1. Industrial microbiology Part 1 Industrial microbiology Part 1 Applied to food and beverages Compiled by Dr. Harbant

2. CO3: Ability to define, describe and utilize microbial growth in fermentation and biological process

3. Topics for discussion discuss the interaction of intrinsic (food-related) and extrinsic (environmental) factors related to food spoilage discuss the interaction of intrinsic (food-related) and extrinsic (environmental) factors related to food spoilage describe the various physical, chemical, and biological processes used to preserve foods describe the various physical, chemical, and biological processes used to preserve foods discuss the various diseases that can be transmitted to humans by foods discuss the various diseases that can be transmitted to humans by foods differentiate between food infections and food intoxications differentiate between food infections and food intoxications discuss the detection of disease-causing organisms in foods discuss the detection of disease-causing organisms in foods

4. Contd. describe the fermentation of dairy products, grains, meats, fruits, and vegetables describe the fermentation of dairy products, grains, meats, fruits, and vegetables discuss the toxins produced by fungi growing in moist corn and grain products discuss the toxins produced by fungi growing in moist corn and grain products discuss the direct use of microbial cells as food by humans and animals discuss the direct use of microbial cells as food by humans and animals list foods that are made with the aid of microorganisms and indicate the types of microorganisms used in their production list foods that are made with the aid of microorganisms and indicate the types of microorganisms used in their production describe probiotics describe probiotics

5. Intrinsic and Extrinsic Factors

6. Intrinsic Factors composition composition pH pH presence and availability of water presence and availability of water oxidation-reduction potential oxidation-reduction potential –altered by cooking physical structure physical structure presence of antimicrobial substances presence of antimicrobial substances

7. Food composition Food composition –Carbohydrates–do not result in major odor –Proteins and/or fats result in a variety of foul odors (e.g., putrefactions)

8. pH pH –low pH allows yeasts and molds to become dominant; higher pH allows bacteria to become dominant; higher pH favors putrefaction (the anaerobic breakdown of proteins that releases foul-smelling amine compounds)

9. Presence and availability of water Presence and availability of water Drying (removal of water) controls or eliminates food spoilage Addition of salt or sugar decreases water availability and reduces microbial spoilage Even under these conditions spoilage can occur by certain kinds of microorganisms Osmophilic–prefer high osmotic pressure Xerophilic–prefer low water availability Oxidation-reduction potential can be affected (lowered) by Oxidation-reduction potential can be affected (lowered) by cooking, making foods more susceptible to anaerobic spoilage

10. Physical structure affects the course and extent of spoilage Physical structure affects the course and extent of spoilage –Grinding and mixing (e.g., sausage and meat burger) increases surface area, alters cellular structure, and distributes microorganisms throughout the food –Vegetables and fruits have outer skins that protect against spoilage; spoilage microorganisms have enzymes that weaken and penetrate such protective coverings

11. Many foods contain natural antimicrobial substances Many foods contain natural antimicrobial substances –coumarins – fruits and vegetables –lysozyme – cow’s milk and eggs –aldehydic and phenolic compounds – herbs and spices –allicin – garlic –polyphenols – green and black teas

12. Extrinsic Factors temperature temperature –lower temperatures retard microbial growth relative humidity relative humidity –higher levels promote microbial growth atmosphere atmosphere –oxygen promotes growth –modified atmosphere packaging (MAP) use of shrink wrap and vacuum technologies to package food in controlled atmospheres use of shrink wrap and vacuum technologies to package food in controlled atmospheres

13. Temperature and relative humidity–at higher relative humidity, microbial growth is initiated more rapidly, even at lower temperatures Temperature and relative humidity–at higher relative humidity, microbial growth is initiated more rapidly, even at lower temperatures Atmosphere–oxygen usually promotes growth and spoilage even in shrink-wrapped foods since oxygen can diffuse through the plastic; high CO2 tends to decrease pH and reduce spoilage; modified atmosphere packaging (MAP) involves the use of modern shrink wrap materials and vacuum technology to package foods in a desired atmosphere (e.g., high CO2 or high O2) Atmosphere–oxygen usually promotes growth and spoilage even in shrink-wrapped foods since oxygen can diffuse through the plastic; high CO2 tends to decrease pH and reduce spoilage; modified atmosphere packaging (MAP) involves the use of modern shrink wrap materials and vacuum technology to package foods in a desired atmosphere (e.g., high CO2 or high O2)

