Enzymatic and Physical Modifications of Starchn630/pdf_full/Starch-6.pdf · Enzyme classification and Nomenclature ... Glucose Maltose syrup syrup Ethanol Purification & crystallization

Enzymatic and Physical Modifications of Starch

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Content Introduction of enzymes Enzymatic starch degradation: MW reduction to small sugars or

oligosaccharides Starch refining Cyclodextrin Maltooligosaccharides and Isomaltooligosaccharides Debranched starch for making resistant starch

Enzymatic starch modifications: no or minor MW reduction, or MW increase Modification by beta-amylase and maltogenic alpha-amylase Increased branching by starch branching enzymes Alpha-glucan chain extension by amylosucrase

Physical starch modifications Hydrothermal treatment Irradiation and microwave High pressure processing

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Enzyme classification and Nomenclature

Enzymes are classified by the type of reactions they catalyze

No. Class Type of reaction catalyzed1 Oxidoreductases Transfer of electrons

2 Transferases Group transfer reactions

3 Hydrolases Hydrolysis reaction (group transfer to water)

4 Lyases Group addition to double bonds, or double bond formation by group removals

5 Isomerases Group transfer within molecules to yield isomeric form

6 Ligases Formation of C-C, C-S, C-O, and C-N bonds among two molecules coupled with ATP cleavage

All enzymes have formal E.C. numbers and names. Most have trivial names

ATP + D-glucose → ADP + D-glucose 6-phosphate (by hexokinase)

E.C. number: E.C. 2 (transferase class).7 (phosphotransferase subclass).1(hydroxyl group as acceptor).1 (D-glucose as acceptor)

Introduction of enzymes

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How Enzymes Work

Enzyme catalyzed reaction Takes place within the confines of a pocket on the enzyme: active site Substrate: molecule bound in the active site and acted upon

Enzymes affect reaction rate, not equilibrium Free energy, G Ground state: contribution to G by an average molecule

Standard free energy change, G O (Biochemical standard energy change, G’ O)

At transition state, decay to S or P state is equally probable

Activation energy, G‡

Catalyst enhance reaction rates by lowering activation energies

Formation of reaction intermediates


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Catalysis by Enzyme

Reaction with no catalysis Enzyme-catalyzed reaction


Lehninger Principles of Biochemistry, Fourth Edition

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Enzyme KineticsEnzyme kinetics To describe how the reaction rate changes in response to experimental

conditions; An approach to understand mechanism of enzyme catalysis Important concepts

Reactions involving enzyme-substrate complex (ES)

Pre-steady-state: during which ES concentration [ES] builds up Steady-state: [ES] remains approximately constant over time

Initial rate (velocity), V0

Maximum rate (velocity), Vmax

Steady-state kinetics: Michaelis-Menten equation

Vmax [S]V0 =

Km + [S]


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Enzyme Kinetics

Km


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Enzyme Kinetics

Determination and interpretation of Michaelis-Menten equation

Lineweaver-Burk equation is used to determine Km and Vmax

1 Km 1= +

V0 Vmax[S] Vmax

Introduction of enzymesLehninger Principles of Biochemistry, Fourth Edition

Double reciprocal plot

Intercept

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Starch Refining

Enzymes used -Amylase hydrolyzes interior -1,4-glucosidic bonds

-Amylase hydrolyzes -1,4-glucosidic bonds from non-reducing ends and release maltose

Glucoamylase (amyloglucosidase) hydrolyzes -1,4 and -1,6-glucosidic bonds and release glucose

Debranching enzymes (isoamylase and pullulanase) hydrolyze -1,6-glucosidic bonds and release linear chains

Products Simple sugars: glucose, maltose, fructose, HFCS

Syrup: DE (dextrose equivalent) >20

Maltodextrin: DE <20

Enzymatic starch degradation

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Starch Refining

Starch slurry

-amylase

Steam

Liquefaction

MaltodextrinProducts of different

dextrose equivalent (DE)Amyloglucosidase

Pullulanase-amylasePullulanase

Maltose syrupGlucose syrup

EthanolPurification & crystallization

Crystallized glucose

Isomerase

High fructose corn syrup (HFCS)

Isomerization

Saccharification

Fermentation


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Cyclodextrin

Starch Liquefaction

Enzyme conversion

Solvent extraction

α-, β-, or γ-CD based on specific solvent used

Cyclodextrin glucanotransferase(CTGase)

α-CD β-CD γ-CD

Szejtli, Chem. Rev. 1998, 98, 1743-1753Enzymatic starch degradation

Β-CD

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Maltooligosaccharides and Isomaltooligosaccharides

Amylases producing enriched maltotriose and maltotetraose AMT 1.2 L (Amano) is a maltotriose forming amylase

Maltotriose was claimed to have the benefits

Resistant to crystallization (humectant)

Preventing starch retrogradation

Transglucosidase (TG) producing isomaltooligosaccharides (prebiotic)

+

+ +

+ +

TG

TG

Β-amylase

TG

Panose

Isomaltose


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Debranched Starch for Making Resistant Starch

Starch (except for high amylose starch)

Liquefaction

Debranching

Retrogradation

Drying

Resistant starch

Pullulanase

Issues: To reduce the viscosity allowing for economic processing To increase the thermal stability of crystalline structure after retrogradation


Beta-amylase & Maltogenic α-amylase Retard Retrogradation

Change of relative PNMR solid

content (∆S’, %) measured

during 4°C storage for isolated

amylopectin after partial β-

amylolysis. ECL values are labeled (Yao et al., J. Agric. Food Chem. 2003,

51, 4066-4071)

Maltogenic α-amylase: Degrades amylopectin & amylose to produce maltose & oligosaccharides

