SPORE REMOVAL BY BACTOFUGATION AND ITS EFFECT ON ULTRA HKGH TEMPERATURE STERILIZATiON ON MILK Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY MANUEL .tOSE TORRES-ANJEL 1968 ‘ r LIBRARY *1!- Michigan State 5; ssssss 11 1111121191111 1111111111 1111 1111 11 1.1% awe r1 1 "QWWQD \¢:‘9a¢ ABSTRACT SPORE REMOVAL BY BACTOFUGATION AND ITS EFFECT ON ULTRA HIGH TEMPERATURE STERILIZATION OF MILK By Manuel José Torres—Anjel The removal of bacterial spores by bactofugation and the resulting effect on sterilization efficiency and milk spoilage were studied. Also, other means were used to attempt to reduce the resistance of spores to heat. Spores were cultured in a solid medium (Modified Fortified Nutrient Agar) after heat shock of the spore inoculum. The obtention of Bacillus subtilis, Bacillus cereus and Bacillus stearo- thermophilus spores by this method was very successful. Spore counts were performed by a modified agar plate technique and by a modified membrane filter technique. In both cases a standard methods agar specially modified for spores was used. Heat resistance of the most important organism in this investigation (B. subtilis Al) was studied (by fraction negative tests in a thermoresistometer. Similar results were obtained for reconstituted skim milk and autoclaved whole milk as substrates and the four different subculture media, dextrose-tryptone-starch broth, ultra-high temperature (UHT) sterilized milk, and aerobic and anaerobic litmus milk. A D121 value of 0.435 to 0.625 min and a 2 value in the ultra high temperature range of 121.1 to 143.3 C (250 to 290 F), of 12 C (21.5 F) were found for Manuel José Torres-Anjel B. subtilis A1. A D121 value of 0.010 min was found for B. cereus 7. Temperature—survivor curves for “.0 sec showed that changes in the temperature in the UHT range were numerically more significant than changes in initial population of spores in relation to spoilage probability. The higher the temperature the greater this effect. Heat shock of 80 C for 15 min did not stimulate a massive germination of B. subtilis Al in milk. A penicillin- penicillinase system technique was tried to determine counts of primary, non-germinated spores but without success. A commercial bactofuge was used for the spore removal trials. The removal of spores from milk with a single bactofugation was more effective at a flow rate of one— third compared to the normal capacity of the machine (~99.9 vs. ~98.0%). Single bactofugation at the slower flow rate gave approximately the same removal percentage as double bactofugation at the faster flow rate. More than two bactofugations were unnecessary. Milk losses in the sludge were approximately four times greater when the one-third flow rate was used compared to the normal rate. The sludge contained practically no fat. Milk temperature within the range of 71.1 to 82.2 C (160 to 180 F) was adequate for efficient removal of spores by bactofugation. When the temperature of sterilization by UHT was reduced to 132.2 C (270 F) and 137.8 C (280 F), with 2 initial populations of spores of >10 to >10u/ml prior to Manuel José Torres-Anjel bactofugation, spoilage was >90% for the non-bactofuged milk and <10% for the bactofuged milk. Thus a small reduction in the common UHT treatments is possible when the initial number of spores is substantially reduced by bactofugation, or at the same temperature the spoilage probability will decrease accordingly. SPORE REMOVAL BY BACTOFUGATION AND ITS EFFECT ON ULTRA HIGH TEMPERATURE STERILIZATION OF MILK By Manuel José Torres-Anjel A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of. MASTER OF SCIENCE Department of Food Science 1968 Copyright by MANUEL JOSE TORRES—ANJEL 1968 ii ACKNOWLEDGMENTS The author wishes to express his appreciation to his major Professor, Dr. T. I. Hedrick, for his patience and help throughout this study. Gratitude is expressed to Dr. L. G. Harmon and Dr. F. R. Peabody who also served on the Committee and for their suggestions in editing this manuscript. Thanks go to the Food and Dairy Microbiology group for allowing the use of their laboratory facilities and for their help: in particular Dr. L. G. Harmon and Dr. R. V. Lechowich and their students, Donald Wallace and Francis Webster. Also thanks are extended to DeLaval Separator Company, Poughkeepsie, New York, who kindly provided the bactofuge and the VTIS unit. This work would not have been possible without the help of Mrs. Carole Burke in the laboratory and Mr. Donald Hepfer and Mr. Victor Armitage in the University Dairy Plant. To them the author will always be owing gratitude for the personal interest they took in this study. Sincere appreciation is acknowledged to Mr. Octavio Mesa and Mr. Gonzalo Roa for their help in the use of the University CDC 3600 Computer. 111 To Amparo, naturally iv TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . iii DEDICATION . . . . . 1V LIST OF TABLES. . . vii LIST OF FIGURES . . . . . 1X INTRODUCTION . . . . . . 1 LITERATURE REVIEW. . . . . . . . 3 Bactofugation . . . . . . . . . . . 3 Bactofugation as a Process. . . . . . 3 Bactofugation of Market Milk . . . . . . 9 Bactofugation of Cheese Milk . . . . . . . l3 Bactofugation of Milk to be Dried . . . . . l6 Bactofugation as a Pre-sterilization Process. . 16 The Sludge . . . . . . . . . . l7 Thermoresistance and Germination. . . . . . 18 Thermoresistance and Ultra High Temperature (UHT) Treatment . . . . . . . . . . l8 Germination. . . . . . . . . . . . 21 EXPERIMENTAL PROCEDURES. . . . . . . . . . 23 Preparation of the Spore Suspension. . . . . . 23 The Organisms Used . . . . . . . . . . 23 Growing the Spores . . . . . . . . . . 2H Harvesting and Cleaning the Spores . . . 25 Examination of the Spores and Preparation .of Suspensions . . . . . . . . . . . 26 The Spore Counting Procedures. . . . . . 27 Agar Plate Count (APC) . . . . . . . . . 27 Membrane Filter Count (MFG) . . . . . 28 Thermoresistance and Germination. . . . . . . 30 The Thermoresistance Experiments. . . . . . 30 The Germination Experiments . . . . . . . 33 Plant Procedures . . . . . . . . . . . . 33 The Bactofuge . . . . . . . . . . . . 33 . The V T I S . . . . . . . 3A The Bactofugation Experiments. . . . . 35 The Bactofugation-Sterilization Experiments . . 38 Page Handling the Samples. . . . . . . . A0 The Statistical Analysis of Data and Calculations . . . . . . . . . . “1 RESULTS AND DISCUSSION . . . . . . . . . . . “3 SUMMARY AND CONCLUSIONS. . . . . . . . . . . 68 APPENDIX. 0 o o o o o o o o o o o o o o 71 LITERATURE CITED . . . . . . . . . . . . . 107 vi Table 10. ll. l2. 13. LIST OF TABLES Bactofuge and VTIS Operating conditions Statistical data on spore counts of milk for the bactofugation experiments . . . . Fortran IV program for the statistical and reduction-per cent calculations. . . Fortran IV program for the calculation of the sludge-—losses per cent . . Results of the trials performed on bactofuga- tion and bactofugation-sterilization Results of the fraction negative thermore— sistance tests for B; subtilis A suspended in reconstituted skim milk. . Results of the fraction negative tests for B. subtilis A suspended in autoclaved whole Elf—fkl.......... Counts of B. subtilis Al spores after treatment at 110 C (230 F) for different intervals of time . . . . . . . Counts of B. subtilis A spores, suspended in autoclaved whole milk, after treatment at 121.1 C (250 F) for different intervals of time. . . . . . . . . . . Counts of B. subtilis Al spores, suspended in reconstituted skim milk, after different temperatures for A. 0 sec . . . . . . Counts of B. subtilis A spores, suspended in autoclaved whole milk, after different temperatures for A. 0 sec . . Counts of B. subtilis spores after heat shock at_80C (I70 F)1 for 15 min, and incubated for different times . Spoilagecfi‘non-bactofuged and bactofuged, UHT treated milk after 8 weeks storage. vii Page 72 75 87 89 90 96 97 98 99 100 101 102 103 Table Page 1“. Spoilage ratio of milk sterilized by UHT at . mlh C. ntial population of spores >10 to >10 /ml 0 o o o o o o o o 10“ 15. Spoilage ratio of milk sterilized by UHT at [”132 C o o o O o o o o o o 105 16. Spoilage of milk sterilized by UHT at ~l38 C with initial spore population of >10u/ml. . 106 viii Figure l. 6. 10. ll. l2. 13. LIST OF FIGURES General pattern of the bactofugation experiments . . . . . . . General pattern of the experiments with bactofugation followed by sterilization. Mean counts of B; subtilis Al spores with one or two bactofugations at ~1800 kg/hr SP0 and spores counted with (trial 12A) and without (trial 123) cleaning the bactofuge bowl between BI and BII . . . . . . . Removal of B; subtilis A spores by bactofuga- tion. Flow rate was $5,400 kg/hr for sub- trial A and m1,800 kg/hr for B and C. The temperature of bactofugation was 71 C except for B11 in subtrial C (82 C) Removal of B. subtilis A spores when BI was at «1,860 kg/hr and All at «5,1100 kg/hr. Removal of B. cereus 7 spores when BI flow rate wasfivl,800 kg/hr and BII m5,U00 kg/hr (left graph). Subtrials A (right graph) were at the faster flow rate and B and C at the slower flow rate Heat activation of B; subtilis A spores at 1000 (212F) according to the data by Ridgway (60). . . . . . . . . Thermoresistance curve of B;_subtilis A1 at UHT in milk . . . . . Survivor curve for B; subtilis A1 at 1100 (230F). . . . . . . . . . . Survivor curve for B; subtilis A1 at 121.10 (250F) in milk . . . . . . Temperature-survivor curves for B. subtilis Al in milk at UHT treatments of_W.0 sec. Comparison in spoilage between bactofuged and non-bactofuged UHT treated milk ix Page 36 39 11 H6 “7 50 53 57 58 60 61 63 66 INTRODUCTION Sterile milk and sterile milk products are increasing in importance in areas where long shelf life under limited or no refrigeration is required. These types of products may contribute to the solution of protein and other nutrient scarcity problems, particularly of infants and children in developing countries. 0f the several sterilization processes, continuous sterilization at ultra high temperatures (UHT) for very short times coupled with aseptic packaging seems to have a very promising future. The high temperatures used in the process are principally to assure the destruction of bacterial spores. Only miner changes occur in nutritional properties during UHT sterilization of milk products. Sterilization temperatures are responsible for some of the undesirable changes in UHT sterilized‘milk." The flavor can be improved compared to pasteurized products. The objec- tional cooked flavor in UHT treated milk is reduced compared to retort sterilization but is not completely eliminated. This research was initiated to study means of removing bacterial spores from milk or reducing their inherent heat resistance. Special attention was given to the removal of spores from milk by bactofugation. Concurrently the influence of both standard and substandard UHT processing 1 was investigated. Bactofugation has been used in the removal of bacterial cells by several investigators, but literature on the specific application for the removal of spores is very limited. No literature was found on the effects of bactofugation as a pretreatment to sterilization. The results of these findings could apply to any milk sterilization process, or to any fluid milk product, fluid imitation product or other liquids subjected to steriliza- tion. Centrifugal removal of microorganisms has been called bacterial ultracentrifggation,'bacterifugation and Bagggf fugation."These'terms have been respected in the literature review. LITERATURE REVIEW Bactofugation Bactofugation gg’a Process Simonart and Debeer (63) reported on "ultracentrifu- gation" as a method to improve the microbiological quality of milk. They suggested that the difference in size between the colloidal particles of milk (maximum 200uu) and the bacterial cell (1 to 2p and more) is sufficient to separate the latter from the former by an adequately regulated centrifugal force. In the early stage of their experiments they used a Sharples high speed centrifuge with a stainless steel clarifying bowl 1H with an interior diameter of 4.