Food Microbiology

1. Food Microbiology Dr M.Altamimi

2. Is it a big deal? Indian police have arrested the headteacher of the school where 23 children died after eating food contaminated with a pesticide last week. World's best restaurant Noma (Denmark) gives 70 customers food poisoning

3. History and Development of FoodMicrobiology Food Fermentation • 1822 C.J. Person named the microscopic organism found on the surface of wine during vinegar production as Mycodermamesentericum. • Pasteur in 1868 proved that this organism was associated with the conversion of alcohol to acetic acid and named it Mycodermaaceti. • In 1898, MartinusBeijerinck renamed it Acetobacteraceti. • 1837 Theodor Schwann named the organism involved in sugar fermentation as Saccharomyces (sugar fungus). • 1838 Charles Cogniard-Latour suggested that growth of yeasts was associated with alcohol fermentation. • 1860 Louis Pasteur showed that fermentation of lactic acid and alcohol from sugar was the result of growth of specific bacteria and yeasts, respectively. • 1883 Emil Christian Hansen used pure cultures of yeasts to ferment beer.

4. Foodborne Diseases • 1820 Justin Kerner described food poisoning from eating blood sausage (due to botulism). Fatal disease from eating blood sausage was recognized as early as A.D. 900. • 1849 John Snow suggested the spread of cholera through drinking water contaminated with sewage. • In 1854, FilippoFacini named the cholera bacilli as Vibrio cholera, which was isolated in pure form by Robert Koch in 1884. • 1856 William Budd suggested that water contamination with faeces from infected person spread typhoid fever and advocated the use of chlorine in water supply to overcome the problem. • In 1800, G. de Morveau and W. Cruikshank advocated the use of chlorine to sanitize potable water.

5. Food Spoilage • 1804 Francois Nicolas Appert developed methods to preserve foods in sealed glass bottles by heat in boiling water. He credited this process to LazzaroSpallanzani(1765), who first used the method to disprove the spontaneous generation theory. • 1819 Peter Durand developed canning preservation of foods in steel cans. • Charles Mitchell introduced tin lining of metal cans in 1839. • 1870 L. Pasteur recommended heating of wine at 145 F (62.7°C) for 30 min to destroy souring bacteria. F. Soxhlet advanced boiling of milk for 35 min to kill contaminated bacteria. Later, this method was modified and named pasteurization, and used to kill mainly vegetative pathogens and many spoilage bacteria. • 1895 Harry Russell showed that gaseous swelling with bad odors in canned peas was due to growth of heat-resistant bacteria (spores).

6. 1885 Theodor Escherich isolated Bacterium coli (later named Escherichia coli) from the feces and suggested that some strains were associated with infant diarrhea. • 1888 A.A. Gartner isolated Bacterium (later Salmonella) enteritidisfrom the organs of a diseased man as well as from the meat the man ate. • In 1896, Marie von Ermengem proved that Salmonella enteritidiscaused a fatal disease in humans who consumed contaminated sausage. • 1894 J. Denys associated pyogenic Staphylococcus with death of a person who ate meat prepared from a diseased cow. • 1895 Marie von Ermengem isolated Bacillus botulinus(Clostridium botulinum) from contaminated meat and proved that it caused botulism.

7. Microbiology Techniques • 1854 Heinrich Schröder and Theodore von Dusch used cotton to close tubes and flasks to prevent microbial contamination in heated culture broths. • 1876 Car Weigert used methylene blue (a synthetic dye) to stain bacteria in aqueous suspensions. • 1877 Ferdinand Cohn showed heat resistance of Bacillus subtilisendospores. • 1878 Joseph Lister isolated Streptococcus (now Lactococcus) lactisin pure culture by serial dilution from sour milk. • 1880s Robert Koch and his associates introduced many important methods that are used in all branches of microbiology, such as solid media (first gelatin, then agar) to purify and enumerate bacteria, Petri dish, flagellarstaining, steam sterilization of media above 100 C, and photography of cells and spores. • 1884 Hans Christian Gram developed Gram staining of bacterial cells.

