Universitatea LUCIAN BLAGA, Sibiu Facultatea de Stiinte Agricole, Industria Alimentara si Protectia Mediului

Alternative food-preservation technologies: efficacy and mechanisms /Tehnologii alternative de conservare a alimentelor: mecanisme si eficacitate

Profesor coordonator: Conf. univ. dr. ing. Victor Nederita

Studenti: Baies Alexandru Bisboaca Vasile Buda Iulian

1

2011 2012SummaryAbstract................................................................3 1.Introduction.......................................................3 2.Food-preservation technologies.........................3 3.Efficacy of alternative preservation....................4 4.Mechanisms of microbial inactivation................5 5. Kinetics of microbial inactivation......................7 6. Microbial resistance to alternative processes...9 7. Enhancing the efficacy.................... ..............11 8.Microorganisms for efficacy testing................11 9. Microorganisms for efficacy testing...............12 10.Conclusion...... .............................................12 Acknowledgements...... .....................................13

2

References...... ..................................................13

Abstract High-pressure processing, ionizing radiation, pulsed electric field and ultraviolet radiation are emerging preservation technologies designed to produce safe food, while maintaining its nutritional and sensory qualities. A sigmoid inactivation pattern is observed in most kinetic studies. Damage to cell membranes, enzymes or DNA is the most commonly cited cause of death of microorganisms by alternative preservation technologies. 1. Introduction Consumers are increasingly aware of the health benefits and risks associated with consumption of food. To meet consumers expectations, the food industry is devoting considerable resources and expertise to the production of wholesome and safe products. Production of safe food includes scrutinizing materials entering the food chain, suppressing microbial growth (e.g. storage at chilling temperature), and reducing or eliminating the microbial load by processing and preventing post-processing contamination. The presence of a processing unit operation aiming at microbial destruction is of primary importance to ascertain safety and stability of food. Heat treatments are traditionally applied to pasteurize or sterilize food, generally at the expense of its sensory and nutritional qualities. As consumers increasingly perceive fresh food as healthier than heattreated food, the industry is now seeking alternative technologies to maintain most of the fresh attributes, safety and storage stability of food . Satisfactory evaluation of a new preservation technology depends on reliable estimation of its efficacy against pathogenic and spoilage food-borne microorganisms. Research on alternative technologies was initially focused on process design, product characteristics and kinetics of microbial inactivation. The success of these new technologies, however, depends on progress in understanding microbial physiology and behavior of microbial cells during and after treatment. Consequently, this article reviews alternative preservation technologies with emphasis on comparing their efficacy with conventional heat treatment, mechanisms of microbial inactivation, patterns of inactivation kinetics, microbial resistance mechanisms and potential causes of underestimation of survivors during food processing by alternative technologies. 2. Food-preservation technologies Thermal pasteurization and sterilization are predominantly used in the food industry for their efficacy and product safety record. Excessive heat treatment may, however, cause undesirable protein denaturation, non-enzymatic browning and loss of vitamins and volatile flavor compounds. Many US consumers, for example, consider the cooked and caramelized flavors of sterilized milk as taste defects . Advances in technology allowed optimization of thermal processing for maximum efficacy against microbial contaminants and minimum deterioration of food quality. High-temperature short-time (HTST) pasteurization and ultra-high temperature (UHT) sterilization, for example, minimize vitamin losses in milk in comparison with batch pasteurization and

3

conventional commercial sterilization, respectively . Products processed by modern thermal technologies, however, still lack the fresh flavor and texture. Non-thermal alternative technologies have been investigated intensively in the past 30 years . These technologies are named according to the main processing parameter leading to cell inactivation. Food treated with high-pressure processing (HPP) is exposed to a high hydrostatic pressure (up to 1000 MPa) for a few minutes . Pulsed electric field (PEF) treatment is based on the delivery of pulses at high electric field intensity (5-55 kV/cm) for a few milliseconds . Gamma radiations and electron beams generate doses of 210 kGy, and are commonly referred to as ionizing radiations . Electron-beam technology is currently developed as a safer alternative to gamma radiation since radioactive isotopes are not used. Ultraviolet (UV) energy is a non-ionizing radiation with germicidal properties at wavelengths in the range of 200-280 nm . Alternative technologies are occasionally described as non-thermal. Food is treated at ambient or refrigeration temperatures and heat generation during the process is not substantial. HPP and gamma radiation are more suitable than other alternative technologies for application in solid foods . The lethal agent must penetrate these foods uniformly without degrading the food texture. The current design of PEF treatment chambers does not allow processing of solid foods. The shielding effect of solid particles restricts applications of UV radiation and electron beams to the treatment of food surfaces . Some alternative technologies are inherently batch processes, while others are adaptable to continuous applications. HPP was designed primarily for processing of packed food, but new designs were developed recently for continuous processes. PEF is a rapid treatment that is well adapted to continuous processing. Maximal PEF treatment intensity is limited by equipment design and foods ability to withstand dielectric breakdown . Maximal treatment intensity in HPP is also limited by equipment design, particularly when adiabatic heating is to be avoided. Irradiation is a batch process that is easily adapted to continuous applications. When irradiated, raw meats and seafood are commonly treated frozen or chilled . methods, compared to heat

3. Efficacy of alternative preservation Most alternative preservation processes achieve the equivalent of pasteurization, but not sterilization . For illustration purposes, results from different studies will be used to compare alternative preservation methods with a mild heat treatment . Substantial reductions in the population of Escherichia coli are possible using alternative technologies; these reductions are comparable to those achieved by heating at 63 C for 16 s. HPP requires a comparatively long treatment time. This process may be combined with heat and applied intermittently for elimination of spores . Treatment of food with PEF is a

4

rapid process for inactivating vegetative cells such as E. coli. The current status of PEF technology does not enable it to be used to inactivate bacterial spores . Gamma irradiation is effective against vegetative and sporulated bacteria . The greatest advantage of alternative processes, particularly HPP and irradiation, is their efficacy against microorganisms at ambient, chilling and freezing temperatures. Holding food at mild temperatures favors rapid multiplication of pathogens, spore germination and toxin production. It is safer, therefore, to apply alternative preservation processes at chilling ( C) or hot (45-60 C) temperatures than at ambient temperatures. 5 Inactivation of Saccharomyces cerevisiae was not observed between 0 and 25 C after 10 min at 180 MPa , probably because cell metabolism was active and cell damage (e.g., membrane non-selective permeability) was repaired quickly ; higher pressures were required to kill this psychrotrophic microorganism at this temperature range. The greatest inactivation was accomplished when S. cerevisiae was treated at 10, 20 and 48 C. Treating food with gamma irradiation and HPP at chilling or freezing temperature maintains heat-labile compounds, which contribute to the fresh attributes of food. Temperatures ranging from 45 to 70 C synergistically enhance the efficacy of HPP or PEF against resistant cells. For example, Bacillus spores are fairly resistant to pressures 200 MPa for several hours at 25 C, but not to a 600 Mpa oscillatory pressurization at 1 5070 C for 30 min . Heat treatment at 70 C would be insufficient to kill these spores without pressure.

