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Introduction
Pouteria sapota (Jacq. H. E. Moore & Stearn) is a tropical species that produces a fruit with sweet, soft, and slightly fibrous flesh, which shows a reddish-orange color due to the presence of carotenoids (Alia-Tejacal et al., 2005a; Zou et al., 2012). In Mexico, the fruit of P. sapota is called mamey sapote and its cultivated area is about 1336 ha (SIAP, 2016); the consumption is mainly in fresh stage or processed into milkshakes, ice cream or desserts.
P. sapota belongs to the climacteric type of fruit. During the ripeness process of climacteric fruit, four stages are defined according to the respiration rate and ethylene production (preclimacteric, climacteric rise, climacteric peak and postclimacteric) and changes in the chemical composition and texture of the flesh occurred throughout the whole process (Watada et al., 1984; Torres-Rodríguez et al., 2011).
The flesh of this fruit contains a high amount of total soluble phenolics (TSP), which diminish with ripening (Torres-Rodríguez et al., 2011). In TSP fraction, flavanols as catechin, epicatechin, gallocatechin, and catechin 3-O-gallate have been identified, in addition to gallic (Torres-Rodríguez et al., 2011; Ma et al., 2004), syringic, p-hydroxybenzoic, and p-coumaric acids (Yahia et al., 2011).
There are no reports of proanthocyanidins (PAs) in P. sapota, although in matured fruit, a slight astringency maybe detected. This astringency could be associated with the presence of PAs, which are oligomers or polymers of polyhydroxy flavan-3-ols with units of epi-catechin (procyanidins) and epi-gallocatechin (prodelphinidins) (Schofield et al., 2001). Their polyphenolic structure and particular degree of polymerization, confers to PAs, biological activities beneficial to human health (Silva et al., 2009; Hellström & Mattila. 2008). The consumption of PAs of high molecular weight has been associated with the reduction of different types of cholesterol (Zou et al., 2012) and with a low damage to the vascular tissue, by atherosclerosis (Gorinstein et al., 2011). In addition, studies with animals have allowed qualify the PAs as promising compounds to prevent the development of degenerative diseases of the nervous system (Lee et al., 2010; Wang et al., 2012).
On the other hand, two of the main enzymes related with fruit browning, are polyphenol oxidase (PPO) and peroxidase (POD) (Luo and Xie, 2012). No published reports were found on the relationship of the activity of these enzymes and the browned flesh of MS that could be observed in fruit at consumption maturity or at over ripened fruit. Since there are no reports about analysis of PAs in mamey sapote and only few reports about enzymatic activity during ripening of mamey sapote fruit, and given the important biological activities attributed to these polyphenols, the objectives of this study were: i) to study proanthocyanidins (PAs) in mamey sapote fruit, ii) to determine the activity of the polyphenol oxidase (PPO) and peroxidase (POD), and to evaluate their changes during the ripening process
Materials and Methods
Plant material
Two hundred mamey sapote fruits were collected at Tuxtla Chico, Chiapas, Mexico (14° 56’ 20” N; 92° 10’ 05” W) at physiological maturity, using as harvest index the presence of a pink-orange tonality in flesh, which was verified by making an incision near the apex. Transportation of fruits to the experimental site, at Texcoco, Mexico (19° 29′ 23″ N, 98°53′37″ W), was carried out by land, using plastic boxes. Within 20 h after harvest, fruits were stored at 12 °C, which is a temperature that can extend the postharvest life of mamey sapote fruits without chilling injury (Alia-Tejacal et al., 2005b).
Fruit firmness
Every three days, three fruits were randomly taken from the storage chamber in order to evaluate flesh firmness (FF) which was measured with a texture analyzer (Stable Micro Systems, TA-XT2i, UK). The fruit was peeled at two points of its equatorial region and with a conical element of 2.6 cm at the base, an angle of 75°, and a rounded tip, the lectures were taken under a routine where the fruit flesh was deformed up to 4 mm at a 3 mm s−1 velocity. FF was expressed in newtons (N). Thereafter, a sample of flesh was taken from each fruit to be placed in liquid nitrogen to freeze and store at -20 ° C for subsequent evaluation of phenolic compounds and enzymatic activity.
