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In general, enzymes are less sensible than microorganisms to HIPEF and their inhi-bition depends on the enzyme itself, the media where they are suspended, and the processing conditions (Martín-Belloso and Elez-Martínez, 2005). The mechanisms involved in the inactivation of enzymes by HIPEF are not fully understood. The effects of electric field on proteins include the association or dissociation of func-tional groups, movements or charged chains, and changes in alignment of α-helix (Tsong and Astumian, 1986). Some studies have evidenced that HIPEF treatments caused a loss of α-helix and an increase in β-sheet content, indicating that HIPEF affects the conformation in the secondary structure of enzymes (Zhang et al., 2007;

Zhong et al., 2007). The impact of HIPEF on quality-related enzymes of fruit juices such as peroxidase (POD), polyphenoloxidase (PPO), lipoxygenase (LOX), hydro-peroxide lyase (HPL), pectin methylesterase (PME), polygalacturonase (PG), and β-glucosidase (β-GLUC) has been studied (Table 5.2). In general, the effectiveness of enzyme inactivation in fruit juices by HIPEF is higher when electric field strength and treatment time increase (Ho et al., 1997; Yeom et al., 2000a, 2002; Van Loey et al., 2002; Elez-Martínez et al., 2007; Aguiló-Aguayo et al., 2008a). POD activities in grape juice treated by HIPEF were depleted as electric field strength and treat-ment time increased (Marsellés-Fontanet and Martín-Belloso, 2007). Elez-Martínez et al. (2006a) and Aguiló-Aguayo et al. (2008a) reported that POD inactivation in

Advances in Fruit Processing Technologies TABLE 5.2

Effects of Pulsed Electric Fields on Food Quality–Related Enzymes in Fruits

Enzyme Fruit Treatment Conditions

Inactivation 

(%) Reference

Peroxidase Orange 35 kV/cm, 1,500 μs, 35°C 100 Elez-Martínez et al. (2006a)

Grape 30 kV/cm, 5,000 μs, 40°C 50 Marselles-Fontanet and Martín-Belloso (2007)

Apple 35 kV/cm, 74°C 100 Schilling et al. (2008)

40 kV/cm, 100 μs, 50°C 68 Riener et al. (2009) Watermelon 35 kV/cm, 1,000 μs, 35°C 99 Aguiló-Aguayo et al. (2010b) Tomato 35 kV/cm, 2,000 μs, 35°C 100 Aguiló-Aguayo et al. (2008a)

Polyphenoloxidase Apple 35 kV/cm, 74°C 93 Schilling et al. (2008)

31 kV/cm, 40,000 μs, 60°C 32 Van Loey et al. (2002) 24.6 kV/cm, 6,000 μs, 15°C 97 Giner et al. (2001) 40 kV/cm, 100 μs, 50°C 71 Riener et al. (2009) Pear 24.6 kV/cm, 6,000 μs, 15°C 62 Giner et al. (2001) Peach 24.3 KV/cm, 5,000 μs, 25°C 70 Giner et al. (2002)

Grape 30 kV/cm, 5,000 μs, 40°C 100 Marselles-Fontanet and Martín-Belloso (2007) Strawberry 35 kV/cm, 2,000 μs, 35°C 97.5 Aguiló-Aguayo et al. (2010a)

169-Intensity Pulsed Electric Field Applications in Fruit Processing 40 kV/cm, 97,000 μs, 45°C 88 Min et al. (2003a)

35 kV/cm, 1,500 μs, 35°C 80 Elez-Martínez et al. (2007) Strawberry 35 kV/cm, 1,000 μs, 35°C 92 Aguiló-Aguayo et al. (2009c) Tomato 24 kV/cm, 8,000 μs, 15°C 94 Giner et al. (2000)

Orange-carrot 35 kV/cm, 340 μs, 35°C 81.4 Rodrigo et al. (2003) 25 kV/cm, 280 μs, 68°C 76 Rivas et al. (2006)

Watermelon 35 kV/cm, 2,000 μs, 35°C 85 Aguiló-Aguayo et al. (2010d) Polygalacturonase Strawberry 35 kV/cm, 1,000 μs, 35°C 28 Aguiló-Aguayo et al. (2009c) Tomato 35 kV/cm, 1,000 μs, 35°C 62 Aguiló-Aguayo et al. (2009c) Watermelon 35 kV/cm, 1,000 μs, 35°C 40 Aguiló-Aguayo et al. (2010d)

Lipoxygenase Tomato 35 kV/cm, 50 μs, 30°C 80 Min et al. (2003c)

35 kV/cm, 1,000 μs, 35°C 20 Aguiló-Aguayo et al. (2009e) Strawberry 35 kV/cm, 1,000 μs, 40°C 35 Aguiló-Aguayo et al. (2008b) Watermelon 35 kV/cm, 1,000 μs, 35°C 52 Aguiló-Aguayo et al. (2010b)

Green pea 20 kV/cm, 400 μs 0 Van Loey et al. (2002)

β-glucosidase Strawberry juice 35 kV/cm, 1,000 μs, 40°C 27 Aguiló-Aguayo et al. (2008b) Hydroperoxide lyase Tomato juice 35 kV/cm, 1,000 μs, 35°C 93 Aguiló-Aguayo et al. (2009e)

orange and tomato juices increased when increasing treatment time. Treatments of longer duration also resulted in higher reductions of PPO activity in strawberry juice. Maximum PPO inactivation of 97.5% was achieved by selecting bipolar treat-ments at 35 kV/cm, frequencies higher than 229 Hz, and pulse widths between 3.23 and 4.23 μs for a constant treatment time of 2000 μs (Aguiló-Aguayo et al., 2010a).

