Chemical fractionation of caseins by differential precipitation: influence of pH,calcium addition,protein concentration and temperature on the depletion in α- and β-caseins

The impact of pH, calcium and casein concentrations, and temperature on the efficiency of the differential precipitation of caseins by calcium affinity was investigated, using native phosphocaseinate as starting material. We adapted one of the most recent methods for the separation of caseins that is based on the addition of calcium at alkaline pH. Increasing the pH to 11 disturbed the micellar structure by enhancing electrostatic repulsion of caseins, leading to a marked viscosity increase and a significant particle size decrease, indicating casein micelle disruption. This pH-driven increase in negative charges enhanced the affinity of individual caseins for calcium, proportionally to the number of phosphate groups carried by each casein. The addition of calcium first led to a progressive increase in the proportion of precipitated caseins, before reaching a plateau. Hence, an optimal calcium/casein molar ratio of about 40 was evidenced to optimise casein precipitation (and fractionation), leading to significant depletion of α-casein (around 80%) and, in a lesser extent, β-casein (around 65%) and κ-casein (around 55%). This method led to relative proportions of caseins significantly differing from the starting material: 31% α-casein, 45% β-casein and 24% κ-casein.     

Effect of α-lactalbumin and β-lactoglobulin on the oxidative stability of 10% fish oil-in-water emulsions depends on pH

The objective of this study was to investigate the influence of pH on lipid oxidation and protein partitioning in 10% fish oil-in-water emulsions prepared with different whey protein isolates with varying ratios of α-lactalbumin and β-lactoglobulin. Results showed that an increase in pH increased lipid oxidation irrespective of the emulsifier used. At pH 4, lipid oxidation was not affected by the type of whey protein emulsifier used or the partitioning of proteins between the interface and the water phase. However, at pH 7 the emulsifier with the highest concentration of β-lactoglobulin protected more effectively against oxidation during emulsion production, whereas the emulsions with the highest concentration of α-lactalbumin were most stable to oxidation during storage. These differences were explained by differences in the pressure and adsorption induced unfolding of the individual protein components.     

Genetic variation in the αS1-casein of Chinese yak (Bos grunniens)

The aim of this study was to investigate diversity of the αS1-casein gene in Maiwa yak. A polymorphism of αS1-casein gene has been identified in Chinese Maiwa yak by PCR single-strand conformation polymorphism protocol. Comparing the X59856 sequence, some non-coding and coding DNA variants of yak CSN1S1 gene were detected in this present study, including KJ397913 (promoter region, 9969A/G, 9984 A/G, 10153 C/T, 10175 A/C, 10261 G/C), KJ397914 (promoter region 10261 G/C), KJ397896 (intron III, 14862 T/C), KJ397897 (intron III, 14862 T/C, and deleted 14944-14946 CTT), KJ397899 (intron IV, 15297 A/C), KJ397901 (intron VIII, 17503 A/C, 17666 A/C), KJ397902 (exon IX, 18901C/A, intron IX, 18970 T/G, 19088 add A, 19120 add A, 19202 add T, 19296 G/T), KJ397903 (intron IX, 18970 T/G, 19088 add A, 19120 add A, 19202 add T, 19296 G/T), KJ397904 (intron IX, 19537 T/G, 19539T/G, 19599G/A, 19638 C/T), KJ397905 (intron XIV, 23402 T/G, 23417 C/T, 23480 T/C, 23511 G/A), and KJ397907 (intron XVI, 25976 G/T; exon XVII, 26181A/G). The polymorphism of αS1-casein gene may be helpful for future studies on genetic variation within and between yak populations or on associated traits.     

Effect of storage conditions on the solubility and viscosity of β-glucan extracted from bread under in vitro conditions

