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Changes in non-structural carbohydrates in developing fruit of Myrciaria jaboticaba
SCIENTIA HORTICuLTuM ELSEVIER
Scientia Horticulturae 66 (1996) 209-215
Changes in non-structural carbohydrates in developing fruit of Myrciaria jaboticaba Raimundo S. Barros a**,Fernando L. Finger b, Marcel0 M. MagalhZes a aDepartamento de Biologia Vegetal, Universidade Federal de Vicosa, 36571400, b Deparfamento de Fitotecnia, Universidade Federal de Vigosa, 36.571400,
Vicosa, MG, Brazil Vicosa, MG, Brazil
Accepted 25 March 1996
Abstract Reducing sugars represented the main non-structural carbohydrate of fruit of Myrciaria juboricubuowing to the high content found in pulp, which also accumulated most of the non-reducing sugars. The contribution of the skin to the amount of total sugar in the berry was negligible. Starch was relatively low in the pulp but more abundant in the seed. Keywords: Jaboticaba; Non-structural
carbohydrates;
Pulp; Reducing
sugars; Seed; Skin; Starch
1. Introduction
Most species of jaboticaba are distributed within the genus Myrciuria, of the family Myrtaceae. They are multi-stemmed, short trees, exhibiting cauliflory, in which the flower cushions and berries are borne directly on the main trunk and old branches. Berries grow rapidly, completing their development within 45-60 days (Andersen and Andersen, 1988). The fruiting season is short; fruit decay and fermentation being observed within 2-3 days of harvesting. The berries are globoid, 20-30 mm in diameter, and red to purple or blackish when ripe. The pulp, the largest component of ripe berries, is whitish and its taste varies from slightly acidic to very sweet. Owing to its flavour, jaboticaba fruit is often consumed fresh; the berries are also used in the production of home-made liquors and jams (Wiltbank et al., 1983). Despite these and potential future uses, the various phases of fruit growth in jaboticaba have been described only recently (MagalhZes et al., 1996). The mineral and organic
* Corresponding
author.
03044238/%/$15.00 Copyright P/I SO304-4238(96)00910-7
0 1996 Elsevier Science B.V. All rights reserved.
210
R.S. Barros et al./ Scientia Horticulturae 66 (1996) 209-215
composition of fruits have not yet been reported, and so their nutritional value is not known. In the present study, changes in the content of reducing sugars, total sugars and starch in the berry, and components thereof, of Myrciaria juboricuba Berg were analysed throughout development. 2. Materials and methods Out-of-season fruits were periodically harvested from mid June to mid August 1988, from field-grown trees in Visosa, Minas Gerais State, Brazil. They were placed in stoppered glass vials that were immersed in an ice and NaCl mixture before being taken to the laboratory for the determination of growth parameters and pigment levels. Fruits and their components were stored at - 20°C until analysis. Four samples, each of 10 g fresh weight, were homogenised in 30 ml of hot 80% ethanol and centrifuged at 2000 x g for 15 min. The pellet was then re-extracted twice as above, and the ethanol removed under vacuum at 45°C. The aqueous phase was clarified with solid lead acetate and excess lead was removed with sodium acetate. Aliquots from clarified extracts were then taken for quantification of reducing sugars by Somogy-Nelson’s method and total soluble sugars using the anthrone reaction (Hodge and Hofreiter, 1962).
F” - __m-In
e
n-
1
-3
I
3. 2
2 400
-2
Ec
ZI 200 n
-I
E 3
.g I
Y E
8
I
75-
5 E 3 =
50-
2
25
z 0 5
o-I
15
Days
after
blossom
Fig. 1.Dry matter accumulation, volume changes and pigment contents in jaboticaba fruit. Four samples of 50 berries each were averaged to constitute a point for dry matter and volume determinations. Four samples were taken for pigment determination. In this and subsequent figures standard errors were smaller than symbols.
