Thermal processing implications on the digestibility of meat, fish and seafood proteins

Abstract

Thermal processing is an inevitable part of the processing and preparation of meat and meat products for human consumption. However, thermal processing techniques, both commercial and domestic, induce modifications in muscle proteins which can have implications for their digestibility. The nutritive value of muscle proteins is closely related to their digestibility in the gastrointestinal tract and is determined by the end products that it presents in the assimilable form (amino acids and small peptides) for the absorption. The present review examines how different thermal processing techniques, such as sous‐vide, microwave, stewing, roasting, boiling, frying, grilling, and steam cooking, affect the digestibility of muscle proteins in the gastrointestinal tract. By altering the functional and structural properties of muscle proteins, thermal processing has the potential to influence the digestibility negatively or positively, depending on the processing conditions. Thermal processes such as sous‐vide can induce favourable changes, such as partial unfolding or exposure of cleavage sites, in muscle proteins and improve their digestibility whereas processes such as stewing and roasting can induce unfavourable changes, such as protein aggregation, severe oxidation, cross linking or increased disulfide (S‐S) content and decrease the susceptibility of proteins during gastrointestinal digestion. The review examines how the underlying mechanisms of different processing conditions can be translated into higher or lower protein digestibility in detail. This review expands the current understanding of muscle protein digestion and generates knowledge that will be indispensable for optimizing the digestibility of thermally processed muscle foods for maximum nutritional benefits and optimal meal planning.

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Received:  February  Revised:  June  Accepted:  June 
DOI: ./-.
COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY
Thermal processing implications on the digestibility of
meat, fish and seafood proteins
Zuhaib F. Bhat1James D. Morton2Alaa El-Din A. Bekhit3Sunil Kumar1
Hina F. Bhat4
Division of Livestock Products
Technology, SKUAST of Jammu, India
Department of Wine Food and
Molecular Biosciences, Faculty of
Agriculture and Life Sciences, Lincoln
University, Christchurch, Lincoln, New
Zealand
Department of Food Sciences, University
of Otago, Dunedin, New Zealand
Division of Biotechnology, SKUAST of
Kashmir, India
Correspondence
James D. Morton, Department of Wine
Food and Molecular Biosciences, Faculty
of Agriculture and Life Sciences, Lincoln
University,Lincoln , Christchurch,
New Zealand.
Email: James.Morton@lincoln.ac.nz
Abstract
Thermal processing is an inevitable part of the processing and preparation of
Q1
Q2
meat and meat products for human consumption. However, thermal process-
ing techniques, both commercial and domestic, induce modifications in muscle
proteins which can have implications for their digestibility. The nutritive value
of muscle proteins is closely related to their digestibility in the gastrointesti-
nal tract and is determined by the end products that it presents in the assim-
ilable form (amino acids and small peptides) for the absorption. The present
review examines how different thermal processing techniques, such as sous-vide,
microwave, stewing, roasting, boiling, frying, grilling, and steam cooking, affect
the digestibility of muscle proteins in the gastrointestinal tract. By altering the
functional and structural properties of muscle proteins, thermal processing has
the potential to influence the digestibility negatively or positively, depending
on the processing conditions. Thermal processes such as sous-vide can induce
favourable changes, such as partial unfolding or exposure of cleavage sites, in
muscle proteins and improve their digestibility whereas processes such as stew-
ing and roasting can induce unfavourable changes, such as protein aggregation,
severe oxidation, cross linking or increased disulfide (S-S) content and decrease
the susceptibility of proteins during gastrointestinal digestion. The review exam-
ines how the underlying mechanisms of different processing conditions can be
translated into higher or lower protein digestibility in detail. This review expands
the current understanding of muscle protein digestion and generates knowledge
that will be indispensable for optimizing the digestibility of thermally processed
muscle foods for maximum nutritional benefits and optimal meal planning.
KEYWORDS
boiling, frying, gastrointestinal digestion, grilling, microwave, muscle proteins, protein
digestibility, roasting, steam cooking, stewing, sous-vide, thermal processing
Compr Rev Food Sci Food Saf. ;–. ©  Institute of Food Technologists R
1wileyonlinelibrary.com/journal/crf

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2T P  D
1 INTRODUCTION
Cooking is an integral part of processing and prepara-
tion of meat and meat products meant for human con-
sumption and has a significant impact on the quality and
sensory characteristics, such as texture and flavour, of
the finished products (Gómez, Janardhanan, Ibañez, &
Beriain, ). Thermal processing of the meat and meat
products improves microbial safety by inactivating food-
borne pathogens and spoilage microorganisms (Pathare
& Roskilly, ). Thermal processing is a critical step to
ensure food safety and public health and is achieved by
cooking meat and meat products to a specific core tem-
perature. The cooking temperature-time regime used to
render meat and meat products safe for human consump-
tion depends on several factors such as cooking method
employed, legislation of a country and the product to be
cooked. For example, the United States Department of
Agriculture (USDA) and United States Food and Drug
Administration (US FDA) recommends that steaks, roasts
and chops (fresh beef, veal and lamb) to be cooked to a
minimum core temperature of ◦C (with a  min rest
time), fresh pork and fresh ham to ◦C (with a  min rest
time), fish to ◦C, ground meat (beef, pork, veal, lamb) to
◦C, ground turkey and chicken to ◦C, chicken breasts
to .◦C and whole chicken to ◦C(USFDA,;
King & Whyte, ). Likewise, the Canadian govern-
ment recommends cooking ground meats (burgers, meat-
balls and sausages of beef, veal, lamb or pork) to a mini-
mum core temperature of ◦C, poultry products [burgers,
meatballs, sausages, frozen raw breaded chicken products
(nuggets, fingers, strips, burgers), pieces (wings, breasts,
legs, thighs)] to ◦C, whole chicken to ◦C, fish to ◦C,
shellfish (shrimp, lobster, crab, scallops, clams, mussels or
oysters), hot dogs and game meat to ◦C (Government of
Canada, ). The European Food Information Council
(EUFIC, ) recommends cooking fresh pork, poultry,
and ground products, such as burgers, sausages, or fish-
cakes, to a minimum core temperature of ◦C for at least
 min. The UK Food Standards Agency recommends cook-
ing meat and meat products to a core temperature of ◦C
for at least  min or similar effective time-temperature
regimes (◦Cformin,
◦C for  s or ◦Cfors)(UK
Food Standards Agency, ).
In addition to sensory quality and microbial safety, ther-
mal processing can alter the digestibility and bioavail-
ability of nutrients in the meat during gastrointestinal
digestion. These effects depend on the peak tempera-
ture attained. Higher processing temperatures can induce
unfavourable structural changes in meat proteins (Kaur,
Maudens, Haisman, Boland, & Singh, ). Processing
meat at temperatures above ◦C can cause modifications
such as formation of intermolecular aggregates and cross-
links in proteins which reduce their susceptibility to diges-
tive enzymes. This decreases the digestibility and subse-
quent release of peptides and amino acids (Kaur et al., ;
Santé-Lhoutellier, Astruc, Marinova, Greve, & Gatellier,
; Morzel, Gatellier, Sayd, Renerre, & Laville, ).
The heating rate and the final temperature are probably the
most important factors in determining the rate and extent
of protein denaturation (Qian et al., ) and aggregation.
Cooking also promotes oxidation of the muscle proteins
and extensive oxidation is considered as the primary cause
of reduced protein digestibility by increasing the resistance
of proteins to digestive enzymes (Du et al., a, b;Shan-
lin, Stocker, & Davies, ). Oxidation of muscle proteins
is accelerated by heat and the presence of transition met-
als (Estévez & Xiong, ; Luna & Estévez, )andcan
be mediated through formation of disulfide bonds between
nearby polypeptides or via reactive oxygen species (Mar-
tinaud et al., ). Heating at high temperature (≥◦C)
has been shown to induce intense protein oxidation and
severe aggregation, thereby reducing the digestibility of
muscle proteins (Zhao et al., ). Higher cooking temper-
atures can also induce modifications at amino acids level
such as formation of amide bonds, disulphide linkages, or
dityrosine bridges. These reduce the protein’s susceptibil-
ity to gastrointestinal enzymes and can slow the release
of amino acids during digestion (Bhat, Morton, Mason,
Jayawardena, & Bekhit, ; Bhat, Kumar, & Bhat, ;
Kaur et al., ). Depending on the amino acid residues
involved, the mechanisms of oxidation could favour the
formation of different types of bonds, such as S-containing
amino acids methionine and cysteine favour the formation
of disulfide cross-links, tyrosine favours specific dityrosine
cross links and arginine, lysine, and proline favour forma-
tion of carbonyls on their side chains (Estévez & Xiong,
; Estévez & Luna, ).
Texture of meat is a complex trait that is influenced by
several factors including animal, environmental and man-
agemental factors, post-mortem treatments, and process-
ing (Bhat, ; Bhat Morton, Mason, & Bekhit, e;
Kumar et al., ). The texture of meat is highly affected
by heating temperature and cooking time. Meat cooked at
high temperatures such as ◦Cor
◦C is harder than
meat cooked at lower temperatures such as ◦Cor
◦C
(Palka, ) due to the excessive denaturation of myofib-
rillar proteins and connective tissues at temperature over
◦C. Thermal processing of meat at higher temperatures
causes both transverse and longitudinal shrinkage of mus-
cle fibres and induces coagulation of some meat proteins
that makes the muscle tissue denser and increase its hard-
ness. This cooking induced contraction of muscle fibres
and intramuscular connective tissue results in the cooking
losses, the intensity of which depends on the cooking tem-
perature (Ježek, Kameník, Macharáčková, Bogdanovičová,

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T P  D 3
& Bednář, ). Connective tissues tend to gelatinize at
◦C and form a gel that fills the spaces in between mus-
cle fibres (Roldan, Antequera, Martín, Mayoral, & Ruiz,
). These cooking-induced changes increase almost lin-
early with temperature up to ◦C (Baldwin, )and
can reduce the digestibility of meat proteins by reduc-
ing the diffusion of digestive enzymes into the compact
myofibrillar structure (Kaur et al., ). Thermal process-
ing of meat has been reported to reduce the susceptibility
of myofibrillar proteins to gastrointestinal enzymes under
in vitro gastric and duodenal simulated digestion (Santé-
Lhoutellier et al., ).
Giving the fact that digestive enzymes (such as pepsin
and trypsin) act more effectively on unfolded and dena-
tured proteins (Gong et al., ;Kauretal.,),
processing of meat at lower temperatures can induce
favourable changes at protein level and enhance the
digestibility of meat proteins. Denaturation causes the
loss of thermodynamic stability [quantitatively defined
by the Gibbs free-energy change upon unfolding (Luke,
Higgins, & Stafshede, )] and the unfolding of proteins
(Dominguez-Hernandeza, Salaseviciene, & Ertbjerg, )
that can increase the exposure of hydrolytic sites to diges-
tive proteases. Mild oxidation and partial denaturation
of the muscle proteins have been shown to increase the
digestibility of muscle proteins by enhancing their sus-
ceptibility to digestive proteases (Du et al., b;Shanlin
et al., ). Myofibrillar proteins, which provide structure
to the muscles, begin to denature at different temperatures
(Yu, Morton, Clerens,& Dyer, ). While the myofibrillar
proteins from beef and lamb exhibited denaturation peaks
of –◦C (Kemp, North, & Leath, ), the myosin
and α-actinin begin to denature from –◦C(Wright
&Wilding,; Cheng & Parrish, ), titin from ◦C
(Pospiech et al., ) and actin from ◦C (Wright et al.,
). SDS-PAGE analysis showed denaturation of myosin
and actin in the range of –◦C (Hassoun, Aït-Kaddour,
Sahar, & Cozzolino, ). No distinctive denaturation
peaks were exhibited by the sarcoplasmic proteins of beef,
lamb, or pork (Kemp et al., ), however, the maximum
heat capacity values suggested that denaturation occurred
between  and ◦C. Connective tissue proteins, such as
collagens, denature at comparatively higher temperatures
and begin to shrink at –◦C (Schmidt, )andgela-
tinize at a temperature higher than ◦C (Bejerholm et al.,
). Gelatinization of collagen involves disintegration
of the helical structures into random coils and forms
a water soluble gelatin, although, its formation is very
limited below ◦C (Lawrie & Ledward, ). Most
skeletal muscle proteins lose their native structure above
◦C (Yu et al., ). Further, fibrous proteins contract
on heating whereas globular proteins expand on heating
(Tornberg, ). Denaturation of meat proteins is a
combination of time and temperature effects; therefore,
proteins can denature even below standard temperatures
when heated for longer holding periods (Dominguez-
Hernandeza et al., ; Zielbauer, Franz, Viezens, &
Vilgis, ). Compared to conventional heating methods,
novel methods such as microwave and ohmic heating have
been reported to induce less denaturation of meat proteins
when higher heating rates are used (Hassoun et al., ;
Li, Tang, Yan, & Li, ; Roberts, & Lawrie, ).
2MEAT PROCESSING AND MUSCLE
PROTEIN DIGESTIBILITY
Meat processing involves turning the raw meat into con-
sumable products (Bhat et al., a) and varies from the
simple processes of mincing, ageing, or packaging of meat
(Sharma et al., a,b; Kalem et al., a,b,c,;
Noor et al., a,b,;Bhatetal.,a;Singhetal.
a,b;Dilnawazetal.,a,b) to the complex produc-
tion of cultured meat products (Bhat, Bhat, & Kumar, ;
Bhat, Morton, Mason, Bekhit, & Bhat, ; Bhat, Bhat, &
Pathak, ; Bhat & Bhat, ; Pathak et al., ). Pro-
cessing fulfils some marketing and industrial needs such as
improving shelf-life or storage quality (Bhat et al., d,
; Bekhit et al., ;Kauretal.,,,a,b,
c,d; Mahajan et al., a,b,a, b; Dua et al., ,
a,b,c;Zargaretal.,,; Singh et al., a; Bhat,
Kumar, & Kumar, a, b,;Jamwaletal.,;Kumar
et al., a,b,,,,a,b,; Ahmed et al.,
), production of designer products (Singh et al., b;
Bhat&Pathak,a;Bhatetal.,a,b,c,d; Bukhari
et al., ,,,;Bhatetal.,; Bhat & Pathak,
; Pathak et al., a, b) and tenderization using novel
technologies such as pulsed electric field (PEF) (Bhat, Mor-
ton, Mason, & Bekhit, ,a,b;Bhatetal.,b,c;
Singh et al., ). Therefore, before being ready in a final
consumable form, muscle proteins undergo several man-
ufacturing processes. All these processes have the poten-
tial to affect the bioavailability and quality of the proteins
present in meat and meat products. Emerging technolo-
gies, such as PEF (Bhat et al., a; Bhat, Morton, Mason,
& Bekhit, c,d; Bhat, Morton, Mason, Bekhit, &
Mungure, ), and non-thermal processes, such as cur-
ing and ripening (Bhat et al., a), have been reported
to affect the digestibility of muscle proteins. The impact
of both emerging technologies and non-thermal processes
on the digestibility of muscle proteins have been reviewed
recently (Bhat et al., a,b). Thermal processing has
a similar potential to induce changes, such as disulfide
bond formation, cross linking, oxidation, microstructural
alterations, protein aggregation, and denaturation of vary-
ing levels often determined by the processing conditions

