Process-induced amino acid side chain modifications

Food processing can involve extremes of temperature and pH. Under these conditions some amino acid sidechains are susceptible to undergoing chemical reactions^{(Reference Gerrard, Lasse and Cottam14,Reference McKerchar, Clerens and Dobson43)} that cause permanent loss of bioavailability^{(Reference Moughan and Rutherfurd44)}. Amino acid side chains can undergo acid-/alkali- and water-catalysed reactions such as oxidation and deamination, or can react with other amino acids, leading to non-native cross-links^{(Reference McKerchar, Clerens and Dobson43)}. Side chains can react with other food components, for example Maillard reactions with reducing sugars, leading to glycation and further reactions^{(Reference Al Jahdali and Carbonero45)}.

The indispensable amino acids histidine, lysine, methionine, threonine and tryptophan are particularly reactive, and reactions catalysed by heat, oxidising conditions or alkaline pH will compromise their bioavailability^{(Reference Gilani, Xiao and Cockell46,Reference Rutherfurd, Montoya and Moughan47)} and may give rise to toxic derivatives^{(Reference Gilani, Xiao and Cockell46,Reference Kolpin and Hellwig48)} . Special precautions are required when analysing lysine in processed food or feed, because Maillard-reacted lysine (which is not bioavailable) reverts to lysine during the acid hydrolysis step in amino acid analysis^{(Reference Rutherfurd49)}.

Nyakayiru et al. ^{(Reference Nyakayiru, Van Lieshout and Trommelen50)} measured the effect of glycation on lysine peripheral bioavailability with milk protein powders that had minimal glycation (3%), or glycation of 20% or 50% after dry heating at 50°C. In 15 healthy volunteers, glycation of 20% and 50% reduced post-prandial plasma lysine iAUC (incremental AUC) by 35% and 92%, respectively (Figure 2). Although the level of lysine glycation was artificially manipulated in this study, site-specific glycation rates of ≥20% have been observed in commercial milk products whose manufacture involves severe heating^{(Reference Meltretter, Wüst and Pischetsrieder51)}, and this is thought to be responsible for reduced lysine bioavailability^{(Reference Rutherfurd and Moughan52)}.

Fig. 2. Post-prandial plasma lysine concentrations for volunteers consuming milk protein powders with lysine glycation of 3% (open circles), 20% (grey circles) or 50% (black circles). After Nyakayiru et al. ^{(Reference Nyakayiru, Van Lieshout and Trommelen50)}, reproduced with permission. *Significantly lower concentrations following ingestion of 50% glycation than 3% glycation (P<0·001). †Significantly lower concentrations following ingestion of 20% glycation than 3% glycation (P≤0·029). ‡Significantly lower concentrations following ingestion of 50% glycation than 20% glycation (P<0·001).

The effect of Maillard reactions on lysine bioavailability was also seen by Rérat et al. ^{(Reference Rérat, Calmes and Vaissade53)} in pigs after feeding heat-treated skim milk powder (51% lysine blockage). The cumulative appearance of lysine in portal blood was reduced by 60% relative to unheated skim milk powder. Valine absorption was also reduced (−34%) by heat treatment, but cystine absorption increased (+37%). Other in vitro and in vivo studies of processing-induced amino acid modifications to dairy proteins were reviewed by van Lieshout et al. ^{(Reference van Lieshout, Lambers and Bragt54)}.

Relatively little is known about process-induced amino acid modifications in foods other than dairy products, partly due to the diversity of reaction products and the complexity of mass spectrometric analysis. In a survey of commercial soybean meal ingredients, Troise et al. ^{(Reference Troise, Wiltafsky and Fogliano55)} reported that 26 out of 80 samples had ≥20% blocked lysine. Lassé et al. ^{(Reference Lassé, Deb-Choudhury and Haines56)} reported that boiling egg white protein created a wide range of oxidative and other amino acid modifications. In blanched navy beans, Deb-Choudhury et al. ^{(Reference Deb-Choudhury, Cooney and Brewster57)} found lysinoalanine cross-links and heat-induced amino acid modifications.

