P. Chen, S. Deng, Y. Cheng, X. Lin, L. Metzger and R. Ruan,     University of Minnesota, USA

1.1 Introduction

Many consumers enjoy the robust, natural flavor and taste of unpasteurized/raw apple juice or cider. However, due to associated outbreaks of foodborne illnesses, unpasteurized fruit juice has become mostly a thing of the past. In 1998, FDA adopted a regulation that forced fresh juice processors to either pasteurize their products to inactivate 5 logs of pathogenic microorganisms or attach the label ‘WARNING: this product has not been pasteurized and, therefore, may contain harmful bacteria which can cause serious illness in children, the elderly and persons with weakened immune systems’ (FDA, 1998). In 2001, FDA adopted the ruling to implement the Hazard Analysis and Critical Control Point (HAACP) procedures for the Safe and Sanitary Processing and Importing of Juice, effective February, 2002 (FDA, 2001). Apple juice production and consumption in the United States has been in decline for many years, which puts tremendous pressure on the fruit juice industry to boost consumption, while ensuring safety and retaining freshness and nutrients. In order to retain full flavor of their products, some companies have adopted a tight sanitation and HACCP program to achieve a 5-log reduction in production of unpasteurized apple juice/cider. A few producers even accept the warning label on some products, and others have combined ‘light’ or ‘ultralight’ pasteurization with HACCP, thus minimizing the decrement of flavor. However, most of the companies prefer to choose pasteurization to assure the safety of their products.

Methods of pasteurization have changed from conventional treatments used in the past. Until recently, thermal processes, especially ultra high temperature (UHT) and high temperature short time (HTST) have been the most commonly used methods in the food industry to increase shelf-life and maintain food safety. However, studies have shown that heat degrades product color, flavor, and nutrients because of protein denaturation and the loss of vitamins and volatile flavors (Processors, 1998). Therefore, there is increasing demand for alternative methods for fresh food pasteurization that ensure safety while decreasing product degradation.

Non-thermal methods provide such an option because they reduce overprocessing to result in more fresh-like foods featuring greater retention of color, flavor, and nutrients. Currently, there are several methods having the ‘nonthermal’ claim for liquid food product pasteurization: (1) pulse electric field (PEF), (2) high hydrostatic pressure (HHP), (3) irradiation, and (4) UV light. Two emerging processes; namely, cold or non-thermal plasma (NTP), and concentrated high intensity electric field (CHIEF), are under development. In this chapter, we will provide a brief description of each of their mechanisms of microbial inactivation, technological characteristics, and current application status of these processes. Some of these alternative processes have been studied extensively for at least two decades, but none of these alternative processes is in large-scale commercial practice for fruit juice and milk pasteurization due to technical issues or, more often, economic disadvantages. The high resistance of enzymes and bacterial spores to these processes is a major problem. Efforts are needed to improve these processes or develop new processes. It is also suggested that combinations of these processes and other methods, which are termed ‘hurdle technology’, may present potential benefits and practical uses of these processes.

1.2 Pulsed electric field

High intensity pulsed electric field (PEF) processing (Fig. 1.1) involves the application of short pulse (1–10 μs) of high voltage (typically 20–80 kV/cm) to food materials located between two metal (usually stainless steel) electrodes (Qin et al., 1996; Vega-Mercado et al., 1997). Studies of exposure of microorganisms to electric fields have indicated that electric field can cause changes to cell membranes (Pothakamury et al., 1997; Barbosa-Cánovas et al., 1999). When a voltage is applied to a cell, a sufficiently high transmembrane potential is induced across the cell membrane, causing the membrane to rupture (direct mechanical damage, the electric breakdown theory), or destabilizing the lipid and proteins layers of cell membranes, resulting in pores (electroporation theory). The damaged cell membrane loses its selective semi-permeability, which allows water to enter the cell, and results in excessive cell volume swelling, and ultimately leads to cell rupture and inactivation of the organism. Some studies have provided microscopic evidence to support this theory (Harrison et al., 1997; Calderón-Miranda et al., 1999). Recent studies showed increased membrane permeability after PEF treatment (Aronsson et al., 2005; García et al., 2007).

