GAS 201 FERMENTED FOOD PRODUCTS Prof. Dr. Mehmet D. Öner Grading Attendance 10%

1. GAS 201 FERMENTED FOOD PRODUCTS Prof. Dr. Mehmet D. Öner Grading Attendance 10% Midterm exam 40 % Final exam 50 %

2. 1- Principles of Fermentation Technology by P F STANBURY , A. WHITAKER, S. Hall , Butterworth-Heinemann;2 edition(May 3, 1999) 2- Microbiology andTechnology of Fermented Foods by Robert W. Hutkins IFT press, Blackwell Publishing, 2006

7. DEFINITION OF FOOD FERMENATION: Microbial(1) activities(2), usually anaerobic(3), on suitable substrate under controlled(4) or uncontrolled(5) conditions resulting(6) in the production of desirable(7) foods or beverages which are characteristicaly more stable(8), palatable(9) and nutritious(10) than the raw substrate.

10. Biochemical Pathways

12. Cell Inputs Outputs Sugars Amino acids Small molecules Oxygen CO2, NH4, H2S, H2O Energy Protein Large molecules

13. Metabolic processes • Catabolic = Breakdown: • generation of energy and reducing power from complex molecules • produces small molecules (CO2, NH3) for use and as waste products • Anabolic = Biosynthesis: • construction of large molecules to serve as cellular components such as • amino acids for proteins, nucleic acids, fats and cholesterol • usually consumes energy

14. ATP consists of three phosphate groups, ribose, and adenine. Adenine Phosphate groups Ribose

17. Requirements for Microbial Growth

18. Water:Major componentof all fermentation process is consistent, clean water. • PH, dissolved salts and microbial contamination are important factors. • Long before plants are set up near by good water sources. • Now water treatment is applied. There might be different characteristic water requirement for different type of food production.

19. Carbon source: • -CarbohydratesCarbohydrates are capable of being used by all microorganisms,. • Glucose is the most readily metabolized sugar. • Most common carbohydrate is starch( from maise, cereals and potatoes) Sugar cane, sugar beet. • -Hydrocarbons: petroleum products • -Lipids

20. Energy source: • either from light(photosynthesis)or oxidation of mediumcomponents • Most industrial organisms are chemoorganotrophs( common energy source organic materials.

21. Nitrogen sources • Inorganic nitrogen sources: ammonia gas and ammonium nitratesammonium salts • Organic nitrogenamino acids, proteins, urea Vitamins and growth factors There is considerable species variation in the requirements of vitamins and related factors by other microorganisms.

22. Chemical elements and inorganic ions • Mineral nutrients required by microorganisms are species dependent but consists generally of Fe, K, Mg, Mn.

23. NADH NADH CO2+NADH GTP CO2+NADH GDP+Pi FADH2 Glycolysis Glucose Glucose 6-Phosphate Fructose 6-Phosphate Dihydroxyacetone phosphate Fructose 1,6-Bisphosphate Glyceraldehyde 3-Phosphate 2-Phosphoglycerate Phosphoenolpyruvate Pyruvate Lactate TCA cycle Acetyl CoA Acetate Ethanol Citrate Oxaloacetate Isocitrate Malate Fermentation a-Ketoglutarate Fumarate Succinate

24. Fermentation

25. The major carbohydrate-metabolizing pathways are: Embden–Meyerhof–Parnas (EMP) pathway, also called glycolysis • Entner–Doudoroff (ED) pathway • pentose phosphate (PP) pathway. • The three pathways differ in many ways, but two generalizations can be made: • 1. All three pathways convert glucose to glyceraldehyde 3-phosphate (GAP) by different routes. • 2. The GAP is converted to pyruvate via reactions that are the same in all three pathways.

26. Oxidation of Pyruvate to Acetyl Coenzyme A In all the major carbohydrate catabolic pathways, pyruvate is a common product,

27. The Tricarboxylic Citric Acid Cycle To finish the respiratory metabolism of glucose, acetyl coenzyme A enters the tricarboxylic acid (TCA) cycleto produce carbon dioxide, water, reduced coenzymes and ATP

28. Other Substrates as Sources for Metabolic Activity Living organisms can use a variety of substrates for growth: almost every naturally occurring organic compound can serve as a source for cell carbon or energy.

