Kinetics of Tomato Pectin Methylesterase Inactivation by Temperature and High Pressure

ABSTRACT: In this study we investigated the inactivation of endogenous pectin methylesterase (PME) in tomato juice during combined high-hydrostatic pressure (ambient to 800 MPa) and moderate temperature (60 to 75 °C) treatments under isobaric and isothermal processing conditions. PME inactivation rates increased with increasing processing temperature, with the highest rate obtained during processing at 75 °C and ambient pressure. Inactivation rates were dramatically reduced as soon as processing pressure was raised. High inactivation rates were again attained when processing pressure exceeded a value of about 700 MPa. Such a behavior was described by considering two parallel mechanisms of inactivation, each one following first order kinetics with its own kinetic parameters.     

超高压与重组果胶甲酯酶抑制剂联合应用对鲜榨橙汁果胶甲酯酶活性及品质的影响

为了探究超高压与重组果胶甲酯酶抑制剂(recombinant pectin methylesterase inhibitor,rPMEI)联合处理对鲜榨橙汁中果胶甲酯酶(pectin methylesterase,PME)活性及品质的影响,研究了超高压(400、500和600 MPa,5 min,20 ℃)与重组果胶甲酯酶抑制剂对橙汁微生物、PME酶活、色泽和V_C含量的影响。结果表明:超高压处理条件为500 MPa/5 min,rPMEI添加浓度为0.06 mg/mL时,橙汁中的菌落总数、霉菌与酵母菌数均能达到农业行业标准《NY/T 434-2016绿色食品、果蔬汁饮料》所规定的要求,同时PME被完全钝化;橙汁色泽变化显著小于热处理组(ΔE^*=1.22<2.26);V_C保留率为85.1%,显著高于热处理组(保留率=8.33%)。     

Thermal and High-Pressure Inactivation of Tomato Polygalacturonase: A Kinetic Study

ABSTRACT: Polygalacturonase (PG) was extracted from tomato fruit. The inactivation kinetics of the PG crude extract was investigated under isothermal and isobaric-isothermal conditions. Thermal inactivation of tomato PG in the temperature range 58 to 65 °C could be described by a fractional conversion model, and in the temperature range from 70 to 90 °C by a biphasic model. Pressure-temperature inactivation (range 300 to 600 MPa/5 to 50 °C) of tomato PG could be described by a fractional conversion model, which points to first-order inactivation kinetics of a pressure-sensitive enzyme fraction and to the occurrence of a pressure-stable PG fraction. A mathematical model describing the influence of pressure and temperature on the inactivation rate constant was formulated.     

高压二氧化碳和超高压对多酚氧化酶和果胶甲酯酶结构的影响

为考察高压二氧化碳(HPCD)和超高压技术钝化多酚氧化酶(PPO)和果胶甲酯酶(PE)活性的机理,采用SDS-PAGE电泳、红外光谱和荧光光谱对处理前后酶蛋白的结构进行比较研究。结果表明：经过不同压强HPCD处理和超高压处理60min后,PPO和PE的活性均随着处理压强的增大而逐渐降低。SDS-PAGE电泳分析显示处理后酶蛋白的肽链不发生断裂,但红外光谱和荧光光谱分析显示酶蛋白的二级和三级结构均发生变化。因此,HPCD和超高压技术通过改变PPO和PE酶蛋白的结构达到钝化酶活性的目的。     

低盐腌渍仔姜果胶甲酯酶保脆工艺的优化

采用果胶甲酯酶对低盐腌渍的仔姜进行保脆处理。采用单因素试验研究葡萄糖酸钙的添加量、果胶甲酯酶的添加量和保脆处理的温度与时间对低盐腌渍仔姜的影响。以硬度为指标,通过正交试验优化低盐腌渍仔姜的保脆工艺。结果表明,低盐腌渍生姜的最佳保脆工艺条件为果胶甲酯酶添加量0.8%,处理温度40℃,处理时间25min,葡萄糖酸钙添加量0.4%。在此条件下,保脆后的低盐腌渍仔姜片的平均硬度为3209.224g。     

Improved Firmness in Calcified Diced Tomatoes by Temperature Activation of Pectin Methylesterase

