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Fig. 7.1 HPP-treated meat product manufactured by a Spanish meat company

Fig. 7.2 NC Hyperbaric industrial equipment for HPP up to 650 MPa located at IRTA-CENTA (Monells). About 120 L capacity, 300 mm inner diameter hour could be carried out. Products treated by HHP do not need specific label in United States. The technology is well accepted in Europe as an alternative technology, despite the general lack of consumer awareness of "high-pressure processing" (HPP). Baron et al. (1999) reported 67% acceptability by consumers from three different European countries (France, Germany and United Kingdom). In more recent studies, the results of a conjoint analysis to assess the importance of various product and marketing factors on consumer interest in foods processed by emerging technologies showed that HPP has a strong positive influence on consumer interest, compared with irradiation and genetic modification (Cardello, Schutz, & Lesher, 2007).

General Effects of HHP

Food enzymes can undergo reversible or irreversible pressure-induced changes resulting in partial or complete activation or inactivation (Cheftel, 1995). The denaturation of proteins by pressure seems to allow the destabilization of non-covalent interactions in the tertiary structure (Pittia, Wilde, Husband, & Clark, 1996; Tedford, Kelly, Price, & Schaschke, 1999) and although these proteins structurally retain much of their secondary structure, the small degree of unfolding that exposes the hydrophobic regions of the protein could be the cause of protein aggregation (Mozhaev et al., 1996; Tedford et al., 1999). But the nutritional value, vitamins, and the majority of small substances responsible for the flavors of the products are retained. This is viewed as an important benefit for the food industry (Hoover, Metrick, Papineau, Farkas, & Knorr, 1989; Smelt, 1998; Tellez, Ramirez, Perez, Vazquez, & Simal, 2001) and in general minimal modifications in the sensory characteristics of the product are introduced, especially in cooked and cured meat products. Nevertheless, it has been reported that the pressurization of post-mortem beef meat could modify the enzymatic system (Homma, Ikeuchi, & Suzuki, 1994; Jung, De Lamballerie-Anton, & Taylor, 2000), the texture and ultrastructure (Macfarlane, 1985; Suzuki, Kim, Homma, Ikeuchi, & Saito, 1992), the gelation properties of myofibrillar proteins (Ikeuchi, Tanji, Kim, & Suzuki, 1992), and the microbiological quality of meat (Cheftel & Culioli, 1997). During the last decade, to overcome these drawbacks, different applications of high-pressure-low-temperature combinations were investigated, including high-pressure freezing or thawing and storage at subzero temperatures under pressure (Otero, 1999; Sanz, 2005). Low-temperature pressurization of frozen meat was patented as a system to prevent color degradation (Arnau et al., 2006).

Effect of HHP on Microorganisms

High pressure induces several changes in the cell membrane and cell wall of microorganisms, including separation of the cell membrane from the cell wall, contraction of the cell membrane, compression of gas vacuoles, cell lengthening, and release of intracellular material (Patterson, 2005). Ribosome dissociation was also shown to limit cell viability at high pressures (Abe, 2007).

Moderate levels of pressure decrease the rate of growth and reproduction, whereas very high pressures cause inactivation, the threshold depending on the microorganism and species. Yeasts and moulds are relatively pressure sensitive; however, ascospores of heat-resistant moulds such as Byssochlamys, Neosartorya, and Talaromyces are generally considered to be extremely HHP resistant (Chapman, Winley, & Fong, 2007; Smelt, 1998). In general, Gramnegative bacteria and cells in lag phase are more sensitive than Gram-positive and stationary phase cells. Vegetative pathogens like Vibrio and Yersinia are relatively sensitive to pressure and can be inactivated at pressures less than 350 MPa, whereas Staphylococcus aureus needs pressures higher than 500 MPa (Chen, Guan, & Hoover, 2006). Nevertheless and according to Schreck, Layh-Schmidt, and Ludwig (1999) barotolerance could not be correlated with the Gram type and the presence of cell wall. Most pressure sensitive bacteria are rod or spiral shaped, whereas the most resistant ones are spheres. Medium sensitive bacteria exhibit a mixed assortment of forms between short rods and cocci (pleomorphic shape). Ludwig, van Almsick, and Schreck (2002) even concluded that the presence of a cell wall might be disadvantageous for a bacteria species when exposed to high pressure. More recently, Hartmann, Mathmann, and Delgado (2006) confirmed that pressure load on the cell wall induces severe non-hydrostatic stress which might interact with inactivation mechanisms such as denaturation of membrane-bound proteins.

Endospores, when compared with vegetative cells, tend to be extremely HHP resistant, Clostridium endospores being more pressure resistant than Bacillus. However, bacterial spores can be inactivated by first inducing spore germination using relatively low pressure, followed by complete inactivation and death of the spores using relatively mild heat treatments (Smelt, 1998) or subsequent pressure treatments (Wuytack, Boven, & Michiels, 1998). Different combinations of temperature, time, pressure, and cycling treatments were studied and it was reported that the complete efficacy for achieving spore inactivation depends on several factors (Farkas & Hoover, 2000; Torres & Velazquez, 2005).

Generally, the prions associated to neurological disorders are even more difficult to destroy than bacterial spores. Some prions are affected by pressure combined with a simultaneous heat treatment at 60°C (Garcia et al., 2004). Pressure resistance of viruses varies considerably; HHP can cause damage to the virus envelope preventing the virus particles from binding to cells or even complete dissociation of virus particles, which may be either fully reversible or irreversible (Hogan, Kelly, & Sun, 2005).

