Intragastric restructuring dictates the digestive kinetics of heat-set milk protein gels of contrasting textures Siqi Li a,1,2, Tanyaradzwa Mungure b,1,3, Aiqian Ye a,*, Simon M. Loveday a,b,4, Ashling Ellis b,2, Mike Weeks b,*, Harjinder Singh a a Riddet Institute, Massey University, Private Bag 11 222, Palmerston North 4442, New Zealand b Smart Foods & Bioproducts Group, AgResearch Ltd, Te Ohu Rangahau Kai, Massey University, Palmerston North 4442, New Zealand A R T I C L E I N F O Keywords: Milk protein gels In vitro digestion Food structure Gastric restructuring Protein digestion A B S T R A C T The gelation of milk proteins can be achieved by various means, enabling the development of diverse products. In this study, heat-set milk protein gels (15 % protein) of diverse textures were made by pH modulation and two gels were selected for dynamic in vitro gastric digestion: a spoonable soft gel (SG, pH 6.55′ Gʹ of ~100 Pa) and a sliceable firm gel (FG, pH 5.65; Gʹ of ~7000 Pa). The two gels displayed markedly different structural changes and digestion kinetics during gastric digestion. The SG underwent substantial structural compaction during the first 120 min of gastric digestion into a denser and firmer gastric chyme (26.3 % crude protein, G* of ~8500 Pa) than the chyme of the FG (15.7 % crude protein, G* of ~3000 Pa). These contrasting intragastric structural changes of the gels reversed their original textural differences, which led to slower digestion and gastric emptying of proteins from the SG compared with the FG. The different intragastric pH profiles during the digestion of the two gels likely played a key role by modulating the proteolytic activity and specificity (to κ-casein) of pepsin. Preferential early cleavage of κ-casein in SG stimulated coagulation and compaction of solid chyme, whereas rapid hydrolysis of αS- and β-caseins in the FG weakened coagulation. This study provided new insights into controlling the structural development of dairy-based foods during gastric digestion and modulating digestion kinetics. 1. Introduction The gelation function of milk proteins allows the development of milk-based foods with diverse structural and textural properties, such as cheese and yoghurt. Gelation of the milk proteins is achieved by means that destabilize their colloidal stability (Lucey, 2020). Rennet-induced gelation of the casein micelles in milk is caused by the specific enzy- matic cleavage of κ-casein, which provides steric and electrostatic sta- bilization at the casein micelle surface. Acidification of milk towards the isoelectric point of the caseins at pH 4.6 reduces the negative charge and collapses the κ-casein surface layer of the casein micelles, causing their aggregation and gelation. Heat-induced gelation of the whey proteins, primarily β-lactoglobulin (β-LG), arises from the unfolding of their native globular structure and their subsequent aggregation via disulphide bonding and hydrophobic interactions. Research interest in the heat-induced gelation of the micellar casein has increased recently (Nicolai & Chassenieux, 2021). Concentrated dispersions of the casein micelles form a gel under heat treatment. pH, and salts, and the presence of whey proteins plays important roles in modulating the rheological properties of the resulting gels (Balakrishnan et al., 2018; Ji et al., 2016; Nicolai & Chassenieux, 2021). For example, firmer heat-set milk protein gels form at low pH, which has been attributed to the effect of pH in altering the structure of whey proteins and their interactions with the caseins during heat treatment (Ji et al., 2016). This provides the opportunity to develop heat-set milk protein gels with high protein content and diverse textural and sensory profiles. Sufficient protein intake is crucial for the healthy ageing of senior citi- zens, alleviating sarcopenia and its associated risks (Wolfe et al., 2008). * Corresponding authors. E-mail addresses: A.M.Ye@massey.ac.nz (A. Ye), mike.weeks@agresearch.co.nz (M. Weeks). 1 S.L. and T.M. contributed equally to this work. 2 Current address: Fonterra Research and Development Centre, PO Box 11 029, Palmerston North 4442, New Zealand. 3 Current address: School of Agriculture and Food, Faculty of Sciences, University of Melbourne, Parkville, VIC 3010, Australia. 