MEAT SCIENCE Meat Science 74 (2006) 303–311 www.elsevier.com/locate/meatsci
Effect of irradiation of frozen meat/fat trimmings on microbiological and physicochemical quality attributes of dry fermented sausages I. Chouliara a, J. Samelis b, A. Kakouri b, A. Badeka a, I.N. Savvaidis a, K. Riganakos a, M.G. Kontominas a,* a
Laboratory of Food Chemistry and Technology, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece b National Agricultural Research Foundation, Dairy Research Institute, Katsikas, 45221 Ioannina, Greece Received 12 December 2005; received in revised form 24 March 2006; accepted 24 March 2006
Abstract Changes in microbiological and physicochemical quality attributes resulting from the use of irradiation in the production of Greek dry fermented sausage were investigated as a function of fermentation/ripening time. Results showed that irradiating meat/fat trimmings at 2 or 4 kGy prior to sausage production eliminated natural contamination with Listeria spp., and reduced pseudomonads, enterococci and pathogenic staphylococci, and enterobacteria, to less than 2 and 1 log cfu g 1, respectively. Pseudomonads were very sensitive (>3.4 log reduction) to either radiation dose. Yeasts were the most resistant followed by inherent lactic acid bacteria; their reductions on the trimmings were radiation dose-dependent. Residual effects of irradiation were noted against enterococci, but not against gram-negatives which died off fast during fermentation even in non-irradiated samples. Growth of the starter bacteria, Lactobacillus pentosus and Staphylococcus carnosus, inoculated in the sausage batters post-irradiation was unaffected by the 2 or 4 kGy pre-treatment of the trimmings. Irradiation had little or no effect at the end of ripening period (28 days) on pH, moisture content and color (parameters L*, a*, and b*). Changes in TBA values were small but statistically significant with irradiated samples having higher TBA values than control samples. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Fermented sausage; Salami; Irradiation; Quality; Hygiene
1. Introduction An increased incidence of recent outbreaks of illness (Bremer et al., 2004; CDC, 1995; Moore, 2004; Sauer, Majkowski, Green, & Eckel, 1997) as well as many challenge studies (Cosansu & Ayhan, 2000; Farber, Daley, Holley, & Usborne, 1993; Glass, Loeffelholz, Ford, & Doyle, 1992; Lahti, Johansson, Honkanen-Buzalski, Hill, & Nurmi, 2001; Nissen & Holck, 1998; Thevenot, Delignette-Muller, Christieans, & Vernozy-Rozand, 2005) have shown that important meat–borne pathogens, such as Escherichia coli O157:H7, Salmonella and Listeria mono-
*
Corresponding author. Tel.: +30 2651098342; fax: +30 2651098795. E-mail address:
[email protected] (M.G. Kontominas).
0309-1740/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2006.03.021
cytogenes, may survive and pose a health risk in dry fermented sausages. As a response to the 1994 Washington– California hemorrhagic colitis outbreak first linked with salami consumption in the US (CDC, 1995), the USDA/ FSIS established the requirement of a 5-log reduction in E. coli O157:H7 for fermented sausages. This mandate and its updates (Billy, 1997) have affected traditional procedures of producing fermented sausages, which include the maintenance of a low pH and low water activity achieved both by fermentation and drying to control spoilage and pathogenic microorganisms (Lucke, 2000). Numerous validation studies, recently reviewed by Getty, Phebus, Marsden, Fung, and Kastner (2000), have clearly shown that traditional processes for fermented sausages can not deliver a 5-log reduction in E. coli O157:H7 unless additional processing steps, such as a post-fermentation thermal
304
I. Chouliara et al. / Meat Science 74 (2006) 303–311
treatment, are employed. However, post-process thermal treatments may adversely affect the sensorial quality of the final product (Johnson, Sebranek, Olson, & Wiegand, 2000), while neither such treatments can be applied pre-fermentation due to substantial denaturation of meat proteins in fresh batters (Fernandez-Martin, Fernandez, Caballo, & Colmenero, 1997). Overall, thermal processes are not desired in, and cannot be accepted for, the production of high quality traditional salamis produced in many European countries (Lucke, 2000), including Greece (Samelis, Metaxopoulos, Vlassi, & Pappa, 1998). Thus, alternative non-thermal processes are needed to achieve an equivalent level of product safety; the use of properly decontaminated, pathogen-free raw materials for dry sausage manufacture may provide a feasible option. However, chemical meat decontamination technologies, such as spraying with organic acids, currently applied commercially in the US (Huffman, 2002) are still not permitted in EU. On this basis, physical technologies, such as irradiation or high hydrostatic pressure, may be better options for pathogen control on raw meat trimmings or sausage batters compared to chemical decontamination (Prochaska, Ricke, & Keeton, 1998). Although irradiation has been studied extensively over the past decades and its bactericidal effects have been demonstrated in both fresh and cooked meat products (Farkas, 1997, 2001; Lee, Sebranek, Olson, & Dickson, 1996; Maxcy, 1983; Sommers et al., 2004), very limited information is available on its use in fermented sausages, mainly focused on E. coli O157:H7 control (Dickson & Maxcy, 1985; Johnson et al., 2000; Samelis, Kakouri, Savvaidis, Riganakos, & Kontominas, 2005). Although irradiation can provide pathogen-free raw materials, its application in fermented sausage production may be hampered by its known adverse effects on quality characteristics of fresh or processed meats, including color defects, generation of volatile sulfur compounds and hydrocarbons, production of free radicals and the potential for oxidative reactions resulting in lipid oxidation, and off-odors and off-flavors (Ahn & Lee, 2004; Brewer, 2004; Sommers et al., 2004). Similar defects may be caused in fermented sausages made from pre-irradiated raw materials, and this potential limitation needs to be addressed in parallel studies with pathogen control (Johnson et al., 2000). The present work, which constitutes part of previous research (Samelis et al., 2005), evaluated changes in both microflora and selected physicochemical and quality attributes as a result of pre-irradiation of raw materials used for Greek dry fermented sausage production. 2. Materials and methods 2.1. Fermented sausage production Sausage samples made from irradiated (2 or 4 kGy) or non-irradiated meat/fat trimmings were produced in a local meat processing plant (VIKI S.A., Filippiada, Greece), as described previously (Samelis et al., 2005).
