Enzyme Inactivation in Food Processing using High Pressure Carbon Dioxide Technology

High pressure carbon dioxide (HPCD) is an effective non-thermal processing technique for inactivating deleterious enzymes in liquid and solid food systems. This processing method avoids high temperatures and exerts a minimal impact on the nutritional and sensory properties of foods, but extends shelf life by inhibiting or killing microorganisms and enzymes. Indigenous enzymes in food such as polyphenol oxidase (PPO), pectin methylesterase (PME), and lypoxygenase (LOX) may cause undesirable chemical changes in food attributes, showing the loss in color, texture, and flavor. For more than two decades, HPCD has proved its effectiveness in inactivating these enzymes. The HPCD-induced inactivation of some microbial enzymes responsible for microbial metabolism is also included. This review presents a survey of the published knowledge regarding the use of HPCD for the inactivation of these enzymes, and analyzes the factors controlling the efficiency of HPCD and speculates on the underlying mechanism that leads to enzyme inactivation.     

Quality-Related Enzymes in Fruit and Vegetable Products: Effects of Novel Food Processing Technologies,Part 1: High-Pressure Processing

The activity of endogenous deteriorative enzymes together with microbial growth (with associated enzymatic activity) and/or other non-enzymatic (usually oxidative) reactions considerably shorten the shelf life of fruits and vegetable products. Thermal processing is commonly used by the food industry for enzyme and microbial inactivation and is generally effective in this regard. However, thermal processing may cause undesirable changes in product's sensory as well as nutritional attributes. Over the last 20 years, there has been a great deal of interest shown by both the food industry and academia in exploring alternative food processing technologies that use minimal heat and/or preservatives. One of the technologies that have been investigated in this context is high-pressure processing (HPP). This review deals with HPP focusing on its effectiveness for controlling quality-degrading enzymes in horticultural products. The scientific literature on the effects of HPP on plant enzymes, mechanism of action, and intrinsic and extrinsic factors that influence the effectiveness of HPP for controlling plant enzymes is critically reviewed. HPP inactivates vegetative microbial cells at ambient temperature conditions, resulting in a very high retention of the nutritional and sensory characteristics of the fresh product. Enzymes such as polyphenol oxidase (PPO), peroxidase (POD), and pectin methylesterase (PME) are highly resistant to HPP and are at most partially inactivated under commercially feasible conditions, although their sensitivity towards pressure depends on their origin as well as their environment. Polygalacturonase (PG) and lipoxygenase (LOX) on the other hand are relatively more pressure sensitive and can be substantially inactivated by HPP at commercially feasible conditions. The retention and activation of enzymes such as PME by HPP can be beneficially used for improving the texture and other quality attributes of processed horticultural products as well as for creating novel structures that are not feasible with thermal processing.     

Quality-Related Enzymes in Plant-Based Products: Effects of Novel Food Processing Technologies Part 2: Pulsed Electric Field Processing

Pulsed electric field (PEF) processing is an effective technique for the preservation of pumpable food products as it inactivates vegetative microbial cells at ambient to moderate temperature without significantly affecting the nutritional and sensorial quality of the product. However, conflicting views are expressed about the effect of PEF on enzymes. In this review, which is part 2 of a series of reviews dealing with the effectiveness of novel food preservation technologies for controlling enzymes, the scientific literature over the last decade on the effect of PEF on plant enzymes is critically reviewed to shed more light on the issue. The existing evidence indicates that PEF can result in substantial inactivation of most enzymes, although a much more intense process is required compared to microbial inactivation. Depending on the processing condition and the origin of the enzyme, up to 97% inactivation of pectin methylesterase, polyphenol oxidase, and peroxidase as well as no inactivation have been reported following PEF treatment. Both electrochemical effects and Ohmic heating appear to contribute to the observed inactivation, although the relative contribution depends on a number of factors including the origin of the enzyme, the design of the PEF treatment chamber, the processing condition, and the composition of the medium.     

