Acid-induced Coagulation Research Articles

Influence of Ethanol on the Acid-Induced Flocculation of Casein Micelles

The influence of ethanol (0–10%, v/v) on the acid-induced flocculation of casein micelles was examined using diffusing wave spectroscopy. For this purpose, samples containing 10% (w/w) reconstituted skim milk powder and 0–10% (v/v) ethanol were acidified with glucono-delta-lactone and acid-induced coagulation was monitored by diffusing wave spectroscopy. The pH at which acid-induced flocculation of the casein micelles commenced (pHf) increased near-linearly with increasing ethanol content, whereas the rate of flocculation was not affected by ethanol. The results are discussed in terms of the steric stabilisation of casein micelles by a polyelectrolyte brush in a medium of high ionic strength. Ethanol-induced increases in pHf are probably primarily due to an ethanol-induced reduction in solvent quality; an ethanol-induced reduction in dissociation of carboxylic acid groups in the brush is likely to contribute.

Monitoring of acid-induced coagulation of dromedary and cow's milk using different complementary analytical techniques

This study investigates the acid-induced coagulation of dromedary and cow's milk using rheological, light backscattering, and FT-MIR spectroscopy techniques. Throughout the acid-induced coagulation process, dromedary milk exhibited higher values of pH than cow's milk. Indeed, pH values of milk attained values of 3.94 and 3.84 at the end of the coagulation for dromedary and cow's milks, respectively. Rheology measurements showed that, throughout the acid-induced coagulation, dromedary milk significantly presented lower values of storage modulus (G′) and loss modulus (G″) than cow's milk. The type of milk significantly affects the curd firmness of acid milk gels. Indeed, the lowest curd firmness of dromedary (8.71 Pa) and cow's milk (11.37 Pa) was observed at pH = 3.8, and 3.94, respectively. The delta backscattering (ΔBS) showed a sigmoidal increase at the initial steps of acid-induced coagulation of dromedary milk.

Monitoring of acid-induced coagulation of dromedary and cows' milk by untargeted and targeted techniques

The monitoring of acid-induced coagulation of dromedary and cow milk was investigated using both conventional (viscosity, conductivity, acidification kinetics) and non-destructive (turbidity, fluorescence spectroscopy) methods. Maximum of viscosity (36.66 ± 0.01 mPa s) for dromedary milk was observed at a pH of 3.5. Throughout acid-induced coagulation, dromedary milk exhibited lower viscosity than cow milk (maximum 36.66 ± 0.01 versus 57.67 ± 0.05 mPa s, respectively). The addition of HCl induced an increase of the electrical conductivity reaching values of 9.49 and 6.16 mS at the end of coagulation for dromedary and cow milk, respectively. Results obtained by turbiscan showed that the change in ΔBS was more steady for dromedary milk than for cow milk allowing the formation of a uniform gel. The structural and conformational changes in casein micelles during acid coagulation induced a decrease in the fluorescence intensity of tryptophan as the gel formation progressed for both milk samples.

Vibrations as a cause of texture defects during the acid-induced coagulation of milk – Fluid dynamic effects and their impact on physical properties of stirred yogurt

Abstract Vibrations during yogurt fermentation have been reported to disturb the gelation by inducing large, visible particles. Such vibrations are generated by the machinery used for the industrial manufacture and can cause structural excitation of the fermentation tanks. To study the effect of vibrations on yogurt structure, an experimental setup was constructed consisting of a cylindrical tank (height: 600 mm). The bottom of the tank was excited by a shaker, resulting in a vertical vibration propagation. Skim milk was vibrated during fermentation at 30 Hz and different amplitudes. Vibrated gels showed syneresis up to ∼5%. Samples were taken at different height positions of the fermenter, processed into stirred yogurt, and analyzed. The number of large particles was primarily related to the set vibration amplitude, however, particle formation was more pronounced in the lower and upper area. Yogurts with a high particle number exhibited a reduced water-holding capacity and apparent viscosity. Moreover, the fluid dynamic effects of vibrations were investigated by particle image velocimetry. Vibrations led to uniform to-and-fro motions, whereas chaotic flow was observed at the surface. Finally, the impact of vibrations on the gelation mechanism is discussed.