Briefly, for each irradiation treatment, 8 kg of mixed trimmings made from frozen pork (40%) and beef (30%) meat and pork back fat (30%) were divided in 2-kg portions in plastic bags, vacuum packaged, pressed to form flat blocks of approximately 6 cm thickness and stored in a freezer at 25 °C for 1 week prior to irradiation. Then, the blocks were transferred to the irradiation unit (EL.VIO.NI. Co., Mandra, Attiki) and irradiated frozen under a Cobalt-60 radiation source at 2 or 4 kGy. In each experiment, a series of non-irradiated (0 kGy) blocks served as control. After irradiation, the trimmings were returned to the meat plant and formulated into sausages of 45 mm in diameter at pilot-scale, according to standard manufacturer practices and with the use of a starter culture (Bactoferm T-SL, Chr. Hansen, Pohlheim, Germany). This starter, consisting of Lactobacillus pentosus and Staphylococcus carnosus, was inoculated in the trimmings at a concentration of ca. 7 log cfu g 1; the addition of curing agents and other ingredients, mixing, stuffing, fermentation, and ripening of the sausages were according to Samelis et al. (2005). No special cleaning and sanitizing measures were taken during sausage manufacture in the pilot plant to simulate commercial conditions. Precautions were only that the equipment used was rinsed thoroughly between treatments, and the sausage batches were formulated in the order from the highest (4 kGy) to the lowest (0 kGy) irradiation dose to avoid false results due to accidental microbial contamination of the more with less severe treatments. 2.2. Sampling and analyses Meat/fat trimmings were analyzed microbiologically before and after irradiation. Sausages manufactured from trimmings irradiated at 0, 2, and 4 kGy were analyzed microbiologically and physicochemically at 0, 4, 7, 14, 21, and 28 days after formulation. For microbiological analysis, 25 g of sample were transferred aseptically to 225 ml of sterile 0.1% (w/v) bacteriological peptone (LAB M, Bury, Lancashire, UK) water, and homogenized in a stomacher (Lab Blender, Seward, London, UK) for 1 min at low speed and 1 min at high speed at room temperature. Serial decimal dilutions in 0.1% peptone water were prepared and duplicate 1 ml or 0.1 ml samples of appropriate dilutions were poured or spread on all purpose and selective agar plates. Unless otherwise stated, all media and supplements were purchased from LAB M (Bury, UK). Total viable counts (TVCs) were determined on tryptone soy agar with 0.6% yeast extract, incubated at 30 °C for 72 h; lactic acid bacteria (LAB) on de Man, Rogosa, Sharpe (MRS) agar, incubated at 30 °C for 72 h under anaerobic conditions (GasPack System, BBL, Becton Dickinson, Sparks, MD, USA); pseudomonads on Pseudomonas agar base supplemented with cephalothin-fucidin-cetrimide (CFC; supplement X108), incubated at 25 °C for 48 h; total enterobacteria on violet red bile glucose agar, overlayed with 5 ml of the same medium and incubated at 37 °C for
I. Chouliara et al. / Meat Science 74 (2006) 303–311
24 h; enterococci on kanamycin aesculin azide agar, incubated at 37 °C for 48 h; Micrococcaceae on mannitol salt phenol-red agar (Merck, Darmstadt, Germany), incubated at 30 °C for 72 h; pathogenic staphylococci on Baird–Parker agar base supplemented with egg yolk tellurite (supplement X085), incubated at 37 °C for 48 h; yeasts on rose bengal chloramphenicol agar (Merck), incubated at 25 °C for 5 days. The selectivity of some of the above microbiological media was checked with appropriate rapid tests to ensure microbial enumeration data (Samelis et al., 1998). Specifically, the agar surface of CFC plates was flooded with the Kovacs’s oxidase test reagent solution, and only the oxidase-positive (purple-colored) colonies were counted. The presence of Micrococcaceae on mannitol salt agar was tested by microscopic colony examination followed by flooding of the plates with a 3% hydrogen peroxide solution (Merck) to ensure a catalase-positive reaction for the colony types enumerated. Staphylococcal colonies showing lecithinase activity on Baird–Parker’s medium were confirmed with a rapid agglutination test (Staph Microscreen, Microgen Bioproducts, Camberley, UK). Also, the presence of Salmonella and Listeria spp. was determined in the trimmings before and after irradiation by culture enrichment of 25 g samples in appropriate broth media followed by streaking on selective agar media and confirmation of 3–5 characteristic colonies by the API 20E or the API Listeria identification kits (BioMerieux, Marcy l’ Etoile, France), respectively, as reported previously (Samelis et al., 1998). The pH of each sausage sample was determined by immersing the pH electrode in the stomacher bag after samples were plated. A Crison digital pH meter (model Micro-pH 2001, Alella, Spain) equipped with a glass electrode was used for the measurement. The moisture content of the sausages was determined according to AOAC (1995). TBA was determined according to Kirk and Sawyer (1991) while color was evaluated using a HunterLab model DP 25 L colorimeter (HunterLab Assoc., Reston, VA, USA) according to Johnson et al. (2000).
