From lab-based to in-line: Analytical tools for the characterization of whey protein denaturation and aggregation-A review.

Whey protein denaturation and aggregation have long been areas of research interest to the dairy industry, having significant implications for process performance and final product functionality and quality. As such, a significant number of analytical techniques have been developed or adapted to assess and characterize levels of whey protein denaturation and aggregation, to either maximize processing efficiency or create products with enhanced functionality (both technological and biological). This review aims to collate and critique these approaches based on their analytical principles and outline their application for the assessment of denaturation and aggregation. This review also provides insights into recent developments in process analytical technologies relating to whey protein denaturation and aggregation, whereby some of the analytical methods have been adapted to enable measurements in-line. Developments in this area will enable more live, in-process data to be generated, which will subsequently allow more adaptive processing, enabling improved product quality and processing efficiency. Along with the applicability of these techniques for the assessment of whey protein denaturation and aggregation, limitations are also presented to help assess the suitability of each analytical technique for specific areas of interest.

Enhancing the Techno-Functional Properties of Lentil Protein Isolate Dispersions Using In-Line High-Shear Rotor-Stator Mixing.

In response to global challenges such as climate change and food insecurity, plant proteins have gained interest. Among these, lentils have emerged as a promising source of proteins due to their good nutritional profile and sustainability considerations. However, their widespread use in food products has been impeded by limited solubility. This study aimed to investigate the potential of high-shear mixing, a resource-efficient technique, to enhance lentil protein solubility and its functional properties. Red lentil protein isolate powders were rehydrated and subjected to a semi-continuous in-line high-shear treatment at 10,200 rpm for a timespan ranging from 0 to 15 min. The results highlighted a significant (p < 0.05) increase in solubility from 46.87 to 68.42% after 15 min of shearing and a reduction in particle size as a result of the intense shearing and disruption provided by the rotor and forced passage through the perforations of the stator. The volume-weighted mean diameter decreased from 5.13 to 1.72 µm after 15 min of shearing, also highlighted by the confocal micrographs which confirmed the breakdown of larger particles into smaller and more uniform particles. Rheological analysis indicated consistent Newtonian behaviour across all dispersions, with apparent viscosities ranging from 1.69 to 1.78 mPa.s. Surface hydrophobicity increased significantly (p < 0.05), from 830 to 1245, indicating exposure of otherwise buried hydrophobic groups. Furthermore, colloidal stability of the dispersion was improved, with separation rates decreasing from 71.23 to 24.16%·h-1. The significant enhancements in solubility, particle size reduction, and colloidal stability, highlight the potential of in-line high-shear mixing in improving the functional properties of lentil protein isolates for formulating sustainable food products with enhanced techno-functional properties.

Physicochemical properties of micellar casein retentates generated at different microfiltration temperatures.

Processing temperature has a significant influence on the composition and functionality of the resulting streams following microfiltration (MF) of skim milk. In this study, MF and diafiltration (DF) were performed at 4 or 50°C to produce ß-casein (ß-CN)-depleted and nondepleted (i.e., native casein profile) micellar casein isolate retentates, respectively. Microfiltration combined with extensive DF resulted in a 40% depletion of ß-CN at 4°C, whereas no ß-CN depletion occurred at 50°C. Microfiltration at 4°C led to higher transmission of calcium into permeates, with retentate generated at 4°C containing less total calcium compared with retentate generated at 50°C, based on the volume of retentate remaining. Higher heat stability at 120°C was measured for retentates generated at 4°C compared with those at 50°C, across all pH values measured. Retentates generated at 4°C also had significantly lower ionic calcium values at each pH compared with those generated at 50°C. Higher apparent viscosities at 4°C were measured for retentates generated at 4°C compared with retentates generated at 50°C, likely due to increased voluminosity of ß-CN-depleted casein micelles. The results of this study provide new information on how changing the composition of MF retentate, by appropriate control of processing temperature and DF, can alter physicochemical properties of casein micelles, with potential implications for ingredient functionality.

The effect of whey source on heat-induced aggregation of casein and whey protein mixtures of relevance to infant nutritional product formulation.

