u —< .Jnu-fi.‘ A.- -u .m.... ‘ " 2"“: 'v': 'V'“| A r . um ‘ .z vb. “.r_'.‘.n rn‘ ., fl”... . ......... u,“ PROTEOLYTIC ENZYMES PROBUCED BY . Pseudomonas parolens; THElR CHARACTER AND ACTION ~ ‘ OH MUSCLE PROTEINS ., ~ Thesis for {he Begree'af Ph. D.- f " r MCH'IGANSTATE UNWERSEFY . (exams m BUCKLE? . r ' . - ‘. r ‘ . , . 1-972: _ 7 v LIBRARY Michigan State University This is to certify that the thesis entitled Proteolyt ic Enzymes Produced by Pseudomonai merolens; Their Character and Action on Muscle Proteins presented by Denis Joe Buckley has been accepted towards fulfillment of the requirements for PhoDo degreein FOOd Science JAM/W Major professor Date July 22, 1972 0-7639 LE ABSTRACT PROTEOLYTIC ENZYMES PRODUCED BY Pseudomonas perolens; THEIR CHARACTER AND ACTION ON MUSCLE PROTEINS by Denis Joe Buckley During the past decade there has been renewed interest in the area of bacterial contamination and spoilage of fresh meats under refrigerated storage. The psychrophilic Pseudomonas have been implicated as being the most prevalent group of bacteria involved in fresh meat spoilage. Proteases elaborated during growth of certain of these bacterial species appear to be potential hazards to the integrity of the primary muscle proteins. It was with this information in mind that this study was planned. Of the three species of Pseudomonas 088d initially, Pseudomonas perolens AICC 10757 was chosen for use in all the subsequent experiments. In a preliminary study, the effect, if any, of these bacteria on the primary protein solubility changes on sterile ground porcine muscle was determined at 10°C. The major changes observed were increases in the myofibrillar and non protein nitrogen fractions. Large increases in pH and bacterial numbers were observed. Enzyme production was first detected on the 11th day of storage coinciding with the initial increase in pH. Attempts to' extract and purify the enzyme(s) from the muscle were not very fruitful and enzyme production on a non protein medium facilitated further study. Koser's citrate plus 0.5 g/l of calcium chloride was the medium developed for growth and enzyme production. A crude enzyme _ solution was prepared by centrifugation of the growth medium at 10,000Xg for 20 minutes, after appreximately 35 hours incubation. Denis Joe Buckley The following parameters were established for the crude enzyme(s) (1) Optimum storage temperature was found to be 3°C. (2) Optimum assay temperature was 35°C. (3) Optimum stability occurred within the pH range 6.5 to 9.0. (4) Maximum activity over a 15 minute period was obtained using a 0.5% w/v casein solution as substrate. (5) There was a linear increase in activity with time of assay up to 20 minutes. (6) Enzyme concentrations of up to 80 pg protein per ml resulted in linear increases in reaction rates. (7) Collagen and bovine salt soluble proteins resulted in the highest initial reaction rates. All of the above parameters were determined using 1 ml of enzyme(s) solution reacted with l‘ml of 22 w/v casein solution at 35°C for 15 minutes periods in a shaking incubator. The crude enzyme(s) solution was injected into an eye of round meat portion which brought about up to a 30% increase in tenderness. However, it did result in an undesirable potato-like odor. A purification system involving ultrafiltration and gel filtration of the crude enzyme solution resulted in a 20 fold increase in purification and a 12% yield. The three criteria used for purity were disc gel elec- trophoresis, homogeneity of protein and specific activity. Eluate fractions from a K 25/45 G-lOO Sephadex column were measured for protein concentra- tion and enzyme activity. The resultant coincident nature of these two parameters indicated the presence of a homogeneous protein in the eluate. Specific activity values of those eluate fractions containing the highest enzyme activity were found to be similar. Denis Joe Buckley 0f the four inhibitors and/or activators used, only cysteine had a slight activation effect. The chelating agent, ethylenediaminetetraacetic acid had a 50% inactivation effect at 10-4M concentration and reactivation of 822 of the initial activity was obtained on adding lO-ZM calcium chloride. The purified enzyme fraction appeared to break down collagen but did not result in the release of hydroxyproline as free amino acids. It also caused considerable changes in the primary protein solubility over a 25 day storage period at 3°C. The major changes were (1) a large decrease in the stroma protein and (2) a large increase in the myofibrillar protein fractions after 4 days of storage. A concurrent experiment using the purified enzyme solution and a bacterial solution on aseptically sampled unground porcine muscle was carried out to observe any ultrastructural changes taking place. The enzyme treated samples showed loss of Z and M lines at 4 days of storage. Loss of the I band and some A band changes were observed after 8 days. The bacterial treated samples showed no changes at day 4 but localized I band disruption occurred after 8 days of storage. Both the enzyme solution and bacterial inoculum showed some disruption of the myofibrillar protein fraction. PROTEOLYTIC ENZYMES PRODUCED BY Pseudomonas perolens; THEIR CHARACTER AND ACTION ON MUSCLE PROTEINS By Denis Joe Buckley A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1972 ACKNOWLEDGEMENTS I wish to express my sincere appreciation and thanks to my advisor Dr. J. F. Price in helping to make my stay at Michigan State University a happy and rewarding experience. His ideas and suggestions during my program and in the preparation of this thesis are greatly appreciated. I am deeply indebted to the other members of my guidance committee, namely, Drs. R. A. Merkel, T. I. Hedrick, D. R. Dilley, W. G. Bergen and A. M. Pearson for the use of laboratory facilities. I also wish to thank Gary L. Gann for providing all the electron microscopy data used in this study. Thanks are also due to those who helped prepare this thesis, Mrs. Bea Eichelberger for her excellent typing, Dr. Ian Gray for reading and my dear wife, Nuala, for all her help and encouragement during the past three years. The financial support provided by the Kellogg Foundation, University College Cork, and the Department of Food Science and Human Nutrition (M.S.U.) is gratefully appreciated. I would also like to thank my Pro- fessor, T. O'Mullane, University College Cork, for making it all possible. Finally, to those jovial souls in the Graduate Office and to the Meat Laboratory staff, I extend a very special thank you for everything. 11 TABLE OF CONTENTS Page INTRODUCTION 0 O O O I O O O O C O O O O O O O O I O O O O O O O O 1 LITERATURE REVIEW ’. . . . . . . . . . . . . . . . . . . . . . . . 4 Meat Microbiology 0 O I I C O O O O O O O O O O O O O I O C O 4 Sources of micro-organisms . . . . . . . . . . . . . . . 4 Antemortem sources . . . . . . . . . . . . . . . . 4 Postmortem sources . . . . . . . . . . . . . . . 5 Types Of micro-organisms o o o o o o o o o o o o o o o o 6 PSYChrophilic Bacteria O O O O O D O O O O O O O O O O O O O 8 Psychrophilic Pseudomonas . . . . . . . . . . . . . . . . . . 9 Psychrophiles and Meat Spoilage . . . . . . . . . . . . . . . 10 Bacterial Proteases . . . . . . . . . . . . . . . . . . . . . 12 Classification of Proteases . . . . . . . . . . . . . . . . . 14 Role of Calcium . . . . . . . . . . . . . . . . . . . . . . . 15 Tenderizers . . . . . . . . . . . . . . . . . . . . . . . . . 16 Muscle Proteins . . . . . . . . . . . . . . . . . . . . . . . l7 Sarcoplasmic proteins . . . . . . . . . . . . . . . . . l7 Myofibrillar proteins . . . . . . . . . . . . . . . . . 18 Stroma proteins . . . . . . . . . . . . . . . . . . . . 19 Non protein nitrogen . . . . . . . . . . . . . . . . . . 20 Ultrastructure of Bacteria . . . . . . . . . . . . . . . . . 21 EXPERIMENTAL METHODS I I O O O O O I O O O O O O O O O O O O O O O 22 Pure Culture Propogation and Maintenance . . . . . . . . . . 22 CUltures used 0 O O O O O O O 0 O O O O O I O O O O O I 23 iii Media Preparation . . . . . . . . . . . . . . . Assays O O O O O O O O O O O O O O O O O O O 0 Enzyme assay . . . . . . . . . . . . . . . Protein assay . . . . . . . . . . . . . . Preparation of Crude Enzyme Solution . . . . . Replacements for Calcium Chloride in Enzyme Production Parameters of the Crude Enzyme Solution . . . . Temperature of storage . . . . . . . . . . Temperature of assay . . . . . . . . . . . pH . . . . . . . . . . . . . . . . . . . . Time . . . . . . . . . . . . . . . . . . . Substrate concentration . . . . . . . . . Enzyme concentration . . . . . . . . . . . Substrate specificity . . . . . . . . . . Purification Procedures . . . . . . . . . . . . Ultrafiltration . . . . . . . . . . . . . Gel filtration . . . . . . . . . . . . . . Absorbance readings . . . . . . . . . . . Disc gel electrophoresis . . . . . . . . . Tenderization Effects of the Crude Enzyme Solution Enzyme solution preparation . . . . . . . Meat sample preparation . . . . . . . . . Meat injection . . . . . . . . . . . . . . Cooking methods . . . . . . . . . . . . . Tenderness measurements . . . . . . . . . Procedure for Obtaining Sterile Porcine Muscle Slaughter O O O O O O O O O O O O O O O 0 Muscle sample procurement . . . . . . . . Growth and Enzyme Production on Porcine Muscle Muscle sample preparation and inoculation Enzyme extraction . . . . . . . . . . . . Analysis . . . . . . . . . . . . . . . . . pH . . . . . . . . . . . . . . . . . Bacterial counts . . . . . . . . . . Disc gel electrophoresis . . . . . . Enzyme assay . . . . . . . . . . . . iv Page 23 24 24 25 25 26 26 26 27 27 28 28 28 28 30 30 31 32 32 33 33 33 34 34 35 35 36 36 37 37 37 38 38 38 38 38 Protein Changes due to Enzyme Action . . Sample preparation . . . . . . Enzyme solution and inoculation . . Protein extraction . . . . . . Sarcoplasmic protein . . Myofibrillar protein . . Non protein nitrogen . . Total protein nitrogen . Stroma protein . . . . . Protein determination . . . . Effect of Inhibitors and/or Activators . Effect of Enzyme Solution on Collagen . . . . . . Electron MicroscOpic Examination of Meat Spoilage Sample preparation and incubation . Sample fixation and embedding Sectioning and staining . . . Photography of sections . . . RESULTS AND DISCUSSION . . . . . . . . . Preliminary Study on the Effects of ATCC 10757 on Muscle Tissue . Bacterial growth . . . . . . . pH . . . . . . . . . . . . . . Enzyme production . . . . . . Primary protein changes . . . Pseudomonas perolens Development of a Medium for Growth and Enzyme Pseudomonas perolens ATCC 10757 Growth on Koser's citrate medium . . Medium for enzyme production . Role of calcium chloride . . . Effect of foaming and anti foaming . Replacement of calcium chloride by Enzyme Unit Definitions . . . . . Enzyme activity . . . . . . . Specific activity . . . . . other Production with metal Page 39 39 39 40 4O 40 40 41 41 41 41 42 44 44 44 46 46 47 47 47 50 51 51 55 55 56 62 69 7O 72 72 73 Crude Enzyme Parameters . . . . . . Temperature of storage . . . . . . Temperature of assay . . . . . . . Assay time . . . . . . . . . . . . pH . . . . . . . . . . . . . . . . Effect of substrate concentration Effect of enzyme concentration . . Substrate specificity . . . . . . Purification of the Enzyme Fraction . . Ultrafiltration . . . . . . . . . Gel filtration . . . . . . . . . . Disc gel electrophoresis . . . . . Effect of Inhibitors and/or Activators Hydroxyproline Released by Enzyme Tenderization Effect of the Crude Action on Collagen Enzyme Fraction . . Muscle Protein Solubility Changes due to Enzymes Action Sarcoplasmic proteins . . . . . . Myofibrillar proteins . . . . . . Stroma proteins . . . . . . . . . Non protein nitrogen fraction . . Changes in Muscle Ultrastructure . . . SUMMARY . B IBL IOGWHY O O O I I O O O O O O O O O O 0 APPENDIX . vi Page 73 73 75 75 78 78 82 84 84 84 86 91 94 95 96 100 100 102 103 103 105 121 124 135 LIST OF TABLES Table Page 1 Summary of reported microbial action on the primary muscle protein fractions . . . . . . . . . . . . . . . . . . . . 52 2 Primary protein solubility changes due to the action of Pseudomonas perolens ATCC 10757 . . . . . . . . . . . . . 53 3 Effect of calcium replacements in the developed medium on growth of Pseudomonas perolens and enzyme production . . 72 4 Enzyme purification using PM-10 and UM-10 membranes . . . 86 5 Specific activity values for the enzyme containing eluate fractions obtained from the K 25/45 and K 15/90 columns . 91 6 Purification data for protease from Pseudomonas perolens 93 7 Effects of inhibitors and/or activators on enzyme activity 94 8 Inhibition and reactivation of the enzyme fraction using EDTA and calcium chloride, respectively . . . . . . . . . 95 9 Tenderization effect of the crude enzyme fraction . . . . 98 10 Data obtained on the solubility changes in the primary proteins due to the 6-100 K 25/45 enzyme fraction . . . . 104 vii LIST OF FIGURES Figure Page 1 Growth of three species of the genus Pseudomonas in Koser's citrate medium, pH 7.5, at 10°C in a rotary shaker at 160 rpm I O O O C O C C O C O O O O O O C C O C C O O C O O 48 2 The action of Pseudomonas perolens ATCC 10757 on porcine muscle during a 20 day storage period at 10°C . . . . . . . 49 3 Growth cycle of Pseudomonas perolens ATCC 10757 in Koser's citrate medium, pH 7.5, at 10°C in a rotary shaker at 160 rpm 0 O O O O O O C O O C O O C O O I O O O I O O O O O 57 4 Growth of Pseudomonas perolens ATCC 10757 in Koser's citrate medium, pH 7.5 at 10°C in a rotary shaker at 160 rpm with the citrate being replaced by amino acids . . . . 58 5 Growth of Pseudomonas perolens ATCC 10757 on Koser's citrate medium pH 7.5 at 10°C in a rotary shaker at 160 rpm with the citrate being replaced by dipeptides . . . . . . . 59 6 Growth of Pseudomonas perolens ATCC 10757 and enzyme production in Koser's citrate medium, pH 7.5, at 10°C in a rotary shaker at 160 rpm with the citrate being replaced by a complex mixture of amino acids and dipeptides dissolved in different solvents . . . . . . . . . . . . . . . . . . . 61 7 Growth of Pseudomonas perolens ATCC 10757 in Koser's citrate medium with and without citrate being replaced by other carbon sources. Calcium chloride (0.5 g/l was added in each case, pH was 7.5 at a temperature of 10°C . . . . . 63 8 Growth of Pseudomonas perolens ATCC 10757 and enzyme production in Koser's citrate medium with varying concen- trations of calcium chloride added, pH 7.5, at 10°C . . . . 65 9 Growth of Pseudomonas fragi ATCC 4973 and enzyme production in Koser's citrate medium with varying concentrations of calcium chloride added, pH 7.5, at 10°C . . . . . . . . . . 66 10 Effect of calcium chloride concentration on enzyme pro- duction by Pseudomonas perolens ATCC 10757 and Pseudomonas fragi ATCC 4973 grown on Koser's citrate medium, pH 7.5, at 10°C for 55 hours . . . . . . . . . . . . . . . . . . . 67 viii Figure Page 11 Growth of Pseudomonas perolens ATCC 10757 and enzyme pro— duction in Koser' s citrate medium plus 0. 5 g/l calcium Chloride’ pH 7I 5, at 10°C I I I I I I I I I I I I I I I I I 68 12 Effect of foaming and an antifoam agent on growth and enzyme production by Pseudomonas perolens ATCC 10757 in Koser's citrate medium plus 0.5 g/l of calcium chloride, pH 7.5, at 10°C . . . . . . . . . . . . . . . . . . . . . . 71 13 Stability of the crude enzyme fraction at various storage temperatures I I I I I I I I I I I I I I I I I I I I I I I 74 14 Effect of assay temperature on enzyme activity . . . . . . 76 15 Effect of assay time on enzyme activity at the optimum assay temperature of 35°C . . . . . . . . . . . . . . . . . 77 16 Effect of pH, using various buffer systems on enzyme actiVity I I I I I I I I I I I I I I I I I I I I I I I I I 79 17 Effect of pH on enzyme activity using a combination buffer system (phosphoric acid-acetic-acid-boric acid with pH range from 5 I O to 9 I o I I I I I I I I I I I I I I I I I I I 80 18 Effect of casein concentration, as substrate on enzyme actiVity I I I I I I I I I I I I I I I I I I I I I I I I I 81 19 Effect of enzyme concentration on enzyme activity . . . . . 83 20 Enzyme action on various proteins as substrates . . . . . . 85 21 The relationship between enzyme activity and absorbance at 216 nm of the eluate fractions obtained from a G-75 Sephadex K 25/45 c01m I I I I I I I I I I I I I I I I I I I I I I 89 22 The relationship between enzyme activity, absorbance at 216 nm, and protein concentration of the eluate fractions obtained from a G-lOO Sephadex K 25/45 column . . . . . . . 90 23 Disc gel electrophoresis of the enzyme fractions obtained during the various purification steps . . . . . . . . . . . 92 24 Hydroxyproline released as measured after hydrolysis of the trichloroacetic acid soluble enzyme treated collagen, pH 7I5 and 35°C I I I I I I I I I I I I I I I I I I I I I I 97 25 Effect of the enzyme fraction obtained from G-100 Sephadex K 25/45 column on porcine primary muscle proteins stored at 3°C I I I I I I I I I I I I I I I I I I I I I I I I I I I 101 ix Figure 26 27 28 29 30 31 32 33 34 35 36 Page Electron micrograph of myofibrils from uninoculated pig muscle incubated at 3°C for 4 days in sterile Koser's Citrate medium I I I I I I I I I I I I I I I I I I I I I 107 Electron micrograph of myofibrils from bacterial inoculated pig muscle incubated at 3°C for 4 days . . . . . . . . . 108 Electron micrograph of myofibrils from enzyme treated pig muscle incubated at 3°C for 4 days . . . . . . . . . . . 109 Electron micrograph of myofibrils from uninoculated pig muscle at 3°C for 8 days in sterile Koser's citrate medium 110 Electron micrograph of myofibrils from enzyme treated pig muscle incubated at 3°C for 8 days . . . . . . . . . . . 112 Electron micrograph of myofibrils from bacterial inoculated pig muscle incubated at 3°C for 8 days . . . . . . . . . 113 Electron micrograph of myofibrils from bacterial inoculated pig muscle incubated at 3°C for 8 days . . . . . . . . . 114 Electron micrograph of myofibrils from bacterial inoculated pig muscle incubated at 3°C for 8 days . . . . . . . . . 115 Electron micrograph showing localized bacterial growth on pig muscle at 8 days of storage at 3°C . . . . . . . . . 116 Electron micrograph showing bacterial growth at a distance from the myofibrils of pig muscle at 8 days of storage at 3°C I I I I I I I I I I I I I I I I I I I I I I I I I 117 Electron micrograph of a bacterial cell section showing a bleb like formation at 8 days of growth on pig muscle incubated at 3°C I I I I I I I I I I I I I I I I I I I I 118 will [IE/ll LIST OF APPENDIX TABLES Appendix Page A Composition of solutions used in this study . . . . . . 135 B Summary of the data obtained and the media used in this study I I I I I I I I I I I I I I I I I I I I I I I I I 139 xi INTRODUCTION Meat as a form of food can be traced back many thousands of years. In the Upper Paleolithic times (approx. 60,000 years ago) the Aurignacians, for example, were mainly flesh eaters. During the mesolithic period (approx. 