“ cm. It was operated at 30,000 rpm. The capacity of the machine was 6 liters/hr. In the experiments they used milk with normal flora and artificially contaminated milk containing Streptococcus lactis, Escherichia coli, Micrococcus aureus, Proteus vulgaris, Pseudomonas fluorescens, Bacillus subtilis, and Bacillus mycoides. The authors concluded "in general the flagellated bacteria (B. fluorescens, B. vulgaris and B. ggli) are less easily eliminated than the non-flagellated bacteria, which, on the other hand, con- stituted the group that most easily agglutinates." The ideal centrifugal force was around 10,000 x g. For spores 3 the removal was >98% while for bacterial cells it was generally <90%. Simonart and his coworkers at the University of Louvain in Belgium continued to improve the laboratory process and to convert it into a commercial process (6H, 65, 67, 72, 73). Most of their findings were summarized by Simonart (62) in a lecture given at the Netherlands Institute of Dairy Research (N.I.Z.O.), Ede. The term supercentrifugation instead of ultracentri- fugation was used in this work to describe the semi- industrial centrifugation of milk (8,000 to 20,000 x g, “5 to 200 liters/hr) that promotes the removal of a high proportion of bacterial cells. But "the separating power of the centrifuge decreases after the bowl has been running for 15 to 20 min. However, when a hole of 0.35 mm is drilled in the side wall of the bowl, the separating power could be kept at a satisfactory level indefinitely." Simonart (62) described also the industrial centrifugal- pasteurization process (6,000 liters/hr, 9,000 x g) at 72 to 76 0 (161.6 to 168.8 F), which had an efficiency of about 91% in the removal of bacterial cells. He applied the term bacterifugation to this process. The "bacterifu— gation effect" (called bactofugation effect by the manufac- turers of the commercial equipment) according to his description, "gives, as a percentage of the bacterial popu— lation of the raw milk, the sum of the bacteria eliminated by the centrifugal force and those killed by the thermal treatment." A report from Russia on the removal of bacteria from milk by high speed centrifugation (M9) utilizing 12,000 to 1A,000 rpm and a throughput of 70 liters/hr showed at 30 to A0 0 (86 to 10“ F) 85.5% removal at the highest speed and 79% at the lowest. When the throughput was lowered, a maximum removal of 96.5% was obtained. A regular clarifier operating at 8,000 rpm removed A6% of the bacteria. The work by Surkov and Schmidt (79) was of interest since it was the only work available referring to the theoretical basis of bactofugation. They explain that the determining factor of the process is expressed by the equation: T = T2 (1) in which T stands for the time of the passing of the 1 liquid through the centrifuge rotor and T2 for the time needed for the sedimentation of particles (bacteria) in the rotor. From this equation may be derived the following formula (6): l = 9 (R2- r0) n (2) v 2pAw r mean 1 stands for the length of the centrifuge (cm) R stands for the inner radius of the rotor (m) rO stands for the inner radius of the liquid layer (m) V stands for the capacity of the centrifuge (m3/sec) n stands for the viscosity of the liquid (m2 /sec. ) p stands for the size of the sedimented particle (m) A stands for the difference in the density of the dispersed ghase and that of the dispersing medium (kg /m2) W stands for the angular velocity of the rotor rotation (r/sec) By grouping the construction, biological and regime factors, the equation (2) will read: 2 2 A Z [l x r mean] x [ p ] x [n x V x n ] — 0.0725 construction biological regime factors factors factors For milk the following relation of % and the temperature (0) is valid: A _ H - 0.29 t Then: 2 [l x r” 2 Jxo' 2] x [V x t x n g 0.25 me an Using the same centrifuge the construction factors remained unchanged. The biological factor was not regu- lated and was determined by the microflora of milk. The authors described the characteristics of size and shape of several microorganisms, and commented that the milk plasma containing microorganisms is not a mono component system, which levels down the summary values of the curves of bacteria distribution in the medium. These authors also commented very pertinently that bacteria are living beings and are in motion, the intensity of the latter depending on the conditions of the "bacteriofugal" process (tempera- ture for example). Another very interesting phase of their work was the use of two capacities (100% and 50%) in the tubular centrifuge. The speed (30,000 rpm) and the accel— eration (22,600 x g) remained the same. Bacteria were, in general, removed more efficiently at the lower capacity. Another of their observations was the formulation of the interdependence between the quantity of separated micro- organisms and the temperatures of milk during centrifugation: y = Kt + C in which y is the quantity of centrifuged microorganisms (in %). t stands for the temperature (0) K and 0 are coefficients depending on the capacity. These same authors, Surkov and Schmidt (78), using E. coli claimed that the percentage of separated micro— organisms rises with the increase of the concentration. When the content of microorganisms was changed by 10 times, the effect of bactofugation changed 4 to 5%. Panchenko (50) utilizing an ASG-lA laboratory clari— fier showed that an increase in the operating rate from 12,000 to 16,000 rpm caused the mean percentage of bacteria removed to rise from 62.4 to 92.4% (double processing). The acid value of the non—bactofuged milk (3.84 x 105 bacteriological count/ml) in 24 hr increased from 17.7°T to 22.5°T at 19 0 (66.2 F). In the case of bactofuged milk, it increased from l7.2°T to l8.7°T. Houran (27, 28) explained how the bactofuge utilized the difference in specific gravity and size between bacteria and the constituents of milk: Sp Gr Size Bacteria 1.07 - 1.13 0.5 — 8p Milk (Skim) 1.035 Casein particles 1.066 500 — 800uu Actually, as mentioned by Dahlstedt (11), the difference in specific gravity between skim milk and bacteria is less than the density difference between skim milk and milk fat (0.93). This small difference made separation diffi- cult and explains why the problem was not undertaken until specialized centrifuges and high speed centrifugation were developed. 'Moreno and Kosikowski (46) described the high efficiency of the process in removing specific pathogens (coagulase positive Staphylococcus aureus 98.5%, members of the Enterobacteriaceae 99.8%). Surkov and Dukochaev (76), utilizing a l3-disk centrifuge (30 liters/hr) studied the influence of reducing or increasing the outer diameter of the disks. Reduction resulted in increased butterfat content of the skim milk, impaired efficiency of clarification and removal of bacteria. Increasing the diameter to a limited extent, had none of these adverse effects. Surkov EE.§£° (77), utilizing a Volga separator and aqueous suspensions of Bacillus megaterium and B. lactis studied the separating effect of the peripheral area of the bowl of disk separa- tors and concluded that the concentration of microorganisms in the suspension was similar to the original at the bottom of the bowl and similar to that of the clarified effluent at the top. They utilized continuous sampling by welding four hollow needles at different levels into the wall of the bowl. Peripheral separation during bactofugation required continuous removal of the sludge layer. Bactofugation of Market Milk Dahlstedt (11) described the first commercially operated plant located outside Brussels. Operation was started in January, 1962, after 6 months of experimental trials. The milk processing procedure was as follows: "raw milk is preheated by regeneration in a plate heat exchanger and then passes through the pasteurizing section 10 and on to the two Alfa—Laval high speed centrifuges con— nected in series. After leaving these, the milk is homo— genized and led back to the heat exchanger where it passes through the regenerative section and the sections cooled by means of water and ice water respectively." The bactofugation plant has a capacity of 6,000 liters/hr (approx. 13,000 HL/hr). The results have shown (11): a) High removal of microorganisms (above 99.99%). The use of 75 C (167 F) heat assures the destruction of any remaining pathogens. b) The possibility of reducing the pasteurization heat treatment. The bactofugation temperature of 75 0 (167 F) is a sufficient heat treatment. c) Improved keeping quality (approximately doubled). d) Absolutely natural taste and flavor which is expected with the reduction in heat treatment and the lack of significant change in chemical composition of bactofuged milk. This was even more marked when condensed bactofuged milk was compared to conventional condensed milk. Similar plants exist in Mexico and France (39, 40). Simonart gB_aB. (68) reported on trials at 70 to 75 C (158 to 167 F), 6,000 liters/hr, 9,000 X g whereby double centrifugation was used. The removal of Streptococcus, Micrococcus, Microbacterium, Pseudomonas and coliforms ranged from 98.58 to 99.97% of the initial counts. ll Simonart 22 BB. (69) studied the bactofugation of summer milk and the flora changes in milk during the hot season. They described three basic operations in the industrial process: a) Preheating to 75 0 (167 F) b) Bactofugation at this temperature, and c) A second bactofugation at the same temperature. They studied the relative proportion of several genera of bacteria in the total count of raw milk, pasteur- ized, bactofuged and double bactofuged milk. They worked with milk of very low bacteriological grade (2.2 to 2.4 x 107 SPC/ml. The proportional number increased in the case of Microbacterium, Micrococcus and Lactobacillus and decreased in the case of Alcaligenes, also when Pseudomonas were mixed with coliforms. This difference reflected different proportional removal since the total number always decreased. Simonart SE §l° (70) described the bactofugation of milk in a commercial plant near Brussels. They used two different stains for microscopic counts, aniline—oil— methylene blue (AOM) and periodic acid-bisulfite-toluidine (PST), as well as plate counts. The following reductions in counts were observed: a) AOM test (cells that did and did not stain after heating) 99.25% 12 b) PST test 1) When using milk with initial counts < 2.0 x 107/ml 98.97% 2) When using milk with initial counts > 2.0 x 107/ml 99.37% Scarpari (61), in Italy, studied the inclusion of a bactofuge in the pasteurization cycle of market milk. In comparing milk that had been both bactofuged and pasteurized and milk which had been pasteurized only, the author reported no significant difference in acidity, taste and aroma, but the keeping quality of the former was "slightly superior" and the total bacterial count was lower. Reduction in total count from highly contaminated raw milk (1.62 x 107 bacteria/m1) was 94.8% when bactofuged at 40 0 (104 F) and 96% when bactofuged at 70 0 (158 F). The thermophilic count (ll7/ml in raw milk) was reduced by 88.9% at 70 C (158 F). The density of the sludge was 1.064 and 1.047 after bactofugation at 70 0 (158 F) and 40 0 (104 F), respectively. Langeveld and Galesloot (35) studied the influence of bactofugation on the keeping quality of pasteurized milk as well as on the occurrence of the "bitty cream" defect. The experiments comprised both homogenized (clarifixated) and nonhomogenized milk. They were mainly concerned with the elimination of Bacillus cereus spores from the milk. 0n the average, bactofugation reduced the number of B. cereus spores in milk by 98%. 