Preparation of Culture Medium

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

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

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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

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• For use
• Basic medium
• Enriched medium
• Differential medium
• Selective medium

• Manufacture intention
• Seed medium
• Ferment medium

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Nutrient sources for industrial fermentation
Carbon energy source nitrogen source O2
other requirements ? Biomass Product
byproducts CO2 H2O heat
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Fermentation media
Nutrient Raw material
Carbon molasses, starch
Nitrogen corn steep liquor, soybean meal, pure ammonia or ammonium salts, urea, nitrate salts, phosphate salts
Vitamins and growth factors biotin, yeast extract, beef extract, corn steep liquor, wheat germ meal
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• 1. Carbon sources
• (1) Starch (corn, wheat, potato )
• Widely used in fermentation industry
• Starch ? ?dextrin ?glucose
• Advantages cheaper than glucose

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• (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

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• 2. Nitrogen source
• (1) Inorganic nitrogen source
• Ammonia, ammonium and 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

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

corn steep liquor powder
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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
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Corn steep Liquor, CSL

• Corn steep liquor is the water extract by-product
  resulting from the steeping of corn during the
  commercial production of corn starch and other
  corn products.

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Different medium for different food products
Chinese distilled spiritsolid medium Fruit
spiritfruit juice or fruit sauce
Beerwort Alcohol starch mash
Amino acid starch mash Citric acidstarch
mash Lactic acidstarch mash
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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.
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• Starch found in grains, potatoes, beans, peas,
  cassava
• Typically 20-30 amylose, remainder amylopectin

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• 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.

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starch granule
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Starch Amylose

• Amylose is a linear glucan with ?1,4 glycosidic
  linkages

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Starch Amylopectin

• Amylopectin is a branched glucan with ?1,4 and
  ?1,6 glycosidic linkages

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Starch Amylopectin

• ?1,6 branch points result in tree-like
  structure

• less extended (more compact) than amylose
• larger MW

?1,6 linkages
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• 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.

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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
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Due to heat and acid, there are Glucose
combination reaction and
decomposition reaction
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starch
glucose
oligosaccharide colour content (
difficult to utilize )
(difficult to decolor) Total lost of
glucose 7 lt1
acid
Decomposition reaction
Combination reaction
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• How to avoid combination and
• decomposition reactions occur?
• Concentration of starch
• Concentration of hydrolysis acid
• Reaction temperature

not too high
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• Advantage
• Simple
• Time saving
• Disadvantage
• More by-product, DE value only 90
• Requiring fine starch
• Need corrosion resistant materials
• Need more energy for heating

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Extent of hydrolysis measured using dextrose
equivalents (DE ) value reducing
sugars DE value ? 100
total solids
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• 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.

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A B C
DE
time
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• 2. Enzyme hydrolysis method
• Which enzyme is used to break down starch
  into sugar syrup?
• Amylase
• Glucoamylase
• Also called double enzyme hydrolysis

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Endoenzymes

• amylase
• attacks 1,4 bonds to produce various
  oligosaccharides

Lower MW!

• pullulanase
• attacks 1,6 bonds to produce various
  oligosaccharides (amylose fragments)

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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!
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Enzymes in processing starch
Enzyme Source Action 
a-amylase Bacillus subtilis Only a-1,4-oligosaccharide links are cleaved to give dextrins with maltose and up to 50 (w/w) glucose 
ß-Amylase Malted barley Only a-1,4-links are cleaved, from non-reducing ends, to give limit dextrins and maltose
Glucoamylase Aspergillus niger a-1,4 and a-1,6-links are cleaved, from the non-reducing ends to give glucose
Pullulanase B. acidopullulyticus Only a-1,6-links are cleaved to give straight-chain maltodextrins
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There are two basic steps in enzymatic starch
conversion liquefaction and saccharification
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Production of Glucose Syrups from Starch
Corn Starch Slurry
Liquefaction Thermostable a-amylase
Gelatinization (105C, 5 min) Dextrinization
(95C, 2h)
Liquefied Starch DE 10-15
Saccharification Glucoamylase (60C, pH
4.0-4.5, 24-72 h)
Glucose Syrups DE 95-96
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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. 
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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 a-amylase End-point DE vaule
20?30(why?) iodide ?brown
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• Why should be two steps, liquefaction and
  saccharification?
• Low DE value
• Retrogradation (ageing)
• Substance size

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QWhats the difference between gelatinization
and retrogradation?
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2) Saccharification Involving the production of
glucose and a little maltose by further
hydrolysis. Enzyme glucoamylase End-point DE
is the highest
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• Advantage
• Higher quality
• Save energy
• Protect environment
• Disadvantage
• Complex operation
• Time consuming

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• How to make high-fructose corn syrup?
• Need 3 steps and 3 enzymes
• alfa-amylase-gtshortening the starch
  (Liquefaction)
• glucoamylase-gtproduces glucose (saccharification)
• glucose isomerase-gt convert to fructose
  (isomerization)

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• 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.

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

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

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Production of Glucose from Starch
Items acid acid/enzyme enzyme
DE value 91 95 98
Color 10 0.3 0.2
Processing condition HTP HTP NPT
Energy consuming high high low
By-product more moderate little
Cycle time short moderate long
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Review Questions
1.Which kind of carbon source and nitrogen source
usually used in industry? Cite the advantages
and disadvantages of the different choices. 2.
What is the definition of growth factor and its
main source? 3. What is the significance of
getting sugar by hydrolyzing starch? 4. Compare
the three methods to produce glucose from starch
and the difference between combination reaction
and decomposition reaction, liquefaction and
saccharification.