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Andean blackberries (Rubus glaucus Benth) quality as affected by harvest maturity and storage conditions
Scientia Horticulturae 226 (2017) 293–301
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Andean blackberries (Rubus glaucus Benth) quality as affected by harvest maturity and storage conditions
MARK
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Sandra Horvitza, , Diana Chanaguanoa, Iñigo Arozarenab a b
Food Science and Engineering Faculty, Universidad Técnica de Ambato, Campus Huachi, Av. Los Chasquis y Rio Payamino, Ambato, Ecuador Food Technology Department, Universidad Pública de Navarra, Campus Arrosadía s/n 31006, Pamplona, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords: Antioxidant activity Blackberries Cold storage Polyphenols Postharvest quality
Maturity stage at harvest and storage conditions are critical factors determining fruit postharvest quality. Physicochemical (fruit size, mass loss, color, firmness, pH, total soluble solids, titratable acidity), microbiological (total aerobic mesophiles, psychrotrophes, and yeasts and molds) and sensory quality of Andean blackberries harvested at two maturity stages and stored under room (18 ± 2 °C) and cold storage (8 ± 1 °C) was studied. Total phenolic compounds, anthocyanins and organic acids content, and antioxidant activity were also evaluated. The more mature fruit was classified as “big”, according to the Ecuadorian Standard and showed lower acidity and higher total soluble solids, anthocyanins content and sensory scores compared with the fruit harvested earlier, whilst maturity at harvest did not affect the microbial counts of any of the groups studied. Cold storage was effective in delaying weight loss, softening and microbial growth and also in maintaining a better sensory quality of the blackberries. What’s more, under refrigeration it was possible to extend the shelf-life of the fruit to up to 8 d. The main limiting factors for shelf-life were microbial growth and loss of firmness at room storage and cold storage, respectively. Based on these results, it would be advisable to harvest the fruit at maturity stage 5 in order to achieve an appropriate fruit size, a high anthocyanin concentration, a better sugars/ acids equilibrium, and a better sensory quality and the fruit should be maintained under refrigerated storage.
1. Introduction Andean blackberries (Rubus glaucus Benth) are native to the Andes and grow year round, mainly in cold and temperate climates in South America (Martínez et al., 2007). The fruit are consumed fresh or processed as pulp, juices, jams or desserts and are appreciated for their color, flavor, bioactive compounds, and nutritional attributes, due to their high content in polyphenols, anthocyanins, vitamins, and minerals (Garzón et al., 2009; Junqueira-Gonçalves et al., 2016). Blackberries are non-climateric fruit and though, they must be harvested at full maturity, usually when they present a bright, dark purple/black color and optimum firmness (Bejarano, 1992). At this stage, also the best flavor quality is obtained (Skrovankova et al., 2015). Maturity at harvest is one of the main contributing and determining factors to small fruit quality during transport and commercialization, due to its direct relation with postharvest shelf-life (Perkins-Veazie and Collins, 2002). Immature fruit will not reach appropriate organoleptic characteristics whilst shelf-life of over-mature fruit is generally very short as the susceptibility to decay also increases (García, 2001). During ripening, berries soften and become darker in color, and also
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chemical changes like accumulation of sugars, reduction in organic acids, and an increase in anthocyanins content occur (MikulicPetkovsek et al., 2015). Different maturity indexes (color, firmness, total soluble solids, and days after full bloom, among others) are commonly used in order to determine the optimum maturity stage for fruit and vegetables harvest. In the case of blackberries, the Ecuadorian Quality Standard NTE-2427 (INEN, 2010) establishes that harvest can be done once the fruit reaches, at minimum, the maturity stage 3, defined as light red. However, Ayala-Sánchez et al. (2013) concluded that the berries should be harvested at stage 5, as at that stage, the fruit presented an adequate size, the characteristic shape, and appropriate acids and soluble solids content. In Ecuador, there are no accurate methods for determining when to pick the berry fruit and this can result in inappropriate quality of marketable product (Mikulic-Petkovsek et al., 2015). What’s more, due to agro-climatic conditions, fruit quality may differ in fruit harvested in different locations, even if their external color is similar. In addition, most of the research done on this fruit is focused on yield and quality comparisons among cultivars, and on fruit quality and determination of chemical compounds and antioxidant activity under
Corresponding author. E-mail address: [email protected] (S. Horvitz).
http://dx.doi.org/10.1016/j.scienta.2017.09.002 Received 2 March 2017; Received in revised form 30 August 2017; Accepted 1 September 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
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to a standard white tile (X 79.80, Y 84.97, Z 90.74) and using the CIELab color space (CIE, 1976), the illuminant D65 and the 10° observer as a reference. The measurements were done on two opposite points of the equatorial zone and on the basis of each fruit and the hue angle was calculated. Flesh firmness was measured with a Texture Analyzer (Model CT3, Brookfield Engineering Labs, Inc., USA) and the TexturePro CT V1.2 Build Software. Each fruit was punctured once in its equatorial zone with a stainless steel, flat plunger (3 mm diameter) and the results were expressed as the maximum force (N) necessary to break the flesh in each fruit.
different storage conditions whilst less attention has been paid to maturity stage at harvest and its relation with quality and commercial requirements (Ayala-Sánchez et al., 2013). Another problem with blackberries is that they are highly perishable fruits, with a usual shelf-life that does not exceed 2–3 d at refrigerated temperatures, what implies great limitations for fresh market and storage. In effect, blackberries have a very thin and fragile skin and thus, are very susceptible to water loss, softening, mechanical injuries and postharvest diseases like gray mold, caused by Botrytis cinerea, and Rhizopus rot (Junqueira-Gonçalves et al., 2016). Hence, the use of techniques that can improve shelf-life duration without altering the physical, sensorial, and nutritional characteristics of the fruit is of vital importance to reduce the postharvest losses, which in some cases can be as high as 60% of the production (Sora et al., 2006). In this sense, one of the main factors affecting the storage shelflife and quality of fruit and vegetables is temperature, as it regulates the rate of all the metabolic processes that occur in these products. Low temperatures slow down fungal growth and at the same time, reduce respiration rate and water loss and therefore, delay ripening and senescence processes (Oliveira et al., 2013a, 2013b). As blackberries are insensitive to chilling injury, the recommended optimum temperature for this fruit is at 0–5 °C (Joo et al., 2011). The aim of this work was to characterize the physicochemical, microbiological and sensory quality, and the postharvest performance of Andean blackberries, harvested at two maturity stages and stored under room temperature and refrigerated storage. Bioactive compounds (total polyphenols, individual anthocyanins), organic acids and antioxidant activity were also studied.
2.3.1.3. pH, total soluble solids (TSS) and titratable acidity (TA). These parameters were determined on the juice obtained from each sample. The pH of the blackberries was measured with a pH-meter (ORION VersaStar, Thermo Scientific, Denmark). TSS were determined with a hand refractometer (Atago, Japan) and titratable acidity was determined by titration with 0.1 N NaOH to pH 8.1 (AOAC, 2000) and expressed as g citric acid 100 g−1 fruit (fresh weight). 2.3.2. Extraction and determination of organic acids A 2-g sample of lyophilized fruit was homogenized with 20 mL of distilled water and shaken for 30 min at 100 rpm in a bench-top orbital shaker (Infors HT, Switzerland). Afterwards, the solution was centrifuged (15 min, 2100g) at room temperature using a Medifriger BL-S centrifuge (J.P. Selecta, Spain). The extraction process was repeated twice, the supernatants were combined and the volume was made up to 50 mL with distilled water. Citric, malic, and ascorbic acids were determined by HPLC, following the method described by Scherer et al. (2012), with some modifications. For this purpose, a liquid chromatograph Waters 2695 coupled to a Waters 996 PDA detector and a LiChroCART Purospher RP-18 column (250 mm × 4 mm, 5 μm, Merck, Germany), with a Purospher precolumn (4 mm × 4 mm, 5 μm) were employed. The extracts (20 μL) were injected into the chromatograph after filtration with 0.45 μm PVDF filters (Análisis Vínicos S.L., Spain). An isocratic elution procedure was used with a 0.01 M KH2PO4 buffer solution, at a flow rate of 0.8 mL per minute, with the column kept at 25 °C. The peaks were identified by comparison of their retention times and UV-VIS spectra with those of standards. An external standard curve of each organic acid was used for quantification, at 250 nm for ascorbic acid, and at 210 nm for malic and citric acids.
2. Materials and methods 2.1. Plant material Andean blackberries (Rubus glaucus Benth) were hand-harvested in Tungurahua Province, Ecuador at maturity stages 3 (light red) and 5 (dark purple) in January and July 2016. Maturity stages were defined by the external color of the fruit, according to the Ecuadorian Standard (NTE-INEN 2427 (INEN, 2010)). Immediately after harvest the fruit were transported to the Technical University of Ambato for analyses. Fruit of uniform size and color, and free from damage and injuries were selected for the study. 2.2. Packing and storage
2.3.3. Extraction and determination of anthocyanins, total polyphenols and antioxidant activity 2.3.3.1. Extraction. 1-g of lyophilized fruit was homogenized with 20 mL of ethanol:distilled water:formic acid (50:48:2; v:v:v) and shaken for 30 min at 100 rpm in a bench-top orbital shaker (Infors HT, Switzerland). Afterwards, the solution was centrifuged (15 min, 2100g) at room temperature using a Medifriger BL-S centrifuge (J.P. Selecta, Spain). The extraction process was repeated twice, the supernatants were combined and the volume was made up to 50 mL with ethanol:distilled water (v:v).
200 ± 10 g of blackberries were packaged in transparent polyethylene terephthalate (PET) plastic containers and stored at 18 ± 2 °C (RT) and under refrigerated storage, at 8 ± 1 °C. 2.3. Quality attribute analyses Quality parameters were evaluated on the harvest day and every 3 d during the storage period, with the exception of fruit size that was determined only on day 0.
