Changes in phenol metabolism of minimally processed ‘ baby cassava ’ under different temperatures : An alternative to commercialization

1 Departament of Plant Biology, Universidade Federal de Viçosa MG, S/N 36570-000, Viçosa, Brazil. 2 Fazenda Saco, Unidade Acadêmica de Serra Talhada, Universidade Federal Rural de Pernambuco, Serra Talhada, Brazil. 3 Department of Agronomy, Universidade Federal do Ceará, Campus do Pici, 60455-900. Fortaleza, CE, Brazil. 4 College of Agronomic Engineering, Universidade Federal do Pará, 68372-040 Altamira, PA, Brazil.


INTRODUCTION
Cassava crop (Manihot esculenta Crantz) has high economic and nutritional value.According to FAO data, the production in 2013 was 263 thousand tons in a harvested area of approximately 20.5 million hectares (FAO, 2014).Harvest and market are responsible for economic loss of the crop.Cassava roots are sold commonly in streets, markets, and in natura as a whole or in pieces.They are kept in high temperatures or sold as frozen in supermarkets.In all cases, they undergo few processing and stored for marketing, leading to high risk to consumers" health.This contributes to high perishability after harvest, either in whole or cut form.In addition, improper handling makes them susceptible to rapid postharvest physiological deterioration (PPD), within 48h (Iyer et al., 2010;Sánchez et al., 2013).However, there is no real time for minimally processed form.
PPD is characterized by a series of physiological phenomena that reduce the shelf life of cassava, which also results in an increase of enzymes activity like polyphenol oxidase (PPO) and peroxidase (POD) (Rickard, 1985;Uarrota et al., 2015).As oxidative enzymes, PPO catalyzes two kinds of reactions in the presence of molecular oxygen (co-substrate): first, there is hydrolysis of monophenols to o-diphenol and further oxidation of o-diphenols to o-quinones.These quinones can undergo cyclization or self-polymerization (nonenzymatic reactions with amino acids and proteins) forming melanins, which are variegated colored compounds (red, brown or black pigmentation) (Buckow et al., 2009;Holderbaum et al., 2010).
PODs are oxide-reducing enzymes.They contain iron as a prosthetic group, which catalyzes the reduced reaction of hydrogen peroxide (H 2 O 2 ) using electron donors (phenolic compounds, alkaloids, aromatic amines, auxins) for the formation of water (Almagro et al., 2009).Appropriate handling can minimize these reactions, especially with temperature control, from harvest to processing and marketing.
An alternative that adds value and maintains the organoleptic characteristics of cassava is the application of minimal processing technology.In minimal processing, cassava roots are physically modified by a sequence of steps such as peeling, cutting, slicing, cleaning, centrifuging and packaging, which present the sensory characteristics of a fresh product with high food safety, ready for consumption (Ghidelli and Pérez-Gago, 2016).
However, this handling causes injuries to the tissues which may shorten the shelf life of the product due to PPD.Such tissue damage can be reduced by using cold chain in the processing where temperature is one of the most influencing factors, in the maintenance of quality of minimally processed products (Donegá et al., 2013;Fagundes et al., 2013).The use of cold chain is required in all procedures, since all physical alterations cause a cellular injury by activating oxidative enzymes, generating browning, tissue degradation and sensory changes (Artés, 2004), which vary according to temperature.
The conservation of minimally processed products for marketing is generally done in refrigerated shelves, whose temperatures are between 5 and 10°C.In addition, the minimally processed cassava can be transported for long periods at room temperature (around de Brito et al. 929 25°C) until it is consumed.This can be an extremely strong inducer for anticipating darkening, by an increase of the activities of enzymes cited, increasing the susceptibility of microbial growth.The changes in the biochemical markers associated to phenolic compounds can be an important tool to coordinate browning and shelf life of minimally processed cassava.
In this work, the proposed "baby cassava" used is recently developed (Freire et al., 2015); it has potential market due to its added value; it makes consumers" life easier by making cooking fast and does not require pressure cooker and has attractive shapes.Thus, understanding the changes in the PPD and biochemical markers in the first hours after minimal processing simulates transport and market.Therefore, this study aimed to evaluate the changes in the phenolic oxidation mediated for PPO and POD in the quality of minimally processed "baby cassava", during the first hours of conservation at different temperatures

Plant material and minimal processing
Cassava cv.Recife harvested was acquired in a street market, in the city of Serra Talhada -PE 12 months prior to the study.The roots were transported to the experimental kitchen of the Unidade Acadêmica de Serra Talhada of the Universidade Federal Rural de Pernambuco.They were selected, and washed in running water with the aid of a brush; they were kept in a refrigerator at 8 ± 2°C for 24 h.
Minimal processing was performed according to the method described by Brito et al. (2013).Roots were cross-cut into pieces of 3.0 cm and peeled.They were longitudinally cut into pieces called "baby cassava" with a stainless knife.The "baby cassava" was initially rinsed by immersion for 10 s in water at 5 ± 2°C; it was sanitized in chlorinated water solution at a temperature of 5 ± 2°C and 200 mg L -1 of active chlorine (sodium dichloroisocyanurate dihydrate) for 10 min.Then, it was finally rinsed in 5 mg L -1 chlorinated solution for 10 min.