PEF has been used to process fruit juices (Jin and Zhang, 1999), dairy products (Reina et al., 1998), and eggs (Dunn, 1996). Research found that apple juice processed with PEF at 50 kV/cm, 10 pulses, pulse width of 2 μs, and initial temperature controlled at 45 °C had a shelf-life of 28 d compared to a shelf-life of 21 d for untreated, fresh-squeezed apple juice. PEF processed apple juice showed no physical or chemical changes in ascorbic acid or sugar contents. PEF also demonstrated advantages over heat pasteurization for orange juice in terms of vitamin C, flavor, and color retention without inducing sedimentation like thermal treatments (Yeom et al., 2000). A majority of studies involving PEF focused on its effect on milk and dairy products due to the importance of the dairy industry. Model aqueous suspensions similar to milk ultrafiltrate, pasteurized milk, and raw milk have been used in those studies. Different levels of microbial inactivation were achieved with PEF treatment depending on the type of samples, type of microbe, the field strength, and the number of pulses applied during the process (Martin et al., 1997; Pothakamury et al., 1997; Bai-Lin et al., 1998; Qin et al., 1998). The inactivation of enzymes by PEF is limited, although the effect of PEF on enzymes has been shown to vary with the electric field intensity, the number of pulses applied during the process, and the intrinsic characteristics of the enzyme (Bendicho et al., 2003; Kambiz et al., 2008).

There are a limited number of studies on the effect of PEF on the nutrients and sensory quality of milk. Bendicho et al. (2002) found that PEF-treated milk showed no changes in the contents of most vitamins, except for ascorbic acid (Vitamin C), which reduced slightly. Grahl and Märkl (1996) reported that the ascorbic acid content of milk was reduced considerably (90%, data not shown) by PEF treatment, whereas the content of vitamin A and the flavor showed no significant changes. Zulueta et al. (2007) reported that high intensity PEF treatment slightly changed the amounts of total fat, saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids contained in an orange juice-milk beverage fortified with n-3 fatty acids and oleic acid; however, these changes were not considered significant. They concluded that changes in molecular composition of the orange juice-milk beverage were negligible from a nutritional viewpoint (Zulueta et al., 2007). Other research found that PEF has little influence on the physical, chemical and sensory properties of milk (Qin et al., 1995).

Most of the recent studies on PEF tend to adopt an approach combining multiple factors such as the addition of heat (Craven et al., 2008; Noci et al., 2009; Riener et al., 2008; Wouters et al., 1999; Yu et al., 2009), antimicrobial compounds (Sobrino-Lopez and Martin-Belloso, 2008; Sobrino-Lopez et al., 2009), and thermosonication (Noci et al., 2009). Positive synergistic effects on bacteria and enzyme inactivation were demonstrated. Other research has also found PEF useful in facilitating the extraction of juice and other compounds from plant tissues (Bazhal et al., 2001; Fincan et al., 2004; Knorr and Angersbach, 1998; El-Belghiti and Vorobiev, 2005; Schilling et al., 2007).

According to a fact sheet posted on The Ohio State University (OSU) Extension website (Ramaswamy et al., 2005), the first commercial scale continuous PEF system is located in OSU’s Department of Food Science and Technology. Diversified Technologies Inc. (Bedford, MA) manufactures high voltage, high power pulse modulators, DC power supplies and control systems, builds commercial PEF systems with the PEF treatment chambers supplied by the Ohio State University. However, commercial applications of PEF in food pasteurization have been limited so far.

