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Ⅳ. 연구개발결과
제 1 절 식물공장을 활용한 브로콜리 새싹으로부터 기능성 성분의 분리․동정 및 표준화
1. LED 광원에 따른 브로콜리 새싹의 이화학적 특성
○ LED광원에 따른 브로콜리 새싹의 일반성분을 분석한 결과, 광원에 따라 유의적인 차이를 나타냈으며, 구성당은 glucose가 0.22~3.12%로 가장 많았고, fructose는 0.10~2.04% 검출되어, 총 2종이 검출되었다.
○ 유기산은 총 3종의 유기산이 검출되었고, LED 광원에 따라 각각 달랐으며, citric acid. malic aci

Ⅳ. 연구개발결과
제 1 절 식물공장을 활용한 브로콜리 새싹으로부터 기능성 성분의 분리․동정 및 표준화
1. LED 광원에 따른 브로콜리 새싹의 이화학적 특성
○ LED광원에 따른 브로콜리 새싹의 일반성분을 분석한 결과, 광원에 따라 유의적인 차이를 나타냈으며, 구성당은 glucose가 0.22~3.12%로 가장 많았고, fructose는 0.10~2.04% 검출되어, 총 2종이 검출되었다.
○ 유기산은 총 3종의 유기산이 검출되었고, LED 광원에 따라 각각 달랐으며, citric acid. malic acid는 모든 시료에서 검출되었다. citric acid는 241.48-3292.59 mg%로 건조방법별 차이는 나타났으나 blue광원에서 가장 높게 측정되었고, malic acid는 183.76-2477.48 mg%로 red광원에서 가장 높은 함량을 보였으며, 건조방법별로 차이를 나타냈다. oxalic acid 는 42.14-331.31 mg%로 Blue광원에서 가장 높게 측정되었으며 열풍건조에서는 검출이 되지 않았다. 이는 건조과정에서 산화작용을 통한 저분자화로 그 함량이 감소하거나 휘발된 것으로 사료 된다.
○ LED 광원에 따른 브로콜리 새싹의 무기질은 총 7종의 성분이 검출되었고, 동결건조한 브로콜리 새싹의 무기질 조성이 가장 높게 측정 되었으며, Fe, Mn, Cu, Zn의 함량은 10㎎/100g 이하로 매우 낮게 측정되었다. K 함량은 73.55-669.04㎎/100g, Mg 49.71-585.35㎎/100g, Na 27.02-394.13㎎/100g 으로, LED 광원을 쬐인 브로콜리 새싹과 브로콜리 잎의 무기질 성분 및 함량에 약간의 차이가 있음을 알 수 있었다.
○ 비타민 A와 C 및 E 함량을 측정한 결과, 비타민 A의 함량은 FLR 212.62㎍RE/100g, HDR 860.62㎍RE/100g, FDR 4633.1㎍RE/100g 로 광원별 함량에서 Red광원을 쬐인 브로콜리 새싹에서 가장 높은 함량을 보였으며, 비타민 C의 경우 생잎(FL)브로콜리에서는 측정이 되지 않았고 HDR 134.57㎎/100g, HDB 137.05㎎/100g, HDW 119.87㎎/100g, FDR 136.96㎎/100g, FDB 84.19㎎/100g, FDW 8.53㎎/100g으로 열풍건조 브로콜리가 높게 측정되었다. 비타민 E 경우는 0.93-16.83 IU의 함량을 나타내었으며, 동결건조 브로콜리에서 가장 높게 나타났다.
2. LED 광원에 따른 생리활성
○ 식물공장에서 재배한 브로콜리 새싹의 광원에 따른 생리활성 효과를 비교한 결과, 총 페놀 함량은 생 브로콜리 1.51 mg/mL, 저온 열풍 건조 브로콜리 1.6 mg/mL, 동결건조 브로콜리 1.46 mg/mL로 측정되었으며, SOD 유사활성은 생 브로콜리 37.99%, 저온 열풍 건조 브로콜리 63.04%, 동결건조 브로콜리 48.22%로 나타났다. 전자공여능은 생 브로콜리 29.15%, 저온열풍건조 브로콜리 38.81%, 동결건조 브로콜리 15.02% 였으며, Hydroxyl radical 소거능은 생 브로콜리 61.37%, 저온열풍건조 브로콜리 67.76%, 동결건조 브로콜리 60.58%로 나타났다.
3. 브로콜리 추출물의 분획별 생리활성
○ 브로콜리 분획물들의 SOD 유사활성 및 hydroxyl radical 소거 능이 매우 높았고, 분획 물에 따라 약간의 차이를 나타내었으나, EDA(전자공여 능)이 다른 분획 물보다 ethyl acetate와 butanol에서 높게 측정되었다. 따라서, 활성이 대체적으로 높은 Butanol 분획 층을 이용하여 활성물질을 분리․정제 하였다.