高静压对桃汁杀菌、钝化酶活性的效果

研究在不同处理压力和时间条件下,高静压加工技术对桃汁中微生物(细菌总数、霉菌、酵母菌、大肠菌群)以及酶(多酚氧化酶、果胶甲基酯酶、脂肪氧化酶)的影响。结果表明:经400MPa、5min高静压处理即可完全杀灭桃汁中的微生物;在400MPa和500MPa条件下,桃汁中的多酚氧化酶和脂肪氧化酶的活性出现了不同程度的激活现象,但在600MPa时,随着处理时间的延长,其活性逐渐降低,经30min处理后,分别被钝化了0.7662和0.641。而果胶甲基酯酶在400、500、600MPa条件下,出现了不规律的激活或钝化现象。另外,研究表明在高静压加工前增加漂烫工艺,可以有效杀灭桃汁中的微生物及钝化酶活性。     

Thermosonication: a potential technique that influences the quality of grapefruit juice

Thermosonication (TS) is an emerging nonthermal processing technique used for the liquid food preservation and is employed to improve the quality and acceptability of grapefruit juice. In this study, fresh grapefruit juice samples were subjected to TS treatment in an ultrasonic cleaner with different processing variables, including temperature (20, 30, 40, 50 and 60 °C), frequency (28 kHz), power (70%, 420 W) and processing time (30 and 60 min) for bioactive compounds, inactivation of enzymes pectin methylesterase (PME), peroxidase (POD) and polyphenolase (PPO) and micro‐organisms (total plate count, yeasts and moulds). The micro‐organism activity was completely inactivated in the treatment (60 °C for 60 min). The TS treatment at 60 °C for 60 min exposure reduced PME, PPO and POD activity by 91%, 90% and 89%, respectively. Results indicate that the advantages of TS for grapefruit juice processing at low temperature could enhance the inactivation of enzymes and micro‐organisms and it can be used as a potential technique to obtain better results as compared to alone .     

Carrot pectin methylesterase and its inhibitor from kiwi fruit: Study of activity, stability and inhibition

Carrot pectin methylesterase (PME) and its inhibitor (PMEI) from kiwi fruit were successfully purified by affinity chromatography. Enzyme and inhibitor activity and stability and PME–PMEI complex formation, as influenced by intrinsic product factors (pH and NaCl) and extrinsic process factors (temperature and pressure), were studied. The effect of temperature- or pressure-induced denaturation of PME and PMEI on their respective activities was assessed by estimating inactivation kinetic parameters. PME inactivation obeyed first-order kinetics. The enzyme was rather heat-labile but pressure-stable. PMEI inactivation was best described by a model taking into account a processing-stable PMEI intermediate. The behavior of PME and the PME–PMEI complex at elevated temperature or pressure in the presence of pectin was explored by following methanol formation as a function of treatment time. PME catalytic activity was stimulated up to a certain temperature or pressure level before declining. No conclusive evidence was obtained for a temperature-induced dissociation of the PME–PMEI complex, whereas high pressure exposure caused the complex to separate.

Industrial relevance

PME activity control is a major point of interest in the quest of obtaining high quality plant-derived food products. The current study demonstrates that both traditional thermal processing and novel high hydrostatic pressure processing allow stimulation as well as inactivation of PME and, hence, directing the PME-catalyzed pectin hydrolysis. An alternative or additional approach to control endogenous PME activity (e.g. to obtain cloud-stable juices) is through enzyme inhibition using kiwi PMEI. In this context, pH and NaCl boundaries for application were established, the existence of a temperature- and pressure-stable PMEI intermediate was shown and the PME–PMEI complex was proven not to be dissociated at mild temperature and pressure levels. These observations endorse the possibility of inhibiting undesirable PME activity remaining after mild processing.     

