1 the Effects of Sous Vide Cooking on Tenderness and Protein

The Effects of Sous Vide Cooking on Tenderness and Protein Concentration in

Young Fed Beef and Cow Semitendinosus Muscles

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in

the Graduate School of The Ohio State University

Victoria R. Trbovich

Graduate Program in Animal Science

The Ohio State University

2017

Thesis Committee:

Dr. Lyda G. Garcia, Advisor

Dr. Eric M. England

Dr. Francis L. Fluharty

Dr. Macdonald P. Wick

Copyrighted by

Victoria R. Trbovich

2017

Abstract

The beef industry is continuously seeking ways to increase consumer demand, while improving eating quality and consistency of beef products through added value products. Thus, industry leaders are have introduced various “value cuts” to the marketplace through innovative cutting and muscle profiling in the last ten years at a moderate price. However, there has been little innovation in adding value through methods of cooking. Therefore, two experiments were designed to add value to an undervalued tough cut of beef through sous vide cooking. The first experiment was conducted to investigate the utility of sous vide cooking in beef tenderness, to ultimately add value to undervalued cuts of meat. The second experiment was performed to determine the effects of different temperature and time combinations on tenderness and to understand the effects of sous vide cooking on meat proteins.

In the first experiment, six whole beef semitendinosus muscles (ST) were used representing two age groups: fed (n = 3) and cow (n = 3). Three roasts were obtained from each ST muscle that were cooked via sous vide at 55C for 2, 10 and 30 h. Cook loss (%), internal cooked color and WBSF was measured following cooking cycles.

Cooking time had a significant effect on cook loss, as the percent cook loss increased at longer cooking times (P = 0.001). WBSF values decreased as cooking time increased as roasts cooked for 10 and 30 h were significantly more tender (P < 0.005) than roasts ii cooked for 2 h. Altogether, cooking ST muscles via sous vide at low temperatures for long cooking times resulted in a similar tenderness when compared to tender cuts of meat cooked at shorter times.

In the second experiment, whole beef semitendinosus muscles (ST; n = 40; IMPS

171C) represented two age groups: young fed beef (< 30 mo. of age; n = 20) and cow beef (> 42 mo. of age; n = 20). Semitendinosus muscles were then portioned into 6 cm thick roasts which were weighed and individually vacuum packaged. Roasts were cooked in a recirculating water bath and assigned one of four different temperatures (55, 60, 65, and 70C) using four different times (2, 4, 6, and 8 h). Percent cooking loss, raw and cooked color, Warner-Bratzler shear force (WBSF), protein concentration of cooked liquid and cooked ST were evaluated.

There was an interaction (P = 0.028) between temperature and age with WBSF values. Fed ST roasts cooked at 60C were significantly tougher than cow ST roasts at

60C (P < 0.05). Additionally, cooking losses (%) increased as temperature and time increased (P < 0.001). Semitendinosus roasts cooked to temperatures of 55, 60, 65, and

70C at 2 h. had the lowest percent cooking loss (P < 0.05) compared with roasts cooked at 70C for 6 and 8 h. (P < 0.05). Furthermore, L* values increased as temperature and time increased, whereas a* decreased. Redness values (a*) for fed ST at 60C cooked for

4 and 8 h was significantly higher (redder) than cow ST at 60C for 8 h (P < 0.05).

Roasts cooked lower than 65C and less than 6 h had significantly higher b* (increased yellowness) values than those cooked at higher temperatures for longer periods of time.

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Moreover, soluble protein fractions decreased in concentration with increasing temperature, showing greatest concentrations occurring at 55C for 2, 4, 6, and 8 h when compared to all other temperature and time points (P < 0.05). Additionally, at 60C, fed

ST had significantly higher concentrations of soluble protein than cow ST (P = 0.020).

Total protein concentrations of the cooked liquid revealed a three-way interaction of age, temperature, and time (P = 0.021). Total protein concentrations of cooked ST resulted in a temperature effect (P = 0.005) as temperature increased, total protein concentration slightly decreased. Altogether, cooked roasts originating from fed beef had a higher concentration of total protein than cow (P = 0.001). SDS-PAGE gel patterns of cooked liquid soluble proteins exhibited similarities to sarcoplasmic proteins from muscle exudate found in previous studies that suggested sarcoplasmic proteins might play a role in determining tenderness and quality attributes.

Sous vide cooking of ST roast achieved WBSF values that met consumer acceptability of “tender” for both fed and cow ST. However, longer cooking times may be needed to achieve significant tenderization in tough cuts of meat like the semitendinosus muscle. Also, additional research using different muscles varying from tender to tough might also help to better understand the effects of sous vide cooking on tenderness and other sensory attributes.

Dedication

To my mother and grandfather:

Julie K. Kennedy and John H. Kennedy

Acknowledgments

First and foremost, I would like to thank my advisor Dr. Lyda Garcia. I have immensely valued your support, mentorship, and dedication to my education through my master’s degree. I appreciate all the opportunities you provided me by allowing me to work alongside you on various extension/outreach programs, carcass show adventures, and teaching your classes. I have learned so much about the meat and beef industries, and life from you! Thank you for having confidence in me and most importantly, your friendship. Your love for teaching and coaching is contagious and the skills I learned from you will be treasured for the rest of my life.

Secondly, thank you to Dr. Eric England and Dr. Mick Wick. Your patience and support throughout this process has been much appreciated. I am grateful for your guidance, challenging questions, and patience teaching me various lab techniques. You have greatly contributed to my growth as a scientist. I have enjoyed having the opportunity to work with both of you.

Next, I would also like to thank Dr. Francis Fluharty for being on my committee and for all his help with my project. Additionally, I would like to extend a thank you to

Dr. Luis Moraes, for the many meetings to help me better understand JMP and statistics. I also want to thank the University Meat Lab manager, Ron Cramer, for his guidance not

vi only with my project but throughout my undergraduate and graduate career and his friendship.

I owe a huge thank you to graduate students, Trey Garza and Karli Feicht, for their help with my project and support. Trey Garza, thank you for helping me get through the tough times and for always making me laugh, whether it was with you or at you!

Karli, I will always be grateful for our friendship and your continued support through school and life. I will never forget all the jam sessions we had in the lab to help us get through the long days of lab work, you always did it with a smile. I would not have made it through without their friendship, support and guidance!

To Morgan Foster, Kristen Cavoli, Paige McAtee, and Michelle LeMaster, thank you for all your help with collecting data and lab work, it would not all be possible without you. Audrey, thank you for always being a phone call away. You have been the

‘behind the scenes’ support and provided me with faith when I needed it the most.

Lastly, the biggest thank you is owed to my parents, Julie Kennedy and Okie

Trbovich, and brother Tyler, for their never-ending encouragement that I could achieve anything as long as I remained disciplined and hardworking. Mom, you have instilled in me a sense of integrity and strong work ethic. You have been my constant voice of reason, my number one fan, and my rock. Your support though this journey has meant more than you will ever know.

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Vita

February 12th, 1993………………………Born in Canton, Ohio

May 2015…………………………………B.S. Animal Science, The Ohio State

University, Columbus, Ohio

August 2015 to present……………………Graduate Research Associate, Department of

Animal Sciences, The Ohio State University

Fields of Study

Major Field: Animal Science

Emphasis: Meat Science

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Table of Contents

Abstract ...... ii

Dedication ...... v

Acknowledgments...... vi

Vita ...... viii

Table of Contents ...... ix

List of Tables ...... xi

List of Figures ...... xii

Chapter 1 Introduction ...... 1

Chapter 2 Literature Review ...... 4

Chapter 3 Pilot study: Investigating the effects of sous vide cooking on tenderness in fed and cow semitendinosus muscles ...... 26

Abstract ...... 26

Introduction ...... 27

Materials and Methods...... 28

Results and Discussion ...... 30

Conclusion ...... 32

Chapter 4 Effects of sous vide cooking on tenderness and protein concentrations in fed and cow semitendinosus muscles ...... 36

Abstract ...... 36

Introduction ...... 38

Methods and Materials...... 39

Results and Discussion ...... 44

Conclusion ...... 51

Chapter 5 Conclusions and Future Directions ...... 53

References ...... 63

List of Tables

Table 3.1. Least-square means on the effects of cooking time on cooking loss1 (%), internal cooked color, and Warner- Bratzler shear force5 (WBSF) of ST roasts...... 34 Table 3.2. Least-square means on the effects of cooking time on Warner-Bratzler shear force (kg) of cooked ST1, LL2, and PS3 muscles...... 35 Table 4.1. Simple means of uncooked semitendinosus (ST) weights and instrumental color of age1 groups...... 54 Table 4.2. Least-square means on the effects of temperature1 and time on cooked instrumental color parameters of age2...... 56 Table 4.3. Least-square means on the effects of temperature1 and time on soluble2 and total3 protein concentrations of the cooked liquid...... 58

List of Figures

Figure 4.1. Least-square means on the effects of temperature and time on percent cooking loss. a-gMeans without a common letter are significantly different (P < 0.05)...... 55 Figure 4.2. Least-square means on the effects of age and temperature (C) on Warner- Bratzler shear force (WBSF) values. a-bMeans without a common letter are significantly different (P < 0.05)...... 57 Figure 4.3. Least-square means on the effects of age, temperature and time on total (soluble and insoluble) protein concentration of the cooked liquid. a-dMeans without a common letter are significantly different (P < 0.05)...... 59 Figure 4.4. Least-square means on the effects of temperature on total (soluble and insoluble) protein concentration of the cooked whole ST muscle. a-bMeans without a common letter are significantly different (P < 0.05)...... 60 Figure 4.5. SDS-PAGE gel image of soluble proteins extracted from the cooked liquid at different temperature and time points...... 61 Figure 4.6. SDS-PAGE gel image of muscle proteins from cooked ST roasts at different temperature and time points...... 62

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Chapter 1

Introduction

The amount of beef purchased by American consumers at the foodservice and retail levels in 2016 was 25.7 billion lbs., with 55.7 lbs. consumed per capita (NCBA,

2017). The beef industry continuously seeks ways to increase consumer demand, while also improving the eating quality and consistency of beef products. Attributes that determine beef palatability are often described as the combination of three factors: tenderness, flavor, and juiciness. The relationship of these three factors contribute to the consumers’ perception of taste and/or the overall liking of a product. Several consumer surveys suggest that taste (flavor) is the primary driver of purchasing decisions and consumer satisfaction (Tatum, 2015; Corbin et al., 2014; Brooks et al., 2000).

Conversely, other studies suggest that tenderness is the most important qualitative characteristic in meat (Lepper-Blilie et al., 2014; Miller et al., 2001; Huffman et al.,

1996; Miller et al., 1995; Savell et al., 1987). Meanwhile, other studies have shown flavor is equally important and in some instances, more important than tenderness when determining overall palatability (Corbin et al., 2014; O’Quinn et al., 2012; Killinger et al., 2004). Even though the beef industry has made great strides to improve consistencies in beef products, inconsistencies still exist pertaining to tenderness, specifically in undervalued beef cuts. In accounting for the lack of background knowledge of anatomical

1 location and function of muscle cuts, American consumers often will express their dissatisfaction of eating quality across all muscle cuts while viewing them as equal.