11,000 years ago), Europeans used large mammal herds of rhino, and later bison, reindeer and elk. It was during this period also that sheep, goats, cattle, pigs and dogs began to become domestiCated. With the development of pottery and other utensils and with the onset of urbanization, surpluses of perishable foods were preserved and stored as dried salted meats, cooked meats, and fish in sesame oil. As far back as 2,000 B.C. the Jews had food restrictions and tolerances which had to meet with a specific code called the Code of Moses. The Romans had meat shops which were controlled by market police, who saw to it that all condemned meat and sausage were discarded into the river Tiber. In northern Europe, ice caves and snow were not uncommon for pre- serving foods. Apicius who lived during the reign of Tiberius had many captions and preservation formulas in his writings, such as, "to keep meat without salt for any length of time, use honey as a preservative" or "to keep cooked sides of pork, beef or tenderloins use a pickle of mustard, vinegar, salt, and honey". Abattoirs, stock yards and meat regulations appear around the year 1080 (Jensen, 1945). Until some 100 years ago, man was dependent upon the perishable food supplies produced within a close radius from his home. The meat packing industry was operated only during the cold winter season. With the advent of mechanical refrigeration and the refrigerated rail car, the perishable food industry, in particular the meat industry, took on a whole new outlook. Throughout history, one sees that preservation of perishable foods was indeed a big problem, and still remains a problem for some food pro- ducts. Methods to prevent and retard spoilage of meat and meat products have always been an area for intense study. Measures to minimize bacter- ial contamination and growth on and in animal tissues have been developed and are used prior to slaughter and throughout the slaughtering and dress- ing operations. Ayres (1955) has extensively reviewed the microbial implications in the handling, slaughtering, and dressing of meat animals. The fast increasing use of frozen and chilled foods in recent years and prolonged storage periods prior to consumption have placed much importance on the psychrophilic group of micro-organisms in the food industry. Large losses of meat, fish, poultry and dairy products occur due to the many undesirable odors and off flavors that can be produced by these low temperature micro-organisms. It has long been established that species of the genus Pseudomonas are predominant in this group of low temperature spoilage bacteria. Recently, species of the genera Pseudomonas and Achromobacter have been reported as being responsible for the spoilage of beef (Jay and Kontou, 1967). These workers concluded that microbial growth was supported by low molecular weight compounds such as amino acids and nucleotides with resultant spoilage. Lerke gt a1. (1967) using a species of Pseudomonas and fish muscle reported similar findings. The primary muscle proteins have been shown to change with microbial growth and spoilage by Borton (1970) and Tarrant (1971). Proteases elaborated during the growth of one of the psychrophilic Pseudomonas have been shown to be responsible for the primary protein breakdown (Dutson g£_§l., 1971; Tarrant g£_§l,, In press). Thus the properties of the proteases from the Pseudomonas species which may influence both production and activity are of interest. The objectives of this study were to (l) to determine whether enzyme(s) were produced and under what condi- tions. (2) Also to determine the effect of these enzyme(s) on porcine muscle and muscle components. LITERATURE REVIEW ‘Meat Microbiology Sources of micro-organisms Antemortem sources. The normal body defense mechanisms, which in- clude skin and mucous membranes, gastric juices, inflammatory processes involving lymphatic and phagocytic cells, are credited with the maintenance of sterile tissues. However, many kinds and large numbers of micro-organisms can exist on the hoofs, hides and hair as well as in the intestinal tract of animals. Empey and Scott (1939a) reported 105 to 3X106 aerobes per cm2 from unwashed hides of cattle and Jensen and Hess (1941) recovered 105 to 106 aerobes per cm2 and 108 anaerobes per cm2 on hog necks. The former workers also found considerable variation in the extent of contamination of hides from different animals and in the various areas of the same hide, probably due to the extent of hide contact with soil and the amount of moisture present. Ruminant animals have bacteria in the rumen, the actual numbers depending on the type of feed and the stage in the digestion cycle (Empey and Scott, 1939a; Grant, 1953). Generally however, these bacteria are destroyed in the stomach with only small numbers escaping into the small intestine where they can multiply rapidly. Jensen and Hess (1941) showed that muscle tissue and bone marrow of live hogs are sterile but many bacteria can be isolated after slaughter. The lymph nodes have been shown to contain many different species of bacteria (Lepovetsky £3 21,, 1953; Weiser g£_§1,, 1954). Animal fatigue prior to slaughter has been shown to have some effect on the defense mechanisms and thus the possi- bility of passage of micro-organisms into the muscle tissues from the intestines (Charrin and Roger, 1890; Ficker, 1905). Jensen (1945) re- ported that struggling at slaughter causes muscle to undergo early rigor mortis and to putrefy rapidly. The antemortem conditions in relation to postmortem changes in glycogen and lactic acid seems to be important in microbial spoilage. Bates-Smith (1948) and Ingram (1948, 1949) each concluded that the ultimate pH was indeed important in that slight acidi- fication markedly reduced the growth rate of many bacteria. The slaughter floor and environment at the time of killing can be important sources of contamination as well as the sticking knife, workers'hands and any other types of equipment being used (Ayres, 1955). Postmortem sources. Skinning is the first operation postmortem in the case of cattle and sheep, or dehairing for hogs. Empey and Scott (1939a) found that bacterial numbers of 104 to 105 per cm2 were trans- ferred from the hide, hoofs, knives, workmen's hands and clothing to the carcass surfaces. Similar transfer of microbes to carcass parts occurs during evisceration (Ayres, 1955; Empey and Scott, 1939a, 1939b; Haines, 1933a). Present practices in meat handling involve the quick transfer of carcasses to the chill room which helps delay bacterial growth and con- sequent changes (Jensen, 1945). Sources of contamination in the chill room include the air, walls, workmen and equipment (Richardson gt 21,, 1954). On cutting up carcasses, many sources of contamination are again possible such as knives, saws, tables and workmen. Most important during the process is the fact that greater areas of cut surfaces are exposed and.the juices released support the growth of large numbers of bacteria, which become redistributed during further cutting (Ayres and Adams, 1953). Types of micro-organisms Of all the sources of micro-organisms mentioned in the previous sections, postmortem contamination on the slaughter floor and in the chill room during the cutting operation seem to be the most important. During the slaughtering process the soil and water-borne type micro-organisms are predominant. These include species of the genera, Bacillus, Achromo- bacter, Micrococcus, Pseudomonas and many others (Empey and Scott, 1939a; Mallmann 2; 31., 1940; Jensen and Hess, 1941). In addition, during evis- ceration and washing, Staphlococcus, Escherichia, Proteus, Sarcina and others appear. The airborne contamination depends on the ventilation system and the amount of dirt and dust accumulation (Ayres, 1955). Predominant in this initial contamination are the mesOphilic types of micro-organisms which grow rapidly at room temperatures but not at chill room temperatures, where they are effectively retarded. However, at low temperatures the psychrophilic type micro-organisms become pre- dominant. Haines (1934) and Empey and Scott (1939a) reported that less than 12 of the microbial population growing on beef surfaces at 20°C were viable at -1°C. Species of the genus Pseudomonas have been reported by Jensen (1944), Ayres (1951) and Kirsch £5 31. (1952) to be the most common psychrophilic types of bacteria contained in the initial flora. Many had previously been reported as Achromobacter (Empey and Vickery, 1933; Empey and Scott, 1939a). However, with reclassification (Breed gt 31., 1948) they were deemed to be species of the genus Pseudomonas. Slime forming bacteria from the cut surfaces of meat have been re- ported by Glage (1901), Haines (1933b) and Empey and Scott (1939a). These workers also thought them to be of the genus Achromobacter, being aerobic, motile and growing at 2°C with optimum growth at 10 to 12°C. Character- istic odors which changed with increasing growth were also observed. More recently, Jensen (1944), Ayres (1951) and Kirsch £5 31, (1952) have indicated that species of the genus Pseudomonas are more important in slime formation. Bone taint is a form of putrefactive souring which occurs in the deep tissues of the larger meat pieces. Jensen and Hess (1941) catalogued some of the souring types as Clostridia, Micrococcus, Achromobacter, and Pseudomonas. These were mostly anaerobic and grew well at 3°C (Haines and Scott, 1940). Ayres (1955) reported the principal types of changes occurring in refrigerated meats were: 1. Off odors and slime. 2. Bone taint. 3. Mold discoloration. 4, Fat rancidity. Psychrophilic Bacteria The term psychrophile is somewhat misleading in that it literally means "cold loving", yet it is usually used to describe those bacteria that are capable of growth at 0° to 5°C, but with optimum growth at about 20°C. Mossel and Zwart (1960) and Eddy (1960) have suggested that the term psychrotroph (i.e., cold tolerant) would more aptly describe these bacteria growing at 5°C or less. Forster (1887) was first to isolate pure cultures capable of growing at 0°C. He found them in water, milk, meat and soil. More recently, psychrophiles have been isolated from dairy products (Hiscox, 1936; Erdman, 1951; Boyd Efiugio, 1953). and chilled and frozen meats (Empey and Scott, 1939a; Sulzbacher, 1950; Brown and Weidemann, 1958). Ingraham and Stokes (1959) concluded that the ability to grow at 0°C is possessed by a limited group of bacteria, which are mainly gram negative non spore- forming rods. Within this group, species of the genus Pseudomonas are predominant along with some strains of Achromobacter, Flavobacterium and Micrococcus. The distinguishing characteristic of psychrophilic bacteria 18 their ability to grow at low temperatures. Sulzbacher (1950) and others have isolated psychrophiles, mainly Pseudomonas, capable of growth down to -10°C. Ingraham and Stokes (1959) stated that bacteria do not readily adapt to growth at lower temperatures. Many of the psychrophilic bacteria also produce pigments. Hess (1934), Jezeski and Macy (1946) and Ayres g£_al, (1950) have reported pigments produced on fish extract, butter and chilled poultry, respect- ively. Many other biochemical activities such as fermentations, indole formation and protein decomposition have been extensively reviewed by Ingraham and Stokes (1959). In 1959, Castell and Greenough reported on a number of strains of Pseudomonas, capable of producing strong off odors in fish muscle, which were non proteolytic. However, they did note that proteolytic strains could have been missed due to the method used for culture selection. These non-proteolytic strains were capable of pro- ducing off odors when inoculated into sterile fish muscle. Psychrophilic Pseudomonas As already indicated, psychrophilic Pseudomonas have been implicated in most all reports of bacterial contamination at low temperatures. Ayres g£_§l, (1950) described the formation of off odors and slime on cut-up poultry at temperatures from 0°C to 10°C as; "formation of trans- lucent, moist colonies in large numbers on the cut surfaces, giving the appearance of water droplets, becoming larger, white or creamy in color, often coalescing into uniform slimy layers". Initially with colony formation he reported sweet smelling rancid odors which, in the final stages, changed to a pungent ammoniacal odor. In a review, Ayres (1963) cited up to 20 references indicating the importance of the Pseudomonas species in the production of slime on flesh meats. Tomlinson and Cambell (1963) observed the accumulation of free ammonia as the catabolic pro- duct of the nitrogenous compounds by Pseudomonas species. 10 Brown and Weidemann (1958) concluded that the green pigment producing psychrophiles reported by Empey and Scott (1939a) were all Pseudomonas. However the growth of non pigmented Pseudomonas, as observed by Barnes and Shrimpton (1958) explained the poor correlation between the develop- ment of off odors and the appearance of pigmentation. Lipase activity from Pseudomonas fragi at 15°C has been reported by Nashif and Nelson (1953). Proteolytic activity from a Pseudomonas strain has been shown by Greene and Jezeski (1954). Psychrophiles and Meat Spoilage Thus it appears that the Pseudomonas strains constitute the predom- inant group involved in fresh meat spoilage at low temperatures. Ockerman £5 31. (1964) and Ockerman and Cahill (1967) describe procedures for obtaining sterile muscle tissue samples, thereby providing the preliminary step towards elucidating the basic mechanism by which meats undergo bacterial spoilage at low temperatures. Jay (1964) reported a lack of breakdown of major beef proteins by low temperature spoilage flora when fresh beef was held until spoilage occurred. Davey and Gilbert (1966) have shown a lack of proteolysis under conditions of aseptic postmortem incubation of beef muscles. There has been much discussion as to whether the major meat proteins are involved in the early stages of spoilage. Some conflicting reports have appeared as to the role of muscle proteins in the spoilage process. Jay (1964) has shown that low molecular weight compounds of the sarcoplasm constitute one source of readily utiliz:able nitrogenous compounds. Jay (1966) concluded that lowttemperature spoilage 11 occurs in the absence of significant proteolysis and that ultimate break- down of primary proteins in beef is caused by cathepsins released due to bacterial action. Jay and Kontou (1967) and Lerke‘etpal, (1967) have concluded that meat spoilage micro-organisms do not utilize meat and fish muscle proteins for growth when low molecular weight non protein nitrogen sources are available. Ockerman Ethel, (1969) found changes in sarcOplasmic proteins and non protein nitrogen in late storage of sterile beef tissue inoculated with Pseudomonas and Achromobacter species. Borton £5 21. (1970) found that Pseudomonas fragi caused increases in water soluble or myofibrillar proteins. Hasegawa g£_§l, (1970) reporting on a number of bacteria commonly associated with meat spoilage found high proteolytic activity. Tarrant £5 51. (1971) found considerable salt soluble protein degradation in sterile pork muscle inoculated with Pseudomonas fragi. Over a 20 day storage period at 10°C large increases in non protein nitrogen corres- ponding to the salt soluble protein decreases were observed. Considerable extracellular proteolytic activity was also observed, most of which 3 occurred immediately after the onset of spoilage. Some activity was ob- served prior to spoilage. Liberation of extracellular proteolytic activity had previously been reported by Juffs st 51. (1968). Dutson ££“§£- (1971) working on the ultrastructural changes that occur in the muscle proteins during bacterial spoilage have shown almost complete absence of material in the H zone, marked disruption of the A band and some loss of the dense material from the 2 line, all indicating that marked proteolysis had occurred. In a later study, Tarrant 25 il- (in 12 press) using partially purified proteolytic enzyme solution prepared from a synthetic growth medium have shown preferential myofibrillar breakdown. Dutson (1971) postulated that the enzyme or enzymes may be secreted into blebs which he found on the bacterial cell surface and later form glo- bules. These globules then release their contents into the muscle tissue. Bacterial Proteases The proteases which are of interest for use in the food industry are mostly extracellular enzymes of plant and microbial origin. Auto- lysis of bacteria during enzyme elaboration cannot,however,be ruled out (Pollock, 1962). Reed (1966) reported that all commercially important proteases are essentially excretions of the living organisms into the surrounding medium. Isolation and purification of proteases from various microbial sources have received much attention in recent years. Bacterial proteases elaborated from mesophiles (Rappaport g£”§1., 1965; Feder and Lewis, 1967; Fabian, 1970), from thermophiles (Ohta g£_al,, 1966) and from psychro- philes (Nunokawa and McDonald, 1968) along with many others have been reported. Extracellular proteases have been reported for some of the Pseudomonas species, including Pseudomonas putrgfggigng, (Van Der Zant, 1957), Pseu- dnmnnaa.£lngxg§g§n§ (Peterson and Gunderson, 1960), Pseudomonna.myxogenes (Morihara, 1956) and Pseudomonas aeruginosa (Morihara, 1963). More re- cently Juffs £5 31. (1968) reported on liberation of proteolytic enzymes from several Pseudomonas species under different conditions of temperature and nutrition. l3 Pollock (1962) reporting on the problem of cell autolysis and cri- teria for extracellularity suggested that the following precautions be taken to determine whether or not an enzyme is truly extracellular. 1. Enzyme liberation should be followed in young cultures pre- ferably during the logarithmic phase of growth. 2. If an enzyme is found in a culture supernatant the cells themselves should be tested for activity. 3. Cell autolysis can be looked for by testing the culture supernatant for the presence of substances normally present within the cell. Peterson and Gunderson (1960) reported that the maximum proteolytic enzyme elaboration from Pseudomonas fluorescens occurred very soon after growth was initiated and while the bacterial population was still low. Enzyme liberation was found to be a maximum at 0°C and decreased with increasing temperatures. They also investigated the influence of temperature on the enzyme activity after elaboration and found that it increased linearly up to 25°C and decreased with further temperature increases. Juffs gt 21, (1968) have reported similarvresults for at least six other species of the genus Pseudomonas. Gale (1943) stated that the factors affecting enzyme production were the chemical constituents of the cultural medium, the physicochemical conditions during growth and the age of the culture. The elaboration of extracellular proteases seems to require the presence of organic nitrogen in the form of amino acids and dipeptides in the growth medium, none being elaborated in a mineral salts medium containing inorganic nitrogen plus either citrate or casein (Juffs g£_§l,, 1968; Tarrant et_ ‘21,, in press). 14 It has been shown that calcium is an essential component for the production of extracellular enzymes from Bacillus proteus vulgaris in a synthetic medium (Merrill and Clark, 1928). Others to report similar effects of calcium included Wilson (1930), Haines (1931, 1932, 1933), Gorini (1950) and Morihara (1956). Morihara (1957b) in his studies on the specific action of a crystalline protease obtained from Pseudomonas myxogenes has shown that the enzyme might be classified as a collagenase. Adamcic .ggual. (1970) reporting on the bacteria induced biochemical changes in chicken skin indicated that most of the pigmented Pseudomonas associated with spoilage of whole poultry possess collagenolytic enzymes. Adamcic and Clark (1970) found ninhydrin positive materials from soluble collagen on incubation with two strains of pigmented Pseudomonas after a lag period of two to six days. This lag period was eliminated on using cells previously adapted to utilizing collagen. Classification of Proteases The earliest classifications were based on the origin of the enzymes and their names ended in "in", for example, papain, ficin, bromalin from plants, trypsin from the pancreas. The classification in general use today is based on the scheme suggested by Bergmann and Fruton (1941) and Bergmann (1942), dividing proteases into exopeptidases and endopepti- dases. More recently proteases are being classified by the chemical nature of their active site. This method seems to be most useful for the characterization of an enzyme in a preparation of unknown origin. 15 The first group consists of those enzymes whose activity depends on the presence of sulfhydryl groups at the active site. Binding of the thiol group by oxidizing agents, heavy metals and alkylating agents inhibit such enzymes. Plant proteases and some cathepsins fall into this group. The second group called metallo enzymes consists of enzymes whose activity depends on the presence of a metal. These metals can be loosely or tightly bound. Removal of the metal removes enzymatic activity and re-addition of the metal usually restores it. Chelating agents such as ethylenediaminetetraacetic acid are used to effect such removal of metals. Some bacterial proteases are included in this group. There is a third group of enzymes not inhibited by reagents react- ing with thiol groups or with metals, but they are strongly inhibited by diisopropylphosphofluoridate (D.P.F.). Trypsin and chymotrypsin fall into this group (Reed, 1966). Role of Calcium Merrill and Clark (1928) and Haines (1931, 1932, 1933) proposed that calcium participated in protein synthesis. Gorini (1951) proposed that calcium acted as a firmly bound prosthetic group which affected enzyme stability. Morihara (1959) suggested that calcium may be required in any of the synthesizing steps from precursor to the actual enzyme as shown below. Unstable enzyme Amino acids +_precursor enzyme + inactive enzyme.l + Enzyme Cell | Medium ..._.._. __.