m INFLUENCE OF WLK FA? cm THE THERMAL assmucrzw a? Psgupomoms FRA'G! Them for flu Daqroc of DH. D. MICHIGAN STATE UNIVERSITY Lloyd 0-. Luedecke 1962 m {is .m This is to certify that the thesis entitled The influence of milk fat on the thermal destruction of Pseudomonas fragi presented by Lloyd 0. Luede cke has been accepted towards fulfillment of the requirements for Ph. D. Food Science degree in Major ofessor Date January 17, 1962 0-169 L I B R A R Y Michigan State University “ W‘s, THE INFLUENCE OF MILK FAT ON THE THERMAL DESTRUCTION OF PSEUDOMONAS FRAGI by Lloyd 0. Luedecke AN ABSTRACT OF A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY College of Agriculture 1962 ABSTRACT THE INFLUENCE OF MILK FAT ON THE THERMAL DESTRUCTION OF PSEUDOMONAS FRAGI by Lloyd 0. Luedecke The thermal destruction rates of Pseudomonas fraqi were investigated in skimmilk, milk containing 10 per cent milk fat, half-and-half and cream containing 20 per cent milk fat. 2, fragi_was also heated in reconstituted skimmilk which contained 0.1 per cent by weight of one of the following fatty acids: butyric, lauric or oleic. All of the survivor curves prepared from the data ob- tained in this investigation showed a lag during the initial intervals of the heating trials. The slope of the straight line portion of the survivor curves was determined by linear regression and the D values were determined from the calculated slopes. A z of 10 was obtained with the cells grown in half—and- half at 25° c. for 20 hours and heated in half-and—half at 48°, 500 and 520 C. A z of 13 was obtained when the cells were grown in half-and-half at 70 C. for 7 days and heated in half-and-half at 48°, 50° and 52° c. The cells grown at o 7 C. required longer heating times to reduce the initial Lloyd 0. Luedecke cell population by 99.9999 per cent than did the cells grown at 250 C. When cells were heated in skimmilk, milk containing 10 per cent milk fat and in cream containing 20 per cent milk fat, the 2 values were 14, 11 and 22, respectively. At 480 and 500 C. the cells grown and heated in skimmilk required longer heating times to destroy 99.99 per cent of the original cell population than did the cells grown and heated in cream containing 20 per cent milk fat. At 520 C. the times were nearly the same. The above 2 values were obtained by heating the cells in a medium which contained the same concentration of milk fat as the medium in which they were grown. Heating trials were also conducted in which the cells were heated in a medium in which the concentration of milk fat differed from the concentration in which they were grown. The data from these trials indicated that the medium in which the cells were heated influenced the 2 values, but the medium in which the cells were grown did not seem to influence the 2 values. The largest 2 values were obtained when the cells were heated in cream containing 20 per cent milk fat and the 2 values were approximately of the same magnitude ii Lloyd 0. Luedecke when the cells were heated in skimmilk and in milk con— taining 10 per cent milk fat. The growth medium seemed to influence the times required to reduce the original cell population by 99.99 per cent, but the times did not seem to be affected by the medium in which the cells were heated. The longest times were required with the cells grown in skimmilk and the shortest times were re- quired with the cells grown in cream containing 20 per cent milk fat. The 2 values ranged from ll to 14 when 2, fragi_was heated in the skimmilk-fatty acid mixtures. The similarity of the 2 values would seem to indicate that they were not influenced by the carbon chain length or the character- istics of saturation and unsaturation of the fatty acids used. Heating trials were conducted with and without adjustment of the pH to 6.7. When the pH of the skimmilk— butyric acid mixture was at 5.85 the times required to reduce the original cell population by 99.99 per cent were longer than those required when the pH was at 6.7. With lauric and oleic acid the heating times were approximately the same when the pH of the skimmilk-fatty acid mixtures were 6.7 and less than 6.7. iii THE INFLUENCE OF MILK FAT ON THE THERMAL DESTRUCTION OF PSEUDOMONAS FRAGI BY Lloyd 0. Luedecke A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY College of Agriculture 1962 ii ACKNOWLEDGMENTS The author wishes to express his sincere gratitude to Dr. L. G. Harmon for his patience and guidance throughout this graduate program and for his helpful suggestions and assistance in preparing this manuscript. The counsel of Dr. 0. W. Kaufmann was most helpful dur- ing the course of the research program and the careful read- ing of this manuscript was certainly appreciated. The author wishes to acknowledge Dr. R. N. Costilow, Dr. C. M. Stine, Dr. T. I. Hedrick and Dr. H. A. Lillevik for their valuable suggestions while serving on the author's guidance committee and for their reading and subsequent sug- gestions in the preparation of this manuscript. The advice and suggestions of Dr. L. D. McGilliard and Dr. R. C. Nicholas were most helpful in the statistical anal— yses of the data in this manuscript. Acknowledgement is also due to the Agricultural Experi- ment Station of Michigan State University for the financial support received throughout the entire graduate program. Grateful acknowledgment is due to the author's wife, Doris, for her unending encouragement and understanding throughout the course of this graduate program and for her assistance in preparing this manuscript. iii TABLE OF CONTENTS Page IMRODUCTI ON 0 O O O O O O O O O O O O O O 0 O O O O 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . 3 Thermal Destruction of Psychrophiles in Dairy Products . . . . . . . . . . . . . . . . . . 3 Influence of Fat on the Thermal Destruction of Microorganisms . . . . . . . . . . . . . . . . 7 Influence of Fatty Acids on the Growth of Microorganisms . . . . . . . . . . . . . . . . ll EXPERIMENTAL PROCEDURES . . . . . . . . . . . . . . 13 Preparation of Cells . . . . . . . . . . . . . . 15 Preparation of Half—and-half as a Heating Medium 0 O O O O O O O O O O O O O O O O O O O 18 Preparation of Fresh Skimmilk and Cream Con— taining 20 Per Cent Milk Fat as Heating Media. 19 Preparation of Reconstituted Skimmilk, Reconsti- tuted Milk Containing 10 Per Cent Milk Fat and Reconstituted Cream Containing 20 Per Cent Milk Fat as Heating Media . . . . L . . . . . 20 Preparation of Reconstituted Skimmilk Contain— ing Selected Fatty Acids as Heating Media . . 20 Heating Procedure 0 o o o o o o o o o o o o o ‘o 22 Determination of Thermal Destruction Rates . . . 23 RESULTS 0 O O O O O O O O O O O O O O O O O O O O O 26 Thermal Destruction of P, fragi in Half-and— half . . . . . . . . . . . . . . . . . . . . . 26 Grown in half-and-half at 25° c. for 20 hours and heated in half-and-half . . . . 26 iv Page Grown in half—and—half at 7° c. for 7 days and heated in half—and-half . . . . . . . 29 Thermal Destruction of P. fragi in Fresh Skim- milk and Cream Containing 20 Per Cent Milk Fat 32 Grown and heated in skimmilk . . . . . . . . 32 Grown and heated in cream containing 20 per cent milk fat . . . . . . . . . . . . . . 36 Thermal Destruction of g. fragi in Reconsti- tuted Skimmilk, Reconstituted Milk Containing 10 Per Cent Milk Fat and Reconstituted Cream Containing 20 Per Cent Milk Fat . . . . . . . 36 Grown and heated in reconstituted skimmilk . 39 Grown and heated in reconstituted milk containing 10 per cent milk fat . . . . . 39 Grown and heated in reconstituted cream containing 20 per cent milk fat . . . . . 44 Thermal Destruction of g, fragi Cells in a Medium Containing a Different Concentration of Milk Fat Than the Medium in Which the Cells were Grown . . . . . . . . . . . . . . . 48 Grown in reconstituted skimmilk -— heated in reconstituted milk containing 10 per cent milk fat . . . . . . . . . . . . . . 48 Grown in reconstituted skimmilk -- heated in reconstituted cream containing 20 per cent milk fat . . . . . . . . . . . . . . 48 Grown in reconstituted milk containing 10 per cent milk fat -- heated in reconsti— tuted skimmilk . . . . . . . . . . . . . 53 Grown in reconstituted milk containing 10 per cent milk fat -— heated in reconsti- tuted cream containing 20 per cent milk fat . . . . . . . . . . . . . . . . . . . 56 Grown in reconstituted cream containing 20 per cent milk fat -- heated in reconsti- tuted skimmilk . . . . . . . . . . . . . . Grown in reconstituted cream containing 20 per cent milk fat -- heated in reconsti- tuted milk containing 10 per cent milk fat Thermal Destruction of P, fragi in Reconstituted Skimmilk Containing Selected Fatty Acids . . . In reconstituted skimmilk containing lauric aCid O O O I O O O O O O O O O I O I O O In reconstituted skimmilk containing oleic acid . . . . . . . . . . . . . . . . . . . In reconstituted skimmilk containing butyric acid . . . . . . . . . . . . . . . . . . . DISCUSSION 0 O O O O O O O O O O O O O O O O O O O 0 Survivor Curves . . . . . . . . . . . . . . . . Thermal Destruction of g. fragi in Half-and- half 0 O O O O O O O O O O O O O O O O O O O 0 Thermal Destruction of g. fragi in Fresh Skimmilk and Cream Containing 20 Per Cent Milk Fat 0 O O O O O O O O O O O O O O O O O 0 Thermal Destruction of g, fragi in Reconstitu- ted Skimmilk, Reconstituted Milk Containing 10 Per Cent Milk Fat and Reconstituted Cream Containing 20 Per Cent Milk Fat . . . . . . . Thermal Destruction of g, fragi in a Medium Containing a Different Concentration of Milk Fat Than the Medium in Which the Cells were Grown . . . . . . . . . . . . . . . . . . . . Thermal Destruction of g; fragi in Reconsti— tuted Skimmilk Containing Selected Fatty Acids Page 56 61 65 65 68 75 83 83 86 88 91 94 97 F===:*i SUMMARY AND CONCLUSIONS LITERATURE CITED vi Page 100 103 Table TABLES Number of 2, fragi per milliliter surviving heat treatment in half-and-half after growth in half-and-half at 250 C. for 20 hours . . . . Number of g, fragi per milliliter surviving heat treatment in half-and-half after growth in half- and-half at 70 C. for 7 days . . . . . . . . . . Number of g, fragi per milliliter surviving heat treatment in skimmilk after growth in skimmilk at 25° C. for 20 hours . . . . . . . . . . . . . Number of g. fragi per milliliter surviving heat treatment in cream containing 20 per cent milk fat after growth in cream containing 20 per cent milk fat at 250 C. for 24 hours . . . . . . . . . Number of g. fragi per milliliter surviving heat treatment in reconstituted skimmilk after growth in reconstituted skimmilk at 250 C. for 20 hours. Number of g, fragi per milliliter surviving heat treatment in reconstituted milk containing 10 per cent milk fat after growth in reconstituted milk containing 10 per cent milk fat at 250 C. for 20 hours . . . . . . . . . . . . . . . . . Number of g, fragi per milliliter surviving heat treatment in reconstituted cream containing 20 per cent milk fat after growth in reconstituted cream containing 20 per cent milk fat at 250 C. for 24 hours . . . . . . . . . . . . . . . . . . Thermal destruction of g, fragi when grown and heated in various dairy products containing 0 to 20 per cent milk fat . . . . . . . . . . . . . . Number of g, fragi per milliliter surviving heat treatment in reconstituted milk containing 10 per cent milk fat after growth in reconstituted skimmilk at 250 C. for 20 hours . . . . . . . . . vii Page 27 3O 33 38 4O 42 45 47 49 viii Table Page 10. Number of g, fragi per milliliter surviving heat treatment in reconstituted cream containing 20 per cent milk fat after growth in reconstituted skimmilk at 250 C. for 20 hours . . . . . . . . . 52 11. Number of g, fragi per milliliter surviving heat treatment in reconstituted skimmilk after growth in reconstituted milk containing 10 per cent milk fat at 25° C. for 20 hours . . . . . . . . . 55 12. Number of P. fragi per milliliter surviving heat treatment in reconstituted cream containing 20 per cent milk fat after growth in reconstituted milk containing 10 per cent milk fat at 250 C. for 20 hours . . . . . . . . . . . . . . . . . . 57 13. Number of P. fragi per milliliter surviving heat treatment in reconstituted skimmilk after growth in reconstituted cream containing 20 per cent milk fat at 25° C. for 24 hours . . . . . . . . . 59 14. Number of g. fragi per milliliter surviving heat treatment in reconstituted milk containing 10 per cent milk fat after growth in reconstituted cream containing 20 per cent milk fat at 25° C. for 24 hours . . . . . . . . . . . . . . . . . . 62 15. Thermal destruction of g. fragi in a medium con- taining a concentration of milk fat which was variant from that in the medium in which the cells were grown . . . . . . . . . . . . . . . . 64 16. Number of P, fragi per milliliter surviving heat treatment in reconstituted skimmilk containing 0.1 per cent by weight of lauric acid at pH 6.6 . 66 17. Number of P. fragi per milliliter surviving heat treatment in reconstituted skimmilk containing 0.1 per cent by Weight of lauric acid and neutralized to pH 6.7 . . . . . . . . . . . . . . 69 18. Number of g. fragi per milliliter surviving heat 'treatment in reconstituted skimmilk containing 0.1 per cent by weight of oleic acid at pH 6.45 .; 71 ix Table Page 19. Number of §,_fragi per milliliter surviving heat treatment in reconstituted skimmilk containing 0.1 per cent by weight of oleic acid and neutral- ized to pH 6.7 . . . . . . . ... . . . . . . . . 72 20. Number of E, fragi per milliliter surviving heat treatment in reconstituted skimmilk containing 0.1 per cent by weight of butyric acid at pH 5.85 . . . . . . . . . . . . . . . . . . . . . . 76 21. Number of E, fragi per milliliter surviving heat treatment in reconstituted skimmilk containing 0.1 per cent by weight of butyric acid and neutralized to pH 6.7 . . . . . . . . . . . . . . 80 22. Thermal destruction of E, fragi after growth in reconstituted skimmilk and heated in reconstitu- ted skimmilk containing 0.1 per cent by weight of butyric, lauric or oleic acid . . . . . . . . 81 23. Time required to destroy 99.99 per cent of the E, fragi cells when heated in the medium and at the temperatures indicated . . . . . . . . . . . 82 FIGURES Figure 1. 