Digestion of food proteins: the role of pepsin

“However, excessive calcium had a negative effect on curd formation due to charge repulsion or reduced κ-casein solubility (Udabage, McKinnon, and Augustin 2001; Choi, Horne, and Lucey 2007; Fox et al. 2017; Lucey and Fox 1993; Patel and Reuter 1986).”

“These tools leverage databases such as MEROPS and PRIDE to enhance the characterization of cleavage sites, while advanced algorithms, including recurrent neural networks and transformers, capture sequence and structural dependencies (Rawlings, Barrett, and Bateman 2010; Jones et al. 2006).”

“This is expected, as α S1 - and α S2 -caseins are poorly structured proteins with no defined tertiary structure, and therefore exposed to proteases, such as pepsin, trypsin, and chymotrypsin (Swaisgood 2003; Astwood, Leach, and Fuchs 1996).”

“Myoglobin is a sarcoplasmic protein that contributes to meat color (Suman and Joseph 2013).”

“The prolamins found in wheat are called gliadins, while the nomenclature of the prolamins in other cereals is based on their Latin names; zein in maize, hordein in barley, secalin in rye etc. (Shewry and Tatham 1990).”

“(Réhault-Godbert, Guyot, and Nys 2019).”

“Most thermal, non-thermal, and emulsification processes can affect the structural properties of egg proteins; the gelation mechanism of egg white protein is typically described as follows: native protein → denatured protein → soluble aggregated protein → gel or curd (Yoshinori Mine 1995).”

“The individual casein molecules lack a tertiary structure, but together with colloidal calcium phosphate (CCP), can assemble as large structures called casein micelles, with κ-casein bound to the micelle surface layer (Fox 1993; Dalgleish and Corredig 2012).”

“Glutelin, which consists of α and β subunits linked through disulfide bonds, represents the dominant dietary protein fraction in rice (Amagliani et al. 2017).”

“The aggregation and gelation of sarcoplasmic proteins begin at approximately 40 °C and finish at approximately 60 °C (Baldwin 2012).”

“The stomach secretes an average of 2–3 L of gastric fluid each day, containing water, electrolytes, hydrochloric acid, enzymes, mucus, and glycoprotein intrinsic factor (Arora et al. 2005; Bornhorst and Paul Singh 2014).”

“…few decades, different models have been proposed to characterize the internal and external structures of casein micelles, including the submicelle (Walstra 1990), dual binding (Horne 2006), and nanocluster (de Kruif and Holt 2003; Douglas G Dalgleish 2011) models. α S1 -casein, α S2 -casein,…”

“…of casein micelles, including the submicelle (Walstra 1990), dual binding (Horne 2006), and nanocluster (de Kruif and Holt 2003; Douglas G Dalgleish 2011) models. α S1 -casein, α S2 -casein, β-casein, and κ-casein have been classified as important cow milk allergens by the World Health…”

“However, depending on heating conditions, excessive heat could cause the formation of large protein aggregates, which block enzymatic reactions (Sá, Moreno, and Carciofi 2020).”

“However, excessive calcium had a negative effect on curd formation due to charge repulsion or reduced κ-casein solubility (Udabage, McKinnon, and Augustin 2001; Choi, Horne, and Lucey 2007; Fox et al. 2017; Lucey and Fox 1993; Patel and Reuter 1986).”

“Cooking legumes has been shown to cause protein denaturation, reducing their resistance to enzymatic attack (Alajaji and El-Adawy 2006; Wiesinger et al. 2018).”

“…have been proposed to characterize the internal and external structures of casein micelles, including the submicelle (Walstra 1990), dual binding (Horne 2006), and nanocluster (de Kruif and Holt 2003; Douglas G Dalgleish 2011) models. α S1 -casein, α S2 -casein, β-casein, and κ-casein have been…”

“Milk from different species responds differently to pepsin and acid (Roy et al. 2020b).”

“…broad specificity for hydrolyzing bonds adjacent to aromatic and hydrophobic amino acids, such as phenylalanine, tyrosine, tryptophan, and leucine (Dunn 2002; Inouye and Fruton 1967), with an optimum pH of approximately 2.0 (Kondjoyan, Daudin, and Santé-Lhoutellier 2015; Pletschke, Naudé, and…”

“When pres-ent as an adsorbed layer on emulsion surfaces, the change in conformation due to unfolding at the emulsion interface exposes peptic hydrolysis sites, substantially decreasing the resistance of β-Lg to pepsin (Sarkar et al. 2009; Nik, Wright, and Corredig 2010; Peram et al. 2013).”

“…the internal and external structures of casein micelles, including the submicelle (Walstra 1990), dual binding (Horne 2006), and nanocluster (de Kruif and Holt 2003; Douglas G Dalgleish 2011) models. α S1 -casein, α S2 -casein, β-casein, and κ-casein have been classified as important cow…”

“Actin and myosin heavy chains are rapidly degraded within a short period of time under simulated gastric digestion conditions (Liu et al. 2011); whereas tropomyosin is relatively resistant to pepsin digestion (Liu et al. 2011) and even after heat treatment (Leung et al. 1994; Shanti et al. 1993).”

“Most ingested proteins are first digested by pepsin, which produces large polypeptides, a few smaller peptides, and a few free amino acids in the stomach (Joye 2019).”