Consumers indicate that they would be willing to pay a premium price in the marketplace for beef that is guaranteed tender (Zapata et al., 2009; Miller et al., 2001; Brooks et al.,

2000). Over the past decade, the Beef Innovations Group has introduced 13 new beef cuts through innovative cutting and muscle profiling, thus, introducing new beef cuts to the marketplace. These new cuts, known as value cuts, are fabricated primarily from the round and chuck. More recently, the Beef Innovations Group has introduced a new value cut, known as the merlot cut. Originating from the heel muscle of the round, the merlot cut consists of a finer grain texture and a certain tenderness unlike other cuts from the round (Thomson, 2014). With the introduction of new value cuts, more steaks and roasts are offered to consumers at the retail level at a moderate price, while helping improve overall profitability in the beef industry. However, there has been little innovation in the area of adding value through methods of cooking. Sous vide cooking is a method where vacuum-packaged food is immersed in a water bath at a precisely controlled temperature.

This method of cooking preserves sensory attributes associated with eating quality

(Baldwin, 2012; Vaudagna et al., 2002). Yet, its use in commercial processing of meat is currently limited.

Therefore, the present study was designed to understand the effects of sous vide cooking and more specifically, how it might be advantageous with tough cuts of meat, such as beef semitendinosus. The specific objective of the study was to investigate the effects of temperature and time combinations of sous vide cooking on tenderness in

2 young fed and cow beef semitendinosus muscles, to ultimately utilize lower valued muscle cuts from beef, particularly cow, while also guaranteeing consumers an enjoyable eating experience.

Chapter 2

Literature Review

Introduction

Skeletal muscle is composed of many highly organized proteins that serve different functions. Proteins identified in muscle will fall in one of the following categories: myofibrillar, sarcoplasmic, or stromal (Yu et al., 2017). Meat is derived from skeletal muscle that has undergone various physical and biochemical changes.

Understanding the structure and the biochemical functions of skeletal muscle is critical to understanding the conversion of muscle to meat, the effects of cooking meat, and the overall quality of meat (Aberle et al., 2012).

Maturity of the animal at time of harvest will play a large role in the overall quality of meat. As an animal ages physiologically, quality characteristics decrease in relation to customer eating satisfaction. Beef cuts originating from a young fed beef (<30 mo. of age) compared to a cull cow (>42 mo. of age) will result in greater value strictly due to physiological maturity at time of harvest.

With a growing population in the U.S. and variability in supply and demand, the

U.S. beef industry continues to strive to meet these demands. A main area of interest to the meat industry is value added products. This concept pertains to adding value to undervalued beef cuts, such as semitendinosus, and matching it with the appropriate cooking method. The ultimate goal of creating added value through a more consistently

positive eating experience should benefit beef producers involved in the market and cull cow arena, beef plants that are involved in harvesting, to marketing meat products, and the end user, the American consumer. Several factors can easily influence the production efficiency of lean muscle that will in turn impact food quality.

The objectives of this literature review are to provide a background on the structure of meat and the factors affecting tenderness. In addition, the literature review will provide an overview of the literature regarding the effects of cooking meat and the impacts this has on tenderness and overall eating satisfaction.

Meat proteins and structure

Muscle is organized into several units working together to provide muscle structure and function. Whole muscle is comprised of: muscle bundles, muscle fibers, myofibrils, and myofilaments. Each whole muscle has a layer of connective tissue over the external surface, called the epimysium (Kerth, 2013). Within each whole muscle are bundles of muscle fibers. These bundles are surrounded by a layer of connective tissue, called the perimysium. Additionally, the endomysium surrounds each individual muscle fiber (Palka et al., 1999). Connective tissue consists of fibrous proteins, collagen and elastin (Purslow, 2002). Collagen is the major structural protein in connective tissue. Its distribution among muscles is not uniform, as muscles of locomotion tend to have higher amounts of collagen then those not typically used for motion (Purslow, 2014).

Skeletal muscle fibers are long, multinucleated cell that are also referred to as a muscle cell or myofiber (Lee et al., 2010). Additionally, each muscle fiber contain many myofibrils (Aberle et al., 2012). Myofibrils are long, thin, rods that extend the length of the muscle fiber. A myofibril is made up repeating units known as sarcomeres, which

gives a striated appearance when viewed under a microscope (Huff-Lonergan et al.,

2010). A sarcomere is the basic contractile unit in muscle, and is composed of structural elements needed to perform contraction (Tonino et al., 2010). According to Fraterman et al. (2007) there are over 65 proteins that make up the structural components of a sarcomere. The striations of a sarcomere result from the banding patterns of alternating proteins within the cell (Kerth, 2013). A-bands are protein dense where I-bands are less dense. However, both bands are bisected by a thin, dense line, known as a Z-line (Aberle et al., 2012; Huff-Lonergan et al., 2010). A single sarcomere is defined by two Z-lines on either end, averaging 2.4 m in length (Tonino et al., 2010). Therefore, a sarcomere contains one A-band and two half I-bands. A-bands are made up of thick filaments and some overlapping thin filaments, whereas I-bands are made up thin filaments (Kerth,

2013).

Thick filaments extend 1.5 m long, making up the A-band of the sarcomere

(Ertbjerg et al., 2017). The predominate protein found within the thick filament is myosin, which can also be referred to as the myosin filaments.

Thin filaments extend about 1.0 m long, making up the I-band of the sarcomere and extends into the A-band (Ertbjerg et al., 2017). Thin filaments are known as actin filaments, as actin is the predominate protein within the thin filament. During contraction, the thick and thin filaments interact, forming a cross-bridge with the myosin head and actin, known as actomyosin (Huff-Lonergan et al., 2010) . As a result from contraction, sarcomeres can become shortened from 2.4 m to about 1.5 m in length (Kerth, 2013).

In addition to the primary components that make up a sarcomere (myosin, actin and the Z-line), there are many other proteins found within skeletal muscle. Skeletal

muscle proteins are generally classified based on their solubility: myofibrillar, sarcoplasmic, and stromal (Lee et al., 2010). Myofibrillar proteins are proteins located within the myofibril and can be further classified into subgroups: contractile proteins, myosin and actin; regulatory proteins, which initiate and aid in contraction; and cytoskeletal proteins, which provide myofibrils with structural support and stability (Yu et al., 2017). In brief, sarcoplasmic proteins are soluble proteins such as enzymes and myoglobin, whereas stromal proteins consist of connective tissue proteins such as collagen and elastin (Yu et al., 2017; Aberle et al., 2012; Lawrie, 2006).

Contractile proteins

Myosin and actin are the two major contractile proteins that make up the myofibrils. Myosin is the most abundant myofibrillar protein (45% of the myofibril) and the major component of thick filaments (Tornberg, 2005). Myosin filaments are made up of 250 – 300 individual molecules of myosin that are bundled together by C-protein

(Ertbjerg et al., 2017). Each thick filament is then surrounded by six thin filaments. Actin is the second most abundant myofibrillar protein (20% of the myofibril), which is found within the thin filaments (Aberle et al., 2012; Huff-Lonergan et al., 2010). Actin is a globular protein known as G-actin, the single molecule form of actin with a molecular weight of about 42 kDa (Lawrie, 2006). G-actin monomers are then linked together to form the actin filament, F-actin (fibrous actin) (Kerth, 2013). Two fibrous strands of F- actin are coiled together to form a helix. These myofibrillar proteins play an important role in the degree of tenderness and overall palatability. The degree of the contracted state of the myofibrils post-rigor can largely affect the shear and tensile force used to

chew (Lawrie, 2006). In addition to contracted state, the type of muscle and cooking temperature can also reflect the effect of tenderness and palatability.

Regulatory proteins

Regulatory proteins, tropomyosin and troponin, lie alongside the actin filament

(Aberle et al., 2012). Tropomyosin constitutes approximately 5% of myofibrillar proteins, is a protein that spans the length of the thin filament. Tropomyosin has a molecular weight of 37 kDa and consists of two peptide chains coiled together that lies alongside the grooved surface of the helixes of F-actin (Ertbjerg et al., 2017). Its function, along with troponin, is to regulate the binding site for myosin on actin during contraction and relaxation. When contraction is initiated tropomyosin is moved to uncover the binding site, allowing myosin to bind to actin. A single strand of tropomyosin reaches the length of 7 G-actin.

Troponin, approximately 5 % of myofibrillar proteins, is found on the thin filament alongside tropomyosin. Troponin is composed of three subunits: troponin-T (30 kDa), troponin-I (20 kDa), and troponin-C (18 kDa), also known as the troponin complex

(Ertbjerg et al., 2017). Specifically, troponin-T binds tropomyosin to the troponin complex, while troponin-I binds F-actin and troponin-C is the binding site for calcium, causing a conformational shift of tropomyosin during contraction (Kerth, 2013).

Cytoskeletal proteins

Cytoskeletal proteins consist of titin, nebulin and C-protein and H-protein. Titin is the third most abundant protein (10% of the myofibrillar proteins) and is the largest protein (approximately 3,700 kDa) (Huff-Lonergan et al., 2010; Tornberg, 2005). It is known to play a role in alignment, structural integrity of the myofibril, maintain elasticity

and prevent overstretching during contraction (Huff-Lonergan et al., 1996; Koohmaraie,

1994; Wang et al., 1991). Titin is located from the Z-line to the middle of the sarcomere

(M-line), spanning half the length of the sarcomere (Kerth, 2013). The end of titin attached to the Z-line is spring like, so it can stretch and shorten without breaking during contraction. Proteolysis of titin has been documented to be correlated to tenderness in meat (Sawdy et al., 2004). Due to its function in living muscle, degradation of titin would weaken the structure of the sarcomere, leading to improvement in tenderness (Robson et al., 1997; Koohmaraie, 1994).

Nebulin is also a large protein (600-900 kDa) representing 4% of the myofibrillar proteins (Huff-Lonergan et al., 2010). Nebulin reaches the entire length of the thin filament to the Z-line (Kerth, 2013). Its role is to anchor the thin filament to the Z-line, and serve as a ruler for the thin filament during development (Tonino et al., 2010). Like titin, degradation of nebulin can weaken the anchor between thin filaments and the Z- line, leading to postmortem tenderization (Huff-Lonergan et al., 1996).

Additionally, alpha-actinin, a 97 kDa protein, is the primary component of the Z- line and accounts for 2 % of the myofibrillar proteins (Ertbjerg et al., 2017). C-protein constitutes approximately 2 % of myofibrillar proteins (Aberle et al., 2012). There are seven bands of C-protein that encircles and stabilizes each thick filament. H-protein, 1% of myofibrillar proteins, is present in the portion of the sarcomere where the thin and thick filaments do not overlap and aid in the organization of thick filaments. Proteins associated with the Z-line or intermediate filaments comprises < 1 % of myofibrillar proteins which include: desmin (52 kDa), filamin (~300 kDa), paranemin, and synemin

(Ertbjerg et al., 2017). All, are believed to aid in the lateral alignment of myofibrils.

Dystrophin, talin, and viniclin, accounting for < 1 % of myofibrillar proteins, connects the cytoskeletal protein network to the extracellular matrix outside of the cell (Aberle et al., 2012).

Sarcoplasmic proteins

Sarcoplasmic proteins represent 30-35% of the total muscle proteins (Yu et al.,

2017; Tornberg, 2005). Several of these proteins are comprised of mostly glycolytic enzymes, mitochondrial enzymes, myoglobin, cytochromes and flavo-proteins (Yu et al.,

2017; Przybylski et al., 2016). Studies have reported sarcoplasmic proteins are correlated to meat quality traits such as water holding capacity, color, sensory attributes, and postmortem aging (Przybylski et al., 2016). Furthermore, understanding muscle structure and function is important when evaluating the effects of heating on eating quality, protein degradation and the structure of muscle fibers.

