EMULSIFYlNG AND OTH’éR PROPERHES 0F PORUNE MUSCLE YESSUE AS RELATED TO MICROBML CONTAMINATION 'i'hesis for the Degree cf M. S. MKHWAN STATE UNIVERSITY Rem-said James Barton 19636 “,4:qu LIBRARY Michigan Stan! University ABSTRACT EMULSIFYING AND OTHER PROPERTIES OF PORCINE MUSCLE TISSUE AS RELATED TO MICROBIAL CONTAMINATION by Ronald James Borton For many years it has been known that bacteria caused spoilage of meat and meat products, but the affect of bacteria on the chemical pro- perties of meats has not been studied very extensively. The purpose of this research was to study the influence of bacteria on some properties of muscle tissue through a 17 day storage period. In order to accomplish this, a method of obtaining a sample relatively free of bacteria was develoPed. Some contamination of the samples obtained by this method was noted but the contamination was slight when compared to the inoculated sample or to normal fresh meats. Protein extractability was studied as protein is important in emul- sification of fats in sausage products and nutritionally in fresh meats. Results indicated the protein extractability changed due to the factor of time but changed very little due to bacteria. The sarcoplasmic protein fraction tended to decrease during the period of rapid bacterial growth, but the bacteria had little or no effect on the myofibrillar, stroma, and nonprotein nitrogen (NPN) fractions. The emulsifying capacity was also studied as amulsification of fat in a sausage product is important to sausage manufacturers. The emulsi- fying capacity appeared to decrease due to the bacteria growth in the sample. Ronald James Borton The Extract Release Volume (ERV) was studied as it was reported to be an indicator of the amount of bacterial spoilage. Results of this study indicated that the ERV decreased inversely to the increase in bacteria numbers. Hewever, the ERV appeared to be related to growth of bacteria rather than bacterial numbers as there was little difference between control and inoculated samples at O and 1 day of storage. Sta- tistical analysis indicated there was a significant difference at P < .01 between the treatments as related to numbers of bacteria present at var- ious storage periods. EMULSIFYING AND OTHER.PROPERTIES 0F PORCINE MUSCLE TISSUE AS RELATED TO MICROBIAL CONTAMINATION By RONALD JAMES BORTON A THESIS Submitted to Michigan State University in partial fulfilhment of the requirements for the degree of MASTER.0F SCIENCE Department of Food Science 1966 ACKNOWLEDGEMENT The author wishes to express his appreciation to Professor L. J. Bratzler for his guidance throughout the research and for his assistance in the preparation of this manuscript. He also wishes to thank Dr. Neil B. webb (Director of Technology, Eckert Packing Company) for his advice, encouragement, and aide in accomplishing this research. The author is grateful to his parents for their continual support and encouragement. The author is indebted to the Eckert Packing Company, Defiance, Ohio, for the SUpport furnished him by a special assistantship and providing space, equipment and materials for this project. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . .. . . . . .. . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . . . . . . 3 Microorganisms . . . . . . . . . . . . . . . . . . . . . . 3 Spoilage . . . . . . . . . . . . . . . . . . . . . . . 3 Extract Release Volume (ERV) . . . . . . . . . . . . . 7 Aseptic Muscle Sampling . . . . . . . . . . . . . . . 8 Proteins . . . . . . . . . . . . . . . . . . . . . . . . . 10 Sarc0plasmic proteins . . . . . . . . . . . . . . . . 10 Myofibrillar proteins . . . . . . . . . . . . . . . . 13 Stroma proteins . . . . . . . . . . . . . . . . . . . l6 Nonprotein nitrogen (NPN) . . . . . . . . . . . . . . 17 Emulsifying Capacity . . . . . . . . . . . . . . . . . . . 18 EXPERIMENTAL METHODS . . . . . . . . . . . . . . . . . . . . . . 21 Slaughter . . . . . . . . . . . . . . . . . . . . . . . . . 21 Excising the Muscle Sample . . . . . . . . . . . . . . . . 21 Inoculation of the Sample . . . . . . . . . . . . . . . . . 22 Inoculum Preparation . . . . . . . . . . . . . . . . . . . 23 Bacterial Numbers .I. . . . . . . . . . . . . . . . . . . . 24 Protein Extraction . . . . . . . . . . . . . . . . . . . . 24 Emulsion Capacity . . . . . . . . . . . . . . . . . . Extract Release Volume . . . . . . . . . . . iii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . .. . . . . .. . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . . . . . . 3 Microorganisms . . . . . . . . . . . . . . . . . . . . . . 3 Spoilage . . . . . . . . . . . . . . . . . . . . . . . 3 Extract Release Volume (ERV) . . . . . . . . . . . . . 7 Aseptic Muscle Sampling . . . . . . . . . . . . . . . 8 Proteins . . . . . . . . . . . . . . . . . . . . . . . . . 10 Sarc0plasmic proteins . . . . . . . . . . . . . . . . lO Myofibrillar proteins. . . . . . . . . . . . . . . . . 13 Stroma proteins . . . . . . . . . . . . . . . . . . . 16 Nonprotein nitrogen (NPN) . . . . . . . . . . . . . . l7 Emulsifying Capacity . . . . . . . . . . . . . . . . . . . 18 EXPERIMENTAL METHODS . . . . . . . . . . . . . . . . . . . . . . 21 Slaughter . . . . . . . . . . . . . . . . . . . . . . . . . 21 Excising the Muscle Sample . . . . . . . . . . . . . . . . 21 Inoculation of the Sample . . . . . . . . . . . . . . . . . 22 Inoculum Preparation . . . . . . . . . . . . . . . . . . . 23 Bacterial Numbers .I. . . . . . . . . . . . . . . . . . . . 24 Protein Extraction . . . . . . . . . . . . . . . . . . . . 24 Emulsion Capacity . . . . . . . . . . . . . . . . . . ExtractReleaseVolune.................. 27 iii pH Determination Protein Determination . Moisture Determination Statistical Analysis RESULTS AND DISCUSSION . Bacterial . Extract Release Volume Proteins Emulsifying Capacity SUMMARY AND CONCLUSIONS BIBLIOGRAPHY . APPENDIX . Sarc0plasmic . Myofibrillar . Stroma . Nonprotein Nitrogen Page 28 28 28 28 29 29 33 36 36 39 41 43 45 47 50 52 57 LIST OF TABLES Table Page 1 Ratio of bacterial types in inoculum cultures . . . . . . 32 2 Analysis of variance of ERV among two treatments and Six time periOdS O O 0 O I O O O O O O O C O O O O O O O 35 3 Ranking of means with indication of significance by Duncan's New Multiple Range Test . . . . . . . . . . . . 36 4 Analysis of variance of sarcoplasmic protein nitrogen among two treatments and six time periods . . . . . . . . 38 LIST OF FIGURES Figure Page 1 Outline of protein fractionation . . . . . . . . . . . . 25 2 Relationship of the log of bacterial numbers/gram of control and inoculated samples . . . . . . . . . . . . . 3O 3 Relationship of the ERV of control and inoculated smples O O O O O O O O O O C O O O O O O O O O I O O O 34 4 RelationShip of the sarcoplasmic protein nitrogen of control and inoculated samples . . . . . . . . . . . . . 37 5 Relationship of the myofibrillar protein nitrogen of control and inoculated samples . . . . . . . . . . . . . 40 6 Relationship of the stroma protein nitrogen of control and inoculated samples . . . . . . . . . . . . . . . . . 42 7 Relationship of the nonprotein nitrogen of control and inoculated samples . . . . . . . . . . . . . . . . . . . 44 8 Relationship of emulsifying capacities of control and inoculated samples . . . . . . . . . . . . . . . . . . . 46 9 Relationship of pH of control and inoculated samples . . 48 vi Appendix Appendix Appendix Appendix Appendix Appendix Appendix Appendix Appendix Appendix LIST OF APPENDIX TABLES Page Log of the bacterial numbers per gram.of sample for control and inoculated samples plus log of bacterial numbers per ml of inoculum . . . . . . . . . . . . 57 Pfilligrams of sarcoplasmic protein nitrogen per 100 milligrams of total protein nitrogen for control and inoculated samples . . . . . . . . . . . . . . . . 58 IMilligrams of myofibrillar protein nitrogen per 100 milligrams of total nitrogen for control and inoculated samples . . . . . . . . . . . . . . . . 59 Milligrams of stroma protein nitrogen per 100 milligrams of total nitrogen for control and inoculated samples . . . . . . . . . . . . . . . . 6O Nfilligrams of nonprotein nitrogen (NPN) per 100 milligrams of total nitrogen for control and inoculated samples . . . . . . . . . . . . . . . . 61 Emulsifying capacity of control and inoculated samples m1 of soybean oil per 100 mg of protein . . 62 Nfilliliters of extract release volume (ERV) for control and inoculated samples . . . . . . . . . . 63 pH of control and inoculated samples . . . . . . . 64 The percent moisture in the control and inoculated smples O O O O O O O I O O O O O O O O O O O O O O 65 The percent protein in the control and inoculated smples O O O 0 O O O O O O O O O O O O I O O 0 vii INTRODUCTION ZMicroorganisms have caused spoilage problems for the meat packer since the beginning of the meat packing industry. Ayres (1955) reviewed the work of many others concerning the spoilage problem. His review indicated that the spoilage organisms have been identified, the effects of microorganisms on physical properties such as odor, texture, flavor, water binding, sliminess, etc., have been established, and the origins of the Spoilage microorganisms have been found. Very little work has been expended to determine the effects of microorganisms on the chemical prOperties of muscle tissues. Davis (1965) pr0posed a method of aseptic slaughter and sample procurement involving surgical isolators which could provide control samples to compare with bacterially contaminated samples. He found he could hold control samples for at least 35 days at 2-5°C without any evidence of bacterial contamin- ation. When he compared these aseptic samples with inoculated samples, his results indicated that bacteria may influence chemical prOperties as well as physical properties of muscles. One chemical proPerty which may be altered in protein extractability. Previous work indicated that one of the primary meat spoilage genera, We, (Kirsh, again 1952; Ayres, 1955, 1956; Wolin 3531., 1957; and Halleck 25 31,, 1958) is a proteolytic organism (Camp and Van Der Zent, 1957; Van Der Zent, 1957; Peterson and Gunderson, 1960; and Witter, 1961) which would affect the extractability of the proteins. -1- -2- A second chemical property which.may be altered is the emulsifying capacity of the meat tissue. The emulsifying capacity is dependent on the amount and type of protein present (Swift‘g£”§1,, 1961; Swift and Sulzbacher, 1963; Hagerty 25 31., 1963; and Trautman, 1964). Thus, if the protein content is altered, the emulsifying capacity could be altered also. A third proPerty, the Extract Release Volume, which could be consi- dered a chemical property, could also change with bacterial growth (Jay, 1964; and Jay and Kontou, 1964). This study was initiated to determine: 1. If a more economical yet efficient means of obtaining a sample rela- tively free from microbial contamination could be found. 