Moreover, Marsellés-Fontanet and Martín-Belloso (2007) observed that PPO activ-ity in grape juice treated by HIPEF was lessened as electric field strength and treat-ment time increased. Consistently, Schilling et al. (2008) observed a similar trend in the inactivation of PPO of HIPEF-treated apple juice. In addition, other HIPEF criti-cal variables such as pulse frequency, pulse width, and pulse polarity are also impor-tant in defining HIPEF treatment conditions necessary to adequately reduce enzyme activity (Elez-Martínez et al., 2006a; Aguiló-Aguayo et al., 2008a). Aguiló-Aguayo et al. (2008a) observed that it is feasible to maximize POD inactivation selecting pulse width higher than 5.5 μs in bipolar mode at a frequency of 200 Hz, keeping the electric field strength constant at 35 kV/cm and treatment time at 1500 μs. Higher POD inactivation in orange juice was achieved when pulse frequency increased, and residual activities of 6.9% were obtained when applying frequencies of 450 Hz (bipo-lar pulses of 4 μs at 35 kV/cm for 600 μs) (Elez-Martínez et al., 2006a). Elez-Martínez et al. (2007) reported 80% of PME inactivation when orange juice was treated at 35 kV/cm for 1500 μs by applying bipolar pulses of 4 μs at 200 Hz. Moreover, Aguiló-Aguayo et al. (2009c) observed that PME and PG activities in tomato juice were depleted by increasing pulse frequency and/or pulse width, irrespective of the treat-ment polarity. However, PME activity in strawberry juice was not affected when a HIPEF treatment was applied in mono- or bipolar mode.

The medium in which the enzyme is suspended has a significant effect on the inactivation level reached. POD from grape juice required stronger HIPEF treatment conditions than POD from watermelon juice to reach at least 50% of the degree of inactivation (Marsellés-Fontanet and Martín-Belloso, 2007; Aguiló-Aguayo et al., 2010b). An 80% reduction of LOX activity was observed when tomato juice was exposed to HIPEF at 35 kV/cm for 50 or 60 μs (3-μs pulse width) (Min et al., 2003c).

In contrast, LOX in pea juice was not affected after applying 400 pulses of 1 μs at a field strength of 20 kV/cm and frequency of 1 Hz (Van Loey et al. 2002). Differences in the secondary or tertiary structure among enzymes may result in diverse sen-sitivity to HIPEF (Ho et al., 1997). Aguiló-Aguayo et al. (2008b) observed high resistance of LOX and β-GLUC to HIPEF in strawberry juice. They reported maxi-mum LOX inactivation of 35% when processing the juice at 35 kV/cm for 1000 μs by applying 4 μs monopolar pulses at 150 Hz, whereas β-GLUC was activated up to 110% at the same treatment conditions. Tomato juice HPL was inactivated around 93% when juice was processed at 35 kV/cm for 1000 μs with 7 μs pulse width and 250 Hz, whereas the highest tomato juice LOX inactivation (20%) was obtained in monopolar mode at 150 Hz for 1 μs (Aguiló-Aguayo et al., 2009c).

Because of the resistance of several enzymes to HIPEF, treatments combining HIPEF with other hurdles such as mild heat or the use of some additives were studied on different fruit juices with promising results (Hodgins et al., 2002; Riener et al., 2008, 2009). The synergistic effects between HIPEF and thermal treatments in the inactivation of PME in orange juice have shown that an increase in electric field

strength could cause greater levels of PME inactivation with an increase in tempera-ture during the treatment (Yeom et al., 2000b).

Studies concerning enzyme activity stability during the storage of HIPEF-treated fruit juices revealed variations of the initial inactivation values reached after pro-cessing along storage.

A progressive decay of the initial residual values of LOX and PG has been observed in tomato, strawberry, and watermelon juices processed by HIPEF (35 kV/cm and treatment times up to 1727 μs) for 56 days of storage at 4°C (Aguiló-Aguayo et al., 2008c, 2009b, 2010c). However, other enzymes such as POD and PME were less affected than others by storage time. Thus, Elez-Martínez et al. (2006b) reported that POD activity of HIPEF-treated (35 kV/cm for 1000 μs; bipolar 4 μs pulses at 200 Hz) orange juice remained inactivated for 56 days, indicating that the changes induced in the enzyme structure were irreversible. In the same way, HIPEF-treated tomato and strawberry juices kept values of residual POD activities below 20%

throughout storage (Aguiló-Aguayo et al., 2008c, 2010c). Residual PME activities in strawberry and orange juices remained below 10% and 20% during 56 days of storage (Elez-Martínez et al., 2006b; Aguiló-Aguayo et al., 2009b). Hence, the efficiency of HIPEF in inducing irreversible enzymatic changes has been dem-onstrated since no activation of enzyme has been reported in storage of HIPEF-treated fruit juices.