Structure and quality of meat on cooking

The cooking of meat has a large effect on many aspects like structural and chemical changes as well as mechanical properties of meat (García-Segovia et al., 2007;

Palka et al., 1999; Cheng et al., 1979). In addition, cooking is an important step in achieving a safe and palatable product. However, the application of heat during the cooking process will have a large impact on meat tenderness. It is important to understand the effect of cooking on the mechanical properties of meat and its effect on eating experience. According to Davey et al. (1974), cooking is defined as the heating of meat to a sufficiently high temperature to denature proteins causing conformational changes. These conformational changes due to protein denaturation lead to structural changes within the meat and, therefore, can impact the overall eating experience. Several

studies have investigated how the application of heat influences the mechanical properties of meat and overall eating experience (Christensen et al., 2011; García-

Segovia et al., 2007; Tornberg, 2005; Obuz et al., 2004; Christensen et al., 2000; Palka et al., 1999; Cheng et al., 1979; Davey et al., 1974; Bouton et al., 1972). Christensen et al.

(2000), Davey et al. (1974) and Bouton et al. (1972) confirmed that toughness increased in two phases with increasing cooking temperature. The first phase was observed between 40 and 50C and the second phase occurred at temperatures above 60C. Studies from Christensen et al. (2000) and Bouton et al. (1972) are in agreement that the first phase of toughening was due to increased strength of the perimysium upon heating leading to only partial denaturation of the collagen fibers. The second phase due to myofibrillar protein denaturation. However, Davey et al. (1974) reported the opposite; the first phase is due to myofibrillar denaturation and the second is connective tissue denaturation. Myofibrillar protein denaturation of myosin and actin occur between 40-

60C and 66-73C respectively (Palka et al., 1999) and collagen denaturation occurs between 53C and 63C (Tornberg, 2005). Additionally, Christensen et al. (2000) reported a decrease in muscle fiber breaking strength occurred between the two toughening phases, at 50C and 60C, likely due to an increase in the shrinkage of collagen fibers and the continuation of collagen solubilization. According to Baldwin

(2012), muscle fibers start to shrink between 35-40C and continues to increase linearly up to 80C. However, collagen shrinkage begins at 60C and contracts when temperatures exceed 65C. At temperatures of 60C to 70C, Tornberg (2005) noted that the intramuscular collagen, mostly from the perimysium, and muscle fibers simultaneously shrink longitudinally.

Moreover, the shrinkage of muscle structure components and protein denaturation also impacts the water holding capacity of meat. Christensen et al. (2011) concluded that shrinkage of meat during cooking results in the expelling of water from the structural components in the meat. Palka et al. (1999) found that shrinkage occurs during cooking at

40-60C is due to the transverse shrinkage of muscle fibers and at 60-90C longitudinal shrinkage of collagen and muscle fibers occur. Water is expelled due to the forces exerted by the shrinking collagen and muscle fibers. Christensen et al. (2011) concluded that collagen shrinkage and denaturation negatively impacted the ability to retain water within the meat during cooking. Additionally, it was reported that the denaturation of sarcoplasmic proteins had little effect on water holding capacity and structural changes

(Christensen et al., 2011; Tornberg, 2005). However, it was suggested that extended cooking, up to 20 h, resulted in higher cooking losses due to the denaturation of actin

(Christensen et al., 2011).

Therefore, the rate of denaturing of proteins increases with higher temperatures

(Baldwin, 2012; Baldwin, 2008). Temperatures above 55C resulted in denaturation more rapidly, however, tenderness was affected at temperatures 70C and above (Powell et al.,

2000). Therefore, heat causes toughening and tenderization depending on several factors: rate of increase in temperature, amount of collagen, frequency of heat-stable collagen crosslinks, cooking method and a combination of different times and temperatures

(Aberle et al., 2012). The application of heat not only affects meat structure and proteins but also the external and internal cooked color of meat.

Color

During the cooking process, the color of beef changes from red to different shades

of pink to a dull brown color. These color changes are used as a subjective way to determine various degrees of doneness, such as: rare, medium, and well done. The cooked pigment is denatured myoglobin. Myoglobin is the protein within the muscle that plays a large role in beef color. Myoglobin consists of a protein (globin) and a non- protein (heme ring) (Aberle et al., 2012). The heme ring is of importance as the color of meat is dependent upon the oxidation state of the iron within the ring. The chemical state of myoglobin when uncooked beef is not exposed to oxygen is deoxymyoglobin (DMb), resulting in a purplish-red color. When exposed to oxygen, DMb changes to a bright cherry-red color, referred to as oxymyoglobin (OMb). When the iron becomes oxidized the color of beef results in a brown color, known as metmyoglobin (MMb). When beef is cooked (regardless of the state of myoglobin), the globin is denatured thus, unable to return back to the red pigment (Claus, 2007). Davey et al. (1974) noted that meat begins to change color at 43C to 44C. Roldan et al. (2013) reported that the denaturation process of myoglobin takes place between 55C and 65C and continues until 75C or

80C. According to Lawrie (2006) myoglobin is one of the most heat stable sarcoplasmic proteins, and is almost completely denatured at temperatures of 80C and above. Thus, the degree of myoglobin denaturation is highly dependent on the end-point temperature when cooking. Beef cooked to an internal temperature of 60C (rare) has a bright red interior pigment; 63C to 71C (medium) has a warm pink center; and an internal temp cooked to 77C (well done) or higher has a greyish-brown center (AMSA, 2008). Thus, as cooking temperatures increase, myoglobin denaturation also increases (Roldan et al.,

2013).

Other factors that contribute to the external color of meat is the caramelization

and the Maillard reaction. Caramelization is a reaction of sugars within meat when heat is applied. This reaction involves the thermal degradation of sugars which produce caramel aromas and browned-caramel colors. Caramelization occurs at temperatures above

120C. The Maillard reaction involves reducing sugars and amino acids to interact when heat is applied. Along with color, Maillard reaction provides various flavors and aromas

(Lawrie, 2006).

Cookery Methods

Cookery method can affect overall palatability and the amount of cooking loss of meat products. With the various types of cooking methods, selection of which to use can be difficult. However, the most common cookery methods consist of two categories: dry and moist. Dry methods use dry, high heat (> 160C) whereas moist methods use either liquid or humidity to cook at low temperatures (< 95C) (Kerth, 2013). Application of either dry or moist cookery methods on various meat products can have a significant impact on the overall palatability (Jeremiah et al., 2003). Therefore, the initial tenderness, available cooking equipment, the amount of time allowed for preparation, and the desired quality are all criteria that should be used when selecting the correct cooking method

(AMSA, 2017).

Dry

Tender cuts of meat are most suitable for dry cooking methods. However, less tender cuts can be cooked via dry heat if marinated prior to cooking. Ideal tender retail cuts are steaks and chops from muscles like: psoas major (tenderloin), longissimus dorsi

(ribeye/ striploin) and the infrasprinatus (top blade/ flat iron). When using dry cookery methods duration of cooking is critical due to the use of high temperatures, especially

when cooking thin cuts, making it easy to exceed the appropriate doneness. Dry cooking methods consist of: broiling, pan-broiling, grilling and oven roasting. Broiling is a method where meat is cooked by direct high heat. Tender beef steaks of at least ¾ inch thick are best when broiling (AMSA, 2017). Marinated, less tender cuts like the semimembranosus (inside top round) may also be broiled. Marinating meat can help to improve tenderness in tough cuts, however, its effects are limited (AMSA, 2017). These same cuts can also be pan-broiled. It is important to consider the thickness of the cut before using this method. Meat cuts that are thicker than ¾ of an inch tend to overcook on the outside before the center has reached the desired internal temperature. When using this method, it is important to turn or rotate the meat, as heat only radiates from one direction (AMSA, 2016). Grilling uses high heat (> 200C) by searing the external side of the meat cooked at a fast pace. Grilling can be accomplished on a rack over a bed of coals, or an open fire. Additionally, grilling methods can be done indoors with electric clamshell grills and range tops. Oven roasting is usually accomplished at 150C to

175C, where meat is cooked by convection, high heat circulated by fans (AMSA, 2016;

Aberle et al., 2012). In addition, studies have shown that dry heat cookery methods impart a desirable flavor and aroma compare to moist heat (Jeremiah et al., 2003; Hood et al., 1955). Meat recommended for roasting includes larger cuts of beef.

Moist

Moist heat cooking methods are best for cuts of meat that are less tender, such as, semitendinosus (eye of round), semimembranosus (inside top round), and biceps femoris

(outside round) (Cover, 1941). The application of moist heat aids in decreasing the amount of surface drying in cuts requiring long cooking times (AMSA, 2017).

Additionally, cooking temperatures are low when using moist heat methods, as heat penetration is faster that dry-heat due to steam and water conducting heat rapidly (Cover,

1941). Nonetheless, with moist heat, some water is lost from the meat into the cooking liquid, but not totally if consumed in stews or soups (AMSA, 2017). Braising and cooking in liquid are examples of moist heat cookery. An example of braised meat is pot roast, which could be achieved using oven cook-in bags. Cook-in bags create an environment where no additional water is needed, due to the moisture being drawn out of the meat within the cook-in bag. Cooking in liquid involves covering less tender cuts of meat with liquid and simmered at temperatures < 90.5C (195F) until tender (AMSA,

2017). The advantage of moist-heat cooking is the reduction in surface drying with longer cooking times.

Sous vide

Sous vide cooking is a method immersing vacuum-packaged food in a water bath at a precisely controlled temperature (Baldwin, 2012; Baldwin, 2008). Temperature regulation allows for greater control and consistency to achieve a greater degree of doneness than traditional cooking methods. This cooking technique was originally developed in France in the 1970’s by Chef Georges Pralus (Baldwin, 2013). Chef Pralus initially used sous vide as a means to optimize cooking and minimize the costly shrinkage in foods. Since then this method has quickly expanded to elite chefs worldwide. In 2005, sous vide initially made its way into the U.S landing in four star restaurants (Hesser,

2005). Today, sous vide is used worldwide in the catering industry and is being used in various restaurants and homes (Baldwin, 2013; Sanchez Del Pulgar et al., 2012). Sous vide cooking provides chefs and restaurants with a method that is easily applicable to raw

and precooked foods (Armstrong, 2000). Typically chefs use a low temperature-long time relationship when cooking meat via sous vide, resulting in a unique textural characteristic

(Sanchez Del Pulgar et al., 2012). Previous studies have shown that sous vide cooking preserves sensory attributes associated with quality by reducing shear force, the loss of flavor volatiles, while also decreasing moisture loss (Sanchez Del Pulgar et al., 2012;

Baldwin, 2012; García-Segovia et al., 2007; Vaudagna et al., 2002; Church et al., 2000;

Schellekens, 1996). Roldan et al. (2013) concluded that lamb loins cooked for 24 h were significantly tender compared to shorter cooking times of 6 and 12 h. Sanchez Del Pulgar et al. (2012) found that pork cooked at 80 for 12 h resulted in improved tenderness however, moister loss also increased. As sous vide cooking temperature and time increased, García-Segovia et al. (2007) determined that the amount of cooking losses increased. Therefore, cooking temperature and time seems to play a role on the cooked characteristics of sous vide meat. Furthermore, Roldan et al. (2013) concluded that sous vide temperature had the greatest effects on weight and moisture loss, color and textural properties.

Other authors have reported a reduction in lipid oxidation as result in the absence of oxygen as well as different color characteristics due to a change in the Millard reaction combined with myoglobin denaturation (García-Segovia et al., 2007). However, even with the previous studies, the effects of sous vide cooking, and its potential benefits regarding meat eating quality has yet to be further investigated. More specifically, research is needed to determine the temperature and time combination to optimize tenderness in sous vide cooked meat, while also maintaining or reducing moisture loss.

