**4.2.2 High pressure carbon dioxide (HPCD)**

High pressure carbon dioxide (HPCD) is another upcoming treatment that is being extensively used as a non-thermal technique for food pasteurization. The process is not only environmentally friendly due to the non-toxic nature of carbon dioxide but also involves application of lower CO2 pressure as compared to those employed for HPP. The use of lower pressures makes this technique an energy-saving process. The major factor involved in the destruction is CO2 although pressure helps in greater penetration of CO2 in the cells. Lethality imparted by pressurized CO2 is a result of disassociation of CO2 (in foods with high water content) into reactive ions such as carbonates (CO3 2-), bicarbonates (HCO3 -) and hydrogen (H+). These reactive ionic species can then have an effect on the permeability of the cell membrane and properties of cell constituents. In addition, generation of carbonic acid (H2CO3) in the water present in food products further results in a reduction in the pH of the food products enhancing the penetration of CO2 (Wei et al., 1991).

Studies involving the use of HPCD for the inactivation of *S.* Typhimurium (Kim et al., 2007; Erkmen and Karaman, 2001; Erkmen 2000; Wei et al., 1991) have clearly reported the microbial strain, pressure applied, pH of the medium, type of medium and temperature to be important factors for the inactivation. *S.* Typhimurium in orange juice was effectively reduced by 5-6 logs when subjected to continuous dense phase carbon dioxide (DPCD) for 10 min at 21–107 MPa and 25 °C (Kincal et al., 2005) whereas in another study reduction as high as 8 logs was achieved when the growth media was changed to physiological saline (PS) or phosphate buffer solution (Kim et al., 2007). Kim et al. (2007) also analyzed the structural changes in *S.* Typhimurium cells upon the application of super-critical CO2. A

Recent Advances in the Application

**4.3 Pulsed electric field (PEF)** 

the range of 3-4 logs.

**4.4 Natural antimicrobials** 

**4.4.1 Extracts from vegetables** 

of Non Thermal Methods for the Prevention of *Salmonella* in Foods 295

Pulsed electric field (PEF) is another non-thermal technology that can be used to inactivate bacterial cells at ambient temperatures. The process involves placing the food material between two electrodes and passing pulses of high electric field (1-50 kV/cm) strengths. Since the pulses are applied for short durations (2μs to 1 ms) the negative impact on food quality due to heat processing is highly diminished (Barbosa-Cánovas et al., 2001). The technique is more suitable for liquid or semi-liquid foods which can be easily pumped. It can be used to increase the shelf life of soups, milk, whole liquid eggs and fruit juices. PEF as a non-thermal preservation method has been implemented by Genesis Juices, Oregon, USA. The application of electric field results in cellular death due to generation of pores (electroporation) in the bacterial cell membrane without having an effect on enzymes or proteins present in foods (Wouters et al., 2001). The effectiveness of the technique will strongly depend on the treatment time, electric field strength and specific energy of the pulses. For instance, Monfort et al., (2010) achieved an inactivation of 4 log for *Salmonella* Typhimurium when 45 kV/cm of electric field was applied for 30 μs. Higher number of pulses and electric field was reported to be a stronger factor for reducing the number of *S.*  Typhimurium population in orange juice (Liang et al., 2002) whereas in another study on melon and water melon juices, treatment time was found be a more important factor (Mosqueda-Melgar et al., 2007). Treatment of watermelon and melon juice with PEF resulted in a reduction of 4.27 log (at 2000 μs and 100 Hz) and 3.75 log (at 1250 μs and 175 Hz) of *S.* Enteritidis, respectively (Mosqueda-Melgar et al., 2007). In contrast, Liang et al. (2002) reported a 5 log reduction of *S.* Typhimurium in orange juice exposed to a PEF of 90 kV/cm at a temperature of 55 °C. However, the higher reduction could be a result of combination of higher acidity of orange juice in addition to relatively higher temperature and high intensity of the PEF applied. Although the technique is useful, inactivation has only been achieved in

Since ancient times, spices and herbs have been used for preventing food spoilage and deterioration, and also for extending food shelf life. The antimicrobial effect of these components is a result of an increase in the permeability of the cytoplasmic membrane which leads to the loss of cellular constituents. At the same time, plant secondary metabolites such as essential oils and natural plant extracts have also been reported to have antibacterial, antifungal and anti-insecticidal properties. Extracts from capsicum, seaweeds and green tea have been found to inhibit the growth of *Salmonella* spp. in-vitro. Studies are also available wherein inhibitory effect of plant extracts was evaluated against *Salmonella* inoculated in minced beef, salad vegetables, fresh cut apples and minced sheep meat.

Vegetable extracts have shown a good potential when applied under laboratory conditions in culture media. For instance, application of 6% seaweed extract was shown to result in complete inhibition of *S. abony* whereas 3% extract resulted in 93% inhibition (Gupta et al., 2011). In contrast, 2.8% methanolic extract from Irish York cabbage was shown to result in only 64% inhibition of *S. abony* (Jaiswal et al., 2011). Xu et al. (2007) reported a minimal inhibitory concentration (MIC) of 15μl of grapefruit seed extract to

complete loss of colony forming activity was observed for the treated cells with a formation of veins and small vesicles on the surface. TEM images showed the inner areas to be highly disrupted accompanied by a membrane deformation. In addition, shrinking and uneven dispersion of cytoplasmic materials was also observed (Figure 1). Liao et al. (2010) obtained a remarkable reduction of 5 logs for *S.* Typhimurium when carrot juice was subjected to DPCD treatment. Both temperature and pressure had a noticeable effect as the inactivation was enhanced with increasing pressure at a constant temperature or increasing temperature at a constant pressure. In contrast, inactivation of *S*. Typhimurium in PS or PS containing 10% brain–heart infusion (PS-BHI) broth was completed in 35 min in PS whereas it took 140 min in the case of PS-BHI (Erkmen, 2000). Besides, the previous study reported the presence of two phases during the destruction characterized by a slow rate of reduction in the cell number which increased sharply at the later stage. Erkmen and Karaman (2001) observed that the exposure time required to achieve the same level of *Salmonella* inactivation was drastically reduced as the pressure during the inactivation increased. Complete inactivation of *Salmonella* was reported in egg yolk, 94-98% in chicken meat strips and limited inactivation in whole egg at a pressure of 13.7 MPa at 35 °C for 2 h (Wei et al., 1991). The variation in the results clearly indicates the complex nature of food systems. A treatment of 14 MPa at 45 °C for 40 min resulted in a 34.48% and 32.74% reduction for *S.* Typhimurium in soy sauce and hot-pepper paste marinated pork products, respectively (Choi et al., 2009a). However, the technique is more suitable for liquid foods as the diffusion of CO2 into solid samples becomes a limitation due to the absence of agitation in solid foods. Also, high concentrations of CO2 can cause darkening of color of certain animal products due to the formation of metmyoglobin. Due to the complex nature of foods conflicting results are available on the effect of HPCD on sensory, chemical and physical properties of foods. In spite of the potential advantages of HPCD more research is needed to monitor and quantify sensory and chemical characteristics of foods undergoing this preservation technique.

Fig. 1. Scanning electron micrograph (upper; magnification: 20,000) and Transmission electron micrograph (lower; magnification: 50,000) images of *S*. Typhimurium cells (left: untreated; right: treated) upon application of super critical carbon dioxide at 35°C and 100 bar for 30 min (Kim et al., 2007)
