SALT CONCENTRATION AND SPECIES AFFECTS PROTEIN EXTRACTABILITY AND PROCESSED MEAT CHARACTERISTICS
BY
BRIANNA DAWN KEEVER
THESIS
Submitted in partial fulfillment of the requirements for the degree of Master of Science in Animal Sciences
in the Graduate College of the University of Illinois at Urbana-Champaign, 2011
Urbana, Illinois
Adviser:
Professor Anna Dilger
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Abstract
Excessive sodium consumption is one contributor to high blood pressure and
cardiovascular disease, and the Institute of Medicine has recommended that salt levels be
reduced in food, including processed meats. As such, it is critical to understand the interaction of
salt level and meat species with regards to characteristics of further processed meat. Salt-soluble
protein extraction, important in forming meat emulsions, is dependent on several factors
including species of protein source. As such, the purpose of our first study was to quantify the
amount of salt-soluble proteins in various muscles of three species commonly used in meat
products - beef, pork, and chicken. Pork and beef serratus ventralis (dark) and semitendinosus
(light) muscles, and chicken pectoralis major muscles (light) and the entire thigh (dark) were
used. Samples were analyzed for salt-soluble protein content at salt (NaCl) concentrations
ranging from 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, to 3.5%. Overall, there was an increase in
extractable salt-soluble protein content as the salt concentration increased. The interaction of
species and salt showed all species were different (P < 0.05) from one another when compared at
the same salt concentration when corrected for fat and moisture content with chicken showing
the greatest extractability. In chicken, extractable salt-soluble protein was increased (P < 0.01)
in light muscles compared with dark muscles. In industry, most processed products include 1.5-
3.5% salt, and at these levels we have demonstrated that chicken meat had more salt-soluble
proteins to aid in the processing of meat products. Given the advantage of chicken overall and in
chicken light muscles compared with dark muscles, the purpose of the second study was to
evaluate characteristics of products manufactured from pectoralis major muscle (light) and the
entire thigh (dark) of chicken. The study was designed as a complete randomized design in a
2x4 factorial arrangement with factors of muscle type (light and dark) and salt concentration
(0.5%, 1%, 1.5%, and 2%). Bologna was manufactured and analyzed for salt-soluble protein
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content, proximate analysis, cooking yield, pH, Minolta L*, and binding strength. In thigh
muscle bologna batches, raw and cooked moisture content, as well as cooked fat content, was
increased over breast muscle batches (P < 0.01). Increased retention of moisture in thigh muscle
bolognas contributed to improved cooked yields compared to breast muscle bolognas (P = 0.01).
We were especially interested in whether characteristics of low-salt (≤ 1%) light muscle
bolognas were similar or superior to higher salt (≥ 1.5%) dark muscle bolognas. Therefore, we
used a single degree of freedom contrasts to compare these products. Low salt breast bolognas
exhibited slightly higher (0.05 percentage units) salt-soluble protein extractability (as a
percentage of crude protein) compared with higher salt thigh meat bolognas. The increase in
salt-soluble protein content, however, was not reflected in a higher break strength value for low-
salt breast bologna compared to high-salt thigh bologna (P < 0.01). Hardness increased in low-
salt breast bologna compared to high-salt thigh bologna (P < 0.01). Low salt breast bologna
showed acceptable processing characteristics and textural properties, therefore it can be used to
manufacture low salt products.
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Table of Contents
Chapter I Review of Literature ...................................................................................................... 1
Introduction ............................................................................................................................. 1
Protein Extraction .................................................................................................................... 2
Formation of Meat Emulsions ................................................................................................. 3
Gelation ................................................................................................................................... 4
Sensory .................................................................................................................................... 6
Objectives ................................................................................................................................ 6
References ............................................................................................................................... 8
Chapter II Extraction of Salt-soluble Proteins in Meat is Affected by Species and Muscle Type....................................................................................................................................................... 11
Abstract .................................................................................................................................. 11
Introduction ........................................................................................................................... 11
Material and Methods ............................................................................................................ 13
Results and Discussion .......................................................................................................... 15
Conclusions ........................................................................................................................... 18
References ............................................................................................................................. 19
Tables and Figures ................................................................................................................. 21
Chapter III The Effects of Varying Salt Concentrations on Protein Solubility and Product Characteristics of Bolognas Manufactured from Chicken Muscles ............................................. 25
Abstract .................................................................................................................................. 25
Introduction ........................................................................................................................... 25
Materials and Methods .......................................................................................................... 26
Results and Discussion .......................................................................................................... 30
Conclusions ........................................................................................................................... 34
References ............................................................................................................................. 35
Tables and Figures ................................................................................................................. 38
1
Chapter I
Review of Literature
Introduction
Today’s consumer devotes less time to preparing meals, so they look to ready-to-eat
products to reduce time spent in the kitchen. However, these products traditionally tend to be
higher in sodium (975mg sodium/100g), than unprocessed meat, such as steaks or roasts (50-90
mg sodium/100g) (Romans, Costello, Carlson, Greaser, and Jones, 1994). In 2010, the Dietary
Guidelines for Americans listed reducing sodium as a major goal (USDA and USDHHS, 2010).
In addition, the Institute of Medicine recommends that intake of sodium not exceed 2300 mg per
day for people over the age of 14. At this level of intake, sodium is not likely to trigger adverse
health effects, such as increased risk of developing hypertension (Dahl, 1972). However,
average sodium intake for Americans over the age of two is approximately 3400 mg per day,
nearly 1.5 times more than recommended levels (USDA-ARS and USDHHS-CDC, 2006). Thus,
it is imperative that consumers lower sodium intake as one way to ensure a healthy lifestyle. In
response to desires for lowered sodium, meat processors offer products with reduced sodium
levels. The challenge for the industry, however, is to create high-quality products that remain
acceptable to the consumer despite reductions in salt.
Salt, defined in this paper as sodium chloride (NaCl), was initially used by people to
preserve meat. Currently, salt is the most widely used non-meat ingredient in processed foods
and functions to enhance the flavor and texture of meat products. Terrell (1983) described the
following to be advantages of salt addition to meat products: extraction of proteins to increase
hydration and water binding capacity, increased binding properties of proteins to improve
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texture, increased viscosity of meat batter facilitating the incorporation of fat to form stable
batters, increased pH of meat systems, flavor enhancement and bacteriostatic effects.
Protein Extraction
Muscle protein is categorized into three classes of proteins, differing in function and their
ability to be solubilized in salt solutions. The first class, sarcoplasmic proteins, account for
approximately 30% of total muscle proteins and are soluble at low ionic strength (>0.3mM)
(Scopes, 1964). Proteins from the sarcoplasm, including myoglobin and metabolic enzymes
make up this class of proteins. The second class of proteins is the myofibrillar proteins, which
comprise approximately 50-60% of muscle proteins and are soluble at salt concentrations greater
than 0.3M. Myosin and actin contribute 45 and 20%, respectively, to the total myofibrillar
protein content making these two proteins the most important when considering protein
extraction in further processed products (Scopes, 1964). The third class of proteins is known as
the stromal proteins, which are not soluble in salt solutions unless heat is added. These stromal
proteins constitute approximately 10-20% of the total muscle proteins and consist of collagen
and elastin.
Further processed meat products typically begin with a primary comminution (reduction
of particle size) phase. Comminution is essential in disrupting membranes and sarcolemma of
muscle in order to free myofibrillar proteins and allow them to swell. After comminution, salt is
added, increasing the water-binding capacity of myofibrillar proteins by creating a downward
shift in the isoelectric point of these proteins, resulting in a larger net negative charge. Hamm
(1961) theorized that salt caused a repulsion of peptide chains on myofibrillar protein, allowing
water to enter this space. As the concentration of salt incorporated in products increases, the
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amount of myofibrillar protein extracted increases until a threshold is reached at 1.2 M NaCl
(Munasinghe and Sakai, 2004). Salt extraction of proteins is affected by pH, extraction volume,
and homogenization time (Lan et al., 1993). The ideal extraction for beef and pork occur at a pH
of 6.0 with an extraction volume of 15 X sample weight and a homogenization time of 90 to 120
seconds (Lan et al., 1993). Extraction also differs between species and muscles within species.