Extraction of phenolic compounds (proanthocyanidins)
Fifteen grams of frozen flesh were ground in a domestic blender, using a small volume container. One gram of the homogenized material was weighed and mixed with 20 mL of solvent (acetone:water:acetic acid, 75.0:24.5:0.5 v/v/v) in an Erlenmeyer flask (Prior et al., 2010). The mixture was placed in an ultrasonic bath (Branson (Model 2510, USA) for 30 min and shaken on a horizontal equipment (Gyratory shaker, model G10, USA) for one additional hour. The sample was centrifuged (Universal 32, Hettich Zentrifugen, Germany) at 2200 × g for 10 min at 20 °C, and the supernatant was recovered, filtered with Whatman No. 1 paper, and brought to a volume of 20 mL with the extraction solvent. This extract was divided into two equal volumes; one of them was concentrated at 40 °C in a rotary evaporator (Heidolph, Laborata 4010, Germany), brought to 10 mL with distilled water, and subjected to liquid-liquid extraction with 15 mL of ethyl acetate. The organic phase was recovered and the liquid-liquid extraction repeated for three times. The collected organic phases were pooled and concentrated to dryness in a rotary evaporator. The residue was re-suspended in 2 mL of methanol grade HPLC and used for the HPLC-DAD analysis. The other volume of 10 mL was used for quantitation of phenolic compounds by several methods.
Quantification of total soluble phenolic compounds by Folin-Ciocalteu method
The colorimetric method of Folin-Ciocalteu (Singleton & Rossi, 1965) was used. A standard curve of gallic acid (Sigma, MN) was prepared to express the concentration of phenols in mg gallic acid equivalents (GAE) per 100 g of fresh tissue (FM).
Proanthocyanidins Method of butanol/HCl
The method of Porter et al. (1986) was used with a volume of 500 μL of extract that was placed in test tubes with bakelite stopper. The butanol/HCl method confirms the presence of condensed tannins or proanthocyanidins, when an intense red color is developed by heating the sample, because oxidation of the leucoanthocyanins (Schofiel et al., 2001). For running the test, 3 mL of concentrated butanol/HCl solution (95:5 v/v) (JT Baker S.A. de C.V., Mexico) were mixed with 100 μL of ferric reagent (2 % ammonium ferric sulphate in 2 N HCl). The tubes were sealed and placed in a boiling water bath at 92 °C for 60 min. Thereafter, the absorbance of the sample was read at 550 nm in a spectrophotometer (Perkin Elmer, Lambda 25, USA) that was adjusted to cero with the blank prepared with all the solvents and 500 µL of solvent extraction. Sorghum flour (Dykes & Rooney. 2006) and calyxes of jamaica were used as positive and negative control tests, respectively. In both cases the extraction of PAs was prepared out as did with mamey sapote flesh. Due to the lack of chemical standards of condensed tannins adequate for mamey sapote fruit, the results are reported as values of absorbance.
Method of DMAC (4 - (dimethylamino) cinnamaldehyde)
The DMAC colorimetric method (Wallace & Giusti, 2010) was used, placing 2380 μL of methanol HPLC grade in test tubes with an aliquot of 20 μL of the extract of phe-nols and 100 μL of DMAC reagent prepared at 2 % in 6 N sulphuric acid in methanol. Methanol HPLC grade was used as blank. A standard curve with catechin (Sigma, MN) solubilized in the solvent used for extracting PAs. The PAs concentration was expressed in mg of catechin equivalents (CE) per100 g fresh weight (FW).
Method of vanillin
The method proposed by Sun et al. (1998) was used. A volume of 2500 μL of vanillin 1 % was placed in test tubes with 1000 μL of extract. Thereafter, 2500 μL of 8 % HCl solution was added and tubes were shaken on a vortex for 20 s and centrifuged at 2200 × g for 10 min. The reaction was allowed to stabilize for 10 min in darkness. A standard curve of proanthocyanidin B2 (Fluka, MN) were prepared. The standard was solubilized in the solvent used for extracting PAs. The concentration of PAs was expressed as mg of proanthocyanidin B2 equivalents (PB2E) per 100 g FW.