305
2.3. Statistical analysis Experiments were replicated twice by analyzing duplicate samples per replicate. Microbiological counts were converted to log cfu g 1 and subjected to analysis of variance (Statistical Graphics Corp., Rockville, MD, USA). Independent variables included treatments and time, as well as treatment–time interactions. Means and standard deviations were calculated, and, when F-values were significant at the P < 0.05 level, mean differences were separated by the least significant difference (LSD) procedure. 3. Results and discussion 3.1. Effect of irradiation on the microflora of meat/fat trimmings Mixed meat/fat trimmings contained a TVC level of 6 log cfu g 1 before irradiation, with pseudomonads being the most predominant followed by LAB and yeasts (Table 1). This suggested that preparation of the trimmings in the plant’s fermented sausage area might have been contaminated with ca. 1-log higher populations of spontaneous LAB and yeasts than those normally found on fresh meat. The hygienic quality of the trimmings before irradiation was good, as total enterobacteria, enterococci and pathogenic staphylococci were slightly above 2 log cfu g 1, while Salmonella were absent. However, Listeria spp. were present in non-irradiated trimmings (Table 1), confirming previous findings on the high incidence of listeriae in Greek meat processing plants, and in fermented sausage batters in particular (Samelis & Metaxopoulos, 1999; Samelis et al., 1998). In this study, 2 out of 10 characteristic Listeria colonies isolated after streaking of enriched non-irradiated samples on PALCAM plates were identified as L. monocytogenes, while the others as L. innocua (data not shown). Irradiation of trimmings significantly reduced (P < 0.05) populations of all types of natural contaminants; pseudomonads showed the highest sensitivity while yeasts were
Table 1 Effect of ionizing radiation doses of 2 or 4 kGy on the populations (log cfu g 1) of natural contaminants present in meat/fat trimmings used for Greek dry salami production before and after irradiation Contaminant type
No irradiation
Irradiated at 2 kGy
Irradiated at 4 kGy
Total viable count Lactic acid bacteria Micrococcaceae Yeasts Pseudomonads Total enterobacteria Enterococci Pathogenic staphylococci Listeria spp. Salmonella spp.
6.1 ± 0.1 4.8 ± 0.1 3.5 ± 0.4 4.4 ± 0.2 5.4 ± 0.5 2.6 ± 0.2 2.4 ± 0.3 2.3 ± 0.5 + –
4.5 ± 0.5 3.6 ± 0.3 2.0 ± 0.1 4.3 ± 0.4 <2.0 a <1.0 a <2.0 a <2.0 a – –
3.9 ± 0.2 a 2.3 ± 0.2 a <2.0 a 3.7 ± 0.1 a <2.0 a <1.0 a <2.0 a <2.0 a – –
c c c b b b b b
b b b ab
Each value is mean ± SD of two replicate experiments with two samples analyzed per replicate (n = 4). Means with different lowercase letters in the same row are significantly different (P < 0.05). +, Positive in 25 g samples by culture enrichment; -, negative in 25 g samples by culture enrichment.
306
I. Chouliara et al. / Meat Science 74 (2006) 303–311
the most resistant followed by LAB (Table 1). Most importantly, irradiation at either dose reduced populations of enterobacteria, enterococci, and pathogenic staphylococci to levels below 1, 2 and 2 log cfu g 1, respectively. In addition, Listeria spp. became undetectable in 25 g of irradiated trimmings. Thus, irradiation at 2 or 4 kGy had a significant decontamination effect on, and improved the hygienic quality of, the trimmings. In particular, it is encouraging that a radiation dose of 2 kGy was sufficient to eliminate inherent listeriae. In challenge experiments (Samelis et al., 2005), irradiation could not ensure a 5-log reduction of a mixed five-strain inoculum of L. monocytogenes/innocua; reductions were 1.3 and 2.4 logs only on trimmings treated at 2 and 4 kGy, respectively, while survival occurred after 28 days of ripening. We opinioned though (Samelis et al., 2005) that the above degree of inhibition by irradiation may be sufficient in practice because natural contamination of L. monocytogenes commonly encountered in the raw materials for sausages is 6100 cfu g 1 (Samelis & Metaxopoulos, 1999; Samelis et al., 1998; Thevenot et al., 2005). The present findings are in support of this.
3.2. Effect of irradiation on growth of technological flora during sausage fermentation After formulation (day 0), sausages contained ca. 7 log cfu g 1 of TVC (Table 2), irrespective of previous irradiation (P > 0.05), which was due to the post-irradiation inoculation of all samples with L. pentosus plus S. carnosus in a similar manner. Accordingly, similarly high initial populations consisting of the above starter bacteria were also enumerated on MRS (Table 3) and mannitol salt agar (Table 4), respectively. When the microbiological data in Table 1 are compared with those of day 0 in Tables 2–4, it can be concluded that irradiation reduced populations of natural contaminants transferred to the batter from the trimmings, and therefore, the dominance of the starter bacteria over the inherent flora should have been higher in irradiated as compared to non-irradiated samples. On this basis, pre-irradiation of raw sausage materials may assist starter cultures to govern the process by reducing competitive effects of natural flora, especially at the onset of fermentation.
Table 2 Growth (log cfu g 1) of total mesophilic bacteria during fermentation and ripening of Greek dry salami manufactured from irradiated meat/fat trimmings Sausage treatment
Fermentation – ripening (days) 0
4
7
14
21
28
No irradiation 2 kGy 4 kGy
6.9 ± 0.0 Aa 6.9 ± 0.1 Aa 6.9 ± 0.1 Aa
8.7 ± 0.1 Ea 8.7 ± 0.1 Da 8.7 ± 0.2 Ca
8.6 ± 0.1 DEa 8.4 ± 0.1 Ca 8.4 ± 0.3 BCa
8.4 ± 0.3 CDa 8.4 ± 0.2 Ca 8.3 ± 0.4 BCa
8.4 ± 0.2 Ca 8.3 ± 0.1 Ca 8.4 ± 0.3 BCa
8.1 ± 0.1 Ba 8.1 ± 0.1 Ba 8.1 ± 0.2 Ba
Each value is mean ± SD of two replicate experiments with two samples analyzed per replicate (n = 4). Means with different uppercase letters in the same row are significantly different (P < 0.05). Means with different lowercase letters in the same column are significantly different (P < 0.05).
