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Mechanism of soybean (Glycine max L. Merrill) degreening related to maturity stage and postharvest drying temperature
Postharvest Biology and Technology 38 (2005) 269–279
Mechanism of soybean (Glycine max L. Merrill) degreening related to maturity stage and postharvest drying temperature Patr´ıcia Sinnecker a , Nelson Braga b , Eduardo L.A. Macchione c , Ursula M. Lanfer-Marquez a,∗ a
Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of S˜ao Paulo, Av. Prof. Lineu Prestes, 580, Bl. 14, 05508-900 S˜ao Paulo, SP, Brazil b Agronomic Institute of Campinas, Av. Bar˜ ao de Itapura, 1481, 13020-902 Campinas, SP, Brazil c Institute of Physics, University of S˜ ao Paulo, P.O. Box 66318, 05315-970 S˜ao Paulo, SP, Brazil Received 28 April 2005; accepted 2 July 2005
Abstract The degreening of soybean seeds was studied relative to their maturity stage, moisture content at harvest and postharvest drying temperature. Studies were performed on Brazilian soybean over its whole maturation period (R6 –R8 ) according to the Fehr and Caviness scale. Chlorophyll and its colored derivatives were quantified as a function of three drying conditions: slow drying at room temperature (25 ◦ C) and oven drying at 40 and 75 ◦ C in order to quantify degreening and pigments produced under these temperatures. Pathways for chlorophyll degradation, enzymatic, chemical or both, could be elucidated by this experimental design. Pigments were quantified by HPLC and identities were confirmed by spectral characteristics, retention times and plasma desorption mass spectrometry (PDMS). Postharvest drying at 25 ◦ C allowed almost complete degradation of chlorophyll in seeds harvested at maturity stage R6 (Fehr scale) or later, with no green pigment detected, which mimics maturation in the field. Fast drying at 40 or 75 ◦ C blocked the breakdown process at all stages of maturity and only seeds harvested at R8 lost their green color. At 40 ◦ C, chemical and/or enzymatic mechanisms of degradation seemed to have occurred, the former is supported by high levels of pheophytins and the latter, by the appearance of small amounts of chlorophyllides and pheophorbides. At 75 ◦ C, considerable levels of only chlorophylls and pheophytins were observed probably due to the inactivation of enzymes. So, chemical pheophytinization was the primary mechanism of degradation. It was concluded that the degree of maturation at harvest time and the temperature of postharvest drying significantly affect the chlorophyll content of soybeans. In order to avoid retention of chlorophyll and to guarantee marketing quality of seeds, harvesting at full maturity followed by fast or slow drying is suggested. If premature harvesting is necessary, the drying should be performed at temperatures lower than 40 ◦ C or seed quality can be compromised. © 2005 Elsevier B.V. All rights reserved. Keywords: Soybean; Degreening; Maturation; Temperature of drying
∗
Corresponding author. Tel.: +55 11 30913684; fax: +55 11 38154410. E-mail address: [email protected] (U.M. Lanfer-Marquez).
0925-5214/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2005.07.002
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1. Introduction The phenomenon of chlorophyll degradation in degreening plants has been a topic of great interest (M¨uhlecker et al., 1997; Oberhuber et al., 2001). However, only a few years ago, the general degradation pathway was established and since then, different catabolic products have been identified in senescent leaves and during fruit ripening of several plant species (H¨ortensteiner et al., 1998). Difficulties in extracting and identifying the chlorophyll catabolic derivatives may explain that the breakdown mechanism is still not fully understood. Particularly, in soybean, this phenomenon has a remarkable significance since the disappearance of the green color affects the market quality of grains. The presence of chlorophyll or other greenish pigments in canola and soybean not only imparts an undesirable dark color and promotes oxidation in the presence of light, it also poisons the catalyst during the hydrogenation process of oils (Usuki et al., 1984; Abraham and DeMan, 1986; Johnson-Flanagan and Thiagarajah, 1990; Ward et al., 1992, 1995; Gomes et al., 2003). The presence of green grains can sometimes be observed as a consequence of hot weather during the maturation period, which causes water stress (excessive water loss) or high rainfalls, which force farmers to anticipate the harvest of the crops in order to avoid losses. These abnormal climate conditions have been observed in the southeast and central west regions of Brazil. Prematurely harvested seeds require postharvest drying in order to reduce the moisture to a maximum of 13%, which is the upper limit considered for safe storage (Adam et al., 1983; Pritchard, 1983). Chlorophyll breakdown is currently described as a multi-step mechanism. The first group of reactions produces greenish derivatives, while the more advanced steps produce colorless compounds. According to H¨ortensteiner et al. (2000), the whole process is as complex as chlorophyll biosynthesis. The main changes occurring in the first group of reactions correspond to the release of Mg by displacement with two H under acidic conditions and/or by the action of Mgdechelatase and the cleavage of the phytol chain by the enzyme clorophyllase, which produces greenish intermediates, such as pheophytins, chlorophyllides and pheophorbides, all of them showing an intact tetrapyr-
role ring (Heaton and Marangoni, 1996; Mangos and Berger, 1997). The second group of reactions is responsible for degreening by the rapid formation of colorless and polar derivatives, due to the opening of the tetrapyrrole ring by the action of pheophorbide a monoxygenase, which seems to act specifically on pheophorbide a (M¨uhlecker et al., 1997; Rodoni et al., 1997; Oberhuber and Kr¨autler, 2002). Although the general mechanism of degradation has already been established, there is no information about the external environmental influences on the mechanism involved in chlorophyll degradation in soybeans during maturation and postharvest storage (Ginsburg and Matile, 1993; Takamiya et al., 2000). The objective of this study was to investigate the degreening of soybean seeds relative to their maturity stage and postharvest drying temperature by analyzing the chlorophyll catabolites formed in each case.
2. Material and methods 2.1. Experimental design and plant material Two Brazilian soybean (Glycine max L. Merr) cultivars, IAC-17 and IAC-22, were grown at the Agronomic Institute of Campinas, S˜ao Paulo, Brazil. The factorial experimental design was 2 × 6 × 3 (two cultivars × six maturation stages × three drying conditions). Seeds were planted as described before (Gomes et al., 2003) and harvested at six developmental stages, from R6 to R8 , according to the scale proposed by Fehr and Caviness (1977). The first swath occurred at physiological maturation (R6 ), nearly 100 d after planting and continuing until commercial maturation (full maturity or R8 ), at nearly 126 d after planting. For better monitoring of the colored derivatives formed during the degreening, three more intermediate steps R6 –R7 (I), R6 –R7 (II) and R6 –R7 (III) with intervals of approximately 5 d were added. Analysis of variance was performed by using the SAS 6.0 statistical software package and significance was determined at the 0.05 level. The results were similar for both cultivars and data presented in graphs show mean values of triplicates performed in each experimental condition.