13 They reported an improvement in the keeping quality of both clarifixated and non—clarifixated milk: at 20 to 21 0 (68 to 70 F), more than 15 hr for bottled milk and more than 20 hr for milk in plastic containers. The formation of "flecks" in the cream layer of nonhomogenized, pasteurized milk was reduced by bactofugation independent of the presence or absence of post pasteurization contamina— tion. Bactofugation of Cheese Milk Perhaps the most studied application of bactofugation of milk is as a pretreatment process in the manufacture of cheese. Simonart and Debeer (63) in their first study suggested it as one of the most prominent possible appli- cations of the process. In Poland, Jakubowsky (29), studied centrifugation of cheese milk using an ordinary separator (500 liters/hr, 7,500 rpm). Kaolin added to the milk assisted the removal of bacteria. Reductions of 75 to 99% of bacteria and 99% of bacterial spores, molds and yeasts were obtained. Improved‘eye formation of Trappist cheese and somewhat impaired renneting capacity of cheese milk were observed. Kosikowski and O'Sullivan (34) described the use of the process to treat low grade milk for the manufacture of Cheddar cheese. Reduction ranged from 95.8 to 99.8% for total counts (original counts 2.7 to 9.8 x 107/ml) and 7 94.1 to 99.3% for coliforms (original counts 5.6 x 10 to 14 1.10 x 106/m1). The composition of the cheese was not affected by bactofugation provided the sludge solids were pasteurized and reincorporated. The quality of the cheese was predominantly "atypical" in the case of the cheese made from non—bactofuged milk and always typical for the cheese made from the bactofuged milk. Syrjanen (80, 81, 82) described the application of bactofugation in the manufacture of several specific types of cheese. In 1963 (80) he studied the effect of the process on the number of bacteria and the properties of milk for cheese making. He reported a 70% removal of bacteria at 4,400 liters/hr, 9,000 x g and 7,000 rpm and an "entire removal" of spores with double bactofugation. No changes in buffer capacity, clotting time of milk, or acid formatiOn by starter bacteria were observed. In 1964 he studied its application in the manufacture of Edam cheese (81) and in the manufacture of Emmental cheese (82). For both types of cheese he obtained a reduction of 97 to 98% of Clostridium in the milk, a good reduction in coliforms (73.2%) and a better flavor. No changes in the manufacturing procedure were necessary. Peltola and Syrjanen (51) also investigated the application of bacto— fugation to milk for Emmental cheese-making. The effect was found to be insufficient to prevent butyric acid formation. The 10-fold reduction in spores of Clostridium left enough organisms to produce a "glaesler" defect in 15 most cheeses which suggested that the milk was highly con- taminated. Kosikowski and Fox (32, 33) studied the removal of B. coli and Aerobacter aerogenes organisms from Cheddar cheese milk by bactofugation. The milk was inoculated with B. coli and B. aerogenes and held overnight at 50 0 (122 F). Populations of coliforms prior to treatment ranged from 5 x 105 to 1.5 x 107. Both the non-bactofuged control and the milk to be bactofuged were heated to 55 0 (130 F). The control milk gave Cheddar cheese with an "unclean" flavor. In 1966 Simonart gB_a;. (71) described the effect of bactofugation on the flora of Gouda cheese. Similarly to Seranen's work, the Spectacular results obtained comparing cheese made with bactofuged and non-bactofuged milk were due mostly to the removal of Clostridia: 99.12% after single and 99.7% after double bactofugations. The bacto- fugation was after preheating at 78 0 (171 F). The non— bactofuged milk was pasteurized at 80 0 (176 F) prior to the cheese making. The starter, predominantly B. cremoris, was added after bactofugation or pasteurization. During the ripening of the cheese no undesirable flora changes occurred. In Sweden, Lodin (37) and Lodin EE.§l' (38) observed that the reduction in total counts with the use of bacto- fugation and pasteurization was 99.8% and 97%, respectively. The corresponding reduction in spore counts was 98% and 11%. 16 The reduction in the butyric acid bacteria was 96.0 to 99.1%. Texture problems in the cheese from bactofuged milk were negligible. Moller—Madsen (45) worked with a commer— cial bactofuge (9,400 to 10,900 x g) at 54 to 57 C (12.2 to 13.9 F) and obtained the following reductions: Total bacteria 84% Acid producers 86% Micrococci 95% Propionic acid bacteria 84% Coliforms 88% Lactobacilli 97% Anaerobic sporeformers 94% Rennet coagulation time for bactofuged, pasteurized milk were an average of 6 sec longer than for non-bactofuged milk. Bactofugation of Milk to be Dried Reduction of Bacillus cereus spores is of particular importance when producing certain types of milk powder used for baby foods (Made, 40). Made predicted other advantages of bactofugation in relation to the dry milk industry (40): a. Reduction of the total bacterial count, especially when producing low heat powder. b. Keeping the cell count (viable or non-viable) within tolerable limits. Bactofugation as a Pre- sterilization Process In early work Simonart and Debeer (63) mentioned the particular value of bactofugation for milks destined for l7 sterilization. Simonart SE il- (66) used milk inoculated with Bacillus subtilis and studied the effect of bactofuga- tion at 12,200 x g following preheating at 70 0 (159 F). They found that the removal was approximately five times greater for the spore formers than for the non-spore formers. They used centrifugal force of 12,200 x g for 4 to 5 sec. 31 The Sludge The centrifuge sludge contains a high number of microorganisms and, according to several authors, practically .J no fat. Kosikowski and O'Sullivan (34) and Kosikowski and V Fox (32), when working with cheese milk, found that the protein content of the sludge was 8 to 12% and Moreno and Kosikowski (46) found it to be 8%. These authors agreed that the sludge constituted 3.0% of the volume processed at each centrifugation or 6% after the double process. Scarpari (61) found the density of the sludge was 1.047 to 1.064, depending on the temperature of bactofugation (no G, 104 F or 70 c, 158 F). In the market milk industry it is advisable to discard the sludge from the first bactofugation while the second which has <10% of the bacterial content of the first may be returned to the raw milk for reprocessing. By this method the milk solid losses are cut in half (27). A recent patent by Alfa Laval AB (1) illustrates a system in which milk is heated to 72 0 (161 F) and fed continuously into a bactofuge; the bactofuged milk is then l8 pasteurized while the sludge is sterilized at 130 to 140 0 (266 to 284 F) for l to 4 sec and recombined with the milk. In the cheese industry the reincorporation of the sludge is more critical to reduce losses and to avoid changes in the fat-casein ratio which would affect the body of the cheese. Kosikowski and O'Sullivan (34) pasteurized the sludge and incorporated it back into the bactofuged milk. The original amount of protein in the milk was closely but never completely regained, probably because of losses in the bowl and sampling. Kosikowski and Fox (32) devised a method by which, prior to reintroduction, the sludge was treated by the hydrogen peroxide—catalase method to destroy coliform and other bacteria. Another possibility (28) is "to ignore it [the sludge] completely and substitute spray dry milk powder in the cheese vat in an amount equivalent in weight to the protein carried by the sludge." This leads to "the added expense of paying for the powder but it too produces a cheese with good body" (28). Thermoresistance and Germination Thermoresistance and Ultra High Temperature (UHT) Treatment The thermal destruction of microorganisms has been extensively studied. The excellent work by Pflug and Schmidt (54) thoroughly reviews the subject. The l9 thermoresistance of B. subtilis Al was studied at water boiling temperatures by Ridgeway (60) who first isolated the organism. He found a heat activation even after 30 min at 100 0. His data were plotted and the curve is shown in Figure 8. From the three B, subtilis strains isolated by Ridgeway (60) from sterilized milk, strain A was the only one that showed such an activation. Edwards 22.21: (15, 16) utilized this organism in their thermal inactiva- tion (15) and heat injury studies in skim milk (16) at UHT. They used a modification of the survivor curve method in a capillary tube system and in a UHT unit. This was a steam injection system of industrial capacity. They com— pared two culture media, with and without sodium dipicoli- nate (CNA and FNA respectively). They found 2 values of 8.9 0 (16 F) to 18 C (33 F) in the range from 113 0 (235 F) to 135 C (275 F) when utilizing FNA as a recovery medium. Lower z values were observed when the CNA medium was used: 6.7 0 (12 F) in the 113 C (235 F) to 127 C (260 F) range and 13 0 (24 F) in the 127 C (260 F) to 135 0 (275 F) range. .The latter medium gave higher D values at all temperatures. The operation of large scale UHT equipment does not facilitate the use of extended holding times. Therefore the temperature-survivor curve, a plot of the number of surviving spores against temperature, best illustrated the thermal inactivation of their system (15). 20 Little precedent for this graphical presentation could be found in the literature (8, 18, 19). This type of curve may become more meaningful znrd useful with the present trend toward UHT processing of milk (7, 59, 74). Studies on evaluation of UHT processing systems are abundant (7, 8, 18, 19, 59, 74). All were based on the evaluation of the h. sporicidal effect on spore populations in water or milk ‘1 and comprised both laboratory and plant trials. B. subtilis strains were the most used species of -1 microorganism for these UHT system tests. g In 1962 Arph and Hallstrom (4) described the vacu- therm instant sterilizer (VTIS) system as a "package plant" for the UHT treatment of milk. The direct steam injection system is used. Lindgren and Swartling (36) studied the sterilizing efficiency of the VTIS using B. subtilis and_Bacillus stearo— thermophilus strains. They found logarithmic reductions of >9 for B, subtilis and >7 for' B. stearothermophilus obtained at temperatures of 130 to 140 0 (266 to 284 F). The reduction was expressed in terms of sterilizing effi- ciency defined by Galesloot (21) as follows: initial spore count final spore count sterilizing efficiency = log Unfortunately, these authors and those following 1did not express heat resistance of organisms in terms of D and z values. 21 Thomé 22 ii: (83) published a work that covered the engineering, bacteriological, chemical, taste, enzymatic and nutritive aspects of the VTIS. Their work was con- ducted on a laboratory model and on a full scale model. Modifications during their work on the latter resulted in the VTIS commercial unit. The two principal changes in flavor noted were "cooked" and "chalk" due to steriliza- tion. No changes in color were observed. Germination The germination of spores has been studied by several authors. These studies have been based on changes that occur in the cell upon germination. Pulvertaft and Haynes (58) utilized changes in the microscopic properties of the cells, mainly the loss of refractability and the darkening of the spore as examined by phase contrast microscopy. Changes in the form and structure of the cell, which lead to changes in the optical density of the suspension of cells, have also been studied (22, 47). Other aspects of cell changes which have been studied include increase in stainability (55), the loss of spore components such as the release of dipicolinic acid (DPA) (85), and the reappearance of glucose oxidation (24, 41) and other metabolic activities. The loss of resistance to heat and chemical agents also has been a useful indication of the occurrence of germination. 22 The most used method for studying germination and the one that is easiest to apply is based on optical density (transmittance). It has been used in studying germination of spore formers related to milk (42) but because of the characteristics of milk this technique is not applicable for studies of germination in milk and similar substrates. The reduction of heat resistance has been successfully used as a spore germination index in milk (31, 43). Many substances have been studied as possible germina- tion agents. Glucose, L-alanine (and 18 other amino—acids), lactose, sucrose, pyruvate, succinate, fumarate, malate and phosphates, have been found to be effective germination triggers for B, subtilis (22, 23, 25, 56). No specific work on germination of B. subtilis Al was found. Of the physical conditions studied, heat shock is the most effective germination trigger for most bacterial spores, among them B. subtilis (l7). Penicillin acts by principally blocking peptoglycan synthesis in the cell wall of growing cells and thus does not affect resting bacterial cells (10, 26). Davis (12) utilized this property of penicillin to isolate auxotrophic mutants. EXPERIMENTAL PROCEDURES Preparation of the Spore Suspension The Organisms Used For most experiments B. subtilis A a strain provided 1, by Dr. Z. John Ordal of the Department of Food Science, University of Illinois, Urbana, was used. This was the .1 same as B. subtilis Type A, which was isolated from milk :J by Ridgeway (60).. This strain was selected because it was isolated from sterilized milk and showed the highest heat resistance in comparison with the other strains (B. subtilis B and 0) and the other species (Bacillus licheni- formis) which he studied. Some work has been done on the thermal inactivation and heat injury of this organism at UHT in skim milk (l5, 16). For several experiments Bacillus cereus 7 was also used. This strain was isolated and provided by Dr. E. M. Mikolajcik at Ohio State University (31, 42). For a few experiments B. stearothermophilus NCA 1518 was used. It was provided by Dr. D. H. Ashton from North Carolina State University who has studied its inhibition by milk components (5). 