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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
Introduction of enzymes
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Catalysis by Enzyme
Reaction with no catalysis Enzyme-catalyzed reaction
Introduction of enzymes
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]
Introduction of enzymes
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Enzyme Kinetics
Km
Introduction of enzymes
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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
Enzymatic starch degradation
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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
Enzymatic starch degradation
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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
Enzymatic starch degradation
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
Enzymatic starch modifications
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
Enzymatic starch modifications
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
Enzymatic starch modifications
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
Physical starch modifications
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Irradiation
Physical starch modifications
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
Physical starch modifications
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Microwave Heating
Physical starch modifications
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
Physical starch modifications
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Corn starch 95 C, left: native, right: microwaved
Waxy corn starch 75oC, left: native, right: microwaved
Microwave Treated Starch
Physical starch modifications
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
Physical starch modifications
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HPP Treated Starch
Physical starch modifications
Bauer and Knorr. Journal of Food Engineering 2005, 68: 329–334