Effects of polyphenol oxidase and peroxidase activity, phenolics and lignin content on the browning of cut jicama

Postharvest Biology and Technology 33 (2004) 275–283

Effects of polyphenol oxidase and peroxidase activity, phenolics and lignin content on the browning of cut jicama Elia N. Aquino-Bolaños a , E. Mercado-Silva b,∗ a

b

Universidad Autónoma del Estado de Hidalgo, Centro de Investigación en Ciencia y Tecnolog´ıa de Alimentos, Instituto de Ciencias Agropecuarias, Tulancingo Hidalgo, Mexico Universidad Autónoma de Querétaro, Facultad de Qu´ımica, Departamento de Investigación y Posgrado en Alimentos, Centro Universitario Cerro de las Campanas S/N, P.O. Box 76010 Querétaro, Qro., Mexico Received 22 October 2003; accepted 13 March 2004

Abstract Jicama (Pachyrizus erosus L. Urban) is a plant native to Mexico and Central America where its root is eaten for its succulence and its sweet starchy taste. During its commercialization, as a whole or cut root, it is easily damaged, and brown areas appear on the root. This browning has been attributed to polyphenol oxidase (PPO) which acts on the phenols; nevertheless, the participation of other enzymes in the process has not been evaluated. The objective of this work was to relate the activity of PPO and peroxidase (POD) and the phenolics and lignin content during the development of browning on cut jicama stored at 10 and 20 ◦ C for a week. Their color changes, phenolics and lignin content and the activities of PPO and POD on external as well as internal tissue were analyzed daily. After a week at 20 ◦ C, the phenolic content, expressed as gallic acid in fresh tissue, increased from 0.37 to 1.04 g kg−1 while the lignin content, expressed as coumaric acid, increased from 16.50 to 52.22 mg kg−1 . The lignin values were correlated with color changes expressed by chroma (R2 = 0.8765). PPO and POD activities were induced by damage and were greater in the damaged external tissue than in the internal tissue; they were also influenced by temperature. POD was at a maximum on the sixth day of storage at 20 ◦ C (7500 units of activity (UA) kg−1 ). Coumaric, caffeic and ferulic acids, coniferaldehyde and coniferyl alcohol (precursors in lignin synthesis) proved to be good substrates for POD with Km of 40.0, 89.4, 150.0, 44.1 and 580 ␮M, respectively. Results suggest that the browning of cut jicama at 20 ◦ C is related to the process of lignification in which the peroxidase enzyme plays an important role. © 2004 Elsevier B.V. All rights reserved. Keywords: Jicama; Pachyrhizus erosus; Browning; Peroxidase; Phenols

1. Introduction Horticultural products are susceptible to mechanical damage during harvest, transport, storing (Uritani, ∗ Corresponding author. Tel./Fax: +11-52-442-1921307ext.5579. E-mail address: [email protected] (E. Mercado-Silva).

1999) or minimum processing. Jicama, native to both Mexico and Central America, generates a succulent, sweet, starchy root that is eaten fresh. It is currently cultivated in different tropical and subtropical areas around the world (Sorensen, 1996); it is easily damaged during harvest due to the scuffing of the periderm against the soil. Nevertheless, during commercialization it also experiences physical damage