Table 3 Growth (log cfu g 1) of lactic acid bacteria during fermentation and ripening of Greek dry salami manufactured from irradiated meat/fat trimmings Sausage treatment
No irradiation 2 kGy 4 kGy
Fermentation – ripening (days) 0
4
7
14
21
28
7.0 ± 0.1 Aa 7.0 ± 0.1 Aa 7.0 ± 0.1 Aa
8.7 ± 0.1 Da 8.6 ± 0.1 Da 8.6 ± 0.2 Da
8.6 ± 0.1 Da 8.5 ± 0.1 CDa 8.5 ± 0.3 CDa
8.4 ± 0.2 Ca 8.4 ± 0.3 Ca 8.2 ± 0.4 BCa
8.4 ± 0.1 Ca 8.4 ± 0.2 Ca 8.3 ± 0.2 BCDa
8.1 ± 0.1 Ba 8.1 ± 0.1 Ba 8.0 ± 0.1 Ba
Each value is mean ± SD of two replicate experiments with two samples analyzed per replicate (n = 4). Means with different uppercase letters in the same row are significantly different (P < 0.05). Means with different lowercase letters in the same column are significantly different (P < 0.05).
Table 4 Growth (log cfu g 1) of Micrococcaceae during fermentation and ripening of Greek dry salami manufactured from irradiated meat/fat trimmings Sausage treatment
No irradiation 2 kGy 4 kGy
Fermentation – ripening (days) 0
4
7
14
21
28
6.4 ± 0.4 Ba 6.3 ± 0.2 ABa 6.3 ± 0.3 ABa
6.9 ± 0.2 Ca 6.4 ± 0.7 ABa 6.4 ± 0.8 Ba
6.5 ± 0.6 BCa 6.7 ± 0.2 ABa 6.5 ± 0.3 Ba
6.4 ± 0.4 Ba 6.8 ± 0.4 Ba 6.8 ± 0.5 Ba
6.4 ± 0.2 Ba 6.3 ± 0.2 ABa 6.3 ± 0.4 ABa
5.5 ± 0.3 Aa 6.2 ± 0.2 Ab 5.7 ± 0.2 Aa
Each value is mean ± SD of two replicate experiments with two samples analyzed per replicate (n = 4). Means with different uppercase letters in the same row are significantly different (P < 0.05). Means with different lowercase letters in the same column are significantly different (P < 0.05).
I. Chouliara et al. / Meat Science 74 (2006) 303–311
Within the first four days of fermentation, a vigorous growth (P < 0.05) of LAB (Table 3), also reflected in the increases of TVC (Table 2), and a slight growth of Micrococcaceae (Table 4) occurred. Thereafter their populations remained almost constant for 2 weeks, and decreased during the last week of ripening. There were no significant differences (P > 0.05) in TVC, LAB and Micrococcaceae populations between irradiated and non-irradiated samples (Tables 2–4). This indicated that growth of beneficial bacteria, originating mainly from the starter culture, followed a similar pattern in the sausages, irrespective of treatment. Thus, irradiation of meat/fat trimmings had no major stimulating or retarding effect on growth of technological flora during sausage fermentation and ripening. It should be stressed, however, that the above conclusion is based on bacterial enumeration only. With regard to potential differences in the types of LAB and Micrococcaceae among treatments, it was generally observed that non-irradiated samples contained a more diversified technological flora than that of the irradiated samples, especially on mannitol salt agar from days 0 to 7 of fermentation. Dickson and Maxcy (1985) reported that irradiating fresh batters at 500 Krad resulted in a 70% dominance of the starter inoculated post-irradiation in the fermenting sausages as compared to only 1% presence in the non-irradiated controls. In this study, such a major shift was not evident probably because competitive effects of the inherent flora in non-irradiated samples were suppressed by the high (7-log) inoculation of the starter. Additional studies are required to evaluate potential increases in prevalence of L. pentosus and S. carnosus during sausage production attributed to the inactivation or injury caused to the inherent meat LAB and Micrococcaceae by irradiation. Such studies are useful because irradiation may increase the assurance of dominance and reduce the necessary inoculum level of the starters in commercial production of fermented sausages (Dickson & Maxcy, 1985). Meat yeasts, mainly Debaryomyces spp., are known to play a secondary beneficial role in fermented sausages, behind that of LAB and Micrococcaceae, and are often used as starter cultures in Germany and other countries (Lucke, 2000; Samelis & Sofos, 2003), but not in Greece. During this study, because yeasts were less affected by irradiation (Table 1), sausages made from trimmings treated at 2 and 4 kGy had slightly lower (P > 0.05) initial (day 0) yeast populations than the controls (Table 5). In non-irra-
307
diated sausages, yeasts tended to increase between days 4 and 14 of fermentation/ripening, but their populations decreased (P < 0.05) to ca. 3 log cfu g 1 during late ripening. Conversely, yeasts in irradiated sausages did not show any major growth probably due to injury caused by irradiation, but survived without death. Thus, irradiation at 2– 4 kGy had an insignificant (P > 0.05) effect on yeast populations throughout ripening. Yeasts are known to resist irradiation, and therefore, they may increase at high numbers or even dominate at spoilage in irradiated fresh or cured meat products (Samelis & Sofos, 2003). In this study, however, yeasts were not favored by irradiation because their growth was controlled by the active starter bacteria in the sausages. 3.3. Effect of irradiation on growth of undesirable bacteria during sausage fermentation Populations of pathogenic staphylococci were <2 log cfu g 1 on day 0 and remained below this level in the sausages with or without irradiation throughout ripening (data not shown). Thus, no differences in pathogenic staphylococci among treatments occurred to allow us to evaluate residual effects of irradiation, which were more pronounced against enterococci (Table 6). Indeed, in non-irradiated samples, populations of enterococci, which were as low as 2.1 log cfu g 1 in the batters, grew by 1.5 log cfu g 1 from days 0 to 4 (P < 0.05), and remained at a level of 3 log cfu g 1 throughout the entire ripening period. In contrast, populations of enterococci in samples irradiated at 2 kGy, which were <2 log cfu g 1 on day 0, increased slightly above this level and remained ca. 1 log lower than in non-irradiated sausages during ripening. The higher irradiation dose (4 kGy) was even more effective in controlling enterococci, the populations of which remained <2 log cfu g 1 throughout ripening (Table 6). There is still considerable scientific controversy with regard to the technological and hygienic importance of enterococci in dry sausages. In fact, enterococci are often present at population levels of 5 log cfu g 1 or higher in naturally fermented sausages, and may positively contribute to the hygienic and sensory quality of these products by bacteriocin production and their glycolytic and proteolytic activities, respectively (Callewaert, Hugas, & De Vuyst, 2000; Leroy, Verluyten, & De Vuyst, 2006; Samelis et al., 1998). However, several enterococci are hemolytic and
Table 5 Growth (log cfu g 1) of yeasts during fermentation and ripening of Greek dry salami manufactured from irradiated meat/fat trimmings Sausage treatment
No irradiation 2 kGy 4 kGy
Fermentation – ripening (days) 0
4
7
14
21
28
4.0 ± 0.1 BCa 3.3 ± 0.6 Aa 3.5 ± 0.5 Aa
4.7 ± 0.7 Cb 3.3 ± 0.8 Aa 3.7 ± 0.5 Aab
3.6 ± 0.2 ABa 3.3 ± 0.6 Aa 3.5 ± 0.5 Aa
4.3 ± 0.8 BCa 4.0 ± 1.0 Aa 4.8 ± 1.1 Aa
3.2 ± 0.1 Aa 3.7 ± 0.3 Ab 3.4 ± 0.4 Aab
3.2 ± 0.5 Aa 3.7 ± 0.6 Aa 3.4 ± 0.2 Aa
Each value is mean ± SD of two replicate experiments with two samples analyzed per replicate (n = 4). Means with different uppercase letters in the same row are significantly different (P < 0.05). Means with different lowercase letters in the same column are significantly different (P < 0.05).