8. CURRENT STATUS Food Spoilage Food Fermentation /Probiotics FOOD MICROBIOLOGY Foodborne Diseases FOOD MANAGEMENT(HACCP)

9. YOUR ROLE AS FOOD MICROBIOLOGIST • Determine microbiological quality of foods and food ingredients by using appropriate techniques • Determine microbial types involved in spoilage and health hazards and identify the sources • Design corrective procedures to control the spoilage and pathogenic microorganisms in food • Learn rapid methods to isolate and identify pathogens and spoilage bacteria from food and environment

10. • Identify how new technologies adapted in food processing can have specific microbiological problems and design methods to overcome the problem • Design effective sanitation procedures to control spoilage and pathogen problems in food-processing facilities • Effectively use desirable microorganisms to produce fermented foods • Design methods to produce better starter cultures for use in fermented foods and probiotics • Know about food regulations. • Understand microbiological problems of imported foods

11. Characteristic of predominant microorganisms in food Bacteria, yeasts, molds, and viruses are important in food for their ability to: • cause foodborne diseases. • food spoilage. • produce food and food ingredients.

12. Classification microorganisms With nucleus Without nucleus Molds yeasts Eubacteria (bacteria) Archaea Gram stain Protein profile Base composition C+G % (10%) DNA RNA hybridization (90%) Nucleotide sequence. Morphology Reproduction Metabolic patterns

13. Yeast vs Molds Yeasts are widely distributed in nature. The cells are oval, spherical, or elongated, about 5–30 X 2–10 µm in size They are nonmotile. The cell wall contains polysaccharides (glycans), proteins, and lipids. The wall can have scars, indicating the sites of budding. The membrane is beneath the wall.. The nucleus is well defined with a nuclear membrane. Molds are nonmotile, filamentous, and branched . The cell wall is composed of cellulose, chitin, or both. A mold (thallus) is composed of large numbers of filaments called hyphae. An aggregate of hyphae is called mycelium. A hypha can be vegetative or reproductive. The reproductive hypha usually extends in the air and form exospores, either free (conidia) or in a sack (sporangium). Shape, size, and color of spores are used for taxonomic classification.

14. Bacteria • unicellular, most ca. 0.5–1.0 X 2.0–10 µm in size. • have three morphological forms: spherical (cocci), rod shaped (bacilli), and curved (comma). • The ribosomes are 70S type and are dispersed in the cytoplasm. • The genetic materials (structural and plasmid DNA) are circular, not enclosed in nuclear membrane, and do not contain basic proteins such as histones.

15. On the basis of Gram-stain behavior, bacterial cells are grouped as Gram-negative Gram-positive. • Gram-negative cells have a complex cell wall containing an outer membrane (OM) and a middle membrane (MM). • OM is resistant to many antibiotics, hydrophilic compounds and enzymes.

16. IMPORTANT MICROORGANISMS IN FOOD 1- Molds Fusarium. Many types are associated with rot in citrus fruits, potatoes, and grains. They form cottony growth. Aspergillus. It is widely distributed and contains many species important in food. Able to grow in low Aw such as grains, causing spoilage. They are also involved in spoilage of foods such as jams, and fruits and vegetables. Produce aflatoxin, some can be food additives. Rhizopus. They cause spoilage of many fruits and vegetables. Rhizopusstolonifer is the common black bread mold. Penicillium. form conidiophores on a blue-green,brushlike conidia head. Some species are used in food production, such as Penicilliumroquefortii and Pen. camembertii in cheese.

17. 2-Yeast Genera Saccharomyces. Cells are round, oval, or elongated. Saccharomycescerevisiae are used in baking for leavening bread and in alcoholic fermentation. They also cause spoilage of food, producing alcohol and CO2. Zygosaccharomyces. Cause spoilage of high-acid foods, such as sauces, ketchups, pickles, mustards, mayonnaise, salad dressings, especially those with less acid and less salt and sugar (e.g., Zygosaccharomycesbailii). Candida. Many species spoil foods with high acid, salt, and sugar and form pellicles on the surface of liquids. Some can cause rancidity in butter and dairy products (e.g., Candida lipolyticum).

18. 3-viruses • Hepatitis A,B,C. • Enteric virus. • Poliovirus. • Bacteriophage.