The kinetics of inactivation of pectin methylesterase and polygalacturonase in tomato juice by thermosonication

The ultrasonic inactivation kinetics of polygalacturonase (PG) and pectin methylesterase (PME) in tomato juice were studied at a frequency of 20 kHz, amplitude of 65 μm and temperatures between 50 and 75 °C. Thermal treatments at the same temperatures were conducted to separate the effects of heat and ultrasound. The thermal inactivation of PG was described by a fractional conversion model with PG 1 remaining stable, whereas the inactivation of PG by combined ultrasonic and heat treatment (thermosonication) was best described by first order biphasic kinetics, with both PG1 and PG2 inactivated at different rates. The thermal and thermosonication inactivation of PME was described well by first order kinetics. The inactivating effect of combined ultrasound and heat was synergistic. Thermosonication enhanced the inactivation rates of both PME and PG. The inactivation rate of PME was increased by 1.5–6 times and the inactivation rate of PG2 by 2.3–4 times in the temperature range 60–75 °C, with the highest increase corresponding to the lowest temperature.     

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     

Impact of pH and Total Soluble Solids on Enzyme Inactivation Kinetics during High Pressure Processing of Mango (Mangifera indica) Pulp

This study was undertaken with an aim to enhance the enzyme inactivation during high pressure processing (HPP) with pH and total soluble solids (TSS) as additional hurdles. Impact of mango pulp pH (3.5, 4.0, 4.5) and TSS (15, 20, 25 °Brix) variations on the inactivation of pectin methylesterase (PME), polyphenol oxidase (PPO), and peroxidase (POD) enzymes were studied during HPP at 400 to 600 MPa pressure (P), 40 to 70 °C temperature (T), and 6‐ to 20‐min pressure‐hold time (t). The enzyme inactivation (%) was modeled using second order polynomial equations with a good fit that revealed that all the enzymes were significantly affected by HPP. Response surface and contour models predicted the kinetic behavior of mango pulp enzymes adequately as indicated by the small error between predicted and experimental data. The predicted kinetics indicated that for a fixed P and T, higher pulse pressure effect and increased isobaric inactivation rates were possible at lower levels of pH and TSS. In contrast, at a fixed pH or TSS level, an increase in P or T led to enhanced inactivation rates, irrespective of the type of enzyme. PPO and POD were found to have similar barosensitivity, whereas PME was found to be most resistant to HPP. Furthermore, simultaneous variation in pH and TSS levels of mango pulp resulted in higher enzyme inactivation at lower pH and TSS during HPP, where the effect of pH was found to be predominant than TSS within the experimental domain.     

Thermal and High-Pressure Stability of Pectinmethylesterase, Polygalacturonase, β-Galactosidase and α-Arabinofuranosidase in a Tomato Matrix: Towards the Creation of Specific Endogenous Enzyme Populations Through Processing

The thermal and pressure stability of tomato pectinmethylesterase (PME), polygalacturonase (PG), β-galactosidase (β-Gal), and α-arabinofuranosidase (α-Af) were investigated in situ. Enzyme inactivation by thermal and high-pressure processing (respectively 5 min at 25–95 °C at 0.1 MPa and 10 min at 0.1–800 MPa at 20 °C) was monitored by measuring the residual activity in crude enzyme extracts of treated tomato purée samples. PME was completely inactivated after a 5-min treatment at 75 °C. Only 30 % of the pressure stable PME was inactivated after a treatment at 800 MPa (20 °C, 10 min). A 5-min treatment at 95 °C and a treatment at 550 MPa (20 °C, 10 min) caused complete PG inactivation. β-Gal and α-Af activities were already reduced significantly by thermal treatments at 42.5–52.5 °C and 45–60 °C, respectively. These enzymes were, however, rather pressure resistant: treatments at respectively 700 and 600 MPa were necessary to reduce the activity below 10 % of the initial value. Assuming that first-order, fractional conversion or biphasic inactivation models could be applied to the respective enzyme inactivation data, inactivation rate constants and their temperature or pressure dependence for the different enzymes were determined. Based on differences in process stability of the enzymes, possibilities for the creation of specific “enzyme populations” in tomato purée by selective enzyme inactivation were identified. For industrially relevant process conditions, the enzyme inactivation data obtained for tomato purée were shown to be transferable to intact tomato tissue.     

