Manufacturing-Processing

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1 Manufacturing-Processing DEXTRANS AND DEXTRANASE R. H. Tilbury Tate & Lyle Ltd., Research Centre Keston, Kent, England ABSTRACT Information was sought on the cause, effects, and control of dextran in sugar manufacture in Jamaica. Storage experiments on manually harvested, burnt, whole-stalk cane revealed that Leuconostoc mesenteroides was the predominant spoilage organism. Its growth was accompanied by significant increases in dextran content and juice viscosity. However, these parameters. did not increase linearly with storage time. "Dextran" was frequently detected in fresh cane by the haze analysis technique but it was thought to differ from true dextran. Measurements of the occurrence and frequency distribution of dextran in factory mixed juice indicated that much of the cane was sour or stale and likely to cause processing difficulties. A small but significant correlation existed between L. mesenteroides count and dextran content of mixed juice (r =.320, p =.01). The viscosity of mill syrups was correlated with dextran content (r =.633, P =.01), but no correlation was observed between c-axis crystal elongation and dextr.an content of c-massecuites. Final molasses purity was correlated with dextran content of mixed juice in corresponding weeks (r =.557, P =.02), but this may have been due in part to a decline in cane quality. Treatment of mixed juice with dextranase significantly reduced the dextran content. At an enzyme concentration of 3 units/100 ml juice, 68.5% of the initial dextran was removed within 20 min at 40 C. Microbial amylases were less effective than dextranase. It was concluded that the harmful effects of dextran on sugar manufacture may be successfully eliminated by treatment with dextranase, either alone or with other enzymes. INTRODUCTION Recent expansion of mechanised harvesting in Queensland has stimulated interest in the harmful effects of post-harvest deterioration of sugarcane (Egan, 1968). It was shown by Egan (1965) that sour storage rot of mechanically harvested, chopped-up cane is caused by bacterial infection with Leuconostoc mesenteroides (Cienkowski) van Tieghem. This organism has been associated with processing problems in the sugar industry for many years. It possesses the ability to multiply rapidly in dilute sucrose solutions and toform dextran from sucrose (Tilbury, 1968). The injurious effects of dextran on raw sugar manufacture were recently summarised by Foster (1969a). Some of the effects are: a) an increase in the viscosity of process materials, which leads to a decrease in factory capacity; b) a decrease in sucrose crystallisation rate; c) formation of needle-shaped sucrose crystals elongated along the c-axis (Keniry, Lee and Davis, 1967a; Sutherland and Paton, 1969; Leonard and Richards, 1969); d) interference with analyses; e) poor clarification; and f) greater loss of sucrose to final 1444

2 R. H. TILBURY 1445 molasses (Keniry et al., 1967a). Furthermore, recent work in Japan showed that "gums" (including dextran) are occluded in raw sugar crystals derived from deteriorated cane (Ando et al., 1968). Such sugar exhibits poor filterability (Yamane et al., 1968) and yields needle-shaped crystals in the refinery (Kamoda et al., 1968)., Financial losses incurred by the presence of dextran in sour cane have not been assessed. However, it is apparent that high dextran levels may result in significant economic losses in both raw sugar factories and refineries. The relative absence of research on microbiological deterioration and dextran formation in whole-stalk cane prompted an investigation in Jamaica. Here, sour cane and processing difficulties are frequently encountered when manually harvested, whole stalk, burnt cane is crushed in the rainy season. It was shown that this cane is almost universally infected with L. mesenteroides. Furthermore, the degree of infection and deterioration is enhanced by wet climatic conditions (Tilbury, 1969a, 1970). Attempts to control sour cane by. chemical treatment and other methods were unsuccessful. It was concluded that the only practical control measure is to minimise post-harvest deterioration losses by improvements in the logistics of cane transportation. However, a preliminary study showed that the dextran content of mill juices also could be significantly reduced by treatment with dextranase (Tilbury, 1969a). This approach appeared promising. At least 2 polysaccharides other than starch are of economic significance in harvested cane (Tilbury, 1969a). Sour cane principally contains dextran produced by L. mesenteroides infection. Dextran is a homologous glucose polymer containing mainly a 1-6 glucosidic linkages. It causes both c-axis crystal elongation and increases in viscosity. Stale cane appears to form a glucan polymer with predominantly a 1-4 glucosidic linkages in the absence of microbial infection (Nicholson and Lilienthal, 1959; Bruijn, 1966). This polymer, named sarkaran by Bruijn (1970), is different from starch and pullulan. It causes increased viscosities but does not produce elongated sucrose crystals (Sutherland and Paton, 1969). Sarkaran is partially hydrolysed by pullulanase and salivary amylase, but it is not attacked by bacterial a-amylase (Bruijn, 1970).. The present paper describes work at Frome Estate, Jamaica, on the formation of dextran in harvested, burnt, whole-stalk cane; levels of dextran entering the factory in mixed juice; some effects of dextran on sugar manufacture; and further work on the enzymic removal of dextran in the mill. MATERIALS AND METHODS Fields chosen for study of the effects of storage on harvested cane were part of the narmalestate reaping programme and consisted of mature cane burnt the previous day. A sample of 200 whole; healthy stalks was manually cut from 8 random locations in.the field. Harvested cane was immediately transported to the factory where it was mixed and divided into 8 25-stalk samples. Each bundle was weighed and laid on grass in the open. Initially, and at daily intervals up to 10 days storage time, a bundle was re-weighed and crushed by passing 3 times through a sterilized, small 3-roller mill at constant pressure. Cane juice was collected aseptically and analysed immediately for L. mesenteroides count, dextran content and specific viscosity. Viable plate counts on sucrose

