Measurement of water-holding capacity in fermented milk using near-infrared spectroscopy combined with chemometric methods.

We investigated the use of near-infrared spectroscopy (NIR) for measuring water-holding capacity (WHC) in fermented milk. Increased WHC ensures improved texture and decreased syneresis in fermented dairy products and also improves cheese yield. NIR combined with partial least-squares-discriminant analysis (PLS-DA) was found to be a promising rapid and non-invasive method with no pretreatment of the samples for prediction of WHC in fermented milk samples. Analysis of the chemical bonds in the region 10 700-4500 cm-1 (935-2200 nm) of the electromagnetic spectrum was able to distinguish between samples with high vs. low WHC. This technique was successfully used to screen different strains of lactic acid bacteria for their ability to provide fermented milk with increased WHC, which is of great importance for use in various dairy products.

Impact of Alginate Mannuronic-Guluronic Acid Contents and pH on Protein Binding Capacity and Complex Size.

Alginates, serving as hydrocolloids in the food and pharma industries, form particles at pH < 4.5 with positively charged proteins, such as ß-lactoglobulin (ß-Lg). Alginates are linear anionic polysaccharides composed of 1,4-linked ß-d-mannuronate (M) and α-l-guluronate (G) residues. The impact of M and G contents and pH is investigated to correlate with the formation and size of ß-Lg alginate complexes under relevant ionic strength. It is concluded, using three alginates of M/G ratios 0.6, 1.1, and 1.8 and similar molecular mass, that ß-Lg binding capacity is higher at pH 4.0 than at pH 2.65 and for high M content. By contrast, the largest particles are obtained at pH 2.65 and with high G content. At pH 4.0 and 2.65, the stoichiometry was 28-48 and 3-10 ß-Lg molecules bound per alginate, respectively, increasing with higher M content. The findings will contribute to the design of formation of the desired alginate-protein particles in the acidic pH range.

Contrasting Assemblies of Oppositely Charged Proteins.

Oppositely charged proteins can form soluble assemblies that under specific physical chemical conditions lead to liquid-liquid phase separation, also called heteroprotein coacervation. Increasing evidence suggests that surface charge anisotropy plays a key role in heteroprotein complexation, and coacervation. Here, we investigated complexation of an acidic protein, ß-lactoglobulin (BLG), with two basic proteins, rapeseed napin (NAP) and lysozyme (LYS), of similar net charge and size but differing in surface charge distribution. Using turbidity measurements and isothermal titration calorimetry, we confirmed that LYS binds BLG as expected from previous studies. This interaction leads to two types of phase separation phenomena, depending on pH: liquid-solid phase separation in the case of strong electrostatic attraction and liquid-liquid phase separation for weaker attraction. More interestingly, we showed using dynamic light scattering that NAP interacts with BLG, resulting in formation of assemblies in the nanometer size range. The formation of assemblies was also evident when modeling the interactions using Brownian dynamics for both BLG + NAP and BLG + LYS. Similarly, to DLS, BLG and NAP formed smaller assemblies than BLG with LYS. The molecular details rather than the net charge of BLG and NAP may therefore play a role in their assembly. Furthermore, simulated BLG + NAP assemblies were larger than those experimentally detected by DLS. We discuss the discrepancy between experiments and simulations in relation to the limitations of modelling precisely the molecular characteristics of proteins.

The influence of pH, protein concentration and calcium ratio on the formation and structure of nanotubes from partially hydrolyzed bovine α-lactalbumin.