The viscosity and solubility of β-glucan in muffins have been shown to be reduced by certain storage conditions, though the effect of storage on bread fortified with barley β-glucan concentrate has not been investigated. Therefore, this study investigated the effect of storage temperature and time (23 °C for 1, 4, and 7 d, 4 °C for 4, 7, and 14 d, and -20 °C for 1, 2, 4, and 8 wk) on the solubility and viscosity of β-glucan upon incorporation into bread at levels corresponding to 0 or 1.5 g β-glucan/serving, with or without vital gluten addition. The firmness and moisture content of bread following each storage treatment were also evaluated. The highest moisture and lowest firmness values were found in fresh bread, though these parameters were still maintained at appreciable levels upon room temperature storage of the 1.5 g β-glucan/serving bread with added gluten and at either room temperature or frozen storage for the 1.5 g β-glucan/serving bread for 4 d. If it is desirable to store bread for 7 d or more, frozen storage should be utilized in order to best maintain bread moisture and firmness levels. It is recommended that β-glucan-fortified bread be consumed fresh for greatest β-glucan solubility and viscosity, though β-glucan solubility of approximately 40% is still achievable upon frozen storage of the bread for up to 2 wk. It is still unclear, however, as to what extent of reductions in the solubility and viscosity of β-glucan would lower its physiological effectiveness. PRACTICAL APPLICATION: Previous research has demonstrated that solubility and thus viscosity of β-glucan, which is an important property associated with its health benefits can be impacted by different storage conditions applied to some bakery products, like muffins. This study demonstrates the extent of changes in the solubility and viscosity of β-glucan incorporated into bread. Therefore, storage time and temperature should be optimized to minimize changes in β-glucan for maintaining its efficacy for its health benefits.     

Effect of high-pressure treatment on denaturation of bovine β-lactoglobulin and α-lactalbumin

The effect of high-pressure treatment on denaturation of β-lactoglobulin and α-lactalbumin in skimmed milk, whey, and phosphate buffer was studied over a pressure range of 450–700 MPa at 20 °C. The degree of protein denaturation was measured by the loss of reactivity with their specific antibodies using radial immunodiffusion. The denaturation of β-lactoglobulin increased with the increase of pressure and holding time. Denaturation rate constants of β-lactoglobulin were higher when the protein was treated in skimmed milk than in whey, and in both media higher than in buffer, indicating that the stability of the protein depends on the treatment media. α-Lactalbumin is much more baroresistant than β-lactoglobulin as a low level of denaturation was obtained at all treatments assayed. Denaturation of β-lactoglobulin in the three media was found to follow a reaction order of n = 1.5. A linear relationship was obtained between the logarithm of the rate constants and pressure over the pressure range studied. Activation volumes obtained for the protein treated in milk, whey, and buffer were −17.7 ± 0.5, −24.8 ± 0.4, and −18.9 ± 0.8 mL/mol, respectively, which indicate that under pressure, reactions of volume decrease of β-lactoglobulin are favoured. Kinetic parameters obtained in this work allow calculating the pressure-induced denaturation of β-lactoglobulin on the basis of pressure and holding times applied.     

Effect of genotype and cultivation location on β-sitosterol and α-, β-, γ-, and δ-tocopherols in sorghum

This study aims to examine variations in β-sitosterol and α-, β-, γ-, and δ-tocopherol composition and content in sorghum grains with different genotypes and cultivation areas (Wonju and Miryang in Korea). β-Sitosterol content was found to be higher in Wonju sorghum; however, total tocopherol content was found to be higher in Miryang sorghum. Moreover, the β-sitosterol content did not correlate with physical characteristics, including 100-seed weight and color values (L, a, and b), but it was negatively correlated with total tocopherol content. β-Tocopherol was a major tocopherol in sorghum, constituting approximately 40%–46% of the total tocopherol content, and it was highly correlated with the total tocopherol content. Furthermore, the α-tocopherol content also increased with increasing γ-tocopherol content. Among the 4 tocopherols, only δ-tocopherol content correlated with physical characteristics of sorghum grain. This study deepened our knowledge of the variations in β-sitosterol and α-, β-, γ-, and δ-tocopherol content in sorghum with respect to genotype and cultivation location. In addition, this study demonstrated a correlation between some physical characteristics of sorghum and β-sitosterol/tocopherol content, and this information can be useful for breeding programs that develop or breed sorghum varieties or manufacture sorghum-based foods containing high amounts of β-sitosterol and tocopherols.     

Membrane fractionation of a β-lactoglobulin tryptic digest: Effect of the pH

The tryptic digestion of β-lactoglobulin leads to the release of a large range of biologically active peptides. Ultrafiltration/nanofiltration technology can be used to fractionate protein hydrolysates in order to obtain permeate products with increased functionality and free from intact proteins and enzymes. The influence of the pH in the fractionation of the hydrolysate through a polyethersulfone membrane (MWCO 5 kDa) was investigated in this work. In this case peptide transmission was mainly governed by charge mechanisms and reached its maximum value when the pH value is close to the peptide isoelectric point. Almost complete rejection of acidic peptides was achieved at basic pH values due to electrostatic repulsive forces with the negatively charged membrane and, in agreement with Donnan theory, positively charged peptides shown lower transmission than the neutral species. The highest peptide recovery and the best separation factor between bioactive and non-bioactive peptides were obtained following nanofiltration at pH 8.0.     