Early Maillard reaction products appear to have low digestibility, but they are absorbed to some extent and excreted in urine^{(Reference Rérat, Calmes and Vaissade53,Reference Somoza, Wenzel and Weiß58)} . Low digestibility may derive from the inability of trypsin to recognise lysine-adjacent cleavage sites (according to Keil rules^{(Reference Rodriguez, Gupta and Smith59)}) when lysine is glycated, or digestive enzymes may be sterically hindered from accessing cleavage sites near glycated and/or cross-linked lysine^{(Reference van Lieshout, Lambers and Bragt54)}. Alkali exposure induces cross-linking of certain amino acids with the ϵ-NH₂ group of lysine via elimination–addition reactions^{(Reference Friedman60)}, as well as amino acid racemisation^{(Reference Friedman60)}. Alkali-induced cross-links may similarly obstruct digestive enzymes, and lysinoalanine does not appear to be absorbed in rats^{(Reference Somoza, Wenzel and Weiß58)} or humans^{(Reference Erbersdobler, Lohmann and Buhl61)}, but can be absorbed by ruminants^{(Reference Friedman and Brandon62)}. D-amino acids are utilised by humans to different extents, but D-lysine is not utilised^{(Reference Friedman63)}.

Collectively, these data suggest that certain food processes involving extreme heating in the presence of reducing sugars and/or alkaline pH can modify amino acid side chains in ways that impair bioavailability. In contrast, more moderate heat treatments can improve protein digestibility through a combination of inactivating enzyme inhibitors^{(Reference Gilani, Xiao and Cockell46)} and denaturing proteins so that they are more susceptible to enzymatic cleavage^{(Reference Salazar-Villanea, Hendriks and Bruininx64)}.

Aggregation and gelation

Many food proteins will gel under certain conditions, i.e. they will aggregate into supramolecular protein–protein networks that percolate across an entire sample and give it a self-supporting semi-solid form. Familiar foods such as cheese, yoghurt, gelatin jelly and tofu are examples in which proteins are responsible for the gelled texture.

A series of studies by a group of French scientists^{(Reference Barbé, Ménard and Le Gouar65–Reference Dupont, Le Feunteun and Marze68)} illustrated the magnitude of gelation effects on plasma amino acid kinetics using a pig model of human digestion. In these studies, heated milk (90°C 10 min) was gelled with rennet (cheese-like texture), gelled with acid (set yoghurt texture), or gelled with acid and then stirred (stirred yoghurt texture).The kinetics of amino acid appearance in the blood were strongly altered by gelation (Figure 3), and the different gel types produced significant differences. The effects of gelation and gel type were attributed to alteration of gastric emptying rate.

Fig. 3. Post-prandial plasma leucine kinetics in pigs after consuming milk in liquid or gelled forms. After Dupont et al. ^{(Reference Dupont, Le Feunteun and Marze68)}, reproduced with permission.

Particulate or coagulated foods may retain their microstructure in the stomach, and gastric conditions may catalyse coagulation of non-particulate foods, e.g. by destabilising colloidal casein micelles^{(Reference Ye10)}. Particles or coagula that are too large to pass through the gastric pylorus are gradually eroded by the actions of pepsin and peristaltic mechanical shear. Mechanical breakup of protein coagula depends on properties such as elasticity and brittleness, and the rate of pepsin diffusion into protein coagula depends on microstructural factors such as pore size and degree of cross-linking^{(Reference Thévenot, Cauty and Legland69,Reference Bayrak, Mata and Raynes70)} . Thus, the rate of gastric emptying is determined by the competing dynamics of microstructure formation versus mechanical/enzymatic erosion in gastric digesta. In the aforementioned studies with milk gels, gelling in different ways was thought to retard the erosion of gastric coagula to differing extents^{(Reference Barbé, Ménard and Le Gouar66,Reference Gaudichon, Roos and Mahe71)} .

In a study with adults aged 66·7±4·3 y, Horstman et al. ^{(Reference Horstman, Ganzevles and Kudla72)} reported a small but significant difference in peak serum amino acid concentration after consuming stirred yoghurt or low-fat ultra-high temperature treated (UHT) milk. The maximum concentration of serum essential amino acids was 13% higher with yoghurt (P<0.005), but area under the curve and other parameters were not significantly different.