Sweet and, to a lesser extent, acid whey protein ingredients can be used for the formulation of infant nutritional products. Unlike acid whey, sweet whey contains caseinomacropeptide (CMP), a heat-stable peptide liberated from κ-casein during cheese and rennet casein manufacture. Four protein systems-sweet whey (SW) and acid whey (AW), with or without standardization for CMP protein content-were added to skim milk (50/50, wt/wt) and unheated or heated to 85 or 110°C. These 12 samples were assessed for physicochemical stability in the presence of added calcium at pH 6.8. The effect of CMP content on the physicochemical properties of the protein systems was also assessed. Without preheat treatment, mixtures of AW and skim milk (SM) were more heat stable than SW and SM, demonstrating the effect of whey protein type on heat stability. Preheat treatment of the SW in the presence of SM significantly improved the heat stability of the resultant protein systems on subsequent heating. All of the protein systems had significantly lower heat stability with the addition of Ca, although the reduction was significantly smaller for the heated protein systems than the unheated controls. The findings can help identify heating parameters and ingredients for optimizing processing stability and physicochemical characteristics of nutritional beverages such as infant formulations.

Formulation and Physical Stability of High Total Solids Lentil Protein-Stabilised Emulsions for Use in Plant Protein-Based Young Child Formulae.

The demand for high-quality plant protein products is increasing and the aim of this work was to evaluate the impact of increasing the total solids content on the formation and stability of lentil protein stabilised oil-in-water emulsions. A series of emulsions were formulated using different proportions of total solids: 23, 26, 29, 32, and 35% (w/v). The emulsions were formulated using three ingredients-lentil protein, sunflower oil, and maltodextrin-which made up 15.85, 27.43, and 56.72% (w/w) of the total solids, respectively. The changes in apparent viscosity, particle size distribution, and colour during thermal processing were evaluated, with the physical stability investigated using an analytical centrifuge. The apparent viscosity of the solutions increased with total solids content (25.6 to 130 mPa.s-1), as did redness colour intensity (a* value increased from 5.82 ± 0.12 to 7.70 ± 0.09). Thermal processing resulted in greater destabilisation for higher total solids samples, as evidenced by greater changes in particle size, along with decreased redness colour. These results bring a better understanding of high total solids plant protein emulsions and factors affecting their stability, which could be used for the development of cost-effective and sustainable processing solutions in the production of plant protein young child formulae.

The Protein Composition and In Vitro Digestive Characteristics of Animal- versus Plant-Based Infant Nutritional Products.

The protein composition and digestive characteristics of four commercially available infant formulae (IF) manufactured using bovine (B-IF), caprine (C-IF), soy (S-IF), and rice (R-IF) as a protein source were examined in this study. Plant-based formulae had significantly higher crude protein and non-protein nitrogen (NPN) concentrations. Static in vitro gastrointestinal digestion of these formulae, and subsequent analysis of their digestates, revealed significantly higher proteolysis of B-IF at the end of gastrointestinal digestion compared to the other formulae, as indicated by the significantly higher concentration of free amine groups. Furthermore, differences in structure formation during the gastric phase of digestion were observed, with formation of a more continuous, firmer coagulum by C-IF, while R-IF demonstrated no curd formation likely due to the extensive hydrolysis of these proteins during manufacture. Differences in digestive characteristics between formulae manufactured from these different protein sources may influence the bio-accessibility and bioavailability of nutrients, warranting additional study.

Plant-Based Alternatives to Cheese Formulated Using Blends of Zein and Chickpea Protein Ingredients.

In this study, zein protein isolate (ZPI) and chickpea protein concentrate (CPC) ingredients were used to formulate five plant-based cheese alternatives. Ingredient ratios based on protein contributions of 0:100, 25:75, 50:50, 75:25 and 100:0 from ZPI and CPC, respectively, were used. Formulations were developed at pH ~4.5, with a moisture target of 59%. Shea butter was used to target 15% fat, while tapioca starch was added to target the same carbohydrate content for all samples. Microstructural analysis showed differences among samples, with samples containing ZPI displaying a protein-rich layer surrounding the fat globules. Schreiber meltability and dynamic low amplitude oscillatory shear rheological analyses showed that increasing the proportion of ZPI was associated with increasing meltability and greater ability to flow at high temperatures. In addition, the sample containing only CPC showed the highest adhesiveness, springiness and cohesiveness values from the texture profile analysis, while the sample containing only ZPI exhibited the highest hardness. Furthermore, stretchability increased with increasing ZPI proportions. This work will help understanding of the role and potential of promising plant-protein-ingredient blends in formulating plant-based alternatives to cheese with desirable functional properties.