0925-5214/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2004.03.009

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from the rubbing of roots against each other, cuts and cracking (Mercado-Silva and Cantwell, 1998; Mercado-Silva et al., 1998), and this facilitates the entry of microorganisms which cause decay, water loss and the appearance of brown areas that decrease the quality of the root (Cantwell et al., 1992). Mechanical damage causes physical stress, which damages the plant tissue and alters the phenolic metabolism (Rhodes and Wooltorton, 1978). Mechanical damage to jicama pieces at 5 and 10 ◦ C caused surface browning associated with increased phenolic content and activity of the phenylalanine ammonia lyase enzyme (Aquino-Bolaños et al., 2000). In plant tissues, browning induced by mechanical damage has been attributed to PPO activity acting on phenolic compounds, causing their oxidation and polymerization with the consequent development of a brown color (Vámos-Vigyázó, 1981; Macheix et al., 1990; Lee and Whitaker, 1995). Nevertheless, there are reports that demonstrate little or no correlation between browning and PPO activity in fruit (Amiot et al., 1992; Cheng and Crisosto, 1995). Hyodo et al. (1978) also indicated that he found no relation between increased PPO activity and the development of russeting spots on lettuce (Lactuca sativa) leaves exposed to ethylene. Similarly Larrigaudiere et al. (1998) reported the same regarding the development of brown heart and its relation to PPO activity in pears stored in a controlled atmosphere. To counteract mechanical damage, plants create a physical barrier to prevent tissue destruction, including the synthesis of polyphenols such as lignin and suberin (Walter et al., 1990). Lignin is a complex polymer composed of phenyl propane units derived from three cinnamyl alcohols (monolignols), p-coumaryl, coniferyl and sinapyl (Whetten et al., 1998). From a functional point of view, lignin supports and strengthens the cell wall, facilitates water transport and prevents degradation of cell wall polysaccharides acting as a line of defense against attacks by pathogens and insects, etc. (Hatfield and Vermerris, 2001). The peroxidases are involved in several metabolic plant processes such as the catabolism of auxins, the formation of bridges between components of the cell wall and oxidation of the cinnamyl alcohols before their polymerization during lignin and suberin formation (Quiroga et al., 2000). These promote the oxidative coupling of monolignols by means of a reaction

of free radicals dependent on H2 O2 in the last stage of lignin biosynthesis (Campa, 1991). Since lignins or their intermediaries, formed by the oxidation of monolignols, have reddish-brown colorations, the browning of some fresh cut products may be due to the presence of these compounds produced by the tissue as part of a healing mechanism, contrary to the established concept that browning is due to uncontrolled phenol oxidation by the PPO enzyme. The former hypothesis is based on previous results, which reported that PPO activity did not completely account for the browning of pieces of jicama (Aquino-Bolaños et al., 2000), meaning that the lignification process could be occurring on the surface of damaged areas, thus accounting for the browning of the tissue. The objective of this study was to evaluate the role of the PPO and POD enzymes and the changes in phenolic and lignin content during the development of browning in jicama cylinders stored at 10 and 20 ◦ C.

2. Materials and methods 2.1. Biological and chemical materials Jicama roots (Pachyrhizus erosus L. Urban, cv. Cristalina) were cultivated in the experimental fields of the Instituto Nacional de Investigaciones Forestales, Agr´ıcolas y Pecuarias (INIFAP), located in Celaya, Guanajuato, Mexico, during the 1999 and 2000 production cycles. They were harvested by hand upon reaching commercial size (0.7–1.0 kg) and taken to the laboratory where roots free of mechanical damage or decay were selected. They were washed with fresh water and disinfected in a sodium hypochlorite solution of 200 ␮l l−1 (pH 7) for 15 min and drained at room temperature. All reagents used were from the Sigma Chemical Co. 2.2. Preparation and storage of cylinders The ends of each root were removed, leaving an equatorial section approximately 5 cm thick from which cylinders were obtained (1.8 cm in diameter and 5 cm long) using a stainless steel borer. The cylinders were placed on a tray over ice and covered with cheesecloth to prevent dehydration; the cylinders were handled with care to avoid, as far as possible,

