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Thermal death of bacterial pathogens in linguiça smoking
Food Control 22 (2011) 668e672
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Thermal death of bacterial pathogens in linguiça smoking Maha N. Hajmeer 1, Mehrdad Tajkarimi 2, Edward L. Gomez 3, Nathaniel Lim 4, Maryam O’Hara 5, Hans P. Riemann 6, Dean O. Cliver* Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616-8743, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 April 2010 Received in revised form 15 July 2010 Accepted 20 July 2010
Linguiça, a smoked sausage originally from Portugal, is often made in small quantities in California, without inspection. Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica serotype Newport, and Yersinia enterocolitica were added individually to batter representing California linguiça. Batter (1 mm thick), heated at 50, 55, and 60 C, showed decimal reduction times ranging from >10 min for most trials at 50 C to <2 min at 60 C. Pork casings, stuffed with the batters to a diameter 3 cm, length 10 cm, and weight 75e80 g, were hot smoked; sausage centers were at 60 C for 90 min. Contaminant levels in the batters (three experiments/pathogen) ranged from 2.3 106 to 3.0 1010 CFU/g in various runs; reductions were 5 log10 in all cases. These experiments indicate a reasonable margin of safety for products processed in this way. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Bacterial pathogens Linguiça sausage Hot smoking
1. Introduction Linguiça is a Portuguese-type sausage containing pork and is prepared either fresh or smoked. According to the United States Department of Agriculture (USDA), the product excludes other meat and meat by-products and usually contains nonfat dry milk, vinegar, cinnamon, cumin seed, garlic, red pepper, salt, and sugar (USDA, 2005). Paprika and cures are also acceptable ingredients in this commodity. Linguiça is well known in the Latin communities. Mexican-style versions are available in the US, and the sausage is popular in ethnic markets, particularly in California. In California, products such as linguiça, manufactured and sold in local ethnic markets, present a potential food safety concern. Most of the establishments preparing the products are small local operations that do not fall under USDA inspection and are not held to the same food safety regulations as larger facilities. Operators of
* Corresponding author. Tel.: þ1 530 754 9120; fax: þ1 530 752 5845. E-mail address: [email protected] (D.O. Cliver). 1 Present address: Emergency Response Unit, Food and Drug Branch, California Department of Public Health, Sacramento, CA 95899-7435, USA. 2 Present address: Food Microbiology and Biotechnology Laboratory, North Carolina A & T State University, 171-B Carver Hall, Greensboro, NC 27411-1064, USA. 3 Present address: Stuart Cellars, 33515 Rancho California Rd., Temecula, CA 92591, USA. 4 Present address: PDL Virology Core, California National Primate Research Center, University of California, Davis, CA 95616, USA. 5 Present address: School of Veterinary Medicine, University of California, Davis, CA 95616, USA. 6 Deceased. 0956-7135/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2010.07.027
such small-scale establishments may not be aware of potential food safety risks; if the product is contaminated with pathogens, this may cause foodborne illness. Limited information is available in the literature on the microbiological safety of linguiça. A study on the microbiological quality of mixed (pork and beef), fresh linguiça showed that 18% of the samples analyzed were contaminated with salmonellae and 4% with fecal coliforms (Silva et al., 2002). A follow-up study showed that Listeria spp. was present in all of 41 samples; 29.3% was Listeria monocytogenes (Silva et al., 2004). In 1999, there was a recall of cooked linguiça sausage for possible Listeria contamination (FSIS, 1999). In 2000, the USDA’s Food Safety and Inspection Service (USDA-FSIS) warned of a possible health hazard with linguiça from a California factory (USDA, 2000). Factors that may affect the growth or survival of foodborne pathogens in sausage products such as linguiça include water activity (aw), pH, temperature (e.g., storage, cooking), use of starter cultures (if the product is a fermented type), use of ingredients with antimicrobial activity (e.g., smoke), and packaging materials and methods. Smoking of foods, although mainly used for flavoring, has been shown to have antimicrobial effects (Faith, Yousef, & Luchansky, 1992; Maga, 1998; Sofos, Maga, & Boyle, 1988),. However, temperature of smoking plays a role as well. Sunen (1998) indicated that the temperatures used in cold smoking are not sufficient to kill microorganisms. This study evaluated the survival of Campylobacter jejuni, Escherichia coli O157:H7, L. monocytogenes, Salmonella enterica serotype Newport, and Yersinia enterocolitica in linguiça under a number of specified matrix conditions, including pH, aw (salt
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content), and temperature over time. All of these agents are identified by Mor-Mur and Yuste (2010) as bacterial pathogens of concern in meat and poultry products. The study was undertaken at the request, and with the support, of the California Department of Food and Agriculture (CDFA). Its goals were to provide: (1) needed food-safety-related information on the survivability of bacterial foodborne pathogens in linguiça and (2) a scientific basis for any guidelines for the safe production of linguiça that might be issued to makers in California and the nation. This study would also be a valuable precedent for rule-making that would apply to other ethnic products formulated on the basis of the makers’ and customers’ tastes, rather than an arbitrary standard of identity. 