According to Figure 3, acid gelation of pre-heated milk as part of yoghurt making slows the digestion of milk proteins, and this effect is partially reversed by stirring, which breaks up large aggregates. The findings of Horstman et al. ^{(Reference Horstman, Ganzevles and Kudla72)} indicate that the protein in stirred yoghurt is still digested and absorbed faster than the protein in UHT milk, despite the semi-solid form of stirred yoghurt. This is probably the result of the more severe heat treatment during yoghurt manufacture (e.g. 90°C for 10 min) compared with UHT treatment (e.g. 140°C for 5 s), which results in greater whey protein denaturation^{(Reference Horstman, Ganzevles and Kudla72–Reference Ye, Liu and Cui74)}.

Some protein-rich food ingredients are deliberately aggregated as part of their manufacture, either as a way to concentrate and fractionate proteins with different properties (e.g. casein and whey) or to modify their functionality for food manufacture^{(Reference Ji, Fitzpatrick and Cronin75)}. Protein-rich food ingredients usually take the form of uniform fine powders, but with diverse microstructures that have measurable effects on protein digestion and absorption.

Trommelen et al. ^{(Reference Trommelen, Weijzen and van Kranenburg76)} measured amino acid uptake from three casein-derived food ingredients: calcium caseinate, a form of casein that has been acid precipitated and resolubilised with calcium hydroxide; micellar casein, which is produced by microfiltration of milk and retains its native micelle structure; and sodium caseinate cross-linked with the enzyme transglutaminase. The kinetics of amino acid appearance in blood plasma were similar for calcium caseinate and micellar casein, but markedly different for cross-linked sodium caseinate (Figure 4). The C_max and iAUC for branched-chain and essential amino acids were significantly higher for cross-linked sodium caseinate.

Fig. 4. Plasma leucine kinetics in volunteers after consuming different milk protein solutions. Mi-Cas, micellar casein; Ca-CAS, calcium caseinate; XL-CAS, cross-linked sodium caseinate. After Trommelen et al. ^{(Reference Trommelen, Weijzen and van Kranenburg76)}, reproduced with permission.

The materials used in this study differed widely in apparent solubility after 2 h of stirring: 99%, 53% and 5% soluble for cross-linked sodium caseinate, calcium caseinate and micellar casein, respectively^{(Reference Trommelen, Weijzen and van Kranenburg76)}. In comparison with other studies^{(Reference Horstman, Ganzevles and Kudla72,Reference Boirie, Dangin and Gachon77)} , all casein ingredients in this study gave plasma leucine time courses (Figure 4) more characteristic of whey protein than of casein. This can be understood by considering the behaviour of the three test materials under gastric conditions.

Sodium caseinate powder dissolves rapidly^{(Reference Ji, Fitzpatrick and Cronin75)} and coagulates slowly under gastric conditions to form a loose coagulum^{(Reference Ye10,Reference Horstman, Ganzevles and Kudla72)} . Cross-linking with transglutaminase slows down coagulation^{(Reference Bönisch, Heidebach and Kulozik78)}. The rapid absorption of transglutaminase cross-linked sodium caseinate in Figure 4 probably reflects rapid dissolution followed by emptying from the stomach before coagulation can occur.

Calcium caseinate powder dissolves slowly and incompletely: even after 2 h of stirring at 25°C most material remains as suspended particles 10–100 μm in diameter^{(Reference Ji, Fitzpatrick and Cronin75,Reference Moughal, Munro and Singh79)} , i.e. two to three orders of magnitude larger than casein micelles^{(Reference Dalgleish, Boland, Golding and Singh80)}. Particles in this size range sediment slowly under centrifugal force, and may appear soluble. Suspended calcium caseinate particles do not coagulate under gastric conditions^{(Reference Horstman, Ganzevles and Kudla72)}, probably because of low collision frequency (high viscous drag^{(Reference O’Melia, Hahn and Chen81)}) and/or low collision efficiency^{(Reference Taboada-Serrano, Chin and Yiacoumi82,Reference Salvador, Acosta and Zamora83)} due to relatively large size, so they will also empty from the stomach rapidly. The overall bioavailability of the calcium caseinate in the study of Trommelen et al. ^{(Reference Trommelen, Weijzen and van Kranenburg76)} was lower than that of cross-linked sodium caseinate, which may be because a fraction of calcium caseinate particles remained partially undissolved in intestinal fluid and was therefore inaccessible to digestive enzymes.