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2.2. Drying conditions Swathed seeds, in their intact pods of each maturation stage, were divided into four groups and three of them submitted to three drying temperatures until their moisture contents had decreased to 13%. Slow-dried seeds were obtained by air drying at room temperature (25 ± 5 ◦ C) for approximately 10 d and fast-dried seeds were obtained by dehydration in an air-circulation oven at 40 ± 2 and 75 ± 2 ◦ C, respectively. Thereafter, seeds were threshed by hand and stored at 2 ◦ C until analysis of moisture and pigments. The fourth group was analyzed immediately after harvesting. 2.3. Moisture determination The moisture content of recently harvested seeds was determined by oven drying 10 g samples at 105 ◦ C until reaching constant weight. Residual moisture in fast- or slow-dried seeds was determined by drying 2 g of powdered soybean seeds at 105 ◦ C until reaching constant weight (AOAC, 1995). 2.4. Pigment extraction Five grams of dried seeds were ground in a laboratory mill (Polymix KCH-Analytical mill A10, Kinematica AG, Luzern, Switzerland), mixed with 80% cold acetone in a proportion of 1–6 (w/v) and filtered through a sintered plate funnel under vacuum. Then, the precipitated pellet was extracted twice until the residue was colorless. The combined filtrates were transferred to a separating funnel in 60 mL of petroleum ether and 20 mL cold deionized water. The aqueous layer was discarded after vigorous shaking and standing. The washing procedure was repeated 3–4 times to remove acetone and the ether phase containing the pigments was dehydrated with sodium sulfate anhydride. This solution was filtered, transferred to a beaker and dried in a rotary evaporator (Heidolph Elektro GmbH & Co., WB2000, Kelheim, Germany). The residue was dissolved in 4–5 mL acetone (HPLC grade, Carlo Erba) and filtered on a 0.45 m membrane filters (PTFE, Millipore) before HPLC analysis. 2.5. HPLC pigment analysis A triple pump with a ternary gradient (LC10ADVP), computer-controlled (Class-VP 5.032) sys-
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tem (Shimadzu, Tokyo, Japan Class-M10A) was used for HPLC analysis. Separation was achieved using a Nucleosil ODS 100 column (5 m, 250 mm × 4 mm i.d., pre-column 11 mm × 4 mm i.d., Macherey-Nagel, D¨uren, Germany) and a 1 mL/min flow rate. The UV–vis diode array spectrophotometric detector (SPD-M10AVP) was set at 432 nm for chlorophylls and chlorophyllides, 409 nm for pheophytins and pheophorbides according to their maximal absorbance and 669 nm for all catabolites simultaneously (Breemen et al., 1991). Additionally, UV–vis absorption spectra were recorded in the 380–670 nm wavelength range with an 8 nm bandwith. The solvent system consisted of: (A) methanol/ 1.0 M ammonium acetate buffer (pH 7.0) (80:20, v:v); (B) methanol:acetone (80:20, v:v); (C) acetone. The separation started linearly from 100% A to 100% B for 20 min, followed by an isocratic hold for 2.5 min, then linearly for 7.5 min to 100% C and was finished by conditioning the column for 4 min with starting eluent A (Mangos and Berger, 1997). An aliquot of 10–50 L of each sample was injected in three replications. The isolated pigments were identified by their retention times and by their absorption spectra in comparison with their respective standards as well as literature data (Johnson-Flanagan and Thiagarajah, 1990). 2.6. Preparation of pigment standards Chlorophylls (a and b) were purchased from Sigma Chemical Co. (St. Louis, MO). As chlorophyll derivatives are not available in the market, the greenish chlorophyll catabolites were prepared from spinach. Fresh spinach (200 g) leaves obtained from local markets were washed, drained, chopped and blended with 100 mL of distilled water for 4 min, so that a homogeneous puree was obtained and then freeze dried (Supermodulyo, Edwards High Vacuum International, NY, USA), powdered and stored at −20 ◦ C, prior to usage. Five grams of the dehydrated spinach was extracted with 80% acetone as previously described (Section 2.4) and brought to a final volume of 5 or 10 mL acetone. Chlorophylls (a and b) present in the spinach extract were separated and collected in a preparative HPLC system (Fraction Collector Module, FRC-10A, Shimadzu, Kyoto, Japan). The separation was achieved by applying an isocratic run with methanol/acetone
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(80:20, v/v) and a 10 mL/min flow rate. Fractions of chlorophylls a and b were collected, concentrated under vacuum until dryness and stored in a known volume of acetone. Pheophytins (a and b) were prepared by dropwise addition of 1.0 M HCl to the collected fractions of chlorophylls (a and b) and conversion was completed in approximately 15 min (Schwartz et al., 1981; Breemen et al., 1991) changing their color from green to olive brown. Extracts containing chlorophyllides (dephytylated derivates) were obtained by incubating 5 g of dehydrated spinach with 100 mL acetone/0.2 M Tris–HCl buffer pH 8.0, 1:1, v:v) at 40 ◦ C for 2 h in the dark by the action of the endogenous chlorophyllase on chlorophylls (a and b) (H¨ortensteiner et al., 1995). Then, the chlorophyllides present in the water/acetone phase were separated by adding diethyl ether in a separatory funnel. The aqueous phase was discarded after the extract had been washed twice and the organic layer containing the pigments was collected and evaporated under vacuum after removing residual moisture over anhydrous sodium sulfate. The chlorophyllides (a and b) were dissolved in acetone and analyzed by HPLC. Pheophorbides (a and b) were prepared from the chlorophyllides by HCl acidification in a similar way as described for obtaining pheophytins from chlorophylls. Pheophorbides (a and b) and chlorophyllides (a and b) were separated and collected in a preparative HPLC system in an isocratic run with methanol/1.0 M ammonium acetate buffer (pH 7.0)/acetone (80:10:10, v:v:v). All handling procedures were carried out under dim light and cold solvents were used to avoid degradation. The calibration curves of chlorophylls (a and b), pheophytins (a and b), chlorophyllides (a and b) and pheophorbides (a and b) were linear over the range of 2.5–100 mg kg−1 (correlation coefficient = 0.99). In addition to HPLC analysis, the identity of the synthesized standards was confirmed by plasma desorption mass spectrometry (PDMS) (Macfarlane and Torgerson, 1976). All pigments previously separated and collected by HPLC were concentrated and suspended to a volume of acetone (approximately 200 mL, concentration 2 g L−1 ). Aliquots of 5 mL of each solution were dropped on an aluminized polyester foil (1 m) and covered with a thin nitrocelullose film. After being dried with a stream of N2 , the samples were
immediately inserted into the spectrometer. The equipment was operated at 9.33 × 10−5 Pa and the acceleration potential voltage was +13.5 kV. The time of flight information about secondary ions was accumulated for 106 start events (Macchione, 1998). A mass spectrum of each compound was determined in duplicate.
3. Results 3.1. Synthesis of chlorophyll derivative standards It was necessary to synthesize pheophytins, pheophorbides and chlorophyllides a and b due to the lack of commercially available chlorophyll derivatives. Fresh spinach leaf extracts were successfully employed and the identities of the synthesized compounds were confirmed by their spectral characteristics in 80% acetone over a 400–800 nm range, as shown in Fig. 1. The spectra obtained were similar to those reported by Johnson-Flanagan and Thiagarajah (1990). As expected, the spectra of chlorophyllides a and b were similar to those of their respective parents chlorophylls a and b because the only difference in molecular structure is lack of a phytol chain in chlorophyllides. Similarly, the spectra of pheophorbides a and b may be compared to pheophytins a and b, respectively. Additionally, chlorophylls a and b and pheophytins a and b had their identities confirmed by plasma desorption mass spectrometry. Table 1 shows the molecular ions and the major fragments obtained by mass spectrometry in comparison with the calculated molecular weights. The observed molecular weights of the pigments perfectly matched their calculated values. The standards obtained in the present work were used to identify the pigments present in soybeans over the whole experiment. Table 1 Molecular weights of chlorophyll derivatives isolated from soybeans calculated and determined by plasma desorption mass spectrometry Pigments
M•+
Mass spectra most abundant fragment ion
Calculated molecular weight (mass units)
Chlorophyll a Chlorophyll b Pheophytin a Pheophytin b
893.0 907.2 871.0 884.9
481.2 496.0 419.7 473.0
893.5 907.5 871.2 885.2
615.6 629.1 592.5 606.1
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Table 2 Retention times (min) of pigments present in soybean seeds Pigments
λmax (nm)
Retention time (min) ± S.D.