R.S. Barros et al./Scientia
Horticulturae
66 (1996) 209-215
211
Starch analysis was carried out as described by Hubber and Nevins (19771, with modifications. The pellet from the ethanolic extractions was washed twice with distilled water and boiled in 10 ml of 0.1 M KOH for 15 min to facilitate starch extraction. After cooling, the pH was adjusted to 6.9 with acetic acid, and 20 ml of 50 mM phosphate buffer, containing 10 mM Cl-, added. This was then incubated with 12 or 24 units of ol-amylase (EC 3.2.1.1, Sigma) at 40°C for 24 h, with stirring. The reaction was terminated by boiling and the mixture centrifuged at 1200 X g for 15 min. The pellet was extracted twice more with hot water and aliquots of the supernatants from three centrifugations taken for the anthrone reaction. Identical results were obtained when samples-were extracted as described by McCready et al. (1950). 3. Results and discussion During the initial stages of berry development, dry matter accumulation was low, increasing from about 20 days after blossom (DAB). Fruit size, however, increased
250
-
? -2 = 1502 Cn E
T
50-
250-
5
15
25
35
Days after
45
55
65
blossom
Fig. 2. Time course of accumulation and relative content, on a DW basis, of non-structural jaboticaba fruit. TS, total sugars; RS, reducing sugars; ST, starch.
carbohydrates in
212
R.S. Barros et al./ Scientia Horticulturae 66 (1996) 209-215
40
45
50
Days after
55
60
65
blossom
Fig. 3. Time course of accumulation and relative content, on a DW basis, of non-structural pulp of jaboticaba fruit. TS, total sugars; RS, reducing sugars; ST, starch.
carbohydrates
in
much later, but reached its maximum earlier (Fig. 1). During ripening, when the chlorophyll content fell dramatically and anthocyanins accumulated in the skin, dry matter was still increasing. Most of the dry matter was partitioned into the pulp, with a distribution ratio of 0.5; for skin and seeds the ratios were 0.3 and 0.2, respectively. On a fresh weight basis, the proportion of pulp was even larger. The accumulation of non-structural carbohydrates increased slowly from 30 DAB (Fig. 2). Substantial increments coincided with a marked increase in volume (Fig. 1). By 50 DAB, fruit size was maximal, but as with dry matter, soluble sugars continued to accumulate until near the end of fruit development. Fig. 2 also shows that the increase in total sugar was mainly due to an increase in concentration. The decreases in the total amount and concentration of sugars at the end of fruit development were probably associated with the final stages of ripening. Similar patterns of sugar accumulation have been reported for grape berry (Nii and Coombe, 1983), apple (Beirtiter, 1985) and mango (Laksminarayana et al., 1970). The high amounts of soluble sugars in the berry were almost entirely due to the sugar content of the pulp (Fig. 3), as also observed for grape (Nii and Coombe, 1983). The sugar content of the pulp of ripe berries was about 600 g kg-’ dry weight (DW), 490 g kg-’ being reducing sugars (Fig. 3). At this stage, the amount of sugars in pulp was over 7- and 27-fold higher than in skin and seed, respectively. The patterns of sugar accumulation in the pulp and skin were similar (Figs. 2 and 4), but this trend was not
R.S. Barros et al./ Scientia Horticulturae 66 (19%) 209-215
213
4orr
0 -’ 40
45
50
55
60
Days
after
blossom
65
Fig. 4. Time course of accumulation and relative content, on a DW basis, of non-structural skin of jaboticaba fruit. TS, total sugars; RS, reducing sugars; ST, starch.
carbohydrates
in
observed in seed (Fig. 5). In both the skin and seed of ripe berries, the amount of reducing sugars was higher than that of non-reducing sugars. In berries, the starch content remained low throughout fruit development (Fig. 2). After an initial increase, the total amount remained constant from 47 DAB onwards. Rapid fruit growth resulted in a decrease in the percentage of starch from that time until ripening, a situation also observed for apple (Beirliter, 1985) and banana (Marriott, 1980; Stover and Simonds, 1987). The starch content of pulp was low, the seeds containing the highest amounts (Figs. 3-5). Maximal amounts of starch in the berry were attained by 47 DAB (Fig. 21, owing to increases in the skin content (Fig. 4); these amounts were subsequently maintained owing to increased seed contents (Fig. 5). Quantitative relationships among non-structural carbohydrates in jaboticaba berry are depicted in Fig. 6. Reducing sugars were by far the dominant fraction throughout berry development (Fig. 6). From 41 DAB at least, this was due to storage of sugars in the pulp (Fig. 3). The non-reducing sugar fraction was considerable in the berry (Fig. 6), owing to the amounts stored in skin (Fig. 4) and pulp (Fig. 3). In seed it constituted a minor fraction, being detected only from 47 DAB onwards (Fig. 5). The starch fraction in fruit increased slightly until 41 DAB (Fig. 61, and represented a large reserve in the seed (Fig. 51, likely to be used during germination. Owing to small seed size (Magalhks et al., 1996) and high sugar content in the pulp (Fig. 31, starch contribution to total non-structural carbohydrate fell after 41 DAB (Fig. 6).
