Introduction

The varieties of commercial importance for the production of nopal in Mexico are Milpa Alta, Atlixco, COPENAS and Valtierrilla (^{Flores et al., 1995}. The international quality standard nopal, ^{CODEX STAN 185-1993}, provides that the tender cladodes can be marketed when they present from 9 cm to 30 cm in length (^{FAO-WHO, 1993)}. Cladodes 20 cm in length and weight of 90-100 g, are marketed as whole stalk (^{Cantwell, 1999)}; while smaller cladodes (12 cm) or longer (about 30 cm) are used for the production of fresh-cut produce (^{Rodriguez, 2002)}.

The enzymatic browning is one of the main problems limiting the marketing of nopal, both in their whole form as fresh-cut (^{Rodriguez, 2002)}. This deterioration is of great importance for its visual impact affecting the organoleptic acceptance and merchantable quality and reduce the nutritional value of fruits and vegetables (^{Artes et al., 2007)}. The enzymatic browning occurs on the surface of cuts and wounds and is caused by the enzyme polyphenol oxidase (PPO). This enzyme in the presence of oxygen phenolic substrates acts on forming quinones. These compounds react with each other or with other compounds (amino acids, peptides and reducing sugars) melaninas forming compounds, red or black coffee (^{Artes et al., 20079}. However, the formation of quinones is reversible in the presence of reducing agents such as ascorbic acid, which are capable of reducing quinones to native phenolic compounds; while the formation of melanin is irreversible (^{Barbagallo et al., 2009)}. Enzymatic browning occurs when phenolic substrates, PPO and molecular oxygen under appropriate conditions bind pH, temperature and water activity (^{Artes et al., 1998)}.

There are differences in the degree of dimming, the content and type of phenolic compounds, PPO activity and the ascorbic acid content between varieties of vegetables such as potatoes, artichoke and other (^{Barbagallo et al., 2009}; ^{Cabezas et al., 2009a b)}. Additionally, it has been observed that there are changes in the activity of PPO, content of phenolic compounds, darkening and ascorbic acid content during the development of leafy vegetables like kale (Brassica oleracea var. Acephala) (^{Korus, 2011)})9 and lettuce (^{Chutichudet et al., 2011)}. ^{Aguilar et al. (2007)} evaluated PPO activity and browning potential in cladodes of 21 varieties of Opuntia, finding significant differences between varieties, presenting 'Chicomostoc', 'Negrito' and 'Jade', the lowest values of dimming.

However, PPO activity values of these three varieties was similar to other varieties had a higher potential blackout. These authors did not evaluate other variables such as ascorbic acid (reducing quinones), total phenols, or the content of PPO substrates, which can influence the development of darkening. It has been observed that the darkening of the fabric occurs when the ascorbic acid content is exhausted (^{Adams and Brown, 2007)}. Additionally, it indicated that ascorbic acid may inhibit PPO with decreasing pH of the cytosol (^{Yoruk and Marshall, 2003)}. ^{Degl'Innocenti et al. (2007)} concluded that the high content of ascorbic acid of arugula (Eruca sativa) is highly associated with resistance to darkening shown during storage of this vegetable in its fresh-cut form, while ^{Bottino et al. (2009)} observed similar behavior in spinach.

Although it has been evaluated PPO activity and browning of different varieties of cactus, it is unknown whether there are changes in the content of phenolic compounds and ascorbic acid during the development of the cladodes of commercial sizes established by the standard of the Codex Alimentarius (^{CODEX STAN 185-1993)}. Therefore, the objective of this work was to study the activity of polyphenol oxidase (PPO), the content of phenolic compounds and ascorbic acid, and the degree of dimming during the development of commercial varieties of nopal.

Materials and methods

Experimental Material

The nopal was used (Opuntia ficus indica) varieties COPENA F-1, COPENA V-1, Milpa Alta and Atlixco, a commercial plantation located in Hermosillo, Sonora, Mexico (29° 11' 27.53" north latitude, 110° 51' 17.13" west longitude). The four varieties of nopal were under the same conditions of commercial cultivation, same type of soil, climate and crop management. Cladodes harvest of the four varieties and sizes are manually performed the same day and at the same time, in july. Tender cladodes harvested, had leaves, and immediately transported to the laboratories of the institution, where firm and free of defects cladodes were selected. They were classified into six developmentbased on length, including cladodes 9-30 cm in length, sizes considered commercial by the international quality standard nopal, ^{CODEX STAN 185-1993} (^{FAO-WHO, 1993)}. They were then measured length, weight and thickness (Table 1), and stored at -20°C.

