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A. .. 3 3 , 3 _ . n . 34 : 3 . . . . 3 3 . . V 3 . a. . 3 . . . . . . 3 t. 1 ~ 3; c 3 . t c vl. o . 3 I h n. 3 s: . . . .. 3 r. .. . . I . . u. . . . K. , u v 3 3 O. . c . . n . . A... 3 3 3.- r V . 4— 3 3 .r. 133 g... .. 13.3.53 . malfiutu... 3. . ‘1 J' I .A - A33? 3 a; b.3391 :7 3. . .. §§é§§§§§§§ llllllll llllllllll 3 1293 0107 LIBRARY Mnch' ‘8" a Sub University ‘ llllllllllllll l l0.110 This is to certify that the thesis entitled Influence of Bacterial Growth on Porcine Muscle Ultrastructure and Headspace Volatiles presented by Maxwell Todd Abbott has been accepted towards fulfillment of the requirements for Ph.D. degree in FOOd SCience ‘1 l 3 13/ fi '24(/, (22.1; tarmv/ Major professor Date#i_~ 0-7639 Eu: 0 4 35:49; ABSTRACT INFLUENCE OF BACTERIAL GROWTH 0N PORCINE MUSCLE ULERASTRUCTURE AND HEADSPACE VOLATILES By Maxwell Todd Abbott The present investigation was undertaken to provide information concerning changes in muscle ultrastructure and headspace volatiles during spoilage of meat by psychro- tolerant and food poisoning microorganisms. In addition. the influence of muscle fiber type on bacterial growth and muscle degradation was studied. The red and white portions of aseptic porcine semi- tendinosus muscle were sliced to a thickness of approximately 3 mm. dip inoculated, and incubated with pure cultures of either W m; (10°C), was pumilus (10°C ). Staphylococcus auregs (15°C). or W W (30°C). Samples were taken after 0, 24. 48. 96, and 168 hrs incubation for pH measurements, total bacterial counts. and electron microscopic examination. Aseptic samples of jporcine longissimus dorsi muscle were ground. inoculated. and incubated with either nggdomonas {gagi or a mixed culture isolated from commercial hamburger. Samples of ground. inoculated and uninoculated aseptic tissue were analyzed by sensory and gas-chromatographic headspace Maxwell Todd Abbott analysis after incubation for 0, 24, #8, 72 and 96 hrs. Bacterial growth and ultrastructural degradation were not influenced by fiber type. Changes in tissue pH were related to the amount of bacterial growth which had occurred in the tissue and were not influenced by fiber type. Extensive ultrastructural degradation was observed in tissues inoculated with Pseudomonas fra i. Staphylococcus aureus. and Clostridium perfringeng. Growth of gapillpg pumilus caused no detectable ultrastructural change. Myo- fibrillar degradation followed the same pattern for all three organisms. Degradation appeared to start with I-band breakage. after which the I-band-Z-line material became diffuse and finally indistinguishable. These results indi- cate that the A-band region of the myofibril is the most resistant to microbial breakdown. Nuclei were degraded by Clostridium pgrfringens and Pseudomonag fpggi but were not effected by growth of either Bacillus ppmilus or Staphylococcus aurepg. Mitochondria appeared to be degraded mainly by autolysis, except in the case of Clostridium perfringeng. which caused disappearance of the cristae. Ultrastructural observations and pH values combined with the published pH optima of catheptic enzymes suggest that pH levels near neutrality resulting from growth of either Pseudomonas gpagi or Staphylococcus aureus caused greater mitochondrial resistance to degradation. Ultrastructural observations of aseptic control tissues Maxwell Todd Abbott showed the presence of vesicular structures in the degraded areas of the myofibril. The vesicles ranged in size from 0.01 to 1.4hu. with the larger vesicles being confined to the red fibers. The proximity of the vesicles to the degraded areas suggests that they may be related to autolytic degradation. It was also noted that intermyofibrillar mitochondria were more stable to autolysis than intramyo- fibrillar mitochondria. This led to greater disruption of the red fibers due to their high mitochondrial content, although fiber type did not appear to influence the rate of degradation of individual mitochondria. Analysis of the headspace volatiles from aseptic control tissue resulted in tentative identification of acetone. lactic acid, and acetaldehyde. Tissue inoculated with Pseudomonas fgggi produced chromatograms similar to the con- trols except for the presence of a small ethyl acetate peak. However. tissue inoculated with a mixed culture isolated from commercial hamburger produced peaks tentatively identified as lactic acid, acetaldehyde. ethyl acetate. ethanol, and propionic acid. With the exception of ethanol. the appear- ance of chromatographic peaks did not correlate with off- odor development. These results suggest that gas-chroma- tography may prove useful in detecting the onset of meat spoilage. but further work with more sophisticated systems will be required to make the data useful. INFLUENCE OF BACTERIAL GROWTH 0N PORCINE MUSCLE ULTRASTRUCTURE AND HEADSPACE VOLATIIES BY Maxwell Todd Abbott A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR 0F PHIIDSOPHY Department of Food Science and Human Nutrition 1976 ACKNOWLEDGEMENTS The author wishes to express his appreciation to his major professor, Dr. A. M. Pearson, for his guidance. suggestions. and support throughout the research program and for his assistance in the preparation of the disser- tation. Appreciation is expressed to Dr. L. L. Bieber, Dr. K. E. Stevenson, Dr. J. R. Brunner, and Dr. D. R. Heldman for serving on the guidance committee. Special thanks are expressed to Dr. J. F. Price and Dr. R. A. Merkel for their assistance in obtaining aseptic tissue. The author is especially grateful to his wife. Leslie. and children. Bret. Chandra. and Ian, for their continuous understanding and encouragement. ii TABLE OF CONTENTS INTRODUCTION 0 I I O O O O O O O O O O O O O O O O 0 LITERATURE REVIEW . . . . . . . . . . . . Microbiology of Meats . o . . . . . . . . Classification of spoilage bacteria Bacterial growth patterns on meat . Aesthetic changes in meat caused by bacterial grOWth o o o o o o o o o o o Evaluation of bacterial spoilage . . . Chemical changes in meat due to bacterial growth . . . . . . . . . . . . . . . . . Ultrastructural changes caused by bacterial growth . . . . . . . . . . . Red and White Muscle Fibers . . . . . . . . Characterization of red and white muscle fibers.... o o o oooooo Protein differences between red and white fibers . . . . . . . . . . Ultrastructural differences between red and white muscle fibers . . . . . . . Autolysis of Muscle Tissues . . . . . . . . . Protein and nonprotein nitrogen changes Stmcmral Changes 9 o o o o O Catheptic enzyme and lysosomal activity METHODS AND MATERIALS . . . . . . . Preparation of Stock Cultures . . . Slaughter and Tissue Preparation . Slaughtering procedures . . . . . Preparation of muscle for ultrastructu anaIYSiSoooo ooooooooo Preparation of muscle for headspace analySiSooooooooooooo Tissue Inoculation and Incubation . . . . Tissue for ultrastructural analysis Tissue for headspace analysis . . . Preparation of control tissue for ult structural and headspace analysis Bacterial Counting Procedures . . . . . . pHMGasurement ooooooooooooo Histochemical Analysis . . . . . . . . . ral a ooooHoooo oooo iii Tissue preparation . . . . . . Determination of fiber type . Electron Microscopy . . . . . . . . Fixation and embedding . . . . Sectioning and staining . . . Observation and photography of mu cle seCthHSoooooooo oo ooo Gas-Liquid Chromatography of Headspace Vapors Sampling of headspace vapors . . . . . . Gas- ~liquid chromatography . . Gas Chromatography-Mass Spectrometry (CC-MS) OfHeadSpace Vapors o o o o o o o o o o o o o . o o o 8 RESULTS AND DISCUSSION Ultrastructural Changes Due to Microbial Growth Histochemical analysis of semitendinosus mUSCIGooooooooooooooooo Changes in aseptic control tissue . . . . Bacterial growth and pH changes . . . Changes in the ultrastructure of aerobic control tissues . . . . . . Changes in the ultrastructure of anaerobic control tissue . . . . . Changes in tissue inoculated with Pseudomonas fragl o o o o o o o o o Bacterial growth and pH changes . Changes in the ultrastructure of inoculated tissue . . . . . . . O 0 Changes in tissue inoculated with Bacillus BumiIUSoooo oo oooooo Bacterial growth and pH changes . . . Changes in the ultrastructure of inoculated tissue . . . . . . . . Changes in tissue inoculated with Staphylococcus aureus . . . . . . Bacterial growth and pH changes . . . Changes in the ultrastructure of inoculated tissue . . . . . . . . . Changes in tissue inoculated with Clostridium perfringens . . . . . . . . Bacterial growth and pH changes . . . Changes in the ultrastructure of inoculated tissue . . . . . . Changes in Meat Headspace Volatiles Due to Microbial Growth . . . . . . . . . . Bacterial growth, pH changes. and off-odor developmentoooooooooooooo Gas-chromatographic analysis of headspace vapors................. "‘ Standard compounds o o o o o o o o o Analysis of headspace vapors from uninoculated control tissue . . . . iv 45 #5 46 46 57 60 6O 67 79 79 84 81+ 84 92 97 97 102 115 115 118 118 121 Page Analysis of headspace vapors from tissue inoculated with a mixed culture..............131 Analysis of headspace vapors from tissue inoculated with Pseudomonas fragi.......o....o..136 Analysis of headspace vapors using GC'MSooooooooooooooo136 SUMMARY 0 o o o o o o o o o o o o o o o o o o o o o o 114‘3 B IBLIOGR APHY o o o o o o o o o o o o o o o o o o o o 1L} 6 APPENDIX o o o o o o o o o o o o o o o o o o o o o o 160 Table LIST OF TABLES Page Preinoculation, Experimental. and Enumeration Conditions for Bacterial cultures 0 I O O I I O I I C I O O O O O O O 36 Odor Evaluation of Porcine Longissimus Dorsi Muscle Inoculated with Pseudomonas fragi or a Mixed Culture and Incubated at 10° 0 O O O O O O O I C I C O O O O O O O l 15 Standard Compound Adjusted Retention Times on 3% Apiezon L and 10% Carbowax 20M Columns...................121 vi LIST OF FIGURES Figure Page 1 Aseptic Control Mitochondria from the Red Portion of Porcine Semitendinosus Muscle at 0 hr Incubation (24 hrs post- mortem)ooooo0ooooooooooooo “'8 2 Aseptic Control Nucleus from the White Portion of Porcine Semitendinosus Muscle at 0 hr Incubation . . . . . . . . . . . . . 48 3 Aseptic Control Nucleus from the White Portion of Porcine Semitendinosus Muscle ‘ After 192 hrs of Incubation at 10°C . . . . . 50 4 Aseptic Control Red Fiber and Intramyofi- brillar Mitochondrial Vesicles After 24 hrs Incubation at 10°C o o o o o o o o o o o o o 50 Aseptic Control Red Fiber at 0 hr . . . . . . 5h Aseptic Control White Fiber at 0 hr Incu- bation . . . . . . . . . . . . . . . . . . . 5h 7 Aseptic Control White Fiber After 168 hrs of Incubation at 10°C Showing Typical Type of White Fiber Degradation . . . . . . . . . 56 8 An Atypical Aseptic Control White Fiber at OTimeooooooooooooooooooo 56 9 Aseptic Control Red Fiber After 168 hrs of Incubation at 10°C . . . . . . . . . . . . . 59 10 Aseptic Control Red Fiber After 96 hrs of Incubation at 10° C . . . . . . . . . . . . . 59 11 An Anaerobically Stored Aseptic Control Red Fiber After 2# hrs Incubation at 30°C . . . . 