Proanthocyanidins analysis by HPLC-DAD
A high-performance liquid chromatograph (HPLC; PerkinElmer Series 200, USA) was used with an UV-DAD detector, that was controlled by a computer through the TotalChrom™ software. For the analysis of the ethyl acetate fraction, a Phenomenex Luna C18 analytical column (250 x 4.6 mm, 5 mm) was employed. The method described by Cabrera et al. (2003) was used. The mobile phase was acetic acid (0.1 %) in water (A) and methanol (B), with flow rate of 1 mL min-1. The composition of the mobile phase started with 100 % A for 1 min, increased linearly to 63 % B in 27 min, returning to initial conditions in 5 min. The detection was performed at 280 (catechins) and 360 nm (flavonols). The column temperature was set at 25 °C. An injection volume of 10 μL was used. Samples were filtered through a nylon membrane of 0.45 μm (Millipore™) before they were injected in the equipment. All solvents were HPLC grade. Standard curves of gallic acid, catechin, epicatechin, gallocatechin, and epigallocatechin-3-gallate (Sigma-Aldrich, MN) were prepared at concentrations of 5 to 20 ppm. The curves were run at 280 nm and had R2 values higher than 0.997. The identification of the peaks was performed through comparison of pure standards retention times and the UV spectra obtained.
Enzymatic activity
Forty grams of flesh of mamey sapote fruit were mixed with 50 mL of cold acetone (15 °C). The mixture was homogenized in a domestic blender (Man LMU 9013, Mexico) for 1 min and filtrated with vacuum to separate the solid phase, which was named acetone powder. This procedure was repeated until the powder took a white color. Finally, the acetone powder was allowed to dry at room temperature and was stored at 20°C for subsequent evaluation. The methods of Lamikanra (1995) and Flurkey & Jen (1978) were applied to evaluate polyphenol oxidase (PPO; EC. 1.14.18.1) and peroxidase (POD; EC. 1.11.1.7) enzyme activities, respectively, both with 0.2 g of acetone powder that were mixed with 5 mL of Tris-HCl 100 mM pH 7.1 (TH) that contained polyvinylpyrrolidone (1 %). Homogenization was applied (Ultra Turrax T25 equipment, IKA Labortechnik, Staufen, Germany) for 30 s and centrifugation (Sorvall centrifuge RC-5B, GMI, USA) for 20 min at 10000 x g and 4°C. To evaluate PPO, an assay was performed with 2 mL of supernatant that were mixed in a quartz cell with 3 mL of catechol 60 mM dissolved in TH. The change of absorbance was evaluated at 420 nm with a Genesys 10 spectrophotometer (Thermo Fisher Scientific Remel Inc., USA) using TH as blank. In the case of POD, 0.05 mL of supernatant were mixed with 2.6 mL of TH, 0.25 mL of guaiacol 0.1 M, and 0.1 mL of hydrogen peroxide 0.25 %. The change of absorbance was also evaluated at 470 nm for 3 min. Assays were conducted at 20 °C and the enzymatic activity was reported as U g1, where an enzymatic unit (U) was defined as 1 μmol of obenzoquinone formed per min for PPO or 1 μmol of tetraguaiacol min1 for POD.
In addition to the enzymatic activity, the color of P. sapota fruit flesh was measured during the storage at 12°C with the purpose of detecting enzymatic browning. The equipment used was a MiniScan XE Plus colorimeter (Hunter-Lab, model 45/0-L). The CieLab scale was used and the color parameters L*, a* and b* were obtained. Three fruits were analyzed for each sampling day.
Statistical analysis
A completely randomized design was used to perform an analysis of variance and routines of means comparison (Tukey, ≤ 0.05), with data of fruit firmness and phenolic compounds with the purpose of comparing the two stages of the maturation process (pre-climacteric and horticultural maturity). The experimental unit consisted of three fruits taken randomly on each sampling day from the storage. Pearson correlation analysis was performed with the data obtained from the variables analyzed. The statistical software SAS (SAS, 2002) was used.