23 24 Growing the Sppres A general method and medium were devised that gave high yields of spores for the various strains used, includ- ing B. stearothermophilus. The medium was a modification of that described by Edwards 22.21- (15) and Kim and Naylor (30). As soon as the strains were received a spore crop was produced, cleaned as described below, and kept under refrigeration. After examination the first crop was labeled as the stock spore supply. Whenever a new spore suspension was needed, a few ml of the stock suspension were heat shocked at 80 0 (176 F) for 15 min for B, subtilis and B. cereus 120 C (248 F) for approximately 3 min for B, stearothermophilus. One ml of the heat shocked suspension was inoculated into a tube of dextrose tryptone starch broth* (Special Difco, Control 17.1041). Transfers (a loopful) were repeated every 4 to 6 hr into tubes of the same medium until heavy growth in 3 hr was observed. Four milliliter of this subculture were used as the inoculum into a 32 oz prescription bottle (GK-32, Armstrong) which contained a layer of Modified, Fortified Nutrient Agar (MFNA).** After >95% sporulation v.11“ ”fr-9‘ F f v ‘3" ‘ *Dextrose 10 g, tryptone 5 g, starch 5 g, bromcresol purple 0.04 g per liter. **Nutrient broth 8 g, bacto agar 20 g, yeast extract (Difco) 5 g, NaCl 8 g, Ca012-2H20 0.089 g, dextrose 0.10 g, MnSou (sol. 300 ppm) 30 ml, dist. H20 970 ml. 25 was attained (a maximum of 48 hr for B. subtilis Al and B. cereus 7, up to 1 week for B. stearothermophilus NCA 1518) the spores were washed from the agar by flooding the plates twice with 25 ml of chilled distilled water. Immediately after heat shocking, aliquots of 3 m1 of this suspension were used as inoculum for each of 10 to 30 I1 bottles containing MFNA. The use of 2% agar in this 2‘ medium, rather than the usual 1.5%, improved moisture retention during incubation at high temperature and facili— J tated washing of the spores from the agar surface (30). 5 After approximately 48 hr >95% sporulation was attained. Incubation temperatures were 45 C (113 F) for B. subtilis A 37 C (98.6 F) for B. cereus 7 and 55 C (131 F) for l, B. stearothermpphilus NCA 1518. Excellent growth of B. cereus 7 was attained also when incubated at 45 0 (113 F). Additional incubation for 24 hr and refrigeration of the culture bottles for 48 to 72 hr induced complete liberation of the spores from the remaining vegetative cell structures. Harvesting and 01eaning_the Spores To harvest the spore crop, each bottle was flooded with 25 m1 of chilled, sterile distilled water. The operation was repeated twice and the suSpension thus obtained was filtered through three "Rapid Flow" (Johnson and Johnson) milk filters, collected in 250 ml polystyrene centrifuge bottles, each containing No. 10 26 glass beads and a magnetic stirring bar. The suspension in the different bottles was concentrated by centrifugation under refrigeration (Sorvall RC-2 centrifuge, 16,300 x g using the 5.75—inches head, for 10 min), resuspended in about 20 ml of sterile distilled water, magnetically stirred for 10 min and all bottles pooled into two bottles. F] These two bottles were submitted to four consecutive I“ centrifugations (650 x g for the initial one and 1,465, . 2,520 and 4,080 x g for the three subsequent ones) for 20 E’ min each time (15). After each centrifugation the spore g pellets were resuspended in distilled water by vigorous magnetic stirring. After the final washing, the spore pellets were resuspended in 20 m1 of water, the content of the two bottles pooled together and the final suspension filtered (57) through two sterile lOu polypropylene membrane filters (Gelman Instrument 00., Ann Arbor, Mich.). Examination of the Spores and Preparation of Suspensions Microscopical examination (phase contrast) showed a clean spore suspension with no clumps and very few vegeta- tive cells. A microscopic count (Petroff-Hauser chamber) utilizing a dry 100x dark phase objective, and plate counts were performed on the suspension. The chamber counts were performed using a diluted suspension. The number of spores in 20 squares were counted and the average count per square used for the following calculation: 27 Average count/sq x 400 x 50 x 1,000 = spores/ml, since Area of square is 1/20 x 1/20 = 1/400 mm2, and Depth of square is 0.02 mm = 1/50 mm 1 ml = 1,000 mm3 If any dilution was necessary this result was multiplied by the factor of the dilution. The counts by the two methods differed by <10%. Sterile distilled water was added to adjust the concentration of suspension to approximately 8 x 108 spores/m1. The Spore Counting Procedures Agar Plate Count (APC) The procedures described by the twelfth edition of the Standard Methods for the Examination of Dairy Products (3) for both thermoduric and thermophilic bacteria were found inadequate because of the difficulty encountered in counting Spreaders and the lack of starch in the medium. This ingredient is of definite importance for the growth of spores. Olsen and Scott (48) postulated that starch inactivates inhibitory substances from the medium, and that unsaturated fatty acids are probably involved as inhibitors. This was confirmed by Wynne and Foster (86). Several approaches were tried to reduce the difficulty of Spreaders. Milk and milk dilutions were plated on a thin layer of hardened agar, and a second layer of agar 24 El 28 (approximately 10 ml) was added and mixed with the sub- strate. A third layer of agar (approximately 3 to 5 ml) was used on top to avoid spreading. The increase of agar to 2% in the medium helped to retain the moisture when high temperatures of incubation were used. The addition of 0.85% of salt decreased the spreading problem and increased the counts. To recognize colonies in plating low dilutions of milk bromcresol purple was added. It gives a yellow color at a low pH. This dye is in Difco's m-dextrose tryptone broth, dextrose tryptone broth (special) and in Stumbo's (75) medium. These media are recommended for culturing sporeformers. The same amount used in Difco's media (0.04g/1iter) was selected which is double that (0.02g/1iter) recommended by Stumbo (75). The medium was a Standard Methods Agar modified for spores (SMAS).* Membrane Filter Count (MFC) The spreading problem, the difficulty in counting colonies when milk is plated, and several reports on the inhibition of sporeformers by milk components (5, 9) led to the trial of the Membrane Filter Technique. The pro- cedures for this method basically were those suggested for coliform counts in milk (44) and dairy equipment (2). *Plate count agar (Difco) 23.5 g, bacto agar 5.0 g, soluble starch 5.0 g, NaCl 8.5 g, bromcresol purple 0.04 g per liter. 29 Regular petri dishes instead of the special small ones, and SMAS instead of broth absorbed in pads were used. For the differentiation of colonies on the membrane after growth, several approaches were tried: a) Staining the membrane filter with malachite green solution as described in the Standard Methods 1 b) Using prestained membrane filters (Green-6, Grid, n1 Catalogue No. 5013, Gelman Instrument Co.) c) Using SMAS with pH indicator dye and placing the -‘ membrane filters upside down on the bottom layer 1 of agar. This was the preferred procedure since it gave distinct yellow colonies on a purple back- ground and allowed the use of white grid membrane filters (GA-6, Grid, Gelman). These filters showed better autoclavable properties than other brands tried. Everytime that MFC was tried, identical replicate samples were plated following the regular APC procedure. Neither B. subtilis Al nor B. cereus 7 counts showed a significant difference when the plates contained from 1 to %100 colonies. Only data from the agar plate counts were used for the cal- culations. Samples were plated after heat shocking. The tempera- tures were 80 0 (176 F) for B. subtilis Al and B. cereus 7, and 100 C (212 F) for B. stearothermophilus. The time was 15 min. Incubation conditions were as previously mentioned. 30 Thermoresistance and Germination The Thermoresistance Experiments Small scale thermoresistance experiments were conducted in the thermoresistometer designed and described by Pflug (52). For a few experiments thermal-death time cans in miniature retorts were used. a The thermoresistometer cups described and studied by LA Pflug and Esselen (53) were utilized as a substrate holder. ”J They concluded that 0.01 ml samples in the open cup gave a ! negligible lag correction factor so this volume of sample was used for the experiments. To measure the samples of a Gilmont micrometer syringe of 2.0 m1 capacity (smallest division 0.002 ml) was used. The plastic model was pre— ferred because of its relatively low price, ease of auto- claving and accuracy. For the few experiments with minia- ture retorts the cups were placed into special cans (10 per can); filter paper, impregnated with distilled water, was also enclosed. Miniature retorts were used when long treat— ments were necessary since the thermoresistometer was impractical. In an effort to reproduce the plant conditions two different types of substrate were utilized. Difco dry skim milk which is a standardized medium and free of inhibitors was used. It was reconstituted to 10% by weight allowing for the addition of 1 m1 inoculum. After reconstitution the milk was left overnight under 31 refrigeration to help rehydration of the particles and next day was centrifuged (10,000 rpm, 16,300 x g, RC-2 Sorvall centrifuge) to remove nonsoluble particles. Tests indicated that 0.2% of the milk solids were lost by this procedure. The milk was filtered in 100 ml amounts utilizing a Seitz filter with an S-l (0.5u) asbestos pad (31). All trials utilizing membrane filters (0.45u) failed because the flow of milk stopped after a few milliliters. Chlorphenol—red (84) in 7.5 ppm amounts was added when the better detection of growth was desired. The second substrate was low spore count whole milk. Milk that contained <1 spore/10 ml was dispensed in 9 ml amounts in tubes, autoclaved at 10 psi for 5 min and incubated at 45 C for 48 hr. Negative tubes were used as substrate (pH 6.8). Subculture in several milk preparations was used in some experiments and the results compared with those in DTS broth. The first one, autoclaved litmus milk, was used as aerobic and anaerobic substrate. To obtain anaerobic con- ditions sodium thioglycollate in amounts of 4 ppm was added to the milk. Sealing was accomplished by adding 2 m1 of a mixture of paraffine-vaseline-mineral oil (1:1:4) (75). The second subculture medium consisted of UHT sterilized milk that did not spoil after 1 week at 45 C (113 F). Negative samples were distributed into sterile tubes, and rechecked for another week. The negative tubes were used as recovery substrate for thermoresistometer experiments. 32 The thermoresistance experiments were always performed with 10 replicates (10 cups with 0.01 inoculated substrate) prepared immediately before use as described by Eder (14) but without drying. Five cups at each time were exposed to the desired temperature-time combination in the thermo- resistometer. The initial number of spores were determined for each trial by placing random inoculated cups into dilution blanks (10 or 100 ml according to concentration), heat shocking for 15 min and shaking manually for 15 min before plating. The experiments for the survivor curves and the temperature- survivor curves also necessitated plate counts. The above procedure was followed. However in these cases no heat shocking was necessary When "fraction-negative" (FN) tests were performed, after the heat treatment the cups were immediately placed in 8 ml of DTS broth contained in 20 m1 tubes with resilient plastic foam plugs. These tubes were incubated at 45 C (113 F) for 1 week. Growth was evidenced by characteristic visual changes. The results of the fraction-negative results were processed according to the Stumbo, Murphy and Cochrane method described by Pflug and Schmidt (54). The D values obtained by this method were plotted semilogarith— mically vs.temperature. From the resulting thermoresistance (TR) curves, z values were determined graphically. 