Ascorbic acid and 4-hexylresorcinol effects on pear PPO and PPO catalyzed browning reaction

ABSTRACT:  The effects of ascorbic acid (AA) and 4-hexylresorcinol (4-HR) on pear polyphenoloxidase (PPO) activity and stability have been investigated in vitro . AA does not interact directly with PPO but prevents browning by reducing oxidized substrates. The 4-HR exerts a dual role on PPO. If no substrates are present, it interacts preferably with the deoxy form of PPO inactivating it. If substrates and 4-HR are both present they compete for the catalytic site. The 4-HR behaves then as a canonical enzyme inhibitor, binding to the met form of PPO. Simultaneous addition of 4-HR and AA has synergistic inhibition or inactivatory effects depending on the presence or the absence of PPO substrates.     

PPO酶促诱发因素对苹果多酚非酶褐变的影响

为探究苹果多酚氧化酶(PPO)酶促诱发因素对苹果多酚参与的非酶褐变的影响,以绿原酸和根皮苷为底物,以磷酸缓冲液为苹果汁模拟体系,通过控制PPO酶促诱发条件,进行酶促诱发反应,然后钝化酶,模拟体系于60℃进行非酶聚合反应,每隔24 h用色差计测定L*值、a*值、b*值和ΔE*值。结果表明,酶促诱发因素对绿原酸和根皮苷参与的非酶褐变的影响存在较大差异。p H在3.5~6.0,含有绿原酸和根皮苷的模拟体系,其褐变程度随酶促诱发p H升高而增大;酶促诱发温度20~60℃,诱发体系温度对绿原酸参与的非酶褐变影响不显著,而根皮苷在40℃时褐变程度较大;酶促诱发时间分别为40 min和30 min时绿原酸和根皮苷参与的非酶褐变程度较大;酶活14.25~171 U,酶活的增大加速两种多酚参与的非酶褐变。结果同时表明,根皮苷非酶褐变与果汁后浑浊有关,且较高的p H、温度、PPO酶活以及较长的酶促诱发时间加速果汁后浑浊形成。     

Effects of argon enriched low-oxygen atmospheres and of high-oxygen atmospheres on the kinetics of polyphenoloxidase (PPO)

Abstract: The reported benefits of enrichment of air atmospheres with argon or oxygen for control of enzymatic browning were investigated by determining the effects of these atmospheres on PPO kinetics. Kinetics of purified apple PPO and a commercially available mushroom PPO were studied in an in vitro model system. Enrichment with argon produced greater inhibitory effects than the current industry practice of enrichment with nitrogen. Km^app values (mM) for apple PPO in 3%O₂/97%Ar, 3%O₂/97%N₂, and air, were 133, 87, and 48, respectively. The data indicate that inhibition by both gases is competitive, and also support the hypothesis that the greater inhibitory effect of argon was proportional to the size of the Van der Waals radius of argon against nitrogen (1.91Å against 1.54Å). Much smaller inhibitory effects were observed in the presence of 80% O₂ (Km^app 57mM), and the nature of this inhibition was less clear. The results suggest that the benefits of argon enrichment may be relatively small, and may require critical enzyme, substrate, and gas levels to be successful. However, these benefits may be exploitable commercially in some fresh‐cut products, and may allow less anoxic atmospheres to be used. Practical Application: Control of enzymatic browning without sulfites continues to be a challenge in some fresh‐cut products. While sporadic benefits of these atmospheres in control of enzymatic browning have been reported, results have been inconsistent in commercial practice. The results suggest that the benefits of argon enrichment may be relatively small, and may require critical enzyme, substrate, and gas levels to be successful. However, these benefits may be exploitable commercially in some fresh‐cut products, and allow less anoxic atmospheres to be used.     

INHIBITION STUDIES ON BURDOCK POLYPHENYL OXIDASE (PPO) ACTIVITY

The inhibitory effect of 4-hexylresorcinol (HR), ascorbic acid (AA), citric acid (CA), and bisulfites on polyphenol oxidase (PPO) of burdock roots was studied. HR was an effective inhibitor at low concentrations used in this study for crude PPO from root slices. A binary mixture of HR and AA at 1.0% concentration for 5 min achieved more than 90% inhibition of crude PPO. AA also showed high antibrowning activity for the purified PPO comparable to that of bisulfites, whereas its activity was much lower than that of HR in the crude extract. The inhibitory effect of the combined use of inhibitors was overall in the decreasing order of HR+AA, HR+CA and AA+CA, irrespective of the crude PPO or the purified PPO from burdock slices.     