{ Nitrogen source Calcium 16 He showed that resting cells could produce enzyme on a carbon source plus calcium, indicating that the cellular nitrogen had contributed to enzyme synthesis. Morihara (1960) observed that protein secretion from cells grown in a carbon containing medium was reduced to 1/5th in the absence of calcium. Thus he concluded that the incorporation of calcium occurs in the completely synthesized calcium free proteinase. Tenderizers The role of digestive enzymes in the breakdown of food proteins prior to absorption and assimilation has been elucidated. The role of proteolytic enzymes added to foods in the process of manufacture are many, including the action of rennin in cheese manufacture, papain in chill proofing of beer, microbial proteolytic enzymes in cheese ripening and the use of plant enzymes for tenderizing fresh meats. In this latter case, proteolytic enzymes in the form of powders or in solution act on muscle fibers or connective tissue components during the early phases of the cooking process to increase tenderness (Wang St 21,, 1957). While it appears that only a small proportion of the total protein tissue needs to be altered to provide significant increase in tenderness as measured by taste panels, large changes are required to create obser- vable structural changes. Dutson (1971) and Tarrant 35 213 (in press) observed changes in structure due to bacterial and enzymatic activities, respectively, with meat spoilage. However, no attempt has been made to correlate this with tenderization. 17 Muscle Proteins In defining muscle as "a composite of delicately balanced protein suspensions in dilute salt solutions containing various amounts and kinds of lipids, carbohydrates and formed particles", Briskey and Fukazawa (1971) have described a complex machine in simple but very meaningful terms. The proteins of muscle may be categorized into sarcoplasmic or water soluble, myofibrillar or salt soluble, and stromal or insoluble proteins. Sarcoplasmic Proteins The sarcoplasmic proteins have been described as those muscle pro- teins which are soluble in water or low salt concentrations. They have been characterized as globular, of low viscosity, and low molecular weight proteins such as, myogen, myoalbumin and myoglobulin (Helander, 1957). Whitaker (1959) described them in a similar manner but included the muscle enzymes associated with glycolysis. The amount of sarcoplasmic proteins extracted varies with the different species and Hill (1962) reported that they accounted for 15-202 of total nitrogen in bovine muscle, 20-252 in porcine and 25% in ovine muscle. The varia- tions within species are due to environmental and physiological conditions such as temperature of storage, carcass age and pH. During aging or storage the extractable sarcoplasmic protein nitrogen in bovine muscle has been shown to decrease (Goll g£_al,, 1964; Davis, .1965; Aberle and Merkel, 1966). Decreases were also reported in porcine unuscle (Sayre and Briskey, 1963; McLaughlin, 1963). 18 The pH of post rigor muscle is usually below 6.0, and the pH of at death muscle is near 7.0, so it is very important to use a buffered ex- tracting solution to prevent differences due to pH of the extracted sample (Goll e£_§1,, 1970). It has been shown that sarcoplasmic proteins do. not undergo large changes in composition during postmortem storage at 5°C or lower (Aberle and Merkel, 1966; Lawrie £5 31., 1963; Scopes, 1964). This suggests that the sarcoplasmic proteins do not experience extensive postmortem proteolysis and that myofibrillar and stroma pro- teins are not proteolytically degraded (Goll e£_§1,, 1970). Myofibrillar Proteins The myofibrillar proteins have been described as those muscle pro- teins which are insoluble in water or low salt concentrations, but are soluble in high salt concentrations. They can be characterized as fibrous, highly viscous. and high molecular weight proteins (Helander, 1957). The components have been identified as the contractile proteins such as myosin, actin, actomyosin, tropomyosin and other less abundant proteins (Whitaker, 1959). Hill (1962) reported that the myofibrillar fraction comprised approximately 55% of the protein nitrogen in bovine muscle, 56% in porcine muscle and 53% in ovine muscle. Again, within species differences do occur due to the environment and physiological conditions. McIntosh (1967), Aberle and Merkel (1966) found increased extractability of myofibrillar protein up to two weeks postmortem. Davey and Gilbert (1968a) also reported an increase in the myofibrillar protein extract from unaged to aged beef and rabbit muscle, 19 indicating a weakening of the linkage with the stromal protein or disin- tegration of the stroma itself. Goll g£_§1, (1970) indicated that the solubility of the myofibrillar proteins may be complicated by the fact that substances such as ATP and KI have a marked effect on solubility. Recently, it has been shown that myofibrillar proteins undergo at least two kinds of specific alterations during postmortem storage: (1) a loss of 2 line structure and in some cases complete removal of both the Z and M line (Stromer and G011, 1967; Stromer gt 51,, 1967; Davey and Gilbert, 1967, 1969; Henderson e£_§l,, 1970). Further evidence that the bonds holding actin filaments to the Z line are weakened has been shown by Chaudhry EEHEL' (1969), Davey and Gilbert (1968a, b) and Penny (1968). (2) The second type of postmortem alteration is the modification of the actin-myosin interaction. Fujimaki g£_al, (1965a) and Fujimaki e£_§l, (1965b) were first to report increased actomyosin ATPase activity with storage. However, the nature of the change causing this increased activity remains unclear. Okitani g£_§1, (1967) have indicated that myosin B stored at pH 5.7 postmortem required less ATP for dissociation than the same myosin B before storage. They concluded that prolonged exposure below pH 6.0 may have caused this modification that has been observed in the actin- myosin complex with storage. Ultrastructurally it has been observed by Gothard g£_al, (1966) and Stromer §£_al, (1967) that rigor shorten- ing and post rigor lengthening occur by the sliding of the interdigita- ting filaments past one another as a result of the weakening of the interaction between myosin cross bridges and actin filaments. Stroma Proteins The stroma proteins have been described as those muscle proteins Vfllich are insoluble in either water or high salt concentrations (Helander, 1957). Whitaker (1959) identified the stroma proteins as collagen, 20 elastin, and ground substance. Hill (1962) reported 12-182 total nitrogen in bovine muscle consisted of stroma proteins. Weirbicki 25 31. (1954) and Weirbicki g£_a1, (1955) reported that the alkali insoluble protein content of bovine muscle remained unchanged between 3 and 15 days post- mortem storage. This has also been shown in poultry muscle by Khan and Van der Berg (1964) and Sayre (1968), although some studies (Herring 35 31,, 1967; McClain g£_§1,, 1965) indicate that postmortem aging may result in some increase in collagen solubility at temperatures above 37°C. This increase appears to be very subtle and is probably limited to changes in the number or strength of the cross bridges between the connective tissue proteins (Goll gt 51., 1970). Non Protein Nitrogen (NPN) Non protein nitrogen components may be described as those low molecular weight substances such as amino acids, peptides, nucleic acids present in muscle, but not part of the protein material. Hill (1962) reported that NPN content of the total nitrogen in beef, pork and lamb muscle was between 11 and 132. Many workers, including Sharp (1963) using rabbit and beef muscle, and Aberle and Merkel (1966), Ockerman §£_al, (1969), each using aseptic beef muscle have reported slight in- creases in the NPN content during postmortem storage at low temperatures. Borton (1966) also reported slight NPN increases in aseptic porcine muscle held at 3°C for 35 days. 21 Ultrastructure of Bacteria A brief review of the ultrastructure of bacteria (Dutson, 1971) indicates that Pseudomonas appear to have an irregular undulant cell wall and a planar cell membrane. Within this «membrane are contained loosely packed ribonucleoprotein particles distributed throughout the periphery of the cytoplasm, as well as some axially disposed DNA (Wiebe and Chapman, 1968a, b). These workers also reported that some strains of Pseudomonas contained bleb like evaginations from the cell wall. These evaginations have also been shown by Dutson st 21. (1971) in their work using Pseudomonas fragi on porcine muscle. Their results indicate that the bleb like evaginations are formed at the cell wall surface due to growth on the muscle tissue. They postulated that the proteolytic activity was responsible for the myofibrillar disruption observed and that the enzymes involved may have been released from these bleb like evaginations. Other workers to report these blebs or protrusions on various bacterial surfaces include Knox g£_al, (1966) working with E, coli and Hitchins and Sadoff (1970) with Azotobacter vinlandii. EXPERIMENTAL METHODS Pure Culture Propogation and Maintenance Stock cultures were obtained from the American Type Culture Collection and stored in a cooler at 3°C. Culture propogation was performed as follows: the sealed outer vial was removed by breaking, using heat and water to cause cracking of the glass. The pure culture contained in the Iinner vial in powder form was dissolved in 0.5 m1 sterile water. This solution was then transferred aseptically to a screw cap test tube con- taining 10 ml of sterile nutrient broth (Difco Manual). Incubation was carried out at room temperature (26°C) for 36 hours at which time growth was indicated by turbidity of the solution. Stock cultures for subsequent use were then prepared by inoculating test tubes containing 10 m1 of sterile nutrient broth with two loopfuls of the initial solution and allowed to incubate at room temperature until slight turbidity was ob- served. These were then stored in a freezer (Kelvinator) at ~23°C until required. Culture maintenance for media inoculation was performed by thawing the stock culture at room temperature, inoculating a test tube containing sterile Koser's citrate medium with a 1 m1 aliquot and incubating at room temperature (26°C) for 36 hours. This was repeated a second time. A final transfer was likewise made except that incubation was carried out at 10°C in a rotary shaker at 160 rpm until turbidity, as measured by optical density at 660 nm reached 1.0 or greater. Cultures were maintained 22 23 by repeated transfers every 48 hours as described until a new stock cul- ture was prepared, usually once every month. Cultures Used Pseudomonas perolens ATCC 10757 Pseudomonas fragi_ATCC 4973 Pseudomonas aeruginosa ATCC 10145 All three cultures were grown at 10°C in Koser's citrate medium to observe degree of growth as measured by reading optical density at 660 nm. Media Preparation All media were prepared using deionized distilled water and placed in 500 ml Erlenmeyer flasks. Absorbent cotton and aluminum foil were used as stopper and cover respectively during sterilization whibh was carried out at 15 psi, or 121°C for 15 minutes. The sterile medium was cooled to 10° to 15°C prior to inoculation. Koser's citrate (Difco Manual) was the medium chosen as the basal medium for growth of the micro-organisms and its composition is as follows: Sodium Ammonium Phosphate 1.5 g Sodium Phosphate - monobasic 1.0 g Magnesium Sulfate 0.2 g Sodium Citrate 3.0 g and made to 1 liter with deionized distilled water. In the development of a medium suitable for production of enzyme(s), the sodium citrate was 24 replaced as the sole carbon source by individual amino acids and dipep- tides and combinations of these. One hundred fifty ml of medium in a 500 m1 Erlenmeyer flask was used for incubation purposes to prevent ex- cessive foaming. A 1% inoculum was used in all cases. Assays Enzyme assay The assay used was a modification of the method described by Anson (1938). One ml of enzyme solution was reacted with 1 m1 of 22 w/v casein (Hammerstein - Nutritional Biochemical Corporation) solution as substrate. The casein solution was prepared by mixing 2.0 g in 100 m1 of 0.03 M phosphate and pH was adjusted to 7.5 using 1 N sodium hydroxide solution. Preincubation time for all solutions was 3 minutes at the assay temperature. Incubation was carried out in a water bath shaker (Eberbach) at speed number 5 (150 rpm) for a prescribed period of time. The reaction was terminated by addition of 2 m1 of a 5% w/v solution of trichloroacetic acid (TCA). This was allowed to stand for 15 minutes prior to filtering of the precipitate through Whatman number 2 filter paper. One ml of the filtrate was taken to determine the enzyme activity using the method of Lowry gt a_1_. (1951), which measured the tyrosine equivalents released. Blank samples were run with each test sample in a similar manner, except that the casein solution was not added to the enzyme solution until after the TCA had been added to stop the enzyme action. A standard curve was established using L-tyrosine. 25 Protein assay The method of Lowry gt 21. (1951) was used for protein determinations. One ml of the protein solution was mixed with 5 m1 of Lowry reagent C (Appendix A.1.) and allowed to stand for 20 minutes, before addition of 0.5 m1 of Phenol reagent (folinciocalteau, Fisher Scientific Company) and water (1:1). This was allowed to stand for at least 45 minutes with mixing at intervals before reading at a wavelength of 660 nm on a Beckman Model DU SpectrOphotometer. Distilled water was used to zero the instru- ment before making the readings. Blanks were prepared for each test sample in a similar manner, except that the protein solution was replaced by deionized distilled water. Preparation of Crude Enzyme Solution The medium developed for growth of baceeria and enzyme production was the basal Koser's citrate plus calcium chloride (0.5 g/l). This medium was sterilized and cooled prior to incubation. A 1% inoculum was used and tubes were incubated at 10°C for 45 to 48 hours in a rotary shaker. The crude enzyme solution was prepared by centrifugation at 10,000Xg for 20 minutes (Sorvall RCZ-B) at 0°C to remove the cells and the supernatant or crude enzyme solution stored at 3°C until required for use. Various levels of calcium chloride from 0.1 g to 1.0 g per liter were used in the Koser's citrate medium to determine optimum levels for enzyme production. 26 Replacements for Calcium Chloride in Enzyme Production Various metal salts were investigated as possible replacements for calcium chloride in the medium used for enzyme production. The medium was Koser's citrate plus 0.5 g/l of the following metal salts individually. Cobaltous chloride Zinc chloride Sodium chloride Cuprous chloride Barium chloride Manganese sulfate Sterilization, inoculation and incubation were as previously described. Samples were taken at zero hour and twice daily to determine growth as measured by reading optical density at 660 nm. Enzyme assays were carried out on these samples as previously described. Parameters of the Crude Enzyme Solution Temperature of storage The four temperatures chosen for storage were: room temperature 26°C cooler temperature 3°C freezer temperature -23°C freezer temperature -29°C In the case of freezer storage, two methods of freezing were investigated, one being a slow normal freeze and the other a fast freeze obtained by placing the enzyme solution in a dry ice and acetone mixture at -80°C for 1 minute. Each test sample contained 6 ml of enzyme solution in a screw 27 cap test tube with specific activity of 0.05. Freezer samples were thawed by a slow thaw at cooler temperatures and also by a fast thaw at room temperatures. Room temperature samples were assayed daily for a period of 3 days. Cooler and freezer samples were assayed twice per week for a 25 day period. Temperature of assay Assay temperatures at 5°C intervals from 15°C to 50°C were used. The enzyme solution (specific activity of 0.05) had been stored overnight at 3°C, and preparations for the assay were also performed at 3°C. Assay conditions were as previously described. The temperatures were maintained in the water-bath shaker and checked at intervals using a standardized thermometer. Incubation time was 15 minutes at all temperatures. pH Five buffers were used to give a suitable range and degree of overlap for effective analysis of pH and its effects on enzyme activity. Citrate-phosphate - pH range 2.6 to 7.0 (McIlvaine, 1921) Phosphate - pH range 5.7 to 8.0 (McIlvaine, 1921) Tris (Hydroxymethyl) amino methane - pH range 7.2 to 9.0 (Tris-Sigma) Barbital - pH range 6.8 to 9.2 (Michaelis, 1930) Carbonate bicarbonate - pH range 9.2 to 10.7 (Delory & King, 1945) Buffers were prepared in a 22 casein solution and the various pH ranges were as shown in appendix 8.16. The assay was performed in the usual manner following a 3 minute incubation period of all the solutions. One 28 set of the reaction mixtures was used to check pH values at the end of reaction time. Time The relationship of assay time to enzyme activity was followed from zero time up to 90 minutes. All preparations were made in the cooler at 3°C. The enzyme solution had a specific activity of 0.06. Substrate concentration Casein (Hammerstein, N.B.C.) was the substrate used, with the follow- ing amounts of a 22 w/v solution made to 1 ml with deionized distilled water: 0.01, 0.02, 0.04, 0.06, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 ml. The enzyme solution used had a specific activity of 0.06. The assay time period was 11 minutes. Enzyme concentration The following amounts of the enzyme solution (sp. activity 0.06) were used: 0.1, 0.2, 0.4, 0.6, 0.8, and 1 m1 aliquots and made to 1 m1 total in each case with deionized distilled water, assay time being 11 minutes at 35°C. Substrate specificity The following substrates were used to check the specificity of the enzyme solution: I [I‘ll l.l"' lll'l'l'llxlll‘ 29 Casein - A 2% w/v casein solution was prepared in 0.03 M phosphate buffer and pH adjusted to 7.5 with 1 N NaOH. Collagen - A 2% w/v collagen (N.B.C.) was prepared as described. Egg albumin - A 2% w/v egg albumin (N.B.C.) was prepared as described. water soluble or sarcoplasmic proteins - A modification of the method by Helander (1957) was used for extraction. Five grams of powdered low fat beef tissue was extracted with.50 m1 of 0.03 M phosphate buffer, pH 7.4 for 30 minutes at 3°C with stirring action. The residue was centri- fuged off at 10,000Xg for 20 minutes. Supernatant was filtered through 4 layers of cheese cloth and the filtrate used as the water soluble protein fraction. Protein concentrations of 6.5% (approx. 6.0 mg/ml) were obtained using the micro Kjeldahl method (The American Instrument Company, 1961). Salt soluble or myofibrillar proteins. The residue from the water soluble extract was further extracted with 50 ml of 1.1 M KI and 0.1 M phosphate buffer solution, pH 7.4, for 1 hour. The residue was centri— fuged off at 10,000Xg for 20 minutes and the supernatant filtered through four layers of cheese cloth and the filtrate used as the salt soluble protein solution. Protein concentrations of 9.5% were obtained using the micro Kjeldahl method. To prevent interference from the high salt con- centration present on the Lowry assay method, the protein solution was first dialyzed against 0.03 M phosphate buffer, pH 7.4 to remove these salts. The jelly—like mass which remained was diluted to 60 ml with deionized distilled water to facilitate handling and to give approximately 6.0 mg/ml. I!!! 30 The enzyme solution used had a specific activity of 0.05. Preincu- bation of 3 minutes was used before beginning the assay for all substrates. Enzyme activity was followed from zero time to 64 minutes with samples being withdrawn at 2, 4, 8, 16, 32 and 64 minutes. Purification Procedures The crude enzyme solution was prepared as previously explained by centrifugation of cells. Ultrafiltration A process of selective molecular separation utilizing the Amicon ultrafiltration system, model 402 (Amicon publication number 403) was used. Diaflo membranes with the following retention characteristics were used: PM-30-capable of retaining molecules of approximately 30, 000 molecular weight and greater. UMelo-capable of retaining molecules of approximately 10,000 molecular weight and greater. UM-2-capable of retaining molecules of approximately 1,000 molecular weight and greater. Configuration and size of the molecules would effect the actual retention in any particular case. The PM-30 membrane was first used with a nitrogen pressure of 15 to 20 psi to remove all remaining cells and other large molecules. UM-lO and UM-2 membranes with pressures of 30 psi and 70 psi respectively were used to concentrate the enzyme solution, the filtrate being discarded. The concentrated solution was next applied to Sephadex gels. 31 Gel filtration Sephadex gels (Sephadex booklet - Gel filtration in theory and practice) were used in all cases, the molecules being eluted in order of decreasing molecular size. The two gel types used were the Sephadex G-75, medium, which has a dry particle diameter of 40-120 u, fractiona- tion range of 3,000 to 70,000 M.W. and bed volume of 12-15 m1/g dry Sephadex. G-100 Sephadex has a dry particle diameter of 40-120 u, fractionation range of 4,000 to 150,000 M.W. and a bed volume of 15-20 m1/g dry Sephadex. The G-75 was dissolved in water and allowed to swell at room temperature for 1 day. G—100 was treated likewise but allowed to swell for 3 days. The gels were poured into Sephadex K-25/45 columns with a gel reservoir attached. A flow rate of approximately 15 ml per hour was allowed until the slurry had packed. The gel reservoir was then removed and converted to a mariotte flask. To this was added 0.03 M phosphate buffer, pH 7.5 as the eluent. The operating pressure was set at approximately 30 cm difference between inlet and outlet and the flow rate was maintained at 15 ml per hour for 24 hours at room temperature. When the column had been equilibrated, it was removed to 3°C cooler where all experiments were carried out. The crude enzyme concentrate was added in 15 ml aliquots to the G-75 Sephadex K 25/45 column and eluted with 0.03 M phosphate buffer, pH 7.5. The eluate was collected in 6 ml aliquots with an L.K.B. fraction collector, the attached recorder showing the chromatogram as measured at 260 nm (The enzyme was eluted directly after void volume). The G-lOO K 25/45 column 32 was used in a similar manner to obtain better separation. A G-100 Sephadex K 15/90 column was also used to attempt better separation. Absorbance readings The enzyme solution obtained from the Sephadex G-75 Sephadex K 25/45 column was read over the wavelength range 200 nm-800 nm in a model DU Spectrophotometer (Beckman). The deuterium lamp was used over the range 200 nm to 400 nm and the tungsten lamp from 400 nm to 800 nm. Disc gel electrophoresis A modification (Rampton, 1970) of the method reported by Davis (1964) was used. The gels consisted of a runner gel which contained a final con— centration of 6.5% cyanogum, this replacing the acrylimide-bis-acrylimide used by Davis (1964), and a spacer gel containing 5.0% cyanogum (appendix A.2). Both were added to the glass tubes and polymerized by fluorescent light for 20 minutes. These were placed in a Buchner electrophoresis apparatus using a tris-glycine solution, pH 8.0 in the buffer tanks. Bromothymol blue was used to form a tracer band. Various concentrations of enzyme solution from 25 pg to 150 ug protein were applied to the gel surfaces beneath the tank buffer. A current of 2 mA per gel was main- tained during the electrophoretic separation on applying 200 volts at 3°C. Electrophoresis was terminated when the leading bromothymol blue band reached the end of the gel. The gel was removed from the glass tube by inserting a hypodermic needle along the inside surface and forcing water |IPI .