Micellar casein powder also dissolves slowly, but ultimately dissolves completely, e.g. within ∼30 min at 25°C^{(Reference Ji, Fitzpatrick and Cronin75)}. It gave a peak in plasma leucine at 30 min, and overall bioavailability was intermediate between those of calcium caseinate and cross-linked sodium caseinate^{(Reference Trommelen, Weijzen and van Kranenburg76)}. The timing of the leucine peak suggests rapid emptying of most micellar casein from the stomach, i.e. failure to coagulate significantly under gastric conditions. The slightly higher leucine bioavailability at 150–300 min may reflect either partial coagulation in the stomach with more advanced pH decrease, or slower dissolution of this specific micellar caseinate powder compared with that tested by Ji et al. ^{(Reference Ji, Fitzpatrick and Cronin75)}.

All of the materials tested by Trommelen et al. ^{(Reference Trommelen, Weijzen and van Kranenburg76)} were substantially different to native casein micelles in liquid milk, which coagulate rapidly and effectively under gastric conditions^{(Reference Ye10)}. Dissolution is a necessary precursor of coagulation due to hydrodynamic considerations. If a test meal consists of a slow-dissolving protein powder suspended (rather than dissolved) in water, then dissolution may not occur during gastric residence, and coagulation is thereby impaired.

Casein coagulation under gastric conditions is partly driven by the presence of calcium, and partial removal of calcium using zeolite treatment during casein manufacture can weaken the ability of caseins to coagulate^{(Reference Huppertz and Lambers84)}. In the work of Chan et al. ^{(Reference Chan, D’Souza and Beals85)}, calcium-depleted milk protein concentrates (mMPC) gave significantly higher plasma concentrations of essential amino acids at 45–90 min post-prandially, compared with conventional MPC or calcium caseinate (Figure 5A). The MPC versus mMPC difference was attributed to weaker gastric coagulation with mMPC as a result of calcium depletion, and led to significantly higher insulin at 60 min (Figure 5B).

Fig. 5. Plasma concentrations of essential amino acids (EAA) (a) and plasma insulin concentration (b) after consumption of milk protein concentrate (dotted line, circles), mineral-modified milk protein concentrate (solid line, squares) or calcium caseinate (dashed line, diamonds). After Chan et al. ^{(Reference Chan, D’Souza and Beals85)}, reproduced with permission.

Aggregation and/or gelation as a result of food processing alters the physical form of food proteins, and thereby affects food digestion processes. The susceptibility of protein to enzymatic proteolysis may be increased or decreased, depending on aggregate structure. Gastric coagulation slows protein digestion, and processing that affects gastric coagulation significantly impacts protein digestion rates.

In a comprehensive overview of milk protein digestion studies, Horstman and Huppertz^{(Reference Horstman and Huppertz86)} suggested that the main factors controlling the rate of milk protein digestion are the potential of a given milk protein material to coagulate in the stomach and the energy content of a meal. Horstman and Huppertz^{(Reference Horstman and Huppertz86)} noted that not all forms of casein will coagulate under gastric conditions. This often-overlooked nuance can have a substantial effect on plasma amino acid kinetics, as seen in the studies by Trommelen et al. ^{(Reference Trommelen, Weijzen and van Kranenburg76)} and Chan et al. ^{(Reference Chan, D’Souza and Beals85)} (Section 3.2). Homogenising milk causes casein micelles to adsorb to milk fat globule interfaces, and this substantially alters the structure and mechanical properties of coagula formed under gastric conditions^{(Reference Ye, Cui and Dalgleish87)}. It remains to be seen what the consequences of homogenisation are for milk protein digestion and absorption rates.

Heat processing of milk

Heating is a common way to kill pathogens that may be present in raw milk, and heat treatments also extend the shelf life by killing non-pathogenic microorganisms that cause spoilage. In the dairy industry, the most widespread heat treatments for liquid milk are high temperature, short time (HTST) pasteurisation, e.g. 72°C for 15–20 s, and UHT heat treatment, e.g. 140°C for 5 s. The major effect of heat processing milk at >80°C is to denature whey proteins, which adsorb to the surface of casein micelles and weaken gastric coagulation of casein^{(Reference Vasbinder, Rollema and de Kruif97)}. Extreme heating can also cause amino acid side chain modifications, as discussed in Section 3.1.