Other factors influencing threshold of inactivation are the pressure applied, the time of processing, the composition of the food, temperature, pH and water activity (Tewari, Jayas, & Holley, 1999). In addition, pressure resistance of microorganisms would be reinforced in rich nutrient media (Hoover et al., 1989). Carbohydrates, proteins, and lipids have a protective effect (Simpson & Gilmour, 1997). This indicates that validation processes in real products are required. Because the costs of high-pressure processing and throughput are related to treatment pressure, time, and temperature, further studies are needed to help food processors to select optimum processing conditions to be commercially viable. Cell death increases as pressure level increases but not following a first-order kinetics, as a tail of inactivation is sometimes recorded (Garriga, Aymerich, Costa, Monfort, & Hugas, 2002; Kalchayanand, Sikes, Dunne, & Ray, 1998b). Sublethally injured cells recovered during storage and grew (Aymerich, Jofre, Garriga, & Hugas, 2005; Chen & Hoover, 2003; Garriga et al., 2002; Patterson, Quinn, Simpson, & Gilmour, 1995). Depending on the food product, the preservation conditions, and duration, the microbial proliferation could reach very high proportions and could also cause the formation of high levels of biogenic amines (Ruiz-Capillas & Jimenez-Colmenero, 2004). Some biogenic amines (tyramine and histamine, directly, or putrescine and cadaverine, indirectly) can pose health risks due to toxicological effects, when ingested in large quantities. Furthermore, there is some evidence that these technologies can condition the amine profile produced. Therefore, more in-depth studies during the shelf-life of the products are necessary to determine how the different factors associated with these technologies can affect the formation of these compounds and the recovery of microbial cells.

High Hydrostatic Treatments to Improve the Food Safety of Different Types of Meat Products

Raw Meats

In order to extend the shelf-life, increase the food safety and quality of raw meat, alternative non-thermal technologies such as high hydrostatic pressure alone or combined with active packaging and natural biopreservatives have been studied.

HHP was reported as being able to reduce 6-7 log CFU/g the total counts in meat homogeneate and more than 4 log CFU/g in minced beef muscle and mechanically recovered poultry meat, when a ca. 400 MPa treatment was assayed (Carlez, Rosec, Richard, & Cheftel, 1994; Shigehisa, Ohmori, Saito, Taji, & Hayashi, 1991; Yuste et al., 2001). When a higher treatment (500 MPa) was applied in poultry sausages, a total count reduction equal to the pasteurization process was obtained (Yuste, Pla, & Mor-Mur, 2000). Toxoplasma gondii cysts were inactivated in a ground pork meat with an HHP of 300 MPa (Lindsay, Collins, Holliman, Flick, & Dubey, 2006). A 700 MPa treatment was able to reduce 5 log CFU/g the counts of E. coli O157:H7 in raw minced meat (Gola, Mutti, Manganelli, Squarcina, & Rovere, 2000).

Marinated beef loin is a raw meat product with high aw (0.98), low level of salt (1%), and a mixed microflora with an important initial contamination, around 6 log CFU/g (Garriga, Grebol, Aymerich, Monfort, & Hugas, 2004). HHP treatment of sliced marinated beef loin at 600 MPa for 6 min was very effective in reducing all the microbial groups investigated, achieving a reduction of 4 log cycles after treatment for aerobic, psycrotrophic, and LAB counts and nearly 3 log CFU/g for Enterobacteriaceae. No further recovery of survivors were recorded during 120-day storage at 4°C, while untreated samples reached 8 log CFU/g after 30 days of storage. Regarding pathogens, 9 out of 15 control samples (untreated) showed presence in 25 g of L. monocytogenes and Salmonella spp. Moreover all of the HPP samples (n=15) recorded absence of either L. monocytogenes or Salmonella in 25 g during the whole 4°C storage period (120 days) (Garriga et al., 2004). From a safety point of view these results confirm that HPP is a powerful tool to control risks associated with these pathogens in raw meats. In fact challenge tests performed in our laboratory showed that pressurization at 600 MPa for 6 min was capable of reducing from ca. 3.5 log CFU/g the initial spiked counts of Salmonella, L. monocytogenes, C. jejuni, Y. enterocolitica to values below the detection limit during the whole 4°C storage (120 days) of treated marinated beef loin slices.

Morales, Calzada, and Avila (2008) investigated the effect of single- and multiple-cycle HHP treatments at 400 MPa on the inactivation of E. coli O157:H7 inoculated (ca. 7 log CFU/g) into ground beef. The authors concluded that multiple-cycle HHP treatments achieved a higher lethality than did single-cycle treatments for the same total length of treatment (including come up and depressurization times) or the same lethality for a shorter total length of treatment. Changes in the color and texture of ground beef caused by single- and multiple-cycle HP treatments of the same lethality (i.e., one 20-min cycle and four 1-min cycles) were similar. Luscher, Balasa, Frohling, Ananta, and Knorr (2004) and Luscher, Sunderhoff, Urrutia Benet, and Knorr (2005) reported a 2-3 log-cycle bacterial reduction in frozen minced beef and in frozen suspensions, respectively, due to the mechanical stress associated to phase transitions (ice I to ice II or III).

Several authors observed undesirable color modifications in pressurized samples: in marinated beef loin at 600 MPa (Garriga et al., 2004), in minced beef muscle treated at pressures higher than 350 MPa (Carlez, Veciana-Nogues, & Cheftel, 1995), and in shear forces and hardness of semitendinosus (ST) muscle between 100 and 500 MPa (Lee, Kim, & Lee, 2007). Nevertheless, no differences in consumers' acceptance of ready-to-eat low-fat pastrani, Strassburg and Cajun beef were reported (Hayman, Baxter, O'Riordan, & Stewart, 2004). Contractile myofibrillar proteins are thought to be primarily responsible for differences in the textural properties of HHP-treated meat. An increase in the hardness of beef muscle treated at 20°C with pressure levels up to 400 MPa and a slight decrease at higher pressures were reported (Ma & Ledward, 2004), ultrastructural changes in myofibrils becoming visible at pressures above 325 MPa (Jung, De Lamballerie, & Ghoul, 2000). On the other hand, HHP treatment affects the integrity of lysosomes (D. S. Jung et al., 2000) and increases cathepsin D and acid phosphatase activities in pressurized beef, influencing its textural characteristics (D. S. Jung et al., 2000).

