EFFECT 6? pH ANSI 88MB CWCENTMTION ON GR'QWTH AN!) W BWRUCTION OF PA 35?? EN PRGCESSED CHEESE SPREAD Thesis kw {In Duqm of ”I. D. EECEEESAN 3‘1”ka HIKWER'SITY Johm.2£.laynes n 1960 I ! This is to certify that the thesis entitled EFFECT OF pH AND BRINE CONCENTRATION ON GROWTH AND THERMAL DESTRUCTION OF PA 3679 IN PROCESSED CHEESE SPREAD presented by John A. Jaynes has been accepted towards fulfillment of the requirements for _E2D_degree 111M; @fiw Major proffi Date June 1, 1960 0-169 LIBRARY Michigan State University w ..... v' v "I“ EFFECT OF pH AND BRINE CONCENTRATION ON GROWTH AND THERMAL DESTRUCTION 0F PA 5679 IN PROCESSED CHEESE SPREAD by John A. Jaynes AN ABSTRACT Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1960 Approved 1W Saw ,. 6% a 7 J - U ABSTRACT JOHN A. JAYNES The effect of pH and brine concentration was deter- mined on the growth and thermal destruction of spores of PA 3679 of a known heat resistance in a processed cheese Spread. The spores used had a D250 of 0.98 minutes and a z of 17.5° F. Most growth tests were conducted by sealing the in- oculated standardized processed cheese Spread in TDT-cans and incubating at 37° C. for various periods of time. Growth was evaluated by measuring the expansion occurring in the cans with a dial micrometer. The amount of skim milk powder used in the produc- tion of processed cheese Spread effected the growth of the test organism in the product. Profuse growth occurred when a 10.0 per cent concentration was used, but no growth was observed when only 1.0 per cent was used. The amount of spore inoculum, pH, and brine concentration were the same in both lots of processed cheese Spread. Both pH and brine concentration influenced the growth of the test organism in the processed cheese spread. There was no growth at or below a pH of 5.6 and no growth at or above a brine concentration of 7.6 per cent. The two methods utilized for the recovery of viable spores after the thermal process were the subculturing in stratified liver infusion broth of 0.01 gram of product from each TDT-can, and the incubation of the processed can- ii ABSTRACT ' JOHN A. JAYNES ned product with periodical examinations for swells. Studies conducted to determine the effect of the pH and brine concentration of the processed cheese Spread on the thermal process required to destroy the test organism in the product revealed that the pH had an effect, but the brine concentration did not. The average D235 values of PA 3679 in the processed cheese Spread at pH 5.50, 6.25, and 7.00 were 4.55, 6.90, and 8.25 minutes, respectively. The 2 values remained constant at 18° F. Tests conducted by incubating the product in the can revealed that as the temperature of the thermal process increased, the minimum number of Spores required to initiate growth in the product decreased. iii/{v EFFECT OF pH AND BRINE CONCENTRATION ON GROWTH AND ' THERMAL DESTRUCTION OF PA 5679 IN PROCESSED CHEESE SPREAD by John AT Jaynes A THESIS Submitted to the School for Advanced Graduate Studies of Micigan State University of Agriculture and Applied . Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1960 I; ;?/"??1' {3///é: ACKNOWLEDGMENTS The author wishes to e1press his sincere gratitude to the following: Dr. I. J. Pflug, for his guidance and patience dur- ing the course of this study and during the preparation of this manuscript. Dr. L. G. Harmon, for his meticulous study of the proposed manuscript and his subsequent suggestions. Dr. R. N. Costilow, for his constructive criticism of the interpretation of data in the manuscript. My wife, for her tireless efforts in the preparation of the manuscript. Dr. J. R. Brunner and Dr. 0. w. Kaufmann, for their services as committee members. TABLE OF CONTENTS 00.000.000.00 INTRODUCTION 00.000000000000000.000000000 E‘N ...00.0.0.00.00.00.0000000000000000. LITERATURE REVI 000000000.000000000000000000... Thermal Processing .. Spoilage of Canned Cheese ... 00.000.00.00... Spoilage of Processed Cheese ........ Effect of skim milk powder ................ 0000... 0000.00.00.00000000000.000.0000. Effect of pH ..... Effect of salt .......... 8t Organism eeeeee Putrefactive Anaerobe 3679 as a Te EXPERIMENTAL PROCEDURE 0000000000000000000000 t Resistant Spore SuSpension of 0000000000000000.000.000.00... Preparation of Hea PA 3679 00000000000000 Preparation of growth medium . Growth of organism ................. Of organism ooeeeeeeeeeeeeeeeeoeeo storage of organism .......... Harvesting Standardization and anism eeeeeeeeeeeeeeeeeee Thermal resistance of org Growth of PA 3679 in Processed Cheese Spread ....... f Skim milk powder eeeeeeeeeeeeoee Effect 0 Effect of pH and brine concentration ............. Heat Penetration Studies . Concentration on the Thermal Effect of pH and Brine Resistance of PA 3679 in Processed Cheese Spread ... Effect of pH and Brine Concentration on the Heat Treatment Required to Prevent Growth of PA 3679 in Processed Cheese Spree 000.00.000.00000000000.00. vi Pa ge ‘0 (D 0! g; (g ll 12 15 15 15 15 16 17 ‘18 22 82 24 27 33 36 RESITLTS ......0000000.00.000.000.000.00000000000000... PrOperties of Spore Suspension of PA 3679 .......... Growth of PA 3679 in Processed Cheese Spread ....... Effect of skim milk powder ....................... Effect of pH and brine concentration ............. Effect of pH and Brine Concentration on the Thermal Resistance of PA 5679 in Processed Cheese Spread ... Effect of pH and Brine Concentration on the Heat Treatment Required to Prevent Growth of PA 3679 in Processed Cheese Spread ......0000000000000...... DISCUSSION ........................................... Test Organism ...................................... Growth of PA 5679 in Processed Cheese Spread ....... Effect of skim milk powder ....................... Effect of pH and brine concentration ............. Effect of pH and Brine Concentration on the Thermal Resistance of PA 3679 in Processed Cheese Spread ... Effect of pH and Brine Concentration on the Heat Treatment Required to Prevent Growth of PA 3679 in PrOCOSBCd Cheese Spread eeeeeeeeeeeeeeeeeeeeeeeee Smfl‘lARY 000.00.00.000000000000000.0000000000000000000. LITERATURE CITED 0.0.0.0....00000000000000.000000000.. vii Page 38 58 44 44 45 5O 62 469 69 69 69 70 72 75 '77 79 TABLES Page Effect of fill-weight and position of the can on the lag correction factor (to) of a processed Cheese Spread in TDT‘canS eeeeeeeeeeeeeeeeeeeee II. Results of preliminary tests for the thermal resistance of spores 0 PA 3679 in the thermo- resistometer (3.3 I 10 Spores per cup, samples subcultured in liver broth and incubated at 370 C0) 000.00.0.00000000000000.0000000 III. Results of tests for the thermal resistance of Spores of PA 3679 in the thermoresistometer (3.3 X 104 Spores per cup, samples subcultured in liver broth and incubated at 37° C.) ....... 41 of Skim milk powder on the growth of With a pH Of 600 at I. 39 IV. Effect PA 3679 in cheese Spread the time of inoculation and incubated 8 days at 37° C. (measured by gas production in glass tubes) .00....00.000.00.000.0.0000000000000000. V. Effect of pH and brine conce growth of PA 3679 in a cheese Spr 14 days at 37° C. (measured by gas pr in 81888 tubBS) oeeeeeeeeeeeeeeeeeeeeeeeee 44 ntration on the sad incubated oduction 00000 46 oncentration on the growth of pread with a pH of 6.2, C0 000.000.00.000 VI. Effect of brine 0 PA 3679 in a cheese s when incubated 14 days at 37 47 VII. Effect of pH and brine concentration on the lag 3679 in a cheese Spread incubated phase of PA at 37° C. (measured by the swelling of eeeeeeeeeeeeeeee 51 TDT-cans) ..... VIII. Results of preliminary tests for the thermal death rate of spores of PA 3679 in a cheese Spread (3.3 X 106 Spores in each of two TDT- cans per trial, with 0.01 g. from each can subcultured in liver broth and incubated at 57° C.) ........00000000.0000000000000000000... 53 II. Results of tests for thermal death rate of e Spread at pH 5.50 spores of PA 3679 in chees TDT-can, 0.01 g. from (3.3 X 106 Spores per each can subcultured in liver broth incubated at 370 C.) ..eeeeeeeeeeeeeeeeeeeeeeeeeeeoeeeeee 55 X. XI. XII. XIII. XIV. XV. Results of tests for thermal death rate of spores of PA 3679 in cheese spread at pH 6.25 (3.3 X 106 spores per TDT-can, 0.01 g. from each can subcultured in liver broth incubated at 37° C.) ............OOOOOOOOO......OOOOOOOO. Results of tests for thermal death rate of spores of PA 3679 in cheese spread at pH 7.00 (3.3 X 106 spores per TDT—can, 0.01 g. from each can subcultured in liver broth incubated at 37° C.) ...OOOOOOOOOOOOOOOOOOOO......OOOO... Effect of pH and brine concentration on D235, F255, and 2 values for TDT-cans of cheese 5 spores of PA 3679 spread containing 3.3 X 10 per can (0.01 g. from each can subcultured in liver broth and incubated at 37° C.) .......... Effect of heat treatment (U) at different retort temperatures on the eXpansion of TDT- cans of cheese spread at several pH and brine concentrations (3.3 X 106 sgores of PA 3679 per can and incubated at 37 0) 0.00.00.00.00. Effect of pH and brine concentration on F 35 and 2 values for TDg-cans of cheese epreai containing 3.3 X 10 spores of PA 3679 per can (cans incubated at 37° C. and examined periodically for swells) ...................... Calculated number of Spores in cans showing growth (+) at longest heating time and no growth (-) at shortest heating time, as influenced by pH, brine concentration, and prOceSSing temperature ooooooaoooooooocoooooooo ix Page 56 57 61 63 64 65 1. 2. 3. 4. 5. 6. 9. 10. 11. 12. FIGURES Dial micrometer and TDT-can ..................... TDT-can and c0pper-constantan thermocouple ...... Probability curves for determining L.D.5O time (preliminary tQSt) oooooooooooo0.0000000000000900 PrObability curves for determining L.D.50 time ‘final test) 0.0.0.0...OOOOOOOOOOOOOOOOQOOOO...O. D values with 95% confidence limits and the resulting thermal resistance curve for spores of PA 3679 suSpended in neutral phOSphate buffer ... Effect of brine concentration on the gas pro- duction of PA 3679 in a cheese spread at pH 6.0 as measured by the swelling of TDT—cans incubated at 37° C. (each value represents on average of 10 cane) ......OOOOCOOOOOOOOOOO......OOOOOOOOOOC. Effect of brine concentration on the gas pro- duction of PA 3679 in a cheese Spread at pH 7.0 as measured by the swelling of TDT-cans incubated at 37° 0. (each value represents an average of 10 cans.) ......OOOOOOOOOCOO......OOOCOOOOOOOO... Effect of brine concentration on the gas pro- duction of PA 3679 in a cheese spread at pH 6.0 and 7.0, as measured by the swelling of TDT-cans incubated 60 days at 37° C. ..................... End-point destruction curve of spores of PA 3679 in a cheese spread (each point represents dupli- cate determinations) ............................ Thermal resistance curves of spores of PA 3679 in cheese Spread at pH 5.50 oooooocooooooopooocooooo Thermal resistance curves of spores of PA 3679 in Cheese Spread at pH 6.25 oooooooooooooocoocoooooc Thermal resistance curves of Spores of PA 3679 in Cheese Spread at pH 7.00 oooooooooooooooooooo.o.. Page 26 28 4O 42 43 48 49 52 54 58 59 60 Page 13. Effect of the thermal process (F go) on the ex- pansion of TDT-cans containing c eese spread inoculated with 3.3 X 106 Spores of PA 3679 and incubated at 37° C. for 60 days ................. 66 14. Graphical summary showing thermal death time, inhibition, and color curves .................... 