Formation of nanotubes from partially hydrolysed α-lactalbumin (α-La) was investigated at five pH values, two concentrations of α-La and two calcium levels. Nanotubes were formed under almost all combinations of the investigated factors, and for the first time the formation of nanotubes at low pH (4.0) and low protein concentration (10 g l-1) was observed. Only one sample (10 g l-1, calcium ratio 2.4, and pH 7.5) formed mainly fibrils instead of nanotubes. By altering the three investigated factors, fibrils and/or aggregates were sometimes formed together with nanotubes resulting in transparent, semi-transparent, or non-transparent gels, or sediments. However, structural modelling based on small-angle X-ray scattering data indicated that the formed nanotubes were only to a minor degree affected by the investigated factors. The majority of the nanotubes were found to have an outer diameter of around 19 nm, an inner diameter of 6.6 nm and a wall thickness of 6.0 nm, except for three samples at low α-La concentrations and high calcium levels which exhibited slightly smaller dimensions. These three factors affected the hydrolysis as well as the self-assembly rate, resulting in the observed differences. However, these factors did not influence the architecture of the self-assembled nanotubes, and the lateral spacing of the individual parallel ß-sheet motifs was found to be 1.05 ± 0. 03 nm for all nanotubes. This study provides novel fundamental knowledge of the formation and structure of α-La nanotubes under different conditions, which will facilitate future application of these nanotubes in food and pharmaceutical areas.

Effect of starter cultures on properties of soft white cheese made from camel (Camelus dromedarius) milk.

This experiment was conducted to investigate the effect of starter cultures on the physicochemical properties, texture, and consumer preferences of soft white cheese (SWC) made from camel (Camelus dromedarius) milk. The experiment was laid out in a completely randomized design with 5 treatments [starter cultures; i.e., 1 thermophilic (STI-12), 2 blended (RST-743 and XPL-2), and 2 mesophilic (R-707 and CHN-22) cultures]. Starter cultures STI-12 and RST-743 were inoculated at 37°C, whereas XPL-2, R-707, and CHN-22 were inoculated at 30°C. Camel milk inoculated using STI-12 and RST-743 cultures resulted in faster acidification than XPL-2, R-707, and CHN-22 cultures. Camel milk SWC made using STI-12 and CHN-22 cultures gave lower pH (4.54) and titratable acidity (0.59), respectively, whereas R-707 culture resulted in high cheese yield (13.44 g/100 g). In addition, high fat (20.91 g/100 g), protein (17.49 g/100 g), total solids (43.44 g/100 g), and ash (2.40 g/100 g) contents were recorded for SWC made from camel milk made using RST-743 culture. Instrumental analysis of cheese texture revealed differences in resistance to deformation in which camel milk SWC made using RST-743 culture gave higher firmness (3.20 N) and brittleness (3.12 N). However, no significant difference was observed among camel milk SWC adhesiveness made using different starter cultures. Consumer preference for appearance, aroma, taste, and overall acceptances of SWC were affected by inoculation of starter cultures. Considering curd firmness, cheese yield, compositional quality, and textures using STI-12, RST-743, and R-707, these cultures were found to be better for the manufacture of camel milk SWC.

Revealing the Dimeric Crystal and Solution Structure of ß-Lactoglobulin at pH 4 and Its pH and Salt Dependent Monomer-Dimer Equilibrium.

The dimeric structure of bovine ß-lactoglobulin A (BLGA) at pH 4.0 was solved to 2.0 Å resolution. Fitting the BLGA pH 4.0 structure to SAXS data at low ionic strength (goodness of fit R-factor = 3.6%) verified the dimeric state in solution. Analysis of the monomer-dimer equilibrium at varying pH and ionic strength by SAXS and scattering modeling showed that BLGA is dimeric at pH 3.0 and 4.0, shifting toward a monomer at pH 2.2, 2.6, and 7.0 yielding monomer/dimer ratios of 80/20%, 50/50%, and 25/75%, respectively. BLGA remained a dimer at pH 3.0 and 4.0 in 50-150 mM NaCl, whereas the electrostatic shielding raised the dimer content at pH 2.2, 2.6, and 7.0, i.e., below and above the pI. Overall, the findings provide new insights into the molecular characteristics of BLGA relevant for dairy product formulations and for various biotechnological and pharmaceutical applications.

Casein-Based Powders: Characteristics and Rehydration Properties.