“黑美人”马铃薯多酚氧化酶的特性研究

以邻苯二酚为底物,在413nm处测定黑美人马铃薯多酚氧化酶(PPO)的活性,研究了温度、pH、底物浓度对其活性的影响,并建立了酶促褐变反应动力学方程,探讨了L-半胱氨酸、抗坏血酸、柠檬酸、EDTA及亚硫酸氢钠五种抑制剂对酶促褐变的抑制效果。结果表明:黑美人马铃薯多酚氧化酶最适反应pH为6.3;最适反应温度为35℃;酶促褐变反应动力学符合米氏方程描述的单底物酶促反应动力学,以邻苯二酚为底物,Km=0.0011mol/L,Vmax=142.23U/min.g;动力学方程为V=142.23[S]/(0.0011+[S]);五种抑制剂均对PPO酶促褐变具有抑制作用,其强弱依次为亚硫酸氢钠>L-半胱氨酸>抗坏血酸>EDTA>柠檬酸。     

有机酸复合护色液对猕猴桃果脯PPO活性的影响

研究了柠檬酸、酒石酸、植酸3种有机酸复合护色液对猕猴桃果脯PPO(多酚氧化酶)活性的影响。运用混料回归设计试验,得出复合护色液中3种有机酸的最佳配比,并确定复合护色液总浓度及护色浸泡时间。结果表明,最佳护色工艺为:柠檬酸、酒石酸、植酸配比(质量分数)为1∶0.51∶0.51,复合护色液总浓度为0.5%.护色时间为2h。     

果酒中多酚氧化酶及其性质的研究

对山楂酒中含有的六种分子量不同的多酚在多酚氧化酶作用下,测定氧化前后其涩度的变化.发现多酚物质的氧化作用能够加重果酒的苦涩味.并对山楂中的多酚氧化酶进行提取,测定其基本特性,确定了底物、温度、pH值对其活性的影响,并进一步测定其酶动力学曲线,发现多酚氧化酶催化的最适温度是25℃,其最适pH是6.5,其作用的最适底物是儿茶酚,其米氏常数是1.077×10-2M.     

鸡腿蘑多酚氧化酶特性研究

本文采用分光光度法对鸡腿蘑多酚氧化酶(PPO)的催化特性、最适温度、最适pH值、热稳定性等性质进行研究;同时考察了EDTA-2Na、NaHSO₃、抗坏血酸、柠檬酸四种抑制剂对鸡腿蘑多酚氧化酶活力的影响。结果表明:该酶的最适pH为6.0;最适温度为45℃,80℃水浴处理5min,95℃水浴处理90s该酶全部钝化。四种抑制剂对该酶均表现出不同的抑制效果,抑制效果强弱次序为抗坏血酸>柠檬酸>NaHSO₃>EDTA-2Na。     

Polyphenoloxidase (PPO) activity and osmotic dehydration in Granny Smith apple

Osmotic dehydration (OD) permits the preservation of foods via a decrease in water content and an increase in solute concentration. Osmotically dehydrated fruits such as apple are suitable for the manufacture of desserts, cakes, salads, yoghurts, etc. Different microstructural and engineering aspects of OD are already known, but its effects on enzymatic activity are still unknown. This study analyses the activity of polyphenoloxidase (PPO) in fresh Granny Smith apples and the effect that OD by immersion in sucrose‐saturated syrup has on this activity. The low PPO activity found in the edible parenchyma of osmotically dehydrated apples is attributed to penetration by the osmotic agent and flooding of the intercellular spaces, which produces a low moisture content and a limited O₂ concentration in the immediate environment of the enzyme. These results show that OD prevents enzyme–substrate interaction. Thus the low PPO activity would reduce browning of this type of product. Copyright © 2005 Society of Chemical Industry     

桑叶多酚氧化酶活性的研究

采用分光光度法和扫描电子显微镜法对桑叶多酚氧化酶(PPO)的结构、活性以及不同抑制剂的抑制效果进行研究.结果表明:桑叶多酚氧化酶为纤维状蛋白;它的最适pH值为7.0;最适温度为40℃;在相同抑制剂浓度下,总体抑制效果为:抗坏血酸>亚硫酸氢钠>柠檬酸>氯化镁>氯化钠.     

