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Cara Mullen
• Starch and Enzyme Stuff
Indiana University alumni
Corn Composition
• Endosperm – This is the part where the starch comes from. Part of the gluten also comes from here. 65-75% of the kernel is comprised of this.
• Germ – this is the part of the corn that oil is made from. About 5% of the kernel is comprised of germ.
• Pericarp – This is the outer layer of the kernel. It comprises 5% of the kernel.
• Tip Cap – where the kernel obtains nutrients to grow.
• Other components – Moisture comprises about 15% and ash and other solubles are about 6%.
Starch Granules
Polarized Starch Granules
•Microscopic View
• Top View (400X)
Starch Granule (crystals)
Size (5 to 40 µm)
Dippled Centers
Variable Shapes
• Bottom View (Polarized Light)
• Crystalline Structure clearly shown by Maltese Cross
Starch belongs to a group of chemical compounds called carbohydrates which are energy storage units in plants
such as corn.
They are called this because they contain only carbon, hydrogen, and oxygen or sugar (i.e. glucose) molecules.
These glucose molecules are connected together and can be broken down by
enzymes then used as “food” for yeast cells.
Starch is a series of sugar molecules linked together in corn it has two main parts:
amylose and amylopectin.
Amylose is a straight or linear chain of sugar molecules linked together.
Amylopectin is a branched chain of sugars called dextrins.
Starch exists in two different forms - an unbranched chain form called amylose and a branched form called
amylopectin.
Enzymes that digest starch are called amylases.
Tradename: SPEZYME® XTRAAlpha amylaseLiquefying enzymeSlurry enzyme
Enzyme randomly hydrolyzes α-1,4-glucosidic bonds to reduce the viscosity of gelatinized starch producing soluble dextrins and
oligosaccharides.
Alpha Amylase is a catalytic protein (enzyme) which is
harvested from genetically modified bacteria. Most alpha amylases are “thermostable”
meaning tolerating higher heat which increases enzyme half-life and reaction rate.
Optimum pH ~ 5.8Temperature ~ 175-185º
Genencor industrial enzymes• Controlled fermentation of non-pathogenic microorganisms.
• Most industrial enzymes are made from bacteria, yeast and fungi.
• Usually sold as concentrated, partially purified.
Fermentation process for enzyme production
MEDIAPREPARATIONTypical Raw materials:
Protein (CSL, Soy)Salts (PO4, SO4)
Carbon (Corn syrup)
AIRNH3
Culture propagation
SEEDFERMENTATION
Tank Controls:pH, Pressure,Temperature,
Aeration, Agitation, Off gas Mass Spec, Dissolved O2
MAINFERMENTATION
Tank Controls:pH, C-source Feed, Pressure,
Temperature,Aeration, Agitation, Off gas Mass Spec, Dissolved O2
AIRNH3
Continuous Feed System
General Enzyme (Catalytic Proteins) Properties
In general, enzymes share the properties of globular proteins:
• Soluble in water.
• Insoluble in organic solvents.
• Denature and lose their catalytic properties due to heat, pH, some chemicals, and heavy metals.
• Stabilized by ions such as sodium, potassium and calcium.
• One distinctive property of enzymes versus chemicalcatalysts is their thermal sensitivity to acids and bases.
Enzymes that break down dextrins are called
Glucoamylases
Glucoamylaseis a catalytic protein (enzyme) which is
harvested from fungi.
Best pH 3.5 - 6.5 at
Fermentation temperature 88 - 94ºF
Tighter pH 4.0 – 5.0 at
Saccharification temperature 140 - 150ºF
DP = Degree of Polymerization
number of dextrose molecules linked together examples: DP1,
DP2, DP3, DP4…DP10…
Degree of Polymerization by HPLC
DP
DP1=glucoseDP2=maltoseDP3=maltotriose DP4=maltotetraose DP5=maltopentaoseDP6=maltohexaoseDP7=maltoheptaose DP8=maltooctaose DP9=maltononaose DP10=maltodecaose
HPLC
The extent of starch hydrolysis is expressed as Dextrose Equivalent (DE). DE is the percentage of glucosyl bonds that have been broken.
Dextrose is another word for D-glucose.
So, DE will be low (4 - 6) at the beginning of the process such as the Slurry tank outlet.
Meaning: Mostly large molecules exist and not many molecules of glucose.
DEXTROSE EQUIVALENT (DE)When the DE is 100, all of the bonds between glucose units in the starch have been hydrolyzed. About 10% of the starch bonds are broken by the end of Conversion and up to half of the bonds are broken coming out of Saccharification.
If half of the starch bonds are hydrolyzed the average DP is 2 and the DE = 50
example: 25% DP1 + 25% DP3 + 50% DP2 = 50 DE
If a fourth of the starch bonds are broken the average DP is 4 and the DE = 25.DE determines the average DP length of the liquefied mash.
Estimation of DE from HPLC results
%DP1 * 100 + %DP2 * 50 + %DP3*33+ %DP4*10
________________________________________
% Solids
= est. DE
MillingMixing
GelatinizationCooking
ConversionSaccharificationFermentation
Distillation
Milling: Grind size effects– A large particle size can be more difficult to gelatinize since enzyme cannot
react with the substrate efficiently. This can result in yield loss (up to 10%).
– A small particle size (< 200 mesh) can also cause problems:
Mixing of floured grain in water may be more difficult.
Small particles will also cause problems in centrifugation, evaporation, and drying.
May have some positive effect on theoretical yield due to enzymes’ ability to react with starch.
Typical Size : Mill grain to 100% thru 16 mesh
Slurry: Gelatinization
The mash is cooked at 170 – 185 degrees to gelatinize most of the starch and break the viscosity enough to be pumpable.
Alpha-amylase and water molecules together are randomly breaking down (or hydrolyzing) the amylopectin (branches) part of the starch
structure.
The alpha amylase produces a mixture of short sugar molecules (e.g. maltose, maltotriose), and some dextrin molecules (longer molecules of
sugar).
4 - 6 DE mash
LiquefactionAlpha amylase is denatured in Liquefaction
primarily because of heat.
The heat further breaks down starch molecules for the second addition of alpha
amylase.
DE of 12 – 14
Purpose of Saccharification:
• To break down dextrin chains to maltose or glucose molecules, using Glucoamylase which specifically attacks 1-4 linkages, one glucose unit at a time off non-reducing end. Also hydrolyzes 1-6 bonds using Pullulanase.
Purpose of Fermentation:
To produce ethanol through the anaerobic conversion of glucose by
yeast.
51% of the glucose is converted to ethanol and 49% is converted to
carbon dioxide
Ethanol Level at end of Fermentation is typically 12 - 15% v/v.
This is what is happening through Fermentation?
Glucoamylase and water molecules together are randomly breaking down (or hydrolyzing) the mixture of short sugar molecules (e.g. maltose, maltotriose), and dextrin molecules (longer molecules of sugar are also called oligosaccharides).
This produces individual molecules of glucose. Yeast metabolize (or ferment) the glucose. The theoretical byproducts are CO2 and Ethanol. In reality the yeast also produces some glycerol, succinic acid, and new yeast.
Any Questions??