412
Journal o f Food Protection, Vol. 77, No. 3, 2014, Pages 412-418 doi: 10,4315/0362-028X.JFP-13-322 Copyright © , International Association for Food Protection
Analysis of Microbiological Contamination in Mixed Pressed Ham and Cooked Sausage in Korea MYOUNG-SU PARK,1 JUN WANG, 1 JOONG-HYUN PARK, 1 FEREIDOUN FORGHANI, 1 JIN-SAN MOON,2 a n d DEOG-HWAN OH1* 1Department o f Food Science and Biotechnology and Institute o f Bioscience and Biotechnology, Kangwon National University, Chuncheon, Gangwon 200-701, Korea; and departm ent o f Animal Disease Control & Quarantine, Animal and Plant Quarantine Agency, Anyang, Gyeonggi, 430-824, South Korea MS 13-322: Received 4 August 2013/Accepted 17 October 2013
ABSTRACT The objective of this study was to investigate the microbial contamination levels (aerobic bacteria plate count [APC], coliforms, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes) in mixed pressed ham and cooked sausage. A total of 180 samples were collected from factories with and without hazard analysis critical control point (HACCP) systems at four steps: after chopping (AC), after mixing (AM), cooling after the first heating process, and cooling after the second heating process. For ham, APCs and coliform and E. coli counts increased when ingredients were added to the meat at the AC step. Final product APC was 1.63 to 1.85 log CFU/g, and coliforms and E. coli were not detected. 6. aureus and L. monocytogenes were found in nine (15.0%) and six (10.0%) samples, respectively, but only at the AC and AM steps and not in the final product. Sausage results were similar to those for ham. The final product APC was 1.52 to 3.85 log CFU/g, and coliforms and E. coli were not detected. S. aureus and L. monocytogenes were found in 29 (24.2%) and 25 (20.8%) samples at the AC and AM steps, respectively, but not in the final product. These results indicate that the temperature and time of the first and second heating are of extreme importance to ensure the microbiological safety of the final product regardless of whether a HACCP system is in place. Microorganism contamination must be monitored regularly and regulations regarding sanitization during processing should be improved. Education regarding employee personal hygiene, environmental hygiene, prevention of cross-contamination, ingredient control, and step-by-step process control is needed to reduce the risk of food poisoning.
Foodbome diseases caused by microbiological hazards in meat products are a major public health concern for the food industry and consumers. Meat is one of the most widely consumed foods in the world. Meat products have become one of the main foods in Korea and may be an important vehicle of foodbome diseases. Therefore, basic information on the microbiological quality of the processed meat products available to consumers is needed. Listeria monocytogenes and Staphylococcus aureus cause a large number of outbreaks associated with the consumption of contaminated foods, especially meat products. Listeriosis is caused by L. monocytogenes. This disease occurs most often in individuals with weak immune systems and has recently become a serious health problem (17). L. monocytogenes can survive and grow over a temperature range of —0.4 to 45°C and under low pH (as low as 4.4) and high salt conditions (19). This widespread pathogen has been isolated from meat products in retail markets. Products such as pork meat are extremely vulnerable to contamination with L. monocytogenes and are generally considered to be at a higher risk for L. monocytogenes contamination than other foods (16). * Author for correspondence. Tel: 82-33-250-6457; Fax: 82-33-250-6457; E-mail: [email protected].
S. aureus has been identified in ready-to-eat food products in various parts of the world, especially in Southeast Asia (1). Food products usually become contam inated by S. aureus during preparation and processing and can subsequently cause foodbome disease. About 10.8% of 33,353 patients with foodbome illness in Korea from 2001 to 2005 were sickened by S. aureus enterotoxins (3). Staphylococcal enterotoxins have been identified as the cause of staphylococcal foodbome illness (15). From 1969 to 1990, 53% of the staphylococcal foodbome illness cases in the United Kingdom were caused by meat products, especially ham, which was frequently implicated (18). Meat products such as ham and sausages can be a good growth medium for S. aureus and therefore have been implicated in staphylococcal foodbome illness (13). Staphylococcal enterotoxin A in canned meat can cause illness even after the canning process because the toxin is heat resistant. In 2005, the U.S. Department of Agriculture, Food Safety and Inspection Service (14) developed a hazards and controls guide for meat and poultry that can be used to evaluate all aspects of a company’s system for producing processed meat and poultry products. The core goal was to reduce the risk of foodbome illness associated with the consumption of meat and poultry products and provide suitable and feasible measures for improving each step in
J. Food Prot., Vol. 77, No. 3
MICROBIOLOGICAL CONTAMINATION IN HAM AND SAUSAGE
413
FIGURE 1. Process flow fo r mixed pressed ham and sausage in the target factories. AC, after chopping; AM, after mixing; FH, during cooling after first heating process; SH, during cooling after second heating process.
