Microbial inactivation by new technologies of food preservation

Abstract

The increasing consumer demand for ‘fresh-like’ foods has led to much research effort in the last 20 years to develop new mild methods for food preservation. Nonthermal methods allow micro-organisms to be inactivated at sublethal temperatures thus better preserving the sensory, nutritional and functional properties of foods. The aim of this review is to provide an overview of the microbiological aspects of the most relevant nonthermal technologies for microbial inactivation currently under study, including irradiation, high hydrostatic pressure, pulsed electric field and ultrasound under pressure. Topics covered are the mechanisms of inactivation, sensitivity of different microbial groups and factors affecting it and kinetics of inactivation. Micro-organisms are the main agents responsible for food spoilage and food poisoning and therefore food preservation procedures are targeted towards them. Food preservation methods currently used by the industry rely either on the inhibition of microbial growth or on microbial inactivation. Methods which prevent or slow down microbial growth cannot completely assure food safety, as their efficacy depends on the environmental conditions such as, for instance, the maintenance of the chill chain. Thermal treatment is the most widely used procedure for microbial inactivation in foods. However, heat causes unwanted side-effects in the sensory, nutritional and functional properties of food. This limitation together with increasing consumer demand for fresh-like foods has promoted the development of alternative methods for microbial inactivation, among which ionizing irradiation, ultrasound under pressure, high hydrostatic pressure (HHP) and pulsed electric field (PEF) are attracting much interest. The irradiation process involves the application of electromagnetic waves or electrons to foods. Radiation sources are either gamma rays from cobalt-60, electron beams or X-rays, and the amount of irradiation absorbed by a food is measured in kGy (1 Gy = 1 J kg−1). Commercial application of ionizing radiation (IR) treatment on foods was started at the beginning of the 1980s, but its success has been prevented by consumer concerns. Nowadays, social perception of IR is changing and this technology is being re-examined. Ultrasound is defined as sound waves with frequencies above the threshold for human hearing (>16 kHz). Although ultrasound was initially discarded for food preservation because of its weak lethal action, the application of an external hydrostatic pressure of up to 600 kPa [manosonication (MS)] increases substantially the lethality of the treatment. In addition, a combination of MS with temperature [manothermosonication (MTS)] has been proposed (Raso et al. 1998a). The HHP involved the application of pressures from 100 to 1000 MPa. The first studies on the lethal effect of HHP were conducted at the end of the 19th century, but it has been in recent years when commercial applications of this procedure have started. Finally, PEF technology consists in the application of short duration (1–100 μs) high electric field pulses (10–50 kV cm−1) to a food placed between two electrodes. Like ultrasound under pressure, PEF technology is not yet being used to preserve food commercially. This review discusses the state of the art of the research of the microbial aspects of the four technologies described above. The successful implementation of a novel technology for food preservation relies on the progress in the field of mechanisms of inactivation. An adequate knowledge of the physiological behaviour of micro-organisms towards inactivation agents is essential for the development of safe foods. It is necessary for understanding the effect of environmental factors on resistance and also for identifying critical factors. It would help to interpret kinetics of inactivation and to develop mathematical models based on parameters with a biological meaning and therefore more useful and able to predict microbial inactivation in a wider range of conditions. A better knowledge of the effect of the preservation agents on the micro-organisms would lead to a more rational design of processes. Cell death has been associated with either structural damage or physiological dysfunctions. Among structural damage, disruption of the envelopes, DNA conformational changes, ribosome alterations or protein aggregation are the most frequently described. Also physiological disorders, such as membrane selective permeability alterations or loss of function of key enzymes have been proposed as events leading to cell death (Gould 1989). Perhaps the most important difficulties that researchers encounter in this area is that several of these lesions may occur simultaneously when the cells are subjected to an agent and it is therefore difficult to attribute the loss of viability of the cell to a single event. Nevertheless, it must be kept in mind that also multitarget inactivation is feasible, this being the addition of several lesions that together cause death. It is also possible that the