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Effects of Packaging System, Fat Concentration and Carbon Monoxide on Microbiology, Sensory and Physical Properties of G...

Permanent Link: http://ufdc.ufl.edu/UFE0022208/00001

Material Information

Title: Effects of Packaging System, Fat Concentration and Carbon Monoxide on Microbiology, Sensory and Physical Properties of Ground Beef Stored at Plus or Minus 1 Degree Celsius for 25 Days
Physical Description: 1 online resource (70 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: atmosphere, beef, carbon, color, enhancement, ground, modified, monoxide, packaging
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The overall appearance of a fresh meat product at the time of purchase is important to consumers as it may affect their purchasing decisions. The objective of this study was to evaluate the effects of three packaging treatments (modified atmosphere MAP containing 0.4% carbon monoxide CO and 30% carbon dioxide CO2, vacuum packaging VP and polyvinyl chloride PVC overwrap), three fat treatments (10, 20 and 30% fat) and storage time on microbiology, sensory and physical properties of ground beef stored at 4 plus or minus 1 degree Celsius for 25 days. The three packaging and three fat treatments were analyzed for objective color, pH, microbiology (total aerobes, total psychrotrophs, Gram negatives, E.coli O157:H7, total coliforms and generic E. coli) and thiobarbituric acid reactive substances (TBARS). Lactic acid bacteria analysis was conducted only in the MAP and VP treatments. Headspace and residual CO analyses were conducted only in the MAP treatments. Consumer panels were conducted to evaluate color and off-odors. The CO concentrations of the package and the meat, respectively, were deemed harmless at the levels detected in this study. There were no significant increases (P > 0.05) in TBARS for the MAP and VP treatments throughout the storage time. Modified atmosphere and vacuum packaging treatments showed a significant decrease (P < 0.05) in pH values by Day 14, whereas PVC treatments had significantly higher (P < 0.05) pH values by Day 7. Modified atmosphere and vacuum packaging had a bacteriostatic effect on Gram negative microorganisms. The growth of aerobic, psychrotrophic and lactic acid bacteria was not inhibited by MAP or VP. Within the MAP and VP groups, the 10% and 30% fat treatments, respectively, reached aerobic plate counts and total psychrotroph counts above spoilage levels ( > 6 log CFU/g). Furthermore, consumer color and off-odor results revealed that discoloration and off-odors were detected in the 10% and 30% fat treatments within the MAP and VP groups, respectively. Objective color analysis revealed that significant decreases (P < 0.05) in a* values of MAP treatments had occurred by Day 21. However, consumer color scores were similar (P > 0.05) for all MAP treatments when compared to Day 0. Consumers should rely on 'use by' dates on the package and not on the color of the meat as indicators of freshness.
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, 2008.
Local: Adviser: Williams, Sally K.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022208:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022208/00001

Material Information

Title: Effects of Packaging System, Fat Concentration and Carbon Monoxide on Microbiology, Sensory and Physical Properties of Ground Beef Stored at Plus or Minus 1 Degree Celsius for 25 Days
Physical Description: 1 online resource (70 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: atmosphere, beef, carbon, color, enhancement, ground, modified, monoxide, packaging
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The overall appearance of a fresh meat product at the time of purchase is important to consumers as it may affect their purchasing decisions. The objective of this study was to evaluate the effects of three packaging treatments (modified atmosphere MAP containing 0.4% carbon monoxide CO and 30% carbon dioxide CO2, vacuum packaging VP and polyvinyl chloride PVC overwrap), three fat treatments (10, 20 and 30% fat) and storage time on microbiology, sensory and physical properties of ground beef stored at 4 plus or minus 1 degree Celsius for 25 days. The three packaging and three fat treatments were analyzed for objective color, pH, microbiology (total aerobes, total psychrotrophs, Gram negatives, E.coli O157:H7, total coliforms and generic E. coli) and thiobarbituric acid reactive substances (TBARS). Lactic acid bacteria analysis was conducted only in the MAP and VP treatments. Headspace and residual CO analyses were conducted only in the MAP treatments. Consumer panels were conducted to evaluate color and off-odors. The CO concentrations of the package and the meat, respectively, were deemed harmless at the levels detected in this study. There were no significant increases (P > 0.05) in TBARS for the MAP and VP treatments throughout the storage time. Modified atmosphere and vacuum packaging treatments showed a significant decrease (P < 0.05) in pH values by Day 14, whereas PVC treatments had significantly higher (P < 0.05) pH values by Day 7. Modified atmosphere and vacuum packaging had a bacteriostatic effect on Gram negative microorganisms. The growth of aerobic, psychrotrophic and lactic acid bacteria was not inhibited by MAP or VP. Within the MAP and VP groups, the 10% and 30% fat treatments, respectively, reached aerobic plate counts and total psychrotroph counts above spoilage levels ( > 6 log CFU/g). Furthermore, consumer color and off-odor results revealed that discoloration and off-odors were detected in the 10% and 30% fat treatments within the MAP and VP groups, respectively. Objective color analysis revealed that significant decreases (P < 0.05) in a* values of MAP treatments had occurred by Day 21. However, consumer color scores were similar (P > 0.05) for all MAP treatments when compared to Day 0. Consumers should rely on 'use by' dates on the package and not on the color of the meat as indicators of freshness.
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, 2008.
Local: Adviser: Williams, Sally K.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022208:00001


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1 EFFECTS OF PACKAGING SYSTEM FAT CONCENTRATION AND CARBON MONOXIDE ON MICROBIOL OGY, SENSORY AND PHYSICAL PROPERTIES OF GROUND BEEF STORED AT 4 PLUS OR MINUS 1 DEGREE CELSIUS FOR 25 DAYS By NICOLAS ARMANDO LAVIERI 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 2008

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2 2008 Nicolas Armando Lavieri

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3 ACKNOWLEDGMENTS I would lik e to show my grat itude to Dr. Sally K. Williams, my supervisory committee chairperson for the opportunity and support. I al so extend my gratitude to the other committee members, Dr. Hordur Kristinsson and Dr. Keith Schneider, for their collaboration and advice. I also wish to extend my appreciation to Mrs. Alba Ruiz, Ms. Noufoh Djeri and Mr. Frank Robbins for their friendship, support and en couragement. To the employees of the Meat Science laboratory and fellow gr aduate students, I offer recogn ition for the assistance provided. Appreciation and love are expressed to Armando and Yleny Lavieri, my parents, and to my siblings Maria, Amanda a nd Victor Lavieri for their cons tant support. I thank Juliana Campos for her love, friendship, support and advice.

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4 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................3 LIST OF TABLES................................................................................................................. ..........6 ABSTRACT.....................................................................................................................................8 CHAP TER 1 INTRODUCTION..................................................................................................................10 2 REVIEW OF LITERATURE.................................................................................................12 Carbon Dioxide in Modified Atmosphere Packaging............................................................ 12 Effects of Carbon Dioxide on the Microflora of Fresh Meat..........................................12 General Properties of Carbon Dioxide............................................................................14 Carbon Monoxide in Modified Atmosphere Packaging......................................................... 17 Effects of Carbon Monoxide on th e Microflora of Fresh Meat ....................................... 17 General Properties of Carbon Monoxide.........................................................................18 Oxygen in Modified Atmosphere Packaging......................................................................... 21 Effects of Oxygen on the Microflora of Fresh Meat....................................................... 21 General Properties of Oxygen.........................................................................................21 Nitrogen Gas in Modified Atmosphere Packaging.................................................................23 Effects of Nitrogen on the Microflora of Fresh Meat ......................................................23 General Properties of Nitrogen........................................................................................ 24 Regulatory Status for the Use of Carbon M onoxide in Modified Atm osphere Packaging Systems...............................................................................................................................24 Current Applications of Carbon Monoxide in the Food Industry ........................................... 25 Disadvantages of Modified Atmosphere Packaging Systems................................................25 3 MATERIALS AND METHODS........................................................................................... 28 Phase 1: Formulation, Packagi ng and Storage of Ground Beef ............................................. 28 Formulation.................................................................................................................... .28 Sample Treatment............................................................................................................28 Packaging, Gas Injection a nd Storage of Ground Beef ...................................................29 Phase 2: Evaluation of Shelf Life Parameters........................................................................ 29 Objective Color...............................................................................................................30 Microbial Analyses.......................................................................................................... 30 Analysis of pH.................................................................................................................32 Headspace Carbon Monoxide.......................................................................................... 32 Residual Carbon Monoxide.............................................................................................33 Thiobarbituric Acid Reactive Substances....................................................................... 34 Consumer Sensory Panel Analysis.................................................................................. 34 Statistical Analysis.......................................................................................................... 35

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5 4 RESULTS AND DISCUSSION............................................................................................. 37 Evaluation of Ground Beef Stored in Po lyvinyl C hloride Overwrap for 7 d......................... 37 Microbiological Analyses................................................................................................37 Aerobic plate counts................................................................................................. 37 Gram negative counts............................................................................................... 37 Psychrotroph counts.................................................................................................38 Analysis of pH.................................................................................................................38 Evaluation of Ground Beef Stored in Modi fied Atmosphere, Vacuum Packaging and Polyvinyl Chloride Overwrap for 25 d................................................................................ 38 Microbiological Analyses................................................................................................38 Aerobic plate counts................................................................................................. 38 Gram negative counts............................................................................................... 39 Lactic acid bacteria counts....................................................................................... 40 Psychrotroph counts.................................................................................................40 E. coli O157:H7 counts ............................................................................................41 Total coliforms and E. c oli counts...........................................................................41 Product Analyses.............................................................................................................42 Analysis of pH..........................................................................................................42 Thiobarbituric acid reactive substances...................................................................44 Objective Color Evaluation............................................................................................. 45 L* values..................................................................................................................45 b* values...................................................................................................................46 a* values................................................................................................................... 46 Consumer Sensory Evaluation........................................................................................ 48 Color.........................................................................................................................48 Off-odors..................................................................................................................49 Carbon Monoxide Analyses............................................................................................50 Headspace carbon monoxide.................................................................................... 50 Residual carbon monoxide....................................................................................... 51 5 SUMMARY AND CONCLUSIONS.....................................................................................61 LIST OF REFERENCES...............................................................................................................63 BIOGRAPHICAL SKETCH.........................................................................................................70

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6 LIST OF TABLES Table page 2-1 Advantages and disadvantages of modifi ed atm osphere packaging systems as they apply to fresh meat products.............................................................................................. 27 3-1 Treatments evaluated during a 25-d storag e p eriod to determine the effects of packaging system, fat concentration and carbon monoxide on the storage stability of ground beef........................................................................................................................36 3-2 Gas Chromatograph settings used for analyzing packaged ground beef for carbon monoxide gas headspace and residuals in the m eat........................................................... 36 4-1 Mean total aerobic counts for ground beef stored at 4 1C fo r 7 days in polyvinyl chloride overwrap ..............................................................................................................53 4-2 Mean total Gram negative counts for ground beef stored at 4 1 C for 7 days in polyvinyl chloride overw rap.............................................................................................. 53 4-3 Mean total psychrotroph counts for ground beef stored at 4 1C for 7 days in polyvinyl chloride overw rap.............................................................................................. 53 4-4 Mean pH values for ground beef stored at 4 1C for 7 days in polyvinyl chloride overwrap ............................................................................................................................54 4-5 Mean total aerobic counts for ground beef stored at 4 1C for 25 days ......................... 54 4-6 Mean Gram negative counts for grou nd beef stored at 4 1C for 25 days .....................55 4-7 Mean total lactic acid bact eria counts for ground beef stored at 4 1C for 25 days ....... 55 4-8 Mean total psychrotroph counts for gr ound beef stored at 4 1C for 25 days ................ 56 4-9 Mean pH values for ground beef stored at 4 1C for 25 days........................................ 56 4-10 Mean thiobarbituric acid reactive substances values for ground beef stored at 4 1C for 25 days..........................................................................................................................57 4-11 Mean L* values for ground beef stored at 4 1C for 25 days.........................................57 4-12 Mean b* values for ground beef stored at 4 1C for 25 days.........................................58 4-13 Mean a* values for ground beef stored at 4 1C for 25 days.........................................58 4-14 Consumer sensory panel color scores fo r evaluating ground beef stored at 4 1C for 25 days..........................................................................................................................59

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7 4-15 Consumer sensory panel off-odor scores fo r evaluating ground beef stored at 4 1C for 25 days..........................................................................................................................59 4-16 Mean headspace carbon monoxide concentra tion values for ground beef stored at 4 1C for 25 days..................................................................................................................60 4-17 Mean residual carbon monoxi de concentration values for ground beef stored at 4 1C for 25 days ..................................................................................................................60

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8 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF PACKAGING SYSTEM FAT CONCENTRATION AND CARBON MONOXIDE ON MICROBIOL OGY, SENSORY AND PHYSICAL PROPERTIES OF GROUND BEEF STORED AT 4 PLUS OR MINUS 1 DEGREE CELSIUS FOR 25 DAYS By Nicolas Armando Lavieri May 2008 Chair: Sally K. Williams Major: Animal Sciences The overall appearance of a fresh meat product at the time of purchase is important to consumers as it may affect their purchasing decisi ons. The objective of this study was to evaluate the effects of three packaging treatments (modified atmosphere [M AP] containing 0.4% carbon monoxide [CO] and 30% carbon dioxide [CO2], vacuum packaging [VP] and polyvinyl chloride [PVC] overwrap), three fat treatments (10, 20 an d 30% fat) and storage time on microbiology, sensory and physical properties of ground beef stored at 4 plus or minus 1 degree Celsius for 25 days. The three packaging and three fat treatme nts were analyzed for objective color, pH, microbiology (total aerobes, total psychrotrophs, Gram negatives, E.coli O157:H7, total coliforms and generic E. coli ) and thiobarbituric acid reactive substances (TBARS). Lactic acid bacteria analysis was conducted only in the MA P and VP treatments. H eadspace and residual CO analyses were conducted only in the MAP treat ments. Consumer panels were conducted to evaluate color and off-odors. The CO concentrations of the package and the meat, respectively, were deemed harmless at the levels detected in this study. There were no significant increases (P > 0.05) in TBARS for the MAP and VP treatments throughout the stor age time. Modified atmosphere and vacuum

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9 packaging treatments showed a significant decrease ( P < 0.05) in pH values by Day 14, whereas PVC treatments had significantly higher ( P < 0.05) pH values by Day 7. Modified atmosphere and vacuum packaging had a bacteriostatic eff ect on Gram negative microorganisms. The growth of aerobic, psychrotrophic and la ctic acid bacteria was not inhi bited by MAP or VP. Within the MAP and VP groups, the 10% and 30% fat treatment s, respectively, reache d aerobic plate counts and total psychrotroph counts above spoilage levels (> 6 log CF U/g). Furthermore, consumer color and off-odor results revealed that discolor ation and off-odors were de tected in the 10% and 30% fat treatments within the MAP and VP groups, respectively. Obje ctive color analysis revealed that significant decreases ( P < 0.05) in a* values of MAP treatments had occurred by Day 21. However, consumer color scores were similar ( P > 0.05) for all MAP treatments when compared to Day 0. Consumers should rely on use by dates on the package and not on the color of the meat as indicators of freshness.