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4T P  D
FIGURE 1 Mechanisms of thermal processing for affecting the digestibility of muscle proteins [Bhat, Morton, Zhang, Mason, and
Bekhit (); Kehlet, Mitra, Carrascal, Raben, and Aaslyng (); Nguyen, Jones, Kim, Martin-Gonzalez, and Liceaga (); Bax et al.
(b); Ferreira, Morcuende, Madruga, Silva, and Estévez (); Rakotondramavo et al. (); Li et al. (a); He et al. (); Luo, Taylor,
Nebl, Ng, and Bennett (); Bax et al. (b); Gatellier and Santé-Lhoutellier ()]
which can affect the proteolysis of muscle proteins during
digestion. However, no review has been published on the
effect of thermal processing on the digestibility of muscle
proteins.
Ideally, human in vivo trials should be performed to
obtain reliable and accurate results related to the mus-
cle protein digestion. These are very difficult and expen-
sive experiments and simulating this process with in vitro
digestion systems has become widely popular and accepted
during last few decades for studying the health aspects of
foods and assessing the impact of processing on protein
digestion (Bhat et al., a). The in vitro systems can mea-
sure the initial rate of hydrolysis or maximal digestibility
values to assess protein digestibility using enzymes having
specificities similar to those present in the human digestive
tract. The digestibility of muscle proteins has been assessed
by several available in vitro digestion methods includ-
ing filtration methods and closed enzymatic incubations
within closed systems using single or multiple enzymes.
The digestibility of proteins is assessed by measurements
of dissolved nutrients, one of the separated fractions after
centrifugation or on the filtrate or on un-solubilized mate-
rial collected after filtration. The pepsin digestibility assay,
pancreatin digestibility assay or multi-enzymatic assay are
the most popular examples of the closed enzymatic meth-
ods which simulate one, two or all stages of the human
digestive process using one, two or more enzymes (Bryan
& Classen, ). Another popular method known as pH-
stat/drop method is based on the change in the pH of the
reaction media due to the release of protons from cleaved
peptide bonds during hydrolysis of proteins by digestive
enzymes (Bhat et al., a). Studies discussed in this
review have assessed the digestibility of the muscle pro-
teins using different parameters, such as the degree/rate
of hydrolysis, soluble protein fraction, pH drop method,
the optical density of the digested samples (colorimet-
ric assay), peptide analysis of the digestion products and
SDS-PAGE. Table presents the impact of different ther-
mal processes on digestibility of muscle proteins. Figure 
is a summary presentation of common mechanisms of
how different thermal processes affect the muscle protein
digestibility during gastrointestinal digestion. Low temper-
ature cooking generally improves the protein digestibil-
ity by inducing structural and conformational changes
and mild oxidation, which lead to partial unfolding of the
polypeptides and can expose active sites for further inter-
actions with digestive enzymes. Collectively, these changes
can enhance the accessibility of digestive proteases to
cleavage sites. On the other hand, high temperature cook-
ing induces changes such as severe oxidation, disulphide
bond formation and protein aggregation which can bury
the active sites deep into the structure thereby decreasing
their accessibility to digestive enzymes. Sous-vide has been
reported to increase the digestibility of muscle proteins by
inducing protein structural and microstructural changes
and through endogenous enzymatic proteolysis.

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T P  D 5
TABLE 1 Thermal processing techniques which have an impact on the digestibility of muscle proteins
Methods of processing
Effect on protein
digestibility
Muscle proteins and processing
temperature References
Methods studied to assess the
digestibility
Microwave processing ↑Rainbow trout proteins (–◦C) Nguyen et al. () Degree of hydrolysis, protein solubility
↓Shrimp proteins
(–◦C)
Dong et al. () Total soluble protein, in vitro protein
digestibility
Sous-vide cooking ↑Beef proteins
( and ◦C)
Pork proteins
(◦C)
Bhat, Morton, Zhang et al.
();
Kehlet et al. ()
Total soluble protein, in vitro protein
digestibility, amino acid profile,
SDS-PAGE
No effect Beef proteins
(◦C, in vivo study)
Oberli et al.,  True ileal digestibility
Stewing ↓Pork proteins (◦C)
Chicken proteins
(–◦C)
Beef proteins ( K)
Li et al. (a);
He et al. ();
Qi et al. ();
Kaur et al. ()
Degree of hydrolysis, SDS-PAGE, particle
size measurement, peptide analysis of
digestion products
Oven-roasting/baking ↑Pork proteins
( and ◦C)
Chicken proteins
( and ◦C)
Hairtail fish proteins
(◦C)
Bax et al. (b);
Ferreira et al. ();
Tava res et al. ( )
Degree of hydrolysis, amino acid analysis,
half-life time, maximal degradation
(Continues)

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6T P  D
TABLE 1 (Continued)
Methods of processing Effect on protein
digestibility
Muscle proteins and processing
temperature
References Methods studied to assess the
digestibility
↓Prawn proteins (◦C)
Oyster proteins (◦C)
Beef proteins
( and ◦C)
Pork proteins
( and ◦C)
Rhea myofibrillar proteins (◦C)
Luo et al. ();
Bax et al. (b);
Gatellier and Santé-Lhoutellier
();
Santé-Lhoutellier et al. ();
Filgueras et al. ()
Degree of hydrolysis, amino acid analysis,
half-life time, maximal degradation
Steam cooking ↑Pork proteins
(◦C)
Rakotondramavo et al. () Solubility of muscle proteins, Rate of
digestion, half-life time
Boiling/cooking in water bath ↑Hairtail fish proteins
(◦C)
Pork proteins (◦C)
Tava res et al. ( );
Zhao et al. ()
In vitro protein digestibility, free amino
acids, proteome analysis of digestion
products, SDS-PAGE
↓Pacific oyster proteins
(–◦C)
Pork proteins (◦C)
Zhang et al. (a);
Zhao et al. ()
In vitro protein digestibility, free amino acids
Frying ↑Hairtail fish proteins
(◦C)
Tava res et al. ( )In vitro protein digestibility, free amino
acids, proteome analysis of digestion
products, SDS-PAGE
↓
(compared to boiling)
Rabbit proteins (Frying at ◦C, boiling at
◦C)
Zhang et al. () Nitrogen release rate, amino acid analysis,
SDS-PAGE
Grilling ↑Chicken proteins
(◦Cformin)
Ferreira et al. ()In vitro protein digestibility, free thiol groups
↑=Increased/higher protein digestibility.
↓=Decreased/lower protein digestibility.

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T P  D 7
3MICROWAVE PROCESSING
Used commonly for defrosting and reheating of precooked
meat at the domestic level, microwave cooking is a popular
method that converts electromagnetic energy into thermal
energy and is available at industrial scale for processing
of meat products, such as bacon, meat balls and patties
(Orsat, Raghavan, & Krishnaswamy, ). The volumetric
temperature increase achieved in the microwave process
leads to more uniform coagulation of meat proteins
and produces high quality product (Orsat et al., ).
Microwaves are electromagnetic waves within a frequency
range of .– GHz and frequency of  and  MHz
are used to heat foods through molecular friction and
vibration of water molecules and ionic components as a
result of rapid change in the electromagnetic field (Bhat,
Pathak, & Fayaz, ; Bhat & Pathak, ,a, Bhat,
Pathak, Ahmad, Bukhari, & Kumar, ). Water and polar
compounds contents are important factors for microwave
cooking. Microwave heating has several applications in
the food industry due to its favourable characteristics such
as high heating rates, ease of the operation, safe handling,
and low maintenance (Zielinska, Ropelewska, Xiao,
Mujumdar, & Law, ) and may cause less damage to the
flavour and nutritional quality of a food compared to con-
ventional heating (Vadivambal & Jayas, ). Combined
microwave and convection based industrial scale ovens
used in meat industry have also been reported to have
some key benefits for food manufacturers, e.g. low energy
consumption and higher product yield and sensory quality
in case of bacon (Orsat et al., ; James, Barlow, James,
&Swain,). Given that microwave heating is a type of
dielectric heating and proteins and peptides have higher
dielectric constant, microwave processing can have a
significant impact on their structural and functional prop-
erties (Plagemann, von Langermann, & Kragl, )and
has been recently used as a novel thermal processing to
induce certain effects on the food proteins, such as reduced
allergenicity in eggs and fish (Zhu, Vanga, Wang, & Ragha-
van, ; Ketnawa & Liceaga, ). Table presents the
main findings of studies on the impact of microwave
processing on the digestibility of muscle proteins.
Dong, Wang, and Raghavan () studied the effects of
high temperature microwave processing (. GHz, 
W, –   ◦C for – min) on the secondary structures, in
vitro protein digestibility and microstructural characteris-
tics of shrimp proteins. A significant effect of microwave
treatment was observed on the digestion characteristics
where the total soluble protein content and in vitro pro-
tein digestibility were decreased by –% and –%,
respectively. The lowest protein digestibility was recorded
for the samples treated at ◦C for  min. Results of scan-
TABLE 2 Effect of microwave processing on the digestibility of muscle proteins
Authors Aspects studied Processing parameters Main findings
Dong et al. () Studied the impact of high temperature
microwave on in vitro digestibility of shrimp
proteins
. GHz,  W, –◦Cfor–
min
The total soluble protein content and protein digestibility
decreased significantly by –% and –%, respectively.
Lowest protein digestibility was recorded for the samples
treated at ◦C for  min
Nguyen et al. ()Studied the impact of microwave assisted heating
on enzymatic hydrolysis of rainbow trout
by-products in comparison to slow
conventional heating
% power with % duty cycle at 
W, –   ◦C during hydrolysis
The degree of hydrolysis and protein solubility was
significantly higher (P <.) for microwave heated
samples compared to conventional heating
Luo et al. () Studied the impact of microwave cooking on
protein digestibility of four animal (chicken,
beef, pork, and kangaroo) and four aquatic
(trout, salmon, prawn, and oyster) meats
Microwave cooking for  s Compared to raw meat, no effect of microwave cooking was
observed on the digestibility of any meat.
Correlation matrix analysis revealed a negative corelation
of in vitro protein digestibility with total protein (P <.).
This inverse relationship was confirmed by negative
corelations with several individual amino acids

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8T P  D
ning electron microscopy revealed holes, fragments, and
strips in treated sample, especially in the samples treated
at ◦C. Microwave processing induced modifications in
the secondary structure of shrimp proteins, including an
increase in β-sheets and a loss in the α-helix structure
and turns. The highest percentage of β-sheets and the
lowest percentage of the α-helix structure and turns were
observed in the proteins treated with microwave at ◦C.
A decrease in the protein digestibility was also observed
by a decrease in peptide content of some of the treated
shrimp digests. This negative effect of the microwave treat-
ment on the peptide content was explained by protein
denaturation and polymerization. Processing of proteins
under high temperature affects its structure, pulling out
the hydrophobic groups which interact through covalent
bonds and form aggregates which are less susceptible to
enzymatic digestion (Bogahawaththa, Chau, Trivedi, Dis-
sanayake, & Vasiljevic, ).
A significant decrease in the protein digestibility of
microwave treated food proteins has been reported. For
example, Kamble, Singh, Kaur, Rani, and Upadhyay ()
recorded a significant decline in in vitro protein digestibil-
ity of microwave treated wheat protein ( W for  min)
and Xiang, Zou, Liu, and Ruan () observed a signifi-
cant decrease in in vitro protein digestibility of microwave
treated gluten. Contrary to these studies, several other
studies have reported a positive impact of microwave pro-
cessing on in vitro protein digestibility of food proteins,
such as Vagadia, Vanga, Singh, Gariepy, and Raghavan
() in microwave treated soymilk ( W, –◦Cfor
– min), Zhu, Vanga et al. () in microwave processed
egg white protein (-◦C for – min), and Alajaji and El-
Adawy () in microwave treated chickpea proteins. The
increase in the protein digestibility in these studies was
attributed to the reduction in the enzyme inhibitor activ-
ity by microwave induced protein denaturation and the
proceeding digestion (Vagadia, Vanga, Singh, Gariepy, &
Raghavan, ). This suggestion might explain the lower
digestibility in refined proteins from wheat where both the
samples and controls will lack the confounding effects of
protease inhibitors.
It seems that the effect of microwave processing on
the hydrolysis of proteins depends on the origin of pro-
teins and the processing conditions. Nguyen, Jones, Kim,
Martin-Gonzalez, and Liceaga () studied the effect of
microwave assisted heating on enzymatic hydrolysis and
functional properties of rainbow trout by-products in com-
parison to slower conventional heating. The degree of
hydrolysis and protein solubility was significantly higher
(P <.) for the samples heated with microwave (%
power with % duty cycle at  W, –◦C during
hydrolysis) compared to conventional heating. Studies
involving microwave assisted hydrolysis have reported a
higher degree of hydrolysis due to the rapid heat trans-
fer that accelerates the cleavage of proteins by proteases
(Izquierdo, Peñas, Baeza, & Gomez, ;Pramaniketal.,
). By inducing rapid heating within the proteins due
to the presence of polar constituents, microwave induces
faster unfolding of proteins, thereby increasing the rate
of hydrolysis (Uluko et al., ). Microwave heating has
been reported to increase the susceptibility of peptide
bonds to enzymatic hydrolysis by increasing the initial rate
of hydrolysis and the catalytic efficiency of the enzymes
(Uluko et al., ; Uluko et al., ).
While studying the effects of microwave heating on
microstructure and properties of yak meat, Li, Tang, Yan,
and Li () recorded several changes such as protein
denaturation, destruction of cell membranes, solubiliza-
tion of connective tissue, and presence of large gaps
between meat fibres and muscle bundles. Higher muscle
structural damage was observed in meat samples cooked in
boiling water in comparison to microwave heated samples
and this effect was attributed to the differences in cooking
time ( min vs. – s). Prior studies have also reported
that microwave cooking caused less muscle structural
damage compared to boiling or roasting (Hsieh, Cornforth,
Pearson, & Hooper, ). Microwave processing has the
potential to influence protein hydrolysis and digestibility
and the result can go either way, however, one has to keep
in mind that microwave heating favours protein unfolding,
aggregation and cross-linking to a lower extent than con-
ductive heating (Rombouts et al., ) and this knowledge
can be exploited commercially for industrial applications.
Microwave processing has been reported to induce sev-
eral modifications in food proteins at high temperature
such as modifications in secondary structures, increased
hydrophobicity and cross-link formation, and even the
occurrence of glycation through Maillard reaction (Caban-
illas & Novak, ). Microwaves, when applied at high-
temperature and for long-time, can effectively increase
the kinetic energy of protein molecules through vibra-
tions of polar groups and affect the secondary structure of
proteins (Wang & Chi, ) by exposing the hydropho-
bic regions and disruption of the hydrogen bonds (Zhu,
Vanga et a l., ). Microwave treatment of gluten-induced
intermolecular and intramolecular cross-linking between
amino acids, resulted in increased aggregation, which led
to an increase of high molecular weight peptides and
decreased the total soluble amino acids and in vitro pro-
tein digestibility (Xiang et al., ). All these changes
can result in physical and covalent aggregation of proteins,
which can reduce the efficacy of digestive enzymes and
compromise the release and bioavailability of amino acids
and peptides (Morzel et al., ).
Based on the results of all the studies discussed in
this review, it appears that microwave processing has a