‘ [ml ll"! t [lll‘l‘ ll'l I.\l..l‘ ‘._III III [I‘l‘fl’ l.t._llltlolllu1.1l\ l I 33 containing photoflow solution between the glass wall and gel. The gel was next placed in a solution of 0.4% Buffalo Black NBR dye consisting of water, methanol and glacial acetic acid (5:5:1) and allowed to stain for 30 minutes. The gels were washed twice in a 7% glacial acetic acid solu- tion and allowed to destain for 24 to 36 hours in 7% glacial acetic acid solution. Gels were also stored in fresh glacial acetic acid solution at 3°CI Tenderization Effects of Crude Enzyme Solution Enzyme solution preparation The crude enzyme solution for tenderization studies was prepared as previously outlined, except that centrifugation was as follows: 9,000Xg for 15 minutes with the supernatant being decanted and recentrifuged again at 9,000Xg for 15 minutes for a second and a third time. This supernatant solution was used for injection of the meat samples. Meat sample preparation Two eye of the round portions of beef (approximately 4.2 pounds each) were purchased uncut from Michigan State University food stores and stored at 3°C for 24 hours. Prior to injection both portions of beef were split evenly into two 1 pound samples and the opposite halves from each portion paired. One paired set was used as a control and the second set was used as the treatment. 34 Meat injection The treatment consisted of a 10% injection using the crude enzyme solution and an automatic pipetting machine (Brewer, model number 40) with an injection volume of 1 ml per stroke. The injection process was performed evenly over the entire surface of the meat samples. The con- trol samples were similarly injected with a sterile solution of Koser's citrate medium. All samples were weighed before and after injection to determine the exact amounts injected. Samples were stored at low temper- atures for 3 days to facilitate further diffusion of the injected solutions and finally reweighed prior to cooking. One inch thick steaks were taken from the cut surface of each sample, and these plus the remain- ing portions (to be roasted) were weighed. Cooking methods The steaks were cooked in an air convection oven as follows: meat temperature before cooking 45°F meat temperature at end of cooking-internal 153°F temperature of oven 250°F Cooking time 50 minutes The roasts were cooked in a General Electric range (double oven) as follows: meet before cooking 37°F temperature of the small oven 350°F meat temperature after cooking-internal 160°F 35 cooking time 1 1/2 hours temperature of the large oven 200°F meat temperature after cooking-internal 155°F cooking time 3 hours All temperatures were measured using thermocouples and recorded by a potentiometer (Honeywell model). Internal temperatures of the meat por- tions were recorded by placing the thermocouples in the sample centers. On removal from the ovens, the samples were allowed to cool and stored overnight at 3°C, covered with tinfoil to prevent dehydration. Tenderness measurements Tenderness values were obtained using the warner-Bratzler shear device. Cores of 1/2 inch in diameter were obtained from the steaks and three values were taken per core. The roasts were cut into 1 inch thick slices and treated like the steaks. Approximately 12 to 15 cores were obtained from each slice. The shear force values from each control and treatment were averaged and the decrease in treatment shear force values in comparison to corresponding control average values were reported as percentage increase in tenderness. Procedure for Obtaining Sterile Porcine Muscle Porcine muscle samples required for bacterial inoculation and for the effect of partially purified enzyme solution on the primary proteins were obtained from the longissimus dorsi muscle from a 200 pound hog purchased from.Michigan State University Farms. 36 Slaughter The hog was killed without prior stunning. The sticking area of the neck.was washed with a 2% warm bactericidal solution. A knife which had been held in a boiling water bath for 10 minutes was used to stick the hog. The stick area was rewashed after bleeding had finished and the hog was then scalded, dehaired, washed with bactericidal soap and eviscerated. The unsplit carcass was again washed with the bactericidal solution, followed by an alcohol rinse and flaming to dry. The carcass was stored for 24 hours in a 1°C cooler prior to sample removal from the longissimus dorsi muscle. All equipment used in the excision procedure was sterilized for 15 minutes at 121°C (15 psi). Muscle sample procurement The shoulders were removed from the unsplit carcass after 24 hours in the cooler at 1°C. The carcass was placed on a stainless steel table previously steamed and washed with alcohol and flamed to dry. Sterile, disposable rubber gloves were worn by the workers who were to obtain the samples. Sterile knives were first used to strip the backfat which was rolled back to expose the longissimus dorsi muscle. With a second knife, the muscle was sliced and aseptically removed to sterile containers, each container having approximately 1,000 g of muscle sample. The sample from one container was fed through a sterilized grinder having a 2 mm grinder plate and collected in the same container. The sample was reground a second time and stored in sterile glass jars (approximately 100 g per jar) 37 at -23°C until required for use in a subsequent experiment to determine the effect of the enzyme solution on the primary protein fractions. The muscle sample in the second container was used for preliminary experiments to detect enzyme production from Pseudomonas perolens ATCC 10757. Growth and Enzyme Production on Porcine Muscle Pseudomonas perolens was prepared by growing in nutrient broth at 25°C for 36 hours. A 20 fold dilution was made in sterile 0.01 M phos- phate buffer, pH 7.0. Muscle sample preparation and inoculation The muscle sample was obtained as previously outlined and divided into two batches. The first batch or control was ground in a sterilized grinder with 1% solution of sterile 0.01 M phosphate buffer added during the grinding operation. A second grinding was also carried out. Approxi- mately 100 g were added to sterilized glass jars with screw on lids. The second batch.was treated in a similar manner, except that a 1% inoculum of ngudomonas perolens was used. All jars were appropriately identified and the lids were loosely replaced. Storage was at 10°C in a walk-in type cooler. Enzyme extraction One control and one treated sample were removed daily. Twenty gram samples were placed in 250 ml plastic centrifuge bottles plus 30 grams of 38 0.01 M phosphate buffer, pH 7.6. This was stirred gently for 15 minutes and then centrifuged off at 10,000Xg for 30 minutes. The supernatant was removed and dialyzed in 1/4 inch diameter dialysis tubing against 0.03 M phosphate buffer, pH 7.2 at 3°C for 18 hours. Analysis The following tests were carried out on both samples daily for up to 20 days. EE.‘ 1 g of sample plus 1 ml of deionized distilled water were mixed to form a slurry and pH was read using a semi-micro combination electrode (Corning). Bacterial counts - Standard plate counts were performed using 1 g of sample in 99 m1 of 0.01 M phosphate buffer (American Public Health Asso- ciation Inc., 1966). Disc gel electrOphoresis - as before. Enzyme assay - The method used was a modification of Anson (1938). A 1% solution of hemoglobin (NBC — standardized for protease assay) in 0.03 M phosphate buffer was used as substrate. One ml of enzyme solution at 35°C in a shaking incubator at speed number 5 (or 150 rpm) was reacted for the desired time period. The reaction was stapped by adding 2 m1 of 5% TCA. Blanks were run in a similar manner, except that the enzyme solution was incubated without substrate which was added after TCA preci— pitation of the enzyme. All were allowed to stand for 15 minutes before 39 centrifugation of the precipitate at 3,000Xg for 5 minutes and pouring the supernatant through Whatman number 2 filter paper. The optical density of the filtrate was read at 280 nm and the difference between blank and assay readings was reported as enzyme activity. Protein Changes due to Enzyme Action Sample preparation One day prior to the study of the effect of enzyme solution on the primary proteins, the frozen ground samples were thawed at 3°C and powdered in a sterilized waring blender previously cooled by liquid nitrogen. The thawed samples were refrozen in a liquid nitrogen solution to facilitate powder formation. Enzyme solution and inoculation The enzyme solution was freshly prepared and purified by G-lOO Sepha- dex in a K 25/45 column. Specific activity of the enzyme solution was 0.97. Sterilized vials (6 cm long x 2 1/2 cm in diameter) containing 4 g of the powdered muscle samples were inoculated with 2 m1 of the enzyme solution and mixed to form a slurry. Aseptic technique was used in all operations which were performed at 3°C. Control samples were prepared in a similar fashion except that sterilized Koser's citrate medium was used to form the slurry. All samples were stored at 3°C with shaking every 24 hours to remix the slurry. Control and treated samples were extracted 40 and analyzed at 0, 4, 8, 12, 25 days for sarcoplasmic,myofibrillar, and non protein nitrogen. Protein extraction Sarcoplasmig_protein - A modification of the method by Helander (1957) was used. Two grams were removed from the incubation vial into a 250 ml plastic centrifuge bottle plus 50 ml of 0.03 M phosphate buffer, pH 7.4. This was allowed to extract with moderate stirring action using a magnetic stirrer at 3°C for 20 minutes. The residue was centrifuged off at 5,000Xg for 20 minutes (Sorvall RCZ-B) and the supernatant filtered through 6 layers of cheese cloth. The residue was re-extracted a second time and the two filtrates combined to give the number of ml of sarcoplasmic pro- tein extract from 2 g of sample using 100 m1 of buffer. Myofibrillar protein - The residue from the sarcoplasmic protein ex- traction was further extracted with 50 m1 of 1.1 M potassium iodide, 0.1 M phosphate buffer, pH 7.4 at 3°C with moderate stirring action for 1 hour. The residue was re-extracted and the filtrates combined to give the number of ml of myofibrillar protein extract from 2 g of sample using 100 ml of extraction buffer. Non protein nitrogen - To determine the non protein nitrogen fraction, 15 ml aliquots were taken from the water soluble protein fraction. To this, 5 m1 of 10% trichloroacetic acid was added and the mixture was shaken and allowed to stand for 15 minutes. The precipitate was centrifuged off 41 at 5,000Xg for 20 minutes, and the supernatant was decanted off and used for nitrogen analysis. The results were recorded as non protein nitrogen. Total protein nitrogen - Total nitrogen was obtained by subjecting 0.5 g of muscle sample, weighed on nitrogen free paper, to nitrogen analysis. Stroma protein - Stroma nitrogen was found by subtracting the amounts contained in the sarcoplasmic and myofibrillar and NPN fractions from the total nitrogen per gram. Protein determination The micro Kjeldahl method as outlined by the American Instrument Company (1961) was used. Effect of Inhibitors and/or Activators The following solutions were made to 2.5 x 10-3M using deionized dis- tilled water, except for phenyl methyl sulfonyl fluoride which was dissolved in 8% 2-pr0panol and all were adjusted to pH 7.5 with 0.1 N NaOH: Cysteine Sodium bisulfite Iodoacetic acid Phenyl methyl sulfonyl fluoride Two m1 of enzyme solution (specific activity = 0.6) were mixed with 2 m1 of each of the above solutions and allowed to stand for 15 minutes. One ml of each mixture was in turn assayed for enzyme activity in the 42 usual manner. A control using deionized distilled water plus enzyme solu- tion was also run and the activity reported as 100% initial activity. To determine the effect of the Chelating agent, ethylenediaminetetraacetic acid (EDTA) on the enzyme solution, 0.5 m1 of enzyme solution plus 0.5 ml 4M, 2x10'5M, of the following concentrations of EDTA, 2x10-2M, 2x10-3M, 2x10- 2x10-6M, plus 0.1 m1 of deionized distilled water were mixed and allowed to stand at room temperature for 15 minutes. These were then assayed for enzyme activity in the usual manner. Blanks were also run and all tests done in duplicate. At the same time 0.1 m1 aliquots of 0.1 M calcium chloride were added to a second set of tubes which had been prepared with the first set and these also were assayed for enzyme activity after standing for a further 15 minutes. A 10 minute incubation period at 35°C was used in all cases. Effect of Enzyme Solution on Collagen A 2% w/v collagen solution (N.B.C.) with pH adjusted to 7.5 was used as substrate. One half m1 of enzyme solution plus 0.5 m1 of substrate were assayed in the usual manner using incubation time periods of 1, 2, 3, 6, 10 minutes at 35°C. However, instead of determining protein as a measure of enzyme activity, the hydroxyproline procedure of Parrish st 31. (1962) was used to detect collagen breakdown. In one case, this procedure was applied directly to the filtrates obtained after filtration of the TCA precipitates. In another, the filtrates were hydrolyzed in covered stainless steel centrifuge tubes containing 3 m1 of filtrate plus 12.5 ml 43 of 5 N NaOH and autoclaved for 5 hours at 121°C. The samples were then cooled and neutralized with 10 N hydrochloric acid, filtered and diluted to 50 ml. The hydroxyproline assay procedure was as follows: 0.1 ml of 0.1 M phosphate buffer (pH 7.0), 1.0 m1 of each sample were placed in a 10 m1 volumetric flask, plus 2.5 m1 of benzene. Just prior to trans- ferring the flasks to a 70°C i 1°C, water bath, 0.2 ml of cold 0.3 M ninhydrin in methyl cellosolve was added and then shaken vigorously for 5 minutes in the water bath. Samples were then immediately cooled to room temperature in an ice-water bath, diluted to 10 ml with benzene and thoroughly shaken. The organic layer was poured into test tubes contain- ing 200 mg of anhydrous sodium sulfate and again mixed before reading absorbancies at 570 nm and 550 nm on the model DU spectrophotometer. Hydroxyproline standards were also run. Since both hydroxyproline and proline react with ninhydrin, the following equation derived by Troll and Cannon (1953) was used to determine the absorbance values for the hydroxy- proline content of the samples: H.0. - 1.46 O.D. - 0.592 O.D. 570 570 550 where O'D'SSO - optical density at 550 nm O.D.57o - optical density at 570 nm H.0.570 - hydroxyproline at 570 nm The hydroxyproline standard was prepared using 0, 2, 4, 8 ug solutions. Blank samples were also run. 44 Electron Microscopic Examination of Meat Spoilage Sample preparation and inoculation To avoid any possible changes as a result of grinding to the porcine muscle, a pork loin roast which had been previously cut without any special aseptic precautions and stored in freezer at -18°C for 6 weeks was used. The pork loin was allowed to thaw at 3°C for 24 hours before aseptically removing small muscle samples (approximately 1 cm in length and 2 to 4 mm in diameter) from the roast center using sterile knives and aseptic tech- nique. These small muscle samples were then distributed as follows: one piece into each of two vials (4.5 cm in length and 1.5 cm in diameter) containing sterile Koser's citrate medium to act as controls; one piece into each of two similar vials containing the enzyme fraction obtained from G-100 Sephadex K 25/45 column as the enzyme treated samples; finally one piece into each of two other vials containing Pseudomonas perolens cells (approximately 103 cells/m1) to act as bacterial treated samples. The total of 6 vials were sealed and stored at 3°C until required for sampling to determine ultrastructural changes at day 4 and day 8. Sample fixation and embedding At day 4 and 8, one vial of each treatment and a control were re— moved and samples taken for fixation and embedding. A modification of the procedure described by Sjvstrand (1967) was used for tissue fixation. The small muscle tissues cut from the incubated samples were fixed inéi 45 1.25% gluteraldehyde solution, made up in sodium phosphate buffer as shown in Appendix A.3. The pH of the fixation solution was 7.4 and the osmolarity was 415 milliosmolar. Fixation was complete after two hours and the samples were then washed in a phosphate buffer (preparation shown in Appendix A.3) three times for 20 minutes each time. The pH of this washing buffer was 7.4 and the osmolarity was approximately 440 milliosmolar. The samples were transferred from the washing solution to a 2% osmium tetroxide solution in veronal acetate buffer, pH 7.4, and allowed to fix for 1 hour (see Appendix A.3. for fixative preparation). The samples were next dehydrated by placing them in a graded alcohol series (25%, 50%, 75%, 95% ethanol) for 10 minutes in each. Further dehy- dration was carried out in absolute (100%) ethanol for two 15 minute periods. The dehydrated samples were placed in propylene oxide for two 30 minute periods prior to the final transfer into a 1:1 mixture of propy- lene oxide and epon for 12 hours. This final step was carried out in a vacuum desiccator. All transfers were at room temperature. After the tissues were removed from the propylene oxide-epon mixture, they were trimmed to approximately 1 mm in length and 0.5 mm in diameter, and then transferred to "00" gelatin capsules containing pure epon 812. (see Appendix A.3. for epon preparation). The gelatin capsules containing the samples were placed in a desiccator under slight vacuum at room temperature for 12 hours, prior to polymerization in a 60°C oven for 48 hours. The hardened epon blocks containing the muscle samples were stored under vacuum in a desiccator at room temperature until required for U88 0 46 Sectioning and staining The epon embedded tissue blocks were trimmed by hand using a razor blade prior to sectioning on glass knives to a thickness of 600 to 800 A using a LKB Ultratome l. The sections (grey to silver) were collected from the knife boat on 300 mesh uncoated copper grids. The tissue sections were stained by floating the grids on a 5% aqueous uranyl acetate solution for 30 minutes and rinsing with distilled water. Sections were then stained in lead citrate solution for 15 seconds (Spink, 1971, see also Appendix A.3). The stained sections were then washed with 0.02 N sodium hydroxide followed by distilled water and then dried. Photography of sections The dried grids were placed in an electron microscope (Philips EM—300) and observed at an accelerating voltage of 60 K.V. An average of 5 grids were sectioned and observed for each sample and representative photographs were taken of each on Kodak 3 1/4 x 4 inch electron microscope film (Estar Thick Base). The 3 1/4 x 4 inch negatives were printed on Ilford contin- uous tone 8 x 10 inch photographic paper utilizing a Durst S-45 EM enlarger on an Ilford 1501 rapid processor. The prints were fixed in a Kodak fixer, washed and finally dried on a ferrotype dryer. The magnification used in all micrographs was 16X as calibrated using the electron microscope. To overcome inherent discrepancies in the calibration system, the magnification on the final micrographs were cor- rected by making the A-bands equal in length in all cases. RESULTS AND DISCUSSION Preliminary Study on the Effects of Pseudomonas perolens ATCC 10757 on Muscle Tissue Bacterial growth 0f the three species of the genus Pseudomonas available, two of them, namely Pseudomonas fragi ATCC 4973 and Pseudomonas perolens ATCC 10757 grew readily at 10°C. The third, namely Pseudomonas aeruginosa ATCC 10145 grew more slowly, as shown in figure 1 (Appendix B.1). Pseu- domonas perolens ATCC 10757 was chosen for use in the preliminary study and all subsequent studies. The purpose of this preliminary study, which involved the growth of Pseudomonas perolens on aseptic porcine muscle stored at 10°C was to determine if any or all of the following occurred. Microbial growth Enzyme(s) production Primary protein changes pH changes Relationships among these above mentioned parameters The changes observed during the course of this experiment are summarized graphically in figure 2 (Appendix B.2). The bacterial numbers, as ex- pected, remained low initially, corresponding to the lag phase of growth. During this period the new cells produced were approximately equal to the number of the old cells dying off. The logarithmic phase of growth is 47 48 .Emu 00H um poxmcm mumuou m Cw UooH um .m.n ma .Eofiwma oumuuwo m.ummox Ca mmcoEowsmmm macaw mzu mo mmfiomom mouse mo Luzouu .H musmwm 2.: m6; oa om. om on o 1 Nd .. v.0 . nv nlmwnv nu .0 w .. md O U m 1 0.. o m < 1 N._ mqaoa UUH< omocwwnumm mmcoaovowmm n o mnmq 0084 ammuw mmaoaowsomm u m mmmoa 0084 mamaoumm mmoosovoomm a < 1 ¢._ 49 33 t. 2' '00 Bacterial Growth 8 .. 