10. A schematic diagram of the procedure used in preparing the cells for thermal destruction studies . . . . . . . . . . . . . . . . . . . . Rate of destruction of g, fragi in half-and-half after growth in half-and-half at 250 C. for 20 hours . . . . . . . . . . . . . . . . . . . . . Thermal resistance curve of P, fragi in half-and- half after growth in half-andrhalf at 25° C. for 20 hours . . . . . . . . . . . . . L . . . . . . Rate of destruction of P, fragi in half-and—half after growth in half-and-half at 70 C. for 7 days . . . . . . . . . . . . . . . . . . Thermal resistance curve of g, fragi in half-and- half after growth in half-andehalf at 70 C. for 7 days I O ; O O I O O O O O O O O O C O O I. O 0 Rate of destruction of g, fragi in skimmilk after growth in skimmilk at 250 C. for 20 hours . . . Thermal resistance curve of P, fragi in skimmilk after growth in skimmilk at 250 C. for 20 hours . A comparison of the thermal death time curves of .3. fragi in media containing 0, 11 and 20 per cent milk fat with the milk pasteurization curve. (The thermal death time curves are a plot of temperature versus the number of minutes required to bring about a 99.9999 per cent reduction in the original cell population) . . . . . . . . . Rate of destruction of g, fragi in cream con— taining 20 per cent milk fat after growth in cream containing 20 per cent milk fat at 250 C. for 24 hours . . . . . . . . . . . . . . . . . Thermal resistance curve of g. fragi in cream containing 20 per cent milk fat after growth in cream containing 20 per cent milk fat at 250 C. for 24 hours . . . . . . . . . . . . . . . . . Page 16 28 28 31 31 34 34 35 37 37 Figure 11. 12. 13. 14. 15. 16. 17. 18. 19. Rate of destruction of g, fragi in reconsti- tuted skimmilk after growth in reconstituted skimmilk at 25° C. for 20 hours . . . . . . . . . Thermal resistance curve of g, fragi in recon- stituted skimmilk after growth in reconstituted skimmilk at 25° c. for 20 hours . . . . . . . . . Rate of destruction of P, fragi in reconstituted milk containing 10 per cent milk fat after growth in reconstituted milk containing 10 per cent milk fat at 250 C. for 20 hours . . . . . . . . . . . Thermal resistance curve of g, fragi in recon- stituted milk containing 10 per cent milk fat after growth in reconstituted milk containing 10 per cent milk fat at 250 C. for 20 hours . . . . Rate of destruction of g. fragi in reconstituted cream containing 20 per cent milk fat after growth in reconstituted cream containing 20 per cent milk fat at 25° C. for 24 hours . . . . . . Thermal resistance curve of g, fragi in recon- stituted cream containing 20 per cent milk fat after growth in reconstituted cream containing 20 per cent milk fat at 250 C. for 24 hours . . . Rate of destruction of P. fragi in reconstituted milk containing 10 per cent milk fat after growth in reconstituted skimmilk at 250 C. for 20 hours . . . . . . . . . . . . . . . . . . . . Thermal resistance of g, fragi in reconstituted milk containing 10 per cent milk fat after growth in reconstituted skimmilk at 250 C. for 20 hours . . . . . . . . . . . . . . . . . . . . Rate of destruction of P. fragi in reconstituted cream containing 20 per cent milk fat after growth in reconstituted skimmilk at 25° C. for 20 hours . . . . . . . . . . . . . . . . . . . . xi Page 41 41 43 43 46 46 50 50 51 xii Figure Page 20. Thermal resistance curve of g. fragi in recon— stituted cream containing 20 per cent milk fat after growth in reconstituted skimmilk at 250 C. for 20 hours . . . . . . . . . . . . . . . . . . 51 21. Rate of destruction of g. fragi in reconstituted Skimmilk after growth in reconstituted milk con- taining 10 per cent milk fat at 250 C. for 20 hours . . . . . . . . . . . . . . . . . . . . . . 54 22. Thermal resistance curve of g, fragi in recon- stituted skimmilk after growth in reconstituted milk containing 10 per cent milk fat at 250 C. for 20 hours. . . . . . . . . . . . . . . . . . . 54 23. Rate of destruction of P. fragi in reconstituted cream containing 20 per cent milk fat after growth in reconstituted milk containing 10 per cent milk fat at 25° c. for 20 hours . . . . . . 58 24. Thermal resistance curve of g, fragi in recon- stituted cream containing 20 per cent milk fat after growth in reconstituted milk containing 10 per cent milk fat at 25° c. for 20 hours . . . . 58 25. Rate of destruction of g, fragi in reconstituted skimmilk after growth in reconstituted cream containing 20 per cent milk fat at 250 C. for 24 hours . . . . . . . . . . . . . . . . . . . . . . 6O 26. Thermal resistance curve of g, fragi in recon- stituted skimmilk after growth in reconstituted cream containing 20 per cent milk fat at 250 C. for 24 hours . . . . . . . . . . . . . . . . . . 6O 27. Rate of destruction of P. fragi in reconstituted milk containing 10 per cent milk fat after growth in reconstituted cream containing 20 per cent milk fat at 250 C. for 24 hours . . . . . . 63 28. Thermal resistance curve of P. fragi in recon- ’stituted milk containing 10 per cent milk fat after growth in reconstituted cream containing 20 per cent milk fat at 250 C. for 24 hours . . . 63 xiii Figure Page 29. Rate of destruction of g. fragi in reconstituted skimmilk containing 0.1 per cent by weight of lauric acid at pH 6.6 . . . . . . . . . . . . . . 67 30. Thermal resistance curve of g, fragi in recon- stituted skimmilk containing 0.1 per cent by weight of lauric acid at pH 6.6 . . . . . . . . . 67 31. Rate of destruction of P: fragi in reconstituted skimmilk containing 0.1 per cent by weight of lauric acid and neutralized to pH 6.7 . . . . . . 7O 32. Thermal resistance curve of g, fragi in recon- stituted skimmilk containing 0.1 per cent by weight of lauric acid and neutralized to pH 6.7 . 70 33. Rate of destruction of P. fragi in reconstituted skimmilk containing 0.1 per cent by weight of oleic acid at pH 6.45 . . . . . . . . . . . . . . 73 34. Thermal resistance curve of 2, fragi in recon- stituted skimmilk containing 0.1 per cent by weight of oleic acid at pH 6.45 . . . . . . . . . 73 35. Rate of destruction of 2, fragi in reconstituted skimmilk containing 0.1 per cent by weight of oleic acid and neutralized to pH 6.7 . . . . . . 74 36. Thermal resistance curve of g, fragi in recon- stituted skimmilk containing 0.1 per cent by weight of oleic acid and neutralized to pH 6.7. . 74 37. Rate of destruction of g, fragi in reconstituted skimmilk containing 0.1 per cent by weight of butyric acid at pH 5.85 . . . . . . . . . . . . . 77 38. Thermal resistance curve of g, fragi in recon- stituted skimmilk c0ntaining 0.1 per cent by weight of butyric acid at pH 5.85 . . . . . . . . 77 Figure 39. 40. Rate of destruction of P. fragi in reconstituted skimmilk containing 0.1 per cent by weight of butyric acid and neutralized to pH 6.7 . . . . . Thermal resistance curve of g, fragi in recon- stituted skimmilk containing 0.1 per cent by weight of butyric acid and neutralized to pH 6.7. xiv Page 79 79 INTRODUCTION The spoilage of dairy products by psychrophilic bacteria has become an important problem to the dairy industry during recent years. This problem has resulted primarily from the present methods of handling and manufacturing dairy products. These methods often involve holding the dairy products at refrigeration temperatures for several days. At these temperatures the psychrophilic organisms are able to increase in numbers and will eventually cause spoilage by producing off-flavors and odors. The literature contains numerous reports that indicate psychrophiles are destroyed in milk and skimmilk by pasteurization. Most of the work reported has been limited to the results obtained by laboratory pasteurization of samples of whole milk which contained inoculated or naturally occurring psychrophilic organisms. After pasteurization these milk samples were examined to determine if survivors were present. These investigations did not determine the rates at which psychrophiles are destroyed during heating. The work reported herein was performed to investigate the thermal resistance of a psychrophile, Pseudomonas fraqi, in dairy products which contain more milk fat than occurs in whole milk. Literature reports indicate that in some instances bacterial cells and spores receive a limited amount of thermal protection from fat or oil. The influence of the following variables on the thermal destruction rates of E, fragi were investigated; (a) the concentration of milk fat in the environment in which the cells were heated, (b) the concentration of milk fat in the environment in which the cells were grown and (c) the presence of butyric, lauric and oleic acids in the medium in which the cells were heated. .. an—rw=-.§-- ”gum—5“); ..’,_—m ,1--.— LITERATURE REVIEW Thermal Destruction gf_Psychrophiles in_Dairy_Products The destruction of psychrophilic bacteria in milk at pasteurization temperatures has been reported by many investigators. The psychrophilic species found most commonly in dairy products belong to the genera: Pseudomonas, Achromobacter, Flavobacterium, Alcaliqenes, Escherichia and Aerobacter. Most of the species found in dairy products belong to the genus Pseudomonas. Hussong §£_§1, (19) stated that Pseudomonas fraqi, which is a common psychrophilic contaminant in dairy products, was killed by "relatively low pasteurization exposures." According to Seleen and Stark (42) no Pseudomonas survived heating in milk at 600 C. for 30 minutes. Thomas and Sekhar (44) and Chandrasekhar (7) reported that laboratory pasteuri- zation was sufficient to destroy those organisms which develop in milk at 30 to 50 C. Thirty minutes at 62.50 C. was sufficient to prevent a significant bacterial increase in milk during storage for 15 days at 4.40 to 7.30 C. (3). A total of 305 psychrophilic cultures were isolated from milk and none of the cultures survived laboratory pasteurization (44, 47, 48). Thomas _£__l, (45) and Rogick and Burgwald (41) were unable to isolate psychrophilic organisms when they examined samples of milk that had been removed asepti- cally from vat and high-temperature-short-time (HTST) pas- teurizing systems. Parker §£_§1, (36) heated 10 psydhrophilic cultures that had been incubated 64 hours at 250 C. and found that all of the cells were destroyed when heated in milk at 61.70 C. for 30 minutes. Each culture contained approxi- mately 1,000,000 to 5,000,000 cells per milliliter. Olson .;§._1. (35) reported that psychrophiles were destroyed when heated at pasteurization temperatures in thermal death time tubes. Only 6 of 41 cryophilic (psychrophilic) cultures isolated from butter by Jezeski and Macy (21) survived laboratory pasteurization at 1500 F. (65.60 C.) for 30 minutes. Davis and Babel (11) observed that bacteria capable of forming slime on cottage cheese were destroyed by 2.5 minutes of exposure at 62.50 C. Thomas _§_§1, (46) stated that all types of psychrophilic bacteria commonly found in milk, especially those able to grow and multiply at tempera- tures between 00 and 70 C., are destroyed by pasteurization. In contrast to the above data, Kennedy and Weiser (25) reported that all but one of 15 psychrophilic cultures iso- lated from milk survived when heated in milk at 1450 F. (62.80 c.) for 30 minutes. Powell (37) reported on the destruction of organisms that . . 0 caused a bitter flavor in cream stored at 35 F. (1.70 C.). He found that when cream containing the organisms was heated O to 165°, 175° and 185° F. (73.9°, 79.5 and 85° c.) for 30 to 45 seconds the bitter flavor did not develop during 10 days of storage at 35° F. (1.7° C.). Erdman and ThornUal(l6) found that bacteria capable of growth at 10.50 C. did not survive commercial pasteurization in homogenized milk containing 10 per cent milk fat. These workers were not concerned with specific psychrophiles, therefore, they used milk that was known to contain a large number of psychrophiles representing a natural mixed flora. El Sedek and Richards (15) reported on the destruction of the lipolytic flora found in raw cream. They found that one day of storage in "cool conditions" permitted the Pseudomonas and Achromobacter genera to increase in number, but this increase could be nullified by pasteurization. None of the investigations mentioned above included a study of the thermal destruction rate of the psychrophiles. The studies were limited to experimental work in which the organisms were heated at a specific temperature for a specific length of time in various dairy products. The products were examined after heating to determine whether survivors were present. Kaufmann and Andrews (24) were the first to report on the thermal destruction rate of psychrophiles in dairy products. They used Pseudomonas viscosa and an organism tentatively identified as Pseudomonas mephipiga. The authors found 2 values of 14.3 and 6.7 with g. viscosa and 2, mephiticav respectively. They compared the two thermal death time curves to the milk pasteurization curve and noted that neither of the death curves crossed the pasteurization curve, indicating the lethality of the pasteurization process was sufficient to inactivate these two organisms, although the margin of safety was much less with g, viscosa. These workers Suggested that on the basis of their results, some of the psychrophiles might survive HTST pasteurization in whole milk. Normally the margin of safety is less with the HTST process. Also, bacteria that are trapped in the fat or oil of a product may be protected from thermal destruction. Other workers (5,8) have studied the effect of the cottage cheese cooking operation on common psychrophilic contaminants. Bonner and Harmon (5) found that none of the 17 cultures isolated from spoiled cottage cheese survived when heated to 48.90 C. for 15 minutes in whey at pH 4.55. Chaudhary t al, (8) reported that three species of Pseudomonas failed to survive cooking at 1200 F. (48.90 C.) for 30 minutes. These workers found 2 values of 12.4, 8.8 and 8.0 when E, fragi, E, viscosa and E, fluorescens, respectively, were heated in skimmilk. They concluded that none of these organisms would survive pasteurization in skimmilk or whole milk. Influence 9f_§a£_9g_the Thermal Destruction 9f_Microorganisms Many workers have studied various factors influencing the thermal resistance of both spore-forming and non—spore-forming organisms. Some of the factors affecting the heat resistance of cells or spores are: (a) inherent resistance of the organism, (b) the conditions under which the cells or spores were formed and (c) the nature of the medium in which the cells or spores are heated. Inherent resistance is illustrated by the fact that different strains of the same species will have different resistances even when propagated under the same conditions. Incubation temperature and composition of the growth medium are environmental influences that are active during the growth of the cells or formation of the spores of an organism. The nature of the heating medium will affect the thermal resistance of cells or spores. The presence of fat in the heating medium