14. Microbial Growth and Food Spoilage

15. Spoilage in meat and diary products Meats and dairy products are ideal environments for spoilage by microorganisms because: of their high nutritional value and the presence of easily utilizable carbohydrates, fats, and proteins; of their high nutritional value and the presence of easily utilizable carbohydrates, fats, and proteins; proteolysis (aerobic) and putrefaction (anaerobic) decompose proteins; proteolysis (aerobic) and putrefaction (anaerobic) decompose proteins; unpasteurized milk, favors microorganisms growth unpasteurized milk, favors microorganisms growth

16. Spoilage in plant material Fruits and vegetables have much lower protein and fat content then meats and dairy products and undergo different kind of spoilage: the presence of readily degradable carbohydrates in vegetables favors spoilage by bacteria; the presence of readily degradable carbohydrates in vegetables favors spoilage by bacteria; high oxidation–reduction potential favors aerobic and facultative bacteria; high oxidation–reduction potential favors aerobic and facultative bacteria; molds usually initiate spoilage in fruits. molds usually initiate spoilage in fruits.

17. Frozen citrus products are minimally processed and can be spoiled by lactobacilli and yeasts Frozen citrus products are minimally processed and can be spoiled by lactobacilli and yeasts

18. Spoilage in cereals and nuts Grains, corn, and nuts can spoil when held under moist conditions; this can lead to production of toxic substances: Grains, corn, and nuts can spoil when held under moist conditions; this can lead to production of toxic substances: –Ergotism is caused by hallucinogenic alkaloids produced by fungi in corn and grains –Fumonisins—contaminants of corn; cause disease in animals and esophageal cancer in humans; disrupt synthesis and metabolism of sphingolipids

19. Aflatoxins in food 1.Aflatoxins—planar molecules that intercalate into DNA and act as frame shift mutagens and carcinogens; 2.Aflatoxins can appear in milk if consumed by dairy cows, 3.Have also been observed in beer, cocoa, raisins, and soybean meal; 4.Aflatoxin sensitivity can be influenced by prior disease exposure (e.g., hepatitis B infection increases sensitivity)

20. Spoilage in sea food Shellfish and finfish can be contaminated by algal toxins, which cause a variety of illnesses in humans Shellfish and finfish can be contaminated by algal toxins, which cause a variety of illnesses in humans

21. Controlling Food Spoilage

22. Removal of microorganisms— filtration of water, wine, beer, juices, soft drinks and other liquids can keep bacterial populations low or eliminate them entirely Removal of microorganisms— filtration of water, wine, beer, juices, soft drinks and other liquids can keep bacterial populations low or eliminate them entirely Low temperature—refrigeration and/or freezing retards microbial growth but does not prevent spoilage Low temperature—refrigeration and/or freezing retards microbial growth but does not prevent spoilage

23. High temperature High temperature –Canning Canned food is heated in special containers called retorts to 115°C for 25-100 minutes to kill spoilage microorganisms Canned food is heated in special containers called retorts to 115°C for 25-100 minutes to kill spoilage microorganisms Canned foods can undergo spoilage despite safety precautions; spoilage can be due to spoilage prior to canning, underprocessing during canning, or leakage of contaminated water through can seams during cooling Canned foods can undergo spoilage despite safety precautions; spoilage can be due to spoilage prior to canning, underprocessing during canning, or leakage of contaminated water through can seams during cooling

24. Pasteurization kills pathogens and substantially reduces the number of spoilage organisms Pasteurization kills pathogens and substantially reduces the number of spoilage organisms –Low-temperature holding (LTH)—62.8°C for 30 minutes –High-temperature short-time (HTST)—71°C for 15 seconds –Ultra-high temperature (UHT)—141°C for 2 seconds –Shorter times result in improved flavor and extended shelf life

25. Heat treatments are based on a statistical process involving the probability that the number of remaining viable microorganisms will be below a certain level after a specified time at a specified temperature Heat treatments are based on a statistical process involving the probability that the number of remaining viable microorganisms will be below a certain level after a specified time at a specified temperature

