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High-intensity Sweeteners, Polyols, and Non-chemical Modifications of

Yuan Yao Whistler Center for Research October 3, 2017 Do not duplicate

Sweetness • Hypothesis: sweetness perception is initiated by an interaction of a sweet molecule with a receptor site in the taste buds • The actual receptor site has not been isolated • Once a sweet molecule interacts with the receptor site, a series of reactions occurs and the taste signal is sent to brain • Various models have been proposed, and the most widely accepted one is the three-point attachment theory (AH-B-X) • Relationship between chemical structure and the ability to initiate sweetness is not well understood • Most sweeteners were discovered by trial and error Do not duplicate Sweetness

http://www.elmhurst.edu/~chm/vchembook/549receptor.html • AH+ area contains functional groups with hydrogen to form hydrogen bond with partially negative atom on the sweet molecule (acid group COO- on ) • B- area contains partially negative groups available to form hydrogen bond with hydrogen on the sweet molecule (amine group NH3+ on aspartame) • X area is a hydrophobic (lipophilic) area interacting with the non-polar area on the sweet molecule (benzene ring on aspartame) Do not duplicate Sweeteners

High intensity sweeteners Acesulfame K Alitame Aspartame Cyclamate Steviol glycoside

Mogroside V Polyols Erythritol Hydrogenated starch hydrolysates Sorbitol Mannitol Do not duplicate High Intensity Sweeteners

Acesulfame K (200 X sweetness) •5,6-dimethyl-1,2,3-oxathiazine-4(3H)-one 2,2- dioxide, a sweet tasting compound, was incidentally discovered in 1967 by Clauss and Jensen •Among different substitutions, 6-methyl- 1,2,3-oxathiazine-4(3H)-one 2,2-dioxide was demonstrated to be the most favorable one •Generic name was registered by the world Health organization (WHO) •Acesulfame K is a white crystalline powder, dissolving readily in water

http://www.elmhurst.edu/~chm/vchembook/549 acesulfame.html • “The additive shall be used in accordance with current good manufacturing practice in an amount not to exceed that reasonably required to accomplish the intended effect” (FDA 21CFR172.800) • Applications: low-calorie products, diabetic , sugarless products, oral hygiene preparations, pharmaceuticals, and animal feeds Do not duplicate High Intensity Sweeteners

Aspartame (160 - 220 X) • 1-methyl N-L-[alpha]-aspartyl-L- , a dipeptide containing L- and the methyl ester of L- phenylalanine • Incidentally discovered in 1965 by GD Searle and James Schlatter • Under certain conditions, the ester bond is hydrolyzed, forming methanol and dipeptide, which is ultimately hydrolyzed to individual amino acids • Maximum stability at around pH 4.3. Very high temperature reduces stability http://www.elmhurst.edu/~chm/vchembook/549 aspartame.html • Slightly soluble in water (~1.0%)

• The label of any containing the additive shall bear: Phenylketonurics: Contains Phenylalanine. When the additive is used in a for table use, its label shall bear instructions not to use in cooking or baking (21CFR172.804) Do not duplicate High Intensity Sweeteners

Sucralose (600 X) (Splenda) • 1,6-dichloro-1,6-dideoxy-[beta]-D- fructofuranosyl-4-chloro-4-deoxy-[alpha]- D-galactopyranoside • Discovered in 1970s by Hough and his coworkers, with the support of Tate & Lyle • Selective chlorination of • A white, crystalline, nonhydroscopic powder • Highly soluble in water and ethanol • Dry sucralose may show increased discoloration at higher temperature • Sucralose liquid concentrates show high http://www.elmhurst.edu/~chm/vchembook/549 storage stability sucralose.html

• The additive may be used as a sweetener in foods generally, in accordance with current good manufacturing practice in an amount not to exceed that reasonably required to accomplish the intended effect (21CFR172.831) Do not duplicate High Intensity Sweeteners Steviol glycosides (30-300 X) • Steviol: (5β,8α,9β,10α,13α)-13- Hydroxykaur-16-en-18-oic acid • Found in S. rebaudiana, with stevioside (120 X) most abundant, followed by rebaudioside A (250 X, better tasting) • Reb-A: stable at pH 4-8, with reduced stability in more acidic systems; stable Steviol structure. Steviol is the aglycone to steviol with common thermal treatment: retorting, glycosides (Different units at R1 and R2) (J.M.C. UHT, pasteurization; lasting sweetness Geuns, 2003, Phytochemistry 64: 913–921) perceived longer than sucrose