Under ideal extraction conditions, pork longissimus dorsi muscle yielded greater values for salt
soluble proteins as a percent of the sample weight when compared to that of beef longissimus
dorsi muscle and semimembranosus muscle (Lan et al., 1993). The content of extractable salt-
soluble proteins was highly variable due to differences in pH and collagen content among red
meats commonly used in the manufacturing of further processed meats. Cow meat had the
highest extractability (8.19%) and meat classified as “rework”, the lowest (1.23%) (Saffle and
Galbreath, 1964). However, many studies have established that greater amounts of salt-soluble
proteins could be extracted from breast muscles of poultry (turkeys, chickens, and ducks) when
compared to the leg muscles of the same animals (Khan, 1962; Hudspeth and May, 1967; Maurer
et al., 1969; McCready and Cunningham, 1971; Prusa and Bowers, 1984; Richardson and Jones,
1987). Constrained by the type of product being manufactured, the extractability of salt-soluble
proteins is highly variable and dependent on several processing factors, such as pH and time.
Therefore, it is important to characterize the extractability of different species and muscles to
identify where they can best be used in further processed products.
Formation of Meat Emulsions
The ability of salt to solubilize myofibrillar proteins is one of the most important factors
in the processing of emulsified meat products like bologna and frankfurters. These products are
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formed by blending a mixture of lean meat, fat, water, salt and other ingredients into an emulsion
forming a nearly homogenous product. Meat emulsions form a two-phase system with the
dispersed phase containing fat particles and the continuous phase consisting of suspended
proteins and water containing dissolved salts (Forrest et al., 2001). In this system, solubilized
proteins add stability to the emulsion, acting as emulsifying agents and reducing surface tension
formed when fat comes into contact with water (Schut, 1976). Solubilized protein molecules
coat fat particles through protein’s affinity for both hydrophilic and hydrophobic particles.
Protein molecules, once unfolded, are oriented around fat particles with hydrophobic side chains
in contact with fat and hydrophilic side chains in contact with water (Forrest et al., 2001).
Increasing the amount of solubilized protein will lead to an increase in the emulsion stability of
the product, which is a measure of the ability of the emulsion to retain fat and water (Zorba et al.,
1993). Also, increasing the solubilized protein concentration will lead to an increase in the
emulsion capacity of the product, which is a measure of the ability of the emulsion to
encapsulate fat (Swift and Sulzbacher, 1963). Although, bologna and frankfurters are classified
as emulsions, they are not true emulsions. When making frankfurters not all of the fat particles
in the system were uniformly surrounded by solubilized proteins leading to a more
heterogeneous mixture than a true emulsion (Swasdee et al., 1982).
Gelation
After the emulsified product is placed into a casing, it is thermally processed allowing for
muscle protein gelation to occur. The gelation of proteins in comminuted products is responsible
for adhesion of meat to meat as well as binding fat particles within the meat. The process of
forming this gel matrix takes place in three steps: the initial denaturing of proteins; the
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aggregation of these individual proteins, and the crosslinking of the protein aggregates to form a
continuous network after heating (Xiong, 2004). The ability of these myofibrillar proteins to
form a stable gel matrix is influenced by structure, size, concentration and pH of the protein. It
has been demonstrated that intact myosin contributes to the binding properties of gels and that
actin alone cannot influence these properties (Samejima et al., 1969). However, the presence of
actin at a myosin to actin ration (w/w) of 24 increased rigidity of gels when compared to gels
prepared in the absence of actin (Morita and Ogata, 1991). The balance of proteins within a
muscle also influences gel stability. Muscles containing large amounts of connective tissue are
detrimental to the formation of a stable gel matrix due to their poor emulsifying properties and
the propensity to shrink during the heating process. Therefore, the amount of myofibrillar
proteins extracted in relation to the amount of collagen will influence the quality of the finished
product.
Muscle type and pH influence gel stability and these factors interact with each other.
Myofibrillar proteins from white muscles are able to form firmer gels than those from red
muscles due to the differences in myosin isoforms (Xiong, 1994). Gels formed from chicken and
turkey pectoralis muscles (predominantly white) were more rigid and less influenced by pH than
gels formed from chicken leg muscles (predominantly red) (Foegeding, 1987; Xiong and
Blanchard, 1994). Chicken breast muscles formed the strongest gels at a pH of 6.30, while the
thigh muscles were strongest at 6.00 (Xiong and Brekke, 1991; Lesiow and Xiong, 2003). Pork
longissimus dorsi muscles (predominantly white) were also able to form stronger gels when
compared to pork serratus ventralis muscles (predominantly red) (Robe and Xiong, 1993). In
contrast, beef semimembranosus muscles (predominantly white) and vastus intermedius muscles
6
(predominantly red) gels showed no differences when compared at similar pH (Vega-Warner et
al., 1999). The ability to target the use of muscles at their optimum gelation pH and salt
extraction level in further processed products would enhance the emulsion stability.
Sensory
One of the challenges to reducing the inclusion level of salt in further processed products
is consumer acceptability of lower sodium products. Consumers may not find the new flavor or
textural properties to be similar enough to the products they are accustomed to eating. This is
further complicated by the dual pressure to reduce the fat content of processed products as the
combination of fat and salt in further processed products contributes many of the sensory
characteristics found within these products (Ruusenen et al., 2003). Salt reduction, however, is
possible while maintaining sensory properties. Panelists found the same degree of saltiness in
hams containing 1.7, 2.0, and 2.3% salt (Ruusenen et al., 2001). Similarly, frankfurters could be
manufactured with 1.3% salt and still maintain sensory hardness scores deemed previously to be
acceptable to consumers (Matulis et al., 1995) and with no adverse effects to the physical
properties of the product (Puolanne and Terrell, 1983a, 1983b). However, salt inclusions of less
than 1.0% in frankfurters yielded poorly due to a lack of water retention (Puolanne and Terrell,
1983a, 1983b). The ability to reduce salt and maintain textural properties has been shown to a
degree, however, to continue the reduction of salt and maintain acceptability gradual reductions
may need to be made over a period of time.
Objectives
The purpose of this study was to identify species or muscles that would allow for salt
reduction while maintaining acceptable protein extraction and product characteristics. The
7
species of interest are beef, pork and chicken because these species are commonly used by the
meat industry for further processed products. It is expected that chicken will yield the highest
amount of total extractable salt soluble proteins based on previous literature, however it is
unclear what differences will be observed between beef and pork. Within a species, differences
between muscle types and their suitability for further processing will also be determined. By
identifying differences in extractability of salt soluble proteins that may exist between common
livestock species such as beef, pork and chicken or between muscles, meat processors could
choose protein sources that allow maximum myofibrillar protein extraction at reduced salt
inclusion levels.
The objectives of this study were:
1) Quantify the amount of salt-soluble proteins in beef, pork, and chicken.
2) Determine further processed characteristics of products from white muscle and
dark muscles over a range of salt inclusion levels.
8
References
Dahl, L. K. (1972). Salt and hypertension. The American Journal of Clinical Nutrition, 25, 231-244.
Foegeding, E. A. (1987). Functional properties of turkey salt-soluble proteins. Journal of Food
Science, 52, 1495-1499.
Forrest, J.C., Aberle, E.D., Gerrard, D.E., and Mills, E.W. (2001). Principles of Meat Science
Fourth Edition edn. Dubuque, IA, Kendall/Hunt Publishing Company.
Hamm, R. (1961). Biochemistry of meat hydration. In C.O. Chichester and E.M. Mrak, Advances
in Food Research (pp. 355-380) Academic Press.
Hudspeth, J. P., and May, K. N. (1967). A study of the emulsifying capacity of salt soluble proteins of poultry meat. Food Technology, 21, 1141-1142.
Khan, A. W. (1962). Extraction and fractionation of proteins in fresh chicken muscles. Journal
of Food Science, 27, 430-434.
Lan, Y. H., Novakofski, J., Carr, T. R., and McKeith, F. K. (1993). Assay and storage conditions affect yield of salt soluble protein from muscle. Journal of Food Science, 58, 963-967.
Lesiów, T., and Xiong, Y. L. (2003). Chicken muscle homogenate gelation properties: effect of pH and muscle fiber type. Meat Science, 64, 399-403.
Matulis, R. J., McKeith, F. K., Sutherland, J. W., and Brewer, M. S. (1995). Sensory characteristics of frankfurters as affected by fat, salt, and pH. Journal of Food Science, 60, 42-47.
Maurer, A. J., Baker, R. C., and Vadehra, D. V. (1969). The influence of type of poultry and carcass part on the extractability and emulsifying capacity of salt-soluble proteins. Poultry
Science, 48, 994-997.
McCready, S. T., and Cunningham, F. E. (1971). Salt-soluble proteins of poultry meat 1. composition and emulsifying capacity. Poultry Science, 50, 243-248.
