Introducction

Avocado is a fruit of high demand in Mexico and the world for its nutritional and sensory characteristics. Mexico is the main producer and global consumer of this fruit. However, only 16% of the total production is industrialized. Handling operations, processing and storage can induce undesirable changes in quality and appearance of fresh fruits and vegetables as a result of oxidative browning mediated by the enzyme polyphenol oxidase (PPO; EC1.14. 18.1). PPO catalyzes the oxidation of a wide variety of phenolic compounds, generating reactive quinones that participate in subsequent reactions producing colored pigments. Because of the potential health effects caused by the use of sulfites as anti-browning agents, alternative chemical treatments have been studied as an (Manolopoulou and Varzakas, 2011; Sulaiman et al., 2015; Ali et al., 2016). Biochemical treatments aimed at countering enzymatic browning in some fruits and vegetables has been studied (Ma et al., 2010; Bustos et al., 2015). Some biochemical agents act directly as inhibitors of PPO, while others induce a non-favorable environment for the browning reaction and, others reac with the reaction products of PPO before they become colored pigments (Chiabrando and Giacalone, 2012; Sulaiman et al., 2015).

Various inhibitor agents have been studied, not only individually but in binary mixtures, enhancing their action, with the idea of achieving a synergistic effect and better PPO inhibition results. The PPO inhibitor 4-hexylresorcinol (Martin-Belloso and Soliva-Fortuny, 2010) is known as GRAS; it is used in the prevention of shrimp melanosis (Nirmal and Benjakul, 2010) and it is also effective for the control of enzymatic browning in apple slices (Jokić et al., 2009). Compounds containing thiol (-SH) have been reported as effective reducing agents with potential to reduce enzymatic browning (Ioannou, 2013), L-Glutathione is the most abundant non-protein thiol compound present in living organisms and is used as an ingredient in pharmaceuticals, as well as a food additive and in cosmetics (Li et al., 2004). Ascorbic acid and its analogs, which are attributed acidifying and chelating properties (Holzwarth et al., 2013) have been widely used in the food industry, with acceptable microbial results and sensory preservation of mushrooms slices (Cliffe-Byrnes and O’berne, 2008). The industrial application of enzymatic inhibitors that preserve avocado slices requires a polymeric support with high water solubility and without off-lavor addition. Gellan gum (Rojas-Grau et al., 2006, 2008) was chosen as the polymeric matrix for the incorporation of chemicals antioxidants to browning inhibition in fresh-cut ‘Fuji’ apple slices.

This study aimed to determine the kinetic behavior of the PPO enzyme in avocado (Persea americana Mill.) in the presence of 4-hexylresorcinol, L-glutathione, sodium D-isoascorbate and sodium hexametaphosphate, incorporated to a polymeric matrix based on gellan gum, in order to assess their effect on the visual quality and physicochemical properties preservation of avocado slices.

Materials and Methods

Biological material

Avocado fruits (Persea americana Mill.) cv. Hass, were purchased in Xalisco, Nayarit, Mexico (21º26’53”N, 104º54’0”O) of consumption ripeness and uniform appearance and size (classification: premium, according to Mexican Standards NMX-FF-008 171-210).

Chemicals

The chemical agents used in the study were gellan gum (Gelzan™ Sigma, USA), sodium D-isoascorbate (D-ISO), 4-hexylresorcinol (4HSR) and L-glutathione (LG) from Sigma Aldrich, Germany, and sodium hexametophosphate (HAS) from Jalmek Scientific, Mexico.

PPO enzyme extraction

PPO extraction was performed according to a modified Soliva et al. (2001) method. An avocado purée (15g) was mixed with Mcllvaine buffer (1:1; pH adjusted to 6.5), and 0.1ml NaCl 1M and polyvinylpyrrolidone 5% (w/v) were added. Mixture was homogenized and subsequently centrifuged at 12000 rpm for 30min at 4ºC. The supernatant was collected and filtered through a paper filter Whatman # 1. The collected permeate was used immediately. Chemical agents were applied individually and mixed (Table I).