4. Mechanisms of microbial inactivation

5

Microorganisms are inactivated when they are exposed to factors that substantially alter their cellular structure or physiological functions. Structural damage includes DNA strand breakage, cell membrane rupture or mechanical damage to cell envelope. Cell functions are altered when key enzymes are inactivated or membrane selectivity is disabled. A preservation technology, e.g. heat, may cause cell death through multiple mechanisms. Limited information is available about the mechanisms of inactivation of microorganisms by alternative preservation technologies. To speed up the implementation of these technologies, research on mechanisms of cell injury and inactivation is urgently needed. Microbial growth is retarded at pressures in the range of 20-180 MPa; these pressures also inhibit protein synthesis . Loss of cell viability begins at approximately 180 MPa, and the rate of inactivation increases exponentially as the pressure increases. Lethal highpressure treatments disrupt membrane integrity and denature proteins . The irreversible denaturation of proteins above 300 MPa corresponds to the range of pressure necessary for the inactivation of most vegetative cells and bacteriophages, i.e. protein-coated viruses. Lipid-coated viruses such as the Sindbis virus can resist up to 700 MPa . Membrane disruption is likely responsible for the changes in morphology observed in HPP-treated cells . The formation of pores in spore coats during treatment at 50-300 MPa may indicate that HPP induces spore germination. No germination was observed at higher pressures, likely because the environmental conditions were potentially lethal to germinating spores . Membrane structural or functional damage is generally accepted as the cause of cell death during exposure to high-voltage electric field. Zimmermann suggested that PEF temporarily increases the trans-membrane potential of cells by accumulating compounds of opposite charges in the membrane surroundings. High trans-membrane potential exerts pressure on the cell membrane; this pressure decreases membrane thickness and ultimately causes pore formation. On the other hand, Tsong introduced the poration theory to explain the mechanism of cell death by PEF. Electroporation in protein channels and lipid domains results in osmotic swelling of the cell and thus membrane weakening until the cell bursts. In both hypotheses, excessive pore formation causes irreversible loss of membrane functions. In addition, other researchers suggested different mechanisms for the biocidal action of electricity. Electric discharge in liquid media may generate small amounts of microbicidal agents such as chlorine, free radicals and H2O2 that alter the DNA and cytoplasmic activity during the treatment . Ionizing and UV radiations damage microbial DNA and to a lesser extent denature proteins . Potentially lethal DNA lesions are scattered randomly through the cell population during ionizing and UV radiations. Cells that are unable to repair their radiation-damaged DNA die. Sublethally injured cells are often subject to mutations. Ionizing radiations generate hydroxyl radicals from water, which remove hydrogen atoms from the sugar and the bases of the DNA strands. UV energy at 254 nm induces the formation of pyrimidine dimers; this distorts the DNAhelix and blocks cell replication. In addition, UV radiation cross-links aromatic amino acids at their carboncarbon double bonds. The resulting denaturation of proteins contributes to membrane depolarization and abnormal ionic flow . Irradiation with long-wave UV (320-400 nm) causes the formation of hydroperoxide radicals in the membranes unsaturated fatty acids, which induces changes in membrane permeability. Exposure of shell eggs to UV light (254 nm) at

6

4350 W/cm2 for 15 min reduced the aerobic microbial population by 3 log10 units .

5. Kinetics of microbial inactivation The most widely recognized theories explaining the death behavior of cell populations in response to exposure to lethal factors are the vitalistic and the mechanistic concepts . The former considers permanent phenotypic variations in the degree of resistance of individual cells within a population. A symmetric, but not normal, distribution of the sensitivity of the population to the treatment is assumed. High treatment intensity is therefore needed to kill the most resistant cells. In contrast, the mechanistic concept considers that each cell of a population has the same degree of resistance, but only a fraction of the population is affected by the lethal agent at a given time. When microorganisms are treated with heat, the logarithm of cell population normally decreases linearly with the treatment time for constant treatment intensity. The resulting straight logarithmic plots support the mechanistic concept. The vitalistic concept describes a logarithmic inactivation only when process intensity is high compared to the variations in resistance among the cells within the population. Alternative technologies were also believed to inactivate microorganisms logarithmically, as found for Staphylococcus aureus pressure-inactivation in milk . The decimal reduction time (D-value) corresponds to the treatment time required to reduce the microbial population by 90% at constant treatment intensity. The D-value is calculated from the following equation:

7

with X1 and X2 corresponding to the viable counts after treatment times t1 and t2, respectively. Survivor plots in alternative preservation technologies commonly exhibit a shoulder and/or a tail . The initial delay in inactivation of Listeria monocytogenes in milk and S. aureus in poultry meat during HPP for 5 and 10 min, respectively, are typical shoulders. The intensity of the deleterious agent (e.g. heat) must exceed a threshold value before microbial inactivation happens, which would explain the shoulder. The constant numbers of S. aureus in poultry meat and E. coli in a buffer solution during extended HPP treatment correspond to the tailing effect . The mechanistic concept considers the shouldering and the tailing effects as an artifact due to factors including sublethal injury, cell clumping, and heterogeneous treatment zones. The presence of low electric field regions during the PEF treatment of liquid food has not been clearly established . On the other hand, presence of resistant subpopulations explains biphasic curves in the vitalistic concept. A highly resistant sub-population remains viable over a long period of treatment time, causing a substantial decrease in the efficacy of the process observed at low cell concentration. From a practical point of view, the intensity of the treatment should be high enough to ascertain a virtual absence (i.e. undetectable levels) of targeted pathogens in ready-to-eat food with minimal impact on the products quality and production costs. Numerous models have been developed to fit sigmoid patterns of inactivation; these have been reviewed by Van Gerwen and Zwietering . Inactivation by HPP is initiated during the pressure come-up time, i.e. the time needed to raise the pressure to the targeted level. The pressure come-up time depends on the equipment and the headspace in the package . Lethality during come-up time should not be included in constructing survivors plots. Kinetics in HPP, therefore, is based on inactivation while the product is at the targeted pressure . Doseresponse models are derived from kinetic data to predict the efficacy of the preservation treatment . These models depict the relationship between treatment intensity and a population resistance parameter. Treatment intensity corresponds to the temperature, pressure, electric field intensity and radiation dose in the case of thermal, high pressure, PEF and radiation treatments, respectively. The D-value is a commonly used population resistance parameter. Heat treatments sufficient to pasteurize or sterilize food are traditionally predicted from thermal death time plots. In linear models, the z-value is an estimate of the treatment intensity necessary to decrease tenfold the D-value:

with I corresponding to the intensity of the preservation treatment. Doseresponse models describing the PEF treatments are based on sigmoid inactivation plots. The survival fraction during the PEF process (X1/X0) at a fixed electric field (E) and a treatment time can be predicted from Fermis equation

8

where k' is the slope of the steepest segment of the survival plot, and Ec, the critical electric field value for 50% survival. For industrial applications, however, both the treatment time (t) and the intensity of the electric field (E) should be considered. The Hulsheger empirical equation takes into account these two process parameters :

with tc, the time for inactivation threshold. Hulshegers model takes into account the minimum treatment time without loss of viability; therefore, the model describes well the inactivation plots with shoulders. Good fits have been found at high electric strength for both Fermis and Hulshegers models, probably because the shoulder is reduced at high treatment intensity . Models of Fermi and Hulsheger should be used cautiously, as resistance of microorganisms to PEF increases with cell concentration, which could be due to a shadowing effect, cell clusters or the presence of low electric field regions . To ascertain food safety, an adequate dose-response model must include a safety margin for cell heterogeneity and stress-adaptation mechanisms.