19. 4-G (-) bacteria. Brucella. Coccobacillii, mostly single; nonmotile. Different species cause disease in animals, including cattle, pigs, and sheep. They are also human pathogens and have been implicated in foodborne brucellosis. Brucellaabortus causes abortion in cows. Campylobacter. Two species, Campylobacter jejuni and Cam. coli, are foodbornepathogens. microaerophilic, helical, motile cells found in the intestinal tract of humans, animals, and birds. Escherichia. Straight rods; motile or nonmotile; mesophiles. Found in the intestinal contents of humans, warm-blooded animals, and birds. Many strains nonpathogenic, but some strains pathogenic to humans and animals and involved in foodborne diseases. Used as an indicator of sanitation: Escherichia coli. Pseudomonas. aerobes; motile rods; psychrotrophs(grow at low temperatures). Acetobacter. Ellipsoid to rod-shaped, occur singly or in short chains; motile or nonmotile; aerobes; oxidize ethanol to acetic acid; mesophiles. Cause souring of alcoholic beverages and fruit juices and used to produce vinegar.

20. Salmonella. Medium rods ; usually motile; mesophiles. There are over 2000 serovarsand all are regarded as human pathogens. Found in the intestinal contents of humans, animals, birds, and insects. Major cause of foodborne diseases. Species: Salmonella enterica ssp. enterica. Vibrio. Curved rods ; motile; mesophiles. Found in freshwater and marine environments. Some species need NaCl for growth. Several species are pathogens and have been involved in foodborne disease (Vibriocholerae,Vib. parahaemolyticus, and Vib. vulnificus), whereas others can cause food spoilage (Vib. alginolyticus). Shigella. Medium rods; nonmotile; mesophiles. Found in the intestine of humans and primates. Associated with foodborne diseases. Species: Shigelladysenteriae.

21. 5- G(+) bacteria Enterococcus. Spheroid cells; occur in pairs or chains; nonmotile; facultative anaerobes; some strains survive low heat (pasteurization); mesophiles. Normal habitat is the intestinal contents of humans, animals, and birds, and the environment. Can establish on equipment surfaces. Used as an indicator of sanitation. Important in food spoilage. Species: Enterococcusfaecalis. Staphylococcus. Spherical cells ; occur singly, in pairs, or clusters; nonmotile; mesophiles; facultative anaerobes; grow in 10% NaCl. Staphylococcus aureus strains are frequently involved in foodborne diseases. Sta. carnosus is used for processing some fermented sausages. Main habitat is skin of humans, animals, and birds. Streptococcus. Spherical or ovoid ; occur in pairs or chains; nonmotile; facultative anaerobes; mesophiles. Streptococcus pyogenes is pathogenic and has been implicated in foodborne diseases; present as commensals in human respiratory tract. Str. thermophilus is used in dairy fermentation; can be present in raw milk; can grow at 50 C.

22. Lactococcus. Ovoid elongated cells ; occur in pairs or short chains; nonmotile; facultative anaerobes; mesophiles, but can grow at 10 C; produce lactic acid. Used to produce many bioprocessedfoods, especially fermented dairy foods. Species: Lactococcuslactis subsp. lactis and subsp. cremoris; present in raw milk and plants and several strains produce bacteriocins, some with a relatively wide host range against Gram-positive bacteria and have potential as food biopreservatives. Leuconostoc. Spherical or lenticular cells; occur in pairs or chains; nonmotile; facultative anaerobes; heterolacticfermentators; mesophiles, but some species and strains can grow at or below 3o C. Some are used in food fermentation. Psychrotrophic strains are associated with spoilage (gas formation) of vacuum-packaged refrigerated foods. Found in plants, meat, and milk. Species: Leuconostocmesenteroidessubsp. mesenteroides, Leu. lactis, Leu. carnosum. Leu. Mesenteroidessubsp. dextranicum produces dextran while growing in sucrose. Several strains produce bacteriocins, some with a wide spectrum against Gram-positive bacteria, and these have potential as food biopreservatives.

23. Pediococcus. Spherical cells; form tetrads; mostly present in pairs; nonmotile; facultative anaerobes; homolacticfermentators; mesophiles, but some can grow at 50C; some survive pasteurization. Some species and strains are used in food fermentation. Some can cause spoilage of alcoholic beverages. Found in vegetative materials and in some food products. Species: Pediococcusacidilactici and Ped. pentosaceus. Several strains produce bacteriocins, some with a wide spectrum against Gram-positive bacteria, and they can be used as food biopreservatives.

