Effects of Chilling Methods on Bacterial Recovery and Reducing Bacteria on Pork Carcasses

By Vivian Chang

 
This research project was funded by the National Pork Board

 

Outline

• Foodborne Pathogens Associated with Pork
• Pork Processing
• Low temperatures
• Conventional- vs. Blast-Chilling
• Hypotheses
• Objectives
• Experimental Designs
• Results
• Conclusions
• Future Research

 

I. Foodborne Pathogens Associated with Pork

 

Salmonella Typhimurium: 54.4% tested positive from pork carcasses surfaces samples
Listeria monocytogenes: 52% tested positive from pork carcasses surfaces samples
Campylobacter spp.: 79.5% tested positive from pork carcasses surface samples
(Nationwide Pork Microbiological Study, 1996)

 

Salmonella Typhimurium

 

• Rod shaped
• Gram negative
• Facultative anaerobe
• Non spore forming
• Symptoms: diarrhea, fever and vomiting
• Mildly sensitive to heating and freezing
• Associated with pork, poultry and dairy products

 

Listeria monocytogenes

 

• Rod shaped
• Gram positive
• Facultative anaerobe
• Non spore forming
• Symptoms: vomiting, diarrhea and fever
• Resistant to effects of drying, freezing, heating, low pH and high salinity
• Associated with pork, milk, cheese, ready to eat meats, water and vegetables

 

Campylobacter coli

 

• Gram negative
• Microaerophilic organism
• Non spore forming
• Sensitive to heating, freezing and drying
• Symptoms: abdominal pain, fever and watery/bloody diarrhea
• Associated with pork

 

 

 

II. Pork Processing

 

• Stunning
  • Mechanical
  • Electrical
  • Loss of consciousness
• Bleeding
  • Six inch knife
  • Seven pounds of liquid blood
  • Nine minutes
• Scalding
  • Immerse in water at 60°C
  • 4 minutes
  • Loosen hair follicle
• Dehairing
• Remove of Head
  • Tongue
  • Tonsils
  • Esophagus
  • Trachea
  • Jowl
• Evisceration
  • Prevent cut or tear
  • Small and large intestine
  • Stomach
  • Liver
  • Spleen
• Splitting
  • Removal of kidneys and leaf fat
  • Trim blood clots and loose lymph glands
• Washing
  • water and/or 1-3% lactic/acetic acid
• Prepare Carcass for Chilling
  • Blast chill
  • Conventional chill

 

 

III. Low Temperatures

 

• Chilling temperature (10 to 15°C)
• Refrigeration temperature (0 to 10°C)
• Conventional-chilling (air temperature 1 to 4°C)
• Blast-chilling (air temperature –7 to -40°C)

(Mountney & Gould, 1988)

• Cold is used to preserve food for long periods of time
• Is not an effective means of destroying microorganisms in foods
• Maintains the food in a good physical state

(Brown, 1982)

 

Effects of Freezing

 

• Fast versus slow freezing
  • Size of the ice crystal
• Injury of microorganisms
  • Inability to replicate in selective environments
• Type of organism
  • Gram positive – Listeria monocytogenes
  • Gram negative – Salmonella Typhimurium and Campylobacter coli

 

 

 
Cold Shock
 

• Rapid decline of the temperature
• No adaptation to low temperatures
• Loss of selective permeability of the cellular membrane

(Rosset, 1982)

 

 

IV. Conventional- vs. Blast-Chilling

 

Conventional-chilling

• Temperature 1 to 4°C
• Air flow velocity 90 – 180 f/m
• 24 – 48 hrs
• Advantages
  • Commonly used in the pork industry
  • Less expensive
• Disadvantages
  • Requires additional cooler/storage space
  • Results in substantial evaporative weight loss
  • Time consuming – Increases incidence of pale soft exudative (PSE) pork

 

