November, 1931
ISDUSTRIAL AND ENGINEERING CHEMISTRY
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Mannite and Dextran in the Jellying of Molasses from Juice of Frozen and Deteriorated Cane',' C. F. Walton, Jr., and C. A. Fort BUREAUOF CHEXISTRY AND SOILS,U. S. D ~ P A R T X E N OFT AGRICULTURE, WASHINGTON, D. C.
The needle-shaped crystals observed in solidified In the attempt to isolate LARIFICATION, molasses made from juice of deteriorated cane have the substance or substances sugar boiling, and cenbeen identified as the sugar alcohol d-mannitol (manprincipally responsible f o r trifugal work are well nite). It is believed that this fermentation product is the trouble, juice was purknown to be difficult operalargely responsible for the jellying of such molasses. p o s e l y t a k e n from cane of tions in the production of The molasses contained also an abnormally high such extremely poor quality cane s u g a r in L o u i s i a n a percentage of dextrorotatory gums. The methods of that it had been abandoned whenever a freeze followed isolation from molasses and properties of d-mannitol in t h e field. T h e juice b y w a r m w e a t h e r h a s inand the gums are described, and the effects of these possessed an initial acidity of jured the cane. The work substances on sugar manufacture are briefly discussed. 14.0 cc. (0.1 N sodium hvreuorted in this paper was undroxide per 10 cc. of juice). dekaken to ascertain what particular fermentation products in the juice are principally The apparent purity was found to be 27 or 37.7, depending on responsible for the slow and difficult working of the juice the analytical method used. By comparing the composition and intermediate products. The investigation forms a part of this juice, which was heated to the boiling temperature of a project of the Carbohydrate Division, Bureau of Chemis- soon after the cane was milled, with that of juice which in try and Soils, to investigate the chemical and enzymic changes earlier investigations was allowed to ferment in the laboratory, in sugar cane, to determine the best method of handling the an interesting point for discussion and further investigation is crop in the field under varying climatic conditions, to mini- suggested. mize loss of sucrose and facilitate juice clarification and, if possible, to develop an improved process for clarification of the Previous Work on Mannitic Fermentation of Cane Juice juice. I n 1892 Horton (7) , during February of a very long grinding During the latter part of the 1929 grinding season in Louisi- season a t Belair plantation, made a comparative study of the ana, very pronounced factory-operating trouble was experi- composition of juice from the top joints, middle portions, and enced. The clarified juice was of poor quality, as judged by butts of windrowed cane. I n this work Horton determined customary standards; sugar boiling was found to require Brix, sucrose, reducing sugars, purity, and acidity. He then fully twice as much time as usual, even for sirup strikes; and allowed the juices to ferment in the laboratory from February the grain did not purge satisfactorily, the yield often being a 16 to March 8, A t the end of which period the acidity had small fraction of what had been expected. Sugar boilers increased to 20 cc. (0.1 N sodium hydroxide per 10 cc. of everywhere reported needle grain, which is commonly as- juice). Mannite was crystallized from this fermented juice, sociated with slow-boiling pans. The molasses in some in- and a gum was also formed, the properties of which resembled stances actually jellied and became very difficult to pump from those of dextran. magmas to tank cars. Some factories paid so little for cane Microscopic studies of the same gum by Taylor (9) led when the juice acidity reached 3.5 cc. (0.1 N sodium hydroxide to the isolation of a microorganism which he called " B u c t e n h n per 10 cc. of juice) that it was no longer profitable for the sacchuri" and which he found to be a very powerful sugar grower to deliver this cane t o the mill. The cane in the field destroyer and gum former. Taylor concluded that this was topped lower and lower, fully half of the stalk being left in organism was closely connected with Leuconostoc mesenteroides the field in the attempt to render the butts acceptable to the Cien. He reported that it is not positively known whether factory. Finally, entire fields of cane were abandoned. Bacterium