4. Sephadex LH-20에 의한 분획물의 생리활성
○ 브로콜리 분획 추출물의 총 페놀 함량을 측정한 결과 EtOA (3.03 mg/mL) > BuOH (2.49 mg/mL) > CHCl₃ (1.62 mg/mL) > H₂O (1.15 mg/mL) > Hexane (1.05 mg/mL) 순으로 나타났으며, SOD 유사활성은 EtOA (94.91%) > BuOH (91.69%) > CHCl₃ (71.27%) > H₂O(50%) > Hexane (25%)의 활성을 보였으며, 전자공여능은 EtOA (92.64%) > BuOH (82.82%) > CHCl₃ (42.86%) > H₂O (21.06%) > Hexane (17.07%) 로 나타났고, Hydroxyl radical 소거능은 BuOH (91.08%) > EtOA (89.59%) > H₂O (87.54%) > Hexane (86.10%) > CHCl₃ (85.76%)순이었으며, Hydrogen radical 소거능은 H₂O (71.40%) > BuOH (71.37%) > Hexane (70.66%) > EtOA (69.11%) > CHCl₃ (55.24%)의 소거능을 보였다.
5. 활성이 높은 브로콜리 정제 추출물의 분리조건 확립 및 활성물질 분리
TLC 분석은 항산화 활성이 높았던 EtOA, BuOH 분획 추출물을 가지고 BuOH : MeOH : H₂O (4 : 1 : 2, v/v/v), CHCl₃ : Hexane : MeOH : H₂O : AcOH - 4 : 2 : 2 : 2 : 1 : 0.5 v/v/v/v/v/v), CHCl₃ : BuOH : MeOH : H₂O : AcOH - 2 : 4 : 1 : 2 : 1, (v/v/v/v/v)을 전개 용매로 TLC를 실시하였다. TLC 결과 모든 분획 추출물에서 공통적으로 Rf치가 같은 동일선상의 물질을 확인할 수 있었다. 확인된 물질이 항산화 활성 및 헬리코박터-위궤양 개선에 효과가 있는 미지의 물질일 것으로 사료된다.
6. HPLC 분석 및 Sulforaphane 함량 분석
브로콜리 새싹 추출물의 용매 분획별 설포라판 함량을 분석한 결과, CHCl₃ (56.76 mg/100g)> BuOH (49.16 mg/100g) > H₂O (5.67 mg/100g) > n-hexane (2.18 mg/100g)의 함량을 나타내었다. 분석결과, 처리방법과 추출용매에 따라 유효성분인 설포라판의 용출 양에 영향을 미치는 것으로 사료된다.
7. 구조분석
Positive ion에서 GC/MS상 분자량 측정 결과, 177.03의 분자량을 갖는 것으로 나타났다(m/z; 177.03 [M+H]+(Fig. 6). 1H-NMR(in CHCl₃-d6) 측정결과와 13C-NMR의 결과를 종합하여 구조를 결정하였으며, C 6개, H 11개 그리고 NOS₂ 1개를 갖는 분자식 C₆H₁₁NOS₂ 분자량 177.03인 Isocyanate계 Sulforaphane 으로 동정 되었다.
제 2 절 식물공장을 활용한 브로콜리 새싹의 기능성 소재 생산체계 구축 및 고부가가치 기능성 식품 개발
1. 브로콜리 새싹의 생산방법 및 대량생산체계 구축
가. 원적외선 음이온수 장치(Dileka)를 이용하여 브로콜리 새싹을 생산하는 방법
○ 전자와 원적외선이 부여된 활성수를 공급하고, LED 광원을 12시간씩 조사하였으며, 매 1시간 마다 2분씩 수분을 분사하여 재배하였다.
나. 브로콜리 최적 생육조건 확립
○ LED 청색광원을 발아 2일(암조건), 4일 동안 하루에 12시간씩 조사시켰으며, 온도범위는 21~23℃, 습도는 45~60%로 조절하여 브로콜리 새싹을 재배 하였다. 이러한 결과 LED 광원에 따른 영양소 함량 및 유효성분 함량에도 큰 차이를 보여, 브로콜리 새싹의 성분을 향상시키기 위해 가장 적절한 수확 시기는 발아 후 4일째 LED 청색광원으로 재배하는 것이 적절한 것으로 사료된다.
다. 브로콜리 새싹의 추출 최적화 방법 확립
○ 수확한 브로콜리 새싹을 열풍건조기를 이용하여 40~60℃로 24시간 건조한 후 70% EtOH 용매로 40℃에서 12시간씩 3반복 추출하여 수율을 측정한 결과, 백색광 처리구 11.87%, 적색광 처리구 7.65%, 청색광 처리구 14.02%로 청색광 처리구에서 생 추출물에 비해 10배 이상의 수율을 얻을 수 있었다.