6. Microbial resistance to alternative processes Bacterial spores are generally the most resistant to inimical processes, followed by Gram-positive and Gramnegative bacteria . The higher resistance to HPP and PEF of Gram-positive, compared to Gram-negative, bacteria may be linked to the rigidity of the teichoic acids in the peptidoglycan layer of the Gram-positive cell wall. Bacteria of small size and coccoid in shape are generally more resistant to HPP than the large rod-

9

shaped ones . Reduced cell surface area in contact with the environment may limit cell leakage at a given treatment intensity, and thus minimize the effect of the treatment. Resistance to high pressure, however, was greater in S. cerevisiae than bacteria . This finding indicates that other factors than the shape and size of the cell are involved in cell resistance to alternative preservation treatments. The nature of the cell membrane influences cell resistance to preservation processes . High membrane fluidity increases the resistance to HPP and low temperatures. Membranes with relatively high fluidity are rich in unsaturated fatty acids. Pressureresistant cells also have low diphosphatidylglycerol content . A specific porin has been associated with increase in pressure resistance . The enhanced resistance of bacterial spores to physical stress, solvation and ionization is linked to the protective effect of the membranes and coat layers surrounding the core, the low-water activity in the spore core and the presence of dipicolinic acid . Microorganisms are more likely stressed or injured than killed in food processed by alternative preservation technologies. Adaptation of microorganisms to stress during processing constitutes a potential hazard. Sub-lethal stress induces the expression of cell repair systems . For instance, exposure of cells to UV induces enzymatic photorepair and expression of excision-repair genes that may restore DNAintegrity. E. coli that survives UV exposure has an activated htpR gene, i.e. a transcriptional regulator involved in multiple stress resistance. The chaperones GroEL and DnaK, which are under the control of htpR, were isolated post-irradiation . Strains surviving HPP have a similar type of enhanced gene-regulated resistance to both HPP and other stresses . Pressure-induced proteins are produced in a transitory manner, some of which are also induced by heat and cold shocks . The production of high levels of shock proteins is typical of gene-controlled resistance to stress and may contribute to the bacterial stress hardening effect, i.e. the expression of cross-protection mechanisms against multiple types of environmental stresses . Stress-adapted cells are particularly challenging to the food industry; they may survive processes combing several preservation factors (i.e. hurdle technology). In addition, repetitive exposure of bacterial contaminants to the preservation process can select for highly resistant mutants . Most alternative preservation technologies affect DNA structure and expression. Reversible DNA supercoiling protects cells against osmotic pressure . Slow replication under sub-optimal growth conditions increases the time allowed for cell repair activity and thus favors recovery of sub-lethally injured microorganisms . The highest sensitivity of cell populations is when they are in midexponential phase of their growth cycle. This applies to all preservation treatments. Transcriptional regulators such as rpoS are not expressed at this stage of bacterial growth. The growth region of yeast cells during budding was found particularly sensitive to PEF . The shift in gene expression and the higher sensitivity of the cell membrane during division therefore contribute to the susceptibility of actively growing cells.

10

7. Enhancing the efficacy Adaptation of pathogens to environmental and processing stresses constitutes a serious challenge to the food industry . Alternative preservation technologies should inactivate unusually resistant contaminants and prevent or minimize stress adaptation. Improved efficacy was reported with the addition of antimicrobial agents . Nisin has a synergistic effect with PEF treatment, and an additive effect with HPP treatment . Incorporation of a Nisin molecule through the membrane may be facilitated during HPP and PEF treatments, which may cause the formation of higher numbers of permanent pores. Ozone contributes to the breakdown of cells during mild PEF treatments . The addition of antimicrobial agents does not, however, eliminate the tailing effect exhibited by the resistant cell sub-population .

8. Measurement of cell survival An important issue in food safety is to ascertain that alternative preservation processes kill targeted microorganisms rather than merely cause sub-lethal injury or phenotype transition into a resistant dormant state. This minimizes the risk of cell resuscitation and expression of virulence during food storage or after food ingestion . Injured L. monocytogenes, Salmonella typhimurium and E. coli O157:H7 were detected

11

after HPP, when comparing viable counts on non-selective and selective media . The heterogeneity of the cell population after processing is likely due to variations in levels of injury, metabolic activity and viability among the cells. Vital staining with fluorescent dyes showed the presence of viable sub-populations that do not form colonies and are therefore not detected on agar media. It has not been demonstrated whether such viable but non-growing cells are the lineage of scarce injured cells. If this hypothesis were true, an extended lag phase would allow the restoration of the original membrane permeability and metabolic activity . Several methods are available to directly assess the extent and nature of cell injury within a microbial population. These include culturing on selective agar media and cell staining using epifluorescence techniques. Propidium iodide was used to detect cells that lost selective permeability after HPP . Surface hydrophobicity and ATPase activity may help identify the nature of the membrane damage. The combination of fluorescent dyes in flow cytometric analyses can provide information on different types of cell injury, simultaneously. For instance, ethidium bromide, bis-oxonol and propidium iodide can be used to quantify, in a single experiment, the populations that are reproductively viable, metabolically active and/or with intact polarized membrane . Staining with acridine orange differentiates viable from dead cells, based on their relative proportion of DNA and RNA . The combination of propidium iodide and SYTO 9 in the Live/Dead BacLightTM system gives a similar indication . These dyes are potentially useful in rapid detection of cell viability and injury during treatment with alternative processes. 9. Microorganisms for efficacy testing Effective food-preservation processes eliminate hazardous pathogens and decrease the loads of spoilage microorganisms. The canning industry targets Clostridium botulinum and uses heat treatment sufficient to eliminate 12-log of spores (i.e. a 12-D process). Safe pasteurized milk is virtually free of Mycobacterium sp. Challenge and validation studies using food-borne pathogens should not be carried out in the food-processing facility. Surrogate biological indicators have been proposed as alternatives to pathogens in these studies . Surrogate microorganisms should be slightly more resistant than the targeted pathogens, in order to conservatively estimate the level of pathogens remaining in the treated food. The rapid detection of low levels of pathogens or their corresponding nonpathogenic surrogates may be facilitated when the microorganisms are tagged with a selectable marker such as bioluminescence or resistance to antibiotics, provided these mutations are unlikely to be transmitted to other microorganisms in the factory . The Clostridium sporogenes spore is the conventional indicator to predict the heatinactivation of C. botulinum spores. Listeria innocua is a non-pathogenic strain that grows in environments similar to those suitable for L. monocytogenes and this surrogate is suitable for studying efficacy of milk pasteurization. Surrogate organisms for alternative preservation processes are yet to be clearly defined. 10. Conclusion HPP, PEF, ionizing radiations (gamma and electron beams) and UV light can inactivate food-borne microorganisms without substantially heating the food. These technologies are developed to produce safe food with high sensory and nutritional values.

12

The choice of a technique for industrial application depends on food properties and process design. High pressure and irradiation are the most frequently used alternative technologies, partly because of suitability for solid and liquid food applications. UV radiation and electron beams are limited to surface decontamination applications. PEF allows the processing of liquid foods rapidly and in a continuous fashion. Antimicrobial agents such as Nisin synergistically enhance the PEF treatment. The combination of HPP or PEF with heat (45-70 C) increased their efficacy and reduced bacterial spore populations. Alternative preservation technologies, however, may not be suitable for food sterilization. Thermal sterilization has remained nevertheless the simplest and most effective method for spore inactivation. Microbial lethality by HPP and PEF is mainly attributed to changes in the membrane structure and functionality. Nucleic acids are the primary target of ionizing radiations and UV light. Most kinetic studies on alternative technologies reveal a sigmoid inactivation pattern. The tailing effect and cell injury are some of the factors that should be elucidated for improving the efficacy of these new technologies. The role of cell structure, physiology, and gene regulation in microbial resistance to alternative preservation technologies should be investigated. Acknowledgements Funds for compiling this article were provided by the Center for Advanced Processing and Packaging Studies and the US National Science Foundation. References [1] R. Ahvenainen, New approaches in improving the shelf life of minimally processed fruit and vegetables, Trends Food Sci. Technol. 7 (1996) 179187. [2] M.R. Blake, B.C. Weimer, D.J. McMahon, P.A. Savello, Sensory and microbial quality of milk processed for extended shelf life by direct steam injection, J. Food Prot. 58 (1995) 10071013. [3] C. Lavigne, J.A. Zee, R.E. Simard, B. Bellveau, Effect of processing and storage conditions on the fate of vitamins B1, B2, and C and on the shelf-life of goats milk, J. Food Sci. 54 (1989) 3034. [4] J. Farkas, Irradiation as a method for decontaminating food, Int. J. Food Microbiol. 44 (1998) 189204. [5] D.G. Hoover, C. Metrick, A.M. Papineau, D.F. Farkas, D. Knorr, Biological effects of high hydrostatic pressure on food microorganisms, Food Technol. 43 (1989) 99107. [6] J.P.P.M. Smelt, Recent advances in the microbiology of high pressure processing, Trends Food Sci. Technol. 9 (1998) 152158. [7] B.L. Qin, U.R. Pothakamury, G.V. Barbosa-Canovas, B.G. Swanson, Nonthermal pasteurization of liquid foods using high-intensity pulsed electric fields, Crit. Rev. Food Sci. Nutr. 36 (1996) 603627. [8] F.L. Kuo, S.C. Ricke, J.B. Carey, Shell egg sanitation: UV radiation and egg rotation to effectively reduce populations of aerobes, yeasts, and molds, J. Food Prot. 60 (1997) 694697.

