*2.2. Analisys*

Brown stain index (BSI) was always performed by the same analyst by rating carrots based on visual appearance. Scores ranged from 1 to 3, being 1 = roots free of stains; 2 = roots with light or few stains; 3 = roots with a large amount and/or darker stains (Figure S1). The number of carrots that received each score was converted into a percentage and the result was expressed as % of roots. The shelf life limit was determined when there were no more carrots rated as 1. They were discarded at that time.

Color was evaluated by using a MINOLTA CHROMA METER CR-400 and the luminosity L\*, a\* and b\* values were determined. Value a\* is a red/green coordinate (+a indicates red and −a indicates green) and value b\* is a yellow/blue coordinate (+b indicates yellow and −b indicates blue) [8]. Three

measurements were taken on the external surface of each of the seven carrots per repetition, one in the upper area, one in the middle area, and one in the lower area.

To determine the effects of these edible coatings on the carrots, periderm anatomical analyses were performed after 15 days of storage. An optical microscopy analysis was performed in both healthy and damaged carrots on cross-sectional cuts made with a sliding microtome Leica SN 2000 R (Leica Biosystems, Heidelberg, Germany), clarified in a bleach solution 10%, and washed with distilled water. Histochemical tests were conducted with Sudan IV for lipid substances [9] and double color with iodine green (greenish-blue color for lignin) and Congo red (red color for cellulose) [10]. Images were captured by a trinocular Leica DM LB microscope (Leica Microsystems, Wetzlar, Germany) connected to a Leica DC 300 F video camera. External images of the carrots were captured by aM205C (lens) microscope (Leica Microsystems, Wetzlar, Germany) connected to a Leica DFC 295 video camera.

The BSI and color results were submitted to a variance analysis (ANOVA) and the averages were compared by the Tukey test (*P* ≤ 0.05), using the statistical software SISVAR, version 5.6 [11].

#### **3. Results and Discussion**

## *3.1. Visual Quality*

To determine the effect of edible coatings on the quality of yellow carrots, changes in visual characteristics were observed. General appearance was taken as a marketing criteria. Overall, the use of edible coatings on yellow carrots was not viable, irrespective of treatment. The 12% carnauba wax 12% brown stains on the periderm right after the application and the intensity of these stains increased during storage (Figure 1). Carnauba wax at the lower concentrations (0.5% and 1%) and all chitosan coatings did not cause stains on the day of application. Except for the control treatment, all treated carrots presented periderm stains on the seventh day of storage. On this day, all the carnauba treatments and 3% chitosan 3% presented more severe stains, receiving a score of 3. Treatments with 1% and 5% chitosan exhibited light or few stains after seven days of storage. On the 15th day of storage, carrots coated with carnauba wax were discarded due to the severity of the stains. For 1%, 3%, and 5% chitosan-coated carrots, only 27%, 10%, and 13% did not have stains, respectively. The control treatment exhibited 7% of the carrots with light stains. On the 30th storage day, despite the concentration, 100% of the carrots coated with chitosan were stained and thus not marketable.

**Figure 1.** Brown stain index of yellow carrots treated with different edible coatings and stored at 2 ◦C 95–100% RH. Score: 1 = roots without stains;2=roots with light or few stains; 3 = roots with a large amount of stains or with darker stains.

The color of yellow carrots was affected by the application of coatings, confirming the data obtained from the visual rating. The luminosity decreased during cold storage for all treatments (Figure 2A). Except for 1% chitosan, all treatments presented lower luminosity values than the control samples. The decline in luminosity was proportional to the increase of the coating concentration. The reduction on the L\* values indicated the darkening of the surface caused by the brown stains.

**Figure 2.** Luminosity (**A**) and values a\* (**B**), and b\* (**C**) of yellow carrots treated with different edible coatings and stored at 2 ◦C 95–100% RH. Vertical bars represent the standard error of the mean (*n* = 5).

The control treatment did not show significant variation for the a\* values during cold storage, always exhibiting the lowest values among the different treatments (Figure 2B). All of the other samples presented a\* values that increased during storage, which indicated a change in the characteristic yellow color of this variety. The higher the a\* value, the closer the sample is to a reddish hue. Brown stains in these treatments left a reddish tint to the carrots, which is directly related to the increase in the values of a\*.

Control, 1% chitosan 1%, and 5% carnauba treatments showed an increase in the values of b\* during storage. Chitosan at 1% interfered the least on the color of the yellow carrots, not differing from the control (Figure 2C). Carrots coated with 3% chitosan and 12% carnauba showed a reduction in the b\* values from the beginning to the end of the storage period. This reduction showed a loss in the yellow color saturation, especially for the carrots coated with 12% carnauba, which also exhibited the highest degree of brown stains. Carrots coated with 5% chitosan and 1% carnauba 1% did not show any variation; however, their b\* values were lower than the control.