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10 CHAPTER 1 INTRODUCTION The overall appearance of a m eat product at the time of purchas e is important to consumers. Several characteristics of the produc t are evaluated before the purchase is made. Some of these characteristics are: color, smell, texture and flavor (if applicable). Of these characteristics, the color of the product may be the one most heavily relied upon when making the purchase, given that the packaging of most meat products in their raw state prevents consumers from smelling, touching, or tasting them Thus, methods for the enhancement of the color of meat products and its stability during storage have been studied and developed by the meat industry and retailers. Some of the methods for color enhancement us ed in todays meat industry make use of a modified atmosphere system that contains either low carbon monoxide (CO) levels or high oxygen (O2) levels. This practice is commonly referred to as modi fied atmosphere packaging (MAP). Furthermore, the gas mixture of MAP systems does not generally contain CO and/or O2 alone. Other gases such as carbon dioxide (CO2) and nitrogen (N2) are generally included in the mixture for a series of particular reasons that will be discussed later. Definitions : Modified atmosphere packaging has been defined as a process whereby a perishable product is placed in a package, regu lar air is removed by vacuum or flushing, and the package is filled with a pre-determined gas or mi xture of gases with a composition different than air (regular air is composed of about 0.03% CO2, 78% N2 and 21% O2 [66] ), followed by sealing of the package (34) To paraphrase, MAP consis ts of getting rid of an original atmosphere that would eventually affect the shelf-life of th e product negatively and re placing it with another atmosphere pre-formulated so as to optimize, maximize, and preserve the shelf-life of the product. Modified atmospheres for meat and pou ltry are dynamic and will change with time.

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11 Product and microbial metabolism, absorption of gases by the product, and diffusion of the gas or gas mixture through the barrier film will cause th e atmosphere inside the package to vary over time (66) Controlled atmosphere packaging (CAP), by contrast, is a sim ilar packaging method wherein the gas atmosphere is kept relatively constant duri ng the life of the package (66) Vacuum Packaging (VP), a commonly used packaging technique for fresh and cured meats, consists of the removal of all air followed by sealing of th e product in a barrier film that will not allow for diffusion of gases in or out of the package. Vacuum packaging is a form of modified atmosphere packaging because, in th e case of fresh meats, microbial and muscle metabolism will utilize residual O2 to produce CO2 and, as a result of this, a modified atmosphere that achieves signi ficant shelf life extension is created. Modified atmosphere packaging can be differentiated from vacuum packaging by the presence of a headspace into which a larger volume of gases can be introduced into the package and also by the lack of the physical pressure that occurs when a product is vacuum packaged (66)

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12 CHAPTER 2 REVIEW OF LITERATURE Todays m eat industry has to deal with several issues. One of the most relevant issues has to do with the timely and efficient distribution over long distances of fresh meat products that are fabricated at or near centrali zed locations. Thus, the shelf lif e of the fresh product must be protected and enhanced in order to ensure optimal customer and, eventually, consumer satisfaction. Vacuum and modified atmosphere packaging are just two of the techniques developed by meat scientists and used by meat processors and retailers to achieve the ultimate goal of enhancing the sh elf-life of meat products. Both VP and MAP systems provide a greater enhancement of shelf-life when compared to polyvinyl chloride (PVC) overwrap packaging (66) The predominant concern in terms of shelf lif e is bacterial growth, which is most often the limiting factor. In addition to microbial growt h, the shelf life of a meat product is affected by other quality attributes such as color, odor, flavor and texture. Thus, an important objective of MAP systems is to minimize the changes that th ose attributes experience throughout the storage of the product (66) Carbon Dioxide in Modified Atmosphere Packaging Effects of Carbon Dioxide on the Microflora of Fresh Meat Carbon dioxide, a colorless gas with a slightly pungent odor (46) is the centerpiece of MAP systems due to its ability to in hibit a wide range of microorganisms (66) Furthermore, Dixon and Kell (9) determined that CO2 has a greater inhibitory effect on Gram negative bacteria, which grow rapidly on fresh meat, than it does on Gram positives. However, the mechanism by which CO2 exerts its inhibitory effect on bacteria is not yet fully understood (66) It is important to mention that carbon dioxide ga s has been utilized as a preservative for fresh

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13 meat and poultry for over 100 years and, consequen tly, its use in MAP systems has been studied extensively (66) Although the pathway through which CO2 exerts its inhibitory e ffect on bacteria is not yet fully understood, it is known that the gas dissolv es readily in water and will produce carbonic acid (H2CO3) in solution (23) Carbonic acid will then cause a drop in meat pH and, in turn, negatively affect microbial growt h. All of this occurs despite th e buffering ability of fresh meats (66) The antimicrobial effect of CO2 stems from its ability to affect the chemical quality of the meat (23) Dixon and Kell (9) determined that the lo wering of meat pH is a result of CO2 absorption and production of carbonic acid that dissociates to bicarbon ate and hydrogen ions. A decrease in meat pH has been shown to negatively affect the rate of oxidation processes such as pigment and lipid oxidation as well as water holding capacity (WHC) of meat (29) Microflora in VP and MAP meat systems with high levels of CO2 will change from an aerobic to an anaerobic microflora which is the main factor responsible for extending the storage life of the products (81) Mixtures of 20-100% CO2 balanced with N2 are normally used (23) The inhibitory effect of CO2 increases with increasing CO2 concentration in cuts packaged in O2depleted atmospheres (19) Primal cuts in CO2 atmospheres containi ng very low residual O2 levels and stored at -1.5 0.5C have a shelf life of 10-15 weeks or more (27, 81) Carbon dioxide concentrations greater th an 20-30% in MAP systems of fr esh meats have been shown to exert little additional inhibitory effect on the pr edominant spoilage flora in aerobic environments (21) Jakobsen and Nertelsen (23) determined that anaerobic high (> 20-30%) CO2 packaging of retail cuts can also be an advantage ous practice. With low levels of residual O2 and a strict

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14 temperature control of -1.5C, the storage pe riod was 2-3 times longer than for aerobically packaged retail cuts. Upon exposure to O2 the cuts will bloom and will be able to withstand a few days of aerobic display without further handling. In addition, when packaging in atmospheres with concentrations greater than 20-30% CO2, long storage life can be achieved for both primal and retail cuts as long as certain precautions are taken. A high in itial hygiene is necessary, but other critical factors are temperature and O2 control. General Properties of Carbon Dioxide When high CO2 levels are introduced into the headspace of a package, the concentration will decline as CO2 is absorbed into the meat (23) It has been shown that CO2 dissolves in both muscle and fat tissue until saturation or equilibrium is reached (17) In MAP studies in which CO2 was used, it has been shown that its effects are usually related to the concentration of CO2 in the food and not to its concentration in the packag e headspace. Thus, the gas that affects the food and the microorganisms is the one that dissolves in the food and not the one in the headspace (8, 39) As a result, the full preservative potential of CO2 will only be accomplishe d if the amount of CO2 added to the headspace is greater than the amount required to saturate the meat (20) How much CO2 a meat product can absorb is related to biological factors of the meat such as pH, water content and fat content (17) Furthermore, packaging and storage conditions such as temperature, CO2 partial pressure and headspace to meat volume ratio will also affect the rate of absorption (89) Gill (17) concluded that the solubility of CO2 in muscle tissue of pH 5.5 at 0C was approximately 960 mL/kg of tissue. The solubility increased with increasing tissue pH by 360 mL/kg for each pH unit. The solubility decreased with increasing temperature by 19 mL/kg for each 1C rise. Solubilities in beef, por k and lamb muscle tissue were comparable. Gill (17) also concluded that the solubility of CO2 in fat tissues initially increased as the temperature was raised above 0C, but then declined at higher temperatures.

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15 To achieve maximum storage life, meat ha s to be stored at the lowest possible temperature without freezing the pr oduct. Even relatively small in creases in storage temperature result in meaningful reductions in storage life. For example, an increase in storage temperature of 1C results in at least 10% reduc tion in storage life, proving temper ature control to be the most critical factor (25) Storage temperature has a significant effect on weight loss, color stability, lipid oxidation (49) and CO2 absorption, as previously mentioned. In order to prevent discoloration of the product, complete removal of all O2 from the package headspace is necessary. In addition, an impermeable film that prevents O2 from reentering the packag e should be used (25) Oxygen has been shown to accelerate discoloration even at low partial pressures (36) Reducing spoilage caused by bacterial growth is one of the main concerns that MAP systems in fresh meats address. However, as previously mentioned, an attr active red color of the displayed product is also desired, and, therefore, MAP systems must also address the color of the product. After studying different pre-slaughter physiologi cal conditions in pigs and their effect on color and lipid stability duri ng storage, Juncher et al. (29) concluded that ultimate meat pH was the single most important factor affecting pr oduct quality. A low ultimate pH had a negative effect on product quality in terms of color, weight loss and lipid oxi dation. Several studies investigating low levels of CO2 (20-25%) agree that there is no effect of CO2 on meat color (1, 2, 3, 6, 37, 38, 51) However, the same cannot be said for st udies that investigated the use of higher CO2 levels. Numerous research projects have been c onducted using atmospheres containing high levels of CO2, and concluded that a decrease in pH of 0.05-0.35 units was a direct consequence

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16 (22, 36, 64, 69, 76) Rousset and Renerre (64) conducted research on normal and high pH beef packaged in 100% CO2 and found a larger decrease in the pH of high pH meat (0.35) than of normal pH meat (0.1). The researchers attributed this effect to a la rger solubility of CO2 at higher pH, which agrees with the results obtained by Gill (17) Thus, the possibility that MAP systems using high CO2 concentrations will cause discolorati on in the product is a logical conclusion. Another major concern surrounding the use of high levels of CO2 in MAP systems is its potentially negative effect on pr oduct weight. Seideman et al. (68) concluded that packaging fresh meats in 100% CO2 will result in greater weight loss as compared to packaging in 100% N2. The researchers concluded that CO2 may decrease the water holding capacity of meat proteins due to its ability to bind and structurally affect the proteins and their ability to retain moisture. OKeffe and Hood (49) showed that packaging in 100% N2 is superior to 100% CO2 in retaining water due to the possible pH lowering effect of absorbed CO2. However, other studies (53, 54) have found a lower weight loss for meat products packaged in CO2 than for products packaged in N2. The varying physical compression of th e meat during the packaging process is also responsible for pa rt of the weight loss (55) and therefore, complicates the comparison of studies, according to Jakobsen and Nertelsen (23) Another potential problem with high CO2 packaging observed in some studies is the development of a porous/fissured ap pearance of the meat when it is cooked. These fissures may be caused by the rapid release of carbon dioxide gas from the meat as it is exposed to increased temperatures during cooking (23) Bruce et al. (4) determined that carbon dioxide present in either an aqueous state or bound to proteins will evol ve rapidly during c ooking. The researchers concluded that the increase in temperature decreases CO2 solubility and denatures proteins, causing CO2 to evolve rapidly. Furthermore, the researchers also concluded that the

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17 semitendinosus muscle of beef exhibited greater fissure formation than the psoas major when packaged in 100% CO2 and stored at 3-4C, indicating that there are muscle-to-muscle differences in terms of susceptibility to fissure development. Fissure development has been reported as a problem in beef (4, 56) However, Jeremiah et al. (26) reported no fissures in pork upon cooking. Penney (56) suggested that CO2 packaging atmospheres should result in minimal fissure de velopment and still provide sufficient microbial control. However, after conducting research on the subject, Penney (56) found that for all storage times between 4-16 weeks, beef developed fissu res when packaged in atmospheres of 20-100% CO2. It was also determined that fissure development increased with increasing CO2 level. Because CO2 is the active antimicrobial agent in both VP and MAP, there has been significant interest in utilizing increased concentr ations of this gas in MAP systems. However, although beneficial from a microbi al standpoint, using high levels of this gas when packaging fresh meats has not been a common practice because of the discoloration that occurs at more than about 30% CO2 (66) Nevertheless, research that investigat ed the use of CO in MAP systems has shown that as much as 99.5% CO2 will not cause discoloration if combined with 0.5% CO (33) Carbon Monoxide in Modified Atmosphere Packaging Effects of Carbon Monoxide on the Micro flora of Fresh Meat Packaging fresh meat products in atmosphe res containing only CO has been shown to have an indirect effect on microbi al growth by removing all of the O2 from the package. Viana et al. (84) packaged fresh pork loins using four diffe rent packaging atmospheres that included: vacuum; 99% CO2/1% CO; 100% O2 and; 100% CO followed by vacuum after one hour of exposure. The loins were stored for 25 d at 5 0.5C. The researchers reported that packaging fresh pork loins in 100% CO resulted in similar ba cterial growth when compared to the other O2free atmospheres. Except fo r loins exposed to 100% O2, aerobic and anaerobic psychrotrophs

