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Critical Reviews in Food Science and Nutrition
ISSN: 1040-8398 (Print) 1549-7852 (Online) Journal homepage: https://www.tandfonline.com/loi/bfsn20
How processing may affect milk protein digestionand overall physiological outcomes: A systematicreview
Glenn A. A. van Lieshout, Tim T. Lambers, Marjolijn C. E. Bragt & Kasper A.Hettinga
To cite this article: Glenn A. A. van Lieshout, Tim T. Lambers, Marjolijn C. E. Bragt &Kasper A. Hettinga (2019): How processing may affect milk protein digestion and overallphysiological outcomes: A systematic review, Critical Reviews in Food Science and Nutrition, DOI:10.1080/10408398.2019.1646703
To link to this article: https://doi.org/10.1080/10408398.2019.1646703
© 2019 The Author(s). Published withlicense by Taylor & Francis Group, LLC
Published online: 22 Aug 2019.
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REVIEW
How processing may affect milk protein digestion and overall physiologicaloutcomes: A systematic review
Glenn A. A. van Lieshouta, Tim T. Lambersa, Marjolijn C. E. Bragta, and Kasper A. Hettingab
aFrieslandCampina, Amersfoort, the Netherlands; bFood Quality & Design Group, Wageningen University & Research Centre, Wageningen,the Netherlands
ABSTRACTDairy is one of the main sources for high quality protein in the human diet. Processing may, how-ever, cause denaturation, aggregation, and chemical modifications of its amino acids, which mayimpact protein quality. This systematic review covers the effect of milk protein modifications as aresult of heating, on protein digestion and its physiological impact. A total of 5363 records wereretrieved through the Scopus database of which a total of 102 were included. Although thedegree of modification highly depends on the exact processing conditions, heating of milk pro-teins can modify several amino acids. In vitro and animal studies demonstrate that glycationdecreases protein digestibility, and hinders amino acid availability, especially for lysine. Otherchemical modifications, including oxidation, racemization, dephosphorylation and cross-linking, areless well studied, but may also impact protein digestion, which may result in decreased aminoacid bioavailability and functionality. On the other hand, protein denaturation does not affectoverall digestibility, but can facilitate gastric hydrolysis, especially of b-lactoglobulin. Proteindenaturation can also alter gastric emptying of the protein, consequently affecting digestive kinet-ics that can eventually result in different post-prandial plasma amino acid appearance. Apart fromprocessing, the kinetics of protein digestion depend on the matrix in which the protein is heated.Altogether, protein modifications may be considered indicative for processing severity. Controllingdairy processing conditions can thus be a powerful way to preserve protein quality or to steergastrointestinal digestion kinetics and subsequent release of amino acids. Related physiologicalconsequences mainly point towards amino acid bioavailability and immunological consequences.
KEYWORDSDairy; protein quality;glycation; denaturation;aggregation; bioavailability
1. Introduction
Milk is an important source for high quality protein in thehuman diet. The high nutritional quality of milk proteinsoriginates from both its high level of essential amino acids,as well as it high bioavailability. This high bioavailability ofmilk proteins compared to plant proteins is due to its highdigestibility, which is partly due to the absence of anti-nutri-tional factors and different ways of processing (Schaafsma2012). Industrial dairy processing can however change thestructure of milk proteins in several ways, depending on theconditions under which it has been processed. The mainprotein modifications occurring during processing aredenaturation and aggregation of the protein and chemicalmodifications of its amino acids. These processing-inducedprotein modifications may change digestion and the overallphysiological impact the consumption of these proteinshave. The most studied physiological consequences of theeffect of heating on proteins are its digestibility and bioavail-ability. However, the protein modifications may also causechanges along the gastrointestinal tract (e.g. related tomicrobiota, epithelial physiology and immune responses) or
have other physiological consequences, which could beeither locally or systemic. Figure 1 gives an overview of thedifferent processes and their potential consequences.
Aggregation of protein can be a consequence of heat-induceddenaturation, where internal disulfide bridges within proteinmolecules are broken followed by intermolecular disulfidebridge reformation, although also other chemical modificationsmay lead to protein aggregation. These chemical modificationsmay occur during heating of milk, and will have an impact onthe protein as such, as well as increase the tendency of the pro-tein to aggregate. Most previous research has focused on glyca-tion, also known as the Maillard reaction, which is the reactionbetween reducing sugars and the primary amines (NH2) of aprotein. In milk, the Maillard reaction mainly occurs betweenthe reducing sugar lactose and the NH2-group of the essentialamino acid lysine, which is abundant in all milk proteins. Thisreaction proceeds through multiple stages, with specific early,advanced and late products often used as marker, as schematic-ally depicted in Figure 2. Besides the chemical modification ofthe lysine residues, the Maillard reaction can also lead toMaillard-induced aggregation of protein (Van Boekel 1998).
CONTACT Kasper A. Hettinga [email protected] Food Quality & Design Group, Wageningen University & Research Centre, P.O. Box 17, 6700 AA,Wageningen, the Netherlands.� 2019 The Author(s). Published with license by Taylor & Francis Group, LLC.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITIONhttps://doi.org/10.1080/10408398.2019.1646703
Next to the Maillard reaction, other chemical modifica-tions can occur during heating of milk, for example oxida-tion, cyclization and racemization of amino acids (Liardonand Hurrell 1983; Meltretter et al. 2007).
Several reviews already describe the effect of heating onfood proteins in general (Friedman 1999; Gerrard et al. 2012;Mauron 1990). However, with milk being one of the mainprotein sources in the Western diet (EFSA 2012), and the solesource of protein for formula fed infants, it is important tospecifically understand the role of industrial (heat) processingon milk proteins. Another reason to specifically focus on milkproteins is that the major class of milk proteins, the caseins,respond differently to heating than most other food proteins,as will be explained in more detail below.
Existing reviews on milk protein’s response to industrial(heat) processing mainly focus on the direct physico-chem-ical effects on the milk protein itself (Ferrer et al. 1999;Mehta and Deeth 2016; Pellegrino, Cattaneo, and De Noni2011), and only give a limited perspective on the physio-logical relevance. This review will focus more specifically onthe effect of dairy protein modifications, as a result of indus-trial heating, on its digestion and the overall physiologicalrelevance of these modifications.
1.1. Dairy proteins
Milk proteins are classified as the casein fraction, which rep-resents approximately 80% of all protein in bovine milk, and
the whey protein fraction, representing approximately 20%of all protein in bovine milk (Walstra, Wouters, and Geurts2006). These proteins have different physico-chemical prop-erties and therefore behave differently upon heating.
The casein fraction of bovine milk consists of four differ-ent casein molecules, as1-, as2-, b-, and j-casein. The indi-vidual casein molecules lack a tertiary structure, but occurin milk as large (approximately 100–200 nm) structurescalled casein micelles consisting of 1000’s of individualcasein molecules. Because of a lack of tertiary structure,caseins don’t show the typical denaturation and aggregationupon heating. Caseins are however sensitive to processing-induced chemical modifications and aggregation (Pellegrino,Cattaneo, and De Noni 2011). When looking at the upperpart of Figure 1, the caseins thus only follow the right partof the figure with chemical modifications, and the associatedaggregation.
The whey proteins, on the other hand, both denatureand aggregate upon heating and can be chemically modi-fied. They can thus follow all pathways indicated in Figure1. An exception is casein macropeptide (CMP), a casein-derived peptide in cheese whey that does not denature.Besides forming aggregates amongst each other, whey pro-teins also aggregate with casein micelles. The ratio betweenfree whey protein aggregates and casein-whey aggregatesdepends on the pH, with the majority of the whey proteinbinding to the casein micelle surface at the natural pH ofmilk (Vasbinder and de Kruif 2003). The interaction
Native milk protein
Denatured protein Chemically modified protein
Denatured & chemically
modified protein
Protein aggregateAggregate of chemically
modified protein
Kinetics of digestion and digestibility
Physiological consequences (bioavailability,
immunology, etc.)
Figure 1. Schematic overview of the reactions that can occur to milk proteins upon heating and how the state of the protein may have physiological consequencesthrough differences in digestion.
Milk protein NH2
Reducing sugar
Early Maillard
reaction (e.g.
Amadori products)
Advanced Maillard
products (e.g.
CML)
Late Maillard
products (e.g.
melanoidins)
Figure 2. Stages of the Maillard reaction between milk proteins and reducing sugars and commonly used markers to monitor each stage.
2 G. A. A. VAN LIESHOUT ET AL.
between whey proteins amongst each other and betweenwhey proteins and casein micelles is mainly driven bydisulfide bridge formation. With the main whey protein,b-lactoglobulin, having a free SH-group within its tertiarystructure, this protein is important in driving milk proteinaggregation upon heating. Above a critical temperature(approximately 75 �C) irreversible conformational changesoccur as a result of initiated aggregation processes(Loveday et al. 2014). The binding between whey proteinsand casein micelles is mainly through interaction betweenthe SH-groups of b-lactoglobulin and j-casein (Donatoand Guyomarc’h 2009).
1.2. Dairy processing
The ratio in which different reactions (denaturation,aggregation, chemical modification) occur, depends toa large extent on the exact processes to which the milk issubjected (Pellegrino, Cattaneo, and De Noni 2011; Walstra,Wouters, and Geurts 2006). This causes liquid and dry dairyproducts to differ in their degree of protein modifications.It is also important to consider that dairy products maybe heated multiple times depending on the specific product,possibly combining liquid and dry dairy processes. Theextent to which these processing induced modifications,including denaturation, aggregation, glycation, oxidation,etc. occur and their overall impact on digestion and physio-logical outcomes are thus determined by differences in (andcombinations thereof) processing. A schematic overview ofdairy processing steps and their effects on milk proteinmodification is given in Figure 3.
1.2.1. Liquid dairy processingFor industrial heat processing of liquid milk and dairyproducts, a distinction is commonly made between pasteur-ization and sterilization conditions, the two heat treatmentsthat are most commonly applied (Walstra, Wouters, andGeurts 2006).
Pasteurization can be further subdivided in low and highpasteurization. Low pasteurization refers to a very mild heattreatment of 72 �C for around 15 seconds or 63 �C for30minutes, which both hardly affect milk proteins. Onlysome enzymes are denatured and thereby inactivated, butthe major milk proteins remain in their native state. Highpasteurization is less well defined, but is usually both athigher temperature (>80 �C) and longer (up to severalminutes) compared to low pasteurization. Such more intenseheating can lead, depending on the precise heat load, topartial or full denaturation of the whey proteins (Walstra,Wouters, and Geurts 2006). Under such circumstances, thewhey proteins mainly aggregate on the casein micelles,where the linkage between b-lactoglobulin and j-caseinforms the basis, as discussed above. Caseins are not directlyaffected by pasteurization conditions, besides the abovemen-tioned binding of whey proteins to the casein micelle(Pellegrino, Cattaneo, and De Noni 2011; Walstra, Wouters,and Geurts 2006).
When it comes to sterilization, different conditions canbe distinguished, with the main distinction being the rela-tive long in-package sterilization and UHT sterilization.With in-package sterilization, the cumulative heat load isvery high, leading to full whey protein denaturation aswell as extensive chemical modification of the aminoacids, especially glycation of lysine. The most apparentresult of this glycation is the discoloration and specific
Farm:
Cows, milking & milk storage
Distribution over dairy factories
Liquid dairy factory
Liquid processing Homogenization
Dairy powder factory
Cheese making
Packaging
Sterilisation Pasteurisation
Packaging
Whey Cheese
Storage Storage
Evaporation
Drying
Storage
MTPfossol,noitadixo,noitazilcyc,noitazimecaR
, etc
.
Denaturation, S-S aggregation
Maillard reaction & -induced aggregation
?
Figure 3. Schematic overview of different type of dairy processes and the ensuing dairy protein modifications occurring during these processes. Product flowindicated with solid arrows, protein modifications indicated with dotted boxes and dotted arrows. PTM, post-translation modifications.