2. Materials and methods 2.1. Bacterial pathogens Five bacterial pathogens considered relevant to linguiça were included in the study. Each was adapted (specifics below) for antibiotic resistance and synthesis of blue or green fluorescent protein (BFP and GFP, respectively). The antibiotic resistance of these strains obviated use of selective media for recovery of the microorganisms, thus reducing the potential for viable-notculturable artifacts (Doherty et al., 1998). C. jejuni: The selected strain of C. jejuni had been modified for kanamycin (KAN) resistance and synthesis of BFP; it was provided by Dr. Michele Jay Russell, then of the California Department of Public Health (CDPH). The pathogen was grown in modified atmosphere (5% O2, 10% CO2) at 37 C on sheep-blood agar þ kanamycin (SBA þ KAN)(M. Jay Russell, personal communication). E. coli O157:H7: E. coli O157:H7 carrying a plasmid that codes for ampicillin (AMP) resistance and the synthesis of GFP was used. The pathogen was grown by methods described in the literature (Hew, Hajmeer, Farver, Glover, & Cliver, 2005b), in brain-heart infusion broth (BHIB)þampicillin (AMP) or on brain-heart infusion agar (BHIA)þAMP at 37 C. L. monocytogenes: L. monocytogenes strain 108M was used. This strain was modified with a plasmid that codes for AMP resistance and the synthesis of GFP; it was provided by Dr. Linda J. Harris, Department of Food Science and Technology, University of California (UC), Davis. The pathogen was grown by methods described in the literature (Hew, Hajmeer, Farver, Glover, & Cliver, 2005a), in BHIB þ AMP or on BHIA þ AMP at 37 C. S. enterica serotype Newport: S. Newport carrying a plasmid that codes for AMP resistance and the synthesis of BFP was grown by methods described in the literature (Hew et al., 2005b), in BHIB þ AMP or on BHIA þ AMP at 37 C. Y. enterocolitica: A strain of Y. enterocolitica that had been modified for resistance to tetracycline (TET) and GFP production was provided by Dr. Glenn M. Young of the Department of Food Science and Technology, UC, Davis; the strain was grown in BHIB þ TET or on BHIA þ TET at 25 C (G. M. Young, personal communication). The inoculum for each pathogen was harvested from growth on a spread plate (media as described earlier), suspended in 10 ml of 0.1% peptone water and added to 90 g of linguiça batter. Contaminant levels in the batters ranged from 2.3 106 to 3.0 1010 CFU/g in various runs.
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batter was a consensus of various formulations registered by linguiça makers in California, with guidance from the late Earl Willis of the CDFA. Twenty pounds (9.1 kg) of approximately 80% lean meat and 20% trim (fat) was ground through a 3/16 inch (4.8 mm) grinder plate with the linguiça spice mixture: 1 lb (455 g) red wine vinegar, 1 lb (455 g) red wine, 4 oz (114 g) sugar, 3.25 oz (92 g) table salt (NaCl), 1 oz (28 g) sodium nitrite, 1 oz (28 g) paprika, 1 oz (28 g) cayenne pepper, 0.5 oz (14 g) ground black pepper, 0.25 oz (7 g) ground dry ginger, and 0.08 oz (2.3 g) fresh garlic. The resultant batter was bagged in 2 lb (0.91 kg) quantities, vacuum-sealed, frozen at 20 C, and transferred to the Food Safety Laboratory, where it was stored at 20 C. A 10 g sample of the batter was mixed with 90 ml of deionized water, and the pH of the suspension was recorded (Orion pH meter model 410A, Thermo Fisher Scientific, Inc., Waltham, MA). Three replicate 5 g samples of the batter were used to measure aw (Aqua Lab Aw meter model Series 3TE, Decagon Devices, Inc. Pullman, WA). 2.3. Thermal kills in batter A suspension of 10 ml of pathogen in 0.1% peptone water plus 90 g of batter was mixed in a KitchenAid Ultrapower mixer (KitchenAid, St. Joseph, MI), and 1 g of the mixed material was transferred to each of 21 6 oz (w168 ml) Whirl-Pak bags (Nasco, Fresno, CA) with filters. The mixture in the bags was compressed to a thickness of 1 mm, and the bags were sealed with the built-in closure. The sealed bags were completely immersed in a circulating water bath containing w15 L of water at the selected temperature (50, 55, or 60 C). At selected intervals, a bag was removed; 9 ml of 0.1% peptone water at 4 C was added, and the bag was pummeled for 30 s in a Seward Stomacher model 80 (Seward Ltd., Thetford, Norfolk, UK). A 0.1 ml sample was taken from the filtered suspension and assayed by spread plating 10-fold dilutions (in 0.1% peptone water) on the agar medium specified for that pathogen in Section 2.1. 2.4. Sausage making To prepare sausage links, one or more 2 lb (0.91 kg) packages of frozen linguiça batter were thawed in cold tap water to 22 C (1e2 h). A 500 g quantity in a stainless steel container was inoculated with 10 ml suspension of the pathogen and mixed with a stainless steel balloon whisk. No inoculum was added to the other w500 g control batter. Among the pathogens studied, C. jejuni presented a special case, as difficulty was experienced achieving high enough initial levels of CFU/ml to demonstrate a 5 log kill; therefore, 40 ml of the C. jejuni suspension were added to 360 g of batter in this case only. Both inoculated and un-inoculated batters were sampled to determine a time-zero inoculum level, after which the batters were used to make linguiça links. The non-inoculated batter was first placed into the sausage-stuffer attachment of a KitchenAid food processor, stuffed into natural-pork sausage casings (Oversea Casing Company, Seattle, Wash.) and twisted into 10 cm links with a greatest diameter of 3 cm and a fresh weight of 75e80 g. The inoculated batter was then stuffed into additional sausage casings in the same manner. A similar method of preparation was described by Mattick, Bailey, Jorgensen, and Humphrey (2002), who described the products as “handmade sausages”.
2.2. Linguiça batter
2.5. Initial pathogen analysis of sausage links
The linguiça batter was formulated at the UC Davis Cole Meat Laboratory and transported frozen to the UC Davis Food Safety Laboratory for bacterial inoculation and challenge studies. The final
One representative link each of inoculated and un-inoculated sausage was taken for analysis. The links were plunged into boiling water for 5 s to pasteurize the surface, and the interior of the
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sausage was sampled for the target pathogen. Assays were done as described in the batter-heating experiments, above.
