J P'ASTEURillNG AND THERMAL PROCES-SiNG HBGH MOESTURE HELD CORN Thais for the Degrees a5 M. 5. MICHIGAN STATE UNIVERSITY Harris M. Gi‘é‘lin €962 LIBRARY Michigan State University PASTEURIZING AND THERMAL PROCESSING HIGH MOISTURE FIELD CORN by Harrls M. Gitlln AN ABSTRACT Submitted to Mlchlgan State Unlverslty in partlal fulfillment of the requlrements for the degree of MASTER OF SCIENCE Department of Agricultural Engineering l962 Approved WM‘2M ABSTRACT PASTEURIZING AND THERMAL PROCESSING HIGH MOISTURE FIELD CORN Harris M. Gitlln The F- and z-values of high moisture field corn, based on tests with thermal death time cans, were 0.27 minutes and l7° respectively. In addition, corn was given pasteurizing treatments below 2l2°F and stored at 40° and 86°F. The treatment and storage temperature influenced the pH that developed in the grain. The results are described and Compared with previous work. The significance of ‘ngstgigi m Qgtujjggm is discussed and it is pointed out that the process determined above should not be used until control of this organism is developed. Some suggestions are made where the processes might be used after further investigation. ii PASTEURIZING AND THERMAL PROCESSING HIGH MOISTURE FIELD CORN By Harris M. Gltlin A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department Of Agricultural Engineering l962 SUMMARY Using thermal death time cans the F - value of the natural flora of shelled field corn in the moisture range 'Of 22% to 32% w.b. was found to be 0.27 minutes with a z-value of l7°F. These values are similar to some pre- viously reported for similar products at higher moisture contents. No determinations were made for the presence of Clgstgidium bgtulinum, but the hazard of its presence is emphasized in the literature. The F -»value obtained is below the minimum of 2.45 minutes absolutely necessary for anaerobic packaging and room temperature storage. Bac- terial counts were made, but not identification, and found to be as high as any reported in the literature for similar products. ~ Corn was also pasteurized at l70°F, I90°F, and 209°F for periods ranging from I to l6 minutes, aseptically canned in hermetically sealed Q pint Jars and stored at 40° 1 and 86°F. Most samples stored at 40°F produced no gas pressure and retained a fresh corn odor and appearance for the first 9 months. All treatments stored at 86°F produced gas pressure and had a pleasant ensiled odor at 9 months. The pH values of the corn stored at 86°F was generally above 4.5. It is certain that neither the F-value or the pas- teurlzing and storage process would completely destroy iv SUMMARY - CONT'D Clostriglgm botulinum. Since there is no assurance that Clostrigigm,bgtulinum is destroyed, and that toxin production could not occur, these processes are not recommended. Further investigations are suggested that might take advantage of the processes. ACKNOWLEDGMENTS The work described here was made possible through the help and efforts of many people. The author wishes to acknowledge particularly the help of Dr. Carl w. Hall, Agricultural Engineering Department, whose direction, advice, continued encouragement and suggestions were essential to the conduct of this investigation. The author is indebted to Dr. Irving J. Pflug, Food Science Department, who not only made the laboratory facilities, supplies and equipment available, but whose suggestions, counsel, and key questions have had an important bearing on this work. The writer wishes to express appreciation to Professor Donald J. Renwick, Mechanical Engineering Department, for the opportunity to discuss the many problems encountered. The discussions, and the suggestions made in them, aided in resolving the problems. Acknowledgment is extended to Dr. A. W. Farrall, Head of the Agricultural Engineering Department, for his confidence, encouragement, and support which was essential for the work. Encouragement and assistance were received from all members of the staff of the Agricultural Engineering vi ACKNOWLEDGMENTS - CONT'D Department, including the many other graduate students. The help and ideas provided by Mr. James Cawood and Mr. Glen Shiffer are recognized. The author wishes to express his appreciation to Dr. 0. w. Kaufmann, Department of Microbiology and Public Health, for his counsel and suggestions in the microbiological aspects of this work; and to recognize Andre Brillaud, graduate assistant, who made the count of microorganisms reported here. Anything written here would be inadequate to express appreciation to my wife Margaret and son Robert. They not only helped with the work but sacrificed so much that it might be accomplished. vii Section INTRODUCTI REVIEW OF LITERATURE A. B. C. D. E. F. EXPERIMENTAL PROCEDURE AND RESULTS Test Test Test Test Test Test Test Test DISCUSSION CONCLUSION ON Significant relationships Population of microorganisms Methods of reducing the number of microorganisms The importance of glgptridium bgtulinum TABLE OF CONTENTS Control of Enzymes . Chemical preservation . No. No. No. No. No. No. NO. NO. NOW-PU“) O 8 . OF RESULTS 8 SUGGESTIONS FOR FURTHER STUDY REFERENCES viii Page UIKNU ll 2| 22 28 29 30 30 3| 3| 32 32 35 Al 55 56 Section APPENDIX I Method of calculation APPENDIX 11 Details of all tests ix Page 60 63 LIST OF TABLES Table Page I. Results Of all tests . . . . . . . . . 42 2. Examination of pasteurized corn after two years . . . . . 47 3. Data for lethal rate curve, 250°F . . . . . 73 4. Data for lethal rate curve, 232°F . . . . . 74 5. Data for lethal rate curve, 2l4°F . . . . . 75 LIST OF FIGURES Figures I Page I & 2 Photograph of equipment . . . . . . . 2S 3 & 4 Photograph of equipment . . . . . . . 27 5 Thermal death time curve . . . . . . . 34 6 Heating curve, 250°F retort . . . . . . 49 Heating curve, 232°F retort . . . . . . 50 Heating curve, 2|4°F retort . . . . . . 5i Lethal rate curve, 250°F retort . . . . 52 0000-4 Lethal rate curve, 232°F retort . . . . 53 ll Lethal rate curve, 2l4°F retort . . . . 54 xi INTRODUCTION Shelled field corn (533 gala; L.) is conventionally stored in one of two ways: as high moisture corn in a silo, or as dry corn in a bin. It is desirable to commence corn harvest as soon as the corn is mature, and to harvest rapidly. Field losses increase steadily with delay in har- vesting after maturity. Ensiling corn is a way of satisfying the need for early, rapid harvest. As a feed, however, shelled corn silage has certain limitations. The product can be fed only on the farm, or nearby. it must be fed at a rate sufficient to prevent surface spoilage. Total feed value losses, expressed as heat energy losses in the ensiling process, may amount to l5$ or more of the original feed value (Barnett, l954). Accidental losses due to molding, surface spoilage, etc. are common. Drying corn for bin storage also has its problems. The cost of drying may well equal l0} of the value of the crop. .Drylng as rapidly as it is possible to harvest may cost even more in fixed equipment. A search for alternate methods of storing high moisture corn was instituted. Three modes of degradation of such a product were immediately apparent: (I) that due to microorganisms, (2) that due to enzymes. (3) and that due to natural oxidative processes, or other natural chemical processes. The conditions for high moisture corn storage, then, are similar to those encountered in the food industry, so the technology and technique of the food industry were applied to the problem. Various methods Of preserving crops are listed by Hall (l957). Among these methods are: (l) thermal pro- cessing, (2) freezing, (3) freeze drying, and dehydration and freeze drying, (4) the use of pH control, along with some other conditions, (5) additives to control osmotic pressure in the product, (6) antifungal and antibacterial agents, added as a gas, liquid, or solid, and (7) ionizing radiations. Rahn (l945) summarized the effects of many of these methods on microorganisms. While some of these methods may be satisfactory for microorganisms they are not also effective for enzyme destruction. The work reported here is concerned with item (l) thermal processing of field corn. REVIEW OF LITERATURE A. Significant Relationships. Microbiologists, food scientists, and others have developed methematical methods for evaluating the effect of heat on bacterial populations (Ball,l957,and Tischer, l954). Two parameters are used in describing the effect of heat, 2 and F. The 2 is related to QlO. Both F and z are func- tions of the specific microorganism and substrate. Also, the number of such organisms in the product, and the rate at which these numbers are reduced by the temperature used affect F. These parameters are related as follows: (I) F (1:252) F250 = T x lO 2 This expression relates the effect of heat treat- ment at any time and temperature to the equivalent time at 250°F. (2) LogIO fl = -:1 N. D This relates the number of microorganisms remaining after a process to the time of heating at some temperature T. The symbols used throughout this report are: (Ball l957) 3 B - The total elapsed time in which heat was applied to a product, minutes. Note that no temperature of the pro- duct is implied, although a temperature of the applied heating medium is usually also stated. 