key target is only affected when a secondary structure is previously damaged. For instance, heat causes membrane damage, loss of nutrients and ions, ribosome aggregation, DNA strand breaks, inactivation of essential enzymes, protein coagulation, etc. (Gould 1989). In other words, almost every cellular structure is somehow affected by elevated temperatures, and it is very difficult to discern which events are leading to cell death. In fact, this is only possible if a direct relationship between the degree of inactivation and the degree of modification of a given target under different environmental conditions is found. Regarding novel inactivation technologies, progress on inactivation mechanisms research is heterogeneous. In this way, sound hypotheses have been put forward to describe the effect of IR and ultrasound on micro-organisms, but much research is still needed to completely explain the way PEF and HHP can inactivate bacterial cells. Table 1 summarizes the relevant events related to bacterial inactivation by the four technologies. It is well established that the critical target for irradiation is the chromosome (Moseley 1989). The effects of ionizing irradiation on bacterial cells are classified as direct and indirect. Direct actions comprise the events caused by the absorption of radiation energy by the target molecules, whereas indirect actions are those derived from the interaction between the reactive species formed by the radiolysis of water, such as the hydroxyl radical, and the target molecules. The hydroxyl radical OH• is able to react with the sugar-phosphate of the DNA to the of from the This causes the of the and of single strand strand occur when two single strand in of the at a are also by the by but it is not this is relevant to cell death (Moseley 1989). The of inactivation of bacterial cells by ultrasound under pressure has also been described. that the is responsible for the lethal effects of ultrasound et al. et al. 1998a). under an very high pressures and temperatures are and and are formed therefore inactivate bacterial cells in a as that described for However, radical have lead to the that the possible effect of is in with that of the effects by et al. et al. 1998a). et al. the effect of MS and of on the degree of cell disruption and that whereas cells cellular MS cells were completely cells a degree of that ultrasound inactivate microbial cells in an or are aspects still not the of of In and with bacterial a effect of MS and heat has been This is the of et al. cells of et al. and cells in a et al. The for the sensitivity of these cells to a treatment are still not It has been that elevated temperatures would cause a effect on cell envelopes, the disruption of the cell by waves et al. This effect would have for as are by heat at In addition, a effect of MS and heat has been described for bacterial (Raso et al. cause the of and from the et al. It has also been that MS of to (Raso et al. it has been that waves damage the external of the its and its heat In to the mechanisms of inactivation proposed for irradiation and a much more for high hydrostatic pressure inactivation. research has been in this but is still the key target leading to cell death by high pressure. on of inactivation of HHP the membrane as the key target and most researchers still with that been of bacterial being a target for HHP inactivation are pressure causes of the the of and membrane from to in a way as a temperature Although of membrane is not lethal to it has been that the and state of the bacterial cell membrane to pressure treatment bacterial resistance to HHP et al. with a more with a degree of are more et al. It is not a more membrane a more cell to The pressure at which would be in cells with a more but it is not in which if cell damage is to to the membrane has also been loss of and and of et al. et al. and and loss of and of and of et al. et al. and The loss of function of including the or has also been described et al. et al. et al. a direct relationship between loss of membrane and loss of viability has been for cells and and However, it has also been that and membrane is to et al. and and and and that cells of may a membrane in cells and and other the cell have also been proposed as key for inactivation by have between cell inactivation and protein kinetics by HHP et al. and in the of the and protein have been described et al. et al. and et al. a direct relationship between loss of viability in and ribosome damage, by of cells in a which is to have a effect on ribosome the to the The that other factors together with ribosome for cell death and that the loss of essential a membrane be the ribosome et al. loss of cells of of the with the loss of caused by the cell HHP treatment. It that of these cellular lesions DNA and protein are not lethal and and are if the cell a functional membrane and the environmental conditions are In HHP inactivation to be multitarget in is a key but in events such as loss protein coagulation, key inactivation and ribosome conformational changes, together with also needed to are to being able to up to 1000 for treatment