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.

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.

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.

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.

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.

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.

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.

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.

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.

Influence of desialylation of caseinomacropeptide on the denaturation and aggregation of whey proteins.

The effect of the addition of caseinomacropeptide (CMP) or desialylated CMP on the heat-induced denaturation and aggregation of whey proteins was investigated in the pH range 3 to 7 after heating at 80°C for 30 min. The rate and temperature of denaturation, the extent of aggregation, and the changes in secondary structure of the whey proteins heated in presence of CMP or desialylated CMP were measured. The sialic acid bound to CMP favored the denaturation and aggregation of whey proteins when the whey proteins were oppositely charged to CMP at pH 4. A transition occurred at pH 6, below which the removal of sialic acid enhanced the stabilizing properties of CMP against the denaturation and aggregation of the whey proteins. At pH >6, the interactions between desialylated CMP and the whey proteins led to more extensive denaturation and aggregation. Sialic acid bound to CMP influenced the denaturation and aggregation behavior of whey proteins in a pH-dependent manner, and this should be considered in future studies on the heat stability of such systems containing CMP.

Effects of milk heat treatment and solvent composition on physicochemical and selected functional characteristics of milk protein concentrate.

Milk protein concentrate (MPC) powders (∼81% protein) were made from skim milk that was heat treated at 72°C for 15 s (LHMPC) or 85°C for 30 s (MHMPC). The MPC powder was manufactured by ultrafiltration and diafiltration of skim milk at 50°C followed by spray drying. The MPC dispersions (4.02% true protein) were prepared by reconstituting the LHMPC and MHMPC powders in distilled water (LHMPCw and MHMPCw, respectively) or milk permeate (LHMPCp and MHMPCp, respectively). Increasing milk heat treatment increased the level of whey protein denaturation (from ∼5 to 47% of total whey protein) and reduced the concentrations of serum protein, serum calcium, and ionic calcium. These changes were paralleled by impaired rennet-induced coagulability of the MHMPCw and MHMPCp dispersions and a reduction in the pH of maximum heat stability of MHMPCp from pH 6.9 to 6.8. For both the LHMPC and MHMPC dispersions, the use of permeate instead of water enhanced ethanol stability at pH 6.6 to 7.0, impaired rennet gelation, and changed the heat coagulation time and pH profile from type A to type B. Increasing the severity of milk heat treatment during MPC manufacture and the use of permeate instead of water led to significant reductions in the viscosity of stirred yogurt prepared by starter-induced acidification of the MPC dispersions. The current study clearly highlights how the functionality of protein dispersions prepared by reconstitution of high-protein MPC powders may be modulated by the heat treatment of the skim milk during manufacture of the MPC and the composition of the solvent used for reconstitution.

Short communication: Multi-component interactions causing solidification during industrial-scale manufacture of pre-crystallized acid whey powders.

Acid whey (AW) is the liquid co-product arising from acid-induced precipitation of casein from skim milk. Further processing of AW is often challenging due to its high mineral content, which can promote aggregation of whey proteins, which contributes to high viscosity of the liquid concentrate during subsequent lactose crystallization and drying steps. This study focuses on mineral precipitation, protein aggregation, and lactose crystallization in liquid AW concentrates (∼55% total solids), and on the microstructure of the final powders from 2 independent industrial-scale trials. These AW concentrates were observed to solidify either during processing or during storage (24 h) of pre-crystallized concentrate. The more rapid solidification in the former was associated with a greater extent of lactose crystallization and a higher ash-to-protein ratio in that concentrate. Confocal laser scanning microscopy analysis indicated the presence of a loose network of protein aggregates (≤10 µm) and lactose crystals (100-300 µm) distributed throughout the solidified AW concentrate. Mineral-based precipitate was also evident, using scanning electron microscopy, at the surface of AW powder particles, indicating the formation of insoluble calcium phosphate during processing. These results provide new information on the composition- and process-dependent physicochemical changes that are useful in designing and optimizing processes for AW.

Addition of sodium caseinate to skim milk increases nonsedimentable casein and causes significant changes in rennet-induced gelation, heat stability, and ethanol stability.

The protein content of skim milk was increased from 3.3 to 4.1% (wt/wt) by the addition of a blend of skim milk powder and sodium caseinate (NaCas), in which the weight ratio of skim milk powder to NaCas was varied from 0.8:0.0 to 0.0:0.8. Addition of NaCas increased the levels of nonsedimentable casein (from ∼6 to 18% of total casein) and calcium (from ∼36 to 43% of total calcium) and reduced the turbidity of the fortified milk, to a degree depending on level of NaCas added. Rennet gelation was adversely affected by the addition of NaCas at 0.2% (wt/wt) and completely inhibited at NaCas ≥0.4% (wt/wt). Rennet-induced hydrolysis was not affected by added NaCas. The proportion of total casein that was nonsedimentable on centrifugation (3,000 × g, 1 h, 25°C) of the rennet-treated milk after incubation for 1 h at 31°C increased significantly on addition of NaCas at ≥0.4% (wt/wt). Heat stability in the pH range 6.7 to 7.2 and ethanol stability at pH 6.4 were enhanced by the addition of NaCas. It is suggested that the negative effect of NaCas on rennet gelation is due to the increase in nonsedimentable casein, which upon hydrolysis by chymosin forms into small nonsedimentable particles that physically come between, and impede the aggregation of, rennet-altered para-casein micelles, and thereby inhibit the development of a gel network.

