Cysteines in ß-lactoglobulin affects its interfacial adsorption and protein film stabilization.

HYPOTHESIS: Disulfide bonds in proteins are strong chemical bonds forming the secondary and tertiary structure like in the dairy protein ß-lactoglobulin. We hypothesize that the partial or complete removal of disulfide bonds affects the structural rearrangement of proteins caused by intra- and intermolecular interactions that in turn define the interfacial activity of proteins at oil/water interfaces. The experimental and numerical investigations contribute to the mechanistic understanding of the structure-function relationship, especially for the interfacial adsorption behavior of proteins. EXPERIMENTAL AND NUMERICAL: Systematically, the 5 cysteines of ß-lactoglobulin were recombinantly exchanged by alanine. First, the protein structure of the variants in bulk was analyzed with Fourier-transform-infrared-spectroscopy and molecular dynamic simulations. Second, the structural changes after adsorption to the interface have been also analyzed by molecular dynamic simulations. The adsorption behavior was investigated by pendant drop analysis and the interfacial film properties by dilatational rheology. FINDINGS: The structural flexibility of ß-lactoglobulin with no cysteines encourages its unfolding at the interface, and accelerates the interfacial protein film formation that results in more visco-elastic films in comparison to the reference.

(Non)linear Interfacial Rheology of Tween, Brij and Span Stabilized Oil-Water Interfaces: Impact of the Molecular Structure of the Surfactant on the Interfacial Layer Stability.

During emulsification and further processing (e.g., pasteurizing), the oil-water interface is mechanically and thermally stressed, which can lead to oil droplet aggregation and coalescence, depending on the interfacial properties. Currently, there is a lack of insights into the impact of the molecular structure (headgroup and FA chain) of low molecular weight emulsifiers (LME) on the resulting interfacial properties. Additionally, the crystallization/melting of the oil/the emulsifier is often neglected within interfacial rheological experiments. Within this study, the stability of interfaces formed by Tween, Span or Brij was determined as a function of their molecular structure, taking crystallization effects of the LME into account. The headgroup was kept constant while varying the FA, or vice versa. The interfacial film properties (viscoelasticity) were investigated at different temperatures using dilatational and interfacial shear rheology. Both the headgroup and the FA chain impacted the interfacial properties. For the same FA composition, a rather small hydrophobic headgroup resulted in a higher packed interface. The interfacial elasticity increased with increased FA chain length (C12 to C18). This seemed to be particularly the case when the emulsifier crystallized on the interface among cooling. In the case of a densely packed interface, network formation due to chain crystallization of the LME's FA chains occurs during the cooling step. The resulting interface shows predominantly elastic behavior.

Electrostatic spray drying: A new alternative for drying of complex coacervates.

Complex coacervation can be used for controlled delivery of bioactive compounds (i.e., flaxseed oil and quercetin). This study investigated the co-encapsulation of flaxseed oil and quercetin by complex coacervation using soluble pea protein (SPP) and gum arabic (GA) as shell materials, followed by innovative electrostatic spray drying (ES). The dried system was analyzed through encapsulation efficiency (EE) and yield (EY), morphological and physicochemical properties, and stability for 60 days. Small droplet size emulsions were produced by GA (in the first step of complex coacervation) due to its greater emulsifying activity than SPP. Oil EY and EE, moisture, and water activity in dried compositions ranged from 75.7 to 75.6, 76.0-73.4 %, 3.4-4.1 %, and 0.1-0.2, respectively. Spherical microcapsules were created with small and aggregated particle size but stable for 60 days. An amount of 8 % of quercetin remained in the dried coacervates after 60 days, with low hydroperoxide production. In summary, when GA is used as the emulsifier and SPP as the second biopolymer in the coacervation process, suitable coacervates for food applications are obtained, with ES being a novel alternative to obtain coacervates in powder, with improved stability for encapsulated compounds. As a result, this study helps provide a new delivery system option and sheds light on how the characteristics of biopolymers and the drying process affect coacervate formation.

Microencapsulation of flaxseed oil in pea protein-gum arabic complex coacervates delays lipid digestion in liquid yoghurt.

