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chapter three

Protein instability
Ronald E. Barnett and Hie-Joon Kim

Aging of meat
Dairy products
Interaction with additives
Reducing agents
Hydrolyzed vegetable proteins
Nonenzymatic browning
Case study
Protein bioavailability
Reactive lysine loss
Sensory attributes

Proteins in foods, those that are unprocessed or minimally processed as well as those that
are processed to destroy spoilage and pathogenic microorganisms, are susceptible to
chemical changes brought about by indigenous or exogenous components and influenced
by various conditions of treatment and storage. They can be degraded by hydrolysis of
the peptide linkages brought about by enzymes and corresponding to proteolysis, they
can undergo aggregative or degradative reactions as well as modifications to substituent
moieties that are effectively brought about by interactions with food additives, and they
can undergo aggregation and modification due to amine group reactions with indigenous
or added components with carbonyl groups such as reducing sugars. Some of the more
prevalent and significant reactions occurring during refrigerated or ambient storage are
described below, with a focus on proteolysis, additives, and nonenzymatic browning.

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This section covers proteolytic changes occurring in foods that have low but discernible
enzymatic activity. Postharvest physiological changes, which is covered in Chapter 2, and
proteolysis due to spoilage microorganisms will not be discussed. The main areas of
interest are meat tenderization associated with aging and protein changes associated with
storage in some dairy products.

Aging of meat
Lean meat is about 22% protein by weight, most of which is myofibrillar protein and can
be extracted with 0.3 M potassium chloride. These myofibrilar 1 proteins are responsible
for the development of rigor mortis. The other protein fractions are the sarcoplasmic, or
water soluble, proteins and the connective tissue, or insoluble, proteins.
The principal proteins in the myofibrils are myosin, actin, troponin, and tropomyosin.
Myofibrils contract when the level of Ca2+ increases in the muscle cell, with the energy
for the contraction coming from the hydrolysis of adenosine triphosphate (ATP). ATP also
provides the energy to pump the Ca2+ back into the sarcoplasmic reticulum to allow the
muscle to relax. In the living organism, the ATP levels are replenished by respiration in
the mitochondria of the muscle cell with sugar, glycogen, and fat being oxidized to water
and carbon dioxide. With death of the animal, respiration ceases. However, up to 1% of
the muscle cell is glycogen, which can provide energy anaerobically by glycolysis to
produce ATP. During glycolysis, the glycogen is converted to lactic acid, which can ulti-
mately reach a concentration in the muscle of 0.1 M, or 9 g/kg. The lactic acid causes the
pH to fall from near 7.0 to a value between 5.3 and 5.8, depending upon the species, the
type of muscle, and the slaughter conditions. Typically, the time required is 6–12 h for
pork and 18–40 h for beef.1
When the glycogen is exhausted, the ATP concentration falls and rigor mortis occurs,
during which the thin and thick filaments of the myofibrils strongly interact to form
actomyosin. At this stage, the meat is firm, stiff, and tough. Since the pH is near the
isoelectric point of the myofibrillar proteins, protein–protein interactions are strong, with
water being excluded from the fibrils. This decrease in water holding capacity causes the
meat to drip.
The meat will tenderize with aging, which is presumably due to the action of lyso-
somal and other proteases.2 It has also been suggested that catheptic enzymes in muscle
tissue phagocytic cells contribute to postmortem tenderization.3 The time required for
aging depends upon the storage temperature, pH, and species. It varies from 1–2 days
for poultry to 10–20 days for beef. During aging, protein hydrolysis is occurring, as is
evidenced by the appearance of free amino acids.4 Since no free hydroxyproline is formed,
collagen breakdown is not part of the tenderization process.5 Proteolysis alone may not
account for all of the changes occurring during aging. Levels of both salt-extractable
protein and water binding increase, which may be related to dissociation of the actomy-
osin complex.6 Poultry tenderization normally occurs with 24 h and aging is more effec-
tive in ice water than at room temperature, which also implies a more complicated
mechanism than simple proteolysis.7
Until breakage of the lysosomes occurs, any proteolytic tenderization must be due to
free proteases within the muscle cell. Since little degradation of myosin or actin occurs
during aging, the substrate specificity of the proteases responsible for tenderization must
reflect the actual spectrum of proteins degraded.8 A possible candidate is the Ca2+-depen-
dent protease, CAF, which is unique in being unable to degrade myosin and actin.

