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THE EFFECT OF CONCENTRATION ON THE DETERIORATIVE ACTIVITY OF MOLD SPORES IN SUGAR1 By Nicholas Kopeloff, S. Byall and Lillian Kopeloff DEPARTMENT OF BACTERIOLOGY, LOUISIANA SUGAREXPERIMENT STATION, NEWORLEANS,LA.
It is common knowledge t h a t t h e reduction of t h e moisture content of sugar is responsible for a diminution of deterioration. This is tantamount t o stating t h a t an increase in concentration of t h e films surrounding the individual particles in a n y given mass of sugar produces t h e same effect. The influence of this factor on t h e activities of bacteria has been t h e subject of thorough investigation by Owen,2 and our recent work3 with molds has led t o t h e conclusion t h a t “the factor of safety for sugars well infected with molds would appear to be lower t h a n is generally supposed.” The full import of such a consideration depends t o a considerable degree upon t h e fact t h a t molds may be responsible for t h e inversion of sucrose where only spores are present as well as when mycelia are developed. Furthermore, it has been shown4 t h a t t h e invertase activity of mold spores is exhibited a t concentrations of sugar solutions varying from I O t o 7 0 per cent, with maximum activity at 50 t o 6 0 per cent. I n t h e same connection it was observed t h a t a n increase in t h e number of mold spores is responsible for increased invertase activity a t any definite concentration-including saturated solutions. While these might be considered sufficient grounds for inference regarding t h e deteriorative activity of mold spores in sugars of known composition, it was deemed advisable t o carry o u t such a n investigation on a more practical scale.
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rubber stoppers, paraffined three times at short intervals, were later used. All flasks were incubated at 2 8 ’ t o 30’ C. I n Table I is presented t h e summary of t h e per cent increase in reducing sugars over check as resulting from t h e inoculation of molasses b y molds, all values representing t h e averages of closely agreeing triplicate determinations. These d a t a will appear in greater detail in bulletin form.* It will be observed t h a t with each mold used the per cent increase in reducing sugars is made greater with a decrease in concentration of t h e molasses medium a t t h e end of a 4-month incubation period. There was a corresponding decrease in sucrose Clerget in every sample. While this increase is striking, i t does not follow a rigid mathematical progression, due t o certain discrepancies which result from such a method of inoculation. It will be seen from Table I t h a t Aspergillus Sydowi Bainier and Aspergillus niger exhibit t h e maximum inversion, closely followed b y Penicillium e x p a m u m . This corroborates previous conclusions concerning t h e deteriorative activity of these organisms both in t h e mycelial and spore stage.2 Since such definite increases were obtained when molasses of known concentrations were used, i t might be expected t h a t refined sugar coated with such different molasses would yield similar results. T h a t this is practically t h e case may be judged from t h e d a t a in Table 11, which represents a summary of t h e
TABLE11-SUMMARYSHOWING PER CENTINCREASE IN REDUCING SUGARS OB INOCULATED SUGARS AFTER ONE MONTH’S INCUBATION Aspergillus Aspergillus Penicillium Moisture CONCENTRATION niger S.Bainier expansum ratio . . . . . 3 1 .0 142.9 95.2 0.20 Blackstrap. . TABLEI-SUMMARY SHOWING PER CENT INCREASE IN REDUCING SUGARS 4/6 B. S. 2/6 sirup.. . . 75.0 195.0 170.0 0.08 IN INOCULATED MOLASSES AFTER 4 MONTHS’ INCUBATION 5/6 B. S. 1/6 sirup.. . 70.4 270.4 170.4 0.14 Asper- Asfier- Peni- Synce- Asper- Mois95.7 391.1 239.1 0.18 3/6 B. S. 3/6 sirup.. Clados- gillus gillus czllium phala- gillus ture 3/6 B. S. 3/6 sirup.. . , 104.6 404.5 218.2 0.20 CONCENTRATION porium pavus S. Bain. expans. strum niger ratio per cent increase in reducing sugars over check as reBlackstrap. 18.4 9.8 4 . 7 0.44 5/6 B. S. 1 / 6 6irup. .. 5 . 8 19.7 22.3 . .. 0.49 sulting from an inoculation with mold spores at t h e 4/6 B . S. 2/6sirup 38.3 12.3 53.2 56.7 . 3 6 . 1 0.57 rate of IOO,OOO per g after one month’s incubation. It 3/6B.S. 3/6sirup ... 53.8 33.7 83.6 71.6 9 . 5 7 3 . 6 0 . 6 3