Tryptic hydrolysis of bovine αS2-casein: identification and release kinetics of peptides

Bovine α_S2-casein was purified by anionic exchange and hydrophobic interaction chromatographies. The purified α_S2-casein was hydrolysed with trypsin; the resulting peptides were identified by amino acids composition and on line reversed-phase high performance liquid chromatography coupled with electrospray mass spectrometry. A study on the kinetics of peptide release enabled discrimination between different protein areas according to their surface-accessibility. Data from the kinetic study were reconciled with secondary structure predictions and a hypothetical model of the structural organisation of purified bovine α_S2-casein in solution was proposed.     

Effect of pulsed-light processing on the surface and foaming properties of β-lactoglobulin

Pulsed-light processing was used to treat β-lactoglobulin (BLG) solutions. The impact of pulsed light (PL) on the structural properties of this protein was explored through far-UV, CD spectral analysis, size exclusion chromatography, surface hydrophobicity and NMR spectroscopy. Changes on these physicochemical properties were related to surface rheology, surface tension, foam stability and foam capacity of the non-treated and treated BLG to elucidate adsorption mechanism and consequences on foaming capacity. Conformational modification of BLG was related with PL total fluence as important conformational changes increased when total fluence was higher. Consequently, adsorption rate of treated BLG at the air/water interface was faster than native BLG. Additionally, treated BLG formed highly elastic interfaces. This was found to have an impact on the foam stability. Pulsed-light treatment seemed to enhance the overall strength of the interface, resulting in more stable foams.     

Effect of glycation on the gastrointestinal digestibility and immunoreactivity of bovine β-lactoglobulin

Immunoreactivity of bovine β-lactoglobulin (β-Lg) hydrolysates obtained after a simulated gastrointestinal digestion and previously glycated via Maillard reaction with galactose, tagatose, and dextran of 10 or 20 kDa has been determined, with a view to study the effect of glycation and aggregation degree of β-Lg on its residual immunoreactivity. High levels of glycation impaired β-Lg proteolysis and, consequently, increased the IgG- and IgE-reactivities of hydrolysates, regardless of the carbohydrate used. Protein aggregation during the advanced stages of Maillard reaction had a masking effect on β-Lg epitopes, counteracting the negative effect of the lower digestibility of glycated protein on its allergenicity. Finally, the use of polysaccharides as glycation agents did not contribute to enhancement of the masking effect of the attached carbohydrate on β-Lg epitopes. These findings stress the importance of evaluating the impact of glycation on protein gastrointestinal digestibility prior to investigation of the immunoreactivity of protein Maillard complexes.     

Effect of different β-glucans on the gelatinisation and retrogradation of rice starch

Four β-glucan preparations, i.e., curdlan (CL), oat (OG), barley (BG) and yeast (YG) β-glucans, were compared for their effects on the gelatinisation and retrogradation of rice starch (RS). Rapid visco-analysis (RVA) showed that addition of any of these β-glucans significantly increased the peak, breakdown, final, and setback viscosities of RS, whereas the pasting temperatures were significantly decreased by OG or CL addition, but were unaffected by BG or YG addition. Differential scanning calorimetry (DSC) demonstrated that all the β-glucans had a negligible effect on the onset (T_o), peak (T_p), and conclusion (T_c) temperatures but slightly decreased the gelatinisation enthalpy (△H₁) of RS. Storage of all the gels at 4 °C resulted in a marked decrease in the T_o, T_p, T_c, and melting enthalpy (△H₂) values. The retrogradation ratio (△H₂/△H₁) and the phase transition temperature range (T_c–T_o) of all the gels increased with storage time. Dynamic viscoelastic measurements revealed weak gel-like behaviour of all the gels, in which their storage modulus (G′) increased and their loss tangent (tan δ) decreased during storage. Steady flow tests illustrated time-dependent shear-thinning (thixotropic) behaviour of all the gels. The hysteresis loop area and the gel hardness increased with storage time. However, the rate and extent of retrogradation and the rheological and textural changes of the RS gels were reduced by addition of any of these β-glucans. The extent of the aforementioned effects differed among the different β-glucan preparations, generally in the order OG ≈ BG > CL ≈ YG.     