食品中酶的微波钝化技术研究进展

食品中的酶对食品质量具有正反两方面的影响,因此在食品加工过程中,有效控制食品中酶的活性显得尤为重要。传统热处理是钝化食品中酶的主要方式,而近年来一些新型的钝化酶技术已成为新的研究热点。文中就微波钝化酶技术的原理与微波钝化酶的影响因素进行了综述,同时概述了微波钝化酶技术在食品中的应用状况,最后对微波钝化酶技术发展趋势提出了展望。     

Enzyme inactivation of tomato juice by ohmic heating and its effects on physico‐chemical characteristics of concentrated tomato paste

Fresh juice of fully ripe tomato was subjected to ohmic heat (OH) treatment (90°C for 1 min) and the effectiveness of treatment was compared with conventional hot break (CT) treatment (90°C for 5 min). PG (Polygalacturonase) and PME (Pectin methyl esterase) enzyme inactivation achieved by the OH (1 min.) was similar as compared to CT of 5 min. During the kinetic analysis it was observed that the inactivation of PME & PG enzyme and degradation of ascorbic acid followed first order trend in ohmic as well as conventional treatment of tomato juice, however total color change (DE) was found to follow least‐squares non‐linear parameter algorithm behavior. Thermal treatments leads to the increased release of phyto‐chemicals from the matrix which results in a significant (p<0.01) increase in lycopene content during the early phases of the treatments. The Paste (28±0.5 °Brix) obtained after pre‐treatment was analyzed for lycopene, ascorbic acid content and apparent viscosity and color. OH Paste was found more viscous than CT treatment with maximum viscosity of 2.33×103 mPa‐s. The color of OH treated paste was bright red as compared to CT treatment, however the lycopene and ascorbic acid content of paste were found similar in OH and CT. Based on results of present study it is concluded that the ohmic treatment may be applied as an efficient alternative to the conventional method of enzyme inactivation in tomato juice.     

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.     

Principles and recent applications of novel non-thermal processing technologies for the fish industry—a review

Thermal treatment is a traditional method for food processing, which can kill microorganisms but also lead to physicochemical and sensory quality damage, especially to temperature-sensitive foods. Nowadays consumers’ increasing interest in microbial safety products with premium appearance, flavor, great nutritional value and extended shelf-life has promoted the development of emerging non-thermal food processing technologies as alternative or substitution to traditional thermal methods. Fish is an important and world-favored food but has a short shelf-life due to its extremely perishable characteristic, and the microbial spoilage and oxidative process happen rapidly just from the moment of capture, making it dependent heavily on post-harvest preservation. The applications of novel non-thermal food processing technologies, including high pressure processing (HPP), ultrasound (US), pulsed electric fields (PEF), pulsed light (PL), cold plasma (CP) and ozone can extend the shelf-life by microbial inactivation and also keep good sensory quality attributes of fish, which is of high interest for the fish industry. This review presents the principles, developments of emerging non-thermal food processing technologies, and also their applications in fish industry, with the main focus on microbial inactivation and sensory quality. The promising results showed great potential to keep microbial safety while maintaining organoleptic attributes of fish products. What’s more, the strengths and weaknesses of these technologies are also discussed. The combination of different food processing technologies or with advanced packaging methods can improve antimicrobial efficacy while not significantly affect other quality properties under optimized treatment.     

Inactivation of tomato pectic enzymes by manothermosonication

 The resistance of tomato pectic enzymes to manothermosonication (MTS), a combined treatment of heat and ultrasound under moderate pressure, was studied. Pectinmethylesterase (PMF) and polygalacturonases (PG) I and II were inactivated much more efficiently by MTS than by simple heating. In MTS inactivation of these enzymes, the effect of heat and ultrasonic waves was synergistic. D values [the time required for the (original) enzyme activity to decrease by 90%] for PME heat inactivation at 62.5  °C were reduced 52.9-fold by MTS and those for PG I at 86  °C and PG II at 52.5  °C, 85.8-fold and 26.3-fold, respectively. Received: 23 January 1998 / Revised version: 23 March 1998     