is one factor which may influence the thermal destruction of microorganisms. There is a lack of agreement among workers concerning the amount of protection that cells or spores receive from the presence of fat in the heating medium and also, if the organisms are protected, there is lack of agreement concerning the mechanism of protection. For example, Lang (27) suggested that the poor conductivity of oil media resulted in increased heat resistance. Jensen (20) reported the results of an experiment with streptococci which indicated large differences in the heat resistance of the cells in fatty and aqueous media. The streptococci were destroyed in "moist melted butter" after 15 minutes at 1000 C., whereas in "dry butter," 50 minutes at 1150 C. were required and at 1200 C., 20 minutes were required. In milk the organisms were destroyed in 30 minutes at 61.70 C. Bartlett and Kinne (4) reported that Bacillus subtilis, Bacterium anthracis (Bacillus anthracis) and Bacillus vitalis were significantly more heat resistant in glycerine, olive oil, cottonseed oil and paraffin than in water. Rogacheva (40) suggested that the heat resistance of non-spore-forming bacteria was not increased by heating in fats and oils and CO C. erA. Nam I’ll I. ...:H an Lo. mb that the heat resistance of spores from the (Bacillus) subtilis-mesentericus group would increase only if the fats and oils were dehydrated. Nichols (33) reported that when milk or cream was used as a heating medium, an increase in the fat content failed to increase the resistance of Bacillus subtilis spores and that non-sporulating bacteria may have been more subject to thermal destruction as the milk fat content was increased. Daoust §§_al, (l3) heated Corynebacterium diphtheriae in whole milk, 40 per cent cream and ice cream mix and found that the cells were the least resistant in 40 per cent cream. However, Brown and Peiser (6) observed that-Bacterium lactis acidi (Streptococcus lactis) and Bacterium coli (Escherichia 2211) were protected by milk fat from thermal destruction. Mudd and Mudd (31) developed a method for determining the behavior of various cells, including bacteria, at an oil- water interface. These workers found that cells with hydro- phobic surfaces readily pass from the aqueous phase into the oil phase, whereas cells with hydrophilic surfaces usually remain at the interface. The behavior of bacteria at an oil-water interface can be influenced by various materials, such as fatty acids and soaps which enhance the passage of 10 bacteria into oils. Jensen (20) reported that an aqueous solution containing 0.05 per cent sodium oleate allowed all of the microorganisms tested to pass into the oil. In discussing the preparation of canned meats, he suggests that fatty acids and soaps should be eliminated as far as possible because the long chain fatty acids enhance the passage of bacteria into fat. Mellon (30) postulated that calcium ions bring the lipoidal phase to the external surface of the bacterial cell wall, as can be shown by the fact that organisms will pass from an aqueous suspension to lipoidal solvents. This does not occur in the presence of the sodium ion. Later Jones and Lorenz (22) showed that in oil-water mixtures containing bacteria, calcium ions facilitate the passage of bacteria into the oil layer. The layer of oil around the cell may protect the organisms from destruction, but there is also evidence that this same layer of oil may trap the surviving cells. Consequently, cell development may not occur due to the lack of an aqueous environment. Thus, the food product may not spoil even though surviving cells are present. The above theory is based on the assumption that the cells receive thermal protection while they are coated with a layer of fat or oil. 11 Inflpence 9§_Fatty Acids gpjthe Growth 9;.Micrgorganisms Nieman (34) reviewed the influence of trace amounts of fatty acids on the growth of microorganisms. He reported that fatty acids in very low concentrations may inhibit or stimu- late the growth of bacteria. The amount of inhibition or stimulation is dependent on the concentration and nature of the fatty acids and on the bacterial species involved. Generally only Gram positive organisms are susceptible to the influence of minute amounts of fatty acids, although influ- ences on Gram negative organisms have been observed. Both saturated and unsaturated fatty acids may influence the growth of microorganisms. The antibacterial activity of unsaturated fatty acids increases with the number of double bonds in the molecule and the natural cis-forms are generally more inhibitory than the trans-isomers. Antibacterial activity of saturated fatty acids is optimal when the chain length is approximately 12 carbon atoms. Nieman (34) concluded his review by pointing out that a completely satisfactory theory had not yet been advanced explaining growth inhibition by fatty acids, but the most logical explanation is attributed to the changes in cell 12 permeability caused by fatty acids adsorbed at the cell membrane. Costilow and Speck (10) tested the effect of all the fatty acids common to milk and found that the growth of Streptococcus lactis was inhibited by caprylic, capric and lauric acid. Nashif and Nelson (32) studied the effect of fatty acids on the lipase production of E, fragi, They found that the addition of small amounts of tricaprylin, caprylic and capric acid to vitamin-free casamino acids or peptone media caused a pronounced increase in lipase production. However, caprylic acid was the only one of the three that increased both cell population and lipase production. Lauric acid was found to be inhibitory to the strain of E, £3§gi_used by these workers. 13 EXPERIMENTAL PROCEDURES This investigation was conducted to determine the influence of milk fat and selected fatty acids on the thermal resistance of Pseudomonas fraqi, a common psychrophilic contaminant of dairy products which grows well at refrigera- tion temperatures, but as is the case with most psychrophilic bacteria, the optimum growth temperature is 200 to 25° C. The studies on thermal destruction were divided into four different sections which involved heating the cells in: I II (a) (b) III (a) (b)... (C) IV commercial half-and—half fresh fluid skimmilk cream containing 20 per cent milk fat which was prepared by blending fresh fluid skimmilk and pasteurized whipping cream reconstituted skimmilk reconstituted milk containing 10 per cent milk fat which was prepared by blending reconstituted skimmilk and cream containing 42 per cent milk fat reconstituted cream containing 20 per cent milk fat which was prepared by blending reconstituted skimmilk and cream containing 42 per cent milk fat reconstituted skimmilk containing 0.1 per cent by weight of a selected fatty acid 14 The heat resistance of cells grown at 7° C. was compared to the heat resistance of cells grown at 25° C. when commercial half-and-half was used as the heating medium. The influence of varying concentrations of milk fat on the heat resistance of E, fragi was investigated using the two media listed in II above. The data obtained by heating the cells in these two media were compared with the data obtained from the cells which were grown in half-and-half at 250 C. After analyzing the data obtained from using these three media, it seemed that a second study should be made using media which were prepared from the same milk fat and serum solids. The three media listed in III above were pre- pared from non-fat-dry-milk-solids and cream containing 42 per cent milk fat. These three media were used to compare the influence of the heating medium on the thermal resis- tance of 2, fragi cells and also to determine the influence of the growth medium on the heat resistance of E, fragi. This was accomplished by growing the cells in a medium with a specific concentration of milk fat and heating the cells in media which contained different concentrations of milk fat. For example, cells grown in reconstituted skimmilk were heated in (a) reconstituted skimmilk, (b) reconstituted milk containing 10 per cent milk fat and (c) reconstituted cream 15 containing 20 per cent milk fat. Utilizing these three media, tests were performed on cells subjected to all of the nine possible combinations of growing and heating the cells. The thermal destruction studies involving selected fatty acids were performed by growing the cells in reconstituted skimmilk and then heating the cells in reconstituted skim— milk containing 0.1 per cent by weight of each fatty acid. The fatty acids were butyric, lauric and oleic. Preparation 2; Cells The procedure used to culture 3, §£§3i_was carefully standardized to obtain cells with the same heat resistance and also to obtain approximately the same number of cells per milliliter each time they were propagated. Figure l is a schematic diagram of the procedure followed in growing the cells for thermal destruction studies. The procedure as shown was used to grow the cells in skimmilk and in milk containing 10 to 11 per cent milk fat. When the cells were grown in cream containing 20 per cent milk fat the procedure was modified slightly. In growing the cells a tube of trypticase soy broth was inocu- lated and incubated 24 hours at 25° C. After 24 hours of incubation, a loopful of culture was transferred to a 16 Ucmum mHAB Hommoo m >Q Houmuwmm Uflamcmmfi map mo mummusm may m>onm monocfl N paw; mmB .Hmn mGHHHHum Uflumcmmfi m mcficflmucoo A} u 0 mmDOI ON ‘1‘} ;:._Au mmDOI gm 1’1!) .::_.O wusumuwmfima mafiummn Umuflmmp may um Esflpmz .mmwpsum cofluosnumwp Hmfiuonu How mHHmo map mcflummonm cH poms ouscmuoum wnu mo EmHmMHp capmfimfiom < .H .mfim sump ooH .xmmam Hmwmficmanm one .Edflpme £u3oum pmHHmmp on“ no .HE om L L)! ‘ £9089 mom mmmofiummne pcmam Home assoc mumHm n no musuaso H50: em manor cw p g JDuQOOJ L 11 mmDOI gm )I‘I‘] I; ‘\\;_) ' a ...... fl 0 17 second tube of broth and after additional incubation at 25° C. for 24 hours, 0.1 ml. of inoculum was placed into 50 ml. of the desired medium. The inoculated medium was agita- ted continuously with a magnetic stirrer throughout incuba— tion at 25° C. for 20 hours. After the 20 hours, 0.1 m1. of culture was transferred to a second flask containing the same medium. This inoculated medium was also incubated with agitation for 20 hours at 25° C., after which the cells were used in the heating trials. To minimize conduction of heat from the motor of the agitator into the contents of the flask, a small copper wire stand was used to provide a two inch air space between the flask and the top of the agitator. The cells propagated in cream containing 20 per cent milk fat were grown as illustrated in Figure 1, except agitation was not used as it resulted in churning of the milk fat; the incubation time was increased to 24 hours without agitation. The cells grown in half-and—half at 7° C. were also grown according to the procedure in Figure 1, except the time between transfers was 7 days. Agitation . . 0 was not used in grow1ng the cells at 7 C. because of the 18 reason given above. Preliminary studies showed that the cells would be in the same growth phase after incubation under the following conditions: (a) 20 hours with agitation at 25° C., (b) 24 hours without agitation at 25° C. and (c) 7 days without agitation at 7° C. After the final incubation period the cultures were placed in an ice bath and held at 0° C. until one milliliter portions were removed for use in the heating trials at 48°, 50° and 52° c. Placing the cells in the ice bath stopped their growth and assured having cells that were in the same growth phase at the time of heating at each of the three temperatures. Preparation Q£_Half-and-half a§_§_Heating7Medium Commercial half-and-half was used as a heating medium in the preliminary part of this research program. The half-and- half was purchased as needed from the Michigan State University Dairy Store. Two-hundred milliliter quantities of this heating menstrum.wa£ placed in stainless steel heating chambers (24) and covered with parchment paper. The heating chambers and contents were sterilized and allowed to cool before the sterile closure was attached to the heating 19 chamber. Allowing the menstrum to cool before attaching the closure prevented the formation of.a vacuum which pulled the rubber gasket into the chamber. Preparation p£_Fresh Skimmilk and Cream Containing 29_Per Cent Milk Fat as Heating Media The need for a standardized heating medium became quite evident when half-and-half was used as the heating medium. Therefore, the skimmilk and 20 per cent cream were prepared in sufficient quantities to adequately provide the needs of this experiment. These media were prepared from fresh, pasteurized skimmilk and whipping cream. The 20 per cent cream was heated to 60° C. and homogenized in a commercial three piston homogenizer at 2500 pounds per square inch (psi). Only one stage of the homogenizer was used, consequently the 20 per cent cream was very viscous. The skimmilk and 20 per cent cream were dispensed in 200 m1. quantities into 8 ounce jars with screw-cap lids, steril- ized at 121° C. for 15 minutes, and stored at 4° C. Immediately before the heating trial the desired heating medium was aseptically transferred into a sterilized heating chamber and sealed. . :a'Y'V‘ 3x . v“. 