The amino acid composition of the proteinaceous components of konjac mannan, seed endosperm galactomannans and xanthan gum

Food Hydrocolloids vol .I no.2 pp.95-99. 1986

The amino acid composition of the proteinaceous components of konjac mannan, seed endosperm galactomannans and xanthan gum D .M.W .Anderson. LfHowlett! and e.G.A.McNab Chemistry Department, The University. Edinburgh EH9 311. and IFood Science Division. Ministry of Agriculture. Fisheries and Food . Horseferry Road. London SWI P 2AE , UK Abstract. Analytical data are presented for the amino acid composition s of co mmerci al samples of konja c mannan, locust bean (carob) gum, tar a gum , Sesbania (Dhaincha) gum and xanth an gum. Konjac mannan contains high proportions of aspartic acid , glutamic acid and glycine . Major amino acids in xanthan gum are alani ne. glutamic acid . aspart ic acid and glycine. There are similarities in the amino acid compositions of the seed endosperm galactomann ans studied; all contain major amount s of glutamic acid, aspartic acid and glycine and similar proportions of all the other amino acids, with the except ion of tara gum and Sesbania gum, which contain high proporti ons of alanine and histidine respectively . All the gums studied have very low hydroxyproline contents, in marked contrast to the values reported previ ously for the tree exudates from the leguminous genera Acacia (gum arabic), Astragalus (gum tragacanth) and Prosopis (gum mesquite) .

Introduction

Within the period 1982-1984, the Ministry of Agriculture, Fisheries and Food sponsored a project 'The Characterisation of the Proteinaceous Components of Edible Gums' . Its main objectives have been described (I ) . This paper presents the data obtained for the amino acid composition s of commercial samples of xanthan gum , konjac mannan , and the seed endosperm galactomannans locust bean (carob) gum, tara gum and Sesbania gum. A summary of the data published (2) for guar gum is included to facilitate comparisons between the galactomannans of commercial importance. Locust bean gum (E41O) , defined (3) as the ground endosperm of the seeds of the carob tree , Ceratonia siliqua (L. ) Taub. (Family Leguminosae), should not contain (3) more than 7% protein (N X 6.25) , a value also adopted (4) by JECFA in 1983 in place of that (8%) specified earlier (5). The AD! ' not specified' was allocated by JECFA (6) in 1981. Tara gum, the ground endosperm of the seeds of Caesalpinia spinosa (Family Leguminosae) , is not approved for foodstuffs use within the EEC, but specifications for its identity and purity published by JECFA in 1975 and 1978 were revised (7) in 1984, when a temporary ADI 'not specified' was reduced to a temporary ADI of 0-12 .5 mg/kg/day and an earlier request for the submission of additional toxicological evidence of safety was confirmed (8). Tara gum must not contain more than 3.5 % (%N x 5.7) of protein (7) . Sesbania gum , the ground endosperm of the seeds of Sesbania bispinosa (Jacq.) W.F .Wight (Family Leguminosae) has been widely publicized (9, 10) under the Hindi vernacular name ' Dhaincha' as a useful industrial hydrocolloid; there are as yet no accepted criteria for identity or purity, or toxicological evaluation s, and it is not permitted in foodstuffs. Konjac mannan , the acetylated glucomannan derived from Amorphophallus konjac tubers, is used in Japan as a form of dietary fibre and as a foodstuffs 95

D.M. W.Anderson, J.F.Howlett and C.G.A.McNab

delicacy, but it is not included in the positive lists approved by JECFA or the EEC. It is of interest, however, as one of the limiting cases (% galactose = - 0 %) in studies

