The efficacy of different postharvest treatments on physico-chemical characteristics, bioactive components and microbiological quality of fresh blueberries during storage period

Email: valentina.chiabrando@unito.it eISSN: 2550-2166 / © 2017 The Authors. Published by Rynnye Lyan Resources Food Research 1 (6) : 240 248 (December 2017) Journal homepage: http://www.myfoodresearch.com F U L L P A P E R The efficacy of different postharvest treatments on physico-chemical characteristics, bioactive components and microbiological quality of fresh blueberries during storage period


Introduction
Blueberry is a popular and attractive fruit with high visual appeal and nutritional values. Blueberries are rich in bioactive compounds such as anthocyanins and other flavonoids (Bunea et al., 2013, Chiabrando andGiacalone, 2015) but are highly susceptible to water loss, mechanical damage, physiological disorders, rapid spoilage and fungal proliferation during post-harvest storage period (Cantín et al., 2012). The short postharvest life limits their fresh market potential and consumer access (Duan et al., 2011). Several preservation technologies, including cold storage (Connor et al., 2002), high oxygen atmospheres storage (Zheng et al., 2003), ozone (Chiabrando et al., 2006) modified atmosphere packaging (Cantín et al., 2012), hot water treatments (Fan et al., 2008) and edible coatings (Chiabrando and Giacalone, 2016) have been studied to maintain fruit firmness, reduce deterioration and prolong the shelf-life of fresh blueberries.
Chlorine and chlorine-containing compounds are the products most frequently used to sanitize in the fruit industry. However, chlorine reacts with organic compounds and forms carcinogenic trihalomethanes and haloacetic acids, which are dangerous to people and environment (Keskinen et al., 2009). Compared to chlorine, chlorine dioxide has a lower oxidative potential but higher oxidation capacity and sterilizing power, which is found to visibly extend the postharvest life of numerous fruits and vegetables , Fangxu et al., 2016. Unlike chlorine, chlorine dioxide does not oxidize organic matters to producing toxic molecules (Gómez-López et al., 2009), so the use of ClO 2 was approved by the Food and Drug Administration to sanitize fruit and vegetables. In current years, it has been studied that aqueous ClO 2 is well used to reduce microorganisms and preserve the postharvest quality of many fruits like strawberries, plums, melon and apples (Du et al., 2002;Shin et al., 2012;Aday et al., 2013;Guo et al., 2013;Aday and Caner, 2014).
Electrolyzed water (EW), both acidic and neutral types, is a recent alternative sanitizing and antimicrobial treatment utilized in agriculture and in food industry (Artés et al., 2009). EW is obtained by adding NaCl to tap water in a chamber with a separating polyester membrane and conducting an electric current across an anode and a cathode. The EW shows antimicrobial properties due to its low pH (2.3-2.7), its high oxidationreduction potential (>1000 mV) and because it also eISSN: 2550-2166 © 2017 The Authors. Published by Rynnye Lyan Resources contains active oxidizers like hypochlorous acid (Kim et al., 2000). The main advantage of EW is its safety, in fact, it is not corrosive to skin, mucous membranes and organic material. Thus, it is environmentally friendly and not potentially dangerous for human health (Huang et al., 2008). The efficacy of EW in reducing microorganism as well as the main foodborne pathogens associated with fresh-cut fruit and vegetables has been reported (Hung et al., 2010;Rahman et al., 2011;Gómez-López et al., 2013).
Some innovative research showed that extract of bamboo incorporated in edible coatings or dissolved in dipping solution improves the storage life of food, due to its antibacterial and antioxidative activity (Jun et al., 2004;Quitain et al., 2004). In particular, the active ingredient is 2,6-dimethoxy-1,4-benzoquinone showed a strong antibacterial effect (Tanaka et al., 2011;Afrin et al., 2012;Tanaka et al., 2013).
Considering the many post-harvest treatments applicable to blueberries, the aim of this work was to evaluate the effects of aqueous chlorine dioxide, electrolyzed water and bamboo extract (new commercial product) on physico-chemical characteristics, bioactive components and microbiological quality of fresh blueberries stored under standard commercial conditions.