Compound R1 R2

Stevioside -glc- -glc--glc-

Rebaudioside A -glc- (-glc)2--glc-

Rebaudioside B H (-glc)2--glc- Rebaudioside C -glc- (-glc-, -rha-)--glc-

Rebaudioside D -glc--glc- (-glc)2--glc-

http://dalberg.com/blog/wp- content/uploads/2011/08/Peru-2010-067.jpg Do not duplicate High Intensity Sweeteners

Mogroside V (250-400 X) • Luo Han Guo (LHG, Siraitia grosvenorii ) has been used in Asia as medical herbs and teas • LHG contains a number of mogrosides (mogrol glycosides), such as mogroside III, mogroside V, mogroside VI, mogroside IIE, mogroside IIIE, neomogroside, siamenside I, and 11-oxo- http://www.sanherb.com/products/corsvenor-momordica- fruit/luo_han_guo_extract.html mogroside V • Primary sweet component is Mogroside V o GRAS for LHG fruit extracts containing mogroside V o Thermally stable (UHT, retorting) o Stable at pH 3-7 • LHG extracts can be used in combination with others, e.g. http://www.scbt.com/datasheet- 280990-mogroside-v.html rebaudioside A, to offer a more balanced taste profile o Reb-A: quick onset Structure of Mogroside V: 5 glucosyl units attaching to the aglycone mogrol o LHG: slower build Do not duplicate Polyols

• Definition and natural occurrence

• Structure and manufacture

• Physicochemical and organoleptic properties

• Nutritive value and health benefit

• Applications Do not duplicate Definition and Natural Occurrence What are polyols? Are they natural? Definition • “Sugar alcohol” and “polyol” are synonyms • Carbonyl group (>C=O) in the and moieties of mono-, di-, oligo-, and is replaced by alcohol group (>CH-OH) • Polyols generally carry the suffix ‘itol’ in place of the suffix ‘ose’

Natural occurrence Polyol Natural occurrence Erythritol Wine, sake, soy sauce, melons, pears, grapes, etc. Xylitol Fruit, vegetables, intermediate in Sorbitol Rowan, pears, cherries, plums, apricots, apples, etc. Mannitol Tree exudates, manna ash, marine algae, fresh mushroom Maltitol Lactitol Isomalt Polyglycitol Do not duplicate Molecular Structure Structures of polyols are similar: Carbonyl group is replaced by alcohol group

CH2OH CH2OH CH2OH CH2OH O OH OH OH OH Maltitol OH CH2OH HO O HO OH OH OH OH CH2OH CH OH OH 2 OH Erythritol CH2OH Lactitol CH2OH OH CH OH OH O 2 Sorbitol O OH OH CH2OH CH2OH

CH2OH OH CH2OH OH HO HO OH HO HO HO HO OH CH OH CH2OH OH 2 OH OH OH O O OH OH CH OH 2 OH OH CH2OH O O Xylitol HO HO Mannitol OH OH Isomalt: 50%GPM + 50% GPS Do not duplicate Industrial Manufacturing

Polyols industry is established

Starch Xylan Sucrose

Liquefaction Hydrolyzation Enzymatic conversion

Saccharification

Fermentation Hydrogenation Hydrogenation Hydrogenation Hydrogenation

Purification Purification Purification Purification Purification

Erythritol Sorbitol Xylitol Lactitol Isomalt Mannitol Maltitol Polyglycitol Do not duplicate Physicochemical Properties

Polyols have diversified properties

Solubility Melting Hygro- Heat stability Acid stability Polyol/sugar g/100g H O point (°C) scopicity 2 (°C) (pH) (25°C) Erythritol 126 Very low 37-43 >160 2-12 Xylitol 94 High 63 >160 2-10 Mannitol 165 Very low 18-22 >160 2-10 Sorbitol 97 Median 70-75 >160 2-10 Maltitol 150 Median 60-65 >160 2-10 Isomalt 145-150 Very low 25-28 >160 2-10 Lactitol 122 Low 55-57 >160 >3 Hydrolyzes at Sucrose 190 Low 67 <160-186 acidic/alkaline pH