0 - The time required to reduce the population of a parti- cular microorganism to lOf of its original numbers at some specified temperature, minutes. This value is specific for a microorganism, the temperature of treat- ment, and the substrate, including pH. F - The time at 250‘F required to obtain 'commercial ster- ility', minutes. This condition is achieved when no further growth or development of microorganisms will occur under normal conditions of storage and handling. The F-as used in this report refers to the time of treatment at which no swelling was observed in the thermal death time cans, ans swelling was observed at any longer time. FQBOThe time of heat treatment at 250°F that would have the same lethal value as some other time, FT, would have at temperature T, minutes. rT- The lethal time of a process at some temperature T, minutes. N - The number of viable microorganisms of a specific type at any time. The number is commonly expressed as number per unit weight or volume. N°- The initial number of a specific microorganism that are . viable, commonly expressed as number per unit weight or volume. T - The temperature of a process, degrees F. 2' The slope of the thermal death time curve, expressed as degrees F required to reduce the time of an equivalent process to l0% of its original value. From equation (2) it will be seen that the number of microorganisms will never reach zero, but may get very small. When N is less than one its value is related to the proba- bility of finding a viable organism in any one container, or unit weight or volume. In commercial processing it is nec- essary to have this N-value extremely small. In the tests reported here only a relatively small number of containers were used. Based on these results the probability of find- ing a viable microorganism when a large amount of corn is processed may be higher than desirable. B. ngulatigns 21_Microorganism§. One of the first considerations in establishing a time and temperature for thermal processing of corn would be the numbers, type, and location of microorganisms common in corn. Christensen (l948) cites evidence of almost uni- versal occurrence of fungus mycelium in the outer layers of grains, with some evidence of growth into the endosperm and embryo. The deeper penetration of fungi is resisted by an inner membrane of the corn covering. A list of fungi found internally is included in the article, along with a descrip- tion of the types of microorganisms found on the corn sur- face. Mold counts made on corn meal varied from 3500 to 400,000 per gram. Bacterial counts on commercially available corn meal varied from 5,000 to 60,000 per gram, and from i to 600,000 per gram in freshly prepared corn meal. When moisture was submlnimum for growth the mold count declined with time as much as 90% in two months. Manns (l92l) was interested in fungi causing corn damage and carried inside seeds. His study required a sur- face disinfection of the grain for study. The point of the kernel contained the greatest infection. The pOint area included a segment of l/S to l/6 inches back from the point. Most fungi had gained entrance to the kernel only in the cavity under the cap at the point of the kernel, or had penetrated only short distances into the pericarp. Various numbers of corn kernels from many U. 8. states were checked in the test. The variation was large, but approximately 50$ of the kernels showed internal infection of one Or more fungi. Boruff (I938) made a study of bacterial populations of grains received at a distillery. An example of the data follows: Date Number of bacteria Treatment per gram I936 October l5,000 Kiln and natural dried November 35,000 Kiln and natural dried December 2,400,000 Kiln and natural dried l937 January 2,000,000 Kiln and natural dried February 300,000 Only kiln dried corn March 3,500,000 Only natural dried April l,500,000 Only natural dried May 900,000 Only natural dried June 200,000 Only natural dried The range of counts for June (l937) had a maximum count of 800,000 and a minimum of 30,000 with an average of 240,000 counts per gram. The effect of kiln drying on bac- terial counts was shown as follows: Type of Number of drying samples Bacteria/gram Moisture %,w.b. Natural 20 3,725,000 l7.3 Kiln l2 990,000 I4.5 The difference in count appears large. In terms of thermal processes required, however, the difference is less than one log cycle and hence the process time would be less than one D-value more for the larger population. The effect of damaged kernels on bacterial count in corn was also shown by Boruff: Material Count/gram Ratio of count no damage : damage damaged l8,560,000 no damage 40,000 i : 285 damaged 6,464,000 no damage 45,000 I : I39 Christensen (I948) made mold counts on l945 corn received at 5 midwest terminals. The results are summar- ized as follows: Max. moisture Number of Range of Average Grade content, % w.b. samples counts count I I4.0 9 0-48,000 l2,000 2 l5.5 l7 l450-522,000 96,000 3 l7.5 2| 0-I,479,000 262,000 4 20.0 l6 5,000-I.350,000 390,000 5 23.0 l8 25,000-2,270,000 940,000 Sample 39 0-4,375,000 830,000 James (I928) made a study of bacteria prevalent in sweet corn for canning. The initial bacterial count made on ears freshly cut in the field was about 30,000 organisms per kernel when grown on plates at 30°C, and_an average of l or- ganism per kernel when grown on plates at 55°C. Dilution counts made from corn as it passed through the cannery processes showed the following change: Number at 30°C Number at 55°C When sampled incubation~ lncubation~ original l35.000 per kernel 2 per kernel final 22,500,000 per kernel 2 per kernel James also reported the following observations and measurements: (I) 5A pre-heat of l85°F reduced the 30°C count to 40, but did not affect the 55°C count. . (2) Corn with husks was piled in a bin. In four days the temperature reached 5l°C in the corn. At 50°C there were 580,000 viable organisms per kernel in the corn. (3) Corn remaining in a farmer's wagon overnight in a rain reached a temperature of 55°C and had a 30°C plate 6 count of IO X lo per kernel. (4) Eighteen groups of microorganisms were identified and listed. The largest number were a subtilis type. Thir- teen of the l8 groups formed spores readily. Some of these groups were shown to be present in fresh corn, to multiply during storage, and to be unaffected by a pre-heat Of l85°F. (5) Thermal death point determinations were made on all 57 spore forming cultures, using only organisms surviving 240°F pre-treatment for I5 seconds. Suspensions of IO6 spores per cc. were tested at pH 7.0.. The heat treat- ment and survivor data were listed. In general, 2 to 3 minutes at 250°F were required to get negative re- sponses. ‘ It is evident that there is a wide variation in the numbers and types of microorganisms that may be encountered in corn samples. From equation (2) it can be seen that the time of treatment in a thermal process is some function of the population and type of microorganism, among other fac- tors. The variation in count and types of organisms found on corn led to a search for possible ways to reduce the pop- ulation to some minimum and more uniform value. 0. Mgthgds g1_reducing he numbe: gjlmicroogganismg. Thermal processing is a method of reducing the pop- ulation of microorganisms. The literature search was con- fined to methods that might lend themselves to a pre-treat- ment, and rapidly and inexpensively reduce the population count. A physical treatment, common to all food processing, is to wash the produce. Chemical means are limited to those materials that would not be toxic to livestock. Charlton (l935) made some observations on the germi- cidal efficiency of chloramine-T and calcium hypochlorite. The germicidal value was found to vary with the time, pH, iO temperature, and available chlorine. At a fixed pH and tem- perature the time for reduction vs. log of remaining popula- tion was nearly a straight line. These results would imply that the order of destruction of the population in chlorine is similar to that in heat. As the pH was changed from 6.0 to 7.5 the time for reduction to If of the original popula- tion varied from l0 to 70 hours. At a fixed pH of 8.7 the time required to reduce the population to l% of the original was rapidly reduced with increase in temperature. The fol- lowing table shows the germicidal effectiveness of calcium hypochlorite at 25°C on Bacillus metiens spores. PPM available chlorine Reaction pH Time to destroy 90% IOOO Il.3 64 minutes lOOO 7.3 less than 20 sec. IOO l0.4 70 minutes 20 8.2 5 minutes It was concluded that pH had the most significant effect on the time required to reduce the spore population. A rise of I8°F decreased killing time by about 82% if ini- tial pH was 6.0 and 7l.5% if initial pH was 8.7. Doubling the strength of chlorine, at initial pH and temperature, reduced killing time 40-60%. LaBree (I960) studied the effect of chlorine on spores of Bacillus coggulans. His primary interest was the effect of temperature on the action of chlorine. Innocu- Iations of l0,000 spores per ml. were treated at three pH values (4.5, 6.8, 7.8) using three concentrations of chlorine (5,l0,20 ppm) and four temperatures (l5,20,30,60°C). The data were reported as time to reach 90% reduction in popula- tion, or a reduction of one log cycle of the original popu- lation. The following data are taken from the work cited: Temp.°C Chlorine Reduction Time, in minutes at pH - PPM % 7.8 6.8 4.5 IS 20 90 I3.0 9.0 4.0 60 20 90 l.0 0.25 0.30 It was further demonstrated that very little loss of chlorine occurred at 60°C. It is evident that small concentrations of chlorine at elevated temperatures can reduce bacterial populations in a relatively short time; this is particularly true at the pH of 6.5 to 7.0 expected in water containing shelled corn. 0. The Importance 21,0lostridium botulinum. Experience in food processing has resulted in the identification of certain microorganisms whose D-value is high, and whose destruction is particularly important. The destruction of these microorganisms frequently determine the extent of a process. Clostridium botulinum commonly determines safe pro- cessing time for two major reasons: (I) the D-values are relatively high; (2) minute quantities of the toxin of this microorganism are fatal. A literature search was made to obtain some evidence of the effect of this microorganism on livestock, and its distribution in corn. Bergey's manual (l957) lists four types of l2 Clostridium botulinum. Two of the four, 8 and C, are con- sidered pathogens, while two, 0 and E, are pathogenic on injection only. Graham (l92l) established that cattle are variously susceptable to botulism. Some cattle die, some do poorly or become ill. Clostgidium bgtulinum was definitely located in silage used to feed cattle that had died or were doing poorly. The particular places where the organism was located in the silage were associated with scarcely visible mold or yeast. _ Tanner (I944) postulated that the development of Clostridium botulingm in acid foods may be due to pockets of alkaline areas caused by molds, as in silage. Clostridium botulinum was identified as the cause of 'duck sickness' by Shaw (I936). Thousands of wild ducks were dying in their usual swamp feeding grounds along their flyways. Later, Quortrup (l94l) identified potential toxin (9!, botulinum) producing areas in the western North Ameri- can duck marshes. The areas were identified by having prac- tically 0% oxygen in the water. If decaying vegetation was present in the water then Clostridium bgtuligum toxin could be present. It was noted that aerobic bacteria quickly destroyed ore-formed toxin. Later Quortrup (I943) described some ecological relations between Pgeudgmongs geruginosa and Clgstgidium botulinum type C. The oxygen consuming and alkali producing capacity of fig. aeruginosg were observed. A definite symbiotic relationship with Clostridium bgtulinum was demonstrated using broths and swamp weed substrates. l3 Eschericia cgli did not support Clostridium botulinum even though the oxygen content of the water was reduced. This observation was attributed to a reduction in the pH that was also caused by _E_. gm. An intensive survey of the distribution of Clostri- glgm,bgtullnum in all states of the United States and Canada was reported by Meyer (I922). In California there was evi- dence of the microorganism on fruits and vegetables, in man- ured and cultivated soils, in virgin soils, and deep in the earth uncovered by rock slides. The organism was also found in pastures, in hay, and in sewage. Clostgidium botulinum was found in every state in the United States, including Hawaii and Alaska. There was more frequent evidence of the organism in the western states than in the central states. Examples where Clostridium bgtulingm was found include: corn roots and leaves in Minnesota, silage from Michigan State University, and cornstalks in Louisiana and Mississippi. Michigan was listed as having a low spore index. In the total survey Clgstgidium bgtulinum was found in 7.5% of the cultures made from corn husks, leaves and stalks, compared to 20% of the cultures made from silage. Considering the distribution and lethal character of Clostridium botullngm (Dack, l943) a literature search was made for further information about the conditions re- quired for toxin production. It was reasoned that the pro- duction of toxin could be prohibited under storage condi- tions adequate for livestock feed where it could not be l4 done for human food. Studies of the metabolism of Clostridium botulinum in various media were made by Wagner, et al (l925). They found evidence that the production of gas by this organism depended upon crabohydrate utilization. Type 8 did not produce toxin in specific preparations using glucose, al- though this was not true for type A. Some interest was shown in the effect of the presence of air on the microorgan- ism. It appeared that the composition of the medium deter- mined the degree of anaerobiosis necessary for the growth of an obligate anaerobe. Lewis, et al, (l947) searched for media and environ- mental conditions for producing highly toxic cultures of Clostridium botulinum, type A. The optimum media consisted of corn steep liquor, glucose and powdered milk. The im- portance of pH was noted in this work. Production of toxin was reduced as pH dropped below neutral. It was also noted that those cultures which were shaken did not produce as much toxin as the undisturbed. The reduced production was attributed to air incorporated in the media by shaking. A schematic of events leading to final toxicity of culture filtrates of Clostridium bgtulinum was presented by Bonventre (I960). The effect of pH on growth of Clostgidium bgtuiigum was studied by Townsend, et al, (I954). No growth was ob- served below pH 4.7. In corn steep liquor there was growth at pH 4.98 but none at 4.77. and there was no toxin produced '5 at the latter pH. They also found that toxin would be pre- sent at a pH lower than that at which gas was formed. Sognefest, et al, (l948) determined the effect of pH on thermal process requirements of canned foods. Thermal death time cans were used in their tests and sweet corn was included in the foods tested. Selections of their data are included here: pH White corn Yellow corn F (min) 2 (°F) F (min) 2 (°F) 5.0 l.80 l8 0.45 l4 5-5 3.70 I? 3.30 I? 6.0 - 6o} 506 20 7090 20 605 - 608 4o40 '7 900 2' It should be noted that these data are not entirely clear on the exact conditios of the tests, including innocu- lation numbers. Spores of Clostridium botulinum were germinated by Wynne and Foster (l948) in atmospheres of natural gas and in air. There was a greater delay'in germination of the spores in air than in the natural gas, but germination took place in both environments. The lag period was described as: L = 0 Where: L - Is the lag period of the Log I spores germinated in air, hours. 0 - is a constant from 95 to lOl. I - is the number of spores per ml. in the innoculum. There is considerable evidence in literature to support the following observations: l6 l. Clostridium botulinum is toxic to livestock. 2. It is almost universally distributed. 3. Corn provides a good substrate for toxin production. 4. Toxin can be produced in the presence of some air. 5. Toxin production can be eliminated at low pH values. Thermal processing will destroy Clostgidium botulinum, and considerable study has been made on this subject for about forty years. Esty and Meyer (l922) reported determinations of the heat resistance of Clostridigm_botglinum. The following values were determined under optimum growth conditions: Time to destroy all growth in a fixed number of spores, minutes. Temperature, °c. 4 l20 33 llO 330 I00 Another part of this work involved counting the spores remaining after various times of treatment at l00°C. Although the concept of D-value had not been proposed at- this time, the logarithmic order of death was noted, as well as the relation between time and temperature of treatment, 2. Esty, et al, also reported that as much as IOO days of incubation at 35°C may be required before evidence of growth of the remaining spores will appear. One sample germinated 378 days after treatment when stored at 36-37°C. Esty pointed out the influence of concentration of microorganisms upon time required for destruction and I? found variations with the strain selected. It was further observed that a reduction in pH reduced the time required to destroy the organism. For example, there was very little difference in the time required to destroy spores in ripe olives at pH 7.93, corn at pH 6.35, and spinach at pH 5.05. In food juices with pH below 4.5, however, there was marked increase in destruction rate. According to the work of Townsend et al (I954) there may be some question whether it is a change in destruction rate or inhibition of growth. The interaction of substrate and pH might have been inferred from the work of Esty and Meyer. Tanner and McCrea (l923) reported that it took ten minutes at l20°C and pH 6.8-7.4 to destroy Clostridium ggtulinum: however, no count of the numbers was made. Further studies of the thermal death time of Cl 8- tridium bgtullnum spores by Dickson, et al, (I925) included a test using 37,000 tubes of spores and covered a period of 28 to 39 months. The problem of skips and delayed germina- tion was reported. Once case of germination of heat treated cultures was delayed 37 months. Townsend, et al, (l938) made extensive tests to determine the heat resistance of Clostridium botglinum. One object of the work was to locate some less toxic anaerobe that had similar thermal characteristics. Two types of Clostridium botulinum spores were used, type 62A and 2I3B, along with a putrefactlon anaerboe labeled PA 3679. Some of the data are included here because of their bearing on the I8 work reported. Product Organism Populagion Tem erature of treatment i0 5 5 C IlO‘C Surv ve Destroy Surv ve-Desboy min. min. min. min. “aw yellow 62A 75 40.0 45.0 4.0 5.0 oantam corn 2l3B 25 75.0 80.0 4.0 O.0 As a composite of both types of Clogtcigium bgtglinum the following values were reported: Substrate F, 2, Min. °F. P04buffer l.9 l7.6 Asparagus 0.39 l5.0 Spinach 0.70 l5.5 Peas 0.87 l3.6 Whole milk 0.55 l4.5 Retort temperatures for these tests included IOO, l05, ll0, ll5, and l20°C. Different numbers of organisms were involved in the above data. Examples of variations using the same number of organ- isms but different substrates are shown in the following data: Type organism Population Substrate F 2, Min. °F. 62A ID x lo6 Asparagus 0.l9 l4.3 Spinach 0.33 l5.l 20 x IO6 Peas 0.30 l3.5 Spinach l.05 l8.0 75 X l06 PO buffer 0.40 l6.5 ra corn I.l4 l8.5 canned corn l.79 20.8 '9 Type organism Population Substrate F 2 Min. °F. 2:33 l2 x :06 Peas 0.38 I3.4 Asparagus 0.27 l4.2 2 x l08 Spinach 0.68 l5.5 Peas - 2.l5 l8.2 25 x l06 Canned corn l.38 l7.2 Raw corn 0.90 l4.8 POABUffEI‘ 20 53 200 4 P048Uffer 0030 I308 Comparisons of composite F-and z-values for all runs and all suspensions are included in the selected values below: Substrate 62A 2l3B F 2 F 2 Min- °F. Min. °F. P0 buffer l.70 l6.4 2.00 l8.0 Asgaragus 0.39 l5.0 0.09 l5.0 Peas 0.30 l3.4 l.40 l5.6 Spinach 0.45 l4.7 0.50 l4.3 Values of F for Clgstridium botulinum in corn pro- ducts were reported by Tanner (l944), and include the selection below: Product pH F value or heating time in minutes l94°F. 203°F. 2l2°F. 22l°F. 230°F. 239°F. Hominy 6.95 600 495 345 I20 35 l0 Corn 6.45 555 465 255 l05 30 I5 The following information is taken from a study re- ported by Reed, et al (l95l) on organisms of significance in processing: 20 Population Time to destroy 99.999% of Type A Clostridium botulinum spores in corn at 2|2°F, minutes 2,500,000 78 l.750.000 37.2 l25,000 20.7 100,000 9.4 In the work an F-value for Clostridium botulinum in fresh corn was established as l.l2 minutes with a z of l7.7°F. Considerable variation in D-values was experienced, primarily due to erratic nature of recovery of viable spores near the end of the treatment. This irregularity reduced any utility of D-values in determining thermal death time curves. The erratic behavior was attributed to particular resistances or weaknesses In cultures or organisms used. Dack (l943) pointed out that some cultures of Clostridium botulinum produced gas and some did not: and that cattle are susceptable to these toxins. The four toxins listed were all destroyed at 80°C for I to 6 minutes, at 72°C for 2 to l8 minutes, and at 65°C for l0 to 85 minutes. was demonstrated by Evans (l960). Spores of Qiggtngng goutulinum, NCA 3679, and four other bacteria were stored for 40 months at 30°C in buffer solutions at pH of 5.0, 6.0, and 8.0. A pre-heat treatment of 85°C for IS minutes or l00°C for IO minutes was given a portion of the spores of each bacteria. In the case of Clostridium bgjulingm and NCA 3679. the viability of the spores was practically 2i unchanged over the storage period. Their capacity to sur- vive was not appreciably influenced by the pro-heat treat- ment. Spores of the other four bacteria varied greatly in their viability over the