Results and Discussion
Total soluble phenolics and PAs during fruit ripening
The mamey sapote fruit ripening at 12 °C was monitored as a function of the flesh firmness. It had two well defined stages. The first one (stage 1) had a duration of 15 days and was characterized by firmness values between 49.3 and 39.6 N. This stage is equivalent to the preclimacteric phase mentioned by Watada et al. (1984). The second one (stage 2), had a duration of six days. In this stage the firmness decreased drastically between days 15 and 16, to values of 2.7 -1.2 N (Figure 1). Although the respiration rate of fruit was not monitored in this study, Torres-Rodríguez et al. (2011) mentioned that the sharp decrease in firmness coincides with the climacteric peak, in which the highest respiration rate is reached. In stage 2 the fruit was in horticultural maturity, according to that described by Watada et al. (1984). The fruit firmness profile was similar to that described by Alia-Tejacal et al. (2002) under storage at 15 °C. The content of total soluble phenols (TSP) ranged from 2,195.5 to 1,017.6 mg GAE 100 g-1 FW between days 1 and 15 of storage (stage 1), respectively (Figure 1). Upon completion of 16 days of storage (DS) the TSP fell to 395.3 mg GAE 100 g-1 FM. This change was equivalent to a reduction of approximately 2.1 times, relative to the content at the beginning of the storage. This reduction was lower than that reported by Alia-Tejacal et al. (2002) who observed a reduction of 3.2 times in the content of TSP in mamey sapote fruit stored at room temperature (20 °C and 50-60 % relative humidity) when they went through ripening.
Figure 1 Total soluble phenols (TSP) and flesh firmnes of fruit of mamey sapote during ripening at 12 °C. Bars represent standard deviation from the mean of three fruits
The average TSP values at preclimacteric stage (stage 1) and horticultural maturity (stage 2) were similar to those found by Saucedo-Veloz et al. (2001) who reported 1.53 % for fruit at stage 1 and 0.21 % at stage 2, but they were lower than the values of 1653 and 646 mg GAE kg-1 FM found by Alia-Tejacal et al. (2005a) for the same stages of maturation. In this regard, it has been reported that the content of TSP in mamey sapote fruit is affected by temperature (Alia-Tejacal et al., 2002), ripeness degree (Saucedo-Veloz et al., 2001), geographical origin (Torres-Rodríguez et al., 2011; Ma et al., 2004), and the process of extraction and solvent used (Yahia et al., 2011).
The mamey sapote is a fruit with values of total soluble solids between 20 and 30 °Brix. Sugars accumulation occurs in the first two weeks after harvest (Torres-Rodríguez et al., 2011) thus, the TSP quantified by the Folin-Ciocalteu method may have high interference of sugars, which also react in the assay (Singleton et al., 1999). In plant tissues with high content of direct reducing sugars, it is convenient to remove them before to run Folin-Ciocalteu assay (Stratil et al., 2007).
By applying the butanol/HCl method to the acetone extracts of mamey sapote, the presence of polymeric PAs or condensed tannins in the fruit was confirmed, whose pattern during the ripening process was similar to that observed in TSP (Figure 2).
Figure 2 Condensed tannins content (absorbance) and firmness of the flesh in mamey sapote fruits during ripening at 12 °C. Bars represent the mean ± standard deviation of n = 3.
Immature fruits commonly have high amounts of condensed tannins as a tool of plants to safeguard the species survival. The content and types of these phenolic compounds decrease with fruit maturity. However, some fruits maintain high amounts of particular condensed tannins at consumption maturity, which give them their particular taste and flavor.
The behavior of PAs by vanillin and DMAC methods showed the same pattern as that of the TSP, when fruits passed from preclimacteric stage (stage 1) to horticultural maturity (stage 2) through ripening, however the values of PAs were lower, and in general, they were less variable than TSP, particularly in the period between days 1 and 15 of storage. The values of PAs by the vanillin method were higher and more variable than that of DMAC (Figure 3).
Figure 3 Proanthocyanidins content with the methods of vanillin (A) and DMAC (B) in mamey sapote fruits stored at 12 °C. Bars represent the mean ± standard deviation of n = 3 fruit.
