Abstract: Objective: The main objective of this study was to extend shelf life of Cambuci peppers stored under refrigeration or at room temperature by using biodegradable films with Spirulina platensis as coating. Materials and Methods: Peppers were washed, sanitized and separated into four groups. Two groups were coated with biodegradable films and the other two groups were used as control ones. Storage occurred at both room and refrigeration temperatures. All groups were analyzed regarding their general appearance, pH, titratable acidity, soluble solids, loss of mass, color and instrumental texture at the beginning of the experiment and on the 3rd, 6th, 8th and 14th days of storage. Data were analyzed by the analysis of variance and Fisher’s LSD test (p<0.05). Results: There was no significant difference (p>0.05) in luminosity among samples at room temperature. Peppers showed less accelerated maturation at refrigeration temperature. The sample with coating application, stored at room temperature, had less mass loss by comparison with its control. Reduction in soluble solid values was significant (p<0.05) for all samples under analysis. Conclusion: The combination of low temperature and application of coating was beneficial for extension of shelf life of Cambuci peppers.
INTRODUCTION
Biodegradable films, whose composition may vary according to the needs of their application, have been studied as an innovative proposal for coatings and active food packaging. Several biomolecules, such as proteins, lipids and polysaccharides, have the ability to form films in isolation and in combination. However, the use of these components has some limitation due to their high absorption of moisture which leads to consequent changes in properties. Processing conditions of biodegradable coating, besides its composition and storage temperature, are factors that influence the performance of compounds with antimicrobial potential1.
Spirulina sp., is a filamentous microalgae that has anti-inflammatory, immunomodulatory and anti-allergic properties2. Its antioxidant power has been attributed to some of its constituents, such as fatty acids, phenolic compounds, chlorophyll, phycobilins, phycoerythrin, allophycocyanin and phycocyanin3,4.
Fruits and vegetables undergo rapid deterioration, due to their high metabolic activity, which can be delayed by means of layers of edible coatings5. Among methods of food preservation, temperature lowering is important since it slows down various enzymatic and chemical reactions, as well as microbial growth6,7.
The application of coating and reduction in temperature can be effective to maintain the quality of the products, since they act in synergy. The cold chain reduces respiration and perspiration, thus, slowing senescence8. Therefore, studies that take into account the inherent particularity of plants and the specific composition of coatings are necessary to determine the feasibility of coating application. This study aimed at evaluating biofilm application to Cambuci (Capsicum sp.) peppers by immersion, so as to extend their shelf life at both room and refrigeration temperatures.
MATERIALS AND METHODS
Materials: The experiment was conducted at the State University of Ponta Grossa, located in Ponta Grossa, Paraná (PR), Brazil, in the second half of 2016. Peppers (Capsicum sp.) were bought in a local market. Commercial cassava starch and cassava bagasse from the Lorenz® cassava processing industry in Umuarama, PR, Brazil, besides dehydrated, colorless commercial gelatin (Apti®), commercial Spirulina platensis biomass (Tamanduá®), with application to Cambuci peppers, were used for producing the coatings. Reagents were glycerol P.A (Reactif®), NaOH (0.01 N) and phenolphthalein.
Methods
Preparation of peppers: Cambuci (Capsicum sp.) peppers were selected for the experiment. They were firm and homogeneous in relation to their color and average size (6°Brix and 5 cm in diameter). They were washed in running water, immersed in chlorinated solution (200 ppm) for 3 min, rinsed and dried at room temperature.
Elaboration of filmogenic solutions: Coverage was obtained by hydrating the raw materials (starch (4%), cassava bagasse (0.66%), Spirulina platensis (0.66%), gelatin (0.66%) and glycerol (1%)) in distilled water (93%). The solution was then heated on an asbestos plate, stirred until it reached boiling point and kept in this condition for 15 min.
