Evaluation of Pseudomonas graminis CPA-7 as a biopreservation method for fresh-cut pear: Physicochemical, enzymatic, and nutritional quality

Biological preservation methods with bacterial antagonists have emerged as alternatives to chemical sanitizers for extending shelf-life and reducing the population of pathogenic microorganisms. In addition, calcium plays an important role in maintaining the quality of fruit, and postharvest calcium treatments might determine the potential of fruit for processing. The objective of this work was to evaluate the effect of the postharvest application of calcium and biopreservation with the CPA-7 strain of Pseudomonas graminis on the quality parameters of fresh-cut pears. After harvest, whole pears were dipped in calcium chloride solution (1%, w/v) or water (control) for 10 min at 25 ℃ and stored for five months at temperatures ranging from 0 to −0.5 ℃. Both batches of fruit were minimally processed and dipped in a solution containing CPA-7 and an antioxidant solution or kept untreated, and both groups were stored at 4 ℃ for six days. The postharvest calcium treatment had no remarkable effect on the quality of the whole and fresh-cut pears. The enzymatic activities (PPO, PME and PG) related to browning and softening were constant in fresh-cut pears after storage, and the application of P. graminis CPA-7 had a positive effect on the activity of PPO. Finally, a combined effect of the biocontrol agent and calcium treatment was not demonstrated.


Highlights
• Postharvest calcium treatment had a minimal effect on the quality of whole pear • Postharvest calcium treatment and the addition of Pseudomonas graminis CPA-7 maintained the PPO activity of fresh-cut pears • The firmness and activity values of PME and PG remained stable in fresh-cut pears under storage at 5 ± 1 °C Introduction Pears (Pyrus communis L.) have low protein, lipid and glucose contents and are rich in other sugars such as fructose, sorbitol, and sucrose.Pears also contain micronutrients, such as vitamins, minerals, and antioxidants (Colás-Medà et al., 2015).The consumption of pears in Spain, which is currently 5.49 kg/year per person, decreased by 11.9 % in 2015 (MAGRAMA, 2016).In 2014, 373,900 tons of pears were produced in Spain, 50.4 % of which were produced in Lleida (MAPAMA, 2017).
In recent years, the society's lifestyle changes have caused an increase in the consumption of minimally processed fruits and vegetables.These products have desirable attributes such as freshness, low calorie contents, desirable nutritional composition and convenience (Colás-Medà et al., 2015).The main problem with fresh-cut fruits and vegetables is their short shelf-life due to the damage caused during minimal processing by operations such as peeling, coring, slicing and shredding.These lead to an increase in respiration, water loss, biochemical changes and microbial spoilage (Ngamchuachit et al., 2014;Plaza et al., 2016).
The acceptability of fresh-cut fruits and vegetables is determined by appearance and texture.
Therefore, it is essential to understand which processes produce changes in the appearance and texture of these products to develop novel strategies for maintaining their quality (Toivonen and Brummell, 2008).
Surface treatments are necessary to delay physiological decay in fruit tissues, thus stabilizing the surface of the fruit and preventing degradative processes that limit the quality of the product (Soliva-Fortuny and Martín-Belloso, 2003).Currently, the main chemical compounds used for these treatments are calcium salts with calcium chloride being the most common.The application of calcium has been studied in postharvest for whole fruit (Manganaris et al., 2007;Ghafir et al., 2013;Sajid et al., 2014;Kou et al., 2015;Zhao and Wang, 2015) and fresh-cut fruit (Silveira, Aguayo and Artés, 2010;Ngamchuachit et al., 2014), which is usually applied by dipping.The objective of the postharvest application of calcium to whole fruit has been to reduce disorders such as brown spots in pears (Kou et al., 2015) or bitter pits in apples (Sajid et al., 2014).However, in fresh-cut fruit, calcium salts are usually used in combination with ascorbic acid or organic acids such as citric acid as antibrowning agents because they can inactivate enzymes such as polyphenol oxidase (PPO, EC 1.10.3.2) and peroxidase (POD,11.1.1.x)and substrates from damaged cells (Soliva-Fortuny, Elez-Martínez and Martín-Belloso, 2004;Gomes et al., 2010).Moreover, those solutions have been effective in maintaining the firmness of the fruit during storage due to its indirect effect on the activity of polygalacturonase (PG, EC 3.2.1.67)and pectin methyl esterase (PME, EC 3.1.1.11)(Gomes et al., 2010;Ngamchuachit et al., 2014).In addition, the use of calcium salts increases the content of calcium in fresh-cut fruit (Manganaris et al., 2007).
Biological preservation has been presented as a promising alternative to chemical sanitizers to extend the shelf-life of fresh-cut fruit and to reduce the population of pathogenic microorganisms (Plaza et al., 2016).Recently, the CPA-7 strain of Pseudomonas graminis has been isolated from apple and has been demonstrated to be effective for the reduction of the population of Listeria monocytogenes and Salmonella in fresh-cut apples, peaches and melon (Alegre, Viñas, Usall, Anguera, et al., 2013;Alegre, Viñas, Usall, Teixidó, et al., 2013;Abadias et al., 2014) and in the maintenance of the nutritional quality and enzymatic activity in fresh-cut melon (Plaza et al., 2016).Current studies using yeasts or bacterial antagonists are focused on pathogen control, and few of them focus on nutritional quality and enzymatic activity.
There have been few reports on the effect of postharvest calcium application to whole fruit and the effects of biological preservation on the quality, nutritional content and antioxidant properties of minimally processed fruit during refrigerated storage.The general objective of the present study was to investigate the effectiveness of postharvest calcium treatment and Pseudomonas graminis CPA-7 against the main postharvest fruit and vegetable pathogens and maintenance of the quality of whole and fresh-cut pears.We have divided this objective into two parts.The first (Iglesias et al., 2018) addresses the effect of these treatments on foodborne pathogens and volatile compounds.In that paper was demonstrated that neither CPA-7 nor CaCl2 postharvest treatment were found to have an effect against Salmonella enterica under the same conditions.However, CPA-7 significantly reduced (approximately 1-log unit) the population of Listeria monocytogenes.
The second involves their effects on physicochemical, enzymatic and nutritional qualities of the pears.Thus, the objective of second part was to evaluate the effect of postharvest treatment with calcium and the antagonistic CPA-7 strain of P. graminis on the physicochemical parameters, enzymatic activities (PPO, POD, PME and PG), vitamin C content, total phenolic content, antioxidant activity and calcium content of minimally processed pears throughout their shelf-life.