There are a number of drawbacks in the application of PEF technology to foods. First, ohmic (electro-resistive) heating occurs during the PEF discharge, which causes the temperature of the sample to rise, and hence a cooling system has to be in place in order to maintain as closely as possible the initial, lower temperature of liquid samples. Therefore, a significant amount of energy is dissipated by the unwanted heating up and necessary cooling of the liquids. Second, since the electrodes have to be immersed in the liquid, they contribute a major source of contamination to the liquid food due to the erosion of the electrodes that occurs during discharge. Finally, the initial equipment investment is very capital-intensive and presents a major obstacle for the commercial application of PEF technology.

1.3 High hydrostatic pressure

High hydrostatic pressure (HHP) refers to the application of hydrostatic pressure ranging from 100 to over 800 MPa to foods for the purpose of inactivating spoilage and pathogenic microorganisms (Sangronis et al., 1997; Spilimbergo et al., 2002; Smelt, 1998). Liquid or solid food, with or without packaging, is placed in the pressure vessel (Fig. 1.2). The closed pressure vessel is filled with a pressure transmitting fluid, which is usually water or a dilute aqueous solution. The liquid is compressed usually by a pump or pressure intensifier, and the hydrostatic pressure distributes uniformly throughout the pressure vessel and equally in all directions of the food surfaces. Treatment times, once constant high pressure levels are achieved, can range from a millisecond pulse to over 20 min, and initial treatment temperatures can range from 0 to 90 °C. HHP generally produces better results for the pasteurization of foods when combined with initial temperatures around 45–50 °C.

A number of mechanisms of microbial inactivation by HHP have been proposed. HHP is believed to cause pressure sensitive non-covalent bonds (hydrogen, ionic, and hydrophobic bonds) to break. Since non-covalent bonds are present chiefly in large molecules such as proteins, polysaccharides, lipids, and nucleic acids, the breakdown of non-covalent bonds will lead to significant damage to enzymes, membranes, and genetic molecules of microbes, therefore inhibiting the metabolism, growth, and reproduction of microorganisms. The disruption of membranes and inactivation of enzymes, including those responsible for DNA replication and transcription, are believed to be the main mechanisms of pressure-induced microbial injury and death (Hoover et al., 1989).

HHP is used widely for the pasteurization of post-processing refrigerated salads and entrées, avocado fruit, apple sauce, ham, and oysters. HHP also appears to be a very attractive method for pasteurization of fruit juices and milk products. In fact, HHP processed fruit juices are commercially available in Japan. Some researchers reported 5–7 log reductions of bacteria in apple and other fruit juices (Jordan et al., 2001). Linton et al. (2001) used HHP (200–700 MPa for 15 min at 20 °C) to process UHT skim milk inoculated with different strains of E. coli. The strains varied in their sensitivity to HHP. The least sensitive strains could be completely inhibited at 600 MPa for 30 min. Gao et al. (2006) demonstrated 6-log cycle reduction of L. monocytogenes in milk at 448 MPa, 41 °C, and 11 min. In addition to inactivating foodborne microorganisms to prevent spoilage or to ensure food safety, there is also interest in understanding the effects of HHP on proteins in milk. Johnston et al. (1992) studied the extent of conformational and other changes in skim milk proteins caused by the application of HHP at pressures less than 600 MPa. They found that pH and Ca^2 + ion activity were unaffected, but the lightness of the color (L*) of milk decreased by HHP treatments less than 300 MPa. HHP treatments caused interior hydrophobic groups to become exposed, indicating irreversible unfolding of proteins. There are also studies to demonstrate the combined effects of HHP, mild heat treatment, and antimicrobial compounds (Patterson and Kilpatrick, 1998; Garcia-Graells et al., 1999; Haiqiang and Hoover, 2003; Gao et al., 2006; Bilbao-Sainz et al., 2009). HHP is less efficient for inactivating bacterial spores in low acid foods, requiring relatively higher initial temperatures ( ~90 °C) to achieve sterilization temperatures and inactivate resistant endospores during processing. While HHP preserves the sensory quality and nutritional value of liquid foods to create a significant advantage for commercial products, equipment costs are capital-intensive and are only available as batch processes, making HHP an unlikely alternative to conventional pasteurization methods for low value foods for the near future.

