Impact of high-pressure treatments on enzyme activity of fruit-based beverages: an overview

With an ever-increasing demand for clean label products, there is a greater need for efficient and environmentally friendly processes to compete the conventional thermal or chemical treatments. For instance, high-pressure processing (HPP) has been widely studied in the fruit industry from the last two decades. HPP can inactivate or activate different enzymes in fruit juices, pulp, and purées. HPP treatment inactivates the enzymes by the alterating the conformation of the protein structure and the active site. Depending on the enzyme, pressure, pH, temperature and treatment time, HPP can increase enzyme activity due to the release of membrane-bound enzymes and also due to changes in protein conformation and active site that facilitate interaction with the substrate. Furthermore, the combination of high pressure, temperature and reduced treatment times offered greater inactivation of enzymes in fruit beverages. This study aimed to investigate the inactivation kinetics of endogenous enzymes in fruit beverages.     

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.     

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.     

High pressure processing of cocoyam,Peruvian carrot and sweet potato: Effect on oxidative enzymes and impact in the tuber color

High pressure processing (HPP) is a non-thermal technology used to activate or inactivate enzymes. This study investigated the effects of HPP (600 MPa for 5 or 30 min at 25 °C) on cocoyam, Peruvian carrot and sweet potato color, and the polyphenoloxidase (PPO) and peroxidase (POD) activities in tuber cubes, puree, and enzyme extract subjected to HPP. The results showed enzyme inactivation by HPP in cocoyam (up to 55% PPO inactivation in puree and 81% POD inactivation in extract) and Peruvian carrot (up to 100% PPO and 57% POD inactivation the extract). In contrast, enzyme activation was observed in sweet potato (up to 368% PPO and 27% POD activation in puree). The color results were compatible to enzyme activity: the color parameters remained unchanged in cocoyam and Peruvian carrot, which showed high PPO and POD inactivation after HPP. Furthermore, the impact of HPP on the enzymes was influenced by the matrix in which HPP was carried out, evidencing that the enzyme structure can be protected in the presence of other food constituents.Industrial relevanceThe enzymes PPO and POD are an important concern for vegetable processing, due its ability to induce browning after vegetables are cut. The HPP at 600 MPa for 5 or 30 min can be used to inactivate these enzymes in cocoyam and Peruvian carrot, guaranteeing the color and freshness of the tubers similar to the fresh cut vegetable.     

Effects of Ultraviolet Light and High‐Pressure Processing on Quality and Health‐Related Constituents of Fresh Juice Products

Fresh juices are highly popular beverages in the global food market. They are perceived as wholesome, nutritious, all‐day beverages. For a fast growing category of premium juice products such as cold‐pressed juices, minimal‐processing nonthermal techniques such as ultraviolet (UV) light and high‐pressure processing (HPP) are expected to be used to extend shelf‐life while retaining physicochemical, nutritional, and sensory characteristics with reduced microbial loads. Also, UV light and HPP are approved by regulatory agencies and recognized as one of the simplest and very environmentally friendly ways to destroy pathogenic organisms. One of the limitations to their more extensive commercial application lies in the lack of comparative effects on nutritional and quality‐related compounds in juice products. This review provides a comparative analysis using 92 studies (UV light: 42, HPP: 50) mostly published between 2004 and 2015 to evaluate the effects of reported UV light and HPP processing conditions on the residual content or activity of bioactive compounds such as vitamins, polyphenols, antioxidants, and oxidative enzymes in 45 different fresh fruit and vegetable juices (low‐acid, acid, and high‐acid categories). Also, the effects of UV light and HPP on color and sensory characteristics of juices are summarized and discussed.     

Residual polyphenol oxidase and peroxidase activities in high pressure processed bok choy (Brassica rapa subsp. chinensis) juice did not accelerate nutrient degradation during storage

