The aptitude of roots of Witloof chicory for chicon production studied by their carbohydrate composition

Scientia Horticulturae, 13 (1980) 125--134

125

Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

THE APTITUDE OF ROOTS OF WITLOOF CHICORY FOR CHICON PRODUCTION STUDIED BY THEIR CARBOHYDRATE COMPOSITION

V. FIALA and E. JOLIVET Laboratoire d'Etude du MStabolisme lnterm~diaire et de Nutrition min~rale, Centre I.N.R.A., Route de St-Cyr, 78000 Versailles (France)

(Accepted for publication 28 January 1980)

ABSTRACT Fiala, V. and Jolivet, E., 1980. The aptitude of roots of Witloof chicory for chicon production studied by their carbohydrate composition. Scientia Hortic., 13: 125--134. The variations of ethanol- and water-soluble carbohydrates in Witloof chicory roots (Cichorium intybus L.) during growth and cold storage were studied by chromatographic

methods. The carbohydrate contents and refractive index values of these ethanol and water extracts were also measured. An inverse evolution of carbohydrate fractions or refractive index values in these extracts was observed. This enables the different physiological phases of root development to be determined and consequently the results of forcing can be forecast.

INTRODUCTION Chicons are produced by forcing the roots of Witloof chicory ( C i c h o r i u m i n t y b u s L.). Over the past few years, important progress in the field of chicon production has been made. Recent evolution has mainly been carried out in methods of obtaining chicons and their production is now increasingly based on hydroponic forcing in conditioned rooms (Bannerot et al., 1977). Nevertheless, with both modern and traditional forcing, the yield of good commercial quality chicons remains largely influenced by the physiological state o f the roots at the start of forcing; that is to say, by the variations of their biochemical composition during the period of growth in the field and during subsequent low-temperature storage. To this effect, we have shown t h a t the variability of chicon production is related to the activity of r o o t metabolism determined by gazeous 14CO2 fixation, by variation of amino and organic acids (Jolivet et al., 1971) and by the variation of reducing power (Jolivet et al., 1976). However, even if it is possible to forecast the time from which chicon production will improve by means of these different biochemical determinations, it is n o t possible to use t h e m to forecast the m o m e n t chicon quality will decline. In an earlier study (Fiala et al., 1976), we showed t h a t chicory production was related to the variations in the inulin/low molecular weight fructosans ratio, thus corroborating the ro~ults obtained by Rutherford and

126

Phillips (1975) and Rutherford (1977). We have elaborated these studies, and the present paper gives the results we have recently obtained. MATERIAL AND METHODS Plan t material Chicory roots, early cultivar 'Zoom', hybrid F~ I.N.R.A., were obtained from plants which were sown on 21 April and 25 May (at I.N.R.A., Versailles) and fertilized normally (N--P--K--Mg: 40--150--320--32 U/ha). During the period of growth in the field, from 3 October until 1 November, samples of roots were removed every 2 weeks. All the roots of these 2 sowings were lifted on 18 November 1977. At lifting, the trimmed roots were stored in a cold dark r o o m at 0.5 + I°C at 95% relative humidity in paUox o f 0.96 m 3 volume. During storage, the roots were removed for analysis and forcing at 4 week intervals. The roots were trimmed to approximately 17 cm in length and their diameter at the collar varied from 30 to 50 mm. Before forcing, a sample of 10 roots was used for carbohydrate analysis and refractometry measurements, and 100-root groups were subsequently forced hydroponically for 21 days at 18°C. Carbohydrate extraction and fractionation Slices of roots, 25 mm thick, were taken from the zone located between 10 and 35 mm from the collar. Ten such slices were used to prepare a sample of 15 g fresh weight for analysis of carbohydrate. The slices were placed in ethanol 96°G.L. and immediately extracted by homogenising with a Sorval Omnimixer twice a minute at 10 000 revs./min. The resulting homogenate was extracted for 3 h on a magnetic stirrer and then filtered on a Millipore filter. The ethanolic filtrate was brought to a volume of 100 ml and the pellet was then stirred for 3 h in ethanol 80% (v/v). After filtering, the extract was also brought to a volume of 100 ml. The same operations were carried o u t successively in the presence of ethanol 60% (v/v) and distilled water. These 4 extracts, 3 water--ethanol extracts and 1 water extract, were kept separately and used for the following analyses. Identification and quantitative determination o f carbohydrates. -- The carbohydrate c o n t e n t was determined in each of the 4 extracts. The free fructose and total fructosan contents were determined colorimetrically with thiobarbituric acid (Percheron, 1962). The m e t h o d of gel permeation chromatography for the separation of homologous fructose oligomers according to their degree of polymerisation (D.P.) was used. We used a Bio-Gel P-2 (minus 400 mesh) to separate inulin and oligofructosans. Samples were eluted from a 0.9 X 150 cm column with distilled water (with 0.02% NAN3) at a flow rate of 40 ml/h, and a temperature o f 45°C. A differential refractometer (Knauer) was used as a detector.