PPO enzymatic activity

In order to determine enzyme activity, 3 ml of catechol 0.05M, 75ml of enzyme extract and 75ml of the chemical inhibitor were placed into a 4ml quartz cell. The tests were performed at 25ºC, absorbance was monitored for 3min and data recorded every 25s, at 410nm wavelength (UV-visible spectrophotometer Varian Cary 50 Bio). Slopes were obtained from the linear portion of the resulting curves. One activity unit was considered as the amount of enzyme required for the formation of 1µmol of benzoquinone/min. A completely randomized experimental design was used and the data were analyzed using SAS software system 2015.

Kinetics of PPO inhibition

Catechol (Sigma-Aldrich, Germany) as substrate in concentrations of 0.0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11 and 0.120M was prepared, and 3ml of each concentration was placed into a quartz cell along with 75ml of each inhibitor or their combinations. Reaction was started by adding 75ml of enzyme extract and absorbance at 410nm was followed through 2min with data recorded every 10sec for each individual reaction. The kinetic parameters Km, Vmax and inhibition coefficients (Ki) were determined through Michaelis-Menten and Lineweaver-Burk plots (Dogan, 2004).

In vivo inhibitory effects of the agents added to gellan gum

Chemical agents that exhibited significant effect on PPO activity were incorporated into a gellan gum matrix 0.5% w/v; glycerol 1% (v/v) was added as a plasticizer and polyoxyethylene (20) sorbitan monooleate 0.5% v/v as a surfactant, in order to formulate edible coatings (Lin and Zhao, 2007). Avocado fruits were washed by immersion in water with sodium hypochlorite (100ppm) during 3min. Subsequently, the peel and seed were removed and 1cm thick longitudinal slices were obtained. The edible coating formulations were applied by immersion during 1min and excess solution was drained in a sieve for 5min.

Six hundred avocado slices with and without coating formulations were manually packaged in bags Cryovac® Europe (Grace S.A., Sant Boi de Llobregat, Spain), of low oxygen permeability (15cm3· m-2/24h at 23ºC and 0% RH). Air was removed with nitrogen; then, the bags were subjected to vacuum and passed through a food freezer tunnel (CAS Function ABI Co., Japan) at -45ºC during 40min. Finally, samples were stored at -20ºC until analysis. Analyses were carried out in triplicate.

Quality assessment of slices exposed to environmental conditions

Samples were taken out of the freezer and the avocado slices were exposed to room environment conditions (25ºC and 70% RH). This moment was considered as time zero.

Color changes evaluation

The color changes were registered by a colorimeter (Konica Minolta CR-400 Chroma Meter), in terms of the parameters L, a and b. Five random measurements were performed on the surface of avocado slices at 1h intervals after time zero. The total color change was calculated by Eq. 1.

(1)

Sensory evaluation

A duo-trio sensory test by comparison with 15 untrained judges (each judge had extensive experience in the production line and supervision of finished avocado products) was performed. Three sets of coded samples were presented to the judges, one of the samples pertains to a reference or standard (product line for export), identified as R, and judges were asked to respond which of the two samples was equal to the reference sample (R). The data were treated using c2 for determining the presence or absence of significant taste difference between the samples at time zero (Pedrero, 1996; Olivas et al., 2009).

Microbiological analysis

Samples with and without coating formulations were analyzed for total mesophiles and coliforms (mesophilic aerobiccount, ISO 4833, 2003; total coliform count plate, Standard ISO 4832, 1991). The microbiological analyses were performed immediately after defrosting (to) and after 8h (t8) and reported as UFC/g.

Statistical analysis

All experiments were done in triplicate and the results were expressed as mean values ±standard deviation. Analysis of variance ( P<0.05) and Tukey’s range test (α= 0.05) were used to assess significant differences between samples. The statistical analysis was carried out using Statistica v.10 from StatSoft, Inc.

Results and Discussion

Inhibitory effect of chemical agents on avocado PPO extract

The individual effect of 4-hexylresorcinol (4HSR) and sodium hexametophosphate (HAS) did not reduce significantly (p<0.05) the avocado PPO activity in vitro in the concentrations evaluated in this study. Similar results were obtained by Ghidelli et al. (2013), who evaluated the effect of 4HSR on the activity of artichoke PPO precipitates; they found that 4HSR in concentrations of 0.002% and 0.005%, were the least effective among the treatments applied; they also noted that increasing the concentration of 4HSR was accompanied with a decrease of the color L values, in conjunction with an increase of a, indicating tissue damage at concentrations >0.005%. On the other hand, HAS was not effective against enzymatic browning even at 50mol·ml-1. The effect of HAS as anti-browning agent has been reported as effective in mixtures with other agents such as citric and ascorbic acid in acid pH, on PPO activity in apple slices (Pilizota and Sapers, 2004).