62 12 An Anaerobically Stored Aseptic Control White Fiber After 96 hrs Incubation at 30°C I O O C O O I O O I C O I O C O O O O O 62 vii Figure 13 19 15 16 17 18 19 20 21 22 23 24 25 Anaerobically Stored Aseptic Control Intermyofibrillar Mitochondria from the Red Portion of the Semitendinosus Muscle After 21" hrs Incubation at 30°C o o o o o o o Bacterial Growth of Pseudomonas fragi on Both the Red and White Portions of Porcine Semitendinosus Muscle . . . . . . . . . . . . Changes in pH During the Incubation of the Red and White Portions of Porcine Semiten- dinosus Muscle Inoculated with Pgeudomonas fragioooooooooooooooooooo Typical Nucleus from the Red Portion of Porcine Semitendinosus Muscle Incubated for 2h hrs at 10°C with Pseudomonas fragi . . . . Typical Red Fiber Incubated for #8 hrs at 10 C with Pseudomonas frag' . . . . . . . . . White Fiber Incubated for 96 hrs at 10°C with P§eUdom0nanragioooooooooooooo White Fiber Incubated for 96 hrs at 10°C With Pseudomonag fragl o o o o o o o o o o o Typical Field from the Red Portion of Porcine Semitendinosus Muscle After 168 hrs of Incubation at 10°C with Pseudomonas fragi I I I I I I I I I I I I I I I I I I I I Typical Field from the White Portion of Porcine Semitendinosus Muscle after 168 hrs Incubation at 10°C with Pseudomonas fragi I I I I I I I I I I I I I I I I I I I I Bacterial Growth of Bacillus pumilus on Both the Red and White Portions of Porc1ne Semi- tendinosus Muscle at 10°C . . . . . . . . . . Changes in pH During the Incubation of the Red and White Portions of Porcine Semi— tendinosus Muscle Inoculated with Bacillus Qumilus...............o... Typical Red Fiber After 96 hrs Incubation With BaCillus Qumilus at 10°C o o o o o o o o Typical White Fiber After a 168 hr Incubation at 10°C with Bacillus pumilus . . . . . . . . viii Page 64 66 69 72 72 75 75 77 77 81 83 86 86 Figure 26 27 28 29 30 31 32 33 34 35 36 3? Bacterial Growth of Staphylococcus aureus on the Red and White Portions of Porcine Semitendinosus Muscle at 10°C . . . . . . . . Changes in pH During the Incubation of the Red and White Portions of Porcine Semitendin- osus Muscle Inoculated with Staphylococcus aureus I I I I I I I I I I I I I I I I I I I Nucleus from the White Portion of Porcine Semitendinosus Muscle After a 48 hr Incuba- tion with Staphylococcus aureus at 15°C . . . Nucleus from the White Portion of Porcine Semitendinosus Muscle After a 96 hr Incubation at 15°C with Staphylpcppgpg aureusooooooooooooooooooo Intermyofibrillar Mitochondria from the Red Portion of Porcine Semitendinosus Muscle after a 168 hr Incubation with Staphylococ- 9_‘J_.§_ aQrGUSo I I I I I I I I I I I I I I I I I Typical Red Fiber after a 96 hr Incubation with Staphylococcus aureus at 15°C . . . . . Typical Red Fiber after a 168 hr Incubation with Staphylococcus aureus at 15°C . . . . . Bacterial Growth of Clostridium perfringens on the Red and White Portions of Porcine Semitendinosus Muscle . . . . . . . . . . . . Changes in pH During the Incubation of the Red and White Portions of Porcine Semiten- dinosus Muscle Inoculated with Clostridium pgrfringens . . . . . . . . . . . . . . . . . Typical Nucleus from the Red Portion of Porcine Semitendinosus Muscle after a 24 hr Incubation at 300C with Clostridium QgrfrlngenS-.oc.............. Typical Area from the Red Portion of Por- cine Semitendinosus Muscle after a 24 hr Incubation at 30°C with Clostridipm er- fringens I I I I I I I I I I I I I I I I I I I Typical Break in the I-band Region of a White Fiber after a 24 hr Incubation at 30°C with Clostridium perfringens . . . . . . ix Page 87 91 94 94 96 99 99 101 104 10? 107 110 Figure 38 39 40 41 42 43 an 45 46 1+7 48 Degraded Area from a White Fiber after a 24 hr Incubation at 30°C with Clostridium perfringens . . . . . . . . . . . . . . . . . Typical Clostridium pgrfringens Cell after a 24 hr Incubation at 30°C in the Red Por- tion of Porcine Semitendinosus Muscle . . . . Clostridipp,pgrfringens Cell with Surrounding Debris after a 24 hr Incubation at 30°C in the Red Portion of Porcine Semitendinosus “1115019 I I I I I I I I I I I I I I I I I I I Typical Field from the White Portion of Por- cine Semitendinosus Muscle after a 96 hr In- cubation at 30°C with glostridium perfrin- gens I I I I I I I I I I I I I I I I I I I I Bacterial Growth of Pseudomonas fragi and of a Mixed Culture on Porcine Longissimus Dorsi Muscle Stored at 10°C . . . . . . . . . . . . Changes in the pH of Longissimus Dorsi Muscle Inoculated with Pseudomonas fragi or a Mixed Culture and Incubated at 10°C. . . Chromatogram of the Headspace Vapors for Uninoculated Control Porcine Longissimus Dorsi Muscle at 0 Time on a 10% Carbowax 20“: C o lumn I I I I I I I I I I I I I I I I I Chromatogram of the Headspace Vapors of Uninoculated Porcine Longissimus Dorsi Muscle at 0 Incubation Time on a 3% Apiezon L Column . . . . . . . . . . . . . . Chromatogram of the Headspace Vapors of Uninoculated Control Porcine Longissimus Dorsi Muscle Incubated for 48 hrs at 100 C Run on a 10% Carbowax 20M Column . . . . . . Chromatogram of the Headspace Vapors of Uninoculated Porcine Longissimus Dorsi Muscle Incubated for 48 hrs at 10°C Run on a 3% Apiezon Column . . . . . . . . . Chromatogram of the Headspace Vapors of Porcine Longissimus Dorsi Muscle Incubated for 48 hrs at 10°C with a Mixed Culture Run on a 10% Carbowax 20M Column. . . . . . . Page 110 112 112 114 117 120 123 125 128 130 133 Figure 49 50 51 Chromatogram of Headspace Vapors of Porcine Longissimus Dorsi Muscle Incubated for 48 hrs at 10°C with a Mixed Culture Run on a 3% Apiezon Column . . . . . . . . . . Chromatogram of the Headspace Vapors of Porcine Longissimus Dorsi Muscle Incubated for 48 hrs at 10°C with Pseudomonas fragi Run on a 10% Carbowax Column Chromatogram of the Headspace Vapors of Porcine Longissimus Dorsi Muscle Incubated for 48 hrs at 10°C with Pseudomonas fragi Run on a 3% Apiezon Column xi Page 135 138 140 Table 10 11 LIST OF APPENDIX TABIES Bacterial Counts on Meat Inoculated with ngpdomonas fragi Using a Dip Technique . . . Effect of Either Random or Spread Packing Technique on the Growth of Pseudomonas iragi Dip Inoculated on Porcine Muscle. . . . . . Schedule for the Preparation of the Histo- chemical Incubating Medium. . . . . . . . . . Schedule for the Preparation of 1.25% Glutaraldehyde Fixative Solution. . . . . . . Schedule for the Preparation of Glutaralde- hydeWaShBuffer............... Schedule for the Preparation of a 1% Osmium Tetroxide Fixative Solution . . . . . . . . . Schedule for the Preparation Epon-Araldite Reginoooooooooooooooooooo Schedule for the Preparation of Reynolds Lead CitrateStainoooooooooooooooo Schedule for the Preparation of Uranyl Ace- tateStainooooooooooooooooo Proportion of Red, White, and Intermediate Fibers in the Red Portion of Porcine Semi- tendinosus Muscle Inoculated with Pseudomon- fifiagiI I I I I I I I I I I I I I I I I I I Proportion of Red, White, and Intermediate Fibers in the White Portion of Porcine Semi- tendinosus Muscle Inoculated with Pseudom- onas fragi. . . . . . . . . . . . . . . . . . xii Page 160 160 161 161 161 162 163 163 164 165 165 Table Page 12 Proportion of Red, White. and Intermediate Fibers in the Red Portion of Porcine Semi- tendinosus Muscle Inoculated with Bacillus Qumil'USooooooooooooooooooo 166 13 Proportion of Red, White and Intermediate Fibers in the White Portion of Porcine Semi- tendinosus Muscle Inoculated with Bacillus Qum11USooooooooooooooooooo 166 14 Proport1on of Red. White. and Intermediate Fibers in the Red Portion of Porcine Semi- tendinosus Muscle Inoculated with Staphylo- coccusaurGUSoooooooooooooooo 167 15 Proportion of Red. White. and Intermediate Fibers in the White Portion of Porcine Semi- tendinosus Muscle Inoculated with Staphylo- coccus aureus . . . . . . . . . . . . . . . . 167 16 Proportion of Red, White. and Intermediate Fibers in the Red Portion of Porcine Semi- tendinosus Muscle Inoculated with Clostri- dium perfringens . . . . . . . . . . . . . . 168 1? Proportion of Red, White. and Intermediate Fibers in the White Portion of Porcine Semi- tendinosus Muscle Inoculated with Clostri- dium perfringen . . . . . . . . . . . . . . 168 18 Summary of the Growth of Pseudomonas fragi on Porcine Semitendinosus Muscle Stored at 10°C I I I I I I I I I I I I I I I I I I I I 169 19 Summary of the Change in pH During the Incubation of Porcine Semitendinosus Muscle Inoculated with Paaudomonas fragi and StoredatlooCooooooo ooooooo 170 20 Summary of the Growth of Bacillus pumilus on Porcine Semitendinosus Muscle Stored at 10°C I I I I I I I I I I I I I I I I I I I I 17 l 21 Summary of the Change in pH During the Incubation of Porcine Semitendinosus Muscle Inoculated with Bacillus pumilus and StoredathOC..........o..o. 172 22 Summary of the Growth of Staphylococcaa aureus on Porcine Semitendinosus Muscle StoredathOC.............o. 173 xiii Table 23 24 25 26 27 28 29 30 31 32 Summary of the Change in pH During the Incubation of Porcine Semitendinosus Muscle Inoculated with Staphylococcus aureus and Storedatloocoooooooooooooo Summary of the Growth of Clostridium per- fringena on Porcine Semitendinosus Muscle StoredatBOOC.............. Summary of the Change in pH During the Incubation of Porcine Semitendinosus Muscle Inoculated with Clostridium perfripgens and StoredatBOOCoooooooooooooo Bacterial Growth Data for P eudomonas fpagi or a Mixed Culture on Por01ne Longissimus Dorsi Muscle Incubated at 10°C . . . . . . Changes in the pH Values of Longissimus Dorsi Muscle Inoculated with Pseudomonaa fragi or a Mixed Culture from Hamburger and Incu— batedatIOOCooooooooooooooo Retention Time of Chromatographic Peaks of the Headspace Vapors of Uninoculated Porcine Longissimus Dorsi Muscle after Incubation at 10°C Run on a 3% Apiezon Column . . . . Retention Time of Chromatographic Peaks from the Headspace Vapors of Uninoculated Porcine Longissimus Dorsi Muscle after Incubation at 10°C Run on a 10% Carbowax Column . . . Retention Time of Chromatographic peaks from the Headspace Vapors of Porcine Longissimus Dorsi Muscle Incubated with a Mixed Culture from Hamburger at 10°C Run on a 3% ApieZOn COlumnoooooooooooooooooo Retention Time of Chromatographic Peaks from the Headspace Vapors of Porcine Longissimus Dorsi Muscle Incubated with a Mixed Culture from Hamburger at 10°C Run on a 10% Carbowax COlumnoooooooooooooooooo Retention Time of Chromatographic Peak from the HeadSpace Vapors of Porcine Longissimus Dorsi Muscle Incubated with Pseudomonas fragi at 10°C Run on a 10% Carbowax Column xiv Page 174 175 176 177 178 179 179 180 180 181 Table Page 33 Retention Time of Chromatographic Peaks from the Headspace Vapors of Porcine Longissimus Dorsi Muscle Incubated with Paeudomonaa fragi at 10°C Run on a 10% Carbowax Column . . . . . . . . . . . . . . . 181 XV INTRODUCTION Spoilage of meat has been a public health problem since muscle was first used as a food. While the role of bacteria in meat spoilage is well recognized, as yet the mechanism of spoilage is not fully understood. Like- wise. an adequate method of monitoring meat spoilage has not been developed. The Pseudomonas and Achrgmobacter groups have been shown to be the dominant flora of refrigerated meat and are also responsible for producing most of the of- fensive aesthetic changes in meat during spoilage. Re- cent work (Hasegawa EI.El-n 1970a, b; Dutson a; a1., 1971) has shown that several psychrophilic species of bacteria are capable of degrading meat proteins. giga- tridium perfringens. a food poisoning organism, has also been shown to be capable of meat proteolysis (Hapchuk. 