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microbiological contamination. Groups of six jicama cylinders were formed, and each group was placed in a 250 ml plastic container. Each container was considered as a sample unit. Each sample unit was covered with cheesecloth, and a group of 12 units was placed in a 20 l glass container with a flat top having two glass tubes. A flow of humidified air of 0.3 l min−1 (>95% relative humidity) was introduced through one of the tubes and the same air flow came out the other to prevent CO2 accumulation. Three different glass containers were placed in each cold room maintained at 10 or 20 ◦ C. Daily samples were taken of the jicama cylinders. Their color was measured and the external tissue (<2 mm deep) and internal tissue of each cylinder were separated to analyze total phenol content and PPO and POD activity. Another portion of the external tissue was freeze dried and its lignin content was analyzed. 2.3. Methods 2.3.1. Color measurement Color was measured at the base of each cylinder using a color meter (Minolta CM 2002), with a D65 illuminator and a 10◦ observer and registering the values of L∗ , a∗ , b∗ , chroma (C∗ = (a∗2 + b∗2 )1/2 ) and hue value defined as artg(b∗ /a∗ ) as indicated by McGuirre (1992). 2.3.2. Total phenol content Quantification of the total soluble phenolic compounds was carried out using the method proposed by Singleton and Rossi (1965). Five grams of finely diced jicama tissue were homogenized (Ultra-Turrax T25) with 20 ml of 80% ethanol for 1 min; the homogenized mix was then filtered through two layers of cheesecloth, and the filtered liquid was centrifuged at 10,000 × g for 15 min. One ml of the supernatant liquid was mixed with 1 ml of Folin Ciocalteu reagent and 10 ml of sodium carbonate (7%). This was increased to 25 ml with distilled water and left to settle for 1 h. The absorbance was then read at 750 nm. A standard curve of gallic acid (0–0.12 g l−1 ) was used for quantification. 2.3.3. Lignin content Lignins are polymers of phenolic compounds insoluble in all solvents; therefore, it was necessary to break

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them down chemically in order to analyze their structural components. Thioacidolysis is a method used for breaking down lignins in which the 8-0-4 aryl-ether structures are broken down (Önnerud et al., 2002). Based on the conditions described by Bruce and West (1989), the freeze-dried tissue (0.5 g) was homogenized with 150 ml of 80% ethanol for 4 min. The mixture was vacuum filtered (Whatman GF/A), the residue washed with 20 ml of 80% ethanol and then dried at 50 ◦ C for 24 h. Fifteen millilitres of HCl 2 mol l−1 and 1 ml of thioglycolic acid were added to the dry residue which was then boiled with stirring for 4 h; after it was cooled and centrifuged at 10,000 × g for 15 min. The residue (lignin thioglycolate) was washed with 10 ml of water, suspended again in 20 ml of NaOH 0.5 mol l−1 with stirring for 18 h at room temperature and centrifuged; 4 ml of HCl concentrate was added to the supernatant liquid. The lignin thioglycolic acid was precipitated at 4 ◦ C for 4 h, centrifuged (10,000 × g, 15 min) and the residue was dissolved in 10 ml of NaOH 0.5 mol l−1 ; its absorbance was read at 280 nm. Quantification was carried out using a standard coumaric acid curve. 2.3.4. PPO activity The method proposed by Montgomery and Sgarbieri (1975) was followed. Five grams of tissue were homogenized with 0.6 g of PVPP and 20 ml of 50 mM (pH 7) phosphate buffer. This was then filtered and centrifuged at 10,000 × g at 4 ◦ C for 15 min. The activity was measured with 2.85 ml of 0.2 mM (pH 7) phosphate buffer, 50 ␮l of catechol (60 mM) as a substrate and 100 ␮l of enzymatic extract. The mixture was maintained at 25 ◦ C and the change in absorbance was read over 3 min at 420 nm. Activity was expressed as units of activity (UA) in which one unit of PPO was defined as the change in one unit of absorbance per second. 2.3.5. POD activity Peroxidase enzyme activity was assayed following the method described by Childs and Bardsley (1975). Crude extracts of the enzyme were obtained from 6 g of homogenized tissue with 0.6 g of PVPP and 20 ml of a phosphate buffer (50 mM, pH 7). The homogenized mixture was centrifuged (10,000 × g, 15 min at 4 ◦ C) and the supernatant liquid was used as an extract of the enzyme. The

