CONSIDERATIONS IN PROTEIN INGREDIENT USE: THE IMPACT OF PROCESSING AND MOLECULAR INTERACTIONS Baraem (Pam) Ismail Associate Professor Department of Food Science and Nutrition University of Minnesota May 6 th, 2015 + +
PROTEIN INGREDIENTS The global demand for protein ingredients, including plant and animal sources, is expected to reach 4.6 million tons by 2020 The global protein ingredient market revenues are expected to reach 28.9 billion dollars by 2020, growing at rate of 6.5% from 2014 to 2020 Since 2005, the global yield of PROTEIN HYDROLYSATES increased 6.4% annually http://nutritionsuccess.org/blog/2013/04/homemade-protein-boost-powder/
PROCESSING AND SHELF-LIFE CHALLENGES Moisture-induced protein/peptide aggregation affects $4 billion baby formula (dry food) market $3 billion nutrition/energy bar (intermediate-moisture food) market $20 billion functional beverages market www.cornucopia.org www.runnersworld.com www.getfilings.com
PROTEIN ISOLATE (PI): PROCESSING AND SHELF-LIFE CHALLENGES Moisture-induced protein/peptide aggregation causes Caking in dry food Hardening in intermediate-moisture food (bar hardening) Turbidity and precipitation in high protein beverages http://www.powderbulksolids.com Week 0 Week 6 www.thenakedkitchen.com
PROTEIN HYDROLYSATES (PH): WHY? Enhanced digestibility Reduced allergenicity Improved functionality Enhanced bioactivity (bioactive peptides: antihypertensive, anticancerous, antidepressant, promote satiety, etc.) http://www.workswithwater.co.uk/pages/help-blood-pressure-clinical-studies.aspx
PROTEIN HYDROLYSATES (PH): CAN AGGREGATE Prior to enzymatic hydrolysis After enzymatic hydrolysis Aggregation upon heating http://biology-pictures.blogspot.com/2011/11/denaturation-of-proteins.html
PROTEIN HYDROLYSATES (PH): PROCESSING CHALLENGES Moisture and heat-induced protein/peptide aggregation Irreversible interactions with other peptides or carbohydrate in the system, via disulfide linkage or Maillard reaction Loss of bioactivity of peptides Heat
PROCESSING AND SHELF-LIFE CHALLENGES Thermal (Denaturation) Protein Aggregation
PROCESSING AND SHELF-LIFE CHALLENGES Moisture-induced protein/peptide aggregation lead to Reduced shelf-life Economical loss Thus the need to Characterize the moisture-induced protein/peptide aggregation in simple and complex matrices Investigate ways to limit aggregation, one promising approach: Maillard-induced glycation
PROCESSING AND SHELF-LIFE CHALLENGES Moisture-induced protein/peptide aggregation Contributing factors in dry and semi-moist food Thermal treatment o Denaturation Humidity and heat during transport and storage o Aggregation ---caking, hardening Hydrophobic interactions Disulfide linkages Maillard-induced polymerization (presence of reducing sugars)
Effect of moisture content on the temperatures of protein denaturation (T d ) and glass transition (T g ), and the denaturation enthalpy (ΔH d ) of HEW and ovalbumin in DEW. Rao and Labuza, Food Chemistry, 2012
Zhou et al., Journal of Food Science, 2013
Zhou et al., Journal of Food Science, 2013
% Remaining Free Amino Groups 105 100 95 90 85 80 75 70 65 60 a ab ab abc abc bc c c c 0 10 20 30 40 50 60 70 80 90 Days of Storage at 45ºC and 0.79 a w SPI c % Remaining Free Amino Groups 105 100 95 90 85 80 75 70 65 60 a ab abc bc c bc c bc 0 20 40 60 80 100 bc SPH Days of Storage at 45ºC and 0.79 aw bc Gillman, 2014
% FI/g protein 1000 900 800 700 600 500 400 300 200 100 0 0.79 a w 0.71 a w 0.59 a w 0.05 a w 0 20 40 60 80 Days of Storage of SPH at 45ºC Gillman, 2014
% Solubility of SPI (A), SPH (B), and 50/50 (C) at 45ºC after 77 days of storage at various water activities as compared to the solubility prior to storage (original). Gillman, 2014
% Solubility at 28, 56, and 77 days at selected water activities for SPI (A), SPH (B), and 50/50 (C) at 45ºC as compared to % solubility prior to storage (original). Gillman, 2014
Effects of Accelerated Storage on the ACE Inhibitory Activity of SPH 0.4 0.35 IC 50 (mg/ml) 0.3 0.25 0.2 0.15 0.1 0.05 Accelerated storage at 45ºC up to 3 months at a w 0.05-0.79 0 Original 0.06 0.05 0.59 0.79 a w Gillman, 2014
PROCESSING AND SHELF-LIFE CHALLENGES Moisture-induced protein/peptide aggregation Contributing factors in beverages Thermal treatment o Denaturation ph Nature of the protein Ionic strength Protein concentration
PROCESSING AND SHELF-LIFE CHALLENGES Whey Protein Heat Cys Disulfide linkage β-lactoglubulin Trp Disulfide linkage His Disulfide linkage Denatured β-lactoglubulin Disulfide linkage Disulfide linkage His Trp Trp His Disulfide linkage Hydrophobic interaction and di-sulfide interchange Disulfide linkage Aggregation