2.3.3.2. Anthocyanins. Anthocyanins were determined by HPLC following the method of Vasco et al. (2009), with some modifications. The equipment and column were the same previously described for the analysis of organic acids. Mobile phases were 5% formic acid in water (A) and methanol (B). The flow rate was 0.8 mL min−1. The column was kept at 40 °C. The proportion of mobile phase B was progressively incremented through the following elution gradient: 5–10% in 5 min, 10% during 10 min, 10–42% in 20 min, and return to the initial conditions in 5 min. The extracts (20 μL) were injected into the chromatograph after filtration with 0.45 μm PVDF filters (Análisis Vínicos S.L., Spain). Detection was made within 250 and 550 nm. The different anthocyanin compounds detected were quantified using an external standard curve with
2.3.1. Physicochemical attributes 2.3.1.1. Fruit size and mass loss. The individual weight (g), the diameter (mm), and the length (mm) of 30 fruit were measured using an analytical balance (Radwag AS 310.R2, Poland) and a caliper, respectively. For mass loss, three trays were randomly selected and individually weighed at the beginning of the experiment, and on a daily basis during the storage period, until decay symptoms were visually observed. Results were expressed as percentage of mass loss relative to the initial mass. 2.3.1.2. Color and firmness. Color was determined with a HunterLab spectrophotometer (MINISCAN EZ 4500S, HunterLab, USA) calibrated 294
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were taken, totaling 30 measurements for each of these parameters. Data were subjected to a one-way analysis of variance (α = 0.05) using the IBM SPSS Statistics Version 21 software (IBM Corporation, USA). When significant differences were observed, mean treatments were compared using Tukey’s test.
cyanidin-3-rutinoside (Extrasynthese, France), which is the main anthocyanin compound in Rubus glaucus blackberries. 2.3.3.3. Total phenolic content (TPC). TPC was determined with the Folin–Ciocalteu colorimetric method modified for measuring in 96-well microplates (Bobo-García et al., 2015). Absorbance of the samples was measured at 750 nm, using a Multiskan GO spectrophotometer (Thermo Fisher Scientific, Denmark) and the results were expressed as mg gallic acid equivalent 100 g−1 on a fresh weight basis.
3. Results and discussion As no significant differences were observed between the two harvests (January and July 2016) for any of the parameters studied, the data were pooled. The evaluations were continued until symptoms of decay were detected on the fruit. So, storage period was of 3 and 9 d, at 18 and 8 °C, respectively.
2.3.3.4. Antioxidant activity. Antioxidant activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, following the method described by Bobo-García et al. (2015) for microplates. The absorbance of the samples was measured at 515 nm, using a Multiskan GO spectrophotometer (Thermo Fisher Scientific, Denmark) and the results were expressed as μmol Trolox equivalent 100 g−1 on a fresh weight basis.
3.1. Physicochemical attributes 3.1.1. Fruit size Fruit size increased with ripening. The fruit harvested at stage 5 reached an average weight of 6.25 ± 1.49 g, and 21.8 ± 0.22 and 26.6 ± 0.32 mm diameter and length, respectively, which corresponded to the category “big”, according to the Ecuadorian Standard. The fruit harvested at maturity stage 3 was classified as “medium”, with an average weight of 4.46 ± 1.22 g and a diameter and length of 18.9 ± 0.23 and 23.5 ± 0.28 mm, respectively. Consumers associate larger fruit with better quality and are usually willing to pay more for this kind of fruit. So, by harvesting when the fruit reaches stage 3, the fruit obtained could be unacceptable for the market.
2.3.4. Microbiological analyses Microbiological analyses were performed on days 0, 3, 6, and 9 of storage. Also, the containers were visually controlled daily in order to detect symptoms of microbial development. At each evaluation date, 3 samples/treatment were analyzed for total aerobic mesophiles, psychrotrophes, and yeasts and molds. For the analyses, 10 g of fruit were aseptically transferred to a filter stomacher bag and homogenized in 90 mL sterile buffered peptone water (Difco, USA) for 120 s at 200 rpm, using a Stomacher 400 circulator (Seward, UK). Serial decimal dilutions of each homogenized sample were made in peptone water. From each dilution, 1-mL aliquots were aseptically pour-plated for mesophiles and psychrothrophes and 0.1 mL was surface-plated for molds and yeasts analyses. The following media and culture conditions were used: (1) Plate count agar (PCA, Difco, USA) incubated at 35 ± 2 °C for 48 h and at 7 °C for 7 days, for total mesophilic (FDA, 1995) and psychrotrophic (ICMSF, 1982) microorganisms, respectively and (2) Sabouraud-glucose-agar plus chloramphenicol media (Acumedia, USA) incubated at 25 °C ± 2 for 5 days for yeasts and molds. All the samples were analyzed in duplicate, and microbial counts were expressed as log10 (cfu g−1) of tissue.
3.1.2. Mass loss Mass loss increased constantly during storage in the blackberries from both maturity stages, with maximum values of around 9% after 10 d of refrigerated storage and 4–5% after 3 d at RT. No significant differences were observed between the mass loss of the more mature and the more immature blackberries but, at every evaluation date, the mass loss was lower in the fruit stored at 8 °C in comparison with the fruit stored at RT. These results are similar to those reported by AyalaSánchez et al. (2013) and Kim et al. (2015), who attributed the greater mass loss at RT to higher respiration and transpiration rates of the fruit under these conditions. The maximum admissible mass loss for blackberries marketing has been reported as 5% (Salgado and Clark, 2016). This limit was exceeded after 3 d at RT and after 6 d under refrigerated storage. In effect, the use of low temperatures reduces metabolic processes and retards microbial growth which results in better fruit quality maintenance and longer shelf-life of the fruit (Joo et al., 2011).
2.3.5. Sensory analyses During storage, three samples per treatment were evaluated on days 0, 3, 6, and 9 by a sensory panel (5 males and 5 females) using a descriptive test. Before the experiments, the panelists were familiarized with the product and scoring methods, in training sessions in which appropriate scores for each parameter were agreed. The analyses were carried out in individual booths, and the samples from the different treatments (blackberries harvested at different maturity stages and stored at different temperatures) were presented in groups, coded with random numbers. Visual quality (color uniformity, injuries and general appearance) and overall impression were rated from 1 (worst) to 7 (best quality). Firmness was evaluated after softly pressuring the fruit between the thumb and the index fingers, from 1 (very soft) to 7 (very firm). For characteristic aroma, the scale ranged from 1 (none) to 7 (fully typical aroma) and for fruitś color evaluation a color chart (NTE 2427, INEN, 2010) that classifies the fruit into 7 classes, from 0 (light green) to 6 (dark purple/black) was used. Panelists were also asked to characterize off-odors, in case they detected any, and scores below 4 in any of the attributes indicated the rejection of the product. The shelf-life of the fruit was established as 1 d before the appearance of symptoms of microbial growth, sensorial rejection or a combination of both.
3.1.3. Color and firmness Regardless of storage temperature and maturity stage at harvest, the °hue of the blackberries remained stable, with slight but no significant decreases throughout the storage period. The hue of the fruit harvested at maturity stages 3 and 5 was of 23.75 ± 5.01 (light red) and 11.67 ± 6.20 (dark red), respectively. In this sense, it should be considered that a great variability in the superficial color of the blackberries was found, which in turn, may mask differences among the evaluation dates and storage temperatures. The luminosity (L*) was significantly higher in the more immature fruit (25.02 ± 2.89) in comparison with the blackberries harvested at a more advanced maturity stage (17.62 ± 2.17). This parameter remained unchanged in all the treatments, except the most mature fruit, which showed a significant decrease in the L* value after 9 days of cold storage, which indicates a loss of brightness in the fruit, associated with senescence. At each evaluation date, the fruit harvested at maturity stage 3 was firmer than the blackberries harvested at maturity stage 5. During the storage period, a significant loss of firmness was registered in all the samples and for both maturity stages (Fig. 1A and B), the softening was greater in the blackberries stored at RT in comparison with cold storage.
2.3.6. Statistical analyses The experiment was repeated twice (6 months apart between harvests) and the analyses were done in triplicate, considering each container as the experimental unit. For color and firmness, 10 subsamples 295
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Fig. 1. Firmness evolution of blackberries harvested at maturity stages 3 (A) and 5 (B) during 3 and 9 days of storage at 18 ± 2 °C and 8 ± 1 °C, respectively. Values represent the mean of 60 measurements for each storage temperature and evaluation date. Error bars represent the confidence interval (95%) for the mean.
