Retrospective Theses and Dissertations
1984
Effect of sodium chloride on some of the chemical
reactions of sodium nitrite in cured meat
Taekyu Park
Iowa State University
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Park, Taekyu
EFFECT OF SODIUM-CHLORIDE ON SOME OF THE CHEMICAL REACTIONS
OF SODIUM-NITRITE IN CURED MEAT
Iowa State University
University
Microfilms
Internstionâl 300 N. Zeeb Road, Ann Arbor, Ml 48106
PH.D. 1984
Effect of sodium chloride on some of the chemical
reactions of sodium nitrite in cured meat
by
Taekyu Park
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of the
Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Department:
Major;
Animal Science
Meat Science
Signature was redacted for privacy.
IA' Chgrge bf Major Work
Signature was redacted for privacy.
For the Major Department
Signature was redacted for privacy.
For the Gr
Iowa State University
Ames, Iowa
1984
ii
TABLE OF CONTENTS
Page
REVIEW OF LITERATURE
1
Introduction
1
Salt
2
Properties
pH
Water holding capacity and binding properties
Flavor
Oxidation
Health concerns for salt
Sodium Nitrite
Reactivity of nitrite in meat curing
pH effect on nitrite
Temperature effect on nitrite
Other factors influencing nitrite
Salt and Nitrite Interaction
2
3
3
7
7
9
10
10
12
15
18
26
OBJECTIVES OF THE STUDY
30
MATERIALS AND METHODS
31
Experimental Design
31
Meat and Formulation
31
Production
32
Experiment I (Control)
32
Experiment II (Reduction of Product pH)
33
Experiment III (Effect of Reductant)
34
Experiment IV (Different Internal Finishing Temperatures)
34
Experiment V (Effect of Muscle Type)
34
Chemical and Physical Analysis
35
pH
Salt
35
36
iii
Pigment
Residual sodium nitrite
Product yield
Statistical Analyses
RESULTS AND DISCUSSION
Experiment I (Control)
pH
Salt
Cured color
Residual nitrite
Experiment II (Reduction of Product pH)
pH
Cured color
Residual nitrite
Experiment III (Effect of Reductant)
pH
Cured color
Residual nitrite
Experiment IV (Effect of Internal Finishing Temperature)
pH
Cured color
Residual nitrite
Experiment V (Effect of Muscle Type)
pH
Cured color
Residual nitrite
36
36
37
37
38
38
38
40
40
47
51
51
56
62
68
68
71
74
74
74
78
88
92
92
95
99
SUMMARY
102
CONCLUSIONS
104
BIBLIOGRAPHY
105
ACKNOWLEDGMENTS
118
iv
LIST OF TABLES
Page
Table
1.
Cooking schedule
33
Table
2.
Formula:
Experiment I (control)
33
Table
3.
Formula:
Experiment V (with turkey muscle)
35
Table 4, Effect of nitrite and salt on pH of a meat mixture
Table
Table
5.
6.
39
Nitrite and salt effect on cured color of a meat
mixture
42
Effect of salt on the residual nitrite of a meat
mixture
48
Table
7.
Effect of nitrite, salt, and 1.5% GDL on pH
52
Table
8.
Effect of nitrite, salt, and 0.2% GDL on pH
54
Table
9.
Effect of nitrite, salt, and 1.5% GDL on cured color
57
Table 10.
Effect of nitrite, salt, and 0.2% GDL on cured color
60
Table 11.
Effect of nitrite, salt, and 1.5% GDL on residual
nitrite
64
Effect of nitrite, salt, and 0.2% GDL on residual
nitrite
67
Effect of nitrite and salt on pH of product without
erythorbate
69
Effect of nitrite and salt on cured color of product
without erythorbate
72
Effect of nitrite and salt on residual nitrite of
product without erythorbate
75
Effect of nitrite, salt and internal finishing
temperature on pH
77
Effect of nitrite, salt and internal finishing
temperature on cured color
81
Effect of internal finishing temperature on cooking
yield
85
Table 12.
Table 13.
Table 14.
Table 15.
Table 16.
Table 17.
Table 18.
V
Table 19.
Table 20.
Table 21.
Table 22.
Effect of nitrite, salt and internal finishing
temperature on residual nitrite
89
Effect of nitrite and salt on pH of product with
white muscle
93
Effect of nitrite and salt on cured color of product
with white muscle
96
Effect of nitrite and salt on residual nitrite of
product with white muscle
100
vi
LIST OF FIGURES
1.
2.
3.
4.
5.
6.
7.
8,
9,
10,
11
12
13
14
15
Effect of salt and nitrite on pH of control
product during storage
41
Salt content of products from various saltnitrite formulations
45
Effect of salt and nitrite on nitrosyl heme pigment
of control product during storage
46
Effect of salt and nitrite on residual nitrite of
control product during storage
49
Effect of salt and nitrite on pH in products with
1.5% GDL during storage
53
Effect of salt and nitrite on pH in products with
0.2% GDL during storage
55
Effect of salt and nitrite on nitrosyl heme pigment
in products with 1.5% GDL during storage
58
Effect of salt and nitrite on nitrosyl heme pigment
in products with 0.2% GDL during storage
61
Effect of salt and nitrite on residual nitrite in
products with 1.5% GDL during storage
63
Effect of salt and nitrite on residual nitrite in
products with 0.2% GDL during storage
66
Effect of salt and nitrite on pH in products
without reductant during storage
70
Effect of salt and nitrite on nitrosyl heme pigment
in products without reductant during storage
73
Effect of salt and nitrite on residual nitrite in
products without reductant during storage
76
Effect of salt, 156 ppm nitrite and internal
finishing temperature on pH during storage
79
Effect of salt, 75 ppm nitrite and internal
finishing temperature on pH during storage
80
vii
Figure 16.
Figure 17.
Figure 18.
Figure 19.
Figure 20.
Figure 21.
Figure 22.
Figure 23.
Figure 24.
Figure 25.
Effect of salt, 156 ppm nitrite and internal
finishing temperature on nitrosyl heme pigment
during storage
82
Effect of salt, 75 ppm nitrite and internal
finishing temperature on nitrosyl heme pigment
during storage
83
Effect of salt, 156 ppm nitrite and internal
finishing temperature on pigment conversion
during storage
86
Effect of salt, 75 ppm nitrite and internal
finishing temperature on pigment conversion
during storage
87
Effect of salt, 156 ppm nitrite and internal
finishing temperature on residual nitrite during
storage
90
Effect of salt, 75 ppm nitrite and internal
finishing temperature on residual nitrite during
storage
91
Effect of salt and nitrite on pH in products with
white muscle during storage
94
Effect of salt and nitrite on nitrosyl heme pigment
in products with white muscle during storage
97
Effect of salt and nitrite on pigment conversion
in products with white muscle during storage
98
Effect of salt and nitrite on residual nitrite in
products with white muscle during storage
101
1
REVIEW OF LITERATURE
Introduction
Sodium chloride and sodium nitrite are two of the primary ingredients
used for meat curing.
Sodium chloride has been used for centuries as a
flavoring agent and as a preservative, and has provided the main method
for preserving meat in the past.
Sodium chloride performs an additional
function in the current production of processed meats and sausages by
solubilizing muscle proteins.
The dissolved protein assists in binding
meat, moisture, and fat together, in the formation of desirable gel tex­
ture, and in increasing cooking yields.
On the other hand, sodium
chloride consumption has become a major issue in the food industry, pri­
marily because the sodium ion seems closely linked to human hypertension.
Nitrite has also been used for centuries to preserve meats.
The
use of sodium nitrite for meat curing has undergone considerable review
in recent years which has shown the role of nitrite to be multifaceted.
Color, flavor, protection from oxidation and inhibition of Clostridium
botulinum are the major contributions of nitrite to cured meats.
How­
ever, the safety of nitrite has been questioned, because of nitrite's
role in the formation of nitrosamines.
Considering the importance of the functions of these ingredients
and their potential relationship to human health problems, it is very
important to understand the chemical behavior of sodium chloride and
sodium nitrite, both individually and in combination.
Meat curing is
a very complicated chemical system so even though the same compounds
may be used, chemical behavior can change according to their physical
2
and chemical state.
The end products may be altered by minor variations
in reacting and/or interfering compounds.
Some research has been done concerning interaction of salt and
nitrite, but most of these studies were conducted using model systems
which are different from actual meat systems.
The results from these
model systems have been inconsistent and even contradictory.
The fol­
lowing review will consider some of these discrepancies while discussing
the general properties of salt and nitrite.
Salt
Properties
Crystalline sodium chloride is composed of a positively charged
sodium atom surrounded by six negatively charged chloride atoms and
each chloride atom is in turn surrounded by six sodium atoms.
Strong
electrostatic forces hold this crystal together and high temperatures
are required to cause melting or volatilization.
Organic metal salts are also held together by electrostatic forces,
but these bonds are not as strong as those of inorganic salts.
Dif­
ferences in bond strength provide a basis for explaining differences in
water solubility between organic and inorganic salts.
When water mole­
cules (dipolar) come between the sodium and chloride ions of sodium
chloride, they decrease the electrostatic attraction between these ions-.
As a result, the ions spread out and NaCl dissolves in the water.
Principles of ion charge, size, and strength of electrostatic
bonding are used to explain the various effects of chloride salts in
3
meat systems relative to protein hydration. Collectively, these effects
have been called processing properties and include such measurements as
pH and water holding capacity (WHC).
£H
The addition of sodium chloride to meat has been shown to increase
the pH slightly (Berman and Swift, 1964).
This pH change has been
shown to be less prior to cooking than after cooking.
Puolanne and
Terrell (1983) found that preblends with 4.0% salt had higher pH than
those made with 2.0% salt.
Klotz (1950) reported that the positive
charge of amino acid residues would decrease with increasing pH, re­
sulting in a decrease in the masking effect by anions.
This masking ef­
fect has been shown to be consistent with the law of mass action (Briggs,
1940).
By increasing the overall negativity, electrostatic repulsion of
proteins is believed to occur (Hamm, 1971).
These protein effects would
be contributing to the changes in overall pH at the same time.
Water hoIding capacity and binding properties
It is well-known that salt increases the pH and increases water
holding capacity of meat which affects the properties of the subsequent
processed products.
Water holding capacity of meat is greatly affected
by pH and is at a minimum at the isoelectric point (I.P.) of meat pro­
teins.
The I.P. is the pH at which all ionizable groups are charged.
At this point, equal positive and negative charges on the protein re­
sult in a maximum number of salt bridges between peptide chains and a net
charge of zero.
The WHC of meat is at a minimum at this pH which is in
the range of 5.0-5.4 for meat. Increasing or decreasing the pH away
4
from the I.P. will result in increased WHC by creating a charge im­
balance.
A predominance of either positive or negative charges will
result in a repulsion of charged protein groups of the same charge and
increased capacity for water retention.
As early as 1937, Smorodinsev and Bysstrov (as cited by Hamm, 1960)
reported that sodium chloride increased swelling and water holding
capacity of meat. Specific effects of different chloride salts, in
terms of pH dependency and individual effects on WHC, were reported by
Hamm (1957).
He claimed that at pH 6.4, the following order of magnitude
existed for WHC; sodium salts of I > CNS > Br ^ CI ^ F and chloride salts of
Li > Na > I > Mg > Ba > Cu > Zn.
All salts except Zn at pH 6.4 increased
WHC, whereas at pH 3.5, all sodium and all chloride salts decreased WHC.
Schellman (1953) mentioned that there are many unexplained features
of ionic binding in proteins; 1) simple ions like chloride are readily
bound by proteins, though there is little tendency for them to associate
with either free ions or amino acids in aqueous solution and 2) it is
not known what characterizes a protein charge as a binding site since
there seems to be little connection between the number and nature of
known protein charges and the number and strength of binding sites.
He
suggested a series of mathematical equations to solve for an "association
constant" based on protein binding ability of ammonium chloride and
emphasized the importance of the dielectric constant as a component of
the equation.
The WHC of a meat system is greatly affected by the addition of salt
but the salt ions must be absorbed by the protein to affect WHC.
Hamm
(1957) reported that electrostatic binding of chloride ion causes in­
5
creased repulsion of the peptide chains with subsequent absorption of
water by capillary condensation.
Sherman (1961) claimed that ions were
absorbed to meat by electrostatic forces and that absorption was en­
hanced by increasing the ionic strength (or concentration of the salt)
in the meat system.
Increasing ionic strength also increases the
solubility of proteins by dissociating myosin aggregates (Ishioroshi
et al., 1979).
Hamm (1960) stated that increasing pH caused the binding of cations
to increase and of anions to decrease. Hamm (1971) showed that salt in­
creased the WHC of meat and thus not only reduced cooking loss, but
also drew the structural proteins through the sarcolemma so that they
were available for binding. Hamm (1981) also found that WHC may be en­
hanced by salting prerigor or postrigor meats and in order to effectively
increase WHC, salt content of prerigor beef had to be at least 1.8%.
Wierbicki et al. (1957) found that salt not only aided meat particle
binding but also reduced fluid loss during heating.
Schnell et al. (1970) demonstrated that the addition of salt in­
creased the cooked yield of poultry loaves and that salt and phosphate
increased binding in the loaf slices.
Maesso et al. (1970) investigated
the effect of salt, phosphate and heating on the binding ability of
poultry meat and found that treatments which reduced the amounts of cookout generally increased the binding strength.
Wierbicki et al. (1976)
found that reduced shrink was obtained in fresh ground ham with increased
salt levels, an effect that tended to level off as the salt level of the
meat reached 3-5%.
Such results would indicate that the improved texture
was due to the enhanced water holding capacity of the meat or more
6
specifically of the meat myofibrillar proteins.
The high salt levels
probably resulted in increased migration of the salt soluble proteins
to the surface of the meat allowing greater bonding of the pieces of
meat and greater binding of water during heating.
Randall and Voisey
(1977) found that salt in the amounts commercially used had a marked
effect on the tenderness of canned ham and was a major factor contributing
to the characteristic texture associated with this product.
Huffman and Cordray (1981) indicated that salt addition linearly
increased Instron slicing strength values, juiciness, and textural
properties in restructured pork chops.
The addition of salt also de­
creased raw color evaluations, Instron shear values and cooking losses
in restructured pork chops. Salt has been shown to improve the binding
qualities of flaked and formed pork (Mandigo et al., 1972) and of bologna­
like products (Swift and Ellis, 1956).