Flaxseed oil coacervates were produced by complex coacervation using soluble pea protein and gum arabic as shell materials, followed by either spray or electrostatic spray drying and their incorporation to yoghurt. Three yoghurt formulations were prepared: yoghurt with spray-dried microcapsules (Y-SD); with electrospray-dried microcapsules (Y-ES); with the encapsulation ingredients added in free form (Y). The standardised semi-dynamicin vitrodigestion method (INFOGEST) was employed to study the food digestion. The structure was analysed by confocal laser scanning microscopy and particle size distribution. Protein and lipid digestion were monitored by cumulated protein/free NH2 release and cumulated free fatty acids release, respectively. Stable microcapsules were observed during gastric digestion, but there was no significant difference in protein release/hydrolysis among samples until 55 min of gastric digestion. Formulation Y showed less protein release after 74 min (40.46 %) due to the free SPP being available and positively charged at pH 2-4, resulting in interactions with other constituents of the yoghurt, which delayed its release/hydrolysis. The total release of protein and free NH2 by the end of intestinal digestions ranged between 46.56-61.15 % and 0.83-1.57 µmol/g protein, respectively. A higher release of free fatty acids from formulation Y occurred at the end of intestinal digestion, implying that coacervates promoted the delayed release of encapsulated oil. In summary, incorporating protein-polysaccharides-based coacervates in yoghurt enabled the delay of the digestion of encapsulated lipids but accelerated the digestion of protein, suggesting a promising approach for various food applications.

Chemical Composition and Geographic Variation of Cold Pressed Balanites aegyptiaca Kernel Oil.

With the increasing impacts of climate change, establishing more sustainable and robust plants such as desert dates (Balanites aegyptiaca) seems to be necessary. Known for its resilience in arid conditions, this tree has the potential to become a more important food source, particularly for its potential to yield edible oil. This study characterized Balanites kernel oil (BKO) as a promising oil source in arid regions, studying the influence of geographical origin and environmental factors. Moroccan and Sudanese BKO samples were analyzed and compared with Mauritanian BKO. In the fatty acid profile, unsaturated fatty acids constituted over 70% of the BKO profile, with a predominance of linoleic acid (Li), oleic acid (Ol), palmitic acid (Pa), and stearic acid (St). Consequently, the predominant triacylglycerols were PaLiLi, PaLiOl, LiLiOl, OlLiOl, and StLiOl. α-Tocopherol dominated the tocochromanol composition (324 to 607 mg/kg), followed by γ-tocopherol (120 to 226 mg/kg), constituting 90% of the total tocochromanols. The total phytosterol content in BKO ranged from 871 to 2218 mg/kg oil, with ß-sitosterol dominating (58% to 74%). Principal Component Analysis revealed that the geographical origin significantly influences BKO composition, emphasizing environmental factors, particularly water deficit and/or temperatures. Notably, Moroccan BKO collected from an area characterized by high aridity and relatively low winter temperatures, showcased a unique profile in fatty acid, phytosterols, and tocochromanols. The valorization of BKO presents an opportunity for local agricultural development in arid regions and a role model for plant development and agricultural practices in other parts of the world.

Formation of amyloid fibrils from ovalbumin under Ohmic heating.

Ohmic heating (OH) is an alternative sustainable heating technology that has demonstrated its potential to modify protein structures and aggregates. Furthermore, certain protein aggregates, namely amyloid fibrils (AF), are associated with an enhanced protein functionality, such as gelation. This study evaluates how Ohmic heating (OH) influences the formation of AF structures from ovalbumin source under two electric field strength levels, 8.5 to 10.5 and 24.0-31.0 V/cm, respectively. Hence, AF aggregate formation was assessed over holding times ranging from 30 to 1200 sunder various environmental conditions (3.45 and 67.95 mM NaCl, 80, 85 and 90 °C, pH = 7). AF were formed under all conditions. SDS-PAGE revealed that OH had a higher tendency to preserve native ovalbumin molecules. Furthermore, Congo Red and Thioflavin T stainings indicated that OH reduces the amount of AF structures. This finding was supported by FTIR measurements, which showed OH samples to contain lower amounts of beta-sheets. Field flow fractioning revealed smaller-sized aggregates or aggregate clusters occurred after OH treatment. In contrast, prolonged holding time or higher treatment temperatures increased ThT fluorescence, beta-sheet structures and aggregate as well as cluster sizes. Ionic strength was found to dominate the effects of electric field strength under different environmental conditions.