2. 활성물질의 Open column 에 의한 분획
가. Open column에 의한 브로콜리 새싹의 수율, 활성 및 설포라판 함량
○ 70% 에탄올 브로콜리 열풍건조 추출물을 다공성 입자를 가진 HP-20 resin (H = 26, Ø =10)에 흡착시켜 에탄올 농도별 (0, 20, 40, 60, 80, 100%)로 용출한 결과, 수율은 20% 에탄올 용출 분획물 (BHP-20)에서 높았지만, 유효성분인 sulforaphane의 함량은 40% 에탄올 용출 분획물 (BHP-40)에서 가장 높은 함량을 보였다.
○ 브로콜리 항헬리코박터 활성이 가장 우수한 BHP-20 분획물과 생리활성이 우수한 BHP-40 분획물로부터 유효성분을 분리하기 위하여 open column chromatograph에 sephadex LH-20(H = 58, Ø = 1)을 4 x 50 cm까지 충진시키고, HP-20, HP-40 분획물중 2 g을 loading하였으며, 전개용매는 HP-20은chloroform : Methanol (100 : 0 - 0 : 100), HP-40은 70% MeOH(100)을 사용하여 용출하였다. Sephadex LH-20 (H = 58, Ø = 1)을 순차적으로 진행하여 최종적으로 HP-20은 8개의 subfractions를 분리하였고, HP-40은 3개의 subfractions을 분리하여 활성이 가장 우수한 fraction으로부터 active compound를 분리․정제하여 구조 분석을 하였다.
○ 항헬리코박터 활성 및 생리활성이 높은 정제된 분획물로부터 얻은 Fr1~Fr8, LH1~LH3의 활성을 측정한 결과, Fr1,Fr3,Fr5, LH1,3의 subfraction에서 대체적으로 활성이 높았으며, 이물질을 이용하여 구조분석을 실시하였다.
3. 구조분석
가. 활성물질의 TLC 분석 패턴
○ 생리활성이 높았던 BHP-40, Fr1, Fr5, LH1,3 정제 추출물을 가지고 CHCl₃ : n-hexane : BuOH : MeOH : H₂O : AcOH (4 : 2 : 2 : 2 : 1 : 1, v/v/v/v/v/v)을 전개용매로 TLC를 실시하였으며, Fr1,Fr5,LH1 정제 추출물에서 공통적으로 Rf치가 같은 동일선상의 물질을 확인할 수 있었고, BHP-20, 40, 60 부분정제 추출물과, LH3에서 또 따른 형광의 물질을 확인 하였다. 확인된 물질이 생리활성 및 항헬리코박터에 활성이 있는 미지의 물질일 것으로 사료된다.
나. HPLC분석 및 설포라판 함량
○ HPLC (μ-Bondapak C18 3.9×300 mm; flow rate 0.8 ㎖/min; inject vol., 10μL; detector, 254 nm)를 통해 유효성분인 Sulforaphane 함량을 측정한 결과, HP-20은 Fr3 (240 mg/100g) > Fr1 (193 mg/100g) > Fr5 (130 mg/100g) 순으로 나타났으며, HP-40은 LH1(168 mg/100g) > LH₂ (135 mg/100g)의 함량을 보였다.
실험결과 HP-20의 경우, Sephadex LH-20 (H = 58, Ø = 1)으로 정제하기 전에는 설포라판이 검출되지 않았으나, 순차적인 용매에 의한 정제․추출로 설포라판이 용출되는 것을 확인할 수 있었으며, HP-40의 경우, 순차적으로 정제를 하면서 유효성분인 설포라판 함량이 감소하는 것으로 나타났다. LH3의 정제추출물에서 확인된 다른 형광물질도 분리․정제 중에 있다.
다. GC/MS분석
○ GC/MS로 분자량과 피크의 분리를 확인하기 위해서 전자에 충격을 주어서 fragmentation 을 확인한 결과, 1H-Indol-3-propanol 의 adipate와 93%의 유사성을 가지고 있는 것을 확인할 수 있었다.
라. 구조분석(NMR 구조분석)
○ Positive ion에서 GC/MS상 분자량 측정 결과, 175.10의 분자량을 갖는 것으로 나타났다
(m/z ; 175.10 [M+H]+. 1H-NMR(in CHCl₃-d6) 측정결과와 13C-NMR의 결과를 종합하여 구조를 결정하였으며, C 11개, H 13개 그리고 NO1개를 갖는 분자식 C11H13NO 분자량 175.10인 Indol계 1H-Indol-3-propanol 으로 동정 되었다.
4. 브로콜리 새싹 추출물의 항 헬리코박터 및 위궤양 개선 효능평가
가. 헬리코박터균에 대한 항균 및 위염 개선 효능 평가
○ 본 연구는 브로콜리 새싹 추출물(broccoli sprout extract, BSE)의 헬리코박터(Helicobacter pylori)균에 대한 항균 및 위염/위궤양 개선효능을 평가하기 위해 수행되었다.