2. The effect of microorganisms on protein extractability. 3. The effect of microorganisms on emulsifying capacity. 4. The effect of microorganisms on the Extract Release Volume (ERV). LITERATURE REVIEW Microorganisms Spoilage Ayres'(l955) review of meat microbial studies indicated that muscle tissues from or on the carcasses of bovine, porcine, and ovine species provide most of the nutrients required by microorganisms. Also, he indicated the sources of microbial contamination are such that microbial growth is a serious problem to the meat packer. In his review, he listed the normal defenses of an animal against microbial infection as: 1. Skin and mucous membranes; 2. Hair and cilica; 3. Gastric juice; 4. Digestion; and 5. Localization of an infection if it begins. After death of the animal, these defensive mechanisms are lost and microbial contamination can take place. The same author indicated animals carry heavy loads of microorganisms on the hide, hair, skin, and hooves. Two studies he reviewed indicated the skin carried an average of 3.91 million aerobes, 100 million anaerobes, and 100 yeasts and molds per sq. cm. of skin surface. Jensen and Hess (1941) and Ayres (1956) reported similar results. Another source of contamination indicated by Ayres (1955) was the rumen and intestines of the bovine and ovine species and the intes- tines of the porcine species. If workers were careless when eviscerating the animals, the carcass became contaminated. Contamination of the carcass by the workmen and the equipment used in processing the carcass was also found by Ayres (1955, 1956). Every handling of the product increased the microbial load on or in the carcass. Jensen and Hess (1941) reported the sticking knife carried bacteria into the blood stream. The organisms were either contaminants of the knife or were on the skin and hair surrounding the sticking area and forced into the animal with the sticking knife. This contamination by the sticking knife was also reported by Ayres (1955, 1956). Jensen and Hess (1941) also reported that the scalding tank provided another means of contamination of hogs. They reported the deep tissues became more easily contaminated if the heart was still beating when the hog was drOpped into the scalding vat-as the microbially loaded water would be circulated in the animal's system. Ayres (1955, 1956) also found the scalding tank a source of contamination. Other sources of contamination listed by Ayres (1955, 1956) include the air in the packing plants, the water used in processing, and the sawdust used on the cooler floors. Kirsh ggugl. (1952) reported that ground beef purchased from retail stores had been contaminated so that the total aerobic counts ranged from 1-95 million per gram of tissue. When these bacteria were classified, it was found that the non-pigmented Pseudomonas-Aerobacter group dominated the flora with Lactobacillus and cocci organisms also present. The find- ings were similar to those of Ayres (1955, 1956), WOlin'ggngl. (1957), and Halleck gtngl. (1958). 'Woliniggugl. (1957) found that the initial contamination was primarily Gram-positive rods but after incubation at 20°C under moist conditions this changed to non-pigmented, Gram-negative, aerobic rods having polar flagella which are characteristic of Pseudomonas. This trend was also noted by Halleck gtual. (1958). Lepovetsky 35:31, (1953) studied the bacteria found in the deep tissues. They studied 23 beef animals and found bacteria in 15 of 23 lymph nodes, 3 of 23 bone marrow samples, and 2 of 23 muscle samples. The lymph nodes act as a filtering system in an animal and thus trap bacteria found in an animal's system. Twelve different genera of bacteria were isolated from the lymph nodes including Aerobacter and Pseudomonas. The three marrow samples with bacteria contamination were contaminated by bacteria of the genera Aerobacter, Corynebacterium, and Pseudomonas. The two contaminated muscle samples had isolates of the genera Pseudom- .gngg and Streptococcus. This study indicated that deep spoilage of a tissue probably will start at the lymph nodes as they are the primary source of bacteria in the deep tissues. Pseudomonas and Aerobacter bacteria seem to be the primary source of meat Spoilage (Ayres, 1960). Kirsh g; 31. (1952) noted a sour odor in beef after 8-12 days of refrigerated storage. The aerobic count when the odor was noted was 500,000,000 per gram which was close to the criti- cal slime value of 6 x 107 reported by Ayres (1960) and substantiated by Jay and Kontou (1964). Halleck ggugl. (1958) reported a foul odor when Pseudomonas-Aerobacter bacteria were incubated on veal infusion agar. As Pseudomonas and Aerobacter seemed to be the primary meat Spoilage organisms, a brief review of their characteristics would be helpful. Witter (1961), in a review of psychrOphilic bacteria, found Pseudom- ‘gggg and Aerobacter primarily soil contaminants, i.e., they are normally soil organisms. He reported psychrOphiles do not survive pastuerization, -5- are inhibited more extensively by hypochlorites than quarternary ammonia compounds, can grow in salt concentrations of 4 percent, are sensitive to antibiotics, and are sensitive to pH reduction. Pseudomonas bacteria are proteolytic bacteria as reported by many workers. Peterson and Gunderson (1960) found Pseudomonas fluorescens to have extracellular and endocellular proteolytic enzymes. They found at lower temperatures of incubation that the extracellular enzymes reacted more slowly as less enzyme was produced due to a slower rate of growth. The enzymes were very active during the logarithmic phase of growth. Van Der Zant (1957) reported extracellular enzymes were most active when the viable cell count was highest. Also he reported a pH of 6.5 to 8.0 resulted in the greatest activity. When the enzymes were heated to 60°C for 3 minutes they were inactivated. The greatest enzymatic activity was noted at temperatures of 30-50°C. Peterson and Gunderson (1960) found the endo- cellular enzymes to be 3 times more active at 10°C than at 35°C. Camp and Van Der Zant (1957) reported optimum endocellular enzyme activity at a pH of 7.0 to 8.0, but disagreed with Peterson and Gunderson's results as they had Optimum activity at 35°C and a decrease below 25°C. Camp and Van Der Zant (1957) reported endocellular enzymes were not influenced by heating for 8 minutes at 60°C at a pH of 7.0. 'Hurley‘gngg, (1963) reported the presence of ferrous ion increased the proteolytic activity of Pseudomonas.fluorescens. Ayres (1960) found Pseudomonas grew well up to a temperature of 25°C. He also reported at temperatures below 10°C pseudomonads were the only bacteria growing on meat samples. Extract Release Volume (ERV) Jay and Kontou (1964) evaluated a method for the rapid determination of the microbial quality of fresh meat. They found a high correlation between the extract-release volume (ERV) and the number of bacteria per gram. Jay (1964b) found the ERV decreased in a straight line relation- ship as the bacteria numbers increased. He found a mean ERV value of 24.6 ml corresponded with the onset of spoilage detectable by organolep- tic means. The bacterial numbers at this point averaged 3 x 108 bacteria per grams Jay and Kontou (1964) found the mean ERV value of sample rejection by a trained panel was 30.4 ml which correSponded to 7 x 107 bacteria per gram. Price gtual. (1965) found the onset of spoilage occurred when the ERV values were between 30 and 40 m1 and below 30 ml Spoilage was definite and bacterial numbers were very high. Jay (1964a) reported that an ERV was obtained at temperatures vary- ing from 7-37°C. He also found the maximum ERV was obtained at pH values between 5.0 and 5.8 while no ERV was obtained below pH 4.9 or above pH 11.0. Price gtngl. (1965) found the environmental and slurry tempera- tures varied the ERV. They reported the temperature was controlled most efficiently by running the procedure at cooler temperatures (2-6°C). Fat content below 20% did not affect ERV values in beef according to Jay (1964a). However, high amounts of fat tended to increase the ERV value. Price,gt a1. (1965) found the fat content and storage time of beef had an interaction which influenced the ERV value. They also stated the fat content of pork had no significant effect on ERV values. Aseptic Muscle Sampling weiaeriggugl, (1954) used antibiotics to reduce the bacterial numbers to prevent deep spoilage of beef tissue. They infused the beef rounds with antibiotics by a method similar to arterial pumping of hams and thus reduced the bacteriallnpulation to practically zero. The bacteria in the lymph nodes were reduced from 100 millions recorded in the controls to ten thousands in the infused rounds. Also a method of infusing the antibiotic in the animal via the jugular vein during the slaughter Opera- tion was very successful. .Aureomyocin was the antibiotic used and it degenerated during storage and was not present in the tissue when it reached the consumer. Ayres (1956) and Zender gtual. (1958) found normal muscle tissue in the living animal was sterile or relatively free of living microorganisms. Earlier workers (Jansen and Hess,f194l; and Lepovetsky gtflal,, 1953) found bacteria in muscle samples. The low numbers recovered, however, and the methods used for slaughter indicated a greater possibility of contamination than the microorganisms being present in the living muscle tissue. The genm-free work being conducted is based on the fact that the embryo of the various animals are free of microorganisms. Landy gtugl. (1961) claimed any laboratory animal used had bacteria, fungi, viruses, etc. in its system. These pe0ple were especially concerned with the di- gestive tract. Meyer