Polyphenol oxidase (PPO) and peroxidase (POD) cannot be fully inactivated by commercial high pressure processing (HPP) operations, and their residual activities may accelerate nutrient degradation during storage. This study hence aimed to establish the effect of residual enzyme activity on nutrient preservation in bok choy (Brassica rapa subsp. chinensis) juice. Bok choy juice was treated at 600 MPa for up to 20 min and enzyme inactivation, nutrient retention immediately after treatment and nutrient preservation during storage were determined. High residual PPO (85.1 ± 2.6%) and POD (68.5 ± 1.0%) activities remained after 20 min of treatment. Increasing the pressure holding time to enhance enzyme inactivation did not compromise total antioxidant capacity, vitamin C, carotenoids, isothiocyanates and vitamin K levels. Neither did it significantly reduce the vitamin C degradation rate during refrigerated storage. Maximising enzyme inactivation may thus not be necessary for nutrient preservation during the storage of HPP-treated bok choy juice.Industrial relevance textWith HPP, an increase in pressure or holding time is required to achieve higher levels of enzyme inactivation. Without the need to maximize PPO and POD inactivation, juice processors may employ the minimum pressure and holding time required for microbial inactivation. As vegetative bacteria are typically less resistant to HPP inactivation than these enzymes, this could translate to reduced energy costs and increased throughput.     

Effects of high‐pressure processing on fungi spores: Factors affecting spore germination and inactivation and impact on ultrastructure

Food contamination with heat‐resistant fungi (HRF), and their spores, is a major issue among fruit processors, being frequently found in fruit juices and concentrates, among other products, leading to considerable economic losses and food safety issues. Several strategies were developed to minimize the contamination with HRF, with improvements from harvesting to the final product, including sanitizers and new processing techniques. Considering consumers’ demands for minimally processed, fresh‐like food products, nonthermal food‐processing technologies, such as high‐pressure processing (HPP), among others, are emerging as alternatives to the conventional thermal processing techniques. As no heat is applied to foods, vitamins, proteins, aromas, and taste are better kept when compared to thermal processes. Nevertheless, HPP is only able to destroy pathogenic and spoilage vegetative microorganisms to levels of pertinence for food safety, while bacterial spores remain. Regarding HRF spores (both ascospores and conidiospores), these seem to be more pressure‐sensible than bacterial spores, despite a few cases, such as the ascospores of Byssochlamys spp., Neosartorya spp., and Talaromyces spp. that are resistant to high pressures and high temperatures, requiring the combination of both variables to be inactivated. This review aims to cover the literature available concerning the effects of HPP at room‐like temperatures, and its combination with high temperatures, and high‐pressure cycling, to inactivate fungi spores, including the main factors affecting spores’ resistance to high‐pressure, such as pH, water activity, nutritional composition of the food matrix and ascospore age, as well as the changes in the spore ultrastructure, and the parameters to consider regarding their inactivation by HPP.     

The role of water in the impact of high pressure on the myrosinase activity and glucosinolate content in seedlings from Brussels sprouts

In this study, it was shown that the amount of available water was found to influence the high pressure processing (HPP) effect on both myrosinase activity and total glucosinolate concentration in Brussels sprouts seedlings. Brussels sprouts seedlings with different water content (w_c = 4.8–89.4%) and water activity (a_w = 0.17–0.97) were pressurized at selected pressures between 200 and 800 MPa (5 °C and 3 min), thereby affecting pressure-induced enzyme denaturation, molecular diffusion, and cell permeability differently. The myrosinase activity and intact glucosinolate content in the dry seedlings (w_c < 14%) was not affected by the applied pressure treatments. Water adsorption (duration of 5 days) prior to HPP resulted in a decreased initial myrosinase activity due to changed plant cell permeability. Myrosinase was inactivated in seedlings with high water availability (w_c = 45–89%) after HPP, this inactivation is interpreted to be both pressure-induced and result from glucosinolate product catalyzed inactivation facilitated by enhanced cell permeability.Industrial relevanceHigh pressure processing (HPP) is increasingly applied in the food industry. The treatment is acknowledged for the ability to give products longer shelf life concomitant with a high nutritional quality and fresh appearance. Upon HPP of cruciferous plants it is important to have a special focus on the glucosinolate-myrosinase system, since sub-optimal pre-treatment and/or processing parameters can affect the food quality negatively. The present study provides valuable results regarding the significance of water content and activity on the sensitivity of the myrosinase-glucosinolate system in Brussels sprouts seedlings towards HPP. Thus, providing a tool for designing different types of HPP products with respect to levels of active myrosinase and intact glucosinolates by adjustment of water content, water activity and HPP level.     

The effect of high pressure processing on nutritional value and quality attributes of Cucumis melo L.