3 1446 MANUFACTURING-PROCESSING tryptone agar + thallous acetate were performed on serial dilutions of juice according to the method of Tilbury (1969a). Dextran was determined by the haze analysis technique of Keniry, Lee and Davis (1967a) and Keniry, Lee and Mahoney (1969). Dextran is here defined as the material precipitated from a starch-free, protein-free solution by 50% ethanol. The method was standardised against pure food-grade dextran, M.W million (Koch-Light Ltd., Colnbrook, England). Reaction times of, 20 min were used. Optical densities were determined.in a Klett-Summerson photoelectric colorimeter using a red filter ( mp,). Dextran contents were expressed as percent brix (refractometer). Absolute viscosities of cane juice were determined by the capillary tube method in a calibrated Ostwald viscometer at 25 C. Specific viscosities (7] spec). were calculated from the expression: 7]spec = (7] -7]0) 7]0 '. ' where 7] = absolute viscosity of test juice in centipoises and 7]0 = absolute viscosity of a pure sucrose solution at the same brix and temperature as the test juice, from standard tables. Relative viscosities of syrups were determined by the falling sphere method in a Hoppler viscometer at constant temperature. All syrups were diluted to 55 degree brix prior to test. The method of Keniry, Lee and Davis, (1967a) was used to measure c-axis crystal elongation of c-massecuites, Freeze-dried dextranase, specific activity 12 international unitsjmg, was prepared from Penicillium [uniculosuni and kindly 'donated by Dr. W. Bowen (Dental Caries Research Unit, Downe, Kent, England). Dextranase' was freshly prepared in 0.2 M acetate buffer, ph 5.3, at xloo the desired final concentration. One ml enzyme solution was added to 100 ml mixed juice and incubated at 40 C in a water bath, with occasional shaking. After the desired incubation time, the reaction was stopped by addition of 0.2 volumes 10% trichloracetic acid. Controls were treated with buffer solution without enzyme. Dextran contents of enzyme treated solutions were assayed by haze analysis and compared with those of the controls. The following sources of amylases were utilized: Fungal a-amylase (E.C.3.2.I.I.) -Novo II (Globe Products Ltd., Accrington, Lancs., England). Glucoamylase (E.C.3.2.I.3) -Ambazyme PC25 (ABM Industrial Products Ltd., Stockport, Cheshire, England. Bacterial a-amylase (E.C.3.2. I. I.) - Nervanase MT (ABM Industrial Products Ltd.). The method of studying the effect of amylases and dextranases on the dextran content of mixed juice was similar to that described above for dextranase, except that an empirical enzyme concentration of 10 ppm on mixed.ijuice was used. Optimum reaction conditions for each enzyme were selected asfollows: ~''';J~ i,>j,

