THE ROLE OF PSYCKROFHILIC BACTERIA IIT THE KEEP IITG QUALITY OF COMMERCIALLY PASTEURIZED AND HO?’ !0USUI ZED MILK By James Cecil Boyd A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1953 ProQ uest Number: 10008266 All rights reserved IN FO RM ATIO N TO A LL USERS The quality o f this reproduction is dependent upon the quality o f the copy subm itted. In the unlikely event that the author did not send a com plete m anuscript and there are m issing pages, these will be noted. Also, if m aterial had to be removed, a note will indicate the deletion. uest ProQ uest 10008266 Published by ProQ uest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This w ork is protected against unauthorized copying under Title 17, United States Code M icroform Edition © ProQ uest LLC. ProQ uest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 ACKNOWLEDGMENTS The author wishes to express' his sincere appreciation to Dr. G-. M. Trout, Research Professor of Dairying, for his interest in planning and directing this investigation; for his help in scoring of the milk samples used in this investigation and for his guidance in the preparation of this manuscript. The sincere appreciation of the writer is also ex­ pressed to Professor J. M. Jensen, Assistant Research Professor of Dairy­ ing, for his suggestions and for his help in scoring of the milk samples used in this study. Grateful acknowledgment is also expressed to Dr. W. L. Mallmann, Research Professor of Bacteriology and to Dr. C. K- Smith, Instructor of Bacteriology and Public Health for their suggestions and advice relative to many of the bacteriological procedures used in this investi­ gation. The writer is most grateful to Mr. D. K. Weber, Superintendent of Heatherwood Farms, Inc. and to Mr. R. D. Spencer, Assistant Superintendent of Heatherwood Farms, Inc. for their interest in the study and for their efforts to provide the author with part-time work and thus financial aid during the course of this study. Last, but by no means least, the author wishes to express his sin­ cere appreciation to his wife, Marianne, for her encouragements which were so greatly appreciated and for the many sacrifices that she and the children so willingly made in order that this work might be possible. 347169 THE ROLE OF PSYCHRQPHILIG BACTERIA IN THE KEEPING QUALITY OF 0OMMERCIALLY PASTEURIZED AND HOMOGENIZED MILK By lames C* Boyd AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy Year Approved 19 53 THE ROLE OF PSYCHROPHILIC BACTERIA IN THE KEEPING QUALITY OF COMMERCIALLY PASTEURIZED AND HOMOGENIZED MILK by James C* Boyd The role of psychrophilic bacteria In the keeping quality of commercially pasteurized and homogenized milk, collected and stored at 33 and 40° F. during the summer, fall, and winter months from four dairy plants has been studied. Laboratory pasteurized milk stored under the same conditions was studied also* The average keeping quality of commercially pasteurized and homogenized milk stored at 40° F* was found to be 13 to 18 days based on 37/ flavor score and 8 to 11 days based on the 50,000per-ml, bacterial standard. When duplicate samples were stored at 33° F, the average keeping quality was extended an additional 11 to 14 days. The average keeping quality of laboratory pasteurized milk, not subject to post-pasteurization contamination when stored at 40° F* was 28 to 42 days. When duplicate samples were stored at 33° F, the average keeping quality was extended an additional 14 to 28 days. The flavor deterioration was correlated with the growth of psychrophilic bacteria in all cases, except the laboratory pas­ teurized milk stored at 33° F, The keeping quality of the commercially pasteurized milk studied in July was superior to that of November and December, regardless of the storage temperature* No oxidized flavors were encountered in the commer­ cially pasteurized and homogenized milk. The organisms growing on a standard plate prepared from freshly pasteurized milk were found to be largely thermoduric. Pure cultures of these organisms grew at 50° P. but not at 40° S’. The majority of the organisms isolated from the commercially pasteurized milk having the poorest keeping quality when spoiled were gram-negative rods. Studies showed that most of these organisms were killed by laboratory pasteurization at 143° P. for 30 minutes. However, the commercially pasteurized milk which had the best keeping quality at either storage temperature was spoiled eventually by the growth of a heat-resistant, sporeforming rod. These organisms were found to belong to the Aerobacter, Alcaligenes, Achromobacter and Clostridium groups. The A e r o ­ bacter group was observed to be a common spoilage organism In milk stored at 40° P . , but not at 33° P. A gram-negative coccus (Micrococcus lipolyticus. Stark and Schieb, 1936) was encountered In one sample. The majority of the organisms isolated from the laboratory pasteurized milk, after extended storage, were gram-positive rods, possibly Corynebacterium. A gram-negative coccus (Micrococcus lipolyticus) was encountered also. When these organisms were grown at 50° P., they were killed by pasteurisa­ tion temperatures of 143° P. for 3 0 minutes, but when grown at room temperature they were not. Standard plate, collform, and/or psychrophilic bacteria counts on freshly pasteurized milk were found to be of little value in predicting the keeping quality of the milk when held at either 33 or at 40° P. A comparison of incubation temperatures of 50° and 40^ F. for agar plates prepared from milk stored at 40° F. for extended periods of time showed that the incubation temperature of 50° F. resulted in the detection of a group of thermoduric organisms, which were not detected at 40° F. A comparison of bacteria counts obtained after incubation of the agar plates for 7, 10, 15 and SO days, showed that at no point during the incubation at 50° F. did the bacteria counts coincide with those obtained by incubating the plates at 40° F. The counts were always higher when the plates were incubated at 50° F, These studies showed: a) the futility of standard plate counts in predicting the keeping quality of milk; b) the necessity of standardizing the technique for eveluating the presence of psychrophilic bacteria in milk; p) the beneficial effect of low temperatures (33° F. vs. 40° F . ) on the keeping quality of milk; d) the seriousness of post pasteurization contamination on the keeping quality of milk; and e) the need for more studies on seasonal effects on the keeping quality of pasteurized milk, which exists but seems not to have been explained satisfactorily. TABLE OF C0IIT5ICTS Page I"TRODUCTI O N . 1 REVIEW OF LITERATURE ........................................... 5 Introduction . . ................ • ......................... 5 The Keeping Quality of Raw Milk when Stored at Low Temperatures ............................................... 6 Effect of Storing Raw Milk at Low Temperatures on Pasteurization Efficiency ................................. * 9 The Keeping Qpality of Pasteurized M i l k .................. 1. Keeping quality based on bacterial standards . . . . 10 a) Bacterial standards .......................... 10 b) Early s t u d i e s ................................. 12 c) Effect of refrigerated storage on standard plate c o u n t ................................... 12 d) Rate at which the bacterial content increases in pasteurised milk stored at refrigeration t e m p e r a t u r e s ................................. 13 e) Effect of initial quality on bacterial growth in pasteurized milk stored at refrigeration temperatures . . . . . . ... 15 2.Keeping quality based on flavor and other aspects. Effect 10 . of Season of Year on Keeping Q u a l i t y ....... lb . Definitions and Methods of Determining Psychrophilic Bacteria in M i l k ........................................... IS IS 1. Definitions of psychrophilic bacteria 2. Op til.rum growth temperatures and the range in temperatures for growth of psychrophilic bacteria. . 20 Incubation temperatures and time used forthe de­ termination of psychrophilic bacteria in milk . . . 21 3- ............ IS iv Page Ability of Psychrophilic Bacteria found in Milk to Withstand Pasteurization................................... 22 Flavor Defects Produced by Psychrophilic Bacteria in Dairy P r o d u c t s ............................................. 25 Discoloration of Dairy Products Caused by Psychrophilic B a c t e r i a ................................................... 26 Physical Defects of Dairy Products Caused by Psychrophilic B a c t e r i a ...................................... 27 Psychrophilic Bacteria Common to Dairy Products and their S o u r c e ............................................... 27 Miscellaneous Observations onPsychrophilic Bacteria . . . . 30 Quality Tests for the Prediction of the Keeping Quality of Pasteurized M i l k ....................................... 30 S u m m a r y ................................................... 31 SCOPE OF INVESTIGATION AND PRESENTATIONOF D A T A ............... 33 EXPERIMENTAL PROCEDURE......................................... 35 Source and Handling of the Commercially Pasteurized and Homogenized Milk S a m p l e s ................................... 35 Source and Handling of the Laboratory Pasteurized Milk S a m p l e s ................................................... 36 Frequency of Analysis 36 ..................................... Bacteriological A n a l y s i s ................ 37 Flavor A n a l y s i s ........................................... 4l Growth Rates for Certain Organismsat 33 anh ^0° F .......... 42 Analysis of D a t a ........................................... 43 R E S U L T S .................................. SECTION I. THE EFFECT OF VARIOUS TEMPERATURES AND INCUBATION PERIODS FOR AGAR PLATES ON THE DETERMINATION OF TIE PSYCHROPHILIC BACTERIA INM I L K ..................... 45 45 V Page Comparison of Bacteria Counts Obtained When Agar Plates Were Incubated at 50° F. for 20, 25, and 30 D a y s .......... 1. kb Discussion of r e s u l t s ............................. ^7 Comparison of Bacteria Counts Obtained when Agar Plates Were Incubated at 50° F. for 7, 1°, 15, and 20 Days........ 51 1. Discussion of r e s u l t s ............................. 53 Comparison of Bacteria Counts Obtained when Agar Plates Were Incubated at 4o° F. for 10, 15, and 20 D a y s .......... 06 1. Discussion of r e s u l t s .................. 68 Some Characteristics of the Organisms that Develop Between the 10th and 20th Day of Incubation at 50° F • whenthe Milk was Stored 10 Days or Less at U0° F ........................ 77 Comparison of 50 a^d ^0° F. Incubation Temperature for Agar Plates Prepared from Commercially Pasteruized Milks of Different A g e s ......................................... 79 SECTION II.' THE' KEEPING- QUALITY OF COMMERCIAL AND LABORATORY PASTEURIZED MILKS WHEN STORED AT 33° F, AND A T '§0° F. FOR "EXTENDED PERIODS OF T I M E ................ S3 Keeping Quality of Commercially Pasteurized and Homogenized Milk Based on Flavor Score .................... 84 1. Milk stored at ^40°.F ............................... gU 2. Milk stored at 33°.F ............................... 85 3« Discussion of keeping quality at 33° F. vs. 40° F. based on f l a v o r .............................. Keeping Quality of Commercially Pasteurized and Homogenized Milk Based on Bacterial Standards ............ 8b 93 1 . Milk stored at Uo° F ......................... 93 2. stored at 33° F ............................... 9^ Discussion of the keeping quality at 33° F. and 40° F. based on bacterial c o n t e n t ................ 9^- 3* Milk Relationship of Psychrophilic Bacteria Content to Flavor Score of Commercially Pasteurized and Homogenized Milk . . . 99 vi Page 107 Keeping Quality of Laboratory Pasteurized Milk Summary 110 SECTION III. SOME CHARACTERISTICS OF THE ORGANISMS COMMONLY FOUND IN FRESH AND DETERIORATED COIP ER.ClALLY PASTEURIZED AID HOMOGENIZED MILK AND IN LABORATORY PASTEURIZEDMILK. . . 