BSE는 70% 에탄올로 추출한 후 20%와 40% 에탄올로 부분 정제하여 각각 20BSE, 40BSE 및 70BSE로 명명하였다. In vitro 시험에서 10% fetal bovine serum (FBS)와 20BSE, 40BSE 및 70BSE의 최고농도를 첨가하여 serial dilution agar를 제작하고, H. pylori 접종 72시간 뒤의 균체 성장이 완전히 억제된 최소증식억제농도(minimal inhibitory concentration, MIC)를 확인하였다. 20BSE에서는 64 μg/mL 농도 이상의 구간에서, 40BSE와 70BSE에서도 각각 125μg/mL와 1,000 μg/mL 농도 이상의 구간에서 억제능을 나타내어 20BSE > 40BSE > 70BSE의 순으로 항균효능이 우수하였다. 또한 urease 억제효능을 평가하기 위하여 H. pylori 균주(1 x 108 CFU/mL)에 20BSE, 40BSE 및 70BSE를 농도별로 가하고, 6시간 동안 배양한 후 흡광도의 변화를 측정하여 urease의 활성도를 확인하였다. 20BSE, 40BSE 및 70BSE 모두 농도 의존적으로 효소활성도를 억제하였는데, 각각 350, 700 및 >2,000 μg/mL의 중간억제농도(median inhibitory concentration, IC50)를 나타내어 항균효능 순위와 일치하였다.
In vivo 마우스 감염모델에서 위점막에 대한 CLO kit 및 직접적인 균체확인 결과, 20BSE, 40BSE 및 70BSE 모두 균체제거 효과를 보여 주었는데, 40BSE > 70BSE ≥ 20BSE의 순으로 효과적이었는데, 특히 40BSE는 100 mg/kg에서 30 mg/kg pantoprazole과 유사한 효능을 보여 주었다. 위산분비에 대한 영향에 있어서 BSE는 위액량, pH, free HCl 및 total acidity에 전혀 영향을 미치지 않아 proton pump inhibitor인 pantoprazole과는 다른 기전을 가지고 있는 것으로 확인되었다. 흥미롭게도, BSE는 위산분비에 영향을 미치지 않음에도 위산과다로 인한 위궤양을 탁월하게 예방하였는데, in vitro 항균효능이 탁월한 20BSE나 in vivo 균체제거 효과가 뛰어난 40BSE보다 70BSE가 더 우수하였다. 이는 20BSE가 직접적인 항균효능을, 40BSE가 균체의 위점막 침투 억제효과를, 그리고 70BSE가 위점막 보호효과를 나타내는 서로 다른 기전을 가지고 있음을 의미한다. 따라서 브로콜리 새싹 에탄올 추출물은 추출용매에 따라 서로 다른 기전을 통해 복합적인 효능을 발휘하는 것으로 판단된다.
나. 기전별 위궤양 개선 효능 평가
Ethanol, indomethacin, WIRS 등 서로 다른 원인의 위궤양에 대해 브로콜리 새싹 추출물의 개선효능을 비교한 결과, 기존의 시판품인 J-BSE는 indomethacin 유도 위궤양에만 특이적으로 좋은 효능을 보여 주어 위조직 내로의 혈류저하로 인한 위궤양에 효과적인 것으로 나타났다. 이에 비해 70BSE는 indomethacin과 WIRS 유도 위궤양에 모두 좋은 효과를 나타내어 혈류개선과 위산분비 차단을 통해 효능을 발휘하는 것으로 J-BSE보다 적용범위가 확대되었음이 확인되었다. 흥미롭게도 40BSE는 70BSE에 비해 indomethacin 및 WIRS 유도 위궤양에 대한 효능이 상대적으로 낮았지만 최적용량이 30 mg/kg 이하로 내려가는 경향을 보여 이들 모델에서도 효능이 소실된 것이 아니라 유효용량 재설정이 필요함을 시사하고 있으며, 특히 ethanol 유도 위궤양에 대해서는 효능이 크게 상승하여 폭넓은 적응증이 확보되었음이 확인되었다. 한편, 지표물질 겸 유효물질로 추정된 sulforaphane, indole-3-carbinol 및 3-indolepropanol에 대한 유효성 평가 결과 indole-3-carbinol의 효과는 미약하였지만 sulforaphane과3-indolepropanol은 0.2-0.6 mg/kg의 아주 적은 용량에서로 pantoprazole (30 mg/kg)에 버금가는 효능을 발휘함으로써 브로콜리 새싹 추출물을 활용한 기능성 식품소재 개발에 좋은 지표가 될 것으로 기대된다.
5. 브로콜리 새싹 농축액 원료를 이용한 발효유 식품 (대량생산 공정)
가. 원료 생산 과정
○ 브로콜리 종자를 식물공장 시스템과 디레카 장치를 활용하여 평균 4~5시간 버블링하고, 평균 120g 씩 파종하여 온도 21~23℃, 습도 45~60%로 조절하여 2일동안 암조건하에 발아를 유도함.
○ 발아가 진행되면, LED Blue 광원을 12시간씩 조사하였으며, 매 1시간 마다 2분씩 수분을 분사하여 4일 동안 재배한 후 수확함.