4 Current address: Commonwealth Scientific and Industrial Research Organisation (CSIRO), Agriculture and Food, Werribee, Australia. Contents lists available at ScienceDirect Food Research International journal homepage: www.elsevier.com/locate/foodres https://doi.org/10.1016/j.foodres.2024.114944 Received 11 June 2024; Received in revised form 12 August 2024; Accepted 20 August 2024 Food Research International 195 (2024) 114944 Available online 27 August 2024 0963-9969/© 2024 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). mailto:A.M.Ye@massey.ac.nz mailto:mike.weeks@agresearch.co.nz www.sciencedirect.com/science/journal/09639969 https://www.elsevier.com/locate/foodres https://doi.org/10.1016/j.foodres.2024.114944 https://doi.org/10.1016/j.foodres.2024.114944 http://crossmark.crossref.org/dialog/?doi=10.1016/j.foodres.2024.114944&domain=pdf http://creativecommons.org/licenses/by/4.0/ However, decreased food intake and dysphagia are prevalent in the elderly population, which makes meeting nutritional requirements difficult. The heat-set milk protein gel has great potential to deliver high protein in a small-portion snack format for senior citizens. The stomach serves an important role during food digestion, including the secretion of gastric acid and the digestive enzyme pepsin, the size reduction of food particles by gastric motility and control of the gastric emptying into the duodenum (Gallier et al., 2013; Guo et al., 2020). The texture of foods influences their gastric breakdown and emptying and consequently affects the overall digestive dynamics and nutrient absorption (Loveday, 2022; Mulet-Cabero, Mackie, et al., 2020). Previous studies have shown that dairy foods with a hard texture are digested more slowly compared with their softer counterparts, e.g. cheese compared with yoghurt (Horstman et al., 2021; Lamothe et al., 2017) and a hard whey protein gel compared with a soft whey protein gel (Guo et al., 2015). These observations have been attributed to the greater resistance to physical breakdown and the limited access to digestive enzymes of the harder gel matrices. However, structural changes of the food bolus in the stomach are not limited to breakdown and emptying. Previously broken-down food particles, molecules or hydrolysates may undergo “restructuring” behaviours, such as bolus formation, coagulation and phase separation, when subjected to the gastric physiological environment, i.e., ionic composition, acid, enzyme and gastric motility (Guo et al., 2020; Acevedo-Fani & Singh, 2021). A classic example of intragastric restructuring is ruminant milk, which coagulates in the stomach before further digestion and emptying (Huppertz & Chia, 2020; Li, Pan, et al., 2022; Ye, 2021). This is mainly driven by the hydrolysis of κ-caseins at the casein micelle surface by pepsin in the gastric fluid and it occurs early during gastric digestion in 10–20 min. Furthermore, casein-based gels were reported to digest differently in the stomach depending on the means of inducing gelation. Rennet- and acid-induced milk gels with similar firmness and visco- elastic properties displayed different gastric digestion behaviours in a dynamic in vitro model (Qazi et al., 2021) and a pig model (Barbé et al., 2014); the rennet gels were digested more slowly because of their structural compaction in the stomach. The lower pH of acid gels ( 0.05). In contrast, the moisture content of the SG solid chyme decreased significantly from 20 to 120 min of digestion, reaching Fig. 1. In situ gelation profiles of milk protein gels made in the rheometer. Black lines indicate the storage modulus values of the firm gel made at pH 5.65 (●) and the soft gel made at pH 6.55 (✳✳). The red line indicates temperature. Images show the textural differences between the firm gel and the soft gel. S. Li et al. Food Research International 195 (2024) 114944 4 the lowest level of all samples (70.0 %), reflecting its dry appearance as presented in Fig. 2. The moisture content of the SG solid chyme increased from 120 to 240 min of digestion to 75.3 %. At both 120 and 240 min, the SG solid chyme contained significantly less moisture than the FG solid chyme (P < 0.05). The crude protein percentages in the solid chymes displayed the opposite trend to their moisture contents (Fig. 3D). For the FG, crude proteins made up 17.9 % of the chyme weight at 20 min and demonstrated an insignificant (P > 0.05) decreasing trend during digestion to 15.7 % at 120 min and 14.0 % at 240 min. In contrast, the SG solid chyme contained 13.5 % crude pro- teins at 20 min, which almost doubled at 120 min (26.3 %) and then decreased to 21.3 % at 240 min. 3.4. Rheological properties of digested milk protein gels The G* values of the milk protein gel boluses following simulated oral processing and of the solid chymes at 20, 120 and 240 min of gastric digestion were analysed to indicate their consistency (Fig. 4). As expected, the G* of the masticated FG bolus (727 Pa) was markedly higher than that of the masticated SG bolus (31 Pa) before gastric digestion. The G* of the FG solid chyme was 980 Pa at 20 min of digestion, slightly higher than at 0 min, increased to about 3000 Pa at 120 min and decreased to 1160 Pa at 240 min of digestion. In contrast, the G* of the SG solid chyme at 20 min of digestion (166 Pa) was five times greater than that of the gel bolus at 0 min (Fig. 4); it increased dramatically to over 8500 Pa at 120 min of digestion, markedly higher than that of its FG counterpart at 120 min. At 240 min, the G* of the SG solid chyme (4785 Pa) was still higher than that of the FG solid chyme. 