13

[9] T. Bintsis, E. Litopoulou-Tzanetaki, R.K. Robinson, Existing and potential applications of ultraviolet light in the food industry a critical review, J. Sci. Food Agric. 80 (2000) 637645. [10] R. Gervilla, V. Ferragut, B. Guamis, High pressure inactivation of microorganisms inoculated into ovine milk of different fat contents, J. Dairy Sci. 83 (2000) 674682. [11] R. Jeantet, F. Baron, F. Nau, M. Roignant, G. Brule, High intensity pulsed electric fields applied to egg white: effect on Salmonella enteritidis inactivation and protein denaturation, J. Food Prot. 62 (1999) 13811386. [12] Q. Zhang, G.V. Barbosa-Canovas, B.G. Swanson, Engineering aspects of pulsed electric field pasteurization, J. Food Eng. 25 (1995) 261281. B.H. Lado, A.E. Yousef / Microbes and Infection 4 (2002) 433440 439 [13] I. Hayakawa, T. Kanno, M. Tomita, Y. Fujio, Application of high pressure for spore inactivation and protein denaturation, J. Food Sci. 59 (1994) 159163. [14] T. Grahl, H. Maerkl, Killing of microorganisms by pulsed electric fields, Appl. Microbiol. Biotechnol. 45 (1996) 148157. [15] J.Y. DAoust, C.E. Park, R.A. Szabo, E.C. Todd, D.B. Emmons, R.C. McKellar, Thermal inactivation of Campylobacter species, Yersinia enterocolitica, and hemorrhagic Escherichia coli 0157:H7 in fluid milk, J. Dairy Sci. 71 (1988) 32303236. [16] B. Garin-Bastuji, B. Perrin, M.F. Thorel, J.L. Martel, Evaluation of -ray irradiation of cows colostrum for Brucella abortus, Escherichia coli K99, Salmonella dublin and Mycobacterium paratuberculosis decontamination, Letters Appl. Microbiol. 11 (1990) 163166. [17] R. Gervilla, X. Felipe, V. Ferragut, B. Guamis, Effect of high hydrostatic pressure on Escherichia coli and Pseudomonas fluorescens strains in ovine milk, J. Dairy Sci. 80 (1997) 22972303. [18] M. Von Konietzko, H. Reuter, Abttung von Bacillus stearothermophilus whrend der Ultrahocherhitzung von Vollmilch, Milchwissensch. 41 (1986) 222225. [19] A.E. Hashisaka, J.R. Matches, Y. Batters, F.P. Hungate, F.M. Dong, Effects of gamma irradiation at 78 C on microbial populations in dairy products, J. Food Sci. 55 (1990) 12841289. [20] M.F. Patterson, M. Quinn, R. Simpson, A. Gilmour, Sensitivity of vegetative pathogens to high hydrostatic pressure treatment in phosphate-buffered saline and foods, J. Food Prot. 58 (1995) 524529. [21] C. Hashizume, K. Kimura, R. Hayashi, Kinetic analysis of yeast inactivation by high pressure treatment at low temperatures, Bioscience 59 (1995) 14551458. [22] K.J.A. Hauben, D.H. Bartlett, C.C.F. Soontjens, K. Cornelis, E.Y. Wuytack, C.W. Michiels, Escherichia coli mutants resistant to inactivation by high hydrostatic pressure, Appl. Environ. Microbiol. 63 (1997) 945950. [23] K.J.A. Hauben, E.Y. Wuytack, C.C.F. Soontjens, C.W. Michiels, Highpressure transient sensitization of Escherichia coli to lysozyme and nisin by disruption of outer-membrane permeability, J. Food Prot. 59 (1996) 350355. [24] U. Zimmermann, Electrical breakdown, electropermeabilization and electrofusion, Rev. Physiol. Biochem. Pharmacol. 105 (1986) 175256. [25] T.Y. Tsong, Electroporation of cell membranes, Biophys. J. 60 (1991) 297306.

14

[26] H. Hulsheger, J. Potel, E.G. Niemann, Killing of bacteria with electric pulses of high field strength, Radiat. Environ. Biophys. 20 (1981) 5365. [27] L. Lucht, G. Blank, J. Borsa, Recovery of food-borne microorganisms from potentially lethal radiation damage, J. Food Prot. 61 (1998) 586590. [28] B.E.B. Moseley, in: D.E. Johnston, M.H. Stevenson (Eds.), Food Irradiation and the Chemist, Royal Society of chemistry, Bristol, 1990, pp. 97108. [29] O. Cerf, Tailing survival curves of bacterial spores, J. Appl Bacteriol. 42 (1977) 119. [30] T. Ohshima, K. Sato, H. Terauchi, M. Sato, Physical and chemical modifications of high-voltage pulse sterilization, J. Electrostat. 42 (1997) 159166. [31] S.J.C. Van Gerwen, M.H. Zwietering, Growth and inactivation models to be used in quantitative risk assessments, J. Food Prot. 61 (1998) 15411549. [32] B.A. Mertens, High pressure equipment for the food industry, High Pressure Res. 12 (1994) 227237. [33] G.V. Barbosa-Canovas, M.M. Gongora-Nieto, U.R. Pothakamury, B.G. Swanson (Eds.), Preservation of Foods with Pulsed Electric Fields, Academic Press, Washington, 1999. [34] G. Arroyo, P.D. Sanz, G. Prestamo, Response to high-pressure, low-temperature treatment in vegetables: determination of survival rates of microbial populations using flow cytometry and detection of peroxidase activity using confocal microscopy, J. Appl. Microbiol. 86 (1999) 544556. [35] N.J. Russell, R.I. Evans, P.F. Steeg, J. Hellemons, A. Verheul, T. Abee, Membranes as a target for stress adaptation, Int. J. Food Microbiol. 28 (1995) 255261. [36] W.J. Timson, A.J. Short, Resistance of microorganisms to hydrostatic pressure, Biotechnol. Bioeng. 7 (1965) 139159. [37] G.C. Walker, Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli, Microbiol. Rev. 48 (1984) 6093. [38] A. Benito, G. Ventoura, M. Casadei, T. Robinson, B. Mackey, Variation in resistance of natural isolates of Escherichia coli O157 to high hydrostatic pressure, mild heat, and other stresses, Appl. Environ. Microbiol. 65 (1999) 15641569. [39] T.J. Welch, A. Farewell, F.C. Neidhardt, D.H. Bartlett, Stress response of Escherichia coli to elevated hydrostatic pressure, J. Bacteriol. 175 (1993) 71707177. [40] Y. Lou, A.E. Yousef, Adaptation to sub-lethal environmental stresses protects Listeria monocytogenes against lethal preservation factors, Appl. Environ. Microbiol. 63 (1997) 12521255. [41] L. Lucht, G. Blank, J. Borsa, Recovery of Escherichia coli from potentially lethal radiation damage: characterization of a recovery phenomenon, J. Food Safety 17 (1997) 261271. [42] A.J. Castro, G.V. Barbosa-Canovas, B.G. Swanson, Microbial inactivation of foods by pulsed electric fields, J. Food Process. Preserv. 17 (1993) 4773. [43] I.E. Pol, H.C. Mastwijk, P.V. Bartels, E.J. Smid, Pulsed-electric field treatment enhances the bactericidal action of Nisin against Bacillus cereus, Appl. Environ. Microbiol. 66 (2000) 428430. [44] D. McDougald, S.A. Rice, D. Weichart, S. Kjelleberg, Nonculturability:

