Antwoord: A polymer of any sugar. A glucan is a polymer of glucose units.
2.
What is the difference between a 1-3 and 1-4 linkage?
Antwoord: 1-4 linkage gives a stiff, inflexible molecule which can easily interact with other molecules.
1-3 linkage gives more flexible, extended structure which is better soluble
3.
What is the difference between a molecule with more or less OH groups?
Antwoord: More OH groups = more branches so less interaction itselve and other molecules = better solubility
4.
What is a O-CH3 group?
Antwoord: A methyl ether
5.
What is a C-CH3-O group?
Antwoord: An acetyl group
6.
What is the effect of methyl ethers or acetyl groups on your molecule?
Antwoord: It adds hydrophobic sites
7.
What is the effect of adding sulphate, phosphate and pyrophate?
Antwoord: They all add charge which increases the hydrodynamic volume and thus viscosity
8.
What is the effect of substituents on enzyme activity?
Antwoord: The enzymes can be less active
9.
What are the 4 main steps in carbohydrate research?
Antwoord: 1. Sample preparation
2. Extraction
3. Fractionation of extracted polysaccharides
4. Analysis of the polysaccharides
10.
What are the steps in sample preparation?
Antwoord: 1. Drying and grinding --> homogeneous sample
2. Heat to inactivate carbohydrate modifying enzymes
3. Defat the sample using hexane or chloroform
4. Optional: remove starch by gelatinzation alpha-amylase and amyloglucosidase
5. Remove proteins by protease
6. Remove glucose, small oligosaccharides, amino acids and peptides from interesting polysaccharides by dissolving in 80% ethanol
7. Remove fibres by centrifugation
Voorbeeld van de inhoud
Summary Advanced Food Chemistry - Carbohydrates
❖ Endogenous polysaccharides: naturally occurring in the raw material → storage
polysaccharides (starch granules) and cell wall polysaccharides (=dietary fibre! pectin, cellulose
and hemicellulose)
❖ Exogeneous polysaccharides: when the endogenous polysaccharide is extracted and used as
ingredient in food.
❖ Digestible polysaccharides: mono and disaccharides, polysaccharides (for instance starch),
sugar alcohols
❖ Non-digestible polysaccharides: di- and oligosaccharides (e.g. inulin, FOS, GOS),
sweeteners (lactitol), cell wall polysaccharides ((hemi)cellulose, pectin), resistant starch
❖ Glycan: polymer of ANY sugar
❖ Glucan: polymer of only GLUCOSE units
Structure elucidation
Parameters that determine the structure of carbohydrates
• Nature and number of constituent monosaccharides: homopolysaccharide or
heteropolysaccharide
• Type and anomeric configuration of glycosidic linkages:
o Cellulose contains beta 1-4 link → not digestible because no enzyme for beta linkages.
o 1-4 linkage → stiff, inflexible molecule in 1 plane → easy to interact because layering
o 1-3 linkage → more flexible, extended structure → better soluble and less aggregation
• Presence of hydroxyl groups:
o Branches so less interaction with itself and other molecules = better solubility
o
• Molecular weight and 3D conformation:
o Monodisperse or polydisperse
• Presence, number and distribution of substituents:
o Methyl esters (CH3) in pectin neutralize charges
o Methyl ethers (O-CH3, arabinoxylan) or acetyl groups (C-CH3-O, not charged!) add
hydrophobic sites
o Phosphate (amylopectin), sulphate (carrageenan, gives negative charge at all food
pH’s) and pyrophate add charge → increases hydrodynamic volume and thus viscosity.
o More substituents = less activity of degrading enzymes.
Step 1: sample preparation for analysis .
• Drying and grinding so you have a homogeneous sample
• Fruit or vegetables: heat to inactivate carbohydrate modifying enzymes
• Defat the sample by using hexane or chloroform
• Remove proteins by protease enzyme (e.g. trypsin)
• Optional: remove starch by gelatinization and alpha amylase or amyloglucosidase.
• Separate glucose, small oligosaccharides, amino acids and peptides from polysaccharides by
dissolving the small compounds in 80% ethanol.