to their antioxidant activity against free radicals (Mikulic-Petkovsek et al., 2015). Citric acid was the predominant acid found in the fruit, representing nearly 90% of total organic acids, followed by malic and ascorbic acids (Table 2). These results are similar to those reported by Mikulic-Petkovsek et al. (2015) in different berry species. On the contrary, Kafkas et al. (2006) found malic as the main acid, followed by ascorbic whilst citric acid was not detected in any of the cultivars studied. In this sense, the main organic acid and the vitamin C content in these fruit varies considerably among cultivars, ripeness, growing conditions and light intensity, and day/night temperatures (Van de Velde et al., 2016). The concentration of organic acids was higher than those previously reported by Kafkas et al. (2006) and Guedes et al. (2013) in different blackberry cultivars and are among the ranges of 5–30 mg 100 g−1 FW for ascorbic acid (Skrovankova et al., 2015) and 87.5–603 and 569–1892 mg 100 g−1 FW for malic and citric acids, respectively (Fan-Chiang, 1999; Gazioglu-Sensoy et al., 2015). What’s more, to the best of our knowledge, there are no previous reports on organic acids content in Andean blackberries. During storage, some differences between the behaviour of the individual acids studied were observed. Ascorbic acid content decreased, regardless of maturity stage at harvest and storage temperature. However, the losses were greater in the more mature fruit and in the blackberries stored at RT. After 9 d of cold storage, the citric acid presented and increase of 28.5% in the fruit harvested at stage maturity 3 and of 114.51% in the fruit harvested at stage maturity 5. In the blackberries stored at room temperature, there was also an increase of
Our results are similar to those obtained by Kim et al. (2015) and Oliveira et al. (2014). These authors reported a decrease in firmness with maturation process and attributed the softening to the physiological and biochemical changes that occur during ripening like starch to sugars conversion, biosynthesis of volatiles responsible for odor and taste, and changes in cell wall structure, due to the breakdown of cellular substances such as pectin, cellulose, hemicellulose, and other polysaccharides through hydration. 3.1.4. pH, total soluble solids and titratable acidity The pH, total soluble solids content, and titratable acidity of the blackberries harvested at two maturity stages and stored at room temperature and cold storage are shown in Table 1. The more mature fruit presented higher TSS, lower acidity and similar pH than the fruit from stage 3 and these differences could be attributed to acid to sugar conversion during ripening (Ayala-Sánchez et al., 2013). The three parameters remained unchanged during storage, either at RT or refrigerated storage and the values were similar to those reported by Reyes-Carmona et al. (2005) and Carvalho and Betancur (2015) for different blackberries cultivars harvested in different locations and at different maturity stages. 3.2. Organic acids Together with sugars, organic acids are responsible for blackberries’ flavor and they also have positive effects on human diet and health due
Table 1 pH, soluble solids content and titratable acidity evolution of blackberries harvested at maturity stages 3 and 5 and stored at room temperature (18 ± 2 °C) and in cold storage (8 ± 1 °C) during 3 and 9 d, respectively. pH T (ªC)
Day
MATURITY STAGE 3
8
18
0 3 6 9 0 3
Titratable aciditya
Total Soluble Solids (%)
2.29 2.62 2.63 2.68 2.29 2.66
5 ± ± ± ± ± ±
0.05A 0.38A 0.40A 0.41A 0.05A 0.43A
2.79 2.87 2.92 2.92 2.79 2.90
3 ± ± ± ± ± ±
0.34B 0.42A 0.45A 0.38A 0.34B 0.49A
9.63 9.17 9.33 9.22 9.63 9.70
± ± ± ± ± ±
0.69A 0,23A 0.61A 0.39A 0.69A 0.40A
5
3
11.00 ± 1.67B 10.60 ± 0.88B 11.50 ± 0.84B 12.78 ± 1.96B 11.00 ± 1.67B 9.93 ± 1.17B
3.80 3.72 3.69 3.64 3.80 3.63
5 ± ± ± ± ± ±
0.05A 0.34A 0.13A 0.20A 0.05A 0.23A
2.78 2.57 2.58 2.58 2.78 2.59
± ± ± ± ± ±
0.21B 0.22B 0.16B 0.16B 0.21B 0.19B
Values are the mean ± standard deviation (n = 6). For each storage temperature and evaluation date, different capital letters indicate significant differences between the maturity stages (p < 0.05). a g citric acid 100 g−1 on a fresh weight basis.
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Table 2 Ascorbic, malic and citric acids content in blackberries harvested at two maturity stages and stored under room temperature (18 ± 2 °C) and refrigerated (8 ± 1 °C) storage during 3 and 9 d, respectively. ORGANIC ACIDS (mg 100 g−1)a Ascorbic T (ªC)
Day
Malic
MATURITY STAGE 3
8
18
0 3 6 9 0 3
Citric
8.24 8.42 6.63 5.78 8.24 7.38
± ± ± ± ± ±
0.50Aa 1.83Aa 1.51Aab 1.46Ab 0.50Aa 1.53Aa
5
3
11.62 ± 3.65Ba 8.25 ± 1.04Aab 7.72 ± 1.38Ab 5.17 ± 1.79Ab 11.62 ± 3.65Ba 7.33 ± 2.78Ab
478.10 385.18 340.55 294.09 478.10 187.55
5 ± ± ± ± ± ±
35.69Aa 52.31Aab 86.44Ab 72.56Ab 35.69Aa 7.92Ab
295.73 145.43 180.43 263.44 295.73 207.18
3 ± ± ± ± ± ±
021.04Ba 030.54Ba 075.09Bab 084.44Ab 021.04Ba 108.17Ab
5
1004.26 1110.66 1278.02 1290.27 1004.26 1388.49
± ± ± ± ± ±
76.91Aa 273.95Aab 223.21Ab 75.56Ab 76.91Aa 18.72Ab
1739.52 1993.13 1040.60 1586.40 1739.52 1084.45
± ± ± ± ± ±
251.29Ba 156.67Aa 102.13Ba 422.17Ab 251.29Ba 170.46Bb
Values are the mean ± standard deviation (n = 6). For each storage temperature and evaluation date, different capital letters indicate significant differences between the maturity stages (p < 0.05). For each storage temperature and maturity stage, different lower case letters indicate significant differences among evaluation dates (p < 0.05). a Fresh weight basis.
xilorutinoside, respectively (Arozarena et al., 2012; Garzón et al., 2009). Fig. 2 shows how the relative content of the three compounds changed during maturation, particularly in the case of the peaks 1 and 2. While peak 1 increased from 17 at stage 3–26% at stage 5, peak 2 decreased from 18 to 10%. The relative amount of cyanidin-3-rutinoside also diminished, but in a lower extent, from 65 to 62%. Besides this, two additional peaks (4 and 5) were detected in the more mature blackberries. They accounted only the 2% of the total anthocyanin content. Both peaks had a maximum absorbance wavelength around 500 and 505 nm and a pronounced shoulder in the 400–450 nm region. These spectral characteristics have been noted to be typical of pelargonidin derivatives, which have been also detected in Andean blackberries (Garzón et al., 2009). Although the effect of maturation on the relative composition of anthocyanins was significant, its effect on the absolute content was more remarkable (Table 3). Blackberries harvested at stage 5 had a total anthocyanin content (TAC) around 5 times higher than the blackberries at stage 3. The concentration of anthocyanins at stage 5 was consistent with values observed in blackberries from different geographical locations (Probst, 2015). The production and accumulation of anthocyanins during ripening have been observed in different berry fruit
this acid, of 38.3% and 46.6% in the more immature and more mature blackberries, respectively. Finally, regardless of storage temperature, a significant decrease and increase in malic acid content was observed in the fruit harvested at stage maturity 3 and 5, respectively. Several studies were found reporting the organic acids content in different blackberries cultivars at harvest. However, no references were found regarding the behaviour of these acids during the storage period of this fruit. 3.3. Anthocyanins, total polyphenol content and antioxidant activity HPLC-DAD analysis revealed three major anthocyanin compounds in the blackberries (peaks 1, 2 and 3, Fig. 2). Most of the anthocyanins previously found in Andean blackberries were cyanidin glycosides (Vasco et al., 2009). Peak 3, identified as cyanidin-3-rutinoside was largely the most important anthocyanin, with a relative content of 62–65%. This is an outstanding characteristic of Rubus glaucus (Arozarena et al., 2012; Garzón et al., 2009; Mertz et al., 2007) that distinguishes it from other blackberry cultivars, in which cyanidin-3glucoside usually prevails. According to the relative retention times and the spectral characteristics of peaks 1 and 2, they could presumably correspond to the cyanidin-3-glucoside and the cyanidin-3-
Fig. 2. Chromatograms at 520 nm of anthocyanin extracts of Andean blackberries harvested at maturity stages 3 and 5. Retention times, maximum absorption wavelengths and relative amounts of the peaks detected.
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Table 3 Total anthocyanin and cyanidin-3-rutinoside content evolution of blackberries harvested at maturity stages 3 and 5 and stored at room temperature (18 ± 2 °C) and in cold storage (8 ± 1 °C) during 3 and 9 days, respectively. Cyanidin 3-rutinoside (mg 100 g−1)a T (°C)
Day
MATURITY STAGE 3
8
0 3 6 9 0 3
18
Total Anthocyanins (mg 100 g−1)a
20.3 31.0 45.5 41.9 20.3 36.9
± ± ± ± ± ±
4.4 2.1 5.0 8.8 4.4 9.5
Aa Ab Ac Ac Aa Ab
5
3
5
100.0 ± 18.5 Ba 110.7 ± 10.1 Bab 125.1 ± 17.1 Bbc 143.9 ± 9.0 Bc 100.0 ± 18.5 Ba 84.8 ± 44.8 Ba
30.8 46.2 67.1 64.5 30.8 61.2
± ± ± ± ± ±
6.3 Aa 2.8 Ab 6.1 Ac 13.0 Ac 6.3 Aa 19.1 Ab
175.7 174.9 195.7 228.0 175.7 137.1
± ± ± ± ± ±
36.9 14.8 27.4 14.7 36.9 75.4
Ba Ba Bab Bb Ba Ba
Values are the mean ± standard deviation (n = 6). For each temperature and evaluation date, different capital letters indicate significant differences between the blackberries at the two maturity stages (p < 0.05). For each maturity stage and storage temperature, different lower case letters indicate significant differences among days (p < 0.05). For each maturity stage and evaluation date no differences were detected between blackberries at different temperatures. a Fresh weight basis.
maturity stages, and observed that the strong increment of the anthocyanin content was accompanied by a decline on the levels in ellagitannins and flavonols, with the consequence that the total phenolic content did not vary in a specific way. So, the small effect of maturation on the TPC and AA levels in our Andean blackberries could be explained through the same mechanism. The accumulation of anthocyanins during ripening would have been counteracted by the fall in ellagitannins. No changes were observed either on the TPC or the AA levels during storage (Table 4), regardless the maturity stage of the fruit and the temperature used. Previous results are inconclusive. Whilst PerkinsVeazie and Kalt (2002) pointed that storage at 2 °C had no influence on AA of blackberries, Kim et al. (2015) observed a significant increment on the phenolic content of blackberries after 15 d at 1 °C, or after 13 d at 1 °C plus 2 d at 20 °C and Wu et al. (2010) showed different evolution patterns on the anthocyanin content, TPC and AA depending on the blackberry cultivar considered. These authors speculated that this could be related to the different phenolic composition, the polyphenol oxidase activity, and the ripeness of the fruit.
