Inulin production by Vernonia herbacea as influenced by mineral fertilization and time of harvest

Inulin production by Vernonia herbacea as influenced by mineral fertilization and time of harvest¹

MARIA ANGELA MACHADO DE CARVALHO², MARCOS MECCA PINTO³ and RITA DE CÁSSIA LEONE FIGUEIREDO-RIBEIRO²

(recebido em 20/01/98; aceito em 21/05/98)

ABSTRACT - (Inulin production by Vernonia herbacea as influenced by mineral fertilization and time of harvest). The underground organs of Vernonia herbacea (Vell.) Rusby, known as rhizophores, acumulate 80% of their dry mass as fructans of the inulin type. In view of the growing industrial use of fructans as dietetic and general food products, and of their medical application, the present investigation aimed at evaluating the effect of mineral fertilization and period of cultivation on the production of these carbohydrates in field trials. Plants used in the experiments were obtained by vegetative propagation from rhizophores collected from plants growing in natural areas of the cerrado, and cultivated for two years. Fertilization consisted of N:P₂O₅:K₂O (80:200:150 kg.ha^-1) plus 80 kg.ha^-1 nitrogen as dressing. Soil fertilization did not stimulate biomass or inulin production, but in the second year of cultivation a dramatic gain in biomass and inulin was detected in both treated and control plants. Inulin production varied from 113 to 674 kg.ha^-1 which corresponds to 43% of the rhizophore dry mass. The composition of fructans was not altered by fertilization, although treated plants had a higher proportion of sucrose and fructans with degree of polymerization 3-8 in the second year of cultivation. The results identify this species as a fructan source similar to other commercial crops and recommend further agronomic studies, aimed at increasing the production of this polysaccharide.

RESUMO - (Influência da fertilização mineral e do tempo de cultivo na produção de inulina em Vernonia herbacea). Os órgãos subterrâneos de Vernonia herbacea (Vell.) Rusby, denominados rizóforos, acumulam frutanos do tipo inulina como principal carboidrato de reserva, que podem atingir até 80% da massa seca em condições naturais do cerrado. Em vista do uso crescente da inulina na indústria de produtos dietéticos e alimentícios em geral e de sua aplicação médica, o presente trabalho teve por objetivo avaliar a produção desse carboidrato em condições de campo sob o efeito da adubação mineral e tempo de cultivo. As plantas utilizadas no experimento foram obtidas por multiplicação vegetativa a partir de rizóforos coletados de plantas crescendo em condições naturais e, em seguida cultivadas em área de cerrado natural por dois anos. A adubação básica consistiu de N:P₂O₅:K₂O (80:200:150 kg.ha^-1) com uma adubação nitrogenada de cobertura (N=80 kg.ha^-1). Os resultados mostraram que a adubação não estimulou o aumento da biomassa e nem a produção de inulina. O tempo de cultivo, entretanto, afetou positivamente a produtividade de V. herbacea, havendo um ganho expressivo em biomassa e em frutano total (inulina), no segundo ano de cultivo. A produção de inulina variou de 113 a 674 kg.ha^-1, correspondendo a 43% da matéria seca do rizóforo. A composição básica dos frutanos não foi alterada, embora as plantas adubadas tenham apresentado maior proporção de sacarose e frutanos com grau de polimerização de 3 a 8 no segundo ano de cultivo. Os resultados obtidos até o momento indicam ser esta espécie uma fonte de inulina comparável a outras culturas comerciais, sendo recomendado, portanto, a realização de estudos agronômicos, visando ao aumento da produção desse polissacarídeo.

Key words - Asteraceae, fructans, cerrado, mineral fertilization

Introduction

The main global sources of commercial inulin are the fleshy roots of Dahlia sp. and Cichorium intybus L. (chicory) and the tubers of Helianthus tuberosus L.(Jerusalem artichoke). All of these crops are included in the Asteracee family and store inulin at about 75% of the reserve organ dry matter. However, chicory has been shown to be the most efficient since its estimated potential yield of inulin is 12 t.ha^-1 (Meijer et al. 1993). Inulin from chicory has been processed into fructose-rich syrups in Belgium, France and the Netherlands. Besides its importance in medical and food applications, this polysaccharide is regarded as a promising starting material for chemical conversions and non-food utilizations including alcohol production (Fuchs 1991).