Chlorophyllide b Chlorophyllide a Pheophorbide b Pheophorbide a Lutein Chlorophyll b Chlorophyll b Chlorophyll a Chlorophyll a Pheophytin b Pheophytin b Pheophytin a Pheophytin a
432, 669 409, 432, 669 432, 654 409, 669 446, 472 432, 651 432, 651 410, 432, 669 410, 432, 669 432, 654 432, 654 409, 669 409, 669
7.69 9.79 12.60 13.64 19.25 22.09 22.64 23.77 24.29 27.60 28.07 28.91 29.25
± ± ± ± ± ± ± ± ± ± ± ± ±
0.67 0.89 0.38 0.22 0.26 0.26 0.25 0.43 0.51 0.48 0.38 0.59 0.32
n = 108; S.D., standard deviations.
3.2. Separation and identification of soybean pigments by HPLC Pigments observed in samples’ chromatograms were identified according to their absorption spectra and retention times in comparison with the respective standards produced in our laboratory and also to data previously published (Johnson-Flanagan and Thiagarajah, 1990; Mangos and Berger, 1997). Table 2 presents the retention times and the wavelengths of maximum absorbance (λmax ) of all pigments isolated according to the procedures and chromatographic conditions described. The results express the mean retention times accompanied by their standard deviations of all samples analyzed (n = 108). As can be observed in Table 2, the pigments were eluted according to their decreasing polarity, starting at 7.70 min with chlorophyllide b and ending at about 30 min with pheophytin a . All pigments were well separated except for the epimers chlorophyll a and b and pheophytin a and b which eluted shortly after their corresponding parents. Epimers were identified by comparison with data reported in literature (Mangos and Berger, 1997). 3.3. Pigment changes during soybean maturation Fig. 1. Visible absorption spectra of pigment standards in 80% acetone over the range of 400–800 nm, analyzed by HPLC and scanned in the stop-scan mode.
The HPLC chromatograms shown in Fig. 2A–C represent changes in types and amounts of pigments that occur in freshly harvested seeds over the entire
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Fig. 2. HPLC chromatograms of pigments extracted from soybean seeds: (A) harvested at R6 (green seeds); (B) harvested at R6 –R7 ; (C) harvested at R8 (yellow seeds).
field maturation period. Seeds harvested at very early stages of maturation, such as R6 (Fig. 2A), but not submitted to any postharvest drying process presented a complex pattern of pigments. The more prevailing pigments found were lutein, chlorophylls a and b, pheophytins a and b and the less prevailing ones were identified as chlorophyllides a and b, pheophorbide a, the epimers chlorophyll b , pheophytins a and b , as well as some unidentified xantophylls. At the intermediate stages between R6 and R7 (Fig. 2B), a gradual disappearance of both types and amounts of pigments
was observed and at advanced stages of maturation (R8 ) (Fig. 2C), only lutein could be detected in very low amounts and a few unidentified small peaks of xantophylls. 3.4. Relationship between major green pigments and moisture contents in freshly harvested seeds Fig. 3 shows the changes in total chlorophyll, pheophytin and moisture contents of freshly harvested seeds during the maturation period. The first date of swathing
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Fig. 3. Total chlorophyll, total pheophytin and moisture content of recently harvested soybean seeds over the whole maturation period.
occurred at 100 d after planting and corresponded to stage R6 according to the scale of Fehr and Caviness (1977). Seeds reached the stage R7 at 120 d after planting and reached full maturity (R8 ) at 126 d after planting. Additionally, three samples were also swathed between R6 and R7 corresponding to intermediate stages, noted as R6 –R7 (I, II and III). At early stages of maturation, the moisture content decreased slowly and linearly from 71.6% at R6 (100 d after planting) to 62.1% at 113 d after planting. Thereafter, there was a rapid decrease to 13.3% at full maturity (R8 ). In the beginning of the maturation period when seeds contained approximately 70% moisture, the level of total chlorophyll was nearly 500 mg kg−1 . As maturation progressed, there was an exponential decline of chlorophyll down to its almost complete disappearance. Considering the initial content of 500 mg kg−1 chlorophyll at R6 as 100%, there was a reduction of 92%, reaching 48 mg kg−1 at full maturity. Only small amounts of pheophytins were detected in each stage, representing less than 10% of the original chlorophyll contents. Dephytylated derivatives (pheophorbides and chlorophyllides) were only found in some samples in trace amounts despite the degradation of chlorophyll and low levels of pheophytins (results not shown).
3.5. Effect of postharvest drying on chlorophyll degradation Fig. 4 shows the contents of the main pigments found in seeds from R6 to R8 (chlorophylls a and b and pheophytins a and b), after drying at 25, 40 and 75 ◦ C. Seeds dried at 25 ◦ C had their green pigments almost completely degraded, except when harvested at early maturation stage (R6 ). The chlorophyll content of dried seeds at 25 ◦ C harvested at R6 was about 50 mg kg−1 and decreased to 3 mg kg−1 at full maturity. Pheophytins were also present in low levels, starting at 6 mg kg−1 and ending at undetectable levels. Chlorophyllides and pheophorbides were not detected in these samples. Seeds submitted to oven drying at 40 ◦ C with circulating air presented high levels of chlorophylls and an accumulation of pheophytins could also be observed. Both pigments decreased over the maturation process, though. At early swaths, total chlorophyll and pheophytin contents were about 300 and 250 mg kg−1 , respectively. As maturation progressed, these amounts decreased to 18 and 13 mg kg−1 , respectively. At this drying temperature, very small amounts of dephytylated pigments (pheophorbides and chlorophyllides) were detected but not quantified.
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Fig. 4. Chlorophyll (a and b) and pheophytin (a and b) content in soybean seeds swathed on six dates, after postharvest drying at three temperatures.
At 75 ◦ C, high amounts of chlorophylls and pheophytins were observed, though lower than those in seeds dried at 40 ◦ C. Another remark is that the pheophytin/chlorophyll ratio changed in comparison to 40 ◦ C, since less pheophytins were found.
