Chapter 2 Preparation of Culture Medium

1. Chapter 2 Preparation of Culture Medium

2. Unit 1 Industrial Fermentation Medium The basic ingredients of media include carbon source、nitrogen source、inorganic salt 、growth factor and distilled water etc.

3. Medium Development • To maintain economic competitiveness, low-cost crude materials are frequently used • Levels of minerals and growth factors may be critical

4. Kinds of culture media • The source of media constituent • Complex media • Synthetic or defined media • Semi-defined medium • Physics states • Liquid medium • Solid medium • Semi-solid medium

5. For use • Basic medium • Enriched medium • Differential medium • Selective medium • Manufacture intention • Seed medium • Ferment medium

6. Nutrient sources for industrial fermentation Carbon & energy source + nitrogen source + O2 + other requirements → Biomass + Product + byproducts + CO2 + H2O + heat

7. Fermentation media

8. 1. Carbon sources • (1) Starch (corn, wheat, potato ) • Widely used in fermentation industry • Starch dextrin glucose • Advantages: cheaper than glucose

9. (2) Molasses • Byproduct of cane or beet sugar production • A dark viscous syrup containing 50% -75% fermentable sugars (mainly sucrose) with 2% nitrogen, vitamins and minerals • Cheaper

11. 2. Nitrogen source • (1) Inorganic nitrogen source • Ammonia, ammoniumand nitrate • Microbes can utilize it faster. • After it is utilized, the pH of medium will be changed. • (NH4)2SO42NH3 + H2SO4 • NaNO3 + 4H2 NH3 + 2H2O +NaOH

12. (2) Organic nitrogen sources • Urea • Yeast extract • Peptones • Corn steep liquor • Soybean cake powder • Peanut powder • Bran hydrolysis liquid corn steep liquor powder

13. 3. Trace elements Fe, Mn, B, Zn, Cl, Mo, Cu 4. Growth factors Small amount of organic compounds necessary for microbial growth. e.g. amino acids, vitamins, biotin Sources: normally organic nitrogen source e.g. corn steep liquor

14. Corn steep Liquor, CSL • Corn steep liquor is the water extract by-product resulting fromthe steepingof corn during the commercial production of cornstarch and other corn products.

15. Different medium for different food products • Chinese distilled spirit：solid medium • Fruit spirit：fruit juice or fruit sauce • Beer：wort • Alcohol: starch mash • Amino acid:starch mash • Citric acid：starch mash • Lactic acid：starch mash

16. Unit 2 Preparation of Starch Sugar Starch is a large molecule that is made of branching chains of glucose molecules. It can be broken down into glucose by hydrolysis.

17. Starch found in grains, potatoes, beans, peas, cassava • Typically 20-30% amylose, remainder amylopectin

18. Characteristics of starch • Insoluble in hot water • Blue complex compound by reaction with iodine. • Insoluble in 30% of ethanol solution or above. • Hydrolyzed by acid and enzyme, the ultimate product is glucose.

19. starch granule

20. Starch: Amylose • Amylose is a linearglucan with [1,4]glycosidic linkages

21. Starch: Amylopectin • Amylopectin is a branchedglucan with [1,4] and [1,6]glycosidiclinkages

22. Starch: Amylopectin • [1,6] branch points result in tree-like structure • less extended (more compact) than amylose • larger MW [1,6] linkages

23. Why should starch be hydrolyzed to glucose? • Most microbes could not utilize starch directly. • 2. To obtain sugar from the starch of many different plants, rather than just sugar beets or sugar cane.

24. Conversion of starch to fermentable sugars 1. Acid hydrolysis method Starch slurry is acidified to a pH value and then heated in a converter under pressure. (C6H10O5)n → (C6H10O5)x →C12H22O11 →C6H12O6 amylum → dextrin → oligosaccharides→glucose

25. Due to heat and acid, there are… Glucose combination reaction and decomposition reaction

26. starch glucose oligosaccharide colour content ( difficult to utilize ) (difficult to decolor) Total lost of glucose 7％ <1％ acid Decomposition reaction Combination reaction

27. How to avoid combination and • decomposition reactions occur? • Concentration of starch • Concentration of hydrolysis acid • Reaction temperature not too high

28. Advantage • Simple • Time saving • Disadvantage • More by-product, DEvalue only 90％ • Requiring fine starch • Need corrosion resistant materials • Need more energy for heating

29. Extent of hydrolysis measured using dextrose equivalents (DE ) value reducing sugars DEvalue＝ 100％ total solids

30. The higher the DE, the more sugars and less dextrins are present. • The DE value of starch is zero and that of dextrose is 100.

31. A B C DE time

32. add sodium bicarbonate remove after Starch and hydrochloric acid minutes test with Benedict’s cool test sample with iodine

33. 2. Enzyme hydrolysis method • Which enzyme is used to break down starch into sugar syrup? • Amylase • Glucoamylase • Also called: double enzyme hydrolysis

34. Endoenzymes • amylase • attacks [1,4] bonds to produce various oligosaccharides Lower MW! • pullulanase • attacks [1,6] bonds to produce various oligosaccharides (amylose fragments)

35. Exoenzymes(non-reducing end) • -amylase • attacks [1,4] bonds at non-reducing end to produce maltose Sweet! • glucoamylase • attacks [1,4] bonds at non-reducing end to produce glucose High MW!

36. Enzymes in processing starch

37. There are two basic steps in enzymatic starch conversion: liquefaction and saccharification

38. Production of Glucose Syrups from Starch Corn Starch Slurry Liquefaction Thermostable a-amylase Gelatinization (105°C, 5 min) Dextrinization (95°C, 2h) Liquefied Starch DE 10-15 Saccharification Glucoamylase (60°C, pH 4.0-4.5, 24-72 h) Glucose Syrups DE 95-96

39. 1) Liquefaction Higher temperature hydrolysis of starch involve a starch gelatinization process, dissolution of the nanogram-sized starch granules (swells and bursts)to form a viscous suspension. 

40. During liquefaction, long-chained starch molecules are partial hydrolysis into smaller branched and linear chains of glucose units (dextrins) , with concomitant loss in viscosity. Enzyme: α-amylase End-point : DE vaule 2030(why?) iodide →brown

41. Why should be two steps, liquefaction and saccharification? • Low DE value • Retrogradation (ageing) • Substance size

42. Q:What’s the difference between gelatinization and retrogradation?

43. 2) Saccharification Involving the production of glucose and a little maltose by further hydrolysis. Enzyme: glucoamylase End-point : DE is the highest

45. Advantage • Higher quality • Save energy • Protect environment • Disadvantage • Complex operation • Time consuming

46. How to make high-fructose corn syrup? • Need 3 steps and 3 enzymes • alfa-amylase->shortening the starch (Liquefaction) • glucoamylase->produces glucose (saccharification) • glucose isomerase-> convert to fructose (isomerization)

47. 3.Acid-enzyme combination method • (Two-stage hydrolysis) • Slurry is only partially converted by acid and then treated with an appropriate enzyme or enzymes until the conversion is complete.

48. 1) Acid-enzyme hydrolysis • Acid--- liquefaction • Glucoamylase----- saccharification • Suitable for: starch granule is hard

49. 2) Enzyme- acid hydrolysis • α-Amylase (thermostable) --- liquefaction • Acid----- saccharification • Suitable for: starch granule size is irregular

50. Production of Glucose from Starch