are in the state at a (Gould and This has been the for the design of a treatment in which are to in a first and inactivated in a by a combination of mild heat and pressure (Raso and structural or functional is as the cause of cell death by and that the inactivation was the of the direct effect of PEF on the membrane because of the temperature or the can be defined as the of in cells and and the most to explain it is that proposed by et al. the cell membrane to a to in the and of the membrane a of an external electric field is as in PEF a amount of of at membrane in of the the external electric field a critical or the membrane is to the and are The and amount of on the electric field and the duration of the treatment. that the is the of of the membrane under an electric field has also that the of would lead to a that be responsible for the of and in the on electron of several and have alterations disruption of in the etc. and et al. et al. However, between the frequencies of of alterations with loss of viability has been and have been to is a relationship between membrane damage and microbial inactivation by and of researchers have the of membrane in to cell death and et al. et al. et al. have described a relationship between the of cells and the of the PEF the of membrane being the cause of cell inactivation. An and almost of PEF is the of The way as it with high pressure and a of cell PEF treatment but to a in the addition of to the treatment as a of of the have that the degree of of cells was as that when the was PEF treatment. would that a of cells were able to their PEF treatment. Micro-organisms the lethal of preservation agents may be able to the damage and only if the environmental conditions are The of sublethal has two main cells not be when selective conditions are used for of This can lead to an of the lethality of the treatment. the of sublethal in that a between treatment and of if is prevented by the combination of preservation agents that with cellular the cell not be able to and the inactivation be and of sublethal by novel preservation technologies is essential for the of mild methods with a lethal effect on Among the four methods for microbial inactivation only in the of ultrasound under pressure have cells been of MS of and cells in with are to those in a et al. This the of membrane damage and is in with the of the or of the cell as the of inactivation of waves on bacterial cells. or has been for and cells. For agent the of inactivation is and is the and the of the sublethal on sublethal cell and of cells have on It that the sensitivity of the different microbial groups depends not only on their to the direct and indirect of irradiation but also on their of the single and strand several actions (Moseley 1989). not among in a and the in an cell may be damage to other has been have the of cells to selective al. et al. but and that irradiation not membrane damage as by It is not the secondary would its either towards the DNA as by and with with DNA or at other are the main target for sublethal in HHP cells. of the and membrane has been described and and and of HHP and in the such as and with efficacy for and micro-organisms have been proposed et al. et al. the of the membrane of cells is and a functional membrane is et al. the membrane damage is and and protein et al. An membrane is essential for the maintenance of the under environmental and from a of of HHP with or also a lethal effect on micro-organisms et al. et al. et al. et al. The of sublethal cell damage in cells is a of have not the of sublethal the selective et al. et al. et al. et al. it was that bacterial inactivation by PEF was an However, recent in et al. have the of sublethal membrane damage in a of the of cells of by of research that the in may from the that the of sublethal membrane damage by PEF depends on the of the and on the bacterial In this way, several a resistance to PEF at with that was to the to the which was damaged. For cells the was the resistance to the of membrane damage and the was at the cells more and damaged. It is of that cells under conditions viability This from a of that if adequate conditions are the of for HHP and be affecting the microbial of the a degree of be have also the of sublethal damage to the membrane in by The of the membrane in PEF inactivation provide new useful that to the understanding of mechanisms of membrane in bacterial cells. also to the environmental under which PEF with other for food preservation. is in It is well that micro-organisms can develop and when to sublethal and which may have for food In the last years much research has been towards the of bacterial mechanisms and The modification of factors to is the most important in bacterial cells and in the of more involved in resistance to and among et al. The of this in to when cells the of and also when cells are subjected to other and In and an alternative with physiological has been described and et al. it that a for the of resistance in and cells. The of the of these on bacterial resistance to novel preservation has not yet been Nevertheless, it is that the resistance to