3.5. Emptied digesta – appearance, composition and pH profile The milk components were emptied in different forms and quantities during the gastric digestion of the SG and the FG, as illustrated in the photographs of the emptied digesta (Fig. 5A); this reflected the crude protein percentages of the digesta (Fig. 5B). The SG digesta at 20 min Fig. 2. Photographs and confocal laser scanning microscopy images of the firm gel (FG) and the soft gel (SG) at different stages of dynamic gastric digestion (numbers indicate the digestion time). Samples at 0 min were the gel boluses following simulated oral processing; samples at 20, 120 and 240 min were the solid gel chymes retained in the stomach chamber by a 1-mm sieve. S. Li et al. Food Research International 195 (2024) 114944 5 had the most opaque appearance, indicating the presence of a large quantity of free proteins. The SG digesta rapidly became transparent at 40 min of digestion with a small amount of sediment particles (Fig. 5A), corresponding to the sharp drop in protein content in the digesta (Fig. 5B); this was largely maintained throughout the digestion. At 240 min, the SG digesta appeared to be slightly cloudier and the crude protein percentage in the digesta increased (Fig. 5A and 5B). In com- parison, the FG digesta at 20 min was more transparent (Fig. 5A), cor- responding to its lower protein content (Fig. 5B); it became cloudier and contained more sediment particles than the SG digesta from 40 to 120 min of digestion (Fig. 5A). This suggested greater gastric emptying of the milk components as small particles during the digestion of the FG, which was supported by its higher crude protein percentage than that of the SG during this period (P < 0.01, Fig. 5B). At 180 min, the FG digesta was cloudier than the SG digesta but they contained similar amounts of sediment. At 240 min of digestion, slightly fewer particles were found in the FG digesta than in the SG digesta, again in line with their crude protein percentages. The gradual decreases in pH during the digestion of the SG and the FG were measured in their emptied digesta (Fig. 5C). The shape of the pH–time curve during dynamic gastric digestion in the HGS is indicative of the structural changes (e.g. coagulation or breakdown) of ingested foods (Li, Ye, et al., 2022; Ye et al., 2016). As expected, the SG had both a higher initial pH (6.5 ± 0.1) and a higher final pH (2.3 ± 0.1) than the FG (5.6 ± 0.1 at 0 min; 1.7 ± 0.2 at 240 min). However, from 80 to 120 min of digestion, the pH did not differ significantly between the SG and the FG (P ≥ 0.1). This resulted from the different rates of pH decrease during the early stages of digestion (Fig. 5C). The pH of the SG digesta decreased more rapidly than that of the FG digesta from 20 to 60 min, and then decreased linearly. In contrast, FG digesta has a slow rate of pH decrease during early digestion, which then increased over time. This difference was probably because of the depletion of proteins (the main buffering component) from the SG digesta at 40 min of digestion; in contrast, FG broke down faster from 40 to 120 min of digestion that increased the buffering capacity of the liquid phase (Fig. 5B). During the later stages of digestion (from 160 min), the more rapid decrease in pH of the FG digesta than the SG digesta (Fig. 5C) was probably the result of the lower retention of the FG chyme in the gastric chamber (Figs. 2 and 3) that reduced buffering capacity. 3.6. Emptied digesta – microstructure and particle size distribution There were microstructural differences in the CLSM images of the emptied digesta (Fig. 6A). The FG digesta was composed of loose protein aggregates from 20 to 120 min of digestion, which were smaller and tighter at 60 and 120 min. At 180 and 240 min of digestion, the FG digesta contained mainly small particles, although some large but loose structures were also found at 240 min (Fig. 6A). In contrast, SG digesta had a greenish background during early digestion, similar to that of the SG bolus before gastric digestion (Fig. 2), indicating the presence of free proteins or small protein aggregates. At 60 and 120 min of digestion, the SG digesta contained large and highly compact particles; they became smaller towards the end of digestion (180 and 240 min), but were larger and more compact than those in their FG