Visual assessment (general appearance)
The "baby cassava" was evaluated based on its overall appearance, by a trained panel of ten.A subjective scale of grades ranging from 5 (best grade) to 1 (worst grade) was used.Score 3 was set as an acceptance limit and the overall score corresponds to the average of the scores for each "baby cassava" (Freire et al., 2015).

Total soluble phenolics (TSP)
TSP was determined as described by Reyes et al. (2007), and as used for cassava by Freire et al. (2015).One gram of the surface portion (± 3 mm from the equatorial region of the "baby cassava") was collected and macerated in a mortar containing 10 mL of pure methanol.The extract was kept in the dark at a temperature of 5°C for 24 h.They were centrifuged at 7690 g, for 21 min and 2°C (Hettich centrifuge, Model Universal 320 R).
For determination of the phenolic compounds, reaction with 150 µL of methanolic extract was performed and diluted in 2400 µL of distilled water with 150 µL of Folin Ciocalteu reagent (0.25 N) added.After 3 min of homogenization of the mixture, 300 µL of sodium carbonate (1N) was added and stirred for 1 min.It was kept in the dark at 20 ± 2°C for 2h.Readings were taken with a spectrophotometer (Biochrom, Model British S8) at a wavelength of 725 nm.The TSP content was quantified from the gallic acid standard curve.
Extraction and assay of polyphenol oxidase (PPO -EC 1.14.18.1) and peroxidase (POD -EC 1.11.1.7)isozymes PPO and POD isozymes were extracted using the methodology of Freire et al. (2015) for cassava.It was collected at 0.25 g of superficial tissue (± 3 mm from the equatorial side of the "baby cassava").This material was macerated in a mortar containing sodium phosphate buffer (0.2 M, pH 6.0), and was centrifuged at 7690 g and 4°C for 23 min (Hettich centrifuge, Model Universal 320 R).
The PPO assay was performed according to Freire et al. (2015), wherein a mixture was made by adding 100 µL of the enzymatic extract (supernatant) and 1.5 mL of sodium phosphate buffer (0.1 M, pH 6).This was added to catechol (89.65 mM), and diluted in phosphate buffer (0.1 M, pH 6.0).The reaction was observed in a spectrophotometer (Biochrom, Model Libra S8) with change in absorbance at 425 nm, for a period of 2 min.Readings were recorded for 10 s, at 20 ± 2°C.For blank sample, 100 µL of sodium phosphate buffer (0.2 M, pH 6) was used replacing the enzymatic extract.
The POD assay was performed following the method of Freire et al. (2015), with some modifications.A mixture containing 300 µL of an enzymatic extract (supernatant) was made with 1 mL of sodium phosphate buffer (0.2 M, pH 6). 100 µL of hydrogen peroxide (0.08%) and guaiacol (0.5%) were added (both diluted in 0.2 M phosphate buffer) and the reaction was monitored in a spectrophotometer with a change in absorbance 470 nm, for a period of 2 min.For white sample, 300 µL of sodium phosphate buffer (0.2 M, pH 6) was used to replace the enzymatic extract.The results of PPO and POD were expressed in Enzyme Unit (EU -0.001absorbance per minute) per gram of fresh weight per minute.

Experimental design and statistical analysis
This design was completely randomized (CRD); it was divided into two factorial schemes with four replications, each with 5 pieces in package: the first factorial scheme was 2x8: two temperatures (refrigerated at 5 ± 2°C and 25 ± 2°C) and seven storage periods (0, 2, 4, 6, 8, 10 and 12 h after minimal processing) used for evaluations of FWL, appearance, TSP and PPO and POD activity.
The second factorial scheme was 2x6: two temperatures (refrigerated at 5 ± 2°C and 25 ± 2°C) and six storage periods (0, 2, 4, 6, 8 and 10 days) used only for FWL and appearance assessments; due to the deterioration stage of the "baby cassava" kept at room temperature, biochemical analyses (TSP, PPO and POD activities) were not performed from the fourth day.