20 Preparation pf Reconstituted Skimmilk, Reconstituted Milk Containingng_Per Cent Milk Fat and Reconstituted ggeam Containing 29_Per Cent Milk Fat §§_Heatipg_Media These three heating media were prepared from reconstituted skimmilk containing 8.30 per cent total solids and raw 42 per cent cream. This skimmilk was used as a heating medium and also to standardize the other two media to the desired con- centration of milk fat. All three media were prepared in sufficient quantity to provide media for all of the experi- ments. After standardization each medium was treated as described in the previous section. These media were used in two separate experiments: (a) the growth and heating media contained the same concentration of milk fat and (b) the growth and heating media contained different concentrations of milk fat. Preparation pf_Reconstituted Skimmilk Containing Selected Fatty Acids §§_Heatinq Media Reconstituted skimmilk was used as a base to which the fatty acids were added. The lauric and oleic acids were added directly to the skimmilk to give a 0.1 per cent concentration by weight. These skimmilk-fatty acid mixtures were then homoqenized at 2500 psi in a single piston pilot 21 plant homogenizer. Since lauric and oleic acids are essentially insoluble in water, homogenization was necessary to obtain uniform dispersion in the skimmilk. After homoqenization the mixuues were dispensed in 200 m1. quanti- ties into 8 ounce jars and sterilized. Prior to steriliza- tion nitrogen was bubbled for one minute through the skim- milk containing oleic acid to minimize oxidation of the fatty acid. The skimmilk-fatty acid mixtures were held at 4° C. until the time of use. The heating menstrum contain- ing butyric acid was prepared by aseptically adding sterile butyric acid to sterile skimmilk immediately before begin- ning the heating trial. This menstrum was prepared in this manner since a skimmilk-butyric acid mixture would coagulate during sterilization. When the skimmilk contained butyric, lauric or oleic acid in a 0.1 per cent concentration, the pH was reduced to 5.85, 6.6 and 6.45, respectively. Therefore, in order to eliminate differences due to pH, heating trials were conducted with and without the pH adjusted to 6.7 which is normal for skimmilk. In the trials requiring pH adjustment, a sterile saturated solution of trisodium phosphate (Na3PO4) was used. The trisodium phosphate was added aseptically to the menstrum in the heating chambers immediately before beginning the heating trial. ' ‘ ' ..z'w'jtrw J 22 HeatinggProcedure The thermal destruction studies were made using equip- ment and methods similar to those described by Kaufmann and Andrews (24). This method permits tempering of the heating menstrum to the desired temperature before adding the cells. A copper-constantan thermocouple and recording potentiometer were used to determine when the heating men- strum was at the desired temperature. When the menstrum was at the desired temperature a sterile 2 m1. glass hypo- dermic syringe fitted with a sterile 2-inch, l6-gauge needle was used to inject 1.0 m1. of the prepared culture into the heating chamber. At intervals throughout the heating period samples were taken with sterile 5 ml. glass hypodermic syringes fitted with 6-inch, l6-gauge needles. Approximately eight seconds were required to remove a sample and place it in a sterile chilled test tube. The tube was shaken in an ice bath for an additional 20 seconds to further aid in cooling. At the end of 20 seconds the sample temperature was near 0° C. The samples were held in an ice bath until plated; this holding time was normally 30 to 45 minutes. Triplicate plates were prepared using tryptone glucose extract agar _-q. a. ‘3"n-“7Fv'fi ' a". .. 23 containing 0.2 per cent yeast extract (TGEY). The plates were incubated for 48 to 72 hours at 25° C. The samples that were heated at 48° and 50° C. were incubated 48 hours, while those heated at 52° C. were incubated 72 hours. The organisms heated at 52° C. were much slower in recovering and required the additional time to form visible colonies. Determination gf_Thermal Destruction Rates The thermal resistance of E, fragi was determined at 48°, 50° and 52° C. in nineteen different sets of experi- ments which involved variations in pre-incubation of the cells and variations in the media in which the cells were grown and heated. In each of these nineteen different studies, at least two to four heating trials were conducted at each of the heating temperatures. The data obtained from the heating trials at each of the three temperatures were pooled and the statistical method of linear regression was used to prepare a single destruction curve which represented the pooled data. The organisms were not destroyed at a logarithmic rate during the initial interval of the heating period; therefore, the data obtained during the initial intervals were not included in the pool. The approximate shape of the curve during the initial interval 24 of heating is shown as a "broken line." The linear regression calculations determined the slope of each individual destruction curve. The "D" value of each curve was obtained by determining the reciprocal of the calculated slope. The definition of "D'l is the number of minutes required to reduce the cell population by 90 per cent during the logarithmic death rate. Confidence limits at the 95 per cent level were also applied to the D values. The slope of the thermal resistance curve for each of the different experiments was determined by applying the statistical method of least squares to the D values. The "z" value of the thermal resistance curve was obtained by determining the reciprocal of the calculated slope. The definition of "z" is the number of degrees Fahrenheit necessary to cause a ten-fold change in the time required to destroy 90 per cent of the cells. The thermal resistance curve is a phantom thermal death time curve in that it has shape but not position. In order to prepare thermal death time curves that could be compared to the milk pasteurization curve, the time required to decrease the initial cell population by 99.9999 per cent at o o o . . 48 , 50 and 52 C. was determined. These times were 25 determined from the data obtained with the three media listed under I and II on page 13. The 99.9999 per cent destruction times were obtained by extrapolation from the survivor curves when they were extended to 99.9999 per cent destruction. The times required to reduce the initial cell populations by 99.9999 per cent are longer than six D values because the survivor curves always contained a lag period during the initial interval of the heating trials. Thermal death time curves were not prepared from the data obtained when 3, fragi was heated in the media listed under III and IV on page 13. When 2, fragi was heated in these media the times required to reduce the initial cell population by 99.99 per cent were determined. This amount of destruction could normally be determined from the survivor curves without extending the lines. 26 RESULTS Thermal_Destruction pf_§, fragi ip_Half—and-half Grown ip_half-and-half §p_2§° Q, fp£_gg_hours and heated ip_half-and-half. A number of trials were made in which the thermal resistance of B, fragi was measured when commercial half-and-half was used as a growth medium and as a heating medium. The data in Table 1 show the average number of E, fragi per milliliter of sample at various time intervals during heat treatment in half-and-half at 48°, 50° and 52° C. The average was determined from triplicate plates. The "straight line" portion of each survivor curve shown in Figure 2 was prepared by the linear regression method. During the initial interval of heating a lag in cell destruction occurred, consequently the survivor curves show an initial deviation from linearity. This portion of the curve was not considered in the analyses; a broken line has been used to indicate the approximate-shape of the curves. The D values calculated from the straight line portion of the survivor curves in Figure 2 were 5.4, 3.0 and 1.0 at o 48 , 50° and 52° C., respectively. The slope of the thermal resistance curve shown in Figure 3 was determined 27 TABLE 1. Number of g, fragi per milliliter surviving heat treatment in half-and-half after growth in half- and-half at 250 C. for 20 hours g Temperature (0 C.) 48 50 52 Time Survivors Time Survivors Time Survivors (Min. ) (Min. ) (Min. ) 0 *‘k 0 *9: O *‘k 4 2,100,000 2.5 410,000 3 200,000 8 350,000 6 220,000 4 72,000 12 140,000 8 50,000 5 6,900 16 ** 10 5,700 6 490 20 2,400 12 1,000 7 60 24 500 14 110 8 30 0 6,300,000 0 6,300,000 0 6,300,000 4 6,500,000 3 6,100,000 3 1,300,000 8 4,100,000 6 1,400,000 4 300,000 12 1,600,000 8 630,000 5 43,000 16 100,000 10 150,000 6 120 20 ** 12 21,000 7 20 24 4,600 14 3,800 0 2,900,000 0 2,900,000 0 2,900,000 4 3,800,000 3 4,100,000 3 1,300,000 8 3,500,000 5 2,400,000 4 430,000 12 1,200,000 8 520,000 5 73,000 20 130,000 12 74,000 7 6,200 24 7,600 14 11,000 8 360 * Counts represent the average of triplicate plates ** Laboratory accident- 28 E u 8 Q m k c > "S k a U) m. o g 3 P 48°C. q o=|.o 50°C. 2 s. 52°C. I 1 l 1 _l 0 IO 20 30 40 Minutes of Heating Fig. 2. Rate of destruction of E, fragi in half—and-half after growth in half-and-half at 250 C. for 20 hours. l.0 - s" E Q ‘4. o ‘3 .2 - a: - \ O - o -.2 1 1 I g] ”8 IZO I22 l24 l26 Temperature (°F.) Fig. 3. Thermal resistance curve of g, fragi in half-and-half after growth in half-and-half at 250 C. for 20 hours. 29 from the D values using the method of least squares. The 2 value obtained from this thermal resistance curve was 10. The 95 per cent confidence limits of the D values are shown in Figure 3 and in Table 8. The times required to reduce the initial cell population by 99.9999 per cent were 38, 18 and . o o o . 6 minutes at 48 , 50 and 52 C., respectively. 0 Grown ip_ha1f-and-half §p_Z_ p, for Z_days and heated £9 half-and-half. The data presented in Table 2 are the number of E, fragi surviving at various time intervals during the heating trials. These data are shown in the form of survivor curves in Figure 4. The D48’ D50 and D52 values obtained from these curves were 10.0, 5.0 and 2.7, respectively. The 2 value obtained from the thermal resistance curve shown in Figure 5 was 13. The extrapolated survivor curves showed that 99.9999 per cent of the initial cell population was destroyed in 65 minutes at 48° C., 40 minutes at 50° C. and 19 minutes at 52° C. 3O TABLE 2. Number of E, fragi per milliliter surviving heat treatment in half~and4half after growth in half- and-half at 7°C. for 7 days* Temperature (0 C.) 48 5o 52' Time Survivors Time Survivors Time Survivors (Min.) (Min.) (Min.) 0 4,100,000 0 4,100,000 0 4,100,000 12 730,000 4 2,700,000 3 1,500,000 18 180,000 8 830,000 6 250,000 24 51,000 12 190,000 8 62,000 30 11,000 16 20,000 10 6,100 35 3,000 20 2,700 12 1,000 40 1,200 24 660 14 180 0 11,000,000 0 11,000,000 12 1,400,000 4 5,300,000 18 330,000 8 1,400,000 24 84,000 12 340,000 30 29,000 16 61,000 35 7,200 20 9,200 40 2,700 24 1,700 0 3,600,000 0 3,600,000 0 3,600,000 12 430,000 4 1,500,000 3 1,200,000 18 68,000 8 380,000 6 210,000 24 9,900 12 64,000 8 31,000 30 2,100 16 4,400 10 3,500 35 580 20 560 12 330 40 240 24 120 14 130 0 4,100,000 0 4,100,000 12 1,200,000 3 1,800,000 18 390,000 6 620,000 24 150,000 8 200,000 30 33,000 10 53,000 35 8,800 12 11,000 40 3,900 14 3,000 * Counts represent the average of triplicate plates Log of Survivors per ml. Fig. 4. O 31 52°C. 1 IO 1 I l 20 30 40 Minutes of Heating Rate of destruction of g, fragi in half¥and-ha1f after »growth in halféand-half at 7° C. for 7 days. Log ofD (mm) 0 iv ’9 a: ho '0 Fig. 5. H8 1 l l J IZO l22 |24 |26 Temperature (°F.) Thermal resistance curve of g, fragi in half—and-half after growth in half-andéhalf at 7° C. for 7 days. 32 Thermal Destruction p§_£, fragi ip_Fresh Skimmilk and Cream Containinqng_Per Cent Milk Fat In these studies the media were prepared in sufficient quantity to permit the use of the same batch throughout the heating trials desired in this section. Grown and heated ip skimmilk. The average number of survivors per milliliter at various time intervals during . . . . o o o , , heating in skimmilk at 48 , 50 and 52 C. is shown in Table 3. These data were used in preparing the survivor curves shown in Figure 6. At 48° C. the initial lag period continued for approximately 25 minutes after which the cells were destroyed in a logarithmic order. The D values at 48°, 50° and 52° C. were 12.5, 6.2 and 1.9, respectively. Figure 7 contains the thermal resistance curve prepared from the D values and a z of 9 was noted. The time required to destroy 99.9999 per cent of the population was 95, 48 and 18 minutes 0 o o . . at 48 , 50 and 52 C., respectively. In Figure 8, the thermal death time curve is compared to the milk pasteuri- zation curve. Also included in Figure 8 are the thermal death time curves that were prepared from the data obtained when the cells were grown at 25° C. in half-and-half and grown in cream containing 20 per cent milk fat. 33 TABLE 3. Number of g. fragi per milliliter surviving heat treatment in skimmilk after growth in skimmilk at 250 C. for 20 hours* Temperature (0 C.) 48 50 52 Time Survivors' Time Survivors Time Survivors (Min.) (Min.) (Min.) 0 ** 0 *1: O ** 8 6,200,000 5 5,700,000 3 6,900,000 16 4,700,000 10 2,800,000 6 2,800,000 24 3,800,000 15 510,000 8 880,000 32 1,100,000 20 210,000 10 210,000 40 230,000 25 13,000 12 14,000 48 46,000 30 1,500 14 690 0 3,500,000 0 3,500,000 0 3,500,000 8 3,300,000 5 3,500,000 3 3,700,000 16 2,200,000 10 1,400,000 6 830,000 24 1,500,000 15 210,000 8 220,000 32 290,000 20 37,000 10 36,000 40 53,000 25 3,800 12 2,300 48 21,000 14 130 0 9,800,000 5 9,000,000 10 5,200,000 15 1,000,000 20 120,000 25 30,000 30 2,100 * ** Counts represent the average of triplicate plates Laboratory accident 34 D=l2£5 E k Q) Q to B 48°C. .3 S K 3 m .3 50°C a. 3 ’ ' 3 52°C. 2 - I l l I l #1 0 IO 20 30 4O 50 Minutes of Heating Fig. 6. Rate of destruction of g, fragi in skimmilk after :growth in skimmilk at 250 C. for 20 hours. 2.0 '- I A Log of 0 (min.) as o . ' |l8 IZO |22 |24 |26 Temperature (°F.) J .Fig; 7. Thermal resistance curve of E, fragi in skimmilk after gr-owth in skimmilk at 250 C. for 20 hours. .. x. a». \\\ \. 35 l00. _ so. »- A 80. — 70. r- 60 - 50 ~ A 40 F 30 ~ . O 20 r A IO \ 9'. P 8. r D 7. - 6. l- 5. - U) 4m- 2 3 — E 2.— LC t 0.9 0.8 t 0.7 r- 0 Standard curve for 0.6 - the pasturization of milk 0-5 " D Pfrogi cells grown and O 4 _ heated in half-and-halt A P. fragi cells grown and 0-3 - heated m skimmilk 0 P. fragi cells grown and O 0.2 c heated in 20% cream OJ 1 l 1 l J 30 4O 50 60 7O 80 . Temperature (°C.) Fig. 8. A comparison of the thermal death time curves of g. fraqi in media containing 0, 11 and 20 per cent milk fat with the milk pasteurization curve. (The thermal death time curves are a plot of temperature versus the number of minutes required to bring about a 99.9999 per cent reduction in the original cell population) 36 Grown and heated ip cream containing gg_per cent milk Egg, The destruction rate curves shown in Figure 9 were prepared from the data in Table 4. When the cells were heated at 48°, 50° and 52° C. the D values derived from the destruction curves were 4.3, 2.4 and 1.4, respectively. The initial lag periods in these survivor curves are not as long as those obtained with the cells grown and heated in skimmilk. The thermal resistance curve is shown in Figure 10 and a z of 15 was calculated from the slope. At 48°, 50° and 52° C., 99.9999 per cent of the original cell popu- lation was destroyed in 28, 18 and 11 minutes, respectively. The thermal death time curve prepared from these times is shown in Figure 8. Thermal Destruction pf_§, fragi ig_Reconstituted Skimmilk, Reconstituted Milk Containipg_ 19_Per Cent Milk Fat and Reconstituted Cream Containing 29 Per Cent Milk Fat Skimmilk powder and cream containing 42 per cent milk fat ‘were utilized in preparing the following three-media: (a) reconstituted skimmilk, (b) reconstituted milk containing 10 ‘per cent milk fat and (c) reconstituted cream containing 20 per cent milk fat. 7 r 37 6 \ E t 5‘ 8 0:4.3 3 2 4 .8 4 _ /°‘ ' s K 3 (I) 3 50°C 8‘ 3 LD=I.4 ~l 52°C. 48°C. 2 .