Citation
Sous Vide Beef Cookery

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Title:
Sous Vide Beef Cookery
Creator:
Griffing, Derek A
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (9 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Animal Sciences
Committee Chair:
CARR,CHARLES C
Committee Co-Chair:
JOHNSON,DWAIN
Committee Members:
SCHNEIDER,KEITH R
Graduation Date:
8/9/2014

Subjects

Subjects / Keywords:
Beef ( jstor )
Collagens ( jstor )
Cooking ( jstor )
Flavors ( jstor )
Food ( jstor )
Food preparation ( jstor )
Low temperature ( jstor )
Meats ( jstor )
Steak ( jstor )
Water temperature ( jstor )
Animal Sciences -- Dissertations, Academic -- UF
cooking -- cow -- grilling -- ltlt -- mechanical-tenderization -- semitendinosus -- sous-vide -- tenderness
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Animal Sciences thesis, M.S.

Notes

Abstract:
The objective of this research was to evaluate the effect of sous vide cooking on dairy cow semitendinosus muscles (ST) (IMPS # 171C). Each of the ST muscles (n = 50) was further fabricated into steaks according to the specified, randomly assigned treatment in one of three experiments. Experiment 1 indicated steaks sous vide cooked at 62.8 degrees celsius had lower SSF values (P < 0.03) than all other treatment steaks cooked to 51.7 degrees celsius or 57.2 degrees celsius and had numerically lower SSF values than grilled steaks at the same cooking temperature. Steaks sous vide cooked to 62.8 degrees celsius exhibited the greatest percentage of solubilized collagen (P less than or equal to 0.04). As sous vide cooking temperature increased (P less than or equal to 0.04), there was a linear decrease in the moisture percentage. The 51.7 degrees celsius treatments exhibited the lowest fat percentages (P less than or equal to 0.04). Experiment 2 showed mechanically tenderized steaks had lower SSF values (P less than or equal to 0.01) than intact steaks at 51.7 degrees celsius and 57.2 degrees celsius, and an increase in cooking loss was observed (P less than or equal to 0.01) as cooking temperature increased, regardless of mechanical tenderization. The trained sensory panel evaluation complemented our findings for the slice shear force and cook loss analysis. Experiment 3 revealed steaks subjected to the I/62.8 degrees celsius treatment had the lowest (P less than or equal to 0.04) SSF values. All sous vide par-cooked treatments had a greater (P less than or equal to 0.04) cooking loss percentage when compared to all grilled steak treatments. Based on the trained sensory panel evaluations, juiciness was the only category that was significantly different across all treatments (P < 0.01). ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2014.
Local:
Adviser: CARR,CHARLES C.
Local:
Co-adviser: JOHNSON,DWAIN.
Statement of Responsibility:
by Derek A Griffing.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Resource Identifier:
968131572 ( OCLC )
Classification:
LD1780 2014 ( lcc )

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1 SOUS VIDE BEEF COOKERY B y DEREK ANDREW GRIFFING A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2014

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2 © 2014 Derek Andrew Griffing

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3 To my family and loved ones

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4 ACKNOWLEDGMENTS First , I would like to thank the members of my committee including my committee chair Dr. Chad Carr, Dr. Dwain Johnson, and Dr. Keith Schneider for their guidance both personally and professionally. I am extremely grateful to Dr. Chad Carr for all he has done for me during leadership and communication skills. Next, I want to take this opportunity to thank all of the graduate and undergraduate students, as well as faculty at the University of Florida for your help with my research. Specifically, the support I have received from Tommy Estevez, Byron Davis, Ryan Dijkhuis, Larry E ubanks, and all members of the UF meat processing crew has been unwavering. Without their help, I would not have been able to complete my research nor would I have been able to teach students about the fundamentals of meats judging . I am especially glad to have made a friend and fishing buddy in Tommy Estevez. Also, I would like to extend my gratitude to Dr. Gretchen Mafi for her guidance and continued support. She believed in my abilities as a person and encouraged me to take on new challenges. She has bee n an important person in my life and I would not have had many of my opportunities without her. Lastly, and most importantly, I must thank my family and loved ones who have surrounded me with encouragement and love. Their support has pushed me broaden my h orizons and to achieve my goals. I am very blessed to have them in my life.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 7 LIST OF FIGURES ................................ ................................ ................................ ......................... 8 ABSTRACT ................................ ................................ ................................ ................................ ..... 9 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................. 11 2 REVIEW OF LITERATURE ................................ ................................ ................................ . 14 Muscle Characteristics and Factors Effecting Palatability ................................ ..................... 14 Traditi onal Cooking Effects on Whole Muscle Palatability ................................ .................. 16 Traditional Cooking Methods ................................ ................................ .......................... 16 Tenderness ................................ ................................ ................................ ....................... 16 Cook Loss and Juiciness ................................ ................................ ................................ .. 18 Flavor ................................ ................................ ................................ ............................... 19 Sous vide Cooking Method ................................ ................................ ................................ .... 19 Mechanism of Tenderization ................................ ................................ ........................... 20 Time and Temperature Effects ................................ ................................ ........................ 20 Cook Loss ................................ ................................ ................................ ........................ 21 Flavor ................................ ................................ ................................ ............................... 22 Color ................................ ................................ ................................ ................................ 22 Food Safety ................................ ................................ ................................ ............................. 23 Pathogens of Concern ................................ ................................ ................................ ...... 23 Salmonella ................................ ................................ ................................ ................ 23 Escherichia coli O157:H7 ................................ ................................ ........................ 24 Listeria monocytogenes ................................ ................................ ............................ 24 Clostridium perfringens ................................ ................................ ........................... 25 Par Cooking ................................ ................................ ................................ ............................ 26 Tenderness ................................ ................................ ................................ ....................... 26 Cook Loss ................................ ................................ ................................ ........................ 26 Mechanical Tenderization ................................ ................................ ................................ ...... 27 Tenderness ................................ ................................ ................................ ....................... 28 Cook loss ................................ ................................ ................................ ......................... 28 Food Safety ................................ ................................ ................................ ...................... 28 3 SOUS VI DE BEEF COOKERY ................................ ................................ ............................ 30 Materials and Methods ................................ ................................ ................................ ........... 30

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6 Raw Samples ................................ ................................ ................................ ................... 30 Sampl e Preparation ................................ ................................ ................................ .......... 30 Experiment 1: The Effect of Cooking Method and Endpoint Temperature on Characteristics of Cow Semitendinosus Steaks ................................ ........................... 31 Thermal processing ................................ ................................ ................................ .. 31 Slice shear force analysis ................................ ................................ ......................... 32 Moisture analysis ................................ ................................ ................................ ...... 32 Fat analysis ................................ ................................ ................................ ............... 33 Collagen analysis ................................ ................................ ................................ ...... 33 Experiment 2: The Effects of Mechanical Tenderization on Sous vide Cooked, Cow Semitendinosus Steaks. ................................ ................................ ................................ 35 Thermal processing ................................ ................................ ................................ .. 35 Cook loss analysis ................................ ................................ ................................ .... 35 Slice shear force analysis ................................ ................................ ......................... 35 Trained sensory evaluation ................................ ................................ ....................... 35 Experiment 3: Sous vide Par cooking ................................ ................................ ............. 36 Thermal processing ................................ ................................ ................................ .. 36 Cook loss analysis ................................ ................................ ................................ .... 37 Slice shear force analysis ................................ ................................ ......................... 38 Trained sensory evaluation ................................ ................................ ....................... 38 Statistical Analysis ................................ ................................ ................................ .......... 38 Results and Discus sion ................................ ................................ ................................ ........... 39 Experiment 1: The Effect of Cooking Method and Endpoint Temperature on Characteristics of Cow Semitendinosus Steaks. ................................ .......................... 39 Slice shear force and collagen analysis ................................ ................................ .... 39 Moisture and fat ................................ ................................ ................................ ....... 40 Experiment 2: The Effects of Mechanical Tenderization on Sous vid e Cooked, Cow Semitendinosus Steaks. ................................ ................................ ................................ 41 Slice shear force and cook loss ................................ ................................ ................ 41 Trained sensory panel ................................ ................................ ............................... 42 Experiment 3: Sous vide Par cooking ................................ ................................ ............. 43 Slice shear force and cook loss ................................ ................................ ................ 43 Trained sensory pa nel ................................ ................................ ............................... 44 Implications ................................ ................................ ................................ ............................ 45 LIST OF REFERENCES ................................ ................................ ................................ ............... 54 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ......... 63

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7 LIST OF TABLES Table page 3 1 Experiment 1 treatments for cow semitendinosus steaks. ................................ ................. 46 3 2 Effect of cooking method and endpoint temperature on collagen, moisture, and fat characteristics on cow semitendinosus steaks 1 ................................ ................................ .. 49 3 3 Effect of mechanical tenderization on Slice Sh ear Force and cook loss on sous vide cooked 1 cow semitendinosus steaks 2 ................................ ................................ ................. 50 3 4 Trained sensory panel values for mechanically tenderized, sous vide cooked 1 cow semitendinosus steaks 2 . ................................ ................................ ................................ ...... 51 3 5 Effect of sous vide par cooking 1 on Slice Shear Force and cook loss on cow semitendinosus steaks 2 . ................................ ................................ ................................ ...... 52 3 6 Trained sensory panel va lues for sous vide par cooked 1 cow semitendinosus steaks 2 . ..... 53

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8 LIST OF FIGURES Figure page 3 1 Experiment 2 and 3 sample preparation. ................................ ................................ ............ 47 3 2 Effect of cooking method and endpoint temperature on Slice Shear Force values of cow semitendinosus steaks. ................................ ................................ ............................... 48

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9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science SOUS VIDE BEEF COOKERY By Derek Andrew Griffing August 2014 Chair: Chad Carr Major: Animal Sciences The objective of this research was to evaluate the effect of sous vide cooking on dairy cow semitendinosus muscles (ST) (IMPS # 171C) . Each of the ST muscles (n = 50) was further fabricated into steaks according to the specified, randomly assigned treatment in one of three experiments. Experiment 1 indicated steaks sous vide cooked at 62.8°C had lower SSF values (P < 0.03) than all other treatment steaks cooked to 51.7 °C or 57.2 °C and had numerically lower SSF values than grilled steaks at the same cooking temperature. S teaks sous vide cooked to 62.8°C exhibited the greatest percentage of As sous vide cooking . T he 51.7°C treatments ). Experiment 2 showed m echanically tenderiz 57.2°C , and an increased, regardless of mechanical tenderization. The trained sensory panel evaluation comple ment ed our findings for the slice shear force and cook loss analysis. Experiment 3 revealed s . A ll sous vide par e when

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10 compared to all grilled steak treatments . B ased on the trained sensory panel evaluations, juiciness was the only category that was significantly different across all treatments (P < 0.01).

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11 CHAPTER 1 INTRODUCTION Mechanical tenderization and need le injection are processes that are utilized extensively in the United States to improve the palatability of whole mus cle meat. An estimated 10.5% of fresh beef product sold in the United States is mechanically tenderized totaling to approximately 2.6 billion pounds (Muth et al. , 2012). An additional 5.3% has been needle injected or marinated (Muth et al., 2012). Drastic changes are expected to occur in the food service industry on January 1, 2016 as it relates to the cooking of mechanically tenderized beef. S ince the year 2000, six outbreaks resulting in 176 illnesses with 32 hospitalizations and one death were linked to mechanically tenderized beef products. Furthermore, follow up investigations revealed that the contributing factor to these illnesses was fai lure to fully cook the mechanically tenderized beef products. As a result, on Monday, June 10, 2013, the U.S. Department of Agriculture Food Safety and Inspection Service (FSIS) submitted a proposed issuance of rules and regulations ignation for Needle or Blade Tenderized (Mechanically Tenderized) following changes would occur as it relates to the cooking of non intact beef cuts: Mandatory with an accurate description of the beef component with both using the same style, color, and size on a single color contrasting background. The inclusion of validated cooking inst ructions on labels of raw or partially cooked mechanically tenderized beef products destined for use in homes, restaurants, or similar institutions specifying a method of cooking, minimum internal temperature parameters , and a dwell time prior to consumpt ion to ensure the non intact beef product is fully cooked. Raw or partially cooked mechanically tenderized beef products destined to be fully cooked at an official establishment would not be required to have the descriptive designation on the label. For th e validated cooking instructions, the establishment would be required to obtain scientific and technical support for the judgments made in designing the cooking

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12 instructions and validation proving that the establishment is achieving the critical operationa l parameters as documented by the scientific and technical support. Currently, the guidance for validated cooking instructions draws heavily f rom studies by Luchansky et al. (2011; 2012) on Shiga toxin producing O157:H7 and n on O157:H7 Escherichia coli cells within blade tenderized beef steaks after cooking on a commercial open flame gas grill. Lu chansky et al. (2012) reported a 2.0 to 4.1 log CFU/g and 1.5 to 4.5 log CFU/g reductions in ECOH and STEC levels when cooked to internal temperatures of 48.9, 54.4, 60.0, 65.6, and 71.1°C; however, even after reaching an internal temperature of 71.1°C, some cells were still present. Furthermore, as reported by Goodfellow and Brown (1978), the FSIS is recommending on its website to cook mechanically tend erized beef products to an internal temperature of 62.8°C with a 3 min dwell time as this will result in a 5.0 log unit reduction of Salmonella . As of 2011, an estimated 555 beef processing facilities in the U.S. utilize mechanical tenderization (Muth et a l., 2012). Not only would the proposed rule would have an effect on domestic processing establishments, but there would be a global effect on the beef processing industry as foreign establishments that manufacture and export mechanically tenderized be ef pr oducts to the U.S. would have to follow the designated labeling requirements. If the proposed rule were to become final, the estimated total cost to the food processing industry for label changes would be 1.57 million dollars as the labels would include th designation as well as designation relating to enhancement if the beef pro duct had added solutions (Muth et al. , 2012). Furthermore, the estimated cost to the food service industry for the validation studies of the cooking param eters could range in costs from 5,000 to 10,000 dollars per product line with a single fo rmulation (Muth et al., 2012). Labeling compliance would be mandatory on January 1, 2016 as it would be included in the new meat and poultry product

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13 labeling regulatio ns that occur in 2 year increments. Moreover, it is expected that the final mechanically tenderized beef rule will be implemented during the same time period. Under the proposed rules, consumers would not be able to order mechanically tenderized beef produ establishments would be required to heat mechanically tenderized beef products to an internal temperature of 71.1°C or 62.8°C with a 3 min hold as stated by the FSIS . Consum ers prefer steaks cooked to a lower degree of doneness as juiciness and tenderness scores decreased as the degree of doneness increased (Lorenzen et al., 1999; Neely et al., 1999). Furthermore, a survey comprising of 3, 554 consumers, spanning nine restaura nts revealed that the average consumer ordered their steaks cooked to medium, and that the consumers believed steaks cooked to rare or medium rare degrees of doneness were more tender and flavorful leading to greater overall satisfaction and an overall hig her intent to repurchase (Cox et al., 1996). Previous research by Creed (2001) has indicated the sous vide method can provide many opportunities to satisfy several groups of consumers with regard to nutritional, sensory, convenience and safety requirements . Thus, to meet consumer needs in the food service industry, the use of previously under utilized, innovative cooking methods such as sous vide par cooking should be evaluated as an alternative to mechanical tenderization to ensure acceptable tenderness in less palatable beef steaks and roasts. Therefore, the objective of this research was to evaluate sous vide cooking as a comparable alternative to mechanical tenderization.

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14 CHAPTER 2 REVIEW OF LITERATURE Muscle Characteristics and Factors Effecting Pala tability Anatomically, muscles vary in composition and use. Muscles of locomotion such as the bovine semitendinosus that is used for forward movement are compositionally different than muscles of support such as the bovine psoas major or longissimus dorsi found in the loin and rib. Research has indicated some variable fiber type composition within each primal as the bovine semitendinosus is composed of type I fibers in its deepest portions and type IIB fibers in the most outer regions (Totland et al. , 1988) . Furthermore, the bovine semitendinosus and longissimus dorsi are both highly glycolytic in nature despite their differences in anatomical locations and functions (Kirchofer et al. , 2002). Nonetheless, muscles of locomotion are less tender than muscles of support specifically when comparing round subprimals and rib and loin subprimals as indicated by data from three national beef tenderness surveys (Brooks et al., 2000; Guelker et al., 2013; Morgan et al., 1991). Morgan e t al. (1991) reported 56.4% of semi tendinosus steaks had Warner Bratzler shear (WBS) force values greater than 4.6 kg, the threshold WBS value the average consumer associates as a tough steak. In the sub sequent survey by Brooks et al. (2000), 55.9% of semitendinosus steaks had a WBS force s core greater than 3.9 kg as compared to ribeye and top loin steaks which had no values greater than 3.0 kg. And in th e 2010 survey by Guelker et al. ratings and the lowest tenderness level r atings. A major component contributing to the toughness of bovine semitendinosus is connective tissue as it influences the qualitative characteristics of meat; specifically related to background tenderness (Forrest et al. , 1975). Connective tissue surround s muscle at every level of organization; around the entire muscle as epimysium, around the muscle bundle as perimysium,

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15 and around the muscle fiber as endomysium. The bovine semitendinosus contains almost 12 mg/g of collagen (Jones et al., 2004). As the an imal ages, the percentage of soluble collagen in the bovine semitendinosus significantly decreases leading to a greater percentage of stronger, more stable crosslinks known as insoluble collagen (Nishimura et al. , 1996). As a result, the toughness of bovin e semitendinosus increases linearly with age; doubling from 0 months to 32 months of age (Nishimura et al., 1996). The bovine semitendinosus has the same amount of fat as the leanest muscle in the chuck and half the amount of fat as the longissimus dorsi (Jones et al., 2004). The lower percentage of intramuscular adipose tissue directly influences the toughness of the bovine semitendinosus as intramuscular adipose tissue weakens the structure of perimysial connective tissue by breaking away the collagen bu ndles as well providing lubricat ion during mastication (Brooks and Savell, 2004; Nishimura et al. , 1999). Juiciness is a function of both moisture content and intramuscular fat. Moreover, muscles from chuck and loin, with the exception of the gluteus med ius, tend to exhibit greater juiciness than those from the round (Carmack et al., 1995). The bovine semitendinosus is similar to most ot her lean beef cuts at 73% water; and when combined with lack of intramuscular fat, the cooked bovine semitendinosus is p erceived as slig htly dry (Jones et al., 2004). Furthermore, in the 2010 National Beef Tenderness Survey, cuts from the bottom and top round received the lowest 3 and 5.2 respectively for juiciness level on a 10 point scale (Guelker et al., 2013). Additionally, the bovine semitendinosus was ranked as the blandest muscle with a high degree of off f lavor (Brickler, 2000; Calkins and Hodgen, 2007).

