Evaluation of High Pressure Processing As an Additional Hurdle

JFS M: Food Microbiology and Safety

Evaluation of High Pressure Processing as an Additional Hurdle to Control Listeria monocytogenes and Salmonella enterica in Low-Acid Fermented Sausages BEGONYA MARCOS, TERESA AYMERICH, AND MARGARITA GARRIGA

ABSTRACT: Low-acid fermented sausages (fuet and chorizo) were manufactured to evaluate the combined effect of high pressure processing (HPP) and ripening on foodborne pathogens. Raw sausages inoculated with a three-strain cocktail of Salmonella ser. Derby, London, and Schwarzengrund, and a three-strain cocktail of L. monocytogenes ser. 1/2 c and 4b were pressurized at 300 MPa for 10 min at 17 °C. Afterwards, sausages were ripened at 12 °C and 80% RH for 27 d. Salmonella counts decreased in all studied sausages during ripening. However, the application of HPP as an additional hurdle to the ripening process produced a greater decrease in the Salmonella population, showing lower counts (3 MPN/g) in ripened sausages. By contrast, lower values of L. monocytogenes counts were obtained in non-treated (NT) than in pressurized sausages due to the delay of pH drop caused by HPP inactivation of endogenous lactic acid bacteria. After pressurization of raw sausages at 300 MPa, a discoloration of sausages was observed, coinciding with an increase in L* values. Keywords: low-acid fermented sausages, ripening, high pressure processing, Salmonella, L. monocytogenes

Introduction L. monocytogenes is a ubiquitous microorganism with an impor- ermentation has traditionally been used to achieve a preserva- tant occurrence in meat and meat products (Farber and Peterkin Ftion effect in foodstuffs. During fermentation, meat products 1991; Farber and Daley 1994; EC 1999). Moreover, the prevalence become more stable and increase their safety as a consequence of of L. monocytogenes has been detected in 10% of 60 surface sam- different hurdles (Leistner 1995). Acid fermented sausages can gen- ples investigated from Spanish meat industries after cleaning erally be considered low-risk products as a consequence of reduced (Garriga and others 2004). Although there is no epidemiological water activity and pH (4.8 to 5.0) that inhibit pathogenic bacteria evidence of the involvement of fermented sausages in recent even at ambient temperature (Barbuti and Parolari 2002). Among outbreaks of listeriosis (Talon and others 2004), L. monocytogenes Mediterranean countries, there is a preference for dry sausages with may survive in meat fermentations (Johnson and others 1988; a limited sour taste. Low-acid fermented sausage technology is based Glass and Doyle 1989; Junttila and others 1989). The Agence on small diameter (Յ 30 to 40 mm) sausages, and low ripening tem- Française de Sécurité Sanitaire des Aliments (2000) reported 12% peratures (Յ10 to 12 °C) to prevent an intense and rapid acidification to 16% Listeria-positive isolations in French fermented sausages. (Sanz and others 1998). The final pH values of this kind of product Aymerich and others (2003a) also reported the prevalence of low are about 5.3 to 6.2 (Aymerich and others 2003a). numbers of L. monocytogenes (4 MPN/g) in 29.4% of low-acid fer- Over the last few years, there has been an important increase in mented sausages studied. infections by foodborne pathogens, especially in cases of Salmonel- The prevalence of pathogens in fermented meats would require la and L. monocytogenes food poisoning. In Spain, the cases of in- the use of more hurdles to pathogen growth as suggested by Leist- fection by L. monocytogenes and Salmonella have increased since ner (2000). Research dealing with newly developing technologies, 1996 by 158% and 71%, respectively (CNE 1997, 2004). including hydrostatic pressure, would provide potential benefits to

The risk of Salmonella in fermented meats is generally consid- the fermented meat industry. M: Food Microbiology & Safety ered low (EC 2003). However, the survival of Salmonella in this type High pressure processing is a non-thermal food preservation of product has been demonstrated (Smith and others 1975; Levine method, appropriate for foods whose nutritional and sensorial and others 2001; Moore 2004). Several outbreaks due to Salmonella characteristics are thermosensitive (Carlez and others 1994). High in sausage-like products were reported (Van Netten and others pressure disrupts secondary and tertiary structures of macromol- 1986; Cowden and others 1989; Pontello and others 1998). Recently ecules, such as proteins and polysaccharides, and alters their struc- an outbreak linked to consumption of fermented sausages with a tural and functional integrity in a pressure-dependent way. Micro- short fermentation period was described (Bremer and others 2004). organisms are killed owing mainly to membrane damage (Kalchayanand and others 1998). In this study, high pressure processing was applied to raw sau- MS 20040738 Submitted 11/9/04, Revised 5/16/05, Accepted 6/2/05. Authors are with Inst. for Food Research and Technology (IRTA), Meat Technology sages inoculated with S. enterica and L. monocytogenes. The objec- Center, Granja Camps i Armet, 17121 Monells, Spain. Direct inquiries to tive was to evaluate the combined effect of pressurization and rip- author Garriga (E-mail: [email protected]). ening on pathogen survival and to consider its commercial use.