Vernonia herbacea (Vell.) Rusby is also an Asteraceae that grows naturally in cerrado areas. The underground reserve organ, rhizophore, accumulates high amounts of fructans of the inulin type (Carvalho & Dietrich 1993) and is successfully used for vegetative propagation, since reproduction by seeds is difficult. Inulin from V. herbacea was found to be the most suitable substrate for inulinase obtained from the filamentous fungus Penicillium janckzewskii Zalesky isolated from the rhizosphere of these plants (Cordeiro-Neto et al. 1997). Additionally, experiments of glomerular filtration in rats showed this inulin is comparable to that from Dahlia, commercialized by Sigma with respect to the rate of filtration (Dias-Tagliacozzo et al. 1996). This information indicates that inulin from V. herbacea can be used for the production of dietetic products such as high fructose syrups as well as for studies on kidney disorders in humans.

Earlier studies reported that plants of V. herbacea obtained from segments of rhizophores were cultivated for two consecutive years. After six months new rhizophores had developed which contained about 40% of total fructan on a dry matter basis. At the end of the first year of cultivation, fructo-oligo and polysaccharides (inulin) added up to 80% of the dry weight (Carvalho et al. 1997), even though seasonal and phenological variation were shown to occur both in total fructan and in the relative molecular mass of the polysaccharide (Carvalho & Dietrich 1993).

Experiments on irrigation with nutrient solution under greenhouse conditions showed that nutrient treatment affects the pattern of fructan accumulation in rhizophores of V. herbacea and promotes shoot elongation. The low amount of fructan found in those plants was interpreted as an indication of fructan mobilization to support the intense growh of the aerial organs (Teixeira et al. 1997).

The present work was aimed at extending the investigation on the effects of mineral fertilization and time of harvest on the accumulation of inulin in Vernonia herbacea cultivated under natural conditions in the cerrado. It was also of interest to evaluate its potential use as a new source of inulin for practical purposes.

Material and methods

Plants of Vernonia herbacea (Vell.) Rusby (SP 169567) were obtained from fragments of rhizophores of adult plants randomly collected in preserved natural areas of cerrado near Moji-Guaçu, São Paulo, Brazil (22º15’ - 22018’S and 47º08’ - 47012’W). Four month old plants were transplanted to the field located in the same cerrado area and monitored from 1992 to 1994. Chemical composition of the soil where the experiment was carried out is shown in table 1. Climate descriptions of the same area were presented by De Vuono et al. (1986) and Mantovani & Martins (1988).

Table 1.
Initial chemical composition of the soil from the cerrado where the experiment was carried out, according to Seção de Fertilidade do Solo e Nutrição de Plantas, Instituto Agronômico de Campinas, Brazil.

O.M. - organic matter; H⁺+Al³⁺ - potencial acidity; SB - sum of bases; CEC - cation exchange capacity: SB + (H⁺+Al³⁺); V - saturation of bases: (SB/CEC) x 100.

The experiment was set in a split-plot design in randomized blocks with six replicates, considering plots as treatments with or without fertilization and subplots for periodic harvests. The planting was done in rows 50 cm apart and distance between plants was of 25 cm. Nitrogen, phosphate and potassium fertilizer application was based on soil sampling according to recommendation for potato plants, consisting of N:P₂O₅:K₂O (80:200:150) kg.ha^-1 as the basic supply. Further, 80 kg.ha^-1 nitrogen was applied as dressing. To prevent the crop from suffering from water stress, it was occasionally irrigated.

Two harvests were scheduled for April 1993 and 1994 because at this time of the year plants enter dormancy and the fructan content in the rhizophores reach maximal values (Carvalho & Dietrich 1993). At each harvest 18 plants were sampled for each treatment and the fresh and dry matter of rhizophores and aerial parts were determined according to Teixeira et al. (1997).

Soluble carbohydrate extractions - Fructans were extracted in triplicate according to Pollock & Jones (1979) from samples (5 g each) of fresh rhizophores obtained from three plants collected randomly from each block. Tissue was sliced and boiled in 80% aqueous ethanol for 3 min for enzyme denaturation. The mixture was then ground in a mortar with a pestle. The homogenate was placed in a water bath at 80°C for 15 min and centrifuged at 1,000 g for 15 min. The residue was reextracted once as above and then submitted twice to water extraction for 30 min at 60°C. The pooled supernatants were concentrated, frozen, thawed and centrifuged at 9,000 g for 20 min at 5°C. The pellet containing the high molecular weight fructans (fructo-polysaccharides) was resuspended in water.