To determine the effect of cultivar on high pressure processing (HPP) performance three commercial melon varieties were assessed before and after HPP for vitamin C and β-carotene by HPLC and for ferric ion reducing capacity (FIRC) using the Ferric Reducing Ability of Plasma (FRAP) assay. Total titrable acids (TTA), °Brix and colour were also recorded for fresh,−HPP (material cut and packaged) and +HPP samples (material cut, packaged and subject to HPP). The HP process was non-thermal so as to determine the effect of pressure alone on these phytochemicals. There were significant differences between cultivars in vitamin C, β-carotene, TTA, °Brix and colour parameters in fresh samples prior to HPP. HPP did not have an effect on TTA or °Brix, but colour was adversely affected. FIRC and vitamin C concentrations were decreased by HPP and these losses were cultivar dependent for vitamin C. Levels of β-carotene were significantly increased. Cultivar was identified as an important parameter in raw material selection for HPP and retention of vitamin C as a good measure of both quality and cultivar suitability.Industrial relevanceThere is an increasing consumer demand for fresh, natural and healthy fruit and vegetable products with an extended shelf life. This demand is driving industry to look at alternative preservation technologies. HPP has the potential to deliver safe, preserved fruit and vegetables through enzyme inactivation of microbe destruction. HPP removes the need for additives or preservatives and the process is therefore viewed as closer to “natural” by consumers. We show that HPP results in minimal loss of sensorial properties and health-promoting phytochemicals; thus providing consumers a high quality, healthy product with extended shelf life. The introduction of non-thermal processing techniques has the potential to move the focus of the Australian food processing industry from safety to the dual aims of safety and health, resulting in an increase of health-promoting phytochemicals in highly consumed processed foods.     

Optimization of high hydrostatic pressure processing for the preservation of minimally processed peach pieces

Consumers demand fresh-cut fruits, free from additives and with fresh appearance. However, the alteration caused by the tissue processing limits their shelf life. The aim of this work was to optimize the pressure level (from 400 to 600 MPa) and the holding time (from 1 to 9 min) of the high pressure processing (HPP) to achieve enzyme inactivation while preserving texture and color of minimally processed peaches. Peach cylinders were processed by combining dipping in organic acid solution, with vacuum packaging and HPP at room temperature. Results showed that higher pressure levels were more effective to inactivate enzymes and to preserve color than longer times. In addition, long treatments affected the microstructure and the texture of the tissues more seriously. Finally, a desirability study and a principal component analysis were performed. These showed that the optimal treatment would be 585 MPa and 1 min and that the best treatment of the ones studied was 600 MPa for 5 min.Industrial RelevanceThere is an increasing demand for minimally processed fruits as a result of their convenience and fresh-like characteristics. Although consumers are familiar with the consumption of canned peaches, the nutritional profile of this product is far from being optimal, and therefore minimal processing offers the unique advantage of maintaining the original quality of the fresh fruit. However, this product is prone to suffer alterations such as browning and softening. High pressure processing (HPP) is proposed as a non-thermal technology able to suitably preserve minimally processed peaches. This study aimed to optimize the conditions of the HPP treatment, to achieve enzyme inactivation while maintaining texture and color. The promising results obtained can help promote the use of HPP as an alternative to preserve the quality and extend the shelf life of minimally processed fruits.     

Effects of high pressure processing on lipid oxidation: A review

High pressure processing (HPP) is an alternative mild-technology used in the past decades to sterilize and pasteurize food matrices such as meat and seafood. HPP obeys thermodynamic principles, namely Le Chatelier's law of equilibrium and the isostatic rule, both of which account for microbial inactivation. HPP has the advantage of ensuring reduction of pathogens and spoilage in foods, and preserving the organoleptic characteristics of the product that are compromised in traditional heat treatments. However, high pressure changes the thermodynamic equilibrium of chemical reactions. This is the case of lipid oxidation, in which kinetics is accelerated in the presence of high hydrostatic pressure.In recent years, there has been increasing focus on the response of lipid components to HPP, especially considering the deleterious outcomes that secondary products of oxidation have on the final product. The objective of this work is to review the literature on the effect of this “mild-technology” in the degradation of lipid fraction of foods. We discuss qualitative and quantitative determinations, as well as the thermodynamic and chemical interpretations underlying the phenomenon.Industrial relevanceIn this work we reviewed the literature concerning the effect of high-pressure processing (HPP) on lipid oxidation. Since 1990s HPP has been used as an alternative to thermal treatments to pasteurize and sterilize food products, such as meats and seafood. Many of these raw materials have a high content of lipids (among them trialglycerols and cholesterol-derivative) that are susceptible to oxidation. During the last decade, there has been increasing interest on the response of lipid components to HPP, especially considering the deleterious outcomes that secondary oxidation-derivative molecules have on the final product. This review intends to summarize and discuss the data reported in literature, contextualizing the oxidation within the broad transformation of biological structures due to hydrostatic pressure. A better understanding of the underlying phenomena could lead to the development of predicting models which could be use in food industry.     