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T P  D 9
FIGURE 2 Mechanism of thermal processing for affecting the digestibility of muscle proteins
potential to induce both favourable and unfavourable
changes in the digestibility of muscle proteins. The temper-
ature of the processing has a role in determining the over-
all impact on the protein digestibility. Those studies which
used less intense processes reported a positive impact on
protein digestibility of muscle as well as other proteins.
Future studies should compare the impact of a wider range
of mild, medium, and intense processing conditions.
4SOUS-VIDE COOKING
Sous-vide is a novel way of cooking in which meat is
sealed in a plastic pouch under vacuum and cooked in a
water bath to achieve a uniform controlled temperature
for a set time to retain more moisture, flavour and natu-
ral state i.e. no Maillard reaction products formation, com-
pared to common cooking methods. This French method
of cooking is carried out at a temperature range of –
◦C for extended periods of time, usually – h in the
meat industry (Baldwin, ). This method of cooking
has become increasingly popular during the last decade
(Roascio-Albistur & Gámbaro, ; Sun, Sullivan, Strat-
ton, Bower, & Cavender, ) due to the superior sen-
sory (such as superior tenderness, juiciness, and colour)
and technological characteristics (such as better oxidative
stability) of the end products and the decreased loss of
health-related compounds such as antioxidants, vitamins,
and minerals (Roascio-Albistur & Gámbaro, ). Vac-
uum packaging used during sous-vide prevents evapora-
tive losses of volatile flavour substances and moisture dur-
ing cooking and inhibits oxidation of food components
(Baldwin, ). This premium tag attached to the qual-
ity of sous-vide cooked products has made this a popu-
lar new method both in home kitchens and in restaurants
particularly for the processing of meat (Sun et al., ;
Clausen, Christensen, Djurhuus, Duelund, & Mouritsen,
). One of the significant advantages of this method is
its ability to produce acceptable quality products by adding
value to inferior raw materials such as meat from culled
animals or non-primal cuts and can offer a promising
option for older people for inclusion in texture-modified
diets (Botinestean, Keenan, Kerry, & Hamil, ). Figure 
shows the pictorial presentation of the possible mecha-
nisms of how sous-vide processing improves the digestibil-
ity of muscle proteins. Table presents the main find-
ings of studies on the impact of sous-vide cooking on the
digestibility of muscle proteins.
Bhat, Morton, Zhang, Mason, and Bekhit () stud-
ied the effect of sous-vide processing on the quality and in
vitro digestibility of Semitendinosus (silverside) from culled
dairy cows. Meat samples were vacuum packaged and
were cooked in a water bath at ◦C for . h or ◦C
for  h or ◦C until a core temperature of ◦Cwas
attained (average time of  min) and were subjected to
an in vitro gastrointestinal digestion simulation. A signifi-
cant (P <.) effect of sous-vide processing was recorded
and the mean values of the digests obtained from sous-vide
cooked samples were significantly (P <.) higher for sol-
uble protein (%), protein digestibility (%), and release of
free amino acids and minerals. Sous-vide processing not
only improved the sensory attributes and acceptance of
the meat from culled dairy animals but also had a signifi-
cant positive influence on digestion kinetics during in vitro
digestion simulation that led to a greater and faster diges-
tion of proteins. Sous-vide cooking has been reported to
disrupt the secondary structure of proteins resulting into
unfolding of proteins (Dominguez-Hernandez, Salasevi-
ciene, & Ertbjerg, ;Baldwin,) that can increase
the exposure of hydrolytic sites to digestive proteases.
Meat proteins undergo several modifications during
cooking, such as cross-linking, aggregation, oxidation,
and changes in conformation (Yu et al., ), which
have potential to decrease their solubility and affect their

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10 T P  D
TABLE 3 Effect of sous-vide cooking on the digestibility of muscle proteins
Authors Aspects studied Processing parameters Main findings
Bhat, Morton, Zhang et al.
()
Studied the impact of sous-vide processing on in vitro
digestibility of Semitendinosus from culled dairy cows
◦C for . h or ◦C for  h and control
samples were cooked to a core temperature
of ◦C
Sous-vide processing showed a positive influence on
protein profile (SDS-PAGE) of meat digests.
Significantly (P <.) higher soluble protein (%),
protein digestibility (%) and release of free amino
acids was recorded in the digests of processed
samples
Baugreet et al. ()Studied the digestion patterns of sous-vide cooked
protein-enriched restructured beef steaks during an
in vitro gastrointestinal digestion simulation
Cooked at ◦C for  h in a water bath in
sous-vide pouches
SDS-PAGE analysis revealed a significant impact on
hydrolysis of proteins during in vitro gastrointestinal
digestion.
Muscle fibre separation was observed during gastric
digestion whereas fibre breakdown was recorded
after intestinal digestion.
A lower production of small molecular weight
peptides (.- kDa and < Da) was recorded
Kehlet et al. () Studied the effect of sous-vide cooking and holding
time on digestibility of pork Semitendinosus during in
vitro simulated digestion
◦C for  min or  h Sous-vide at ◦C for  min improved the digestibility
of pork Semitendinosus.
No effect of holding time was recorded on the
digestibility
Prodhan et al. ()Studied the effect of cooking (pan-fried or sous-vide) on
digestibility and circulatory amino acids in healthy
adult human males (n =)
Beef steak was grilled for  min on a preheated
pan (◦C) on one side and for  minutes
on other side.
Sous-vide cooking was done at ◦Cforh
No differences were observed in circulating amino acid
concentrations and protein digestion between the
two cooking methods

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T P  D 11
susceptibility to gastrointestinal enzymes during digestion
(Bax et al., ). Thus, method of cooking can directly
influence the release and bioaccessibility of nutrients dur-
ing digestion. Depending on the processing conditions and
the temperature attained during cooking, different meat
proteins, such as myofibrillar, sarcoplasmic, and connec-
tive, can present different behaviours and changes (phys-
ical changes, water-binding, rheological behaviour and
temperature-dependent denaturation). Modifications such
as denaturation and the Maillard’s reaction can produce
components that will not be absorbed and utilized by
the human body (Sgarbieri, ). Some of the products
of Maillard’s reaction decrease the active binding sites
between protein nitrogen and metals and reduce their
bioaccessibility (Cozzolino, ). Since most of the min-
erals are associated with proteins in meats, the digestion
pattern of proteins is likely to influence the digestion and
bioaccessibility of minerals. Recently, da Silva et al. ()
reported an increase in the bioaccessibility of certain min-
erals (Ca, Cu, Fe, Zn and Mg) from bovine liver cooked
by sous-vide method (◦C) during in vitro gastrointestinal
protein digestion.
Baugreet et al. () studied the digestion patterns of
sous-vide cooked protein-enriched restructured beef steaks
during an in vitro gastrointestinal digestion simulation. A
significant hydrolysis of proteins was observed during gas-
trointestinal digestion as revealed by the analysis of SDS-
PAGE. Muscle fibre separation was observed during gas-
tric digestion whereas fibre breakdown as well as pro-
tein re-aggregation was recorded after intestinal digestion.
Regardless of the treatment, most soluble peptides cor-
responded to the peptides of < amino acids and had a
molecular weight of < Da. Pronounced aggregation
was observed in digested samples as studied by confocal
laser scanning microscopy. Addition of plant proteins to
restructured beef steaks caused agglomeration of proteins
during the gastric phase of digestion which underwent
denaturation during the intestinal phase and produced
smaller molecules or clusters. In vitro digestates showed a
lower production of small molecular weight peptides (.–
kDaand< Da) and both formulation and process-
ing were suggested to influence the bioaccessibility. Zhu,
Kaur, Staincliffe, and Boland () studied the impact
of application of actinidin followed by sous-vide cooking
on microstructure and protein digestibility of beef brisket
under simulated gastric conditions. The optimal process-
ing of meat was achieved by vacuum tumbling and cooking
under sous-vide conditions at ◦C for  min. The sous-
vide cooking method was used for thermal inactivation of
the enzyme to prevent over tenderization.
Kehlet, Mitra, Carrascal, Raben, and Aaslyng ()
studied the effect of cooking method (sous-vide ◦Cvs.
oven-cooking ◦C) and holding time ( min vs.  h)
on the protein digestibility of pork using an in vitro sim-
ulated digestion model. The study also included a human
trial to validate the effect in in vivo. A significant effect of
cooking method and holding time was recorded on the pro-
teolytic rate during the gastric phase of digestion whereas
no effect of the pork structure was observed on the diges-
tion parameters. The meat samples cooked by sous-vide
method (◦C) for  min showed higher rate of proteoly-
sis during the early gastric phase of digestion as compared
to other samples (◦C for  h and oven cooking at ◦C
for  min and  h). The authors concluded that sous-vide
method of cooking at ◦C for  min seemed to enhance
in vitro protein digestibility of pork Semitendinosus.The
results are in agreement that reported cooking temperature
modulates the protein digestion rate during in vitro as well
as in vivo experiments in animals (Bax et al., ; Bax et al.,
a). Cooking at ◦C has been demonstrated to increase
protein digestibility in comparison to ◦Corabovedue
to denaturation, which is believed to increase the acces-
sibility of cleavage sites to gastrointestinal proteases (Bax
et al., ). The slower proteolytic rate of the oven-cooked
pork during in vitro digestion was explained on the basis of
protein aggregation which limits the accessibility of cleav-
age sites to gastrointestinal enzymes. Cooking of meat for
a prolonged time or at a higher temperature could result in
protein–protein interaction leading to aggregation of pro-
teins (Bax et al., ; Roldan, Antequera, Armenteros, &
Ruiz, ).
Another important finding observed in the study was
a decline observed in the proteolytic rate of pepsin dur-
ing early phase of digestion with increased holding times
( h vs.  min). Meat proteins undergo several modifica-
tions during cooking and are subject to oxidative changes
and protein aggregation due to increased protein surface
hydrophobicity (Santé-Lhoutellier et al., ). Indepen-
dent of cooking temperature, a long cooking time of up
to  h was demonstrated to increase the protein car-
bonylation of sous-vide cooked lamb loins (Roldan et al.,
). These cooking induced protein modifications could
decrease the protein digestibility by decreasing the bioac-
cessibility of meat proteins to pepsin (Santé-Lhoutellier
et al., ).
The results reported in the study above (Kehlet et al.,
) were inconsistent with the hypothesis that an
increased holding time for cooking would increase the pro-
tein digestion rate. These results were in contrast with the
findings of Bhat, Morton, Zhang, Mason, and Bekhit ()
who observed a higher protein digestibility for the beef
samples cooked for  h (◦C) in comparison to samples
cooked at . h (◦C) during an in vitro gastrointestinal
digestion simulation. Several processes with antagonistic
effects on protein digestibility, such as protein unfolding,
aggregation, and endogenous enzymatic cleavage, seem to

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12 T P  D
co-exist during cooking. Aggregation of proteins during a
 h long cooking time might have reduced access to the
cleavage sites on unfolded proteins and reduced the enzy-
matic activity and digestion rate. Future studies should
consider all these factors and include a wider range of pro-
cessing conditions and different species to gain an in-depth
insight.
One of the limitations in the design of the study (Kehlet
et al., ) was that oven-cooking was chosen to com-
pare with sous-vide, a method during which tempera-
tures are precisely controlled, seems to be an inappropri-
ate comparison. However, authors have stated that oven
roasting was chosen because that is how meat is typi-
cally heated. Further, all the meat samples were pan-fried
beforeserving,tomimicarealisticmeal,whichwouldhave
drastically changed the surface proteins and overridden
any positive effect of sous-vide during in vivo trial. The
structural and physicochemical properties of meat pro-
teins are highly influenced by heat (Bax et al., ;Torn-
berg, ) and oven cooking and panfrying would have
resulted in a mixture of modifications in the meat pro-
teins. Lipid oxidation; carbonylation; denaturation; poly-
merisation and peptide scissions; irreversible modifica-
tions of essential amino acids; increased surface hydropho-
bicity; protein aggregation; and Schiff base formation; all
these have been associated with high-temperature cook-
ing and could improve or reduce the protein digestion rate
(Nuora et al., ; Soladoye, Juárez, Aalhus, Shand, &
Estévez, ; Traore et al., ; Estévez, ;Park&
Xiong, ). In a similar in vivo study involving healthy
adult human males (Prodhan et al., ), the subjects con-
sumed a beef steak sandwich, in which the beef was cooked
by either a sous-vide or pan-fried method. No differences
were observed in circulating amino acid concentrations
and protein digestion between cooking methods. The study
had some limitations that prevented a clear understanding
for the observed findings. Although, same cut of beef was
used as a source of meat in a randomized manner, authors
assumed that the raw amino acid composition was identi-
cal and differences as a consequence of cooking methods
were not analysed, thus, the amino acid composition of the
ingested meat could have differed.
Cooking of the meat is not the only factor that affects
the digestibility of meat proteins in human gut, age of
the subjects has also been reported to determine the
digestibility and assimilation of meat proteins. In an in vivo
study involving young adults (Oberli et al., ), protein
digestibility (true ileal digestibility) of well-done bovine
meat cooked at a high temperature for  min (◦C)
decreased moderately in comparison to rare-cooked meat
that was cooked at a lower temperature for  min (◦C).
The reduction in protein digestibility was attributed to
indigestible protein aggregates formed during high tem-
perature cooking which are subsequently fermented by
the microbiota and can generate potentially harmful com-
pounds for the colonic mucosa. The cooking method did
not affect postprandial exogenous protein metabolism in
young adults (Oberli et al., ) whereas in the elderly,
the bioavailability and assimilation of meat proteins from
the rare-cooked meat (◦C for  min) was lower than that
from fully-cooked (◦C for  min) meat (Buffière et al.,
). A lower concentration of indispensable amino acids
(P <.) and a lower entry rate of leucine (P <.) in the
plasma along with a lower contribution of meat nitrogen to
plasma amino acid nitrogen (P <.) was observed for
rare-cooked meat.
Comparing the results of the above studies suggests
that sous-vide processing has a positive impact on the
digestibility of muscle proteins by inducing favourable
changes in the structure of muscle proteins, thereby
increasing the availability of cleavage sites for enzymatic
hydrolysis. However, all the studies which showed a posi-
tive influence on protein digestibility used moderate tem-
peratures (,  and ◦C) for long cooking times, pecu-
liar to sous-vide cooking. Future studies should validate
the positive effect of sous-vide using in vivo studies.
5 STEWING
Stewing is a moist heat process in which the meat is totally
immersed in liquid during cooking and the meat is served
in the resultant gravy which may also include other ingre-
dients such as vegetables. This slow and long-time process
of cooking is suitable for tougher cuts of meat which con-
tain higher amounts of collagen, such as round and chuck,
and is one of the most popular methods of cooking meat,
particularly in Asian and African countries (Qi et al., ).
As a high temperature long time cooking process, stew-
ing induces changes in muscle proteins and affect their
digestibility. Table presents the main findings of studies
on the impact of stewing on the digestibility of muscle pro-
teins.
Li et al. (a) studied the protein digestibility and
digestion patterns of stewed pork. Strips ( cm width) of
pork Longissimus dorsi were blanched in boiling water for
 min and sliced into cubes ( ×× cm). These cubes
were pan-fried (◦C) for  min using soybean oil (
g/kg of meat), turned twice at an interval of  s (skin
side not fried) and then cooked in boiling water for  min
(water: meat =:) and finally, stewed at ◦C for 
min. The stewed pork samples were digested by in vitro
gastrointestinal digestion model and the digestion patterns
were compared with three other pork products (cooked
pork, dry-cured pork, and emulsion-type pork sausage).
The samples were first digested with pepsin which was