Plasma Kallikrein Contributes to Ellagic Acid-Induced Coagulation in the Absence of FXI By Activating Factor IX

Background - Factor XI (FXI) plays a critical role in thrombosis, as its inhibition reduces clot formation in animal models, and human congenital FXI deficiency appears to protect against venous thromboembolism and stroke. In contrast, in both settings, the impact on hemostasis is rather low, especially compared to FIX deficiency. Little is known about the cause of this difference. As thrombin generation can be observed in FXI-deficient plasma, we speculated that intrinsic activation of FIX is not solely dependent on FXI(a). Aim - Evaluate whether plasma kallikrein contributes to FIX activation and subsequent thrombin generation in FXI deficiency. Methods - Kaolin-initiated coagulation and fibrin formation was assessed in different plasma types in the presence or absence of an active site inhibitor of human plasma kallikrein (PKa) using the activated partial thromboplastin time (aPTT). Thrombin generation was measured in ellagic acid-induced contact activation in human FXI-deficient plasma in the presence or absence of the PKa inhibitor. Levels of FIXa-antithrombin (FIXa-AT) or FXa-AT were determined at defined time points using in-house developed ELISAs. The role of thrombin and its substrate FVIII in ellagic acid-induced contact activation in the absence of FXI was investigated using dabigatran or a human monoclonal FVIII antibody and assessing the FIXa-AT and FXa-AT plasma levels. In in vivo experiments, F11-/- mice were treated with ellagic acid in the presence or absence of the PKa inhibitor and FXIa-AT levels were assessed 30 min after administration. Results - Plasma kallikrein inhibition dose-dependently prolonged kaolin-triggered clotting time in human FXI-deficient plasma but not in normal pooled or plasma prekallikrein depleted plasma, indicating a role for PKa in coagulation in the absence of FXI. In FXI-deficient plasma, ellagic acid activated the coagulation system, as could be demonstrated by measurement of thrombin generation, and FIXa-AT and FXa-AT levels, which immediately reached the upper detection limit of 1.6 nM suggesting a FXI bypass route towards FIX activation. Addition of a PKa inhibitor caused a delayed and diminished FXa-AT and FIXa-AT complex formation and led to a reduction in thrombin peak height (EC50: 3 µM). This indicates that PKa might contribute to FXI-independent coagulation by activating FIX. In the presence of dabigatran FIXa-AT complex formation in FXI-deficient plasma was completely inhibited, which could be reversed by addition of purified thrombin. In the presence of purified thrombin, a FVIII antibody inhibited thrombin-dependent FIXa-AT complex formation suggesting that the activation of FVIII by thrombin is essential for FIX activation by PKa or to support the activity of FIXa. In line with this, thrombin generation in FXI-defcient plasma was inhibited by the FVIII antibody. In ellagic acid-treated F11-/- mice, FIXa-AT complex formation was significantly increased compared to controls (612 pM vs. 121 pM; P < 0.001), and was attenuated upon inhibition of PKa. Conclusions - We demonstrate that, activation of FXII leads to thrombin generation via FIX activation by plasma kallikrein in the absence of FXI. In addition, the activation of FVIII by thrombin is essential for the FXI bypass route towards FIX activation. These findings support the idea that FXI and FIX have distinctly different roles within the intrinsic coagulation cascade upon FXII activation and may contribute to variations in bleeding in FXI-deficient subjects due to different prekallikrein plasma levels. Disclosures Visser: Bayer AG: Employment. Laux:Bayer AG: Employment. Heitmeier:Bayer AG: Employment.

Rheological and physical properties of camel and cow milk gels enriched with phosphate and calcium during acid-induced gelation.

The rheological properties of acid-induced coagulation of camel and cow milk gels following the addition of calcium chloride (CaCl2) and hydrogen phosphate dehydrate (Na2HPO4*2H2O) were investigated using a dynamic low amplitude oscillatory rheology. For a considered condition, the final values of storage modulus (G') and loss modulus (G″) of camel milk gels were significantly lower than those of cow milk gels. The increase of the added CaCl2 levels improved significantly the gelation properties of camel and cow milk gels, since a reduction in the gelation time and an increase in the gel firmness were observed. Following the addition of Na2HPO4*2H2O at 10 and 20mM, no significant effect on the gelation rate and the firmness of camel milk gels was observed, while, a significant decrease in the gelation rate and firmness were observed for cow milk gels.

Bovine chromosomal regions affecting rheological traits in acid-induced skim milk gels