“…slower rate of proteolytic digestion; therefore, its consumption results are nil or not physiologically relevant, much lower yield of β-casomorphin-7 (Boutrou et al. 2013; Haq, Kapila, and Kapila 2015; De Noni 2008) and substantially less severe gastrointestinal symptoms for individuals who…”

“Heating at 80–90 °C induces structural changes in β-Lg that improve susceptibility to pepsin activity (Reddy, Kella, and Kinsella 1988).”

“Its complex triple-helical structure (Exposito et al. 2010) and high levels of glycine and (hydroxyl) proline reduce the flexibility of the protein back-bone, which affects the binding of enzymes to active sites on proteins (Kaur et al. 2010).”

“However, extensive aggregation of proteins can decrease proteolytic susceptibility and hinder protein digestion (Lee et al. 2021), for example, excessive heating (Santé-Lhoutellier et al. 2008), or dry curing (Du et al. 2018).”

“These tools leverage databases such as MEROPS and PRIDE to enhance the characterization of cleavage sites, while advanced algorithms, including recurrent neural networks and transformers, capture sequence and structural dependencies (Rawlings, Barrett, and Bateman 2010; Jones et al. 2006).”

“…(>100 °C) can induce oxidation, disulfide bond formation, and protein aggregation or gelation, which can bury the active sites deep into the tertiary structure, thereby decreasing accessibility to pepsin (Sante-Lhoutellier, Aubry, and Gatellier 2007; Marie-Laure Bax et al. 2012; He et al. 2018).”

“Emerging machine learning and deep learning models, such as DeepCleave and PROSPER, improve predictive accuracy by training on extensive cleavage datasets and incorporating sequence and structural features (Li, Chen, et al. 2020; Song et al. 2012).”

“Collectively, these changes enhance pepsin accessibility to hydrolysis sites and improve the digestibility of myofibrillar proteins (Marie-Laure Bax et al. 2012).”

“However, excessive calcium had a negative effect on curd formation due to charge repulsion or reduced κ-casein solubility (Udabage, McKinnon, and Augustin 2001; Choi, Horne, and Lucey 2007; Fox et al. 2017; Lucey and Fox 1993; Patel and Reuter 1986).”

“Poor digestibility is associated with increased allerge-nicity when intact proteins or large fragments are exposed to the gut immune system, thereby inducing IgE-mediated food allergic responses (Astwood, Leach, and Fuchs 1996; Untersmayr and Jensen-Jarolim 2008).”

“The stomach secretes an average of 2–3 L of gastric fluid each day, containing water, electrolytes, hydrochloric acid, enzymes, mucus, and glycoprotein intrinsic factor (Arora et al. 2005; Bornhorst and Paul Singh 2014).”

“…of the variants (A1, A2, B, and I) was subjected to in vitro digestion using pepsin at pH 2.0, Petrat-Melin et al. (2015) observed minimal intact β-casein presented using SDS-PAGE after 60 min of pepsin digestion (with little difference between different variants), following Dupont et al. (2010).”

“Alves and Tavares (2019) reported that animal and plant protein-mixed food systems are efficient in modulating the texture of protein gels, forming low-cost edible films, and producing stable emulsions and foams.”

“Connective tissues tend to gelatinize at 80 °C and form a gel that fills the spaces between muscle fibers (Roldán et al. 2013); this can decrease the digestibility of meat proteins by reducing the diffusion of digestive enzymes into the compact myofibrillar structure (L. Kaur et al. 2014).”

“A comparison of the primary structures of typical mammalian pepsinogens and pepsins has been reported by Kageyama (2002).”

“Previous studies on protein digestion fall into four categories: in vivo human studies, in vivo animal studies, static in vitro studies, and (semi)-dynamic in vitro studies (van Lieshout et al. 2020).”

“…of 2S albumins to pepsin digestion in simulated gastric fluid due to their compact structure, a high number of disulfide bonds, and their capacity for self-association (Moreno et al. 2005; Murtagh et al. 2003; Pantoja-Uceda et al. 2004; Koppelman et al. 2005; Carbonaro, Maselli, and Nucara 2015).”

“However, humans show wide variations in gastric and pancreatic secretions, which vary with the type of food consumed (da Silva Gomes et al. 2003; Armand et al. 1996).”

“Actin and myosin heavy chains are rapidly degraded within a short period of time under simulated gastric digestion conditions (Liu et al. 2011); whereas tropomyosin is relatively resistant to pepsin digestion (Liu et al. 2011) and even after heat treatment (Leung et al. 1994; Shanti et al. 1993).”

“Furthermore, adsorption-induced changes in β-Lg facilitate the hydrolysis of peptides by pepsin (Macierzanka et al. 2009).”

“According to Jinsmaa and Yoshikawa (1999) and Haq, Kapila, and Kapila (2015), pepsin hydrolyzes the Leu 58 -Val 59 peptide bond of β-casein, and leucine aminopeptidase removes valine from the N-terminus.”

“…β-Lg aggregate through thiol/disulfide interchange, hydrophobic interactions, and electrostatic and ionic interactions with themselves and with κ-casein, leading to so-called whey protein/κ-casein complexes (Guyomarc’h, Law, and Dalgleish 2003; Jang and Swaisgood 1990; Singh and Creamer 1991).”

“The initial step in protein digestion is degradation at the acidic pH in the stomach and hydrolysis by the action of pepsin with varying substrate specificities (Whitecross et al. 1973; Silk, Grimble, and Rees 1985).”

“However, the production of animal-derived protein sources is associated with higher greenhouse gas emissions, whereas plant-based protein sources are considered environmentally sustainable (Gorissen and Witard 2018).”