Plant materials and postharvest treatment
'Conference' pears were obtained from a local farmer (Grealó, Lleida) at commercial maturity (62.6 N; 12.6 % soluble solids; 2.46 g malic acid L -1 ).Fruit free of visual defects and uniform in colour and size were selected and divided into two lots.The postharvest treatments consisted of dipping the fruit at 25 °C for 10 min in water (control) or 1 % CaCl2.Then, the fruit were air dried at room temperature before storage at temperatures ranging from 0 to -0.5 °C for 5 months under controlled atmospheric conditions (2 % O2 and 1 % CO2).

Processing, packaging and storage
As previously described by Iglesias et al. (2018), pears were cleaned, sanitized by immersion into a 0.1 g L -1 NaOCl solution adjusted to pH 6.5 using citric acid, rinsed, and dried prior to cutting.Pears were peeled and cut into 10 wedges using a handheld apple corer and slicer.Then, the cut pieces of fruit were dipped into an ice bath.The pears treated postharvest with calcium (1 % CaCl2), and those left untreated were divided into 2 batches each.Each batch was dipped (1:2 w/v) for 2 min at 150 rpm (horizontal shaker) in cold water and in an antioxidant solution (2 % ascorbic acid, 2 % sodium citrate and 1 % calcium chloride).Two of these batches were treated with P. graminis CPA-7 (10 7 CFU mL -1 ), and the results were compared to those of a negative control (without CPA-7 treatment).The treated fruit were air dried at room temperature.Approximately 110 g of cut pear samples was placed in recycled polyethylene (rPET) trays (120 x 120 x 55 mm, Plus Pack, Denmark) and heat-sealed with a film consisting of biaxially oriented polyester (OLAF) anti-fog inner layer and an oriented polypropylene (OPP) outer layer which contained lines of holes of 60-80 µm 75 mm apart from each other.Afterwards, the pear trays were stored at 5 ± 1 °C for 6 d.At days 0, 2 and 6, a portion of each sample was frozen with liquid nitrogen, pulverized and stored at -80 °C for enzymatic assays, vitamin C analysis, phenolic content analysis, antioxidant activity and calcium determination.