Cured Meat Products

The microflora of dry-cured ham due to their low water activity (0.89) and high salt content (4.6%) are mainly composed of GCC+ and yeasts, which are also present in the product after slicing. Garriga et al. (2004) reported a 2 log cycle decrease of total bacteria counts after an HHP treatment at 600 MPa for 6 min of vacuum-skin-packaged dry-cured ham slices. The counts maintained around 3 log CFU/g till the end of storage (120 days at 4°C). Psychrotrophs showed higher pressure sensitivity compared to mesophiles not recovering their ability to grow during storage. Regarding yeasts, although no growth was observed in non-treated samples during storage, the counts of HHP-treated samples maintained the levels achieved after treatment (<1 log CFU/g) during the whole period studied. Salmonella and Campylobacter were not detected in any samples neither control nor HHP treated, whereas L. monocytogenes were present in one of the control samples but absent in all HHP-treated samples during the whole storage period studied (Garriga et al., 2004). In a challenge test, when Salmonella and L. monocytogenes were spiked between dry-cured ham slices both pathogens were highly inactivated after pressure treatment (600 MPa 6 min)

and the counts were reduced from ca. 3.5 log CFU/g to <10 CFU/g (Jofre, Aymerich, Monfort, & Garriga, 2008). In a previous work at the same pressure/ time different inactivation levels after HHP treatment were observed in dry-cured ham spiked with L. monocytogenes, depending on the equipment used (Hugas, Garriga, & Monfort, 2002).

Treatment of sliced Iberian and Serrano hams at 450 MPa for 10 min significantly reduced the population of L. monocytogenes Scott A, spiked at ca. 6 log CFU/g. After 60 days at 4°C or 8°C, the counts were 3.24 and 4.70 log CFU/g in HPP and control, respectively, for Iberian and 2.73 and 5.07 log CFU/g for Serrano ham (Morales, Calzada, & Nunez, 2006). The color parameters L* and a* were not influenced by high-pressure treatment, and parameter b* increased only in Iberian ham. By contrast, a few studies have reported pressure-induced color changes in both lightness (increased) and redness (decreased) when applied to Parma ham (Tanzi et al., 2004) and Iberian dry-cured ham (Andres, Adamsen, Moller, Ruiz, & Skibsted, 2006). Moreover, Tanzi et al. (2004) reported some texture and flavour changes in the pressurized samples. An enhanced perception of saltiness was reported by Saccani, Paro-lari, Tanzi, and Rabbuti (2004) after a treatment of 600 MPa for 9 min.

Serra, Grebol, et al. (2007) described the effect of HPP (400 and 600 MPa) applied to frozen hams at different stages of the drying process. HHP-treated hams showed slightly lower visual color intensity than the control ones. In general, pressurization did not have a significant effect on the flavour characteristics of the final product as reported by other authors (Morales et al., 2006). The 600 MPa-hams from the ERS process (at the end of the resting stage) showed significantly lower crumbliness and higher fibrousness scores than the control and the 400 MPa, without negatively affecting the overall sensory quality of the hams. An increase in lightness L was only observed in the biceps femoris muscle from green hams (at the early stages) at both pressures studied. Generally, only a little or no decrease in redness has been reported after pressurization, because of the protective action of nitric oxide on myoglobin, i.e., the nitrosylmyoglobin formation protects the pigment against oxidation, thus preserving the cured color (Carlez et al., 1995; Cheftel & Culioli, 1997; Farkas et al., 2002). Serra, Sarraga, et al. (2007) demonstrated that high-pressure treatment (400 and 600 MPa) slightly reduced antioxidant enzyme activity in dry-cured hams.

Another traditional Spanish product, manufactured similarly to dry-cured ham is Cecina de Leon, an intermediate moisture beef meat product with the typical red color, smoked flavor and slightly salty taste. At the end of drying the microbial counts are in general low, around 3 log CFU/g, but after slicing and packaging operations cross-contamination leads to an increase of the total counts which reduces the expected shelf-life of this product. Rubio, Martinez, Garcia-Gachan, Rovira, and Jaime (2007a) studied the application of a 500 MPa pressure treatment for 5 min in order to extend the shelf-life of Cecina de Leon. A delay of the growth of spoilage flora was achieved with a subsequent extension of the shelf-life to 210 days, compared to the 90 days usually expected.

However, the treatment did not avoid sensory changes during storage, limiting the optimum storage time to 90 days. This result agrees with those of Andres, M0ller, Adamsen, and Skibsted (2004) which were unable to detect any differences in TBA values between untreated and HPP (400 MPa and 15 min) Iberian ham. However, Cava, Tarrega, Ramirez, Mingoarranz, and Carrasco (2005) pointed out TBA values on Iberian ham treated with high pressure (200-300 MPa and 15-30 min) increased, although after storage for 90 days, similar TBA values were found on treated and untreated samples. On the contrary, Saccani et al. (2004) reported that the HPP (600 MPa during 3, 6, or 9 min) modified the sensory parameters (loss of color intensity, saltier taste and greater firmness) of dry-cured hams that had undergone 14 and 18 months of ripening.