gt 21. (1964) found pigs could be taken from the sow under sterile conditions and reared using sterile isolators, equip- ment, food, and water so there was no evidence of bacteria, fungi, viruses, etc. in any of the pigs' physiological systems. Ockerman gig. (1964) slaughtered and eviscerated germ-free mice in a sterilized isolator with sterilized equipment. The mouse carcasses were stored in sealed steri- lized tubes and no bacterial contamination was found. However, the carcasses did deteriorate due to proteolysis with those carcasses stored at higher temperature deteriorating at a more rapid rate. Zender EEEA- (1958) followed a very precise and aseptic means of excising muscle samples from rabbits and lambs. Using this method, they obtained samples which were practically void of contamination. Davis (1965) combined the methods of Zender EEEL (1958) and Ockerman 2.23—1- (1964). He slaughtered beef aseptically using sterilized sticking knives, one to cut the shaved, bacteriocidally washed skin, and the second to sever the carotid arteries. Other slaughter and eviscerating procedures were normal except a large patch of the hide was left intact over the loin area. This area was scrubbed with warm water and a bacteriocidal soap, rinsed, scrubbed again, and finally rinsed with 907. ethanol before storing the unsPlit carcass in a cooler. After the carcass had cooled sufficiently, the whole rough loin, after another alcohol rinse, was placed on a cart and a sterile isolator was attached to it and secured. Then by working through the gloves attached to the isolator a slit was made in the isolator and through the hide so the hide could be rolled from the exposed muscle. The muscle was excised, ground, and placed in sterile containers with all Operations taking place in the' isblauors. The samples were then stored up to 35 days at a temperature of 2-5°C with no evidence of bacterial contamination. -10... Proteins Skeletal muscle consists of fibers enclosed in a sheath of perimyeium penmeated with fat deposits and connective tissues. The contractile element is the fiber portion of the muscle. The fibers are multi- nucleated and are composed mainly of the myofibrillar proteins, myosin, actin, actomyosin, and tropomyosin. The perimysium sheath, fatty deposits, connective tissues, nerve tissues, and vascular tissues are composed of proteins which are known as the stroma proteins. The intercellular 'material or sarcoplasma is a liquid material containing proteins which are known as the sarcoplasmic proteins (Helander, 1957; and Whitaker, 1959). Sarc0plasmic Proteins Helander (1957) identified sarcoplasmic proteins as those muscle proteins which are soluble in water or low concentrations of salt and are characterized as globular, low viscosity, and low molecular weight proteins. He stated the primary sarcoplasmic proteins were myogen, myoalbumin, globulin X, and myoglobin. Whitaker (1959) stated the sarco- plasmic proteins were primarily enzymes. Sc0pes and Lawrie (1963) found 35 bands on starch gel electrophoresis from beef sarc0plasmic fractions. During the process of rigor mortis some of the bands were lost. Fujimdki and Deatherage (1964) fractionated sarcoPlasmic protein of beef skeletal ‘muscle on an ion-exchange cellulose chromatography column and found at least 14 different fractions. They also noted a loss in the number of peaks and the level of the peaks during storage and freeze-drying. Aberle -11.. and Merkel (1966) found 15 definable bands in starch gel electrophoretic patterns from the sarcoplasmic fraction of bovine muscle. Their results indicated that the bands increased in intensity and new zones appeared during aging. Hill (1962) found sarc0plaamic protein accounted for 15-20% of the total nitrogen in bovine muscle, 20-25% of the total nitrogen in porcine muscle, and approximately 25% of the total nitrogen in ovine muscle. Hegarty (1963) found similar results for bovine muscle as did Davis (1965). Topel (1965) found similar results for porcine muscle. The amount of sarc0p1asmic protein extracted depended on many environmental and physio- logical conditions including temperature, pH, age of carcass, degree of rigor mortis, etc. Seven different articles have reported that with aging sarc0p1asmic proteins decrease in all types of muscle. Scharpf and Marion (1964) reported this decrease in turkey muscle. Khan and Van Den Berg (1964a) found a decrease in the amount of sarc0p1asmic proteins in chicken muscle due to aging. Porcine muscle also showed this decrease according to Sayre and Briskey (1963) and McLoughlin (1963). Fujimaki (1962), Goll gt 31. (1964) and Davis (1965) also found beef muscle sarcoplasmic protein decreased in amount during aging. Hegarty (1963) found an in- crease in the amount of sarc0p1asmic protein from 0 to 24 hours post 'mortem and little change in the amount from 24 hours to 7 days post mortem in beef muscle. Aberle and Merkel (1966) reported a decrease in the sarcoplasmic fraction from 0 hour throughout a 336 hour storage post -12- mortem for the semi-tendinosus muscle of beef. However, they reported no change in the amount of sarc0p1asmic protein of the beef longissimus 'dgggiymuscle. Scopes and Lawrie (1963) and Scopes (1964) reported evi- dence of a sarcoplasmic precipitation on the myofibrillar proteins such that some of the sarc0p1asmic proteins were extracted with the myofibrillar fraction. Scharpf and Marion (1964) reported some sarc0p1asmic proteins were carried from the muscle sample with the exuded moisture. 'Most workers incorporated the exuded moisture into the muscle samples before testing. The pH of the sample influenced the amount of sarc0p1asmic protein extracted (Sc0pes, 1964). At pH values below 5.0, less than 10% of the protein was extractable. pH values of 5.6-5.7 gave the highest total extractability (sarcoplasmic and myofibrillar fractions) with little change at higher pH values. Helander (1957) found a pH of 7.4 was best for extracting sarcoplasmic proteins. Sc0pes and Lawrie (1963) reported that the pH fall associated with the onset of rigor mortis could account for part of the sarc0p1asmic precipitation noted in the aging of the muscle. Scopes (1964) reported storage temperatures near 37°C caused some denaturation of proteins and thus a loss in extractability. Khan and Van Den Berg (1964) reported there was no variation due to the influence of temperatures ranging from 0° to 5°C. TOpel (1965) reported pork muscles which were extremely soft and watery had a lower portion of extractable sarcoplasmic proteins than did the normal pork muscles. Khan and Van Den Berg (1964a) and Scharpf and -13.. Marion (1964) reported higher sarc0p1asmic fractions in the lighter muscles than in the darkeeruscles of chicken and turkey. This relation- ship held true for the "red meats" as reported by Hill (1962). The darker beef had a lower sarcoplasmic content than the lighter pork and lamb. Davis (1965) found that bacterially inoculated beef muscle had a higher sarcoplasmic content than the aseptic sample throughout the 35 day storage period. At the end of the 35 day storage period, the mean difference was 4 mg of protein per gram of fresh sample. Myofibrillar Proteins Helander (1957) identified the myofibrillar proteins as those muscle proteins not soluble in water or low salt concentrations but soluble in high salt concentrations. He characterized these proteins as fibrous, highly viscous, high molecular weight muscle proteins. They are the contractile proteins; myosin, actin, and their aggregation, actomyosin, plus trapomyosin and X-protein (Whitaker, 1959). Hill (1962) reported the myofibrillar protein fraction comprised approximately 55% of the total nitrogen of beef muscle, 53% of the total nitrogen of lamb muscle, and 56% of the total nitrogen of pork muscle. Davis (1965) found similar results for beef while Hegarty (1963) reported myofibrillar proteins accounted for about 66% of the total nitrogen in beef. Topel (1965) reported results similar to Hill's for pork. The amount of myofibrillar protein extractable from a muscle sample depends on environmental and physiological conditions similar to the sarcoplasmic proteins. It should be pointed out thatImuohTof the work -14- done concerning proteins of the muscle reports on the extractable pro- teins which would include the sarc0p1asmic and myofibrillar protein fractions. This work will be discussed in this section as 70-80% of the extractable proteins are myofibrillar proteins. The length of time a sample was stored had very little effect on the myofibrillar fraction according to Goll _e_t_:_ a_l_. (1964). Kronman and Winterbottom (1960) reported that 10-30% of the soluble nitrogen was lost during a 7 day aging period for beef. 'Moorjani (1962) found a loss of soluble protein when storing fish in crushed ice. The loss in solu- bility was thought to be due to a protein-protein interaction. Khan and Van Den Berg (1964a) found a slight decrease in the solubility of the myosin fraction but no change in the actomyosin fraction. Davis (1965) reported the myofibrillar fraction increased during the first 10 days of storage and then decreased slowly until the 35th day of storage when the myofibrillar content was the same as 48 hours post mortem or 0 days storage. The state of rigor mortis influences the extractability of the myo- fibrillar fraction. Saffle and Galbreath (1964) found pre-rigor beef had 50% more salt-soluble proteins than beef at 48 hours post mortem. Acto- myosin is formed at the onset of rigor mortis according to Wierbicki gt 313(1956) and Whitaker (1959). This is supported by Connell (1962) who found a reduction in the extractability of myosin during the formation of the actinrmyosin bond in rigor mortis. Baliga‘ggflgl. (1962) found a decrease in the salt-soluble fraction during the first 5 days post mortem -15- which was COUpled with the onset of rigor. This first decrease was recovered as the resolution of rigor took place. Then at 16 days a de- crease in the salt-soluble fraction was noted again which was not recoverable. This reaction was due to denaturation of the salt-soluble proteins. Khanand Van Den Berg (1964b) found similar results in poultry except the process was quicker due to a faster onset of rigor. They found a minimum anomt of extractability 4-8 hours post