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16 Traditional Cooki ng Effects on Whole Muscle Palatability Traditional Cooking Methods Dry heat cookery and moist heat cookery are considered traditional cooking methods. Furthermore, grilling is often depicted as the conventional dry heat cooking method; while braising, sim mering, and stewing are considered conventional moist heat cookery methods. In an in home, beef top round cookery survey spanning four major American cities, consumers in Philadelphia, San Francisco, and Houston preferred dry heat cookery such as grilling and pan frying as opposed to Chicago which preferred simmering and stewing (Neely et al., 1999). Moreover, these cooking methods can be further subdivided into long time, low temperature or short time, high temperature. Dry heat cookery for a short time at a high temperature is often discouraged for cuts with high connective tissue content due to the negative effects on palatability such as myofibrillar protein hardening. However, dry heat cookery at a low temperature for a long period of time has been show n to improve the palatability of cuts from the round as compared to water added, moist heat cookery in an oven with a subsequent dry heat finish at 260°C, which produced the highest percentage of undesirable roasts with the lowest ratings for initial and o verall tenderness, juiciness, and overall palatability (Jeremiah and Gibson, 2003). Thus, both dry heat and moist heat cooking methods influence the palatability of muscles from the round depending on both the time and temperature of the cookery. Tendernes s Resear ch has indicated over 50% of consumer s believe tenderness is the most important attribute, over flavor or juiciness, when consuming a steak in home or at a foodservice establishment (Huffman et al., 1996). Cooking can positively or negatively affec t the tenderness of meat depending on the composition of the meat and as a result of the subsequent changes that take place due to cooking (Forrest et al., 1975).

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17 Kolle et al. (2004) showed semitendinosus steaks cooked to an internal temperature of 71°C us ing moist heat cookery, water added to the bottom of aluminum pan to produce a moist environment in forced air convection oven set to a temperature of 93°C, were more tender as shown by a greater WBS force percentage reduction as compared to semitendinosus steaks cooked to an internal temperature of 71°C using dry heat cookery; a clam shell grill set to a surface temperature of 189°C. Previous research on the bovine semitendinosus by Christensen et al. (1999) linked the effect of cooking temperature on the mechanical properties of meat quality. Some changes will occur rapidly, while other changes are much slower. As a result of heat, muscle fibers will rapidly shrink transversely and lo ngitudinally beginning at 35 40 °C with a linear increase up to 80 °C (Bald win, 2012). The slow changes improve tenderness by dissipating collagen into gelatin and reducing muscle fiber adhesion to surrounding connective tissue with d enaturation increasing above 55 °C (Baldwin, 2012). Lastly, in regard to enzymatic activity, high heat inactivates the enzymes responsible for myofibrillar degradation (Forrest et al., 1975). Proteolytic enzymes are stable between the temperatures of 20 35°C; and as the temperature increases above 40°C, a rapid decrease in activity will occur leading t o minimal activity at 60°C (Whitaker, 1996). The degree of doneness refers to the end point temperature of meat. The beef steak color guide published by the American Meat Science Association (1995) lists the degrees of doneness and corresponding end point temperatures for beef steaks: very rare 55°C, rare 60°C, medium rare 63°C, medium 71°C, well done 77°C, and very well done 82°C. The degree of doneness has a significant effect on palatability; as end point temperature increases from 60°C to 80°C, te nderness, juiciness, and overall acceptability all decrease while total cook loss dramatically

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18 increases (Parrish et al. , 1973). Steaks cooked to a very well done degree of don eness have almost 10 % greater cook loss values than steaks cooked to a rare degr ee of doneness (Parrish et al., 1973). Additionally, in home consumer evaluations from four major cities revealed steaks from the top round cooked to the degree of doneness medium rare or less received greater numerical tenderness ratings, juiciness rating s, and overall like ratings when compared to higher degrees of doneness ranging from medium to very well done (Neely et al., 1999). In addition to heat, time contributes to tenderness. Specifically when cooking the bovine semitendinosus, lower temperatures (56 58°C) showed the greatest impac t on tenderness after 60 min ; whereas higher temperatures (72 74°C) showed greatest impact on tenderness after just several minut es; however, as time increased with higher temperatures so did th e resistance to shear (Mac hlik and Draudt, 1963). The increased myofibrillar rigidity of meat cooked for a prolonged period of time at high temperatures is known as protein hardening. Cook L oss and Juiciness Moisture loss occurs due to evaporation and drip loss, and will increase with end point cooking temperature (Forrest et al., 1975). Jeremiah and Gibson (2003) showed eye of round roasts rapidly roasted to an internal temperature of 71°C in an oven at a temperature of 260°C with water added had a cook loss value of 8.1 % higher t han roasts cooked to 71°C in an oven at a temperature of 140°C with no moisture added. Furthermore, Bowers et al. (2012) and Obuz et al. (2004) further depicted that an increase in endpoint temperature led to lower cook yields regardless of cookery method. Thus, an increase in temperature results in greater cook loss due to moisture loss and fat solubilization. Time influences cook loss as well. Garcia Segovia et al. (2007) showed a significant increase in cook loss as time increased over a constant tempera ture using dry cookery.

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19 Flavor Numerous compounds contribu te to the flavor of beef. Water soluble components and lipids are the major precursors of meat flavor (Mottram, 1998). Moreover, lipids are responsible for specific species flavor as aldehydes are a major product of lipid degradation (Mottram, 1998). As a result of direct dry heat cookery; such as grilling, non enzymatic browning otherwise known as the Maillard reaction occurs between amino acids and reducing sugars as well as the thermal degradat ion of lipids resulting in the primary components tha t contribute to taste (Calkins and Hodgen, 2007; Mottram, 1998). The Maillard reaction will not occur without direct heat; thus, the meat taste will be bland. Sous vide Cooking Method The term sous vide Sous vide cooking is a non traditional long time, low temperature cooking method in which food is vacuumed sealed in heat stable, fo od grade plastic pouches and cooked fully submerged in a water bath using precisely controlle d heating (Baldwin, 2012). Thus, sous vide cooking is a cook in bag system utilizing variations of cook hold, cook serve, cook chill, or cook freeze technologies. As cited by Baldwin (2012) in reference to Church and Parsons (2000); vacuum sealing allows f or a more efficient transfer of heat from the water to the food, it eliminates the risk of recontamination during storage, and it inhibits off flavors from oxidation as well as prevents losses of flavor volatiles and moisture during cooking. Furthermore, s ous vide cooking allows for precise temperature control resulting in a highly reproducible product (Baldwin, 2012). As concluded by Vaudagna et al. (2002) the advantages related to palatability traits and microbiological stability make sous vide cooking a suitable process for adding value to a low cost raw material.

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20 Mechanism of Tenderization Current research by Christensen et al. (2013) and Suriaatmaja and Lanier (2014) on sous vide cooking has indicated that collagen solubilization is the primary mechanis m responsible for tenderness improvement. Work by Suriaatmaja and Lanier (2014) showed a clear reduction of toughness at temperatures above 51.5°C through the use of sous vide cookery. Previous research has also linked a significant reduction in connective tissue specifically between the temperatures 50°C to 60°C through the use of long time, time low temperature cookery (Laakkonen et al., 1970). At typical sous vide cooking temperatures between 55°C and 60°C most proteolytic enzymes are denatured (Tornberg , 2005). Furthermore, Christensen et al. (2013) showed the activity of cathepsins B and L in young bull and cow semitendinosus decreased with increased time and temperature. However, recent research has indicated slight catheptic ac tivity above 60°C (Chris tensen et al., 2011). Christensen et al. (2011) reported catheptic activity in sous vide slaughter pig and sow longissimus dorsi and semitendinosus cooked for a long period of time at a temperature of 63°C. Time and Temperature Effects The sous vide cook ing method is a variation of long time, low temperature moist heat cookery. Studies have indicated that both time and temperature are significant to achieving a reduction in toughness when using the sous vide cooking method; however, temperature is the mos t significant and should be kept high enough to solubilize collagen, but low enough to prevent myofibrillar hardening (Christensen et al., 2013; Garcia Segovia et al., 2007; Vaudagna et al., 2002). Current research by Suriaatmaja and Lanier (2014) revealed no increase in tenderness for meat samples sous vide at 50°C up to 30 h , but saw a significant difference in tenderness once the temperature was raised to 51.5°C; thus, temperature had the most significant impact on tenderness . Sous vide research by Rolda n et al. (2013) on lamb loins indicated a loss

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21 of connective tissue integrity at 60°C and the formation of protein gel in the endomysial space at 70°C with the lowest average shear force val ues at 60°C. Christensen et al. (2013) noted tenderness of the sem itendinosus from young animals improved as cooking temperature increased from 53°C to 55°C with a cooking duration of 2.5 h and when time was increased from 2.5 to 7.5 h at 53°C, but a coo king temperature of 58°C for 2.5 h or a cooking duration of 19.5 hou rs at 53°C was needed to significantly improve the tenderness of cow semitendinosus; suggesting the cow semitendinosus exhibited greater cross linking resulting in greater thermal strength of the connective tissue. Nonetheless, after 19.5 h at 63°C, both t he young bull and cow meat samples were equ ally tender. Christensen et al. (2011) reported that increasing the end point sous vide temperature from 53°C to 60°C increased the percentage of soluble collagen in the longissimus dorsi and semitendinosus from s laughter pigs and sows leading to more tender muscles. Cook Loss Cook loss or the lack of retention of the meat juices in the bag is clearly the most notable disadvantage of the so us vide cooking method (Church and Parsons, 2000; Szerman et al., 2012). So me previous research on sous vide cooking has indicated both time and temperature have a significant im pact on cook loss (Christensen et al. , 2011; Garcia Segovia et al., 2007). Furthermore, other research on sous vide cooking has indicated that although t ime has an effect, temperature has the greatest impact on cook loss (Christensen et al., 2011, 2012; Roldan et al., 2013; Vaudagna et al., 2002) Moreover, studies have shown an increase in cook loss as temperature increased as well as a negative correlatio n between juiciness and tenderness (Christensen et al., 2012; Vaudagna et al., 2002). Additionally, previous research has indicated that sous vide cooking results in higher cook loss percentages as compared to traditional grilling methods across the same e nd point temperatures (Obuz et al., 2004). Work by Obuz et al. (2003)

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22 showed 9 % increase in cook loss when using the sous vide cooking method as compared to a belt grill. The slower cooking rate was explained as the reason for the increase in cook loss bet ween the two cooking methods as the belt grill provides direct heat transfer and sears the surface of the meat. Flavor Similar to other long time, low temperature cooking techniques, sous vide cooking results in meat with low flavor intensity; thus, leadi ng to a bland flavor (Christensen et al., 2012). In recent sensory evaluation work on the flavor and odor intensity of sous vide bovine semitendinosus; Szerman et al. (2012) characterized the flavor and odor as both slightly intense. The flavor of meat is result of volatile compounds generated at high temperatures; thus, the lack of high cooking temperatures when using the sous vide cooking method is directly responsible for the low flavor intensity (Christensen et al., 2012; Mottram, 1998). Color Both time and temperature affect the appearance of long time, low temperature cooked beef and pork as heat induced conversions in myoglobin occur (Christensen et al., 2012). An increase in both time and temperature give sous vide cooked meat a lighter more yell ow color leading to an overall more greyish appearance (Garcia Sergovia et al., 2007). The loss of redness is indicative of greater myoglobin degradation as the temperature increases (Rouldan et al., 2013). Additionally, on a cooked beef color ratings scal e from 1 to 7 (1: Very red, 7: Brown) and a color uniformity scale from 1 to 5 (1: No variation, 5: Extreme amount of variation); sous vide cooked semitendinosus was given a rating of 5.58 for cooked beef color and a color uniformity score of 2.73 (Szerman et al., 2012).

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23 Food Safety As with all raw meat products, initial microbial load is a determinant of the survival of the pathogens during processing (Nyati, 2000). Thus, raw material inspection is critical. Specifically to sous vide cookery; pouches must be inspected for leaks as to prevent post processing contamination as well as the proper training of food service professionals to ensure proper food hygiene and handling (Nyati, 2000). Pathogens of Concern Salmonella Salmonella is a heat resistant, gram negative, rod shaped bacilli responsible for an estimated 1.2 million illnesses in the U. S . , with roughly 23,000 hospitalizations and 450 deaths ( CDC , 2014). Raw foods of animal origin can potentially carry Salmonella . The FSIS (2013) recommends that all r aw beef steaks and roasts be cooked to a minimum internal temperature of 62.8°C. Thus, Salmonella would be a pathogen of interest as sous vide processing temperatures can be lower than 62.8°C. As cited by Baldwin (2012) in reference to Snyder (1995); a 3 l og 10 unit reduction in Salmonella species should be sufficient for healthy individuals; whereas, a 6.5 7 log 10 unit reduction should be sufficient for immunocompromised individuals. Work by Goodfellow and Brown (1978) indicated that water bath cooking was capable of reducing Salmonella to undetectable levels in beef rounds at temperatures of 54.4°C and 57.2°C. Very low levels of Salmonella were present after 7 h at a temperature of 51.6°C; and Salmonella was undetectable at a water bath temperature of 54.4° C if the beef rounds were held at this internal tempera ture for a minimum of 30 min . Furthermore, at a water bath temperature of 57.2°C , undetectable levels were seen when the beef rounds w ere held for a minimum of 3 min at this internal temperature. Moreo ver , Salmonella was undetectable at all internal temperatures above 57.2°C during water -

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24 bath cooking. Sous vide microbial research by Juneja and Marks (2003) showed varying the time at which the target tem perature is reached can result in significant chang es in the ability of cells to survive a heat treatment at the target temperature. Additionally, the use of whole muscle as compared to ground products allows for the use of lower temperatures with less time to achieve reductions in Salmonella (Orta Ramirez et al. , 2005). Escherichia coli O157:H7 Escherichia coli O157:H7 is a deadly shiga toxin producing, pathogenic strain of the Escherichia coli bacteria with an extremely low infectious dose resulting in an estimated 2,138 hospitalizations annually ( CDC , 2 012 ; 2013). Determined by a linear regressi on, work by Juneja et al. (1997) showed ground bee f cooked in a water bath at 55, 57.5, 60, 62.5 , and 65°C for predetermined lengths of time had respective D values of 21.13, 4.95, 3.17, 0.93 and 0.39 min . Furthe rm ore, the D values will decrease as the percentage of fat decreases; thus, a whole muscle such as the bovine semitendinosus wi ll have a lower D value (Ahmed et al., 1995). Listeria monocytogenes Listeria monocytogenes is a primary pathogen of concern in sous vide processing as it is an anaerobic organism that is both resistant to high temperatures and is psychrotolerant in nature (Hansen and Knochel, 1996). As cited by Hansen and Knochel (1 996) in reference to Kim et al. (1994) and Stephens et al. (1994); the heat resistance of L . monocytogenes can increase due to slowly rising temperatures. Thus, L. monocytogenes is a pathogen of concern for sous vide cooking due to slow rising temperatures over an extended period of time. Work by Bolton et al. (2000) sho wed sufficient inactivation of L. monocytogenes for beef in vacuum packaging at water bath tempe ratures of 50°C for 36.1 min, 55°C for 3.1 min , and at 60°C for 0.15 min. Furthermore, Vaudagna et al. (2002) reported higher temperatures reduced