Morita, J., and Ogata, T. (1991). Role of light chains in heat-induced gelation of skeletal muscle myosin. Journal of Food Science, 56, 855-856.
Munasinghe, D. M. S., and Sakai, T. (2004). Sodium chloride as a preferred protein extractant for pork lean meat. Meat Science, 67, 697-703.
9
Prusa, K. J., and Bowers, J. A. (1984). Protein extraction from frozen, thawed turkey muscle with sodium nitrite, sodium chloride, and selected sodium phosphate salts. Journal of Food
Science, 49, 709-713.
Puolanne, E. J., and Terrell, R. N. (1983a). Effects of rigor-state, levels of salt and sodium tripolyphosphate on physical, chemical and sensory properties of frankfurter-type sausages. Journal of Food Science, 48, 1036-1038.
Puolanne, E. J., and Terrell, R. N. (1983b). Effects of salt levels in prerigor blends and cooked sausages on water binding, released fat and pH. Journal of Food Science, 48, 1022-1024.
Richardson, R. I., and Jones, J. M. (1987). The effects of salt concentration and pH upon water-binding, water-holding and protein extractability of turkey meat. International Journal of
Food Science and Technology, 22, 683-692.
Robe, G. H., and Xiong, Y. L. (1992). Phosphates and muscle fiber type influence thermal transitions in porcine salt-soluble protein aggregation. Journal of Food Science, 57, 1304-1304.
Ruusunen, M., Simolin, M., and Puolanne, E. (2001). The effect of fat content and flavor enhancers on the perceived saltiness of cooked bologna-type sausages. Journal of Muscle
Foods, 12, 107-120.
Ruusunen, M., Vainionpää, J., Puolanne, E., Lyly, M., Lähteenmäki, L., Niemistö, M., and Ahvenainen, R. (2003). Physical and sensory properties of low-salt phosphate-free frankfurters composed with various ingredients. Meat Science, 63, 9-16.
Saffle, R. L., and Galbreath, J. W. (1964). Quantitative determination of salt-soluble protein in various types of meat. Food Technology, 18, 119-120.
Samejima, K., Hashimoto, Y., Yasui, T., and Fukazawa, T. (1969). Heat gelling properties of myosin, actin, actomyosin and myosin-subunits in a saline model system. Journal of Food
Science, 34, 242-245.
Schut, J. (1976). Meat emulsions. In S. Friberg, Food Emulsions (pp. 385-400). New York: Marcel Dekker Inc.
Scopes, R. K. (1964). The influence of post-mortem conditions on the solubilities of muscle proteins. Biochemical Journal, 91, 201-207.
Swasdee, R. L., Terrell, R. N., Dutson, T. R., and Lewis, R. E. (1982). Ultrastructural changes during chopping and cooking of a frankfurter batter. Journal of Food Science, 47, 1011-1013.
10
Swift, C. E., and Sulzbacher, W. L. (1963). Comminuted meat emulsions: factors affecting meat proteins as emulsion stabilizers. Food Technology, 17, 224-226.
Terrell, R. N. (1983). Reducing the sodium content of processed meats. Food Technology, 37, 66-71.
U.S. Department of Agriculture, Agricultural Research Service and U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. (2006). What we eat in America, NHANES.
U.S. Department of Agriculture and U.S. Department of Health and Human Services. (2010). Dietary guidelines for Americans. 7th edn. Washington, DC, U.S. Government Printing Office.
Vega-Warner, V., Merkel, R. A., and Smith, D. M. (1999). Composition, solubility and gel properties of salt soluble proteins from two bovine muscle types. Meat Science, 51, 197-203.
Xiong, Y. L. (2004). Muscle proteins. In R. Y. Yada, Proteins in food processing (pp. 106-110). Cambridge, England: Woodhead Publishing Limited.
Xiong, Y. L. (1994). Myofibrillar protein from different muscle fiber types: Implications of biochemical and functional properties in meat processing. Critical Reviews in Food Science
and Nutrition, 34, 293-320.
Xiong, Y. L., and Blanchard, S. P. (1994). Dynamic gelling properties of myofibrillar protein from skeletal muscle of different chicken parts. Journal of Agricultural and Food
Chemistry, 42, 670-674.
Xiong, Y. L., and Brekke, C. J. (1991). Protein extractability and thermally induced gelation properties of myofibrils isolated from pre- and postrigor chicken muscles. Journal of Food
Science, 56, 210-215.
Zorba, O., Gokalp, H. Y., Yetim, H., and Ockerman, H. W. (1993). Model system evaluations of the effects of different levels of K2HPO4 NaCl and oil temperature on emulsion stability and viscosity of fresh and frozen turkish style meat emulsions. Meat Science, 34, 145-161.
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Chapter II
Extraction of Salt-soluble Proteins in Meat is Affected by Species and Muscle Type
Abstract
Salt-soluble protein extraction, important in forming meat emulsions, is dependent on
several factors including species of the protein source. The purpose of this experiment was to
quantify the amount of salt-soluble proteins in various muscles of three species commonly used
in meat products - beef, pork, and chicken. Pork and beef serratus ventralis muscles (dark) and
the semitendinosus muscles (light), and chicken pectoralis major muscles (light) and the entire
thigh (dark) muscles were used. Samples were analyzed for proximate composition and for salt-
soluble protein content at salt (NaCl) concentrations of 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, and
3.5%. Overall, there was an increase in extractable salt-soluble protein content as the salt
concentration increased. Chicken had the highest amount of extractable salt-soluble protein
compared with beef or pork at all salt concentrations, when samples were corrected for fat and
moisture content (P < 0.05). Chicken light muscles were able to extract more salt-soluble protein
at most salt concentrations, except 0 and 2.5%, compared with chicken dark muscles (P < 0.01).
This study demonstrates that chicken meat has increased amounts of extractable salt-soluble
protein content compared to beef or pork.
Introduction
Extraction of myofibrillar proteins, commonly called salt-soluble proteins, is crucial in
manufacturing of further processed meat products such as frankfurters and bologna. Once
extracted, these proteins serve as emulsifying agents in meat batter and contribute to emulsion
stability. Extraction is achieved through the addition of sodium chloride to meat. Recently,
however, concerns have emerged in the medical community about excessive sodium intake and
12
its relation to cardiovascular disease (Page, 1976; Porter, 1983; Gleibermann, 1973; Tobian,
1997). Therefore, it is in the interest of meat processors to acceptably reduce sodium content in
meat products.
One way to reduce the amount of sodium chloride included in further processed products
is to target meat sources that are enriched in salt-soluble proteins. Greater amounts of salt-
soluble proteins can be extracted from breast muscles of poultry (turkeys, chickens, and ducks)
when compared with leg muscles of the same animals (Khan, 1962; Hudspeth and May, 1967;
Maurer et al., 1969; McCready and Cunningham, 1971; Prusa and Bowers, 1984; Richardson
and Jones, 1987). Under ideal extraction conditions, pork longissimus dorsi muscle yielded
greater values for salt soluble proteins as a percent of the sample weight when compared with
beef longissimus dorsi and semimembranosus muscles (Lan et al., 1993). There were no
differences, however, in salt-soluble protein extraction between beef semimembranosus or
longissimus dorsi muscles (Lan et al., 1993). Our objective was to determine differences in salt-
soluble protein extraction between three species commonly used for further processing – beef,
pork, and chicken and to determine effect of muscle type on protein extraction. For each species,
a predominantly red fiber type muscle (serratus ventralis and entire thigh) and a predominantly
white fiber type muscle (semitendinosus and pectoralis major) were analyzed for differences
within species.
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Material and Methods
Sample Collection
Five muscle samples (approximately 500 grams) were obtained from each of the
following treatment combinations: serratus ventralis (dark) and semitendinosus (light) muscles
from beef and pork, and from pectoralis major muscle (light) and the entire thigh (dark) from
chicken two days post-harvest resulting in 30 total samples. Meat was trimmed of external fat,
homogenized, vacuumed packaged and stored (-29°C) until analyses were conducted.
Proximate Analysis
The moisture and lipid content was determined for each sample using the method
described by Novakofski et al. (1989). In preparation, all samples were trimmed of external fat
and connective tissue, and homogenized using a Cusinart Food Processor. Moisture content was
determined after samples had been placed in a drying oven at 110°C for 48h. Lipid content was
then determined after extraction by washing samples in a mixture of chloroform and methanol.