storage period. It is evident that considerable variation in F-and z-values may be expected in dealing with microorganisms. In the case of Clostridium botulinum it is reasonable that only maximum values determined could be accepted because of the hazard involved. E. Contgol 91,33gymes. Enzymes as well as microorganisms must be controlled to preserve a food or feed product. Sizer (l943) noted that inactivation of the majority of enzymes is marked at 50 - 60°C. Sumner and Somers (l947) indicate that nearly all enzymes are irreversibly destroyed by heating to 80°C. According to Blanck (I955) it is customary to pasteurlze all juices and acid food at temperatures not less than l90°F for 30 to 90 seconds to definitely eliminate enzymes as a source of deterioration. Processes at temperatures of 250°F and below have generally destroyed enzymes in the time designed for micro- organisms. Recently, high temperature-short time processing has resulted in difficulty with enzyme inactivation in the product due to the high z-value of enzymes. Guyer and Holmquist (I954) were interested in the regeneration of the enzyme peroxidase. Peroxidase was 22 chosen because it is easily identified and difficult to des- troy by heat. They concluded that the curve of destruction of peroxidase was flatter than that of most bacteria. F- values for peroxidase destruction were obtained, but the concept of z-values for enzymes was not presented. Farkas, et al (I956) determined that peroxidase in stock pea filtrate had an F-value of 6 minutes and a z-value of 48°F. Further work by Zoueil and Esselen (l959) in- cluded a study of the mechanism and rate of regeneration of peroxidase in green beans and turnips. The following table shows F-and z-values determined as sufficient to destroy peroxidase activity (a) immediately after treatment and (b) after storage up to l2 weeks. Product Immediately after After storage treatment 250°F. z, 250°F. 2, Min. °F. Min °F. Green beans 0.48 4l.0 3.0 47.0 Turnips l.8 23.0 Il.3 46.0 The concept of D-value as the time to destroy 90% of the peroxidase activity is shown in this work. F. Chemical preservgtion. Food and feed have been preserved by chemical addi- tives under conditions where Clostridium botglinum is not a hazard. Salt concentrations up to l0% have failed to prevent survival and growth of Clostridium bgtulinum according to Wyant and Normington (l920). The conclusion 23 of this study was that pH is the best method of control. Seven compounds were tested on sixteen common food spoilage fungi by Klis,et al, (l959). Myprozlne and rim- ocidin were found to be effective against all fungi at l0 ppm. Sorbic acid at 500 ppm was also effective on fungi in this test, but York and Vaughn (I955) had found that sorbic acid at l.0% did not destroy or inhibit growth of Clostridium botulinum. Gases are also used to control microorganisms. Lloyd and Thompson (I956) present a detailed account of the use of ethylene oxide in various combinations as a sterllant. Foods and surgical materials were sterilized with the gas. They found that the gas may be considered a bactericide, fungicide, viricide, and possibly a sporiclde, but it could not be called an inhibiting agent. 24 FIGURE I Type of jars and cans used in the tests.' Special 2. 3. 4. 5. Thermal death time can cover, and inverted can showing 'dimple' in the bottom. Thermal death time can with corn and water ready to close. A closed thermal death time can. A thermal death time can that has swelled, showing arrangement of Ames dial gage for measuring thickness. A half-pint jar of corn used in the pasteur- izing tests, showing the two-piece lid. FIGURE 2 equipment used in the pasteurizing tests. 2. 3. 4. 5. The screened cage, holding over 2% pints of corn. The handle of the cage showing the valve operating rods. The location of the.two valves that assured dumping the desired quantity in each jar. The sheet steel palette holding five-i-pint jars. The electromagnetic wand, with magnet resting on a jar lid: note location of switch at handle on other end. Type of jars and cans used in the tests. Fig. l. . .... V . . \ WA...4 . _ is. 1' Special equipment used in the pasteurizing jars. Fig. 2. 25 26 FIGURE 3 The retort and extension used in pasteurizing corn. 2. 3. 4. The lid of the retort extension showing glass window. The handle of the screened cage extending from the slot in the retort extension. The valve controlling live steam access to the retort extension. The upper portion of the retort. FIGURE 4 The miniature retorts used to process the TDT cans 2. 3. 4. 5. 6. 7. Miniature retort with packing gland for admission of thermocouple wires. To the right Is a petcock, in the retort cover, used to vent during processing. The 'qulck-on' steam supply valve. Valve to drain, also slightly opened during processing. The huick-on' cooling water supply valve. Above is the 'quick-on' retort top drain valve used to outlet water during cooling. Retort cover clamp. Fig. 3. The retort and extension used in pasteruizing corn. Fig. 4. The miniature retorts used to process the TDT cans. 27 EXPERIMENTAL PROCEDURE AND RESULTS The experimental work was largely exploratory in nature, each phase being planned after results of the pre- vious phase were known. The requirements for heat processing occupied most of the experimental work, and will be described in the order performed. A close approximation of an F250 and z-value for high moisture field corn was desired. The majority of tests were made with thermal death time (TOT) cans (Sognefest,et al, I944) using miniature retorts. A detailed description of each test is included in Appendix II. After each heat process the cans were incuba- ted at 86°F. Distinct swelling of the cans was taken as evidence that some microorganisms survived the process, and this was recorded as (x). No swelling of the cans was inter- preted as satisfactory destruction of the microorganisms, and was recorded as a (-). 'Distinct swelling' was inter- preted as 0.030 inches or more, while 'no swelling' was considered as less than 0.0l0 inches. Any swelling between 0.0l0 and 0.030 was recorded as (?). Sensory examination of swelling in this latter range gave no evidence of any microbiological activity. 'In most tests 8 grams of corn and 9% ml. of tap water were placed in each TDT can. Initial corn moisture was determined for each test and recorded as percent, wet 28 29 basis. In general, the moisture content varied between 22 to 32%. Several times during the series of tests, and at each temperature used, a TDT can was equipped with a thermo- couple and the temperature history recorded. These data were used to determine the equivalent time at any tempera-' ture, using the method described in Appendix I. The equip- ment used is shown in Figures l to 4. The combined results of all TDT can tests are shown on Table l. The results of the pasteurized tests are shown on Table 2. A brief description of each test by number is given to assist in an understanding of the Tables I and 2; the details are given in Appendix II. TEST NO. I: Whole kernel corn of the l959 crop was.immersed in water at the temperatures and times shown in Table 2. The corn was placed in half-pint jars for storage. Ten jars were filled at each temperature and time, five were stored at 40°F and five at 86°F. After 9 months of storage no mold was I found in the I30 jars treated, including the control jars that had no process. Very little change in color was ob- servable at this time. After 9 months storage one jar from each storage temperature was opened from the five that had been pas- teurized 8 minutes at l90°F. The corn from 40° storage had a good fresh corn odor, and a pH of approximately 6.0. The 3O corn stored at 86° had a good ensllage odor and a pH of 4.5. Lithmus paper was used to determine pH at this time. TEST NO. 2: Early indications from preliminary tests suggested that an F of 0.I minutes, or slightly greater, may be ade- quate if a z of l8° is assumed. This test was designed to get a wider range 0f values for estimating both F and 2. Whole kernel corn of the l959 crop and water was processed in TDT cans. In all the temperatures above 200°F the times shown in the Table l are calculated time at retOrt temperature based on a z-value of l7°F. Originally a z of l8° was assumed, but the times were changed when further data was available. At temperatures below 200° the times are actual times of immersion in water. - The results of this test Indicated that an F of o.l6 minutes and a z of l8° were the approximate values to use in planning future tests. In all tests to this date the corn, which had been stored at 0°F, and held for about 2 hours at room temperature before the test started. Thereafter, all corn under test was held for l8 hours at room temperature before test. TEST NO. 2: This test was made to check the approximate F-and z-values assumed from previous tests before planning more comprehensive tests. The influence of a longer holding 3i period could be observed by a brief test. The results in- dicated that F may be slightly higher than supposed from Test No. 3, and that 2 was certainly not higher than l8°F. TEST NO. 4: This test was made to avoid the possible errors in temperature determinations and their significance in the very small F-values assumed. In this test hot water was used and the times were large enough so that come-up time was not significant. This would also be a check on z-values assumed. It became evident that 2, F, or both would have to be altered. TEST N0. 