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reaction was carried out at 30 ◦ C with 1.34 ml of 0.1 M (pH 6) phosphate buffer, 30 ␮l of a 50 mM solution of 2,2 -azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) and 50 ␮l enzymatic extract; the reaction started with the addition of 80 ␮l of 0.92 mM hydrogen peroxide. The changes in absorbance were read at 414 nm for 3 min using a double beam UV-Vis spectrophotometer (Perkin-Elmer Lamda 40). POD activity on acids, caffeic and ferulic, as well as on coniferaldehyde and coniferyl alcohol was carried out using the same method and using these compounds as substrates in place of ABTS; absorbance was registered at 404, 368, 340 and 330 nm, respectively. At these wavelengths, maximum absorbance was obtained due to the disappearance of the substrate (coniferyl alcohol) or due to product appearance. Enzyme activity was expressed as POD UA in which one unit corresponded to the change in one absorbance unit for 1 s. 2.3.6. Histochemical assay PPO and POD enzyme activity in living tissue subjected to browning was observed through histochemical assays. Jicama cylinders were stored for 6 days at 20 ◦ C; after this, the external tissue (2 mm deep) was eliminated and disks 3 mm thick and 1.4 cm in diameter were obtained from the internal tissue. These were placed in Petri dishes with a pH 7 phosphate buffer (control group) or with the same buffer in the presence of 1 mM catechol (PPO activity) or in a pH 4.5 citrate phosphate buffer, 0.05 mM hydrogen peroxide by itself or in the presence of coumaric or ferulic acid (0.5 g l−1 ) for POD activity. Color changes were observed within a few minutes and up to 2 days later at room temperature.

Fig. 1. Color changes in the surface of jicama cylinders stored at 20 ◦ C.

stant until the end of storage. The increase in chroma value and the decrease in lightness and hue in the jicama cylinders (with values within the first quadrant of the color space) indicated color changes ranging from white tones to yellowish colors. Samples stored during the same period at 10 ◦ C showed no significant color changes. Fig. 2 shows the variation in total phenol content in the internal and external tissue of the cylinders stored at 10 and 20 ◦ C. The initial phenolic content (as gallic acid) in fresh jicama tissue was 0.3730 g kg−1 . The cylinders stored at 10 ◦ C remained nearly constant in phenol content in both the internal and external parts, with a slight decrease in the external tissue on day 8 (0.3730–0.3089 g kg−1 ) or to 0.3016 g kg−1 in internal tissue. The same behavior was observed in the internal tissue of pieces stored at 20 ◦ C. On the contrary, the external tissue of samples stored at 20 ◦ C showed

3. Results The most important color changes in the jicama cylinders were observed during the first 4 days of storage at 20 ◦ C (Fig. 1). Lightness decreased from 71.5, at the outset of the experiment, to 51.2 on day 10. Similarly, the hue also decreased during the first 4 days. It then remained constant until day 7 when it again decreased at the end of storage with a color change toward reddish-yellow. The chroma increased from 7.5 to 20.26 during the first 3 days and then remained con-

Fig. 2. Changes in total phenol content of internal and external tissue of jicama cylinders stored at 10 and 20 ◦ C.

20

40 30

16 12 8

20 10

(A)

279

24

50

Chroma

Coumaric acid, mg kg-1

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4

0

2

4

6

Days after wounding

0

(B)

2

4

6

Days after wounding

Fig. 3. Changes in lignin content (A) and chroma value (B) in external tissue of jicama cylinders stored at 20 ◦ C.