Inactivation of Staphylococcus aureus by high pressure processing: An overview

Food safety is a major concern of consumers, food industry and governments, with 25 million foodborne diseases occurring annually worldwide. Staphylococcus aureus, is an extremely versatile opportunistic pathogen being responsible for staphylococcal food poisoning due to enterotoxic strains. With increasing demands for safer food, new food preservation technologies are increasingly gaining interest. In the last two decades, high pressure processing (HPP) appeared as an alternative non-thermal food preservation method promoting inactivation of some spoilage and pathogenic microorganisms, while maintaining food characteristics. Factors that modulate its efficiency will be revised, firstly based on the state-of-the art described for bacteria in general and afterwards, when studies exist, for S. aureus specifically. S. aureus inactivation by HPP, like in other microorganisms, is conditioned by cell structures and biomolecules, matrix, HPP processing conditions, the use of antimicrobials and is also dependent of the strain and growth phase. Cell membrane is the most pressure sensitive structure of S. aureus, being the lipids and proteins the most important target molecules. However, monomeric proteins such as staphylococcal enterotoxins (SE) are not affected by HPP, and strains with SE appear to be more efficiently inactivated than those without. Other phenotypic and genotypic characteristics of S. aureus strains, such as pigmentation and the presence of σ^B factor are extremely important factors determining the efficacy of HPP treatments. Inactivation of S. aureus by HPP to ensure food safety still remains a current challenge regarding the understanding of its particular barotolerance and its inactivation kinetics profile that often deviates from the simpler first order decay. Thus, this review provides state-of-the-art information for researchers interested in studying HPP inactivation of S. aureus.Industrial relevanceThis review gives an insight on the importance of Staphylococcus aureus as a foodborne versatile opportunistic pathogen and its importance from the food safety point, its barotolerance and the main reasons for this resistant behavior to high pressure processing and the mechanisms of S. aureus inactivation by HPP.     

Compression heating influence of pressure transmitting fluids on bacteria inactivation during high pressure processing

Compression heating characteristics of different pressure transmitting fluids [three different concentrations (75:25, 50:50, 25:75) of water–glycol mix and sodium benzoate (2%) solutions] and their influence on inactivation of spores of Bacillus subtilis in phosphate buffer (0.067 M, pH 7.0) during high pressure processing (HPP) were studied. Experiments were conducted using a pilot scale food processor. Pressure transmitting fluids containing highest percentage of glycol (25:75 water–glycol mix) showed highest temperature increase while 2% sodium benzoate solution showed least temperature increase during high pressure processing. The target pressure, holding time, compressibility, initial temperature, and the rate of heat loss to the surroundings primarily influenced the apparent temperature increase of pressure transmitting fluid in a vessel during HPP. The temperature change was further influenced by the fluid properties such as viscosity, specific heat and thermal conductivity. Use of sodium benzoate solution as pressure-transmitting fluid resulted in highest inactivation of B. subtilis spores. Change in pressure transmitting fluid temperature as a result of compression heating and subsequent heat transfer should be considered in inactivation of bacterial spores by HPP.     

Inactivation Kinetics of the Most Baro-Resistant Enzyme in High Pressure Processed Litchi-Based Mixed Fruit Beverage