5: Variations in the number of microorganisms present in a container will change the F required. The 8 grams of corn in each TOT can Included about 25 corn kernels. It was possible that extreme variations in the microbiological population occurred in any set of replicates. The object of this test was to create a more uniform distribution of surface microorganisms throughout the entire corn sample. Sufficient corn was placed in a jar for the entire test. The jar was then filled with the amount of water‘ needed for the test and the total shaken thoroughly for 5 minutes. The water was then drained off and placed in an- other jar. The two components, corn and water, were then added in measured amounts in the TDT cans. This procedure 37 evidence that toxin production is inhibited. In the present state of knowledge this latter process cannot be recommended. It was found that corn could be readily dried_after each of these processes, if desired. Corn taken from the TDT cans some weeks after a sterilizing process was readily dried and had a good appearance and odor. This was true also f0r pasteurized corn stored at 40°F. However, the pasteurized corn stored at 86°F retained an odor, after drying, that was unpleasant. . . There are several harvesting and storage systems wherein the results of these tests may be utilized. The process time is short, far shorter than the time required to dry corn at 20 to 30% moisture. It may be preferable to process wet corn as fast as the harvestcan proceed, rather than dry it. The product can then be fed in its moist state when desired, but should not get into commercial channels. If normal storage was desired the corn could be dried later at a slower and more efficient rate without fear of spoilage in the meantime. According to the I954 Census of Agriculture, over 60% of the corn produced in the U.S.A. did not get into commercial channels. The corn could be pasteurized and maintained at 40°F, or lower. This type of process could be one that might economically precede a slower, more efficient drying operation: in the meantime harvest could continue at a rapid rate since pasteurizing would not take much time. 32 was presumed to create greater uniformity in the distributkm of the microbiological population in each can. The test indicated no advantage for the attempts at uniformity, and indicated that F may be somewhat above 0.l6 minutes If 2 W38 '8‘. TEST N9. 6: The I960 corn crop was being harvested and It was desired to compare the fresh crop with the frozen l959 corn that had been previously used. The sample of I960 corn in this test had been kept at 40°F for several days. The pro- cedure used was the same as that of the previous test except that the water treatment was not repeated. The test showed that the F of I960 corn was about the same as that of the l959 corn. TEST NO. 1: Bacterial counts, but not identification, were made on the l959 and I960 corn samples at the Department of Microbiology and Public Health with the following results: a. After l8 hours at room temperature, non-heat shock count: l959 corn 65 x l06 organisms per gram I960 corn l3 x lo6 organisms per gram b. From the same originally frozen sample, after l8 hours at room temperature: non-heat shock count (regeat) l959 corn 34 x l0 per gram heat shocked (l0 min. at6I00°C) l959 corn l6 x lo per gram 33 In the magnitude of IO6 counts the plate counts re- ported here may vary as much as one log cycle without much significance. Test No. 7 was made from corn samples in the same storage bag, and processed at 232°F after l8 hours at room temperature. The results indicated little difference in F between l959 and I960 corn. TEST NO. §: The test was conducted primarily to see if a re- peat of test No. 7 could be made. The data shows that a higher F-value would have to be used. The data of this test are used as the critical point in establishing the line for the thermal death time curve in Figure 5. From Figure 5 a z-value of l7°F and F- of 0.27 minutes was established. Figures 6, 7, and 8-show the heating curves for water and corn In the TDT cans at retort temperatures of 2l4°, 232°, and 250°F respectively. Figures 9, l0, and II are the lethal rate curves from which actual t-values were determined for the 2l4°, 232°, and 250°, respectively. The basis for plotting and reading these curves is explained in Appendix I. MINUTES TIME 4O 3O -' 20" -‘ iO—- _ 5" _. L0-- .2 L _. (l5h- _- s-so- i- — - B-TESTNO. 8-60-i- 59-CORN YEAR 0., I l l I 230 250 ZIO TEMPERATURE - ° F FIGURE 5 Thermal death time curve for field corn 34 DISCUSSION OF RESULTS In view of the logarithmic order of death of micro- organisms it is clear that variations in the populations in a can will vary the time for destruction of this population. In the usual manner of performing TDT tests a sterile medium is innoculated with a known number of knowm microorganisms. By contrast, the corn used in this test had a heterogeneous population of unknown numbers. Each type of microorganism had a population of its own, with and F-and z-value. The last organisms to be destroyed were not necessarily of the type with the highest F or the highest number, but the one with the highest combination of N,, F, and 2. It was noted that the colonies counted in the spore count all seemed to be one type of organism. It is possible that the F-and z-values shown here are due to some one organism, and to the one in the greatest numbers. In the literature cited previously there were cases where the F-and z-values for Qigstridium bgtulinum were similar to the F- and z-values for corn found in the tests reported here. There were also cases of much higher F-values determined for Clostridium botulinum. Townsend, et al, (I938) place a minimum F- of 2.45 minutes as abso- lutely necessary for anaerobic packaging and room tempera- ture storage of products with a pH near 7.0. An F- of 4 to 35 36 5 minutes is more commonly accepted in order to be safe. This latter value should certainly be considered minimum for processing field corn. There are microorganisms that would require a higher F-value and could be encountered in cOrn. Specifically, the flat-sour organism with high heat resistance such as NCA l5l8. Vetter, et al, (l957) Injected steam at 268°F into sweet corn directly. The corn was in No. 2 cans (307 x 409). The cans had been innoculated with 5 x I06 spores of NCA No. l5l8 per can. An F250 of l6.4 minutes was In- sufficient to stop acid production while 26.l minutes stopped all acid production. The temperature inside a corn kernel was also measured. The kernel interior reached retort temperature in about 45 seconds, while surface mea- surements throughout the can (4 places) all took 25 seconds to reach this temperature. The lag between surface and kernel Interior temperatures is comparable to the results reported here (see Figure 8). The tests described in this report demonstrate that high moisture field corn may be preserved by thermal pro- cessing. Two procedures for processing are demonstrated, sterilizing and pasteurizing. Both of the processes assume hermetic sealing. Pasteurizing assumes aseptic packaging. Sterilizing results In a product that will keep at room temperatures while pasteurizing will not prevent reduction in pH even at 40°F, in 2 years. The latter case it is certain that Clostrigium bgtullnum is not destroyed, and no 38 Some form of pasteurizing, possibly along with innoculation with a desired organism, could be used to enhance the quality of normally enslled shelled corn. A mechanism for pasteurizing is not difficult to visualize. A temperature near the boiling point of water and a holding time of 2 to 4 minutes is sufficient. The full process, 250°F for 5 minutes, followed by aseptic storage would be more difficult. The following are some of the general considerations that might apply to this problem: I. The heat energy requirements for processing would be considerably less than those required for drying. 2. The time for processing would be far less than that for drying. 3. Some techniques of food processing may apply to feed: 3 a. Wash the product before processing. b. Chlorinate at high temperatures before processing. It may be that feeds can be chlorinated at higher rates than foods. c. Inject steam directly into the corn for rapid processing. 0. Package aseptically in large packages of IOOO pounds or more. Pallet boxes lined with plastic might be used and the packages would require no special stor- age structure. 39 It is recognized that the presence of Clostridium Qgtulinum toxin is the major hazard in any of these pro- cesses. In this connection there is one small note in the literature that might bear further investigation. Quortrup and Holt (l94l) noted that aerobic bacteria quickly pre- formed toxin of Clostridium botulinum. Is it possible that in the handling of animal food some use may be made of this fact, and the problem of Clostridium botulinum.eliminated? It was found that the corn used in this test would not germinate after storage at 0°F before processing, which might be expected in high moisture corn. For that reason the effect of processing on the germinative ability of corn is not known. Full thermal processing would surely destroy the germ but some of the pasteurizing processes may not. An inspection of the pattern of spoilage in TDT cans gave rise to the following possiblity: The range of processes included treatments suf- ficient to destroy vegetative cells but not Sufficient to activate spores. Longer pro- cessing activated the spores but did not des- troy them. This would explain a reduction in can swelling, followed by an increase In percent of swells before reducing to no swells. To check this hypothesis a number of TDT cans were selected because they seemed to fall in the category of insufficient heat to activate the spores. These se- lected cans were treated at 2l2°F for l0 minutes and checked for swelling. None of the cans showed any swelling after this test, indicating that the hypothesis was 4O probably incorrect. The history of the cans selected for this test is given in Appendix II. CONCLUSIONS l. Shelled field corn in the moisture range of 22% to 32% may be preserved by thermal processing sufficent to destroy Clostridigm bot linum, followed by aseptic packaging and hermetic sealing. The F1 and z-values determined in this work are below the minimum recommended for Clostridium botulinum and hence cannot be used until some other means of controlling the organism is developed. 2. A pasteurizing process given high moisture field corn before hermetic sealing will result in a higher pH value of the final product, compared to no process. In general, lower temperatures of storage also result in higher pH values. Since pH values are generally above 4.5, there is a hazard of the presence of Clostgidium bgtulinum In the product. Pasteurizing may provide a desirable control over the ensiling of shelled corn, but further work will be required to establish the method. 4| 42 Table l. Experimental results after 9 months storage Corn Test Moisture, Temp., B F 0 No. Description _%g,b. °F. _Min. MIR. anditlon l. Pasteurized In 22.6 I70 2 - X X X X X i-pint jars 4 x X X X x and stored at 8 - X X X X x 86°F l6 - X X X X x I90 I - XXXXX 2 - XXXXX 4 - XXXXX 8 - XXXXX 2IO I - XXXXX 2 - X X X X X 4 - X X X X X 8 - XXXXX Stored at 40°F I70 2 - X X - - X 8 - ----- '6 - ----- 2 - ---x- 4 - ----- 8 - -‘--- 2 - ----- 4 - ----- 8 - ----- x can swelled more than 0.030 inches ? can swelled from 0.0l0 to 0.030 inches - can swelled less than 0.0l0 inches 43 Experimental results after 9 months storage - cont'd Table l. nditi n 0 Th. F M 8 Min. Temp., OF. w.b. Corn Moisture 32.0 1 tion Whole corn in water Descri Test N . 