the food production process where hazards could or do occur. The hazard analysis critical control point (HACCP) system is designed to identify all hazards (biological, physical, or chemical), identify the critical control points and critical limits in the process (e.g., specific sanitation procedures, prevention of cross-contamination, product-formulation con trols, and employee and environmental hygiene), monitor procedures, and keep records (4). In Korea, there are two types of meat product factories: those with HACCP systems and those without such systems. In HACCP factories, the critical control points (such as chopping, mixing, and heat treatment) control the product quality and eliminate most of the biological hazards. However, according to previous studies, the microbial contamination levels in meat products from both HACCP and non-HACCP processing factories in Korea have not been reported. The objective of this study was to evaluate the microbial contamination levels (aerobic bacteria plate counts [APCs], coliforms, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes) in mixed pressed ham and cooked sausage from HACCP and non-HACCP factories in Korea. MATERIALS AND METHODS Collection and preparation of food samples. A total of 180 samples were analyzed. Five 300-g food samples were collected at each sampling point from four different steps: after chopping, after mixing, during cooling after the first heating process, and during cooling after the second heating process. Samples of cooked sausage were collected from three HACCP factories and three nonHACCP factories, and mixed pressed ham samples were purchased from three HACCP factories. Figure 1 shows the processing chain and the sampling points for mixed pressed ham and sausage in the target factories. The collected samples were stored at 5 + 3°C in and icebox and transported to the laboratory within 24 h.
FIGURE 2. Aerobic plate counts (APCs) and counts o f coliforms and E. coli in mixed pressed ham at each processing step in three HACCP factories (A, B, and C). ♦ , APC; A. coliforms; O, E. coli. AC, after chopping; AM, after mixing; FH, during cooling after first heating process; SH, during cooling after second heating process.
Microbiological analysis. Each 25-g sample was mixed with 225 ml of 0.1% sterile peptone water in a stomacher bag and pummeled (Lab-Blender 400, Seward Medical, London, UK) for 2 min at 200 rpm at room temperature (23 + 2°C). A I-ml al iquot of the homogenate was serially diluted in 9 ml of 0.1% sterile peptone water and used for microbiological analysis. The APC was determined according to the method of the Animal, Plant and Fisheries Quarantine and Inspection Agency (11). A 100-pl of the sample suspension was plated in duplicate onto plate count agar (Difco, BD, Detroit, MI), and 1 ml was added to duplicate Petrifilm E. coh'-coliform count plates (3M, St. Paul,
414
PARK ET AL.
FIGURE 3. Aerobic plate counts (APCs) and counts o f coliforms and E. coli in cooked sausage at each processing step in three HACCP factories (A, B, and C) and three non-HACCP factories (D, E, and F). ♦ , APC; A, coliforms; O , E. coli. AC, after chopping; AM, after mixing; FH, during cooling after first heating process; SH, during cooling after second heating process.
J. Food Prot., Vol. 77, No. 3
Manufacturing Process (A)
Manufacturing Process (B)
AC
Manufacturing Process (E)
MN), and the agar plates were incubated at 35 °C for 24 to 48 h. APCs and E. coli counts were then determined, and the mean population from each treatment was calculated from three replicates of each experiment. S. aureus was detected and enumerated according to Korean Food and Drug Administration guidelines (12). Each 25-g sample was homogenized in 225 ml of tryptic soy broth (Difco, BD) in a stomacher (Lab-Blender 400) for 2 min and incubated at 35°C for 16 h. After incubation, 0.1 ml from each homogenate was directly streaked onto Baird-Parker agar base (Difco, BD) supplemented with 50 ml of egg yolk-tellurite solution and incubated at 35°C for 24 h. Suspected colonies (black, shiny, convex, and surrounded by clear zones) from each plate were confirmed as Staphylococcus spp. by microscopic examination and biochemical tests after restreaking on tryptic soy agar (TSA; Difco, BD) with 0.6% yeast extract. Strain identification was confirmed with an API Staph diagnostic kit (bioMerieux, Marcy l’Etoile, France). L. monocytogenes was detected and enumerated according to the ISO 11290-1:1996 and ISO 11290-2:1998 methods, respec tively (6-9). Each 25-g sample was homogenized in 225 ml of sterile University of Vermont medium modified Listeria broth (Difco, BD) in a stomacher (Lab-Blender 400) for 2 min and incubated at 20°C for 1 h. After this resuscitation step, 0.1 ml from each homogenate was directly streaked onto Oxford medium agar (Difco, BD) and incubated at 30°C for 24 to 48 h. From each plate of the selective medium, colonies presumed to be Listeria spp. were restreaked onto TSA with 0.6% yeast extract and incubated
AM
FH
SH
Manufacturing Process (F)
for 24 h at 37°C. Black colonies were selected for further analysis, and their identity was confirmed as L. monocytogenes with an API Listeria diagnostic kit (bioMerieux). Statistical analysis. The SPSS statistics 20 program (IBM Corporation, New York) was used for data analysis. Data were expressed as the mean + standard deviation and analyzed with an analysis of variance. Mean values were compared with Tukey’s test, and differences were considered significant at the 0.05 level.