4. Discussion The presence of unacceptable high levels of immature green seeds is sometimes the reason for downgrading Brazilian oilseeds. The present study aimed to get a better understanding of the degreening process during seed maturation and to evaluate the influence of postharvest drying temperatures. The greenish
pigments derived from chlorophyll degradation, analyzed and quantified individually by HPLC were the main concern. To accomplish this objective, the solvent extraction procedures usually applied to green leaves had to be optimized for seeds, containing only small amounts of green pigments. The chromatographic procedures reported by Mangos and Berger (1997) for the quantification of chlorophyll derivatives in spinach were successfully employed for soybeans. Fig. 2A–C shows the chromatogram of pigments extracted from fresh green seeds harvested at different stages of development and analyzed immediately after harvest. The extracts from R6 presented a complex mixture of pigments with a broad spectrum of polarities, from the least polar phytyl esters of chlorophylls
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and pheophytins to the most polar dephytylated derivatives (chlorophyllides and pheophorbides). As maturation progressed, pheophytins were not accumulated and dephytylated derivatives (chlorophyllides and pheophorbides) could only be detected in trace amounts in some samples. At full maturity, only lutein could be found in significant amounts. These results are similar to those reported by Johnson-Flanagan and Thiagarajah (1990) and Cenkowski et al. (1989a,b; Cenkowski and Jayas, 1993) in studies on canola. Therefore, under ideal conditions of soybean growth, chlorophyll degrades completely and there is no accumulation of any greenish derivatives, which could be explained by the mechanism of chlorophyll breakdown proposed by Ginsburg and Matile (1993), H¨ortensteiner (2004), H¨ortensteiner et al. (1995, 1998) and Kr¨autler (2001). According to these authors, chlorophylls are continuously degraded to colorless compounds by a multi-step pathway, which involves simultaneous action of enzymes, weak acids, oxygen, light and heat. The loss of green color in the end of the degradation process is a consequence of the oxygenolytic opening of the porphyrin macrocycle of chlorophyll catalyzed by the joint action of two enzymes, pheophorbide a oxygenase and red chlorophyll catabolite reductase. The elution with a ternary gradient proved to be essential to achieve satisfactory separation of each component in the mixture. In addition, diode array detection was a useful tool for examining the spectral characteristics of each eluted peak. In Fig. 3, the relationship between the main green pigments (total chlorophyll and total pheophytins) and moisture content can be observed. During field maturation, without any postharvest drying, total chlorophyll disappeared in an exponential pattern and a complete degreening was outstanding, since no pheophytins were accumulated. Seed moisture declined slowly at the beginning of maturation and rapidly at the end. Although chlorophyll breakdown and moisture loss occur simultaneously during maturation, they do not appear to control each other and moisture content cannot be used to predict chlorophyll content. Chlorophyll degradation showed quite different results after postharvest drying, depending on the temperature used. Local room temperature air drying at 25 ◦ C, until moisture content reached 13%, resulted in an almost
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complete disappearance of green color, even when seeds were swathed at early stages of development. This process mimics field maturation under adequate climatic conditions. At this temperature, the mechanism of degradation involves the action of all the enzymes responsible for chlorophyll disappearance, including pheophorbide a monoxygenase. This key enzyme catalyzes the cleavage of the porphyrin structure originating colorless compounds, which explains the absence of any kind of green derivatives, and consequently, yellowing of seeds. Therefore, slow drying at low temperatures (25 ◦ C), independent of the maturation stage at harvest time, reduced moisture and chlorophyll levels below 13% and 22 mg kg−1 , respectively, meeting the requirements established for soybean trading. On the contrary, when the crops were dried at 40 ◦ C or higher, the degreening process was blocked and easily seen at early stages of maturation. It was also evidenced by a significant retention of greenish derivatives, providing some insight into possible degradation pathways. At this temperature, although a significant part of the original amount of chlorophylls had changed to pheophytins, chlorophyll was still the most abundant pigment. The high levels of pheophytins seemed mainly to have been formed by chemical degradation, but the action of Mg-dechelatase, which might be still active at some extent at this temperature, cannot be excluded. On the other hand, the high amounts of remaining chlorophyll may be the consequence of excessive, rapid water loss. This might have compromised the activity of the degrading enzymes activity as well as the reduction of water/acid mobility and even not allowed the chemical degradation of chlorophyll to pheophytin. Since small amounts of chlorophyllides and pheophorbides could be detected, chlorophyllase seemed also to be partially active. On the other hand, the pheophorbide a monoxygenase seemed to have been inactivated at this temperature causing the accumulation of green derivatives and stopping the breakdown to more advanced stages. At 75 ◦ C, the seeds maintained the green color, but significantly less chlorophyll and pheophytin were accumulated when compared to those obtained at 40 ◦ C. Also, it was observed that the pheophytin/chlorophyll ratio at 75 ◦ C was lower than at 40 ◦ C, showing that postharvest drying at 75 ◦ C blocked the degradation, but did not support
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pheophytinization. These results can be explained by the hypothesis that when a tissue is dried, the cellular water is removed and the remaining water molecules get tightly bound to a macromolecular surface and lose their solvent properties since water mobility decreased sharply. In this case, with the dehydration progress, there was insufficient water to allow pheophytinization to proceed. Moreover, the high temperature might have promoted pigment degradation by other chemical mechanisms, such as Maillard and Strecker reactions, impairing their extraction. The absence of chlorophyllides and pheophorbides was a clear indication that chlorophyllase was not active anymore.
5. Conclusions Results from this study indicate that both the degree of maturation at harvest time and the temperature of postharvest drying influence significantly the chlorophyll content of soybeans. Early swaths and fast drying at high temperatures produced seeds with high levels of green pigments and blocked the breakdown process. In order to avoid retention of chlorophyll and to guarantee marketing quality, the suggestion is to harvest soybean at stage R8 , which can be followed by either fast or slow drying. When it is necessary to harvest grains before full maturity, drying should be performed at temperatures below 40 ◦ C, otherwise, the high amounts of chlorophyll that will be retained may not meet the grading standards.
Acknowledgements We are grateful to Fundac¸a˜ o de Amparo ao Ensino e Pesquisa do Estado de S˜ao Paulo (FAPESP), processes 02/01145-8 and 03/08733-5, for financial support.
References Abraham, V., DeMan, J.D., 1986. Hydrogenation of canola oil as affected by chlorophyll. JAOCS 63, 1185–1188. Adam, C.A., Fjerstad, M.C., Rinne, R.W., 1983. Characteristics of soybean seed maturation: necessity for slow dehydratation. Crop Sci. 23, 265–267. AOAC, 1995. Official Methods of Analysis of the Association of Official Analytical Methods, 40th ed., Washington.
Breemen, R.B., Canjura, F.L., Schwartz, S.J., 1991. Identification of chlorophyll derivatives by mass spectrometry. J. Agric. Food Chem. 39, 1452–1456. Cenkowski, S., Sokhansanj, S., Sosulski, F.W., 1989a. Effect of harvest date and swathing on moisture content and chlorophyll content of canola seed. Can. J. Plant Sci. 69, 925–928. Cenkowski, S., Sokhansanj, S., Sosulski, F.W., 1989b. The effect of drying temperature on green color and chlorophyll content of canola seed. Can. Inst. Food Sci. Technol. J. 22, 383–386. Cenkowski, S., Jayas, D.S., 1993. Potential of in-field and lowtemperature drying for reducing chlorophyll contents in canola (Brassica napus L.). J. Sci. Food Agric. 63, 377–383. Fehr, W.R., Caviness, C.E., 1977. Stage of Soybean Development. Ames, Iowa State University of Science and Technology, p. 11. Ginsburg, S., Matile, P., 1993. Identification of catabolites of chlorophyll-porphyrin in senescent rape cotyledons. Plant Physiol. 102, 521–527. Gomes, M.S., Sinnecker, P., Tanaka, R.T., Lanfer-Marquez, U.M., 2003. Effect of harvesting and drying conditions on chlorophyll levels of soybean (Glycine max L. Merr). J. Agric. Food Chem. 51, 1634–1639. Heaton, J.W., Marangoni, A.G., 1996. Chlorophyll degradation in processed foods and senescente plant tissues. Trends Food Sci. Technol. 