Thermophilic Bacteria in Refined Cane Sugars WM. L. OWEN AND R. L. MOBLEY,P. 0. Box 1345, Baton Rouge, La.
S
INCE the publication of the work of Cameron and Williams (5) on the thermophilic flora of sugars, an increasing amount of attention has been given this subject, both by sugar refiners and canners using this article in their canning operations. The pioneer work of the National Canners’ Laboratories, followed by that of James of the United States Department of Agriculture (11), fully established the reality of the presence of substantial numbers of thermophilic spores in the most highly refined granulated sugars, and the resulting hazard to corn canners in particular, of the use of sugar of unknown thermophilic content. Cameron and Yesair (6) indisputably established the fact that to sugar, more than to any other factor, the spoilage of canned corn is due, The literature on the role of thermophiles in canning has already become quite voluminous, but mention should be made of the work of Barlow ( I ) , Weinzirl ( I 4 ) , Cheney (7), Bigelow and Esty @),Esty and Stevenson (9), Cameron and Esty (4),James (IO), Werkman and Weaver (I6),and Werkman (15). I n this paper will be considered the problem of thermophiles in sugars and their significance from the viewpoint of the refiner rather than of the consumer of sugars. I n this particular field there have, as far as the writers are aware, been very few contributions, for reasons which are not difficult to explain. The refiners have been primarily interested in producing sugars that will conform to the standards of the National Canners’ Association, and have published very little on the means that they have employed in attaining this highly desirable end, while investigators in the National Canners’ Laboratories have concerned themselves principally with looking after the interests of their clients and seeing that sugar supplied them does not constitute a menace to the success of their canning operations. More recently, however, the latter investigators have carried out a thorough research into the sources of infection of sugars with thermophiles during refining operations; the contribution of Cameron and Bigelow (3) on “Elimination of Thermophilic Bacteria from Sugar” has given a clear insight into the role played by the different refining operations in the elimination of thermophiles during intermediate stages of manufacture. There are still some questions that present themselves, however, with increasing frequency to refiners; those which have seemed most important to the writers have been the following: (1) Can the chemical and physical purity of the sugar be correlated with its thermophilic content?’ (2) Is the raw sugar used in its manufacture the only source of thermophiles in granulated sugar? (3) Are the thermophilic spores entirely confined to the surface of the sugar crystals, or do they at any time occur within the crystal structure itself?
refined cane sugars that they examined contained flat sour thermophiles; of these, 40 per cent showed an average infection of 50 spores per 10 grams and below. James found 75 per cent of the refined cane sugars contained flat sour thermophiles, whereas 35 per cent carried the thermophilic anaerobes producing hydrogen sulfide. I n beet sugars James found that 21 per cent contained flat sour organisms, and only 3 per cent contained the sulfur spoilage type. Of eleven samples of confectioners’ sugar containing starch, the average number of thermophiles per 5-gram sample was found to be 358. Of these eleven samples, six (54.5 per cent) contained more than 100 thermophiles per 5 grams. Of thirteen samples of confectioners’ sugar not containing starch, the average number of thermophiles per 5 grams was 133, and only three contained more than 100 per gram. TABLEI. SUMMARIZED DATAON THERMOPHILES
GRADE OF SUGAR Fine granulated Coarse granulated Powdered Raw Starch Sacks
PERPERPERAv. FLAT PERCENTAGE CENTAQE CENTAGE SOURS CENTAGE F R E ~ O PHATING HIS PER 10 EXCEEDING FLAT ANPROGRAMS LIMIT SOURS ASROBESDUCERS 13.00 3.1 39.0 14.0 12 8.5 0.0 10.0 33 10 00 50 50 191.0 100.0 70.0 70.0 00 28.0 00 131.9 83 00 50 33 6.5 sq. in. 40 (infected).
.
..
..