Structure, controlled release mechanisms and health benefits of pectins as an encapsulation material for bioactive food components.

Encapsulation of food and feed ingredients is commonly applied to avoid the loss of functionality of bioactive food ingredients. Components that are encapsulated are usually sensitive to light, pH, oxygen or highly volatile. Also, encapsulation is also applied for ingredients that might influence taste. Many polymers from natural sources have been tested for encapsulation of foods. In the past few years, pectins have been proposed as emerging broadly applicable encapsulation materials. The reasons are that pectins are versatile and inexpensive, can be tailored to meet specific demands and provide health benefits. Emerging new insight into the chemical structure and related health benefits of pectins opens new avenues to use pectins in food and feed. To provide insight into their application potential, we review the current knowledge on the structural features of different pectins, their production and tailoring process for use in microencapsulation and gelation, and the impact of the pectin structure on health benefits and release properties in the gut, as well as processing technologies for pectin-based encapsulation systems with tailor-made functionalities. This is reviewed in view of application of pectins for microencapsulation of different sensitive food components. Although some critical factors such as tuning of controlled release of cargo in the intestine and the impact of the pectin production process on the molecular structure of pectin still need more study, current insight is that pectins provide many advantages for encapsulation of bioactive food and feed ingredients and are cost-effective.

Confined flow behavior under high shear rates and stability of oil/water high internal phase emulsions (HIPEs) stabilized by whey protein isolate: Role of protein concentration and pH.

High shear rheometry was used to investigate the rheological behavior of high internal phase emulsions (HIPEs) stabilized by whey protein isolate (WPI). The physical stability of HIPEs was tested at extremely high shear rates generated by decreasing the gap height between parallel plates. Viscosity and yield stress, at narrow gaps, increased with protein concentration due to tighter packing of smaller droplets. Structural breakdown and recovery of HIPEs were affected by protein concentration and pH. The hysteresis behavior of HIPEs was either thixotropic or anti-thixotropic and was determined by protein concentration, whereas pH affected the magnitude of anti-thixotropy. At pH 3, emulsions showed greater stability against extreme shear and creaming due to higher deformability of oil droplets and increased interdroplet interaction compared to neutral pH. Challenging the physical integrity of concentrated emulsions under high shear is an effective way to characterize microstructural changes and stability of HIPEs in foods.

The type of gum arabic affects interactions with soluble pea protein in complex coacervation.

Complex coacervation is an encapsulation process involving two oppositely charged biopolymers. Since different compositions of gum arabic may affect its interaction with protein, we studied the complex coacervation of two types of gum arabic (GA) (Acacia senegal-GA1 and Acacia seyal-GA2) with soluble pea protein (SPP) through Zeta potential, turbidity, morphology, the secondary structure of SPP, UV/vis absorbance and thermodynamic parameters. The maximum formation of coacervates occurred at SPP:GA 3:1 (w/w) and pH 3.5-4.0 with changes in the secondary structure of SPP. GA1 combination resulted in higher binding constant, implying a stronger affinity between SPP and GA1. Entropy of 0.7 and 0.5 kJ/mol.K, and enthalpy of -151 and -95.5 kJ/mol were obtained for SPP:GA1 and SPP:GA2. The complex coacervation was spontaneous as proved by the negative values of the Gibbs free energy. GA1 resulted in stronger interactions with SPP, offering new alternatives for encapsulation of bioactive compounds.

Zinc availability from zinc-enriched yeast studied with an in vitro digestion/Caco-2 cell culture model.