68 xi INTRODUCTION During the production of processed cheese Spreads the batch is usually cooked to approximately 160° F. The finished product is normally packed in hermetic containers and moves through the marketing channels at room tempera- ture to the consumer. Since the cooking temperatures used in the manufacture of the product do not render it sterile, the prevention of epoilage is largely dependent upon the microbial inhibitory nature of the product. The changes in consumer preference have resulted in an evolution in processed cheese spreads. The pH is higher and the brine concentration lower. These changes in pro- cessed cheese spreads have decreased the microbial inhibi- tory prOperty of the product, resulting in an increased susceptibility to Spoilage. A heat treatment to sterilize the product may be utilized to compensate for the increased susceptibility to spoilage. The heat treatment required to prevent food spoil- age by a spore forming organism is a function of the heat resistance and number of bacterial Spores in the food, and the rate of heating of the food product. The heat resis- tance of a Specific bacterial Spore is a function of the substrate in which it is heated, making it necessary to determine experimentally the heat resistance of the Spore in each different substrate. The objective of this research was to determine the thermal process requirements of a processed cheese Spread for one food Spoilage organism under varying conditions of pH and brine concentration. The problem was studied in three parts: (I) The effect of pH and brine concentration on the growth of the Spoilage organism; (2) The effect of pH and brine concentration on the thermal destruction of the Spoilage organism; and, (3) The effect of pH and brine concentration on the heat treatment required to prevent growth of the Spoilage organism. It is haped that the re- sults of this study will aid industry in the design of thermal processes for processed cheese Spreads. LITERATURE REVIEW Thermal Processing Cameron (16) classified foods into two general cata- gories; "acid" foods, pH below 4.5, and "low-acid" foods, pH 4.5 or above. His classification was based primarily upon the premise that Clostridium botulinum would not grow at a pH of less than 4.5. Most pathogenic microorganisms either will not germinate or will not grow in acid foods. Most of the work in the heat preservation of canned foods has been done with the low-acid foods because a relatively severe process is required to reduce the potential Spoilage and health hazard of these foods. Tanner (52) stated that a commercially sterile canned food is one which has been processed by heat in such a manner that it will not Spoil under ordinary market con- ditions, even though it may not be completely sterilized. Design of the thermal process to which a particular canned food should be subjected requires two categories of infor- mation, thermal resistance data and heat penetration data. Bacteria follow a logarithmic order of death when subjected to heat. Rahn (38) pointed out that regardless of the explanation for the existence of the legarithmic order of death, theory permits us to compute death rates and draw conclusions. These death rates make possible the quantitative evaluation of the effect of environmental factors such as pH and brine concentration upon heat sterilization. Bigelow and Cathcart (12) described a curve relat- ing time and temperature for the complete destruction of a bacterial pOpulation under stated conditions, as a thermal death time (TDT) curve. Bigelow (10) further stated that the plotting of thermal death time data for bacteria on semi-logarithmic paper resulted in a straight line. Ball (7) suggested the phantom thermal death time curve which would relate the time and temperature required to reduce the bac- terial pepulation by 90 per cent. Stumbo (49) recommended that the thermal death time curve relate the time and temperature necessary for a known reduction of the bacterial pOpulation. Schmidt (45) applied the name of thermal re- sistance curve to the type of curve suggested by Ball (7). The time required to reduce a known bacterial pOpu- lation by 90 per cent at a specific temperature is defined as the D value. The D value may be determined by measuring the s10pe of the survivor curve which relates the number of survirors and time at a Specific temperature for a stated set of conditions, or it may be calculated from the results of multiple Sample thermal resistance determinations. The methods of Stumbo (50), Stumbo gt 2l° (51), and Schmidt (45) are the methods in general use today for the calculation of D values. Confidence limits can be calculated by the Schmidt method. Kethods of conducting multiple sample thermal resis- tance determinations in general use today include the mul- tiple tube method of Bigelow and Esty (13), the TbT-can method of Townsend gt 31. (2,57), and the thermoresistometer method of Stumbo (50). The bacterial suSpension is usually heated in approximately two milliliters of a phosphate buf- fer solution in the multiple tube method, in approximately 18 g. of the food substrate in the TDT-can method, and in 0.01 ml. of a phOSphate buffer solution in the thermoresis- tometer method. The design of a thermal process for a canned food necessitates the integration of thermal resistance and heat penetration data. Thermal processes may be calculated by the graphical method of Bigelow et al. (11) and the mathema- tical method of Ball (5). The nomogram alignment chart method of Olson and Stevens (31) is a solution of the math- matical method of Bali. Some of the early heat penetration work, as recorded by Thompson (56), was conducted with an apparatus which consisted of a COpper-constantan thermocouple located in the center of the can, a cold junction located in a cracked ice bath, and a galvanometer calibrated by reference to a mer- cury thermometer. A few refinements (8) such as the auto- matic recording potentiometer and special fittings have taken place since that time, but basically the apparatus remains unchanged. Townsend gt al. (57, 58) determined that the rate of heat penetration was affected by the amount of fill and the head-Space. Sognefest and Benjamin (46) conducted heat penetration tests in TbT-cans and found that the point of slowest heating was "located between the side and center midway between the ends due to the dimple in the bottom of the can". A comparison of heat penetration rates with the can in the flat position and with it on its side revealed a greater fh, defined by Bell (5), when the can was on its side if, heating took place by convection. when heating took place by conduction, similar fh values were noted for both positions. These tests were conducted on a 13 g. fill at a retort temperature of 250° F. Sognefest and Benjamin (46) calculated lag correction factors (to) which is the time that must be added to the time at retort temperature (U) to obtain the process time (B). The lag correction factors included both the correction for come-up time and the heat penetration lag because timing was started as soon as steam was admitted to the retort. They concluded that a lag cor- rection factor based on a retort temperature of 250° F. could be used at any retort temperature from 230 to 270° F. This conclusion results from the fact that the lag correc- tion factor is a function of the difference between the re- tort temperature and the initial temperature of the canned product, which is sufficiently great that a change of plus or minus 20 degrees makes very little difference in the re- sulting lag correction factors. Spoilage of Canned Cheese Doane (20) reported that cheese could not be canned because the product would deve10p a putrid flavor and odor. The Eighteenth Annual Report of the Council for Scientific and Industrial Research (4) stated that although process cheese packaged in tin normally kept well, even under ex- treme conditions of temperature, several cases of severe Spoilage had been investigated. Palmer and Sly (32) con- cluded that the cooking temperature in the manufacture of processed cheese was the most important factor in prevent- ing fermentation but Albus and Ayers (1) reported that Spore forming anaerobes survived the cooking temperatures. According to Pette and Liebert (34) gaseous fermentation did not occur in processed cheese that was cooked to tem- peratures greater than 70° C. Ledabyl (26) stated that cooking temperatures in excess of 90° C. were necessary to destroy Spore formers. In tests conducted with Clostridium sporogenes in processed cheese Spreads Csiszar (18) recovered viable organisms after 72 hours at 600 C., 24 hours at 70° C., and 2.5 hours at 800 0.; he was unable to recover viable Spores when the cheese Spread was subjected to 90° C. for 50 minutes or 1000 C. for 6 minutes. He concluded from this work and further investigations (19) that Spores of 21. .§nggggggg§, which produced huffed (swollen by gas) pasteur- ized cheese, could not be destroyed by temperatures used to make a satisfactory product. Meyer and McIntire (28) stated that acceptable pro- cessed cheese Spread could be produced by heat processing the packaged Spread in a conventional retort at 240° F. for 75 minutes. Meyer 33 31. (27) produced a high quality pro- cessed cheese Spread by heating the product to 292° F. in a heat exchanger, holding for 20 seconds, cooling to 135° F. and aseptically packaging. This procedure resulted in an F0 of 90 (thermal process equivalent to 90 minutes at 250° F. with a z of 180 F.). High temperature storage failed to produce any Spoilage. Ball (6) pointed out that high tem- peratures have less effect on quality impairment, in pro- portion to their destructive effect on bacteria, than low temperatures. Spoilage of Processed Cheese Numerous incidents of processed cheese Spoilage have been recorded. Albus and Ayers (1) reported that pro- cessed cheese is a favorable medium for the growth of anae- robes Since the product is packaged hot and contains little oxygen. Also, processed cheese contains very few organisms other than Spore forming anaerobes because of the cooking temperatures during manufacture. Griffiths (22) isolated an anaerobic Spore forming organism from a processed cheese which had deve10ped a serious off-flavor accompanied by a penetrating putrefactive odor within two or three weeks after the cheese was produced. Hood and Bowen (24) investigated spoilage in proces- sed cheese which contained large gas holes and possessed a very obnoxious, penetrating and putrefactive odor and iden- tified the causative organism as a variant of Q}; EQPPOSGQEE Csiszar (17) reported that of the samples of huffed proces- sed cheese examined, 94 per cent were caused by El. gpggg- ggggg. The Eighteenth Annual Report of the Council for Scientific and Industrial Research (4) attributed huffed and putrid odors in processed cheese to Cl. sporogenes and Cl. welchii. Processed cheese Spoilage has also been at- tributed to Q1. butyricum (34, 42, 64). Effect 2; Skim milk powder. Skim milk powder is a common ingredient of processed cheese Spreads. Templeton and Sommer (55) obtained a satisfactory processed cheese Spread by using as high as 10 per cent skim milk powder in the batch. ieyer and McIntire (28) observed that signifi- cant levels of carbohydrate (skim milk powder is approxi- mately 50 per cent carbohydrate) reduced the keeping quality of processed cheese Spread. Hood and Bowen (24) initiated Spoilage in eXperimental batches of processed cheese by adding nutrients such as skim milk powder, casein digest, and mature cheddar cheese. They found that with each in- crease in the amount of skim milk powder added, the time required for subsequent Spoilage decreased. Van Slyke and Price (61) stated that the addition of skim milk powder decreased