Cooked Meat Products

Due to its composition, pH, water activity, and lack of endogenous microflora, sliced cooked meat products may not represent a major hurdle for microbiological growth during refrigerated storage if recontamination during slicing and packaging occurs. Its shelf-life depends on good manufacturing practices, the use of white rooms, and the post-pasteurization process. In this sense, HPP may represent an efficient alternative post-processing technique to increase the shelf-life of these products without significant sensory modifications.

The effect of physico-chemical and sensorial changes were mainly studied in cooked ham. No color and no pH changes have been reported in cooked ham treated by HHP (Carpi et al., 1999; Cheftel & Culioli, 1997; Hayman et al., 2004; Lopez-Caballero, Carballo, & Jimenez-Colmenero, 1999). Even when cooked ham was stored for 8 weeks at 4-6°C after a 300-600 MPa/ 10-30 min/room temperature treatment, no changes in texture or color of cooked ham were reported throughout storage (Karlowski, Windyga, & Fon-berg-Broczek, 2002). Moreover, Hugas et al. (2002) reported that the overall physico-chemical composition of cooked ham was not significantly affected after a treatment of 600 MPa for 10 min at 30°C. The non-proteic nitrogen fraction and aminoacid content were equivalent, fatty acid composition, and cholesterol content were kept and contents of vitamins from group B were not modified. Mineral composition was similar and only a decrease of the calcium content was observed. No changes in bioavailability of nutrients and no increase in the solubility of cytoplasmatic proteins were observed.

In vacuum-packaged-cooked sausages, Mor-Mur and Yuste (2003) also reported that color attributes did not change when the product was treated at 500 MPa for 5 or 15 min at mild temperature (65°C). When color, texture, and yield of pressure-treated sausages were compared to sausages treated with a conventional heat pasteurisation (80-85°C for 40 min), pressurised sausages were more cohesive and less firm than heat-treated sausages. HHP induced higher yield than heat treatment. Sensory analysis did not detect differences between both types of sausages; and even when there were differences, pressurized samples were preferred in more occasions because of their better appearance, taste and, especially, texture. The effects of an HPP at 600 MPa, 10 min, 20°C on the quality of cooked pork ham prepared with two different levels of curing ingredients in brine and stored in refrigeration (4-6°C) for 8 weeks have been also evaluated by Pietrzak, Fonberg-Broczek, and Mucka (2007). HPP causes significant improvement of shelf-life of vacuum-packed ham, including the samples with reduced level of curing ingredients in brine to 8 weeks in refrigerator conditions. HPP did not affect the texture or color of ham, but it increased the drip loss during storage in the packed samples. This may indicate that HPP has negative effects on water holding capacity of cooked products.

Concerning microbiological food safety, different assays have been performed in meat models and different food matrices. In a meat model system, Garriga et al. (2002) reported the application of HHP treatment of 400 MPa for 10 min at 17°C. E. coli displayed a 4-5 log cycle decline after 24 h of pressuriza-tion but it recovered and grew to 106-107 CFU/g at the end of storage at 4°C. A 6 log reduction after treatment was observed for Salmonella, L. monocytogenes, slime producing LAB (Lactobacillus sakei and Leuconostoc carnosum) but while Salmonella was not able to recover during refrigerated storage, the other challenged bacteria quickly recovered after treatment, reaching initial inoculated counts. S. aureus was the species least sensitive to the HPP treatment.

In sliced-cooked ham several assays have been performed by different authors to assess the effectiveness of different high hydrostatic treatments at different temperatures of treatment and shelf-storage and interleaver application to avoid release of meat juices and fat, on naturally contaminated and artificially spiked spoilage and pathogenic microorganisms. Lopez-Caballero et al. (1999) studied the efficiency of a treatment of 200-400 MPa for 5 and 20 min at 7°C in prepackaged naturally contaminated sliced-cooked ham when stored at 2°C for 35 days during post-processing, slicing, and packaging. The treatment at 400 MPa for 20 min was able to reduce total viable counts in 2 log CFU/ g, keeping these levels until the end of the storage. LAB were not detected until day 21 and GCC+ were under the detection limit at day 35. The 400 MPa 5 and 20 min treatments were also better than the 200 MPa 20 min treatment to extend the detection of Enterobacteriaceae and Brochothrix thermosphacta, respectively, until the day 35 when compared to the 7th and 21st days of detection of the 200 MPa treatment. In a second trial, the same authors (Lopez-Caballero, Carballo, Solas, & Jimenez-Colmenero, 2002) assayed the effect of combined treatments of high pressure (300 MPa, for 15 min) and temperature (5, 20, 35, and 50°C) on microbial inactivation (total bacterial count, LAB, Baird Parker microflora, Pseudomonas sp., and Enterobacteriaceae) and color, in naturally contaminated sliced cooked ham. Greater pressure-induced loss viability was observed in Gram-negative bacteria. Microbial inactivation was more pronounced when pressurization was applied at 50°C. Microbial sensitivity to the HPP was conditioned by the processing temperature, the microorganism group, and the type of meat product. The effectiveness of a higher pressure treatment, 600 MPa 6 min at 31°C, to avoid growth of endogenous, non-inoculated yeasts and Enterobacteriaceae, for delaying the growth of LAB and to reduce the risks associated to Salmonella and L. monocytogenes in sliced cooked ham were also reported by Garriga et al. (2004). Later, Aymerich et al. (2005) reported the effect of a treatment of 400 MPa 10 min 17°C on Salmonella and L. monocytogenes artificially spiked in vacuum-packaged sliced cooked ham. The treatment was able to diminish the spiked cells (3x102 CFU/g) under 4 MPN/g for Salmonella and the growth of L. monocytogenes inhibited until 40 days of refrigerated storage at 1 or 6°C. After that period, and at 6°C, L. monocytogenes was able to grow until counts similar to that of non-pressurized samples (ca. 8 log CFU/g), while at 1°C kept to the low levels achieved after pressurization. The effect of the presence of an interleaver to avoid release of meat juices and fat in spiked sliced cooked ham with L. monocytogenes and Salmonella at 3x 104 CFU/g was also studied by Jofre, Garriga, and Aymerich (2007) and Jofre, Aymerich, and Garriga (2008). A three-layer interleaver was placed between the slices, vacuum packaged, and HHP treated at 400 MPa. While in non-pressurized samples, L. monocytogenes grew until ca. 108 CFU/g under refrigerated storage at 6°C, in pressurised samples at 400 MPa 10 min 17° C, an initial decontamination of the pathogen of 1.76 log CFU/g was observed and counts progressively increased after day 30 to levels of 6.5 log CFU/g. Salmonella diminished under 10 CFU/g, a value that was maintained for 3 months of storage at 6 C. The efficiency of an HPP (400 MPa for 10 min 17°C) in sliced cooked ham was also evaluated after a cold chain break when combined with different refrigeration temperatures (Marcos, Jofre;, Aymerich, Monfort, & Garriga, 2008). The treatment caused an immediate reduction of L. monocytogenes counts in a range of ca. 3 log CFU/ g but regrowth, specially at 6°C, was recorded. Levels after the cold chain break reached the same high levels (8 log CFU/g) as without pressurization. At 1°C, a slight regrowth was observed after pressurization but, even with a cold chain break, the counts did not exceed the initial counts and the treatment achieved ca. 2 log CFU/g lower counts than without pressurization. The presence of high-stressed cells that were not able to achieve the same growth rate as at 6°C may be the cause. Afterwards, the effectiveness of higher pressure treatments at 600 MPa was evaluated in sliced cooked ham spiked with 4 log CFU/g of Salmonella sp., L. monocytogenes, and S. aureus, after 3 months of storage at 1° and 6°C (Jofre, Garriga, & Aymerich, 2008). The application of pressure reduced the levels of Salmonella and L. monocytogenes to levels below 10 CFU/ g. These levels continued until the end of storage at both 1 and 6°C. HPP reduced the counts S. aureus by less than 1 log cycle.