Table 1 Decimal reduction times (DRTs) for thin-layer in linguiça batter experiments. Pathogen
Pa
Campylobacter jejuni
0.0028
50 55 60
11.65 4.09 1.56
2.71 0.20 0.24
64.12 30.70 4.50
Escherichia coli O157:H7
0.0706
50 55 60
(12.50)c (5.00)c 1.45
0.28
4.40
C
DRT (min)
2.6. Sausage smoking Sausages were smoked in a 20 lb (9.1 kg) capacity, non-insulated, stainless-steel smoker (Model 41300, The Sausage Maker, Inc., Buffalo, NY; 1000 W, 110 v) that was chosen to fit within our laboratory. The unit was pre-heated to 88 C (measured with a dial thermometer whose stem was inserted 12.5 cm into the interior through a port in the door), and eight to ten sausage links were hung from wooden dowels inside the smoker. In most runs, a thermocouple (Atkins Technical, Inc., Gainesville, FL) was placed inside a randomly selected link of un-inoculated sausage, which was positioned near the center of the array. The smoker was again heated to an internal temperature of w88 C, and a stainless steel pan with 250 g hickory wood sawdust moistened with 400 ml water was placed on the electric coil. Internal sausage and smoking-space temperatures were recorded every 15 min. After the sawdust’s placement in the smoker, the internal smoker temperature was decreased to an average of 80 C, controlled by the setting of the top vent. The internal temperature of the sausage was slowly raised to 60 C and kept at or above that temperature for 90 min. After smoking, the sausage was cooled for 15 min and tested as described above for the presence of the target pathogen. 2.7. Proximate analyses Representative samples from three batches of linguiça, before and after smoking, were submitted to a commercial laboratory (JL Analytical Services, Modesto, CA) for proximate analyses (Crude proteindAOAC 990.03; Crude fatdAOAC 960.39; Crude fiberd AOAC 962.09; AshdAOAC 942.05). Also, samples of raw batter and corresponding smoked sausages were held at 60 C for 24 h in containers devised to let moisture evaporate and fatty drippings to separate from the solid material. Weights of the samples before and after treatment, as well as the weights of the collected fat, were recorded. 2.8. Statistics Numerical data were log-transformed to assure normal distribution and were subjected to analysis of variance (ANOVA) using PROC GLM. Assumption checks were done with PROC UNIVARIATE, and the confidence limits were calculated using PROC MEANS. Statistical assistance was provided by the UC Davis Statistics Laboratory, using the statistical software SAS Version 9.1. Logtransformed data were used to plot thermal death curves by the linear-least-squares method. Decimal reduction times (DRTs), or D-values, were derived from the slopes of the curves, and z-values (number of degrees change that would cause a 10-fold increase or decrease in the DRT) estimated from the DRTs at the chosen temperatures (Hajmeer & Cliver, 2002).
Mean
Upperb
Listeria monocytogenes
0.0618
50 55 60
10.73 4.19 2.15
2.18 0.32 0.51
52.71 55.02 2.48
Salmonella Newport
0.0018
50 55 60
87.36 3.95 1.08
2.62 2.34 0.57
2911.80 6.67 2.02
Yersinia enterocolitica
0.0054
50 55 60
10.44 1.69 (0.22)c
5.42 0.29
92.29 11.79
a b c
Probability that within-pathogen differences occurred by chance. Limits of 95% confidence interval. Based on a single experiment, rather than three.
in response to temperature (P ¼ 0.1223) were significant, the DRT for Y. enterocolitica at 60 C was judged too short to be measured accurately by the present method, so only one trial was performed. Only rough estimates of z-values are possible from these limited data; they are: C. jejuni, 9.9 C; E. coli O157:H7, 9.0 C; L. monocytogenes, 11.7 C; S. enterica, 1.0 C; and Y. enterocolitica, 5.7 C. The z-value for S. enterica seems improbably low: a meta-analysis by Doyle and Mazzotta (2000) calculated a “generic” z-value of 5.3 C for S. enterica. The thin-layer experiments were intended to provide nearinstantaneous temperature adjustments, so that time-at-temperature could be accurately estimated. A similar procedure was used by Sallami et al. (2006) with Listeria and Salmonella in bologna batter, except that their batter was 2 mm, rather than 1 mm, thick. The results indicate that the linguiça batter was not an exceptional matrix for the thermal death of these pathogens. The USDA Pathogen Modeling Program (USDA, 1999) predicts that, at 60 C, pH 5.8, and 1% NaCl, a 5 log reduction of E. coli O157:H7 in simulated beef gravy would occur in 8.6 min (confidence limits 6.8e10.9 min), and of L. monocytogenes in ground beef, in 20.8 min (confidence limits 14.9e29.2 min). Juneja, Snyder, and Marmer (1997) reported D-values for E. coli O157:H7 in ground beef of 21.13 0.25 min at
Table 2 Shrinkage due to smoking, by proximate analysis, assuming 36% total weight loss. Batch
Treatment
Weighta (g)
A
Raw Smoked
77 49
B
Raw Smoked
C Pooled
3. Results and discussion 3.1. Thermal kills in batter DRTs from the thin-layer experiments were measured in minutes and were very short at 60 C (Table 1). There may have been deviations from linearity; the observations were not extensive enough to explore this. For the E. coli O157:H7 experiment, data from all but one trial at 50 C and at 55 C were discarded for technical complications. Although neither the among-species differences overall (P ¼ 0.1584) nor the among-species differences
Lowerb
Moisture
Crude protein
Crude fat
%
g
%
g
%
g
64.9 44.0
50.0 21.6
16.2 24.0
12.5 11.8
17.7 27.9
13.6 13.7
77 49
61.6 39.9
47.4 19.6
16.3 26.6
12.6 13.0
19.0 28.6
14.6 14.0
Raw Smoked
77 49
61.6 40.3
47.4 19.7
16.3 25.7
12.6 12.6
19.0 29.3
14.6 14.4
Raw Smoked
77 49
62.7 41.4
48.3 20.3
16.3 25.4
12.6 12.4
18.6 28.6
14.3 14.0
a These weights were arbitrarily selected for illustrative purposes. Sausage weights before smoking averaged w77 g, and after-smoking weights averaged w49 g, whereas raw samples submitted for proximate analysis were of batter, rather than stuffed sausages.