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25 the time for the thermal destruction of L. monocytogenes . Mor e recent work by Rouldan et al. (2013) found less than log 1 CFU/g of L. monocytogenes at the lowest sous vide he at treatment of 60°C for 6 h indicating a healthy individual would not become ill. This was pos sibly due to low initial counts; nonetheless, growth was not seen across any of the heat treatments. Temperature abuse must be limite d due to L. s heat resistance and ability to recover from heat shock. Previous research by Jorgensen et al. ( 1999) indicated that temperature abuse in the logarithmic growth phase increased the thermotolerance of L. monocytogenes in water bath cooked minced meat at 60°C. Additional work by Hansen and Knochel (2001) showed an increase in the log reduction times of L. monocytogenes in the late logarithmic phase when meat samples were subjected to a heat pre treatment of 46°C for 30 min prior to a sous vide heat treatment of 60°C. Clostridium perfringens Clostridium perfringens is an anaerobic, spore forming bacteria found throughout the environment in the soil, water, and air that produces an enterotoxin (Granum, 1990; Huang, 2003). As a result, the organism can potentially contaminate the surface of raw meat. Furthermore, C. perfringens has an infectious dose of (> 10 6 ) vegetative cells or (>10 6 ) spores/g of food ( FDA , 2012). Clostridium perfringens is an organism of concern as it relates to sous vide cooking due to the survival of anaerobic, heat resistant spores in the vacuum sealed pouch and since the vegetative cel ls have an estimated maximum growth temperature of 53.5°C (Golden et al., 2009). Clostridium perfringens is a viable indicator of the thermal efficacy of vegetative cell destruction (Vaudagna et al., 2002). Research by Juneja and Marmer (1998) indicated th ermal destruction of vegetative cells could be achieve d at 55°C for 21.6 ± 0.1 min, 57.5°C for 10.2 min , 60°C for 5.3 min , and at 62.5°C for 1.6 min . Additional thermal inactivation research on

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26 vegetative cells by Byrne et al. (2006) revealed D values of 0 .9 min at 65°C to 16.3 min at 55°C with the recommendation to cook meat to an internal temperature of 70°C for 1.3 mi n to achieve a 6 log unit reduction of C. perfringens vegetative cells. As a precaution, to prevent sporulation of surviving vegetative cel ls after subsequent heat treatment, food should be held at or below 2.5°C and re heated to an internal temper ature of 65°C for 1 min (Baldwin, 2012; Juneja and Marmer, 1998; Meng and Genigeorgis, 1994). Par Cooking Par cooking is a process that involves th e use of two separate heat treatments. The first heat treatment; usually a method of long time, low temperature cookery, is responsible for cooking the raw meat product to an internal temperature below the desired end point temperature. The par cooked prod uct is then held at refrigerated temperatures until further processing. The second heat treatment; usually a method of dry heat cookery such as grilling, is responsible for cooking the product to a final end point temperature prior to consumption. Furtherm ore, finishing at food service is common for meat products. Searing by the direct heat of a grill or blow torch is popular as browning adds significant flavor as well as a grilled appearance which is preferred by consumers (Baldwin, 2012; Creed, 2001). Te nderness Research by Obuz et al. (2003) showed bovine biceps femoris and longissimus lumborum steaks previously held i n a water bath up to 30 min at temperatures of 57°C or 62°C and then reheated on a belt grill to a final internal end point temperature of 70°C had significantly higher WBS force values as compared to steaks not reheated possibly due to myofibrillar hardening. Cook L oss In a study by Obuz et al. (2003); steaks reheated on a belt grill to an internal temperature of 70°C after being held in a water bath had a significantly greater percentage of cook loss, roughly

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27 7 % higher, than steaks not reheated. Furthermore, the combination of increased holding time at higher temperatures combined with reheating further increased the percentage of coo k loss . As cited by Obuz et al. (2003 ) in reference to Hearne et al. (1978) and Lawrence et al. (2001); the increase in cook loss was due to fluids being forced out as a result of collagen shrinking and the shortening of sarcomeres. Mechanical Tenderization Mec hanical tenderization is a post harvest practice for improving the tenderness of meat. Primal and subprimal cuts are passed through machines that contain reciprocating rows of extreme ly sharp blades or needles that are responsible for disrupting the struct ural integrity of myofibrils, muscle fibers, and muscle bundles as well as severing fibrils of connective tissue (Smith et al. , 2008). An estimated 10.5 % or 2.6 billion pounds of beef product sold in the U.S. is mechanically tenderized (Muth et al., 2012). As indicated in a recent beef mechanical tenderization survey submitted to 241 members of the North American Meat Processors Association with 90 total respondents; mechanical tenderization was used on cuts from the round, sirloin, loin, rib, and chuck wit h 86.9% of top sirloin butts and 85.1 % of strip loins receiving a mechanical tenderi zation treatment (George Evins et al., 2000). Moreover, cuts from the chuck and round received the greatest number of passes through the machine at 2.1 and 1.9 respectively ; whereas, cuts from the loin and rib received the least number of passes through the machine. Additionally, cuts from lower quality grades tended to receive a mechanical tenderizat ion treatment as only 35 % of prime cuts were mechanically tenderized as c om pared to 86.8 % of both Select and Standard. Lastly, re spo ndents indicated 75.1 % of the mechanically tenderized beef was destined f or food service and 13 .3 % was processed for retail.

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28 Tenderness Well established research has indicated both decreased shear f orce values and amounts of organoleptically detectable connective tissue with the use of m echanical tenderization (Savell et al., 1977; Seidman et al., 1977). Recent research by Pietrasik et al. (2010) and Pietrasik and Shand (2011) revealed a significant decrease in WBS force values as a result of mechanical tenderization as well as significantly higher sensory panel scores for overall tenderness and scores indicating a significant decrease in the amount of detectable connective tissue. In addition, work b y Jeremiah et al. (1999) declared mechanical tenderization could be effectively utilized to reduce the variability in and improve both tenderness and palatability in muscles from the hip. Mechanical tenderization reduced the percentage of unpalatable insi d e ro und samples from 36 % to 8 % and from 80 % to 40 % of samples from the eye of round. Cook loss Current research is not in agreement on whether blade tenderization has an effect on cook loss. Work by Pietrasik et al. (2010) found mechanically tenderized bo vine semitendinosus steaks had greater cooking loss as compared to control steaks. Furthermore, research by Obuz et al. (2014) showed comparable results between intact versus non intact steaks from the loin. However, other studies have indicated mechanical tenderization did not affect cook loss (Je remiah et al., 1999; Pietrasik and Shand, 2011). The conflicting results may be attributed to the specific muscles that were used in the studies as certain muscles are more susceptible to greater cook loss with th e use of mechanical tenderization (Jeremiah et al., 1999; Savell et al., 1976). Food Safety The act of mechanically tenderizing meat is of great food safety concern to the FSIS as pathogens may be potentially internalized in the meat product. Research by G ill and McGinnis

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29 (2005) found the contamination of deep tissues increased significantly as a result of mechanical tenderization with increasing numbers of aerobic bacteria on meat surfaces. However, a greater number of passes through the machine did not in crease deep tissue contamination. Furthermore, recent studies have shown microbial translocation depths of several centimeters (Luchansky et al., 2012; Ray et al., 2010). Escherichia coli O157:H7 is a major pathogen of concern to the food industry as it r elates to no n intact beef products. Phebus et al. (2000) showed blade tenderization translocated roughly 3 to 4% of E . coli O157:H7 from the surface of subprimals, regardless of the initial surface contamination. Furthermore, the study found oven broiling at temperatures of 60°C or greater resulted in the complete thermal destruction of E. coli O157:H7 in mechanically tender ized subprimals. However, Gill et al. (2009) showed complete thermal inactivation was not reached until 65°C wit h no hold and at 65°C w ith a 2 min hold at respective growth conditions of 35°C and 42°C simulating temp erature abuse. Luchansky et al. (2012) reported a 2.0 to 4.1 log CFU/g and 1.5 to 4.5 log CFU/g reductions in ECOH and STEC levels when cooked to internal temperatures of 48.9, 54.4, 60.0, 65.6, and 71.1°C; however, even after reaching an internal temperature of 71.1°C, some cells were still present. Studies have indicated the thickness and weight of mechanically tenderized beef affect the thermal destruction of E. coli O15 7:H7 (Adler et al. , 2012; Phebus et al., 2000).

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30 CHAPTER 3 SOUS VIDE BEEF COOKERY Materials and Methods Raw Samples Beef semitendinosus muscles (ST) (IMPS # 171C; n = 50) were removed and collected from USDA Commercial and Utility dairy cow carcasses from a commercial beef processing facility 24 h after slaughter. Selection was made by trained, University of Florida personnel by utilizing carcass weight, subjective external fat thickness and color, and carcass conformation. External fat was not removed prio r to individual packaging in vacuum sealed bags at the commercial beef processing facility. Whole muscles were shipped under refrigeration to the University of Florida Meat Processing Center and aged for 7 d at a temperature of 2°C ± 1°C prior to fabricati on. Sample Preparation After aging, all muscles were denuded free of exterior fat and epimysium. The tail portion of each muscle was then removed. Each of the 50 ST muscles was randomly assigned an identification number. Ten muscles were cut into (n = 7) 2 .54 cm steaks for Experiment 1 (Table 3 1). The remaining muscles were bisected into a posterior and anterior half. One half of each muscle was needle tenderized two times using a 48 knife, Jaccard Meat Tenderizing Machine (Jaccard Corporation, Rochester, NY). To ensure randomization during needle tenderization, the end of the roast, anterior or posterior, was alternated between samples. Muscles were then cut into 5.1 cm steaks (n = 6) with 20 muscles being used for Experiment 2 and 20 muscles for Experimen t 3 (Figure 3 1). All steaks were then individually placed in vacuum bags (BQ6620; Cryovac, Duncan, SC) and vacuumed sealed. All vacuum sealed steaks were frozen at 40°C until further testing.

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31 Experiment 1: The Effect of Cooking Method and Endpoint T emper ature on Characteristics of Cow Semitendinosus S teaks The seven semitendinosus steaks per muscle randomly assigned to 1 of 7 treatments; (n = 1) uncooked, (n = 3) sous vide cooked at 51.7°C, 57.2°C, or 62.8°C, or the same respective endpoint cooking temperatures for grilling (n = 3). Thermal p rocessing Sous vide cooking was done using a constant temperature water bath (Magni Whirl; Blue M Electric Company, Blue Island, IL). A come up time of 1 h was allotted in order for the water temperature to rea ch operating condition. The water agitation function was used for all treatments. Samples were thawed 24 h prior to the cooking treatment. Samples with an initial internal temperature of 6°C ± 2°C were placed into the water bath once the operating temperat ure had been reached. All vacuum packaged samples from each of the 10 muscles, for each respective cooking temperature, were placed in the water bath at 51.7°C, 57.2°C, or 62.8°C for a total of 8 h each. Fully submerged samples were checked for leaks. Wate r bath temperature was monitored periodically using a water proof thermometer (4039 Traceable; Control Company, Friendswood, TX). After cooking, samples were chilled in an ice bath and then placed in a walk in cooler at 4°C ± 2°C until further testing. The process was repeated for all three sous vide cooking treatments. An open hearth, variable heat grill (Model 31605 AH, Hamilton Beach/Proctor Silex Inc., Southern Pines, NC) was used for all grilling treatments. Samples were thawed 24 h prior to the cookin g treatment. The steaks were placed on the pre heated grills and turned once at an internal temperature of 35°C; and then allowed to finish cooking to 51.7°C, 57.2°C, and 62.8°C, respectively. Temperature was monitored using copper constantan thermocouples (Omega Engineering, Inc., Stanford, CT) placed in the geometric center of each steak connected to a

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32 recording thermometer (Measurement Computing Corp., Norton, MA) and recorded using DASYLab 12.0 in Windows 7 (Measurement Computing Corp., Norton, MA). Aft er cooking, samples were placed in a walk in cooler at 4°C ± 2°C. Once chilled, steaks were placed in a bag (BQ6620; Cryovac, Duncan, SC) and vacuumed sealed. Steaks were then placed back in a walk in cooler at 4°C ± 2°C until further testing. The process was repeated for all three endpoint temperatures. Slice shear force a nalysis After completion of cooking and chilling, a slice shear force analysis was performed on each individual steak (n = 60) at a chilled temperature. The slice shear force analysis was conducted using the 90° box as stated in the USMARC Slice Shear Force Procedure for Beef ST made available by the USDA Agriculture Research Service (ARS) (2012). Following the proper orientation of the steak as depicted in the protocol; cuts one and two were made from the sides, the steak was rotated 90° counter clockwise and then placed in the 90° box. Three slices were obtained representing the top, center, and bottom with equal spacing. Each slice was sheared once perpendicular to the muscle fibers usi ng a slice shear head attached to an Instron Universal Testing machine (Model 3343; Instron Corporation, Canton, MA) with a cross head speed of 500mm/min. All trim, including sheared slices, from each treatment steak was individually placed in identified b ags for further analysis and subsequently refrigerated at 4°C ± 2°C. Moisture a nalysis A food processor (# 306 Handy Chopper Plus, Black and Decker, Towson, MD) was used to process samples from each steak. All sous vide and grilled treatment steak samples (n=60) plus raw steak samples (n = 10) were analyzed. All samples were analyzed using the Moisture by Oven Drying protocol (AOAC Official Method 24.003, 1984). Dry aluminum pans were labeled and placed in the drying oven set to a temperature of 105°C. Afte r 12 h, the aluminum pans were

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33 removed from the drying oven and placed in a desiccator for 30 min . The dried aluminum pans were weighed, with weight being recorded as C. Approximately 3 to 6 g of each steak sample was placed, in duplicate, into the dried a luminum pan, and the weight was recorded as A. Those samples were then placed in the drying oven set to a temperature of 105°C for 18 h. After drying, samples were removed from the drying oven and placed in a desiccator to cool. Dried samples were re weigh ed, with weight recorded as B. Moisture percentage was calculated by the difference of A and B over the difference of A and C, and multiplying that value by 100. All moisture analysis samples were discarded after calculations were complete. Fat a nalysis S amples from each steak were processed using a food processor (# 306 Handy Chopper Plus, Black and Decker, Towson, MD). All sous vide and grilled treatment steak samples (n=60) plus raw steak s amples (n = 10) were analyzed. A 1.7 g ± 0.2 g portion of each s ample was placed, in duplicate, into a labeled filter bag, heat sealed, and weight was recorded as initial weigh t. Those samples were placed in a drying oven for 12 h, and then weighed, wit h weight being recorded as W2. Total lipid content was measured usi ng hexa ne in an Ankom XT15 Extractor. After extraction, samples were again placed in a drying oven for 15 min, and then weighed, wi th weight being recorded as W3. Fat percentage was calculated by the difference in W2 and W3, divided by initial weight × 100 . All fat analysis samples were discarded after calculations were complete. Collagen a nalysis After cooking, a collective sample gre ater than 5 g was taken from each individual steak (n = 70) for a collagen analysis. All samples were pulverized using liqu id nitrogen and stored at 20°C prior to the analysis. Hydroxyproline content was determined by following a modified Hill Method (AOAC Official Method 990.26, 2005). Approximately 1 g of the pulverized, freeze -

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34 dried sample was weighed out and transferred i nto a 25mm x 150mm glass, Teflon lined screw cap tube. A 12 mL aliquot then placed in a water bath set to 77°C and allowed to incubate for 80 min with shaking at 80 rpm. The tube was then rem oved from the water bath and centrifuged at 3000 rpm for 12 min at 20°C to separate the soluble and insoluble fractions. Supernatant was transferred to a separate identic into the tube with the insoluble fraction. The tube was then vortexed and subsequently centrifuged at 3000 rpm for 12 min at 20°C. Next, 3.0 mL of concentrated H2SO4 was added to the insoluble fraction; both were vortexed and all tubes were subsequently placed in a drying oven set to 105°C for 16 to 20 h. Once cooled for a minimum of 30 min, samples were filtered, by gravity, through a Whatman 541 (hardened, ashless, fast filter sp eed) filter paper cone and diluted with deionized water to 250 mL for the soluble fraction and 500 mL for the insoluble fraction. Ten milliliters of the sample was transferred to 15 mL glass containers and immediately analyzed using a hydroxyproline assay. Hydroxyproline determination was carried out following the procedures outlined by Bergman and Loxley (1963) using a BioTek Eon spectrophotometer (Winooski, VT) to read absorbance at 558 nm. The spectrophotometer was calibrated using a distilled water blan k sample, and readings were quantified by standard curves prepared for each day of analysis. Total and fractional collagen content was determined by multiplying the hydroxyproline content of the soluble fraction by 7.25 and the insoluble fraction by 7.52 ( Cross et al., 1973).