3’ 8.0 - (5 c b E O #3 6.0 - . C .Q a _ O -J 4.0 - 9'0 - pH Change I H Control a 7'0 _ -<>——o- Inoculated 5.0L E 0.5}. Enzyme Production (3 - 8 H Control 0' 0'3 b -°——°- Inoculated C5 .. <3 O.| - s o W I ' ; U I I I : fl? 1 I #41— 0 3 6 9 :2 :5 l8 2: Storage Time (days) Figure 2. The action of Pseudomonas perolens ATCC 10757 on porcine muscle during a 20 day storage period at 10°C. 50 next during which a linear increase in cell numbers occurs with time. A maximum of 109 cells per gram was reached at day 14 of storage. Tarrant 25 El- (1971) in a similar type of experiment using Pseudomonas fragi re- ported a maximum bacterial count at between 6 and 8 days. This difference can be explained by the fact that the zero day Pseudomonas fragi counts were 105 per gram, as compared to the zero day counts of 103 per gram of porcine muscle in the case of Pseudomonas perolens. Ockerman g£_§1, (1969) and Borton gt 51. (1970) are among others who have reported on the growth of bacteria in bovine and porcine muscle respectively. pH The initial pH value of the aseptic control and the inoculated samples was 5.3 (figure 2, Appendix B.2) and this remained constant during the 20 day storage period. The pH of the inoculated samples remained con- stant at 5.3 until the 10th day and then gradually increased to pH 8.2 at day 19. These results were in general agreement with the results of Borton 25 El: (1970) and Tarrant g£_§l, (1971) using Pseudomonas fragi on porcine muscle and those of Ockerman g£_21, (1969) with a general type inoculum and a species of each of the genera Pseudomonas and Achro- mobacter on beef muscle. Thus, species of the genus Pseudomonas generally appear to grow well at 10°C with resultant increases in pH values in porcine and beef muscle during storage at low temperatures. Tarrant 35 .51. (1971) reported that the increase in pH was due to the production of ammonia. Ammonia was detected by odor evaluation during the course of our experiment . 51 Enzyme production Enzyme production was first detected on the 11th day, coinciding with the initial increase in pH, which in turn occurred during the second half of the bacterial logarithmic phase (figure 2, Appendix B.2.). This is an important fact since it appears that cell numbers of 107 per gram of tissue or greater need to be present before any measurable changes occur. Hasegawa gt_al, (1970) and Tarrant 35 21° (1971) have indicated similar findings in their data. It should be noted that enzyme production in this section was measured by reading absorbance at 280 um and in the following section on Medium Development was determined using the method outlined by Lowry E£.§l' (1951) and reported as absorbance changes measured at 660 nm. Primary protein changes Microbial action on primary muscle proteins has been reported by many workers including Jay (1967), Lerke g£_313 (1967), Ockerman gt_al, (1969), Borton §£_§1, (1970), Hasegawa (1970), Rampton g£_§l, (1970) and Tarrant £5 21. (1971). Table 1 summarizes the bacteria used by these workers and the protein changes observed, if any. The changes in the primary protein solubilities observed in this pre- liminary experiment are summarized in Table 2. 52 uHoooa oaHouom mmmmmmmm when cm x mommauooa momouuoun monouuoou amused oz mmnoaovsomm UoOH an endow humoHaHHuum «Hanna uaHouom vommuuooH commouooH fume ow um undone oz .mmmmw when cm x UoOH .mhov w HoHanH onunu newness has no uooovH>o oz omooaovnumm AHNaHV uncanny oHomoa ocHouom venomous when a uuuwo when cu womoouooH vumuououn case p vumeouoaH mom mummw x .ooOH .ooN unmouonH HoHuHoH, endeavor HmHuHaH moooaovoomm nemmHv couuom . Hummnnann uHomna unHuuom I I undone 91 I Bonn when cm x can ounano motz. Acanv commas“ oHomna moon umuououv .voooouoow pauomnoaouno< when nm N can voououonH womanhoon Heaven» ounu mHHunmouw nonu was AmomHv ummouonH HoHuHcH_ hHHuHanH uouunooo monoaovnumm .mm:mm.nasuuxoo .mmcnodaoo uanua umHnouHoa_3oH mo ooHuoNHHHuo muuoonoH I .vuuuoooo umeHoom I noHuooum nHououn nod mzu mo aoHuHuvm no I .oonHoeo on nH voanmau uoHHomm monoaovoomm + noHuooum oHuuoum I oHomsa anm meme m x o. n lanemmV .Ho no uxuua .cho nuance HaHuouoon HoHanH :mH: nuHsfi uuuoonoaounod oHomna moon muvHuooHonn mam mvHoo onHam mo monks uonHonm Home was when NH x can use auHuonnv use nH monouuoom .ouo :uHa vumoouoon mmnoaovoomm AnoaHv mun .z.m.z maouum uoHHHunHmohz oHemoHnooumm «Huuuoom harem .mcoHuooum aHououn oHonna humaHue can no nOHuoo HanoHUHe couscous mo humseom .H «Hana 53 Table 2. Primary protein solubility changes due to the action of Pseudo- monas perolens ATCC 10757. % Protein 0 Day 18 Day Sarcoplasmic - Control 6.02% 4.1% - Inoculated 6.07% 3.3% Myofibrillar - Control 7.9% 10.7% - Inoculated 7.9% 13.0% Nonprotein nitrogen - Control 2.6% 3.06% - Inoculated 2.57% 4.09% The sarcoplasmic protein extracts of both the control and inoculated samples show decreases at the 18th day of storage, with a larger decrease in the bacterial treated sample. The observed decrease in sarcoplasmic solubility is in agreement with the results of other workers including Ockerman gt_al, (1969) and Borton g£_al, (1970), obtained during storage of beef and porcine muscle, respectively. The latter group, however, did find an increase in the sarcoplasmic protein extractability due to micro— bial action. Myofibrillar protein extractability of both control and inoculated samples were found to increase at day 18 (table 2); the increase in the inoculated sample being almost double that of the control sample. The increase in solubility in the control may be explained by the fact that during storage (as in aging) the more insoluble myofibrillar proteins become more soluble (Sharp, 1963). The larger myofibrillar solubility increases are in agreement with the findings of Ockerman e£_§13 (1969), 54 Borton 22 El: (1970) and Tarrant g£_§l, (1971). These workers, however, reported gradual decreases in myofibrillar extractability after the initial increase. These differences may be explained by the fact that different strains and species of Pseudomonas were used as well as different tech- niques and storage temperatures. 1kn1.protein nitrogen values for the control sample did increase slightly with storage time in comparison to the large increase obtained for the inoculated sample (table 2). These findings agree with those of other workers (Ockerman _e_t_:_ 91., 1969; Borton £31., 1970; Tarrant 9391., 1971). In summary, substantial increases in the myofibrillar and non protein nitrogen extracts of the inoculated samples over the corresponding control samples were found after 20 days of storage at 3°C. Small changes were observed on comparison of control and inoculated samples in sarco- plasmic protein solubilities. Based on these findings it was reasonable to conclude that the solubilization of the insoluble myofibrillar and/or stroma protein fraction by the action of Pseudomonas perolens resulted in the observed solubility increases. Ockerman g£_§1, (1969) has shown that significant decreases in the stroma fraction did occur on inoculation of beef muscle with a general type inoculum of Pseudomonas and Achromobacter and lesser decreases with inoculation of the individual genera. One important criticism of this experiment was the type of storage jar used and the amount of sample therein relative to the total space. The jars (7 cm long x 6 cm in diameter) were filled to approximately three- fourths of the total capacity, resulting in poor aerobic conditions 55 throughout the bulk of the sample and thus poor bacterial growth. A large surface to volume ratio is required for optimum bacterial growth, as species of the genus Pseudomonas are aerobic and grow rapidly only on the surface. It was observed, however, after 20 days that the inocu- lated samples had spoiled throughout the entire mass with a resultant gum like texture. This may indicate the release of enzymes by the surface bacteria, capable of diffusing inwards with time. The main objectives of this preliminary study were successfully accomplished in that enzyme production was detected. This was coincident with bacterial growth and pH changes and resulted in substantial primary protein solubility changes. Subsequent experiments designed to extract and purify the enzyme(s) detected in the spoiled porcine samples resulted in very low yields, possibly due to the rather long and tedious procedures involved. Based on these findings, the development of a synthetic low protein medium.which would support growth of Pseudomonas perolens and allow enzyme production was undertaken to facilitate the purification of the enzyme(s) in question. Development of a Medium for Growth and Enzyme Production with Pseudomonas perolens ATCC 10757 Growth on Koser's Citrate Medium Koser's citrate was selected as the basal medium with which to work. It had sodium ammonium phosphate as the sole nitrogen source and sodium citrate as the sole carbon source required for growth. This medium 56 supported growth of Pseudomonas perolens at 10°C and pH 7.5 as shown in figure 3 (Appendix B3). However, no enzyme production was detected. Medium for Enzyme Production The citrate in the Koser's citrate or basal medium was replaced by a variety of amino acids (Appendix B.4) which resulted in different de~ grees of bacterial growth. No enzyme production, however, was detected (figure 4). Similar results were obtained upon replacing the citrate in the basal medium with a variety of dipeptides as shown in figure 5 (Appendix B.5). Finally, the use of combinations of these dipeptides and amino acids to replace the citrate in the basal medium resulted in no enzyme production being detected. Different degrees of bacterial growth from the different combinations were obtained. The different degrees of bacterial growth observed in figures 4 and 5 can be explained by the fact that some of the amino acids or dipeptides used are not readily utilized by the bacterial cells orcthey may have a slight inhibitory effect on growth. The lack of enzyme production up to this point was thought to be due to the absence of some minerals in the growth medium. This was in- vestigated using a complex medium used by Tarrant 25 El. (1971) which contained 11 amino acids plus 2 dipeptides in place of citrate in Koser's citrate medium (Appendix 3.6). This was made up in ordinary tap water (well water) presumably containing minerals. A typical bacterial growth curve was obtained on incubation with Pseudomonas perolens as shown in l.2 l.O E C.’ o 0.0 (1') U.) c5 0' 0.6 <3 0.4 0.2 0 Figure 3. 57 A—Lag Phase 8 ~Logariihmic Phase C-Siaiionary Phase C: 'n. " J I l I I 20 4O 60 DO lOO IZO incubation Time (hrs) Growth cycle of Pseudomonas perolens ATCC 10757 in Koser's citrate medium, pH 7.5, at 10°C in a rotary shaker at 160 rpm. 58 L4 “- A I2 [- A - Arginine B = Glutamic acid C - Histidine [O .. D - Cysteine ' E - L-aspartic acid :8 . 0.8 C3 C5 <3 0.6 B \m .. 0.4 .- H E 0.2 ' . D a o A ' 20 4o 60 so IOO l20 Time (hr) Figure 4. Growth of Pseudomonas perolens ATCC 10757 in Koser's citrate medium, pH 7.5 at 10°C in a rotary shaker at 160 rpm with the citrate being replaced by amino acids. 59 .moowuoooHo an ooomHaou onon ououuHo ecu nuHa any ocH um uoxnnm mumuou m nH voOH um m.m ma aonua uumuuHo m.uumoM no nmnOH ooa< mouHouom monoaoooumm mo nuaouu .m unstm CE mEC. OWN om. o 1 _ , o Nd . V no .0 .0 % $6 0 U m m oaHohHmIg HhohHu I m I m 0 ucmeummmmIH HzohHo u a unHonoHIH HaomHu n o onHHo>IH HhohHo I m oanmHmIH HhomHo I < I.AV; 6O figure 6. Enzyme production was also detected at approximately 30 hours of growth. Calcium chloride was also added to a similar medium made up in deionized distilled water and inoculated and incubated in the usual manner. Enzyme production was again detected and results are shown in figure 6. A control medium similar to the previous two media in composition but with— out calcium chloride was made up with deionized distilled water and inocu- lated with Pseudomonas_perolens. The typical bacterial growth curve was again obtained (figure 6). However, no enzyme production was detected. The differences in lag times in figure 6 can be explained by slight differ- ences in the amount of inoculum used in each case. From these results one may conclude that the basal medium with citrate replaced by a complex amino acid plus dipeptide mixture and with deionized distilled water as solvent did not support the production of enzyme(s). Both tap (or well) water and deionized water with calcium chloride added were conducive to the production of enzymes. Consequently, calcium chloride appeared to be important for enzyme production under the above conditions. In an attempt to determine the importance of the amino acids and dipeptides in the basal medium for enzyme production a series of different media was set up using deionized distilled water as solvent. Koser's citrate or basal medium, Koser's citrate with citrate replaced by amino acids, dipeptides and combinations of these together with 0.5 g of calcium chloride added were tested for ability to induce enzyme production (Appendix B.7). Figure 7 shows that the basal medium, with the citrate replaced by a complex mixture of amino acids and dipeptides plus 0.5 of calcium chloride per liter, again induced 61 .muco>Hom unopaMMHo cH mu>HommHv monuaonHo mam mvwom oCHam wo uuouxHa meoaoo m an omomHmuu maHon uumuuHo unu nuH3 Emu ooH um uuxmnm mumuou m aH oooH um .m.n ma .asHoma oumuuHo m.uomoM cH oOHuonoouo weaned one nmNOH UUH¢ mcoHopmm,mmaoaoosmmm mo nu3ouu E V as _ a. on ow ow ow om om o. o 0 w...- HHIIII-m-HI-II ..-. .I. uw-H-Ih n-II-n-II. .IMIIIIII-m-II .- \\.\ mil-l- m 9\ ooHHOHno anHono Cw To + amass 8:336 ooNHaoHuo co nOHuonooua ushuao n m sauna emu co aoHuooooud ushncu a a sauna ooHHHumHo quHooHoo u o uoHuoHso aoHono .. H\w m.o + amuse ouHHHumHo ouNHaono u m Iwmumu sauna emu 1 .. 9.0 .. md .. 0.. < m cozoauoca mEEm I--- O chafioeo .o oustm [)‘}lllllilllll ll lllllllllllll III 62 enzyme production. The basal medium plus calcium chloride also induced greater enzyme production. The basal medium with citrate replaced by amino acids or dipeptides did not induce enzyme production even in the presence of calcium chloride, However, this may be explained by the fact that much lower bacterial growth.was noted in both these cases. In summary then, it did seem that calcium chloride was necessary for enzyme production by Pseudomonas perolens ATCC 10757 in synthetic media. Enzyme production was induced by the nonprotein containing Koser's citrate or basal medium with calcium chloride added in the same amounts or greater in comparison to the basal medium with citrate replaced by the complex amino acid and dipeptide mixture used by Tarrant 35:31, (in press). Other reports indicating the necessity of calcium ions in synthetic media for the production of extra- cellular proteases by various bacterial strains have been published by Merrill and Clark (1928), Wilson (1930), Gorini (1950) and Morihara (1956). Role of Calcium Chloride While one can only speculate as to the exact role of calcium chloride in the mechanism of enzyme production, it did seem that the calcium ions were absorbed by the bacterial cell to activate one or more of the enzyme precursor steps which are involved. Morihara (1960) has indicated that the amount of protein present in the supernatant of a centrifuged cell suspension containing no calcium is approximately 20% of that amount of protein found in a similar suspension having calcium in the original growth medium. A similar type experiment carried out during this study indicated similar results. However, this line of study was not pursued further. l.2 LO A 0.0. 660nm 0 .0 as on .0 A Figure 7. 63 —-—-—- Growth ----- Enzyme Production 20 4O 60 Time (hr) Growth of Pseudomonas perolens ATCC 10757 in Koser's citrate medium with and without citrate being replaced by other carbon sources. Calcium chloride (0.5 g/l was added in each case, pH was 7.5 at a temperature of 10°C. Growth; A = Koser's citrate; B = Amino acids; C = Dipeptides; D - Amino acids + dipeptides; Enzyme production in E = Koser's citrate and F = Enzyme production on amino acids + dipeptides. 64 To determine the effect of calcium chloride concentration on bacter- ial growth and enzyme production, concentrations ranging from 0.1 g. to 1.0 g of calcium chloride per liter in the basal medium were tested with two psychrophiles, namely, Pseudomonas perolens and Pseudomonas fragi. Therrelationship between bacterial growth and enzyme production at various concentrations of calcium chloride is illustrated in figure 8 (Appendix 3.8) for Pseudomonas perolens and figure 9 (Appendix B.9) for Pseudomonas fragi. Increases in concentration of calcium chloride have been shown to depress growth of Pseudomonas frag_. However, the same trend does not appear for Pseudomonas perolens. Enzyme production was also affected by increasing concentrations of calcium chloride for both bacterial strains, but optimum levels were noted in each case, as shown in figure 10 (Appendix B.10). Pseudomonas perolens had optimum enzyme production at 0.5 g of calcium chloride per liter of basal medium, whereas Pseudomonas fragi_had optimum enzyme production at 0.2 g of calcium chloride per liter of basal medium. This indicated that the different strains of Pseudomonas have different requirements of calcium chloride for optimum enzyme production. The medium developed at this point in the study, namely Koser's citrate plus 0.5 g of calcium chloride dissolved in 1 liter of deionized distilled water was chosen for use in the production of enzyme for all sub- sequent experiments. A typical relationship between the growth of Pseudomonas perolens and enzyme production in the developed medium is shown in figure 11 (Appendix B.11). Enzyme production was first detected at 47 hours of incubation time which corresponded to the latter portion of 65 .ooOH um .m.n mm .moovm moHuOHno uumuuHo m.uowoM 6H nOHuonvoum ushnno one nmNOH UUH< mcoHonm.mmooaooaumm mo nuaouu E: as; 2w 2» is o H I m m o I u m o .. a O nOHuoovoum sauna .H\w «.0 I m d. . . is o; u n - is To a o is no u m 0 . is so .- a. m cozoauota osxscm II... 5320 III .m ouome O N6 0.— 66 . .H\w o.H u m was H\w m.o u o "H\m ~.o I m “H\m H.o I m "aoHuoovoue ushunm H\w o.H I a "H\w m.o I o .H\w ~.o I m mH\w H.o I < ususouw .ooOH um .n.n mm .mooom uoHuOHno asHono mo ncoHumuuaoonoo wcHhum> £uH3 ESHvoa auouuwo m.uomoM oH coHuosvoun demand one mhmq uoH< Huaum mmaoaomoomm mo nubouo c5 .2: om om ov ON 0 I-IIII-lllllq 1 u IIHIHII\m-J\ \\ a ‘l . mfl-uuuu-I-I- III:- Inn-IX.- 0'llllllllbu|t\\b\\ \\\ ’ll'l‘lll Illa-I \ a .- -.- I No I. v.0 o .. $6 0 I md m_. I 0.. <. cozoanocn. 9535 I-..- .. N. 530.5 .m oustm WU099 'CI'O V . I II .I .l‘ l [\IIIIJI" ‘ .[ l I 1 I A . . 4 4 0.3 .0 N A 0.0. 660 nm .0 0 Figure 10. 