(Skrovankova et al., 2015). In the case of blackberries, increments of 2–4 (Siriwoharn et al., 2004) to 7-fold (Acosta-Montoya et al., 2010) of anthocyanin concentration have been reported. During the 9 d of cold storage, TAC significantly augmented, regardless the maturation stage of the fruit (Table 3). Previous results regarding the behavior of anthocyanin compounds of blackberries during refrigerated storage are not conclusive. Kim et al. (2015) observed that TAC increased after 15 d storage at 1 °C, or after 13 d at 1 °C plus 2 d at 20 °C. On the contrary, Joo et al. (2011) described a decline after 18 d at 3 °C, while Wu et al. (2010) did not see a clear tendency in the evolution of anthocyanins during 7 d at 2 °C. Table 4 shows the total phenolic content (TPC) and antioxidant activity (AA) of the blackberries. TPC values are comparable to values previously described for blackberries (Probst, 2015). The comparison of AA results more difficult due to the great variety of analytical methods and expression units founded in the literature. TPC and AA evolved in a similar way, and were significantly correlated (r = 0,891). The maturation process did not have a strong influence on them, although slightly lower values were observed in the more mature blackberries, in contrast to what happened with anthocyanins. However, it must be noted that no significant correlation was observed between TAC and AC. Although anthocyanins are antioxidants, AA could be more related to other phenolic compounds. It has been shown that ellagitannins and ellagic acid derivatives are largely the main phenolic compounds in Andean blackberries (Rubus glaucus Benth.), followed by anthocyanins and, in a minor extent by other phenolic families, such as flavonols, flavanols and phenolic acids (Mertz et al., 2007; Vasco et al., 2009). Acosta-Montoya et al. (2010) studied the phenolic composition of tropical highland blackberries (Rubus adenotrichus Schltdl.) during three
3.4. Microbiological analyses Yeasts and moulds were the main microbial group causing fruit decay. In effect, the high water and sugar content and the low pH of the fruit may limit the growth of many bacteria and at the same time, enhance fungal growth (Oliveira et al., 2013a, 2013b). The microbial counts observed on day 0 ranged between 4.86 and 5.08 (mesophiles), 0.00 and 1.35 (psychrotrophe), and 4.91 and 4.58 (yeasts and moulds) log (cfu g−1) for the blackberries harvested at
Table 4 Total polyphenol content and antioxidant activity of blackberries harvested at maturity stages 3 and 5 and stored at room temperature (18 ± 2 °C) and in cold storage (8 ± 1 °C) during 3 and 9 days, respectively. Total polyphenol content (mg galic acid 100 g−1)a T(°C)
Day
MATURITY STAGE 3
8
18
0 3 6 9 0 3
Antioxidant activity (μmol Trolox 100 g−1)a
560 517 618 736 560 613
5 ± ± ± ± ± ±
38 34 41 49 38 40
Ba Aa Aa Ba Ba Aa
446 502 555 558 446 585
3 ± ± ± ± ± ±
27 32 36 34 27 37
Aa Aa Aa Aa Aa Aa
6009 5756 6597 6956 6009 6586
5 ± ± ± ± ± ±
408 377 441 472 408 435
Ba Aa Aa Ba Ba Ba
5264 5170 5490 5846 5264 5594
± ± ± ± ± ±
318 328 354 355 318 355
Aa Aa Aa Aa Aa Aa
Values are the mean ± standard deviation (n = 6). For each temperature and evaluation date, different capital letters indicate significant differences between the blackberries at the two maturity stages (p < 0.05). For each maturity stage and storage temperature, different lower case letters indicate significant differences among days (p < 0.05). For each maturity stage and evaluation date no differences were detected between blackberries stored at different temperatures. a Fresh weight basis.
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Table 5 Microbial growth on Andean blackberries harvested at two maturity stages and stored at room temperature (18 ± 2 °C) and under refrigeration (8 ± 1 °C), during 3 and 9 days, respectively. Mesophiles Maturity stage
Day
Psychrotrophes
STORAGE TEMPERATURE (°C) 8
3
5
0 3 6 9 0 3 6 9
Moulds and yeasts
4.86 4.14 3.80 3.69 5.08 4.23 3.85 2.75
± ± ± ± ± ± ± ±
0.37 1.11 0.54 0.32 0.16 0.46 0.12 0.22
Aa Ab b b Aa Ab c d
18
8
4.86 ± 0.37 Aa 5.59 ± 0.42 Bb
0.00 0.00 1.94 3.25 1.35 1.45 1.87 2.06
5.08 ± 0.16 Aa 5.24 ± 0.23 Ba
± ± ± ± ± ± ± ±
0.00 0.00 1.44 0.42 1.46 1.53 1.40 1.52
Aa Aa b c Aa Aa a a
18
8
0.00 ± 0.00 Aa 3.62 ± 0.47 Bb
4.91 4.94 5.17 5.17 4.58 4.66 4.85 5.06
1.35 ± 1.46 Aa 2.60 ± 1.92 Aa
18 ± ± ± ± ± ± ± ±
0.15 0.43 0.45 0.70 0.59 2.18 0.13 0.13
Aa Aa a a Aa Aa a a
4.91 ± 0.15 Aa 6.27 ± 0.41 Bb
4.58 ± 0.59 Aa 6.96 ± 0.56 Bb
Values are the mean ± standard deviation (n = 6). For each maturity stage and evaluation date, different capital letters indicate significant differences between the storage temperatures (p < 0.05). For each maturity stage and storage temperature, different lower case letters indicate significant differences among evaluation dates (p < 0.05).
maturity stages 3 and 5, respectively (Table 5). Regardless of maturity stage at harvest, an increase in the microbial counts of all the groups studied was observed during storage at RT. On the contrary, under refrigerated storage, mesophiles’ counts decreased, yeasts and moulds remained unchanged and psychrotrophe increased only in the more immature fruit after 6 d of storage with no significant changes in the fruit harvested at maturity stage 5 (Table 5). What’s more, on day 3, all the microbial counts were higher in the fruit stored at room temperature in comparison with the fruit stored at 8 °C. These results are similar to those of Kim et al. (2015) and de Arruda Palharini et al. (2015), who reported that cooling was an efficient technique to reduce microbial growth. What’s more, handling the fruit carefully, together with rapid cooling after harvest is essential to reduce fruit damage and delay the growth of microorganisms like Botrytis, Cladosporium, Penicillium, Alternaria, and Fusarium (Perkins-Veazie et al., 1999).
3.5. Sensory analyses On day 0, the fruit harvested at stage maturity 5 presented higher scores than the more immature fruit in all the sensory parameters studied, with the exception of firmness (Fig. 3). However, the firmness of the fruit harvested at stage 3 was described as “too firm” by the panelists, who preferred softer fruit. During the storage period and regardless of harvest maturity and storage temperature, a progressive decrease in the scores of the visual quality, firmness and global impression was recorded (Figs. 4 A,B; and 5 A, B). On the contrary, color and aroma remained unchanged in the
Fig. 4. Sensory quality attributes of blackberries harvested at maturity stages 3 (A) and 5 (B) and stored during 9 days at 8 ± 1 °C.
more mature fruit whilst in the fruit harvested at stage maturity 3, an increase and decrease were obtained for color and aroma scores, respectively. However, even at day 9, the scores of these parameters were below 4, the limit of acceptance established for all the sensory attributes. Finally, the use of cold storage was effective in maintaining fruit quality, especially for the most mature fruit. In effect, on day 3, the blackberries stored under 8 °C presented higher scores for the visual quality, aroma, firmness and global impression than the blackberries kept at room temperature.
4. Conclusions The Ecuadorian Quality Standard establishes stage maturity 3 as the minimum maturity for blackberries harvest. However, at this stage and according to the sensory panelists, the fruit was still “too firm”, did not present the expected dark, bright purple color and did not develop its
Fig. 3. Initial sensory quality attributes of blackberries harvested at maturity stages 3 and 5.
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Fig. 5. Sensory quality attributes of blackberries harvested at maturity stages 3 (a) and 5 (b) and stored during 3 days at 18 ± 2 °C.
full typical aroma. What’s more, the titratable acidity doubled the maximum of 1.8% allowed in the same Standard, and the anthocyanin content was around five-fold lower than at maturity stage 5. Though, as fruit quality is influenced by agro-climatic conditions and may differ in fruit harvested in different locations, even if their external color is similar, it would be advisable to revise and adapt the Standard to the local conditions. Refrigerated storage was effective in delaying weight loss, softening and microbial growth and in maintaining better sensory quality, mainly of the fruit harvested more mature. At the same time, it did not affect either the total phenolic content or the antioxidant activity of the fruit. When cold storage was used it was possible to extend the shelf-life of the blackberries from 3 d at RT to up to 8 d. The main limiting factors for shelf-life were microbial growth and loss of firmness at RT and cold storage, respectively. Based on these results, it would be advisable to harvest the fruit at maturity stage 5 in order to achieve an appropriate fruit size, a high anthocyanin concentration, a better sugars/acids equilibrium, and a better sensory quality and the fruit should be maintained under refrigerated storage.
Acknowledgements Special thanks to the Secretaría de Educación Superior, Ciencia y Tecnología e Innovación (SENESCYT) of the Republic of Ecuador for the financial support of Dr. Horvitz as Prometeo Researcher.