— l l l l J 0 IO 20 30 40 Minutes of Heating Fig. 9. Rate of destruction of g. fragi in cream containing 20 per cent milk fat after growth in cream containing 20 per cent milk fat at 250 C. for 24 hours. l.O *- Log of0(mln) . . '03 '1 /0| O l l I I ”8 IZO |22 |24 l26 Temperature (F°.) Fig. 10. Thermal resistance curve of g. fragi in cream containing 20 per cent milk fat after growth in cream containing 3:20 per cent milk fat at 25° C. for 24 hours. ca— 3- 38 Table 4. Number of E, fragi per milliliter surviving heat treatment in cream containing 20 per cent milk fat after growth in cream containing 20 per cent milk fat at 250 c. for 24 hours* Temperature (0 C.) 48 50 52 Time Survivors Time Survivors Time Survivors (Min.) (Min.) (Min.) 0 2,400,000 0 2,400,000 0 2,400,000 4 1,300,000 3 1,400,000 3 900,000 8 130,000 6 97,000 4 330,000 12 3,500 8 9,200 5 67,000 16 2,100 10 1,600 6 13,000 20 110 7 3,700 24 70 8 280 0 1,500,000 0 1,500,000 0 1,500,000 4 920,000 3 790,000 3 410,000 8 80,000 6 68,000 4 190,000 12 8,000 8 4,100 5 30,000 16 1,000 10 880 6 3,300 20 320 7 310 24 80 8 50 0 1,700,000 0 2,100,000 0 2,100,000 4 890,000 3 1,500,000 3 870,000 8 110,000 6 230,000 4 240,000 12 6,600 8 34,000 5 41,000 16 690 10 4,500 6 9,600 7 2,700 8 420 Counts represent the average of triplicate plates 39 Grown and heated ip_reconstituted skimmilk. The average counts obtained from triplicate plates are shown in Table 5. These data are depicted as destruction rate curves in Figure 0 o o 11 and the D values at 48 , 50 and 52 C. are 6.2, 3.6 and 1.9, respectively. The 2 calculated from these D values was 14 (Figure 12). The initial cell populations were reduced by 99.99 per cent in 46, 27 and 12 minutes at 48°, 50° and o . 52 C., respectively. Grown and heated ip_reconstituted milk containing lg_per cent milk fat. The data in Table 6 show the average number of survivors per milliliter at various time intervals during , o o o . heating at 48 , 50 and 52 C. The surVivor curve prepared from the data obtained at 48° C. has a lag period that continues for approximately 45 minutes (Figure 13) and a total of 75 minutes was required to destroy 99.99 per cent of the initial cell population. At 50° and 52° C., 99.99 per cent of the initial cell population was destroyed in 24 ea d 11 minutes, res ecti l . Th D , n p ve y e 48 D50 and D52 values rare 10.5, 5.3 and 2.4, respectively. The thermal resistance curve prepared from these D values is shown in Figure 14 and -the 2 value is 11. 40 TABLE 5. Number of E, fragi per milliliter surviving heat treatment in reconstituted skimmilk after growth in reconstituted skimmilk at 25° C. for 20 hours* Temperature (0 C.) 48 50 52 Time Survivors Time Survivors Time Survivors (Min.) (Min.) (Min.) 0 8,200,000 0 8,200,000 0 8,200,000 10 7,800,000 5 7,400,000 3 ** 20 ** 10 4,400,000 6 1,700,000 30 1,600,000 15 2,200,000 8 810,000 40 110,000 20 750,000 10 210,000 50 1,100 25 100,000 12 32,000 60 43 30 3,300 14 1,100 0 600,000 0 600,000 0 600,000 10 420,000 5 180,000 3 440,000 20 350,000 10 ** 6 150,000 30 13,000 15 66,000 8 8,900 40 300 20 5,100 10 500 25 220 12 20 0 4,100,000 0 4,100,000 0 4,100,000 10 7,100,000 5 2,800,000 3 1,900,000 20 3,300,000 10 750,000 6 210,000 30 130,000 15 150,000 8 78,000 40 4,300 20 54,000 10 6,500 50 90 25 3,600 12 740 30 94 14 60 * ** Counts represent the average of triplicate plates Laboratory accident 41 Log of Survivors per ml. 52°C. 50°C. l l L I 0 IO 20 30 4O 50 60 Minutes of Heating Fig. 11. Rate of destruction of g. fragi in reconstituted skimmilk after growth in reconstituted skimmilk at 25° C. for 20 hours. Log ofD(mrn) ' in in l / no: O 1 1 l J ”8 l20 l22 l24 I26 Temperature (°F.) Fig. 12. Thermal resistance curve of g. fragi in reconstituted skimmilk after growth in reconstituted skinunilk at 25° C. for 20 hours. 42 TABLE 6. Number of g, fragi per milliliter surviving heat treatment in reconstituted milk containing 10 per cent milk fat after growth in reconstituted milk containing 10 per cent milk fat at 250 C. for 20 hours* Temperature (0 C.) 48 50 52 Time Survivors Time Survivors Time Survivors (Min.) (Min.) (Min.) 0 6,500,000 0 5,000,000 0 5,000,000 10 5,800,000 5 1,700,000 3 930,000 20 3,400,000 10 600,000 6 34,000 30 2,400,000 15 64,000 9 3,000 40 640,000 20 3,000 12 200 50 85,000 25 500 60 5,500 30 60 0 4,400,000 0 750,000 0 2,700,000 10 8,000,000 4 2,000,000 3 550,000 20 8,300,000 8 640,000 5 54,000 30 5,400,000 12 210,000 7 3,800 40 1,900,000 16 33,000 9 ** 50 370,000 20 3,000 11 70 60 30,000 24 750 0 900,000 0 5,500,000 15 1,400,000 3 2,000,000 30 850,000 6 580,000 45 490,000 8 480,000 60 6,000 10 230,000 75 150 12 85,000 14 30,000 0 900,000 0 750,000 15 5,200,000 3 1,100,000 30 5,400,000 6 32,000 45 970,000 9 3,000 60 38,000 12 90 75 990 ** Counts represent the average of triplicate plates Laboratory accident Log of Survivors per ml. 52°C. 1 l 43 48°C Log of 0 (min) 20 30 4O . 50 60 Minutes of Healing c .o ‘0 - 2:“ .8 - .. 2 .6 - \ .4 - §‘ .2 b O l l l J ”8 I20 I22 I24 I26 Temperature (°F.) 44 Grown and heated ip_reconstituted cream containing 29 ,pg; cent milk fat. The data presented in Table 7 show the number of cells surviving heating exposures at 48°, 50° and 52° C. The survivor curves shown in Figure 15 were prepared from the data in Table 7. The calculated D48’ D50 and D52 values were 4.2, 2.5 and 2.0, respectively. The initial cell population was reduced by 99.99 per cent in 25, 17 and 12 minutes at 48°, 50° and 52° C., respectively. The 2 value of P, fgagi_when heated in the 20 per cent cream was 22 (Figure 16). In Table 8 a summary is presented of the results reported thus far. In each case the cells were heated in a medium which contained the same concentration of milk fat as the medium in which they were propagated. The media in which the cells were grown and heated were dairy products con— taining 0 to 20 per cent milk fat. The calculated D values and the calculated 2 value obtained with each of the media -are presented. The 95 per cent confidence limits of the I) values also are presented. 45 Counts represent the average of triplicate plates 'FABLE 7. Number of g. fragi per milliliter surviving heat treatment in reconstituted cream containing 20 per cent milk fat after growth in reconstituted cream containing 20 per cent milk fat at 250 C. for 24 hours* Temperature (0 C.) 48 50 52 Time Survivors Time Survivors Time Survivors (Min.) (Min.) (Min.) 0 5,300,000 0 5,300,000 0 5,300,000 5 4,300,000 3 6,000,000 3 5,100,000 10 410,000 6 3,200,000 5 1,900,000 15 16,000 9 650,000 7 340,000 20 400 12 65,000 9 32,000 25 60 15 3,100 11 1,900 18 140 13 200 0 3,600,000 0 3,600,000 0 3,600,000 5 3,600,000 3 5,200,000 3 3,300,000 10 2,300,000 6 3,000,000 5 1,200,000 15 630,000 9 1,000,000 7 210,000 20 31,000 12 140,000 9 7,400 25 2,300 15 11,000 11 710 30 270 13 41 0 1,900,000 0 1,900,000 0 1,900,000 5 3,700,000 3 4,400,000 3 4,300,000 10 660,000 6 1,800,000 5 2,000,000 15 38,000 9 280,000 7 370,000 20 2,200 12 31,000 9 49,000 25 170 15 12,000 11 6,600 18 35 13 770 0 7,300,000 0 7,300,000 5 5,200,000 3 6,500,000 10 2,500,000 5 2,400,000 15 260,000 7 360,000 20 8,700 9 60,000 25 600 11 4,000 30 15 9" v33." '1." .1: 7 46 L§\ \\\ \\\ \ \\ 6 " ‘ \ \\ E k a 5 (A k 0 2 E 4 " 0:4 2 a a) g. 3 r- s] 48°C. 2 _ 52°C. 50°C. ' I I I I 0 IO 20 30 40 Minutes of Healing Fig. 15. Rate of destruction of g. fragi in reconstituted cream containing'20 per cent milk fat after growth in reconstituted cream containing 20 per cent milk fat at 25° C. for 24 hours. o I Z=22 \ IOI \ Log ofD (mm) N '4: as m 'T /|OI IOI O I I I I ”8 120 :22 I24 I26 Temperature (°F.) FLg. 16. Thermal resistance curve of _P_. fragi in reconstituted Stream Qantaining 20 per cent milk fat after growth in reconstituted cream containing 20 per cent milk fat at 25° C. for 24 hours. m" «as. {Viie‘sr v . --: ..___ -- -.. _ ___ 47 muses cm son .0 0mm .msso a son .0 on mason om now .o.omm um £3080 maamo ammum pm c3oum maaoo «mmnm um czoum mHHmu Amway mldlml (0.0 mm N.m I m.a o.m m.m I m.N m.m m.¢ I m.m N.¢ Aumm Romy Emwuo ocmusuflumcoomm Ha ©.N I N.N v.m m.m I m.v m.m m.mH I N.m m.oa Aumm Xoav xHHE mcmusuflumcoomm ca m.m I h.a m.H v.v I H.m o.m H.b I ©.m N.© MHHEEme acousuflumcooom ma @.H I m.H g.a m.m I m.N g.m o.m I m.m m.v ofiuwm Romv Emmuu m o.m I win 0...” v.0 I H.® N.® 0.09.. I 0.0 m.NH MHHEEHMm nmmum n ma H.m I v.m h.m m.m I m.¢ o.m m.HH I w.@ 0.0H QwamnIUCMImem OH N.H I.¢m. O.H h.m I ©.N o.m h.© I m.¢ v.m MMHMSIUQMIMHME A.m V .U Omm .0 Com .0 Omv o uuscoum n masses o masses o masses o OUCODHMGOU Xmm OUCOGHMEOU Xmm OUCOUHmGOU *mm umm xHflE Dado mom on Op 0 mcwcfimucoo muOSUOHm muflmc msoflnm> GH cause: can czoum c033 ammuw..m.mo cofluosnumop Hasnwna .ngq as. 48 Thermal Destruction pf g. fragi Cells ip.§_Medium Contagping g_Diffepent Concentpation p£_Milk Fat Than the Medium 13 Which the Cells WEre Grown The data presented in the previous sections were obtained with cells grown and heated in media containing the same con- centration of milk fat. In the trials reported in this sec- tion the concentration of milk fat in the growth medium was not the same as that of the heating medium. Grown ip_reconstituted skimmilk -- heated lg reconstituted milk containing 19 per cent milk fat. The data in Table 9 show the average number of g, fgggi_per milliliter surviving heat treatment in milk containing 10 per cent milk fat. The destruction curves shown in Figure 17 were constructed from these data and the D values obtained were 5.0, 2.3 and 1.4 at 48°, 50° and 52° C., respectively. The 2 calculated from the slope of the thermal resistance curve was 13 (Figure 18). A 99.99 per cent reduction in population occurred after 40, 20 and 8 minutes of exposure at 48°, 50° and 52° C., respectively. Grown ip reconstituted skimmilk -- heated ip reconstituted _ggeam containing gg_per cent milk fat. The curves presented irl Figure 19 show the destruction rate of_g. fragi when heated in 20 per cent reconstituted cream after propagation in recon- stjltuted skimmilk. The data in Table 10 were utilized in _ 49 TABLE 9. Number of g. fragi per milliliter surviving heat treatment in reconstituted milk containing 10 per cent milk fat after growth in reconstituted skim- milk at 250 C. for 20 hours* 0 Temperature ( C.) 48 50 52 Time Survivors Time Survivors Time Survivors (Min.) (Min.) (Min.) 0 4,300,000 0 4,300,000 0 4,300,000 6 660,000 4 430,000 3 350,000 12 470,000 8 190,000 5 130,000 18 160,000 12 40,000 6 7,600 24 23,000 16 6,400 7 1,500 30 560 20 150 8 1,100 36 16 9 310 0 3,700,000 0 3,700,000 0 3,700,000 6 380,000 4 150,000 3 160,000 12 160,000 8 61,000 5 2,400 18 40,000 12 7,100 7 200 24 3,000 16 300 30 100 '* Counts represent the average of triplicate plates «WM... ......h‘fl‘s ...-w .4. , .. . ...r _ _ , warring 3 .. ‘ 7 50 Log of Survivors per ml. 52°C. 50°C- l I l I 0 IO 20 30 40 Minutes of Heating Fig. 17. Rate of destruction of p. fragi in reconstituted milk containing 10 per cent milk fat after growth in reconstituted skimmilk at 250 C. for 20 hours. Log of 0 (min) O I I I I “8 I20 l22 I24 l26 Temperature (°F.) ‘ Fig‘ '18. Thermal resistance curve of 3. fragi in reconstituted ““115 containing 10 per cent milk fat after growth in reconstituted skimmilk at 25° c. for 20 hours. W. r». .1» 39* ,"T » w 4?— 3' 51 E K G Q. U) K 0 .> s K .3 45 \~ 0 8‘ 48°C. 4 50°C. I I I I I 0 I0 20 30 40 Minutes of Healing Fig. 19. Rate of destruction of E, fragi in reconstituted cream containing 20 per cent milk fat after growth in reconstituted skimmilk at 25° c. for 20 hours. LOI- .§ _ 2:22 5 .8 Q .6 :° .. ‘0. - o h- e '4 - 3. ".2_- ‘ o I I 4 J ”8 l20 I22 I24 |26 Temperature (°F.) -Eji‘g. 20. Thermal resistance curve of 2, fragi in reconstituted C5331“ Qantaining 20 per cent milk fat after growth in reconstituted Skimmilk at 25° c. for 20 hours. 52 TABLE 10. Number of g. fragi per milliliter surviving heat treatment in reconstituted cream containing 20 per cent milk fat after growth in reconstituted skim- milk at 250 C. for 20 hours* 0 Temperature ( C.) 48 50 52 Time Survivors Time Survivors Time Survivors (Min. ) (Min. ) (Min. ) 0 2,200,000 0 2,200,000 0 2,200,000 8 6,600,000 5 5,800,000 3 7,300,000 16 ** 10 ** 6 1,800,000 25 1,200,000 15 800,000 9 180,000 32 150,000 20 130,000 12 5,100 40 1,800 25 4,200 15 70 48 41 30 90 0 5,300,000 0 5,300,000 0 5,300,000 7 5,300,000 5 4,100,000 3 5,200,000 14 2,300,000 10 1,100,000 5 1,600,000 21 300,000 15 170,000 6 1,200,000 28 10,000 20 3,400 7 480,000 35 50 25 52 8 ** 9 55,000 12 900 * Counts represent the average of triplicate plates ** laboratory accident 53 preparing these curves and the D48' D50 and D52 values calculated from the slope of these curves were 4.5, 2.8 and 2.1, respectively. A z of 22 was calculated from the slope of the thermal resistance curve shown in Figure 20. The initial cell population was reduced by 99.99 per cent . . o o o in 38, 25 and 14 minutes at 48 , 50 and 52 C., respec- tively. Table 15 contains a summary of the data obtained from cells grown in reconstituted skimmilk and heated in (a) reconstituted skimmilk, (b) reconstituted milk contain- ing 10 per cent milk fat and (c) reconstituted cream con- taining 20 per cent milk fat. Grown ip_reconstituted milk containing lg_per cent milk fat —- heated ig_reconstituted skimmilk. The destruction rate curves shown in Figure 21 were constructed from the data presented in Table 11. The D values calculated from the straight line portion of these curves were 5.0, 3.0 and , o o o . 1.2 minutes at 48 , 50 and 52 C., respectively. When the entire survivor curve was considered the populations were reduced by 99.99 per cent in 32, 18 and 8 minutes at 48°, 50° and 52° C., respectively. The 2 value of the thermal resistance curve shown in Figure 22 is 12. Log of Survivors per ml. 50°C. 52°C 48° C. | I I I J 0 IO 20 3O 40 Minutes of Heating Fig. 21. Rate of destruction of g. fragi in reconstituted skimmilk after growth in reconstituted milk containing 10 per cent milk fat at 250 C. for 20 hours. 1.0 - .E' E .8 \— o .6 ‘4. 