Sept., r g ~ g
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
t h a t the second treatment with copperas and lime is superfluous. After the fermentation and distillation of the abovedescribed mash, i t was deemed advisable t o ferment several molasses mashes of larger volume. There were successfully fermented four mashes of 2000 gal. each. From a portion of one of these mashes we recovered IOO lbs. of dynamite glycerin which is now on exhibition in the laboratory. Following is a description of the major fermentation of a typical 2 0 0 0 gal. molasses mash; there is also included the analysis of the resulting material. On April 12, 1919, a molasses mash was macle containing 4856 Ibs. “black strap” sterilized, and 1 1 lbs. of ammonia chloride added The final volume was 2142 gal., Balling 21.7O, at 25O C . At 12 p. m. this was sowed with 200 gal. yeast mash, Yeast 657, at 32O C. Then the following treatment was carried out: Soda Ash Added Temperature Date Time Lbs. Deg. C. 124 30 April 13, 1918 2 : 15 A.M. 4 : 30 A.M. 186 32 April 13, 1918 248 32 April 13, 1918 6 : 30 A.M. 10 : 30 A.M. 248 35 (Lowered to 31.5) April 13, 1918 5 ; 00 P . M . 186 33.5 (Lowered t o 30.0) April 13, 1918
The mash before fermentation contained 16.5 g. per I O O cc. of sugar. It must be borne in mind t h a t there is considerable alcohol produced in these fermentations. At the present price of alcohol and raw materials i t is safe to say t h a t the value of the alcohol balances the cost of all materials and overhead charges entering into the production of t h e fermented mash. This being true, then the slop from t h e alcohol distillation which contains the glycerin is obtained free of cost, so the only cost t o be considered for the glycerin would be t h a t of purification and distillation. This should not be great. No attempt has been made as yet t o recover the alcohol, i t being deemed a matter offering no difficulty. Laboratory tests show t h a t foaming is the only trouble t h a t will be encountered and this can be overcome by reducing the alkalinity with sulfuric acid of about 0.3 per cent. F E R M E N T A T I O N O F CORN AND CANE SUGAR
SOLUTIONS
It was deemed expedient t o pursue some experiments on a larger scale t h a n 2 0 gal. with corn and cane sugar solutions. These sugars contained little or no impurities and it was believed t h a t no great difficulty would be encountered with the glycerin recovered from such soIutions if the fermentations could be carried out properly with yeast nutrients which could be removed easily. Experiments showed, however, t h a t in order t o carry on the fermentation in such a manner t h a t the glycerin would be produced one would have had t o add t o solutions of corn and cane sugars yeast foods in kind and amounts t h a t would have deleteriously influenced the purification of the glycerin; therefore i t was concluded t h a t corn or cane sugar possessed no superiority over molasses for the fermentation for glycerin. ACKNOWLEDGMENT
We wish t o express appreciation of the interest shown b y our immediate chief, Mr. A. B. Adams, towards this experiment from its instigation t o its
845
close. A t the same time we wish t o s a y t h a t thesuccessful issue of our process is in great measure due t o Mr. J. M. Doran, chemist in our laboratory, owing t o his diligent search of the literature. LABORATORY O F THE I N T E R N A L REVENUE BUREAU WASHINGTON, D. C .
THE DETERIORATION OF MANUFACTURED CANE SUGAR BY MOLDS1 B y NICHOLAS KOPELOFF A N D LILLIAN ROPELOFF Received February 3, 1919
The deterioration of food products has assumed an added significance during the war which i t seems likely t o maintain. This applies t o sugar for the familiar reasons as well as the complicating problems of equalization, distribution, and reserve. When i t is remembered t h a t the bulk of sugar is sold on t h e basis of its polarization test, a small fraction of one per cent would represent an economic depreciation of considerable importance. According t o the interesting discussion by Browne,2 “allowing an average loss of 0.I per cent sucrose during transit of the Cuban sugar importation would be a deficiency of $320,342 a t Cuban prices, which would make the total calculated loss from deterioration for the 1 9 1 6 Cuban sugar nearly $ I , ~ O O , O O O . ” HISTORICAL
Since the original observations of Payen3 and Dubrunfaut4 this problem has been considered by numerous investigators to be of a microbiological nature. Their contributions have been comprehensively reviewed by Owen5 and others, so t h a t it is unnecessary a t this point t o do more than note t h a t the emphasis in such investigations has been, t o a great degree, on the bacterial flora involved. More recently, however, there has been an increasing tendency t o regard the fungi (or molds as they are more commonly termed) a;s playing an important r81e in the inversion of sucrose which occurs in manufactured cane sugar. Payen,G as far back as 1851, noted irregular ridges and cavities on sugar crystals in which he found filaments and spores of two different molds. Shorey7 isolated Penicillium glaucum from Hawaiian sugars, which he regarded as responsible for the deterioration of the latter. Kamerlings found in dried sugar a mold flora chiefly related t o Penicillium. Over 2 0 varieties were observed, and not less than 19 belonged t o this group. The author claims t h a t during the course of deterioration of raw sugar, the first attack is made by the molds. 1 This material was presented in part before the Societies of Bacteriologists and Phytopathologists, Baltimore, Md., 1918. 2 “The Deterioration of Raw Cane Sugar,” THISJOURNAL, 10 (1918), 178-190. 8 “Note sur une Vegetation Microscopique que attaque le Sucre Solide,” Comet. rend., 33 (1851), 393. 4 Ibid., 68 (1869), 663. 5 Louisiana Bulletin, 16.2 (1918). 6 The authors are indebted t o Dr. C. A. Browne for this abstract of “Note sur une Vegetation Microscopique que attaque le Sucre Solide,” Compt. rend., 33 (1851), 393. 7 J . SOC.Chem. Ind., 17 (1898), 555-558. 8 Proejstat. v . Suiker. West Java, Kagok, Verslag, 1899, 97-104.