Casein-based powders are gaining industrial interest due to their nutritional and functional properties, but they are also known to have poor rehydration abilities. The fundamental physical and chemical mechanisms involved in the rehydration of these powders are essential for determining the critical steps in the manufacturing processes and for developing casein powders with improved rehydration properties. A number of analytical methods have been developed to measure the rehydration ability of powders, but criteria for the selection of methods for casein-based powders have not been provided. This review article provides an overview of the characteristics and methods for the production of casein-based powders, methodologies to measure their rehydration properties, and it summarizes the current state of understanding regarding rehydration. Advancements have been made in the field; however, a fundamental understanding enabling improvement of the rehydration properties of these powders is still lacking.

Revealing the Compact Structure of Lactic Acid Bacterial Heteroexopolysaccharides by SAXS and DLS.

Molecular structures of exopolysaccharides are required to understand their functions and the relationships between the structure and physical and rheological properties. Small-angle X-ray scattering and dynamic light scattering were used in conjunction with molecular modeling to characterize solution structures of three lactic acid bacterial heteroexopolysaccharides (HePS-1, HePS-2, and HePS-3). Values of radius of gyration RG, cross-sectional radius of gyration RXS, approximate length L, and hydrodynamic diameter were not directly proportional to the molar mass and indicated the HePSs adopted a compact coil-like rather than an extended conformation. Constrained molecular modeling of 15000 randomized HePS-1 conformers resulted in five best-fit structures with R factor of 3.9-4.6% revealing random coil-like structure. Φ and Ψ angle analysis of glycosidic linkages in HePS-1 structures suggests Galf residues significantly influence the conformation. Ab initio scattering modeling of HePS-2 and HePS-3 gave excellent curve fittings with χ2 of 0.43 and 0.34 for best-fit models, respectively, compatible with coil-like conformation. The findings disclose solution behavior of HePS relevant for their interactions with biomacromolecules, for example, milk proteins.

Effect of homogenisation in formation of thermally induced aggregates in a non- and low- fat milk model system with microparticulated whey proteins.

The objective of the research presented in this paper was to investigate how different characteristics of whey protein microparticles (MWP) added to milk as fat replacers influence intermolecular interactions occurring with other milk proteins during homogenisation and heating. These interactions are responsible for the formation of heat-induced aggregates that influence the texture and sensory characteristics of the final product. The formation of heat-induced complexes was studied in non- and low-fat milk model systems, where microparticulated whey protein (MWP) was used as fat replacer. Five MWP types with different particle characteristics were utilised and three heat treatments used: 85 °C for 15 min, 90 °C for 5 min and 95 °C for 2 min. Surface characteristics of the protein aggregates were expressed as the number of available thiol groups and the surface net charge. Intermolecular interactions involved in the formation of protein aggregates were studied by polyacrylamide gel electrophoresis and the final complexes visualised by darkfield microscopy. Homogenisation of non-fat milk systems led to partial adsorption of caseins onto microparticles, independently of the type of microparticle. On the contrary, homogenisation of low-fat milk resulted in preferential adsorption of caseins onto fat globules, rather than onto microparticles. Further heating of the milk, led to the formation of heat induced complexes with different sizes and characteristics depending on the type of MWP and the presence or not of fat. The results highlight the importance of controlling homogenisation and heat processing in yoghurt manufacture in order to induce desired changes in the surface reactivity of the microparticles and thereby promote effective protein interactions.

Functional and technological properties of camel milk proteins: a review.

This review summarises current knowledge on camel milk proteins, with focus on significant peculiarities in protein composition and molecular properties. Camel milk is traditionally consumed as a fresh or naturally fermented product. Within the last couple of years, an increasing quantity is being processed in dairy plants, and a number of consumer products have been marketed. A better understanding of the technological and functional properties, as required for product improvement, has been gained in the past years. Absence of the whey protein ß-LG and a low proportion of к-casein cause differences in relation to dairy processing. In addition to the technological properties, there are also implications for human nutrition and camel milk proteins are of interest for applications in infant foods, for food preservation and in functional foods. Proposed health benefits include inhibition of the angiotensin converting enzyme, antimicrobial and antioxidant properties as well as an antidiabetogenic effect. Detailed investigations on foaming, gelation and solubility as well as technological consequences of processing should be investigated further for the improvement of camel milk utilisation in the near future.