sacchari is found in the freshly expressed juice of At the bureau's sugar experiment station, established in the cane, although it is abundant in the air and is present in 1929 near Houma, La., a part of the chemical research carried great abundance in every sugarhouse. Taylor found that on during the first year was a preliminary investigation of this organism was very resistant to heat, since neither a temthe frozen-cane problem. Clarification of juice having vary- perature of 300' F. (148.9' C.) nor boiling with 90 per cent ing degrees of excessive or abnormal acidity was studied. alcohol destroyed it. The results obtained by light liming were contrasted with those Maxwell (8) found that when juice fresh from the cane was obtained by relatively heavy liming. A light degree of sul- allowed to ferment in the laboratory in a closed flask confuring was compared with heavy sulfuring before liming. nected with an air-tight receiver, the fermentation mas partly The uses of phosphoric acid, sodium fluoride, and sodium alcoholic and partly mannitic. When the same juice was carbonate as adjuncts of clarification were also investigated. first heated to 200' F. (93.3' C.) and allowed to cool before The data resulting from these clarification studies are to be it was put into the fermentation flask, the results were notably reported separately. This work in general is mentioned now different. A study of the fermentation products showed no to call attention to an observation made a t the time that, mannite or other crystallizable bodies, but a class of gums apparently regardless of the method of clarification, certain different from those yielded by the unheated mill juice. fermentation products in juice from deteriorated cane remain Browne ( 4 ) has reported an extensive investigation of the in the clarified juice to cause trouble during subsequent steps enzymes in sugar cane and the fermentation of sugar-cane of the factory process. products. The presence of invertase in sugar cane, especially Presented before the Sugar Division at the 1 Received July 6, 1931. the tops, and the inversion of sucrose by this enzyme under 80th Meeting of the American Chemical Society, Cincinnati, Ohio, Sepdifferent windrowing conditions is discussed. Oxidizing and tember S to 12. 1930. reducing enzymes are also discussed in some detail. The 2 Contribution No. 102 from the Carbohydrate Division, Bureau of question as to whether fermenting bacteria occur normally Chemistry and Soils.
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I N D U S T R I A L A N D ENGINEERING CHEMISTRY
in freshly expressed cane juice, or whether the juice later becomes inoculated, appears to be answered by experiments in which the bacteria were counted a t once after milling and subsequently a t various time intervals. Browne states: "The living plant appears to protect itself against the invasion of microscopic parasites by forming toxic products. I n case the sugar cane is killed and split open, as sometimes happens during a freeze, this power of protection is lost. The formation of toxic products does not go on; hordes of bacteria invade the stalk, and finding no resistance start a fermentation which soon renders the cane worthless for milling." Browne further states that the toxic oxidation products, which are deadly to microorganic life, do not react unfavorably upon the cane itself because those toxic products are reduced by reducing enzymes. Regarding the specific formation of mannite and dextran by the viscous fermentation, Browne states as follows: "The viscous fermentation exerts a powerful reducing action upon the cane juice, and, as a consequence of this reduction various deoxidation products, the most common of which is mannite, are formed. * * * It was first supposed that mannite was the product of a special organism, but this is a mistake, for mannite may be formed in any fermentation of sugar where a reducing action takes place." Browne also gives methods for the separation of mannite and dextran from juices which have undergone the viscous fermentation, and gives the composition and some of the properties of these substances. The results of the present investigation confirm Browne's conclusions that mannite and dextran are sometimes formed within standing and windrowed cane under certain conditions of physiological deterioration. This point is of such importance that it can well be called to the attention of Louisiana sugar producers again. To give additional data on the properties of mannite and dextran, especially as regards the effect of these substances on clarification and sugar recovery, and to emphasize the fact that these fermentation products sometimes occur in such quantity as to cause serious factory trouble, may be considered the occasion for this publication. Experimental Work