Salt-Soluble Protein Content
The extractability of both sarcoplasmic and myofibrillar proteins were determined using
the method described by Boler et al. (2011). A master mix extraction buffer of 0.01M 2-[N-
Morpholino]ethanesulfonic acid was used to make the following eight NaCl extraction buffers
0% (0M), 0.5% (0.09M), 1% (0.17M), 1.5% (0.26M), 2% (0.34M), 2.5% (0.43M), 3% (0.51M)
and 3.5% (0.6M) by volume. All extraction solutions were buffered to a pH of 6.0 according to
Lan et al. (1993). Ground muscle samples (0.1 g) were placed in 2.0 mL microcentrifuge tubes
with 4mm stainless steel beads and 1.5 mL extraction buffer. Samples were homogenized
(TissueLyser II, Qiagen Inc., Valencia, CA) for 1 minute. Homogenates were centrifuged
14
(Microfuge® 18 Microcentrifuge, Beckman Coulter Inc., Germany) at 15000 x g for 20 min at 4°
C. Supernatants were collected for each sample, diluted 10 fold, and protein content quantified
using a BCA protein assay kit (Thermo Scientific Pierce Protein Research Products, Rockford,
IL). The amount of solubilized protein was determined using a second order polynomial
equation. Data were expressed as a percentage of wet weight and as the percentage of solids
weight (weight minus fat and moisture).
pH
Meat (5g) was placed in a 50 mL conical tube with 20 mL of deionized water and
homogenized (Model PT 10/35, Brinkmann Instruments Co., Switzerland) for 30 seconds. pH
was measured with an automatic temperature compensation epoxy body probe attached to a
bench meter (Model 917001, Model 501, Orion Research Inc., Jacksonville, Florida) that had
been calibrated with pH 4.00 and 7.00 buffers.
Statistical Analysis
Data were analyzed as a randomized complete design in a 3x2 factorial arrangement with
salt as a continuous variable. The statistical model included the main effects of species, salt,
muscle type (light or dark) and their interactions. Differences between moisture, fat, and pH
were determined using MIXED procedure of SAS (SAS Inst. Inc., Cary, N.C.) and means
separated using the PDIFF option with a Bonferroni adjustment. Salt-soluble protein content
were analyzed as repeated measures using an unstructured covariance structure with sample
serving as the subject. Means of salt-soluble protein content were separated with the PDIFF
option with a Bonferrroni adjustment. Differences were deemed significant at P < 0.05.
15
Results and Discussion
Proximate Analysis and pH
For moisture content, the main effects of species and muscle type were significant (P <
0.01), however, there was an interaction of species and muscle type (P < 0.01). In beef and
chicken, moisture content was increased in light muscles compared with dark muscles, but in
pork, dark muscle had more moisture compared to light muscles (Table 1). Similarly, for fat
content, the main effects of species and muscle type were significant (P < 0.01), however, there
was an interaction of species and muscle type (P < 0.01). In beef and chicken, fat content was
increased in dark muscles compared with light muscles, but in pork, light muscles had more fat
compared to dark muscles. There is an inverse relationship seen between moisture and fat
content, but this relationship is different for the muscles of pork when compared to beef and
chicken muscles. Generally, it has been accepted that glycolytic (light) muscles contain less fat
when compared to oxidative (dark) muscles since the main energy source of light muscles are
carbohydrate glycogen (Leseigneur-Meynier and Gandemer, 1991). The observed values of
chicken light and dark muscles and beef light and dark muscles were consistent with other
findings (Xiong et al., 1993; Seggern et al., 2005). However, pork muscles did not follow this
trend, perhaps due to the fact that metabolic pathways were more intermediate in the two
muscles examined; still, the observed trend was substantiated by other work (Beecher et al.,
1965).
For pH, the main effects of species and muscle type were significant (P < 0.01), however,
there was an interaction of species and muscle type (P < 0.01). For chicken and beef, pH was
lower in light muscles compared to dark muscles, which was expected due to the method of
metabolism, associated with each muscle type; however, there was no difference between pork
16
muscles. Ultimate pH is dependent on lactate, the final product of glycolysis, which builds up in
muscles after harvest due to stopped blood circulation. Muscles, whose metabolic pathway is
primarily glycolytic like that of light muscles, will produce more lactate, which will drive
ultimate pH downward (Lee et al., 2010). The values observed for pH of chicken, beef, and pork
were consistent with the literature, in that light muscles reported lower values compared to dark
muscles (Xiong et al., 1993; Seggern et al., 2005; Gil, et al., 2008). However, differences in pH
were controlled for when running the salt-soluble protein assay by buffering the solution to pH
of 6.0 for all samples.
Salt Soluble Protein Content
For salt-soluble protein content as a percentage of wet weight, the main effects of species,
salt, and muscle type were significant (P < 0.01), however, there was an interaction of species
and muscle type (P < 0.01), species and salt (P < 0.01), and species, muscle, and salt (P < 0.01).
Overall, increasing amounts of protein were extracted as salt concentration increased (Figure 1).
The increase in extractable salt-soluble protein content as the salt concentration increases is
known as a salting-in effect, and for sodium chloride this effect is seen when salt is added at
concentrations of 0.3 to 1.0 M (Hultin et al., 1995). In chicken light muscles, extractable salt-
soluble protein content increased (P < 0.01) at a more rapid rate with increasing salt levels
compared with all other species and muscle combinations resulting in the interaction of species,
muscle, and salt (Figure 1). Thus, the effect of increasing salt concentrations on the solubility of
muscle proteins is not only dependent on species, but also on muscle type being extracted. The
greater extractability of chicken light muscle compared to chicken dark muscle was in agreement
with other researchers (Khan, 1962; Hudspeth and May, 1967; Maurer et al., 1969; McCready
and Cunningham, 1971; Prusa and Bowers, 1984; Richardson and Jones, 1987). The lack of
17
differences between pork and beef muscles may be due to the increase in the degree of similarity
between muscle fiber types within the muscles, as opposed to the chicken muscles, which were
predominantly type I or type IIb fibers. The interaction of species and salt showed similar
trends, in that, all species were significantly different (P < 0.05) from one another when
compared at the same salt concentration, except for beef and chicken at 0% (Figure 2A). Also of
importance, chicken muscles were able to extract the same amount of salt-soluble proteins at 1%
salt as beef muscles extracted at 2.5% salt, and that pork muscles never equaled this amount even
at 3.5% salt (Figure 2A).
Comparing salt-soluble protein content as a percentage of solids weight (weight minus
fat and moisture), the main effects of species and salt were significant (P < 0.01), however, there
was a significant interaction of species and muscle type (P < 0.01) and species and salt (P <
0.01). The interaction of species and muscle type was due to the increased salt-soluble protein
content of chicken light muscle compared to chicken dark muscle and the lack of differences
within beef and pork muscles (Figures 3A, B, C). The interaction of salt and species was due to
the differences (P < 0.05) of all species, when compared at the same salt concentration (Figure
2B). When comparing the same salt concentration, chicken muscles expressed higher contents
of salt-soluble proteins, followed by beef and finally pork (Figure 2B). Also of importance,
chicken muscles were able to extract the same content of salt-soluble proteins at 1% salt as beef
muscles at 3% salt; an amount that was not able to be extracted from pork even at 3.5% salt
(Figure 2B). Within species, chicken light muscles demonstrated increased (P < 0.02) values in
salt-soluble protein content compared to dark muscles at all salt concentrations, except 0 and
2.5% (Figure 3B). However, pork muscles were only different at 3.5% (P < 0.01)(Figure 3A),
and beef muscles were different at 1% (P < 0.02), 2.5% (P < 0.05), and 3% (P < 0.05) (Figure
18
3C). The values for salt-soluble protein content as a percentage of solids weight may be more
accurate in determining differences in the amount of protein available in these meat sources since
it is corrected for the moisture and fat content.
Identifying salt-soluble protein content of meat sources is important because these
proteins act as emulsifiers in further processed products (Hudspeth and May, 1967). In industry,
most processes included of 1.5-3.5% salt, and at these levels we demonstrate chicken as a species
will have more salt-soluble proteins than pork or beef to aid in the processing of meat products.
Also, chicken light muscles would be even more beneficial due to the advantages in
extractability over chicken dark muscles.