Effect of coagulation temperature on cooking integrity of heat and acid-induced milk gels.

Heat and acid-induced milk gels do not melt or flow upon heating and thus show great potential as a dairy-based protein source for cooking, e.g. for stews. Understanding how processing, e.g. acidification, affects the cooking behavior of these gels is therefore of great industrial interest. The cooking integrity of gels produced by rapidly acidifying milk using citric acid at temperatures of 60, 75, and 90 °C, was determined by analyzing composition, texture, and spatial water distribution before and after cooking. Increasing the acidification temperature from 60 to75 °C resulted in a significant reduction of yield, due to decreased moisture content of the gels. With increasing content of solids, the gels grew harder and denser, as observed by texture profile analysis and low-field Nuclear Magnetic Resonance. Upon cooking the 60 °C gel lost a significant amount of moisture, due to the contraction of the porous protein network. The more compact gels, prepared at 75 and 90 °C, did not lose mass indicating good cooking integrity, i.e. a gel that keeps its structure during cooking. Acidification temperature thus greatly influenced cooking integrity. The effect was mainly ascribed to the density of the gel texture, a result of the speed of protein aggregation and calcium recovery.

Simulated gastrointestinal digestion of protein alginate complexes: effects of whey protein cross-linking and the composition and degradation of alginate.

Alginate and whey protein are common additives in food production improving storage stability, texture and nutritional value. Alginate forms complexes with whey protein and inhibits proteolysis by pepsin and trypsin, but the influence of alginate protein complexation on digestion is poorly understood. This study shows that whey protein cross-linking by microbial transglutaminase dramatically decreased particle size (2-fold) and viscosity of alginate protein complexes. The INFOGEST in vitro simulated gastrointestinal digestion of whey protein was increased by cross-linking (16%) and suppressed by alginate, most pronounced with high mannuronic acid and least with high guluronic acid content. Sizes of alginate whey protein particles increased during gastric digestion, whereas for cross-linked whey protein complexes the size initially increased, but returned to their initial size at the end of gastric digestion. While alginate is not degraded by human enzymes, a few gut bacteria were recently found to encode lyases and other enzymes metabolizing alginate. Alginate lyase added to the intestinal phase enhanced digestion (9%) as controlled by alginate composition and enzyme specificity. Thus we provide evidence that use of hydrocolloids and processing of protein strongly influence digestion and should be considered when using food additives.

In situ SAXS study of non-fat milk model systems during heat treatment and acidification.

Small-angle X-ray scattering (SAXS) was used to monitor structural changes induced by heat treatment and acid gelation in milk matrices with added whey protein concentrates (WPCs) and nano-particulated whey protein (NWP). In general, heat treatment was found to mainly affect whey protein components while pure casein micelles remained largely unaffected. Conversely, acidification mainly affected caseins while leaving pure whey protein components intact. In mixed systems, the overall behaviour could be understood as a combination of the above effects, however, the type of the added whey protein components influenced the resulting structure formation and dynamics. NWP led to formation of larger structures compared to WPC components during heat treatment, although the latter showed faster aggregation dynamics. During acidification the NWP containing samples exhibited structural changes at slightly higher pH values than the WPC samples. The modeling of pure liquid whey protein (LWPC) samples showed that the heat induced denaturation and resulting aggregation of individual whey proteins is mainly a surface effect leaving the overall protein shape and dimensions unaffected. Schematic diagrams based on the current SAXS data and previous studies were constructed to illustrate the suggested interaction mechanisms between casein and whey proteins during both heating and acidification.