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A series of sugars with films of known composition were made in t h e laboratory b y coating large crystals of sterilized sugar with sterilized blackstrap molasses a n d 60” Brix sugar sirup in definite proportions, and purging in t h e centrifugal, a method previously employed with success.6 These sugars and corresponding molasses were aseptically inoculated with mold spores b y adding 1 5 g. of sugar containing approximately 1000 spores per g. (prepared as previously described)6 t o 135 g. of sugar in tightly corked Erlenmeyer flasks which were paraffined. T h e molasses was inoculated with a single scoopful of mold. Solid
will be seen that an increase in moisture ratio Moisture M. R. = I 00 - Polarization ’ which means a decrease in concentration, is responsible for raising the per cent increase in reducing sugars in most instances, except where blackstrap films were employed. The latter phenomenon may be accounted for b y t h e fact t h a t t h e molasses film was relatively thin, due t o too prolonged purging. However, the other data, despite negligible discrepancies, are substantial proof of t h e direct effect of concentration on t h e deteriorative activity of molds. This has been proven t o be a case of t r u e inversion by t h e fact t h a t a n increase in reducing sugars has almost invariably been accompanied b y a decrease in sucrose Clerget in t h e same sample. As in all previous work, Aspergillus Sydowi Bainier exhibits t h e greatest power of inversion. If t h e results in Table I1 are compared with those
1 Read at the 58th Meeting of the American Chemical Society, Philadelphia, Pa., September 2 to 6, 1919. 2 Owen, Louisiana Bulletin, 162. J Kopeloff and Kopeloff, Louisiana Bulletin, 166 (1919). 4 Kopeloff and Byall, “The Invertase Activity of Mold Spores as Affected by Concentration and Amount of Inoculum.” Read at the 58th Meeting of the American Chemical Society, Philadelphia, Pa., September 2 to 6, 1919, to appear in Jour. Agr. Res., February 15, 1920. 5 Owen, LOC. cit. 1 Kopeloff, “Biological Factors Affecting the Deterioration of Cane 0 Kopeloff and Kopeloff, “Do Mold Spores Contain Enzymes?’’ Read at the 57th Meeting of the American Chemical Society, Buffalo, N. Y., Sugar,” Louisiana Bulletin (in preparation). 2 Kopeloff, et al., LOC. cit. April 7 t o 1 1 , 1919, and printed in J . Agu. Res., [41 18 (1919), 195.
Mar., 1920
T H E J O U R N A L OF I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y
presented in Table I; i t may be noted t h a t there was a greater deteriorative activity revealed in t h e former case where t h e moisture ratio was actually much lower. This would seem, upon the surface, t o contradict t h e conclusion t h a t a lowering of t h e concentration of t h e medium increases t h e deteriorative activities of molds. However, i t must be remembered t h a t t h e deteriorative activity of molds in any single medium is a resultant of a t least two variables, i. e., concentration and amount of inoculum. We have shown in another paper1 t h a n an increase in amount of inoculum is responsible for an increase in inversion in any sugar solution of definite concentration. T h a t this principle applies t o manufactured sugars is indicated here, for t h e inoculum used in Table I1 was approximately IOO times as large as t h a t employed in Table I. Consequently, this experiment shows t h a t a decrease in concentration causes increased deteriorative activity in mold-infected sugars, all other things being equal. Nevertheless, t h e effect of other variables cannot be overlooked. A similar experiment was repeated over a period of 4 mo. and t h e d a t a gave similar evidence with moisture ratios varying from 0 . 1 8 t o 0.29. Considering t h e importance of t h e moisture ratio in predicting t h e keeping quality of sugar in storage under what is known as t h e “factor of safety” rule, i t is of special interest t o note t h a t here is presented evidence of t h e deterioration of manufactured cane sugars with moisture ratios from 0.08 to 0 . 2 9 when sufficiently infected with mold spores; and, furthermore, t h a t this deterioration occurs even in films of t h e highest concentrations, namely, of blackstrap molasses. I n sugar solutions we have already arrived at t h e resultant effect of t h e combined variables of concentration and amount of inoculum. I n other words, in saturated sugar solutions upwards of 5000 spores per g. are required t o effect inversion.’ We have experiments nearing completion which should establish similar criteria for sugar coated with films of known concentration as herein described. I n this way it is our purpose t o arrive a t a satisfactory method of judging t h e keeping quality of a sugar from t h e standpoint of mold infection, which we have shown t o be capable of inducing serious economic losses i n sugar as a result of i t s deteriorative activity. We wisli t o acknowledge our indebtedness t o t h e Station staff for their kind assistance. SUMMARY
I-A decrease in concentration of molasses inoculated with molds is responsible for a progressive increase in reducing sugars and a decrease in sucrose Clerget when incubated at 30’ C. for 4 mo. 2-A decrease in t h e concentration of films in inoculated laboratory-made sugars having films of known concentration and moisture ratios of 0 . 0 8 t o 0 . 2 0 , caused a n increase in reducing sugars (and a decrease in sucrose Clerget) which gave evidence of active deterioration. These sugars were incubated a t 30’ C. for one month, and similar results followed a like incubation of 4 mo. 1
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3-Aspergillus Sydowi Bainier, followed by Aspergillus niger, and Penicillium expansztm, in t h e order named, effected t h e greatest deteriorakion in both molasses and sugar. 4-There is evidence t h a t an increase in inoculum is responsible for an increase in inversion at definite concentration. This investigation with laboratorymade sugars corroborates previous results obtained with sugar solutions. THE CAUSE OF DETERIORATION AND SPOILING OF CORN AND CORN MEAL’ By J. S. McHargue KENTUCKY AGRICULTURAL EXPERIMENT STATION, LEXINGTON, KENTUCKY
Considering t h e great economic importance of Indian corn in t h e world’s commerce, it is evident t h a t t h e proper conditions for t h e preservation of this grain should be well understood and closely adhered t o under all circumstances. T h a t such is not t h e case, however, is shown by t h e fact t h a t every year thousands of tons of sound corn are subjected to conditions under which the grain deteriorates or spoils during storage in bins or while in transportation in cars or ships. The American Cooperative Manager states t h a t t h e loss t o shippers, in €our months of one year, through the deterioration of corn arriving in t h e Chicago market, amounted t o more t h a n $4,000,000. It is also well known t h a t tremendous losses are sustained in t h e exportation of corn t o European countries. The problem of keeping meal made from t h e whole grain of corn in a fresh, sweet state and fit for human consumption has long puzzled both miller and consumer. Partly as a war measure and also on account of its economical importance in times of peace, an investigation was suggested by t h e Director of the Kentucky Agricultural Experiment Station, about a year ago, the object of which was t o determine t h e underlying causes of t h e deterioration and spoiling of corn and corn meal while in t h e channels of commerce. Thus far our experiments have been confined t o t h e laboratory and were therefore carried out on small portions of material. However, t h e conditions under which t h e results have been obtained were so planned as t o represent what may be considered a duplication of those under which much larger quantities of corn spoil. For t h e experiments in this investigation a quantity of sound and thoroughly air-dry grains of corn of t h e crop of 1917, which contained 1 2 per cent of moisture, was selected. This was divided into t,wo equal parts, one of which was ground into meal, t h e other remaining unground. The unground grains and t h e meal were further subdivided into 1000-g. lots, each of which was stored separately in glass jars, sealed t o exclude air and moisture. CORN GRAINS
bIoIsTuRE ABSORPTION-FOr a n experiment on t h e hygroscopic property of corn containing 12 per cent 1 An abstract of this paper was read before the Division of Agriculture and Food, 58th Meeting of the American Chemical Society, Philadelphia, Pa., September 2 to 6, 1919.