Furumoto and Stadelman (1980)
found that regardless of rigor state, the addition of salt significantly
improved tenderness of pork, chicken, turkey and beef.
Tenderness of
meat was generally improved by the application of sodium chloride prior
to cooking (Near and Mandigo, 1974; Oblinger et al., 1976), Kardouche
and Stadelman (1978) reported that mixing of 1.5% NaCl with pre- or
postrigor, light or dark, turkey meat chunks for 30 minutes resulted in
a more tender formed roll than mixing for only 10 minutes.
Palladino
and Ball (1979) observed that addition of salts, with the exception of
those containing calcium, had a tenderizing effect on spent hen muscle.
Sodium chloride, because of its widespread use, was recommended as a
tenderizer.
7
Flavor
Salt which is widely used to enhance the flavor of meat, also re­
duces the sourness of acid flavors and increases the sweetness of sugar
(Johnson and Peterson, 1974).
Schwartz and Mandigo (1976) found that as
the salt content of restructured fresh pork was increased from 0 to
2.25%, taste panel scores peaked at 1.5%.
Marquardt et al. (1963) re­
ported no difference in consumer preference of hams where salt level
was varied from 1,5 to 3%.
Neer and Mandigo (1977) found that cured
restructured meat products became more palatable as the salt content
was increased from 0 to 3%.
They suggested that more salt is acceptable
in cured meat products than fresh products because consumers expect
cured meats to be salty.
Gardze et al. (1979) studied beef-soy patties and found that salt
caused few changes in meaty and cereal-like aroma but generally increased
meaty flavor, saltiness and desirability scores and decreased cereal
flavor.
Rakosky (1974) found that adding salt to soy products had a
masking effect on soy flavor but the amount of salt needed for the
masking effect was not stated.
Oxidation
The pro-oxidant effect of NaCl on the triglycerides in meat is one
of the most puzzling influences in food science.
Observations reported
sometimes seem contradictory, and it is possible that a number of factors
can operate jointly.
Banks (1937) and Lea (1939) suggested that NaCl
was not itself a pro-oxidant, but promoted the activity of lipoxidase
in meats.
However, Banks (1944) and Tappel (1952, 1953) showed that
8
there was no lipoxidase in meat, and it was concluded that the activity
promoting autoxidation was due to heme pigments.
Separately, the pig­
ments of meat are powerful catalysts of fatty acid and triglyceride
autoxidation.
However, frozen fresh meat is relatively stable despite
the presence of such catalysts.
There is no evidence of a direct specific role of salt in oxidation
catalyzed by myoglobin or hemoglobin.
Chang and Watts (1950) found that
salt did not accelerate oxidation in clear meat extracts or hemoglobin
solutions.
However, Lea (1939) observed a pro-oxidant effect of salt
on oxidation caused by expressed meat juice.
In this instance, salt
may have acted on the tissue cells or fibers in the press fluid,
Olson
and Rust (1972) found that flavor of dry cured hams was preferred when
a salt with low heavy metal content was used instead of regular salt,
indicating that trace metals may have caused the pro-oxidant effects
rather than the NaCl.
Salt has a powerful influence on meat proteins and pigments (Cole­
man, 1951; Grant, 1956).
Gibbons et al. (1951) reported that rancidity
of bacon sides developed more rapidly as freezer storage temperature
was lowered.
Labuza (1971) theorized that NaCl may affect the water
activity, and in turn, the oxidative stability.
It was concluded that
the freezing process was similar to a dehydration process.
As an
example of the effect of physical conditions, Mabrouk and Dugan (1960)
found that autoxidation of aqueous emulsions of methyl linolate were
inhibited by an increase in dissolved salt.
Ellis et al. (1968) re­
ported that sodium nitrite caused greater oxidation of frozen cured
bacon than NaCl and concluded that the oxidative effects of NaCl and NaN02
9
were independent and additive in cured products.
They also studied
various chloride salts and found that possible direct effects of NaCl
on oxidation did not appear to involve a reactive chloride ion.
Health concerns for salt
Salt is essential to the normal function of the human body; how­
ever, there has been considerable concern for the amount of salt in the
diet.
Much of this concern centers on questions about dietary sodium and
hypertension, or high blood pressure.
Since the sodium ion plays a major role in the physiological regula­
tion of body fluids, it is reasonable to assume that it is capable of
influencing blood pressure.
Hypertension has been ranked first as a
risk factor for cardiovascular disease and a diagnosis of hypertension
doubles the risk of death from stroke, heart attack, or kidney disease
(DHHS, 1979).
Freis (1976) summarized four principal sources for implicating
salt as a contributor to hypertension: 1) epidemiological studies in
unacculturated people showing that the prevalence of hypertension is
inversely correlated with the degree of salt intake; 2) hemodynamic
studies suggesting that the development of chronic experimental hyper­
tension is a homeostatic response to a maintained increase in extra­
cellular fluid volume (ECF); 3) evidence that the ECF of "salt eaters"
is expanded in comparison to that of "no-salt eaters;" and 4) studies
of hypertensive patients receiving either diets greatly restricted in
salt or continuous diuretic therapy both of which show a fall in blood
pressure correlated with a reduction in ECF.
10
There are many reports concerning human health and sodium intake
(Senate Select Committee on Nutrition and Human Needs, 1977a, b; CAST,
1977; HEW-FDA, 1978; AM, 1978; FDA-USDA-FTC, 1979; Marsden, 1980;
Kolari, 1980; Sebranek et al., 1983).
These papers have concluded that:
1) hypertension afflicts a portion of the U.S. population which is
genetically predisposed, but which makes up some 20% or more of the
population; 2) daily intake of sodium chloride by North American con­
sumers is about 10-12 g (3,900-4,700 mg of sodium) which is 20-25 times
the physiological requirement; 3) of this daily intake, about 3 g of
salt is naturally present in foods, 3 g are added in cooking (discretionary
intake) and some 4-6 g are added during commercial processing of foods;
4) about 43% of the respondents to FDA hearings and 26% of the
respondents to food labeling surveys (FDA-USDA-FTC, 1979) reported that
they avoided foods which had salt listed on the label.
Consequently,
those people had a very real concern for how their food affected their
health and well-being.
Sodium Nitrite
Reactivity of nitrite in meat curing
The term nitrite is used genetically to denote both the anion, NO",
and the neutral nitrous acid, HNO^, but it is the nitrous acid which forms
nitrosating compounds.
The acid dissociation constant for the nitrous
acid/nitrite equilibrium is 3.98 x 10 ^ (PK^ = 3.4); at the pH of meat
(5.5-6.5), the concentration of the reactive acid is less than 1% of the
total nitrite (Challis and Butler, 1968). Nitrite can serve as both a
11
reducing agent and an oxidizing agent (Bard and Townsend, 1971).
The basic chemistry of the reactions of nitrite can be found in two
major reviews (Ridd, 1961; Challis and Butler, 1968) which discuss
nitrosation, diazotization, and deamination.
Nitrosation is of greatest
interest to meat curing.
Meat appears to be in a static state but actually it is a very dy­
namic tissue with respect to curing chemistry.
Not only processing
parameters but also animal treatments before and after slaughter should
be taken into account when considering cured meat chemistry.
It is
difficult to control composition and properties of raw meat, but the
interaction of meat quality and processing parameters should be
recognized since the natural properties of meat may be utilized for some
control of residual nitrite. Lee et al. (1976) showed that samples
made with white muscle had lower residual nitrite than those from red
musdie.
They found that this difference resulted from the high pH
of the red muscle.
When they adjusted the pH of white (or red) to
that of red (or white), the white portion had a higher residual nitrite.
It appeared to be due to the fact that red muscle has more myoglobin
leading to a lower residual nitrite.
I'Shler and Scheerer (1979a)
reported that binding of nitrite was greater for light than for dark
muscle.
A number of reactive species may be formed from nitrous acid,
thus leading to a complex chemistry.
According to Bonnett and Martin
(1976), the possibilities for such reactive species are:
12
(Nitrous acid)
HNO^ +
^2^^2 (^^trous acidium ion)
^2^*^2^^2^ ^ NO^ (Nitrosonium ion)
2HNO2 + h"*"^ H^O + NgOg (Dinitrogen trioxide)
SHNOg % HgO + HNO^ + 2N0 (Nitric acid and nitric oxide)
If nitrous acid is consumed via reaction (by various reactive species)
with components in cured meat, nitrite will continue to decrease because
of an equilibrium shift toward nitrous acid formation in the reaction,
NOg +
TZLHNOg. Dahn et al. (1960) reported that the order of reactivity
of these species is:
NO^ > H2NO2 > ^2*^3' Boyland (1972) stated that
the nitrosating agent is probably neither nitrous acid nor the nitrite
ion, but a derivative, which can be a nitrosyl halide or other nitrosyl
compounds.
Ridd (1961) reported that nitrous acid and hydrochloric
acid will generate nitrosyl chloride (NOCl) which is a more powerful
nitrosating species than dinitrogen trioxide (N^O^). Formation of
nitrosyl chloride in meat as opposed to formation of dinitrogen trioxide
could have a number of important consequences.
pH effect on nitrite
The influence of pH on residual nitrite levels is quite wellestablished.
Greenwood (1940) reported that when a peptone solution
was heated with nitrite, loss of nitrite was greater at lower pH than
at higher pH.
Rose and Peterson (1953) found that recovery of nitrite
from a suspension of pork was decreased when the pH was lowered.
Nordin
13
(1969) showed that the rate of nitrite depletion in canned ham was
exponentially related to pH and temperature.
Loss of nitrite from
pickling brines in storage increased with low pH (Jacquet et al., 1972).
Sebranek (1974) stated that greater loss of nitrite occurred in aqueous
solutions at lower pH when held at room temperature.
in pH can be quite important.
A small decrease
Fox et al. (1967) indicated that a pH de­
crease of 0.2 unit in meat will double the rate of color formation due to
nitrite-myoglobin interaction. Generally, as pH decreases from 6.5 to
5.5, cured color development is more rapid and complete (Fox and Thomson,
1963).
Residual nitrite that remains in the tissue after cure processing
(including cooking) serves as a reservoir for nitric oxide for continued
stabilization of the color pigment and other components of muscle.
Olsman and Krol (1972) and Olsman (1974) developed kinetic data
describing nitrite depletion with respect to pH,
Replacing the
equilibrium equation of nitrous acid,
[H+][Nop
d log [NaNO^]
dt
d(log [HNOg] + log
gives
"^
+pH)
^
= K
and because both pH and the equilibrium constant
are independent
of storage time t,
d log [NaNO^]
d log [HNO^]
=
dt
K
dt
Therefore, we can estimate nitrite depletion as a function of nitrous
acid.
At a given nitrous acid concentration c, the logarithmic deple­
14
tion rate is
d log [HNOg]
V =
c
dt
= [HNOg] = K[H^],
c
where t is the storage time and K is a concentration dependent factor.
At a high pH, this factor is nearly constant over a broad range of
concentrations giving linearity to the depletion rate.
the change of
In this case,
concentration with storage time will be
d[HNO ]
= K[H ][HNO^] = K'[H l^fNaNO^]
However, at a low pH, the linearity of the depletion rate is lost because
d log [HNO^]
the change in the slope
—
with storage time progressively
increases with decreasing pH.
As a result, the kinetics of nitrite
loss with respect to pH is between first and second order.
The idea of lowering pH to reduce residual nitrite has been de­
veloped and utilized in meat products in attempts to inhibit the forma­
tion of nitrOS amines (Goodfellow, 1979).
A drastic reduction of residual
nitrite was achieved by lowering the pH in a fermented sausage using
a starter culture (Zaika et al., 1976).
Another area where pH effects
may become evident is in a variety of smoke applications.
Sink and
Hsu (1977) showed a lowering of residual nitrite in a liquid smoke dip
process for frankfurters when the pH also was lowered.
They suggested
that phenolic compounds from smoke may contribute to lowering the pH of
the products as well as the residual nitrite.
The effects of smoke in
nitrite reduction seem to be a combination of pH decrease and direct
nitrosation of phenolic compounds (Knowles, 1974).
Some chemical
acidulants such as acetic acid, glucono-delta-lactone, citric acid
15
and sodium acid pyrophosphate have been used to reduce pH (Goodfellow,
1979).
The van Slyke reaction (nitrous acid reacts with the alpha-
amino group) has been suggested as being responsible for nitrite
decomposition in the presence of acids (Bard and Townsend, 1971).
Temperature effect on nitrite
Many workers have shown that heat treatment causes depletion of
nitrite.
The depletion increases with increasing temperature as with
increasing time at a given temperature (Brooks et al., 1940; Greenwood,
1940; Rose and Peterson, 1953; Pivnick et al., 1967; Johnston et al.,
1969; Olsman and Krol, 1972; Sebranek et al., 1973; Raseta et al., 1978).
However, Greenwood (1940) concluded that the cooking temperature was a
more important factor than the cooking time in causing the destruction
of nitrite.
Brooks et al. (1940) suggested that the loss of nitrite during
heating was not due entirely to the thermal decomposition of sodium
nitrite (3HNO2 -* HNO^ + 2N0 + H^O).
Since in separate experiments,
both nitrogen and nitric oxide were found to be evolved, the formation
of nitrogen suggested that nitrite reacted with amino groups in the
tissue.
Greenwood (1940) also proposed that the loss of nitrite with
heat was due to the decomposition of nitrite by reaction with meat, and
the destruction might well be due to aliphatic diazo reaction.
The
reactions for loss of nitrite in cured meat upon heating suggested by
the author were:
(1) RCHNH^COOH + HONG
RCHOHCOOH +
+ H^O
(2) Amino or hydroxy acids + HONO -* CO^ + X + Y + Z
16
(3) ZHNO^ ^ H^O + NO + NO^
Rose and Peterson (1953) showed that loss of nitrite, from a pork
suspension, increased with increasing temperature until protein de­
naturation became marked.
An explanation might be that increased re­
ducing enzyme activity caused more nitrite depletion until denaturation
occurred.
However, Walters and Taylor (1963) showed that the ability
of pork muscle mince to reduce nitrite to nitric oxide was decreased
with increasing temperature.
Cavlek et al. (1974) reported that nitrite
decomposition was found to be closely related to nitrosomyoglobin forma­
tion in beef upon heating. Lee and Cassens (1976) showed that heated
myoglobin had twice as much
as unheated samples when labelled
NaNOg was incubated with myoglobin.
