THE ORGANIC ACID COMPOSITION OF SOME COMMERCIAL. Adauritius Sugar Industry Research Irzstitute CANE VARIETIES GROWN IN MAURITIUS D. H.

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1 THE ORGANIC ACID COMPOSITION OF SOME COMMERCIAL CANE VARIETIES GROWN IN MAURITIUS D. H. PARISH Adauritius Sugar Industry Research Irzstitute The principal points of illformation required by the technologist about the juices entering the processing stream, are the solids content and the amount of sucrose in the solids. These two determinations, Brix and purity, are the only analyses norinally made, and yet many other analyses could be carried out, particularly on the organic components. The chemical and physical complexity of juice is such that many factors must affect final sugar recovery. Traditionally, the organic constituent on which blame has fallen most frequently is the reducing sugar content, but many other materials are potential or actual problems. The main factors known to affect the Brix and purity of juice are the minimum temperature during the ripening period, moisture regime, nitrogen fertilization, and maturity. In view of the fact that detailed infornlatio~l is lacking on the organic composition of juice, particularly from cane grown under varying conditions, the opportunity was taken to determine amino-acids and non-nitrogenous acids in the juice of samples of cane grown under a wide range of agronomic variables. EXPERIMENTAL Samples of cane were taken from replicated trials laid down in a sub-humid district and a super-humid district, 60" and rzo", respectively. The trials consisted of 6 varieties, B 34104, B 3337, B 37161, B 37172, Eb6ne 1/37 and M , receiving 3 levels of nitrogenous fertilizer at the following rates: NI Low level of nitrogen appllcatio~ 20 kgs N/arpent. Nz Medium,,,,,,.. 40,, N3 High,,,,,,,, 60,, The maturity factor in Mauritius is not important, as apart from plant canes whichrepresent onlyabout aneighthof the total crop, canes are harvested yearly. What is important, however, is the growing period of the ratoons. The crushing season lasts some 5 months and the ratoon canes may therefore have grown between the periods July- July to November-November. The experimental plots were thus harvested at the end of July, September and, November. These trials covered an extremely wide range of growing conditions and, as they were on a field scale, the cane samples represented the limits which could normally be expected in cane delivered to any factory in Mauritius. The samples were shredded in a "Cutex" shredder, squeezed under hydraulic pressure, and the juice filtered through cotton wool.

2 348 FACTORY a. Amino-Acids ANALYTICAL METHODS Initially, the method used to determine the total content of these substances was the WIGGINS et al7 modification of the ninhydrin method of MOORE and STEIN^, but this method sometimes gave erratic results and an alternative was sought. Finally it was decided that as the nitrogenous material soluble in 80 % v/v alcohol included all the amino-acids, together with smaller quantities of low molecular weight nitrogenous compounds, then this fraction would be satisfactory in giving an indication ol tllelevel of "noxious" nitrogen. The sugarbeet industry uses a similar fractionation as a routine procedure on beet juices. Chromatography was carried out as described by PARISH^. b. Non-nitrogenous A cids For paper chromatography, the acids were separated by adsorption on "Deacidite E" resin and eluted with hydrochloric acid. The solvent system was the supernatant layer of a mixture of equal parts of 5 M formic acid and amyl alcohol. For the quantitative determination of aconitic acid, the silica-gel column procedure of ROBERTS and MARTIN^ as slightly modified by PARISH= was used. RESULTS Fig. I shows a chromatogram of samples of juice of the variety M receiving 3 levels of nitrogenous fertilization. Each sample has been run 4 times, the Flg. I. Chromatogram of the juice of M fertilized with 3 levels of nitrogen. amount of material applied being doubled each time, thus giving a wide concentration range. The resulting chromatograms then show quite clearly any minor as well as major changes occurring in the composition.

3 Fig. 3. Chromatogram showing the non-nitrogenous organic acids of the juice of M Fig. z shows the levels of 80 % alcohol soluble nitrogen occurring in the juice of B and Ebene 1/37, and the means for the 6 varieties tested when nitrogenous fertilization, time of harvest, and site are varied. Fig. 3 is a chromatogram of the non-nitrogenous acids of a sample juice of M. 134/3z, aconitic acid is the control spot. Table I gives the effect of the agronomic variables studied in this work on the aconitic acid content of the juices from M and Ebhne 1/37. References p. 351.