Modified: L. Nabors. Alternative Sweeteners. Third edition, 2001, Marcel Dekker, Inc, NY Do not duplicate Organoleptic Properties Polyols are sweet and cool

Sweetness Compared with Sucrose

100

80

60

40

20

0 Erythritol Xylitol Sorbitol Mannitol Maltitol Isomalt Lactitol Sucrose

Sweetness 65 100 60 50 90 40 35 100

Cooling Effect

0

-10 -20

-30 -40

-50 Erythritol Xylitol Sorbitol Mannitol Maltitol Isomalt Lactitol Sucrose

Heat of Solution (cal/g) -42.9 -36.6 -26.5 -28.9 -5.5 -9.4 -13.9 -4.3 Do not duplicate In the Gastrointestinal Tract

Polyols are not fully digested in the body

Carbohydrate Not metabolized Kidneys Urine Absorbed Small intestine

Metabolized Not Energy absorbed CO2 Volatile fatty acids Fermented Large intestine CH4/H2 Not Biomass fermented

Feces Feces Do not duplicate Glycemic Response

Polyols are low in glycemic response

Glucose Xylitol Sucrose Sorbitol Maltitol Isomalt Erythritol Lactitol Mannitol

Glycemic curves for glucose, sucrose, maltitol, isomalt, lactitol, xylitol, sorbitol, erythritol, and mannitol in normal individuals. Data from several publications were pooled to yield curves representative of 25 g doses (20–64 g for erythritol) (Adapted from G. Livesey. Nutrition Research Reviews. 2003, 16, 163 –191) Do not duplicate Colon Health

Polyols are beneficial to colon health

• Fermented by microflora, polyols benefit colon health • Enables saccharolytic anaerobes and aciduric organisms to grow in preference over putrefying, endotoxic, pathogenic, and procarcinogen-activating aerobic organism • Acidic conditions may normalize epithelial functions • Lactic acid is generated from fermentable • Butyric acid generated from polyols is beneficial to maintaining a healthy colonic epithelium and improving inflammatory conditions of the colonic mucosa Do not duplicate Dental Health: Non-cariogenic

Polyols are non-cariogenic

Starch & derivatives Fermentable

Salivary Plaque (mutans buffering, washing, Inhibit Produce streptococci and neutralizing lactobacilli) Acid Enamel crystal Partly dissolved Carbonated apatite Demineralization crystal

Calcium, Phosphate, Remineralization Fluoride Crystal with Salivary flow new surface

Polyols are not fermentable. No acid is generated. So polyols are non-cariogenic Do not duplicate Regular Food Applications

Polyols are widely used in foods

Polyols Specific properties Main applications Cooling effect, compressibility, Crystalline Chewing gum, tablets, surimi cryoprotective Sorbitol Micro-crystallization, surface Deposited high boiled sweets, biscuits, cakes, Syrup crystallization, humectancy pastry High sweetness, non-hygroscopic, high Crystalline Chocolate, coated chewing gum, bakery melting point, crystallization Maltitol Plasticity of massecuites, anti-crystallizing, High boiled sweets, chewy sweets, jellies, Syrup plasticizer, HFS substitute chewing gum, gums, bakery Crystalline mannitol Non-hygroscopic Dusting powder, chewing gum, chewy sweets High sweetness, cooling effect, Crystalline xylitol Jellies, chewing gum, coated chewing gum crystallization Chewing gum, chocolate, confectionery, Lactitol Low-hygroscopic, mild cooling effect bakery, ice-cream, tablets Non-calorie, non-hygroscopic, cooling Low-calorie beverages, chocolate, chewing Erythritol effects, gum, dehydrated fruit, bakery Chocolate, high-boiled candy, chewing gum, Isomalt Low cooling effect pan-coated products Non-reducing, cryoprotective, heat/acid Corn syrup substitute, confectionery, ice- Polyglycitol (HSH) resistant cream, hard candies, bakery 21 Do not duplicate Starch Structure

Kernels and starch granules of two wx mutants

wx

ae wx 22 Do not duplicate Structure of Starch Granules

We are going here!

Gallant et al, 1997, Carbohydrate Polymer, 32:177-191 23 Do not duplicateFine Structure and Chain Length Distribution

Why study “starch fine structure”?

