We used Andean blackberry (Rubus glaucus Benth) harvested in the municipality of Granada (Antioquia) with degree of maturity 5 [19], selected with a uniform size, without mechanical damage and visual fungal contamination, and then they underwent a washing process and disinfection with NaClO solution at 50 ppm [10]. The ingredients of the EC were CS, WP, BW, G, SA and CH (Sigma Aldrich) and acetic acid as solvent of CH (Merck KGaA).

The development of EC formulation was carried out based on an experimental design of a face-centered response surface (α=1) with three factors: CS (3.00, 3.25 and 3.50% p/p), WP (0.5, 1.0 and 1.5% w/w) and BW (0.00, 0.25 and 0.50% w/w) (Table 1) and considering the dependent variables WL and firmness. Additionally, three controls based on CS (3.00, 3.25 and 3.50% w/w) plus G according to the G/CS ratio of 1/2 were made in order to consider not including WP, BW and SA components.

Table 1

Source: The Authors

For the preparation of each complete EC, colloidal dispersions of CS and WP were initially prepared in distilled water, homogenized at 13000 rpm for 3 min (Ultraturrax, digital IKA T25), then, G was added according to the G/(CS+WP) of 1/2 and homogenization was continued for 3 more minutes. This dispersion was heated up to 85 °C with constant stirring in an IKA C-MAG HS 4 heating plate, when it reached 70 °C the BW and SA were added, corresponding to a BW/SA ratio of 5/1. The emulsion prepared was cooled to 35 °C, homogenized at 21000 rpm for 1 min and finally degassed in a vacuum chamber at 7,4 kPa for 45 min before its application on Andean blackberry fruits.

On the other hand, CH was incorporated to the EC formulation selected in the experimental optimization process as antifungal agent at 3 levels (0.50, 0.75, 1.00% w/v), dissolving it as follows: 1 g of CH was diluted in 100 mL of a 1% v/v glacial acetic acid solution in distilled water and heated for 2 h at 40 °C [20, 21]. The formulations of EC with CH were prepared in a similar way to the formulations of experimental design. Superficial impregnation of EC in classified, washed and disinfected berries was done by immersion for 90 s, then it was left to drain for 90 s more and dried in a tunnel dryer (Centricol, Series 0803) at 30 °C, 1500 rpm 1 hour. Berries with EC were packed in perforated polypropylene boxes and stored in Memmert ICH 256 climate chamber at 65% relative humidity and 25 ºC; and in a conventional refrigerator at 4 ºC.

The WL was determined by gravimetric method [10,20] using an analytical balance (Ohaus PA 2014) and considering the difference of weights in time between Andean blackberries+EC of 15 formulations and their respective day 0. It was applied in a similar way for the three controls and for the samples evaluated with CH. WL was reported as a percentage (eq. 1), where W _i is day 0 weight and W _f is weight in the evaluation time.

The firmness of the fruit was made in the equatorial zone of fruits according to the methodology described by Ramírez et al, (2013) [11] modified, using a Stable Micro System TA.XT2i texture analyzer, accessory P/5, speed and penetration distance of 2 mm/s and 15 mm respectively.

Physicochemical properties of Andean blackberries such as acidity, °Bx, pH, moisture, a_w, total phenolic and antioxidant activity was calculated to OP-CH₂ and control treatments.

Acidity was determined by potentiometric titration with NaOH until reaching a pH of 8.2 by diluting 5 g in 50 mL of distilled water [10,21]. Results were expressed as % malic acid. The ºBrix were determined by refractometric reading at 20 ºC [19]. The pH was determined in a Hanna pH 211 pH-meter [10]. Moisture was determined by 930.15/90 method of AOAC, 1997 [7,22,23] modified: sample was weighed in a metal dish, dried in a vacuum oven (Memmert VO 200) at 60 °C and 1 kPa for 24 h. The a_w was determined with a dew point hygrometer at 25 °C (Aqualab 3TE series, Decagon, Devices, Pullman, WA, USA) 938.18 of AOAC, 2012 [24].

Total phenols (T_p) and antioxidant activity (DPPH and ABTS) were determined from the extract obtained as follows: 0.3 g of pulp and 9 mL of a methanol: water mixture (70:30) were weighed, stirred during 20 min and centrifuged at 8000 rpm in a Hettich Universal 320 R equipment for 10 min and 20 °C, then the supernatant was removed and filtered, shaking for 20 min and centrifuging again under the same conditions. The T_p were determined using the Folin-Ciocalteu reagent and according to the methodology of Patras et al., (2009) [25] modified: 20 μL of methanolic extracts and 1250 μL of 20% Na₂CO₃ were added in 480 μL of distilled water under vortexing was allowed to stand for 5 min; 250 μL of Folin-Ciocalteu reagent diluted in distilled water (ratio 1/1) was added, vortexed and stored in dark for 2 h. Absorbance was measured in a (Thermo Fisher Evo 60) spectrophotometer at a wavelength of 760 nm and T_p were expressed in mg of gallic acid per g of sample (mg GA/g). The ABTS and DPPH methods were determined according to the methodology described by Khandpur and Gogate, (2015) and Da-Silva et al., (2014) [26,27] modified. For ABTS method, 20 μL of methanolic extract and 2 mL of ABTS solution were taken, stirred for 1 min, left in dark for 7 min and read at 734 nm; for DPPH, 20 μL of extract and 1980 μL of DPPH solution were taken, left to stand for 30 min and measured at 517 nm. Results of ABTS and DPPH were expressed in mg of Trolox per 100 g.