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35 Experiment 2: The Effects of Mechanical Tenderization on Sous vide Cooked, Cow Semitendinosus S teaks. The six semitendinosus sections (n = 3 needled and n = 3 intact) per muscle were allotted to the three sous vide cooking treatments as discussed in Experiment 1. Thermal p rocessing All samples were thawed, cooked, and chilled as described in Experiment 1 for sous vide cooking. Cook loss a nalysis A total cook loss analysis was performed on all steaks (n = 120). Initial weights were tak en for each steak prior to thermal processing. After cooking and cooling, steaks were blotted dry with a paper towel, prior to being reweighed for the calculation of cooking loss percentage by taking the difference in weights as a percentage of initial wei ght × 100. Slice shear force a nalysis After completion of cooking and chilling, a slice shear force analysis was performed on each individual steak (n = 120) at a chilled temperature. Prior to analysis, each 5.08 cm steak was cut by bisecting the long axis into two 2.54 cm steaks for slice shear force analysis and trained sensory panel evaluation. The slice shear force analysis was then performed as described in Experiment 1. Trained sensory e valuation Samples were prepared as described for sous vide cookin g in Experiment 1. A total of 60 steaks from 10 muscles were evaluated over 10 taste panel sessions spa nning 6 d. Panelists evaluated six steak samples representing the six possible treatments (mechanically tenderized or intact; sous vide cooked at three d ifferent temperatures) from the same identified, randomly selected cow semitendinosus per session. Each steak was cut into multiple 1.27 cm 3 cubes; and

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36 one cube per sample was served at a chilled temperature in a positive pressure ventilated room with ligh ting and cubicles designed for objective meat sensory analysis. A 7 12 member sensory panel trained according to AMSA sensory evaluation guidelines (AMSA, 1995) evaluated each cow semitendinosus sample for juiciness (1 = extremely dry, 2 = very dry, 3 = mo derately dry, 4 = slightly dry, 5 = slightly juicy, 6 = moderately juicy, 7 = very juicy, 8 = extremely juicy), beef flavor intensity (1 = extremely bland, 2 = very bland, 3 = moderately bland, 4 = slightly bland, 5 = slightly intense, 6 = moderately int ense, 7 = very intense, 8 = extremely intense), overall tenderness (1 = extremely tough, 2 = very tough, 3 = moderately tough, 4 = slightly tough, 5 = slightly tender, 6 = moderately tender, 7 = very tender, 8 = extremely tender), connective tissue (1 = ab undant, 2 = moderately abundant, 3 = slightly abundant, 4 = moderate amount, 5 = slight amount, 6 = traces amount, 7 = practically, 8 = none detected), and off flavor (1 = extreme off flavor, 2 = strong off flavor, 3 = moderate off flavor, 4 = slight off f lavor, 5 = threshold; barely detected, 6 = none detected). A randomly selected sample was provided for an initial panelist warm up, and water and unsalted crackers were provided to panelists between samples for palate cleansing. Experiment 3: Sous vide P ar cooking Five of the 6 ST steaks, per muscle, made at fabrication were allocated into 1 of 5 treatments: 1. Intact/Grilled 62.8°C (IG), 2. Needled/Grilled 62.8°C (NG), 3. Needled/Sous vide par cooked 51.7°C (N/51.7 °C ) , 4. Intact/Sous vide par cooked 57.2°C (I/ 57.2°C ) , and 5. Intact/Sous vide par cooked 62.8°C (I/ 62.8°C ) . All sous vide par cooked samples were finished on a grill to an endpoint temperature of 62.8°C. Thermal p rocessing s the grilling procedure described in Experiment 1. For the sous vide par cooking portion of the experiment,

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37 steak samples designated to their respective sous vide cooking treatments (n = 60) were initially cooked in a constant temperature water bath (Magn i Whirl; Blue M Electric Company, Blue Island, IL) for 8 h. A come up time of 1 h was allotted in order for the water temperature to reach operating condition. The water agitation function was used for all treatments. Submerged samples were checked for lea ks; and the water bath temperature was monitored periodically using a water proof thermometer (4039 Traceable; Control Company, Friendswood, TX). After the completion of the three initial sous vide cooking treatments, steak samples were removed from the wa ter bath and allowed to cool. Samples were then placed on preheated, open hearth, variable heat grills (Model 31605 AH, Hamilton Beach/Proctor Silex Inc., Southern Pines, NC) for the finishing portion of the par cooking process. Temperature was monitored a nd recorded using both copper constantan thermocouples (Omega Engineering, Inc., Stanford, CT) placed in the geometric center of each steak and DASYLab 12.0 in Windows 7 (Measurement Computing Corp., Norton, MA). Steaks were turned once at an internal temp erature of 35°C; and subsequently allowed to finish cooking to an endpoint internal temperature of 62.8°C. After the final cooking process, samples were placed in a walk in cooler at 4°C ± 2°C to cool. Once chilled, identified steaks were individually plac ed in a vacuum bag (BQ6620; Cryovac, Duncan, SC) and vacuumed sealed. Steaks were then placed back in a walk in cooler at 4°C ± 2°C until further testing. Cook loss a nalysis ) as described in Experiment 2. A total and secondary cook loss analysis was conducted on all sous vide par cooked steaks (n = 60). Initial weights were taken for each steak prior to thermal processing and recorded as W1. The sous vide steaks were removed from the water bath upon completion of the cooking treatment and allowed to cool. Steaks were blotted with a dry paper

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38 towel and then re weighed and recorded as W2. Following the final grilling portion of the par cooking procedure, steaks were removed from the grill and allowed to cool. After cooling, steaks were blotted with a dry paper towel, prior to being re weighed and recorded as W3. Total cook loss was calculated by dividing W3 into W1; then subtracting the value from 1 and finally multiplying the va lue by 100 to determine a percentage. The secondary cook loss, due to grilling, was calculated by first; taking the difference between W2 and W3 and then dividing by W2. The value was then multiplied by 100 to determine a percentage. Slice shear force a nal ysis After completion of cooking and chilling, a slice shear force analysis was performed on each individual steak (n = 100) at a chilled temperature. Prior to analysis, each 5.08 cm steak was cut by bisecting the long axis into two 2.54 cm steaks for slic e shear force analysis and trained sensory panel evaluation. The slice shear force analysis was conducted as described in Experiment 1. Trained sensory e valuation A total of 25 steaks from five muscles were evaluated over five taste panel sessions spanning 3 d. During each taste pane l session, panelists evaluated five steak samples representing the five possible treatments from the same identified, randomly selected cow semitendinosus. The trained sensory evaluation was then performed as described in Experi ment 2. Statistical Analysis All data was analyzed using the using the mixed model procedure of SAS (SAS Inst., Inc., Cary, NC). Steak location within roast was used a s a random variable for slice shear force and sensory assessment for all experiments. Le ast square means were calculated and then separated

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39 statistically using pair wise t tests (P DIFF option in SAS). If statistically significant (P < 0.05), F test was discerned. The standard error for the means (SEM) was also reported. Results and Discussio n Experiment 1: The Effect of Cooking Method and Endpoint Temperature on Characteristics of Cow Semitendinosus S teaks. Slice shear force and collagen a nalysis Consumers believe tenderness is the most important attribute in determining the eating quality of meat (Huffman et al., 1996). Cow semitendinosus (ST) steaks sous vide cooked to 57.2°C had lower SSF values (P < 0.03) than all steaks cooked to 51.7°C and steaks grilled to 57.2°C (Figure 3 2). Also, steaks sous vide cooked at 62.8 °C had lower SSF values (P < 0.03) tha n all steaks cooked to the two lower cooking temperatures and had numerically lower SSF values than grilled steaks at the same cooking temperature. These results are different tha n those observed by Obuz et al. (2004) that concluded water ba th cooking did not improve the tenderness of muscles with high collagen content. However, the current findings are in agreement with current work by C hristensen et al. (2013), who reported cow semitendinous muscles sous vide cooked at 51.7 °C had the greate st Warner Bratzler peak force (WB PF) values of those evaluated, whereas semitendinosus muscles sous vide cooked to 62.8 °C displayed the lowest WB PF values. It is well established that intramuscular connective tissue contributes to the backgro und tender ness of meat (Forrest et al. , 1975). The findings for slice shear force are largely explained by the findings for steaks sous vide cooked to 62.8 °C which exhibited the greatest percentage of g and temperature combinations (Table 3 2). Established work by Machlik and Draudt (1963) and Lawrie and Ledward (2006) has indicated the solubility of collagen increases with temperature and at

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40 temperatures between 60°C to 64°C the collagen shortens and i s converted into a more soluble form. Current sous vide research by Suriaatmaja and Lanier (2014) has shown collagen solubilization to be the primary mechanism responsible for changes in tenderness and that semitendinosus toughness was reduced at temperatu res above 51.5°C. This can be explained by the slow changes that occur during long time, low temperature cooking methods. Collagen dissolves into gelatin and muscle fiber adhesion is reduced leading to an increase in tenderness without causing protein hard ening (Baldwin, 2012). Moisture and f at A s sous vide cooking ) , there was a linear decrease in the moisture percentage of the cooked product (Table 3 2) . This trend in moisture loss can be explained by the structural changes that occur in meat as a result of cooking (Offer, 1984; Torn berg, 2005). At 40 °C to 60 °C , the gap between the muscle fiber and the surrounding endomysium widens due to muscle fiber shrinkage. Furthermore, the rate of collagen shrinkage increases at temperatures above 60 °C ; thus leading to pressure placed upon the a queous solution in the gap between the fiber and surrounding endomysium . As a result of the exerting pressure, the water is forced out of the muscle. Our collagen analysis further supports this phenomenon. Among the grilled treatments, temperature had no e ffect on moisture loss (P > 0.30; not shown in table). This could be due to the rate of heat transfer since water bath cooking requires a greater amount of time to reach the endpoint temperature (Obuz et al. , 2003 ; Yancey et al. , 2011 ). Time values in Tabl e 3 1 are in agreement with this conclusion. When compared to the raw mean value, steaks sous vide or grilled at the two higher treatment temperatures had higher numerical mean fat percentage values as opposed to the 51.7°C treatments, which exhibited the lowest numerical mean fat percentages (P ; Table 3 2 ). Research has sho wn, as the degree of doneness increases, the percentage of fat increases

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41 coupled with an increase in cooking loss (Parrish et al., 1973 ; Smith et al., 2011 ). We concluded the diff erences in fat percentages among treatments were due to the increase in moisture loss as temperature increased since water dilutes the meat constituents. Experiment 2: The Effects of Mechanical Tenderization on Sous vide Cooked, Cow Semitendinosus S teaks. Slice shear force and c ook loss Well established research has demonstrated both a decrease in shear force valu es a nd the amount of organoleptically detectable connective tissue with the use of mechanical tenderization (Savell et al., 1977; Seidman et al., 1977). However, to date, no studies have specifically evaluated the effect of mechanical tenderization used in conjunction with sous vide cooking on cow semitendinosus steaks. Mechanically tenderized steaks had lower SSF values (P ) than intact stea ks at 51.7 °C and 57.2 °C , but had only numerically lower SSF values at 62.8 °C (Table 3 3). We think the SSF reduction as a result of mechanical tenderization was minimized for steaks cooked at 62.8 °C due to the substantial increase in the percentage of solu bilized collagen as seen in Experiment 1. Studies have concluded cook loss or the lack of retention o f the meat juices in the bag is the most notable disadvantag e of sous vide cooking (Church and Parsons, 2000; Szerman et al., 2012). The present study reve aled a comparable conclusion (Table 3 3 ). A significant increase in cook ing loss was observed (P 0.01) as cooking temperature increased, regardless of mechanical tenderization . These findings are in agreement with other sous vide research by Christensen et al. (2011; 2012), Roldan et al. (2013), and Vaudagna et al. (2002) , which showed an increase in sous vide cooking temperature resulted in greater cook ing loss . Nonetheless, based on the moisture and fat analysis values obtained in Experiment 1, those re spective values would not seem to constitute such a dramatic increase in cook loss as treatment temperature increased.

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42 F urthermore, research is not in agreement on whether mechanical tenderization has an effect on cook loss ; however, t he conflicting result s may be attributed to the specific muscles that were used in the studies as certain muscles are more susceptible to greater cook loss with the use of mechanical tenderization ( Savell et al., 1976; Jeremiah et al., 1999 ; Pie trasik et al., 2010; Pietrasik a nd Shand, 2011; Obuz et al., 2014) . Trained sensory p anel Previous studies on sous vide cookery indicated an inverse relationship between juiciness and tenderness (Christensen et al., 2012; Vaudagna et al., 2002). The present study yielded comparable resu lts (Table 3 4 ). In general, steaks sous vide cooked to 51.7°C had the greatest juiciness values with the lowest tenderness values; whereas, steak samples sous vide at 57.2° C and 62.8°C had the lowest juiciness values with the greatest tenderness values (P ). Moreover, these results complement our findings for sl ice shear force as well as the cook loss analysis. Additionally , mechanical tenderization did no t have an effect on (P 0. 9; not shown in table ) trained panelist values for tenderness and con nective tissue when compared to intact steaks (Table 3 4). Panelists detected the greatest amount of connective tissue in samples sous vide cooked at 51.7°C as compared to the treatment steaks sous vide at 57.2 and 62.8, which had detectible connective tis mean values (P ; Table 3 4 ). These findings are comparable with those observed in the collagen analysis conducted in Experiment 1. Furthermore, all treatments were had a mean flavor were d etected as indicated by the P value of the model (Table 3 4 ). Overall, samples sous vide cooked at a temperature of 57.2 °C produced the best combination of juiciness and tenderness.

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43 Experiment 3: Sous vide P ar cooking Slice shear force and c ook loss Due to the drastic changes that are expected to occur in the food service industry as a result or Blade Tenderized not be able to order, no r could food service establishments serve, mechanically tenderized steaks at rare or possibly medium rare degrees of doneness. Currently, followi ng research by Luchansky et al. (2011; 2012); the FSIS is proposing mechanically tenderized beef products be co oked to an internal temperature of 71.1°C or to 62.8°C with a three minute hold. Studies have indicated satisfaction of juiciness and tenderness decrease as the degree of doneness increases (Lorenzen et al., 1999; Neely et al., 1999). Additionally, a consu mer survey found the average consumer believes steaks cooked to rare or medium rare degrees of doneness are more tender and flavorful leading to greater overall satisfaction and an overall higher intent to repurchase (Cox et al., 1996). F ew studies have e valuated sous vide par tial cooking as an alternative to mechanical tenderization. Specifically, work by Suriaatmaja and Lanier (2014) showed a controlled sous vide par cook, with the addition of pre injection to enhance moisture retention, yielded a tender and succulent grilled steak. Results from this experiment were comparable in terms of tenderness; however, pre injec tion was not included (Table 3 5 ). S teaks subjected to the I/62.8 °C , and st eaks designated to the I/57.2 °C treatment had lower SSF values than IG steaks as well as numerically lower SSF values when compared to NG. T hus , sous vide par cooking could be a viable alternative to mechanical tenderization in terms of tenderne ss enhancement for tough muscles. However, a total cook loss analysis revealed all sous vide par cooked treatments had a greater cooking loss percentage when compared to all grilled steak treatments (Table 3 5). Overall,

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44 I/62.8 °C treatment steak s had the greatest percentage of cook ing loss ( ). Furthermore, as a result of the grilled, finishing portion of the par cooking process, N/51.7 °C treatment steaks had the greatest ( ) percentage of secondary cook ing loss ; whereas I/62.8°C tr eatment steaks had the lowest ( ) percentage of secondary cook ing loss (Table 3 5 ). These results were co mparable to work by Obuz et al. (2003), which showed meat previously cooked in a water bath and then reheated on a gril l exhibited a 7 % increase in cooking loss when compared to steaks that were not reheated on a grill. Based on the cook loss analysis in Table 3 3 and Table 3 5, we observed a similar finding with steaks sous vide cooked at 51.7 °C exhibiting the greatest increase in total cook loss due to the increased temperature of the finishing portion as compared to the initial water bath temper ature. Furthermore, Obuz et al. (2003) found all water bath cooking treatments that were subsequently reheated yielded greater cooking loss as compared t o all grilled only treatments due to the slower rate of heat penetration . Time values in Table 3 1 are in agreement with this conclusion. Trained sensory p anel Based on the trained sensory panel evaluations, juiciness was the only category that was signif icantly different across all treatments (P < 0.01; Table 3 6 ). When compared to NG treatment steaks , both I/57.2 °C and I/62.8 °C treatment steaks ). The analysis in this experiment. Although no statistical difference wa s observed in the model (P > 0.1; Table 3 6 ), all sous vide p ar cooked treatments received higher mean tenderness scores than the NG treatment. Additionally, the mean values for beef intensity flavor and off flavor, indicated all sous vide par cooked treatments as well as the NG treatment produced a cow semitendinos flavor that was barely detectable (Table 3 6) .

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45 Implications The present studies have indicated a reduction in the toughness of cow semitendinosus steaks can be achieved through the use of sous vide c ookery at lo w temperatures for 8 h . The inclusion of mechanical tenderization with sous vide cookery enhanced tenderness for steaks cooked at temperatures below 62.8°C. These findings indicate sous vide par cooking could be used as an alternative to mechanical tenderiz ation for tenderness improvement for muscles with high connective tissue content. However, cook loss is a major issue as all sous vide par cooked steaks exhibited significantly greater cook loss as compared to the traditional, mechanically tenderized, gril led steak samples. Further research should examine methods to reduce cook loss associated with sous vide cookery. Lastly, further research is needed to validate the microbiological safety of the sous vide par cooking process.