Table 1 Length, weight and thickness during the development of nopal cladodes varieties of COPENA F-1. COPENA V-1, Milpa Alta and Atlixco. 

*Los datos representan el promedio y su desviación estándar, n= 30.

They were subsequently cleaved again and stored at -20°C. In the frozen tissue analyzes degree of blackening and ascorbic acid were performed; and acetone powders prepared for analysis PPO activity. Additionally, they were taken in triplicate, 150 g of nopal frozen, lyophilized in a freeze dryer trays Freezemobile UNITOP 600L. The lyophilized vials nopal placed in hermetically sealed plastic, which were stored at -20°C, these lyophilized samples were used in the determination of total phenols. All determinations were performed in triplicate.

Methodology

Extraction and specif ic activity of PPO. Preparation of acetone powder extraction and PPO were performed according to the procedure indicated by ^{Sciancalepore and Longone (1984)}. The 200 g of finely chopped nopal weighed, were added 400 mLofcoldacetone (-20°C) and 1.4 g of polyethyleneglycol, homogenized in a blender. The homogenate obtained was filtered with vacuum using Whatman No.1. To the residue were performed 2 to 3 washes with 200 mL of cold acetone, until a white powder (acetone powder) was obtained, it was allowed to dry at room temperature for 24 h, placed in plastic bottles sealed and they stored at -20°C. For extraction of PPO 0.3 g of acetone powder they are weighed and added 40 mL of potassium phosphate (K₃PO₄) 0.05 M containing KCl 1 M to pH 8.5. This mixture was subjected to constant agitation for 1 hour at 5 °C. Subsequently, the sample was centrifuged at 4 101 x g at 4°C for 45 min time. The supernatant was filtered with a organza fabric. The filtrate obtained represented the enzyme extract, which was divided into two parts which were used for protein analysis and measurement of specific activity of PPO.

The specificactivity of the PPO was determined by measuring initial rate of formation of quinone following the methodology proposed by ^{Sciancalepore and Longone(1984)}. AonemL of enzyme extract was added 1 mL of 4-methylcatechol in 0.02 M citrate buffer 0.1 M pH 3.5 (as defined in preliminary tests the optimum pH for activity nopal of PPO). The initial rate of formation of quinone indicated by the increase in absorbance at 395 nm was measured. The slope of the initial part of the curve was calculated by expressing the results as units of activity per milligram protein. One unit of enzyme activity is defined as the amount of enzyme which causes a change in absorbance of 0.01/min.

The soluble protein content in enzyme extracts was determined by the ^{Bradford method using bovine serum albumin as standard (Bradford, 1976)}.

Total phenolic. In 500 mg of lyophilized nopal three successive sonication extractions were carried out with methanol to 80% according to the proposed Escarpment and Gonzalez (2000) and ^{Kähkönen et al. (1999)}. The determination of total phenols was carried out according to the procedure of ^{Singleton and Rossi (1965)}. The increase in absorbance at 765 nm is related to the concentration of total phenolic with a standard curve prepared with gallic acid. The results were expressed as mg of gallic acid equivalents (EAG)/g dry weight.

Total ascorbic acid. Extraction was performed on 30 g of frozen sample was homogenized with 100 mL of buffer solution (30 g of phosphoric acid, 80 mL of acetic acid in one liter of water), filtered in organza fabric and subsequently Millipore filters 0.22 microns. The determination was performed according to the technique ^{Doner and Hicks, (1981)} in a HPLC Varian 9012 with a μBondapak NH₂ column at a wavelength of 268 nm. An external standard of L-ascorbic 0.05 mg/mL acid was used. The dithiothreitol (DTT) was used to reduce dehydroascorbic acid to L-ascorbic acid. The results were expressed as mg total ascorbic acid/100 g fresh weight.