Effects of maturity on meat quality

In addition to the application of heat and cookery method, animal maturity also plays a significant role in determining palatability and overall eating experience. Animal maturity is described by the physiological age of the animal; most commonly known as the visible degree of aging in animal tissues. Age can be determined by dentition, bone ossification, and color and texture of the muscle (Tatum, 2007; Kinsman et al., 1994). A common method in determining age in beef carcasses is through ossification along the vertebral column along the outer tips dorsal to each vertebra. Ossification is the conversion of cartilage to bone. Cartilage is abundant in young animals, most originating between vertebrae, at the ends of dorsal vertebrae, and along the sternum bone appearing pearly white in color (Aberle et al., 2012). As an animal ages, cartilage is gradually replaced with bone beginning from the posterior end of the animal to the anterior end, changing from soft, pearly-white cartilage to hard, porous bone.

Maturity has been correlated to beef tenderness and palatability (Acheson et al.,

2014; Tatum, 2011; Boleman et al., 1996; Kinsman et al., 1994; Berry et al., 1974). Meat from animals of advanced maturity is less tender, when compared to animals younger at time of harvest (Berry et al., 1974). This difference in tenderness is largely affected by the amount of connective tissue in the muscle. As maturity increases, the amount of connective tissue also increases (Lepetit, 2007; Kinsman et al., 1994). Consequently, to account for the effects of carcass maturity on tenderness, maturity is assessed at time of harvest via evaluation of dentition. On December 23rd, 2003, the United States

Department of Agriculture (USDA) revealed the first known case of Bovine Spongiform

Encephalopathy (BSE) discovered in an adult Holstein cow in the state of Washington

(CDC, 2017). Since then, beef slaughter facilities have been required to use dentition at time of harvest to initially segregate beef carcasses into two age groups: < 30 mo. and 

30 mo. (FSIS, 2004). Cattle showing 3 or more permeant incisors breaking the bottom gum line are deemed 30 mo. of age or older; whereas cattle arriving with less than 3 permeant incisors are identified as less than 30 mo. of age (FSIS, 2004). Upon harvest and a 36 to 48 hr. chill, beef carcasses are sent towards the grade stand where they are initially ribbed between the 12th/13th ribs, then follow the “bloom” chain for and estimated 20 min. A USDA grader will assess the skeletal maturity, back fat, muscling, marbling, and lean color in the cut surface to the 12th rib, to assign the appropriate USDA

Quality and Yield grades (USDA, 2016). Age groups for beef carcasses are divided into five skeletal maturity classifications: A (9 to 30 mo. of age), B (30 to 42 mo. of age), C

(42 to 72 mo. of age), D (72 to 96 mo. of age), and E (greater than 96 mo. of age)

(Tatum, 2007).

Young fed beef

Over ninety percent of young fed beef harvested in United States originates from young, grain-fed, steers and heifers (fed beef) harvested between 12 to 24 mo. of age

(Tatum, 2011). The National Beef Quality Audit (NBQA) Executive Summary (2017) reported, that of the steers and heifers harvested (n=7,379), 98.8% of the carcasses were classified as <30 mo. of age by dental age classification, indicative of a young animal

(“A” maturity). Beef carcasses of “A” maturity, are eligible for USDA quality grades of

Prime, Choice, Select and Standard. USDA quality grades are determined using visual assessments of the degree of marbling in the longissimus dorsi at the 12th/13th ribs in relation to the maturity of the carcass (USDA, 2016).

In 2016, the NBQA reported a distribution of USDA quality grades of 3.8%

Prime, 67.3% Choice, 23.2% Select and 5.6% received other grades (NBQA, 2016).

Several studies have investigated the effectiveness of the USDA quality grading system for categorizing differences in the overall palatability (O’Quinn et al., 2015; Hunt et al.,

2014; Acheson et al., 2014; Emerson et al., 2013; McKenna et al., 2004; Neely et al.,

1998; Shackelford et al., 1995; Smith et al., 1988; Smith et al., 1982; Breidenstein et al.,

1968). A recent study by Emerson et al. (2013) reported that quality grades effectively identified differences in tenderness, juiciness, and flavor attributes in cattle less than 30 mo. of age. Similarly, Acheson et al. (2014) reported that marbling scores effectively classified carcasses according to differences in palatability, however, no differences were detected in palatability from A maturity carcasses and B-C maturity carcasses.

Additionally, Boykin et al. (2017) noted a correlation of carcasses <30 mo. of age, classified by dentition, had improved USDA quality grades along with larger ribeye areas and decreased maturity, compared to carcasses older than 30 mo. of age. No differences were reported in palatability between ossification classification within dental age groups, consistent with the findings from other researchers (Semler et al., 2016; Acheson et al.,

2014).

Carcasses originating from young fed beef contain immature, soluble collagen within the muscle (Hill, 1966; Goll et al., 1964). Immature collagen is characterized by a low number of heat-stable cross-linkages, which are easily broken (Aberle et al., 2012).

Therefore, during cooking, fed beef collagenous cross-links form a gelatin, which aid in reducing the effects of toughening due to collagen (Tatum, 2011).

Advanced maturity

In the beef industry, cows are routinely culled or removed from operations due to poor reproduction, productivity, and efficiency (Woerner, 2010). According to Woerner

(2010) culled cows make a up 17 to 19% of the United State beef production. Due to the advanced maturity, most culled cows are not eligible for the typical USDA quality grades of fed beef. However, cows that are greater than 42 mo. of age can qualify for USDA quality grades of: Commercial, Utility, Cutter, and Canner. In most commercial plants, carcasses of C, D, or E maturity are referred to as “hardbone,” due to their advanced ossification, and typically do not receive a USDA quality grade (“no roll”)(Tatum, 2011).

Carcasses are priced based on a grid pricing system of premiums and discounts. For instance, on October 17th, 2017, beef carcasses were awarded a premium of $16.77 per hundred weight for USDA Prime, whereas, cattle of the Low Choice grade receive no premiums nor discounts. Grades of Select and Standard received discounts of $4.90 and

$17.88, respectively, per hundredweight. Beef carcasses 30 mo. of age or older are discounted up to $16.36 per hundredweight (USDA-AMS, 2017). In addition, beef carcasses of hardbone characteristics received significant price discounts in the industry,

$33.66 per hundredweight (USDA-AMS, 2017; Tatum, 2011).

In addition to price discounts to carcasses of advanced maturity, several studies have indicated that there are differences in tenderness and overall palatability between youthful carcasses and to very mature carcasses (Hilton et al., 1998; Miller et al., 1983;

Smith et al., 1982; Bouton et al., 1978; Berry et al., 1974; Tuma et al., 1962). Tuma,

Henrickson, Stephens, and Moore (1962) found that tenderness greatly decreased with age in Herford females aging from 18, 42, and 90 mo. of age. More specifically, Tuma et al. (1962) stated that 18 and 42 mo. age groups revealed a greater decrease in tenderness

than 42 and 90 mo. Hilton et al. (1998) reported differences in tenderness from carcasses representing A and B to C, D and E. Additionally, Berry et al. (1974) concluded that samples from E maturity carcasses were inferior in tenderness when compared to youthful carcasses. However, carcasses from A and B maturity groups were similar in palatability (Berry et al., 1974).

When compared to young fed carcasses, cow carcasses are known to contain mature collagen, which is characterized by an increase in the number or strength of heat- stable cross-linkages (Hill, 1966; Goll et al., 1964; Goll et al., 1963). Hill (1966) reported that during cooking, less collagen is solubilized in meat from older animals resulting in increased toughness. Boleman et al. (1996) reported decreased tenderness in whole muscle cuts from cows that were correlated with high levels of insoluble collagen found within the muscle. Alternatively, Miller et al. (1983) found that total intramuscular collagen was higher for mature carcasses; however, no differences were reported in tenderness or collagen solubility between maturity groups (A-B maturity and C-D maturity).

In addition to an increase in toughness, cow carcasses also naturally exhibit a darker lean color than in fed beef carcasses. Studies have shown that as cattle mature, the pigment of lean tissue becomes darker red in color (Acheson et al., 2014; Romans et al.,

1965). This is primarily due to a higher concentration of myoglobin in the muscle, which gives muscle its pigment. Myoglobin concentrations increase as an animal ages due to a decrease in the affinity of oxygen, ultimately causing a darker lean color. Other factors like muscle pH, fat and water content may also play a role (Romans et al., 1965).

Acheson et al. (2014) reported that B-C maturity carcasses presented a significantly

darker colored lean and a redder color lean than A maturity carcasses. Similarly, Romans et al. (1965) reported that color differences were significant only at the A maturity level, no differences were reported between the B, C, and D levels.

Sensory Evaluation

Sensory evaluation is used to measure and analyze products through the responses of human senses: sight, smell, touch, taste, and hearing. The human senses determine how consumers perceive palatability, while also influencing purchasing decisions. Color, price, packaging, brand names etc. all have influences on the initial purchasing decision.

However, after the purchase, the consumer experiences the sensory attributes that determine palatability and overall eating satisfaction. Sensory attributes that determine beef palatability are often described as the combination of three factors: tenderness, flavor, and juiciness. The relationship between these three factors contribute to the consumers’ perception of taste and/or the overall liking of a product. Several consumer surveys have suggested that taste (flavor) is the primary driver of purchasing decisions and consumer satisfaction (Tatum, 2015; Corbin et al., 2014; Brooks et al., 2000).

Conversely, in other studies consumers ranked tenderness as the most important qualitative characteristic in meat and would be willing to pay a premium in the marketplace if it were guaranteed to be tender (Lepper-Blilie et al., 2014; Miller et al.,

2001; Huffman et al., 1996; Miller et al., 1995; Savell et al., 1987). Meanwhile, other studies have shown that flavor is equally important, and in some instances, more important than tenderness when determining overall palatability (Corbin et al., 2014;

O’Quinn et al., 2012; Killinger et al., 2004). Sensory attributes can be evaluated by objective methods (instrumental or trained sensory panels) or through subjective methods

using consumer panels. Trained panels undergo a series of trainings using various scales, ranging from one extreme to the other, based on the characteristic being scored.

Consumer panels strictly evaluate overall likeness; juiciness, and satisfaction of a product.

Tenderness

It is important that consumers are able to distinguish different levels of tenderness in order to establish a value of tenderness, segregating tender from tough muscle cuts

(Boleman et al., 1997). This could provide the beef industry with an economic incentive to segregate beef of varying levels of tenderness, as consumers would be willing to pay premiums if guaranteed tender (Miller et al., 2001). Tenderness can be evaluated through instrumental objective measurements such as the Warner-Bratzler shear force (WBSF) method (AMSA, 2016). This method measures the peak load or maximum shear force required to completely shear a sample. Shear force is used to determine the degree of tenderness, as it mimics the force used during chewing. There have been several studies where the Warner-Bratzler shear force method was used to evaluate beef tenderness and compare values to consumer satisfaction (Igo et al., 2015; Hunt et al., 2014; Emerson et al., 2013; Platter et al., 2003; Miller et al., 2001; Wheeler et al., 1999). Consumers identified and reported that the acceptable tenderness values were < 3.0 - 4.3 kg resulting in a  86% consumer eating satisfaction rating (Miller et al., 2001). Furthermore, Miller et al. (2001) reported WBSF values > 4.9 kg resulted increased consumer dissatisfaction.