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Table 3 Effect of smoking linguiça on bacterial pathogens. Pooled reduction (log)a
Pb
>5 >8 >8
7.43
0.0190
w6 >6 >7
6.62
0.0042
3.5 102 <10 <10
>7 >6 >6
7.06
0.0045
9.8 106 7.7 107 1.9 106
<10 <10 <10
>5 >6 >5
6.05
0.0059
8
<10 <10 <10
>7 >6 >7
7.06
0.0025
Pathogen
Run
Pre-smoke (CFU/g)
Post-smoke (CFU/g)
Kill (log)
Campylobacter jejuni
1 2 3
2.3 106 2.4 109 3.6 109
<10 <10 <10
Escherichia coli O157:H7
1 2 3
7
2.2 10 7.0 107 1.7 108
3.5 10 <10 <10
Listeria monocytogenes
1 2 3
3.0 1010 1.3 107 8.4 107
Salmonella enterica
1 2 3 1 2 3
Yersinia enterocolitica
a b
3.7 10 2.4 107 1.7 108
1
Calculated with <10 CFU/g data assigned a value of 5 CFU/g. Probability that this reduction could have occurred by chance alone.
55 C and 3.17 0.18 min at 60 C, which would predict 5 log destructions in w106 min and w16 min at those temperatures. The largest D-values reported by Doherty et al. (1998) for L. monocytogenes in ground beef at 50, 55, and 60 C were 43.5, 3.53, and 0.33 min, respectively; for Y. enterocolitica, these were 26.3, 1.24, and 0.11 min, respectively. Murphy, Beard, Martin, Duncan, and Marcy (2004) reported D-values for E. coli O157:H7, L. monocytogenes, and Salmonella in ground pork at 60 C that were roughly twice what we observed; the authors suggested that their high D-values were probably associated with the 40.2% fat content of their pork. Mattick et al. (2002) inoculated their “handmade” sausages with Salmonella Typhimurium at a level of 280 CFU/g and found some surviving salmonellae after barbecuing the sausages to an internal temperature of 66 C. 3.2. Smoking effects The smoking process reduced the weight of the linguiça: raw weights ranged roughly 75e80 g, whereas smoked weights were generally 48e51 g. Moisture was removed, primarily due to smoking heat, but fat may or may not have been reduced, depending on the mode of analysis (Table 2). After 24 h at 60 C, weight loss from raw (un-smoked) batter totaled 61.9% (7.7% attributed to fat loss, and 54.1% to moisture loss). Weight lost from smoked-sausage material was 51.6% (0.1% attributed to fat loss, and 51.5% to moisture loss). The mean aw was 0.974 before and 0.962 after smoking. Among 20 batter samples, the mean pH value was 5.58 0.07, with a range of 5.44e5.71. Among 20 samples from smoked sausages, the mean pH was 5.66 0.19, with a range of 5.52e5.93. Smoking was found to be effective against all five of the tested pathogens (Table 3), presumably because of the high temperatures employed. Reductions were found to be similar among the pathogen species (P ¼ 0.5742). The present study has few counterparts in the literature; these generally lack details as to the proximate composition of the sausage and the specifics of the smoking process. Smith, Huhtanen, Kissinger, and Palumbo (1975) reported that smoking beef-pork pepperoni to an internal temperature of 60 C reduced experimentally inoculated S. Dublin from 1.6 102 CFU/g to an undetectable level. Buncic (1991) found L. monocytogenes in 21% of Yugoslavian vacuum-packaged, hotsmoked sausages, but not in hot-smoked sausages heated to
70e75 C after they had been smoked. Ferreira et al. (2006) detected E. coli (not described as enterohemorrhagic) in 16 of 20 and Listeria spp. in 10 of 15 market samples of alheira, a sausage of variable composition produced in Portugal by fermentation followed by smoking. More recently, Karakolev (2009) found L. monocytogenes in 12 of 140 Bulgarian market samples of “rawsmoked” sausages. If not raw, linguiça is sold in California as “not fully cooked” (must attain an internal temperature of at least 144 F [62 C]) or “fully cooked” (must attain an internal temperature of at least 155e158 F [68e70 C]) (Earl Willis, personal communication). Minimum time-at-temperature is not specified, but smoking times are said typically to be 12e18 h or more for not-fully-cooked linguiça and 8e12 h for fully-cooked. Our smoked sausage spent 1e2 h at temperatures 60 C, often with a final attained temperature as high as w71 C; all five of the tested pathogens were inactivated at least 99.999%. This indicates that linguiça, formulated and smoked as was done here, is a relatively safe productdbarring subsequent mishandling.
Acknowledgements We thank the late Earl Willis of the CDFA for providing documentation of California linguiça making, as well as guidance during and after the study. We thank Matthew Livingston of the Cole Meat Laboratory, University of California-Davis (UCD) for preparing the batter and for expert advice on processing. Dr. James Glover, of the CDPH, was a source of both information and inspiration. We are grateful to Dr. Neil H. Willits of the UCD Statistical Laboratory for the statistical analyses. Dale Francis of the CDFA also provided valuable information. This study was supported by the Center for Food Animal Health, UCD, with funds from the CDFA.