Coating: After the filmogen solution was cooled at room temperature, fruits were immersed in it for 2 min. The solution was then drained by suspending the fruits through the stalk. Fruits were left drying at room temperature for 3 h. Afterwards, both treatment and control groups were conditioned on trays at 5±2°C (Prosdócimo 340 L refrigerator, Brazil). Follow-up examinations were carried out for 14 days, i.e., at the beginning of the experiment and on the 3rd, 6th, 8th and 14th days of storage. Samples were denominated as follows: R (A): With coating at room temperature, R (R): With coating at refrigeration temperature, C (A): Control at room temperature and C (R): Control at refrigeration temperature.
Color: Color was measured by a portable spectrophotometer (Hunterlab, Miniscan EZ, USA), which operated in the CIELab system (L*a*b*). Absolute values of coordinates L* (lightness), a* (green to red) and b* (blue to yellow) were obtained. Coordinates a* and b* were used for calculating chromaticity (the higher the value, the more intense the color) and the Hue angle9,10. Equation 1 calculates the chroma whereas Eq. 2 determines the Hue angle.
| (1) |
| (2) |
General appearance: During the storage period (14 days), samples were photographed so that their general appearance could be compared at different stages of maturation.
Mass loss: Fruit mass loss was determined in agreement with a methodology adapted from Mohebbi et al.11, by an analytical balance with precision of 0.0001 g. Five fruits per test were weighed during 14 days of storage.
Texture profile: Texture evaluation was performed by a texturometer, the TA-XT plus Texture Analyzer (Stable Micro Systems, Godalming, U.K.), which operated at 25°C. In the compression test, a 2 mm diameter probe and a 25 kg load cell, at 1.5 mm sec‾1, were used in the central part of the fruit. Compression distance was 40 mm and contact force was 5.0 g. The parameter in Newton’s (N) was evaluated five times during 14 days of storage. Tests were done in six replicates. Firmness was stipulated as the value obtained at the peak of the analysis.
Titratable acidity: Titratable acidity was determined with small adaptations12 (g citric acid/100 g sample). One gram sample was added in 100 mL distilled water and three drops phenolphthalein, was put into Erlenmeyer flasks. Samples were titrated with 0.01 N NaOH solution until they reached pink staining.
pH: One gram sample diluted in 100 mL distilled water was weighed in an Erlenmeyer flask. Reading was performed by a calibrated digital bench top pH meter (pHmeter-PHS-3B).
Determination of total soluble solids: The sample was added drop wise (2) to the prism of the ATAGO portable refractometer (N-1α Brix 0-32%) for direct reading, in agreement with the manufacturer's instructions. Reading was done in degrees Brix.
Statistical analysis: Data were analyzed by the one way analysis of variance (ANOVA) and Fisher’s LSD test13 in order to verify the existence of significant differences among the conditions under study at 90 or 95% confidence level (p <0.10 or 0.05, respectively). The statistical treatment of the data was done by the Excel Action 2.8 supplement (Microsoft, São Paulo, Brazil).
RESULTS
Color: Color is an important attribute of quality since it influences consumer’s acceptability and indicates deterioration due to senescence, chemical and enzymatic reactions and microbial growth14. As a result, several synthetic dyes have been added to food, even though there is consumer’s appeal for the use of natural compounds15.
The use of low temperatures slows enzymatic activity whereas coatings protect against excessive exposure to oxygen, thus, helping to reduce the loss of the green color which is typical of Cambuci peppers16. Data obtained by the analysis of the color of peppers are shown in Table 1.
| Table 1: | Color analysis of peppers with and without coating, stored at room and refrigeration temperatures |
*Mean±Standard Deviation, different upper case letters represent significant differences in the row and different lowercase letters represent significant differences in the column, according to Fisher’s LSD test (p<0.05), R (A): With coating at room temperature, R (R): With coating at refrigeration temperature, C (A): Control at room temperature and C (R): Control at refrigeration temperature | |
| Fig. 1: | General appearance of peppers stored at room temperature A: Coated samples, B: Control samples (uncoated), 0, 3, 6, 8 and 14 days of storage |
| Fig. 2: | General appearance of peppers stored at refrigeration temperature C: Coated samples, D: Control samples (uncoated), 0, 3, 6, 8 and 14 days of storage |
General appearance: During the follow-up period, peppers matured and the application of the coating aimed at decelerating this process. The visual analysis showed that coated samples had a satisfactory appearance until the 8th day while control samples, which began to lose turgidity on the 3rd and 6th days, had an inadequate appearance for consumption at room temperature (19.8±2.8°C). The apparent deterioration is consistent with the loss of mass during storage. Images of uncoated and coated samples at room temperature are shown in Fig. 1 whereas the ones at refrigeration temperature are depicted in Fig. 2.