Platter et al. (2003) observed that when WBSF values increased from 3.0 to 5.5 kg there was a decreased probability of consumer acceptance. More recently, a study by Igo et al.

(2015) concluded that there should be four classifications of tenderness based on

consumer satisfaction ratings: “very tender” < 31.4 N (3.2 kg), “tender” 31.4 – 38.3 N

(3.2 – 3.9 kg), “intermediate”38.3 – 45.1 (3.9 – 4.6 kg) and “tough” > 45.1 (>4.6 kg).

Alternatively, Hunt et al. (2014) noted that there was not a significant correlation between the consumer’s overall liking and WBSF values.

Despite the rising beef prices, beef remains a favorite protein of choice, with 77% of consumers listing beef as their first top choice of protein (CBI, 2017). According to the

2015 Consumer Beef Index (CBI), 73% of consumers surveyed say steaks in the retail case are priced just right or “expensive but worth it,” which supports the findings of consumers’ willingness to purchase premium beef (CBI, 2015). Therefore, when cost is factored in, consumers ultimately believe the price reflects the value of the beef product.

Conclusion

The beef industry has made great strides to improve consistencies in beef products, inconsistencies still exist pertaining to tenderness, specifically in undervalued beef cuts. Thus, sous vide cooking may provide consumers, processors, and food service an alternative method for improving consistency in beef tenderness. However, further investigation is needed to evaluate the utility of sous vide cooking in beef tenderness, to ultimately add value to undervalued cuts of meat, specifically cow beef.

Chapter 3

Pilot study: Investigating the effects of sous vide cooking on tenderness in fed and

cow semitendinosus muscles

Abstract

The beef industry continuously seeks ways to increase consumer demand, while improving eating quality and consistency of beef products through added value products.

Therefore, the present study was designed to add value to undervalued tough cuts of beef through sous vide cooking. Whole beef semitendinosus muscles (ST; n = 6) were used representing two age groups: fed (n = 3) and cow (n = 3). Three roasts were obtained from each ST muscle that were cooked via sous vide at 55C for 2, 10 and 30 h.

Additionally, fed beef longissimus lumborum (n = 4) and psoas major (n = 4) steaks were cooked via sous vide at 55C for 2 h. Cook loss (%), internal cooked color and WBSF was measured following cooking cycles. Cooking time had a significant effect on cook loss, as the percent cook loss increased at longer cooking times (P = 0.001). In addition, cooking time also had significant effects on L* and a* values (P < 0.034), as roasts maintained a darker, redder internal degree of doneness when cooked for 2 h. WBSF values decreased as cooking time increased as roasts cooked for 10 and 30 h were significantly more tender (P < 0.005) than roasts cooked for 2 h. Altogether, cooking ST muscles via sous vide at low temperatures for long cooking times resulted in a similar tenderness when compared to tender cuts of meat cooked at shorter times.

Introduction

In the last six years, the beef industry has made strides to find alternative methods for adding value to undervalued muscle cuts, particularly those originating from the locomotive areas such as the chuck (shoulder) and round (leg) areas. Cow carcasses have received considerable attention due to the challenges of the drought across the United

States and subsequent herd reductions. Cows are significantly price discounted due to their advanced age at time of slaughter. Studies have shown negative correlations between age and palatability (tenderness, flavor, and juiciness) and percent saleable meat

(yield), which subsequently results in an undervalued carcass. There is a need to identify new, lean muscle cuts from cows that can be used as value-added beef products.

However, there has been little innovation in the area of adding value through methods of cooking.

Sous vide cooking is a method where vacuum-packaged food is immersed in a water bath at a precisely controlled temperature. Today, sous vide is used worldwide in the catering industry and is being used in various restaurants and homes (Baldwin, 2013;

Sanchez Del Pulgar et al., 2012). In addition, sous vide cooking provides chefs and restaurants with a method that is easily applicable to raw and precooked foods

(Armstrong, 2000). Sous vide cooking has been known to offer several advantages over traditional cooking methods. Previous studies have shown that sous vide preserves sensory attributes associated with quality by reducing shear force, the loss of flavor volatiles, while also decreasing moisture loss (Sanchez Del Pulgar et al., 2012; Baldwin,

2012; García-Segovia et al., 2007; Vaudagna et al., 2002; Schellekens, 1996). Therefore, a preliminary experiment was conducted to investigate the utility of sous vide cooking in

beef tenderness, to ultimately add value to undervalued cuts of meat. The objectives of the experiment were to determine the temperature and time required to improve tenderness while maintain juiciness in cooked semitendinosus muscles.

Materials and Methods

Muscle preparation

Whole beef semitendinosus muscles (ST; n = 6) were sourced from two processing facilities representing two age groups: facility 1 provided young fed beef (<

30 mo. of age; n = 3) and facility 2 provided cow beef (> 42 mo. of age; n = 3). Whole

ST muscles were portioned into three roast (approximately 6.0 cm thick), individually weighed, vacuum packaged, and stored overnight under refrigerated conditions (4C).

Additionally, fed beef longissimus lumborum (LL; n = 4) and psoas major (PM; n = 4) steaks were sourced from The Ohio State University Meat Laboratory. Steaks were individually vacuum packaged, and stored overnight under refrigerated conditions (4C).

Cooking treatment

Roasts were cooked (in a vacuum sealed bag) in a recirculating water bath (Anova

Culinary Sous Vide, Inc., San Fancisco, CA) at 55C using three different times (2, 10, and 30 h). Longissimus lumborum (LL) and psoas major (PM) steaks were cooked (in a vacuum sealed bag) in a recirculating water bath (Anova Culinary Sous Vide, Inc., San

Francisco, CA) at 55C for 2 h. Upon completion of cook cycles, roasts and steaks were removed from the water bath and stored under refrigerated conditions (4C) overnight.

Cook loss

Upon refrigeration (4C), roasts were removed from the package and re-weighed.

The cooking loss was calculated using the following equation:

(raw weight - cooked weight) Cooking loss (%) = x 100 raw weight

Cooked objective color score

After cooking and chilling, roasts were sliced 1-centimeter below the original cut surface of both sides of the roast to expose the interior and given 10-min. to bloom at

4C, then re-measured for objective color on both cut surfaces. The standardization of the

Minolta was done using a white plate (Y=93.5, x=0.3132, y=0.3198). The average value of duplicate readings was used for statistical analysis.

Warner-Bratzler shear force

Six 1.27-centimeter cores were obtained from each roast parallel to the longitudinal orientation of the muscle fibers (Wheeler et al., 1996). Cores were sheared once with a Warner-Bratzler attachment using a TA-XT Plus Texturometer (Stable Micro

Systems Ltd., Godalming, UK) with a V-shaped shear blade at a cross head speed of 200 mm/min. An average was then determined of the six cores to determine a represented shear force value for each individual roast (Wheeler et al., 2016).

Statistical analysis

Data were analyzed with a mixed model in SAS JMP. Main effects (age and time) were tested in a mixed model as fixed effects. Least square means were compared by

Tukey’s HSD (honest significant difference) using pairwise comparisons with a significance level of P < 0.05.

Results and Discussion

Cook loss (%)

The effects of cooking time on cook loss (%) are presented in Table 1. No significant age x time interaction (P = 0.810) was found. Cooking time had a significant effect on cook loss, as percent cook loss increased with longer cooking times (P = 0.001).

Percent cook loss at 2 h (13.97%) was significantly lower (P = 0.002) when compared to cooking times of 10 h (19.41%) and 30 h (19.49%). These results are in agreement with previous sous vide cooking studies by (Roldan et al., 2013; García-Segovia et al., 2007;

Vaudagna et al., 2002), who found increased cooking losses with longer cooking times at different temperatures. Therefore, it appears sous vide cooking of less than 10 h would result in lower cooking losses.

Internal cooked color

Table 3.1 presents the effects of time on internal color. There was no significant age x time interaction (P > 0.393) for L*(lightness), a*(redness) and b*(yellowness).

Cooking time had significant effects on L* and a* values (P < 0.034). However, there was no significant effects of time on b* values (P = 0.648). Lightness values (L*) increased as time increased (P = 0.002), as 30 h had a significantly lighter internal cooked color when compared to 2 and 10 h. Studies by Roldan et al. (2013) and Sanchez

Del Pulgar et al. (2012) also observed higher L* when sous vide cooking lamb or pork at various temperatures for prolonged times.

Redness values (a*) significantly decreased as time increased (P = 0.034), as roasts cooked for 2 h had a significantly redder internal degree of doneness than roasts cooked to 10 and 30 h. Results from the present study are in agreement with Becker et al.

(2016),Roldan et al. (2013), and García-Segovia et al. (2007), who reported both time and temperature influenced the degree of doneness or redness. The red color of cooked meat is determined by the degree of the denaturation of myoglobin (Suman et al., 2016;

Tornberg, 2005). Therefore, it is no surprise degree of doneness decreases as cooking time and endpoint temperature increases.

Overall, internal cooked color analysis results suggest that sous vide cooked roasts were lighter (higher L*) and decreased in redness (lower a*), whereas b* values

(yellowness) was not affected.

Warner-Bratzler shear force

Results of the effects of time on Warner-Bratzler shear force (WBSF) values can be found in Table 3.1. Cooking time had a significant effect on tenderness (P = 0.001) as

WBSF values decreased as cooking time increased. Roasts cooked for 10 h (1.88 kg) and

30 h (1.49 kg) were significantly more tender (P < 0.005) than roasts cooked for 2 h (3.26 kg). Roldan et al. (2013) observed a decrease in shear force values with prolonged cooking times up to 24 h. The observed improvement in tenderness at low temperatures for longer periods of time could be due to greater collagen solubilization (Roldan et al.,

2013; Christensen et al., 2013; Baldwin, 2012; Christensen et al., 2011; Laakkonen et al.,

1970). Therefore, it can be concluded from the pilot study, that an improvement in tenderness can be achieved via sous vide cooking with cooking times between 2 and 10 h.

Moreover, Table 3.2 displays the effects of time on WBSF values of cooked ST muscles representing each age group (fed vs cow), as well as steaks from the LL and PS muscles. Warner-Bratzler shear force values from fed ST roasts significantly increased as cooking time increased (P < 0.001). Surprisingly, ST roasts from the cow age group had

no significant differences (P = 0.073) in WBSF values as cooking time increased.

However, numerically WBSF values of the cow ST age group indicate an improvement in tenderness occurred at prolonged cooking times. Additionally, ST roasts representing both age groups cooked for 10 h had similar WBSF values when compared to LL and PS steaks that were cooked for 2 h. Therefore, cooking tough cuts of meat, like ST, at low temperatures for 10 h will result in a similar tenderness as in tender cuts of meat.

Conclusion

In conclusion, cooking at low temperatures for prolonged times up to 30 h resulted in an improvement of tenderness. In addition, cooking ST muscles via sous vide at low temperatures for extended cooking times resulted in a similar tenderness values when compared to tender cuts of meat cooked at shorter times. Although tenderness was improved, cooking losses were negatively impacted when cooked for extended periods of time. Results from this study suggest that sous vide cooking times between 2 and 10 h at

55C would provide consumers with a tender product while still maintaining cooking loss and a consistent degree of doneness.

In totality, sous vide is an alternative cooking method that is capable of transforming tough cuts of beef into tender products. Our research could provide consumers and restaurants with an innovative way to produce a consistently cooked product that satisfies consumers palatability through sous vide cooking. Additionally, we can utilize under-valued tough beef cuts, such as the semitendinosus, to achieve a guaranteed tender product. This process gives hope to the meat industry in adding value to undervalued beef cuts. Additionally, cafeterias and buffets would benefit most in

purchasing inexpensive product and receiving a higher profit value simply by utilizing this method of cooking, sous vide. Thus, we can increase the utilization of undervalued cuts from cull cows that consumers typically do not purchase due to being extremely tough, and ultimately increase the overall demand for all beef products.