67 0.25 0.50 0.75 LOO COCIZ ( g/ I) Effect of calcium chloride concentration on enzyme production by Pseudomonas perolens ATCC 10757 (B), and Pseudomonas fragi ATCC 4973 (A) grown on Koser's citrate medium, pH 7.5, at 10°C for 55 hours. 68 .o.oa so .m.s ma .oeauoaau assuamu axe m.o mafia Samoa mum-3H0 m.uumoM cH aoHuooooua cause one 3.3... 0094 maoHouum monoaovaomm mo 1,339.5 .HH ouomHm 25 95... o - Nd .. . V no 0 .O . w 1 mo 0 U m. .. md PI 0 cozoaooi oechm III-o.- .. 0.. 3259.0 IQIIIOI 69 the logarithmic growth phase. These data compared favorably to the data obtained for bacterial growth and enzyme production on porcine muscle in a preliminary study (see figure 2). In addition, the fact that enzyme production began to fall off rapidly as the bacterial cells enter the stationary phase of growth (at which time cell autolysis would be greatest resulting in liberation of the intracellular enzymes from these cells into the medium) indicated that the enzyme(s) being detected may well be truly extracellular. Effect of Foaming and Antifoaming In all the experiments up to this point, a small scale bacterial growth system of 150 ml medium per 500 ml Erlenmeyer flask was used. In an attempt to use larger volumes of medium for bacterial growth and thus increased enzyme production, a larger scale bacterial growth system of 500 ml medium per 2 liter Erlenmeyer flask was used. This system, however, resulted in large amounts of foaming with reduced enzyme production per unit volume. (The change in optical density at 660 nm, using the Lowry method for determining enzyme activity was 0.06 per 1 m1 of enzyme solu- tion in the large scale system as compared to 0.3 using the small original system of 150 ml medium per 500 ml Erlenmeyer flask). A third system using 300 ml medium per 2 liter Erlenmeyer flask was investigated for the effect of foaming and the action of added antifoam, namely silicone. Bacterial growth and enzyme production using this system with and without added silicone, was compared to growth and enzyme production using the small 70 scale system (150 m1 medium/500 m1 Erlenmeyer flask). The results are illustrated in figure 12 (Appendix B.12). Faster generation times were found with the presence of antifoam, although final cell yields were lower. However, very low enzyme production resulted when the antifoam was present in the medium using the two liter flasks. Without antifoam, enzyme pro- duction in these flasks was approximately 25% of the amount normally pro- duced in the smaller flasks. It was concluded that the addition of anti- foam does reduce enzyme production by almost 100% and that foaming reduces enzyme production and cell yields. All future experiments were performed using enzyme(s) produced on the original small scale system of 150 ml medium per 500 m1 Erlenmeyer flask. It is conceivable that large scale production could be carried out using an automatic fermentor (New Brunswick), having either built in agitators or air flow systems to facilitate aeration without causing excessive foaming. Replacement of Calcium Chloride by Other Metal Salts Various metal salts were used to replace calcium chloride in the developed medium as possible inducers for enzyme production. A summary of the results obtained is shown in table 3. Koser's citrate medium and Koser's citrate medium with calcium chloride, sodium chloride, barium chloride individually added, supported good bacterial growth. Slow bac- terial growth was noted using Koser's citrate medium with either zinc chloride or manganese sulfate, while cobaltous and cuprous chloride each inhibited any bacterial growth up to 90 hours of incubation, at which time the experiment was terminated. Bacterial growth.was measured as change in 71 .oooH um .m.n ma .uoHuoHno EsHono mo H\w m.o moHa EsHoma oumuuHo m.uumox cH nmnoH UUH<.mawHouwm wmcoaoooomm an coHuonoouQ uehsou one £u3ouw co unomm EmOmHucm cm mom wcHamow mo uoowwm .NH ouanm :5 .2: om Om 0v ow * q d H m J map: a...- I”"” \\\ IIIIQ\ amowwucm + xmmHm H ~\HS omm 3mmHm H N\HE omm m xmmHm HE oom\Ha omH a "aoHuosooun magnum m . m smeaaucm + amass H ~\Ha omm u m swash H «\Ha omm u o amuse Ha oom\se owe a a "nusouu 5.832.. 253.5 I..- - c..,ocmV .IIIII AV nu. 72 Table 3. Effect of calcium replacements in the developed medium on growth of Pseudomonas perolens and enzyme production. Bacterial Enzyme Basal medium Salts (0.5,g/l) (growth 4production Koser's citrate None Positive Negative Koser's citrate Calcium chloride Positive Positive Koser's citrate Sodium chloride Positive Negative Koser's citrate Barium chloride Positive Negative Koser's citrate Zinc chloride Positive slow Negative Koser's citrate Cobaltous chloride Negative Negative Koser's cutrate Cuprous chloride Negative Negative Koser's citrate Manganese sulfate Positive slow Negative optical density at 660 nm. A green pigmented appearance which was always observed during growth of Pseudomonas perolens in the presence of calcium chloride in the basal medium was seen only in the presence of one of the other salts during bacterialpgrowth, namely, barium chloride. Enzyme production was detected only in the presence of calcium chloride and was measured using the Lowry method and reported as the change in absorbance at 660 nm. Based on these observations, there may be some association be- tween enzyme production and pigmentation. However, subsequent purifica- tion studies did show that removal of the green pigment from the enzyme solution resulted in an increase in enzyme specific activity rather than a decrease. Enzyme Unit Definitions Enzyme Activity Enzyme activity is the change in absorbance measured at 660 nm (A O.D. 660 nm) using the method of Lowry g£_al, (1951), due to 0.25 ml of the 73 enzyme solution at 35°C for 15 minutes using a 2% w/v casein solution as substrate. One unit is expressed as the number of ug of tyrosine equivalents released per 1 ml of enzyme solution per minute at 35°C using a 2% w/v casein solution. Specific Activity One unit is expressed as the number of units of enzyme activity per ug of protein. Crude Enzyme Parameters Temperature of Storage The effects of storage temperatures over a 25 day period are shown in figure 13 (Appendix B.13). Storage at room temperature (26°C) resulted in a rapid loss of initial activity to approximately 7% remaining after 64 hours of storage. Freezer storage temperatures of -23°C and -29°C re- sulted in small decreasesin the initial activity to approximately 83% and 93% remaining, respectively, after 25 days of storage. It may be that the initial freezing effect and/or thawing effect prior to assaying resulted in this decrease in activity. Storage conditions at 3°C were most suitable for retention of enzyme activity, 100% of the initial activity remaining even after 25 days of storage. The first two values for percent initial activity remaining on the 3°C curve (figure 13) were low due to improper preheating conditions prior to assaying for activity. 74 .mousumuoasuu aonOum moOHum> um :OHuomuu cameos moons ago mo muHHHnmum $.83 mgr”...- emu-.05 mm Hum ON m. N. m d 4 Uole I Q UoMNI I U 1 com I m UOON I 4 ON 0..» C) (O C) 00 00. no shaman 38 AHMPV lolllul °/o bUIquuI 75 The effect of a fast freeze (acetone and dry ice mixture at -80°C) as compared to a slower freeze (at freezer temperatures of -23°C and -29°C) on the enzyme(s) in solution did not result in any major differ- ences in percent remaining activity during the storage period. A fast thaw at room temperature (30 minutes at 26°C) in comparison to a slow thaw at 3°C overnight had no effect on the percent remaining activity. Temperature of Assay The optimum assay temperature for enzyme activity was found to be 35°C as shown in figure 14 (Appendix B.14) which also shows the typical bell-shaped curve. The gradual increase in enzyme activity as a result of increases in temperatures from 15°C to 35°C can be explained by the increases in reaction rates. The rapid decrease in activity at tempera- tures above 35°C was thought to be due to enzyme denaturation. Thus, an assay temperature of 35°C was selected for all subsequent assays. Assay Time Enzyme activity, as measured by pg of tyrosine equivalents released was linear with time up to 20 minutes and thereafter gradually decreased up to 90 minutes, the time of final measurements (figure 15, Appendix B.15). A zero order reaction, independent of substrate concentration was indi- cated up to 20 minutes of reaction time, with a resultant rate constant (k) of 6.25 ug tyrosine equivalents per minute. The decrease in reaction rate at longer times may be due to a number of factors including a lower- ing of substrate concentration and/or inhibition by the end products of the reaction. 76 A>H=vo onHmouzy wnv .muH>Huom messes co assumpudaau momma mo uoowwm 80. 228359.. on mu mm mm m. H H 1 H fiéln 00 "7 q- .QH uuome KIIAIIOV euMzug 77 400 - 300 L- 200 - Enzyme Activity lOO I.--¢---———-_— . so so Assay Time (min) 1 40 no C3 0 Figure 15. (Effect of assay time on enzyme activity (ug Tyrosine equiv) at the optimum assay temperature of 35°C. iOO 78 pH A series of five buffer systems, namely citrate-phosphate, phosphate, tris (hydroxymethyl) aminomethane, barbital and carbonate-bicarbonate was used to give a combined range of pH from 5.0 to 10.0, with a suitable degree of overlap. Figure 16 (Appendix B.16) illustrates that the enzyme solution is stable over a broad range of pH values from 6 to 10 with maximum stability between pH 7.0 and 9.0. The observed differences in enzyme activity between buffer systems at any particular pH value may be explained by the fact that those buffers resulting in low activity have inhibitory effects. For example, phosphate and tris buffer systems at pH 7.5 resulted in lower enzyme activity values in comparison to barbital. It should also be mentioned that the molarities of the buffer systems differed; the phosphate buffer was 0.1M whereas tris, barbital and bicar- bonate were 0.05M. A subsequent experiment on the effect of pH on the partially purified enzyme solution using a combination phosphoric acid, acetic acid and boric acid buffer (CochFrugoni, 1957) also indicated the optimum pH range to be between 6.5 and 9.0 (figure 17, Appendix B.17). Effect of Substrate Concentration As expected, increasing the substrate concentration resulted in corresponding increases in enzyme activity up to a certain substrate con- centration. Figure 18 (Appendix B.18) illustrates the effect of substrate concentration on enzyme activity. A 1 m1 aliquot (5.0 mg/ml) of casein 79 .7 A = Citrate phosphate B - Phosphate C = Tris D = Barbital _ E = Carbonate-bicarbonate 6 /HD x x o rt" ‘3 0 <1 a: f" Er: .. r; o~ I: LU ii 2L— E | L O A L‘ I .1- J l "I __.1_ b 6 f 8 9 [0 ll pH Figure 16. Effect of pH, using various buffer systems on enzyme activity. (ug Tyrosine equiv) 80 .o.m Ou o.m Scum swamp ma nuHB oHom oHuonIoHum oHuoom IoHom oHeondmozmv eaummm summon coHumcHaaoo o wcho muH>Huom saunas do me no uoomwm In. Om Qm OH- o.m On 0% l l l O O o “3 tr cu I 0 co EUIUIDwea MMHGV IDIIIUI °/. .NH shaman 81 A>Hovo achopme wnv .muH>Huom ushuco no oumuumnom mm .aOHumuuaooaoo cHummo mo uoomwm .wa shaman 2.595 22.3% ON m. N. m v o N 3 U v n w a V m m.- w .m- w I I o_ 82 solution, pH 7.5, when reacted with 1 ml of the crude enzyme (specific activity of 0.05) gave maximum enzyme activity. Casein concentrations greater than 5.0 mg/ml resulted in a slight decrease in activity. This slight inhibitory effect at substrate concentrations greater than 5.0 mg/ml in comparison to a 2% substrate concentration (20 mg/ml) used in all other experiments did not warrant a lowering of the concentration to 5.0 mg/ml in the subsequent studies. Effect of Enzyme Concentration Figure 19 (Appendix B.19) illustrates the effect of increasing the enzyme concentration on the rate of enzyme activity (pg tyrosine equiva- lents released per m1 of enzyme per minute at 35°C). The data indicated that crude enzyme concentrations of up to 80 ug protein per ml resulted in a linear increase in the rate of enzyme activity. Beyond this con- centration, there was a slight decrease in the activity rate, possibly due to some inhibitory effects from the reaction end products. The curve showing the effect of enzyme concentration on the rate of activity did not go through the origin, but cut the abscissa which may be due to the impure enzyme solution used. Subsequent studies using the partially purified enzyme fraction, obtained from a G-100 Sephadex K 25/45 column also resulted in a linear increase in enzyme activity with in- creasing concentrations of up to 12.5 ug/ml of enzyme solution (figure 19, Appendix B.19). This curve intersected the origin and may indicate a degree of purity in comparison to the crude enzyme, also shown in figure 19. 83 :63... 952m oo. om om 9. o N ¢ w m nsoHoo soomsmow OOHIU aoum aOHuomuu saunas oonHusm I m Av— coHuoHoo ushnao ovouo I d N. I V. .A>Hovo uaHmouma wnv >uH>Huom sexuao no aHa\cHououe wnv COHumuucoocoo saunas mo uoomwm .mH ouome KIIAIIOV suMzug 84 Substrate Specificity As might be expected, the crude enzyme(s) had different initial reaction rates on incubating with the various substrates as shown in figure 20 (Appendix B.20). Casein which was the substrate used through- out the study elicited a linear response up to 20 minutes after initia- tion of the reaction. Casein did not, however, produce the fastest initial reaction rate. Both collagen and the salt soluble proteins of bovine muscle resulted in the highest initial reaction rates with egg albumin and the water soluble proteins of bovine muscle giving the lowest. It seems unlikely that these low rates were due to lack of substrate, but probably were the result of the inability of the enzyme(s) to act on these substrates or a low content of tyrosine and tryptophan. The action of the enzyme(s) on collagen was most interesting and some work on the re- lease of hydroxyproline was carried out later in this study using the partially purified enzyme fraction, obtained from the G-100 Sephadex K 25/45 column. Purification of the Enzyme Fraction Ultrafiltration The first stage in the purification procedure involved the use of ultrafiltration membranes. Of the three types of membranes used (PM-10, UM-lO and UMZ), PM-lO, with an approximate exclusion limit of 30,000 M.W. allowed the enzyme(s) to pass through and retained the larger molecules such as bacterial cells. Data listed in table 4 show an increase 85 .moumuumnsm :wououe msowum> so A>w=vm oawmouha may >ufi>fiuom oahuam .ON «woman 3E me: m w. m N _ O u u a u q .1. . d 400 p. «o ON O ‘1‘ l O (D o co Mgnuov auMzug maaououa umaaaunwmohz msaououe oaammaeooumm u oaaanam wwm u nomeHoo cfiommo a» .0 E .0 >‘ _ z 3 20 0.2 m UU 0) CD :3 :5 - 3 I0 L- J 0.: 5 - O l L l J l l 00 70 75 80 85 9O 95 Eluate Fraction (ml) Figure 21. The relationship between enzyme activity (A) (pg Tyrosine equiv) and absorbance at 216 nm (B) of the eluate fractions obtained from a G-75 Sephadex K 25/45 column. 90 I50 - IZO — . I2 .55 ' l1) .2! O a: 90 ~ 8 J 0.8 34‘ l> .EE ,0 g 06 '0 I- i - m 60 g E. o E‘ " 23 “J a - 0.4 30 - ~ 0.2 0 + . J , " L 0.0 60 72 84 96 I08 I20 Eluate Fraction (ml) Figure 22. The relationship between enzyme activity (A) (ug Tyrosine equiv), absorbance at 216 nm (B) and protein concentration (C) of the eluate fractions obtained from G-lOO Sephadex K 25/45 column. 91 due to an overlapping with the protein fractions detected before and after the enzyme fraction was eluted from the column. To overcome this slight protein overlap, a K 15/90 column was packed with G-lOO Sephadex and the UM-lO concentrate eluted through. Similar results to those shown in figure 22 were obtained, specific activity values being listed in table 5. Table 5. Specific activity values for the enzyme containing eluate fractions, obtained from the K 25/45 and K 15/90 columns. Eluate Specific Eluate Specific Column fraction activity Column fraction activity, 72 - 77 1.17 60 - 65 1.28 78 - 83 1.84 66 - 71 1.48 K 25/45 84 - 89 1.79 K 15/90 72 - 77 1.56 90 - 95 1.57 78 - 83 1.03 95 - 101 1.34 Based on these results, it seemed that the eluate fractions with similar specific activity values were indeed pure. Disc Gel Electrophoresis Disc gel electr0phoresis was performed on the enzyme fractions cor- responding to the various stages in the purification procedure. In the original crude enzyme solution, 15 different bands were visible as shown in gel A, figure 23. Gel B shows the UM-Z concentrate. The enzyme fractions obtained from the K 25/45 and the K 15/90 G-lOO Sephadex columns each resulted in three bands as shown in gels C and D, respect- ively. Finally, the eluate fractions 60-65 and 66-71, obtained from the K 15/90 column each appear as single bands in gels E and F. Figure 23. 92 ‘ 3‘ .’ WWW‘ MW‘E-mfivm Disc gel electrophoresis of the enzyme fractions obtained during the various purification steps. A = Crude enzyme solution B - UM-2 concentrate C K 25/45 enzyme fraction D K 15/90 enzyme fraction E a K/90 eluate fraction 60—65 F a K/90 eluate fraction 66-71 93 Based on the single bands obtained in gels E and F (figure 23), in addition to the results contained in figure 22, illustrating the coinci- dent nature of the parameters, namely, enzyme activity, protein concen- tration and absorbance at 216 nm and the specific activity values listed in table 5, it may be concluded that the enzyme fraction eluted from either the G-lOO Sephadex K 25/45 and K 15/90 columns was a pure enzyme, particularly those eluate fractions with similar specific activity values. A summary of the data obtained during the purification of the protease produced by Pseudomonas perolens ATCC 10757 is contained in table 6. Table 6. Purification data for protease from Pseudomonas perolens. Specific Total Total Total activity volume protein activity (units/pg Recovery Purification Treatment (ml) (ug) (units); 4prot.) 2 fold Crude enzyme 3,880 380,000 27,056 0.0712 100 1 UM-Z filtrate 3,770 113,100 2,614 0.0231 9.8 0.32 UM-2 concentrate 30 141,000 7,240 0.0513 26.7 0.72 Sephadex G-100 49 2,208 3,270 1.48 12.1 20.8 (K 25/45) A 20 fold purification was obtained overall, with a final yield of approximately 122. The greatest loss in yield resulted from the use of the UMrZ membrane during ultrafiltration. 94 Effect of Inhibitors and/or Activators A short study on the effect of inhibitors and/or activators on the purified enzyme fraction obtained from the G-lOO Sephadex K 25/45 column was carried out. A summary of the results is shown in table 7. These data indicated that none of the substances used had any inhibitory effect on the enzyme fraction. However, some slight activation resulted from the cysteine solution. Morihara (1957a) also showed this activation effect of cysteine. Table 7. Effects of inhibitors and/or activators on enzyme activity. Inhibitors or activators Molarity Z Remaining activity Cysteine 10-3 M 1122 Sodium bisulfite 10"3 M 1052 Iodoacetic acid 10"3 M 1002 Phenyl methyl sulfonyl fluoride 10‘3 M 100% water -- 100% The Chelating agent ethylenediaminetetraacetic acid (EDTA) has been employed as a prototype for the mode of action of this group of agents. Failure to inhibit an enzyme with EDTA is frequently interpreted as an indication of the absence of a metal in an enzyme (Vallee and Wacker, 1970). Once a mixed metalloenzyme-inhibitor complex has formed, a criti- cal excess of the metal ions should reverse the inhibition by competing with the metalloenzyme for the inhibitor bound to its metal. The data . obtained from the addition of EDTA and the readdition of calcium chloride 95 and their effects on enzyme activity are listed in table 8. Increasing concentrations of EDTA resulted in corresponding decreases in the Z 4 M EDTA. initial activity remaining to ‘ 50% . on addition of 10- On addition of 10-2 M calcium chloride to the EDTA treated samples, re- activation of the enzyme was observed. However, it varied from 752 to 96% reactivation depending on the EDTA concentration used (table 8). This may have been due to insufficient calcium ions available for com- plete reactivation. Table 8. Inhibition and reactivation of the enzyme fraction using EDTA and calcium chloride, respectively. 