7, 8–15. H¨ortensteiner, S., 2004. The loss of green color during chlorophyll degradation—a prerequisite to prevent death. Planta 219, 191–194. H¨ortensteiner, S., Vicentini, F., Matile, P., 1995. Chorophyll breakdown in senescent cotyledons of rape, Brassica napus L.: enzymatic cleavage of phaeophorbide a in vitro. New Phytol. 129, 237–246. H¨ortensteiner, S., W¨uthrich, K.L., Matile, P., Ongania, K.H., Kr¨autler, B., 1998. The key step in chlorophyll breakdown in higher plants: cleavage of pheophorbide-a macrocycle by a monoxigenase. J. Biol. Chem. 273, 15335–15339. H¨ortensteiner, S., Rodoni, S., Schellenberg, M., Vicentini, F., Nandi, O.I., Qui, Y.L., Matile, P., 2000. Evolution of chlorophyll degradation: the significance of RCC reductase. Plant Biol. 2, 63–67. Johnson-Flanagan, A.M., Thiagarajah, M.R., 1990. Degreening in canola (Brassica napus, c.v. Westar) embryos under optimum conditions. J. Plant Physiol. 136, 180–186. Kr¨autler, B., 2001. Unravelling chlorophyll catabolism in higher plants. Biochem. Soc. Trans. 30, 625–630. Macchione, E.L.A., 1998. Espectrˆometro de massa por tempo de vˆoo para an´alise de macromol´eculas. Institute of Physics, University of S˜ao Paulo, S˜ao Paulo, Brazil, p. 126. Macfarlane, R.D., Torgerson, D.F., 1976. Californium-252 plasma desorption mass spectrometry. Science 191, 920–925. Mangos, T.J., Berger, R.G., 1997. Determination of major chlorophyll degradation products. Lebensm Unters Forsch A 204, 345–350. M¨uhlecker, W., Ongania, K.H., Kr¨autler, B., Matile, P., H¨ortensteiner, S., 1997. Tracking down chlorophyll breakdown in plants: elucidation of the constitution of a fluorescent chlorophyll catabolite. Angew. Chem. Int. Ed. Engl. 36, 401–404. Oberhuber, M., Berghold, J., M¨uhlecker, W., H¨ortensteiner, S., Kr¨autler, B., 2001. Chlorophyll breakdown on a nonflures-
P. Sinnecker et al. / Postharvest Biology and Technology 38 (2005) 269–279 cent chlorophyll catabolite from spinach. Helv. Chim. Acta 84, 2615–2627. Oberhuber, M., Kr¨autler, B., 2002. Breakdown of chlorophyll: electrochemical bilin reduction provides synthetic access to fluorescent chlorophyll catabolites. Chembiochem 1, 104–107. Pritchard, J.R., 1983. Oilseed quality requirements for processing. J. Am. Oil Chem. Soc. 60, 174–184. Rodoni, S., M¨uhlecker, W., Anderl, M., Kr¨autler, B., Moser, D., Thomas, H., Matile, P., H¨ortensteiner, S., 1997. Chlorophyll breakdown in senescent chloroplasts. Plant Physiol. 115, 669–676. Schwartz, S.J., Woo, S.L., Von Elbe, H.J., 1981. High performance liquid chromatography of chlorophylls and their derivatives in fresh and processed spinach. J. Agric. Food Chem. 29, 533–535.
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Takamiya, K., Tsuchiya, T., Ohta, H., 2000. Degradation pathway(s) of chlorophyll: what has gene cloning revealed. Trends Plant Sci. 5, 426–431. Usuki, R., Suzuki, T., Endo, Y., Kaneda, T.K., 1984. Residual amounts of chlorophylls and pheophytins in refined edible oils. J. Am. Oil Chem. Soc. 61, 785–788. Ward, K., Scarth, R., Daun, J., McVetty, P.B.E., 1992. Effects of genotype and environment on seed chlorophyll degradation during maturation in four cultivars of oilseed rape (Brassica napus). Can. J. Plant Sci. 72, 643–649. Ward, K., Scarth, R., Daun, J., Vessey, J.K., 1995. Chlorophyll degradation in summer rape and summer turnip rape during seed maturation. Can. J. Plant Sci. 75, 413–420.
Mechanism of soybean (Glycine max L. Merrill) degreening related to maturity stage and postharvest drying temperature Patr´ıcia Sinnecker a , Nelson Braga b , Eduardo L.A. Macchione c , Ursula M. Lanfer-Marquez a,∗ a
Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of S˜ao Paulo, Av. Prof. Lineu Prestes, 580, Bl. 14, 05508-900 S˜ao Paulo, SP, Brazil b Agronomic Institute of Campinas, Av. Bar˜ ao de Itapura, 1481, 13020-902 Campinas, SP, Brazil c Institute of Physics, University of S˜ ao Paulo, P.O. Box 66318, 05315-970 S˜ao Paulo, SP, Brazil Received 28 April 2005; accepted 2 July 2005
Abstract The degreening of soybean seeds was studied relative to their maturity stage, moisture content at harvest and postharvest drying temperature. Studies were performed on Brazilian soybean over its whole maturation period (R6 –R8 ) according to the Fehr and Caviness scale. Chlorophyll and its colored derivatives were quantified as a function of three drying conditions: slow drying at room temperature (25 ◦ C) and oven drying at 40 and 75 ◦ C in order to quantify degreening and pigments produced under these temperatures. Pathways for chlorophyll degradation, enzymatic, chemical or both, could be elucidated by this experimental design. Pigments were quantified by HPLC and identities were confirmed by spectral characteristics, retention times and plasma desorption mass spectrometry (PDMS). Postharvest drying at 25 ◦ C allowed almost complete degradation of chlorophyll in seeds harvested at maturity stage R6 (Fehr scale) or later, with no green pigment detected, which mimics maturation in the field. Fast drying at 40 or 75 ◦ C blocked the breakdown process at all stages of maturity and only seeds harvested at R8 lost their green color. At 40 ◦ C, chemical and/or enzymatic mechanisms of degradation seemed to have occurred, the former is supported by high levels of pheophytins and the latter, by the appearance of small amounts of chlorophyllides and pheophorbides. At 75 ◦ C, considerable levels of only chlorophylls and pheophytins were observed probably due to the inactivation of enzymes. So, chemical pheophytinization was the primary mechanism of degradation. It was concluded that the degree of maturation at harvest time and the temperature of postharvest drying significantly affect the chlorophyll content of soybeans. In order to avoid retention of chlorophyll and to guarantee marketing quality of seeds, harvesting at full maturity followed by fast or slow drying is suggested. If premature harvesting is necessary, the drying should be performed at temperatures lower than 40 ◦ C or seed quality can be compromised. © 2005 Elsevier B.V. All rights reserved. Keywords: Soybean; Degreening; Maturation; Temperature of drying
∗
Corresponding author. Tel.: +55 11 30913684; fax: +55 11 38154410. E-mail address: [email protected] (U.M. Lanfer-Marquez).
0925-5214/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2005.07.002
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1. Introduction The phenomenon of chlorophyll degradation in degreening plants has been a topic of great interest (M¨uhlecker et al., 1997; Oberhuber et al., 2001). However, only a few years ago, the general degradation pathway was established and since then, different catabolic products have been identified in senescent leaves and during fruit ripening of several plant species (H¨ortensteiner et al., 1998). Difficulties in extracting and identifying the chlorophyll catabolic derivatives may explain that the breakdown mechanism is still not fully understood. Particularly, in soybean, this phenomenon has a remarkable significance since the disappearance of the green color affects the market quality of grains. The presence of chlorophyll or other greenish pigments in canola and soybean not only imparts an undesirable dark color and promotes oxidation in the presence of light, it also poisons the catalyst during the hydrogenation process of oils (Usuki et al., 1984; Abraham and DeMan, 1986; Johnson-Flanagan and Thiagarajah, 1990; Ward et al., 1992, 1995; Gomes et al., 2003). The presence of green grains can sometimes be observed as a consequence of hot weather during the maturation period, which causes water stress (excessive water loss) or high rainfalls, which force farmers to anticipate the harvest of the crops in order to avoid losses. These abnormal climate conditions have been observed in the southeast and central west regions of Brazil. Prematurely harvested seeds require postharvest drying in order to reduce the moisture to a maximum of 13%, which is the upper limit considered for safe storage (Adam et al., 1983; Pritchard, 1983). Chlorophyll breakdown is currently described as a multi-step mechanism. The first group of reactions produces greenish derivatives, while the more advanced steps produce colorless compounds. According to H¨ortensteiner et al. (2000), the whole process is as complex as chlorophyll biosynthesis. The main changes occurring in the first group of reactions correspond to the release of Mg by displacement with two H under acidic conditions and/or by the action of Mgdechelatase and the cleavage of the phytol chain by the enzyme clorophyllase, which produces greenish intermediates, such as pheophytins, chlorophyllides and pheophorbides, all of them showing an intact tetrapyr-
role ring (Heaton and Marangoni, 1996; Mangos and Berger, 1997). The second group of reactions is responsible for degreening by the rapid formation of colorless and polar derivatives, due to the opening of the tetrapyrrole ring by the action of pheophorbide a monoxygenase, which seems to act specifically on pheophorbide a (M¨uhlecker et al., 1997; Rodoni et al., 1997; Oberhuber and Kr¨autler, 2002). Although the general mechanism of degradation has already been established, there is no information about the external environmental influences on the mechanism involved in chlorophyll degradation in soybeans during maturation and postharvest storage (Ginsburg and Matile, 1993; Takamiya et al., 2000). The objective of this study was to investigate the degreening of soybean seeds relative to their maturity stage and postharvest drying temperature by analyzing the chlorophyll catabolites formed in each case.