counterparts. The particle size distributions of the FG digesta and the SG digesta developed differently during digestion (Fig. 6B and 6C). The particle size of the FG digesta decreased consistently throughout the digestion. The main population shifted gradually from 200–300 µm at 20 min to 10–20 µm at 240 min of digestion. In contrast, the changes in the particle size distribution of the SG digesta over time displayed a coagulation phase followed by a breakdown phase. The SG digesta at 20 min was composed Fig. 3. Weight and composition of the solid chyme retained at different stages during the dynamic gastric digestion of the soft gel and the firm gel: wet weight (A), crude protein (B), moisture content percentage (C) and crude protein percentage (D) in the solid chyme weight. Different letters (a, b) indicate sig- nificant differences between the gels at the same time point. Fig. 4. Complex modulus (G*) of milk protein gels following oral processing (0 min) and the solid chymes at 20, 120 and 240 min of gastric digestion. Error bars represent standard deviations. Different letters (a, b) indicate significant differences between the gels at the same time point. No statistical analysis was conducted for Firm gel–240 min because there was sufficient sample in only one run. S. Li et al. Food Research International 195 (2024) 114944 6 mainly of particles of ~20 µm. It was also the only sample that had a population at ~0.3 µm, which probably indicated native or small ag- gregates of casein micelles, in line with the micrograph (Fig. 6A). The main population in the SG digesta gradually increased to around 200 µm from 40 to 120 min of digestion. At 180 and 240 min of digestion, the population of large particles of >100 µm remained, but the population of small particles at 10–20 µm again increased. 3.7. SDS-PAGE profiles Fig. 7 presents the protein profiles of the solid chyme and the emptied digesta as analysed using SDS-PAGE. Major differences already occurred in 20 min of digestion. New peptide bands resulting from casein hydrolysis (15–25 kDa) appeared in the solid chymes of both gels (enlarged lanes, Fig. 7). The FG solid chyme contained less intact αS- casein and β-casein and more peptides than the SG solid chyme. The emptied digesta showed an even greater difference. The FG digesta at 20 min contained much more peptides (including small peptides of ~10 kDa) and fewer intact proteins than the SG digesta. The results indicated greater proteolysis of the FG than the SG at 20 min of digestion. Comparing the SG digesta and the SG solid chyme at 20 min of digestion (enlarged lanes, Fig. 7), the κ-casein band was more pronounced in the SG digesta than in the solid chyme. Staining intensity analysis estimated that the proportion of intact κ-casein in the digesta was 28 % higher than that in the solid chyme. To lesser extents, other intact proteins were also proportionally more abundant in the SG digesta than in the solid chyme at 20 min. From 40 min of digestion, the SDS-PAGE profiles of the two gels became more similar, both composed of more peptides while intact proteins were gradually hydrolysed. Smaller peptides (≤12 kDa) made up a much higher proportion in the SG solid chyme and digesta than in the FG counterparts (Fig. 7). This indicated that proteins and larger peptides were better retained in the SG solid chyme and only the highly digested peptides were emptied. In contrast, the FG was more easily emptied without extensive proteolysis. Regarding whey proteins, β-LG appeared to be more resistant to pepsin digestion in the FG than in the SG. β-LG was present in higher proportions in the FG digesta than in the SG digesta at each time point from 40 to 120 min. From 120 min of digestion, β-LG band was visible in both the solid chyme (dashed rectangles, Fig. 7) and the digesta of FG but had disappeared from the SG. In contrast, α-lactalbumin (α-LA) behaved similarly in both gels, largely disappeared from 120 min from both solid chyme and emptied digesta, when the pH had decreased to about 4.0. This is in agreement with previous studies that α-LA becomes susceptible to pepsin hydrolysis at pH < 4.0 (Li et al., 2021; Roy et al., 2021). Fig. 5. Photographs (A), crude protein contents (B) and pH profiles (C) of the emptied digesta sampled during the gastric digestion of the soft gel and the firm gel. The pH at 0 min was measured in the gel bolus following simulated oral processing. Different letters (a, b) indicate significant differences between the gels at the same time point. S. Li et al. Food Research International 195 (2024) 114944 7 4. Discussion 4.1. Different structural development during gastric digestion of the milk protein gels The structural and compositional analyses consistently demonstrated two