112 The Effect of Various Plating Procedures on the Bacteria Counts Obtained from Commercially Pasteurized and Homogen­ ized Milk Stored at 33 an0 to 4o pounds greater than during the period of 19^9 to 1942 (U.S. Dept. Agr., 1951)* This has been accomplished despite the fact that since about 1942 milk deliveries to the home have been on an "every-other-day” or "three-times-a-week" delivery basis. In effect, then, fluid milk is no longer a strictly perishable food product, but rather a semi-perishable one. By improving the keeping quality of fluid milk, it has been pos­ sible to reduce considerably the cost of processing and distribution, and thus make available to the consumer a reasonably priced product in comparison with other food products. This cost factor is no doubt re­ sponsible, at least to some degree, for the present day consumption. Some of the more important changes in processing and distribution which have resulted in reducing the costs of these operations, but have had the effect of lengthening the holding period for the milk ares a) "every-other-day” or Hthree-times-a-week,fl delivery to the home; b) hold­ ing of the afternoon deliveries of raw milk overnight for early morning pasteurization, thus enabling more efficient use of labor; c) operation of dairy plants on a six-day week necessitating the holding over of raw and pasteurized milk to take care of the seventh day; and d) the use of bulk tanks on the farm which in some instances may result in every-other-day pick up of milk. Due to the cost-saving features of the above mentioned practices a further trend in the direction of longer storage periods for milk may be deemed advisable in the future. labor might be expected. A forty-hour week for dairy plant In such a case a five-day milk plant operation may become desirable, which will necessitate an even longer holding period for the raw and/or processed products. Additional savings in processing and distribution costs might be effected by further reducing the frequency of milk deliveries to the home and milk collections from the farm. Such reductions in milk deliveries to the home and collections from the farm may be forced on the industry in case of an all-out war. Considering these growing trends, the keeping quality of milk is becoming increasingly important. Several studies have been made of the keeping quality of pasteurized milk when it has been stored at Uo to ^5° IT. These temperatures are considered to be approximately those found in the average household refrigerator. Little attention-has been paid, however, to storage temperatures below Uo° F, yet it is not unreasonable to expect, that lower storage temperatures in the milk plant may be ad­ vantageous or that the manufacturers of household refrigerators would produce refrigerators that maintain lower temperatures, if lower temperartures were shown to be beneficial. In general, those studies which have used storage temperatures of 40° F. or above, have shown that most commercially pasteurized fluid milk will not deteriorate materially in flavor or increase greatiy in "bacterial content during four days of storage. However, most investi­ gators conclude that after seven days of storage, at these temperatures the flavor has deteriorated and/or the bacterial content increased to a point where it is no longer possible to consider the milk to be of good quality. these studies have been made on what is commonly Many of referred to as "cream line" milk, and often the deterioration in flavor was due to the development of the oxidized flavor. Research has shown that homogenized milk is not subject to the development of the copperinduced oxidized flavor. In addition, homogenized milk is outselling the cream line milk as a beverage today. Studies on the keeping quality of pasteurized milk stored at 40 to ^5° F . have shown that the deterioration of the flavor of the milk is, in many cases, accompanied by an increase in the bacteria content. Thus, the role of psychrophilic bacteria (those bacteria which grow at low temperatures) would appear to be of major importance in the keep­ ing quality of the fluid milk distributed under the system used in the United States today. Several studies have pointed out that the psychrophilic bacteria found in fluid milk do not survive ordinary pasteurization temperatures and thus it is assumed that they gain entrance to the milk after pas­ teurization. However, there is a lack of agreement on this point. Some investigators ha/ve pointed out that organisms may become adapted to grcwth at temperatures which have been assumed to be sufficiently low to inhibit decomposition processes. There are numerous reports relative to the psychrophilic bacteria content of milk, but there has been little uniformity in the procedures 4 used in making counts of this group of organisms. Thus, some fundamen­ tal studies on 1 ) the keeping quality of fluid milk at different stor­ age temperatures; 2 ) the methods of determining the numbers of tht psychrophilic bacteria in milk; and 3) the relationship of these organ­ isms to the deterioration of the milk at low temperatures appear to b© timely. REVIEW OP LITERATURE Introduction The terra 11quality” in reference to milk has been used with various meanings, however the definition cited by Dahlberg (1953^ which states that ’'quality is the combination of attributes of a product that have significance in determining the degree of acceptability of the product to the user’1, seems to be a good one. Trie term, ’keeping quality' then, shall in this paper mean the period of time that the product maintaiqed those attributes of acceptance under given storage conditions when meas­ ured in terms of flavor and bacteria content of the product.That the natural lipase of milk which is responsible for the ran­ cid flavor is effectively inactivated by pasteurization temperatures of 1^3° F. for 30 minutes or l6l° F. for 16 seconds has been shown by nu­ merous investigators, only a few of whom are Rogers (I90U), Rogers, Berg and Brooke (1912), Doan (1933), and Krukovsky and Herrington (19^-2). The oxidized flavor in milk was found by Burgwald and Josephson (19^7), and Dahlberg (1953) to be an important factor in determining the keeping quality when non-homogenized milk was used. That chemical changes in the butterfat, resulting in the development of oxidized flavors, are retarded or inhibited by homogenization of milk has been shown by the following investigators, the list of whom is by’no means complete; Tracy, Ramsey and Ruehe (1933); Thurston, Brown and Dustrasn (1936); Ross (1937); Hahle and Palmer (1937); Bahle (1938); and Trout and Gould (193^0 • 6 Thus, deterioration in the quality of milk used in this experiment might well be expected to be due to off flavors or other conditions pro­ duced as a result of bacteriological activity. The literature was re­ viewed with these facts in mind. The Keeping Quality of R aw M ilk when Stored at Low Temperatures While this study was concerned with the keeping quality of pas­ teurized milk the results of a few of the early studies on the bacterial activities in raw milk held at low temperatures seemed applicable and are included herein. Conn (1903 ) stated that when raw milk was stored at 50° F. the groups of lactic organisms1did not grow well, but that miscellaneous species of organisms did grow and produce putrifaction. He concluded from this that old mil k was never wholesome even though it was sweet and uncurdled. Conn and Esten (1904) emphasized the fact that in raw m i l k the development of lactic acid bacteria, which are harmless, served to check the growth of other species and thus, conditions which favored the growth of lactic acid bacteria made milk wholesome, while conditions which checked the growth of lactic bacteria actually rendered the milk less wholesome. The experimental results of these workers are of inter­ est . Ordinary market milk (raw) containing about 20,000 bacteria per ml. was divided into several samples. of the following temperatures: One sample was stored at each 37» 20, 10 and 1° C. were then made at appropriate intervals. Bacteria counts They found that there was no increase in the bacteria count for a certain period of time, regardless 7 of the storage temperature, hut that the length of this period depended upon temperature. At 1 o C. this period of time was 6 to 8 days, hut at the end of 6 days the bacteria content of the m ilk was less than at th© beginning. This period of bacterial inactivity was always followed by an increase in bacteria content, but the types that developed dependo ed upon temperature. At 37 C* o ant* 20 C. the development of the lactic bacteria prevented and finally killed other types of bacteria, a n d milk curdled in about 40 hours. At 10° C. there was a delay in the development of any bacterial types for 2 to 3 days after which all types developed uniformly. The milk finally soured, but the lactic, neutral, and liquifying bacteria all became abundant. bacteria never predominated. The lactic acid For milks stored at 1° C. the picture was essentially the some as for the milk stored at 10° C; except the rate of growth was slower. They found that curdling of the m ilk was independent of the numbers of bacteria present. For example, at 37° C. eight million organisms per ml. caused milk to curdle, whereas at lower temperatures four billion organisms per ml. did not cause the m ilk to cur dl e . In another extensive study of the bacterial growth in raw milks stored at low temperatures, Pennington (1908) found that bacteria grew in mil k stored at 29 to 31° F. even when it was semi solid with ice crystals. Also, that although the bacteria content of the m i l k was in the hundreds of millions per ml., no off taste or odor was observed, nor did the m i l k curdle upon heating. Not until the bacterial numbers began to decrease and putrifaction set in did off flavors and odors "become evident. He found that after a few weeks at 0° C. that a higher acidity was reached in the milk than normally required for coagulation, hut that coagulation did not occur. He found also that as much as 50 percent of the casein was finally changed to soluble compounds, and that the change was pronounced at the end of two weeks of storage at 0° C In a later study, Pennington, et_ al. (1913) showed that the proteolysis of milk and cream stored at 0° C. was due primarily to bacterial activity while the breakdown of albumin was due to the native enzymes of the milk. However, the native enzymes and bacterial flora by combined ac­ tion gave rise to a more rapid proteolysis than either alone. Apparently these workers were the first to use low temperatures (0° C.) for the in­ cubation of the agar plates used to determine the bacteria content of the milk stored at low temperatures* Ravenal, Hastings, and Hammer (1910) obtained similar results and concluded that cold storage was identical to pasteurization as it elim­ inated the growth of the lactic organisms that kept the harmful species from growing. Bernstein (1909-) stated that the most healthful milk was that in which the bacteria were destroyed by pasteurization and was innoculated with a culture of lactic bacteria. Ayers, Cook, and Clemmer (1918) pointed out that the rate of bacter­ ial increa.se in raw milk stored at 9-.9° C. was influenced by the quality of the milk stored, which they believed was to be expected. They showed that clean milk contained a considerable portion of organisms from the interior of the udder which grow comparatively slow. Horth (1918) showed that raw milk which had a bacteria count of 9-,000 per ml. as 9 received at the processing plant, had a bacterial content of 8 ,5 0 0 per ml. after 29 hours of storage at 38° F. Frandsen and Glickstein (1938) showed that organisms causing "ropy" milk grew more rapidly than milk-souring organisms at 50 to 60° F., while Hammer (1938) pointed out that milk stored at low temperatures was charecterized by proteolysis and bitter flavors. More recently, Frayer (1930) and Morris (1992) showed that psychrophilic bacteria might develop in raw milk stored at 9° C. and might be responsible for a quicker reduction of methylene blue. Effect of Storing Raw Milk at Low T emperatures on Pasteurization Efficiency The observation by Sherman, Stark and Stark (19^9)* though