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Tenderization of meat can be enhanced by the use of exogenous enzymes such as
papain, ficin, and bromelain. This enhancement can be achieved by injecting the
enzyme(s) into the live animal prior to slaughter (the Swift process9) or topical applica-
tion of the enzyme(s) as a solution or powder. Care must be taken to avoid excess
localized proteolysis, which will produce mushy tissue. Unlike the meat aging process,
the exogenous enzymes attack all the muscle proteins including connective tissue.10–12
The shelf life of the meat may also be shortened because of continued proteolytic activity.
Microbial growth will also be enhanced because of the greater availability of low molec-
ular weight substrates.
Meat that is frozen and then thawed will normally deteriorate rapidly even at refrig-
eration temperatures. This deterioration may be due to the breakage of any intact lysos-
omes, thereby releasing a wide spectrum of active proteases. Since membranes throughout
the tissue will be broken, other compartmentalized proteases may also be released. They
will cause the meat to become mushy because of extensive proteolysis, and the resultant
low molecular weight substrates will facilitate microbiological growth.
Fish quality deteriorates after rigor mortis, with proteolytic activity being a causal
factor. If ground herring meat is treated with a potato extract, which contains protease
inhibitors for a broad spectrum of proteases, the production of histamine, putrescine,
tyramine, and cadaverine is reduced, as is total volatile nitrogen.13 The primary source of
these compounds is from arginine, tyrosine, and lysine, which are produced by proteolytic
action. The bacterial count is also reduced, presumably due to a decrease in the availability
of substrates for growth. Proteolytic deterioration of fish meat continues even in the frozen
state if the storage temperature is not low enough, with some proteins having a half-life
of as little as 5 days.14

Dairy products
Cheese manufacture begins with the use of proteolytic enzymes such as rennet to cause
milk to coagulate to produce cheese curd. Coagulation is the result of proteolysis of the
milk casein. “Casein” is a generic term for a family of related proteins. In milk, the
caseins form aggregates of 0.02 to 0.3 µm in diameter called micelles, which are stabilized
by β-casein.15 Rennet causes fragmentation of the β-casein, which leads to destabilization
of the micelles. In the presence of Ca2+, the caseins aggregate to form the curd. The
cheese curd is then treated in various ways appropriate to the variety of cheese being
produced. During aging, continued proteolysis contributes to flavor and texture devel-
opment. For some cheese types residual protease activity can have an adverse effect on
quality. For example, the tensile strength of mozzarella cheese decreases logarithmically
with storage time due to protease activity.16
UHT-processed milk will eventually gel upon storage and develop off-flavors even
though there is no microbial growth. One of the proteases responsible for is plasmin,
which probably enters the milk from blood in the form of its precursor, plasminogen.17 In
fresh milk, most of the enzyme is present as the precursor. Increased plasmin activity is
observed after UHT treatment, and plasminogen levels decrease while plasmin activity
increases upon storage.18

Interaction with additives

Polyphosphate salts are polyanions that can participate in polyelectrolyte interactions with
proteins and gums in food systems. They are good chelators of metal ions19,20 and can also

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increase the amount of water binding in a food product.21 The polyphosphates tend to
hydrolyze in food systems, the rate of hydrolysis being dependent upon the pH, temper-
ature, the presence of phosphatases, and the types of metal ions bound.
The addition of sodium hexametaphosphate, (NaPO3)n, to milk converts it to a
translucent, yellow-green fluid. The effect is not a consequence of calcium ion chelation,
since ethylene diamine tetraacetic acid (EDTA) does not produce the same result. It
appears that a large stable complex is formed between casein and polyphosphate.22,23 If
an excess of the polyphosphate is added to the milk, the casein micelles become com-
pletely dispersed and there is an accompanying loss of opacity. Sodium hexametaphos-
phate also stabilizes milk against gelation24; since the hexametaphosphate rapidly hydro-
lyzes to pyrophosphate, the pyrophosphate may be the active agent that causes protein
unfolding and dispersion of the casein. The polyphosphate treated milk may also be
more digestible.25,26
Polyphosphates can also be used as cheese emulsifying salts.27 They form complexes
with the calcium ions and the cheese proteins to stabilize the protein on the surface of the
fat globules to prevent coalescence. The bound phosphate also increases the water-binding
capacity of the protein.
Polyphosphates can have dramatic effects on the tenderness and flavor of meat.28–33
They cause actomyosin to dissociate to actin and myosin and increase the water-binding
capacity of the proteins. As is the case in milk stabilization by polyphosphates, hydrol-
ysis to pyrophosphate may be necessary. Calcium ion chelation is probably not the cause
of the effects of polyphosphates on meats,34–37 but it is more likely the result of pH
alteration and associated protein unfolding and of protein–protein dissociation due to
phosphate binding.