1974). It, therefore, seems likely that other food poisoning organisms may be able to utilize muscle pro- teins for growth. Although several workers have studied bacterial proteolysis of meat using electrophoretic techniques. only a few reports employing electron microsc0py are found in the literature. Almost all of these studies used ground muscle tissue. and consequently. were unable to detect changes in nuclei, mitochondria, or membranes, which are disrupted during the grinding process. Gauthier (1970) pointed out that failure to recog- nize differences in muscle fiber type has often resulted in incorrect interpretation of experimental data. It has also been noted (Dutson at al., 1971; Gann, 1974) that ultrastructural changes due to aging may be related to fiber types. This suggests that the rate and degree of bacterial spoilage may also be associated with fiber types. Guarino and Kramer (1969) theorized that food head- space vapors could be used to identify the bacteriological flora during spoilage. However. on using meat products they were unsuccessful due to interference from the vapors normally present in the tissues. Other workers (Miller a} al.. 1973a, b) have identified some of the volatiles produced by the growth of Pseudomonas species on fish muscle. As yet, methods have not been developed to cor— relate aesthetic changes in meat with changes in the headspace volatiles occurring during the onset of spoilage. This study was undertaken to provide information concerning changes in ultrastructure and headspace vapors occurring in meat during the growth of known spoilage and food poisoning organisms. The major ob- jectives of the study were: (1) to identify and compare the sequence of ultrastructural changesin meat inocu- lated and incubated with various meat spoilage and food poisoning microorganisms: (2) to determine the relative susceptibility of red and white muscle fibers to bac- terial degradation: (3) to determine the relationship of pH and bacterial counts to the ultrastructural changes occurring during spoilage; and (4) to monitor the vola- tile components of meat as bacterial spoilage ensues. LITERATURE REVIEW Microbiology of Meats Classification of Spoilage Bacteria Glage (1901) first reported that the moist surfaces of meat stored at low temperature and high humidity be— came covered with bacterial colonies. He found these organisms to be oval to rod shaped with rounded ends and occasionally in chains. They were motile aerobes that slowly liquified gelatin and turned litmus milk al- kaline. Although the indigenous bacteria grew well at 2°C, their Optimum temperature for growth was 10 to 12°C. Haines (1933) observed that with the exception of a few Pseudomonaa and Proteus strains almost all bacteria growing on meat in cold storage belonged to the Apppp- mobacter group. Independently, Empey and Vickery (1933) found that 95% of the initial flora of beef capable of growth at 1°C were members of the Achromobacter genus. with the other 5% being various species of Pseudomonas and Micrococcpa. Later, Empey and Scott (1939) isolated bacteria growing at low temperatures on fresh meat and found Achppmobacter to comprise 90%. Micrococcus 7%, Flavobacterium 3%, and Pseudomonas 1% of the total flora. Ayres _p_a1. (1950). Kirsch ap_a1. (1952). and Wolin at al. (1957) reported that Pseudomonads are the dominant bacteria found on meat stored at low tempera- tures. These workers recognized that the discrepancy between their results and those of earlier investigators were due to changes in the nomenclature system. mainly as a consequence of the priority accorded the position of flagella. Due to changes in nomenclature, Brown and Weideman (1958) re-evaluated a large number of the Achrppppactep isolated by Empey and Scott (1939) and found most to be polarly flagellate species of Pgapdompnas. Subsequent studies (Thornley. 1967; Ayres, 1960; and Jay, 1967) have shown that the predominant meat spoilage bacteria at low temperatures are of the Pseudomonas genus. Due to a proposal to do away with the Achromo- bacter classification by Hendrieap a, (1974), all members of the Achromobactep genus are currently undergoing char- acterization studies with the possibility of reclassi- fication. Regardless of the animal specie or muscle, the pre- dominant bacterial flora remains relatively the same. Ayres (1960) reported that freshly slaughtered meat contained an approximately equal number from the genus Micrococcus and Psepdomonas. with lesser populations of Achrpppbacter, Flavobacteriam, Microbacterium, A1— pagigenes. Aeromppes. Bacillaa, Clostridipm, and Streptococcus. Organisms commonly found on poultry carcasses include both pigmented and nonpigmented strains of Eaapgppppaa and strains of Apipaxppagta; (Barnes and Impey. 1968). Ostovar a; a1. (1971) found Pagpdomopaa. Achrgmobactgr. and Flavobaptarium to be the dominant psy- chrotolerant genera isolated from deboned poultry meat. They also found only 4 of 54 samples were contaminated with Clostridium perfringgns. Halleck a: a1. (1958) reported that Achromobacter and Psgpgpmonaa species to- gether represented 60 and 51% of the flora for prepackaged fresh lamb and beef. respectively. Bacterial Growth Patterns pn Meat While enormous and varied microbial populations are associated with living meat animals (Lechowich. 1971). the bacterial species responsible for the spoilage of refrigerated meat seem to be quite restricted. Although Ayres (1960) found that more than 80% of the total micro- bial population on fresh ground beef consisted of chromo- genic bacteria. molds. yeasts. and sporeforming microorganisms. examination of spoiled meat revealed a microflora consisting primarily of single. paired. or short-chained. motile. Gram-negative. nonsporeforming rods. One of the first reports on changes in the relative numbers of bacterial species during cold storage was by Empey and Vickery (1933). who noted an increase in the relative numbers of Aghrompbacter and P eudomona . while the proportion of Micrococcps decreased. Kirsch a3 a_. (1952) reported a significant p0pulation of Lactobacillus and Micrococcus on meat at the beginning of storage. but found that Pseudomonas species predominated after spoilage. Wolin.ap_a1. (1957) reported that spoiled meat contained nonpigmented, Gram-negative. aerobic. polarly flagellate rods. with half of the isolated strains being capable of liquifying gelatin. Halleck ap_a1. (1958) examined prepackaged meat and found that during the first 2 weeks of storage the predominant organisms were of the nonpigmented Achromo- bacter-Pseudomonas type. but that pigmented types be- came predominant during subsequent storage. Adams ap_a;. (1964) reported that Pseudomonas and Achromobacter were the only groups in which the percentage of the total population increased during spoilage. Stringer 2£.§$- (1969) reported that Pseudomonas fra i. Pseudomonas geniculata. and Micrococcus luteus were the predominant organisms on beef carcasses. but only the Pseudomonas strains were found on meat at the retail level. Using pure cultures. Barnes and Impey (1968) found that chicken breast muscle and leg muscle differed in their ability to support bacterial growth. They reported good growth of Pseudomonas cultures on both muscle types. but gaipatobacter grew more readily on the leg muscle. 0n the other hand. they reported that Pseudomonas putrefaciens grew much faster on leg than on breast muscle. Examination of the ecological parameters effecting microbial growth in or on refrigerated meat revealed that the incubation temperature is the most important single factor influencing growth, preventing growth of all but a few genera (Jay. 1972). Green and Jezeski (1954) found that when the incubation temperature was raised, the proteolytic activity of Pseudomonads decreased. Al- ford and Elliott (1960) showed that elevated temperatures inhibited lipase production by Pseudomonaa fluorescens. but not the activity of the lipolytic enzymes. Alford (1960) found greater lipolytic and proteolytic activity when various Psapgomonas and Achromgpacter strains were incubated at temperatures lower than that for their op— timal growth. Ingram (1962) found that temperature. humidity. carbon dioxide. and oxygen levels all influence the bacterial flora of meat. Jay (1972) noted that the relatively low pH of fresh meat completely prevents growth of some bacteria. but only slows the growth rate of others. Aesthetic Chapges in Meat Caused by Bacterial Growth One of the first reports on aesthetic changes in meat due to bacterial growth was that of Mace (Circa 1900. cited by Tissier and Martelly. 1902). who observed that meat spoils in essentially two phases; first. by proliferation of aerobic sporeformers. which produce a flat odor, and secondly. by growth of Gram-negative bacteria. which cause development of a clearly putrid odor. Tissier and Martelly (1902) subsequently confirmed these observations. Ayres a§_a1. (1950) identified a characteristic rancid. sweetly aromatic. ester-like odor. which developed in poultry meat. and noted that production of this off-odor preceded slime formation. They also reported a pungent ammonia~like odor during the later stages of spoilage. Ayres (1960) isolated microorganisms from refrigerated beef and found that at 10°C or lower. the bacteria respon- sible for slime production were almost without exception pseudomonads. Various workers (Schmid. 1931: Empey and Vickery. 1933; Haines. 1937; Kraft and Ayres. 1952; Ayres, 1959. 1960) have attempted to relate bacterial numbers to slime formation. and hence. meat salability. Their results have been somewhat variable. with slime first appearing at bacterial counts ranging from 1 X 10°/cm2 (Kraft and Ayres. 1952) to 5 X 10°/cm2 (Schmid. 1931) of the meat surface area. Ogilvy (1950) obtained good agreement in comparing off-odor development or slime production with either bacterial load or an increase in carbon dioxide produc— tion. Ayres (1959. 1960) concluded that off—odor devel- Opment in eviscerated poultry and packaged beef occurs whenever microbial populations exceeded 107 cells/cm2 of surface area. Ayres (1960) pointed out that many of the meat 10 contaminating bacteria are mesophiles and are not res- ponsible for the development of off-odors. off-colors. or slime formation. Shewan at al. (1960) reported that Pseudompnas species are largely responsible for spoilage odors in fish; whereas. Achromobacter strains have little effect. Adams ap_a1. (1964) suggested that even though the extent of participation of individual members of a bac- terial population to spoilage has commonly been attributed to the preponderance of certain genera at the time of spoilage this is not always the case. They then theorized that all bacteria or bacterial groups are not equally active in spoilage. They concluded that most of the bacteria capable of causing spoilage characteristics in fish were confined to the Psepgomgnas—Achromobacter groups. but that only a small portion of those two groups were actually "spoilers". There was no selective in- crease for "spoilers" as storage progressed. Upon inocu- lation of sterile raw fish press juice with cultures from spoiled fish, they found that the initial load of "spoilers" was consistently below 10% of the total bacterial popula- tion. They also noted that even though Pseudpmonaa and Achromobacter strains accounted for almost 100% of the bacterial population by the end of spoilage. the percentage of "spoilers" had not increased. Subsequently. Herbert at a__l_. (1971) reported similar results for marine fish. McMeekin and Patterson (1975) found that only a ll restricted number of bacteria are capable of producing detectable amounts of hydrogen sulfide from meat. McMeekin (1975) tested the ability of pure cultures to produce off-odors in poultry meat and found strains of both pigmented and nonpigmented Pseudomonaa organisms to be capable of off-odor production. His data showed that 73% of nonpigmented Paeudomonas produced off-odors compared to only 22% for the pigmented Pagudoponaa strains. Bacterial growth on meats has been observed to cause changes in the pH of the tissue. Ockerman at a_. (1969) and Adamcic and Clark (1970) reported an increase in the pH values of beef and poultry tissue upon being inoculated and incubated with Achppmobactgr and Paapgpmonaa cultures. Lobben and Lee (1968) obtained similar results with fish muscle. Several authors (Hasegawa a; al.. 1970a. b; Borton a; al.. 1970a: Tarrant a; al.. 1973) have reported that growth of Paepdomonas fpag; on porcine muscle may raise the pH of the tissue to alkaline levels. Hasegawa 23 a_. (1970b) reported that growth of Clostridiup parfpingana caused an increase in the pH of porcine tissue. but noted no change in pH due to growth of Aghroppbapter ligpafacieng. Evaluation of Bacterial Spoilage Recognition of the end point of meat salability' as spoilage proceeds has been a continual problem of the meat industry. Eber (1892. cited by Turner. 1960) first proposed ammonia detection 12 as a means of assessing meat spoilage. Shortly thereafter. several workers (Ottolenghi. 