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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
corn cobs, a way would be open for the use of a very cheap and abundant waste product. When one realizes t h a t the best sources for nearly all the sugars are t o be found among raw products and plants t h a t occur abundantly in America, most of them being of distinctly American origin, the poet’s lines, “My Country ’tis of Thee Sweet land of liberty,” seem peculiarly appropriate in a novel sense. DSPARTMENT O F AGRICULTURE BUREAUOP CHEMISTRY WASHINGTON, D. C.
THE DETERIORATION OF RAW CANE SUGAR: A PROBLEM IN FOOD CONSERVATION By C. A. BROWNE Received January 4, 1918 INTRODUCTION
T h e changes in composition of food products between manufacture and consumption involve some of t h e most interesting problems of agricultural-chemical research. The problems are also of great economic importance, the financial losses, which result from deterioration of food materials during transportation or storage, amounting each year t o many millions of dollars. I n t h e case of cane sugar, of which there is a t present so serious a shortage, calculations based upon careful analytical and statistical data show t h a t the losses from the deterioration of Cuban sugars alone probably exceed one million dollars per year. The chief ingredient responsible for t h e deterioration of sugars is moisture. As far back as three centuries ago, when sugars began t o be shipped from the R e s t Indies t o Europe, i t was observed t h a t moist sugars reached their destination in a much damaged condition. T h e need of excluding moisture was quickly recognized. Ligon,l one of t h e earliest writers upon t h e subject, i n 1673 pointed out t h e necessity of keeping sugar “drie in good casks, t h a t no wet or moist aire come t o it.” But while early observers were agreed t h a t moisture played an important part in deterioration, the actual cause of t h e phenomenon was for centuries unknown. It was believed by some t h a t t h e trouble was due t o a deliquescence produced by t h e action of chlorides and other saline impurities upon t h e sugar; as late as 1848 Wray2 stated t h a t in his belief it was possible for “this deliquescence t o continue, until the whole mass of sugar is decomposed” and suggested as a possible remedy for deterioration t h e precipitation of chlorides from cane juices b y means of silver nitrate. A more common belief was t h a t deterioration resulted from t h e action of a glutinous fecula or ferment which occurred naturally in t h e cane and, if clarification was imperfect, passed into the sugar. T h e true explanation was not forthcoming until after t h e work of Pasteur, when “History of the Island of Barbadoes,” London, 1678, 111. “The Practical Sugar Planter,” London, 1848, 342-343. It is interesting t o note that Pekalharing (International Sugav Jouvnal, 3, 434) as late as 1900 found it necessary to combat the idea that deterioration was due to the salts contained in sugars, 1 2
Vol.
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Dubrunfautl about 1869 discovered in a deteriorating sugar microorganisms similar t o t h e alcohol and lactic acid producing organisms of Pasteur. After this t h e deterioration of sugars began t o be studied with increasing interest from t h e standpoint of infection b y germs, until t h e subject has now become one of t h e most important fields of research in industrial mycology. After t h e invention of t h e polariscope, some threequarters of a century ago, it became possible to determine t h e keeping power of sugars with an exactness undreamed of by earlier observers. The refiners of sugar, who were t h e first t o p u t t h e polariscope t o practical use, employed this instrument not only for determining t h e value of purchases and for controlling factory operations, b u t they also used i t for following t h e keeping power of stored sugars. With the accumulation of analytical data, which all such establishments acquire, i t was soon observed t h a t other factors beside moisture played a n important r61e in t h e keeping of sugars. It was noticed t h a t impure molasses sugars of high moisture content might keep perfectly when highgrade white sugars of much lower moisture content would rapidly deteriorate. I n other words, it became evident t h a t t h e impurities or non-sucrose constituents of raw sugars must be considered i n connection with t h e moisture content before a reliable forecast could be formed as t o keeping power. Various tables and rules were devised, in fact, towards this end, although but little of t h e valuable information thus gathered was published. The best known of these rules is t h e so-called “factor-of-safety” of t h e Colonial Sugar Refining Company of Australia, according t o which t h e moisture of a sugar must not be more t h a n half the nonsugar if t h e product is t o keep. I n other words, if W is t h e percentage of water and S t h e percentage of W sucrose, the quantity must not exceed 100-
s-
0 . 5 ; or simplified, t h e quantity
mi
W ~
100-
s
must
not
exceed 0.333. EXPERIMENTAL PART A-CHEMICAL
OBSERVATIONS
I n a report published two years ago t h e author2 called attention to t h e value of the “factor-of-safety” of t h e Colonial Sugar Refining Company, and his more recent investigations show t h a t t h e rule is one which can be relied upon in t h e great majority of cases. The 1 ComPt. rend., 68 (1869), 663. The classic observation of Dubrunfaut upon the deterioration of sugars is worth translating. In commenting upon the fact noted so many times since, that raw beet sugars, which were not made by an alkaline clarification, failed t o keep, Dubrunfaut wrote as follows’ “By means of the microscope we were able t o detect in impure beet sugars the presence of those lower organisms, so accurately described by M. Pasteur, and which are the living causes of the alcoholic and lactic fermentations. Nothing can be more simple, therefore, than to arrive a t a n immediate understanding of the formation of the glucoses and of the acid reaction in sugars which were not made by the old traditional sugar-house process known under the name of the alkaline process.” Dubrunfaut attributed the deterioration of refined sugars to the impurities, ferments, etc., introduced into the factory by the raw sugar. It is remarkable how here, as in so many other instances, the opinions of this great French investigator (to whom the sugar industry is indebted for more discoveries than to any other chemist) have been confirmed by subsequent workers. 2 “The Deterioration of Raw Sugar Samples,” Louisiana Planter, 64 (1915), 281-2.
T H E JOURNAL OF INDUSTRIAL A N D ENGINEERING CHEMISTRY
Mar., 1918
author’s experiments indicate, however, t h a t , for Cuban a n d Porto Rican sugars a t least, t h e constant 0.333 is a little too high, t h e value 0.3 for
s being
100-
more nearly correct. This is shown b y t h e following series of experiments begun i n 191j: Eight sugars were selected of t h e average Cuban a n d Porto Rican t y p e with factors of s a f e t y ranging between 0.2j and 0.3j. F o r t y glassstoppered bottles of 8 oz. capacity were then filled with t h e well mixed samples (making eight sets of five bottles each) a n d t h e stoppers sealed hermetically with wax. Periodic analyses were t h e n made, one set of eight bottles being analyzed a t t h e beginning a n d t h e other sets put aside for future comparison. T h e four sugars whose factors ranged from 0.3 13 t o 0.346 deteriorated, while t h e four samples whose factors ranged from 0 . 2 5 3 t o 0.289 suffered no appreciable loss in sucrose. T h e results obtained upon t h e four deteriorating samples are given in Table I. Date
of
An+ ysis May 1915
TABLE I-PERIODIC ANALYSESOF BAD-KEEPINGSUGARS Water Sucrose Invert Undeter(S) Sugar Ash mined Polariby Clerget Per Per Per Sample zation cent Per cent cent cent cent 100-S 1.25 A96.15 96.39 0.91 0.47 0.98 0.346 B 94.85 1.65 95.18 1.22 0.62 1.33 0.343 95.85 1.18 96.34 C 1.13 0.56 0.79 0.322 D 95.55 1.31 95.82 0.86 0.70 1.31 0.313
&Ti
w
- - - - - - -
AVERAGE95.60 95.55 October A 1915 B 94.35 C 95.15 94.65 D
1.35 1.27 1.65 1.09 1.41
95.93 95.75 94.70 95.54 95.09
AVERAGE94.93 January A 95.55 1916 B 94.10 C 94.95 D 94.55
1.35 1.34 1.55 1.12 1.39
95.27 95.92 94.65 95.59 95.10
AVERAGE: 94.79 A 94.30 B 93.10 C 93.85 D 93.05
1.35 1.40 1.58 1.23 1.59
AVERAGE93.58
1.45
--
-
1.03 1.61 2.01 2.04 1.83
-
0.59 0.45 0.61 0.54 0.65
-
1.10 0.92 1.03 0.79 1.02
1.87 1.64 2.12 2.02 1.90
0.56 0.46 0.61 0.55 0.67
0.95 0.64 1.07 0.72 0.94
0.285 0.328 0.289 0.254 0.284
95.31 94.61 93.72 94.80 94.06
1.92 2.30 2.61 2.65 2.69
0.57 0.43 0.67 0.57 0.68
0.85 1.26 1.42 0.75 0.98
0.288 0.260 0.252 0.237 0.268
94.30
2.56
0.59
1.10
0.254
- - - - _ _ _ _ -
August 1917
_ _ - _ _ - - - -
0.331 0.298 0.311 0.244 0.287
__
T h e results obtained upon t h e four samples which did not deteriorate are given i n Table 11. As there was b u t little change in composition only two of the periods are given. T h e results indicate t h a t t h e limiting factor for good-
w
rTr
keeping is about
100-
s
= 0.3.