SOURCES OF THERMOPHILES IN GRANULATED Suom The preceding paragraph has partially answered question 2, since James found such large numbers of thermophiles in confectioners’ sugars containing starch. Evidently here is a source of infection in the refinery entirely independent of the raw sugar that is being refined. Again referring to Table I, the average flat sour content of a large number of fitarch samples was almost twice that of raw sugars. I n order to determine the position in the refinery where there was the greatest concentration of thermophilic infection in the dust, 100-gram portions of a refined granulated sugar were exposed in a refinery a t various locations in the plant from the raw-sugar dump to the refined warehouse. The sugar was spread out in thin layers and exposed for several hours; i t was then collected in sterile bottles and subjected to analysis for flat sour thermophiles, with the results shown in Table 11. The greatest infection of the dust with thermophiles was around the starch mixer and powder mill. These and many other similar tests have shown that much of the thermophilic infection of sugars is entirely exotic as regards its source, and that the problem of producing thermophilefree sugars must include the prevention of this exotic infection as well as the elimination of that which is brought in with the raw sugars.
CORRELATION OF PURITY OF SUQAR WITH ITSTHERMOPHILIC TAB^ 11. FLAT s o m THERMOPHILES IN EXPOSED SWQARS CONTENT FLAT~OURSPER FLATSOUREPEB The first of these questions is answered by referring to Table I, showing averages of many samples of various grades of sugars obtained from a great many different sources. It is seen that powdered sugars contain more thermophiles than raws, which indicates that chemical and physical purity have nothing to do with thermophilic purity of the sugars. Cameron and Williams found approximately 85 per cent of the
POSITION EXPOSED 10 GRAMS Granulated stack 20.00 Raw-sugar dump 40.00 Starch mixer 230.00 Powder mill 106.00
POSITION EXPOSED Warehouse soales Sugar hopper Centrifugals
10 GRAME 20.00 60.00 45.00
Table I shows that the number of thermophiles in the samples of starch used in powdered sugars is approximately twice as great as in the raws used for refining. This same
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INDUSTRIAL AND ENGINEERING CHEMISTRY
condition is also reflected in the examination of hundreds of samples of refined sugar bags, especially the toweling grade of bags used as containers for all classes of granulated refined sugars. Of hundreds of such samples examined, over 40 per cent showed flat sour thermophilic infection, and the average infection was 5.5 spores per square inch. I n making this examination, the sacks were taken from the interior of the bundles with sterile forceps, and sections were cut with sterile scissors, placed in flasks of sterile water, and boiled as in making thermophilic determinations of sugar. These results show convincingly the presence of thermophilic bacteria, both in the sizing of the bags and in the starch used in powdered sugars; they demonstrate that, even if raw sugars were entirely free from thermophiles, the finished product would probably acquire some infection of this kind, either from the bags or from the starch dust in the refinery. Owing to the presence of flat sour thermophiles in the sizing in sugar sacks, the results of analyses of the sugar as it comes from the hoppers in the refinery will usually be found to be lower than if taken from the sack, provided the container is a toweling bag.
which the finished product is subsequently exposed. Sugars, therefore, are in this case merely the carriers or “receptors” of an infection from which it suffers no deteriorative changes whatever. There may be rare instances in which the thermophiles occurring in the refined sugar may have developed during some of the subsequent stages of manufacture, but these cases are extremely rare; as a matter of fact, these species would scarcely find the sugar liquors and intermediate product any more suitable to their needs than the finished sugars. For the most part they are entirely exotic and merely represent the uneliminated contamination of the original raw sugars, or the acquired contamination from the dust of the refinery. A continuous study of the variation of infection in a refinery has shown that it varies from hour to hour. VARIATION I N INFECTIOK AT DIFFEREKTTIMESO F DAY Samples drawn a t 5-minute intervals showed a very sharp variation; the average numbers of thermophiles per 10 grams in sugars collected a t different periods are as follows: Mormng