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 3
flavor of the finished product. UHT sterilization on theother hand is done at higher temperature (above 130 �Cinstead of 110 �C for in-package sterilization), but formuch shorter heating times (seconds). At these condi-tions, the whey proteins also denature, but the level ofchemical modification is much more limited, making thiscurrently the most popular mode of sterilization(Pellegrino, Cattaneo, and De Noni 2011; Walstra,Wouters, and Geurts 2006).
1.2.2. Dry dairy processingThe reduced water activity during production of dried dairyproducts leads to a different balance between the proteinmodifications that occur. Dry heating enhances chemicalmodifications of amino acids, like glycation and oxidation,while at the same time, the rate of denaturation is lowerthan in liquid dairy products (Mehta and Deeth 2016;Pellegrino, Cattaneo, and De Noni 2011).
Dry dairy products have usually undergone prior heattreatments in a liquid state, which can range from low pas-teurization to intense sterilization, to ensure safety and/orachieve specific functionality of the end product. When itcomes to protein denaturation in dried dairy products, themain driver is thus the heating intensity of the liquid pro-cess, with limited or no further denaturation occurring dur-ing the drying process itself (Morgan et al. 1999; Mehta andDeeth 2016; Singh and Creamer 1991).
While denaturation happens mostly during the liquidprocessing, chemical modifications mainly occur during thedrying process itself. Especially the effect of drying parame-ters on glycation has been studied in detail (Guyomarc’het al. 2000). The protein modification in the final dried dairyproducts thus depends on a combination of the intensity ofthe heating of the liquid process combined with the parame-ters of the drying process.
1.2.3. StorageBesides processing-induced modifications, changes may alsooccur during storage of the final product. Especially steri-lized or dry dairy products stored for a prolonged period athigh ambient temperatures are sensitive towards changes intheir milk protein. Most research on storage-inducedchanges in protein modification has been done on drieddairy products and to a lesser extent sterilized products(Erbersdobler 1989; Ferrer et al. 1999; Mehta and Deeth2016; Rutherfurd and Moughan 2008). Protein denaturationis not affected by storage, but protein aggregation andchemical modification of amino acids may occur duringstorage. The most apparent change that occurs is glycation,that also leads to browning of dairy products stored for pro-longed times. The extent of modification during storage is acombination of storage conditions (time, temperature) andproduct conditions (predominantly water activity)(Guyomarc’h et al. 2000).
1.3. Consequences for digestion
The different states of the protein after heat processing, asdescribed in Figure 1, can have a large impact on theirdigestion. This is modulated through both the structure ofthe protein and the chemical modifications, which may bothinfluence the accessibility of the amino acid bonds to theproteolytic enzymes. Generally, the unfolding of proteinsincreases accessibility of amino acids to proteolytic enzymes,whereas aggregation and chemical modifications, dependingon the specific amino acid being modified, reduces theaccessibility to proteolytic enzymes. Differences in productand processing conditions may thus lead to different effectson protein digestion.
1.4. Physiological consequences
Modification of milk proteins, and a consequentially altereddigestion, may have further physiological consequences to theconsumer. Different kinetics of protein digestion can, forexample, lead to different postprandial absorption kinetics ofamino acids. Also, the presence of peptides of different size,and different chemical modifications of these sequences, mayhave effects on e.g. the gastro-intestinal tract itself or theimmune system (Nowak-Wegrzyn and Fiocchi 2009). This isespecially relevant in early life for formula fed infants, wherebovine milk proteins are typically the sole source of food pro-teins at a period where, amongst others, the digestive andimmune system are not yet fully developed.
1.5. Aim of this review
As milk proteins play an important role in human nutrition,and can undergo changes during industrial processing, itis important to better understand how this may influencethe digestion of milk proteins and their subsequent overallphysiological activity. As bovine milk is the main milkindustrially processed around the world, this review focuseson this milk specifically. This systematic review will providean overview of the current state-of-the-art in this field,as well as indicate gaps that could be the focus forfuture research.
Better understanding of the relation between processingof dairy proteins, its digestibility, and subsequent physio-logical impact is also relevant for application in industry,where it may be applicable for designing optimal processeswith a minimum impact on protein quality.
2. Methods
A systematic review on the effect of protein modificationson protein digestion in dairy was performed using severalterms as tested in a search query. All searches were doneusing the Scopus database. Ultimately, four terminologygroups were defined in relation to the research question, asspecified in Table 1. Other terms that have been assessed fortheir inclusion were: protein, process�, modifi�, CML,Amadori, hydroly�, lysine, cross-link� and transport�. Those
4 G. A. A. VAN LIESHOUT ET AL.
terms were excluded because they were either too general(protein, process�, hydroly�), did not include new publica-tions (Amadori) or did not include additional publicationsrelevant to the research question (modify�, CML, lysine,cross-link� and transport�). Since a substantial number ofanimal studies relevant to the research question were pub-lished between 1990 and 2000, a publication date within thelast 30 years was chosen for the final search.
The final search query was defined as (TITLE-ABS-KEY(milk� OR dairy OR (infant AND formula) OR whey ORcasein� OR lact�) AND TITLE-ABS-KEY (glycat� ORMaillard� OR heat� OR furosine OR brown� OR oxidat�OR therm� OR aggregat� OR denat�) AND TITLE-ABS-KEY (digest� OR absor� OR proteoly�) AND TITLE-ABS-KEY (gastr� OR intestin� OR availab� OR metabol� OR (inAND vitro) OR allerg� OR grow�)) AND PUBYEAR >1987. On 24 September 2018, the final search was completedusing Scopus.
The identified articles were screened on their eligibility.Because of limited information available in titles, articles wereincluded when they could be associated with at least two outof the four grouped terms. First, the titles were screened fortheir eligibility by one of the authors. Subsequently, abstractsof included titles were screened by all authors independentlyusing the below described criteria.
Studies were included when they compared differentlyprocessed dairy proteins. Only the major bovine dairy pro-teins were included, being a-lactalbumin, b-lactoglobulin,a-caseins, b-casein and j-casein. Both isolated proteins andproteins in a dairy matrix were included. Studies wereincluded when digestion was involved that is (partly) repre-sentative for gastrointestinal digestion in humans, either invivo or in vitro. Outcome measures related to protein digest-ibility and absorption that were included were nitrogen bal-ance calculations, protein hydrolysis, SDS-PAGE, LC/MS-MS data, or possible physiological consequences like aminoacid bioavailability, growth, and allergenicity/immunology.
Articles not in English were excluded. In addition, non-experimental publications were excluded, including conferencepapers, editorials and reviews. Some reviews were used as partof the introduction. Studies that focus on non-bovine milk(e.g. human milk) or hydrolyzed protein were also excluded.Furthermore, studies were excluded when no (simulation of a)dairy heat process was used, e.g. homogenization and enzym-atic cross-linking, or when no comparison was made betweendifferently heated samples. When no human-like gastrointes-tinal digestion or no outcome measure related to digestion orphysiology was used, studies were excluded as well. Lastly,
studies were excluded when the focus was on development ofa new analytical methodology.
After screening for abstracts, full-text articles weregrouped based on four major topics; glycation, denaturationand aggregation, other modifications and physiologicaleffects. In turn, topics were assigned to different authorsand full-text articles were assessed for eligibility according tothe in- and exclusion criteria described above. For introduc-tion purposes, and for further discussion of overall physio-logical relevance of findings from individual studies, anumber of references were added manually.
3. Results and discussion
In total, 5386 records were identified through database search-ing and through manual addition. After screening for title, 235abstracts were included, of which 116 were selected to assessfull-text eligibility. Ultimately, 103 articles were included basedon the criteria described in section 2. An overview of the pro-cess is given in Figure 4. No quantitative comparison of indi-vidual studies was done as only a limited number of studiesquantified both modification of the product and differences inprotein hydrolysis. Moreover, those studies varied in theirmethods and outcome measures used, making it unfeasible tocombine data in a quantitative synthesis.
A limitation of this systematic review is that search termswere already biased towards glycation, denaturation, andaggregation related modifications, despite inclusion of heat-ing in general. In addition, the number of search termsrelated to physiological consequences was limited and there-fore, relative to other areas of interest, it appeared necessaryto manually include articles to complement the discussion.
Included studies mainly involved in vitro and animaldigestion studies. A limited number of studies includedproducts that were not heat processed but processed inanother manner (e.g. homogenized). In those studies, onlythe data and discussion on non-heated versus heated studyarms were used for this review. Most studies investigatedthe effect of glycation, denaturation and aggregation, or acombination thereof, on protein hydrolysis. A limited num-ber of studies explored the effect of other modifications ondigestion and/or overall physiological effect. Those topicswere discussed separately.
Several methods are used to study the effect of heat-induced modifications on protein digestion, either inhumans, animals or in vitro. A single study assessed theeffect of heat treatment on postprandial kinetics in humansusing intrinsically labeled milk proteins. In animal studies,nitrogen balance methodology is often used, in which a dietcontaining either heated or unheated protein is fed afterwhich nitrogen absorption is measured. Both labeled andnon-labeled nitrogen are used. Absorption can be measuredat three levels. First, nitrogen is analyzed in feces to calculateapparent digestibility. Second, a correction for endogenousprotein secretion can be done to calculate true digestibility.Third, a possible effect of colonic fermentation can beexcluded by measuring nitrogen in the ileum to calculate(true or apparent) ileal digestibility. A few animal studies do
Table 1. Search terms.
Product Modification Digestion Physiological
Milk� Glycat� Digest� Gastr�Dairy Maillard� Absor� Intestin�Infant formula Heat� Proteoly� Availab�Whey Furosine Metabol�Casein� Brown� In vitroLact� Oxidat� Grow�
Therm� Allerg�Aggregat�Denat�
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 5
not use nitrogen balance methodology, but determine pro-tein hydrolysis in samples taken from the stomach or intes-tine. In in vitro studies, gastrointestinal digestion issimulated, mostly based on adult or infant conditions as dis-cussed in literature. In vitro experiments simulate GI diges-tion in a static or dynamic manner. In case of staticexperiments, the food is incubated with simulated digestivejuices and samples are taken at the end of gastric and/orintestinal digestion. Dynamic models include the gradualaddition of enzymes and acid, and gastric emptying of thefood. This enables investigation of different gastric behaviorand emptying of dairy products after heating. After simu-lated digestion, protein hydrolysis is characterized by analyt-ical methods like available amino-group measurements, gelelectrophoresis or mass spectrometry.
For clarification purposes, we defined protein hydrolysisas the reaction where proteins or peptides are cleaved intosmaller peptides or amino acids. Protein hydrolysis is some-times used to calculate protein digestibility. Protein digest-ibility is defined as the amount of nitrogen that becomesavailable for absorption compared to the total amount ofnitrogen ingested. Finally, when peptides or amino acidsbecome available for human metabolism after both digestionand absorption in the gut, this is defined as bioavailability.
3.1. Glycation
Glycation of milk proteins as a result of heat treatment, dry-ing in particular, is suggested to impair protein quality.
Studies investigating an effect of glycation on protein digest-ibility are listed in Table 2. A wide variation of products isused with a diverse range of heat processes and tempera-tures to induce glycation. Most studies use whey protein,casein or isolated milk proteins together with lactose or glu-cose. A few studies use GOS and FOS, being a mixture of(reducing) carbohydrates, while others use pectin and dex-tran to demonstrate effects specifically of larger carbohy-drates. In addition to extreme heating conditions,methylglyoxal or 3-deoxyglucosone are used to obtain moreadvanced glycation products. In general, unheated andheated carbohydrate-protein mixture are compared. Severalstudies use mixtures heated at various temperatures or wateractivities to cover a range of process variations. Ultimately,heating temperature, timing, the ratio of carbohydrate andprotein, and the water activity together determine the extentof the Maillard reaction in the product, as explained in sec-tion 1.2 (Guyomarc’h et al. 2000).
Different methods are used to characterize the Maillardreaction products (MRPs), with the most common markersshown in Figure 2, although some studies do not character-ize the Maillard reaction at all. Methods used include a shiftin molecular weight, a decrease in available amino-groups,or a higher browning after heating. This limits informationabout the different MRPs formed, their amount and there-fore the exact stage of the Maillard reaction. Other studiesdirectly quantify MRP-related parameters like unmodifiedlysine, also referred to as available or reactive lysine, modi-fied lysine, also referred to as blocked lysine, furosine, and
Figure 4. Database search results according the PRISMA statement (Moher et al. 2009).