Conclusions
Overall, the largest quantity of salt-soluble proteins was extracted from chicken followed
by that of beef and finally pork. Chicken light muscles exhibited higher extractability at low salt
concentrations (0.5-2%) with light muscles containing more extractable salt-soluble proteins
compared to dark muscles as a percentage of solids weight. There were no differences in pork
muscles at these low salt concentrations (0.5%-2%). Beef muscles did exhibit a difference at
1%, when comparing within this low salt range (0.5%-2%), with dark muscles being greater than
light muscles. This study suggests that chicken light muscles could be used at lower salt
concentrations compared to chicken dark, or beef and pork muscles due to the increase in amount
of salt-soluble protein extracted that is needed to manufacture further processed products.
19
References
Beecher, G. R., Cassens, R. G., Hoekstra, W. G., and Briskey, E. J. (1965). Red and white fiber content and associated post-mortem properties of seven porcine muscles. Journal of Food
Science, 30, 969-976.
Boler, D. D., Holmer, S. F., Duncan, D. A., Carr, S. N., Ritter, M. J., Stites, C. R., Petry, D. B., Hinson, R. B., Allee, G. L., McKeith, F. K., Killefer, J. (2011). Fresh meat and further processing characteristics of ham muscles from finishing pigs fed ractopamine hydrochloride. Journal of Animal Science, 89, 210-220.
Gil, M., Delday, M. I., Gispert, M., i Furnols, M., Maltin, C. M., Plastow, G. S., Klont, R., Sosnicki, A. A., Carrión, D. (2008). Relationships between biochemical characteristics and meat quality of Longissimus thoracis and Semimembranosus muscles in five porcine lines. Meat Science, 80(3), 927-933.
Gleibermann, L. (1973). Blood pressure and dietary salt in human populations. Ecology of Food
and Nutrition, 2, 143-155.
Hudspeth, J. P., and May, K. N. (1967). A study of the emulsifying capacity of salt soluble proteins of poultry meat. Food Technology, 21, 1141-1142.
Hultin, H. O., Feng, Y., and Stanley, D. W. (1995). A re-examination of muscle protein solubility. Journal of Muscle Foods, 6, 91-107.
Khan, A. W. (1962). Extraction and fractionation of proteins in fresh chicken muscles. Journal
of Food Science, 27, 430-434.
Lan, Y. H., Novakofski, J., Carr, T. R., and McKeith, F. K. (1993). Assay and storage conditions affect yield of salt soluble protein from muscle. Journal of Food Science, 58, 963-967.
Lee, S. H., Joo, S. T., and Ryu, Y. C. (2010). Skeletal muscle fiber type and myofibrillar proteins in relation to meat quality. Meat Science, 86, 166-170.
Leseigneur-Meynier, A., and Gandemer, G. (1991). Lipid composition of pork muscle in relation to the metabolic type of the fibres. Meat Science, 29, 229-241.
Maurer, A. J., Baker, R. C., and Vadehra, D. V. (1969). The influence of type of poultry and carcass part on the extractability and emulsifying capacity of salt-soluble proteins. Poultry
Science, 48, 994-997.
McCready, S. T., and Cunningham, F. E. (1971). Salt-soluble proteins of poultry meat 1. composition and emulsifying capacity. Poultry Science, 50, 243-248.
20
Novakofski, J., Park, S., Bechtel, P. J., and McKeith, F. K. (1989). Composition of Cooked Pork Chops: Effect of Removing Subcutaneous Fat Before Cooking. Journal of Food Science, 54, 15-17.
Page, L. B. (1976). Epidemiologic evidence on the etiology of human hypertension and its possible prevention. American Heart Journal, 91, 527-534.
Porter, G. A. (1983). Chronology of the sodium hypothesis and hypertension. Annals of Internal
Medicine, 98, 720-723.
Prusa, K. J., and Bowers, J. A. (1984). Protein extraction from frozen, thawed turkey muscle with sodium nitrite, sodium chloride, and selected sodium phosphate salts. Journal of Food
Science, 49, 709-713.
Richardson, R. I., and Jones, J. M. (1987). The effects of salt concentration and pH upon water-binding, water-holding and protein extractability of turkey meat. International Journal of
Food Science and Technology, 22, 683-692.
Seggern, D. D. V., Calkins, C. R., Johnson, D. D., Brickler, J. E., and Gwartney, B. L. (2005). Muscle profiling: Characterizing the muscles of the beef chuck and round. Meat Science, 71, 39-51.
Tobian, L. (1997). Dietary sodium chloride and potassium have effects on the pathophysiology of hypertension in humans and animals. The American Journal of Clinical Nutrition, 65, 606S-611S.
Xiong, Y. L., Cantor, A. H., Pescatore, A. J., Blanchard, S. P., and Straw, M. L. (1993). Variations in muscle chemical composition, pH and protein extractability among eight different broiler crosses. Poultry Science, 72, 583-588.
21
Tables and Figures
Table 1. Effects of species and muscle on proximate composition and pH of raw materials
Light Dark Light Dark Light Dark SEM Species Muscle Interaction
Moisture, % 76.12 73.57 74.60 66.86 73.86 74.36 0.15 <0.01 <0.01 <0.01
Fat, % 1.79 7.08 3.58 15.87 6.34 5.71 0.19 <0.01 <0.01 <0.01
pH 5.98 6.40 5.76 6.11 5.71 5.72 0.01 <0.01 <0.01 <0.01
P - valuesChicken Beef Pork
21
22
Figure 1. Salt-soluble protein content of light and dark muscles of pork, beef and chicken (as a percentage of wet tissue weight). Within a level of salt, asterisks indicate a difference at P < 0.05 between chicken light and all other species and muscle combinations.
0
1
2
3
4
5
6
7
0 0.5 1 1.5 2 2.5 3 3.5
% W
et W
eigh
t
% Salt
Pork Dark Pork Light Beef Dark Beef Light Chicken Dark Chicken Light
Species * Muscle * Salt P = 0.01
* * *
* * * *
23
Figure 2. Salt-soluble protein content of chicken, beef, and pork (A) (as a percentage of wet tissue weight) (B) (as a percentage of solids weight basis (weight minus fat and moisture)). a, b, c
Within a level of salt, means with different letters are different at P < 0.05.
0.0
1.0
2.0
3.0
4.0
5.0
6.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
% W
et W
eigh
t
% Salt
Chicken Beef Pork
0.00
4.13
8.25
12.38
16.50
20.63
24.75
28.88
33.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
% S
olid
s W
eigh
t
% Salt
Chicken Beef Pork
a a a a
b a a b b
a bb
c
b
c c
b
c c c c
a
b
a a a
b
a
b b
a a
b b b
a
c c c b
c c c c
b
a
c
A
B
Species * Salt P < 0.01
Species * Salt P < 0.01
a a
24
1
Figure 3. Salt-soluble protein content of light and dark pork muscles (A), chicken muscles (B), and beef muscles (C) (as a percentage on solids weight basis (weight minus fat and moisture)). Means lacking a common superscript are different at P < 0.05.
0.00
4.13
8.25
12.38
16.50
20.63
24.75
28.88
33.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
% S
olid
s W
eigh
t
% Salt
Pork Dark Pork Light
0.00
4.13
8.25
12.38
16.50
20.63
24.75
28.88
33.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
% S
olid
s W
eigh
t
% Salt
Chicken Dark Chicken Light
0.00
4.13
8.25
12.38
16.50
20.63
24.75
28.88
33.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
% S
olid
s W
eigh
t
% Salt
Beef Dark Beef Light
* * * * *
*
*
* * *
Species * Muscle P < 0.01
Species * Muscle P < 0.01
Species * Muscle P < 0.01
A
C
B
25
Chapter III
The Effects of Varying Salt Concentrations on Protein Solubility and Product
Characteristics of Bolognas Manufactured from Chicken Muscles
Abstract
This experiment evaluated product characteristics of bolognas manufactured from
pectoralis major (light) and the entire thigh (dark) muscles of chicken at various salt levels. The
study was designed as a complete randomized design in a 2x4 factorial arrangement with factors
of muscle type (light and dark) and salt concentration (0.5%, 1%, 1.5%, and 2%). Bologna was
manufactured and analyzed for salt-soluble protein content, proximate analysis, cooking yield,
pH, Minolta L*, and binding strength. Increased retention of moisture in thigh muscle bologna
contributed to improved cooked yields compared with breast muscle bolognas (P = 0.01). The
comparison of low-salt breast bologna to high-salt thigh bologna indicated a difference (P <
0.01) 0.05 percentage units extractable salt-soluble proteins as a percentage of total crude
protein, with low-salt breast exhibiting higher values. The increase in salt-soluble protein
content was reflected in higher hardness scores in low-salt breast bologna compared to high-salt
thigh bologna (P < 0.01). This study suggests that incorporating chicken light muscles into
processed products would reduce the amount of salt needed to produce products with acceptable
processing characteristics.