Mass loss: Reduction in mass loss of samples under analysis occurred at refrigeration temperatures during 14 days of storage as shown in Fig. 3. Results of samples kept at room temperature (coated and control) and at refrigeration temperature (coated and control) were similar. Samples with coating application had lower mass loss than their respective controls. Loss of mass among samples and throughout the days of the analyses (p<0.10) was significant.
Texture profile: Determination of the texture of fresh and processed fruits and vegetables is a parameter which has been used for establishing product quality and consumers’ acceptability17. In general, plant tissues have higher values of firmness at pH below 4; however, firmness decreases with increasing pH18. Metabolic processes can cause starch degradation and conversion of acids to sugars, leading to changes in firmness19 (Table 2).
Titratable acidity, pH and total soluble solids (°Brix): Reduction in acidity is associated with the natural process of fruit and vegetable ripening, since in respiration, organic acids are metabolized in other non-acidic compounds20.
| Table 2: | Texture of Cambuci pepper samples with and without coating at room and refrigeration temperatures during storage |
Mean±Standard Deviation, different upper case letters represent significant differences in the row and different lowercase letters represent significant differences in the column, according to Fisher’s LSD test (p<0.05), R (A): With coating at room temperature, R (R): With coating at refrigeration temperature, C (A): Control at room temperature and C (R): Control at refrigeration temperature | |
| Table 3: | Monitoring of acidity, pH and soluble solids of Cambuci pepper samples with and without coating stored at room and refrigeration temperatures |
| *Mean±Standard Deviation, different upper case letters represent significant differences in the row and different lowercase letters represent significant differences in the column, according to Fisher’s LSD test (p<0.05), R (A): With coating at room temperature, R (R): With coating at refrigeration temperature, C (A): Control at room temperature and C (R): Control at refrigeration temperature |
| Fig. 3: | Mass loss of Cambuci pepper samples R (A): With coating at room temperature, R (R): With coating at refrigeration temperature, C (A): Control at room temperature, C (R): Control at refrigeration temperature |
In this study, acid contents of all samples reduced during storage.
Refrigerated samples (coated and control) showed significant reduction in pH in the 14 day period (p<0.05). Values of pH found for samples at room temperature were consistent with their acidity and had significant difference (p<0.10) in the 14 day analysis, except coated samples maintained at room temperature, which maintained their pH throughout this period. Samples maintained at room temperature showed increase in the pH value during storage.
Reduction in soluble solids (°Brix) was significant (p<0.05) for all samples under analysis. The highest reduction in these values occurred for control samples stored at room temperature (Table 3).
DISCUSSION
Regarding the luminosity parameter (L), refrigerated samples were observed to have higher values by comparison with those stored at room temperature, although there was no significant difference between them, neither at the beginning of the experiment nor on the 6th and 8th days. Luminosity did not show any significant difference among samples with and without coating stored at room temperature from the beginning of the experiment to the 14th day (p>0.10). It shows that color was maintained by the presence of the film in this condition. The maturation process occurred at different speeds, a fact evidenced by the luminosity on the pepper surfaces on the 3rd and 8th days.
A study by Carvalho et al.21 of Physalis peruviana L., coated with alginate films found no significant difference among the fruits regarding the staining parameter during refrigerated storage (21 days). On the other hand, L* values and the Hue angle were low in all samples with polysaccharide films enriched with dietary fibers and applied to apples22.
The control sample stored at refrigeration temperature did not have any statistical difference (p> 0.10) concerning the Hue angle during 14 days of storage. On the last day of the analysis (14th), a significant difference (p> 0.10) regarding the Hue angle was established among all samples under analysis. In a study of the application of emulsion-based films in apples23, showed that coated apples had a higher color parameter L* than uncoated apples during the storage period.