2. . .XT nesx- .TX- X.X? xxx- 2355 000.... 232 xxxxx XXXTX XX??X XXXTX xxxxx I6228 III-23h. 2|5 xxxxx X??xx xxxxx XXTX? xxxxx 00000 O O O O 0 50522 Iain/.3 I97 xxx xxx xxx xxx xxx l6.0 32. 48. ISO X.-? X... XX?- XTX? X.X. 0.05 0.I2 0.I? O.i7 32.0 250 Corn thawed l8 hours Whole corn in I959 crop water. 3. .- X? xx .x .- I.O i.25 232 XXX xxx xxx xox xxx 000 O O O 680 2I4 xxx XX. 9.th xx- 9.xx 80 I00 l9? 6O 44 Table l. Experimental results after 9 months storage - cont'd Corn Test Moisture, Temp., B F 50 N9. Description .% w.g: °F. Min. MT . Condition 40 Same as 30 F based on 2 of i8°. |96 l00 - X X X X X - I25 - X X X X X I50 - X X X X x 209 I9 - X X X - X 23.8 - - X X X X 280 - X °" '° "° - 3303 00.75 - - X - X 5. Whole corn in 23.8 232 l.0 - PX X x x x water. I959 WX X X x X crop. 2.0 .0l8 PX X X X X P-Normal process WX X X x X W-Washed to equal- ize organisms. 4.0 .l6 P- - X - - W- ? ? - - 6.0 .36 P- - 8.0 .56 P- - - - - I6.0 l.36 P- - - - - 20.0 l.76 P- - - - - 3000 2076 P- ' - - - 45 Table l. Corn Moisture, Temp., 0 g w.b. F. 30.0 232 Test N00 6. Description Whole crop in water I960 crop. 7. I959 corn 24.9 232 I960 corn 8 Min. 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 —o 0 -0 U'I-k #‘U UN [0" O O O O OU'I OU'I OUI GUI OUT OU‘I OU'I OUT OUT OUT UHF- k?‘ UN i0- its? 0.055 0.085 0.l29 0.l7l 0.205 0.249 0.293 0.337 0.002 0.009 0.027 0.055 0.085 0.l29 0.l7l 0.205 0.249 0.002 0.009 0.027 0.055 0.085 0.l29 0.I7i 0.205 0.249 Experimental results after 9 months storage - Cont'd anditiog ---X-X-XX- ------ ---X --X--?--?- --x-----7- not run XXXXXXXXXX -----XXXXX X?X---X-XX XXXXXXXXXX XXXXXXXXXX - x-------- ------ ‘2--- 46 Table l. Experimental results after 9 months storage - Cont'd Corn Test Moisture Temp., B F N2. Description 5_% w.b. °F. Min. M??? Condition 8 I959 corn 25.4 250 (Secs. 45 0.0i5 XXXX-XXXXX 55 0.035 -XXXXXXXXX 65 0.082 ??---?-??X 78 0.l75 ??X?-XX-?? 85 0.240 ----X-X-XX 99 0 0 40 ------- ‘2'- I20 0.68 ---------- l960 corn 26.5 250 Secs. 45 0.0I5 X?XXXXXXXX 55 0.035 XXXX?XXXX- 65 0.082 ---------- 78 0.l76 --x------- 85 o g 240 ---------- 99 0.40 -----?---- I I 0 O 0 55 ....... --'° I 20 0'0 68 ......... - l959 corn 25.4 2l4 Min. 7 0.05 X?X???X-X? i2 0.I0 ??--X--??- I 7 0 0 I 5 ---------- 27 0.25 -x--?----- 47 0.45 ---------- I960 corn 26.5 2l4 7 0.05 --X-----X- l2 0.l0 ?--------- I 7 o. '5 --------x- 27 0 0 25 ---------- 2.7 Table 2. Evaluation of pasteurized corn tests after two years of 86°F storage treatment Odor & aggearance lid pH _pH lid Odor & aggearance storage. Test no. & 40°F storage Sweet N l70°F Slightly sweet N 2 Min. Slightly butyric N l70°F Slightly sweet N 4 Min. Slightly sweet P l70°F Very slightly sweet P 8Min. ' Sweet N l70°F Slightly sweet N l6 Min. Sweet N l90°F Neutral V l Min. Slight acid V l90°F Slight acid V 2 MIn0 Ensiled V l90°F Good enslled odor V 4 Min. 0‘10!) (071(0) comm) (D110?) 011m) comm) 010:) N - No net pressure In jar P - Pressure in jar V - Vacuum in jar 5.l 5.6 .2 N Sweet,kernels dark 4.8 N Sweet,kernels dark 5.2 5.3 4.7 V Slightly,sweet,dan< 4.6 N Slightly,sweet,daik 5.l 5. 4.6 P Sweet,kernels dark 4.6 P Sweet,kernels dark 5.5 5.2 5.0 P Sweet,kernels dark 4.6 P Ensile,kernels dan< 5.3 5.6 5.l N Sweet & ensilepark 4.9 N Ensile,sl.butyric 5. 5.6 4.8 v Sl.ecid&flsh,dark 5.2 N Sl.ensile,some dk. 5.9 5-9 4.7 P Ensiled 4 8 P Ensiled Ensile - common odor of ensilage Acid - usually acetic Sweet - Similar to Butane or Ethanol 48 Table 2. Evaluation of pasteurized corn tests after two years of storage - Cont'd Test no. & 40°F storage 86°F storage ent Odor & earance lid pH pH lid Odor & appearance A Slight corn odor P 5.4 C l90°F Corn odor V 5.8 g F 8 Min. 5.0 P Ensiled H 4.9 P Ensiled A No odor v 5.8 B 2l0°F Slight corn odor V 6.0 F l Min. 5.0 P Slight acid G 4.6 P Acid,sl.butyric A Slight corn odor V 5.9 B 2l0°F Good corn odor V 5.4 F 2 Min. 5.3 N Top moldy,bpened) G 6.l V Cooked corn odor C Slight corn odor N 5.l D 2l0°F Slight acid ? 5.9 F 4 Min. 5.l P Strong corn &ensile G 5.0 N Strong corn &ensile B No odor V 6.2 C 2l0°F No odor V 6.l F 8 Min. 4.8 P Cooked corn G 5.3 P Cooked corn A Good ensile sl. dk. P 5.0 B Control Good ensile sl. dk. P 4.9 I 4.3 V Strong acid, dark J 4.3 N Acid,all kernels dark 249 245 IIIII 240 ~ I I 200 TEMPERATURE ‘ ° F llll I50- 50 I I l I I I 0 50 I00 I50 TIME . SECONDS FIGURE 6 Heating curve for thermal death time cans. Retort temperature - 250°F 49 °F TEMPERATURE ' 23l l 227 [Hill N N N I l82 illlll I32 32 l I I I l I I I l I O 50 IOO I50 2 00 250 TIME " SECONDS FIGURE 7 Heating curve for thermal death time cans. Retort temperature 232°F. 50 °F TEMPERATURE " 2|3 l I l I I I l / I I I '\ . \. , \ l l I 209. s 111111 jlilil .\ ' 204 I \ I l I l l \ I i64 IIIIII lllll \ Ii4 '4, I I l I l I l I 0 50 '00 I50 2 00' TIME ' 'SECONDS FIGURE 8 Heating curve for thermal death time cans. Retort temperature 2I4°F. 5i |.O 0.9 0.8 0.7 0.6‘ 0.5 TIME " MINUTES FIGURE 9 Lethal rate curve for 250° F retort temperature. Unit area is I minute at 250‘ F. 52 //// / q, ? O 04 / / ' ? é 0.3 élfl UNIT AREA ? 0.2 J? é “ //////// TIME ‘ MINUTES FIGURE 9 Lethal rate curve for 250°F retort temperature. Unit area Is I minute at 250°F. 52 \ \\\\\\ \ s \ \ m UNIT AREA OOI \ E s e \ 0. a) 0. Ix. C) (D O. o. 0. 0 o q- ro N ' X e.0I 3 FIGURE ll Lethal rat eeeeee f rrrrrrrrrrrrrrrrrrr of 2l4°F. Unit areasla 0.0l mlnutee at 2I4°F. TIME - MINUTES SUGGESTIONS FOR FUTURE WORK I. A measure of feeding value should be made along with any future work in thermal processing. 2. The value of pasteurizing corn before ensiling could be investigated as a possible aid to ensiling the pro- duct. This investigation could include: (i) the possibility of innoculation of the corn after ensiling to enhance proper biological action; (2) the process of adding an acid to create surface pH desired; (3) the possible aerobic destruc- tion of the toxin of Clgstridium botulinum if conditions would permit its formation. 3. A study of mechanizing thermal processing of corn, including aseptic packaging, if the process itself shows promise. 4. An investigation of processing could be made, including heat and particle irradiation. 5. A study of the thermal properties of corn is needed. 6. Investigations are being made of chemical means of preserving corn, and may provide a method of storing high moisture corn. 55 SELECTED REFERENCES Anderson, J. A., and Alcock, A. w. ‘ l954. Storage of Cereal Grains and their Products. t. aul, M nnesota: Amer. Assoc. Cerea Chemists. 5l5 pp. Ball, C. 0., and Olson, F. C. W. l957. Sterilization in Food Technolo . New York, N.Y.: McGraw-Hill Corp. 353 PP. Barnett, A. J. G. I954. Sila e Fermentation. London, England: Academic Press Inc. 203 pp. Blanck, F. C. I955. Handbook of Fgod gng Agricultuge. New York, N.Y.: e nhoid ub. Corp. 99 PP. Bonventre, P. F., and Kempe, L. L. I960. Physiology of toxin production by Clgstgldium ggtulinum types A and B, IV. Act vat on o the toxin. Jour. Bact. 79: 24-32. Boruff C. 8., Claasen, R. 1., and sotier, A. L. l93é. A study of the bacterial population of grains used in a distillery. Cereal Chemistry l5: #EI-A56e Breed, R. 8., BMurray. E. 6.0., and Smith, N. R. I957. Mgg x 8 Manual of Determinative Bacteriglogy. Baltimore, Md: The Wi liams and w k ns 00., 7th ed. Charlton, D. 8., and Levine, Max I935. Some observations on the germicidal efficiency of chloramine-T and calcium hypochlorite. Joure BaCte 30: '63-'7'0 Christensen, 0. B., and Gordon, D. R. I948. The mold flora of stored wheat and corn. Cereal Chemistry 25: 40-5l. Dack, G. M. I943. Fogd Poisoning. Chicago, Ill.: University of Chicago ress. 234 pp. 56 57 Dickson, E. C., Burke, Georgina 8., Beck, Dorothy, and Johnston, Jean l925. , Studies on thermal death time of spores of CI str dium bgtuligum. Jour. Infect. Dis. 3 3 2" 3e Esty, J. R., and Meyer, K. F. I922. The heat resistance of the spores of g, botulinus and allied anaerobes. Jour. Infect. Evans, F. R., and Curran H. R. I960. Influence of pre-heating and holding tempera- - ture upon viability of bacterial spores stored for long periods in buffer substrates. Jour. Bact. 79: 36%—368. Farkas’ D. F., GO'dbiith’ 8. A0. and PrOCtor, B. E. l956. Stopping storage off-flavors by curbing peroxidase. Food Engineering 28 (I): 52. Graham, R. 8., and Holmquist, J. w. I954. Enzyme regeneration in high temperature-short time sterilized canned foods. Food Tech. 8 (I2): 547'5500 HEII, c. w. I957. Drying Farm 0592 . Ann Arbor, Michigan: Edwards Brothers Inc., 336 pp. James, L. H. . l928. Bacteria prevalent in sweet corn canning. JOUI'. BaCto l3:l‘09-#'8e Klis, J. B., Witter, L. 0., and Ordal, Z.J. I959. The effect of several antifungal antibiotics on the growth of common food spoilage fungi. Food Tech. l3: l24-l28. LaBree, T. E., Fields, M. L., and Desrodler, N. w. I960. Effect of chlorine on spores of Bacillus ggagulan . Food Tech. l4 (l2): 632-334. Lewis K H., and Hill, E. V. I947. Practical media and control measures for pro- ducing highly toxic cultures of Clos%ridium botulinum, type A. Jour. Bact. 53: 2 3-230. Lioyd, R. 8., and Thompson, E. L. l956. Gaseous sterilization with ethylene oxide; a supplement to chapter 2|, Pgingiglgs and Methods of Sterilizatlgn. John J. Perk ns and Char es C. Thomas, publisher, Springfield, Ill. 58 Manns, T. F., and Adams, J. F. l92l. Prevalence and distribution of fungi internal of seed corn. Science 54:385-387. Meyer, K. F., and Dubovsky, Bertha J. I922. The distribution of B. botulinus in California. Jour. Infect. Diseases 3|:54I-555. Quortrup, E. R., and Holt, A. L. ' I94I. Detection of potential botulinus-toxin- producing areas in western duck marshes, with suggestions for control. Jour. Bact. 4I: 353-372- Quortrup, E. R., and Sudheimer, R. L. I943. Some exological relations of P8 ud man s aeruginosa to Clostridium b tuIin m, type C. Jour. Bact. 45: 55I-554. Rahn, Otto. l945. Physical methods of sterilization of micro- organisms. Bacteriological Reviews 9: I-47. Reed, J. M., Bohrer, 0. w., and Cameron, E. J. l95l. Spore destruction rate studies on organisms of significance in the processing of canned foods. Food Research I6: 383-408. Shaw, R. M., and Simpson, Gretta. I936. Clostridium botulinum type C In relation to duck sickness In the province of Alberta. JOUI". BaCte 32:79-88e ' Sizer Irwin w. l943. Effects of temperature on enzyme kinetics. Advances in Enzymology 3:35-62. Sognefest, P., and Benjamin, H. A. I944. Heating lag in thermal death time cans and tubes. Food Research 9 (3): 234-243. Sognefest, P., Hays, G. L., Wheaton, E., and Benjamin, J.A. 48. Effect of pH on thermal process requirements of canned foods. Food Research l3 (5):400-4l6. Sumner, J. 8., and Somers, F. G. I947. Chemistrygand Methods of Enz¥me§. New York, N. Y.: Academ c ress nc. 5 pp. Tanner, F. W.. and McCrea, F. D. I923. Clostridium bgtulinum IV. Resistance of spores to moist heat. Jour. Bact. 8: 269-276. 