Metabolic Activity and Physiological Characteristics of Wheat and Barley Grass Juices Extracted by a Household Extractor Equipped with a Twin Gear
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Abstract:
본 연구는 대표적인 식량 작물인 밀과 보리의 새싹을 쌍기어를 장착한 가정용 저속 녹즙기로 착즙하여 제조한 각각의 새싹 주스에 대해 주스의 품질 특성을 조사하고 주스 섭취 후 소화 과정에 따른 폴리페놀 함량 변화와 그에 따른 항산화 활성 및 ACE 저해능 분석을 통해 밀싹과 새싹보리의 대사활성 변화를 조사하기 위해 수행되었다. 밀싹과 새싹보리의 착즙을 통해 분쇄되는 입자의 평균 크기는 117.7 μm 및 203.1 μm로 미세한 입자들이 주스에 분포되어 있음을 확인 하였다. 그뿐만 아니라 밀싹과 새싹보리 주스에는 chlorophyll과 protease 효소를 포함한 다양한 영양성분이 풍부하게 함유되어 있으며, 특히 비타민 C와 폴리페놀 및 플라보노이드를 풍부하게 함유하고 있어 SOD 유사 활성 및 지질과산화 억제능 시험에서 높은 항산화 활성을 보였다. 이들 주스의 섭취에 따른 대사활성 변화를 조사하기 위해 위장 및 소장 소화를 in vitro digestion model을 통해 재현하였다. 가장 먼저 소화 과정에 따른 폴리페놀 및 플라보노이드 함량을 분석한 결과 위장 소화 단계를 거치면서 함량이 크게 증가하는 것을 확인하였으며, 이에 따라 밀싹과 새싹보리 주스의 환원력과 ABTS 라디칼 소거능 또한 크게 증가하는 것을 확인하였다. 폴리페놀 및 플라보노이드 함량 증가와 FRAP 및 ABTS 소거능 간의 상관계수는 모두 0.78 이상으로 높은 양의 상관관계를 보였다. 또한 소화 과정에 따른 ACE 저해 활성을 조사한 결과 위장 소화 단계에서 저해능이 매우 증가하는 것을 확인하였으나 소장 소화를 거치면 오히려 감소하는 것을 확인하였다. 따라서 본 연구에서는 밀싹과 새싹보리의 주스 품질 특성을 확인하였으며 주스 섭취에 따라 위장 및 소장 소화와 같은 대사 변화를 통해 생리활성이 크게 증가할 수 있음을 확인하였다.Keywords:
ABTS
Extractor
Different assays have been developed to measure the antioxidant activity of foods and biological samples. The most popular assays adapted to foods are the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)/Trolox equivalent antioxidant capacity (TEAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and oxygen radical absorbance capacity (ORAC). This chapter discusses ABTS and TEAC in more detail. ABTS was used for the determination of fecal occult blood and also for the determination of glucose as a reagent in the glucose oxidase/peroxidase test. Simultaneously, ABTS was used as a peroxidase substrate for kinetic studies due to its excellent physical and chemical properties. The first use of ABTS for determining antioxidant activity was reported by Miller and his team in the TEAC assay, although a similar assay had already been used by Arnao group's. The TEAC assay is based on the generation of ABTS•+ using metmyoglobin, while the inhibition of ABTS•+ production due to antioxidant compounds in the sample is measured at a fixed time.
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The ABTS/H2O2/HRP decoloration method is capable of determining both hydrophilic (in buffered media) and lipophilic (in organic media) antioxidant properties in complex samples. Now, we have adapted this method for on-line chromatographic determination. The easy, rapid and controlled generation of the ABTS radical and its great stability in buffered and organic media were important characteristics in the measurement of antioxidant activities. The HPLC-ABTS method used two pumps (one for isocratic eluting-phase and the other for preformed ABTS radical) and an UV-VIS diode array detector. The dual analysis of samples – conventional (with UV-VIS detection) and ABTS-scavenging (at 600 nm) – provided valuable on-line information about the correspondence between the presence of a determined compound and its possible antioxidant activity, and was applicable to both hydrophilic and lipophilic antioxidants (HAA and LAA). A comparison between HAA and LAA determined by the end-point method and by the on-line HPLC method is presented. The application to juices showed that both methods are suitable, sensitive and selective, gave similar values, and the HPLC-ABTS method contributed additional information about the antioxidant activity profile.
