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ABSTRACT: This research used a customized laboratory setup to compare the filtration performance and fouling buildup during microfiltration with polymeric membranes of skim milk using 2 diafiltration media: ultrafiltration permeate and ultrapure water. Two filtration modes were evaluated: in stage 1, the diafiltration media was added in a 1:1 ratio, with the collection of permeate continuing until the initial protein concentration was restored. In stage 2, retentates and permeates were recycled to simulate fouling accumulation in a steady-state without altering the retentate composition. Utilizing water as the diafiltration medium resulted in higher flux and lower resistance values compared with using ultrafiltration permeate, irrespective of the filtration mode. The concentration had a significant effect on membrane resistance, with no noticeable time-dependent effect on fouling layer development after 60 min of filtration when the retentate composition remained constant. The protein composition of the permeate and extracted foulants were comparable between the 2 media, with caseins predominating in the fouling layer.

The aim of this work was to investigate the impact of the addition of salivary α-amylase on starch hydrolysis in protein-containing dispersions during an in vitro digestion process. In vitro digestion provides useful insights on the fate of nutrients during gastro-intestinal transit in complex food matrices, an important aspect to consider when developing highly nutritious foods. Many foods contain polysaccharides, and as their disruption in the gastric stage is limited, salivary α-amylase is often neglected in in vitro studies. A reference study on the effect of salivary α-amylase using one of the most advanced and complex in vitro digestion models (INFOGEST) is, however, not available. Hence, this work reports the gastrointestinal breakdown of three mixed dispersions containing whey protein isolate with different polysaccharides: potato starch, pectin from citrus peel and maize starch. The latter was also studied after heating. No polysaccharide or salivary α-amylase-dependent effect on protein digestion was found, based on the free NH2 and SDS-PAGE. However, in the heat-treated samples, the addition of salivary α-amylase showed a significantly higher starch hydrolysis compared to the sample without α-amylase, due to the gelatinization of the starch granules, which improved the accessibility of the starch molecules to the enzyme. This work demonstrated that the presence of different types of polysaccharides does not affect protein digestion, but also it emphasizes the importance of considering the influence of processing on food structure and its digestibility, even in the simplest model systems.

Concentrated casein micelle suspensions show an altered balance between the colloidal and soluble phases compared with native skim milk. The objective of this research was to probe the role of such a change on the chymosin-driven destabilization of casein micelles. Skim milk was gently concentrated by osmotic stressing to increase the volume fraction of the micelles while maintaining a constant ionic composition. In situ turbidity measurements demonstrated that the secondary stage of gelation was hindered in the concentrated suspensions. Addition of ionic calcium overcame this inhibition, whereas restoring the original concentration by redilution did not. This work clearly demonstrated that calcium plays a major role in decreasing electrostatic repulsion in casein micelles, but also showed the importance of noncolloidal proteins in altering the gelling functionality of casein micelles in concentrated milk. Additional calcium induces aggregation of these soluble caseins, restoring gelation in concentrates.