2. Material and methods 2.1. Experimental design and plant material Two Brazilian soybean (Glycine max L. Merr) cultivars, IAC-17 and IAC-22, were grown at the Agronomic Institute of Campinas, S˜ao Paulo, Brazil. The factorial experimental design was 2 × 6 × 3 (two cultivars × six maturation stages × three drying conditions). Seeds were planted as described before (Gomes et al., 2003) and harvested at six developmental stages, from R6 to R8 , according to the scale proposed by Fehr and Caviness (1977). The first swath occurred at physiological maturation (R6 ), nearly 100 d after planting and continuing until commercial maturation (full maturity or R8 ), at nearly 126 d after planting. For better monitoring of the colored derivatives formed during the degreening, three more intermediate steps R6 –R7 (I), R6 –R7 (II) and R6 –R7 (III) with intervals of approximately 5 d were added. Analysis of variance was performed by using the SAS 6.0 statistical software package and significance was determined at the 0.05 level. The results were similar for both cultivars and data presented in graphs show mean values of triplicates performed in each experimental condition.
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2.2. Drying conditions Swathed seeds, in their intact pods of each maturation stage, were divided into four groups and three of them submitted to three drying temperatures until their moisture contents had decreased to 13%. Slow-dried seeds were obtained by air drying at room temperature (25 ± 5 ◦ C) for approximately 10 d and fast-dried seeds were obtained by dehydration in an air-circulation oven at 40 ± 2 and 75 ± 2 ◦ C, respectively. Thereafter, seeds were threshed by hand and stored at 2 ◦ C until analysis of moisture and pigments. The fourth group was analyzed immediately after harvesting. 2.3. Moisture determination The moisture content of recently harvested seeds was determined by oven drying 10 g samples at 105 ◦ C until reaching constant weight. Residual moisture in fast- or slow-dried seeds was determined by drying 2 g of powdered soybean seeds at 105 ◦ C until reaching constant weight (AOAC, 1995). 2.4. Pigment extraction Five grams of dried seeds were ground in a laboratory mill (Polymix KCH-Analytical mill A10, Kinematica AG, Luzern, Switzerland), mixed with 80% cold acetone in a proportion of 1–6 (w/v) and filtered through a sintered plate funnel under vacuum. Then, the precipitated pellet was extracted twice until the residue was colorless. The combined filtrates were transferred to a separating funnel in 60 mL of petroleum ether and 20 mL cold deionized water. The aqueous layer was discarded after vigorous shaking and standing. The washing procedure was repeated 3–4 times to remove acetone and the ether phase containing the pigments was dehydrated with sodium sulfate anhydride. This solution was filtered, transferred to a beaker and dried in a rotary evaporator (Heidolph Elektro GmbH & Co., WB2000, Kelheim, Germany). The residue was dissolved in 4–5 mL acetone (HPLC grade, Carlo Erba) and filtered on a 0.45 m membrane filters (PTFE, Millipore) before HPLC analysis. 2.5. HPLC pigment analysis A triple pump with a ternary gradient (LC10ADVP), computer-controlled (Class-VP 5.032) sys-
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tem (Shimadzu, Tokyo, Japan Class-M10A) was used for HPLC analysis. Separation was achieved using a Nucleosil ODS 100 column (5 m, 250 mm × 4 mm i.d., pre-column 11 mm × 4 mm i.d., Macherey-Nagel, D¨uren, Germany) and a 1 mL/min flow rate. The UV–vis diode array spectrophotometric detector (SPD-M10AVP) was set at 432 nm for chlorophylls and chlorophyllides, 409 nm for pheophytins and pheophorbides according to their maximal absorbance and 669 nm for all catabolites simultaneously (Breemen et al., 1991). Additionally, UV–vis absorption spectra were recorded in the 380–670 nm wavelength range with an 8 nm bandwith. The solvent system consisted of: (A) methanol/ 1.0 M ammonium acetate buffer (pH 7.0) (80:20, v:v); (B) methanol:acetone (80:20, v:v); (C) acetone. The separation started linearly from 100% A to 100% B for 20 min, followed by an isocratic hold for 2.5 min, then linearly for 7.5 min to 100% C and was finished by conditioning the column for 4 min with starting eluent A (Mangos and Berger, 1997). An aliquot of 10–50 L of each sample was injected in three replications. The isolated pigments were identified by their retention times and by their absorption spectra in comparison with their respective standards as well as literature data (Johnson-Flanagan and Thiagarajah, 1990). 2.6. Preparation of pigment standards Chlorophylls (a and b) were purchased from Sigma Chemical Co. (St. Louis, MO). As chlorophyll derivatives are not available in the market, the greenish chlorophyll catabolites were prepared from spinach. Fresh spinach (200 g) leaves obtained from local markets were washed, drained, chopped and blended with 100 mL of distilled water for 4 min, so that a homogeneous puree was obtained and then freeze dried (Supermodulyo, Edwards High Vacuum International, NY, USA), powdered and stored at −20 ◦ C, prior to usage. Five grams of the dehydrated spinach was extracted with 80% acetone as previously described (Section 2.4) and brought to a final volume of 5 or 10 mL acetone. Chlorophylls (a and b) present in the spinach extract were separated and collected in a preparative HPLC system (Fraction Collector Module, FRC-10A, Shimadzu, Kyoto, Japan). The separation was achieved by applying an isocratic run with methanol/acetone
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(80:20, v/v) and a 10 mL/min flow rate. Fractions of chlorophylls a and b were collected, concentrated under vacuum until dryness and stored in a known volume of acetone. Pheophytins (a and b) were prepared by dropwise addition of 1.0 M HCl to the collected fractions of chlorophylls (a and b) and conversion was completed in approximately 15 min (Schwartz et al., 1981; Breemen et al., 1991) changing their color from green to olive brown. Extracts containing chlorophyllides (dephytylated derivates) were obtained by incubating 5 g of dehydrated spinach with 100 mL acetone/0.2 M Tris–HCl buffer pH 8.0, 1:1, v:v) at 40 ◦ C for 2 h in the dark by the action of the endogenous chlorophyllase on chlorophylls (a and b) (H¨ortensteiner et al., 1995). Then, the chlorophyllides present in the water/acetone phase were separated by adding diethyl ether in a separatory funnel. The aqueous phase was discarded after the extract had been washed twice and the organic layer containing the pigments was collected and evaporated under vacuum after removing residual moisture over anhydrous sodium sulfate. The chlorophyllides (a and b) were dissolved in acetone and analyzed by HPLC. Pheophorbides (a and b) were prepared from the chlorophyllides by HCl acidification in a similar way as described for obtaining pheophytins from chlorophylls. Pheophorbides (a and b) and chlorophyllides (a and b) were separated and collected in a preparative HPLC system in an isocratic run with methanol/1.0 M ammonium acetate buffer (pH 7.0)/acetone (80:10:10, v:v:v). All handling procedures were carried out under dim light and cold solvents were used to avoid degradation. The calibration curves of chlorophylls (a and b), pheophytins (a and b), chlorophyllides (a and b) and pheophorbides (a and b) were linear over the range of 2.5–100 mg kg−1 (correlation coefficient = 0.99). In addition to HPLC analysis, the identity of the synthesized standards was confirmed by plasma desorption mass spectrometry (PDMS) (Macfarlane and Torgerson, 1976). All pigments previously separated and collected by HPLC were concentrated and suspended to a volume of acetone (approximately 200 mL, concentration 2 g L−1 ). Aliquots of 5 mL of each solution were dropped on an aluminized polyester foil (1 m) and covered with a thin nitrocelullose film. After being dried with a stream of N2 , the samples were
immediately inserted into the spectrometer. The equipment was operated at 9.33 × 10−5 Pa and the acceleration potential voltage was +13.5 kV. The time of flight information about secondary ions was accumulated for 106 start events (Macchione, 1998). A mass spectrum of each compound was determined in duplicate.