24. Spore forming Bacillus. Rod-shaped, straight cells; (thick or thin); single or in chains; motile or nonmotile; mesophiles or psychrotrophic; aerobes or facultative anaerobes; all form endospores that are spherical or oval and large or small (one per cell), spores are highly heat resistant. Includes many species, some of which are important in foods, because they can cause foodborne disease (Bacillus cereus) and food spoilage, especially in canned products (Bac. coagulans, Bac. stearothermophilus). Enzymes of some species and strains are used in food bioprocessing (Bac. subtilis). Present in soil, dust, and plant products (especially spices). Many species and strains can produce extracellular enzymes that hydrolyze carbohydrates, proteins, and lipids. G positive

25. Clostridium. Rod-shaped cells that vary widely in size and shape; motile or nonmotile; anaerobes (some species extremely sensitive to oxygen); mesophiles or psychrotrophic; form endospores (oval or spherical) usually at one end of the cell, some species sporulate poorly, spores are heat resistant. Found in soil, marine sediments, sewage, decaying vegetation, and animal and plant products. Some are pathogens and important in food (Clostridium botulinum, Clo. perfringens) and others are important in food spoilage (Clo. tyrobutyricum, Clo. saccharolyticum, Clo. laramie). Some species are used as sources of enzymes to hydrolyze carbohydrates and proteins in food processing.

26. Gram-Positive, Non sporulating Regular Rods G negative spore forming Lactobacillus. Rod-shaped cells that vary widely in shape and size, some are very long whereas others are coccobacilli, appear in single or in small and large chains; facultative anaerobes; most species are nonmotile; mesophiles (but some are psychrotrophs); can be homo- or heterolacticfermentors. Found in plant sources, milk, meat, and feces. Many are used in food bioprocessing (Lactobacillus delbrueckii subsp. bulgaricus, Lab. helveticus, Lab. plantarum) and some are used as probiotics (Lab. acidophilus, Lab. reuteri, Lab. casei subsp. casei). Some species can grow at low temperatures in products stored at refrigerated temperature (Lab. sake, Lab. curvatus). Several strains produce bacteriocins, of which some having a wide spectrum can be used as food biopreservatives. Desulfotomaculum. One species important in food is Delsufatomaculumnigrificans. The medium-sized cells are rod shaped, motile, thermophilic, strictly anaerobes, and produce H2S. Endospores are oval and resistant to heat. Found in soil. Cause spoilage of canned food.

27. Gram-Positive, Nonspore forming Irregular Rods Corynebacterium. Slightly curved rods; some cells stain unevenly; facultative anaerobes; nonmotile; mesophiles; found in the environment, plants, and animals. Some species cause food spoilage. Corynebacteriumglutamicum is used to produce glutamic acid Bifidobacterium. Rods of various shapes; present singly or in chains; arranged in V or star-like shape; nonmotile; mesophiles; anaerobes. Metabolize carbohydrates to lactate and acetate. Found in colons of humans, animals, and birds. Some species are used in probiotics(Bifidobacteriumbifidum, Bif. infantis, Bif. adolescentis).

28. Why ? An understanding of the sources of microorganisms in food is important to develop methods to control access of some microorganisms in the food, develop processing methods to kill them in food, and determine the microbiological quality of food, as well as set up microbiological standards and specifications of foods and food ingredients.

29. How ? Plants (Fruits and Vegetables) Proper methods used during growing (such as use of treated sewage or other types of fertilizers), damage reduction during harvesting, quick washing with good qualitywater to remove soil and dirt, and storage at low temperature before and after processing can be used to reduce microbial load in foods of plant origin. Animals, Birds, Fish, and Shellfish Prevention of food contamination from these sources needs the use of effective husbandry of live animals and birds, which includes good housing and supply of uncontaminated feed and water. Also, testing animals and birds for pathogens and culling the carriers are important in reducing the incidence of pathogenic microorganisms in foods.