仙人掌多酚氧化酶的特性及抑制研究

本文研究了仙人掌中多酚氧化酶(PPO)的特性以及一些抑制剂对其活性的影响。结果表明:仙人掌中PPO的最适温度为50℃,最适pH有两个,分别为pH4.0和pH7.0,因而在仙人掌中的PPO存在同工酶。柠檬酸、抗坏血酸及EDTA对仙人掌PPO均有一定的抑制效果,抑制能力顺序为:柠檬酸>抗坏血酸>EDTA。对各种抑制剂的作用机理进行了探讨。EDTA主要是通过螯合PPO中的辅酶因子Cu2+而起作用,抗坏血酸螯合Cu2+的能力比EDTA弱,但抗坏血酸的还原能力较强,而柠檬酸则是通过降低pH和螯合作用抑制PPO。     

酶钝化处理对菊苣茎PPO活性和绿原酸含量的影响

通过水浴、蒸汽、微波3 种烫漂方式处理新鲜菊苣茎,钝化多酚氧化酶（PPO）,为提高干燥菊苣茎中绿原酸含量提供依据。烫漂处理后立即测定菊苣茎PPO相对活性,同时测定经烫漂处理后再经空气干燥茎中绿原酸含量,分析二者之间的相关性;测定不进行烫漂处理直接进行空气干燥的菊苣茎在干燥过程中PPO相对活性和绿原酸含量的变化。结果表明：空气干燥过程中,PPO始终保持一定的相对活性（≥20.02%）;水浴、蒸汽和微波烫漂处理可有效抑制PPO相对活性,且在以上3 种处理方式中绿原酸含量与PPO相对活性均呈显著负相关（P＜0.05）;4 种方法处理后风干茎中绿原酸的含量依次为：直接空气干燥＜水浴烫漂后干燥＜蒸汽烫漂后干燥＜微波烫漂后干燥。在实验条件下,微波（600 W）烫漂鲜茎样品4.67 min时,风干菊苣茎中绿原酸含量（8.11 mg/g）最高,与鲜茎相比只降低16.82%,是直接空气干燥茎中绿原酸含量的2.17 倍。     

Increased ethylene biosynthesis elevates incidence of chilling injury in cold‐stored ‘Amber Jewel’ Japanese plum (Prunus salicina Lindl.) during fruit ripening

Mature ‘Amber Jewel’ Japanese plum fruit were stored at 0 or 5 °C for 3 and 6 weeks to investigate their effects on ethylene (C₂H₄) biosynthesis during ripening at ambient temperature in the development of chilling injury (CI) and fruit quality. CI (internal breakdown and browning) and fruit softening were higher during ripening, in the fruit stored at 5 °C than 0 °C, irrespective of storage period (SP). C₂H₄ production and activities of 1‐aminocyclopropane‐1‐carboxylic acid synthase (ACS) and 1‐aminocyclopropane‐1‐carboxylic acid oxidase (ACO) enzymes, and 1‐aminocyclopropane‐1‐carboxylic acid (ACC) content were higher during ripening in fruit stored at 5 °C than 0 °C. The fruit stored at 5 °C also exhibited higher respiration rate and higher soluble solids concentration/titratable acidity ratio. In conclusion, increase in storage temperature and SP elevates the activities of ACS and ACO enzymes and consequently C₂H₄ production which leads to the development of CI in plum fruit with advancement of ripening.     

砀山酥梨多酚氧化酶酶学特性及抑制效应的研究

多酚氧化酶是酶促褐变的关键酶,其特性与对其抑制效应研究一直是果蔬酶促褐变生理生化研究的重要内容,本试验以邻苯二酚为底物,采用分光光度法对砀山梨多酚氧化酶的酶学特性及不同抑制剂对多酚氧化酶活性的影响进行了研究。结果表明：砀山梨PPO的最适pH为4.5,最适温度为34℃;短时间高温能显著抑制PPO活性;PPO催化的酶促褐变反应动力学符合米氏方程,该酶促反应的最大速率为178.57U/min,酶反应速度为最大反应速度1/2时的底物浓度为0.125mol/L;柠檬酸、L-半胱氨酸、抗坏血酸,亚硫酸氢钠较柠檬酸能较好地抑制PPO活性,随着浓度的升高,抑制效应逐渐增强,但综合研究表明L-半胱氨酸抑制效应较好。     

桑叶多酚氧化酶分离及活性研究

采用分光光度法和扫描电子显微镜对桑叶多酚氧化酶(PPO)的结构、活性以及不同抑制剂的抑制效果进行研究。结果表明:桑叶多酚氧化酶为纤维状蛋白;其最适pH值为7.0,最适温度为40℃;在相同抑制剂浓度下,总体抑制效果为:抗坏血酸>亚硫酸氢钠>柠檬酸>氯化镁>氯化钠。     

Purification and characterisation of polyphenol oxidase (PPO) from eggplant (Solanum melongena)