214
R.S. Barros et al./Scientia
40
Horticulturae 46 (1996) 209-215
45
50
55
Days
after
blossom
60
65
Fig. 5. Time course of accumulation and relative content, on a DW basis, of non-structural carbohydrates in seed of jaboticaba fruit. TS, total sugars; RS, reducing sugars; ST, starch.
0.8 i? t
II
0.6
.E 2 .s u e IL
0.4
0.2
0 5
15
25
Days Fig. 6. Changes in fractions of each non-structural jaboticaba
fruits.
NR, non-reducing
reducing sugars; ST, starch.
sugars,
35
after
45
55
65
blossom
carbohydrate group as related to their total amounts (1 .O) in estimated from difference of total and reducing sugars; RS,
R.S. Barros et al./ Scientia Horticuburae
66 (1996) 209-215
215
In summary, most of the increase in the content of non-structural carbohydrates occurred when volume growth began and immediately before the maximal rates of dry matter accumulation (Figs. 1 and 2). When fruit growth reached its maximum, carbohydrate accumulation was accelerated; soluble sugars were accumulated even after full berry size was reached. Storage of reducing sugars in the pulp (Fig. 31, which may be related to fruit dispersion by animals, is also one of the reasons underlying liquor preparation and fruit consumption in natura. Excess sugar in the berry could also be associated with the rapid decay and fermentation, and thus to the very short postharvest shelf-life of the fruit.
Acknowledgements CAPES and CNPq are gratefully acknowledged for the fellowships granted to MMM and RSB, and FINEP for financial support during the execution of this experiment.
References Andersen, 0. and Andersen, V.U., 1988. As Fruteiras Silvestres Brasileiras, 2nd edn. Editora Globo, Rio de Janeiro, 203 pp. Beiriiter, J., 1985. Sugar accumulation and changes in the activities of related enzymes during development of apple fruit. J. Plant Physiol., 121: 331-341. Hodge, J.E. and Hofreiter, B.R., 1962. Determination of reducing sugars and carbohydrates. In: R.L. Wilster and M.L. Wolfram (Editors), Methods in Carbohydrate Chemistry, Vol. 1. Academic Press, New York, pp. 380-394. Hubber, D.J. and Nevins, D.J., 1977. Preparation and properties of B-D-glucanase for the specific hydrolysis of B-D-ghtcans. Plant Physiol., 60: 300-304. Laksminarayana, S., Subhadra, M.V. and Subramanyan, H., 1970. Some aspects of the developmental physiology of mango fruit. J. Hortic. Sci., 45: 133-142. MagalhHes, M.M., Barros, R.S. and Lopes, N.F., 1996. Growth relations and pigment changes in developing fruits of Myrciaria jaboticaba. J. Hortic. Sci., in press. Marriott, J., 1980. Bananas: physiology and biochemistry of storage and ripening for optimum quality. Crit. Rev. Food Sci. Nutr., 13: 41-88. McCready, R.M.J., Guggolz, J., Silveira, V. and Owens, H.H., 1950. Determination of starch and amylose in vegetables. Anal. Chem., 22: 1156-l 158. Nii, N. and Coombe, B.G., 1983. Structure and development of the berry and pedicel of the grape Vitis uinifera L. Acta Hortic., 139: 129- 140. Stover, R.H. and Simonds, N.W., 1987. Bananas, 3rd edn. Longman Scientific and Technical, New York, 468 PP. Wiltbank, W.J., Chalfun, N.N.J. and Andersen, Sci., Tropical Region, 27A: 57-69.