High values represent bright or intense colors24. There was no significant difference (p>0.10) among the 4 samples under analysis on the days of the analyses. Control sample C (A) had increased staining intensity at the end of the 14 days of storage. The other samples remained constant.
Control samples stored at refrigeration temperature had high color intensity after the 14 day follow-up period while samples kept at room temperature had the lowest chroma values. In the case of coatings produced with fruit and vegetable residues, chroma values ranged from 59-625. In general, refrigerated storage time had a significant influence on the fruit color (p<0.10) and the treatments with coatings did not affect the color parameters in a remarkable way. This fact can be explained by the decrease in reaction rates due to the effect of temperature, thus inhibiting post-harvest metabolism26.
Mechanical damage and cuts during transport and storage of quality fruits and vegetables decrease their quality and commercialization generating large losses in the production chain27. In order to store these products for a longer period of time, post-harvest refrigeration is
fundamental, in addition to proper handling practices28. The quality of the marketed product depends on the maintenance of some parameters, such as appearance, color and firmness, which are affected by deterioration caused by ripening and microorganisms21.
The use of coating visually interfered in the samples from the 6th day on. Changes were not as noticeable as in samples at room temperature due to reduction in surface wrinkling. Changes were observed in peppers from the control group from the 6th day on, whereas alterations in the coated samples were only observed on the 14th day.
Loss of water from coated fruits occurs due to an accelerated evaporation of surface water caused by low relative humidity (between 40 and 50%)16. In this study, samples were stored at 50±12% relative humidity. Even though mass loss occurred in peppers, moisture was maintained due to the formation of water from the respiratory process29. After 17 days of storage, as ripening proceeded, the weight loss increased by 8.3-9.7% in melatonin-treated tomato fruits30. At refrigeration temperature (3.6±1.6°C), peppers showed less accelerated maturation than samples kept at room temperature (19.8±2.8°C). It confirms that the combination of preservation methods (coating and low temperature) is positive for fruits and vegetables.
The result of the physiological processes may correspond to the tissue sensitivity to ethylene, rather than exclusively to the increase in its production. Fruit and pepper seeds show uniformity in their development but indeterminate growth31. Therefore, fruits in different stages of maturation may be found in the same plant, a fact which makes harvest difficult32.
Perspiration and respiration of fruits can be reduced by applying coatings, which act as a cover on the food, thus filling stomata and lenticels33. CO2 produced as a result of O2 consumption in the respiratory process, is maintained even at low rates and modifies the storage atmosphere19. The atmosphere formed and retained by the film may not be tolerated by peppers and cause physiological disorders, such as weight loss. Since films can extend shelf life due to protection of the food against the external environment, there may be condensation of water from the respiration, a fact that enables microbial development and reduction in product preservation33.
Mass loss is one of the limiting factors of the use of biodegradable coatings, since they have low barriers to moisture and water vapor, besides problems related to mechanical properties34. Weight loss and aroma in fruit is related to respiration, which is a physiological process that results in the consumption of sugars and starch35. If it is a non-climacteric fruit, respiration decreases gradually during maturation. Weight reduction implies loss of quality for consumption, related to texture36,37. Higher losses make fruits and vegetables unfit for consumption.
In some products, color changes are related to surface dehydration, which occurs due to moisture losses in the form of vapor38. In this study, as well as in the study by Xing et al.27, storage temperature influenced the weight loss of peppers, showing that the use of refrigeration is able to control this parameter. Coated peppers stored at refrigeration temperature showed decrease in transpiration by comparison with control samples in the same conditions. Likewise, a study of green chillies showed that they were benefited by the use of refrigeration and high relative humidity16.
High concentrations of chitosan and gum arabic in films applied to carambolas caused great loss of mass during the storage period, a fact that may be explained by increase in anaerobic respiration39. Alginate films applied to minimally processed watermelon gave good results in relation to mass loss during the storage period, by comparison with the control sample40.
Minimally processed products must go through the sanitation stage-to reduce pathogenic microorganisms-use of packaging and refrigeration system41. Plastic films (PVC), modified atmosphere and low temperatures have been commonly used in fruits for export42. These factors interfere in the mass loss of marketed products.