© 2005 Institute of Food Technologists Vol. 70, Nr. 7, 2005—JOURNAL OF FOOD SCIENCE M339 Further reproduction without permission is prohibited Published on Web 8/19/2005 High pressure in low-acid sausages . . .

Materials and Methods A PCR pretreatment was applied. Two milliliters of enriched culture were centrifuged at 4 °C for 5 min at 12500 × g. The pellet was Sausage manufacture suspended in 300 ␮L 6% Chelex® 100 (Bio-Rad Laboratories, Her- Two types of low-acid fermented sausages, fuet and chorizo, were cules, Calif., U.S.A.), incubated at 56 °C for 20 min, boiled for 10 min, manufactured. Both products were made with 50% lean pork meat and cooled on ice. Mixing between each step was needed. Cell debris and 50% pork back fat. The ingredients of the fuet formulation were were centrifuged at 4 °C for 5 min at 24600 × g. Twenty microliters of as follows (g/kg): sodium chloride 20, black pepper 2.5, potassium the supernatant were used for PCR reaction. nitrate 0.1, sodium nitrite 0.1, dextrose 1, and sodium ascorbate 0.5. The ingredients of the chorizo formulation were (g/kg): sodium chlo- PCR reactions ride 20, cayenne pepper 15, paprika 15, dextrose 1, and garlic 3. A validated PCR identification protocol of Salmonella and L. Batters were inoculated with 6 × 102 colony-forming units (CFU)/ monocytogenes was used (European Commission FOOD-PCR: g of a three-strain cocktail of Listeria monocytogenes (L. monocyto- D’Agostino and others 2004; Malorny and others 2003). genes ser. 1/2 c CTC1010, L. monocytogenes ser. 1/2 c CTC1011, L. Twenty microliters of either colony suspended in distilled water monocytogenes ser. 4b CTC1034) and 6 × 102 CFU/g of a three-strain or MPN origin were mixed with 2 ␮L of buffer 10x (Invitrogen, Carls- ␮ ␮ cocktail of Salmonella enterica (S. enterica sp. enterica ser. Derby bad, Calif., U.S.A.), 1.5 mM MgCl2, 150 M of each dNTP, and 0.3 M CTC1022, S. enterica sp. enterica ser. London CTC1003, S. enterica of each primer (139 and 141 for Salmonella: Rahn and others 1992; sp. enterica ser. Schwarzengrund CTC1015). All strains were isolated Lip1 and Lip2 for L. monocytogenes: Simon and others 1996), 1 mg/ from meat products. L of BSA, and 1 U of Platinum Taq DNA polymerase (Invitrogen). Lean pork meat and pork back fat were minced in a meat mincer Amplification was performed in a GeneAmp PCR System 2700 (Ap- with an adjustable plate set at a hole diameter of 6 mm, mixed with plied Biosystems, Foster City, Calif., U.S.A.) with the following pro- other ingredients in a kneading machine and stuffed into collagen gram: 30 s at 95 °C, 30 s at 64 °C, and 30 s a 72 °C during 38 cycles, casings. with a final extension of 4 min at 72 °C. The reaction was visualized on an agarose gel (1.5% agarose and 0.5 ␮g/mL ethidium bromide) High pressure processing (HPP) after 30 min of electrophoresis at 100 V. Raw sausages were vacuum packed in polyamide-polyethylene bags (Sacoliva, Castellar del Vallè, Spain) and subjected to pres- pH and water activity measurements sure, the day after stuffing. Pressure was applied in an industrial pH determinations were taken using a Crison Basic 20 pH- hydrostatic pressurization unit (Alstom, Nantes, France) with a meter, equipped with a Crison penetration 52-32 electrode (Crison chamber of 320 L volume and 280 mm diameter. Sausages were Instruments, Alella, Spain). Water activity was measured at 25 °C treated at 300 MPa for 10 min at 17 °C. The come-up time was about using a Novasina Thermoconstanter TH-500 (Novasina, Pfäffikon, 9 min and the pressure release time was 1.5 min. Switzerland). After pressure treatment, plastic bags were removed and both non-treated (NT) and pressurized (HPP) sausages were ripened Instrumental color analysis under the same conditions. Color measurements were performed using a Minolta Chroma- meter CR200 (Minolta, Tokyo, Japan). C illuminant and 0° standard Ripening conditions observer were chosen. L* (brightness), a* (redness), and b* (yellow- Sausages (fuet and chorizo) were hung in a climate chamber ness) color values were determined in the 1976 CIELab system. The Sanyo MLR-350 H, and dried for 27 d at 12 °C and 80% RH. At select- colorimeter was calibrated before each series of measurements ed times—after stuffing (day 0), after pressurization (day 1), and using a white ceramic plate. Each measurement was repeated at during ripening (days 6, 13, and 27)—3 different sausages from least 6 times. each treatment (pressurized and non-treated) were analyzed. M: Food Microbiology & Safety Statistical analysis Microbial analysis Data were subjected to analysis of variance of the General Linear At each selected time, 20 g sausage were 10-fold diluted in ster- Model procedure of SAS software (SAS System for Windows NT, re- ile 0.1% peptone water (PW) (Difco Laboratoriess, Detroit, Mich., lease 8.1, SAS Inst., Cary, N.C., U.S.A.) where product, treatment, U.S.A.) and 0.85% Na Cl (Merck, Darmstadt, Germany). The solu- time, and interaction between these parameters were included as tion was homogenized for 1 min in a Masticator (IUL Instruments, fixed effects. Level significance was set for P < 0.05. Barcelona, Spain) and serially diluted in PW. Enumeration was done in selective media: lactic acid bacteria Results and Discussion (MRS agar, Merck) incubated anaerobically at 30 °C for 72 h; L. monocytogenes (PALCAM agar base and PALCAM Listeria Selective Water activity and pH