Some other products such as pork marengo, Morcilla de Burgos, and Frankfurters have been considered for HPP. The improvement of microbial quality of pork marengo (a low acidic particulate meat product) after a high-pressure treatment of 400 MPa for 30 min at 20° or 50°C was evaluated by Moerman (2005). Several Clostridium spp. and Bacillus spp. survived the treatment, and the Gram-positive cocci Enterococcus faecalis and S. aureus were revealed to be more pressure resistant than Saccharomyces cerevisiae and the Gram-negative bacteria Pseudomonas fluorescens and E. coli. In commercially sterile sausage, Chung, Vurma, Turek, Chism, and Yousef (2005) reported the effect of HPP (600 MPa, 28°C, 5 min) against barotolerant L. monocytogenes inoculated at 106-107 CFU/g. Inactivation was evaluated after sample enrichment to detect the viability of the pathogen after the treatments. HPP caused a modest decrease in the number of positive samples.

In Morcilla de Burgos, the most traditional and famous blood sausage in Spain, Diez, Santos, Jaime, and Rovira (2008) studied the effect of HPP of 300-600 MPa 15°C 10 min, during the chilled storage (28 days). A decrease of Enterobacteriaceae and Pseudomonas spp. counts below the detection level, <102 and <10 CFU/g, respectively, was achieved for all the pressures applied, but a treatment of 600 MPa was necessary to reduce the LAB counts in 1 log CFU/g. These microbiological changes seemed sufficient to reduce the sour taste, presence of slime, and vacuum loss until day 15 and to improve the shelf-life of morcilla de Burgos to 28 days of vacuum-packed storage at 4°C, in comparison with control samples, possibly due to the injury provoked by the treatment and the storage conditions. LAB recovered after day 7 and reached values of 108 CFU/g at day 35. LAB species were differentially affected by HPP at 600 MPa (Diez, Urso, & Rantsiou, 2008) as shown by DNA and RNA-DGGE (PCR-denaturing gradient gel electrophoresis) microbial dynamics analysis, Leuconostoc mesenteroides was completely inactivated by the HPP treatment while Weissella viridescens was able to recover and carry out the typical spoilage of the product.

In vacuum-packaged Frankfurters, Ruiz-Capillas, Jimenez-Colmenero, Carrascosa, and Muñoz (2007) reported the effect of HPP on the formation of polyamines, microorganism inactivation, and physico-chemical characteristics on the product. The consequences of these treatments were also evaluated throughout chilled storage (2°C) for up to 141 days. Pressurization (400 MPa 10 min 30°C) caused decreases in the levels of total viable and LAB counts by ca. 2 log CFU/g and kept stable and no growth was observed until the end of the 141 days of chilled storage. Enterobacteriaceae were kept below the detection limit (<1 log CFU/g). A significant decrease was observed in hardness and chewiness throughout storage. No changes in polyamines were observed.

From the results obtained in the different research studies presented we could conclude that HPP could be recommended as a suitable treatment after post-processing to improve food safety of cooked meat products, without significantly altering sensorial properties.

