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 cul- ture 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).