나. 브로콜리 새싹 농축액 제조공정
○ 수확한 브로콜리를 브로콜리 무게의 5배의 물을 사용하여 깨끗이 수세함.
○ 수세한 브로콜리 생물은 식물세포가 살아있기 때문에 건조과정을 거쳐 세포를 파괴시켜 성분이 최대한 추출되도록 함.
○ 건조과정을 거친 것을 성분추출의 최적화를 위해 세절함.
○ 성분의 파괴를 방지하기 위해 100℃가 아닌 추출온도를 80℃를 유지하며, 장시간 추출함.
○ 추출성분이 아닌 이물질을 함유하지 않도록 30㎛ 필터로 1차 여과하고, 2차로 4 ㎛필터로 제균 여과하여 브로콜리의 성분만을 함유하게 함.
○ 희석된 성분을 고농도로 생산하기 위해 저온 감압 농축함.
○ 농축 시 휘발되는 향을 포집하여 액을 다시 농축액에 혼합시켜 농축함으로써 브로콜리 고유의 향을 함유시킴. (에센션 성분으로 생리활성 강함)
○ 농축액을 20 kg 프라스틱 통에 충진 포장함.
○ 충진 포장한 것을 출고 전에 표준품과 비교 시험 함.(성상, 물리시험)
○ 합격품을 출고 시킴.

Abstract ▼

IV. Results of Research & Development
Section 1. Simultaneous determination and standardization of functional substances from broccoli sprouts cultivated in plant factory
1. Physical and Chemical Features and Components of Broccoli sprouts by LED light sources
○ Sugar:
According to a res

IV. Results of Research & Development
Section 1. Simultaneous determination and standardization of functional substances from broccoli sprouts cultivated in plant factory
1. Physical and Chemical Features and Components of Broccoli sprouts by LED light sources
○ Sugar:
According to a result of chemical composition analysis of broccoli sprouts by LED light sources, it shows that chemical composition were significantly different by light sources, respectively. In component sugar analysis, there were 2 kinds of monosaccharides. In details, 0.22%-3.12% of glucose was found and 0.10%-2.04% of fructose was detected.
○ Organic Acid:
There were 3 kinds of organic acid in broccoli sprouts samples and its contents were different by LED light conditions. Citric acid, malic acid were detected from all the samples. Range of citric acid was 241.48-3292.59 mg%. Content of citric acid was different by drying methods, also high content of citric acid was detected in broccoli sprouts by blue LED light. Regarding malic acid, the range was 183.76-2477.48 mg%.
And high content of malic acid was detected by red LED. The content also was different by drying method, In case of oxalic acid, the range of content was 42.14-331.31 mg% and high content of the acid was detected by blue LED light condition. However, oxalic acid was not detected in the hot-air drying sample. It was assumed that content of oxalic acid was decreased or volatilized because of low-molecularization through drying process.
○ Minerals:
Total 7 kinds of minerals were detected from broccoli sprouts samples by different
LED conditions. Also, high component of minerals was in the freezing dried broccoli sprouts. Contents of Fe, Mn, Cu, Zn were less than 10mg/100g. K was 73.55 - 669.04 mg/100g, Mg was 49.71 - 585.5 mg/100g and Na was 27.02 - 394.13mg/100g. Mineral ingredients and its content were slightly different between broccoli sprouts and broccoli leaf exposed by LED lights.
○ Vitamins:
Content of Vitamin A of broccoli sprouts sampels was FLR 212.62㎍RE/100g, HDR 860.62㎍RE/100g. In the samples by red LED, Vitamin A was highly contained.
Content of Vitamin C was HDR 134.57㎎/100g, HDB 137.05㎎/100g, HDW 119.87㎎ /100g, FDR 136.96㎎/100g, FDB 84.19㎎/100g, FDW 8.53㎎/100g. Vitamin C was highly detected from hot-air drying sample while it was not contained in sample of broccoli leaf (FL). In case of Vitamin E, the content was 0.93-16.83 IU. Also, Vitamin E was most highly detected from freezing dried sample.
2. Bioactive effect of active substances in broccoli sprouts with different drying methods
○ Bioactive effect of active substances in broccoli sprouts with different drying methods were investigated.
- Total phenol contents were as below:
In fresh broccoli sprouts: 1.51 mg/mL,
In low temperature drying broccoli sprouts: 1.6 mg/mL, And In freezing dried broccoli sprouts: 1.46mg/mL.
- SOD (Superoxide dismutase)-like activity were as below:
In fresh broccoli sprouts: 37.99%, In low temperature drying broccoli sprouts: 63.04%, And In freezing dried broccoli sprouts: 48.22%
- Electron-Donating Abilities (EDA) were as below:
In fresh broccoli sprouts: 29.15%,
In low temperature drying broccoli sprouts: 38.81%, And In freezing dried broccoli sprouts: 15.02%
- Hydroxyl radical scavenging activities were as below: In fresh broccoli sprouts: 61.37%, In low temperature drying broccoli sprouts: 67.76%, And In freezing dried broccoli sprouts: 60.58%
3. Bioactive effect of active substances among broccoli sprouts fractions
○ SOD-like activities and hydroxyl radical scavenging activities of broccoli sprouts fractions were very high. EDA(Electron-Donating Abilities) of ethyl acetate fraction and butanol fraction were high comparing other fraction. Therefore, it was tried that the active substances were isolated and refined by butanol fraction which had high activities.