308
I. Chouliara et al. / Meat Science 74 (2006) 303–311
Table 6 Growth (log cfu g 1) of enterococci during fermentation and ripening of Greek dry salami manufactured from irradiated meat/fat trimmings Sausage treatment
No irradiation 2 kGy 4 kGy
Fermentation – ripening (days) 0
4
7
14
21
28
2.1 ± 0.2 Ab <2.0 Aa <2.0 Aa
3.6 ± 0.2 Cb 2.0 ± 0.1 Ba 2.1 ± 0.2 Ba
3.4 ± 0.4 BCc 2.2 ± 0.4 Bb <2.0 Aa
2.9 ± 0.5 Bc 2.1 ± 0.2 Bb <2.0 Aa
2.8 ± 0.5 Bc 2.1 ± 0.2 Bb <2.0 Aa
3.0 ± 0.6 BCc 2.1 ± 0.2 Bb <2.0 Aa
Each value is mean ± SD of two replicate experiments with two samples analyzed per replicate (n = 4). Means with different uppercase letters in the same row are significantly different (P < 0.05). Means with different lowercase letters in the same column are significantly different (P < 0.05).
mented sausages; if hygiene is poor, raw materials of otherwise high hygienic quality, as they were here the trimmings post-irradiation (Table 1), may become heavily contaminated, and this may lead to deviations in meat fermentation and faulty or unsafe final products. Overall, the present bacteriological data are in agreement with previous meat studies reporting an increased effectiveness of irradiation at 2–5 kGy against pseudomonads, enterobacteriaceae, and staphylococci while LAB are much less affected (Dickson & Maxcy, 1985; Farkas & Andrassy, 1993; Hastings & Holzapfel, 1987; Kanatt, Chander, & Sharma, 2005; Lacroix, Ouattara, Saucier, Giroux, & Smoragiewicz, 2004). 3.4. Effect of irradiation on physicochemical parameters No significant differences (P > 0.05) in pH reduction during fermentation and ripening between irradiated and non-irradiated sausages were observed (Fig. 1). The pH of the sausages, which was 6.3–6.4 at the time of formulation (day 0), displayed a rapid decline to 4.8–4.9 by day 4, declined somewhat further (pH 4.5–4.6) by the end of the fermentation (day 7) and remained at this level during ripening. These final pH values are ca. 0.5 pH unit lower than the pH values previously reported for Greek dry sausages made without starters (Samelis et al., 1998). Commercial starters acidify sausages more rapidly and extensively, as the present one did. The similar pattern of pH drop in all treatments (Fig. 1) was confirmatory of that 7 cn 2kGy 4kGy
6 pH
opportunistic pathogens, while they may also enhance formation of biogenic amines in salamis and other foods by their amino acid decarboxylase activities. For the latter reasons, enterococci have only been tested experimentally in meat fermentations (Callewaert et al., 2000), and are still not included in commercial starter culture preparations. Based on the results of this study, if manufacturers so desire, growth of enterococci in fermented sausages may be controlled by previous decontamination of the raw materials by irradiation. After formulation (day 0), populations of undesirable pseudomonads were 5.0 ± 0.4, 2.2 ± 0.2 and 2.7 ± 0.8 log cfu g 1 and those of enterobacteria were 2.6 ± 0.2, <2.0 and 3.2 ± 0.1 log cfu g 1 in the sausages made from trimmings irradiated at 0, 2, and 4 kGy, respectively (data not shown). Thus, sausages from trimmings irradiated at 4 kGy contained unexpectedly higher initial populations of enterobacteria than sausages without irradiation or irradiated at 2 kGy, as well as higher initial pseudomonad counts than sausages irradiated at 2 kGy. This was the case despite the fact that irradiated (2 or 4 kGy) trimmings had <1.0 and <2.0 log cfu g 1 of enterobacteria and pseudomonads, respectively (Table 1). This paradoxical finding was due to the order the sausage treatments were processed, which, as mentioned, was from the highest to the lowest irradiation dose. Probably the 4-kGy sausages processed first were subjected to an accidentally higher environmental contamination with enterobacteria, which might have been present on the equipment or other sites in the pilot plant, and/or on the bowls and other utensils taken from normal processing areas to handle the experimental batters. For the same reason, initial pseudomonad counts were higher in the sausages irradiated at 4 kGy than at 2 kGy, but still lower than in the control sausages since pseudomonads were above 5 logs in non-irradiated trimmings (Table 1). Nonetheless, enterobacteria declined <1 log cfu g 1 and pseudomonads <2 log cfu g 1 in all sausages on day 7 of fermentation and were undetectable during ripening (data not shown). These results indicated successful monitoring of the intrinsic and extrinsic factors in the sausages, which in combination with the antagonistic LAB flora (Table 3) eliminated undesirable bacteria, irrespective of irradiation. Although gram-negative bacteria competed poorly and died off under the conditions of this study, the observed cross-contamination of the irradiated batters underscores the importance of processing hygiene in production of fer-
5
4 0
5
10
15
20
25
30
Days of storage Fig. 1. Changes in pH of Greek dry salami manufactured from irradiated (2 or 4 kGy) meat/fat trimmings as a function of fermentation/ripening time (n = 6; SD range 0.2–0.5).