“Native ovotransferrin and lysozyme can be rapidly hydrolyzed by pepsin (Liu et al. 2017; Liu et al. 2018; Yoshino et al. 2004), whereas ovotransferrin is more susceptible to pepsinolysis than ovalbumin and lysozyme (Liu et al. 2017; Fu, Abbott, and Hatzos 2002; Moreno 2007).”

“Consequently, milk coagulation time is inversely dependent on enzyme concentration (Figure 3B3) (Dalgleish 1993; Carlson, Hill, and Olson 1987a; López, Lomholt, and Qvist 1998; Foltmann 1959; Hyslop, Richardson, and Ryan 1979).”

“Whey proteins mainly consist of β-lactoglobulin (β-Lg) and α-lactalbumin (α-La) (ratio ca. 3:1) in milk (Visser, Slangen, and Rollema 1991).”

“Collagen is the main component of the endomysium and perimysium, which determines meat tenderness and digestibility (Purslow 2018).”

“Egg yolk proteins can be hydrolyzed by food-grade proteinases from Bacillus sp. (Sakanaka et al. 2004).”

“The major proteins observed in egg yolk include low-density lipoprotein (68%), high-density lipoprotein (16%), phosvitin (4%), and livetin, which exist in a homogeneously emulsified fluid (Anton et al. 2003). α-Livetin (Gal d 5) is commonly associated with allergies (Dona and Suphioglu 2020).”

“Physicochemical processes and pretreatments, such as pulsed electric fields, high-pressure processing, and cooking, affect digestive processes, thereby affecting the release and bio-availability of amino acids (Gong et al. 2020; Bhat et al. 2021b).”

“Curd at a lower pH is more prone to microsyneresis (Mellema et al. 2002), with larger pores in the microstructure (Yang et al. 2022).”

“Ogawa et al. (1987) demonstrated that the 13 kDa prolamin band in raw rice persisted after pepsin digestion, even as glutelin subunit bands (37– 39 kDa and 22–23 kDa) disappeared, suggesting that prol-amin is an indigestible protein.”

“Egg proteins are highly digestible: the true ileal digestibility of cooked whole egg proteins has been observed to be 90.9% in humans (Evenepoel et al. 1998).”

“Studies on milk digestion, especially milk protein digestion, have recently attracted extensive attention (Thom Huppertz and Chia 2021; Ye 2021; Miranda and Pelissier 1983; Wada and Lönnerdal 2014; Tunick et al. 2016; Ye et al. 2016b; Mulet-Cabero et al. 2020).”

“The products generated during protein digestion largely depend on the structural properties of the food, including protein solubility, accessibility of digestive enzymes, protein sources, and the processing history of foods (Kaur et al. 2022).”

“Egg yolk is widely used as a functional and nutritional ingredient in food products (Juneja and Kim 2018; Y Mine 2002).”

“A “hard” whey protein-based gel structure can slow down the disintegration of the protein matrix, whereas a “soft” whey protein-based gel structure can be completely broken down after 4 h of digestion (Guo et al. 2014; N. Luo et al. 2021).”

“Studies on milk digestion, especially milk protein digestion, have recently attracted extensive attention (Thom Huppertz and Chia 2021; Ye 2021; Miranda and Pelissier 1983; Wada and Lönnerdal 2014; Tunick et al. 2016; Ye et al. 2016b; Mulet-Cabero et al. 2020).”

“As temperature affects pepsin activity (Zhao et al. 2011) and enzyme-induced milk coagulation behavior (Nájera, De Renobales, and Barron 2003; Dalgleish 1983; Horne and Lucey 2014), milk temperature has a profound effect on the structural organization of pepsin-induced curd.”

“In the soft curds formed from heated milk (72 °C for 15 s or 140 °C for 4 s), casein proteins are hydrolyzed faster than in hard clots formed from unheated milk (Ye et al. 2016b, 2017; Ye, Liu, et al. 2019; Miranda and Pelissier 1983).”

“Generally, three different phases of protein digestion can be distinguished throughout the gastrointestinal tract (Erickson and Kim 1990): cephalic, stomach, and intestinal.”

“Different hydrolysis and digestion behaviors have been observed under gastric environmental conditions because of the structural differences between casein and whey proteins (Wang, Ye, et al. 2018), which affect allergenicity (Bernard et al. 1998; Busse et al. 2002; Cocco et al. 2003).”

“When pres-ent as an adsorbed layer on emulsion surfaces, the change in conformation due to unfolding at the emulsion interface exposes peptic hydrolysis sites, substantially decreasing the resistance of β-Lg to pepsin (Sarkar et al. 2009; Nik, Wright, and Corredig 2010; Peram et al. 2013).”

“Studies on milk digestion, especially milk protein digestion, have recently attracted extensive attention (Thom Huppertz and Chia 2021; Ye 2021; Miranda and Pelissier 1983; Wada and Lönnerdal 2014; Tunick et al. 2016; Ye et al. 2016b; Mulet-Cabero et al. 2020).”

“…consume only A2 β-casein (Sheng et al. 2019). κ-casein has nine variants, containing galactose, N-acetylgalactosamine, and N-acetylneuraminic acid or sialic acid, which occur as either trisaccharides or tetrasaccharides attached to threonine residues in the C-terminal region (Vreeman et al. 1986).”