only in­ directly concerned with the present investigation, is of interest. These investigators showed that milk stored at low temperatures had a lower percentage destruction of the bacteria by pasteurization than milk stored at temperatures that permitted multiplication of the bacteria. This was explained on the bases that the age of the cells decreased their sensitivity to heat. Prior to the report of Sherman et_ al. (1929), Sherman and Albus (1923) and Robertson (1927) reported that young cells were more easily killed by heat than older cells. Using pure cultures of three organisms important in dairy products, Stark and Stark (1929) found that young cells were more rapidly destroyed at pasteurization exposures than were old cells. Fabian and Coulter (1930) an(l Hammer and Hussong (1931) 10 also report that young cells are more rapidly killed by heat than older ones. Using E. c o l i , Elliker and Frazier (1938) found that during the period of most active reproduction the heat resistance declined to its lowest point. phase, As the cultures entered the maximum stationary growth their heat resistance again rose. Hammer (1998) pointed out that commonly organisms are more heat resistant when grown at their optimum than when grown at lower tempera­ tures. This is in agreement with Olson, Macy and Halvorson (1952 ) who report that the thermal death-time of E. coli increased from 22 minutes at 140° F., when the culture was grown at 20° C., when the culture was grown at 37° C. to 7^ minutes at 140° F., Anderson and Meanwell (193&) de­ scribed a Streptococcus which showed increased resistance to heat as its temperature of growth was reduced below' the optimum. found that raw milk held at 20° C. (b8° F.) for 9 Hucker (1928) hours before pasteur­ ization contained mostly Strep, thermophilus in the pasteurized products, but that when the same raw milk was held at 10° C. (50° -F.) for the same period of time, Strep, thermophilus was rarely found in the pas­ teurized product. The Keeping (feialit.y of Pasteurized Milk 1• Keeping quality based on bacterial standards. a) (1939) Bacterial standards. The United States Public Health Service stated that Grade A, pasteurized milk should not contain over 30,000 bacteria per ml. as determined by the standard plate count. 11 This was not a universal standard, hut was given by the U.S.F.H.S. as a reasonable standard to which local authorities might refer in plan­ ning local milk ordinances. The Michigan Milk Ordinance (Michigan Allied Dairy Association, 19^9) allowed 50,000 bacteria per ml. of pas­ teurized milk. While the United States Public Health Service Milk Ordinance and Code (1939). does not contain any bacteriological standards relative to the presence of the coliform group of organisms in the pasteurized milks, such standards are a part of many local ordinances. According to Dahl- berg et_ al. (1953) standards for coliform organisms in freshly pasteur­ ized milk are included in the 19^9 edition of the USFKS Milk Ordinance and Code. The presence of even a few coliform organisms per 100 ml. of freshly pasteurized bottled milk indicates, however, improper processing, subsequent contamination from equipment, or unsterile bottles according to the American Public Health Association (I9 U8 ). This has been estab­ lished because it had been shown by McGrady and Langevin (1932), Chilson, Yale, and Eglinton (1936) Long, Hedrick and Hammer (19^*0* Tiedeman and Smith (19^5)* an(l Eahlberg ejb al. (1953) that practically all organisms of the coliform group in milk or cream are killed by pasteurization. Several investigators, Sherman and Wing (1933). Dahlberg (l94ba, 194bb, 19^9) Olsen, Willoughby, Thomas, and Morris (1952), and Dahlberg, Adams, and Held (1953) have shown, however, that the coliform organisms would grow at refrigeration temperatures (45 to 59° ^ •) and, thus, the presence of coliform organisms in milk stored for several days was not a good 12 indication of post-pasteurization contamination. Id) Early studies. Ayers and Johnson (1913) gave some considera­ tion to “bacterial growth in pasteurized milks stored at low temperatures in their early studies of the “bacteria which survive pasteurization. Flasks of milk pasteurized in the laboratory which had initial bacteria counts on the pasteurized milk of 25,000 per ml. were stored at 70 to 75° F. and the counts were found to be in the millions in 2 and 3 days. 1 Other flasks were stored at 4b to 500 F. and the bacteria counts were found to be under 100,0 00 in 3 to 6 days. Aside from this observation, the keeping quality of pasteurized milk did not command the attention of scientific workers again until about 1930. c) Effect of refrigerated storage on standard plate count. Leet (1930) showed that fresh, bottled milk contained about 13 percent more bacteria than the same milk from the pasteurizer. After storage at 40° F. for 5 days, 2 7 .2 percent of the samples showed an increase in bacteria content and at 45° and 50° percent a decrease. When the milk was stored , 37 smd 73*8 percent respectively, of the samples showed increases in bacterial content. He pointed out that when bac­ teria counts are to be used as a measure of storage conditions for milk, care must be taken to account for variations in the temperature of stor­ age. His data was obtained, however, by incubating agar plates at 37° 0* (98° F.). Palmer and McCutchen (1930) reported in the same year that on 105 samples of bottled milk stored at 42° F. for 6 and 24 hours that the 13 standard plate counts (incubation of plates at ^8° F.) decreased in 80 percent of the cases after 6 hours of storage, and in 67*5 percent of the cases after 2b hours. The observation that standard plate counts of milk decreases when the milk is first placed in refrigerator storage has been observed also by other investigators, namely, Frazer (1934), Dahlberg (19**5)» Burgwald and Josephson (19^7) and Dahlberg et al. (1953)* The observation of Sherman and Cameron (193*0 that bacteria cultures cooled suddenly from 45° C. to 10° C. resulted in the death of over 90 percent of the cells, might explain, in part, these results. In a sim­ ilar study on S. coli, Hegarty and Weeks (19*4)) showed that when the cultures were suddenly cooled from 37° 0. to 0° C. the mortality was in­ creased with the rate of growth of the culture. d) Ra te at which the bacterial content increases in pasteurized milk stored at refrigeration temperatures. Robinton, Borman, and Michel ^1941), working with market cream, showed that bacteria counts obtained, using incubation temperature of 37° 0. and 8° C., differed. The 8° C.- incubation temperature for 4 days resulted in the detection of 8 per­ cent more cream samples that contained more than 500,000 bacteria per ml. than did the 37 o C.-incubation temperature. Bressler, Anderson, Clark and Bilenner (1943)* who studied alternateday deliveries of milk found the bacterial content, as determined by a standard plate count, increases about 1/3 every 24 hours when the milks were stored at 33 to 68° F. However, Doetseh and Scott (1951)* in studying the ba.cterial aspects of milk as consumed in the household, 14 found that, out of *+9 samples 2-days old, 20 samples had bacteria counts of 3,000 or less, and 3 4 a d counts of over 3 million. old, For milk 7-days the counts ranged from 3,000 to 2,500,000 per ml. derson (1949) Weese and Hen­ showed there was a decided increase in bacteria counts of m i l k stored at refrigerator temperatures for 5 a n d b days over the same milk stored 3 and 4 days. Kennedy and Weiser (1950) found that the psychrophilic bacteria counts, on 33 samples of pasteurized milk, as determined by incubation of the plates at 10° C., increased from an average of 1 per ml. to 300 per ml. In J2 hours. Dahlberg (1945) showed significant increases in psychrophilic bac­ teria in milk from the Hew York area in 4 days of storage at 35 to 40° F., while Burgwald and Josephson (1947) reported that the psychrophilic bacteria did not start to increase until the 4th or 5tb day of storage of the milk at 39 “to 42° F. In a raore recent study covering milk from 8 cities located in dif­ ferent parts of the United States, Dahlberg, Adams and Held (1953), observed that the psychrophilic bacteria count averaged 1 per ml. in the fresh milk. After 4 days of storage at 44° F . , the standard plate counts averaged 1 8 ,9 0 0 , the psychrophilic counts 7 *700, and the coli­ form count 14 per ml. However, after 7 days of storage at 44° F. the average psychrophilic and/or Standard plate counts, were 946,000, with 3 cities having average counts of over 2,000,000 per ml. The coliform counts on the milk stored J days averaged 2,000,000 per ml. 15 Based on “bacterial counts, either psychrophilic or standard plate counts, most investigators, Sherman, Cameran and White (l94l), Dahlberg (1945, 194bb, 1949, et a l . 1953) ar*d Burgwald and Josephson (1947), agreed that good quality pasteurized milk would retain good bacterio­ logical qualties under proper refrigeration commonly available in the home for 4 days, but that at the end of 7 days the bacteriological qualities might be questioned. e) Effect of initial quality on bacterial growth in pasteurized mil k stored at refrigeration temperatures. Mott and Mazer (1942), using certified pasteurized milk, found the psychrophilic bacteria content to average 8 per ml. when fresh, but, after the milk had been stored at 40° F. for 5 days, the psychrophilic count had increased to 770 per ml. Using Grade A pasteurized milk, the psychrophilic counts averaged 300 per ml. on the fresh milk and 1 ,300,000 per ml. on the 7-day old milk. ■Grade B pasteurized milk averaged 6,000 psychrophilic bacteria per ml. when fresh and 1,700,000 when J days old. Chaffee (1952) found that, at refrigeration temperatures, in good quality pasteurized milks held the bacteria content did not increase ap­ preciably in 120 hours, but that poor quality pasteurized milks deter­ iorated materially in the same period of time. Olson, Willoughby, Thomas and Morris (1952). This was shown also by They found that milk from a plant with a history of high coliform counts showed excessive psychro­ philic counts, which increased with storage of the milk. However, milk from another dairy which had a history of low coliform counts, had low psychrophilic counts, which did not increase greatly upon storage of the milk* 16 2. Keeping quality based on flavor and other aspects. Thurston and Olsen (1973) reported that pasteurized milk stored at 45 F. did not develop objectionable flavors or odors until the 7 th day, when slightly stale and unclean flavors developed. They no­ ticed, however, a decided tendency for the milk to develop a "cappy" flavor after 2 days of storage. "Cappy" is often used to describe what is more commonly referred to as oxidized flavor. Nickolas and Anderson (1942) showed that pasteurized and homogenized milk might be stored as long as two weeks at 40° F. before spoilage oc­ curred. Bressler, Anderson, Clark and Bilenner (1943) found pasteurized milk to keep at least 4 days in mechanical refrigerators, Weese and Henderson (1949) found, that good quality milk stored in household refrigerators would retain its good flavor 3 to 4 days but after the 4th day there was a decided drop in flavor score. Dahlberg (1945) and Burgwald and Josephson (1947) showed that the flavor of pas­ teurized milk stored at 35 to 45° F. remained of good quality for 7 da.ys of storage. In most cases, however, Burgwald and Josephson (194-7) found that oxidized flavors developed in a number of samples, whereas, other samples maintained their good flavor for as long as 20 days. Dahlberg et_ al. (1953), in a more extensive study involving milk from 8 cities located in different parts of the United States, reported that 75 percent of the fresh pasteurized milk samples were criticized for feed flavor, but that feed, cooked, cowy and barny flavors tended to decrease with storage of the milk at 44 and 33° ^ • Oxidized flavors increased with storage and were present in 24 percent of the samples 17 stored, at 44° F. and 67 percent of the samples stored at 33 end of 7 days. I1- at the Oxidized flavor was shown to he the predominant flavor in 7-day old milk stored at 44o F. Lacking freshness, stale and un­ clean flavors were detected in a limited number of samples and were most prevalent in the milks stored at 33° I1- Acid flavors developed in 8 percent of the samples stored at 44° F., but did not develop in the samples stored at 33° F. The highest flavor scores (3 points above the average) were found in milks that contained 500,000 to 5 .0 0 0 ,0 0 0 psychrophilic bacteria per ml. Milk with psychrophilic counts of less than 500,000 had on the average flavor scores 1 point below the average for all the milk. Milk containing over 5 ,000,000 psychrophilic bacteria per ml. had flavor scores that averaged 4 points below the average for all the milk. Dahl­ berg et a l . (1953) concluded that the unclean flavors that developed after 7 days of storage were not of bacterial origin, nor could he cor­ relate oxidized flavors with bacterial counts. Burgwald. and Josephson (1947) and Dahlberg (1949) concluded that, based on bacterial content and flavor, good quality commercially pas­ teurized milk would maintain a good flavor and bacteriological quality at refrigeration temperatures for 4 days, but that at the end of J days, the quality might be questioned. Burgwald and Josephson (1947) pointed out that storage of the milk did not change the riboflavin content, but that the ascorbic acid was depleted rapidly; only insignificant amounts remained after 1 day of storage. 