Anyone who has studied t h e keeping quality of sugars can no doubt report numerous exceptions t o such a rule as t h e above. Examples can be cited of sugars with a factor far beyond t h e limit for safe-keeping which keep perfectly well a n d of sugars with a factor well within t h e safety limit which deteriorate rapidly. T h e exceptions of t h e first class need not detain us long TABLE 11-PERIODIC Date of Anslysis
May 1915
Sample
E
F G H
Polarization 96.70 96.15 96.35 96.45
ANALYSES O F GOOD-KEEPING SUGARS Sucrose (S) Water by Invert Undeter(W) Clerget Sugar Ash mined Per Per Per Per Per aT cent cent cent cent cent 100- S 0.88 96.96 0.66 0.55 0.95 0 289 1.02 96.42 1.40 0.50 0.66 0.285 0.94 96.53 0.92 0.78 0.83 0.271 0.81 96.80 1.05 0.51 0.83 0.253
- - - - - - - -
AVSRAGB96.41 January E 96.75 1916 F 96.15 G 96.05 H 96.45
0.91 0.85 0.99 0.92 0.75
- - - -0.88_
AVERAGE96.35
96.68 97.12 96.71 96.37 96.98
96.80
1.01 0.65 1.12 0.94 1.05
0.59 0.53 0.50 0.79 0.50
0.82 0.85
0.94
0.58
0.81
0.68 0.98 0.72
0.274 0,295 0.300 0.253
0.24s - - - ~
0.275
I79
for t h e probabilities i n t h e case of moist sugars which keep are t h a t t h e organisms which produce deterioration are either absent or t h a t t h e conditions of t e m perature, alkalinity, etc., are unfavorable for their development INFLUEh’CE O F T E M P E R A T U R E U P O N DETERIORATIOlX-
T h e author has noted moist sugars which kept perfectly well i n t h e climate of New York from October t o May. T h e organisms producing deterioration, however, were present and with t h e approach of warm weather in May, t h e conditions became favorable for their development and t h e sugars suddenly began t o undergo a rapid decrease in polarization. As a n example of such a n influence of temperature upon deterioration t h e following analyses are given of a soft refined sugar with high factor.
. . . ., . .... .. .. .. .. .. ..
January 17.. . . . March 18.. June 10.. . .
Polarization 94.35 94.35 92.70
Moisture Per cent 3.90
....
4.07
Sucrose by Clerget Per cent 94.42 94.42 93.10
W 100 s 0.699
~
-
.....
0.590
I n t h e cool season between January and March there was no deterioration, b u t sometime between March and June a very rapid destruction of sucrose began. Cool weather m a y not only retard t h e commencement of deterioration, b u t i t m a y also check t h e process after it has once begun. This can be seen from Table I, which shows a n average loss of 0.66 per cent sucrose between May a n d October of 191j. I n January 1916t h e sucrose h a d undergone no further diminution a n d t h e process of deterioration had apparently come t o a standstill. But t h e organisms producing t h e destruction of sucrose resumed their activity in t h e following summers so t h a t we find i n August 1917a further loss of 1.00per cent sucrose. A correlation of analytical a n d meteorological d a t a shows t h a t t h e destruction of cane sugars b y microorganisms does not usually t a k e place until t h e daily maximum temperature exceeds 2 0 ’ C., which for t h e climate of New York is from about t h e middle of M a y t o t h e first of October. R a w cane sugars of a n y class can be stored without serious risk when t h e maximum temperature in t h e warehouse is below zoo C. But if sugars are t o be kept for t h e season when t h e temperature maximum exceeds 20’ C., then only such sugars should be selected as have a factor of safety below 0.3. DETERIORATION
WITHOUT
LOSS IiX POLARIZATION-
Attention should be called a t this point t o a condition of not infrequent occurrence, where a sugar during storage in a warehouse undergoes no loss i n polarization and yet is steadily deteriorating. This circumstance arises from t h e fact t h a t t h e sugar during storage is losing moisture and t h a t t h e loss in polarization from destruction of sucrose is counterbalanced by t h e drying out of t h e product. T h e custom of making spot tests f r o m time t o time i n order t o see if stored sugar is holding up is therefore of little value unless such polarizations are controlled b y moisture or invert sugar determinations. T h e author has found t h e periodic analysis of sealed samples t o be a most useful criterion of what is taking place in t h e warehouse. He has had excellent opportunity of making such comparisons in connection with t h e analysis of sugars for t h e New York Coffee Exchange where t h e same lots of sugar in t h e warehouse
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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
are resampled and retested with every change of ownership. I n every case where sugars in the sealed bottle lost in polarization, deterioration was observed in the stored sugar. Although the polarization of the latter frequently showed no falling off, yet deterioration was advancing as was indicated by the steady increase in invert sugar. T h e case of Sugar B in Table I is a n illustration of this. This sugar was part of a large lot t h a t was stored in a New York warehouse in May 1915. A sample t a k e n from t h e bags in the warehouse the following October polarized 94.80 as compared with 94.85 when the sugar was stored. This was t a k e n by t h e owner as a n evidence t h a t the sugar was undergoing no deterioration, although a sealed sample of this sugar, kept from the previous May, polarized only 94.35. A complete analysis of the October warehouse sample showed, however, t h a t inversion was taking place. Sucrose
Polarization 94.85 Sealed Sample, May 94.35 Sealed Sample, Oct WarehouseSample. Oct.. 94.80
... . .......
Water (W) Per cent 1.65 1.65 1.21
(S)
by Invert Clerget Sugar Per Per cent cent 95.18 1.22 94.70 2.01 95.19 1.76
UndeterAsh mined Per Per W cent cent 100-S 0 . 6 2 1.33 0.343 0 . 6 € 1 . 0 3 0.311 0 . 6 6 1.18 0.251
T h e October warehouse sample shows a n increase of 0.54 per cent invert sugar: deterioration is t h u s plainly indicated, b u t is concealed, when only a polarization or sucrose determination is made, owing t o t h e loss of 0.44 per cent water through drying out of the sugar. Shrinkage in weight, without the a t t e n d a n t increase in test, caused the owner of t h e sugar a considerable financial loss. U N E V E N DISTRIBUTION OF MoIsTuRE-The exceptions of t h e second class, where sugars of low factor deteriorate, are usually found upon examination t o confirm rather t h a n to nullify the factor-of-safety rule. The deterioration of a raw sugar is confined entirely t o the thin films of molasses which adhere t o the crystals of sucrose. T h e cases of low-moisture sugars which deteriorate result nearly always from uneven distribution of moisture; t h e average percentage of moisture is low b u t there are zones of sugar in the bag whose percentage of moisture is relatively high. Uneven distribution of moisture in the bag may result from mixing together sugars of varying moisture content a t the factory, b u t it seems t o be produced more commonly by the migration of moisture after the sugar is bagged, with the result t h a t the liquid films become more concentrated on some grains of sucrose and more dilute upon others. Fermentation will then set in where t h e films are more dilute, the result being t h a t the average mixed sample of the lot shows deterioration, although the average moisture content may appear t o be well within t h e limit for good keeping. SWEATING-The sweating of raw sugar due t o warm packing or t o unfavorable storage conditions is one of t h e chief causes of moisture migration. T h e danger of bagging sugar as soon as it is emptied from the centrifugals has long been recognized. It is very evident t h a t when warm sugar is packed in a bag, there will be a n expulsion of water from t h e center towards the cooler surface. Zones of high moisture content are formed
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which, with the favoring warmth from the interior of the bag, become exceedingly favorable for the development of microbrganisms. An examination of such sugars when the bags are opened shows t h a t deterioration is not evenly distributed b u t is confined t o zones, the polarization of sugar from different parts of t h e bag showing variations sometimes of several per cent. If such sugar be imperfectly mixed, extremely wide variations may be noted in the composition of duplicate samples. T h e following is a n actual case of three samples of a deteriorated sugar taken from the same mix, drawn from 2420 bags of one mark in a New York warehouse in December 1910. Polarization
Sample No. 1 . . Sample No. 2 . . Sample No. 3 . .
