1. -by- Jovana Kovačević Food Spoilage: “Stinkies”, “Slimies” and Biofilms October 22, 2009

2. 10/22/20092 Outline Spoilage of Food oSpoilage Signs oMeat, Fish, Dairy oWhat do laboratory tests tell you about spoilage? Biofilms oProperties and formation oWhy are they a problem? How do we deal with spoilage and biofilms?

3. 10/22/20093 Food Spoilage Undesirable deterioration of food quality that may result in changes in odor, taste and appearance of food.

4. 10/22/20094 Food Spoilage Types of spoilage:  Microbial:  Bacteria, yeasts, molds  Non-microbial  Foreign materials, enzymes Changes not necessarily harmful

5. 10/22/20095 Microbial Food Spoilage Each food unique microbial environment  unique spoilage agents for each Why do microorganisms spoil our food?  food composition ideal source of nutrients for microbes  availability of water and oxygen  suitable pH Factors that affect spoilage:  Oxygen, temperature, pH, a w, humidity

6. 10/22/20096 Spoilage organisms Similar microflora emerge in different foods under the same conditions Affected by the: oType of food oProcessing oPreservation oStorage conditions Domination of specific spoilage microbes: oInitial counts present oNutrient composition oChemical and physical parameters

7. 10/22/20097 Spoilage Signs Odor:  Breakdown of proteins – putrefaction e.g. “rotten egg” smell Sliminess Discoloration  Mold on bread, blue and green mold on citrus fruit and cheese

8. 10/22/20098 Spoilage Signs… Souring  Production of acid e.g. sour milk from production of lactic acid Gas formation  Meat becomes spongy  Swollen or bubbling packages and cans

9. 10/22/20099 Food Types: Based on rate of spoilage 1) Highly perishable  meat  fruit  milk  vegetables  eggs 2) Semi perishable  potatoes  nuts 3) Stable  rice  flour  dry beans WET DRY

10. 10/22/200910 Inadequate storage temperatures Prolonged storage times Improper ventilation Cross contamination Excessive delays between receiving and storing Inadequate food safety standards Common Causes of Food Spoilage

11. 10/22/200911 Meat Spoilage

12. 10/22/200912 Meat spoilage… Cutting board contamination Conveyor belts Temperature Delay between storage and distribution Fecal contamination from intestines

13. 10/22/200913 Meat Spoilage Continued Food Types of Spoilage Spoilage Microorganisms MEAT Fresh Putrefaction Clostridium, Pseudomonas, Proteus, Alcaligenes, Chromobacterium Souring Chromobacterium, Lactobacillus, Pseudomonas Cured Mouldy Penicillium, Aspergillus, Rhizopus Souring Pseudomonas, Micrococcus, Bacillus Greening Lactobacillus spp., Streptococcus, Pediococcus Slimy Leuconostoc Vacuum Packed Souring Greening Lactobacillus, Carnobacterium, Leuconostoc PoultryOdor, Slime Pseudomonas, Alcaligenes, Xanthomonas

14. 10/22/200914 Meat Spoilage Spoilage microflora is dominated by:  Pseudomonas spp.  P. fragi, P. lundensis and P. fluorescens  Acinetobacter  Psychrobacter immobilis When surface is left to dry:  Micrococci  Yeasts  Molds

15. 10/22/200915 Meat Spoilage Vacuum – packed:  Lactic acid bacteria  Enterobacteriaceae  Brochothrix thermosphacta (occasionally)  Clostridia  Shewanella putrefaciens (meat with ↑ pH)

16. 10/22/200916 Fish and Eggs Spoilage

17. 10/22/200917 Fish and Eggs Spoilage Continued Fish  Polluted waters  Transportation vehicles, boxes Poultry and Eggs  Human contact  Penetration by bacteria  Cracks TEMPERATURE PHYSICAL DAMAGE INTRINSIC FACTORS

18. 10/22/200918 Fish & Eggs Spoilage Continued Food Types of Spoilage Spoilage Microorganisms FISH Discoloration Pseudomonas Putrefaction Chromobacterium, Halobacterium, Micrococcus EGGS Green rot Pseudomonas Colorless rot Pseudomonas, Alcaligenes, Chromobacterium Black rot Coliforms Fungal rot Proteus, Penicillium, Mucor

19. 10/22/200919 Spoilage of Milk and Dairy Products http://farm3.static.flickr.com/2313/2181694147_12ce5cd763.jpg