• To differentiate (and starch derivatives) at molecular levels

• To define and document unique starches or starch derivatives

• To monitor product profiles and maintain product consistency

• To control important properties (digestibility, retrogradation, solubility, water adsorption, viscosity, gel strength, stability) of starches and starch derivatives

• To define goals for starch modifications

• To guide new product development 24 Do not duplicateFine Structure and Chain Length Distribution

Starch fine structure is usually characterized by chain length distribution using cluster model

Starch materials

Fully dispersed

Completely debranched

Chain length distribution characterized

Chains are non-randomly clustered Structural parameters

Thompson. Carbohydrate Polymers, 2000, 43: 223-239 constructed 25 Do not duplicate Chain Length Distribution

Chain length distribution of normal and ae corn starch

• These figures (by HPSEC) are called “chain length distribution (profile)” • Usually, the chain length profiles are compared among different starches to differentiate starches at molecular level • Starch molecules need to be debranched to release chains 26 Do not duplicate Chain Length Distribution

Analysis of chain length distribution

• Three types of debranched starch material are usually prepared

• Debranched starch

• Debranched

• Debranched beta-limit

• Three types of separation methods are often used to describe the chain length distribution of these materials

• Size exclusion chromatography (SEC or HPSEC)

• Fluorophore-assisted carbohydrate electrophoresis (FACE)

• Anion exchange chromatography (HPAEC) 27 Do not duplicate Chain Length Distribution

Size exclusion chromatography (SEC or HPSEC)

By Dr. Shulamit Levin, http://www.forumsci.co.il/HPLC/modes/modes14.htm 28 Do not duplicate Chain Length Distribution

• Eluent from HPSEC columns passes through a refractive index (RI) detector for quantifying the mass of carbohydrate molecules

• Molecular standards are used to calibrate columns and determine the molecular weight of samples

• Eluent may pass through a multi-angle laser light scattering (MALLS) detector for molecular weight determinations 29 Do not duplicate Chain Length Distribution

Fluorophore-assisted carbohydrate electrophoresis (FACE): labeling of using 1-aminopyrene- 3,6,8-trisulfonate (APTS)

- O3S NH2

NH - NaBH3CN SO3 +

- - - - O3S SO3 O3S SO3

Oligosaccharide APTS APTS adducts Excitation 488 nm Emission 520 nm

Courtesy of Beckman-Coulter 30 Do not duplicate Chain Length Distribution

APTS adducts of carbohydrate molecules have 2 properties

- NH SO3

- - O3S SO3

• The APTS adducts are negatively charged, so may migrate in an electric field of electrophoresis

• The APTS adducts may release detectable fluorescent emission with laser excitation at 488 nm 31 Do not duplicate Chain Length Distribution

Molecular weight is determined by migration time Number of molecules is determined by fluorescent signal

- NH SO3

- - Longer migration time due to larger CHO molecule O3S SO3

- Weaker fluorescent signal due to fewer molecules NH SO3

- - O3S SO3

- - NH SO3 NH SO3

- - - - Shorter migration time due to smaller CHO molecule O3S SO3 O3S SO3

- - Stronger fluorescent signal due to more molecules NH SO3 NH SO3

- - - - O3S SO3 O3S SO3 32 Do not duplicate Chain Length Distribution

Electrophoregram of FACE

APTS FU 30

3

2 10 40

50 80 1 20

0 0 10 20 30

Courtesy of Beckman-Coulter 33 Do not duplicate Chain Length Distribution

Comparison between HPSEC and FACE

HPSEC FACE 34 Do not duplicate Chain Length Distribution

High performance anion-exchange chromatography equipped with pulsed amperometric detector (HPAEC-PAD)

Sample molecules, different types of Mobile phase, charge or neutral negatively charged

Stationary phase, positively charged

Negatively charged sample molecules adsorbed by stationary phase may be desorbed by mobile phase

Courtesy of Amersham Biosciences 35 Do not duplicate Chain Length Distribution

The adsorption/desorption equilibrium is governed by:  The amount of negative charge of sample molecules  The ionic strength of mobile phase

The greater the net charge, the The greater the net charge, the higher the stronger the adsorption salt concentration required for desorption