It has been shown that the amount
of nitric oxide bound to myoglobin increased with increasing temperature
and time (Renerre and Rougie, 1979).
MShler and Scheerer (1979b) re­
ported that appreciable binding of nitrite to heme pigment-free meat
samples was observed after heating at 75°C for 100 min, but not in nonheated samples.
Christiansen et al. (1973) reported that cooking had little or no
effect on nitrite levels in canned comminuted pork cooked at 77°C to
an internal temperature of 68.5°C,
Again, Greenberg (1972) also reported
recoveries of nitrite in ground pork after heat processing to be similar
to values found after formulation and before heat processing.
These
results may have been due to less time required to heat the product
because of the size of the can used (Kolari and Aunan, 1972).
17
The overall loss of nitrite due to heat treatment probably results
from a combination of several factors including heat decomposition of
nitrite itself, reduction of nitrite by increased activity of reducing
systems and increased reaction of nitrite with meat components which
became more available for reaction due to heat.
The most comprehensive
work concerning the effects of temperature on depletion of nitrite was
done by Nordin (1969).
The author used various temperatures from -2.2°C to
97.20C to derive the relationships between temperature, nitrite deple­
tion, product pH and time.
The rate of depletion appeared to be ex­
ponentially related to both pH and temperature and the equation could
be expressed as follows:
(1) log NaN02 = a + bt
where a = log initial concentration;
b = constant depending on pH and temperature;
t = time.
(2) log (1/2 life) = 0.65 - 0.025 (temperature °C) 4- 0.35 (pH),
It was pointed out that the rate was not affected by heat denaturation of meat and the relationship did not apply at room temperature.
At room temperature, depletion was unexpectedly rapid, indicating that
bacterial action was probably a factor.
In practical aspects, the effect of temperature has been used in
various ways to reduce the residual nitrite in the final product.
Sebranek (1979) pointed out that bellies pumped at a higher temperature,
resulting in increased brine retention, showed less residual nitrite
after processing than bellies pumped at low temperature.
He suggested
18
that the higher temperature may lead to increased nitrite utilization
by the tissue to more than overcome the effect of increased brine
retention.
In addition, a prolonged smokehouse process at low tempera­
ture was recommended to lower the level of residual nitrite in bacon
(Paul, 1979).
This might be related to sensitivity of nitrite reducing
ability in pork to cooking temperature (Walters and Taylor, 1963).
After
cooking, slow chilling of the finished product was proposed for additional
reduction of nitrite (Meat Industry, 1979).
Other factors influencing nitrite
Additives with reducing properties or with properties that lower
the pH value generally cause a reduction in the residual nitrite content
(Mirna and Coretti, 1977).
A very common additive in cured meat has
been ascorbic acid or sodium ascorbate. Sauter et al. (1977) claimed
that addition of sodium erythorbate to the curing mixture had no signifi­
cant effect on the rate of nitrite disappearance in cured pork.
However,
it is generally accepted that erythorbate has similar properties to
ascorbic acid in all aspects except antiscorbutic (vitamin C) activity
(Ranken, 1974).
In cured meat, ascorbic acid (or the sodium salt) has been added
to insure increased yields of nitric oxide from nitrite.
However, the
compatibility of sodium ascorbate and sodium nitrite in meat curing
mixtures prior to and during the curing has been recognized as a prob­
lem because of nitrite loss.
In curing solutions, high pH and salt
concentration in addition to low temperature lowered the rate of reaction
between nitrite and ascorbate (Hollenbeck and Monahan, 1955).
The fact
19
that ascorbic acid, ascorbate or erythorbate added to cured meat
accelerates nitrite loss has been substantiated by many workers (Ando
and Negata, 1969; Grau, 1969; Mirna, 1972; Raevuori et al., 1973; Fox
and Nicholas, 1974; Rahelic et al., 1974; Woolford and Cassens, 1977;
Mathey, 1979; Scheid and Lordt, 1979).
The mechanism of reaction between nitrite and ascorbic acid has
been studied since the early 1930s, mainly by German researchers with
emphasis on kinetics of the reaction.
Karrer and Bondas (1934) reported
first that ascorbate, in a boiling reaction medium at pH 6.0 or below,
reduced nitrite to nitric oxide, which accumulated under anaerobic
conditions.
Evans and McAuliffe (1956) found that when nitrite and
ascorbic acid were incubated anaerobically at a ratio of 1/50 at 26°G
for 30 minutes, NO, N^O and N^ were produced.
The reaction with ascorbate
occurred at pH values as high as 6 and increased rapidly with decreasing
pH.
The reduction of nitrite by ascorbate has been extensively studied
by Dahn et al. (1960), who have found that the kinetics of the inter­
action was markedly dependent on the pK over the range of 0 to 5.
At
the acid extreme (pH 0-1), the reaction occurred between nitrous acidium
ion or nitrosonium ion and undissociated ascorbic acid.
The reaction
4+
species in this range would be H2NO2 or NO for nitrous acid and the PK^
of ascorbic acid is 4.1.
The rate of reaction was first order in terms
of both nitrous acid and ascorbic acid concentration.
The rate of reac­
tion at pH 3-5 was independent of ascorbic acid concentration and was
second order in terms of nitrous acid concentration.
This means that
reaction probably occurred between dinitrogen trioxide and the mono
anion of ascorbic acid.
In the acidity range (pH 1.5-2.5) between
20
the two mentioned above, the reaction occurred between dinitrogen
trioxide and various proportions of either the undissociated form or
the mono anion form of ascorbic acid.
Fox et al. (1981) reported the presence of intermediates by spectral
analysis of reacting solutions of nitrite and ascorbate.
With electron
spin resonance measurements, they found that the reaction proceeded with­
out the formation of any detectable ascorbyl radical.
Anion exchange
column chromatography of the intermediates showed that they were
nitrosoascorbic acid and nitrodiketogulonic acid.
A few other additives have been studied in terms of their effect
on residual nitrite in cured meat.
Compounds such as acetic acid,
lactic acid, citric acid, glucono-delta-lactone and sodium acid pyro­
phosphate are used for direct acidulation of cured meat (Goodfellow,
1979); in general, they reduce the residual nitrite level due to lower
pH in the final product.
When such compounds were used in combination
with ascorbate, the effect on residual nitrite may be intensified.
For
example, a highly significant reduction of residual nitrite resulted
from the use of trisodium citrate and sodium ascorbate (Scheid and
Lordt, 1979).
Citric acid and sodium erythorbate were recommended as
well to reduce the residual nitrite in sausage products (Raevuori et al.,
1973).
The effect of phosphates may depend upon their ability to change
pH of the product when they were used in curing (Sebranek, 1979).
Mori
et al. (1972), for example, mentioned that addition of phosphate and
sucrose to nitrite treated meat resulted in a high residual nitrite
level.
This was probably because phosphate increased pH.
However,
21
Neer and Mandigo (1977) reported that residual nitrite was found to be
significantly decreased with increasing sodium triphosphate.
This may
have been caused by sodium triphosphate 's ability to reduce metal ions
(Inklaar, 1967), and could result in increased cured color evaluations,
as reported by Volovinskaya et al. (1959).
Glucono delta lactone has been reported to be effective in reducing
the residual nitrite in ham (Pate et al., 1971) but a comparatively
small amount of nitrite disappeared as nitric oxide when this compound
was added during the mixing of brines (Mathey, 1979).
Some food additives are effective in reducing the residual nitrite
only when they are used in the presence of ascorbate.
Ando (1974)
reported that the addition of glutamine, succinate, nicotinic acid or
nicotinamide appreciably enhanced the decomposition of nitrite during
cooking only in the presence of ascorbate at pH 5.0.
The use of
nicotinamide in preparation of sausage was also studied.
The addition
of nicotinamide to cured sausage products reduced nitrite content by
a factor of 3 (Shalushkova and Ivanov, 1976).
This may be interpreted
to mean that meat has some endogenous ascorbic acid.
However, reduced
nicotinamide adenine dinucleotide (N^H) caused a decrease in the nitrite
content in meat slurries without the addition of ascorbate (Fox and
Nicholas, 1974).
Sofos et al. (1979) reported that the presence of sorbic acid in
the formulation decreased the disappearance rate of nitrite in chicken
frankfurter type emulsion, whereas no effect of sorbic acid on the rate
of nitrite depletion was observed in bacon (Sofos et al., 1980).
However,
sorbate enhanced the effect of ascorbate on the decomposition of nitrite
22
in model systems and combination of both additives was more effective
in decomposition of nitrite in sausage than either of them alone.
This
was probably due to the effect of sorbate which resulted in a pH decrease
of 0.2-0.4 units (Ando and Negata, 1969).
Ando (1974) observed that ferrous ion significantly decomposed
nitrite in the absence of ascorbate, and Mg
I
H**!*
I ,|.
I II
, Ca , Zn
and Fe
enhanced the decomposition of nitrite in the presence of ascorbate,
Olsman (1977) used meat emulsions and found that addition of Fe"*^
at 0.1 or 1.0 |Jmole/Kg resulted in a higher content of protein-bound
nitrite.
The effect was pronounced at pH 6.2, but absent at pH 5.1.
Tompkin et al. (1978) have suggested that iron is critical to the inhibi­
tion of Ç. botulinum in perishable canned cured meat.
Their hypothesis
is that nitric oxide reacts with an essential iron-containing compound
within the germinated botulinal cell and prevents outgrowth; an excess
of "available" iron negates the inhibitory effect of nitrite.
The influence of meat systems on nitrite reactions can also be
considered in more general terms.
Two points can be made; first, meat
contains an enormous number and variety of chemicals; and second, these
chemical constituents are compartmentalized within or by complicated
morphological structures of the muscle.
Goutefongea et al. (1974)
reported that when myofibrils from Longissimus dorsi of pig were incubated
with NaNO^ and thermally processed, about 10% of the initial nitrite
was lost.
Tinbergen (1974) reported that a water-soluble dialyzable
fraction of beef muscle strongly reduced nitrite content in a heated
anaerobic system, the activity being pH dependent.
When amino acids
and small peptides were removed from that fraction, the nitrite content
23
slowly decreased suggesting the possible reducing activity of a nonamino acid compound.
Ando (1974) showed similar results in which some
low molecular weight fraction of sarcoplasm of porcine muscle was active
in decomposing nitrite,,
Different proteins have different amino acid compositions, thus
contain a different number of potential reaction sites with nitrite.
It is well-known that nitrite reacts with sulfhydryl groups to form
nitrosothiols, and a number of researchers have studied nitrosothiol
formation in meat.
Mirna and Hofmann (1969) showed that the decrease
of nitrite was paralleled by a decrease of sulfhydryl groups in minced
meat.
Blocking of the sulfhydryl groups resulted in a decrease of the
nitrite loss.
However, at pH of 6.27, the effect was unexpectedly small
(Olsman, 1974).
Reaction of sulfhydryl groups with nitrite was greatly
influenced by pH and temperature, but Rubbered et al. (1974) observed
that formation of nitrosothiols accounted for only a small portion of
the nitrite lost in normal cured meat.
Fox and Nicholas (1974) showed
that the addition of cysteine to meat slurries caused a loss of
nitrite.
This might be because the added cysteine acted as a reducing
agent and was more available to nitrite for reaction.
The fact that
nitrous acid reacted preferentially with surface residues (Kurosky and
Hofmann, 1972) can be another explanation.
When mixtures of 19 amino
acids, which contained cystine instead of cysteine, were incubated with
nitrite at 37°C, no decrease in nitrite was observed.
However, some
depletion of nitrite occurred when the medium was heated at 121°C,
It was postulated that the loss of nitrite on reaction with cystine
was due to the reaction of cystine to cysteine during autoclaving with
subsequent reaction of nitrous acid with the free sulfhydryl group
(Riha and Solberg, 1973).
The alpha-amino group of amino acids, the epsilon-amino group of
lysine, the ring nitrogen of tryptophan, and tyrosine could react with
nitrous acid (Kurosky and Hofmann, 1972), thus depleting nitrite.
Reac­
tion of tyrosine with nitrous acid has been shown by many workers
(Philpot and Small, 1938; Means and Feeney, 1971; Bard and Townsend,
1971; Knowles, 1974; Woolford et al., 1976).
However, the addi­
tion of tyrosine to meat slurries did not cause any loss of nitrite
(Fox and Nicholas, 1974).
Nakai et al. (1978) reported that the condi­
tion for nitrosation of tryptophan is unfavorable in most cured meat.
When tryptophan was added to meat slurries and incubated for 300 minutes
at 60°C, no loss of nitrite resulted (Fox and Nicholas, 1974).
Histidine
caused loss of nitrite while lysine did not when they were added to meat
slurries and incubated at 60°C (Fox and Nicholas, 1974).
Since the
effect of histidine on nitrite loss was observed only in systems con­
taining meat, it would appear that histidine acts only as an inter­
mediary or catalyst in some other reaction.
Reaction may occur between
nitrite and proline to form nitrosoproline (Kushnir et al., 1975;
Bharucha et al,, 1979), thus proline may contribute to the loss
of nitrite.
Even though most cured meat products contain high amounts of
lipid, the study on the reaction of lipids with nitrite leading to
nitrite loss in cured meat has been neglected. Woolford and Cassens
(1977) reported that both protein and lipid in bacon play a role in
the reduction of nitrite level. Goutefongea et al. (1977) studied
25
directly the interaction of nitrite with adipose tissue, and found that
nitrite reacted with the connective tissue component, extracted lipid
and also with unsaturated carbon-carbon bonds when conditions are
similar to those for meat curing.
Waldman et al. (1974) showed that codium nitrite concentration de­
creased as storage time increased in preblended sausage meat, but after
manufacturing the sausage, there was not much difference in residual
nitrite between products with or without preblending.
Puolanne et al.
(1978) reported that meat precured with nitrite had less residual nitrite
than normal cured meat.
These results might be related to a greater ni­
trite reducing ability in raw tissue as compared with cooked.
Chyr (1978)
found that an uncooked braunschweiger mixture contained about 1/3 as
much residual nitrite after 3 days of storage as the same mixture
that had been cooked.
Radiation caused considerable loss in residual nitrite during
storage (six months) of canned pork (Hougham and Watts, 1958). Wierbicki
and Heiligman (1974) also showed that after one month of storage, nonirradiated ham cured with 200 ppm of nitrite and 550 ppm of ascorbate
had more residual nitrite than that of irradiated sample.