Additionally, for samples with EC+CH an observation of the superficial fungal infection in fruit was made during its storage [28]. Fruits were considered infected when any signal of micellar growth was observed, evaluating in triplicate 15 fruits for each treatment. Data were expressed as percentage of infected fruit.

Data obtained for a response surface design were analyzed by analysis of variance with a confidence level of 95% in Statgraphics Centurion XVI statistical package. Properties of the product were determined in triplicate for each treatment.

The experimental optimization of the EC formulation applied in Andean blackberry was determined from the following criteria: maximize the surface firmness of the fruit and minimize WL during storage; additionally, weights and impacts were fixed as described in Table 3. Statgraphics Centurión XV II software application generated the following optimal formulation: CS (3.50%), WP (1.16%) and BW (0.47%). A residual error is observed between 15.0 and 20.0% of experimental samples and those obtained by the mathematical model, which is supported by the desirability found (0.69) in the optimization of multiple responses.

Table 3

Source: The Authors

Fig. 3 shows the evolution over time of WL and visual infection of blackberry with EC, without and with the addition of Q→0% (OP), 0.5% (OP-Q1); 0.75% (OP-Q2) and 1.0% (OP-Q3), and control Andean blackberry (blackberry in fresh state), stored at 25 and 4 ºC. For both storage conditions, WL and visual infection of Andean blackberries showed significant differences (p <0.05) with respect to time and treatment.

Figure 3 Source: The Authors

An increase of WL in Andean blackberries was observed with the increase of storage time, being higher the WL in Andean blackberries in fresh state (11.4-13.6% and 10.2-12.4% at 25 and 4 ºC respectively), this due to the physiology of the fruit itself that does not contain EC and in addition, it is enhanced with the increase of temperature; additionally, there is a significant effect of the presence of CH in EC, conferring a lower WL than blackberry with EC-OP (without CH) (7.4-8.4% and 5.9-6.5% at 25 and 4 °C respectively) and a decrease in superficial visual infection (7.5-19.1% and 20.0-40.0% at 25 and 4 °C respectively).

In general, a positive impact of CH is highlighted, especially in samples stored at 4 ºC where WL were less than 2% at times ( 8 days and at 10 days no visual infection was observed in samples OP-CH2 and OP-CH3 (yellow and blue lines respectively). However, in day 12 and 14 control times this is not maintained. This phenomenon is attributed to the fact that CH improves water vapor permeability properties of ECs, preventing their migration during storage of the fruit [35]. Similar behaviors of WL have been reported in strawberry with EC based on hydroxypropyl methylcellulose and carboxymethyl cellulose enriched with CH [36], and in strawberries with EC based on BW and CH [28]. On the other hand, the reduction of visual infection is attributed to the antimicrobial capacity of CH, which causes damage at cellular level in fungi [14,18,28,36].

In this context and due to cost effects, it was considered that the most adequate storage conditions were 4 ºC and 10 days, with EC formulated at CH2 concentration (0.75%). Table 4 presents physico-chemical characterization of Andean blackberries with EC+CH2 and Andean blackberries without EC (control).

Table 4

Treatment	Xw (%)	aw	°Brix (%)	pH	TA (%)	Tp (mg GA/100g)	Antioxidant activity (Trolox mg/100g)		Firmness (N)
							DPPH	ABTS
OP-CH2	85.80 a (0.37)	0.946 a (0.003)	7.43 a (0.06)	3.03 a (0.01)	2.35 a (0.02)	8.24 a (1.07)	889.42 a (36.3)	556.81 to (44.2)	7.12 a (0.48)
Control	85.16 a (0.60)	0.955 b (0.003)	7.63 a (0.15)	3.09 b (0.01)	2.26 b (0.02)	9.37 a (0.98)	921.44 a (25.4)	778.21 b (107.9)	2.04 b (0.15)

Source: The Authors

ANOVA showed significant differences (p <0.05) in the parameters aw, pH, TA, ABTS, and firmness. However, it is observed that changes in pH and TA are not very relevant despite the addition of acetic acid for solubilization of CH in EC. On the other hand, a similar situation occurs with aw, whose changes are very low but their values are very high (aw ( 0.950), corresponding to high moisture values, where microbiological stability of products during storage makes them perishable and also, favorable to other deterioration reactions [8]. In all cases, products are within the ranges established in Colombian Technical Standard 4106 [19].

Antioxidant activity by ABTS showed higher levels in fresh state, being higher by 28.4% with respect to Andean blackberry with EC+CH2, which could be attributed to thermal degradation of non-polar compounds with antioxidant activity present and they could be affected during drying applied at 30 ºC. Although Tp and DPPPH did not present significant differences, a similar situation arose as ABTS, where the reduction in Andean blackberry with EC corresponded to 12.1%, 3.5% respectively. This behavior of Tp, DPPH and ABTS is similar to that reported by some researchers in blueberry Duke and Elliot variety [35].

One of the greater importance parameters in the investigation was superficial firmness, which showed an important influence in Andean blackberries with EC+CH2, where the mechanical resistance increased approximately 250% with respect to Andean blackberry in fresh state and of 28% with respect to Andean blackberries with EC without CH (OP). This result reflects an important contribution of the research to post-harvest management of the fruit (harvest, packing, transport, sale and processing for transformation), and to the improvement in useful life that the product could have.