18 Effect of Season of Year on Keeping Quality Dahlberg (19^5) found, no difference in the keeping quality of milk collected, in February and October* However, he suggested further study on the effect of season of the year on the keeping quality of milk, Burgwald and Josephson (19^+7) showed that winter samples showed somewhat better keeping quality than summer samples, but off flavors were more prevalent in the winter samples* Also, winter samples showed the presence of the oxidized flavor more often. Dahlberg ejt al* (1953) showed that the keeping quality of the winter milk was poorer than that of summer milk, when the milks were stored at F, for 7 days; his explanation being that in the winter months the milk was contaminated with organisms accustomed to growing at the low temperatures at which the milks were stored, whereas in the summer months the organisms con­ taminating the milk were adapted to growing at higher temperatures* Definitions and Methods of Determining Psychrophilic Bacteria in Milk 1* Definitions of psychrophilic b a c t er i a * Psychrophilic bacteria have been defined somewhat differently by different authors. Roadhouse and Henderson (19U1) defined psychrophilic bacteria as those organisms that would grow within the temperature range of 3? with an optimum growth range of 68 to 70° F. This agrees fairly well with the definition of Salle (19^3) and Michell (1951), who described them as organisms which did not develop even at room tem­ perature and which had an optimum growth temperature of 18 to 20° C. (bh.5 to 68° F.) Salle (19^3) gave the following temperature relationship 19 for organisms classified according to their minimum,maximum and opti­ mum growth temperatures; Growth Temperature Minimum Psychrophilic Mesophilic Thermophilic Optimum Maximum 0° C* 15 to 20° G. 30° C* 5 to 250 C. 37° C. ^3° C. 25 to ^5° C. 50 to 55° C. 60 to 90° C. Tanner (193^) , Standard Methods for the Examination of Dairp y Products (APHA 19*+&) and Erdman and Thornton (1951a), however, described psychrophilic organisms as those organisms that had an optimum growth temperature of around 5 to 10° C. (hO to 50° Tanner (193^) stated that their minimum temperature for growth was just above 0° C* or where the medium in which they were found remained liquid* Erdman and Thorn­ ton (1951a) pointed out that these organisms did not grow at 35° C. Kennedy and Weiser (1950) described them as organisms with an optimum growth temperature of 5 to 25° C., but pointed out that the possibility of mesophilic bacteria with optimum temperature ranges of 25 to U50 C. becoming adapted to growth at lower temperatures, must be accepted* This view was also expressed by Prescott, Bates and Needle (1931)* Porter (19^6) and Lampert (19^+7) defined psychrophilic bacteria as those that grew best below 20° G. (68° 3T-), but Porter (19^6) pointed out that the boundaries of such groupings were indistinct and overlap­ ping* 20 2. Optimum growth temperatures and the range in temperatures for growth of psychrophilie bacteria. The optimum temperature for growth of psychrophilic bacteria has been reported by Jezeski and Macy (19^6), Kennedy and Weiser (1950)* and Watrous, Doan and Josephson (1952) to be between 5 ^nd. 25° C. respectively. Zobell and Conn (l9*+0) found maximum growth at IS to 22° C. when working with organisms from sea water, while Morrison and Hammer (19^1), working with organisms isolated from deteriorated butter, found optimum growth at 20 to 2?° C. Lampert (19^7) reported the optio mum temperature for psychrophilic bacteria to be below 20 C, This view was supported by Standard Methods for the Analysis of Dairy Prod­ ucts (APHA, 19^8 )f Tanner (193^)* Erdman and Thornton (1951a), Burgwald and Josephson (19^7)» Dahlberg et al. (1953) who reported optimum temperatures for the growth of psychrophilic bacteria to be approximately 4.5 to 10.5° C. The range in temperatures for the growth of psychrophilic bacteria was reported by Roadhouse and Henderson (19^+1) and Tanner (193^) as 0 to 30° 0. Salle (19^3) and Porter (19^6) gave the minimum temperature of growth as -5° C. and the maximum of 30° C. while Erdman and Thornton (1951a) and Watrous, Doan and Josephson (1952) reported that the maximum temperature for growth was 35° C* Many of the variations noted in the optimum growth temperatures given for this group of organisms may have been caused by methods used to evaluate optimum growth temperatures. Dorn and Hahn (1939) stated that in bacteriological literature optimum growth temperatures usually refer to the temperature at which organisms grow most rapidly as 21 measured by protoplasm produced, Porter (19^6) suggested, however, or numbers or minimum generation time. that total crop yield of bacterial cells was a better criteria for determining the optimum temperatures of psychrophilic bacteria than their growth rate during the logarithmic phase, because of their slow rate of growth. Unfortunately most authors do not state which of the methods of evaluating optimum growth temperatures were used. It was pointed out, however, by Dorn and Rahn (1939)» Wilson (1922) and Michell (1951) that the rate of net viable growth did not coincide with that of total growth. Wilson (1922) stated that this was true because some organisms ceased to be able to grow, but remained intact in the culture. Foster and Rahn (1936), Dorn and Rahn (1939) an(l Michell (1951) stated that the optimum temperature for total crop was about 10° C. below that for maximum growth rate. 3- Incubation temperatures and time used for the determination of psychrophilic bacteria in milk. Watrous, Doan and Josephson (1952) used a 10-day incubation period, with an incubation temperature of 5° C.» while Burgwald and Josephson (19^7)* an G-aleshoot (1951) Lactobacillus group Raw milk Erdman and Thornton (1951b) Fs. group Floors and sewers, raw milk, defective dairy products, cottage cheese and water, raw milk Hammer and Olsen (19I+I) Morris (19^-2) Meyer (1950) Green and Jezeski (1951) Erdman and Thornton (195113) 29 TABLE (Cont.) Name of organism Source Described by Ps. Eluorescens Decomposed butter Derby and Hammer (1931) Ps. Eragi Ice cream, butter, rancid dairy products, cottage cheese, raw milk, defective dairy products Harrison and Assoc. (1929) Hussong, Long, Hammer (1937) Hammer (1948) Elliker, Smith, Parker (1951) Morrison, Hammer (I94l) Ps. Putrifaciens Soil, Earm well water, creamery water, raw and pasteurized milk and cr earn, abno rmal but ter Wagenaar (1952') Ps. nigrificans Abnormal butter White (1940) Ps. (N. sp) Abnormal butter Hiscox (1936) Ps. viscosa Deteriorated cottage cheese, soil, farm well water Elliker, Smith/ and Parker (1951) Milk, swiss cheese Sherman and Stark (1931) Strep, glycerinaceus Milk, swiss cheese Poster and Rahn (1936) Strep, lactis Milk Haw milk Yawger and Sherman (1937) Erdman and Thornton (1951b) Strep, licpiefaciens Milk, swiss cheese Sherman and Stafrk (1931) Streps fecalis , 30 at 34 to 35 o 0. Hiscox (193&) found that certain Pseudomonas groups did not grow at 37° 0. G-reene and Jezeski (1951) observed that Aero— genes cultures did not produce gas at 30° C. and Pseudomonas groups did not grow at 30° 0. Thus, routine water analysis likely would miss these groups of organisms* Provan (19^1) suggested that plate counts at 22 C. supplement routine water analysis, while Doetsch and Scott (1951) advised the de­ termination of fat-splitting and proteolytic bacteria in water, in ad­ dition to the routine analysis. Schutt (1929) recommended pasteurization of suspected water supplies as a safeguard against contamination of the dairy products* Doetsch and Scott (1951)* Elliker, Smith, and Parker (1951). and Wagenaar (1952) pointed out that chlorination of water supplies to the extent of 2 to 3 p.p.m. of available chlorine, was effective in preventing contamina^ tion of dairy products from water. Miscellaneous Observations on Psychrophilic Bacteria Hammer, and Olson (19^+1) have shown that members of the Pseudomonas, E. coli, FIavobacterium and Alcaligenes groups of bacteria are capable of producing phosphatase. Thus the phosphatase test, as a measure of adequate pasteurization might be of less value on products where growth of these organisms takes place. Quality Tests for the Prediction of the Keeping Quality of Pasteurized Milk Burgwald and Josephson (19^7). an te 'S o d •H (1) d O £ OT 4) +» oS rH Pi d 4) d .H •iH 9 PQ < E< H a Td 0) N •tH u PD < +> to oS Pi o o S o * Eh ® «h a •H •Td +* ® d Cm *H O 6 -( *» (/) P o d C(J o Td d oj O d +» CO Td +» &p 5 d *rl © d © «S to +3 cC , < d qj 3? n o d «n ^ rd • ^ Ph ; td o © o , d LO. I *cH8 +> d o . • sCO pH LOi O o IO at n 0) Td O CM © > cd o *rl E d +3 •H d o d o o o at at- t " - rH CM rH f H rH LO CM O O CM CM CO ro HD f lt . « o Io !d r f O o o at Sf r-~ O o o o at d" C7> O o o ■h MO IO CM O O O o CM L T \ at cm o o o o o ir* Ot o o to O0 6 O O O rH rH o» i— 1 Ok O o CO o at rH O O o Ok MO 1 — O ctm o o o o o o o at no to CM iH O rO CM mO at O o o at CO cr> rH ■» o O o o o CM i — o o o o o c r> O O O O O to CM 3 o o o o o o o o o \o O O at at at at at • pH O O d O o o o o rH <0 S Td H O C O CSM r— CM at ro CM • u r\ pH o o LTv to I dS at o O IO o> CVJ • •p» ! o o cd •H d © +> o dd © kA O o MO at c— CM ro o at C3M at at at o o o at (=1 o MO CM O HD ON o o o 5 CO at O at OM rH o L T \ to .d O o o o o o o cT 8 cT tn -p 0& S3 S o d rH O > d o CO CM d CM SECTION II THE KEEPING- QUALITY OF COMMERCIAL AND LABORATORY PASTEURIZED MILKS WHEN STORED A2L 11° II- A M A5L L- H22: extended periods OF TIME 84 Keeping Q.ual i ty of Commercially Pasteurized, and Homogenized Ifilk Based on Flavor Score 1. Milk stored at 40° F_. Data in Table XXIII show the keeping quality of the milk from each plant at some different seasons of the year. These data show that, "based upon the length of time in days that the milk retained its good flavor (flavor score of 37 or above), the milk from Plant A had a keep­ ing quality of 24 days in July, l4 days in September, 17 days in Novem­ ber and 7 days in December, with an average of 15*5 days; milk from Plant B had a range in keeping quality extending from 10 to 17 days, with an average of 13-2 days, which is slightly lower than the milk from Plant A; whereas, milk from Plants C_ and D had keeping qualities which ranged from l4 to 21 days with averages in both cases of 17*3 days. The average keeping qualities of the milk collected in July ranged from l4 to 24 days, and averaged 18.3 days. For the milk collected in September the keeping qualities were l4 days for milk from three of the plants, and 21 days for that from the fourth plant. The average keeping quality for the milk collected from all the plants in September was 15-8 