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46 Table 3 1. Experiment 1 tre atments for cow semitendinosus steaks. Cooking Me thod Water Bath Temperature (°C) 2 Time 3 (min) Time 4 (min) Sous vide 1 I 51.7 480 80±10 II 57.2 480 60±10 III 62.8 480 40±10 Grilled 1 Endpoint Temperature (°C) 2 Time 4 (min) I 51.7 10±1 II 57.2 15±1 III 62.8 21±1 1 Cooking method. 2 Water bath was maintained at the specified temperature for entire cooking period. Steaks were removed from grill once the internal temperature reached the specified endpoint temperature. 3 Time of e ntire cooking treatment 4 Time to reach the same temperature for both water bath and internal temperature. Time to reach specified endpoint temperature.

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47 Whole Cow Semitendinous 51.7°C 1 Needled 2 57.2°C Needled 62.8°C Needled 51.7°C Control 57.2°C Control 62.8°C Control Figure 3 1. Experiment 2 and 3 sample preparation. Notes: 1 Water bath temperatures for sous vide cooking treatments. 2 Mechanically tenderized. 5.08 cm steaks with corresponding treatments

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48 Figure 3 2. Effect of cooking method and endpoint temp erature on Slice Shear Force values of cow semitendino sus steaks. Notes: Grilled = Cooked to specified endpoint temperature on grill; Sous vide = Cooked in a water bath under vacuum at specified temperature for 8 h. Slice Shear Force measurements taken a t 4°C ± 2°C. A,B,C,D Values lacking common superscript differ (P < 0.0246).

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49 Table 3 2 . Effect of cooking method and endpoint temperature on collagen, moisture, and fat characteristics on cow semitendinosus steaks 1 Raw 2 Sous vide cooking temperature 3 G rilled cooking temperature 3 SEM P value Trait --51.7 °C 57.2 °C 62.8 °C 51.7 °C 57.2 °C 62.8 °C Total collagen, mg/g 16.2 d 21.2 a 19.8 abc 21.4 a 19.1 bc 18.2 c 20.5 ab 0.9 < 0.001 Insoluble, % 95.3 a 93.9 abc 93.4 bc 88.9 d 94.2 ab 93.9 abc 92 c 0.7 < 0. 001 Soluble, % 5.0 d 6.6 cd 7.1 bc 13.0 a 6.2 cd 6.6 cd 8.9 b 0.9 < 0.001 Moisture, % 74.3 a 69.8 b 68.3 c 66.0 d 67.8 c 67.1 cd 67.2 cd 0.5 < 0.001 Fat, % 2.7 ab 2.1 c 2.9 a 2.5 abc 1.3 d 2.3 bc 2.3 bc 0.3 < 0.001 1 All values represent steaks from 10 different roasts 2 Uncooked. 3 Cooked in a water bath under vacuum at specified temperature for 8 h; Cooked to specified endpoint temperature on grill

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50 Table 3 3 . Effect of mechanical tenderization on Slice Shear Force and cook loss on sous vide cooked 1 cow semitendinosus steaks 2 Cook ing Temperature of I ntact Cooking Temperature of T enderiz ed 3 SEM P value Trait 51.7 °C 57.2 °C 62.8 °C 51.7 °C 57.2 °C 62.8 °C SSF, kg 4 50.1 a 35.2 c 30.3 cd 45.0 b 29.9 d 27.4 d 1.2 <0.0001 Cook loss, % 20.3 c 25.1 b 33.3 a 20.0 c 24.7 b 32.8 a 0.7 <0.0001 1 Cooked in a water bath under vacuum at specified temperatu re for 8 h 2 All values represent steaks from 20 different roasts . 3 Needled twice. 4 Slice Shear Force measurements taken at 4°C ± 2°C. Values lacking common superscript differ ( P < 0.0038)

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51 Table 3 4 . Trained sensory panel values for mechanically tende rized, sous vide cooked 1 cow semitendinosus steaks 2 . Cooking Temperature of Intact Cooking Temperature of Tenderized 3 SEM P value Trait 51.7 °C 57.2 °C 62.8 °C 51.7 °C 57.2 °C 62.8 °C Juiciness 4 5.7 a 5.2 b 4.4 c 5.8 a 5.4 b 4.2 c 0.1 <0.0001 Beef Flavor I ntensity 5 4.3 b 4.6 ab 4.7 a 4.3 b 4.7 a 4.8 a 0.2 <0.0165 Tenderness 6 4.0 c 5.7 ab 5.5 b 4.2 c 6.0 a 5.6 b 0.2 <0.0001 Connective Tissue 7 3 d 5.1 c 5.7 ab 3.2 d 5.5 bc 6.0 a 0.2 <0.0001 Off flavor 8 5.8 5.9 5.9 5.8 5.9 5.9 0.1 <0.9867 1 Cooked in a water bath under vacuum at specified temperature for 8 h 2 All values represent steaks from 10 different roasts. 3 Needled Twice. 4 Juiciness: 1 extremely dry, 2 very dry, 3 moderately dry, 4 slightly dry, 5 slightly juicy, 6 moderately juicy, 7 very juicy, 8 extremely juicy. 5 Beef flavor intensity: 1 extremely bland, 2 very bland, 3 moderately bland, 4 slightly bland, 5 slightly intense 6 moderately intense, 7 very intense 8 extremely intense. 6 Tenderness: 1 extremely tough, 2 very tough, 3 moderately tough, 4 slightly tough , 5 slightly tender, 6 moderately tender, 7 very tender, 8 extremely tender. 7 Connective tissue: 1 abundant amount, 2 moderately abundant, 3 slightly abundant, 4 moderate amount, 5 slight amount, 6 traces amount, 7 practically none, 8 none detected. 8 Off flavor: 1 extreme off flavor, 2 strong off flavor, 3 moderate off flavor, 4 slight off flavor, 5 threshold, barely detected, 6 none detected. All samp les served at a temperature of 4°C ± 5°C. Values lacking common superscript differ ( P < 0.0416).

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52 Table 3 5 . Effect of sous vide par cooking 1 on Slice Shear Force and cook loss on cow semitendinosus steaks 2 . Treatments 3 IG NG N/ 51.7 °C I/ 57.2 °C I/ 62.8 °C SEM P Value SSF, kg 4 39.2 a 36.5 bc 37.4 ab 34.5 c 29.6 d 1.1 <0.0001 Total Cook loss, % 24.8 d 29.3 c 34.1 b 33.9 b 37.5 a 0.7 <0.0001 Secondary Cook loss, % 5 16.6 a 13.8 b 8.9 c 0.4 <0.0001 1 Sous vide cooked to specified endpoint temperatures prior to grilling to an internal temperature of 62.8 °C. 2 All values represent steaks from 20 di fferent roasts 3 IG: Intact/Grilled 62.8 °C , NG: Needled/Grilled 62.8 °C , N/51.7 °C : Needled/Sous vide 51.7 °C , Intact/Sous vide 57.2 °C , Intact/Sous vide 62.8 °C 4 Slice Shear Force measurements taken at 4°C ± 2°C. 5 Cook loss as a result of grilling to an interna l temperature of 62.8 °C after sous vide cooking treatments. Values lacking common superscript differ ( P < 0.0449)

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53 Table 3 6 . Trained sensory panel values for sous vide par cooked 1 cow semitendinosus steaks 2 . Treatment 3 IG NG N/ 51.7 °C I/ 57. 2 °C I/ 62 .8 °C SEM P value Juiciness 4 5.0 a 4.8 ab 4.5 bc 4.4 cd 4.1 d 0.1 <0.0001 Beef Flavor Intensity 5 4.9 4.9 4.4 4.5 4.8 0.2 <0.0768 Tenderness 6 4.4 4.7 5.0 4.9 5.0 0.3 <0.1497 Connective Tissue 7 4.8 5.4 5.2 5.3 5.6 0.4 <0.2338 Off flavor 8 5.9 5. 8 5.9 5.9 5.8 0.1 <0.6904 1 Sous vide cooked to specified endpoint temperatures prior to grilling to an internal temperature of 62.8 °C. 2 All values represent steaks from 5 different roasts 3 IG: Intact/Grilled 62.8 °C , NG: Needled/Grilled 62.8 °C , N/51.7 °C : Needled/Sous vide 51.7 °C , Intact/Sous vide 57.2 °C , Intact/Sous vide 62.8 °C 4 Juiciness: 1 extremely dry, 2 very dry, 3 moderately dry, 4 slightly dry, 5 slightly juicy, 6 moderately juicy, 7 very juicy, 8 extremely juicy. 5 Beef flavor intensity: 1 extreme ly bland, 2 very bland, 3 moderately bland, 4 slightly bland, 5 slightly intense 6 moderately intense, 7 very intense 8 extremely intense. 6 Tenderness: 1 extremely tough, 2 very tough, 3 moderately tough, 4 slightly tough, 5 slightly tender, 6 moderately tender, 7 very tender, 8 extremely tender. 7 Connective tissue: 1 abundant amount, 2 moderately abundant, 3 slightly abundant, 4 moderate amount, 5 slight amount, 6 traces amount, 7 practically none, 8 none detected. 8 Off flavor: 1 extreme off flavor, 2 st rong off flavor, 3 moderate off flavor, 4 slight off flavor, 5 threshold, barely detected, 6 none detected. All samp les served at a temperature of 4°C ± 5°C. Values lacking common superscript differ ( P < 0.0331).

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57 Granum, P.E. (1990). Clostridium perfringens toxins involved in food poisoning. International Journal of Food Microbiology , 10, 101 112. Guelker, M.R., Haneklaus, A.N., Brooks, J.C., Carr, C.C., Delmore Jr., R.J., Griffin, D.B., . . . Savell, J.W. (2013). National beef tenderness survey 2010: Warner Bratzler shear force values and sensory ratings for beef steaks from United Sates retail and food service establishments. Journal of Anima l Science , 91, 1005 1014. Hansen, T.B., & Knochel, S. (1996). Thermal inactivation of Listeria monocytogenes during rapid and slow heating in sous vide cooked beef. Letters in Applied Microbiology , 22, 425 428. Hansen, T.B., & Knochel, S. (2001). Factors i nfluencing resuscitation and growth of heat injured Listeria monocytogenes 13 249 in sous vide cooked beef. International Journal of Food Microbiology , 63, 135 147. Hearne, L.E., Penfield, M.P., & Goertz, G.E. (1978). Heating effects on bovine semitendinos us: shear, muscle fiber measurements, and cooking losses. Journal of Food Science , 43, 10 12. Hill, F. (1966). The solubility of intramuscular collagen in meat animals of various ages. Journal of Food Science , 31, 161 166. Huang, L. (2003). Growth kinetics of Clostridium perfringens in cooked beef. Journal of Food Safety , 23 (2), 91 105. Huffman, K.L., Miller, M.F., Hoover, L.C., Wu, C.K., Brittin, H.C., & Ramsey, C.B. (1996). Effect of beef tenderness on consumer satisfaction with steaks consumed in the ho me and restaurant. Journal of Animal Science , 74, 91 97. Janz, J.A.M., Aalhus, J.L., & Price, M.A. (2006). The effect of empimysial connective tissue on factors related to tenderness of beef semitendinosus . Journal of Muscle Foods , 17, 43 55. Jeremiah, L.E ., Gibson, L.L., & Cunningham, B. (1999). The influence of mechanical tenderization on the palatability of certain bovine muscles. Food Research International , 32, 585 591. Jeremiah, L.E., & Gibson, L.L. (2003). Cooking influences on the palatability of ro asts from the beef hip. Food Research International , 36, 1 9. Jones, S.J., Guru, A., Singh, V., Carpenter, B., Calkins, C.R., & Johnson, D. (2004). Bovine Myology and Muscle Profiling. Retrieved from http://bovine.unl.edu.

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59 O157:H7 Shiga toxin producing Escherichia coli in brine injected, gas grilled steaks. Journal of Food Protection , 74 (7), 1054 1064. Luchansky, J.B., Porto Fett, A. C. S., Shoyer, B. A., Call, J. E., Schlosser, W., Shaw, W., Bauer, N., & Latimer, H. (2012). Fate of Shiga toxin producing O157:H7 and non O157:H7 Escherichia coli cells within blade tenderized beef steaks after cooking on a commercial open flame gas grill. Journal of Food Protection , 75 (1), 62 70. Machlik, S.M., & Drau dt, H.N. (1963). The effect of heating time and temperature on the shear of beef semitendinosus muscle. Journal of Food Science , 28 (6), 711 718. Meng, J., & Genigeorgis, C.A. (1994). Delaying toxigenesis of Clostridium botulinum by sodium Letters in Applied Microbiology , 19, 20 23. Morgan, J.B., Savell, J.W, Hale, D.S., Miller, R.K., Griffin, D.B., Cross, H.R., & Shackelford, S.D. (1991). National beef tenderness survey. Journal of Animal Science, 69, 3274 3283. Mottram, D. S. (1998). Flavour formation in meat and meat products: a review. Food Chemistry , 62 (4), 415 424. Muth, M.K., Michaela, M.B., & Coglaiti, C. (2012). Expert elicitation on the market shares for raw meat and poultry products containing added solutions and m echanically tenderized raw meat and poultry products. Retrieved from http://www.fsis.usda.gov/wps/wcm/connect/3a97f0b5 b523 4225 8387 c56a1eeee189/Market_Shares_MTB_0212.pdf?MOD=AJPERES Muth, M.K., Ball, M.J., Coglaiti, M.C., & Karns, S.A. (Oct 2012). Mode l to estimate costs of using labeling as a risk reduction strategy for consumer products regulated by the Food and Drug Administration. Contract No. GS 10F 0097L, Task Order 5. Revised final report: Prepared for U.S. Food and Drug Administration, Center fo r Food Safety and Applied Nutrition. Research Triangle Park, NC: RTI International. Neely, T.R., Lorenzen, C.L., Miller, R.K., Tatum, J.D., Wise, J.W., Taylor, J.F., Buyck, M.J., Reagan, J.O., & Savell, J.W. (1999). Beef customer satisfaction: cooking meth od and degree of doneness effects on the top round steak. Journal of Animal Science , 77, 653 660. Nishimura, T., Ojima, K., Liu, A., Hattori, A., & Takahashi, K. (1996). Tissue and structural changes in the intramuscular connective tissue during the develo pment of bovine semitendinosus muscle. Tissue and Cell , 28 (5), 527 536. Nishimura, T., Hattori, A., & Takahashi, K. (1999). Structural changes in intramuscular connective tissue during the fattening of Japanese black cattle: effect of marbling on beef ten derization. Journal of Animal Science , 77, 93 104.

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60 Nyati, H. (2000). An evaluation of the effect of storage and processing temperatures on the microbiological status of sous vide extended shelf life products. Food Control , 11, 471 476. Obuz, E., Dikeman, M .E., & Loughin, T.M. (2003). Effects of cooking method, reheating, holding time, and holding temperature on beef longissimus lumborum and biceps femoris tenderness. Meat Science , 65, 841 851. Obuz, E., Dikeman, M.E., Grobbel, J.P., Stephens, J.W., & Loughi n, T.M. (2004). Beef longissimus lumborum , biceps femoris , and deep pectoralis Warner Bratzler shear force is affected differently by endpoint temperature, cooking method, and USDA quality grade. Meat Science , 68, 243 248. Obuz, E., Akkaya, L., Gok, V., & Dikeman, M.E. (2014). Effects of blade tenderization, aging method, and aging time on meat quality characteristics of Longissimus lumborum steaks from cull Holstein cows. Meat Science , 96, 1227 1232. Offer, G. (1984). Progress in the biochemistry, physiolo gy and structure of meat. Proceedings from the 30th European meeting of meat research workers (87) . Bristol, UK . Orta Ramirez, A., Marks, B.P., Warsow, C.R., Booren, A.M., & Ryser, E.T. (2005). Enhanced thermal resistance of Salmonella in whole muscle com pared to ground beef. Journal of Food Science , 70 (7), 359 362. Parrish Jr., F.C., Olson, D.G., Miner, B.E., & Rust, R.E. (1973). Effect of degree of marbling and internal temperature of doneness on beef rib steaks. Journal of Animal Science , 37, 430 434. Phebus, R. K., Thippareddi, H., Sporing, S., Marsden, J. L., & Kastner, C. L. (2000). Escherichia coli O157:H7 risk assessment for blade of progress 850 (125 126). Manhattan, KS: Kansas State University. P ietrasik, Z., Aalhus, J.L., Gibson, L.L., & Shand, P.J. (2010). Influence of blade tenderization, moisture enhancement and pancreatin enzyme treatment on the processing characteristics and tenderness of beef semitendinosus muscle. Meat Science , 84, 512 517 . Pietrasik, Z., & Shand, P.J. (2011). Effects of moisture enhancement, enzyme treatment, and blade tenderization on the processing characteristics and tenderness of beef semimembranosus muscle. Meat Science , 88, 8 13. Ray, A.N., Dikeman, M.E., Crow, B.A., Phebus, R.K., Grobbel, J.P., & Hollis, L.C. (2010). Microbial translocation of needle free versus traditional needle injection enhanced beef strip loins. Meat Science , 84, 208 211.

