Comparative study of the chemical composition of germ meals from carob, guar and tara seeds

Comparative study of the chemical composition of germ meals from carob, guar and tara seeds

Food Hydrocolloids YoU no.2 pp. 149-156, 1989 Comparative study of the chemical composition of germ meals from carob, guar and tara seeds Bethy L.Del...

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Food Hydrocolloids YoU no.2 pp. 149-156, 1989

Comparative study of the chemical composition of germ meals from carob, guar and tara seeds Bethy L.Del Re-Jimenez and Renato Amado! Swiss Federal Institute of Technology, Department of Food Science, ETH-Zentrum, CH-8092 Zurich, Switzerland ITo whom all correspondence should be addressed Abstract. The processing of the seeds from carob (Ceratonia siliquai, guar (Cvamopsis tetragonoloba) and tara (Caesalpinia spinosa) plants to yield the corresponding endosperm involves the removal of the germ fraction. Whereas the ground endosperms arc commercially important thickening agents, the germs arc recovered as a by-product and are mainly used as animal feed or as a source for protein hydrolysates. The present investigation was undertaken in order to characterize the germ fractions of the three Leguminosac seeds. The results obtained indicate that the dry matter as well as the ash content of the three germ meals arc similar. On the other hand, differences were found in the lipid content, the protein content and the amino acid composition. Isoelectrie focusing and SDS gel electrophoresis of the water-soluble protein fractions showed considerable variations in the protein composition. The determination of the isoeleetric points as well as the molecular weights of the main protein bands could be used for the identification of carob, guar and tara germ meals.

Introduction

The seeds of the Leguminosae carob (Ceratonia siliqua L.), guar (Cyamopsis tetragonoloba L.) and tara (Caesalpinia spinosa L.) are used as starting material for the preparation of commercially important gelling- and thickening agents (1-3). The processing of the seeds to yield the corresponding gums, all belonging to the galactomannans, from the endosperm involves the removal of the hulls and of the germ fractions. The germs are recovered as by-products during galactomannan production and are mainly used, after milling and heat treatment, as animal feed or as a source for hydrolyzed proteins (4). Relatively little is known about the chemical composition of the germs of legumes other than that they are rich in proteins and lipids. Studies on the protein fraction of guar germs have been published (5,6). Furthermore, evidence for the presence of anti-nutritive compounds such as trypsin-inhibitors in guar and carob germs (7-9) as well as saponins in the guar lipid fraction (10) has been presented. The aim of the present investigation was the chemical characterization of guar, tara and carob germ meals. Particular attention was given to the protein fraction, which was analyzed by means of electrophoretic methods. Materials and methods

Raw materials

Germ meals of carob, originating from Spain and Morocco, and guar were obtained from Polygal AG (Marstctten, Switzerland). The germ meal of carob had been isolated by acid treatment of the seeds and removal of the endosperm.

© IRL Press

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Guar germ meal on the other hand was isolated from the kernels by mechanical treatment. Tara seeds were obtained from Unipektin AG (Eschenz, Switzerland). The germ fraction was separated from the hull and endosperm by hand. By this procedure a pure germ meal was obtained, whereas the germ meals of carob and guar , which were obtained by using technological procedures, were slightly contaminated with endosperm. Chemical methods

The moisture content of the germ meals was determined gravimetrically after heating the material (1 g) in an oven at 105°e for 6 h to constant weight. Lipids were extracted from the germ meals (1 g) in a Soxhlet extractor for 6 h (8-10 syphon cycles/h), using petroleum ether (boiling range: 40-60°C). The solvent was removed by evaporation at 70-80o e, and the extracted lipids were heated at 95 ± 2°e in an oven to constant weight and determined gravimetrically. The ash content of the germ meals (2-3 g) was determined by dry mineralization at 480 e for 1 h. The crude protein content was estimated by the Kjeldahl procedure, using a Kjeltec-System I (Tecator AB, Hoganas, Sweden) for mineralization and distillation. The amino acid composition of the germ meals was determined after hydrolysis in a nitrogen atmosphere with 6 N hydrochloric acid at 110 e for 24 h and automated amino acid analysis on a Liquimat III amino acid analyzer (Kontron AG, Zurich, Switzerland) using a four-buffer, single column system as described by Amado et al. (11). 0

0

Isolation of water-soluble proteins

The water-soluble proteins of the germ meals were extracted with distilled water at room temperature. The slurry (10 mg meal/ml water) was shaken for 30 min and the mixture centrifuged at 2500 g in an MSE-Minor centrifuge (Zivy AG, Oberwil, Switzerland). The supernatant was freeze-dried and the yield of watersoluble proteins determined gravimetrically. Electrophoretic analyses