Supplement according to Van Netten and others 1989, Merck) incu- The water activity (aw) of sausages decreased throughout the bated at 30 °C for 72 h; Salmonella (BGA agar, Difco) incubated at 37 drying process from an initial value of 0.98 to values of 0.83 to 0.86

°C for 24 to 48 h. Five presumed colonies of L. monocytogenes and (data not shown). The decrease of aw was as expected for this kind Salmonella from respective media were retained for polymerase of product (Baracco and others 1982; Lizaso and others 1999). No

chain reaction (PCR) confirmation. The most probable number significant differences (P > 0.05) were found between aw values of (MPN) technique was used when counts under 10 CFU/g were ex- non-treated (NT) and pressurized sausages during drying. There- pected. Serial dilutions of 3 or 5 PW tubes of 3 successive dilutions fore, high pressure treatment did not affect the normal evolution of

were incubated at 30 °C for 48 h. Peptone water was used as non- aw during sausage ripening. selective medium to recover the sublethally injured bacteria with- From the beginning of the process lower pH values were obtained out underestimating the counts. The growth of L. monocytogenes in chorizo than in fuet, which can be related to the acidity of paprika and Salmonella in MPN tubes was determined by validated species- and cayenne pepper added to chorizo (data not shown). The pH of specific PCR assays. non-treated sausages decreased from an initial range of 5.7 to 5.8 in

