본 연구는 칡뿌리의 isoflavone생물 전환 조절을 위한 배지 조건 최적화를 위해 수행되었다. Isoflavone 은 phytoestrogen 이라고 불리우는 식물 유래 대사물질이다. 이는 estrogen 과 유사한 구조를 가지며, ...
본 연구는 칡뿌리의 isoflavone생물 전환 조절을 위한 배지 조건 최적화를 위해 수행되었다. Isoflavone 은 phytoestrogen 이라고 불리우는 식물 유래 대사물질이다. 이는 estrogen 과 유사한 구조를 가지며, cancer, inflammation 및 cardiovascular 예방에 효과적이라고 알려져있다. 이와 같은 isoflavone 은 본질적으로 conjugated glucoside 의 형태이며, 발효를 거치면 isoflavone 유래 유용물질인 aglucone 의 함량이 높아진다. 대표적인 식물로는 대두와 칡뿌리가 있다.
실험은 칡뿌리의 aglucone 의 최적 비율을 도출하기 위해 3 가지 레벨의 각기 다른 조성으로 수행하였으며, 실험의 설계는 Box Behnken design 을 이용하여 구성하였다. 높은 glucosidase 활성을 가진 균주 발효는 isoflavone 의 aglucone 함량을 증진시킬 수 있으며, isoflavone 섭취에 따른 이익 또한 높인다. 따라서 발효에 사용할 높은 glucosidase 활성을 가진 젖산균주 및 효모 21 종을 선발하였고, 대표적으로 Lactobacillus plantarum, Pediococcus pentosaceus 및 Saccharomyces cerevisiae 로 조사되었다.
Thin layer chromatography (TLC) 및 high performance liquid chromatography (HPLC)를 이용하여 대두와 칡뿌리의 isoflavone 을 분석하였다. TLC 분석을 위해 통계적으로 최적화된 유기용매 비율은 각각 chloroform (55%), butanol (5%), acetic acid (38%) 및 water (2%)로 조사되었다. 칡뿌리에서 높은 isoflavone 농도를 나타내었으며, 대두는 칡뿌리보다 isoflavone spot 이 작게 나타났다. HPLC 결과, 칡뿌리가 대두에 비해 높은 농도(91.5%)의 glucoside 함량을 나타내었으며, 대두는 칡뿌리에 비해 높은 농도(20.2%)의 aglucone 를 함유한 것으로 조사되었다.
Box Behnken design 을 이용하여 가장 효과적인 배지 혼합 조건을 도출하였고, β-glucosidase activity 와 aglucone 함량을 기준으로 하였다. 이에 최적 혼합 배지조건은 yeast extract (5g/L), sodium acetate (5 g/L), manganese sulphate (0.05 g/L), fructose (10 g/L), molasses (20 g/L) 및 ammonium sulphate (5 g/L)로 설정되었다. 통계 모델 예측을 통해 칡뿌리 최대 Aglucone 함량은 0.21 mg/g 으로 조사되었다.
본 연구는 칡뿌리의 isoflavone 생물 전환 조절을 위한 배지 조건 최적화를 위해 수행되었다. Isoflavone 은 phytoestrogen 이라고 불리우는 식물 유래 대사물질이다. 이는 estrogen 과 유사한 구조를 가지며, cancer, inflammation 및 cardiovascular 예방에 효과적이라고 알려져있다. 이와 같은 isoflavone 은 본질적으로 conjugated glucoside 의 형태이며, 발효를 거치면 isoflavone 유래 유용물질인 aglucone 의 함량이 높아진다. 대표적인 식물로는 대두와 칡뿌리가 있다.
실험은 칡뿌리의 aglucone 의 최적 비율을 도출하기 위해 3 가지 레벨의 각기 다른 조성으로 수행하였으며, 실험의 설계는 Box Behnken design 을 이용하여 구성하였다. 높은 glucosidase 활성을 가진 균주 발효는 isoflavone 의 aglucone 함량을 증진시킬 수 있으며, isoflavone 섭취에 따른 이익 또한 높인다. 따라서 발효에 사용할 높은 glucosidase 활성을 가진 젖산균주 및 효모 21 종을 선발하였고, 대표적으로 Lactobacillus plantarum, Pediococcus pentosaceus 및 Saccharomyces cerevisiae 로 조사되었다.