RESULTS AND DISCUSSION APCs and coliform and E. coli counts in mixed pressed ham and sausage. Meat product processing usually involves a wide range of physical and chemical treatments, including cutting, chopping, mixing, curing, utilization of spices, stuffing, fermentation, drying, heat treatment, and packing. For ham and sausage in Korea, the technique applied in most meat factories is shown in Figure 1. A total of 180 samples were analyzed for APCs and counts of coliforms, E. coli, and the two foodbome pathogens S. aureus and L. monocytogenes. APCs in the materials of mixed pressed ham were 3.93 to 6.49 log CFU/ g and increased to 5.55 to 7.68 log CFU/g after mixing with other food ingredients (Fig. 2). After two heat treatments,
415
MICROBIOLOGICAL CONTAMINATION IN HAM AND SAUSAGE
J. Food Prot., Vol. 77, No. 3
TABLE 1. Levels of S. aureus and L. monocytogenes contamination in mixed pressed ham at each production step in three
HACCP factories Contamination level (log CFU/g)°
Factory A
Pathogen S. aureus
L. monocytogenes
B
S. aureus
L. monocytogenes
C
S. aureus
L. monocytogenes
After chopping
After mixing
Cooling after first heating
Cooling after second heating
Journal o f Food Protection, Vol. 77, No. 3, 2014, Pages 412-418 doi: 10,4315/0362-028X.JFP-13-322 Copyright © , International Association for Food Protection
Analysis of Microbiological Contamination in Mixed Pressed Ham and Cooked Sausage in Korea MYOUNG-SU PARK,1 JUN WANG, 1 JOONG-HYUN PARK, 1 FEREIDOUN FORGHANI, 1 JIN-SAN MOON,2 a n d DEOG-HWAN OH1* 1Department o f Food Science and Biotechnology and Institute o f Bioscience and Biotechnology, Kangwon National University, Chuncheon, Gangwon 200-701, Korea; and departm ent o f Animal Disease Control & Quarantine, Animal and Plant Quarantine Agency, Anyang, Gyeonggi, 430-824, South Korea MS 13-322: Received 4 August 2013/Accepted 17 October 2013
ABSTRACT The objective of this study was to investigate the microbial contamination levels (aerobic bacteria plate count [APC], coliforms, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes) in mixed pressed ham and cooked sausage. A total of 180 samples were collected from factories with and without hazard analysis critical control point (HACCP) systems at four steps: after chopping (AC), after mixing (AM), cooling after the first heating process, and cooling after the second heating process. For ham, APCs and coliform and E. coli counts increased when ingredients were added to the meat at the AC step. Final product APC was 1.63 to 1.85 log CFU/g, and coliforms and E. coli were not detected. 6. aureus and L. monocytogenes were found in nine (15.0%) and six (10.0%) samples, respectively, but only at the AC and AM steps and not in the final product. Sausage results were similar to those for ham. The final product APC was 1.52 to 3.85 log CFU/g, and coliforms and E. coli were not detected. S. aureus and L. monocytogenes were found in 29 (24.2%) and 25 (20.8%) samples at the AC and AM steps, respectively, but not in the final product. These results indicate that the temperature and time of the first and second heating are of extreme importance to ensure the microbiological safety of the final product regardless of whether a HACCP system is in place. Microorganism contamination must be monitored regularly and regulations regarding sanitization during processing should be improved. Education regarding employee personal hygiene, environmental hygiene, prevention of cross-contamination, ingredient control, and step-by-step process control is needed to reduce the risk of food poisoning.