BACKGROUND: Organic zinc sources for the treatment of zinc deficiency or as a supplement to a specific diet are increasingly needed. Zinc-enriched yeast (ZnYeast) biomass is a promising nutritional supplement for this essential micronutrient. However, these products are not yet authorized in the European Union and a clear position from the European Food Safety Authority on the use of ZnYeast as a zinc supplement is pending, demanding more data on its bioavailability. OBJECTIVE: The study aimed to produce a ZnYeast based on a Saccharomyces genus (S. pastorianus Rh), characterize its zinc enrichment quota, cellular distribution of zinc, and evaluate its zinc bioavailability after human digestion by comparing it to commonly used inorganic and organic zinc supplements (ZnO, ZnSO4, zinc gluconate, and zinc aspartate). METHOD AND MAIN FINDINGS: The zinc-enriched S. pastorianus Rh contained 5.9 ± 1.0 mg zinc/g yeast, which was predominantly localized on the cell surface according to its characterization on the microscale with scanning electron microscopy (SEM) with energy-dispersive X-ray (EDX). Combined experiments with a human in vitro digestion model and the in vitro intestinal cell model Caco-2 showed that intestinal zinc bioavailability of digested yeast biomass was comparable to the other zinc supplements, apart from ZnO, which was somewhat less bioavailable. Moreover, zinc released from digested ZnYeast was available for biological processes within the enterocytes, leading to mRNA upregulation of metallothionein, a biomarker of intestinal zinc status, and significantly elevated the cellular labile zinc pool. CONCLUSIONS: Our findings demonstrated that ZnYeast represents a suitable nutritional source for organically bound zinc and highlighted optimization strategies for future production of dietary ZnYeast.

Advances of plant-based structured food delivery systems on the in vitro digestibility of bioactive compounds.

Food researchers are currently showing a growing interest in in vitro digestibility studies due to their importance for obtaining food products with health benefits and ensuring a balanced nutrient intake. Various bioactive food compounds are sensitive to the digestion process, which results in a lower bioavailability in the gut. The main objective of structured food delivery systems is to promote the controlled release of these compounds at the desired time/place, in addition to protecting them during digestion processes. This review provides an overview of the influence of structured delivery systems on the in vitro digestive behavior. The main delivery systems are summarized, the pros and cons of different structures are outlined, and examples of several studies that optimized the use of these structured systems are provided. In addition, we have reviewed the use of plant-based systems, which have been of interest to food researchers and the food industry because of their health benefits, improved sustainability as well as being an alternative for vegetarian, vegan and consumers suffering from food allergies. In this context, the review provides new insights and comprehensive knowledge regarding the influence of plant-based structured systems on the digestibility of encapsulated compounds and proteins/polysaccharides used in the encapsulation process.

Formation of Secondary and Tertiary Volatile Compounds Resulting from the Lipid Oxidation of Rapeseed Oil.

The lipid oxidation of fats and oils leads to volatile organic compounds, having a decisive influence on the sensory quality of foods. To understand formation and degradation pathways and to evaluate the suitability of lipid-derived aldehydes as marker substances for the oxidative status of foods, the formation of secondary and tertiary lipid oxidation compounds was investigated with gas chromatography in rapeseed oils. After 120 min, up to 65 compounds were detected. In addition to secondary degradation products, tertiary products such as alkyl furans, ketones, and aldol condensation products were also found. The comparison of rapeseed oils, differing in their initial peroxide values, showed that the formation rate of secondary compounds was higher in pre-damaged oils. Simultaneously, a faster degradation, especially of unsaturated aldehydes, was observed. Consequently, the formation of tertiary products (e.g., alkyl furans, aldol adducts) from well-known lipid oxidation products (i.e., propanal, hexanal, 2-hexenal, and 2-nonenal) was investigated in model systems. The experiments showed that these compounds form the new substances in subsequent reactions, especially, when other compounds such as phospholipids are present. Hexanal and propanal are suitable as marker compounds in the early phase of lipid oxidation, but at an advanced stage they are subject to aldol condensation. Consequently, the detection of tertiary degradation products needs to be considered in advanced lipid oxidation.

Influence of the Carboxylic Function on the Degradation of d-Galacturonic Acid and Its Polymers.

Past investigations have shown high browning potential during the caramelization of sugar acids in comparison to reducing sugars. However, no approaches to elucidate the chemical mechanisms have been made. Therefore, this study aims to clarify the reasons for the high browning potential by measuring the mutarotation velocity and the elimination of CO2 during the heat treatment of uronic acids. Performed polarimetric experiments show that the mutarotation velocity of d-galacturonic acid exceeds that of d-galactose by a factor of nearly 4.5. However, the ring opening velocity is not the only parameter that differs between the two carbohydrate structures. Measurements of the release of CO2 of heated d-galacturonic acid at 60 °C show a steady increase, and after 48 h, 6% of degraded d-galacturonic acid has eliminated CO2. CO2 release was also found during the heating of pectin, indicating a decarboxylation reaction during thermal degradation. One of the degradation reactions postulated for the release of CO2 leads to α-ketoglutaraldehyde, which is responsible for the formation of several chromophoric substances.