Functions like an endo-enzyme

Retard starch retrogradation by shortening external chains

Enzymatic starch modifications

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Some Enzymes for Bread Making

Fungal -amylase

Maltogenic -amylase

Xylanase, pentosanase, & hemicellulase

Lipase

Glucose oxidase

Breadmaking

Acting on damaged starch, producing sugar, increasing volume, flavor, and crust color

Acting mostly on amylopectin, reducing external chain length, reducing retrogradation, and extending shelf life

Partially hydrolyzing pentosan, reducing negative effect of insoluble pentosan, and increasing dough machinability & stability and crumb structure & volume,

Dough conditioning: larger volume, more uniform crumb structure, possibly forming linkage between gliadin and glutenin

Resulting in stronger dough, mechanism not clear


Increased Branching by Starch Branching Enzymes (SBE)

O O OO OO

O

OO O

SBEReaction I

O O OOO

O O OO OO

OO O

O O OOO

O

+

+

SBEReaction II

OH

OH

OH

OH

OH

OH

Potentials: Reduce starch retrogradation by shortening external chain length Reduce digestibility by forming more branches, since α-1,6 linkages are much

less susceptible to glucoamylase than 1,4 linkages


Alpha-glucan Chain Extension by Amylosucrase

Sucrose + (α-1,4-D-glucosyl)(n) = D-fructose + (1,4-α-D-glucosyl)(n+1) Used to synthesize amylose with certain length

Potocki-Veronese, G. et al, Biomacromolecules 2005, 5,1000

Low concentration

Highconcentration


Amylosucrase Forms Dendritic Nanoparticles

a. The surface chains of an initial glycogen particle (IGP) are extended by amylosucrase to form linear glucan chains (LGC)

b. The chains are further elongated, forming a corona around the glycogen core c. The elongated chains form double helical segments and crystallites, resulting in a

shrinkage of the corona and an increase in density and crystallinity

Putaux, J-L. et al. Biomacromolecules 2006, 7,1720.Enzymatic starch modifications

Hydrothermal Treatment

Annealing: starch incubated in excess or intermediate water content (>40%) at a temperature above the glass transition temperature but below the gelatinization temperature

Heat-moisture treatment: moisture level is low (<35%), temperature is above the glass transition temperature and may reach up to 100oC

Both lead to substantial change in starch properties: lower peak viscosity and setbacks, greater swelling consistency

Annealing leads to increased and narrower gelatinization temperature, whereas heat-moisture treatment results in broader ones

Annealing and heat-moisture treatment at low moisture content have no impact on starch crystallinity, whereas heat-moisture treatment at higher moisture content leads to partial gelatinization

Physical starch modifications

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Hydrothermal Treatment to Prepare Resistant Starch

High amylose starch Annealing ~100oC

Swelling Debranching Annealing

Partial acid hydrolysisAnnealing

Heat-moisture treatment

Effect of amylose chain length on RS DP<100: not long enough to form resistant crystallites DP100-260: RS increases to a maximum DP>300: too long, not easily reach the required alignment of chains

for resistant crystallites

Resistant starch


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Irradiation


Radiation: transmission of energy through space Electromagnetic waves (EM):

E = h = hc/ : frequency, c: speed of light, : wavelengthh:Planck's constant

Microwave (10-1 - 10-3m)

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Decrease of degree of polymerization of starch molecules

Radiolytic end product is similar irrespective of the type of starch used

Degradations of amylose and amylopectin lead to change of properties

Starch Modification by Irradiation

Left: Starch pasting

results for rice samples

receiving different γ-ray

irradiation doses.

Yu & Wang, Food Research International , 2007, 40: 297–303


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Microwave Heating


By passing non-ionizing* microwave radiation at a frequency of 2.45 GHz (or 0.915 GHz for industrial oven) through food materials

Water, fat, and other substances absorb energy by dielectric heating

Many molecules (e.g. water) are electric dipoles, i.e. they have a positive charge at one end and a negative charge at the other. They rotate to align themselves with the alternating electric field of the microwaves

This molecular movement creates heat

Microwave heating is more efficient on liquid water than on fats and sugars (which have less molecular dipole moment)

* Non-ionizing radiation: electromagnetic radiation that does not carry enough energy per quantum to ionize atoms or molecules (i.e. to completely remove an electron from an atom or molecule. Non-ionizing radiation is not mutagenic.

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Brabender viscosity curves for 8% solution of corn starch (left) and waxy corn starch (right)

Lewandowicz et al. Carbohydrate Polymers 2000, 42: 193–199

Microwave Treated Starch

Microwave condition: 30% moisture, 60 min, 2.45 GHz, 0.5 W/g energy


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Corn starch 95 C, left: native, right: microwaved

Waxy corn starch 75oC, left: native, right: microwaved

Microwave Treated Starch


Lewandowicz et al. Carbohydrate Polymers 2000, 42: 193–199

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High Pressure Processing (HPP)

Also known as High Hydrostatic Pressure (HHP) processing

Food material subjected to 100-900 MPa (commercially 400-700 MPa)

Pressurization carried out in a pressure vessel containing a fluid (e.g. water) as the pressure transmitting medium

Pressure applied isostatically (equally applied in all directions)

HPP processing system consists of pressure vessel, pressurization system, temperature control, and product handling device


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HPP Treated Starch


Bauer and Knorr. Journal of Food Engineering 2005, 68: 329–334

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Enzymatic and Physical Modifications of Starchn630/pdf_full/Starch-6.pdf · Enzyme classification and Nomenclature ... Glucose Maltose syrup syrup Ethanol Purification & crystallization