impact of wet distillers grains plus solubles and antioxidants on a basic mechanism of beef

195 Pages·2017·2.78 MB·English

University of Nebraska - Lincoln
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Theses and Dissertations in Animal Science Animal Science Department
Spring 5-2015
IMPACT OF WET DISTILLERS GRAINS PLUS
SOLUBLES AND ANTIOXIDANTS ON A
BASIC MECHANISM OF BEEF
TENDERIZATION
Michael D. Chao
University of Nebraska-Lincoln, [email protected]
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Chao, Michael D., "IMPACT OF WET DISTILLERS GRAINS PLUS SOLUBLES AND ANTIOXIDANTS ON A BASIC
MECHANISM OF BEEF TENDERIZATION" (2015).Theses and Dissertations in Animal Science. 105.
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IMPACT OF WET DISTILLERS GRAINS PLUS SOLUBLES AND ANTIOXIDANTS
ON A BASIC MECHANISM OF BEEF TENDERIZATION

by

Michael Chao

A DISSERTATION

Presented to the Faculty of
The Graduate College at the University of Nebraska
In Partial Fulfillment of Requirements
For the Degree of Doctor of Philosophy

Major: Animal Science

Under the Supervision of Professor Chris R. Calkins

Lincoln, Nebraska

May, 2015
IMPACT OF WET DISTILLERS GRAINS PLUS SOLUBLES AND ANTIOXIDANTS
ON A BASIC MECHANISM OF BEEF TENDERIZATION

Michael Chao, Ph.D.
University of Nebraska, 2015

Adviser: Chris R. Calkins
Feeding high levels of wet distillers grains plus solubles (WDGS) increases
polyunsaturated fatty acid (PUFA) levels in beef. Perhaps, WDGS in feedlot diets
increases PUFA concentration in the SR membrane, thereby altering membrane integrity,
resulting in more rapid calcium leakage and improved tenderness. Feeding antioxidants
may mitigate such effects. In the first study, effects of feeding 50 % WDGS on SR
membrane composition, free calcium concentration and tenderness were studied. The SR
membrane from steers fed WDGS were more tender, had higher free calcium
concentration, had more PUFA, more phosphatidylcholine (PC), less
phosphatidylethanolamine (PE) and less total phospholipids when compared to SR
membrane from steers fed corn-only (P < 0.05). In the second study, effects of feeding
30% WDGS and two antioxidants on muscle tissue fatty acid profiles, color and lipid
oxidation of beef were studied. Steaks from cattle fed WDGS had increased muscle tissue
PUFA, were not different in color, and were less oxidized when compared to steaks from
steers fed corn-only (P < 0.05). Supplementing vitamin E (E) was very effective in
reducing discoloration and lipid oxidation, and increasing muscle tissue E levels
compared to the diets without E supplementation (P < 0.05). Feeding Agrado Plus (AG)
had minimal effect of discoloration and lipid oxidation, but significantly increased
muscle tissue ethoxyquin level compared to diets without AG supplementation. In the
third study, effects of feeding WDGS and the two antioxidants on SR membrane
composition and proteolysis were studied. Again, feeding WDGS increased total PUFA
and PC, but decreased PE in the SR membrane (P < 0.05). Conversely, E
supplementation in WDGS diet prevented such shifts, while supplementing AG in the
corn-only diet created similar effects as the WDGS-only diet for SR membrane
phospholipid profile. At 2 d postmortem, steaks from steers fed WDGS had more
troponin-T degradation compared to steaks from steers fed corn-only or either diet
supplemented with E only (P < 0.05). Feeding WDGS induced SR membrane
phospholipid shift and clearly contributed to cattle SR membrane instability, while E
supplementation may prevent such phenomenon.