Physicochemical assays
Physical (colour and firmness) and chemical (titratable acidity and soluble solids content) assays were performed according to methods described by Abadias et al. (2014) with minor modifications as detailed by Plaza et al. (2016).Soluble solids contents were expressed as % and titratable acidity as g of malic acid L -1 .Three determinations were performed per treatment and sampling time.Firmness evaluation was carried out by determination of the maximal strength necessary for a cylindrical probe 4 mm in diameter and plane basis to penetrate 10 mm into a pear sample using a TA-TX2 Texture Analyser (Stable Micro Systems Ltd., Surrey, England).Briefly, colour was measured with a CR-200 Minolta Chroma Meter (Minolta, INC., Tokyo, Japan) with illuminant D65 and a 10° observer angle. .L* defines the lightness, and a* and b* define the red-greenness and blue-yellowness, respectively.These values were used to calculate the browning index (BI) described by Liu et al. (2016).

Assessment of overall visual quality
The assessment was carried out as described by Altisent et al. (2014).The visual quality was determined by an untrained panel based on the following hedonic scale: 9 = excellent; 7 = very good; 5 = good, limit of marketability; 3 = fair, limit of usability; and 1 = poor, inedible.

PPO and POD activity
The PPO and POD activities were measured as described by Plaza et al. (2016).A PPO unit was defined as 0.01 absorbance units per minute, and PPO activity was defined as the change in absorbance per second per kilogram on a fresh weight basis.A POD unit was defined as 0.01 absorbance units per minute, and POD activity was defined as the change in absorbance per second per kilogram on a fresh weight basis.

PME and PG activity
PME activity was determined according to the method of Plaza et al. (2003) with some modifications.Briefly, the enzyme was extracted by the homogenization of 5 g of frozen sample with 10 mL of an extraction solution (1 mol L -1 NaCl in 0.2 mol L -1 sodium phosphate buffer, pH 7.5).The resulting mixture was shaken for 10 min at 4 °C, centrifuged at 13523 × g for 20 min at 4 °C and then filtered.The supernatant was then collected and stored at -80 °C until the determination of enzyme activity.PME activity was measured titrimetrically at 25 °C.Briefly, 2 mL of the enzymatic extract were mixed with 40 mL of a pectin-salt substrate solution (0.35 % pectin and 0.1 mol L -1 NaCl).The solution was adjusted to pH 7.5 with 1 mol L -1 NaOH.After the pH reached 7.5, 0.1 mL of 0.05 mol L -1 sodium hydroxide was added.The time elapsed until the pH of the solution regained pH 7.5 was measured.A PME unit was defined on a fresh weight basis as the amount of enzyme required to release 1 mol of carboxyl groups per second.
PG activity was determined according to the method of Van linden et al. (2008) with some modifications.Six grams of frozen sample were homogenized with 9 mL of cold deionized water.The mixture was adjusted to pH 3.0 using HCl, stirred for 5 min and centrifuged at 13523 x g for 20 min at 4 °C.The pellet was recovered and resuspended in 6 mL of cold 1.2 mol L -1 NaCl, the solution was adjusted to pH 6.0 and stirred for 5 min and then centrifuged at 13523 x g for 20 min at 4 °C.Finally, the collected extract was stored at -80 °C until PG determination.Reaction mixtures consisted of 350 µL of a 0.2 % (w/v) buffered PGA solution containing 0.04 mol L -1 Na acetate at pH 4.4 and 50 µL of PG extract.The reaction mixtures were then incubated at 40 °C for 10 min.Two millilitres of cold borate buffer (pH 9.0, 0.1 mol L -1 ) were added to each sample to stop the reaction, and 0.4 mL of a 1 % 2-cyanoacetamide solution were added.The mixture was incubated for 10 min at 100 °C and then immediately cooled in an ice bath.The absorbance was determined using a Genesys 10S UV-Vis spectrophotometer (Thermo Scientific TM , Spain) at 276 nm and 22 °C.A standard calibration curve was generated at 276 nm using known concentrations of galacturonic acid (ranging from 0 to 3 x 10 -3 mol L -1 final volume).The enzyme activity was calculated on a fresh weight basis as the release of reducing groups per second.

Vitamin C analysis
The assay was carried out as described by Altisent et al. (2014).The reduction of dehydroascorbic acid to ascorbic acid using TCEP as the reducing agent was used to determine the total vitamin C content.Total vitamin C was determined using high-performance liquid chromatography (HPLC).The results were expressed on a fresh weight basis as g of ascorbic acid per kg.