the acidity and rendered the cheese Spread more favorable for the growth of gas-forming organiSms. The Eighteenth Annual Report of the Council for Scientific and Industrial Research (4) pointed out that Skim milk powder had been used in the manufacture of all cheese which was Spoiled and swollen. The report also stated that the pre- 10 sence of skim milk powder necessitated lower processing temperatures to avoid a browning reaction of the carbohy- drate and that the lower FO employed did not destroy the Spore forming Spoilage organisms present. Effect g£_p§. Under the Federal Food, Drug and Cosmetic Act (60), the pH of processed cheese spread may not be less than 4.0. Barker (9) pointed out that the keeping quality of a processed cheese Spread is determined by the amount of acid present. Sommer and Templeton (48, 53, 54) recommended acidifying processed cheese Spreads to a pH of 5.8 to 6.3 to improve keeping quality. Wearmouth (65) observed that the majority of processed cheese had a pH of 5.9 to 6.6. Pette and Liebert (33) stated that a pH of less than 5.7 was necessary to prevent putrefactive Spoilage. Hood and Smith (25) and Ritchie (41) found that putrefactive Spoilage was completely eliminated at a pH of less than 5.4. In 1920 Bigelow and Esty (13) observed that as the pH was decreased the time required for complete destruction of a Spore suSpension by heat decreased. Bigelow and Cathcart (12) stated that the lower the pH of the food, the lower the Fo required for sterilization. Segnefest g3 31. (47) observed that the prcgressive increase in the resis- tance of spores to heat was slight from pH 5.5 to 9.0 in comparison to the progressive increase in heat resistance from pH 5.0 to 5.5. Ball and Olson (8) stated that gener- ally, the thermal resistance of Spores is greatest at a pH of 7.0. Vas and Proszt (62) attributed the need of a re- 11 latively small F0 for the preservation of acid foods to the inhibition of Spore germination rather than to the low heat resistance of the Spores. Schmidt (44) stated that spores of Cl. sporogenes would germinate at a pH of 5.3 but would not grow. Effect 33 salt. Esty and heyer (21) reported that Spores of 91. botulinum and allied anaerobes exhibited no reduction in heat resistance in the presence of salt until a total concentration of 8.0 per cent was reached. Salt concentrations of 0.5 and 1.0 per cent resulted in greater heat resistance'than concentrations of 2.0 and 3.0 per cent. According to Viljoen (63) salt increases the heat resistance of bacterial spores. This protective influence was detect- able up to concentrations of 3.5 per cent but the greatest protection was exhibited between concentrations of 1.0 and 2.5 per cent salt. Townsend 33,31. (59) stated that salt was protective up to concentrations of 4.0 per cent and that higher levels lowered heat resistance. In studies with comminuted pork Bulman and Ayres (14) showed that when the Spore inoculum (PA 3679) remained con- stant the shelf-life of the product increased with increases in the salt level. They concluded that the critical level of salt in the product for prevention of Spoilage was around 4.0 per cent. In eXperiments with brain-liver-heart medium ‘and processed cheese, Hood and Smith (25) demonstrated the inhibitory effect of salt on the growth of £1. Spongenes. They concluded that processed cheese would not Spoil if it 12 contained more than 3.5 per cent salt regardless of the pH. 91. sporogenes was reported to have germinated in 8.0 per cent salt but failed to grow under this condition (44). Yesair and Cameron (68) reported that a comparison of ther- mal death times of spores of Cl. botulinum in salted and unsalted media demonstrated the inhibitory effect of curing salts, but when the heated media were subcultured there was no destructive influence. The resistance determined by subculturing after heat treatment was approximately the same in the salted and unsalted product. Putrefactive Anaerobe 3679 as a Test Organism PA 3679 is typical of the non-toxic, heat—resistant, meSOphilic anaerobes which grow at normal storage tempera- tures (39) and is widely used by laboratories connected with the canning industry in eXperiments on canned foods (57). The Canngd_Food Referengg hanual (2) states that Spores of PA 3679 are more heat resistant than Spores of Cl. botulinum and that a process based on the destruction of PA 3679 is more than adequate to destroy Spores of Ql°.EEEE' lgggg. SOgnefest et al. (47) stated that a process based upon a suspension of PA 3679 having an F0 of 3.65 to 4.15 minutes in neutral phOSphate, should insure a commercially sterile product for non-acid foods. Townsend.§t'§l. (57) advocated the comparison of the heat resistance of differ- ent suSpensions of Spores in a standard phOSphate buffer 13 consisting of equal amounts of m/is NagHPO4 and M/lfi KH2P04 mixed to give a pH of 7.0. Reed £2.2l- (59) reported that a 2 (OF. required for the thermal resistance curve to transverse one 10g cycle) of 18° F. for PA 5679 was a valid approximation. Reynolds gt al. (40) observed 2 values ranging from 14.5 to 27.50 F. for PA 5679 when the curves were based upon data derived from incubating the food substrate. Schmidt (44) pointed out that Spores which survived drastic killing influences were more exacting in their nu- tritional requirements; therefore, enrichment was necessary for accurate enumeration of survivors. Sognefest gt al. (47) stated that the subculturing of samples frequently resulted in data too erratic to obtain a straight line thermal resis- tance curve, as contrasted to the straight line curves us- ually obtained from data from products incubated in TDT-cans. Townsend gt_gl, (59) stated that TDTLcans were difficult to subculture and were not suitable for conducting tests where the test organism did not grow readily in the food. They reported that with uncultured TDT-cans, F values were gen- erally lower and 2 values were generally different from those obtained by other methods. Delayed germination was reported to be charcteristic of PA 5679 (29). ‘5 Laboratory manual for the Canning Industry (59) suggests that samples containing PA 3679 be incubated at least three months and at least one month after the last positive sample deve10ps. The survival of PA 3679 is usually l4 determined by noting gas procuction and characteristic odor in the subculture medium (15). Liver broth is an excellent medium for subculturing PA 5679 (39, 44, 59). EXPEHIKEETAL PROCEDURE Previous investigations have proven the feasibility of preserving canned processed cheese Spreads by heat treat- ing the product. The experimental procedure employed in this investigation was designed to study the effect of pH and brine concentration on the growth and heat destruction of one spoilage organism (PA 5679) in the product. Preparation of Heat Resistant Spore Suspension of PA 5679 Preparation g£_gpowth medium. Ten liters of pork extract were prepared according to the method of Reed £3 31. (59) for growth of the organism. This procedure is described below: 1. 10 lb. ground lean pork mixed with 10 1. of water. 2. Boil 1 hour. 5. adjust pH to 7.4 with NaOH. 4. Press out meat and dry for later use. 5. Cool over-night and remove fat. 6. flake up to 10 l. with water. 7. Add 50 g. bacto-peptone, 15 g. bacto-tryptone, 10 g. dextrose and 12.5 g. KgHPO4. 8. Adjust pH to 7.4. 9. DeSpense and autoclave 50 minutes at 15 lbs. pressure. Growth g£_organism. A culture of PA 5679 of known history was obtained from Wyeth Company in Kason, kichigan. Using the pork extract and the following schedule, six liters 15 of 1. 16 Spores of PA 5679 were grown: Transfer 2 ml. of culture into each of three tubes con- taining dried pork and 10 ml. of extract. Stratify (two parts mineral oil to one part paraffin) and in- bate at 37° C. When the tubes show good growth as indicated by gas pro- duction (usually one day) transfer 2 ml. into each of three tubes containing dried pork, two nails, and 10 ml. of extract. Incubate one day at 57° C. Transfer the contents of each of the three tubes into each of three flasks containing dried pork, five nails, and 50 ml. extract. Incubate two days at 57° 0. Transfer the contents of each of the three flasks into each of three large flasks containing dried pork, 12 nails, and 2 l. of extract. Incubate one week at 57° c. and then two weeks at 30° c. Harvesting of organism. The Spores were harvested washed according to the method suggested by Townsend £2 (59) and is briefly described as follows: Filter the material through a layer of fiber glass with cheese cloth on both sides. Fill international centrifuge bottles (250 ml.), centri- fuge (50 minutes at 1500 r.p.m.) and decent repeatedly until all Spores are concentrated in the bottom of six bOttleSo Decant, add glass beads, and fill with sterile distilled water. Shake five minutes and centrifuge. Decant, fill bottles one-half full with sterile dis- tilled water, shake five minutes, combine into four bottles, and centrifuge. Decant, fill bottles one-half full with sterile distilled water, shake five minutes, combine into two bottles, and centrifuge. Decant, fill bottles one-half full with sterile distilled water, shake five minutes, combine into one bottle, and centrifuge. Decant, fill bottle three-fourths full with neutral phOSphate buffer (equal amounts of M/15.KH2PO4 and M/l5 F82HPO4), shake five minutes, and centrifuge. 8. Repeat step seven three times except do not centrifuge last time. 9. Stgre concentrated spore suspension in capped bottle at 40 F. Standardization and storage g; organism. The spore suspension was standardized by determining the most probable numbers (MPH) of Spores in the concentrated suspension and then diluting with neutral M/l5 phOSphate buffer to obtain approximately 106 Spores per milliliter. The diluted sus- pension was stored at 40° F. The KPN and all subsequent subculturing was carried out in liver infusion broth (59, 44, 59); stratified with two parts mineral oil to one part paraffin (59). The KPN was conducted using five tubes at each dilution. Two liters of liver infusion broth were prepared by the method of Reed st 31. (59) as outlined be- low: 1. Boil 1000 g. of coarse ground beef liver in 2000 ml. of water for one hour. 2. Adjust pH to 7.2 with NaOH and boil for an additional 10 minutes. 5. Filter material through cheese cloth and cotton. 4. Air dry solid portion and make fluid up to 2000 ml. with water. 5. Add 20 g. peptone and 2 g. K2HPO4. 6. Dispense into tubes, each of which contains a pinch of dfiied liver. 7. Stratify and autocalve 50 minutes at 15 lb. pressure. 