33 The Germination Experiments In the germination experiments only B. subtilis Al spores were used. Replicate bottles of 100 ml were innoculated with >102 to >10“ spores of B. subtilis per ml. The initial population was determined by plating 1 ml from each of the heat shocked milk replicates. The replicates were incubated at 45 C (113 F). After time intervals of O, 3, 6, 18, 24 and 48 hr samples were taken. After refrigeration for 24 hr or more these samples were heat shocked and plated. The use of a penicillin-penicillinase system was tried to inhibit outgrowth of the germinated spores that could cause the formation of secondary spores. Penicillin G was added in 100 units/ml of milk. Duplicate controls containing no penicillin were tested. Refrigeration after incubation allowed the penicillin to act further on the growing cells. The action of penicillin was stopped before plating by adding a penicillinase suspension either to the bottles or to the agar. The suspension contained enough penicillinase to inactivate three times the amount of penicillin. Plant Procedures The Bactofugg A Type D3187M Bactofuge provided by De Laval Separator Company was used for the experiments. It was much like a 34 hermetic milk clarifier but had two 0.3mm holes in the bowl for sludge outlets. The milk was fed in at the bottom of the machine, passed through the distributor and flowed into the disc stack through the holes in the discs. It passed into the center of the bowl and was discharged at the top free from foam. The sludge containing bacteria was gathered in the bowl casing which was equipped with a special groove for the removal of the sludge and cooling air. Air and sludge were separated from each other in an attached cyclone. The contaminated air may be directed back into the hood frame for recirculation but for the experiments it was exhausted into the atmosphere. The maximum capacity of the machine was 6,000 liters/hr (approx. 13,000 lb./hr). The machine operated with a centrifugal force of about 9,000 x g. Two revolution counting devices were provided on the machine, a tachometer and a pulsing revolution counter. The VTIS The sterilization equipment consisted of a size A VTIS provided by the De Laval Separator Company. A centrifugal pump fed the milk to a plate heat exchanger for preheating to 57.2 C (135 F) by means of vapors from the vacuum chamber. Then the milk flowed to a similar unit in which it was heated indirectly to 76.7 C (170 F) with steam. A timing pump controlled the flow of the milk to the steam injection head. The temperature 35 was raised immediately to the desired sterilization tempera- ture within the range of 136 C (270 F) to 149 C (300 F). The time the product was in the holding tube was calculated to be 3.8 sec. The flow diversion valve was set according to the temperature of sterilization used. In forward flow the milk was flash cooled in the first vacuum chamber; its temperature dropped to approximately 136 C (270 F) in order that the same amount of water would be flashed off as was previously condensed during direct steam heating. The temperature of the milk in the vacuum chamber was controlled by the vacuum regulator in the vapor line from the vacuum chambers. The system had a diverting chamber if the tempera- ture was below 140.6 C (285 F). A centrifugal pump removed the milk from the vacuum chamber and directed it to the aseptic homogenizer. It was then cooled to 10 to 21 C (50 to 70 F) in a specially designed aseptic plate cooler. The Bactofugation Experiments The general procedure used in the trials is shown in Figure 1. Approximately 780 kg (210 gal) of raw milk from the University herd were held for llor 2 days at 4.4 C (40 F). The milk had a fat content of 3.25 to 3.50% and a pH of 6.65 to 6.75. Raw samples were taken and immediately inoculated with the spores while cold (approximately 4.4 C; 40 F), and agitated in a vat for not less than 30 min. The small ® & approx. 390 kg. Heat up to bactofugation (71.10) i Bactofugation 1 (fast rate) temp. Reheat to bactofugation temp. (71.10) Bactofugation 1 (fast rate) I Reheat to bactofugation (82.20) i Bactofugation 1 (fast rate) temp. 4 1 : >10 **Set 2 : >102 Fig. 11 \J‘ to >10 spores/ml 36 m-w 11k-———+> limp,e: .' .. Q . 1- \ (1L -iLr) .. 7‘ V" VI ‘1 ' ’7 I": . - ~ igyl'wk- 1' 1 (11::- vVu’ i L I] l inwculntion 1ppl'fl: . 3}” r’ . 14% k; “fr «.1 1* 1 l . BIDS" - . { 7 I ‘3' ‘ I ‘ s .’ '«L’vz‘lz . 1 15 , -‘ ‘ ‘ 1"- 1, ‘ w," (V ‘ . A‘.’ ., 4. A z _ ‘ _i,“J rat. I 11‘ o‘ ‘ '*) l Hell up to bactofuyiticn Heat up to b , . /'71 15‘“. »,. " " .'R.p. (71.1») 151.13. ((1.1‘V Bactofugation I : *o.u£ufll (slow -<1e) (almw rate) 1 I i Reheat to temp. (71. 'u _, . ' , tciualtlwn V oac 1;) Bactofugation 11 slow rate for set 1*) fast rate for set 2**) Reheat V8 to pa" 0‘131‘1 . temp. (71.10) Tictofukaticn 1:1 (slow rate) spores/m1 Reheat a ( LO ( 5‘3 1 bactofu 3 . 9(7) ’71:. 1,13 1.11.511) 1 on I 1 S 1 L 1:; w l.-—General pattern of the bactofugation experiments. rate) Q. .1 .L\ A; :J 1 (3 . rate) ‘v 5 \ ) toiuretion o bactofugati~ 1:1 37 volume of inoculum prepared, as explained previously, was always placed in about 1 liter of cold raw milk and thoroughly mixed by stirring with a glass rod before being Slowly poured into the bulk tank. After the mixing period, samples of the raw inoculated milk were aseptically taken. The milk to be bactofuged was then heated up to the bacto— fugation temperature of 71.1 C (160 F) in the double jacketed vat and pumped centrifugally into the bactofuge. The tachometer and pulsating revolution counter readings were recorded. By weighing milk collected in a lO-gal can and recording the time with a stop watch the flow rate was calculated. From the line at the outlet of the bactofuge samples were taken aseptically during the operation. Also, samples of the sludge were taken. The tOtal weight of the sludge at each subtrial was recorded. After the first run through the bactofuge, the milk was reheated to 71.1 or 82 0 (160 or 180 F) and a second run was made through the bactofuge. Sampling, flow rate determination and revolution and pulse counts as well as sludge weights were recorded. In some trials the process was repeated a third time. The first set of trials was conducted to compare the influence of the flow rate temperature, and a third bactofu— gation, on the bactofugation effect. This set comprised Subtrials A, B and C in eaCh trial. The influence of flow rate was investigated in the second set of trials. Milk 38 containing 1/100 less inoculated spores than on the regular trials was used. This second set had only two subtrials, A and B. Figure 1 shows the general flow scheme for these two sets of trials. Table 1 (Appendix) shows the detailed features of the operation of the bactofuge. The slow flow rate was 1,540 to 2,010 kg/hr (3,400 to 4,400 1b./hr) and the fast flow rate was 4,850 to 6,000 kg/hr (11,700 to 13,200 lb./hr). The process time was 3 to 30 min. The pressure imparted by the feeding pump was -7 to +4 psi. The tachometer readings were quite uniform, from 1,650 to 1,800 rpm and always slightly lower for the second (and third) bactofugation. The Bactofugation—Sterilization Experiments This group of experiments was designed to study the effect of bactofugation upon the keeping quality of UHT sterilized milk. The procedure is outlined in Figure 2, except that after sampling, the inoculated milk was divided into two equal batches, control milk and milk to be bactofuged. After bactofugation the milk was cooled to about 4.4 to 21.1 C (40 to 70 F), with the temperature depending on the time elapsed between bactofugation and VTIS treatment, usually 0.5 to 3.0 hr. The VTIS unit was sterilized at 144.4 to 145.6 C (292 to 294 F). Bactofuged (B) milk was processed first, followed by the non-bactofuged (NB) control milk. 39 Raw Mi lké Sample S (in.tank) i Inoculation with spores suspension @ ® Heat up to bactofugation temperature (in double jacketed tank) 71.10 Bactofugation I—-> Sludge I-—9 Samples FSamples Reheat up to bactofugation temperature (in double jacketed tank) 71.10 Bactofugation II—e Sludge 11—6 Samples Samples Tank .1 VTIS treatment (BII milk first, NB second) and aseptic homogenization «L Multiple replicate samples 1 Storage at different temperatures and compared spoilage rates Fig. 2.--General pattern of the experiments with bactofugation followed by sterilization. 40 Replicate samples were taken after sterilization. Index samples were procured by continuously sampling during sterilization of the milk. Table 1 (Appendix) shows the operating conditions of the VTIS unit during milk sterilization. Handling the Samples Samples of the raw (R) milk were taken before 7 inoculation. Generally five samples of the inoculated raw milk (I) were obtained and plated in duplicate. The same applied to the bactofuged samples (BI, B11 and BIII). These samples were taken by the same procedure and at about the same time during the different trials. Also two samples of each of the sludges (SI, SII, SIII) produced during each bactofugation were taken. Spore plate counts were carried out as described previously. Samples 1, B1, B11, and BIII were plated in duplicate, at different dilu— tions. Spore count platings of each of the duplicate samples were made for R, SI, 811, and SIII at two different dilutions. Standard plate counts of one of the replicates of each sample, selected at random, was carried out each time. Samples from the VTIS were taken utilizing a sterilized chamber with attached rubber gloves for hands and arms. Ethylene oxide gas was the sterilization agent. The ster— ilizing effect of ethylene oxide was checked with filter paper strips or copper paper clips inoculated with about 108 41 Spores of B. subtilis A or B. subtilis var. globigii, 1 In all cases the tests were negative, indicating complete sterilization. Sixty samples were taken for each trial batch (30 for NB + VTIS, 30 for B + VTIS). Each sample was at 1/2 pint or approximately 200 ml of milk with a sterile cap and aluminum foil on top. Ten samples of each batch were incubated at 45 or 37 0 (113 or 98.6 F), 32 C (89.6 F) and 21 0 (79 F) for 8 weeks. Samples were checked visually for spoilage at l, 2, 4 and 8 weeks and confirmed by micro- scopic examination, phase contrast microscopy, and by isolation of the organisms used. The Statistical Analysis of Data and Calculations A FORTRAN IV program was designed to make statistical analysis of data and calculate the reduction precentages in the University's CDC 3600 digital computer. The mean (AMEAN) and standard deviation (STD) of the replicate sample counts (XI) were calculated (13). The program pro- vided for the elimination of any replicate count which deviated from the mean by more than two standard deviations. For the purified data (XAD) the means (AMEANA), standard deviation (STDAD) and 95% confidence limits (L951 and L952) were calculated. The statistical data are shown in Table 2 (Appendix). These refined data were used to calculate the per cent reduction for each bactofugation, initial and total (cumulative). 42 Another FORTRAN IV computer program was designed to calculate the per cent losses of sludge (PTSl, PTS2 and PTS3). This was calculated on the basis of the known initial volume (TV) converted to weight (TW), and the weight of the sludge (81, 82 and S3) at each process. These results are shown in Table 2 (Appendix). The programs are Shown in Tables 3 and 4 (Appendix). RESULTS AND DISCUSSION Data in Table 5 (Appendix) Show the reduction in mean spore counts as well as the reduction in standard plate counts for the inoculated control (I) and the bacto- fuged samples (BI, BII, BIII). Data showing the spore and standard plate counts for the milk prior to inoculation (R), as well as the sludge content for the one, two and/or three bactofugations (81, S2, 83), are also in Table 5. The first trial (117) involved non-inoculated milk which had very low initial numbers of spores (4.25/m1); consequently it was very difficult to evaluate the remaining population after bactofugation. Figure 3 gives the results using the slow flow rate for both BI and B11. A11 trials indicated a definite pattern of reduction of the Spore population, an efficient removal for the first bactofugation (99.24 to 99.76%) and a much lower removal for the second bactofugation (0 to 43.21%). There were some trials where the increase in mean count after BII in relation to B1 suggested an "anti- reduction" effect. However, this apparent increase in the counts after BII might also have been caused by statistical variation or experimental error in counting. These reasons may account for the negative reduction values occasionally shown in Table 5. 