Protein Flashcards
Purpose of protein analysis
Labelling - legal requirement to state nutritional content if a health claim is made - however commonplace to state nutrition currently - hence need to know total protein content
Product development - to understand the composition of competitors products - ie meat in pie
Quality control - species differentiation - post clean pcr tests no check for contamination
How to calculate total protein
Kjeldahl method - determine total protein from nitrogen content then mutiply by 6.25 - the method is to digest the sample with conc H2SO4 - digest the made alkali with conc NaOH and steam distil - then titrate with 0.1 HCl - use a selenium catalyst to reduce the time from 16h to 1
This is a reference method and reliable - however it also measures non-protein nitrogen and it is not very sensitive so gram quantities are required
Reasons for detection:
Species determination - possible to detect horsemeat by isoelectric focusing which gives different bands for horse compared with beef - however DNA testing is more sensitive and specific
Wheat proteins - aim to identify wheat species which corralate with good baking properties
Flour properties - gluten forming proteins = 85% - need to detect for coeliac disease - gluten
Direct spectrophotometric methods
UV absorption at 280nm for proteins in aqueous solution - due to the phenolic groups of tyrosine and the indole group of tryptophan - simple method and only a small sample is required - however protein to protein variation due to differences in amino acid composition - distortion due to light scattering from turbid samples
Indirect colourimetric methods
Biuret - gives a purple colour (540-560nm) when peptide linkages react with cupric ions at alkaline pH - advantages = little dependence on amino acid composition and simple, disadvantages = lower sensitivity than the lowry method
Lowry - similar - produces a blue compound at 750nm - simple and can be automated for milk - very sensitive - very widely used - disadvantages - other compounds can interfer eg. lipids, sucrose - variation between proteins due to tyrosine and typtophan - pH and temp dependent
Dye binding assays
negatively charged dye with positively charged proteins (lysine and arginine) under lo pH –> insoluble dye protein complex - dye = Coomassie Brilliant BLue - rapid and sensitive - less interferrence
Amino acids - chromatographic method
step 1 - hydrolysis - then step 2 is seperation and detection of amino acids - use an ion exchange column with a ninhydrin dye detection method - elution by pH gradient - use amino acid standards to quantify
HPLC - amino acids
fast and sensitive - hydrolysis, precolumn derivatisation with PITC o form PTC - analysis by reverse phase HPLC - no need for dedicated instrument just use any HPLC - increased sensitivity just need 1mg sample - decreased analysis time 30 mins not 1 hour
Functional properties
viscometers - rheometers - texture analysers, foaming, emulsification
Characteristics relating to geltaion, foaming and emulsification
Disulphide /sulphydryl groups
Specific proteins - enzyme assays
Phospholipase activity is an indication of milk pasteurisation - peroxidase is an indicator of blanching efficiency
ELISA
Enzyme linked immunosorbent assay - colour compound formed
Electrophoresis
powerful technique which can be used to detect different protein or DNA in complex food products - small quantities of samples can be used- can be used for GMO - however lengthy procedure - use of toxic acrylamide - not suitable for factory floor QC
Detection of soya protein in meat
Polyacrylamide gel electrophoresis - meat homogenised, freeze-dried, defatted and ground into powder - protein extracted and used on the gel - advantage - detection of soya protein in raw ad heated meat products at 0.2-0.6% - difficult to detect soya in heated meat products using immunoassays as the antibody may not recogise he denatured antigen