Degree of darkening. This method is based on measuring the absorbance of the quinone formed due to oxidation of phenolic compounds by PPO in the presence of oxygen. The wavelength of maximum absorbance of the quinone formed from catechol and L-DOPA is 390 and 480 nm, respectively (^{Nicolas et al., 1994)}. The 20 g samples were homogenized with 100 ml of buffer pH 4.46 citrate (pH average nopal vegetables), incubated in a water bath at 35°C and constant stirring for 48 h. Subsequently, the samples were filtered with organza fabric and the filtrate was centrifuged for 10 min at 18 500 rpm in a refrigerated centrifuge Beckman Model J2-21. In preliminary tests of the extract absorbance at 380 to 510 nm it was measured and found that the highest absorbance at 420 nm was obtained. The absorbance of the supernatant at 420 nm was read on a Perkin Elmer Lambda 3A (UV-VIS). Results were expressed as absorbance values at 420 nm (^{Sapis et al., 1983)}.

Experimental design and statistical analysis. An experimental design was used in blocks with factors variety and state of development, in which the range factor was used as a block. An analysis of variance was performed by general linear models (GLM) at a significance level of 0.05. When there were significant mean comparisons were made by theTukey test. All data were processed in the computational statistical package NCSS (^{Hintze, 1997)}.

Results and discussion

The morphological characteristics of the four varieties of nopal studied are different. Cactus cladodes COPENA F-1 are characterized by long and thin with many thorns; COPENA while the V-1 are discoid and few bones. COPENA F-1 cladodes had the highest length and COPENA V-1 of the minor, Milpa Alta and Atlixco had similar lengths (Table 1). The Atlixco variety had the highest weight and variety COPENA V-1 minor; while COPENA F-1 and Milpa Alta presented a similar weight. The thickness of the cladodes COPENA V-1 was higher and those of Milpa Alta was the lowest.

PPO specific activity. In the four varieties of nopal studied, the specific activity of PPO showed a tendency to increase significantly (p< 0.05) as advanced development status cladode, with values of 36.83 units of activity per milligram protein in the state reaching values 62.08 units of activity per milligram of protein in the state VI; which represents a 168% increase (Figure 1). Comparing the specific activity of PPO of different varieties no effect (p> 0.05) of the variety found in the activity of this enzyme, showing these average similar values of 48.71, 46.81, 49.15 and 47.43 units of activity per milligram of protein, for COPENAF-1, COPENAV-1, Milpa Alta and Atlixco, respectively.

Figure 1 Specific activity of polyphenol oxidase during the development of nopal cladodes of COPENA F-1, COPENA V-1, Milpa Alta and Atlixco varieties. One unit of activity (U) was defined as the amount of enzyme which causes an increase in absorbance of 0.01/min. The vertical bars indicate the standard deviation for each stage of development, n= 3. 

These results are consistent with that published by ^{Aguilar et al. (2007)} who found no significant differences in the activity of PPO cladodes about 20 cm of varieties COPENA F-1, Atlixco and Milpa Alta. It has been shown that the pH causes conformational changes in the enzyme and affects the kinetics of PPO activity. The pH optimum plant PPO vary, with optimum pH values between 4 and 8. It is estimated that the optimal pH of grape and apple PPO is 3.5 to 4.0 (^{Yoruk and Marshall, 2003)}. In this study it was determined that 3.5 is the optimal pH for PPO activity nopal, this value is similar to the optimum pH of PPO activity apple and grape. In the present study the results of specific activity of PPO were expressed as activity units per mg protein, while Aguilar et al. (2007) results expressed as activity units per g fresh weight, the above is not possible to compare the activity values of this study with those of these authors. The increase in PPO activity observed during the development of nopal consistent with that observed by ^{Korus (2011)}, who showed that PPO activity increased as they advanced the state of maturity of the leaf kale.

Total phenolics. The state of development had a significant effect on total phenol content of cactus pads of the four varieties studied (p< 0.05), finding that the cladodes in the development stage II had a higher content than cladodes of V states and VI (Figure 2). Also, significant differences (p< 0.05) were found in the content of these compounds in different varieties, observing that COPENAF-1 had the highest content (19.89 mg EAG/g dry weight), followed by Milpa Alta (16.06 mg EAG/g dry weight) presented similar values to COPENA V-1 (13.85 mg EAG/g dry weight) and finally Atlixco had the lowest content (12.12 mg EAG/g dry weight), although similar to COPENA V-1.

