Industrial microbiology Part 1

1. Industrial microbiologyPart 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 • describe the various physical, chemical, and biological processes used to preserve foods • discuss the various diseases that can be transmitted to humans by foods • differentiate between food infections and food intoxications • discuss the detection of disease-causing organisms in foods

4. Contd. • 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 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 • describe probiotics

5. Intrinsic and Extrinsic Factors

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

7. 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 • 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 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 cooking, making foods more susceptible to anaerobic spoilage

10. 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 • 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 • lower temperatures retard microbial growth • relative humidity • higher levels promote microbial growth • atmosphere • oxygen promotes growth • modified atmosphere packaging (MAP) • 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 • 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; • proteolysis (aerobic) and putrefaction (anaerobic) decompose proteins; • 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; • high oxidation–reduction potential favors aerobic and facultative bacteria; • molds usually initiate spoilage in fruits.

17. 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: • 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 • Aflatoxins—planar molecules that intercalate into DNA and act as frame shift mutagens and carcinogens; • Aflatoxins can appear in milk if consumed by dairy cows, • Have also been observed in beer, cocoa, raisins, and soybean meal; • 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

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 • Low temperature—refrigeration and/or freezing retards microbial growth but does not prevent spoilage

23. High temperature • Canning • 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

24. 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

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

27. 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 • 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; • 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

29. Microbial product-based inhibition • 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

30. Food-borne Diseases

31. 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 • 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 • transmitted by uncooked or poorly cooked poultry products, • raw milk and red meats; • thorough cooking prevents transmission

34. Listeriosis • transmitted by dairy products • 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 • 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 • 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 • 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 • 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 • 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 • 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

39. 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 • 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)

42. Microbiology of Fermented Foods

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

45. 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 • these fermentations include kefir, which is made by the action of yeasts, lactic acid bacteria, and acetic acid bacteria • 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 • Meat and Fish • 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)

48. Production of Alcoholic Beverages

49. Wines and champagnes • Grapes are crushed and liquids that contain fermentable substrates (musts) • Musts are separated and fermented immediately, but the results can be unpredictable; • 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; • 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” • 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