6 G. A. A. VAN LIESHOUT ET AL.
Table2.
Literature
overview
ofstud
iesinvestigatingtheimpact
ofglycationon
dairy
proteindigestibility.
Reference
Metho
dProteinsource
Carboh
ydrate
source
Glycatio
nmetho
dStud
yarms
Maillard
prod
ucts
Effect
glycationon
digestibility
Animal
stud
ies
(Alamiret
al.2
013)
Nitrog
enbalancein
rats
Sodium
caseinate
Powderedbiscuit
ingredients
Biscuitextrusion
Non
-extruded
Extrud
edCM
Lform
ation
Nosign
ificant
effect
onapparent
digestibility
(Gilani
and
Sepehr
2003)
Nitrog
enbalancein
rats
SMP
Lactosein
SMP
Heatin
g121� C
for1h
Unh
eated
Heated
Not
measured
Apparent
proteindigestibility
was
redu
cedin
oldand
youn
grats
(Lacroixet
al.2
006)
Nitrog
enbalancein
rats
15Nlabeledmilk
Lactosein
milk
Spray-dryinlet250� C,
outlet87
� CNon
-heated
Heated
Early
Maillard
Nitrog
enavailabilitywas
lower
forspray-dried
(Larsenet
al.2
010)
Nitrog
enbalancein
kittensandrats
Lacticcasein
Dextrose
Heatin
gat
121� C
for2h
Non
-heated
Heated
Early
and
advanced
Maillard
Bioavailablelysine
was
redu
ced
(Mou
ghan,G
all,and
Rutherfurd
1996)
Nitrog
enbalance
inpigs
Sodium
caseinate
Glucose
Heatin
gat
121� C
for
1min
andat
70� C
for1day
Unh
eated
Heated
Early
and
advanced
Maillard
Lower
apparent
ileal
digestibility
forlysine
andAA
s
(Mou
ghan
and
Rutherfurd
1996)
Nitrog
enbalancein
rats
Lacticcasein
Lactose
Heatin
gat
121� C
for3.5min
Unh
eated
Heated
Early
and
advanced
Maillard
Lower
true
ileal
digestibility
for
lysine
andAA
s(R� erat
etal.2
002)
Nitrog
enbalance
inpigs
SMP
Lactosein
SMP
Roller-drying
Freeze-dried
Roller-dried
Early
Maillard
Decreased
absorptio
nof
lysine
andTA
As(Rutherfurdand
Mou
ghan
2008)
Nitrog
enbalancein
rats
SMP Hydrolyzed-
lactoseSM
P
Lactosein
SMP
Hydrolyzedlactose
inSM
P
Storageat
30,3
5and
40� C
upto
18mon
ths
Differentstoragetim
esEarly
and
advanced
Maillard
Lower
true
ileal
digestibility
forlysine
(Rutherfurdand
Mou
ghan
1997)
Nitrog
enbalancein
rats
SMP
Lactosein
SMP
Heatin
gat
121� C
for1,
3,5and10
min
Unh
eated
Heated
Early
and
advanced
Maillard
Lower
true
ileal
digestibility
for
lysine
andmostAA
s(Sarri� a,L� op
ez-Fandi~ no
,andVaqu
ero2000)
Nitrog
enbalancein
rats
Infant
form
ula
Lactosein
IFIF
prod
uctio
nPowder
UHT
In-bottle
sterilized
Early
and
advanced
Maillard
Apparent
digestibility
was
lower
(Sarwar,P
eace,B
ottin
g,andBrul� e,
1989)
Nitrog
enbalancein
rats
SMP
Lactosein
SMP
‘Overheated’
Unh
eated
Heated
Not
characterized
Apparent
crud
eandtrue
protein
digestibility
werelower
and
true
lysine
digestibility
was
lower
Invitrostud
ies
(Cattaneoet
al.2
017)
Infant
invitrodigestion
Infant
form
ula
mod
elsystem
sLactose(L)
Maltodextrin
(M)
Inbatchsterilizatio
n,110� C
38min
Commercial
Non
commercial
NoLþM
LþM
Furosine
and
pyrralineform
ation
Proteolysiswas
increased
anddecreased
(Corzo-M
art� ın
ezet
al.2
010)
Adultin
vitrodigestion
b-lactog
lobu
linGalactose
Tagatose
Dextran
10kD
aDextran
20kD
a
Heatin
gat
40/50/60
� Cwith
orwith
out
pyrid
oxam
ine
Not
heated
Heated
Early
andadvanced
Maillard,
cross-linking
Lower
hydrolysisof
intact
b-lactog
lobu
linwith
high
erglycation.
(Culverand
Swaisgood1989)
48h2-step
GIin
vitrodigestion
Sodium
caseinate
Glucose
Storageat
30or
60� C
for0-30
days
Heatedat
30or
60� C
Not
measured
Proteindigestibility
was
decreased
(Desrosiersand
Savoie
1991)
Adultin
vitrodigestion
WPC
Lactose
Heatin
gat
75,1
00and
121� C
for50,5
00and5000
s
Unh
eated
Heated
Early
and
advanced
Maillard
AAdigestibility
was
decreased
(Desrosierset
al.1
989)
Adultin
vitrodigestion
WPC
Lactose
Heatin
gat
75,1
00and
121� C
for50,5
00and5000
s
Unh
eated
Heated
Early
and
advanced
Maillard
Lysine
digestibility
was
decreased
(Hellwig
and
Henle
2013)
72h3-step
GIin
vitrodigestion
b-casein
3-Deoxyglucoson
eHeatin
gat
70� C
for10,
30,6
0and180min
Unh
eated
Heated
Pyrralineform
ation
Proteindigestibility
was
decreased
(Hiller
and
Lorenzen
2010)
Pancreatin
invitrodigestion
Sodium
caseinate
WPI
SMP
Glucose
Lactose
Pectin
Dextran
Heatin
gat
70� C
for0-
240hat
65%
RHUnh
eated
Heated
Short-term
andlong
-term
Maillard
Proteindigestibility
was
decreasedforsodium
caseinateandincreased
forWPI
(continued)
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 7
carboxymethyllysine (CML). This enables a better under-standing of the MRPs that differ between the tested productsand the exact stage of the Maillard reaction. When aminoacid analyses are used to calculate digestibility, it is of majorimportance to distinguish between total and reactive lysinelevels as standard methodology may not always distinguishbetween both, leading to an overestimated lysine content thatcould result in an underestimated lysine digestibility coeffi-cient (Moughan and Rutherfurd 1996). The complicated reac-tion pathways, and the many resulting MRPs, likely explainswhy no study was found that investigates a possible quantita-tive relation between glycation and digestibility.
3.1.1. The effect of glycation on protein digestionThe overall result, taking all studies in Table 2 together, is thatglycation decreases protein digestibility. From 24 studies, onlya single study does not demonstrate an effect of glycation onmilk protein digestion, 3 studies show an increase and decreasein protein digestibility depending on protein or carbohydratesource, and 20 studies show a decrease. Three possible mecha-nisms are mentioned that could explain the observed decreasein protein digestibility: 1) direct blocking of lysine residues,preventing it from recognition and cleavage by digestive pro-teases; 2) indirect blocking by glycation of lysine residues thatare positioned near the cleavage site of enzymes; 3) glycationinduced cross-linking that decreases accessibility of cleavagesites for proteases. When initial trypsin digestion is inhibited,this may hinder digestion by other intestinal proteases includ-ing brush border membrane proteases as one of the final stepsbefore amino acid absorption, overall thus affecting the releaseamino acids during digestion. The decreased enzyme accessibil-ity as a result of a different structure is also termed steric hin-drance. In contrast, three studies show that glycation is able toincrease digestibility. This was seen for WPI and a-lactalbumin(Cattaneo et al. 2017; Hiller and Lorenzen 2010; Joubran,Moscovici, and Lesmes 2015). Unfolding of the globular wheyproteins as a result of heating and carbohydrate binding mayhave made hidden cleavage sites better accessible. Thus, forwhey proteins, a balance between protein unfolding and sterichindrance, depending on the exact processing conditions, willdetermine whether an increase or decrease in digestibility isfound after glycation. Most studies show that this balance leansmore towards inaccessible cleavage sites, resulting in an overalllower digestibility.
The extent of the Maillard reaction differs between andwithin studies, but the number of studies that distinguishbetween early and advanced Maillard reaction products orpossible Maillard reaction-induced cross-linking is limited.An exception is the study by R�erat et al. (2002) who useda heated skimmed milk powder (SMP) representative of theearly Maillard reaction. After ingestion of the early MaillardSMP, plasma appearance of lysine and total amino acids wasdecreased compared to a SMP low in MRPs, suggestingthat already early Maillard reaction events have an effect onprotein digestion and absorption. In another study, Hellwigand Henle (2013) focused on the specific conversion oflysine to the AGE pyrraline. A higher pyrraline contentresulted in a decreased formation of small peptides whichTa
ble2.
Continued.
Reference
Metho
dProteinsource
Carboh
ydrate
source
Glycatio
nmetho
dStud
yarms
Maillard
prod
ucts
Effect
glycationon
digestibility
(Jou
bran,M
oscovici,
andLesm
es2015)
Infant
invitrodigestion
a-lactalbu
min
Fructose
FOS
Heatin
gat
60� C
for12
and24
h(FOS)
or12
and36
h(Fru)at
79%
RH
Unh
eated
Heated
HigherMWsandcolor
developm
ent
Lower
hydrolysisof
intact
a-lactalbu
min
with
increasedglycation
(Jou
bran
etal.2
017)
Infant
andin
vitrodigestion
a-lactalbu
min
Glucose
Galactose
GOS
Heatin
gat
60� C
for12
and24
h(GOS)
or12
and36
h(Glu
and
Gal)at
79%
RH
Unh
eated
Heated
HigherMWsandcolor
developm
ent
Glu,G
alandGOS-24hincreased
proteolysis.GOS-12h
decreasedproteolysis.
(Pinto
etal.2
014)
Adultin
vitrodigestion
b-casein
b-lactog
lobu
linA
Glucose
Heatin
gsolutio
nat
90� C
upto
24h
Unh
eated
Heated
Advanced
Maillard
Lower
hydrolysisof
intact
b-casein
andb-lactog
lobu
lin(Luz
Sanz
etal.2
007)
Adultin
vitrodigestion
b-lactog
lobu
linGOS
Heatin
gat
40� C
for16
days
atAw
0.44
Unh
eated
Heated
Early
and
advanced
Maillard
Lower
hydrolysisof
intact
b-lactog
lobu
lin(Zhao,
Le,etal.2
017)
Adultin
vitrodigestion
b-casein
b-lactog
lobu
linMethylglyoxal
Glyoxal
Butanedion
e
Heatedat
95� C
for1h
Unh
eated
Heated
Advanced
Maillard
Decreasein
degree
ofhydrolysis
(Zhao,
Li,etal.2
017)
Adultin
vitrodigestion
b-casein
b-lactog
lobu
linMethylglyoxal
Heatedat
98� C
for2h
Unh
eated
Heated
Advanced
Maillard
Decreasein
hydrolysisof
intact
proteins
andreleaseof
free
AAs
CML,carboxym
ethyllysine;A
A,am
inoacids;TA
A,totala
minoacids;WPI,W
heyproteinisolate;SM
P,Skimmed
milk
powder;IF,Infantform
ula;WPC
,Wheyproteinconcentrate;RH
,relativehu
midity.