0 o, .4 0 ~l .2 O O l I I I us I20 :22 I24 |26 Temperature (°F.) Fig. 22. Thermal resistance curve of P. fragi in reconstituted skimmilk after growth in reconstituted milk containing 10 per cent milk fat at 250 C. for 20 hours. i TABLE 11. Number of g, fragi per milliliter surviving heat treatment in reconstituted skimmilk after growth in reconstituted milk containing 10 per cent milk fat at 250 c. for 20 hours* 0 Temperature ( C.) 48 50 52 Time Survivors Time Survivors Time Survivors (Min.) (Min.) (Min.) 0 6,000,000 0 6,000,000 0 6,000,000 8 820,000 5 750,000 3 2,400,000 16 360,000 10 110,000 6 49,000 24 14,000 15 3,500 9 100 34 130 20 80 0 9,100,000 0 9,100,000 0 9,100,000 6 660,000 4 1,700,000 3 2,100,000 12 220,000 8 190,000 5 190,000 18 53,000 12 40,000 7 3,100 24 4,100 16 2,200 9 50 30 100 20 33 36 22 * Counts represent the average of triplicate plates 56 Grown ip_reconstituted milk containing 19_per cent milk fat -- heated ip reconstituted cream containing gg_per cent milk fat. The data in Table 12 show the number of survivors per milliliter in the samples taken during heating at 48°, 50° and 52° C. The D48' D50 and D52 values obtained from the survivor curves shown in Figure 23 were 3.3, 2.3 and 1.4,, respectively. The survivor curves show that 99.99 per cent of the original cell population was destroyed in 25 minutes at 48° C., 15 minutes at 500 C. and 8 minutes at 52° C. The 2 calculated from the D values was 20 (Figure 24). A summary of the data obtained with the cells grown in milk containing 10 per cent milk fat is shown in Table 15. Grown ip_reconstituted cream containing gg'per cent milk fat -— heated lg reconstituted skimmilk. The data in Table 13 were used to prepare the survivor curves in Figure 0 o o 25. When the cells were heated at 48 , 50 and 52 C. the D values were 5.0, 2.9 and 1.4, respectively. The 2 value calculated from these D values was 13 (Figure 26). The times irequired to reduce the initial cell population by 99.99 per 0 o o c-ent at 48 , 50 and 52 C. were 20, 15 and 8 minutes, re spectively. 57 TABLE 12. Number of g, fragi per milliliter surviving heat treatment in reconstituted cream containing 20 per cent milk fat after growth in reconstituted milk containing 10 per cent milk fat at 250 C. for 20 hours* 0 Temperature ( C-) 48 50 52 Time Survivors Time Survivors Time Survivors (Min.) 7 5 (Min.) (Min.) 0 2,900,000 0 2,900,000 0 2,900,000 7 1,000,000 4 710,000 3 590,000 14 110,000 8 130,000 5 110,000 21 330 12 2,600 7 6,200 9 60 0 1,500,000 0 1,500,000 0 1,500,000 6 3,800,000 4 1,600,000 3 1,800,000 12 1,200,000 8 950,000 5 150,000 18 310,000 12 23,000 6 73,000 24 20,000 16 1,000 7 21,000 30 440 8 3,600 9 2,100 * Counts represent the average of triplicate plates 7 58 Log of Survivors per ml. A T D= L4 50°C 48°C. 0 I0 20 30 40 Minutes of Heating Fig. 23. Rate of destruction of p. fragi in reconstituted cream containing 20 per cent milk fat after growth in reconstituted milk containing 10 per cent milk fat at 25° C. for 20 hours. l.0 - Log of 0 (min) 0 N b (D 00 I / 0| 0‘ 4* I I I “8 l20' l22 I24 I26 Temperature (°F.) Fig. 24. Thermal resistance curve of g. fragi in reconstituted cream containing 20 per cent milk fat after growth in reconstituted milk containing 10 per cent milk fat at 25° C. for 20 hours. - Z..-v"’.~p‘ fuzz—é. s. 55‘ -’1- :93... ___‘ ~12 I}. A) 71?. - '1‘?!“ 52:53 3.3”!" 3 .".'.~ 'Hil‘" 54"" I" * "Vs-"E 59 TABLE 13. Number of g, fragi per milliliter surviving heat treatment in reconstituted skimmilk after growth in reconstituted cream containing 20 per cent milk fat at 250 C. for 24 hours* Temperature (° C.) 48 50 52 Time Survivors Time Survivors Time Survivors (Min.) (Min. ) (Min.) 0 3,600,000 0 3,600,000 0 3,600,000 8 120,000 5 350,000 3 580,000 16 2,800 10 15,000 6 62,000 24 100 15 200 9 400 32 40 0 1,400,000 0 1,400,000 0 1,400,000 7 110,000 4 380,000 3 120,000 14 800 8 14,000 5 5,100 21 30 12 200 7 100 * Counts represent the average of triplicate plates gr *FnW'EJ 6.:- 3"“ 60 Log of Survivors per ml. 50°C. I I I I 0 IO 20 30 40 Minutes of Healing Fig. 25. Rate of destruction of p. fragi in reconstituted skimmilk after growth in reconstituted cream containing 20 per cent milk fat at 25° C. for 24 hours. I.0 - ‘2 ' Z=I3 E .8- Q .6-_ - ‘5 o u .4 ~ - - o " .2 e O I 1 I J ' ll8 l20 l22 I24 l26 Temperature (°F.) Fig . 26. Thermal resistance curve of p. fragi in reconstituted Skimmilk after growth in reconstituted cream containing 20 per cent,milk fat at 25° C. for 24 hours. 61 Grown ip reconstituted cream containipg 29 per cent milk fat -- heated ip_reconstituted milk containing lg_per cent mflk fat. The data in Table 14 show the average number of p, fragi per milliliter surviving at various time intervals . . o o o , during heating at 48 , 50 and 52 C. Figure 27 shows the survivor curves prepared from these data and the D value of o o 0 each curve. At 48 , 50 and 52 C. the D values were 3.7, 2.3 and 0.9, respectively. Figure 28 shows the thermal resistance curve prepared with these D values and a z of 11 was calculated from the slope of the curve. A 99.99 per cent reduction in population occurred in 18, 12 and 8 . o o o , minutes at 48 , 50 and 52 C., respectively. Tabl 15 t ' th D , D d D ' e con ains e 48 50 an 52 values obtained with cells grown and heated in media containing the same concentration of milk fat, and also includes values obtained when the cells were grown and heated in media in which the concentration of milk fat was varied. The z values and the 95 per cent confidence limits of the D values also are included in Table 15. 62 TABLE 14. Number of g, fragi per milliliter surviving heat treatment in reconstituted milk containing 10 per cent milk fat after growth in reconstituted cream containing 20 per cent milk fat at 250 C. for 24 hours* o Temperature ( C.) 48 50 52 Time Survivors Time Survivors Time Survivors (Min.) (Min.) (Min.) 0 1,700,000 0 1,700,000 0 1,700,000 7 100,000 4 190,000 3 160,000 14 200 8 2,500 5 10,000 21 20 7 100 0 3,500,000 0 3,500,000 0 3,500,000 6 730,000 4 890,000 3 830,000 12 12,000 8 13,000 5 120,000 18 300 16 16 7 400 8 70 * Counts represent the average of triplicate plates 63 E s Q U) k 0 > 'S 2.3 s In 0: .9 k 0 o. O \I 48°C. I 50°C. 52°C. I 1 l 1 _I 0 IO 20 30 40 Minutes of Heating Fig. 27. Rate of destruction of g. fragi in reconstituted milk containing 10 per cent milk fat after growth in reconstituted (cream containing 20 per cent milk fat at 250 C. for 24 hours. 8 .6 Log of 0 (min) In n 2 l 1 1 J ' ll8 l20 l22 I24 I26 . Temperature (° F.) Fig. 28. Thermal resistance curve of g. fragi in reconstituted 'milk containing 10 per cent milk fat after growth in reconstituted cream containing 20 per cent milk fat at 25° C. for 24 hours. 64 NN N.N I m.H o.m m.N I m.m m.m m.¢ I m.m m.¢ ON ON HH mm. Imm. hm. m.m I m.a m.N m.m I h.m b.m OH om ma m.m I o.H v.H n.m I v.N m.N m.m I 0.m o.m 0 ON ON m.H I H.H g.H 0.N I H.m m.m m.¢ I h.m m.m om OH Ha 0.N I N.N ¢.m m.m I m.¢ m.m m.NHI m.m m.oa OH OH NH 0.H I o.H N.H o.v I ¢.N o.m m.m I 0.m o.m o OH mm m.m I m.H H.N o.¢ I N.N m.m m.m I N.m m.g .ON 0 ma h.H I N.H v.H o.m I m.H m.m m.m I m.¢ o.m OH 0 ma m.N I h.H m.H ¢.¢ I H.m 0.m H.m I m.m «.0 o o . . . ESHCmE .Eswpmfi U on 0 00m 0 00¢ maflpmmn nmsoum A.m VN muHEHH Q muflEHH Q mufiEHH O :H umm mocmwwmcoo Xmm 00:60:00 .83 cocooflsoo ans 0:? usoo nos czoum mums maamo mnu £0H£3 CH Eswpus esp CH ups» Baum unmanm> 003 £0fl£3 pom Made 00 sawumuucwocoo m mcflcwnucoo EchmE m CH Amman .M.mo coauusnunup HmEHoSB .m._.. magma. 65 Thermal Destruction g§_g, fragi ip_Reconstituted Skimmilk Containipg Selected Fatty Acids In these studies the cells were propagated in recon— stituted skimmilk at 25° C. for 20 hours and then heated in reconstituted skimmilk which contained 0.1 per cent by weight of an individual fatty acid. The pH of the skim- milk decreased with the addition of the fatty acids, but since the pH did not decrease by the same amount with each of the fatty acids it was necessary to adjust the pH to a common level. A pH of 6.7 was selected since this is normal for skimmilk. Heating trials were also conducted without adjustment of the pH in order to determine what effect the fatty acids would have on the thermal resistance of P._§£§gi at a pH of less than 6.7. ;p_reconstituted skimmilk containing lauric acid. A 0.1 per cent concentration of lauric acid in the skimmilk reduced the pH from 6.7 to 6.6. The data in Table 16 show the average number of survivors per milliliter at various time intervals during heating when the pH of the medium was 6.6. The D48' D50 and D52 values at this pH were 6.7, 3.2 and 1.8, respectively. The survivor curves from which tfliese D values were obtained are shown in Figure 29. The triermal resistance curve prepared from these D values is 66 TABLE 16. Number of g, fragi per milliliter surviving heat treatment in reconstituted skimmilk containing 0.1 per cent by weight of lauric acid at pH 6.6* 0 Temperature ( C.) 48 50 52 Time Survivors Time Survivors Time Survivors (Min.) (Min.) (Min.) 0 12,200,000 0 12,200,000 0 12,000,000 10 8,700,000 5 6,900,000 3 7,000,000 20 4,700,000 10 3,300,000 6 1,200,000 30 890,000 15 660,000 8 170,000 40 100,000 20 100,000 10 65,000 50 1,600 25 9,300 12 4,800 60 41 30 150 14 49 0 11,000,000 0 1,900,000 0 1,900,000 10 7,500,000 5 1,300,000 3 1,800,000 20 2,200,000 10 480,000 6 190,000 30 180,000 15 110,000 8 23,000 40 3,900 20 1,300 10 300 50 120 25 70 12 20 0 1,600,000 0 11,000,000 0 1,600,000 9 5,200,000 5 6,600,000 3 410,000 18 2,400,000 10 2,900,000 5 180,000 27 870,000 15 430,000 7 29,000 36 140,000 20 86,000 9 4,300 45 5,800 25 3,700 11 250 54 240 30 48 13 32 * Counts represent the average of triplicate plates Log of Survivors per ml. 67 52°C. 50°C. '48°C. I I I I l l ’1 0 I0 20 30 40 50 60 Minutes of Heating Fig. 29. Rate of destruction of g. fragi in reconstituted skimmilk containing 0.1 per .LHI6}6. cent by weight of lauric acid at |.O ' E e - Q .6 - - ‘6 2 a .4 " .. 3 0‘ .2 - " .0 ' ‘ ' 4 HS l20 :22 I24 |26 Temperature I °F.) Fig. 30. Thermal resistance curve of g. fragi in reconstituted skimmilk containing 0.1 per pH 6.6. cent by weight of lauric acid at 68 shown in Figure 30 and the z value was 12. When the pH of the heating medium was 6.6 the cell populations were reduced by 99.99 per cent in 49 minutes at 48° C., 25 . o . 0 minutes at 50 C. and 11 minutes at 52 C. The data in Table 17 were obtained when p, fragi was heated in skimmilk-lauric acid mixtures at pH 6.7. The destruction rate curves in Figure 31 were prepared from the data in Table 17. The D values of the curves were 6.2, 4.3 o o o . . and 1.5 at 48 , 50 and 52 C., respectively. In Figure 32 the thermal resistance curve is shown and the 2 value was 12. The times required to decrease the cell population by . o o 0 99.99 per cent were 39, 22 and 8 minutes at 48 , 50 and 52 C., respectively. Summaries of the data obtained at these two pH levels are presented in Tables 22 and 23. Also included for com- parison purposes are the data obtained with butyric and oleic acids. lg reconstituted skimmilk containing oleic acid. A 0.1 per cent concentration of oleic acid in the skimmilk decreased the pH to 6.45. The data in Tables 18 and 19 indicate the average number of survivors when the pH of the heating medium was 6.45 and 6.7, respectively. Figures 33 and 35 69 TABLE 17. Number of g, fragi per milliliter surviving heat treatment in reconstituted skimmilk containing 0.1 per cent by weight of lauric acid and neutralized to pH 6.7* Temperature (0 C.) 48 50 52 Time Survivors Time Survivors Time Survivors (Min.) (Min.) (Min.) 0 5,400,000 0 5,400,000 0 5,400,000 9 ** 4 1,800,000 3 290,000 18 1,200,000 8 570,000 5 120,000 27 74,000 12 180,000 7 9,100 36 400 16 12,000 9 430 45 ** 20 290 24 48 0 8,900,000 0 8,900,000 0 8,900,000 9 3,900,000 4 1,500,000 3 850,000 18 710,000 8 290,000 5 95,000 27 140,000 12 51,000 7 5,800 33 17,000 16 4,800 8 1,900 39 1,700 20 1,200 9 460 45 80 24 42 10 61 0 1,600,000 0 1,600,000 0 5,400,000 9 1,100,000 5 1,200,000 3 210,000 18 330,000 10 700,000 5 140,000 27 36,000 15 180,000 7 4,000 36 1,900 20 30,000 8 1,400 45 31 25 1,400 9 210 30 30 10 55 * ** Laboratory accident Counts represent the average of triplicate plates 70 Log of Survivors per ml. 52°C. 50°C. l I I l L I 0 IO 20 50 4O 50 Minutes of Healing Fig. 31. Rate of destruction of p. fragi in reconstituted skimmilk containing 0.1 per cent by weightorf lauric acid and neutralized to pH 6.7. .5 8w Z=|2 .5 - " E .6- 2 c ‘5 .4- g) _ q 2- 2 O I t 1 J IIB l20 I22 |24 I26 . Temperature (°F.) Fig. 32. Thermal resistance curve of g. fragi in reconstituted skimmilk containing 0.1 per cent by weight of lauric acid and neutralized to pH 6.7. 71 TABLE 18. Number of g. fragi per milliliter surviving heat treatment in reconstituted skimmilk containing 0.1 per cent by weight of oleic acid at pH 6.45* Temperature (0 C.) 48 50 52 Time Survivors Time Survivors Time Survivors (Min.) (Min.) (Min.) 0 5,500,000 0 5,500,000 9 530,000 4 310,000 18 240,000 8 160,000 27 190,000 12 110,000 33 63,000 16 63,000 39 9,100 20 15,000 45 1,100 24 680 0 ** 0 8,400,000 0 8,400,000 9 1,500,000 5 5,000,000 3 2,200,000 18 720,000 10 3,400,000 6 1,100,000 27 170,000 15 980,000 8 360,000 33 79,000 20 150,000 10 28,000 39 15,000 25 1,900 12 1,200 45 2,400 0 4,700,000 0 4,700,000 10 1,200,000 3 760,000 20 780,000 6 100,000 30 540,000 8 140,000 40 90,000 10 16,000 50 3,600 12 1,200 60 140 14 59 * Counts represent the average of triplicate plates ** Laboratory accident 72 TABLE 19. Number of g. fragi per milliliter surviving heat treatment in reconstituted skimmilk containing 0.1 per cent by weight of oleic acid and neutralized to pH 6.7* Temperature (0 C.) 48 50 52 Time Survivors Time Survivors Time Survivors (Min.) (Min.) (Min.) 0 6,200,000 0 6,200,000 0 6,200,000 10 780,000 5 2,400,000 3 740,000 20 880,000 10 450,000 6 90,000 30 180,000 15 120,000 8 30,000 40 16,000 20 6,300 10 2,700 50 190 25 80 0 4,400,000 0 4,400,000 0 4,400,000 9 4,200,000 4 710,000 3 90,000 18 760,000 8 300,000 5 10,000 27 110,000 12 100,000 7 1,400 33 18,000 16 4,400 8 450 39 440 20 150 9 430 45 50 0 35 0 6,000,000 0 6,000,000 0 6,000,000 10 1,200,000 5 850,000 3 810,000 20 1,000,000 10 320,000 6 400,000 30 210,000 15 180,000 8 99,000 40 7,600 20 52,000 10 15,000 50 310 25 590 12 710 30 35 * Counts represent the average of triplicate plates 73 7 -‘---—-~‘~ 6- \:\\\ ‘ \\ \\ a- I \ E 5_ \ 3 \ o. (A k 2 .S 4 '- k 2 m ‘k 0 g 3" D=l 8 ‘1 2- 52°C. ' 50°C. 48°C. I 1 l 1 I l 0 I0 20 30 4O 50 60 Minutes of Heating Fig. 33. Rate of destruction of P. fragi in reconstituted skimmilk containing 0.1 per cent by weight of oleic acid at pH 6.45. ..ol_ :- .8-‘3 -~ Z=l E 2 \. 6.. Q - °5 4'- o 6. . ‘ 3 .2- " O J 1 l I II8 l20 l22 I24 |26 Temperature (°F.) Fig. 34. Thermal resistance curve of p. fragi in reconstituted skimmilk containing 0.1 per cent by weight of oleic acid at pH 6.45. 