.. .. .... .... .. . .. . .. .. ,. 92.75 94.50 .. . . . . . . . . . . . 93.65 ,
,
Moisture 1.80 1.46 1.18
Migration of moisture may t a k e place not only within the bag b u t may proceed from one bag t o another. I n the case of sweat-damaged sugars in the hold of a ship or in a warehouse, the moisture from the lower tiers may condense upon the ceiling overhead and fall back in a shower upon the upper layer of sacks. A careful manufacturer, who makes sugars t h a t conform to the rules of safe keeping, may t h u s have his product deteriorate through the negligence of other people. DEDUCTIONS FROM THE “FACTOR-OB-SAFETY”
RULE-
If a fixed ratio between moisture and non-sucrose i s the governing factor in the keeping quality of raw cane sugars, there are a number of deductions or corollaries which must follow from such a proposition. The first corollary which we will consider is t h a t slight fluctuations in moisture content have a much greater influence upon t h e keeping quality of high-grade t h a n of low-grade sugars. Thus 0.1 per cent increase in moisture will raise the factor of a 90’ sugar with 0.28 per cent moisture from 0.28 t o 0.35, b u t will raise t h e factor of a 90’ sugar of 2.80 per cent moisture from 0.28 t o only 0.29. I n other words, a high-grade sugar of good-keeping quality can be made unfit for storage by the absorption of only 0.1per cent moisture, while the keeping quality of a low-grade ,sugar having the same factor will not be sensibly affected. This conclusion is abundantly confirmed not only b y laboratory tests but by practical experience. T h e storage of high-grade raw sugars or of moist refined sugars has always been regarded as hazardous. Even white granulated sugar has been found t o deteriorate in a humid atmosphere, owing t o the absorption of moisture. Low purity sugars, on the other hand, can be subjected t o considerable variations in moisture content without loss of keeping quality. A second deduction, which results from the factor-ofsafety rule,is t h a t displacement or saturation of moisture by non-sucrose constituents should render a questionable sugar fit for storage. This conclusion has also been confirmed b y practical experience. It is customary with some factories t o wash t h e sugars in t h e centrifugals with low-grade molasses instead of with water. A superintendent, who had long followed molasses washing, upon being asked why he did t h i s replied t h a t sugars t h u s treated never went back i n
Mar., 1918
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 B E M I S T R Y
storage. This superintendent, who knew a n d cared nothing about “factors-of-safety,” was yet unconsciously making a practical application of t h e rule. LIMIT O F DETERIORATION-A third corollary, t o which t h e author called attention a few years ago, is t h a t sugars which are prevented from absorbing moisture, as in a sealed container} can deteriorate only t o a certain limit. I n other words deterioration will continue until t h e quantity
W 100-
s
becomes less
t h a n 0.3
when t h e process must come automatically t o a standstill. This deduction has been repeatedly confirmed b y making periodic analyses of deteriorating samples t h a t were contained in sealed bottles. T h e deterioration, after a few months or years, depending upon t h e moisture content of t h e sample, came gradually t o a stop and subsequent analyses, even after several years, showed no change in composition. T h e value of -
W IO0 -
s at
which a deteriorating sugar ceased t o lose
in polarization was found usually t o be nearer 0.25 t h a n 0.30. This would seem t o indicate t h a t although t h e destructive organisms might not multiply under conditions when the safety factor was 0.3, yet if a sufficient number of organisms were already present i n a s t a t e of great activity they might continue for a time t o exert a n inverting action upon t h e sucrose dissolved in t h e liquid films. It will be noted, for example, in Table I t h a t t h e average factor of 0.331 i n May 1915, after remaining t h e following winter a t 0.288, underwent a further decrease to 0.254. T h e average factor of 16 sugars a t t h e end of deterioration, determined by t h e writer1 in 1914, was 0.251. The fact t h a t a sugar in an active s t a t e of deterioration may continue t o undergo inversion, even though its factor of safety be under 0.3, helps to explain why many sugars of apparently good keeping quality fail t o hold up. A sugar may have been made with a factor of 0.33 and, beginning a t once t o deteriorate, have had a factor of 0.29 a t t h e time of its arrival in New York. T h e purchaser of this sugar, unaware of previous conditions, might therefore be misled as t o its keeping quality, for t h e sugar being in a n active s t a t e of fermentation had not yet reached t h e limit of deterioration. T h a t there is a certain limit of deterioration has been intimated b y previous investigators. L. LewtonBrain and Noel Deerr2 make t h e following statement: “Another point of interest t h a t requires further investigation is whether there is a definite maximum of deterioration for each bacillus, for each percentage of water or whether t h e deterioration will go on indefinitely merely varying i n rapidity according t o temperature a n d moisture conditions. The probability is t h a t it will go on indefinitely, b u t there is also a possibility t h a t a n accumulation of by-products might inhibit further activity when a certain point has been reached.” “The Deterioration of Raw Sugar Samples,” Lodsiunu Pluntn,
64,282.
* Hawaiian Sugar Planters’ Association,
9 (1909), 32-3.
Division of Pathology, Bdletin
181
In answer t o a letter requesting his present opinion upon t h e subject, Mr. Deerr makes t h e following additional statement: “I now think there is a final maximum of deterioration for a percentage of water, b u t if t h e sugar is free t o absorb water, deterioration will continue t o far limits. If t h e sugar is in a stoppered bottle, I think the deterioration is limited.” This opinion of Mr. Deerr coincides with t h e results of t h e author’s experience. I n 1 9 1 5 t h e writer’ suggested t h e explanation t h a t t h e limit of deterioration in a sealed sample was reached when t h e liquid films were saturated with non-sucrose ingredients, a t which limit “the dissolved sucrose is practically all inverted and no more sucrose can pass into solution from t h e underlying crystal.” Subsequent studies of t h e liquid films a t t h e end-point of deterioration show them, however, t o contain a considerable amount of uninverted sucrose. Experiments made t o reproduce t h e conditions in a deteriorating sugar by coating fine glass beads with films of a molasses undergoing deterioration likewise showed t h a t all t h e sucrose could not be destroyed under such conditions. Unless, therefore, a sugar can absorb moisture from t h e outside or produce moisture during fermentation, deterioration never destroys t h e whole of t h e sucrose originally present in t h e liquid films. T h e possibility, suggested b y Lewton-Brain and Deerr, t h a t a n accumulation of by-products may inhibit the activity of t h e organisms which produce deterioration, derives considerable support from t h e fact t h a t after a few years fermented samples of sugar in many cases fail t o produce colonies upon agar or gelatine plates. It was only in cases where deterioration was produced by organisms which formed resistant spores or where water seemed t o be formed as a fermentation by-product t h a t t h e author was able t o obtain colonies from old fermented sugars. T h e death of t h e organisms in old sugars may be due t o t h e formation of substances actually toxic or t o a concentration of invert sugar which by its plasmolytic action2 causes t h e destruction of life. A B N O R M A L FERMENTATIONS-over 9 0 per Cent Of t h e cases of deterioration of sugars studied by t h e author correspond t o t h e examples given in Table I, in which t h e polarization and sucrose regularly diminish and t h e invert sugar increases until t h e limit of deterioration is reached, t h e percentage of moisture remaining practically constant throughout. A number of cases have been noted, however, in which t h e fermentation followed a different course. I n some instances a n increase in polarization was observed which might afterwards be followed by a progressive decrease in test. Examples of this type of fermentation have been 1
”The Deterioration of Raw Sugar Samples.” LO%iS%aMA Plantfl,
I C (1915), 281-2. 2 Prof. W. L. Owen, Bacteriologist of the Louisiana Sugar Experiment Station, in a recent conversation with the author, suggests that the degree of osmotic pressure necessary t o produce plasmolysis in the cells of the organisms which inhabit the sirupy films, may represent the limit t o which sugars can deteriorate. Osmotic pressure may, perhaps, be the basis of the factor-of-safety rule.