mortem which corresponded with the onset of rigor mortis. After rigor mortis was resolved in approximately two days the extractability reached a maximum and held fairly constant for ten days of aging. Helander (1957) reported satisfactory myofibrillar extraction at pH values ranging from 6.5 to 9.0. A low ultimate pH of meat resulted in lower nitrogen content in the myofibrillar extract according to'McLoughlin (1963) and Sayre and Briskey (1963). Both papers also reported that higher muscle pH caused less loss of extractability due to changes in rigor mortis and storage time. Kronman and Winterbottom (1960) found that during freezing of a bovine muscle sample the myofibrillar fraction decreased due to denaturation of the protein. Khan.gtugl. (1963) also indicated that the proteins denatured during frozen storage of chicken 'muscle caused a loss in myofibrillar solubility. At temperatures of 0-5°C, Khan.and Van Den Berg (1963a) found little difference in the ex- tractability of myofibrillar proteins. In beef samples, a high temperature (above 35°C) resulted in a severe loss of the myofibrillar fractions (Sayre and Briskey, 1963). T0pel (1965) found that pork tissue which -16.. was extremely soft and watery had a significantly lower quantity of myofibrillar protein. Davis (1965) reported that aseptic tissue had a slightly lower myofibrillar protein content for the first 10-15 days of storage than inoculated tissue. However, by the 35th day of storage the myofibrillar content of the aseptic tissue was higher than that of the inoculated tissue. Stroma Protein The stroma proteins are the connective tissues, nerve tissues, and vascular systems of the muscle tissue (Helander, 1957). He identified these proteins as being insoluble in either water or high salt concentra- tions. Whitaker (1959) identified the stroma proteins as collagen, elastin, reticulum, and ground substance. Hill (1962) reported that the stroma fraction contained 12-18% of the total nitrogen of bovine muscle, 8-12% of the total nitrogen of ovine muscle, and 7-10% of the total nitrogen of porcine muscle. Hegarty (1963) found the stroma content of bovine muscle to be slightly lower at 8% of the total nitrogen. Davis (1965) reported results similar to Hill's for bovine muscle. Topel (1965) reported similar results for porcine muscle. Very few reports are available on the effect of environmental and physiological changes on the stroma fractions. Davis (1965) found the stroma fraction decreased in bacterially inoculated beef muscle during a 35 day storage period while the stroma fraction of the aseptic sample remained constant or increased slightly. iMost other workers studied -17- exclusively the effect of various conditions on the myofibrillar, sarco- plasmic, and non-protein nitrogen fractions. Non-protein Nitrogen (NPN) Some of the nitrogen present in a muscle sample is not incorporated as a part of the protein material. This nitrogen is present as amino acids, ammonia, peptides, etc. All of these compounds are water soluble so they are fractionated with the sarc0p1asmic proteins. To determine the amount of NPN, the sarc0p1asmic fraction is treated with a 20% tri- chloracetic acid solution which percipitates the proteins. The nitrogen content determined on the remaining extract is the NPN. Hill (1962) found that NPN. accounted for 11-13‘7. of the total nitro- gen in beef, pork, and lamb. Hegarty (1963) found beef to have 9-10% of the total nitrogen in the NPN fraction. Davis (1965) found NPN values in these ranges for beef. Topel (1965) reported results similar to those of Hill for pork. NPN is influenced primarily by proteolytic activity. There are two sources of proteolytic activity in a muscle sample; proteo- lytic enzymes found in the muscle and proteolytic activity of enzymes from outside sources such as bacteria. Khangtgl. (1963), Van Den Berg 2511. (1963), and Khan and Van Den Berg (l963a,b) reported the NPN con- tent of chicken muscle increased during a storage time of 5 weeks with bacterial growth kept to a minimum with chemical treatment. Scharpf and Marion (1964) obtained the same results with turkey muscle. Sharp (1963) found during storage of rabbit and beef muscle there was an increase in the amount of NPN. Aberle and Merkel (1966) also found this increase in -13- beef. Davis (1965) found aseptic beef samples had an increase in NPN during a 35 day storage period but bacterially inoculated beef had a greater increase in NPN content. Emulsifying Capacity An emulsion is a dispersion of one liquid in another, the liquids being immiscible (Jirgensons and Straumanis, 1962). An emulsion can be prepared by shaking the liquidatogether causing a dispersion of one liquid into the other. However, this emulsion would be very unstable, so emulsifying agents would be used to increase stability. These agents lower interfacial tension and aid the formation of stable draplets which are then surrounded by the continuous phase of the emulsion. A.meat emulsion may not be a true emulsion in that some solid mater- ial is present in the muscle tissue. Hansen (1960) found that a meat emulsion was essentially a fat or oil dispersed in water with the protein of the muscle tissue acting as the emulsifying agent. Studies by Pearson Iggflgl. (1965) indicated that protein from extenders such as nonfat dry milk, soy sodium proteinate, and potassium caseinate, though they emulsify fat provided very little emulsifying capacity to a meat emulsion contain- ing them. Meyer gt 2;! (1964) found the emulsifying agents used in chemical work, such as the diglycerides and some of their derivatives, do not aid in the fbrmation of a meat emulsion. Actually these workers found such emulsifiers incorporated in a meat emulsion hindered the formation of a stable emulsion. They thought there were two reasons for the lack of stability. First, the emulsifiers caused the fat droplets -19- to become so small the protein present in the emulsion could not cover the dr0plets. Second, they found evidence of a reaction between the protein and the emulsifiers causing denaturation of the protein and thus the emulsion broke down. Since Hansen (1960) has found that proteins are the primary emulsi- fying agents in.meat emulsions, others have studied this aspect of sausage emulsions. Swift 25 El. (1961), while devising a method for determining emulsifying capacity, found the salt soluble proteins were efficient emulsifying proteins. Fukatawa (1961) found the salt-soluble myosin A was essential in the emulsification of fat in a sausage. Traut- man (1964) reported pre-rigor meat had higher emulsifying capacities than post rigor meat. He studied this thoroughly and found pre-rigor meat had a higher amount of salt-soluble protein which accounted for the greater emulsifying capacity. He found the water-soluble proteins formed weak emulsions which were readily separated into oil and water phases. These results were not in agreement with those of Hegarty gtflgl. (1963). They found the sarc0p1asmic proteins formed a very stable emulsion at the pH of normal fresh meat. They also reported salt-soluble proteins produced very stable emulsions. Swift 2; 31. (1961) found the emulsifying capacity of meat increased with an increasing concentration of salt. Swift and Sulzbacher (1963) carried their work a little further and reported that the water-soluble proteins had increases in emulsifying capacity with increasing concentra- tions of salt. Vold and Groot (1962, 1964) studied this phenomena in laboratory emulsions and reported that the salt is adsorbed at the inter- face forming a more stable emulsion. Thus salt is important in the forma- tion of meat emulsions. -20- Chapping temperature is another important factor in the formation of meat emulsions. Hansen (1960) used a slow chop and found at tempera- tures below 11°C the protein matrix was not formed, and above 23°C the matrix broke down by denaturation. Chopping temperatures around 15-l9°C proved to form more stable emulsions with the desired size of fat dr0plet. Helmer and Saffle (1963) found similar results using a high Speed chopper. Hansen (1960) and Helmer and Saffle (1963) reported that chapping for extended periods of time caused a decrease in the size of the fat gldbule and thus a weakening of the emulsion. This weakening was due to the overextension of the protein. Davis (1965) found the emulsifying capacity of beef inoculated with microorganisms had a slight increase during a 35 day storage period at 2-5°C. This inoculated muscle also had a higher emulsion capacity than the aseptic tissue after the 5th day of storage. The aseptic tissue had a constant emulsifying capacity throughout the storage period. EXPERIMENTAL METHODS Slaughter The 180-230 lb. pigs used in this study were produced by the Michigan State University Farms and brought into the Meat Laboratory the evening before or on the morning of slaughter. The animals were stunned with an electric stunner and then hoisted by one rear leg. The sticking area of the neck was scrubbed thoroughly with a warm solution of pHisdHex bacter- iocidal soap. An alcohol-flame sterilized knife was used to stick the hog which was then allowed to bleed to death by heart action. The hog was placed in the scalding tank to loosen the hair and then transferred to the dehairer where the hair was removed. The evisceration, dr0pping the head, and further cleaning of the carcass were accomplished in a normal manner. The carcass was not Split, however. Before placing the carcass in the cooler, it was thoroughly rinsed with alcohol. The slaughter procedure was similar to that used by Davis (1965) except shav- ing the neck was not accomplished due to the difficulties encountered. Excising the Muscle Sample The carcass was chilled in the cooler for 20 hrs after which it was removed and again rinsed with alcohol. The carcass was placed on a kraft paper covered table where the shoulders and hams were removed at normal locations from the unaplit middle. The middle was then placed on the table so the midline of the external fat cover was easily accessible. Using a sterilized knife, a cut was made down the midline of the loin -21- -22- backfat cover, then two cuts were made perpendicular to the midline cut and about 5-8 cm.in from each end. The backfat was then stripped and rolled back to expose