I. Chouliara et al. / Meat Science 74 (2006) 303–311
10 cn 2kGy 4kGy
8 mg MA/kg
growth of the starters, mainly L. pentosus, in the sausages was unaffected by irradiation of the trimmings. Conversely, differences were noted in the dehydration rate, as estimated by % moisture loss from the sausages (Table 7). After formulation, the mean moisture content of non-irradiated samples (48.6%) was lower (P < 0.05) than that of sausages irradiated at 2 and 4 kGy, which was 52.3% and 53.2%, respectively. However, the moisture content was equilibrated between all treatments after 7 days of fermentation (P > 0.05), indicating that non-irradiated sausages (37.9%) tended to lose water at a lower rate than sausages irradiated at 2 kGy (35.8%) or 4 kGy (38.1%). This may indicate that the functionality of meat proteins might have been affected by irradiation in a manner that water binding capacity was reduced and thus water loss was enhanced. No significant differences were noted in the final (day 28) moisture content of the sausages, which was ca. 25% and ca. 5% lower than the moisture content previously reported for naturally fermented Greek salamis (Samelis et al., 1998). TBA data expressed as mg malondialdehyde kg 1 sausage are given in Fig. 2. TBA values for control samples increased from 2.97 (day 0) to 6.95 (day 14) and then decreased to a final (day 28) value of 2.43 mg malondialdehyde kg 1. Respective values for samples irradiated at 2 kGy were 3.54, 7.71, and 2.01 and for samples irradiated at 4 kGy were 3.43, 7.87, and 2.37. Thus, there were small but statistically significant (P < 0.05) differences in TBA values between control and irradiated (2 and 4 kGy) samples up to day 14 of ripening while these differences leveled off between days 14 and 28 of ripening. Higher initial TBA values for irradiated samples may be attributed to the higher oxidation potential of radiation energy resulting in the formation of a large amount of free radicals in the sausage substrate. Present TBA values are 10-fold higher than those reported for irradiated pepperoni (3 kGy) after a period of 5 months under refrigeration (Johnson et al., 2000). However, such large differences can not be attributed to irradiation since relatively large TBA values were also recorded for the non-irradiated samples (Fig. 2). This result suggests that the trimmings used for sausage production in this study might have an increased TBA value, probably due to the mixed pork back fat at 30%. Nonetheless, some increase in lipid peroxidation as measured by TBA assay
309
6 4 2 0 0
5
10
15
20
25
30
Days of storage Fig. 2. Changes in TBA values of Greek dry salami manufactured from irradiated (2 or 4 kGy) meat/fat trimmings as a function of fermentation/ ripening time (n = 6; SD range 0.1–0.3).
was observed in irradiated (3 kGy) pork salami under chilled storage, which, however, did not affect the sensory attributes of the product (Kanatt et al., 2005). Color measurement data are presented in Table 8. The HunterLab colorimeter objectively measured the color giving L* (0–100 with 100 being perfect white), a* (positive numbers increasing with intensity of redness) and b* (positive numbers increasing with intensity of yellowness) values. L* values decreased with time of fermentation/ ripening ranging between 38.00 and 30.76 for the control sample, 39.19 and 30.28 for the irradiated at 2 kGy and between 38.31 and 30.43 for the irradiated at 4 kGy sample indicating the darkening of the sausage color with time. There were no statistically significant differences (P > 0.05) in L* values between control and irradiated samples. a* (redness) values were more or less constant with time with no significant (P > 0.05) differences between control and irradiated samples. Finally b* (yellowness) values decreased both in the control and the irradiated samples with time. Present color results are in general agreement with those of Johnson et al. (2000) for irradiated pepperoni and show that by using irradiation, lightness, yellowness and, to a lesser degree, redness will be somewhat modified resulting in a darker colored product. A decrease in redness as a result of irradiation (3 and 5 kGy) and storage time was observed in sausages stored at 25 °C (Kuo & Chen, 2004). In contrast, Ahn and Lee (2004) reported an
Table 7 Changes in moisture content (%) of Greek dry salami manufactured from irradiated (2 or 4 kGy) meat/fat trimmings as a function of fermentation/ ripening time Sausage treatment
No irradiation 2 kGy 4 kGy
Fermentation – ripening (days) 0
4
7
14
21
28
48.60 ± 0.54 Da 52.25 ± 0.80 Fb 53.24 ± 0.62 Eb
46.67 ± 0.63 Dc 44.18 ± 0.72 Eb 40.75 ± 1.24 Da
37.94 ± 0.54 Cb 35.83 ± 0.37 Da 38.07 ± 0.48 Cb
36.21 ± 0.68 DCb 34.50 ± 0.54 Ca 37.02 ± 0.80 Cc
31.78 ± 4.64 Ba 29.27 ± 0.71 Ba 30.69 ± 0.75 Ba
25.65 ± 0.85 Aab 24.60 ± 0.49 Aa 26.39 ± 0.73 Ab
Each value is mean ± SD of two replicate experiments with two samples analyzed per replicate (n = 4). Means with different lowercase letters in the same row are significantly different (P < 0.05). Means with different uppercase letters in the same column are significantly different (P < 0.05).