PAGE 18

18 were the dominant microbiota reaching 7 log CFU/ g (colony forming units) after 20 d of storage for all modified atmospheres treatments. The counts for loins packaged in 100% O2 reached numbers greater than 8 log CFU/g. Pseudomonas counts reached a maximum level of 5 log CFU/g in all modified atmosphere treatments except vacuum and 100% O2 during the 25 d of storage. Gee and Brown (14) investigated the effects of diff erent concentrations of CO on pure bacterial cultures of Pseudomonas, Achromobacter and E. coli species. It was concluded that 1530% CO had an inhibitory effect on the growth of bacteria. These levels, however, far exceed the levels legally allowed for use in the packaging of red meat and poultry products (83) General Properties of Carbon Monoxide Carbon m onoxide is used in a MAP system ma inly because it has the ability to form a stable bright red or cherry red color in meat, ev en in very low concentrat ions. Concentrations of 0.4 1.0% CO can be regarded as sufficient an d suitable for color purposes in MAP of meat (78) Improved but still limited color stabilit y is achieved by packaging meat in high O2 atmospheres, with a minimum of 60% O2. Thus, MAP systems using CO in the gas mixture have become more widely used by processors and retailers. Myoglobin is the most abundant pigment found in fresh meats, and it can be found in different forms depending on the O2 status of the environment. The different forms or states are: reduced myoglobin (Mb), oxymyoglobin (MbO2), and metmyoglobin (MMb +), and the actual color of meat will vary depending on the presence of these three derivatives on the surface of the product. Reduced myoglobin is the form of myogl obin associated with meat immediately after it has been cut, and also with fresh meat p ackaged in anaerobic conditions, such as VP. Oxymyoglobin, secondly, is responsible for the t ypical bright red color of fully oxygenated meat,

PAGE 19

19 and it forms once reduced myoglobin is exposed to O2. Thirdly, the brown metmyoglobin is formed by oxidation of the pigment to its ferric (Fe3+) form (47) According to Srheim et al. (78) the main driving force for the use of CO in MAP systems of meat is the development of a stable, bright red color as a re sult of the strong binding of CO to reduced myoglobin and the formati on of carboxymyoglobin (MbCO). This pigment and its relative stability to oxida tion are responsible for the red color retention under MAP systems containing CO. Carboxymyoglobin is much more stable towards oxida tion than oxymyoglobin due to the stronger affinity of CO for the ir on porphyrin site on the myoglobin molecule. Thus, the addition of CO to MAP systems at low levels to counteract discoloration issues brought about by CO2 has received significant attention fro m meat processors and retailers (47) It has been known for more than 50 years that the color spectrum of carboxymyoglobin is very similar to that of oxymyoglobin (79) What is more important in terms of color stability is the fact that carboxymyoglobin is more re sistant to oxidation than is oxymyoglobin (35) Without CO present, atmospheres cont aining a gas mixture comprised of CO2 and N2 are vulnerable to discoloration by m yoglobin oxidation due to residual O2. Discoloration due to myoglobin oxidation occurs in beef when less than 0.1% O2 is present (18) and/or due to a decreased meat pH in the case of high CO2 packaging, as previously discussed. Research has shown that the use of CO at levels between 0.1 2.0% improve meat color and color stability. These reports include beef, pork, and poultry. The color improvement by CO seems to be valid if the other gases in the atmosphere are CO2, N2 and O2 (78) However, when the CO concentration was increased to 2.0%, the color was characterized as too artificial by a sensory panel (61)

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20 Fresh beef steaks stored in CO concentrati ons of 0.5% in MAP systems remained red for 8 weeks in a study conducted at Utah State University (24) The study used the Hunter Lab Miniscan portable colo rimeter (Reston, VA) for the objectiv e measurement of color in ground beef stored with varying CO concentrations in MAP systems. Using this system, larger hue angle values are associated with less red color, where hue-angle 0 = red and hue-angle 90 = yellow. Fresh beef steaks stored in MAP systems that contained 0.5% CO had lower hue angle values compared with 5% CO in VP system s and steaks stored in PVC overwrap (24) It was concluded that fresh ground beef stored in 0.5% CO-MAP sy stems remained red for the full 8-week length of the study, while the red color was lost within the first week of storage of ground beef in PVC. The ground beef in 0.5% CO-MAP systems had lowe r hue angle values th an did the ground beef in PVC. Researchers at the Universidad de Zaragoza (41) objectively evaluate d the color on the surface of meat samples using a reflectance spectrophotometer (Minolta Chroma Meter CM2002) 30 minutes after opening the package. This device yielded color measurements in the form of L*, a*, and b* values. A total of six different gas mixtures were used in the experimental units. These consisted of CMA (Control Modified Atmosphere): 70% O2 + 20% CO2 + 10% N2 LO-CO 0.1: 24% O2 + 50% CO2 + 25.9% N2 + 0.1% CO LO-CO 0.25: 24% O2 + 50% CO2 + 25.75% N2 + 0.25% CO LO-CO 0.5: 24% O2 + 50% CO2 + 25.5% N2 + 0.5% CO LO-CO 0.75: 24% O2 + 50% CO2 + 25.25% N2 + 0.75% CO LO-CO 1: 24% O2 + 50% CO2 + 25% N2 + 1% CO The study revealed that a* values incr eased as CO concentration increased, demonstrating that redness was in fluenced by the concentration of CO used. The values of a* for beef steaks did not differ ( P > 0.05) within the same group, while differences were significant ( P < 0.01) amongst groups (41) The researchers concluded that th e three types of atmospheres that

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21 could be established according to their increasing ability for main taining red color in the meat included Type 1: Packages stored in the LO-CO 0.1 a nd LO-CO 0.25 experimental units that did not increase red color stability with re spect to steaks stored in the CMA. Type 2: Packages stored in the LO-CO 0.5 experimental unit that increased color stability. Type 3: Packages stored in the LO-CO 0.75 a nd LO-CO 1 experimental units that greatly increased red color stability. Oxygen in Modified Atmosphere Packaging Effects of Oxygen on the Microflora of Fresh Meat It is logical to c onclude that if O2 is present in the package aerobic bacteria will proliferate and cause the spoilage of the product. This is due to th e fact that aerobic bacteria are commonly responsible for food spoilage due to their higher growth rates with respect to anaerobic bacteria under refrigeration conditions. This conc lusion is supported by research projects that investigated the combined effects of storage temperature and O2 concentration on the shelf life of different types of fish and fishery products (58, 59, 60, 85) and meat products (84) General Properties of Oxygen Oxygen is a colorless, odorless gas that has a rela tively low solubility in water, supports combustion (is explosive) and is very reactive with a wide variety of biological compounds (46) Oxygen, given its reactive nature, is involved in many of the reactions that are detrimental to the quality of food products. These reactions includ e browning, rancidity development, fat oxidation and pigment oxidation (66) The main reason for the inclusion of O2 in MAP systems of fresh meat is the development of the bright red or ch erry-red color and its maintenance. These are two factors that are considered esse ntial to the display and acceptan ce of fresh meat. Even though the

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22 red oxymyoglobin pigment is formed readily under normal atmospheric O2 pressure, an elevated O2 concentration (65-80%) in MAP systems helps to form a deeper la yer of oxymyoglobin pigment that will extend the time the color ap pears attractive. However, the presence and increased level of O2 will also promote the growth of rapidly-proliferating, aerobic microorganisms. Oxygen in MAP systems for fresh meat is usually combined with 20-25% carbon dioxide to achieve improved microbial control (34) Fresh meat is particularly susceptible to discoloration when low levels of O2 are present in the packaging environment. A partial O2 pressure in the range of 5-10 mm. of mercury (normal atmospheric partial pressure of O2 is 159.2 mm Hg) will quickly cause the myoglobin pigment in meat to convert to metmyoglobin, which is brown. Low levels of residual O2 in MAP packages of fresh meat will result in at least some metmyoglobin formation (66) Therefore, it is recommended that the residual O2 in fresh meat MAP systems be no more than 0.01% (10 ppm) immediately after packaging, and essentia lly 0% within 24 hours after packaging (74) Atmospheric or greate r concentration of O2 results in an attractive red color for fresh red meat. Complete elimination of O2, as is the case in VP, prev ents color deterioration. Upon exposure to O2, however, formation of the attractive re d color will occur. If a package has air leaking into it due to a poor seal or if low O2 levels remain in it due to poor flushing, it is likely to discolor quickly. Low O2 (> 0.01% O2) levels can also be a problem in cooked or cured cooked meats where color fading and rancidity may occur as a result. In this case, 0.5% or less O2 in package atmospheres is recommended (66) The problems of excess residual O2 can sometimes be solved if O2 scavengers or absorbers that react with any residual O2 that may remain in the p ackage are used. Iron powders in small packages are most often used for this purpose and can be frequently found in packages

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23 of highly O2-susceptible products such as dried snack sticks and jerky and other ready-to-eat products (66) Another problem created by exposing beef to high O2 atmospheres is called premature browning. The phenomenon develops when the beef is cooked and turns brown at lower-thanusual cooking temperatures (31, 70, 71) In this case, meat that is cooked to a medium degree of doneness (71.1C internal) or less appears to have been cooked to a well-done degree of doneness (66) Research has shown that premature browning can occur in ground beef even when cooked to internal temperatures as low as 49C (28) Furthermore, ground beef patties stored in atmospheres containing 80% O2 at 2C were shown to develop premature browning in nearly 100% of the patties evaluated (28) From a microbial standpoint, this creates a food safety concern due to the fact that many consumers use cooked color as an indicator of the temperature achieved during cooking (doneness) (66) Nitrogen Gas in Modified Atmosphere Packaging Effects of Nitrogen on the Microflora of Fresh Meat In addition to CO2 and O2, N2 is the other gas used in significant amounts in MAP systems. Nitrogen is an inert gas that is colorless, odorless and tasteless (46) Some of its characteristics include lower density than air, low solubility in water and fat and it is nonflammable. Research demonstrates that N2 can affect meat product shelf life indirectly because when N2 is used to completely displace O2, the atmosphere will not allow growth of aerobic microorganisms. Nitrogen is used to replace oxygen in packages to retard oxidative rancidity and inhibit growth of aerobic microorgani sms, as an alternative to VP (73) However, no direct effect of N2 on microbial growth has been observed and, as a result, it has no impact on anaerobic bacteria (66)

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24 General Properties of Nitrogen Pack collapse, which can occur in MAP systems as a consequence of absorption of CO2 into meat tissue, is usually avoided by using N2 in combination with CO2 as an inert filler (5) Nitrogen acts as an inert filler due to its very low solubility in meat. As mentioned previously, N2 has no antibacterial properties a nd does not affect meat color (47) High concentrations (usually 100%) of N2 are often used in packages of cooked, cured meats, particularly sliced items wher e slice adhesion is to be prevented (65) In these packages, given the cooked and cured nature of the products, it is recommended that O2 be reduced to 0.5% or less to ensure cured color stability (45) When dealing with unc ured, cooked products, the elimination of O2 in order to retard rancidity development and diminish flavor losses is of utmost importance. Due to the fact that cooked products are expected to have low microbial loads, prevention of flavor changes during storage is ofte n more critical to shelf life of these products than microbial inhibition. In this case, the inclus ion of a gas with bacterio cidal or bacteriostatic properties is not necessary. Instead, the use of 100% N2 can extend shelf life of the products by preventing the chemical changes and fla vor losses brought about by exposure to O2 (66) Regulatory Status for the Use of Carbon Monoxide in Mo dified Atmosphere Packaging Systems The approved use of CO in the packaging of fresh meat, poultry and fish products is a relatively new practice in the US. However, requ ests from processors to regulatory agencies began to appear as early as 1985 (88) and in 1999 the most significant patent for fish applications was issued (32) A patent for the use of CO in fresh red meats was issued in 2001 (72) The United States Food and Dr ug Administration (FDA) approved master-bag packaging with 0.4% CO in 2002 (82) In 2004 this approval was extended to retail, case-ready packaging (83) In other parts of the world, however, governme nt acceptance of the use of CO is not as

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25 high. In fact, the use of CO in MAP systems in other countries is very limited. Norway, for example, used CO in MAP systems from 1985 (77) until trade agreements between it and the European Union caused the use of CO in MAP systems to cease in 2004 (66) Current Applications of Carbon Monoxide in the Food Industry Today CO is being applied to a variety of seafood as a single gas with variable concentrations, as a component in filtered or t asteless sm oke (TS), and as so-called artificialfiltered smoke based on gas blends to exemplify tasteless smoke. Commercial use is expanding primarily with fish from either traditional harvests or culture operations in most seafood producing nations around the world. The primary market driving this trend is base d in the US due to particular market acceptance, regulatory al lowances, and the necessity for frozen products due to extensive transportation distances (52) The relatively recent approval for the use of CO in the packaging of fresh meats in the US is expected to increase the percentage of low-O2 packaging formats and also to increase retail acceptance of case ready at retail stores throughout the country (10) Modified atmosphere packaging of fresh meats would qualify under the case ready category. A study conducted in 2004 to audit and report trends in fresh meat packaging at retail level concluded that 60% of the packages audited were case ready (up from 49% in 2002). Modified atmosphere packages increased by 4% from 2002 to 2004 from 9% to 13%, respectively. Conversely, the traditional Styrofoam tray with PVC overwrap decreased from 51% in 2002 to 47% in 2004 (44) Disadvantages of Modified Atmosphere Packaging Systems The m ajor hurdle that MAP systems, especially those that make use of CO in the gas mixture, will have to clear before they become wi dely used by processors in the US is consumer acceptance. Several companies throughout the count ry have already asked the FDA to prohibit the use of CO in the packaging of fresh meat s. In February of 2006, Kalsec, Inc. (Kalamazoo,

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26 MI) officially petitioned that the FDA ban the us e of CO in fresh meats because the use of carbon monoxide deceives consumers and crea tes an unnecessary risk of food poisoning by enabling meat and ground beef to remain fresh-looking beyond the point at which typical color changes would indicate ageing or bacterial spoilage. If not banne d, consumers should at least be notified through labeli ng if a meat product has been treated with carbon monoxide (87) Another major disadvantage of the use of MAP systems, including those that make use of CO, is the costs associated with this practice. Specialty packaging films with specific gas permeability rates are required, and state-of-the-art equipment is also required when packaging a fresh meat product in a modifi ed atmosphere of any kind.