O., 1983. The jaboticaba
in Brazil. Proc. Am. Sot. Hortic.
Scientia Horticulturae 66 (1996) 209-215
Changes in non-structural carbohydrates in developing fruit of Myrciaria jaboticaba Raimundo S. Barros a**,Fernando L. Finger b, Marcel0 M. MagalhZes a aDepartamento de Biologia Vegetal, Universidade Federal de Vicosa, 36571400, b Deparfamento de Fitotecnia, Universidade Federal de Vigosa, 36.571400,
Vicosa, MG, Brazil Vicosa, MG, Brazil
Accepted 25 March 1996
Abstract Reducing sugars represented the main non-structural carbohydrate of fruit of Myrciaria juboricubuowing to the high content found in pulp, which also accumulated most of the non-reducing sugars. The contribution of the skin to the amount of total sugar in the berry was negligible. Starch was relatively low in the pulp but more abundant in the seed. Keywords: Jaboticaba; Non-structural
carbohydrates;
Pulp; Reducing
sugars; Seed; Skin; Starch
1. Introduction
Most species of jaboticaba are distributed within the genus Myrciuria, of the family Myrtaceae. They are multi-stemmed, short trees, exhibiting cauliflory, in which the flower cushions and berries are borne directly on the main trunk and old branches. Berries grow rapidly, completing their development within 45-60 days (Andersen and Andersen, 1988). The fruiting season is short; fruit decay and fermentation being observed within 2-3 days of harvesting. The berries are globoid, 20-30 mm in diameter, and red to purple or blackish when ripe. The pulp, the largest component of ripe berries, is whitish and its taste varies from slightly acidic to very sweet. Owing to its flavour, jaboticaba fruit is often consumed fresh; the berries are also used in the production of home-made liquors and jams (Wiltbank et al., 1983). Despite these and potential future uses, the various phases of fruit growth in jaboticaba have been described only recently (MagalhZes et al., 1996). The mineral and organic
* Corresponding
author.
03044238/%/$15.00 Copyright P/I SO304-4238(96)00910-7
0 1996 Elsevier Science B.V. All rights reserved.
210
R.S. Barros et al./ Scientia Horticulturae 66 (1996) 209-215
composition of fruits have not yet been reported, and so their nutritional value is not known. In the present study, changes in the content of reducing sugars, total sugars and starch in the berry, and components thereof, of Myrciaria juboricuba Berg were analysed throughout development. 2. Materials and methods Out-of-season fruits were periodically harvested from mid June to mid August 1988, from field-grown trees in Visosa, Minas Gerais State, Brazil. They were placed in stoppered glass vials that were immersed in an ice and NaCl mixture before being taken to the laboratory for the determination of growth parameters and pigment levels. Fruits and their components were stored at - 20°C until analysis. Four samples, each of 10 g fresh weight, were homogenised in 30 ml of hot 80% ethanol and centrifuged at 2000 x g for 15 min. The pellet was then re-extracted twice as above, and the ethanol removed under vacuum at 45°C. The aqueous phase was clarified with solid lead acetate and excess lead was removed with sodium acetate. Aliquots from clarified extracts were then taken for quantification of reducing sugars by Somogy-Nelson’s method and total soluble sugars using the anthrone reaction (Hodge and Hofreiter, 1962).
F” - __m-In
e
n-
1
-3
I
3. 2
2 400
-2
Ec
ZI 200 n
-I
E 3
.g I
Y E
8
I
75-
5 E 3 =
50-
2
25
z 0 5
o-I
15
Days
after
blossom
Fig. 1.Dry matter accumulation, volume changes and pigment contents in jaboticaba fruit. Four samples of 50 berries each were averaged to constitute a point for dry matter and volume determinations. Four samples were taken for pigment determination. In this and subsequent figures standard errors were smaller than symbols.