• Fibres can removed by centrifugation
Step 2: extraction of polysaccharides
Extraction of pectin
• Extract water soluble pectins (HM and branched pectin) → hot water and buffer solutions
• Extract calcium bound pectins (LM-pectin) → solution with chelating agent (EDTA) to
chelate calcium ions to destroy the calcium-pectin gel and release the LM-pectin
• Extract alkali soluble pectins (pectins bound by ester linkages and hydrogen bonding) →
extraction with cold diluted sodium hydroxide (0.05 M NaOH, 0℃, 4-16 h) hydrolyse ester
bonds and breaks H-bonds to make complex pectins soluble. Important: temperature should be
low to prevent beta-elimination at this high pH. You don’t want beta-elimination (chain
hydrolysis) because you want to look at the intact polysaccharides.
• You only do the last step if you don’t want to study the separate fractions.
Extraction of hemicellulose
• Hemicellulose: group of insoluble polysaccharides that are bound to cellulose
• Starting point extraction: cell wall material without pectin (end point pectin extraction)
• Extract arabinoxylan (easy) → 1 M NaOH
, • Extract xyloglucan (difficult) → 4 M NaOH (or use 6 M NaOH so cellulose fibrils swell so
strongly bound pectins and hemicelluloses become more soluble.
• Residue contains both cellulose and lignin (polyphenol that is a lot in wheat straw, strengthens
the cell wall)
• Treat residue with sulphuric acid to degrade the cellulose (not useful anymore), to analyse the
lignin.
Step 3: fractionation of the extracted polysaccharides
Preparative chromatographic techniques
• Size exclusion chromatography (SEC): separation based on size (for instance more or less
degraded by enzymes). Column has defined pore size through which larger molecules are not
able to penetrate → larger molecules will elute earlier compared to smaller molecules. Light
scattering as detector to see how material you have. Carbohydrates do not show absorption in
UV.
• Anion exchange chromatography (AEC): separate neutral from charged/heavily charged
molecules. Charged molecules will interact with the positive column so elute later. pH or salt
gradient leads to elution of molecules with low charge and after that also elution of those with
a high charge. For instance to separate pectins with more or less methyl esterification. More
esterified = less charge = earlier elution
Specific precipitations
• Separation based on branches: increase the amount of ethanol or ammonium sulphate →
lowers the quality of the solvent → so molecules that are bad soluble (linear) become even
worse soluble and precipitate. You end up with a solution of the most branched molecules.
• Separation based on methyl groups on pectin: increase the amount of copper-acetate →
precipitation
Step 4: Analysis of the polysaccharides
Sugar composition analysis
• Gives information about number and type of monosaccharides
• Convert polysaccharides to monosaccharides by acid hydrolysis
o Strong acid + heat → cleavage of glycosidic bonds between monosaccharides
o 6 ring is stronger than 5 ring. Beta linkages are stronger than alpha. Soluble
polysaccharides are hydrolysed easier than insoluble ones.
o Xylan (polymer of xylose) is a 6 ring so stronger than arabinan (polymer of arabinose)
which is a 5 ring. Cellulose is beta 1-4 linked which is stronger than starch (alpha 1-4).
Uronic acids (for instance galacturonic acid) are hard to hydrolyse.
o Cellulose > galactan > xylan > arabinan
o Not too mild conditions (not all bonds would be cleaved)
o Not too harsh conditions (to prevent hydrolysis of monosaccharides)
o Break neutral and water soluble polysaccharides → 2M TFA 1 hour 120℃ → TFA is
mild, volatile, can be easy removed
o Disrupt fibrous material → 12M sulphuric acid (78%), 1 hour, room temperature
+ 1M sulphuric acid, 3 hours 100℃
o Break polysaccharides with acidic sugar residues (pectins) → methanolysis (2M HCl in
methanol, 16 hours at 80℃) → releases methylglycosides
o Analyse the monomers by GC or HPAEC
• Gas chromatography
o Sugars should be made volatile first → reduction to their alditol, followed by
acetylation of all free OH groups
o You can compare the peak area of the alditol acetates with standards to quantify.
o Acidic sugars → are not made volatile, colour assay is done OR they are made volatile
by converting them to TMS derivatives but the GC pattern is then difficult to interpret
o GC: sample preparation not accurate and quantity of sugar may be underestimated if
the reduction to alditols or the acetylation was not completed.
• HPAEC
o HPLC for carbohydrates. At high pH (12), the OH groups are – charged so they react
with the column. Changing pH changes charge and thus they will elute.
o Advantages: acid hydrolysis can be checked in one run, easy sample preparation, short
analysis time needed.
o PAD detector: separation and quantification of monosaccharides with low detection
limit. You need specific standards because response varies per component.
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