a rapid increase in phenol content, reaching the maximum value of 1.042 g kg−1 on day 8, 2.8 times more than the initial content of these compounds. This behavior clearly indicated a phenol synthesis process in the external area of root damaged, in contrast to the internal tissue which manifested no important changes. The initial lignin content (as coumaric acid) in fresh external tissue was 16.6 g kg−1 . During storage of the cylinders at 20 ◦ C, these values increased 1.7, 2.9 and 3.1 times on days 2, 4 and 6, reaching values of 28.2, 47.5 and 52.2 g kg−1 , respectively (Fig. 3A). The increase in the lignin content was directly related to color change, expressed through the chroma parameter (Fig. 3B); this relation was approximately linear with a correlation coefficient of R2 = 0.8765. PPO activity (UA) was greater at 20 ◦ C and much higher in the external tissue (Fig. 4A) where it reached values of 81.67 UA kg−1 ; the internal tissue also manifested these increases with a maximum of only 36.67 UA on the sixth day of storage. At 10 ◦ C, the activity of this enzyme was similar to that registered at the beginning of the experiment (23.33 UA kg−1 ). These results indicated an association of mechanical damage with the activity of this enzyme. POD activity was also affected by temperature; at 20 ◦ C it was greater than at 10 ◦ C. It was also observed that activity was different according to tissue location; activity in the external tissue was greater than in the internal tissue, which indicated that the activity of this enzyme was also directly related to mechanical damage of the tissue. External tissue samples stored at 20 ◦ C showed maximum activity on day 6 (7 500 UA kg−1 ). At this time, internal tissue also

manifested the same maximum activity, although the values were 5 333 UA kg−1 (Fig. 4B). At 10 ◦ C, activity was noticeably less, although a slight increase in activity was found between days 5 and 6 (2067 and 2302 UA in internal tissue and 2547 and 2675 UA in external tissue). Also greater activity was observed in the external tissue, although the difference was slight. The results of PPO and POD activity suggests evidence of the participation of these enzymes during the damage process. To compare the action of POD and PPO in the browning process, experiments were

Fig. 4. Changes in polyphenol oxidase (A) and peroxidase (B) in internal and external tissue of jicama cylinders stored at 10 and 20 ◦ C.

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carried out in vivo in order to observe the activity of the two enzymes. After 6 days of storage of the cylinders at 20 ◦ C under the conditions described in the methods section, approximately 1 or 2 mm of the surface was eliminated, and portions of the remaining internal tissue were placed in Petri dishes with phosphate buffer (control), with buffer and the presence of catechol (PPO test) or buffer and H2 O2 (POD test), as described in the methods section. After a few minutes, a slightly reddish color was produced on the surface of cylinders placed in H2 O2 ; no color changes were observed in the control pieces or in those placed in the presence of catechol. After 48 h, the pieces placed in the H2 O2 solution, formed a reddish-brown product on the surface, while those placed in the catechol solution developed only a slight yellowish color. PPO and POD activities may be related to the processes of lignification or damaged tissue repair; since the lignin content increased during the damage process, it was then possible that these two enzymes could use as substrates those phenolic compounds which are precursors of lignin synthesis; such as coumaric, caffeic, ferulic and sinapic acids, coniferyl and sinapyl alcohols and sinapaldehyde. In the presence of H2 O2 , caffeic and ferulic acids, as well as coniferaldehyde and coniferyl alcohol proved to be good POD enzyme substrates and indicated classic saturation kinetics of enzymatic reactions; similarly, a strict requirement for H2 O2 was found. On the contrary, no POD activity was detected with sinapic acid, synapaldehyde and sinapyl alcohol. In the case of PPO, only caffeic acid was used for this enzyme. From the data of the activity curves, the values of Km and Vmax were obtained (Table 1); these show that the greatest affinity of the enzyme was for coumaric acid and coniferaldehyde, with an intermediate affinity for caffeic and ferulic acids. The least affinity was for coniferyl alcohol. Nevertheless, coniferyl alcohol had the greatest Vmax value (567 UA kg−1 ). The importance of the participation of these intermediary compounds in lignin synthesis was investigated by means of histochemical tests in which jicama cylinders (6 days at 20 ◦ C), with the surface portion eliminated, were put in a pH 4.5 buffer in the presence of different precursors of lignin synthesis and H2 O2 . All compounds showing activity with POD also showed browning after 24 h of exposure.