“…β-Lg aggregate through thiol/disulfide interchange, hydrophobic interactions, and electrostatic and ionic interactions with themselves and with κ-casein, leading to so-called whey protein/κ-casein complexes (Guyomarc’h, Law, and Dalgleish 2003; Jang and Swaisgood 1990; Singh and Creamer 1991).”

“The temperature of a human gastric environment is approximately 37 °C in a fasting state but can change depending on the temperature of ingested food (Sun et al. 1988).”

“Connective tissues tend to gelatinize at 80 °C and form a gel that fills the spaces between muscle fibers (Roldán et al. 2013); this can decrease the digestibility of meat proteins by reducing the diffusion of digestive enzymes into the compact myofibrillar structure (L. Kaur et al. 2014).”

“Legume proteins become less soluble after heating and the resulting thermal aggregation may impair their digestibility (Carbonaro et al. 2000; Ruiz et al. 2016).”

“Studies on milk digestion, especially milk protein digestion, have recently attracted extensive attention (Thom Huppertz and Chia 2021; Ye 2021; Miranda and Pelissier 1983; Wada and Lönnerdal 2014; Tunick et al. 2016; Ye et al. 2016b; Mulet-Cabero et al. 2020).”

“…for hydrolyzing bonds adjacent to aromatic and hydrophobic amino acids, such as phenylalanine, tyrosine, tryptophan, and leucine (Dunn 2002; Inouye and Fruton 1967), with an optimum pH of approximately 2.0 (Kondjoyan, Daudin, and Santé-Lhoutellier 2015; Pletschke, Naudé, and Oelofsen 1995).”

“As temperature affects pepsin activity (Zhao et al. 2011) and enzyme-induced milk coagulation behavior (Nájera, De Renobales, and Barron 2003; Dalgleish 1983; Horne and Lucey 2014), milk temperature has a profound effect on the structural organization of pepsin-induced curd.”

“However, excessive calcium had a negative effect on curd formation due to charge repulsion or reduced κ-casein solubility (Udabage, McKinnon, and Augustin 2001; Choi, Horne, and Lucey 2007; Fox et al. 2017; Lucey and Fox 1993; Patel and Reuter 1986).”

“Emerging machine learning and deep learning models, such as DeepCleave and PROSPER, improve predictive accuracy by training on extensive cleavage datasets and incorporating sequence and structural features (Li, Chen, et al. 2020; Song et al. 2012).”

“Plant proteins are divided into legume and cereal proteins, which are abundant, relatively low-cost, sustainable, less allergenic, and widely accepted by consumers (Goldstein and Reifen 2022).”

“Goat milk-based infant formulas form smaller protein aggregates, leading to faster hydrolysis of proteins compared with those from cow’s milk formula (Maathuis et al. 2017; Ye, Cui, et al. 2019).”

“During in vitro gastric digestion, pepsin hydrolyzes the ovomucoid almost completely, producing free amino acids and di-and tri-peptides (Jiménez-Saiz et al. 2011; Matsuda et al. 1985; Kovacs-Nolan et al. 2000; Abeyrathne et al. 2015).”

“In plants, globulins are present as storage proteins in both, dicots and monocots, making them the most common group of storage proteins (Rasheed et al. 2020).”

“Studies on milk digestion, especially milk protein digestion, have recently attracted extensive attention (Thom Huppertz and Chia 2021; Ye 2021; Miranda and Pelissier 1983; Wada and Lönnerdal 2014; Tunick et al. 2016; Ye et al. 2016b; Mulet-Cabero et al. 2020).”

“Four different egg white gel structures with different combinations of pH and ionic strength before heating were prepared by Nyemb et al. (2016), who reported that the overall extent of protein digestion, ranging from highest to lowest, was granular-spongy, intermediate, smooth-rigid, and breakable…”

“However, excessive calcium had a negative effect on curd formation due to charge repulsion or reduced κ-casein solubility (Udabage, McKinnon, and Augustin 2001; Choi, Horne, and Lucey 2007; Fox et al. 2017; Lucey and Fox 1993; Patel and Reuter 1986).”

“The diffusion of digestive enzymes can be impacted as a consequence of different protein structures formed during digestion, i.e., inhibition of pepsin diffusion within a solid compared to a liquid matrix (Q. Luo et al. 2017).”

“…results are nil or not physiologically relevant, much lower yield of β-casomorphin-7 (Boutrou et al. 2013; Haq, Kapila, and Kapila 2015; De Noni 2008) and substantially less severe gastrointestinal symptoms for individuals who consume only A2 β-casein (Sheng et al. 2019). κ-casein has…”

“According to Liu et al. (2017), differences in the digestibility were observed in the two systems (i.e. colloidal dispersion versus gel), where enzyme diffusion was a decisive factor in the gel network (Luo, Boom, and Janssen 2015; Nyemb et al. 2016).”

“This improvement results from heat-induced alterations to the three-dimensional protein structure and reductions in trypsin inhibitors, phytic acid, and tannins (Habiba 2002).”

“Goat milk-based infant formulas form smaller protein aggregates, leading to faster hydrolysis of proteins compared with those from cow’s milk formula (Maathuis et al. 2017; Ye, Cui, et al. 2019).”

“The acidity of the infant stomach is much lower, with a pH of approximately 4, which may lead to the poor and slow degradation of allergenic epitopes (Martos et al. 2010).”

“…have determined the milk coagulation properties with different apparatus, which are summarized by Troch et al. (2017) and Lucey (2002), including rheology, microscopy (Everett 2007 Gilbert 2009; Gilbert 2019; Adams et al. 2019; de Kruif 2014; Gilbert 2023; Bayrak et al. 2021; Yang et al. 2025).”