8. Store at 40° F. Before use, the tubes of liver infusion broth were exhausted by flowing steam in an autoclave for 20 minutes to remove 18 excess oxygen and melt the stratifying cap. The tubes were then tempered to 45° C. in a water bath and inoculated with the prOper dilution of spores. If a KPH was being deter- mined, the tubes were heated for 10 minutes at 800 C. to heat-shock the Spores and then incubated at 37° 0. Positive tubes were identified by the presence of gas which was indicated by the elevation of the stratifying cap above the surface of he broth. host positive tubes became apparent within the first five days of incubation. Thermal resistance 33 organism. The thermal resis- tance of the suSpension was determined by eXposing samples of Spores to different heat treatments in a thermoresisto- meter and then subculturing in tubes of stratified liver infusion broth. The results, in numbers of positive and negative tubes at each time-temperature combination, were used to calculate a values, which in turn were used to plot a thermal resistance curve. The thermoresistometer used was similar to the one described by Stumbo (51) and Pflug and Esselen (57). It is an apparatus which essentially consists of a pressure chamber which can be maintained at‘a Specified temperature and a series of valves and pistions which introduce small cups containing a known number of spores into the steam chamber for a Specified length of time, after which the cups are automatically withdrawn and drOpped into tubes of subculture broth. Five cups were tested at a time. 19 The sample cups (11 mm. outside diameter by 8 mm. deep and formed from tinplate 0.008 in. thick) were soaked and washed in alcohol, placed in petri dishes and sterilized in an autoclave for 15 minutes at 15 lb. pressure. The standardized suSpension of spores was shaken 15 minutes and a small portion transferred to a 50 ml. flask. The flask was placed on a magnetic stirrer which kept the suspension constantly agitated. The cups were loaded with Spores us- ing a micro syringe-burette which held 0.25 ml. calibrated in 0.0001 ml. The micro syringe-burette was filled from the flask and then clamped to a ring stand. Each cup was removed from the petri dish with sterile tweezers, 0.01 ml. of suspension measured into it, and the cup returned to the petri dish. The calibration of the syringe was checked be- fore using by weighing delivered samples of distilled water on an analytical balance. Twenty-five cups could be loaded each time the syringe was filled. The petri disnes of loaded cups were placed in a desiccator and held at least 24 hours prior to use to remove most of the moisture before the cups were introduced into the high temperature chamber. The procedure for making a test with the thermore- sistometer follows: 1. AdJust pressure controller to obtain desired temperature. 2. Set exposure time on automatic cycle timer. 3. Asaptically place loaded cups and subculture tubes in apparatus. Tubes containing PA 5679 should be strati- fied and steamed at least 20 minutes. 4. actuate timing cycle and rotate trip bar 180 degrees. 5. After the cups are automatically withdrawn and drOpped into the subculture tubes; remove and flame tubes, in- sert sterile plugs, and place in a 57° 0. incubator. 6. Reset support pins for next trial. The complete Operation, excluding the time the cups are in the chamber, takes approximately one minute. Two trials were made at each time and temperature, resulting in 10 re- plicate tubes. All tubes were incubated at least three months. At the end of the incubation period a D value and 95 per cent confidence limits were calculated for each of the test temperatures, using the method described by Schmidt (45). Using this procedure a curve was prepared for each temperature condition by plotting the value of probability (P) as a function of time (U) on arithmetic probability paper and determining the L.D.50 point which is the time (U) at which P equals 0.50, r the time at which one—half of the samples are positive and one-half are ne- gative. The probability (P) was calculated using the equation P g n + l + n + m 2 (1) where n : cumulated samples not surviving each exposure time. m : cumulated samples surviving each eXposure time. The D value for each temperature was calculated using the L.D.5O value and the equation D : LOD050 Iog i + 0.16 (a) 21 where i = the initial no. of organisms per tube. The 95 per cent confidence limits of the L.D.5O value were calculated using the equation 95; 01 z 1.96 x 25 Vfifi‘ (5) where 28 3 difference between the time (U) when P : 0.16 or 16 per cent of the tubes would have been negative, and the time (U) when P : 0.84 or 84 per cent of the tubes would have been negative, as determined from the probability curve. N = total no. of tubes in the groups showing partial survival. The 95 per cent confidence limits of the D value were cal- culated using the equation 95% CLD ; L.D.50 I 95% Cl _Tea A + 0.I5 (4) Preliminary trials were conducted at 240 and 250° F., the D values calculated, and a preliminary thermal resis- tance curve plotted using the two points. The final ther- mal resistance tests were conducted at 240, 245, 250, 255, and 2600 F. The range of time over which testing was to be conducted was determined and then the actual testing times (U) were found by dividing the range by units of one-seventh of a log cycle. After incubation the D values and 95 per cent confidence limits were calculated and the thermal re- sistance curve for the Spore suSpension was plotted. 22 Growth of PA 3679 in Processed Cheese Spread Effect 23 skim milk pgwder. a preliminary 5 lb. batch of processed cheese spread was prepared to test the effect of skim milk powder on the growth of PA 5679. The batch was divided into two lots and enough skim milk powder was added to give a 1.0 per cent concentration in one lot and a 10.0 per cent concentration in the other. A beaker of each lot of processed cheese Spread was placed in a water bath and tempered to 65° c. to obtain complete fluidity. Enough Spores of PA 5679 were mixed into the processed cheese spread to give 1.84 X 105 spores per gram and the pH was adjusted to 6.0. Initially the pH was determined by using both a glass and a quinhydrone electrode system. These determin- ations revealed no significant difference in the pH when using the two different electrode systems; therefore, the pH was determined with the glass electrode system only, during the latter part of the project. All pH determinations were made at 650 C. After the addition of the Spores and the adjustment of pH, the processed cheese Spread was dispensed into test tubes. To facilitate this step, a metal tube (0.7 cm. in- side diameter and 15 cm. long) was soldered to a 2 oz. "dose syringe". This instrument made it easy to fill the tubes to the prOper level without incorporating air pockets or smearing the product on the upper inner surface of the tube. 25 Each tube was stratified, plugged, heated for 10 minutes at 80° C. to heat-shock the Spores, tempered to 37° 0., and incubated eight days at 57° C. At the end of the incubation period each tube was examined for the presence of gas. The stratifying plug was melted and decanted from all tubes showing gas production and the odor noted. A Gram stain was made of the processed cheese Spread and a 100p full of the product subcultured in stratified liver in- fusion broth which was incubated at 57° C. After the preliminary test was completed a large batch of processed cheese Spread was produced for use through- out the remainder of the project. The ingredients included 45 lb. of aged cheddar cheese, 50 lb. of unsalted green cheese curd, 22 lb. of water, 10 lb. of 55 per cent cream, 10 lb. of skim milk powder, and 3 lb. of sodium citrate. The mixture was cooked to 160° F. in a Kojonnier processing kettle, drawn off into No. 10 cans (605 X 700) and the cans sealed. The cans of processed cheese Spread were cooled and stored in a 40° F. refrigerator. The amount of fat and moisture in the processed cheese Spread was determined by the Kojonnier method (50). The salt content was determined by the method outlined by Wilster gt al. (67). The equation for brine concentration is: brine conc. (%); pg. salt X 100 g. saft’+ g. water (5) 24 which is equivalent to: brine conc. (fl) 3 5 salt X 100 *‘Tffim (b) To determine the total amount of salt necessary to give a specified brine concentration in the processed cheese Spread, equation 6 was rearranged as follows: g. salt : fibrine X_g. H20 100 - % brine (7) where % brine : brine concentration desired. The brine concentration in the processed cheese spread was standardized by calculating the amount of salt present in the sample, determining the amount of salt necessary to give the desired brine concentration using equation 7, and then adding the difference because when the brine concen- tration was standardized, it was always increased. In us- ing equation 7, the grams of water added with the Spore inoculum were taken into consideration. Brine concentra- tion throughout the project is defined by equation 6. Effect pf pg and brine concentration. Several tests were conducted to determine the effect of pH and brine con- centration of the processed cheese Spread on the growth of PA 3679. A sealed can of processed cheese Spread was re- moved from the refrigerator and allowed to temper over-night at room temperature. The can was then placed in a covered water bath at 650 C. for one-half day. The can was Opened 25 and the desired amount of processed cheese Spread weighed into a beaker which was placed in the 65° C. water bath. Enough Spore inoculum was added to result in 1.84 X 105 Spores per gram and the brine concentration standardized. The pH was determined and standardized using NaOH or HCl as necessary. Growth tests were conducted both in test tubes and in TDT-cans. The tubes were stratified, plugged, heated for 10 minutes at 80° C. to heat-shock the Spores, tempered, and incubated at 37° C. The TDT-cans contained 18 g. of the standardized processed cheese spread and 1 ml. of Spore suSpension. The sealed cans were treated in the same man- ner as the plugged tubes. The test tubes and a few of the TDT-cans of proces- sed cheese Spread were checked visually for evidence of growth, gas bubbles in the tubes‘and eXpansion of the TJT- cans. The expansion of most of the TDT-cans of product was determined at pre-selected intervals by a dial micro- meter (Figure 1). By using the TDT-can and the dial micrometer, the effect of pH and brine concentration on the growth lag and amount of gas produced by the Spores was determined. Ten TDT—cans were used for each set of variables and the arith- metic average of the can eXpansion calculated. All measure- ments were made in a constant temperature 57° C. walk-in incubator to avoid changes in can thickiess due to tempera- ture differences. 26 Figure l. Dial micrometer and TDT—can. 27 Heat Penetration Studies The lag correction factor, which is the difference between the total heating time and the time at retort tem- perature, is evaluated by heat penetration studies. These studies were conducted to determine the effect of fill- weight and the position of the ThT-can on the rate of heat- ing and consequently the lag correction factor of TDT-cans of processed cheese Spread. COpper—constantan thermocouples were mounted in 12 TDT-cans (Figure 2). The cans were heated in a miniature retort similar to the one described by Townsend 33 al. (57). Using the data obtained, heat penetration curves were plotted as described by Ball and Olson (8) and the lag correction factors calculated. The c0pper-constantan thermocouples, made from No. 50 B & S gauge wire, were positioned between the side and center midway between the ends of the ThT-can. The thermo- couple was held in position by passing the wire through a hole drilled in a No. 00 rubber stopper and the lead wire was brought out through a hole punched in the lower side of the TDT-can. An electrical insulating resin (Epocast) was used to secure the stopper in place and seal the hole where the lead wire was brought out of the can. Heat penetration data were collected with the TDT- can in the flat position, and with it standing on its edge. Fills of 16, 18, and 20 a. of processed cheese Spread were U tested in both positions. One milliliter of water was Figure 2. TDT-can and copper-constantan thermocouple. 