Therefore, we can add value to tough, undervalued cuts of beef like cow semitendinosus muscles. Applying this cooking method in an industry setting could offer processors a means to sell pre-cooked products, like semitendinosus, while guaranteeing a tender, satisfying product at a moderate price.

Table 3.1. Least-square means on the effects of cooking time on cooking loss1 (%), internal cooked color, and Warner- Bratzler shear force5 (WBSF) of ST roasts. Time (h) Item n 2 h 10 h 30 h SE P-value CL%1 6 13.97b 19.41a 19.49a 0.89 0.002 L*2 6 56.00b 58.04b 60.50a 0.77 0.002 a*3 6 21.04a 19.35b 18.38b 0.53 0.034 b*4 6 6.70 6.00 6.51 0.54 0.648 WBSF5, kg 6 3.26a 1.88b 1.49b 0.20 0.001 a,b Least-square means within a row without a common letter are significantly different (P < 0.05). 1 CL% = Percent cooking loss 2 L* = Lightness 3 a* = Redness 4 b* = Yellowness 5 WBSF = Warner-Bratzler shear force values, kg n = Number of observations

Table 3.2. Least-square means on the effects of cooking time on Warner-Bratzler shear force (kg) of cooked ST1, LL2, and PS3 muscles. Time (h) Item 2 h 10 h 30 h SE P-value Fed4 ST 3.14a 1.55b 1.09c 0.08 <0.001 Cow5 ST 3.39 2.22 1.80 0.39 0.073 LL 1.56 PS 2.04 a-c Means within a row without a common letter are significantly different (P < 0.05). 1 ST = Semitendinosus 2 LL = Longissimus lumborum 3 PS = Psoas major 4 Fed = < 30 mo. of age 5 Cow =  42 mo. of age

Chapter 4

Effects of sous vide cooking on tenderness and protein concentrations in fed and

cow semitendinosus muscles

Abstract

Whole beef semitendinosus muscles (ST; n = 40; IMPS 171C) represented two age groups: young fed beef (< 30 mo. of age; n = 20) and cow beef (> 42 mo. of age; n =

20). Semitendinosus muscles were then portioned into 6 cm thick roasts which were weighed and individually vacuum packaged. Roasts were cooked (in a vacuum sealed bag) in a recirculating water bath at four different temperatures (55, 60, 65, and 70C) using four different times (2, 4, 6, and 8 h). Percent cooking loss, raw and cooked color,

Warner-Bratzler shear force (WBSF), protein concentration of cooked liquid and cooked

ST were evaluated.

There was an interaction (P = 0.028) between temperature and age with WBSF values. Fed ST roasts cooked at 60C were significantly tougher than cow ST roasts at

60C (P < 0.05). Additionally, cook loss (%) increased as temperature and time increased

(P < 0.001). Semitendinosus roasts cooked to temperatures of 55, 60, 65, and 70C at 2 h. had the lowest percent cooking loss (P < 0.05) compared with roasts cooked at temperatures of 70C for 6 and 8 h. (P < 0.05). Furthermore, L* values increased as

temperature and time increased, whereas a* decreased. Redness values for fed ST at 60C cooked for 4 and 8 h was significantly higher (redder) than cow ST at 60C for 8 h (P <

0.05). Roasts that were cooked at temperatures lower than 65C and less than 6 h had significantly higher b* (increased yellowness) values than those cooked at higher temperatures for longer periods of time.

Moreover, soluble protein fractions decreased in concentration with increasing temperature, with the greatest concentrations occurring at 55C for 2, 4, 6, and 8 h when compared to all other temperature and time points (P < 0.05). Additionally, at 60C, fed

ST had significantly higher concentrations of soluble protein than cow ST (P = 0.020).

Total protein concentrations of the cooked liquid revealed a three-way interaction of age, temperature, and time (P = 0.021). Total protein concentrations of the whole cooked ST muscle showed a temperature effect (P = 0.005) as temperature increased the total concentration of cooked muscle revealed a slight decrease. Altogether, cooked roasts originating from fed beef had a higher concentration of total protein than cow (P =

0.001). SDS-PAGE gel patterns of cooked liquid soluble proteins exhibited similarities to sarcoplasmic proteins from muscle exudate found in previous studies suggesting sarcoplasmic proteins might play a role in determining tenderness and quality attributes.

Introduction

As stated in the previous pilot study, sous vide cooking resulted in an improvement in tenderness at prolonged times up to 30h. However, it was concluded that prolonged cooking time greater than 10 h led to higher cooking losses and a less desirable degree of doneness. Cooking meat for prolonged times up to 30 h may also lead to negative impacts on texture and palatability, by imparting a grainy or mealy texture

(Becker et al., 2016), creating a negative eating experience for consumers. Therefore, a balance between achieving consumer acceptable tenderization and cooking loss needs to be determined to obtain a product of ultimate value.

Moreover, as previously mentioned in chapter 2, sous vide is a unique cooking method which utilizes vacuum sealed packaging during cooking, allowing cooking losses to remain in the bag, resulting in no evaporative losses (Baldwin, 2012). Therefore, the containment of the cooked liquid during cooking allowed for the liquid to be collected and analyzed out of curiosity. To our knowledge there is no previous studies or literature found on the cook liquid collected from sous vide vacuum sealed bag and its effects on sous vide cooking.

The primary aim of the present study was to determine the effects of different temperature and time combinations on tenderness in fed and cow semitendinosus roasts.

Secondly, we tried to understand the effects of sous vide cooking on meat proteins by analyzing the cook liquid (purge).

Methods and Materials

Muscle preparation

Whole beef semitendinosus muscles (n = 40; IMPS 171C) were sourced from two beef packing plants representing two age groups: plant 1 provided USDA Choice from young fed beef (< 30 mo. of age; n = 20) and plant 2 provided cow beef (> 42 mo. of age; n = 20). Whole beef ST were purchased and shipped upon 7 days post-harvest to the Ohio

State University Meat Laboratory. Upon arrival, product was randomly removed from the shipping container. Subprimals were individually weighed, including the vacuum package, then removed from the vacuum package and weighed independently followed by the weight of the bag after it was rinsed and dried. Subprimals then were portioned into roasts (6.0 cm thick) using a cutting template resulting in a total of four roasts which were produced per whole ST subprimals. Roasts were individually weighed and randomly assigned a three-digit code.

Raw objective color score

Uncooked roasts were objectively evaluated for color ( L* (lightness), a*

(redness), and b* (yellowness)) upon a 30-min. bloom time at 4C using a Konica

Minolta CR-410 Chroma colorimeter with a 50mm aperture (Wheeler et al., 2016). The standardization of the Minolta was done using a white plate (Y=93.5, x=0.3132, y=0.3198). The average value of duplicate readings was used for statistical analysis.

After raw color measurements were obtained roasts were individually vacuum packaged, and then stored overnight under refrigerated conditions (4C).

Cooking treatment

Roasts were cooked (in a vacuum sealed bag) in a recirculating water bath (Anova

Culinary Sous Vide, Inc., San Francisco, CA) at four different temperatures (55, 60, 65, and 70C) using four different times (2, 4, 6, and 8 h). Each time point was replicated five times per temperature treatment for each age group (fed and cull cow). Upon completion of cook cycles, cooking was arrested by placing roasts in an ice bath for 15 mins. Roasts were then stored under refrigerated conditions (4C) overnight (Hunt et al.,

2012) until further analysis.

Cook loss

As stated in the previous study, cooking loss was calculated using the following equation:

(raw weight - cooked weight) Cooking loss (%) = x 100 raw weight

Cooked objective color score

After cooking and chilling, roasts were sliced 1-centimeter below the original cut surface of both sides of the roast to expose the interior and given 10-min. to bloom at

4C, then re-measured for objective color on both cut surfaces. The standardization of the

Minolta was done using a white plate (Y=93.5, x=0.3132, y=0.3198). The average value of duplicate readings was used for statistical analysis.

Warner-Bratzler shear force

Systems Ltd., Godalming, UK) with a V-shaped shear blade at a cross head speed of 200 mm/min. An average was then determined of the six cores to determine a represented

shear force value for each individual roast (Wheeler et al., 2016). A 1-cm x 1-cm x 1-cm cube of ST was removed, placed in Whirl-pak bags and stored at -80C until subsequent analyses was conducted.

Protein concentration of cook liquid

The cooked liquid (purge) produced in the vacuum sealed package was collected after each roast was removed and re-weighed. Cooked liquid was pipetted into a 1.5 mL microcentrifuge tubes and stored at -80C until protein analysis was conducted. Cook liquid samples were analyzed for total and soluble protein concentrations. Samples analyzed for total protein concentrations were solubilized in a buffer containing 8 M urea,

2 M thiourea, 3% SDS (wt/vol), 75 mM dithiothreitol, and 0.05 M Tris-HCl (pH 6.8) and placed on a heating block at 95C for five min. Samples were then diluted to a 1:10 ratio with deionized water. Total protein concentration determination from the cook liquid followed the methods of the Bio-Rad RC DC protein assay (Bio-Rad Laboratories,

Hercules, CA, USA). Bovine serum albumin (BSA) was used to set a standard curve

(Fisher Scientific, Pittsburg, PA). Samples were evaluated using a spectrophotometer

(Thermo Scientific Multiskan FC) at 750 nm.

Cook liquid samples to be analyzed for soluble protein concentrations were solubilized in a buffer containing 120 mM Tris-HCl and 4% SDS (wt/vol) and centrifuged for 3 min. at 9, 200 x g. The supernatant was collected and diluted to a 1:10 ratio with deionized water. Soluble protein concentration determination from the cook liquid followed the methods of the Thermo Scientific Pierce BCA protein assay and

Smith et al (1985) with a few modifications. BSA was used to set a standard curve

(Fisher Scientific, Pittsburg, PA). The Thermo Scientific Pierce BCA protein assay is a

detergent-compatible formulation that uses bicinchonic acid (BCA) to detect and quantify protein through colorimetric detection using a spectrophotometer at 570 nm.

Protein concentration of cooked muscle tissue

Frozen 1-cm cubes of cooked ST roasts were powdered using a liquid nitrogen cooled mortar and pestle. Duplicate 50 mg samples of ground muscle tissue from the frozen cubes were placed inside of 2 ml microcentrifuge tubes for total protein concentration analysis and gel electrophoresis. Upon analysis, the frozen ground muscle tissue samples were solubilized in a buffer containing 8 M urea, 2 M thiourea, 3% SDS

(wt/vol), 75 mM dithiothreitol, and 0.05 M tris-HCl (pH 6.8) and vortexed. Samples were then place in the heating block at 95C for 5 min. then diluted to a 1:10 ratio with deionized water. Total protein concentration analysis followed the methods of the BioRad

RC DC protein assay (BioRad Laboratories, Hercules, CA, USA). Bovine serum albumin

(BSA) was used to set a standard curve (Fisher Scientific, Pittsburg, PA, USA). Samples were evaluated using a spectrophotometer set at 750 nm.

Gel electrophoresis sample preparation

Cooked liquid samples were centrifuged for 10 min. at 9, 200 x g to obtain the soluble fraction. The soluble fraction was solubilized in a buffer containing 8 M urea, 2

M thiourea, 3% SDS (wt/vol), 75 mM dithiothreitol, and 0.05 M Tris-HCl (pH 6.8), and placed on a heating block at 95C for 5 min. until the fraction was completely solubilized. Samples were mixed with 0.05% bromophenol blue and stored at -20C until further analysis.