Lacroix et al. ^{(Reference Lacroix, Bon and Bos98)} reported post-prandial nitrogen flows in humans after consuming milk treated by heat pasteurisation, UHT treatment or microfiltration (a low-temperature pasteurisation method). Post-prandial plasma amino acids were elevated above baseline levels for all treatments, but the type of milk processing did not produce significant differences. However, a higher rate of deamination and a lower rate of retention was seen with UHT milk, relative to microfiltered milk. The authors suggested that the different nitrogen utilisation pattern for UHT milk could be a result of more rapid gastric emptying and/or heat-related lysine damage^{(Reference Lacroix, Bon and Bos98)}. Lysine damage in UHT milk is minor^{(Reference Rutherfurd and Moughan52)}, and in vitro simulated digestion studies support the hypothesis of more rapid gastric emptying for UHT milk as a result of weaker gastric coagulation^{(Reference Ye10)}.

In another study focused on dairy product processing, Horstman et al. ^{(Reference Horstman, Ganzevles and Kudla72)} reported slightly higher post-prandial plasma amino acid levels following consumption of low-fat UHT milk, compared with low-fat pasteurised milk (P=0.066 for the iAUC comparison). In this case, the UHT treatment was applied by direct steam injection, and Horstman et al. ^{(Reference Horstman, Ganzevles and Kudla72)} suggested that the observed differences may be even larger with conventional indirect UHT treatment, which exposes milk to high temperatures for a longer time.

Barbé et al. ^{(Reference Barbé, Ménard and Le Gouar65)} also examined the effect of a more severe heat treatment (90°C for 10 min) on post-prandial plasma amino acid kinetics in pigs after consumption of liquid milk. Consuming heated milk resulted in slightly higher aminoacidaemia than consuming unheated milk, but the differences were not significant.

Other in vivo and in vitro studies of milk protein digestion have been reviewed by Horstman and Huppertz^{(Reference Horstman and Huppertz86)} and Li et al. ^{(Reference Li, Ye and Singh99)}. In vitro studies report differences in proteolysis rates between UHT-treated and pasteurised milks^{(Reference Ye, Liu and Cui74)}, but in vivo studies have found only small differences in amino acid bioavailability.

Cooking and mincing of beef

Cooking transforms raw meat into an edible form, as well as eliminating pathogens and enhancing flavour. Meat undergoes extensive microstructural and biochemical changes during cooking, including partial or complete denaturation of myofibrillar, sarcoplasmic and connective tissue proteins, shrinkage and expulsion of water, amino acid side chain modifications^{(Reference Deb-Choudhury, Haines and Harland100)} and Maillard reactions between proteins and sugars. These cooking-related changes affect post-prandial nitrogen flows, including plasma amino acid kinetics.

One illustration of cooking effects on post-prandial amino acid kinetics is the study by Bax et al. ^{(Reference Bax, Buffière and Hafnaoui101)} using pigs that were fed beef samples cooked for 30 min in a water bath at 60°C, 75°C or 95 °C. Plasma amino acid levels showed significant differences over the first 1.5 h (Figure 7). Kinetic parameters were not significantly different, but it was clear that heating at 75°C led to a faster increase in plasma indispensable amino acids, and heating at 95°C resulted in slower aminoacidaemia.

Fig. 7. Post-prandial kinetics of indispensable amino acids in pigs fed beef cooked for 30 min at 60°C, 75°C or 95°C. After Bax et al. ^{(Reference Bax, Buffière and Hafnaoui101)}, reproduced with permission.

Based on the results of a parallel simulated in vitro digestion study with pork^{(Reference Bax, Aubry and Ferreira102)}, Bax et al. ^{(Reference Bax, Buffière and Hafnaoui101)} proposed that low-temperature cooking led to protein denaturation, which enhanced proteolysis by exposing more cleavage sites. Cooking at a higher temperature led to protein aggregation, which made cleavage sites less accessible to proteases.

In a comparison of beef cooked at 55°C for 5 min with that cooked at 90°C for 30 min, Oberli et al. ^{(Reference Oberli, Marsset-Baglieri and Airinei103)} reported higher ileal nitrogen flow and slightly lower digestibility in healthy young adults for the higher cooking temperature, but plasma amino acid kinetics were not significantly different. The authors hypothesised that the chosen cooking treatments gave rise to meat microstructures that resisted digestion to a similar extent, either through retained native microstructure (cooking at 55°C) or extensive protein aggregation (cooking at 90°C), as suggested by Bax et al. ^{(Reference Bax, Aubry and Ferreira102)}.