1913; Falk a3 a1.. 1919; Tillmans apfla1.. 1921; Schmidt. 1928) attempted to cor- relate aesthetic changes in stored meat with bacterial loads. but the relationships were low. As microbiological techniques for sampling and enumeration improved. the relationship between bacterial counts and off-odor pro- duction and/or slime formation gave a more accurate assessment of spoilage (Schmid. 1931; Empey and Vickery, 1933; Haines. 1937; Kraft and Ayres. 1952; Ayres. 1959. 1960). A number of workers (Proctor and Greenlie. 1939; Johns. 1944; Straka and Stokes. 1957; Ferguson. 1958; Wells. 1959; and Walker a3 al.. 1959) have correlated bacterial numbers with resazurin dye reduction time. Kurtzman and Snyder (1960) developed a freshness test for iced shrimp based on an increase in the turbidity of a picric acid extract. which they reported to be associated with a decrease in organoleptic scores and an increase in total bacterial counts. Saffle ap,a1. (1961) com- pared the resazurin dye reduction and picric acid methods. as well as a ninhydrin method. in an attempt to determine the shelf—life of meat. They concluded that the resazurin reduction method gave the best correlation between odor scores and bacterial counts. Kraft at_a1. (1956) employed lead acetate impreg- nated filter paper strips to estimate the extent of 13 hydrogen sulfide production as an in-package indicator for growth of meat spoilage bacteria. Jay (1972) pro- posed that meat spoilage organisms alter the hydration capacity of muscle proteins. and subsequently deve10ped procedures for measuring muscle hydration. which he claimed 7 could be used to estimate the extent of spoilage. The extract-release volume (Jay. 1964a. b). water holding capacity (Jay. 1965) and meat swelling and viscosity (Jay. 1969) were all reported to correlate with organo— leptic and bacterial papulation data. On the other hand. Miller and Price (1971) found that the correlation between extract-release volume and bacterial numbers did not re- liably predict the bacteriological soundness of pork. Shelef and Jay (1970) also developed a titrimetric tech- nique for estimating spoilage. which is based upon the alkalinization of meat during bacterial growth. Fleming‘ap‘al. (1969) noted that the volatiles from a food may include the metabolic products of the various microorganisms growing within the food. Previous workers (O'Brien. 1966; Bassette at_al.. 1967) have shown that some bacterial species can be identified by analysis of the vapors produced when grown on culture media. Specifically. Escherichia 921;. Epterobacpap aerogenes. Pseudomonas aeppginosa (O'Brien. 1966). Streptococgpa faecalis. Streptocopgus lactis. Streptococpus diacetilactis. Lactobacillus acidophilus. Lactobacillps paaai. Achromo- pappap 1ypolyticum. and Pseudomonas fpagi (Bassette a; a1.. 14 1967) were reported to produce gas chromatograms varied enough to allow identification of the bacterial species. Guarino and Kramer (1969) speculated that analysis of headspace vapors for a food could be used as a rapid method for identifying the microbiological flora. Although they were able to identify members of the Enterobacteriaceae family. difficulty was encountered in differentiating bacterial species on hamburger due to the interference of the vapors normally present. While studying bacterial metabolism. Keenan a} a1. (1967) observed that pseudomonads have the ability to reduce acetaldehyde to ethyl alcohol. Subsequently, Reddy a: a1. (1969) noted that Psepgpmonas fpagi grown in milk con- verted ethyl alcohol to ethyl butyrate and ethyl hexanoate. These findings prompted Miller at_a1. (1973a. b) to study the volatiles produced by Pseudomonas species grown on fish muscle. Miller ap al. (1973a) first reported that Paga: domonas parolens produced methyl mercaptan. dimethyl di— sulfide. dimethyl trisulfide. 3-methyl-l-butanol. butanone, and 2-methoxy-3-isopropy1pyrazine. Later. Miller 23.él- (1973b) reported that Pseudomonas fragi produced dimethyl sulfide. acetaldehyde. ethyl acetate. ethyl alcohol. and dimethyl disulfide. Chemical Changes in Meat Due to Bacterial Growth Jay and Kontou (1967) reported that fresh beef allowed to undergo microbial spoilage at 7°C showed decreases in 15 the quantity and types of amino acids as well as decreases in nucleotide levels. Based on the decreases in the simple nitrogenous components. these authors concluded that beef spoilage bacteria do not attack the primary muscle proteins. However. careful examination of the chromatograms presented by Jay and Kbntou (1967) suggest that they had incorrectly interpreted the data. The chromatogram of tissue inoculated with a mixed culture of meat spoilage organisms showed large increases in several of the amino acids while the chromatogram from the sterile control revealed no such increases. Jay (1967) noted that not all bacteria capable of degrading gelatin were able to break down muscle proteins and concluded that any degradation of muscle proteins was probably due to the action of catheptic enzymes. He further stated that low-molecular weight compounds support the growth of spoilage bacteria and that the salt-soluble proteins are essentially untouched by bacterial proteolysis. Rampton apflal. (1970) reported that porcine myofibrillar proteins were not degraded by Achropobacter ligaafagiena. Micrpgpccua 1 t . gagiopoccufi gargviaiae. Eaaaggapaaa flaopagana. Streptpcoccus faecalia. or a mixed culture from spoiled meat. It should be noted. however. that these authors used sucrose density centrifugation. which has since been found to be insensitive to the protein changes occurring during meat spoilage. However. the authors reported poor growth of all cultures except Aphromobacter liguefaciens. which may account for their failure to find 16 degradation of myofibrillar proteins. In contrast. Ockerman 23.§l- (1969) found that beef muscle inoculated and incubated with Pseudomonas and Achromobacter cultures decreased slightly in stromal pro- tein. increased in nonprotein nitrogen. and had a greater emulsifying capacity than control uninoculated beef muscle. Adamcic a; a1. (1970) reported that Achromobacter and non- pigmented Pseudomonas cultures reduced the amount of amino acids On inoculated chicken skin during the early log phase of growth. whereas. pigmented Pseudomonas cultures caused a marked increase in amino acid levels during the late log phase. Borton.ap al. (1970a) found that growth of Pseudoppnas fgagi increased the water-soluble proteins in porcine muscle. They also noted increases in nonprotein nitrogen and salt-soluble protein levels when the tissue was inocu- lated with Pseudomonas fragi. Pedicoccus cereviaiaa. Micrococcus luteus. or Leuconostoc mesentergiaaa. Sub- sequently. Borton ap_a1. (1970b) reported changes in the electrophoretic pattern of porcine salt-soluble proteins caused by growth of the same four bacterial species. Samples inoculated with Pseudgppnas fragi showed a marked decrease in a number of protein bands indicating that pro— teolysis of salt-soluble proteins had occurred. Growth of the other bacterial species caused no detectable changes in the electrophoretic patterns of the tissue extracts. l7 Hasegawa a; al. (1970a) compared starch gel electro- phoretic patterns of extracts from aseptic and inoculated porcine and rabbit muscles. Pseudomonas fragi showed the greatest amount of proteolytic activity on the sarcoplasmic fraction of both rabbit and pig muscle. with lower but significant amounts of activity on the urea-soluble pro- teins. Leuconostoc mesenteroides caused alteration of the sarcoplasmic proteins of both rabbit and porcine muscle. but lg££2223£3§_ luteus degraded only the rabbit muscle proteins. Pedicoccpa cerevisiae was found to break down the urea—soluble proteins from both rabbit and porcine muscle. This organism also degraded the sarcoplasmic pro— teins from rabbit muscle. but had no effect on porcine sarcoplasmic proteins. Further analysis showed that Pseudompnas fpagi decomposed aldolase. glyceraldehyde phos- phate dehydrogenase. lactic dehydrogenase. creatine kinase and hemoglobin from pig muscle. and creatine kinase. phosphofructokinase. phosphoglycerate kinase. phospho- pyruvate hydratase. and hemoglobin from rabbit muscle. Thus. results indicated that different microorganisms pref- erentially utilized specific proteins from rabbit and por- cine muscle. Hasegawa a: al. (1970b) reported a similar study using Clostridium perfringaps. Salmonella anteritidis. Achpgmpbacter liquefacians. Streptococcus faecalis. and Karthia aopfii. They noted that Clostridium perfringens caused extensive breakdown of the sarcoplasmic and urea-soluble 18 proteins from pig and rabbit muscle. while Streptococcus faecalis and §a1ppnel1a entapipidis effected only myo- globin. Achromobacter liquefaciens and Kurthia zopfii had no measurable proteolytic activity on muscle tissue. Tarrant a: a1. (1971) inoculated and incubated por- cine muscle with Pseudomonas fragi. They noted a large decrease in the salt-soluble protein fraction with a corresponding increase in nonprotein nitrogen. Disc-gel electrophoretic patterns indicated significant proteolysis of the salt-soluble proteins by the 10th day of storage and almost complete breakdown by the 20th day. Protec— 1ytic activity was found to increase significantly during the late log phase of growth. Tarrant a; a1. (1973) isolated a proteolytic enzyme fraction from Pseudomonas fragi which rapidly degraded a myofibrillar protein preparation. This enzyme preparation also broke down G-actin and myosin more slowly and hy— drolyzed the sarcoplasmic proteins at even slower rates. They noted that the enzyme preparation displayed optium activity at neutral pH and 35°C. Porzio and Pearson (1975) purified and characterized a proteolytic enzyme isolated from Pseudomonas fragi. They found it to be Zn+2 activated and Ca+2 stabilized. Further characterization showed the enzyme preparation to have a molecular weight of 40.000-50.000 daltons. Buckley (1972) studied a protease fraction from Pseudomonas perolens and found that it degraded collagen l9 and myofibrillar proteins. but had little effect on the sarcoplasmic proteins. This enzyme preparation was re— ported to have an Optimum pH range of 6.5 to 9.0 and an Optimum temperature of 35°C. He found that the enzyme(s) were inactivated by EDTA and theorized that they were Ca+2 activated. Hapchuk (1974) studied the degradation of meat by the food poisoning organism. Clostridium perfpingens. She observed that Clostridium perfringens caused an in- crease in nonprotein nitrogen and a decrease in total sarcoplasmic nitrogen. troponin. actin. and trOpomyosin. She concluded that Clostridium perfringens was capable of degrading both the sarcoplasmic and myofibrillar pro- teins. However. a proteolytic enzyme fraction isolated from cultures of Clostridium perfringens exerted its major action on the sarcoplasmic proteins. leading to the conclusion that Clostridipm perfripgens presumably elab- orates more than one protease. Ulppastructural Changes Caused by Bacterial Growth Walker (1969) reported that the structural proteins of beef remained essentially intact for several weeks after incipient spoilage. On the other hand. Dutson ap_a1. (1971) reported that porcine tissue inoculated with Pseudomopaayfpag; was extremely disrupted after 8 days incubation at 10°C. These workers observed an almost complete absence of material in the H-zone. marked 20 disruption of the A-band. and some loss of dense material from the Z-line. They noted that myosin appeared to be the most susceptible protein to degradation. The ultra- structure of Pseudomonas fragi organisms observed in de- graded muscle tissue showed formation of bleb-like evaginations on the outer cell wall. This led the authors to theorize that the blebs may be the mechanism for pro- tease transport from the bacterial cell into the muscle tissue where a source of nutrients is available. Tarrant ap_al. (1973) inoculated porcine muscle with an enzyme preparation from Pseudomonas fragi. They re- ported evidence of some dissolution of the Z-line after 6 hours incubation. and almost complete loss of the dense material from the Z-line after 72 hours incubation. The other portions of the myofibril did not seem to be affected by the enzyme(s). Buckley a; a1. (1974) reported that porcine muscle inoculated and incubated with Pseudomonas perolens showed little ultrastructural change until after 8 days of storage, at which time marked Z-line fragmentation and myofibril separation were noted. On incubating the muscle with a proteolytic enzyme preparation from Pseudomonas perolens. the M-line and Z-line were completely removed by day 4. and by day 8 disruption of the actin-myosin complex be- came evident. Thus. the effects of the enzyme(s) and the organism pap §§ were not identical. Hapchuk (1974) reported that Clostridium perfringens 21 caused degradation of the thin filaments in the I-band region. She also noted a loss of material from the M-line which could be attributed to either proteolysis by the bacteria or the relatively high (37°C) incubation temperature. She also observed that Clostridium perfgingens degraded the white fibers more readily than the red fibers. especially in being more active in degrading the thin fila- ments. The degradation of the thin filaments observed by electron microsc0py confirmed the electrophoretic evidence for the breakdown of troponin. which is a component of the thin filament (Maruyama and Ebashi. 