59 Tanner, F. W. I944. Microbiolo of Foods. Champaign, Ill: Garrard Press, 2nd edition, 768 pp. Tischer, R. G. and Hurwicz, H. I954. Thermal characteristics of bacterial populations. Food Research I9 (I): 80-9l. Townsend, C. T., Esty, J. R. and Baselt, F. C. I938. Heat resistance studies on spores of putre- factlve anaerobes in relation to determination of safe processes for canned foods. Food Research 3: 323-346. Townsend, C. T., Yee, Lois, and Mercer, W. A. I954. Inhibition of the growth of Clostridium gotulinum by acidification. Food Research 9: 53 ‘5420 Vetter, J. L., Nelson, A. 1., and Stelnberg, M. P. I957. Direct steam injection for high temperature- short time sterilization of whole kernel corn. Food Technology II (5): 27I-274. Wagner, E., and Meyer, K. F., and C.C. Dozier. I925. Studies on the metabolism of B. gotulinus in various media. Jour. Bact. IO: 32 - 2. Wyant, Z. N., and Normington, Ruth. I920. The influence of various chemical and physical agencies upon bacillus Botuligus and its spores. Jour. Bact. 5: 553-55 . Wynne E. 8., and Foster, J. W. l9 . Physiological studies on spore germination with special reference to Clostridium bgtglingm. I. Development of a quantitative method. Jour. Bact. 55: 6l-68. York, G. K., and Vaughn, R. H. I955. Resistance of Clostridium botuli um to sorbic acid. Food Research 20: 60- 5. Zoueil, M. E., and Esselen, W. B. l959. Thermal destruction rates and regeneration of peroxidase In reen beans and turnips. Food Research 24 (I : II9-l33. APPENDIX I Method of calculations For determination of lethality at different temper- atures reference is made to Bail (I957). A. When processed in hot water: Most of the process times were large so that come-up time was not considered significant since cooling time was also not counted. Thus the B-value was also the FTvalue reported. The total immersion time was used in the equation: F 1-250 F250, Equivalent time at 250°F = T'I'o z (3) Where T is the temperature of the water and 2 was orginally l8°F but later corrected for l7°F. B. When processed by steam: A trial run was made at the desired steam tem- perature using a TDT can containing a thermo- couple connected to a recording potentiometer. A table of equivalent times at 250’F was made for temperatures above 200°F and for times of 5 second duration, which was the cycling time of the temperature recorder. From each recorded temperature the equivalent time at 250' was 60 6i determined, using equation (3) above. A z-value is required for this purpose. In the original work a z of l8° was assumed. When a z of l7' was finally established the values used were corrected. In this manner it was possible to determine the actual retort time required to give the Interior of a TDT can any desired process based on time at 250°F. C. For lethal rate values: The heating curves, Figures 6, 7, and 8 were first determined. The method of getting these data is described in Appendix II. Tables were made up from the heating curves listing the temperatures at each l0 seconds of time. A calibration of the recording potentiometer indicated that approxi- mately Q degree should be added to all tempera- tures below 225 degrees F and l degree to all temperatures above this value. These corrected temperatures were used in the final plotting of the heating curves. Referring to equation (3), let 'Equivalent time at 250" be equal to F. Then 1F___ is the ratio: T - 250 5222, = E¥¥lvalent time at 25%“ = l0 2 FT me at emperature F The value of‘; was calculated from the heating curves for each l0 seconds. These data are shown on Tables 62 3, 4, and 5. The ratio E was plotted on the ordinate of rec- tangular coordinates against t on the abscissa, where t: FT. This was done in Figures 9, I0, and II. The area under these curves at any time Is : g' X t = F, or equivalent time at 250°F. The unit areas blocked out in each of the above figures represents the labeled number of minutes at 250° equivalent. A planlmeter can be used to measure the area under the curve for any heating time t, and the equivalent number of minutes at 250° immediately determined. The F-values shown in Table l, and plotted in the thermal death time curve, Figure 5, were determined in this manner. It should be noted that the F used in the explanation above is a simplification (In print) of F250 as defined in this report. Also, t is used for FT for the same reason. This simplification was made to conform with the explanation by Ball (l957). APPENDIX II TEST N9. l. l959 corn at 22.6% moisture w.b. This test Involved processing corn at temperatures below 2l2°F by direct immersion into hot water. The bulk corn, after immersion, was aseptically loaded Into half- pint jars and hermetically sealed. The process times were short enough so that this process may be called pasteurizing. A cage of l/8 inch hardware cloth was made with a volume slightly more than 5 half-pint jars. (Figure 2, No.l). The bottom of the cage was fitted with a funnel to fit over the jars, along with two sliding valves. (Figure 2, No.3).' The valves permitted selection of approximately one half pint from the bulk In the cage and trapping it between the two valves. This selected quantity was then dropped into a jar. The valves were operated from rods extending along the handle of the screened cage. (Figure 2, No. 2). The entire loaded cage was immersed In the water in a retort that was maintained at the desired temperature. Since the retort contained over 20 gallons of water there was no significant change in the temperature of the water when the charge was submerged. The cage was vigorously moved around during the immersion period. To approach aseptic conditions during filling of the jars a sheet steel extension of the retort was made, 63 64 extending vertically up from the retort, and fitted with a removable lid. (See Figure 3). A slot In the wall of this extension permitted the passage of the handle of the screened cage. A sheet steel palette was made to fit inside the extension, covering less than half of the surface area of the retort. This palette held the five jars. The removable lid of the extension permitted intro- duction of the palette and screened cage into the retort extension, with the handle of the cage fitting out through the slot. A glass in the lid, along with a light above the glass, provided visibility through the slot Into the exten- sion for filling the jars. A steam line and valve permitted the introduction of live steam into the extension during processing. The procedure began by autoclaving 5 jars and lids at 250°F for I5 minutes. The lids were placed askew on the jars during this process. When processed, the lids were placed over the jars by means of electromagnetic wand, touching only the top of the lids. The jars were then lo- cated in the palette and allplaced in the retort extension. The required amount of corn was weighed into the screened cage and immersed In the water of the retort for the desired time. While immersed, the cage was continuously stirred, and the steam was turned on in the extension. At the proper time the cage was pulled out of the water, the steam turned off in the extension, and loading of the jars accomplished. The loading operation took about I minute. 65 The electromagnetic wand was used to remove and replace the jar lids. All manipulation of the wand and the cage was accomplished from the handles of each projecting through the slot of the extension. When all 5 jars were loaded and covered the entire paletter with the jars was quickly removed from the extension and placed in water at 60°F. The jars remained In the water for at least one hour. The times and temperatures used are listed in the main body of this report. A repeat of this entire operation was made using thermocouples to determine corn surface tem- peratures. It was found that regardless of the treatment temperature, the corn temperatures were in the area of l50°F by the time the jars were all filled. After being placed In the cold water, cooling in the center of the jars proceeded at about 3 to 5 degrees per minute, at least down to l00°F. However, the outside kernels in the jar received the least process and cooled qulte rapidly. TE§T N0. 2, I959 corn, 26.2% moisture. Ten grams of corn and lo cc. water were used per can. Miniature retorts were used in temperatures above 2l2°F (Figure 4) and a hot water retort in temperatures be- low 2l2°F. A thermocouple in a TDT can with water only was used to determine corn surface temperatures. The thermocouple was first checked in distilled Ice water, using a Brown 66 Electronik recording potentiometer (A.E. 668). The times shown In Table l are based on equivalent time at the tem- perature shown, with correction for a l7°F as noted pre- viously. In all cases where the miniature retorts were used the temperature of the steam was determined from a mercury thermometer In the steam supply tank. The well of the ther- mometer was vented slightly. The automatic control was adjusted to maintain this steam pressure (related to the temperature desired) and permitted variations of fi’F or less. The retorts were vented for l0 seconds after the steam was first turned on, and then a very slow but contin- uous venting held. TEST N9, 3, I959 corn, 32$ moisture. An Instrumented TDT can was used, after checking the thermocouple at 0°F. Calculations again were based en 2 of l8°, and later corrected. The corn in this test had been at room temperature for l8 hours prior to the test. In previous tests the corn was allowed to thaw at room temperature for about 3 hours. Ten grams each of corn and water, were used per can. TEST N9. 