The juice was freshly expressed from cane that had been frozen and deteriorated in the field. It was not permitted to ferment subsequently in the laboratory. In the preparation of the sample, no lime a t all was used for clarification, as it had been learned by a preliminary test that the molasses jellied regardless of whether the juice had been treated with heavy liming or had been clarified with heat alone. The degree & clarification accomplished could be considered practically nil, as very little mud or scum resulted on simply heating to boiling and settling. The juice was centrifuged, however, a solid basket being used to remove as much of the finely suspended matter as possible. The juice was then evaporated in a laboratory-size glass vacuum pan a t 20.5inch (52.07-em.) vacuum and an average temperature of 71" C. Circulation was so poor in this case that the sample was evaporated to a density of only 70" Brix. Even a t this low density for a molasses, the sample jellied or solidified after standing overnight a t about 20" C. A mass of very small, needle-shaped crystals could easily be seen to be distributed throughout the jellied product. The crystalline material was apparently not the same substance as the needle grain, or elongated sucrose crystals, often observed by sugar boilers in slow-boiling sirup strikes. (According to the true meaning of the term, this product was a sirup, but its composition, as given later, justifies calling it molasses.) The condensed vapors obtained from the evaporation of the sour juice to 70" Brix titrated 10.3 cc. of 0.1 N sodium hydroxide per 10 cc. of condensate. As judged by comparing
Vol. 23, No. 11
the acidity of the juice before evaporation with that of the resulting molasses, approximately 60 per cent of the acidity was removed by evaporation. The volatile acids were neutralized with lime, and the solution was concentrated t o small volume. Subsequently, Nelsons identified the volatile acid by the preparation of the silver salt, finding it to consist entirely of acetic acid. The proximate analysis of the molasses, on a solids basis, is as follows: * Sucrose Ash Gums Reducing sugars
3 2 . 1 per cent 4 . 4 8 per cent 6 . 2 4 per cent 3 3 . 9 0 per cent
The viscosity of a 50" Brix solution a t 30" C. was 9.75" Engler, as contrasted with 1.55"for a normal sirup. ISOLATION OF SEEDLE GRAIK-TWO methods were successfully used to separate gums from the crystalline materialultra-filtration and alcohol precipitation. Although the use of alcohol to remove the gums was found to be the easiest method for preparing the crystalline material alone, most of the molasses was ultra-filtered in order to obtain the gums in an unaltered condition for a study of their properties. For ultra-filtration, the molasses was diluted to 20" Brix and subsequently washed with an equal volume of water. Use was made of very slow-filtering collodion membranes, about 3 days being required for filtering a n d washing. The filtrate, which contained only 0.3 per cent of gums, was concentrated in vucuo to 70" Brix at which density the crystals were larger than those obtained a t greater concentration. The filtrate crystallized nearly solid after standing a t from 30" to 35" C. for 48 hours. It was then mixed with 3 parts of glacial acetic acid, centrifuged, the supernatant liquor decanted, and this treatment once repeated. The crystalline material was transferred to a Ruchner funnel and washed twice with glacial acetic acid and five or six times with absolute alcohol. The crystals were then air-dried a t room temperature. The yield of somewhat impure material was 6.9 per cent, based on molasses solids. For recrystallization, the product was dissolved in a small volume of water and treated with 2 per cent of decolorizing carbon and enough Filter-Cel to give a clear filtrate. This was evaporated on the steam bath a t a temperature of from 60" to 70" C. to a density of 25-30 per cent solids as determined by refractometer. It was found later that, in the case of this product, the per cent solids