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
182
previously reported b y Watts and Tempanyl in the West Indies, by Deerr and Norris2 in Hawaii, and b y other observers. I N C R E A S E O B POLARIZATION I N ABNORNAL FERMENTA-
TIONS-An initial increase in t h e polarization of stored sugars is usually due t o a partial drying out of t h e product. This increase, however, may take place without the sugar losing moisture, in which case there must be either a production of some new dextrorotatory substance such as dextran or a destruction of some levorotatory constituent of t h e sugar such as fructose. The author3 has studied the production of dextran in fermenting sugar-cane juice but has not been able t o observe its formation in raw sugars in any instance among the several hundred cases of deterioration which he has investigated. It seems more probable t h a t the increase in polarization of stored sugars, where loss of moisture does not occur, is due t o the fermentation of fructose as suggested by Watts and tern pan^.^ The destruction of reducing sugars in stored samples of sugar is in fact not unusual. Watts and Tempany4 report a n instance where a muscovado sugar between May 6 and June 2 1 underwent a decrease in reducing sugars from 3.58 per cent to 0.65 per cent and an increase in polarization from 88.8 t o 91.0. Deerr and Norris4 also report the case of a sugar which, after four months storage, underwent a decrease in reducing sugars from 1.65 to 0.22 per cent and an increase i n polarization from 93.7 t o 95.2. I n a case observed by the author a sugar on June I j , 1915, polarized 94.05 and contained 1.90 per cent invert sugar and 1.54 per cent water; on January IO, 1916, a duplicate sample of t h e same sugar, which had been hermetically sealed, polarized 95. j5 and contained 0.89 per cent invert sugar and 1.73 per cent water. One part of fructose conceals t h e rotation of 1.4 parts of sucrose, and if we assume t h a t the loss of 1.01 per cent invert sugar was due t o fermentation of fructose there should be an increase in polarization of 1.41, which agrees fairly well with t h e 1.5 increase observed. ABNORMAL CLERGET VALVES-If the fructose be fermented away from a mixture of sucrose and invert sugar, the residue of sucrose and glucose should give a Clerget value lower t h a n t h e direct polarization instead of higher as is usually t h e case. The above sample which polarized 95. j j gave a Clerget value of 95.26. An independent analysis, performed a t t h e author's request by Mr. A. H. Bryan, gave a polarization of 95.60 and a Clerget value of 9j.36. The results indicate a destruction of fructose in this sample by fermentation. FORMATIOX
OF
WATER
(VOLATILE
MATTER)
IK AB-
the case of abnormal fermentation previously cited, t h e water, or volatile matter, increased from 1.54 per cent t o 1.73 per cent. This difference of 0.19 per cent may have been due
NORNAL FERimNTATIoNs-In
1
"Fermentation Changes Occurring in Muscovado Sugars,"
West
Zndzan Bulletin No. 11, 7 (1906). 226-36. 2 "The Deterioration of Sugars on Storage," Experiment Station of the Hawaiian Sugar Planters' Association, Bulletin 24. a "The Fermentation of Sugar Cane Products," J. Am. Chem. S O C , %E, 453-469. 4 LOC.
czt.
Vol.
IO,
No. 3
partly t o some experimental error and partly to t h e formation of alcohols, esters, or other volatile products. The increase in water, or volatile matter, seems t o be associated in some way with this type of fermentation. As another example t h e following case of a Cuban sugar is given. Four jars of the well-mixed sample were filled and hermetically sealed in April 1915. Periodic analyses of the samples showed the following: Water Date of Analysis Apr.6, 1915 Jan. 10, 1916.. , , , Aug. 1, 1917
......
Polarization 96.00 96.05 95.85
......
Sucrose (S) by
Invert
Per cent 96.43 96.36 95.47
Per cent 1.17 1.05 0.87
(W) Clerget Sugar
Per cent 1.14 1.11 1.51
Ash Per cent 0.51 0.54 0.54
Undetermined Per W cent 100 S 0.75 0.316 0.94 0.304 1.56 0.338
-
But little change is noted between t h e April and January tests. The August 1917 analysis, however, shows an increase of 0.37 per cent in water, or volatile matter, a decrease of 0.30 per cent in invert sugar, a decrease of 0.15 in polarization, and a decrease of 0.96 per cent in sucrose. I n t h e case of the first two tests the Clerget value is higher t h a n t h e polarization, while i n t h e August analysis i t is lower, as is usually t h e case with this type of fermentation. The undetermined matter shows a marked increase, as does also the factor of safety. The fact t h a t a r a k sugar can undergo a serious loss in its sucrose content with but little change in polarization is only an additional illustration of t h e inadequacy of an uncontrolled polariscope test. VOLATILE
DECOMPOSITION
PRODUCTS
OF
STORED
Alcohols and Esters-As shown by Table I t h e chief chemical change in the deterioration of sugars is the inversion of a part of the sucrose. With a n average loss of 1.63 per cent sucrose there was a n average gain of 1.j3 per cent invert sugar which is only 0.18per cent below t h e theoretical. This unknown loss may be taken as the average amount of sucrose converted into gums, acids, alcohols, esters, and other fermentation products. The inversion of 1.63 per cent sucrose involves t h e loss of 0.08 per cent water; the results of Table I, however, show an average increase of 0.10 per cent water, or volatile matter, SO t h a t the unknown loss of 0.18 per cent must consist almost wholly of volatile constituents. The quantity of samples was not sufficient t o determine whether t h e latter were of an alcohol, aldehyde or acid nature. Nearly all the samples, upon opening, gave off a perceptible rum-like odor, so t h a t it seems safe t o assume t h a t alcohols and esters make u p a certain part of t h e volatile decomposition products of stored sugars. The strong odor of esters, which is noticed upon entering a warehouse or the hold of a ship, where raw sugar is stored, is sometimes regarded as an evidence of deterioration, but this is not necessarily the case. Sealed samples of raw sugar may develop an intense rum-like odor without showing t h e slightest loss in sucrose. Carbon Dioxide-The volatile decomposition product which is given off in greatest quantity b y stored sugars is carbon dioxide. This gas seems always t o be produced, whether or not the sugar is undergoing deterioSUGARS.
M a r . , 1918
T H E J O U R P A L 0 F I N D U S T RI A L A ATD E-VGILVEERING C H E M I S T R Y
ration. While t h e q u a n t i t y of carbon dioxide in sealed bottles of raw sugars is usually highest where t h e loss of sucrose is greatest, this is not always true. I n the case of six samples of raw sugar which had undergone deterioration t h e carbon dioxide content of the air in the bottle after two years was found t o be 6 2 j , 2 7 4 , 310, 33, 1 0 2 a n d 494 times t h e amount present in the laboratory air. I n t h e case of five samples of raw sugar which had undergone no loss in sucrose similar figures for carbon dioxide after one year were 2 2 6 , 51, 26, 1 7 1 and 309. I n several instances where t h e air was drawn off from bottles of deteriorating sugar t h e oxygen was found t o be almost completely displaced b y carbon dioxide. I t was thought a t first t h a t t h e exhaustion of the oxygen supply might be t h e cause of the d e a t h of t h e organisms in t h e old samples of sugar b u t this condition of oxygen exhaustion was observed in only a few cases a n d there seemed t o be no connection between t h e phenomenon a n d the limit of deterioration. I n the case of a soft refined sugar undergoing deterioration t h e oxygen of t h e air in t h e bottle was 91 per cent consumed after 8 months a n d 95 per cent consumed after 19 months. T h e number of microorganisms per gram after 19 months was j50,oOo and t h e sugar was still undergoing inversion. T h e evolution of carbon dioxide from sugar in t h e hold of a ship is sometimes so great t h a t workmen have been overcome u p o n removing the hatches. S P O K T A N E O U S C O M B U S T I O N OF SuGARs-The absorption of oxygen a n d the evolution of carbon dioxide b y a stored sugar resemble t h e process of respiration which fruits, vegetables, grains, tobacco a n d other products undergo in storage. Under certain unusual conditions, which are not perfectly understood, this absorption of oxygen may proceed with sufficient intensity t o cause spontaneous combustion of t h e product. There are, in fact, well authenticated cases where this has happened t o sugar in bulk. Schonel mentions a n instance where 1000 tons of raw beet sugar in a German factory underwent spontaneous combustion with almost explosive violence. Wasilieff2 also mentions a similar occurrerice with raw beet sugar i n a Russian factory. M a n y of the cases where cargoes of raw cane sugar have mysteriously caught fire no d o u b t resulted from spontaneous combustion. Certain sugar-containing products, such as molasses feeds, are particularly susceptible t o this p h e n ~ m e n o n . ~T h a t t h e heating of a mass of moist sugar is produced b y yeasts or other organisms is well known, b u t how these organisms can elevate t h e temperature far above t h e point a t which t h e y c a n exist has seemed a contradiction. It has been held b y some physiologists t h a t in t h e fermentation of sugar t o alcohol or lactic acid, certain unsaturated intermediate products are produced. I n t h e interior of a fermenting mass of sugar, when t h e supply of oxygen is used up, these unstable unsaturated products may possibly be formed in considerable a m o u n t . If outside air sud1
Deut. Zuckevind., 36 (1911), 608.
2
2. Ver. Zuckerind., 1902, 864.
a Report of the Chief Inspector, Bureau for the Safe Transportation of Explosives, etc., B. E. Report No. 7 , p. 47, discusses the spontaneous combustion of alfalfa-molasses mixtures. Spontaneous combustion of bagassemolasses feeds has occurred in ships.