4 R. H. TILBURY 1447 Enzyme Reaction temperature C ph of acetate buffer Reaction time in minutes Dextranase Fungal a-amylase Glucoamylase Bacterial a-amylase RESULTS AND DISCUSSION Dextran Formation in Harvested, Whole-Stalk Cane Forty-five replicated storage trials on harvested cane were completed during April-July Thirty-nine trials utilised variety B4362, and 6 trials utilised variety B 51129, which constituted 73 and 12% of the harvested crop, respectively. Trials with B 4362 were divided into 4 experimental groups, A, B, C and D, based on climatic data and degree of maturity of the cane in the period studied (Table 1). For various reasons the 1969 crop extended into the rainy season, which resulted in progressive overmaturity and decline in cane quality. Table 1. Climatic data and distribution of 45 experiments on the storage of harvested cane, Frome Estate, Mean Mean period Mean Total no. of weekly temperaturevv age of replicate storage Experi- No. total (OF) cane at experiments mental wet rainfall" " harvest period Date days" (in.) Max Min (months) B II29 A 5th April th May B 17th May st May C 7th June- 2 l.l th June ]) 5th July- I II 2nd August " Days which received more than 0.1" rain ** Recorded in the factory (storage) area Results forl mesenteroides count, dextran content and specific viscosity of harvested cane are summarised graphically in Fig. 1 and 2.,L. mesenteroides was detected in juice from freshly harvested cane juice in every replicate experiment. Initial counts were usually between 5 X 10 4 and 5 X 105/ml (Fig. 1). The organism multipled rapidly in harvested cane and exhibited a characteristic exponential growth curve. Maximum counts of 10 1 to 108/ml were obtained 3,4

5 105, Iii i j o 2 "4 6 If I STORAGE TIMEIN DAYS Fig. 1. The effect of storage time on the count of Leuconostoc mesenteroides in juice from harvested cane, Frome Estate, Key Experimental period Variety Experimental period Variety A 5th April-10th May B 4362 D 5th July-2nd Aug B 4362 B 17th May-31st May " E 5th April-2nd Aug C 7th June-28th June " F 5th April-2nd Aug B days after harvest, followed by a slight decline. No significant differences in the mean initial count were detected between periods or between varieties. The mean maximum count was highest in period B, probably due to the high rainfall and moist ambient conditions which prevailed in that period. In individual trials the level of infection of cane with L. mesenteroides varied widely. However, L. mesenteroides was the predominant spoilage organism in almost every case. Since the specific viscosity of cane juice is probably governed by its dextrancontent, results for these 2 parameters may conveniently be discussed together (Fig.. 2). In general, both dextran content and specific viscosity were directly proportional to time of storage. However, the,relationships were non-linear, and it was not possible to derive simple regression equations for these parameters, The curves for B 4362 all season (E) showed peaks for dextran content atdays

6 R. H. TILBURY 1449 A 1.5 B 1.0 0,5 o C 1,5 D 1.0 DEXTRAN %BRIX OR SPECIFIC VISCOSIT'J1.5 (xio) o E 1.5 F 1.0 0, STORAGE TIME IN DAYS Fig. 2. The effect of storage time on the dextran content and specific viscosity of juice from harvested cane, Frome Estate, Open circles-dextran % brix; closed circles-specific viscosity (X 10). Key Experimental period Variety Experimental period Variety A 5th April-10th May B 4362 D 5th July-2nd Aug B 4362 B 17th May-Slat May E 5th April-2nd Aug " C 7th June-28th June F' 5th April-znd Aug B and 6, and troughs at days 5 and 7. In contrast, specific viscosity showed peaks and troughs displaced by a period of one day prior to those for dextran. These fluctuations may be partly explainedin terms of dextran production by L. mesenteroides. It is know that pure cultures of L. mesenteroides in a liquid sucrose medium produce dextran, which causes an Initialviscosity increase followed by a decrease (Jeanes, 1965). However, the lack of correlation between dextran and viscosity is not readily explained., Increases in dextran content and specific viscosity beyond 7 days storage time, when counts of L. mesenteroides are actually declining, may have several explanations, e.g., continued activity of extracellular Leuconostoc dextransucrase; formation of polysaccharides by certain aciduric lactobacilli (Tilbury, 1970), or formation of a non-microbial polysaccharide such assarkaran. A disadvantage of the haze analysis technique for dextran determination is its lack of specificity

