Microbiology of Canning W. D. BIGELOW AKD E. J. CAMERON, Research Laboratories, National Canners hssociation, Washington, D. C
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temperature limits and resistance H E scope of this paper on Presercation by canning depends upon the to heat. The obligate thermothe microbiology of can“effective” sterilization by heat of the food material philes are, as a class, more resistning will, for the most and subsequent protection against contact with a ant than the facultative thermopart, be limited to the presentacontaminated environment. B y “effective” steril iphiles. tion of information available on zation is meant the destruction of all microThe three principal thermocanned vegetables and f r u i t s . philic groups are the “flat sour” The principles involved in the organisms capable of growth in the product under thermophiles (facultative anaersterilization of these p r o d u c t s conditions io which the canned product would obes) and two dissimilar groups of have been firmly established, ordinarily be subjected. Effective sterilization of thermophilic anaerobes. One of and the essential microbiological acid products is accomplished by destruction of these anaerobic groups is characinformation upon which they are only the vegetutive bacteria because of the inability terized by the production of hybased has been generally acdrogen sulfide gas unaccomcepted. With respect to the of spores to germinate in this acid environment. panied by appreciable proteolytic nonacid products, there is the I n cegetable products there must be destruction of or saccharolytic changes. The further limitation that this inall spore forms that can grow under usual condiresultant condition in foods is formation applies primarily to t ions of temperature. T h e necessary “process” known as sulfide spoilage. The products that have been procf o r effective sterilization depends upon the initial remaining group is characteressed or sterilized in accordance ized by p r o d u c t i o n of hydrowith commercial practice. contamination of the product, and recent work gen a n d c a r b o n d i o x i d e i n Strictly s p e a k i n g , all vegehas shown the necessity and means for control of varying amounts, together with tables and fruits are acid to some contaminntion sources. concomitant p r o d u c t i o n of degree, but for convenience we volatile acids of which butyric group them as nonacid, semiacid, and acid. The nonacid products, such as peas and acid is predominant. The flat sour bacteria have been studied by Donk (7) and corn, are in the range of pH 6.0 to 7.0. The semiacid products, such as string beans, spinach, and asparagus, are commonly by Cameron and Esty (4). Donk has given the name B. between pH 4.5 and 6.0, most of them higher than p H 5.0. stearothermophilus to the obligate thermophilic flat sour types. The upper limit of the acid range may be taken a t p H 4.5. As the result of study of 214 cultures of nongas-forming sporeThis is about the point below which spore-forming bacteria forming thermophiles, Cameron and Esty define two large cease to be factors in spoilage, and it becomes unnecessary to thermophilic groups as causes of flat sour spoilage. Spoilage by flat sour bacteria is characterized by the producuse a process sufficiently severe to destroy them. Kecessarily these groupings are more or less arbitrary, but tion of acid without gas, resulting in souring without swelling there are microbiological similarities within each group and of the container. Lactic acid is probably the principal end distinct differences between the groups. Consequently, product, although complete biochemical data are lacking. this classification offers a convenient basis for specific dis- The heat resistance of flat sour bacteria has been reported by cussion. Bigelow and Esty (1). Material difference in range of resistance has been noted in this and in subsequent work. SONACID CANNEDFOODS Owing to the variation in resistance, i t is difficult to give Spoilage in nonacid canned products may result from under- typical values. It may be said, however, that, while as a sterilization or from leakage of the container after the product group the facultative thermophilic flat sour bacteria are has been processed. As would be expected in nonacid somewhat less resistant than the obligate thermophiles, vegetables, the bacterial flora differs according to the cause of representative strains of both groups have been found to spoilage. Where spoilage has resulted from leakage, many resist more than 100 minutes a t 115” C. free living types may gain entrance, and mixed cultures of The distribution of flat sour bacteria and the means by nonspore-forming bacteria are commonly encountered. Usu- which they gain entrance to canned products will be discussed ally, some of these nonresistant bacteria are gas formers and later in this paper. cause swelling of the container. Spoilage from. this cause, The acid-producing gas-forming thermophilic anaerobes, however, is a mechanical rather than a biological problem as a group and in relation to spoilage in canned foods, have not and, as such, does not call for extended comment here. A been thoroughly studied, and the authors know of no available study of this problem has been reported by Esty and Steven- reports regarding them. It is a difficult group to study. son (S). An ideal propagating medium has not as yet been found in Thermophilic bacteria are economically most important this laboratory and it has been difficult to induce sporulation as causes of spoilage resulting from understerilization. Three by laboratory methods. Until proper growth requirements principal spoilage groups have been defined. They have a are satisfied, it will not be possible to say that this group common characteristic in the production of spores of high contains facultative and obligate thermophilic members in resistance to heat. Their optimum growth temperatures the sense that these terms are applied to the flat sour group. appear to lie between 50” and 60” C. Some strains will not Spoilage by the acid-producing thermophilic anaerobes is grow in canned foods a t temperatures below 40” C , and, for characterized by swelling of the container a s d souring of the practical purposes, are known as obligate, as opposed to product. The odor is not unpleasant and usually may be facultative, thermophiles. Whether these temperature rela- described as cheesy. tionships represent fixed characteristics is not known, but Because of the difficulty of producing spores of this group there appears to be a group relationship between the growth in the laboratory, no direct heat-resistance figures are arailI
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able. Canning tests, however, utilizing material known to contain these bacteria, indicate a resistance approaching that of the flat sour group. The hydrogen sulfide-producing thermophilic anaerobes have been studied by Werkman and Weaver (14) and Werkman ( I S ) , and the name C1. nigrificans has been proposed. Spoilage by Cl. nigrificans is characterized by a hydrogen sulfide odor and, usually, blackening of the product. I n canned corn the black iron sulfide is most intense in the germ, Owing to solution of the gas and the formation of iron sulfide, the can remains flat. Spoilage by Cl. nigrijicans has been rare, but there have been a few instances of severe losses from sulfide spoilage. Aside from thermophilic bacteria, the remaining group of outstanding importance is the mesophilic group of putrefactive anaerobes. I n this group, anaerobes, resembling CZ. sporogenes but probably not identical with it, have been found to be of greatest resistance (30 to 40 minutes at 115" C.) and have been discovered as causes of spoilage in peas and corn. The spoilage manifestations are swelling of the container and a putrid odor resulting from proteolytic action. Spoilage by putrefactive anaerobes is less common than thermophilic spoilage. A&robic spore-formers have infrequently been found as causes of spoilage. Some members of this group are able to produce spores of a resistance sufficient to resist processes in common use. I n most instances the anaerobic condition maintained in the can will prevent growth. The aerobic spore-formers are not recognized as spoilage agents in canned vegetables, but have been known to produce spoilage in canned milk (Morrison and Rettger, 10). The present authors have studied spoilage in dry-pack shrimp, which was evidently caused by aerobic spore-forming bacteria. Miscellaneous types, such as Cl. welchii and members of the amylobacter group, have been encountered in products which were obviously underprocessed, but such bacteria have not been considered important as causes of spoilage because of the extreme infrequency with which they are found. Summarizing this information for the nonacid products, the thermophilic bacteria (principally the flat sour type) are regarded as the most important causes of spoilage in nonacid vegetable products. The putrefactive types of spoilage are met with less frequently, and spoilage by aerobic spore-formers is noted rarely. Effective sterilization of nonacid vegetables requires the destruction of all spore-forming types except the obligate thermophiles and, possibly, the aerobic spore-formers. The obligate thermophiles are controlled or prevented from producing spoilage by adequate cooling after the food is processed, and these bacteria will not grow under usual conditions of temperature. The aerobic spore-formers will, a t least in canned vegetables, be prevented from growth by lack of oxygen. It is probable, however, that the resistance of most spores produced by aerobic spore-formers is materially less than that of other types that must be killed in order to protect against spoilage in canned vegetables. SEMIACID PRODUCTS The microbiology of the semiacid products differs from that of the nonacid principally in the following respects: I n the thermophilic group the acid-producing thermophilic anaerobes appear to be of greater importance as spoilage agents than the flat sour bacteria. The resistant thermophilic anaerobes will grow well in the acidity range of pH 5.0 to 6.0, while, for the most part, only the relatively low-resistant flat sour bacteria have been observed here to produce spoilage under those conditions. It has also been observed that the flat sour bacteria that will act as facultative thermophiles in the
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nonacid products will fall into the obligate thermophilic category in the semiacid products. Putrefactive anaerobes are regarded as potential causes of spoilage in semiacid products, but are rarely encountered. When found, the characteristic swelling of the container and putrefactive decomposition may be lacking. It appears here that not only is the acid environment a disadvantage to the organism, but the substrate is lacking in protein nutrient. ACID PRODUCTS