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27 Table 2-1. Advantages and disa dvantages of modified atmosphere packaging systems as they apply to fresh meat products Advantages Disadvantages Increase in shelf life Controve rsy surrounding the use of CO (consumer perception) Reduced economic losses due to increased shelf life Added costs of production (special equipment and trained personnel) Centralized packaging and greater portion control Gas mixture is productdependent (one gas mixture for each product category) Improved presentation and limited or no need for chemical preservatives Increased volume of the package will increase transportation costs Packaging films will prevent crosscontamination of the product Loss of benefits once the package has been opened Convenient packaging Spoilage of the product may be masked

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28 CHAPTER 3 MATERIALS AND METHODS The purpose of this study was to evaluate th e effects of three packaging treatm ents (MAP containing 0.4% CO, VP and PV C overwrap), three fat treatm ents (10, 20 and 30% fat) and storage time on shelf life attributes of ground beef stored at 4 1C. The shelf life attributes evaluated were: microbial counts, objective color, sensory color and odor, 2-thiobarbituric acid reactive substances (TBARS), pH, headspace CO and residual CO. Two trials were conducted, each of which consisted of two phases. Phase 1 consisted of formulating, blending, grinding, packaging and storage of the meat. Phase 2 involv ed the evaluation of the different parameters associated with the shelf life of ground beef and also with CO levels in both the meat and the headspace of the packages. Phase 1: Formulation, Packaging and Storage of Groun d Beef Formulation Beef trimmings were obtained from the University of Florida Animal Science USDA inspected meat processing facili ty (EST. 6537), located in Gaines ville, FL. Beef trimmings of different fat compositions (50/50 and 90/10) were blended in the appropriate proportions using a Pearsons square to obtain 10, 20 and 30% fat. After blending, the meat was ground through a 1/8 (0.3175 cm) grinding plate, p ackaged in the appropriate syst ems and stored at 4 1C. Sample Treatment A total of nine treatments were evaluate d throughout the study (Table 3-1). Following mixing and grinding of the beef trimmings, approximately 454 g portions were packaged under the three packaging atmospheres.

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29 Packaging, Gas Injection and Storage of Grou nd Beef After grinding, the ground beef was divided into three equal aliquots and packaged in either Genpak 2 Styrofoam trays and over wrap ped with one layer of PVC film (Companions, product#: 12073), vacuum packaged in 9 x 18 Cryovac B4770 barrier bags (0.5-0.6 g/100 in2/24 hr @ 100F, 100% relative humidity [RH] fo r water vapor transmission rate, and 1 cm3/m2/24 hr atm @ 40F at 0% RH for O2 transmission rate, Simpsonville, SC), or vacuum packaged in Cryovac B4770 barrier bags contai ning a manually, aseptically installed septum valve (ColeParmer, Vernon Hills, IL 60061-1844, catalog#: 00095XR) to allow for the injection of the gas blend into them. After sea ling of these bags, they were transpor ted to the University of Floridas Food Science and Human Nutrition (FSHN) Department in Styrofoam coolers with ice, where each package was injected with the gas blend. Th ree (3.0) L of a custom gas blend containing 0.4% CO, 30% CO2 and 69.6% N2 (Airgas Specialty Gases, Gainesville, FL 32609) were measured using a mass flow meter (Alicat Scientific, Inc., Tucson, AZ 85745, model M50SLPM-D) and injected into each bag. After the injection of the gas blend was completed, all bags were transported to the University of Floridas Department of Animal Sc iences (ANS) for storage in a walk-in cooler for 25 d at 4 1C. The temperature was monitored daily and continuously using a circular-chart thermometer installed in the cooler (P artlow, Elizabethtown, NC 28337, model RTF) thermometer throughout the duration of the study. The lights inside th e cooler were kept on at all times. The type of light bulbs inside the cooler emitted fluorescent light. Phase 2: Evaluation of Shelf Life Parameters Different parameters associated with ground beef shelf life were evaluated throughout the study. Those parameters were: objective color, microbial counts, sensory color and odor, rancidity, pH, headspace CO and residual CO.

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30 Objective Color Except for the PVC overwrap treatments, a ll treatments were evaluated on Days 0, 1, 3, 5, 7, 14, 21 and 25 for color. The PVC overwrap treatments were analyzed on Days 0, 1, 3, 5 and 7 due to spoilage of the meat. To objectively eval uate the effects of the different treatments used on the color of the ground beef, a Minolta colorimeter (Minolta Corp., Ramsey, NJ, model CR310 with 50 mm aperture) was employed. On each sampling day, two bags of ground b eef per treatment were removed from the storage cooler and evaluated for objective colo r. Instrumental data of the ground beef was measured using the L* a* b* color spectrum. This spectrum includes L* (lightness), which is a measure of total light reflect ed on a scale ranging from 0 = black to 100 = white. The a* (red/green) value is a measure of the red (positive values) and green (negative values) colors of the sample. As the value of a* increases, the samp le has an increase in red tones. As the value of a* decreases, the sample has an increase in green tones. The b* (blue/ye llow) value is a measure of the yellow (positive values) and blue (negative values) colors of a sample. As the value of b* increases, the sample takes on a more yellow co loration. As the value of b* decreases, the sample takes on more of a blue coloration. The colorimeter was calibrated as describe d in the users manual on each sampling day. Furthermore, since two different packaging film s were used (PVC and Cryovac B4770 film), the colorimeter was calibrated with each film separately before the color measurements were conducted. After calibration, two m easurements per treatment were taken and averaged. Each treatment was evaluated in duplicates. Microbial Analyses Except for the PVC overwrap treatments, a ll treatments were evaluated on Days 0, 1, 7, 14, 21 and 25 for microbial counts. The PVC overw rap treatments were analyzed on Days 0, 1,

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31 3, 5 and 7 due to spoilage of the meat. All treatments were analyzed for the following microorganisms: total aerobes, to tal psychrotrophs, Gram negativ es, lactic acid bacteria, total coliforms, generic E. coli and E. coli O157:H7. All media (Difco Laboratories, Detroit, MI 48232) and materials used for the cultivation and maintenance of the bacteria were purchased from Fisher Scientific (Pittsburgh, PA 15238). Twenty-five grams of ground beef from each treatment were placed in sterile 18 x 30 cm Fisherbrand stomacher bags (400 mL, Fischer Scientific, Pittsburgh, PA 15238) along with 225 mL of sterile 0.1% peptone water (Cat. No. DF1807-17-4). Each treatment was done in duplicate. The stomacher bags were massaged by hand for two minutes to loosen up any surface ba cteria. One mL of the sample mixture was transferred to a test tube containing 9 mL of sterile 0.1% peptone water, from which 10-1 to 10-6 (or more if needed) serial dilutions were prepared for each treatment. One L of the dilutions was pipetted and sp read (using a glass hockey stick previously flame-sterilized) BBL Trypticase Soy Agar (TSA Cat. No. B11043) for total aerobe and total psychrotrophs counts, Sorbitol MacConkey agar (SMAC, Cat. No. OXCM0813B) supplemented with a Cefixime-Tellurite supplem ent (CT, Cat. No. OXSR0172E) for E. coli O157:H7 counts, Lactobacilli MRS agar (MRS, Cat. No. DF0882170) for lactic acid bacteria counts and GN Broth (GN, Cat. No. DF0486-17-4) with added granul ated agar (Cat. No. DF0145-17-0) for Gram negative bacteria counts. One mL of each dilution was added onto 3M Petrifilm E. coli/Coliform Count Plates (3M Company, St. Paul, MN 55144, Ca t. No. 6414) for the evaluation of generic E. coli and total coliforms. The TSA plates for total aerobic counts, GN and MRS plates were incubated at 35 1C for 48 hr, CT-SMAC plates were incubated at 37 1C for 24 hr, 3M Petrifilm E. coli/Coliform Count Plates were incubated at 35 1C for 24 hr and TSA plates for total psychrotrophs counts

PAGE 32

32 were incubated at 7 1C for 10 d. After incuba tion, suspected colony forming units (CFU) were counted, recorded and averaged. Analysis of pH Except for the PVC overwrap treatm ents, all treatments were evaluated on Days 0, 1, 7, 14, 21 and 25 for pH. The PVC overwrap treatments were analyzed on Days 0, 1, 3, 5 and 7 due to spoilage of the meat. Analysis of pH wa s conducted immediately af ter the microbiological analyses were completed. An Accumet Basic pH meter (Fisher Scientific, Pittsburgh, PA 15238, model AB15) was used and pH measurements were recorded for all treatments. The probe was standardized using standard buffer solutions of pH 4.0 and pH 7.0. After standardization, the probe was placed inside the sample homogenate and allowed to reach equilibrium for 1 minute before the reading was taken. The probe was ri nsed with distilled water and dried with a Kimwipe (Kimberly-Clark Corporation, Roswell, GA, Cat. No. 06-666) between each sample measurement. Headspace Carbon Monoxide On Days 1, 7, 14, 21 and 25 of the experiment, the CO concentration in the headspace of the MAP treatments was analyzed. Two bags of ground beef per treatment were removed from the storage cooler, placed in Styrofoam coolers c ontaining ice, and transported to the University of Floridas FSHN Department for analysis. The CO concentration in the headspace and CO residuals in the meat was determined using an Agilent Technologies 6890N Network Gas Chromatograph (GC) System. The GC was equipped with a flame ionization detector (FID ), a Supelco 80/100 Porapak Q column (1.82 m long) and a hydrogen aided nickel catalyst (to convert carbon m onoxide and carbon dioxide into methane and make it detectable for the FID). The GC settings used for CO evaluation are listed in Table 3-2.

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33 Two 100 L samples from each bag were injected into the GC using a 100 L Hamilton syringe (Cat. No. 14-815-80) and analyzed. One p eak was obtained for each 100 L sample that was injected into the GC. The two peaks per ba g of ground beef were th en averaged and plugged into the following formulas to determine CO c oncentration in the headsp ace of the package: % CO = 0.000196 X 0.001458 (where X = Average peak area) ppm CO = % CO 1,000,000 Residual Carbon Monoxide On Days 0, 1, 3, 5, 7, 9, 11, 14, 21 and 25 of the experiment, the CO concentration per gram of beef in the MAP treatments was m easured. A 6 g sample from each treatment was collected in duplicate on each of the sampling days, placed in 60 mL I-Chem Economy 100series tubes (Cat. No. 05-719-398) and frozen at -20 1C for no more than 30 d before sampling. Samples were thawed overnight at 4 1C and transported to the University of Floridas FSHN department for CO analysis. Th e following protocol was followed to determine ppm CO/g meat: 1. Transfer a 6 g core sample into a 60 mL I-Chem bottle. 2. Add 3 drops of octanol (antifoaming) (Cat. No. S80109). 3. Add 12 mL of 10% sulfuric acid (H2SO4) (Cat. No. 815032). 4. Shake mixture for 10 sec by hand. 5. Incubate for 5 min at 40C. 6. Shake at room temperature for 15 min in a shaker. 7. Inject 100 L into GC and analyze. Two 100 L samples from each tube were inj ected into the GC using a 100 L Hamilton syringe (Cat. No. 14-815-80) and analyzed. One p eak was obtained for each 100 L sample that was injected into the GC. The two peaks per tu be were then averaged and plugged into the following formulas to determine ppm CO/g meat:

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34 % CO = 0.000196 X 0.001458 (where X = Average peak area) g CO/g Meat = (0.000001145 % CO 430)/6 Adjusted g CO/g Meat = (6 g CO/g meat)/Weight of Sample ppm CO/g Meat = Adjusted g CO/g meat 1,000,000 Thiobarbituric Acid Reactive Substances Except for the PVC overwrap treatments, a ll treatments were evaluated on Days 0, 1, 3, 5, 7, 14, 21 and 25 for rancidity in the form of TBARS. The PVC overwrap treatments were analyzed on Days 0, 1, 3, 5, 7 and 9 due to spoilage of the meat. Samples were frozen at -20 1C for no more than 30 d in sterile 18 x 30 cm Fisherbrand stomacher bags (400 mL, Fischer Scientific, Pittsburgh, PA 15238) and allowed to thaw overnight in a cold room at 4 1C before they were analyzed. The TBARS distillation pr ocedure for meat and poultry was adapted using procedures from Tarladgis et al. (80) Rhee (62) and Ke et al. (30) In the adapted procedure, the sample was read against the blank at the optical wavelength of 535 nm. In addition, 66% recovery was obtained, compared to 69% in Tarladgis et al. (80), resulting in a variation in K (distillation value). Two bags per treatment were analyzed on each of the days mentioned above. Two absorbance readings per sample were taken and then averaged. Consumer Sensory Panel Analysis Except for the PVC overwrap treatments, a ll treatments were evaluated on Days 0, 1, 3, 7, 9, 14, 21 and 25 for consumer sensory pane l color and off-odors. The PVC overwrap treatments were analyzed on Days 0, 1, 3, 7 and 9 due to spoilage of the meat. The sensory

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35 facilities at the University of Floridas Animal Science Meat Research laboratory were used. Samples of ground beef were removed from thei r initial packaging atmo spheres, placed in transparent Zip-lock bags and placed on white countertops for panelist evaluation. The panelists were instructed to evaluate all samples for color and off-odors. The color scale used was an 8 point hedonic scale where 1 = extremely dark brown, 2 = very dark brown, 3 = dark brown, 4 = dark red, 5 = slightly dark re d, 6 = cherry red, = 7 moderately light cherryred and 8 = light cherry-red. The scale used to evaluate off-odor s was a 7 point hedonic scale where 1 = none, 2 = barely perceptible, 3 = pe rceptible, 4 = slightly strong, 5 = moderately strong, 6 = very strong and 7 = very strong. A hedoni c scale is a rating scale that measures the level of liking or disliking of food products (50) Statistical Analysis The statistic al analysis for this pr oject was performed using SAS Windows (65) A splitplot design with three packaging treatments and three fat treatments was used for evaluating all the parameters associated with the shelf life of ground beef described above. The analysis of variance of the Mixed Proce dures (PROC MIXED) of SAS software and the LSMEANS procedure for generating standard e rrors of the mean (SEM) were us ed to analyze trial, day, fat, packaging, day by fat, day by packaging, fat by packaging and day by fat by packaging interaction. Variations in data were accounted for by eight treatment effects: trial, day, fat, packaging, day*fat, day*packaging, fat*pack aging and day*fat*pack aging. Any significant differences were analyzed by the multiple comparison procedure of Duncans Multiple range test, using a level of si gnificance of alpha = 0.05.

