Summary Advanced Food Chemistry - Proteins part. See also my other summaries for the other parts.
Wageningen Univeristy - FCH-30806
Contains the following:
- Occurrence, isolation and properties of commonly used proteins
- Composition of isolated proteins
- Structural properties of commonly u...
Summary Advanced Food Chemistry – Proteins part
Occurrence, isolation and properties of commonly used proteins
Functionality
• Bio functional properties: muscle proteins (provide structure to the tissue), milk proteins
(source of nitrogen and essential amino acids for the animal), storage proteins in plants
(provide nitrogen and amino acids for the new plant), protease inhibitors (protect the plant
from attack by fungi or animals)
• Techno-functional properties: thickening, gelling, emulsifying. Properties can be influenced by
processing and storage → enzymatic and chemical reactions.
• Chemical and structural properties: affected by the system conditions. Other compounds can
react with proteins.
Commonly used food proteins
• Animal proteins: whey, egg white, blood (globular), caseins (random coil), gelatin (fibrillar)
• Plant proteins: soy, lupin, sunflower proteins (all globular)
o Legumin seeds: albumins, globulins, prolamins (determine by Osborne)
• Novel (plant) proteins: potato, algae, leafy proteins (all globular)
• Fibrillar proteins are in muscle tissue to provide structure → should be insoluble under neutral
pH. Globular proteins are in milk or egg → should be soluble under that pH and ionic strength
to provide the young animal with nitrogen and amino acids
• Isolates contain more protein = more expensive but less volumes marketed
• You can choose a protein based on plant or animal origin. However, it says nothing about
functionality. In food you want no colour, free of taste and good solubility.
Isolation of proteins
• Removing as much impurities as needed while retaining as much of proteins as possible
• Proteins intrinsically insoluble (gluten) → separate by wind sifting, shear separation
• Proteins intrinsically soluble → aqueous extraction
• Crude separation (separate proteins from other compounds). Mass balance is important →
there can be a very low protein content in a fraction, but if there is a lot of that fraction, there
might be a lot of protein in this fraction.
• After crude separation, removal of other compounds to obtain higher purity
Production of soy protein concentrate
• Mill soybeans and defat them
• Suspend the defatted soybean meal in an aqueous solution of pH 4-5 (proteins are not soluble
so will precipitate, soluble carbohydrates will dissolve)
• Centrifuge to remove the precipitated proteins and insoluble carbohydrates from supernatant
Production of soy protein isolate
• Mill soybeans and defat them
• Suspend the defatted soybean meal in an aqueous solution of pH 8-9 (proteins and soluble
carbohydrates are soluble (both – so no interactions), non-dissolved parts are starch and cell
wall polysaccharides. However, they might hydrolyse because of high pH)
o pH should not be too high because then chemical hydrolysis of proteins = irreversible
changes in quaternary structure
o pH should not be too low because then proteins are + and they interact with carbs
• Centrifuge to remove insoluble carbohydrates
• Change pH to 4.5 (IEP so proteins will precipitate)
• Centrifuge to remove precipitated proteins → higher purity because no insoluble carbohydrates
Presence of other compounds
• Enzymes (proteases and PPO are also enzymes so are also isolated)
o Discolouration (for instance enzymatic browning by PPO)
o Change in taste / smell (proteases may form aromatic peptides by hydrolysis,
lipoxygenase is active in soy protein processing → off taste with low threshold)
o Change in digestibility
• Non-protein compounds (carbohydrates, phenolic compounds etc.)
o Complexation with proteins (phenolics becomes quinones under influence of PPO,
quinones can covalently link to proteins → less soluble because proteins are crosslinked
to aggregates by phenols)
, o Modification of the proteins (for instance by lactosylation)
o Discolouration (when phenolics are bound to proteins)
o Changes yield (non-protein nitrogen is also recognized by Dumas)
• Ash: only small amount but can shield charges so has influence of protein properties. Less
charge = more aggregation so lower solubility
• Starch: present in large granules. You can centrifuge them out if proteins are solubilized.
Anti-nutritional factors
• Protease inhibitors from plants inhibit the digestive enzymes (proteases:
trypsin/chymotrypsin) in the gastro-intestinal tract. Proteins cannot be digested and taken up
by the body..
o You don’t get the nutrients so you are going to eat more but then you also eat more
protease inhibitors.
o Potato contains a lot of it → take into account when making an isolate because not
healthy.
o Indirect effect: more proteases are formed in the body → loss of endogenous protein
o Kunitz trypsin inhibitor and Bowman-Birk trypsin inhibitor. Last one is a small protein
with a lot of disulphide bridges = very heat stable protein. Difficult to inactive.. And
when you heat them, you also unfold your proteins of interest = less soluble.
• Lectins interact with epithelial cells → loss of nutrient uptake. You can inactivate them by
heating but then you also affect the good proteins.
• Allergenic proteins (for instance gluten or milk proteins)
• Tannins decrease protein digestibility by complexation with proteins → less soluble and less
susceptible to enzymatic hydrolysis
Composition of isolated proteins
Osborne fractionation
• Several types of proteins can be in one isolate
• Wash with water → albumins are extracted
• Wash with NaCl → globulins are extracted
• Wash with ethanol → prolamins are extracted
• Not extracted: glutelins (together with prolamins the gluten fraction)
• For instance the albumins are a group of proteins. They only have the same solubility!
Protein heterogeneity
• The albumins is still a group of proteins → fractionate based on charge or size
• Ion-exchange chromatography (IEC): separation based on charge
o Charged molecules bind to the column
o You can remove the non-charged molecules. Elute charged compounds by changing
conditions.
• Size-exclusion chromatography (SEC): separation based on size
o Calibration set with standard proteins of which the M is known. Plot the Kav (by
formula) against log M.
▪ Kav = (elution volume – V0) / (Vc – V0)
o You measure the elution time. V0 = void volume, all compounds that are larger than
the void volume don’t go into the pores and thus will elute at V0.
o Vc = column volume. This is the included volume.
o You can calculate the Kav for your samples and with the calibration curve you can
determine the M
• SDS page (type of electrophoresis) → separation based on size
o SDS breaks non-covalent interactions in the protein. The proteins goes from folded to
denatured state.
o Reducing conditions (you use DTT) → disulphide bonds are broken. For instance the
acidic and basic subunit in soy glycinin is broken so 2 bands on the gel.
o Not very detailed. When a protein contains an extra glucose unit, you cannot see that
• Iso-electric focussing → separation based on iso-electric point
o Voltage is applied on the gel. There is a + and – pole. The charge of SDS makes the
proteins go from – to +. You can also apply a pH gradient, the proteins will move over
the gel until they do not have charge anymore.
o If the proteins are – charged, they go to the + pole. At their IEP, they will stop moving
• Example: potato protein patatin
o Separate patatin protein by ion-exchange chromatography from the potato fruit juice
o Separate the fraction from IEC by SEC → only take the fraction with the size of patatin
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