Sample preparation
The blueberries (Vaccinium corymbosum, cv Duke) were harvested at commercial maturity in Peveragno, Cuneo Province, Italy, and transported to University (DISAFA; University of Turin) lab immediately. For the study, fully ripe fruits were selected for their uniform color, size, and absence of physical damage. The samples were subsequently treated, dried, packed and kept at 0°C for storage conservation. Three replicates from each treatment were taken on day 1, 5, 10, 15 and 15+3 for analysis and the average value is reported.

Treatments preparation
A commercially available brand of ClO 2 powder was dissolved in mineral water to prepare the solution according to the manufacturer's instructions (SuperTab, Air Aqua, The Netherland). Considering other experimental results (Pao et al., 2007;Colgecen and Aday, 2015), 20 mg L −1 was selected as the aqueous ClO 2 treatment concentration.
Electrolyzed water (MEDL, Tehcno3, Italy) was generated with a fow-type electrolysis apparatus (Hyperoxidation Equipment, Model MEDL Serie A, Gruper). This equipment is capable of producing up to 1.500 liters of neutral water electrolyzed in 10 working hours, with a concentration of 2.000 PPM oxidizers agents.
Berry Very® (Takex-labo, Osaka, Japan) is a commercial product based on Bamboo extract obtained from the crushed sheath of the stem of a gramineous Moso bamboo grown in China and Japan, using ethanol extraction.

Postharvest treatments
Four groups of blueberries were respectively treated as follow: (1) mineral water (electrical conductivity 76 µS/cm, pH 7.6) as control, (2) aqueous ClO 2 (20 mg L −1 ), (3) Berry Very, and (4) electrolyzed water. The berries were sprayed all over the surface for about 30 seconds with the aqueous solutions. Samples were then air-dried for 30 min and about 130-150 g of blueberries were put into plastic baskets, clamshell type (13.5 X 9.0 X 2.5 cm, perforated, polyethylene terephthalate) and stored at 0°C.

Determination of weight loss
Samples were numerated at the start of the experiment. Weight loss was measured calculating the difference between the initial and final weight of fruits containers. Results of weight loss were reported as a relative percentage and calculated as Weight loss (%) = ((Initial weight − Final weight)/Initial weight)×100.

Determination of skin color
The external color was measured on 30 berries per treatments using a Minolta colorimeter CR-400 (Chroma Meter, Minolta, Japan). Chromatic analyses were carried out following the CIELAB system. Values of L* (brightness), a* (redness+, greenness−) and b* (yellowness+, blueness−) were measured to describe a three-dimensional color space. The color of blueberries was also expressed as chroma or saturation index (SI = (a 2 + b 2 ) 1/2 ) and hue angle (hue angle = tan−1 (b*/a*).

Determination of firmness
Berries samples were randomly selected for analysis and the sample size was of 30 berries each testing, according to Doving et al. (2005). Each berry was considered a replicate. Before the analysis, samples were warmed to room temperature (20°C) for 3 h, because most fruits and vegetables showed decreasing firmness with increasing temperature (Bourne, 1980 Texture Analyzer TaxT2i® (Stable Micro System, Godalming, UK). The penetration test was performed in the equatorial part of the blueberry, at a cross-head speed of 3 mm/s and with a 3mm diameter punch (Chiabrando et al., 2009). A 30-kg load cell was used for firmness determination and the probe was programmed to penetrate 3 mm into the sample. The maximum penetration force (N), which is related to the firmness of the samples, was the parameter selected for further statistical analysis.

Determination of decay incidence
The percentage of decay incidence was evaluated after 1, 5, 10 and15 days of storage and calculated as Decay incidence (%) = (weight of decayed fruits/total weight)×100.

Determination of total soluble solid content, titratable acidity, and pH
Total Soluble Solid Content (TSSC, °Brix) of blueberry was determined in the juice using a handheld Atago digital refractometer (model PR-32, Atago, Italia, Milan, Italy). Titratable Acidity (TA) and pH were determined by titrating the juice with 0.1 M NaOH to pH 8.1 using an automatic titrator (Titration workstation Titralab AT1000 Series, Radiometer Analytical, Villerbaunne, France). Results were expressed as meq/l.