The differences in the content of PAs between the two methods may be due to the predominance of different types of PAs in each maturity stages and to the particular affinity of each method for specific types of PAs. The DMAC method detects more accurately monomers of flavan-3-ols, and reacts only with terminal units, so it does not discriminate between monomers, dimers or trimers (Hummer & Schreier, 2008). Catechin and epicatechin react easily with the DMAC reagent in comparison with that of oligomeric PAs (Li et al., 1996) which have more affinity for the vanillin reagent, especially that with m-oriented hydroxyl groups (Hageman et al., 1997). Neither of both methods is specific for condensed tannins (proanthocyanidins polymer), since any monomer of flavanol reacts in the assay (Dykes & Rooney, 2006).
The values of PAs obtained with the DMAC method are similar to those reported for apple ‘Red Delicious’(162 ± 10 mg 100 g-1 fresh fruit) and red grapes (54 ± 4.2 mg 100 g-1 of fresh fruit) (Hellström & Mattila, 2008). According to the values of PAs obtained with vanillin method, mamey sapote contains more PAs than the aforementioned fruits. In addition, the values of PAs found in mamey sapote were similar to those reported in the fruit of persimmon, that ranged from 61.2 to 91.5 mg of catechin equivalents per gram of dry weight (DW) with DMAC method, and from 3.15 to 4.03 mg of catechin equivalents per gram DW, with vanillin method (Zou et al., 2012). It is noteworthy that in mamey sapote the content of PAs determined with the vanillin method is much higher than that with the DMAC method at ripening stage 2. This pattern is contrary to what has been reported for persimmon. These results suggest that in mamey sapote the oligomeric PAs, associated with diverse health benefits (Gorinstein et al., 2011; Zou et al., 2012; ), could represent a rich source of these phytochemicals in fruit which are not commonly predominant in other fruit species in which monomeric PAs predominate as persimmon.
Table 1 summarizes the average results of dry matter, fruit firmness and phenolic compounds content for the two ripening stages studied. Dry matter of the flesh of mamey sapote fruit slightly increased with ripeness which means that it was less juicy. In stage 1, the fruit of mamey sapote had high values of TSP and PAs, but in horticultural maturity (stage 2) these values decreased significantly. From stage 1 to stage 2, TSP was reduced 2.1 times; PAs content determined by DMAC was reduced 2.8 times, while PAs with the vanillin method was reduced 1.6 times. Considering the specificity of DMAC and vanillin methods toward a particular type of PAs, these results mean that at horticultural maturity (stage 2), the mamey sapote fruit has predominance of oligomeric PAs. However, it is necessary to run new studies to determine the chemical structures of these compounds, and even more important, to evaluate their biological activities.
Table 1 Dry matter, firmness of pulp, and content of phenolic compounds in mamey sapote fruit during ripening at 12 ± 1°C.
| Proanthocyanidins (PAs) | ||||||
|---|---|---|---|---|---|---|
| Ripening stages | % Dry matter | Firmness (N) | TSP¶ | DMAC§ | Vanillin‡ | |
| Preclimacteric stage | ||||||
| Stage 1 (1-15 days) | 26.4±5.1 | 47.8 a | 1298.0 a | 223.5 a | 486.0 a | |
| horticultural maturity | ||||||
| Stage 2 (16-21 days) | 30.4±7.2 | 2.9 b | 622.5 b | 79.9 b | 307.1 b | |
| 3.1 | 386.9 | 58.9 | 108.9 | |||
¶: TSP= fenoles solubles totales obtenidos con el método de Folin-Ciocalteau (mg GAE 100 g-1 FM). §: expresados en mg CE 100 g-1 peso fresco (FW). ‡: expresados en mg PB2E 100 g-1 FW. HSD: diferencia significativa honesta.