ABSTRACT: The effects of temperature and calcium on pectin methylesterase (PME) activity and texture in tomato pericarp material were examined. Heating thin slices of pericarp to temperatures between 50°C and 75°C led to the rapid evolution of methanol from the material, indicating an activation of PME. This activity was further stimulated when CaCl₂ (up to 2.0% w/v) was added. When applied to half-inch diced tomato pericarp, the same conditions that led to the activation of PME also improved firmness. Diced tomatoes treated for 5 min with 0.5% CaCl₂ at 70°C were 2.5 times firmer than diced tomatoes treated with CaCl₂ at room temperature. This improvement in texture by treating with CaCl₂ at elevated temperatures was only apparent when the tomatoes received a subsequent 100°C treatment. Heating tomatoes to 70°C either before or after the CaCl₂ treatment also improved firmness through a subsequent high-temperature treatment, but to a lesser extent than heating during the CaCl₂ treatment. These results are consistent with the model that heating to 70°C greatly increases PME activity, leading to extensive pectin de-esterification and increased calcium cross-linking of the pectins in the middle lamella. Production of thermally processed diced tomatoes with improved firmness should be possible by increasing the temperature during and after calcium treatment.     

Tomato Product Quality from Transgenic Fruits with Reduced Pectin Methylesterase

Juice from transgenic tomato (Lycopersicon esculentum cv Rutgers) fruits with reduced levels of pectin methylesterase (PME) activity due to the expression of a PME antisense gene exhibited improvement in quality. The percentage increase in juice from transgenic fruits over that of juice from wild type Rutgers ranged between 5.1–5.3 for total solids, 3.8–6.1 for soluble solids, 70–80 for efflux viscosity, 180–220 for serum viscosity and about 50 for precipitate weight ratio. Time of harvest had no effect on quality of juice. Ketchup prepared from transgenic fruit juice had a lower Bostwick value, reduced serum separation and high serum viscosity compared to ketchup from parental Rutgers.     

果蔬加工中果胶甲基酯酶活性抑制的研究进展

果胶甲基酯酶（pectin methylesterase,PME）是一种广泛存在于高等植物、真菌和细菌的果胶降解酶,其残余活性会导致果蔬汁和鲜切果蔬等产品产生分层沉淀、析水结块、质构软化等品质劣变。因此,在果蔬加工过程中实现对PME活性的有效抑制是加工的重要目的。目前生产中对于PME活性的抑制主要以热加工方式为主,但这会对热敏性果蔬产品品质造成不可逆转的不良影响。随着人们生活水平和健康意识的提高,为了满足人们对更高品质产品的追求,一些新型热加工和非热加工技术以及PME抑制剂逐渐被研究并应用于PME活性的抑制。本文综述当前热、非热加工方式和抑制剂对于PME的抑制效果及其机理,并列举不同加工方式联合处理或加工手段与抑制剂联合处理在PME抑制方面的应用情况,以期为未来更高品质果蔬产品的加工提供理论指导。     

果胶甲酯酶抑制剂的异源表达及其在果酒降甲醇中的应用

该文从猕猴桃中克隆出果胶甲酯酶抑制剂（Pectin Methylesterase Inhibitor,PMEI）基因,经EcoRⅠ/XbaI双酶切后连接到表达载体pPICzαA上,电转化到毕赤酵母GS115筛选出阳性菌株GS115/pPICzαA-PMEI,并将重组酵母表达的PMEI浓缩纯化后应用于果酒发酵。研究结果表明：重组毕赤酵母表达菌株GS115/pPICzαA-PMEI成功构建,PMEI的发酵表达量为35.38 mg/L。将PMEI应用到果酒发酵中,桔子酒甲醇降低47.54%,含量由189.75 mg/L降低到99.55 mg/L;苹果酒甲醇降低21.52%,含量由118.15 mg/L降低到91.20 mg/L;葡萄酒甲醇变化不显著,含量均低于20.00 mg/L,表明PMEI对果胶丰富且内源性果胶酶强的桔子具有明显的降甲醇效果。桔子酒发酵工艺优化的PMEI最低抑制质量浓度8.84 mg/L,最适温度18 ℃,此时桔子酒中的甲醇降低66.40%到89.10 mg/L。这为低甲醇果酒的发酵提供了新的技术路线。     