3. Results 3.1. Synthesis of chlorophyll derivative standards It was necessary to synthesize pheophytins, pheophorbides and chlorophyllides a and b due to the lack of commercially available chlorophyll derivatives. Fresh spinach leaf extracts were successfully employed and the identities of the synthesized compounds were confirmed by their spectral characteristics in 80% acetone over a 400–800 nm range, as shown in Fig. 1. The spectra obtained were similar to those reported by Johnson-Flanagan and Thiagarajah (1990). As expected, the spectra of chlorophyllides a and b were similar to those of their respective parents chlorophylls a and b because the only difference in molecular structure is lack of a phytol chain in chlorophyllides. Similarly, the spectra of pheophorbides a and b may be compared to pheophytins a and b, respectively. Additionally, chlorophylls a and b and pheophytins a and b had their identities confirmed by plasma desorption mass spectrometry. Table 1 shows the molecular ions and the major fragments obtained by mass spectrometry in comparison with the calculated molecular weights. The observed molecular weights of the pigments perfectly matched their calculated values. The standards obtained in the present work were used to identify the pigments present in soybeans over the whole experiment. Table 1 Molecular weights of chlorophyll derivatives isolated from soybeans calculated and determined by plasma desorption mass spectrometry Pigments
M•+
Mass spectra most abundant fragment ion
Calculated molecular weight (mass units)
Chlorophyll a Chlorophyll b Pheophytin a Pheophytin b
893.0 907.2 871.0 884.9
481.2 496.0 419.7 473.0
893.5 907.5 871.2 885.2
615.6 629.1 592.5 606.1
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Table 2 Retention times (min) of pigments present in soybean seeds Pigments
λmax (nm)
Retention time (min) ± S.D.
Chlorophyllide b Chlorophyllide a Pheophorbide b Pheophorbide a Lutein Chlorophyll b Chlorophyll b Chlorophyll a Chlorophyll a Pheophytin b Pheophytin b Pheophytin a Pheophytin a
432, 669 409, 432, 669 432, 654 409, 669 446, 472 432, 651 432, 651 410, 432, 669 410, 432, 669 432, 654 432, 654 409, 669 409, 669
7.69 9.79 12.60 13.64 19.25 22.09 22.64 23.77 24.29 27.60 28.07 28.91 29.25
± ± ± ± ± ± ± ± ± ± ± ± ±
0.67 0.89 0.38 0.22 0.26 0.26 0.25 0.43 0.51 0.48 0.38 0.59 0.32
n = 108; S.D., standard deviations.
3.2. Separation and identification of soybean pigments by HPLC Pigments observed in samples’ chromatograms were identified according to their absorption spectra and retention times in comparison with the respective standards produced in our laboratory and also to data previously published (Johnson-Flanagan and Thiagarajah, 1990; Mangos and Berger, 1997). Table 2 presents the retention times and the wavelengths of maximum absorbance (λmax ) of all pigments isolated according to the procedures and chromatographic conditions described. The results express the mean retention times accompanied by their standard deviations of all samples analyzed (n = 108). As can be observed in Table 2, the pigments were eluted according to their decreasing polarity, starting at 7.70 min with chlorophyllide b and ending at about 30 min with pheophytin a . All pigments were well separated except for the epimers chlorophyll a and b and pheophytin a and b which eluted shortly after their corresponding parents. Epimers were identified by comparison with data reported in literature (Mangos and Berger, 1997). 3.3. Pigment changes during soybean maturation Fig. 1. Visible absorption spectra of pigment standards in 80% acetone over the range of 400–800 nm, analyzed by HPLC and scanned in the stop-scan mode.
The HPLC chromatograms shown in Fig. 2A–C represent changes in types and amounts of pigments that occur in freshly harvested seeds over the entire
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Fig. 2. HPLC chromatograms of pigments extracted from soybean seeds: (A) harvested at R6 (green seeds); (B) harvested at R6 –R7 ; (C) harvested at R8 (yellow seeds).
field maturation period. Seeds harvested at very early stages of maturation, such as R6 (Fig. 2A), but not submitted to any postharvest drying process presented a complex pattern of pigments. The more prevailing pigments found were lutein, chlorophylls a and b, pheophytins a and b and the less prevailing ones were identified as chlorophyllides a and b, pheophorbide a, the epimers chlorophyll b , pheophytins a and b , as well as some unidentified xantophylls. At the intermediate stages between R6 and R7 (Fig. 2B), a gradual disappearance of both types and amounts of pigments
was observed and at advanced stages of maturation (R8 ) (Fig. 2C), only lutein could be detected in very low amounts and a few unidentified small peaks of xantophylls. 3.4. Relationship between major green pigments and moisture contents in freshly harvested seeds Fig. 3 shows the changes in total chlorophyll, pheophytin and moisture contents of freshly harvested seeds during the maturation period. The first date of swathing
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Fig. 3. Total chlorophyll, total pheophytin and moisture content of recently harvested soybean seeds over the whole maturation period.
occurred at 100 d after planting and corresponded to stage R6 according to the scale of Fehr and Caviness (1977). Seeds reached the stage R7 at 120 d after planting and reached full maturity (R8 ) at 126 d after planting. Additionally, three samples were also swathed between R6 and R7 corresponding to intermediate stages, noted as R6 –R7 (I, II and III). At early stages of maturation, the moisture content decreased slowly and linearly from 71.6% at R6 (100 d after planting) to 62.1% at 113 d after planting. Thereafter, there was a rapid decrease to 13.3% at full maturity (R8 ). In the beginning of the maturation period when seeds contained approximately 70% moisture, the level of total chlorophyll was nearly 500 mg kg−1 . As maturation progressed, there was an exponential decline of chlorophyll down to its almost complete disappearance. Considering the initial content of 500 mg kg−1 chlorophyll at R6 as 100%, there was a reduction of 92%, reaching 48 mg kg−1 at full maturity. Only small amounts of pheophytins were detected in each stage, representing less than 10% of the original chlorophyll contents. Dephytylated derivatives (pheophorbides and chlorophyllides) were only found in some samples in trace amounts despite the degradation of chlorophyll and low levels of pheophytins (results not shown).