days. All the milk had a keeping quality of 17 days in November; for that collected in December the keeping qualities ranged from J to 17 days, with an average of 12.8 days. These results show that the keeping quality of the milk from Plant A varied, while that of Plant B was rather more consistent, but showed a slightly lower average; also, that the keeping qualities of the milk from Plants C and D are almost identical, and somewhat better than the 85 milks from Plants A and B. These results also show that, whereas there is no great seasonal variation, the trend is toward a poorer keeping quality in the fall and winter. Milk stored at 5_5° H* Data in Table XXIV show the results for duplicate samples of milk collected in September, November, and December when the milk samples o were stored at 33- ® ie keeping quality of the milk from Plant A varied from 42 days in September to 21 days in December, with an average for the 3 samples of 3^*3 days. For the milk from Plant B the keeping quality varied from 42 days in September to 28 days in November and De­ cember, with an average of 3^*7 days. For the milk from Plant £ the keeping quality was the best in November (49 days) and the poorest (35 days) in December, with an average for'the 3 samples of 42.0 days. The milk from Plant D showed the greatest variation, with a keeping quality of 42 days in September, and only 14 days in December, with an average of 2 5 .7 days. The keeping qualities by months show an average of 42 days in September, 3^*5 days in November, and 24.5 days in December. Data in Table XXV show that if a flavor score of 3^ to 3h (fairflavor milk) had been picked as the end-point in determining the keeping quality of milk instead of a flavor score of 37 to 39 (good-flavor milk), only 3 of the 16 samples would have reflected extended keeping quality. Whereas, this analysis for the milk stored at 33° table form the data are essentially the same. is n°t shown in So Data in Table XXVI show that the most coipmon criticisms of the flavor of the milk stored at 40° F. when scoring 37 above was " cook, ed", "feed", "flat", and "lacks freshness" in that order. However, the most common criticisms of the flavor of the milk scoring 3b. 5 or less were "unclean", "bitter", "high-acid", and "sour". cid" and "fruity" flavor was encountered. An occasional "ran­ The same conditions were found in the milk stored at 33° F . , although not shown in table form. Interestingly the oxidized flavor was never encountered. 3• Discussion of keeping quality at 33° F. vs. 40° F. based on flavor. These results show that the milk from Plant D, which had one of the best average keeping qualities when stored at 40° F. had the poorest average keeping quality when stored at 33° F. This was due mainly to the poor keeping quality at the 33° F. storage temperature for the milk collected from this dairy in November and December. This can be ex­ plained in part at least by the organisms which predominate in this milk. These will be discussed in Section III. Also, a more pronounced seasonal variation in the milk stored at 33° F. was noted than in the milk stored at 40° F. quality of the milk stored at 33 o The average keeping F. decreased 17 days between September and December, while the keeping quality of duplicate samples of milk stored at 40° F. decreased only 3 days. The data in Table XXVII show, however, that by lowering the storage temperature of the milk from 4o to 33° F. the keeping quality based on flavor was increased on the average from 9*7 to 20 days, depending upon the dairy from which the milk was collected. On the average the keeping 27 quality was increased 26 days for the milk collected in September, but only 9 .2 5 days for the milk collected in December. Interestingly, the keeping quality of the milk from Plant D col­ lected in December was 17 days for that stored at 40° P., but only l4 days for that stored at 33° F. Actually this might have been the case, or it might have been due to the inability of the judging panel to agree upon the identity of this flavor, or to the degree to which the off flavors were objectionable, thereby, disagreeing on the flavor score. Variations in different peoples' ability to identify and evaluate the intensity of some flavors are recognized. It is also of interest to note that the keeping quality is not significantly longer where an end point for evaluating keeping quality is taken as a fair-flavored milk (flavor score 3^-36) over an end point of good-flavored milk (flavor score 37~39)• As it will be shown, the keeping quality of these milk samples was closely related to the bacteri­ al activity in the milk this is to be expected, as the bacterial growth is logarithmic in nature. Thus, once an off flavor due to bacterial action starts to develop it progresses rapidly. The common flavors encountered in the milk stored at 40° P. or 33° F . scoring 37 or above were "cooked", "feed", "flat", and "lacks freshness", whereas the most common flavors encountered when the milk was no longer considered of good flavor (flavor score 3b or less) were "unclean", "bit­ ter", "high-acid" and "sour". 88 TABLE XXIII The keeping quality of commercially pasteurized and homogenized milk as shown by the flavor score when the milk was stored at 4o° F. for various periods of time. Length of time (days) milk retained its good flavor* when collected and stored during month of Milk from Plant July September November December Average A 2b ib 17 7 15-5 B lb lb 17 10 13.8 C ib 21 17 17 17.3 D 21 lb 17 17 17*3 is.3 15.8 17 12 .s Averages i>y months * Flavor score 37» or above. 83 TABLE XXIV The keeping quality of commercially pasteurized and homogenized milk as shown by the flavor score when the milk was stored at 33° ^ • for various periods of time Length of time (days) rnilk retained its good flavor* when collected and stored during the month of Milk from Plant September November December Average A 42 28 21 30.3 B 42 28 28 32.7 C 42 49 35 42.0 D 42 21 lb 25*7 Averages by months 42 31.5 24.5 * Flavor score 37» or above 90 TABLE XXV Increased keeping quality of milk stored at *40 F. when the end point was taken as flavor score 3^ to (fair-flavor milk) as against 37 to 39 (good-flavor milk) Milk from Plant Increased length of time (days) milk retained a "fair" flavor, as against a "good" flavor t when collected and stored at 40° F . during the month of July September November December A 0 7 0 3 B 0 0 0 0 C 0 0 0 0 D 0 0 0 3 91 TABLE XXVI The distribution of flavor criticisms among the various flavor score groups for commercially pasteurized and homogenized milk stored at 4o° F. for various periods of time Flavor criticism Number of samples of each flavor criticism that fell in eachl flavor- score group 39 38 37 3b 35 34 33 32 31 30 0 10 24 l - - - - - - - - Feed 2 9 - - ■= - - - - - - Flat 1 4 2 1 - - - - - - - 7 1 - - - -- - - - - Cooked Lacks freshness Unclean _ 4 5 3 - 1 2 2 - i 2 Bitter - - - - - - 1 1 - i 9 High-acid - - - 1 2 - 1 1 - 4 l Sour - - - - - - - - - - 12 Rancid - - - - - - - 1 - - - Fruity - - - - - - - 1 - 13 4S 9 5 2 1 4 6 0 Total no* samples 1 6 25 92 TABLE XXVII The increased keeping quality of commercially pasteurized and homogen­ ized milks as shown by flavor score when the milk was stored at 33° as against storage at Uo° F.: Milk from Plant Increased length of time (days) milk retained its good flavor when stored at 33° 3P. as against Uo° F. when collected and stored during month of Sept ember November December Average A 28 11 ib 17.7 B 28 11 18 1 9 .0 C 21 32 8 20.3 D 28 h -3 9*7 Average by months 26.3 1U .5 9 .2 5 93 Quality of Commercially Pasteurized and Homogenized Milk Based on Bacterial Standards 1. M i l k stored at Uo° F. Data in Table XXVIII show the keeping quality of commercially pas­ teurized an d homogenized milk, as determined by a maximum bacterial standard of 50,000 when the milk was stored at Uo° F. for various per­ iods of time. The bacteria content was determined by the Standard Plate count (APHA, 19*48) except for the mil k collected in September, when use of the standard plate count was not available. The psychro- philic bacteria count was used in this case. These data show that, based on the length of time in days before a bacteria content of 50,000 per ml. was reached, the milk from Plant A h a d a bacterial keeping quality of 10 days in July, and 3 days in Septernber, November and December, with an average keeping quality of *4.7 days. The mil k from Plant B h a d a bacterial keeping quality of 10 days for the m i l k collected in July, September, and November, and a keeping quality of 7 days for the m i l k collected in December. The average bacterial keeping quality for the milk from Plant B was 9-2 days. The m i l k from Plant £ had a bacterial keeping quality of 10 days in July, September, a nd December, with an and a keeping quality of 1*4 days in November, average keeping quality of 11 days. The milk from Flant D had a uniform bacterial keeping quality of 1*4 days for all of the samples. The bacterial keeping quality as effected by season is shown (Table XXVIII) to range from an average of 11 days for the mil k collected in July down to 8.2 days for the m i l k collected in December. 94 It should be pointed out, however, that for the milk collected in September, November, and December, that 3 otLt of 4 of the plantshad milk which had keeping qualities which ranged from 10 to l4 daysin all but one case. If the milk from Plant A were eliminated, the average keeping quality, based on bacterial content, would be considerably higher for the milk collected during these months. Milk stored at 33° F,* Data in Table XXIX show the keeping qualities of milk collected in November and December, based upon a bacterial standard of 50*000 per ml. Bacterial data for the milk collected and stored at 33° in July and September were not obtained. The data show that the milk from Plant A had the poorest bacterial keeping quality, ranging from J to l4 days, and averaging 10.5 days. The milk from Plant B had a bacterial keeping quality of 28 days for both the milk collected in September and December. The milk from Plant C had the best bacterial keeping quality, ranging from 35 to 49 days, and averaging 42 days. The milk from Plant D had a keeping quality based upon bacterial content of 21 days for both samples. The keeping quality of the November samples averaged 28 days, while that of the December samples averaged 22.5 days. 3* Discussion of the keeping quality at 35° F. and 4o° F . based on bacterial content. The limited data show that the keeping quality of the milk from Plant D, based on bacterial standards, was one of the best when the milk was stored at 40° F . , but one of the poorest when stored at 33° 95 However, the data in Table XXX show that by lowering the temperature of storage from 40° F. to 33° F. the keeping quality, based on bacteria 1 content, is increased from 7 20 days, depending upon the source of the milk, and that the increase is greater for the milk delivered in November (15 days) than for the milk delivered in December (ll.8 days). \ 96 TABLE XXVIII The keeping quality of commercially pasteurized and homogenized milk as shown by the bacterial content when the milk was stored at bO° F. for various periods of time Milk from Plant Length of time (days) before a bacterial content of 50.000 per ml. was reached when the milk samples were collected and stored during the months of December July September A 10 3 3 3 b*7 B 10 10 10 7 9.2 C 10 10 ib 10 11 D 1^ ib ib ib ib Averages 1^ months 11 3.2 November 1 0 .2 8.2 Average 97 TABLE XXIX The keeping quality of commercially pasteurized and homogenized milks as shown by the bacterial content when the milk was stored at 33° F. for various periods of time Milk from Plant Length of time (days) before a bacterial content of 50,000 per ml. was reached when the milk samples were collected and stored during the months of November December Average 10 .5 A li+ 7 B 28 28 28 C bs 35 k2 D 21 21 21 28 22.5 Average months 98 TABLE XXX The increased keeping quality of commercially pasteurized and homogen­ ized milk as shown hy "bacterial content when the m i l k m s stored at 33° F. as