Multi-Pulsed High Hydrostatic Pressure Treatment of Foods

Multi-pulsed high hydrostatic pressure (mpHHP) treatment of foods has been investigated for more than two decades. It was reported that the mpHHP treatment, with few exceptions, is more effective than the classical or single-pulsed HHP (spHHP) treatment for inactivation of microorganisms in fruit juice, dairy products, liquid whole egg, meat products, and sea foods. Moreover, the mpHHP treatment could be also used to inactivate enzymes in foods and to increase the shelf-life of foods. The effects of the mpHHP treatment of foods are summarized and the differences between the mpHHP and spHHP are also emphasized.


Introduction
High hydrostatic pressure (HHP) treatment is an effective technique to destroy microorganisms and inactivate enzymes in order to enhance safety and shelf-life of foods. Therefore, HHP has become a reality in the food industry and has spread world wide [1]. After 2000, the number of installed HPP machines for the food industry increased exponentially [2].
Classical HHP or single-pulsed HHP (spHHP) treatment can be applied as: compression to target pressure, holding for a certain period of time at the target pressure, and decompression to atmospheric pressure ( Figure 1). On the other hand, it may be also possible to apply successive application of HHP in which more than one compression, holding, and decompression periods exist ( Figure 2). This type of treatment is called multi-pulsed HHP (mpHHP). The mpHHP treatment, for the same holding time, is more effective than the spHHP treatment for enzyme [3][4][5], yeast cells [6] bacterial cells [7,8], and bacterial spores [9][10][11] inactivation. It was also reported that there was less recovery from injury for Escherichia coli for the mpHHP treatment compared to the spHHP treatment [12]. However, some researchers reported that the use of the mpHHP treatment did not considerably enhance pressure inactivation of virus [13], and bacteria [14,15] as compared to the sHHP treatment.
The mpHHP treatment inactivation of microorganisms in laboratory media, foods, blood plasma, vaccines, and drugs is well documented by a recent review [16]. This review, however, provides information not only about microorganisms but also enzymes, food quality and shelf-life.

Process Parameters of the mpHHP Treatment
It is known that pressure, temperature, and (holding) time are the most important process parameters of the spHHP treatment. However, more parameters should be taken into account before applying the mpHHP. Pressure and temperature are also the most important parameters for the mpHHP treatment. Besides, pulse duration, i.e., pulse holding time, number of pulses, off-pressure time (duration between the pulses), compression and decompression rates or times, and pulse shape (ramp, square, sinusoidal) may also affect the outcomes of the mpHHP treatment ( Figure 2). The effect of these process parameters on microbial inactivation was given by Buzrul [16] and will not be deeply investigated here.

Fruit Juices
Studies on fruit juices by the application of mpHHP began about two decades ago. The mpHHP treatment was reported to be more effective than the spHHP treatment for the same holding time for the inactivations of Saccharomyces cerevisiae in pineapple juice [17], Byssochlamys nivea ascospores in apple and cranberry juices [18]. On the other hand, Alemán et al. [17] observed no inactivation of S. cerevisiae in pineapple juice after 40-4000 fast sinusodial pulses (10 cycles/s) at 4-400 s total holding time in the range of 235-270 MPa (total processing time was 0.39-39 min) indicating that pulse shape is (step pressure pulse was effective, but sinusodial pulses had no effect on inactivation of yeasts in fruit juice) also an important parameter for the mpHHP treatment.
Donsì et al. [19] found that efficiency of the mpHHP treatment depends on the combination of pulse holding time and number of pulses for the inactivation of S. cerevisiae in pineapple and orange juices. They also observed higher reduction for slow compression rate (2.5 MPa·s −1 ) than that of faster compression rates (10.5 and 25 MPa·s −1 ) if several pulses (3 to 10 pulses) were applied. Buzrul et al. [20] found that increasing the pulse number did not effect the inactivation of Escherichia coli and Listeria innocua to great extends in kiwifruit juice (high inactivations were already obtained by application of the spHHP treatment in kiwifruit juice); however, in pineapple juice especially after 5 pulses inactivation increased significantly for both bacteria.
The mpHHP treatment up to 3 pulses with no holding time, i.e., compression followed by decompression was also applied to inactivate pectin methyl esterase (PME) in single strength and concentrated orange juices [21]. The mpHHP has a significant contribution to inactivation of PME in both juices.
A comprehensive study by Donsì et al. [22] indicated that the effectiveness of the mpHHP treatment on apple and orange juices depends on the combination of pressure, temperature, and pulse number. Optimum conditions applied to apple (300 MPa, 50 °C, 6 pulses × 1 min) and orange (250 MPa, 45 °C, 6 pulses × 1 min) juices resulted in a minimum shelf-life of 21 days at 4 °C.

Dairy Products
Milk, cheese and yogurt are the dairy products treated with the mpHHP. The mpHHP was considerably more effective than the spHHP for the same total treatment time for inactivation of E. coli in skim milk [23], E. coli and L. innocua in whole milk [24,25].
The mpHHP treatment up to 4 pulses with no holding time was applied to inactivate E. coli O157:H7 and L. monocytogenes in raw milk cheese [26]. Significant microbial and enzyme inactivation could be possible by the application of the mpHHP (at room temperature, 3 pulses × 5 min) at higher pressures (600 and 800 MPa) in three different types of cheese which were at different ripening stages [27]. Storage of cheeses at 5 °C for 12 weeks revealed that microorganisms inactivated by the mpHHP were also absent during storage. López-Pedemonte et al. [28] obtained low inactivation (about 1.6 log10) for spores of Bacillus cereus in cheese by the mpHHP inactivation with 2 pulses (first pulse with low pressure (60 MPa) to germinate the spores and the second one is with high pressure (400 MPa) to inactivate the vegetative cells).