M340 JOURNAL OF FOOD SCIENCE—Vol. 70, Nr. 7, 2005 URLs and E-mail addresses are active links at www.ift.org High pressure in low-acid sausages . . . the meat batter to about 5.3 in the final product (Figure 1). Minimum Carlez and others (1993) reported no variation in a* values at pH values of 5.17 (chorizo) and 5.32 (fuet) were reached at day 6 of pressures below 400 MPa, and no variation of b* values at any pres- ripening. High pressure processing had a significant effect (P < 0.05) sure applied; however, Shigehisa and others (1991) observed a on the sausage pH. pH values increased 0.13 (fuet) and 0.15 (chorizo) moderate decrease of a* values between 100 and 200 MPa that points as a consequence of pressurization at 300 MPa. Mandava and progressed up to 600 MPa. Ananth and others (1998) did not detect others (1994) reported an increase of pH in raw sausages pressurized changes of any of the color parameters studied when pressurizing above 200 MPa, as a consequence of protein denaturation. Macfar- raw loin pork for 13 min at 414 MPa and 25 °C. lane and others (1980) attributed the pH rise to a loss of protons as a consequence of increased ionization by HPP and consequent redis- Microbial results tribution of ions. Interactions between treatment and time were sig- Figure 3 illustrates the evolution of lactic acid bacteria (LAB) pop- nificant (P < 0.05), suggesting that HPP affected not only the pH ulation during ripening. At day 6 of drying, coinciding with mini- values but also the evolution of pH during ripening. Minimum pH mum pH values, LAB reached maximum growth levels (about 109 values of HPP sausages 5.32 (chorizo) and 5.47 (fuet) were obtained CFU/g) that were maintained until the end of the process. High at d 13 and stayed at this level until the end of ripening. initial counts of endogenous LAB (about 106 CFU/g), close to the level usually inoculated as starter cultures, assured the rapid and Instrumental color analysis intense pH decrease in NT sausages during the first day of drying. NT fuet showed the typical red color from raw meat, while the LAB population experienced an immediate reduction of 3 and 4 red color of non-treated chorizo was provided by paprika and cay- log CFU/g in fuet and chorizo, respectively, after treatment at 300 enne pepper. Pressurization (300 MPa) after stuffing induced visu- MPa. Carlez and others (1994) detected lower inactivation of lactic al changes. A discoloration toward a pale pink color was observed in acid bacteria (1 log CFU/g) when processing minced meat at 300 fuet, and coloration of chorizo also became paler. Carlez and others MPa (20 min, 20°C), while pressurization at 400 MPa (20 min, 20°C) (1995) also reported the red color of minced beef meat turning into showed a 4- to 5-log reduction. a much paler pink color at pressures ranging between 200 to 350 After pressurization, a rapid recovery of LAB viability was ob- MPa. The “whitening” effect of pressure was attributed to globin served (Figure 3). At day 6, LAB counts of HPP sausages were about denaturation and/or to heme displacement or release. 7 log CFU/g, and by day 13, they had already reached the same The observed discoloration was numerically confirmed by an levels as NT sausages. Similarly, Garriga and others (2002) reported increase in brightness (L*) (P < 0.001) as a consequence of pressur- the recovery of LAB to levels close to initial ones during chilled stor- ization (Figure 2). Goutefongea and others (1995) related the in- age of pressurized meat homogenates (400 MPa for 10 min at 17 °C). crease of L* values by pressure either to a loss of active pigment or The growth of L. monocytogenes, inoculated at a level of 6 × 102 to protein coagulation, affecting structure of the sample and sur- CFU/g, was prevented in all sausages (NT and HPP) during drying. face properties, that, in turn, may have increased the share of re- The growth limit of L. monocytogenes in foods has been fixed at flected light versus absorbed light. An increase in L* values was also values of aw below 0.95 and pH below 5.5 (FSIS 2002). Both values reported by Carlez and others (1995) in minced beef meat at pres- were reached during ripening, thus the physiochemical conditions sures ranging between 200 and 350 MPa. Shigehisa and others during the process prevented the growth of L. monocytogenes. (1991) observed that L* values of pork muscle homogenates started Ripening reduced L. monocytogenes counts in NT sausages by 2- to increase between 100 and 200 MPa, to reach a maximum L* val- log units (Figure 4), a reduction mainly due to the rapid decrease of ue between 300 and 400 MPa. No further increase was observed pH values. After 27 d of drying (end product) L. monocytogenes lev- even at 600 MPa. els were below 3 MPN/g in fuet and absence in 25 g was reached in a* (redness) values were not altered (P > 0.05) as a consequence chorizo. The absence of L. monocytogenes found in chorizo could be of HPP. b*(yellowness) values of fuet were reduced significantly explained by the presence of factors with antilisterial activity in (P < 0.05) by pressure, whereas no changes were observed in cho- such products. In fact, 95% of enterococci isolated from non-treat- rizo (P > 0.05). Variations of a* and b* values have been related to ed chorizo showed activity against L. monocytogenes in vitro, while changes in the chemical state of myoglobin. The presence of red col- none of the enterococci isolated from non-treated fuet showed this orants in chorizo could have hidden the possible alterations of myoglobin occurring in the meat. M: Food Microbiology & Safety

Figure 2—Color parameters, L* (brightness), a* (redness), and b* (yellowness), of NT and HPP sausages after treatment at 300 MPa (day 1). Different letters indicate signifi- Figure 1—pH values of NT and HPP sausages during rip- cant differences (P < 0.05) between NT and HPP in fuet, ening. Bars indicate standard deviation of triplicates. (a and b), and in chorizo (y and z).