However, ir­
radiated samples were stored at ambient temperature (25 + 3°C) while the
nonirradiated samples were stored at -29°C.
Fiddler et al. (1981)
showed that irradiation sterilization with Co^O reduced more residual
nitrite in bacon than nonirradiated bacon,
Wedzicha et al. (1982) reported that the intermediates of nonenzymatic browning reactions caused a significant loss of nitrite at
26
either pH 4.7 or pH 7 when they were heated with nitrite in a model
system at 41°C.
Salt and Nitrite Interaction
Reaction of salt with sodium nitrite has been studied by several
researchers.
Greenwood (1940) heated a peptone solution with nitrite
and observed that as concentration of salt was increased, an increased
loss of nitrite occurred. However, Goutefongea et al. (1974) showed
that when myofibrils were treated with nitrite and 0 or 3% sodium
chloride, there was no difference in free nitrite, and Olsman and Krol
(1972) found that during the storage of meat products, the content of
sodium chloride had no effect on the rate of depletion of nitrite.
Lee
and Cassens (1980) reported on the effect of sodium chloride on
residual nitrite in a model system, in ground meat and in bacon.
The results showed that residual nitrite was lower in the presence of
larger amounts of sodium chloride.
However, if the effect of NaCl in
decreasing the pH and the effect of loss of water during cooking and
storage were excluded, then sodium chloride did not cause a significant
decrease in measurable residual nitrite.
Chloride catalysis of diazo pigment formation in the Griess method
for nitrite determination shows a reagent specific effect (Hildrum,
1979).
Different salt levels resulted in different amounts of Griess
pigment being formed, depending on the particular reagent combination
used.
That is, apparent nitrite values were increased by chloride
levels when sulfanilic acid was used as the nitrosatable species before
27
coupling to form the dye (Fox et al., 1981; Fox et al., 1982; Reith and
Szakaly, 1967a, b).
When sulfanilamide was used, the reaction was in­
sensitive to chloride concentrations.
Thus, some of the earlier attempts
to measure residual nitrite may have included some error due to chloride
affecting the analytical method.
Comparisons of the effect of chloride
and reductants on measurable nitrite in a pH 5.6 meat slurry using
several analytical approaches (Griess method, chemiluminescent detection,
and differential pulse polarography) for measuring nitrite have been
made (Fox et al., 1981; Fox et al., 1982).
Although there is some dif­
ference as a result of analytical methodology, a relative reduction of
measurable nitrite with increasing sodium chloride was almost universally
demonstrated.
An analysis of variance showed a significant three-way
interaction between salt, reductant, and heat processing; the authors
attributed the differences in loss of nitrite to formation of NOCl.
An accompanying model system which contained only buffer, nitrite,
ascorbate, and salt did not show the chloride effect observed in the meat
slurry.
The chloride effect in these model meat systems, therefore,
must involve tissue components, modified by ascorbate.
Since the
fastest nitrosation reaction is with ascorbate, the tissue component
reactions must involve secondary reactions with nitrosoascorbate inter­
mediates or reaction products thereof.
In addition to measurable nitrite, another indication of formation
of NOCl and increased nitrosating ability would be enhanced production
of nitrosyl heme pigments due to increased production of the nitrosoreductant intermediates.
If significant NOCl is formed, increased
nitrosating activity should result in increased nitric oxide and
28
subsequent nitrosyl heme formation.
Reith and Szakaly (1967a) reported
that salt had an adverse effect on the color intensity and formation of
nitrosylmyoglobin in model systems consisting of myoglobin, nitrite,
ascorbate, and sodium chloride, whereas Fujimaki et al. (1975) showed
that sodium chloride increased residual nitrite, enhanced development
of color, and decreased the level of nitrate in model systems with the
same components as mentioned previously but at different molar ratios.
Fox et al. (1980) studied the ultrastructure of fresh and cured meat
and reported a unique structural change in the interfiber spaces caused
only by a combined treatment of sodium chloride and sodium nitrite.
Scanning electron microscopy (SEM) of the ultrastructure of cured beef
has shown that neither sodium chloride nor sodium nitrite alone
caused changes in the interfiber spaces.
The authors also demonstrated
a five-fold increase in the reaction rate constant for formation
of nitric oxide in 3M (~ 24%) NaCl as compared to no NaCl.
The
combined effect of nitrite and chloride was attributed to catalysis
of nitrosation reactions through the formation of the more powerful
nitrosating species, NOCl.
Hildrum et al. (1975) reported that sodium chloride (IM) activated
nitrosation of proline in strongly acidic media, whereas it produced an
inhibiting effect on nitrosation in mildly acidic or neutral food
systems, but Fiddler et al. (1973) found that sodium chloride (2.5%)
had no effect on the formation of dimethyInitrosamine.
Mirvish at al.
(1973) reported that the reducing effect of sodium chloride on Nnitrosation of sarcosine was not seen in lower concentrations when
sodium chloride was 0.05, 0.15 and 0.5M because chloride ion concentration
29
was low.
Thei1er et al. (1981) studied N-nitrosamine formation in meat
and concluded that low sodium chloride levels led to greater nitrosopyrolidine formation during cooking.
suggested to account for this effect.
No specific mechanism was
However, it seems clear that
the precursors for nitrosamine formation are present and will react with
nitrite when conditions are conducive to the reaction (Spinelli-Gugger
et al., 1981).
Considering the above review, sodium chloride could play a direct
role in the reactions of nitrite or could act indirectly.
Changing
chloride levels could alter some or all of the several chemical reaction
characteristics of nitrite during meat curing depending on whether
chloride ions interact significantly with nitrite.
The nitrite and
chloride interaction has not been well-addressed, with the limited
available information appearing to be somewhat indecisive:
More­
over, sodium chloride is undergoing some reduction at the present
time in processed foods and knowledge of potential changes in nitrite
reactions, as a result, would be an important element in maintaining
product quality and safety.
30
OBJECTIVES OF THE STUDY
The project had the following objectives;
1.
To determine the nature and extent of chemical interaction
between sodium nitrite and sodium chloride in a cured meat product.
2.
To determine the effect of pH on sodium nitrite and sodium
chloride interaction.
3.
To determine the effect of different internal finishing
temperatures on sodium nitrite and sodium chloride interaction.
4.
To determine the effect of different muscle types on sodium
nitrite and sodium chloride interaction.
31
MATERIALS AND METHODS
Experimental Design
The interaction of various combinations of salt and nitrite was
investigated by using five different product variables.
These variables
included acidity, presence or absence of reductant, different internal
finishing temperatures, and presence of white muscle (turkey breast
muscle).
Measurements of pH, salt content, nitroso pigment, total pigment,
and residual nitrite were conducted at four time intervals (0-day,
1-day, 15-day, and 30-day) after the completion of product manufacturing.
Zero-day measurements were made on raw samples before cooking, and 1-day
samples were evaluated 24 hours after heat processing.
Yields after
cooking were monitored in the experiment evaluating different cooking
temperatures.
All measurements consisted of three replications.
Meat and Formulation
All meats were obtained from and processed in the Iowa State
University Meat Laboratory.
pork trimmings were used.
Frozen boneless 90% lean beef and 50% lean
These meat ingredients were thawed in a 4°C
cold room for a period of 36-48 hours.
Each meat ingredient was ground
through a commercial meat grinder (Weiler & Company Grinder, Model 6)
fitted with a 3/8" plate.
After grinding, the meat was divided into
appropriate batching units, placed in plastic bags and stored in a
-20°C (-4°F) freezer.
Twenty-four hours before production, all meat
was taken from the freezer and tempered to 0°C (32°F).
32
Production
Production was conducted in a refrigerated room held at 3°C (37.4°F).
A Hobart Laboratory Chopper (Model 8418 ID) with a 480 mm (18.9 in.)
diameter bowl was used.
The lean beef portion was chopped for 30
seconds to reduce particle size before the addition of salt.
The
mixture was then chopped for one minute before addition of all of the
remaining ingredients after which the batter was chopped until IZ^C
(53.6°?) was reached.
The batter was then placed in an E-Z Pakmobile
Stuffer (E-Zuber Engineering and Sales, Minneapolis, Minnesota) fitted
with a 16 mm diameter stuffing horn, and the product was stuffed into
2 in. X 16 in. fibrous casings that had one end previously clipped
(Tipper Clip-Model 4057).
After stuffing, the casing was
hung on a smoke truck and cooked in a Mauser and Sohn Allround
System oven.
Cooking schedules are given in Table 1.
Finally, the
cooked product was chilled (2°C) overnight (18 hours) prior to vacuum
packaging.
Experiment I (Control)
The initial experiment was designed to study an emulsion type
product made with five levels of salt (0%, 0.5%, 1%, 2%, and 4%),
and two levels of sodium nitrite (75 ppm and 156 ppm).
will be referred to hereafter as the "control."
formula of products produced.
This experiment
Table 2 shows the
33
Table 1.
Time
(min)
Cooking schedule
Service
temp (OC)
Program
30
Drying
50
30
Hot air finish
71
—
Cook scalding
71
Cold shower
—
5
Table 2.
Formula:
Moist I
55
Moist
impulse
Core
temp (OC)
85
85
68
Experiment I (control)
Ingredients
Meat
Beef
Pork
Spice^
Sodium nitrite:
Salt (NaCl):
Weight (g)
75 ppm
156 ppm
0.5%
1.07=
2.0%
Erythorbate:
4.0%
550 ppm
1134.0
1134.0
14.18
0.17
0.354
11.34
22.68
45.36
90.72
1.24
^ixon #244-N Wiener Seasoning.
Experiment II (Reduction of Product pH)
This experiment was designed to evaluate the effect of product pH
and consisted of two different experimental comparisons. Each ex­
periment had twenty treatments.
Ten treatments were the same as the
control (Experiment I) while the other ten included addition of glucono
34
delta lactone (GDL) to increase acidity.
The first group utilized
1.5% glucono delta lactone which reduced the pH drastically.
The second
group included less glucono delta lactone (0.2%) in order to produce a
less drastic alteration of the pH.
Experiment III (Effect of Reductant)
The effect of reductants was studied by leaving erythorbate
out of the formulation.
Erythorbate had been included in all
of the previously described products.
The experimental design
was the same as that of the 0.2% glucono delta lactone treatment
(Experiment II).
Experiment IV (Different Internal Finishing Temperatures)
This experiment was designed to compare products cooked to different
final core temperatures (52°C, 60°C, and 68°C).
The core temperature probe
in the oven was programmed to 68°C and each experimental batch was taken
out of the smokehouse when the desired target temperature (52°C or 60°C)
was reached.
Consequently, the cold shower was not included for any of the
three treatments.
This experiment included three levels of salt (0%,
1%, and 3%) and two levels of sodium nitrite (75 ppm and 156 ppm).
Experiment V (Effect of Muscle Type)
The design of this experiment was identical to that of Experiment I
except that turkey breast meat and pork back fat were utilized as meat
35
raw ingredients.
The turkey breast meat was obtained from a commercial
processing company and pork back fat was obtained from the Iowa State
University Meat Laboratory.
for previous experiments.
Table 3.
Formula:
The meat was again prepared as described
The formulation used is given in Table 3.
Experiment V (with turkey muscle)
Ingredients
Meat
Turkey breast
Pork back fat
Spicea
Sodium nitrite:
Salt (NaCl):
Erythorbate:
75 ppm
156 ppm
0.5%
1.0%
2.0%
4.0%
550 ppm
Weight (g)
1587.6
680.4
14.18
0.17
0.354
11.34
22.68
45.36
90.72
1.24
^ixon #244-N Wiener Seasoning.
Chemical and Physical Analysis
£H
The pH of the samples was determined by homogenizing ten gram por­
tions of the meat product with 100 ml of deionized, distilled water in
a Virtis homogenizer for 60 seconds.
The pH of the slurry was deter­
mined using a standardized, Fisher Accumet Selective Ion Analyzer
Model 750 (pH meter) fitted with a combination pH electrode.
36
Salt
The salt (NaCl) content of the sample was measured using a Fisher
Accumet Selective Ion Analyzer Model 750, fitted with an Orion chloride
electrode (Model 94-17) and a double junction reference electrode.
The salt determination was done according to procedures developed by
Orion Research, Inc. (1980).
A 5.00 (+ 0.01) g sample of the meat product was added to a 150 ml
beaker.
Small aliquots of the extracting solution (6.3 ml nitric acid
diluted to 1 liter with dis tilled/deionized water) were stirred into
the sample with a glass stirring rod until a homogeneous slurry was
formed.
The balance of the extraction solution (100 ml total) and boiling
beads were then added to the slurry.
The beaker was covered with a watch
glass and heated to boiling on a hot plate.
for 5 minutes.
A slow boil was maintained
The contents were then allowed to cool to room tempera­
ture prior to determining the chloride ion concentration.
The Selective Ion Analyzer (Fisher Scientific) was standardized
with 0.5% and 5.0% NaCl solutions before samples were analyzed.
Pigment
Quantitation of total and of nitrosyl pigment was performed ac­
cording to Hornsey (1956).
Pigment conversion was expressed as the per­
centage of total heme pigments converted to nitric oxide hemochrome.
Residual sodium nitrite
The residual sodium nitrite concentration of the samples was
determined spectrophotometrically by using a modification of the
AOAC (1980)/Fox et al. (1981) procedure.
Hildrum (1979) reported that
37
chloride catalysis of diazo pigment formation occurs in the Griess
method with a reagent specific effect.
Fox et al. (1981) found that
chloride increased the amount of pigment formed from a given amount of
nitrite when sulfanilic acid was used, but had no effect on the amount
of pigment produced from sulfanilamide.
Although 1-naphthylamine
has been placed on the list of suspected carcinogens, it is the best
coupling reagent studied in terms of rapidity, conversion, freedom from
extraneous reactions, and pigment stability (Fox, 1979).
After considering
the above results, a modified Griess reagent was made by dissolving 9.5 g
sulfanilamide in 150 ml of 15% acetic acid and adding a solution of 0.1 g
O-naphthylamine (dissolved by boiling) in 20 ml H^O.
The two solutions
were mixed, filtered if necessary, and stored in a brown glass bottle.
Product yield
The product batches were weighed (Fairbanks Model 4190-7601) before
and after heat processing and yield was calculated by use of the fol­
lowing formula:
Flashed weight ^
Raw weight
= % product yield
Statistical Analyses
Statistical analyses of all data were performed using the Statistical
Analysis System's (SAS Institute, 1979) analysis of variance (ANOVA)
procedure.