15

adaptation or debilitation? FEMS Microbiol. Ecol. 25 (1998) 19. [45] N. Kalchayanand, T. Sikes, C.P. Dunne, B. Ray, Hydrostatic pressure and electroporation have increased bactericidal efficacy in combination with bacteriocins, Appl. Environ. Microbiol. 60 (1994) 41744177. [46] G. Bogosian, P.J.L. Morris, J.P. ONeil, A mixed culture recovery method indicates that enteric bacteria do not enter the viable but non-culturable state, Appl. Environ. Microbiol. 64 (1998) 17361742. [47] T.V. Votyakova, A.S. Kaprelyants, D.B. Kell, Influence of viable cells on the resuscitation of dormant cells in Micrococcus luteus cultures held in an extended stationary phase: the population effect, Appl. Environ. Microbiol. 60 (1994) 32843291. [48] G. Nebe von Caron, P. Stephens, R.A. Badley, Assessment of bacterial viability status by flow cytometry and single cell sorting, J. Appl. Microbiol. 84 (1998) 988998. [49] L. Boulos, M. Prevost, B. Barbeau, J. Coallier, R. Desjardins, LIVE/DEAD BacLightt: application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water, J. Microbiol. Meth. 37 (1999) 7786. [50] P.M. Foegeding, N.W. Stanley, Listeria innocua transformed with an antibiotic resistance plasmid as a thermal-resistance indicator for Listeria monocytogenes, J. Food Prot. 54 (1991) 519523

Cuprins

16

Abstract.................................................................3 1.Introducere........................................................3 2.Tehnologiile de conservare a alimentelor...........3 3.Eficacitatea conservarii alternative.....................4 4.Mecanisme de inactivare microbiene..................5 5.Cinetica de inactivare microbiana.......................7 6. Rezisten microbian la procese alternative.....9 7. mbuntirea eficacitii.................... ............11 8. Msuri de supravietuire a celulei......................11 9. Microorganisme pentru testarea eficacitii......12 10.Concluzii.........................................................12 Mulumiri.............................................................13 Referinte....... ...................................................13

17

Abstract Prelucrare la presiune nalta , radiatiile ionizante, camp electric pulsatoriu si radiatiile ultraviolete sunt tehnologii de conservare in curs de dezvoltare concepute pentru a produce alimente sigure, pastrand in acelasi timp calitatile sale nutritionale si senzoriale. Un model de inactivare de tip sigmoid este observat in majoritatea studiilor cinetice. Distrugerea membranelor celulare, enzimelor sau ADN-ului sunt cauzele cele mai des citate in cazul distrugerii microorganismelor de alternativele tehnologiilor de conservare. 1.Introducere Consumatorii sunt tot mai constienti de beneficiile sanatatii si de riscurile asociate cu consumul de alimente. Pentru a face fata asteptarilor consumatorilor, industria alimentara dedica resurse considerabile si expertiza pentru productia de produse sanatoase si sigure. Productia de alimente sigure include examinarea materialelor care intra in lantul alimentar, suprimarea cresterii microbiene si reducerea sau eliminarea incarcaturii microbiene prin prelucrarea si prevenirea post-procesare contaminarii. Prezenta procesarii operatiilor unitare care vizeaza distrugerea microbiana este de o importanta primara sa verifice siguranta si stabilitatea produselor alimentare. Tratamentele termice sunt in mod traditional aplicate pentru a pasteuriza sau steriliza produsele alimentare, in general, in detrimentul calitatilor sale senzoriale si nutritionale. Consumatorii percep din ce in ce mai des mancarea proaspata ca fiind mai sanatoasa decat cea tratata termic, industria cautand acum tehnologii alternative pentru a mentine cele mai multe dintre atribute proaspete, sigure si stabile la depozitare. Evaluarea satisfacatoare a unei tehnologii noi de conservare depinde de estimarea fiabila a eficacitatii impotriva patogenelor si alterarea produselor alimentare suportate de microorganisme. Cercetarea tehnologiilor alternative a fost initial concentrata asupra procesului de proiectare, caracteristicilor produsului si inactivarii cineticii microbiene. Succesul acestor noi tehnologii, oricum, depinde de progresele inregistrate inregistrate in intelgerea fiziologiei microbiene si comportamentul de celule microbiene in timpul si dupa tratament. In consecinta acest articol revizuieste alternativa tehnologiei de conservare cu accent pe (i) comparand eficacitatea lor cu tratamentul termic conventional, (ii) mecanisme de inactivare microbiene, (iii) modele de inactivare cinetica, (iv) mecanisme de rezistenta microbiana si (v) cauze potentiale de subapreciere a supravietuitorilor in timpul prelucrarii produselor alimentare cu tehnologii alternative. 2. Tehnologii de conservare a alimentelor Pasteurizarea si sterilizarea termica sunt predominant utilizate in industria alimentara pentru eficacitatea lor si inregistrarea sigurantei produselor. Tratamentul cu caldura excesiva poate, oricum, provoca denaturarea proteinei nedorite, non-enzimatice si pierderea de vitamine si arome volatile compuse. Multi consumatori din SUA, de exmplu, considera aromele gatite si caramelizate ale laptelui sterilizat ca gust defect. Progresele tehnologice au permis optimizarea prelucrarii termice pentru eficacitate maxima impotriva contaminarii microbiene si deteriorarea minima a calitatii alimentelor. Temperaturi ridicate de scurta durata (HTST) de pasteurizare si temperatura ultra-inalta (UHT) de sterilizare, de

18

exemplu, minimizeaza pierderile de vitamina in lapte in comparatie cu lotul de pasteurizare si sterilizare conventionale in scop comercial. Produse prelucrate cu tehnologii termice moderne au inca lipsa unei arome si textura proaspata. Tehnologiile alternative non-termice au fost investigate intens in ultimii 30 de ani (tabelul 1). Aceste tehnologii sunt denumite in functie de parametrii prelucrarii principale care duce la inactivarea celulelor. Alimentele tratate si prelucrate la inalta presiune sunt expuse la o presiune hidrostatica (pana la 1000 MPa) pentru cateva minute. Tratamentul cu impulsuri de camp electric este bazate pe livrarea de impulsuri la o intensitate mare de camp electric (5-55 kV/cm) pentru cateva milisecunde. Radiatiile gamma si fasciculele de electroni pot genera doze intre 2-10 kGy si sunt de obicei denumite radiatii ionizante. Tehnologia fasciculului de electroni este in prezent dezvoltata ca o alternativa mai sigura a radiatiilor gamma de cand izotopii radioactivi nu ami sunt utilizati. Energia UV este o radiatie non-ionizanta cu proprietati germicid cu lungimi de unda cuprinse in intervalul 200-280 nm. Tehnologiile alternative sunt uneori descrise ca non-termice. Hrana este tratata la temperatura ambientala sau de refrigerare si caldura generata in timpul procesului nu este substantiala. HPP si radiatile gamma sunt mai potrivite decat alte tehnologii alternative pentru aplicarea in mancarurile solide. Agentul letal trebuie sa patrunda in aceste produse alimentare uniform fara a degrada textura lor. Proiectarea actuala a camerelor de tratament PEF nu permite prelucrarea de alimente solide. Efectul de ecranare a particulelor solide limiteaza cererile de radiatii UV si fasciculele de electroni pentru tratarea suprafetelor alimentare. Unele tehnologii alternative sunt in mod inerent procese de lot, in timp ce altele sunt adaptabile aplcatiilor continue. HPP a fost conceput in primul rand pentru a procesa mancarea ambalata, dar noi modele au fost create recent pentru procesare continua. PEF este un tratament rapid care este bine adaptat la procesarea continua. Intensitatea maxima a PEF este limitata de echipamente de proiectare si de capacitatea mancarii de a suporta defalcarea dielectrica. Intensitatea maxima a tratamentului este de asemenea limitata de designul echipamentelor, in special cand caldura adiabatica trebuie sa fie evitata. Iradierea este un proces lot care este usor de adptat aplcatiilor continue. Cand sunt iradiate, carnea si fructele de mare sunt tratate de obicei congelate sau refrigerate.