用GAP启动子在毕赤酵母中组成型表达重组 猕猴桃果胶甲酯酶抑制剂

目的构建组成型表达重组猕猴桃果胶甲酯酶抑制剂(kiwi pectin methylesterase inhibitor, kwPMEI)的毕赤酵母(P．pastoris)GS115工程菌株,探索碳源(葡萄糖、甘油、甲醇)对重组菌表达kwPMEI的影响,纯化kwPMEI并鉴定其对番茄果胶酶的抑制活性。方法应用PCR方法从P．pastoris GS115染色体中扩增了三磷酸甘油醛脱氢酶启动子(pGAP),以其取代诱导型表达载体pPIC9K-kwPMEI上的醇氧化酶启动子(pAOX1),构建了组成型表达载体pGAP9K-kwPMEI,并转化至GS115中。用Tricine-SDS-PAGE和Western blot分析目的蛋白表达情况,镍柱亲和层析纯化目的蛋白,并用凝胶扩散方法鉴定其抑制活性。结果重组毕赤酵母工程菌株成功组成型表达了kwPMEI,48h即达到最大表达水平,表达量约为66 mg/L。并且以甘油为碳源时kwPMEI表达量最高。成功分离纯化了kwPMEI,并经凝胶扩散方法检测表明其具有抑制活性。结论成功构建了组成型分泌表达kwPMEI的毕赤酵母菌株,为kwPMEI在果蔬汁中的进一步应用奠定了基础。     

Inactivation of pectin Methylesterase and Lactobacillus Plantarum by ohmic heating in pomelo juice

The use of ohmic heating (OH) to inactivate damaging elements in pomelo juice was evaluated. The work investigated the effect of frequencies (50–20 000 Hz) on pectin methylesterase (PME) and Lactobacillus plantarum (L. plantarum) in pomelo juice at 30 V cm⁻¹ of electric field strength. The results show that frequency does not affect PME inactivation (P > 0.05) but affects bacterial reduction and has the most efficiency at 60 Hz, followed by 50 Hz, and in the frequency range at or higher than 500 Hz (P < 0.05). Additionally, the inactivation kinetics of PME and L. plantarum and the impact of nonthermal factors were given. The nonthermal element has contributed to the inactivation enhancement of L. plantarum and PME to reduce the necessary treatment temperature and time. The major spoilage bacteria and enzyme destruction in pomelo juice occurred faster under OH than under conventional heating (CH) suggesting that OH can be effectively used to pasteurise pomelo juice.     

Kinetic Study of Antibrowning Agents and Pressure Inactivation of Avocado Polyphenoloxidase

Pressure inactivation at room temperature (25 °C) of avocado polyphenoloxidase (pH 5.0) in absence or presence of 10 mM ethylene diaminetetraacetic acid (EDTA), NaCl, or benzoic acid or 0.05 mM 4-hexylresorcinol or glutathione could be described by a fractional conversion model. There appeared to be a resistant enzyme fraction (∼10% to 20%), necessitating about 825 MPa for inactivation in absence of antibrowning agents. EDTA addition resulted in sensitization of the pressure-sensitive enzyme fraction, which was attributed to the acid effect. Benzoic acid or NaCl addition resulted in a marked stabilization, whereas addition of glutathione resulted only in a minor stabilization of the pressure-sensitive enzyme fraction. 4-Hexylresorcinol displayed a sensitizing effect below and a stabilizing effect above 700 MPa. The pressure dependency of the inactivation rate constant was altered (p < 0.05) by addition of 4-hexylresorcinol or benzoic acid.     