3.5. Effect of postharvest drying on chlorophyll degradation Fig. 4 shows the contents of the main pigments found in seeds from R6 to R8 (chlorophylls a and b and pheophytins a and b), after drying at 25, 40 and 75 ◦ C. Seeds dried at 25 ◦ C had their green pigments almost completely degraded, except when harvested at early maturation stage (R6 ). The chlorophyll content of dried seeds at 25 ◦ C harvested at R6 was about 50 mg kg−1 and decreased to 3 mg kg−1 at full maturity. Pheophytins were also present in low levels, starting at 6 mg kg−1 and ending at undetectable levels. Chlorophyllides and pheophorbides were not detected in these samples. Seeds submitted to oven drying at 40 ◦ C with circulating air presented high levels of chlorophylls and an accumulation of pheophytins could also be observed. Both pigments decreased over the maturation process, though. At early swaths, total chlorophyll and pheophytin contents were about 300 and 250 mg kg−1 , respectively. As maturation progressed, these amounts decreased to 18 and 13 mg kg−1 , respectively. At this drying temperature, very small amounts of dephytylated pigments (pheophorbides and chlorophyllides) were detected but not quantified.
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Fig. 4. Chlorophyll (a and b) and pheophytin (a and b) content in soybean seeds swathed on six dates, after postharvest drying at three temperatures.
At 75 ◦ C, high amounts of chlorophylls and pheophytins were observed, though lower than those in seeds dried at 40 ◦ C. Another remark is that the pheophytin/chlorophyll ratio changed in comparison to 40 ◦ C, since less pheophytins were found.
4. Discussion The presence of unacceptable high levels of immature green seeds is sometimes the reason for downgrading Brazilian oilseeds. The present study aimed to get a better understanding of the degreening process during seed maturation and to evaluate the influence of postharvest drying temperatures. The greenish
pigments derived from chlorophyll degradation, analyzed and quantified individually by HPLC were the main concern. To accomplish this objective, the solvent extraction procedures usually applied to green leaves had to be optimized for seeds, containing only small amounts of green pigments. The chromatographic procedures reported by Mangos and Berger (1997) for the quantification of chlorophyll derivatives in spinach were successfully employed for soybeans. Fig. 2A–C shows the chromatogram of pigments extracted from fresh green seeds harvested at different stages of development and analyzed immediately after harvest. The extracts from R6 presented a complex mixture of pigments with a broad spectrum of polarities, from the least polar phytyl esters of chlorophylls
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and pheophytins to the most polar dephytylated derivatives (chlorophyllides and pheophorbides). As maturation progressed, pheophytins were not accumulated and dephytylated derivatives (chlorophyllides and pheophorbides) could only be detected in trace amounts in some samples. At full maturity, only lutein could be found in significant amounts. These results are similar to those reported by Johnson-Flanagan and Thiagarajah (1990) and Cenkowski et al. (1989a,b; Cenkowski and Jayas, 1993) in studies on canola. Therefore, under ideal conditions of soybean growth, chlorophyll degrades completely and there is no accumulation of any greenish derivatives, which could be explained by the mechanism of chlorophyll breakdown proposed by Ginsburg and Matile (1993), H¨ortensteiner (2004), H¨ortensteiner et al. (1995, 1998) and Kr¨autler (2001). According to these authors, chlorophylls are continuously degraded to colorless compounds by a multi-step pathway, which involves simultaneous action of enzymes, weak acids, oxygen, light and heat. The loss of green color in the end of the degradation process is a consequence of the oxygenolytic opening of the porphyrin macrocycle of chlorophyll catalyzed by the joint action of two enzymes, pheophorbide a oxygenase and red chlorophyll catabolite reductase. The elution with a ternary gradient proved to be essential to achieve satisfactory separation of each component in the mixture. In addition, diode array detection was a useful tool for examining the spectral characteristics of each eluted peak. In Fig. 3, the relationship between the main green pigments (total chlorophyll and total pheophytins) and moisture content can be observed. During field maturation, without any postharvest drying, total chlorophyll disappeared in an exponential pattern and a complete degreening was outstanding, since no pheophytins were accumulated. Seed moisture declined slowly at the beginning of maturation and rapidly at the end. Although chlorophyll breakdown and moisture loss occur simultaneously during maturation, they do not appear to control each other and moisture content cannot be used to predict chlorophyll content. Chlorophyll degradation showed quite different results after postharvest drying, depending on the temperature used. Local room temperature air drying at 25 ◦ C, until moisture content reached 13%, resulted in an almost
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complete disappearance of green color, even when seeds were swathed at early stages of development. This process mimics field maturation under adequate climatic conditions. At this temperature, the mechanism of degradation involves the action of all the enzymes responsible for chlorophyll disappearance, including pheophorbide a monoxygenase. This key enzyme catalyzes the cleavage of the porphyrin structure originating colorless compounds, which explains the absence of any kind of green derivatives, and consequently, yellowing of seeds. Therefore, slow drying at low temperatures (25 ◦ C), independent of the maturation stage at harvest time, reduced moisture and chlorophyll levels below 13% and 22 mg kg−1 , respectively, meeting the requirements established for soybean trading. On the contrary, when the crops were dried at 40 ◦ C or higher, the degreening process was blocked and easily seen at early stages of maturation. It was also evidenced by a significant retention of greenish derivatives, providing some insight into possible degradation pathways. At this temperature, although a significant part of the original amount of chlorophylls had changed to pheophytins, chlorophyll was still the most abundant pigment. The high levels of pheophytins seemed mainly to have been formed by chemical degradation, but the action of Mg-dechelatase, which might be still active at some extent at this temperature, cannot be excluded. On the other hand, the high amounts of remaining chlorophyll may be the consequence of excessive, rapid water loss. This might have compromised the activity of the degrading enzymes activity as well as the reduction of water/acid mobility and even not allowed the chemical degradation of chlorophyll to pheophytin. Since small amounts of chlorophyllides and pheophorbides could be detected, chlorophyllase seemed also to be partially active. On the other hand, the pheophorbide a monoxygenase seemed to have been inactivated at this temperature causing the accumulation of green derivatives and stopping the breakdown to more advanced stages. At 75 ◦ C, the seeds maintained the green color, but significantly less chlorophyll and pheophytin were accumulated when compared to those obtained at 40 ◦ C. Also, it was observed that the pheophytin/chlorophyll ratio at 75 ◦ C was lower than at 40 ◦ C, showing that postharvest drying at 75 ◦ C blocked the degradation, but did not support
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pheophytinization. These results can be explained by the hypothesis that when a tissue is dried, the cellular water is removed and the remaining water molecules get tightly bound to a macromolecular surface and lose their solvent properties since water mobility decreased sharply. In this case, with the dehydration progress, there was insufficient water to allow pheophytinization to proceed. Moreover, the high temperature might have promoted pigment degradation by other chemical mechanisms, such as Maillard and Strecker reactions, impairing their extraction. The absence of chlorophyllides and pheophorbides was a clear indication that chlorophyllase was not active anymore.
5. Conclusions Results from this study indicate that both the degree of maturation at harvest time and the temperature of postharvest drying influence significantly the chlorophyll content of soybeans. Early swaths and fast drying at high temperatures produced seeds with high levels of green pigments and blocked the breakdown process. In order to avoid retention of chlorophyll and to guarantee marketing quality, the suggestion is to harvest soybean at stage R8 , which can be followed by either fast or slow drying. When it is necessary to harvest grains before full maturity, drying should be performed at temperatures below 40 ◦ C, otherwise, the high amounts of chlorophyll that will be retained may not meet the grading standards.
Acknowledgements We are grateful to Fundac¸a˜ o de Amparo ao Ensino e Pesquisa do Estado de S˜ao Paulo (FAPESP), processes 02/01145-8 and 03/08733-5, for financial support.
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