Determination of the total phenolic content
The total phenolic content was determined by the Folin-Ciocalteu method (Singleton, Orthofer and Lamuela-Raventós, 1999) employing the modifications described by Altisent et al. (2014).
The results were expressed on a fresh weight basis as g of gallic acid equivalent per kg.

Determination of antioxidant activity
The antioxidant activity was determined by two different methods, namely, the 2,2diphenyl-1-picrylhydrazyl (DPPH • ) radical scavenging and ferric reducing antioxidant power (FRAP) assays.The extraction and assays were carried out according to the methods described by Plaza et al. (2016).The results were expressed on a fresh weight basis as moles of ascorbic acid equivalents per kg.

Determination of calcium
The determination of calcium was performed using a HORIBA Jobin Yvon ICP Spectrometer Model Activa inductively coupled plasma optical emission spectrometry (ICP-OES) equipped with a Meinhard nebulizer.The instrument was calibrated using a singleelement atomic absorption standard solution of calcium (1000 mg L -1 ).The sample pretreatment (digestion procedure) was carried out using an MLS 1200 mega microwave (Milestone, Milan, Italy) with HNO3 (700 mL L -1 ), H2O2 (300 mL L -1 ) and nanopure water.The optical emission was measured at 393 nm.The results were expressed on a fresh weight basis as g of calcium per kg.

Statistical analysis
The results were expressed as the means ± standard deviation.Differences among treatments were evaluated by using one-way analysis of variance (ANOVA) and were significant at P<0.05 (95 % confidence level).Tukey's test and t-tests methods were used to determine differences between means.JMP 8 software was used to conduct ANOVA statistical analyses (SAS Institute Inc., Cary, NC, USA).

Physical and chemical parameters and overall visual quality
The results of the physical and physicochemical parameters of whole and fresh-cut pears are shown in Table 1 and Table 2, respectively.The postharvest application of 1 % CaCl2 to whole pear did not affect the colour or soluble solids content (SSC) after a storage period of 5 months.However, the results showed that calcium treated whole pears presented higher acidity and lower firmness than non-treated samples probably due to long-storage time (5 months).
Moreover, Trentham et al. (2008) suggested that the concentration of calcium needed in the tissues to exert an effect on the firmness of the fruit should be 0.8-1.0g kg -1 .Sajid et al. (2014) observed that pears cv.Le-Conte treated with 3 % CaCl2 for 5, 10 or 15 min did not present SSC values that were significantly different from those of a control.However, Zhao and Wang (2015) reported that apples (Malus domestica Borkh.cv.Shouhong) dipped in 5 % CaCl2 for 10 min presented higher SSC and firmness values than the corresponding values of untreated apples.Similar results were obtained by Ghafir et al. (2013) who reported that 'Red Delicious' (Malus domestica) apples treated with 1 % CaCl2 presented higher firmness and SSC values and lower total acidity than untreated apples.
Overall, neither calcium chloride nor Pseudomonas graminis CPA-7 affected the firmness of fresh-cut pears (Table 2).Moreover, there were no significant differences at any point in the storage period for either treatment.However, fresh-cut pears subjected to only a postharvest calcium treatment presented higher BI (26.01 ± 15.09) compared to un-treated samples (17.78 ± 5.85) after a 6-day storage period at 5 ± 1 °C.In samples treated with P. graminis CPA-7, postharvest treatment with calcium had no effect in their BI after a 6-day storage period at 5 ± 1 °C.In addition, the BI of fresh-cut pears increased during storage at 5 ± 1 °C in the case of all treatments.Oms-Oliu et al. (2010) suggested that formulations composed of calcium and ascorbic were not completely effective in controlling the enzymatic browning of fresh-cut fruits, since once the ascorbic acid is completely oxidized to dehydroascorbic acid, the o-quinones were no longer reduced and browning might appear.
The results of visual quality analysis obtained for fresh-cut samples were indirectly related to their BI (Fig. 1).As the BI value increased, the visual quality score of the sample decreased.After 6 d of storage at 5 ± 1 °C, the highest visual value (5.2 ± 1.0) was obtained for fresh-cut pears that had not been treated with either calcium or CPA-7.This was the only sample that was still marketable after six days of storage, and that limited their shelf-life (6 d).