Kinetic study of high pressure microbial and enzyme inactivation and selection of pasteurisation conditions for Valencia Orange Juice

Keeping quality of fresh orange juice is highly dependent on pectinolytic enzyme activity and the growth of spoilage microorganisms. The inactivation kinetics of indigenous pectin methylesterase (PME) and of the two more pressure resistant species of spoilage lactic acid bacteria (LAB) Lactobacillus plantarum and L. brevis in freshly squeezed Valencia orange juice under high hydrostatic pressure (100–500 MPa) combined with moderate temperature (20–40 °C) was investigated. PME inactivation followed first order kinetics with a residual PME activity (15%) at all pressure–temperature combinations used. The values of activation energy and activation volume were estimated at each pressure and at each temperature, respectively. Values of 90 kJ/mol and ?30 mL/mol at reference pressure of 300 MPa and reference temperature of 35 °C were estimated respectively. The corresponding z_T and z_P values of LAB inactivation were also estimated at all conditions tested. Values of 19.5 °C and 95 MPa at reference pressure of 300 MPa and reference temperature of 30 °C were estimated respectively for L. plantarum, while the corresponding values for L. brevis were 40 °C and 82 MPa, respectively, at the same reference conditions. Pressure and temperature were found to act synergistically both for PME and LAB inactivation. The PME and LAB inactivation rate constants were expressed as functions of the temperature and pressure process conditions. These functions allow the determination of the pressure/temperature conditions that achieve the target enzyme and microbial inactivation at a selected processing time. The process conditions of 350 MPa at 35 °C for 2 min are proposed as effective for Valencia orange juice cold pasteurisation.     

Inactivation Kinetics of Listeria monocytogenes by High-Pressure Processing: Pressure and Temperature Variation

The enhanced quasi-chemical kinetics (EQCK) model is presented as a methodology to evaluate the nonlinear inactivation kinetics of baro-resistant Listeria monocytogenes in a surrogate protein food system by high-pressure processing (HPP) for various combinations of pressure (P= 207 to 414 MPa) and temperature (T= 20 to 50 °C). The EQCK model is based on ordinary differential equations derived from 6 "quasi-chemical reaction" steps. The EQCK model continuously fits the conventional stages of the microbial lifecycle: lag, growth, stationary phase, and death; and tailing. Depending on the conditions, the inactivation kinetics of L. monocytogenes by HPP show a lag, inactivation, and tailing. Accordingly, we developed a customized, 4-step subset version of the EQCK model sufficient to evaluate the HPP inactivation kinetics of L. monocytogenes and obtain values for the model parameters of lag (λ), inactivation rate (μ), rate constants (k), and "processing time" (tp). This latter parameter was developed uniquely to evaluate kinetics data showing tailing. Secondary models are developed by interrelating the fitting parameters with experimental parameters, and Monte Carlo simulations are used to evaluate parameter reproducibility. This 4-step model is also compared with the empirical Weibull and Polylog models. The success of the EQCK model (as its 4-step subset) for the HPP inactivation kinetics of baro-resistant L. monocytogenes showing tailing establishes several advantages of the EQCK modeling approach for investigating nonlinear microbial inactivation kinetics, and it has implications for determining mechanisms of bacterial spore inactivation by HPP. Practical Application: Results of this study will be useful to the many segments of the food processing industry (ready-to-eat meats, fresh produce, seafood, dairy) concerned with ensuring the safety of consumers from the health hazards of Listeria monocytogenes, particularly through the use of emerging food preservation technologies such as high-pressure processing.     

High-Pressure Processing of Orange Juice: Kinetics of Pectinmethylesterase Inactivation

ABSTRACT: A kinetic study of pectinmethylesterase (PME) inactivation in orange juice was conducted. Juice samples were subjected to combinations of high pressure (400, 500, 600 MPa) and thermal (25, 37.5, 50 °C) treatments for various time periods. PME inactivation followed a first-order kinetic model with a residual activity of pressure-resistant enzyme remaining. Calculated D-values ranged from 4.6 min to 117.5 min at 600 MPa/50 °C and 400 MPa/25 °C, respectively. Pressures in excess of 500 MPa resulted in sufficiently fast inactivation rates for economic viability of the process.     