The production of fermented milk products has increased worldwide during the last decade and is expected to continue to increase during the coming decade. The quality of these products may be optimized through breeding practices; however, the relations between cow genetics and technological properties of acid milk gels are not fully known. Therefore, the aim of this study was to identify chromosomal regions affecting acid-induced coagulation properties and possible candidate genes. Skim milk samples from 377 Swedish Red cows were rheologically analyzed for acid-induced coagulation properties using low-amplitude oscillation measurements. The resulting traits, including gel strength, coagulation time, and yield stress, were used to conduct a genome-wide association study. Single nucleotide polymorphisms (SNP) were identified using the BovineHD SNPChip (Illumina Inc., San Diego, CA), resulting in almost 621,000 segregating markers. The genome was scanned for putative quantitative trait loci (QTL) regions, haplotypes based on highly associated SNP were inferred, and the additive genetic effects of haplotypes within each QTL region were analyzed using mixed models. A total of 8 genomic regions were identified, with large effects of the significant haplotype explaining between 4.8 and 9.8% of the phenotypic variance of the studied traits. One major QTL was identified to overlap between gel strength and yield stress, the QTL identified with the most significant SNP closest to the gene coding for κ-casein (CSN3). In addition, a chromosome-wide significant region affecting yield stress on BTA 11 was identified to be colocated with PAEP, coding for β-lactoglobulin. Furthermore, the coagulation properties of the genetic variants within the 2 genes were compared with the coagulation properties identified by the patterns of the haplotypes within the regions, and it was discovered that the haplotypes were more diverse and in one case slightly better at explaining the phenotypic variance. Besides these significant QTL comprising the 2 milk proteins, 3 additional genes are proposed as possible candidates, namely RAB22A, CDH13, and STAT1, and all have previously been found to be expressed in the mammary gland. To our knowledge, this is the first attempt to map QTL regions for acid-induced coagulation properties.

Genetic parameters for rennet- and acid-induced coagulation properties in milk from Swedish Red dairy cows

Milk coagulation is an important processing trait, being the basis for production of both cheese and fermented products. There is interest in including technological properties of these products in the breeding goal for dairy cattle. The aim of the present study was therefore to estimate genetic parameters for milk coagulation properties, including both rennet- and acid-induced coagulation, in Swedish Red dairy cattle using genomic relationships. Morning milk samples and blood samples were collected from 395 Swedish Red cows that were selected to be as genetically unrelated as possible. Using a rheometer, milk samples were analyzed for rennet- and acid-induced coagulation properties, including gel strength (G′), coagulation time, and yield stress (YS). In addition to the technological traits, milk composition was analyzed. A binary trait was created to reflect that milk samples that had not coagulated 40min after rennet addition were considered noncoagulating milk. The cows were genotyped by using the Illumina BovineHD BeadChip (Illumina Inc., San Diego, CA). Almost 600,000 markers remained after quality control and were used to construct a matrix of genomic relationships among the cows. Multivariate models including fixed effects of herd, lactation stage, and parity were fitted using the ASReml software to obtain estimates of heritabilities and genetic and phenotypic correlations. Heritability estimates (h2) for G′ and YS in rennet and acid gels were found to be high (h2=0.38–0.62) and the genetic correlations between rennet-induced and acid-induced coagulation properties were weak but favorable, with the exception of YSrennet with G′acid and YSacid, both of which were strong. The high heritability (h2=0.45) for milk coagulating ability expressed as a binary trait suggests that noncoagulation could be eliminated through breeding. Additionally, the results indicated that the current breeding objective could increase the frequency of noncoagulating milk and lead to deterioration of acid-induced coagulation through unfavorable genetic associations with protein content (0.38) and milk yield (−0.61 to −0.71), respectively. The outcome of this study suggests that by including more detailed compositional traits genetically associated with milk coagulation or by including milk coagulation properties directly within the breeding goal, it appears possible to breed cows that produce milk better suited for production of cheese and fermented products.

Electrical conductivity and thermal stability of polypropylene containing well-dispersed multi-walled carbon nanotubes disentangled with exfoliated nanoplatelets

The morphology, crystallization behavior, electrical conductivity, and thermal stability of polypropylene (PP) modified with disentangled multi-walled carbon nanotubes (MWCNTs) is reported. Slightly oxidized MWCNT clusters were disentangled in solution by mild sonication in the presence of exfoliated α-zirconium phosphate nanoplatelets. The disentangled MWCNTs were isolated using acid-induced coagulation to precipitate the nanoplatelets, and were subsequently reacted with octadecylamine. The recovered functionalized MWCNTs (F-MWCNTs) are disentangled and easily dispersed in a commercial PP matrix, and serve as more efficient nucleating agents than the untreated MWCNTs. The PP/F-MWCNT composites exhibit an extremely low percolation-like transition in electrical conductivity, which is attributed to the preservation of a random dispersion of disentangled F-MWCNTs upon cooling from the melt. The thermal stability of PP in air is also substantially enhanced at loadings below the percolation threshold due to the tremendous interfacial area between the polymer chains and the free radical scavenging F-MWCNTs. The present approach provides an efficient and potentially scalable route for commercial production of conductive semi-crystalline thermoplastics. The method may be adapted to uniformly disperse MWCNTs in other polymer matrices by appropriate selection of surface functionality.