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T P  D 13
TABLE 4 Effect of stewing on the digestibility of muscle proteins
Authors Aspects studied Processing parameters Main findings
Li et al. (a) Compared the digestion patterns of stewed pork
with three other pork products (cooked pork,
dry-cured pork, and emulsion-type sausage)
during in vitro gastrointestinal digestion
Longissimus dorsi cubes were stewed at
◦Cformin
Stewed pork showed the lowest protein digestibility and
the largest particle size compared to all other pork
products during pepsin as well as trypsin digestion
He et al. ()Compared stewed pork with other pork products
including raw meat. Total proteins were
extracted and were subjected to digestion by
trypsin
Longissimus dorsi strips were stewed at
◦Cformin
The sulfydryl (SH) content of stewed pork was
significantly lower than raw pork.
The stewed pork proteins were very less digested
compared to cooked pork proteins
Qi et al. () Studied the impact of stewing time on in vitro
digestibility of chicken proteins
The chicken meat was cooked for ,  or  h
at –◦C
The digestibility decreased significantly by .% (from
. to .%) after  h of stewing.
The digestibility of the control and the samples
cooked for  h was comparable
Kaur et al. ()Studied the impact of cooking conditions as used
in stews and curries on protein digestibility of
beef proteins
Beef Semitendinosus cubes were vacuum
packaged and cooked at  K in a boiling
water bath for  and  min
A significant decrease was observed in the amino N
released from the samples cooked for  min.
SDS-PAGE analysis revealed that most of the small
peptides (< kDa) in the samples cooked for  min
were partially digested
followed by trypsin digestion. Stewed pork showed the
lowest protein digestibility and the largest particle size
compared to all other pork products during pepsin as well
as trypsin digestions. High-temperature cooking for longer
times induces severe protein aggregation and oxidation
which can decrease the digestibility of muscle proteins
during gastrointestinal digestion (Wen et al., a, b).
Moderate denaturation happens to meat proteins at ◦C,
however, at ◦C or higher temperatures severe protein
oxidation and aggregation occur (Promeyrat et al., a).
Among the undigested samples, the lowest particle sizes
were presented by stewed pork. High temperature cook-
ing has been reported to induce severe protein aggregation
accompanied with a decrease in number and size of the
particles (Promeyrat et al., a,b).
Based on results of SDS-PAGE, particle size and LC-MS
spectra, stewed pork showed greater resistance to pepsin
digestion. Very few of the characterized peptides were
derived from myofibrillar proteins during pepsin digestion
whereas most of the derivable peptides were from enzymes
involved in energy-metabolism (i.e., from the sarcoplas-
mic fraction). These results indicated that long-time and
high-temperature cooking of meat may increase the resis-
tance of myofibrillar proteins to pepsin digestion. During
trypsin digestion, the number of the peptides increased
in the digests of stewed pork particularly from myosin
and from other high abundance proteins such as alpha-
chain, tropomyosin, -phosphofructokinase, and phospho-
rylase. These results indicate that high-temperature and
long-time cooking may change the conformation of pro-
teins but only slightly affect the cleavage sites of trypsin
(Wen et al., a, b).
Among undigested (whole proteins) samples, most of
the bands from stewed pork had lowest intensities on the
gel (SDS-PAGE) which was attributed to the aggregation of
proteins as cross-linked proteins failed to enter the –%
SDS-PAGE gel. Similar findings have been reported in pre-
vious studies (Wen et al., a, b).
While studying the effects of meat processing meth-
ods on changes in disulfide bonding, protein structures
and protein digestion products, He et al. () compared
stewed pork with other pork products including raw meat.
Strips of pork Longissimus dorsi were blanched in boil-
ing water for  min and sliced into pieces ( ××
cm). These pieces were pan-fried (◦C) for  min using
soybean oil ( g/kg of meat), turned twice at an inter-
val of  s and then cooked in boiling water for  min
(water: meat =:)andfinally,stewedat
◦C for  min.
Total proteins were extracted using % sodium dodecyl sul-
phate (SDS) and homogenates were centrifuged and super-
natants obtained were subjected to digestion by trypsin.
The sulfhydryl group (SH) content of stewed pork was
comparable with that of cooked pork [cooked at ◦Cby

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14 T P  D
steam-cooking (core temperature of ◦C) for  min],
however, it was significantly lower than raw pork. Pro-
tein structure is greatly influenced by the cooking temper-
ature and results in the formation of irreversible oligomers
at ◦C due to unfolding of meat proteins whereas at
◦C or above, the proteins are further modified by oxida-
tion which promotes aggregation (Philo & Arakawa, ).
Long cooking times and high temperatures during stewing
induced the formation of disulphide bonds, thereby pro-
moting the protein aggregation (Bax et al., ). This leads
to a decrease in the amount of SH groups and increases
surface hydrophobicity (Liu, Xiong, & Chen, ;Lee&
Lanier, ). Surface hydrophobicity of stewed pork was
significantly higher than raw meat whereas it was com-
parable with cooked pork. Cooking increased the surface
hydrophobicity of meat proteins dramatically and exposed
some embedded hydrophobic residues due to unfolding of
proteins and other changes (Bax et al., ). The ordered
structure of the protein was transformed into a loose and
disordered structure with increased cooking time which
led to the breakage of hydrogen bonds and the exposure of
aliphatic amino acid residues, such as leucine, valine, and
isoleucine, at the surface of the protein molecules, thereby
increasing the protein hydrophobicity (Gao et al., ).
Processing induced changes were recorded in secondary
and tertiary structure of the proteins. In comparison to
raw pork, proteins were disrupted and had a loose con-
formation with more exposed hydrophobic groups in both
cooked pork and stewed pork. Fifty common proteins
(excluding uncharacterized proteins) were identified in
the digestion products of the untreated (without DTT)
samples during LC-MS/MS analysis. The myofibrillar pro-
teins from stewed pork were least digested whereas those
from cooked pork were well digested. The comparison
of digested products among samples showed that cooked
pork proteins were highly digested compared to the stewed
pork proteins prior to DTT treatment whereas the stewed
pork proteins were highly digested after DTT treatment.
These results indicated that abundant disulfide bonds were
formed in stewed pork proteins. While a significant impact
of processing was recorded on disulfide bond formation,
structural changes, and protein digestion, proteins from
different muscle sources may behave differently after pro-
cessing.
Qi et al. () studied the impact of stewing time on
in vitro digestibility of chicken proteins. The chicken meat
was cooked by stewing method for ,  or  h and the
temperature of both soup and chicken was maintained at
–◦C. A significant impact of stewing time was found,
and the protein digestibility was decreased by .% (from
. to .%) after  h of stewing. The digestibility of the
control (raw) and the samples cooked for  h was compara-
ble. Similar results have been reported by previous studies
(Santé-Lhoutellier, Astruc, Marinova, Greve, & Gatellier,
;Kauretal.,) who have observed a decline in pro-
tein digestibility with cooking time. The authors attributed
this decrease in protein digestibility to various amino acid
modifications resulting in formation of disulphide link-
ages, amide bonds, and dityrosine bridges, reducing the
susceptibility of chicken proteins to enzymatic hydrolysis
during gastrointestinal digestion. These stewing-induced
modifications in the proteins were further supported by the
results of SDS-PAGE where protein aggregates were found
above the uppermost protein bands. Further, increased
stewing time also resulted in changes in meat microstruc-
ture including decreased fibre diameter (by . μm), trans-
verse and longitudinal shrinkage of muscle fibres and the
degradation of the myosin heavy chain.
Kaur et al. () studied the impact of cooking condi-
tions as used in stews and curries on microstructure and
protein digestibility of beef proteins. Beef Semitendinosus
were cut into small blocks ( ××cm
), vacuum pack-
aged and cooked at ◦C in a boiling water bath for  and
 min to study the effects of cooking time and tempera-
ture typically used during stew and curry preparations. It
is important to mention here that traditionally stews and
curries are cooked in liquid medium (such as broths, soups,
or water) and there is mass transfer between meat and the
cooking medium which can affect the digestibility of meat.
Cooking of meat in a water bath after packaging under vac-
uum is closer to sous-vide than stewing.
The meat samples, both cooked and raw, were subjected
to in vitro gastrointestinal digestion simulation. A signifi-
cant impact of cooking was recorded on protein digestibil-
ity and the amount of ninhydrin-reactive amino N released
during the digestion showed a significant decrease on pro-
longed cooking ( min). This was also supported by the
results of SDS-PAGE as most of the small peptides (<
kDa) in the samples cooked for  min were partially
digested. However, the proteins and polypeptides with a
molecular weight of > kDa from cooked meat samples
showed faster and greater digestion compared to raw sam-
ples, particularly during gastric phase of digestion. Fur-
ther, prolonged cooking also caused a decrease in the
amounts of major identified amino acids such as trypto-
phan, tyrosine, and phenylalanine with highest amounts
detected in the raw samples. The authors suggested that
some ‘limited peptides’ were formed during prolonged
stewing which were resistant to the hydrolysis by digestive
enzymes and were not broken down to free amino acids.
The decrease in free amino acids and amino N in cooked
samples was also attributed to various cooking induced
modifications in amino acids such as formation of disul-
phide bonds, amide bonds or dityrosine bridges, affect-
ing the release of amino acids by reducing the suscepti-
bility of proteins to enzymatic hydrolysis (Morzel et al.,

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T P  D 15
; Santé-Lhoutellier et al., ). Further, cooking has
been reported to induce oxidation related modifications
in muscle proteins which can reduce their digestibility by
affecting the hydrolytic sites available for pepsin and chy-
motrypsin (Liu & Xiong, ). The specificities overlap
with pepsin prefers cleavage of peptide bonds adjacent to
aromatic amino acids such as phenylalanine or tyrosine
residues (Mikita & Padlan, ) and chymotrypsin prefers
cleavage of peptide bonds adjacent to tyrosine, phenylala-
nine, and tryptophan. All these amino acids are highly
sensitive targets for free radicals (Stadtman, )andcan
therefore reduce the available hydrolysis sites for pepsin
and chymotrypsin on cooked meat proteins.
The prolonged cooking seemed to have induced severe
structural changes resulting in a compact fused muscle
mass so much so that the sarcomere structure remained
intact even after  min of gastric digestion whereas the
raw meat samples showed a complete loss of basic sar-
comere structure. It appeared that the enzymes action was
more random in the raw meat whereas in case of cooked
meat, the enzymes progressed from the edges towards the
centre of the myofibrils, indicating the difficulty enzymes
had in diffusing into the compact cooked myofibrillar
structure. While raw meat appeared more susceptible to
enzymatic hydrolysis, the fused myofibril mass formed in
the raw meat as the gastric digestion progressed made it
resistant to further pepsin action. Similar results were also
found by Palka and Daun () who observed the forma-
tion of similar dense protein structures in beef proteins,
such as titin, during cooking due to the shrinkage of mus-
cle fibres and increased exposure of hydrophobic residues,
allowing intra- and inter-protein interactions (Straadt, Ras-
mussen, Andersen, & Bertram, ).
At the end of the gastrointestinal digestion, some undi-
gested collagen fibrils and electron-dense aggregates were
observed in the cooked meat samples assumed to be the
remains of undigested fused myosin thick filaments. For-
mation of similar aggregates has been reported in cooked
beef muscles (Astruc, Gatellier, Labas, Lhoutellier, & Mari-
nova, ) and farmed cod and salmon samples heated to
such cooking temperatures (Ayala et al., ;Ofstadetal.,
). These studies reported that the aggregates were
formed due to the disintegration of coagulated contractile,
sarcoplasmic, and sarcolemma proteins (Ayala et al., ).
Collagen, in contrast to these proteins, is quite resistant to
hydrolysis by digestive enzymes due to its high levels of
proline and hydroxyproline, which reduce the flexibility of
protein backbone that influences the binding of enzymes
to the active sites on proteins (Kaur, Rutherfurd, Moughan,
Drummond, & Boland, ).
While stewing is an effective method of improving
the tenderness of tougher cuts of meat, it significantly
reduces digestibility of their proteins. The results of all the
above studies indicate that stewing has an overall negative
impact on the digestibility of muscle proteins by induc-
ing unfavourable changes in the structure of the muscle
proteins, thereby decreasing the availability of the total
cleavage sites for enzymatic hydrolysis. This is in contrast
to sous-vide that improves the muscle protein digestibil-
ity of meat cooked in the liquid medium inside vacuum-
packages at moderate temperatures. Future studies should
compare the cooking of meat emersed in liquids at moder-
ate temperatures with sous-vide cooking.
6 ROASTING/BAKING OR
OVEN-COOKING
Roasting is a dry-heat cooking method during which meat
is cooked by hot air inside an oven through convective heat
transfer to the surface of the meat. This method of cook-
ing is more suitable for tender cuts of meat and is widely
utilized in commercial processing and foodservice opera-
tions (Mora, Curti, Vittadini, & Barbanti, ). Oven tem-
peratures of higher than ◦Caregenerallyusedandcan
be up to ◦C (Pathare & Roskilly, ). Temperatures
usually employed for slow roasting of meat is ◦Canda
temperature of –◦C is applied for moderate roasting
(Bhat & Pathak, b,b). The time required for any
kind of roasting usually depends on the weight and size of
the cut being roasted. High cooking temperatures result in
rapid rate of heating which reduces the total cooking loss
of meat and improve the quality attributes and microbial
safety of products (Goñi & Salvadori, ; Palka & Daun,
). However, roasting has been reported to induce sev-
eral changes in meat proteins such as shrinkage, dehydra-
tion, denaturation and aggregation of the myofibrillar pro-
teins and shrinkage of intramuscular collagen, and other
structural changes which can have a significant impact on
the digestibility of meat proteins during gastrointestinal
digestion (Suleman et al., ; Pathare & Roskilly, ;
Silva, Ferreira, Madruga, & Estévez, ; Bailey & Light,
). Table presents the main findings of studies on the
impact of roasting/baking on the digestibility of muscle
proteins.
Luo, Taylor, Nebl, Ng, and Bennett () studied the
effect of cooking conditions (raw, ◦C oven for  or 
min,  s microwave) on protein digestibility of four animal
(chicken, beef, pork, and kangaroo) and four aquatic meats
(trout, salmon, prawn, and oyster). The lean meat sam-
ples were minced, cooked and both raw as well as cooked
samples were subjected to in vitro gastrointestinal diges-
tion. A significant effect of cooking was observed and in
vitro protein digestibility of cooked chicken and pork (oven
cooked for  min) increased significantly whereas a sig-
nificant decrease was recorded in the protein digestibility