determined by drying agreed closely with the per cent solids determined by refractometer. The 30 per cent solution was allowed to crystallize a t room temperature for about 48 hours, these conditions being found to give large crystals and to facilitate efficient washing. The material was washed on a Buchner funnel with 50 per cent alcohol and dried in vacuo a t 70" C. The mother liquor was concentrated and worked for another crop of crystals. The yield of product once recrystallized was 5.7 per cent of the original molasses solids. When the alcohol-precipitation method for removing gums was used, a similar procedure was followed for obtaining a crystalline product from the filtrate. The yield in this case was 6.7 per cent of crude material. IDEKTIFICATION OF d-MANNIToL-The purified crystals had the following properties, which identify the substance as mannite, or d-mannitol: 1-Melting point, 166' C. (3). 2-Polarization in borax s o h . (5 grams d-mannitol and 9 grams borax in 100 cc. at 20" C. in a 2-dm. tube), $7.48' V. 3-Sp. rotation in borax soln., +25.9" (5). 4-Approx. solubility in water at 30" C., 22 grams in 100 cc. 5-Melting point of tribenzal derivative, 220" C. 6-Optical properties: Food Research Division, Bureau of Chemistry and Soils. Determined by G. L. Keenan, Food Control Division, Microanalytical Laboratory, Food and Drug Administration. 8
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November, 1931
INDUSTRIAL A N D ENGINEERING CHEMISTRY
( a ) Mannitol (This material apparently crystallizes in the rhombic system, consisting of clear, glassy prisms when crystallized from water), refractive indices: no = 1.520 (on irregular fragments and crosswise on rods), ng = 1.555 (crosswise on rods), n y = 1.558 (lengthwise on rods), ny - no = 0.038, all ~ 0 . 0 0 3 ; axial angle 2E = 68-70' ; sign - ; parallel extinction, positive elongation. (This material appears to correspond with the "6-modification" described by Groth ( 6 ) who gives 2E = 71' 30', the sign - ) ( b ) Tribenzal mannitol (This material, crystallized from toluene, consists of a felty mass of fine needles), no = 1.580 (lengthwise), n,g = 1.640 (crosswise), n-, = 1.656 (crosswise), all * 0.003; extinction parallel, elongation negative.
E. K. Nelson3 who prepared the tribenzal derivative gives the best method of preparation as follows: It was found that if 5 grams of mannitol and 8 7 grams of benzaldehyde were well mixed, and 1 gram of zinc chloride and 10 cc. of concentrated hydrochloric acid were added, the mixture soon solidified. The product was drained on a Buchner funnel, washed with water and alcohol, and recrystallized from boiling benzene. The compound was also made from a kiiown specimen of mannitol.
Although mannitol has been reported previously as an anabrobic fermentation product (4,the possibility that it may occur in standing and windrowed cane, and in such quantity as to cause serious factory-operating difficulties, does not seem to have been sufficiently emphasized. h/IETHOD O F SEPARATION AND PROPERTIES OF COLLOIDAL MATERIAL-The colloidal material retained on the ultrafilter was dialyzed for 5 days against running tap water and then for 3 days against daily renewed distilled water. The a-naphthol test then showed the absence of sugar. Toluene was added as a preservative, and various portions of the colloidal material were taken for a study of its composition. An aliquot dried a t 70" C. and 27-inch (68.6-em.) vacuum showed a yield of 4.05 per cent total colloidal material, based on total molasses solids. Filtration of the water suspension, resulting from ultra-filtration and dialysis, through sufficient Filter-Cel to give good clarity, removed about one-third of the solids (subsequently found to contain most of the ash and protein). The determination of solids and polarization of the filtrate, without further purification, gave a specific rotation of + 1 5 i o . This indicates the possibility of the gums being largely dextran, which has a specific rotation of approxiAfter acid hydrolysis, however, the specific mately +ZOO". rotation was found to be only +Is", which is lower than would be expected if the gums consisted only of dextran. The identity of the gums will be further investigated. The composition of the total colloidal material was partially determined as follows: Ash, 3.9 per cent
Gums (estimated as e uivalent to 90 per cent of the reducing sugars formed b y hydrolysi8.63.0 per cent Protein (6.26X N ) , 8.9 per cent