The agents sodium D-isoascorbate (D-ISO; 0.094M) and L-glutathione (LG; 0.05M) showed effect on avocado PPO activity individually, achieving a reduction from 148.45μmol·min-1 to 2.86 and 1.74μmol·min-1, respectively. The combination of these chemicals affects the enzymatic activity of PPO in a similar manner (Table I); it reduced the reaction rate to just 0.99μmol·min-1, but also showed a Km value of 3.49, the highest observed in this study. Results suggest that there is a difficulty in the formation of the enzyme substrate complex, and also a synergistic effect of this combination is exhibited on the enzymatic activity of PPO avocado cv. Hass. Similar results of the effect of LG on the inhibition of PPO activity in apple have been reported (Billaud et al., 2004). Treatments using D-ISO also showed a significant reduction of PPO activity in peach (Nogueira et al., 2011). No reports with regard to the combined effect of D-ISO (0.094M) and LG (0.05M) were found; however, the synergistic effect of other combinations of anti-browning agents has been reported. Rojas-Grau et al. (2006) studied the effect of four anti-browning agents (4HSR, GL, N-acetylcysteine and D-ISO) on minimally processed Fuji apples. Synergistic effects of antioxidant combinations (4HSR/N-acetylcysteine and N-acetylcysteine/GL) were reported by these authors, who proposed that the synergism is probably due to the combined effect of the ability of the competitive agents 4HSR and GL to bind with the available enzyme at the active site, in conjunction with substrate blockade through N-acetylcysteine (Altunkaya and Gökme, 2009).

In addition, the synergistic combinations of RC promoting a modified atmosphere with the PPO inhibitors have been reported in various pre-cut products (Laurila et al., 1998; Gorny et al., 2002; He and Luo, 2007, Rojas Grau et al., 2008).

TABLE I
AVOCADO CV. HASS PPO ENZYMATIC ACTIVITY IN PRESENCE OF INHIBITORS(SINGLE AND BINARY COMBINATIONS) AND KINETIC PARAMETERS

Inhibition mechanisms

The combinations of LG (0.05M) + D-ISO (0.094M), D-ISO (0.094M) + 4HSR (0.025M) and LG (0.05M) + 4HSR (0.025M) and LG (0.05M) led to an increase in the reaction constant Km, which reveals a difficulty in forming the enzyme-substrate complex, while Vmax was not affected (Table I). A Km increase accompanied with unmodified Vmax is characteristic of competitive inhibition, which usually occurs when the inhibitor has a chemical similarity with the substrate of the enzyme (catechol in this case) and competes with the enzyme are busy, situation that occurs in competitive inhibition because active sites are occupied by substrate but also by the inhibitor. Moreover, the Lineweaver-Burk and Eadie-Hofstee graphs confirm a pattern of competitive inhibition (Figure 1).

Figure 1.
Kinetic behavior of the browning reaction mediated PPO in presence of L-glutathione (LG) 0.05M, 4-hexylresorcinol (4HSR) 0.025M, D-isoascorbate (D-ISO) 0.094M catechol (0.0 to 100mM) and 75ml of enzyme extract in McLlvaine buffer (pH 6.5), total volume of 3ml.

The combination of LG (0.05M) + D-ISO (0.094M) allowed a greater increase of Km to 3.45, which indicated a high difficulty to form the enzyme-substrate complex, probably due to the ability of L-glutathione to act as a competitive inhibitor, as it is a strong reducing and chelating agent ( Witschi et al. 1992; Jiang and Fu, 1998). It may interact with the two copper atoms located at the active site of the enzyme, and thus inhibit PPO activity. The kinetics of PPO in the presence of D-ISO does not f it a typical Michaelis-Menten kinetics, since it shows an asymptotic behavior on the abscissa, corresponding to a total or linear inhibition (Fersh, 1985), be- cause the slope is equal to zero, as well as Km/Vmax.