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Thermal death of bacterial pathogens in linguiça smoking Maha N. Hajmeer 1, Mehrdad Tajkarimi 2, Edward L. Gomez 3, Nathaniel Lim 4, Maryam O’Hara 5, Hans P. Riemann 6, Dean O. Cliver* Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616-8743, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 April 2010 Received in revised form 15 July 2010 Accepted 20 July 2010
Linguiça, a smoked sausage originally from Portugal, is often made in small quantities in California, without inspection. Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica serotype Newport, and Yersinia enterocolitica were added individually to batter representing California linguiça. Batter (1 mm thick), heated at 50, 55, and 60 C, showed decimal reduction times ranging from >10 min for most trials at 50 C to <2 min at 60 C. Pork casings, stuffed with the batters to a diameter 3 cm, length 10 cm, and weight 75e80 g, were hot smoked; sausage centers were at 60 C for 90 min. Contaminant levels in the batters (three experiments/pathogen) ranged from 2.3 106 to 3.0 1010 CFU/g in various runs; reductions were 5 log10 in all cases. These experiments indicate a reasonable margin of safety for products processed in this way. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Bacterial pathogens Linguiça sausage Hot smoking
1. Introduction Linguiça is a Portuguese-type sausage containing pork and is prepared either fresh or smoked. According to the United States Department of Agriculture (USDA), the product excludes other meat and meat by-products and usually contains nonfat dry milk, vinegar, cinnamon, cumin seed, garlic, red pepper, salt, and sugar (USDA, 2005). Paprika and cures are also acceptable ingredients in this commodity. Linguiça is well known in the Latin communities. Mexican-style versions are available in the US, and the sausage is popular in ethnic markets, particularly in California. In California, products such as linguiça, manufactured and sold in local ethnic markets, present a potential food safety concern. Most of the establishments preparing the products are small local operations that do not fall under USDA inspection and are not held to the same food safety regulations as larger facilities. Operators of
* Corresponding author. Tel.: þ1 530 754 9120; fax: þ1 530 752 5845. E-mail address: [email protected] (D.O. Cliver). 1 Present address: Emergency Response Unit, Food and Drug Branch, California Department of Public Health, Sacramento, CA 95899-7435, USA. 2 Present address: Food Microbiology and Biotechnology Laboratory, North Carolina A & T State University, 171-B Carver Hall, Greensboro, NC 27411-1064, USA. 3 Present address: Stuart Cellars, 33515 Rancho California Rd., Temecula, CA 92591, USA. 4 Present address: PDL Virology Core, California National Primate Research Center, University of California, Davis, CA 95616, USA. 5 Present address: School of Veterinary Medicine, University of California, Davis, CA 95616, USA. 6 Deceased. 0956-7135/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2010.07.027
such small-scale establishments may not be aware of potential food safety risks; if the product is contaminated with pathogens, this may cause foodborne illness. Limited information is available in the literature on the microbiological safety of linguiça. A study on the microbiological quality of mixed (pork and beef), fresh linguiça showed that 18% of the samples analyzed were contaminated with salmonellae and 4% with fecal coliforms (Silva et al., 2002). A follow-up study showed that Listeria spp. was present in all of 41 samples; 29.3% was Listeria monocytogenes (Silva et al., 2004). In 1999, there was a recall of cooked linguiça sausage for possible Listeria contamination (FSIS, 1999). In 2000, the USDA’s Food Safety and Inspection Service (USDA-FSIS) warned of a possible health hazard with linguiça from a California factory (USDA, 2000). Factors that may affect the growth or survival of foodborne pathogens in sausage products such as linguiça include water activity (aw), pH, temperature (e.g., storage, cooking), use of starter cultures (if the product is a fermented type), use of ingredients with antimicrobial activity (e.g., smoke), and packaging materials and methods. Smoking of foods, although mainly used for flavoring, has been shown to have antimicrobial effects (Faith, Yousef, & Luchansky, 1992; Maga, 1998; Sofos, Maga, & Boyle, 1988),. However, temperature of smoking plays a role as well. Sunen (1998) indicated that the temperatures used in cold smoking are not sufficient to kill microorganisms. This study evaluated the survival of Campylobacter jejuni, Escherichia coli O157:H7, L. monocytogenes, Salmonella enterica serotype Newport, and Yersinia enterocolitica in linguiça under a number of specified matrix conditions, including pH, aw (salt
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content), and temperature over time. All of these agents are identified by Mor-Mur and Yuste (2010) as bacterial pathogens of concern in meat and poultry products. The study was undertaken at the request, and with the support, of the California Department of Food and Agriculture (CDFA). Its goals were to provide: (1) needed food-safety-related information on the survivability of bacterial foodborne pathogens in linguiça and (2) a scientific basis for any guidelines for the safe production of linguiça that might be issued to makers in California and the nation. This study would also be a valuable precedent for rule-making that would apply to other ethnic products formulated on the basis of the makers’ and customers’ tastes, rather than an arbitrary standard of identity. 