Polyphenol oxidase (PPO) and peroxidase (POD) assays
The postharvest application of 1 % CaCl2 had no effect on the PPO and POD activities in whole pear after 5 months of storage under CA (Table 1).Overall, in fresh-cut pears, the PPO activity decreased or remained constant during the 6-day storage period at 5 ± 1 °C regardless of the applied treatment (Table 2).In calcium-untreated fresh-cut pears, the presence of CPA-7 could reduce the PPO activity during the 6-d storage period.However, Collazo et al. (2018) reported that PPO activity of fresh-cut apples increased drastically in response to CPA-7 in MAP reaching almost 8-fold the activity showed by the non-inoculated control stored in the same conditions.Moreover, Plaza et al. (2016) showed that after 8 d of refrigerated storage at 5 °C, fresh-cut melons inoculated with CPA-7 and packaged under MAP did not present significant changes with regard initial values.The inactivation of PPO could be associated with the antioxidant solution used in this study, which contained 2 % ascorbic acid (Altunkaya and Gökmen, 2008) more than the action of the antagonistic microorganism.Indeed, Arias et al. (2007) demonstrated that ascorbic acid did not inactivate pear-derived PPO, but it did decrease the activity of PPO by reacting with the product of the PPO-catalysed reaction.
Overall, the POD activities of both treated and untreated samples increased during the 6 d of storage at 5 ± 1 °C (Table 2).The same trend was observed in other studies with different food matrices and conservation treatments.For example, Xu et al. (2015) studied the effect of high-pressure nitrogen treatments on the activity of POD in fresh-cut pear (Pyrus nivalis), and the results suggested that the activity increased during 14 d of storage at 4 °C.Plaza et al. (2016) reported that the POD activities in untreated fresh-cut melon (C.melo L.) and those treated with P. graminis CPA-7 increased after 8 d of storage at 5 °C.Moreover, Liu et al. (2016) observed that the POD activity increased during storage in fresh-cut apples treated with ozone.Rojas-Graü et al. (2008) did not observe any influence from the 1 % ascorbic acid antibrowning solution on the POD activity of fresh-cut 'Fuji' apple (Malus domestica Borkh.).
The POD activity was significantly lower after storage in samples treated with P. graminis CPA-7 regardless of the calcium treatment.In the recent study carried out by Collazo et al. (2018), POD activity was inhibited immediately after P. graminis CPA-7 inoculation in freshcut 'Golden delicious' apples.However, in that study after 6 d of storage time in MAP, POD activity increased in the P. graminis CPA-7 inoculated samples (2.6-folder higher) respect to control.
The increase in the BI of fresh-cut pears observed in the present study during storage at 4 °C could be related to the increase in the POD activity found during the same period.Browning in fresh-cut fruits and vegetables has been associated with the PPO activity; however, several studies suggested that POD could also play a key role (Toivonen and Brummell, 2008).The increase observed in BI could also be due to secondary browning, which is usually associated with the germination of fungal spores on the cut surface (Toivonen, 2006).

Pectin methyl esterase (PME) and Polygalacturonase (PG) assays
Postharvest treatment with CaCl 2 produced a significant decrease (P<0.05) in the PME activity of whole pear, but no effect was observed in the PG activity (Table 1).However, some authors suggested that PME activity should increase in the presence of calcium salts (Alandes et al., 2006(Alandes et al., , 2009;;Quiles et al., 2007).The results contradict those obtained by Manganaris et al. (2007) who reported that the PME activities of peach samples (Prunus persica, cv.'Andross') dipped in calcium chloride, lactate, or propionate were higher than those of the control.Ortiz et al. (2011) suggested that postharvest dips in 2 % CaCl2 for 5 min did not enhance the PME activity in 'Golden Reinders' apples (Malus x domestica Borkh., cv.Golden Reinders) perhaps due to less intense effects from ethylene production.Moreover, they suggested that the PME activity was not directly related to the softening of the fruit.Therefore, the reduction in the firmness values obtained for whole pear (Table 1) might be more related to the PG activity than the PME activity.However, the demethylation of pectin (polymer of galacturonic acid) by PME is necessary for the depolymerization by PG in pear softening (Toivonen and Brummell, 2008;Ortiz, Graell and Lara, 2011).
In fresh-cut pears (Table 2), the lowest PME activities (0.84 ± 0.14 and 1.14 ± 0.03 x 10 -5 mol s -1 kg -1 ) were observed in pears subjected to postharvest calcium treatments at sampling day 0.Although the PG activity was higher in fresh-cut pears treated with calcium and P. graminis CPA-7 (1.91 ± 0.42 x 10 -5 mol s -1 kg -1 ) than in untreated samples (0.93 ± 0.21 x 10 -5 mol s -1 kg -1 ), no significant differences were observed.The maintenance of the PME and PG activities in all fresh-cut samples throughout the storage period could be related to the preservation of their firmness values.The dipping treatment used in the fresh-cut experiment consisting of ascorbic acid, sodium citrate and calcium chloride might have had a strong effect on these results by rinsing enzymes and substrates away from injured cells at the cut surfaces (Soliva-Fortuny and Martín-Belloso, 2003;Toivonen and Brummell, 2008).