74 E K Q) Q U) K O -2 2 0:6.2 3 U) k 0 Q 0 s] 48°C. 52°C. 50°C. I I l 1 I J 0 IO 20 30 4O 50 Minutes of Heating Fig. 35. Rate of destruction of _13. fragi in reconstituted skimmilk containing 0.1 per cent by weight of oleic acid and :neutralized to pH 6.7. n.0- E -8 z=I3 E .s Q - ‘6 .4- _ Q 0 3 .2- .. O 1 I 1 J ”8 l20 I22 l24 l26 Temperature (°F.) Fig. 36. Thermal resistance curve of P. fragi in reconstituted skimmilk containing 0.1 per cent by weight of oleic acid and neutralized to pH 6.7. 75 show the survivor curves and D values calculated from the data presented in Tables 18 and 19, respectively. At pH 6.45 the D48' D50 and D52 values were 7.4, 2.5 and 1.8, respectively. When the pH was 6.7 the D D and D 48' 50 52 values were 6.2, 3.8 and 1.8, respectively. At pH 6.45 and 6.7 the z values were 12 and 13, respectively (Figures 34 and 36). The original cell populations were decreased by . . o o 0 99.99 per cent in 53, 26 and 13 minutes at 48 , 50 and 52 C., respectively, when the pH of the heating medium was 6.45. At pH 6.7 the same amount of destruction occurred in . o o o , 46, 25 and 10 minutes at 48 , 50 and 52 C., respectively. Summaries of the data obtained with oleic acid are presented in Tables 22 and 23. lg reconstituted skimmilk containing butyric acid. A 0.1 per cent concentration of butyric acid in the skimmilk de- creased the pH to 5.85, The heating temperatures at this 0 o o . . . pH were 50 , 52 and 54 C. The data in Table 20 indicate the average number of survivors per milliliter at various times throughout the heating trials with the pH at 5.85. The survivor curves shown in Figure 37 were prepared from the data obtained at pH 5.85. The D values calculated from 0 these survivor curves were 9.1, 5.4 and 1.9 at 50°, 52 and 54° C., respectively. The 2 calculated from the slope of 76 TABLE 20. Number of g. fragi per milliliter surviving heat treatment in reconstituted skimmilk containing 0.1 per cent by weight of butyric acid at pH 5.85* Temperature (0 C.) 50 52 54 Time Survivors Time Survivors Time Survivors (Min.) (Min.) (Min.) 0 1,400,000 0 1,400,000 0 1,400,000 7 800,000 3 430,000 3 180,000 14 370,000 6 150,000 4 86,000 21 150,000 9 84,000 5 14,000 28 33,000 12 25,000 6 5,800 35 3,500 15 3,800 7 2,600 42 700 18 1,000 8 740 0 7,300,000 0 7,300,000 0 7,300,000 7 3,700,000 3 1,100,000 3 ** 14 1,000,000 6 230,000 4 150,000 21 150,000 9 60,000 5 61,000 28 51,000 12 12,000 6 7,900 35 5,200 15 ** 7 1,800 42 310 18 1,300 0 ** 0 800,000 3 3,800,000 3 380,000 6 1,200,000 4 230,000 9 410,000 5 64,000 12 230,000 6 6,900 15 19,000 7 9,600 18 5,800 8 2,000 l J * Counts represent the average of triplicate plates ** Laboratory accident E K Q) Q. U) K O '> s K 3 In K O 8‘ q 50°C. 2.— 54°C. I I I I I I 0 l0 - 20 30 4o 50 Minutes of Heating Fig. 37. Rate of destruction of g. fragi in reconstituted skimmilk containing 0.1 per cent by weight of butyric acid at pH 5.85. ‘ I.3 - '0 Z=ll .s' .8 _ E 2 c3 £5- ‘5 . ,4? - Di 0 0 ~‘ .2- - O I 1 I L l2I l23 I25 I27 I29 Temperature (°F.) Fig. 38. Thermal resistance curve of g. fragi in reconstituted Skimmilk containing 0.1 per cent by weight of butyric acid at ;pHI5.85. 78 the thermal resistance curve was 11 (Figure 38). A 99.99 per cent reduction in population occurred in 45, 22 and 8 . o o o . . minutes at 50 , 52 and 54 C., respectively (Table 23). When the pH of the heating menstrum was 6.7, the heating 0 o o temperatures were 48 , 50 and 52 C. and the_D values at these temperatures were 7.1, 3.4 and 2.1, respectively. The survivor curves from which these D values were derived are shown in Figure 39. Table 21 contains the data which were used in constructing these curves. The slope of the thermal resistance curve gave a z of 14 (Figure 40). The original cell populations were reduced by 99.99 per cent in 43, 20 and 10 minutes at 48°, 50° and 52° C., respectively (Table 23). --. T‘i’d 79 E K Q) Q. t 0 .5 s K 3 U) K o - u 0-7.I 0 s] 52°C. 50°C. 48°C. I I I I I J 0 I0 20 30 40 50 Minutes of Heating Fig. 39. Rate of destruction of g, fragi in reconstituted sskimmilk containing 0.1 per cent by weight of butyric acid and rieutralized to pH 6.7. N I.O - .s' E 8 \, Q 6 to. 0 g, 4 _\ 7' .2 - O I i I J “8 I20 I22 l24 I26 Temperature (°F.) Fig. 40. Thermal resistance curve of g. fragi in reconstituted skimmilk containing 0.1 per cent by weight of butyric acid and neutralized to pH 6.7 ;‘v-'.‘ — 1|. I! [III 8O TABLE 21. Number of g, fragi per milliliter surviving heat treatment in reconstituted skimmilk containing 0.1 per cent by weight of butyric acid and neutralized to pH 6.7* o Temperature ( C.) 48 50 52 Time Survivors Time Survivors Time Survivors (Min.) (Min.) (Min.) 0 1,500,000 0 1,500,000 0 1,500,000 10 5,200,000 5 6,000,000 3 3,300,000 20 1,200,000 10 440,000 6 390,000 30 53,000 15 30,000 8 9,500 40 100 20 100 10 1,300 50 10 0 ** 0 ** 0 6,700,000 9 1,500,000 4 3,800,000 3 1,600,000 18 550,000 8 460,000 5 280,000 27 130,000 12 140,000 7 160,000 33 11,000 16 32,000 8 62,000 39 320 20 2,400 9 24,000 45 34 24 160 10 4,000 0 6,700,000 0 8,000,000 9 4,900,000 3 3,500,000 18 1,600,000 6 320,000 27 460,000 8 85,000 33 78,000 10' 7,100 39 16,000 12 660 45 2,600 14 48 W * Counts represent the average of triplicate plates ** Laboratory accident ~47»: . I 81 44 4.4 i 6.4 6.4 4.6 I 6.4 4.6 6.64 I 4.6 4.6 66.6 6406 o4ssesm .0 044 .o 046 .o 066 44 6.4 I 6.4 4.4 4.4 I 4.4 4.4 6.44 I o.4 4.4 4.6 6404 6444656 44 4.4 u 6.4 6.4 6.4 u 6.4 4.4 4.4 l 4.6 4.6 6.6 6466 644564 44 4.4 I 4.4 6.4 6.4 i 6.4 4.4 4.4 I 4.6 4.6 4.6 6406 644:64 44 6.4 l 4.4 64.4 4.4 i 4.4 6.4 4.6 I 4.6 4.4 64.6 6466 o4o4o 44 4.4 u 4.4 . 6.4 6.4 u 4.4 6.4 4.4 I 6.4 4.6 4.6 6406 o4o4o A.m VN .0 0mm .0 00m .0 000 mm MO 0000 40m 4.0 c 604844 a 644844 a 604844 a 4540 44488446 60:6646600 444 60:6646600 466 mosoc4usoo 464 6464 04040 no 044504 .Uaumusn 00 0:0403 an 0000 40m 4.0 mcwcwmucoo MHHEEme 000904000000H.- :4 00000: 006 xHHEEme 00usuflumcoo0u :4 £03040 H0000 Mdmmm.xm.mo 00400040000 4084038 .NN mamme 82 TABLE 23. Time required to destroy 99.99 per cent of the g, fragi cells when heated in the medium and at the temperatures indicated* Temperature (0 C.) Heating medium pH 48 50 52 54 Time in minutes Skimmilk (control) 6.7 46 27 12 ** Skimmilk plus lauric acid (neutralized) 6.7 39 22 8 ** Skimmilk plus lauric acid (not neutralized) 6.6 49 25 ll ** Skimmilk plus oleic acid (neutralized) 6.7 46 25 10 ** Skimmilk plus oleic acid (not neutralized) 6.45 53 26 13 ** Skimmilk plus butyric acid (neutralized) 6.7 43 20 10 ** Skimmilk plus butyric acid (not neutralized) 5.85 ** 45 22 8 * Average of two or three trials ** Not determined 83 DISCUSSION Survivor Curves All of the survivor curves prepared from the data ob— tained in this investigation had a "lag period" during the initial interval of the heating trials. The duration of this interval depended on the heating temperature and the substrate. The D values were determined by calculating the reciprocal of the slope of the "straight line" portion of the survivor curves. Thus the lag portion of the curve did not influence the D value. Other workers (2, 9, 12, 14, 17, 18, 23, 24, 26, 29, 38, 39, 43, 49, 50) have reported survivor curves with a similar lag period. A few of the reasons offered in explanation for the non-linear survivor curves are: clumping of the cells or spores, poor tech- niques, erratic data and a non-logarithmic rate of death. The latter reason has been a subject of controversy. Recently Humphrey and Nickerson (18) tested thermal death data for significant non-logarithmic behavior. They analyzed data obtained from heating Bacillus stearother— mgphilus and found that the spores were not destroyed at a logarithmic rate. They analyzed their data by the method of least squares and also by the second polynomial 84 (y = a + bx + cx2) of least squares. The least squares method determined the position of a straight line; this line represented a logarithmic behavior of death. The second polynomial of least squares determined the position of a curved line through the data; this line represented a non-logarithmic behavior of death. An analysis of variance test was made on the "fit" of these two lines to the data. From the statistical test the hypothesis of linear regression (logarithmic behavior) was rejected and Humphrey and Nickerson (18) concluded that there was significant curvi-linearity (non-logarithmic behavior) in the regression representing the data. On the basis of their data, it would seem that additional research would be necessary to determine whether the second degree polynomial of least squares would apply to all survivor curves. This then raises the question of how one might analyze data obtained from heating bacterial cells and spores. The usual method of determining D and 2 values is convenient and was used throughout the investigation reported herein. The survivor curves derived from the data obtained in this investigation showed that the time involved in the lag 85 period and in the overall heating period decreased as the heating temperature increased. In preliminary analyses of the data during the progress of this research project, survivor curves were prepared by plotting log of survivors per milliliter versus time and drawing a line through the points by the "best fit" method. It was noted that at each of the three heating temperatures a fairly constant ratio was obtained when the time in the lag period was compared to the time required to destroy 99.99 per cent of the initial cell population. This was true only when the same cell suspension was used at all three temperatures. When different cell suspensions were compared a difference in the magnitude of the ratio was noted. Unfortunately, the survivor curves shown in the results section (prepared from the pooled data) do not have a constant ratio. This is attributed to the fact that the original cell populations were not the same. Further investigation would be necessary to determine whether a constant ratio is obtained with other organisms and under different heating conditions. The 3, fragi_cells were heated at 480, 500 and 520 C. which includes the minimum and maximum temperatures which could be used conveniently. Excessively long lag periods 86 would have been encountered at temperatures of less than 480 C. and even at 480 C. long lag periods were occasion- ally encountered. For example, in Figure 13, the survivor curve representing the data obtained with reconstituted skimmilk at 480 C. contains a lag of approximately 45 minutes. At temperatures greater than 520 C. the organisms were destroyed at such a rapid rate accurate sampling could not be achieved with the apparatus used. Thermal Destruction 2f 2. fragi in Half-and-half This study was undertaken to determine the thermal resis- tance of g, figggi in a dairy product which contained a higher percentage of milk fat than is normally found in whole milk. The product selected in which to grow and heat the cells was "half-and—half" which contains approximately 11 per cent milk fat. A comparison of the thermal resistance of g, §£§Qi_cells grown at 70 and 250 was included because raw milk and cream held at refrigeration temperatures often contain a high psychrophilic population and it was desired to determine whether a low growth temperature influenced the heat resis- tance of the organism.- According to reports in the litera- ture all psychrophilic organisms are destroyed during 87 pasteurization, but none of these reports contained data on the thermal resistance of psychrophilic cells that had been propagated at refrigeration temperatures. Work with other organisms has shown that the thermal resistance of cells usually decreases as the growth temperature decreases. However, Lawton and Nelson (28) observed that incubation o o o . . temperatures of 5 , 10 and 25 C. did not influence the heat resistance of g, fluorescens, g, ovalis, g, geniculata and an unidentified species of Pseudomonas. When the cells were grown in half-and-half at 70 and 250 C. the z values were 13 and 10, respectively. When the times required to destroy 99.9999 per cent of the original cell population were compared, it was observed that the cells grown at 70 C. required 58, 45 and 42 per cent more heating time at 480, 500 and 520 C., respectively, than did the cells grown at 250 C. These longer heating times were not due to the lag period since all of the D values obtained with cells grown at 70 C. were approximately twice as large as those obtained with cells grown at 250 C. The reason(s) for the slightly larger 2 value and the longer heating times obtained with the cells grown at 70 C. is not apparent. Approximately the same number of cells per milliliter were obtained after incubation at 70 C. for 7 88 days or at 250 C. for 20 hours. The conditions under which the cells were grown and heated were similar except the cells grown at 250 C. were agitated during incubation whereas those grown at 70 C. were not. Therefore, the difference in incubation temperature seems to be the most logical reason for the difference observed in the 2 values and in the time required to destroy 99.9999 per cent of the original cell population. Thermal Destruction 9£_§, fragi in Fresh Skimmilk and Cream Containing gQ_Per Cent Milk Fat When 3, fragi cells were grown and heated in different batches of half-and-half reproducible results were diffi- cult to obtain. The reproducibility of the curves in replicate experiments improved when the skimmilk and the cream containing 20 per cent milk fat were prepared in sufficient quantities to provide the anticipated needs of the experiments. The data obtained from heating the cells in these two media were compared to the data obtained when the cells were grown in half-and-half at 250 C. and then heated in half-and-half. When the cells were grown and heated in media containing 0, 11 and 20 per cent milk fat the 2 values were 9, 10 and 15, respectively. These 2 values would seem to indicate 89 that a 20 per cent concentration of milk fat was sufficient to increase the size of the 2 value whereas a 10 per cent concentration was not sufficient. There was no apparent difference in the z value when the cells were grown and heated in milk containing 0 per cent fat or 10 per cent fat. A comparison of the times required to destroy 99.9999 per cent of the original cell population showed that the cells grown and heated in skimmilk (Figure 6) required approximately twice as much heating time as did the cells grown and heated in half-and—half (Figure 2) and in 20 per cent cream (Figure 9). These 99.9999 per cent destruction times were used to prepare thermal death time curves for comparison with the milk pasteurization curve (Figure 8). Pasteurization is considered adequate when the thermal death time curve is to the left or below the pasteurization curve. The thermal death time curve shifts toward the pasteurization curve as the required level of destruction increases. However, when the z is approximately 7 the thermal death time curve is almost parallel to the milk pasteurization curve and the margin of safety is adequate although the cells require a long heat treatment. Since the thermal death time curves in Figure 8 are to the left of the pasteurization curve, this would indicate that 9O pasteurization would normally be sufficient to destroy g. fragi in media containing 0, 11 and 20 per cent milk fat, but the margin of safety would be less when the cells are heated in media containing 20 per cent milk fat. The 2 values were nearly the same when the cells were grown and heated in skimmilk (z = 9) and grown and heated in half-and-half (z = 10), but the cells heated in skim- milk required longer periods of exposure to destroy 99.9999 per cent of the original cell population. This was due primarily to the lag period during the initial portion of the heating trials. These two media contained different concentrations of milk fat and also they were not prepared from the same serum solids. Thus the differences observed in the heating exposures required to achieve the same level of cell destruction would seem to be due to either the milk fat or the serum solids. To eliminate the serum solids as a variable, media containing 0, 10 and 20 per cent milk fat were prepared from identical serum solids. The milk fat in the media containing 10 and 20 per cent milk fat was also identical. 