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36 Table 3-1. Treatments evaluate d during a 25-d storage period to determine the effects of packaging system, fat concentration and carbon monoxide on the storage stability of ground beef 1Modified atmosphere, 2Vacuum packaging, 3Polyvinyl chloride Table 3-2. Gas Chromatograph settings used for analyzing packaged ground beef for carbon monoxide gas headspace and residuals in the meat Parameters Settings Injection temperature 100C Carrier gas and flow rate Helium at 26.9 mL/min (splitless) Nickel catalyst temperature 375C Oven temperature 30C isotherm Runtime 3 min Detector temperature 250C Column 80/100 Porapak Q packed column (1.82 m) 1 MAP1 10% fat 4 VP2 10% fat 7 PVC3 10% fat 2 MAP 20% fat 5 VP 20% fat 8 PVC 20% fat 3 MAP 30% fat 6 VP 30% fat 9 PVC 30% fat

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37 CHAPTER 4 RESULTS AND DISCUSSION The objectives of this study were to evaluate the effects of three packaging atm ospheres (MAP containing 0.4% CO, VP a nd PVC overwrap) and three fa t percentages (10, 20 and 30%) on pH, color, TBARS, microbiological and se nsory characteristics of ground beef, and to determine the concentration of CO in the headsp ace of the package and in the meat during 25-d storage at 4 1C. Evaluation of Ground Beef Stored in Polyvinyl Chloride Overw rap for 7 d Microbiological Analyses Aerobic plate counts Total aerobic counts in PVC treat ments significantly increased ( P < 0.09) on Day 5 compared to Day 0 regardless of fat level (Table 4-1). However, a slight but not significant decrease ( P > 0.05) in log CFU/g was detected on Day 7 compared to Day 5. These results indicate that, once maximum levels were attained, aerobic bacteria ente red the death phase of their growth curve. An alternat ive conclusion would be that, as shown by other microbiological analyses, the slight decrease in Gram negativ e counts was caused by competitive inhibition. No fat effect was detected ( P > 0.05). Gram negative counts Gram negative microorganisms in all PV C treatments significantly increased ( P < 0.05) on Day 5 compared to Day 0 regardless of fat level (Table 4-2). Furthe rmore, a decrease of almost 1 log CFU/g was detected in all treatm ents between Day 5 and Day 7, suggesting that Gram negative microorganisms had entered the death phase of their growth curve. Gram negative counts for the 30% fat treatment were significantly higher ( P < 0.05) than those in the 20% fat treatment on Day 1. However, no overall fat effect was detected ( P > 0.05).

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38 Psychrotroph counts Psychrotroph counts in all PVC tr eatments significantly increased ( P < 0.05) by Day 5 compared to Day 0 regardless of fat level (Tab le 4-3). In the 30% fa t treatment, however, a significant increase ( P < 0.05) in psychrotroph counts was detected as early as Day 1 compared to Day 0. In both the 20 and 30% fat treatments, psychrotroph counts slightly decreased ( P > 0.05) by Day 7 compared 5, indicating that psychr otrophic microorganisms had entered the death phase of their growth curve by Day 7. Psychrot roph counts for the 30% fat treatment were significantly higher ( P < 0.05) than all other fat levels on Day 1. However, no overall fat effect was detected ( P > 0.05). Analysis of pH A significant increase (P < 0.05) in pH values for all PVC treatments was detected by Day 7 compared to Day 0 regardless of fat leve l (Table 4-4). Although no overall fat effect was detected (P > 0.05), the pH values for the 30% fat treatment were significantly higher ( P < 0.05) than all other fat treatments on Day 5. Evaluation of Ground Beef Stored in Mo dified Atmosphere, Vacuum Packaging and Polyvinyl C hloride Overwrap for 25 d Microbiological Analyses Aerobic plate counts Aerobic microorganisms (APCs) were less th an 7 log CFU/g for all treatments through 25 d storage (Table 4-5). In gene ral, APCs increased for all trea tments as storage time increased. In the MAP group, significant ( P < 0.05) increases in APCs were detected by Day 14 when compared to Days 0 and 1 for all fat treatments. However, for the 20 and 30% fat treatments, significant increases ( P < 0.05) in APCs were detected by Day 7. Counts were similar ( P > 0.05) for all MAP treatments from Days 14-25 and re mained less than 6 log CFU/g. This suggested

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39 that aerobic microorganisms in all MAP treatment s had entered the stationary phase of their growth curve by Day 14. Similar results were observed in the VP group. Significant ( P < 0.05) increases in APCs were detected by Day 14 when compared to Days 0 and 1 regardless of fat level. Counts were similar ( P > 0.05) from Days 14-25. This suggested that aerobic microorganisms in all VP treatments had entered the stationary phase of their growth curve by Day 14. The APCs for the PVC group significantly ( P < 0.09) increased by Day 7 when compared to Day 0 for the 10 and 20% fat levels. However, APCs were similar ( P > 0.05) for the 30% fat treatment through 7-d storage. Counts within each day were similar ( P > 0.05) throughout the storage time for all treatments. However, the 20% fat treatment in the PVC group on Day 1 had significantly lower ( P < 0.05) APCs than all other treatments. Gram negative counts Gram negative microorganisms were less th an 4 log CFU/g for all treatments through 25d storage (Table 4-6). No significant ( P > 0.05) increase in Gram negative counts was detected in MAP and VP treatments during storage. In addition, Gram negative counts were similar ( P > 0.05) within and between all MA P and VP treatments. These results suggested that both packaging methods had a bacteriostatic effect on Gram negative microorganisms. Gram negative counts for the PVC group increased significantly ( P < 0.05) by Day 7 when compared to Day 0 regardless of fat leve l. These results suggest ed that exposure to O2 promoted the growth of Gram negative microorganisms. Gram negative counts for the 10 and 30% fat treatments in th e PVC group were higher ( P < 0.05) than all MAP and all VP treatments on Day 7.

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40 Lactic acid bacteria counts Lactic acid bacteria counts were less than 6 log CFU/g for all treatm ents throughout 25-d storage (Table 4-7). Similar results were observe d for all MAP and VP treatments. Lactic acid bacteria counts significantly increased ( P < 0.05) in all treatments by Day 7 when compared to Day 0 regardless of fat level and packaging method. In addition, lactic acid bacteria counts significantly increased ( P < 0.09) in all treatments by Day 1 when compared to Day 0 regardless of fat level and packaging method. Lactic acid bacteria counts remained similar ( P > 0.05) from Days 7 through 25. No significant ( P > 0.05) differences were detected between treatments on each individual day throughout the storage time. Psychrotroph counts Psychrotroph counts were less than 7 log CFU/g for all treatment s through 25-d storage (Table 4-8). In general, psychrotroph counts increased for al l treatments as storage time increased. All treatments in the MAP group had significantly higher ( P < 0.05) counts on Day 14 when compared to Day 0. Counts on Days 21 and 25 were significantly higher ( P < 0.05) when compared to Day 0 for the 10 and 20% fat treatments, but similar ( P > 0.05) for the 30% treatment. The 10 and 20% fat treatments in the VP group showed significantly higher ( P < 0.05) levels of bacteria by Day 21 compared to Day 0. In the 30% fat treatment, however, no significant changes ( P > 0.05) were detected throughout the storage time. The 10 and 20% fat treatments in th e PVC group showed significantly higher (P < 0.05) levels of bacteria by Day 7, when compared to Day 0. In the 30% fat treatment, however, no significant changes ( P > 0.05) were detected throughout the storage time. On Day 7, counts in the PVC group were all above 5 log CFU/g regardle ss of fat level, whereas they did not exceed 4.61 log CFU/g for all MAP and VP treatments. Fu rthermore, the 10% fat treatment in the PVC

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41 group had significantly higher ( P < 0.05) counts compared to all VP treatments and to the 20 and 30% fat treatments within the MAP group. E. coli O157:H7 counts No E. coli O157:H7 was detected thr oughout the duration of the study. Total coliforms and E. coli counts No E. coli was detected throughout the duration of the study. Total coliform s were detected only in the first trial conducted. Thus, the statistical analysis coul d not be carried out. Anaerobic conditions have been shown to inhibit the growth of aerobic bacteria and increase microbiological sh elf life of meat products (16, 57, 63, 67) Increased bacterial inhibition occurs with CO2 levels of 20% or more in MAP systems (11, 41) Vacuum packaging is a form of MAP because, in the case of fres h meats, microbial and muscle metabolism will utilize residual O2 to produce CO2 (66) Low levels (0.1 1.0%) of CO, on the other hand, have little effect on meat microflora (40) Results from this study indicated that MAP and VP had a bacteriostatic effect on Gram negative microorganisms (Table 4-6). Dixon and Kell (9) determined that CO2 has a greater inhibitory effect on Gram negative bacteria, wh ich grow rapidly on fresh meat, than on Gram positives. The growth of aerobic (Table 4-5), psychr otrophic (Table 4-8) and lactic acid bacteria (Table 4-7), on the other hand, was not inhi bited by MAP or VP. Significant increases (P < 0.05), compared to Day 0, in aerobic (Day 14), psyc hrotrophic (Day 14) and lactic acid bacteria (Day 7) in all MAP treatments were obs erved. Similarly, significant increases ( P < 0.05), compared to Day 0, in aerobic (Day 14), psychrotrophic (Day 21 [ P < 0.09]) and lactic acid bacteria (Day 1) in all VP treatments were also observed. Mean log CFU/g of total aerobes and lactic acid bacteria generally did not significantly increase ( P > 0.05) after Day 7 of storage, while total psychrotroph counts did ( P < 0.05). It is important to mention that, within the MAP

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42 (10% fat, Day 21) and VP (30% fat, Day 25) groups, two of the six treatments reached aerobic plate counts and total psychrotr ophs counts above spoilage levels (> 6 log CFU/g). Consumer panel sensory color (Table 4-14) and off-odors (Tab le 4-15) results suggested that discoloration and off-odors were detected in thos e treatments on those days. Packaging of ground beef in PVC overwrap favors the growth of aerobic microorganisms due to its O2 permeability. Bacterial growth is favored in PVC-wrapped meats compared with vacuum packaged meats (67) Vacuum packaging favors the growth of lactic acid bacteria, whereas Pseudomonads spp. are generally the dominant spoi lage microflora of PVC-wrapped meats (16, 57, 63) These conclusions are in agreement w ith results obtained during this study. Packaging ground beef in PVC overwrap allowed for significant increases ( P < 0.05) in Gram negative (Table 4-2), total aerobes (Table 4-1) and psychrotrophic (Table 4-3) bacteria by Day 5 of storage. In addition, total psychrotroph coun ts for the 20 and 30% fat treatments in the PVC group reached spoilage levels (>6 log CFU/g) by Day 5 of storage. Consumer panel sensory color (Table 4-14) and off-odors (Table 4-15) re sults suggested that di scoloration and off-odors were detected in those treat ments as early as Day 3. Product Analyses Analysis of pH The pH values varied between 5.00 and 6.17 (T able 4-9). The pH values for all MAP and VP treatments decreased significantly ( P < 0.05) by Day 14 when compared to Day 0 regardless of fat level. These results agree with those observe d in the microbiological analysis of lactic acid bacteria, whose levels rose significantly ( P < 0.05) by Day 7 compared to Day 0 regardless of fat level and packaging method (Table 4-7). The pH values for the PVC group increased significantly ( P < 0.05) by Day 7 when compared to Day 0. Furthermore, pH values for the PVC group on Day 7 were significantly

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43 higher ( P < 0.05) than those for all MAP and all VP treatments regardless of fat level. No significant differences ( P > 0.05) were detected between fat levels throughout the storage time regardless of packaging method. The modified atmosphere used in this study contained 30% CO2, the active antimicrobial agent in both anaerobic MAP and VP (66) When CO2 is introduced into the headspace of a package, its concentration will decline as CO2 is absorbed into the meat (23) It has been shown that CO2 dissolves in both muscle and fat tissue until saturation or equilibrium is reached, and the rate at which it is absorbed by tissues is aff ected by initial meat pH, temperature and water activity (17) It is also known that the gas dissolv es readily in water and will produce H2CO3 in solution (23) Carbonic acid will then cause a drop in meat pH and, in turn, negatively affect microbial growth. Numerous research projec ts have been conducted using atmospheres containing high levels of CO2, and concluded that a decrease in pH of 0.05-0.35 units was a direct consequence (22, 36, 64, 69, 75) Results from this study show ed that a significant decrease ( P < 0.05) in pH occurred after Day 14 of storage for all MAP and VP tr eatments (Table 4-9). Thus, the absorption of CO2 into the meat could have been responsible for this significant drop in pH values. However, as discussed previously, log CFU/g of lactic acid bacteria significantly increased ( P < 0.05) by Day 7 of storage regardless of p ackaging method and fat level (T able 4-7). The absence of O2 from the packaging system, as is the case in anaerob ic MAP and VP, has been shown to favor the growth of lactic aci d-producing bacteria (16, 57, 63) This production of lactic acid may have led to the significant decrease in pH values obs erved in all MAP and VP treatments by Day 14. Results observed in the PVC group contrasted from those observed in the MAP and VP groups in that a significant increase ( P < 0.05) in pH was observed by Day 7 of storage (Table 4-