3.Eficacitatea metodelor de conservare alternative, in comparatie cu caldura Cele mai multe procese alternative ating echivalentul pasteurizarii dar nu si sterilizarii. In scopul de a ilustra, rezultate din diferitevstudii vor fi folosite pentru a

19

compara metode alternative de conservare cu tratament termic usor. (tabelul 2 si 3). Reduceri susbstantiale in populatia de Escherichia coli sunt posibile folosind tehnologii alternative; aceste reduceri sunt comparabile cu cele obtinute prin incalzire la 63 grade C timp de 16 secunde. HPP necesita un timp relativ lung de tratament. Acest proces poate fi combinat cu caldura si aplicat intermitent pentru distrugerea bacteriilor sporulate. Tratamentul alimentelor cu PEF este un proces rapid pentru inactivarea celulelor vegetative cum ar fi E. coli. Starea actuala a tehnologiei PEF nu ar putea fi utilizata pentru a inactiva bacteriile sporulate. Iradiatiile gamma sunt eficiente impotriva bacteriilor sporulate vegetative. Cel mai mare avantaj de procese alternative, in special HPP si iradiere , este eficacitatea lor impotriva microroganismelor din ambient, la temperaturi de refrigerare si de congelare. Exploatatia alimentelor la temperaturi blande favorizeaza multiplicarea rapida de agenti patogeni, germinarea sporilor si a producerii de toxine. Este mai in siguranta sa se aplice procesul de conservare alternativa la temperaturi reci (1200MPa pentru cateva ore la 25 grade C, dar nu si la presiune oscilatorie de 600 MPa la temperatura de 50-70 grade C timp de 30 minute. Tratamentul cu caldura la 70 de grade C ar putea fi insuficient pentru a ucide acesti spori fara presiune.

20

4. Mecanisme de inactivare microbiene Microorganismele sunt inactivate atunci cand acestea sunt expuse la factori care modifica substantial structura celulalra a lor sau functiile fiziologice. Distrugerile structurale includ ruperea tesuturilor ADN-ului, membranei celulare sau distrugerea mecanica a invelisului celular. Functiile celulei sunt modificate atunci cand enzimele sunt inactivate sau membrana selectiva este nefunctionala. O tehnologie de conservare, de exemplu caldura, poate provoca moartea celulelor prin mecanisme multiple. Informatii limitate sunt disponibile cu privire la mecanismele de inactivare a microorganismelor de tehnologiile alternative de conservare. Pentru a accelera punerea in aplicare a acestor tehnologii, este nevoie urgenta de o cercetare asupra inactivarii si asupra mecanismului celulei . Cresterea microbiana este intarziata la presiuni cuprinse in intervalul 20-180 MPa, aceste presiuni de asemenea inhiba si sinteza proteinelor. Pierderea viabilitatii celulare incepe de la aproximativ 180 MPa, iar rata de inactivare creste direct proportional cu presiunea. Tratamentele cu presiune inalta letala perturba integritatea membranei si denaturarea proteinei. Denaturarea ireversibila de proteine mai mare de 300 MPa corespunde la gama de presiune necesara pentru inactivarea mai multor celule vegetative si bacteriofagi adica proteinele filmate , virusurile lipidic filmate cum ar fi virusul Sindbis poate rezista pana la 700 MPa . intreruperea membranei este responsabila pentru modificarile ce sunt observate in morfologia HPP tratate cu celule. Formarea porilor in straturi de pori in timpul tratamentului la 50-300MPa poate indica faptul ca HPP duce la germinarea sporilor . Nici o germinare nu a fost observata la presiuni inalte probabil din cauza conditiilor din mediu potential letale pentru germinarea sporilor. Distrugerile din membrana structurala sau functionala sunt in general acceptate ca o cauza a distrugeri celulelor in timpul expunerii intr-un camp de inalta tensiune. Potentialul inalt al trans-membranei exercita o presiune asupra membranei celulare; aceasta presiune scazand grosimea membranei si in cele din urma cauzand formarea porilor. Pe de alta parte, Tsong a introdus teoria porilor care explica mecanismul moartii celulare de catre PEF. Electroportarea in canalele proteinelor si domeniul lipidelor obtinute in urma umflarii osmotice a celulei In ambele ipoteze, formarea excesiva a porilor cauzeaza pierderea

21

ireversibila a functiilor membranei. In plus alte cercetari au sugerat diferite mecanisme de actiune biodestructiva a energiei electrice. Descarcarea electrica in mediu lichid poate genera mici cantitati de substante active microbicide cum ar fi clorul, radicali liberi si H2O2 care altereaza ADN-ul si activitatea citoplasmatica in timpul tratamentului. Radiatiile ionizante si UV deterioreaza ADN-ul microbian si intr-o mai mica masura proteinele. Leziunile potential letale ale ADN-ului sunt imprastiate aleatoriu prin populatia de celule in timpul radiatiilot ionizante si UV. Celulele care nu isi pot repara ADN-ul, mor. Celulele lezate subletal sunt adesea obiectul unor mutatii. Radiatiile ionizante genereaza radicali de hidroxil din apa, care elimina atomii de hidrogen din zahar si bazele ADN-ului. Energia UV la 254 nm induce formarea dimeri piramidinici; aceasta denatureaza spirala ADN-ului si blocheaza raspunsul celulei. In plus radiatiile UV apropie legaturile aromatice ale aminoacizilor la legaturile duble de carbon-carbon. Denaturarea rezultata a proteinelor contribuie la depolarizarea membranei si la debitul anormal ionic. Iradierea cu unde lungi UV (320-400 nm) cauzeaza formarea radicalilor de hidroperoxid in membrana acizilor grasi nesaturati, care induce schimbari in permeabilitatea membranei. Expunerea oualelor la UV scazute (254 nm) la 4350 W/cm2 timp de 15 minute reduce populatia microbiana aeroba cu 3 log10 unitati.

5. Cinetica de inactivare microbiana Cele mai recunoscute teorii explica moartea populatiilor de celule ca raspuns la expunerea la factori letali care sunt concepte vitalice. Prima considera ca variatiile fenotipice permanente descresc cu gradulde rezistenta a unei populatii. Simetric, dar nu

22

normal, distributia sensibilitatii populatiei fata de tratament este asumata. Prin urmare intensitatea tratamentului e astfel necesara pentru a omora cele mai rezistente celule. In contrast conceptul de mecanisme considera ca fiecare ca fiecare celula a unei populatii are acelasi grad de rezistenta, ci doar o fractiune din populatie este afectata de catree agentul letal la un moment dat. atunci cand microorganismele sunt tratate cu caldura , in mod normal logaritmul populatiei de celule scade liniar cu timpul de tratament pentru tratament intensitate. Conceptul descrie o inactivare logaritmica numai atunci cand intensitatea procesului este mare in comparatie cu variatiile de rezistenta printre celule in randul populatiei. se crede ca tehnologiile alternative au fost folosite pentru inactivarea logaritmica la Staphylococcus aureus sub presiune inactivare in lapte. Reducerea zecimala a timpului corespunde cu timpul de tratament necesar pentru reducerea populatiei microbiene cu 90% la intensitatea constanta a tratamentului. Valoarea lui D este calculata de la urmatoarea ecuatie