2. Materials and methods 2.1. Bacterial pathogens Five bacterial pathogens considered relevant to linguiça were included in the study. Each was adapted (specifics below) for antibiotic resistance and synthesis of blue or green fluorescent protein (BFP and GFP, respectively). The antibiotic resistance of these strains obviated use of selective media for recovery of the microorganisms, thus reducing the potential for viable-notculturable artifacts (Doherty et al., 1998). C. jejuni: The selected strain of C. jejuni had been modified for kanamycin (KAN) resistance and synthesis of BFP; it was provided by Dr. Michele Jay Russell, then of the California Department of Public Health (CDPH). The pathogen was grown in modified atmosphere (5% O2, 10% CO2) at 37 C on sheep-blood agar þ kanamycin (SBA þ KAN)(M. Jay Russell, personal communication). E. coli O157:H7: E. coli O157:H7 carrying a plasmid that codes for ampicillin (AMP) resistance and the synthesis of GFP was used. The pathogen was grown by methods described in the literature (Hew, Hajmeer, Farver, Glover, & Cliver, 2005b), in brain-heart infusion broth (BHIB)þampicillin (AMP) or on brain-heart infusion agar (BHIA)þAMP at 37 C. L. monocytogenes: L. monocytogenes strain 108M was used. This strain was modified with a plasmid that codes for AMP resistance and the synthesis of GFP; it was provided by Dr. Linda J. Harris, Department of Food Science and Technology, University of California (UC), Davis. The pathogen was grown by methods described in the literature (Hew, Hajmeer, Farver, Glover, & Cliver, 2005a), in BHIB þ AMP or on BHIA þ AMP at 37 C. S. enterica serotype Newport: S. Newport carrying a plasmid that codes for AMP resistance and the synthesis of BFP was grown by methods described in the literature (Hew et al., 2005b), in BHIB þ AMP or on BHIA þ AMP at 37 C. Y. enterocolitica: A strain of Y. enterocolitica that had been modified for resistance to tetracycline (TET) and GFP production was provided by Dr. Glenn M. Young of the Department of Food Science and Technology, UC, Davis; the strain was grown in BHIB þ TET or on BHIA þ TET at 25 C (G. M. Young, personal communication). The inoculum for each pathogen was harvested from growth on a spread plate (media as described earlier), suspended in 10 ml of 0.1% peptone water and added to 90 g of linguiça batter. Contaminant levels in the batters ranged from 2.3 106 to 3.0 1010 CFU/g in various runs.
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batter was a consensus of various formulations registered by linguiça makers in California, with guidance from the late Earl Willis of the CDFA. Twenty pounds (9.1 kg) of approximately 80% lean meat and 20% trim (fat) was ground through a 3/16 inch (4.8 mm) grinder plate with the linguiça spice mixture: 1 lb (455 g) red wine vinegar, 1 lb (455 g) red wine, 4 oz (114 g) sugar, 3.25 oz (92 g) table salt (NaCl), 1 oz (28 g) sodium nitrite, 1 oz (28 g) paprika, 1 oz (28 g) cayenne pepper, 0.5 oz (14 g) ground black pepper, 0.25 oz (7 g) ground dry ginger, and 0.08 oz (2.3 g) fresh garlic. The resultant batter was bagged in 2 lb (0.91 kg) quantities, vacuum-sealed, frozen at 20 C, and transferred to the Food Safety Laboratory, where it was stored at 20 C. A 10 g sample of the batter was mixed with 90 ml of deionized water, and the pH of the suspension was recorded (Orion pH meter model 410A, Thermo Fisher Scientific, Inc., Waltham, MA). Three replicate 5 g samples of the batter were used to measure aw (Aqua Lab Aw meter model Series 3TE, Decagon Devices, Inc. Pullman, WA). 2.3. Thermal kills in batter A suspension of 10 ml of pathogen in 0.1% peptone water plus 90 g of batter was mixed in a KitchenAid Ultrapower mixer (KitchenAid, St. Joseph, MI), and 1 g of the mixed material was transferred to each of 21 6 oz (w168 ml) Whirl-Pak bags (Nasco, Fresno, CA) with filters. The mixture in the bags was compressed to a thickness of 1 mm, and the bags were sealed with the built-in closure. The sealed bags were completely immersed in a circulating water bath containing w15 L of water at the selected temperature (50, 55, or 60 C). At selected intervals, a bag was removed; 9 ml of 0.1% peptone water at 4 C was added, and the bag was pummeled for 30 s in a Seward Stomacher model 80 (Seward Ltd., Thetford, Norfolk, UK). A 0.1 ml sample was taken from the filtered suspension and assayed by spread plating 10-fold dilutions (in 0.1% peptone water) on the agar medium specified for that pathogen in Section 2.1. 2.4. Sausage making To prepare sausage links, one or more 2 lb (0.91 kg) packages of frozen linguiça batter were thawed in cold tap water to 22 C (1e2 h). A 500 g quantity in a stainless steel container was inoculated with 10 ml suspension of the pathogen and mixed with a stainless steel balloon whisk. No inoculum was added to the other w500 g control batter. Among the pathogens studied, C. jejuni presented a special case, as difficulty was experienced achieving high enough initial levels of CFU/ml to demonstrate a 5 log kill; therefore, 40 ml of the C. jejuni suspension were added to 360 g of batter in this case only. Both inoculated and un-inoculated batters were sampled to determine a time-zero inoculum level, after which the batters were used to make linguiça links. The non-inoculated batter was first placed into the sausage-stuffer attachment of a KitchenAid food processor, stuffed into natural-pork sausage casings (Oversea Casing Company, Seattle, Wash.) and twisted into 10 cm links with a greatest diameter of 3 cm and a fresh weight of 75e80 g. The inoculated batter was then stuffed into additional sausage casings in the same manner. A similar method of preparation was described by Mattick, Bailey, Jorgensen, and Humphrey (2002), who described the products as “handmade sausages”.
2.2. Linguiça batter
2.5. Initial pathogen analysis of sausage links
The linguiça batter was formulated at the UC Davis Cole Meat Laboratory and transported frozen to the UC Davis Food Safety Laboratory for bacterial inoculation and challenge studies. The final
One representative link each of inoculated and un-inoculated sausage was taken for analysis. The links were plunged into boiling water for 5 s to pasteurize the surface, and the interior of the
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sausage was sampled for the target pathogen. Assays were done as described in the batter-heating experiments, above.