4, I949 corn, 32% moisture. Corn remaining from the package used for test No. 3 above was placed at 0°F after the test. Five days later the same corn was thawed for 3% hours at room temperature and 67 used in this test. Eight grams of corn and I2 grams of water per can were used In this test. An extra 5 replicates at I96°F for IOO minutes was made using l0 grams of corn and ID cc. of water. All cans swelled, but the time for swelling was greater in the case of the ID grams of water. TEST N9. 5, I959 corn, 23.8% moisture. Eight grams of corn and I2 grams of water were placed in each TDT can. Ten cans, including 5 replicates of each of two conditions were processed at one time. Treatment labeled P (in Table I) was treated normally; treatment labeled W was shaken In water as described under the Procedure. The cans were processed in one stack, I0 high, alternating replicates. It should be noted here that the method of stacking in the retort and the method or reporting are related. In Table l the first can reported (reading from left to right) Is the can that was on top. The last can was on the bottom. This is the only test (no.5) where alternate stacking occurred. TEST N0. 6, I960 corn kept at 40°F for 5 days, 30% moisture. Eight grams of corn and lo grams of water were used In each can. An instrumented can was used following the test to determine the F value of the treatment in the method previously described. In all cases the ID replicates were stacked in the retort as described above. 68 I§§IMNQ;_1, l959 corn at 24.9% moisture and I960 corn at 25.4% moisture. Eight grams of corn and ID cc. of water were used in each can. The ten replicates each of l959 and I960 csrn were placed In two parallel stacks in the retort at the same time. Again, replicates ran from A to J In top to bottom order. The F values were based on a subsequent run with an Instrumented TDT can. Both I959 and l960 corn had been kept at 0°F. A period of l8 hours at room temperature was allowed before processing. As an additional factor in this test, l959 and I960 corn samples sufficient for l0 replicates each were soaked In water with 200 ppm chlorine at approximately 70°F. The water still gave Indication of approximately 200 ppm chlorine after the 3 minutes allowed for soaking. The corn was then washed twice before filling the TDT cans. Water fromthe second washing showed 25 ppm chlorine residual. This ppm was determined by indicating paper. The initial 200 ppm was based on 2 cc of Roman Cleanser to 500 cc water. Process time for the chlorine treated corn was at least as great as that for the untreated corn In this single test. The results of the chlorine treated corn are not shown In the results in Table I, primarily because the F-value of these replicates was uniformly greater than the normally treated. Since these results are distinctly con- trary to the literature they either represent an error or 69 the entire procedure should be more carefully Investigated. The investigation obviously required is not within the scope of the work at this time. Igor N0. 8, I959 corn at 25.4% moisture and I960 corn at 26.5% moisture. Test No. 7 provided data sufficient to assume an F-value in a range that indicated a z-value when related to previous data. This test was conducted to see if the re- sults could be repeated, and to use points (250' and 2l4°) such that z-value would be verified in the same test that an F was established. The procedure in this test was the same as that for No. 7, except that no further work was done with chlorine solutions. From Table I It will be seen that the results indicated a higher F than that of previous tests and a slightly different 2 (l7°). This was the last test made. Methgd of dgtermlglng tempergtures in TDT cgng. The methods described in the Individual tests above Involved the use of a single thermocouple located in the water in the can to determine the surface temperature his- tory of the corn. The method described here was used to get both surface and corn interior temperatures. The heating curves, Figures 6, 7, and 8 are based on the method described below. Two copper-Constantan thermocouples, 30 gauge wire, lacquer coated and fiber-glass covered were used in a TDT 70 can. The entrance to the can was made In a l/l6 inch hole at the bottom bend of the can. The fiber-glas Insulation was stripped off the wires from the point of entrance to the can on to the thermocouple itself. The entrance was sealed with Epocast IOF epoxy resin, about 2 cc. The can was filled with 6 grams of corn and sufficient water to reach the same level as the water in the normally filled cans. One of the two thermocouples was passed through a short piece of rubber tube, so that the contact area was miantalned in the water. The other thermocouple was in- serted In the center of a corn kernel and sealed with DuPont Duco cement. The leads of this couple were arranged to lie above the water in the can, while the leads to the couple in the water passed through the water. The stripped portion of the wires in the can were painted with resin to reduce the possibility of shorts. A twelve couple recording potentiometer (A.E. 2Il4) was used with the thermocouples. The leads were alternated so that all odd numbers recorded water temperatures and even numbers recorded kernel interior temperatures. The instrumented can was first checked at 32°F (distilled ice water) with a Leeds Northrup potentiometer (A.E. 2036X). Later the recording potentiometer was checked against the Leeds and Northrup potentiometer. Corrections were made to the data to account for the difference noted. For example, below 225°F, §° was added to each temperature noted, while above 225°F a l° increase was given in each 7i temperature noted. Description of the cocn uged in the tests: The corn used in these tests, l959 and I960, came from experimental hybrid plots of Michigan State University Agronomy Department. The plots were harvested with a field picker-sheller. The corn used was actually a mixture of an unknown number of hybrids. A field mixed sample of about 2 bushels was packaged In on-quart polyethylene bags and sealed. The bagging operation took place at room tempera- ture and lasted about 2 hours. Following bagging the corn was stored in a 0°F freezer. An exception was made In test No. 7 where fresh corn from I960 was kept at 40°F for 5 days before being used for a test. In the discussion it was noted that some TDT cans were later selected for further heat processing. A description of these cans and their process history is given below: Original Original Test treatment time of _Iig. °F tr_e_a_i._m_ggt Repl i caie 2 250 l0 sec. 0 2 250 ID sec. E 2 232 I2 sec. D 2 232 l8 sec. D 2 232 l min. D 2 232 I min. E 2 2I5 3.2 min. 0 Original Original Test treatment time of fNo. °F treatment Replicate 2 l97 l5 min. 2 I97 I5 min. 3 250 4.8 sec. D 3 232 48 sec. A 3 232 48 sec. B 3 2l4 8 min. 0 3 l97 60 min. C 3 I97 I00 min. D 4 209 I9 min. 0 6 232 ‘2.5 min. A 6 232 2.5 min. B 7 232 l.5 min. I 7 232 l.5 min. J 7 232 l.5 min. 0 7 232 l.5 min. D 7 232 2.0 min. A 7 232 2.0 min. l959 & I960 D 7 232 2.0 min. E 8 250 65 sec. l959 & I960 A 8 250 65 sec. I959 & I960 B 72 TABLE 3 Data for lethal rate curve - 250°F Time Temp. 250 - T __t__ __I-'_ Sec. Min. °F #250 - T 2 F t O 000 70. ' 05 '00' - - '0 00.67 '4905 ' 05 509 - - 20 0.333 I94.5 .5 3.27 I863. 0.000538 30 0.50 2I5. .0 2.06 IIS. 0.0087 0.667 228. O l.295 I9.72 0.0508 0.833 236.5 5 0.795 6.237 0.l6l l.00 24I 0 0.529 3.38I 0.296 l.l67 243.5 5 0.382 2.4I 0.4l5 l.333 246 0 0.235 l.7l8 0.582 I.50 247 0 0.l765 l.502 0.667 l.667 247.5 5 0.I47 l.403 0.7l4 l.833 248 0 0.ll75 l.3ll 0.763 2.00 248.5 5 0.0882 l.227 0.8l5 I30 2.l67 249 l.0 0.0588 I.l45 0.875 I40 2.333 249.2 0.8 0.047l l.ll5 0.900 l50 2.50 249.4 0.6 0.0353 l.085 0.92I I60 2.667 249.5 0.5 0.0294 l.070 0.935 I70 2.833 249.6 0.4 0.0235 l.056 0.943 I80 3.00 249.7 0.3 0.0I77 l.042 0.960 3.l67 249.75 0.25 0.0I47 l.035 0.966 3.333 249.82 0.I8 0.0I06 l.025 0.975 3.50 249.87 0.I3 0.0077 l.0l8 0.982 3.667 249.9 0.I0 0.0059 l.0l4 0.987 TABLE 4 Data for lethal rate curve - 232°F Time Temp. 250 - T __t_ _f__ flares MIN. .F £50 - T 4 F t 40 0.667 I94 56 3.30 I996. 0.000502 50 0.833 203 47 2.76 575.5 0.00l74 60 l.00 2l2 38 2.23 I69.9 0.00588 70 l.l67 2I8.5 3I.5 l.85 70.80 0.0l4l 80 l.333 22l.5 28.5 l.68 47.87 0.0208 90 l.50 223. 27. I.59 38.9I 0.0257 l00 l.667 224. 26. I.53 33.89 0.0294 IIO l.833 225.5 24.5 l.44 27.55 0.0363 I20 2.00 227. 23. l.35 22.39 0.0446 I30 2.l67 228.5 2l.5 l.26‘ l8.20 0.0549 I40 2.333 229. 2|. l.23 I6.99 0.0588 |50 2.50 229.5 20.5 l.2l I6.22 0.06I7 '60 20667 2300 200 '0'8 '50.4 0.0662 I70 2.833 230.5 I9.5 l.l5 I4.l3 0.0709 l80 3.00 23l. I9. l.l2 l3.l9 0.0758 I90 3.l67 23I.2 I8.8 I.I05 ‘ I2.74 0.0787 200 3.333 23l.35 l8.65 l.095 I2.45 0.0803 2I0 3.50 23I.5 l8.5 l.087 l2.22 0.0820 220 3.667 23I.6 l8.4 l.083 I2.II 0.0825 230 3.833 23l.67 I8.33 l.077 II.94 0.0840 240 4.00 23l.74 l8.26 l.073 II.83 0.0845 250 4.I57 23l.79 l8.2l l.07l II.78 0.0849 260 4.333 23I.83 I8.I7 l.068 II.70 0.0855 270 4.50 23I.87 I8.I3 l.066 II.64 0.0859 280 4.667 23I.90 I8.I0 l.064 II.59 0.0863 290 4.833 23I.92 I8.08 l.063 II.53 0.0867 300 5.00 23I.935 I8.065 l.062 II.5I 0.0869 74 §§§§§§ 855383 9‘95 TABLE 5 Data for lethal rate curve - 2l4°F. Time Temp. 250 - T _t_ .___ SJ_ Mine .Fe 2 0 - __E F t 30 0.50 l54.5 95.5 5.62 4I6900 0.0024 40 0.667 I70. 80.0 4.7l 5l200 0.0l95 50 0.833 l82.5 67.5 3.97 9333 0.l072 60 l.00 l9l.5 58.5 3.44 2754 0.3630 70 l.l67 I97.5 52.5 3.09 I230 0.8I30 80 l.333 20l.5 48.5 2.85 780 l.282 90 l.50 205. 45.0 2.64 436.5 2.290 IOO l.667 206.5 43.5 2.56 363.l 2.754 llO l.833 207.5 42.5 2.50 3I6.2 3.l63 I20 2.00 208.8 4l.2 2.43 269.2 3.7l4 I30 2.l67 209.8 40.2 2.365 23l.7 4.323 I40 2.333 2IO.5 39.5 2.33 2l3.8 4.689 l50 2.50 2ll 39.0 2.295 l97.2 5.07l I60 2.667 2ll.5 38.5 2.265 l84.l 5.422 I70 2.833 2l2.0 38.0 2.23 I69.8 5.893 l80 3.00 2l2.35 37.65 2.2l5 l64.l 6.093 I90 3.l67 2l2.6 37.40 2.l95 l56.7 6.382 200 3.333 2l2.85 37.l5 2.l80 l52.4 6.56l 2l0 3.50 2l3.05 36.95 2.l70 I47.9 6.780 220 3.667 2l3.25 36.75 2.l60 l44.5 6.920 230 3.833 2l3.35 36.65 2.l50 l4l.3 7.077 240 4.00 2l3.47 36.53 2.l45 l39.6 7.l73 250 4.l67 2l3.57 36.43 2.l43 , l39.0 7.l94 260 4.333 2l3.64 36.36 2.l38 I37.4 7.267 270 4.50 2l3.70 36.30 2.l35 I36.5 7.325 280 4.667 2l3.75 36.25 2.l32 I35.5 7.379 290 4.833 2l3.80 36.20 2.l30 l34.9 7.4I2 300 5.00 2l3.83- 36.l7 2.l27 l34.0 7.463 3l0 5.l67 2l3.86 36.l4 2.l26 l33.7 7.479 320 5.333 2l3.88 36.l2 2.l25 l33.4 7.496 330 5.50 2l3.9 36.l0 2.l23 l32.7 7.558 75 FIE GR. L33. IIIIIIII II 06 3 1293 0 l l Ill IIII II II IIll ||| Ill l l I‘ll l l l l i ll l l l l Ill l Ill l i l l l Illll