”
T H E JOURNAL OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY
846
TAELEI-PER
CENT
=I
8
.B
‘D
c
‘ I I
1
18 3 16 5 11 5 12 8
100 100 89 40 82 100
100 100
98
100
100 95 20 78 80 92 100 90
17 33
0
0 0 20 8
25 14
Greig-Smith’ isolated Aspergillus glaucus from smallgrained, moist, refined sugar, but did not consider its presence t o be of as much significance as the potato group of bacilli. Schone2 emphasized the importance of molds and torulae in inversion and noted the presence of Pcnicillia, especially P. glaucum, and Mucor in deteriorating sugar. He investigated the acidity produced by pure cultures of these organisms inoculated into sterilized sugar solution. I n another connection he notes the isolation of Penicillium and Rhizopus. Scott3 studied the development and inverting power of Aspergilli and Penicillia and cautions against permitting sugar t o become infected with these organisms. Amons4 has detected mycelial threads of mold in sugar as did Scott, and isolated Aspergillus and a Rhizogus as well as Penicillium glaucum and purpurogenum. By means of inoculation experiments he proved t h a t Penicillium glaucum is capable of producing an appreciable deterioration. He believes with Browne and Noel Deerr t h a t no single organism can be made universally responsible. Our evidence tends t o corroborate this view. Browneb has recently isolated two Monilia from Cuban raw sugar which are capable of inverting sucrose in sugar solutions of high concentration. He also mentions the presence of other molds, Penicillia, etc., and notes their possible importance. Owen6 has isolated a number of Aspergilli from a large number of Louisiana sugars and has studied their activity in pure culture on sugar solutions of high concentration. He regards the molds as the most dangerous group of micro6rganisms in sugar, because of their strong inverting power, their ability t o exercise this power in highly concentrated solutions of sucrose of varying reaction, and also on account of their ability t o develop on media which is very deficient in nutrients. Blake6 has recently made a contribution t o the deterioration of raw sugar, as has Prinsen Geerligs.’ They are especially interesting in t h a t they represent observations made from the factory rather than from the laboratory standpoint. 1
Intern. Sugar J . , 4 (1902), 430.
* Deut. Zuckerind., 31 (1906),
1338; 33 (1908), 638; 36(1911), 247. Intern. Sugar J . , 14 (1912), 582. 4 Med. v . h. Proefsla. 8 . d. Java Suikerind. Chem. Ser., 1917, No. 5. LOG. G i t . 8 Louisiina Planter, 61 (1918), 316-317. 7 Intern, Sugar J . , 20 (1918), 543-546. 8
No. 9
*
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k
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17 0 0 0 0 20 0 25 9
6 0 13
39 67
28 33
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80 42 62 38
9 40 33 25 23
z
.-
8
SUQAR TYPE
................ ......... ...................... .................. ......................... .................................. ......................... .................................. ...............
II,
OF SAXPLES SEOWINQ M O L D
r‘
Plantation Granulated. ; Plantation Granulated, undried. Yellow Claritied. Standard Granulated. Cuban Raw.. 96 96O Washed.. 2nd AVERAGE,RELATIVE.,
Vol.
0 0 0
0
5
0 0 0
-r QI
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5 0 0
VI 67 0 56 40 0 40 8 12 29
2 82
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22 0 37 0 0 60 50 8 24
2 f
2
T 28 67 68 0 36 80 25 75 50
OCC,U&RENCE OF MOLDS
It is evident from this brief review of the previous investigations t h a t there is an informing body of d a t a which makes i t imperative t o subject the molds t o closer scrutiny with regard t o their ability t o deteriorate manufactured cane sugar. With this end in view, a comprehensive survey of the mold flora of cane sugars was undertaken. The samples under investigation included a variety of types of sugar from different sources and represented a considerable range in composition, age, and keeping quality. These representative sugars were plated in triplicate on eight different media (using a 1 7 Brix ~ sugar solution as dilution water t o prevent plasmolysis). Czapek’s agar maintained the highest relative efficiency with regard t o the largest variety of molds isolated, while a modification introduced by the authors was responsible for a more rapid colony development and therefore proved highly satisfactory. The composition of this medium (modified) is as follows: Tap Water.. .............................. Sucrose.. .................................
..................................
.........................
.........
1000 cc. 50 g. 1 . 0 g.
20.0g.
Reaction Sterilization at 15 lbs. for 15 min.