28 29 added to each TDT-can of product of simulate the added Spore suspension. The TDT-can of processed cheese spread was placed over a boiling water bath long enough to melt’ the product and allow it to settle into the can around the thermocouple Junction. The TDT-can was sealed at atmos- pheric pressure and placed in the desired position in the miniature retort. The thermocouple wire was brought out through a stuffing box in the side of the retort and con- nected to the temperature recording potentiometer. A pres- sure controller was used to adjust the temperature of a ballast tank to 250° F. and the retort lid was clamped closed. The following precedure was used in making a test: The vent was Opened, the recording potentiometer turned on, and, after waiting 10 to 30 seconds to get an initial tem- perature reading, the steam valve was Opened. The retort was vented for 15 seconds to insure complete air removal. When the temperature recording potentiometer indicated that the contents of the TLT-can had reached retort temperature, the steam was shut off, the vent and drain Opened, and the cooling water turned on. When the temperature recording potentiometer indicated that the contents of the TDT-can had reached the cooling water temperature, the water was turned off and the retort Opened. The position of the TDT-can was changed and the sequence repeated. Each time the position f the TLT-can was changed, the product was 30 heated to approximately 210° F. to melt the processed cheese Spread and allow it to seek a level position after which it was cooled back to the cooling water temperature before the next trial was started. When the TDT-can was placed on edge the thermocouple junction was located in the bottom half of the can. The heat penetration curves were plotted and the lepe (fh) and lag factors (3) for each curve were calcu- lated by the method of Ball (5). The fh and 3 values ob- tained from the duplicate tests were used in the calculation of the lag correction factors (to). The lag correction factor (to) that must be evaluated is the difference between the total heating time (B) and the lethality (U) at the exposure or retort temperature. A relatively simple method of calculating lag correction factors from heat penetration data was deve10ped from equa- tion 8 by Fflug and Bsselen (37) using Ball's (6) formula. B : fh (log 31 - 105 s) (9) where fh : SlOpe of heat penetration curve. J I lag factor. I : RT - IT. (10) g - the difference between RT temperature and great- est temperature attained by the point of slowest heating in the can (assumed to be 0.1). If g : 0.1, then the 10g of g ; - 1.0. Using a log g : - 1.0 51 and assuming a z of 180 F. and using the curve in Ball and Olson (8) éh : 0.58 Rearranging equation 11, substituting equations 9 and 12 into equation 8, assumin Spread would be heated from 56 to 2500 F. tc fh (log 31 - log g) - 1.725 fh 8 105 8 : - 1.0 and solving. .0 : rh (10g jl - 0.725) (11} (12) (13) It was assumed that the TDT-cans of processed cheese On the basis of this assumption, lag correction factors (to) were calculated for use results in all subsequent thermal processing studies. are shown in TABLE I. TABLE I The Effect of fill-weight and position of the can on the lag correction factor (to) of a processed cheese Spread in TDT—cansa Cangposition Fill-wt. Edge Flat gramsb chmin. ,jp tappin. chmin. 43 tetfiin. 16 1.440 1.290 2.41 1.870 0.895 2.83 18 1.740 1.200 2.82 1.995 1.055 5.16 20 1.830 1.205 3.00 2.040 1.120 3.28 a. Assumed 1 = RT - 1T g 194, z = 18° F. b. 1 ml. water added to each can. 32 Ball and Olson's (8) table of P values does not go above a z of 26° F.; therefore, it is necessary to use Ball and Olson's (8) new method, or the method of Pflug (35) which uses Hick's (23) data to calculate a lag correction factor (tc) for a z of 37° F. The calculation of the lag correction factor (to) for a z of 37° F. follows: From Hicks tables (23) U - f X 195 7 h _ 0 I30 (14) and rearranging, Uh ; 1.960 fh (15) The ratio of the lethality accumulated during heating and cooling is 0.93 (35). as H O to ()3 (16) Rearranging equation 16 and substituting it into equation 15, U : 2.107 rh (17) substituting equation 9 on page 30 and equation 17 into equation 8 on page 30, t0 : fh (log 31 - log g) - 2.107 rh (18) 33 assuming 10g g : - 1.0 and solving, to : fh (log 31 - 1.1.07) (19) By using the same data used to calculate the lag correction factor (tc) for an 18 g. fill with the TDT-can in the flat position (3.16 minutes, TABLE I) and equation 19, to : 10995 (10g 204.67 ‘ 1.107) tc : 2.40 minuteS‘ The difference in the lag correction factor (to) for a z of 18° F. and a z of 37° F. is 3.16 - 2.40 - 0.76 minutes. USing equation 2; D - 0.76 3 0.11 minutes therefore, if the thermal resistance curve exhibited a z of 0 37° F. instead of 18 F. when the lag correction factor (to) used was based on a z of 180 F., the calculated D values would be 0.11 minutes too small. Effect of pH and Brine Concentration on the Thermal Resistance of FA 3679 in Processed Cheese Spread To determine the range in which to heat treat the TDT-cans, several lots of processed cheese Spread at vari- ous pH and brine concentrations were treated at arbitrarily 34 selected times and temperatures. The first two temperatures were 230 and 2400 F. After obtaining these results and as- suming a z of 180 F., tests were also made at 220, 225, 235, and 245° F. Two TDT-cans containing 1.84 X 105 Spores per gram of processed cheese Spread were processed at each temperature and time. These results were used to plot an end-point destruction curve. To construct this curve the temperature was plotted on the arithmetic scale against the time (U) on the log scale of semi-logarithmic paper. The positive and negative results secured from the above pro- cedure were placed on the graph and a straight line drawn above all positive points and below as many negative points as possible. By using this curve as the center of a range, the times (U) at each Specific temperature were chosen for the thermal resistance tests. The actual testing times (U) were found by dividing the range by units of one-tenth of a log cycle. The prOper lag correction factor (tc) was added to the times (U) to obtain the heating times (B). TDT-cans containing 18 g. of standardized processed cheese Spread and 1 ml. of Spores were used in all tests to determine the effect of pH and brine concentration on the heat destruction of PA 3679 in processed cheese Spread. The cans were prepared, heated, incubated over-night, and then 0.01 g. of product from each can was subcultured in liver infusion broth. The 0.01 g. sample was taken by stab- bing the center of the Opened TDT-can of product in three different places with a calibrated hot 100p. After the 35 third stab the 100p of product was introduced into the sub- culture medium. A subjective evaluation of the color of the product was made when it was subcultured. A11 tubes were incubated at least three months. Tests for the ther- mal resistance of PA 3679 were Conducted by heating five TDT-cans at each time and temperature. The TDT-cans were prepared as follows: 1. Temper the sealed can of processed cheese Spread at room temperature over-night. 2. Flace can in 650 0. water bath one-half day. 3. Open can and weigh desired amount into a mix-master bowl which was then placed on the mix-master setting in 65° 0. water bath. 4. Mix in the desired amount of Spore suSpension (1.84 X 105 spores per gram) and salt. 5. Standardize the pH by adding NaOH or H01 as necessary. 6. Heigh 19 g. of mixture into each can and seal. Batches were standardized at pH 5.50 and a brine concentra- tion of 2.0 per cent, pH 5.50 and a brine concentration of 4.4 per cent, pH 6.25 and a brine concentration of 2.0 per cent, pH 6.25 and a brine concentration of 4.4 per cent, pH 7.00 and a brine concentration of 2.0 per cent, and pH 7.00 and a brine concentration of 4.4 per cent. D values were calculated for each temperature according to the Schmidt method (45) and the thermal re- sistance curves were plotted. 36 Effect of pH and Brine Concentration on the Heat Treatment Required to Prevent Growth of PA 3679 in Processed Cheese Spread The effect of pH and brine concentration on the heat treatment required to prevent growth of PA 3679 in processed cheese Spread was determined by sealing 18 g. of standardized processed cheese Spread and 1 ml. of the spore suSpension in TDT-cans, heat-treating, and incubating the cans at least five months. The number of swollen TDT-cans was noted and the data were used to plot end-point inhibi- tion curves. Preliminary data were used to aid in the selection of the range to be used in heat treating the product. The times (U) were determined by dividing the range into units of approximately one-fifth of a log cycle. The lag cor- rection factor (tc) was added to determine the heating times (B). The wide spacing of U values resulted from a shortage of processed cheese Spread. Batches were standardized at pH 6.0 and a brine concentration of 1.6 per cent, pH 6.0 and a brine concen- tration of 2.4 per cent, pH 7.0 and a brine concentration of 1.6 per cent, and pH 7.0 and a brine concentration of 2.4 per cent. The TDT-cans were heated in miniature re- torts at the pre-Selected times and temperatures, cooled, and placed in a 37° 0. incubator. Each TDT-can was measured every day for the next three days using the dial micrometer to determine the ini- 37 tial thickness of the can. all TOT-cans were measured again at the end of 60 days of incubation to determine which ones had eXpanded due to gas production by FA 3679, and how much. An arithmetic average of the amount of eXpansion of the five TDT-cans at each time and temperature was computed. To determine if the cans had stopped expanding after 60 days, they were measured once a month for three more months. Using the data from the positive and negative TDT- cans, end-point inhibition curves were plotted. The num- ber of viable Spores remaining in the cans following heat treatment, which Showed growth at the longest heating time and no growth at the shortest heating time were calculated for each temperature using equation 2. The thermal process received by each group of five TDT-cans was equated to the time at 2500 F. using the 2 value obtained from the end-point inhibition curves and the equation for lethality rate (2). r; U _ log 'I 250 - RT (20) Z where F ; equivalent process time at 2500 F. U : time at retort temperature. RT : retort temperature. 2 ; slope of the thermal resistance curve. With the thermal processes placed upon a common reference, the effect of the thermal process and brine concentration on TDT-can expansion at pH values of 6.0 and 7.0 could be compared. RESULTS The primary objective of this investigation was to determine the effect of pH and brine concentration upon the growth, thermal resistance, and the heat treatment required to prevent growth of PA 3679 in processed cheese Spread. The results of this investigation are reported in the fol- lowing order: (1) Preperties of the Spore suSpension of PA 3679, (2) Growth of PA 3679 in processed cheese Spread, (3) Effect of pH and brine concentration on the thermal resistance of PA 3679 in processed cheese Spread, and (4) Effect of pH and brine concentration on the heat treatment required to prevent growth of PA 3679 in processed cheese Spread. 1 PrOperties of Spore SuSpension of PA 3679 The Spores from 4 1. of culture media were concen- trated to 220 ml. with a MPH of 52.8 x 106 Spores per mil- liliter. This suspension was diluted to a volume of 3520 ml. with M/15 phosphate buffer (pH 7.0) to give a MPH of 3.3 X 106 Spores per milliliter. The diluted suspension was stored at 400 F. in large flasks containing glass beads. TABLE II shows the results of preliminary tests of the thermal resistance of spores of PA 3679 as determined by the thermoresistometer technique. The L.D.5O value for each temperature was obtained from the curves in Figure 3. A preliminary thermal resistance curve was constructed 38 TABLE II Results of preliminary tests for the thermal resistance 39 f Spores of PA 3679 in the thermoresistometer (3.3 I 102 Spores per cup, samples subcultu broth and incubated at 37 sed 12 .) liver n U N0. tube}: N0.’ N0.- 2 P 11.13.50 D 240° F. 5.0 10 10 o 22 0 0.041 7.0 10 8 2 12 2 0.187 10.0 10 4 6 4 8 0.642 9'3 1‘98 14.5 10 0 10 0 18 0.950 250° F. 1.4 10 8 2 25 2 0.105 2.0 10 6 4 17 6 0.280 2.8 10 6 4 11 10 0.478 5.0 0.64 4.0 10 5 5 5 15 0.727 5.6 10 0 10 0 25 0.962 a. Data Show only the results involved in calculations. MO r T 1 AP >— 240° F. I38 3. — a 5 in» . §4_ % i— o J .2 3a .5 9_ 1.0.50.9.3 y E 2,“ 0 .E r— — g ‘_ . k 1.. . 7— ~ + L 4 E s 1 1 x 1 a 1 .. 1 ; __ . . : 601 0.1 ‘8 so 95 99.9 0.0l 0.: 5 50 95 99.9 Probability of sterility Probability of sterility Figure 3. Probability curves for determining L.D.BO time (preliminary test). using the DEMO and D250 values. The curve had a 2 value of 20.20 F. and a D250 of 0.64 minutes. The results of the series of tests for the thermal resistance of the suspension are tabulated in TABLE III. The L.D. 50 value for each temperature was obtained from the curves in Figure 4. The D values from TABLE III and their 95 per cent confidence limits were plotted on semi- logarithmic paper and the thermal resistance curve was drawn by eye (Figure 5). This curve has a 2 value of 17.50 F. and a D250 of 0.98 minutes. 41 TABLE III Results of tests for the thermal resistance of Spores of PA 3679 in the thermoresistometer (3.3 X 104 Spores per cup, samples subcultured in liver broth and incubated at 37° C.)8 U No. tubes N0.+ N0.- m n P L.D.SO D 240° F. 5.00 10 10 0 57 0 0.025 7.00 10 9 1 27 1 0.066 10.00 10 9 1 18 2 0.156 7 7 14.00 10 8 2 9 4 0.555 1 ’50 3° 4 20.50 10 1 9 1 15 0.875 29.50 10 0 10 o 25 0.960 245° F. 7.00 10 10 0 14 0 0.062 10.00 10 3 7 4 7 0.615 8.50 1.82 14.00 10 1 9 1 16 0.894 20.00 10 0 10 0 26 0.964 250° F. 2.40 10 10 0 21 o 0.045 3.50 10 9 1 11 1 0.142 4.50 0.98 4.90 10 2 8 2 9 0.769 7.00 10 0 10 0 19 0.952 255° F. 1.20 10 10 0 25 0 0.040 1.70 10 9 1 15 1 0.125 2.40 0,51 2.50 10 4 6 4 7 0.615 5.50 10 0 10 0 17 0.947 260° F. 0.61 10 10 0 24 0 0.058 0.88 10 8 2 14 2 0°166 1.22 0.26 1.20 10 6 4 6 6 0-500 1.80 10 0 10 0 16 0.944 a. Data show only the results involved in calculations. Time in minutes, U Time in minutes, U 42 f T fi 20 T 240‘ F. / 25'- ‘ I6 D 20— O — 5 12 a 28 I I175 L.D. 50: I25 E .5- q .5 8 232/].2 L.D.50-8.5 e 5 L. IO'- -4 41—- 5 1 W 1 1 1 o g 1/ 1 1 1 0.0l OJ 5 50 95 99.9 0.0! OJ 5 50 95 99,9 Probability of sterility Probability of sterility i0 1 r I i I 5 1 1 i T I 250°E 255°F 8'. 4*- -d __ D i 6— '5 3- f 1 '5 £924 0 -‘ E L.D. I . 25'2-35 Loco-4.6 c 4» 4 '- 2» « O 1- 1 _§ 3.. 2- 4 1- — 0 l 1 L 1 _1_ 0 l 1 4 1 0.0I 0| 5 50 95 99.9 0.0| OJ 5 5O 95 99.9 Probability of sterility Probability of sterility 2 Time in minutes, U ? L__ 1 2580.68 L.D.50-l.22 l_ L O l 0.0l OJ 5 50 95 99.9 Probability of sterility Figure 4. Probability curves for determining L.D.SO time (final test). 0 value, min. 43 '0-0 I 7 1 fl T 1 _ a. _a _ . _" L0 :: :: _. e —. e 0' '_ O ——1 _ 1+——-—-Z=I25 F: A : V L_ ... (10' l IJ i. J l J 235 240 245 250 255 260 265 270 Temperature, °F. Figure 5. D Values with 95% confidence limits and the resulting thermal resistance curve for spores of PA 3679 suspended in neutral phosphate buffer. 44 Growth of PA 3679 in Processed Cheese Spread Effect 2£.£E$E.Ell5 powder. In tests to determine the effect of skim milk powder no visually detectable gas was found in the tubes of processed cheese spread contain- ing 1.0 per cent skim milk powder: whereas, the tubes of Spread containing 10.0 per cent skim milk powder were honey combed with large pockets of gas (TABLE IV). This gas had a putrid odor typical of PA 3679. A Gram stain of the con- tents revealed large Gram positive rods. Tubes of strati- fied liver infusion broth inoculated from these positive tubes of processed cheese spread showed gas production within three days. TABLE IV Effect of skim milk powder on the growth of PA 3679 in cheese Spread with a pH of 6.0 at the time of ino- culation and incubated 8 days at 37° 0. (mea- sured by gas production in glass tubes) Cheege Spread plus Cheese a read plus Tube no. 141$ NFDM 10.0 NFDM 1 - e 2 - + 3 - + After completion of the preliminary test a large batch of processed cheese Spread containing 10.0 per cent skim milk powder was produced for use throughout the re- mainder of the project. This batch of processed cheese 45 Spread had a moisture content of 46.04 per cent, a fat con- tent of 22.34 per cent, a pH of 6.20, and a salt content of 0.775 per cent. The brine concentration was 1.65 per cent. Effect 25 pg 339 bring ggncentration. TABLE 7 shows the results, recorded as either positive or negative tubes, of the effect of pH and brine concentration on the growth of PA 3679 in processed cheese Spread. There was grgwth above and no growth at or below pH 5.6 at the brine concentrations tested (2.8 to 6.8 per cent). In another test where both tubes and TDT-cans were used, the growth of PA 3679 in tubes of processed cheese spread with a pH of 6.2 was completely inhibited at a brine concentration equal to or greater than 7.6 per cent (TABLE VI). The results in TABLE VI indicate that growth which could not be subjectively detected was taking place in the TDT-cans of processed cheese Spread. The limitation of sub- Jective evaluation led to the develOpment and use of the dial micrometer for measuring the expansion of TDT-cans. Figures 6 and 7 Show the effect of brine concentra- tion on the gas production by PA 3679 in the processed cheese Spread, as measured by the SXpansion of TDT-cans. An in- crease in the brine concentration resulted in an increase in the lag time for gas production, a decrease in the total amount of gas produced, a decreased rate of gas production, and an increase in the time during which gas was produced. There was no measureable gas production after 60 days of incubation. The lag time was always greater at pH 6.0 than 46 TABLE V Effect of pH and brine concentration on the growth of PA 3679 in a cheese Spread incubate'd 14 days at 37°C. (measured by gas production in glass tubes) Brine concentration,_% v_ DH 2.8 3.6 404 502 6.0 6e8 5.2 5.4 5.6 5.8 6.0 6.2 e-e-e e-e-e e-ete +-ee- «es-e e»+1e TABLE VI 47 Effect of brine concentration on the growth of PA 3679 in s cheese Spread with a pH of .2 when incubated 14 days at 37 C. Brine concentration Visual examination % ‘_GIass tubes TDT-Cans_ 2.0 +e+++ e 2.8 ’9‘. - 306 +9 " 4.4 ++ - 5.2 Q - 6eo + _ 6.8 + - 7.6 - - 8.4 - _ II ( nac0t\ s t°.\mtoqx u Expansion, Inches 0.07 r 1 g , 1 r (106L- ° ° _flfl’//) 4 ° L€596 5’ O (a I 1 O O b r e ( ’ E“ m t o\° 0.03 - 4 e #{I—r 4-0 CL02 - , — - 141996 0.0! - _ ' ' 1 1 L, 1 1 0 IO 20 30 40 50 60 Days Figure 6. Effect of brine concentration on the gas pro— duction of PA 3679 in a cheese spread at pH 6.0, as measured by the expansion of TDT-cans incubated at 37°C. (each value represents an average of 10 cans). Expansion , Inches L19 (L07 *1 1 1 1 1 *1 01361- a I.6% 0.05 L W —1 . 0 e . . Q 443 - 49* “I 0.04 _ . k— ... 2.8% 0.03 — ' . ' — O 0 C102 - 0 fl ‘ 4.0% / 0.0' ’- . o .1 ' a 1 1 i 1 0 IO 20 30 40 50 60 Days Figure 7. Effect of brine concentration on the gas pro- duction of PA 3679 in a cheese spread at pH 7.0, as measured by the expansion of TDT-cans incubated at 37°C. (each value represents an average of 10 cans). 50 at pH 7.0 (TABLE VII). Figure 8 illustrates the effect of brine concentration at pH 6.0 and 7.0 on the gas production by PA 3679 in processed cheese spread after 60 days of in- cubation. Effect of pH and Brine Concentration on the Thermal Resistance of PA 3679 in Processed Cheese Spread TABLE VIII shows the results of preliminary tests for the thermal resistance of Spores of PA 3679 in the pro- cessed cheese Spread. The times (U) at the different tem- peratures were so widely Spaced that both duplicates were either positive or negative; the results were the same for all pH and brine concentrations tested. Using data from TABLE VIII, an end-point destruction curve was plotted (Figure 9). The end-point destruction curve was used as the center of the range in the selection of times (U) at Specific temperatures for the final thermal resistance tests. TABLES IX, X and XI show the results of tests for the thermal resistance of spores of PA 3679 in processed cheese Spread at pH 5.50, 6.25, and 7.00, reSpectively, with brine concentrations of 2.0 and 4.4 per cent. Data from TABLES IX, X, and XI were used to plot Figures 10, 11, and 12, reapectively. The D235, F235, and 2 values from each of the curves in Figures 10, 11, and 12 are tabulated in TABLE XII. The F335 values are based on commercial sterility. An increased D235 value with each increase in pH was noted. The D235 values were essentially 51 TABLE VII Effect of pH and brine concentration on the lag phase of PA 3679 in a cheese Spread incubated at 37° 0. (measured by the swelling of TDT-cans) Brine concentration- Lag time in days 1.6 4 3 2.0 4 3 2.4 6 4 2.8 6 4 3.2 7 6 3.6 10 7 4.0 12 10 4.4 l5 l2 52 pH'lO assasssssm assasssssssau .\\\\\\\\\\\\\\\\\\\\\\\\sm §§m _ _ _ _ pflSA) _— §M §m .\\\\\\\\\\\\\\\\\\.u .\\\\\\\\\\\\\\\\\\\\\\.u w\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\m ammfimxxwxwwwwwwxxm\m 0.07 _ m Q _ _ _ m. m. m 0 0 «235 66.3.3an I O O. O. 0 .0 _ 2 O O assasswu Brine concentration, % Effect of brine concentration on the gas pro- d 7.0 6 in a cheese spread at pH 6.0 an , y3t£2 expansion of TDT-cans incubated 60 days duction of PA as measured b at 37°C. Figure 8. 53 TABLE VIII Results of preliminary tests for the thermal death rate of Spores of PA 3679 in a cheese Spread (3.3 X 106 Spores in each of two TDT-cans per trial, with 0.01 g. from each can subcultured in liver broth and incubated at 37° C.) 245° F. No.4 U n0.+ 240° F:— Bo.+ U 255‘ F. N0.+ U 2505*F. No.+ U 220° F. $2250 F. U ‘16.. U pH 5.5 and a brine concentration of 2.0% 99.8 2 51.8 2 14.0 2 16.8 2 3.7 2 3.7 2 141.8 0 71.8 0 27.0 2 21.8 0 7.1 2 5.2 2 199.8 0 91.8 0 51.0 0 31.8 0 13.5 0 7.0 0 98.0 0 26.0 0 pH 5.5 and a brine concentration of 4.4% 99.8 2 51.8 2 14.0 2 16.8 2 3.7 2 3.7 2 141.8 0 71.8 0 27.0 2 21.8 0 7.1 2 5.2 2 199.8 0 91.8 0 51.0 0 31.8 0 13.5 0 7.0 0 98.0 0 26.0 0 pH 7.0 and a brine concentration of 2.0% 99.8 2 51.8 2 14.0 2 16.8 2 3.7 2 3.7 2 141.8 0 71.8 0 27.0 2 21.8 0 7.1 2 5.2 2 199.8 0 91.8 0 51.0 0 31.8 0 13.5 0 7.0 0 98.0 0 26.0 0 pH 7.0 and a brine concentration of 4.4% 99.8 2 51.8 2 14.0 2 16.8 2 3.7 2 3.7 2 141.8 0 71.8 0 27.0 2 21.8 0 7.1 2 5.2 2 199.8 0 91. 0 51.0 0 31.8 0 13.5 0' 7.0 0 98.0 0 26.0 0 l000 i I I Time in minutes, U 54 l00 l0 | .1 l l J L J 1 2'5 220 225 230 235 240 245 250 Temperature, °F. Figure 9. End-point destruction curve of spores of PA 3679 in a cheese spread (each point represents dupli- cate determinations). TABLE IX 55 Results of tests for thermal death rate of Spores of PA 3679 in cheese Spread at pH 5.50 (3.3 X 106 Spores per TDT-can, 0.01 g. from each can subcultured in liver broth incubated at 37° C.) U N0. tubes No.+ No.- m__ n P L.D.50 D Brine concentration, 2.0% __ 225° F. __. 58.8 5 5 0 7 0 0.111 48.8 5 2 5 2 5 0.571 48.00 14.05 59.8 5 0 5 ., 0 8 0.900 ,__ 250° F. __ 26.8 5 5 0 5 0 0.142 52.8 5 0 5 0 5 0.857 29°30 3‘71 fi_ 2555 F. ‘ 14.5 5 5 0 7 0 0.111 I 17.8 5 2 5 2 5 0.571 17.50 5.15 21.8 5 0 5 o 8 0.900 Ii fl 2403—111. w 5.2 5 5 0 9 '0 0.090 6.8 5 2 5 4 5 0.444 8.8 5 1 4 2 7 0.727 7.60 2.22 11.5 5 1 4 1 11 0.857 14.5 5 0 5 0 16 0.944 _ 2453'F. 3.8 5 5 0 12 g 8°200 ‘ .9 5 4 1 . 