In addition, Duncan's Multiple Range Test (Steel and Torrie,
1980) was performed when more than two treatments were being compared.
38
RESULTS AND DISCUSSION
Experiment I (Control)
£H
The effects of five levels of salt on pH of the meat mixture are
shown in Table 4.
These results indicate that the pH of the meat product
was influenced by the amount of salt incorporated.
As the salt con­
tent was increased, there was a corresponding increase in pH.
Similarly,
as shown in Table 4, an increase in sodium nitrite concentration appeared
to result in some increase in pH but the effect was not so pronounced
as with addition of salt.
Table 4 shows that after cooking, the product
pH values were slightly increased. In general, cooking resulted in an
increase in pH.
Fox et al. (1967) reported that the pH of frankfurters
during cooking rose from an initial value of 5.45 to a constant value
of 5.90 when cooked to an initial temperature of 65.6°C (150°F),
Hamm and Deatherage (1960) determined the acidic and basic groups in
muscle protein after heating at different temperatures.
They suggested
that there was little or no change in basic groups but a stepwise de­
crease in the amount of the acidic groups of muscle protein during
cooking resulted in an increase of pH.
Forrest et al. (1975) reported
that when muscle proteins are subjected to heat, a slight elevation in
pH (approximately 0.3 unit) is believed to result from the exposure of
a reactive group on the amino acid, histidine.
The addition of sodium chloride has been shown to increase the pH
of meat slightly (Berman and Swift, 1964).
This pH change has been shown
to be less when the initial pH prior to cooking was greater on the basic
39
Table 4.
Effect of nitrite and salt on pH of a meat mixture^
Treatments
Cooked samples (N = 45)
Nitrite
All samples (N = 60)2
75 ppm
156 ppm
S.E.
5.89*
5.93*
5.91
+ 0.008
5.95*
+ 0.008
Salt
All samples (N = 24)2
Cooked samples (N = 18)
0%
0.5%
5.76^
1.0%
5.86=
5.90%
2.0%
4.0%
S.E.
5.96^
5.99*
+ 0.013
5.78^
5.91^
5.95%
6.02
6.05
+ 0.013
Mean values in a column followed by different letters were
significantly different (p < .05).
All samples = data include raw emulsion.
40
side of the I.P. (Hamm and Deatherage, 1960).
Puolanne and Terrell (1983)
found that 4.0% salt in preblends resulted in a higher pH than preblends
made with 2.0% salt.
Waldman et al. (1974) found that meats premixed
with sodium nitrite had a significantly (p < .01) higher pH than those
mixed with salt alone or salt in combination with sodium isoascorbate.
Figure 1 shows the interaction of salt, nitrite, and storage time
on the pH of the products.
Higher salt and nitrite levels gave higher
pH values than lower levels.
The interaction was not significant
(p < .05) during storage because most of the treatments resulted in
parallel lines.
Ockerman and Crepso (1982) studied precured beef
blends and found that pH values were not affected by the amount of added
salt.
Mahon (1961) reported that adding salt to meat lowers the pH
slightly because ion exchange liberates some hydrochloric acid.
These
results differ from those of the present study. It appears from the
current work that chloride ion and nitrite ions change ionic strength
and protein charges in meat resulting in a shift of pH.
However, because
limited work has been done and the results have been contradictory,
further work is needed.
Salt
Figure 2 shows the salt content in the products.
The figure
indicates that actual salt levels corresponded well with the formulation
targets.
Cured color
The effects of salt and nitrite on cured color are given in Table 5.
As expected, the higher nitrite level gave significantly (p < .05)
6. 5
SALT
o0
A 0.5
6. 3
(%)
Nitr^ite
Cppm)
75
156
•1
O2
6. 1 - o 4
()
11
/V
5. 9
8
5. 7
5. 5
Q 1
15
DAYS
Figure 1.
Effect of salt and nitrite on pH of control product during storage
30
42
Table 5.
Nitrite and salt effect on cured color of a meat mixture^
Treatments
Nitrosyl heme
pigment (ppm)
Nitrite
All
samples
(N = 60)
75 ppm
156 ppm
S.E.
64.2%
75.4*
± 1.4
90.2
± 1.7
All
samples^
(N = 24)
Cooked
samples
N = 18)
Salt
0%
0.57=
1.0%
2.0%
4.0%
S.E.
iii
::::
76.7
± 2.3
Total
pigment (ppm)
Pigment
conversion (%)
Cooked
All
samples
samples
(N = 45)
Cooked
samples
All
samples
Cooked
samples
143.1*
144.2*
+ 2.2
144.7a
144.3
± 2.5
44,7a
52.5
± 1.1
56.6^
62.7*
± 1.2
All
samples
Cooked
samples
All
samples
Cooked
samples
86.4*'%
143.4*
146.3*
151.5,
81.2^
148.8142.3
140.7,
143,5
± 3.5
46.
46.2b
81.Sg
90.0*
± 2.7
142.3139.3143.3*
+ 3.9
48.5b
48.753.7
± 1.7
^Mean values in a column followed by different letters were
significantly different (p < .05).
2
All samples = data include raw emulsion.
a
59.0,
57.9a
60.058.562,7
± 1.9
43
greater nitrosyl heme pigment (11 ppm more) than that of the low
nitrite level.
The highest salt level also resulted in significantly
(p < .05) greater nitrosyl heme pigment than that of the other salt
levels.
There is no difference in total pigment at any of the levels
of salt and nitrite and consequently, the 156 ppm nitrite and 4%
salt combination gave significantly (p < .05) higher pigment conversion.
However, salt concentration effects appeared to differ between emulsion
and cooked product for pigment conversion.
A high salt level converted
significantly more pigment than low salt before cooking but after cooking,
there was no significant (p < =05) difference in pigment conversion.
The highest salt level still resulted in somewhat greater pigment con­
version, though the difference was nonsignificant.
The data in Figure 2 demonstrate the salt and nitrite interaction
o.n nitrosyl heme pigment during storage.
Most of the treatments
resulted in relatively constant nitrosyl heme pigment during storage.
Products with 156 ppm nitrite levels showed a decrease in nitrosyl
heme pigment at 30 days while the lower nitrite levels gave significantly
lower nitrosyl heme pigment at 15 days.
The pigment levels remained
constant for the rest of the storage period with both nitrite groups
having comparable values.
Fox (1966) stated that the rate of cured color development and the
amount of nitroso hemochrome formation are somewhat pH-dependent.
Generally, as pH decreases from 6.0 to 5.5, cured color development is
more rapid and complete (Fox and Thomson, 1963). In this study, salt
resulted in increased pH but also seemed to increase the pigment conversion
and nitrosyl heme pigment. It appears that the catalytic effect
44
of chloride continues to accelerate nitrosation and favor high nitrosyl
heme pigment in spite of the somewhat elevated pH.
Another possibility
is that chloride ions change the globin moiety of the pigment and
give more room for nitric oxide on the molecule.
Myoglobin is a conjugated protein and the heme group is the actual
source of meat color; any physical or chemical change in the heme will
alter the color observed (Sebranek, 1981; Giddings, 1977). Sebranek
(1981) suggested that changes in residual nitrite or nitrosyl heme
pigment may result from changes in chloride levels.
Added chloride
increased nitrosyl heme pigment formation in a model system (Fujimaki
et al., 1975) and five-fold increase in the reaction rate constant
has been noted for the formation of nitric oxide in 3M (~ 24%) NaCl
as compared to no addition of NaCl (Fox et al., 1980).
On the other hand, Reith and Szakaly (1967a, b) claimed that at
relatively high salt concentrations, final color intensity of the nitro­
syl heme was decreased.
Several investigators (Sebranek, 1979, 1981;
Gassens et al., 1979; Fox et al.. 1980, 1981, 1982) have discussed
potential nitrosyl chloride formation in mixtures containing sodium
nitrite and sodium chloride at a pH typical of meat.
Fox et al. (1980)
suggested that nitrosyl chloride formation was responsible for a
morphological change in the interfibrillar spaces of cured beef muscle.
The effect was not observed when either sodium chloride or nitrite was
used alone.
The overall effect of chloride levels on nitrite, however, may depend
not only on the suggested catalytic role of chloride, but also on the
A (]. 5
Nitnite
(ppm)
75
156
H-
_J
<
CO
•pUl
•1
DAYS
Figure 2.
Salt content of products from various salt-nitrite formulations
120
e 100
Q
QCL
::::::::
'-
-P
c 80
(U
E
m
•r*i
CL
60
(U
SALT
£
(%)
QJ
%
I—1
40
A 0. 5
X
CO
0
L
-P
z
Nitnit e
75
156
20
0
0 1
15
DAYS
Figure 3.
Effect of salt and nitrite on nitrosyl heme pigment of control product during storage
47
various competing reactions in the system (Sebranek, 1979, 1981), in­
cluding reductants, proteins and pigments.
In this study, the somewhat increased nitrosyl heme pigment and
pigment conversion as chloride levels increased suggest that catalysis
of nitrosation reactions through the formation of NOCl may be significant
in meat systems.
Residual nitrite
The effect of formulated nitrite and salt on the residual nitrite
is shown in Table 6.
nitrite.
The 0% salt level generally gave the lowest residual
This is not significantly different from the 4.0% salt level but
is significantly (p < .05) lower than the other salt levels.
The overall
trend indicates that the residual nitrite is related (p < .05) to in­
creasing the salt concentration; from 0.5% to 4.0% residual nitrite is
decreased.
Figure 4 shows the interaction of the salt and nitrite on the residual
nitrite during storage.
The data indicate that nitrite values decreased
during storage when the initial nitrite concentration was high; however,
when the initial nitrite concentration was low, it did not change signifi­
cantly.
Changes in measurable residual nitrite appear to be related to changes
in chloride levels in this study; however, other studies in this area which
have been reported in the literature have been contradictory.
Lee and
Cassens (1980) indicated that residual nitrite was lower in the presence
of larger amounts of sodium chloride in a model system.
However, if the
effect of decreasing the pH (an effect of sodium chloride in model systems)
48
Table 6.
Effect of salt on the residual nitrite of a meat
mixture ^
Residual nitrite
Treatments
Nitrite
75 ppm
156 ppm
S.E.
Salt
07o
0.5%
1.0%
2.0%
4.0%
S.E.
All samples (N = 60)2
36.7%
71.1
± 0.65
All samples (N = 24)2
II
53.of
± 1.03
Cooked samples (N = 45)
31,7^
61.3
± 0.62
Cooked samples (N = 18)
44.6^
44.9b
+ 0.98
^Mean values in a column followed by different letters were
significantly different (p <0.5).
2
All samples = data include raw emulsion.
120
SALT
Q 100
• j A.
Ûvx
(U
-P
ri
Nitnit e
•PP m .
75
156
o0
A 0. 5
ê;
Q-
(%)
n 1
80
û 2
L
-P
o4
'
•H
4>
vo
60
0
"ô:
D
A
8:::::::
O
"D
•H
Q
0)
Û
QJ
o
a:
40
5:::o
A
20
. 0 1
^
I
I
1
1
I
_i
1
1
I
1
1 S L .1
J
1
J
1
L_
_l
I
I
,15
DAYS
Figure 4.
Effect of salt and nitrite on residual nitrite of control product during storage
I
1
30
50
and the effect of loss of water during cooking and storage were•excluded,
then sodium chloride did not cause a significant decrease in residual
nitrite in the model system.
While the experiments with model systems
demonstrate valuable relationships, they cannot give decisive information
because conditions in meat are different.
As shown in the earlier data,
rather than a decrease in pH due to sodium chloride as occurs in model
systems, in meat systems, an increase in pH was observed as the sodium
chloride content increased.
The influence of pH on residual nitrite levels is quite wellestablished.
Zaika et al, (1976) reported that a reduction in pH
will rather drastically lower residual nitrite.
Results of the current
study indicate that sodium chloride decreased the residual nitrite even
though the pH was elevated.
A difference in residual nitrite concentra­
tion due to sodium chloride might result from the well-known solubility
effect of sodium chloride on muscle proteins.
The greater availability
of proteins, in high compared to low sodium chloride conditions, may
make them more susceptible to reaction with nitrite thereby lowering
the residual nitrite concentration.
Fox et al. (1982) also reported that increasing chloride in a pH
5.6 meat slurry showed a relative reduction of residual nitrite
measured in several different ways.
In combination, nitrous acid and hydrochloric acid will generate
nitrosyl chloride which is a powerful nitrosating species (Ridd, 1961).
Increased nitrosating ability would result in enhanced production of
nitrosyl heme pigments through increased production of nitrous reductant
intermediates and the resulting nitric oxide.
This sequence would, in
51
turn, result in decreased residual nitrite.
The present study suggested a chloride effect on residual nitrite;
that is, chloride ion appears to react with nitrite and generate
nitrosyl chloride.
One result is a small depletion of residual nitrite.
Experiment II (Reduction of Product pH)
£H
The data in Table 7 show the effect of nitrite, salt and glucono
delta lactone on the pH of the meat product.
The higher nitrite and
salt levels again resulted in a significantly higher pH than that of the
lower levels as noted previously.
Addition of glucono delta lactone
to the meat product resulted in a significantly lower pH value (p < .05)
but resulted in no interaction among the additives.
effect of the salt and nitrite on pH during storage.
Figure 5 shows theAt 0 day, with
the product at the uncooked stage, high pH values were observed, but
after cooking, the pH decreased significantly (p < .05).
The effect of salt, nitrite and glucono delta lactone on the overall
pH values of the meat product are shown in Table 8.
As mentioned
earlier, salt and nitrite increased the pH value significantly (p < .05);
however, GDL treatments decreased the pH significantly (p < .05).
The data of Figure 6 show the result of a salt and nitrite inter­
action on pH during storage.
pH was increased after cooking and became
constant for the duration of the storage period.
Sair (1963, 1965) found that GDL produced little effect when added
to meat at low temperature (4.4°C), but decreased the pH when meat was
52
Table 7.
Effect of nitrite, salt, and 1.5% GDL on pH^
Treatments
Nitrite
75 ppm
156 ppm
S.E.
pH
All samples (N = 120)2
Cooked samples (N = 90)
5.02^
5.05
+ 0.007
5.05%
5.07
+ 0.008
Salt
2
All samples (N = 48)
Cooked samples (N = 36)
0%
0.5%
1.0%
2.0%
4.0%
S.E,
4.89®
4.96^
5.03f
5.10%
5.20
+ 0.01
All samples (N = 120)2
GDL
0%
1.5%
S.E.