HHP of cells is the of the of the protein in and in et al. et al. A range of and physiological on has been described for and cells that for the in pressure resistance et al. It can be that these also have an on bacterial resistance to ultrasound and irradiation, but up to are on the of the on the resistance to these technologies. by the the heat which consists of a and of and The application of sublethal HHP the of several heat et al. a direct of the heat in HHP In fact, it has been that a heat may cells HHP and et al. In addition, et al. have described that the of several heat or is in et al. The of a sublethal heat on PEF resistance has been but and have that to be in that the to from PEF damage is also research is needed in this The application of a heat not to a MS treatment et al. This that the possible that heat may in cell envelopes, if are not relevant to ultrasound Although is the of other of on bacterial resistance to novel preservation from it can be that in most resistance is et al. have described the to and in et al. have the of irradiation resistance of when cells were previously and heat also to this to PEF and In of micro-organisms to environmental conditions a that not be research is still needed to the and of microbial involved in to food preservation processes. inactivation by irradiation, ultrasound under pressure, HHP and PEF has been to on factors. of in is by the of and conditions by the different Nevertheless, this to an overview on the most relevant factors affecting resistance to novel technologies. The factors are classified process microbial and A of the most important factors for technology is given in Table process parameters are to technology and can be For instance, the of an irradiation treatment is given by the irradiation as the radiation energy is et al. parameters for ultrasound under pressure are treatment of the waves and external pressure (Raso et al. 1998a). HHP efficacy depends on treatment and pressure et al. PEF lethality with parameters such as electric field and from treatment et al. process parameters that are for the four technologies. This is the for treatment a as the temperature is the lethality of the four technologies increases (Raso and The lethal effects of irradiation on micro-organisms are more when the treatment is at elevated irradiation in the state the sensitivity of the micro-organisms in by a of et al. and this has been to the of The lethality of ultrasound under pressure is almost not by an in temperature lethal temperatures are in which an lethal effect is in the lethal effect has been to be et al. et al. The effect of temperature on high pressure inactivation is of HHP with mild treatment temperature lead to a lethal effect and et al. and this has been to a degree of damage on et al. Also treatment temperatures inactivate cells et al. and this effect has been proposed to be the of the of the Regarding the effect of treatment temperature on PEF increases in at and lethal the efficacy of the treatment. This effect has been related to a of the membrane that would cells more to and on the resistance of micro-organisms to irradiation, ultrasound under pressure, HHP and PEF are in Table inactivation with technology on factors such as and food would to that of is by the different treatment etc. in the are for a bacterial are the most micro-organisms to and et al. et al. et al. are more and this has been to the of their of and is In are more to irradiation cells and the are more to PEF and this is at electric field as the electric field threshold needed to inactivate is and and are to be to but among have been Regarding inactivation, are among the most radiation micro-organisms, and the irradiation is not to be in and to the HHP resistance of depends on their which is heterogeneous. and A are inactivated to safe with of and and et al. The inactivation of by PEF has been but and in of human PEF of kV for are on the resistance of to In addition, cell and to have a in resistance to The the cell the the resistance to HHP and PEF et al. et al. cells to be more cells. are to these It be that in is of et al. in to HHP of more among of et al. between 1 and of in the of cells among subjected to irradiation and pressure in resistance to PEF has also been with and and et al. the of has been et al. as an adequate target be for safe processes. In addition, the of relationship between the of a given to the of the most for This of resistance among of the species has not been for ultrasound under pressure that in their heat were almost in their resistance to MS et al. resistance to different agents depends not only on the resistance of the micro-organisms but also on their physiological It is well that bacterial heat resistance widely on the growth growth temperature and to and in the are more to of agents the of growth temperature are and in It to be an important HHP but its effect is different in and cells et such as of the treatment or addition of the resistance of micro-organisms to The of such factors on microbial resistance to novel technologies depends on their mechanisms of factors of in a technology have