contrasting processes of restructuring and destructuring during the gastric digestion of the FG and the SG. The FG gradually broke down and was emptied as particles from the beginning of the gastric digestion whereas the SG underwent coagulation and firming of the structures during the first 120 min of gastric digestion before apparent breakdown took place in the last 120 min. The main difference in the structural development of the two gels occurred in the first 120 min of digestion, resulting in the SG forming a much larger and firmer solid chyme than the FG (Figs. 2 and 4), which reversed their initial textural differences. The intragastric coagulation and compaction of the SG resulted in a pronounced increase in the percentage of crude protein in the solid chyme from 20 to 120 min (Fig. 3D), which fused into a compact structure (Fig. 2) that expelled moisture (Fig. 3C) and had the highest consistency (Fig. 4). For the FG, its structure, composition and consistency did not change markedly during digestion, except for a smaller increase in consistency at 120 min (Fig. 4). Analyses of the emptied digesta indicated that the compaction of the SG limited gastric emptying of proteins in the first 120 min of digestion, when major destructuring and emptying occurred for the FG (Fig. 5). From the FG solid chyme, 64.2 % of the total ingested protein was released from the stomach from 20 to 120 min. Meanwhile, after gastric emptying at 20 min, 33 % of the total protein was present in the SG liquid chyme, much higher than in the FG liquid chyme (4 %). Most of these liquid-phase proteins had presumably been incorporated into the SG solid chyme later, resulting in the sudden decrease in emptied pro- teins at 40 min (Fig. 5), and the increased percentage of protein in the SG solid chyme from 20 to 120 min (Fig. 3D). The compact structures of the SG solid chyme and the SG particles (Figs. 2 and 6) probably limited the access to pepsin and resisted physical breakdown, contributing to their delayed gastric digestion and emptying. The results highlighted that the digestive dynamics of casein-based milk protein gels can be dictated by their structural changes during gastric digestion rather than their initial structures. 4.2. Possible mechanisms for the contrasting gastric digestion behaviours 4.2.1. Role of pH profile during gastric digestion We propose that the pH contrast between the SG (pH 6.55) and the FG (pH 5.65) played a key role in modulating their structural changes during the first 120 min of digestion, via its impacts on the proteolytic activity and the specificity (to κ-casein) of pepsin and the calcium equilibrium. The proteolytic activity of pepsin is highly pH-dependent. Pepsin is barely active at pH > 6.0, starts to increase in activity at pH < 5.5, reaches a major activity peak at pH 4.0–4.5 (70 % of the maximum) and exhibits maximal activity at pH 2.0 (Piper & Fenton, 1965; Salelles et al., 2021). As a result, different pH profiles during dynamic gastric digestion can play a crucial role in determining the rate of digestion. Previous studies have reported that the more rapid gastric digestion of acid- Fig. 6. Confocal laser scanning microscopy images (A) and particle size distributions (B and C) of the digesta emptied at different time points (from 20 to 240 min) during gastric digestion of the firm gel (FG) and the soft gel (SG). Proteins are indicated in green in the confocal images. S. Li et al. Food Research International 195 (2024) 114944 8 induced milk gels (pH 4.0–4.6) than of rennet milk gels (pH > 6) arises partially from the greater pepsin activity at pH 4 or below, which is reached much earlier during the digestion of acid gels (Barbé et al., 2014; Qazi et al., 2021). However, in the current study, the FG had a pH of 5.65, considerably higher than that of typical acid milk gels. The pHs of the SG digesta and the FG digesta did not differ significantly during 80–120 min of digestion when the pH of both gels decreased from around 4.6 to around 4.0 (Fig. 5C). This indicated that the time to reach the peak of pepsin activity at pH 4.0 was not a key contributor to the contrasting digestion behaviours during early digestion. As well as affecting the overall pepsin proteolytic activity, pH is also crucial for the specificity of pepsin to κ-casein (Tam & Whitaker, 1972; Yang et al., 2022). Pepsin can completely hydrolyse the κ-casein in milk during prolonged incubation at pH 6.0 while not hydrolysing other milk proteins (Yang et al., 2022). The specific rate of pepsin hydrolysis of κ-casein is slightly lower at pH 6.3 and is significantly lower at pH 5.3 (Yang et al., 2022). The preferential hydrolysis of κ-casein by pepsin at a fairly high pH (>6.0) is responsible for the gastric