“Hemoglobin digestive activity was determined at pH 2.0 (Foltmann 1992; nielsen and Foltmann 1995; tang et al. 1959). c assay at pH 6.3 (Foltmann 1992; tang et al. 1959). the value of porcine pepsin B is estimated from a “caseogram” assay (Foltmann, szecsi, and tarasova 1985). the highest value is…”

“During in vitro gastric digestion, pepsin hydrolyzes the ovomucoid almost completely, producing free amino acids and di-and tri-peptides (Jiménez-Saiz et al. 2011; Matsuda et al. 1985; Kovacs-Nolan et al. 2000; Abeyrathne et al. 2015).”

“Its complex triple-helical structure (Exposito et al. 2010) and high levels of glycine and (hydroxyl) proline reduce the flexibility of the protein back-bone, which affects the binding of enzymes to active sites on proteins (Kaur et al. 2010).”

“To explore the impact of protein substrates on the pH dependence of pepsin activity, a comparation of degree of hydrolysis with initial rates of pepsinolysis was reported in Figure 1 (Salelles, Floury, and Le Feunteun 2021).”

“…amino acids, and preferably aromatic amino acids, such as tryptophan, tyrosine, and phenylalanine (Mohseni-Shahri, Moeinpour, and Nosrati 2018; Inouye and Fruton 1967; Fujimoto et al. 2004), whereas it does not hydrolyze bonds containing valine, alanine or glycine (Monogioudi et al. 2011).”

“Different hydrolysis and digestion behaviors have been observed under gastric environmental conditions because of the structural differences between casein and whey proteins (Wang, Ye, et al. 2018), which affect allergenicity (Bernard et al. 1998; Busse et al. 2002; Cocco et al. 2003).”

“The major proteins observed in egg yolk include low-density lipoprotein (68%), high-density lipoprotein (16%), phosvitin (4%), and livetin, which exist in a homogeneously emulsified fluid (Anton et al. 2003). α-Livetin (Gal d 5) is commonly associated with allergies (Dona and Suphioglu 2020).”

“Para-κ-casein from sheep and goat milk differs from that of cows by ten and eight amino acids, respectively (Jollès et al. 1974; Mercier et al. 1976).”

“As temperature affects pepsin activity (Zhao et al. 2011) and enzyme-induced milk coagulation behavior (Nájera, De Renobales, and Barron 2003; Dalgleish 1983; Horne and Lucey 2014), milk temperature has a profound effect on the structural organization of pepsin-induced curd.”

“Egg proteins are generally considered a valuable protein resource that provides all essential amino acids for human nutritional requirements (J. Wang, Chi, et al. 2018).”

“Classical methods of CMP analysis include electrophoresis and chromatography (Manso and López-Fandiño 2004).”

“Additionally, protein aggregation is markedly dependent on temperature (Dybowska and Fujio 1996; Gunasekaran and Ay 1996), with a temperature coefficient ( Q 10 ) of approximately 12 (McMahon and Brown 1984).”

“The degree of unfolding before aggregation is the main factor affecting the digestibility of ovalbumin, with the lowest unfolding recorded in spherical agglomerated aggregates and the highest in linear aggregates (Nyemb et al. 2014; Van der Plancken et al. 2003).”

“Different hydrolysis and digestion behaviors have been observed under gastric environmental conditions because of the structural differences between casein and whey proteins (Wang, Ye, et al. 2018), which affect allergenicity (Bernard et al. 1998; Busse et al. 2002; Cocco et al. 2003).”

“The effects of different methods of processing on the digestibility of egg proteins and allergenicity have been reviewed by Bhat et al. (2021a) and Farjami et al. (2021).”

“Studies on milk digestion, especially milk protein digestion, have recently attracted extensive attention (Thom Huppertz and Chia 2021; Ye 2021; Miranda and Pelissier 1983; Wada and Lönnerdal 2014; Tunick et al. 2016; Ye et al. 2016b; Mulet-Cabero et al. 2020).”

“…in hydrolyzing peptide bonds between hydrophobic amino acids, and preferably aromatic amino acids, such as tryptophan, tyrosine, and phenylalanine (Mohseni-Shahri, Moeinpour, and Nosrati 2018; Inouye and Fruton 1967; Fujimoto et al. 2004), whereas it does not hydrolyze bonds containing valine,…”

“The effect of heating on the digestibility of myofibrillar proteins under static in vitro digestion conditions is shown in Figure 6 (Chen et al. 2023).”

“When β-casein from each of the variants (A1, A2, B, and I) was subjected to in vitro digestion using pepsin at pH 2.0, Petrat-Melin et al. (2015) observed minimal intact β-casein presented using SDS-PAGE after 60 min of pepsin digestion (with little difference between different variants), following…”

“Different hydrolysis and digestion behaviors have been observed under gastric environmental conditions because of the structural differences between casein and whey proteins (Wang, Ye, et al. 2018), which affect allergenicity (Bernard et al. 1998; Busse et al. 2002; Cocco et al. 2003).”

“At pH 6.0, pepsin has the fastest initial rate of hydrolysis on κ-casein, followed by α s1 -casein and β-casein (Tam and Whitaker 1972).”

“…consisting of an eight-stranded antiparallel β-hydrophobic barrel unit with an α-helix, accounting for the resistance to acid hydrolysis and pepsin action in the gastrointestinal tract (Dalgalarrondo et al. 1995; Chobert et al. 1995; Reddy, Kella, and Kinsella 1988; Schmidt and Poll 1991).”