Fermented Meat Products

In acidic fermented sausages, the fermentation and acidification process that happens during fermentation as a result of LAB growth, together with the additives added and the decrease of water activity during ripening, is enough to avoid undesirable microbial growth and transform raw meat into a stable product. Nevertheless, in traditional slightly fermented sausages, the pH barrier is not present and thus some pathogens may grow or simply survive. Moreover, consumption of these traditional meat products marketed after slicing has increased in recent years, contamination of the final product just immediately prior to packaging together with the required longer shelf-life have to be considered. Research is ongoing into new technologies to preserve the products' high nutritional and sensory qualities and their comparability with similar untreated products, while assuring microbiological safety. In that sense, an additional hygienic treatment after processing like HPP seems to have gained potential application in the meat industry to increase safety of these products.

Several authors have tested the efficiency of high hydrostatic processing against pathogenic microorganims and quality markers. Krockel and Muller (2002) reported the effect of HPP (200-800 MPa for 10 min at 0°C) and further storage at 7°C for 44 days in vacuum-packaged sliced Bologna-type sausages, Gelbwurst (a "diet bologna'' without nitrite) and Lyoner (nitrite-containing sausage). After HPP, the bacterial counts were markedly decreased at 400 MPa and above and were below the detection limit at 600 MPa and higher. However, a complete inactivation of all inoculated bacteria (L. monocytogenes, S. aureus, Serratia marcescens) was not achieved even at 800 MPa. After enrichment, S. marcescens was detected in all samples. At pressures of 400 MPa and higher, the type of sausage-influenced survival, recovery, and subsequent growth of the microorganisms during cold storage. Although bacterial counts directly after treatment were slightly higher for Lyoner than for Gelbwurst, recovery and growth were much more restricted in Lyoner-sausage.

In slightly fermented sausages (fuet and chorizo) Garriga et al. (2005) reported that when a treatment of 400 MPa 10 min 17°C was applied, the LAB or GCC+ population, neither Enterococcus populations were affected, whereas the treatment was able to control the growth of L. monocytogenes, to reduce Enterobacteriaceae, and kept the biogenic amine content stable. HHP was necessary to ensure absence of artificially spiked Salmonella. The low aw may contribute to the protection of several bacterial groups. The high hydrostatic treatment did not modify the TBARS or color parameters although a slight increase in cohesiveness, springiness, and chewiness was observed (Marcos, Aymerich, Guardia, & Garriga, 2007). When an HPP of 200 MPa was applied to the meat batter after stuffing and just before sausage fermentation, the treatment did not interfere with the ripening performance in terms of acidification, drying, and proteolysis as the inoculated LAB decarboxylase-negative strains were not significantly affected. The treatment also prevented Enterobacteriaceae growth and subsequently a strong inhibition of diamine (putrescine and cadaverine) accumulation was observed although not tyramine (Latorre-Moratalla et al., 2007). Nevertheless, when 300 MPa was applied to the meat batter after stuffing, a reduction of the spiked Salmonella was observed but a partial inhibition of the endogenous LAB delayed the pH drop and thus

L. monocytogenes growth was favored. Moreover, a discoloration of sausages reflected by an increase in the L* value (lightness) was observed (Marcos, Aymerich, & Garriga, 2005).

Microbiological, physico-chemical, and sensory properties of three types of sausages, with different composition of fats (control, high oleic, and high linoleic salchichon), and their evolution over 210 days of storage under refrigeration after a treatment of 500 MPa for 5 min at 18°C were studied by Rubio, Martinez, Garcia-Cachan, Rovira, and Jaime (2007b). No clear relationship could be established between fatty acid composition and the effectiveness of the treatment. HPP had a slight inhibitory effect on some groups of microorganisms, especially yeasts and moulds, psychrotrophic, and anaerobic bacteria. After treatment, LAB counts showed 1 log-cycle reduction, enterococci only slightly diminished in the high oleic batch while Micrococcaceae counts were unaffected. During storage, no clear differences in enterococci, LAB, and Micrococcaceae counts were observed between treated and non-treated samples. HPP did not show differences in the physico-chemical and sensory properties of the salchichon, even though this product is rich in monounsaturated or polyunsaturated fatty acids, the treatment did not exert an enhancing effect on oxidation during storage. No differences in the color parameters were observed. This is a crucial point, if we consider that color is important in consumer acceptance. Moreover, in Spanish fermented sausage chorizo, Ruiz-Capillas, Carballo, and Jimeenez-Colmenero (2007) reported that HPP (350 MPa 15 min 20°C) did not affect pH or water activity and reduced by <1 log unit the LAB counts that were kept until 160 days of storage at 2°C. The HPP caused a significant reduction of tyramine, putrescine, and cadaverine levels, while there was a significant increase in spermidine. Enterobacteriaceae remained below the detection limit.

While high hydrostatic pressure processing could not be recommended at the initial steps of the production, it could be a technology of choice to improve the food safety of fermented meat products, without significant changes in sensory characteristics when pressure is applied on the ripened product.

Combined Hurdles: Antimicrobials and High Hydrostatic Pressure

The effectiveness of mild preservation technologies is based on the combination of different processes or antimicrobial factors in the so called hurdle technology (Leistner & Gorris, 1995). When microorganisms are confronted with multiple antimicrobial factors the likelihood for survival decreases due to an increase in the energy costs that leads to cell exhaustation and death. In addition, the synergy between different factors may permit a decrease in their dose (Galvez, Abriouel, Lopez, & Omar, 2007). The use of generally recognized as safe (GRAS) LAB or the antimicrobial compounds they produce (i.e., bacteriocins) is a promising ongoing development in food preservation. In general, antimicrobials provide an excellent opportunity to incorporate them into a combined preservation system. Synergistic effects with HPP have been reported with antimicrobials, low pH, carbon dioxide, organic acids, vacuum packaging, and chilled storage. The effect of lactate, nisin, enterocins, and sakacin together with chilled storage and HHP treatment on the inactivation of L. monocyto-genes and other food-borne pathogens was reported (Smid & Gorris, 2007). Different modi of application, addition to the meat batter, surface application, and active packaging have been studied. Also the effectiveness of selected starter cultures and high hydrostatic pressure after ripening was evaluated.