43 Number of Spores/m1. 44 10"» 1031. 121 11 123 124 1021» 120 119 126 127 122 101 A , NB(I) BI BII Number of Bactofugations Fig. 3.--Mean counts of B. subtilis Al spores with one or two bactofugations at «.1800 kg7fir'."""" “5 To eliminate the possibility that the sludge in the bowl could have been responsible for the low efficiency of removal observed during the second bactofugation, the bowl was disassembled and cleaned between BI and BII. Figure 4 shows the results of an identical procedure, except for the washing of the bowl between bactofugations. No sig— nificant increase in the removal for the second bactofuga— 1 tion was demonstrated by cleaning the bowl. Figure h also shows the reduction curves for SPC. Data in Table 5 indicate that in all trials the reduction in SPC followed the same trend as the removal of spores. However, because of the low SPC of the raw milk and the high spore inoculum, the spores contributed considerably to the SPC which was performed only as a guide. Because of the heat resistance of the spores and the counting procedure one may assume that the spore reductions were mainly by removal while SPC reduction comprised the total bactofugation effect which consists of centrifugal force and lethality of the bactofugation temperature. Figure 5 shows the effect of changes in the flow rate upon the removal of spores. Two different sets of trials were carried out. In the first set, besides changes in flow rate and temperature of B11, a third bactofugation took place. The results of trials 132, 133, 152 and 154 were averaged and are illustrated in Figure 5. Bl, BII and 8111 Count/ml 1:6 10% A Spore counts/ml Q SPC/ml 103» 123 12“ 1024f ” 123 ~.-‘~fi~“‘ 101 % 1. W- NB(I) BI BII Number of Bactofugations F1 . H.--SPC and spores counted with (trial 12“) and without %trial 123) cleaning the bactofuge bowl between BI and BII. Number of Spores/ml High Inoculum “7 Low Inoculum __A._B+_Q_. A. B 10“» “ A 103» i A A 102“ C A +- B B B c ._._,,a____ 101 4, 1‘ + : .r ¢ NB(I) BI BII NB(I) BI BII Fig. 5.--Removal of B. Number of Bactofugations subtilis AJ spores by bactofugation. Flow rate was m5,HOO kg/hr—for subtrial A and m1,800 kg/hr for B and C. in subtrial C (82 C). The temperature of bactofugation was 71 C except for 811 10 10 10 48 had the faster flow rate in subtrials A and the slower in subtrials B and C. In the trials with the higher spore population (>10u/m1), subtrial A showed a lower per cent removal for BI (98.14 to 98.52%) than in subtrials B and C (99.87 to 99.89%). The per cent removal for BII was in contrast considerably higher in A (92.93 to 94.30%) in comparison to B and C (38.65 to 79-00%). However the per- centage of spores remaining after BII in the subtrial A was approximately equal to that remaining after only B1 in the B and C subtrials. Thus by single centrifugation at the slow flow rate (one—third the maximum flow rate) approximately the same effect was obtained as when double bactofugation at the fast flow rate took place. Commer- cially, however, there are other considerations, for example, sludge losses. The percentage removal of spores for BII was 44-18 to 60% for subtrial A and 18.18 to 22.73% for subtrials B and C. In general BIUIwas found to be too inefficient and therefore it was considered unnecessary. In the trials with the lower spore population (>102/m1) the situation was similar to the trials with a higher inoculum, although in subtrial A the per cent removal for BI (98.88 to 99.30%) was not as low and the per cent removal for BII (69.33 to 71.26%) was not as high as in the trials with the higher inoculum. In subtrial B the removal was 99.79 to 99.81% for BI and 60.87 to 68.12% for BII. 49 Thus, these two sets of trials show that when the faster and the slower flow rates were compared during BI a higher efficiency was obtained with the slower flow rate. For the subsequent bactofugations (BII and BIII), however, although performed at different flow rates in the different subtrials, the differences in efficiency were due more to the different percentage of spores remaining after BI (for BII to act upon) than to the flow rate. In the high inoculum trials changes in the temperature of BII from 71.1 C (160 F) to 82.2 C (180 F) as recommended by Simonart (62), did not show any significant effect on the percentage of removal of spores of B. subtilis Al. The percentages removed were, for BII 78.65 to 78.96% at the lower temperature and 72.06 to 79.00% at the higher temperature, and for BIII 18.18% and 22.73%. Figure 6 shows the removal of spores in 12 trials. The trials represented in this figure differ from those in Figure 5. BI took place at the slow flow rate and BII at the fast flow rate. The percentage removal was 99.72 to 99.98% for BI with the high inoculum and 99.68 to 99.82% with the low inoculum; for BII 48.19 to 80.13% with the high inoculum and 42.50 to 60.00% for the low inoculum. Again, the initial population did not seem to affect significantly the efficiency of removal, although the low inoculum gave initial populations of about the same magni— tude as the population remaining after BI, when the high Number of Spores/ml 50 . Low Inoculum High Inoculum u quir v 102 ‘r 101 1034 .102‘r 10° 101 l L g m 5 : 10'] NB(I) BI BII NB(I) BI BII Number of Bactofugation: Fig. 6_.--Removal 01‘}; mm kg/hr. and BII at ~5,400 kg/hr. ° A1 spores when BI was at ~1,800 51 inoculum was used. Figure 6 and also Figure 5 indicate that in a particular pOpulation of Spores the "removability," which is directly related to the specific gravity of the spores (Stokes law), follows a normal distribution among the cells. Thus, some of the spores were very removable, the majority were moderately removable, while some were only slightly removable, under the same conditions. A similar type of distribution is also true of the heat resistance of spores (20). BI would then have removed the highly removable and the removable Spores while, no matter how many bactofugations were conducted, a percentage of the least removable Spores will remain in the milk. The magnitude and importance of the number remaining depends on the initial number. This contention would explain the low efficacy of BII (and BIII) since only the less remov- able spores would have remained. One way to improve efficacy under these conditions would be to increase the centrifugal force (gravities) of the process. Commercial raw milk in developing countries probably would have ~102 spores/ml which is similar to the counts on the samples with the low inoculum presented in Figures 5 and 6. By carrying out the second bactofugation at the faster flow rate in the trials illustrated in Figure 6, the "anti— reduction effect" disappeared. The phenomenon of apparent or real increase had been observed in a preliminary group 52 of trials (Figure 3) in which BI and BII were conducted at the slower flow rate. An explanation is the use of a fast flow rate seemed to give, in some cases, a slightly better reduction in the counts of milk bactofuged twice and/or three times. This reduction might have been due to the fact that a fast flow rate accelerated the exit of the sludge and bactofuged milk from the bowl, and reduced the time of contact between the bactofuged milk and the sludge on the wall of the bowl. Such change might have prevented reincorporation of spores from sludge before they leave the bowl. Because of the small number of remaining spores, a slight variation in the counts significantly affected the percentage of removal. The reintroduction of spores might have also occurred during BI especially when slow flow rate was used but the removal effect was so great that small changes in the counts did not affect the results. Nevertheless, the phenomenon of antireduction was only of academic interest, and in practice the small difference in percentage when translated into number of spores would not be significant. Figure 7 shows the results of four trials involving the use of B. cereus 7. The removal pattern in these trials followed the same general trend as when B. subtilis Al was used. BI was carried out with the slower flow rate, except for subtrial A (trial 134) shown on the left half of the graph. The percentage removal was high (98.53 to Number of Spores/ml 53 A, B, c . High_ Q Inoculum ‘ 10“ 4» ”'10“ Low Inoculum 103” A. #103 B 102. C .u02 A B -- -- a -1 1 4 J AAAAAAA _ '4 J L 1 .0 j , - . ~ - r 10 ‘ NB(I) BI BII NB(I) BI BII Number of Bactofugationa .Fig. 7.--Removal of B. cereus 7 spores when BI flow rate was ~1,800 kg/hr and B11 N5,4OO'kg/hr (left graph). slower flow rate. Subtrials A (right graph) were at the faster flow rate and B and C at the 54 99.79%) for BI with the higher inoculum and was 98.87% for the lower inoculum but was 26.00 to 72.77% for BII for both high and low inoculum. BII in the lower inoculum trial and in subtrial A, shown on the left half of the graph, was with the faster flow rate. BI with the faster flow rate in subtrial A gave a slightly lower per cent removal (98.65%) than with the slower flow rate (99.82%) and consequently a higher per- centage of remaining spores; thus BII per cent removal (93.61%) for A was higher than normal. The per cent removal with BII was exceptionally high for B (90.29%) and c (98.14%) at 71.1 c (160 F) and 82.2 c (180 F) respectively. This suggested the possibility of lethal effect during bactofugation due to longer subjection to heat for the much less resistant species involved. The trial with B, stearothermophilus NCA 1518 (131) was successful only for BI. The very low count after BII caused its evaluation to be difficult even with MPN techniques. However, the total removal of spores was >99.5% since the removal for BI was 99.5%. The spore reductions were, in general, slightly higher than reported in the literature (37, 38, 39, 51, 63, 66, 80, 81, 82). Except for the work by Simonart 22 El. (66) no specific spore counting techniques were described in the literature reviewed. Several authors (37, 38, 51, 80, 81, 82) referred to reductions of >90% of anaerobic (Clostridium) _L._—_.i 55 spores. Syrjanen (80) reported the "entire removal" of spores which seems very improbable. None of these authors, except Simonart SE §l° (66), studied aerobic spores removal specifically, and his equip- ment was at a semi-industrial level with a flow rate of 400 kg/hr. He utilized only one species. Kosikowski and O'Sullivan (34) also worked with reduced flow rate due to limitations of their heating equip— ment. They commented on a possible increase of the effi- ciency of removal and mentioned similar results by Simonart in a personal communication, but provided no data. Surkov and Schmidt (79) working with a laboratory centrifuge reported an increase in the removal of bacteria and spores by utilizing half the maximum flow rate but they did not conduct experiments on the influence of this higher removal on the efficiency of the second bactofugation. The flow rate had a very marked influence on the amount of sludge eliminated from the milk by bactofugation. At the slower flow rate the sludge losses ranged from 5.28 to 6.98% by weight (Table 1). At the fast flow rate it constituted 1.31 to 2.59% of the milk which was approxi- mately three to four times less than the slower flow rate. Other authors (32, 33, 34, 46), using the same type of machine at 50% normal flow rate, also found per cent losses of a higher magnitude (2.5 to 3.5%) than those reported for the normal flow rate (1.35 to 1.85%). As reported by 56 the same and other (80) authors, the per cent fat observed in the sludge was negligible. Although the flow rate determined the volume of sludge, and consequently the concentration of microorganisms, there is a proportional relationship between the spore counts of sludge and their reduction in the bactofuged milk. The sludge counts, however, are two, sometimes three, log cycles greater than the count of the corresponding bactofuged milk in the majority of the trials. The results of the fraction negative (FN) tests are shown in Tables 6 and 7 (Appendix). Collateral experiments were performed to ascertain the differences in FN results by using different subculturing substrates such as DTS broth, litmus milk and milk with anaerobic conditions. These tests were performed at 143.3 C (290 F). No signifi- cant differences were found. Figure 8 shows the heat activation curve of B. subtilis A resulting from plotting the data of l Ridgeway (60). Figure 9 shows the thermoresistance (TR) curve plotted from data in Tables 6 and 7 (Appendix). The 2 value for this curve is 12 C (21.5 F). Data in the two thermoresistance experiments are so compatible that the curve passes between the two Sets of data, indicating almost the same slope. The 2 values individually calculated were 11.1 and 12.2 C (20 and 22 F). Below 121.1 C (250 F) Number of Spores 105 v4 10 103 (212F) according to the data by R 57 63.5 6’35 min. k A A ‘ v ' 5 10 175 20 Time (min.) Fig.8.