The composition of the ash of the total colloidal material was 11.9 per cent ferric 34.6 per cent silica, 15.2 per cent P205, oxide, no calcium oxide. Comparison of these figures with the composition of the ash of the original molasses shows that a large proportion of the silica, iron, and phosphate are present in colloidal condition. The composition of the colloidal material filtered through Filter-Cel was as follows:
Influence of These P r o d u c t s in Sugar Factory
As abnormally high viscosity of the sirup and molasses is considered one of the causes of poor circulation and slow boil-
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ing in vacuum pans, the effects of mannitol and gums in this c'onnection were investigated by H. G. Hill of this division. By using the method of Bingham and Green ( 2 ) ,it was found that substituting a concentration of 5 per cent mannitol for the same concentration of pure sucrose gave a viscosity of 0.609 poise, as compared with a viscosity of 0.700 poise for a pure sucrose sirup of the same total solids content (73 per cent solids). The presence of mannitol, therefore, does not increase the viscosity from the standpoint of substitution of mannitol for sucrose and cannot be considered responsible for the slow boiling of sirup strikes, except from the standpoint of reducing the purity and necessitating a higher density in order to obtain the requisite sucrose supersaturation. Moreover, the web-like mass of very small interlacing mannitol crystals found in molasses should not be confused with the needle-shaped or elongated sucrose crystals often observed in slow-boiling sirup strikes. The mannitol becomes concentrated in the molasses, and when crystallization ensues to a material extent, the molasses jells or sets, because of the characteristic crystallizing habit of mannitol in such a medium-the formation of web-like masses of small interlacing needle-like crystals. It is believed, moreover, that this behavior may cause a plastic effect in the boiling of molasses strikes, thereby contributing to poor vacuumpan circulation. Although mannitol was obtained in greatest proportion from excessively acid and badly deteriorated cane, it was present in sufficient quantity to cause difficulty in handling the molasses of a large factory that was operating late in the season (early January, 1930). By using modifications of analytical methods (1, 4 ) which will be described more fully later, the percentage of mannitol in a sample of the effect sirup obtained from this factory was found to amount to 3.0 to 4.0 per cent on Brix solids. The presence of mannitol in this effect sirup was confirmed by removing sucrose and obtaining a small quantity of mannitol by crystallization. By the same analytical method, the presence of a total of 15 per cent of mannitol was indicated in the juice from badly deteriorated cane, and from this juice about 7 per cent was actually recovered by crystallization. The influence of the gums on viscosity was also investigated by Hill. Substituting for pure sucrose, 1.3 and 4.0 per cent of the colloidal material previously described, increased the viscosity from 0.455 poise t o 0.691 poise and to 0.732 poise, respectively. (These values are for solutions having the same total-solids content.) The presence of gums in deteriorated juice is therefore assumed to increase the viscosity materially during sugar boiling. Gums also reduce the true purity somewhat, particularly if present in considerable proportion, thereby necessitating higher density to obtain the requisite sucrose supersaturation. Although the viscosity of the molasses is materially increased as a result of concentration of the gums in the molasses, thereby causing difficult working of low-grade products, it is believed that mannitol is principally responsible for the jellying or setting of the molasses. Literature Cited Badreau, J . pharm. chim., 24, 12 (1921). Bingham, "Fluidity and Plasticity," McGraw-Hill, 1922. Braham, J. Am. Chem. Soc.. 41, 1707 (1919). Browne, Ibid., 88, 453-69 (1906); La. Agr. Expt. Sta.. Bull. 91, 17, 96 (1907). Gilmour, J.'Chcm. SOL.,181, 1333 (1922). Groth, "Chemische Krystallographie," Vol. 3, p. 432, W. Engelmann, Leipzig, 1910. Horton, La. Planter Sugar Mfr., 8, 210-11, 369 (1892); La. Sugar Expt. Sta., Bull. 38, 1339 (1895). Maxwell, Ibid., 38,1408 (1895). Taylor, Ibid., S8, 1334-40 (1895).