Thin layer chromatography (TLC) 및 high performance liquid chromatography (HPLC)를 이용하여 대두와 칡뿌리의 isoflavone 을 분석하였다. TLC 분석을 위해 통계적으로 최적화된 유기용매 비율은 각각 chloroform (55%), butanol (5%), acetic acid (38%) 및 water (2%)로 조사되었다. 칡뿌리에서 높은 isoflavone 농도를 나타내었으며, 대두는 칡뿌리보다 isoflavone spot 이 작게 나타났다. HPLC 결과, 칡뿌리가 대두에 비해 높은 농도(91.5%)의 glucoside 함량을 나타내었으며, 대두는 칡뿌리에 비해 높은 농도(20.2%)의 aglucone 를 함유한 것으로 조사되었다.
Box Behnken design 을 이용하여 가장 효과적인 배지 혼합 조건을 도출하였고, β-glucosidase activity 와 aglucone 함량을 기준으로 하였다. 이에 최적 혼합 배지조건은 yeast extract (5g/L), sodium acetate (5 g/L), manganese sulphate (0.05 g/L), fructose (10 g/L), molasses (20 g/L) 및 ammonium sulphate (5 g/L)로 설정되었다. 통계 모델 예측을 통해 칡뿌리 최대 Aglucone 함량은 0.21 mg/g 으로 조사되었다.
Isoflavones belong to a group of compounds exclusively manufactured by plants called phytoestrogens. Because of their similarity in structure to the human hormone, estrogen, they are associated with a number of health benefits such as protection against cancers, inflammation and cardiovascular d...
Isoflavones belong to a group of compounds exclusively manufactured by plants called phytoestrogens. Because of their similarity in structure to the human hormone, estrogen, they are associated with a number of health benefits such as protection against cancers, inflammation and cardiovascular diseases. Isoflavones are intrinsically found in the conjugated glucoside form, which is less easily absorbed than their aglucone counterparts. Although fermentation decreases the total level of isoflavones, it breaks the glycosidic bond to release a sugar residue and thereby increases the levels of aglucones; which are more metabolically active.
A three-level approach was taken to optimize levels of aglucones in kudzu root: selecting a highly active glucosidase activity from microorganisms, selecting a source with high isoflavone content and finally optimizing media for bioconversion using statistical modelling- Box Behnken design. A total of 21 lactic acid producing bacteria and 1 yeast laboratory isolates were cultured in de Man, Rogosa and Sharpe (MRS) broth for 24 h before investigating their β-glucosidase capabilities using p-nitrophenyl β-D-glucopyranoside as substrate. All strains exhibited some β-glucosidase activity, although it was strain-dependent and differed within species (p<0.0001). Lactobacillus plantarum, Saccharomyces cerevisiae and Pediococcus pentosaceus were initially selected for high glucosidase activity. S. cerevisiae was later excluded due to viability challenges. L. plantarum showed optimal enzyme activity at 45 °C and P. pentosaceus at 32°C. Both bacteria showed stability over a wide range of pH; 4 to 9. L. plantarum was ultimately selected because it exhibited higher enzyme activity than P. pentosaceus, even at lower numbers. High glucosidase is likely to increase isoflavones aglucone content, and thus increase health benefits associated with isoflavone consumption.
Soy beans and kudzu root are widely known to contain high amounts of isoflavones. We compared their isoflavone content using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) for qualitative and quantitative analysis. A statistically optimized solvent consisting of chloroform (55%), butanol (5%), acetic acid (38%) and water (2%) was used for TLC. The glucosides; daidzin and genistin, and their aglucones; daidzein and genistein, were separated for both soybean and kudzu root. Contrary to literature, kudzu root showed higher concentrations of isoflavones on the TLC plates through darker spots. This was confirmed by HPLC, kudzu root had a higher percentage of glucosides at 91.5% compared to that of soybean of 71.3%. Soy, however, contained 20.2% more aglucones. Kudzu root was therefore chosen as the candidate for further bioconversion experiments due to its higher glucoside and total isoflavone content.