5.5 5 5 2 5 5 0.500 5‘05 1’47 ___6.8 5 0 5 0 8 0.900 __ Brine concentration, 4.4% 2255_F. 51.8 5 5 0 9 0 0.090 58.8 5 4 1 4 1 0.285 41.60 12.16 48.8 5 0 5 0 6 0.875 .1_ I“ 250° F. . " 21.8 5 5 0 8 0 0.100 'I“ 26.8 5 5 2 5 2 0.428 27.50 8.04 __52.8 5 0 5 0 7 0.888 255° F. “— 11.5 5 5 0 7 0 0.111 14.5 5 2 5 2 5 0.571 14.10 4.12 17.8 5 0 5 0 8 0.900 __. 240‘F. I“ 6.8 5 5 0 8 o 0.100 ““ 8.8 5 5 2 5 2 0.428 9.10 2.66 __11.5 5 0 5 2 7 0.888 _I 245° Fe ‘ 3'3 5 5 ‘2’ 9 2 8'33? . 5 5 4 . 5.5 5 1 4 1 6 0.777 4'90 1°43 _i 6.8 5 0 5 0 11 0.925__ ‘— I. Data show only the results involved in calculations. cheese spread at pH 6.25 (3.3 X 106 TABLE X Results of tests for thermal death rate of Spores of PA 8679 in spores per TDT-can, 0.01 g. from each can subcultured in liver broth incubated at 37° C.)‘ 56 __, U No. tubes No.+ No:: m n P L.D.50 D ‘— Brine concentration, 2.0% "‘ 225° F. 58.8 5 5 o 14 0 0.062 48.8 5 4 1 9 1 0.166 59.8 5 2 3 5 4 0.454 54,00 18.71 71.8 5 2 5 5 7 0.666 86.8 5 1 4 1 11 0.857 104.8 5 o 5 0 16 0.888 2365—F. 59.8 5 5 0 5 o 0.142 48.8 5 0 5 o 5 0.857 44°40 12'98 ____255° F. 4:: 17.8 5 5 0 9 0 0.090 21.8 5 4 1 4 1 0.285 25.00 6.72 26.8 5 0 5 o 6 0.875 .1 240° F. 11.5 5 5 0 5 o 0.142 14.5 5 0 5 0 a 5 0.857 12°80 3'74 __ fi_ 245° F. 5.5 5 5 0 9 o 0.090 6.8 5 4 1 4 1 0.285 7.50 2.15 8.8 5 0 5 0 6 0.875 Brine concentration, 4.4% __ 225° F. A 59.8 5 5 o 10 0 8.283 71.8 5 4 1 5 1 . 5 86. 5 1 4 1 5 0.750 83'00 24'26 104.8 5 0 5 0 10 0.916 250° F. 7*1 52.8 5 5 0 10 0 0.280 59.8 5 4 1 5 1 o. 50 48.8 5 1 4 1 5 0.750 45‘00 13'15 __58.8 5 o 5 0 10 0.916_ 255°F. ___ 17.8 5 5 o 6 0 0.125 21.8 5 1 4 1 4 0.714 21.00 6.14 __26.8 5 0 5 0 9 0.909 __1 240° F. 11.5 5 5 0 5 o 0.142 *" 14.5 5 o 5 0 5 0.857 12°30 3.74 __ _fi 245° F. _“ 6.8 5 5 0 5 o 0.142 __ 8.8 5 o 5 0 5 0.857 7'80 2'28 a. Data show only the results involved in calculations. TABLE XI Results of tests for thermal death rate of spores of BA 3679 in cheese spread at pH 7.00 (3.3 X 106 from each can subcultured in liver broth incubated at 37° C.)3 57 spores per TDT-can, 0.01 g. _— Brine concentration, 2.0% 2256_F. 5 0 5 0.142 0 5 0 ‘i 0.857 27'43 256U F. 5 5 0 6 0.125 5 1 4 1 0.714 16.57 5 0 5 0 0.909 _1 255° F. 5 5 o 5 0.142 5 0 5 o 0.857 8'71 240° F. 5 5 0 6 0.125 5 o 5 1 0.750 _ 5 1 4 1 0.855 4‘32 5 0 5 o 1 0.957 245° F. 5 5 0 5 0.142 5 0 5 o 0.857 2°38 Brine concentration, 4.4% 225° F. —~:: 5 5 0 8 o 0.100 5 5 2 5 2 0.428 51.45 5 0 5 o 7 0.888 250° F. 5 5 o 10 o 0.085 5 4 1 5 1 0.250 5 1 4 1 5 0.750 15‘°8 5 o 5 0 10 0.916 255° F. 5 5 0 15 o 0.066 ‘* 5 4 1 8 1 0.181 7 66 5 4 1 4 2 0.575 ‘ 5 0 5 o 7 0.888 240° F. 5 5 o 9 o 0.090 5 2 5 4 5 0.444 5 2 5 2 6 0.700 4°41 5 o 5 0 11 0.925 245° F. 5 0 6 0.125 ‘— 1 4 1 0.714 1.90 0 5 o 0.909 Data show only the results involved in calculations. .om.m mm as @mmsmm mmmmso CH mbmm p§o mommpmammp HFELmSB mom .oH magmas ......822an.... Ohm new 03.. nnm onm nmw o-._.o . . . . . . ...? nmd. uN III... 1 0.. 0 A w. n w m. .u I l 0.0. RON 60.34335... 2:5 . . . . _ _ odo. .onrn..a 9 5 .mm.© mm um GMmLQm mmmmno CH mwmm <0 00 non mad In mmsoam 00 mm>230 mocwpmammp HmEsmLB .HH maswflm .u. 6.3235: .u o .32anth 0mm MVN 0¢N onN 0mm ONN CNN mom 00m OVN OVN omN OmN ONN CNN—.0 . . a . . . ..0 . . _ . . _ we and. uNIIIIi 1 0.. we 0.5.0. uN ‘4 l 0.. a a m m w M m. m w w T J 0.0. I l 0.0. o .\. e... .8..§.§§6 2.4m 8 cu .8..§8686 2.4m . _ _ 4 _ . o.oo. _ _ _ _ _ o.oo. 6O .OO.N ma um Ummsam mmmmno CH mpwm am 00 mmpogm mo mm>250 womapmfimmh HmEamSB nnN ....o . 05.2an.... 0..“ new OVN nnm 0mm emu o-. fl « 1 1 ...? 00K. 5N Ill: o\. Q... 60:23.88 veto _ _ .0 0.. 0.0. 0.00. .unXN 'ugw ‘ onion 0 v.a nnN .... ...-5.235.» own new 03.. nnm on~ mum 0- 5 q . 4 . J “l0 nNNsIN J «a 0N 632.528 320 .ma mssmfim ..0 0.. mm ‘enlon a 0.0. 0.00. Effect of pH and brine concentration on D235, F335, for TDT-cans of cheese spread containing 3.3 I 10 PA 5679 per can (0.01 g. from each can subcultured in TABLE XII liver broth and incubated at 37° 0.) 61 and 2 values 5 spores of Brine concentration.wfi 2.0 4.4 pH 235,min. Ea§5,min. 2,6F. Dg§§,m1n. F335,m1n. 2, F. 5.50 4.6 24.2 18.25 4.5 25.7 19.25 6.25 6.8 35.8 18.75 7.0 36.8 18.25 7.00 8.4 44.2 17.75 8.1 42.6 17.00 8. F235 values representing commercial sterility calculated using Schmidt's equation (45). 62 the same at both brine concentrations. The average z value for all pH and brine concentrations is 18.20 F. The color curve in Figure 14 was obtained from a subjective evaluation of the processed cheese spread in the TDT—cans. TDT-cans of product that were subjected to a time-temperature process above the curve were unacceptably brown while those below the curve possessed an acceptable color. Effect of pH and Brine Concentration on the Heat Treatment Required to Prevent Growth of PA 3679 in Processed Cheese Spread The results of the effect of different heat treat- ments on the gas production of PA 3679 in processed cheese spread at several pH and brine concentrations, as measured by the eXpansion of TDT-cans are shown in TABLE XIII. End- point inhibition curves were plotted from these data. The F235 and 2 values, based on resistance studies of inoculated TDT-cans, are tabulated in TABLE XIV. The theoretical number of viable spores remaining in the canned processed cheese Spread following heat treat- ment may be calculated using equation 2. The calculated number of spores in the cans showing growth at the longest heating time and no growth at the shortest heating time, at each temperature, are tabulated in TABLE XV. Figure 13 shows the effect of the sterilizing value of the thermal process (FEED) on the eXpansion of TDT-cans , containing processed cheese spread inoculated with 3.3 X 106 63 TABLE XIII Effect of heat treatment (U) at different retort temperatures on the expansion of TDT—cans of cheese spread at several pH and brine concentrations (3. 3 X 106 spores of PA 3679 per can and incubated at 57° c. )3 ...2200 F. 22553- mi“. - M U EuLan.‘6 U £11ij U Eugen;l3 U Exm . pH 6.0 and a brine concentration of 1.6% 7.6 0.112 5.4 0.100 4.0 0.115 2.9 0.107 12.5 0.095 9.0 0.089 6.6 0.094 4.8 0.098 20.0 0.022 14.5 0.025 10.5 0.051 7.8 0.057 33.0 0.000 24.0 0.000 18.0 0.000 #13.0 0.000 pH 6.0 and a brine concentration of 2.4% 4.6 0.063 3.4 0.061 2.5 0.048 1.8 0.041 7.6 0.031 5.4 0.040 4.0 0.039 2.9 0.029 12.5 0.017 9.0 0.017 6.6 0.015 4.8 0.018 20.0 0.000 14.5 0.000 10.5 0.000 7.8 0.000 pH 7.0 and a brine concentration of 1.6% 12.5 0.094 9.0 0.103 6.6 0.108 4.8 0.104 20.0 0.073 14.5 0.067 10.5 0.087 7.8 0.087 33.0 0.030 24.0 0.015 18.0 0.036 13.0 0.041 55.0 0.000 40.0 0.000 29.0 0.000 21.0 0.000 pH 7.0 and a brine concentration of 2.4% 7.6 0.065 5.4 0.057 4.0 0.065 2.9 0.061‘ 12.5 0.045 9.0 0.044 6.6 0.047 4.8 0.044 20.0 0.026 14.5 0.027 10.5 0.017 7.8 0.026 33.0 0.000 24.0 0.000 18.0 0.000 13.0_ 0.000 a. Data show only the results involved in calculations. b. Can eXpansion in inches. 64 TABLE XIV Effect of pH and brine concentration on F235 and 2 values for TDT-cans of cheese spread containing 3.3 X 10 spores of PA 3679 per can (cans incubated at 37° C. and examined eriodically for swells a Brine concentration,j£ 1.6 2.4 _ApH Faggimin. 2:3F. _§g§§£min. z.°F. 6.0 7.8 37 4.6 37 7.0 13.0 37 ' 7.8 37 8. F2 5 values based on resistance studies of inocup laged TDT-cans. TABLE IV 65 Calculated number of spores in cans showing growth (+) at longest heating time and no growth (-) at shortest heating time, as influenced by pH, brine con- concentration, and processing temperature [g No. of spores in thousands _j I Brine concentration I _Temperature, °F. I 1.0% ] zggfl gj pH 6.0 - + - + 220 589 1290 1100 1620 225 316 795 759 1290 230 126 490 502 1020 235 43 246 246 661 DE 700 - 4 - + 220 389 914 912 1510 225 162 537 576 1100 230 48 235 234 693 235 2 74 85 372 Expansion, inches 66 m2 pH 6.0-brine cone. l.6% (MO 008 006 004 002 pH 7.0-brine cone. l.6% 0'00 Us ' D 5.12 3.09 5.72 8.55 o.I2_ 9" 5.0- btine cone. 2.4 ‘7. CHO- 008- 006- 004 002 pH 7.0- brine conc. 2.4% ' ""1350: '- ' 04.4.4.6 1.‘ 0'00 0.7 118 1.94 3.09 5J2 (J8 37 F 250 Figure 13. Effect of the thermal process (Eggo) on the expansion of DT-cans containing cheese spread inoculated with 3.3 X 10 spores of PA for 60 days. 3679 and incubated at 370 C. 67 Spores per can and incubated at 37° C. for 60 days. A graphical summary of the thermal death time, in- hibition, and color curves is sh0wn in Figure 14. The thermal death time curves are based upon the data in TABLE XII; the inhibition curves are based upon the data in TABLE XIV, and the color curve is based upon a subjective evalua- tion of the processed cheese Spread after heat treatment. 63 “”00 I i i i I i i I ~pH 20 neutral buffer '— / , ...—pH 7.0 brine 4.4% 8 //—pH 7.0 brine 2.0% Thermal ’/ . death time //—pH 6.25 brine 4.4% curves /—pH 6.25 brine 2.0% / ——-< / //—pH 5.5 brine 4.4 % / pH 5.5 brine 2.0 % IOOO .__J F value, min. 5 o - . J pH 7.0 brine 1.6%? 10+- pH 6.0 brine i.6% Inhibition ~~~ i pH 7.0 brine 2.4% curves \\ \ e—pH 6.0 brine 2.4% J: \ 0.: l 1 l l l J L A 220 225 230 235 240 245 250 255 260 255 Temperature, °F. Figure 14. Graphical summary showing thermal death time, inhibition, 0nd color curves. DISCUSSION Test Organism The suspension of PA 3679 possessed a D350 of 0.98 minutes with a 2 value of 17.50 F. The F250 value, based upon a lethality of five D values, is 4.9 minutes. Townsend 33 al. (59) stated that the ideal suspension of PA 3679 should have an F350 of 3.6 to 4.2 minutes but the F250 of most suspensions of this organism is 5.0 to 5.6 minutes. According to Sognefest (47), a process based upon a suspen- sion of PA 3679 with an F350 of 3.65 to 4.15 minutes should insure a commercially sterile product for low-acid foods. The 2 value of 17.50 F. for the Spore suspension agrees in general with the findings of many authorities (39, 45, 46, 47, 57, 59). Growth of PA 3679 in Processed Cheese Spread Effect 2; EElE.Ell§ powder. The results of the preliminary test shown in TABLE IV indicate a relationship between the amount of skim milk powder used in the produc- tion of processed cheese spread and the ability of PA 3679 to grow in the product. Profuse growth was obtained in the product containing 10.0 per cent skim milk powder while no growth was observed in the product containing 1.0 per cent skim milk powder. The difference between growth and no growth in the product cannot be attributed to an increase 69 70 in pH resulting from the addition of skim milk powder as reported by Van Slyke and Price (61); because, in the work reported herein, the processed cheese spread was adjusted to pH 6.0 prior to incubation regardless of the amount of skim milk powder used. There may be some factor associated with skim milk powder which stimulates the growth of PA 5679 in processed cheese spread. Further work should be done to elucidate this phenomenon. The Eighteenth Annual Report of the Council for Scientific and Industrial Research (4) states that the use of skim milk powder in processed cheese necessitates lower cooking temperatures, which fail to destroy the Spore form- ing spoilage organisms present. Cooking temperatures and times used in the production of processed cheese spread do not destroy spores responsible for putrefactive spoilage. If the TDT curve labeled I‘pH 6.25 brine 2.0 per cent" in Figure 14 is extrapolated to 160° F. (the temperature nor- mally attained in cooking processed cheese spreads), the F150 (commercial sterility) would be 450,000 minutes or _7,500 hours. These results confirm the conclusion of Csiszar (19) that spores of