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61 Roldan, M., Antequera, T., Martin, A., Mayoral, A.I., & Ruiz, J. (2013). Effect of different temperature time combinations on physicochemical, microbiological, textural and structural features of sous vide cooked lamb loins. Meat Science , 93, 572 578. Savell, J.W., Carpenter, Z.L., & Smith, G.C. (1976). Mechanical Tenderization of three kinds of beef. Journal of Animal Science , 43, 253. Savell, J.W., Smith, G.C., & Carpenter, Z.L. (1977). Blade tenderization of four muscles from three weight grade groups of beef. Journal of Food Science , 42 (4), 866 870. Seidman, S.C., Smith, G.C., Carpenter, Z.L., & Marshall, W.H. (1977). Blade tenderization of beef psoas major and semitendinosus muscles. Journal of Food Science , 42 (6), 1510 1512. Smith, G.C., Tatum, J.D., Belk, K.E., & Scanga, J.A. (2008). Mechanical T enderization. Post Harvest Practices for Enhancing Beef Tenderness (9). United States of America: Smith, A.M., Harris, K.B., Haneklaus, A.N., & Savell, J.W. (2011). Proximate composition and energy content of beef steaks as influenc ed by USDA quality grade and degree of doneness . Meat Science , 89 (2), 228 232. Snyder, O. P. (1995). The applications of HACCP for MAP and sous vide products. In J.M. Farber & K. L Dodds (Eds.), Principles of Modified Atomosphere and Sous Vide Product Pac kaging (325 383). Technomic Publishing Co, Inc. Stephens, P.J., Cole, M.B., & Jones, M.V. (1994). Effect of heating rate on thermal inactivation of Listeria monocytogenes . Journal of Applied Bacteriology , 77, 702 708. Suriaatmaja, D., & Lanier, T.C. (2013) . Mechanism of Meat Tenderization by Long Time Low Temperature Heating. Masters Thesis, North Carolina State University, Raleigh, NC. Szerman, N., Gonzalez, C.B., Sancho, A.M., Grigioni, G., Carduza, F., & Vaudagna, S.R. (2007). Effect of whey protein con centrate and sodium chloride addition plus tumbling procedures on technological parameters, physical properties and visual appearance of sous vide cooked beef. Meat Science , 76 (3), 463 473. Szerman, N., Gonzalez, C.B., Sancho, A.M., Grigioni, G., Carduza, F., & Vaudagna, S.R. (2012). Effect of the addition of conventional additives and whey protein concentrates on technological parameters, physicochemical properties, microstructure and sensory attributes of sous vide cooked beef muscles. Meat Science , 90, 701 710. Tornberg, E. (2005). Effect of heat on meat proteins implications on structure and quality of meat products. Meat Science , 70, 493 508.

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62 Totland, G.K., Kryvi, H., & Slindle, E. (1988). Composition of muscle fibre types and connective tissue in bov ine M. semitendinosus and its relation to tenderness. Meat United States Department of Agriculture [USDA] Agricultural Marketing Service [ARS] . (2012). USMARC slice shear force procedure for beef semi tendinosus (ST). Retrieved from http://www.ars.usda.go v/sp2UserFiles/Place/54380530/protocols/SSF_PROCEDURE_ST. pdf United States Department of Agriculture [USDA] Food Safety and Inspection Service [FSIS] . (2013). Beef from Farm to Table. Retrieved from http://www.fsis.usda.gov/wps/portal/fsis/topics/food saf ety education/get answers/food safety fact sheets/meat preparation/beef from farm to table/ct_index Vaudagna, S.R., Sanchez, G., Neira, M.S., Insani, E.M., Picallo, A.B., Gallinger, M.M., & Lasta, J.A. (2002). Sous vide cooked beef muscles: effects of low temperature long time (LT LT) treatments on their quality characteristics and storage stability. International Journal of Food Science and Technology , 37, 425 441. Whitaker, J.R. (1996). Enzymes. In O.R. Fennema (Ed.), Food Chemistry (3rd ed.) (431 526). N ew York, NY: Marcel Dekker, Inc. Yancey, J.W.S., Wharton, M.D., & Apple, J.K. (2011). Cookery method and end point temperature can affect the Warner Bratzler shear force, cooking loss, and internal cooked color of beef longissimus steaks . Meat Science , 88 (1), 1 7.

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63 BIOGRAPHICAL SKETCH Derek Griffing was born in Tulsa, Oklahoma in 1989 ; the son of John and Linda Griffing . He was raised northeastern Oklahoma. He graduated from Broken Arrow High School, Broken Arrow, Oklahoma in May 2008 . While working on hi s undergraduate degree at Oklahoma State University, he participated on the intercollegiate meats judging team in 2010 , a nd was a member of 2011 national champion, meat animal evaluation team. Derek also worked at the Food and Agriculture Products Center ( FAPC) meats laboratory and the Dairy Cattle Center while attending Oklahoma State University. Furthermore , he was a c arcass m erchandizing intern for the summer of 2011 at Tyson Foods Inc. in Amarillo, Texas. He received his Bachelor of Science degree in a n imal s ciences in May 2012 . He is currently a graduate teaching and research assistant finishing a Master of Science degree in the Department of Animal Sciences at the University of Florida.



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Relationshipbetweenmeattoughnessandpropertiesofconnectivetissuefromcows andyoungbullsheattreatedatlowtemperaturesforprolongedtimesLineChristensena,PerErtbjergc ,,HanneLøjeb,JensRisboa,FransW.J.vandenBerga,MetteChristensenaaDepartmentofFoodScience,UniversityofCopenhagen,Rolighedsvej30,DK-1958FrederiksbergC,DenmarkbDTUNationalFoodInstitute,TechnicalUniversityofDenmark,SøltoftsPlads,Building227,DK-2800KgsLyngby,DenmarkcDepartmentofFoodandEnvironmentalSciences,UniversityofHelsinki,FI-00014UniversityofHelsinki,Helsinki,Finlandabstract articleinfoArticlehistory: Received28June2012 Receivedinrevisedform13November2012 Accepted3December2012 Keywords: Sous-vide Beef Collagen Hyper-spectralimaging Cathepsin DenaturationTheaimofthecurrentstudywastoelucidatewhetherco wsandyoungbullsrequiredifferentcombinationsof heatingtemperatureandheatingtime toreducetoughnessofthemeat.Thecombinedeffectofheatingtemperatureandtimeontoughnessof semitendinosus musclefromthetwocategoriesofbeefwasinvestigatedandthe relationshiptopropertiesofconnectivetissuewasexamined.Measurementsoftoughness,collagensolubility, cathepsinactivityandproteindenaturationofbeef semitendinosus heatedattemperaturesbetween53°Cand 63°Cforupto191/2hwereconducted.Theresultsreveal edthatslightlyhighertemperaturesandprolonged heatingtimeswererequiredtoreducetoughnessof semitendinosus fromcowstothesamelevelasinyoung bulls.Reducedtoughnessof semitendinosus asaresultoflowtemperatureforprolongedtimeissuggestedtoresult fromweakeningoftheconnectivetissue,causedpartlyby denaturationorconformationalchangesoftheproteins and/orbysolubilizationofcollagen. ©2012ElsevierLtd.Allrightsreserved.1.Introduction Heattreatmentatlowtemperaturesbetween50°Cand60°Cfor prolongedtimes(LTLT)increasestendernessofbeef( Beilken,Bouton,& Harris,1986;Bramblett,Hostetler, Vail,&Draudt,1959;Bramblett& Vail,1964;Laakkonen,Wellington ,&Sherbon,1970;Machlik&Draudt, 1963 )andpork( Christensen,Bertram,Aa slyng&Christensen,2011; Christensen,Ertbjerg,Aaslyng,& Christensen,2011;Christensen, Gunvig,Tørngren,Aaslyng,Knøchel,&Christensen,2012 ).TheLTLTtreatmentmethodisincreasinginpopularityinthecateringindustryand amongchefsduetothepossibilityofincreasedconsistencyalongthe muscleaswellasappealingtextureandcolorofthemeat( Christensen etal.,2012;Mortensen,Frøst,Skibsted,&Risbo,2012 ).Textureand colorarethemaindriversoflikinginLTLTtreatedmeat,whilethe avor hasbeenevaluatedasneutral( Christensenetal.,2012 ).Connectivetissue contributessigni cantlytothetoughnessofmeatbutalsotendernessof cutshighinconnectivetissuesuchas semimembranosus , deeppectoralis and semitendinosus canbeimprovedbyLTLTtreatment( Beilkenetal., 1986;Bouton&Harris,1981 ). Livestockcattleproductionforspeci cpurposessuchasmilkor meatcreatesvariabilityintheeatingquality.Dairycowsusedformilk productionareoftenemaciatedafterseveralyearsofmilking,while bullsusedformeatproductionareslaughteredwhentheyarestill young( b 36months).Theagedifferencesofanimalsbelongingtothe twocategoriesofbeefareparticularlyre ectedintheintramuscular fatcontent( Maltin,Sinclair,Warriss,Grant,Porter,Delday,&Warkup, 1998 )andthethermalstabilityofcollagen( Allain,LeLous,Bazin, Bailey,&Delaunay,1978;Lepetit,2007 ).Thethermalstabilityofcollagenisdeterminedbythedegreeofinternalheat-stablecross-linkages whichincreaseswithanimalageatslaughter( Lepetit,2007 ).LTLT treatmentshavebeensuggestedtoaffectchemicalandphysicalpropertiesofconnectivetissueanditisthereforerelevanttoevaluatewhether beeffromdifferentcategories,havingdistinctdifferencesinthecompositionofconnectivetissue,requiredifferentLTLTtreatmentsinorderto becometender. SeveralstudieshavesuggestedthatweakeningoftheconnectivetissuemayberesponsibleforthetenderizationofmeatduringLTLTtreatmentofbeef( Beilkenetal.,1986;Bouton&Harris,1981;Christensen, Purslow,&Larsen,2000;Laakko nen,Sherbon,&Wellington,1970; Laakkonen,Wellington,etal.,1970 )andpork( Christensen,Ertbjerg,et al.,2011 ). Christensenetal.(2000) foundthatthedecreaseinwhole meattoughnessofbeef semitendinosus between50°Cand60°Cwas associatedwithadecreaseinthestrengthofperimysialconnective tissue. Brüggemann,Brewer,Risbo,andBagatolli(2010) usedsecond harmonicgeneration(SHG)microscopytoinvestigateheat-induced denaturationofcollageninpork.Theauthorsshowedthatcollagen shrinkageoccurredat57°Candat5 9°Ccollagendenaturedasthe SHGsignalfromcollagenvanished,suggestingthatthecollagentriple helixstructureunfolded.Still,stud ieselucidatingthecombinedeffect oflowheatingtemperaturesandprolongedheatingtimesonthephysicalandchemicalpropertiesofconnectivetissuearelacking. MeatScience93(2013)787 … 795 Correspondingauthor.Tel.:+358919158458;fax:+358919158460. E-mailaddress: per.ertbjerg@helsinki. (P.Ertbjerg). 0309-1740/$ … seefrontmatter©2012ElsevierLtd.Allrightsreserved. http://dx.doi.org/10.1016/j.meatsci.2012.12.001 Contentslistsavailableat SciVerseScienceDirectMeatSciencejournalhomepage:www.elsevier.com/locate/meatsci

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Proteolyticenzymessuchascollagenase( Laakkonen,Sherbon,etal., 1970 )andcathepsinsBandL( Christensen,Ertbjerg,etal.,2011; Ertbjerg,Christiansen,Pedersen,&Kristensen,2012 )havebeenfound tobeactiveintheLTLTtime … temperaturerange ,andmaytherefore contributetotheincreasedtendernessofLTLTtreatedmeat.Contrary, calpainswhicharewidelystudiedandbelievedtocontributetoincreased degradationofthemuscleproteinspostmortem( Koohmaraie,1992 ), werefoundinactivatedafteronly2minofheatingat55°C( Ertbjerget al.,2012 ).Colorchangeshavenot,totheauthor'sknowledge,beenstudiedinbeefduringLTLTtreatment. Theaimofthecurrentstudywastoelucidatewhether semitendinosus musclefromcowsandyoungbullsrequiredifferentcombinationsof heatingtemperatureandheatingtimetoreducetoughnessofthemeat byinvestigatingthecombinedeffectofheatingtemperatureandtime. Inaddition,therelationshiptopropertiesofconnectivetissuewasexaminedbymeasurementsofdenaturationandconnectivetissuestrength. Furthermore,thecombinedeffectofheatingtemperatureandtimeon thechangesincolorofthemeatwasevaluated. 2.Materialsandmethods 2.1.Rawmaterials Twentyfourcows(4 … 6yearsold)and24youngbulls(10 … 12months old)wereslaughteredat2conventionalslaughterhouses(Hadsund Kreaturslagteri,Hadsund,DenmarkandMogensNielsenKreaturslagteri, Herlufmagle,Denmark).Fromeachanimalone semitendinosus (ST)musclewasexcised3days postmortem .pHwasmeasuredandusedasa selectioncriteriaforthemuscles(pH5.4 … 5.8).Themuscleswerevacuum packagedafterselectionandfurth erstoredfor7daysat5°C.After storage,3samplesfromeachmusclewe recutintosizesofapproximately 5×7×10cm,vacuumpackagedandfrozenat 20°C. 2.2.Experimentaldesign Afullfactorialdesignwith4heati ngtemperaturesand3heatingtimes wasused.The12LTLTtreatmentswer eassignedtosamplesfromdifferentanimalsandfromdifferentlocationsonthemuscleinanincomplete blockdesign.EachLTLTtreatmentwasrepeated6timesforbothcows andyoungbulls. 2.3.Heattreatmentsandsampling Sampleswerethawedfor24hat4°Cpriortoheattreatment.Heat treatmentsofvacuumpackagedmeatsampleswereperformedin waterbaths(ICC  Roner Ž ,FrinoxAps,Hillerød,Denmark)setat53°C, 55°C,58°Cand63°C.Heattreatmentswerecarriedoutat3time points:21/2h(timetoreachthetemperatureofthewaterbathinthe centerofthesample),71/2h(holdingtime=5h)and191/2h(holdingtime=17h).Heatingrateswerecalculatedfromthelinearpartof theheatingpro lestobe1.12°C/minat53°C,1.27°C/minat55°C, 1.18°C/minat58°Cand1.38°C/minat63°C(n=3).Heatingwas arrestedbyplacingthesamplesinicewaterfor10min. Sampleswereweighedandcookinglossinpercentageofmeat weightbeforeheattreatmentwascalculated.Then,cookinglosswascollected,centrifugedfor15minat5072× g at4°Candsubsequentlyfrozen at 80°Cforinvestigationofcathepsinactivityandcollagensolubility. Heattreatedsampleswerestoredinvacuumbagsovernightat4°C. Warner … Bratzlersheartestwasthenperformed,andsubsequently sampleswerefrozenat 20°CforfurtherinvestigationbyDifferential ScanningCalorimetry(DSC)andHyperspectralImaging. 2.4.Hyper-spectralimaging VideometerLabhyper-spectralimages(VideometerA/S,Hørsholm, Denmark)werecollectedtoevaluatechangesincolorandmyoglobin formsintheheattreatedmeatsamples.Imageswerecapturedfrom asliceofmeatfromeachtreatmentplacedintheVideometerLab, whichwasequippedwith18colorchannelscenteredatfollowing wavelengths:430,450,470,505,565,590,630,645,660,700,850,870,890,910,920,940,950and970nm.Priortomeasurementsan intensitycalibrationwasperformedusingarangeofstandardcolors spanningthecolor-spaceregardingthecolorsoffreshandcookedbeef. Sampleswererepresentedasthe50%trimmedmeanintensityvalueof 350×400pixelsintheheartoftheobject(correspondingtoapproximately12cm2),resultinginaspectrumof18valuesforeachsample. Trimmingwasperformedtofocusonthemeatpartandnotinclude whitefatareasordarkercracksintheproduct.Themeasurements wererepeatedon6samplesfromeachtreatmentoncowsand3samples fromeachtreatmentonyoungbulls. 2.5.Warner – Bratzlershearforce Sixblocksof1×1×6cmwerecutfromtheheattreatedsamples.The Warner … Bratzlersheartestwasconducted3timesoneachblockbyan InstronequippedwithatriangularWarner … Bratzlertestcell.Theparametersmeasuredfromtheobtainedforce-deformationcurveswerepeak force(PF,themaximumforce)andinitialyield(IY,the rstmajorin ectionatthecurve).ThedifferencebetweenPFandIY(PF … IY)wasthen calculated. 2.6.ActivityofcathepsinsBandLincookingloss ActivityofcathepsinsBandLincookinglosswasanalyzedintriplicateasdescribedin Christensen,Ertbjerg,etal.(2011) .Inshort,15 l thawedcookinglosswasmixedwith135 lofbuffer(340mMsodium acetate,60mM100%aceticacid,4mMEDTA,0.1%Brij35,pH4.3)and incubatedwith100 lsubstrate(12.5 MZ-Phe-Arg-Nmec(Sigma)) for10minat40°C.Thereactionwasarrestedbyadding1mlbuffer (100mMNaOH,30mMsodiumacetate,70mM100%aceticacid, 100mMchloroaceticacid,pH4.3).Theexcitationandemissionwavelengthsof355nmand460nm,respectively,weremeasured.Standards weremadeby7-amino-4-methyl-coumarin(Sigma).Theactivitywas expressedin U/gmeatwhere1unit(U)wasde nedas1 molproduct producedperminuteat40°C. 2.7.Solublecollagenincookingloss Theamountofhydroxyprolinewasmeasuredinthecookingloss asdescribedin Kolar(1990) and Christensen,Ertbjerg,etal.(2011) . Fourreplicatesweremadeoneachsample,giving24replicatesper heattreatment.Afactorof7.14wasusedtocalculatetheamountof collagenfromhydroxyproline.Bymultiplyingtheconcentrationof collagenincookinglosswiththevolumeofcookingloss,theamount ofsolublecollagenincookinglosswasexpressedasmgsolublecollagen/gmeat. 2.8.Differentialscanningcalorimetry Differentialscanningcalorimetry(DSC)wasconductedinduplicate onheattreatedsamplesasdescribedin StabursvikandMartens(1980) and Christensen,Bertram,etal.(2011) .Brie y,approximately700mg samplewasplacedintoaDSCsamplecellandplacedintheDSC (MicroDSCIII,Setaram,Caluire,France)togetherwithareferencecell containingwater.DSCscanswererecordedfrom25°Cto90°Cata rateof1°C/min.ThethermogramswereanalyzedinCalistoversion 1.061(AKTSAB,Siders,Switzerland)wheretheenthalpyofdenaturation, Htotal(J/g),wasobtainedbyintegrationaftersubtractionof appropriatebaselines.Inordertoseparatethepeakscorrespondingto theenthalpies, H68°Cand H75°C,thethermogramsweredividedat 70°Cpriortointegration.788 L.Christensenetal./MeatScience93(2013)787 – 795