Foodbome diseases caused by microbiological hazards in meat products are a major public health concern for the food industry and consumers. Meat is one of the most widely consumed foods in the world. Meat products have become one of the main foods in Korea and may be an important vehicle of foodbome diseases. Therefore, basic information on the microbiological quality of the processed meat products available to consumers is needed. Listeria monocytogenes and Staphylococcus aureus cause a large number of outbreaks associated with the consumption of contaminated foods, especially meat products. Listeriosis is caused by L. monocytogenes. This disease occurs most often in individuals with weak immune systems and has recently become a serious health problem (17). L. monocytogenes can survive and grow over a temperature range of —0.4 to 45°C and under low pH (as low as 4.4) and high salt conditions (19). This widespread pathogen has been isolated from meat products in retail markets. Products such as pork meat are extremely vulnerable to contamination with L. monocytogenes and are generally considered to be at a higher risk for L. monocytogenes contamination than other foods (16). * Author for correspondence. Tel: 82-33-250-6457; Fax: 82-33-250-6457; E-mail: [email protected].
S. aureus has been identified in ready-to-eat food products in various parts of the world, especially in Southeast Asia (1). Food products usually become contam inated by S. aureus during preparation and processing and can subsequently cause foodbome disease. About 10.8% of 33,353 patients with foodbome illness in Korea from 2001 to 2005 were sickened by S. aureus enterotoxins (3). Staphylococcal enterotoxins have been identified as the cause of staphylococcal foodbome illness (15). From 1969 to 1990, 53% of the staphylococcal foodbome illness cases in the United Kingdom were caused by meat products, especially ham, which was frequently implicated (18). Meat products such as ham and sausages can be a good growth medium for S. aureus and therefore have been implicated in staphylococcal foodbome illness (13). Staphylococcal enterotoxin A in canned meat can cause illness even after the canning process because the toxin is heat resistant. In 2005, the U.S. Department of Agriculture, Food Safety and Inspection Service (14) developed a hazards and controls guide for meat and poultry that can be used to evaluate all aspects of a company’s system for producing processed meat and poultry products. The core goal was to reduce the risk of foodbome illness associated with the consumption of meat and poultry products and provide suitable and feasible measures for improving each step in
J. Food Prot., Vol. 77, No. 3
MICROBIOLOGICAL CONTAMINATION IN HAM AND SAUSAGE
413
FIGURE 1. Process flow fo r mixed pressed ham and sausage in the target factories. AC, after chopping; AM, after mixing; FH, during cooling after first heating process; SH, during cooling after second heating process.
the food production process where hazards could or do occur. The hazard analysis critical control point (HACCP) system is designed to identify all hazards (biological, physical, or chemical), identify the critical control points and critical limits in the process (e.g., specific sanitation procedures, prevention of cross-contamination, product-formulation con trols, and employee and environmental hygiene), monitor procedures, and keep records (4). In Korea, there are two types of meat product factories: those with HACCP systems and those without such systems. In HACCP factories, the critical control points (such as chopping, mixing, and heat treatment) control the product quality and eliminate most of the biological hazards. However, according to previous studies, the microbial contamination levels in meat products from both HACCP and non-HACCP processing factories in Korea have not been reported. The objective of this study was to evaluate the microbial contamination levels (aerobic bacteria plate counts [APCs], coliforms, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes) in mixed pressed ham and cooked sausage from HACCP and non-HACCP factories in Korea. MATERIALS AND METHODS Collection and preparation of food samples. A total of 180 samples were analyzed. Five 300-g food samples were collected at each sampling point from four different steps: after chopping, after mixing, during cooling after the first heating process, and during cooling after the second heating process. Samples of cooked sausage were collected from three HACCP factories and three nonHACCP factories, and mixed pressed ham samples were purchased from three HACCP factories. Figure 1 shows the processing chain and the sampling points for mixed pressed ham and sausage in the target factories. The collected samples were stored at 5 + 3°C in and icebox and transported to the laboratory within 24 h.
FIGURE 2. Aerobic plate counts (APCs) and counts o f coliforms and E. coli in mixed pressed ham at each processing step in three HACCP factories (A, B, and C). ♦ , APC; A. coliforms; O, E. coli. AC, after chopping; AM, after mixing; FH, during cooling after first heating process; SH, during cooling after second heating process.