26. Water availability—dehydration procedures (e.g., freeze-drying) remove water and increase solute concentration Water availability—dehydration procedures (e.g., freeze-drying) remove water and increase solute concentration

27. Chemical–based preservation Chemical–based preservation Regulated by the U.S. Food and Drug Administration (FDA); preservatives are listed as “generally recognized as safe” (GRAS); include simple organic acids, sulfite, ethylene oxide as a gaseous sterilant, sodium nitrite, and ethyl formate Regulated by the U.S. Food and Drug Administration (FDA); preservatives are listed as “generally recognized as safe” (GRAS); include simple organic acids, sulfite, ethylene oxide as a gaseous sterilant, sodium nitrite, and ethyl formate Effectiveness depends on pH; nitrites protect against Clostridium botulinum, but are of some concern because of their potential to form carcinogenic nitrosamines when meats preserved with them are cooked Effectiveness depends on pH; nitrites protect against Clostridium botulinum, but are of some concern because of their potential to form carcinogenic nitrosamines when meats preserved with them are cooked

28. Radiation—non-ionizing (ultraviolet or UV) radiation is used for surfaces of food- handling utensils, but does not penetrate foods; Radiation—non-ionizing (ultraviolet or UV) radiation is used for surfaces of food- handling utensils, but does not penetrate foods; ionizing (gamma radiation) penetrates well but must be used with moist foods to produce peroxides, which oxidize sensitive cellular constituents (radappertization); ionizing (gamma radiation) penetrates well but must be used with moist foods to produce peroxides, which oxidize sensitive cellular constituents (radappertization); ionizing radiation is used for sea foods, fruits, vegetables, and meats ionizing radiation is used for sea foods, fruits, vegetables, and meats

29. Microbial product-based inhibition Microbial product-based inhibition Bacteriocins—bactericidal proteins produced by bacteria; active against only closely related bacteria (e.g., nisin) Bacteriocins—bactericidal proteins produced by bacteria; active against only closely related bacteria (e.g., nisin) Bacteriocins function by several mechanisms, including dissipation of proton motive force, formation of hydrophobic pores in membranes, or inhibition of protein and RNA synthesis Bacteriocins function by several mechanisms, including dissipation of proton motive force, formation of hydrophobic pores in membranes, or inhibition of protein and RNA synthesis

30. Food-borne Diseases

31. Food-borne illnesses impact the entire world; Food-borne illnesses impact the entire world; –are either infections or intoxications; –are associated with poor hygiene practices

32. Food-borne infections Due to ingestion of microorganisms, followed by growth, tissue invasion and/or release of toxins

33. Salmonellosis Salmonellosis –caused by a variety of Salmonella serovars; –commonly transmitted by meats, poultry, and eggs; –can arise from contamination of food by workers in food-processing plants and restaurants Campylobacter jejuni Campylobacter jejuni –transmitted by uncooked or poorly cooked poultry products, –raw milk and red meats; –thorough cooking prevents transmission

34. Listeriosis Listeriosis –transmitted by dairy products Enteropathogenic, enteroinvasive, and enterotoxigenic Escherichia coli Enteropathogenic, enteroinvasive, and enterotoxigenic Escherichia coli –Spread by fecal-oral route; found in meat products, in unpasteurized fruit drinks, and on fruits and vegetables –Prevention requires prevention of food contamination throughout all stages of production, handling, and cooking

35. Viral pathogens Viral pathogens –usually transmitted by water or by direct contamination by food processors and handlers; –recently Norwalk-like viruses have been involved in major outbreaks on several large cruise ships Variant Creutzfeld-Jakob disease Variant Creutzfeld-Jakob disease –transmitted by ingestion of beef from infected cattle; –transmission between animals is due to the use of mammalian tissue in ruminant animal feeds; –prevention and control is difficult