Effect of pH on betalain–anthocyanin mixture in bayberry juice: influences on pigments,colour, and antioxidant capacity

Betalains (mainly betanin, isobetanin, and phyllocactin) extracted from red–purple pitaya were blended with Chinese bayberry juice (rich in anthocyanins) to explore variation trends of two natural pigments in one system. The mixture showed higher anthocyanin contents at pH 4, 4.5, and 5. In this process, redness and colour saturation were also maintained to a greater extent compared to the control. The coexisting betalains and anthocyanins increased colour acceptability of the juice matrix and expanded the pH tolerance range. Interestingly, colour change (ΔE) showed the opposite trend. Besides, betalains showed the function of improving the antioxidant activity of juice matrix at pH 3, 4, and 4.5, which confirmed their biological activity. Betalain-added bayberry juice obtained a satisfactory ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging capacity for 62.52% at pH 5.     

Effect of prolactin, β-lactoglobulin, and κ-casein genotype on milk yield in East Friesian sheep

The effect of prolactin (PRL), β-lactoglobulin (β-LG), and κ-casein (CSN3) on milk yield was estimated in an East Friesian dairy sheep population from Old Chatham Sheepherding Company, New York. Genotypes were determined by PCR amplification followed by digestion with HaeIII and RsaI for PRL and β-LG, respectively, and by PCR amplification for CSN3. Monthly milking records and pedigree information were used to evaluate the effect of each polymorphism on milk yield. Results indicated that PRL genotype had a significant effect on milk yield. Ewes carrying one A allele produced 110.6 g more milk per day than ewes with no A alleles. There was no statistical difference between ewes with only one A allele and ewes with 2 A alleles. No association among polymorphisms at the β-LG and CSN3 loci and milk yield was found. The results presented in this study indicate that the PRL gene is a potential marker that could be used in selection programs for improving milk yield in dairy sheep.     

Effect of CSN1S1-CSN3 (α(S1)-κ-casein) composite genotype on milk production traits and milk coagulation properties in Mediterranean water buffalo

The aim of this study was to estimate effects of CSN1S1-CSN3 (α_S1-κ-casein) composite genotypes on milk production traits and milk coagulation properties (MCP) in Mediterranean water buffalo. Genotypes at CSN1S1 and CSN3 and coagulation properties [rennet clotting time (RCT), curd firming time (K₂₀), and curd firmness (A₃₀)] were assessed by reversed-phase HPLC and computerized renneting meter analysis, respectively, using single test-day milk samples of 536 animals. Alternative protein variants of α_S1-CN and κ-CN were detected by HPLC, and identification of the corresponding genetic variants was carried out by DNA analysis. Two genetic variants were detected at CSN1S1 (A and B variants) and 2 at CSN3 (X1 and X2 variants). Statistical inference was based on a linear model including the CSN1S1-CSN3 composite genotype effect (7 genotypes), the effects of herd-test-day (8 levels), and a combined days in milk (DIM)-parity class. Composite genotype AB-X2X2 was associated with decreased test-day milk yield [?0.21 standard deviation (SD) units of the trait] relative to genotype BB-X2X2. Genotypes did not affect milk protein content, but genotype AB-X1X1 was associated with increased fat content compared with genotype BB-X2X2 (+0.28 SD units of the trait) and AB-X1X1 (+0.43 SD units of the trait). For RCT, the largest difference (+1.91 min; i.e., 0.61 SD units of the trait) was observed between genotype AA-X1X2 and AB-X1X1. Direction of genotype effects on K₂₀ was consistent with that for RCT. The maximum variation in K₂₀ due to genotype effects (between AA-X1X2 and AB-X1X1 genotypes) was almost 0.9 SD units of the trait. Magnitude of genotype effects was smaller for A₃₀ than for RCT and K₂₀, with a maximum difference of 0.5 SD units of the trait between genotype AA-X1X2 and AA-X1X1. The B allele at CSN1S1 was associated with increased RCT and K₂₀ and with weaker curds compared with allele A. Allele X2 at CSN3 exerted opposite effects on MCP relative to CSN1S1 B. Because of linkage disequilibrium, allele B at CSN1S1 and allele X2 at CSN3 tend to be associated and this likely makes their effects cancel each other. This study indicates a role for casein genes in variation of MCP of buffalo milk. Further studies are necessary to estimate the effects of casein genetic variants on variation of cheese yield.     