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16 T P  D
TABLE 5 Effect of roasting/baking and steam cooking on the digestibility of muscle proteins
Authors Aspects studied Processing parameters Main findings
Roasting/baking
Luo et al. ()Studied the impact of oven cooking on
protein digestibility of four animal
(chicken, beef, pork, and kangaroo) and
four aquatic (trout, salmon, prawn, and
oyster) meats
◦C for  or  min Compared to raw samples, digestibility of cooked chicken
and pork (cooked for  min) increased significantly
whereas the digestibility of prawn and oyster (cooked for
 min) decreased significantly.
An inverse relationship of protein digestibility with several
individual amino acids was established
Bax et al. (b) Studied the impact of cooking on in vitro
protein digestion of pork Longissimus
dorsi
The samples were heated for  min in a dry
bath at ◦C, ◦Cor
◦C
The rate of digestion (initial slope and maximal rate) of the
samples cooked at ◦C was higher compared to raw meat
samples during pepsin digestion, however, a decrease was
observed in the digestion rate (%) of the samples cooked
at ◦C and ◦C
Gatellier et al. () Studied the impact of cooking on beef
protein digestion using a developed
semi-automatic flow procedure with
photometric detection
Beef muscle samples were heated at ◦C
for , ,  and  min
A significant decrease was observed in Amax values with
increasing cooking time, indicating a negative impact of
cooking on intensity of proteolysis.
The highest decline (%) was recorded in the samples
cooked for  min compared to raw meat samples
Santé-Lhoutellier et al. () Studied the impact of cooking on the
digestibility of beef myofibrillar proteins
extracted from beef Rectus abdominis
during in vitro digestion simulation
Myofibrillar proteins were heated at ◦C
for , , , , and  min or at ◦Cfor
min
The activity of pepsin (proteolytic rate) decreased
significantly after cooking both at ◦C and ◦C
compared to raw control.
No significant impact of cooking was observed on the
activity of pancreatic proteases both at ◦C and ◦C
Tava res et al. ( )Studied the impact of baking on in vitro
protein digestibility of hairtail fillets
The fish fillets were baked at ◦Cfor,
 or  min
The protein digestibility of the baked samples was
significantly higher than raw samples during both pepsin
and pepsin/trypsin digestion.
No impact of cooking time was recorded on the
digestibility of the samples
Ferreira et al. () Studied the impact of protein oxidation on
the digestibility and nutritional value of
chicken patties
The products were roasted in an oven to a
core temperature of ◦C for  min
A significant increase was observed in the protein
digestibility of roasted patties compared to raw patties
Filgueras et al. ()Studied the impact of cooking on digestion
rate of rhea meat proteins aged for ,  and
 days at ◦C
Steaks from Gastrocnemiusparsinternawere
heated at ◦C in a dry bath for  min
The rate of proteolysis (∆OD/h) of rhea myofibrillar proteins
showed a significant decrease after cooking during pepsin
digestion
Bax et al. () Studied the digestion of pork Longissimus
dorsi heated to , , and ◦Cwithin
polypropylene test tubes in a dry bath for
 min.
Longissimus dorsi was heated to , , and
◦C in a dry bath for  min
Heating at ◦C increased initial slope compared to raw meat
during digestion and decreased thereafter with increasing
temperature.
Highest mean values for maximal rate of digestion were
recorded for the samples heated at ◦C
Steam cooking
Rakotondramavo et al. () Studied the effect of steam cooking on in
vitro digestibility of cooked ham prepared
from pork Longissimus dorsi
Steam cooked to a core temperature of ◦C Cooking increased the overall digestibility (pepsic +trypsic
digestion) and rate of digestion (half-life time decreased)
compared to raw meat

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T P  D 17
of prawn and oyster (oven cooked for  min) compared
to raw samples. No effect of roast cooking was observed
on other meats, in contrast to the decreasing (P >.)
trend recorded for microwave cooking. Correlation matrix
analysis revealed a negative corelation of in vitro protein
digestibility with total protein (P <.) for microwave
cooking. This inverse relationship of protein digestibility
with total protein was further confirmed by negative core-
lations with several individual amino acids, such as Iso,
Leu, Lys, Phe, Val, Arg, Thr, Pro, Tyr, Ala, and Ser, for both
microwave and oven cooking. These results suggest that
both total protein and particular amino acids were respon-
sible for reduction in digestion rate during cooking. A sig-
nificant decrease was observed in total peptide counts of
final digests of cooked beef, chicken, pork, kangaroo, and
salmon compared to raw digests. A significant decrease
was also observed for total peptide count of oven cooked
( min) oyster compared to raw digests. No effect of cook-
ing was recorded on total peptide counts of trout and prawn
muscle proteins.
Bax et al. (b) studied the effect of cooking on in vitro
protein digestion of pork Longissimus dorsi. The samples
were placed in polypropylene test tubes and heated for 
min in a digital temperature controlled dry bath at three
different temperatures viz. ◦C, ◦Cand
◦C. The
samples were subjected to in vitro gastrointestinal simula-
tion using pepsin, trypsin, and α-chymotrypsin. An effect
of cooking was observed and the rate of digestion (initial
slope and maximal rate) of the samples cooked at ◦C
was higher compared to raw meat samples during pepsin
digestion, however, a decrease was observed in the diges-
tion rate (%) of the samples cooked at ◦Cand
◦C.
The other digestion parameters (half-life time, maximal
degradation and time of maximal rate of digestion) were
increased steadily during digestion. While cooking influ-
enced all digestion parameters during digestion by trypsin
and α-chymotrypsin, no difference was observed among
the samples cooked at different temperatures.
Gatellier and Santé-Lhoutellier () developed a semi-
automatic flow procedure with photometric detection for
studying the protein digestion of meat. Beef muscle sam-
ples were placed in sealed polypropylene test tubes and
heated at ◦C in a digital temperature controlled dry
bath for , ,  and  min, emulating meat oven cook-
ing where similar core temperatures can be reached. Both
raw and cooked meat samples were subjected to gastroin-
testinal digestion in the developed system. To determine
the digestive kinetic constants, a mathematical model was
established. The results showed that the cooking caused a
significant decrease in the protein digestibility of the beef
proteins by proteases of the developed system. A signifi-
cant decrease was observed in Amax values with increas-
ing cooking time, indicating a negative impact of cooking
on intensity of proteolysis. The highest decrease (%) was
recorded in the samples cooked for  min (◦C) com-
pared to raw meat samples. These results were supported
by a parallel increase in t/ (half-life of the reaction), indi-
cating slowing down of the digestion process. This effect
was significant for the samples cooked for  min and an
increase of % was recorded compared to raw meat sam-
ples. This negative effect of cooking on the digestibility of
myofibrillar proteins during digestion by pepsin has been
previously reported (Santé-Lhoutellier et al., ).
Santé-Lhoutellier et al. () studied the impact of
cooking on the digestibility of beef myofibrillar proteins
during in vitro digestion simulation. Myofibrillar proteins
were extracted from beef Rectus abdominis andweresub-
jected to two types of heat treatment. Samples were placed
in sealed polypropylene tubes which were heated at ◦C
in a dry bath for , , , , and  min, emulating meat
cooking in an oven for which a similar temperature can be
reached at core. Other samples were placed in aluminium
tubes and were heated at ◦C for  min in an oil bath,
emulating meat frying during which meat is subjected to
a high temperature for a brief time. A significant effect
of cooking temperature was observed and the activity of
pepsin (proteolytic rate) decreased significantly in the sam-
ples cooked at ◦Cand
◦C compared to raw control.
A sharp decrease (P <.) of % was recorded in the
pepsin activity for the samples cooked at ◦Cformin
and the activity continued decreasing slowly afterwards
with increasing cooking time and dropped to % for 
min. A sharp decrease was also found in the pepsin activ-
ity for the fast cooking at ◦C and resulted in a decline
of similar magnitude as that of cooking at ◦Cfor
min. While a similar decrease was also reported in the
pepsin activity of chemically oxidized myofibrillar proteins
(Santé-Lhoutellier, Aubry, & Gatellier, ), no significant
impact of refrigerated storage ( week) was recorded on the
digestibility of lamb myofibrillar proteins by pepsin (Santé-
Lhoutellier, Engel, Aubry, & Gatellier, ). These results
clearly indicate that the processes which can substantially
induce oxidation of muscle proteins can reduce their pro-
teolysis by pepsin, which is the first step of the mammalian
protein digestion process.
No significant impact of cooking was observed on the
activity of pancreatic proteases (trypsin +α-chymotrypsin)
on myofibrillar proteins, previously digested with pepsin,
both at ◦Cand
◦Ccomparedtorawcontrol.Forthe
samples cooked at ◦C, a biphasic response was recorded
with a -fold increase of activity for the samples cooked for
 min compared to raw control and decreased thereafter
with cooking time with a maximum drop (%) recorded
after  min of cooking (Santé-Lhoutellier et al., ).
A similar biphasic response has been previously reported
during proteolysis of oxidized myosin by trypsin and

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18 T P  D
chymotrypsin (Liu & Xiong, ). An increase (P >.)
was also recorded in the protease activity of the samples
cooked at ◦C compared to raw control and of the same
order as that of the samples cooked at ◦C for  min.
In addition to an impact on activity of proteases, a signif-
icant impact of oven cooking was also recorded on the car-
bonyl content and aggregation of the myofibrillar proteins
by Santé-Lhoutellier et al. (). An increasing trend was
observed in the carbonyl content with increasing cooking
time and significantly higher values were recorded for the
samples cooked at ◦C for  min compared to raw sam-
ples. The samples cooked at ◦C also showed a signif-
icant increase compared to raw samples. A significant (P
<.) increase was observed in the protein aggregation
of myofibrillar proteins cooked both at ◦Cand
◦C
compared to raw control. The mean values of the samples
cooked at ◦Cand
◦C were comparable to each other
for all the parameters. Results of the correlation study
revealed a direct and quantitative relationship of cooking
induced aggregation (P <.) and protein carbonylation
(P <.) with proteolytic susceptibility to pepsin, indi-
cating the impact of these phenomena on the recognition
of proteins by enzymes. However, such correlations were
not observed for trypsin and α-chymotrypsin. A signifi-
cant increase was also reported in the surface hydropho-
bicity of myofibrillar proteins heated both at ◦Cand
◦C compared to raw control and was attributed to con-
formational changes resulting in unfolding of the myofib-
rillar proteins and increased exposure of non-polar amino
acids on protein surface. Interestingly, no correlations were
established between surface hydrophobicity of myofibril-
lar proteins and pepsin activity which was in contrast
with the findings of Santé-Lhoutellier et al. ()who
reported a significant impact of surface hydrophobicity
on the pepsin activity. Correlation matrix analysis also
revealed that the protein surface hydrophobicity and the
formation of disulfide bridges were mainly responsible for
the aggregation process of proteins whereas the carbonyl
groups seemed to have no role in the formation of protein
aggregates. These results were surprising given that car-
bonyl groups have been implicated in the formation of pro-
tein aggregates through their reaction with non-oxidized
amino groups, resulting in the formation of amide bonds
(Santé-Lhoutellier et al., ; Morzel et al., ). The
authors stated that the fact that carbonyl groups always
increase while protein hydrophobicity reaches its maxi-
mum suggest that oxidation is not the only reason for cook-
ing induced structural changes. The major reason for pro-
tein unfolding during heat treatment was attributed to the
rupture of hydrogen bonds.
It is important to note here that Santé-Lhoutellier et al.
() and Gatellier and Santé-Lhoutellier ()used
extracted myofibrillar proteins and homogenized meat
proteins, respectively, and not intact meat during digestive
simulations. The meat matrix provides some resistance to
the diffusion of digestive enzymes and does not allow an
unimpeded access to proteins. Further, the meat matrix
can also play a role and allow slow exchange of heat during
cooking.
Sobral, Casal, Faria, Cunha, and Ferreira () stud-
ied the impact of culinary practices including the effect
of oven/microwave cooking on lipid and protein oxida-
tion of chicken meat burgers during in vitro gastrointesti-
nal digestion. Oven/microwave cooking can induce lipid
and protein oxidation which are not restricted to cook-
ing process but are likely to be continued along the gas-
trointestinal digestion in the gut. Studies have reported the
oxidation of lipids during in vivo (Gobert et al., )and
in vitro digestion conditions (Van Hecke, Ho, Goethals, &
De Smet, ; Steppeler, Haugen, Rodbotten, & Kirkhus,
; Tullberg et al., ). The stomach acts like a bioreac-
tor for many biochemical and chemical reactions (Tirosh,
Shpaizer, & Kanner, ), such as lipid peroxidation and
consequent oxidation of other dietary constituents, such
as proteins. Meat has been reported to generate free rad-
icals inside the stomach promoting further ProtOx and
LipOx reactions (Gorelik, Ligumsky, Kohen, & Kanner,
; Kanner & Lapidot, ). These oxidation promot-
ing reactions can induce denaturation process of digestive
enzymes and reduce their activity and affect the digestibil-
ity of muscle proteins by reducing the bioavailability of
amino acids or even contribute to the production of toxic
compounds.
The in vitro digestion of muscle proteins was recorded
to increase oxidative markers such as malondialdehyde,
carbonyls, and Schiff base structures regardless of the
culinary practices whereas -hydroxy--nonenal and hex-
anal contents showed a decline. A massive increase was
observed in carbonylation after in vitro digestion regard-
less of the addition of ingredients and the same behaviour
was recorded after cooking. This increase in the values of
the carbonyls observed after digestion agreed with previ-
ous studies (Hu et al., ).Alossoffreeaminoacids
which were susceptible to oxidation, such as Thr, Phe, and
His, was also recorded along with an increase of cystine
(dimer of cysteine) which can be used as a measure of
oxidation because the loss of free thiol groups has been
previously used in studies as a protein marker of oxida-
tion after cooking and during storage and in vitro digestion
(Hu et al., ). Increased release of free amino acids is a
general expectation during digestion due to the proteoly-
sis of muscle proteins by digestive enzymes. While amino
acids such as Tyr, Met, Val, Trp, Leu, Ile, and Lys showed
an increase after digestion compared to raw and cooked
samples, however, for some of the free amino acids the
balance between digestion/oxidation was low. Complexa-