183
denly gained access to t h e interior of such a mass, the intense absorption of atmospheric oxygen might easily elevate t h e temperature t o the point of combustion. T h e conditions of moisture, air supply, etc., which favor t h e spontaneous combustion of sugars are, however, of very unusual occurrence, a n d t h e financial losses from this cause are slight in comparison with t h e slower a n d less spectacular process of inversion which, after all has been said, is responsible for t h e greater part of the losses from deterioration of the Cuban crop. B-
M Y C 0L 0 G I C A L
0 B S E R V A T 10 N S
P R E V I O U S INVESTIGATIOKS-Since t h e time Of the first observation b y D u b r u n f a u t , fifty years ago, various writers have referred t o t h e action of microorganisms in producing t h e deterioration of sugars. I t is only, however, within t h e last t w e n t y years t h a t t h e specific behavior of these organisms a n d t h e method of preventing their action upon sugars have been subjected to serious study. I n 1898 Shoreyl detected the mould Pelzicillium glaucum in samples of deteriorated Hawaiian sugars a n d suggested t h a t t h e inverting action of this fungus was a common cause of deterioration. Shorey be1;eved infection t o t a k e place through spores drawn into the sugar by t h e current of air i n t h e centrifugals, a n d made t h e observation t h a t the sugars which showed most deterioration were usually made in dusty localities where such spores would be most easily scattered. AS a protection against infection Shorey recommended t h a t the sugar in the centrifugal, during t h e process of curing, be sterilized b y a current of d r y steam. I n 1 9 0 2 Greig-Smith a n d Steel2 in Australia discovered a sugar-destroying organism, related t o t h e heatresisting so-called “potato” bacilli, which was found t o occur in raw sugar from Australia, J a v a , hlauritius, E g y p t , Peru, Fiji, France, Germany a n d Russia. F r o m its conversion of sucrose into t h e levorotatory gum levan, Greig-Smith a n d Steel named their organism Bacillus levaniformans a n d from its wide distribution expressed the belief t h a t i t , a n d not the mould of Shorey, was “responsible for t h e bulk of t h e deterioration of sugar during transit a n d in store, which has been noted from various parts of t h e world.” As a remedy against t h e infection of sugars b y this organism, GreigSmith a n d Steel recommended thorough cleanliness of apparatus a t all stages of manufacture a n d s t e a m sterilization in t h e centrifugals. I n 1909 Lewton-Brain a n d Deerr3 made a s t u d y of t h e “Bacterial Flora of Hawaiian Sugars’’ a n d came t o t h e same conclusion a s Greig-Smith a n d Steel t h a t moulds are not t o be considered as a cause of deterioration. Lewton-Brain a n d Deerr isolated five different kinds of sugar-destroying bacteria, two of which produced a gum similar to levan: these authorities came, therefore, t o t h e conclusion t h a t t h e production of levan cannot be held as characteristic of one particular 1 “The Deterioration of Raw Cane Sugar in Transit or Storage,” J . SOC.Chem. I n d . , 17 (1898), 5 5 5 . 2 “Levan. A New Bacterial Gum from Sugar,” J. SOC.Chem. Ind., 2 1 (1902), 1381. 8 Experiment Station of the Hawaiian Sugar Planters’ Assoc., Division of Pathology and Physiology, Bullelin 9.
'84
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
bacterium. According t o Lewton-Brain ' and Deerr "the best safeguard against deterioration is the maintenance of the factory itself in as aseptic condition as possible, t h e avoidance of t h e introduction of bacteria, as for instance in t h e use of unclean water a t or about the centrifugals, a n d t h e disinfection of the factory during t h e off season." I n 191r Owen,' in Louisiana, came t o the conclusion t h a t the deterioration of sugars is not due to inversion b u t to a gum fermentation which is formed by a slightly alkaline reaction. This gum fermentation, according t o Owen, is produced b y a group of bacteria, comprising t h e so-called potato bacilli, t h a t cause t h e destruction of sucrose b y an enzyme ~ C W I W X C , * which is extracellular in its action a n d breaks down sucrose according to the equation: C ~ ~ H ~= ~O CaHisO6 IL CsHioOs Sucrose Glucose Levan The potato bacilli, which occur widely distributed in t h e soil, can easily he introduced into the sugar factory by dirt adhering to the cane, a n d the great resistance of t h e spores of these organisms t o heat offers a means of their passing through all t h e stages of manufacture into the sugar. Owen in fact showed b y experiments in Louisiana t h a t while 98 per cent of t h e organisms occurring in cane juice are destroyed i n t h e process of clarification, in no stage of t h e manufacture is t h e product entirely free from microbrganisms a n d t h a t there is a remaining z per cent of heat-resisting spores, which escape destruction and find their way into t h e final sugar. As a remedy against deterioration Owen recommends t h e use of antiseptic washes for the mills and tanks of the sugar house and t h e exercise of greater care in drying t h e sugars. While t h e observations of t h e authorities just mentioned may in large measure he true for t h e respective countries where their work was performed, it has seemed t o the author entirely too sweeping t o assert t h a t t h e deterioration of sugar is never due t o moulds or t h a t i t is nearly always produced b y one specific bacillus or specific class of bacilli. Table I , which is typical, shows t h a t the deterioration of Cuban sugars, a t least, is mainly a process of inversion and t h a t t h e formation of levan a n d other gums is not of usual occurrence. According t o cultural experiments carried out by the author, t h e organisms most prevalent in Cuban raw sugars are not bacteria b u t certain organisms belonging t o the budding fungi. The occurrence of such fungi in deteriorating sugars has in fact been previously indicated by Schbne. I n a sample of "farine" (powdered sugar) which had undergone a loss of nearly 8 per cent sucrose, Scb6nd observed a large number of yeast-like cells mixed together with the spores and mycelia of moulds. T h e deterioration, in Schbne's opinion, was started by a budding fungus of the Monilia class and then con-
+
1 "The Bacterid Deterioration of Sugar." Lauisionn Buiiefin 1%; "A Recently Discovered Bacterial Decomposition of SUEm8e,'' TSIE JounaAL, 3 (1911).481. It should be noted in pOSing that levan is a polyssccharide (C6Hio0dn aod. therefore. could not be formed from sueroe by the hvdiolvtlc action
Vol.
IO, No.
3
tinued by t h e moulds. In a second sample of deteriorated "farine" Schbne isolated another budding fungus of the T O ~class. Q PRESENT I N V E S T I G A T I O N S
UEDIA-In the experiments performed by the author a medium was prepared b y boiling a 3 0 per cent solution of raw cane sugar of t h e ordinary 96" type with a little salt-free alumina cream, filtering, and diluting t h e cold filtrate t o a concentration of 20" Brix. This stock raw sugar solution was sterilized and kept, with the usual precautions against infection, i n a large flask. The agar medium for plating was prepared by dissolving 1 5 g. of agar-agar i n 1000 cc. of t h e stock raw sugar solution in a sterilizer a n d filtering through a hot water funnel. The sterilized agar medium was kept in test tubes and flasks, with t h e usual precautions against infection. m m I o n OF PLATING-I g. of raw sugar was dissolved in I O cc. of sterile dist;lled water and 0.5 cc. of t h e solution was mixed with I O cc. of t h e agar meP R E P A R A T I O N OP CULTURE
no.1 x
2
dium, previously liquefied a t about 55' C., and poured into a warm petri dish. After t h e agar had set, the petri dish was placed in an incubator a t ahout 30' C. At t h e end of three or four days t h e colonies were counted and examined under the microscope. If t h e colonies were too numerous, a new plate was prepared from a raw sugar solution of lower concentration. Typical growths were selected from the agar plates and inoculated into measured amounts of t h e stock raw-sugar solution in order t o study the specific action of each organism. The behavior of the latter was also tested upon concentrated raw sugar syrups and upon sterile sugars. I t is not possible in a chemical paper t o give in detail the results of all experiments and only a few of t h e more typical observations are described. N U M B E R OF ORGANISMS-The average number of colonies produced upon raw sugar-agar plates by the method just described was 144,ooo per gram for Cuban sugars as delivered in New York, the number varying
Mar., 1918
T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY
from 3,500 t o j71,ooo. During storage this number of organisms might increase for a time if the sugar was i n a state of deterioration, and samples have been noted which produced over r,ooo,ooo colonies per gram of sugar. On very long storage, however, the number of microBrganisms undergoes a decrease; samples of raw sugar after two years are frequently found t o be almost sterile. An example of the colonies obtained from o.oj g. of a Cuban raw sugar upon an agar plate is shown in Fig. I . K I N D S OF M I C R C O R C A N I S M S . Torula CommunisT h e most abundant organism observed i n Cuban raw sugars was a non-inverting Torula. A similar organism was also found in raw sugars from the British West Indies 2nd in soft refined sugars.. It appears to be one of t h e most widely distributed of the microscopic flora which thrive in cane sugar factories, 2nd for this reason has been named by the author Torula commuuis.'
I 8j
GROWTH I N RAW SUGAS SoLUTIONS-TO?ulU communis grows readily in raw cane-sugar solutions from the most dilute to t h e most concentrated. It forms a granular sediment of cells and, a t later stages of growth, a thin
ao,3 x lono Magnified cells of Torulo communir.
marginal film. A slight evolution of gas takes place, b u t never with froth or foam as with yeast. A strong, fruity, ester-like odor is also characteristic. A C T I O N U P O N R A W SUGAR SoLuTIoNs-The action Of Torula communis upon raw cane-sugar solutions consists principally in a destruction of invert sugar, fructose being t h e ingredient mostly affected. Sucrose is not inverted. T h e following fermentation experiment upon j o cc. of a solution of 64O Brix was conducted for 21 days a t 28' C.
so
ORICMU. CC. SOLOTlON CONTAMS
SOLUTZON
DILUIBD 10 200 Cc.