LOCATION OF THERMOPHILIC CONTAMINATION The question of whether the thermophilic‘contamination of sugars is confined to the surface of the crystals or is sometimes incorporated within the crystal structure itself has received considerable attention here, and so far the results point conclusively to the assumption that the infection is entirely a superficial one. By washing the crystals thoroughly with absolute alcohol, negative results have been obtained from subjecting the washed sugar to thermophilic analysis, while the washings have shown virtually the same infection as was obtained on the original sugars. Occasionally a few spores have been obtained from the washed crystals, but these were within the limits 0.f accuracy of the method; negative results were never obtained from the washings obtained from a sugar known to be infected. Of the phylogenetic relationship of the thermophilic bacteria in sugars we have as yet too meager data to specifically classify all of the species occurring under this group. The flat sour bacteria have been studied by Donk and by Cameron and Esty, and have been given the name of B. stearothermophilus by Donk (8); the hydrogen sulfide-producing anaerobes have been assigned the name of Clostridium nigrificans by Werkman and Weaver. While the writer (IS) in his investigations of the bacterial flora of raw sugars, in connection with his former study of the deterioration of sugars in storage, found that many of the levan-forming bacteria occurring in sugars are facultative thermophiles, it mould appear that they are rarely numbered among the thermophiles included in the tests to which these samples are now subjected in the thermophilic determinations. That many of these thermophilic species occur as part of the epiphytic flora of cane seems very probable in the light of Kuhr’s pioceer work (1.2)’ which establishes the existence of such a flora on the growing cane similar to that which occurs on grain, Carpenter and Bomonti’s work on the occurrence of a sporeforming thermophilic organism in hot clarified juices in sugar factories indicates that a t times this organism ma.y also be included in the thermophiles found in finished sugars. It should be remembered, however, that the occurrence of these bacterial spores in sugars is in no sense indicative of any activity on their part a t any stage in the manufacture of the raw sugar or during its conversion into the refined product. These spores are as exotic to the sugar as if they were some extraneous particles of trash or inert debris, and the significance of their presence is the indication of the incompleteness of the refining operations and the uncleanliness of the air to
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P. M.
7.5 30.00
Afternoon Night
40.00
20.00
The percentage of infection a t different times is as follows: Morning sugars Afternoon sugars Night sugar0
71.00
75.00
66.00
I n this refinery in particular the greatest percentage of infection and the greatest concentration of infection occurred in the afternoon production. These figures are the averages of a great number of samples; they indicate that the chances of infection with thermophiles are variable, and that the production of thermophile-free sugars depends upon a very thorough analysis of the sources of this infection and of the efficiency of the factors operating for its elimination.
PREVALEKCE OF THERMOPHILES IN SUQARS The production of thermophile-free sugars, or even of sugars that consistently conform to the standards of the National Canners’ Association, cannot possibly be attained without the most systematic effort on the part of the refiner, and cannot be guaranteed to the consumer without regular tests of the bacteriological purity of the product. There is a tendency on the part of some of the refiners to discredit the demonstrated importance of sugars in the role of spoilage producers in canned corn, but all of the large refiners have established bacteriological control and have found that i t is necessary for the production of thermophile-free sugars. Only by unceasing vigilance in the refinery can the output be free of thermophiles, or even consistently within. the limits required by the National Canners’ Association. While refined sugars have for many decades constituted the purest article of the daily diet from a chemical standpoint, no thought had been given to their bacteriological purity until the National Canners’ Association so ably proved that even the highest grades of these products were frequently contributive to one of the most troublesome spoilage problems in the canning industry. It is quite possible that still other spoilage conditions may be found in the food industry that we may attribute quite justly to sugar, which has been the last of all of the ingredients to come under suspicion. LITERATURE CITED (1) Barlow, B., Univ. Illinois, Master of Science Thesis, 1913. (2) Bigelow, W.D., and Esty, J. R., J. Infectious Diseases, 27, 602 (1920). (3) Cameron, E.J., and Bigelow, W. D., IND. ENG.CHEM.,23, 1330 (1931).
IKDUSTRIAL AND ENGINEERING CHEMISTRY
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(4) Cameron, E. J., and Esty, J. R., J . Infectious Diseases, 39, 89105 (1926). (5) Cameron, E. J., and Williams, C. C., Centr. Bakt. Parasitenk., I1 Bbt., 76, 28-37 (1928). ( 6 ) Cameron, E. J., and Yesair, J., Canning Age, 12, 239 (1931). (7) Cheney, E. W., J. Med. Research, 40, 177 (1919). (8) Donk, P. J., J. Bact., 5 , 373 (1920). (9) Esty, J. R., and Stevenson, A. E., J . Infectious Diseases. 36,486 (1925). (10) James, L. H., J . Bact., 13, 409 (1927). (11) James, L. H., Food Industries, p. 265 (1928).