Proanthocyanidins analysis by HPLC
In the ethyl acetate fraction (EAF), the phenolic compounds identified were: gallic acid (GA), catechin (CAT), epicatechin (EPC), and epicatechin 3-gallate (EPCG). The contents of GA, CAT, EPC and EPCG did not show statistical differences (p<0.05) between preclimacteric stage (stage 1) and horticultural maturity (stage 2) (Table 2). However, a compound of low polarity and therefore with a high retention time, was observed in a high proportion in relation to the identified phenolic compounds. This compound, named P6, was the most abundant in the EAF and showed a gradual increase throughout the ripening process, with values in stage 2 that were almost double than those in stage 1 (Table 2). P6 was tentatively identified as a dimer of epicatechin/ chatechin according to its UV spectrum and its retention time (26.5 min). Previously, Yahia et al. (2011) identified a dimer of epicatechin in fruit of mamey sapote through mass spectrometry analysis, which could be the compound, P6, identified in our study. None of the samples analyzed in this study contained gallocatechin that was reported to be in high amounts in mamey sapote fruit cultivar Magaña (Ma et al., 2004). The lack of information about the cultivar or genotype used in the majority of the studies on sapote mamey fruit makes it difficult to assess reliably a comparison of results.
Table 2 Content of phenolic compounds in two stages of fruit ripening of mamey sapote fruit analyzed by HPLC-DAD.
| Ripening stages | GA† | CAT | EPGC | EPC | P6 |
|---|---|---|---|---|---|
| Preclimacteric stage | |||||
| Stage 1 (1 -15 days) | 4.37 a | 4.26 a | 0.72 a | 3.00 a | 26.90 a |
| Horticultural maturity | |||||
| Stage 2 (16 - 21 days) | 7.75 a | 5.45 a | 0.58 a | 5.14 a | 49.92 b |
| HSD | 2.61 | 3.35 | 0.67 | 4.00 | 16.17 |
†:GA: gallic acid, CAT: catechin, EPGC: epicatechin 3-gallate, EPC: epicatechin, P6: Peak with retention time of 26.5 min; data is expressed in µg g-1 FW. HSD: honest significant difference.
One should note that with the exception of GA, the PAs CAT, EPC and EPGC quantified by HPLC would be preferentially determined by the DMAC method. The fact that total PAs analyzed by the DMAC method showed a significant decrease during the transition from stage 1 to stage 2, might suggest that additional PAs compounds not quantified in the current work might be responsible for the observed changes.
Enzymatic activity
The activity of the PPO enzyme had a continuous increment during storage, rising from 61.6 (±27.2) U g1 at preclimacteric stage (stage 1) to 182.2 (±59.4) U g1 at horticultural maturity (stage 2). In the case of POD, the activity remained at a low value during stage 1, but showed a notable increment during stage 2, changing from 633.6 (±183.9) U g1 at preclimacteric stage to 7235 (±1139.3) U g1 at horticultural maturiy (Figure 4). PPO catalyzes the conversion of phenols to o-diphenols through hydroxylation and conversion of o-dihydroxyphenols to o-quinones through oxidation, resulting in browning of plant tissue (4). POD is another oxidoreductase enzyme involved in plant tissue browning, since it also oxidizes diphenols as substrates (Mdluli, 2005), and could act synergistically with PPO due to formation of hydrogen peroxide during the PPO oxidation activity (Luo & Xie, 2012).
Figure 4 Variation in the activity of the polyphenol oxidase (PPO) and peroxidase (POD) enzymes during the ripening process of mamey sapote fruit stored at 12 °C. Bars represent the mean ± standard deviation of n = 3.
Of the color parameters monitored during the ripening process of mamey sapote fruit, only the luminosity showed changes during the ripening process. This variable exhibited a reduction from day 15 of storage (data not showed) that coincided with the increase in POD activity.
Conclusions
The degree of maturity of the mamey sapote based in flesh firmness of the fruit is related to the content and type of proanthocyanidins. According to the results obtained by DMAC and vanillin analytical methods, zapote mamey is an important source of proanthocyanidins, with predominance of the oligomeric type. Of the enzymes analyzed during the maturation process, the polyphenol oxidase gradually increased its activity, while the peroxidase detonated its activity from the fall of the firmness of the fruit. A significant inverse correlation (r = -0.4385) was observed between the content of total soluble phenols and the activity of the polyphenol oxidase but the correlation with peroxidase was not significant.
