Structural evolution of pea-derived albumins during pH and heat treatment studied with light and X-ray scattering.

Pea albumins are found in the side stream during the isolation of pea proteins. They are soluble at acidic pH and have functional properties which differ from their globulin counterparts. In this study, we have investigated the aggregation and structural changes occurring to pea albumins under different environmental conditions, using a combination of size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALS) and small-angle X-ray scattering (SAXS). Albumins were extracted from a dry fractionated pea protein concentrate by precipitating the globulin fraction at acidic pH. The albumins were then studied at different pH (3, 4, 4.5, 7, 7.5, and 8) values. The effect of heating at 90 °C for 1, 3, and 5 min on their structural changes was investigated using SAXS. In addition, size exclusion of the albumins showed 4 distinct populations, depending on pH and heating conditions, with two large aggregates peaks (∼250 kDa): a dimer peak (∼24 kDa) containing predominantly pea albumin 2 (PA2), and a monomer peak of a molar mass of about 12 kDa (PA1). X-ray scattering intensities as a function of q were modeled as polydisperse spheres, and their aggregation was followed as a function of heating time. Albumins was most stable at pH 3, showing no aggregation during heat treatment. While albumins at pH 7.5 and 8 showed aggregation after heating, solutions at pH 4, 4.5, and 7 already contained aggregates even before heating. This work provides new knowledge on the overall structural development of albumins under different environmental conditions, improving our ability to employ these as future ingredients in foods.

In situ single-droplet analysis of emulsified fat using confocal Raman microscopy: insights into crystal network formation within spatial resolution.

Fat crystallization is one of the predominant factors influencing the structure and properties of fat-containing emulsions. In the present study, the role of emulsifiers on fat crystallization dynamics within droplet multiphase systems was evaluated via single-droplet analysis, taking advantage of the non-destructive properties of confocal Raman microscopy. Palm oil droplets dispersed in water were used as a model system, due to palm oil's well-known crystallization properties. Emulsion droplets of the same size were generated using two different emulsifiers (Whey Protein Isolate and Tween 60), at various concentrations. Fast and slow cooling treatments were applied to affect fat crystallisation and network formation as well as droplet morphology, and crystallization dynamics. Raman imaging analysis demonstrated that the chemical structure and concentration of the emulsifier significantly influenced both crystal nucleation within the droplets, as well as the spatial distribution and morphology of the fat crystal network. Additionally, analysis of the spectra of the crystallized phase provided essential information regarding the impact of the emulsifiers on the microstructure, degree of structural order, and structural arrangements of the fat crystal networks. Furthermore, by performing single droplet analysis during cooling it was possible to observe shape distortions in Tween 60 stabilized droplets, as a consequence of the formation of a three-dimensional network of fat crystals that strongly interacted with the interface. On the other hand, the droplets retained their shape when whey proteins were absorbed at the interface. Confocal Raman microscopy, in combination with polarized light microscopy, is, therefore, a well-suited tool for in situ, single-droplet analysis of emulsified oil systems, providing essential information about emulsified fat crystallization dynamics, contributing to better understanding and designing products with enhanced structure and function.

Lupin protein-stabilized oil droplets contribute to structuring whey protein emulsion-filled gels.

This work aimed to understand the role of lupin protein or mixed lupin-whey protein stabilized oil droplets on the texture and microstructure of a heat-induced whey protein gel. Protein-stabilized emulsions were compared to surfactant-stabilized emulsions to investigate the potential of their interfacial interactions to impart unique structures in the filled gels. The structure development was followed in situ using rheology and the final heat-induced gels were characterized by small and large amplitude oscillatory rheology and confocal microscopy. The development of the gel modulus as well as the final gel properties were linked to the type of interactions between the whey protein matrix and the protein adsorbed at the oil interface. The final gels were selectively dissolved in various buffers, and the results showed that replacing interfacial whey protein with lupin protein resulted in a reduced amount of disulfide bridges, explaining the softer gel in the lupin containing gels compared to those with whey protein. Non-covalent interactions were the main forces involved in the formation of actively filled droplets in the gel network. This work demonstrated that by modulating the interfacial composition of the oil droplets, differing gel structures could be achieved due to differences in the protein-protein interactions between the continuous and the interfacial phase. There is therefore potential for the development of innovative products using lupin-whey protein mixtures, by careful control of the processing steps and the matrix composition.