Effect of temperature on the weight loss and visual appearance in minimally processed cassava
The "Baby cassava" kept at room temperature had greater weight loss than those kept under refrigeration (Figure 1A).It was found that, at 12 h after minimal processing, the fresh weight loss was approximately 0.035 and 0.002% for the "baby cassava" maintained at 25 ± 2 and at 5 ± 2°C, respectively (Figure 1A to i).Nevertheless, this dehydration was not enough to generate losses in the visual integrity of the pieces (Figure 2).
Furthermore, the pieces kept at room temperature for long had severe dehydration reaching 3.14% at 10 days (Figure 1A).Unlike the refrigerated pieces, values were within 0.05% in the same period (Figure 1A).The refrigerated "baby cassava", during the first 12 h after minimal processing, maintained maximum values according to the visual scale (Figure 1B).The pieces were kept at room temperature, and a decrease was noticed in the appearance values (Figure 1B to i) at 12 h.Even with lower values, these were still above 3 (acceptance limit) (Figure 1B and 2), reaching its value in two days (Figure 1B and 2).From the second day until the end of the experiment, the most observed features were: pronounced browning of the surface; exudation of liquid appearance of a totally contaminated product and loss of firmness.
The severe dehydration of the "baby cassavas" kept at room temperature, in relation to the refrigerated ones, can be explained partly by the relative humidity, which was around 75 ± 5% for the refrigerated cassavas and 69 ± 5% for those kept at room temperature.In the latter, the atmosphere was drier, increased water loss and respiration as observed for minimally processed radish (Aguila et al., 2006) and pumpkin (Sasaki et al., 2014).
Browning rates in cassava roots may vary according to the various conditions relating to plant material and minimal processing."Baby cassava" has smaller signs of browning, when conserved for more than 8 days (Brito et al., 2013;Freire et al., 2015).This is due to the removal of the first layers of the periderm in which it presents Figure 1.Weight loss (A) and visual appearance (B) in minimally processed cassava in the 'baby cassava' format for 12 h (i) or for 10 days (ii) at 5 ± 2°C (refrigerated) and 25 ± 2°C (ambient) after processing.The dotted line has in visual appearance (B) which represent the acceptance limit (note 3).secondary phloem, being therefore more metabolically active, as seen in "cassava sticks" during 12 days of storage (Freire et al., 2015).
These results indicate that, the minimally processed cassava can be marketed without the use of refrigeration, provided that the consumption is fast, less than 12 h (Figures 1 and 2).This is important as in the case of the institutional market, that is, industrial kitchens, schools, companies, among others, in which consumption takes place in a few hours or even after a short period of transportation at room temperature.Although, these pieces had lower grades and good characteristics, with light to medium severe symptoms of browning are observed (Figure 1B, and Figure 2), provided the product is correctly sanitized.
Thus, these results suggest that dehydration of nonrefrigerated "baby cassava", after 12 h of storage slightly reduced its visual quality.The type with minimal dehydration can be accepted in the market.On the other hand, in retail market in which time is needed, the 'baby cassava' needs to be refrigerated.