This research used a customized laboratory setup to compare the filtration performance and fouling buildup during microfiltration with polymeric membranes of skim milk using 2 diafiltration media: ultrafiltration permeate and ultrapure water. Two filtration modes were evaluated: in stage 1, the diafiltration media was added in a 1:1 ratio, with the collection of permeate continuing until the initial protein concentration was restored. In stage 2, retentates and permeates were recycled to simulate fouling accumulation in a steady-state without altering the retentate composition. Utilizing water as the diafiltration medium resulted in higher flux and lower resistance values compared with using ultrafiltration permeate, irrespective of the filtration mode. The concentration had a significant effect on membrane resistance, with no noticeable time-dependent effect on fouling layer development after 60 min of filtration when the retentate composition remained constant. The protein composition of the permeate and extracted foulants were comparable between the 2 media, with caseins predominating in the fouling layer., (© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Rapid quantification of plastic contaminants, particularly microplastics (MPs), in foods is a challenge. This study introduces a novel method using Fourier transform infrared spectroscopy coupled with thermogravimetric (TGA-FTIR) and chemometric analysis for the quantification of MPs in foods. A model study was performed using polystyrene (PS) MPs (1 μm) added to various foods, namely, water, milk, and coffee without any pretreatment. Foods were spiked with PS microbeads at different concentrations, heated in a TGA, and FTIR spectra of the gases evolved from the TGA were collected over time. The FTIR spectral data were used to construct a Gram-Schmidt profile and identify the characteristic PS peak. The spectrum corresponding to the peak maxima was extracted to represent the specific PS concentration. A dataset of selected spectra and their associated PS concentrations was preprocessed prior to calibration and cross-validation using PLS regression models, for each food matrix studied. The results showed that the PLS models reliably predicted the PS content in water, milk, and coffee with R 2 CV above 0.96, and RMSECV between 0.045 and 0.07 mg. Multivariate detection limit intervals (LOD min /LOD max ) were 0.041/0.085 mg for water, 0.061/0.128 mg for milk and 0.06/0.101 mg for coffee. This method is simple to operate, relatively rapid, and most importantly, does not require sample pretreatment. This research also suggests that the analysis is applicable to a broad range of food samples, and it is suitable for quantifying MPs and nanoplastics regardless of size and shape. The chemometric approach also shows its potential for automation in daily detection and quantification of MPs in food safety control., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hypothesis: The structural details of foams made with pea albumins are affected by the pH of the initial solution and followed heat treatment., Experiments: An in situ, time-resolved investigation of foams prepared with pea albumins was conducted using small-angle neutron scattering (SANS) in combination with imaging and conductance measurements. Solutions were tested at pH three pH values (3, 4.5, and 8) before and after heating (90 °C for 1 and 5 min)., Findings: The characteristic structures present in the foam from the nano to the meso-scale differed during drainage depending on solution pH. Foams obtained at pH 3, had the largest bubble radius and thinnest plateau border, as well as the highest extent of liquid drainage. At pH 4.5, close to the isoelectric point of the proteins, foams displayed similar bubbles' behavior to those at pH 8, but with the largest film thickness. In this case, the proteins were extensively aggregated. Heating of the solutions prior to foaming did not significantly affect the foam aging regardless of pH. The quantification of specific surface areas and film thickness over time without sample disruption shows to be a powerful approach to designing foam structures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

Membrane filtration technologies have shown great potential as a gentle and effective method for concentrating and fractionating proteins for food applications. However, the application of this technology to plant-derived protein streams is in its infancy. In this study, an aqueous rapeseed protein concentrate was obtained with wet milling, and its performance during ultrafiltration with two distinct molecular weight cut-offs (10 and 100 kDa) was tested. All rapeseed proteins were retained during filtration. The addition of pectinase during extraction prior to filtration caused important structural modifications to the extract, resulting in increased permeate fluxes, increased carbohydrate permeation and a reduction in irreversible fouling. Lager pore sizes led to more pronounced fouling. FTIR analysis of the spent membranes showed that proteins and lipids are causing irreversible fouling.

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.