Reducing agents
The most common reducing agents in food systems are ascorbic acid (vitamin C) and
erythorbic acid, which is an isomer of ascorbic acid. A major use of these is as color
preservatives in cured meats (see Nitrites below). Ascorbic acid is also used as a dough
conditioner in the baking industry where the primary effects are oxidative,38 catalytically
resulting in disulfide bond formation in the gluten proteins. The overall mechanism
appears to be the following39:

ascorbic acid + 1/2 O 2 → dehydroascorbic acid

dehydroascorbic acid + 2 RSH → ascorbic acid + R ± S ± S ± R

Ascorbic acid or dehydroascorbic acid can also react with the amino groups of proteins
and lead to protein crosslinking.40–42

Sulfites are widely used as antimicrobials and to preserve color in a variety of food systems.
In the U.S., sulfites cannot be used in meats, although its use is allowed in some foreign
countries. The U.S. prohibition is a result of the effect of sulfites on hemoglobin and
myoglobin, in which an S-heme complex is formed that has the same bright red color as
oxyhemoglobin and oxymyoglobin. Consequently, sulfite-treated meat would have the
appearance of fresh meat regardless of its age, and so discolored meat could be adulterated
to appear to be fresh. Sulfites also destroy any thiamine in the product. Most of the

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chemistry of the sulfites depends upon the multiple equilibria among the various forms
of sulfite and the nucleophilic properties of the bisulfite ion:

SO 2 + H 2 O = H 2 SO 3
H 2 SO 3 = H + + HSO 3 – pK = 1.86 43
HSO 3 – = H + + SO 3 2 – pK = 7.18 44
2 HSO 3 – = S2 O 5 2 – + H 2 O K = 0.076 45

With the exception of the reactivity of SO2 toward heme proteins, the reactive species is
bisulfite (HSO3–), which is a potent nucleophile. Bisulfite reacts reversibly with aldehydes
and some ketones to form hydroxysulfonates and irreversibly with α , β-unsaturated
carbonyl compounds. This is the mechanism by which Maillard browning is inhibited.
The primary effect on proteins is to react with disulfide bonds46:

R ± S ± S − R + HSO 3 ± → R − S − SO 3 ± + HS ± R

The main uses of nitrites in food systems are as antimicrobials and for curing of processed
meats. Of interest here is the chemistry of the curing process and any other chemistry that
involves food proteins. The nitrite ion is relatively stable and unreactive, but nitrous acid
is highly reactive and unstable:

HONO → NO 2 ± + H + pK = 3.23 47

Nitrous acid can react with itself and halides to give powerful nitrosating agents48,49:

2HONO → N 2 O 3 + H 2 O

2HONO + HX → NOX + H 2 O

These nitrosating agents can react at a number of sites in meat tissue, and can decompose
to nitric oxide, nitrogen dioxide, and nitric acid. In a typical cured meat, the nitrite nitrogen
ends up distributed as follows: myoglobin (5–15%), sulfhydryl groups (5–15%), lipid
(1–5%), protein (20–30%), nitrate (1–10%), nitrite (5–20%), and gaseous products (1–5%).50
The characteristic color of cured meats comes from reactions between myoglobin and
nitrite.51–54 Myoglobin (red) is oxidized with assistance from nitrite to metmyoglobin
(brown). Metmyoglobin forms a complex with nitrite that is then reduced to nitric oxide
myoglobin (red). Ascorbate and erythorbate accelerate this step. Heating denatures the
nitric oxide myoglobin to form the stable pink pigment nitrosylhemochrome. The pigment
contains two nitric oxide ligands.55
As pointed out above in the case of sulfite reaction with hemoglobin and myoglobin,
excessive use of nitrite could lead to fresh-looking but microbiologically unsafe meat prod-
ucts. Recently, higher total plate counts were found in intensely red-colored Chinese-style
sausage sold in traditional markets and containing higher residual nitrite levels than in
similar but less intensely colored products sold in supermarkets and containing less nitrite.56