4. Bioactive effect of broccoli sprouts fractions by Sephadex LH-20
○ Bioactive effect of broccoli sprouts fractions by Sephadex LH-20 were investigated.
- Total phenol contents were followed by (as below):
EtOA (3.03 mg/mL) > BuOH (2.49 mg/mL) > CHCl3 (1.62 mg/mL) > H2O (1.15 mg/mL) > Hexane (1.05 mg/mL)
- SOD-like activities were followed by (as below):
EtOA (94.91%) > BuOH (91.69%) > CHCl3 (71.27%) > H2O (50%) > Hexane (25%)
- EDA were followed by (as below):
EtOA (92.64%) > BuOH (82.82%) > CHCl3 (42.86%) > H2O (21.06%) > Hexane (17.07%)
- Hydroxyl radical scavenging activities were followed by (as below): BuOH (91.08%) > EtOA (89.59%) > H2O (87.54%) > Hexane (86.10%) > CHCl3 (85.76%)
- Hydrogen radical scavenging activities were followed by (as below): H2O (71.40%) > BuOH (71.37%) > Hexane (70.66%) > EtOA (69.11%) > CHCl3 (55.24%)
5. Determination of bioactive substances and Establishment of isolation condition for broccoli sprouts extract which has high performance
○ Thin layer chromatography (TLC) analysis was conducted with EtOA, BuOH fractions which antioxidant activities were high. In the TLC analysis, developing solvents were BuOH : MeOH : H2O (4 : 1 : 2, v/v/v), CHCl3 : Hexane : MeOH : H2O : AcOH - 4 : 2 : 2 : 2 : 1 : 0.5 v/v/v/v/v/v), CHCl3 : BuOH : MeOH : H2O : AcOH - 2 : 4 : 1 : 2 : 1, (v/v/v/v/v). Result of the analysis showed that Refention factor (Rf) values were same. It was found that all fractions were collinear substances. It was suggested that verified substance might be an unknown chemical which had antioxidant antivity, anti-helicobacter pylori and improvement of gastric ulcer.
6. HPLC Analysis and Sulforaphane Quantitative Analysis
○ Sulforaphane contents of solvent fractions of broccoli sprouts extracts were followed by CHCl3 (56.76 mg/100g) > BuOH (49.16 mg/100g) > H2O (5.67 mg/100g) > n-hexane (2.18 mg/100g). Result showed that processing methods and extraction solvents could influence elution of active substance sulforaphane.
7. Structural Analysis
○ Molecular weight was measured by using Gas Chromatography Mass Spectrometry (GC/MS). M.W. at positive ion on GC/MS was 177.03 [M+H]+. Structure was determined with combination of the results of 1H-NMR(in CHCl3-d6) and13C-NMR. In our study, sulforaphane (molecular fomular: C6H11NOS2, M.W. 177.03) was isolated from broccoli sprouts fraction.
○ Japanese BSE (J-BSE) exerted a high anti-ulcer effect in only indomethacin-induced ulcers, indicative of its improving activity on the local blood flow. In comparison, 70BSE exhibited high efficacies in both indomethacin- and WIRS-induced ulcers, suggesting that it improves local blood flow and inhibits gastric acid secretion as seen in pantoprazole. Notably, 40BSE displayed an excellent anti-ulcer activity in ethanol-induced ulcers, in addition to the decreased optimal dosages in indomethacinand WIRS-induced ulcers, implying that the target causative factors were expanded. As active ingredients, sulforaphane and 3-indolepropanol were found to improve the ethanol-induced ulcers at low doses (0.2-0.6 mg/kg), whereas the effect of indole-3-carbinol was negligible. The results indicate that BSE, prepared by stepwise ethanol extraction, attenuates gastric ulcers induced by various ulcerative factors via diverse action mechanisms. Therefore, it is suggested that 40BSE could be a good candidate for the improvement of gastric function, and that sulforapahne and 3-indolepropanol might be useful markers as active ingredients in 40BSE.
Section 2. Establishment of mass production of broccoli sprouts utilizing plant factory and development of high value added functional food
1. Production Methods of broccoli sprouts and establishment of mass production
A. Production method of broccoli sprouts using an infrared and electron generating device for water (DILEKA)
○ In order to cultivate broccoli sprouts, infrared and electron generated water was supplied in the plant factory system. The water was injected for 2 minutes per 1 hour during cultivation period. Also, the sprouts were exposed by LED lights 12 hours per day.
B. Optimization of growth conditions for broccoli sprouts
○ Germination of broccoli seeds was conducted 2 days in the dark place with temperature 21~23℃ and humidity 45~60%. After germination, LED light exposed 12 hours during 4 days.