Seasonal variation in the composition and processing characteristics of herd milk with varying proportions of milk from spring-calving and autumn-calving cows.

The study investigated the seasonal changes in the compositional, physicochemical and processing characteristics of milk from a mixed-herd of spring- and autumn-calving cows during the year 2014-2015. The volume proportion of autumn-calving milk (% of total milk) varied with season, from ~10-20 in Spring (March-May), 5-13 in Summer (June-August), 20-40 in Autumn (September-November) and 50-100 in Winter (December-February). While all characteristics varied somewhat from month to month, variation was inconsistent, showing no significant trend with progression of time (year). Consequently, season did not significantly affect many parameters including concentrations of total protein, casein, whey protein, NPN, total calcium, pH, rennet gelation properties or heat stability characteristics. However, season had a significant effect on the concentrations of total P and serum P, levels of αs1- and ß-caseins as proportions of total casein, casein micelle size, zeta potential and ethanol stability. The absence of a significant effect of season for most compositional parameters, rennet gelation and heat-stability characteristics suggest that milk from a mixed-herd of spring- and autumn-calving cows is suitable for the manufacture of cheese and milk powder on a year-round basis, when the volume proportion of autumn milk, as a % of total, is similar to that of the current study.

Effect of hydrolyzed whey protein on surface morphology, water sorption, and glass transition temperature of a model infant formula.

Physical properties of spray-dried dairy powders depend on their composition and physical characteristics. This study investigated the effect of hydrolyzed whey protein on the microstructure and physical stability of dried model infant formula. Model infant formulas were produced containing either intact (DH 0) or hydrolyzed (DH 12) whey protein, where DH=degree of hydrolysis (%). Before spray drying, apparent viscosities of liquid feeds (at 55°C) at a shear rate of 500 s(-1) were 3.02 and 3.85 mPa·s for intact and hydrolyzed infant formulas, respectively. On reconstitution, powders with hydrolyzed whey protein had a significantly higher fat globule size and lower emulsion stability than intact whey protein powder. Lactose crystallization in powders occurred at higher relative humidity for hydrolyzed formula. The Guggenheim-Anderson-de Boer equation, fitted to sorption isotherms, showed increased monolayer moisture when intact protein was present. As expected, glass transition decreased significantly with increasing water content. Partial hydrolysis of whey protein in model infant formula resulted in altered powder particle surface morphology, lactose crystallization properties, and storage stability.

The Impact of pH on Fouling and Related Physicochemical Properties of Skim Milk Concentrate during Heat Treatment Using a Laboratory-Scale Fouling Rig.

The objective of this study was to investigate the effect of pH (6.1, 6.3, 6.5, and 6.7) on heat-induced changes in concentrated skim milk as related to fouling in heat exchangers. Skim milk (30%, w/w, total solids) was recirculated in a laboratory-scale fouling rig at an initial target temperature of 85 °C for 90 min to simulate thermal processing and preheating of evaporated liquid concentrate feeds in dairy processing. This study investigated key changes in relevant physicochemical properties, such as viscosity, particle size, and sedimentation, as major contributors to fouling at lower pHs (6.1 and 6.3). Additionally, protein aggregation and calcium phosphate precipitation were identified as significant contributors to fouling deposits. Possible strategies to mitigate fouling were determined, including optimizing pH and adjusting heat treatment parameters to minimize protein denaturation and mineral deposition. The findings indicate that carefully controlling pH and processing parameters can greatly enhance the efficiency of milk concentration by evaporation and tailor finished product quality. Moreover, this study showed that monitoring of CIP solutions for protein content and turbidity provides valuable information on the intensity of fouling and the efficiency of cleaning.

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.

The impact of complexation or complex coacervation of lactoferrin and osteopontin on simulated infant gastrointestinal digestion, intestinal inflammation, and in vivo bone development.

Lactoferrin (LF) and osteopontin (OPN) are bioactive milk proteins which can form heteroprotein complexes and complex coacervates. This research studied the effect of LF-OPN complexation and complex coacervation on the simulated infant gastrointestinal digestion of LF with subsequent examination of gut and bone health bioactivities in preclinical models. In an infant digestion model, the proteolytic profile of LF was unaltered by the pre-association of LF and OPN. Gastric proteolysis of LF was increased when the model gastric pH was reduced from 5.3 to 4.0, but less so when complexed with OPN. In a model of intestinal inflammation, undigested (79% inhibition) and gastric digestates (26% inhibition) of LF, but not gastrointestinal digestates, inhibited lipopolysaccharide (LPS)-induced NF-κB activation in intestinal epithelial cells. LF-OPN complexation sustained the inhibitory effect (21-43% of the undigested effect, depending on the type of complex) of LF after gastrointestinal digestion, suggesting that the peptides produced were different. In a neonatal rodent model used to study bone development, coacervating LF and OPN improved bone structures with a significant increase of trabecular proportion (BV/TV increase by 21.7%). This resulted in an 11.3% increase in stiffness of bones. Feeding the LF and OPN proteins in coacervate format also increased the levels of OPN, P1NP and M-CSF in blood, signifying a more pronounced impact on bone development. This research demonstrated that LF-OPN complexation and complex coacervation can delay simulated infant gastrointestinal digestion of LF and protect or improve the bioactivity of the proteins.

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.