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44 9). Analyses of aerobic (Table 4-1), Gram negativ e (Table 4-2) and psychrotrophic (20 and 30% fat treatments only [Table 4-3]) bact eria showed a slight decrease ( P > 0.05) in log CFU/g counts on Day 7 compared to Day 5, suggesting that th e organisms in question had entered the death phase of their growth curves. T hus, the increase in pH observed in all PVC treatments may have been caused by the by-products of bacterial cell death. Thiobarbituric acid reactive substances There were no signi ficant increases (P > 0.05) in TBARS for the MAP and VP treatments throughout the storage time when compared to Day 0 (Table 4-10). These results suggested that the MAP and VP systems have an antioxidant e ffect on ground beef regardless of fat level. Significant increases ( P < 0.05) in TBARS for the PVC group were observed for both the 10 and 20% fat treatments by Day 7 when compar ed to Day 0. Thiobarbituric acid reactive substances values for the 30% fat treatment were similar ( P > 0.05) over time. Furthermore, on Days 3, 5 and 7, the 20% fat treatment within the PVC group yielded si gnificantly higher ( P < 0.05) TBARS values than all VP treatments. These levels were also higher ( P < 0.05) when compared to the values observed in the 10 and 30% fat treatments within the MAP group on those days, suggesting that exposure to O2 accelerates lipid oxidation. It is also important to me ntion that, within the MAP group, the 30% fat treatment yielded higher ( P < 0.05) TBARS values than the 10% fat treatment on Days 1, 7, 9 and 25, results which suggest that fat level has an effect on lipid oxidation. These results, however, were not significant ( P > 0.05) when they were compared to those from the 20% fat treatment. A low ultimate pH has been shown to have a negative effect on product quality in terms of color, weight loss and lipid oxidation (29) However, TBARS values did not significantly increase ( P > 0.05) for all MAP and VP treatments thr oughout the duration of the study (Table 4-

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45 10) even though the pH values for those treatm ents had significantly decreased by Day 14 of storage (Table 4-9). Since rancidity is an oxidative process, CO can be viewed as an antioxidant. Oxygen, given its reactive na ture, is involved in many of the reactions that are detrimental to the quality of food products. These reactions include browning, rancidity development, fat oxidati on and pigment oxidation (66) Results from this study showed significant increases ( P < 0.05) in TBARS in the PVC group for both the 10 and 20% fat treatments by Day 7 when compared to Day 0. Th iobarbituric acid reactive substances values for the 20% fat treatment in the PVC group were significantly higher (P < 0.05) than all VP treatments and the 10 and 30% fat treatments within the MAP group on Days 3, 5 and 7. These results emphasize the shorter shelf life achieve d by packaging fresh meats in PVC overwrap compared to both anaerobic MAP and VP. Objective Color Evaluation L* values L* values, which measure total light reflecte d on a scale ranging from 0 = black to 100 = white, did not change ( P > 0.05) over time in all MAP and PV C treatments (Table 4-11). These results suggest that both of these packaging methods had similar effects on L* values. Conversely, L* values for all VP tr eatments significantly decreased ( P < 0.05) by Day 1 when compared to Day 0 regardless of fat level. L* values for all VP treatments remained constant ( P > 0.05) after Day 1. Fat treatment within each packag ing treatment had an effect ( P < 0.05) on L* values. On Days 0, 1, 3, 5 and 7, L* values for the 30% fat treatment within each packaging treatment were significantly higher ( P < 0.05) than those observed for the 10% fat treatment. Similar results were observed on Days 14 and 21, where L* values for the 30% fat treatment within both the MAP and the VP groups were significantly higher ( P < 0.05) than those observed in the 10% fat

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46 treatment. These results suggest that fat level influences the br ightness scores of ground beef under the conditions used in this study. L* values for the 10% fat treatment with in the VP group were significantly lower ( P < 0.05) from all MAP and PVC treatments on Days 1, 3, 5, 7 and were also lower ( P < 0.05) than all MAP treatments on Days 14, 21 and 25. These results suggest that VP had the opposite effect on L* values than fat level did under the conditions used in this study. b* values Table 4-12 summarizes the b* values obtai ned during this study. Within the MAP and the VP treatments, all fat levels showed significant decreases ( P < 0.05) in b* values by Day 1 compared to Day 0. No significant changes ( P > 0.05) were detected af ter Day 1 for any of these treatments. In the PVC group, similar results were obser ved. The b* values si gnificantly decreased ( P < 0.05) by Day 1 in the 10 and 20% fat treatments and by Day 3 in all treatments when compared to Day 0. All PVC treatments showed significantly higher ( P < 0.05) b* values on Day 1 when compared to all MAP and all VP treatments. However, on Days 3, 5 and 7, b* values for all PVC treatments did not differ significantly ( P > 0.05) from all MAP treatments while they remained higher ( P < 0.05) than those observed in all VP treatments. All MAP treatments showed significantly higher ( P < 0.05) b* values than those observed in all VP treatments regardless of fa t level on Days 3, 5, 7, 14, 21 and 25. These results indicated that the MAP system used in this study had a similar effect on b* values of ground beef as did conventional PVC overwrap regardless of fat level and under the conditions of this study. a* values A significant decrease ( P < 0.05) in a* values was observed in the 10 and 20% fat treatments within the MAP group by Day 21 when compared to Day 0 (Table 4-13). All fat

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47 treatments within the MAP gr oup, however, showed significant ( P < 0.05) decreases in a* values on Day 21 when compared to Day 1. In addition, a* values within the MAP group reached their highest levels on Day 5 regardless of fat level. Vacuum packaging caused a significant decrease ( P < 0.05) in a* values by Day 1 compared to Day 0 regardless of fat leve l. After Day 1, however, no significant ( P > 0.05) changes were observed. The a* values for a ll VP treatments were significantly lower (P < 0.05) than all MAP treatments on Days 1, 5 and 7. All treatments within PVC overwrap group, on the other hand, showed significant decreases ( P < 0.05) in a* values by Day 3. Furthermore, a* values for all tr eatments within the PVC group were significantly lower ( P < 0.05) than all MAP and all VP treatments on Days 3, 5 and 7. Red color can be maintained in low-CO tr eated meats that have spoiled, emphasizing the need for adherence to label instructions for product shelf life and the use of odor and overall appearance as spoilage indicators (7) As results from this study indicate, a significant decrease ( P < 0.05) in a* values was observed in the 10 and 20% fat treatments within the MAP group by Day 21 when compared to Day 0. In addition, all fat levels within the MAP group showed significant ( P < 0.05) decreases in a* values on Day 21 when compared to Day 1, suggesting that the appealing red color associated with fresh ground beef was lost after 21 days of storage. It is general consensus that consumer accep tance of vacuum packaged retail beef has been low because of its dark reddish-purple color (43) Reduced myoglobin is the form of myoglobin associated with fresh meat packaged in anaerobic conditions, such as VP. Results from this study indicated that ground beef that was vacuum packaged was darker in appearance

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48 as shown by the initial decrease in a* values and lower L* and b* values when compared to that ground beef in the MAP system. Fresh meat is particularly susceptible to discoloration when low levels of O2 are present in the packaging environment. A partial O2 pressure in the range of 5-10 mm of mercury (normal atmospheric partial pressure of O2 is 159.2 mm Hg) will quickly cau se the myoglobin pigment in meat to convert to metmyoglobin, which is brown. Low levels of residual O2 in MAP packages of fresh meat will result in at least some metmyoglobin formation (15, 66, 75) Results from this study suggested that exposure to O2 had a negative effect on redness values (Table 4-13). These results agree with previous research projects that concluded that packaging fresh meats in PVC overwrap allows rapid surface pigment oxygenation and red color development, but brown color discol oration occurs within 1-7 days (42) Consumer Sensory Evaluation Color The average consumer panel color scores for the 10% fat treatment within the MAP group were similar ( P > 0.05) on any day compared to Day 0 (Table 4-14). However, a clear drop in the consumer panelist scores was noti ced on Day 21. This drop in color scores was significant ( P < 0.05) when compared to Day 14. Similar results were obtained with the 20% fat treatment within this group ( P < 0.05). The 30% fat treatment, however, did not show a clear drop in average consumer panel scores until Day 25. These results allow us to conclude that the modified atmosphere used in this study was able to enhance the color of the meat until Day 14 of storage under the conditions used in this study. Panelists rated the color of all ground beef in the VP group significantly higher ( P < 0.05) on Day 3 when compared to Day 0 regardless of fat level. The color of all samples was rated similar ( P > 0.05) for Days 9-25.

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49 Color scores signifi cantly decreased (P < 0.05) for the PVC group on Day 3 when compared to Days 0 and 1 regardless of fat level. In addition, color scores for all PVC treatments were significantly lower than all VP treatments and the 20 and 30% fat treatments within the MAP group on Days 3 and 7 ( P < 0.05). Color scores for all PV C treatments were significantly lower (P < 0.05) than all VP and all MAP treatments on Day 7. Off-odors Off-odor consumer panel scores for MAP treatments were significantly higher ( P < 0.05) on Days 21 and 25 when compared to Days 0 and 1 regardless of fat level (Table 4-15). These results indicate that the modifi ed atmosphere used in this study prevented the development of off-odors up until Day 14. Within the VP group, all treatments showed higher ( P < 0.05) off-odor scores on Days 7, 14, 21 and 25 compared to Day 0 regardless of fa t level. Prior to Day 7, however, no significant increases ( P > 0.05) in off-odor scores were detected. All PVC treatments showed significant increases ( P < 0.05) in off-odor scores after Day 3 of storage regardless of fat level. Further increases ( P < 0.05) in off-odors were shown on Day 9, indicating that PVC overwrap prevented the development of off-odors for only 1 d after packaging under the conditions used in this st udy. In addition, all PVC treatments showed significantly higher ( P < 0.05) off-odor scores than a ll MAP treatments on Days 7 and 9 regardless of fat level. The 10% fat treatment within the MAP group reached spoilage leve ls of psychrophilic bacteria by Day 21 of storage (T able 4-4). Although by this day consumer panel color scores were significantly lower (P < 0.05) than those for Day 14, they did not significantly differ ( P > 0.05) from Day 0. These results indicate packag ing fresh ground beef in an atmosphere

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50 containing 0.4% CO may mask spoilage from a visual sta ndpoint. Cornforth and Hunt (7) after reviewing literature on the use of carbon monoxide in fresh meats, reached the same conclusion. After comparing a* values obtained during this study (Table 4-13) to consumer panel color scores (Table 4-14), a clear discrepancy ca n be seen. The a* values for the 10 and 20% fat treatments within the MAP group significantly decreased ( P < 0.05) by Day 21 compared to Day 0. Panelists, conversely, were not ab le to detect this decrease ( P > 0.05). Off-odor consumer panel scores, conversel y, showed that all MAP treatments had developed off-odors by Day 21 (P < 0.05). Thus, it can be conclude d that, even though consumer panel color scores did not significantly decrease ( P > 0.05) throughout the storage time, panelists were still able to detect the onset of off-odors. Panelist color scores for all VP treat ments did not significantly decrease ( P > 0.05) over time. Off-odor scores, convers ely, significantly increased (P < 0.05) after 7-d storage, which coincided with a significant increase ( P < 0.05) in lactic acid bacter ia levels (Table 4-7). These results suggested that the growth of lactic acid-producing ba cteria within the packaging environment may lead to the development of off-odors. The rapid onset of discoloration and off-odor s in all PVC treatments further supported the conclusion that exposure to O2 has detrimental effects on shelf life attributes such as color and odor. Carbon Monoxide Analyses Headspace carbon monoxide A significan t decrease ( P < 0.09) in CO concentration was detected on Day 14 compared to Day 1 in both the 20 and 30% fat treatments (Table 4-16). The 10% fat treatment, conversely, did not show significant changes ( P > 0.05) in CO levels throughout the duration of the study.

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51 Although fat level did not appear to have an effect on the CO concentration in the headspace of the package, the 20 and 30% fat treatments showed significantly higher ( P < 0.05) concentrations of CO than the 10% fat treatment on Day 1. These re sults suggested that fat level has an effect on how rapidly CO is absorbed into the ground beef. Residual carbon monoxide Significantly higher ( P < 0.05) residual levels of CO were detected on Days 5, 7, 9, 11, 14, 21 and 25 compared to Day 0 regardless of fat level (Table 4-17). Although no overall fat effect was detected (P > 0.05), the residual levels of CO in the 20 and 30% fat treatments were significantly lower ( P < 0.05) than those in the 10% fat treatment on Days 11 and 14. Residual levels of CO peaked on Days 11, 21 and 25 for the 10, 20 and 30% fat treatments, respectively. These re sults suggested that fat level had an effect on how rapidly maximum levels of residual CO were r eached under the conditions of this study. Although the concentration of CO in the headsp ace of the bags (Table 4-12) appears to be high enough to present a health ri sk to humans, the quantity of CO in one or several packages would still be too low to cause health c oncerns when packages are opened at home (7). It has been concluded that, for MAP packages contai ning 0.4% CO with a headspace of 1.5 L, opened in a typical room with a volume of 150 m3, opening of 216 packages would be required to exceed the Environmental Protection Agency (EPA ) National Ambient Air Quality Standard of 9 ppm for 8 hr (13) for a typical person inhaling 5 m3 air/8 hr (7) Residual CO levels in the 20 and 30% fat treatments peaked on Days 21 and 25, respectively. The a* values (Table 3-13) for t hose fat levels on Days 21 and 25, however, were significantly lower ( P < 0.05) than those for previous days suggesting that discoloration may occur even when the carboxymyoglobi n complex is still present.

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52 Residual levels of CO found in the meat are unlikely to present a health risk to consumers. If one consumed a large meat meal (8.8 oz; 0.25 kg), and all the CO in a typical COMAP package remained bound to the meat after cooking, one would consume only 3.5% of the EPA 8-hr maximum safe level (7, 12, 13) In reality, one would consume even less CO per meal because it is known that only 15% of bound CO remains with the meat after cooking (86). Also, it is unlikely that all ingested CO is absorbed. Thus, CO exposure from consumption of COpackaged meat is well below EPA safety standards.