Table 1 Decimal reduction times (DRTs) for thin-layer in linguiça batter experiments. Pathogen
Pa
Campylobacter jejuni
0.0028
50 55 60
11.65 4.09 1.56
2.71 0.20 0.24
64.12 30.70 4.50
Escherichia coli O157:H7
0.0706
50 55 60
(12.50)c (5.00)c 1.45
0.28
4.40
C
DRT (min)
2.6. Sausage smoking Sausages were smoked in a 20 lb (9.1 kg) capacity, non-insulated, stainless-steel smoker (Model 41300, The Sausage Maker, Inc., Buffalo, NY; 1000 W, 110 v) that was chosen to fit within our laboratory. The unit was pre-heated to 88 C (measured with a dial thermometer whose stem was inserted 12.5 cm into the interior through a port in the door), and eight to ten sausage links were hung from wooden dowels inside the smoker. In most runs, a thermocouple (Atkins Technical, Inc., Gainesville, FL) was placed inside a randomly selected link of un-inoculated sausage, which was positioned near the center of the array. The smoker was again heated to an internal temperature of w88 C, and a stainless steel pan with 250 g hickory wood sawdust moistened with 400 ml water was placed on the electric coil. Internal sausage and smoking-space temperatures were recorded every 15 min. After the sawdust’s placement in the smoker, the internal smoker temperature was decreased to an average of 80 C, controlled by the setting of the top vent. The internal temperature of the sausage was slowly raised to 60 C and kept at or above that temperature for 90 min. After smoking, the sausage was cooled for 15 min and tested as described above for the presence of the target pathogen. 2.7. Proximate analyses Representative samples from three batches of linguiça, before and after smoking, were submitted to a commercial laboratory (JL Analytical Services, Modesto, CA) for proximate analyses (Crude proteindAOAC 990.03; Crude fatdAOAC 960.39; Crude fiberd AOAC 962.09; AshdAOAC 942.05). Also, samples of raw batter and corresponding smoked sausages were held at 60 C for 24 h in containers devised to let moisture evaporate and fatty drippings to separate from the solid material. Weights of the samples before and after treatment, as well as the weights of the collected fat, were recorded. 2.8. Statistics Numerical data were log-transformed to assure normal distribution and were subjected to analysis of variance (ANOVA) using PROC GLM. Assumption checks were done with PROC UNIVARIATE, and the confidence limits were calculated using PROC MEANS. Statistical assistance was provided by the UC Davis Statistics Laboratory, using the statistical software SAS Version 9.1. Logtransformed data were used to plot thermal death curves by the linear-least-squares method. Decimal reduction times (DRTs), or D-values, were derived from the slopes of the curves, and z-values (number of degrees change that would cause a 10-fold increase or decrease in the DRT) estimated from the DRTs at the chosen temperatures (Hajmeer & Cliver, 2002).
Mean
Upperb
Listeria monocytogenes
0.0618
50 55 60
10.73 4.19 2.15
2.18 0.32 0.51
52.71 55.02 2.48
Salmonella Newport
0.0018
50 55 60
87.36 3.95 1.08
2.62 2.34 0.57
2911.80 6.67 2.02
Yersinia enterocolitica
0.0054
50 55 60
10.44 1.69 (0.22)c
5.42 0.29
92.29 11.79
a b c
Probability that within-pathogen differences occurred by chance. Limits of 95% confidence interval. Based on a single experiment, rather than three.
in response to temperature (P ¼ 0.1223) were significant, the DRT for Y. enterocolitica at 60 C was judged too short to be measured accurately by the present method, so only one trial was performed. Only rough estimates of z-values are possible from these limited data; they are: C. jejuni, 9.9 C; E. coli O157:H7, 9.0 C; L. monocytogenes, 11.7 C; S. enterica, 1.0 C; and Y. enterocolitica, 5.7 C. The z-value for S. enterica seems improbably low: a meta-analysis by Doyle and Mazzotta (2000) calculated a “generic” z-value of 5.3 C for S. enterica. The thin-layer experiments were intended to provide nearinstantaneous temperature adjustments, so that time-at-temperature could be accurately estimated. A similar procedure was used by Sallami et al. (2006) with Listeria and Salmonella in bologna batter, except that their batter was 2 mm, rather than 1 mm, thick. The results indicate that the linguiça batter was not an exceptional matrix for the thermal death of these pathogens. The USDA Pathogen Modeling Program (USDA, 1999) predicts that, at 60 C, pH 5.8, and 1% NaCl, a 5 log reduction of E. coli O157:H7 in simulated beef gravy would occur in 8.6 min (confidence limits 6.8e10.9 min), and of L. monocytogenes in ground beef, in 20.8 min (confidence limits 14.9e29.2 min). Juneja, Snyder, and Marmer (1997) reported D-values for E. coli O157:H7 in ground beef of 21.13 0.25 min at
Table 2 Shrinkage due to smoking, by proximate analysis, assuming 36% total weight loss. Batch
Treatment
Weighta (g)
A
Raw Smoked
77 49
B
Raw Smoked
C Pooled
3. Results and discussion 3.1. Thermal kills in batter DRTs from the thin-layer experiments were measured in minutes and were very short at 60 C (Table 1). There may have been deviations from linearity; the observations were not extensive enough to explore this. For the E. coli O157:H7 experiment, data from all but one trial at 50 C and at 55 C were discarded for technical complications. Although neither the among-species differences overall (P ¼ 0.1584) nor the among-species differences
Lowerb
Moisture
Crude protein
Crude fat
%
g
%
g
%
g
64.9 44.0
50.0 21.6
16.2 24.0
12.5 11.8
17.7 27.9
13.6 13.7
77 49
61.6 39.9
47.4 19.6
16.3 26.6
12.6 13.0
19.0 28.6
14.6 14.0
Raw Smoked
77 49
61.6 40.3
47.4 19.7
16.3 25.7
12.6 12.6
19.0 29.3
14.6 14.4
Raw Smoked
77 49
62.7 41.4
48.3 20.3
16.3 25.4
12.6 12.4
18.6 28.6
14.3 14.0
a These weights were arbitrarily selected for illustrative purposes. Sausage weights before smoking averaged w77 g, and after-smoking weights averaged w49 g, whereas raw samples submitted for proximate analysis were of batter, rather than stuffed sausages.