coagulation of casein micelles (Huppertz & Chia, 2020; Ye, 2021). For milk clotting enzymes, a high ratio of milk clotting activity (for specific κ-casein hydrolysis) to their overall proteolytic activity is crucial for their coagulating proper- ties (Guinee & Wilkinson, 1992). The porcine pepsin used in in vitro digestion studies has greater proteolytic activity than rennet (Guinee & Wilkinson, 1992) and thus is prone to result in excessive hydrolysis of αS- and β-caseins, which impairs coagulation. Therefore, the time for which the pH remains in the range for preferential κ-casein hydrolysis of pepsin is crucial for the structural changes during gastric digestion. In the present study, the pH of the SG digesta was 6.2 at 20 min and remained above pH 5.5 at 40 min, whereas the pH of the FG digesta was already reduced to 5.2 at 20 min (Fig. 5C). The higher pH during the early stages of digestion of the SG probably favoured preferential hydrolysis of κ-casein while not markedly hydrolysing the other caseins as demon- strated with SDS-PAGE (Fig. 7). The high concentration of intact caseins in the SG digesta at 20 min (Fig. 7) provided the potential for further coagulation, which occurred, as evidenced by the sharp decrease in the protein content in the digesta at 40 min (Fig. 5B) and the greater per- centage of proteins in the SG solid chyme at 120 min (Fig. 3D). In contrast, the pH during the digestion of the FG quickly fell out of the optimal range that favours coagulation and much greater proteolysis of other caseins occurred during the first 20 min of digestion (Fig. 7). Consequently, the FG lacked the driving force for strong compaction during the early stages of digestion, as indicated by the smaller change in structure and texture of the solid chyme (Figs. 2 and 4) and the in- crease in gastric emptying from 20 to 60 min (Fig. 5). The pH difference between the SG and the FG would also have affected the calcium equilibrium, i.e. the dissolution of CCP. Around 70 % of milk calcium is insoluble at pH 6.7, which decreases to 22 % at pH 5.6 (Choi et al., 2007). The SG with its higher pH would have contained a greater quantity of CCP, which probably contributed to its greater intragastric compaction during early digestion. This is supported by the study of Huppertz and Lambers (2020), who reported that a model in- fant formula made from unadjusted skim milk (pH 6.7) underwent coagulation during gastric digestion, but a model infant formula made from skim milk equilibrated at pH 5.7 to partially remove the CCP did not. From 120 to 240 min of digestion, all the results consistently showed that both milk protein gels underwent breakdown and protein digestion. This can be explained primarily by the markedly higher proteolytic ac- tivity of pepsin during this period when the pH was below 4.0 (Fig. 5C). Furthermore, there may have been swelling of the solid chyme structure Fig. 7. Sodium dodecyl sulphate polyacrylamide gel electrophoresis profiles of the gastric solid chyme and the emptied digesta at different time points (min) during the dynamic gastric digestion of the soft gel (SG) (top) and the firm gel (FG) (bottom). Lanes of three samples at 20 min of digestion are enlarged on the right-hand side for better comparison. Peptide bands referred to in the text are labelled in the enlarged images. S. Li et al. Food Research International 195 (2024) 114944 9 as the pH decreased further away from the isoelectric point of the pro- teins (pH 4.6–5.3) (van der Sman et al., 2020). It could have contributed to the higher moisture content and lower consistency of the solid chyme (Figs. 3C and 4) towards the end of digestion. 4.2.2. Role of whey proteins Whey proteins can contribute to the structure of heat-set gels formed by casein micelles even without gelling themselves (Ji et al., 2016; Nicolai & Chassenieux, 2021). It was interesting to observe a markedly higher proportion and longer survival of β-LG in the FG solid chyme and digesta than in the SG counterparts, whereas α-LA behaved similarly during the digestion of the two gels (Fig. 7). The greater emptying of β-LG from the SG at 20 min of digestion (Fig. 7) probably contributed to the lower retention of β-LG in the SG solid chyme. However, this cannot explain the greater resistance of β-LG to pepsin hydrolysis in the FG than in the SG. It is well known that heat-induced denaturation renders β-LG susceptible to gastric digestion by exposing the cleavage sites of pepsin (Deng et al., 2020; Kitabatake & Kinekawa, 1998; Li et al., 2021). The pH of heat treatment is known to affect the structures of heat-induced whey protein gels (Langton & Hermansson, 1992), which can affect their susceptibility to gastrointestinal digestion (Macierzanka et al., 2012). When