“…1987; Carlson, Hill, and Olson 1987b, 1987d); this was due to partial charge neutralization of the negatively charged para-casein micelles and an increase in the concentration and activity of calcium ions in milk serum at lower pH (van Hooydonk 1987; Zoon 1988; Choi, Horne, and Lucey 2007).”

“Most meat proteins are susceptible to digestion, whereas myoglobin and collagen are less digestible (Li, Zhao, et al. 2020; Zhang et al. 2020).”

“…and pH in the gastric environment (Culioli and Sherman 1978; Kameswaran and Smith 1999; Mohamed A Mehaia and El-Khadragy 1998), heat treatment of proteins (Wilson and Wheelock 1972; Anema, Kim Lee, and Klostermeyer 2007), and the type of mammals from which the milk is obtained (Roy et al. 2020b).”

“Ovomucoid comprises three tandem domains, each cross-linked by three intradomain disulfide bonds, which lead to resistance to proteolytic digestion and heat denaturation (Matsuda et al. 1985; Kovacs-Nolan et al. 2000; Kosti et al. 2013; Shin, Han, and Ahn 2013).”

“However, recent work indicates that structural changes induced by alkali extraction improve the in vivo digestibility of prolamin (Kubota et al. 2010; Kumagai et al. 2006; Kubota et al. 2014).”

“…of 2S albumins to pepsin digestion in simulated gastric fluid due to their compact structure, a high number of disulfide bonds, and their capacity for self-association (Moreno et al. 2005; Murtagh et al. 2003; Pantoja-Uceda et al. 2004; Koppelman et al. 2005; Carbonaro, Maselli, and Nucara 2015).”

“A decrease in prolamin protein digestibility upon cooking is different amongst the various grain varieties and does not correlate with uncooked digestibility values (Nunes et al. 2004).”

“Legumins (11S) are degraded more by pepsin than by trypsin, leading to a small decrease in IgE binding capacity (van Boxtel et al. 2008; Bavaro et al. 2018; Mattison, Grimm, and Wasserman 2014; Orruño and Morgan 2011).”

“These proteins contain a high proportion of hydrophobic amino acids (e.g., proline, leucine, alanine) and some polar amino acids (e.g. glutamine) (Taylor, Anyango, and Taylor 2013).”

“Legume proteins become less soluble after heating and the resulting thermal aggregation may impair their digestibility (Carbonaro et al. 2000; Ruiz et al. 2016).”

“Numerous studies have been based on the Michaelis–Menten model (Ruan, Chi, and Zhang 2010; Luo, Zhan, et al. 2018; Luo, Chen, et al. 2018), which allows prediction of the reaction kinetics by considering the effects of the enzyme-to-sub-strate ratio, the affinity of the protease for the substrate,…”

“Studies on milk digestion, especially milk protein digestion, have recently attracted extensive attention (Thom Huppertz and Chia 2021; Ye 2021; Miranda and Pelissier 1983; Wada and Lönnerdal 2014; Tunick et al. 2016; Ye et al. 2016b; Mulet-Cabero et al. 2020).”

“…Haq, Kapila, and Kapila 2015; De Noni 2008) and substantially less severe gastrointestinal symptoms for individuals who consume only A2 β-casein (Sheng et al. 2019). κ-casein has nine variants, containing galactose, N-acetylgalactosamine, and N-acetylneuraminic acid or sialic acid, which occur…”

“Physicochemical processes and pretreatments, such as pulsed electric fields, high-pressure processing, and cooking, affect digestive processes, thereby affecting the release and bio-availability of amino acids (Gong et al. 2020; Bhat et al. 2021b).”

“Compared with animal proteins, plant proteins are believed to have lower digestibility, principally because of the presence of anti-nutritional factors in plant proteins (Mariotti 2017).”

“…digestion following heating at lower temperatures (55 °C) compared with that at higher temperatures (70–90 °C); this arises due to protein denaturation causing a loss of solubility and reduction in interactions among sarcoplasmic or myofibrillar proteins (Sayd, Chambon, and Santé-Lhoutellier 2016).”

“Hemoglobin digestive activity was determined at pH 2.0 (Foltmann 1992; nielsen and Foltmann 1995; tang et al. 1959). c assay at pH 6.3 (Foltmann 1992; tang et al. 1959). the value of porcine pepsin B is estimated from a “caseogram” assay (Foltmann, szecsi, and tarasova 1985). the highest value is…”

“…of 2S albumins to pepsin digestion in simulated gastric fluid due to their compact structure, a high number of disulfide bonds, and their capacity for self-association (Moreno et al. 2005; Murtagh et al. 2003; Pantoja-Uceda et al. 2004; Koppelman et al. 2005; Carbonaro, Maselli, and Nucara 2015).”

“…of 2S albumins to pepsin digestion in simulated gastric fluid due to their compact structure, a high number of disulfide bonds, and their capacity for self-association (Moreno et al. 2005; Murtagh et al. 2003; Pantoja-Uceda et al. 2004; Koppelman et al. 2005; Carbonaro, Maselli, and Nucara 2015).”

“…(>100 °C) can induce oxidation, disulfide bond formation, and protein aggregation or gelation, which can bury the active sites deep into the tertiary structure, thereby decreasing accessibility to pepsin (Sante-Lhoutellier, Aubry, and Gatellier 2007; Marie-Laure Bax et al. 2012; He et al. 2018).”