Introduction
Today, there is growing concern in the medical community that excess salt consumption
is leading to the development of disease. Nearly 16.7 million deaths worldwide and 685,000
U.S. deaths annually are attributed to cardiovascular disease (Mackay and Mensah, 2004; Hoyert
et al., 2006). Hypertension, a chronic, abnormally high systolic and diastolic arterial blood
26
pressure (Lopez et al., 2006) has been identified as a contributing risk factor to the development
of cardiovascular disease (JNC VI, Working Group). In 1972, Dahl described the link between
dietary sodium intake and the development of hypertension, which other researchers have further
demonstrated (Page, 1976; Porter, 1983; Gleibermann, 1973; Tobian, 1997). Therefore,
reducing sodium intake, including that in processed meats is crucial to improving human health.
The need to reduce sodium levels in food products is evident; however, the use of sodium
chloride in the further processing of meat products is essential. Salt disrupts the myofibrillar
structure within muscle and allows myofilaments to swell and solubilize (Hamm, 1961).
Increasing sodium chloride concentration increases salt-soluble protein extraction, resulting in
increased amounts of fat and water that can be bound in products (Swift and Sulzbacher, 1963).
As the amount of salt-soluble proteins acting as binding agents increases in a product, the
binding strength of that product also increases (Acton, 1972). Break strength measures the
texture profile of the cooked product and indicates ability to be handled for packaging and
slicing (Herrero et al., 2008). In chicken loaves prepared entirely from either chicken breast or
chicken thigh muscle, breast meat was superior in binding qualities when comparing break
strength (Maesso et al., 1970). The purpose of this study was to determine the processing yield
and textural properties of emulsified products manufactured from chicken breast and thigh
muscles at various low salt (0.5-2%) concentrations.
Materials and Methods
Raw Materials
Approximately 120 kg of fresh, boneless and skinless chicken breast and thigh meat was
obtained from a commercial poultry plant two days postharvest. Breast muscles were trimmed
27
to isolate pectoralis major muscles. Approximately 50 kg each of pectoralis major muscles and
entire thighs were ground separately through a half-inch plate and mixed in a Hobart mixer to
form a homogenate sample. For each muscle type, eleven (30 g) samples were collected to
determine the proximate composition. While proximate composition analysis was being
conducted, ground meat was stored in vacuumed packaged bags (4°C).
Proximate Composition
The moisture and lipid content was determined for each sample using the method
described by Novakofski et al. (1989). In preparation, all samples were trimmed of external fat
and connective tissue and homogenized using a Cuisinart Food Processor. Moisture content was
determined after samples had been placed in a drying oven at 110°C for 48h. Lipid content was
then determined after washing samples in a mixture of chloroform and methanol. Results of
proximate composition analysis reported in Table 1.
Formulation
Two muscle combinations (pectoralis major muscle and thigh composite) and four
sodium chloride concentrations (0.5%, 1%, 1.5%, and 2%) with four replicates per treatment
were formulated for a total of 32 batches of bologna. Results of the proximate composition
analyses were used to determine the appropriate fractions of raw materials for an 80:20 lean to
fat ratio (Table 3). Pork back fat trim, free of visible lean, was added to reach the defined fat
inclusion levels. Chicken was placed in a bowl chopper (Talsa, Windsor Food Machinery LTD,
Kent, England) with seasonings and 1.5 pounds of ice and was allowed to mix for two
revolutions. Pork fat trim was added and the mixture was chopped on a low blade speed for 1
minute and then on high for 1 minute. Batter was removed from the bowl chopper, and four
samples (30g) were collected for proximate analysis, pH, and salt-soluble protein analysis.
28
Bologna batter was vacuumed stuffed into 5 3/8 inch collagen casings using a Handtmann stuffer
(Model# VF 80, Biberach, Germany). After stuffing, chubs were weighed and individually
identified. Product was allowed to rest at 2°C overnight before cooking. Bologna was cooked in
an Alkar smokehouse (Lodi, Wisconsin) to an internal temperature of 66.1°C and chilled at 2°C
for 24 hr. After chilling, the bolognas were weighed to determine cook loss and then peeled
from casings. Bolognas were cut into six 2.54 cm pieces for texture analysis, proximate
composition, pH, color.
Salt-Soluble Protein Content
The extractability of both sarcoplasmic and myofibrillar proteins were determined using
the method described by Boler et al. (2011). A master mix extraction buffer of 0.01M 2-[N-
Morpholino]ethanesulfonic acid was used to make the following eight NaCl extraction buffers
0% (0M), 0.5% (0.09M), 1% (0.17M), 1.5% (0.26M), 2% (0.34M), 2.5% (0.43M), 3% (0.51M)
and 3.5% (0.6M) by volume. All extraction solutions were buffered to a pH of 6.0 according to
Lan et al. (1993). Ground muscle samples (0.1 g) were placed in 2.0 mL microcentrifuge tubes
with 4mm stainless steel beads and 1.5 mL extraction buffer. Samples were homogenized
(TissueLyser II, Qiagen Inc., Valencia, CA) for 1 minute. Homogenates were centrifuged
(Microfuge® 18 Microcentrifuge, Beckman Coulter Inc., Germany) at 15000 x g for 20 min at 4°
C. Supernatants were collected for each sample, diluted 10 fold, and protein content quantified
using a BCA protein assay kit (Thermo Scientific Pierce Protein Research Products, Rockford,
IL). The amount of solubilized protein was determined using a second order polynomial
equation. Data were expressed both as a percentage of wet weight and as a percentage of total
crude protein.
29
pH
Meat (5g) was placed in a 50 mL conical tube with 20 mL of deionized water and
homogenized (Model PT 10/35, Brinkmann Instruments Co., Switzerland) for 30 seconds. pH
was measured with an automatic temperature compensation epoxy body probe attached to a
bench meter (Model 917001, Model 501, Orion Research Inc., Jacksonville, Florida) that had
been calibrated with pH 4.00 and 7.00 buffers.
Binding Strength and Compression Analysis
Binding strengths of bologna slices were analyzed using a cross bar and platform
attached to a Texture Analyzer TA.HD Plus (Texture Technologies Corp., Scarsdale, NY/Stable
Microsystems, Godalming, UK) (Souza et al., 2011). The crossbar speed was 10 mm/sec and the
load cell capacity was 100 kg. The crossbar descended until the sample was fractured and the
peak force necessary to fracture the sample was recorded. Two samples were fractured and the
average peak force was reported for each bologna.
The Bourne analysis (Bourne, 1978) was used to evaluate hardness of bologna. Four
2.54 cm cores were collected from each sample and compressed on Texture Analyzer TA.HD
Plus (Texture Technologies Corp., Scarsdale, NY/Stable Microsystems, Godalming, UK). A 5.08
cm diameter plate compressed each core in two consecutive cycles to 75 percent of the samples
original height with 2 s intervals between cycles. The cross-head moved at a constant speed of 5
mm/s. A force-time curve was plotted and the peak force of the first compression was used to
determine hardness. The values for the 4 cores were averaged and reported as hardness of each
bologna.
30
Color
Objective color scores (L*) were obtained using a Minolta CR-300 at a D65 light source
and a 0° observer immediately from the cut surface of each bologna. For each sample, one
measurement was taken from each half of the slice and the average of the two values was
reported for each parameter.
Statistical Analysis
Data were analyzed as a randomized complete design in a 2x4 factorial arrangement.
The statistical model included the main effects of muscle type (light or dark) and salt (0.5%, 1%,
1.5%, and 2%) and their interaction. Differences between moisture, fat, pH, textural properties
and color were determined using MIXED procedure of SAS (SAS Inst. Inc., Cary, N.C.). Means
for moisture, fat, pH, and color were separated using the PDIFF option and adjusted using
Tukey’s W Procedure to control for experimentwise error. Salt-soluble protein content was
analyzed as repeated measures using a first order auto regressive covariate structure with sample
serving as the subject. Single degree of freedom contrast statements were used to compare low-
salt (0.5-1%) breast bologna and higher-salt (1.5-2%) thigh bologna for salt-soluble protein
content, hardness, and break strength. Differences were deemed significant at P < 0.05.