The aim of this study was to develop a small-scale model system resembling the micro- and meso-structure of butter, namely having a water droplet size distribution and water content close to that of commercially produced butter. Although it is possible to churn cream on a small scale, matching the microstructure of butter is a challenge. A 2-step churning process was introduced with the application of a kitchen mixer. The resulting microstructure was evaluated using confocal laser scanning microscopy. In addition, low-field nuclear magnetic resonance was used to determine the water droplet size distribution. Results demonstrated that a water content of 16% to 19% could be obtained with the proposed procedure, close to the standard water content of 16%. Average water droplet size ranged between 2.5 to 4.3 µm and did not depend on batch-to-batch variations, nor storage-induced differences in the cream. In conclusion, the proposed method could be employed to prepare water-in-oil emulsions with a microstructure similar to that of butter and opens new opportunities for studying microbial growth, flavor release, and texture formation., (© 2024.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Microplastics (MPs) and nanoplastics (NPs) pervade both the environment and the food chain, originating from the degradation of plastic materials from various sources. Their ubiquitous presence raises concerns for ecosystem safety, as well as the health of animals and humans. While evidence suggests their infiltration into mammalian and human tissues and their association with several diseases, the precise toxicological effects remain elusive and require further investigation. MPs and NPs sample preparation and analytical methods are quite scattered without harmonized strategies to exist at the moment. A significant challenge lies in the limited availability of methods for the chemical characterization and quantification of these contaminants. MPs and NPs can undergo further degradation, driven by abiotic or biotic factors, resulting in the formation of cyclic or linear oligomers. These oligomers can serve as indicative markers for the presence or exposure to MPs and NPs. Moreover, recent finding concerning the aggregation of oligomers to form NPs, makes their analysis as markers very important. Recent advancements have led to the development of sensitive and robust analytical methods for identifying and (semi)quantifying these oligomers in environmental, food, and biological samples. These methods offer a valuable complementary approach for determining the presence of MPs and NPs and assessing their risk to human health and the environment., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The aim of this work was to investigate the impact of the addition of salivary α-amylase on starch hydrolysis in protein-containing dispersions during an in vitro digestion process. In vitro digestion provides useful insights on the fate of nutrients during gastro-intestinal transit in complex food matrices, an important aspect to consider when developing highly nutritious foods. Many foods contain polysaccharides, and as their disruption in the gastric stage is limited, salivary α-amylase is often neglected in in vitro studies. A reference study on the effect of salivary α-amylase using one of the most advanced and complex in vitro digestion models (INFOGEST) is, however, not available. Hence, this work reports the gastrointestinal breakdown of three mixed dispersions containing whey protein isolate with different polysaccharides: potato starch, pectin from citrus peel and maize starch. The latter was also studied after heating. No polysaccharide or salivary α-amylase-dependent effect on protein digestion was found, based on the free NH 2 and SDS-PAGE. However, in the heat-treated samples, the addition of salivary α-amylase showed a significantly higher starch hydrolysis compared to the sample without α-amylase, due to the gelatinization of the starch granules, which improved the accessibility of the starch molecules to the enzyme. This work demonstrated that the presence of different types of polysaccharides does not affect protein digestion, but also it emphasizes the importance of considering the influence of processing on food structure and its digestibility, even in the simplest model systems., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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.

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 β-casein found in the serum phase (23-33% of total β-casein). In addition, there was an uneven dissociation behavior of the various genetic β-casein variants, whereof A2 was more readily released with cooling. In skim milk, approximately 22%, 18%, and 14% of κ-, α S2 , and α S1 -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-, α S2 , and α S1 -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., (© 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

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.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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.

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.

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 Mg 2+ concentration (P < 0.05), in addition to primer quality. The optimal Mg 2+ 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 × 10 0 PFU/15 g, 8.3 × 10 1 PFU/50 g, 8.3 × 10 0 PFU/5 g, and 8.3 × 10 0 PFU/40 mL, respectively. The developed RT-LAMP-BART was an effective, simple, sensitive, and robust method for foodborne HAV detection., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tsochatzis, Theodoridis and Corredig.)

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 β-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., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

With raising consumer demand for plant-derived proteins, there has been an increased interest in the utilization of pea ingredients in food formulations. It was hypothesized that differences in processing history and composition affect their colloidal properties and their breakdown during in vitro simulated gastrointestinal digestion. The gastrointestinal fate of three different commercial pea protein ingredients, two protein isolates and one less refined concentrate was compared. The concentrate dispersion showed greater solubility, different protein composition and smaller particle size than the reconstituted pea protein isolates. When heat-treated, the release of free amino groups decreased for the isolates, but increased for the concentrate dispersions. LC-TQMS of free amino acids in the intestinal digestates indicated a significantly higher release of methionine (limiting amino acid in pea protein) in the concentrates than in the isolates. This work highlights the influence of processing and composition on techno-functional and digestion properties of pea ingredients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