○ The result showed that contents of nutrients and active substances were different by cultivation conditions of broccoli sprouts. It was suggested that optimized harvest time of broccoli sprouts was 4 days after germination and cultivation with blue LED light.
C. Establishment of optimized extract methods for broccoli sprouts
○ Harvested broccoli sprouts was dried in the hot air drying device with 40~60℃, 24 hour. The dried sprouts were extracted 3 times by 70% EtOH solvent, 40℃, 12 hours.
Yield rate of broccoli sprouts with white light 11.87%, red light 7.65% and blue light 14.02%. It showed that yield rate of broccoli sprouts cultivated with blue light were more than 10 times comparing fresh broccoli sprouts.
2. Active substance from broccoli sprouts fraction by open column
A. Yield rates, activities and sulforaphane contents of broccoli sprouts by open column ○ Hot-air dried broccoli sprouts were extracted by 70% EtOH. And the broccoli sprouts extract was absorbed in particle size selectable HP-20 resin (H = 26, Ø = 10). And the extract was eluted by different EtOH concentrations (0, 20, 40, 60, 80, 100%).
The yield rate was most high at 20% EtOH eluted fraction (BHP-20). However, sulforaphane was most highly contained at 40% EtOH fraction (BHP-40).
○ BHP-20 fraction was excellent activity of anti-Helicobactor pylori effect. Also, HP-40 was superior in bioactive effect. In order to isolate bioactive substances from BHP-20 and BHP-40. The fractions inserted into 4 x 50 cm of sephadex LH-20 (H = 58, Ø = 1) for open column chromatograph. And 2 g of HP-20 and HP-40 fraction were loaded, respectively. In the elution, developing solvent of HP-20 was chloroform : Methanol (100 : 0 - 0 : 100) and the solvent of HP-40 was 70% MeOH(100). From HP-20 fraction, 8 kinds of subfractions were isolated using Sephadex LH-20 (H = 58, Ø = 1). And, 3 kinds of subfractions were isolated from the HP-40 fraction. Also, active compound from superior fractions was isolated and refined. And the structural analysis was also in progress.
○ Fr1~Fr8 subfractions were obtained from BHP-20 fraction which had anti-Helicobacter pylori effect. LH1 ~ LH3 subfractions were also obtained from BHP-40 fraction which had superior bioactive effect. The results for subfractions analysis, activities were high at the Fr1, Fr3, Fr5 and LH1,3 subfractions. Using this substances, structural analysis were conducted.
3. Structural Analysis
A. TLC Analysis patterns for active substances
○ Thin layer chromatography (TLC) analysis was conducted using BHP-40, and Fr1, Fr5, LH1,3 refined extracts which had superior bioactive effect. In the TLC analysis, developing solvents were CHCl3 : n-hexane : BuOH : MeOH : H2O : AcOH (4 : 2 : 2 : 2 : 1 : 1, v/v/v/v/v/v). Result of the analysis showed that Retention factor (Rf) values were same. It was found that all fractions of Fr1, Fr5 and LH1 were collinear substances. Also, other fluorescence substances were found at BHP-20, 40, 60 and LH3 refined extracts. It was suggested that verified substances might be an unknown chemical which had bioactive effect and anti-Helicobacter pylori effect.
B. HPLC Analysis and Sulforaphane content
○ Bioactive substance sulforaphane were analyzed by HPLC (μ-Bondapak C183.9×300 mm; flow rate 0.8 ㎖/min; inject vol., 10μL; detector, 254 nm). Sulforaphane contents of HP-20 were followed by Fr3 (240 mg/100g) > Fr1 (193 mg/100g) > Fr5 (130 mg/100g). And the content of HP-40 were followed by LH1 (168 mg/100g) > LH2 (135 mg/100g). In case of HP-20, sulforaphane was not detected before refining by Sephadex LH-20 (H = 58, Ø = 1). However, it showed that sulforaphane was eluted through extraction and refinement by solvents. Regarding HP-40, content of sulforaphane was decreased through refinement. Other fluorescence materials from refined LH3 subfractions were also being isolated and refined
C. GC/MS Analysis
○ In order to measure molecular weight and identify peak, Gas Chromatography Mass Spectrometry (GC/MS) was used. After electron impact fragmentation, it showed that peak was 93% similar with adipate of 1-H-indol-3-propanol.
D. NMR Structural Analysis
○ Molecular weight at positive ion on GC/MS was 175.10 [M+H]+. Structure was determined with combination of the results of 1H-NMR(in CHCl3-d6) and 13C-NMR.
In our study, 1H-Indol-3-propanol (molecular formula C11H13NO, M.W. 175.10) was isolated.