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Nitrosation of the protein itself also occurs.57–59 After enzymatic hydrolysis the follow-
ing amino acids have been identified: 6-hydroxynorleucine, 2-nitrotyrosine, 3,4-dihydroxy-
phenylalanine, and S-nitrosocysteine. The 6-hydroxynorleucine came from nitrosation of
lysine, and the two aromatic amino acids resulted from nitrosation of tyrosine. Up to 25%
of the nitrite added to cured meats can end up as S-nitrosocysteine.
N-nitroso derivatives of secondary amines are carcinogenic, as shown in animal feed-
ing studies.60 The levels of nitrite that are approved for curing meats produce no more
than trace levels, which probably represent no health risk. Furthermore, ascorbate or
erythorbate, which are usually included in curing formulas, inhibits the formation of N-
nitroso compounds. N-nitrosation of the peptide bond occurs, but the products are unsta-
ble and would not persist in a cured meat product.61

Hydrolyzed vegetable proteins

Vegetable proteins are hydrolyzed in 10–20% HCl at atmospheric or elevated pressures
and then neutralized with NaOH to pH 6.62 After filtration and charcoal treatment, the
hydrolyzed vegetable protein (HVP) is sold as is or spray-dried. HVP is high in sodium
chloride and glutamate, and several amino acids are modified or destroyed during hydrol-
ysis, namely, tryptophan, serine, threonine, cysteine, cystine, and methionine. HVP
imparts meat-like flavor to products, and is sometimes used to enhance flavor in cured
meats and sausages.63 Because of the high ionic strength, buffering capacity, and pH of
HVP, it will increase water binding capacity of the meat and dissociate protein–protein
aggregates, increasing tenderness and storage stability.

Nonenzymatic browning
In this section the physicochemical changes taking place in proteins during long-term
storage of foods will be discussed. Chemical changes in proteins often lead to changes in
color and texture. The changes can also lead to decreased digestibility and the loss of
reactive amino acids such as lysine. Therefore, one key question is whether the deterio-
rative change in the sensory attribute or the decreased nutritive value of the foods is the
limiting factor in the shelf-life of the protein-rich foods.
Common physical and chemical changes in proteins during cooking and storage have
been extensively reviewed.64–69 Initially, denaturation without chemical changes takes
place upon heating. For example, myofibrillar proteins in meat upon mild heating become
unextractable with salt solution, but they can be extracted with guanidine-HCI and show
the same protein profile by sodium docecylsulfate–polyacrylamide gel electrophoresis
(SDS-PAGE) as the unheated meat. Chemical changes in food proteins take place upon
further heating. Chemical changes under alkaline conditions are irrelevant during thermal
processing or storage, because most foods are at neutral or slightly acidic pH. The extent
of racemization and similar reactions would be relatively small under typical processing
and storage conditions.
One of the most common reactions in foods is the nonenzymatic browning reaction,
the Maillard reaction. It can involve reducing sugars and the amino groups on the proteins.
Consequent changes in color and texture of the proteins can occur under abusive storage
conditions, because its Q10, the increase in the rate constant as temperature is raised by
10°C, is 3–4. It can also lead to decrease in nutritive values with respect to reduced
digestibility and the loss of lysine, due to the crosslinking of the proteins as demonstrated
in freeze-dried meat.70 The relative significance of these deteriorative changes during

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Table 1 The Effect of Temperature on Browning, Texture Score, and
Extractable Protein of Chicken-a-la-King after 3-Year Storage
Storage Extractable Proteinc
Temperature Browninga Textureb (mg/mg dry sample)
(°C) (A420) Score w/ DTT w/o DTT
Controld 0.11 6.53 0.91 0.91
4 0.47 5.45 0.36 0.21
21 0.68 5.18 0.27 0.14
30 1.02 4.36 0.14 0.09

a Portions (50 g) of pulverized freeze-dried chicken meat were digested with

2% pronase in 2 ml 0.1 M ammonium bicarbonate buffer (pH 8.0) overnight
at 37°C. The digest was centrifuged and the lipid in the supernatant was
extracted with 1 ml chloroform. Absorbance of the clear aqueous phase was
determined at 420 nm. Average of duplicate determinations.
b From M. Klicka, Ration Design and Evaluation Branch, Food Engineering