8 G. A. A. VAN LIESHOUT ET AL.
could be explained by the modification of lysine to pyrralinethat is not recognizable for trypsin. Moreover, Corzo-Mart�ınez et al. (2010) used pyridoxamine (PM) to partiallyinhibit aggregation and cross-linking in the advanced stagesof the Maillard reaction. Addition of PM resulted inan increased peptide formation and disappearance of intactb-lactoglobulin after 15 minutes intestinal hydrolysis ofb-lactoglobulin conjugated with galactose or tagatose, sug-gesting an increased hydrolysis when aggregation and cross-linking is inhibited. Lastly, Sarri�a, L�opez-Fandi~no, andVaquero (2000) compared consumption of different formsof infant formula in rats and discussed that advanced MRPsare mostly excreted through feces, indicating reducedprotein digestibility. Early MRPs were suggested to bepartially absorbed, but metabolic utilization was minimalbecause most of it was excreted via urine. Altogether, itappears that early MRPs decrease amino acid bioavailability,and when absorbed, metabolic utilization is minimal, whileadvanced MRPs and Maillard-induced cross-linking furtherdiminish overall protein digestibility.
3.1.2. Glycation in combination with other proteinmodifications
While the aforementioned studies focus on glycation inparticular, various studies include heating processes whereboth glycation and denaturation or aggregation of theproteins occurred. For example, protein digestibility ofpowdered infant formula was significantly higher than liquidconcentrates of the same manufacturer in a rat balancestudy (Sarwar, Peace, and Botting 1989). Likewise, when sev-eral infant formulas were digested using an infant in vitromodel, it was concluded that digestibility of protein wasgenerally highest for powdered formula, followed by liquidconcentrate and ready-to-feed formula suggesting that proc-essing does have an effect (Liusuwan and L€onnerdal 1996).Wada and L€onnerdal (2014, 2015) conducted two studieswhere differently heated milk products were digested bothin vitro and in rats. In the first, raw milk was either pasteur-ized, plate UHT treated, steam infused, steam injected, orin-can sterilized, while in the second, enteral formulas weresteam injected or in-can sterilized. This resulted in productsthat differed in glycation, denaturation and aggregation.Overall, they suggested that denaturation can improve pro-tein digestibility, while formation of aggregates or formationof lactulosyllysine (early MRP) can reduce digestibility(Wada and L€onnerdal 2014, 2015). Differences in glycationand aggregation may also result in different peptides formedduring digestion of WPC powders heated with lactose (Liuet al. 2016). This is in line with the results of Pinto et al.(2014), who showed that native b-lactoglobulin remainedintact during gastric digestion, while heated b-lactoglobulinwas digested and addition of glucose limited this heat-induced increase in hydrolysis. This illustrates that the bal-ance of denaturation and glycation affects the digestion ofb-lactoglobulin. For this reason, it is important to distin-guish between different protein modifications when evaluat-ing digestion of a heated product. For example, Nooshkamand Madadlou (2016) suggested that Maillard conjugation
could improve gastric digestibility of whey proteins, whilethis effect is likely more related to denaturation of the wheyproteins. Besides an effect via the Maillard reaction, otherprotein modifications after heating a more complex matrixcould affect protein hydrolysis and digestibility by othermeans. These include protein-lipid interactions or differentformation of coagulates in the stomach (Rudloff andL€onnerdal 1992; Wada and L€onnerdal 2014). The variousmodifications that occur in parallel make it hard todistinguish the effect of a single modification specificallywhich may be an interesting topic for further research.
3.2. Denaturation and aggregation
An overview of studies that investigated the impact ofdenaturation and aggregation on dairy protein digestion islisted in Table 3. Studies were divided in four categories: ahuman study, animal studies, static in vitro studies and(semi)-dynamic in vitro digestion experiments. These differ-ent methods enable to distinguish between the effect of heat-ing on protein digestion and protein digestion kinetics,although direct and quantitative comparability of differentstudies becomes more complex and the full complexity of invivo digestion is not simulated in these in vitro models.Furthermore, it is important to note that most studies useheating as a way to induce denaturation and aggregation,but do not actually measure the level of these modificationsor other modifications that may occur in the proteinsamples used prior to digestion.
3.2.1. The impact of denaturation and aggregation ongastric protein hydrolysis and gastric behavior
3.2.1.1. Whey proteins and caseins. Zooming into thedigestion of the major milk proteins, casein is almost com-pletely digested in the stomach, while native whey proteinsare more resistant to hydrolysis and are still intact aftergastric digestion (Tunick et al. 2016). The resistanceof whey proteins to gastric proteolysis is thought to relate totheir conformation, which is required to exert specificphysiological functions, whereas the open casein structure ismainly thought to relate to nutritional purposes, i.e. supplyof amino acids (Guo et al. 1995). Most in vitro studiesfocused on hydrolysis of b-lactoglobulin. Generally, andin different matrices, it is described that heated (>75 �C),denatured b-lactoglobulin becomes more digestible bypepsin in vitro, due to unfolding and the consequentialincreased accessibility of protein cleavage sites (Guo et al.1995; Kitabatake and Kinekawa 1998; S�anchez-Rivera et al.2015; Wang et al. 2018). Rat studies confirm in vitro resultsby showing intact b-lactoglobulin in the rat stomach fornative b-lactoglobulin, while heated b-lactoglobulin did notshow intact b-lactoglobulin in the stomach (Kitabatake andKinekawa 1998). When using a dynamic gastric digestionmodel, where gastric emptying is included, only intactb-lactoglobulin was emptied into the intestinal compartmentfrom non-heated whey protein isolate, whereas peptideswere emptied during digestion of heated whey protein
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 9
Table 3. Literature overview of studies investigating the impact of denaturation and aggregation on dairy protein digestibility.
Reference Method Protein source
Study arms withdenaturation/aggregation method Outcome measure
Effect on digestibility anddigestion kinetics
Human study
(Lacroix et al. 2008) In vivo, randomizedintervention studyusing 15Nlabled milk
15N labeled milk Microfiltered milkPasteurized milk (72�C, 20 sec)UHT milk (140�C, 5 sec)
Serum AAconcentrationPostprandialretention
No effect on overall serum AAconcentrations; more sustainedhyperaminoacidemia in themicrofiltered group than UHT;more rapid kinetics for UHT;lower protein retention for UHT
Animal studies
(Barb�e et al.2013, 2014)
In vivo, cross-overdigestion in 18-month-old adultmini pigs
Ultra-lowheat SMP
Raw liquid milk derivedfrom ultra-low heatskim milk powderHeated liquid milk(10 mins, 90 �C)
Protein hydrolysis(SDS-PAGE),Peptides (nano-RPLC MS/MS),cleavage score
Heat treatment increased plasmaIAA bioavailability; madeb-lactoglobulin more susceptiblefor hydrolysis; increased stomachretention time
(Efigenia, Povoa, andMoraes-Santos 1997)
In vivo digestion inWeanler Holtzmanmale rats
Whole milk Raw milkPasteurized milk(72 �C for 15 sec)Boiled milk (30 secboiling)UHT milk (135-150 �Cfor few sec).
Apparent proteindigestibility,growth
No differences in apparent proteindigestibility. UHT milk impairedgrowth of weaning rats, whichcorrelated with food intake.
(Kitabatake andKinekawa 1998)
Static in vitro GIdigestion and in vivodigestion inWistar rats
b-lactoglobulin Heated (80 �C, 1 h) andunheatedb-lactoglobulin
Protein hydrolysis(SDS-PAGE), Sizeexclusionchromatography
Heating enabled pepsin hydrolysis ofb-lactoglobulin in vitro and invivo. Similar extent of pancreatichydrolysis of native and heatedb-lactoglobulin, but heatedb-lactoglobulin more rapidly invitro. Complete intestinalhydrolysis of heated and un-heated b-lactoglobulin
(Lacroix et al. 2006) Nitrogen balance in rats 15N labeled milk Microfiltered milkHTST milk, 72 �C, 20 sHHST milk, 96 �C, 5 sUHT milk, 140 �C, 5 s
Digestibility,biological valueand netpostprandialprotein utilization
Digestibility and biological valuewere similar between all milkproducts, and no correlationbetween temperature andnitrogen availability.
(Moughan et al. 1989) Nitrogen balance inyoung pigs and invitro flocculation test
SMP Heated 74 �C, 121 �C95 secHeated 70 �C, 85 �C105 sec, 102 �C 1 sHeated 85 �C, 118 �C1 sHeated 77 �C,102 �C 1 s
Mean apparent fecaland ilealdigestibility,biological valueand growth;flocculation
Higher levels of heat treatment ledto a longer time to flocculation invitro. Curd was firmer for the twoleast denatured samples. Noeffect of diet on apparent fecaland ileal digestibility, dietarynitrogen utilization and growth.
(Mpassi et al. 2001) In vivo digestion ingrowing pigs
15N labeled milkand yoghurt
MilkYogurtHeat-treated yoghurt(80 �C for 10min)
15N aminonitrogenportal absorption
No differences in absorption rate(�80%) between the milkproducts. The kinetics differed,absorption of N for milk andheat-treated yoghurt mainlyoccurred during the first 2 hours,while for yoghurt it was moreevenly spread over 4 hours.
Static in vitro studies
(Carbonaro et al.,1997) In vitro static enzymaticdigestion withporcine pancreatictrypsin, bovinepancreaticchymotrypsin,porcine intestinalpeptidase, followedby bacterial protease
Whole milkWheyproteinextracts
Raw milkPasteurized milk(15 sec at 72, 75, 78,80 �C)UHT treated milk (3and 5 sec at 145 �C),Sterilized milk (3 and5 sec at 145 �Cfollowed byautoclaving for12min at 118 �C or10min at 116 �C)
In vitro proteindigestibility, wheyprotein solubility,whey amino acidcomposition
Digestibility increased from raw topasteurized to UHT or sterilizedwhole milk. Digestibility of wheyprotein extracts decreased withincreasing heat treatment.
(Dupont, Boutrou,et al. 2010)
Static in vitro infant GIdigestion model
Ultra-lowheat SMP
Heating at 80 �C, 20 sHeating at 85 �C,180 sHeating at105 �C, 60 s
SDS-PAGE, WesternBlotting and LCMS-MS
b-lactoglobulin was still presentafter digestion in all samples.High processed samples showhigh resistance of intact caseinsto digestion.
(Dupont, Mandalari,et al. 2010)
Static in vitro infant GIdigestion model
Whole milk Raw milkPasteurized milk (30 s
Pasteurization and sterilizationincreased gastric hydrlysis of
(continued)
10 G. A. A. VAN LIESHOUT ET AL.
Table 3. Continued.
Reference Method Protein source
Study arms withdenaturation/aggregation method Outcome measure
Effect on digestibility anddigestion kinetics
/ 82 �C)Sterilized milk(10min / 120 �C)
SDS-PAGE, WesternBlotting and LCMS-MS
b-lactoglobulin. Intactb-lactoglobulin was present afterintestinal digestion for raw milkand pasteurized, not for sterilized.Sterilized milk showed higherresistance of casein peptides.
(Guo et al. 1995) Static in vitro digestionwith pepsinor trypsin
b-lactoglobulin b-lactoglobulin heatingat 70 to 100 �C for 5or 10minutes at pH6.8 or pH 3.0
Protein hydrolysis(SDS-PAGE)
Native b-lactolgobulin resistant topeptic digestion, but heating at>80 �C enhanced proteolysis withincreasing temperature.
(Lamothe et al. 2017) Static in vitroGI digestion
Whole milkheated to50 �C andskimmed,adding back2% of fat
Pasteurized milk (65 �C,30min)Milk doublehomogenized andheated at 65 �C for30minMilk doublehomogenized andheated at 95 �Cfor 5minutes.
Matrix degradation,protein hydrolysis
Heating at 95 �C increased hydrolysisin first 30minutes of gastricdigestion compared to 65 �C.Proteolysis was similar inintestinal phase, so after wholedigestion, no difference inhydrolysis depending on heating.
(Lindberg et al. 1998) In vitro static digestionwith duodenal juicefrom adults
WPCs WPC freeze-dried: 0%denaturationWPC spray-dried:26% denaturationWPC heated 80 �C15 sec, freeze-dried:55% denaturationWPC heated 80 �C30min, freeze-dried:97% denaturation
Digestion viapolyacramidegradient gelelectrophoresis,electroimmunoassay,nonproteinnitrogen
Digestibility of whey proteinsdecreased with higher degree ofprotein denaturation.