the longissfimus dorsi muscle which was then excised in 3 cm pieces and placed in sterile containers. The second muscle was excised following the same procedure. The person handling the knife wore sterile disposable plastic gloves during the sample procurement. The gloves were changed and the sterilized knife replaced by another between excising the first and second muscles. The tissue excised from each side of the loin was kept separate but was treated similarly during the excising Operations. The excised samples were transferred to a 4-6°C cooler where they were ground through a sterilized pre-chilled grinder (4-6°C) into sterilized Mason quart jars. A two millimeter plate was used for grinding the sample. The jars con- taining the ground sample were placed in an insulated plastic bag with dry ice and then transported to the Eckert Packing Company Laboratory, Defiance, Ohio, for further study. The excising procedure was similar to that used by Zender g£_§L. (1958) Davis (1965) used a comparable method but the excising and grinding took place in sterilized isolatOrs. Inoculation of the Sample One loin sample was removed from.the Mason jar and placed on a sterile tray. Using an alcohol-flame sterilized atomizer, 50 ml of ster- ile APT broth were Sprayed over the sample and mixed into the sample with a sterilized fork. This sample was then divided into six sterilized -23- sample bottles, each containing approximately 200 grams of sample. Five of the bottles were placed in a refrigerator case and stored at a temper- ature of 4-6°C. Caps were placed on the bottles but they were not tightly sealed. The samples were removed and analyzed at l, 5, 9, 13, and 17 days of storage considering the 6th sample taken immediately after Spraying as control sample, day 0. The other ground loin sample was removed from the Mason jar and placed on the same tray used for spraying the control sample. This sample was then Sprayed with 50 ml of the inoculum and handled the same as the control samples. This sample was the inoculated sample. Inoculum Preparation The inoculum was prepared by placing 15 grams of pork trim, taken from the packing house, in 150 ml of sterilized APT broth (Difco) which was held at 4-6°C in a loosely capped, flat, 500 milliliter bottle which was laid on the flat surface. After 5 days of incubation, 50 ml of the mixture was removed and Sprayed on the inoculated sample. In an attempt to have the inoculum contain the same type and relative number of microorganisms, the remaining inoculum was held at 4-6°C in an upright position for 23 days. At that time 15 ml of the inoculated broth were removed and placed in 150 ml of freshly prepared and sterilized APT broth. This was then held 5 days in the flat position at 4-6°C and 50 ml of the resultant broth were used to inoculate a succeeding loin sample. -24- Bacterial Numbers The method outlined by the American Public Health Association (1958) was used. Eleven grams of sample were blended in a Sterile blender with 99 ml of sterile water. This slurry was then appropriately diluted and 1.0 ml or 0.1 ml pipetted into sterile petri dishes. Tryptone glucose 25". extract agar was used as the incubating media. The plates were incu- : bated 72 hours at 32°C after which the colonies were counted and reported 3 as the number per gram of sample. Protein Extraction @' The method used for extracting the different protein fractions was Similar to that used by Helander (1957) and is outlined in Figure 1. Davis (1965) and TOpel (1965) also used modifications of Helander's method and provided sources of information for the method outlined. All extractions were accomplished at 4-6°C. Ten grams of the meat sample were weighed and blended with 35 m1 Of 0.03M phosphate buffer, pH 7.4, for 30 seconds. The slurry was transferred to a 300 m1 centrifuge bottle. The blender was then rinsed with successive 15 ml, 25 m1, and 25 ml of 0.03M phosphate buffer with all rinses being placed in the centrifuge bottle. A Magna-Stirrer bar was added to the bottle and the bottle was placed on a Magus-Stirrer where the solution was gently agitated for 30 minutes. The solution was then centrifuged 20 ‘minutes at 1400 x G. Supernatant I was filtered through glass wool into a 250 m1 flask. One hundred ml of 0.03M phosphate buffer were added to the centrifuge bottle and mixed with Residue I. This was -25- Muscle Sample 10 gms. blended in 35 m1 of 0.03M phosphate buffer, pH 7.4 Transfer to 300 ml centrifuge bottles by 15, 25, and 25 m1 rinses of .03M buffer Extract for 30 minutes with gentle agitation Centrifuge 20 min @ 1,400 X G and filter through glass wool [47 I Resxdue I Supernatant I I__ add 100 m1 of .03M buffer mix, reextract for 30 min. centrifuge 20 min @ 1400 X G and filter through glass wool l i Residue II SupernaEant II add 100 ml of 1.1M KI, Combine SUpernatant mix, extract for 1 hr I and II centrifuge 20 min @ 1400 X G and filter through glass wool Make to 250 m1 f 1 volume Residue III Supernatant III repeat as for Fraction I residue II I fi fi Residue IV Supernatant IV 50 ml 50 ml aliquot l aliq t J repeat as for 50 1 of 20% residue II TCA for 12 hr I 1 Total nitrogen @ 2-6°C Residue V Sgpgrngggnt y L_ analysis _L_ rr’ I combine SUper- Serotplasmic & NPN ppt Filtrate Total Nitrogen natant LII, IV, V analysis make to 350 m1 sol. 50 m1 aliquot 50 ml 20% TCA Stroma protein Fraction II Total Nitrogen (NPN) 50 m1 aliquot Total Nitrogen analysis Fibrillar protein Figure 1. Outline of Protein Fractionation. -26.. agitated for 30 minutes and again centrifuged and filtered. Supernatants I and II were combined and diluted to 250 ml. Protein analysis was then made on Fraction I and recorded as sarc0p1asmic protein nitrogen and non- protein nitrogen. Residue II was suspended in 100 m1 of 1.1M KI, 0.1M phOSphate buffer solution. This mixture was agitated for 1 hour and was then centrifuged for 20 minutes at 1400 X G. Supernatant III was filtered through glass wool and held in a 500 ml flask. Residue III was suSpended in 100 ml of 1.1M KI, 0.1M phOSphate buffer solution, agitated for 1 hour, centrifuged 20 minutes at 1400 X G, and Supernatant IV was filtered through glass wool and held with Supernatant III in a 500 m1 flask. Residue IV repeated the same process as Residue III. Supernatants III, IV, and V were com- bined and diluted to 350 ml (Fraction II) and the fibrillar protein was determined on a 50 ml aliquot of Fraction II. Residue V was added to a Kjeldahl flask for protein determination and designated as the stroma protein. The non-protein nitrogen (NPN) was determined by taking a 50 ml aliquot of Fraction I and adding 50 m1 of 20% trichloroacetic acid solu- tion. This was held for 12 hours at 4-6°C and was then filtered through Whatman NO. 1 filter paper. A.50 ml aliquot of the filtrate was used for determining the amount of NPN. The amount of NPN was subtracted from the amount of nitrogen found in Fraction I and the remainder was desig- nated as the sarc0p1asmic protein. ‘me I 3?. .Lxmfla-fifiaj ‘ . . '1 a, -27- Emulsion Capacity The method used was Similar to that reported by Swift (1961). Twenty-five grams of sample were blended in 100 m1 of cold (2-6°C) 1.0M NaCl solution in a Wering blender for 2 minutes. 6.25 grams of the re- sultant slurry were placed in a Mason quart jar. Then 37.5 ml of cold 1.0M NaCl solution and 25 m1 of once-refined soybean oil (from Central Soya Co.) were added to the jar. The mixture was stirred at approximately 1750 rpm with a Lightnin Model F mixer equipped with an Open 3 bladed- prOpeller. Once-refined soybean oil was added at a rate of approximately 1 m1/sec from.a 750 ml separatory funnel. An emulsion was formed which was noted by an increasing viscosity and a fine honeycomb-like appearance. The endpoint was reached when the viscosity was suddenly lost and the mixture had an oil-like appearance. The amount of oil used was then measured by pouring oil into the separatory funnel from a 500 ml graduated cylinder recording the amount required to refill the funnel plus the 25 m1 added as the emulsion was started. The emulsifying capacity per 100 milligrams of total protein was then calculated. Extract Release Volume (ERV) The procedure followed was first reported by Jay (1964a). Twenty- five grams of sample tempered at 32°C for at least 1 hour were blended with 100 m1 of distilled water for 2 minutes. The slurry was poured into a funnel equipped with a piece of Whatman No. 1 filter paper folded thrice to yield eight Sides. The filtrate was collected in a 100 m1 graduated cylinder by running the filtrate down the side of the graduated cylinder. -28.. After 15 minutes, the volume of the filtrate was recorded as the Extract Release Volume (ERV). The filtration was carried out in a constant temperature chamber at 32°C. pH Determination - The pH of the samples was determined by blending 10 grams of sample in 100 ml of distilled water for 30 seconds. The pH was then taken using a Beckman Zero-matic pH meter. Protein Determination The Kjeldahl method was used as outlined by the A.0.A.C. (1960). A pre-mixed package of the catalysts, called Kelpac No. 5 was used in the digestion flask. The nitrogen content was multiplied by 6.25 to get the protein content. Moisture Determination The A.0.A.C. (1960) method of drying 2.5-3.0 grams of sample for 3 hours at 125°C was used. Statistical Analysis Analysis of variance was done as outlined by Guenther (1964). The data which indicated a significant difference by analysis of variance were further analyzed by ranking and comparing means by Duncan's new multiple range test (Steel and Torrie, 1960). RESULTS AND DISCUSSION Bacterial Zender gtgal. (1958) obtained lamb and rabbit muscle samples which were practically sterile. Ockerman st 31. (1964) processed mice so that bacteria-free carcasses were Stored and studied. These two studies, however, did not compare aseptic samples with contaminated samples as did Davis (1965) in his work. He used sterile isolators to Obtain asep- tic beef samples and stored these samples for 35 days without evidence of bacterial contamination. The study reported here was undertaken to determine if aseptic samples could be obtained using methods not requiring isolators and to use such samples to compare with bacterially inoculated samples. Figure 2 Shows the control samples were found to be slightly contam- inated using the methods outlined previously. However, the controls had a bacterial count of approximately 10 on day 0 and day l which is a very low count for any meat sample. By day 9 the control had a bacterial count of slightly more than 100 which was much less than the 100 million counted in the inoculated samples. From the 9th day of storage, the bacterial numbers of the control increased to a count of 1 million at the 17th day of storage. This count was about the same as or less than the 1-95 million bacteria per gram reported by Kirsh ggual. (1952) in fresh ground beef purchased at retail stores. Also the 1 million bacteria per gram of the control was less than the critical slime or rejection values of 60-500 -29- -30- vouoHaooea no. aouuooo mo aluw\-uoea=a gowuouoon mo mod «no mo manuaoeuofiou van-:83 0.. .0 3.380 I awaken. mo amen . w .nodeauo .N Ouamam N 1 ~c ".18 red orequnu oraeuooq J0 801 1 Q -31- million reported by Kirsh 25 a1. (1952), Ayres (1960), and Jay and Kontou (1964). The value was also less than the 450 million recorded in the inoculated sample.