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Table 3 Effect of smoking linguiça on bacterial pathogens. Pooled reduction (log)a
Pb
>5 >8 >8
7.43
0.0190
w6 >6 >7
6.62
0.0042
3.5 102 <10 <10
>7 >6 >6
7.06
0.0045
9.8 106 7.7 107 1.9 106
<10 <10 <10
>5 >6 >5
6.05
0.0059
8
<10 <10 <10
>7 >6 >7
7.06
0.0025
Pathogen
Run
Pre-smoke (CFU/g)
Post-smoke (CFU/g)
Kill (log)
Campylobacter jejuni
1 2 3
2.3 106 2.4 109 3.6 109
<10 <10 <10
Escherichia coli O157:H7
1 2 3
7
2.2 10 7.0 107 1.7 108
3.5 10 <10 <10
Listeria monocytogenes
1 2 3
3.0 1010 1.3 107 8.4 107
Salmonella enterica
1 2 3 1 2 3
Yersinia enterocolitica
a b
3.7 10 2.4 107 1.7 108
1
Calculated with <10 CFU/g data assigned a value of 5 CFU/g. Probability that this reduction could have occurred by chance alone.
55 C and 3.17 0.18 min at 60 C, which would predict 5 log destructions in w106 min and w16 min at those temperatures. The largest D-values reported by Doherty et al. (1998) for L. monocytogenes in ground beef at 50, 55, and 60 C were 43.5, 3.53, and 0.33 min, respectively; for Y. enterocolitica, these were 26.3, 1.24, and 0.11 min, respectively. Murphy, Beard, Martin, Duncan, and Marcy (2004) reported D-values for E. coli O157:H7, L. monocytogenes, and Salmonella in ground pork at 60 C that were roughly twice what we observed; the authors suggested that their high D-values were probably associated with the 40.2% fat content of their pork. Mattick et al. (2002) inoculated their “handmade” sausages with Salmonella Typhimurium at a level of 280 CFU/g and found some surviving salmonellae after barbecuing the sausages to an internal temperature of 66 C. 3.2. Smoking effects The smoking process reduced the weight of the linguiça: raw weights ranged roughly 75e80 g, whereas smoked weights were generally 48e51 g. Moisture was removed, primarily due to smoking heat, but fat may or may not have been reduced, depending on the mode of analysis (Table 2). After 24 h at 60 C, weight loss from raw (un-smoked) batter totaled 61.9% (7.7% attributed to fat loss, and 54.1% to moisture loss). Weight lost from smoked-sausage material was 51.6% (0.1% attributed to fat loss, and 51.5% to moisture loss). The mean aw was 0.974 before and 0.962 after smoking. Among 20 batter samples, the mean pH value was 5.58 0.07, with a range of 5.44e5.71. Among 20 samples from smoked sausages, the mean pH was 5.66 0.19, with a range of 5.52e5.93. Smoking was found to be effective against all five of the tested pathogens (Table 3), presumably because of the high temperatures employed. Reductions were found to be similar among the pathogen species (P ¼ 0.5742). The present study has few counterparts in the literature; these generally lack details as to the proximate composition of the sausage and the specifics of the smoking process. Smith, Huhtanen, Kissinger, and Palumbo (1975) reported that smoking beef-pork pepperoni to an internal temperature of 60 C reduced experimentally inoculated S. Dublin from 1.6 102 CFU/g to an undetectable level. Buncic (1991) found L. monocytogenes in 21% of Yugoslavian vacuum-packaged, hotsmoked sausages, but not in hot-smoked sausages heated to
70e75 C after they had been smoked. Ferreira et al. (2006) detected E. coli (not described as enterohemorrhagic) in 16 of 20 and Listeria spp. in 10 of 15 market samples of alheira, a sausage of variable composition produced in Portugal by fermentation followed by smoking. More recently, Karakolev (2009) found L. monocytogenes in 12 of 140 Bulgarian market samples of “rawsmoked” sausages. If not raw, linguiça is sold in California as “not fully cooked” (must attain an internal temperature of at least 144 F [62 C]) or “fully cooked” (must attain an internal temperature of at least 155e158 F [68e70 C]) (Earl Willis, personal communication). Minimum time-at-temperature is not specified, but smoking times are said typically to be 12e18 h or more for not-fully-cooked linguiça and 8e12 h for fully-cooked. Our smoked sausage spent 1e2 h at temperatures 60 C, often with a final attained temperature as high as w71 C; all five of the tested pathogens were inactivated at least 99.999%. This indicates that linguiça, formulated and smoked as was done here, is a relatively safe productdbarring subsequent mishandling.
Acknowledgements We thank the late Earl Willis of the CDFA for providing documentation of California linguiça making, as well as guidance during and after the study. We thank Matthew Livingston of the Cole Meat Laboratory, University of California-Davis (UCD) for preparing the batter and for expert advice on processing. Dr. James Glover, of the CDPH, was a source of both information and inspiration. We are grateful to Dr. Neil H. Willits of the UCD Statistical Laboratory for the statistical analyses. Dale Francis of the CDFA also provided valuable information. This study was supported by the Center for Food Animal Health, UCD, with funds from the CDFA.
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