R.S. Barros et al./Scientia
Horticulturae
66 (1996) 209-215
211
Starch analysis was carried out as described by Hubber and Nevins (19771, with modifications. The pellet from the ethanolic extractions was washed twice with distilled water and boiled in 10 ml of 0.1 M KOH for 15 min to facilitate starch extraction. After cooling, the pH was adjusted to 6.9 with acetic acid, and 20 ml of 50 mM phosphate buffer, containing 10 mM Cl-, added. This was then incubated with 12 or 24 units of ol-amylase (EC 3.2.1.1, Sigma) at 40°C for 24 h, with stirring. The reaction was terminated by boiling and the mixture centrifuged at 1200 X g for 15 min. The pellet was extracted twice more with hot water and aliquots of the supernatants from three centrifugations taken for the anthrone reaction. Identical results were obtained when samples-were extracted as described by McCready et al. (1950). 3. Results and discussion During the initial stages of berry development, dry matter accumulation was low, increasing from about 20 days after blossom (DAB). Fruit size, however, increased
250
-
? -2 = 1502 Cn E
T
50-
250-
5
15
25
35
Days after
45
55
65
blossom
Fig. 2. Time course of accumulation and relative content, on a DW basis, of non-structural jaboticaba fruit. TS, total sugars; RS, reducing sugars; ST, starch.
carbohydrates in
212
R.S. Barros et al./ Scientia Horticulturae 66 (1996) 209-215
40
45
50
Days after
55
60
65
blossom
Fig. 3. Time course of accumulation and relative content, on a DW basis, of non-structural pulp of jaboticaba fruit. TS, total sugars; RS, reducing sugars; ST, starch.
carbohydrates
in
much later, but reached its maximum earlier (Fig. 1). During ripening, when the chlorophyll content fell dramatically and anthocyanins accumulated in the skin, dry matter was still increasing. Most of the dry matter was partitioned into the pulp, with a distribution ratio of 0.5; for skin and seeds the ratios were 0.3 and 0.2, respectively. On a fresh weight basis, the proportion of pulp was even larger. The accumulation of non-structural carbohydrates increased slowly from 30 DAB (Fig. 2). Substantial increments coincided with a marked increase in volume (Fig. 1). By 50 DAB, fruit size was maximal, but as with dry matter, soluble sugars continued to accumulate until near the end of fruit development. Fig. 2 also shows that the increase in total sugar was mainly due to an increase in concentration. The decreases in the total amount and concentration of sugars at the end of fruit development were probably associated with the final stages of ripening. Similar patterns of sugar accumulation have been reported for grape berry (Nii and Coombe, 1983), apple (Beirtiter, 1985) and mango (Laksminarayana et al., 1970). The high amounts of soluble sugars in the berry were almost entirely due to the sugar content of the pulp (Fig. 3), as also observed for grape (Nii and Coombe, 1983). The sugar content of the pulp of ripe berries was about 600 g kg-’ dry weight (DW), 490 g kg-’ being reducing sugars (Fig. 3). At this stage, the amount of sugars in pulp was over 7- and 27-fold higher than in skin and seed, respectively. The patterns of sugar accumulation in the pulp and skin were similar (Figs. 2 and 4), but this trend was not
R.S. Barros et al./ Scientia Horticulturae 66 (19%) 209-215
213
4orr
0 -’ 40
45
50
55
60
Days
after
blossom
65
Fig. 4. Time course of accumulation and relative content, on a DW basis, of non-structural skin of jaboticaba fruit. TS, total sugars; RS, reducing sugars; ST, starch.
carbohydrates
in
observed in seed (Fig. 5). In both the skin and seed of ripe berries, the amount of reducing sugars was higher than that of non-reducing sugars. In berries, the starch content remained low throughout fruit development (Fig. 2). After an initial increase, the total amount remained constant from 47 DAB onwards. Rapid fruit growth resulted in a decrease in the percentage of starch from that time until ripening, a situation also observed for apple (Beirliter, 1985) and banana (Marriott, 1980; Stover and Simonds, 1987). The starch content of pulp was low, the seeds containing the highest amounts (Figs. 3-5). Maximal amounts of starch in the berry were attained by 47 DAB (Fig. 21, owing to increases in the skin content (Fig. 4); these amounts were subsequently maintained owing to increased seed contents (Fig. 5). Quantitative relationships among non-structural carbohydrates in jaboticaba berry are depicted in Fig. 6. Reducing sugars were by far the dominant fraction throughout berry development (Fig. 6). From 41 DAB at least, this was due to storage of sugars in the pulp (Fig. 3). The non-reducing sugar fraction was considerable in the berry (Fig. 6), owing to the amounts stored in skin (Fig. 4) and pulp (Fig. 3). In seed it constituted a minor fraction, being detected only from 47 DAB onwards (Fig. 5). The starch fraction in fruit increased slightly until 41 DAB (Fig. 61, and represented a large reserve in the seed (Fig. 51, likely to be used during germination. Owing to small seed size (Magalhks et al., 1996) and high sugar content in the pulp (Fig. 31, starch contribution to total non-structural carbohydrate fell after 41 DAB (Fig. 6).