36. Foods transported and consumed in uncooked state are increasingly important sources of food-borne infection, especially as there is increasingly rapid movement of people and products around the world Foods transported and consumed in uncooked state are increasingly important sources of food-borne infection, especially as there is increasingly rapid movement of people and products around the world –Sprouts can be a problem if germinated in contaminated water –Shellfish and finfish can be contaminated by pathogens (e.g., Vibrio and viruses) found in raw sewage –Raspberries are often transported by air to far-away markets; if contaminated, outbreak occurs far from source of pathogen

37. Food intoxications Ingestion of microbial toxins in foods Ingestion of microbial toxins in foods Staphylococcal food poisoning is caused by exotoxins released by Staphylococcus aureus, which is frequently transmitted from its normal habitat (nasal cavity) to food by person’s hands; improper refrigeration leads to growth of bacterium and toxin production Staphylococcal food poisoning is caused by exotoxins released by Staphylococcus aureus, which is frequently transmitted from its normal habitat (nasal cavity) to food by person’s hands; improper refrigeration leads to growth of bacterium and toxin production Clostridium botulinum, C. perfringens, and B. subtilis also cause food intoxication Clostridium botulinum, C. perfringens, and B. subtilis also cause food intoxication –Botulism, caused by C. botulinum –C. perfringens is a common inhabitant of food, soil, water, spices and intestinal tract; upon ingestion, endospores germinate and produce enterotoxins within the intestine; this causes food poisoning; often occurs when meats are cooked slowly –Bacillus cereus food poisoning is associated with starchy foods

38. Detection of Food-borne Pathogens Methods need to be rapid; therefore, traditional culture methods that might take days to weeks to complete are too slow Methods need to be rapid; therefore, traditional culture methods that might take days to weeks to complete are too slow identification is also complicated by low numbers of pathogens compared to normal microflora identification is also complicated by low numbers of pathogens compared to normal microflora chemical and physical properties of food can make isolation of food-borne pathogens difficult chemical and physical properties of food can make isolation of food-borne pathogens difficult

39. Molecular methods are valuable for three reasons Molecular methods are valuable for three reasons –They can detect the presence of a single, specific pathogen –They can detect viruses that cannot be conveniently cultured –They can identify slow-growing or non- culturable pathogens

40. Some examples DNA probes can be linked to enzymatic, isotopic, chromogenic, or luminescent/ fluorescent markers; are very rapid DNA probes can be linked to enzymatic, isotopic, chromogenic, or luminescent/ fluorescent markers; are very rapid PCR (Polymerase Chain Reaction) can detect small numbers of pathogens (e.g., as few as 10 toxin-producing E. coli cells in a population of 100,000 cells isolated from soft cheese samples; as few as two colony- forming units of Salmonella); PCR systems are being developed for Campylobacter jejuni and Arcobacter butzleri PCR (Polymerase Chain Reaction) can detect small numbers of pathogens (e.g., as few as 10 toxin-producing E. coli cells in a population of 100,000 cells isolated from soft cheese samples; as few as two colony- forming units of Salmonella); PCR systems are being developed for Campylobacter jejuni and Arcobacter butzleri

41. Food-borne pathogen fingerprinting is an integral part of an initiative by the Centers for Disease Control (CDC) to control food- borne pathogens; The CDC has established a procedure (PulseNet) in which pulse-field gel electrophoresis is used under carefully controlled and standardized conditions to detect the distinctive DNA patterns of nine major food pathogens; these pathogens are being followed in an surveillance network (FoodNet) Food-borne pathogen fingerprinting is an integral part of an initiative by the Centers for Disease Control (CDC) to control food- borne pathogens; The CDC has established a procedure (PulseNet) in which pulse-field gel electrophoresis is used under carefully controlled and standardized conditions to detect the distinctive DNA patterns of nine major food pathogens; these pathogens are being followed in an surveillance network (FoodNet)

42. Microbiology of Fermented Foods

43. Fermented milks Fermented milks –at least 400 different fermented milks are produced throughout the world; –fermentations are carried out by mesophilic, thermophilic, and therapeutic lactic acid bacteria, –as well as by yeasts and molds

44. Mesophilic Mesophilic –acid produced from microbial activity at temperatures lower than 45°C causes protein denaturation (e.g., cultured buttermilk and sour cream) Thermophilic Thermophilic –fermentations carried out at about 45°C (e.g., yogurt)