29. Carbohydrates Glucose is not the only carbohydrate that can be converted to pyruvate by glycolysis

30. Anaerobic Breakdown of Carbohydrates • The terms glycolysis and fermentationhave been applied to the anerobic decomposition of carbohydrate to the level of lactic acid. • The final product in some organisms is lactic acid; in others, the lactic acid is further metabolized anaerobically to butyric acid, butyl alcohol, acetone and propionic acid. • The two most common forms of fermentation are lactic and alcoholic.

31. Fermentation • Fermentation is a major source of energy for those organisms that can only survive in the absence of air (obligate anaerobes). • Other fermentative organisms that can grow in the presence or absence of air (facultative anerobes) use fermentation as a source of energy only when oxygen is absent.

32. Pyruvate is a sort of Grand Central Station, in that it is the point of arrival and departure of a wide variety of metabolic substrates and products. • Pyruvate is reduced to lactic acid. • It may also be decarboxylated and reduced to ethyl alcohol.

33. Lactic Fermentation The products of glucose fermentation by all species of Streptococcus, many species of Lactobacillus and several other species of bacteria are mainly lactic acid with minor amounts of acetic acid, formic acid and ethanol.

34. Several species of Streptococcusproduce more than 90% of lactic acid based on the sugar used, and hence this type of fermentation is referred to as homolactic fermentation. • , e.g. Lactobacillus casei, Streptococcus cremoris and pathogenic streptococci, a heterolactic fermentation converts only half of each glucose molecule to lactate. • Both these fermentations are responsible for the souring of milk and pickles.

35. The heterofermentativemetabolic sequence found in Leuconostoc and some species of Lactobacillus ferments glucose according to the equation: Glucose→Lactate+Ethanol+CO2

36. Alcoholic Fermentation • The major substrates yielding ethanol are the sugars which in yeasts are degraded to pyruvateby the EMP or glycolytic pathway. • There is a net yield of one ATP for each pyruvate formed from glucose.

37. Mixed Acid (Formic) Fermentation by Escherichia coli.

38. General pathways for the formation of fermentation products from glucose by various organisms.

39. Overview of fermentation products formed from pyruvic acid by different bacteria.

40. Metabolic regulators added to media Precursors : Some intermediary chemicals, when added to fermentation medium directly incorporated to product Example : phenylethylamine, phenylacetic acid in penicillin fermentation Inducers : Compounds that increase production of final products during fermentation.( Common in enzyme fermentations) Example: To induce alphaamylase production via fermentation add starch( an inducer and substrate for the enzyme )

41. Inhibitors: When certan inhibitors are added to fermentations more of a specific product may be produced or a metabolic intermediate which is normally metabolised is acumulated Example : Ethanol fermentation may be modified to produce glycerol by adding sodiumbisulphite ( acetaldehyde is electron acceptor in forming NAD+ from NADH2 , if sodiumbisulphite is added it forms complex with acetaldhyde and it is no longer electron acceptor, instead dihydroxyacetonephosphate act as electron acceptor then when it is reduced glycerol is formed)

42. MICROBIAL GROWTH

43. Typical growth curve for a bacterialpopulation

44. Batch Growth • A batch fermentation can be considered to be a closed system. • At time t=0 the sterilized nutrient solution in the fermentor isinoculated with microorganisms and incubation is allowed to proceed. • In the course of the entire fermentation, nothing is added, except oxygen (in case of aerobic microorganisms), an antifoam agent, and acid or base to control the pH.

45. Lag phase A period of adaptation for the cells to their new environment • New enzymes are synthesized. • A slight increase in cell mass and volume, but no increase in cell number • Prolonged by low inoculum volume, poor inoculum condition (high % of dead cells), age of inoculum, nutrient-poor medium

46. Exponential growth phase In this phase, the cells have adjusted to their new environment and multiply rapidly (exponentially)

47. Typical growth curve for a bacterial population

48. Batch Growth Kinetics Deceleration growth phase Very short phase, during which growth decelerates due to either: • Depletion of one or more essential nutrients • The accumulation of toxic by-products of growth (e.g. Ethanol in yeast fermentations) • Period of unbalanced growth: Cells undergo internal restructuring to increase their chances of survival

49. Typical growth curve for a bacterial population

50. Stationary Phase: With the exhaustion of nutrients (S≈0) and build-up of waste and secondary metabolic products • Cells may have active metabolism to produce secondary metabolites. Primary metabolites are growth-related: ethanol by S. cerevisae. Secondary metabolites are non-growth-related: antibiotics, pigments.