The present study reports the biodegradation of polystyrene (PS) by mealworm (Tenebrio molitor) following different feeding regimes. Changes in lipids and protein were studied to evaluate possible differences in the growth and metabolic pathways of the insects depending on the diets. Thermo-gravimetric analysis of the excretions (frass) revealed a decrease in the molecular mass of the PS polymers. The insects' biomass contained less protein when PS was part of the diet, suggesting that the insects undergo a certain level of stress compared to control diets. The frass also contained lower amount of nitrogen content compared to that from insects fed a control diet. NH 4 + and other cations involved in biochemical processes were also measured in insects' frass, including potassium, sodium, magnesium, and calcium, combined with a small pH change. The decrease in the mineral content of the frass was attributed to increased cellular activity in PS-fed insects. A higher amount of ceramides and cardiolipins, biomarkers of apoptosis, were also found in association with PS consumption. It was concluded that the insects could metabolize PS, but this caused an increase in its stress levels., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The raising consumer demand for plant-derived proteins has led to an increased production of alternative protein ingredients with varying processing histories. In this study, we used a commercially available potato protein ingredient with a nutritionally valuable amino acid profile and high technological functionality to evaluate if the digestibility of a suspension with the same composition is affected by differences in the structure. Four isocaloric (4% protein, w / w ) matrices (suspension, gel, foam and heat-set foam) were prepared and their gastrointestinal fate was followed utilizing a semi-dynamic in vitro digestion model. The microstructure was observed by confocal laser scanning microscopy, protein breakdown was tested by electrophoresis and free amino acids after intestinal digestion was estimated using liquid chromatography/triple-quadruple-mass spectrometry (LC-TQMS). The heat-treated samples showed a higher degree of hydrolysis and lower trypsin inhibitory activity than the non-heat-treated samples. An in vitro digestible indispensable amino acid score was calculated based on experimental data, showing a value of 0.9 based on sulfur amino acids/valine as the limiting amino acids. The heated samples also showed a slower gastric emptying rate. The study highlights the effect of the food matrix on the distribution of the peptides created during various stages of gastric emptying.

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 × 10 0  PFU/15 g, 8.3 × 10 1  PFU/50 g, and 8.3 × 10 0  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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 H 2 O 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.

Concentrated casein micelle suspensions show an altered balance between the colloidal and soluble phases compared with native skim milk. The objective of this research was to probe the role of such a change on the chymosin-driven destabilization of casein micelles. Skim milk was gently concentrated by osmotic stressing to increase the volume fraction of the micelles while maintaining a constant ionic composition. In situ turbidity measurements demonstrated that the secondary stage of gelation was hindered in the concentrated suspensions. Addition of ionic calcium overcame this inhibition, whereas restoring the original concentration by redilution did not. This work clearly demonstrated that calcium plays a major role in decreasing electrostatic repulsion in casein micelles, but also showed the importance of noncolloidal proteins in altering the gelling functionality of casein micelles in concentrated milk. Additional calcium induces aggregation of these soluble caseins, restoring gelation in concentrates., (© 2021.)

In the present study, the structuring and breakdown of a 5% protein dispersion prepared with commercial fractions of pea and rice isolates (PPI and RPI, respectively) were monitored by in vitro digestion. These proteins were blended in a 2 : 1 ratio of pea and rice, respectively, as this would deliver a high amino acid score. The effect of heating at 90 °C for 15 min on the digestion behavior was evaluated not only for the blend, but also for the respective protein isolate fractions, using the INFOGEST international consensus, semi-dynamic in vitro gastric model. Digesta were characterized by gel electrophoresis, light scattering, confocal laser scanning microscopy and size exclusion HPLC. Heating increased the solubility of PPI from 15.7% to 26.6% at pH 7.4. RPI showed low solubility (a maximum of 2.6% at pH 2), regardless of the treatment. Confocal microscopy observations evidenced major differences in the aggregates formed during digestion, with larger aggregates for heated PPI. While the unheated pea protein dispersions precipitated near the isoelectric pH, the heated counterpart formed macro-aggregates under the same conditions. In the case of RPI, there were no differences in structuring behaviour between unheated and heat treated reconstituted powder, due to their low solubility. Rice prolamins showed resistance to hydrolysis by pepsin and pancreatic enzymes. In the heated blend, macro-aggregates formed, but with a smaller size compared to heated pea protein alone, suggesting that pea protein aggregation was hindered by the presence of rice proteins. These results demonstrate how the composition of protein isolates can affect their in vitro digestion. However, pre-treatment of plant protein blends, such as heating, can modulate the rate and mechanism of digestion.