In a meat model system, Garriga et al. (2002) observed the importance of nisin addition to the reduction of the less sensitive genera to HPP, S. aureus, and to the inhibition of the regrowth of E. coli after HHP treatment. To keep L. monocytogenes under 102 CFU/g the addition of sakacin K, enterocins, or pediocin (ALTA 2351Tm, Quest International) was needed. An additive effect between HHP treatment (400 MPa 10 min 17°C) and different antimicrobials (lactate, nisin) applied to the meat batter was observed in sliced cooked ham (Aymerich et al., 2005). Nisin and lactate allowed the regrowth of L. monocytogenes at 6°C, while lactate exerts a bacteriostatic effect during the whole storage period (three months) at 1°C. The most effective treatment for L. monocytogenes was the combination of HPP, lactate, and low-temperature storage. Absence of Salmonella in 50% of the samples was only achieved in the batches where HPP and nisin (800 AU/ml) were applied. When two different antimicrobials (enterocins and lactate-diacetate) were tested (Marcos et al., 2008), lactate-diacetate exerted a bacteriostatic effect against L. monocytogenes during the whole storage period (three months) at 1 and 6°C, even after temperature abuse. Nevertheless, the combination of low storage temperature (1°C), HPP, and the addition of lactate-diacetate was necessary to reduce the levels of L. monocytogenes during storage by 2.7 log CFU/g. The combination of enterocins at 2,400 AU/g, HPP, and 6°C storage temperature was not able to inhibit the growth of L. monocytogenes after the cold chain break. On the contrary, at 1°C the combination of HPP with enterocins was more effective than with lactate-diacetate, being able to reduce the population of the pathogen to final counts of 4 MPN/g after 3 months of storage, even after the cold chain break.

The effectiveness of the combination of HPP (600 MPa) with the natural antimicrobials nisin and potassium lactate applied in the meat batter of cooked ham has been evaluated in slices artificially inoculated with 4 log CFU/g of Salmonella sp., L. monocytogenes, and S. aureus after 3-month storage at 1° and 6°C (Jofre, Garriga, et al., 2008). In non-HPP sliced cooked ham, the addition of nisin plus lactate inhibited the growth of L. monocytogenes during the entire storage period, while the refrigerated storage inhibited the growth of Salmonella sp. and S. aureus. The application of HPP was necessary to reduce the levels of Salmonella and L. mono-cytogenes to <10 CFU/g, levels that were kept until the end of storage at both 1 and 6°C. HPP only reduced the counts of S. aureus less than 1 log cycle and the combination with nisin and refrigeration at 6°C was necessary to decrease the counts of S. aureus by 2.4 log CFU/g after 3 months of storage.

Differential efficiency of sprayed surface bacteriocin application combined with an HPP of 600 MPa were assessed when cooked ham was compared to cured meat for risk associated to Salmonella, L. monocytogenes, and S. aureus (Jofre, Aymerich, Monfort, et al., 2008). The decrease of L. monocytogenes counts was higher for cooked than cured meat products and for nisin than enterocins A and B and sakacin. Salmonella was not affected by the bacterio-cins. After 7 days of storage at 4°C, absence of both pathogens was achieved in all batches and kept until the end of storage, even in abusive temperature (15°C). For S. aureus, reductions were lower and only the application of nisin in dry-cured ham produced a significant reduction in the counts. Thus, at the end of storage, while S. aureus counts were <1 log CFU/g in all dry-cured ham batches, only nisin, as previously reported, could inhibit its growth in cooked ham. Cooked and dry-cured meat products have a similar pH but diferent aw. Water activity is recognized as playing an important role on HHP inactivation.

A different and promising way to apply antimicrobials is active packaging, an innovative concept that could be defined as a packaging system where the pack, the product, and the environment interact and change the condition of packed food, extending the shelf-life and improving the food safety or the sensorial properties of the product thus preserving its quality (European Commission, 2004; Suppakul, Miltz, Sonneveld, & Bigger, 2003; Vermeiren, Devlie-ghere, & Debevere, 2002). Application of enterocins A and B, sakacin K, nisin A, potassium lactate, and nisin plus lactate as interleaver together with an HHP treatment at 400 MPa was also evaluated in sliced cooked ham (Jofre; et al., 2007; Jofre, Aymerich, & Garriga, 2008). HHP produced an important reduction in Salmonella; however, the elimination of the pathogen was only achieved when nisin was absorbed in the interleaver. The interleaver application of nisin had a more long-lasting effect on L. monocytogenes than through its application to meat batter (4.5 log CFU/g at the end of 3-month storage when applied in the meat batter and only 1.2 log CFU/g when applied in the interleaver). On the contrary, potassium lactate was much more efficient when applied in meat batter than through interleavers (Fig. 7.3). Therefore, the antimicrobial complement of HHP treatment may depend on its application form and refrigerated storage. It is important to consider this when several hurdles must be applied in order to obtain value-added ready-to-eat products with a safe long-term storage. A further synergistic effect of the bacteriocins enterocins and HHP (400 MPa) against L. monocytogenes was observed when the antimicrobials were applied in biodegradable active packaging such as alginate films (Marcos et al., 2008). Three lots of cooked ham were prepared: control, packaging with alginate films, and packaging with antimicrobial alginate films containing enterocins (2000 AU/cm2). After packaging, half of the samples were pressurized. Sliced cooked ham stored at 6°C experienced a quick growth of L. mono-cytogenes. Both antimicrobial packaging and pressurization delayed the growth of the pathogen. However, at 6°C the combination of antimicrobial packaging and HPP was necessary to achieve a reduction of inoculated levels without recovery during 60 days of storage. Further storage at 6°C of pressurized

days

Fig. 7.3 Differential effect of antimicrobial application modi (nisin and lactate) against L. monocytogenes together with an HHP treatment of 400 MPa 10 min 17°C N, nisin; L, lactate; mb, meat batter; i, interleaver.