4 FACTORY TABLE I Locality: Bonne Veine 120" Locality: Bon Espoir 60" Variety: M. 134/32 Variety: M Izg N Early Mid Late kg AT Early Mid. Late Variety: Ebene 1/37 TTaviety : Ebene 1/37 Iig. N Early Mid. Late, Kg. N Early Mid. Late pev arpent (a) Anzino acids DISCUSSION Whilst amino-acids have been suspected for a long time of exercising some important influence on the limitation of the crystallization of sucrose from molasses, ICELLYI proposed that when an organic solute other than sucrose reaches saturation point, then the crystallization of sucrose ceases. Amino-acids have fairly low solubilities and therefore, despite their low concentrations, they may exert a controlling influence on crystallization. Changes in the amino-aclds of juice could be either quantitative or qualitative. PARISH^ has shown for the 6 varieties nsed in this work that, as far as the major amino-acid components are concerned, varietal differences are slight, whilst the chromatogram Fig. I shows clearly that the effect of nitrogenous fertilization on these amino-acids is also unimportant. This work implies that a figure for the total amino-acids is all that is needed in comparing the different juices studied, and, as has already been pointed out, the alcohol-soluble nitrogen is probably a satisfactory guide to this. Fig. 2 shows the alcohol-soluble nitrogen in the B and the Ebene 1/37samples together with the means of all varieties. These 2 varieties were selected because the former shows the largest variation in the amount of soluble nitrogen, whilst Ebhne 1/37 shows the narrowest and lowest range. The curves show clearly that the dominant factor deterinining the level of low molecular weight nitrogen compounds in a juice is the growth site. In almost every case, the hlgh ralnfall trial gave levels almost twice the correspondiilg levels of the drier area plots The high rainfall area trials, unlilte the low rainfall trials, did not respond to nitrogenous fertilization in terms of cane yleld, and this could be the priilcipal cause of the high levels of nitrogenous constituents, rather than the climatic differences. Nitrogen fertilization itself increases the amount of alcohol-soluble nitrogen markedly, whilst the effect of advancing harvest date is to lower the level slightly. Varietal effects are not marked as regards the average content, but, from the polut of view of the range of levels occurring, they are important, Ebene 1/37 havmg qulte a narrow range and B a wide range. From the figures for B , it is seen that the content of 80% alcohol-soluble nitrogenous consltuents of juice, in other words, those nitrogenous materials of potential interest during manufacture, can vary s~xfold. Variations of ths order, of substances profoundly affecting sugar recovery, could cause large variations in the exhaustion of molasses. The known factory data do not show such variations, and this would seem to imply that the actual level of amino-acids occurring in juice cannot be a critical factor in exhaustion. WIGGINS? has shown that under severe drought conditions the amino-acid level of Juice increases markedly, and this fact was offered as an explanation for the poor clarification of juice from canes harvested during a drought in Tr~nldad.

5 ; (b) Non-Nitrogenous Organic Acids D. H. PARISH The acids, malic, citric, succinic, tartaric and oxalic, are widely distributed in plants. Less widely spread are iso-citric and aconitic acids, and it is interesting that the principal acid of sugar cane should be aconitic acid. The non-nitrogenous acids are present in all juices and account for a significant proportion of the non-sugar solids. That they have an important effect on clarification and sugar recovery is generally accepted, MARTIN^ considering'them harmful because their solubility and reactivity make them potential sources of mellasigenic substances. An additional point of interest in the case of aconitic acid is its potential value as a synthetic chemical. The aconitic acid content of tropical cane is known to be much lower than that of ~o;isiana cane where aconitic acid is recovered as a bye-product of the sugar industry. The first analyses carried out therefore, were to determine the relative amounts of the various non-nitrogenous acids present in local cane juice. Fig. 3 is a chromatogram of the acids separated from a typical juice run at three strengths in the ratio I : 2 : 4. This shows that aconitic acid, despite the low levels at which it occurs in Mauritian juices, is still the principal acid constituent of this fraction. Above the aconitic acid spots are those of mallic and then citric acids. Since aconitic acid is the principal non-nitrogenous acid of local juice, and since the quantitative determination by the decarboxylation method, when it is present in only small amounts, is unreliable, the method of ROBERTS and MARTIN^ was used after minor modifications6. Only 2 varieties were studied, M , which grows well all over Mauritius, and Ebkne 1/37, which grows well only in districts receiving more than 80" rain/annuin. The results which are given in Table I show that there is no apparent relationship on a field scale between the aconitic acid content and the harvest date, and no change in content which could be ascribed to increasing nitrogenous fertilization. Differences in content of this acid between the 2 varieties in the Bonne Veine samples are slight, but there is some indication that Ebbne I 137, when grown under conditions to which it is not suited, gives a higher aconitic acid content. The average aconitic acid content of local juice is around 0.2% D.M., compared with the figure of 1.26% D.M. for Louisiana juicese. At this low level, it is extremely unlikely that the acid has any commercial potential. The results obtained show that the variations occurring in the levels of aconitic acid, and therefore of the non-nitrogenous organic acids in general, with differing agronomic conditions are slight. Under Mauritian conditions, any effect these materials have on the sugar manufacturing process will, therefore, not be subject to large variations. SUMMARY The variations of low molecular weight nitrogenous compounds, including amino-acids, with nitrogen fertilization, variety, growing period, and site, have been examined. Nitrogen fertilization increases the level of these constituents markedly, whilst rainfall and/or response of the crop to nitrogen can also be important. Varietal characteristics are slight compared with these other factors. Organic acids on a field scale gave no marked trends with any of the variables. Unless a cane is abnormal, e.g., the cane is suffering from drought, the role of the amino-acids in sugar manufacture cannot be critical, as their concentration varies far more than the known factory performances, whilst any effect the non-nitrogenous acids have will be fairly constant. REFERENCES KELLY, F. H. C. and THOMPSON, G. P., Amino-acid constituents of sugar cane molasses. Intern. Sugar J., 57 : MARTIN, L. F., Principles of Sugar Technology , Elsevier, London. MOORE, S. and STEIN, W. H., Photometric ninhydrin method for use in the chromatography of amino-acids. J. Biol. Clzem., 176: 367. PARISH, D. H., I. The amino-acid and nitrogen contents of cane juice. Mauritzus Sugar Industry Research Institute. Annl. Rep : PARISH, D. H., A study of the organic acid constituents of juice. Mauritius Sugar Industry Research Instztute. Annl. Rep. : ROBERTS, E. J. and MARTIN, L. F., Identification and determination of non-nitrogenous organic acids of sugar cane by partition chromatography. Anal. Chem., 26 : ' WIGGINS, L. F. and WILLIAMS, J. HOWARTH, Amino-acid content of West Indies sugar cane. J. Agr. Food Chem., 3 :