Unaided efficient transglutaminase cross-linking of whey proteins strongly impacts the formation and structure of protein alginate particles.

There is a dogma within whey protein modification, which dictates the necessity of pretreatment to enzymatic cross-linking of ß-lactoglobulin (ß-Lg). Here microbial transglutaminase (MTG) cross-linked whey proteins and ß-Lg effectively in 50 mM NaHCO3, pH 8.5, without pretreatment. Cross-linked ß-Lg spanned 18 to >240 kDa, where 6 of 9 glutamines reacted with 8 of 15 lysines. The initial isopeptide bond formation caused loss of ß-Lg native structure with t1/2 = 3 h, while the polymerization occurred with t1/2 = 10 h. Further, cross-linking effects on protein carbohydrate interaction have been overlooked, leaving a gap in understanding of these complex food matrices. Complexation with alginate showed that ß-Lg cross-linking decreased onset of particle formation, hydrodynamic diameter, stoichiometry (ß-Lg/alginate) and dissociation constant. The complexation was favored at higher temperatures (40 °C), suggesting that hydrophobic interactions were important. Thus, ß-Lg was cross-linked without pretreatment and the resulting polymers gave rise to altered complexation with alginate.

Proteolytic activity of selected commercial Lactobacillus helveticus strains on soy protein isolates.

Soy protein isolates were fermented by three commercial Lactobacillus helveticus strains for a maximum of seven days to investigate the resulting proteolysis. The proteolytic activity of the most active strain (LH88) was further analyzed (LC-MS/MS and GC-MS) and it was shown that the ß-conglycinin α subunit 1, ß-conglycinin α' subunit, glycinin G1, and 2S albumin were specifically degraded. Peptigram analysis and visualization of the crystal structure showed that the hydrolysis sites of ß-conglycinin α subunit, α' subunit, and the glycinin G1 were located on the surface of the molecule or at the mobile disordered region, hence being highly accessible for the proteinase of LH88. The proteins were partially further degraded to free amino acids, and subsequently catabolized to volatile compounds. However, most of the proteins remained native, even after seven days of fermentation, thus additional modification of protein structure or adjustment of fermentation conditions are required for effective generation of flavor compounds.

Stabilization of directly acidified protein drinks by single and mixed hydrocolloids-combining particle size, rheology, tribology, and sensory data.

BACKGROUND: High methoxyl pectin and carboxymethylcellulose (CMC) can be used as a stabilizer for directly acidified protein drinks (DAPDs). Use of pectin or CMC together with other polysaccharides and their impacts on product's rheological properties and tribological behavior are still largely unknown. This project investigated the impact of pectin and CMC, alone or in combination with guar gum, locust bean gum (LBG), and gellan gum when preparing DAPDs. The particle size distributions, rheological properties, tribological properties, and sensory properties were determined. RESULTS: Pectin and CMC were dominating in the mixed system with other stabilizers. Increasing the concentration of hydrocolloids resulted in higher viscosity and better lubrication (lower friction coefficient). The sensory viscosity, smoothness, coating, and stickiness intensified as the concentration of hydrocolloids increased. The type and amount of hydrocolloids had a strong effect on the sensorial texture perception, but the flavor perception was only slightly affected. CONCLUSION: Use of combined stabilizers may contribute to providing an effective viscosity enhancement without affecting the flavor in acidified milk beverages.

Metagenomic analysis of bacterial community composition in Dhanaan: Ethiopian traditional fermented camel milk.