Moreover, the intersection of the ordinate is zero then 1/ Vmax= 0, since Vmax corresponds to the time of the reaction when all the active sites of the enzyme are saturated forming enzyme-substrate complexes (Voet and Voet, 1990; Bell and Bell, 1998). The results suggest that the reaction rate at any point of the reaction is zero. It is known that sodium D-isoascorbate is an isomer of ascorbic acid and it reduces the O-quinones formed in the reaction catalyzed by PPO, returning them to its form of O-diphenols, avoiding in this manner, the formation of colored pigments (Golan-Goldhirsh and Whitaker, 1984). Since the absorbance measured corresponds to the product of the reaction catalyzed by PPO, the speed of the O-quinones synthesis could be equal to the rate at which these were returned to their O-diphenols form by the reducing action of D-sodium isoascorbate (Janovitz- Klapp et al., 1990), then it is possible that Vmax values were not detected in the reaction of PPO when it acts in the presence of D-ISO. Additionally, the inhibition coefficient Ki obtained in this study (0.07 to 3m M, Table I) is similar to that obtained by Serap et al. (2006), who studied the kinetics of inhibition using PPO extract from mushrooms with cinnamic acid as an inhibitor, obtaining Ki in the range of 0.064 to 14.09mM.

Effect of inhibitor agents incorporation to the edible coating on visual quality and color

Avocado slices exposed at room temperature (Figures 2 and 3) treated with formulations of gellan gum matrix containing 0.05M LG, and the combination of inhibitors LG 0.05M + D-M ISO 0.094M and D-ISO 0.094M + 0.025M 4HSR, showed L values of 55 units that were maintained through 8h, whereas slices treated with formulations of gellan gum containing combination of agents D-ISO 0.094M+4HR 0.025M and the control, showed an important reduction of L values, decreasing below 50 units after a 3h exposure at room temperature. These results show the correlation between the values of L and visual quality observed in surfaces of avocado slices.

Figure 2.
Visual quality of avocado slices treated with inhibitors incorporated into gellan gum matrix at 0h, and after 3 and 8h at room temperature. Formulation A: Gellan gum, L-glutathion 0.05M, polysorbate 80 al 0.5% v/v, glycerol 1.0% v/v); Formulation B: Gellan gum, L-glutathion 0.05M, D-isoascorbate 0.094M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v; Formula C: Gellan gum, D-isoascorbate 0.094M + 4hexylresorcinol 0.025M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v; Formulation D: Gellan gum, D-isoascorbate 0.094M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v; Formulation E: control.

Figure 3.
Brightness as L values of avocado cv. Hass slices through 8h exposed to room temperature. Formulation A: Gellan gum, L-glutatión 0.05M, 0.5% v/v polysorbate 80, glicerol 1.0% v/v); Formulation B: Gellan gum, L-glutatión 0.05 M, D-isoascorbate 0.09 M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v; Formulation C: Gellan gum, D-isoascorbate de sodio 0.094M + 4hexylresorcinol 0.025M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v, Formulation D: Gellan gum, D-isoascorbate 0.094M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v; E: control.

The analysis of the color parameters a (Figure 4) and b (Figure 5) showed that during the initial two hours, the avocado slices treated with all formulations still possess the characteristic yellow and green avocado colors, which correspond to negative values of a (-7 to -8) and positive values of b (27-30).

Figure 4.
a values of avocado cv. Hass slices through 8h exposed to room temperature. Formulation A: Gellan gum, L-glutatión 0.05M, 0.5% v/v polysorbate 80, glicerol 1.0% v/v); Formulation B: Gellan gum, L-glutatión 0.05 M, D-isoascorbate 0.09 M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v; Formulation C: Gellan gum, D-isoascorbate de sodio 0.094M + 4hexylresorcinol 0.025M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v, Formulation D: Gellan gum, D-isoascorbate 0.094M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v; E: control.

Figure 5.
b values of avocado cv. Hass slices through 8 h exposed to room temperature. Formulation A: Gellan gum, L-glutatión 0.05M, 0.5% v/v polysorbate 80, glicerol 1.0% v/v); Formulation B: Gellan gum, L-glutatión 0.05 M, D-isoascorbate 0.09 M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v; Formulation C: Gellan gum, D-isoascorbate de sodio 0.094M + 4hexylresorcinol 0.025M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v, Formulation D: Gellan gum, D-isoascorbate 0.094M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v; E: control.