Figure 2 Contents of total phenols during the development of nopal cladodes varieties COPENA F-1, COPENA V-1, Milpa Alta and Atlixco. EAG: gallic acid equivalents. The vertical bars indicate the standard deviation for each stage of development, n= 3. 

As in this study, ^{Guevara et al. (2010)} found significant differences in total phenol content of 10 varieties of nopal (8 wild and 2 grown) collected in different regions of Mexico, showing the highest values (19.9 mg EAG/g dry weight) in the Morado variety and cladodes minors in the range plug (less than 2 mg EAG/g dry weight). These authors attribute the differences between the varieties to differences in climatic conditions. However, in the present study they evaluated the four varieties were grown under the same conditions and were sampled on the same day, suggesting that the differences are intrinsic to the variety. The total phenol content of cladodes COPENA F-1 is similar to that found by ^{Guevara et al. (2010)} in the wild variety Morado (19.9 mg EAG/g dry weight). The total phenolic contentof the four varieties tested in this study is greater than the commercial variety of O. ficus indica, Blanco had a content of 5.25 mg EAG/g dry weight (^{Guevara et al., 2010)}. However, the values found in Milpa Alta cladodes of this study are lower at 32.20 mg EAG/g dry weight shown by ^{Corral et al. (2008)}, which could be attributed to differences in climatic conditions growing regions.

The total phenol content of nopal ranged from 10.63-24.23 mg equivalent gallic acid/g dry weight in the various development stages of the varieties studied. These values are higher than those of other horticultural products such as onion, cucumber, carrot, tomato and potato, which showed values of 0.4-6.6 mg of EAG/g dry weight (^{Kähkönen et al., 1999)}.

Total ascorbic acid. The content of total AA in the four varieties studied decreased signif icantly (p< 0.05) as advanced development cladode, with values of 13.50 to 7.41 mgAA/100 g fresh weight in cladodes states I and VI, respectively (Figure 3). This indicates a decline of almost 50% of the content. significant differences (p< 0.05) in AA content between varieties studied were found, it was observed that COPENA F-1 had a higher content (11.90 mg AA/100 g fresh weight), followed by COPENA V-1 (10.72 mg AA/100 g fresh weight); while Milpa Alta and Atlixco presented the lowest content (8.79 and 8.19 mg AA/100 g fresh weight, respectively). These results show that the cladodes COPENAF-1 had anAAcontent of 40% higher than those of Milpa Alta and Atlixco.

Figure 3 Total ascorbic acid content during the development of nopal cladodes varieties COPENA F-1, COPENA V-1, Milpa Alta and Atlixco. The vertical bars indicate the standard deviation for each stage of development, n= 3. 

^{Rodriguez and Cantwell (1988)} found no consistent pattern of change in the content of ascorbic acid during the development of cladodes of three species of Opuntia, with values of 7-18 mg AA/100 g fresh weight, although it should be noted these authors used a colorimetric method unlike the present study using HPLC technique. These results agree with those of ^{Rodriguez and Villegas (1997)} who reported values of 12 mg AA/100 g fresh weight in cladodes of 20 cm length of two varieties of nopal vegetables (COPENA F-1 and COPENA V-1). ^{Corral et al. (2008)} showed a content of 22.2 mgAA/100 g fresh weight cladodes in MilpaAlta grown in the State of Mexico, Mexico; higher than the values of this study. These differences could be attributed to differences in the methodology used in the determination or the effect of the climatic conditions of the cultivation sites, since it has been shown that both the light intensity and ambient temperature inf luence the content of AA (^{Lee and Kader, 2000)}.

The highest content of AA in the early development stages of cactus cladodes agrees with that shown by ^{Parsons and Fry (2012)}, who indicated that high concentrations of ascorbic acid are characteristic of tissue rapidly growing, despite these also they have high activities of ascorbate oxidase, an enzyme that initiates ascorbic acid catabolism.