Palaiiiation Blank ..., +76.60 Tordo comm~nis.. +76.85
.,.___..
FIO. 2 X .. 2 Colonies oi Torula communi* in different stage3 of growth. O n the left is n!amall star-shaped cyst, the nucleua of the future colony; above it are two colonies forming around their nuclear cysts. The l v z e circles are idly ~~
~
developed colonies. The colonies ere white but sppesr black by trammitted tight.
CoLoNrEs-The colonies upon raw sugaragar a t t h e beginning have the appearance of minute white cysts which under the microscope show a n angular contour of boat-shape or arrow-head form. T h e cysts increase in size t o a diameter of 0 . 2 - 0 . 5 mm. until the surface of the ayar is reached, when the organisms spread out in all directions from the point of emergence. The colony gradually assumes a circular or heart-shaped form of grayish white color, varying in diameter from 3 t o I O mm. and retaining the original cyst as a dense white nucleus (Fig. 2 ) . With very old colonies a brownish color appears. MICROSCOPIC APPEARANCE-under a high power Of t h e microscom Torula commuuis is seen t o consist of single cells, yeast-like in appearance and without mycelium (Fig. 3). * Owen (Louiriono Plonlsr, 56, 173) mentiom amoog the mieroarganilimr F O R M OF
of unrefined sugars a "on-invening T O ~ ~ which O , is no doubt the same organirm 89 the o m described by the author BO Torula commwis. The spedfie names, remmunis, nrzro, fusco, etc., employed by the author in thio and the following cases are used provisionally until the exact relatiomship of the organisms to their 6enera can be determined.
Clueet Value
76.78
16.56
sueiose Invert S U * P I Grams Gram 39.9256 n.8o8s 39.8112 0.2338
I n the above experiment the blank solution lost 0,3458 g. in weight and t h e Torulu-inoculated solution 0.7535 g., the difference of 0.4077 g. representing t h e loss due t o evolution of carbon dioxide and other volatile products. A second fermentation experiment was conducted upon j o cc. of a clarified supersaturated raw sugar solution of 78" Brix for 2 7 days a t 30° C. T h e excess of sucrose crystallized out during the exueriment. leaving a solution of about 69' Brix. I
50L"lZOlr
Dnmm m 200 Cc. Pelarization Clerget Value Biaok +98.70 98.86 Torwlo romntundr.. +98.95 98.27
.......__...
In the second experiment the loss due t o evolution of carbon dioxide, alcohols, esters, etc., after correcting for t h e loss in weight of the blank solution, was 0.6118 g. The distribution of this loss over t h e 27-day ueriod is shown in the following It will be - diaaram. -
Days seen t h a t the greatest intensity of fermentation was between the 9th and 15th days. The two experiments show a marked destruction of invert The "lective action Of communis upon fructose is seen by t h e increase in polarization and
186
T I l E J O l ~ R , V A L O F I N D C S 2 R I A L A N D EAVGIA7EERING C B E X I S T R Y
b y t h e lowering oi the Ciergct value beloiv tlie polarization. The changcs of this character. pKcYioUsl)r notcd in the.casc of certain stored sugars, ar? n o doubt due t o this organism. The inability t o in\-ert sucrose is characteristic of many Torulue,' aithough such inability does not prevent these varieties from subsisting a t the expense of sucrose. h destructivc action of this kind is shown in the previous results. w!iere thcre was a loss of 0.8144 g. sucrose in the first experiment and a loss of 0.2968 g. sucrose in t h e secoiid. The experiments show how a ram sugar may gain in polarization and yet undergo an actual ioss in sucrose. Although r e l a t i d y harmless, in comparison with t h e organisms which invert sucrose: the non-inverting Toruloc may become deieterious in the case of sugar stored for a considerable period. Among t h e most destructive organisms found by t h e author in Cuban raw sugars were two varieties of M o n i l i a c , u,liicli from the difference in color of their colonies upon agar have heen named Monilia nigra and Afoizilia justa,.
Vol.
10,
No. 3
tains a diameter varying from I mm. t o 1 5 mm. the ends of the hyphae pi-ojccling beyond the bud-cell conglomerate and yeast iiims usually break up into ciusters ol dark conidia (irequently of branched treelike form) which give t h e colony a jet-black coior.
smaii ~
PIC. 5 x 2 ~or ~ o ni i i i a ~ in v~iious ~ stages i or~grOwth. ~
From the latter circumstance the organism has been named Monilio iiigra. Somet,imes t h e colony stops growing before t h e conidia stage is reached, i n which case the white color remains unchanged. The latter is particularly a p t t o occur when tlie coionies are so nunierons as t o coaiesce; thc surface of the agar may then be covered with a dcnse white gromtli of bud-cells which a t first glance might be mistaken for yeast colonies. Variations in composit,ion oi the agar and in temperature of incubation cause such differcnce in t h e shape and appearance of the colonies t h a t the latter
J P*a. 4
x
2
3,arge rdonies of Monilia nigra. The radiating growths consist7d hyphae covered with bud~cells. T h e tiiitrd terminal growths a m conidia.
Monilia- nigra-Some of the raw sugars examined by the author, when plated out, gave practicaliy pure cultures of this organism. I n the case of one fermented sample (A, Table I ) which had been sealed owx two years, rgoo colonies of this Monilia were produced from I g. of sugar (Fig. 4). F O R M OF c o ~ o x ~ ~ s - T l i colonies e upon raw sugaragar have a t first the appearance of smaii star-shaped white dots, which under the microscope are seen t o consist of radial hyphae. The latter throw off a conglomerate oi bud-cells, the mass oi which increasing in thickness soon gives t h e colony a starfish appearance. This primary g r o r t h of thc colony is usually succeeded hy a secondary growth, due t o t h c propagation of the burl-cells, which, without t h e formation oi hyphae, germinate like yeast and cover thc center oi the colony with a white amoeba-like film. When t h e colony at-
,
or
F~~rusther pnrticu~arse%to the action the ~~~~i~~~~~ sUCrOEe and other swarr see Ld.aiar's Terhnisihc rMykdotic, a (1907). 717, or Sdter's ~ r ~ . ~ a (1911). ~ ~ 296. ~ t i ~ ~ ,
FIG.6 X 50 Magnified ~ o l o o yof Monilia niaro. The radiating growth. c~nsist01 hyphae, covered with bud-cells. The black terminal Erowihs are conidis. The secondary growth of.bud-cclls forms the circillar film.
might appear due t o differcnt organisms. The appearance of the colonies is shown in Figs. 4, 5 and 6 . YICROSCOPIC APPEARAXCE-The polymorphic characteristics of Monilia. nigra are also shown under the microscope. T h e hyphae are sometimes smooth, of
T I I E J O U R i V A L OF I.VDUSTRIAI,
Mar., 1918
t h e ordinary branched type, b u t are more often studded with clusters of bud-cells. T h e latter are irregularly elliptical in shape a n d when detached, depending upon conditions, produce new hyphae or propagate like yeast. When t h e mycelium approaches its maximum growth t h e hyphae hegin to break u p a t the ends into dark thick-walled conidia. The latter often occur as twin spores in which case they are produced b y a process of cell division. T h e disintegration of t h e hyphae into thick-walled cells may also occur a t other points t h a n t h e ends i n which case they often have the appearance of oidia. T h e various cell forms of M o d i a uigra contain large numbers of oil globules. The microscopic appearance of the various cells is shown in Fig. 7. G R O W T H I N R A W S U G A R SOLCTIoNS--i~OfZilia nigra grows readily in raw cane sugar solutions excepting the most concentrated. T h e solution becomes turbid with a growth of fibrous mycelia while the walls of the flask about 2 mm. above t h e liquid often become coated, after several days, with a margin of dark conidia I cm. or more in width. There is a very slight formation of gas; a mild, ester-like odor is also perceptible.
A N D ENGINEERING CEEMISTRY
181
A third fermentation experiment was attempted with a saturated raw sugar solution of 69" Brix, but the organism was unable t o thrive in a solution of this concentration and no change i n composition could be detected after four weeks incubation a t 30' C.
FIG. 8 X 2 Colonies of Monilia furco in YBIious stages of growth
u-
P.0. 7
Y
SM)
ACTION U P O N R A W S U G A R SoLuTroNs-The action Of Monilia nigra upon raw cane-sugar solutions consists principally in an inversion of sucrose. T h e following fermentation experiment upon j o cc. of a solution of 21' Brix was conducted for three weeks at 28' C.