7 1450 MANUFACTURINc;;.-PROCESSING for true dextran. Although starch is excluded, other high-molecular weight, alcohol-precipitable polysaccharides may be detected by this method. The rate of increase of dextran and viscosity was significantly greater during the rainy season (Period B) than in other periods. This coincides with a greater degree of infection of cane with L. mesenteroides. It is significant that many samples of freshly hanrested cane contained dextran. The maximum figure obtained was 0.51% brix late in the season. Mean initial dextran contents for the various periods were: A = 0.13% brix; B = 0.16% brix; C = 0.20% brix; D = 0.22% brix; overall mean = 0.18% brix. Within variety B 4362 there was a small but probably significant increase in initial dextran content throughout the season which may be related to the degree of cane overmaturity, The presence of dextran in fresh cane contravenes the findings of Keniry et al. (1967b), but confirms reports by Foster (1969b) for burnt, chopped cane in Queensland. However, it is probable that this dextran is a natural cane polysaccharide such as sarkaran rather than L. mesenteroides dextran. Dextran content and specific viscosity may be suitable indicators of the processing quality of harvested cane (Keniry et ai., 1967a). However, they are less satisfactory as indicators of the age of cane post-harvest, owing to their nonlinear relationship with time of storage and the presence of dextran in varied amounts in fresh cane. Determination of dextran by haze analysis is preferable to viscosity measurement for use as a routine quality control test in the mill because it is more rapid, sensitive and accurate. Dextran Content of Mixed Juice One hundred and seventy samples of mixed juice were taken at random from the mill during Feb-March 1970 and analysed for dextran content (Table 2). The overall mean dextran content was 0.34% brix, which is significantly. Table 2, Dextran content of mixed juice, Frome Estate, 1970, Tandem No. I Tandem No.2 Overall results --- Mean Mean Mean Week No, dextran No. dextran No. dextran commencing tested (% brixj tested (% brix) tested (% brix) Feb 23rd March 2nd March 9th March 16th March 23rd 15 Al Overall less than 0.41% brix for the period April-July 1969 (Tilbury, 1969a). Dry climatic conditions during the 1970 test period may have reduced the incidence of sour cane and accounted for this difference. Juice from No.1 tandem had a higher JI1.ean dextran content (0.37% brix) than that from No.2 tandem (0.32% brix). This confirms 1969 observations.

8 R. H.TILBURY 1451 A possible explanation is that the mean age of cane _post-harvest is greater on No.1 tandem, which grinds mainly farmers' cane, than on No.2 tandem, which grinds mainly Estate-grown cane. It is apparent that cane ground at the mill is not fresh. From the relationship between age of cane post-harvest and dextran content (Fig. 2,E.) it may be estimated that the mean age of cane ground at the mill in March 1970 was 48 hr. This exceeds the mean bill-to-mill time of 36.2 hr determined for 135 samples of cane in However, this figure was obtained for Estate-grown cane, which comprised only 50% of the crop. It excludes the effect of farmers' cane, which is' probably more stale. The frequency distribution of dextran content (Fig. 3) is similar to that of 1969 (Tilbury, 1969a). Of the mixed juice samples 34% had dextran contents 25 r-r- 20 ~ 15 % FR~QUENCY I-- l- I- 10. l-.-- I-- Wl.-, ro ~ , DEXTRAN % BRIX Fig. 3. Frequency distribution of dextran content of 170.samples of mixed juice, Frome Estate, 23rd Feb-26th March in excess of 0.4% brix. It was shown.by Keniry eta!' (1967a) that c-massecuites with a dextran content of 0.2% brix exhibited double thee-axis crystal elongation of dextran-free samples. Similar results were obtained by Sutherland and Paton (1969)' for purified dextran in laboratory-treated syrups. Therefore it seems likely that the dextran content of mill juices at Frome is sometimes sufficient to cause severe harmful effects in the factory. It was postulated that if dextran in cane juice is due to infection of cane with L. mesenteroides, there may be a positive correlation between the L. mesenteroides count and dextran content of mixed juice. A study of 62 random samples of mixed juice taken during Feb-April 1970 revealed a small but significant correlation between these parameters (r ;= 0.320, p =0.01) (Fig. 4). Three