The content of total AA of nopal vegetables obtained in this study is similar to that reported for bananas, fresh potatoes and fresh tomatoes; although it is lower than reported for blackberry, melon, grapes, kiwi, tangerine, orange, raspberry, broccoli, cabbage, caulif lower and spinach (^{Lee and Kader, 2000)}. Additionally it was observed that in nopal, L-ascorbic acid is the main component of the total ascorbic acid, approximately 80% on average for the different stages of development and varieties; this behavior is similar to that shown by ^{Lee and Kader (2000)} in different fruits and vegetables.

An inverse relationship between ascorbic acid and specific activity of PPO with correlation coefficients of -0.80, -0.93, -0.88 and -0.87 in varieties COPENA F-1, COPENA V-1, Milpa Alta and Atlixco, respectively was found. This behavior shows that by decreasing the content of total ascorbic acid increases the specific activity of PPO.

Darkening. Values darkening displayed by cladodes nopal of different varieties and stages of development were low, even after 48 h with absorbance at 420 nm of 0 to 0.2 (Figure 4), finding that no significant changes (p> 0.05) in the degree of darkening at 420 nm during the development of the cladodes of the four varieties tested. However, significant differences (p< 0.05) in the darkening between different varieties were found, showing that COPENA F-1 and Milpa Alta had the highest values, followed by varieties COPENA V-1 and Atlixco that had similar values.

Figure 4 Degree of obscuration (Abs 420 nm) during the development of nopal cladodes varieties COPENA F-1, COPENA V-1, Milpa Alta and Atlixco. The vertical bars indicate the standard deviation for each stage of development, n= 3. 

The results of this study are consistent with those shown by ^{Aguilar et al. (2007)}, who evaluated PPO activity and browning potential in cladodes 30 days old of 21 varieties of Opuntia, finding significant differences between varieties analyzed, presenting 'Chicomostoc', 'Negrito' and 'Jade', the lower values of dimming. However, PPO activity values of these varieties were similar to other varieties had a higher potential blackout. ^{Artes et al. (1998)} indicated that the species and variety are among the most important factors that influence susceptibility to browning of fruits and vegetables, noting that potato and apple susceptibility to darkening depends on the variety.

Heads et al. (2009a) evaluated five potato varieties (Agata, Agria, Almera, Marabel and Vivaldi), finding differences in the darkening between varieties, showing that Marabel was characterized by a low content of total phenols, low PPO activity and lower incidence dimming, while Almera had the highest total phenol content, PPO activity and browning incidence. Similarly, ^{Cabezas et al. (2009b)} they found differences in susceptibility to darkening of five varieties of artichoke (C3, Catanese, Tema, Violetto Foggiano and Violetto Sardo), indicating that C3 showed the highest total phenol content and PPO activity and the highest incidence dimming; while Catanese showed the highest content of total ascorbic acid and the lowest incidence of darkening.

The results of this study show that as indicated in potato and apple (^{Artes et al., 1998)}, potato (^{Cabezas et al., 2009a}) and artichoke (^{Cabezas et al., 2009b)} variety is the factor that more influences susceptibility to darkening of nopal cladodes as the susceptibility to darken cladodes was similar between the various stages of development. However no differences were observed specific activity of PPO in the four varieties tested. The cladodes COPENA F-1 showed a greater tendency to darken and even showed the highest content of ascorbic acid compound that reduces the quinones, also showed the highest content of total phenols, which are substrates of PPO enzyme. Atlixco cladodes showed less tendency to darken and a lower content of ascorbic acid and total phenolic compounds.

Conclusions

The specific activity of PPO cladodes of four varieties of Opuntia ficus indica significantly increased with advancing development, reaching maximum values in the state VI; and a reverse behavior occurred in the content of ascorbic acid, which decreased to advance the state of development. Differences in total ascorbic acid content, total phenol and darkening of the varieties were found COPENA F-1, COPENA V-1, Milpa Alta and Atlixco, presenting cladodes COPENA F-1 the highest content of total ascorbic acid, total darkening and phenols, while Atlixco showed the lowest values. These results suggest that from the point of view darkening the cladodes of Atlixco may be more suitable to be used for the production of cut fresh cactus.