30. Air Microbial contamination of food from the air can be reduced by removing the potential sources, controlling dust particles in the air (using filtered air), using positive air pressure, reducing humidity level, and installing UV light. Soil Removal of soil (and sediments) by washing and avoiding soil contamination can reduce microorganisms in foods from this source. Sewage To reduce incidence of microbial contamination of foods from sewage, it is better not to use sewage as fertilizer. If used, it should be efficiently treated to kill the pathogens. Also, effective washing of foods following harvesting is important.

31. Water Chlorine-treated potable water (drinking water) should be used in processing, washing, sanitation, and as an ingredient To overcome the problems, many food processors use water, especially as an ingredient, that has a higher microbial quality than that of potable water. Food Ingredients The ingredients should be produced under sanitary conditions and given antimicrobial treatments. In addition, setting up acceptable microbial specifications for the ingredients will be important in reducing microorganisms in food from this source.

32. Humans Proper training of personnel in personal hygiene, regular checking of health, and maintaining efficient sanitary and aesthetic standards are necessary to reduce contamination from this source. Equipment Proper cleaning and sanitation of equipment at prescribed intervals are important to reduce microbial levels in food. In addition, developing means to prevent or reduce contamination from air, water, personnel, and insects is important. Finally, in designingthe equipment, potential microbiological problems need to be considered.

33. Discussion scenario 1 • A batch of turkey rolls (10 lb — about 4.5 Kg — each) were cooked to 73.8⁰cinternal temperature in bags, opened, sliced, vacuum-packaged, and stored at 4.4⁰c. The product was expected to have a refrigerated shelf life of 50 days. However, after 40 days, the packages contained gas and 10 7bacterial cells/g of meat. The bacterial species involved in the spoilage was found to be Leuconostoccarnosum, which is killed at 73.8⁰ c. What could be the sources of the bacterial species in this cooked product?

34. Normal microbiological quality of food understanding of the microbial types (and their levels where possible) that can be expected under normal conditions in different food groups. RAW AND READY-TO-EAT MEAT PRODUCTS Normally, carcasses contain an average of 101–3 bacterial cells/in Salmonella serovars, Yersiniaenterocolitica, Campylobacter jejuni, Escherichia coli, Clostridium perfringens, and Staphylococcus aureus,

35. Ground meat can have 104–5 microorganisms/g Salmonella can be present at 1 cell/25 g. As indicated before, the frequency of the presence of Salmonella is higher in chicken than in red meats. • If the products are kept under aerobic conditions, psychrotrophic aerobes will grow rapidly, especially Gram negative rods, such as Pseudomonas. • Under anaerobic packaging, growth of psychrotrophic facultative anaerobes and anaerobes (e.g., Lactobacillus, Leuconostoc,)

36. Heat treatment, especially at an internal temperature of 73o C or higher, kills most microorganisms, except some thermodurics, which include Micrococcus, some Enterococcus, and maybe some Lactobacillus and spores of Bacillus and Clostridium. • The microbial level can be 101–2/ g.

37. RAW AND PASTEURIZED MILK • The predominant types from inside a healthy udder are Micrococcus, Streptococcus, and Corynebacterium. • Normally, raw milk contains <103 microorganisms/ml. • During refrigerated storage (at dairy farms and processing plants) before pasteurization, only psychrotrophs can grow in raw milk. They include Pseudomonas,coliforms and Bacillus spp.

38. In the U.S., the standard plate counts of raw milk for use as market milk are 1–3 X 105/ml, and for use in product manufacturing are 0.5–1 X 106/ml. • Grade A pasteurized milk can have standard plate counts of 20,000/ml and ≤10 coliforms/ml. SHELL EGG AND LIQUID EGG 107 bacteria Pseudomonas, Esc. coli, Enterobacter, Enterococcus, Micrococcus, and Bacillus. They can also have Salmonella from fecal contamination. Infected ovaries of laying hens can be the source of Salmonella Enteritidis in the yolk.

39. FISH AND SHELLFISH • Muscles of fish and shellfish are sterile, but scales, gills, and intestines harbor microorganisms. • 103–8 bacterial cells/g. • Pseudomonas,Enterococcus, Micrococcus, coliforms. • Fish and shellfish harvested from water polluted with human and animal waste can contain Salmonella, Shigella, Clo. perfringens, Vib. cholerae, and hepatitis A and Norwalk-like viruses. • Following harvest, microorganisms can grow rapidly in fish and crustaceans because of high Aw and high pH of the tissue.