URLs and E-mail addresses are active links at www.ift.org Vol. 70, Nr. 7, 2005—JOURNAL OF FOOD SCIENCE M341 High pressure in low-acid sausages . . .

effect. This finding is consistent with reports of several authors who The reduction of L. monocytogenes in pressurized fuet reflects described the antilisterial activity of bacteriocins produced by en- that the addition of nitrate and nitrite influenced L. monocytogenes terococci (Aymerich and others 1999; Callewaert and others 2000). survival during sausage fermentation, as observed by other au- HPP reduced L. monocytogenes counts by 1 log CFU/g (Figure thors (Junttila and others 1989; Duffy and others 1994). In addi- 4). A similar reduction (1.5 log CFU/g) was reported by Styles and tion, the nitrate and nitrite effect would have been maximized by others (1991) in Ultra High Temperature (UHT) milk treated at 340 high pressure, which makes sublethally injured cells more sensitive MPa (20 min, 23 °C), whereas much higher inactivation (7 log CFU/ to the presence of antimicrobial agents (Kalchayanand and others g) was observed in phosphate buffer. A rapid recovery of L. mono- 1994). cytogenes was observed after pressurization. From day 1 (after HPP) In the ripened sausages, counts of L. monocytogenes (P < 0.05) to day 6 of ripening L. monocytogenes counts increased 0.9 log were lower in NT than in HPP sausages. This fact could be attributed CFU/g in fuet and 1.8 log CFU/g in chorizo. The recovery of L. to the delay of pH drop in HPP sausages due to lactobacilli inacti- monocytogenes after pressurization has been reported in other vation. HPP also affected endogenous enterococci with reduction meat products. Garriga and others (2000, 2002) detected a recovery of antilisterial activity from 95% (NT chorizo) to 27% (HPP chorizo). of L. monocytogenes, even in chilled storage, after HPP of meat Thus, high pressure processing of raw sausages in the studied con- homogenates at 400 MPa. ditions had a counter-productive effect on L. monocytogenes con- There was a decrease in L. monocytogenes during ripening of trol as a consequence of LAB inactivation. The addition of a bacte- pressurized fuet from initial counts of 2.26 log CFU/g to final values riocin producer starter culture resistant to pressure would prevent of 1.42 CFU/g. By contrast, no changes in L. monocytogenes (P > inactivation of LAB by HPP and would improve the reduction of L. 0.05) were detected in pressurized chorizo after 27 d of ripening. monocytogenes counts. This result suggests that higher pressure levels should be applied The population of Salmonella decreased significantly during the to eliminate L. monocytogenes cells in the end product. Carlez and manufacture of both pressurized and non-treated sausages (Fig- others (1993) reached complete inactivation of L. innocua in minced ure 5). Minimal growing conditions of Salmonella are pH of 4 to 4.5 beef muscle at 400 MPa, and Yuste and others (1999) reported sig- and water activity of 0.95 (ICMSF 1996; EC 2003). Thus, inhibition nificant inactivation of L. innocua in poultry sausages pressurized of Salmonella in the manufacturing conditions (pH > 5.0) was main- above 350 MPa. ly obtained by drying and not by acidification as usually happens in acid fermented sausages (Talon and others 2004). During ripening of NT sausages, Salmonella population decreased 1 logarithm, from 2.45 log CFU/g to 1.35 CFU/g in fuet and from 2.67 log CFU/g to 1.62 log CFU/g in chorizo. Until day 6, Sal- monella counts were higher in chorizo, with no added nitrite and nitrate, compared with fuet. From day 13 until the end of the process no differences in Salmonella (P > 0.05) were found between both sausage types. Bard and Townsend (1976) attributed a short life to the nitrite hurdle, because of the microbial nitrite depletion during fermentation. After pressurization, no immediate reduction of Salmonella counts was observed. By contrast, inactivation of S. enteriditis (2-log reduction) was reported by Aymerich and others (2003b) in cooked ham pressurized at 400 MPa (10 min, 17 °C). A higher inactivation of S. enteriditis (5-log reduction) was observed by Patterson and others (1995) when pressurizing inoculated phosphate buffer at M: Food Microbiology & Safety 450 MPa. This suggests the baroprotective effect of food constitu- Figure 3—Lactic acid bacteria counts of NT and HPP sau- ents, which increase the ability of bacteria to survive under high sages during ripening. Bars indicate standard deviation of pressures (Hoover and others 1989; Ponce and others 1998). triplicates. The pressure effect was observed later, when dehydration was

Figure 4—L. monocytogenes counts of NT and HPP sausages during ripening. Bars indicate standard deviation of Figure 5—Salmonella counts of NT and HPP sausages dur- triplicates. ing ripening. Bars indicate standard deviation of triplicates.