Microcentrifuge tubes containing 50 mg of ground tissue were solubilized in a buffer 8 M urea, 2 M thiourea, 3% SDS (wt/vol), 75 mM dithiothreitol, and 0.05%

bromophenol blue, 0.05 M tris-HCl (pH 6.8) and then homogenized. Mixture was heated to 60C for 10 min., vortexed and centrifuged for 5 min. at 9,200 x g and then stored at -

20C until further analysis.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Proteins were separated using a sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis (PAGE). A 1 mm x 12 mm x 14 mm polyacrylamide slab gel consisting of 10% resolving gel and 3% stacking gel was used to separate the solubilized protein samples. A PageRuler Broad Range Unstained Protein Ladder (Thermo

Scientific, Product ID# 26630) consisting of 11 proteins (5 kDa to 250 kDa) was used as a size standard. Each gel was loaded with one lane containing 10l of protein ladder and the remaining lanes containing 10l of protein sample. Separation was carried out with a running buffer at a consistent voltage of 170 V for ~70 min. or until the dye front reached the bottom of the gel. After separation, gels were stained in a Coomassie Brilliant Blue

R-250 Gel Stain (50% methanol, 10% acetic acid, 0.1% Coomassie Blue) overnight, then de-stained using 10% acetic acid solution and stored in deionized water at room temperature until imaging.

Image analysis

De-stained gel images were acquired and digitalized using an Azure Biosystem c600 (with NIR capabilities. Images were then normalized and individual protein bands were analyzed for molecular weights relative to the protein standard using TotalLab software (Cleaver Scientific Ltd, Warwickshire, UK).

Statistical analysis

All data were analyzed using statistical package JMP Pro 12.2.0 (SAS, Institute

Inc., Cary, NC). Main effects (age, time and temperature) were tested in a mixed model as fixed effects. Animal number was defined as a random factor and time was treated as a repeated measure by animal. Least square means were compared by Tukey’s HSD

(honest significant difference) using pairwise comparisons with a significance level of P

< 0.05.

Results and Discussion

Uncooked semitendinosus data

Table 4.1 shows the simple means of uncooked semitendinosus (ST) muscle weights and instrumental color by age group. Mean whole muscle weights between fed and cow age groups were not statistically different (P = 0.463). However, when separated in to roasts, mean weights differed specifically with roasts representing the fed group resulting in heavier weights (P = 0.002). It was noted that ST muscle originating within the fed age group varied in muscle shape and thickness from anterior to posterior.

Regardless of these differences, overall whole ST muscles (fed) were bigger and thicker compared to ST muscles in the cow age group; which explains weight differences between age groups. This parallels previous research who found muscle size differences between fed and cow muscle cuts (Hilton et al., 1998).

Uncooked color score values are presented in Table 4.1. Uncooked color did not differ between age groups for lightness (L*; P = 0.685). However, redness (a*), and yellowness (b*) values were significantly increased (P < 0.001) in the cow age group

when compared to the fed age group. It is suggested that the difference in redness is primarily due to a higher concentration of myoglobin in the muscle, as myoglobin concentrations increase as an animal ages due to a decrease in the affinity for oxygen, ultimately causing a darker lean color (Acheson et al., 2014; Romans et al., 1965).

Cook loss (%)

Results for cooking loss (%) due to the effects of temperature x time can be found in Figure 4.1. There were no significant differences (P > 0.05) in cooking loss across time points when cooked to 55C. This could be due to proteins not fully denaturing at lower temperatures, such as 55C. However, percent cooking loss significantly increased at higher temperatures (60C to 70C) between 2 and 6 h (P < 0.05). This could be due to denaturation of myofibrillar proteins causing muscle fiber shrinkage (Tornberg, 2005).

Thus, cooking for longer time periods at higher temperatures increases denaturation and shrinkage of structural components, resulting in excess liquid being released from the roast (Christensen et al., 2011). Results from the present study are in agreement with other sous vide studies reported by Vaudagna et al. (2002), García-Segovia et al. (2007) and Christensen et al. (2013) for beef, Sanchez Del Pulgar et al. (2012) and Christensen et al. (2011) for pork, and Roldan et al. (2013) for lamb. Increasing sous vide cooking temperatures to 70C and above would lead to increased cooking losses than at lower temperatures of 60C for longer periods of time (Roldan et al., 2013; García-Segovia et al., 2007). From a practical stand point, sous vide cooking times of less than 6 h at temperatures ≤65C would be most advantageous lower cooking losses.

Internal cooked color

Table 4.2 displays a three-way interaction for objective color score (L* (lightness, a* (redness), and b*(yellowness)) for age x temperature x time (P = 0.006). Lightness data represented in Table 4 reveals small differences in lightness values between age groups thus, it can be suggested that cooking time and temperature had greater effects.

Fed and cow ST roasts cooked at 55C for 2 h had a significantly darker (lower L* value) internal cooked color compared to roasts cooked at 65C for 4 h or longer (P < 0.05). It is speculated that the extent of protein denaturation, cooking loss, and muscle fiber shrinkage might contribute to the differences in lightness (Hughes et al., 2014).

Increasing temperature and time may lead to an overall increase in lightness as structural changes occur due to shrinkage and water loss; hence, contributing to the scattering of light (Christensen et al., 2011; García-Segovia et al., 2007). Thus, these results indicate that with increased temperature and time comes higher L* values; which is supported by

Hughes et al. (2014); Roldan et al. (2013).

Internal redness (a*) intensity in cooked ST roasts also revealed a three-way interaction of age x temperature x time (Table 4.2; P = 0.020). Small differences in a* values are noted between age groups thus, suggesting cooking temperature and time had greater effects on internal cooked color. The degree of redness was significantly redder at lower temperatures, such as 55C and 60C (indicative of a rare degree of doneness), when compared to roasts cooked at 65C and 70C (medium degree of doneness; P <

0.05). The loss of redness with an increase of temperature and time can be strongly related to the degree of denatured of myoglobin (Suman et al., 2016; Tornberg, 2005) that begins to change in color at approximately 44C (Davey et al., 1974). Roldan et al.

(2013) reported that myoglobin begins to denature between 55C and 65C and continues until reaching 75C or 80C, aligning with the current results of statistical and visual degree of redness differences between 55C and 70C. Therefore, these results are in accordance with the findings of other sous vide studies: Vaudagna et al. (2002) cooked beef at 50-65C for as long 12 h.;García-Segovia et al. (2007) cooked beef samples at 60-

80C from 15-60 min; and Roldan et al. (2013) cooked lamb loins at 60-80C for 6-24 h, that increasing sous vide temperature and time results in an increase of the degree of doneness due to myoglobin denaturation. Additionally, other methods of cooking can influence the rate of internal cooked color. Yancey et al. (2011) compared the internal color of beef steaks cooked using various cooking methods: convection oven, clam-shell grill, gas fired grill, and electric griddle. Steaks cooked via convection oven resulted in the reddest internal color, whereas the clam-shell grill presented the least redness (brown in color). Thus, cooking method, temperature, and time can all impact the development of internal color during cooking.

Unlike L* and a* values, a two-way interaction was found for yellowness (b*) in

Table 4.2 for temperature x time (P < 0.001). Internal b* (yellowness) values decreased as cooking temperature and time both increased. Roasts that were cooked less than 6 h at temperatures ≤ 60C had significantly higher b* (increased yellowness) values than those cooked longer than 2 h at temperatures ≥65C Other authors have reported just the opposite: higher b* values with increasing temperature and time (Botinestean et al., 2016;

Roldan et al., 2013; Christensen et al., 2011; García-Segovia et al., 2007). However, the relationship of the degree in yellowness to quality and sensory attributes is not well understood (Botinestean et al., 2016).

Warner-Bratzler shear force

Results for Warner-Bratzler shear force values (WBSF) on the effects of age x temperature (P = 0.028) can be found in Figure 4.2. There were no statistical differences in tenderness when comparing fed and cow at temperatures of 55, 65 and 70C. Fed ST roasts had a significantly higher WBSF value at 60C (4.30 kg) than ST roasts from cows cooked at 60C (3.29 kg; P = 0.049), resulting in a 1.01 kg difference. It is suggested that this small numerical difference in tenderness would not be detected by consumers.

Although, no significant differences were found in tenderness, when the four tenderness classifications (very tender, tender, intermediate, and tough) was applied to the current study, both fed and cow age groups resulted below the threshold of “tough” (>4.6 kg)

(Beef-Checkoff, 2016; Igo et al., 2015; Belew et al., 2003). Therefore, it can be concluded that sous vide cooking prevents the effects of toughening during cooking, and value can be added to tough cuts of meat, like the ST, through sous vide cooking.

Soluble protein concentration of cooked liquid

Results for the effects of temperature x time on soluble and total protein concentrations are found in Table 4.3. Overall, a decrease in soluble concentrations occurred as temperature increased (P < 0.001). However, time had a more complex effect as soluble concentrations decreased between 2 and 4 h (P < 0.001). As temperature and time increased, the soluble protein fraction decreased in concentration, with the greatest concentrations occurring at 55C for 2, 4, 6, and 8 h when compared to all other temperature and time points (P < 0.05). During cooking, different proteins denature at different rates, and cause shrinkage of muscle fibers as well as interact with other proteins (Tornberg, 2005). Davey et al. (1974) concluded that the aggregation of

myofibrillar and sarcoplasmic proteins during cooking might contribute to the formation of a gel within the structural elements of the meat and effecting the consistency of the meat (Tornberg, 2005). It could be suggested that the decrease in protein solubility in the cook liquid as temperature and time increases is due to the aggregation of proteins within the muscle fiber. However, the effect of temperature and time on soluble protein found within the cook liquid is unclear, as there has been no previous research on this topic.

Total protein concentration of cooked liquid

Statistical analysis resulted in a three-way interaction for total protein concentration in cooked liquid on the effects of age x temperature x time (P = 0.021) represented in Figure 4.3. ST roasts cooked at 70C for 4 and 8 h had significantly lower total protein concentration than 55C for 2, 4, and 6 h, and 60C for 2, 4, and 8 h (P <

0.05). Furthermore, fed ST roasts cooked at 70C for 8 h exhibited significantly lower concentrations when compared to fed ST: at 55C for 2, 4, 6 h, 60C for 2, 4, and 8 h,

65C for 8 h; and cow ST: at 55C for 2 and 6 h, and 60C for 2 and 8 h (P < 0.05).

Decreasing total protein concentrations could be due to an aggregation of proteins within the meat structure as temperature and time increases. Another explanation for total protein concentration decreasing within the cooked liquid may be due to a dilution effect.

An increase in the expulsion of water from the meat as temperature and time increases, causes the concentration of protein to become diluted with increased water. As stated in the previously, the effects of cooking on the proteins within the cook liquid is unknown as there has been no previous research on cook liquid from sous vide.

Total protein concentration of cooked muscle tissue

Figure 4.4 shows total protein concentrations of the cooked muscle tissue on the effect of temperature (P = 0.005) No two-way or three-way interactions were significant

(P > 0.05). As temperature increased the total protein concentration of the cooked tissue revealed a slight decrease. There were no significant differences between 55C and 60C, nor 65C and 70C. Yet, there was a significant decrease in total protein concentration of the cooked ST between 60C and 65C (P < 0.027). Altogether, cooked roasts originating from fed beef had a higher concentration of total protein than cow (P = 0.001). Therefore, protein concentrations within the ST decreases which suggests that proteins denature with increasing temperatures, while some proteins may also be expelled out of the meat.

