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??