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Skrovankova, S., Sumczynski, D., Mlcek, J., Jurikova, T., Sochor, J., 2015. Bioactive compounds and antioxidant activity in different types of berries. Int. J. Mol. Sci. 16, 24673–24706. Sora, A.D., Fischer, G., Flórez, R., 2006. Almacenamiento refrigerado de frutos de mora de Castilla (Rubus glaucus Benth.) en empaques con atmósfera modificada. Agron. Colombiana 24, 306–316. Van de Velde, F., Grace, M.H., Esposito, D., Pirovania, M.E., Lila, M.A., 2016. Quantitative comparison of phytochemical profile, antioxidant, and anti-inflammatory properties of blackberry fruits adapted to Argentina. J. Food Compos. Anal. 47, 82–91. Vasco, C., Riñen, K., Ruales, J., Kamal-Eldin, A., 2009. Phenolic compounds in Rosaceae fruits from Ecuador. J. Agric. Food Chem. 57, 1204–1212. Wu, R., Frei, B., Kennedy, J.A., Zhao, Y., 2010. Effects of refrigerated storage and processing technologies on the bioactive compounds and antioxidant capacities of ‘Marion’ and ‘Evergreen’ blackberries. LWT-Food Sci. Technol. 43, 1253–1264.
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Andean blackberries (Rubus glaucus Benth) quality as affected by harvest maturity and storage conditions
MARK
⁎
Sandra Horvitza, , Diana Chanaguanoa, Iñigo Arozarenab a b
Food Science and Engineering Faculty, Universidad Técnica de Ambato, Campus Huachi, Av. Los Chasquis y Rio Payamino, Ambato, Ecuador Food Technology Department, Universidad Pública de Navarra, Campus Arrosadía s/n 31006, Pamplona, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords: Antioxidant activity Blackberries Cold storage Polyphenols Postharvest quality
Maturity stage at harvest and storage conditions are critical factors determining fruit postharvest quality. Physicochemical (fruit size, mass loss, color, firmness, pH, total soluble solids, titratable acidity), microbiological (total aerobic mesophiles, psychrotrophes, and yeasts and molds) and sensory quality of Andean blackberries harvested at two maturity stages and stored under room (18 ± 2 °C) and cold storage (8 ± 1 °C) was studied. Total phenolic compounds, anthocyanins and organic acids content, and antioxidant activity were also evaluated. The more mature fruit was classified as “big”, according to the Ecuadorian Standard and showed lower acidity and higher total soluble solids, anthocyanins content and sensory scores compared with the fruit harvested earlier, whilst maturity at harvest did not affect the microbial counts of any of the groups studied. Cold storage was effective in delaying weight loss, softening and microbial growth and also in maintaining a better sensory quality of the blackberries. What’s more, under refrigeration it was possible to extend the shelf-life of the fruit to up to 8 d. The main limiting factors for shelf-life were microbial growth and loss of firmness at room storage and cold storage, respectively. Based on these results, it would be advisable to harvest the fruit at maturity stage 5 in order to achieve an appropriate fruit size, a high anthocyanin concentration, a better sugars/ acids equilibrium, and a better sensory quality and the fruit should be maintained under refrigerated storage.
1. Introduction Andean blackberries (Rubus glaucus Benth) are native to the Andes and grow year round, mainly in cold and temperate climates in South America (Martínez et al., 2007). The fruit are consumed fresh or processed as pulp, juices, jams or desserts and are appreciated for their color, flavor, bioactive compounds, and nutritional attributes, due to their high content in polyphenols, anthocyanins, vitamins, and minerals (Garzón et al., 2009; Junqueira-Gonçalves et al., 2016). Blackberries are non-climateric fruit and though, they must be harvested at full maturity, usually when they present a bright, dark purple/black color and optimum firmness (Bejarano, 1992). At this stage, also the best flavor quality is obtained (Skrovankova et al., 2015). Maturity at harvest is one of the main contributing and determining factors to small fruit quality during transport and commercialization, due to its direct relation with postharvest shelf-life (Perkins-Veazie and Collins, 2002). Immature fruit will not reach appropriate organoleptic characteristics whilst shelf-life of over-mature fruit is generally very short as the susceptibility to decay also increases (García, 2001). During ripening, berries soften and become darker in color, and also
⁎
chemical changes like accumulation of sugars, reduction in organic acids, and an increase in anthocyanins content occur (MikulicPetkovsek et al., 2015). Different maturity indexes (color, firmness, total soluble solids, and days after full bloom, among others) are commonly used in order to determine the optimum maturity stage for fruit and vegetables harvest. In the case of blackberries, the Ecuadorian Quality Standard NTE-2427 (INEN, 2010) establishes that harvest can be done once the fruit reaches, at minimum, the maturity stage 3, defined as light red. However, Ayala-Sánchez et al. (2013) concluded that the berries should be harvested at stage 5, as at that stage, the fruit presented an adequate size, the characteristic shape, and appropriate acids and soluble solids content. In Ecuador, there are no accurate methods for determining when to pick the berry fruit and this can result in inappropriate quality of marketable product (Mikulic-Petkovsek et al., 2015). What’s more, due to agro-climatic conditions, fruit quality may differ in fruit harvested in different locations, even if their external color is similar. In addition, most of the research done on this fruit is focused on yield and quality comparisons among cultivars, and on fruit quality and determination of chemical compounds and antioxidant activity under
Corresponding author. E-mail address: [email protected] (S. Horvitz).
http://dx.doi.org/10.1016/j.scienta.2017.09.002 Received 2 March 2017; Received in revised form 30 August 2017; Accepted 1 September 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
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to a standard white tile (X 79.80, Y 84.97, Z 90.74) and using the CIELab color space (CIE, 1976), the illuminant D65 and the 10° observer as a reference. The measurements were done on two opposite points of the equatorial zone and on the basis of each fruit and the hue angle was calculated. Flesh firmness was measured with a Texture Analyzer (Model CT3, Brookfield Engineering Labs, Inc., USA) and the TexturePro CT V1.2 Build Software. Each fruit was punctured once in its equatorial zone with a stainless steel, flat plunger (3 mm diameter) and the results were expressed as the maximum force (N) necessary to break the flesh in each fruit.
different storage conditions whilst less attention has been paid to maturity stage at harvest and its relation with quality and commercial requirements (Ayala-Sánchez et al., 2013). Another problem with blackberries is that they are highly perishable fruits, with a usual shelf-life that does not exceed 2–3 d at refrigerated temperatures, what implies great limitations for fresh market and storage. In effect, blackberries have a very thin and fragile skin and thus, are very susceptible to water loss, softening, mechanical injuries and postharvest diseases like gray mold, caused by Botrytis cinerea, and Rhizopus rot (Junqueira-Gonçalves et al., 2016). Hence, the use of techniques that can improve shelf-life duration without altering the physical, sensorial, and nutritional characteristics of the fruit is of vital importance to reduce the postharvest losses, which in some cases can be as high as 60% of the production (Sora et al., 2006). In this sense, one of the main factors affecting the storage shelflife and quality of fruit and vegetables is temperature, as it regulates the rate of all the metabolic processes that occur in these products. Low temperatures slow down fungal growth and at the same time, reduce respiration rate and water loss and therefore, delay ripening and senescence processes (Oliveira et al., 2013a, 2013b). As blackberries are insensitive to chilling injury, the recommended optimum temperature for this fruit is at 0–5 °C (Joo et al., 2011). The aim of this work was to characterize the physicochemical, microbiological and sensory quality, and the postharvest performance of Andean blackberries, harvested at two maturity stages and stored under room temperature and refrigerated storage. Bioactive compounds (total polyphenols, individual anthocyanins), organic acids and antioxidant activity were also studied.
2.3.1.3. pH, total soluble solids (TSS) and titratable acidity (TA). These parameters were determined on the juice obtained from each sample. The pH of the blackberries was measured with a pH-meter (ORION VersaStar, Thermo Scientific, Denmark). TSS were determined with a hand refractometer (Atago, Japan) and titratable acidity was determined by titration with 0.1 N NaOH to pH 8.1 (AOAC, 2000) and expressed as g citric acid 100 g−1 fruit (fresh weight). 2.3.2. Extraction and determination of organic acids A 2-g sample of lyophilized fruit was homogenized with 20 mL of distilled water and shaken for 30 min at 100 rpm in a bench-top orbital shaker (Infors HT, Switzerland). Afterwards, the solution was centrifuged (15 min, 2100g) at room temperature using a Medifriger BL-S centrifuge (J.P. Selecta, Spain). The extraction process was repeated twice, the supernatants were combined and the volume was made up to 50 mL with distilled water. Citric, malic, and ascorbic acids were determined by HPLC, following the method described by Scherer et al. (2012), with some modifications. For this purpose, a liquid chromatograph Waters 2695 coupled to a Waters 996 PDA detector and a LiChroCART Purospher RP-18 column (250 mm × 4 mm, 5 μm, Merck, Germany), with a Purospher precolumn (4 mm × 4 mm, 5 μm) were employed. The extracts (20 μL) were injected into the chromatograph after filtration with 0.45 μm PVDF filters (Análisis Vínicos S.L., Spain). An isocratic elution procedure was used with a 0.01 M KH2PO4 buffer solution, at a flow rate of 0.8 mL per minute, with the column kept at 25 °C. The peaks were identified by comparison of their retention times and UV-VIS spectra with those of standards. An external standard curve of each organic acid was used for quantification, at 250 nm for ascorbic acid, and at 210 nm for malic and citric acids.
2. Materials and methods 2.1. Plant material Andean blackberries (Rubus glaucus Benth) were hand-harvested in Tungurahua Province, Ecuador at maturity stages 3 (light red) and 5 (dark purple) in January and July 2016. Maturity stages were defined by the external color of the fruit, according to the Ecuadorian Standard (NTE-INEN 2427 (INEN, 2010)). Immediately after harvest the fruit were transported to the Technical University of Ambato for analyses. Fruit of uniform size and color, and free from damage and injuries were selected for the study. 2.2. Packing and storage
2.3.3. Extraction and determination of anthocyanins, total polyphenols and antioxidant activity 2.3.3.1. Extraction. 1-g of lyophilized fruit was homogenized with 20 mL of ethanol:distilled water:formic acid (50:48:2; v:v:v) and shaken for 30 min at 100 rpm in a bench-top orbital shaker (Infors HT, Switzerland). Afterwards, the solution was centrifuged (15 min, 2100g) at room temperature using a Medifriger BL-S centrifuge (J.P. Selecta, Spain). The extraction process was repeated twice, the supernatants were combined and the volume was made up to 50 mL with ethanol:distilled water (v:v).