(Lorieau et al. 2018) In vitro staticGI digestion
Gels made bystirring andheating anemulsion ofnative wheyproteinand cream
Liquid native wheyHard gel (70 �C for36.5min at pH 6.4),Processed gel (75 �Cfor 19min at pH 5.9),Whipped gel (75 �Cfor 80min at pH 5.4)
Protein hydrolysis(SDS-PAGE),release of aminogroups (OPA),amino acidconcentration
Whey proteins were resistant topepsin hydrolysis. Proteinhydrolysis and amino acid releasewas higher in whey gelscompared to the liquidnative whey.
(Loveday et al. 2014) Static in vitrogastric digestion
b-lactoglobulin Heated b-lactoglobulinat 78 �C and 90 �Cfor 0, 5, 10, 20, 30,40, 50, 60, 90and 120min
Peptide formationafter hydrolysis(SDS-PAGE andRP-HPLC)
Native b-lactoglobulin shows initialslow digestion and rapiddigestion after �4 hours. Heatedb-lactoglobulin gives highlydigestible non-native monomers,highly resistant dimers andb-lactoglobulin monomersbonded to peptides that aremoderately digested. Distributionof those is influenced byheating time.
(Peram et al. 2013) Static in vitrogastric digestion
b-lactoglobulin b-lactoglobulin heatedat 90 �C for0-120min
Protein and peptidecharacterization(SDS-PAGE andsize exclusionchromatography
Heating increased initial hydrolysisof b-lactoglobulin with acorrelation between hydrolysisand heating duration.
(Rahaman, Vasiljevic,andRamchandran 2017)
Static in vitroGI digestion
b-lactoglobulin b-lactoglobulin samplesprepared withdifferent pH (3, 5, 7),temperature (roomtemperature, 120 �C)and shear(0 s�1, 1000 s�1)
Degree of hydrolysis,peptide formation(SDS-PAGE),antigenicity assay
Unheated b-lactoglobulin wasresistant to peptic digestion, butwas susceptible to pancreaticdigestion. Heating increasedgastric and pancreatic digestionand resulted in lower antigenicity
(Rinaldi et al. 2014) Static in vitroGI digestion
Commercialdairy products
Commercial pasteurizedmilkCommercialsterilized milk
Protein content andsolubility, peptideanalysis (SDS-PAGE), freeamino acids.
No significant difference in solubleprotein and free amino acidcontent between products aftergastric and duodenal digestion.Faster digestion of caseins andwhey in gastric phase in sterilizedmilk compared to pasteurizedmilk. b-lactoglobulin and A-lacdegraded faster in sterilized induodenal phase.
(Singh andCreamer 1993)
Low-heat SMP Whey protein/k-caseincomplexes after
SDS-PAGE Unheated b-lactoglobulin is resistantto proteolysis by pepsin. Degree
(continued)
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 11
Table 3. Continued.
Reference Method Protein source
Study arms withdenaturation/aggregation method Outcome measure
Effect on digestibility anddigestion kinetics
Static in vitro digestionwith pepsin, trypsinor chymotrypsin
heating at 90 �Cfor 5min
of hydrolysis by trypsin orchymotrypsin was much lower forunheated b-lactoglobulin than inthe heated complexes. Hydrolysisof j-casein was higher for thecomplexes compared to unheatedas well.
(Singh et al. 2014) Static in vitroGI digestion
WPI WPIWPI gels heated at90 �C for 10min atpH 6.8 and 4.6
Protein and peptideanalysis (SDS-PAGE andRP-HPLC)
In the gastric phase, whey proteinsin gels are faster hydrolyzed thanin native solution. Intestinalhydrolysis of WPI in solutionwas slowest.
(Stender et al. 2018) Static in vitroGI digestion
Serum proteinconcentrate
Heat denatured SPC(1 hour, 85 �C)
TCA soluble peptides Heat treatment lowered gastricdigestion rate. No difference inhydrolysis in the intestinal phase.
(Tunick et al. 2016) Static in vitroGI digestion
Bovine milk Raw unprocessed milkRaw skimmed milkRaw whole milkHTST pasteurizedmilkUHT processedskimmed milk
Protein analysis(SDS-PAGE) andparticle sizedistribution
In the raw samples, caseins anda-lactalbumin were broken downin gastric phase, b-lactoglobulinwas resistant. This was similar forpasteurized milk. UHT showedhydrolysis of b-lactoglobulin.During intestinal digestion, bandsfor b-lactoglobulin faded within15min for raw and UHT and30min for pasteurized.
(Wada andL€onnerdal 2014)
Static in vitro GIdigestion and in vivodigestion in rat pups
Milk and liquidenteral formula
Pasteurized milk (73 �Cfor 15 sec)UHT milk (140 �C for2 sec)Steam infused milk(135 �C for 2 sec)Steam injected liquidenteral formula milk(151 �C for 4 sec)In can sterilizedliquid formula(121 �C for 20min)
Protein and peptideanalysis (SDS-PAGE, Kjeldahl)
Protein hydrolysis of casein,a-lactalbumin and b-lactoglobulinwas rapid for in can sterilized andUHT milk, due to denaturation.No differences in proteindigestibility.
(Semi)-dynamic in vitro studies
(Mulet-Caberoet al. 2019)
Semi dynamic in vitrogastricadult digestion
Bovine milk Raw milkRaw homogenizedmilkPasteurized milkPasteurizedhomogenized milkUHT milkUHThomogenized milk
Gastric behavior,nutrients emptiedand proteindigestion(SDS-PAGE)
Raw milk forms a firmer coagulum,whereas heated samples and UHTin particular give a more opencoagulum with more pores.Pasteurized and raw milk showedb-lactoglobulin present duringgastric digestion, whileb-lactoglobulin could only beobserved in the two first gastricemptying points for UHT. UHThad a higher protein release inthe early stages of digestion.
(S�anchez-Riveraet al. 2015)
Dynamic in vitrogastric digestion
SMP Non-heated skimmedmilk prepared fromSMPHeated (90 �C,10min)skimmed milk
Protein and peptideanalysis (SDS-PAGE, LC MS/MS),immunoreactivity
Heating increased resistance topepsin hydrolysis for casein, whileb-lactoglobulin became moresusceptible to pepsin hydrolysis.Time of release of peptides wasdifferent between heatedand unheated.
(Wang and Zhao 2017) In vitro dynamicgastric digestion
WPI Whey protein isolateHeated whey proteinisolate (90 �Cfor 20min)
Curd weight, pH,protein hydrolysis(SDS-PAGE)
No aggregation in native WPI overwhole digestion, whileaggregation occurred in heatedWPI in the stomach. Forunheated WPI, b-lactoglobulinremained intact during wholedigestion and decreased graduallywith time. b-lactoglobulin forheated WPI was much less anddisappeared. Peptides werevisible in emptied digests fromheated WPI, but not for unheatedWPI, where native b-lactoglobulinremained intact. a-lactalbumin didnot show any difference betweenunheated and heated WPI.
(continued)
12 G. A. A. VAN LIESHOUT ET AL.
isolate (Wang et al. 2018). On the other hand, gastric diges-tion of heated serum protein concentrate was also found tobe delayed compared to its non-heated comparator, mostlikely by formation of whey protein aggregates and increasedparticle size in this particular setting (Stender et al. 2018)although it is likely that other reactions may have occurredsimultaneously (see section 4.3).
Three different forms of b-lactoglobulin can be formeddepending on the exact heating conditions. Dimers areformed with shorter heating times (i.e. 5minutes, 90 �C)that are mostly still undigestible by pepsin. To a certainextent, the native confirmation may still be present in thosedimers, thereby preventing accessibility by pepsin. With lon-ger heating times (i.e. 60 to 120minutes, 90 �C), other aggre-gates are formed, including non-native aggregates linked bydisulfide-bonds that are rapidly digested by pepsin. Lastly,intermediates may be formed by disulfide-bonding ofb-lactoglobulin monomers with peptides, giving a structurethat is digested slower by pepsin than its unbound form(Loveday et al. 2014; Peram et al. 2013). Overall, this sug-gests that b-lactoglobulin may not be affected in low-tem-perature pasteurization processes, but high pasteurizationand sterilization will increase its gastric hydrolysis.
Impact of heating on digestion of casein is less well studiedcompared to whey proteins. In general, hydrolysis of casein bypepsin in the stomach is high because of its open structure.However, a single study found that heating increased the resist-ance of casein to gastric hydrolysis in heated vs unheatedskimmed milk, which was attributed to the formation of aggre-gates between caseins and whey proteins (S�anchez-Rivera et al.2015). The resulting peptides formed from caseins differedbetween unheated and heated skimmed milk (S�anchez-Riveraet al. 2015). On the other hand, it has been argued that com-plexes of whey proteins with kappa-casein are better digestibleby pepsin, trypsin and chymotrypsin than when they do notform complexes (Singh and Creamer 1993).
Overall, the available evidence indicates that induction ofmilk protein denaturation by heating seems to enhance gas-tric protein hydrolysis (mainly for whey proteins). However,it cannot be ruled out that aggregate formation betweenwhey proteins themselves, between whey and casein, andpotential interactions with lipids (Rudloff and L€onnerdal
1992) can alter gastric hydrolysis. For casein, its coagulationbehavior at low pH in the stomach may alter digestive kinet-ics, as discussed in the next paragraph.
3.2.1.2. Milk matrix. Static in vitro experiments show thatheating milk at higher temperatures increased the formationof peptides within the first 30minutes of gastric digestion(Lamothe et al. 2017). This may be explained by theobservation that heat treatment of milk results in a morefragmented and crumbled coagulum under gastric condi-tions, leading to a greater rate of gastric protein hydrolysis(Mulet-Cabero et al. 2019; Ye et al. 2017). The less consist-ent coagulum as a result of heating is suggested to be causedby: 1) binding of denatured whey proteins to the caseinmicelles, thereby blocking micelle aggregation; 2) denaturedwhey proteins and j-casein forming soluble complexes,incorporating whey proteins in the coagulum, and 3) cal-cium concentration being reduced in the serum, changingthe ion equilibrium, and thereby the coagulum structure(Mulet-Cabero et al. 2019). The increased hydrolysis isconfirmed in commercial milk samples where intact wheyproteins, and b-lactoglobulin in particular, in (in-can) steri-lized or UHT-treated milk are better digested by pepsinthan in pasteurized milk (Rinaldi, Gauthier, et al. 2014;Tunick et al. 2016; Wada and L€onnerdal 2014). Caseins anda-lactalbumin were hydrolyzed independent of heattreatments in all samples (Tunick et al. 2016). In contrast,heating skimmed milk for 10 minutes at 90 �C resulted inintact caseins that were still present after up to 50 minutesof gastric digestion. This could be due to the formation ofaggregates between caseins and whey proteins (S�anchez-Rivera et al. 2015), as also discussed in section 1. The widelydiffering heat treatments amongst the studies discussed may,at least to a certain extent, explain the variation in results.
Although the full complexity of human digestion cannotbe simulated, dynamic in vitro models and animal modelsenable to give a more reliable picture of protein digestion kin-etics over time. The different kinetics are mainly caused inthe stomach, because heating may result in different gastricbehavior and emptying. While this may not influence overalldigestibility, differences in kinetics of digestion may be rele-vant for their physiological consequences (see section 7).
Table 3. Continued.
Reference Method Protein source
Study arms withdenaturation/aggregation method Outcome measure
Effect on digestibility anddigestion kinetics
(Ye et al. 2017) In vitro dynamicgastric digestion
Whole milk Unprocessed raw wholemilk HomogenizedmilkHeat-treatedhomogenized wholemilk (90 �Cfor 20min)
Curd weight, pH,protein hydrolysis
Homogenization and heat treatmentleads to more fragmented andcrumbled curds leading to greaterrates of protein hydrolysis.Emptied digests from heated milkcontained only small amounts ofintact caseins and whey, whileunheated samples showedemptying of intactb-lactoglobulin anda-lactalbumin.