Courtesy of Amersham Biosciences 36 Do not duplicate Chain Length Distribution

The retention time of a molecule is determined by  Its net negative charge, which may be affected by pH  Elution power, which is controlled by gradient elution

Courtesy of Amersham Biosciences 37 Do not duplicate Chain Length Distribution

Carbohydrates can be separated via anion-exchange

• In basic solution (high pH), carbohydrates are negatively charged

• Thus, there exists an adsorption/desorption equilibrium of carbohydrate molecules with stationary phase

• The higher DP of carbohydrate molecule, the greater net negative charge it possesses, the higher ionic strength (salt concentration) needed to desorb the molecule

• Using gradient eluent, carbohydrate molecules with different DP are eluted at different retention time and separated 38 Do not duplicate Chain Length Distribution

HPAEC-PAD gives a chromatogram similar to FACE

0.160 礐

0.125

0.100

0.075

0.050

0.025

min -0.003 0 25 50 75 100 125 150 175 200

Chain length distribution of debranched amylopectin of sorghum starch 39 Do not duplicate Chain Length Distribution

Pros and cons of HPSEC, FACE, and HPAEC

• HPSEC provides information of a broad range of molecular weight (>DP10,000) but with relatively low resolution >DP5

• FACE provides baseline resolution up to DP100, but unable to (with current techniques) give a chain length profile with broader DP range

• HPAEC is similar to FACE 40 Do not duplicate Genetic Starch Modifications

What can genetic starch modifications do for us?

• Improve the starch yield of major crops

• Acquire starches with desirable functionalities and high value

• Suitable starting materials for chemical and enzymatic modifications

• Modified digestibility or degradability as food, feed, and industrial raw materials

• Retarded or enhanced retrogradation after cooking, leading to extended shelf life or unique functionalities

• Unique granular and nano structures for high-end uses, e.g. as carriers for controlled release 41 Do not duplicate Genetic Starch Modifications

Successful genetic starch modification is based on the knowledge of starch biosynthesis

• Where are starch granules synthesized?

• What enzymes are involved in starch biosynthesis?

• What are the functional behaviors of these enzymes?

• Can we tailor starch structure for desirable functions? 42 Do not duplicate Enzymes Synthesizing Starch

Starch biosynthesis in a cell of maize endosperm

Starch debranching enzyme AMYLOPLAST starch granules D-enzyme? amylopectin + Starch branching enzyme

Granule-bond starch synthase Soluble starch synthase ADP ADP-Glc Glucosyl primer (UDP-Glc:protein transglucosylase?) ADP-Glc transporter

ADP-Glc PPi ATP ADP-Glc pyrophosphorylase Glc-1-P Phosphoglucomutase ADP UTP UDP-Glc pyrophosphorylase ATP PPi + UDP-Glc Sucrose synthase UDP CYTOSOL Sucrose 43 Do not duplicate Enzymes Synthesizing Starch

ADP-glucose pyrophosphorylase (AGPase)

ATP PPi

O P AGPase ADP

ADP-glucose 44 Do not duplicate Enzymes Synthesizing Starch

Starch synthase (SS) Isoforms identified: GBSSI, SSI, SSIIa, SSIIb, & SSIII

+ OH ADP O O O O

SS

ADP

OH O O O O O

• Soluble starch synthase (SSS) responsible for amylopectin synthesis • Granule-bond starch synthase (GBSS) responsible for amylose synthesis 45 Do not duplicate Enzymes Synthesizing Starch Starch branching enzyme (SBE) Isoforms identified: SBEI, SBEIIa, & SBEIIb

OH O O O O O O

Reaction SBE I

O

OH O O O

OH O O O O O O +

OH O O O O O

Reaction SBE II

OH O O O

O +

OH O O O O O 46 Do not duplicate Enzymes Synthesizing Starch

Starch debranching enzyme (DBE) Isoforms identified: Isoamylase-like (SU1) & Pullulanase-like (ZPU1)