-—Beat activation of ii: id subtilis gway (60). A l 25 spores at 1000 30 5E3 Average curve \ \ ‘ 0 \ \ \ + \ \ \ \ \ \ \ \ 1024 \ \ \ 16.5F \ \ d m U) 8 l. 3.10 * «s > O z 21.5F + Skim milk 9 Whole milk 10 (p 23'0 ' 250 27.0 280 290 Temperature Treatment (F) Fig. 9.--Thermoresistance curve of B; subtilis A1 at UHT in milk. 59 the curve has another slope and a 2 value of 9.2 C (16.5 F), giving a concave TR curve. Although the D values in skim milk are lower than in whole milk except at 143.3 C (290 F), the difference is not significant when the D values in seconds are converted into minutes. (The initial number of spores in skim milk was approximately 10 fold more than the initial number in whole milk.) This difference diminishes as the temperature increases. Skim milk and whole milk as supporting sub- strates thus gave approximately the same heat resistance results. The heat resistance data and the TR curves are not significantly different from those reported by Edwards (15) for skim milk, although his technique was different. He also found concave curves with lower 2 values at the lower temperatures. The 2 values are between those found by him for FNA and CNA recovery media. The survivor curves for 110 C (230 F) and 121.1 C (250 F) are shown in Figures 10 and 11 (data in Tables 8 and 9, Appendix). These trials were conducted only with whole milk. D is similar to the average D found by FN 121 tests. D is considerably higher for the curve. 110 Temperature-survivor data for skim milk and whole milk may be seen in Tables 10 and 11 (Appendix). The temperature-survivor curves for these cannot be combined nor averaged since the counts in one are ten times greater than in the other. Thus the temperature-survivor curves Number of Survivors 103 {p 60 D23o 2,010 sec 33.5 min L A I 4 A L f V U I A V y f V V 300 600 900 1200 1500 1800 1950 2100 Time (sec.) Fig. 10.--Survivor curve for B; subtilis A1 at 110C (230F). Number of Survivors 61 TH-4C +- 10“ ‘" 103'r 103.. 102 4 4 c s : o 15 30 us 60 75 Time (sec.) Fig. ll.--Survivor curve for B; subtilis A1 at 121.1C (250F) in milk. 62 for each of the two trials were plotted (Figure 12). The two curves are similar and follow the same pattern of the curves of Edwards 22 21° (15). The heat induced increase in spore count between the control and the lowest temperature treatment is worth noting. This phenomenon was checked by repeated experi- H ments comparing counts performed after 80 C (176 F) for 15 min and counts after 110 C (230 F) for 4.0 sec. The results were similar. ‘fleB—_"~' The thermoresistance of B, cereus 7 was also studied. No detectable survival occurred with the minimum holding times at 132.2 C (270 F) and 143.3 C (290 F). A very low D (0.632 sec) was observed. 121 The germination trials were to study the possibility of reducing heat resistance of spores present in milk by stimulating their germination before sterilization. Heat shock at temperatures lower than 80 C (176 F) required a long heating time, and higher temperatures were not practical because of a cooked flavor problem. The result of these experiments was negative since no significant decrease in the spore counts occurred during incubation up to 48 hr (Table 12, Appendix). After this period of time an increase was apparent. This;1ndicated that secondary spores were being produced from the vegetative cells germinated from primary spores. The use of a penicillin- penicillinase was tried to avoid the formation of secondary Number of Survivors 63 Heccnstituted skim milk Autoclaved whole milk 4 \ x \ . 1 105 J)- ‘._\ 4.104 r \ \\ 1 . \ I \ lo“ 1* 0105 103 *r 1LW’ 102 1» \ 0101 1 hr xl O 10. g o o o o o oj\ a o o o o 000 (:10 LIN \O [\- CO ’ KO N 0\ Control N (:0 (\I (\J (\l (\1 F1} (\J g :13 N N (\1 0.1 Control Temperature of Treatment (F) " at Fig. 12.-—Temperuture—survivor curves far 2' :uh‘ilis A1 in milk UHT treatments of 4.0 sec. 64 spores. Several problems must be solved before the penicillin—penicillinase technique is practical. Adequate sterilization of the penicillinase solutions without chang- ing its effectiveness is one. Precise standardization of the penicillin sensitivity to the strain used and the penicillin inactivation during the incubation also are ‘ necessary. The results of the germination experiments with whole milk incubated for 0 to 48 hr confirmed the observa- tions made by the author (unpublished data). He worked mun-e flux ' with reconstituted skim milk and incubation periods of 0 to 3 hr. The pH of milk after BII was 6.65 to 6.75. The acidity of the substrate has a marked influence on the lethal effect of heat, particularly on the acid side. Milk is a low acid food although it was not included in Cameron's original grouping (20). Table 13 (Appendix) shows the results of the storage trials. In many cases the samples were observed for 12 weeks but no significant spoilage was observed after 8 weeks. The most significant spoilage occurred during the first 2 weeks. The high sterilization temperatures (above 146 C, 295 F) in the first group of bactofugation-sterilization trials did not allow for the observation of very definite differences between the spoilage ratio of the NB and BII milk. The spoilage averages and their ratios are shown in Table 14. Neverthe- less a slight difference in spoilage was observed at the three storage temperatures. 65 Table 15 (Appendix) shows the spoilage when the UHT was approximately 132 C (270 F). In the case of the high initial population the difference in the average spoilage was not significant because it was high in NB and BII milk. In the similar trials with a lower initial population of ~100 fold, the resulting count after BII allowed for very significant differences in the averages of spoilage. At 45 C (113 F) the difference was 100%. All NB samples spoiled but none of BII samples spoiled. Significant dif— ferences at all storage temperatures were also observed when UHT of approximately 138 C (280 F) and high population were used (Table 16, Appendix). The spoilage of NB was 18, 50 and 4 times greater than for BII milk at 21, 32 and 45 C. The spoilage ratios at 45 C for all these trials are shown in Figure 13. The number of spores in milk prior to UHT steriliza- tion had a marked influence on the spoilage. Bactofugation reduced by 100 to 1,000 times the initial number of spores in milk. The decrease in spore population decreased the probability of spore survival after UHT sterilization. Although changes of a few degrees in the heat treatment have a more marked influence upon the probability of sur— vival and spoilage, as shown in the temperature-survivor curves (Figure 12), the population of spores also has influence, allowing for small reductions in the UHT treat- ment necessary to obtain a given per cent of spoilage. ‘ in. 0"- y -""-“r 66 Lonesz .xaae cosmos» 9:: emmzoooomp Icoc new cowsmouomn cmmzpmn mwmafioom :H comHLmQEoo||.mH .me Hmfise m2 4. HHm m2 A, HHm m2 4c HHm 4. HHm A,HHm mwmwm ‘ m2 mma HE\mmBoum NQHA mmH H ommam omH < wfimfiuusm .m Ema mad Ha\mmsoow ommam mqfi and mfiaflspsm [I .m etdmeg 1‘ v setdmeg JO‘ON 19101/ 'ON pettods JO 67 Any temperature reduction obviously will depend upon number and thermoresistance of spores as well as other conditions. Because of the characteristics of the thermal death of microorganisms, generalizations cannot be made. Also by using the regular UHT treatments the reduction in the number of spores by bactofugation will improve the efficiency of sterilization if other factors remain the same . V SUMMARY AND CONCLUSIONS 1) Bactofugation removed >99.5% bacterial spores of Bacillus subtilis, Bacillus cereus and Bacillus stearothermo— philus from whole milk. The species of microorganism did not have any significant influence on the bactofugation efficiency. SPC reductions followed similar patterns. 2) Single bactofugation at‘m30% of the normal flow rate of the machine gave approximately the same efficiency as double bactofugation at the normal flow rate. This reduction in flow rate gave a three to four—fold increase in sludge losses. Spore counts in the sludge were propor— tional but 100 to 1,000 times greater than the spore counts in the corresponding bactofuged milk. 3) More than two bactofugations were unnecessary because of the low efficiency of removal (18 to 60%) upon< the low number of spores that remained after one or two bactofugations. 4) Changes in the temperature of milk for bactofuga- tion from 71 to 82 C (160 to 180 F) did not give signifi- cant differences in the removal capacity. One trial with B. cereus 7 was an exception- 5) Cleaning of the bactofuge bowl did not improve the relative efficiency of the second bactofugation. 68 .r e . Paul: . 69 6) The percentage of spores removed was not signifi— cantly affected by the initial number of spores (from >101 to >10u/ml) but was affected by the percentage of spores remaining in milk after a first bactofugation. 7) Bactofugation by effectively reducing the initial number of spores in milk reduced up to 100 times the ratio T1, of spoilage in sterilized milk when UHT treatments of approximately 132.2 C (270 F) and 134.8 C (280 F) were used for milk inoculated with >102 to >10“ spores/m1. 8) D values for B. subtilis Al were similar when a the spores were suspended in reconstituted skim milk or in autoclaved whole milk: a) 7.350 to 12.350 min at 110 C (230 F) b) 0.435 to 0.625 min at 121.1 C (250 F) c) 0.064 to 0.116 min at 132.2 C (270 F) d) 0.020 min at 137.8 C (280 F) and e) 0.0065 to 0.0072 min at 143.3 C (290 F) A 2 value of 12 C (21.5 F) was found for the UHT range of 121.1 to 143.3 C (250 to 290 F). At 121.1 C (250 F) a D value of 0.010 min was obtained for B. cereus 7 suspended in whole milk. 9) The spores of B. subtilis Al did not lose heat resistance in milk after a heat shock of 80 C (170 F) for 15 min followed by incubation at 45 C (113 F) for 0 to 48 hr. 7O 10) Temperature-survivor studies in the thermoresis- tometer showed that reduction in the initial number of spores (10“ to 105/ml) present in milk to be sterilized had an influence on the Spoilage probability although numerically, changes in the UHT range of 110 to 143.3 C (230 to 290 F) had a much greater influence. In conclusion bactofugation of milk will effectively remove spores to low levels. This process decreases the probability of spoilage when the common UHT treatments are used or may permit a small decrease in sterilization temperature by the UHT method. 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Trial No. Countc Adjusted V0110)l x10" x10" x10" x10” 3.70 0.70 7.80 7.90 8.13 I U.)0 0.90 6.86 1.95 to 5.70 5.70 5.59 5.3 5.20 x10‘ x10a x101 x10l x102 1.30 1.20 1.00 1.00 1.10 BI 0.95 0.95 9.76 1.59 to 0.31 0.1) 0.55 0. 0'9 0.5-'1 x10l x101 x101 x100 x10l 5.00 3.00 5.00 3.00 9.93 311 9.90 9.10 9.90 5.55 to 9.00 9.00 3.95 3.30 3.150 137 x10J x103 x103 x10‘ x103 1.50 1.50 1.50 I 1.10 1.10 1.20 2.65 to 1.00 1.00 0.90 x101 x10l x10l x100 x10l 1.70 1.70 . 1.69 81 1.20 1.20 1.35 3.09 to 1.15 1.15 1.00 x101 x10l x101 x10l x10l BII 1.00 1.00 (MPN) 1.00 1.00 1.00 0.00 1.00 1.00 1.00 139 x10“ x10" x10“ x104 x10" 8.50 8.50 8.78 I 8.30 8.30 7.20 1.61 to 7.00 7.00 5.62 5.00 5.00 x102 x102 x102 x10l x102 1.90 1.90 1.92 BI 1.20 1.20 1.20 1.95 to 1.01 1.01 0.98 {I . P - 4. ”'1 ‘T! .;._... .. .. TABLE 2.