Blast-Chilling

• Temperature 7 to – 40 °C
• Air velocity 600 – 960 f/m
• 30 – 90 minutes
• Conventional-chilling
• Advantages
  • Reduces chilling time by 30 – 50%
  • Reduces incidence of pale soft exudative (PSE) pork
• Disadvantages
  • Expensive
  • Air fans in order to obtain high air flow velocity rates
  • Space requirements for the tunnels

 

 

Previous studies found:

• Blast-chilling decreases levels of carcass contamination and improves keeping quality (Price et al., 1976)
• Conventional-chilling is more detrimental to the psychrotophic cells than blast-chilling on pork carcasses (Brown, 1982)
• Blast-chilling reduced Campylobacter spp. on pork to below detectable levels (Oosterom et al., 1982)
• No difference between blast- and conventional-chilling for reduction of mesophilic bacterial populations on pork carcasses. (James et al., 1983)
• Blast-chilling on pork surfaces with fat tissue produced lower counts of coliform and Staphylococcus spp. (Carr et al., 1998)

 

 

V. Hypotheses

 

Differences exist between blast- and conventional-chilling of pork with regard to:

• Skin surface (skin-on vs. skin-off)
• Inoculation level (10⁵ vs. 10³ CFU/cm²)
• Type of microorganisms (Salmonella Typhimurium, Campylobacter coli, Listeria monocytogenes, Escherichia coli, Coliform and mesophilic bacteria)

 

 

 

VI. Objectives

 

• To determine the best recovery method for pathogens associated with cell suspensions and pork surfaces following freeze-thaw cycle.
• Use the most efficient recovery method to determine whether conventional- or blast-chilling effectively reduces bacterial levels associated with fecal contamination and pathogen contamination on skin-on and skin-off pork surfaces.

 

 

VII. Experimental Design

 

• Recovery Methods Experiments
• Blast- and Conventional-Chilling Experiments

 

Materials and Methods

• Listeria monocytogenes Scott A
  • Non-selective (trypticase soy agar, TSA)
  • Selective (modified oxford, MOX)
• Salmonella Typhimurium ATCC 14028
  • Non-selective (trypticase soy agar, TSA)
  • Selective (xylose lysine decarboxylase , XLD)
• Campylobacter coli ATCC 33559
  • Non-selective (brucella agar, Bru)
  • Selective (Campylobacter blood-free selective medium, CCDA)
• Recovery Methods
  • Non selective (NS)
  • Selective (S)
  • Overlay method (OV)
  • Thin agar layer method (TAL)
  • Lutri plate (LP)

 

 

 

 

Recovery Method Experiments

• 4 replications
• Pathogens (Listeria monocytogenes, Salmonella Typhimurium and Campylobacter coli)
• Cell suspension
• Pork loin roast experiments (covered with adipose tissue)

 

Cell Suspension Experiments Protocol

• Overnight culture
• Place tube in a freezer at -15 °C for 24 h
• Thaw at 4°C for 4h
• Serially dilute in BPW
• Plate on 5 different recovery methods
• Incubate
• Count plates

 

Statistical Analyses for Recovery Methods on Cell Suspensions

• Analysis of variance (ANOVA)
• General linear model procedure
• Tukey pairwise comparison test

 

 

 

 

Conclusions

 

• The TAL, OV and LP methods were not statistically different (P > 0.05) as compared to the NS method
• The TAL method was easier to perform and allowed for improved isolation of single colonies

 

Pork Loin Roast Experiments Protocol

• Overnight culture
• UV sterilized pork surfaces, mark 2, 10 x 10 cm areas
• Inoculate the areas with pathogen, for 15 min at 25°C
• Excise sample, stomach and plate (control sample)
• Place inoculated meat in a freezer at -15°C for 24h
• Thaw at 4°C, 4 h
• Excise sample, stomach
• Serially dilute, plate and incubate
• Count

 