Structural, Binding and Functional Properties of Milk Protein-Polyphenol Systems: A Review.

Polyphenols (PP) are linked to health benefits (e.g., prevention of cancer, cardiovascular disease and obesity), which are mainly attributed to their antioxidant activity. During digestion, PP are oxidised to a significant degree reducing their bio-functionality. In recent years, the potential of various milk protein systems, including ß-casein micelles, ß-lactoglobulin aggregates, blood serum albumin aggregates, native casein micelles and re-assembled casein micelles, to bind and protect PP have been investigated. These studies have yet to be systematically reviewed. The functional properties of the milk protein-PP systems depend on the type and concentration of both PP and protein, as well as the structure of the resultant complexes, with environmental and processing factors also having an influence. Milk protein systems protect PP from degradation during digestion, resulting in a higher bioaccessibility and bioavailability, which improve the functional properties of PP upon consumption. This review compares different milk protein systems in terms of physicochemical properties, PP binding performance and ability to enhance the bio-functional properties of PP. The goal is to provide a comprehensive overview on the structural, binding, and functional properties of milk protein-polyphenol systems. It is concluded that milk protein complexes function effectively as delivery systems for PP, protecting PP from oxidation during digestion.

Interactions between whey proteins and calcium salts and implications for the formulation of dairy protein-based nutritional beverage products: A review.

Whey-based nutritional beverages are often fortified with calcium (Ca) in order to deliver the recommended intake of Ca. However, technical and product quality challenges are often experienced with Ca fortification of whey protein-based nutritional solutions, such as poor heat stability, high viscosity, colloidal instability, and impaired heat transfer. Understanding of the relationships and interactions between whey proteins and Ca relative to liquid process (e.g., ready to feed products, feed material prior to drying) is essential to designing and formulating nutritional whey-based products with desired physicochemical and colloidal stability properties. This article reviews the interactions between whey proteins and Ca salts used in the formulation of nutritional whey-based products as well as major processing implications associated with Ca fortification of whey-based solutions.

Formulation, pilot-scale preparation, physicochemical characterization and digestibility of a lentil protein-based model infant formula powder.

BACKGROUND: Infant formula is a human milk substitute for consumption during the first months of life. The protein component of such products is generally of dairy origin. Alternative sources of protein, such as those of plant origin, are of interest due to dairy allergies, intolerances, and ethical and environmental considerations. Lentils have high levels of protein (20-30%) with a good amino acid profile and functional properties. In this study, a model lentil protein-based formula (LF), in powder format, was produced and compared to two commercial plant-based infant formulae (i.e., soy; SF and rice; RF) in terms of physicochemical properties and digestibility. RESULTS: The macronutrient composition was similar between all the samples; however, RF and SF had larger volume-weighted mean particle diameters (D[4,3] of 121-134 µm) than LF (31.9 µm), which was confirmed using scanning electron and confocal laser microscopy. The larger particle sizes of the commercial powders were attributed to their agglomeration during the drying process. Regarding functional properties, the LF showed higher D[4,3] values (17.8 µm) after 18 h reconstitution in water, compared with the SF and RF (5.82 and 4.55 µm, respectively), which could be partially attributed to hydrophobic protein-protein interactions. Regarding viscosity at 95 °C and physical stability, LF was more stable than RF. The digestibility analysis showed LF to have similar values (P < 0.05) to the standard SF. CONCLUSION: These results demonstrated that, from the nutritional and physicochemical perspectives, lentil proteins represent a good alternative to other sources of plant proteins (e.g., soy and rice) in infant nutritional products. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Formation and thermal and colloidal stability of oil-in-water emulsions stabilized using quinoa and lentil protein blends.