9 1452 MANUFACTURlNG-PROCESSTNG 10 7 a a 10 6 COUNT PER ML.JUICE o 0 I~ ~ o o 0 0.'l.~.,. Q v //"~;;V',b1, >r 'fl.:.j \0<;\\\)"- ~ DEXTAAN % BRIX Fig. 4. Correlation between Leuconostoc mesenteroides count and dextran content of 62 samples of mixed juice, Frome Estate, 24th February-24th April Key:. Tandem no.l; 0 Tandem no.z. possible causes of this small correlation are suggested: 1) a proportion of the dextran may be a non-microbial polysaccharide such as sarkaran; 2) a viable count does not detect organisms which may have formed dextran and subsequently died; 3) organisms derived from cane are diluted with other microorganisms from soil, trash, etc. during the milling. The Effects of Dextran on Sugar Manufacture C-axis crystal elongation of c-massecuites. Keniry et al. (1967a) demonstrated a significant correlation between dextran content and c-axis crystal elongation of c-massecuites derived from chopped cane in Queensland. This relationship was studied at Frome in 1969 for 23 samples of c-massecuite derived from whole-stalk cane. Samples were taken during June and July, when high dextran contents were recorded inrnixed juice entering the factory. There was no statistically significant correlation between dextran content and crystal elongation of these c-massecuites. However, marked crystal elongation was present in every sample, and dextran contents were high ( % brix). The mean dextran content was 1.30% brix and the mean crystal elongation was 2.3. During March and April 1970 a further 36 samples of c-massecuite were examined for crystal elongation..the mean crystal elongation was 1.8, but dextran content was not determined. However, the dextran content of mill juice during this period was significantly less than that for the period of the 1969 results. Accord-

10 R. H. TILBURY 1453 ing to Keniry et al. (1967a), the c-axis crystal elongation of normal c-massecuites of good processing quality is Despite the lack of correlation between dextran and crystal elongation at Frome, it is probable that high dextran levels caused marked crystal elongation. However, the effects of dextran in c-massecuites derived from whole-stalk cane appear to be much less than in those from chopped cane. This may be due to the presence of non-microbial dextrans in the former case. Syrup viscosity. Examination of 21 samples of syrup in April 1970 revealed a statistically significant correlation between dextran content and viscosity (r = 0.633, P = 0.01) (Fig. 5). The mean dextran content of the samples was 0.56% 80 '" RElATIVE VISCOSITY - TimeIn seecnds o o 60 or.~. 50, ~ ~~., o 000 < DEXTRAN ;ORIX 0.' 0.6 Fig. 5. The effect of dextran content on viscosity of factory syrups, Frome Estate, 20th-24th April brix. The slope of the regression line was sreep. which indicates that dextran content had a marked effect on syrup viscosity. Therefore the entry of high levels of dextran from sour cane into the factory will increase the viscosity of materials and retard factory throughput. Finalmolasses purity. Keniry et al. (1967a) reported a significant positive correlation between the dextran content of syrup and the purity of final molasses made from it. At Frome the mean weekly dextran content of mixed juice was compared with final molassespurityfor the corresponding week, displaced by 4 days to allowfor processing time (Fig. 6). There was a significant positive correlation between these parameters (r = , P = 0.02). It may be concluded that high dextran levels increase the loss of sugar to final molasses. However, during 1969 the cane quality progressively deteriorated due to overmaturity, This caused a decrease in the exhaustibility of fresh cane which may also have contributed to this correlation,

11 MANUFACTURING-PROCESSING DEXTRAN % BRIXOF MIXED JUICE (WeeklyMean)* Fig. 6. Relationship between dextran content of mixed juice and apparent purity of final molasses made from it, Frome Estate. * Weekly period of apparent purity is displaced 4 days beyond that of dextran content. Key: th April-Isth July; th February-28th March. Enzymic Removal of Dextran from Mill Juices Preliminary experiments at Frome in 1969 showed that treatment of mill juice with dextranase reduced the dextran content by 78%. The enzyme concentration was 300 units/ 100 ml juice, and reaction times of min at 43 C were used (Tilbury, 1969a). When the enzyme concentration was reduced to 30 unitsj l Of) ml juice and the reaction time was reduced to 30 min, 76% of the dextran was removed. The purpose of the present work Was to define the optimum conditions for economic removal of dextran from mixed juice by treatment with dextranase. Thework was done at Frome during March-April At an enzyme coricentration of 30 unitsj l Gtl ml juice the effect of a reduction in reaction time to 15 min was examined for 38 samples of mixed juice taken at random from the mill (Table 3). A mean of 79% of the initial dextran was removed. It was concluded that the reaction time. can be reduced to 15 min without loss of enzyme efficiency. Enzyme efficiency did not appear to be influenced by substrate concentration. Most samples showed more than 50% removal of dextran, but in 4 samples the amount of dextran removed was less than 4%. The variations in degree of dextran hydrolysis and occasional weak activity may be explained. by the high specificity of dextranase for a 1-6 glucosidic linkages (Bourne, Hutson and Weigel, 1962). Cane juice may contain a variety of strains of L. mesenteroides, each of which produces characteristic dextrans differing in their degree and type