- Figure 2 Shows the number of bacteria per gram of tissue in the inoculated sample started at approximately 5 million on day 0 but then decreased to approximately 100,000 on day 1. This decrease probably resulted from the need for the bacteria in the inoculum to be- come acclimated to the pork tissue. After the decrease at day l, the bacterial numbers increased rapidly until day 9 which corresponds to a bacterial count of slightly more than 150 million. The bacterial load continued to increase but at a much slower rate as the count at day 17 was approximately 450 million.. An off-odor, slimy, off-color condition was noted between the 5th and 9th days in the inoculated sample, which corresponds to the critical slime value of 60 million reported by Ayres (1960) and Jay and Kontou (1964). Davis (1965) reported slime and Off- Odor at 15 days of storage with a bacterial count of 35 million. However, his initial contamination of 30,000 was less than the 5 million obtained in this study, which could cause a slower onset of Spoilage. Pseudomonas and Aerobacter bacteria have been reported as the pri- mary meat spoilage organisms (Ayres, 1960). In order to determine what organisms were added to the inoculated sample in this study, a portion of an inoculum handled similarly to the ones used on the pork loin samples was sent to Hillt0p Milk and Food Laboratories, Cincinnati, Ohio, for gross identification of the microorganisms present. The inoculum was sampled at 5 days, then 3 additional samples were taken at 4 week inter- vals, or in other words, a culture was sent at the same time a sample -32.. would have been inoculated if this inoculum would have been used. The ratio of the different bacteria types are shown in Table l. The Gram- positive diplococci were identified grossly as bacteria of the genera Leuconostoc and Lactobacillus. Non-pathogenic bacteria of the Clostri- dium genus were the primary Gram-positive spore-forming bacillus organisms. fix “YA The genera found in the Gram-negative rods were Escherichia and Aero- bacter. Therefore, only a small portion of the bacteria added to the inoculated sample was of the genus Aerobacter and none of the genus tr‘i-JC‘Y" Tfi. ‘ Pseudomonas. Kirsh E; El. (1952) reported that Lactobacillus was present in meat samples and Ayres (1960) found Clostridium organisms were also present. The ratio of the different types Of organisms changed during storage of the inoculum but the Gram-positive diplococci were the pre- dominant organism throughout the storage time. One cause Of this pre- dominance was that the APT broth used for preparing the inoculum was for the cultivation of heterofermentative Lactobacillus and other organisms requiring a high thiamine content. Table 1. Ratio of bacterial types in inoculum cultures Bacteria / Culture NO. 1 2 3 4 Gram-positive diplococci 9 18 2000 1500 Gram-positive spore-forming bacillus 5 2 80 280 Gram-negative rods 1 1 l l -33- Extract Release Volume (ERV) The extract release volume (ERV) of the control and inoculated samples are compared in Figure 3. The ERV of the control decreased from 55.0 ml at day 0 to 47.0 ml at day 17. However, the decrease was very slight when compared to the decrease in the ERV of the inoculated sample from 53.0 ml at day 0 to 30.0 ml at day 17. The ERV of the control gradually decreased to approximately 51.0 ml at day 5, then was constant until day 13 at which time it decreased moderately to 47.0 ml at day 17. The ERV of the inoculated sample changed very little from day 0 to day 1. However, from.day l to day 9 there was a very fast decrease from 53.0 ml to 29.0 ml. A Slight increase was noted at day 13 but at day 17 the ERV was again near 29.0 ml indicating a Steadying trend. Jay and Kontou (1964) found a high correlation between the ERV and the number Of bacteria per gram. Jay (1964b) reported the ERV decreased in a straight line relationship as the bacteria numbers increased. This relationship can be seen by comparing Figures 2 and 3. As the bacteria numbers increased rapidly in the inoculated sample from day 1 to day 9, the ERV decreased rapidly. This relationship was also found in the control sample at day 17. Also, as the bacterial growth of the inoculated sample equilibrated from day 9 to day 17, the ERV also tended to hold constant. An analysis of variance was completed on the ERV data and the results are summarized in Table 2. A significant difference was found between the treatments of samples or between the inoculated and control samples at the Significance level of 1%. Also, the results indicated that time -34- .aneao- vouoasooaa new douuooO «0 2mm one no aaaaaoauuaum .m Ouawwm unencuu no when ha ma a n .IM . a d u d OmN nYI .\\ .\kU// \\ 1x \\ 1 o.Nm /O\ / / woulgooaH 0|. IO 1 6.0m #9330 I . z’ ,/ Im.‘Axs . o.ma / MVI: a o.~m 0.0m -35.. Table 2. Analysis of variance of ERV among two treatments and 6 time periods Sum of Degrees of Mean Source squares freedom square F value Treatments 3078.2 1 3078.2 37.73** periods 3224.4 5 644.9 7.90** interaction 1337.3 5 267.5 3.28* error 5874.8 72 81.6 Total l3,514.7 83 **Significant at P < .01 *significant at P < .05 or periods caused a significant variation at the 1% level while a bacteria and time interaction was Significant at the 5% level. However, when the data were analyzed by Duncan's New Multiple Range Test (Steel and Lawrie, 1960), it was found to be divided into two groups (Table 3). Except for control mean, day 17, and inoculated mean, day 5, all of the control means and the inoculated means from day 0 and day 1 are significantly different from the inoculated means day 5 to day 17. These results indicate the number of bacteria present had the greatest influence on the ERV. The addition of a high number of bacteria to the inoculated sample at day 0 did not alter the ERV very much. This indicates the ERV decreased due to the action of the growing bacteria rather than the presence of the bacteria on the sample. This may also partially account for the variation due to time and the time-bacteria interaction. -36- Table 3. Ranking of ERV means with indication of Significance by Duncan's New Multiple Range Test Rank 123456789101112 Sample+ c-o c-1 I-0 1-1 c-5 c-13 c-9 c-17 1-51-131-17 1-9 Mean, m1 53.1 53.6 53.2 52.9 50.9 50.9 50.8 46.9 37.3 32.9 29.9 29.2 +C = control I = inoculated Number = days of storage Those means underlined by the same line are not significantly different at the 1% level. An off-odor, slimy condition was noted between day 5 and day 9 in the inoculated sample which corresponded to an ERV value of 37.3 ml to 29.2 ml. Price 25 31. (1965) found the onset of spoilage occurred when the ERV value ranged between 30 and 40 m1 and the sample was definitely Spoiled when the ERV was below 30 ml. Jay and Kontou (1964) reported a rejection of meat samples by a trained panel when the mean ERV value was 30.4 ml. Therefore, the results of this study were in close agreement with those of other workers in this area. Proteins Sarc0plasmic In this study the amount of sarcoplasmic protein nitrogen in the control and inoculated samples of pork loin decreased with storage time (Figure 4), which agreed with the results reported by Sayre and Briskey (1963) and McLoughlin (1963). The inoculated sample lost sarc0p1asmic protein nitrogen at a more rapid rate than the control sample similar.- £03an wound-coo.“ one Houuaoo mo cowouuac 530nm oaaooHeoouo-w 05 mo 3.3.333} .o 0.33m amououm wo when . a m a 1% umH - W - .1 a 13 wound.- 006H 0:! IO I Hon-coo I .. 6 rx .4 ueSonIu 1330:: En oouueSonpu uyanoxd opmndooxts JO 8H -37- l I i‘ / / a -38- to the results reported by Davis (1965). However, statistical analysis did not indicate any significant difference due to the inoculation. The loss of sarc0p1asmic protein nitrogen due to aging was significant at the 1% level (Table 4). The loss of sarcoplasmic protein nitrogen in the inoculated sample appeared to be most rapid during the period of greatest bacterial growth, day 1 - day 9. After day 9, the sarc0p1asmic protein nitrogen remained relatively constant as did the bacterial numbers. In both samples there was an increase in the sarc0p1asmic protein at day 1, with a greater increase in the inoculated sample. Table 4. Analysis of variance of sarCOplasmic protein nitrogen among two treatments and six time periods Degrees of Mean F Source Sum of squares freedom Square value treatments 24. 9 1 24. 90 1 . 