BACKGROUND: The amino acid composition, and rheological, thermal and colloidal stability of plant protein-based oil-in-water emulsion systems containing 1.90, 3.50 and 7.70 g 100 mL-1 protein, fat and carbohydrate, respectively, using quinoa and lentil protein ratios of 100:0 and 60:40 were investigated. The emulsion containing lentil protein showed lower initial, peak and final viscosity values (22.7, 61.7 and 61.6 mPa s, respectively) than the emulsion formulated with quinoa protein alone (34.3, 102 and 80.0 mPa s, respectively) on heat treatment. RESULTS: Particle size analysis showed that both samples had small particle sizes (~1.36 µm) after homogenization; however, the sample with 60:40 quinoa:lentil protein ratio showed greater physical stability, likely related to the superior emulsifying properties of lentil protein. However, upon heat treatment, large aggregates (~100 µm) were formed in both samples, reducing the physical stability of the samples. This physical stability was increased with the addition of 0.20% sodium dodecyl sulfate (SDS), whereas it was negatively affected by the addition of α-amylase. Addition of α-amylase led to lower viscosity for both emulsion samples, with measured values of 41.8 and 46.0 mPa s for the 100:0 and 60:40 samples, respectively. This suggests that the heat-induced increases in particle size were partially due to hydrophobic interactions between the proteins as SDS disrupts hydrophobic bonds between proteins. CONCLUSION: These results demonstrated that using a mixture of lentil and quinoa proteins positively affected the physical stability of plant protein-based emulsions, in addition to contributing to a more nutritionally complete amino acid profile - both important considerations in the development of plant-based beverages. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Chickpea protein ingredients: A review of composition, functionality, and applications.

Chickpea (Cicer arietinum L.) is a pulse consumed all over the world, representing a good source of protein, as well as fat, fiber, and other carbohydrates. As a result of the growing global population the demand for the protein component of this pulse is increasing and various approaches have been proposed and developed to extract same. In this review the composition, functionality, and applications of chickpea protein ingredients are described. Moreover, methods to enhance protein quality have been identified, as well as applications of the coproducts resulting from protein extraction and processing. The principal dry and wet protein enrichment approaches, resulting in protein concentrates and isolates, include air classification, alkaline/acid extraction, salt extraction, isoelectric precipitation, and membrane filtration. Chickpea proteins exhibit good functional properties such as solubility, water and oil absorption capacity, emulsifying, foaming, and gelling. During protein enrichment, the functionality of protein can be enhanced in addition to primary processing (e.g., germination and dehulling, fermentation, enzymatic treatments). Different applications of chickpea protein ingredients, and their coproducts, have been identified in research, highlighting the potential of these ingredients for novel product development and improvement of the nutritional profile of existing food products. Formulations to meet consumer needs in terms of healthy and sustainable foods have been investigated in the literature and can be further explored. Future research may be useful to improve applications of the specific coproducts that result from the extraction of chickpea proteins, thereby leading to more sustainable processes.

Influence of Transglutaminase Crosslinking on Casein Protein Fractionation during Low Temperature Microfiltration.

Low temperature microfiltration (MF) is applied in dairy processing to achieve higher protein and microbiological quality ingredients and to support ingredient innovation; however, low temperature reduces hydrophobic interactions between casein proteins and increases the solubility of colloidal calcium phosphate, promoting reversible dissociation of micellar ß-casein into the serum phase, and thus into permeate, during MF. Crosslinking of casein proteins using transglutaminase was studied as an approach to reduce the permeation of casein monomers, which typically results in reduced yield of protein in the retentate fraction. Two treatments (a) 5 °C/24 h (TA) and (b) 40 °C/90 min (TB), were applied to the feed before filtration at 5 °C, with a 0.1 µm membrane. Flux was high for TA treatment possibly due to the stabilising effect of transglutaminase on casein micelles. It is likely that formation of isopeptide bonds within and on the surface of micelles results in the micelles being less readily available for protein-protein and protein-membrane interactions, resulting in less resistance to membrane pores and flow passage, thereby conferring higher permeate flux. The results also showed that permeation of casein monomers into the permeate was significantly reduced after both enzymatic treatments as compared to control feed due to the reduced molecular mobility of soluble casein, mainly ß-casein, caused by transglutaminase crosslinking.