Table I shows the per cent of each type of sugar indicating the presence of the more important mo1ds.l This represents a summary of more detailed data which will appear shortly in bulletin form.2 Thus where Aspergillus na’ger was isolated from all 18 samples of Plantation Granulated sugar, its rating for t h a t sugar type is 100. The relative average indicates quite clearly then t h a t Aseergillzls niger and Blue Aspergillusa appear in practically every sample of sugar examined. Cladosporium appears in 90 per cent of the cases. Aspergillus $avus and the Sterile orange mold were isolated in about one-half of the total number of samples, while the other organisms noted appeared in the following order: Unknown sterile I I , Syncephalastrum, Unknown sterile I I I , Penicillium s p . , Unknown sterile I V , Citromyces I I I , P. luteum, Citro1
The authors are indebted to Dr. Charles Thorn and Miss M. Church
of the Bureau of Chemistry, U. S. Department of Agriculture, for the identification of cultures. 2 Nicholas Ropeloff and Lillian Ropeloff, “The Deterioration of Cane Sugar by Fungi,” Louisiana Bulletin, 196, 1919. 8 Blue Aspergillus now identified as Aspergillus Sydowi Bainim.
Sept., 1919
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
myces I , and P . divnrticataim. Those which appeared in only rare instances are not tabulated. While no definite flora could be assigned t o any particular type of sugar, the lower grades of sugar appeared t o have t h e greatest infection with regard t o both number and variety. It is of importance t o establish whether the molds in sugar are present in the spore stage chiefly, or whether mycelium could be detected. I n several instances mycelium was found but the evidence was doubtful or negative in character in the majority of samples. Some striking preparations of mycelium stained with Conn’s rose bengal’ were obtained by Dr. Thom from lumpy samples of Cuban raw sugar and from sugars inoculated with pure cultures of molds which were employed in our later experiments. Thus i t was proven conclusively t h a t molds can grow in such a highly concentrated substratum containing a minimum of available nutrients. DETERIORATION
OF
SUGAR
BY
PURE
CULTURES
OF
MOLDS
Pure cultures of the molds mentioned above were inoculated into sterilized sugar and Io-g. portions of the latter used as a n inoculum for 150-g. portions of sterilized sugar of t h e three principal types, namely: Plantation granulated, refined, and Cuban raw. The Erlenmeyer flasks containing the inoculated sugars were plugged with cotton and incubated a t 27’ t o 30’ C. for 4 mo. Under such conditions the sugars dried t o such an extent as t o make the moisture content negligible. At the end of the incubation period the sugars were analyzed and the results recorded as in Table II.a TABLE 11-ANALYSES OF SUGARS INOCULATED
WITH FUNGI. PLANTATION GRANULATED Presence after Single Reducing No. Fungus 4 mo.1 Polar. Clerget Sugar Moisture 7 Check. 99.2 99.26 0.16 0 10 Check 99.0 98.88 0.16 0 14 Check. 98.9 98.73 0.15 0.07 AVERAGE 99.0 98.96 0.16 0.02 99.06 0.16 0.02 99.1 12 Aspergillus flavus. 98.88 0.16 0 13 Aspcrgillus flavus. 98.9 0.01 99.0 98.97 0.16 AVERAGE 16 Blue Aspergillus.. 98.9 98.27 0.17 0.11 17 Blue Asperg{llus 4- 98.6 98.50 0.16 0.02 18 Blue Aspsrg!llus.. 99.2 99.12 0.15 0.07 20 Blue Aspergrllus.. 99.1 99.22 0.16 0.12 AVERAGE f 98.9 98.78 0.16 0.08 15 Syncephalastrum.. 99.0 98.88 0.13 0.08 21 Syncephalastrum 98.9 98.66 0.14 0.05 23 Synceghalastrum 98.9 98.94 0.15 0.08 25 Syncephalastrum. 98.9 98.59 0.15 0.05 AVERAGE f 98.9 98.77 0.14 0.07 4 Asgergillus n i g n . 99.0 99.06 0.18 0 8 Aspergillus niger.. 99.4 99.25 0.15 0 28 Aspergillus ntger 98.9 98.59 0.13 0.10 29 Asgngillus ntger. 98.8 98.43 0.15 0.04 AVERAGE 99.0 98.83 0.15 0.03 1 Average of triplicate plates with two dilutions.
................. .................. ................. ................. ....... ....... -
................. ........ + .......... ........ ........ ................. ........ .......... + .......... ......... ................. ........ ....... .........
-
........ 2+
.................
In discussing these data, it is well to bear in mind the general concensus of opinion both in the laboraof tory and the field, to the effect that the safety” is a fairly reliable criterion for the keeping quality of a sugar, In other it generally Moisture ceded t h a t the value of ___ should be I oo-Polarization 0.33 t o guarantee the non-deterioration of raw sugars, 1
iV. Y . (Geneva) Tech. Bull. 64 (1918).