91 Thermal Destruction of g. fragi in Reconstituted Skimmilk, Reconstituted Milk Containing 19 Per Cent Milk Fat and Reconstituted Cream Containing gQ_Per Cent Milk Fat In View of the variation which may have been introduced in preparing media from different batches of product, a carefully controlled study was undertaken to compare 2 values and the times required to reduce the original cell population by 99.99 per cent. The experimental design of the study involved using standardized media made from the same basic ingredients. The 2 values calculated from the data obtained by heating the cells in the media containing O, 10 and 20 per cent milk fat were 14, 11 and 22, respectively. These 2 values would seem to indicate that the cells received a limited amount of thermal protection when they were grown and heated in cream containing 20 per cent milk fat. The 2 values of 14 and 11 would seem to indicate that the medium containing 0 per cent milk fat offered the cells as much thermal protec- tion as did the medium containing 10 per cent milk fat. The 2 values of 14 and 11 obtained with media containing 0 and 10 per cent milk fat compare favorably with the z of 12.4 obtained by Chaudhary t al, (8) when they heated g, fragi in skimmilk. 92 A comparison of the times required to destroy 99.99 per cent of the original cell population showed that at 480 C. the longest time was required by the cells grown and heated in media containing 10 per cent milk fat (Figure 13). In this medium approximately 45 minutes of heating time were required at 480 C. before destruction of the cells began to occur at a logarithmic rate. This long lag period was verified by eight different heating trials on eight dif- ferent days and with eight different cell suspensions. The shortest heating time required at 480 C. was with the cells which were grown and heated in cream containing 20 per cent milk fat (Figure 15). The data obtained by heating the cells at 520 C. indicated that the times required to destroy 99.99 per cent of the initial cell population were nearly the same regardless of the concentration of milk fat in the media in which the cells were grown and heated. Other workers who have heated microorganisms in dairy products with different concentrations of milk fat are Nichols (33) and Daoust t al. (13). Nichols (33) reported that the heat resistance of Streptococcus thermophilus, Streptococcus durans and four species of Escherichia did not increase as the concentration of milk fat in the heating substrate increased. In some instances the heat resistance 93 of the cells may have decreased as the per cent milk fat in the heating substrate increased. The decrease in heat resistance could probably be attributed to the increasing concentrations of milk fat in the media in which the cells were heated since all of the cells were grown on agar slants prior to heating. Nichols did not report D or 2 values, but measured the heat resistance of the organisms by observ- ing the number of positive tubes after exposure for various lengths of time at different temperatures. This method of determining heat resistance would be similar to determining the time required to destroy a certain percentage of the cell population. Daoust et al. (13) observed that Corynebacterium diph- theriae cells were less heat resistant in 40 per cent cream than in whole milk or ice cream mix. These workers were basing heat resistance on 2 values. Their observations do not agree with the results reported herein in that the 2 values obtained when the cells were heated in 20 per cent cream were larger than the 2 values obtained when the cells were heated in skimmilk and 10 per cent milk. The lack of agreement between the two experiments may be due to the different genera of bacteria which were used as well as the different conditions under which the cells were grown and heated. 94 On the basis of the data obtained in this investigation and the data presented by others, it is difficult to deter- mine whether milk fat provides thermal protection to non- spore forming bacteria. However, the data obtained in this investigation seem to indicate that the g, fragi_cells received a limited amount of thermal protection in the cream containing 20 per cent milk fat. Thermal Destruction 9; g; fgagi ig_a Medium Containing §_Different Concentration gf.Milk Fat Than the Medium in Which the Cells Were Grown This study was undertaken to determine the influence of the growth medium and the heating medium on the 2 values and the time required to reduce the original cell population by 99.99 per cent. The experimental design of the study in- volved using standardized media made from the same basic ingredients. When the cells were heated in media containing 0 and 10 per cent milk fat the z values ranged from 11 to 14, whereas when the heating medium contained 20 per cent milk fat the 2 values ranged from 20 to 22 (Table 22). The 2 values did not seem to be influenced by the medium in which the cells were grown prior to heating. -- .4443. a." 0.. : ~50 "- .1, as; 24:51,;6Wm‘m :- .. '2 ls-" , - 95 The data obtained by heating the cells at 48° and 50° c. seem to indicate that the growth substrate influenced the time required to reduce the original cell population by 99.99 per cent. Cells grown in 20 per cent cream were reduced to this level in less time than cells grown in reconstituted skimmilk. The medium in which the cells were heated did not seem to influence the times required to destroy 99.99 per cent of the original cell population. As the time required to destroy 99.99 per cent of the original cell population increased, the length of time involved in the lag period also increased. Humphrey and Nickerson (18) have presented a theory describing why the logarithmic order of death does not begin immediately at the onset of heating. They have suggested that organisms may contain four to ten "heat labile reproduction initia- tors" and that these "reproduction initiators" would be "heat-induced" during the initial heating, but would eventually be destroyed by further heating, thus the cells would approach a "logarithmic behavior of death." These workers observed that when the straight line portion of the survivor curves was extrapolated back to zero time, the point of intersection was a population that was nor- mally four to ten times larger than the population which; a... 47% 96 was actually present. This was how these workers obtained the "four to ten" possible "reproduction initiators." If one attempted to relate this theory to the data reported herein, one might postulate that the cells grown in skim- milk may have had a larger number of "reproduction initia- tors" than the cells grown in the media containing milk fat. Also the possibility exists that the "reproduction initiators" may have been more resistant when the cells were grown in the absence of milk fat. This is strictly a hypothetical observation, but merits mentioning since an explanation for the variation in the time required to accom- plish a given amount of destruction is not apparent. In any case, the factor(s) influencing the time required to destroy 99.99 per cent of the original cell population must have been either inCorporated directly into the cell during its formation, or perhaps associated with the material surround- ing and adhering to the cell. In either case the responsible factor(s) were identified with the cells. This can be sub- stantiated by the fact that the medium in which the cells were grown seemed to exert more influence on the time required to obtain 99.99 per cent destruction than did the medium in which the cells were heated. 97 Thermal Destruction gf_§, fragi_in Reconstituted Skimmilk Containing_Selected Fatty Acids 3, fragi is very lipolytic (1, 19, 32), therefore, some of the free fatty acids or other lipid residues in the growth medium may have been responsible for the differences observed in the time required to destroy 99.99 per cent of the original cell population. The free fatty acids and other lipid residues could have been carried along with the cells when they were transferred into the medium in which they were heated. In an attempt to determine whether free fatty acids influenced the time required to destroy 99.99 per cent of the original cell population, cells were heated in reconstituted skimmilk which contained 0.1 per cent by weight of a selected fatty acid. The fatty acids selected were butyric, lauric and oleic. These fatty acids were selected so that a comparison could be made to show the influence of carbon chain length and saturation versus unsaturation on the destruction rates of g, fragi. Each fatty acid was added to the skimmilk on a per cent basis rather than a molar concentration, thus the addition of butyric acid resulted in a greater change in pH than did the addition of lauric or oleic acid. To com— pensate for differences in pH on the thermal resistance "' £1.94, F_ 98 of the cells, heating trials were conducted with and with- out adjustment of the pH to 6.7 with trisodium phosphate. Since very little difference exists in the 2 values obtained with the three fatty acids (11 to 14) it would seem that the carbon chain length and the characteristics of saturation or unsaturation of the fatty acids has little influence on the resultant 2 values. Also the pH levels encountered did not influence the 2 values obtained. When the cells were heated in reconstituted skimmilk without the fatty acids the 2 value was 14, which is within the range of 11 to 14 encountered when fatty acids were added to the skimmilk. Therefore, it appears that the presence of the acids in the skimmilk had little influence on the 2 values. Perhaps a higher concentration of each fatty acid in the skimmilk would have exhibited a more notice— able effect. A comparison of the times required to destroy 99.99 per cent of the original cell population showed that with lauric and oleic acids the heating times required were approximately the same when the pH of the skimmilk-fatty acid mixture was 6.7 and less than 6.7 (Table 23). However, when the skim— milk contained butyric acid the times required at pH 5.85 99 were longer than those required at pH 6.7. There is no apparent explanation for the longer heating times required when the pH was 5.85. lOO SUMMARY AND C ONCLUS I ONS The destruction rates of Pseudomonas fragi were deter- mined on cells heated in media containing different concen— trations of milk fat and in reconstituted skimmilk con— taining selected fatty acids. All of the survivor curves contained a lag period during the initial interval of the heating trials. When cells from the same suspension were heated at 48°, 500 and 520 C., the time involved in the lag period of the survivor curves appeared to be a fairly constant proportion of the time required to obtain a given level of destruction. The 3, fragi cells grown in half-and-half at 70 C. for 7 days and at 250 C. for 20 hours had 2 values of 10 and 13, respectively. The cells grown at 70 C. required longer periods of heating to reduce the initial cell population by 99.9999 per cent than did the cells grown at 25° c. These longer heating time requirements were attributed to the incubation temperature. When cells were heated in media containing O, 10 and 20 per cent milk fat, the largest 2 values were obtained with media containing 20 per cent milk fat. The 2 values were approximately the same when the cells were heated in media 101 containing 0 and 10 per cent milk fat. The cells grown and heated in media containing 20 per cent milk fat required less time to obtain a 99.99 per cent reduction in the initial cell population than did the cells grown and heated in media containing 0 per cent milk fat. Data obtained from heating trials performed with media prepared from the same basic ingredients indicated that the 2 values were influenced by the medium in which the cells were heated, but the 2 values were not influenced by the medium in which the cells were grown. The largest 2 values were observed when the cells were heated in cream containing 20 per cent milk fat. The concentration of milk fat was the primary difference in the heating media, thus it would seem that the milk fat was responsible for the larger 2 values. Apparently the cells were influenced by the medium in which they were grown and this in turn influenced the times required to destroy 99.99 per cent of the original cell population. The cells grown in media containing 0 per cent milk fat required the longest heating times. The medium in which the cells were heated did not influence the times required to destroy 99.99 per cent of the original cell population. The differences observed in the destruction times were attributed to the concentration of milk fat in the growth medium. 102 Little if any difference in the 2 values was noted when 3, fragi was heated in reconstituted skimmilk which con- tained 0.1 per cent by weight of butyric, lauric or oleic acid. 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