“However, extensive aggregation of proteins can decrease proteolytic susceptibility and hinder protein digestion (Lee et al. 2021), for example, excessive heating (Santé-Lhoutellier et al. 2008), or dry curing (Du et al. 2018).”

“…increases (Figure 3B1) (Panthi et al. 2019; Yang et al. 2023a), and coagulation occurs with a lower requirement for the extent of κ-casein hydrolysis at a higher temperature (Figure 3C1), in agreement with chymosin-induced coagulation results (Carlson, Hill, and Olson 1987c; Garg and Johri 1994).”

“As dairy products are a source of high-quality protein and many micronutrients, synthesizing nature-identical milk proteins has been considered a possible solution to replace milk proteins (Hettinga and Bijl 2022).”

“The degree of unfolding before aggregation is the main factor affecting the digestibility of ovalbumin, with the lowest unfolding recorded in spherical agglomerated aggregates and the highest in linear aggregates (Nyemb et al. 2014; Van der Plancken et al. 2003).”

“Legumes, cereals, pseudocereals and oilseeds are important sources of plant protein for humans (Zhang et al. 2024).”

“…have determined the milk coagulation properties with different apparatus, which are summarized by Troch et al. (2017) and Lucey (2002), including rheology, microscopy (Everett 2007 Gilbert 2009; Gilbert 2019; Adams et al. 2019; de Kruif 2014; Gilbert 2023; Bayrak et al. 2021; Yang et al. 2025).”

“Among them, globulin and albumin are mainly observed in legumes (such as soy and pea) while the prolamin and glutenin constituted the major protein fractions in monocotyledonous crops (Adebowale et al. 2007).”

“…for hydrolyzing bonds adjacent to aromatic and hydrophobic amino acids, such as phenylalanine, tyrosine, tryptophan, and leucine (Dunn 2002; Inouye and Fruton 1967), with an optimum pH of approximately 2.0 (Kondjoyan, Daudin, and Santé-Lhoutellier 2015; Pletschke, Naudé, and Oelofsen 1995).”

“Several techniques have been used to evaluate the progress of protein hydrolysis using the concept of degree of hydrolysis, defined as the percentage of hydrolyzed peptide bonds (Navarrete del Toro and García‐Carreño 2003).”

“Myofibrils mainly consist of myosin heavy chain (200 kDa), actin (42 kDa), and other minor proteins, such as α-actinin (100 kDa), tropomyosin, troponins, and myosin light chains (Liu et al. 2011).”

“67 His in the β-casein variant A1 residue is more susceptible than the 67 Pro in β-casein variant A2 residue toward pepsin-induced hydrolysis, with the 66 Ile residue following (Thiruvengadam et al. 2021).”

“Conversely, heat treatment at 100 °C resulted in extensive aggregation, potentially hiding a few hydrolysis sites (Figure 5) (Wang, Qiu, and Liu 2018).”

“Native collagen is resistant to hydrolysis by digestive enzymes (pepsin from porcine gastric mucosa, CAS No. 9001-75-6; pancreatin from porcine pancreas, CAS No. 8049-47-6) (M. Zhang et al. 2020; Li, Zhao, et al. 2020), because of its tight tertiary structure (Ravikumar and Hwang 2008).”

“However, recent work indicates that structural changes induced by alkali extraction improve the in vivo digestibility of prolamin (Kubota et al. 2010; Kumagai et al. 2006; Kubota et al. 2014).”

“Additionally, protein aggregation is markedly dependent on temperature (Dybowska and Fujio 1996; Gunasekaran and Ay 1996), with a temperature coefficient ( Q 10 ) of approximately 12 (McMahon and Brown 1984).”

“Cooking legumes has been shown to cause protein denaturation, reducing their resistance to enzymatic attack (Alajaji and El-Adawy 2006; Wiesinger et al. 2018).”

“A “hard” whey protein-based gel structure can slow down the disintegration of the protein matrix, whereas a “soft” whey protein-based gel structure can be completely broken down after 4 h of digestion (Guo et al. 2014; N. Luo et al. 2021).”

“The reduction of disulfide bonds in β-Lg can increase its susceptibility to pepsin action (Kananen et al. 2000).”

“Native ovotransferrin and lysozyme can be rapidly hydrolyzed by pepsin (Liu et al. 2017; Liu et al. 2018; Yoshino et al. 2004), whereas ovotransferrin is more susceptible to pepsinolysis than ovalbumin and lysozyme (Liu et al. 2017; Fu, Abbott, and Hatzos 2002; Moreno 2007).”

“However, extensive aggregation of proteins can decrease proteolytic susceptibility and hinder protein digestion (Lee et al. 2021), for example, excessive heating (Santé-Lhoutellier et al. 2008), or dry curing (Du et al. 2018).”

“Qazi et al. (2021) reported a faster gastric emptying of peptides produced from an acid-induced (1.6% w/w Glucono-delta-lactone) curd, whereas the restructuring of the rennet-induced curd during gastric digestion reduced the flux of proteins and peptides into the digesta.”

“In general, the unfolding of buried residues through denaturation (e.g. moderate heating) can improve protein digestibility (Bax et al. 2013).”

“…activity was determined at pH 2.0 (Foltmann 1992; nielsen and Foltmann 1995; tang et al. 1959). c assay at pH 6.3 (Foltmann 1992; tang et al. 1959). the value of porcine pepsin B is estimated from a “caseogram” assay (Foltmann, szecsi, and tarasova 1985). the highest value is considered as 100%.”