Table 1 Michaelis–Menten constant (Km ) and maximum speed (Vmax ) for phenolic substrates of peroxidase Substrate

Km (␮M)

Vmax (UA kg−1 )

Coumaric acid Caffeic acid Ferulic acid Coniferaldehyde Coniferyl alcohol Sinapic acid Sinapaldehyde Sinapyl alcohol

40.0 84.0 150.0 42.5 580.0 ND ND ND

138 107 97 133 567 ND ND ND

Km : Michaelis–Menten constant; Vmax : maximum speed, AU: activity units; ND: not detectable.

4. Discussion The color parameters L∗ , chroma and hue best represented the color change in the jicama cylinders during their storage at 20 ◦ C. This concurs with Mercado-Silva and Cantwell (1998) and Mercado-Silva et al. (1998) reports in which they indicated that the browning of the intact jicama root under conditions of chilling injury was most directly expressed with these values. The initial phenol content in fresh tissue (0.3730 g kg−1 ) was similar to the data reported by Paull et al. (1988) for mature jicama roots at the end of the crop cycle or somewhat greater than what was indicated by Paull and Chen (1988) for stored jicama. Tanaka and Uritani (1977) reported that cuts in sweet potato roots (Ipomoea batatas L.) caused intense production of polyphenols in adjacent cells (1 mm deep) and that this production was less in cells farther from the damaged area which concurs with the results found in this study at 20 ◦ C. These researchers described similar behavior for the phenylalanine ammonia lyase enzyme. Also in the case of jicama cylinders, during storage at 20 ◦ C and as a response to damage, the phenolic compounds increased in the external area of the cylinders (<2 mm deep); this suggests that the phenolic compounds were synthesized in the intact cell layers located under the damaged surface as has been reported for sweet potatoes and taro species (Colocasia esculenta L. and C. antiquorum) (Uritani, 1999). This author suggested that the formation of these phenolic compounds is due to the formation of a lignin layer, although this was not a general response in all products,

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Fig. 5. Dyeing of mechanically damaged jicama cylinders with phloroglucinol (lignins) and Sudan IV (suberins).

since cassava (Manihot esculenta L.) did not manifest this formation, but did present vascular streaking, a physiological disorder of the xylem that is manifested in a brown or brownish-green color of the vascular bundles. Aquino-Bolaños et al. (2000), working with jicama cylinders, reported an increase in the activity of phenylalanine ammonia lyase (an enzyme that initiates the phenol synthesis process) previous to the increase in the phenol content, indicating a synthesis of these compounds during storage. Total phenolic content on the surface of the cylinders was highly correlated with the browning measured by means of a decrease in the lightness L∗ (R2 = 0.80) value, although this does not necessarily mean that the synthesized phenols are directly responsible for the development of browning but this indicated that both processes occurred simultaneously. The increase in the lignin content of the external tissue of the jicama cylinders could be a response mechanism to mechanical damage caused by the cut in the tissue. It is known that plants create a natural barrier that includes lignin and suberin synthesis (Vance et al., 1980; Rhodes and Wooltorton, 1978), components directly linked to support systems, vascular bundles, wound healing and the formation of periderm on the roots. In order to visually register the most important fraction of these compounds on the roots of cut jicama, a histochemical assay was carried out in which the recently prepared jicama cylinders and others which had been stored for 6 days, were put in contact with phloroglucinol (which dyes the lignin red) or with Sudan IV (which dyes the suberins with ocher color) (Hammerschmidt, 1985). With Sudan IV, both the recently prepared samples and the stored samples manifested a similar color, while with phloroglucinol the recently prepared samples showed a reddish color