12 R. H. TILBURY 1455 Table 3. The effect of dextranase on the dextran content of mixed juice, Frome Estate, Date Initial dextran (% brix) Removal of dextran (%) (; ;23 : : M Mean of cross-linkage (Tilbury, 1970). It is probable that where the enzyme efficiency was low the samples mainly contained polysaccharides other than L. mesenteroides dextran. In a 2nd series of experiments the effect of a reduction in enzyme concentration was examined. Fourteen samples of mixed juice were treated with dextranase at 12, 6 and 3 units/ioo 'ml juice for reaction times of 10 and 20 min (Table 4). At 10 min the mean percent removal of dextran was less than at 20 min. This effect was most pronounced at the lowest enzyme concentration. However, in a reaction time of 20 min the degree of dextran removal was independent of enzyme concentration. At an enzyme concentration of only 3 units/ 100 ml juice the mean percent removal of dextran was 68%. This result compares favourably with commercial processes for. removal of starch in S. African sugar factories using amylases (Bruijn and Jennings, 1968). Pure amylases 'lie specific for a 1-4 glucosidic linkages. It was thought that some non-dextran polysaccharides'in mill juice may be partially hydrolysed by these enzymes.. Therefore, the effect of some commercially available amylases was compared with that of dextranase for 12 samples of mixed juice (Table 5). Dextranase removed a greater percentage of dextran on average than any of the amylases, but the latter showed significant activity. However, quantitative comparisons cannot strictly be made because each enzyme was tested under different reaction conditions and concentrations. Since starch was absent from the test samples, the activity of the amylases might be explained by the presence of a significant proportion of a 1-4 glucosidic linkages in L. mesenteroides dextran. Alternatively, these enzymes may have contained impurities with dextranase activity. This is likely with glucoamylase, which is known to hydrolyse a 1-6

13 1450 MANUFACTURING-PROCESSING Table 4. The effect of dextranase on the dextran content of 14 samples of mixed juice, Frome Estate, % removal of dextran at following enzyme ~onc (units/ioo ml juice) Initial dextran Date <'10 brix) 10. min 20 min 10 min 20 min 10 min 20 min , J j{ " " " ", " " " " " " tl Mean "Result obtained after 15-min reaction time and excluded from calculations of the mean percent removal of dextran. Table 5. The effect of some amylases and dextranase on the dextran, content of 12 samples of mixed juice, Frome Estate, Initial % removal of dextran with following enzymes Date dextran (April) (% brix) Dextranase Fungal a-amylase Glucoamylase Bacterial a-amylase ' II Mean glucosidic linkages slowly. Since Bruijn (1970) showed that sarkaran is not attacked by bacterial a-amylase, the observed activity of this enzyme cannot be explained by the presence of sarkaran. Further research on the nature, origins and effects of polysaccharides in mill juices should facilitate the most rational selection of enzymes. At present it appears that dextranase, either alone or mixed with other enzymes, offers a