Key words: Beef, distillers grains, antioxidants, sarcoplasmic reticulum.
iv
ACKNOWLEDGEMENTS
I would like to thank my major professor, Dr. Chris Calkins, for giving me the
chance to be a member of the UNL Meat Science Group, an experience that has enriched
my life more than words can describe. I am grateful for his indispensable advice as a
mentor, which not only led to the completion of this dissertation, but also taught me to
always challenge myself to be a better person and scientist that generates positive
influence to the community.
I would also like to thank my supervisory committee: Dr. Donald Beermann, for
serving as the co-chair of my committee during time in need, and always providing
helpful advice when I was lost; Dr. Dennis Burson, for sharing with me his extensive
knowledge on real world issues; Dr. Timothy Carr and Dr. Madhavan Soundararajan for
teaching me everything I know about lipid and protein research methodologies. None of
this would have happened without them.
Big thanks to all the supporting staff: chemistry lab manager Tommi Jones and
her student workers for assisting in my lab work and ordering anything and everything I
needed; meat lab manager Calvin Schrock and his student workers for teaching me how
to slaughter and fabricate and helping me to prepare my samples in the meat lab; the
meats group secretary, Sherri, for keeping everything in line; Dr. Carr’s lab manager
Anjeza Pashaj, for assisting me in my lab work and running all of my fatty acids samples;
Dr. Moreau’s lab manager Mengna Xia, for giving me advices on gel electrophoresis and
Western Blotting.
Furthermore, many thanks to all the past and current graduate students: Lasika
Senaratne-Lenagala, Michelle Semler, Kim Varnold, Derek Schroeder, Katherine
v
Domenech-Perez, Kelby Sudbeck, Amy Redfield, Brandy Cleveland, Chad Bower, Sara
Sieren, Joe Buntyn, Kelly McCarthy, Regan Stanley, Hope Voegele, Alessandra Rosa,
Kenni Nubiato and Emery Kunze for their assistance and company along this project,
even if it involved meeting at 3 am for sample collection. Without them, this trial would
not be such a success. Special thanks to Dr. Steven Jones, Dr. Ty Schmidt and Dr. Gary
Sullivan for their warm hearts, inspiration and suggestions, both academic and personal. I
also want to thank the University of Nebraska Research Council and National
Cattlemen’s Beef Association for partially funding this project.
I want to express gratitude to my parents, Kung and Marjorie Chao, for their
confidence and ongoing support. Most importantly, I want to thank my wife, Ying Chen,
and my son Luke Chao. They were here, giving me love and strength to sustain me in
every circumstance; they had faith in the path I chose and believed unconditionally in my
success. I cherish their patience, kindness, and optimism, which have helped me to reach
the place where I now am. The tears and joys they shared with me have made this journey
truly unforgettable.
vi
TABLE OF CONTENTS
TITLE PAGE........................................................................................................................i
ABSTRACT………………………………………………………………………….…...ii
ACKNOWLEDGEMENTS................................................................................................iv
TABLE OF CONTENTS....................................................................................................vi
INTRODUCTION………………………………………………………………………1
LITERATURE REVIEW…………………………………………………………………4
Sarcoplasmic reticulum (SR): The calcium reservoir..........................................................4
Transverse-tubule (T-tubule) and SR structures......................................................5
Type of SR proteins….............................................................................................5
Luminal calcium-binding proteins……………………..…….................................5
Calcium release channels………………………………….....................................7
Calcium uptake…………………….…….............................................................10
SR protein oxidation may induce rapid calcium release postmortem....................12
Lipid oxidation of muscle food……………......................................................................12
Membrane structure...............................................................................................14
Membrane lipid oxidation......................................................................................17
SR membrane oxidation leading to accelerated calcium leakage..........................18
Role of dietary antioxidants in our hypothesis…………………………………………..21
Type of antioxidants..............................................................................................22
Dietary antioxidants as an approach to prevent membrane lipid oxidation...........25
Myofibrillar protein degradation........................................................................................28
Muscle structure.....................................................................................................28
vii
Proteinase system……………………………………………………...................30
Mechanism of tenderization…………………………………...............................34
Conclusion.........................................................................................................................36
MATERIAL AND METHODS.........................................................................................39
Manuscript 1: Feeding wet distilers grains plus solubles contributes to sarcoplasmic
reticulum membrane instability………………………………………………………….39
Animals…………………………………………………………………………..39
Sample collection and preparation……………………………………………….39
Warner-Bratzler Shear Force (WBSF)…………………………………………...40
Free calcium concentration………………………………………………………41
Lipid oxidation…………………………………………………………………...42
Sarcomere length………………………………………………………………...42
Myofibrillar protein extraction…………………………………………………..43
Protein concentration………………………….…………………………………44
Gel electrophoresis……………………………….………………………..……..44
Western Blotting…………………………………………………………………45
Sarcoplasmic reticulum (SR) membrane extraction……………………………..46
SR membrane lipid extraction…………………………………………………...46
SR membrane fatty acids………………………………………………………...47
SR membrane phospholipids, neutral lipids and total lipids……………………..48
Statistical analysis………………………………………………………………..49
Manuscript 2: Effects of dietary antioxidant supplementation of cattle finished with 30%
wet distillers grains plus solubles on fatty acid profiles and display life……………...…49
viii
Animals…………………………………………………………………………..49
Sample collection and preparation……………………………………………….50
Discoloration……………………………………………………………………..51
Objective color…………………………………………………………………...51
Muscle tissue lipid extraction……………………………………………………52
Muscle tissue fatty acids…………………………………………………………52
Lipid oxidation…………………………………………………………………...52
Vitamin E extraction…………………….……………………………………….53
Ethoxyquin extraction……………………………………………………………53
Statistical analysis………………………………………………………………..54
Manuscript 3: Feeding vitamin E may reverse sarcoplasmic reticulum membrane
instability caused by feeding wet distillers grains plus solubles to
cattle……………………………………………………………………………...………55
Statistical analysis………………………………………………………………..55
LITERATURE CITED…………………………………………………………………..56
MANUSCRIPT 1 – Feeding wet distillers grains plus solubles contributes to
sarcoplasmic reticulum membrane instability………………………………………..….70
Abstract..................................................................................................................71
Introduction...........................................................................................................72
Material and Methods............................................................................................73
Results...………………………………………………………………………….81
Discussion..............................................................................................................82
Implications............................................................................................................87
ix
Literature cited.......................................................................................................89
Tables.....................................................................................................................94
Figures....................................................................................................................97
MANUSCRIPT 2 – Effects of dietary antioxidant supplementation of cattle finished with
30% wet distillers grains plus solubles on fatty acid profiles and display life…………102
Abstract................................................................................................................103
Introduction..........................................................................................................104
Material and Methods..........................................................................................105
Results…………………………………………………………………………..110
Discussion............................................................................................................113
Conclusion...........................................................................................................119
Literature cited.....................................................................................................120
Figures..................................................................................................................124
Tables...................................................................................................................126
MANUSCRIPT 3 – Feeding vitamin E may reverse sarcoplasmic reticulum membrane
instability caused by feeding wet distillers grains plus solubles to cattle………..…….130
Abstract...............................................................................................................131
Introduction.........................................................................................................132
Material and Methods..........................................................................................134
Results…………………………………………………………………………..141
Discussion............................................................................................................143
Conclusion...........................................................................................................151
Literature cited....................................................................................................152