After a 3h exposure to ambient temperature, a values of avocado slices treated with formulations D: Gellan gum, D-ISO 0.094M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v and E (control) change drastically, reaching positive values. A significant decrease in the b values was also observed, corresponding to the loss of green and gain of brown color, a decrease in the visual quality of the product. Avocado slices treated with formulations A: Gellan gum, LG 0.05M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v; B: Gellan gum, LG 0.05M, D-ISO 0.094, 0.5% v/v polysorbate 80, glycerol 1.0% v/v; and C: Gellan gum, D-ISO0.094M + 4HSR 0.025M, 0.5% v/v polysorbate 80, glycerol 1.0%v/v, despite a slight increase inthe a values, this parameterdid not change to positive valuesupon 4h of exposure to room temperature, maintainingthe green-yellow pigmentsduring the 8h of evaluation,preserving the visual quality of the treated samples duringthe period of evaluation (Mendoza-Gómez et al., 2016). This behaviorwas evidenced with thetotal color change (ΔE) foreach treatment from 0h to 8hexposure (Figure 6). Formulations A and C were thosewith greater effectiveness incolor conservation, which indicatesa decrease in PPO activity (Campas-Ríos et al., 2012).

Figure 6.
Total color change (∆E) in avocado cv. Hass slices through 8h exposed to room temperature. Formulation A: Gellan gum, L-glutathion 0.05M, polysorbate 80 al 0.5% v/v, glycerol 1.0% v/v); Formulation B: Gellan gum, L-glutathion 0.05M, D-isoascorbate 0.094M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v; Formula C: Gellan gum, D-isoascorbate 0.094M + 4hexylresorcinol 0.025M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v; Formulation D: Gellan gum, D-isoascorbate 0.094M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v; Formulation E: control.

Microbiological and sensory evaluation

Avocado slices treated with polymeric formulations or without it showed a low microbial count. For mesophilic aerobic and total coliform were found values of 180 ±30 and values <100 UFC/g, respectively. These values are lower than those allowed by international standards (ISO 4833, 2003 for mesophilic aerobic and ISO 4832, 1991 for total coliform).

For samples treated with the formulations A: Gellan gum, LG 0.05M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v; B: Gellan gum, LG 0.05M, D-ISO 0.094, 0.5% v/v polysorbate 80, glycerol 1.0% v/v; and D: Gellan gum, D-ISO 0.094M, 0.5% v/v polysorbate 80, glycerol 1.0% v/v, the data collected from the panelists do not shown significant differences (p˂0.05) in taste, compared against the reference at time zero. The individual components of these formulations have no dominant sensory characteristics that could generate significant differences in treated avocado slices. The samples treated with formulation C: gellan gum, D-ISO 0.094M + 4HSR 0.025M, 0.5% v/v polysorbate 80 and glycerol 1.0% v/v showed statistically significant difference (p>0.05) in flavor compared against the reference. This is probably due to the presence of 4-hexylresorcinol, since Mendoza- Gómez et al. (1993) found that concentrations greater than 0.03% of this compound influenced the taste of apple slices cv. Fuji.

Conclusions

The edible coating formulation with gellan gum, added with the mixture of L-glutathione 0.05M, D-Isoascorbate 0.094M, 0.5% v/v polysorbate 80 and glycerol 1.0% v/v proved effectiveness in inhibiting PPO, preventing enzymatic browning and preserving the visual quality along color parameters (L, a and b) and total color change (∆E*) in avocado slices through 8h at room temperature. This represents an increase in shelf life of 5h compared to the control, which only lasts 3h, or 2.6 times the increase in the commercial time compared to untreated avocado slices. Additionally, the incorporation of the formulation (edible coating) to the avocado slices does not alter their sensory properties. Finally, this edible coating, is highly likely to be effective if it is applied in other fruits sensitive to enzymatic browning caused by the PPO enzyme.

Acknowledgments

The authors thank CONACyT (Mexico) for their support in conducting the work throughout this project PEI.184189 and COCYTEN (Nayarit, Mexico) for the scholarship granted to V. Mendoza-Gomez.

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Author notes