The Box-Behnken design is a highly effective and efficient tool used in optimization experiments. Significant factors were selected from MRS medium, with the addition of carbon and nitrogen sources. β-glucosidase activity and aglucone content were analysed. The optimum medium was composed of yeast extract (5 g/L), sodium acetate (5 g/L), manganese sulphate (0.05 g/L), fructose (10 g/L), molasses (20 g/L) and ammonium sulphate (5 g/L). The model predicted aglucone content to be maximised at 0.21 mg/g kudzu root, and the observed after validation was 0.19 mg/g. By using the Box-Behnken design we were able to statistically optimize the medium for increasing metabolically active aglucones in kudzu root. The outcome verified what was expected, confirming the effectiveness of this statistical tool in optimization experiments. Increased aglucone levels suggest that there will be more health benefits associated with the ingestion of isoflavones. This has potential to significantly impact the feeds and food industry in delivering functional products – foods which offer health benefits beyond inherent nutrition-to consumers.
Isoflavones belong to a group of compounds exclusively manufactured by plants called phytoestrogens. Because of their similarity in structure to the human hormone, estrogen, they are associated with a number of health benefits such as protection against cancers, inflammation and cardiovascular diseases. Isoflavones are intrinsically found in the conjugated glucoside form, which is less easily absorbed than their aglucone counterparts. Although fermentation decreases the total level of isoflavones, it breaks the glycosidic bond to release a sugar residue and thereby increases the levels of aglucones; which are more metabolically active.
A three-level approach was taken to optimize levels of aglucones in kudzu root: selecting a highly active glucosidase activity from microorganisms, selecting a source with high isoflavone content and finally optimizing media for bioconversion using statistical modelling- Box Behnken design. A total of 21 lactic acid producing bacteria and 1 yeast laboratory isolates were cultured in de Man, Rogosa and Sharpe (MRS) broth for 24 h before investigating their β-glucosidase capabilities using p-nitrophenyl β-D-glucopyranoside as substrate. All strains exhibited some β-glucosidase activity, although it was strain-dependent and differed within species (p<0.0001). Lactobacillus plantarum, Saccharomyces cerevisiae and Pediococcus pentosaceus were initially selected for high glucosidase activity. S. cerevisiae was later excluded due to viability challenges. L. plantarum showed optimal enzyme activity at 45 °C and P. pentosaceus at 32°C. Both bacteria showed stability over a wide range of pH; 4 to 9. L. plantarum was ultimately selected because it exhibited higher enzyme activity than P. pentosaceus, even at lower numbers. High glucosidase is likely to increase isoflavones aglucone content, and thus increase health benefits associated with isoflavone consumption.
Soy beans and kudzu root are widely known to contain high amounts of isoflavones. We compared their isoflavone content using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) for qualitative and quantitative analysis. A statistically optimized solvent consisting of chloroform (55%), butanol (5%), acetic acid (38%) and water (2%) was used for TLC. The glucosides; daidzin and genistin, and their aglucones; daidzein and genistein, were separated for both soybean and kudzu root. Contrary to literature, kudzu root showed higher concentrations of isoflavones on the TLC plates through darker spots. This was confirmed by HPLC, kudzu root had a higher percentage of glucosides at 91.5% compared to that of soybean of 71.3%. Soy, however, contained 20.2% more aglucones. Kudzu root was therefore chosen as the candidate for further bioconversion experiments due to its higher glucoside and total isoflavone content.
The Box-Behnken design is a highly effective and efficient tool used in optimization experiments. Significant factors were selected from MRS medium, with the addition of carbon and nitrogen sources. β-glucosidase activity and aglucone content were analysed. The optimum medium was composed of yeast extract (5 g/L), sodium acetate (5 g/L), manganese sulphate (0.05 g/L), fructose (10 g/L), molasses (20 g/L) and ammonium sulphate (5 g/L). The model predicted aglucone content to be maximised at 0.21 mg/g kudzu root, and the observed after validation was 0.19 mg/g. By using the Box-Behnken design we were able to statistically optimize the medium for increasing metabolically active aglucones in kudzu root. The outcome verified what was expected, confirming the effectiveness of this statistical tool in optimization experiments. Increased aglucone levels suggest that there will be more health benefits associated with the ingestion of isoflavones. This has potential to significantly impact the feeds and food industry in delivering functional products – foods which offer health benefits beyond inherent nutrition-to consumers.
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