This study was conducted to evaluate the safety and bacterial profile of Dhanaan (Ethiopian traditional fermented camel milk). The composition of the microbial community in Dhanaan samples was analysed by a metagenomic approach of 16S rRNA gene amplicon sequencing. Metagenomic profiling identified 87 different bacterial microorganisms (OTUs) in six samples analysed. Although the Dhanaan samples contained various lactic acid bacteria (LAB), they also all contained undesirable microorganisms in large proportions. The following LAB genera were identified: Streptococcus, Lactococcus and Weissella. One Streptococcus species represented by OTU-1 (operational taxonomic unit) was found in all Dhanaan samples and the dominating species in four out of six samples. This common isolate was found to be closely related to S. lutetiensis and S. infantarius. Undesirable microorganisms from genera such as Escherichia, Klebsiella, Enterobacter, Acinetobacter and Clostridium were, however, also frequent, or even dominant in Dhanaan samples. Thus, this calls for a change in the Dahnaan manufacturing practice to an improved and safer production system. Starter cultures suitable for Dhanaan production might be developed from the Streptococcus, Weissella and Lactococcus microorganisms identified in this study. However, further safety evaluation and technological characterization need to be conducted on strains defined by OTU-1, OTU-2, OTU-3, OTU-8 and OTU-35 before they can be used as food grade starter cultures.

Isoenergic modification of whey protein structure by denaturation and crosslinking using transglutaminase.

Transglutaminase (TG) catalyzes formation of covalent bonds between lysine and glutamine side chains and has applications in manipulation of food structure. Physical properties of a whey protein mixture (SPC) denatured either at elevated pH or by heat-treatment and followed by TG catalyzed crosslinking, have been characterised using dynamic light scattering, size exclusion chromatography, flourescence spectroscopy and atomic force microscopy. The degree of enzymatic crosslinking appeared higher for pH- than for heat-denatured SPC. The hydrophobic surface properties depended on the treatment, thus heating caused the largest exposure of the hydrophobic core of SPC proteins, which was decreased by crosslinking. The particle size of the treated SPC samples increased upon crosslinking by TG. Moreover, the particle morphology depended on the type of denaturing treatment, thus heat-treated SPC contained fibrillar structures, while pH-denatured SPC remained globular as documented by using atomic force microscopy. Finally, the in vitro digestability of the different SPC samples was assessed under simulated gastric and intestinal conditions. Notably heat-treatment was found to lower the gastric digestion rate and enzymatic crosslinking reduced both the gastric and the intestinal rate of digestion. These characteristics of the various SPC samples provide a useful basis for design of isoenergic model foods applicable in animal and human studies on how food structure affects satiety.

Interaction between structurally different heteroexopolysaccharides and ß-lactoglobulin studied by solution scattering and analytical ultracentrifugation.

Despite a very large number of bacterial exopolysaccharides have been reported, detailed knowledge on their molecular structures and associative interactions with proteins is lacking. Small-angle X-ray scattering, dynamic light scattering and analytical ultracentrifugation (AUC) were used to characterize the interactions of six lactic acid bacterial heteroexopolysaccharides (HePS-1-HePS-6) with ß-lactoglobulin (BLG). Compared to free HePSs, a large increase in the X-ray radius of gyration RG, maximum length L and hydrodynamic diameter dH of HePS-1-HePS-4 mixed with BLG revealed strong aggregation, the extent of which depended on the compact conformation and degree of branching of these HePSs. No significant effects were observed with HePS-5 and HePS-6. Turbidity and AUC analyses showed that both soluble and insoluble BLG-HePS complexes were formed. The findings provide new insights into the role of molecular structures in associative interactions between HePSs and BLG which has relevance for various industrial applications.

Self-assembly of partially hydrolysed alpha-lactalbumin.

Hydrolysis of the whey protein alpha-lactalbumin with a specific serine protease has been shown to result in regular nanotubes of approximately 20 nm in outer diameter and reaching several mum in length. Tubular assembly depends on the concentration of protein as this determines how far the hydrolysis proceeds. A concentration of 30 g L(-1) is a prerequisite for tubular formation, as is a minimum concentration of calcium. At lower protein concentrations calcium-independent formation of linear fibrils (approximately 5 nm in diameter) is favoured. Possible applications of alpha-lactalbumin nanotubes include use as a viscosifier and gelling agent and also pharmaceutical utilization (such as targeted drug release) and use in nanotechnology can be envisioned.