(11) of the interactions with other polysaccharides shown to varying extents by glucomannans and galactomannans e.g. locust bean gum (-20% galactose), tara gum (-2S% galactose), Sesbania gum ( - 30% galactose) and guar gum ( - 38 % galactose) (12). The D-galactosyl substituent groups in galactomannans are apparently distributed (13) in irregular block patterns along the mannan backbone; galactomannans containing greater proportions of galactose (e.g. SO% galactose, 14) exist. Xanthan gum, a high molecular weight polysaccharide produced by pure culture fermentation of a carbohydrate with Xanthomonas campestris (1S), has been approved as a foodstuffs additive within the EEC (E4lS) (16) and by JECFA with an AD! of 0-10 mg/kg/day (17). Further information (18) on residual nitrogen levels was requested by JECFA in 1978. There is an EEC specification (19) and a tentative JECFA Specification for Identity and Purity (20); the former imposes (19) a limit of 1.S% (maximum) on the nitrogen content. Methods The gum samples (sources as detailed below), powdered to pass ISO-mesh, were analysed for moisture content (drying to constant weight at IOS°C) and nitrogen content (PerkinElmer model 240 AutoAnalyser) to permit the weight of gum containing 2 mg of nitrogen to be hydrolysed for amino acid analysis. Hydrolysis was carried out by refluxing the gum sample (plus norleucine added as internal standard) with 6 M hydrochloric acid for 20 h under a continuous stream of oxygen-free nitrogen. Full details of the procedure, and of the preparation of hydrolysates for the determination of cysteine and methionine, have been given (21). The hydrolysates were applied to a column of cation exchange resin; the amino acids, separated by elution with suitable buffers of increasing ionic strength or pH, were detected with ninhydrin in a continuous flow analytical system (Rank-Hilger Chromaspek) and quantified by reference to solutions of calibration standards. Each gum sample was hydrolysed and analysed in duplicate. The entire analytical procedure was repeated if the values obtained for the individual amino acids differed by >S%. Results The data in Tables I and II are mean values, obtained from satisfactory replicate analyses, for commercial samples of locust bean gum, tara gum and xanthan gum supplied by European gum importers. The Sesbania gum was of technical quality, brown in colour, from a small consignment of seeds milled for experimental purposes. The values quoted in Table I for guar gum, for comparative purposes, are the ranges of values obtained (2) for ten commercial guar gum samples. The sample ofkonjac mannan was kindly supplied by Dr E.R.Morris, Unilever Research Laboratory. Discussion The sample of Sesbania gum contained 3.28% nitrogen; incomplete separation of the 96

Proteinaceous components of Konjac mannan

Table I. The amino acid composition (residues per 1000 residues of the proteinaceous components) of konjac mannan and some seed galactomannans. Konjac mannan

%N Alanine Arginine Aspartic acid Cystine Glutamic acid Glycine Histidine Hydroxyproline Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine

0.57 118 48 230 0 153 60 26 4 24 36 24 7 25 46 96 35 16 54

Locust bean gum Sample I

Sample 2

0.84 79

77 94 3 236 85 21 4 34 56 50 8 24 47 59 46 21 53

0.93 80 88 97 4 192 101 23 3 37 63 48 5 26 38 65 51 23 55

Tara gum

Sesbania gum

Guar gum" ranges. n = 10

3.28

0.46-0.70

0.36 99 36 134 4 133 88 25 II 41 70 43 9 30 59 72 58 24 68

70 55 94 4 154 131 74 0 40 66 42 4 29 43 72 47 22 52

72-92 41-71 86-121 2-5 120-134 97-214 23-28 10-18 33-41 55-68 36-59 7-10 26-32 37-55 72-90 47-57 26-33 52-62

"Data from ref. (2). Table II. The amino acid composition (residues per 1000 residues of the proteinaceous content) of commercial xanthan gum. Commercial xanthan sample

B

A

%N Alanine Arginine Aspartic acid Cystine Glutamic acid Glycine Histidine Hydroxyproline Isoleucine Leucine Lysine Methionine Pheny lalanine Proline Serine Threonine Tyrosine Valine

0.76 150 46 95 0 103 90 24 7 41 88 27 28 29 58 57 64

17 76

C 1.01

147 48 94 0 106 90 22 0 41 87 31 17 29 61 61 68 19 79

0.56 143 43 90 I 107 85 25 0 47 88 41 22 28

60 56 61 20 82

97

D.M. W. Anderson , J.F.Howlett and C.G.A.McNab

testa from the endosperm had apparently been achieved prior to milling. Nevertheless it is of interest that its amino acid content, for all amino acids other than glutamic acid, histidine and hydroxyproline, lies within the ranges established (2) for guar gum. The similarity in properties of guar and Sesbania gums has been reported (9,10); increasing supplies of the latter are becoming available as a result of the publicity given (22) to the advantages of Sesbania spp. as underdeveloped resources of economic potential. Konjac mannan has a distinctive amino acid profile by virtue of its very high aspartic acid content and high levels of glutamic acid, alanine and serine. Locust bean gum has an exceptionally high content of glutamic acid, with aspartic acid and glycine as the next most abundant amino acids. Clearly, however, there are similarities between the amino acid profiles of the galactomannans viz. locust bean gum, tara gum, Sesbania gum and guar, which have mannose/galactose ratios approximating to 4/1, 3/1, 3/1 and 2/1 respectively. All four gums have glutamic acid, glycine and aspartic acid as their main amino acid components; tara gum and Sesbania gum also contain characteristically high proportions of alanine and histidine respectively. The relative proportions of all of the other amino acids are remarkably similar. This is of interest because these galactomannans are derived from botanical species placed within different sub-families of Family Leguminosae. Table II shows that the xanthan samples studied, of American and European origins, had closely similar amino acid compositions, the major amino acids being alanine, glutamic acid and aspartic acid. The low proportion of methionine is, nevertheless, higher than found in exudate gums (1,21) and the other gums studied here. All of the gums studied here have low hydroxyproline contents (0-11 residues per 1000 residues). Recent publications have shown that the exudate gums arabic, tragacanth (23), mesquite (24) and many Acacia gums (25) contain very large proportions of hydroxyproline (up to 425 residues per 1000 amino acid residues). In contrast gum karaya (1) and other tree exudates (26) contain < 30 hydroxyproline residues per 1000. It is becoming apparent that a major distinction can be made between gums containing very high or very low proportions of hydroxyproline; it is suspected that the hydroxyproline content may be of structural significance in the former (21,23,25).