45. Therapeutic Therapeutic –fermented milks may have beneficial therapeutic effects –Acidophilus milk contains L. acidophilus; improves general health by altering intestinal microflora; may help control colon cancer –Bifid-amended fermented milk products (containing Bifidobacterium spp.) improve lactose tolerance, possess anticancer activity, help reduce serum cholesterol levels, assist calcium absorption, and promote the synthesis of B-complex vitamins; may also reduce or prevent the excretion of rotaviruses, a cause of diarrhea among children

46. Yeast lactic Yeast lactic –these fermentations include kefir, which is made by the action of yeasts, lactic acid bacteria, and acetic acid bacteria Mold lactic Mold lactic –this fermentation is used to make viili, a Finnish beverage; –carried out by the mold Geotrichium candidum and lactic acid bacteria

47. Cheeses produced by coagulation of curd, expression of whey, and ripening by microbial fermentation; cheese can be internally inoculated or surface ripened Cheeses produced by coagulation of curd, expression of whey, and ripening by microbial fermentation; cheese can be internally inoculated or surface ripened Meat and Fish Meat and Fish Meat products include sausages, country-cured hams, bologna, and salami; these fermentations frequently involve Pediococcus cerevisiae and Lactobacillus plantarum Meat products include sausages, country-cured hams, bologna, and salami; these fermentations frequently involve Pediococcus cerevisiae and Lactobacillus plantarum Fish products include izushi (fresh fish, rice, and vegetables incubated with Lactobacillus spp.) and katsuobushi (tuna incubated with Aspergillus glaucus) Fish products include izushi (fresh fish, rice, and vegetables incubated with Lactobacillus spp.) and katsuobushi (tuna incubated with Aspergillus glaucus)

48. Production of Alcoholic Beverages

49. Wines and champagnes Grapes are crushed and liquids that contain fermentable substrates (musts) Grapes are crushed and liquids that contain fermentable substrates (musts) Musts are separated and fermented immediately, but the results can be unpredictable; Musts are separated and fermented immediately, but the results can be unpredictable; usually must is sterilized by pasteurization or with sulfur dioxide fumigant; usually must is sterilized by pasteurization or with sulfur dioxide fumigant; to make a red wine, the skins of a red grape are left in contact with the must before the fermentation process; to make a red wine, the skins of a red grape are left in contact with the must before the fermentation process; if must was sterilized, the desired strain of Saccharomyces cerevisiae or S. ellipsoideus is added, and the mixture fermented (10 to 18% alcohol) if must was sterilized, the desired strain of Saccharomyces cerevisiae or S. ellipsoideus is added, and the mixture fermented (10 to 18% alcohol)

50. Another important fermentative process that occurs is the malo-lactic fermentation carried out by Leuoconostoc spp.; this fermentation reduces the amount of organic acids (e.g., malic acid) in the wine, improving its flavor, stability, and “mouth feel” Another important fermentative process that occurs is the malo-lactic fermentation carried out by Leuoconostoc spp.; this fermentation reduces the amount of organic acids (e.g., malic acid) in the wine, improving its flavor, stability, and “mouth feel” For dry wine (no free sugar), the amount of sugar is limited so that all sugar is fermented before fermentation stops; for sweet wine (free sugar present), the fermentation is inhibited by alcohol accumulation before all sugar is used up; in the aging process flavoring compounds accumulate For dry wine (no free sugar), the amount of sugar is limited so that all sugar is fermented before fermentation stops; for sweet wine (free sugar present), the fermentation is inhibited by alcohol accumulation before all sugar is used up; in the aging process flavoring compounds accumulate

51. RackingCremoval of sediments accumulated during the fermentation process RackingCremoval of sediments accumulated during the fermentation process Brandy (burned wine) is made by distilling wine to increase alcohol concentration; wine vinegar is made by controlled microbial oxidation (by Acetobacter or Gluconobacter) to produce acetic acid from ethanol Brandy (burned wine) is made by distilling wine to increase alcohol concentration; wine vinegar is made by controlled microbial oxidation (by Acetobacter or Gluconobacter) to produce acetic acid from ethanol For champagnes, fermentation is continued in bottles to produce a naturally sparkling wine For champagnes, fermentation is continued in bottles to produce a naturally sparkling wine