——Continued. E33 Trial No. . Adjusted ” , Standard C.L. and Sample tounts Counts “ed“ ueviation 95% x101 x101 x10l x10O x10l 9.60 9.60 9.61 BII 3.90 3.90 9.03 5.13 to 3.60 3.60 3.95 ”0 x10“ x10“ x10“ 11103 x10“ :3 5.50 5.50 5.98 - - I 9.30 9.30 9.70 6.93 to 9-33 9.50 3.92 i 2 2 2 0 2 I x10 x10 x10 x10 x10 1.91 1.91 1.39 . BI 1.33 1.33 1.33 6.95 to f 1.32 1.32 1.26 ." 1.29 1.29 ‘ x10l x101 x10l x10O x101 2.90 2.90 2.90 BII 2.50 2.50 2.63 2 31 to 2.50 2.50 2.37 191 x105 x105 x105 x103 x105 1.19 1.19 1.18 1.18 1.18 1.18 I 1.19 1.19 1 19 9.69 to 1.11 1.11 1.10 1.08 1.08 x10 x10 x10 x10 x10 1.55 1.55 1.5 1.59 1.56 BI 1.52 1.52 1 93 1 97 to 1.:8 1.23 1.30 1.26 1.2” x101 x101 x10l x10 x10l 5.90 5.90 5.25 5.25 5.90 BII 5.25 5.2 5 03 9 22 to 9.90 9.90 9.66 9.35 9.35 142 x105 x105 x10” x10” x105 1.13 1.13 1.09 1.09 1.08 I 0.99 0.99 9.89 1.03 to 0.90 0.90 0.89 0.86 0.56 ’) x102 x102 ‘ x10“ x102 x102 1.29 1.29 1.21 1.21 1.23 BI 1.16 1.16 1.17 6.89 to 1.16 1.16 1.11 1.06 1.06 X101 X101 x10l x10O 11101 3.90 3.90 3.60 3.60 3.69 BII 3.20 3.2 3.39 3.97 to 3.15 3.15 3.10 3.10 3.10 TABLE 2.——Cont1nued. Trial NO» .1 _.3 11M Lei ' Ctanduwl C.L. and Sample hQUUV” 1!“ 3 L1“ .Cviqtinn 95% I" 1' .3 I" 1‘) 111' x'1l 0' x10“ x10 9.00 _1.W 7.90 7 '1 8.98 1 6.70 a {0 {.19 1.31 to 6 . a. 0 1f. . a1 6 . 00 0.111 0 1'1 . 1 . 2 110 I11 11‘ 219 x10 1.;1 1.31 1.38 B1 1. V 1.‘1 l. 1 9 37 to 1. 3 1. 1.39 1. 1 1.. 9101 :1"!1 x10 x10J x101 .7" ‘1 I 1.‘ :1 6 . LI 9 BII I) 1.11 ‘_1 {1:1 L ,9 9 If j “.7“ (1.10 ()- ‘30 .11.) L; 7111 t I, 1 1“] x10“ 110” 111.2)4 x103 x10“ 7.95 7.95 8.21 1 7.611 7.193 7’ 35 7.19; to 6.13:) (1 {,0 6.99 2 2 2 1 2 x10 x10 «10 x10 x10 1.61 1JL1 1.'2 81 1 ‘2 1.9“ 1.9. '9 to 1 ’1 1 1 1.22 10‘ 1 9 X1» 110‘ x10 {.90 f 90 7,59 B11 {.15 7.1L 6.97 5.99 to 11‘9. 6.3; 6.35 ( 1 I, 1 , 198 1194 213” 2.1.1.i 3108 X10“ {.00 ,.-;:0 7.19 I 13.7..) h 7;? 6.‘ ‘ 5.97 to b 1)“) L) ‘9“ 5 .12) 9 x10‘ x10“ x10“ x10O x102 1.5)) 1.5:) 1.59 BI 1.50 1.56 1.50 2.58 to 1.59 1.59 1.53 1 . 110‘ 110 2101 1.100 x101 1.20 d 30 8.25 B11 0 15 0.15 8.10 1.32 to {.95 (.95 7.95 150 x102 x10‘ x10‘ x10l x102 7.95 7.95 7.30 7. 0 7.27 I 6.90 6.90 6.65 7.12 to 6. 3‘) 6. 35 6.03 5.’ 5 5. ('5 x100 x10O x100 x10O x10O 2.00 2.00 1.50 1.50 1.70 BI 1.00 1.00 1.20 0.57 0.70 5.00 5.00 1.00 1.00 x100 x10O x100 BII 0.69 0.69 0.69 -- -- (MPN) ‘TABLE 2.--ConL1nued. 85 Trial No. ., Adjusted .\ Standard C.L. and Sample Louan Counts Mcan neviution 95% 152“ x10‘ x102 x108 x10 x102 5.$U 5.30 4 70 0.70 “.90 I “.00 ".00 “.30 6.89 _ to 3.90 3.90 3.70 5.00 3.00 x100 x10l x101 x10 100 5.00 5.00 3.00 3.00 “.07 BI 3.00 3.00 3.00 1.23 to (MPN) 2.00 2.00 1.93 2.00 2.00 x100 x10O x100 3 BII 0.92 0.92 0.92 -- -- .g‘ (MPN) a ) 1528 x10“ x10 x102 x10 x102 5 30 5.30 I: 7;) “.70 u .90 I ”.00 H.00 “.30 6.98 to 3.90 3.90 3.70 3.00 3.60 x100 x10‘ x100 BI 0.30 0.30 0.3U -— -— x10L x10O XIOU BII 0.92 0.92 0.92 -- -- (MPN) 153 x102 xlo‘ x10‘ x10 x102 8.110 mm 7.90 (.90 8.19 I 7.80 7.80 7.78 “.66 to 7.70 7.70 7.37 7.10 7.10 x100 x100 x100 x10 x10O 3.00 3.00 3.00 3.00 2.93 BI 2.00 2.00 2.20 0.8“ 1.U7 2.00 2.00 1.00 1.00 x100 x100 x10O BII 0.92 0.92 0.92 —- -- (MPN) ISNA x102 x102 x102 x10 x102 11.20 14.20 “.00 “.00 “.08 I 3.80 3.80 3.72 “.15 to 3.UO 3.UO 3.36 3.20 3.20 x100 x100 x10O x10 x100 u.90 ”.90 11.10 14.10 11.66 BI 3.90 3.90 u.18 0.05 to (MPN) 3.80 3.80 3.70 TABLE 2.-—Cont1nued. 86 Trial No. . - Adjusted Standard C.L. and Sample Louan Counts Mean deviation 9’1 x100 x100 x100 x100 x10O 1.110 1.110 1.30 1.3“ BII 1.20 1.20 1.20 0.16 to (MPN) 1.10 1.10 1.06 1.00 1.00 q 15MB x102 x10‘ x102 x10l x102 “.20 “.20 “.00 “.00 “.08 I 3.80 3.80 3.72 “.15 to 3.u0 3.uo 3.36 3.20 3.20 x100 x10O x10O BI 0.69 0.69 0.69 —— —- (MPN) x100 x100 x100 BII 0.22 0.22 0.22 —— -- (MPN) 155 x102 x102 x102 x10l x102 7.60 7.60 7.50 7.50 7.50 I 7.00 7.00 7.10 “.53 to 6.90 6.90 6.70 6.50 6.50 x100 x10 x10 BI 2.30 2.30 2.30 -— -- (MPN) x10O x100 x10O BII 0.92 0.92 0.92 -- -- (MPN) 599 L) U) 7 program for the statistical and reduction-per cent calculations. 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NIH: x <><><><>< O N V\Dv—4\OO O 00 RD [\ m H 963 'TABLE 6.--Results of the fraction negative thermoresistance tests for B. subtilis A suspended in reconstituted skim milk. —— ———————— 1 r NO *' T t + — r/q Nu=ln 4110310 Nu 10310 /Nu DT(sec.) 1100 120 10 0 0 -- (230F) * 2ND 10 0 0 -- 360 10 0 O -- U80 ' 10 0 0 -_ 900 10 0 0 -- 1,800 10 0 o -- 2,700 l 9 1.11 0.105 -0.9788 6.908 390 2,N00 9 1 10.00 2.303 0.3623 “.5927 "50 3,600 1 9 1.11 0.105 —0.9788 6.908 520 u,800 0 10 1.00 0.000 —- EEO 121.1C 3O 10 0 O __ (250F) ' 9O 10 0 0 -- 150 3 7 l.u3 0.358 —0.MU77 6.3u77 23.30 150 3 7 1.U3 0.358 —0.UN77 6.3U77 23.30 180 2 8 1.25 0.223 -06517 6.5517 27.50 210 1 9 1.11 0.105 -O.9788 6.8788 30.50 290 0 10 1.00 0.000 -— . Av. 26:15— 132.2C 10 3 7 1.U3 0.358 -0.uu77 6.3“77 1.57 (270F) 20 2 8 1.25 0.223 -O.6517 6.5517 3.30 30 2 8 1.25 0.223 -O.6517 6.5517 “.58 NO 1 9 1.11 0.105 -0.9788 6.8788 5.80 ' Av. 3.81 1H3.3C 1.5 2 8 1.25 0.223 —0.6517 6.5517 0.229 (290?) 2.5 1 9 1.11 0.105 —O.9788 6.8788 0.362 3.5 1 9 1.11 0.105 -O.9788 6.8788 0.507 4.5 0 10 1.00 0.00 -— -- Av. O.u28 1H3.3C 1.5* 8 2 5.0 1.609 +0.2066 5.0290 0.298 (29OF) 2.5* 1 9 1.11 0.105 -0.9788 6.21U8 0.322 Aer. litmus 3.5* 1 9 1.11 0.105 -O.9788 6.21MB 0.H85 milk U.5* 0 10 1.00 0.00 —- Av. 0.368 1H3.3C 1.5‘ 8 2 5.0 1.609 +0.2066 5.0290 0.290 (290F) 2.5* 2 8 1.25 0 223 —0.6517 5.8877 0.338 Anaer.11tmus 3.5* O 10 milk “.5* 0 10 1.00 0.00 -- Av. 0.318 #2::17 *Tests where substrates other than DTS broth was used. i“'log NO was: 5.9300 for 110C _ 5.9000 for 121.1, 132.2 and 1N3.3C 5.2uoo for 1&3.3C when other than DTS subcultures were used. 97-- TABLE 7.-—Results of the fraction negative tests for g. §u§tlll§ Al ‘ suSpended in autoclaved whole milk. *. T t + _ r/q Nu=lnP/q loglONu loglONo7Nu DT(sec) 1109 1,200 10 0 0' -— (23OF) 1,800 10 0 O -- 2,u00 10 0 0 -— 3,000 10 0 0 -- 3,600 6 u 2.50 0.916 —0.0381 ”.9831 720 0,500 l 9 1.11 0.105 -0.9788 5.9300 760 5,100 0 10 1.00 0.000 —- -- Av. 740 121.1C 3O 10 O O -- (250F) NO 10 O 0 -- 50 10 0 O -- 60 10 0 0 -_ 90 10 0 0 _- 120 10 O 0 -- ' 150 9 1 10.0 2.303 0.3623 4.6830 32.0 180 6 u 2.5 0.916 -0.0381 5.08u0 35.0 210 5 5 2.0 0.693 -O.1593 5.20u6 “0.0 2ND 2 8 1.25 0.223 -0.6517 5.6970 “2.0 Av. 37.“ 132.20 5 10 0 0 -- (270F) 10 10 O 0 —- 15 10 O 0 -- 20 U 6* 1.67 0.513 -0.2899 5.2452 3.68 30 3 7* 1.43 0.358 -0.NU61 5.U016 5.55 U0 2 8 1.25 0.223 -0.6517 5.6069 7.15 50 l 9 1.11 0.105 -O.9788 5.93u0 8.H2 60 l 9 1.11 0.105 -0.9788 5.9340 10.12 Av. 6.98 137-8C u 10 0 m (280F) 6 6 u 2.50 0.916 -0.0381 u.9933 1.20 8 2 8 1.25 0.223 -0.6517 5.6069 1.u3 10 0 10 1.00 0.000 -- Av. 1.32 1“3-3C 1.5 u 6 1.67 0.513 -0.2898 5.3350 0.28 (290F) 2.0 2 8 1.25 0.223 -0.6517 5.6980 0.35 2.5 1 9 1.11 0.105 -0.9788 6.0230 0.u2 3.0 1 9 1.11 0.105 —0.9788 6.0230 0.50 3.5 0 10 1.00 0.000 -— Av. 0.39 “When others substrates were used results were: 0 J g 20 30 1 9 H103 No was: “.9352 for 110C, 132.2C and 137.8C. 5.0 53 for 121.10 and 153.30. ,-.. n-avag -"r mu. 4.. ‘flhfi l 1 98 TABLE 8.--Counts of Q. subtilis Al spores after treatment at 110 C (230 F) for different intervals of time. T e Adjusted Adjusted Standard C.L. (:20) Counts Counts Mean Deviation 95% x10“ x10” x10" x103 x10Ll NO 8.20 8.20 8.2M > 7.90 7.90 7.62 7.01 to (after 800 7.80 7.80 7.01 15 min). 7.80 7,80 6.40 6.40 300 x10’4 x10” x10” x103 103 7.50 7.50 7.58 6.40 6.40 6.50 9.54 -to 5.60 5.60 5.42 600 x10” x101l XIOLl x10“ x101l 3.90 3.90 3.90 3-70 3.70 3.73 1.53 to 3.60 3.60 3.56 900 x10LI x10" x10Ll x103 x10Ll 3.10 3.10 3.13 2.90 2.90 2.90 2.00 to 2.70 2.70 2.67 1,500 x10” x10“ x10” x103 x10” 2.00 2.00 2.07 1.50 1.50 1.50 5.00 to 1.00 1.00 0.93 1,800 x10“ x10” x10” 1.00 1.00 1.00 1.00 1.00 1.00 1.00 99 TABLE 9.--Counts of BL_subtilis A spores, suspended in autoclaved whole milk, after trea ment at 121.1 C (250 F) for different intervals of time. Time Adjusted Adjusted Standard C.L. (sec) Counts Counts Means Deviation 95% N x10” x10" x10" x103 x10” 0 8.20 8.20 8.2a (after 80C, 7.90 7.90 to 15 min), 7.80 7.80 7.62 7.01 7.01 7080 7080 6.40 6.40 15 x10“ x10” x104 x103 x10Ll «- 2.80 2.80 2.80 2.u0 2.u0 2.53 2.31 to 2.50 2.u0 2.27 30 x10“ x10Ll x10LI x103 x10” ‘ - 1.93 1.93 1.97 1.51 1.51 1.53 3.86 to 1.16 1.16 1.10 as x103 x103 x103 x102 x103 ' 7-50 7.50 7.60 7.90 7.u0 7.30 2.65 to 7.00 7.00 7.00 60 x103 x103 x103 x102 x103 1.23 1.23 1.23 1.00 1.00 1.05 1.65 to 0.91 0.91 8.60 75 x102 x102 x102 x102 x102 ' 7.40 7.40 7.74 6.90 6.90 6.57 1.04 to 5.40 5.40 5.39 100 TABLE 10.--Counts of B. subtilis Al spores, suspended in recon- stituted skim milk, after dI??erent temperatures for 80 sec. Heat Counts Adjusted Adjusted Standard C.L. Treat. Counts Mean Deviation 95% T! N x105 x105 x105 x105 x105 ‘1 o ' 8.90 8.90 8.84 (after 80C, 8.50 8.50 7.85 1.00 to 15 min), 7.20 7.20 6.85 ‘ 6.70 6.80 .y“ 1100 x106 x106 x105 x105 95% 7 (230F) 1.02 1.02 0.89 0.89 9.55 0.83 0.83 8.u0 1.31 to 0.80 0.80 7.25 0.66 0.66 121.10 x105 x105 x105 x10“ x105 (250s) 1.55 1.55 1.u7 1.97 1.50 1.36 1.35 1.35 1.67 to 1.22 1.22 - 1.20 1.15 1.15 132.20 x10“ x10Ll x10“ x102 10“ (270F) 1.60 1.60 1.58 1.58 1.58 to 1.48 1.48 1.50 8.60 1.43 1.42 1.u2 1.u2 1.42 137.80 1/5 x102 x102 x102 x10l x102 (280F) 1.90 1.90 1.67 1.00 1.00 1.13 5.32 to 0.90 0.90 0.60 0.70 0.70 .101 TABLE 11.--Counts of B. subtilis A1 Spores, suspended in auto— claved whole milk, after different temperatures for 4.0.se0.. Heat Adjusted Adjusted Standard C.L. treat. Counts Counts Mean Deviation 95% x10“ x10“ x104 x103 x104 No 8.20 8.20 (after 80C, 7.90 7.90 8.24 15 min.) 7.80 7.80 7.62 7.01 to 7.80 7.80 7.00 6.40 6.40 1 00 x10" x10” x10” x103 x10LI 1 8.90 8.90 (23°F) 8.50 8.50 8.82 8.50 8.50 8.26 6.43 to 8.20 8.20 7.70 7.20 7.20 x10“ x107 x10“ x102 x10“ 121.10 2.06 2.06 2.06 (2500) 1.97 1.97 1.98 7.10 to 1.92 1.92 1.90 x103 x103 x103 x102 x103 132.20 2.08 2.08 2.09 (270F) 1.70 1.70 1.77 2.77 to 1.54 1.54 1.46 x100 x10O x10O x100 100 137.80 9.00 u.00 u.u0 (270E) 3.00 3.00 2.67 1.53 to 1.00 1.00 0.94 143.3C x10O (290F) <1 .1MBN) TABLE 12. --Counts of B. 102 subtilis A1 spores after heat shock at 80 C for 15 min, and incubated for different times. Time Adjusted Standard 95% (hr) Counts Counts Mean Deviation C.L. x102 x102 X102 X101 x102 0 3 90 3.90 3.82 3. 60 3.60 3.43 4.03 to 3. 20 3.20 3.03 3. 00 3.00 x102 x102 X102 X101 x102 3 3.70 3.70 3.54 3.10 3.10 3.20 3.46 to 3.10 3.10 2.86 2.90 2.90 x102 x102 x102 x10l x102 6 3.20 3.20 3.06 2.70 2.70 2.68 3.83 to 2.50 2.50 2.30 2.30 2.30 x102 x102 x102 x10l x102 18 4.90 4.90 4.84 4.70 4.70 6.65 1.92 to 4. 50 4. 50 4.46 4.50 4. 50 x102 x102 x102 x101 x102 24 6.10 6.10 6.14 6.10 6.10 6.00 1.41 to 6.00 6.00 5.86 5.80 5.80 x102 x102 x102 x10l x102 48 9 90 9 90 9.90 ' 9. 50 9. 50 9.63 2.31 to 9 50 9. 50 . 9.37 TABLE 13.--Spoilage of non-bactofuged and bactofuged, UHT treated milk after 8 weeks storage. 103 Storage Trial No. Inoculation Treatment 37 or 45C 32C 21C + — + — + — UHT > 1460 117 None NB 5 1 6 0 6 0 BII 2 4 4 2 1 5 118 E5 §32£2315 A1 ”8 .. Mold contamination Bil 119 B: subtilis A1 NB 5 4 6 5 5 BII 6 4 2 8 1 9 120 B; subtilis Al NB 0 10 0 10 0 10 ‘7 BII 0 10 0 10 O 10 121 B; subtilis Al NB 4 6 2 8 l 9 Bil 7 1 9 4 6 122 B; subtilis Al NB 0 10 0 10 O 10 BII 0 10 O 10 0 10 123 j; subtilis Al NB 0 10 O 10 0 10 B11 0 10 O 10 0 10 124 B; subtilis A1 NB 1 7 O 10 2 8 BII 0 10 1 9 O 10 126 B; subtilis Al AB 0 10 0 10 P 8 BII 0 10 0 10 0 10 127 I; subtilis A] :12.» 0 10 0 10 0 10 BII 0 10 0 *3 0 10 129 E; cereus 7 JB 9 b L3 J 10 B11 H 10 U 10 0 10 130 B; cereus 7 1n 0 10 0 LC 0 10 B11 0 10 0 10 0 10 15:1? 2 1",“? 135 E; subtilis A1 JR 4 o 4 ' Mold surf. 811 ‘y t) H cont. 136 1% subtilis Al NB “ 10 id surf. BII 1 v 2 5 "nt. 137 E; cereus 7 JB 2 10 U 10 0 10 811 0 10 0 10 0 10 139 ’3. subtilis A1 .114. 10 0 10 0 10 0 B11 0 4 10 0 10 0 140 B: subtilis A1 NB 10 0 10 0 1 9 ” B11 10 0 2 d 0 10 WET * 13'\ 141 J; subtilis Al Ab 10 .O 10 0 0 10 BII 1 0 O 10 2 8 142 EL subtilis Al AB 9 1 10 0 7 3 B11 0 10 O 10 0 10 145 B; subtilis Al NB 0 1 O 10 O 10 BII 0 10 0 10 0 10 147 J; subtilis Al NB 9 1 0 10 l 9 BII 0 10 0 10 O 10 UHT > 132C 150 B; subtilis Al NB 10 O 10 O 5 5 BII 0 10 2 8 0 10 153 B; subtilis Al NB 10 0 10 O 6 4 BII O 10 O 10 0 10 155 B; subtilis A1 NB 10 O 3 7 10 0 BII 0 10 0 10 1 9 “4*. 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