80 periods 224.3 5 44.86 3.34** interaction 39.1 5 7.82 .58 error 953.0 71 Total 1241.3 82 **Significant at P < .01 The sarcoplasmic protein nitrogen values ranged from 18-23% which ‘were the same as the 21-23% reported by Hill (1962). -39- Myofibrillar The myofibrillar protein nitrogen of the control and inoculated samples followed a similar pattern until day 17 at which time the myofi- brillar protein nitrogen of the controls increased as the inoculated samples decreased. Figure 5 shows the myofibrillar protein nitrogen was lowest at day 0 then increased until day 9 at which time it decreased to day 13. The low myofibrillar extractability at day 0 or 1 day post-mortem could be due to the state of rigor of the sample as Baliga g; 21. (1962) found a decrease in the salt soluble protein fraction during the onset of rigor, then as the resolution of rigor took place an increase was noted. As this study started 1 day post-mortem, the sample should have been in rigor and thus the low extractability on day 0, then as the resolution of rigor took place the myofibrillar protein nitrogen increased for a period of time. This general pattern was followed by the inoculated and control samples. At day 9 a second decrease was noted in both samples which was similar to results found by Baliga 25 31. (1962) which they re- ported was caused by denaturation of the proteins and was not recoverable. However, the control sample did Show another increase at day 17 indicating the decrease in myofibrillar protein was recoverable. The inoculated sample had a lower myofibrillar protein nitrogen extractability initially but reached a higher peak than the control. However, by the 17th day of storage the control again had a higher myofibrillar protein nitrogen fraction. Davis (1965) found the inoculated tissue in his study had a higher myofibrillar protein fraction for the first 10-15 days of storage .manEdm caudaaoooa one aouuaoo mo cowouuao :aououe uoaawunamoza one mo manmoowuoaum .m ouawwm nouoasoooH 0| :0 Houucoo I -40- l 06¢ o.~¢ [1330131211 {930: Sn: OOI/uOSOJHu upenoxd IRIIIJQIJOA“! J0 3N -41- than the aseptic tissue but by the 35th day of storage the aseptic tissue had a higher myofibrillar protein nitrogen fraction than the inoculated sample. The myofibrillar protein nitrogen values averaged between 38-48% for 7 trials of control and inoculated samples which were much lower than the 56% reported by Hill (1962). Appendix C gives the results from all trials and it can be noted that most results were in the range of 45-55 mg of myofibrillar protein nitrogen per 100 mg of total nitrogen except trial 3 which ranged from 26-31. Samples from trials 6 and 7 also had low myofibrillar protein nitrogen fractions. Stroma The stroma protein nitrogen of the control and inoculated samples followed the same general pattern except for day 0 and day 17 (Figure 6). The stroma protein nitrogen at day 0 was higher in the control sample than in the inoculated sample. However, the control decreased Sharply while the inoculated increased very slightly, thus at day 1 the inoculated sample had a stroma protein nitrogen extractability which was approxi- mately 1 mg stroma protein nitrogen/100 mg total protein nitrogen higher than the control sample which remained constant until day 17 at which time the inoculated sample had a much higher stroma value than the control. These results are not in agreement with those of Davis (1965) who found the stroma fraction decreased during storage in the bacterially inoculated samples. By comparing figures 5 and 6, it appears that the stroma and .noaeeum mundanooaa vac Houocoo mo comouuwc oaououe meouuu one mo ewnmaoauoaum .o ouswaw oweuoua mo choc NH ma . m n H o . J . I m . In H 3N d vouoHaooaH 0| 10. L U [\ H I. Houuaoo I -42- ueSoquu 19303 ;o 8m OOI/uaSoxnru UIBJOld smoaas go li'ii -43- myofibrillar fractions were inversely dependent on each other. This was especially evident at day 17 when the greatest divergence between the control and inoculated samples was noted. In the control sample the myo- fibrillar fraction increased while stroma fraction decreased while the Opposite results were noted in the inoculated samples. The average value of 19.66 mg of stroma protein nitrogen per 100 mg of total nitrogen was much higher than the average of 7-10% found by Hill (1962) in porcine muscle. Appendix D lists the results from each trial and it can be seen that trial 3 had an extremely high stroma fraction and trials 6 and 7 are also rather high. Those 3 runs can account for the high average of stroma protein nitrogen obtained in this study. It also stressed the dependence of the stroma and myofibrillar fractions on each other as the Opposite was true of the myofibrillar fraction. Nonprotein Nitrogen Except for day l the NPN values of the control and inoculated samples were relatively constant (Figure 7). The increase of NPN found at day l in the control sample may be the result of the decrease noted in the stroma fraction. The NPN fraction of both samples increased gradually during the storage period indicating that proteolytic activity was taking place due to aging as reported by many other workers. There was little difference between the control and inoculated samples during the 17 day storage period. As there was little difference, it indicated that pro- teolytic bacteria were not present in the inoculum which had been substan- tiated previously. Therefore, these results do not agree with those of .moaeewm woumHDOOCw poo Hoeucoo mo cowouuac eaoooeecoc one we daemCOHumamm owmeOum mo expo NA ma a a m - -44- - emu-3:09; 0| .6 HOE-coo I .m oeswwm O .—4 N H \1' .—a \D H a) H usfioxgru 19303 50 Sm OOI/USSOJJTu ugajoiduou 50 SN -45- Davis (1965) who found a greater NPN fraction in the inoculated sample than in the aseptic sample. The average NPN value in this study was 13.32 mg of NPN per 100 mg of total nitrogen which was close to the 11-13% NPN reported for pork by Hill (1962). Emulsifying Capacity Figure 8 indicates the relationship of the emulsifying capacities of the control samples to those of the inoculated samples. Both types of samples have increasing emulsifying capacities from day 0 to day 1. From day l to day 5 the emulsifying capacity of the control remained relatively the same while the emulsifying capacity of the inoculated sample.decreased rather sharply." The sharp decrease in the emulsifying capacity of the inoculated sample continued until day 9 as the control decreased sharply from day 5 to day 9. From day 9 the control sample continued to gradually lose emulsifying capacity until day 17. However, during the same time period the emulsifying capacity of the inoculated sample became constant and then increased Sharply. Swift gtual. (1961) found the salt-soluble proteins were the primary emulsifying proteins. By comparing Figures 5 and 8 it can be seen that the emulsifying capacity did increase as the salt-soluble or myofibrillar protein fraction increased between day 0 and day 1. However, from that point the two curves do not follow each other. As the myofibrillar protein nitrogen fraction reached a peak at day 9, the emulsifying capacities were at the lowest point of a sharp decrease. Then at day 17 when the control myofibrillar protein .manawm neuoasuoaa poo Houucoo mo moauaoweoo waazwamHDEo mo nanmsoauoaom .m oeswam owouOum mo when S m . m m a o . \ I. I \ , u \ / .12: w. \ / I \ S m. ex / m B I 6.02 u O T». . / m 6 .., “e m I % Lv.m¢.~ m on / \ d m / - .I / \Jv.OON u / I _ / .SN nouoHDOOCH nYI nAu ‘ / Houuaoo I I led: -47- nitrogen increased, the control emulsifying capacity continued to de- crease, and as the inoculated myofibrillar protein nitrogen decreased, the inoculated emulsifying capacity increased. Hegarty 25 El. (1963) reported that emulsifying capacities were not entirely dependent on the myofibrillar proteins as they formed emulsions with sarcoplasmic pro- teins also. The results of this study also do not agree with those of Davis (1965) who found inoculated tissue had a Slight increase in emul- sifying capacity during storage while the aseptic sample remained the same. The results of this study indicate that during the time the bacteria increased in numbers most rapidly the emulsifying capacity decreased most rapidly. However this relationship was not Significant statistically. pH AS many of the previously discussed subjects, such as protein ex- tractability, emulsifying capacities, and ERV have been reported to be dependent on pH, the pH results Shown in Figure 9 will be discussed. The average pH changed Slightly until day 9 at which time the inoculated tissue had a slight increase in pH while the control remained constant. The change was about 0.7 pH units which Should not have influenced the results as the protein extraction was buffered and the ERV and emulsifying capacities are not that sensitive to pH changes. However, Appendix H gives the results of the individual trials and the changes were not as small as Figure 9 portrays. All of the control samples remained rela- tively constant except Trial 7 which varied almost 1 pH unit. The inocu- lated samples varied, however. Trials 1, 5, 6, 7 tended to have an increasing pH, while Trials 2 and 3 decreased in pH and Trial 4 remained lllllll .Ilulvil 1'} 1. I . II I -43- .moHnEwm mmumHsooaH mam Houuaoo mo in mo aHnmcoHumHmm .0 oustm owmeoum mo when 5 MH m .m H 0 . d a - - 4 4 8 m . noumHaoocH 0| IO see-a..- I 1 8..- L 00. 00.0 00. m -49- relatively constant. The variation in pH is due to the predominant bac- teria genera. It has been previously reported in this manuscript that Lactobacillus was one of the predominant genera in the inoculum. If these bacteria grew rapidly on the sample, the pH would decrease. Thus, the primary growing organisms in Trials 2 and 3 were probably the nggg— bacillus bacteria. Mbst other bacteria reported would cause an increase in the pH of the sample and would be the predominant organisms in Trials 1, 5, 6, and 7. The variation in pH noted in Appendix H could influence the results of protein extractability, ERV and emulsifying capacity. This was eSpecially true of the two trials with the ultimate low pH. The buffering power of the protein extract buffers may have been overcome. The ERV was non-existent or very low with pH below 4.9 (Jay, 1964a), and emulsifying capacity was affected by the insolubility of the protein. Davis (1965) found an increasing pH in his inoculated sample which in- creased 1.5 pH units by the 35th day of storage. SUMMARY AND CONCLUSIONS A procedure for slaughtering and processing pork was used to obtain muscle samples relatively free of bacteria. The procedure did not pro- vide sterile samples but they had very low counts of 10-100 at the time the samples were placed in storage. The samples had counts of 10,000 bacteria per gram or less until the 17th day of storage when the count was over 1 million. By comparing the samples obtained by this method with a dUplicate sample which was inoculated with a bacterial culture such that the ini- tial bacterial count was 5 million per gram at day 0, it was found bacteria had a great influence on the