14 R. H. TILBURY 1457 technically feasible means of removing the deleterious effects of dextran and other polysaccharides on the factory process. Laboratory tests have demonstrated that treatment of dextran-containing sucrose' solutions with dextranase reduces their viscosity. It was also shown that" neither the enzyme nor the reaction conditions cause sucrose loss (Tilbury, 1970). The effect of dextranase treatment on prevention of c-axis crystal elongation has not been studied. However, the principal products of enzymic hydrolysis of dextran are known to be glucose, isomaltose, isomaltotriose, and homologous oligosaccharides of low molecular weight (Bourne et al., 1963). These compounds are not thought to exert any harmful effects on sugar manufacture.. Dextranase is most active at temperatures between C and at ph values between Therefore the most suitable stage for its addition to the process is mixed juice prior to heating and liming. At this stage the polysaccharides will not have interfered with the process. The economic feasibility of this treatment depends on the degree of reduction in factory capacity due to poly.saccharides, the market price of raw sugar, and the cost of the enzyme. 1 the loss in production due to dextran!is 5% and raw sugar sells for 45/ton, the maximum cost of enzyme when used at the rate of 3 units/ioo ml mixed juice is 167 shillings/million units. This price is estimated to be compatible with commercial production of dextranase in the near future. The application of dextranase and its mixture with other enzymes to remove polysaccharides in sugarcane process materials and refineries has been patented (Tilbury, 1969b). ACKNOWLEDGEMENTS I am grateful to the Directors of Tate & Lyle Ltd. and the West Indies Sugar Co. Ltd. for permission to publish this paper. Special thanks are due to Mr. P. D. Smith, and Mr. H. S. Ive and his staff at Frome Estate, for their encouragement and generous assistance. Laboratory facilities were kindly provided by Mr. T. Chinloy and Dr. Barbara Robison of the S.M.A. (Jamaica) Ltd. Research Dept. REFERENCES Ando, T., M. Kamoda, F. Onda, H. Ito, T. Shirasaki, and T. Miki Occlusion of filtrationimpeding substances in sugar crystal. Proc. ISSCT, 13: Bourne, E. J., D. H. Hutson, and H. Weigel Studies on dextran and dextranases. 2. The action of mould dextranases on modified isoma1todextrins and the effect of anornolous linkages on dextran hydrolysis. Blochem. J., 85: Bourne, E. J., D. H. Hutson, 'and H. Weigel Studies on dextrans and dextranases, 3. Structures of oligosaccharides from L. mesenteroides (Birmingham) dextran. Biochem. J., 86:555' Bruijn, J Deterioration of sugar cane after harvesting. Part II. Investigation of the polysaccharide formed. Int. Sug. J., 68: Bruijn, J Deterioration of sugar cane after harvesting, Part III. Enzymatic hydrolysis of the polysaccharide formed. Int. Sug. J., 72: Bruijn, J., and R. P. Jennings Enzymatic hydrolysis of starch in cane juice. Proc. S. Afr. Sug. Technol. Assoc., Egan, B. T A sour storage rot of mechanically harvested chopped-up sugar cane. Proc. ISSCT, 12: :Egan, B. T Post-harvest deterioration losses in sugar cane in Queensland. Proc. ISSCT, 13: Foster, D. H. 1969a. Sugar processing difficulties. Aust. Sug. J., 60:

15 1458 MANUFACTURIN~PROCESSING Foster, D. H. 1969b. Deterioration of chopped cane. Proc.Qd. Soc. Sug. Cane TechnoI. 36: Jeanes, Allene Preparation. of dextrans from growing Leuconostoc cultures. In "Methods in Carbohydrate Chemistry" VoI.V. General Polysaccharides. Edited by R. L. Whistler. Academic Press: New York and London. p.122. Kamoda, M.,.F. Onda, H. Ito, T. Shirasaki,T. Miki, and T. Ando On the formation of needle-shaped sugar crystals. Proc. ISSCT, 13: Keniry, J. S., J. B. Lee, and C. W. Davis. 1967a. Deterioration of mechanically harvested choppedup cane. Part I. Dextran-a promising quantitative indicator of the processing quality Of chopped-up cane. Int. Sug. J., 69: Keniry, J. S., J. B. Lee, and C. W. Davis. 1967b. Deterioration of mechanically harvested choppedup cane. Part II. The rate of dextran formation. Int. Sug. J., 69: Keniry, J. S., J. B. Lee, and V. C. Mahoney Improvements in the detxran assay of cane sugar materials.. Int. Sug. J., 71: Leonard, G. J., and G. N. Richards Polysaccharides as causal agents in production of elongated sucrose crystals from cane juice. Int. Sug. J., 71: Nicholson, R. I., and B. Lilienthal Formation of a polysaccharide in sugarcane: Aust. J. BioI. Sci., 12: Sutherland, D. N., and N. Paton Dextran and crystal elongation: Further experiments. Int. Sug. J., 71: : Tilbury, R. Ft Biodetcrioration of harvested sugarcane. In "Biodeterioration of Materials." Editors: A. H. Walters and J. J. Elphick. Elsevier, London. p Tilbury, R. H. 1969a. The ecology of Leuconostoc meseriteroides and control of post-harvest biodeterioration of sugarcane in Jamaica. Proc. W.I.S.A. Sug. Technol. Meeting. Trinidad. (In press). Tilbury, R. H. 1969b. Improvements in the production of sucrose. British Patent Application No Tilbury, R. H Biodeterioration of harvested sugarcane in Jamaica. PhD. Thesis. Univ. of Aston in Birmingham. Yamane, T., K. Suzuki, T. Kaga, and Y. Takamizawa The detrimental effects of impurities occluded in affined sugars in the sugar r~fining process. Proc. ISSCT, J 3:380-~84.