Statistical Analyses for Recovery Methods

• Analysis of variance (ANOVA)
• General linear model procedure
• Tukey pairwise comparison test

 

 

 

 
Conclusions
 

• Thin agar layer (TAL) method was not significantly different, as compared to the Non-selective (NS) method
• TAL method presents selective isolation of foodborne pathogens

 

Materials and Methods for Blast- and Conventional-Chilling Experiments

• 4 replications of each experiment
• Variables
  • Treatments (untreated, blast- and conventional-chilling)
  • Pathogens (Listeria monocytogenes, Salmonella Typhimurium, Campylobacter coli)
  • Indicator organisms (Escherichia coli, coliforms and mesophilic bacteria)
  • Type of skin (skin-off and skin-on)
  • – Inoculation level for pathogens (10³ and 10⁵ log₁₀ CFU/ml)

 

General Protocol for Blast- and Conventional-Chilling Experiments

• Pork carcass sample with skin-on/off
• With USDA blue edible ink, mark 4, 12 x 12 cm area
• Test one area for natural flora
• Inoculate 3 areas with fresh sterile dilute feces inoculated with pathogen “cocktail” or non sterile dilute feces
• Aseptically excise one area and plate for initial bacterial level (control)
• Place inoculated surfaces in the blast or conventional chiller following industry parameters
• Aseptically excise and sample remaining 25cm²
• Enrichment (qualitative)
• Plate (quantitative)
• Incubate
• Count colonies

 

Statistical Analyses for Blast- and Conventional-Chilling Experiments

• Analysis of variance (ANOVA)
• General linear model procedure
• Tukey pairwise comparison test

 

Results for Indicator Microoganisms

• No statistically significant difference between blast- and conventionalchilling in reducing indicator microorganisms at 10³ and 10⁵ log₁₀ CFU/cm²
• No statistically significant difference between skin-on and skin-off in reducing indicator microorganisms at 10³ and 10⁵ CFU/cm₂

 

Results for Listeria monocytogenes

High inoculation ~ 5 log₁₀ CFU/cm²
Low inoculation ~ 3 log₁₀ CFU/cm²

 

Results for Salmonella Typhimurium

High inoculation ~ 5 log₁₀ CFU/cm²
Low inoculation ~ 3 log₁₀ CFU/cm²

 

Results for Campylobacter coli

High inoculation ~ 5 log₁₀ CFU/cm²
Low inoculation ~ 3 log₁₀ CFU/cm²
*Direct plating method negative, enrichment method negative
+Direct plating method negative, enrichment method positive

 

Conclusions

• Using TAL method, no significant difference between blast- and conventional-chilling in reducing indicator microorganisms
• Blast-chilling was significantly different in reducing pathogen microorganisms at 10⁵ log₁₀ CFU/cm²
• Pork samples inoculated with 3 log₁₀ CFU/cm² of Campylobacter coli and subjected to blast-chilling were reduced to undetectable levels.

 

X. Future Research
 

• To validate our results, commercial processors utilizing blast- or conventional-chilling should be surveyed and samples collected for bacteriological analyses
• If viable but nonculturable cells (VBNC) are present after chilling regimen

 

Acknowledgments

• Dr. Catherine Cutter
• Dr. Stephanie Doores, Dr. Edward Mills, Dr. William Henning
• Dr. Nancy Ostiguy and Robert Guyer
• Personnel at Meats Lab
• Kerry, Barb, Donna, Sergio, Ruben, and Dawn
• Juanita Wolfe

 
Vivian Chang
Vivian Chang, born in Guatemala City in 1975, obtained a B.S in Food Science from Universidad del Valle de Guatemala in 1994. In December 2002, she earned a M.S degree in Food Science from Penn State University and her major advisor was Dr. Catherine Cutter. Ms. Chang’s thesis project was concentrated on pathogens associated with pork and was funded by the National Pork Board. Since March 2002, she has been working at Tyson Foods Inc.