310
I. Chouliara et al. / Meat Science 74 (2006) 303–311
Table 8 Changes in color parameters of Greek dry salami manufactured from irradiated (2 or 4 kGy) meat/fat trimmings as a function of fermentation/ripening time Sausage treatment
Color parameter
Fermentation – ripening (days) 0
4
7
14
21
28
No irradiation 2 kGy 4 kGy
L*
38.00 ± 2.15 Ba 39.19 ± 0.81 Ba 38.31 ± 1.44 Ba
39.04 ± 1.98 Ba 37.62 ± 0.99 Ba 37.77 ± 1.48 Ba
36.07 ± 2.40 ABa 37.39 ± 1.79 Ba 37.28 ± 0.99 Ba
35.23 ± 0.88 ABa 34.68 ± 1.47 ABa 34.64 ± 2.23 ABa
32.97 ± 1.67 Aa 32.02 ± 2.04 Aa 31.98 ± 1.88 Aa
30.76 ± 0.96 Aa 30.28 ± 2.44 Aa 30.43 ± 2.11 Aa
No irradiation 2 kGy 4 kGy
a*
10.95 ± 0.61 Ba 10.66 ± 0.42 ABa 10.43 ± 0.39 Ba
11.63 ± 0.31 Ba 10.97 ± 0.55 Ba 11.40 ± 0.93 Ba
9.35 ± 0.79 Aa 9.62 ± 0.55 Aa 9.57 ± 0.38 Aa
10.84 ± 0.66 ABa 10.23 ± 0.51 ABa 10.11 ± 0.29 ABa
9.56 ± 1.08 Aa 9.89 ± 0.77 Aa 10.01 ± 0.91 ABa
No irradiation 2 kGy 4 kGy
b*
6.49 ± 0.38 Aab 6.81 ± 0.59 Ab 6.02 ± 0.17 Aa
6.23 ± 0.64 Aa 6.59 ± 0.24 Aa 6.72 ± 0.31 Ba
6.03 ± 0.43 Aa 5.96 ± 0.61 Aa 5.98 ± 0.28 Aa
8.47 ± 0.41 Aa 9.68 ± 0.31 Ab 9.78 ± 0.69 ABb 10.97 ± 0.36 Da 11.06 ± 0.71 Ca 11.32 ± 0.59 Da
8.75 ± 0.28 Ca 8.01 ± 0.17 Ba 7.99 ± 0.49 Ca
7.91 ± 0.49 Ba 8.03 ± 0.59 ABa 8.00 ± 0.34 Ca
Each value is mean ± SD of two replicate experiments with two samples analyzed per replicate (n = 4). Means with different lowercase letters in the same row are significantly different (P < 0.05). Means with different uppercase letters in the same column are significantly different (P < 0.05).
increase in a* redness values in irradiated (2.5 and 5 kGy) raw breast meat with no changes being observed in L* and b* values. Increase in redness of irradiated meat products has been related to the formation of carbon monoxide–myoglobin (CO–Mb) complexes; such complexes are more stable than oxymyoglobin because of the strong binding of CO to the iron-porphyrin site on the myoglobin molecule (Ahn & Lee, 2004; Brewer, 2004). On this basis, irradiated fermented sausage batters containing ascorbic acid and other antioxidants present in spices and flavorings are less sensitive to color defects and lipid oxidation than fresh refrigerated meat products without ascorbic acid (Lacroix et al., 2004). 4. Conclusions In addition to enhancing pathogen control (Samelis et al., 2005), irradiation (2–4 kGy) of raw materials prior to the production of dry fermented sausages inactivated undesirable bacteria and/or retarded their growth during fermentation and ripening. Therefore, irradiation contributed to an increased dominance of the starter bacteria at the onset of fermentation, which is critical in dry sausage technology. Gram negative spoilage bacteria, mainly pseudomonads, were the most sensitive to irradiation while yeasts and LAB were the most resistant. Previous irradiation of raw materials did not affect growth of starter bacteria upon their addition to the fermenting sausages post-irradiation, while it resulted in product with quality characteristics closely resembling those of non-irradiated dry fermented sausage. Acknowledgements This work was financed from the Greek Ministry for Development, General Secretariat for Research and Technology in the framework of the Greek–Hungarian Scientific and Technological Research Co-operation programme 2002–2004 (Contract No. 5879/6-6-2002). We thank VIKI
S.A. for providing raw materials and facilities for sausage production, and EL.VIO.NI. Co. for sample irradiation. References Ahn, D. U., & Lee, E. J. (2004). Mechanisms and prevention of off-odor production and color changes in irradiated meat. Irradiation of Food and Packaging: Recent Developments ACS Symposium Series, 875, 4376. AOAC (1995). Official methods of analysis (16th ed.). Washington, DC, USA. Billy, T. J. (1997). Expansion of microbiological monitoring program for ready-to-eat dry and semi-dry fermented sausages. US Department of Agriculture, Food Safety and Inspection Service. Washington. DC. June, 19, 1995 (letter to plant managers). Bremer, V., Leitmeyer, K., Jensen, E., Metzel, U., Meczulat, H., Weise, E., et al. (2004). Outbreak of Salmonella Goldcoast infections linked to consumption of fermented sausage, Germany 2001. Epidemiology and Infection, 132, 881–887. Brewer, S. (2004). Irradiation effects on meat color – A review. Meat Science, 68, 1–17. Callewaert, R., Hugas, M., & De Vuyst, L. (2000). Competitiveness and bacteriocin production of enterococci in the production of Spanishstyle dry fermented sausages. International Journal of Food Microbiology, 57, 33–42. Centers for Disease Control and Prevention (1995). Escherichia coli O157:H7 outbreak linked to commercially distributed dry-cured salami – Washington and California, 1994. Morbidity Mortality Weekly Report, 44, 157–160. Cosansu, S., & Ayhan, K. (2000). Survival of enterohemorrhagic Escherichia coli O157:H7 strain in Turkish soudjouk during fermentation, drying and storage periods. Meat Science, 54, 407–411. Dickson, J. S., & Maxcy, R. B. (1985). Irradiation of meat for the production of fermented sausage. Journal of Food Science, 50, 1007–1009, 1013. Farber, J. M., Daley, E., Holley, R., & Usborne, W. R. (1993). Survival of Listeria monocytogenes during the production of uncooked German, American and Italian-style fermented sausages. Food Microbiology, 10, 123–132. Farkas, J. (1997). Status and prospects of decontamination and preservation of poultry meat/carcasses by ionizing radiation. In P. Colin, & W.A.W. Mulder (Eds.), Pathogenic microorganisms in poultry and eggs