Effect of temperature on the total soluble phenolics, oxidative enzymes activity and fluorescence emission on 'baby cassava'
It was observed that, for both temperatures there was an increase in TSP content expressed in gallic acid content in the first hours after minimal processing, with a peak of 1.7 times at 6 h at 25°C, and of 1.33 times for 8 h at 5°C (Figure 3A).When storage was extended, the 'baby cassava' kept at room temperature was damaged, not being made by TSP quantification in these materials after 48 h.On the other hand, in the refrigerated ones, the TSP continued to rise (Figure 3A).These authors reported that, the rapid increase in TSP biosynthesis is as a result of the mechanism of response to an injury caused in plant tissues (Xu et al., 2013).This increase was anticipated to the synthesis or activation of phenylalanine ammonia-lyase (PAL) enzyme (Beeching et al., 2002;Sanchéz et al., 2013).In this work, the room temperature resulted in larger TSP content, browning and loss of visual quality of 'baby cassava' at room temperature after 48 h (Figure 2 and 3A).
When the "baby cassava" was refrigerated it could be seen that, the phenolic compounds content were increased by 1.6 times in the sixth day of storage (Figure 3C to ii) as compared to the initial day of processing, next to 1.7 times after 6 h at room temperature (Figure 3C to i).This shows the importance of refrigeration in the delay of peak TSP for 'baby cassava' to be less brown.The same trend was observed in cassava roots, over a few days of storage at room temperature (Uarrota and Maraschin, 2015) and also "baby cassava" (Freire et al, 2015), as well as some varieties of potato (Cantos et al., 2002).
The short and long storage in different temperatures of the "baby cassavas" influenced the PPO and POD activities (Figure 3B and C).Enzymatic activities increased for the minimally processed cassava for 10 days under refrigerated storage (5 ± 2°C) (Figure 3B to ii, C to ii).The PPO activity in the "baby cassavas" kept at room temperature slightly increased more than 3 times in its original value in relation to those stored at 5 ± 2°C at the end of 12 h (Figure 3B).Moreover, the POD activity after 12 h at 5 ± 2°C remained stable, while at room temperature, it increased at about 2 times (Figure 3C).In this context, the browning observed for 'baby cassava' occurred after explosion in the enzymatic activities studied, especially those kept at room temperature.Vitti et al. (2011) also reported these parameters, where the initial symptoms of senescence in minimally processed potatoes increase in enzyme activity.The increase of enzymes activities accompanied an increase in the phenols oxidation to 6 h, under room temperature until 6 days at 5°C (Figure 3A).This shows a close association between PPO, POD activities, TSP and darkening in cassava as described by Yingsanga et al. (2008).Increased PPO activity and browning levels in vegetables, which darken are seen in some studies on minimally processed jicama roots (Pachyrhizus erosus) (Aquino-Bolaños and Mercado-Silva, 2004).
Notwithstanding, Vitti et al. (2011) and Cantos et al. (2002) found no direct relationship between the PPO activity and browning of some cultivars of minimally processed potatoes, even with high levels of the enzyme and high susceptibility to darkening in some varieties.The same was reported by Cantos et al. (2001), for minimally processed lettuce leaves.This is not fully elucidated and influenced by plant and its structures.
In another approach, the increase of POD activity in "baby cassava" may be related to the defense mechanism of abiotic stress (Liu et al., 2010).In this work, it was high temperature and cut.Thus, it plays an important role in the lignin synthesis of cassava (Beeching et al., 2002;Canto et al., 2013), and to a lesser extent comparable to other tuberous roots such as taro (Colocasia antiquorum), sweet potato (Ipomoea batatas L. Lam) and jicama (Pachyrhizus erosus L. Urban) (Aquino-Bolaños and Mercado-Silva, 2004).
In this work, it is possible to see the simultaneous action of the enzymes POD and PPO, for both forms of temporary storage.This behavior can be related to the synergistic action of these two enzymes (Cantos et al., 2001).In the reaction catalyzed by PPO, oxidizing the phenolic compounds resulted in the release of hydrogen peroxide (H 2 O 2 ), inducing POD activity.Another factor may also be associated with the de novo synthesis of POD, as observed by Cantos et al. (2001) who obtained a linear increase in POD activity and synthesis of new isoenzymes, attributing tissue repair to this factor (Yingsanga et al., 2008).
It was observed that at 12 h and at the end of 10 days, there was no fluorescence emission on 'baby cassava' at 5°C (Figure 4).On the other hand, at 25 °C, the pieces emitted fluorescence in the ultraviolet light, as was also evident, in later stages: stickiness on the surface, after the second day.Simões et al. (2016) observed Pseudomonas ssp., using fluorescence emission under ultraviolet light, in which it has aerobic metabolism (Sillankorva et al., 2004).The packaging used evidenced a certain aerobic environment, due to high PPO activity (Figure 3) in which it uses O 2 in the catalysis of the reaction.Moreover, some phenolic compounds can emit fluorescence under ultraviolet light (Wulf et al., 2005).In this work, PPO and POD activities and phenolic compounds increased during conservation (Figure 3); perhaps the fluorescence emitted is related to the increase of phenols.
Thus, in the present work, the fluorescent spot observed is associated to surface stickiness, which may show microbiological contamination in the "baby cassava" at 25°C, after two days.However, no quantitative analysis was done.This evidence is important for quick handling and consumption, not more than 12 h of 'baby cassava' at room temperature.
Thus, high POD and PPO activities and accumulation of phenols compounds were biochemical markers that preceded the browning in "baby cassava".This shows that maintenance of minimally processed cassava at low temperatures delayed the metabolism studied, reduced the effects caused by injuries of processing, probably retarding microbial growth and consequently prolonging shelf life of 'baby cassava' (Figure 5).Moreover, the new form of 'baby cassava' made it possible for commercialization and consumption, at room temperature before 12 h.

Conclusion
The minimally processed "baby cassavas" from the roots of cassava cv.Recife remained with high visual quality for 12 days at 5 ± 2°C, and not more than 12 h at 25 ± 2°C.The accumulation of phenolic compounds and the increases in the PPO and POD activities in early hours were more pronounced in the "baby cassavas" kept at 25 ± 2°C, as compared to those kept at 5 ± 2°C in all cases, with browning.

Figure 3 .
Figure 3. Content of total soluble phenols (A); polyphenoloxidase (B) and peroxidase (C) activity in hours(i) or days (ii) after processing for "baby cassava' format, and stored at 5±2°C and 25±2°C for 10 days.

Figure 5 .
Figure 5. Model illustrative of the handling and responses of minimally processed cassava kept at high and low temperatures.Changes in PPO and POD activities with phenolics compound, response to shelf life of 'baby cassava'.The bold letters represent larger enzymatics activities.