ERV. AS the bacteria grew in numbers, the ERV decreased rapidly. Also, the ERV of the control and inoculated samples were similar at day 0 and day 1 indicating that the growth of the bacteria rather than the number of bacteria caused the change in the ERV value. The protein extractability changed with storage time but the bacteria present in the inoculated tissue altered the protein extractability slightly. The sarc0p1asmic protein fraction had a noticeable difference between the control and inoculated samples but the difference was not significant statistically. The emulsifying capacity was lower for the inoculated tissue than for the control tissue throughout the 17 day Storage period. The reason for this is not known as there was no evidence of protein breakdown in the inoculated sample by a greater NPN content. -50... -51- The results of this study are inconclusive in many areas, such as the bacterial influence on protein extractability and emulsifying capacity which exhibited trends but no definite results. ‘MOre study should be directed in the area of controlled bacterial types. It is the contention of this author that if the predominant organism in a meat sample was Pseudomonas there would be protein breakdown by proteolytic activity. Also, Lactobacillus as the predominant organism would yield different changes such as a souring or decrease in pH. This study lacked control in this area and probably caused the varied results especially notice- able when examining the pH of the inoculated trials as given in Appendix H. BIBLIOGRAPHY Aberle, E. D. and Re As Merkel. 1966. Solubility and electrophoretic behavior of some proteins of post-mortem aged bovine muscle. J. 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APPENDIX -57- Log of the bacterial numbers per gram of sample for control and inoculated samples plus log of bacterial numbers per m1 Appendix A. of inoculum Sample / Trial 1 2 3 4 5 6 7 Control - Day 0 0.00 1.18 0.00 -- 0.00 3.49 2.92 Control - Day 1 0.00 0.00 2.00 0.00 0.00 3.58 3.51 Control - Day 5 0.00 -- 0.00 3.70 0.00 5.70 3.02 Control - Day 9 0.00 0.00 2.80 3.95 1.98 3.95 2.67 Control - Day 13 4.53 3.30 3.06 4.71 3.73 3.40 5.70 Control - Day 17 8.70 4.44 4.50 5.41 5.70 6.70 7.70 Pre-inoculated - Day 0 0.00 0.00 2.02 0.00 Inoculated - Day 0 -- 5.70 6.73 -- 5.03 7.51 7.20 Inoculated - Day 1 3.48 6.67 5.37 0.00 6.13 5.60 7.60 Inoculated - Day 5 5.20 -- 7.28 3.59 7.70 7.99 9.70 Inoculated - Day 9 9.70 6.81 7.57 5.41 9.70 9.40 9.30 Inoculated - Day 13 9.46 7.19 6.62 5.70 10.39 9.74 9.33 Inoculated - Day 17 9.96 7.53 7.74 7.03 9.94 8.00 10.06 Inoculum 4.98 8.70 8.33 <3.00 6.98 9.31 10.17 -53- Pfilligrams of sarc0p1asmic protein nitrogen per 100 milli- grams total protein nitrogen for control and inoculated Appendix B. samples. Sample / Trial 1 2 3 4 5 6 7 Average Control - Day 0 25.06 23.46 16.21 25.86 26.49 20.99 23.23 23.04 Control - Day 1 --- 24.84 17.83 29.46 27.53 18.56 21.60 23.30 Control - Day 5 23.85 20.69 17.27 27.73 23.85 20.06 22.56 22.29 Control - Day 9 23.78 21.59 17.52 24.53 21.43 19.98 20.16 21.28 Control - Day 13 21.49 21.30 15.01 24.91 22.46 20.26 22.97 21.20 Control - Day 17 21.87 23.33 14.78 23.90 21.78 20.26 19.01 20.70 Inoculated - Day 0 25.62 22.67 18.62 27.83 23.21 21.91 22.36 23.17 Inoculated - Day 1 28.96 21.71 19.16 30.48 26.66 23.44 21.19 24.51 Inoculated - Day 5 25.83 18.94 14.91 25.31 21.93 22.07 19.58 21.22 Inoculated - Day 9 22.32 16.71 13.14 24.33 22.35 18.58 16.99 19.20 Inoculated - Day 13 20.67 15.04 12.87 23.43 22.71 16.96 18.19 18.55 Inoculated - Day 17 20.83 15.74 11.91 24.02 23.40 17.28 17.15 18.62 -59- Appendix C. IMilligrams of myofibrillar protein nitrogen per 100 milligrams of total nitrogen for control and inoculated samples Sample / Trial 1 2 3 4 5 6 7 Average Control - Day 0 50.24 49.87 26.04 49.99 50.49 30.54 39.99 42.45 Control - Day 1 --- 51.79 26.93 51.38 50.52 40.79 42.66 44.01 Control - Day 5 49.56 54.83 29.37 51.91 53.53 43.03 41.16 46.20 Control - Day 9 53.53 56.89 28.81 52.95 54.49 39.23 46.05 47.42 Control - Day 13 48.77 54.00 26.40 53.27 55.28 41.65 46.68 46.58 Control - Day 17 51.02 51.43 28.21 55.22 56.01 44.62 48.55 47.87 Inoculated - Day 0 51.26 51.98 28.73 52.49 49.72 36.52 37.81 38.67 Inoculated - Day 1 46.49 52.20 31.06 51.95 48.39 32.70 40.41 43.31 Inoculated - Day 5 49.38 55.04 29.26 54.01 52.10 40.20 44.55 46.36 Inoculated - Day 9 54.29 41.87 28.00 51.71 55.91 54.31 51.46 48.22 Inoculated - Day 13 56.19 33.56 22.46 53.48 57.35 52.88 55.13 47.29 Inoculated - Day 17 54.89 23.17 26.23 51.06 55.60 51.37 49.92 44.61 Appendix D -50- . Rfilligrams of stroma protein nitrogen per 100 milligrams of total nitrogen for control and inoculated samples Sample / Trial 1 2 3 4 5 6 7 Average Control - Day 0 12.33 14.24 46.50 10.62 10.74 37.64 23.49 22.27 Control - Day 1 --- 10.07 41.50 7.21 10.33 25.03 23.16 19.55 Control - Day 5 14.77 11.15 40.89 6.46 11.26 23.70 22.04 18.62 Control - Day 9 8.19 8.54 40.45 9.81 11.42 27.59 20.67 18.10 Control - Day 13 16.42 10.44 44.30 10.16 9.18 23.74 16.55 18.68 Control - Day 17 13.18 10.60 41.91 7.91 8.79 19.20 16.72 16.90 Inoculated - Day 0 10.59 12.67 40.51 7.34 13.24 30.46 26.53 20.19 Inoculated - Day 1 12.66 13.55 37.62 7.17 13.90 31.59 25.78 20.32 Inoculated - Day 5 12.29 11.88 43.77 8.02 13.54 23.80 23.17 19.50 Inoculated - Day 9 9.82 27.02 44.24 9.08 9.89 14.95 19.29 19.18 Inoculated - Day 13 9.12 37.90 50.89 10.35 8.01 15.29 13.19 20.67 Inoculated - Day 17 9.51 46.62 11.27 8.79 14.72 17.20 21.89 45.14 ~61- (NPN) per 100 milligrams inoculated samples Milligrams of nonprotein nitrogen of total nitrogen for control and Appendix E. Sample / Trial 1 2 3 4 5 6 7 Average Control - Day 0 12.34 12.45 11.26 13.22 12.30 10.83 13.30 12.24 Control - Day 1 --- 12.81 14.18 11.96 11.63 15.63 12.59 13.13 Control - Day 5 12.33 13.33 12.47 13.91 11.37 13.20 14.25 12.98 Control - Day 9 14.48 12.50 13.23 12.72 13.18 13.21 13.13 13.21 Control - Day 13 16.42 14.27 14.30 11.67 13.09 14.36 13.82 135%! Control - Day 17 13.18 14.66 15.17 12.97 13.43 15.95 15.79‘ 14.45 Inoculated - Day 0 12.53 12.69 12.16 12.35 13.85 11.10 13.31 12.59 Inoculated - Day 1 11.90 12.55 12.17 11.79 11.05 12.27 12.63 12.05 Inoculated - Day 5 12.50 14.15 12.06 12.69 12.93 14.44 13.16 13.13 Inoculated - Day 9 13.58 14.40 14.64 14.90 11.86 12.17 12.28 13.40 Inoculated - Day 13 14.03 14.99 13.78 12.76 11.94 15.38 13.51 13.77 Inoculated - Day 17 14.77 15.96 15.26 13.71 12.23 16.65 15.74 14.90 -62- Appendix F. Emulsifying capacity of control and inoculated samples m1 of soybean oil per 100 mg of protein Sample / Trial 1 2 3 4 5 6 7 Average Control - Day 0 288.1 211.7 147.0 179.4 224.1 208.1 177.9 205.2 Control - Day 1 273.1 185.0 143.6 249.6 239.9 203.5 179.9 210.7 Control - Day 5 216.0 238.9 143.4 177.1 240.7 230.2 225.2 210.2 Control - Day 9 199.4 187.2 191.2 171.1 210.5 201.8 230.5 198.8 Control - Day 13 193.3 171.0 174.4 159.7 205.8 205.5 259.7 194.1 Control - Day 17 165.4 165.4 150.3 226.5 214.5 192.9 215.7 190.1 Inoculated - Day 0 --- 154.8 191.2 206.7 237.5 173.8 195.3 193.2 Inoculated - Day 1 280.3 156.0 169.6 226.7 246.6 200.3 187.3 209.5 Inoculated - Day 5 253.8 140.5 163.7 170.9 210.5 211.4 213.4 194.9 Inoculated - Day 9 141.9 162.2 158.0 170.6 197.2 197.0 245.7 181.8 Inoculated - Day 13 159.1 162.3 159.6 160.8 232.6 209.4 177.5 180.2 Inoculated - Day 17 173.3 161.7 146.0 169.6 231.6 218.6 211.3 188.7 -63.. Appendix G. IMilliliters of extract release volume (ERV) for control and inoculated samples Sample / Trial 1 2 3 4 5 6 7 Average Control - Day 0 60.5 50.0 60.0 48.5 54.5 57.5 54.5 55.1 Control - Day 1 58.5 51 0 55 0 53.5 50.0 55.0 52.5 53.6 Control - Day 5 52.0 48.5 61 5 42.0 43 5 53.5 55.0 50.9 Control - Day 9 50.0 48 0 56 2 46.8 50.5 53.5 50.5 50.8 Control - Day 13 48.0 49 0 58 0 46.5 51 5 52.5 50.5 50.9 Control - Day 17 44.0 39 0 52 0 39.5 51.0 48.5 54.0 46.9 Inoculated - Day 0 55.0 46.0 52.5 48.0 54.5 57.5 59.0 53.2 Inoculated - Day 1 55.5 52.0 52.0 47.0 55.5 51.0 57.0 52,9 Inoculated - Day 5 48.5 8.5 15.0 48.0 49.0 48.0 44.0 37.3 Inoculated - Day 9 32.0 9.0 11.5 54.0 20.5 34.0 43.5 29.2 Inoculated - Day 13 37.0 8.0 43.5 47.0 23.0 22.0 50.0 32.9 Inoculated - Day 17 16.5 19.5 12.0 51.5 9.0 53.5 47.5 29.9 Average -64- pH of control and inoculated samples Sample / Trial Control - Day 0 Control - Day 1 Control - Day 5 Control - Day 9 Control - Day 13 Control - Day 17 Appendix H. 37 Inoculated - Day 0 Inoculated - Day 1 Inoculated - Day 5 Inoculated - Day 9 Inoculated - Day 1 Inoculated - Day 1 -55- Appendix J. The percent moisture in the control and inoculated samples Sample / Trial 1 2 3 4 5 6 7 Average Control - Day 0 66.20 68.40 57.98 62.22 61.68 64.58 63.36 63.49 Control - Day 1 66.00 70.00 59.58 60.20 64.54 62.15 63.23 63.67 Control - Day 5 66.58 70.06 60.76 64.42 59.68 63.24 64.88 64.23 Control - Day 9 65.93 68.11 58.88 62.37 64.95 65.41 62.76 64.06 Control - Day 13 66.37 69.09 61.99 62.38 66.05 63.67 64.77 64.90 Control - Day 17 66.09 68.29 61.10 62.73 67.31 64.54 66.34 65.20 Inoculated - Day 0 --- 69.30 64.98 65.91 60.33 65.09 64.96 65.10 Inoculated - Day 1 65.18 69.45 62.04 63.77 60.36 63.30 66.52 64.37 Inoculated - Day 5 65.42 70.24 63.01 65.87 58.00 64.69 65.75 64.71 Inoculated - Day 9 64.15 69.02 62.43 64.97 65.20 63.34 66.70 65.12 Inoculated - Day 13 63.24 69.16 61.21 63.56 63.78 64.70 68.27 64.85 Inoculated - Day 17 65.48 69.16 62.55 64.91 63.65 66.82 66.98 65.65 -66- Appendix K. The percent protein in the control and inoculated samples Sample / Trial 1 2 3 4 5 6 7 Average Control - Day 0 19.30 18.29 16.54 19.96 18.86 17.49 18.46 18.41 Control - Day 1 18.60 16.65 17.77 18.19 19.59 17.89 18.12 18.12 Control - Day 5 18.85 18.07 17.16 19.65 18.87 17.38 18.03 18.29 Control - Day 9 18.96 18.16 16.53 19.29 18.15 17.84 18.31 18.18 Control - Day 13 18.98 17.78 17.48 19.41 19.05 17.52 18.10 18.33 Control - Day 17 19.10 17.78 17.83 18.19 19.02 17.00 18.17 18.16 Inoculated - Day 0 --- 18.43 17.99 20.22 19.03 18.30 19.15 18.85 Inoculated - Day 1 18.48 17.31 17.76 20.07 18.41 17.67 19.22 18.15 Inoculated - Day 5 18.44 18.59 17.35 19.54 18.05 17.98 19.49 18.49 Inoculated - Day 9 18.60 18.38 19.72 19.58 18.36 17.16 19.86 18.52 Inoculated - Day 13 18.93 18.44 18.04 20.28 18.49 17.29 19.04 18.64 Inoculated - Day 17 18.93 18.18 16.71 20.13 17.96 16.65 19.59 18.31 AAAAAAAA u mun 1111.11111ng 1111'“ ”Whmujujlg 930