1.4 Ionizing irradiation

Food irradiation involves the use of ionizing radiations to inactivate spoilage and pathogenic microorganisms, control insects and parasites, and inhibit post-harvest ripening and sprouting (Sendra et al., 1996; Zehnder, 1988; Burditt, 1982). Radiation sources may include radioactive isotopes of cobalt or cesium or beta rays or x-rays from electron accelerators. During the radiation processing of foods, ionizing radiation may directly damage sensitive macromolecules, such as DNA, making the organisms unable to replicate or reproduce. Irradiation may also generate free radicals which react with molecular constituents in cells, and cause irreversible lethal damage to the cells.

The effectiveness of food irradiation is dosage dependent. Insects and parasites may be killed at low dosages under 0.1 kiloGray (kGy). Medium doses between 1.5 and 4.5 kGy are needed to inactivate most bacterial pathogens. Inactivating bacterial spores and viruses requires doses higher than 10–45 kGy.

The first commercial application of irradiation in food preservation took place in 1957 in Germany, when a spice manufacturer irradiated their products with electrons to improve the hygienic quality. Forty-two countries in the world currently have approved the use of ionizing irradiation on more than 100 foods. In the US, irradiation of some fruits and vegetables, poultry, beef, pork, and lamb has been approved by the FDA. Irradiation of papayas to kill fruit flies in Hawaii is a successful example of the use of irradiation to control the spread of insects via agricultural exports. In the US, irradiation of meat and meat products requires prior approval not only by the FDA, but also the USDA’s Food Safety and Inspection Service. Protein rich foods such as milk are not suited for pasteurization by irradiation because irradiation may induce off-flavor, odor, and discoloration. There were earlier studies on the irradiation of liquid milk to extend shelf-life. Naguib et al. (1974) showed that three species of Brucella (Br. abortus, Br. melitensis and Br. Suis) in skim-milk (approximately 10⁹ organisms/mL) were completely destroyed by exposure to gamma irradiation at dosages greater than 200 Krad. An early study by Scanlan and Lindsay (1967) found the irradiation of liquid milk with a dose of 4.5 Mrad promoted extreme browning and caramelization. When the milk was irradiated in the frozen state at –80 and –185 °C an extremely bitter flavor resulted. All of the irradiated milk samples were regarded as unacceptable by flavor assessment. Fractionating of the irradiated milk separated the bitter flavor and suggested that the bitter component was a protein or a non-dialyzable protein fragment. A more recent study by Naghmoush et al. (1983) showed more favorable results. These researchers treated raw milk from cows, buffaloes or goats with 0.25–0.75 Mrad of gamma-irradiation. The treated samples showed noticeable bacterial count and spore count reduction compared with untreated controls. However, the irradiation treatment decreased the nutritional content (specifically, carotene and vitamin A) and flavor of all three types of milk, and samples became progressively more oxidized with increasing radiation dose; the initially yellowish cows’ milk became progressively whiter.

Although irradiation has gained substantial media attention and has been approved for use on a broad range of foods, consumers still worry about the safety of irradiated food products, and the acceptance of irradiated foods grows, albeit very slowly. The pace of growth varies by country, as different countries have different prevailing consumer attitudes, regulations, and enforcement. Further research on issues such as the radiation resistance of different microorganisms and strains, irradiation-induced chemical reactions in foods, the influence of irradiation on sensory and nutritional losses, the use of irradiation in combination with other hurdle techniques, the interaction with packaging materials, and consumer education and safety awareness will continue to guide the development of food irradiation.