antimicrobial-packed cooked ham resulted in L. monocytogenes levels below the detection limit (day 90). On the other hand, storage at 1°C controlled the growth of the pathogen until day 39 in non-pressurized ham, while antimicrobial packaging and storage at 1°C exerted a bacteriostatic effect for 60 days. All HPP lots stored at 1°C kept the levels of 0.60 log CFU/g achieved after treatment after day 60 (Fig. 7.4). After a cold chain break no growth of

days

Fig. 7.4 Importance of the hurdle technology on L. monocytogenes behavior in sliced cooked ham stored in refrigeration for 60 days

E and ENT-enterocins- alginate film (2,000 AU/cm2 ). 1ENT, enterocins 1 °C; 6ENT, enter-ocins 6°C; 1EHHP, enterocins, HHP (400 MPa) 1°C; 6EHHP, enterocins, HHP (400 MPa) 6°C.

L. monocytogenes was observed in pressurized ham packed with antimicrobial films, showing the higher efficiency of combining both technologies (Marcos et al., 2008). In this system the microbial substance would gradually migrate from the pack (container) to the food through diffusion and partitioning or release through evaporation in the headspace during storage and distribution, thus being able to reduce the post-processing contaminations in the surface of the ready-to-eat products during storage (Han, 2005). Combination of active packaging with HPP may thus be considered as an alternative technique to increase the efficiency of these natural antimicrobials whose activity could be reduced by interaction with the food matrixes. When applied with biodegradable film, the technology could be even more environmentally friendly.

The combination of starter culture and HPP after ripening was recommended to produce low-risk and high-quality slightly fermented sausages (Garriga et al., 2005; Marcos et al., 2007). Starter cultures were able to control the growth of L. monocytogenes, S. aureus, Enterobacteriaceae, Enterococcus, and the biogenic amine content. HHP treatment (400 MPa) promoted an additional reduction of Enterobacteriaceae and L. monocytogenes counts and it was crucial to assess the absence of Salmonella spp. While starter cultures slightly modified the pH values and cohesivenes in fuet and increased in cohe-siveness, springiness, and chewiness but no change in TBARS or color parameters was observed after HHP.

Some other antimicrobials as TBHQ (100-300 ppm) and nisin (100 and 200 ppm) were reported in combination with HHP (600 MPa, 28°C) (Chung et al., 2005) in inoculated commercial sausage samples with 106-107 CFU/g barotolerant L. monocytogenes. Most of the samples treated with nisin, TBHQ, or their combination were positive for L. monocytogenes. HPP alone resulted in a modest decrease in the number of positive samples, but L. monocytogenes was not detected in any of the inoculated commercial sausage samples after treatment with HPP-TBHQ or HPP-TBHQ-nisin combinations. These results suggest that addition of TBHQ or TBHQ plus nisin to sausage followed by in-package pressurization is a promising method for producing Listeria-free RTE products.

Hurdle technology may also be applied to improve physico-chemical and sensorial properties. While raw chicken meat is oxidatively stable, HPP at 600 MPa and above induces lipid oxidation, resulting in off-flavors during subsequent cooking due to the presence of highly unsaturated fatty acids and low tocopherol content. Addition of 0.1% dried rosemary to minced chicken thighs or breasts prior to HPP inhibits lipid oxidation during subsequent cooking and could form the basis for product development (Bragagnolo, Danielsen, & Skibsted, 2007). Sage was also found to protect minced chicken breast processed with HPP up to 800 MPa for 10 min against lipid oxidation during subsequent chilled storage for 2 weeks. Garlic showed prooxidative additive effects especially at moderately high pressure around 300 MPa, an effect partly counteracted by simultaneous addition of sage (Mariutti, Orlien, & Bragagnolo, 2008).

Final Conclusions

High hydrostatic pressure is an alternative and industrially attractive (600 MPa for 5 min) non-thermal technology with higher potential of application to meat products. The technology may be used for hygienization of sliced cooked or cured meat products extending its shelf-life without major changes in sensorial properties. Nevertheless raw products are not so well suited to pressure treatment. HPP at low temperatures, i.e., frozen fresh meat is under study in order to avoid non-desired textural modifications. Moreover, the addition of several natural additives is being considered in order to avoid oxidative reaction as in poultry. However, more in-depth studies must be carried out in order to predict the microbial resistance and the role of bacterial stress during the shelf-life of the product. The treatment must be optimized and accurately defined with a view to legislation. Different stakeholders must interact to convince consumers of their convenience with objective and unbiased data including negative aspects and limitations. Nevertheless, as cells need adaptation before their growth, extension of their lag time by a combination of storage temperature-high-pressure treatment will result in a considerable delay before spoilage, and hence, the shelf-life before opening the package will be extended. Thus, and according to Smelt and Hellemons (1998) sublethal injury of microorganisms could also be a tool in food preservation. Moreover, the results of different investigations suggest that addition of different antimicrobials followed by in-package pressurization may improve the efficiency of the treatment and could be considered a good strategy to produce food-borne pathogen-free RTE products.

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Beauty for Newbies

Beauty for Newbies

Do you feel like an ugly duckling sometimes? Doesn't it seem like everyone else seems to know the best ways to present themselves, from their hair, to their skin, to their makeup?

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