214
R.S. Barros et al./Scientia
40
Horticulturae 46 (1996) 209-215
45
50
55
Days
after
blossom
60
65
Fig. 5. Time course of accumulation and relative content, on a DW basis, of non-structural carbohydrates in seed of jaboticaba fruit. TS, total sugars; RS, reducing sugars; ST, starch.
0.8 i? t
II
0.6
.E 2 .s u e IL
0.4
0.2
0 5
15
25
Days Fig. 6. Changes in fractions of each non-structural jaboticaba
fruits.
NR, non-reducing
reducing sugars; ST, starch.
sugars,
35
after
45
55
65
blossom
carbohydrate group as related to their total amounts (1 .O) in estimated from difference of total and reducing sugars; RS,
R.S. Barros et al./ Scientia Horticuburae
66 (1996) 209-215
215
In summary, most of the increase in the content of non-structural carbohydrates occurred when volume growth began and immediately before the maximal rates of dry matter accumulation (Figs. 1 and 2). When fruit growth reached its maximum, carbohydrate accumulation was accelerated; soluble sugars were accumulated even after full berry size was reached. Storage of reducing sugars in the pulp (Fig. 31, which may be related to fruit dispersion by animals, is also one of the reasons underlying liquor preparation and fruit consumption in natura. Excess sugar in the berry could also be associated with the rapid decay and fermentation, and thus to the very short postharvest shelf-life of the fruit.
Acknowledgements CAPES and CNPq are gratefully acknowledged for the fellowships granted to MMM and RSB, and FINEP for financial support during the execution of this experiment.
References Andersen, 0. and Andersen, V.U., 1988. As Fruteiras Silvestres Brasileiras, 2nd edn. Editora Globo, Rio de Janeiro, 203 pp. Beiriiter, J., 1985. Sugar accumulation and changes in the activities of related enzymes during development of apple fruit. J. Plant Physiol., 121: 331-341. Hodge, J.E. and Hofreiter, B.R., 1962. Determination of reducing sugars and carbohydrates. In: R.L. Wilster and M.L. Wolfram (Editors), Methods in Carbohydrate Chemistry, Vol. 1. Academic Press, New York, pp. 380-394. Hubber, D.J. and Nevins, D.J., 1977. Preparation and properties of B-D-glucanase for the specific hydrolysis of B-D-ghtcans. Plant Physiol., 60: 300-304. Laksminarayana, S., Subhadra, M.V. and Subramanyan, H., 1970. Some aspects of the developmental physiology of mango fruit. J. Hortic. Sci., 45: 133-142. MagalhHes, M.M., Barros, R.S. and Lopes, N.F., 1996. Growth relations and pigment changes in developing fruits of Myrciaria jaboticaba. J. Hortic. Sci., in press. Marriott, J., 1980. Bananas: physiology and biochemistry of storage and ripening for optimum quality. Crit. Rev. Food Sci. Nutr., 13: 41-88. McCready, R.M.J., Guggolz, J., Silveira, V. and Owens, H.H., 1950. Determination of starch and amylose in vegetables. Anal. Chem., 22: 1156-l 158. Nii, N. and Coombe, B.G., 1983. Structure and development of the berry and pedicel of the grape Vitis uinifera L. Acta Hortic., 139: 129- 140. Stover, R.H. and Simonds, N.W., 1987. Bananas, 3rd edn. Longman Scientific and Technical, New York, 468 PP. Wiltbank, W.J., Chalfun, N.N.J. and Andersen, Sci., Tropical Region, 27A: 57-69.
O., 1983. The jaboticaba
in Brazil. Proc. Am. Sot. Hortic.