This work focused on a litchi-based mixed fruit beverage, comprising of coconut water and lemon juice, mixed in an optimized proportion. Based on preliminary studies, three resistant spoilage enzymes were identified in the beverage, viz. polyphenol oxidase (PPO), peroxidase (POD), and pectin methyl esterase (PME). The response surface methodology (RSM) based on central composite face-centered design (FCCD) screened out PPO as the most resistant enzyme within the high pressure processing (HPP) domain of 200–600 MPa/30–70 °C/0–20 min. A detailed kinetic study was conducted on PPO inactivation within the same HPP domain along with a set of thermal treatments (0.1 MPa/30–70 °C). A synergistic effect of pressure and temperature on PPO inactivation was observed, throughout the HPP domain. However, PPO was almost completely inactivated at 500 MPa/70 °C/20 min. The inactivation order (n) values for PPO were 1.10 and 1.25 for thermal and HPP treatments, respectively. For every 10 °C rise in temperature, the inactivation rate constant (k, U^n-1 min^?1) increased approximately by 1.5 times, within 50–70 °C (at 0.1 MPa), while a 10-fold increase was obtained in the case of HPP treatments. The activation energy (E _a ) and the activation volume (V _a), depicting the temperature and pressure dependence of k, was found to decrease slightly, with an increase in pressure and temperature, respectively. The PPO inactivation rate constant was modeled as a function of both temperature and pressure conditions by combining both Arrhenius and Eyring equations.     

A novel approach to predicting microbial inactivation kinetics during high pressure processing

The inactivation kinetics of Escherichia coli (ATCC 25922) during high pressure processing (HPP) was examined from 200 to 400 MPa in 50 MPa increments at 15 degrees C. Although the time course of HPP-induced E. coli inactivation in 0.1% peptone water successfully fitted the Weibull function, this procedure involved curve fitting, and not prediction. The objective of this study was to develop a novel HPP-induced microbial inactivation model to simulate the inactivation kinetics under various pressure conditions. The maximum inactivation rate during HPP was calculated from the inactivation curves at different pressure conditions on a semi-log plot. The relationship between the square root of the absolute value of the inactivation rate (k(max)) and treatment pressure was linear (R(2)=0.99). The linear relationship between k(max) and treatment pressure also successfully described independent data from other studies in the literature. Overall, the newly developed differential equation model, into which was substituted the square root function of the inactivation rate, was capable of simulating the inactivation kinetics during HPP at constant pressure. Additionally, the model could successfully describe the inactivation kinetics during HPP using other researchers' data. The accuracy of prediction of the new model was comparable to that derived from Weibull or modified Gompertz fitting to the observed data. Furthermore, the new model could successfully simulate the inactivation kinetics during dynamic pressure conditions, which included come-up time, changes in holding pressure during treatment, and pressure-release time. Moreover, the effect of pulsed pressure treatment was also simulated successfully using this model. Therefore, the modeling procedure presented in this study will contribute to the advancement of predictive modeling for HPP-induced microbial inactivation.     

Effects of high pressure,microwave and ultrasound processing on proteins and enzyme activity in dairy systems — A review

High-pressure processing (HPP), microwaves (MW) and ultrasound (US) are used for pasteurization with minimum heat input. They also alter physico-chemical properties of milk proteins and enzymes. This article aims at identifying the important changes in milk proteins imparted by these three processing technologies. HPP dissociates casein micelles at low pH (<6.7) and concentrations (<4% w/w), while β-LG is the most pressure sensitive whey protein due to the presence of free thiol groups. Milk enzyme activity is inhibited at higher pressures (>400 MPa). MW treatment denatures whey proteins rapidly, even below their thermal denaturation temperatures. High-power MW treatment (e.g. 60 kW) deactivates enzymes by denaturing them. However, low-power controlled MW irradiation (e.g. 30 W) improves enzyme activity. Ultrasound can homogenize protein aggregates in dairy systems and cause whey protein denaturation. Sonication under applied pressure and heat (e.g. 3.5 kg/cm², 126.5 °C) causes enzyme inhibition while mild sonication conditions can improve enzyme activity.Industrial relevanceHPP, MW and US are gaining popularity in the dairy industry due to their ability to pasteurize and functionalize dairy streams with minimal heat input. This review offers insights into how these technologies can be used in isolation or in combination to alter milk proteins and enzyme activity for different academic and industrial applications. However, to fully understand the potential of HPP, MW and US treatment on dairy systems, further research is required in several areas including health related nutritional changes in milk and milk products caused by these technologies.     

Preservation effect of high pressure processing on ascorbic acid of fruits and vegetables: A review

High Pressure Processing (HPP) is a well‐established nonthermal technology for ensuring microbial safety and nutritional quality of foods. Ascorbic acid (AA) is highly labile antioxidant, susceptible to degradation when exposed to oxygen, change in pH, temperature, or pressure. Preservation of AA in fruit and vegetable products is a prime concern for food processors. This review summarizes recent data on the effect of HPP on AA content of different fruits and vegetables, and their products. In most of the food products, HPP has supported either preservation or better retention of AA after pressurization (400–600 MPa/5–10 min) at lower or room temperature. High pressure processed foods have demonstrated better stability of AA during refrigeration storage as compared to thermally processed ones. These studies establish the positive implications of HPP and justify its potential use as a promising preservation technique to safeguard AA in food products.     