20. 10/22/200920 Spoilage of Milk and Dairy Products Continued FoodTypes of SpoilageSpoilage Microorganisms DAIRY MILK (pasteurized) BitternessPseudomonas spp. SouringLactobacillus thermophilus Sweet curdlingBacillus cereus CHEESE Green discoloration Penicillium Green to black discoloration Cladosporium Black discoloration Candida Sliminess (high pH) Pseudomonas spp. “Gassy” cheese Coliforms, LAB, Clostridia

21. 10/22/200921 Spoilage of Fruits and Vegetables http://timelytidbits.wordpress.com/2008/07/28/the-word-of-god-as-rotting-fruit/

22. 10/22/200922 Spoilage of Fruits and Vegetables Food Types of Spoilage Spoilage Microorganisms FRESH FRUITS AND VEGETABLES Bacterial soft rotErwinia carotovera, Pseudomonas spp. Gray mould rotBotryitis cinerea Rhizopus soft rotRhizopus nigrican Blue mould rotPenicillium italicum Black mould rotAspergillus niger, Alternaria Sliminess and Souring Saprophytic bacteria

23. 10/22/200923 Examples of Food Quality Tests Meat & Fish: pH for vacuum packaging (< 6.0) Visual inspection for leakage / color change  Faulty seals, pin-holes Sensory test Aerobic plate count at 30°C  Indication of hygiene at packaging  Expected refrigerated shelf-life  ↑ APC = improper cleaning of equipment, unacceptable time/temperature history of raw products

24. 10/22/200924 Testing for Microbial Quality Total Viable Count Enterobacteriacea Coliforms Escherichia coli Enterococci Staphilococcus aureus Bacillus spp. Clostridium perfringens Lactic acid bacteria Pseudomonas species Yeasts and molds

25. 10/22/200925

26. 10/22/200926 Examples of Food Submission Forms

27. 10/22/200927 Total Viable Count Total Viable Count also known as:  Heterotrophic plate count (HPC)  Aerobic plate count (APC)  Total plate count (TPC)  Aerobic colony count (ACC)  Aerobic mesophilic count Purpose: The aerobic plate count gives the total number of bacteria able to grow in an oxygenated or aerobic environment. Cooked foods should have little to no bacteria present Result provides a guide for shelf-life of foods  The higher the count: prolonged storage, and/or inadequate cooking. Note: Not a useful indicator for fermented foods or fresh ready-to-eat raw fruits and vegetables.

28. 10/22/200928 Enterobacteriaceae A family of Gram-negative, facultatively anaerobic, rod-shaped bacteria that do not form endospores and ferment variety of sugars Includes:  Pathogenic microbes: e.g. pathogenic E. coli, Proteus, Salmonella, Shigella, Yersinia, Enterobacter, Serratia, Klebsiella  Non-pathogenic microbes: e.g. Citrobacter, Erwinia, Hafnia

29. 10/22/200929 Coliforms and E. coli Coliforms:  Total coliforms  Fecal coliforms: o Found in human and animal intestinal wastes o More precise indicator of the presence of sewage contamination than total coliforms  Four groups of fecal coliforms: oEnterobacter oKlebsiella o Escherichia oCitrobacter

30. 10/22/200930 Escherichia coli Best indicator of fecal sanitation problems Characterized by the fermentation of glucose and lactose Found  In the gut of warm blooded mammals  In the gut of insects and reptiles  As natural soil microbial flora

31. Sanitary Quality Guidelines Food Group Indicator Test Result (CFU/g) SatisfactoryUnsatisfactory Aerobic Colony Count Canned foods & Cooked hot-held food < 5> 5 Cooked chilled food no handling < 100> 1000 Cooked chilled food with handling & Preserved foods < 100,000> 1,000,000 Long shelf life fish products, meat products, fruit and vegetable products < 1,000,000> 100,000,000 Fermented foods & Fresh fruits and vegetables Not applicable Total Coliform Count All foods except fresh fruits & vegetables, or foods containing them <100> 1000 Fecal Coliform Count All foods except fresh fruits & vegetables, or foods containing them < 3> 3 Escherichia coli Count All foods < 3> 3

32. 10/22/200932 Aerobic Plate Count: Environmental Swabs Guidelines for interpretation of bacterial counts from swabs and sponges as means of monitoring the cleanliness of surfaces. Interpretation CFU Count Per Area Swabbed Log Conversion Counts on Surface* (based on 25 cm 2 surface area sampled) Clean< 45 CFU< 1.65< 5 CFU / cm 2 Contaminated140 to 260 CFU2.15 – 2.41~ 5 to 10 CFU / cm 2 Very Contaminated> 260 CFU> 2.41> 10 CFU / cm 2 ** Values will vary depending on size of the surface area sampled. To calculate the number of bacteria per cm 2 take the total CFU and divide by the surface area swabbed.