Directorate, U.S. Army Natick RD&E Center. A 1–9 hedonic scale was used.
c Portions (10 mg) of freeze-dried chicken meat were stirred with 5 ml 8 M

guanidine-HCl solution with or without 20 mM DTT. The extract was filtered

with nitrocellulose membrane filter (Schleicher & Schuell) and the amount of
protein in the filtrate was determined by a dye-binding method with boving
serum albumin as a standard.71 Average of duplicate determinations.
d Without storage.

storage has not been thoroughly investigated in the past. As will be demonstrated here,
using the chicken-a-la-king as representative, consumer acceptance based on sensory
attributes such as color and texture determines the shelf-life of protein-rich foods. It will
also be demonstrated that the decrease in nutritive value of the proteins is not significant
even when the sensory value of the protein food decreased significantly and the product
became unacceptable.

Case study
An earlier version of chicken-a-la-king from the military Meal, Ready-to-Eat (MRE)
showed substantial browning and decreased texture score after a 3-year storage at either
21°C or 30°C. The color measurements, texture score, and the amount of protein extractable
with 8 M guanidine-HCl solution with or without added dithiothreitol (DTT), a reducing
agent, are summarized in Table 1. After 3 years at 30°C the chicken meat became signifi-
cantly dark to the eye, which was also reflected in the increase in absorbance at 420 nm
of the protein digest. A significant drop in the texture score by the technical panel, from
6.53 to 4.36, accompanied the browning reaction. In the 1–9 hedonic scale, a score below
5 is considered unacceptable. Toughness and dryness of the meat were noted for samples
stored for 3 years at either 21°C or 30°C. A significant decrease in extractable protein was
also observed with or without DTT. The decreased extractability with guanidine-HCl
reflects covalent crosslinking of protein molecules as observed before in freeze-dried
meat.70 The difference in the extractability with and without DTT represents the amount
of proteins that became unextractable by disulfide crosslinking. The low extractability of
proteins in the samples stored at 4°C probably reflects crosslinking during the retort
processing of the MRE pouch. It appears that further crosslinking, color, and textural
changes take place during storage at a high temperature. As expected, SDS-PAGE revealed
little protein extractable with 6 M urea–2% SDS–10 mM DTT from chicken meat stored at
all three temperatures.
Since Maillard-type browning has been shown to lead to protein crosslinking with
accompanying textural changes in freeze-dried meat, we suspected that a similar reaction

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Figure 1 Comparison of browning of chicken meat heated with nonfat dry milk (c) and lactose-
free nonfat milk (b). Control was unheated (a).

involving lactose from nonfat dry milk in the chicken-a-la-king might be responsible for
the reaction. To demonstrate the involvement of lactose, the following experiment was
performed. In a beaker containing 40 g precooked chicken meat, 159 ml water, and 0.93 g
table salt, 4.2 g nonfat dry milk (Carnation Co., 53% carbohydrate by weight) or 4.2 g
lactose-free nonfat milk (prepared by dialysis of the nonfat dry milk and containing 0.24%
of original lactose as measured by the phenol-sulfuric acid method) was added and
thoroughly mixed. The mixture was divided into six aliquots and one was analyzed
without heating. The remaining five aliquots were heated in a sealed can for 7 min at
120°C and further heated at 73°C for 0, 1, 2, 4, and 8 days.
Both control samples, treated with water only or with lactose-free nonfat milk, showed
a slight increase in browning. A significant increase in browning was observed from
chicken meat treated with nonfat dry milk containing lactose (Figure 1). In a separate
experiment, it was shown that addition of nonreducing sucrose does not promote brown-
ing as the addition of lactose, a reducing sugar. These results clearly indicate that lactose
is responsible for the browning of the chicken meat through the Maillard-type reaction.
Consequently, lactose was removed from the MRE chicken-a-la-king.