52. Beers and ales Malt is produced by germination of the barley grains and the activation of their enzymes; Malt is produced by germination of the barley grains and the activation of their enzymes; mash is produced from malt by enzymatic starch hydrolysis to accumulate utilizable carbohydrates; mash is produced from malt by enzymatic starch hydrolysis to accumulate utilizable carbohydrates; mash is heated with hops (dried flowers of the female vine Humulus lupulis) to provide flavor and clarify the wort; mash is heated with hops (dried flowers of the female vine Humulus lupulis) to provide flavor and clarify the wort; hops inactivate hydrolytic enzymes so that wort can be pitched (inoculated with yeast) hops inactivate hydrolytic enzymes so that wort can be pitched (inoculated with yeast)

53. Beer is produced with a bottom yeast, such as Saccharomyces carlsbergensis and ale is produced with a top yeast, such as S. cerevisiae; freshly fermented (green) beers are lagered (aged), Beer is produced with a bottom yeast, such as Saccharomyces carlsbergensis and ale is produced with a top yeast, such as S. cerevisiae; freshly fermented (green) beers are lagered (aged), bottled, and carbonated; bottled, and carbonated; beer can be pasteurized or filtered to remove microorganisms and minimize flavor changes beer can be pasteurized or filtered to remove microorganisms and minimize flavor changes

54. Distilled spirits beerlike fermented liquid is distilled to concentrate alcohol; Distilled spirits beerlike fermented liquid is distilled to concentrate alcohol; type of liquor depends on composition of starting mash; type of liquor depends on composition of starting mash; flavorings can also be added; flavorings can also be added; a sour mash involving Lactobacillus delbrueckii mediated fermentation is often used a sour mash involving Lactobacillus delbrueckii mediated fermentation is often used

55. Production of breads Aerobic yeast fermentation is used to increase carbon dioxide production and decrease alcohol production; other metabolic products add flavors Aerobic yeast fermentation is used to increase carbon dioxide production and decrease alcohol production; other metabolic products add flavors Other microorganisms make special breads, such as sourdough Other microorganisms make special breads, such as sourdough Bread products can be spoiled by Bacillus species that produce ropiness Bread products can be spoiled by Bacillus species that produce ropiness

56. Other fermented foods Sufu, fermented tofu (a chemically coagulated soybean milk product) and tempeh, made from soybean mash, are made by the action of molds Sufu, fermented tofu (a chemically coagulated soybean milk product) and tempeh, made from soybean mash, are made by the action of molds SauerkrautCfermented cabbage; involves a microbial succession mediated by Leuconostoc mesenteroides, Lactobacillus plantarum, and Lactobacillus brevis SauerkrautCfermented cabbage; involves a microbial succession mediated by Leuconostoc mesenteroides, Lactobacillus plantarum, and Lactobacillus brevis

57. Pickles are cucumbers fermented in brine by a variety of bacteria; fermentation process involves a complex microbial succession Pickles are cucumbers fermented in brine by a variety of bacteria; fermentation process involves a complex microbial succession Silages–animal feeds produced by anaerobic, lactic-type mixed fermentation of grass, corn, and other fresh animal feeds Silages–animal feeds produced by anaerobic, lactic-type mixed fermentation of grass, corn, and other fresh animal feeds

58. Microorganisms as Foods and Food Amendments

59. Microbes that are eaten include a variety of bacteria, yeasts, and other fungi (e.g., mushrooms, Spirulina) Microbes that are eaten include a variety of bacteria, yeasts, and other fungi (e.g., mushrooms, Spirulina)

60. Probiotics Probiotics –the addition of microorganisms to the diet in order to provide health benefits beyond basic nutritive value also called microbial dietary adjuvants also called microbial dietary adjuvants

61. Prebiotics Prebiotics –oligosaccharide polymers that are not processed until reaching the large intestine; –often combined with probiotics to create a symbiotic system Probiotics Probiotics –are being used with poultry to increase body weight and feed conversion; –also reduce coliforms and Campylobacter; may be useful in preventing Salmonella from colonizing gut due to competitive exclusion