Cold Microfiltration as an Enabler of Sustainable Dairy Protein Ingredient Innovation.

Classically, microfiltration (0.1-0.5 µm) of bovine skim milk is performed at warm temperatures (45-55 °C), to produce micellar casein and milk-derived whey protein ingredients. Microfiltration at these temperatures is associated with high initial permeate flux and allows for the retention of the casein fraction, resulting in a whey protein fraction of high purity. Increasingly, however, the microfiltration of skim milk and other dairy streams at low temperatures (≤20 °C) is being used in the dairy industry. The trend towards cold filtration has arisen due to associated benefits of improved microbial quality and reduced fouling, allowing for extended processing times, improved product quality and opportunities for more sustainable processing. Performing microfiltration of skim milk at low temperatures also alters the protein profile and mineral composition of the resulting processing streams, allowing for the generation of new ingredients. However, the use of low processing temperatures is associated with high mechanical energy consumption to compensate for the increased viscosity, and thermal energy consumption for inline cooling, impacting the sustainability of the process. This review will examine the differences between warm and cold microfiltration in terms of membrane performance, partitioning of bovine milk constituents, microbial growth, ingredient innovation and process sustainability.

Influence of Glycomacropeptide on Rehydration Characteristics of Micellar Casein Concentrate Powder.

Glycomacropeptide (GMP) shows potential for enhancing the rehydration properties of high-protein dairy powders due to its hydrophilic nature. This study involved formulating micellar casein concentrate (MCC) solutions (8.6% final protein content) with 0, 10, and 20% GMP as a percentage of total protein, and investigated the physicochemical and rehydration properties of the resultant freeze-dried powders (P-MCC-0G, P-MCC-10G, and P-MCC-20G, respectively). The surface charges of caseins in the control MCC and 10 or 20% GMP blended solutions were -25.8, -29.6, and -31.5 mV, respectively. Tablets prepared from P-MCC-10G or P-MCC-20G powders displayed enhanced wettability with contact angle values of 80.6° and 79.5°, respectively, compared with 85.5° for P-MCC-0G. Moreover, blending of GMP with MCC resulted in faster disintegration of powder particles during rehydration (i.e., dispersibility) compared to P-MCC-0G. Faster and more extensive release of caseins from powder particles into solution was evident with the increasing proportion of GMP, with the majority of GMP released within the first 15 min of rehydration. The results of this study will contribute to further development of formulation science for achieving enhanced solubility characteristics of high-protein dairy powder ingredients, such as MCC.

The impact of thermal processing on the simulated infant gastrointestinal digestion, bactericidal and anti-inflammatory activity of bovine lactoferrin - An in vitro study.

Lactoferrin (LF) is a multifunctional glycoprotein which, when thermally processed, undergoes significant physicochemical changes. The link between such changes and the bioactivity of LF is not well characterised and requires much research. In this work, bovine LF solutions (1%, w/v, protein, pH 7) were thermally processed using high temperature short time conditions (72, 80, 85 or 95 °C with 15 s holding times). Following this, it was shown that LF and heat induced LF aggregates were largely resistant to simulated infant gastric, but not intestinal, digestion. Also, the efficacy of LF bactericidal activity, and inhibition of lipopolysaccharide-induced NF-κB activation were negatively impacted by thermal processing. This study confirmed that the efficacy of LF bio-functionalities was affected by the extent of heat-induced changes in protein structure whereby processing conditions of least severity (i.e. pasteurisation) had the least impact on bioactivity.

Rheological and Solubility Properties of Soy Protein Isolate.

Soy protein isolate (SPI) powders often have poor water solubility, particularly at pH values close to neutral, which is an attribute that is an issue for its incorporation into complex nutritional systems. Therefore, the objective of this study was to improve SPI solubility while maintaining low viscosity. Thus, the intention was to examine the solubility and rheological properties of a commercial SPI powder at pH values of 2.0, 6.9, and 9.0, and determine if heat treatment at acidic or alkaline conditions might positively influence protein solubility, once re-adjusted back to pH 6.9. Adjusting the pH of SPI dispersions from pH 6.9 to 2.0 or 9.0 led to an increase in protein solubility with a concomitant increase in viscosity at 20 °C. Meanwhile, heat treatment at 90 °C significantly improved the solubility at all pH values and resulted in a decrease in viscosity in samples heated at pH 9.0. All SPI dispersions measured under low-amplitude rheological conditions showed elastic-like behaviour (i.e., G' > G″), indicating a weak "gel-like" structure at frequencies less than 10 Hz. In summary, the physical properties of SPI can be manipulated through heat treatment under acidic or alkaline conditions when the protein subunits are dissociated, before re-adjusting to pH 6.9.