4. Efficacy evaluation of broccoli sprouts extract on anti-Helicobacter pylori effect and improvement of gastric ulcer
○ The study was conducted in the purpose of efficacy evaluation of broccoli sprouts extract (BSE) on anti-Helicobacter pylori effect and improvement of gastritis / gastric ulcer. After BSE was extract by 70% EtOH, the extract was also refined by 20% and 40% EtOH, separately. In vitro, 3 kinds of BSEs such as 20% BSE, 40% BSE and 70% BSE were prepared. 10% of fetal bovine serum (FBS) was added into the high concentrations of 20 BSE, 40 BSE and 70 BSE in order to prepare serial dilution agar. And the agar preparation were inoculated by Helicobacter pylori. After 72 hours of H. pylori inoculation, Minimal inhibitory concentration (MIC) was defined as the lowest concentration of antimicrobial. Inhibition activity of 20 BSE was found over 64 μg/mL concentration. And inhibition activities of 20 BSE and 70 BSE were found over 125 μ g/mL and 1,000μg/mL, respectively. Inhibition activities of BSEs were effective followed by 20 BSE > 40 BSE > 70BSE. To evaluate efficacy of urease inhibition activity, 1 x 108 CFU/mL of H. pylori strain were added on 20 BSE, 40 BSE and 70 BSE. After 6 hour cultivation, urease activity was determined by measuring change of absorbance. All of the BSEs inhibited enzyme activity dose-dependantly. Median inhibitory concentration IC50 of 20 BSE, 40 BSE and 70 BSE were 350, 700, over 2,000 μg/mL. The order is same as those of antimicrobial activity. In vivo, in a mouse infection model, the result of CLO kit analysis showed that all of 20 BSE, 40 BSE and 70 BSE had efficacy of bacteria eradications against gastric mucosa. The efficacy of bacteria eradications were followed by 40BSE > 70BSE ≥ 20BSE. Especially, 100mg/kg of 40 BSE showed similar efficacy of 30mg/kg of pantoprazole. Regarding effect of maximum gastric acid secretion, BSE didn't influence to amount of gastric juice, pH, free HCL and total acidity, so that it had different mechanism comparing with pantoprazole (a proton pump inhibitor, PPI). Although BSEs didn't influence to gastric acid secretion, the BSEs prevented excellently gastric ulcer induced by excess acid in the stomach. In this study, 70 BSE showed superior activity comparing with 20 BSE which had antimicrobial activity (in vitro) and with 40 BSE which had bacteria eradications (in vivo). It was suggested that 3 kinds of BSE had different mechanism, respectively. In details, 20 BSE had direct antimicrobial activity, and 40 BSE had inbibition effect of penetration of bacteria into gastric mucosa . Also, 40 BSE had protective effect on gastric mucosa. Therefore, it was suggested that the broccoli sprouts extracts (EtOH BSEs) had synergistic effects on gastric health by their complex activities with different mechanisms.
○ 70BSE exhibited high efficacies in both indomethacin- and WIRS-induced ulcers, suggesting that it improves local blood flow and inhibits gastric acid secretion as seen in pantoprazole. Notably, 40BSE displayed an excellent anti-ulcer activity in ethanol-induced ulcers, in addition to the decreased optimal dosages in indomethacinand WIRS-induced ulcers, implying that the target causative factors were expanded. As active ingredients, sulforaphane and 3-indolepropanol were found to improve the ethanol-induced ulcers at low doses (0.2-0.6 mg/kg), whereas the effect of indole-3-carbinol was negligible. The results indicate that BSE, prepared by stepwise ethanol extraction, attenuates gastric ulcers induced by various ulcerative factors via diverse action mechanisms. Therefore, it is suggested that 40BSE could be a good candidate for the improvement of gastric function, and that sulforapahne and 3-indolepropanol might be useful markers as active ingredients in 40BSE.
5. Mass production process of broccoli sprouts concentrate for application of a yogurt product
A. Production of broccoli sprouts
○ Broccoli seeds were washed for 4~5 hours. And broccoli sprouts were cultivated in the plant factory system utilizing DILEKA device. Average 120g of broccoli seeds were sown on plates of plant factory. Germination was conducted in the dark place during 2 days with temperature 21~23℃, and humidity 45~60%.
○ After germination, blue LED light exposed 12 hours per day. And the water was injected for 2 minutes per 1 hour during cultivation period. Harvest time of broccoli sprouts was 4 days after germination and cultivation.
B. Process flow of Broccoli sprouts concentrate
○ Cleaning fresh broccoli sprouts with 5 times of water
○ Drying of broccoli sprouts for extraction
○ Cutting dried broccoli sprouts for optimization of substance extraction
○ Extraction with temperature 80℃, long hours.
○ First Filtration with 30㎛ filter for removal of foreign substance
○ Second Filtration with 4㎛ filter for removal of microorganism
○ Concentration of diluted substances using low temperature evaporator
○ Collecting volatile flavour of broccoli sprouts when concentration
○ Mixed flavour into broccoli sprouts concentrate
○ Filling the concentrate into PE 20KG bag
○ Quality Control for broccoli sprouts concentrate
○ Labelling of the product in accordance with Korean Food Sanitation Act