1970). Red and White Muscle Fibers Characterization of Red and White Muscle Fibers It has long been known that mammalian skeletal muscles differ in color (Ciaccio. 1898). and that the fibers com- posing skeletal muscles differ in their microscopic appear— ance (Ranvier. 1874). Even within a given muscle the fibers are known to differ from one another (Grutzner. 1884; Knoll. 1891). and as many as three types of fibers were described over 55 years ago by Bullard (1919). His- tochemical procedures for localizing enzymic activity have confirmed these early observations and extended them by revealing additional differences (Padykula. 1952; Ogata. 1958; Nachmias and Padykula. 1958; Dubowitz and Pearse. 1960). Red fibers have been characterized as those having 22 a highly oxidative metabolism. large mitochondrial numbers (Dubowitz and Pearse. 1960). high succinic dehydrogenase activity (Stein and Padykula. 1962). and elevated myoglobin levels (Chinoy. 1963; James. 1968). They have also been reported to be more resistant to fatigue than white fibers (Burke ap 1.. 1971 and 1973), and have higher levels of triglycerides (Adams ap,a1.. 1962) and lipase activity (Piantelli and Rebello. 1967). Conversely. white fibers predominate in glycolytic metabolism with high levels of phosphorylase and myo— fibrillar ATPase activity (Dubowitz and Pearse. 1960; Engel. 1962). They also are characterized by having faster contraction rates and motor unit response times than red fibers (Barnard at al.. 1971; Peter at a1.. 1972). The original observations on muscle color by Ranvier (1874) and Grutzner (1884) led to the use of nomenclature of "red" or "white" to describe the fibers. Subsequent findings have shown fiber variations running the gamut between the classical definitions of red and white fibers. The diversity has led to the development of numerous classi- fication schemes (Engel. 1974) for determining muscle fiber types. To avoid confusion the classical definitions of red and white fibers will be used throughout this work and intermediate fibers will not be considered. 23 Protein Differences Between Red and White Fibers Seidel ap_a1. (1964) discovered that myofibrils pre— pared from red muscle had a lower ATPase activity than those prepared from white muscle. Subsequently. several t al., 1965; Streter a§_a1.. 1966; Seidel, 1967) reported a lower Ca+2 groups (Barany ~modified and EDTA- modified ATPase activity for red than for white muscle myosin. Streter a; a1. (1966) found that at low ionic strength the ATPase activity of red muscle myosin was ac- tivated by N-ethylamaleidmide. while white muscle myosin was unaffected. Several workers (Seidel. 1967; Guth and Samaha. 1969; Samaha ap_a1.. 1970a) have reported that the Ca+2-modified ATPase activity of red muscle myosin is very labile at alkaline pH (10.5). but relatively stable at acid pH (4.35); conversely. the Ca+2—modified ATPase activity of white muscle myosin is relatively stable at alkaline pH and labile at acid pH. Kuehl and Adelstein (1970) found that red muscle myo— sin had no 3-methyl-histidine. but white muscle myosin contained 2 residues of 3-methyl-histidine per molecule. locker and Hagyard (1968) and Samaha a§_a1. (1970a, b) reported that upon treatment of red and white muscle myosin with p-chloromercuriphenylsulfonate. the red muscle myosin released two electrophoretically distinct proteins not found in white muscle myosin. Seidel (1967) and Gergely a; a1. (1965) reported that red muscle myosin was more resistant to tryptic digestion than white muscle 24 myosin. Suzuki ap_a1. (1973) studied a-actinin from porcine red and white muscles and found no differences in sedi— mentation patterns. circular dichroic spectra. rate of trypsin digestion, or in the ability to increase the Mg+2— modified ATPase activity or rate of turbidity formation in suspensions of either red or white reconstituted actomyosin. However, they did find that apactinin from red muscle contained more aspartic acid than a-actinin from white muscle; this resulted in a—actinin from red muscle having 17 more negatively charged amino acids per 100 residues than white a—actinin. glprastructural Differences Between Red and White Muscle Fibers Using the system of Gauthier (1969. 1970), Dutson at al. (1974). and Suzuki a; a1. (1973) classified porcine muscle fibers as either red or white. They reported that red fibers had wide. dense Z-lines approximately 120 nm in width. while white muscle fibers were characterized by narrow. less dense Z-lines approximately 6.25 nm in width. They also noted that mitochondria from red fibers were very large. showed closely packed cristae. were dense in appearance, and were arranged in groups just beneath the sarcolemma. in intrafibrillar rows. and/Or in pairs at the Z-line. The mitochondria from white fibers were ob- served to be smaller, less dense. and contained fewer cristae. Yhite fiber mitochondria were observed mainly 25 between myofibrils at the level of the Z—line, with a limited number located near the sarcolemma. Dutson a; a1. (1974) reported the sarcoplasmic reticulum of white fibers to have large. Open longitudinal tubules; and in the region of the H-zone. they contained open sac- 1ike structures with some fenestrations. Red fiber longitudinal tubules were found to be narrower. more tortuous and have fewer fenestrations. Terminal cisternae of red fibers appeared dense throughout, whereas. in white fibers the dense areas were primarily next to the transverse tubule. Autolysis of Muscle Tissue Protein and Nonprotein Nitrogen Changes Hoagland ap_a1. (1917) noted an increase in noncoagu- lable nitrogen during low temperature storage of meat and attributed it to autolysis. McCarthy and King (1942) ob- served that meat aged at either cooler or high temperatures (35°C) showed an increase in soluble nitrogen values. Radouco-Thomas ap a1. (1959) found that the release of amino acids in postmortem muscle was inhibited by the in- jection of epinephrine and suggested its use as an anti- autolytic agent. Numerous other workers subsequently have observed increases in the amounts of either nonprotein nitrogen or amino acids during meat aging (locker. 1960; Na 21,§l-: 1961; Sharp. 1963; Davey and Gilbert, 1966: 26 Suzuki a$_a1.. 1967; Field and Chang, 1969; Parrish §£.§l-: 1969a). Several authors (Kronman and Winterbottom. 1960; McIoughlin. 1963; Sayre and Briskey, 1963; Goll 23.31-: 1964; Aberle and Merkel. 1966) have reported the sarcoplasmic proteins to be most soluble immediately after death. fol- lowing which the solubility decreases with increased storage times. The effect of aging upon the myofibrillar proteins is less clear. Some workers (Zender a: al., 1958; McLoughlin. 1963; Sayre and Briskey, 1963) have reported a decrease in myofibrillar solubility with aging. while Goll 22.81; (1964) found no change. Other authors (Hegarty, 1963; Aberle and Merkel. 1966; McIntosh. 1967; Penny. 1968; Davey and Gilbert. 1968a. b) have found an increase in the solubility of the myofibrillar proteins as postmortem time increased. It has been suggested by several groups (Sayre and Briskey, 1963; McLoughlin. 1963; Davey and Gilbert. 1968a) that pH and temperature may determine protein solubility during aging. Structural Chapges Henderson _p‘_;. (1970) found that unrestrained por- cine muscles underwent minimal shortening at 25°C and only slightly more contraction at 2 and 16°C. Gann (1974) working with bovine muscle reported variable degrees of contraction at various intervals throughout 216 hrs of cooler storage. 27 Davey and Gilbert (1969) observed a loss of adhesion in aged bovine myofibrils and alterations of the Z-line. which sometimes led to complete dissolution of the Z—line. Henderson 31 a1. (1970) reported that Z-line degradation occurs more quickly and to a greater extent at storage temperatures of 25°C or above than at temperatures of 16°C or below. Cassens _§_a1. (1963) reported mitochondrial disrup- tion in porcine muscle at 24 hrs postmortem. Dutson 23.§l- (1974) observed that mitochondrial disruption or loss of cristae density occurred in all samples by 24 hrs post- mortem. but was particularly obvious in white fibers. Gann (1974) noted considerable morphological variation in mito- chondria by 48 hrs postmortem. Intermyofibrillar mito- chondria appeared more stable than those encircling the I-band-Z-line area and suggested a fiber type degradation relationship, with red fibers being more stable than white fibers. Dutson a: a1. (1974) working with porcine muscle re- ported that no triads or transverse tubules were apparent in either fiber type by 24 hrs postmortem. Furthermore. by 24 hrs postmortem the sarcoplasmic reticulum was no longer recognizable. Gann (1974) observed that even though both red and white fibers showed disruption of the connection between the thin filaments and the Z-line, white fibers were easily distinguishable from red because of a greater number of 28 breaks. Lehninger (1970) reported an abundant supply of 15 to 30 nm diameter glycogen granules in the intermyofibrillar cytoplasm of muscle at death. However. Gann (1974) noted that glycogen granules were completely absent in bovine muscle by 48 hrs postmortem. Henderson 21.