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T P  D 19
tion with aldehydes, aggregation, crosslinking, and poly-
merization can hinder the enzymatic proteolysis by affect-
ing the protease-active sites and consequently decrease the
release of free amino acids during digestion (Hu et al., ;
Santé-Lhoutellier et al., ). Cooking beef proteins for a
long time (◦C,  min) has been reported to affect the
digestibility of peptides of molecular weight of < kDa
and reducing the bioavailability of amino acids (Kaur et al.,
). Further, the amino acids mostly involved in the pro-
teolytic reactions of pepsin and trypsin [Tyr, Phe, Leu, Met,
ArgandLys(Wenetal.,b)] are highly susceptible to
oxidation which can limit the protease activity and prote-
olysis of muscle proteins (Kaur et al., ).
In addition to the increased oxidative markers, Sobral
et al. () also observed an increase in the fluorescent
intensity of samples after in vitro digestion with a shift
to ∼ nm and the formation of only one peak, which
was attributed to the reaction of amino acids released dur-
ing digestion with the reactive aldehydes produced dur-
ing LipOx. Extreme oxidation and consequent overlap of
peaks has been reported as a cause for the formation of
only one peak (Hu et al., ;Huetal.,). While
previous studies have reported a maximum fluorescence
emission of – nm for the reactions between amino
acids (e.g. lysine and glycine) and unsaturated aldehy-
des (e.g. malonaldehyde and -hexenal) (Veberg, Vogt, &
Wold, ), a positive Pearson correlation has also been
established between TBARS levels and protein carbonyla-
tion (Van Hecke, Goethals, Vossen, & De Smet, ). The
authors concluded that the high Schiff’s base and carbonyl
formation suggest a high ProtOx during digestion.
Tavares, Dong, Yang, Zeng, and Zhao () studied the
impact of baking on in vitro protein digestibility of hair-
tail fillets. The fish fillets were baked at ◦C for , 
or  min which was followed by freeze drying and the
samples were subjected to in vitro gastrointestinal diges-
tion simulation along with raw control. A significant effect
of cooking was observed and the protein digestibility of
the baked samples was significantly higher than the raw
samples during both pepsin and pepsin/trypsin digestion.
No significant effect of cooking time was recorded and the
protein digestibility of the samples cooked for ,  and
 min were comparable. A significant effect of cooking
was found on the content of released free amino groups
and significantly lower values were observed for cooked
samples compared to raw samples. However, an increas-
ing trend was observed in the content of free amino groups
with increasing cooking time. This decrease in the con-
tent of free amino groups observed in baked samples was
attributed to the cooking loss due to the release of free
amino acids in meat juices. Juices released from cooked
beef, pork, chicken and shrimp have been reported to have
higher NHcontent (Totani, Kuzume, Yamaguchi, Takada,
&Moriya,).
A significant impact was recorded on the content of total
carbonyl groups and the sulfhydryl (SH) groups, the most
common protein oxidation markers (Lu et al., ). A sig-
nificant increase was observed in the total carbonyl con-
tent of the baked samples compared to raw samples and
no increase was recorded with increasing cooking time. A
parallel decrease (P <.) was observed in the sulfhydryl
content of the cooked products compared to raw samples.
This increase in the carbonyl content was attributed to
the free radicals and hydroperoxides produced during lipid
oxidation and the decrease in SH groups was attributed to
cooking induced protein aggregation and oxidation. A neg-
ative correlation of in vitro protein digestibility with NH
and carbonyls contents was observed. A negative relation-
ship was also established between SH content and protein
digestibility with pepsin.
While studying the impact of oven roasting on in
vitro protein digestibility of chicken patties, Ferreira, Mor-
cuende, Madruga, Silva, and Estévez () observed a sig-
nificant increase in the protein digestibility of roasted pat-
ties compared to raw patties. The products were roasted in
an oven to a core temperature of ◦C for  min. A signif-
icant decrease was observed in the free thiol content of the
roasted patties whereas the values for carbonyl content of
roasted and raw patties were comparable.
Filgueras, Gatellier, Ferreira, Zambiazi, and Santé-
Lhoutellier () studied the impact of cooking process on
digestion rate of rhea meat proteins. Gastrocnemius pars
interna was excised after  h of chilling and sliced into 
cm thick steaks. Steak samples aged for ,  and  days
at ◦C were sealed in polypropylene test tubes and heated
at ◦C in a dry bath for  min, reflecting meat cooking
in an oven for which similar meat core temperatures can
be reached. The meat samples aged for  days were pack-
aged under vacuum whereas other samples were packaged
in air permeable films. The myofibrillar proteins extracted
from both cooked and fresh samples were subjected to
in vitro gastrointestinal digestion simulation. A significant
impact of cooking was recorded and the rate of proteolysis
(∆OD/h) of the rhea myofibrillar proteins showed a signif-
icant decrease after cooking during pepsin digestion. This
decrease in the proteolysis rate of pepsin was attributed
to the changes induced by protein oxidation which affects
the accessibility of pepsin to proteins and modifies the
recognition sites of amino acids. The aromatic amino acids,
which are preferentially cleaved by pepsin, are one of the
preferred targets for the attack of reactive oxygen species
(Stadtman, ; Berlett & Stadtman, ). Further, oxi-
dation induced changes can hamper the pepsin hydrolysis
by the formation of peptides in which N-terminal amino

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20 T P  D
acids are blocked by α-ketoacyl derivates (Berlett & Stadt-
man, ).
In contrast to the digestion by pepsin, proteolysis by
pancreatin (trypsin +α-chymotrypsin) showed an increase
after cooking and the rate of proteolysis was highest (P <
.) for the samples aged for  days after cooking. While
cooking is known to induce denaturation and oxidation
of muscle proteins, the cooking time and the intensity of
heating determines the extent of the transformations. Fur-
ther, different amino acids undergo different modifications
during oxidation (Berlett & Stadtman, ) and the pro-
teolysis behaviour of the pancreatin enzymes are differ-
ent, while trypsin has preference for amino acids such as
arginine and lysine, chymotrypsin has preference for aro-
matic amino acids and methionine (Nelson & Cox, ).
So, while the oxidation of basic amino acids and pres-
ence of carbonyl groups may negatively affect the proteol-
ysis of trypsin, the oxidation of aromatic amino acids and
heat induced conformational changes, such as increased
protein hydrophobicity, may enhance the activity of α-
chymotrypsin.
In addition to the changes observed in the protein
digestibility, a significant effect of cooking was recorded on
the carbonyl content of the rhea proteins and a significant
increase was recorded in the carbonyl contents of the sam-
ples after cooking. However, no significant increase was
observed in the carbonyl content of meat samples aged for
 days under vacuum.
The loss of antioxidant protection of muscles during
storage and presence of prooxidant agents, such as iron,
play an important role in the oxidation of muscle proteins
(Bekhit, Hopkins, Fahri, & Ponnampalam, ;Hoac,
Daun, Trafikowska, Zackrisson, & Akesson, ). The
high concentration of iron in rhea muscles (Filgueras et al.,
), which can promote the formation of free radicals
in an aerobic environment (Garcia-Segovia, Andres-Bello,
& Martinez-Monzo, ; Purchas, Busboom, & Wilkin-
son, ), may explain the high content of carbonyls in
rhea proteins under air-packaging. The protein surface
hydrophobicity of the myofibrillar proteins, which indi-
cates the unfolding of proteins and exposure of non-polar
amino acids to the surface (Chelh, Gatellier, & Santé-
Lhoutellier, ; Van der Plancken, Van Loey, & Hen-
drickx, ), was markedly affected by the cooking and
the BPB (bromophenol blue) bound content was three
times higher in cooked proteins compared to fresh rhea
proteins. Higher contents of Schiff bases and aggregates
were observed after cooking and were attributed to heat
induced exposition of thiol groups leading to the formation
of hydrogen and disulfide bonds and hydrophobic interac-
tions between proteins and other compounds (Su, He, &
Qi, ).
Bax et al. () studied the digestion of pork Longis-
simus dorsi heated to , , and ◦C in polypropy-
lene tubes in a digital temperature controlled dry bath for
 min. Heating at ◦C increased initial rate of hydrol-
ysis compared to raw meat during pepsin digestion and
decreased it thereafter with increasing temperature. Half-
life time, maximum degradation rate and time to maxi-
mal rate of digestion, all were increased with increasing
temperature with lowest values recorded for raw samples.
Highest mean values for maximal rate of digestion were
recorded for the samples heated at ◦C. This increase in
the digestibility at ◦C was attributed to protein denat-
uration inducing conformational changes that increased
the exposure of hydrophobic residues on the surface and
therefore increased the cleavage sites and bioaccessibility
for pepsin. However, at ◦C and above protein oxidation
processes also kicked in and protein aggregation seemed
to outpace the thermal denaturation process, thereby
decreasing the pepsin digestibility (see Figure ). These
explanations were confirmed with the help of hydrolysis
equations. Similar decrease in the digestibility of myofib-
rillar proteins heated to ◦C has been recorded during
pepsin digestion and was correlated with increased car-
bonyl content (Santé-Lhoutellier et al., ). The results
suggest a greater degradation potential for cooked meat but
required a longer digestion time.
The digestion profile of trypsin and α-chymotrypsin dur-
ing intestinal digestion was similar as that of the pepsin.
Half-life time, maximum degradation rate and time to
maximal rate of digestion, all increased with increasing
temperature with lowest values recorded for raw samples.
Highest mean values for maximal rate of digestion were
recorded for the samples heated at ◦C. These results sug-
gest a greater degradation potential for meat cooked at
◦C for a set time interval. The authors concluded that
cooking temperature had similar effects on both trypsin/α-
chymotrypsin digestion and pepsin digestion, although
trypsin/α-chymotrypsin had a faster digestion rate com-
pared to pepsin. In addition to protein digestibility, myofib-
rillar protein hydrophobicity of the samples increased sig-
nificantly with increasing temperature. The carbonyls of
the samples cooked at ◦C were comparable with that of
raw samples and increased with increasing temperature.
The results of the above studies show that oven-
roasting/baking can induce both favourable and
unfavourable changes in the muscle proteins and can
affect their digestibility positively or negatively. The
temperature of the processing seems to have a limited
role in determining the overall impact on the muscle
protein digestibility. However, it is worth mentioning
that different studies have used different samples (from
intact meat to solubilized proteins) and methods to heat
the samples and as such their results cannot be directly
compared or extrapolated. Future studies should compare
the impact of a wider range of processing covering mild,
medium, and intense heating.

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T P  D 21
7STEAM COOKING
Steam cooking is a moist heat cooking method that causes
a significant improvement in the texture of meat using
steam as a medium of heat (Rinaldi, Chiavaro, & Massini,
). It is a healthy way of cooking meat and is usually
done for – min for small cuts of meat and may take
as long as an hour for a whole leg of lamb (Suleman et al.,
). Compared to a conventional oven, steam cooking
takes less time and ensures uniform distribution of heat
(Pathare & Roskilly, ). This method of cooking similar
to other heating systems induces changes in meat proteins
such as denaturation and protein aggregation which can
impact the digestibility of muscle proteins. An innovative
method of steam cooking, known as superheated steam
cooking, that uses superheated steam alone or in combi-
nation with infrared radiation to cook the meat products
and can be applied to all types of meats (poultry, pork, beef,
and lamb) and meat products. The common temperature
and time used in a superheated steam oven is –◦C
for – min (Suleman et al., ). While superheated
steam cooking has been reported to modify the functional
and structural properties and digestibility of starch (Ma
et al., ;Huetal.,), no study has evaluated its effect
on muscle protein digestibility. Table presents the main
findings of studies on the impact of steam cooking on the
digestibility of muscle proteins.
Rakotondramavo et al. () studied the effect of steam
cooking on in vitro digestibility and the digestion rate of
cooked ham prepared from pork Longissimus dorsi. After
curing and tumbling, the meat was packed under vac-
uum and steam cooked up to a core temperature of ◦C
[increased step-by-step to ◦C(min),
◦C(min)
and ◦C ( min)]. Cooking showed a significant effect
and decreased the gastric digestibility (%) and rate of peptic
digestion (half-life time increased) of the meat proteins in
comparison to cured ham. Muscle proteins undergo con-
formational changes and oxidation during cooking that
can reduce their nutritional value (Promeyrat et al., ).
The results of microcalorimetry analysis of this study
showed that proteins underwent substantial denaturation
during cooking that led to a significant loss of solubility
of muscle proteins. Further, the results of carbonyl and
thiol contents also suggested an increase in temperature
induced protein oxidation during cooking. Both oxidation
and denaturation lead to protein aggregation which might
have masked the cleavage sites on proteins for digestive
proteases. Furthermore, cooking is likely to induce the
structural changes in proteins which can reduce the access
of enzymes to cleavage sites and influence the digestion of
proteins (Santé-Lhoutellier et al., ).
Although cooking reduced the pepsic digestibility as
compared to cured ham, it is important to mention here
that cooking increased the overall digestibility (pepsic
+trypsic digestion) and rate of digestion (half-life time
decreased) of the meat proteins in comparison to raw meat.
The authors explained this digestive behaviour by suggest-
ing that cooking induced denaturation might have exposed
the aromatic amino acids (corresponding to pepsin cleav-
age sites) to the surface of meat proteins, otherwise present
in the interior of native soluble proteins (Santé-Lhoutellier
et al., ). The internal molecular forces, which are
responsible for maintaining the native structure of a pro-
tein molecule, in most cases are affected by increasing tem-
perature and are weakened enough to induce ‘denatura-
tion’ above certain temperatures. During this denatura-
tion, parts of the interior of the previously native molecules
get exposed to the molecular surface due to the unfolding
of the amino acid chains, changing the affinity of molecule
towards others. Thus, a progressive denaturation of the
proteins during the steps of processing (curing, tumbling,
and cooking) increased the bioaccessibility of the proteins
for the digestive enzymes. Cooking of ham was done in
stages and was believed to have induced an irreversible
two-step rearrangement process in solubilized myofibril-
lar proteins (Gravelle, Marangoni, & Barbut, ;Torn-
berg, ). The first step involved partial denaturation and
protein aggregation which was predominantly recorded in
the heavy myosin head groups between  and ◦C. This
was followed by denaturation of the helical light myosin
tail groups above ◦C which led to further protein/protein
interactions and resulted in the formation of a -D gel
matrix (Gravelle et al., ). These cooking induced
changes caused a drastic decrease in protein solubility and
agreed with the findings of Chen et al. () who also
reported a decrease in the solubility of salt-soluble myofib-
rillar proteins from chicken breast heated at –◦C.
While a positive impact of steam cooking was recorded
on overall muscle protein digestion, it would be too early
to conclude so because the study (Rakotondramavo et al.,
) used comparatively low temperature (a core temper-
ature of ◦C) to cook the meat which was packaged under
vacuum. Further studies are required to confirm this posi-
tive impact and should evaluate the impact of high temper-
ature steam cooking/superheated steam cooking on mus-
cle protein digestibility and the impact of packaging.
8BOILING AND COOKING IN WATER
BATH
Boiling is the most traditional method of cooking meat in
water and is still very popular in Middle Eastern, Chinese,
Japanese and Thai Cuisine, such as hot pot (Suleman et al.,
). The small pieces of meat are usually boiled (◦C)
for – min or longer if bigger cuts are used, while