Determination of anthocyanins, total phenolics, and antioxidant capacity
Blueberries samples were extracted with methanol, recovered and stored at -26°C (Chiabrando and Giacalone, 2015). The spectrophotometric pH differential method (Giusti and Wrolstad, 2001) was used to determine total anthocyanins in the methanol extracts. Absorbance was measured using a UV-visible spectrophotometer (U-5100, Hitachi, Japan) at 510 nm and at 700 nm in buffers at pH 1.0 and 4.5, the results were calculated as: The results were expressed as milligrams of cyanidin -3-glucoside equivalent per 100 g of fresh weight and were an average of three replicate measurements per treatment.
The total phenolic content was determined according to the adapted Folin-Ciocalteu method (Waterhouse, 2002). The absorbance was measured using a UV-visible spectrophotometer (U-5100, Hitachi, Japan) at 750 nm. Aqueous solutions of gallic acid were used for calibration. The results are expressed as gallic acid equivalents (GAE)/100 g.
The antioxidant activity of berries was measured using the Ferric Reducing-Antioxidant Power (FRAP) assay. The procedure was based on the report of Kraujalyte et al. (2013). Results were expressed in mmol Fe 2+ * kg -1 fresh fruits.

Yeast and mold evaluation
Total yeasts and molds were examined according to the methods described by the Compendium of Methods for the Microbiological Examination of Foods (Vanderzant and Splittstoesser, 1992). 270 mL of peptone buffered water (Sigma Italiana SRL, Ozzano Emilia, Italy) was added to 30 g of fresh blueberries sample in a Stomacher® bag using a blender (Stomacher®400 Circulator, Seward, Worthing, UK). Appropriate dilutions were equipped. Rose Bengal agar (Sigma Italiana SRL, Ozzano Emilia, Italy) was utilized for the yeast and molds evaluations. All the plates were incubated at 30°C for 5 days. Microbial counts were expressed as log colony forming units g −1 (log CFU/g).

Statistical analysis
The results were expressed as a mean ± standard error of the mean and analyzed using STATISTICA (STATISTICA 8.0 software, 2007). One-way analysis of variance followed by Tukey's post hoc test was used to test for significant differences among means. The differences were considered statistically significant at p≤0.05.

Weight loss
The weight loss of fresh produce is an important parameter because it leads to economic losses. Fruit and vegetable products generally show symptoms of freshness loss with 3-10% weight loss (Ben Yehoshua, 1987). In the case of blueberries, fresh fruits become non -marketable when the weight loss is approximately 5% to 8% (Sanford et al., 1991) and a weight loss higher than 5% is the cause of the reduction in retail value of fruits (Ohta et al., 2002). Taking this into account, in this work all the blueberries samples would have still been marketable during the 15 days storage and showed even a lower weight loss if compared with other works . Weight loss of treated blueberries is shown in Figure 1. Electrolyzed water influenced positively the reduction of weight loss only in the first 10 days of storage, in fact, at the end of the storage period, the better treatment was chlorine dioxide, followed by Berry Very®, electrolyzed water and control, with losses of about 2.5%. Similar results have been reported with aqueous chlorine dioxide for blueberries and cherry tomatoes (Song et al., 2011).

Decay incidence
The postharvest shelf-life of blueberries is also limited by fungal and microbial decay because blueberries are extremely perishable and susceptible. Figure 2 shows the incidence of decay (%) of blueberries treated and stored at 0°C for 15 days. Decay was significantly reduced by all the post-harvest treatments, compared to control berries (Figure 2). In particular, Berry Very® and electrolyzed water treatment significantly (p ⩽ 0.05) delayed the incidence of decay and improved the storage life of the blueberries.