§l- (1970) using porcine muscle found that storage at 37°C had caused the complete disappearance Of the M-line structure. However. Gann (1974) utilizing bovine muscle reported that at cooler temperatures the M-line was unaltered at 48 hrs postmortem. but that by 216 hrs the M~line although still apparent was not discrete or prominent. Catheptic_§nzyme and Lysosomal Activity Much attention has been devoted to isolating a com- ponent or components which would promote protein degrada- tion in postmortem muscle. Balls (1938) isolated and partially purified a cathepsin from muscle which had a pH Optimum of 4.1. Snoke and Neurath (1950) reported the isolation and partial purification of a proteolytic agent from rabbit skeletal muscle with a pH Optimum of 4.0 and activity against crude muscle extracts. Sliwinski ap,al. (1959) purified an enzyme from beef muscle and determined that Optimum activity occurred at pH 4.4 and 37°C. Doty (1950) suggested that many of the changes ob- served in postmortem aging of meat could be attributed to 28 breaks. Lehninger (1970) reported an abundant supply of 15 to 30 nm diameter glycogen granules in the intermyofibrillar cytoplasm of muscle at death. However. Gann (1974) noted that glycogen granules were completely absent in bovine muscle by 48 hrs postmortem. Henderson 93 a1. (1970) using porcine muscle found that storage at 37°C had caused the complete disappearance of the M—line structure. However. Gann (1974) utilizing bovine muscle reported that at cooler temperatures the M-line was unaltered at 48 hrs postmortem. but that by 216 hrs the M~line although still apparent was not discrete or prominent. Catheptic_§nzyme anquysosomal Activity Much attention has been devoted to isolating a com- ponent or components which would promote protein degrada- tion in postmortem muscle. Balls (1938) isolated and partially purified a cathepsin from muscle which had a pH optimum of 4.1. Snoke and Neurath (1950) reported the isolation and partial purification of a proteolytic agent from rabbit skeletal muscle with a pH Optimum of 4.0 and activity against crude muscle extracts. Sliwinski a1_a1. (1959) purified an enzyme from beef muscle and determined that Optimum activity occurred at pH 4.4 and 37°C. Doty (1950) suggested that many of the changes ob- served in postmortem aging of meat could be attributed to 29 proteolytic enzymes of the catheptic group. Balls (1960) in reviewing catheptic enzymes in muscle stated that the quantity of these components was very low and that their optimum activity occurs at a more acidic pH than is attained by postmortem muscle. Subsequently. Koszalka and Miller (1960a. b) purified an enzyme from rat skeletal muscle with Optimum activity at pH 8.5-9.0 on both synthetic and homo- genized muscle substrates. Sliwinski 23.81- (1961) re- ported on a crude bovine enzyme preparation. which was isolated at pH 5.6 and contained three different proteolytic enzymes. Landman (1963) observed that autolytic activity in beef muscle had a dual optima at pH 5.0 and 8.5-9.0. He concluded that cathepsins B and C were responsible for the dual optima. Although proteases have been demonstrated in muscle, the most important aspect of this subject is their activity against muscle substrates. Sharp (1963) studied autolysis of bovine and rabbit muscle and observed an increase in non- protein nitrogen but no change in the fine structure of the tissue. They concluded that the increased quantity of nonprotein nitrogen was due to the degradation of sarco- plasmic proteins. Bodwell and Pearson (1964). using a partially purified bovine catheptic fraction. studied catheptic activity on peptides. synthetic substrates. and four natural sub- strates isolated from muscle. The fraction was found to have no detectable enzymatic action on crude preparations 30 of actin. myosin, or actomyosin. but the sarcoplasmic pro— teins appeared to be readily hydrolyzed. Martins and Whitaker (1968) prepared cathepsin D and found no detec- table activity on actomyosin. In related papers, Suzuki and Fujumaki (1968) and Suzuki §t_al. (1969a, b) reported on the isolation and purification of cathepsin D from rabbit muscle. They found cathepsin D to be most active against the sarc0plasmic pro- teins, followed in order by myosin, actin. and actomyosin. Several investigators (Balls, 1960; Sharp, 1963; Bod— well and Pearson, 196M; Martins and Whitaker, 1968) have raised doubts about the significance of aseptic autolysis of primary meat proteins during aging. Martins and Whitaker (1968) and Caldwell (1970) have suggested that a combina- tion of the various cathepsins (A, B, C, D) may be neces- sary for the degradation of the salt-soluble proteins of meat. Smith (1964) reported that histochemical enzyme markers showed lysosomal activity to be confined to the vascular system of normal muscle. Parrish and Baily (1966, 1967) found that bovine cathepsins were particulate and suggested that at least a portion of the enzymes found in muscle were membrane bound. Pecently, other workers (Moore gt_gl., 1970; Bernacki and Bosmann, 1972; Tokes and Chambers. 1975) have found membrane associated proteases. Bernacki and Bosmann (1972) found cathepsin D-like activity to be associated with the membrane of human erythrocytes. 'hese 31 results suggest a possible role for membrane associated proteases in the autolysis of muscle tissue. METHODS AND MATERIALS Preparation of Stock Cultures Pure cultures of Pseudomonas fragi, Bacillus pumilus (formerly classified as Achromobacter liquefaciens), gtaph- ylococcus aureus, and Clostridium perfringens were obtained from the American Type Culture Collection (Rockville, Mary- land). The specific culture numbers and their incubation conditions are shown in Table l. The Staphylococcus aureus culture was initially incubated at room temperature but was gradually acclimated to 15°C. The Clostridiumyperfringens culture was started at 35°C and gradually reduced to 30°C. A mixed culture from commercial hamburger was pre— pared by blending the tissue with distilled water and in- oculating the slurry into APT broth. Slaughter and Tissue Preparation Slaughterinq_Frocedures Seven market weight pigs (81 to 10h Kg) produced at the Hichigan State University Swine Varm were slaughtered individually over a 6 month period. A modification of the method of Hasegawa g: al. (1970) was employed to ob— tain aseptic muscle samples. 33 The unstunned pigs were suspended by the hind leg and the area of the neck utilized for sticking was scrubbed thoroughly with a bactericidal soap solution. Sticking was carried out with a sterile knife. After conventional dehairing and evisceration, the unsplit carcass was rinsed twice with ethanol and flamed with a dehairing torch. The carcass was then placed in a l to 3°C cooler for approximately 2h hrs. After chilling. the carcass was laid on a steam sterilized stainless steel table in a clean room free from excessive air currents and the ethanol rinse-flame proce- dure was repeated twice. The carcass was then ready for muscle excision. Preparation of Muscle for Ultrastructural Analysis The carcass was positioned on the sterile table so that the hams were exposed. An incision was then made starting at the lower midline, curving inward and finally outward to the hock. The skin and subcutaneous fat were stripped off exposing the upper portion of the semitendin— osus muscle. The semitendinosus muscle was then dissected out from origin to incision and placed in sterile stainless steel containers. The excised muscle was then taken to a filtered-air inoculation room. The predominantly red and white areas of the muscle were then separated and the remainder dis— carded. A sample was taken from both the red and white areas and frozen in liquid nitrogen for subsequent pH 34 and histochemical analysis. The remaining tissue was pre- pared for inoculation by cutting it into 1-3 mm thick slices with a sterilized Sears Model #90 electric knife. During all muscle preparation procedures only sterilized equip- ment was used and all personnel wore sterile disposable gloves. Preparation of Muscle for Headspace Analysis The pig carcasses used to study meat spoilage volatiles were prepared in the same way as the ones used for ultra- structural analysis. except that the longissimus dorsi was used instead of the semitendinosus muscle. This was done by making an incision through the backfat along the dorsal midline. followed by two incisions perpendicular to the midline. The backfat was stripped back from the midline and sections of the longissimus dorsi muscle were excised and placed in a sterile container. The precautions pre- viously described were used in order to avoid contamina— tion of the tissue. Tissue Inoculation and Incubation Tissue for Ultrastructural Analysis Stock cultures of either Pseudomonas fpagi, Bacillus pumilus. Staphylococcus a reus. or Clostridium perfringens were used to inoculate the tissue. This was done by dipping the sliced tissue into a magnetically stirred inoculum for 5 seconds and draining by suspension for 10 35 seconds. The inoculated tissue was then spread across the bottom of disposable petri dishes. covered immediately and placed at the prOper incubation temperature (see Table I). Clostridium perfringens samples were maintained in anaerobic conditions by placing in a National Appliance (Portland, Oregon) incubator, which was then vacuumized and nitrogen flushed three times. A preliminary study showed that the dipped-slice inocu- lation technique yielded uniform bacterial loads per gram of tissue (see Appendix I). Tissue for Headspace Analysis Sections of the longissimus dorsi muscle were ground through a sterilized prechilled grinder. placed in sterile containers, and covered with a loose fitting sterile lid. Inoculum was prepared by diluting loo-fold a #8 hr culture of Pseudomonas fpggi or a mixed culture prepared from com- merical hamburger. During grinding. 50 ml of the inoculum were added to approximately 1000 gm of muscle. The ground tissue was then Spread evenly across the bottom of petri dishes and incubated at 10°C. Samples were removed daily for bacterial counting, sensory evaluation, pH measurement, and headspace analysis. A three member sensory panel was used to detect the onset of spoilage. They were presented with samples of inocu- lated and uninoculated control tissue and asked to indicate the absence or presence of off-odors. 36 .nomusnsdn Hmaohossoo scum auspaso .mmmwmmwmmmwfi umammmmmmmqu mm cmwmwmmmao wanesuomw mm mama am< oH oa swamp am< nun: nmuspaso vex“: oswpomuoomo on In npwj\mmm spams mud on same E on on $83329 :39 melodclilfltom ssfiuaupmoao paws pawn swamp nacpwssms cofimsmsfi mm news sm< ma ma shamansfimmm mmssa mampam msoooooa n «Pm mN paws Bad 0H 0H spoup am< wanna smsafissm msaafiomm mm mews sm< 0H CH group and mum: «mama mwcoaocsmmm oo mommmcmoz on» go Emnwopmaoano .w: oaswwm as I spa>aeancem cfls\as com I epnm scam nae cfls\ae on I swam 30am :mmoaczm sfl£\ae o: I Aaowuhmov ovum 30am sawdom ooomH I enzymummsme whom sofipoowsH oooma I snapsthSoa sopoopom 0000 I ousvmuomEoe so>o 133 mé 0.x. n6 0 - u d .o Tm o 4 J movscfle .mefie seawzmpog .m m .3 d 0.: Tm o.m min. on m; pie To 0 J a W q 4 q q u 1.)? mvznmUHMPmo< 3363 Harem}. .\ 33K. canons :— aocnrpm\q n2 iufiteH Haas 134 Oven Temperature - 60°C 0 Detector Temperature - 190 C Injection Port Temperature - 190°C Helium Flow Rate (carrier) - 40 ml/min Hydrogen Flow Rate - 30 ml/min Air Flow Rate - 500 ml/min Sensitivity - 10 Figure 49. Chromatogram of headspace vapors of porcine longissimus dorsi muscle incubated for 48 hrs at 10°C with a mixed culture. Run on a 3% Apiezon column. Peak Height 135 /‘ Ethanol and (Lactic Acid Air mPropionic Acid O /—— Ace taldehyde Ethyl Acetate I I 1 I I J I I 1 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4. Retention Time. minutes 136 culture from hamburger were the presence and size of the ethanol peak. Analysis at 24 hrs showed that substantial amounts of ethanol were produced before off-odor production. This suggests that ethanol may be an indicator of bacterial growth when acid producers are the dominant species. An 1 sis of Head a Va r from T ue Inoc l d W th Pgepdomonag fpggi. At 0 hrs incubation. there was no dif- ference in the headspace volatiles for tissue inoculated with Pgeudomonag fpggi and those of controls. However. after 48 hrs of incubation three peaks were produced on the Carbowax 20M column. When the same sample was analyzed on the Apiezon L column four distinct peaks were observed. Further incubation caused no changes in the chromatographic pattern produced by either type of column. Upon comparison with the controls at 48 hrs incubation. chromatograms from the Pgeudomonas'fpggi inoculated tissue (Figures 50 and 51) and those from the aseptic controls (Figures 46 and 47) were similar. The only major difference was an additional peak on both Apiezon L and Carbowax 20M columns. Retention times for the unidentified peak on both columns were identical to the ethyl acetate standards. Thus. the headspace vapors showed that ethyl acetate was produced by Paeudomongg gpggi. However. the use of ethyl acetate as an indicator of bac- terial spoilage is questionable due to the small size of the peaks. their incomplete separation. and because their appearance does not coincide with off-odor production. Ana f ds e Va or U in GC-MS. Attempts to 137 Oven Temperature - 60°C Detector Temperature — 190°C Injection Port Temperature - 190°C Helium Flow Rate (carrier) - 40 ml/min Hydrogen Flow Rate - 30 ml/min Air Flow Rate - 500 ml/min Sensitivity - 10 Figure 50. Chromatogram of the headspace vapors of por- cine longissimus dorsi muscle incubated for 48 hrs at 10°C with Pseudomonas fragi. Run on a 10% Carbowax column. Peak Height 138 (Air {Lactic Acid [E thyl Acetate \ 1") Acetaldehyde A L I L I J I I I. O 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Retention Time. minutes ":- . 