33. 10/22/200933 Common Spoilage Microorganisms Shewanella putrefaciens Pseudomonas spp. oGram negative oRod shaped oMotile oAerobic oNon-spore forming oBiofilm formers http://www.lille.inra.fr/lille_eng/unites_et_recherches/nos_unites/ gpta/interfaces_bacteries_aliments_surfaces_solides

34. Pseudomonas aeruginosa http://www.microbelibrary.org/asmonly/details.asp?id=545&Lang=

35. 10/22/200935 Biofilms “Variety of microorganisms arranged in a complex relationship to one another and embedded in a mass of extracellular polysaccharides of their own making” (http://www.personal.psu.edu/faculty/j/e/jel5/biofilms/)

36. Biofilms Continued Communities of bacteria adhering to:  Environmental surfaces  Living tissues  Indwelling medical devices  Industrial or potable water system piping  Natural aquatic systems  Food processing facilities: oFloors, waste water pipes, bends in pipes, rubber seals, conveyor belts, stainless steel surfaces (Kumar and Anand, 1998) FEMS Microbiology Letters. 228:203-210. http://www.microbelibrary.org/

37. 10/22/200937 Environmental Biofilms Sewage treatment bioreactors Water pipes Legume root nodules Dental units Contact lenses Termite, ruminant digestion Fermentations http://www3.sympatico.ca/tania.nicolas/River%20Biofilm.jpg

38. 10/22/200938 Why do bacteria form biofilms? Protection from the environment oSanitizers oAntibiotics Nutrient availability and metabolic cooperativity Acquisition of new genetic traits

39. 10/22/200939 Biofilm formation http://www.personal.psu.edu/faculty/j/e/jel5/biofilms/

40. 10/22/200940 Biofilm formation Figure 2. Formation and development of gram-negative microorganisms’ biofilm. Adapted from Microbial Biofilms: from ecology to molecular genetics by Davey and O’Toole (2000). Microbiol. Molec. Bio. Rev. 64(4):847-867.

41. 10/22/200941 Control of Microbial Spoilage Good manufacturing practices Cleaning and sanitation  Processing environment  Equipment Handling with minimal physical damage Washing (in some case) Proper storage temperature Rapid movement of food through processing plant Treatments…  preservation

42. 10/22/200942 Temperature based: oChilling oRefrigeration oFreezing oPasteurization oCanning Water activity reduction oDehydration oAddition of salts and sugars Irradiation Modified Atmosphere Packaging Fermentation Treatment with chemicals o Sorbic acid, phenylphenates, diphenyl and iodophors, fumigation with sulfur-containing dusts Food preservation

43. 10/22/200943 References 1. Davey and O’Toole (2000). Microbial Biofilms: from ecology to molecular genetics. Microbiol. Molec. Bio. Rev. 64(4):847-867. 2. Forsythe, S.J, and P.R. Hayes. 1998. Food Hygiene Microbiology and HACCP. 3 rd Ed. pp. 434. Aspen Publishers Inc., Maryland. 3. ICMSF. 2005. Microorganisms in Foods. Microbial Ecology of Food Commodities. 2 nd Ed. pp. 763. Kluwer Academic/Plenum Publishers, New York, NY. 4. Marsh, E.J., H. Luo, H. Wang. 2003. A three-tiered approach to differentiate Listeria monocytogenes biofilm-forming abilities. FEMS Microbiology Letters. 228:203- 210. 5. http://www.personal.psu.edu/faculty/j/e/jel5/biofilms/ 6. http://www3.sympatico.ca/tania.nicolas/River%20Biofilm.jpg 7. http://www.lille.inra.fr/lille_eng/unites_et_recherches/nos_unites/ gpta/interfaces_bacteries_aliments_surfaces_solides 8. http://www.personal.psu.edu/faculty/j/e/jel5/biofilms/ 9. http://www.microbelibrary.org/asmonly/details.asp?id=545&Lang= 10. http://timelytidbits.wordpress.com/2008/07/28/the-word-of-god-as-rotting-fruit/ 11. http://farm3.static.flickr.com/2313/2181694147_12ce5cd763.jpg

44. 10/22/200944 Thank you!