Role of the pea protein aggregation state on their interfacial properties.

HYPOTHESIS: Plant protein ingredients from similar sources can vary in functionality not only because of compositional differences, but also because of differences in their structure depending on their processing history. It is essential to understand these distinctions to develop novel food emulsion using plant proteins. It is hypothesized that differing interfacial properties can be attributed to their structures, aggregation, and colloidal states. EXPERIMENTS: The adsorption behavior of a commercial protein isolate, homogenized or non-homogenized, was compared to a mildly extracted isolate to evaluate the effect of aggregation state and structural differences. After characterization of the particle size and protein composition, the interfacial properties were compared. FINDINGS: Atomic force microscopy provided evidence of interfaces packed with protein oligomers regardless of the treatment. Differences in adsorption kinetics and interfacial shear rheology depending on oil polarity suggested different interfacial structures. A polydisperse mixture of protein oligomers resulted in increased rearrangements and protein-protein interactions at the interface. Homogenization of commercial proteins resulted in a lower interfacial tension and less elastic interfaces compared to those of native proteins due to the presence of larger aggregates. This study highlights how the interfacial properties can be related to the protein aggregation state resulting from differences in processing history.

Pectin polysaccharide contribution to oleosome extraction after wet milling of rapeseed.

Oleosomes are lipid composites providing energy storage in oilseeds. They possess a unique structure, comprised of a triglyceride core stabilized by a phospholipid-protein membrane, and they have shown potential to be used as ingredients in several food applications. Intact oleosomes are extracted by an aqueous process which includes soaking, milling, and gravitational separation. However, the details of the complexes formed between oleosomes, proteins and pectin polysaccharides during this extraction are not known. It was hypothesized that pectins play an important role during the oleosome separation, and different proteins will be complexed on the surface of the oleosomes, depending on the pH of extraction. Rapeseed extracts were treated with and without pectinase (Pectinex Ultra SP-L) and extracted at pH 5.7 or 8.5, as this will affect electrostatic complexation. Acidic conditions led to co-extraction of storage proteins, structured as dense oleosome emulsions, stabilized by a network of proteins and polysaccharides. Pectinase intensified this effect, highlighting pectic polysaccharides' role in bridging interactions among proteins and oleosomes under acidic conditions. The presence of this dense interstitial layer around the oleosomes protected them from coalescence during extraction. Conversely, under alkaline conditions, the extraction process yielded more purified oleosomes characterized by a larger particle size, most likely due to coalescence. Nevertheless, pectinase addition at pH 8.5 mitigated coalescence tendencies. These results contribute to a better understanding of the details of the colloidal complexes formed during extraction and can be used to modulate the composition of the extracted fractions, with significant consequences not only for yields and purity but also for the functional properties of the ingredients produced.

Shaping Future Foods through Fermentation of Side Streams: Microbial, Chemical, and Physical Characteristics of Fermented Blends from Sunflower Seed Press Cake and Cheese Whey.

The current food system suffers from the inefficient use of resources, including the generation of side streams of low economic value that still contain nutritional components. One potential approach to reach a more sustainable food system is to reintroduce such side streams into a circular value chain and valorise them in novel food products, preferably in an unrefined or minimally refined manner. Blending side streams from different industries might be a suitable way to improve the nutritional value of the final matrix. In this study, sunflower seed press cake and cheese whey were combined to obtain matrices containing valuable proteins, structuring polysaccharides, as well as lactose and minerals facilitating fermentation with three different co-cultures of lactic acid bacteria and yeasts. Fermentation for 48 h at 26 °C decreased the pH from ~6.3 to ~4.7 and enhanced the storage stability of the blends with no effect on their rheological properties and microstructure. This research demonstrates the potential of fermentation as a mean to stabilise side stream blends while only minimally affecting their physical appearance.

A Shared Perspective on in Vitro and in Vivo Models to Assay Intestinal Transepithelial Transport of Food Compounds.

Assessing nutrient bioavailability is complex, as the process involves multiple digestion steps, several cellular environments, and regulatory-metabolic mechanisms. Several in vitro models of different physiological relevance are used to study nutrient absorption, providing significant challenges in data evaluation. However, such in vitro models are needed for mechanistic studies as well as to screen for biological functionality of the food structures designed. This collaborative work aims to put into perspective the wide-range of models to assay the permeability of food compounds considering the particular nature of the different molecules, and, where possible, in vivo data are provided for comparison.

Compositional Attributes of Blue Lupin (Lupinus angustifolius) Seeds for Selection of High-Protein Cultivars.

Lupin is a promising protein source with a high protein concentration. Breeding efforts have resulted in the development of varieties low in quinolizidine alkaloids. The objective of this work was to evaluate 22 different blue lupin genotypes for a high protein concentration and low content of antinutritional alkaloids. These genotypes were grown under uniform controlled environmental and soil conditions, and the harvested seeds were evaluated for their composition. The low phosphorus content confirmed that the phytic acid presence was low in lupin, especially compared to other legumes. Furthermore, some of the varieties had less than 200 ppm alkaloids. Lupin proteins were rich in leucine and lysine, with the lowest amino acid concentration being methionine. There were significant differences in the protein concentration and recovery. This work demonstrated that an approach for selection of genotypes should be based on not only agronomic yields but also nutritional phenotypes, driving better decision making on future varietal selection.

Compartmentalization of lutein in simple and double emulsions containing protein nanoparticles: Effects on stability and bioaccessibility.

Delivery systems designed through protein stabilized emulsions are promising for incorporating carotenoids in different products. Nevertheless, the versatility in structures of such systems raises questions regarding the effect of the bioactive compound localization on their bio-efficacy, in particular for double emulsions. In this context, the aims of this study were to determine the impact of the localization of lutein in different water/oil/water double emulsions versus a single oil/water emulsion on the stability and in vitro bioaccessibility of lutein, a lipophilic carotenoid. The inner aqueous phase, which contained whey protein isolate (WPI) nanoparticles obtained by desolvation, was emulsified in sunflower oil stabilized by polyglycerol polyricinoleate (PGPR). The primary emulsion was then emulsified in a continuous aqueous phase containing whey protein isolate (WPI) and xanthan gum, the latter to increase the viscosity of the outer phase and delay creaming. Lutein was incorporated using different strategies: (1) lutein entrapped by WPI nanoparticles within the inner water phase of a double emulsion (W-L/O/W); (2) lutein incorporated into the oil phase of the double emulsion (W/O-L/W); (3) lutein incorporated in the oil phase of a single emulsion (O-L/W). All systems contained similar whey protein concentrations, as well as all other stabilizers. W-L/O/W sample showed the lowest lutein stability against light exposure during storage, and the highest lutein bioaccessibility after in vitro digestion, for freshly made samples. Furthermore, the in vitro bioaccessibility of lutein incorporated into the single emulsion was considerably lower than those observed for the double emulsions. The results reinforce the importance of designing appropriate structures for delivering improved stability and bioaccessibility of bioactive compounds.

Novel details on the dissociation of casein micelle suspensions as a function of pH and temperature.

Membrane filtration is a widespread process for fractionation and recombination of milk components. Although the dissociation of micellar caseins has been studied in detail in skim milk, it is important to better understand the dissociation dynamics occurring between the colloidal and noncolloidal fractions in systems of modified composition. This research aimed at understanding the dissociation of casein proteins in micellar fractions depleted of whey proteins. Casein micelle dispersions were tested at neutral pH and pH 6 (using glucono-δ-lactone as acidulant), after incubation at 4°C or 22°C, and compared with skim milk. The ionic composition of the serum phase was measured using inductively coupled plasma-mass spectrometry, and the protein distribution analyzed using reversed phase-HPLC coupled with mass spectrometry. When incubated at 22°C, there were no differences in casein micelle dissociation between skim milk and whey protein-depleted micelles (∼2.6% dissociated casein). No additional dissociation occurred by lowering the pH from 6.8 to 6 at 22°C, albeit there were more soluble ions at low pH (71% Ca and 65% P). At 4°C, there was an increased amount of ß-casein found in the serum phase (23-33% of total ß-casein). In addition, there was an uneven dissociation behavior of the various genetic ß-casein variants, whereof A2 was more readily released with cooling. In skim milk, approximately 22%, 18%, and 14% of κ-, αS2, and αS1-caseins, respectively, were dissociated from the micellar phase upon cooling and acidification to pH 6.0. This was in contrast to whey protein-depleted casein suspensions, in which only 6%, 5%, and 3% of κ-, αS2, and αS1-caseins, respectively, had dissociated. The results suggested that the whey proteins in the serum phase play a role in the equilibrium between colloidal and soluble caseins in milk. This is of great relevance in processes such as cold membrane fractionation, where more attention should be given to the protein composition in the serum phase, especially when concentration is combined with fractionation of the serum proteins.

Analysis of oligomers to assess exposure to microplastics from foods. A perspective.

There is an emerging interest in evaluating the presence of microplastic (MP) and nanoplastic (NP) residues in food. Despite their potential threat to human health, there is still a need for harmonized methods to evaluate and quantify their presence. Incomplete polymerization may occur during the production of plastic. Conversely, oligomers are formed during chemical, mechanical, or enzymatic depolymerization. Oligomers are a few nanometers in size. Recent advances in analytical chemistry have enabled the quantification and identification of these oligomers in various complex biological matrices. Therefore, we propose that the specific nanosized oligomers can be considered markers for the presence of MPs/NPs. This advance may facilitate a broader perspective for the assessment of MPs/NPs exposure, leading to the evaluation of food safety and associated risks to humans.

Transmembrane Pressure during Micro- and Diafiltration of Milk Affects the Release of Non-Sedimentable Caseins.

Membrane filtration, especially in combination with diafiltration, can affect the colloidal structure of casein micelles in milk and concentrated milks. The partial dissociation of casein proteins from the casein micelles into the serum phase has been shown to depend on diafiltration conditions. This dissociation can affect the technological functionality of the milk concentrates. The present study aimed at determining the contribution of the gel layer deposited onto the membrane during filtration in the colloidal equilibrium between soluble and micellar caseins. Skimmed milk was concentrated by microfiltration combined with diafiltration using a cross-flow spiral-wound membrane at two transmembrane pressure (TMP) levels, causing differences in the extent of the gel layer formed. Non-sedimentable casein aggregates were formed to a greater extent at a low TMP compared to a high operating TMP. This difference was attributed to the greater compression of the deposit layer during filtration at a high TMP. This study contributes new knowledge to the understanding of how to modulate the functionality of milk concentrates through the control of processing conditions.

Coupling in vitro food digestion with in vitro epithelial absorption; recommendations for biocompatibility.

As food transits the gastrointestinal tract, food structures are disrupted and nutrients are absorbed across the gut barrier. In the past decade, great efforts have focused on the creation of a consensus gastrointestinal digestion protocol (i.e., INFOGEST method) to mimic digestion in the upper gut. However, to better determine the fate of food components, it is also critical to mimic food absorption in vitro. This is usually performed by treating polarized epithelial cells (i.e., differentiated Caco-2 monolayers) with food digesta. This food digesta contains digestive enzymes and bile salts, and if following the INFOGEST protocol, at concentrations that although physiologically relevant are harmful to cells. The lack of a harmonized protocol on how to prepare the food digesta samples for downstream Caco-2 studies creates challenges in comparing inter laboratory results. This article aims to critically review the current detoxification practices, highlight potential routes and their limitations, and recommend common approaches to ensure food digesta is biocompatible with Caco-2 monolayers. Our ultimate aim is to agree a harmonized consensus protocol or framework for in vitro studies focused on the absorption of food components across the intestinal barrier.

Confocal Raman microscopy to evaluate anisotropic structures and hydration development. Methodological considerations.

Confocal Raman microscopy is a promising technique to study structural complexity of multi-phase foods and soft materials. This technique overcomes the limitations of traditional microscopic techniques, such as the inability to identify water regions or to map the composition of various phases in situ, without sample disruption or the addition of specific dyes. The objective of this work was to carry out a systematic study on a well-understood model food, pizza cheese, establishing a methodology for data acquisition and handling for confocal Raman microscopy studies of anisotropic protein structures. The study demonstrated that conventional confocal microscopy remains an important tool to study the structure of protein networks. However, confocal Raman microscopy brings added value in the observation of components distribution, for example, water distribution in the protein phase during storage, using line scans or area imaging, and to detect spatial heterogeneities. This research compared different means of processing spectroscopic data, and demonstrates the critical importance of data handling, advocating for detailed methodological descriptions to better compare research results.

Molecular structures of high- and low-methoxy water-soluble polysaccharides derived from peas and their functions for stabilizing milk proteins under acidic conditions.

The structural and functional properties of two different pea water-soluble polysaccharides, a high methyl-esterified (HM-SPPS; degree of methyl esterification (DMe): 71.0 %) and low methyl-esterified SPPS (LM-SPPS; DMe: 25.2 %) were investigated. The two extracts did not vary in composition and showed a weight average molecular mass of about 1,000 kDa, as measured by size exclusion chromatography equipped with a multi-angle light scattering detector. Both HM-SPPS and LM-SPPS had similar sugar compositions, with arabinose 42.2-47.1 %, glucose 26.6-31.0 %, and galacturonic acid 17.5-18.0 %, as their main sugars. Their charge varied as a function of pH. The molecular structure was observed by a scanning probe microscope and showed a straight chain structure with small branches. The structure was similar to that already reported for polysaccharides from kidney bean. SPPS molecules interact with acidified milk protein particles at pH < 4.4. There were differences between the two SPPS. LM-SPPS could stabilize a model acidified milk dispersion with minimal aggregation between pH 3.6-4.4, while HM-SPPS showed the presence of bridging flocculation caused by polysaccharide's entanglements. It was concluded that SPPS stabilizes acidified protein by steric and electrostatic repulsion.

Cold ultrafiltered or microfiltered milk retentates: A systematic comparison of the effects of compositional differences on their gelation functionality.

The colloidal stability of casein micelles suspensions prepared using ultrafiltration (UF) and microfiltration (MF) was studied by testing acid- and rennet-induced destabilization. Skim milk and 4× (based on volume reduction) concentrates were obtained by processing under similar conditions, at temperatures below 10°C. Concentrates were subjected to different levels of diafiltration (DF), resulting in samples with comparable casein volume fractions but different amounts of proteins and ions in the serum phase. The novelty of the work is the systematic comparison of MF and UF concentrates of similar history. More specifically, concentrates similar in ionic composition but with or without serum proteins were compared, to evaluate whether whey proteins and ß-casein depletion from the micelles will play a role in the processing properties, or whether these are affected solely by the ionic balance. Microfiltered micelles' apparent diameter decreased by about 50 nm during the specific hydrolysis of κ-casein by chymosin, whereas those in skim milk control showed a decrease of about half that size. All concentrates subjected to extensive DF showed smaller hydrodynamic diameters, with reductions of ∼18 and 13 nm for MF and UF, respectively. Highly diafiltered UF retentates showed a delayed onset of rennet-induced gelation, due to low colloidal calcium, compared with other samples. Low-diafiltered samples showed weak storage modulus (∼1 Pa) after 60 min of onset of gelation. In addition, onset pH increased with diafiltration to ∼5.8 for UF and ∼6 for MF in high-diafiltered samples. These results clearly demonstrated that the functional properties of casein micelles change during membrane concentration, and this cannot be solely attributed to changes in ionic equilibrium.

Rapid Detection of Hepatitis A Virus in Foods Using a Bioluminescent Assay in Real-Time (BART) and Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Technology.

Foodborne hepatitis A infections have been considered as a major threat for public health worldwide. Increased incidences of hepatitis A virus (HAV) infection has been associated with growing global trade of food products. Rapid and sensitive detection of HAV in foods is very essential for investigating the outbreaks. Real-time RT-PCR has been most widely used for the detection of HAV by far. However, the technology relies on fluorescence determination of the amplicon and requires sophisticated, high-cost instruments and trained personnel, limiting its use in low resource settings. In this study, a robust, affordable, and simple assay, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in combination with a bioluminescence-based determination of amplification in real-time (BART), was developed for the detection of HAV in different food matrices, including green onion, strawberry, mussel, and milk. The efficiencies of a one-step RT-LAMP-BART and a two-step RT-LAMP-BART were investigated for the detection of HAV in different food matrices and was compared with that of real-time RT-PCR. The sensitivity of the RT-LAMP-BART assay was significantly affected by Mg2+ concentration (P < 0.05), in addition to primer quality. The optimal Mg2+ concentration was 2 mM for one-step RT-LAMP-BART and 4 mM for two-step RT-LAMP-BART. Compared with cartridge-purified primers, HPLC-purified primers could greatly improve the sensitivity of the RT-LAMP-BART assay (P < 0.05). For detecting HAV in different food matrices, the performance of two-step RT-LAMP-BART was comparable with that of real-time RT-PCR and was better than that of one-step RT-LAMP-BART. The detection limit of the two-step RT-LAMP-BART for HAV in green onion, strawberry, mussel, and milk was 8.3 × 100 PFU/15 g, 8.3 × 101 PFU/50 g, 8.3 × 100 PFU/5 g, and 8.3 × 100 PFU/40 mL, respectively. The developed RT-LAMP-BART was an effective, simple, sensitive, and robust method for foodborne HAV detection.

A model on an absolute scale for the small-angle X-ray scattering from bovine casein micelles.

Casein micelles extracted from milk are 100-400 nm-sized particles, made up of proteins and calcium phosphates, with the latter as colloidal calcium phosphate particles (CCPs) in a size range of 2-4 nm embedded in a protein network. The hierarchical structures give rise to a variation of scattering intensity over many orders of magnitude, which can be measured by small-angle X-ray scattering and static light scattering. Expressions for the scattering intensity of a general simple model for composite particles with polydispersities of overall size and subparticles are derived, and some approximations are checked by generating scattering data for systems generated by Monte Carlo simulations. Based on the simpler models, a new model has been developed for casein micelles, where the scattering is expressed on an absolute scale and where the concentrations of, respectively, protein and CCPs are used as constraints, providing a consistent model. The CCPs are modelled as oblate ellipsoids and the protein as star structures. Correlations between the substructures of CCPs and protein structures are taken into account in terms of partial structure factors. The overall structure as well as some heterogeneities at intermediate length scale are modelled as polydisperse spheres. The model fits the data very well on all length scales and demonstrates that both the scattering from CCPs and protein is important. Thus, the model provides a detailed description of the casein structure, which is consistent with the information available in the literature.

New opportunities for sustainable bioplastic development: Tailorable polymorphic and three-phase crystallization of stereocomplex polylactide by layered double hydroxide.

Stereocomplexation between enantiomeric poly(l-lactide) (PLLA) and poly(d-lactide) (PDLA) is a promising sustainable approach and gaining momentum to overcome the shortcomings of polylactide (PLA) for its use as a replacement for fossil-based plastics. Filler addition in tailoring the crystallization of stereocomplex PLA (SC-PLA) has attracted extensive attention; however, research has primarily focused on the heterogeneous nucleation effect of filler. The impact of filler on the chain behavior of SC-PLA during crystallization has not been exclusively discussed, and the rigid amorphous fraction (RAF) development remains unknown. In this study, the crystallization of PLLA/PDLA blends was modified by low loading of layered double hydroxide (LDH) (≤ 1 wt%) with the proposed local effect of such filler, and additional RAF development was incurred. In the early stage of crystallization, LDH facilitates the pairing of PLLA and PDLA and arrests the ordered SC pairs during the dynamic balance between the separation and pairing of racemic segments. This explains the severely suppressed homochiral (HC) crystallization, promoted SC crystallization, and additional RAF formation driven by the nucleation-induced chain ordering. This work, for the first time, highlights the role of LDH in creating SC-PLA with tailorable polymorphism and RAF, where the mechanism can be extended to other filler-type nucleator systems.