been Table For instance, of the most important factors irradiation sensitivity is the of the treatment The of irradiation has been to lethal effect because of radical et al. a a effect on This has for heat and have been for new technologies, ultrasound under pressure, as for the of factors a very of has been described et al. of the is the environmental that heat resistance in It has been that the of to irradiation is in et al. also resistance to ultrasound under pressure. in a with of a to a MS treatment as that in a with et al. The of this effect is very when with other technologies, but it is of that a in the of the treatment is the environmental of Regarding HHP inactivation, et al. have that the effect of of in depends on the cells HHP inactivation of membrane protein However, the addition of a increasing the of membrane on the of on PEF microbial inactivation are very but that PEF and et al. et al. have that from to the PEF of by an at kV The for this are The of the treatment is of the factors bacterial resistance with a more The combination of with heat has been used for to the of for as resistance of micro-organisms and the it has been that has effect on bacterial resistance to irradiation et al. It not ultrasound under pressure resistance either et al. Regarding HHP inactivation, from it that bacterial cells are more to pressure in et al. but the of the effect is not the the of the treatment has a on PEF but it depends on the species et al. et al. The application of new technology in food preservation a that the inactivation The be able to treatment conditions to of microbial inactivation, the of and safe foods. be and be on parameters based on the physiological of inactivation et al. Thermal parameters have been the first kinetics This that microbial inactivation from the of the lethal agent the key a a of the of the of cells treatment is and and are used to treatment in the last years the of first kinetics for the of heat inactivation, as well as for novel technologies, is being This is because of the of such as and et al. et al. et al. et al. et al. have been proposed to explain these which may be different for and for cells. have been to the of sublethal multitarget inactivation, cell or for are to be the of resistance the either to the bacterial cells or the treatment. are when et al. of irradiation, ultrasound under pressure, HHP and PEF are in that irradiation the of irradiation of treatment have been for bacterial inactivation by irradiation, and it has been to cellular damage in the range (Moseley et al. have also been in such as and and kinetics inactivation is described for ultrasound under pressure. This is in with the of a single key target and an inactivation It also with the of and with the and in resistance to for HHP and PEF in et al. et al. et al. et al. It is that HHP and PEF are the and therefore every cell is subjected to the for irradiation high hydrostatic pressure and pulsed electric field have been proposed to explain the for the of have that this of are and the first inactivation of two microbial in resistance et al. A also be by a of resistance the microbial et al. A of models have been to describe such as the the and models based on the among et al. have been by the most used by several to describe microbial inactivation by PEF and based on the are by their as only two parameters the and also their as can either or their most important is the of biological of the parameters of the which their are of on bacterial inactivation by PEF that have been described the A secondary that the of the parameters of the with the electric field has been described for several micro-organisms et al. this secondary a to the for can be defined that the of microbial at different electric field et al. have also a that treatment conditions necessary to a of inactivation of the bacterial to be the microbial inactivation by HHP has been a but very studies have been This is because of the that with and under conditions the inactivation is the of the effect of pressure and with temperature are in and a studies have the of the to HHP kinetics and secondary models that allow the of the lethality at different pressures and temperatures have been proposed and are and a kinetics be a a of models have been in the last years that can be used to describe inactivation and their application is by that more research in this field is needed to on the inactivation of spoilage and micro-organisms for In addition, research to the causes and that the of be ultrasound under pressure, HHP and PEF are procedures to inactivate micro-organisms in but the high of their as a for food preservation. these novel technologies are applications as that assure food microbial inactivation in high their more research effort is needed to mechanisms of inactivation, for HHP and to better the effect of environmental and the of and sublethal aspects of food of may be useful in and of target for technology is also Finally, development of mathematical models based on physiological to treatment conditions is models the lethal or events leading to the the bacterial and possible of and