I. Chouliara et al. / Meat Science 74 (2006) 303–311 (Chapter 7), COST Action 97. Zoopole, 25–26 November 1996, France. Farkas, J. (2001). Irradiation of minimally processed foods. In Food irradiation: Principles and applications (pp. 273–290). New York: John Wiley and Sons, Inc. Farkas, J., & Andrassy, E. (1993). Interaction of ionizing radiation and acidulants on the growth of microflora of a vacuum packaged chilled meat product. International Journal of Food Microbiology, 19, 145–152. Fernandez-Martin, F., Fernandez, P., Caballo, J., & Colmenero, F. J. (1997). Pressure/heat combination on pork meat batters: Protein thermal behaviour and product rheological properties. Journal of Agricultural and Food Chemistry, 75, 4440–4445. Getty, K. J. K., Phebus, R. K., Marsden, J. L., Fung, D. Y. C., & Kastner, C. L. (2000). Escherichia coli O157:H7 and fermented sausages: A review. Journal of Rapid Methods and Automation in Microbiology, 8, 141–170. Glass, K. A., Loeffelholz, J. M., Ford, J. P., & Doyle, M. P. (1992). Fate of Escherichia coli O157:H7 as affected by pH or sodium chloride and in fermented, dry sausage. Applied Environmental Microbiology, 58, 2513–2516. Hastings, J. W., & Holzapfel, W. H. (1987). Numerical taxonomy of lactobacilli surviving radurization of meat. International Journal of Food Microbiology, 4, 33–49. Huffman, R. D. (2002). Current and future technologies for the decontamination of carcasses and fresh meat. Meat Science, 62, 285–294. Johnson, S. C., Sebranek, J. G., Olson, D. G., & Wiegand, B. R. (2000). Irradiation in contrast to thermal processing of pepperoni for control of pathogens: Effects on quality indicators. Journal of Food Science, 65, 1260–1265. Kanatt, S. R., Chander, R., & Sharma, A. (2005). Effect of radiation on the quality of chilled meat products. Meat Science, 69, 269–275. Kirk, R. S., & Sawyer, R. (1991). Pearsons compostition and analysis of foods (9th ed.). Essex, England: Longman Science and Technical, pp. 642–643. Kuo, J. C. C., & Chen, H. L. (2004). Combination effect of sodium lactate and irradiation on color, lactic acid bacteria, lipid oxidation and residual nitrite in Chinese sausages during storage at 25 °C. Journal of the Science of Food and Agriculture, 84, 903–908. Lacroix, M., Ouattara, B., Saucier, L., Giroux, M., & Smoragiewicz, W. (2004). Effect of gamma irradiation in presence of ascorbic acid on microbial composition and TBARS concentration of ground beef coated with an edible active coating. Radiation Physics and Chemistry, 71, 73–77. Lahti, E., Johansson, T., Honkanen-Buzalski, T., Hill, P., & Nurmi, E. (2001). Survival and detection of Escherichia coli O157:H7 and Listeria
311
monocytogenes during manufacture of dry sausage using two different starter cultures. Food Microbiology, 18, 75–85. Lee, M., Sebranek, J. G., Olson, D. G., & Dickson, J. S. (1996). Irradiation and packaging of fresh meat and poultry. Journal of Food Protection, 59, 62–72. Leroy, F., Verluyten, J., & De Vuyst, L. (2006). Functional meat starter cultures for improved sausage fermentation. International Journal of Food Microbiology, 106(3), 270–285. Lucke, F.-K. (2000). Fermented meats. In B. M. Lund, T. C. BairdParker, & G. W. Gould (Eds.). The microbiological safety and quality of food (Vol. 1, pp. 420–444). Maryland, USA: Aspen Publications. Maxcy, R. B. (1983). Significance of residual organisms in foods after substerilizing doses of gamma radiation. A review. Journal of Food Safety, 5, 2003–2007. Moore, J. E. (2004). Gastrointestinal outbreaks associated with fermented meats. Meat Science, 67, 565–568. Nissen, H., & Holck, A. (1998). Survival of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella kentucky in Norwegian fermented, dry sausage. Food Microbiology, 15, 273–279. Prochaska, J. F., Ricke, S. C., & Keeton, J. T. (1998). Meat fermentation: Research opportunities. Food Technology, 52, 52–57. Samelis, J., & Metaxopoulos, J. (1999). Incidence and principal sources of Listeria spp. and Listeria monocytogenes contamination in processed meats and a meat processing plant. Food Microbiology, 16, 465–477. Samelis, J., & Sofos, J. N. (2003). Yeasts in meat and meat products. In T. Boekout & V. Robert (Eds.), Yeasts in food (pp. 239–265). Hamburg, Germany: Behr’s Verlag. Samelis, J., Metaxopoulos, J., Vlassi, M., & Pappa, A. (1998). Stability and safety of traditional Greek salami – A microbiological ecology study. International Journal of Food Microbiology, 44, 69–82. Samelis, J., Kakouri, A., Savvaidis, I. N., Riganakos, K., & Kontominas, M. G. (2005). Use of ionizing radiation doses of 2 and 4 kGy to control Listeria spp. and Escherichia coli O157:H7 on frozen meat trimmings used for dry fermented sausage production. Meat Science, 70, 189–195. Sauer, C. J., Majkowski, J., Green, S., & Eckel, R. (1997). Foodborne illness outbreak associated with a semi-dry fermented sausage product. Journal of Food Protection, 60, 1612–1617. Sommers, C. H., Keser, N., Fan, X. T., Wallace, F. M., Novak, J. S., Handel, A. P., et al. (2004). Irradiation of ready-to-eat meats: Eliminating Listeria monocytogenes while maintaining product quality. Irradiation of Food and Packaging: Recent Developments ACS Symposium Series, 875, 77–89. Thevenot, D., Delignette-Muller, M. L., Christieans, S., & VernozyRozand, C. (2005). Fate of Listeria monocytogenes in experimentally contaminated French sausages. International Journal of Food Microbiology, 101, 189–200.