Hydrocolloids are often added as functional ingredients in foods, to better design the structure of the matrix and ensure food quality and optimal sensory properties. However, much less is known about their influence on the physical and chemical changes during gastric digestion. In this study, semi-continuous in vitro gastric digestion was applied on a model food system, prepared with milk protein concentrate (MPC) (3% w/v) and 1% alginate, pectin, guar gum, as well as a 1:1 mixture of alginate and pectin. The dynamics during simulated gastric digestion were observed by measuring particle size distributions, structuring at various length scales, as well as by evaluating differences in protein breakdown. Immediately after contact with the simulated gastric fluids, all samples showed extensive aggregation and formation of different structures. MPC control dispersions (no polysaccharide) and MPC containing alginate formed large inhomogeneous aggregates. The lack of structural homogeneity affected the simulated gastric emptying: there were marked differences in the type of aggregates present at various times of emptying depending on the hydrocolloid present in the mixture. MPC containing pectin or guar gum formed macroscopically homogeneous dispersion, with rather small protein aggregates showing a large population of particles between 60 and 100 ​μm of diameter, with marked differences in microstructure. Pectin created large coacervates, while guar microscopic phase separated systems. These dispersions showed a higher extent of protein digestion, due to the larger surface area created for enzyme activity compared to the macroscopically phase separated matrices. In all cases, there was a large undigested fraction at the end point of 140 ​min. SDS PAGE demonstrated differences in the casein peptides distribution depending on the type of polysaccharide present during simulated gastric emptying. This in spite of similarities in cumulative protein emptied. It was concluded that in this semi-continuous in vitro gastric digestion model, structuring with polysaccharides has a significant impact on gastric emptying and protein digestion kinetics., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Pectin nanocapsules were obtained by gelling the inner droplets, in situ, in a multiple emulsion of the water in oil in water (W/O/W) type, using the enzyme Pectin methyl esterase. The inner phase was tested for its encapsulation efficiency by addition of brilliant blue or magnesium chloride. Rheological measurements and light scattering showed similarities for gelled and non gelled droplets containing high methoxy pectin. After one month of storage, the gelled nanocapsules showed a population of larger droplets, not appearing in the other W/O/W emulsions: the non gelled pectin droplets or control with no pectin. Confocal microscopy observations demonstrated the presence of the inner water droplets in all emulsions after one month of storage at 4 °C. Brilliant blue was retained in the inner droplets containing pectin, and the retention was not affected by the gelled inner phase. Magnesium ions were retained in all treatments, even in the case of control W/O/W emulsions, not containing pectin. With both systems studied, the presence of gelled nanocapsules did not cause significant differences in the encapsulation efficiency or stability of the W/O/W emulsions., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

High-moisture Mozzarella is one of the most exported cheeses worldwide, but affected by short shelf-life. Freezing can help to reduce waste, but its effect on quality needs to be considered. In this study, the physico-chemical changes of Mozzarella occurring during frozen storage and subsequent refrigerated storage (after thawing) were evaluated. Frozen cheeses stored at -18 °C between 1 and 4 months showed microstructural damage and different physical, textural, sensory properties. With NMR relaxometry it was possible to observe freeze-related dehydration of caseins, by measuring the changes in water relaxation times within the matrix. These modifications were confirmed by microstructural observations that showed the formation of larger serum channels in samples subjected to freezing, compared with fresh cheeses. Sensory evaluation showed skin peeling off in frozen samples. By observing the changes at various length scales it was therefore possible to identify the critical points affecting HM Mozzarella cheese quality during frozen storage., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)