In a subsequent study using the same cooking treatments, Buffière et al. reported significantly different plasma amino acid kinetics for elderly volunteers^{(Reference Buffière, Gaudichon and Hafnaoui104)}. Plasma leucine and plasma indispensable amino acids had significantly higher peaks for the beef cooked at 90°C than for beef cooked at 55°C, but the time at which plasma amino acid concentrations peaked was unaffected by cooking method. Whole body protein synthesis was higher with the well-cooked meat^{(Reference Buffière, Gaudichon and Hafnaoui104)}. The contrast between the findings of Oberli et al. ^{(Reference Oberli, Marsset-Baglieri and Airinei103)} and those of Buffière et al. ^{(Reference Buffière, Gaudichon and Hafnaoui104)} highlights the effect of age on digestion, i.e. amino acids from rare and well-cooked meat were equally bioavailable for adults, whereas amino acids in well-cooked meat were more bioavailable for elderly volunteers.

Cooked beef was minced in the work of Buffière et al. ^{(Reference Buffière, Gaudichon and Hafnaoui104)}, Oberli et al. ^{(Reference Oberli, Marsset-Baglieri and Airinei103)} and Bax et al. ^{(Reference Bax, Buffière and Hafnaoui101)} to eliminate the effects of inter-individual variation in chewing patterns. Cooking-related differences were attributed to different meat meso- and microstructures. Pennings et al. ^{(Reference Pennings, Groen and van Dijk105)} examined the effect of millimetre-scale meat structure, i.e. beef steak versus minced beef, using L-[1-¹³C]phenylalanine intrinsically labelled beef. Minced beef produced a higher plasma enrichment of L-[1-¹³C]phenylalanine than steak (Figure 8), suggesting more rapid digestion and absorption. Consequently, whole-body protein balance in the subjects (10 men 74±2 y old) was higher with minced beef.

Fig. 8. Plasma enrichment with L-[1-¹³C]phenylalanine following consumption of beef steak (open circles) or minced beef (closed circles). After Pennings et al. ^{(Reference Pennings, Groen and van Dijk105)}, reproduced with permission.

Whereas previous studies compared minced beef cooked at different temperatures and times, or compared minced beef with steak, Prodhan et al. ^{(Reference Prodhan, Pundir and Chiang106)} compared beef steaks cooked with different methods: sous vide cooking at 80°C for 6 h or pan frying for 5 min. In young men taking part in the crossover trial, post-prandial plasma amino acid kinetics and hormone levels were not significantly affected by the cooking method. Results from in vitro studies of cooking effects on meat digestion have been reviewed by Bhat et al. ^{(Reference Bhat, Morton and Bekhit107)}.

As with other food systems, processing of meat influences protein digestion via alterations to microstructure and physical form. Gross alterations such as mincing speed up protein digestion. Mild-to-moderate cooking increases proteolysis rates due to protein denaturation, whereas more extensive cooking can retard proteolysis as a result of extensive protein aggregation

Cooking of egg protein

Egg protein digestion has been studied in vitro ^{(Reference Bhat, Morton and Bekhit108)}, but few in vivo studies have examined how food processing affects egg protein digestion and absorption. Evenepoel et al. ^{(Reference Evenepoel, Geypens and Luypaerts109)} reported that a raw egg meal had substantially lower ileal digestibility than the same meal that had been microwave-cooked (51·3±9·8% versus 90·9±0·8%). Gastric emptying was significantly faster with raw egg, which was attributed to its liquid consistency.

Nau et al. ^{(Reference Nau, Nyemb-Diop and Lechevalier110)} took a more detailed look at gastric processing of cooked egg white in pigs, using egg white gels with different microstructures, which were created by adjusting pH and ionic strength before heating^{(Reference Nyemb, Guérin-Dubiard and Dupont111)}. Gel structure affected gastric acidification rates and gastric emptying kinetics. The differences were attributed to the effect of egg white gels on the rheological properties of gastric chyme. Parallel in vitro studies suggested that egg white gel structure could affect protein hydrolysis rates^{(Reference Nyemb, Guérin-Dubiard and Dupont111)}, as well as the type and amount of peptides released by hydrolysis^{(Reference Nyemb-Diop, Causeur and Jardin112)}.