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22 T P  D
ensuring a core temperature of ◦C. Proteomic analysis
has revealed that boiling of meat induces several modifica-
tions in muscle proteins and amino acids including denat-
uration, protein aggregation, side-chain modifications of
amino acids, oxidation of phenylalanine and formation of
carboxyethyl lysine (Yu et al., ). These cooking induced
changes have potential to affect the digestibility of the mus-
cle proteins and should be considered while choosing the
duration of the cooking. Table presents the main find-
ings of studies on the impact of boiling on the digestibility
of muscle proteins.
Ozvural and Bornhorst () studied the impact of
cooking on the structural and chemical characteristics of
frankfurters during in vitro gastric digestion. Beef frank-
furters were boiled in distilled water for , , ,  and 
min and strained from the water after cooking. The cooked
product was sliced into cubes which were subjected to oral
and gastric digestive simulations along with raw control.
The samples were taken during digestion at , , , ,
, , and  min and were analysed for pH and mois-
ture. Digestion had a significant effect on the moisture con-
tent of the samples and a significant increase was observed
in the moisture content of all the samples, both cooked
and raw, after  min of digestion and thereafter no sig-
nificant increase was noticed till  min of digestion. No
significant (P >.) effect of cooking and cooking time
was observed and the moisture content of raw and cooked
samples was comparable. A significant (P <.) impact
of the digestion was recorded on the pH of the samples
and a significant decreasing trend was observed in the pH
of all the samples, both cooked and raw, throughout the
period of digestion. A significant (P <.) effect of cook-
ing and cooking intensity was also observed on the pH val-
ues of the meat digests. The cooked meat digests showed
higher pH values compared to raw samples and the differ-
ence became larger with increasing cooking time for the
entire digestion period. It is important to mention that the
range of pH recorded for the samples at the beginning of
the digestion was .–. which decreased to a range of
.–. at the end of the digestion and appears to be much
higher than pH traditionally described for gastric diges-
tion (∼.–.) and was attributed to the higher buffering
capacity of the meat products due to their protein and lipid
contents. A similar higher pH has been recorded during
an in vivo study in pigs on protein and fat rich almonds
by Bornhorst et al. () who measured an initial pH of
∼. that decreased to .–. after  min of gastric diges-
tion of raw and roasted almonds in the proximal stomach
region. The relationship between meat buffering capacity
and pH change during gastrointestinal digestion is an area
that requires scientific attention.
While hardness of raw samples decreased significantly
after digestion for  min, only the samples cooked
for  min showed a significant decline in the hardness
after the digestion. This decrease in the hardness of the
samples observed during digestion was attributed to the
added moisture content into the matrix and the structural
breakdown. However, the samples which did not show a
significant decrease in hardness after digestion indicate
that the meat structure did not change significantly during
digestion. Microscopic results showed that cooked and
raw samples were different from each other before and
after digestion. The structure of the raw product was rough
and heterogeneous before digestion with deep crevices
and large holes which were closed after cooking for 
min and the surface became smooth. The surface became
smoother with increasing cooking time, however, small
holes appeared on the surface which were attributed to
the decomposition of the structure during longer cooking
time. The surface of the raw samples appeared less porous
after digestion and the gaps present either shrank or
disappeared. Minimal to no differences were recorded in
the surface microstructure of the cooked samples after
the digestion particularly for samples cooked for  min.
The measured effective diffusivity of water into the raw
samples was higher than that of cooked samples which
was attributed to the changes in microstructure that
became less porous after cooking.
Tavares et al. () studied the impact of boiling on in
vitro protein digestibility of hairtail fillets. The fish fillets
were boiled at ◦C for , , or  min which was fol-
lowed by freeze drying and the samples were subjected to
in vitro gastrointestinal digestion simulation using pepsin
and trypsin. A significant impact of cooking was recorded
and the protein digestibility of the boiled samples was sig-
nificantly higher than raw samples during both pepsin and
pepsin/trypsin digestion. No significant impact of cooking
time was recorded and the protein digestibility of the sam-
ples cooked for ,  and  min were comparable. Inter-
estingly a significant impact of boiling was recorded on
the content of released free amino groups and significantly
lower values were observed for cooked samples compared
to raw samples. However, an increasing trend was observed
in the content of free amino groups with increasing cook-
ing time. This decrease in the content of free amino groups
recorded in boiled samples was attributed to the cooking
loss due to the release of free amino acids to the boiling
water. Juices released from cooked beef, pork, chicken and
shrimp have been reported to have higher NHcontent
(Totani et al., ).
A significant impact of cooking was recorded on the
content of total carbonyl groups and the sulfhydryl (SH)
groups. A significant increase was observed in the total
carbonyl content of the boiled samples compared to raw
samples and showed a significant increase with increasing
cooking time. A parallel decrease (P <.) was observed

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T P  D 23
TABLE 6 Effect of boiling/cooking in water bath on the digestibility of muscle proteins
Authors Aspects studied Processing parameters Main findings
Ozvural and Bornhorst () Studied the impact of boiling on the
characteristics of beef frankfurters during
in vitro gastric digestion
Frankfurters were boiled in water for , , ,
 and  min
A significant decreasing trend was observed in the pH of all
the samples during digestion.
A significant increase was observed in the moisture
content of all the samples after  min of digestion
Tava res et al. ( ) Studied the impact of boiling on in vitro
protein digestibility of hairtail fillets
The fish fillets were boiled at ◦Cfor,,
or  min
The protein digestibility of the boiled samples was
significantly higher than raw samples during both pepsin
and pepsin/trypsin digestion.
No impact of cooking time was recorded
Farouk et al. () Studied the impact of cooking of beef along
with accompaniments on digestibility
during static in vitro gastrointestinal
digestion
Minced beef Semimembranosus with
accompaniments (meat: accompaniment
=:  w/w) were cooked at ◦Cina
water bath
Cooking of the meat at ◦C with protein accompaniments,
such as mushroom and pumpkin, improved the
digestibility of muscle proteins.
Cooking with starchy foods, such as potatoes and rice,
decreased the digestibility
Zhang et al. (a)Studied the impact of heat treatment on in
vitro digestibility of salt-soluble proteins
from Pacific oyster (Crassostrea gigas)
Gradient heating method was used
(–◦C, ◦C per gradient) in a water
bath for  min
The digestibility of oyster proteins decreased significantly
with increasing temperature during both gastric and
intestinal digestion.
The free amino groups content also showed a significant
decline with increasing temperature
Sayd et al. () Studied the impact of cooking on the
digestion kinetics of beef proteins during
gastrointestinal digestion
Semimembranosus was cooked at ◦Cfor
min, ◦C for  min and ◦C for  min
The sarcoplasmic proteins heated at ◦C were more
susceptible to pepsin whereas proteins cooked above ◦C
were more susceptible to trypsin and corresponded mainly
to extracellular structural proteins
Zhao et al. ()Studied the impact of hydrothermal
treatment on the digestibility of pork
actomyosin during in vitro gastrointestinal
digestion
The actomyosin extracted from Longissimus
dorsi was heated to ◦Cor
◦Cfor
min or ◦Cfor,,ormin
Cooking of the samples at ◦C increased whereas cooking at
 ∘C decreased the digestibility of the actomyosin

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24 T P  D
in the sulfhydryl content of the cooked products com-
pared to raw samples. This increase in the carbonyl con-
tent was attributed to the free radicals and hydroperoxides
produced during lipid oxidation and the decrease in SH
groups was attributed to cooking induced protein aggrega-
tion and oxidation. The results of correlation study estab-
lished a negative relationship of in vitro protein digestibil-
ity with hardness, NHand carbonyls contents. A negative
relationship was also established between SH content and
protein digestibility with pepsin. Overall, both protein oxi-
dation and released NHgroups had a great impact on the
digestibility of fish proteins.
Farouk, Wu, Frost, Staincliffe, and Knowles () stud-
ied the impact of cooking of beef along with accompa-
niments such as mushroom, pumpkin, rice and potatoes.
The samples of minced beef Semimembranosus along with
accompaniments (meat: accompaniment =:  w/w) were
cooked at ◦C in a water bath and the samples were sub-
jected to static in vitro gastrointestinal digestion simula-
tion. Unaccompanied meats served as controls. Cooking
of the meat at ◦C with accompaniments rich in pro-
tein, such as mushroom and pumpkin proteins, improved
the digestibility of muscle proteins from animals of all ages
(dairy cattle of  years, – months, and – days). On
the contrary, cooking of meat with starchy foods, such
as potatoes and rice, resulted in a decline of the protein
digestibility.
Zhang et al. () studied the impact of heat treatment
on in vitro digestion of salt-soluble proteins from Pacific
oyster (Crassostrea gigas). The water-soluble oyster pro-
teins were extracted, lyophilized and the developed pow-
der was dispersed in phosphate buffered saline ( mg/mL)
which was heated by gradient method (–◦C, ◦Cper
gradient) in a water bath for  min. A significant (P <
.) impact of heat treatment was observed and the in
vitro protein digestibility of oyster proteins decreased sig-
nificantly with increasing temperature during both gastric
and intestinal digestion and the highest digestibility was
recorded in control group. These results were supported by
the results of free amino groups content which also showed
a significant decline with increasing temperature both in
gastric and intestinal digestion phases. Free amino groups
are released during protein digestion due to the hydroly-
sis of peptide bonds and are used to assess protein diges-
tion level (Bassompierre et al., ; Nielsen, Petersen,
& Dambmann, ). Interestingly, analysis of the diges-
tion products (HPLC) indicated that heat treatment had
no significant impact on digestion products which dif-
fered only in protein content, indicating that heat treat-
ment had a negative effect on the digestion rate without
affecting the digestion sites on oyster proteins. The results
of the proteome analysis revealed that the identified pep-
tides mainly came from myosin heavy chain, paramyosin
and tropomyosin.
Results from gel electrophoresis indicated that the oys-
ter protein was mainly composed of myofibrillar proteins
and the intensity of some of the bands decreased above
◦C, which was attributed to the decrease in protein sol-
ubility. A significant decrease was recorded in the solu-
bility of the oyster proteins with increasing temperature
and lowest values were observed at ◦C. Thereafter pro-
tein solubility increased until ◦C which was attributed
to the thermal degradation of proteins induced by oxida-
tion at higher temperatures (–◦C). A similar pattern
was recorded in the carbonyl content which showed a sig-
nificant increase with increasing temperature (–◦C)
and thereafter decreased slightly (–◦C). Increased
carbonyl content indicates the oxidation of proteins dur-
ing thermal processing (Tavares et al., ). A significant
increase was also observed in the surface hydrophobicity
of oyster proteins with increasing temperature which was
partly held responsible for decrease in protein solubility.
This increased protein oxidation and hydrophobic interac-
tion between denatured oyster proteins at higher temper-
atures resulted in protein cross-linking (Grossi et al., )
and decreased the distance between protein particles. The
oyster protein particles showed a partially clustered state
in the solution at ◦C which caused a decrease in pro-
tein solubility and stability. The authors concluded that the
increase in protein aggregation due to increased oxidation
and surface hydrophobicity with increasing temperature
reduced the digestibility of oyster proteins.
Sayd, Chambon, and Santé-Lhoutellier () studied
the impact of cooking on the digestion kinetics of meat
proteins during gastrointestinal digestion. Beef Semimem-
branosus was cooked within the bags in a water bath at
◦C for  min (rare cooked), ◦C for  min (medium
cooked) and ◦C for  min (well done). A database
of  peptides produced under different cooking condi-
tions from  meat proteins was established along with
their quantification using label free mass spectrometry.
The digestion of meat proteins was affected by the cooking
conditions and depended mainly on the location of pro-
teins in the muscle cell. The sarcoplasmic proteins were
more hydrolysed when heated at lower temperature (◦C)
during gastric digestion compared to higher temperatures
(◦C, ◦C) which was attributed to protein denaturation
causing a loss of solubility and inducing new interactions
among sarcoplasmic or myofibrillar proteins. While sar-
coplasmicproteinsheatedat
◦C were more susceptible to
pepsin, proteins cooked above ◦C were more susceptible
to trypsin and corresponded mainly to extracellular struc-
tural proteins. No direct link appeared to exist between the
number of peptides released and the molecular weight of
the protein.

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T P  D 25
Zhao et al. () studied the impact of hydrothermal
treatment on the digestibility of pork actomyosin dur-
ing in vitro gastrointestinal digestion. The actomyosin was
extracted from Longissimus dorsi and heated to ◦Cor
◦C for  min in a water bath and compared with a
control stored at ◦C. Some other samples were heated
to ◦C for , ,  or  min. The surface hydrophobic-
ity and particle size significantly increased with increas-
ing temperature and lowest values were recorded for raw
samples. These results suggested that the thermal treat-
ment resulted in unfolding and aggregation of actomyosin.
A significant increase was observed in the free SH groups
on heating whereas a significant decrease was recorded
in total SH groups, although the mean values of samples
cooked at ◦C were compared with raw samples. These
results suggested that cooking induced denaturation of the
proteins that resulted in more exposure of free SH groups.
However, cooking at higher temperature (◦C) reduced
the total SH groups due to the formation of disulfide bonds.
Increase in carbonyl groups and the disulfide bonds after
heat treatment indicated the oxidation of specific residues.
The heat induced denaturation/unfolding of the proteins
at ◦C increased the digestibility of the actomyosin in
terms of both number and intensity of the peptides in the
digests identified using LC-MS/MS. However, the disulfide
bond formation seemed to reduce the activity of the diges-
tive enzymes as both the number and intensity of the pep-
tides increased after the addition of DTT (breaks S-S bonds)
in the samples heated at ◦C for  min. Cooking of the
samples at ◦C resulted in a decreased digestibility of
actomyosin due to severe aggregation and intense oxida-
tion which either damaged or buried the cleavage sites for
digestive proteases.
Based on the results of the above studies, it seems
that ‘boiling/cooking in water bath’ has a potential to
induce both favourable and unfavourable changes in the
digestibility of muscle proteins. Once again lower tempera-
tures favour protein digestibility. The low temperature pro-
cessing induces favourable changes in protein structure
resulting in a substantial increase of total cleavage sites for
enzymatic hydrolysis. Studies which have reported a nega-
tive impact of boiling (◦C) on muscle protein digestion
(Zhang et al., a;Zhaoetal.,)haveusedextracted
proteins and not the intact muscles to evaluate the impact
on muscle protein digestibility. Future studies should con-
firm the impact of boiling on muscle protein digestibility
using intact muscle matrices.
9 FRYING
A high temperature method of cooking that uses hot oil
or melted fat in direct contact with the meat as a cooking
medium (Varela, ). Fried meat has a crispy texture and
appealing flavour and taste; however, it has been reported
to induce several chemical and structural changes such
as protein denaturation, crust formation, moisture loss,
oil uptake, protein oxidation, production of aromatic
compounds, and changes in colour via Maillard reaction
(Silva et al., ; Mir-Bel, Oria, & Salvador, ). Vacuum
frying of food products is gaining popularity due to some
of its advantages over conventional frying methods such
as oil uptake, texture, retention of natural colours, flavours
and nutrients, and protection of oil quality (Dehghannya
&Ngadi,). Vacuum frying is a deep-frying method
that is carried out in a closed system under pressures lower
than atmospheric pressure (– kPa) that aids in reduc-
ing the frying temperatures due to depression in boiling
point of oil and of water inside food products (Contardo,
James, & Bouchon, ). Vacuum frying of starch-based
food matrices has been reported to slow the enzymatic
digestibility of the starch during both in vivo and in vitro
studies due to the microstructural changes induced in
starch granules (Contardo, Villalón, & Bouchon, ;
Contardo, Parada, Leiva, & Bouchon, ). The effect of
vacuum frying on the digestibility of proteins, such as mus-
cle proteins, has not been studied and this needs scientific
attention. Table presents the main findings of studies on
the impact of frying on the digestibility of muscle proteins.
Ozvural and Bornhorst () studied the impact of deep
fat frying on the structural and chemical characteristics of
frankfurters during in vitro gastric digestion. Beef frank-
furters were deep-fried for ,  or  min. The cooked prod-
ucts were removed of their crust and sliced into cubes
which were subjected to oral and gastric digestive simu-
lations. The samples were taken during digestion at , ,
, , , , and  min and were analysed for pH and
moisture. Digestion had a significant impact on the mois-
ture content of the samples and a significant increase was
observed in the moisture content of all the samples, both
deep-fried and raw, after  min of digestion and there-
after a significant increase was noticed only in the samples
fried for  min at  min of digestion. While deep-frying
of the samples reduced the moisture content significantly
before digestion with lowest moisture content recorded
for samples cooked for  min, however, no difference was
observed in the moisture content of the samples after 
min of digestion. These results indicate that the samples
with lowest moisture had greater moisture absorption rate
during first  min of digestion. A significant (P <.)
impact of the digestion was recorded on the pH of the sam-
ples and a significant decreasing trend was observed in the
pH of all the samples, both deep-fried and raw, through-
out the period of digestion. A significant (P <.) impact
of cooking and cooking severity was also observed on the
pH values of the meat digests. The cooked meat samples

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26 T P  D
TABLE 7 Effect of frying and grilling on the digestibility of muscle proteins
Authors Aspects studied Processing parameters Main findings
Frying
Ozvural and Bornhorst () Studied the impact of deep-frying on
the characteristics of frankfurters
during in vitro gastric digestion
Beef frankfurters were deep-fried for ,
ormin
A significant decreasing trend was observed in the pH of all the
samples during digestion.
A significant increase was observed in the moisture content of
all the samples after  min of digestion
Tava res et al. ( ) Studied the impact of deep-frying on in
vitro protein digestibility of hairtail
fillets
The fish fillets were deep-fried at
◦C(,,ormin)
The protein digestibility of the fried samples was significantly
higher than raw samples during pepsin and pepsin/trypsin
digestion.
The protein digestibility of the samples decreased significant
with increasing cooking time
Zhang et al. ()Compared the impact of frying and
boiling method of cooking on in vitro
protein digestibility of rabbit meat
Rabbit meat was fried in soybean oil
( ±◦C) for ,  and  min or
boiled in water ( ±◦C) for , 
and  min
In general, the nitrogen release rate was lower for the fried samples
compared to boiled samples during both pepsin and trypsin
digestion.
The nitrogen release rate was higher for the samples boiled for
 and  min and lowest for the samples fried for  min
Grilling
Martini et al. ()Assessed the protein hydrolysis of
grilled pork, beef, chicken and
turkey during in vitro
gastrointestinal digestion
Meat strips ( × ×. cm) were
cooked at ◦Conagrillformin
Comparatively lower proteolysis by pepsin during gastric phase of
the digestion was attributed to higher cooking temperature
Ferreira et al. () Studied the impact of grilling on in
vitro protein digestibility of chicken
burger patties
The patties were grilled to a core
temperature of ◦C for  min on
each side
A significant increase was observed in the protein digestibility of
grilled patties compared to raw patties.