heated in the presence of casein micelles, denatured whey proteins partially associate with them, and the extent of association is greater at a lower pH (Corredig & Dalgleish, 1996; Vasbinder & de Kruif, 2003). The different pHs during the gelation of the SG and the FG may have affected the state of aggregation and the micelle association of denatured β-LG, which resulted in the different susceptibilities of β-LG to pepsin hydrolysis. The greater incorporation of denatured whey proteins in gastric milk clots has been well demonstrated to loosen the clot structure by hin- dering the fusion of the casein matrix (Li et al., 2021; Ye et al., 2017). Hence, the higher proportion of β-LG in the FG solid chyme may have contributed to its looser structure and lower consistency than the SG from 120 min (Figs. 2 and 4). However, rennet milk gels have been re- ported to undergo stronger compaction than acid milk gels during gastric digestion, regardless of whether they are made from unheated (Qazi et al., 2021) or intensely heated (90 ◦C for 10 min) (Barbé et al., 2014) milk, indicating that the involvement of whey proteins did not play the main role. 4.2.3. Role of the initial gel texture The different textures of the SG and the FG and their masticated boluses before gastric digestion may also have affected their gastric digestion behaviours. The mastication of the two gels was simulated differently to reflect their textural differences (Fig. 1), which would influence the particle size of the bolus in the gastric phase and their subsequent digestion. The weaker consistency of the masticated SG bolus and the presence of free proteins and small protein aggregates (Figs. 2 and 4) may have allowed pepsin to access and hydrolyse κ-casein more readily, and the relatively high mobility of intact proteins within the weak gel facilitated optimal coagulation and compaction during early digestion. The impact of the initial gel structures can be further investigated by studying liquid milk systems adjusted to different pH levels similar to those of the SG and the FG. 5. Conclusions The present study demonstrated that the kinetics of protein digestion and gastric emptying of two heat-set milk protein gels with different textures were dominated by their different structural changes under gastric conditions. The FG (pH 5.65) was continuously broken down during gastric digestion. In contrast, the SG (pH 6.55) underwent coagulation and structural compaction during the first 120 min of gastric digestion, forming firmer gastric chyme that retained greater quantities of protein than the FG. This contrast in textural developments resulted in a slower digestion of the SG than the FG. The effect of pH on pepsin activity and specificity probably played a key role, as supported by the more rapid digestion of intact caseins in the FG during early digestion. the findings of this study provided a deeper understanding of the physicochemical leavers to modulate the structural changes of milk protein gels during digestion, which could enable novel food designs of unique texture and digestive outcomes. CRediT authorship contribution statement Siqi Li: Writing – original draft, Visualization, Methodology, Inves- tigation, Formal analysis, Conceptualization. Tanyaradzwa Mungure: Writing – review & editing, Methodology, Investigation, Formal anal- ysis, Data curation. Aiqian Ye: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization. Simon M. Loveday: Writing – review & editing, Methodology, Funding acquisition, Conceptualization. Ashling Ellis: Writing – review & editing, Methodology. Mike Weeks: Writing – re- view & editing, Supervision, Project administration, Funding acquisi- tion, Conceptualization. Harjinder Singh: Writing – review & editing, Resources. 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. Data availability Data will be made available on request. Acknowledgements This work was supported by the New Zealand Ministry of Business, Innovation and Employment (Wellington, New Zealand), via the pro- gramme “New Zealand Milks Mean More” (MAUX1803), and the Ter- tiary Education Commission, via the Riddet Institute–New Zealand Centre of Research Excellence. The Manawatu Microscopy and Imaging Centre (Palmerston North, New Zealand) is acknowledged for accom- modating microscopy imaging. We thank Xiangqian Zhu, Jack Cui (Riddet Institute, Massey University) for conducting the confocal im- aging and Noby Jacob (AgResearch Ltd) for contributing to sample preparation. The authors acknowledge Claire Woodhall for proofreading the manuscript. References Acevedo-Fani, A., & Singh, H. (2021). Biopolymer interactions during gastric digestion: Implications for nutrient delivery. 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