Results and Discussion
Proximate Analysis and pH
In thigh muscle bologna batches, raw moisture content was increased over breast muscle
batches (Table 3). Also, the raw fat content of thigh muscle bologna was increased (P = 0.07)
over breast muscle bologna (Table 3). The interaction of muscle and salt can be attributed to
thigh bologna at 0.5% having a higher raw fat content (P < 0.05), compared to thigh bologna at
31
1.5% (Figure 4A). However, the raw fat content of breast bologna showed no differences at any
of the salt concentrations. Differences in raw fat content between thigh and breast bolognas may
be attributed to formulation error during the addition of fat trim to chicken trim and may have
affected further characteristics.
To account for the variation in raw fat content, this parameter was used as a covariate for
cooked fat content, cooked moisture content and cooked yield.
For cooked moisture content, thigh muscle bologna was increased compared to breast
muscle bologna (P < 0.01); however, there was an interaction of salt and muscle type (P < 0.01).
This interaction was due to thigh bologna having increased (P < 0.05) cooked moisture content at
0.5% salt compared to 1.5% salt (Figure 4B). However, breast bologna showed no differences in
cooked moisture content (Figure 4B). The main effect of muscle (P < 0.01) was significant for
cooked fat content, and there was an interaction of muscle and salt (P= 0.02) (Table 4). The
interaction was due to breast bologna tending to decrease in cooked fat content with increased
salt levels while thigh bologna exhibited the opposite trend (Figure 4C). Overall, thigh bologna
had more cooked fat content compared to breast bologna at all salt concentrations (P < 0.01)
(Table 4). The increase in moisture content and fat content of thigh muscles contributed to the
improved cooked yields of thigh muscle bologna compared to breast muscle bologna. The main
effects of species and muscle were significant (P < 0.01) for cooked yield as a percentage of
product weight (Table 4). Increasing the salt concentration resulted in increased cooked yields
for both bologna muscle types, however, thigh bologna had improved (P < 0.01) cooked yields
compared to breast bologna. The findings of improved cooked yields of chicken products with
the addition of increasing salt concentrations have been observed by other researchers (Acton,
32
1972; Lan et al., 1995; Somboonpanyakul et al., 2005). However, none of these papers
compared the cooked yields of chicken breast and chicken thighs.
The main effect of muscle type was significant (P < 0.01) for raw pH (Table 3). Breast
muscle bologna had lower pH values compared to thigh muscle bologna, which was expected
due to the method of metabolism associated with each muscle type. The increase in lactate
within the breast muscles drove the pH down compared to thigh muscle and values observed
were similar to other findings (Lee et al., 2010; Xiong et al., 1993). Increased values for thigh
pH may have led to an increase in water retention in the final product. However, pH was
buffered for salt-soluble protein content analysis to a pH of 6.0.
Salt-Soluble Protein Content
A single degree of freedom contrast statement was used to evaluate salt-soluble protein
content of low-salt (0.5%-1%) breast bologna and high-salt (1.5-2%) thigh bologna. Comparing
the low-salt breast bologna group to the high-salt thigh bologna group a difference (P = 0.01)
was detected, in that breast bologna extracted 1.05 percentage units more salt-soluble proteins as
a percentage of wet weight compared to thigh bologna (Figure 5A). These differences persisted
when salt-soluble protein content was corrected for total protein content of bolognas. The
comparison of low-salt breast bologna to high-salt thigh bologna indicated a difference (P <
0.01) of only 0.05 percentage units extractable salt-soluble proteins as a percentage of total crude
protein, with low-salt breast exhibiting higher values (Figure 5B). The increased extractability
of breast muscle compare to thigh muscle was consistent with the findings of several researchers
(Khan, 1962; Hudspeth and May, 1967; Maurer et al., 1969; McCready and Cunningham, 1971;
Prusa and Bowers, 1984; Richardson and Jones, 1987). Overall, the increased amount of salt-
33
soluble proteins in low-salt breast muscle bologna should impact the products overall textural
properties resulting in a firmer product when compared to high-salt thigh bologna.
Binding Strength and Compression
Break strength scores were expected to increase as the amount of extractable salt-soluble
proteins increased, due to their role as a binding agent in further processed products. As the
level of salt increased, break strength scores also increased, for both breast and thigh bologna
(Figure 6A). When comparing low-salt breast bologna to high-salt thigh bologna, it took 3.30 kg
more force to break the thigh bologna (P < 0.01) (Figure 6A). Even though it low-salt breast
bologna contained a larger quantity of salt-soluble proteins compared to high-salt thigh bologna,
that did not correlate to higher values for break strength.
Bourne (1978) defined the parameter of hardness to be a measure of the first bite of a
product. The first bite of low-salt breast bologna was increased (P < .01) by 2.19 kg of force
compared to high-salt thigh bologna (Figure 6B). The increase in hardness scores agrees with
previously published results which found that salt-soluble protein content increases corresponded
to rigidity of the product (Somboonpanyakul et al., 2005; Barbut and Mittal, 1989). The
acceptability of this product by the use of a sensory panel was not tested; however, Matulis et al.
(1994) observed the acceptable hardness scores of frankfurters to be approximate six with a
standard deviation of two, which would put low-salt breast bologna within this range.
Color
For L* color scores, the main effect of salt and muscle type were significant (P < 0.01),
however, there was a significant interaction of salt and muscle type (P = 0.05). Increasing salt
concentrations resulted in a decrease in L* color scores for both breast and thigh muscle
bologna; however, at all salt concentrations breast muscle bologna was lighter compared to thigh
34
muscle bologna (Figure 7). This result was similar to other studies, which found that as the pH
of the product measured increased there was a decrease in the recorded L* value due to an
increase in the retention of surface water (Fletcher et al., 2000).
Conclusions
In thigh muscle bologna batches, raw and cooked moisture content as well as cooked fat
content was increased over breast muscle batches (P < 0.01). The increased values for thigh pH
may have led to an increase in water retention in the final product. The increase in retention of
moisture of thigh muscles contributed to the improved cooked yields of thigh muscle bologna
compared to breast muscle bologna (P = 0.01). The comparison of low-salt breast bologna to
high-salt thigh bologna indicated a difference (P < 0.01) 0.05 percentage units extractable salt-
soluble proteins as a percentage of total crude protein, with low-salt breast exhibiting higher
values. The increase in salt-soluble protein content, however, was not reflected in a higher break
strength value for low-salt breast bologna compared to high-salt thigh bologna (P < 0.01).
Although the scores recorded for hardness did show an increase in low-salt breast bologna
compared to high-salt thigh bologna (P < 0.01). Overall, textural properties did not show the
same trend for each group compared and no human analysis was used to determine if these
products would be acceptable. Furthermore, although thigh muscle bologna was improved in
cooked yields and moisture content, it was not tested if breast muscle bologna would still be
deemed acceptable by consumers. The ability of chicken to extract high amounts of salt-soluble
proteins at low salt concentrations could be taken advantage of in the meat industry by
manufacturing products that incorporate both chicken and beef or pork. This would allow for a
variety of low sodium meat products to be manufactured.
35
References
Acton, J. C. (1972). Effect of heat processing on extractability of salt-soluble protein, tissue binding strength and cooking loss in poultry meat loaves. Journal of Food Science, 37, 244-246.
Barbut, S. and Mittal, G.S. (1989). Effects of salt reduction on the rheological and gelation properties of beef, pork, and poultry meat batters. Meat Science, 26, 177-191.
Boler, D. D., Holmer, S.F., Duncan, D.A., Carr, S.N., Ritter, M.J., Stites, C.R., Petry, D.B., Hinson, R.B., Allee, G.L, McKeith, F.K., and Killefer, J. (2011). Fresh meat and further processing characteristics of ham muscles from finishing pigs fed ractopamine hydrochloride. Journal of Animal Science, 89, 210-220.
Bourne, M. C. (1978). Texture profile analysis. Food Technology, 32, 62-66.
Dahl, L. K. (1972). Salt and hypertension. The American Journal of Clinical Nutrition, 25, 231-244.
Fletcher, D. L., Qiao, M., and Smith, D. P. (2000). The relationship of raw broiler breast meat color and ph to cooked meat color and pH. Poultry Science, 79, 784-788.
Gleibermann, L. (1973). Blood pressure and dietary salt in human populations. Ecology of Food
and Nutrition, 2, 143-155.
Hamm, R. (1961). Biochemistry of meat hydration. In C.O. Chichester and E.M. Mrak, Advances
in Food Research (pp. 355-380) New York: Academic Press.
Herrero, A. M., de la Hoz, L., Ordóñez, J. A., Herranz, B., Romero de Ávila, M. D., and Cambero, M. I. (2008). Tensile properties of cooked meat sausages and their correlation with texture profile analysis (TPA) parameters and physico-chemical characteristics. Meat
Science, 80, 690-696.