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Summary The 2, 2 ′ ‐Azino‐bis(3‐ethylbenz‐thiazoline‐6‐sulfonic acid) (ABTS)/Trolox equivalent anti oxidant capacity (TEAC) assay, which measures the ability of compounds to scavenge the ABTS radical cation (ABTS •+ ) in relation to Trolox (TEAC) is one of the most widely used methods to determine anti oxidant capacity. Usually, the measurements are based on a fixed end‐point (4–6 min), which may not take into account the different kinetic behaviour of anti‐oxidants. The aim of this work was to propose a kinetic procedure for ABTS •+ assay, by using the kinetic parameters ED 50 , t EC50 and AE [antiradical efficiency = 1/(ED 50 × t EC50 )], previously applied to the 2, 2‐Diphenyl‐1‐picrylhydrazyl (DPPH • ) assay. It was found that some polyphenols standards – ferulic acid, resveratrol and others‐have not yet completed their reaction at the fixed end‐point. The consideration of kinetic parameters in this method may provide a more thorough understanding of the behaviour of the anti‐oxidants. A comparison of the activity of anti‐oxidants measured by the scavenging of ABTS •+ with that of DPPH • was also carried out.
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Trolox
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Laccase, a blue copper oxidase, in view of its moderate redox potential can oxidise only phenolic compounds by electron-transfer. However, in the presence of ABTS (2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) as a redox mediator, laccase reacts with the more difficult to oxidise non-phenolic substrates, such as benzyl alcohols. The role of ABTS in these mediated oxidations is investigated. Redox interaction with laccase could produce in situ two reactive intermediates from ABTS, namely ABTS++ or ABTS˙+. These species have been independently generated by oxidation with Ce(IV) or Co(III) salts, respectively, and their efficiency as monoelectronic oxidants tested in a kinetic study towards a series of non-phenolic substrates; a Marcus treatment is provided in the case of ABTS++. On these grounds, intervention of ABTS++ as a reactive intermediate in laccase–ABTS oxidations appears unlikely, because the experimental conditions under which ABTS++ is unambiguously generated, and survives long enough to serve as a diffusible mediator, are too harsh (2 M H2SO4 solution) and incompatible with the operation of the enzyme. Likewise, ABTS˙+ seems an intermediate of limited importance in laccase–ABTS oxidations, because this weaker monoelectronic oxidant is unable to react directly with many of the non-phenolic substrates that laccase–ABTS can oxidise. To solve this paradox, it is alternatively suggested that degradation by-products of either ABTS++ or ABTS˙+ are formed in situ by hydrolysis during the laccase–ABTS reactions, and may be responsible for the observed oxidation of non-phenolics.
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Antioxidant activity (capacity or potential) is widely used as parameter to characterize different substances and mixtures, which is able to scavenge or neutralize free radicals. Recently, ABTS assay has been the most widely employed method for estimating antioxidant activity. The method is based on the spectrophotometric measurement of ABTS cation radical (ABTS•+) concentration changes resulting from the ABTS•+ reaction with antioxidants. Yet little is known about factors influencing the kinetics that reaction i.e., about factors affecting the estimation of antioxidant activity of examined compounds'. The paper shows that metal ion type and concentration, water content and pH of the measuring system all significantly influence the estimation of antioxidant activity in ABTS assay and thus make the estimation of correct antioxidant properties of plant and food extracts difficult. Butylhydroxytoluene (BHT) was used as standard antioxidant in the performed experiments. The relationships discussed in this paper indicate the necessity of standardizing the ABTS method and reveal the complexity of estimating adequate antioxidant activity of examined substances.
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In fermentations of lignocelluloses, redox potentials (If not indicated otherwise, redox potentials in Volt are taken versus Normal Hydrogen Reference Electrodes (NHE).) E0 of laccases/plant peroxidases by 0.79/0.95 V enable oxidations of phenolic substrates and transformations of synthetic and substrate-derived compounds to radicals that mediate attacks on non-phenolic lignin (models) by 1.5 V. In consecutive one-electron abstractions, the redox mediator 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) is oxidized by electro- or wet-chemistry to the green cation radical (ABTS•+, 0.68 V) and the red dication (ABTS2+, 1.09 V). The enzyme/ABTS couple generates the stable ABTS•+ whose low E0 cannot explain the couple’s contemporary attack on non-phenolic lignins. This paradoxon indicates the non-confirmed production of the ligninolytic ABTS2+ by the enzymes. During incubations of live sapwood chips in ABTS/H2O2 to prove their constitutive peroxidase, the enzyme catalyzed the formation of the expected green-colored ABTS•+ solution that gradually turned red. Its spectrophotometric absorbance peaks at λ = 515–573 nm resembled those of ABTS2+ at 518–520 nm. It is shown that portions of an ABTS•+ preparation with inactivated enzyme are reduced to ABTS during their abiotic oxidation of low-MW extractives from lignocelluloses to redox mediating radicals. The radicals, in turn, apparently transform the remaining ABTS•+ to red derivatives in the absence of functional oxidoreductases. Ultrafiltration and Liquid-Chromatography suggest the presence of a stable ABTS2+ compound absorbing at 515 nm, red protein/ABTS adducts, and further ABTS moieties. Therefore, ABTS mediated lignin degradations could result from chain reactions of ABTS•+-activated lignocellulose extractives and fissured rather than complete ABTS2+ molecules.
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