against Uo° F. Milk from Plant Increased length of time (days) before a bacterial con­ tent o‘f 5 ^,0 0 0 per ml. was reached when milk was collected and stored at 33° F. against *40° P ., during the month of November December Average A 11 4 7.5 B IS 21 19-5 C 25 15 2 0 .0 D 7 7 Average t>y months 15 11.8 7 99 Relationship of Psychrophilie Bacteria Content to Flavor Score of Commercially Pasteurized and Homogenized Milk Data presented in Table XXXI show the number of milk samples which were placed in each flavor-score classification having psychrophilic bacteria counts that fell within various groupings, when commercially pasteurized and homogenized milk samples are stored at 40° F. for vari­ ous periods of time. These data show that the majority of the milk samples which were scored 38 or 39» had psychrophilic bacteria counts of less than 50,000. However, it will be noted that a number of sam­ ples (6 out of 29) which scored 39 had psychrophilic counts above 50,000 per ml. and that 21 out of *+9 samples which scored 38 had psychrophilic counts of 50,000 or above. However, only an occasional sample that scored 37 °r less had psychrophilic counts of less than 50,000 per ml., and most of these samples had psychrophilic counts of between one mil­ lion and 50 million per ml. Data in Table XXXII show the logarithmic average bacteria counts and average flavor scores for the commercially pasteurized and homogen^ 0 ized milk stored at 40 F. Recognizing that whenever averages are taken, the extremes are leveled off, these data show that the flavor score does not start to decrease until the psychrophilic bacteria con­ tent shows a marked increase. These data show also that an increase in psychrophilic bacteria content of from 11 to 6,390 (a 5^,000 percent increase) is possible without an adverse effect upon the fla­ vor score, but that when the psycrhophilic bacteria content reached 23b, 000 per ml. (a 2 ,1^0 ,0 0 0 percent increase) that the flavor score is adversely affected and the milk is no longer classified as a good 100 quality milk. These data also show that as the psychrophilic bacteria content continues to increase that the flavor score decreases. These data are shown graphically in Figure 3. The relationship of the psychrophilic bacteria content to the flavor score of milk stored at 33° F. is shown in Table XXXIII, and Figure 4. It is recognized that the number of samples of milk stored at 33° F. are too small for significant evaluation, yet the data avail­ able were collected during November and December, a period of poorest keeping quality, as pointed out earlier, and as shown by other investi­ gators (Dahlberg et_ al. 1953)* Thus, the conditions are probably as poor as they will ever be. The data show that the increase in psychrophilic bacteria per ml. of milk is somewhat less than that encountered at 4o° F. at the point that the flavor is adversely affected (flavor score 34.5)* Also, that psychrophilic bacteria content per ml. of the milk does not become as high (2 ,500,000 as against 26,300,000) at the point where the milks are considered unsalable. It is of interest also, that the age of the milk stored at 33° F., when they were no longer considered of good flavor or bacteriological quality, fell between 21 and 23 days. Thus, based on average conditions (recognizing that some milk will have better and some poorer keeping quality), lowering the storage temperature for commer­ cially pasteurized and homogenized milk by 7° F. (4o to 33° F.), the keeping quality is increased 11 to l4 days. It should be pointed out that whereas the comparison of psychrophilic bacteria counts to flavor score does not demonstrate it well, in general, the milk stored at 40° F. showed an increase in bacterial content sufficient to exceed the 50,000 101 per ml. standard, several days before the flavor deteriorated to a point at Which the milk was no longer of good flavor quality. This was less evident in the milk stored at 33° F., than at 40° F. At this storage temperature flavor and bacterial quality of the samples were more nearly equal. 102 TABLE XXXI The number of milk samples in each flavor-score classification that fall within various groupings of psychrophilic bacteria when commer­ cially pasteurized and homogenized milk were stored at 4o° F. for vari­ ous periods of time. Psychrophilic bacteria Number of milk samples falling into each bacterial classification that had flavor scores of groupings 39 38 37 36 35 34 33 32 31 30 0 0- 1 ,0 0 0 19 27 0 0 0 0 0 0 0 0 0 1 ,001- 10 ,0 0 0 3 4 1 0 0 0 0 0 0 0 0 1 0 ,001- 50,00 0 1 6 1 1 0 0 0 0 0 0 0 5 0 ,001- 100,000 1 11 0 0 0 0 0 0 0 0 0 1 0 0 ,001- 1 ,000,0 00 3 4 2 0 0 0 0 0 0 0 0 1 ,00 0,001- 5 ,000,0 00 1 4 4 0 0 0 0* 1 0 0 2 5 ,0 0 0 ,001- 1 0 ,000,000 0 1 1 2 1 1 1 0 0 1 0 1 0 ,0 0 0 ,001- 5 0 ,0 0 0,0 00 1 1 2 1 0 1 1 1 0 3 s 5 0 ,00 0 ,001- 0 0 0 0 1 0 0 1 0 0 4 11 4 2 2 3 3 0 5 15 up Total no. of samples 29 ^ . 103 TABLE XXXII The logarithmic average psychrophilic bacteria counts and average fla­ vor scores for commercially pasteurized and homogenized milk stored at 40° F. for various periods of time Time (days) milk stored 4o° Logarithmic average bacteria counts Average flavor score* 0 11 38.5 3 25 38.5 7 220 38.5 10 b, 390 38.0 l4 236,000 3 6 .6 17 3 ,500,000 35.7 21 9 ,600,000 32.5 24 26,800,000 28.3 * A flavor score of 25 wa-s given any sample considered unsalable. 104 40 MILLIONS f 36 PSYCHROPHILIC 34 32 30 28 Psychrophilic Count 7 Figure 3* 14 42 35 21 28 DAYS MILK STORED 3 3 ° E 49 relationship of the logarithmic average "bac­ teria counts to the average flavor scores of commercially pasteurized and homogenized milk stored at 4o° F. 25 56 SCORE Flavor Score FLAVOR COUNT 38 105 TABLE XXXIII The logarithmic average psychrophilic "bacteria counts and average fla­ vor scores for commercially pasteurized and homogenized milks stored at 33° F* for various periods of time Time (days) milk stored at 330 F. Logarithmic average bacteria counts Av erage f1avo r score** 0 27 38.5 7 26 38.5 1*4 ^95 3S.0 21 4 ,5 0 0 37.0 28 382,000 34.5 35 150,000 3 M 42 1,840,000 30.5 49 ♦ * 56 2 ,500,000 29.5 * ** No usable data available* A flavor score of 25 was given any sample considered unsalable. 106 40 M ILLIO N S 36 20 PSYCHROPHILIC 34 32 30 Psychrophilic Count O Figure 3 7 14 17 21 10 DAYS MILK STORED 4 0 ° F 28 25 24 The relationship of the logarithmic average bacteria counts to the average flavor scores of commercially pasteurized and homogenized milk stored at 33° SCORE F favor Score FLAVOR COUNT 38 107 Keeping Quality of Laboratory Pasteurized Milk Lata in Table XXXIV show that in most cases there was no bacteria growth in the laboratory pasteurized milk samples until the 28th to 42nd day of storage of the milk at 4o° F. The milk varied in this respect with the source, but in 4 out of 5 milk samples, bacterial growth at 40° F. was evident in the milk by the 42nd day of storage. In most cases, this increase in psychrophilic bacteria was accompanied by a deterioration of the flavor. Whereas, this observation may have little or no application to commercial market milk, it may be significant in pointing out that the long time of storage of creams or other products which have been pasteurized at normal temperatures, is limited at stor­ age temperatures of 4o° F. The keeping quality of the samples of laboratory pasteurized milk stored at 33 o F. sistent increase temperature* is shown in Table XXXV. These data show that no con­ in bacteria content through 70 days of storage at this An occasional sample showed a high psychrophilic count, but the high count failed to be encountered at the next analysis, lead­ ing to the belief that the sample which gave the high count was somehow contaminated, possibly through a loosened cap. However, it will be noted, that, although bacterial growth was not evident, the flavor score after 56 days of storage was usually below that which is considered to be of good quality.In one sample of milk, the flavor was considered to be of poor quality (below 36) at the 42nd day of storage. A discussion of the characteristics of the organisms involved in these laboratory pasteurized milk samples will be taken up in the next section. 108 TABLE XXXIV The psychrophilic bacteria counts and flavor scores of laboratory pas­ teurized milk stored at Uo° F. for extended periods of time Time (days) milk stored Psychrophilic count Flavo r score Criticism 38 39 38 38 35 Cooked — * Cooked Cooked Dirty 37 37 38 38 38 Cooked SI. oxid. Cooked 38 Feed — Cooked Musty Dirty Plant 0 i4 28 42 56 10 10 10 10 10 Plant A-2 0 i4 28 42 56 10 10 10 910,000 3 ,260,000 — SI. musty Plant B 10 10 100 b5,000 9,120,000 0 ib 28 42 56 39 38 30 32 Plant C 0 14 28 42 56 10 60 19,600 2,400,000 1 5 ,900,000 39 39 37 35 35 — . Chemical Unclean Unclean Plant D 0 ib 28 42 56 160 io 2b0 4,500,000 91,200,000 * No criticism. 38 39 38 34 1 /2 32 Cooked Cooked Unclean Dirty 109 TABLE XXXV The psychrophilic bacteria counts and flavor scores of laboratory pasteurized m i l k stored at 33° extended periods of time Time (days) m i l k stored Psychrophilic count Flavor score Critici sm Plant A 0 l4 28 42 56 70 10 10 500 100 100 100 38 39 38 38 39 0 Feed Cooked Lack freshness --- - Medicinal Plant B 0 28 10 10 10 42 56 70 33 ,ooo 500 2,300 i4 38 39 38 33 1/2 35 32 Feed -».= Cooked Unclean SI. dirty Old stale Plant C 0 i4 28 42 56 70 10 10 10 39 39 38 36 39 36 35 2,400 350,000 — Cooked Unclean — Old stale Plant D 0 14 150 10 * 28 42 56 70 * No criticism. ** No data. 250 100 240,000 38 39 38 36 38 33 Cooked — - Co oked Unciean — Old-unclean 110 Summary This study showed that the keeping qualities of commercially pas­ teurized and homogenized milk, when stored at 4o° F. for various periods of time, were from 13 to IS days, based on flavor score (37 or above) and S to 11 days when based on bacterial quality (5 0 ,00 0 per ml. stand­ ard). Also, the keeping quality is poorer to the extent of 3 days for milk collected in December than for milk collected in July. When the milk was stored at 33° * the keeping quality, based on flavor score, was from 24 to 42 days, with the best keeping quality in the September milk and the poorest in the December milk. When based on bacterial standards, the keeping quality of the milk stored at 33° was slightly poorer only to the extent of about 2 days. ing of the storage temperature from 4o to 33° (7° Thus, the lower­ ) resulted in an increased keeping quality of from 11 to l4 days, based on flavor and bac­ terial standards. The oxidized flavor was not encountered. There was definite cor­ relation between the keeping quality of commercially pasteurized and homogenized milk and bacterial activity. The deterioration of laboratory pasteurized milk, free of post-pas­ teurization contamination, had a keeping quality at 40° F. of between 28 and 42 days, more than twice that of the commercial products. How­ ever, these milk samples eventually showed bacterial growth, which was correlated with the deterioration of the flavor score. When laboratory pasteurized samples were stored at 33° the keeping quality ranged from 42 to 56 days, but the deterioration of the Ill flavor could not be correlated with bacterial activity. Bacterial enzyme systems may have been responsible, however, as the flavors on the JOVa day of storage (’’stale” , ’’old”, ’’unclean”) are typical of pro­ tein decomposition. SECTION III SOME CHARACTERISTICS OF THE ORGANISMS COMMONLY FOUND IN FRESH AND DETERIORATED COMMERCIALLY PASTEURIZED AND HOMO­ GENIZED MILK AND IN LABORATORY PASTEURIZED MILK 113 The Effect of Various Plating Procedures on the Bacteria Counts Obtained from Commercially Pasteurized and Homogenized Milk Stored at 53 and 4o° F. for Extended Periods of Time Bata in Table XXXVI show the effect of different plating pro­ cedures on the bacteria counts obtained from two commercially pasteur­ ized milk samples stored at 4o° F. These data show that sample A had a considerably lower standard plate count than sample B, and that the rate at which the standard plate counts increased in sample A was consid­ erably faster than in sample B. The data show also that for both samples the psychrophilic bacteria count, obtained