5.78*
4.28^
± 0.007
4.92®
5.06^
5.13%
5.20
+ 0.012
Cooked samples (N = 90)
5.85®
4.26%
+ 0.008
Mean values in a column followed by different letters were
significantly different (p < .05).
All samples = data include raw emulsion.
S/\L.T
o0
4. 8
h ^ 0. 5
(3:)
Nitnite
(ppm)
75
156
• 1
o2
o4
4. 6
o
o.
X
CL
4. 4
à
It. V
o• o-
• <
- <
a•
p•
• • •
4. 2
T.—8
O
o
• 1
Figure 5.
15
DAYS
Effect of salt and nitrite on pH in products with 1.5% GDL during storage
30
54
Table 8.
Effect of nitrite, salt. and 0.2% GDL on pH^
Treatments
Nitrite
75 ppm
156 ppm
S.E.
PH
All samples (N = 120)2
Cooked samples (N = 90)
5.89^
5.93*
± 0.007
5.83^
5.87
+ 0.006
Salt
All samples (N = 48)2
0%
0.5%
1.0%
2.0%
4.0%
S.E.
5.72®
5.80
5.85^
5.9cf
5.96
+ 0.009
• Cooked samples (N = 36)
All samples (N = 120)2
GDL
0%
0.2%
S.E.
5.96^
5.73'
+ 0.006
5.79®
5.87*
5.9lJ
5.96^
6.02*
+ 0.01
Cooked samples (N = 90)
6.03*
5.79^
+ 0.007
Mean values in a column followed by different letters were
significantly different (p < .05).
2
All samples = data include raw emulsion.
6. 5
s i *f *
%
CL
S/\L_T
5. 5
(3%)
Nitnite
Cppm)
75
A 0. 5
156
•
o2
o4
L_
0 1
15
DAYS
Figure 6.
Effect of salt and nitrite on pH in products with 0.2% GDL during storage
30
Ln
Ui
56
heated (66°C). Conversion to gluconic acid is the source of change
in acidity level.
The above result was observed in the drastic decrease
in pH (pH 4.9) of the product (1.5% GDL), while 0.2% GDL product did not
show an increase in acidity after cooking.
Cured color
The data of Table 9 show the effect of nitrite, salt and GDL on cured
color when the pH change was relatively large (pH 4.9).
It is well-known
that GDL gives a more rapid and improved color development (Meester,
1965) by providing a more acid medium.
All three of these compounds
(nitrite, salt and GDL) increased the nitrosyl heme pigment signifi­
cantly (p < .05).
The present experiment shows that GDL affected
the reaction of nitrite by generating more nitrosyl heme pigments before
cooking.
The data of Figure 7 demonstrate that in this case, there is some
interaction between salt and nitrite to form additional nitrosyl heme
pigment during storage.
The results seemed somewhat erratic.
Nitrite and salt in this study did not change total pigment
concentration but the addition of GDL decreased the total pigment
significantly (p < .05).
It was not expected to have an effect on the
total amount of pigment.
It may be that GDL changes the heme moiety
or affects the extraction of the protein resulting in a decreased total
amount of pigment detected.
However, the results are contrary to the
finding of Pate et al. (1971); they found that when hams with or without
GDL were extracted 14 days post cure, the initial total pigment levels
of hams receiving no GDL were significantly (p < .05) lower than hams
57
Table 9.
Effect of nitrite, salt, and 1.5% GDL on cured color^
Treatments
Nitrite
Nitrosyl heme
pigment (ppm)
Cooked
All 2
samples
samples
(N = 120) (N = 90)
Total
pigment (ppm)
All 2
samples
Cooked
samples
Pigment
conversion (%)
All 2
samples
Cooked
samples
75 ppm
156 ppm
S.E,
105.9^
112,0*
± 1.9
120.7*
125,4*
+ 2.2
169.1*
171.3*
+ 2.0
178.8*
180.5*
± 2.2
62.8^
65.1*
+ 0,6
68.8*
70.0*
+ 0.7
Salt
All 2
samples
(N = 48)
Cooked
samples
(N = 36)
All 2
samples
Cooked
samples
All 2
samples
Cooked
samples
60.3^
63.(f
63.5^
64.f
68.9*
± 0.9
65.7^
68.3^'=
69.2^
69.1^
74.7*
± 1.1
All 2
samples
Cooked
samples
50.sf
77.2*
+ 0.6
61.6^
77.2
± 0.7
0%
0.5%
1.0%
2.0%
4,0%
S.E.
GDL
103,1^
106.9*'^
107,3*'b
111.4*'^
116.2*
± 3.1
117.1^ ,
120,7 'J
121.2*'^
125.6*'°
130.6*
- 3-5
Cooked
All 2
samples
samples
(N = 120) (N = 90)
0%
1.5%
S.E.
90.5%
127.4
+ 1.9
111.6%
134.5
+ 2.2
170.4*
169.6*
171.8*
171.9*
167.2*
± 3.1
All 2
samples
± 1.0
171.5^
178.0*
182.3*
182.4*
175.8*
+ 3.5
Cooked
samples
184.3*
174.9^
+ 2.2
Mean values in a column followed by different letters were
significantly different (p < ,05).
2
All samples = data include raw emulsion.
160 r
•T
.. o
•0
- I
O"
o•
—— O "
_.o<b
g
m 130
•A
6
o• A-
Ul
00
£ 120
5ALT
o-
(%)
o0
Nitrite
Cppm) a 0
75
156
5
a 1
Û 2
o4
J
0 1
Figure 7,
U
••I
I
I
I
1
I
u
15
DAYS
Effect of salt and nitrite on nitrosyl heme pigment in products with 1.5/o GDL during
storage
30
59
receiving the GDL treatment.
The reason for the differences is not
clear at this point because little work has been done concerning the
action of GDL on total pigment levels or on pigment solubility in cured
meats.
There were interactions among nitrite, salt, and GDL as measured
by the nitrosyl heme pigment conversion (p < .05).
This may be ex­
plained on the basis that GDL changed the apparent total pigment
concentration, possibly because the pH levels of the 1.5% GDL treatment
were lower (pH 4.9) than most typical cured meats.
The effects of salt, nitrite and GDL on the cured color of a product
with a more modest decline in pH were studied and are shown in Table 10.
In this experiment, 0.2% GDL was added to the meat mixture which re­
duced the pH about 0.2 unit.
As the salt level was increased, the
amount of nitric oxide heme pigment increased significantly (p < .05)
in the raw emulsion but only a slight change was observed after cooking.
The addition of GDL also caused an increase in the amount of nitrosyl
heme pigment (p < .05).
The concentration of the nitrosyl heme pigment
was found to increase linearly with the addition of salt. This supported
the thesis that pH may enhance the synergistic effect of salt and subse­
quently the nitrosation reaction.
Total pigment concentration changed
significantly in this experiment as in the previous GDL experiment. The
addition of nitrite and GDL increased the total pigment significantly
(p < .05). Salt, however, decreased the total pigment significantly
(p < .05) and the 4.0% salt addition gave the lowest total pigment
concentration. It is not clear why total pigment changes; however,
salt may change the globin moiety and hinder the spectral absorbance
60
Table 10.
Treatments
Effect of nitrite, salt, and 0.2% GDL on cured color
Nitrosyl heme
pigment (ppm)
Nitrite
All
Cooked
samples
samples
(N = 120) (N = 90)
75 ppm
156 ppm
S.E.
79.6^
90.0
± 1.2
100.7%
107.7
± 1.5
Total
pigment (ppm)
All
160.5,
165.7'
+ 1.3
Salt
All 2
samples
(N = 48)
Cooked
samples
(N = 36)
All
0%
0.5%
1.0%
2.0%
4.0%
S.E.
80.4a b
101.2*
105.0,
85.2 \
85.4*'t
105.9
104.1
104.6
± 2.4
163.2,
166.1!
166.3a,b
162.8
157.0'
GDL
87.9
± 1.9
Cooked
All 2
samples
samples
(N = 120) (N = 90)
0%
0.2%
S.E.
77.6%
92.0
± 1.2
97.4%
111.0
+ 1.5
+ 2.1
All
Cooked
samples
167.1,
172.8'
+ 1.6
Cooked
samples
a.b
169.3'a
173.7r a
174.2a
170.1b
162.6
+ 2.5
Cooked
samples
166.0'
166 . 1 ,
173.9'
+ 1.3
+ 1.6
160. 2 ,
Pigment
conversion (%)
All 2
samples
Cooked
samples
60.3,
62.3'
48.1,
53.2'
+ 0.5
+ 0.6
All 2
samples
Cooked
samples
b
c
47.9b,c
49.7b c
49.6b
51.Oa
54.9
+ 0.7
64.2
+ 0.9
All 2
samples
Cooked
samples
60.6t
60.8b
58.7,
63.9'
+ 0.5
+ 0,6
Mean values in a column followed by different letters were
significantly different (p < .05).
2
All samples = data include raw emulsion.
140
o-
• D.
•<t>
m 80
g
60
SALT (%)
oG
A 0. 5
N i tn i
75
156
ppr
a 1
o2
o4
i
0 1
Figure 8.
I
I
.i—
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
15
DAYS
Effect of salt and nitrite on nitrosyl heme pigment in products with 0.2% GDL during
storage
30
62
used for quantitation or less protein extraction may occur in this
particular pH range.
Nitrite and GDL gave significantly (p < .05) higher pigment con­
version, which agreed with the nitric oxide heme pigment measurements.
Salt did not cause significant change in nitrosyl heme pigments after
cooking, but pigment conversion was significantly (p < .05) greater
in the cooked products.
This was due to the changes of total pigment
in the cooked products and may not reflect actual conversion dif­
ferences.
Results shown in Figure 9 indicate that in this experiment,
salt and nitrite both influenced nitrosyl heme pigment during storage.
The 4% salt combined with 156 ppm nitrite level gave the highest
value for nitrosyl heme pigment.
Most of the treatments resulted
in relatively constant nitrosyl heme pigment values during storage.
In the present experiment, comparing two pH effects on cured
color showed that drastically decreased pHL resulted in more nitrosyl
heme pigment than that of the slightly decreased pH.
Residual nitrite
The data of Table 11 show the effects of salt and 1,5% GDL on .
residual nitrite concentration.
Salt does not affect the residual
nitrite content significantly, though the 4% salt level resulted in the
lowest residual nitrite content after cooking.
The GDL treatment
decreased the residual nitrite content significantly (p < .05) as
expected,
Meester (1965) found that with the addition of GDL, residual
SALT (%)
20
D 10
0 1
DAYS
Figure 9.
Effect of salt and nitrite on residual nitrite in products with 1.5% GDL during storage
64
Table 11.
Effect of nitrite, salt, and 1.5% GDL on residual nitrite^
Residual nitrite
Treatments
Nitrite
75 ppm
156 ppm
S.E.
All samples (N = 120)2
All samples (N = 48)2
0%
0.5%
1.0%
2.0%
4.0%
S.E.
26.0*
24.8*
26.0*
25.8*
25.2
+ 0.89
All samples (N = 120)2
0%
1.5%
S.E.
13.3^
16.1^
35.1
+ 0.56
Salt
GDL
Cooked samples (N = 80)
41.1*
9.9
+ 0.56
27.5*
± 0.57
Cooked samples (N = 36)
•
21.7^
20.1*
21.0*
20.3*
19.0*
+ 0.90
Cooked samples (N = 80)
32.5*
8.4
+ 0.57
Mean values in a column followed by different letters were
significantly different (p < .05).
All samples = data include raw emulsion.
65
nitrite decreased as cured color increased.
The present study con­
firmed those results indicating that a low pH or GDL itself caused a
significant nitrosation.
The salt and nitrite interaction during storage are indicated from
the data in Figure 10. In general, the depletion of residual nitrite
was not significant during storage because nitrite was greatly depleted
after cooking.
A significant interaction (p < .05) was observed
between nitrite and GDL, however.
Data presented in Table 12 show the effect of salt and 0.2%
GDL on residual nitrite concentration.
In this case, the addition of
salt resulted in a significant difference (p < .05) in residual nitrite
concentrations.
High salt levels showed a significant decrease in the
residual nitrite concentration, which was not evident when the pH was
reduced more drastically.
As expected, GDL treatment by itself lowered
residual nitrite concentration significantly (p < .05).
The effect of
salt on nitrite during storage is evident from the data of Figure 10,
In general, residual nitrite concentration decreased during storage.
It is evident that there was a significant nitrite reaction in the
products during storage.
Fox (1980) reported that nitrite content decreased after processing;
and salt remained constant.
Since the relative concentration of
NgOg and NOCl are dependent on the nitrous acid and chloride concentra­
tions, formation of N^Og will be bimolecular in nitrous acid, and forma­
tion of NOCl will be monomolecular.
In a system where both species
are formed, a doubling of the nitrous acid concentration will result
In four times as much K2O3 ^s NOCl,
Considering the above results,
100
£
80
CL
•H
Nitrite
(ppm)
75
156
n 1
o\
CL
m
-p
SALT (%)
o0
A 0. 5
o2
o4
60
L
-+)
•H
40 ij0
3
"D
•H
(0
QJ
(T
20
i
0
Figure 10.
•
0 1
•
«
«
«
I
1
I
1
1
I
1
-J
i—J
15
DAYS
•
•
•
•
I
I
1
1
1
1
1
1
u.
30
Effect of salt and nitrite on residual nitrite in products with 0.2% GDL during storage
67
Table 12.
Effect of nitrite, salt, and 0.2% GDL on residual nitrite'"
Treatments
Nitrite
75 ppm
156 ppm
S.E.
Salt
0%
0.57=
1.0%
2.0%
4.0%
S.E,
Residual nitrite
All samples (N = 120)2
24.6^
56.2
+ 0.41
All samples (N = 48)2
42.0*
41.5*
42.2*
39.3
38.8^
+ 0.64
All samples (N = 120)2
GDL
0%
0.2%
S.E.
46.7*
34.1^
± 0.41
Cooked samples (N = 90)
20.6^
46.7*
+ 0.46
Cooked samples (N = 36)
35.4*
34.9*
35.4*
32.5b
30.0^
+ 0.73
Cooked samples (N = 90)
40.6*
26.7b
+ 0.46
Mean values in a column followed by different letters were
significantly different (p < .05).
