcggtg 660 aaatgcgtag atatatggaa gaacaccagt ggcgaaggcg gctgtctggt ctgtaactga 720 cgctgaggct cgaaagcatg ggtagcgaac aggattagat accctggtag tccatgccgt 780 aaacgatgat tactaagtgt tggagggttt ccgcccttca gtgctgcagc taacgcatta 840 agtaatccgc ctggggagta cgaccgcaag gttgaaactc aaaagaattg acgggggccc 900 gcacaagcgg tggagcatgt ggtttaattc gaagctacgc gaagaacctt accaggtctt 960 gacatcttct gccaacctaa gagattaggc gttcccttcg gggacagaat gacaggtggt 1020 gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080 ccttattact agttgccagc attcagttgg gcactctagt gagactgccg gtgacaaacc 1140 ggaggaaggt ggggacgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg 1200 ctacaatgga tggtacaacg agtcgcgaaa ccgcgaggtt tagctaatct cttaaaacca 1260 ttctcagttc ggactgtagg ctgcaactcg cctacacgaa gtcggaatcg ctagtaatcg 1320 cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1380 tgagagtttg taacacccaa agccggtggg gtaaccttta ggagctagcc g 1431
Claims (10)
상기 가열 살균액에 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)을 접종하여 접종 조성액을 제조하는 제2단계;와
상기 접종 조성액을 발효하는 제3단계;를 포함하며,
상기 제1단계에서는 새싹브로콜리 추출물 100 부피부에 대하여 부원료 농축액 0.1 내지 20 부피부를 첨가하되, 새싹브로콜리 추출물 농도 8 내지 12°Brix, 부원료 농축액 농도 4 내지 7 °Brix 로 첨가하고,
상기 제2단계에서는 가열 살균액 100중량부에 대하여 신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)을 1 내지 10중량부 접종하며,
상기 제3단계에서는 접종 조성액을 30 내지 45℃에서 50 내지 60시간 발효하고,
상기 제1단계의 부원료 농축액은 바나나 농축액, 복숭아농축액, 망고농축액, 딸기농축액, 토마토농축액 및 당근농축액 중 적어도 어느 하나를 포함하는 것임을 특징으로 하는
신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)를 이용한 새싹브로콜리 유산균 발효물의 제조방법.
A first step of preparing a heat sterilizing solution by heat sterilizing a mixture of a sprout broccoli extract and an auxiliary raw material concentrate; and
A novel lactic acid bacterium Pediococcus acidilactici DU. LAB. A second step of preparing an inoculation composition by inoculating K-1 (KCTC 13963BP); and
Including; a third step of fermenting the inoculation composition;
In the first step, 0.1 to 20 parts by volume of the auxiliary raw material concentrate is added with respect to 100 parts by volume of the sprout broccoli extract, but added at a sprout broccoli extract concentration of 8 to 12 ° Brix, and a concentration of the auxiliary raw material concentrate of 4 to 7 ° Brix,
In the second step, the novel lactic acid bacterium Pediococcus acidilactici DU. LAB. 1 to 10 parts by weight of K-1 (KCTC 13963BP) is inoculated,
In the third step, the inoculation composition is fermented at 30 to 45° C. for 50 to 60 hours,
The auxiliary raw material concentrate of the first step is characterized in that it comprises at least one of a banana concentrate, a peach concentrate, a mango concentrate, a strawberry concentrate, a tomato concentrate, and a carrot concentrate.
A novel lactic acid bacterium Pediococcus acidilactici DU. LAB. Method for producing sprouted broccoli lactic acid bacteria fermented product using K-1 (KCTC 13963BP).
상기 제1단계의 새싹브로콜리 추출물은
분쇄액, 농축액, 건조물, 분말 및 이들의 조합 중 어느 하나를 포함하는 것을 특징으로 하는
신규한 유산균 Pediococcus acidilactici DU. LAB. K-1(KCTC 13963BP)를 이용한 새싹브로콜리 유산균 발효물의 제조방법.
The method of claim 1,
The sprout broccoli extract of the first step is
A pulverized liquid, a concentrate, a dry matter, a powder, and any one of a combination thereof
A novel lactic acid bacterium Pediococcus acidilactici DU. LAB. Method for producing sprouted broccoli lactic acid bacteria fermented product using K-1 (KCTC 13963BP).
A novel lactic acid bacterium Pediococcus acidilactici DU produced by the method of any one of claims 1 or 2. LAB. Broccoli sprouted lactic acid bacteria fermented product using K-1 (KCTC 13963BP).
A novel lactic acid bacterium Pediococcus acidilactici DU comprising the fermented sprout of broccoli sprouts of claim 9. LAB. Sprout broccoli lactic acid bacteria fermented beverage composition using K-1 (KCTC 13963BP).
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