SDS-PAGE

Soluble proteins of the cook liquid

Figure 4.5 is a representative SDS-PAGE gel image of fed and cow ST soluble proteins within the cooked liquid at different temperature and time points. A total of 8 protein bands were detected in a range of 20 – 200 kDa. Although molecular weights were determined by TotalLab software, proteins cannot be accurately identified based strictly off molecular weight. The SDS-PAGE protein band patterns showed that soluble proteins were affected by temperature and time. As temperatures increased above 60C, protein bands begin to weaken or disappear. Protein bands with molecular weights of 87,

80 and 70 kDa disappear at higher temperatures of 65 and 70C for cooking times greater than 2 h. In contrast, the 54, 31 and 26 kDa protein bands begin to fade at lower temperatures. The 54 kDa band disappears after 2 h at 55C, the 31 kDa band disappears after 6 h at 60C, and the 26 kDa band fades at 6 h at 55C thus, suggesting that all three

proteins might be sensitive when exposed to heat for long periods of time as well as high heat at shorter cooking times. An alternative explanation could be that these proteins are not released with the cook loss at higher temperatures due to protein aggregation with other proteins within the muscle fibers. However, it can be hypothesized that the 8 protein bands found in the cook liquid might play a role in predicting tenderness during cooking. Further research is need to determine the identity of each protein in order to accurately conclude the effects of these proteins might have on meat.

Total proteins from cooked muscle tissue

Figure 4.6 is a representative SDS-PAGE gel image of the total proteins from fed and cow cooked ST roast whole muscle at different temperature and time points. A total of 20 bands were detected in a range of 162 – 17 kDa. The SDS-PAGE gel pattern suggests that there are no differences across temperature and time points. However, bands with molecular weights 45, 38, 35, 20, and 17 kDa are suggested to be actin, troponin T, tropomyosin, troponin C and myoglobin, respectively.

Conclusion

In the present study, the effects of increased temperature and time appear to play a major role in beef semitendinosus roasts regardless of age group (fed vs. cow). Beef roasts cooked at increased temperature and time, increased cooking loss (%), L*

(lightness) and b* (yellowness) values, while decreasing a* (redness) values. With the changes in color during cooking, due to the denaturation of myoglobin, consumers can easily mistake degree of doneness regardless of internal temperature. Regardless of age group (fed vs. cow), ST roasts resulted in little to no difference in tenderness as cooking

temperature increased. However, ST roasts representing both age groups met consumer acceptability in tenderness based on previous tenderness surveys (Beef-Checkoff, 2016;

Igo et al., 2015).

Overall, the current study exhibits advantages in sous vide cooking as a method to add value (tenderness) to an undervalued muscle cut, such as semitendinosus originating from cows.

Chapter 5

Conclusions and Future Directions

Results from the preliminary experiment concluded that prolonged cooking times up to 30 h resulted in an improvement of tenderness. Therefore, suggesting that sous vide cooking times between 2 and 10 h at 55C would provide consumers with a tender product while still maintaining cooking loss and a consistent degree of doneness.

However, results from the main study contradicted the results from the primary experiment, as no improvements in tenderness were detected. These contradicting experiments could be due to small sample sizes, specifically in the pilot study, not accurately representing an industry scale.

Collectively, the experimental studies conducted does support that sous vide can be an alternative cooking method that is capable of transforming tough cuts of beef into tender products. Therefore, we can add value to tough, undervalued cuts of beef like cow semitendinosus muscles.

Future research should investigate the effects of prolonged sous vide cooking times at low temperatures on sensory characteristics and consumer acceptability on tenderness. In addition, future research should continue to investigate meat protein found in the cook liquid. Identification of these proteins could lead to understanding the effects of cooking on meat structure, tenderness, cooked color or sensory attributes of cooked meat.

Table 4.1. Simple means of uncooked semitendinosus (ST) weights and instrumental color of age1 groups. Age1 Group Fed Cow Trait n2 Mean SD3 Min Max Mean SD3 Min Max P-value Whole Wt. (g) 40 2614.5 410.1 1569.4 3506.3 2539.3 158.2 2109.2 2771.5 0.463 ST Roast (g) 40 537.9a 94.6 280.7 864.5 498.9b 51.9 303.0 620.5 0.002 L*4 40 41.2 2.5 35.7 46.4 41.4 2.7 37.5 51.2 0.685 a*5 40 27.6b 1.5 24.0 30.5 29.7a 1.2 26.8 32.8 <0.001 b*6 40 10.3b 1.2 7.3 12.5 11.9a 1.1 10.0 15.1 <0.001 a,b Means within a row with different superscript are significantly different (P < 0.05). 1 Age = Fed (< 30 mo.); Cow ( 42 mo.) at time of harvest. 2 n = Number of observations 3 SD = Standard deviation of the mean 4 L* = Lightness 5 a* = Redness 6 b* = Yellowness

35 a a

30 b b bc 25 cd de de de 20 efg ef efg fg efg 2 h g g 4 h 15 6 h

Cooking Loss (%) Cooking 10 8 h

0 55 60 65 70 Temperature (°C)

Figure 4.1. Least-square means on the effects of temperature and time on percent cooking loss. a-gMeans without a common letter are significantly different (P < 0.05).

Table 4.2. Least-square means on the effects of temperature1 and time on cooked instrumental color parameters of age2. Age2 Group Fed Cow Fed Cow Fed Cow L*3 a*4 b*5 Temp.1 (C) Time (h.) (SEM6 = 0.97) (SEM6 =0.81) (SEM6 =0.26) 55 2 50.35e,z 53.38a,xyz 28.16a,m 26.07a,mnop 11.84ab 10.97abcd 4 53.44cde,xyz 55.52a,vwxyz 27.48ab,mn 25.39a,mnop 12.35a 11.55abc 6 52.64de,yz 56.56a,vwxy 26.52ab,mno 24.40ab,mnopq 11.36abc 11.17abcd 8 54.55abcd,vwxyz 54.30a,vwxyz 25.03abc,mnopq 25.42a,mnop 11.88ab 10.85abcd

60 2 53.45cde,xyz 54.94a,vwxyz 27.10ab,mn 25.17a,mnopq 11.76ab 11.62ab 4 54.89abcd,vwxyz 56.82a,vwxy 24.64abcd,mnopq 22.57abcd,opqrs 11.64ab 10.82abcd 6 55.21abcd,vwxyz 56.78a,vwxy 22.04bcd,opqrst 20.89bcde,qrstu 10.90bc 10.58abcd 8 54.09bcde,wxyz 57.25a,vwxy 23.93abcd,mnopqr 19.62cde,rstuv 11.15abc 10.36cd

65 2 57.58abcd,vwxy 55.40a,vwxyz 21.62cd,pqrst 23.43abc,nopqr 11.24abc 11.84a 4 59.08ab,vw 56.00a,vwxy 16.87ef,uvwxy 18.14def,stuvw 10.49bc 11.02abcd 6 59.20ab,vw 56.59a,vwxy 15.45efg,vwxyz 14.52fgh,wxyz 10.08c 10.40bcd 8 59.62a,v 56.58a,vwxy 13.60fg,xyz 14.36gh,wxyz 9.95c 10.11cd

70 2 58.24abc,vwx 56.64a,vwxy 18.70de,stuvw 17.62efg,tuvwx 11.32abc 10.73abcd 4 58.36abc,vwx 55.91a,vwxy 13.54fg,xyz 12.65h,yz 9.92c 9.78d 6 59.25ab,vw 56.13a,vwxy 11.45g,z 12.47h,yz 10.07c 10.09d 8 58.67bc,vwx 55.54a,vwxyz 11.03g,z 11.64h,z 10.05c 9.97d SEM7 0.97 0.97 0.81 0.81 0.26 0.26 P-value 0.006 0.020 0.477 a-h Means within a column with different superscript letters are significantly different between the temperature x time (P < 0.05) m-z Means with different superscript letters are significantly different between the age x temperature x time (P < 0.05). 1 Age = Fed (< 30 mo.); Cow ( 42 mo.) at time of harvest. 2 Temp. = Temperature (C). 3 L* = Lightness 4 a* = Redness 5 b* = Yellowness 6 SEM = Standard error of the mean within the age x temperature x time interaction. 7 SEM = Standard error of the mean within the column representing the temperature x time interaction.

a 4.30 4.30 4.09 4.5 4.09

4.04 4 3.80 b 3.67 3.29 3.5 WBSF(kg)

2.5 55 60 65 70 Temperature (°C) Fed Cow

Figure 4.2. Least-square means on the effects of age and temperature (C) on Warner-Bratzler shear force (WBSF) values. a-bMeans without a common letter are significantly different (P < 0.05).

Table 4.3. Least-square means on the effects of temperature1 and time on soluble2 and total3 protein concentrations of the cooked liquid. Trait Temp.1 Time Soluble2 Total3

(C) (h.) (mg/mL) (mg/mL)

2 52.41a 60.19a

4 28.49c 59.66ab 55 6 25.59cd 56.50ab

8 33.24b 43.04abcd

2 20.89de 59.41ab

4 14.34fgh 47.51abc 60 6 12.79fghi 34.74bcd

8 16.74ef 56.68ab

2 12.53fghi 26.55cd

4 11.46ghi 27.98cd 65 6 10.77hi 38.56bcd

8 10.82hi 45.78abcd

2 16.04fg 33.31cd i d 4 9.06 19.42 70 6 9.33i 40.34abcd 8 11.58ghi 22.05d

SEM4 0.96 4.72

P-value <0.001 0.002 a-i Means within a column with different superscript letters are significantly different between the temperature x time (P < 0.05). 1 Soluble protein concentration of the cook liquid 2 Total protein concentration (soluble and insoluble proteins) of the cook liquid 3 Temp. = Temperature (C) 4 SEM = Standard error of the mean within the column.

90 a 80 ab ab ab ab 70 ab ab ab ab ab 60 abc abcd abcd abcd abcd abcd abcd abcd 50 bcd bcd bcd bcd 2 h 40 bcd bcd bcd bcd bcd bcd bcd 4 h 30 cd bcd d 6 h 20 8 h

10 Total Protein Concentration Protein (mg/mL) Concentration Total 0

Age/Temperature (°C)

Figure 4.3. Least-square means on the effects of age, temperature and time on total (soluble and insoluble) protein concentration of the cooked liquid. a-dMeans without a common letter are significantly different (P < 0.05).

20.00 a 18.00 a

16.00 b b 14.00

12.00

10.00

8.00 Total Protein Concentration Protein (%) Concentration Total 6.00 55 60 65 70 Temperature (°C)

Figure 4.4. Least-square means on the effects of temperature on total (soluble and insoluble) protein concentration of the cooked whole ST muscle. a-bMeans without a common letter are significantly different (P < 0.05)

Figure 4.5. SDS-PAGE gel image of soluble proteins extracted from the cooked liquid at different temperature and time points.

The image is a representation of both age groups (fed and cow) of the soluble protein fraction collected from the cooked liquid at different cooking temperatures (55, 60, 65 and 70C) at times (2, 4, 6, 8 h).

Figure 4.6. SDS-PAGE gel image of muscle proteins from cooked ST roasts at different temperature and time points.

The image is a representation of both age groups (fed and cow) of muscle proteins from the cooked ST muscle at different cooking temperatures (55, 60, 65 and 70C) at times (2, 4, 6, 8 h).

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