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2.9.Statisticalanalysis Analysisofvariancewasperform edusingSASSoftware9.2.Results onyoungbullsandcowswereanalyzedinseparatemodels,andthe modelsincludedtemperature,timeandtheirinteractionas xedeffects, whileanimalwasincludedasrandomeffect.Forcomparisonbetween youngbullsandcows,anothermodeladditionallyincluded xedeffects oftheinteractionswithtypeofanimal.InallanalysesLSmeanswere comparedusingtheBonferroniadjustment.Pearsoncorrelationcoef cientsbetweenthemeasuredparamet erswerecalculated.Fortheanalysisofthehyper-spectralimaging,PCAwasperformedonnormalized (usingStandardNormalVariatescaling)andcolumnmean-centered spectra.AllcomputationswereperformedinMatlabversionR2011b andin-houseroutines. 3.Results Thisstudyinvestigatedthecombinedeffectofheatingtemperatures between53°Cand63°Candheatingtimesbetween21/2hand191/ 2hontoughnessof semitendinosus fromcowsandyoungbullsandthe relationshiptopropertiesofconnectivetissue.Meattoughnesswas examinedbyWarner … Bratzlershearforce,andfromtheforce … deformationcurvesthechangesintheconnectivetissuecomponent wasinterpreted.Theunderlyingmechanismsinheat-inducedweakeningoftheconnectivetissuewerestudiedbyanalysisofcollagensolubilization,activityofcathepsinsBandL,andproteindenaturation (DSC).Inaddition,heat-inducedchangesinmeatcolorwereinvestigatedbyhyper-spectralimaging. Theresultsfromhyper-spectralimagingoftheLTLTtreatedSTfrom cows( Fig.1 )andyoungbulls( Fig.2 )aregivenasprincipalcomponent scoreandloadingplots.Forbothcowsandyoungbulls( Figs.1and2 , left)atendencyforclusteringaccordingtoheatingtemperatureand timewasobserved,however,theeffectwasmostevidentforsamples fromcows.The rstprincipalcomponent(PC1)oftheloadingplotswas primarilyaseparationoftheshort-wavenear-infrared(~900+nm) andthevisiblewavelengths( b 700nm)( Figs.1and2 ,right).The secondprincipalcomponent(PC2)oftheloadingplotsseparated samplesmainlybasedonrednessintensityofthemeatspanningfrom 565nm(absorptionwavelengthfordeoxymyoglobin)and590nm (absorptionwavelengthforoxymyoglobin)to630nm(metmyoglobin absorption).Thus,incows( Fig.1 )themeatbecamelessredwhen heatingtimewasincreasingfrom21/2hto191/2h,whiletheresults onyoungbulls( Fig.2 )showednocleartendencies.Dataanalysisfrom hyper-spectralimagingfortheamountsofthespeci cmyoglobin formsdidnotshowanyeffectsoftimeortemperature(datanot shown). Bothheatingtemperatureandheatingtimesigni cantlyaffected Warner … Bratzlerpeakforce(PF),cathepsinactivityandDSCenthalpies inSTfromyoungbullsandcows( Table1 ).Theamountofcookingloss andheat-solublecollageninSTfromyoungbullswereonlyaffectedby cookingtime.Approximately10%increaseincookinglossappeared whenheatingtimeincreasedfrom21/2hto191/2h( Table2 ).Incontrast,neitherheatingtemperaturenortimeaffectedcookinglossofST fromcows( Tables1and2 )andheat-solublecollagenwasonlysigni cantlyaffectedbyheatingtemperature( Table1 ). Inyoungbulls,WB-PFwassigni cantlyhigherwhenSTwasheated at53°Cfor21/2hcomparedtoallothertreatments( Table2).In cows,WB-PFofSTdecreasedwithincreasingtemperatureandonly minordecreasesofWB-PFwereobservedwithincreasedheatingtime, exceptat55°Cwhereasigni cantdecreaseinWB-PFwasobserved withincreasingtemperaturefrom21/2hto191/2h.Upto58°C, WB-PFwassigni cantlyhigherinSTfromcowscomparedwithyoung bullswhenheatingwasperformedfor21/2and71/2h.At63°Cno signi cantdifferencesinWB-PFbetweenthetwocategoriesofbeef wereobserved( Table2 ).Furthermore,at191/2h,nodifferencesin WB-PFwereobservedat55°C,butat53°Cand58°CWB-PFfromST ofcowsweresigni cantlyhigherthaninyoungbulls. Theinitialyield(IY)oftheforce-d eformationcurvesobtainedduring theWBsheartestwasalsomeasuredontheLTLTtreatedmeat(datanot shown).InyoungbullsIYdecreasedfromapproximately40Nto25N andincowsfromapproximately60Nto40Nwithincreasingtime andtemperature,however,thechangeswerenotsigni cant.Previous interpretationsoftheforce … deformationcurves( Bouton&Harris, 1981;Møller,1981 )haveindicatedthatIYvaluesprimarilyre ectthe strengthofthemyo brillarcomponents,whilePFvaluesrepresent theoveralltoughnessre ectingbothmyo brilsandconnectivetissue. Fig.1. Scoreandloadingplotsofthehyper-spectralimagingdataof semitendinosus fromcowsheatedat53°C,55°C,58°Cand63°Cfor21/2h,71/2hand191/2h. 789 L.Christensenetal./MeatScience93(2013)787 – 795

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Bouton,Harris,andShorthose(1975) arguedthatsubtractingIYfromPF (PF … IY)illustratethecontributionoftheremainingconstituentsofthe meat … connectivetissueandotherstructuresremainingaftertheyield ofthemyo brillarstructure … totheoveralltoughness.Toillustratethe contributionoftheconnectivetissuetothetoughnessofyoungbulls andcows,IYwassubtractedfromPF(PF … IY)andpresentedin Fig.3 , respectively.Inyoungbulls( Fig.3 A),PF … IYwassigni cantlyhigherat 53°Cfor21/2hcomparedtoallothertreatments.Fortemperatures above53°Candheatingtimesabove21/2hthelevelofPF … IYreached verylowlevelsandnosigni cantdifferenceswereobservedforthese treatments.PF … IYofSTfromcowsdecreasedsigni cantlybetween 55°Cand58°Candwasonlyaffectedbyheatingtime(191/2h)at 55°C( Fig.3 B). ActivityofcathepsinsBandLinthecookinglossdecreasedwithboth increasingtimeandtemperature,whilenodifferencesbetweenyoung bullsandcowswereobserved( Fig.3 ).Afterheatingfor21/2htheactivitywassigni cantlyhigherat53°Cand55°Ccomparedwith58°Cand 63°C,andafter71/2htheactivityat55°Cdecreasedsigni cantly.Upon 191/2hofheatingtheactivityat53°Calsodecreasedsigni cantly,and atthistimepointnosigni cantdifferenceswereobservedbetweenthe heatingtemperatures.Thesolubilityofcollagenincreasedwithincreasingtemperaturefrom53°Cto63°Cincowsandincreasingheating timefrom21/2hto191/2hat58°Cinyoungbulls( Fig.3 A).Thecollagensolubilitywasmuchgreaterintheyoungbullscomparedwithcows, especiallyafter191/2hofheattreat ment.ThePearsoncorrelationcoefcientbetweencookinglossandtheamountofsolubilizedcollagenin youngbullswas0.83( P b 0.001),whilenosigni cantcorrelationwas foundincows. PreviousstudiespresentingDSCthermogramsconductedonraw beefmusclesreportendothermicpeaksaround53°C,68°Cand77°C ( Parsons&Pattersons,1986;Stabursvik&Martens,1980 ).DSConLTLT treatedSTfromcowsandyoungbullsrevealed2endothermicpeaksat approximately68°C( H68°C)and75°C( H75°C)( Fig.4 ). Fig.4 showsarepresentativeDSCthermogramonSTfromyoungbullsheated at53°Cfor21/2,71/2and191/2h.Increasingheatingtimeat53°C changedthepatternoftheDSCthermogramstowardssmallerpeaks andafter191/2honlyonepeakwasobservedaround75°C.Thetotal areaofthepeaksisdesignated Htotal.Thechangesinenthalpies, Htotal, H68°Cand H75°C,duringheattreatmentofyoungbullsandcowsare illustratedin Fig.5 AandB,respectively.Incows, H68°C( Fig.5 B)could bedetectedatthe3heatingtimesat53°C,whileat55°Cthepeak wasonlypresentafter21/2hand71/2h,andat58°Cthepeakwas notpresentabove21/2hofheating.At63°C H68°Cwasnotpresent ineithercowsoryoungbulls. H75°Cand Htotal( Fig.5 )decreased withincreasingtemperatureandtime.At53°C H75°Cdecreasedsignificantlywithincreasingtimeinyoungbullsbutnotincows.ThePearson correlationcoef cientbetweencookinglossand H68°Cinyoungbulls was 0.66( P =0.08),whilenorelationshipwasfoundincows. 4.Discussion Amongalterationsinthemeatoccurringduringcookingaretoughnessandcolorchanges,bothofthembeingimportantwhenassessing eatingqualityofmeat.Meatcookedtoaninternaltemperaturebelow 60°Cisgenerallyconsideredrareandwithabrightredinterior.Changes incoloroccurringuponcookingre ectheat-inducedconversionsin Fig.2. Scoreandloadingplotsofthehyper-spectralimagingdataof semitendinosus fromyoungbullsheatedat53°C,55°C,58°Cand63°Cfor21/2h,71/2hand191/2h. Table1 Signi cantmaineffects( P b 0.05)oftemperature,timeandtheircrosseffects (temp time)forWarner … Bratzlerpeakforce(WB-PF),cookingloss,amountofsoluble collagen,activityofcathepsinsBandLandtheDSCenthalpies Htotal, H68°Cand H75°Cin semitendinosus fromcowsandyoungbulls. ParameterYoungbullsCows TempTimeTemp timeTempTimeTemp time WB-PF CookinglossNS NSNSNSNS SolublecollagenNS NS NSNS CathepsinsBandL Htotal H68°C NS H75°C NS NS=notsigni cant. P b 0.001. P b 0.01. P b 0.05. 790 L.Christensenetal./MeatScience93(2013)787 – 795

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themusclepigmentmyoglobin.Thethreeformsofmyoglobin,metmyoglobin,oxymyoglobinanddeoxymyoglobin,differinheatstability, withdeoxymyoglobinbeingmoststableat55°C( Hunt,Sørheim,& Slinde,1999 ).Inthepresentstudy,deoxymyoglobinisconsideredto bethedominantmyoglobinforminthemeatsincesampleswerevacuumpackaged,however,itcannotbeexcludedthatotherformsmight alsobepresentsincetheheattreatedsampleswereexposedtoatmosphericairduringmeasurement.Hyper-spectralimagingofSTfrom cowsandyoungbulls( Figs.1and2 )revealedaseparationofheating temperatureandtimeaccordingtorednessintensity(PC2),and showedthatasheatingtimeandpartlyheatingtemperatureincreased,themeatbecamelessred.ThisisinaccordancewithpreviousstudiesoncolorchangesinLTLTtreatedporcine( Christensen, Ertbjerg,etal.,2011 )andbovinemuscles( Christensenetal.,2012 ). Nocleardifferencesbetweenyoungbullsandcowscanberevealed fromtheseresults. Inthepresentstudy,toughnessofSTfromcowsandyoungbullswas reducedbyapplyingLTLTtreatments.This ndingisinagreementwith severalotherstudiesonprolongedheattreatmentsatlowtemperatures ofbeef(e.g. Beilkenetal.,1986;Christensenetal.,2012 ).Toughness (WB … PF)wassigni cantlyhigherincowscomparedwithyoungbulls atalltreatmentsexceptforthetre atmentsat63°Candafter191/2h at55°C.AdistinctdifferencebetweenSTfromcowsandyoungbullsis thethermalstrengthoftheconnectivetissueduetodifferentdegrees ofcross-linkingofthecollagenmolecules.Theheat-inducedchangesin thermalstrengthofconnectivetissuearere ectedinthePF … IYvalues, whicharetakenasameasureofconnectivetissuetoughness( Møller, 1981 ).From Fig.3 itappearsthataweakeningoftheconnectivetissue takesplaceduringLTLTtreatmentofSTfrombothcowsandyoung bulls.ItisevidentthatSTfromcowsrequireshighertemperaturesand longerheatingtimestoreducethetoughnesstothesamelevelasin youngbulls.After191/2hat63°CPF … IYvaluesapproachedthesame valuesinSTfromcowsasinyoungbulls.Inyoungbulls,PF … IYvalues werealreadymuchlowerafter71/ 2hat53°Cor21/2hat55°Ccomparedto53°Cfor21/2h.Theseresultssuggestthattheconnective tissuestrengthdecreasesmarkedlyinyoungbullsjustbyincreasing thetemperature2°Cfrom53°Cto55°Corincreasingtheheating timefrom21/2to71/2hat53°C. Beilkenetal.(1986) investigated theeffectofprolongedheattreatmentsat50°C,55°Cand60°Con musclesfromvealand17yearsoldsteers.Inveal,PF … IYdecreased drasticallywhenheatingtimewasincreasedupto4hat55°Cand 60°C,butnotat50°C.Inmusclesfromoldsteerstheauthorsobserved thatPF … IYdecreasedsigni cantlywhenheatingtimewasincreasedup to48hat55°Cand60°C,withthedecreasebeingfasterandlargestat 60°C.Inaccordance, BoutonandHarris(1972) foundthattextureattributesofbeefmusclescookedfor1hat40°Cto75°Cwerestronglyaffectedbyanimalageand BoutonandHarris(1981) heatedmeat samplesfromcattleofdifferentagesat50°Cand60°Cfor1or24h. Theauthorsshowedthatadhesionvalues(measureofconnectivetissue strength)ofmeatfrom1yearoldanimalsdecreasedwithincreasing temperaturefrom50°Cto60°C,butnoeffectofheatingtimewasdemonstrated.Resultsofsamplesfromolderanimals(4 … 8years)revealed thatincreasingtemperaturefrom50°Cto60°Chadnosigni canteffect onadhesionvaluesat1h,butat24hofheatingat60°Cadhesionvalues weresigni cantlydecreasedcomparedwith50°C.Theseresultsarein agreementwiththeresultsofthepresentstudy,wherelongerheating timeswererequiredtodecreasePF … IYincowsthaninyoungbulls. BoutonandHarris(1981) alsoinvestigatedtheeffectofheating1or 24hat50 … 65°ConshearvaluesofSTfromveal,steers(2 … 4years) andcows(8 … 15years).Inagreementwiththecurrentstudy Bouton andHarris(1981) observedthatasanimalageincreased,theheating temperaturerequiredtoobtainasigni cantdecreaseinshearvalue alsoincreased. Mortensenetal.(2012) performedsensoryanalysisofeatingqualityandfoundincreasedtendernessofSTfromyoungbullswith increasingheatingtimesfrom3to12hat56°C,58°Cand60°C.In accordance,decreasedshearforcewithincreasingheatingtimewas foundinthepresentstudy.Incontrasttotheresultsofthecurrent studyandpreviousstudiesmeasuringtendernessinstrumentally ( Beilkenetal.,1986;Bouton&Harris,1981;Christensenetal., 2000;Machlik&Draudt,1963 ), Mortensenetal.(2012) foundthat themeatwaslesstenderwhentemperaturewasincreasedupto 60°C.Thisdiscrepancymaybeduetodifferencesinheatingrates betweenthestudies. Mortensenetal.(2012) conductedsensoryanalysis onsmallersampleswhichallowedafasterequalizationofsample temperaturewithwater bathtemperature.Thedisagreementmayalso beduetoaninteractingeffectofsaltandtheheattreatmentappliedin thestudyof Mortensenetal.(2012) ,whereabrinecontaining7%salt and3%sugarwasaddedpriortoheattreatment.Theeffectoflow temperaturelongtimeheattreatmentsonthebehaviorofmeatproteins containingbrineandontheresultingtextureisunknown. Collagencontentsweremeasuredinthecookinglosssinceitwas assumedthatsolubilizedcollagendiffuseoutofthemusclewiththe meatjuiceduringcooking.Solublecollagenincookinglossappearto beagoodindicatorofcollagensolubilityastheamountofsolublecollagenwaslessincowscomparedtoyoungbulls( Fig.3 )inaccordancewith theincreasedthermalstabilityofcollagenfromolderanimals( Lepetit, 2007 ).Collagenisamajorpartoftheconnectivetissueandwhencollagendenatures,it rstshrinkandsubsequentlysolubilizes/gelatinizes. Weobservedincreasedcollagensolubilityduetoincreasedheating timeinyoungbullsandincreasedtemperatureincows,suggestingthat thereducedmeattoughnessafterLTLTtreatmentinvolvessolubilization ofcollagen. Thetemperatureatwhichtheheat-induceddenaturationof collagenoccursisstilldebated,andinLTLTtreatedbeefitseemsto bedependentonbothheatingtemperatureandtime.Inthepresent studyanendothermicpeakwasfoundbyDSCatapproximately 68°C,whichaccordingto StabursvikandMartens(1980) canbe ascribedtoconnectivetissue,sincethedenaturationtemperature ofisolatedconnectivetissuewasfoundbytheseauthorstobe67°C. Thecurrentstudyinvestigatedheatingtemperaturesbetween53°C and63°C,andtheenthalpiesat68°C( H68°C)decreasedwhenheating temperatureandtimewereincreased.Togetherwiththeincreased solubilityofcollagen,theresultssuggestthatdenaturationofconnective tissueinitiatesattemperatureslowerthan68°CinLTLTtreatedmeat.In agreement,porkconnectivetissued enaturationwasreportedtobegin Table2 LSmeansofcookingloss(CL)andWarner … Bratzlerpeakforce(WB-PF)inlowtemperature-longtimetreated semitendinosus fromcowsandyoungbullsat53°C,55°C,58°Cand 63°Cfor21/2,71/2and191/2h.Lettersa … frefertosigni cancebetweentreatments(n=6)andxandyrefertosigni cancebetweenproductionsystemswithintreatments. Time21/2h71/2h191/2hSE Temperature53°C55°C58°C63°C53°C55°C58°C63°C53°C55°C58°C63°C CL(%) Youngbulls21.3d24.2cd22.4d25.4cd28.0bc25.9bcd31.4ab29.4bc31.4ab29.7bc31.0ab35.7a2.0 Cows23.4ab27.1ab19.7b24.0ab26.6ab29.6a23.4ab27.9a27.1ab28.7a24.7ab24.7ab3.0 WB-PF Youngbulls68.6a x36.7bc x34.6bc x35.7bc35.1bc x34.0bc x28.2c x37.1bc31.3bc x28.3c33.6bc x37.8b5.2 Cows106.0ab y103.7ab y68.7cde y57.5def98.9ab y87.0bc y68.3cde y50.5ef115.0a y56.7def69.3cd y40.0f7.1 791 L.Christensenetal./MeatScience93(2013)787 – 795