Structure and adsorption behavior of high hydrostatic pressure-treated ß-lactoglobulin.

HYPOTHESIS: High hydrostatic pressure treatment causes structural changes in interfacial-active ß-lactoglobulin (ß-lg). We hypothesized that the pressure-induced structural changes affect the intra- and intermolecular interactions which determine the interfacial activity of ß-lg. The conducted experimental and numerical investigations could contribute to the mechanistic understanding of the adsorption behavior of proteins in food-related emulsions. EXPERIMENTS: We treated ß-lg in water at pH 7 with high hydrostatic pressures up to 600 MPa for 10 min at 20 °C. The secondary structure was characterized with Fourier-transform infrared spectroscopy (FTIR) and circular dichroism (CD), the surface hydrophobicity and charge with fluorescence-spectroscopy and ζ-potential, and the quaternary structure with membrane-osmometry, analytical ultracentrifugation (AUC) and mass spectrometry (MS). Experimental analyses were supported through molecular dynamic (MD) simulations. The adsorption behavior was investigated with pendant drop analysis. FINDINGS: MD simulation revealed a pressure-induced molten globule state of ß-lg, confirmed by an unfolding of ß-sheets with FTIR, a stabilization of α-helices with CD and loss in tertiary structure induced by an increase in surface hydrophobicity. Membrane-osmometry, AUC and MS indicated the formation of non-covalently linked dimers that migrated slower through the water phase, adsorbed more quickly due to hydrophobic interactions with the oil, and lowered the interfacial tension more strongly than reference ß-lg.

Interfacial properties of milk proteins: A review.

The interfacial properties of dairy proteins are of great interest to the food industry. Food manufacturing involves various environmental conditions and multiple processes that significantly alter the structure and colloidal stability of food materials. The effects of concentration, pH, heat treatment, addition of salts etc., have considerable influence on the surface activity of proteins and the mechanical properties of the interfacial protein films. Studies to date have established some understanding of the links between environmental and processing related parameters and their impacts on interfacial behavior. Improvement in knowledge may allow better design of interfacial protein structures for different food applications. This review examines the effects of environmental and processing conditions on the interfacial properties of dairy proteins with emphasis on interfacial tension dynamics, dilatational and surface shear rheological properties. The most commonly used surface analytical techniques along with relevant methods are also addressed.

FTIR analysis of ß-lactoglobulin at the oil/water-interface.

Knowledge about the critical interfacial concentration of a protein supports our understanding of the kinetic stability of an emulsion. Its determination is currently limited to either invasive or indirect methods. The aim of our study was the determination of the critical interfacial concentration of whey protein ß-lactoglobulin at oil/water-interfaces through fluorescence and pendant drop analysis and the comparison to an in situ Fourier-transform-infrared-spectroscopy (FTIR) method. Exponentially decreasing interfacial tension with increasing ß-lactoglobulin content (0.10-1.00 wt%) in pendant drop analysis could partly be confirmed by fluorescence spectra. A critical interfacial concentration of 0.20-0.31 wt% ß-lactoglobulin (1.80-2.69 mg/m2) in oil/water (5/95)-emulsions was determined via FTIR, analyzing the Amide I/Amide II peak intensity ratio. This was confirmed by the increasing formation of intermolecular ß-sheets, revealed by second derivative spectra. With this FTIR method we expand current options to investigate the interfacial behavior of food proteins by determination of secondary structure elements.

Oat protein concentrate as alternative ingredient for non-dairy yoghurt-type product.