“The interaction between pepsin and egg yolk proteins has not been studied in detail, but a slight increase in susceptibility to hydrolysis of egg white proteins was observed in the presence of egg yolk during in vitro digestion (Martos, López-Fandiño, and Molina 2013).”

“Studies on milk digestion, especially milk protein digestion, have recently attracted extensive attention (Thom Huppertz and Chia 2021; Ye 2021; Miranda and Pelissier 1983; Wada and Lönnerdal 2014; Tunick et al. 2016; Ye et al. 2016b; Mulet-Cabero et al. 2020).”

“Para-κ-casein from sheep and goat milk differs from that of cows by ten and eight amino acids, respectively (Jollès et al. 1974; Mercier et al. 1976).”

“During the coagulation of milk, the curd is mainly formed from the casein matrix with fat globules entrapped within the casein network (Fox et al. 2004; Joshi et al. 2004).”

“Most meat proteins are susceptible to digestion, whereas myoglobin and collagen are less digestible (Li, Zhao, et al. 2020; Zhang et al. 2020).”

“…β-Lg aggregate through thiol/disulfide interchange, hydrophobic interactions, and electrostatic and ionic interactions with themselves and with κ-casein, leading to so-called whey protein/κ-casein complexes (Guyomarc’h, Law, and Dalgleish 2003; Jang and Swaisgood 1990; Singh and Creamer 1991).”

“To explore protein unfolding as a limiting factor in the gastric digestion of proteins, Herman, Gao, and Storer (2006) developed a theoretical approach with two consecutive exponential steps, one for protein unfolding and one for protein hydrolysis by pepsin.”

“These phenomena are affected by several factors, such as the pepsin concentration and pH in the gastric environment (Culioli and Sherman 1978; Kameswaran and Smith 1999; Mohamed A Mehaia and El-Khadragy 1998), heat treatment of proteins (Wilson and Wheelock 1972; Anema, Kim Lee, and Klostermeyer…”

“Legumins (11S) are degraded more by pepsin than by trypsin, leading to a small decrease in IgE binding capacity (van Boxtel et al. 2008; Bavaro et al. 2018; Mattison, Grimm, and Wasserman 2014; Orruño and Morgan 2011).”

“Consequently, milk coagulation time is inversely dependent on enzyme concentration (Figure 3B3) (Dalgleish 1993; Carlson, Hill, and Olson 1987a; López, Lomholt, and Qvist 1998; Foltmann 1959; Hyslop, Richardson, and Ryan 1979).”

“…of 2S albumins to pepsin digestion in simulated gastric fluid due to their compact structure, a high number of disulfide bonds, and their capacity for self-association (Moreno et al. 2005; Murtagh et al. 2003; Pantoja-Uceda et al. 2004; Koppelman et al. 2005; Carbonaro, Maselli, and Nucara 2015).”

“The hydrolysis and aggregation phases differ kinetically but are not fully separated over time (Frederiksen et al. 2011; Yang et al. 2022).”

“The coagulation time decreases as set temperature increases (Figure 3B1) (Panthi et al. 2019; Yang et al. 2023a), and coagulation occurs with a lower requirement for the extent of κ-casein hydrolysis at a higher temperature (Figure 3C1), in agreement with chymosin-induced coagulation results…”

“Numerous studies have determined the milk coagulation properties with different apparatus, which are summarized by Troch et al. (2017) and Lucey (2002), including rheology, microscopy (Everett 2007 Gilbert 2009; Gilbert 2019; Adams et al. 2019; de Kruif 2014; Gilbert 2023; Bayrak et al. 2021; Yang…”

“Numerous studies have been based on the Michaelis–Menten model (Ruan, Chi, and Zhang 2010; Luo, Zhan, et al. 2018; Luo, Chen, et al. 2018), which allows prediction of the reaction kinetics by considering the effects of the enzyme-to-sub-strate ratio, the affinity of the protease for the substrate,…”

“…used model relies on the use of a first-order reaction; for example, a two-parameter kinetic model has been applied to simulate the digestion of milk proteins under conditions of moderate enzyme inactivation (Margot, Flaschel, and Renken 1997; Yang et al. 2022; Carlson, Hill, and Olson 1987a).”

“…used model relies on the use of a first-order reaction; for example, a two-parameter kinetic model has been applied to simulate the digestion of milk proteins under conditions of moderate enzyme inactivation (Margot, Flaschel, and Renken 1997; Yang et al. 2022; Carlson, Hill, and Olson 1987a).”

“…used model relies on the use of a first-order reaction; for example, a two-parameter kinetic model has been applied to simulate the digestion of milk proteins under conditions of moderate enzyme inactivation (Margot, Flaschel, and Renken 1997; Yang et al. 2022; Carlson, Hill, and Olson 1987a).”

“…consisting of an eight-stranded antiparallel β-hydrophobic barrel unit with an α-helix, accounting for the resistance to acid hydrolysis and pepsin action in the gastrointestinal tract (Dalgalarrondo et al. 1995; Chobert et al. 1995; Reddy, Kella, and Kinsella 1988; Schmidt and Poll 1991).”

“Milk from different species responds differently to pepsin and acid (Roy et al. 2020b).”

“Digestive characteristics of protein gels significantly influence the release of nutrients and delivery of bioactive ingredients (M. Yang, Yang, et al. 2025 ).”