Protein levels and protease activity in milk from mothers of pre-term infants: A prospective longitudinal study of human milk macronutrient composition.

BACKGROUND AND AIMS: The composition and enzymology of human milk changes throughout the lactation period, and differ for mothers who give birth prematurely compared to those who deliver at full-term. Understanding the composition of milk from mothers of very low birth weight premature infants is of great significance, and the objective of this study was to evaluate the composition, protein profile and plasmin activity of milk from mothers who delivered infants at different gestational ages. METHODS: Samples of human milk were donated by women (n = 74) in the Cork, Ireland, area who gave birth to full-term (>37 weeks gestation, FT), pre-term (32-37 weeks, PT) and very pre-term (≤32 weeks, VPT) infants. FT milk was collected at 1, 3, 6 and 10 weeks post-partum (PP), while PT and VPT milk was collected weekly until the FT due date of the infant and subsequently followed the FT protocol. RESULTS: Gestational age did not significantly affect lactose or fat content or total energy content of milk. However, protein content, and levels of some individual proteins, were significantly affected by both gestational age at birth and duration of lactation, with significantly higher protein levels in PT or VPT milk samples at 0-7 days and 1-2 months, respectively. Plasmin activity was significantly higher in VPT milk, indicating differences in proteolytic processing in milk. CONCLUSION: Compositional differences between the milk of mothers of term and pre-term infants were greatest in terms of the protein profile, which showed both qualitative and quantitative differences, as well as difference in proteolytic activity.

Dynamic Mechanical Analysis as a Complementary Technique for Stickiness Determination in Model Whey Protein Powders.

The α-relaxation temperatures (Tα), derived from the storage and loss moduli using dynamic mechanical analysis (DMA), were compared to methods for stickiness and glass transition determination for a selection of model whey protein concentrate (WPC) powders with varying protein contents. Glass transition temperatures (Tg) were determined using differential scanning calorimetry (DSC), and stickiness behavior was characterized using a fluidization technique. For the lower protein powders (WPC 20 and 35), the mechanical Tα determined from the storage modulus of the DMA (Tα onset) were in good agreement with the fluidization results, whereas for higher protein powders (WPC 50 and 65), the fluidization results compared better to the loss modulus results of the DMA (Tα peak). This study demonstrates that DMA has the potential to be a useful technique to complement stickiness characterization of dairy powders by providing an increased understanding of the mechanisms of stickiness.

Physical and colloidal stability of conventional and micronised calcium citrate ingredient powders in the formulation of infant nutritional products.

The fortification of food systems with calcium remains difficult; one challenge is to maintain the colloidal stability of insoluble calcium salts during processing and shelf life. Particle size reduction of insoluble salts may result in improved product stability. In this study, insoluble calcium citrate in two different particle sizes, conventional calcium citrate and micronised calcium citrate, were first evaluated in terms of physical and bulk handling properties, followed by protein adsorption and colloidal stability when dispersed in two dairy-based nutritional beverages differing in composition, i.e. infant milk formula stage 1 and 3. Particle size distribution analysis showed micronised calcium citrate (volume-weighted diameter = 5.10 µm) to have significantly smaller (p < 0.05) particle size than conventional calcium citrate (volume-weighted diameter = 88.2 µm). The adsorption of dairy proteins onto particles of calcium citrate resulted in caseins having greater affinity for both salts, followed by ß-lactoglobulin. The smaller particle size of the micronised citrate resulted in higher affinity for casein and greater colloidal stability when dispersed in both infant milk formula solutions compared to conventional calcium salts. The results of this study provide knowledge on the application of micronised insoluble calcium salts in the fortification of nutritional dairy-based products.