BACKGROUND: During the industrial production of ß-glucan, a protein-rich fraction remains as a by-product. Recovery of this protein as oat protein concentrate (OPC) results in a source of cereal protein for food and improves the overall economy of the process. In this study, a yoghurt-type product is developed by lactic acid fermentation of an OPC suspension after subjection to heat treatment to assure starch gelatinization. RESULTS: In detail, the process of yoghurt production involved an initial heating step to 90 °C, followed by 24 h fermentation with a starter culture consisting of Lactobacillus delbrueckii subsp. bulgaricus und Streptococcus thermophilus. The resulting yoghurt-type product was mildly sour (pH 4.2) with a certain amount of lactic acid (3.3 ± 0.2 g kg-1 ) and contained 4.9 × 106 cfu g-1 lactobacillus after 24 h fermentation. Scanning electron microscopy revealed a porous network presumably built up from the gelatinized starch fraction containing aggregated structures, between which were assumed to be aggregated oat proteins. Moreover, to a limited extent, proteolysis occurred during fermentation. Thus some of the proteolytic enzymes present in the yoghurt culture cleaved oat protein and released peptides. However, the effect on essential amino acids was small. CONCLUSION: The results of this study provide a deeper knowledge into the role of starch and protein in fermented OPC yoghurts. The structure of fermented OPC verifies the applicability of oat protein as an alternative source for yoghurt-type products. © 2019 Society of Chemical Industry.

Conformational state and charge determine the interfacial stabilization process of beta-lactoglobulin at preoccupied interfaces.

Amphiphilic properties enable proteins like ß-lactoglobulin to stabilize oil/water-interfaces and provide stability in food-related emulsions. During emulsification, the protein undergoes three stages: (I) migration through bulk phase, (II) adsorption, and (III) interfacial rearrangement at the oil/water-interface - the kinetics of which require further research. Therefore, the aim of our study was the analytical and computational investigation of stage (I) and (II) as a function of the interfacial preoccupation, conformational state and charge of ß-lactoglobulin. For this purpose, the adsorption of ß-lactoglobulin (at pH 7, pH 7 containing 0.1 M NaCl, and pH 9) at increasingly preoccupied oil/water-interfaces has been compared through measuring interfacial tension and ζ-potential and through running molecular dynamics simulations. With increasing interfacial preoccupation, (I) the migration via lag time increased and (II) the adsorption rate decreased. The (II) adsorption rate was highest for ß-lactoglobulin containing NaCl, due to dense packing and electrostatic screening. ß-lactoglobulin at pH 7 reached a lower adsorption rate than the more negatively charged ß-lactoglobulin at pH 9, due to exposure of hydrophobic regions that had a greater effect on adsorption rates than electrostatic repulsion. Our research contributes to a profound understanding of the interfacial stabilization mechanism of proteins at oil/water-interfaces, necessary to characterise and control emulsification processes.

Towards By-Product Utilisation of Pea Hulls: Isolation and Quantification of Galacturonic Acid.

In order to evaluate by-products from food processing as alternative raw materials for pectin extraction, their amount of galacturonic acid (GalA) has to be analysed as a marker for pectin content. In the present study, significant differences in GalA release using different digestion methods are shown for pea hulls, as an example of by-products with a high content of cellulose. Complete digestion of the fibre matrix was assumed for Saeman hydrolysis as a reference protocol. Significantly lower GalA release was achieved by a treatment with trifluoracetic acid (TFA). An alternative treatment with ethylenediaminetetraacetic acid (EDTA) at pH 11 followed by an enzymatic digestion at pH 4.5 using a combination of polygalacturonase (Vegazyme M) and cellulase (Celluclast 1.5L) resulted in a similar release of GalA compared to Seaman hydolysis. Pea hull samples, analysed by this alternative protocol, showed on average a GalA content of 11.2%. Therefore, pea hulls may serve as new raw material for pectin extraction.

Formation of Phenolic Compounds from d-Galacturonic Acid.

Aqueous d-galacturonic acid (d-GalA) model systems treated at 130 °C at different pH values show an intense color formation, whereas other reducing sugars, such as d-galactose (d-Gal), scarcely react. GC-MS measurements revealed the presence of several phenolic compounds: e.g., 3,8-dihydroxy-2-methyl-4 H-chromen-4-one (chromone) and 2,3-dihydroxybenzaldehyde (2,3-DHBA). These phenolic compounds, especially 2,3-DHBA, possess an intense browning potential and cannot be found within heated model solutions of reducing sugars. Investigations regarding the formation of these substances show that α-ketoglutaraldehyde plays an important role as an intermediate product. In addition, MS analysis of model systems of norfuraneol in combination with 2,3-DHBA showed the formation of oligomers that could also be detected in d-GalA model systems, leading to the assumption that, in addition to reductic acid, these compounds are jointly responsible for the strong color formation during the heat treatment of d-GalA.