AA, amino acids; IAA, indispensable amino acids; UHT, ultra-high temperature; WPI, whey protein isolate; SMP, Skimmed milk powder; GI, gastrointestinal; HTST,high temperature, short time pasteurized; HHST, higher temperature, shorter time pasteurized; TCA, trichloroacetic acid; WPC, whey protein concentrate; SPC,serum protein concentrate.
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 13
The lower consistency of the coagulum as a result ofheating influences gastric emptying of protein by increasingprotein release in the beginning of digestion (Mulet-Caberoet al. 2019). Whey proteins that were incorporated in thecoagulum of heated milk were digested together with thecasein. On the other hand, no whey proteins were incorpo-rated in the raw milk coagulum but they were present in theserum phase, resulting in emptying of intact whey proteins(Ye et al. 2017). This confirms the findings from static invitro models that non-heated whey proteins are still intactafter gastric digestion, resulting in intact whey proteins emp-tied in the intestine.
A limited number of animal studies investigated kineticsof digestion and absorption of heated milk. Barb�e et al.(2013, 2014) used cannulas shortly after the pylorus tosample duodenal effluents in minipigs. They showed thatstomach retention time was increased for heated milk com-pared to raw liquid milk. For raw milk, intact caseins onlyappeared in the duodenum in the first minutes of digestion,and were completely hydrolyzed in the following duodenaleffluents. These pattern was similar for heated milk. In con-trast, intact b-lactoglobulin was visible up to 3 h after inges-tion of the raw milk, while it disappeared 50minutes afteringestion of heated milk. This suggests that b-lactoglobulinwas susceptible to gastric digestion after heating the milk,resulting in disappearance of the intact form in duodenaleffluents of the heated milk from 50minutes onwards. Thedifferences in raw and heated milk led to different postpran-dial plasma amino acid concentrations with an increasedmaximal and average indispensable amino acid concentra-tion for heated milk (Barb�e et al. 2013, 2014). This is in linewith another study in pigs, where nitrogen was mainlyabsorbed in the first 2 h postprandial for milk and heatedyogurt, while this was more distributed over 4 h postprandialfor non-heated yogurt. It was suggested that the non-heatedyogurt retained its viscosity, while this was reduced afterheating. In turn, the milk and heated yogurt were emptiedmore rapidly from the stomach resulting in a faster post-prandial absorption, while this was more delayed for themore viscous non-heated yogurt (Mpassi et al. 2001).
Altogether, both in vitro and animal studies show thatheating milk or yogurt could alter gastric behavior andgastric emptying of the milk, thereby affecting the rate ofpost prandial plasma amino acid appearance.
3.2.2. The impact of denaturation and aggregation onintestinal protein digestibility
Heating-induced enhanced proteolysis demonstrated in agastric model does not automatically mean an increasedoverall digestibility. Therefore, including a gastric and intes-tinal phase to assess protein digestibility is of importance.Different results are found after intestinal digestion forindividual proteins and the total milk matrix.
3.2.2.1. b-lactoglobulin. Heating mainly impacts wheyprotein (predominantly b-lactoglobulin) hydrolysis in thestomach. While pancreatic enzymes are able to hydrolyzeb-lactoglobulin completely anyways, it was shown for
b-lactoglobulin that proteolysis by pancreatic enzymes wasfaster when it had been heated at different time-temperaturecombinations (Guo et al. 1995; Kitabatake and Kinekawa1998; Rahaman, Vasiljevic, and Ramchandran 2017). Thiswas shown for whey protein based gels as well (Lorieauet al. 2018; Singh et al. 2014). In contrast, Carbonaro et al.(1997) and Lindberg et al. (1998) showed that severe heatingof whey protein extracts or WPC above a critical tempera-ture (�75 �C) decreased overall digestibility of its proteins,probably due to formation of more compact whey proteinsand aggregates (Carbonaro et al. 1997; Lindberg et al. 1998;Wada and L€onnerdal 2014). However, it cannot be ruled outthat confounding factors including protein glycation andglycation-induced aggregation, as discussed in section 4.3,underlie these observed effects. A single study showedthat intestinal proteolysis was similar for native andheated serum protein concentrate (Stender et al. 2018).Interestingly, the studies that found an increased hydrolysisused isolated b-lactoglobulin, while the studies that founda decreased hydrolysis used whey protein extracts or wheyprotein concentrates and no effect was seen for serumprotein concentrates. This suggests that the production andmatrix of the product may influence the denaturation andaggregation state of the proteins, subsequently resulting ina different digestibility for the different matrices.
3.2.2.2. Casein. Intense processing of skimmed milk pow-ders could result in higher resistance of intact caseins togastrointestinal digestion. This mainly involves as2-caseinand j-casein that are able to form disulfide bridges withdenatured whey proteins. Therefore it was suggested thatwhey-casein aggregates formed during heating may result ina decreased hydrolysis of caseins, although this is in conflictwith the results of a study by Singh and Creamer (1993)(Dupont, Boutrou, et al. 2010; Singh and Creamer 1993).The interaction of caseins with denatured whey proteinsmay thus affect casein digestion, but the resulting effects ondigestion differ between studies.
3.2.2.3. Milk matrix. In a milk matrix, several in vitro studiesshow that, comparable to the gastric hydrolysis results, heat-ing of liquid milk results in (small) increases of proteinhydrolysis by pancreatic enzymes (Carbonaro et al. 1997;Rinaldi et al. 2015). On the other hand, other in vitro studiesshow a similar intestinal proteolysis between raw, pasteurizedand sterilized milk (Lamothe et al. 2017; Tunick et al. 2016).Overall, the effect of heating of liquid milk on intestinalhydrolysis thus seems small or absent.
Also, animal studies show that denaturation after heatingof different dairy matrices does not affect overall digestibil-ity. When comparing milk, yoghurt, and heated yoghurt,overall nitrogen absorption determined by portal labelednitrogen absorption was similar in growing pigs (Mpassiet al. 2001). Milk being raw, pasteurized, UHT sterilized, orboiled also did not affect protein digestibility (Efigenia,Povoa, and Moraes-Santos 1997) or biological value of milkprotein in rats (Lacroix et al. 2006). In young pigs, no effecton apparent fecal and ileal digestibility and dietary nitrogen
14 G. A. A. VAN LIESHOUT ET AL.
utilization was shown for skimmed milk powders that weredifferently heated during the liquid processing. This resultwas found despite a different flocculation behavior foundfor the powders in vitro that could have influenced gastricemptying and digestion kinetics. This indicates thatdenaturation does not affect overall digestibility but canaffect digestion kinetics (Moughan et al. 1989).
Those results are in line with a single human studythat used labeled nitrogen to compare postprandial kineticsof UHT milk with microfiltered (MF) and pasteurized milk(Lacroix et al. 2008). Overall, no significant differences wereshown for different treatments on serum amino acid con-centrations. However, a significant increase in plasma aminoacid levels from baseline was more sustained for UHT milkthan MF milk. Moreover, postprandial dietary protein reten-tion was reduced in the UHT group. Together, this suggeststhat the more rapid digestive kinetics of UHT milk resultedin a higher loss of dietary N compared to MF and pasteur-ized milk (Lacroix et al. 2008).
Altogether, most studies, including animal studies anda single human study, conclude that heating of milk proteinsin a liquid milk matrix does not affect overall protein digest-ibility and bioavailability, but it can affect digestive kineticsmainly through a different stomach behavior, that couldimpact further metabolism.
3.3. Other modifications
Glycation, denaturation, and aggregation could affect proteindigestion and quality, as described in section 3.1 and 3.2.Moreover, other modifications that have been demonstratedto occur during dairy processing, as evidenced by their pres-ence in actual dairy products, could impact protein digestionand quality (Liardon and Hurrell 1983; Meltretter et al.2007). For example, oxidation is demonstrated when isolatedwhey proteins are heated with lactose, while racemizationoccurs in heated milk powders. However, those modifica-tions seem to be less abundant than glycation (Liardon andHurrell 1983; Meltretter et al. 2007). The exact conditions,and in which stage of dairy processing these other modifica-tions actually occur, is not yet known (Figure 3).
3.3.1. OxidationAlthough less studied than Maillard-based modifications,oxidation as a result of industrial processing is anotherchemical amino acid modification that has been demon-strated to affect protein digestion and quality. To date,several studies applying different experimental oxidationstrategies have demonstrated effects of oxidation on digest-ibility and protein quality (Chang and Zhao 2012; Fenget al. 2015; Rutherfurd, Montoya, and Moughan 2014).Oxidation strategies include treatment of the proteins by anoxidative system containing oxidase-enzymes (Chang andZhao 2012), a free radical-generation system with hydrogenperoxide (Feng et al. 2015), or incubation with performicacid (Rutherfurd, Montoya, and Moughan 2014). Rutherfurdand colleagues applied a chemical strategy to oxidize several
protein sources including the milk proteins casein anda-lactalbumin to study true ileal amino acid digestibility(TIAAD) in rats. Oxidation of all protein sources resulted inan almost complete modification of methionine, tyrosine,and tryptophan. Importantly, as these amino acid are con-sidered either essential (methionine and tryptophan) or con-ditionally essential (tyrosine), care should be taken withexcluding these for further ileal digestibility calculations.Nonetheless, the authors excluded these amino acids fromfurther TIAAD calculations because they were destroyedduring the oxidation process of the proteins. For the otheramino acids, it was demonstrated that TIAAD of oxidizedcasein was significantly lowered whereas that of oxidizeda-lactalbumin was unchanged. The decreased overall TIAADfor casein, excluding the oxidized amino acids, indicates thatother amino acids are affected as well, meaning oxidationcan affect overall digestibility of casein. Other studies pro-vided more detailed information about chemical and struc-tural changes as a result of oxidation by using either a freeradical generating system and WPI (Feng et al. 2015) or aperoxidase system and sodium caseinate (Chang and Zhao2012). Despite the different approaches for oxidation of themilk proteins, both studies revealed that oxidation causedphysico-chemical and structural changes, such as loss ofthiol groups, formation of dityrosine and carbonyls,increased surface hydrophobicity, turbidity and particle size,which coincided with an overall decreased in vitro hydrolysisfor both gastric and overall gastrointestinal conditions.
3.3.2. RacemizationRacemization, the conformation of L-amino acids to theirenantiomer D-form, may affect protein quality predomin-ately as the physiological response to L-amino acids may bedifferent than to the D-forms (Friedman 2010). Specificallyfor milk proteins (both caseins and b-lactoglobulin), racemi-zation as a result of heat and alkali treatment has been dem-onstrated to decrease true ileal protein digestibility inminipigs (de Vrese et al. 2000). It is suggested that enzym-atic breakdown and absorption of peptides containing D-amino acids and free D-amino acids is affected, becausethey are not recognized by the peptidases. This may affectother amino acids in the peptides as well, resulting in adecreased ileal protein digestibility (de Vrese et al. 2000).These results are in line with previous findings where race-mization of milk proteins was induced in a more artificialmanner and digestibility values were directly related to thedegree of racemization and to the amount of lysinoalanine(Swaisgood and Catignani 1985). Thus, although studied toa lesser extent than glycation, racemization appears to beanother modification that occurs during processing ofmilk proteins that may decrease overall digestibility andprotein quality.
3.3.3. DephosphorylationMilk proteins, predominantly caseins, have a high degreeof phosphorylation which contributes to their structureand functionality (Huppertz, Fox, and Kelly 2018).
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 15
Phosphorylation has generally been considered to be sensi-tive to heat (Singh 2004) although limited studies deter-mined the impact of milk protein dephosphorylation ondigestion. Fox et al. (2004) discuss that partial dephosphory-lation of casein reduces the size of protein precipitates underacid conditions such as occurring during gastric digestion.This would suggest a faster gastric emptying, at least for thecasein fraction that would normally coagulate and form aprotein network. However, as demonstrated by scintigraphyanalyses in this study, gastric emptying of the dephosphory-lated casein appeared to be faster than the unmodifiedcasein. With respect to direct measurements of digestion,Wada and L€onnerdal (2014) determined that, out of severaltested industrial heating procedures for raw milk, in-cansterilization resulted in the most prominent casein dephos-phorylation in addition to increases in other protein modifi-cations including denaturation, aggregation andlactulosyllysine and carboxymethyllysine formation.Although not directly linked to dephosphorylation, andlikely predominantly an effect of denaturation, these heat-ing-induced modifications increased the degree of digestionas assessed by hydrolysis of major milk proteins by in vitroand in vivo digestion experiments.