All samples = data include raw emulsion.
68
high nitrite level combined with a high chloride level might be expected
to result in enhanced production of nitroso reductant intermediates.
From the data, it appears that there was a significant (p < .05)
interaction between salt and nitrite.
Comparing the two pH treatments, it would seem that nitrosation at
low pH overcome any effect of chloride such that residual nitrite was
decreased only slightly during subsequent storage.
A slight decrease
in the pH resulted in a significant catalytic effect of the chloride ion
during storage and the formation of NOCl during storage may have
contributed to continued depletion of nitrite.
Experiment III (Effect of Reductant)
£H
The data in Table 13 show that pH differences seem quite large as
a result of salt concentration.
In Experiment II, which included
erythorbate and 0.2% GDL, nitrite increased the pH of both the raw emul­
sion and the cooked products significantly (p < .05).
However, in the
absence of erythorbate with GDL present, high nitrite level gave a higher
pH than low nitrite level after cooking.
Also, nitrite depletion was
slow without erythorbate while in the emulsion stage as expected; but
after cooking, there was a definite depletion of nitrite.
The data of Figure 11 show the salt and nitrite interaction on
pH during storage. Immediately after mixing the components (0 day),
pH values were low (pH 5.3), but after cooking, the pH increased
significantly (pH 5.6) and was relatively constant during storage.
69
Table 13.
Effect of nitrite and salt on pH of product without
erythorbate^
pH
Treatments
Nitrite
75 ppm
156 ppm
S.E.
Salt
0%
0.5%
1.0%
2.0%
4.0%
S.E.
All samples (N = 60)'
Cooked samples (N = 45)
a
5.49
5.52
+ 0.011
+ 0.011
All samples (N = 24)'
Cooked samples (N = 18)
5.31=
5.43d
5.50?
5.59%
5.70
+ 0.017
5.37:
5.50^
5.56=
5.64^
5.75*
+ 0.018
Mean values in a column followed by different letters were
significantly different (p < .05).
2
All samples = data include raw emulsion.
5. 8 -
5. 6
%
CL
.
•
:
SALT
(%)
5. 4
A 0. 5
5. 2
Figure 11.
DAYS
Effect of salt and nitrite on pH in products without reductant during storage
71
Cured color
The effects of nitrite and salt in absence of erythorbate on cured
color are shown in Table 14.
As the amount of salt increased, nitric oxide
heme pigment increased significantly (p < .05) in the emulsion.
However,
the addition of 2% salt gave significantly (p < .05) higher nitrosyl heme
pigment and the 0% gave the lowest nitrosyl heme pigment in the cooked
product.
emulsion.
Salt appeared to increase the nitric oxide heme pigment in the
However, after cooking, even though salt increased the nitric
oxide heme pigment, the effect of varying the salt concentration was not as
great as before cooking.
This result was similar to that in the presence
of erythorbate even though the salt effect was more evident in the absence
of erythorbate.
This difference may be due to the low pH and consequential
formation of nitrosyl heme pigment.
Total pigment was not changed in the emulsion but a slight change
was observed after cooking.
The 4% salt gave significantly (p < .05)
lower total pigment and the 0.5% gave the highest total pigment.
As
mentioned earlier, salt may change the globin structure and inhibit the
measurement of pigment.
The higher nitrite concentration resulted in
significantly (p < .05) greater pigment conversion; salt also gave
significantly (p < .05) greater pigment conversion.
This probably means
that the other reductants present in meat -will combine with NOCl so
that chloride still enhances nitrosation in the absence of erythorbate.
The results shown in Figure 12 indicate that salt and nitrite both
affected nitrosyl heme pigment during storage.
At the high nitrite
level, the 0% salt level gave the lowest nitric oxide heme pigment which
72
Table 14.
Treatments
Effect of nitrite and salt on cured color of product with­
out erythorbate
Nitrosyl heme
pigment (ppm)
Nitrite
Cooked
All 2
samples
samples
(N = 60) (N = 45)
75 ppm
156 ppm
S.E.
79.9^
99.0
+ 1.6
99.3^
123.6*
± 2.1
Salt
Cooked
All 2
samples
samples
(N = 24) (N = 18)
07=
0.5%
82.2®
86.8
89.of
94.0^
95.2*
± 2.5
1.0%
2.0%
4.0%
S.E.
105.1^
110.1
111.4*'T)
116.2*
114.4*'^
+ 3.3
Total
pigment (ppm)
All 2
samples
184.8*
182.7*
± 2.3
All 2
samples
183.8*
187.9*
186.2*
184.6*
176.3*
+ 3.7
Cooked
samples
192.0*
189.8*
+ 2.8
Cooked
samples
,b
191.9*
196.5*
193.8*
190.6*
181.8°
+ 4.4
Pigment
conversion (%)
All 2
samples
Cooked
samples
42.2^
22.8*
± 0.5
51.8^
65.2*
± 0.7
All 2
samples
Cooked
samples
43.3d
55.(f
44.7='*
56.ij
57.5b
46.4^
49.9b
61.1*
63.0*
53.1*
+ 0.9
+ 1.0
Mean values in a column followed by different letters were
significantly different (p < .05).
2
All samples = data include raw emulsion.
1 4 0 r-
E
1
f î î î î î î. • • • e
Ê N I I I I I : : : : : : I ;:
120
CL
CL
100
£
Q)
£
m
;9'
80
•H
SALT
CL
QJ
£
(U
60
JZ
X
CO
40
0
L
-P
o0
A 0. 5
1
Figure 12.
(ppm)
75
156
(jj
a 1
û2
o4
20
0
Nitrite
I
01
1
1
I I ' '
L.
1
1
1
L_
15
DAYS
Effect of salt and nitrite on nitrosyl heme pigment in products without reductant
during storage
30
74
increased linearly with increasing salt levels but no interaction
between salt and nitrite during storage could be detected.
Residual nitrite
In Table 15, it can be seen that at 47» salt, a significantly
(p < .05) lower residual nitrite concentration was present in the
emulsion than at 0%, but the residual nitrite concentration is not
greatly different at the other salt levels.
It appears that again
chloride ion generally enhanced nitrosation and depleted the nitrite
content, but the effect is clearly not linear.
An interaction of salt and nitrite during storage is evident from
Figure 13.
At the high nitrite level, high salt concentration re­
sulted in a significantly (p < .05) lower residual nitrite concentration,
but there was no significant difference at the low nitrite level.
There­
fore, salt enhanced nitrosation at 156 ppm nitrite but with 75 ppm
nitrite, the salt effect was not evident in the absence of erythorbate.
Experiment IV (Effect of Internal Finishing Temperature)
£H
The effects of nitrite, salt, and internal finishing temperature
on product pH are shown in Table 16.
The presence of nitrite again
increased the pH significantly (p < .05).
Salt and internal finishing
temperature also increased the pH (p < .05).
As mentioned earlier,
it is well-known that cooking changes the protein resulting in a shift
of the pH to the alkaline side.
The results of the present experi-
75
Table 15.
Effect of nitrite and salt on residual nitrite of product
without erythorbate^
Treatments
Nitrite
75 ppm
156 ppm
S.E..
Salt
07c
0.57=
1.0%
2.0%
4.0%
S.E.
Residual nitrite
All samples (N = 60)^
41.3^
76.5®
± 0.77
All samples (N = 24)^
59.5®
60.8®
60.7®
58.9®
54.6%
± 1.22
Cooked samples (N = 45)
34.4^
65.6
+ 0.51
Cooked samples (N = 18)
49.9*'h
51.2
47.5^
± 0.81
Mean values in a column followed by different letters were
significantly different (p < .05).
2
All samples = data include raw emulsion.
120 ISALT
(%)
o0
A 0. 5
Nitrite
(ppm)
75
156
8:-.
n 1
Û 2
o4
::::
0 1
Figure 13.
DAYS
Effect of salt and nitrite on residual nitrite in products without reductant during
storage
77
Table 16.
Effect of nitrite, salt and internal finishing temperature
on pH^
Treatments
Nitrite
pH
All samples (N = 108)2
Cooked samples (N = 81)
75 ppm
156 ppm
S.E.
5.86^
5.89
+ 0.004
5.93^
5.97
+ 0.005
Salt
All samples (N = 72)2
Cooked samples (N = 54)
0%
1.07=
3.0%
S.E.
5.81^
5.87'
5.95
+ 0.004
+ 0.006
Temperature
All samples (N = 72)2
Cooked samples (N = 54)
52°C
60°C
68°C
S.E.
5.81%
5.89^
5.93
+ 0.004
5.86?
5.97b
6.02
+ 0.006
5.89%
5.94^
6.02*
Mean values in a column followed by different letters were
significantly different (p < .05).
All samples = data include raw emulsion.
78
ment are in agreement.
The results given in Figures 14 and 15 show the interaction of these
treatments during storage.
All the treatments resulted in an increased
pH after cooking which remained relatively constant during storage.
Increasing salt or nitrite gave significantly (p < .05) higher pH
values.
There was no interaction, however, among the treatments.
Cured color
The effect of treatments on cured color are presented in Table 17.
As expected, nitrite increased the nitrosyl heme pigment significantly
(p < .05) while as the salt level was increased, nitrosyl heme
pigment decreased significantly especially after cooking which was
opposite to previous results.
Nitrosyl heme pigment concentration
increased significantly (p < .05) with increasing internal finishing
temperature.
Results showing the interaction of the treatments during storage are
presented in Figures 16 and 17.
It may be observed that internal finishing
temperature affected the nitric oxide heme pigment significantly.
High temperature showed significantly higher nitrosyl heme pigment.
Changes in salt levels do not show any obvious trend; however, there was
a significant (p < .05) interaction between salt and cooking tempera­
ture.
The nitrite level in this case did not affect the total pigment
concentration.
However, changes in salt concentration and changes in
temperature altered the total pigment significantly (p < .05).
The
6. 5
SALT
Figure 14.
(%)
TEMP
(°C)
o•
52
A 1
60
D3
68
DAYS
Effect of salt, 156 ppm nitrite and internal finishing temperature on pH during storage
6. 5
SALT
6
T
û-
5. 5
Figure 15.
(%)
TEMP
(°C)
- 6
•1
DAYS
Effect of salt, 75 ppm nitrite and internal finishing temperature
81
Table 17.
Treatments
Nitrite
Effect of nitrite, salt and internal finishing temperature
on cured color^
Nitrosyl heme
pigment (ppm)
Total
pigment (ppm)
Cooked
All 2
All 2
samples
samples
samples
(N = 108) (N = 81)
75 ppm
156 ppm
S.E.
84.8^
96.8*
± 1.1
Salt
All 2
samples
(N = 72)
0%
1.0%
3.0%
S.E.
94.8*
91.5*
86
± 1.3
GDL
All 2
samples
(N = 72)
520c
60OC
680C
S.E.
78.aP
91.2%
102.3*
± 1.3
110.7^
121.7*
± 1.5
189.7*
187.3*
± 2.2
Cooked
All 2
samples
samples
(N = 54)
124.1*
117.r
107,4'^
+ 1.8
197.1*
- 189.6*
178.7"
± 2.7
Cooked
All 2
samples
samples
(N = 54)
99.8^
116,7^
132.0*
+ 1.8
Cooked
samples
Pigment
conversion (%)
All 2
samples
Cooked
samples
43.6^
50.6*
± 0.4
56.8^
63.1*
± 0.5
Cooked
samples
All 2
samples
Cooked
samples
204.7*
195.1*
180.5
± 3.4
46.3%
47.1*'^
48.0*
+ 0.4
60.3*
59.9*
59.7*
± 0.6
Cooked
samples
All 2
samples
Cooked
samples
43.1^
48.0^
50.2*
+ 0.4
54.4=
61.lb
64.4*
± 0.6
194.4*
192.5*
± 2.8
181.3^
187.O"
197.2*
i 2.7
183.8^
191.3^
205.2*
± 3.4
Mean values in a column followed by different letters were
significantly different (p < .05).
'All samples = data include raw emulsion.
û_
0)
80
SALT
(%)
TEMP
(°C)
E
(U
%
X
®
0
60
40
o0
52
A 1
60
O3
68
L
-P
•H
20
0
Figure 16.
.I
0 1
I
I
i
I
,1 .. I
,1
I
i
i
-I
15
I—I—I—I
I
I
I
I
I
30
I—
DAYS
Effect of salt, 156 ppm nitrite and internal finishing temperature on nitrosyl heme pig­
ment during storage
160
—;z= A
SALT
(%)
TEMP
( C)
0 1
DAYS
Figure 17,
Effect of salt, 75 ppm nitrite and internal finishing temperature on nitrosyl heme pig­
ment during storage
84
47o salt level resulted in the lowest total pigment.
As the temperature
was increased, the total pigment content also increased.
In this
experiment, cooking yield'(Table 18) was measured, which showed
that higher cooking temperature resulted in lower cooking yield; so,
more moisture was lost and all the components were concentrated.
However, even after adjusting for the shrink loss, total pigment was
increased due to the high internal finishing temperature.
Bakes and
Blumer (1975) reported an increase in color when a decreased moisture
content gave greater pigment concentration of country-style hams.
It is assumed that these findings may partly support the present study
even though the product was different.
The high nitrite level gave a significantly greater pigment
conversion (p < .05) than that of the low nitrite level.
Even though
use of a high salt level resulted in less nitric oxide heme pigment,
it showed the highest pigment conversion in the raw emulsion, which
was due to the fact that total pigment was relatively low.
High finishing
temperature showed the highest pigment conversion.
The interaction of salt, nitrite and internal finishing temperature
on pigment conversion during storage are shown in Figures 18 and 19.
At a low finishing temperature, pigment conversion increased during
storage but at high temperature, pigment conversion remained constant
during storage.
There was a significant interaction between salt and
nitrite and between finishing temperature and nitrite (p < .05).
In this experiment, a high salt level gave the lowest nitrosyl
heme pigment and total pigment.
This result is opposite to other re­
sults in previous experiments.
It is not clear at this point but there
85
Table 18.
Effect of internal finishing temperature on cooking yield^
Treatments
Yield (N = 3)
520c
97.6^
60OC
97.1*
68°C
95.7^
S.E.
± 0.17
^Mean values in a column followed by different letters were
significantly different (p < .05).
70 r
—.
—— O -
SALT
u 30
J
0 1
Figure 18.