Microbiological analysis. Each 25-g sample was mixed with 225 ml of 0.1% sterile peptone water in a stomacher bag and pummeled (Lab-Blender 400, Seward Medical, London, UK) for 2 min at 200 rpm at room temperature (23 + 2°C). A I-ml al iquot of the homogenate was serially diluted in 9 ml of 0.1% sterile peptone water and used for microbiological analysis. The APC was determined according to the method of the Animal, Plant and Fisheries Quarantine and Inspection Agency (11). A 100-pl of the sample suspension was plated in duplicate onto plate count agar (Difco, BD, Detroit, MI), and 1 ml was added to duplicate Petrifilm E. coh'-coliform count plates (3M, St. Paul,
414
PARK ET AL.
FIGURE 3. Aerobic plate counts (APCs) and counts o f coliforms and E. coli in cooked sausage at each processing step in three HACCP factories (A, B, and C) and three non-HACCP factories (D, E, and F). ♦ , APC; A, coliforms; O , E. coli. AC, after chopping; AM, after mixing; FH, during cooling after first heating process; SH, during cooling after second heating process.
J. Food Prot., Vol. 77, No. 3
Manufacturing Process (A)
Manufacturing Process (B)
AC
Manufacturing Process (E)
MN), and the agar plates were incubated at 35 °C for 24 to 48 h. APCs and E. coli counts were then determined, and the mean population from each treatment was calculated from three replicates of each experiment. S. aureus was detected and enumerated according to Korean Food and Drug Administration guidelines (12). Each 25-g sample was homogenized in 225 ml of tryptic soy broth (Difco, BD) in a stomacher (Lab-Blender 400) for 2 min and incubated at 35°C for 16 h. After incubation, 0.1 ml from each homogenate was directly streaked onto Baird-Parker agar base (Difco, BD) supplemented with 50 ml of egg yolk-tellurite solution and incubated at 35°C for 24 h. Suspected colonies (black, shiny, convex, and surrounded by clear zones) from each plate were confirmed as Staphylococcus spp. by microscopic examination and biochemical tests after restreaking on tryptic soy agar (TSA; Difco, BD) with 0.6% yeast extract. Strain identification was confirmed with an API Staph diagnostic kit (bioMerieux, Marcy l’Etoile, France). L. monocytogenes was detected and enumerated according to the ISO 11290-1:1996 and ISO 11290-2:1998 methods, respec tively (6-9). Each 25-g sample was homogenized in 225 ml of sterile University of Vermont medium modified Listeria broth (Difco, BD) in a stomacher (Lab-Blender 400) for 2 min and incubated at 20°C for 1 h. After this resuscitation step, 0.1 ml from each homogenate was directly streaked onto Oxford medium agar (Difco, BD) and incubated at 30°C for 24 to 48 h. From each plate of the selective medium, colonies presumed to be Listeria spp. were restreaked onto TSA with 0.6% yeast extract and incubated
AM
FH
SH
Manufacturing Process (F)
for 24 h at 37°C. Black colonies were selected for further analysis, and their identity was confirmed as L. monocytogenes with an API Listeria diagnostic kit (bioMerieux). Statistical analysis. The SPSS statistics 20 program (IBM Corporation, New York) was used for data analysis. Data were expressed as the mean + standard deviation and analyzed with an analysis of variance. Mean values were compared with Tukey’s test, and differences were considered significant at the 0.05 level.
RESULTS AND DISCUSSION APCs and coliform and E. coli counts in mixed pressed ham and sausage. Meat product processing usually involves a wide range of physical and chemical treatments, including cutting, chopping, mixing, curing, utilization of spices, stuffing, fermentation, drying, heat treatment, and packing. For ham and sausage in Korea, the technique applied in most meat factories is shown in Figure 1. A total of 180 samples were analyzed for APCs and counts of coliforms, E. coli, and the two foodbome pathogens S. aureus and L. monocytogenes. APCs in the materials of mixed pressed ham were 3.93 to 6.49 log CFU/ g and increased to 5.55 to 7.68 log CFU/g after mixing with other food ingredients (Fig. 2). After two heat treatments,
415
MICROBIOLOGICAL CONTAMINATION IN HAM AND SAUSAGE
J. Food Prot., Vol. 77, No. 3
TABLE 1. Levels of S. aureus and L. monocytogenes contamination in mixed pressed ham at each production step in three
HACCP factories Contamination level (log CFU/g)°
Factory A
Pathogen S. aureus
L. monocytogenes
B
S. aureus
L. monocytogenes
C
S. aureus
L. monocytogenes
After chopping
After mixing
Cooling after first heating
Cooling after second heating