Similar results were obtained with model enteral formulasby the same group where in-can sterilization resulted inhigher protein modifications including carboxymethyllysineformation and dephosphorylation of serine residues in themajor milk proteins (Wada and L€onnerdal 2015). In thismore complex matrix, in vitro and in vivo experiments inrats show that digestive differences of model enteral formu-las were larger.
In conclusion, although mostly coinciding with othermodifications, available literature suggests that dephosphory-lation of milk proteins may impact digestibility. Morestudies are however required to obtain a more completeunderstanding of dephosphorylation, preferably adoptingmodification strategies that are able to separate dephosphor-ylation from other processing-induced modifications.
3.3.4. Cross-linkingHeating of dairy proteins could also result in the formationof cross-links between different amino acids within a pro-tein. A well-known example is lysinoalanine (LAL) that canbe formed during heating of proteins. Cross-linking couldaffect digestibility of a protein by changing enzyme accessi-bility. However, an in vitro infant digestion of infant for-mula model systems suggested that different protein cross-linking as assessed by LAL content did not change proteinhydrolysis (Cattaneo et al. 2017). In another study, in vitrodigestion of different infant formulas showed no direct cor-relation between digestibility and LAL content, lysine block-age, or HMF content (Pompei, Rossi, and Mare 1988).Moreover, an animal study in minipigs showed that truedigestibility of casein was not related to LAL content, but itwas still suggested that small amounts of LAL could impairdigestion by interfering with enzymatic cleavage (de Vreseet al. 2000). In line with that, LAL formation in a-lactalbu-min resulted in a reduced protein digestibility and protein
quality in rats (Gilani and Sepehr 2003). Thus, availablestudies indicate that cross-linking may or may not affectdigestibility of proteins, thereby showing a need for fur-ther research.
3.4. Physiological consequences
The effects of dairy processing, as described in section 1 ofthis review, can affect milk proteins in many different ways(Figure 3). This can cause changes in their digestibility ordigestion kinetics as detailed in sections 3.1–3.3. In this sec-tion, the possible physiological consequences of these differ-ences in digestion are discussed.
3.4.1. Protein digestion, bioavailability and metabolicutilization
To a certain extent, heating of milk proteins may increasethe speed of digestion as a result of heat induced proteindenaturation and better protease accessibility (predominantlydemonstrated for b-lactoglobulin). However, the discussednon-enzymatic modifications that occur simultaneously willmodify specific amino acids and decrease bioavailability andfunctionality of these amino acids. Furthermore, blocking ofspecific amino acids may hinder further proteolytic activitythereby impairing overall protein digestibility. In addition,cross-linking can occur with formation of novel structuresthat may further inhibit enzymatic hydrolysis and overalldigestibility. As a consequence, non-enzymatic proteinmodification may be considered indicative for a reducedprotein quality as demonstrated by direct comparisons oftheir non-modified forms as discussed in sections 3.1and 3.3.
Intestinal epithelial cells contribute to degradation ofdietary proteins in different ways. Brush border membraneproteases represent the last step of luminal protein digestion,hydrolyzing proteins/peptides in an absorbable form.Furthermore, during transport across the intestinal epithe-lium, enterocytes contribute to further hydrolysis ofabsorbed di- and tri-peptides intracellularly. Epithelial deg-radation and transport of heating induced modified b-lacto-globulin may be affected, as demonstrated by in vitro studiesusing different intestinal epithelial cell-models (Bernasconi,Fritsch�e, and Corth�esy 2006; Rytk€onen et al. 2006).
The fate of modified amino acids remains largely unex-plored to date as most studies investigating digestion ofmodified and non-modified proteins only discuss decreasedbioavailability. Decreased digestibility and absorption ofdietary proteins may increase protein fermentation in thelower gastrointestinal tract with increased production ofmicrobial protein fermentation products, includingbranched-chain fatty acids, ammonia, phenol, p-cresol,indole and hydrogen sulfide (Nyangale, Mottram, andGibson 2012), which may be unfavorable from several per-spectives. In contrast to fecal carbohydrate fermentation,microbiome protein fermentation is less extensivelyresearched and the toxic potential of these compounds ispredominantly derived from in vitro cell-toxicity and animal
16 G. A. A. VAN LIESHOUT ET AL.
studies determining effects of purified individual protein fer-mentation by-products (Verbeke et al. 2015). Minimally irri-tating concentrations were assessed as a marker of localeffects and exceeded 1% for all compounds, which is wellabove the concentrations occurring in vivo in the colon.Moreover, in human studies there is little evidence foradverse effects of protein fermentation metabolites. Furtherstudies are therefore required and especially more chronicexposure of combinations of modified amino acids/digestionproducts and effects on the longer term remain tobe elucidated.
To a certain extent, the microbiome also appears to beequipped to metabolize modified lysine, which may be oneof the explanations why fecal extraction of glycated lysinewas rather low in rat, pig, and human studies, typicallyaround 4% of the ingested amount (Lee and Erbersdobler2005; R�erat et al. 2002). Interestingly, one particular micro-bial specie, Intestinimonas, is able to convert lysine andAmadori products thereof into butyrate and acetate, whichare considered important microbiome derived signaling mol-ecules in the gut (Bui et al. 2015).
Post absorption, nitrogen metabolism may be affected byprocessing-induced protein modifications and result in alarger splanchnic extraction, as demonstrated in rats fed dif-ferentially processed milk protein preparations (Lacroixet al. 2006). Interestingly, in this study lactosylation waslikely the most important driver for increased splanchnicextraction as spray dried soluble milk proteins did notdenature or aggregate (as illustrated by preserved solubilityat pH 4.6) but did show the most prominent effect onsplanchnic extraction. In a study in humans, Lacroix et al.,(2008) showed that UHT milk gives a more rapid appear-ance of amino acids in plasma compared to MF milk, ultim-ately resulting in a lower postprandial nitrogen retention.This suggests that protein digestive kinetics can influencemetabolic utilization of the ingested protein. Additionalwork by R�erat et al. (2002) in pigs reveals that loss of nutri-tional quality as a result of additional heating of milk pow-ders is mainly driven by loss of lysine bioavailability and toa lesser extent by the decrease in digestibility of other essen-tial amino acids. Nonetheless, fecal excretion of almost allamino acids was higher in milk with 50% blocked lysine.Interestingly, in this study fructoselysine was detected post-prandially suggesting that, at least to a certain extent, thegalactose residue of lactuloselysine is released in the gutlumen or brush border. Moreover, urine levels of fructosely-sine recorded over 72 h revealed that urinary excretion ofglycated lysine is around 20% of the ingested amount. Inhumans, urinary excretion of protein-bound fructoselysineranged from 1.4 to 3.5% of the amount ingested as deter-mined using test meals enhanced with fructoselysine (Leeand Erbersdobler 2005).
For advanced glycation end-products (AGE), most avail-able data reflect the health impact of endogenously formedAGE, whereas data on the impact of dietary AGE is limited.Basically, AGE are known to induce effects in the body bytwo distinct mechanisms, including deformation or cross-linking of AGE with endogenous proteins and interactions
with AGE receptors (Poulsen et al. 2013). For AGE recep-tors, studies related to milk proteins are available (discussedin section 3.4.3), whereas deformation and cross-linking ofAGE with endogenous proteins has almost exclusively beenrelated to endogenous formation of AGE, while effects ofdietary AGE are still largely unknown.
During the digestion of milk protein, bioactive peptidesare formed that may induce physiological responses pre-dominantly along the GI tract (Nongonierma and FitzGerald2015). Well known examples in this area are casein-derivedsequences termed casomorphins. Cattaneo et al. (2017)demonstrate that processing may impact the formation ofb-casomorphins. Thus, besides differences in amino acidbioavailability, these result highlight potential additionalmechanisms of action how intense processing of milk pro-teins may affect their overall physiological activity.
Taken together it thus appears that processing-inducedprotein and amino acid modifications may affect digestion,absorption, and metabolism of protein at all levels, althoughparticularly about the fate of modified amino acids post-digestion more data is needed.
3.4.2. Gastrointestinal physiologyBeyond an altered digestion and reduced bioavailability, pro-tein modifications may have additional physiological conse-quences. Proliferation and differentiation responses to foodintake may be changed. Amongst others, this is evident fromin vivo studies on the effect of differently treated milk proteinpreparations in preterm pigs. Immaturity of the gut predis-poses these animals to gut complications and milk proteinpreparations (particular sow milk or minimally processedbovine milk protein preparations and colostrum) have beendemonstrated to reduce the risk for such complications (Li,Ostergaard, et al. 2013; Li et al. 2018; Støy et al. 2016).Preterm pigs fed formula containing differentially processedwhey protein concentrates (WPCs) displayed differences inintestinal structure, function, and integrity as determined bystudying gut maturation events including villus height, disac-charide digestion/absorption, gut permeability, and intestinalinflammatory responses (Li et al. 2018). It was concluded thatoptimization of processing technology may be important topreserve the bioactivity and nutritional value of formula fornewborns. Particular for colostrum (generally containing highlevels of bioactive proteins), pasteurization and spray dryingdecreased the levels of bioactive proteins, including trans-forming growth factor b1 and b2, and increased proteinaggregation (Støy et al. 2016). Although differences wereobserved, all tested preparations still showed trophic and anti-inflammatory activity effects on the intestine in the pretermpig model. Further in vitro work by the same group demon-strates that, at least in part, these observations could beexplained by direct effects of the preparations on intestinalepithelial cells (Nguyen et al. 2016). Likely, this mostly relatesto inactivation of bioactive whey proteins, although it cannotbe excluded that processing-induced protein and amino acidmodifications contribute to these observed effects.
For casein, similar results were obtained (Wang andZhao 2017). In an in vitro setting, Wang et al. demonstrated
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 17
that digests of glycated casein, relative to digests of non-modified casein, show altered proliferative, differentiation,and anti-apoptotic effects on epithelial cells. These resultssuggest that Maillard-based modifications of casein couldhave a direct effect on intestinal cells through mechanismslikely involving casein peptides.
3.4.3. ImmunologyFood processing can alter the allergenic properties of pro-teins by hiding, destroying, or exposing allergic epitopesthrough structural changes. Milk proteins are among thebest studied proteins in this area and several studies haveassessed their antibody reactivity and allergic potential, afterprocessing, in different models for allergy. As most studiesin this area apply different antibodies and different modifi-cation strategies, direct comparison of individual studies is,however, challenging. Generally, for clinical relevance andconclusions around allergenicity, patient-derived sera (singleor pooled) is preferred, whereas animal derived sera shouldonly be employed for characterization of structure-immunereactivity interactions.
Sletten et al. (2008) studied casein and b-lactoglobulinreactivity after processing and simulated digestion usingpolyclonal antibodies and milk-allergic patient sera. In thisstudy, UHT treatment resulted in increased stability of IgEand IgG epitopes to simulated gastric digestion but had noeffect on specific IgE and IgG binding. Specifically, forb-lactoglobulin, digestive stability decreased althoughimmunogenicity was retained after simulated gastric diges-tion in both treated and non-treated milk.
In a similar experimental design employing polyclonalantibodies, Li et al. revealed that glycation of whey proteinisolate with oligoisomaltose reduced b-lactoglobulin anda-lactalbumin reactivity after simulated gastric digestion (Li,Luo, et al. 2013). These results are more or less in line withresults using human derived basophils and sera from cowsmilk allergic patients (Morisawa et al. 2009). This studydemonstrated that heat treatment reduced the allergenicityof b-lactoglobulin by inducing conformational changeswhich increased its susceptibility to enzymatic digestionwhich disrupted B-cell epitopes.