3" L'- Figure 51. 139 Oven Temperature - 60°C Detector Temperature - 190°C Injection Port Temperature - 190°C Helium Flow Rate (carrier) - 40 ml/min Hydrogen Flow Rate - 30 ml/min Air Flow Rate - 50 ml/min Sensitivity - 10 ChromatOgram of the headspace vapors of porcine longissimus dorsi muscle incubated for 48 hrs at 10°C with Pseudoponas fragi. Run on a 3% Apiezon column. Peak Height 140 Acetaldehyde Airq )Lactic Acid I ' I l I I I l l I 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Retention Time. minutes 141 conclusively identify the headspace volatiles of either inoculated or uninoculated tissues by mass-spectrometry were unsuccessful. Since the mass spectrometer requires a very high vacuum to operate. the air injected as a component of the gas sample must be pumped out before any sample can be ion- ized. The relatively short retention times encountered with meat headspace volatiles did not allow enough time for the mass spectrometer to regain sufficient vacuum before the sample came off the column. Thus. the loss of vacuum created by injecting an air sample and the short retention times of the components to be analyzed did not permit mass-spectral analysis. The results of these experiments. although not conclu- sive. indicated that there are differences in the headspace volatiles from fresh and spoiled meat. Although the tech- niques employed were not sensitive enough for the identifi- cation of some of the off-odor volatiles. modification of the procedures may yield more useful results. Although sensory evaluation showed off-odor develOpment. chromatograms at the same time interval showed no corres- ponding changes except for the production of ehtanol in mixed culture inoculated tissues. This was probably due to the insensitivity 01‘ the flame-ionization detector to some of the known off-odor compounds in meat. i.e.. hydrogen sul- fide. dimethyl disulfide. and dimethyl disulfide. Therefore. a chromatograph with a detector system suitable for iden- tifying sulfur compounds. such as a flame photometric 142 detector. might show more conclusive differences as spoilage progresses. Vacuum distillation could also be expected to improve the sensitivity of the technique due to a more com- plete extraction and concentration of the tissue volatiles and the resultant flexibility of using various solvent sys- tems. Thus. this type of modification would probably result in a sample more compatible with the GC-MS system. SUMMARY The red and white portions of aseptic porcine semiten- dinosus muscle were inoculated and incubated with selected bacterial cultures to determine the influence of bacterial growth upon different fiber types. pH levels. and tissue ultrastructure. In addition. the effects of growth by meat spoilage microorganisms (Psgudomonas fpggi and a mixed cul- ture from commercial hamburger) were monitored by gas- chromatographic analysis of headspace vapors from the meat at different time intervals. Pure cultures of Pseudomonas gpggi. Bacillus pumilus. Staphylococcus aureus. and Clogtridium perfpipgens grew equally well on both the red and white portions of porcine semitendinosus muscle. Thus. fiber type did not appear to influence bacterial growth. Tissue pH increased during growth of £3eudogong§ fra i. Staphylococcgs aureps. and Clostridium perfringens. however. growth of Bacillus pppilug did not alter tissue pH. Increases in pH values of the inoculated tissue were not influenced by fiber type but appeared to be related to the amount of bacterial growth which had occurred in the tissue. Ultrastructural damage was extensive in tissues 143 144 inoculated with W Lear 1. We assess. and Clsstridium.pspfpipgsps. whereas Essillss pppilus caused no detectable ultrastructural change. Fiber type did not appear to influence the mechanism or rate of ultrastructural damage caused by bacterial growth. Incubation with Pssudomonas fra i. Staphylococcus aureus. and Clostridium pergringsns resulted in complete disruption of the myofibrillar structure. In each case. degradation appeared to start with I-band breakage. after which the I- band-Z-line material became diffuse and finally indistin- guishable. Thus. the A-band region of the myofibril was the most resistant area to microbial attack. Growth of Clostpidism pgrfpingeps and Pssudoponss gpsgi resulted in degradation of tissue nuclei and nuclear material. whereas. growth of Stspnylococcus sppsss and Bssillss_pnpilss did not appear to alter the nuclei. Mitochondrial cristae disappeared due to growth of W W! Whereas! _..L_L§.Ba 11 ‘1 umilus. W fra i. and Staphylocscsss aursps did not effect the mitochondria during the first 96 hrs of incubation. After 96 hrs of in- cubation. mitochondria in tissue inoculated with Pssud0ponss fpsgi and Staphylocoscus aureus appeared to be more stable than in aseptic controls or in tissue inoculated with Bacillus pumilus. Comparison of pH changes during growth with the published pH Optima of catheptic enzymes suggested that mitochondrial degradation during incubation was probably the result of autolysis. Thus. pH increases caused by the 11.5 growth of Psssd0ponss fragi and Staphylososcns ssreus caused inhibition of the catheptic enzymes. resulting in greater mitochondrial stability. Ultrastructural observations during autolysis of control samples revealed the presence of vesicular structures in the degraded areas of the myofibril. These vesicles ranged in size from 0.01 to 1.45um and appeared to be associated with autolytic degradation. It was also noted that intermyofibrillar mitochondria were more stable to autolysis than intramyofibrillar mitochondria. This resulted in greater disruption of red fibers because of their higher mitochondrial content. However. fiber type psp,ss did not influence the rate of degradation of in- dividual mitochondria. Headspace analysis of uninoculated aseptic control tissue resulted in the tentative identification of acetone. lactic acid and acetaldehyde. Chromatograms from tissue inoculated with an acid producing mixed culture showed peaks for lactic acid. acetaldehyde. ethyl acetate. ethanol. and propionic acid. whereas. Psssdomonas fpsgi produced chromatograms similar to the controls except for the presence of a small ethyl acetate peak. With the exception of ethanol. the appearance of chromatographic peaks did not correspond to development of off-odors. These results suggest that chromatographic profiles of headspace volatiles may prove useful for detecting the onset of meat spoilage. but further work with more refined systems will be needed to make the data useful. REFERENCES REFERENCES Aberle. E. D. and R. A. Merkel. 1966. Solubility and electrophoretic behavior of some proteins of post- mortem aged bovine muscle. J. Food Sci. 31: 151. Adamcic. M. and D. S. Clarke. 1970. 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APPENDICES Appendix I Bacterial Numbers and Growth Using a Dip Inoculation Technique 160 Appendix Table 1 Bacterial Counts on Meat Inoculated with Pseudomonas fragi UsingaDip Technique Sample Number* Dilution 1 2 3 4 10"1 TMTC TMTC TMTC TMTC 10"2 TMTC TMTC TMTC TMTC 10’3 ammo TMTC TMTC TMTC 10"“ 33 35 41 39 10’5 o o 1 o 10"6 o o o o *TMTC = too many to count Appendix Table 2 Effect of Either Random or Spread Packing Technique on the Growth of Pssudomonas fragi Dip Inoculated on Porcine Muscle log Bacterial Number Sample Number Spread Pack Random Pack 1 10.18 10.23 2 10.36 10.26 3 10.36 10.33 4 10.42 10.26 Average 10.33 10.27 Appendix II Schedules for the Preparation of Histochemical and Electron Microscopy Solutions 161 Appendix Table 3 Schedule for the Preparation of the Histochemical Incubating Medium Reagents Amount 0.2M Tris buffer (pH 7.4) 10 ml nitro blue tetrazolium 10 mg NADH 8 mg Appendix Table 4 Schedule for the Preparation of 1.25% Glutaraldehyde Fixative Solution Reagents Amount Final Molarity NaH2P0h°H20 1.80 gm 0.007 NazHPOu-7H20 23.25 gm 0.041 NaCl 5.00 gm 0.043 50% Glutaraldehyde 50.00 m1 Appendix Table 5 Schedule for the Preparation of Glutaraldehyde Wash Buffer Reagents Amount Final Molarity NaHZPOu-HZO 1.80 gm 0.013 NazHP04-7H20 23.25 gm 0.081 NaCl 5.00 gm 0.086 H 0 925.00 ml 2 162 Appendix Table 6 Schedule for the Preparation of a 1% Osmium Tetroxide Fixative Solution A. Stock solution A. Reagents Amount Sodium acetate 9.714 gm Veronal-sodium 14.714 gm Make to 500 m1 final volume with H20. B. Stock solution B. Reagents Amount Sodium chloride 40.25 gm Potassium chloride 2.10 gm Calcium chloride 0.90 gm Make to 500 ml final volume with H20 The solutions are mixed according to the following scheme: Solution A 10.0 ml Solution B 3.4 ml Dilute to 50 ml with H20 0.1N HCl 11 m1 (approx.) Solutions A and B are measured out and made to a 50 ml volume with distilled water. The pH is then adjusted to 7.2-7.4 with 0.1N HCl. To this mixture 0.5 gm of osmium tetroxide is added and stored in a brown glass stappered bottle. 163 Appendix Table 7 Schedule for the Preparation Epon-Araldite Resin Reagents Amount Epon 812 62 m1 Araldite 506 81 ml DDSA (hardner) 100 ml dibutyl-phthalate 4-7 ml UMP-30 1.5-3.0% Mixing solution: Epon 812. Araldite 506. and DDSA are thoroughly mixed after which the dibutyl-phthalate and the DMP-30 are added. After mixing the resin is ready for embedding the tissue. Appendix Table 8 Schedule for the Preparation of Reynolds Lead Citrate Stain Reagents Amount Lead nitrate 1.33 gm Sodium citrate 1.76 gm 1N NaOH 8 ml H20 (freshly boiled) make to 50 ml 164 Appendix Table 9 Schedule for the Preparation of Uranyl Acetate Stain Reagents Amount Uranyl acetate 8 gm H20 (glass distilled) 100 m1 Appendix III Raw Data for the Histochemical Determination of Fiber Type {val 165 Appendix Table 10 Pr0portion of Red. White. and Intermediate Fibers in the Red Portion of Porcine Semitendinosus Muscle Inoculated with Psssdomonas fragi Intermediate Red Fibers White Fibers Fibers Fasciculi # Number % Number % Number % 1 20 71o“ "" "'""' 8 28o6 2 26 70.3 1 2.7 10 27.0 3 25 71.4 1 2.9 9 25.7 L" 20 76o9 "" "" 6 23o]. 