Isoelectric focusing of the soluble proteins was performed according to the instructions given by the manufacturer (12) on an LKB-Multiphor system (LKB Produkter AB, Brornma, Sweden) using Ampholine PAG plates, pH range 3.5-9.5, as support material. The protein bands were stained with Coomassie Brilliant Blue R 250 (Fluka AG, Buchs, Switzerland). SDS gel electrophoresis of the soluble proteins was performed according to the manufacturer's instructions (13), using a Phast system (Pharmacia AB, Uppsala, Sweden). A set of marker proteins with molecular weights in the range of 14 000 to 94000 daltons (electrophoresis calibration kit; Pharmacia AB, Uppsala, Sweden) was used for determination of the molecular weights of the 150

Chemical composition of Leguminosae germ meals

germ meal proteins. The protein bands were stained with Phast Gel Blue R (Coomassie Brilliant Blue R 250) soluble tablets. Results and Discussion

Table I shows the results of the fractionation of the tara seeds into hull, endosperm and germ fraction. These results agree well with data published by Rahanitriniana et at. (14). For comparison, reference values for carob and guar are included in the table. The figures clearly indicate that guar seeds have by far the thinnest hull and the largest germ fraction. On the other hand, tara seeds show the lowest and carob seeds the highest endosperm content. It must be kept in mind that within the different Leguminosae some varietal differences are observed. The overall chemical compositions of the different germ meals are shown in Table II. The moisture and ash contents are similar, whereas large differences can be observed in the lipid content. Carob germs show an unexpectedly low amount of lipids compared to guar and tara germs. As already pointed out, it is known that guar germs contain large amounts of anti-nutritive saponins in their lipid fraction (10). The solvent used in the present work (petroleum ether) only extracts neutral lipids; the figures given in Table II therefore represent the neutral lipid content of the germ meals. Further work, using polar solvents like chloroform/methanol for extraction of the polar lipids, should be done in order to establish the total lipid content of the germs. An accurate analysis of the total lipid fraction will then show if the observed differences are due to a variability of the triglyceride content or if other lipids (free fatty acids, phosphatides, carotenoids, alkaloids) are present in different proportions in the germs. With Table I. Portions of hull, endosperm and germ of tara, carob and guar seeds (in % of fresh wt)

Hull Endosperm Germ

Tara

Carob (1)

Guar (2)

Tara (14)

38 30 32

30-38 42-46 23-25

14-17 35-40 42-47

38-40 30-34 27-30

Table II. Overall composition of germ meals from carob, guar and tara (all figures in % of fresh wt) Carob Morocco

Spain

Guar

Tara

Moisture Lipids (neutral) Ash Crude protein Carbohydrate (by difference)"

6.83 7.98 5.78 52.99 26.42

6.80 8.05 6.59 51.68 26.88

7.63 16.25 5.31 60.08 10.73

6.24 13.93 6.38 54.32 19.13

Water-soluble proteins

28.85

30.54

37.00

ndb

The values given represent the means of triplicate analyses. apolar lipids included. bnd, not determined.