Protein bioavailability
The covalent crosslinking of proteins in the meat is expected to reduce the bioavailability
of the proteins due to the direct involvement of the lysine residues as well as the reduced
digestibility by hydrolytic enzymes. In order to study the effect of protein crosslinking on
the bioavailability, we determined, by gel filtration chromatography, the molecular weight
distribution of the pronase digest of the chicken meat. Pronase is a nonspecific enzyme
and hydrolyzes all peptide bonds except the ones near the crosslinking sites. One can
assume that the amount of amino acids released by pronase is a good approximation of
it released by a combination of proteolytic enzymes in the body. Rayner and Fox reported
a good correlation between in vitro available lysine using pronase digestion and “Silcock
available” lysine.72

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Figure 2 Separation on a Bio-Gel P-2 column of pronase digest from control chicken (upper frame)
and from chicken heated with 0.04 M lactose at 73°C for 8 days. Brown color associated with the
limit peptide was observed in the void volume from the heated chicken, and a 12% decrease in
tryptophan content was found.

Since tryptophan has a high extinction coefficient in the UV region around 280 nm, it
is useful for monitoring the extent of digestion, as demonstrated by Kim and Haering.73
Figure 2 shows the separation, using a Bio-Gel P-2 (Bio-Rad, Richmond, CA) column
(1.5 × 46 cm, 18 ml/h flow rate, 2.3 ml/fraction), of amino acids and peptides released
from the control and browned chicken meat upon pronase digestion (see Table 1 for the
digestion condition). For both the control chicken meat (heated at 73°C for 8 days in water)
and the browned chicken meat (heated the same way in the presence of 0.04 M lactose),
two major peaks associated with absorption at 280 nm were observed in the late fractions
(around fraction 31 and 39).
The compound corresponding to the large peak around fraction 39 was identified as
tryptophan by several methods. First, its UV absorption spectrum was identical to that of
authentic tryptophan. Second, authentic tryptophan eluted at fraction 39 from the same
gel filtration column. Third, when the material in fraction 39 and authentic tryptophan
were dansylated and separated by high-performance liquid chromatography (HPLC),
identical chromatograms were obtained. The identity of the other UV-absorbing com-
pound around fraction 31 was not determined. A dipeptide, glycyl-L-tryptophan, was
found to elute around fraction 23. Therefore, it is unlikely that the material in fraction 31
is a tryptophan-containing peptide.
The chromatogram in Figure 2 for browned chicken meat shows a decrease in
tryptophan (fraction 39) relative to the control chicken. The amount of material in
fraction 31 did not change upon heating. Therefore, it could be used as an internal
standard. The amount of tryptophan released from the browned chicken meat was
approximately 12% less than that from the control chicken. Clearly lactose facilitated
crosslinking of the meat proteins. Some tryptophan is either involved directly in the

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Table 2 The Effect of Lactose Concentration on the Loss of Reactive Lysine
in Chicken Meat Heated at 73°C for 8 Days
Lactose Storage
Conc. Temp. Total Basic His + Arg Reactive Lys
(M) (°C) (mmol/g) (mmol/g) (mmol/g) %
4 1.362 0.673 0.689 100
(0.017) (0.006) (0.018)
73 1.269 0.670 0.599 86.9
(0.029) (0.010) (0.031)

4 1.367 0.679 0.688 100

(0.012) (0.018) (0.022)
73 1.335 0.673 0.662 96.2
(0.013) (0.026) (0.029)

Note: Average of four measurements; standard deviation in parentheses.

crosslinking or in close proximity to the crosslinking site and is not susceptible to release
by the nonspecific pronase.
From the browned chicken meat, a substantial increase in absorption at both 280 and
420 nm was observed near the void volume of the column. Brown color in the pronase
digest observed by the eye was visible only in these early fractions after separation by
size exclusion. Clearly these fractions correspond to the limit peptide pigment derived
from protein crosslinked through condensed sugar moieties described by Clark and Tan-
nenbaum.74 The decrease in free tryptophan measured by absorbance at 280 nm was
roughly accounted for by the increase of the limit peptide pigment in the void volume.