Inactivation kinetics of pectin methylesterase in orange juice as a function of pH and temperature/time process conditions

Pasteurisation of orange juice (OJ) is necessary to prevent spoilage due to microorganisms and enzymes, mainly pectin methylesterase (PME). PME has a higher thermal resistance than the bacteria and yeasts existing in OJ and therefore its inactivation is used as a parameter to define the time/temperature combination of the thermal process. The enzyme has isoforms with different activities and thermal resistances. A three‐parameter model can be used to describe the kinetics of PME inactivation, where the more and less thermally resistant fractions are represented. In this study the thermal inactivation kinetics was evaluated at six pH values (3.6, 3.7, 3.8, 3.9, 4.0 and 4.1), three minimal temperatures (82.5, 85.0 and 87.5 °C) and at least six holding times for each condition. It was found that the thermolabile PME fraction (a) was influenced by pH and processing temperature. A slower reaction rate constant (k₁) was found for juices with pH values of 3.8 and 3.9 at the studied temperatures. The highest inactivation levels were obtained in juices with pH values of 3.6 and 3.7. Copyright © 2006 Society of Chemical Industry     

Potential Application of Hot Rehydration Alone or in Combination with Hydrogen Peroxide to Control Pectin Methylesterase Activity and Microbial Load in Cold-stored Intermediate-moisture Sun-dried Figs

ABSTRACT: Sun-dried figs contain a considerable amount of pectin methylesterase (PME) activity (22 JAM COOH/ min/g). The enzyme causes softening and loss of desired gummy texture in cold-stored intermediate-moisture (IM) sun-dried figs brought to a 28% to 29% moisture range. Partial reduction of PME activity (28%) delayed undesirable textural changes in IM figs rehydrated at 80 °C for 16 min. The heat treatment did not cause a considerable reduction in microbial load. However, the addition of 2.5% H2O2 to the rehydration medium at 80 °C reduced the initial total mesophilic aerobic count of figs by at least 90% and turned the figs from a brown color to a desirable and stable yellow-light brown. The in situ fig catalase remains after rehydration at 80 °C. Thus, by reducing the contact period of figs with H2O2 or by pureeing figs, it is possible to eliminate residual H2O2 and to obtain safe and SO2-free light-colored fig products.     

促进植物内源性果胶甲酯酶催化作用的因素与机制及其在果蔬加工中的应用

植物内源性果胶甲酯酶（pectin methylesterase,PME）存在于天然植物组织中,可以催化高酯果胶脱甲基酯化生成低酯果胶,进而影响果蔬硬度、出汁率等质构及加工品质。一方面,为了避免贮藏期间出现果蔬汁浑浊沉淀、果蔬罐头中果肉变软、调味酱（如辣椒酱）中皮肉分离等不良品质,会在加工过程中抑制PME活性;另一方面,为了提高果蔬汁出汁率、生产低酯果胶等,也可在食品中添加外源PME。目前,对于通过激活内源PME来改善食品加工的研究较少,因此,本综述重点讨论促进植物内源性PME催化作用的因素与机制及其在果蔬贮藏加工中的应用,为植物内源性PME在果蔬加工中的充分利用提供理论参考。     

Demethylation of Pectic Substances: Relationship to Methylesterase Activity during Brine Storage of Cherries

Residual pectin methylesterase activity (60% of initial activity) was present in cherry fruit stored 12 mo in brine. The specific activity of pectin methylesterase increased during the first 4 mo (195% of initial specific activity) but remained relatively stable thereafter. Methanol concentration increased (+144.5%) indicating that pectic substances underwent continuous demethylation during 12 mo storage. Pectin deesterification was probably of enzymatic origin since notable chemical deesterification occurred only at a pH lower than that found in fruit during storage. Residual pectin methylesterase activity introduces the possibility of improving fruit texture by manipulating physico-chemical conditions prior to blanching and candying.     

Combined Pressure—temperature Inactivation of Alkaline Phosphatase in Bovine Milk: A Kinetic Study

A detailed kinetic study on pressure-temperature inactivation of alkaline phosphatase has been performed in the pressure range 0.1 to 725 MPa at temperatures between 25 and 63 °C. Inactivation could be accurately described by a first order kinetic model, allowing D-values to be calculated. According to the thermal death time terminology, z _r- and z _p-values were calculated, expressing temperature and pressure dependence respectively. However, at high temperature, pressure dependence could not be calculated unambiguously. D-values firstly increased with increasing pressure up to 300 MPa and then decreased with further pressure increase, showing thermal inactivation to be counteracted by low pressure. Finally, a global model describing the D-value as a function of pressure and temperature has been formulated.