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Identification of oligofructosans was confirmed by thin-layer chromatography (TLC) on silica gel layers (G 1500 Schleicher and Schiill) using a modification of the technique of Hansen (1974). Aliquots of extracts were separated using isopropanol--acetone--lactic acid (4:4:2) as a solvent. The development was carried out twice for 180 min at 40°C. Refractive index determination. -- Refractive index was measured on the aliquot parts of solutions obtained by mixing, on the one hand, ethanolic extract 96 with extract 80% and, on the other, ethanolic extract 60% with water extract. The aliquot parts (5 ml) of these 2 solutions were evaporated by a stream of compressed air at room temperature. The residue was taken up by the same volume of distilled water. Refractive index of extracts was measured using an Abbe refractometer to fourth decimal precision. RESULTS

Possibilities and limits o f Bio-Gel P-2 gel permeation chromatography

Figure 1 illustrates the possibility of the gel permeation chromatographic technique using Bio-Gel P-2 to separate the various oligofructosans. The D.P.

sucrose

inulin A OF

DP7

DP3

~ ~ope

start

v~

EtOH

60., H2 0

Et OH

: ................................................

96~÷ 80

-M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

|

0

1

2

3

4

5

6

7

hours 8 9

Fig. 1. Separation of fructosans by gel chromatography on Bio-Gel P-2.

128

O

1

2

i

v

Fig. 2. Chromatogram of fructosans on 20 x 20 TLC silica gel plates. Samples of ethanol 60% and water extract (1,2) and ethanol 96 and 80% extract (3,4), fructose and sucrose

(5). was determined for each peak after acid hydrolysis by estimation of the fructose/glucose ratio. By use of this technique, both low molecular weight oligofructosans up to D.P. 6 or 7 and the higher molecular weight fructosans (inulin D.P. 37--38) can be separated. The low molecular-weight fructosans of D.P. ~ 5, fructose + glucose and sucrose were extracted with 96 and 80% ethanol, whereas more polymerized

129

fructosans were extracted by ethanol 60% and water. By the TLC technique, we are able to separate the fructosans up to D.P. = 17 (Fig. 2).

Quantitative variations of carbohydrates During the growth of roots in the field, an inverse evolution of watersoluble carbohydrates (more polymerized) and ethanol-soluble carbohydrates (less polymerized) was observed (Fig. 3). Water-soluble carbohydrate contents were higher than ethanol-soluble carbohydrate contents at the end of September and the beginning of October. They decreased during the growth period, whereas the ethanol-soluble carbohydrate contents increase. The result is that in early November, shortly before lifting the roots, the contents of these 2 fractions became practically equal. During prolonged cold storage, the contents of water-soluble carbohydrates decreased progressively and became minimum and stationary from the end of January. Inversely, the ethanol-soluble carbohydrate contents increased and then remained at a fixed value until the end of March. At this time, the contents started to decrease. It should be noted that the variations of these 2 carbohydrate fractions occurred in a parallel manner for the 2 sowings (Fig. 3). A particular study has been made of the ethanol-soluble fraction which is mainly composed of hexoses (fructose and glucose), sucrose and low molecucv

ZOOM sowing: 21 A p r i l 1977 ~ ethanol o . . . . . o water sowing: 2 5 M a y

mg g fw

1977 ~. . . . .