Vitamin C
The vitamin C content was not affected by postharvest calcium treatment in whole pear (Table 1).In fresh-cut pears (Table 3), the initial vitamin C contents were very high due to the dipping treatment with the antioxidant solution containing ascorbic acid.However, the vitamin C concentration dramatically decreased from day 0 to 2, and then it remained constant for the remainder of the storage period.Ascorbic acid is typically located in the vacuole of the cell and is protected from oxidation by low pH and phenolic flavonoids (Cocci et al., 2006;Hajilou and Fakhimrezaei, 2013).However, exogenous ascorbic acid is probably housed in a different location and is therefore more exposed to O2 (Cocci et al., 2006), enzyme ascorbate oxidase or oxidizing enzymes such as POD (Martíns et al., 2016).Fresh-cut pears treated with antagonist P. graminis CPA-7 presented the lowest vitamin C contents (0.12 ± 0.01 and 0.13 ± 0.01 g kg -1 ) after 6 d of storage at 5 ± 1 °C.However, Collazo et al. (2018) recently reported that vitamin C contents remained constant along storage time (6 d, 5 °C) and there were no differences between P. graminis CPA-7 inoculated and non-inoculated fresh-cut apples at any of the analysed sampling points (0, 1, 3 and 6 d).Moreover, in the study carried out by Plaza et al. (2016), the application of P. graminis CPA-7 to fresh-cut melon had no effect on the vitamin C content.Therefore, P. graminis CPA-7 might use the ascorbic acid in pears as a nutrient.

The total phenolic content and antioxidant activity
Postharvest calcium treatment had no effect on the total phenolic content of whole pears (Table 1).Similar results were obtained by Zhao and Wang (2015) who observed that the phenolic content of apples (Malus domestica Borkh.cv.Shouhong) treated with 5 % CaCl2 for 10 min and stored for 28 d were not different from those of the control.However, Kou et al. (2015) reported that 'Huangguan' pears (P.pyrifolia Nakai cv.Huangguan) dipped in 2 % CaCl2 for 15 min and stored for 8 months showed a higher contents of individual phenolic compounds than untreated pears.Overall, the total phenolic contents of fresh-cut pears decreased (P>0.05), and there were no significant differences between treatments after 6 d of storage at 5 ± 1 °C (Table 3).This might be due to progressive oxidation of the antioxidant solution (calcium chloride, ascorbic acid and sodium citrate).The cyclic structures of ascorbic acid and citric acid are very similar to the structure of phenolic compounds, and they could interfere in the spectrophotometric determination of the total phenolic content (Singleton, Orthofer and Lamuela-Raventós, 1999;Siddiq, Sogi and Dolan, 2013).
For whole pears treated with calcium, after 5 months storage, the antioxidant activity decreased in the DPPH • assay and increased in the FRAP assay (Table 1).The differences observed could be attributed to the different principles of each assay.Both assays are redoxlinked colorimetric methods, but the DPPH • assay is based on accepting hydrogen (H) atoms (Mishra, Ojha and Chaudhury, 2012), and the FRAP assay is indicative of taking electrons from antioxidants (Benzie and Strain, 1996).The antioxidant activity decreased in fresh-cut pears during 6 d of storage at 5 ± 1 °C (Table 3).Moreover, fresh-cut pears subjected to a postharvest calcium treatment presented the highest antioxidant activities for both assays (P<0.05).Freshcut pears treated with P. graminis CPA-7 presented the lowest antioxidant potentials when measured using the FRAP method (1.52 ± 0.01 x 10 -3 and 1.89 ± 0.02 x 10 -3 mol kg -1 ) for those fresh-cut pears with or without postharvest calcium treatment, which is consistent with the vitamin C contents.However, in a previous study carried out by Plaza et al. (2016), no significant differences (P>0.05) were observed between fresh-cut melons (C.melo L.) treated with P. graminis CPA-7 and the control after 8 d of storage at 5 °C.The results suggested that P. graminis CPA-7 could use antioxidant compounds such as ascorbic acid as nutrients.Further studies are needed to confirm this and to study the mode of action of this microorganism, including the competition for nutrients in different food matrices.