It is commercial experience that spore-forming bacteria will not cause spoilage in acid products. Effective sterilization of acid products, therefore, depends upon the destruction of all vegetative or nonspore-forming bacteria, and this may be accomplished by relatively short processing in boiling water sufficient to give a minimum temperature of 82' C. (180" F.) in the coldest part of the contents. I n this connection, such laboratory studies as the authors have made point to the validity of commercial practice, Mold, yeast, and bacteria are all of importance where fruits and tomatoes are concerned. The molds, however, while having importance with respect to the condition of the product before canning, are not causes of spoilage after canning and will not be considered further. Yeasts are rare causes of spoilage in canned fruits and tomatoes, and there is no reliable information as to the extent of spoilage from this cause. I n all likelihood the amount of loss is small. I n recent years remarkably few samples of yeast spoilage, or reports of such spoilage, have come t o this laboratory. Bacterial spoilage in acid products is, for the most part, a problem of tomatoes and tomato products. It is true that there have been reports (notably by Savage and Hunwicke, 19) of bacterial spoilage of fruit, but sterilization of fruit is a simple thing. Indeed, in some unpublished experiments conducted in this laboratory, it has been found that certain bacteria which were able to thrive in tomatoes and in certain unpigmented fruits of much higher acidity, were not able to show anything like parallel growth in other products, such as blueberries and loganberries, which are characterized by the presence of pigment. Bacterial spoilage of tomato products has been comprehensively studied by Pederson (11). His report is most complete and constitutes a valuable contribution in this field. He reported on the examination of 266 cultures of bacteria obtained from eighty-three samples of tomato products, All but a few miscellaneous cultures were classified as belonging to six species. Of the six species, all but one produced gas in glucose broth. It is very probable that the list of organisms identified by Pederson contains the bacteria responsible for the great majority of cases of spoilage in tomato products. I n the authors' own experience, bacteria have been isolated from spoiled acid products which failed to show gas production in a variety of tubed mediums, but which, when reinoculated into canned tomatoes, produced swelling with the production of carbon dioxide; the reverse condition has also been observed. I n other words, nongaseous spoilage has been found in tomato products, from which bacteria were isolated that would produce gas in tubed media. Reinoculation in canned tomatoes again gave acid spoilage, but no gas was developed. There seems to be no explanation for these phenomena, but they point to an attractive field for biochemical study. It may also be that a study of the dissociative tendencies of the tomato-spoilage organisms would throw light on the question. A few years ago flat sour spoilage in tomatoes was almost unknown to the industry. Within the past few years, however, it has repeatedly come to the attention of this laboratory.
The aut.hors have no explanation for the cliariged condition. The bacteria involved do not appear unusually resistant to heat, and, therefore, such spoilage is probably due to escessive contamination of the raw product. Before leaving the subject of spoilage in acid products, it seems desirable to point out that in such products there is not the biological criterion of spoilage through leakage that is available in the case of nonacid products. Ractcriological findings are limited in their significance hy the fact that no specific differences lime been ohserved betwecn tiie flora of imdersterilieed and lealcy cans. Under these circiimstances, a thorough and competent knowledge of paekiiig conditions and of the coiidition of the seams is essential in determining cause of spoilage.
ELIM~NATION OF SPO~IAGI: BA As lias been shown by Bigelow and Esty ( I ) and otircrs, the iiiitial concentration of spoilage bacteria affects the time necessary for stcrilieation. I n other words, it is more difficult to kill 10,000,WO than 100,000 flat sour bacteria or putrefactive anaerobes. Why tliis is so has not been satisfactorily explained. Tlie authors, of course, conceive of substantial variation in the resistance of individual spores even from pure culture propagation, but that is probably not tlie coriiplete explanation. Regardless of the reason, however, this effect of numbers has consistently been p r o d i n hcat-resist:ance studies in this laboratory. The degree of process ne ary to skrilize a product,
F m a L~BOMTORV OF NxnoxAi, C m ~ e n s AssocIxrnm Above. Mototiaed labarstory 88 *st up lor operation staosanningfadoiy. Right. Apparatus se eel UP in the c ~ n o e w . Left to right: table on which media are p r e ~ pared. dry sterilizer. therms1 death time wpamtm fur determining heat resistance of bacteria, autoclave lor sterilising media, and s t e m box.