The authors are indebted to Mr. E. C. Freeland, assistant chemist, for his assistance with the chemical analyses. 2
847
while the fraction is substantially lower t h a n this €or white sugars. A glance a t the column marked “Moisture” in Table I1 indicates the presence of a very low moisture content, and the factor of safety or “moisture ratio” in this series would be considerably lower than 0.I . Therefore marked deterioration would not be anticipated. T h a t such is actually the case may be seen in the polarization, Clerget, and reducing sugar values set forth. Practically all these determinations are within the limit of experimental error. Slight indications of loss of sucrose are perhaps t o be noted in the case of the Blue Aspergillus (Flasks 16 and 17) and Aspergillus lziger (Flasks 28 and 29). The same general negative evidence is t o be gathered from Table 111. However, in the Cuban raw series shown TABLE111-ANALYSESO F SUGARS INOCULATED WITH FUNGI. REFIN$D Presence after Single Reducing No. Fungus 4 mo. Polar. Clerget Sugar Moisture 99.4 99.72 0.09 0.04 2 Check 3 Check 99.7 99.84 0.05 0.02 5 Check. 99.4 99.38 0.05 0.02 6 Check 99.4 99.49 0.04 0.03 AVERAGE 99.5 99.61 0.06 0.03 7 Aspergillus f l a v u s . , 99.3 99.33 0.05 0.03 8 Aspergillus flavus.. 99.76 0.05 0 AVEBAGS 99.52 0.05 0.02 14 Blue Aspergillus.. 99.5 99.90 0.04 0.03 15 Blue Aspergillus 99.5 99.46 0.04 0.05 16 Blue Aspergillus.. 99.3 99.26 0.04 0.04 17 Blue Asgcrgillus 99.3 99.16 0.04 0.04 AVERAGE 99.4 99.44 0.04 0.04 18 Syncephalastrum 99.6 99.61 0.04 0 19 Syncephalastrum 99.7 99.80 0.04 0 99.2 99 04 0.04 21 Syncephalastrum. 0 23 Syncephalastrum 99.3 99130 0.06 0 AVERAGE f 99.4 99.44 0.05 0 26 Asgergtllus ntger 99.7 99.72 0.04 0 27 Aspngrllus ntger.. 99.7 99.80 0.04 0 28 Aspergillus niger... 99.7 99.91 0.05 0 29 Aspergillus niger. 99.6 99.65 0.05 0 AVERAGE 99.7 99.77 0.05 0
.................. .................. ................. .................. ................. ...... ...... ..................
........ .......... ........ .......... .................. .......... .......... ......... .......... .................. ........... ........ ....... ......... .................
+
$ :; $ $ +
+ $
+
-+ $
TABLBIV-ANALYSES OF SUGAR INOCULATED WITH FUNGI. CUBAN RAW Presence after Single Reducing No. Fungus 4 mo. Polar. Clerget Sugar Moisture 95.77 96.27 1.04 0.83 1 Check. 95.79 95.99 1.09 2 Check.. 1.06 95.67 95.97 4 Check 1.04 1.15 95.7 5 Check. 96.05 1.06 1-14 AVERAGE 95.7 96.07 1.06 1.05 95.6 95.93 1.09 1.07 17 Aspergillus flaws.. 95.6 95.78 1.09 1.06 18 Aspergillus flavus. AVERAGE. - 95.6 95.86 1.09 1.07 95.51 1.13 0.96 6 Blue Aspergillus 4- 95.0 7 Blue Aspergillus 95.0 95.51 1.04 1.00 495.1 95.54 1.56 1.14 8 BlueAspevg(l1us 10 Blue Aspergrllus 95.2 95.74 1.19 0.94 95.1 95.48 1.23 1.01 AVERAGE 12 Syncephalastrum 4- 95.7 96.27 1.09 1.11 95.28 1.09 1.06 13 Syncephalastrum 4- 94.8 15 Syncephalastrum 4- 95.4 95.86 1.09 1.10 f 95.6 96.02 1.09 0.99 16 Syncephalastrum AVERAGE 95.4 95.86 1.09 1.07 95.51 1.11 1.09 26 Asperg{llus ntger 495.0 27 Aspergrllus nzger. 95.6 96.01 1.11 1.23 28 Aspergtllus ntger. 95.5 95.93 1.11 1.22 29 Asgergillus niger., 9.5.6 96.01 1.14 1.29 AVERAGE f 95.4 95.86 1.12 1.20
................ ............... ................. ................ ................
+
................. ......... ........ ........ ....... ................
+ +
...... ....... ............... ......... ......... ......... ......... ................ ......... ......... ......... .........
in Table I V , where the moisture content is amxeciable. _though - well below the factor of safety limit, some evidence of the activity in inverting sucrose may be found in the case of the Blue .4spergiLlus, and t o a lesser degree with cultures of Aspergillus niger. This experiment, consequently, may be regarded as lishing the lower limit for the deterioration of manufactured cane sugar by the molds employed. All the sugars were plated out in triplicate on the authors’ modification of Czapek’s agar and the presence or absence of the inoculated organisms indicated in the third column of the foregoing tables. There is no consistency exhibited and there is furthermore no
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
848
multiplication of the molds involved. examinatio'n failed t o establish the presence of any mycelium in these sugars. TABLE V-ANALYSES
OF S U G A R
INOCULATED
WITH
PLANTATION GRANULATED
DIFFERENT PUHGI.
i 0
30 31 32 33 34 36 37 38
............ ....... ....... .....
..... ....... ....... ....... ..... ......