1.5 Ultraviolet radiation

Ultraviolet (UV) processing uses radiation from the UV region of the electromagnetic spectrum to inactivate microorganisms in foods, water, and packaging materials (Koutchma, 2009). The typical wavelength for UV processing ranges from 100 to 400 nm. UV in the 200–280 nm region is believed to be most effective in inactivating bacteria and viruses. During UV irradiation, DNA molecules absorb UV light, which causes crosslinking between neighboring pyrimidine nucleoside bases in the same DNA strand, and this mutation in the DNA base-pairing results in hindered growth and reproduction (Miller et al., 1999).

To inactivate microorganisms effectively, a minimum of 400 J/m² energy in all parts of the product being irradiated must be obtained through UV treatment. The effectiveness of a UV process is a function of ‘the transmissivity of the product, the geometric configuration of the reactor, the power, wavelength and physical arrangement of the UV source(s), the product flow profile, and the radiation path length’ (FDA, 2009).

UV light lacks penetration capability, and therefore lends itself best to surface treatments. The treatment is more efficient for liquid foods that are pre-filtered or clarified. To enhance the lethality of UV treatments for inactivating microorganisms, the UV may be used in combination with other alternative processing technologies, including strong chemical oxidizing agents such as ozone and hydrogen peroxide.

There is considerable interest in using UV irradiation for the pasteurization of milk (Munkacsi and Elhami, 1976; Caserio et al., 1978; Bodurov et al., 1979; Filipov, 1979, 1981; Giraffa and Carini, 1984; Ibarz et al., 1986; Yu et al., 1999; Smith et al., 2002; Chernyh and Yurova, 2006; Matak et al., 2007). Yu et al. (1999) studied the effects of UV radiation time, distance from the UV source, thickness of the treated milk sample during processing, and temperature. They reported that there was a critical thickness of the liquid milk sample, which is apparently limited by the penetrability of UV light. Their study also showed that varying temperature in the range of 0–37 °C did not significantly influence the pasteurization process. Smith et al. (2002) reported that samples from dairy bulk tanks of milk showed no bacterial growth after the samples were exposed to UV light (248 nm) at a dose of 12.6 J/cm². There are some negative effects of UV radiation on milk quality, such as the deterioration of flavor due to an increase in thiobarbituric acid reactive substances and acid degree values which are related to chemical oxidation and hydrolytic rancidity (Matak et al., 2007), delayed rennet coagulation of milk and the development of acidity, and the induction of a slight cooked flavor (Munkacsi and Elhami, 1976). Although there is interest in potentially applying UV radiation to control microbes in liquid milk (Smith et al., 2002), and notwithstanding the fact that FDA has approved the use UV radiation for the treatment of water and food under specific conditions (CFR, 2005), the commercial application of UV radiation for food pasteurization is presently unavailable.

1.6 Non-thermal plasma

Non-thermal plasma (NTP) is electrically energized matter in a gaseous state, and can be generated by passing gases through electric fields (Conrads and Schmidt, 2000). The mean electron energies of NTP, which is about 20 eV, are considerably higher than those of the components of the ambient gas. During NTP generation, the majority of the electrical energy goes into the production of energetic matters rather than into gas heating. The energy in NTP is thus directed preferentially to the electron-impact dissociation and ionization of the background gas to produce NTP species including electrically neutral gas molecules, charged particles in the form of positive ions, negative ions, free radicals and electrons, and quanta of electromagnetic radiation (photons) (Ulrich, 2007). Theses species are very strong oxidizers that can rapidly decompose other inorganic and organic compounds.