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at57°C( Brüggemannetal.,2010 )to60°C( Voutila,Ruusunen,& Puolanne,2008 ).Still,itremainsunclearhowextensivethedenaturation ofconnectivetissueisbelow68°C.Recently, delPulgar,Gazquez,and Ruiz-Carrascal(2012) studiedcollagen bersduringprolongedheat treatmentsat60°Cand80°Cofporkcheeksbyusinghistological analysis.Thestudyshowedthatsamplescookedat60°Cforupto 12hconsistedofagreaterquantityofbroken,notcompletelydenaturedcollagen berscomparedwithsamplescookedat80°Cfor 12h.Furthermore,proteolyticenzymesweakeningtheconnective tissueorchangingtheconformationoftheproteinsduringheat treatmentmayalsoberesponsibleforthelowereddenaturation temperature.Theendothermicpeakat68°Chas,inadditionto collagen,alsobeenascribedtowater-solublesarcoplasmicproteins ( Martens,Stabursvik,&Martens,1982 ).Participationofdenatured sarcoplasmicproteinsinachievingtendermeathasbeenreported by Tornberg(2005) tobedependentonthedenaturedproteins Fig.3. LSmeansofPF … IY(N),solublecollagen(mg/g)andactivityofcathepsinsBandL( U/g)of semitendinosus fromyoungbulls(columnA)andcows(columnB)heatedat53°C, 55°C,58°Cand63°Cfor21/2h,71/2hand191/2h.Barsrepresentstandarderrorofmeans. 792 L.Christensenetal./MeatScience93(2013)787 – 795

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gluingthemeat berstogether.Accordingto Tornberg(2005) the viscous owthenbecomeslower,wherebythemeatbecomesmore brittleandthemeatismoreeasilyfractured. CathepsinsBandLinbeefhavepreviouslybeenshowntobereleased fromtheircontainmentwithinthelysosomeswithdecreasingpHand withincreasingstoragetimeinthetemperatureregionupto30°C ( Ertbjerg,Larsen,&Møller,1999;Ertbjerg,Mielche,Larsen,&Møller, 1999 ).InthepresentstudytheactivityofcathepsinsBandLintheexpelledcookingloss( Fig.3 )washighestafterLTLTtreatmentat53°C for21/2handthendecreasedwithincreasingtemperatureandtime, whichisinagreementwiththeresultsfoundinporcinemusclesafter LTLTtreatments( Christensen,Ertbjerg,etal.,2011 ).Asigni cant amountofcathepsinsBandLactivitywasstillmeasureableevenafter 191/2hat63°C,suggestingthattheseenzymesduringtheentireheat treatmenthadthepotentialtocontributetothemechanicalweakening ofthemuscleproteinsincludingtheconnectivetissue.However,the in uenceofotherenzymesystemssuchascollagenasesontheobserved activitycannotbeexcludedinthecurrentstudy.NodifferencesinactivityofcathepsinsBandLbetweenyoungbullsandcowswereobserved suggestingthatequalpotentialforproteolyticdegradationduringLTLT treatmentexisted.Heat-inducedunfoldingofthecollagentriplehelix structuremayrenderthepeptidechainmoresusceptibletodegradation byproteolyticenzymesandtherebyincreasethesolubility.Cathepsins BandLareendopeptidasescatalyzinghydrolysisofinternalpeptide bondsofproteins( Agarwal,1990 ),andhaspreviouslybeensuggested tobeinvolvedinweakeningofcollagenleadingtoincreasedsolubility ( Christensen,Ertbjerg,etal.,2011 ). Beltran,Bonnet,andOuali(1992) found,byinvestigatingtheeffectofcathepsinBonisolatedconnective tissuefromcalfandsteers,thattheproteolyticeffectofcathepsinB wasgreateronconnectivetissuefromcalf.Theysuggestedthatthe higherthermalstabilityofcollagenfromsteersaccountedforthe lowersusceptibilitytoproteolysisbycathepsinB.Furthermore,incubationofconnectivetissuewithcathepsinBsigni cantlydecreasedthe denaturationtemperatureofconnectivetissuefrombothcalfand steer.Additionally,resultsfrom Burleigh,Barrett,andLazarus(1974) indicatedthatcathepsinBplaysanessentialpartinthedegradationof bothsolubleandinsolublecollagenbyeliminatingtheintermolecular cross-links.TheactionofcathepsinBoncollagenmayincreasethe solubilityoftheinsolublepart,and/ordecreasethedenaturationtemperatureandtherebythetemperatureatwhichsolubilizationoccurs. Additionally,cathepsinBattacksmyo brillarcomponentsofthemeat ( Baron,Jacobsen,&Purslow,2004 )andtherebypossiblycontributes toreducedtoughnessofthemyo brillarcomponentduringLTLT treatments.Previously,calpain( Ertbjergetal.,2012 )andcollagenase ( Laakkonen,Sherbon,etal.,1970;Laakkonen,Wellington,etal.,1970 ) havebeenstudiedduringLTLTtreatmentsofmeat. Ertbjergetal. (2012) foundthecalpainstoberapidlyinactivatedat55°C,whilecollagenasewasactiveupto58 … 60°Cwhenheldforupto6h,whichwasalso thetimetoachieveasigni cantloweringoftheshearforce( Laakkonen, Sherbon,etal.,1970;Laakko nen,Wellington,etal.,1970 ).However,furtherinvestigationsofcollagenaseactivityduringLTLTtreatmentofmeat areneededinordertoelucidatepossibleinvolvementoftheseenzymes inthetenderizationprocess. Inyoungbullscookinglossincreasedwithincreasingheatingtime togetherwithincreasedsolubilizationofcollagen,andthePearsoncorrelationcoef cientbetweencookinglossandsolubilizedcollageninyoung bullswas0.83( Pb 0.001),howeverthiscorrelationwasnotobserved ( P >0.1)incows.Thismaybecausedbythelowdegreeofheat-stable cross-linksinyounganimals( Shimokomaki,Elsden,&Bailey,1972 ) whichallowssolubilizationofcollagenduringtheheattreatment. Cookinglossisregardedtore ectthecontractionofconnectivetissue, butthiscontractionmainlyoccursabove60°C( Lepetit,Grajales,& Favier,2000 )andwouldbeexpectedtobemoresevereforcowsthan youngbullsaccordingtothetheoryofhigherfrequencyofheat-stable cross-linksinolderanimals.Thedegreeofcontractionoftheconnective tissueseemstobelessinLTLTtreatedmeatsamples.Aprerequisitefor thecontractionoftheconnectivetissueisdenaturationofthetropocollagenmolecule,whichaccordingtotheDSCmeasurements( H68°C)do occurduringLTLTtreatment.Theexplanationmightbethatenzymes disintegratedthecollagenstructur ebeforeanycontractionoccurred, andthesubsequentdenaturationofdi sintegratedconnectivetissuein turncausedlesscontractionunderLTLTconditions.Pearsoncorrelation coef cientsbetween H68°Candcookinglossincowsandyoungbulls supportthishypothesis,sinceanegativecorrelation( 0.66; P =0.08) wasfoundinyoungbullswhilenosigni cantrelationshipwasfound incows.Iftheheat-stabilityoftheconnectivetissueishigherincows thaninyoungbulls,thentheconnectivetissueincowsmaydenature athighertemperaturesthaninyoungbulls,suggestingthattheproteolyticenzymeshavemoretimetodisintegratetheconnectivetissuein cowscomparedtoyoungbullspriortocontraction.Underthespecial conditionsofcookingforlongtimeatlowtemperature,theconnective tissuemaytherebycontracttoalowerdegreeincowsandhencein uencethecookinglosslesscomparedtoyoungbulls.However,theconnectivetissueofSTcontainshighamountsofelastin(37%)whichis mechanicallyquitestrong( Bendall,1967;Rowe,1986 ).Thismayhave in uencedtheresultsfromthecurrentstudyandmaythereforenotbe generalanddirectlyapplicableinothermuscles. ThesecondendothermicpeakfromtheDSCpatternaroseat approximately75°C,andthispea khaspreviouslybeenascribedto denaturationofactinandreportedtooccuratapproximately77°C ( Stabursvik&Martens,1980 ).Inthecurrentstudy H75°Cgradually decreasedwithincreasingtemperatureandtime( Fig.5 ),whichsuggests thatactindenaturationinitiateatl owertemperaturesthanpreviously reportedwhensamplesareheatedforprolongedtime.Proteolytic enzymesdegradingmyo brillarproteinsduringheattreatmentmaybe involvedbyweakeningthestructuretobemorevulnerabletowards denaturationorchangetheconformationoftheproteinsandthereby thedenaturationtemperature. Martensetal.(1982) reportedthatsensoryassessedmeat rmnessincreasedbetween63°Cand73°C,andfound thatitcorrespondedtoactindenatur ation.Actindenaturationisalso suggestedtooccurinthepresentstudysinceasigni cantdecreasein H75°Cat53°Cwithincreasingheatingtimeswasobservedinyoung bullstogetherwithasigni cantdecreaseintoughness(PF)from21/ 2hto71/2h.Inaddition, H75°Chadalmostvanishedafter71/2h and191/2hat63°Candwassupportedbyasigni cantincreasein theoveralltoughness(PF)ofyoungbullsafter191/2hfrom55°Cto 63°C. Theendothermicpeaks( H68°Cand H75°C)graduallydisappear withincreasingheatingtemperatureandtime.Thereforebothtemperatureandtimeofheattreatmentareimportantfactorsin uencingthe Fig.4. DSCthermogramsof semitendinosus fromyoungbullsafterheattreatmentat 53°Cfor21/2h,71/2hand191/2h. 793 L.Christensenetal./MeatScience93(2013)787 – 795

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degreeofdenaturation. ParsonsandPattersons(1986) heatedbovine longissimusdorsi tointernaltemperaturesof55°C,60°Cand65°C andalsoobservedtwoendothermicpeaks:at66°Cand79°C.The peakat66°Cvanishedgraduallybetween50°Cand70°C,whilethe peakat79°Chadcompletelyvanishedat80°C.Alsoinbeef Bertola, Bevilacqua,andZaritzky(1994) foundthatthispeakvanished after60minofheatingat68°C.ThethermogramsobtainedbyDSC measurementsmayalsobeaffectedbyheatingrates.Iftheheating rateisslowasinthecurrentstudydenaturationmaytakeplaceat lowertemperatures,whichmightexplaintheslightlylowerdenaturationtemperaturesfoundinLTLTtreatedmeat.Theslowheatingrate mayalsogiverisetoconformationalchangesintheproteins,andtherebychangetheDSCpatternasseenin Fig.5 .However,since Htotaldecreasedwithincreasingtemperatureandtimeitseemsthatsome Fig.5. LSmeansofDSCenthalpies(J/g)( Htotal, H68°Cand H75°C)of semitendinosus fromyoungbulls(columnA)andcows(columnB)heatedat53°C,55°C,58°Cand63°Cfor 21/2h,71/2hand191/2h.Barsrepresentstandarderrorofmeans. 794 L.Christensenetal./MeatScience93(2013)787 – 795

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proteinshadalreadydenatured,orchangedconformationduetoe.g.proteolysis,andtherebypossiblychangedtheirdenaturationtemperature. Insummary,toughnessofSTincowsandyoungbullsdecreaseatdifferenttime … temperaturecombinations.Inordertoreducethetoughness ofSTfromcowstothesamelevelasyoungbullsslightlyhighertemperaturesandprolongedheatingtimesarerequired.Thereducedtoughness ofSTinducedbytheLTLTtreatmentaresuggestedtoresultfromaweakeningoftheconnectivetissue,becausedpartlybydenaturationorconformationalchangesoftheproteinsand/orpartlybysolubilizationof collagen.Thesolubilizationofcollagenmayresulteitherfromheatinducedbreakageoftheheat-labilecross-linksand/orbyincreasedenzymaticweakeningcausedbyproteolyticenzymessuchascathepsinsBand L.Underthespecialconditionsofcookingforlongtimeatlowtemperature,theconnectivetissueappearstocontracttoalowerdegreein cows.Hence,theconnectivetissuein uencethecookinglosslessin cowscomparedtoyoungbulls. 5.Conclusion Increasedheatingtemperaturefrom53°Cto58°Candtimefrom2 1/2to191/2hat55°Csigni cantlyreducedthetoughnessof semitendinosus fromcows.InSTfromyoungbullsasigni cantreduction oftoughnessoccurredwhentemperaturewasincreasedfrom53°Cto 55°Candwhentimewasincreasedfrom21/2hto71/2hat53°C. Areductionoftoughnessincowsrequiredhigherheatingtemperature andlongerheatingtimescomparedwithyoungbulls.After191/2hat 63°Cthemeatfromcowsandyoungbullswereequallytender. Thestrengthofconnectivetissuedecreasedwithincreasingheating temperatureandtime,andsimultaneouslycollagensolubilization increased,suggestingthatweakeningofconnectivetissueoccursduring LTLTtreatmentandpartlyaccountforthedecreasedtoughnessobserved atthesetime … temperaturecombinations.DSCmeasurementsshowinga concomitantdecreasein H68°Csupportedthishypothesis.Inaddition, rednessintensityandactivityofcathepsinsBandLdecreasedwith increasingheatingtemperatureandtime. Acknowledgments TheauthorsthankIdaSørensenandLiseAndersen(Universityof Copenhagen)forlaboratoryassistance,andacknowledgethe nancial supportfromtheDanishMinistryofFood,AgricultureandFisheries fortheprojectentitled  SafetyandgastronomicqualityofnewLTLT treatmentsofmeat Ž . ReferencesAgarwal,S.K.(1990).Proteasescathepsins „ Aview. 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