.......... ..... .. + ...... ....... ..... ........ + + +
I n Table V are presented the analyses of t h e sugars inoculated with t h e various molds which were isolated and which were t o be used for inoculation. I n this Plantation Granulated series, the moisture ratio was uniformly low and the amount of deterioration, in consequence, relatively slight, as will be seen from the values of sucrose Clerget on the d r y basis (which represent the most adequate criterion for comparison). TABLEVI-ANALYSES O B SUGAR INOCULATED CUBANRAW
WITH
DIFFERENTFUNGI.
i
104 20 21 22 23 24 24A 25 26 27 28 29
GRANULATBD
30 31 32 33 34 36 37 38
...... ....... ..... ....... ....... .......
......... ..... .. + ...... + ...... + ........ + ....... + ...... +
ul
............ ............ ............ .............. ..... ..... ..... .............. ...... ...... + + + ...... .............. + .... ...... ...... ............ + ... ... -
1 Check.. 2 Check.. 3 Check.. AVERAGE 4 Aspergillus jlauus 5 Aspergillus jlavus 6 Aspergillus jlavus AVERAGE 7 Blue Aspergillus 8 Blue Aspergillus 9 Blue Aspergillus AVERAGE 10 Syncephalastrum.. 1 1 Syncephalastrum 12 Syncephalastrum AVERAGE.. 13 Asaergillus niger.. 14 Aspergillus niger.. 15 Aspergillus niger AVERAGE..
98.6 98.5 0.25 0.47 0.34 99.0 98.9 98.6 98.3 0.21 0.40 0.28 98.9 98.7 98.5 98.2 0.23 0.45 0.30 98.9 98.6 98.6 98.3 0.23 0.44 0.31 98.9 98.8 98.6 98.3 0.25 0.56 0.38 99.0 98.8 98.5 98.2 0.26 0.42 0.22 98.8 98.6 98.5 98.4 0.25 0.43 0.22 98.8 98.8 98.5 98.3 0.26 0.47 0.31 98.9 98.7 97.2 97.1 0.57 1.00 0.35 98.1 98.0 97.0 97.1 0.75 0.64 0.21 97.6 97.7 97.2 97.3 0.74 1.00 0.37 98.1 98.3 97.1 97.2 0.69 0.90 0.31 97.6 98.0 98.5 98.4 0.26 0.36 0.24 98.8 98.8 98.7 98.6 0.24 0.40 0.30 99.0 99.0 98.6 98.5 0.23 0.35 0.24 98.8 98.8 98.6 98.5 0.24 0.37 0.26 98.9 98.9 97.9 97.7 0.44 0.63 0.30 98.5 98.3 97.9 98.0 0.43 0.63 0.30 98.5 98.1 96.6 96.6 0.44 0.68 0.20 97.2 97.2 j, 97.4 97.4 0.44 0.65 0.27 98.1 97.9
..... ............
TABLEVIII-ANALYSES
OF
SUGARS INOCULATED WITH MOLDS. REPINED
.s* P cd
4 .............
No. Fungus 1 Check. 2 Check AVERAGE.. 4 Aspergillus flavus. 5 Aspergillus jlavus.. 6 Aspergillusjlavus AVERAGE.. 7 Blue Asgergillus 8 Blue Aspergillus. 9 Blue Aspergillus.. , , AVERAGE 10 Syncephalastrum.. 1 1 Syncephalastrum AVERAGE.. 13 Aspergillus niger.. 14 Aspergillus niger.. 15 Aspergillus n i g e r . , , , , AVERAGE..
.............. ..........:. .... ... + ..... +
............ ...... ..... .. .............. .... ...... ............ ... ... ............
............
Check. Cladosporium. A s p . jlauus.. Blue Aspergillus.. ... Penicillium No. 2694 Syncephalastrum Unknown I . . Trichoderma. Citromyces I . A s p . nidulans.. ..... Cztromyces 11 ....... Penicillium pinophilum.. Citromyces 111. Penicillium diuart.. Unknown I I . . Sterile orange. . . . . . . Unknown I I I . A s p . niger.. Unknown I V . Unknown V . .
96.2 96.13 1.09 96.0 95.89 1.09 95.9 95.75 1.09 96.0 95.75 1.09 95.9 95.75 Lost 94.9 95.06 1 . 1 1 96.1 96.05 1.09 96.0 96.01 1 . 1 1 95.6 95.74 1.56
1.26 0.33 97.4 97.3 1.16 0.29 97.1 97.0 1.00 0.24 96.9 96.7 1.30 0.32 97.2 97.0 1.43 0.35 97.3 97.1 1.44 0.28 96.3 96.4 1.33 0.34 97.4 97.3 1.29 0.32 97.3 97.3 1.68 0.38 97.2 97.4
Some of the Unknown sterile molds, especially 11, and the Blue Aspergillus caused a n appreciable inversion. I n the same way in the Cuban Raw series shown in Table VI, where the moisture ratio was considerably higher than in the preceding series, the greatest inversion, indicated by the lowest Clerget values, as well as the greatest amount of reducing sugars, was obtained with the Blue Aspergillus. Similarly, a number of other organisms were more active t h a n in the previous set, accentuating again the im-
No. 9
TABLG VII-ANALYSES OF SUGARS INOCULATJ3D WITH FUNGI. PLANTATION
*
Check. Cladosporium As#. pasus.. Blue Aspergillus Penicillium No. 2694 Syncephalastrum Unknown I . . Trichoderma. Citromyces I . Asp. nidulans.. Citromyces I I . Pdnicillium bino98.8 0.28 0.23 99.0 99.0 0.20 philum. - 99.0 98.83 99.22 0.15 0.11 99.1 99.3 0.17 Citromyces I I I . Penicillium divart. 99.5 99.54 0.19 0.09 99.6 99.6 0.06 Unknown I I . . $. 98.4 98.29 0.14 0.11 98.5 98.4 0.06 99.1 98.76 0.16 0.07 99.2 98.8 0.07 Sterile orange. Unknown I I I . 98.9 98.92 0.23 0.15 98.9 99.1 0.13 Asg. niger.. 99.5 99.50 0.15 0.06 99.6 99.6 0.04 Unknown I V ........ 97.4 97.39 0.31 0.31 97.7 97.7 0.12 Unknown V . . . . . . . . - 98.9 98.77 0.31 0.30 99.2 99.1 0.27
11,
portance of moisture as a limiting factor in t h e deterioration of Sugar by microorganisms. The mycological observations revealed t h e presence of organisms in a larger number of instances than in t h e preceding series where the moisture content was decidedly lower.