O

OH O O O

DBE

OH O O

OH O O O 47 Do not duplicate Mutant Starches from Maize

A list of documented starch mutants of maize

Single mutants double mutants Triple/quadruple mutants • Waxy (wx) • ae wx • ae du1 su1 • Amylose-extender (ae) • sbe1 wx • ae du1 su2 • sbe1 • ae su1 • ae du1 wx • sbe2a • ae su2 • ae su1 su2 • Sugary-1 (su1) • ae du1 • ae su1 wx • Zpu1 • du1 su1 • ae su2 wx • Sugary-2 (su2) • du1 su2 • sbe1 ae wx • Dull-1 (du1) • du1 wx • du1 su1 su2 • Brittle-1 (bt1) • su1 wx • du1 su1 wx • Brittle-2 (bt2) • su2 wx • du1 su2 wx • Shrunken-1 (sh1) • su1 su2 • su1 su2 wx •Do notShrunken duplicate-2 (sh2) • ae du1 su1 wx 48 Do not duplicate Mutant Starches, Single Mutants

Waxy (wx)

• Deficiency of granule-bond starch synthase

• Identified in maize, sorghum, rice, barley, wheat, & potato

• Kernels of wx are full

• Starch and dry weight are equal to normal

• Mutant wx is epistatic to other known mutants, e.g. multiple mutants containing wx has NO amylose

• Dosage effect shown for Wx wx wx

• Broad applications in the food and non-food industries

Do not duplicate 49 Do not duplicate Mutant Starches, Single Mutants

Amylose-Extender (ae)

• Deficiency of starch branching enzyme IIb (SBEIIb) for maize • Identified in maize, rice, peas, & barley • Kernels of ae have a smaller size than normal • Dosage effect exists for ae gene • Amylose concentration usually ranges 50-75% by blue value tests. Chromatographic analysis shows lower amylose value and the presence of intermediate materials • Granules are smaller than normal, and some may be non- birefringent • B-type x-ray pattern is shown for ae starch • Broad applications Do not duplicate 50 Do not duplicate Mutant Starches, Multiple Mutants

Amylose-Extender Waxy (ae wx)

• Deficiency of both SBEIIb and GBSSI

• Reduced in size, dry weight, and starch content (~50%)

• Blue value test indicates 15-26% amylose, but chromatographic separation indicates solely amylopectin

• Both ae and wx functions independently

• Amylopectin has increased proportion of long chains

• Dosage effect exists for both ae and wx genes Do not duplicate 51 Do not duplicate Mutant Starches, Multiple Mutants

Dull-1 Waxy (du1 wx)

• Deficiency of both SSIII and GBSSI

• Kernels weight similar to du1 and wx, slightly less than normal

• Sugar concentrations are higher, and starch concentration is lower than in either normal, du1, or wx

• Gene wx is epistatic to du1, so du1 wx has 100% of amylopectin

• Amylopectin has increased portion of shorter chains

Do not duplicate 52 Do not duplicate Mutant Starches, Multiple Mutants

Amylose-Extender Dull-1 Waxy (ae du1 wx)

• Deficiency of SBEIIb, SSIII, and GBSSI

• Starch concentration is low compared with the component single and double mutants. Sugar contents are several fold higher. Sweetness is between standard sweet corn (su1) and sh2 mutation.

• Amylopectin branching is different from either ae wx or du1 wx, and somewhat intermediate between wx and du1 wx

Do not duplicate 53 Do not duplicate Mutant Starches, Multiple Mutants

Amylopectin of mutants containing ae, wx, and du1

Chain length profile of amylopectin Chain length profile of -limit dextrin

DEGREE of POLYMERIZATION DEGREE of POLYMERIZATION 1000 100 50 20 10 3 1000 100 50 20 10 3 70 60 3 60 50 wx 50 40 du1 wx 40 30 30 ae wx 20 20

NORMALIZED NORMALIZED D.R.I. ae du1 wx 10 10 0 0 10 12 14 16 18 20 10 12 14 16 18 20 RETENTION TIME, min RETENTION TIME, min

• Deficiency of DU1 leads to increased branching •DoThe not presence duplicate or absence of SBEIIb does not eliminate effect of DU1 54 Do not duplicate Mutant Starches, Patents

A partial list of mutant starch patents in last 20 years

• 2004, Method of grain production for heterozygous waxy sugary-2 maize • 1996, Foodstuffs containing a waxy amylose extender starch • 1992, ae du1 batter starch for deep fat fried food • 1989, Foodstuffs containing starch of a waxy shrunken-2 genotype • 1989, Foodstuffs containing starch of an amylose extender sugary-2 genotype • 1988, Food stuffs containing starch of a dull sugary-2 genotype • 1988, Food stuffs containing starch of an amylose extender dull genotype • 1988, Food stuffs containing starch of a dull waxy genotype • 1988, Starch of the duh genotype and products produced therefrom • 1988, Starch of wx fl1 genotype and products produced therefrom • 1988, Starch of the wx sh1 genotype and products produced therefrom • 1986, Bread containing wx su2 genotype starch as an anti-stalent Do not duplicate Enzymatic and Physical Modifications of Starch 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 Catalysis by Enzyme