Whey protein aggregates can be formed by heating ofwhey proteins as discussed in detail in section 3.2, whichmay affect their immunogenicity. Amongst others, this isevident from a study by Delorenzi et al. (2011) who pre-pared different aggregates through heating and speculatedthat structural changes may reduce overall allergenicity ofb-lactoglobulin, either through changes in proteolytic acces-sibility and/or shielding of major epitopes within the aggre-gate structure.
In another study, the impact of typical variations inindustrial processing conditions were studied on as1-, as2-,b- and j-casein binding by monoclonal antibodies (Dupont,Boutrou, et al. 2010). Heat-treatment of milk prior to spray-drying was shown to increase residual casein immunoreac-tivity after digestion, likely as a result of decreased digestionof casein during simulated gastrointestinal digestion and, asa consequence, increased survival of antibody epitopes,
which is in line with another study from the same group(Dupont, Mandalari, et al. 2010). Interestingly, higher heattreatment regimes in this study also led to the formation ofaggregates which may be an important factor to considerregarding uptake of milk proteins by Peyer’s patches andimmunological responses including allergic sensitization.
This has been studied in detail in vivo in two elaboratestudies employing purified milk proteins and a combinationof in vitro and in vivo experiments (Roth-Walter et al. 2008;Stojadinovic et al. 2014). The study by Roth-Walter et al.(2008) aimed at unraveling the mechanisms underlying milkprotein sensitization and the effect of processing, whichidentified that uptake of aggregated milk proteins thoughPeyer’s patches may be one of the underlying mechanismsof milk protein sensitization. Typical pasteurization proc-esses caused aggregation of b-lactoglobulin and a-lactalbu-min, inhibiting their uptake by intestinal epithelial cells bothin vitro and in vivo. Interestingly, in mice uptake of theseaggregates appeared to be redirected to Peyer’s patches andpromoted a higher Th2-associated antibody and cytokineresponse relative to their non-pasteurized counterparts. Inmilk protein sensitized mice, only non-aggregated solublewhey proteins elicited an anaphylactic response whenadministrated orally, where casein and whey protein aggre-gates required a systemic administration to induce anaphyl-axis. Overall, the authors concluded that allergydevelopment may be affected by processing both pre- andpost-sensitization: Initial sensitization by aggregated proteinsthrough uptake of modified protein (aggregates) by Peyer’spatches and eventual uptake of soluble proteins/fragmentswhich induce anaphylaxis. These results are in line withmore recent findings comparing crosslinked and nativeb-lactoglobulin where crosslinking of b-lactoglobulinincreased its sensitizing capacity through increased samplingof b-lactoglobulin through Peyer’s patches (Stojadinovicet al. 2014). Interestingly, in a similar experimental design,non-native b-lactoglobulin was also demonstrated to cause amore intense local immunologic response in the intestinalmucosa than its native form (Rytk€onen et al. 2002), whichcould, speculatively, cause additional local inflammation-associated complications.
In conclusion, processing induced milk protein modifica-tion may thus alter their allergic potential through differentprocesses with relevance for both sensitization and subse-quent allergic response.
Further, modified milk proteins may affect the immunesystem through advanced-glycation end-products. In an invitro setting, Deo et al. (2009) demonstrated that digesteddietary AGE, as prepared by dry-heating casein and glucose,complexed with serum albumin, may regulate expression ofRAGE (receptor for AGE) and downstream inflamma-tory pathways.
Poulsen et al. (2016) compared the effect of diets contain-ing milk protein of which different proportions were inten-sively heated (70 �C for 7 days). Increased intake ofintensively heated milk protein was associated with anincreased expression of the AGE receptors RAGE andAGER1 in whole blood (predominantly when the protein
18 G. A. A. VAN LIESHOUT ET AL.
source was in the form of a milk protein hydrolysate). Thegroup where RAGE and AGER1 was increased also had thehighest urinary excretion of the AGE methylglyoxal-derivedhydroimidazolone, which is associated with age-related dis-eases (Maessen, Stehouwer, and Schalkwijk 2015). In sum-mary, although it is clear that milk protein modification(predominantly glycation) can affect advanced glycationend-product receptor signaling, the overall physiologicalconsequences remain to be identified.
3.4.4. Antioxidant activityMilk proteins may exert antioxidant activity, either as intactproteins or as digest (Corrochano et al. 2018; Nongoniermaand FitzGerald 2015). Most studies in this area employ invitro assays and/or direct cell exposure of proteins and pep-tides. The physiological relevance of these in vitro assays,however, remains to be elucidated. Although some in vivostudies are available that assess the relevance of milk anti-oxidant activity (Fardet and Rock 2018), there is a need forcontrolled human intervention studies in this area to revealthe overall impact on health. Processing, and in particularthe Maillard reaction, may increase the antioxidant and rad-ical scavenging activity of milk protein and its digest, whichis evident from several studies that looked at the antioxidantcapacity of milk products before and after processing (Guet al. 2010; Sanlidere Aloǧlu 2013). Although the increase inantioxidant capacity may possibly be beneficial, other effectsof the Maillard reaction as discussed in this review may beless favorable.
3.4.5. Calcium homeostasisMilk products and proteins are important dietary contribu-tors to human mineral homeostasis by acting as a source ofminerals and regulating their absorption (van den Heuveland Steijns 2018). Milk protein Maillard reaction productsmay reduce mineral solubility and bioavailability duringgastrointestinal digestion which has been discussed for sev-eral food preparations and model Maillard reaction prod-ucts, both in vitro and in vivo (Mes�ıas, Seiquer, and Navarro2009; Sarri�a, L�opez-Fandi~no, and Vaquero 2001).Specifically, for milk and milk proteins, several studies areavailable. In vitro experiments using heated casein-glucose-fructose solutions revealed that calcium solubility was low-ered which resulted in a decreased in vitro absorption of cal-cium (Seiquer et al. 2001). However, in vivo absorptionappeared to be unaffected, although urinary calcium excre-tion was increased compared to non-heated controls. In amore recent study from the same authors, UHT milk wascompared with overheated milk (3 severe sterilization heat-ings, 16minutes at 116 �C) both in vitro and in vivo(Seiquer et al. 2010). Relative to UHT treatment, severeheating resulted in a reduced calcium solubility during invitro digestion, which in turn resulted in a decreased cal-cium absorption and retention in rats.
Overall, further long-term and human intervention stud-ies are needed to investigate the overall effect of processing-modified milk proteins on mineral homeostasis.
4. Conclusion and future perspectives
This systematic review demonstrates that dairy processingsignificantly affects protein quality and can be used as a toolto steer gastrointestinal protein digestion. Glycation as aresult of heat processing decreases protein digestibility andamino acid availability, leading to a decrease in proteinquality. Oxidation, racemization, dephosphorylation, andcross-linking are less well studied chemical modificationsthat may impact protein quality as well as digestibility. Incontrast, protein denaturation does not affect overall digest-ibility, despite that gastric hydrolysis of whey proteins (pre-dominantly b-lactoglobulin) is increased when heated.Nonetheless, protein denaturation can be used as a tool toalter gastric emptying of the protein, eventually resulting ina different post-prandial plasma amino acid appearance,although the matrix will play a role as well. Physiologicaleffects for a change in digestibility or digestion kineticsmainly point towards amino acid bioavailability andimmunological consequences.
Although the effects of processing-induced milk proteinmodifications have been extensively studied, several aspectsremain to be elucidated in future research.
4.1. Human studies
Although many studies highlight the negative impact onprotein digestion and overall protein quality, there is a needfor more human studies to identify overall physiologicalrelevance of digestive differences as a result of processing-induced protein modifications. In particular, the metabolicfate and longer-term impact of modified amino acids is, to alarge extent, still unclear.
For overall relevance to the food industry, it is preferredto adopt commercially relevant processes and products. Thelatter as interactions between different food componentsbeyond protein and reducing sugars are likely to affect theoverall outcome, including level of modification and overallphysiological effects.
4.2. Stages of Maillard reaction and other modifications
As highlighted by a number of studies discussed in thisreview, the impact of early and late stage Maillard reactionproducts on digestion and overall protein quality may differ.Moreover, it is evident that small changes in heating condi-tions may have a large impact. From an industrial perspec-tive, this may be an important area of research as theextreme heating conditions tested in several previous studiesare typically not applied in dairy processing. Especially ofrelevance for the more intense processing conditions,Maillard-based reactions may be coinciding with otherchemical changes as discussed in this review (Figure 3). Inthis respect, there is thus a need to assess effects of the dif-ferent stages of the Maillard reaction separately, and in con-junction with other modifications, both from an analyticaland functional perspective, as in most studies to date this ismostly not clear.
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 19
4.3. Glycosylation stability
Glycosylation of milk proteins (predominantly lactoferrin,immunoglobulins, j-casein, and proteins of the milk fatglobule membrane) contributes to overall milk proteinstructure and functionality (O’Riordan et al. 2014). Inaddition to being a source of glycans, glycosylation ofindividual milk proteins has been associated with antipa-thogenic, prebiotic (mainly bifidogenic), immunomodula-tory and satiety effects, mostly by studies that studyglycosylation-depleted proteins. That milk protein proc-essing can affect protein glycosylation, and thus its diges-tion and potentially associated functionalities, is evidentfrom several studies which investigated whey and glyco-macropeptide glycosylation patterns (Ferron-Baumy et al.1992; Floris et al. 2010; Taylor and Woonton 2009).Variation in heat treatments of milk demonstrated thatthis influenced the level of glycosylation of (cheese-derived) glycomacropeptide and whey protein. Thus, inaddition to direct chemical and structural modification ofamino acids, processing of milk may affect protein glyco-sylation stability and as a results the overall structure andactivity of individual milk proteins. However, effects ofmilk protein deglycosylation on their digestion and over-all functionality remain to be identified, which could thusbe another angle for future research in the field of proc-essing-induced protein modifications.
4.4. In vitro digestion
For in vitro digestion analyses, a harmonized approachwould be recommended as, to date, in vitro digestion proce-dures in the area of milk protein research and processing-induced modifications are not identical. Differences include,for example, proteases and protease concentrations, digestivepH, protein/enzyme ratio’s, incubation time, etc. which to alarge extent will affect overall outcomes. In this respect, it isrecommended to adopt a straightforward harmonizedapproach, as e.g. recently discussed by the INFOGEST net-work (Brodkorb et al. 2019).
Although progress has been made to establish a morequantitative relation between glycation and digestion (Denget al. 2017), a harmonized in vitro digestion procedurewould also allow to assess better the quantitative relationbetween processing-induced protein modifications anddigestion among the different studies and tested processingconditions. Ultimately, quantitative assessments of milk pro-tein modifications and digestion will enable industry to fur-ther develop processes that will have a minimum impact onoverall milk protein modification.
With respect to translatability to overall physiological rele-vance of processing-induced modifications, more complex mod-els may be required. Particularly for studies aimed at unravelingthe fate of modified amino acids and studies employing epithe-lial/immune cells, brush border membrane proteases shouldideally be included in the in vitro digestion procedure.
4.5. Prebiotic activity
The dairy industry is mainly aimed at preventing process-ing-induced protein modifications. Nonetheless, several invitro studies highlight the potential of Maillard based conju-gation of milk proteins and carbohydrates as novel foodingredients with a possible beneficial prebiotic character(Corzo-Mart�ınez et al. 2012; Hernandez-Hernandez et al.2011; Luz Sanz et al. 2007). Corzo-Mart�ınez et al. (2012)specifically focused on the growth of bifidobacterial and lac-tic acid bacteria and demonstrated that both the carbohy-drate and protein source in the conjugates matter, withstrain-specific effects. Hernandez-Hernandez et al. (2011)demonstrated that b-lactoglobulin conjugated with GOSshowed similar fecal fermentation effects as purified GOS.However, from the work of Sanz and colleagues it is alsoevident that GOS conjugation to milk proteins coincideswith a reduced digestibility. Taken together, the overall rele-vance of these postulated novel prebiotics thus need furtherevaluation (Luz Sanz et al. 2007).
ORCID
Kasper A. Hettinga http://orcid.org/0000-0002-9017-4447
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