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53 Table 4-1. Mean total aerobic counts for ground beef stored at 4 1C for 7 days in polyvinyl chloride overwrap 1Colony forming units per gram, 2Polyvinyl chloride. a b means in the same column with different superscripts ar e significantly different (P < 0.05). w z means in the same row with different superscripts ar e significantly different (P < 0.05). Each mean value represents 4 measurements. Table 4-2. Mean total Gram negative counts for ground beef stored at 4 1 C for 7 days in polyvinyl chloride overwrap 1Colony forming units per gram, 2Polyvinyl chloride. a b means in the same column with different superscripts ar e significantly different (P < 0.05). w z means in the same row with different superscripts ar e significantly different (P < 0.05). Each mean value represents 4 measurements. Table 4-3. Mean total psychrot roph counts for ground beef stored at 4 1C for 7 days in polyvinyl chloride overwrap 1Colony forming units per gram, 2Polyvinyl chloride. a b means in the same column with different superscripts ar e significantly different (P < 0.05). w z means in the same row with different superscripts ar e significantly different (P < 0.05). Each mean value represents 4 measurements. Log10 CFU1/g Packaging Fat % Day 0 Day 1 Day 3 Day 5 Day 7 10 3.69a,xz 3.64a,xy4.33a,xyz5.11a,z 4.77a,xyz 20 2.98a,wx 1.86b,x 3.77a,wy5.03a,z 4.83a,yz PVC2 30 3.60a,x 3.92a,xy4.49a,xyz5.27a,yz4.46a,xyz Log10 CFU1/g Packaging Fat % Day 0 Day 1 Day 3 Day 5 Day 7 10 2.51a,w 2.32ab,wx3.31a,wxy4.65a,z3.87a,yz 20 2.36a,xy 1.53a,x 2.71a,y 4.41a,z3.41a,yz PVC2 30 2.67a,x 2.72b,xy 3.54a,xyz 4.51a,z3.83a,z Log10 CFU1/g Packaging Fat % Day 0 Day 1 Day 3 Day 5 Day 7 10 3.33a,x 4.99a,yz 4.31a,xy 5.86a,z5.99a,z 20 3.20a,w 4.06a,wx4.23a,wxy6.14a,z5.29a,xyz PVC2 30 4.06a,y 5.70b,z 5.64a,z 6.31a,z5.39a,yz

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54 Table 4-4. Mean pH values for ground beef stor ed at 4 1C for 7 days in polyvinyl chloride overwrap 1Polyvinyl chloride. a b means in the same column with different superscripts are significantly different ( P < 0.05). x z means in the same row with different superscripts are significantly different ( P < 0.05). Each mean value re presents 4 measurements. Table 4-5. Mean total aerobic counts for ground beef stored at 4 1C for 25 days 1Colony forming units per gram, 2Modified atmosphere, 3Vacuum packaging, 4Polyvinyl chloride. a b means in the same column with different su perscripts are significantly different ( P < 0.05). x z means in the same row with different superscripts are significantl y different ( P < 0.05). Each mean value represents 4 measurements. Storage Time Packaging Fat % Day 0 Day 1 Day 3 Day 5 Day 7 10 5.71a,x 5.60a,y 5.63a,xy5.53a,y6.04a,z 20 5.61a,y 5.53a,y 5.59a,y 5.62a,y6.07a,z PVC1 30 5.67a,x 5.57a,x 5.55a,x 5.95b,y6.17a,z Log10 CFU1/g Packaging Fat % Day 0 Day 1 Day 7 Day 14Day 21Day 25 10 3.68a,xy 3.52a,y4.85a,xz5.72a,z 5.77a,z 5.43a,z 20 2.98a,y 3.35a,y4.80a,z 5.50a,z 5.41a,z 5.29a,z MAP2 30 3.59a,y 3.59a,y4.81a,z 5.54a,z 5.30a,z 5.21a,z 10 3.69a,x 3.89a,x4.48a,xy5.58a,z 5.63a,yz5.54a,yz 20 2.98a,x 3.17a,x4.57a,yz5.51a,z 5.28a,z 5.46a,z VP3 30 3.59a,x 3.72a,x4.60a,xy5.53a,yz5.60a,yz6.11a,z 10 3.68a,yz 3.64a,y4.77a,z 20 2.98a,x 1.86b,y4.83a,z PVC4 30 3.60a,z 3.92a,z4.46a,z

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55 Table 4-6. Mean Gram negative counts for ground beef stored at 4 1C for 25 days 1Colony forming units per gram, 2Modified atmosphere, 3Vacuum packaging, 4Polyvinyl chloride. a e means in the same column with different supe rscripts are significantly different ( P < 0.05). x z means in the same row with different superscripts are significantl y different ( P < 0.05). Each mean value represents 4 measurements. Table 4-7. Mean total lactic aci d bacteria counts for ground beef stored at 4 1C for 25 days 1Colony forming units per gram, 2Modified atmosphere, 3Vacuum packaging. a b means in the same column with different superscr ipts are significantly different (P < 0.05). x z means in the same row with different superscripts are significantly different (P < 0.05). Each mean value represents 4 measurements. Log10 CFU1/g Packaging Fat % Day 0 Day 1 Day 7 Day 14 Day 21Day 25 10 2.51a,yz 2.34ab,xy1.62a,z 2.20a,xyz2.60a,y 2.38a,yz 20 2.35a,z 2.01ab,z 1.82a,z 1.78a,z 2.38a,z 2.43a,z MAP2 30 2.67a,yz 2.80a,yz 2.58ac,yz2.29a,z 3.13a,y 2.75a,yz 10 2.50a,z 2.43ab,z 1.83a,z 2.11a,z 2.12a,z 2.14a,z 20 2.36a,z 1.77ab,z 2.36ae,z 2.32a,z 2.26a,z 2.49a,z VP3 30 2.67a,z 2.68a,z 2.45ae,z 2.47a,z 2.81a,z 2.58a,z 10 2.51a,y 2.32ab,y 3.87bd,z 20 2.36a,x 1.53b,y 3.41cde,z PVC4 30 2.67a,y 2.72a,y 3.83bd,z Log10 CFU1/g Packaging Fat % Day 0 Day 1 Day 7 Day 14Day 21Day 25 10 2.06a,y 2.97a,y 5.06a,z5.63a,z 5.43a,z 5.47ab,z 20 1.99a,x 3.15a,y 5.18a,z5.45a,z 5.50a,z 5.45ab,z MAP2 30 1.98a,x 3.07a,y 4.97a,z5.35a,z 5.32a,z 5.25a,z 10 2.05a,x 3.32a,y 4.60a,z5.45a,z 5.51a,z 5.38ab,z20 1.98a,x 3.49a,y 5.08a,z5.37a,z 5.41a,z 5.62ab,z VP3 30 1.98a,x 3.09a,y 4.77a,z5.35a,z 5.43a,z 5.68b,z

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56 Table 4-8. Mean total psychr otroph counts for ground beef stored at 4 1C for 25 days 1Colony forming units per gram, 2Modified atmosphere, 3Vacuum packaging, 4Polyvinyl chloride. a f means in the same column with different su perscripts are significantly different ( P < 0.05). x z means in the same row with different superscripts are significantl y different ( P < 0.05). Each mean value represents 4 measurements. Table 4-9. Mean pH values for ground beef stored at 4 1C for 25 days 1Modified atmosphere, 2Vacuum packaging, 3Polyvinyl chloride. a b means in the same column with different superscripts are significantly different ( P < 0.05). w z means in the same row with different superscripts ar e significantly different (P < 0.05). Each mean value represents 4 measurements. Log10 CFU1/g Packaging Fat % Day 0 Day 1 Day 7 Day 14 Day 21Day 25 10 3.24a,x 3.35a,x 4.61abd,y5.31abc,yz6.09a,z 5.64a,yz 20 3.14a,x 4.52ab,yz 4.17ad,xy5.76ad,z 5.74a,z 5.56a,yz MAP2 30 3.92a,y 4.06ac,y 4.06ad,y 5.81ae,z 5.34a,yz5.19a,yz 10 3.01a,y 4.92bce,xz 4.06ad,xy4.14bc,xy 5.59a,z 5.62a,z 20 3.39a,y 3.74aef,y 3.68d,y 4.38cde,yz5.47a,z 5.64a,z VP3 30 3.99a,yz 4.47adef,yz4.01ad,z 4.15bc,z 5.46a,y 4.58a,yz 10 3.33a,y 4.99bcf,z 5.99bc,z 20 3.20a,y 4.06ac,y 5.29ac,z PVC4 30 4.06a,y 5.70bd,z 5.39ac,yz Storage Time Packaging Fat % Day 0 Day 1 Day 7 Day 14Day 21Day 25 10 5.63a,w 5.58a,wx5.53a,wx5.15a,yz5.36a,xy5.07a,z 20 5.62a,x 5.59a,x 5.43a,xy5.29a,y 5.07b,z 5.11a,z MAP1 30 5.62a,y 5.58a,y 5.51a,y 5.19a,z 5.09b,z 5.10a,z 10 5.59a,xy 5.64a,x 5.42a,y 5.14a,z 5.00b,z 5.01a,z 20 5.61a,y 5.56a,y 5.52a,y 5.23a,z 5.06b,z 5.08a,z VP2 30 5.68a,x 5.66a,x 5.42a,y 5.21a,yz5.10ab,z5.06a,z 10 5.71a,y 5.60a,y 6.04b,z 20 5.61a,y 5.53a,y 6.07b,z PVC3 30 5.67a,y 5.57a,y 6.17b,z

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57Table 4-10. Mean thiobarbitu ric acid reactive substances values for gr ound beef stored at 4 1C for 25 days mg of malonaldehyde/kg sample Packaging Fat % Day 0 Day 1 Day 3 Day 5 Day 7 Day 9 Day 11Day 14 Day 21 Day 25 10 1.07ab,z 0.85a,z 1.10ab,z 0.88ac,z 0.89a,z 0.77a,z 0.93a,z 0.88ab,z 1.08ab,z 0.94ac,z 20 1.21ab,yz 1.34ab,yz 1.25ab,yz1.41abc,yz 1.25ab,yz 1.12ab,yz 0.85a,z 1.38a,y 1.35ab,y 0.87ac,z MAP1 30 1.83a,w 1.50bc,wyz1.23ab,yz1.21ace,wyz1.52bd,wxz1.39bd,wyz 1.10a,xy0.99ab,y 1.66b,wz1.75b,wz 10 0.96b,z 1.01ac,z 0.85bd,z 0.81a,z 0.94ad,z 0.81ad,z 0.78a,z 0.63b,z 0.80a,z 0.68ac,z 20 1.75a,y 1.08ac,z 1.02bd,z 1.02acd,z 1.10adf,z 1.16ade,yz 1.05a,z 1.12ab,yz1.15ab,yz0.98cd,z VP2 30 1.45ab,z 1.38acd,z 1.15ad,z 1.28acd,z 1.25ab,z 1.23abe,z 1.00a,z 1.12ab,z 1.05a,z 1.18abd,z10 1.10ab,xy 1.05ac,x 1.65ac,y 1.45bc,xy 2.38c,z 2.42c,z 20 1.47ab,y 1.76bd,yz 2.04c,yz 1.93b,yz 2.23ce,z 2.25c,z PVC3 30 1.79a,yz 1.76bd,z 1.30ab,y 1.55bde,yz 1.68bef,yz 1.45be,yz 1Modified atmosphere, 2Vacuum packaging, 3Polyvinyl chloride. a f means in the same column w ith different superscripts are significantly different ( P < 0.05). w z means in the same row with different superscripts are significantly different ( P < 0.05). Each mean value represents 4 measurements. Table 4-11. Mean L* values for ground beef stored at 4 1C for 25 days Storage Time Packaging Fat % Day 0 Day 1 Day 3 Day 5 Day 7 Day 14 Day 21 Day 25 10 50.42af,yz 49.40ad,yz49.85ade,yz51.22ade,yz 51.65ade,yz50.42a,yz 49.62ae,y 51.58ad,z 20 52.46ae,z 52.46ab,z 53.06ab,z 52.61abe,z 53.18ab,z 53.76b,z 52.71a,z 52.87ad,z MAP1 30 54.49bde,z 54.68b,z 54.89bg,z 55.81b,z 55.15b,z 55.00b,z 56.08c,z 56.46b,z 10 47.95f,x 44.24c,yz 43.48c,y 45.17c,yz 44.28c,y 45.52c,xyz44.89d,y 46.94ce,xz20 48.20f,y 45.86ce,z 46.81ef,yz 45.80ch,yz 46.27cf,yz 47.15ac,yz46.43de,yz46.40c,yz VP2 30 51.54ae,y 48.89de,z 49.25efh,yz49.04dfgh,yz48.68df,z 50.03a,yz 50.33ab,yz50.04de,yz10 51.08af,y 49.30ad,z 48.65df,z 49.53ef,yz 49.98ad,yz 20 52.61ae,z 52.36ab,z 51.61adgh,z51.31aeg,z 51.97ab,z PVC3 30 57.34d,x 55.05b,yz 55.09b,xy 53.33ab,z 53.32be,yz 1Modified atmosphere, 2Vacuum packaging, 3Polyvinyl chloride. a h means in the same column w ith different superscripts are significantly different ( P < 0.05). x z means in the same row with different superscripts are significantly different ( P < 0.05). Each mean value represents 4 measurements.