. I
103 20 21 22 23 24 24A 25 26 27 28 29
Vol.
*
+ + + +
-
-
8a
* J
% w
T Q g
8vj
M
'0
!i
Y
2 I W .
3
2 B B
--d u '
Dry Basis
v j v i 99.5 99.4 0.11 0.20 0.40 99.7 99.7 99.6 99.4 0.11 0.12 0.30 99.7 99.5 99.6 99.4 0.11 0.16 0.35 99.7 99.6 98.3 98.2 0.26 0.38 0.23 98.7 98.6 97.2 97.4 0.29 0.72 0.26 97.9 98.1 99.1 99.2 0.18 0.17 0.20 99.1 99.2 98.2 98.2 0.24 0.42 0.23 98.6 98.6 98.0 97.9 0.57 0.56 0.28 98.6 98.4 97.9 97.9 0.57 0.63 0.30 98.6 98.0 97.9 97.8 0.56 0.70 0.33 98.6 98.5 97.9 97.9 0.57 0.63 0.30 98.6 98.3 99.2 99.3 0.23 0.22 0.28 99.4 99.6 99.6 99.4 0.21 0.15 0.40 99.7 99.5 99.4 99.4 0.22 0.19 0.34 99.6 99.6 98.6 98.6 0.43 0.64 0.45 99.2 99.2 98.8 98.9 0.43 0.52 0.43 99.3 99.4 99.0 99.1 0.37 0.36 0.36 99.2 99.3 98.8 98.8 0.41 0.51 0.41 99.3 99.3
I n order t o approximate more nearly the optimum conditions for the activity of these organisms, the following experiment was conducted in a manner similar t o those discussed above, with one important difference, namely, the sterilized sugars (of the three principal types) were permitted t o absorb moisture in the autoclave before inoculation. The incubation period in this case was reduced t o one month, at the end of which time the sugars were analyzed and t h e results recorded in Table VII. It will be readily observed from the closely agreeing duplicate determinations t h a t the Blue Aspergillus and Aspergillus niger are responsible for a considerable inversion as represented b y t h e reduction in Clerget values as compared with the check. This is corroborated by the tripling and doubling, respectively, in amount of reducing sugars. The moisture ratio in this series approaches the critical value arrived a t in the valuable contributions of Browne,' Owen,l and others. Two of the 1LOC.
Cit.
Sept., 1919
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
organisms, on t h e other hand, showed little, if any, power to cause inversion even under these favorable conditions. The Refined series, recorded in Table VIII, yields much the same evidence. Here again, where the moisture ratio is in the vicinity of 0.3 and deterioration is t o be expected, the Blue Aspergillus and Aspergillus niger are active in inversion as evidenced by t h e low Clerget and high values for reducing sugars. Aspergillus flavus appears also t o have some activity in the present series. I n t h e Cuban Raw series in Table I X t h e results parallel those previously noted. The B h e Aspergillus and Aspergillus niger are very active, while Syncephalastrrum exhibits some inclination t o invert the sucrose present. TABLE IX-ANALYSES
OF
SUGARS INOCULATED WITH FUNGI. CUBANRAW
.+p
.a No.
Fungus 1 Check.. 2 Check.. 3 Check AVERAGE.. 4 Aspergillus jlauus 5 Aspergillus Savus.. , , , 6 Aspergillus jlavus.. , AVERAGE.. 7 Blue Aspergillus 8 Blue Aspergillus.. 9 Blui Asgergillus.. AVERAGE 10 Synceghalastrum 1 1 Syncephalaslrum 12 Syncephalaslrum AVERAGE 13 Aspergillus niger.. 14 Aspergillus niger.. 15 Aspevgillus niger.. AVERAGB
............