Intarzierea initiala ininactivarea de Listeria monocytogenes in lapte si S. aureus in carne de pasare in timpul HPP pentru 5 si 10 min respectiv pentru umeri. Intensitatea agentului daunator (caldura) trebuie sa depaseasca un prag de valoare inainte de inactivarea microbiana reiese cea ce ar explica umarul. Numarul constant de S. aureus in carne de pasare si de E. coli intr-o solutie tampon in timpul tratamentului cu HPP corespunde cu efectul de decantare. Conceptul mecanicist considera umerii si efectele de decantare ca un artefact din cauza unor factori inclusiv prejudiciu subletal, agregarea celulei si tratamentul zonei eterogene. Prezenta sarcinilor electrice joase in timpul tratamentului PEF alimentelor lichide nu poate fi stabilita in mod clar. Pe de alta parte, prezenta de subpopulatii rezistente explic curbele bifazice n conceptul vitalist. O sub-populatie extrem de rezistenta ramane viabila peste o lunga perioada de timp de tratament, provocand o substantiala scadere n eficacitatea procesului observata la o concentratie scazuta de celule. Dintr-un punct de vedere practic, intensitatea tratamentului ar trebui sa fie suficient de mare pentru a stabili "o absenta virtuala" (niveluri nedetectabile, de exemplu) a agentilor patogeni vizati in alimente gata-pentru-consum cu impact minim asupra calitaii si costurilor produsului. Numeroase modele au fost dezvoltate pentru a se potrivi modelelor sigmoid de inactivare; acestea au fost revizuite de ctre Van Gerwen i Zwietering. Inactivarea prin CHE este initiata n timpul aparitiei presiunii, adic timpul necesar pentru a ridica presiunea de la nivelul tinta. Timpul aparitiei presiunii depinde de echipamentul folosit si de spatiul gol din pachet. Letalitatea in timpul aparitiei presiunii nu ar trebui inclusa in parcelele pe care se construieste. Cinetica in CHE, prin urmare, se bazeaza pe inactivare n timp ce produsul este la presiunea vizata. Modelele doza-raspuns sunt derivate din datele cinetice pentru a prezice eficacitatea tratamentului de conservare. Aceste modele depind de relatia dintre intensitatea tratamentului si parametrii populatiei rezistente. Intensitatea tratamentului corespunde cu tmperatura ,presiunea , intensitatea campului electric si dozele de radiatii in cazul termic , presiunea inalta, tratamentul cu PEF respectiv tratamentul cu radiatii. De obicei valoarea lui utilizeaza parametrii polulatiei rezistente. tratamentele cu caldura ,suficente pentru a pasteuriza sau

23

steriliza alimentele sunt traditional pezise de moarrtea parcelelor in timp. In model linear valoarea lui Z este o estimare a tratamentului necesar pentru a se reduce de zece ori valoarea lui D :

Modelele doza-raspuns care descriu tratamentele PEF sunt bazate pe loturi inactivate sigmoid. Fractiunea de supravietuire in timpul procesului PEF la un camp electric fixsi un timp de tratament poate fi anticipat din ecuatia lui Fermi.

In cazul in care k este panta abrupta a segmentului de supravietuire complot si Ec valoarea critica de camp electric de 50%de supravietuire Pentru aplicatii industriale cu toate acestea atat timpul de tratamentcat si intensitatea campului electric ar trebui sa fieluate in considerare. Ecuatia empirica Hulsheger ia in considerare acesti doi parametri de proces cu tc pragul timpului de inactivare. Modelul Hulsheger ia n considerare nivelul minim perioada de tratament, fr pierderi de viabilitate, de aceea, modelul descrie i parcelele de inactivare cu umerii

O buna potrivire a fost gasita la o rezistenta electrik mare pentru ambele din modelele lui Fermi si Hulshger, probabil din cauza ca umarul este redus la un tratament cu o intensitate mare. Modelele lui Fermi si Hulsheger ar trebui folosite cu precautie stfel ca rezistenta microorganismelor la PEF sa creasca cu concentratia celulelor , care ar putea fi datorat de umbrirea efectellor, grupurilor de celule sau de prezenta regiunilor de cam electric scazut. Pentru a aigura siguranta alimentara un model doza raspuns adecvat trebuie sa includa o marja de siguranta pentru celulele eterogenesi mecanismelor stres-adaptare.

24

6 Reazistenta microbiana la procese alternative Sporii bacterieni sunt in general cei mai rezistenti la procesul daunator, urmat de bacteria Gram-pozitiv si Gram-negativ. Cea mai mare rezistenta la HPP si PEF al Gramului pozitiv, comparat cu cel negativ, bacteria(Fig 4) poate fi atasata de rifiditatea acizilor. in invelisul peretelui celulei Gram-pozitiv. Bacteriile de marime mica si sunt in general mai rezistente la HPP decat cele largi in forma de ac (bat). Zona redusa a suprafetei celulei in contact cu mediul inconjurator, pot limita atenuarea celulei la o anume intensitate a tratamentului si asa se minimizeaza efectul tratamentului. Rezistenta la presiune mare , cu toate acestea, a fost mai mare decat bacteria. Aceasta descoperire indica faptul ca alti factori, in afara de forma si marime a celulei, sunt implicati in rezistenta celulei la tratamente alternative de conservare Natura membranei celulei influenteaza rezistenta celulei la procesul de conservare. Fluiditatea membranei mari favorizeaza rezistenta la HPP si la temperaturi mici.Membranele cu fluiditate relativ mare, sunt bogate in acizi grasi nesaturati. Celulele rezistente la comprimare au de asemenea continut slab. Un "por" specific a fost asociat cu cresterea in rezistenta la comprimare. Rezistenta amplificata a sporilor bacterieni la stresul fizic, solvatare si ionizare este cuplat de efectul protector al mebranelor si captuseala invelisului ce imprejmuieste nucleul, activitatea slaba a apei, in sporul nucleului si prezenta acidului. Microorganismele sunt mai degraba accentuate sau accidentate decat omorate in mancare procesata cu ajutorul tehnologiilor alternative de conservare. Adaptarea microorganismelor la constrangere in timpul procesului constituie un potential pericol. Constrangerea fatala cauzeaza expresia de sistem de reparare a celulelor. De exemplu, expunerea celulelor la UV induce reparatie [foto] enzimatica si expresia de 25

excizie-reparatie a genelor care pot reface integritatea ADN. Celulele care supravietuiesc expunerii au o gena activata a htpR, i.e.un compensator de transpunere implicat in multiple rezistente la constrangere. Insotitorii Groel si DNAK, care sunt sub controlul HTPR, au fost izolati de iradiere. Deformarile ce supravietuiesc HPP au caracter identic de rezistenta la ambele HPP si alte solicitari.. Proteinele cauzate prin comprimare sunt produse intr-un mod tranzitoriu, unele dintre ele fiind cauzate si prin socuri calde sau reci. Productia de nivel inalt al proteinelor este tipica rezistentei de gena controlata la constrangere si pot contribui la efectul greoi al constrangerii bacteriilor, expresia de mecanism de protectie incrucisata impotriva multiplelor tipuri de constrangeri cauzate de mediul inconjurator. Celulele adaptate la constrangere sunt in mod deosebit competitive in industria alimentara; pot supravietui proceselor combinand mai multi factori de conservare. Pe langa aceasta, dezvelirea repetitiva a impuritatilor bacteriene a procesului de conservare poate selecta Cele mai multe tehnologii alternative de conservare afecteaza structura ADN si exprimarea. ADN convertibil protejeaza celulele impotriva presiunii osmotice. Raspunsul incet sub conditii neoptime de crestere creste timpul permis pentru activitatea de reparare a celulelor si asa se favorizeaza recuperarea microorganismelor deteriorate in mod fatal. Cea mai mare precizie a populatiei celulelor este atunci cand sunt intr-o faza exponentiala a ciclului de crestere. Asta se aplica la toate tratamentele de conservare. compensatori de reproducere precum sporii nu sunt formulati in aceasta faza de crestere a bacteriei. Regiunea de crestere a celulelor de drojdie in timpul devenirii, s-a dovedit a fi, intr-un mod deosebit, sensibila la PEF. Metoda (Schimbul) in exprimarea genelor si marea sensibilitate a membranei celulare in timpul despartirii, contribuie asadar la sensibilitatea(susceptibilitatea) la cresterea activa a celulelor.