Calcium content
The calcium contents in whole pears are listed in Table 1.There were no significant differences (P>0.05) between untreated and treated whole pears.The results contradict those obtained by Kou et al. (2015) who reported that the application of 2 % CaCl2 to 'Huangguan' pears (P.pyrifolia Nakai cv.Huangguan) increased the concentration of calcium.Moreover, Manganaris et al. (2007) observed that the application of calcium salts increased the content of calcium in the peel and flesh of peach (Prunus persica, cv.'Andross').Chardonnet et al. (2003) suggested that 2 % calcium chloride dip was enough to achieve maximum calcium accumulation in the cell wall of apples.'Conference' pears might have reached saturated levels of calcium in their cell walls during formation and ripening and the addition of external calcium had no effect in the total content of calcium.
The calcium content in fresh-cut pears was higher than in whole pears (≈ 2 g kg -1 ) due to the applied antioxidant treatment that contained 1 % CaCl2.The content of calcium was maintained in fresh-cut pears after 6 d of storage at 5 ± 1 °C, except for fresh-cut pears not subjected to postharvest calcium treatment and treated with P. graminis CPA-7 after cutting (Table 3).To the best of our knowledge, in the studies carried out with P. graminis and specifically with P. graminis CPA-7 (Alegre, Viñas, Usall, Anguera, et al., 2013;Alegre, Viñas, Usall, Teixidó, et al., 2013;Abadias et al., 2014;Oliveira et al., 2015;Plaza et al., 2016), there is no mention of the use of calcium as a nutrient.The lowest content of calcium was observed in fresh-cut pears subjected to a postharvest calcium treatment (2.05 ± 0.00 and 2.02 ± 0.01 g kg -1 ) after 6 d of storage at 5 ± 1 °C.The postharvest calcium treatment had no influence on the calcium content of fresh-cut pears.The flesh and peel of pears, where calcium is normally most concentrated, were eliminated during processing, and thus the postharvest calcium treatment might have had no effect on the final calcium content in fresh-cut pears.However, a significant effect on the final calcium content was caused by the antioxidant solution, which contained calcium chloride.

Conclusions
The effects of postharvest calcium treatment and bioconservation using Pseudomonas graminis CPA-7 on the enzymatic activities and nutritional qualities of fresh-cut pears were evaluated.Postharvest calcium application had a minimal effect on the quality of whole 'Conference' pears.Fresh-cut pears could be the most suitable option for increasing the consumption of pears in Spain and throughout Europe.Consumers usually demand fresh-cut fruits or vegetables with no symptoms of browning or softening.In general, firmness values and PME, PG and PPO activity values were maintained in fresh-cut pears after 6 d of storage at 5 ± 1 °C.It was shown that the application of P. graminis CPA-7 had a positive effect on maintaining the PPO activity, which is usually associated with the browning index and limited the shelf-life of fresh-cut products.In this study, a clear synergy between calcium chloride and P. graminis CPA-7 in fresh-cut pears was not observed.Future studies should include the application of P. graminis CPA-7 in extreme conditions (for example without a disinfection step) and the use of ascorbic acid and phenolic compounds as nutrients by bacteria.Moreover, descriptive tests involving a trained panel and emerging sensory methods with consumers are necessary.

Fig. 1
Fig. 1 Effect of postharvest calcium chloride treatment and biopreservation using Pseudomonas

Table 2
Physical parameters and enzymatic activities of fresh-cut pears during storage at 5 ± 1 °C.Values are the means of independent determinations ± 578 standard deviation.Different capital letters in the same treatment indicate significant differences between days (P<0.05).Different lower-case letters in the 579 same storage day indicate significant differences (P<0.05) between treatments.a OD, optical density

Table 3
Vitamin C, phenol, and calcium contents and antioxidant activity of fresh-cut pears during storage at 5 ± 1 °C.Values are the means of independent 582 determinations ± standard deviation.Different capital letters in the same treatment indicate significant differences between days (P<0.05).Different lower-583 case letters in the same storage day indicate significant differences (P<0.05) between treatments 584