Total soluble solid content, titratable acidity, and pH
Soluble solids are relatively high in blueberries (Bushway et al., 1983). During storage, total soluble solids content increased significantly in blueberries due to the water losses (Tab.1). Highbush blueberries are characterized by high succinic and citric acid contents, averaging between 75% and 17% respectively (Ehlenfeldt et al., 1994). Results in titratable acidity levels of 'Duke' treated blueberries during storage are summarized in Table 1. Acidity decreased (p ⩽ 0.05) significantly in all blueberry samples during storage. The decreases were significant after 1 day for Berry Very® treated fruits and after 5 days for all the other treatments but the control that was stable until 10 days of storage. Among treatments, Berry Very® treated fruits showed significantly lower values from the first day and until the end of storage. At the end of the storage period control and electrolyzed water treated berries showed the higher acidity values, followed by chlorine dioxide treatment and Berry Very® treated samples. At day 0, fresh blueberries had a pH value of 3.69 ± 0.06 and the values were almost constant for 15 days of storage for all the treatments evaluated (data not shown).

Color
Skin color is an important factor that affects the visual appearance of fresh fruits and vegetable. Blueberries may lose commodity value if the treatment to reduce microorganisms development leads to a negative color variation (Perkins-Veazie et al., 2008). In this research, the L* values persisted almost unchanged for control blueberries and electrolyzed water treated samples and slightly decreased in chlorine dioxide berries. By contrast, Berries Very® treated fruits showed the highest variations in L* values, with the consequence that samples lost their bright color during storage, becoming darker or losing their waxy bloom.
The h* values of blueberries stored remained almost stable or slightly increased during storage and, after 15 days of storage, reached levels comparable to initial values. The hue of blueberries treated with Berry Very® and stored at 0°C for 15 days increased to approximately 321.06. In this case, the blueberries were more blackdark blue than blue, compared with the other treatments.
Just after the treatment (day 0), Berry Very® treated samples showed significant lower Chroma (2.33) values compared with the other treatments (Table 2). In this case, the color of the blueberries became less vivid after the treatment and during the storage.

Firmness
Blueberries firmness is an important quality attribute and an excessive softening is one of the main factors  reducing fruit quality and limiting its commercialization for fresh consumption (Angeletti et al., 2010). As shown in Table 3, firmness of blueberries declined gradually in all the samples. After 5 days of storage, Berry Very® samples showed significantly lower weight losses compared with other treatments. These results indicated that aqueous treatments could not delay the softening of blueberries during storage, probably because these treatments do not interact positively with the cell wall enzymatic degradation during postharvest storage.

Anthocyanins, total phenolics, and antioxidant capacity
Anthocyanins content increased significantly (p⩽0.05) during storage, but without significant differences among the treatments evaluated. The values at day 0, ranged from 87.75 mg/100 g (cyanidin 3-gluc) to 150 mg/100 g in Berry Very® and electrolyzed water samples, 145 mg/100 g in the control and 132 mg/100 g in chloride dioxide samples after 15 days of storage (Table 4).
The same results were obtained for total phenols content. Phenols increase significantly (p⩽0.05) during storage period from 205.91 mg/100 g (gallic acid) at day 0, to 245 mg/100 g (gallic acid) on average, after 15 days of storage. After 10 and 15 days of storage, control samples showed significantly (p⩽0.05) higher values compared with others treatments, on the contrary, electrolyzed water samples showed the lowest values. Antioxidant capacity values decreased after 1 day of storage and then remained almost stable during (Table  4).

Yeast and mold
Microbial counts of yeasts and molds throughout the storage of treated blueberries are shown in Table 5  Treatment with electrolyzed water showed a higher disinfectant effect on yeasts proliferation compared to the other treatments. On the contrary, chlorine dioxide treatment was active in the reduction of molds, in particular at the end of the storage period, according to Aday and Caner (2014) on strawberries and Zhang et al. (2015) on blueberries.

Conclusion
In this paper, we presented the effect of chlorine dioxide, electrolyzed water and Berry Very® on blueberry quality during the postharvest storage period. Among the individual treatments, at commercial level, Berry Very® and electrolyzed water showed the best results in term of lower weight loss, postharvest decay and microbial counts. On the contrary, if we considered the blueberry quality, Berry Very® affected negatively berries color, acidity level, and firmness. All the treatments did not influence the anthocyanin content, polyphenol, and antioxidant activity. In future, confirmation of this novel treatments efficacy in other fruit and berry fruit will be necessary to gain a better knowledge on their preserving fruit quality and safety for future commercial purposes.