Identification of bitter peptides in aged Cheddar cheese by crossflow filtration-based Fractionation, Peptidomics, statistical screening and sensory analysis.

Despite bitterness being a common flavor attribute of aged cheese linked to casein-derived peptides, excessive bitterness is a sensory flaw that can lead to consumer rejection and economic loss for creameries. Our research employs a unique approach to identify bitter peptides in cheese samples using crossflow filtration-based fractionation, mass spectrometry-based peptidomics, statistics and sensory analysis. Applying peptidomics and statistical screening tools, rather than traditional chemical separation techniques, to identify bitter peptides allows for screening the whole peptide profile. Five peptides-YPFPGP (ß-casein [60-65]), YPFPGPIPN (ßA2-casein [60-68]), LSQSKVLPVPQKAVPYPQRDMPIQA (ß-casein [165-189]), YPFPGPIHNS (ßA1-casein [60-69]) and its serine phosphorylated version YPFPGPIHN[S] (ßA1-casein [60-69])- demonstrated high levels of bitterness with mean bitterness intensity values above 7 on a 15-point scale. In the future, this data can be combined with the microbial and protease profile of the Cheddar samples to help understand how these factors contribute to bitter taste development.

A comprehensive database of cheese-derived bitter peptides and correlation to their physical properties.

Bitterness is a common flavor attribute of aged cheese associated with the peptide fraction, but excessive levels are a defect leading to consumer rejection. Bitterness in cheese has been primarily associated with peptides that arise from the breakdown of casein. The last review of bitter peptides was published in 1992. This updated review compiled information about the bitter peptides published up to 2022. Our comprehensive search of the literature compiled 226 peptides associated with bitterness and cheese protein origins into a database (Supplemental Materials). The influences of a peptide's physical properties, such as molecular weight, average hydrophobicity, peptide length, number of prolines and the presence of hydrophobic amino acids in the peptide's terminus, were assessed for correlation with bitterness threshold values this assessment found that, among variables considered, higher molecular weight had the strongest correlation with higher bitterness among known peptides. Heatmaps of bitter peptides and their bitterness threshold values highlight ß-casein as the primary source of known bitter peptides in cheese. This comprehensive database of cheese protein-derived bitter peptides and this discovery of the correlation of a peptide's physical properties to bitterness will aid future researchers in the identification and discovery of contributors to cheese bitterness.

Thermal stability of reconstituted milk protein concentrates: Effect of partial calcium depletion during membrane filtration.

Milk protein concentrate (MPC) powders are increasingly utilized in manufacturing of protein fortified beverages. Thermal stability of the protein dispersions is of significant importance in such applications. It is known that a decrease in pH can induce partial dissociation of casein micelles and modify the natural equilibrium of calcium and phosphate between the micelles and the serum phase. The presence of soluble casein may improve the rehydration properties of MPC powders, and may impact their thermal stability. The objective of this work was to investigate the effects of partial acidification of milk prior to ultrafiltration on the heat stability of reconstituted MPC dispersions. Milk protein concentrate powders were prepared from skim milk acidified to pH6.0 by addition of glucono-δ-lactone, and then concentrated using ultrafiltration (UF) and diafiltration (DF). The heat stability of the reconstituted MPC dispersions was studied, by determining heat coagulation time, particle size, turbidity, viscosity, soluble and colloidal calcium and phosphate, and non-sedimentable casein both before and after heating at 120°C. Reconstituted MPC powders made with partially acidified skim milk contained lower soluble calcium and phosphate and exhibited very poor thermal stability compared to MPC powders made with skim milk at its natural pH. The thermal stability of the acidified MPC dispersions was not only recovered by restoration of pH and the serum composition through dialysis against skim milk, but it was improved compared to control MPC dispersions. All dialyzed samples had comparable pH, protein content and calcium and phosphate concentration, but the structure of the casein micelles was altered, causing differences in the type of soluble aggregates. It was concluded that the integrity of the casein micelles and the amount of dissociated, non-sedimentable caseins play a major role in determining the thermal stability of the MPC dispersions.

Partial calcium depletion during membrane filtration affects gelation of reconstituted milk protein concentrates.