M342 JOURNAL OF FOOD SCIENCE—Vol. 70, Nr. 7, 2005 URLs and E-mail addresses are active links at www.ift.org High pressure in low-acid sausages . . . more pronounced. By day 6 of ripening, when a was 0.96, the Sal- D’Agostino M, Wagner M, Vazquez-Boland JA, Kuchtat T, Karpiskiva R, Hoorfar J, w Novella S, Cortti MS, Ellison J, Murray A, Fernandes I, Kuhn M, Pazlarova J, monella population of HPP sausages started to decrease. Whereas Heuvelinki A, Cook N. 2004. A validated PCR-based method to detect Listeria monocytogenes using raw milk as a food model—Toward an international counts of NT sausages did not decrease until day 13, when aw was standard. J Food Prot 67(8):1646–55. about 0.92. These results are consistent with the affirmation that Duffy LL, Vanderlinde PB, Grau FH. 1994. Growth of Listeria monocytogenes on sublethally injured cells become more sensitive to low water activ- vacuum-packed cooked meats: effects of pH, aw, nitrite and ascorbate. Int J Food Micro 23:377–390. ity (Smelt 1998). From day 6 and until the end of ripening, Salmo- [EC] European Commission. 1999. Opinion of the scientific committee on veter- nella counts of HPP sausages were lower (P < 0.05) than those of NT inary measures relating to public health on Listeria monocytogenes. Brussels: sausages (Figure 5). Pressurized sausages showed a 2-log reduction EC. Available from: http://europa.eu.int/comm/food/fs/sc/scv/out25_en.pdf. Accessed Dec 1, 2004. of Salmonella levels during the process, showing low counts (3 [EC] European Commission. 2003. Opinion of the scientific committee on veter- MPN/g) in the end product. inary measures relating to public health on Salmonellae in foodstuffs. Brus- sels: EC. Available from: http://europa.eu.int/comm/food/fs/sc/scv/ out66_en.pdf. Accessed Dec 1, 2004. Conclusions Farber JM, Peterkin PI. 1991. Listeria monocytogenes: A food-borne pathogen. Microbiol Rev 55:476–86. he ripening process at 12 °C and 80% RH for 27 d was effective Farber JM, Daley E. 1994. Presence and growth of Listeria monocytogenes in Tin preventing the growth of Salmonella and L. monocytogenes. naturally-contaminated meats. Int J Food Microbiol 22:33–42. [FSIS] Food Safety and Inspection Service. 2002. Microbial sampling of ready- Pressure treatment of raw sausages at 300 MPa (10 min, 17 °C) was to-eat (RTE) products for the FSIS verification testing program. Washington, an additional hurdle to Salmonella control. However, HPP before D.C.: United States Dept. of Agriculture. Garriga M, Aymerich T, Costa S, Monfort JM, Hugas M. 2000. Las altas presiones ripening had a negative effect on L. monocytogenes control as a con- en combinación con bacteriocinas como nueva tecnología de conservación sequence of LAB inactivation. en productos cárnicos. Eurocarne 87:59–63. Pressurization at 300 MPa before ripening is not commercially Garriga M, Aymerich T, Costa S, Monfort JM, Hugas M. 2002. Bacterial synergism through bacteriocins and high pressure in a meat model system during stor- viable because it alters the visual aspect of sausages significantly. age. Food Microbiol 19(5):509–18. Further studies are in progress on pressurization after ripening at Garriga M, Fadda S, Aymerich T, Hugas M. 2004. Evaluation of the hygienic status of traditional dry sausage workshops in Catalonia. Paper presented at the 2nd higher pressure levels, to improve L. monocytogenes control and Intl. Conference on Food Factory of the Future. 2004 Oct 6-8; Laval, France: HPP commercial use. Laval Mayenne Technopole. Glass KA, Doyle MP. 1989. Fate of Listeria monocytogenes in processed meat products during refrigerated storage. Appl Environ Microbiol 55(6):1565–9. Acknowledgments Goutefongea R, Rampon V, Nicolas J, Dumont JP. 1995. 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