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.     

Effect of water activity on inactivation of Listeria monocytogenes and lactate dehydrogenase during high pressure processing

The aim of this study was to investigate the effect of water activity (a_w) on the inactivation of Listeria monocytogenes and lactate dehydrogenase (LDH) during high pressure processing (HPP). For microbial inactivation lyophilized cells of L. monocytogenes 19,115 were left dry or were suspended in 10 ml of 0.1% peptone water, 10 ml of glycerol, or mixtures of glycerol and peptone water. All samples of various a_ws were high pressure (HP) processed at ambient temperature at 600 MPa for 300 s. Following HPP, samples were serially diluted in 0.1% peptone and spread-plated on Tryptic Soy agar supplemented with Yeast Extract. For enzyme inactivation, 4.2 mg of lyophilized LDH was suspended in 2 ml of 100 mM phosphate buffer (pH 7.4), 2 ml of peptone water or glycerol, or in 2 ml mixtures of glycerol and peptone water. A lyophilized sample with no added liquid was also included. All enzyme samples were subjected to HPP as described above. After HPP, LDH was diluted to 0.28 μg/ml in 100 mM phosphate buffer (pH 7.4). LDH activity was assessed by measuring the change in concentration of β-NADH as a function of time. Dynamic light scattering analysis (DLS) was performed to examine the size distribution, polydispersity, and hydrodynamic radius of LDH before and after HPP. No significant difference in CFU/g was observed between lyophilized cells not subjected to HPP and lyophilized cells subjected to 600 MPa for 300 s (P < 0.05). However, lyophilized cells that were suspended in 100% to 60% peptone water showed a ~ 7.5-log₁₀ reduction when subjected to HPP. Survival of L. monocytogenes following HPP significantly increased (P < 0.05) when the peptone water concentration was decreased below 60% (a_w ~ 0.8). DLS results revealed that LDH suspended in buffer underwent aggregation following HPP (600 MPa, 300 s). Inactivation rate constants obtained using a first-order kinetic model indicated that untreated and HP processed lyophilized LDH had similar activities. When LDH was subject to HPP in solutions containing glycerol, enzyme activity decreased as the water content increased (r² = 0.95). Lyophilization completely protected L. monocytogenes and LDH from inactivation by high pressure. Furthermore, enzyme activity and cell survival increased as water activity was decreased. We postulate low a_w results in protein stabilization, which prevents protein denaturation and cell death during HPP.     

Modeling the protective effect of aw and fat content on the high pressure resistance of Listeria monocytogenes in dry-cured ham

High pressure processing (HPP) is a promising food preservation technology as an alternative to thermal processing for microbial inactivation. The technological parameters, the type of microorganism, and the food composition can greatly affect the microbicidal potential of HPP against spoilage and pathogenic microorganisms. Presently, the number of available models quantifying the influence of food characteristics on the pathogen inactivation is scarce. The aim of this study was to model the inactivation of Listeria monocytogenes CTC1034 in dry-cured ham, as a function of pressure (347–852 MPa, 5 min/15 °C), water activity (a_w, 0.86–0.96) and fat content (10–50%) according to a Central Composite Design. The response surface methodology, based on the equation obtained with a stepwise multivariate linear regression, was used to describe the relationship between bacterial inactivation and the studied variables. According to the best fitting polynomial equation, besides pressure intensity, both a_w and fat content exerted a significant influence on HP-inactivation of L. monocytogenes. A clear linear piezoprotection trend was found lowering the a_w of the substrate within the whole range of tested pressure. Fat content was included in the model through the quadratic term and as interaction term with pressure, resulting in a particular behavior. A protective effect due to the presence of high fat content was identified for pressure treatments above ca. 700 MPa. At lower pressure, higher inactivation of L. monocytogenes occurred by increasing the fat content above 30%. The results emphasize the relevant influence of intrinsic factors on the L. monocytogenes inactivation by HPP, making necessary to assess and validate the effectiveness of HPP on specific food products and consequently set process criteria adjusted to each particular food product.