Milk protein concentrate powders (MPC) with improved rehydration properties are often manufactured using processing steps, such as acidification and high-pressure processing, and with addition of other ingredients, such as sodium chloride, during their production. These steps are known to increase the amount of serum caseins or modify the mineral equilibrium, hence improving solubility of the retentates. The processing functionality of the micelles may be affected. The aim of this study was to investigate the effects of partial acidification by adding glucono-δ-lactone (GDL) to skim milk during membrane filtration on the structural changes of the casein micelles by observing their chymosin-induced coagulation behavior, as such coagulation is affected by both the supramolecular structure of the caseins and calcium equilibrium. Milk protein concentrates were prepared by preacidification with GDL to pH 6 using ultrafiltration (UF) and diafiltration (DF) followed by spray-drying. Reconstituted UF and DF samples (3.2% protein) treated with GDL showed significantly increased amounts of soluble calcium and nonsedimentable caseins compared with their respective controls, as measured by ion chromatography and sodium dodecyl sulfate-PAGE electrophoresis, respectively. The primary phase of chymosin-induced gelation was not significantly different between treatments as measured by the amount of caseino-macropeptide released. The rheological properties of the reconstituted MPC powders were determined immediately after addition of chymosin, both before and after dialysis against skim milk, to ensure similar serum composition for all samples. Reconstituted samples before dialysis showed no gelation (defined as tan δ=1), and after re-equilibration only control UF and DF samples showed gelation. The gelation properties of reconstituted MPC powders were negatively affected by the presence of soluble casein, and positively affected by the amount of both soluble and insoluble calcium present after reconstitution. This work, testing the chymosin-induced gelation behavior of various reconstituted MPC samples, clearly demonstrated that a decrease in pH to 6.0 during membrane filtration affects the integrity of the casein micelles supramolecular structure with important consequences to their processing functionality.

Changes in the physical properties, solubility, and heat stability of milk protein concentrates prepared from partially acidified milk.

A limiting factor in using milk protein concentrates (MPC) as a high-quality protein source for different food applications is their poor reconstitutability. Solubilization of colloidal calcium phosphate (CCP) from casein micelles during membrane filtration (e.g., through acidification) may affect the structural organization of these protein particles and consequently the rehydration and functional properties of the resulting MPC powder. The main objective of this study was to investigate the effects of acidification of milk by glucono-δ-lactone (GDL) before ultrafiltration (UF) on the composition, physical properties, solubility, and thermal stability (after reconstitution) of MPC powders. The MPC samples were manufactured in duplicate, either by UF (65% protein, MPC65) or by UF followed by diafiltration (80% protein, MPC80), using pasteurized skim milk, at either the native milk pH (~pH 6.6) or at pH 6.0 after addition of GDL, followed by spray drying. Samples of different treatments were reconstituted at 5% (wt/wt) protein to compare their solubility and thermal stability. Powders were tested in duplicate for basic composition, calcium content, reconstitutability, particle size, particle density, and microstructure. Acidification of milk did not have any significant effect on the proximate composition, particle size, particle density, or surface morphology of the MPC powders; however, the total calcium content of MPC80 decreased significantly with acidification (from 1.84 ± 0.03 to 1.59 ± 0.03 g/100 g of powder). Calcium-depleted MPC80 powders were also more soluble than the control powders. Diafiltered dispersions were significantly less heat stable (at 120°C) than UF samples when dissolved at 5% solids. The present work contributes to a better understanding of the differences in MPC commonly observed during processing.

The impact of antioxidant addition on flavor of cheddar and mozzarella whey and cheddar whey protein concentrate.

Lipid oxidation products are primary contributors to whey ingredient off-flavors. The objectives of this study were to evaluate the impact of antioxidant addition in prevention of flavor deterioration of fluid whey and spray-dried whey protein. Cheddar and Mozzarella cheeses were manufactured in triplicate. Fresh whey was collected, pasteurized, and defatted by centrifugal separation. Subsequently, 0.05% (w/w) ascorbic acid or 0.5% (w/w) whey protein hydrolysate (WPH) were added to the pasteurized whey. A control with no antioxidant addition was also evaluated. Wheys were stored at 3 degrees C and evaluated after 0, 2, 4, 6, and 8 d. In a subsequent experiment, selected treatments were then incorporated into liquid Cheddar whey and processed into whey protein concentrate (WPC). Whey and WPC flavors were documented by descriptive sensory analysis, and volatile components were evaluated by solid phase micro-extraction with gas chromatography mass spectrometry. Cardboard flavors increased in fluid wheys with storage. Liquid wheys with ascorbic acid or WPH had lower cardboard flavor across storage compared to control whey. Lipid oxidation products, hexanal, heptanal, octanal, and nonanal increased in liquid whey during storage, but liquid whey with added ascorbic acid or WPH had lower concentrations of these products compared to untreated controls. Mozzarella liquid whey had lower flavor intensities than Cheddar whey initially and after refrigerated storage. WPC with added ascorbic acid or WPH had lower cardboard flavor and lower concentrations of pentanal, heptanal, and nonanal compared to control WPC. These results suggest that addition of an antioxidant to liquid whey prior to further processing may be beneficial to flavor of spray-dried whey protein. Practical Application: Lipid oxidation products are primary contributors to whey ingredient off-flavors. Flavor plays a critical and limiting role in widespread use of dried whey ingredients, and enhanced understanding of flavor and flavor formation as well as methods to control or minimize flavor formation during processing are industrially relevant. The results from this study suggest that addition of an antioxidant to liquid whey prior to further processing may be beneficial to minimize flavor of spray-dried whey protein.