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58Table 4-12. Mean b* values for ground beef stored at 4 1C for 25 days Storage Time Packaging Fat % Day 0 Day 1 Day 3 Day 5 Day 7 Day 14 Day 21Day 25 10 10.40ac,w 7.16a,xz 6.94a,z 8.20a,x 7.89ac,xz6.91a,z 5.20a,y 6.95a,xz 20 9.94ac,v 6.78ad,wz7.82ab,xz8.44a,x 8.18a,xy 7.76a,wxy 6.52b,z 6.99a,yz MAP1 30 9.69ac,w 7.55a,y 8.74be,wz8.91a,wz8.20a,yz 7.93a,yz 7.88c,yz8.58b,wyz10 9.74a,y 3.89b,z 3.27c,z 3.18b,z 2.97b,z 3.35c,z 3.34d,z 3.51c,z 20 10.14ac,x 5.72d,y 4.06cd,z 3.94bc,z 3.72b,z 3.65c,z 3.96ad,z3.86c,z VP2 30 11.17cd,x 6.31ad,y 4.90d,z 4.73c,z 6.60c,y 4.28c,z 4.98a,z 5.52d,yz 10 12.65b,x 11.20c,y 7.95ab,z 8.17a,z 8.01a,z 20 12.45bd,x 11.15c,y 8.66be,z 9.29a,z 9.16a,z PVC3 30 13.03b,y 11.99c,y 9.64e,z 9.26a,z 9.02a,z 1Modified atmosphere, 2Vacuum packaging, 3Polyvinyl chloride. a e means in the same column w ith different superscripts are significantly different ( P < 0.05). v z means in the same row with different superscripts are significantly different ( P < 0.05). Each mean value represents 4 measurements. Table 4-13. Mean a* values for ground beef stored at 4 1C for 25 days Storage Time Packaging Fat % Day 0 Day 1 Day 3 Day 5 Day 7 Day 14 Day 21 Day 25 10 23.68ad,vx 22.98a,x20.66ab,x 27.52a,w 26.41a,vw 22.21a,x 14.77a,y 11.63a,z 20 21.61ab,w 21.43a,w22.86bd,wy26.01a,x 25.10ab,xy22.31a,wy 16.15ab,z 13.48a,z MAP1 30 18.88b,y 22.52a,x24.12d,wx 26.07a,w 22.94b,x 22.26a,x 17.97bc,y 12.78a,z 10 21.49ab,y 17.82b,z19.63a,yz 19.73d,yz19.23c,yz 19.65ac,yz 19.87c,yz 18.52b,yz20 21.61ab,x 16.47b,y18.60a,xyz 20.20d,xz19.16c,xyz19.30ac,xyz 19.30bc,xyz18.35b,yz VP2 30 21.16ab,y 17.44b,z19.20a,yz 19.71d,yz18.87c,yz 18.70bc,yz 18.48bc,yz 18.26b,yz10 27.11c,x 24.26a,x14.13c,y 7.47b,z 8.32d,z 20 25.87cd,w 22.51a,x15.20c,y 7.51b,z 8.21d,z PVC3 30 24.33ac,y 22.37a,y13.46c,z 10.91c,z 10.40d,z 1Modified atmosphere, 2Vacuum packaging, 3Polyvinyl chloride. a d means in the same column w ith different superscripts are significantly different ( P < 0.05). v z means in the same row with different superscripts are significantly different ( P < 0.05). Each mean value represents 4 measurements.

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59Table 4-14. Consumer sensory panel color scores for evaluating ground beef stored at 4 1C for 25 days Average score1 Packaging Fat % Day 0 Day 1 Day 3 Day 7 Day 9 Day 14 Day 21 Day 25 10 4.69ac,xz 5.36ab,xz4.58ab,xz6.09a,x 5.90ac,xy 5.84ab,xy 3.77a,z 4.17a,xyz 20 4.21ac,x 6.28ac,yz6.94c,y 6.20a,xyz6.14ac,xyz6.42a,y 4.56ab,xz4.36a,xz MAP2 30 5.60ab,yz 6.75a,y 6.72ac,yz7.48a,y 7.15a,y 7.25a,y 6.08b,yz 4.69a,z 10 3.38a,z 3.79bd,xz5.42ac,y 5.43a,xy 3.99bcd,yz3.59b,yz 2.68a,z 4.16a,yz 20 3.88ac,z 3.94bcd,z6.77ac,y 5.46a,yz 5.25ad,yz 4.00b,z 4.20ab,z 4.07a,z VP3 30 3.88ac,z 5.32ad,yz6.15ac,y 6.14a,y 5.43ad,yz 5.50ab,yz 4.08ab,z 4.63a,yz 10 5.99bc,y 4.69ab,y 2.29b,z 2.40b,z 1.97b,z 20 4.98ac,y 5.15ab,y 2.72b,z 2.44b,z 2.26b,z PVC4 30 7.23b,y 6.28ac,y 2.78b,z 2.52b,z 2.55b,z 1Score Scale: 1 = extremely dark brown, 2 = very dark brown, 3 = dark brown, 4 = dark red, 5 = slightly dark red, 6 = cherry red = 7 moderately light cherry-red and 8 = light cherry-red, 2Modified atmosphere, 3Vacuum packaging, 4Polyvinyl chloride. a d means in the same column with different superscripts are significantly different ( P < 0.05). x z means in the same row with different superscripts are sign ificantly different (P < 0.05). Table 4-15. Consumer sensory panel o ff-odor scores for evaluating ground b eef stored at 4 1C for 25 days Average score1 Packaging Fat % Day 0 Day 1 Day 3 Day 7 Day 9 Day 14 Day 21 Day 25 10 1.45a,xz 1.22a,x1.92a,xz 2.26a,z 1.82ab,xz 1.83ac,xz 3.31ab,y3.65a,y 20 1.35a,yz 1.25a,z 1.74a,wyz2.21a,xy 1.53a,yz 1.75ac,wyz 2.54a,wx2.94ab,x MAP2 30 1.55a,y 1.44a,y1.74a,xy 2.14a,xy 1.78ab,xy1.67a,y 3.31ab,z 2.69b,xz 10 1.40a,x 1.86a,x1.71a,x 2.96ac,z 2.79b,z 3.41b,yz 3.93b,y 3.04ab,yz20 1.35a,w 1.44a,w2.39ab,x 2.50a,x 2.27ab,wx2.67ab,xy 3.57ab,z 3.57ab,yz VP3 30 1.35a,w 1.25a,w2.03ab,wx2.86ac,xyz2.38ab,xy2.75bc,xyz 3.35ab,z 3.00ab,yz10 1.55a,w 1.48a,w2.70ab,x 3.88cd,y 5.80c,z 20 1.40a,x 1.71a,x3.08b,y 3.92cd,y 5.48c,z PVC4 30 1.25a,w 1.32a,w2.63ab,x 4.22d,y 5.78c,z 1Score Scale: 1 = none, 2 = barely perceptib le, 3 = perceptible, 4 = slightly strong, 5 = moderately strong, 6 = very strong and 7 = very strong, 2Modified atmosphere, 3Vacuum packaging, 4Polyvinyl chloride. a d means in the same column w ith different superscripts are significantly different ( P < 0.05). w z means in the same row with different superscripts are significantly different ( P < 0.05).

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60Table 4-16. Mean headspace carbon monoxide concentration values for ground beef stored at 4 1C for 25 days 1Parts-per-million of carbon monoxide, 2Modified atmosphere. a b means in the same column with different superscripts are significantly different ( P < 0.05). y z means in the same row with different superscripts are significantly different ( P < 0.05). Each mean value represents 4 measurements. Table 4-17. Mean residual carbon monoxi de concentration values for ground b eef stored at 4 1C for 25 days ppm CO/g1 Packaging Fat % Day 0 Day 1 Day 3 Day 5 Day 7 Day 9 Day 11 Day 14 Day 21Day 25 10 0.38a,r 0.94a,rs 1.23a,st 1.41a,stvw1.53a,svw2.09a,vwy2.87a,z 2.79a,yz 2.40a,yz2.40a,yz 20 0.22a,v 0.69a,vw1.40a,wx 1.43a,wxy1.73a,xyz2.06a,xyz 1.74b,xyz1.85b,xyz2.44a,z 2.40a,z MAP2 30 0.46a,w 0.90a,wx1.22a,wxy1.41a,xyz 1.84a,yz 1.86a,yz 2.01b,yz 1.93b,yz 1.88a,yz2.08a,z 1Parts-per-million of carbon m onoxide per gram of beef, 2Modified atmosphere. a b means in the same column with different superscripts are sign ificantly different (P < 0.05). r z means in the same row with different su perscripts are significantly different ( P < 0.05). Each mean value represents 4 measurements ppm CO1 Packaging Fat % Day 1 Day 7 Day 14 Day 21 Day 25 10 260411a,yz 314171a,y 244993a,yz182056a,z243944a,yz 20 362527b,y 312111a,yz253937a,z 238716a,z253379a,z MAP2 30 366731b,y 305913a,yz278447a,yz254906a,z248263a,z

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61 CHAPTER 5 SUMMARY AND CONCLUSIONS The objectives of this study were to evalua te the effects of thr ee packaging treatments (MAP containing 0.4% CO, VP a nd PVC overwrap) and three fa t treatments (10, 20 and 30% fat) and storage time on pH, color, TBARS, mi crobiological and sensor y characteristics of ground beef, and to determine the concentrati on of carbon monoxide in the headspace of the package and in the meat during 25-d storage at 4 1C. Two trials were conducted, each of which consisted of two phases. Phase 1 consiste d of formulating, blending, grinding, packaging and storage of the meat. Phase 2 involved the ev aluation of the different parameters associated with the shelf life of ground beef a nd also with CO levels in both the meat and the headspace of the packages. Results from this study suggested that MAP and VP had a bacteriostatic effect on Gram negative microorganisms. The growth of aerobic, psychrotrophic and lactic acid bacteria, on the other hand, was not inhibited by MAP or VP. With in the MAP (10% fat, Day 21) and VP (30% fat, Day 25) groups, two of the six treatmen ts reached aerobic pl ate counts and total psychrotrophs counts above spoilage levels (> 6 log CFU/g). Consumer panel sensory color and off-odors results suggested that discoloration (compared to Day 14) and off-odors were detected in those treatments on those Days. Furtherm ore, packaging ground beef in PVC overwrap allowed for significant increases ( P < 0.05) in Gram negative, total aerobes and psychrotrophic bacteria by Day 5 of storage. In addition, to tal psychrotroph counts for the 20 and 30% fat treatments in the PVC group reached spoilage levels (>6 log CFU/g) by Day 5 of storage. Consumer panel sensory color and off-odors resu lts suggested that disc oloration and off-odors were detected in those tr eatments as early as Day 3.

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62 The absence of O2 from the packaging system, as was the case in MAP and VP treatments, favored the growth of lactic acid-pro ducing bacteria. This production of lactic acid may have led to the significant decrease in pH values observed in all MAP and VP treatments by Day 14. The MAP and VP systems had an antioxidant effect on ground beef re gardless of fat level as shown by the TBARS result s. Significant increases ( P < 0.05) in TBARS for the PVC group were observed for both the 10 and 20% fat treatments. A significant decrease ( P < 0.05) in a* values was observed in the 10 and 20% fat treatments within the MAP gr oup by Day 21. Consumer panel colo r scores showed that the modified atmosphere used in this study was able to enhance the color of the meat until Day 14 of storage under the conditions used in this st udy. Furthermore, consumer panel off-odor scores showed that MAP treatments had significantly higher ( P < 0.05) off-odor scores by Day 21. Carbon monoxide concentrati ons in both the package a nd the meat were deemed harmless at the levels detected in this study. Ho wever, as shown by the consumer panel analyses, consumers should rely on use by dates on the package and not on the color of the meat as indicators of freshness.

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63 LIST OF REFERENCES 1. Ada ms, J., and D. Huffman. 1972. Effect of controlled gas atmospheres and temperatures on quality of packaged pork. J. Food Sci. 37:869-872. 2. Asencio, M., J. Ordonez, and B. Sanz. 1988. Effect of carbon dioxide and oxygen enriched atmospheres on the shelf-life of refrig erated pork packed in plastic bags. J. Food Prot. 51:356-360. 3. Brooks, J. 1933. The effect of carbon dioxide on the color changes or bloom of lean meat J. Soc. Chem. Ind. 52:17-19. 4. Bruce, H., F. Wolfe, S. Jones, and M. Price. 1996. Porosity in cooked beef from controlled atmosphere packaging is caused by rapid CO2 gas evolution. Food Res. Int. 29:189-193. 5. Church, P. 1993. Principles and Application of Modified Atmosphere Packaging of Foods, p. 229. R. Parry (ed.), Blackie Academic & Professional, London, UK. 6. Clark, D., and C. Lentz. 1972. Use of carbon diox ide for extending shelf-life of prepackaged beef. Can. Food Sci. Technol. J. 5:175-178. 7. Cornforth, D., M. and Hunt. 2008. Low-oxygen packaging of fresh meat with carbon monoxide: meat quality, microbiology, and safety. Available at: http://www.meatscience.org/Pubs/Wh ite%20Papers/wp_002_2008_CO_ MAP_Packaging.pd f Accessed: 23 January 2008. 8. Devlieghere, F., J. Debevere, and J. Van Impe. 1998. Effect of dissolved carbon dioxide and temperature on the growth of Lactobacillus sake in modified atmospheres. Int. J. Food. Micribiol. 41:231-238. 9. Dixon, N., and D. Kell. 1989. The inhibition by CO2 of the growth and metabolism of microorganisms. J. Appl. Bateriol. 67:109-136. 10. Eilert, S. 2005. New packaging technologies for the 21st century. Meat Sci. 71:122-127. 11. Enfors, S., G. Molin, and A. Ternstrom. 1979. Effect of packaging under carbon dioxide, nitrogen, or air on the microflo ra of pork stored at 4C. J. Appl. Bacteriol. 47:197-208. 12. EPA. 2007a. Air Trends carbon monoxide Nati onal trends in CO le vels. Available at: http://www.epa.gov/airtrends/carbon.htm Accessed: 23 January 2008. 13. EPA. 2007b National ambient air qualit y standards (NAAQS). Available at: http://www.epa.gov/air/criteria.html. Accessed: 23 January 2008. 14. Gee, D., and D. Brown. 1980-81. The effect of carbon m onoxide on bacterial growth. Meat Sci. 5:215-222.

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70 BIOGRAPHICAL SKETCH Nicolas Arm ando Lavieri was born in 1984 in Maracay, Venezuela. The third of four children, he moved to the US at age 17. He earned his Bachelor of Science degree from the Department of Animal Sciences in December 2005 at the University of Florida. He began his masters research in 2006 after receiving a departmental graduate assistantship. He earned his Master of Science degree in May 2008. After gr aduation, Nicolas plans to pursue a Doctor of Philosophy degree at Iowa State University under Dr. Joseph Cordrays supervision.