Inactivation of tomato pectic enzymes by manothermosonication

 The resistance of tomato pectic enzymes to manothermosonication (MTS), a combined treatment of heat and ultrasound under moderate pressure, was studied. Pectinmethylesterase (PMF) and polygalacturonases (PG) I and II were inactivated much more efficiently by MTS than by simple heating. In MTS inactivation of these enzymes, the effect of heat and ultrasonic waves was synergistic. D values [the time required for the (original) enzyme activity to decrease by 90%] for PME heat inactivation at 62.5  °C were reduced 52.9-fold by MTS and those for PG I at 86  °C and PG II at 52.5  °C, 85.8-fold and 26.3-fold, respectively. Received: 23 January 1998 / Revised version: 23 March 1998     

Opportunities and challenges in application of ultrasound in food processing

The demand for convenience foods of the highest quality in terms of natural flavor and taste, and which are free from additives and preservatives, has spurred the need for the development of a number of non-thermal approaches to food processing, of which ultrasound technology has proven to be very valuable. Increasing number of recent publications have demonstrated the potential of this technology in food processing. A combination of ultrasound with pressure and/or heat is a promising alternative for the rapid inactivation of microorganisms and enzymes. Therefore, novel techniques like thermosonication, manosonication, and manothermosonication may be a more relevant energy-efficient processing alternative for the food industry in times to come. This review aims at identifying the opportunities and challenges associated with this technology. In addition to discussing the effects of ultrasound on foods, this review covers various areas that have been identified as having great potential for future development. It has been realized that ultrasound has much to offer to the food industry such as inactivation of microorganisms and enzymes, crystallization, drying, degassing, extraction, filtration, homogenization, meat tenderization, oxidation, sterilization, etc., including efficiency enhancement of various operations and online detection of contaminants in foods. Selected practical examples in the food industry have been presented and discussed. A brief account of the challenges in adopting this technology for industrial development has also been included.     

Pectins in Processed Fruit and Vegetables: Part I—Stability and Catalytic Activity of Pectinases

ABSTRACT: Pectinases catalyze numerous pectin conversion reactions strongly impacting on the quality of fruits, vegetables, and the related intermediate and end products. The effect of processing on the stability and catalytic activity of pectinases is of prime importance to food processors since desirable and/or deleterious reactions can be tailored (accelerated or inhibited) meeting specific quality targets. Of the multiple endogenous enzymes involved in the modification and degradation of pectin, pectinmethylesterase (PME), and polygalacturonase (PG) have been widely investigated in the context of fruit and vegetable processing. This review covers the stability and catalytic activity of endogenous plant PME and PG including the quantitative approaches applied in inactivating and/or boosting the catalytic activity of the enzymes in purified and real food systems. This will be discussed in the context of both traditional and novel food processing technologies.     

Effects of conventional and ultrasound blanching on enzyme inactivation and carbohydrate content of carrots

There is a growing interest in the use of ultrasound (US) as an alternative to conventional processes. Although US has previously been applied as a pretreatment of fruits and vegetables, no investigation has been done on the usefulness of US for carrot blanching, paying special attention to its effect on enzyme inactivation and leaching losses. In the present paper, the influence of US (in bath and with probe) on peroxidase (POD) and pectinmethylesterase (PME) inactivation and on the loss of total soluble solids and carbohydrates by leaching has been evaluated. Results of this preliminary study have also been compared with those obtained after conventional (hot water and steam) blanching of carrots. The highest enzyme inactivation was obtained with the conventional treatments performed at high temperatures and with the US probe treatments with heat generation. Carrots blanched by US probe for 10?min at a temperature up to 60?°C showed characteristics similar to those conventionally treated at 60?°C for 40?min. Although the efficiency of US was limited for total inactivation of POD and PME, this treatment resulted to be advantageous in terms of time for blanching at mild temperatures. US probe treatments could also be considered as an advantageous alternative to low-temperature long-time (LTLT) conventional treatments for those applications in which partial inactivation of PME is required for the better preservation of carrot structure.     

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.