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T P  D 27
showed higher pH values compared to raw samples before
the digestion and no regular trend was recorded during the
digestion period. The range of pH recorded for all the sam-
ples at the beginning of the digestion was .–. which
decreased to a range of .–. at the end of the digestion.
A significant effect of cooking was recorded and the
hardness of the cooked samples was lower than raw sam-
ples (except samples cooked for  min). The lower hard-
ness of the samples deep-fried for more time was attributed
to their higher fat content. While hardness of raw sam-
ples decreased significantly after digestion of  min,
cooked samples showed a non-significant decline in the
hardness after digestion. This decrease was attributed to
the structural breakdown and the added moisture content
into the matrix. However, the fried samples did not show
a significant decrease in hardness after digestion indicat-
ing that the meat structure did not change significantly
during digestion. However, the removal of the crust after
cooking may have influenced these results, which may act
as a barrier for acid and moisture uptake by the samples
and probably increase the hardness. Microscopic results
showed that cooked and raw samples were different from
each other before and after digestion. The structure of the
raw product was rough and heterogeneous before diges-
tion with large holes and deep crevices which tended to
disappear after frying for  min and the surface became
smooth. The surface became smoother with increasing
cooking time and was attributed to the absorption of frying
oil during cooking. The surface microstructure of samples
fried for  min became smoother with fewer cracks after
digestion whereas the samples cooked for  min appeared
more granular and spongier. The recorded effective diffu-
sivity of water into the raw samples was higher than that
of fried samples which was attributed to the changes in
microstructure that became less porous after cooking and
to the higher fat content of the fried samples.
Tavares et al. () studied the impact of deep-frying on
in vitro protein digestibility of hairtail fillets. The fish fil-
lets were deep-fried at ◦C (, , or  min), followed
by freeze drying, and the samples were subjected to in
vitro gastrointestinal digestion simulation along with raw
control. A significant effect of frying was observed and
the protein digestibility of the fried samples was signifi-
cantly higher than raw samples during both pepsin and
pepsin/trypsin digestion. A significant impact of cooking
time was also observed and the protein digestibility of the
samples decreased with increasing cooking time, however,
digestibility of all the fried samples were higher than raw
samples. The authors attributed this decrease in the pro-
tein digestibility to the increased surface hardness of fried
fillets with increasing cooking time. The chilled storage of
meat products has been reported to increase the oxidative
damage and the hardness which was accompanied with
a decline in digestibility of the products (Ferreira et al.,
). A significant impact of frying was also recorded on
the content of released free amino groups and significantly
higher values were observed for fried samples compared
to raw samples. Frying grass carp fillets has been reported
to increase the content of released amino groups (Li et al.,
b) and this increase in the content of free amino groups
during frying of marine fishes was attributed to the hydrol-
ysis of soluble proteins (Erkan, Özden, & SelçUk, ).
Unlike the results recorded for protein digestibility, an
increasing trend was observed in the content of free amino
groups with increasing cooking time.
A significant effect of frying was observed on the total
carbonyl and the sulfhydryl (SH) content, the most com-
mon protein oxidation markers (Lu et al., ). A sig-
nificantly higher total carbonyl content was observed in
the cooked samples compared to raw samples which also
increased with increasing frying time. A parallel decrease
(P <.) was observed in the sulfhydryl content of
the deep-fried products compared to raw samples. This
increase in the carbonyl content was attributed to the
lipid-derived radicals and hydroperoxides produced dur-
ing lipid oxidation which have been reported to signif-
icantly increase protein carbonylation in meat (Estévez,
). These lipid-peroxidation produced free oxygenated
radicals can react with NH or NHmoiety on the side
chains of the amino acids and transform them to carbonyl
groups (Stadtman, ). The decrease in SH groups was
attributed to cooking induced protein aggregation due to
the formation of new disulfide and hydrogen bonds and
hydrophobic interactions (Alipour, Shabanpoor, Shabani,
& Mahoonak, ). Oxidation of free thiol (SH) groups
present at the surface of cysteine residues (Bulaj, )
and formation of other thiol oxidation products after reac-
tion with O(Hu et al., ), both were attributed to this
decrease. The correlation study revealed a negative and a
significant correlation between in vitro protein digestibil-
ity and hardness, NHand carbonyls contents. A negative
relationship was also established between SH content and
protein digestibility with pepsin. Contrary to this study,
Sun, Zhao, Yang, Zhao and Cui () reported a positive
correlation between SH group and in vitro digestibility of
myofibrillar proteins. Overall, both protein oxidation and
released NHgroups had a great impact on the digestibility
of fish proteins.
Zhang, Wang, Wang, and Zhang () compared the
impact of frying and boiling methods of cooking on in vitro
protein digestibility of rabbit meat. Meat from hind legs
of Sichuan rex rabbits was boiled in water ( ±◦C) for
 (equal to medium cooked),  (equal to well done) and
 min (equal to very well done) or fried in soybean oil
( ±◦C) for  (equal to medium cooked),  (equal to
well done) and  min (equal to very well done). In general,

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28 T P  D
the nitrogen release rate was lower for the fried sam-
ples compared to boiled samples during both pepsin and
trypsin digestion. The nitrogen release rate was higher for
the samples boiled for  and  min compared to others
and the lowest rate was recorded for the samples fried for
 min during pepsin digestion. Similar trend was recorded
during trypsin digestion and highest nitrogen release rate
was observed for samples boiled for  min and the low-
est rate was recorded for the samples fried for  min. These
results were supported by the amino acid and SDS-PAGE
analysis. The authors concluded that longer boiling peri-
ods were beneficial for the digestibility of rabbit meat pro-
teins whereas longer frying time reduced the digestibility.
These studies show that frying has a positive impact
on the fish protein digestibility compared to raw samples.
Studies are required to confirm a positive effect in meat
and chicken muscle proteins. While a lower digestibility
was recorded for fried rabbit meat proteins compared to
boiledrabbitmeatproteins(Zhangetal.,), this study
compared two different cooking methods at different tem-
peratures, frying at ◦C and boiling at ◦C. Future stud-
ies should evaluate the impact of vacuum frying on mus-
cle protein digestion and compare the different methods of
cooking at comparable cooking temperatures.
10 GRILLING
A traditional dry heat cooking method that involves apply-
ing heat directly (open flame, charcoal, gas, and infrared)
to the surface of meat from above, below or from sides.
Compared to roasting, which is a slow cooking method
that uses hot air inside an oven (low indirect heat), grilling
is a fast-cooking method that uses intense direct heat to
cook the meat. This method is suitable for tender cuts
of meat which are generally grilled using direct heat at
◦C or a higher temperature for – min, depends
on the cut used and its thickness (Suleman et al., ).
Grilling adds a peculiar flavour, aroma, and smell to the
meat, attributed to various complex reactions and a large
number of volatile compounds such as carbonyls, aldehy-
des, and Maillard intermediates (Arena, Salzano, Renzone,
D’Ambrosio, & Scaloni, ). Since meat is subjected to a
high temperature during grilling, it induces several modifi-
cations in muscle proteins, such as denaturation, carbony-
lation, disulfide bond formation, and cross linking (Silva
et al., ), which have potential to affect the digestibility
of muscle proteins. While grilling is a fast method of cook-
ing meat (hamburgers, hot dogs, tender cuts such as steaks,
chops) at a high temperature, barbecue, a similar method,
involves cooking meat (often large and bone-in cuts such
as pork shoulder, ribs, beef brisket, whole chickens, and
turkeys) low and slow either on a grill or a smoker using
indirect heat. The barbecue method commonly involves
cooking meats that tend to be tougher for hours or even a
whole day. The temperature in the smoker should be main-
tained at –◦F (–◦C) for safety (FSIS-USDA,
). Table presents the main findings of studies on the
impact of grilling on the digestibility of muscle proteins.
Martini, Conte, and Tagliazucchi () assessed the
protein hydrolysis of grilled pork, beef, chicken and turkey
meat during in vitro gastrointestinal digestion. Meat strips
( × ×. cm) were cooked at ◦C on a grill for  min,
homogenized, and subjected to in vitro gastrointestinal
digestion. The hydrolysis of proteins during in vitro diges-
tion was monitored by measuring the amount of released
amino groups. Majority of hydrolysis that occurred dur-
ing gastric digestion occurred during first  min of incu-
bation and no significant impact of gastric digestion was
recorded and the digestibility of all the four different meats
were comparable at the end of gastric digestion. A signif-
icant increase was recorded in the release of free amino
groups in all the meats during intestinal digestion and sig-
nificantly higher values were observed for beef compared
to pork (P <.) which in turn was significantly higher
than turkey meat. While pancreatic digestion of chicken
resulted in the release of higher (P <.) amounts of
amino groups than turkey meat, the amino groups released
from chicken were comparable (P >.)tobothbeef
and pork. The results indicated that the beef and chicken
proteins were hydrolysed faster and more efficiently than
pork and turkey proteins. No differences were recorded in
the amino groups released between different meats dur-
ing control in vitro digestions performed without digestive
enzymes. A higher protein digestibility has been recorded
for chicken and pork proteins compared to beef proteins
during gastric digestion (Wen et al. b), however, the
differences disappeared after the completion of gastroin-
testinal digestion. No difference was recorded between the
protein digestibility of beef, pork and chicken proteins dur-
ing an in vitro gastrointestinal digestion in another study
(Luo, Taylor et al., ). The authors attributed the differ-
ences in the results of these studies to the differences in
the in vitro digestion models, muscle type and the meth-
ods of processing, all of which have an impact on pro-
tein digestibility (Zhou et al., ;Lietal.,a;Wen
et al., a; Bax et al., ). Comparatively poor efficiency
of hydrolysis by pepsin during gastric phase of the diges-
tion was attributed to higher cooking temperature (◦C)
which can reduce the protein hydrolysis by pepsin by pro-
moting protein aggregation (Bax et al., ; Wen et al.,
a).
Ferreira et al. () studied the impact of grilling
method of cooking on in vitro protein digestibility of
chicken burger patties. The chicken burger patties were
grilled to an internal temperature of ◦C for  min on

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T P  D 29
each side. Both raw and grilled chicken patties were stored
under refrigerated conditions for  days. Samples were
taken on day  and  and were subjected to in vitro gas-
trointestinal digestion. A significant increase was observed
in the protein digestibility of grilled patties compared to
raw patties. While a significant decrease was observed in
the free thiol content of the grilled patties, the values for
carbonyls content of grilled and raw patties were compa-
rable. Comparison with different cooking methods, grilled
patties showed significantly higher protein digestibility
compared to boiled (core temperature of ◦Cformin)
and roasted (core temperature of ◦C for  min) patties.
These results were supported by a significantly higher free
thiol content of the grilled patties compared to boiled and
roasted patties.
Grilling seems to have a positive impact on muscle pro-
tein digestion; however, further studies are required to
validate this effect. Future studies should evaluate the
impact of long-time barbecue method on muscle protein
digestibility.
11 CONCLUSIONS
Thermal processing of meat and meat products can have
serious implications on the nutritional value by affecting
the digestibility and bioavailability of muscle proteins
during gastrointestinal digestion. Preparation of meat
products involves several cooking methods which can
have positive or negative effects on protein digestibility.
Generally, the studies on cooking methods that have used
milder conditions of heat have presented a positive impact
whereas studies which have used intense cooking meth-
ods have presented a negative impact on muscle protein
digestibility. Contradictory results reported by some of
the studies are due to the differences in processing of the
samples, digestion models and the digestibility parameters
used. Some studies have directly used meat matrices while
many others have used extracted or solubilized proteins. It
is difficult to extrapolate the results of these pure extracted
proteins to cooked meats. There are discrepancies in heat-
ing and cooking methods used, some studies have heated
samples to much higher temperatures before or after the
actual thermal processing. While some have compared the
effect with raw meat, others have no control at all and have
just compared different methods of cooking. In general, we
can conclude that the mild thermal processes have positive
effects on muscle protein digestibility and can add value to
the final product. However, intense cooking methods can
have a sizable negative impact depending on the process-
ing conditions and necessitates optimization to minimize
the negative impact. Extensive research is required to
validate the impact of these cooking methods on muscle
protein digestibility using in vivo studies. Research is
required to elucidate the underlying mechanisms respon-
sible for inducing the changes in the digestibility of the
muscle proteins. Future studies should focus on compar-
ing the effect of different cooking methods under different
sets of conditions on the muscle protein digestibility and
comparing the underlying mechanisms. Of particular
interest is to determine the critical set of conditions
(temperature/time) for each cooking method beyond
which a negative impact is observed on the digestibility of
muscle proteins. Studies on the impact of novel thermal
processing technologies, such as ohmic heating and radio
frequency heating, on muscle protein digestion are miss-
ing in the literature and need scientific attention. Future
studies should give particular attention to the sampling
procedures [comparing the right portion of the treated
samples, using muscle matrices rather than extracted pro-
teins (which are already partially denatured)] and heating
methods (to replicate the actual cooking methods).
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
AUTHOR CONTRIBUTIONS
Zuhaib F. Bhat and James D. Morton: conceptual-
ization; formal analysis; methodology; writing-original
draft; writing-review and editing. Alaa El-Din A. Bekhit
and Hina F. Bhat: conceptualization; formal analysis;
methodology; visualization; writing-review and editing.
Sunil Kumar: formal analysis; methodology; visualization;
writing-review and editing.
ORCID
Zuhaib F. Bhat https://orcid.org/---
Alaa El-Din A. Bekhit https://orcid.org/--
-
Hina F. Bhat https://orcid.org/---X
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How to cite this article: Bhat ZF, Morton JD,
Bekhit AEl-DA, Kumar S, Bhat HF. Thermal
processing implications on the digestibility of meat,
fish and seafood proteins. Compr Rev Food Sci Food
Saf. ;–.
https://doi.org/./-.