Plasma may kill both vegetative cells and bacterial endospores. The killing mechanisms of NTP are not well established; however, there are some hypotheses. It is well documented that reactive oxygen species (ROS) such as oxygen radicals can produce profound effects on cells by reacting with various macromolecules (Kelly-Wintenberg et al., 1999; Mounir, 2005). Among the cellular macromolecules altered are membrane lipids (Montie et al., 2000), which exhibit sensitivity probably because of their location at the cell surface and their susceptibility to oxidation by ROS. Altering the cytoplasmic membrane lipids results in a release of intracellular substances and the death of cells. Philip et al. (2002) proposed three basic mechanisms, individually or synergistically, responsible for the inactivation of microbial spores in plasma environments. These mechanisms include: (1) destruction of DNA by UV irradiation, (2) volatilization of compounds from the spore surface by UV photons and (3) erosion, or so-called ‘etching,’ of the spore surface by adsorption of reactive species like free radicals.

NTP has been used mostly for water and wastewater treatment, surface sterilization and environmental control (Montie et al., 2000; Mounir, 2005; Ma et al., 2001a, 2001b, 2001c; Ruan and Chen, 2000; Ruan et al., 1999a, 1999b, 2000; Ashikov et al., 2008). There is increasing interest in using NTP to inactivate vegetative foodborne pathogens on different surfaces, such as thin films of agar (Kayes et al., 2007), heat sensitive polyethylene terephthalate (PET) foils (Muranyi et al., 2007), polycarbonate membranes (Yu et al., 2006), culture media (Ashikov et al., 2008), and almond (Deng et al., 2007). Log-reductions ranging from 1 to 7 were reported for vegetative pathogens in these studies. There are relatively few reports on the use of NTP for the pasteurization of liquids (Anpilov et al., 2002; Ashikov et al., 2008).

Ruan and co-workers began their research on the use of NTP for liquid food pasteurization in the early 2000s (Ruan et al., 2003a, 2003b, 2003c; Montenegro et al., 2002). The dielectric barrier discharge NTP systems they developed (Fig. 1.3) were able to produce up to 6-log reductions of E. coli 25922 in water and juice. These systems use an AC power supply and are cheap to construct. They also designed reactors that incorporate a bubbling mechanism to enhance NTP discharges for treating liquids (Fig. 1.4), and they devised a system for processing solid foods (Figs 1.5 and 1.6). Even with these successes, the use of these systems for milk pasteurization encountered difficulties associated chiefly with the poor penetration of NTP species. These processing systems eventually evolved to become concentrated high intensity electric field systems, as described in the following section.

1.7 Concentrated high intensity electric field

Concentrated high intensity electric field (CHIEF) is a new process developed by researchers at the University of Minnesota (Ruan et al., 2008). The process uses a unique treatment chamber (orifice) and electrode configuration where a high intensity electric field is concentrated within the orifice through which liquid is pasteurized. CHIEF bears characteristics similar to those of a dielectric barrier NTP system, which consists of two electrodes separated by two layers of dielectric materials and driven by AC power. However, the mechanisms of microbial inactivation by CHIEF resemble more closely those of PEF than of NTP. In comparison with PEF technology, CHIEF has some unique characteristics:

Our recent studies have demonstrated a 7-log reduction of E. coli 0157 inoculated in orange juice and a 5-log reduction of E. coli 25922 inoculated in milk. In these processes, the temperature rise is minimal (from 16 to 50 °C), and there was no significant physical and chemical changes observed. We consider the CHIEF process to be one of the most promising, and perhaps the leading technology for the non-thermal pasteurization of fresh milk.

1.8 Conclusions

Six non-thermal food processes of industrial and academic significance were reviewed. Most of these processes are still under development. Among these processes, ionizing irradiation is the most mature technology. Further efforts to address some technical issues and to increase consumer acceptance through education and government safety regulations are expected to increase the commercial use of food irradiation. Some commercial PEF and HHP systems are available, but the initial capital investments are costly, and sometimes prohibitively costly, limiting the technologies to high value products. UV is limited by its low penetration capacity. No commercial UV system is available for food processing. NTP and CHIEF are emerging processes with great potential. Substantial research is needed to understand the processes and their limitations, to develop cost effective processing procedures and equipment, and to collect scientific data on a broad spectrum of microbes, enzymes and food systems.

1.9 References