26

7. mbuntirea eficacitii Adaptarea agenilor patogeni de mediu constituie o provocare serioas pentru prelucrarea in industria produsele alimentare. Tehnologii alternative de conservare ar trebui s inactiveze contaminani neobinuit de rezistenti i sa previna sau minimizeze adaptarea la stres. mbuntirea eficacitii a fost raportat cu adaos de ageni antimicrobieni . Nisin are un efect sinergic la tratamentul PEF, i un efect aditiv tratamentului cu HPP. ncorporarea unei molecule Nisin prin membrana poate fi facilitat n timpul tratamentelor HPP i PEF, care pot provoca formarea de unui numr mare de pori permanent. Ozonul contribuie la distrugerea celulelor n timpul tratamentelor uoare cu PEF. Adugarea de ageni antimicrobieni, cu toate acestea,nu elimina efectul de decantare expus de ctre celula de rezistenta la sub-populaie.

8. Msurarea supravietuirii celulare Un aspect important n sigurana alimentar este de a constata c de procese alternative de conservare ucid microorganismele specifice, mai degrab dect pur i simplu

27

ranirea celulelor sau fenotipul de tranziie ntr-o stare latenta rezistenta. Acest lucru reduce riscul de resuscitare de celule i de exprimare a virulenei n timpul stocarii produselor alimentare sau dup ingerarea de alimente. L. monocytogenes vtmate, Salmonella typhimurium i E. coli O157: H7 au fost detectate dup CHE, atunci cand se compara constantele viabile privind media dintre non-selective i selective. Continutul eterogen al populaiei de celule dup prelucrare se datoreaz, probabil, variaiilor n nivelurile de lezare, activitatea metabolic i viabilitatea printre celule. Colorarea vitala cu culori fuorescente au artat prezena de viabile sub-populaii care nu fac colonii i nu sunt, prin urmare, detectate n mediu de agar-agar. Nu a fost demonstrat dac astfel de celule viabile, dar necrescande sunt cauza ranirii de celule. Dac aceast ipotez ar fi adevrat, o faz de laten prelungit ar permiterestabilirea permeabilitii membranei originale i activitatea metabolic. Cteva metode sunt disponibile pentru a evalua n mod direct amploarea i natura ranirii celulei din cadrul unei populaii microbiene. Acestea includ cultur pe medii agaragar selectiv i colorarea celulare care utilizeaz tehnici de epifluorescenta. Iodura de propidiu a fost utilizata pentru a detecta celulele care au pierdut permeabilitatea selectiv dup CHE. Hidrofob de suprafa i activitatea ATP-ului pot ajuta la identificarea daunei de natura a membranei. Combinaia de colorani fluorescenti n analizele de citometrie n flux pot furniza informaii cu privire la diferite tipuri de prejudiciu de celule, n acelai timp. De exemplu, bromur de etidiu, bis-oxonol i iodura de propidium poate fi folosit pentru a cuantifica, ntr-un experiment unic, care populaii sunt viabile reproductiv, metabolic active i / sau cu membrana polarizata intact. Colorarea in portocaliu acridin diferentiaza viabilitatea din celulele moarte, bazat pe proporia lor relativ de ADN i ARN. Amestecul de iodura de propidiu i SYTO 9 n sistemul BacLightTM Live / Dead ofera o indicatie similara. Aceste vopsele sunt potential utile n detectarea rapida a viabilitatii celulare si a prejudiciului n timpul tratamentului cu procese alternative. 9. Microorganisme pentru testarea eficacitatii Procesele de conservare eficiente elimina agentii patogeni periculosi si scade incarcatura de microorganisme de alterare. Industria de conserve urmareste Clostridium botulinum si foloseste un tratament termic suficient pentru a elimina 12-jurnal de spori (adic un proces '12-D "). Laptele pasteurizat n condiii de siguran este "practic liber" de Mycobacterium sp. Studiile de provocare i validare prin utilizarea de ageni patogeni care produc toxiinfecie alimentar nu ar trebui s fie efectuate n instalaia de prelucrare a alimentelor. Indicatori biologici surogat au fost propusi ca alternative la agenii patogeni n aceste studii. Microorganisme surogat ar trebui s fie puin mai rezistente dect agenii patogeni vizati, n scopul de a estima nivelul de ageni patogeni rmasi n produsele alimentare tratate. Detectarea rapid a nivelurilor sczute de ageni patogeni sau surogatele lor corespunztoare nepatogene poate fi facilitat n cazul n care microorganismele sunt etichetate cu un marcator de selecie, cum ar si bioluminescenta sau rezisten la antibiotice, cu condiia ca aceste mutatii sunt puin probabil s fie transmise altor microorganisme n fabric. Sporul Clostridium sporogenes este indicatorul convenional pentru a prezice inactivarea la temperatura a sporilor de C. botulinum. Listeria innocua este o tulpin non-patogena, care crete n mediisimilare cu cele potrivite pentru L. monocytogenes i acest surogat este potrivit pentru a studia eficacitatea de pasteurizare

28

lapte. Organismele surogat pentru procesele de conservare alternative urmeaza s fie clar definite. 10. Concluzii CHE, PEF, radiaii ionizante (gamma si fascicule de electroni) i lumina UV pot inactiva microorganismele de toxiinfecie alimentar , fr a incalzi n mod substanial produsele alimentare. Aceste tehnologii sunt dezvoltate pentru a produce alimente sigure cu valori mari senzoriale i nutriionale. Alegerea unei tehnici pentru aplicaii industriale depinde de proprietile produselor alimentare i a procesului de proiectare. De nalt presiune i de iradiere sunt tehnologiileutilizate cel mai frecvent subsidiar, n parte din cauza cererilor de adecvare pentru alimente solide si lichide. Radiaiile UV i fasciculele de electroni sunt limitate la aplicatiile de decontaminare ale suprafetelor. PEF permite prelucrarea alimentelor lichide rapid i ntr-un mod continuu. Agenii antimicrobieni, cum ar fi Nisin pot spori sinergic tratamentul PEF. Combinarea CHE sau PEF cu cldur (4570 C) a crescut eficacitatea lor si a redus populatiile bacteriene. Tehnologiile alternative de conservare, cu toate acestea, nu pot fi potrivite pentru sterilizare alimente. Sterilizarea termic a rmas totui cea mai simpl metod i cea mai eficient pentru inactivarea sporilor. Letalitatea microbiana de CHE si PEF este atribuit n principal de modificri n structura membranei i funcionalitatea. Acizii nucleici sunt principala int a radiaiilor ionizante si lumina UV. Cele mai multe studii cinetice pe tehnologii alternative sunt facute pentru a descoperi un model de inactivare sigmoid. Efectul de decantare i a prejudiciului de celule sunt unii dintre factorii care ar trebui s fie elucidat pentru mbuntirea eficacitii acestor noi tehnologii. Rolul structurii celulaea, fiziologie, i reglementarea n gena rezistenei microbiene latehnologii alternative de conservare ar trebui s fie investigate. Mulumiri Fondurile pentru elaborarea prezentului articol au fost furnizate de catre Centrul pentru Studii de Procesare Avansata i Ambalare i US National Science Foundation.

29