1
(%)
TEMP
C°C)
o0
52
A 1
60
a3
68
I—I—I—I—I—I—I—I—I—I—I—I—I—'—'—'—'—'—'—'—
15
00
ON
_J
I
I
1
1
L_
30
DAYS
Effect of salt, 156 ppm nitrite and internal finishing temperature on pigment conversion
during storage
70
B;
60
—
C
50
0
0)
L
Q)
40
>
n
I
SALT
u 30
(%)
TEMP
(°C)
00
o0
I 20
m
^ 1
52
60
p3
68
CL
10
0
Figure 19.
I
01
I
I
I
I
I
I
t
I
15
1
30
•AYS
Effect of salt, 75 ppm nitrite and internal finishing temperature on pigment conversion
during s torage
88
may have been a problem in extracting pigment or the different cooking
method may have caused a change in protein structure.
Preliminary
experiments had shown that salt increased the nitric oxide heme pigment
even though shrink loss was not measured.
Residual nitrite
The effect of nitrite, salt, and finishing temperature on residual
nitrite concentrations are shown in Table 19.
Increasing salt resulted
in significantly (p < .05) lower residual nitrite concentrations.
High
finishing temperature resulted in significantly higher residual nitrite
concentration (p < .05).
It is well-known that cooking decreases the
residual nitrite (Brooks et al., 1940; Greenwood, 1940; Rose and Peterson,
1953; Pivnick et al., 1967; Sebranek et al., 1973).
present experiment, the opposite effect resulted.
However, in the
As mentioned in the
cured color discussion, nitrosyl heme pigment and residual nitrite
concentration are usually inversely related.
As the nitrosyl heme
pigment increases during heating, residual nitrite concentration generally
decreases.
Measurement of cooking yields showed that the high finishing
temperature gave significantly (p < .05) greater shrink loss during the
operation.
All of the solutes may have been concentrated to some
degree and as a result, gave higher nitrosyl heme pigment and higher
residual nitrite concentration.
However, when adjusted for the
shrink loss, nitrosyl heme pigment was still high, but residual •
nitrite was decreased by the high internal finishing temperature.
The interaction of nitrite, salt and finishing temperature on re­
sidual nitrite during storage is evident from Figures 20 and 21,
There
89
Table 19,
Effect of nitrite, salt and internal finishing temperature
on residual nitrite
Treatments
Nitrite
75 ppm
156 ppm
S.E.
Salt
0%
1.0%
3.0%
S.E.
Temperature
520c
60OC
680C
S.E.
Residual nitrite
All samples (N = 108)2
26.1^
65.3
± 0.59
All samples (N = 72)2
48.4*
45.1?
43.7
± 0.72
All samples (N = 72)2
44.0%
46.4*
46.7
+ 0.72
Cooked samples (N = 81)
18.4^
50.9
± 0.52
Cooked samples (N = 54)
37.7*
34.0%
32.2^
+ 0.63
Cooked samples (H = 54)
32.3^
35.5a
36.0
+ 0.63
^Mean values in a column followed by different letters were
significantly different (p < .05).
2
All samples = data include raw emulsion.
TEMP
SALT
Ê
CL
CL
w
o0
A 1
D 3
60
.68
\
60
Q)
-P
*rt
L
A>
52
-CZ:
60
Ô
\
riB-"
d«îœ
,,,idu.i «i"i«
60
SALT (%)
50
E
CL
Q.
TEMP C°C)
A 1
52
60
•3
68
o0
40
m
-p
•H
•H
30
0
D 20
-
' O
T)
•iH
••it,
(0
QJ
o:
—o
A
— ^
9:
'é
•m
10
0
Figure 21.
•
0 1
•
I
I—I—I—I—I—I—'—'—
15
•AYS
f
«
'
«
i_ •
I- . I
I
I
I ...J
—
30
Effect of salt, 75 ppm nitrite and internal finishing temperature on residual nitrite
during storage
92
was a significant (p < .05) interaction between salt and nitrite.
In­
creasing salt concentration resulted in lower residual nitrite in all
cases.
However, effects of temperature were not consistent.
At low
nitrite, the low temper ature-high salt was most effective for reducing
residual nitrite while 156 ppm nitrite, the high temperature was more
effective; the reasons for these differences are not clear.
Experiment V (Effect of Muscle Type)
£H
The effect of salt and nitrite on pH are shown in Table 20,
It is
evident that again nitrite and salt increase the pH of the product
significantly (p < .05).
The results given in Figure 22 show that there is an interaction
between salt and nitrite during storage.
The pH value increased
after cooking and increased slightly during the rest of the period.
The 4% salt combined with the high nitrite level gave the highest pH
and 0% salt combined with low nitrite level resulted in the lowest pH.
There was no interaction in the emulsion but after cooking, salt and
nitrite showed significant (p < .05) interaction.
It is generally accepted that white muscle exhibits a lower pH
than red muscle but in the present experiment, the pH values of the
two types were found not to be different.
Nevertheless, the effect of
chloride and nitrite on product pH was again observed as it was in
products utilizing beef muscle as an ingredient.
93
Table 20.
Effect of nitrite and salt on pH of product with white
muscle^
Treatments
pH
Ni tri te
All samples (N = 60)'
75 ppm
156 ppm
S.E.
,b
5.91:
a
5.93
+ 0.006
5.95:
5.98
+ 0.004
Salt
All samples (N = 24)'
Cooked samples (N = 18)
Cooked samples (N
0%
0.5%
1.0%
2.0%
4.0%
S.E.
5.92=
5.97;
5-99a
6.06
+ 0.0087
6.04
a
6.12
+ 0.007
^Mean values in a column followed by different letters were
significantly different (p < .05).
2
All samples = data include raw emulsion.
45)
SALT
(%)
N i tn i te (
75
^ 0. 5
156
X
CL
#/.
5. 5
0 1
DAYS
Figure 22.
Effect of salt and nitrite on pH in products with white muscle during storage
95
Cured color
The effect of nitrite and salt on cured color is presented in
Table 21.
As the salt level was increased, a higher amount of nitrosyl
heme pigment was observed (p < .05) as in previously discussed results.
The results plotted in Figure 23 show that salt and nitrite interact
during storage.
At the high nitrite level, a low salt concentration
gave the highest nitrosyl heme pigment; but a high amount of nitrosyl
heme pigment was observed with a high salt level in conjunction with a
low nitrite level.
Nitrosyl heme pigment was constant during storage
at the low nitrite level, while the high nitrite level showed some
slight changes.
There was no interaction between salt and nitrite
during storage according to the data.
Total pigment again decreased significantly (p < .05) with in­
creasing salt levels but the nitrite level had no effect on the amount
of total pigment analyzed.
It seems that chloride ion may have affected
the myoglobin in white muscle to alter extraction of the pigment as
noted previously.
The addition of salt also increased pigment conversion significantly
(p < .05).
The 4% salt gave the highest pigment conversion but the
increase in this case was not significant.
The presentation of data
in Figure 24 shows the salt and nitrite effect on pigment conversion
during storage.
Pigment conversion was increased significantly
(p < .05) up to 15 days and decreased slightly during the rest of the
storage period.
96
Table 21.
Treatments
Effect of nitrite and salt on cured color' of product with
white muscle'^
Nitrosyl heme
pigment (ppm)
Total
pigment (ppm)
Nitrite
All 2 Cooked
All 2
samples
samples
samples
(N = 60) CN = 45)
75 ppm
156 ppm
S.E.
11.4^
13.0*
+ 0.2
Salt
07,
0.5%
1.0%
2.0%
4.0%
S.E.
12.9^
14,6*
+ 0.3
25.3*
25.3*
+ 0.3
All 2 Cooked
All 2
samples
samples
samples
(N = 24) (N = 18)
11.8^ ,
12.4*^)
12.2^'^
11.7b
13.1^
± 0.4
14.0*
14.4* ,
13.6^'b
12.5%
14.2*
± 0.5
26.9*
25.7^ ^
24.7 '
24.2*
25.0^'^
+ 0.2
Cooked
samples
Pigment
conversion (%)
All 2
samples
Cooked
samples
25,7*
75.5b
26.0*
51.0%
56.33
+ 0.3
51.23
± 1.0
± 1.2
Cooked
samples
All 2
samples
Cooked
samples
27.5*
26.4*'b
25.3°'C
43.4b
47.8^'°
49.2*
48.6*
25 3b,c
+ 0.5
52.6*
—
51.63
54,33
54.23
51.03
56.73
± 1.9
^Mean values In a column followed by different letters were
significantly different (p < ,05).
2
All samples = data include raw emulsion.
SALT
(%)
Nit nit e
75
156
o4
0
1~
0 1
_ji
U-
»
I
,j
1——J
i
I
Ii
.1.
15
DAYS
Figure 23.
Effect of salt and nitrite on nitrosyl heme pigment in products with white muscle
during storage
—
30
70
•*
^
SALT
* • • • ••• % % » » « « # ^ f
(%)
Nitrite
\ ^)
Cppm)
75
156
c 30
01
DAYS
Figure 24.
Effect of salt and nitrite on pigment conversion in products with white muscle during
storage
99
Residual nitrite
The effect of salt on residual nitrite is depicted in Table 22.
It may be seen that salt decreased the residual nitrite concentration
significantly (p < .05). It seems that chloride also enhanced nitrosation in white muscle which has less myoglobin than red muscle.
In
the present experiment, there was a higher residual nitrite concentra­
tion than in Experiment I (control).
It is likely that a low myoglobin
concentration in the white muscle resulted in less depletion of nitrite
because the control and white muscle had essentially the same pH.
The salt and nitrite interaction during the storage is depicted
in Figure 25.
Residual nitrite decreased significantly (p < .05)
during storage, an effect that seems to be encouraged by higher salt
levels.
100
Table 22.
Effect of nitrite and salt on residual nitrite of product
with white muscle^
Residual nitrite
Treatments
Nitrite
75 ppm
156 ppm
S.E.
All samples (N = 60)^
47.6^
93.0
+ 0.52
Cooked samples (N = 45)
41.0^
78.9*
+ 0,60
•
Salt
All samples (N = 24)^
Cooked samples (N = 18)
07,
0.5%
1.07=
2.0%
4.0%
S.E.
70.6^
74.4*
71.6*
69.of
65.7^
± 0.814
61.3%
65.2*
61.of
58.6=
54.1*
± 0.94
Mean values in a column followed by different letters were
significantly different (p < .05).
All samples = measurement includes emulsion.
140 jT
SALT
120
t:
E
CL
(U
-P
•H
L
-P
Nit r it e
•PP m.
75
156
o0
A 0.5
n 1
a.
sv
(%)
100
o2
o4
80 -
0
:
D
-4
•n
-0
•H
(0
QJ
ct:
C3
40
20
<P
•o,.i
0 1
15
J ••,
*
• t - 1
<
T
•<>
30
DAYS
Figure 25.
Effect of salt and nitrite on residual nitrite in products with white muscle during
storage
102
SUMMARY
The interaction of salt and nitrite was studied using five dif­
ferent product variables including different pH values, presence or
absence of reductant at reduced pH, different internal finishing
temperature, and use of white muscle (turkey breast muscle) in addition
to controls.
The product was evaluated for pH, salt content, nitrosyl pig­
ment, total pigment, pigment conversion and residual nitrite.
In ad­
dition, product yield was measured for the different finishing tempera­
ture.
There was some variation depending on the type of product treatment,
but in general, high nitrite levels resulted in a slightly increased
pH, more nitrosyl heme pigment, greater pigment conversion, and
higher residual nitrite.
Salt increased the pH, nitrosyl heme
pigment, and pigment conversion but decreased the residual nitrite.
Addition of salt decreased the total pigment significantly (p < .05)
and affected the pigment conversion ratio in both reduced pH product and
white muscle product.
It is not clear why total pigment changes; however,
salt may change the globin moiety and hinder the spectral absorbance
used for quantitation, or less protein extraction may occur in these
products.
With a relatively large decrease in pH (pH 4.9), salt had no
significant effect on the residual nitrite even though slight decreases
were observed.
Compared to controls (pH 5.76), only the high salt level
(2% and 4%) affected nitrite reactions in a product with a slightly de­
creased pH (pH 5.7). Higher internal finishing temperature resulted in
103
more shrink loss and caused greater pigment and residual nitrite concentra­
tions.
Adjusted for the shrink loss, however, pigment concentrations
were still increased by higher internal finishing temperature while
residual nitrite was decreased.
Salt showed a trend decrease in residual
nitrite except for the treatment with a relatively large decrease in
pH.
Considering these results, salt appears to have catalyzed nitro-
sation reactions which increased the nitrosyl heme pigment and enhanced
the depletion of residual nitrite.
These data lend considerable sup­
port to the hypothesis for formation of NOCl in meat systems, with further
implications yet to be rully realized.
104
CONCLUSIONS
1.
In general, salt and nitrite increased the product pH, nitrosyl
heme pigment content and pigment conversion ratio in meat mixtures.
2.
Products that contained added salt showed a trend toward
decreased residual nitrite with increasing salt concentrations.
High
ingoing nitrite (156 ppm) resulted in higher residual nitrite than the
lower nitrite level (75 ppm).
3.
Salt had no effect on the residual nitrite when product pH
was decreased to a relatively low level (pH 4.9) and this treatment did
not demonstrate the interaction between salt and nitrite.
4.
Compared to the control (pH 5.76), only the high salt levels
(2% and 4%) affected nitrite reactions in a product with a slightly de­
creased pH (pH 5.7).
5. . These data support the hypothesis that NOCl occurs in meat
systems.
This has potential implications for the inhibitory mechanism
of nitrite on bacterial growth and could be important in meat systems
with a reduced salt (NaCl) level.
105
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ACKNOWLEDGMENTS
The author wishes to express his sincere appreciation to his major
professor, Dr. Joseph G. Sebranek, for his guidance, assistance, and
encouragement throughout this graduate study.
The author is also grate­
ful to Dr. E. A. Kline and Dr. D. G. Olson for their guidance during
his graduate studies and for reviewing the manuscript.
Appreciation is
extended to Dr. A. A. Kraft, and Dr. H. W. Walker for serving on the
author's committee and for reviewing the manuscript.
Gratitude is also extended to Mrs. Sharon Colletti, Mr. Randy
Petersohn, and fellow graduate students for their friendship and help
during the author's time at Iowa State University.
The author would
like to express his deepest thaïes to his parents for their love and
encouragement.
Lastly, the author would like to thank his wife,
Heesun, for her assistance and encouragement during his graduate
study.