2 Initial activity 2 Reactivation EDTA Conc. remaining with CaC12_ 10-2 M o -- 10-3 M 39.6 74.9 10-4 M 50.1 81.5 10'5 M 92.6 96.0 10-6 M 100.0 -- It may be concluded that the enzyme produced by Pseudomonas perolens ATCC 10757 is a metal containing enzyme and this metal ion appears to be calcium. The inhibitory effect of EDTA appears to be competitive in nature. Hydroxyproline Released by Enzyme Action on Collagen To determine whether or not the enzyme activity observed using collagen as substrate (figure 20) resulted in the release of free hydroxy- proline, the enzyme fraction obtained from the G-lOO Sephadex K 25/45 was 96 reacted with collagen. No free hydroxyproline was detected in the filtrate obtained from the trichloroacetic acid precipitation following the reaction. In a similar type experiment with hydrolysis of the fil- trate prior to hydroxyproline determination, there appeared to be a definite release of free hydroxyproline between three and ten minutes of incubation time as illustrated in figure 24 (Appendix 323). It can be concluded that collagen was broken down by the enzyme fraction. Howe ever, it did not result in the release of free hydroxyproline as single amino acids. On precipitation of the remaining collagen and other proteins present to terminate the reaction and on hydrolysis of the resultant filtrate, free hydroxyproline was detected. Enzymes are specific for certain amino acids or side chains containing these specific amino acids, which are released due to enzyme action on a protein mole- cule. It is possible that the enzyme produced by Pseudomonas perolens ATCC 10757 did not result in the release of hydroxyproline as single amino acids but rather contained in formed peptides or polypeptides. Hagihara (1960) stressed the fact that in contrast to animal enzymes, all well characterized microbial proteases had very wide ranges of side chain specificity. Some may hydrolyze more than 802 of peptide bonds in pro- teins. Tenderization Effect of the Crude Enzyme Fraction The preliminary study (see table 2) involving the growth of Pseu- domonas perolens ATCC 10757 on sterile ground porcine muscle indicated Reiecsed Hydroxyproiine F9 Figure 24. 97 6»- 5» 4.. 3L. 0 2- o o I _ o O 2 4 6 8 IO Time (min) Hydroxyproline released as measured after hydrolysis of the trichloroacetic acid soluble enzyme treated collagen, pH 7.5 and 35°C. 98 that primary protein changes did occur. Based on these observations, it was decided to determine the tenderizing effect, if any, of the crude enzyme fraction on a large portion of meat. The steaks which were cut from the injected eye of the round meat portions and cooked to an internal temperature of 155°F in a 250°F heated oven showed approximately 7.5% increase in tenderness over corresponding controls as measured by the Werner-Bratzler shear device (table 9). The remaining roast portions, however, showed variable results depending on the temperatures used. The lower oven temperature of 200°F and final internal temperature of 153°F resulted in a 302 increase in tenderness in comparison to the higher oven temperature of 350°F and final internal temperature of 159°F which resulted in no increase in tenderness. However, in this latter case the final internal temperature of the control portion was 163°F or 4°F higher than the corresponding treatment. Table 9. Tenderization effect of the crude enzyme fraction. Oven Internal 9% Increase in Sample temperature temperature tenderness Steak (1) 250°F 155°F 7.5 (11) 250°F 155°F 7.7 Roasts (1) 200°F 153°F 30.6 (11) 200°F 153°F 25.8 Roasts (1) 350°F 159°F None (11) 350°F 159°F None The crude enzyme fraction appeared to bring about an increase in tenderness. However, an odor described as a distinctly bad potato type odor was easily detected from the enzyme treated samples. Whether or not this odor was due to the enzyme fraction or due to other compounds 99 present in the crude enzyme solution was not determined. The data listed in table 9 also indicated that the oven temperatures and/or the internal meat temperature is a major factor in this tenderizing effect. The 200°F heated oven and the 153°F final internal temperature resulted in a 302 increase in tenderness in comparison to the 350°F heated oven and 159°F final internal temperature. These results are in general agreement with other published reports on the effects of plant enzymes on meat tenderness. Reed (1966), reviewb ing some of the earlier reports using the plant enzyme papain, indicated that tenderness was significantly increased when steaks were treated with this enzyme, but roasts were not significantly tenderized. This may have been due to the fact that the enzyme preparation was not injected into the roasts and thus poor penetration resulted. It has been shown that penetration bYaSUCh an enzyme solution was no greater than 5 mm from the surface even after a precooking storage period. Tappel at El: (1956) reported very low penetration of papain into meat held at room tempera- ture prior to cooking. They also stated that temperature of cooking is important, maximum tenderization being obtained at internal meat tempera- tures corresponding to the optimum temperature required for maximum enzyme activity. Moreau and Jankus (1963) using papain as the tenderizing agent in roasts reported significant increases in tenderness at 158°F internal temperature after 2 hours. Therefore, it appears that the internal meat temperature during cooking is most important when using enzyme tenderizing agents. Internal 100 meat temperatures of approximately 100°F (37°C) would be most suitable for maximum tenderizing effects using the enzyme obtained from Pseudomonas perolens ATCC 10757. However, the potato like odor which the crude enzyme fraction emitted leaves in question the suitability of this enzyme for tenderizing purposes. The purified enzyme solution did not have this odor, although it was not tested on meat during this study to determine if it produced the undesirable potato like odor during cooking. Muscle Protein Solubility Changes Due to Enzyme Action This series of experiments was carried out in conjunction with the study on ultrastructural changes in muscle fibers due to the K 25/45 Sephadex G-100 purified enzyme fraction during storage at 3°C. Sarcoplasmic Proteins During a 25 day storage period, the sarcoplasmic protein fraction from the control sample was found to decrease slightly at day 4 and remained constant for the remainder of the period as illustrated in figure 25 (Appendix B.24). These results agree with those reported by Sayre and Briskey (1963), McLoughlin (1963), and Borton g£_§1, (1970) for porcine muscle; G011 £5 31' (1964) and Aberle and Merkel (1966) for bovine muscle. The enzyme treated sample, however, showed a rapid increase of up to 30% in sarcoplasmic protein solubility at 4 days of storage and over 502 in- crease at day 8 and decreased to a constant level at day 12. A comparison with similar type enzyme treated samples 80 60 00 C) O O) 0 mg Protein lg Muscle Sample Figure 101 h n : fl ’4. I \ I” p \ ” ’I \ at I- \ / \ -o— z’ \I--"""" “‘--o’ .—-"' no I- .o’.’” a- ‘- +----*"‘- ‘. L ',a---- ’v I . o Myofibrillar Protein 0 Sarcoplasmic Protein 0 Insoluble Protein ' Non-Protein Nitrogen Control ----- Inoculated l- ’ A~~ l" ~ ~ ‘ s ‘ ;:4‘~:--—o i. ran-v " ‘ ‘«\ \ “ 4 8 l2 IS 20 24 28 Storage Time (days) 25. Effect of the enzyme fraction obtained from G-lOO Sephadex K 25/45 column on porcine primary muscle proteins stored at 3°C. 102 of muscle tissue was not possible due to a lack of such information in the literature. Instead, comparisons will be made with previous reports using bacterial inoculated muscle. Ockerman £5 31, (1969) reported decreases in the sarcoplasmic protein fraction of aseptic and Pseudomonas and/or Achromobacter inoculated samples. Samples inoculated with high bacterial levels had a much slower decrease than either the low level inoculated samples or the aseptic control sample. This indicated that the slower rate of decrease with large numbers of bacteria present was due to some breakdown of the other protein fractions by the bacteria. Borton 35 El: (1970), Hasegawa g£_§l, (1970) and Tarrant ‘ggnal. (1971) using a variety of bacteria all reported similar type re- sults with regard to the changes in solubility of the sarcoplasmic protein fractions of porcine muscle. None were as clearly illustrated as in figure 25. The reported increases have been explained as due to pH in- creases during bacterial growth. However, the present data were obtained using powdered muscle made to a slurry using an enzyme fraction buffered at pH 7.5. This indicated that proteolytic action by bacteria on the insoluble and/or salt soluble protein fractions appears to be more likely. Myofibrillar Proteins The solubility of the myofibrillar protein fraction was unusual in that the control sample showed a gradual decrease during the 25 day storage period from 46 mg to 34 mg/g of sample. This was in contrast to 103 the increases reported by other workers (Borton 35 51., 1970; Hasegawa EELal,, 1970 and Tarrant gt 31., 1971). The enzyme treated sample (figure 25, Appendix 3.24) was found to show a large increase in myo- fibrillar protein solubility at day 4, decreasing slightly during the next 8 days with a final large decrease noted at day 25. These results are in agreement with those of Ockerman 25 31. (1969) using bovine muscle and Barton £5 31, (1970) and Tarrant g£_al, (1971) using porcine muscle. The initial increases may be explained by the increased solubility of the insoluble myofibrillar protein fraction due to weakening of the actin-myosin complex or due to the rapid decrease in the stroma protein fraction (54 mg to 35 mg/g of sample) at day 4. Stroma Protein The large decrease in stroma protein fraction was complete at day 4 and remained constant or increased slightly at day 25. Ockerman 25:51. (1969) did report decreases in the stroma protein fraction on inoculation with bacteria, in particular the low level inoculum of Pseudomonas and Achromobacter. The increases observed in the control stroma protein fraction have also been reported by Ockerman g£_al, (1969). Non Protein Nitrogen Fraction The nonprotein nitrogen (NPN) changes in solubility were as expected; the control fraction remaining constant and the enzyme treated fraction gradually increasing up to day 25,the final day in the storage period. 104 Again these results are comparable with those of Aberle and Merkel (1966) and Ockerman £5 21' (1969) for bovine muscle and Barton g£_§l, (1970) for porcine muscle. Other workers (Khan and Van der Berg, 1964, with chicken muscle and Sharp, 1963, with rabbit and bovine muscle) have reported in- creases in NPN during aseptic storage. However, temperatures of 37°C were used in these experiments whereas 10w storage temperatures were used by all the other workers. A summary of the data obtained in this experiment is given in table 10. Table 10. Data obtained on the solubility changes in the primary proteins due to the G-lOO K 25/45 enzyme fraction. Days 0 4 8 12 25 mg Protein/gram of muscle sample Water soluble proteins Control 23.5 21.6 22.1 22.2 21.9 Inoculated 21.7 28.1 33.1 21.5 20.8 Salt soluble proteins Control 47.1 43.1 38.4 39.0 34.4 Inoculated 50.0 60.0 56.0 54.9 30.3 Non protein nitrogen Control 17.3 17.4 16.3 16.2 17.7 Inoculated 18.1 22.4 24.5 23.5 35.5 Insoluble proteins Control 57.3 64.1 68.3 68.8 70.7 Inoculated 54.4 35.4 37.5 35.2 58.5 In conclusion, the data indicated definite changes in the solubility Of the primary protein fractions as a result of enzyme action, in particu- lar' the stroma protein fraction which was extracted in the other soluble 105 fractions at 4 days of storage. The initial increase in the myofibrillar protein fraction is of importance, in that it coincided with the increased solubility of the stroma protein fraction at day 4 indicating that it did contain at least some of the solubilized stroma protein fraction. The large decrease in the myofibrillar protein fraction from day 12 to 25 coincides with the increase in the non protein nitrogen fraction during the same period. The actual pattern of breakdown may be as follows: (1) the stroma protein fraction is solubilized and is extracted in the sarcoplasmic and myofibrillar protein fractions initially. (2) the myofibrillar protein fraction in turn was being broken down and extracted in the non protein nitrogen fraction, at which time spoilage of the meat sample was occurring. Finally, the changes brought about by the enzyme fraction at pH 7.5 are in agreement with the results reported by other workers including Ockerman g£_al, (1969), Borton g£_al, (1970) and Tarrant g£_al, (1971), all of whom used bacteria and low storage temperature. This indicated that the proteolytic activities of these bacteria are similar to the action of the proteolytic enzyme fraction obtained from Pseudomonas pero- lens ATCC 10757 used in this study. Changes in Muscle Ultrastructure Davey and Gilbert (1967, 1968a, 1968b, 1969) have reported distinct changes occurring within the myofibrillar structure during aging. Chemical and microscopic studies have shown loss of myofibril adhesion, loss of asso- ciation between actin and tropomyosin within the I filaments and loss of material from the 2 lines resulting in breakage at the I band - 2 line junction. Henderson g£_al, (1970) demonstrated that Z line degradation 106 occurred in the intact muscle as a result of postmortem aging. Porcine muscle Z lines were fragmented and amorphous in appearance after 5 days of storage at 3°C. Goll g; a1. (1971) reported a series of similarities between the effects of trypsin and the effects of postmortem aging on muscle cells. Z line degradation was one of these similarities, indicating the possibility of proteolysis being a factor in postmortem alterations in muscle cells. Using an inoculum of Pseudomonas perolens and an enzyme fraction produced by these bacteria as treatments on sterile porcine muscle, the following results were obtained. The photomicrographs shown are those most representative of all the sections observed. No discernable differ- ences were found between the untreated control (figure 26) and a bacterial inoculated sample (figure 27) at 4 days of storage at 3°C. The enzyme treated sample showed marked ultrastructural alterations at day 4 (figure 28). The Z line had been completely removed from the I band as well as the M line from the H zone. On closer observation it appears that the actin filaments have partially disintegrated in the I band region, but no evident changes were observed in the A band region other than the M line disappearance. In the untreated control the actin and myosin filaments, the Z and M lines and the H zones are clearly visible. At day 8 in storage the control sample (figure 29) was still intact. However, the Z line and the I band appeared less dense in comparison to those in the 4 day control (figure 26). This Z line fragmentation has been shown to occur during postmortem aging (Stromer‘gt al,, 1967; Stromer and G011, 1967, among others). The enzyme treated sample (figure 107 Figure 26. Electron micrograph of myofibrils from uninoculated pig muscle incubated at 3°C for 4 days in sterile Koser's citrate medium. A - A band, I - I band, B a H zone, M - M line, Z - Z line, S - one sarcomere length. (X 42,000) 108 Figure 27. Electron micrograph of myofibrils from bacterial inoculated pig muscle incubated at 3°C for 4 days. A - A band, I = I band, H - H zone, M - M line, Z = Z line. (X 42,000) 109 Figure 28. Electron micrograph of myofibrils from enzyme treated pig muscle'incubated at 3°C for 4 days. A - A band, I - I band, B. = H. zone, M = where M line was situated, A =- where Z line was situated. (X 42,000) 110 Figure 29. Electron micrograph of myofibrils from uninoculated pig muscle at 3°C for 8 days in sterile Koser's citrate medium. A-Aband, I= Iband, H- Hzone, Z = Z line, M=M line. (X 42,000) 111 30) had further disintegrated with some loss of the actin filaments in the A bands, indicating disruption of the actindmyosin complex. Wider and more distinct H zones which were also evident would indicate such actin- myosin disruption in comparison with the control sample (figure 29). Almost complete disappearance of the I bands was also observed. The bacterial treated sample at day 8 showed variable ultrastructural changes from very similar to the control sample (figure 31), to a partial loss of the Z line or Z line fragmentation (figure 32), to separation of myofibers (figure 33). This apparently inconsistent pattern may have been due to a number of factors. (1) At 3°C psychrophilic bacteria would grow slowly and only on the muscle sample surface. (2) Localized growth patterns would be expected due to the low level of inoculum.used. (3) The enzyme(s) produced would be localized and therefore any resultant proteolysis would be localized on the surface initially. Electron micrographs showing localized bacterial growth.were obtained (figure 34). Bacteria appear to be at a distance from the myofibrils (figure 35). Bleblike evaginations appear on the bacterial cell surfaces obtained from porcine muscle after 8 days of storage at 3°C (figure 35). A high magnification of such a bleblike formation is shown in figure 36. Dutson 35 a1. (1971) have reported specific disruption of components in the A band region and removal of material from the Z line in ground porcine muscle due to incubation with Pseudomonas fragi_ATCC 4973. Bleb— like evaginations were also reported by these workers. They postulated that proteolytic activity was responsible for the myofibrillar disruption. Figure 30. Electron micrograph of myofibrils from enzyme treated pig muscle incubated at 3°C for 8 days. A - A band, I - I hand, H - H zone, M - where '11 line was situated, Z - where 2 line was situated. (X 42,000) 113 Figure 31. Electron micrograph of myofibrils from bacterial inoculated pig muscle incubated at 3°C for 8 days. A - A band I - I hand, a - a zone, 2 - z line,’M - M line. (X 42.0005 114 Figure 32. Electron micrograph of myofibrils from bacterial inoculated pig muscle incubated at 3°C for 8 days. A = A band, I.- I hand, H a H zone, M = M line, Z - Z line. (X 42,000) 1 "l‘llll 115 Figure 33. Electron micrograph of myofibrils from bacterial inoculated pig muscle incubated at 3°C for 8 days. A I A band, I = I band, H =- H zone, z I Z line, M - M line. (X 42,000) 116 Figure 34. Electron micrograph showing localized bacterial growth, on pig muscle at 8 days of storage at 3°C. Arrows indicate bleb-like formations. (X 40,000) 117 Figure 35. Electron micrograph showing bacterial growth.at a distance from the myofibrils of pig muscle at 8 days of storage.at 3°C CX 28,000). 118- Figure 36. Electron micrograph of a bacterial cell section showing a bleb.- 1ike formation at 8 days of growth on pig muscle incubated at 3°C. (X 400,000) 119 The enzyme(s) may be excreted into the blebs which in turn release their contents into the surrounding environment. This theory would support the postulate of localized disruption in the case of bacterial treated samples (figure 39). The overall disruption caused by the enzyme treated samples indicated that the enzyme(s) were capable of diffusion into the muscle mass (approximately 2 mm in this experiment). In summary, the enzyme treated sample showed Z and M line disappear- ance at 4 days at storage at 3°C. The bacterial treated samples showed no differences from the control. At 8 days of storage the enzyme treated sample showed complete loss of the actin filaments in the I hand. Wider and more distinct H zones may indicate some disruption of the actin- myosin complex. The bacterial treated sample showed some localized dis- ruption at the I band - Z line junction. On comparison of these data with the data obtained from the primary protein solubility changes listed in table 10, the following interpreta- tions were made. It may be that (1) The stroma protein fraction, in particular the connective tissue proteins, is first altered by the enzyme(s). Alterations in the collagen molecular structure during aging have been recently observed (Pfeiffer g£_§l,, in press). (2) The less soluble myofibrillar proteins become more soluble and proteolysis occurs in the I band initially and then the A band. (3) The increases in the sarcoplasmic and non protein nitrogen fractions may be a direct result of this proteolysis. It appears that the protease(s) elaborated by the psychrophilic Pseudo- EQEE§.d° disrupt the muscle fibers, particularly the myofibrillar pro— teins. It is also very interesting to note the observations of Busch 120 e£_al, (1972) who have reported that Z line removal from rabbit muscle was obtained on incubation in the presence of high calcium content plus a specific sarcoplasmic factor. 2 line removal was not observed in the presence of EDTA. These workers suggested that a calcium activated protease was responsible for the Z line removal. The calcium dependent protease obtained from Pseudomonas perolens in this study may be similar in nature to the endogenous protein fraction reported by Busch gt a1. (1972). SUMMARY The psychrophile Pseudomonas perolens ATCC 10757 was chosen for use in this study. In a preliminary study on the effects of this microorganism on ground aseptically procured porcine muscle stored at 10°C, the followh ing changes were observed: (1) Bacterial numbers increased from the initial in culation of 1.5 x 104 to 1.5 x 10 at day 14, decreasing to 6.6 x 10 at day 20. No growth was found in the aseptic control. (2) pH values of both control and inoculated samples were 5.3 initially. The pH of the inoculated sample remained constant until the 10th day and then gradually increased to 8.2 at day 19. ‘The pH of the control sample remained at 5.3 over the entire storage period. (3) Enzyme production was first detected on the 11th day coinciding with the initial increase in pH and high bacterial numbers of 10 per gram of tissue. (4) The major changes in primary protein solubility were increases in the myofibrillar and non protein nitrogen fractions. Attempts to extract and purify the enzyme(s) from the porcine muscle were not completely satisfactory and enzyme production and purification using a synthetic medium facilitated further study. The medium most suitable for enzyme production was found to be a simple non protein medium, namely Koser's citrate with 0.5 g/l of calcium chloride added. To prevent excess foaming with resultant loss of enzyme production, approximately 150 m1/500 ml Erlenmeyer flask was used at 160 rpm in a rotary shaker, in preference to larger volumes in larger flasks. Various other metal salts could not replace calcium chloride which was essential for enzyme(s) production in the Koser's citrate medium. 