Medium molecular mass fructans present in the supernatant were precipitated with three volumes of ethanol and added to the fructopolysaccharide fraction. The remaining supernatant constituted the low molecular weight fructans (fructo-oligosaccharides).

Carbohydrate analyses - Free and combined fructose were measured using a ketose-specific modification of the anthrone reaction (Jermyn 1956), using fructose and inulin from Helianthus tuberosus as standards.

Fructo-oligosaccharides were analysed by thin-layer chromatography (TLC) on silica gel plates as described by Kanaya et al. (1978). Samples were prepared as described in Carvalho & Dietrich (1993). Fructans were visualized by spraying with the ketose-specific urea-phosphoric acid (Wise et al. 1955). Fructo-oligosaccharides of the inulin series from tubers of H. tuberosus were used as reference material.

After deionization through cation (Dowex-50W, Na⁺ form) and anion (Dowex-1, Cl^- form) resins, neutral soluble carbohydrates were also analysed on a 4x250 mm CarboPac PA-1 anion exchange column using a Dionex DX-300 gradient chromatography system with pulsed amperometric detection (HPAEC/PAD). The gradient was established according to Shiomi (1993). Eluent A (150 mM NaOH) and eluent B (500 mM sodium acetate in 150 mM NaOH) were mixed as follows: 0-1 min, 25 mM; 1-2 min, 25-50 mM; 2-14 min, 50-500 mM; 14-22 min, 500 mM; 22-30 min , 25 mM. The flow rate through the column was 1.0 cm³.min^-1. The applied PAD potentials for E1 (300 ms), E2 (120 ms) and E3 (300 ms) were 0.04, 0.60 and 0.80 V, respectively, and the output range was 1 mA. HPAEC/PAD elution patterns were compared to those of reference standards obtained from tubers of H. tuberosus extracted according to Pollock & Jones (1979).

Fructo-polysaccharides were analyzed by gel permeation chromatography (GPC) using a Bio-Gel P-10 column as described by Carvalho & Dietrich (1993). Relative molecular mass (Mr) was estimated according to Andrews (1965).

Data were statistically analysed by the F test at 5% level.

Results and Discussion

The effects of nutrient treatment and time of harvesting of plants of Vernonia herbacea cultivated in the cerrado were compared by the analysis of variance (table 2). Except for the increase in dry mass percentage of the rhizophore, nutrient treatment did not affect the total biomass or inulin production. Nevertheless the values obtained for treated plants were lower than those of control plants, otherwise indicating possible negative effects of fertilization which were not statistically significant probably owing to the high coeficient of variation (table 2).

Table 2. Variation in biomass, inulin content, harvest index and inulin production from plants of Vernonia herbacea treated with NPK (N:P₂O₅:K₂O/80:200:150 kg.ha^-1) in field trials at two harvesting times.

Values followed by different letters in each line are different at 5% probability. ns – not significant (F test). No significant interaction was found. Capital letters refer to nutrient treatment.

The utilization of carbohydrate by the shoot and its accumulation in underground reserve organs depend on various factors, including the level of nitrogen supply. High carbohydrate levels have been associated with low nitrogen availability (Paul & Driscoll 1997). Considering the low levels of nutrients in the soil of the cerrado it is possible that fertilization treatment could have produced changes in the source/sink balance of Vernonia plants, thus reducing both the rhizophore growth and yield of inulin. The present results are in agreement with those previously found by Teixeira et al. (1997) and emphasize the effect of mineral fertilization on carbon partition in V. herbacea by stimulating shoot growth and inhibiting the accumulation of inulin. They also corroborate the hypothesis that favorable growth conditions such as elevated nitrogen supply inhibit fructans accumulation (Pollock 1986).

With respect to time of harvesting, an increase in total biomass, inulin yield and harvest index was found in the second year of cultivation in field conditions, even though the inulin concentration - as a function of dry matter - remained constant (table 2). The dry mass as a percentage of fresh matter of aerial organs and rhizophores decreased in the second year, reflecting an increase in the water content (table 2).

Table 3 shows that the increase of total biomass in the second harvest was accompained by increases in both aerial organs and rhizophores in treated and control plants of Vernonia cultivated in the cerrado. Dry mass partition between above soil organs and rhizophores was practically even in the first year, that is 50% of dry mass was partitioned to the roots in treated plants and 52% in control plants. In the second year, after a certain crop mass had been reached, 59% of the dry mass was distributed to the roots in treated plants while 65% was allocated to roots in control plants (table 3).