Reactive lysine loss

Friedman reviewed various aspects of the nutritional value of proteins.75 Availability of
lysine is an important index of protein quality. The extent of the involvement of lysine in
the crosslinking can be assessed by the dye-binding method.76 In this method the dye-
binding capacity of basic groups (histidine, arginine, and lysine) in the proteins for acid
orange 12 is measured before and after reaction with propionic anhydride to block the
lysine groups. The difference in the dye-binding capacity corresponds to the reactive lysine
content, which is an estimate of the biologically available lysine. The lysine content in
chicken meats heated at 73°C for 8 days in the presence of 0.01 and 0.04 M lactose was
determined by the dye-binding method. The control samples showed 0.69 mmol reactive
lysine per gram of meat (Table 2). When the meat was heated in the presence of 0.01 M
lactose, approximately 4% loss of lysine was observed. When the lactose concentration
was increased to 0.04 M, the lysine loss was increased to 13%.
Incidentally, the percent loss of lysine (13%) is about the same as the percent loss of
tryptophan (12%) discussed above for the same sample. Due to the three-dimensional
nature of the proteins, only a portion of the lysine will be exposed to react with the lactose.
An even smaller portion of the lysine residues will be involved in crosslinking with the
condensed sugars as a bridge. The probability is small of the tryptophan residue being in
the immediate vicinity of the lysine residue modified by lactose and not released by
pronase. However, the crosslinking will decrease the digestibility of the proteins by pro-
nase, as reflected by the 12% decrease in released tryptophan. Using the loss of either the
lysine or the tryptophan, one could estimate that the nutritive value of the proteins in the

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chicken meat heated at 73°C for 8 days with a high concentration of reducing sugar is
about 13%. If Q10 of 4 is assumed for the browning reaction,77 heating for 8 days at 73°C
corresponds to a storage for 22 years at the ambient temperature (23°C), which exceeds
the military shelf-life requirement of 3 years at the ambient temperature. As a matter of
fact, the browning of the meat in the 30°C, 3-year chicken-a-la-king sample in Table 1 is
about the same as that from the chicken meat heated with nonfat dry milk at 73°C for
4 days (Figure 1). Therefore, one can conclude that the loss of the protein nutritive value
under typical storage conditions would be much less than 10%.
There is ample data in the literature on the loss of protein nutritive value in dry or
low-moisture systems,69,73–75 but not much in high-moisture systems. It is well known that
the Maillard reaction is slower at a high moisture content than at an intermediate moisture
content. From an equilibrium consideration, it is not favored at high moisture, because
the advanced Maillard reaction, as well as the early formation of the Schiff base, involves
removal of water.79 For example, when skim milk powder (adjusted to 15% moisture
content) was stored for 32 days at 37°C, 33% loss of total lysine was observed after acid
hydrolysis.73 When UHT milk was stored at the same temperature, it took 3 years to reach
the same level of lysine loss (approximately 31%).80 The results described above suggest
that the loss of the protein nutritive value in chicken meat is less than 10% during a long-
term storage under nonabusive temperature conditions. The slower reaction in the meat
compared to milk is expected, because the lactose concentration inside the muscle will be
lower than that in the milk.

Sensory attributes
A significant deterioration in sensory attributes takes place under conditions similar to
those in which browning and nutritive loss occur. Table 1 shows that the texture score
drops from 6.5 to about 5 after 3 years at ambient temperature. The technical panel score
on appearance for the same MRE chicken-a-la-king samples was 5.6, 5.0, and 3.19 after 3
years at 4°C, 21°C, and 29°C, respectively.81 Clearly, the foods became unacceptable (below
5 in texture and appearance scores). When the nonfat dry milk was replaced by powdered
vegetable shortening in the new MRE chicken-a-la-king, the browning problem was elim-
inated and the product became more acceptable.
Though a significant decrease in the sensory quality of protein foods can occur during
storgage with minimal change in the nutritive value, it can be avoided by a proper selection
of ingredients. Conversely, the nutritional value of the proteins should not be a significant
problem as long as the food is acceptable to the consumer from a sensory point of view.

Some protein instability, such as proteolysis, is biochemical in nature; others, such as
interaction with additives and nonenzymatic browning, are strictly chemical. Some are
advantageous to the food; others are deleterious. Deleterious reactions, even though they
may not affect the nutritional quality of the proteins significantly, often compromise the
sensory quality of the foods and can be minimized by controlling the physicochemical
environment of the proteins.

1. Honikel, K.O., Kim, C. J., Fleischwirtschaft, 1985, 65, 1125.
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