WHEY PROTEIN IN BEVERAGES Neutral ph Whey Beverages ph 6.5-7.5 Aceptic or retort processing, or pasteurized and refrigerated Low clarity, high turbidity Stabilizers or emulsifiers < 3% protein Gelling or precipitation Acidified Whey Beverages ph 2.8 3.4 Mild thermal pasteurization Shelf-stable at room temp High clarity, low turbidity Stabilizers usually not needed below ph 3.5 < 4% protein o According to FDA (21 CFR 101.54 B), beverages of protein concentration at least 4.2 % (w/v) can be claimed as high protein beverages
Wang and Ismail, Intl. Dairy J., 2012
Surface hydrophobicity index E F Heating time at 75ºC At low ph (<3.4) Wang and Ismail, Intl. Dairy J., 2012 E F At higher ph (4.5-5.5)
Maillard initial reaction Glycated protein + +
Initial stage: products colorless Reaction A: Sugar-amine condensation Reaction B: Amadori rearrangement Intermediate stage: products colorless or yellow Final stage: products highly colored Mutagenic compounds formation Off-flavors development Excessive browning
Glycation Must be carried out under very controlled conditions to avoid the later stage color and flavor changes Preferably under dry heating conditions (temperatures less than denaturation onset of proteins) a w less than optimum (a w < 0.5) Use of polysaccharides rather than sugars, to limit reaction rate for better control Partial glycation to minimize nutritional loss while imparting significant physiochemical changes + +
Partial glycation will result in increased net negative charge of the proteins increased surface hydrophilicity reduced denaturation rate reduced disulfide interchange Increased steric hindrance due to bulky polysaccharides thus enhanced stability and reduced protein/peptide interactions + + + +
Formation of Amadori compounds Only 1.4% loss in free amine groups and 0.6% loss in lysine Browning Wang and Ismail, Intl. Dairy J., 2012
Wang and Ismail, Intl. Dairy J., 2012
Maillard-induced glycation delayed onset of denaturation Heat flow endo up (mw) Differential scanning calorimetry of partially glycated whey protein (PGWP) and whey protein isolate (WPI) after purification by hydrophobic interaction chromatography. Solid line: PGWP, dotted line: WPI. Td: denaturation temperature and ΔH: enthalpy. Temperature ( C) Wang and Ismail, Intl. Dairy J., 2012
Maillard-induced glycation delayed protein unfolding Surface hydrophobicity index 3500 3000 2500 2000 1500 1000 500 Surface hydrophobicity index of 5% whey protein isolate (WPI) and partially glycated whey protein (PGWP) solutions heated at 75 C for 10-60 min. Error bar represents standard deviations; n=3. WPI: ; PGWP:. 0 0 10 20 30 40 50 60 Heating time (min) Wang and Ismail, Intl. Dairy J., 2012
Polymerization Maillard-induced glycation limited polymerization β-lg α-la Lane 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 SDS-PAGE gels with Coomassie blue staining; lane 1: molecule weight standard, lane 2: nonheated whey protein isolate (WPI), lanes 3-8: WPI heated at 75 C for 10, 20, 30, 40, 50, and 60 min, lane 9: non-heated partially glycated whey protein (PGWP), lanes 10-15: PGWP heated at 75 C for 10, 20, 30, 40, 50, and 60 min. Lane 1 2 3 SDS-PAGE gel with glycoprotein staining; lane 1: molecular weight standard, lane 2: non-heated PGWP, and lane 3: nonheated WPI. Wang and Ismail, Intl. Dairy J., 2012
Maillard-induced glycation imparted structural rigidity Raman spectra of WPI and PGWP at ph 3.4 (a), 4.5 (b), 5.5 (c) and 7(d) before (nh) and after heating (h) at 75 C for 30 min. Wang, He, Labuza, and Ismail., Food Chem., 2013
Effect of glycation site on structure rigidity MALDI-TOF MS peptide spectra of dextranase/trypsin digested PGWP 1727 5359 Wang and Ismail, Intl. Dairy J., 2012
Effect of glycation site on structure rigidity MKCLLLALAL TCGAQALIVT QTMKGLDIQK VAGTWYSLAM AASDISLLDA QSAPLRVYVE ELKPTPEGDL EILLQKWENG ECAQKKIIAE KTKIPAVFK IDALNENKVLV LDTDYKKYLL FCMENSAEPE QSLACQCLVR TPEVDDEALE KFDKALKALP MHIRLSFNPT QLEEQCHI E F At low ph (<3.4)
Effect of glycation site on structure rigidity F E At higher ph (4.5-5.5)
Effect of glycation site on structure rigidity F E At higher ph (4.5-5.5)
Effect of glycation site on structure rigidity E F At higher ph (4.5-5.5)
Understand PI and PH characteristics Understand the effect of formulations (e.g. presence of reducing sugars, ph, protein concentration) Identify ideal storage conditions Utilize modified protein ingredients with enhanced functionality
Initiati ve
+ + Baraem (Pam) Ismail Associate Professor Department of Food Science and Nutrition University of Minnesota May 6 th, 2015