200 ± 10 g of blackberries were packaged in transparent polyethylene terephthalate (PET) plastic containers and stored at 18 ± 2 °C (RT) and under refrigerated storage, at 8 ± 1 °C. 2.3. Quality attribute analyses Quality parameters were evaluated on the harvest day and every 3 d during the storage period, with the exception of fruit size that was determined only on day 0.
2.3.3.2. Anthocyanins. Anthocyanins were determined by HPLC following the method of Vasco et al. (2009), with some modifications. The equipment and column were the same previously described for the analysis of organic acids. Mobile phases were 5% formic acid in water (A) and methanol (B). The flow rate was 0.8 mL min−1. The column was kept at 40 °C. The proportion of mobile phase B was progressively incremented through the following elution gradient: 5–10% in 5 min, 10% during 10 min, 10–42% in 20 min, and return to the initial conditions in 5 min. The extracts (20 μL) were injected into the chromatograph after filtration with 0.45 μm PVDF filters (Análisis Vínicos S.L., Spain). Detection was made within 250 and 550 nm. The different anthocyanin compounds detected were quantified using an external standard curve with
2.3.1. Physicochemical attributes 2.3.1.1. Fruit size and mass loss. The individual weight (g), the diameter (mm), and the length (mm) of 30 fruit were measured using an analytical balance (Radwag AS 310.R2, Poland) and a caliper, respectively. For mass loss, three trays were randomly selected and individually weighed at the beginning of the experiment, and on a daily basis during the storage period, until decay symptoms were visually observed. Results were expressed as percentage of mass loss relative to the initial mass. 2.3.1.2. Color and firmness. Color was determined with a HunterLab spectrophotometer (MINISCAN EZ 4500S, HunterLab, USA) calibrated 294
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were taken, totaling 30 measurements for each of these parameters. Data were subjected to a one-way analysis of variance (α = 0.05) using the IBM SPSS Statistics Version 21 software (IBM Corporation, USA). When significant differences were observed, mean treatments were compared using Tukey’s test.
cyanidin-3-rutinoside (Extrasynthese, France), which is the main anthocyanin compound in Rubus glaucus blackberries. 2.3.3.3. Total phenolic content (TPC). TPC was determined with the Folin–Ciocalteu colorimetric method modified for measuring in 96-well microplates (Bobo-García et al., 2015). Absorbance of the samples was measured at 750 nm, using a Multiskan GO spectrophotometer (Thermo Fisher Scientific, Denmark) and the results were expressed as mg gallic acid equivalent 100 g−1 on a fresh weight basis.
3. Results and discussion As no significant differences were observed between the two harvests (January and July 2016) for any of the parameters studied, the data were pooled. The evaluations were continued until symptoms of decay were detected on the fruit. So, storage period was of 3 and 9 d, at 18 and 8 °C, respectively.
2.3.3.4. Antioxidant activity. Antioxidant activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, following the method described by Bobo-García et al. (2015) for microplates. The absorbance of the samples was measured at 515 nm, using a Multiskan GO spectrophotometer (Thermo Fisher Scientific, Denmark) and the results were expressed as μmol Trolox equivalent 100 g−1 on a fresh weight basis.
3.1. Physicochemical attributes 3.1.1. Fruit size Fruit size increased with ripening. The fruit harvested at stage 5 reached an average weight of 6.25 ± 1.49 g, and 21.8 ± 0.22 and 26.6 ± 0.32 mm diameter and length, respectively, which corresponded to the category “big”, according to the Ecuadorian Standard. The fruit harvested at maturity stage 3 was classified as “medium”, with an average weight of 4.46 ± 1.22 g and a diameter and length of 18.9 ± 0.23 and 23.5 ± 0.28 mm, respectively. Consumers associate larger fruit with better quality and are usually willing to pay more for this kind of fruit. So, by harvesting when the fruit reaches stage 3, the fruit obtained could be unacceptable for the market.
2.3.4. Microbiological analyses Microbiological analyses were performed on days 0, 3, 6, and 9 of storage. Also, the containers were visually controlled daily in order to detect symptoms of microbial development. At each evaluation date, 3 samples/treatment were analyzed for total aerobic mesophiles, psychrotrophes, and yeasts and molds. For the analyses, 10 g of fruit were aseptically transferred to a filter stomacher bag and homogenized in 90 mL sterile buffered peptone water (Difco, USA) for 120 s at 200 rpm, using a Stomacher 400 circulator (Seward, UK). Serial decimal dilutions of each homogenized sample were made in peptone water. From each dilution, 1-mL aliquots were aseptically pour-plated for mesophiles and psychrothrophes and 0.1 mL was surface-plated for molds and yeasts analyses. The following media and culture conditions were used: (1) Plate count agar (PCA, Difco, USA) incubated at 35 ± 2 °C for 48 h and at 7 °C for 7 days, for total mesophilic (FDA, 1995) and psychrotrophic (ICMSF, 1982) microorganisms, respectively and (2) Sabouraud-glucose-agar plus chloramphenicol media (Acumedia, USA) incubated at 25 °C ± 2 for 5 days for yeasts and molds. All the samples were analyzed in duplicate, and microbial counts were expressed as log10 (cfu g−1) of tissue.
3.1.2. Mass loss Mass loss increased constantly during storage in the blackberries from both maturity stages, with maximum values of around 9% after 10 d of refrigerated storage and 4–5% after 3 d at RT. No significant differences were observed between the mass loss of the more mature and the more immature blackberries but, at every evaluation date, the mass loss was lower in the fruit stored at 8 °C in comparison with the fruit stored at RT. These results are similar to those reported by AyalaSánchez et al. (2013) and Kim et al. (2015), who attributed the greater mass loss at RT to higher respiration and transpiration rates of the fruit under these conditions. The maximum admissible mass loss for blackberries marketing has been reported as 5% (Salgado and Clark, 2016). This limit was exceeded after 3 d at RT and after 6 d under refrigerated storage. In effect, the use of low temperatures reduces metabolic processes and retards microbial growth which results in better fruit quality maintenance and longer shelf-life of the fruit (Joo et al., 2011).
2.3.5. Sensory analyses During storage, three samples per treatment were evaluated on days 0, 3, 6, and 9 by a sensory panel (5 males and 5 females) using a descriptive test. Before the experiments, the panelists were familiarized with the product and scoring methods, in training sessions in which appropriate scores for each parameter were agreed. The analyses were carried out in individual booths, and the samples from the different treatments (blackberries harvested at different maturity stages and stored at different temperatures) were presented in groups, coded with random numbers. Visual quality (color uniformity, injuries and general appearance) and overall impression were rated from 1 (worst) to 7 (best quality). Firmness was evaluated after softly pressuring the fruit between the thumb and the index fingers, from 1 (very soft) to 7 (very firm). For characteristic aroma, the scale ranged from 1 (none) to 7 (fully typical aroma) and for fruitś color evaluation a color chart (NTE 2427, INEN, 2010) that classifies the fruit into 7 classes, from 0 (light green) to 6 (dark purple/black) was used. Panelists were also asked to characterize off-odors, in case they detected any, and scores below 4 in any of the attributes indicated the rejection of the product. The shelf-life of the fruit was established as 1 d before the appearance of symptoms of microbial growth, sensorial rejection or a combination of both.
3.1.3. Color and firmness Regardless of storage temperature and maturity stage at harvest, the °hue of the blackberries remained stable, with slight but no significant decreases throughout the storage period. The hue of the fruit harvested at maturity stages 3 and 5 was of 23.75 ± 5.01 (light red) and 11.67 ± 6.20 (dark red), respectively. In this sense, it should be considered that a great variability in the superficial color of the blackberries was found, which in turn, may mask differences among the evaluation dates and storage temperatures. The luminosity (L*) was significantly higher in the more immature fruit (25.02 ± 2.89) in comparison with the blackberries harvested at a more advanced maturity stage (17.62 ± 2.17). This parameter remained unchanged in all the treatments, except the most mature fruit, which showed a significant decrease in the L* value after 9 days of cold storage, which indicates a loss of brightness in the fruit, associated with senescence. At each evaluation date, the fruit harvested at maturity stage 3 was firmer than the blackberries harvested at maturity stage 5. During the storage period, a significant loss of firmness was registered in all the samples and for both maturity stages (Fig. 1A and B), the softening was greater in the blackberries stored at RT in comparison with cold storage.