El, sporogenes, which produce huffed pasteurized cheese, were not destroyed by temperatures used to make a satisfactory product. Effect ngpg agglggigg concentration. A decrease in pH from 7.0 to 6.0 resulted in an increased lag time for growth and growth was completely inhibited at a pH of 5.6 or below. Most investigators (5, 35, 48, 55, 54) agree 71 that decreasing the pH of processed cheese increases the keeping quality. Hood and Smith (25) and Ritchie (41) re- ported that putrefactive Spoilage is completely eliminated at a pH of less than 5.4. The acidification of the proces- sed cheese spread used in this project to a pH of less than 5.7 resulted in an objectionably crumbly body and texture. End—point results in the form of either growth or no growth of PA 3679 in replicate samples of processed cheese spread may be correlated with expansion of TDT-cans of the inoculated product. It is impossible to correlate the amount of growth with the amount of gas production and the amount of gas production with the amount of TDT-can ex- pansion at different pH values because of variation in the solubility of gas in the product. As the pH of the product increases the solubility of the gas increases; therefore, if the same amount of gas was produced at two different pH levels, the TDT-can of product at the lowest pH would show the greatest eXpansion. Carbon dioxide is approximately ten times more soluble in water at pH 7 than in water at pH 6. The solubility of a gas is also a function of the pres- sure; more gas will be dissolved in the product at high pressures than at low pressures. TDT-cans with the greatest internal pressure have the largest amount of gas dissolved in the product. Correction for this phenomenon would make the differences in the amounts of eXpansion of different TDT-cans even greater than the actual measured differences. 72 The difference in expansion of cans observed at 6.0 compared to pH 7.0 (Figures 6, 7, and 8) cannot be used as a criterion of differences in gas production since differ- enes in expansion due to the greater solubility of gas in the product at pH 7.0 as compared to the solubility at pH 6.0 must be considered. Each increase in the brine concentration of the product resulted in an increased lag time, a decreased rate of gas production, and a decreased amount of gas production. Growth of PA 5679 was completely inhibited at a brine con- centration of 7.6 per cent or greater. The effect of brine concentration on gas production at a specified pH may be determined by the eXpansion of TDT-cans because salt has very little effect on the solubility of gas. The inhibi- tion of the growth of clostridium organisms by salt is widely recognized but very few comparisons can be made because most of the salt concentrations reported in the literature are recorded as the per cent in the whole sample rather than brine concentration. Effect of pH and Brine Concentration on the Thermal Resistance of PA 5679 in Processed Cheese Spread The results in TABLE XII indicate that D235 values decrease with reductions in the pH and that brine concen- tration does not affect the D235 values of PA 5679. There is a significant difference in the thermal resistance of spores at pH 7.0 compared to pH 5.5, as determined from the 73 95 per cent confidence limits. There appears to be a sign- ificant difference in the thermal resistance of Spores at pH 6.25 compared to 5.5. Differences in the thermal resis- tance of the spores between pH 6.25 and 7.0 are not statis- tically significant. There would probably be a significant difference in the thermal resistance of the spores at all three pH levels with a larger number of replications. Thermal resistance curves vary with the methodology used in obtaining them. The error involved in removing 0.01 g. of sample from each TDT-can for subculturing is estimated to be about plus or minus 20 per cent. Since lOgarithims are used in the calculations there would be practically no change in the calculated D values unless the subcultured samples were either at least ten times greater or ten times smaller than 0.01 g. In addition to the error in sample weight, errors due to spare distribution and the determination of the most probable numbers are inherent. The decrease in the thermal resistance of the Spores at reduced pH values verified the findings of other inves- tigators (8, 12, 13, 44, 47). However, Vas and Proszt (62) attributed the adequacy of a mild heat process for acid foods to the inhibition of Spore germination, rather than to the low heat resistance of the spores in the acid sub- strate. Since the samples in this study were subcultured in liver infusion broth after heat treatment, any subsequent inhibition of spare germination was removed; therefore, the decreased D values definitely were due to the decreased heat 74 resistance of the spores at the lower pH values. The lack of influence of brine concentration on the thermal resis- tance of the spores substantiated the results of Yesair and Cameron (68) who reported that the thermal resistance of spores of‘gl. botulinum was unaffected by different concen- trations of salt when the samples were subcultured in an Optimum growth medium. Salt did not show any protective influence in this study which is contrary to work reported by some other workers (21, 59, 65). The relative position of the color curve and the F curves in Figure 14 indicate that the thermal processes at higher temperatures for shorter times are preferable to low- temperature-long-time processes for producing a commercially sterile product with an acceptable color. Under conditions of this study the minimum temperatures that.could be used to commercially sterilize the processed cheese at pH 5.50, 6.25, and 7.00 and still maintain an acceptable color were 257, 24c, and 245° F. respectively. This is true for high- temperature-short-time sterilization and not true for ster- ilization in the container. The above results establish the necessity for using high temperatures for short times to obtain a commercially sterile processed cheese spread with an acceptable color. This conclusion agrees with the observation of Ball (6) who pointed out that, in prOportion to their destructive effect on bacteria, higher temperatures have less effect on quality impairment than lower temperatures. 75 The results of the work reported herein show that an F0 of approximately 5 minutes was required for commercial sterility, which is considerably less than an F0 of 90 min- utes reported by Meyer 21 al. (27). It would be difficult to control the process to obtain an F0 of approximately 5 minutes when using a temperature of 292° F. A 0.1 minute holding time at 292° F. would result in an so of approxi- mately 17 minutes which does not take into consideration the lethality accumulated during heating and cooling. Straight line thermal resistance curves (Figures 10, 11, and 12) are not routinely obtained when plotting data derived from subculturing the sample in a liquid medium. SOgnefest £3 3;. (47) were unable to obtain straight line thermal resistance curves from data obtained by subculturing vegetable purees after heat processing. Effect of pH and Brine Concentration on the Heat Treatment Required to Prevent Growth of PA 5679 in Processed Cheese Spread A comparison of the thermal death time and inhibi- tion curves in Figure 14 reveals that the F values for in- hibition are less, and the 2 values for inhibition are greater than the F and 2 values for thermal death time. The results agree generally with the results of Reynolds (40) who ob- served 2 values ranging from 14.5 to 27.50 F. for PA 5679 when the curves were based on the results of food substrate incubation tests. The results also agree with the observa- tions of Townsend gt'gl. (59) to the extent that F values 76 are lower and z values are different when the spores are incubated in a food substrate which is slightly inhibitory to growth, as compared to the incubation of the spores in an Optimum growth medium. This phenomenon is exPlained by the fact that a relatively large number Of spores may be necessary to ini- tiate growth in the inhibitory food substrate (see TABLE IV). The statistical number of spores necessary to get 50 per cent positive and 50 per cent negative samples is 0.69 when using an Optimum subculture medium. If the food substrate is inhibitory to the growth of the organism, the number of spores necessary to produce 50 per cent positive and 50 per cent negative samples may be several hundred-thousand. An examination of TABLE XV reveals that fewer spores are required to initiate growth in processed cheese spread at pH 7.0 and a brine concentration of 1.6 per cent than at pH 6.0 and a brine concentration of 2.4 per cent. Another interesting phenomenon is the fact that with an increase in the temperature of heat treatment the number of spores necessary to initiate growth in the product decreases. This may be related to the availability of nutrients. SUMMA RY The effect of pH and brine concentration was deter- mined On the growth, thermal destruction, and the heat treat- ment required to prevent growth of spores of PA 5679 of a known heat resistance in a processed cheese Spread. Deter- mination Of the thermal resistance of the spore suspension revealed a D250 of 0.98 minutes and a z of 17.50 F. The amount of skim milk powder used in the produc- tion of processed cheese spread had an effect upon the growth of the test organism in the product. When a 10.0 per cent concentration was used, profuse growth took place, .but when a 1.0 per cent concentration was used, no growth was Observed. The pH, brine concentration, and amount of Spore inoculum were the same under both conditions. A decrease in pH from 7.0 to 6.0 resulted in an in- creased lag time and growth was completely inhibited at a pH of 5.6 or below. The acidification of the processed cheese spread used in this project to a pH Of less than 5.7 resulted in an Objectionably crumbly body and texture. Each increase in the brine concentration of the .product resulted in an increased lag time, a decreased rate of gas production, and a decreased total amount of gas pro- duction. Growth was completely inhibited at a brine con- centration of 7.6 per cent or more. Heat penetration studies were conducted using TDT- cans of processed cheese spread and OOpper-constantan 77 78 thermocouples. It was found that the lag correction factor increased with each increase in the fill-weight and was greater when the TDT-can was processed while setting in the flat position than when it was processed setting on its edge. Studies conducted to determine the effect of the pH and brine concentration of the processed cheese Spread on the thermal process required to destroy the test organism in the product revealed that the pH had an effect, but that the brine concentration did not. The average D235 values of the test organism in the processed cheese spread at pH 5.50, 6.25, and 7.00 were 4.55, 6.90, and 8.25 minutes respectively. The 2 values remained constant at 18° F. A comparison of thermal death time and inhibition curves revealed that the difference in results stemmed from the difference in the number of survivors necessary to pro- duce 50 per cent positive and 50 per cent negative samples when calculating D values. It was also found that as the temperature of the heat treatment increased the minimum number of spores necessary to initiate spoilage in the pro- duct decreased. (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (ll) (12) (13) LITERATURE CIT ED Albus, W. R. and Ayers, S. H. 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