Monilia fusca-Bes;.des the preceding form a second more strongly inverting variety of Monilia has been observed by the author in Cuban sugars. T h e colonies upon raw sugar-agar (Figs. 8 and 9) resemble those of Monilia uigra in some characteristics, b u t are distinguished from t h e latter b y a much greater length of hyphae, by a less pronounced tendency t o form secondary yeast films. a n d by a greenish brown color in t h e conidia stage instead of black. Owing t o this difference in color the organism has been named Monilia fusca. The principal microscopic features are shown in Fig. IO. GROWTH I N R A W S U G A R soLvrIoNs-Mo%ilia fusca grows in raw cane sugar solutions excepting the most concentrated. T h e solutions become turbid and there is a deposit of mycelia and cells. The walls of the
"sa:
onzow.4i.50 cc. SOLUTlON UILCTID 70 1M)
CC.
Polarization cierget valve Blank ............ +40.55 40.94 Monilia nigra , , _ _ , 7.15 15.54
+
SOLVTION CONTAINS
sucrose CramS 10.6444 4.0304
Invert svzar Grams 0.2952 5.8965
9 second fermentation experiment, conducted upon 2 8 ' C.
j o cc. of a solution of 64' Brix for three weeks a t
showed the following results: OazclN*r. 50 SOLUTION
DISUTSDTO 200 Cc. Polarkration Clerzet Value Rlank ............ +76.60 76.78 Jfoniiio nigra.. +72.65 73.83
...
cc.
SoLYIrDN CONTAINS
sucrase Gramr
39.92Sh 38.3916
Invert sugar GramS 0.8085 2.1932
In the second fermentation experiment t h e solution inoculated with Alonilia qzigra lost 0.04j q g. more in w i g h t t h a n the blank, which shows only a very slight evolution of carbon dioxide. T h e experiments show t h a t t h e inverting action of Moizilia nigra is considerably restrained by increasing the concentration of sugar.
PIG.n X 50 Magnified c o l o n y ~ ddfonilio /"sin. with bud-cells s n d dark conidia.
The radiating hyphae are covered
flask, t o a distance of 3 em. or more above the liquid, become coated u,ith a growth of dark conidia. There is a r e r y slight evolution of gas: a pronounced fruity odor is also developed.
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 CEE,MISTRY
188
Vol.
IO,
No. 3
ACTION UPON R A W S U G A R SoLUTIoNs-The fol~owing A C T I O N U P O N R A W S U G A R sOLuTIoNs-The action Of fermentation experiment upon 50 cc. of a solution of the bacterium upon raw cane-sugar solutions consists for 21' Brix was conducted for three weeks a t 28' C. the most part in an inversion of sucrose, from which circumstance the organism has been named Bacterium O B I O I ~ ~5n L cC. SOL"r*O* SoLurroli CONT*lNS inverlens The following fermentation experiment upon DLLUTZD TO inn CC. sucrose Invert susar Polariratioo CIerpet Vdue
......,..... 1-41.30 Monilinfuscn ..... f 3 . 1 5 Blank
41.81
12.60
Grams
Grams
10.8706 3.2760
0,6368 7.1869
A second fermentation experiment conducted upon 5 0 cc. of a solution of 64* Brix for three weeks a t 28'
C. showed t h e followina- results: SOLUTION D!LUTBO YO
zoo
CC.
Polu,zation Clerget Value
...._.....__ f76.60 .._..1-43.75
Blank Moniliofurrs
O B M ~ 50 ~ LcC.
SoLur'on
76.78
52.01
s"cr0Sc Gramn
39.9256
27.0452
CONTAINS
Invert sugar Gram$
0.8085 10.9880
I n the second fermentation experiment the solution inoculated with Monilia fusca lost 0.0346 g. more in weight than the blank, which shows only a very slight evolution of carbon dioxide. The experiments show t h a t Moniliafusca has a much stronger inverting action than Monilia nigra and t h a t the activity of t h e organism is less restrained b y increasing the concentration of sugar. A third fermentation experiment was attempted with a saturated raw sugar solution of 69' Brix, b u t the organism was unable t o thrive in a solution of this concentration a n d no change in composition could be detected after €our weeks incubation at ,yoo C.
50 cc. of a solution of weeks a t 28' C.
Brix was conducted for three OIIOINAL
SOLIIIION
I ~ . ~ I ( BfO D
inn
cC.
Polarization Clerget Valuc
Fie. IO
x SW
Magnified cells of Monilia h s r o . In the middie is a branched part of the mycelium beating 4 bud-cells: two of the latter (one germmatimp) ?re shown at the left. At the risht i? the end o< o w of t h c h y p h s e , brcakmg UP 01 the end into 3 csoidia end m the middie into 2 oidia.
The great variability of t h e Moniliae in habits of growth renders t h e m exceedingly adaptable t o conditions of environment, and for this reason they are t o be counted among the most destructive organisms which thrive in raw sugars. Bacterium invertens-In addition to t h e Torula and Monilia forms just described, plate cultures of Cuban raw sugars frequently exhibit a different type o€ colony. The surface of the agar becomes covered with an exudation of clear colorless drops (Pig. 1 1 ) which sometimes run together and cover a considerable part of the plate. The organism producing this appearance is a bacterium which under the high power of t h e microscope appears as rod-like cells, detached or in chains, surrounded b y a capsule (Fig. 12). G R O W T I I IN R A W S U G A R soLUTIoN-Bacter~um inverlens grows best in raw cane sugar solutions of low concentration. The solution acquires a milky turbidity, a little sediment is formed and there is a slight evolution of gas. A disagreeable putrid odor is also perceptible.
50 CC.
SoLvrrorr C O N T I I N S suerose Iovert sugar Grams Grams 10.8706 0.6368 7.1578 4.0862
A raw sugar solution of 64') Brix was inoculated with Baclerium inverlens and kept in a n incubator for three weeks a t 28' C. T h e organism appeared unable t o thrive i n a solution of this concentration. No perceptible change took place in t h e appearance of the medium and an analysis a t the end of t h e three weeks showed no difference in composition from the blank. T h e four micronrganisms just described were the most common forms observed by the author in Cuban raw sugars. Other organisms, including moulds (such as Penicillium and Oideum) a n d various bacilli and micrococci, were also detected in different sugars but a description of these must be passed over. The conclusions which the author desires t o emphasize are ( I ) t h a t the microorganisms of raw' cane sugars, as regards their action upon sucrose, are in part harmless and in part destructive; ( 2 ) t.hat the destruction of sucrose in deteriorated sugar is not due t o any single organismor classof organisms; moulds and budding fungi, as well as bacteria, must be looked for, FzG. i 2 20(10 when searching for the agents of Mamised cells of Bodr,;urn inan,clir. destruction: and (3) t h a t the fungi and bacteria, which cause t h e inversion of sncrose in raw sugars, are unable t o thrive in saturated solut,ions. The washing of raw sugars in the centrifugals, by diluting the saturated films of sirup t o a point where the inverting organisms can thrive, must therefore be regarded as a leading cause of deterioration.