2.3.6. Statistical analyses The experiment was repeated twice (6 months apart between harvests) and the analyses were done in triplicate, considering each container as the experimental unit. For color and firmness, 10 subsamples 295
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Fig. 1. Firmness evolution of blackberries harvested at maturity stages 3 (A) and 5 (B) during 3 and 9 days of storage at 18 ± 2 °C and 8 ± 1 °C, respectively. Values represent the mean of 60 measurements for each storage temperature and evaluation date. Error bars represent the confidence interval (95%) for the mean.
to their antioxidant activity against free radicals (Mikulic-Petkovsek et al., 2015). Citric acid was the predominant acid found in the fruit, representing nearly 90% of total organic acids, followed by malic and ascorbic acids (Table 2). These results are similar to those reported by Mikulic-Petkovsek et al. (2015) in different berry species. On the contrary, Kafkas et al. (2006) found malic as the main acid, followed by ascorbic whilst citric acid was not detected in any of the cultivars studied. In this sense, the main organic acid and the vitamin C content in these fruit varies considerably among cultivars, ripeness, growing conditions and light intensity, and day/night temperatures (Van de Velde et al., 2016). The concentration of organic acids was higher than those previously reported by Kafkas et al. (2006) and Guedes et al. (2013) in different blackberry cultivars and are among the ranges of 5–30 mg 100 g−1 FW for ascorbic acid (Skrovankova et al., 2015) and 87.5–603 and 569–1892 mg 100 g−1 FW for malic and citric acids, respectively (Fan-Chiang, 1999; Gazioglu-Sensoy et al., 2015). What’s more, to the best of our knowledge, there are no previous reports on organic acids content in Andean blackberries. During storage, some differences between the behaviour of the individual acids studied were observed. Ascorbic acid content decreased, regardless of maturity stage at harvest and storage temperature. However, the losses were greater in the more mature fruit and in the blackberries stored at RT. After 9 d of cold storage, the citric acid presented and increase of 28.5% in the fruit harvested at stage maturity 3 and of 114.51% in the fruit harvested at stage maturity 5. In the blackberries stored at room temperature, there was also an increase of
Our results are similar to those obtained by Kim et al. (2015) and Oliveira et al. (2014). These authors reported a decrease in firmness with maturation process and attributed the softening to the physiological and biochemical changes that occur during ripening like starch to sugars conversion, biosynthesis of volatiles responsible for odor and taste, and changes in cell wall structure, due to the breakdown of cellular substances such as pectin, cellulose, hemicellulose, and other polysaccharides through hydration. 3.1.4. pH, total soluble solids and titratable acidity The pH, total soluble solids content, and titratable acidity of the blackberries harvested at two maturity stages and stored at room temperature and cold storage are shown in Table 1. The more mature fruit presented higher TSS, lower acidity and similar pH than the fruit from stage 3 and these differences could be attributed to acid to sugar conversion during ripening (Ayala-Sánchez et al., 2013). The three parameters remained unchanged during storage, either at RT or refrigerated storage and the values were similar to those reported by Reyes-Carmona et al. (2005) and Carvalho and Betancur (2015) for different blackberries cultivars harvested in different locations and at different maturity stages. 3.2. Organic acids Together with sugars, organic acids are responsible for blackberries’ flavor and they also have positive effects on human diet and health due
Table 1 pH, soluble solids content and titratable acidity evolution of blackberries harvested at maturity stages 3 and 5 and stored at room temperature (18 ± 2 °C) and in cold storage (8 ± 1 °C) during 3 and 9 d, respectively. pH T (ªC)
Day
MATURITY STAGE 3
8
18
0 3 6 9 0 3
Titratable aciditya
Total Soluble Solids (%)
2.29 2.62 2.63 2.68 2.29 2.66
5 ± ± ± ± ± ±
0.05A 0.38A 0.40A 0.41A 0.05A 0.43A
2.79 2.87 2.92 2.92 2.79 2.90
3 ± ± ± ± ± ±
0.34B 0.42A 0.45A 0.38A 0.34B 0.49A
9.63 9.17 9.33 9.22 9.63 9.70
± ± ± ± ± ±
0.69A 0,23A 0.61A 0.39A 0.69A 0.40A
5
3
11.00 ± 1.67B 10.60 ± 0.88B 11.50 ± 0.84B 12.78 ± 1.96B 11.00 ± 1.67B 9.93 ± 1.17B
3.80 3.72 3.69 3.64 3.80 3.63
5 ± ± ± ± ± ±
0.05A 0.34A 0.13A 0.20A 0.05A 0.23A
2.78 2.57 2.58 2.58 2.78 2.59
± ± ± ± ± ±
0.21B 0.22B 0.16B 0.16B 0.21B 0.19B
Values are the mean ± standard deviation (n = 6). For each storage temperature and evaluation date, different capital letters indicate significant differences between the maturity stages (p < 0.05). a g citric acid 100 g−1 on a fresh weight basis.
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Table 2 Ascorbic, malic and citric acids content in blackberries harvested at two maturity stages and stored under room temperature (18 ± 2 °C) and refrigerated (8 ± 1 °C) storage during 3 and 9 d, respectively. ORGANIC ACIDS (mg 100 g−1)a Ascorbic T (ªC)
Day
Malic
MATURITY STAGE 3
8
18
0 3 6 9 0 3
Citric
8.24 8.42 6.63 5.78 8.24 7.38
± ± ± ± ± ±
0.50Aa 1.83Aa 1.51Aab 1.46Ab 0.50Aa 1.53Aa
5
3
11.62 ± 3.65Ba 8.25 ± 1.04Aab 7.72 ± 1.38Ab 5.17 ± 1.79Ab 11.62 ± 3.65Ba 7.33 ± 2.78Ab
478.10 385.18 340.55 294.09 478.10 187.55
5 ± ± ± ± ± ±
35.69Aa 52.31Aab 86.44Ab 72.56Ab 35.69Aa 7.92Ab
295.73 145.43 180.43 263.44 295.73 207.18
3 ± ± ± ± ± ±
021.04Ba 030.54Ba 075.09Bab 084.44Ab 021.04Ba 108.17Ab
5
1004.26 1110.66 1278.02 1290.27 1004.26 1388.49
± ± ± ± ± ±
76.91Aa 273.95Aab 223.21Ab 75.56Ab 76.91Aa 18.72Ab
1739.52 1993.13 1040.60 1586.40 1739.52 1084.45
± ± ± ± ± ±
251.29Ba 156.67Aa 102.13Ba 422.17Ab 251.29Ba 170.46Bb
Values are the mean ± standard deviation (n = 6). For each storage temperature and evaluation date, different capital letters indicate significant differences between the maturity stages (p < 0.05). For each storage temperature and maturity stage, different lower case letters indicate significant differences among evaluation dates (p < 0.05). a Fresh weight basis.
xilorutinoside, respectively (Arozarena et al., 2012; Garzón et al., 2009). Fig. 2 shows how the relative content of the three compounds changed during maturation, particularly in the case of the peaks 1 and 2. While peak 1 increased from 17 at stage 3–26% at stage 5, peak 2 decreased from 18 to 10%. The relative amount of cyanidin-3-rutinoside also diminished, but in a lower extent, from 65 to 62%. Besides this, two additional peaks (4 and 5) were detected in the more mature blackberries. They accounted only the 2% of the total anthocyanin content. Both peaks had a maximum absorbance wavelength around 500 and 505 nm and a pronounced shoulder in the 400–450 nm region. These spectral characteristics have been noted to be typical of pelargonidin derivatives, which have been also detected in Andean blackberries (Garzón et al., 2009). Although the effect of maturation on the relative composition of anthocyanins was significant, its effect on the absolute content was more remarkable (Table 3). Blackberries harvested at stage 5 had a total anthocyanin content (TAC) around 5 times higher than the blackberries at stage 3. The concentration of anthocyanins at stage 5 was consistent with values observed in blackberries from different geographical locations (Probst, 2015). The production and accumulation of anthocyanins during ripening have been observed in different berry fruit
this acid, of 38.3% and 46.6% in the more immature and more mature blackberries, respectively. Finally, regardless of storage temperature, a significant decrease and increase in malic acid content was observed in the fruit harvested at stage maturity 3 and 5, respectively. Several studies were found reporting the organic acids content in different blackberries cultivars at harvest. However, no references were found regarding the behaviour of these acids during the storage period of this fruit. 3.3. Anthocyanins, total polyphenol content and antioxidant activity HPLC-DAD analysis revealed three major anthocyanin compounds in the blackberries (peaks 1, 2 and 3, Fig. 2). Most of the anthocyanins previously found in Andean blackberries were cyanidin glycosides (Vasco et al., 2009). Peak 3, identified as cyanidin-3-rutinoside was largely the most important anthocyanin, with a relative content of 62–65%. This is an outstanding characteristic of Rubus glaucus (Arozarena et al., 2012; Garzón et al., 2009; Mertz et al., 2007) that distinguishes it from other blackberry cultivars, in which cyanidin-3glucoside usually prevails. According to the relative retention times and the spectral characteristics of peaks 1 and 2, they could presumably correspond to the cyanidin-3-glucoside and the cyanidin-3-
Fig. 2. Chromatograms at 520 nm of anthocyanin extracts of Andean blackberries harvested at maturity stages 3 and 5. Retention times, maximum absorption wavelengths and relative amounts of the peaks detected.
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Table 3 Total anthocyanin and cyanidin-3-rutinoside content evolution of blackberries harvested at maturity stages 3 and 5 and stored at room temperature (18 ± 2 °C) and in cold storage (8 ± 1 °C) during 3 and 9 days, respectively. Cyanidin 3-rutinoside (mg 100 g−1)a T (°C)
Day
MATURITY STAGE 3
8
0 3 6 9 0 3
18
Total Anthocyanins (mg 100 g−1)a
20.3 31.0 45.5 41.9 20.3 36.9
± ± ± ± ± ±
4.4 2.1 5.0 8.8 4.4 9.5
Aa Ab Ac Ac Aa Ab
5
3
5
100.0 ± 18.5 Ba 110.7 ± 10.1 Bab 125.1 ± 17.1 Bbc 143.9 ± 9.0 Bc 100.0 ± 18.5 Ba 84.8 ± 44.8 Ba
30.8 46.2 67.1 64.5 30.8 61.2
± ± ± ± ± ±
6.3 Aa 2.8 Ab 6.1 Ac 13.0 Ac 6.3 Aa 19.1 Ab
175.7 174.9 195.7 228.0 175.7 137.1
± ± ± ± ± ±
36.9 14.8 27.4 14.7 36.9 75.4
Ba Ba Bab Bb Ba Ba
Values are the mean ± standard deviation (n = 6). For each temperature and evaluation date, different capital letters indicate significant differences between the blackberries at the two maturity stages (p < 0.05). For each maturity stage and storage temperature, different lower case letters indicate significant differences among days (p < 0.05). For each maturity stage and evaluation date no differences were detected between blackberries at different temperatures. a Fresh weight basis.
maturity stages, and observed that the strong increment of the anthocyanin content was accompanied by a decline on the levels in ellagitannins and flavonols, with the consequence that the total phenolic content did not vary in a specific way. So, the small effect of maturation on the TPC and AA levels in our Andean blackberries could be explained through the same mechanism. The accumulation of anthocyanins during ripening would have been counteracted by the fall in ellagitannins. No changes were observed either on the TPC or the AA levels during storage (Table 4), regardless the maturity stage of the fruit and the temperature used. Previous results are inconclusive. Whilst PerkinsVeazie and Kalt (2002) pointed that storage at 2 °C had no influence on AA of blackberries, Kim et al. (2015) observed a significant increment on the phenolic content of blackberries after 15 d at 1 °C, or after 13 d at 1 °C plus 2 d at 20 °C and Wu et al. (2010) showed different evolution patterns on the anthocyanin content, TPC and AA depending on the blackberry cultivar considered. These authors speculated that this could be related to the different phenolic composition, the polyphenol oxidase activity, and the ripeness of the fruit.