121 122 The supernatant obtained by the centrifugation of the inoculated and incubated (40-45 hours) growth medium at 10,000g x 20 minutes was designated the crude enzyme solution. The following crude enzyme para- meters were established using a 2% w/v casein solution, pH 7.5 as sub- strate. (l) A storage temperature of 3°C was found to be most suitable, 100% of the initial activity remaining up to 25 days of storage. Storage at room temperature resulted in almost complete inactivation after 64 hours. Freezer storage temperatures of -23°C and -29°C resulted in small‘decreases in the 2 initial activity remaining. (2) The optimum assay temperature for enzyme activity was found to be 35°C. (3) Enzyme activity was linear with time up to 20 minutes, thereafter decreasing gradually. (4) Enzyme activity was stable over a wide range of pH values from 6 to 10 with maximum stability between pH 7.0 and 9.0. (5) Maximum enzyme activity resulted from 1.0 m1 of enzyme solution incubated with 1.0 ml of a 0.5% w/v casein solution, pH 7.5 for a 15 minute period. (6) Crude enzyme concentrations of up to 80 ug protein per ml resulted in linear increases in the rate of eniyme activity. Slight decreases were obtained above this concentration. (7) Different initial reaction rates were observed on incubating with different protein substrates. Collagen and the salt soluble proteins resulted in the highest initial reaction rates with the egg albumin and the water soluble proteins giving the lowest. The crude enzyme solutions did bring about increased tenderness in meat but resulted in an undesirable potato-like odor on cooking. A purification procedure involving ultrafiltration and gel filtra- tion resulted in a 20 fold increase in purification and a 12% yield. Disc gel electrophoresis of the enzyme fractions obtained from a G-lOO Sephadex K 25/45 column showed that those fractions having maximum absor- bance at 216 nm and highest protein concentrations had single bands along with very similar specific activities. 123 A slight activation effect was found in the presence of cysteine in the purified enzyme solution with no effect from sodium bisulfite, iodoacetic acid or phenyl methyl sulfonyl fluoride. Increasing concentrations of EDTA resulted in decreases in the % initial activity remaining to 50%. on addition of 10.4 M. Reactivation of up to 82% of the initial activity resulted on the addition of 10.2 M calcium chloride. Collagen appears to be broken down by the purified enzyme fraction. However enzyme treatment did not result in the release of free hydroxy- proline as single amino acids, but rather as peptides or polypeptides. The purified enzyme fractions resulted in considerable changes in protein solubility. The sarcoplasmic protein fraction and the non protein nitrogen were found to increase by 40% and 100%, respectively, over a 25 day storage period at 3°C. The myofibrillar protein fraction increased by 20% at day 4 and gradually decreased to 60% of the original after 25 days. The control sample, however, did decrease to 71% of the original. The stroma protein fraction decreased to 36% of the initial content at day 4, in comparison to a slight increase from the control. Ultrastructural changes due to the purified enzyme fraction were observed at days 4 and 8 of storage at 3°C. At day 4 both Z and M lines had disintegrated. At day 8 breakdown of the I and A bands was observed. 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Composition of solutions used in the Lowry method. Reagents: Lowry A. Sodium carbonate 60 g Sodium hydroxide 12 g Sodium potassium tartarate 0.6 g Water 3,000 m1 Lowry B. Copper sulfate 6 g Water 1,000 ml Lowry C. Lowry A 50 parts Lowry B 1 part prepare fresh daily Phenol reagent - Folin-Ciocalteau phenol 1 part - Water 1 part - prepare fresh daily Procedure: 1. 1 m1 of protein solution (standard or unknown) plus 5 m1 of Lowry C. 2. Incubate 20 minutes at room temperature. 3. Add 0.5 m1 of phenol reagent, jetted in immediately. 4. Allow to stand for 45 minutes at room temperature with occasional mixing. 5. Read O.D. 660 nm. Preparation of protein standard curve Standard serum albumen; - dry bovine serum albumin - stock solution 400 ug/ml - made up in deionized distilled water Data for standard curve Bovine albumin (ugjml) 20 40 60 80 100 120 160 200 240 280 320 360 Change in .051 .096 .152 .192 .245 .282 .364 .451 .531 .607 .674 .731 O.D. 660 400g . 794 Appendix A.2. I. II. III. 136 Composition of solutions used. Sarcoplasmic protein extraction solution. 0.03M phosphate buffer, pH 7.4; and 4.39 g of KZHP04 were dissolved in 1 liter of deionized distilled water Myofibrillar protein extraction solution 1'1 KI, 0.1M phosphate buffer, pH 7.4; 182.6 g of K1, 2.18 g of KH2P04 and 14.63 g of KZHPO4 were dissolved in 1 liter of deionized distilled water. Disc gel solutions Running gel - made by mixing 6.4 m1 of solution 1, 1.6 ml of solution 2, and 2.67 ml of solution 3, for 8 gel tubes. Solution 1, Solution 2, Solution 3, 5 ml of 2N HCl, 7.62 g of Tris, 0.1 ml of TMED, 81.25 ml of 10M urea were mixed and then diluted to 100 ml with deionized distilled water. 43.3 g of cyanogum were dissolved in 25 ml of 10M urea and then diluted to 100 ml with deionized distilled water. l'mg of riboflavin was dissolved in 35 ml of 10M urea and then diluted to 50 ml with deionized distilled water. Spacer gel - made by mixing 1.6 m1 of solution 1, 0.4 ml of solution 2, and 0.67 ml of solution 3 for 8 gel tubes. Solution 1, Solution 2, Solution 3, 5 ml of 2N HCl, 1.25 g of Tris, 0.075 ml of TMED and 81.25 ml of 10M urea were mixed and then diluted to 100 ml with deionized distilled water. 33.3 g of cyanogum were dissolved in 25 ml of 10M urea and then diluted to 100 ml with deionized distilled water. This solution was identical to solution 3 used in the runner gel. Tank buffer - made by dissolving 6.0 g Tris and 28.8 g of glycine in 1 liter of deionized distilled water. 200 m1 of this stock solution was further diluted to 1 liter to provide the buffer used for each electr0phoretic run. 137 Appendix A.3. Composition of solutions used for electron microscopic study 1. Gluteraldehyde fixative Na H2 P04 . 320 1.8 g Na2H P04 . 7 H20 23.25 3 NaCl 5.0 g H20 925 ml 25% commercial gluteraldehyde 100 m1 This stock solution was diluted 1:1 with distilled water to give a 1.25% solution of 415 milliosmolar. Gluteraldehyde washing phosphate buffer Na H2 P04 . H20 1.8 g Nag H P04 . 7 320 23.25 3 NaCl 5.0 g H20 925 m1 Osmium.tetroxide (2%) fixative Stock solution A Sodium acetate 9.714 g Veronal-sodium 14.714 g Distilled water to make 500 m1 Stock solution B Sodium chloride 40.25 g Potassium chloride 2.1 g Calcium chloride 0.9 g Distilled water to make 500 m1 To make a 2% solution of osmium tetroxide, add 10 ml of Stock Solution A, 3.4 ml of Stock Solution B and 26.6 ml of distilled water, adjust pH to 7.4 with 0.1N HCl (approximately 11 m1), and dilute 4% aqueous osmium tetroxide solution 1:1. Epon embedding material Mixture A Dodecenyl succinic anhydride 94 g Epon 812 80 g mix thoroughly Mixture B Nadic methyl anhydride 78 g Epon 812 100 g mix thoroughly 138 Add together 7 g of mixture A, 3 g of mixture B and 0.14 g of dimethyl amino methyl phenol - 30. Mix for 5 minutes. Lead citrate stain l g lead citrate in 50 ml water. Shake. Allow to stand and shake again for 5 minutes. Add 0.5 ml 10N NaOH. Add,one drop at a time, lON NaOH and invert until the solution becomes clear. This method is a modification of the Reynolds method reported by Richard Puffing, technician, the Biology Department, Brookhaven National Laboratory. 139 Appendix B.1. Growth of Pseudomonas pgrolens, Pseudomonas fragi_and Pseudomonas aernginosa in Koser's citrate medium, pH 7.5, at 10°C. Change in optical density at 660 nm Pseudomonas Pseudomonas Pseudomonas hrs perolens hrs fragi hrs aergginosa O 0.0 0 0.0 0 0.0 13 0.007 21 0.141 73 0.089 25 0.143 33 0.781 89 0.175 37 0.95 45 1.224 101 0.297 46 1.116 54 1.219 115 0.577 61 1.158 69 1.131 138 0.967 149 1.019 162 1.062 176 1.055 186 1.064 Koser's citrate or basal medium Sodium ammonium phosphate 1 Sodium phosphate—monobasic 1 Magnesium sulfate 0 Sodium citrate 3 and made to one liter with deionized distilled water and adjusted to pH 7.5. Appendix B.2. 140 The action of Pseudomonas perolens ATCC 10757 on porcine muscle during a 20 day storage period at 10°C. Enzyme activity Bacterial counts (7 O.D. 230) (log nos.) Days Control Inoc. Control Inoc. Control Inoc. 1 - - 0.014 0.035 None 1.5.104 2 - - 0.023 0.029 " 1.75.104 3 5.3 5.25 0.029 0.037 " - 4 5.3 5.3 0.032 0.028 " 4.1.105 5 5.3 5.25 0.034 0.040 " - 6 5.25 5.3 0.051 0.051 " 5.9.105 7 5.2 5.3 0.014 0.019 " - 8 5.3 5.2 0.018 0.018 " 2.6.106 9 5.25 5.3 0.010 0.019 " - 10 5.25 5.35 0.010 0.040 " 2.9.107 11 5.3 5.5 0.010 0.061 " - 12 5.3 5.7 0.010 0.144 " - 13 5.3 5.7 0.02 0.179 " - 14 5.3 6.0 - - " 1.5.109 15 5.3 6.5 0.010 0.267 " - 16 5.35 7.0 0.032 0.303 " - 17 5.3 7.7 0.010 0.414 " - 18 5.3 8.2 0.030 0.462 " - 19 0.051 0.690 " - 20 " 6.6.108 Appendix B.3. Growth cycle of Pseudomonas perolens on Koser's citrate medium, pH 7.5, at 10°C in a rotary shaker at 160 rpm. Time (hours) 19 54 66 77 88 Optical density 660 nm 0.01 0.917 1.156 1.013 0.837 Change in optical density 0.0 0.907 1.146 1.003 0.827 141 Appendix B.4. Growth of pseudomonas perolens in basal medium, pH 7.5, at 10°C the citrate being replaced by amino acids. Chaoge in optical density at 660 nm Time Glutamic L-Asparitic (hours) Arginine acid Histidine Cysteine acid 0 0.0 0.0 0.0 0.0 0.0 17 0.0 0.338 0.0 0.0 0.061 24 0.0 0.872 0.0 0.07 0.371 41 0.74 1.525 0.02 0.115 1.335 52 0.238 1.185 0.119 0.125 1.250 63 0.657 0.64 0.49 0.135 0.523 73 1.255 0.612 1.584 0.19 0.461 88 - - - - 0.450 Media used Sodium ammonium phosphate 1.5 g Sodium phosphatedmonobasic 1.0 g, Magnesium sulfate 0.2 g Single amino acids 3.0 g and made to 1 liter with deionized distilled water. Individual amino acids were added to the basal medium.without citrate at 3.0 g/l. 142 Appendix B.5. Growth of Pseudomonas perolens in basal medium, pH 7.5, at 10°C the citrate being replaced by dipeptides. Time Glycyl Glycyl Glycyl Glycyl Glycyl (hours) L-Alanine L-Valine L-Leucine L-Glycine L-Asparagine 0 0.0 0.0 0.0 0.0 - 18 0.0 0.0 0.0 0.0 26 0.006 0.0 0.0 0.0 42 0.032 0.005 0.0 0.011 50 0.057 0.01 0.0 0.019 64 0.196 0.026 0.004 0.046 88 0.661 0.086 0.006 0.075 97 0.795 0.142 0.012 0.122 116 0.702 0.281 0.013 0.226 123 0.655 0.363 - 0.267 140 0.574 0.517 0.027 0.410 150 0.508 0.604 - 0.525 162 0.445 0.658 0.042 0.604 174 0.388 0.712 - 0.654 185 0.351 0 728 - 0.610 209 - - 0.164 - 221 - - 0.204 - 290 - - - - Individual dipeptides were added to the basal medium without citrate at 3.0 g/l. Appendix B.6. 143 Growth of Pseudomonas perolens and corresponding enzyme production in complex type medium with different solvents. Chaoge in optical density at 660 nm Time Deionized distilled Deionized (hours) Tap water water + Callz distilled water 0 0.0 0.0 0.0 13 0.031 0.018 0.0 24 0.108 0.455 0.047 37 0.580 0.840 0.630 43 0.714 1.003 - :51 0.963 1.110 0.897 61 1.093 1.082 1.140 Basal medium (In 500 m1 deionized distilled H20) amino acids Monopotassium phosphate 0.5 g L-arginine Mcl 0.1 g Magnesium sulfate 0.1 g L-asparagine 0.1 g Sodium ammonium phosphate 0.75 g L-aspartic acid 0.1 g L—cysteine 0.1 g Dipeptides added L-glutamic acid 0.1 g Glycyl glycine 0.2 g L-glutamine 0.1 g Glycyl L-asparagine 0.2 g L-leucine 0.1 g L-lysine 0.1 g L-threnine 0.1 g L-tryptophan 0.1 g 1... O N 144 Appendix 8.7. Growth of Pseudomonas perolens in basal medium and basal medium with citrate replaced by other carbon sources. All media had calcium chloride added. ‘ Change in optical density at 660 nm Basal Citrate replaced Citrate replaced Citrate replaced ' Time medium by amino acids by dipeptides by aminoacids (hours) + CaCl2 + Ca.C12 + CaCl2 dipeptides + Ca.c12' O 0.0 (—) 0.0 (-) 0.0 (-) 0.0 (-) 20 0.045 (—) 0.267 (-) 0.063 (-) 0.824 (-) 45 0.659 (.181) 0.510 (-) 0.078 (-) 1.100 (.092) 51 0.588 (.316) 0.460 (-) 0.085 (-) 0.980 (.176) Enzyme production as measured by change in O.D. at 660 nm using the Lowry method is shown in parentheses. Media used - 0.5 g calcium chloride used in all cases. I. Koser's citrate (Difco manual) IV. Basal medium with citrate replaced by II. Basal medium with citrate re- L-cystine 0.05 g/l placed by L-asparatic acid 0.05 g/l L-cystine 1.0 g/l L-histidine 0.05 g/l L-aspartic acid 1.0 g/l Glycyl L-asparagine 0.05 g/l L-histidine 1.0 g/l Glycyl glycine 0.05 g/l Glycyl L-alanine 0.05 g/l III. Basal medium with citrate re- placed by Glycyl L-asparagine 1.0 g/l Glycyl glycine 1.0 g/l Glycyl L-alanine 1.0 g/l 145 Appendix B.8. Growth of Pseudomonas perolens on basal medium with varying calcium chloride concentrations and corresponding enzyme production at 10°C, pH 7.5. Change in optical density at 660 nm Calcium chloride conc (g/l) 0.04 0.05 0.08 0.1 Time (hours) 13 0.0 (-) 0.0 (-) 0.0 (-) 0.0 (-) 35 0.204 (-) 0.23 (-) 0.214 (-) 0.125 (-) 47 0.660 (.093) 0.676 (.129) 0.559 (-) 0.732 (.027) 59 0.950 (.253) 0.937 (.322) 0.950 (.167) 0.89 (.054) 70 0.890 (.186) 0.936 (.263) 0.93 (.126) 0.81 (.041) 83 0.778 ( 086) 0.948 (.128) 0.91 (.084) 0.75 (.036) Enzyme production as measured by change in optical density at 660 nm using the Lowry method is shown in parentheses. Appendix B.9. Growth of Pseudomonas fragi_ATCC 4979 on basal medium with varying calcium chloride concentrations and corresponding enzyme production at 11°C, pH 7.5. Change in optical density at 660 nm Calcium chloride conc(g/l) 0.01 0.02 0.05 0.1 Time (hours) 0.0 (e) 0.0 (—) 0.0 (-) 0.0 (-) 21 0.14 (-) 0.065 (-) 0.0 (-) 0.0 (-) 33 0.78 (.008) 0.675 (.058) 0.343 (.026) 0.241 (.015) 45 1.22 (.045) 0.895 (.161) 0.7 (.094) 0.487 (.039) 54 1.22 (.05) 0.942 (.168) 0.78 (.126) 0.613 (.066) 69 1.13 (.049) 0.91 (.146) 0.75 (.104) 0.6 (.051) Enzyme production as measured by change in Optical density at 660 nm using the Lowry method is shown in parentheses. 146 Appendix B.10. Effect of calcium chloride concentration on enzyme pro- duction by Pseudomonas perolens and Pseudomonas £9981. grown in basal medium at 11°C and pH 7.5 for an incubation time of 55 hours. Change in optical density at 660 nm Calcium chloride conc Pseudomonas perolens Pseudomonas frngi 0.01 g/l 0.021 0.05 0.02 g/l - 0.168 0.04 3/1 0.253 - 0.05 3/1 0.322 0.126 0.06 g/l 0.320 - 0.08 g/l 0.167 - 0.10 g/l 0.054 0.066 The enzyme production was measured using the Lowry method. Appendix B.11. Growth of Pseudomonas perolens ATCC 10757 and enzyme production on Koser's citrate medium plus 0.05 g calcium chloride per liter at 11°C, pH 7.5. Changes in O.D. Time (hours) at 660 nm 0 13 35 47 59 70 83 Bacteri l growt enzyme 0.0 0.0 0.230 0.676 0.937 0.936 0.948 production 0.0 0.0 0.0 0.129 0.322 0.263 0.128 Enzyme production was measured at 660 nm using the Lowry method. 1“! [5.13 [{[tl’ ll 147 Appendix B.12. Effect of foaming and an antifoam agent on growth and enzyme production of Pseudomonas perolens on developed medium at 11°C and pH 7.5. Changes in optical density at 660 nm Time (hours) Regnlar growth Antifoam added Foaming allowed 0 0.0 (-) 0.0 (-) 0.0 (-) 13 0.0 (-) 0.0 (—) 0.0 H 18 - - 0.055 (-) 0.05 (-) 31 - - 0.62 (0.008) 0.305 (0.039) 35 0.23 (—) - 42 - - 0.668 (0.009) 0.546 (0.091) 47 0.676 (0.129) - 50 - - 0.66 (0.005) 0.592 (0.080) 59 0.937 (0.322) 70 0.936 (0.263) 83 0.948 (0.128) Enzyme production was measured at 660 nm using the Lowry method and is shown in parentheses. Appendix B.13. Stability of the crude enzyme fraction at storage tempera- ture of 26°C, 3°C, -23°C, -29°C. Stornge time Storage temperature (days) 26°C 3°C -23°C -29°C % Initial activity remaining 0 100% 100% 100% 100% l 69% 90% -- -- 12 35% 90% -- 100% 3 7% 100% 86% -- 5 -- 100% -- -- 8 —- 100% 83% 93% 11 -- 100% 83% -- 17 -- 100% -- -- 25 -- 100% 83% 91% 148 Appendix B.14. Effect of assay temperature on enzyme activity. Assay temperature °C ug Tyrosine equivs 15° 20° 25° 30° 35° 40° 45° 50° released/ml/mm 1.2 1.9 2.9 4.26 5.13 4.6 3.06 0.11 Appendix B.15. Effect of assay time on enzyme activity. Assay time °C ug Tyrosine equivs 4 10 15 20 25 30 35 40 50 60 90 released/m1 44.8 64.4 93.6 122.4 142.4 174 198 216 253.2 284 366.4 149 .muscwa you as use wwwmwamu mucoam>wsvm sawmouhu w: onu mm vommmuaxm zufi>auum sexuam e.~ H.m n.w m.q m.o q.m o.m m.H H.0H m.m o.m m.q 5.0 w.¢ ~.o m.~ o.oH om.m q.w m.e w.o mm.q m.o N.q w.m m.m ~.m N m m.n 0.6 H.n o.m 5.9 mm.m o.m q.m w.“ n m H.w o~.q m.n oo.m m.c m.m ~.m o.m «.5 m m m.w o.q m.m H.m a.“ nm.o m.m o.m m.n o q m.w w.m o.w m.c o.n GHE\HE\wn mm. cwa\as\mm, mm GHE\HB\mn mm :Ha\aa\m: mm, aHE\HmMm: mm mumaonumoan Haufinumm mwue mumsmmonm mumsmmonm\wumuuao Imumconumo mummmsm .uomm um zua>fiuom mahnam so mamumxm gunman maowum> maam: mm mo uommmm .oa.m xauawaa< 150 Appendix B.16. (continued) Data for buffer preparations See preparation of buffers for use in enzyme study by G. Gormori Citrate phosphate buffer Stock solutions A; 0.1M citrate acid 1.92 g/100 ml B; 0.2M dibasic sodium phosphate 2.84 g/100 ml Phosphate buffer Stock solutions A; 0.2M monobasic sodium phosphate 2.783/100 m1 B; 0.2M dibasic sodium phosphate 2.84g/100 m1 Tris (hydroxymethyl) aminomethane (tris) buffer A; 0.2M Tris 2.42g/1OO ml B; 0.2M Hydrochloric acid 7.293/100 m1 Barbital buffer A; 0.2M sodium barbital (veronal) buffer 4.13/100 ml B; 0.2M hydrochloric acid 7.29g/100 ml Carbonate-bicarbonate buffers A; 0.2M anhydrous sodium carbonate 2.12g/100 m1 B; 0.2M sodium bicarbonate 1.68g/100 m1 151 Appendix B.17. Effect of pH using a combination buffer system (phosphate -acetic acid-boric acid) with pH range from pH 5.0 to 9.0. pH Z Initial 4.1 5.1 6.1 6.6 7.1 7.5 8.1 8.8 activity remaining 0.0 25.5 78.7 97.7 100 100 100 91.5 Buffer preparation: Phosphoric acid 39.2 g Acetic acid 24.0 g Boric acid 24.8 g and made to 1 liter with distilled water. 100 mls of the above stock solution made to 1 liter to give a 0.04M solution. Appendix B.18. Effect of casein concentration as substrate on enzyme activity. Casein as substratemgjml pg Tyrosine 0.2 0.4 0.8 1.2 2.0 4.0 8.0 12.0 16.0 28.0 equivalent released/ml/min 2.32 4.07 6.2 7.2 8.7 9.45 9.2 8.3 8.0 7.3 Appendix B.19. Effect of enzyme concentration on enzyme activity. Crude enzyme Enzyme concentration (pg protein) pg tyrosine 10 20 4O 60 80 100 equivalents released/ml/min 0.55 1.27 2.73 4.55 5.71 6.55 Purified enzyme Enzyme concentration (ug protein) ug tyrosine 1.0 2.0 4.0 5.0 7.5 10.0 12.5 equivalents released/mllmin 0.8 1.6 3.36 4.26 6.8 9.9 12.9 152 Appendix B.20. Enzyme action on various proteins as substrates. Protein substrate ' Egg Time Casein Collagen albumin Sarcoplasmic Myofibrillar min. pg Tyrosine equivalents released/m1 0.66 4.4 16.0 - - - l 4.8 - 14.0 4.8 21.2 2 17.2 38.8 15.2 13.6 42.8 4 36.8 - 20.8 14.4 65.2 8 71.6 118.8 26.0 15.2 88.4 16 143.6 178.8 30.4 25.2 106.8 32 260.4 225.2 34.0 34.8 151.6 64 412.0 247.2 35.2 49.2 182.4 90 - 256.0 Appendix 8.21. The relationship between enzyme activity and absorbance at 216 nm of the enzyme fraction obtained from G-75 Sephadex K 25/45 column. Eluate fractions gml) 68-70 71-73 74-76 80-82 83-85 86-88 89-91 Absorbance 216 nm 0.033 0.177 0.306 0.267 0.074 0.074 0.033 enzyme activity 9.0 27.6 40.0 39.7 14.3 12.9 11.2 Enzyme activity reported in mg tyrosine equivalents released per ml per min. 0 153 sundae son as non wmmmoaou muoon>Hovo mafimouhu w: m£u no coupons» hufi>fluum mahnom we mm «N mm «m cm Ho mm Hm II n.m N.n m.o« n.0HH m.N¢H w.NHH o.nm ¢.N one. mmm. mmm. Hum. nun. H.H w. mam. me. I Away scan Imuudoocou samuoum I hua>fiuom mahucm mus. a: oHN mononuomn< ONHImHH «Halmoa wOHlmoa Noalnm mmlam omlmw «mums whims «mine owloo mcowuomuw oumoflm .aaoaoo m¢\m~ M autonomm ooalu scum vooamuno moOfiuomum oumaao man a« soaumuuooo taco aaououa was a: cam um mononuomnm .hufi>fiuom saunas cooBuon awanOHumamu 058 .-.m xfiwamaaa 154 Q Appendix B.23. Hydroxyproline released as measured after hydrolysis of enzyme treated collagen trichloroacetic acid filtrate at pH 7.5 and 35°C. Time min. Hg 0 1 2 3 6 10 Hydroxyproline released 0.0 1.1 1.43 1.55 2.6 4.5 93 03082 256 mumaulufljmmmumvIumfimmmmun