Mar., r918
T N E J O G 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 E E M I S T R Y
ORIGIN OF THE INFECTION OF R A W S U G A R S B Y MICRO-
ORcANlsMs-The opinions of various authorities upon t h e infection of raw sugars from the air, from dirt adhering t o the cane, from contaminated wash water a t the centrifugals, from unclean tanks and unsanitary factory conditions have already been mentioned. A few other sources of infection remain t o be considered. INFECTION FROM BAGASSE-Another possible source of infection, t o which attention has been lately directed, is the contamination b y fine bagasse particles (generally called bagacillo from the Spanish diminutive) which find their way, in greater o r smaller amounts, from the cane mills into the raw sugar. T h e contamination may take place through the juicc by reason of imperfect filtration.' or through the air by means of the wind. According t o Owenz t h e only possible ways by which bagacillo can affect the keeping quality of sugar are
Frc. 13 Rottom of pile of sugar bags in a Cubna warehouse. ( I ) by retaining microhrganisms which would otherwise be eliminated, ( 2 ) by providing nutritive material for microorganisms, ( 3 ) by retaining moisture and creating zones favorable far the growth of microorganisms. I n Owen's opinion, whatever merit may reside in t h e bagacillo theory is based solely upon the retention of moisture. The percentage of water-insoluble organic matter, which consists mostly of bagasse particles, in Cuban raw sugars was found from analyscs made i n t h e New York Sugar Trade Laboratory t o vary from 0.01 per cent t o 0.46 pcr cent, the average being 0.17 per cent. T h e average per cent of water-insoluble organic matter
The possibility of deterioration of sugars resulting from infection by bagacillo from high pressure mills through imperfect Kltratioo is discussed by Guijarro in the Lovisiono Planter. 64, 348, and 66, 61. Louiiiann Plant#, 66, 174. 1
189
in sugars which deteriorated was found t o be 0 . 2 2 per cent, and in sugars which did not deteriorate 0.10 per cent. In other words, t h e deteriorated sugars contained twice as much water-insoluble organic matter as t h e sound samples and this apparently would lend support t o the bagacillo theory of contamination. It seems more probable, however, t h a t hagacillo is not so much t h e cause of deterioration as an indication of general carelessness and sloppiness in manufacture. I n other words, if a superintendent is careless in his clarification or filter-press work, he is probably equally careless about protecting his sugars against infection or deterioration. T h e washing of raw sugar in the eentrifugals with water from t h e cooling tower or other infected sources is probably responsible for more losses t h a n the introduction of bagasse particles, Without denying t h e possibility of hagacillo acting as a moisture carrier, i t is only necessary t o point out the case of soft refined sugars, t h e higher grades of which are exceedingly subject to deterioration and yet are absolutely free from bagacillo. INFECTION FROM TIIE COOLING TowER-Olle Of the most dangerous sources of infection for raw cane sugars is t h e cooling tower. In this contrivance the warm condensation water from t h e factory is cooled b y falling in a shower over an outdoor framework into a n exposed basin, from which i t is afterwards returned to the factory. The cooling-tower water, which contains any sugar lost by entrainment, is quickly invaded b y microorganisms, the conditions for infection and growth being exceedingly favorable. The spray from the cooling tower is not only carried into the factory, where it can come into contact with bags and sugars, b u t the cooling-tower water itself is sometimes used for washing the sugars in t h e centrifugals.' All things considered, a more ideal source of infection than the cooling tower can hardly be imagined. INFECTION F R O Y BAGs-Kamerlingz has suggested t h a t deterioration of sugars is produced by organisms introduced from the bags. Although this idea has not found general acceptance, much may he said in its favor. While a mass of solid sugars offers more resistance t o the invasion of germs than does a liquid, the sirupy films which surround the sucrose crystals are in contact and form a continuous medium for the spread of microorganisms. T h e ramifying mycelium of the M o i d i a e also offers a n easy means for this class of organisms t o penetrate t o the interior of a sack of sugar. Infection of bags may take place not only by wetting a-ith spray from t h e cooling tower, b u t it may also occur in the varehouse, or in the hold of a ship. Fig. 13 is a photograph of the bottom of a pile of sugar in a Cuban warehouse. The dark discoloration upon the floor consists of a slimy mass of fermenting molasses a n d sugar dissolved from the bags by rain from a leaky roof. T h e sugar in the bottom bags was in direct cont a c t with this filth and was in a bad state of deterioration. Under such conditions infection might spread 1 An i s t a n c e in Nawaii where deterioration of the manufactured sugar was traced to the use of cooling-tower water for washing at thecsotriiugals, hss been mentioned to the author bg Mr. Noel Deem. 2 Infnnvlionnl Sugar Journal, 8, 484. From Report of the West Java Sugar Experiment Station *'Rezok" for 1900.
THE J O U R N A L OF I N D L ' S T R I A L A:VD BNGIA'EERIiVG C H E M I S T R Y
I90
through a large pile of sugar. Fig. 1.i shows the pile of fermenting slime which had been raked u p after removing the bags of sugar. PREVENTION O F THE DETERIORATIOS OF R A W C A N E
SWGARS-In concluding the mycoiogical part of this paper a few words might be said about t h e means for counteracting the destruction of sugar by microorganisms. I n t h e matter of manufacture i t is necessary ( I ) t o exercise the utmost possible cleanliness and care in order t o diminish infection, ( 2 ) t o control the moisture content of the sugar so t h a t the ratio of non'. sucrose t o water is within the liniits of safety, ( 3 ) t o cool the sugar thoroughly before bagging t o prevent the migration o[ water a n d t h e formation oi zones of high moisture content. I n the matter of storage it is necessary (s) t o keep the sugar perfectly dry in warehouses which are rain-proof, ( 2 ) to keep the warehouse tightly closed i n wet weather to prevent the sugar absorbing moisture from t.he air, ( 3 ) t o construct the warehouse and store the sugar so as to secure in dry weather the maximum ventilation underneath and between t h e bags.
Yo].
IO,
No. 3
I n order t o see how near the manufacturing conditions of Cuba conform t o these requircments t h e following figures are given for t h e year '91.6.
.
Average PFliariratioo of sugsr as =ampled at Ne- York.. ... .. , . . . . 95.80 Average per cent moisture of sugar 8s sampled st New York.. .. .. .. 1.35
The above per cent moisture, however, owing t o drying out of sugar during transportation and during t h e operations of sampling and mixing is about 0.3 lower than when the sugar was made. As a conservative estimate we may accept 1 . 5 per cent moisture and 9 j.6 j polarization as t h e average condition of t h e sugar between manufacture and delivery. For raw cane sugar of this polarization there is an average difference of 0 . 3 5 between polarization and sucrose content, which would make t h e average condition of Cuba sugars between factory and refinery t o be 96.00 per cent sucrose and i . j o per cent moisture. Sugar of this grade has a safety-factor of 0 . 3 7 5 which is considerably above the limit for safe-keeping. Such sugar, if stored for one season, would deteriorate in New York t o a factor of a t least 0.30 a n d in Cuba, where t h e climate is much warmer, t o a factor of 0.25. This would mean t h a t the average Cuban sugar of 96.00 per cent sucrose would deteriorate if stored in New York for one year t o 9 j.00, and if stored in Cuba for one year t o 94.00 per cent sucrose. The average amount of Cuban sugar stored in warehouses a t any one time in 1916 was 163,000 long tons in the United States and 440,000 long tons in Cuba. T h e average price of Cuban sugar per pound for 1916 was 5.786 centsintheUnitedStatesand4.767 centsin Cuba.
- _-
I per Cent loss on 163,000 b a g tom at 5.786 cents per Ib. =$ 2 per cent 10s on 440,000 long tons at 4.767 cents per ib.
211.258(o) 939.671
$1.150.929
Fro. 14 Pile of fermenting dime on Boor of a sugar warehoupe.
These precautions can be carried into effect with comparatively little expense and would result in eliminating much of t h e needless loss which occurs at present between t h e manufacture and refining of cane sugar. ECONOMIC C O N S I D E R A T I O N S
Before concluding this paper upon the deterioration of raw cane sugar there are several economic questions which require discussion. Inasmuch as there is always danger of raw sugars becoming infected, no matter how extreme the conditions of cleanliness in the factory may be, i t is important for the manufacturer always to make the moisture content of his sugars conform t o the rules of safe-keeping. If Ne accept the formula Water = 0.3 (100.- S) as a requirement for safe-keeping, the moisture content of 3ifferent grades of raw sugars should not exceed t h e following percentages: Suerwe Per cent 99.9 99.5 99.0 98.5 98.0 97.5 97.0 96.5
Moistlire Per cent 0.03 0.15 0.30 0.45 0.60 0.75 0.90 1.05
Sucrose cent 96.0 95.5 95.0 94.5 94.0 93.0 92.0 91.0
PET
Moistme Per cent 1.20 1.35
l.5U 1.65 I .80 2.10 2.40 2.70
Suerose Per cent 90.0 89.0 88.0 87.0 86.0 84.0 82.0 80.0
Moisture Per cent 3.00 3.30 3.60 .?.YO
4.20 4.80 5.40 6.00
T h e above calculation does not take into account the loss due t o the deterioration of the 3,000,000 tons of Cuban sugars during transportation. Allowing an average loss of only 0.1' per cent sucrose during transit, there would be a deficiency of $320,342 at Cuban prices which would make t h e total calculated loss from deterioration for the 1916 Cuban sugars nearly $I,~OO,OOO. Reducing t h e moisture content of raw sugars would not only prevent the losses from deterioration b u t would accomplish a considerable saving in the costs of transportation. I n the shipment of Cuban sugars for the year 1917 approximately soo,ooo,ooo Ibs. of water were carried, which, a t the rate of $0.004 per lb., would make an expenditure of ~ 4 0 0 , 0 0 0for transportation of a useless ingredient. While the manufacture of moisturefree sugar is practicable only with the very highest grades, t h e moisture content of t h e ordinary qualities of raw sugar can be reduced nearly one-half without much extra cost of manufacture. I n conclusion the author desires t o thank his assistants Mr. G. H. Hardin and Mr. C. A. Gamble for helpin the analytical work of this paper,and Mr. J.A.Hall,Jr.,of the A. hi. Byers Co., for photographs of Cuban warehouses. NEWYon= S u o m Tame Lnaonnron~,INC. 80 SOUIX SIRRGT, NBWYoax CITY 8 This is a coilrexvative estimate. Cuban ~ u g a frequently i~ show B loss of nearly 1.0 in polaiiiation between the times of Shipment and delivery