(Skrovankova et al., 2015). In the case of blackberries, increments of 2–4 (Siriwoharn et al., 2004) to 7-fold (Acosta-Montoya et al., 2010) of anthocyanin concentration have been reported. During the 9 d of cold storage, TAC significantly augmented, regardless the maturation stage of the fruit (Table 3). Previous results regarding the behavior of anthocyanin compounds of blackberries during refrigerated storage are not conclusive. Kim et al. (2015) observed that TAC increased after 15 d storage at 1 °C, or after 13 d at 1 °C plus 2 d at 20 °C. On the contrary, Joo et al. (2011) described a decline after 18 d at 3 °C, while Wu et al. (2010) did not see a clear tendency in the evolution of anthocyanins during 7 d at 2 °C. Table 4 shows the total phenolic content (TPC) and antioxidant activity (AA) of the blackberries. TPC values are comparable to values previously described for blackberries (Probst, 2015). The comparison of AA results more difficult due to the great variety of analytical methods and expression units founded in the literature. TPC and AA evolved in a similar way, and were significantly correlated (r = 0,891). The maturation process did not have a strong influence on them, although slightly lower values were observed in the more mature blackberries, in contrast to what happened with anthocyanins. However, it must be noted that no significant correlation was observed between TAC and AC. Although anthocyanins are antioxidants, AA could be more related to other phenolic compounds. It has been shown that ellagitannins and ellagic acid derivatives are largely the main phenolic compounds in Andean blackberries (Rubus glaucus Benth.), followed by anthocyanins and, in a minor extent by other phenolic families, such as flavonols, flavanols and phenolic acids (Mertz et al., 2007; Vasco et al., 2009). Acosta-Montoya et al. (2010) studied the phenolic composition of tropical highland blackberries (Rubus adenotrichus Schltdl.) during three
3.4. Microbiological analyses Yeasts and moulds were the main microbial group causing fruit decay. In effect, the high water and sugar content and the low pH of the fruit may limit the growth of many bacteria and at the same time, enhance fungal growth (Oliveira et al., 2013a, 2013b). The microbial counts observed on day 0 ranged between 4.86 and 5.08 (mesophiles), 0.00 and 1.35 (psychrotrophe), and 4.91 and 4.58 (yeasts and moulds) log (cfu g−1) for the blackberries harvested at
Table 4 Total polyphenol content and antioxidant activity of blackberries harvested at maturity stages 3 and 5 and stored at room temperature (18 ± 2 °C) and in cold storage (8 ± 1 °C) during 3 and 9 days, respectively. Total polyphenol content (mg galic acid 100 g−1)a T(°C)
Day
MATURITY STAGE 3
8
18
0 3 6 9 0 3
Antioxidant activity (μmol Trolox 100 g−1)a
560 517 618 736 560 613
5 ± ± ± ± ± ±
38 34 41 49 38 40
Ba Aa Aa Ba Ba Aa
446 502 555 558 446 585
3 ± ± ± ± ± ±
27 32 36 34 27 37
Aa Aa Aa Aa Aa Aa
6009 5756 6597 6956 6009 6586
5 ± ± ± ± ± ±
408 377 441 472 408 435
Ba Aa Aa Ba Ba Ba
5264 5170 5490 5846 5264 5594
± ± ± ± ± ±
318 328 354 355 318 355
Aa Aa Aa Aa Aa Aa
Values are the mean ± standard deviation (n = 6). For each temperature and evaluation date, different capital letters indicate significant differences between the blackberries at the two maturity stages (p < 0.05). For each maturity stage and storage temperature, different lower case letters indicate significant differences among days (p < 0.05). For each maturity stage and evaluation date no differences were detected between blackberries stored at different temperatures. a Fresh weight basis.
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Table 5 Microbial growth on Andean blackberries harvested at two maturity stages and stored at room temperature (18 ± 2 °C) and under refrigeration (8 ± 1 °C), during 3 and 9 days, respectively. Mesophiles Maturity stage
Day
Psychrotrophes
STORAGE TEMPERATURE (°C) 8
3
5
0 3 6 9 0 3 6 9
Moulds and yeasts
4.86 4.14 3.80 3.69 5.08 4.23 3.85 2.75
± ± ± ± ± ± ± ±
0.37 1.11 0.54 0.32 0.16 0.46 0.12 0.22
Aa Ab b b Aa Ab c d
18
8
4.86 ± 0.37 Aa 5.59 ± 0.42 Bb
0.00 0.00 1.94 3.25 1.35 1.45 1.87 2.06
5.08 ± 0.16 Aa 5.24 ± 0.23 Ba
± ± ± ± ± ± ± ±
0.00 0.00 1.44 0.42 1.46 1.53 1.40 1.52
Aa Aa b c Aa Aa a a
18
8
0.00 ± 0.00 Aa 3.62 ± 0.47 Bb
4.91 4.94 5.17 5.17 4.58 4.66 4.85 5.06
1.35 ± 1.46 Aa 2.60 ± 1.92 Aa
18 ± ± ± ± ± ± ± ±
0.15 0.43 0.45 0.70 0.59 2.18 0.13 0.13
Aa Aa a a Aa Aa a a
4.91 ± 0.15 Aa 6.27 ± 0.41 Bb
4.58 ± 0.59 Aa 6.96 ± 0.56 Bb
Values are the mean ± standard deviation (n = 6). For each maturity stage and evaluation date, different capital letters indicate significant differences between the storage temperatures (p < 0.05). For each maturity stage and storage temperature, different lower case letters indicate significant differences among evaluation dates (p < 0.05).
maturity stages 3 and 5, respectively (Table 5). Regardless of maturity stage at harvest, an increase in the microbial counts of all the groups studied was observed during storage at RT. On the contrary, under refrigerated storage, mesophiles’ counts decreased, yeasts and moulds remained unchanged and psychrotrophe increased only in the more immature fruit after 6 d of storage with no significant changes in the fruit harvested at maturity stage 5 (Table 5). What’s more, on day 3, all the microbial counts were higher in the fruit stored at room temperature in comparison with the fruit stored at 8 °C. These results are similar to those of Kim et al. (2015) and de Arruda Palharini et al. (2015), who reported that cooling was an efficient technique to reduce microbial growth. What’s more, handling the fruit carefully, together with rapid cooling after harvest is essential to reduce fruit damage and delay the growth of microorganisms like Botrytis, Cladosporium, Penicillium, Alternaria, and Fusarium (Perkins-Veazie et al., 1999).
3.5. Sensory analyses On day 0, the fruit harvested at stage maturity 5 presented higher scores than the more immature fruit in all the sensory parameters studied, with the exception of firmness (Fig. 3). However, the firmness of the fruit harvested at stage 3 was described as “too firm” by the panelists, who preferred softer fruit. During the storage period and regardless of harvest maturity and storage temperature, a progressive decrease in the scores of the visual quality, firmness and global impression was recorded (Figs. 4 A,B; and 5 A, B). On the contrary, color and aroma remained unchanged in the
Fig. 4. Sensory quality attributes of blackberries harvested at maturity stages 3 (A) and 5 (B) and stored during 9 days at 8 ± 1 °C.
more mature fruit whilst in the fruit harvested at stage maturity 3, an increase and decrease were obtained for color and aroma scores, respectively. However, even at day 9, the scores of these parameters were below 4, the limit of acceptance established for all the sensory attributes. Finally, the use of cold storage was effective in maintaining fruit quality, especially for the most mature fruit. In effect, on day 3, the blackberries stored under 8 °C presented higher scores for the visual quality, aroma, firmness and global impression than the blackberries kept at room temperature.
4. Conclusions The Ecuadorian Quality Standard establishes stage maturity 3 as the minimum maturity for blackberries harvest. However, at this stage and according to the sensory panelists, the fruit was still “too firm”, did not present the expected dark, bright purple color and did not develop its
Fig. 3. Initial sensory quality attributes of blackberries harvested at maturity stages 3 and 5.
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Fig. 5. Sensory quality attributes of blackberries harvested at maturity stages 3 (a) and 5 (b) and stored during 3 days at 18 ± 2 °C.
full typical aroma. What’s more, the titratable acidity doubled the maximum of 1.8% allowed in the same Standard, and the anthocyanin content was around five-fold lower than at maturity stage 5. Though, as fruit quality is influenced by agro-climatic conditions and may differ in fruit harvested in different locations, even if their external color is similar, it would be advisable to revise and adapt the Standard to the local conditions. Refrigerated storage was effective in delaying weight loss, softening and microbial growth and in maintaining better sensory quality, mainly of the fruit harvested more mature. At the same time, it did not affect either the total phenolic content or the antioxidant activity of the fruit. When cold storage was used it was possible to extend the shelf-life of the blackberries from 3 d at RT to up to 8 d. The main limiting factors for shelf-life were microbial growth and loss of firmness at RT and cold storage, respectively. Based on these results, it would be advisable to harvest the fruit at maturity stage 5 in order to achieve an appropriate fruit size, a high anthocyanin concentration, a better sugars/acids equilibrium, and a better sensory quality and the fruit should be maintained under refrigerated storage.
Acknowledgements Special thanks to the Secretaría de Educación Superior, Ciencia y Tecnología e Innovación (SENESCYT) of the Republic of Ecuador for the financial support of Dr. Horvitz as Prometeo Researcher.
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