Eggplant (Solanum melongena) is a very rich source of polyphenol oxidase (PPO), which negatively affects its quality upon cutting and postharvest processing due to enzymatic browning. PPO inhibitors, from natural or synthetic sources, are used to tackle this problem. One isoform of PPO was 259-fold purified using standard chromatographic procedures. The PPO was found to be a 112 kDa homodimer. The enzyme showed very low K_m (0.34 mM) and high catalytic efficiency (3.3 × 10⁶) with 4-methyl catechol. The substrate specificity was in the order: 4-methyl catechol > tert-butylcatechol > dihydrocaffeic acid > pyrocatechol. Cysteine hydrochloride, potassium metabilsulphite, ascorbic acid, erythorbic acid, resorcylic acid and kojic acid showed competitive inhibition, whereas, citric acid and sodium azide showed mixed inhibition of PPO activity. Cysteine hydrochloride was found to be an excellent inhibitor with the low inhibitor constant of 1.8 μM.     

鲜切芦蒿PPO特性的研究

芦蒿去除不可食部分、冰水清洗、切分后用研钵碾成匀浆状于低温(4℃)离心机中离心(10000r/min,30min),获得的粗酶液用于多酚氧化酶(PPO)活力的测定和酶特性的研究。结果表明:鲜切芦蒿PPO适宜的pH值为6.5,温度为40℃。鲜切芦蒿PPO对60℃以下的温度不敏感,而在80℃以上温度下很易失活。鲜切芦蒿PPO对酚底物的亲和力依次为:绿原酸、愈创木酚>焦性没食子酸苯酚>酪氨酸>儿茶酚>间苯二酚>苯酚,且与儿茶酚结合时出现最大活力。抑制鲜切芦蒿PPO活力以焦亚硫酸钠效果最好,当其浓度达到0.33mmol/L(即0.06%)时能使PPO完全失活;亚硫酸氢钠和L-Cys的抑制效果也十分强;而EDTA-2Na浓度达3.0mmol/L对鲜切芦蒿PPO的抑制也仅为29.55%,作用很微弱;植酸、AA、醋酸锌、CA和4-HR的抑制作用介于L-Cys和EDTA-2Na之间。     

香菇多酚氧化酶酶学特性的研究

以没食子酸为底物,以3-甲基-2-苯并噻唑腙盐酸盐(MBTH)与酶作用产物形成粉红色的复合物,通过分光光度法在505nm处测定该复合物的吸收来测定香菇多酚氧化酶(PPO)的活性.分别研究了温度、pH、底物浓度对酶活性的影响,得到了米氏常数Km和最大反应速度Vmax,并探讨了PPO提取液的稳定性.结果表明:香菇PPO活性在20～60℃内逐渐升高,其最适pH为4.6,温度对酶活的影响比pH的影响要大.三种底物与PPO亲和力高低的顺序为L-多巴-水没食子酸邻苯二酚,其米氏常数Km分别为0.98、1.45、3.07mmol/L,最大反应速度Vmax分别为29、49、80U.     

Colonic availability of polyphenols and D-(-)-quinic acid after apple smoothie consumption

Scope : The aim of this study was to determine the amounts of polyphenols and D ‐(?)‐quinic acid reaching the ileostomy bags of probands (and thus the colon in healthy humans) after ingestion of apple smoothie, a beverage containing 60% cloudy apple juice and 40% apple puree. Methods and results : Ten healthy ileostomy subjects each ingested 0.7 L of apple smoothie (a bottle). Their ileostomy bags were collected directly before and 1, 2, 4, 6 and 8 h after smoothie consumption, and the polyphenol and D ‐(?)‐quinic acid contents of the ileostomy fluids were examined using HPLC‐DAD and HPLC‐MS/MS. The total polyphenol and D ‐(?)‐quinic acid content of the apple smoothie was determined to be 1955.6±124.6 mg/0.7 L, which is very high compared to cloudy apple juices. The most abundant substances found in the ileostomy bags were oligomeric procyanidins (705.6±197.9 mg), D ‐(?)‐quinic acid (363.4±235.5 mg) and 5‐caffeoylquinic acid (76.7±26.8 mg). Overall recovery of ingested polyphenols and D ‐(?)‐quinic acid in the ileostomy bags was 63.3±16.1%. Conclusions: The amounts of polyphenol and D ‐(?)‐quinic acids reaching the ileostomy bags are considerably higher after apple smoothie consumption than after the consumption of cloudy apple juice or cider. These results suggest that the food matrix might affect the colonic availability of polyphenols, and apple smoothies could be more effective in the prevention of chronic colon diseases than both cloudy apple juice and apple cider.