by incubation of agar plates at 50° F. for 20 days, were comparable to the standard plate counts on the fresh milk, and usually slightly higher on the milk stored for 10 days or longer at 4-0° F. The psychrophilic bacteria counts obtained by incubation of the agar plates at 40° F. for 20 days were found always to be lower than either the standard plate count or the psychrophilic count obtained at 50° F* sample A the psychrophilic count of the freshly pasteurized milk obtained by incubation of the agar plates at 40° F. was 100 per ml., and this count increased rapidly during the stor­ age of the milk at 4o° F. The coliform count was also significant (8 per ml.) and also increased rapidly during storage of the milk. In sample B, however, the psychrophilic counts of the fresh milk obtained by incubating the agar plates at 4-0° F. was too low to detect in 0.1 ml. of milk until after the milk had been stored for 7 days at 4-0° F. rapidly. After 7 days of storage the psychrophilic counts increased The coliform counts of sample B were negative throughout the nh salable life of the milk. These data, represented the two extremes which were encountered. Bata in Table XXXVII show the effect of various plating proce­ dures on the bacteria counts obtained from commercially pasteurized milk stored at ^0° F. where all of the bacteria counts are grouped into log­ arithmic averages. These data show that the standard plate counts de­ crease slightly when the samples are first placed in storage and that that the psychrophilic bacteria counts obtained, using an incubation temperature of 50° F. for 20 days, are lower than the standard plate count of the fresh milk. However, when the milk samples were stored for 14- days and longer at 4-0° F. the psychrophilic count, using a 50° F.incubation temperature, gave higher bacteria counts than the standard plate count. The psychrophilic counts obtained, using a 4o° F.-incubation temperature, were low averaging 9 per ml. on the fresh milk, but in, creased as the milk was stored at 40 o F. When the milk samples had been stored 17 days or longer the psychrophilic counts obtained using a 40° F.-incubation temperature were higher than the standard plate counts, but never equaled the psychrophilic counts obtained using a 50° F.- in­ cubation temperature. The logarithmic average of the coliform counts is shown but is of no significance, as several of the milk samples had negative coliform tests throughout the storage period. Data in Table XXXVIII show the effect of various plating proce­ dures on the bacteria counts obtained from commercially pasteurized milk 115 stored at 33° F. Milk samples from the same source are used here as in the comparison, using a storage temperature of 40° F. (Table XXXVI). A comparison of these two tables will show that, in the case of sample A, the psychrophilic count obtained, using a 4-0° F.-incubation tempera­ ture, increased almost as fast when the milk was stored at 33° F. as when it wa.s stored at 4-0° F. However, the coliform counts increased at a much slower rate in the milk stored at 33° F. than in the milk sotred at 4o° F. The standard plate counts also showed a slower increase in the milk stored at 33° F. and never reached as high a total count. The data from sample B, however, show about the same results when stored at 33° F. as at 4-0° F . t except that increases in bacteria counts, as determined by the standard pla te count or the psychrophilic count, were much slower. The standard plate count actually decreased until the milk had been stored for 35 days. The psychrophilic count was less than 10 per ml. until the 28th day of storage. Data in Table XXXIX show the effect of various plating procedures on the bacteria counts obtained when all of the samples stored at 33° F. were grouped into logarithmic averages. These data show for the milk stored at 33° F., that the standard plate count decreased until the l4th day of storage of the samples, and then steadily increased throughout the usable life of the milk. The psychrophilic counts obtained using a 4-0° F. incubation temperature was low (27 per ml.) but showed a signifo icant increase on the 14-th day of storage of the samples at 33 F. The psychrophilic count surpassed the standard plate count when the milk had been stored 28 days or longer. The coliform counts were low and did not increase throughout the salable life of the milk. 116 The Relationship of the Bacteria Counts Obtained, on Freshly Pasteurized Milk to the Keeping Qnality Data in Table XXXX show the relationship of the standard plate, psychrophilic, and coliform counts on the freshly pasteurized milk to the keeping quality, when the milk was stored at 33 a-Hd- ^0° F. These data show that the milk samples which had the lowest aver­ age standard plate counts on the freshly pasteurized milk (Plants A and B) had the poorest keeping quality when the milk samples were stored at 4-0° F., and that the milk samples which had the highest average standard plate counts on the fresh milk had the best keeping quality* The same relationship is shown also when the milk samples are stored at 33° F., except for the milk from Plant D. which showed a high average standard plate count on the fresh milk, but a poor keeping quality when the milk was stored at 33° F. These data also show that the milk from Plant A and B which had higher coliform and psychrophilic counts than milk from Plants £ and D, also had the poorest keeping quality when stored at 40° F. The milk from Plants £ and D had negative coliform and low psychrophilic counts (10 per ml.) and better keeping qualities when stored at 4-0° F. This relationship also held true for the milk stored at 33° F. except for the milk from Plant D, which had negative coliform and low psychrophilic counts, but a relatively poor keeping quality* 1. Discussion of results. These data show that: H7 a) the standard plate count decreases when the milk samples are placed in storage at 33 an ^0 anrd transfers at low tempera­ tures over the growth rate at the 1st transfer from room temperature, although the 3rd transfer is some 3^ days after the original inoculation, during which time the organisms have had 30 days of constant exposure to these low temperatures. In fact, the results obtained indicate that successive transfers at low temperatures (33° 54) result in growth rates which are less than the growth rate at the initial transfer from room temperature to 33° 54 These results tend to place a reasonable doubt upon the statements that organisms adapt themselves to rapid growth at low temperatures, and become an important cause of food spoilage. Rather the spoilage is like­ ly due to contamination with a spoilage organism which naturally grows at the temperature of storage. Recognizing their limitation, the data seem to indicate that an organism grows at a fairly definite rate at a given temperature, and that little adaptive processes toward faster 153 growth take place upon exposure of the organism to low temperatures for extended periods of time. However, further study on the ability of im­ portant milk organisms to adapt themselves to growth at low temperatures is needed. OF BACT. LOG 0 2 4 7 9 fI 14 16 18 21 24 27 29 31 33 35 DAYS INCUBATION Figure 8. The rate of growth of Culture No. 9 at incubation temperatures of 33 and U0° F. 9 LOG OF BACT. 8 7 5 — 40° F 4 Cult. No. 4 — 3 3 °F 3rd Trans. 1st Trans. 3 0 2 5 7 9 II 13 0 2 5 7 9 II 13 DAYS INCUBATION Figure 9- The rate of growth of Culture No. 4 at the 1st and ^rd ■ transfer when incubated at 33 and 40° F. 9 LOG OF BACT 8 7 6 5 Cult. No. 14 1st Trans. 3rd Trans. 4 0 2 4 7 9 II 13 16 18 21 24 DAYS INCUBATION Figure 10. The rate of growth of Cul­ ture No. lU at the 1st and 3rd transfer when incubated at 40° F. LOG OF BACT. 9- 4" Cult. No. 14 1st Trans. 3rd Trans. DAYS INCUBATION Figure 11. The rate of growth of Cul­ ture No. 14 at the 1st and 3rd transfer when incubated at 33° F. CD 5 Cult. No. 10 I st Trans. 2nd Trans. e> 0 2 4 7 9 11 13 15 17 2 0 22 24 DAYS INCUBATION Figure 12. The rate of growth of Culture Ho. 10 at tjjie 1st and 3rd transfer when in­ cubated at U0° F. O Cult. No. 10 1st Trans. o — 2nd Trans. 2 «--» 1— * DAYS INCUBATION Figure 13* The rate of growth of Culture Ho. 10 at the 1st a.nd 3rd transfer when in­ cubated at 33° F. h-* 7 o A 00 o u_ O 5 Cult. No 2 2 0 1st Trans. 2nd Trans. o -i 4 0 2 4 7 9 II 14 16 18 21 DAYS INCUBATION Figure 1^. The rate of growth of Culture No. 220 at the 1st and 2nd transfer when incubated at k0° F. < 6 GO Ll 5 Cult. No. 2 2 0 1st Trans. 2nd Trans. DAYS INCUBATION Figure 15* The rate of growth of Culture !To. 220 at the 1st and 2nd transfer when incubated at 33° F. DISCUSSION As pointed out by Erdman and Thornton (1951st) there seems to be no universally recognized definition of psychrophilic bacteria. As applied to milk, the term ,,psychrophilic,, bacteria is usually taken to mean all those bacteria which will grow at some designated refrigera­ tion temperature. These temperatures range from 10-5° C. (Erdman and Thornton (l951& ) t o as low as 0° C. (Pennington, 190S), with 5 to 10° C. being the more common. In addition to the temperature of growth, another factor, namely, the ability of this group of organisms to survive pasteurization ex­ posures, has also been introduced as a characteristic of the psychro­ philic bacteria commonly found in milk. Sherman et a!. (19^1), Clayton (I9h3), Dahlberg (I9^ba), Thomas et al. (1951) ahd Meyer (1950) indicate their belief that the psychrophilic bacteria commonly found in milk are not thermoduric, but gain entrance to the milk after pasteurization. On the other hand, Ayers and Johnson (1913)» Ravenal et al. (1910), Prescott et al. (1931)» Burgwald and Josephson (l9*+7)t Rogick and Burgwald (1950)* Jezeski and Macy (19^6), Egdell and Bird (1950), Kenned;/ and Weiser (1950), Roadhouse and Hen­ derson (19^1 ), Elliker et al. (1951) an(i ^bd at al. (1952) indicate that at least some organisms which grow in milk at refrigeration temperatures are thermoduric. This difference in opinion apparently is due to the different tem­ peratures used to grow these organisms and to the variations in the heat resistance of the organisms as a result of previous environmental growth 159 conditions. Such variations have been pointed out by several inves­ tigators, including Sherman et al. (1929). The study herein reported has shown that most of the thermoduric bacteria isolated from pasteurized milk will grow at 10° C. (50° F.), but not at 5° C. (^0° F.) within a 20-day period. Thus, if the heat resistance of the organism is to be used as a characteristic of psychro­ philic bacteria commonly found in milk, a more limited temperature range for their growth would seem to be necessary. In view of the importance of the psychrophilic organisms to the keeping quality of milk, a standard procedure for detecting their pres­ ence and for interpreting the data would seem to be desirable. Such methods would result in more comparable future studies and less con­ fusing data in the literature. In the light of the results of the in­ vestigation reported herein the present standard method for determining psychrophilic bacteria (APHA, 19^8), which specifies a temperature of 5 to 10° C. for 10 to id days, does not seem to be adequate. Accord­ ingly the following procedure for determining psychrophilic bacteria and for interpretation of results is suggested: The psychrophilic bacteria content of fluid milk should be de­ termined by the preparation of agar plates in accordance with Standard Methods (APHA, 19^3). The temperature of incubation of the plates should be 40° P. (5° for a‘ fc least 1° days, and preferably 20 days. The bacteria count should be interpreted as having been comprised of obligate and facultative psychrophiles. These organisms are most likely responsible for the bacteriological deterioration of milk stored at refrigerator temperatures (Uo° P. or less) and are for the most part_non-thermoduric when normal pas­ teurization exposures are used. /Since this was written Nelson (1953) k3-9 indicated that Standard Methods (APHA, 19^-8) is to be changed and thenext edition will specify an incubation