40. VEGETABLES, FRUITS, AND NUTS • Generally, vegetables have 103–5 microorganisms/cm2 or 104–7/g. • lactic acid bacteria, Corynebacterium, Micrococcus, Enterococcus, and sporeformers. • Alternaria, Fusarium, and Aspergillus. • pathogenic protozoa and parasites. • In general, fruits have microbial populations 103–6/g. CEREAL, STARCHES, AND GUMS Unprocessed products (grains) may contain high bacterial levels (aerobic plate count @104/g, coliform @102/g, yeasts and molds @103/g).

41. CANNED FOODS • The products with a pH of 4.6 or above are given heat treatments to obtain commercial sterility, but those with a pH below 4.6 are given heat treatments 100oC. • In canned products stored at 30oC or below, thermophilic spores do not germinate to cause spoilage. However, if the cans are temperature-abused to 40oC or higher, the spores germinate. • If the canned products are given lower heat treatment (100oC), spores of mesophilic bacteria that include both spoilage (Bac. coagulans, Bac. licheniformis, Clo. sporogenes, Clo. butyricum) and pathogenic types (Bac. cereus, Clo. perfringens, Clo. botulinum), along with the spores of thermophiles, survive.

42. In low-pH products, particularly in tomato products, Bac. coagulans spores can germinate and cells can multiply and cause spoilage. Other sporeformers can germinate and grow in high-pH products SUGARS AND CONFECTIONERIES Sugar can have thermophilic spores of Bac. stearothermophilus, Bac. coagulans, Clo. thermosaccharolyticum, as well as mesophilic bacteria (e.g., Lactobacillus and Leuconostoc), yeasts, and molds

43. When sugars are used as ingredients in food products, the spores can survive and cause spoilage of products. Pathogens are not present in refined sugar unless contaminated. In liquid sugar, mesophiles can grow. • Refinedsugar, used in canned products or to make liquid sugar, has strict microbiological standards (for spores). SOFT DRINKS, FRUIT AND VEGETABLE DRINKS, JUICES, AND BOTTLED WATER. pH of 2.5 to 4.0. Fruit juices (100%) have a pH of 4.0 or below. Vegetable juices (e.g., tomato) can have a pH of 4.5 or above.

44. molds, yeasts, lactic acid bacteria, and acetic acid bacteria, can multiply. • In carbonated beverages, some yeasts being microaerophilic can grow; in beverages with fruit juices, Lactobacillus and Leuconostocspecies can grow. • In noncarbonated beverages, molds(Geotrichum) and Acetobacterand Gluconobacterspp. can also grow. Bottled water should not contain more than 10 to 100 bacteria and >10 coliforms/100 ml.

45. MAYONNAISE AND SALAD DRESSINGS • pH between 3.5 and 4.0. • Microorganisms are introduced into the products through ingredients, equipment, and air. • Normally, their numbers should not exceed 10/g. SPICES AND CONDIMENTS may contain microorganisms as high as 106–7/g. Spores of molds, Bacillus, and Clostridium spp. Also, micrococci, enterococci, yeasts, and several pathogens such as Salmonella spp., Sta. aureus, and Bac. cereus have been found.

46. Microbial Growth= increased number of cells. • Binary fission • Spores. • Asexual and sexual. • Generation time, for bacteria 18-20 min. then yeast and moulds. • Vibrio parahaemolyticus, under optimum conditions can have a generation time as low as 10 to 12 min. • microbial population can be calculated mathematically using logarithmatics (log10)

47. Optimum Growth • Many environmental parameters of food, such as storage temperature, acidity (pH), water activity (Aw), oxidation–reduction (O–R) potential, and nutrients, influence microbial growth rate. • If the growth rate is fastest (or generation time is shortest) at a certain temperature. This temperatureis referred to as the optimumgrowthtemperature. • The area under the two points on both sides of an optimum growth condition where minimum growth occurs is the growth temperature range.

49. Growth Curve • Cell mass, optic density OD, 600 nm • Cell count, every 1 hour. • Cell componenet, DNA, RNA • Cell products, acid, gas, metabolites. • Growth curve= cell(s)/hour.