= ethanol water

lifting IO0 \ %

80

c~ "~,~ - ~.Q ,.-, \

.J

%

40 20

3.i0 1710 21,

22.11

19112

23.01

20.02

20.03 forcing

17.04

23.05

start

Fig. 3. Variations of ethanol-solubleand water-solublecarbohydrate contents during the growth period and cold storage.

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lar-weight fructosans of D.P. 3--5. The increase in ethanol-soluble carbohydrates was due to the parallel increase of each of these carbohydrates, but the sucrose contents were proportionally higher for the 2 sowings during the cold storage of roots (Figs. 4 and 5). cv ZOOM sowing: 21 April 1977

mg :g rw

¢

rifting

¢ SUCROSE

0-----4

FRU,I-GLC

0-~0

DP3

g - - - - - 0 DP~

50

40

3O

20.09 3.10 17.10 2.11 22.11

19.12

23.01 20~2 20.03 forcing start

Fig. 4. Variations of ethanol-soluble carbohydrate contents during the growth period and cold storage. First sowing.

Variations of refractive index The variations observed for carbohydrate fractions mentioned above were found again by measuring the refractive index of the 2 extracts (Fig. 6). The values of the refractive index of water extract of root samples taken during the period of growth were higher than those of ethanolic extract, but in the root samples which were taken at the end of November and in the samples cold-stored until March, the refractive index of water extract became lower

131

than that of ethanolic extract. At the end of the storage period, in April, a temporary increase of refractive index values of water extract was observed. This probably indicates a new modification in biochemical composition of chicory roots.

Correlation between carbohydrate composition and forcing The study of evolution of the 2 carbohydrate fractions, one soluble in water, another soluble in ethanol, allows the determination of the different physiological phases of root development during growth and cold storage. The measurement of refractive index variations of these two extracts gives a simple and quick method for the determination of the physiological state of the roots. cv

mg

ZOOM s o w i n g : 25 M a y 1977

FW ~SUCROSE • .....

• FR U~-GLC DP 3 DP~;

0~0 0----0 50

lifting

40

30

20

10

Y

/'0"%,%

/

,.O.o, -.41( °

.e-..~" .

.

20993.10 17.10 2.11 22.11

19.12

23.01

20J]2

forcing

start

20.

Fig. 5. Variations of ethanol~oluble carbohydrate contents during the growth period and cold storage. Second sowing.

132 cv ZOO M

$ ethanol s o w i n g : 21 A p r i l 1 9 7 7 ~" ~_._~ water

R.

s o w i n g : 25 M a y

1.339Q

1977= • ethanol ~. . . . . , w a t e r

lifting

8O 70

6O

i D

50

_

_

40 30

XX

• ..

"~: ....

31o 1i,lo

2:11

:2.11

19.12

=:=~:::::::~:::::::~ 23.01

20.02

xx

"'%

i

20.03

17:04

forcing

23.05

start

Fig. 6. Ethanolic and water extract refractive index variations during the growth period and cold storage. Consequently, as the results of forcing depend on this physiological state, it is possible to forecast the results of forcing, i.e. the aptitude of roots to produce good quality chicons. The highest percentage of the best quality chicons (Extra) was obtained b y r o o t forcing from the end of November and the beginning of December until the end of March (Table I). During this period, the refractive index of water extract became and remained lower than that of ethanolic extract. These estimations complete the previously described test concerning the start of forcing (Jolivet et al., 1976), which was based on reducing p o w e r variations. We are carrying o u t further studies upon the determination of the physiological state of chicory roots in relation to forcing. ACKNOWLEDGEMENTS

The authors wish to thank Mrs. M.H. Valadier for valuable technical assistance and B. de Coninck (CTIFL-INVUFLEC, Station d'Am61ioration des Plantes, Centre I.N.R.A., Versailles) for cooperation in Witloof chicory-root forcing.

133 TABLE I Chicon yield (100 chicons) and percentage cultivar ' Z o o m ' Forcing start Total (day/month/year) weight (kg)

Extra

I category

Extra + I Cat.