............ .............. ............ ..... -
.. ............ ...... .... .... -
.............. -
...... ...... ...... -
.............. ... ... -
... .............. -
vj
Dry Basis
d
0'
z v j v j
95.2 0.92 1.97 0.40 97.0 97.1 95.2 1 .oo 2.01 0.40 97.0 97.2 95.3 1.00 1.96 0.40 97.1 97.2 95.2 0.97 1.98 0.40 97.0 97.2 95.2 95.2 1.06 1.91 0.40 97.0 97.1 95.3 95.3 1.02 1.84 0.39 97 .O 97.1 95.1 95.2 1.06 1.96 0.40 97.0 97.1 95.2 95.2 1.05 1.90 0.40 97.0 97.1 92.8 94.0 1.89 2.39 0.33 95.1 96.3 93.0 93.5 1.97 2.36 0.34 95.2 95.7 92.2 92.9 2.17 2.75 0.35 94.8 94.8 92.7 93.4 2.01 2.50 0.34 95.0 95.6 94.7 94.9 0.98 2.07 0.39 96.7 96.9 95.3 94.9 1 .OO 2.20 0.48 96.4 97.0 94.7 94.8 1.04 1.83 0.34 96.4 96.6 94.9 94.9 1.01 2.03 0.40 96.5 96.8 94.5 94.7 1.06 2.17 0.39 96.6 96.8 94.5 94.7 1.11 2.01 0.36 96.4 96.7 94.8 94.9 1.09 1.86 0.35 96.5 96.7 94.6 94.8 1.09 2.01 0.37 96.5 96.7
I n considering the deterioration of sugar by the pure cultures of molds employed i t is of paramount importance t o note t h a t the organism possessing the greatest power for causing inversion is the very one isolated with the greatest frequency from the large number of samples studied, namely, the Blue Aspergillus. The mycological examination by plating a t the end of t h e experiment resulted in the recovery of numerous organisms from all of the inoculated flasks, the checks remaining sterile. A microscopical examination revealed t h e presence of mycelia in several instances where deterioration had occurred. However, in other cases where deterioration was noted, no mycelium could be detected and only spores were present. This phenomenon is worthy of further study (which is at present being carried forward), for upon its interpretation depends much t h a t is of economic significance. If i t is possible for t h e spores of molds t o secrete enough invertase t o cause the deterioration of sugar without the development of mycelia, then sugar which has been properly dried and considered safe by virtue of its moisture ratio would, in reality, be likely t o undergo deterioration depending upon the nature and extent of the infection. This would point indubitably, from a new angle, t o t h e necessity for cleanliness in the sugar house. I t leads directly into a n investigation, now in progress, of sterilizing sugar in the centrifugals or reducing the infection by adequate protection a t this point. For example, i t
849
would be advisable, on the basis of the foregoing results, t o have the centrifugals operating in a room having a concrete floor and where strict methods of cleaning and perhaps disinfection could be employed. From the broader viewpoint i t is of prime importance t o industrial mycology t o have determined t o what extent the spores of molds are capable of secreting enzymes without active growth, and i t is hoped t h a t experiments now being conducted in this laboratory will throw some light on this question. For the present, i t may serve t o interpret some peculiar phenomena. For example, in a recent conversation, Dr. C. A. Browne has suggested t h a t it appeared t o be a plausible explanation of t h e fact which he has often noted, namely, that some sugars which start t o deteriorate -at a high moisture ratio continue t o do so in spite of the lowering of this ratio below the critical point of safety. The bearing that such a relationship might have upon the factor of safety rule is obvious and demands further attention. It may be necessary t o introduce a revision of this rule based on infection. MOLDS I N THE SUGAR FACTORY
Having isolated the molds which occur in the marketable cane sugars and determined their power to cause deterioration by inversion, i t was of interest t o trace t h e fate of the molds and bacteria through the process of sugar manufacture. With this in view, a daily bacteriological and mycological examination of each stage in the process was carried on throughout the past grinding season. I n brief, t h e local process consists of crushing the harvested cane t o extract the raw juice. This is treated with sulfur dioxide fumes and later limed t o slight acidity and brought t o a boil. The impurities are put through a filter press, the filtered juice joining the clarified juice in the storage tank. The juice is evaporated in the "effects" t o a sirup by heating at 1 2 0 O t o 185' F. under a vacuum of I O t o 26 in. The sirup goes to the vacuum pan where thesugar isgrained at 130Oto 165' F. under 23 t o 2 5 in. of vacuum. The massecuite (sugar coated with molasses) is run into the centrifugal, which permits the molasses t o be thrown through the brass screen and holds the sugar crystals behind. A jet of water (about I lb.) is played on the sugar ,in the rapidly whirling centrifugal t o remove some of the molasses, which results in a lighter colored sugar. Further methods of refinement were ordinarily not employed in our mill. TABLE X-SUMMARY OF PER CENTOF FUNGI AND BACTERIA IN EACH STAGE
OF THE
SAMPLE Raw juice..
MANUFACTURING PROCESS AVERAGE Fungi Bacteria 100 1.14 0.17 3.3 1.6 0.51 0.0 0.28 24.6 0.10 3 1.1 0.11 91.8 0.06 85.2 0.08 28.0 0.29 0.0 0.13 11.5 0.001 32.8 0.006
... ......................... ..................... ...................... ............................ ........................ ........................ ..................... ......................... ....................... .............. .......
Limed juice Filtered juice.. Settled juice.. Syrup.. Massecuite. R a w sugar.. Washed sugar.. Molasses.. Wash water. Air above centrifugal.. Air below centrifugal..