therefore, must be determined witli regard t u the probable kinds of spoilage bacteria that may be present, and also to their nuinhers. Obviously, these requirements lead to process suggestions that will carry a factor of safety to guard against something more,, qnalitRtively aud quantitatively, than a miriirnum contamination, It has heen t i i e aim of laboratories connected with the canning industry to nialce process suggestions on this basis. But such processes may not he effective where there is grcat contamination. Tliere Iiave been iiistances where recommended processes have failed to protect against spoilage. These failures could not be explained by a so-called sanitary examination of the cannery, raw materials, and equipment. It has appeared in such cases that there were unexplained iuercases in spoilage bacteria in tlie operations wliich were involved. With the hope of finding the infective sources of contamination, this laboratory in 1926 and subsequently has conducted field surveys, fast in factories where spoilage had been experienced, arid later in factories where no spoilage had recently occurred. Tile object was first to obtain infoiniabioii about probable sources of contamination, and later to apply this information in eliminating such foci before the onset of t,rouble. The first studies concerned thermophilic spoilage in peas and corn. The principal findings of these bacteriological field studies in canning have been reported by Cameron, Williams, and Thompson (0). The most iniportant basic finding was that, when spoilage occurs as a result of understerilization in canned peas or canned corn, the cause of the spoilage is independent of the condition of tlie raw product. On the other hand, it was showii definitely that the development of tliermopliilic contamination takes place largely in the cannery itself. Wooden tanks, such as are used in the prepa.ration of brine, were found a t times to be heavily inoculated vritli spores of spoilage tliermopliiles and, tiicrefore, were primary sources of contamination. In one instance, n wooden tank whieh was used only for the storage of liot water was found to be similarly contaminated. I n the canning of peas, blancliers have frequently been found to he contaminat.ed, indicating that, while it has not been consistently possible to isolate thennopliilic bacteria from soil, they must be present in a t least small numbers. I n general, tlie information gaiiieri has indicated that, 1111 heat,cd equipment ufied f or preparing
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nonacid vegetables for canning must be considered as possible sources of thermophilic spoilage bacteria. SUGAR-4s SOURCEOF THERMOPHILIC BACTERIA Reports on the significance of thermophilic contamination of sugar have been made by Cameron and Williams (6)and Cameron and Bigelow (3). The existence of thermophilic spoilage bacteria in this medium has also been noted by James (9). Refined sugar has proved to be an outstanding source of thermophilic spoilage bacteria. Sugar is the only ingredient of canned foods in which spores of such bacteria have been found with any degree of frequency. It appears that granulated sugar is seldom so highly contaminated as t o be a direct cause of spoilage, and its greatest significance appears to come from the fact that it may inoculate brine tanks or other equipment and thus indirectly lead t o massive inoculation. All three significant thermophilic groups have been found in sugar, both beet and cane. The flat sour group has been the one most frequently encountered, but a t times either or both of the thermophilic anaerobic groups have been in predominance. Flat sour bacteria have been found in refined cane sugar to a greater extent than in white beet sugar, but the white beet sugar a t times has been found to be quite highly contaminated with spores of the acid-producing thermophilic anaCrobes. With respect to contamination, neither appears to possess any consistent advantage. The sugar industry has cooperated with the canners in the matter of supplying sugar that is satisfactory with respect to spoilage thermophiles. The National Canners Association has announced Tentative Bacterial Standards, and the sugar industry has used these standards as a guide. The results of the collaborative effort have been most gratifying. COXTROLOF THERMOPHILIC SPOILAGE Based on the foregoing, the following suggestions have been made for the purpose of controlling thermophilic spoilage : 1. Canners using wooden brine tanks or wooden tanks for holding hot water or any other heated substance should have competent tests made to determine the condition of the tanks. Where contamination is found, the tanks should be replaced with hard-surfaced equipment. Where new installations are made, hard-surfaced equipment should be used. 2. Blanchers, corn and pea fillers, corn preheaters, and heated equipment used in preparing the product for canning should be kept cold when not in use in order to prevent undue thermophilic development. Immediately after use, such equipment should be flushed with cold water and cooled as rapidly as possible. Specifically in connection with the blanchers, the product after blanching should immediately be washed with cold water to remove any contamination that may have been picked up in the blancher. 3. It is now possible to buy sugar on specification, and canners have been urged to do this. Where sugar is bought on sample, it is also suggested that the canners have samples of deliveries tested in a laboratory familiar with the work. 4. The canned product should be given an.ade uate process and should subsequently be cooled below the point &ere obligate thermophilic development is possible.