temperature of 5° C. (40° F.) for 10 daysjf 160 This study demonstrated the presence of spore-forming psychrophilic bacteria which were thermoduric in certain milk samples. Some thermo­ duric psychrophilic "bacteria have "been described by other investigators. It is believed, however, that the thermoduric organisms which grow at refrigeration temperatures are of more academic than commercial interest so far as the keeping quality of the milk is concerned* However, there is much that remains to be learned relative to the effect of time and temperature used in the cold storage of raw milk on the efficiency of pasteurization and the significance in terms of keep­ ing quality of the organisms that survive pasteurization. It has been pointed out by several investigators (Sherman et al. 1929; Fabian and Coulter, 1930; Hammer and Hussong, 1931; and others) that young bacterial cells are more ea.sily killed than old cells. Sherman et^ al. (1929) showed that holding milk at cold temperatures prior to pasteurization reduced the efficiency of pasteurization. On the other hand, Hammer (19^+3) and Olson at al. (1952), as well as others, demonstrated that organisms were more heat resistant when grown at their optimum than when grown at lower temperatures. In view of these reports, it seems logical to expect that raw milk, which has been stored at refrigeration temperatures for an extended period of time prior to pasteurization, will have a higher standard plate count.after pasteurization than that milk held at higher tempera­ tures. This supposition is based on the greater heat resistance of the old bacterial cells. The higher count will be due largely to the pres­ ence of the thermoduric bacteria which grow readily at 35° C.» *>ut ho not grow at refrigeration temperatures. On the other hand, those lbl organisms in the raw milk which do grow at refrigeration temperatures (about 4o° F.) likely will have their heat resistance lowered and thus be more susceptible to destruction by pasteurization. Apparently, it is this latter group which is most important in the keeping quality of milk. In view of the prevailing tendency to hold raw milk at refrigera­ tion temperatures for longer periods of time prior to pasteurization, it would be desirable to determine the storage time-and temperature relationship of the raw milk to the pasteurization efficiency in terms of both the thermoduric and the psychrophilic bacteris. Such informa­ tion appears to be needed in order to arrive at the most advantageous storage time and temperature for raw milk. That psychrophilic bacteria common to milk are not thermoduric and that thermoduric organisms do not adapt themselves to growth at refrig­ eration temperatures has been pointed out. Thus, it can be assumed that the thermoduric bacteria play little or no part in the keeping quality of milk stored at refrigeration temperatures. Dahlberg et_ al. (1953) showed that the standard plate count of the fresh milk collected in one city was low in the winter, and high in the summer. However, after storage of the milk at 44° F. for 7 days the winter milk had the highest counts, and the summer milk the lowest. The data collected in the present study show that milk with a high standard plate count on the freshly pasteurized milk had good keeping qualities at 40° F. , while milk samples with low standard plate counts had poorer keeping qualities. 162 Actually, a standard plate count on freshly pasteurized milk for the most part is a thermoduric count. of the coccus type. Most of the viable organisms are This raises the question as to whether the pres­ ence of considerable numbers of cocci (thermoduric organisms) in freshly pasteurized milk has any effect on the growth of psychrophilic bacteria (mostly gram-negative rods) even though the thermoduric organisms show no visible growth at refrigeration teiaperatures. Schalm (1953) recently pointed out that the elimination of Strep­ tococcus agalactiae from the udders of dairy cows by treatment and isolation paved the way for infections caused by Micrococcus pyogenes and that elimination of the micrococci favored infections by gramnegative rods of the coliform variety which was never encountered when the cocci were present. The question might well be raised, in absence of any evidence to the contrary, as to whether the constant effort of the milk sanitarian, farmer, and milk plant operators to produce a pasteurized milk of a low (thermoduric) bacterial content is beneficial to the keeping quality of the milk stored under conditions common in the United States today. It is suggested that possibly the seasonal variations in the keeping quality of pasteurized milk might be explained on the basis of an inhibitory effect of a large thermoduric population, more common in the summer milk, on the rod-type organism which caused spoilage. The suggestion has been made that winter milk is more commonly con­ taminated with organisms accustomed to growing at low temperatures than summer milk, and that this is responsible for the poorer keeping quality 163 of winter milk. However, the data obtained in the present study show that the rate of growth of common gram-negative rod types of organisms in milk at low temperatures does not change materially after exposure of the organism to low temperatures for 20 to 30 days.In fact, for some organisms, the growth rate appears to decrease asthe period of exposure to low temperatures increases. It has been shown in this study and pointed out by several other investigators (Burgwald and Josephson, 19^7; Dahlberg et al.. 1953; an& Olson e_t al. , 1953) that neither the standard plate, coliform, or psychrophilic counts on the freshly pasteurized milk are any indication of the keeping quality of the milk. More recently, Olson et_ a l . (1953) has shown that a past history of high standard plate and coliform counts is a fair indication of keeping quality, but that the absence of coli­ form bacteria cannot be depended upon to indicate a good keeping quality. He emphasized that extremely low psychrophilic counts (less than can be observed in 1 or 2 ml. of milk) are necessary to insure good keeping quality. There seems to be no question but that the bacteriological spoilage of milk stored at low temperatures is due to psychrophilic bacteria and that most of these organisms fall into the classification of the gramnegative rods. The gram-positive organisms, largely thermoduric, ap­ parently play no part. Evaluating the keeping quality of the milk is difficult, however, because the gram-negative organisms are present in the freshly pasteur­ ized milk in such few numbers that present methods are unreliable in detecting their numbers. In addition to being unreliable, present day 164 methods ar= slow, requiring 10 days or longer. However, in this study most of the organisms isolated from the deteriorated milk stored at re­ frigeration temperatures were gram-negative rods which showed growth in brilliant-green— "bile broth and lauryl—tryptose broth, even though they did not produce gas. Possibly a keeping quality test of milk could be developed byworking out a technique whereby certain dyes, possibly brilliant green or sodium lauryl sulfate, might be used to inhibit the growth of the gram-positive organisms, and yet permit the growth of the gram-negative rods. The addition of such a dye in the proper concentration to a milk Sample would permit incubation for several hours at about 7^° F. Then the gram-negative rods might be determined by a plate count, using a media also containing the dye, and a J 2 ° F. incubation temperature for 48 to 72 hours. Presumably, such a procedure would eliminate the interference from the gram-posi tive organisms, but increase the numbers of gram-negative organisms in the milk sample, which are probably psychrophilic, so that they could be more reliably determined by a plate count. A gram-negative count (probably psychrophilic) that met a certain standard under such a determination might be correlated with keeping quali ties. SUMMARY The role of psychrophilic bacteria in the keeping quality of cormmercially pasteurized and homogenized milk, stored at 33 *3md 40° F. dur­ ing the summer, fall, and winter months from four da/ry plants has been studied. The keeping quality of laboratory pasteurized milk stored under the same conditions was studied also. The keeping quality of commercially pasteurized and homogenized milk stored at 4o° F. averaged 13 to 13 days, based on a 37-flavor score; and 8 to 11 days based on the ^0,000-per-ml. bacterial standard. On an average, the keeping quality of duplicate samples stored at 73° F. was extended for an additional 11 to l4 days. The keeping quality of laboratory pasteurized samples not subject to post-pasteurization contamination stored at 4o° F. averaged from 28 to 42 days, based on 37-Flavor score, and/or on the 50,000-per-ml. bac­ terial standard. When duplicate samples were stored at 33° F., the average keeping quality was extended an additional 14 to 28 days. The fla.vor deterioration was correlated withthe growth ofpsychro­ philic bacteria at both storage temperatures in all of the milk samples, o except the laboratory pasteurized samples stored at 33 F. The keeping quality of the milk studied in July was superior to that of the November and December milk, regardless of the storage temper­ ature. Oxidized flavors were not encountered in the commercially pas­ teurized and hoinogeni zed milk. The organisms growing on a standard plate prepared from freshly pasteurized milk were found to be largely thermoduric. lure cultures of these organisms grew at 50° F. , but not at 40° F. The majority of the organisms isolated from the milk having the poorest keeping quality when spoiled, were gram-negative rods. Studies showed that most of these organisms were killed by laboratory pasteuri­ zation. However, the milk which had the best keeping quality at either storage temperature was spoiled eventually by the growth of a gram-nega­ tive, heat-resistant, spore-forming rod. The spoilage organisms were found to belong to the Aerobacter, Alcaligenes, Achroraoba.cter, and Clostridium groups. The Aerobacter group was observed to be a common spoilage organism in milk stored at 4o° F . , but not at 33° F. A gram-negative coccus (Micrococcus lipolyticus. Stark and Schieb, 193&) was encountered in one sample. The laboratory pasteurized milk stored at 4o° F. was spoiled event­ ually by the growth of a gram-positive rod, possibly of the Corynebacterium genus. The gram-negative coccus (Micrococcus lipolyticus) was en­ countered also. These organisms were killed by pasteurization tempera­ tures of 1^ 3° F. for 30 minutes when first isolated from the milk stored at refrigeration temperatures but became heat resistant after cultivation at room temperature. Standard plate, coliform and/or psychrophilic counts on the freshly pasteurized milk were found to be of little value in predicting the keeping quality of the milk when held at either 33 or F. The incubation of agar plates at 50° F. resulted in the detection of a group of thermoduric organisms which were not detected at 40° F. A comparison of bacteria counts obtained after incubation of the plates 167 for 7» 10, 15 , and 20 days showed that at no point during the incubation at 50° F. did the bacteria counts coincide with those obtained by incu­ bating the plates at 40° F. The counts were always higher when the plates were incubated at 50° F. A gram—positive, thermoduric micrococcus was found to be unable to grow as a pure culture in milk at 33 °r ^0° F. over a 35-day incubation period* A Pseudomonas organism from a culture grown at room temperatures did not show any faster growth at the 3r) Dahlberg, A. C194bb. The keeping quality of pasteurized milk. Mew York. (Cornell) Agr. Expt. Sta. Bui. 833. 23 pp. (2b) Dahlberg, A. C. 19 49. The keeping quality of pasteurized milk. Dairy Cong, 83-88 . 12th Internatl. (27) Dahlberg, A. C.; H. S. Adams, and M. E. Held. 1953* Sanitary milk control and its rela.tion to the sanitary, nutritive and other qualities of milk. Natl. Acad. Sci.Natl. Res. Council Pub. 290. Wash. 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