Regarding the nano-laminate coating applied to fresh-cut pear, the coating avoided mass loss, proving the efficiency as barrier43. Since cell membranes act as barriers to solute movements, loss of mass can be attributed to their degradation due to the senescence of the fruit which leads to disintegration and impairment of the control of biochemical and physiological processes of the cells. The purpose of the coatings is to reduce mass losses, but it has not always been observed.
In this study, controls at both temperatures showed decrease in firmness during 14 days of storage. All samples significantly differed among them (p<0.05). Samples with coating application maintained at room temperature (19.8±2.8°C) kept their firmness characteristics with no changes whereas coated peppers kept at refrigeration temperature (3.6±1.6°C) became firmer with the passage of time. At the end of the analysis period, the highest and lowest results of firmness were found for coated and refrigerated samples (3.6±1.6°C) and for control ones at room temperature (19.8±2.8°C), respectively. The use of coating was efficient in maintaining the firmness of the samples under analysis by comparison with control ones.
A study of fresh melon samples showed that they required mean compression force of 35 N to get 50% deformation while chitosan coated samples required strength between 45 and 54 N, thus, indicating that the coating was efficient in reducing fruit softening7. A similar result was obtained by Poverenov et al.20, who’s study of untreated samples of red peppers (Capsicum annuum L. cv. vergasa) showed considerable degradation in texture, whereas samples coated with chitosan and gelatin remained unchanged during storage.
Control samples stored at room temperature showed the highest acidity reduction at the end of the period under study, it was an expected result due to maturation caused by temperature. Reduction in organic acid contents was significant (p<0.05) for all samples, except for the control samples stored at refrigeration temperature, i.e., they maintained organic acids for 14 days. This result indicates that the control sample stored at low temperature, in relation to the acidity parameter, was better preserved. On the last day of the analysis, samples showed no significant difference in titratable acidity.
Total Titratable Acidity (TTA) of minimally processed papaya (climacteric fruit) coated with chitosan film and pectin remained constant during storage by comparison with control samples5. A similar result was found by a study of film coating applied to carrots based on residues of fruits and vegetables. It showed the tendency to increase TTA contents, followed by reduction during storage. At the end of the experiment, there was a significant difference (p<0.05) between both groups (coated and control)25.
Determination of pH or hydrogen ions in solution deals with interferents, such as temperature, pressure and viscosity18. Carrots coated with films from fruit and vegetable residues showed an initial acidic pH by comparison with control ones, during storage, values suffered small variations28. The use of chitosan and pectin coatings applied to papaya did not influence fruit pH, which remained stable during the analysis5.
Reduction in soluble solids (°Brix) demonstrate that the metabolic process followed its natural course, although it was influenced by temperature and coating. Data from the literature showed that coatings applied to carrots had initial 1.8 °Brix while control samples had 2.4 °Brix. These values were linearly reduced during storage28. Different results were also obtained, i.e., total soluble solid contents were higher in the coated samples than in the control ones5. These data enables the peculiarity of maturation among different fruits to be observed.
CONCLUSION
The overall appearance of coated Cambuci peppers was kept close to the fresh fruits at both temperatures (4 and 20°C). At refrigeration temperature the peppers presented less accelerated maturation when compared to the samples kept at room temperature. Coatings aided in the maintenance of color and texture, besides preventing surface wrinkling. It was possible to confirm that the combination of preservation techniques, i.e., application of coating and low temperatures were beneficial for extension of shelf life of Cambuci peppers.
SIGNIFICANCE STATEMENTS
Biodegradable coatings are packaging applied to food for the purpose of preserving the product. In the present study a biodegradable coating containing Spirulina platensis was evaluated for improving the post harvest behavior of Cambuci peppers, contributing to decrease losses of this vegetable.
ACKNOWLEDGMENT
The authors are grateful to the State University of Ponta Grossa for its research infrastructure and to CAPES for granting a research scholarship. The author would like to thank the National Council for Scientific and Technological Development (CNPq/Brazil) for financial support and scholarship through grants No. 445476/2014-1 and 307311/2013-9.