Genome-wide association study for αS1- and αS2-casein phosphorylation in Dutch Holstein Friesian

Phosphorylation of caseins (CN) is a crucial post-translational modification that allows caseins to form colloid particles known as casein micelles. Both α_S1- and α_S2-CN show varying degrees of phosphorylation (isoforms) in cow milk and were suggested to be more relevant for stabilizing internal micellar structure than β- and κ-CN. However, little is known about the genetic background of individual α_S2-CN phosphorylation isoforms and the phosphorylation degrees of α_S1- and α_S2-CN (α_S1-CN PD and α_S2-CN PD), defined as the proportion of isoforms with higher degrees of phosphorylation in total α_S1- and α_S2-CN, respectively. We aimed to identify genomic regions associated with these traits using 50K single nucleotide polymorphisms for 1,857 Dutch Holstein Friesian cows. A total of 10 quantitative trait loci (QTL) regions were identified for all studied traits on 10 Bos taurus autosomes (BTA1, 2, 6, 9, 11, 14, 15, 18, 24, and 28). Regions associated with multiple traits were found on BTA1, 6, 11, and 14. We showed 2 QTL regions on BTA1, one affecting α_S2-CN production and the other harboring the SLC37A1 gene, which encodes a phosphorus antiporter and affects α_S1- and α_S2-CN PD. The QTL on BTA6 harbors the casein gene cluster and affects individual α_S2-CN phosphorylation isoforms. The QTL on BTA11 harbors the PAEP gene that encodes for β-lactoglobulin and affects relative concentrations of α_S2-CN-10P and α_S2-CN-11P as well as α_S1-CN PD and α_S2-CN PD. The QTL on BTA14 harbors the DGAT1 gene and affects relative concentrations of α_S2-CN-10P and α_S2-CN-11P as well as α_S1-CN PD and α_S2-CN PD. Our results suggest that effects of identified genomic regions on phosphorylation of α_S1- and α_S2-CN are related to changes in milk synthesis and phosphorus secretion in milk. The actual roles of SLC37A1, PAEP, and DGAT1 in α_S1- and α_S2-CN phosphorylation in Dutch Holstein Friesian require further investigation.     

Effect of αS1-casein genotype on phenolic compounds and antioxidant activity in goat milk yogurt fortified with Rhus coriaria leaf powder

The aim of this work was to evaluate the phenolic compounds and antioxidant activity of goat milk yogurt characterized by different α_S1-casein genotypes and fortified with Rhus coriaria leaf powder. The α_S1-casein genotype was determined by isoelectric focusing, total phenol content was determined by the Folin–Ciocalteu method, phenolic compounds were identified and quantified by HPLC-UV analysis, and antioxidant activity was measured using 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid and ferric-reducing antioxidant power. The statistical analysis showed a significant effect of the studied factors. Comparing different genotypes it emerged that yogurt from goats with weak alleles at CSN1S1 loci (FF) showed the lowest phenolic compounds and therefore a lower antioxidant activity compared with yogurt from goats with strong alleles at CSN1S1 loci (AA, BB, AB). Rhus coriaria-fortified yogurt showed a significant increase in total phenolic compounds and antioxidant activity in comparison with plain yogurt. The FF-fortified yogurt presented the lowest total phenol content and antioxidant activity. This could be due to a greater capacity of proteins and peptides in this yogurt to form stable complexes with phenols. The different total phenol content detected in R. coriaria-fortified yogurt indicates that the α_S1-casein genotype influenced the amount of added phenols that are bound to the caseins and, therefore, the part that remains free and that affects the biological capacity of the final product.     

Effect of salts on the alkaline degradation of β-lactoglobulin gels and aggregates: Existence of a dissolution threshold

The effect of NaCl and CaCl₂ on the alkaline degradation of β-lactoglobulin gels and aggregates, and particularly on the onset of dissolution, is studied. For gels, measurements of solubility in 0.063–0.5 M NaOH at 20 °C show the existence of a practical dissolution threshold in NaCl concentration, lying between 0.24 and 0.47 M. For aggregates, destruction of soluble β-lactoglobulin in alkali, followed by size exclusion chromatography, yields similar results. Furthermore, during dissolution of a gel in alkali at high NaCl concentrations, the protein aggregates released are very large (e.g. ∼40% are larger than 200 kDa). CaCl₂ is found to cause similar inhibition of dissolution to NaCl, but at concentrations about 30× lower (∼10 mM). The threshold is hypothesised to arise from a combination of physical entanglements caused by the high protein concentration under conditions where little swelling occurs, and hydrophobic/electrostatic interactions between aggregates favoured by the high concentration of salts.