The PEF technology uses a very high voltage field (10-80 kV/cm for 1-100 pis) and is applied preferably in pumpable foods to be processed, such as juices, milk, fermented beverages. The irreversible damage to cell membranes and pore formation caused by electric pulses leads to leakage of intracellular components [80]. Since EOs are membrane-active compounds, the electroporation of target cells by PEF can facilitate the diffusion across the cell membrane thus damaging various functions leading cells to lose viability [8]. However, the impact of combined treatments of PEF and EO is still to known well. No synergy was reported between PEF and carvacrol in apple juice against Leuconostoc sp. and Saccharomyces sp [27] and in buffer (pH 4 and 7) against E. coli 0157:H7 and L. monocytogenes [3]. Nevertheless, the effectiveness is determined by factors such as pH, organic acids, food components, added EO, PEF parameters, and other intrinsic and extrinsic factors [119]. Therefore, specific applications of PEF and EO must be evaluated for each target organism and food type under study. The research studies have examined effects combination of PEF and EO on actual foods. The inactivation of S. typhimurium in skim milk was synergistically enhanced by adding cinnamon and demonstrated maximum synergy at 30 kV/cm-700 ps and 5% w/v [117]. Incorporation of cinnamon bark oil to apple and pear juice (0.1%) and strawberry juice (0.05%) followed by PEF (1575-1700 ps at 35 kV/cm) was enough to destroy 5 log cells of S. enteritidis and E. coli 0157:H7 [105]. The efficacy of combination of PEF and lemon EO in liquid whole egg was dependent on the sequence of application and heat [41]. Further, the stability of EOs to PEF is another aspect to be studied as they can act as additional hurdle following PEF against the surviving bacteria particularly spore formers [119].


High hydrostatic pressure (HHP) is followed for solid or liquid foods as non-thermal process that is applied in either batch or continuous method. In HHP, a static pressure (100 to 1000 MPa at- 20°C to 60°C) is applied to food for a few seconds to minutes [99]. Numerous reports have evaluated combination of EOs and HHP on target bacteria. Allyl-isothiocyanate (AIT: 10 to 80 pg/ml) sensitized the E. coli 0157 to HPP (200 or 250 MPa) [108]. The combination of AIT and cinnamaldehyde showed synergistic effect with HHP in buffer and meat against pathogenic E. coli and L. monocytogenes [89]. These EOs showed no effect with thymol and this difference may be attributed to the thiol-reactive compounds (such as cinnamaldehyde and allyl-isothiocyanate) that inhibit the thioredoxin reductase and glutathione reductase activities helping in oxidative stress tolerance and exert synergism with pressure [49, 108, 133].

The lethality is often correlated with temperature and intensity of applied pressure has been categorized into different modalities of treatment, such as cold HHP pasteurization, HPP-assisted pasteurization, or HHP-assisted sterilization. Application of high-intensity pressure or prolonged time requires high cost and can adversely affect the acceptability by consumers. A combination of citral or lemongrass EO (0.75 mg/ml) reduced pressure from 350 to 150 MPa for 30 min required to inactivate Colletotrichum gloeosporiodes spore in saline solution [113].

Combination of EO-HHP treatments have been successfully applied in foods enabling microbial inactivation at the requirement of lesser pressure or shorter exposure time [113]. In yogurt drink, combination mint EO with HHP synergistically reduced the pressure required or holding time (210 sec) for 5 decimal reductions of Listeria. The pH, aw, and color of drink remained unchanged and no separation of serum protein was observed [47]. Similarly, in apple and orange juice limonene-HPP acted synergistically on E. coli 0157 [40]. The carvacrol and HHP displayed the synergistic effect on L. monocytogenes in milk (3.2 log reduction). However, it was lowered by two-fold compared to that achieved in buffer (> 6 log reduction) [78]. These observations are in support of the requirement of validation of the results of food models for efficacy in real foods. Citral and lemongrass are likely to be organoleptically acceptable antimicrobial for use in citrus- based beverages and juices owing to pleasing fruity flavor [133].

The high levels of destruction of target bacteria seen in the combination method of EOs and HHP in buffer and food are attributed to simultaneous action on cell membranes through a different mode. During pressurization, irreversible damage to cell membranes due to compression causes altered permeability thus leading to leakage of intracellular ions and ATP molecules. The pressurization can also allow easy penetrability of EOs and conversely weakening of cell wall sensitizes the pressure tolerant bacteria to HHP. However, the precise mechanism is not clearly understood. Also, the tailing effect after HHP, which means a small portion of target population survive and remain viable, is continued to be inhibited by EOs and serves as hurdle thereby preventing cells to recover from sub lethal injuries [110]. Perez et al. [115] observed additional 1.38 and 1.8 log cells killing of methicillin resistant S. aureus in rice puddings by HHP in the presence of cinnamon or clove, respectively. Following HPP during one-week storage in refrigerator, total bacteria continued to decrease by 1.5 log cycle in HHP-cinnamon treatment. The citral-HHP combination showed synergistic effect on L. monocytogenes, conversely resulting in sublethal injury after the treatment. Interestingly, the pressure lethality threshold for butyl hydroquinone (BHQ)- HHP against barotolerant E. coli 0157:H7 (200 MPa) was much lower than L. monocyogenes (>300 MPa) [29]. This difference in sensitivity was probably associated with difference in cell wall composition [94].

Bacterial endospore resistance towards HHP intensities is used presently for food processing and it still remains a challenge. However, the induction of germination by HHP renders them susceptible to other treatments [4]. Corthouts et al. [30] also described impact of EO on pressure-induced germination of spore of psychro-tolerant in B. cereus. Use of EO as additional hurdle inhibited induced germination; however, the inactivation was at moderate temperature and pressure. EO combination at high temperatures and pressure may inhibit the release of DPA; and spore rehydration resulted in less inactivation of B. cereus [93].

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