Vol. 24, No. 9
Kuhr, Wohlzogen Arch. Zuckerind., S o . 31 (1923). Owen, W. L., La. Agr. Expt. Sta., Bull. 125 (1911). Weinsirl, J., J . Med. Research, 39,349 (1919). Werkman, C. H., Iowa Agr. Expt. Sta., Station Bull. 117, 16380 (1929). (16) Werkman, C. H., and Weaver, H. J., Iowa State Coll. J . S a . , 2, 57-67 (1927).
(12) (13) (14) (15)
RECEIVED April 8 , 1932. Presented before the Division of Sugar Chemistry a t t h e 83rd Meeting of the American Chemical Society, New Orleans, L a , March 28 t o April 1, 1933.
The Yellowing of Oils 111. Relation between Color and Chemical Constitution of Oxidized Drying Oils A. C. ELMAND G. W. STANDEN, The New Jersey Zinc Company, Palmerton, Pa.
A
NUMBER of investigations (3, 7 ) have established the fact that the yellowing of oils upon interior exposure and in the absence of light is caused by the formation of colored oxidation products of the more highly unsaturated glycerides. The various investigators of this field, however, disagree as to the exact chemical constitution of these oxyns. Elm (2) adopted Scheiber’s theory that the colored compounds are polyketones, whereas Morrell and Marks (8) came to the conclusion that yellowing is caused by the keto-enol tautomerism of ketohydroxy compounds formed from the oil peroxides by a simple intramolecular rearrangement. -4final decision in favor of one or the other hypothesis on the basis of the chemical evidence is extremely difficult, although there are indications that these compounds are present
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1
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able to formulate widely accepted theories as to the relation between the color and chemical constitution of organic compounds. A detailed discussion of the relations between color and molecular structure is unnecessary here, for, ever since it was first discussed by Graebe and Liebermann in 1867, this subject has received so much attention that a t least a brief account of it is found in every good textbook on organic or physical chemistry, as the one by Smiles, for example (9). When comparing the structures of colored compounds with those of colorless derivatives, it is evident that the color of the former is caused by the presence of certain groups, called “chromophores.” The dicarbonyl group, -CO-CO-, is among the more important chromophores and induces yellow color as demonstrated by diacetyl (CHICOCOCH,), b e n d (CJ&CO.COCJ&), and others. Statements as to the chromophoric properties of the ketohydroxy or its tautomeric form -C(OH)= group -COCH,OHC(0H)- in aliphatic compounds could not be found in the literature. Although for reasons of analogy (for example, benzoin CeHsCOCHOHG-Hs, and ketoses) this grouping of atoms is not expected to cause selective absorption of light in the visible region of the spectrum, it was thought advisable to investigate this point, for one of the outstanding theories of yellowing is based on the presence or formation of such compounds in drying-oil films.
EXPERIMENTAL PROCEDURE Ketohydroxy- and diketostearic acids were prepared by progressive oxidation of oleic acid as illustrated by the following scheme: ,?OM
yY)O
4050
J
O Y I U T I O N FREQUENCY
FIGURE1
in oxidized drying oils. Attempts a t their isolation and identification have proved unsuccessful because the chemical reactions necessary to unravel the complicated mixture making up drying-oil films usually result in the disruption of the oxyns. I n the course of the analysis new compounds are formed which make it unusually difficult to draw any definite conclusions as to the exact chemical constitution of the oxidized glycerides as present in the films. The history of organic chemistry is full of similar cases in which, after the usual chemical methods had failed, physical and especially optical measurements formed the basis for the final decision. Especially the dyestuff chemists have made frequent and successful use of these methods and have been
R
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R
R I
AH AH
I R
+
bH.OH
CH OH
AHOH-
L o
R
R
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c=o +
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C=O
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The methods followed are described by Lewkowitch (6) and Holde and Marcusson (4). The transmission spectra of alcoholic solutions of these compounds were determined using a Baly cell ( 5 ) and a Hilger spectrograph (1). The light source was an iron arc, the time of exposure one minute. Photographs were taken of the original (0.2 11‘) and the diluted (0.02 N ) solutions a t a distance between the cell windows of 40,30,20,10, and 5 mm. The results (Figure 1) are set forth in the usual manner-