5 30 65.2 3 6.5 13 28.3 6 16 69o6 "" "" 7 BOoLI' 7 17 68.0 -- --- 8 32.0 8 24 72.7 1 3.0 8 24.2 9 17 63.0 1 3-7 9 33-3 10 17 70.8 -- ~-- 7 29.2 Avg. 21.2 69o9 o? 1.9 8o5 28.2 ’- Appendix Table 11 Proportion of Red. White. and Intermediate Fibers in the White Portion of Porcine Semitendinosus Muscle Inoculated with Psspdomonas fragi Intermediate Red Fibers White Fibers Fibers Fasciculi # Number % Number % Number % 1 3 3.8 60 75.9 16 20.3 2 2 2.7 58 79.5 13 17.8 3 4 4.3 70 76.1 18 19.6 4 --- --- 17 81.0 4 19.0 5 1 1.3 62 79.5 15 19.2 6 2 2.7 58 77.3 15 20.0 7 --- --- 28 82.4 6 17.6 8 --- --- 20 80.0 5 20.0 9 l 3.3 22 73-3 7 23-3 10 1 2.6 29 76.3 8 21.1 Avg. 1.4 2.1 42.4 78.1 10.7 19.8 166 Appendix Table 12 Pr0portion of Red. White. and Intermediate Fibers in the Red Portion of Porcine Semitendinosus Muscle Inoculated with Bacillus pumilus Intermediate Red Fibers White Fibers Fibers Fasciculi # Number % Number % Number % l 9 52.9 2 11.8 6 25.3 2 6 50.0 1 8.3 5 1.7 3 5 45-5 1 9-1 5 45-5 4 10 66.7 --- ---- 5 33.3 5 10 47.6 2 9.5 9 2.9 6 11 64.7 2 11.8 4 23.5 7 6 46.2 1 7.7 6 46.2 8 5 50.0 1 10.0 4 40.0 9 10 50.0 4 20.0 ‘6 30.0 10 12 52.2 3 13.0 8 34.8 Avg. 80“ 5206 107 1001 5.8 3703 Appendix Table 13 Proportion of Red. White. and Intermediate Fibers in the White Portion of Porcine Semitendinosus Muscle Inoculated with Bacillus pumilus w —' Intermediate Red Fibers White Fibers Fibers Fasciculi # Number 5 Number % Number % 1 1 2.6 27 71.1 10 26.3 2 3 10.3 19 65.5 7 24.1 3 --- ---- 19 67.9 9 32.1 1" “" """""" 15 7101‘" 6 2806 5 --- ~--- 21 77.8 6 22.2 6 2 4.9 30 73.2 9 21.9 7 l 3.3 23 76.7 6 20.0 8 1 4.2 19 79.2 4 16.7 9 --- ---- 20 83.3 4 16.7 10 l 2.9 26 76.5 7 20.6 Avg. .9 208 2109 7’4’03 608 2209 167 Appendix Table 14 Pr0portion of Red. White. and Intermediate Fibers in the Red Portion of Porcine Semitendinosus Muscle Inoculated with Staphxlococcus aureus Intermediate Red Fibers White Fibers Fibers Fasciculi # Number % Number % Number % 1 35 81.4 ---- ---- 8 18.6 2 32 72.7 -—-- ---- 12 27.3 3 28 71.8 1 2.6 10 25.6 31 72.1 ---- ---- 12 27.9 5 29 76.3 1 2.6 8 21.1 6 39 69.6 2 3.6 15 26.8 7 31 75.6 1 2.4 9 22.0 8 28 70.0 1 2.5 11 27.5 9 21 75.0 ---- ---- 7 25.0 10 36 75.0 1 2.1 11 22.9 Avg. 3]. 7400 07 106 1003 2‘705 Appendix Table 15 Proportion of Red. White. and Intermediate Fibers in the White Portion of Porcine Semitendinosus Muscle Inoculated with Staphylococcus aureu§ Intermediate Red Fibers White Fibers Fibers Fasciculi # Number % Number % Number % 1 ""'"""’ """'" 20 8000 5 2000 2 2 6.3 25 78.1 5 15.6 3 1 7.7 9 69.2 3 23.1 4 ---- --- 17 81.0 4 19.0 5 ---- --- 20 69.0 9 31.0 6 ~--- --- 20 71.4 8 28.6 7 1 2.9 24 68.6 10 28.6 8 ---- --- 18 75.0 6 25.0 9 ---- --- 23 76.7 7 23.3 10 1 5.3 1 72.7 4 21.1 Avg. .5 202 19 7 03 601 2305 168 Appendix Table 16 Proportion of Red. White. and Intermediate Fibers in the Red Portion of Porcine Semitendinosus Muscle Inoculated with glggtgidium perfringens Intermediate Red Fibers White Fibers Fibers Fasciculi # Number % Number % Number V % l 35 77.8 1 2.2 9 20.0 2 20 71.4 --- --- 8 28.6 3 17 77-3 --- --- 5 22.7 4 31 75.6 1 2.4 9 22.0 5 28 73.7 2 5.3 8 21.1 6 21 7500 "'"" "" 7 2500 7 29 80.6 1 2.8 6 16.7 8 37 72.5 2 3.9 12 22.5 9 19 7600 "" “'“' 6 2 .0 10 23 82.1 --- --- 5 17.9 Avg. 26.0 7602 07 107 705 2202 Appendix Table 1? Proportion of Red. White. and Intermediate Fibers in the White Portion of Porcine Semitendinosus Muscle Inoculated with Clostridium perfringens Intermediate Red Fibers White Fibers Fibers Fasciculi # Number % Number % Number % 1 1 2.0 37 75.5 11 22.4 2 1 2.6 30 76.9 8 20.5 3 2 4.3 35 76.1 9 19.6 4 --- --- 20 76.9 6 23.1 5 q-- --- 27 79.4 7 20.6 6 1 2.6 31 79.5 7 17.9 7 --- --- 20 80.0 5 20.0 8 1 2.3 34 79.1 8 18.6 9 --- --- 21 75.0 7 25.0 10 --- --- 19 70.4 8 29.6 Avg. .6 1.4 27.4 76.9 7.5 21.7 Appendix IV Raw Data on Bacterial Growth and pH Changes 169 Appendix Table 18 Summary of the Growth of Pseudomonas gragi on Porcine Semitendinosus Muscle Stored at 10°C if 10g Bacterial Number Inoculated Tissue Control Tissue Incubation Time Red White Red White 0 5.43 5.85 0.0 0.0 o 5.60 5.82 0.0 0.0 Average 5.51 5.83 0.0 0.0 24 7.18 7.38 0.0 0.0 24 7.03 7.29 0.0 0.0 Average 7.11 7.34 0.0 0.0 48 9.31 9.18 0.0 0.0 48 9.27 9.12 0.0 0.0 Average 9.29 9.15 0.0 0.0 96 10.33 10.14 0.0 0.0 96 10.38 10.18 0.0 0.0 Average 10.36 10.16 0.0 0.0 168 9.89 10.27 0.0 0.0 168 9.96 10.19 0.0 0.0 Average 9.93 10023 000 000 170 Appendix Table 19 Summary of the Change in pH During the Incubation of Porcine Semitendinosus Muscle Inoculated with Pseudomonas fragi and Stored at 10°C pH Value Inoculated Tissue Control Tissue Incubation Time Red White Red White 0 5.75 5.60 5.80 5.63 0 5.83 5.75 5.81 5.69 o 5.80 5.70 5.74 5.64 Average 5.79 5-68 5.78 5-65 24 6.10 5.81 ---- ---- 2’4 601? 5083 "'"""" ""'" 24 6.10 5.85 ---- --—- Average 6.12 5.83 ---- ---- 48 6.68 6'“° ---- ---- #8 6.57 6.21 ____ ____ 48 6.63 6°15 ---- ---- Average 6.63 6'25 ---_ ---_ 96 7.84 7'36 5.75 5.70 96 7.90 7'32 5.81 5.62 96 7.65 7'10 5.83 5.64 Average 7.80 7'26 5-79 5-55 168 8.17 8'0“ 5.93 5.77 168 8.24 7'9" 5.97 5.80 168 8.21 7'98 5.96 5.81 Average 8.21 7'99 5.95 5.79 171 Appendix Table 20 Summary of the Growth of Bacillus pumilus on Porcine Semitendinosus Muscle Stored at 10°C log Bacterial Number Inoculated Tissue Control Tissue Average 0.0 Incubation Time Red White Red White 0 5.31 4.80 0.0 0.0 0 5.28 4.65 0.0 0.0 Average 5.30 #073 000 000 24 5.69 5.04 0.0 0.0 24 5.79 4.84 0.0 0.0 Average 5.74 4.94 0.0 0.0 48 5.79 4.71 0.0 0.0 48 5.79 4.81 0.0 0.0 Average 5.79 4.76 0.0 0.0 96 6.18 5.48 0.0 0.0 96 6.21 5.52 0.0 0.0 Average 6.20 5.50 0.0 0.0 168 6.00 5.85 0.0 0.0 168 5.85 6.00 0.0 0.0 0.0 172 Appendix Table 21 Summary of the Change in pH During the Incubation of Porcine Semitendinosus Muscle Inoculated with Bacillus 2321123 and Stored at 10°C pH Value Inoculated Tissue Control Tissue Incubation Time Red White Red White 0 6.02 5.83 6.15 5.81 0 , 6.21 5.87 6.18 5.87 0 6.19 5.89 6.10 5.88 Average 6.14 5.86 6.14 5.85 24 6.31 6.07 6.14 5.99 24 6.30 6.06 6.27 5.90 24 6.33 6.01 6.16 6.01 Average 6.31 6.05 6.19 5.97 48 6.20 5.96 6.12 5.75 48 6.21 6.99 6.21 5.82 48 6.23 6.01 6.14 5.83 Average 6.21 5.99 6.16 5.80 96 6.25 5.80 6.02 5.72 96 6.27 5.88 6.10 5.71 Average 6.25 5.84 6.09 5.76 168 6.30 5.83 6.01 5.66 168 6.28 5.80 6.05 5.64 168 6.27 5.79 6.11 5.70 Average 6.28 5.81 6.06 5.67 173 Appendix Table 22 Summary of the Growth of §taphylococcgs aureus on Porcine Semitendinosus Muscle Stored at 15 C log Bacterial Number Inoculated Tissue Control Tissue Incubation Time Red White Red White 0 5.36 5.38 0.0 0.0 0 5.32 5.37 0.0 0.0 Average 5.34 5.38 0.0 0.0 24 6.44 6.41 0.0 0.0 24 6.41 6.56 0.0 0.0 Average 6.43 6.44 0.0 0.0 48 7.67 7.87 0.0 0.0 48 7.72 7.83 0.0 0.0 Average 7.70 7.85 0.0 0.0 96 9.73 9.15 0.0 0.0 96 9.79 9.36 0.0 0.0 Average 9.76 9.26 0.0 0.0 168 10.27 9.77 0.0 0.0 Average 10.24 9.83 0.0 0.0 173 Appendix Table 22 Summary of the Growth of Staphylococcus aureu on Porcine Semitendinosus Muscle Stored at 15 C 10g Bacterial Number Inoculated Tissue Control Tissue Incubation Time Red White Red White 0 5036 5038 000 0.0 0 5.32 5.37 0.0 0.0 Average 5.34 5.38 0.0 0.0 24 6.44 6.41 0.0 0.0 24 6.41 6.56 0.0 0.0 Average 6.43 6.44 0.0 0.0 48 7.67 7.87 0.0 0.0 48 7.72 7.83 0.0 0.0 Average 7.70 7.85 0.0 0.0 96 9.73 9.15 0.0 0.0 96 9.79 9.36 0.0 0.0 Average 9.76 9.26 0.0 0.0 168 10.31 9.88 0.0 0.0 168 10.27 9.77 0.0 0.0 Average 10.24 9.83 0.0 0.0 174 Appendix Table 23 Summary of the Change in pH During the Incubation of Porcine Semitendinosus Muscle Inoculated with Sta h lococcus auregg and Stored at 155C pH Value ___ Inoculated Tissue Control Tissue Incubation Time Red White Red White 0 5.97 5.67 5.98 5.68 o 6.00 5.71 5.96 5.64 0 5.99 5.69 6.01 5.70 Average 5.99 5.69 5.98 5.67 24 6.07 5.86 5.97 5.73 24 6.23 5.87 6.01 5.70 24 6.26 5.75 6.00 5.72 Average 6.18 5.83 5.99 5.72 48 6.22 5.88 5.92 5.74 48 6.24 5.79 5.92 5.78 48 6.15 5.85 5.93 5.70 Average 6.20 5.84 5.92 5.74 96 6.31 6.08 5.92 5.71 96 6.29 5.97 5.91 5.78 96 6.44 6.02 5.88 5.76 Average 6.35 6.02 5.90 5.75 168 7.74 6.45 6.08 5.91 168 7.70 6.38 6.11 5.87 168 7.69 6.24 6.10 5.96 Average 7.71 6.36 6.10 5.91 175 Appendix Table 24 Summary of the Growth of Clostridium perfringens on Porcine Semitendinosus Muscle Stored at 30°C log Bacterial Number Inoculated Tissue Control Tissue Incubation Time Red White Red White 0 5.77 5.41 0 0 0 5.74 5.51 0 0 Average 5.76 5.46 0 0 24 8.66 7.71 0 0 24 8.64 7.81 0 0 Average 8.65 7.76 0 0 48 8.65 8.32 0 0 48 8.81 8.38 0 0 Average 8.73 8.35 0 0 96 8.57 8.42 0 0 96 8.59 8.45 0 0 Average 8.58 176 Appendix Table 25 Summary of the Change in pH During the Incubation of Porcine Semitendinosus Muscle Inoculated with Clostridium perfringens and and Stored at 30 C pH Value Inoculated Tissue Control Tissue Incubation Time Red White Red White 0 5-58 5-31 5-53 5.34 0 5.67 5.38 5.62 5.36 O 5.64 503’4‘ 5058 5036 Average 5.66 5.34 5.58 5.35 24 5.98 5-17 5-52 5.39 24 6.00 5.27 5.64 5.32 24 5.95 5.26 5.68 5.25 Average 5.98 5.23 5.65 5.32 48 6.12 5.71 5.39 5.33 48 6.14 5.73 5.55 5.37 48 6.15 5.78 5.62 5.35 Average 6.14 5.74 5.52 5-35 96 6.62 6.11 5.65 5.40 96 6.50 6.08 5.54 5.35 96 6.55 6.09 5.57 5.41 Average 6.56 5-39 ' 42:- ———--l—- Appendix V Raw Data From Headspace Analysis Experiments 177 Appendix Table 26 Bacterial Growth Data for Pseudomonas ggagi or a Mixed Culture on Porcine Longissimus Dorsi Muscle Incubated at 10°C log Bacterial Numbers Incubation Time. hrs. ‘Pseudomonag frgg; Mixed Culture Control 0 6.54 6.96 0 0 6.51 6.90 0 Average 6.53 6.93 0 24 6.72 8.14 0 24 6.69 8.15 0 Average 6.71 8.15 0 48 8.49 8.83 0 48 8.48 8.72 0 Average 8.49 8.78 0 72 9.88 8.69 0 72 9.96 8.64 0 Average 9.92 8.67 0 96 10.26 8.45 0 96 10.25 8.49 0 Average 10.26 8.47 0 178 Appendix Table 27 Changes in the pH Values of Longissimus Dorsi Muscle Inoculated with Pseudomonas fragi or a Mixed Culture from Hamburger and Incubated at 10°C pM'Value Incubation Time. hrs. Pseudomonas fragi Mixed Culture Control 0 5.61 5.63 5.62 0 5.64 5.63 5.65 o 5.62 5.64 5.63 Average 5.62 5.63 5.63 24 5.73 5.51 5.63 24 5.71 5.55 5.65 24 5.70 5-54 5-65 Average 5-71 5.53 5.64 48 6.47 5.09 5.66 48 6.47 5.06 5.64 48 6.49 5.06 5.69 Average 6.48 5.07 5.66 72 7.12 4.95 5.70 72 7.18 4.97 5.65 72 7.17 4.95 5.68 Average 7.16 4.96 5.68 96 8.06 4.91 5.71 96 8.01 4.95 5.79 96 8.10 4.94 5.77 Average 8.06 4.93 5.76 179 Appendix Table 28 Retention Time of Chromatographic Peaks of the Headspace Vapors of Uninoculated Porcine Longissimus Dorsi Muscle after Incubation at 10°C. Run on a 3% Apiezon Column. Retention Time. sec.1 Incubation Time. hrs. 1 2 3 ‘4 5 6 ‘ a 0 9* i 242 - - -- -- -- --- i 48 3 11 71 78 102 l 72 3 11 68 76 100 s 96 3 13 71 79 103 L. lCorrected by subtraction of the air peak retention time. 2 Samples lost due to equipment malfunction. Appendix Table 29 Retention Time of Chromatographic Peaks from the Headspace Vapors of Uninoculated Porcine Longissimus Dorsi Muscle after Incubation at 10°C. Run on a 10% Carbowax Column. Retention Time. sec.1 Incubation Time. hrs. 1 2 3 4 5 0 44 242 -- -- -- _-- --- 48 29 49 125 155 72 27 51 129 160 96 27 50 125 157 1Corrected by subtraction of the air peak retention time. 28amples lost due to equipment malfunction. — 180 Appendix Table 30 Retention Time of Chromatographic Peaks from the Headspace Vapors of Porcine Longissimus Dorsi Muscle Incubated withaMixed Culture from Hamburger at 10°C. Run on a 3% Apiezon Column. Retention Time. sec.1 Incubation Time. hrs. 1 2 3 4 5 6 0 9 242 - __ -- _- _- --- 48 9 12 42 57 72 102 72 8 11 41 57 73 103 96 9 11 43 58 75 106 1Corrected by subtraction of the air peak retention time. 2Samples lost due to equipment malfunction. Appendix Table 31 Retention Time of Chromatographic Peaks from the Headspace Vapors of Porcine Longissimus Dorsi Muscle Incubated with a Mixed Culture from Hamburger at 10°C. Run on a 10% Carbowax Column. 1 Retention Time. sec. Incubation Time. hrs. 1 2 3 4 5 6 7 8 0 43 242 -- -- -- -- -- _-- --- --- 48 30 50 69 90 117 309 339 72 29 49 69 91 110 306 338 96 30 50 71 90 107 307 342 1Corrected by subtraction of the air peak retention time. 2Samples lost due to equipment malfunction. 181 Appendix Table 32 Retention Time of Chromatographic Peaks from the Headspace Vapors of Porcine Longissimus Dorsi Muscle Incubated with Pseudomonas fragi at 10°C. Run on a 3% Apiezon Column. Retention Time. sec.1 Incubation Time. hrs. 1 2 3 4 5 0 9 242 -- __ -- _- __ _- 48 4 12 31 70 74 72 3 11 29 68 77 96 3 11 28 71 75 1 Corrected by subtraction of the air peak retention time. 2Samples lost due to equipment malfunction. Appendix Table 33 Retention Time of Chromatographic Peaks from the Headspace Vapors of Porcine Longissimus Dorsi Muscle Incubated with P eudomonas fragi at 10°C. Run on a 10% Carbowax Column. Retention Time. sec.1 Incubation Time. hrs. 1 2 3 4 0 43 242 -- -- -- -- 48 29 50 68 72 29 51 70 96 28 49 67 1Corrected by subtraction of the air peak retention time. 2Samples lost due to equipment malfunction.