Acknowledgements We thank members of the International Natural Gums Association for Research Ltd (INGAR) for providing commercial samples for analysis. This study was funded by the Food Science Division of the UK Ministry of Agriculture, Fisheries and Food. The authors acknowledge permission from the Ministry of Agriculture, Fisheries and Food to publish this paper.

References I. Anderson,D.M.W., Howlett,J.F. and McNab,C.G.A. (1986) Food Additives Contaminants, 2, 159-164. 2. Anderson,D.M.W., Howlett,J.F. and McNab,e.G.A. (1986) Food Additives Contaminants, 2, 225-230. 3. Council Directive of25 July 1978 (75/663/EEC), Official J. Eur. Commun., No. L 223/7, 14 August 1978. 4. JECFA (1983) Food and Nutrition paper No. 28. Specifications for Identity and Purity. FAG, Rome, pp. 22-24. 5. JECFA (1980) Food and Nutrition Paper No. 17. Specifications for Identity and Purity. FAG, Rome, pp. 26-27.

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Proteinaceous components of Konjac mannan

6. WHO (1981) Technical Report Series No. 669. Evaluation of Certain Food Additives. Geneva, p. 28. 7. JECFA (1984) Food and Nutrition paper No. 3112. Specifications for Identity and Purity. FAO, Rome, pp. 123-124. 8. WHO (1984) Technical Report Series No. 710. Evaluation of Certain Food Additives. Geneva, p. 21. 9. Kapoor,V.P. and Farooqi,M.I.H. (1979) Res. Ind., 24,167. 10. Chandra, V. and Farooqi,M.I.H. (1979) Extension Bulletin No. I. National Botanical Research Institute, Lucknow. 11. Dea,I.C.M., Morris,E.R., Rees,D.A., We1sh,EJ., Barnes,H.A. and Price,J. (1977) Carbohydr. Res., 57, 333-356. 12. Wielinga,W.e. (1984) in Phillips,G.O., Wedlock,DJ. and Williams,P.A. (eds.), Gums and Stabilisers for the Food Industry. Pergamon Press, Oxford, pp. 251- 276. 13. McCleary,B.V., Dea,I.C.M. and C1ark,A.H. (1984) In Phillips,G.O., Wedlock,D.J. and Williams,P.A. (eds), Gums and Stabilisers for the Food Industry. Pergamon Press, Oxford, pp. 33-44. 14. Bose.S, and Gupta,A.K. (1984) In Carbohydrates 1984. Abstracts of the 12th Int. Carbohyd. Symp., Utrecht, p. 398. 15. Pettit,DJ. (1982) In Glicksman.M. (ed.), Food Hydrocolloids. CRC Press, Boca Raton, FL, Vol. 1, pp. 127-149. 16. Council Directive of29 May 1980 (80/597/EEC), Official J. Eur. Commun., No. L 155/23, 23 June 1980. 17. WHO (1974) Technical Report Series No. 557. Evaluation of Certain Food Additives. Geneva, p. 21. 18. WHO (1978) Technical Report Series No. 617. Evaluation of Certain Food Additives. Geneva, p. 31. 19. Council Directive of 12 July 1982 (82/504/EEC), Offic. J. Eur. Commun., No. L 230/35,5 August 1982. 20. FAO (1978) Food and Nutrition Paper No.4. Specifications of 1dentity and Purity. Rome, pp. 56-59. 21. Anderson,D.M.W., Howlett,J.F. and McNab,C.G.A. (1986) Food Additives Contaminants, 2, 153-157. 22. US National Academy of Sciences (1979) Tropical Legumes: Resources for the Future. pp. 287 -289. 23. Anderson,D.M.W .. Howlett,J.F. and McNab,e.G.A. (1986) Food Additives Contaminants. 2. 231-235. 24. Anderson.D.M.W., Howlett,J.F. and McNab.C.G.A. (1986) Phytochemistry, 24. 2718-2720. 25. Anderson,D.M.W. and McDougall,F.J. (1986) Phytochemistry, 24,1237-1240. 26. Anderson,D.M.W., Bell,P.C., om.v.c.t.. McNab,C.G.A. and McDougall,FJ. (1986) Phytochemistry, 25,247-249. Received on /1 July 1985; accepted on 23 August 1985

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