Liquid Whole Egg
A few studies on microbial inactivation in liquid whole egg (LWE) by the mpHHP treatment revealed that the mpHHP treatment showed greater effectiveness than the spHHP treatment for inactivations of Samonella Enteritidis [30][31][32] and E. coli [33].
The effect of temperature during the mpHHP treatment is well documented in these studies. For example, Ponce et al. [33] applied the spHHP (350 MPa, 10 or 15 min) and the mpHHP (350 MPa, 2 or 3 pulses × 5 min) treatments at different temperatures (2, 20, or 50 °C) for the inactivation of E. coli in LWE. The highest reduction was achieved at 50 °C for both treatments; however, at lower temperatures, especially at 20 °C, the mpHHP treatment was clearly more effective than the spHHP treatments. Ponce et al. [30] observed the strongest effectiveness at 50 °C, followed by 20, 2, and −15 °C for the inactivation of S. Enteritidis in LWE after the application of mpHHP (2 or 3 pulses × 5 min at 350 and 450 MPa).

Meat Products
The mpHHP applied to mechanically recovered poultry meat showed that the mpHHP treatment was slightly better than the spHHP treatment for psychrotrophs, but the mpHHP treatment did not offer better results than the spHHP treatment for mesophiles [15,34]. On the other hand, the use of the mpHHP treatment instead of the spHHP treatment showed to be more advantageous for the inactivation of E. coli O157:H7 in ground beef [35] and S. Enteritidis in chicken breast fillets especially at higher pressures [36].
Morales et al. [35] and Del Olmo et al. [37] studied the effect of the spHHP and mpHHP treatments on color and texture of beef patties and chicken breast fillets, respectively. Changes in the color and texture of ground beef caused by spHHP and mpHHP treatments of the same lethality for E. coli O157:H7 (20 min for spHHP and 4 pulses × 1 min for mpHHP) were similar [35]. Color parameters (L *, a * and b *) were significantly higher for both treatments than for vacuum-packaged control fillets. Similarly, the texture of chicken breast fillets was also significantly affected by both treatments [37].

Sea Foods
The effect of the spHHP (400 MPa, 7 °C, 10 min) and the mpHHP (400 MPa, 7 °C, 2 pulses × 5 min) on microbial flora, total volatile bases, pH, and texture of purified and unpurified oysters was studied by López-Caballero et al. [14]. The mpHHP produced no apparent advantages over the spHHP based on any of the indices used.
The mpHHP treatment reduced the microbial load in octopus arm muscle more effectively than the spHHP treatment; however, the mpHHP treatment was not so effective in reducing autolytic activity [38,39]. Inactivations of S. Enteritidis and Staphyloccoccus aureus in sturgeon and trout caviar also studied [40]. Results indicated that the mpHHP treatment (350 MPa for S. Enteritidis and 450 MPa for Staphyloccoccus aureus at room temperature for 3 pulses × 5 min) were as effective as the spHHP treatment (400 MPa for S. Enteritidis and 500 MPa for Staphyloccoccus aureus at room temperature for 15 min).

Other Food Products
Similar results were also obtained for other food products: the mpHHP treatment was more effective than the spHHP treatment for the inactivation of S. cerevisiae in fresh cut pineapple [41], S. Enteritidis in raw almonds [42], E. coli in egg white [43]. Meyer [44] reported sterility in macaroni and cheese with spore load of Clostridium sporogenes and B. cereus by the mpHHP treatment (690 MPa, 90 °C, 2 pulses × 1 min; 1 min pause between the pulses).
A summary of the mpHHP inactivation of microorganisms in foods and a summary of studies on the effect of mpHHP treatment on quality, shelf-life, microbial and enzyme inactivation of foods are provided in Tables 1 and 2, respectively.

Commercial Application of the mpHHP
Although there is now enough evidence that the mpHHP treatment is an effective way of inactivating microorganisms and enzymes, there is no commercial application of the mpHHP treatment up to date. One and most important reason for this is that the mpHHP treatment is a longer application and thus more expensive than the spHHP treatment [8,13,16,25,41]-see Figures 1 and 2. Besides, most probably a more complicated HHP equipment which can withstand fast compression and decompression rates (to reduce the total duration of the treatment) is needed for commercial applications.
However, as the technology improves it may be possible to have faster compression and decompression rates hence it may be possible to reach compatible total treatment times for the commercial applications of the mpHHP treatment. To the best of the author's knowledge there is no study on the cost and optimization of process parameters of the mpHHP treatment. Optimization between pulse number, pulse holding time, pressure level, compression and decompression rates as well as initial and target temperature in a lab scale equipment will accelerate the application of commercial mpHHP treatment. Moreover, the differences in effectiveness of mpHHP and spHHP treatments must be weighed against the design capabilities of added wear on the HHP equipment, and possible additional time required for pulse treatment [45].

Conclusions
The mpHHP treatment could be used to inactivate microorganisms and enzymes in foods. It could also be used to contribute the quality and shelf-life of foods. However, it should be noted that optimization between the pressure, temperature, pulse number, pulse holding time, and compression and decompression rates can increase the effectiveness of the mpHHP treatment. However, more studies are needed especially on the cost of the mpHHP treatment.

Conflicts of Interest
The author declares no conflict of interest.