CHEMISTRY
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bacteria. Yet the hazard is always present, and it requires something more than a superficial examination of the cannery to determine whether spoilage is likely to occur. The sanitary condition of the cannery as judged by usual standards is no index as to the likelihood of thermophilic spoilage. There is no relation between thermophilic contamination and sanitary conditions, and it is interesting t o note that the same statement has been made with regard to the development of thermophilic contamination in pasteurizing equipment in the dairy industry ( 2 ) .
PUTREFACTIVE SPOILAGE Spoilage by putrefactive anaerobes is rare, and, unlike thermophilic spoilage, it probably has relation t o sanitary conditions. Where the authors have studied this type of spoilage, it has resulted through the presence of spores in such things as wooden conveyors and baskets made of porous material. Soil is considered to be the source of these bacteria, but in all probability the direct soil contamination is not sufficient in amount to cause spoilage. Spores of putrefactive anaerobes may be found more generally distributed through the cannery than is the case with the thermophiles, there being no such strict limitation such as confinement t o the heated equipment.
ELIMIXATIO?; O F SPOIL.4GE BACTERIA PRODUCTS
IS
ACID
With the acid products, the condition of the raw product from the standpoint of bacterial contamination probably has a great influence on the subsequent success or failure of the process. It is appreciated, however, that improper handling of the product in preparation for canning may increase the original contamination. Control of spoilage in such products for the most part depends on proper selection of the raw product coupled with competent handling in preparation. LITERATURE CITED Bigelow, 1%'. D., and Esty, J. R., J . Infectious Diseases, 27, 602 (1920). Breed, R. S., Prickett, P. S., and Yale, M. W., J . Bact., 17, 37 (1929). ENQ.CHEY.,23, 1330 Cameron, E. J., and Bigelow, W. D., IND. (1931). Cameron, E. J . , and Esty, J. R., J. Infectious Diseases, 39, 89-105 (1926). Cameron, E. J., and Williams, C. C., Centr. Bakt. Parasitenk., 11, 76,28-37 (1928). Cameron, E. J., Williams, C. C., and Thompson, R. J., S a t l . Canners Assoc., Bull. 25L (1928). Donk, P. J., J . Bact., 5, 373 (1920). Esty, J. R., and Stevenson, A. E., J . Infecfious Diseases, 36, 486 (1925). James, Id. H., Food Industries, 1, 65 (1928). Morrison, E. W., and Rettger, L. F., J . Bact., 20,299 (1930). Pederson, C. S.. N. Y . Agr. Expt. Sta., Tech. Bull. 150 (1929). Savage, W. G., and Hunwicke, R. F., Food Investigation Board, London, Special Rept. 16 (1923). Werkman, C. H., Iowa dgr. Expt. Sta., Research Bull. 117, 163-80 (1929). Werkman, C. H., and Wearer, H. J., Iowa State Coil. J . S C L, 2, 57-67 (1927).
Extensive field studies since 1926 have shown that few canneries are excessively contaminated with thermophilic
R E C E I V Maroh ~D 22, 1932
ONE OF THE FACTORS in the quality of canned peas is the appearance of the liquor. A certain amount of insoluble organic matter is normal to the liquor of good quality canned peas but excessive amounts cause a distinct cloudiness. This insoluble material is a mixture of finely divided peas material (cotyledons) and coagulated pea starch. Some of the starch in the peas is dissolved by the liquid during sterilization and this dissolved starch slowly coagulates to form a milky suspension of fine white
particles. Several weeks may be necessary to complete the separation but a t the end of this period all of the starch originally dissolved is coagulated. Early varieties of peas, such as Alaska, contain more starch than the sweet or wrinkled varieties. The state of maturity of the peas at the time they are harvested also influences their starch content. The use of hard water in canning operations reduces the amount of starch dissolved.
