Lipid co oxidation of proteins Reaction pathways products and

Document Sample
Lipid co oxidation of proteins Reaction pathways products and Powered By Docstoc
					 Lipid co-oxidation of proteins:
Reaction pathways, products, and
    consequences in foods

               Faculty PI: K.M. Schaich
               Students: Y. Dong
                          W.I. Wan Zunair
  Lipid oxidation is not always what it

  Lipids oxidize and produce free radicals,
  hydroperoxides, aldehydes, and epoxides that are
  measured to follow the process and determine extent
  of reaction.
• Each of these intermediates and products also react
  with proteins to broadcast damage while also
  eliminating some footprints of lipid oxidation.
  Lipid oxidation thus can appear to be low when it is
  actually quite rapid and evidence of oxidation is
  clearly visible in browning and texture changes.
Hypothesis and Position Statement

 All intermediates and products of lipid oxidation react with and
 damage proteins, but they do so differentially, each with specific
 By intercepting lipid radical chain reactions and removing reactive
 lipid products, protein co-oxidations may make lipid oxidation
 seem to slow while transferring oxidation potential to different
 molecular sites and change observable system properties.
 Some consequences of protein oxidation have been misattributed
 to lipid oxidation, particularly flavors.
 Thus, co-oxidations must be accounted for when analyzing extent
 of lipid oxidation in any food system
 Limiting co-oxidation of proteins (and other biomolecules) must be
 included in all strategies for stabilizing food quality.
Project Goals

 Determine the absolute and sequential modifications
 in protein properties and chemistry induced by four
 classes of lipid oxidation products -- free radicals,
 hydroperoxides, epoxides, and carbonyl (mostly
 aldehyde) secondary products
 Differentiate the types of reactions and patterns of
 damage induced by each oxidant
 Show where such reactions occur in some specific
 food applications
Experimental Approach
1. Model systems (lyophilized and fluid emulsions
  oxidizing Me Linoleate         EPR – free radicals
                                 13C NMR – struct and funct grp
                    + proteins   PAGE – xlkg and fragmentn.
    ML aldehydes
    ML epoxides                  Ab rxn (protein oxidation)
                                 Protease digestion
                                 LC-MS -- ID modified amino
2. Simultaneously monitor changes in oxidizing lipids to determine
    how the footprint of lipid oxidation changes with protein co-
3. Coordinate model system studies with analyses of protein and
    lipid oxidation in representative foods (e.g. dairy, meats,
    grains/bread products).
Studies with tortilla chips

      Baked             Fried

  Thermal damage   Thermal + lipid oxidation
Studies with tortilla chips

 Extraction    a. Non-reducing
               1.5% SDS, 0.0125 M Borate, pH = 10
               b. Reducing
               Above + 2% 2-mercaptoethanol
               Protein solubility (Bradford)
 analyses      SH/S-S

               Protein fragmentation and crosslinking
  PAGE         12.5% and 15% resolving gel, 4% stacking gel

  Ab rxns     Identification of proteins with C=O products

Lipids reduce protein solubility

S-S doesn’t alter solubility in baked chips
                                   Extractable Protein




                             baked,    baked, non-     fried,   fried, non-
                            reducing    reducing     reducing    reducing

Solubility reduced by both S-S and other mechanisms in fried chips.
 Lipids alter dye-binding amino acids
               Reducing    Non-Reducing
               Fried Baked   Fried Baked
              −L +L −L +L St −L +L −L +L     R vs NR suggest
                                             more S-S
                                             crosslinking than
                                             was shown in
Major loss                                   Bradford assay
of dye
binding to
proteins in
fried chips

                                           Proteins extracted w/
                                           − L: lipid removed
                                           + L: lipid present
CBB binding sites (His, Arg, Lys, Trp)
are major targets for lipid radical attack

Form stable
radicals with
oxidizing lipids
                   Georgiou et al, Anal. Bioanal. Chem. 2008
                        Silver staining of proteins

 Heat alone ↑ S-S crosslinking,
 Lipids crosslink by other mechanisms
                     Reducing         Non-Reducing
Polymeric            Fried Baked      Fried Baked
fractions,            −L +L −L +L    −L +L −L +L       Lost HIGH mw
probably with                                          fractions
bound lipid                                            potentially
                                                       bound to lipids
eliminates S-S
                                                       High, Mod and
crosslinking, but
                                                       low mol wt
glutelin fractions
still missing in
fried chips

     Antibodies reveal extensive oxidation
     (R-CHO) in both baked and fried chips
                Reducing        Non-Reducing

            C     F   C    B     St    F   C   B   C
                                                       Heat alone generates
Glutelins                                              aldehydes in all
                                                       protein fractions.

                                                       Other mechanisms
                                                       must be active in loss
      A                                                of dye binding and
      B                        25 kd                   specific fractions.
       C                       15 kd
       D                                                C: control
                                                        F: Fried
                                                        B: Baked
Further characterization of protein

 • EPR for free radicals
 • PAGE variations for further differentiation – 2D, selective
 staining for lipid-protein and glycoprotein adducts
 • Analysis of specific lipid oxidation products
 • Dye binding to detect lipid adducts to proteins
 • Fluorescence analysis for Schiff bases (RCHO and
     epoxide adducts)
 • Immobilized enzyme hydrolysis to amino acids, LC-MS
     analysis of modified amino acids
 • Storage studies – following protein changes over time, 40 °C
 • Coordinate with model system studies
                       Peanut butter

 Studies of Products in Controlled
 Release Packaging with Antioxidants

               Quercetin + tocopherols
               EVOH                EVA           EVA Control

40 °C

60 °C

        Are these changes from lipid oxidation -- OR --
                          from protein oxidation?
                        Processed Cheese Spread

 Studies of Products in Controlled
 Release Packaging with Antioxidants

              No N2               N2              N2 + tocopherols

40 °C

        Low viscosity       Increased         Normal consistency
        Gritty                cohesiveness     and flavor
        Off-flavors         Less gritty       Smooth

 Are these changes from lipid oxidation -- OR --
                   from protein or starch oxidation?
Lipid co-oxidations →
extensive degradation of food quality

• Texture -- both ↑and ↓ viscosity, so both
               crosslinking and fragmentation
              both ↑and ↓ cohesion
• Color changes – browning and pigment bleaching
• Flavor changes – definitely not all classical lipox
• Loss of essential amino acids
 These damage processes must be elucidated to develop
 more effective stabilization strategies in formulation,
 processing, and packaging.
Research plans now reverse initial

1. Study protein degradation in these real foods,
    identify key protein modifications responsible for
    quality changes over time.
2. Supplement with studies of maize, milk, and peanut proteins
   in model systems with
      actively oxidizing lipids (free radicals)
      isolated LOOH
      lipid aldehydes
      lipid epoxides
  to determine damage kinetics and products associated with
   different lipid species.
3. Use model system results to elucidate co-oxidation pathways
   active in foods.
Thank you for your attention…
Any questions?

Shared By: