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.

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.

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.

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.

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.

Efficient capturing and sensitive detection of hepatitis A virus from solid foods (green onion, strawberry, and mussel) using protamine-coated iron oxide (Fe3O4) magnetic nanoparticles and real-time RT-PCR.

Hepatitis A virus (HAV) continues to be a public health concern and has caused large foodborne outbreaks and economic losses worldwide. Rapid detection of HAV in foods can help to confirm the source of outbreaks in a timely manner and prevent more people getting infected. In order to efficiently detect HAV at low levels of contamination in foods, rapid and easy-to-use techniques are required to separate and concentrate viral particles to a small volume. In the current study, HAV particles were eluted from green onion, strawberry, and mussel using glycine buffer (0.05 M glycine, 0.14 M NaCl, 0.2% (v/v) Tween 20, pH 9.0) and suspended viral particles were captured using protamine-coated magnetic nanoparticles (PMNPs). This process caused a selective concentration of the viral particles, which could be followed by quantitative real-time RT-PCR analysis. Results showed that pH, NaCl concentration, and PMNP amount used for the capturing had significant effects on the recovery efficiency of HAV (P < 0.05). The highest recovery rate was obtained at pH 9.0, 0.14 M NaCl, and 50 µL of PMNPs. The optimized PMNP capturing method enabled the rapid capture and concentration of HAV. A sensitive real-time RT-PCR test was developed with detection limits of 8.3 × 100 PFU/15 g, 8.3 × 101 PFU/50 g, and 8.3 × 100 PFU/5 g of HAV in green onion, strawberry, and mussel, respectively. In conclusion, the PMNP method is rapid and convenient in capturing HAV from complex solid food samples and can generate concentrated HAV sample solutions suitable for high-sensitivity real time RT-PCR detection of the virus.

Freezing as a solution to preserve the quality of dairy products: the case of milk, curds and cheese.

When thinking of the freezing process in dairy, products consumed in frozen state, such as ice creams come to mind. However, freezing is also considered a viable solutions for many other dairy products, due to increasing interest to reduce food waste and to create more robust supply chains. Freezing is a solution to production seasonality, or to extend the market reach for high-value products with otherwise short shelf life. This review focuses on the physical and chemical changes occurring during freezing of milk, curds and cheeses, critical to maintaining quality of the final product. However, freezing is energy consuming, and therefore the process needs to be optimized to maintain product's quality and reduce its environmental footprint. Furthermore, the processing steps leading to the freezing stage may require some changes compared to traditional, fresh products. Unwanted reactions occur at low water activity, and during modifications such as ice crystals growth and recrystallization. These events cause major physical destabilizations of the proteins due to cryoconcentration, including modification of the colloidal-soluble equilibrium. The presence of residual proteases and lipases also cause important modifications to the texture and flavor of the frozen dairy product.

Time-dependent aggregation of casein micelle concentrates.

This research focused on understanding physical and chemical changes occurring to concentrated milk protein suspensions as a function of time. Skim milk (untreated and heat treated at 90°C for 10 min) was concentrated at 6 times the original volume using osmotic stressing, a noninvasive concentration method, maintaining the serum composition as close as possible to that of native milk. A protease inhibitor cocktail, with broad specificity for the inhibition of serine, cysteine, aspartic proteases, and aminopeptidases, was added in selected samples. Within 9 d of storage at 4°C, the apparent viscosity increased markedly for both unheated and heated concentrated milk, but not for those in the presence of protease inhibitors. However, only unheated milk showed a significant increase in the apparent diameter of the casein micelles. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry measurements indicated a significantly lower extent of proteolysis in heated than in unheated samples. The microstructure of the aggregates was observed using field emission scanning electron microscopy, and unheated samples clearly showed aggregation of casein micelles with storage time. In heated samples, aggregation was instead triggered by heat-induced protein-protein interactions.

Gut Microbiome and Degradation Product Formation during Biodegradation of Expanded Polystyrene by Mealworm Larvae under Different Feeding Strategies.

Polystyrene (PS) is a plastic polymer extensively used for food packaging. PS is difficult to decompose and has low recycling rates, resulting in its accumulation in the environment, in the form of microplastic particles causing pollution and harming oceans and wildlife. Degradation of PS by mealworms (Tenebrio molitor) has been suggested as a possible biological strategy for plastic contamination; however, the biodegradation mechanism of PS by mealworms is poorly understood. It is hypothesized that the gut microbiome plays an important role in the degradation of PS by mealworms. This study carried out a comparative analysis of the gut microbiome of Tenebrio molitor larvae under different feeding strategies, and of the formation of degradation compounds (monomers, oligomers). A diet of bran:PS at 4:1 and 20:1 ratios was tested. The diet with the low ratio of bran:PS led to the presence of higher amounts of these compounds, compared to that with the high ratio. In addition, it was demonstrated that the addition of H2O significantly improved the biodegradation of PS monomer and oligomer residues, which could be identified only in the frass. The protein and nitrogen contents in insects' biomass and frass varied amongst treatments. The diets resulted in differences in the gut microbiota, and three potential bacterial strains were identified as candidates involved in the biodegradation of PS.

Effect of frozen and refrigerated storage on proteolysis and physicochemical properties of high-moisture citric mozzarella cheese.

High-moisture mozzarella is one of the most-exported Italian cheeses worldwide, but its quality is affected by storage. Freezing is regarded as a solution to decrease product waste, extend market reach, and increase convenience, but its effect on quality has to be estimated. In this study, the details related to proteolysis, physicochemical properties, and sensory quality parameters of high-moisture mozzarella as a function of frozen storage (1, 3, and 4 mo) and subsequent refrigerated storage after thawing (1, 3, and 8 d) were evaluated. Frozen cheeses stored at -18°C showed a higher extent of proteolysis, as well as different colorimetric and sensory properties, compared with the fresh, nonfrozen control. Sensory evaluation showed the emergence of oxidized and bitter taste after 1 mo of frozen storage, which supports the proteolysis data. The extent of proteolysis of frozen-stored cheese after thawing was greater than that measured in fresh cheese during refrigerated storage. These results help better understand the changes occurring during frozen storage of high-moisture mozzarella cheese and evaluate possible means to decrease the effect of freezing on the cheese matrix.

Diafiltration affects the gelation properties of concentrated casein micelle suspensions obtained by filtration.

Using membrane filtration it is possible to selectively concentrate proteins and, in the case of microfiltration, concentrate casein micelles. During filtration, water is often added and this practice, called diafiltration, causes further release of permeable components and maintains filtration efficiency. Filtration causes changes in composition of the protein as well as the soluble phase, including soluble calcium, which is a critical factor controlling the gelation properties of the casein micelles in milk. It was hypothesized that concentrates obtained using membrane filtration with or without diafiltration would have different gelation behavior. To test this hypothesis, two concentrates of similar casein micelle volume fraction were prepared, using spiral wound polymeric microfiltration membranes with a 800 kDa molecular weight cutoff, with or without diafiltration. The concentrates showed a gelation behavior comparable to that of skim milk, with a similar gelation time and with a higher firmness, due to the higher number of protein linkages in the network. In contrast, the hydrolysis of κ-casein by chymosin and casein aggregation were inhibited in diafiltered casein micelle suspensions. When the concentrates were recombined with the original skim milk to a final concentration of 5% protein, which re-established a similar soluble phase composition, differences in gelation behavior were no longer observed: both treatments showed similar gelation time and gel firmness. These results confirmed that membrane filtration can result in concentrates with different functionality, and that ionic environmental conditions are critical to the aggregation behavior of casein micelles. This is of particular significance in industrial settings where these fractions are used as a way to standardize proteins in cheese making.

Concentration of hepatitis A virus in milk using protamine-coated iron oxide (Fe3O4) magnetic nanoparticles.

Hepatitis A virus (HAV) continues to be the leading cause of viral hepatitis. HAV outbreaks have been linked to the consumption of milk, but methods for HAV detection in milk are very limited. We developed a method to concentrate HAV in milk using protamine-coated iron oxide (Fe3O4) magnetic nanoparticles (PMNPs). In this study, protamine was covalently coated on the surface of the MNPs (20-30 nm) by a three-step chemical reaction. The successful linkage of protamine to the MNPs was confirmed by Fourier transform infrared spectroscopy (FTIR), zeta potential, and transmission electron microscopy (TEM). When used for concentrating HAV from 40 mL of milk, 50 µL of PMNPs were added to the sample and mixed for 20 min by gentle rotation, followed by a magnet capture for 30 min. The captured PMNPs were washed with glycine buffer (0.05 M glycine, 0.14 M NaCl, 0.2% (v/v) Tween 20, pH 9.0) and HAV RNA was extracted using the QIAamp MinElute Virus Spin Kit and quantified by real-time RT-PCR. The method showed a detection limit of 8.3 × 100 PFU of HAV in milk. The whole concentration procedure could be completed in approximately 50 min. The developed method was simple, inexpensive, and easy-to-perform.

Short communication: Determination of the whey protein index in milk protein concentrates.

Milk protein concentrates are common ingredients in the dairy industry, with varying processing histories and composition. The objective of this research was to determine the feasibility of using the whey protein nitrogen (WPN) index, a well-established index for skim milk powder and nonfat dry milk, as a quality parameter for milk protein concentrates. The WPN index is a value based on the moisture-adjusted weight of skim milk powder. We hypothesized that WPN, even when standardized based on protein, may change depending on solubilization conditions of milk protein concentrates because of differences in solubilization conditions or processing history. The WPN was measured for model concentrates with different thermal history or reconstitution conditions. The WPN was not affected by an increased concentration of soluble casein in the dispersions nor after solubilization of the powder at 22 or 60°C. All reconstituted samples were standardized for protein. The WPN was also in full accordance with residual native protein measured by chromatography.

Invited review: Understanding the behavior of caseins in milk concentrates.

The colloidal properties of the casein micelles play a major role in the structural properties of milk protein concentrates. Because of their great technological importance, the structural-functional relationships of casein micelles have been studied for decades in skim milk; however, novel ingredients are now available with higher protein concentrations and varying in composition. The colloidal behavior of caseins in these systems is not fully understood. Concentrates prepared with membrane technologies, and subjected to pre- or post-modifications that affect their technological functionality, have become increasingly widespread. This has created large opportunities for innovation and generation of value-added ingredients. The manner in which caseins interact with themselves and the other components in these concentrates will affect the structure of the final matrix. During concentration by filtration, the interparticle distance between the micelles decreases considerably, increasing their spatial correlation and decreasing their diffusivity. Rearrangements occur due to changes in environmental conditions, such as ionic composition, osmotic stress, shear, pH, or heating temperature. This will have important consequences on bulk viscosity of the concentrates, as well as on the mode of formation of structures' building blocks. This paper aims at highlighting some of the important factors affecting the colloidal structure of casein micelles, their destabilization and network formation, namely, processing history, volume fraction, composition of the serum phase, and ionic equilibrium. Understanding these factors will lead to a better quality control of dairy ingredients and to the development of a new generation of ingredients with targeted functionality.

A comparison of the heat stability of fresh milk protein concentrates obtained by microfiltration, ultrafiltration and diafiltration.

The objective of this work was to evaluate the impact of changes during membrane filtration on the heat stability of milk protein concentrates. Dairy protein concentrates have been widely employed in high protein drinks formulations and their stability to heat treatment is critical to ensure quality of the final product. Pasteurized milk was concentrated three-fold by membrane filtration, and the ionic composition was modified by addition of water or permeate from filtration (diafiltration). Diafiltration with water did not affect the apparent diameter of the casein micelles, but had a positive effect on heat coagulation time (HCT), which was significantly longer (50 min), compared to the non diafiltered concentrates (about 30 min). UHT treatments increased the particle size of the casein micelles, as well as the turbidity of retentates. Differences between samples with and without diafiltration were confirmed throughout further analysis of the protein composition of the unsedimentable fraction, highlighting the importance of soluble protein composition on the processing functionality of milk concentrates.

Protein matrices ensure safe and functional delivery of rosmarinic acid from marjoram (Origanum majorana) extracts.

BACKGROUND: To understand the interactions between carriers and functional ingredients is crucial when designing delivery systems, to maximize bioefficacy and functionality. In this study, two different protein matrices were evaluated as means to protect the extract isolated from marjoram leaves (Origanum majorana), casein micelles from fresh skim milk and soy protein isolate (SPI). RESULTS: Marjoram extract was obtained from pressurization of ethanol and water solvent. Protein dispersions of casein and SPI (5 g L-1 each) with or without marjoram extract (0.1-3 mg mL-1 ) were prepared and homogenized. The physicochemical characterization of charge and entrapment efficiency were conducted. The results demonstrated that entrapment efficiency was highly dependent on the carrier itself where SPI formulations showed 20% higher affinity when compared to casein micelles. To investigate the physiological behaviour of the marjoram-protein dispersions, human macrophages were employed. A non-specific inflammatory response of macrophages stimulated with bacterial lipopolysaccharide was measured for TNF-α, IL-1ß and IL-6 cytokine secretion. CONCLUSION: Casein and SPI protein formulations warranted high bioefficacy of marjoram extract, showing their potential as safe carriers. © 2018 Society of Chemical Industry.

Short communication: Serum composition of milk subjected to re-equilibration by dialysis at different temperatures, after pH adjustments.

The objective of this work was to investigate the properties of casein micelles after pH adjustment and their re-equilibration to the original pH and serum composition. Re-equilibration was carried out by dialyzing against skim milk at 2 different temperatures (4 or 22 °C). Turbidity, the average radius of the casein micelles, and the composition of the soluble phase were measured at different pH values, ranging between 5.5 and 8. Acidification led to the solubilization of colloidal calcium phosphate and decrease of the average radius of the micelles. With re-equilibration, casein dissociation occurred. In milk with pH values greater than 6.0, the average radius was recovered after re-equilibration. At pH values greater than neutral, an increase of the radius of casein micelles and increased dissociation of the casein were found. After re-equilibration, the radius of micelles and soluble protein in the serum decreased. The results were not affected by the temperature of re-equilibration. The changes to the calcium phosphate equilibrium and the dissociation of the micelles will have important consequences to the functionality of casein micelles.

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.