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respect to the carbohydrate contents given in Table II, it must be pointed out that these values have been calculated by difference. Since the polar lipids have not been extracted, it could be that part of what has been assumed to be carbohydrate material in fact is polar lipid. The protein contents of all germs under investigation are high. It is generally thought that the proteinaceous components of seeds function as storage material which on germination is rapidly hydrolyzed to provide a source of reduced nitrogen for the early stages of seedling growth. Besides this, proteins which are essential for the maintenance of normal cell metabolism are also present in the seeds in minor amounts (15). Evidence for the presence of enzymes in germinating seeds (e.g. galactosidases and mannanases) has been given for guar seeds (16,17). Since these enzymes have been isolated from germinating seeds, it may be assumed that they are already present in the dormant seed. It cannot, however, be excluded that the enzymes are synthesized de novo or activated in an unknown manner (zymogenenzyme transformation?) during the first stages of germination. In the present work the potential presence of enzyme activities in the protein fraction of the germs has not been considered. More than 50% of the proteins of tara, carob and guar germs are water-soluble and can be extracted from the germ by a very simple procedure. Because of the high protein content, a potential use of the germs as a protein source for human or animal nutrition is therefore evident. However, it must be kept in mind that Leguminosae seeds are known to contain anti-nutritive substances such as trypsin inhibitors (9); use of germ meals from guar, carob and tara is therefore only possible after treatment (e.g. toasting) to inactivate the toxic components. A possible method for the detoxification of guar germ meal has been described by Khopkar and Rege (18). The amino acid composition of the germ meals is shown in Table III. Only slight differences between the three germs can be observed. From the nutritional point of view, the amount of essential amino acids is very interesting. The essential amino acid content of the germ meals is compared to a FAO/WHO standard protein (19) in Table IV. The results clearly indicate that in carob and guar germ meals methionine and cysteine are the limiting amino acids, whereas in tara germ meal proteins lysine is the limiting amino acid. The chemical score, a measure of the protein quality, is lowest for the carob germ meal of Spanish provenance, tara germ meal giving the highest figure and therefore having the highest nutritional quality. In combination with wheat flour, for example, in which lysine is the limiting amino acid, germ meals of guar, carob and especially tara could be used for improving the protein quality of the former. It is interesting to note the large differences in the chemical score between the two carob germ meals of different origin. This result could be explained by a natural variability of the carob germs under investigation or by an oxidative loss of cysteine and/or methionine during the acid hydrolysis of the protein of the carob germ meal originating from Spain. The pattern of the water-soluble proteins after isoelectric focusing in a pH range of 3.5-9.5 is shown in Figure 1. As expected, the two carob proteins of different origin show a practically identical protein pattern. Guar and tara proteins, on the other hand, differ significantly from the carob proteins. All 152

Chemical composition of Legumlnosae germ meals

Table III. Amino acid composition of carob, guar and tara germ meals (in g amino acid/IOO g of germ meal) Amino acid

Asp + Asn Thr Ser Glu + Gin Pro Gly Ala Cys Val Met He Leu Tyr Phe Lys His Arg

Carob Morocco

Spain

4.01 1.91 2.66 13.34 2.03 2.49 2.18 0.30 1.96 0.72 1.55 3.19 1.40 1.60 2.96 1.38 6.37

3.74 1.73 2.47 12.60 1.99 2.32 2.07 0.25 1.84 0.34 1.44 2.99 1.20 1.48 2.96 1.12 5.80

Guar

Tara

6.27 2.38 3.71 12.16 2.76 3.26 2.51 0.33 2.20 0.94 1.92 3.84 1.76 2.52 2.91 1.73 8.86

4.53 2.07 2.64 1162 2.52 2.85 2.51 0.54 2.54 1.30 1.93 3.84 1.29 1.69 2.18 1.58 8.77

Trp was not determined. The values represent the means of duplicate analyses, the maximal relative deviation between two measurements being ±3%. Table IV. Content of essential amino acids in gcrm meals of carob, guar and tara (in g amino acid/16 g ~') Guar

Tara

FAO/WHO standard protein

3.34 3.56 1.16 2.79 5.78 5.18 5.73

3.97 3.66 2.11 3.20 6.39 7.13 4.84

3.81 4.68 3.39 3.55 7.07 5.49 4.01

4.0 5.0 3.5 4.0 7.0 6.0 5.5

0.33 Met + Cys

0.60 Met + Cys

0.73 Lys

1.0

Carob Morocco

Spain

Thr Val Met + Cys lie Leu Phe + Tyr Lys

3.60 3.69 1.91 2.92 6.03 5.66 5.58

Chern. score Limiting amino acid

0.55 Met + Cys

Amino acid

Trp was not determined. 'Calculated from crude protein content.

samples show three main protein bands with isoelectric points between pH 5.2 and 5.5. In addition, carob shows a few protein bands with isoelectric points in the more alkaline region which are not present in guar. Tara lacks the more acidic protein bands observed in guar as well as in carob in the pH range 4.75-4.85. Finally, carob contains a protein with an isoelcctric point of -5.85 which is not present in the two other samples. The results presented clearly 153

H.L.Del Re-Jtmenez and R.Amado

..

1

2

3

pH 3.5

..