I n Table X is presented a summary of all the data collected, which gives an adequate conception of the fate of the microorganisms in the mill. Considering the number of molds in the raw juice as 100,it will
8$0
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
be seen t h a t there is a decrease of 86 per cent as a result of sulfuring, while the completion of the clarification process renders the juice practically sterile. However, when the massecuite is exposed t o the air, and especially when i t enters the centrifugal, reinfection takes place. The rapidly whirling centrifugal sucks air from below a t great speed and any organisms on the floor or in the mill a t large have an opportunity of gaining an entrance. This is corroborated by the fact t h a t the air below the centrifugal has approximately three times the number of molds t o be found in the air above the centrifugal. It might be well t o keep the centrifugals covered as well as the mixer which brings the massecuite t o t h a t point. The bacterial counts follow the same general trend as the molds. Blue Aspergillus and Cladosporium were the predominating molds appearing under the local conditions. These were likewise found in two of the largest factories in Louisiana. The fact t h a t the Blue Aspergillas is so universal in its habitat and so strong in its deteriorative power makes it advisable to develop methods for its elimination along with the other molds. SUMMARY
I-The molds appearing with the greatest frequency in manufactured cane sugars of different grades belong chiefly t o the Aspergilli and Penicillia. 2-The organism which appeared with the greatest frequency from all sugars, the Blue Aspergillus) also had the greatest deteriorative power. 3-Sterilized sugars inoculated with pure cultures of molds deteriorated rapidly where the moisture content was appreciable. Little, if any, deterioration occurred when the moisture content was reduced t o a minimum. 4-Sugars ordinarily guaranteed against deterioration by virtue of the factor of safety rule are capable of undergoing deterioration if sufficiently infected with molds. 5-Molds cause an inversion of sucrose where only spores are present, as well as when mycelia are developed. It would appear, therefore, t h a t some mold spores, as such, contain invertase. ACKNOWLEDGMENT
The authors are appreciative of the assistance they have so generously received from Dr. C. A. Browne, Mr. W. L. Owen, Dr. Charles Thom, Assistant Director W. G. Taggert, Dr. F. Zerban, and Mr. E. C. Freetand. DEPARTMENT OF BACTERIOLOGY LOUISIANA SUGAR EXPERIMENT STATION NEWORLEANS, LA.
AMERICAN TOMATO SEED OIL1
s.
By GEORGE JAMIESON AND H. s. BAILEY Received March 1 , 1919
The object of this investigation was t o prepare a number of samples of oil from tomato seed grown in various localities in the United States and determine the so-called constants for each sample of oil. Also it was proposed to make a study of the chemical composition of tomato seed oil in order to determine if it 1
Published by permission of the Secretary of Agriculture.
Vol.
11,
No. g
contained any constituents in addition to those already known. I n 1914, a brief report of the work which had been accomplished was made by Bailey and Burnett,l on the extraction and refining of tomato seed oil. It was shown a t t h a t time that the crude oil could be readily refined by the well-known alkali process and t h a t by a subsequent treatment with fuller’s earth, a very pale yellow oil was produced which appeared suitable for use as a salad oil. Since the abovementioned report was made several more samples of oil have been obtained and studied. While making a preliminary investigation of the chemical composition of the oil i t was found t h a t the Renard test indicated the presence of a considerable amount of arachidic acid. However, i t will be shown that tomato seed oil actually contains a very small amount of arachidic acid. The Renard test was repeated several times but in each case the same result was obtained, although great care was taken t o follow the directions given for this test in every detail. These results show t h a t the Renard test when applied t o unfamiliar oils cannot be relied upon t o indicate the quantity of arachidic acid present. I t is hoped t h a t this important observation will be properly emphasized in the future editions of books on oil analysis. Several attempts were made a t various times t o separate the arachidic acid by fractional crystallization from 90 per cent alcohol, of the fatty acids obtained by the Renard test, but without success. Then i t was decided t o make a n exhaustive study in order t o determine if any arachidic acid was present in tomato seed oil, with the result t h a t the completion and publication of these investigations were much delayed. Meanwhile a study on “The Utilization of Waste Tomato Seeds and Skins” was made by Rabak, of the Bureau of Plant Industry, the results of which were published in bulletin form in 1 9 1 7 . ~ This bulletin gives statistics on the amount of tomato seeds and skins, as well as the quantity of oil and press cake produced in Italy, and also indicates the amount of this waste product available in the United States. Since the bulletin gives a satisfactory description of the modern pressure and solvent methods for obtaining the oil on a commercial scale, i t will not be necessary t o discuss them here. The chemical investigation of the tomato seed oil extracted by solvents described in the above-mentioned bulletin showed t h a t it contained 17.54 per cent of solid acids and 75.84 per cent of liquid acids. It was also shown t h a t the oil had the following approximate composition: Per cent 45.00 .............. .............. 34.20
Olein. Linolein
Per cent Palmitin.. ............ 12.47 Stearin 5.89
.............
The remaining small portion consisted of free acids and unsaponifiable matter. I n addition t o these compounds Battaglio3 has shown that tomato seed oil contains a small amount of myristin. A further attempt was made on much larger scale than in the first experiments t o separate the arachidic acid from the palmitic and stearic acids by fractional crystallization. About 36 g. of the solid fatty acids 1 2
3
Science, 39 (1914), 953. U. S. Dept. of Agr., Bull. 63% (Professional Paper). Les Corps gras., 1901, 135