Reaction with no catalysis Enzyme-catalyzed reaction

Lehninger Principles of Biochemistry, Fourth Edition

Introduction of enzymes Starch Refining

Enzymes used

• -Amylase hydrolyzes interior -1,4-glucosidic bonds

• -Amylase hydrolyzes -1,4-glucosidic bonds from non-reducing ends and release

• 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

• : DE <20

Enzymatic starch degradation Starch Refining

Steam Starch slurry

Liquefaction -amylase Products of different Maltodextrin dextrose equivalent (DE) Amyloglucosidase -amylase Pullulanase Saccharification Pullulanase

Glucose Maltose syrup syrup Fermentation Isomerization Isomerase

Purification & High fructose corn Ethanol crystallization syrup (HFCS)

Crystallized glucose

Enzymatic starch degradation Cyclodextrin

Starch Liquefaction Cyclodextrin glucanotransferase (CTGase) β-CD Enzyme conversion

Solvent extraction

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

α-CD β-CD γ-CD

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

TG + TG + + Panose

TG + +

Enzymatic starch degradation Debranched Starch for Making Resistant Starch

Starch (except for high amylose starch)

Liquefaction

Debranching Pullulanase

Retrogradation

Drying

Resistant starch 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

Left: Change of relative PNMR solid content (ΔS’, %) 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 &

•Functions like an endo-enzyme

•Retard starch retrogradation by shortening external chains

Enzymatic starch modifications Some Enzymes for Bread Making

Fungal -amylase Acting on damaged starch, producing sugar, increasing volume, , and crust color

Maltogenic -amylase Acting mostly on amylopectin, reducing external chain length, reducing retrogradation, and extending shelf life

Xylanase, pentosanase, Partially hydrolyzing pentosan, reducing Bread negative effect of insoluble pentosan, and & hemicellulase making increasing dough machinability & stability and crumb structure & volume,

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

Glucose oxidase Resulting in stronger dough, mechanism not clear

Enzymatic starch modifications Increased Branching by Starch Branching Enzymes (SBE)

OH O O O O O O

Reaction SBE I O O O O O H

OH O O O O O O + OH O O O O O Reaction SBE II OH O O O

O + OH O O O O O

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- 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

Low concentration

High concentration

Potocki-Veronese, G. et al, Biomacromolecules 2005, 5,1000 Enzymatic starch modifications Amylosucrase Forms Dendritic Nanoparticles

a. The surface chains of an initial 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 100°C

• 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 Hydrothermal Treatment to Prepare Resistant Starch

High amylose Annealing ~100oC starch

Swelling Debranching Annealing Resistant starch

Annealing Partial acid 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

Physical starch modifications Combined Hydrothermal and Enzymatic Treatment

(a)

NCS

Treatments to stabilize starch pasting behavior

•95°C treatment at 40% moisture NCS0-1 NCS0-2, NCS0-3 •Followed by partial β- NCS-1 amylolysis NCS-2, NCS-3

Hickman et al., 2009, Journal of Agricultural and

Physical starch modifications Starch Treated by Irradiation

• 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

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 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.

Physical starch modifications Microwave Treated Starch

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

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

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

Physical starch modifications Microwave Treated Starch

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

Waxy corn starch 75°C, left: native, right: microwaved

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

Physical starch modifications Porous Starch for Drug Delivery

Starch + ethylene vinyl alcohol + baking 0% soda (blow agent) +H O + Model drug BA 2 2

Microwave treatment

Porous starch 5% BA Drug release test

10% BA

Malafaya et al. J. Biomater. Sci. Polymer edn 2001, 12: 1227-1241 Physical starch modifications High Pressure Processing (HPP)

• Also known as High Hydrostatic Pressure (HHP) processing

• Food material subjected to 100-900 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 HPP Treated Starch

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

Physical starch modifications