#### *3.2. Damage Evaluation with Microscope and Magnifier*

Due to the observation of visual quality losses caused by the coatings, microscopy and magnifying glass techniques were used to investigate the extent of damage.

After 15 days of storage, damage affected the carrot periderm. Carnauba at 12% caused not only stains, but also depressions and superficial viscosity (Figure 3F). Carrots with chitosan presented lighter stains than the carnauba after 15 days of storage. However, at the end of the storage period, the stains were darker, similar to the ones caused by the carnauba.

**Figure 3.** Details of yellow carrots 15 days after the application of different edible coatings and stored at 2 ◦C 95–100% RH. (**A**,**<sup>a</sup>**) Control; (**B**,**b**) chitosan 1%; (**C**,**<sup>c</sup>**) chitosan 3%; (**D**,**d**) chitosan 5%; (**E**,**<sup>e</sup>**) carnauba wax 0.5%; (**F**,**f**) carnauba wax 1%; (**G**,**g**) carnauba wax 12%.

Carnauba wax at 6% (Figure 4A) was also tested (no data presented) and, like 12% carnauba (Figure 4B), it caused brown stains immediately after the coating application. The carrot surface has a periderm full of imperfections, creating a debarking aspect. Apparently, stains were concentrated on these imperfections along the carrot, suggesting that these structures were more sensitive to carnauba than the ones covered by the periderm. As the days advanced, stains spread and damaged the periderm cells, causing bigger injuries. Carnauba at 12% caused browning of the inner part on a significant area of the secondary phloem. The intensity of the injury also induced the occurrence of viscosity over the stains (Figures 3F and 4B) and depressions (Figure 4). Depressions were generated by the compaction of periderm and parenchyma cells (Figure 4C,E). In some cases, there was periderm rupture (Figure 4D,F) and deposition of lignin on the depressed region (Figure 4F). It was possible to observe that the damaged regions frequently occurred close to the secretory canals, which are located longitudinally in the root. This intensifies the depression and brings about the rupture and exposition of these canals (Figure 4F).

Brown stains probably resulted from the oxidation of phenolic compounds and the subsequent formation of darker compounds. The interaction between the coating and the cells seems to have degraded cell structures, and thus this decompartmentalization provided the contact between phenolic compounds and oxidative enzymes. The *o*-quinones, which are products of this reaction, have an undesirable brown color that stands out on the yellow carrot surface. It depreciates its appearance and commercial quality.

The emergence of stains on the treatments with carnauba wax may be related to the basic pH of the product (around 9.0) that remains unaltered after its dilution. Solutions that are very basic may cause injuries on the cell walls, such as burns. Caron et al. [12] also reported the emergence of stains on 'Brasília' carrots. These stains were caused by the application of carnauba coatings undiluted or in concentrations of 1:3 and 1:6, depending on the brand of coating used.

Based on these results, chitosan and carnauba wax coatings at the tested concentrations are not recommended for yellow carrots, as they damaged the carrots appearance, thus making them unmarketable.

**Figure 4.** Damage to yellow carrots promoted by the carnauba wax: (**A**) Carnauba wax 6%; (**B**) carnauba wax 12%; (**C**) carnauba wax 6%; and (**D**) carnauba wax 12%. Photomicrographs of cross sections of superficial region of carrots: (**C**) Depression and compaction of the periderm. (**D**) Rupture and compaction of periderm cells evidenced by lignin deposition. (**E**) Compression of the periderm layers. (**F**) Periderm rupture near the secretory canal. Sc = Secretory canal; Ph = Phloem; Li = Lignin; Pa = Parenchyma; Pe = Periderm (arrows = depressions; \* regions with viscous appearance).

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2311-7524/4/4/31/s1, Figure S1: Score of Brown Stains Index in yellow carrots. 1 = roots without stains; 2 = roots with light stains or few stains; 3 = roots with large amount of stains or with darker stains, Table S1: Color of yellow carrots treated with different edible coatings and stored at 2 ◦C 95–100% RH.

**Author Contributions:** N.D.B., R.A.K. and M.H.F.S. conceived and designed the experiments; N.D.B., M.A.T., A.P.P. and B.T.N. performed the experiments, analyzed the data and wrote the paper. R.A.K. and M.H.F.S. edited and revised the paper.

**Funding:** This research was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp) gran<sup>t</sup> number 2014/07779-6 (first author) and 2015/06378-0 (fourth author).

**Acknowledgments:** The authors acknowledge the company Aruá Comércio e Serviço LTDA. For the donation of carnauba wax.

**Conflicts of Interest:** The authors declare no conflict of interest.