Hoyert, D. L., Heron, M. P., Murphy, S. C., and Kung, H. C. (2006). Deaths: Final Data for 2003. National Vital Statistics Reports, 54, 1-5.
Hudspeth, J. P., and May, K. N. (1967). A study of the emulsifying capacity of salt soluble proteins of poultry meat. Food Technology, 21, 1141-1142.
JNC IV. (1997) Special article: the Sixth Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Archives of Internal
Medicine, 157, 2413-2446.
Khan, A. W. (1962). Extraction and fractionation of proteins in fresh chicken muscles. Journal
of Food Science, 27, 430-434.
36
Lan, Y. H., Novakofski, J., McCusker, R. H., Brewer, M. S., Carr, T. R., and McKeith, F. K. (1995). Thermal gelation of pork, beef, fish, chicken and turkey muscles as affected by heating rate and pH. Journal of Food Science, 60, 936-940.
Lan, Y. H., Novakofski, J., Carr, T. R., and McKeith, F. K. (1993). Assay and storage conditions affect yield of salt soluble protein from muscle. Journal of Food Science, 58, 963-967.
Lee, S. H., Joo, S. T., and Ryu, Y. C. (2010). Skeletal muscle fiber type and myofibrillar proteins in relation to meat quality. Meat Science, 86, 166-170.
Lopez, A. D., Mathers, C. D., Ezzati, M., Jamison, D. T., and Murray, C. J. (2006). Global and regional burden of disease and risk factors, 2001: systematic analysis of population health data. The Lancet, 367, 1747-1757.
Mackay, J., and Mensah, G. A. (2004). The atlas of heart disease and stroke. London, The Hanway Press.
Maesso, E. R., Baker, R. C., and Vadehra, D. V. (1970). The effect of vacuum, pressure, pH and different meat types on the binding ability of poultry meat. Poultry Science, 49, 697-700.
Matulis, R.J., McKeith, F.K., and Brewer, M.S. (1994). Physical and sensory characteristics of commercially available frankfurters. Journal of Food Quality, 17, 263-271.
Maurer, A. J., Baker, R. C., and Vadehra, D. V. (1969). The influence of type of poultry and carcass part on the extractability and emulsifying capacity of salt-soluble proteins. Poultry
Science, 48, 994-997.
McCready, S. T., and Cunningham, F. E. (1971). Salt-soluble proteins of poultry meat 1. composition and emulsifying capacity. Poultry Science, 50, 243-248.
Novakofski, J., Park, S., Bechtel, P. J., and McKeith, F. K. (1989). Composition of Cooked Pork Chops: Effect of Removing Subcutaneous Fat Before Cooking. Journal of Food Science, 54, 15-17.
Page, L. B. (1976). Epidemiologic evidence on the etiology of human hypertension and its possible prevention. American Heart Journal, 91, 527-534.
Porter, G. A. (1983). Chronology of the sodium hypothesis and hypertension. Annals of Internal
Medicine, 98, 720-723.
Prusa, K. J., and Bowers, J. A. (1984). Protein extraction from frozen, thawed turkey muscle with sodium nitrite, sodium chloride, and selected sodium phosphate salts. Journal of Food
Science, 49, 709-713.
37
Richardson, R. I., and Jones, J. M. (1987). The effects of salt concentration and pH upon water-binding, water-holding and protein extractability of turkey meat. International Journal of
Food Science and Technology, 22, 683-692.
Somboonpanyakul, P., Barbut, S., Jantawat, P., and Chinprahast, N. (2007). Textural and sensory quality of poultry meat batter containing malva nut gum, salt and phosphate. Food Science
and Technology, 40, 498-505.
Souza, C. M., Boler, D.D., Clark, D.L., Kutzler, L.W., Holmer, S.F., Summerfield, J.W., Cannon, J.E., Smit, N.R., McKeith, F.K., Killefer, J. (2011). The effects of high pressure processing on pork quality, palatability, and further processed products. Meat Science, 87, 419-427.
Swift, C. E., and Sulzbacher, W. L. (1963). Comminuted meat emulsions: factors affecting meat proteins as emulsion stabilizers. Food Technology, 17, 224-226.
Tobian, L. (1997). Dietary sodium chloride and potassium have effects on the pathophysiology of hypertension in humans and animals. The American Journal of Clinical Nutrition, 65, 606S-611S.
Working Group. (1993). National high blood pressure education program working group report on primary prevention of hypertension. Archives of Internal Medicine, 153, 186.
Xiong, Y. L., Cantor, A. H., Pescatore, A. J., Blanchard, S. P., & Straw, M. L. (1993). Variations in muscle chemical composition, pH and protein extractability among eight different broiler crosses. Poultry Science, 72, 583-588.
38
Tables and Figures
Moisture, % Fat, %Breast 74.44 3.93
Thigh 71.36 11.90
Table 2. Proximate Compostion of breast and thigh muscles
Breast Thigh SEM Muscle Salt InteractionPork Fat Trim, kg 1.13 0.63Chicken Trim, kg 5.67 6.17Raw Moisture, % 70.43 71.60 0.11 < 0.01 0.30 0.63Raw Fat, % 12.81 13.17 0.14 0.07 0.01 0.01Raw pH 5.72 6.27 0.01 < 0.01 0.23 0.58
P-valueBologna
Table 3. Formulation of chicken bologna batches
39
Breast Thigh 0.05% 1% 1.50% 2% SEM Muscle Salt Interaction
Cooked Moisture, % e
66.99 68.29 67.94a
67.98a
67.49a
67.16a
0.28 < 0.01 0.10 < 0.01
Cooked Fat, % f
14.61 15.60 15.19a
15.22a
15.22a
14.81a
0.23 < 0.01 0.38 0.02
Cook Yield, % g
91.36 92.19 89.78c
91.66b
92.64ab
93.02a
0.36 0.01 < 0.01 0.34
a, b, c Means lacking a common superscript are different P < 0.05
d pH of bologna batch after emulsification
e Moisture content of bologna after cooking
f fat content of bologna after cooking
All means were calculated using raw fat content as a covariate
P-valueMuscle
Table 4. Effect of muscle and salt on processing characteristics of bologna
Salt Concentration
g���������� � ����� *100
39
40
Figure 4. Effects of muscle and salt on processing characteristics of bologna. Raw fat content (A), cooked moisture content (B), and fat content as a percentage (C). Means lacking a common superscript are different at P <0.05.
8
9
10
11
12
13
14
15
0.5 1 1.5 2
% R
aw F
at C
onte
nt
% Salt
Breast Thigh
a
abab abbb b ab
64
65
66
67
68
69
70
71
0.5 1 1.5 2
% C
ooke
d M
oist
ure
Con
tent
% Salt
Breast Thigh
aab
bcabc abccc
c
1212.5
1313.5
1414.5
1515.5
1616.5
17
0.5 1 1.5 2
% C
ooke
d Fa
t Con
tent
% Salt
Breast Thigh
bc
aab
abcabcab
bc c
A
B
C
Muscle * Salt P = 0.01
Muscle * Salt P = 0.02
Muscle * Salt P < 0.01
41
Figure 5. Salt-soluble protein content of breast and thigh as a percentage of wet weight (A) and as a percentage of total crude protein (B). Asterisks indicate differences between low-salt (0.5-1%) breast bologna and high-salt (1.5-2%) thigh bologna at P < 0.05.
0
0.5
1
1.5
2
2.5
3
3.5
4
0.5 1 1.5 2
% W
et W
eigh
t
% Salt Breast Thigh
0
4
8
12
16
20
24
0.5 1 1.5 2
% C
rude
Pro
tein
Wei
ght
% Salt Breast Thigh
B
A
Muscle P < 0.01
Muscle P < 0.01
*
*
*
*
42
Figure 6. Force required to break slices of bologna (A) and force required to compress slices of bologna made from chicken breast and thigh muscles (B). Asterisks indicate differences between low-salt (0.5-1%) breast bologna and high-salt (1.5-2%) thigh bologna at P < 0.05.
5
6
7
8
9
10
11
12
0.5 1 1.5 2
Bre
akst
reng
th (
Kg)
% Salt Breast Thigh
4.5
5.5
6.5
7.5
8.5
9.5
10.5
0.5 1 1.5 2
Har
dnes
s (K
g)
% Salt
Breast Thigh
*
*
*
*
B
A
Muscle * Salt P < 0.01
Muscle * Salt P < 0.01