Structural Changes of Milk Components during Acid-Induced Coagulation Kinetics as Studied by Synchronous Fluorescence and Mid-Infrared Spectroscopy

Dynamic oscillatory experiments and front-face synchronous fluorescence spectroscopy and mid-infrared (mid-IR) spectroscopy have been used to investigate structure evolution, at the macroscopic and molecular levels, during milk acidification kinetics. The studies were performed using skim milk, at two different temperatures (30 °C and 40 °C), to which was added glucono-δ-lactone (GDL) to generate different structural changes in casein micelles and gels. Synchronous fluorescence spectra were recorded in the 250-500 nm excitation wavelength range using an offset of 80 nm between the excitation and emission monochromators for each system during the 300 min acidification kinetics. The change in the fluorescence intensity at 281 nm reflects the pH-induced physicochemical changes of casein micelles and, in particular, structural changes in the micelles in the pH range 5.5-5.0. Regarding mid-infrared spectroscopy, the region located between 1700 and 1500 cm(-1), corresponding to the amide I and II bands, and the 1500-900 cm(-1) region, called the fingerprint region, were considered for the characterization of milk coagulation kinetics. Changes in the absorbance at 1063 cm(-1) as a function of pH for kinetics recorded at 30 °C and 40 °C reflected pH-induced phosphate dissolution in the pH range 5.5-5.0. Compared to rheometry, which reveals microstructure changes only in the gel state, spectroscopic methods make it possible to monitor molecular structure changes in micelles throughout the acidification processes.

Structure and stability of nanogel particles prepared by internal cross-linking of casein micelles

Cross-linking all caseins within the casein micelles with the enzyme transglutaminase creates nanogel particles consisting of a covalently linked casein network from which micellar calcium phosphate (MCP) can be removed without compromising structural integrity. These casein nanogel particles show similar light, neutron and X-ray scattering behaviour to native casein micelles, indicating similarity in size and substructure. Casein nanogel particles are more stable to heat-induced coagulation, but less stable to acid-induced coagulation than native casein micelles. Changing the MCP content of casein nanogel particles to levels between 0% and 150% of its original concentration strongly affected colloidal stability of the particles. Stability to both heat- and acid-induced coagulation increased with decreasing MCP content. Casein nanogel particles offer applications not only in traditional dairy products, but also in products and applications where the integrity and biocompatibility of the nanogel particle is important.

Acid Gelation in Heated and Unheated Milks: Interactions between Serum Protein Complexes and the Surfaces of Casein Micelles

The acid-induced interactions between different protein particles in milk (casein micelles and serum protein/kappa-casein complexes) were studied in a series of different mixtures of heated and unheated proteins using diffusing wave spectroscopy (DWS) and small deformation rheology. The measurements were made as functions of pH during acidification by addition of glucono-delta-lactone (GDL). Heat treatment (85 degrees C, 10 min) affected the composition of the serum and the reactivity of casein micellar surface based on the pH at which the casein micelles aggregated during acidification. It was observed that the gel points as defined by DWS and rheology did not always coincide. The experiments showed that all systems containing heated serum proteins gelled at a higher pH than those containing unheated serum proteins. For systems containing heated micelles, an intermediate network can be formed between heat-induced aggregates of serum proteins and kappa-casein formed at the surfaces of the micelles and dispersed as soluble complexes in the serum. This can explain the observation that DWS measurements detected aggregation of casein micelles at an earlier stage than did rheology. For systems containing unheated micelles and soluble complexes from heated milk, the results appear to be explained only by a direct interaction between soluble serum protein complexes and the casein micelles themselves, once the pH has decreased to below about 5.5. Comparison of the different systems studied gives a more complete description of the possible mechanism of interaction of the different protein materials during the acid-induced coagulation of milk-based systems.

Textural Characteristics and Dynamic Oscillatory Rheology of Maturation of Milk Gelatin Gels with Low Acidity

The potential was assessed for using gelatin to form milk gels in the pH range outside that at which acid-induced coagulation of milk takes place, and performance of these gels was compared with aqueous gelatin gels. We studied gels containing two types of gelatin (A and B) at concentrations of 1.5, 3.0, 4.5, and 6.67% and within a pH range of 5.3 to 6.6. Bloom strength, breaking force, and hardness depended principally on the gelatin concentration and were independent of pH values. Type B gelatin produced weaker gels than did type A gelatin. As determined by instrumental texture profile analysis, values for retarded recoverable springiness and cohesiveness were very high for all samples. The values for storage modulus over maturation time were greater for milk gels than for water gels, suggesting a stabilization of the network from the milk components. The maturation mechanism and melting temperatures of milk gels proved to be similar to those of water gels. Gelatin has the potential for use in the preparation of a wide range of milk gels with low acidity and interesting textural properties.