..._pH 9.5

4

Fig. 1. Isoelcctric focusing of the water-soluble proteins of carob. guar and tara germ meals. (1) Carob (Morocco); (2) carob (Spain); (3) guar; (4) tara.

indicate that by using isoelectric focusing of the water-soluble protein fraction it is possible to distinguish between carob, guar and tara germs. SDS gel electrophoresis was performed in order to determine the molecular weight distribution of the water-soluble proteins of the germs. The results are presented in Figure 2. All samples showed a number of similar low-molecularweight proteins (in the region of 14 000 daltons), but here again, significant differences between the proteins of carob, guar and tara germs were demonstrated. The main proteins of carob have molecular weights of 91 000, 51 000 and 25 000 daltons. The main proteins of guar have molecular weights of 35 000, 45000, 57000 and 63000 daltons. Finally, the main tara proteins have molecular weights of 31 000 and 57 000 daltons. The results obtained by SOS gel electrophoresis are in good agreement with the isoelectric focusing experiments, confirming the possible use of electrophoretic methods for the differentiation of the three germ meals under investigation. In conclusion, it can be summarized that the germ meals of carob, guar and tara seeds differ considerably in their chemical composition, the lipid fraction showing the highest variability. The germs of all three leguminous plants are rich in proteins and could be used, after heat treatment, as protein sources for human and animal nutrition. The pattern of the water-soluble proteins is quite different, thus allowing an unequivocal differentiation of the three legumes. 154

Chemical composition of Leguminosae germ meals

Marker proteins (Mw in kDalton)

.....-14.4

.....- 20.1

.....-.43 .....-.67 .--94

1

2

3

Fig. 2. Eva luat ion of the molecular weights of the water-soluble pro teins of carob , guar and tara germ meals by SDS gel electrophoresis . (1) Carob (Morocco); (2) guar: (3) tara .

Ackno wledgements Th is work was fin anci ally sup porte d in part by a stipend of th e Swiss Government (B. D -J) . Prof.N eukorn 's inte res t and helpful discussion s as well as M.Fischer's he lp in pr eparing th e manuscript ar e gratefully ac know ledged.

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Vo!. III , pp. 161-170. 2. Her ald ,C.T. (1986) In Glicksman.M, (ed .), Food Hydrocolloids. CRC Press. Boca Raton. FL, Vol. III , pp . 171-184. 3. Glieksman ,M. (1986) In Glicksrnan.M. (ed .), Food Hydrocolloids. CRC Press, Boca Raton, FL, Vol. III , pp. 185-1 89. 4. Nittn er,E . (1980) In Ncukorn.H. and Pilnik ,W. (eds), Gelier- und Verdickungsmiuel in Lebensm itteln. Forster Publishers, Zurich , pp. 95-1 12. 5. Nath,J.P. , Subra manian.N. and Narasinga Rao ,M.S. (1978) J. Agric. Food Chem ., 26, 1243-1246. 6. Nat h,J .r. , Subramanian .N, and Narasinga Rao .M.S. (1980) J. Agric. Food Chem .• 28, 844-847. 7. Belitz,H .-D . . Lyncn,F. and Weder.K. P .J . (1982) Z . Lebens m . Villers. Forsch .• 174, 442-446. 8. Rajput ,L.P., Murp hy,K .N . and Ram amami ,S. (1987) 1. Food Sci. , 52,1 755- 1757. 9. Wede r,J.K .P . (1986) In Fr iedman ,M. (cd .), Advances in Experimental Medicine and Biology ,

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Vol. 199. Nutritional and Toxicological Significance of Enzyme Inhibitors in Foods. Plenum Press, New York, pp. 239-279. 10. Coxon,D.T. and Wells,J.W. (1980) Phytochemistry, 19, 1247-1248. 11. Amado.R., Rothcnbuhler.E.; Arrigoni,E. and Solms,J. (1983) Mitt. Geb. Lebensm. Hyg., 74, 23-30. 12. Analytical electrofocusing with thin-layers of polyacrylamide gels. Application Note 250, LKB Produktcr AB, Bromma. 13. Phast-system separation technique nr. 110. Laboratory Separation Division, Pharmacia AB, Uppsala. 14. Rahanitriniana,D., Artaud,J., Iatride,M.C. and Gaydou,E.M. (1984) Rev. Franc. Corps Gras, 31,249-252. 15. Higgins,T.J.Y. (1984) Ann. Rev. Plant Physiol., 35,191-221. 16. McCleary,B.Y. and Matheson,N.K. (1974) Phytochemistry, 13, 1747-1757. 17. Shivanna,B.D. and Ramakrishna,M. (1985) J. Biosci., 9,109-116. 18. Khopkar,P.P. and Rcge,D.Y. (1984) Qual. Plant. Foods Hum. Nutr., 34, 135-139. 19. FAO/WHO (1973) Energy and protein requirements, Report of a joint FAO/WHO ad hoc expert committee, WHO Techn. Rep. Ser. 522, WHO, Geneva. Received on February 13, 1989; accepted on April 3, 1989

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