Table 3. Yields of total dry biomass, aerial organs, rhizophores and inulin (kg.ha^-1) from plants of Vernonia herbacea treated with NPK (N:P₂O₅:K₂O/80:200:150 kg.ha^-1) in field trials in two harvesting times.

The inulin yield calculated from rhizophore biomass showed an increase in the second harvest in both treatments, as a result of favorable allocation to the storage organs from the first to the second harvest. This yield varied from 113 to 674 kg.ha^-1, comprising approximately 24% of the total dry mass or an average of 43% of rhizophore dry mass.

Comparing to data presented by Meijer et al. (1993), for Jerusalem artichoke and chicory, biomass allocation in V. herbacea shows a pattern similar to chicory plants, that is, a high percentage of the dry mass is partitioned to the underground organs by the second growth cycle. In addition, similarly to chicory, inulin concentration in V. herbacea can reach as much as 80% of the dry mass (Carvalho & Dietrich 1993).

Chicory has been considered the most efficient inulin crop, producing between 8 and 12 t.ha^-1 inulin, while Jerusalem artichoke, another inulin source, produces from 2.7 to 6.4 t.ha^-1 (Meijer et al. 1993). Considering that V. herbacea is a wild species and that the best plant density and time of planting in the field are not well established, knowledge of the most suitable growth conditions based on physiological and agricultural studies will certainly improve the inulin yield obtained (0.674 t.ha^-1) under field conditions.

Analyses of fructo-oligosaccharides by thin layer chromatography (TLC) and by high performance anion exchange chromatography, with pulsed amperometric detection (HPAEC/PAD) shown in figures 1 and 2, respectively, demonstrate that the relative amounts of fructans with degree of polymerization (DP) 3-8 increased in the second year, mainly in treated plants. These data indicate that, although the total inulin content was not increased in treated plants, fertilization could be affecting the activity of fructan synthesizing enzymes. The mean molecular mass and the degree of polymerization of the inulin fraction, as determined by gel permeation chromatography increased from DP 24 to 28 in treated plants in the second year (data not shown), reinforcing the suggestion of the predominance of synthetic metabolism in plants under nutrient fertilization.

Figure 1.
Thin layer chromatography of fructo-oligosaccharides from rhizophores of Vernonia herbacea in two harvesting times (A,B – one year), (C,D - two years) and fructo-oligosaccharides from tubers of Helianthus tuberosus (H). B,D: treated with NPK; A,C: control NPK. Each lane contained approximately 100 µg fructose equivalents. F - fructose, S - sucrose, K - 1-kestose, N - nystose, DP - degree of polymerization, or. – origen.

Figure 2.
High performance anion exchange chromatography of fructo-oligosaccharides from rhizophores of Vernonia herbacea in two harvesting times (A,B – one year), (C,D – two years) supplied (B,D) or not supplied (A,C) with NPK. Members of the homologous series based on 1-kestose are represented by numbers 5 to 9; G – glucose, F – fructose, S- sucrose, K- 1-kestose, N – nystose.

In general, the amount of carbon transported to and accumulated in storage organs of crop plants determines crop yield. However, the production of inulin by V. herbacea seemed to be limited more by the growth processes of the underground organs than carbon fixation and partition since the inulin concentration remained high and unchanged in the rhizophores of control and treated plants. The presence of high levels of aluminium in the cerrado soil (Goodland 1971) affecting the growth of the underground organs and decreasing the availability of several essential elements such as N, P, and K must be considered in agronomic studies aimed at increasing the rhizophore biomass and the production of inulin.

Acknowledgements - The authors thank Dr Lilian B.P. Zaidan for critical reading of the manuscript.

2. Seção de Fisiologia e Bioquímica de Plantas, Instituto de Botânica, Caixa Postal 4005, 01061-970 São Paulo, SP, Brazil. Research fellow, CNPq

3. Seção de Ecologia, Instituto de Botânica.

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. Supported by FAPESP (Proc. 91/3588-8) and CNPq.

Publication Dates

• Publication in this collection
  25 Feb 1999
• Date of issue
  Dec 1998

History

• Accepted
  21 May 1998
• Received
  20 Jan 1998