III Category

Weight (kg)

%

Weight (kg)

%

Weight (kg)

%

Weight (kg)

%

14.0 8.2 10.8 15.8 12.8 13.0 13.0 8.4

8.6 4.7 5.9 14.0 10.1 10.5 10.9 4.4

61.4 57.3 54.6 88.8 78.9 80.8 83.8 52.4

3.5 2.5 2.7 1.7 2.4 1.9 1.7 2.9

25 30.5 25.0 10.8 18.6 14.6 13.1 34.5

12.1 7.2 8.6 15.7 12.5 12.4 12.6 7.3

86.4 87.8 79.6 99.4 97.7 95.4 96.9 86.9

1.9 0.95 2.2 0.1 0.3 0.6 0.4 0.3

13.6 12.2 20.4 0.6 2.3 4.6 3.1 3.6

16.5 10.8 12.1 14.1 13.3 12.0 11.8 7.3

6.9 9.4 6.7 12.4 11.3 9.1 9.1 3.3

41.8 87.0 53.4 87.9 85.0 75.8 77.1 45.2

6.8 1.1 3.4 1.5 1.9 1.5 2.2 2.7

41.2 10.6 28.1 10.6 14.0 12.5 18.6 37.0

13.7 10.5 10.1 13.9 13.2 10.6 11.3 6.0

83.0 98.1 83.5 98.6 99.0 88.3 95.8 82.2

2.8 0.45 2 0.3 0.1 1.4 0.5 1.3

17.0 4.2 16.5 2.1 0.8 11.7 4.2 17.8

Sown 21 April 1977 17/10/77 02/11/77 22/11/77 19/12/77 23/01/78 20/02/78 20/03/78 17/04/78 Sown 25 May 1977 17/10/77 02/11/77 22/11/77 19/12/77 23/01/78 20/02/78 20/03/78 17/04/78

REFERENCES

Bannerot, H., de Coninck, B. et Lesaint, C., 1977. Situation actuelle en mati~re de for~age hydroponique. 4~me Biennale Internationale de l'Endive, Beauvais, 14--15 octobre 1977, pp. 139--150. Fiala, V., Jolivet, E. and de Coninck, B., 1976. Biochemical approach to forcing Witloof chicory (Cichorium intybus L.) roots by study of variations in its carbohydrate composition. Proc. Eucarpia Meetings Leafy Vegetables, Wageningen, 15--18 march 1976, pp. 18--31. Hansen, S.A., 1974. Thin-layer chromatographic method for identification of oligosaccharides in starch hydrolyzates. J. Chromatogr., 105: 388--390. Jolivet, E., Nicol, M.Z. et Cochet, J.P., 1971. Mise en 6vidence, par fixation de 14CO2 gazeux de modifications dans le m~tabolisme des acides organiques et des acides amines libres dans la racine tub6ris6e de l'Endive (Cichorium intybus L.) au cours de sa croissance et de son d~veloppement. Relation avec la production du chicon; application ~ la recherche d ' u n test pratique permettant d'estimer la p~riode optimum du for~age. Section Horticole d'Eucarpia, Symp. Int. La Chicor6e deBruxelles, Gembloux, 17 et 18 f~vrier 1970, pp. 177--206.

134 Jolivet, E., Lefevre, S. et de Coninck, B., 1976. D6termination de l'6tat physiologique de la racine tub6ris~e de Chicor~e de Bruxelles (Cichorium intybus L.) par son pouvoir r6ducteur ~ l'6gard du 2,6-dichloroph6nol-indoph6noh application au rep6rage de la p6riode optimale de for~;age. Physiol. Veg., 14: 849--863. Percheron, F., 1962. Dosage colorim6trique du fructose et des fructofuranosides par l'acide thiobarbiturique. C.R. Acad. Sci., 255: 2521--2522. Rutherford, P.P., 1977. Changes during prolonged cold storage in the reducing sugars in chicory roots and their effects on the chicon produced after forcing. J. Hortic. Sci., 52: 99--103. Rutherford, P.P. and Phillips, D.E., 1975. Carbohydrate changes in chicory during forcing. J. Hortic. Sci., 50: 463--473.