on the vascular bundles; with the stored samples a reddish color appeared on the entire surface (Fig. 5). This suggested that lignin, and not suberin, was the principal compound produced during the storage of cut tissue. Histochemical evidence gave greater support to the determinations of lignin content which increased 3.1 times during storage. Lignin content was related to color changes by means of chroma (R2 = 0.8765). These results indicated that tissue browning may be due to a lignification process. Browning in plants, induced by mechanical damage, was previously attributed to the activity of the PPO enzyme (Mayer, 1987); nevertheless, studies done with jicama discs stored at 5, 7.5 and 10 ◦ C showed a low correlation between PPO activity and color change during the browning process (Aquino-Bolaños et al., 2000), suggesting that this enzyme was not directly involved in the process. The participation of peroxidase in lignification processes is found in the monolignol polymerization processes at cell wall level through mechanisms that involve the generation of free radicals (Whetten et al., 1998); Hatfield and Vermerris (2001) indicated that in the lignin formation process, monolignols diffuse through the matrix of the cell wall, coming into contact with an anionic peroxidase dependent on H2 O2 in order to form the free radical of the monolignol which unites with a preformed chain that continues growing. Nevertheless, it has also been pointed out that other enzymes such as laccase and catechol oxidase may take part in this polymerization mechanism (Richardson and McDougall, 1997). The increase in POD activity in the superficial tissue of the jicama cylinders, which was dependent on temperature, has also been described elsewhere. Thomas

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and Delincée (1979) reported that slices of potato mechanically damaged and stored for 14 days at 15 ◦ C had an increase in POD activity in a proportion of 10–14 times compared to initial values; this increase was proportional to the temperature. Such behavior coincides with the findings of this paper. Howard and Griffin (1993) also indicated an increase in the activity of this enzyme in carrot sticks, along with an increase in lignins associated with color change. Greater activity of this enzyme in the external tissue, associated with greater lignin content and color changes in the tissue, seem to indicate that browning on the surface of the jicama cylinder may be due to the lignification process or wound healing in the damaged area, as was suggested in the case of minimally processed lettuce by Ke and Saltveit (1989), for mangosteen (Garcinia mangostana L.) by Ketsa and Atantee (1998) and for sweet potato by Uritani (1999). The development of colored compounds in the histochemical tests and in the presence of H2 O2 , as well as their lack of development in the presence of catechol, suggested a more important role of the peroxidase dependent on H2 O2 in the browning process than the PPO. Lagrimini (1991), studying browning caused by mechanical damage in tobacco plant stems (Nicotiana tabacum L.), both in control and over-expressing peroxidase plants, found that both PPO and POD activity increased after damage; nevertheless, the pattern of PPO activity was similar to that of control plants as well as over-expressing POD plants. Browning, however, was greater in the over-expressing POD plants, indicating that this browning could be due to the presence of an anionic peroxidase located in the cell wall. It is important to point out that the optimal pH of POD extracts was 4.5 (data not shown) and that the development of color in the in vivo tests was carried out with this same pH which appears to indicate that the type of peroxidase induced during damage is anionic, as pointed out by Lagrimini (1991) in the above-mentioned study. If POD activity contributes to browning through the lignin biosynthesis process, this enzyme should be able to polymerize the precursors of lignin. The POD enzyme used hydroxicinnamic acids, coniferaldehyde and coniferyl alcohol as a substrate but could not use sinapaldehyde and sinapyl alcohol. This may indicate that jicama lignin mainly contains guaiacyl type units (also called G units). The low Km values for the phe-

nolic substrates studied indicated a great affinity of POD for those substrates. The greater activity (Vmax ) obtained with coniferyl alcohol could be due to the involvement of the POD enzyme in the last step of polymerization of cinnamyl alcohols in the formation of lignin (Imberty et al., 1985).

5. Conclusion The results of this research appear to indicate that the browning process in jicama cylinders is chiefly explained by a process of lignin formation in which a peroxidase, probably of the anionic type, is directly involved in the process.

Acknowledgements This research was carried out with the financial support of the Consejo de Ciencia y Tecnolog´ıa del Estado de Querétaro (CONCYTEQ) and the Consejo Nacional de Ciencia y Tecnolog´ıa (CONACYT) through project 31684-B and Aquino-Bolaños, E.N. doctoral fellowship.

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