Fruit Quality and Composition of Two Advanced North Carolina Strawberry Selections

ABSTRACT

The advanced selections, NCS 10-038 and NCS 10-156, from the North Carolina breeding program were compared to the traditionally used cultivars, Camarosa and Chandler, for storage life and fruit composition in 2014 and 2015. Postharvest quality of NCS 10-038 was similar to that of ‘Camarosa’ and ‘Chandler’ after 8 days storage at 4 °C. NCS 10-156 was significantly worse in overall appearance and had more fruit shrivel, calyx browning, and mold than the other genotypes. However, NCS 10-156 was highest in soluble solids content and was similar in total anthocyanin content and total phenolic content to ‘Camarosa’ and ‘Chandler’. Further analysis of flavonoids by high performance liquid chromatography showed that NCS 10-156 was much lower in total flavonols than the other genotypes and comparable in anthocyanin pigments to ‘Chandler’. NCS 10-038, while similar in pigment profile to ‘Camarosa’, had less total anthocyanin than the other genotypes. NCS 10-038 had a lighter red color than the other genotypes and was similar in postharvest quality to ‘Chandler’ and ‘Camarosa’ and may be suitable for long-distance markets. NCS 10-156 is slightly softer than the other genotypes and more prone to mold while the higher soluble solids content may make it suitable for direct market sales. Both selections show postharvest promise for strawberry production in the humid mid-South region.

KEYWORDS:

• Postharvest
• phenolics
• anthocyanin
• pelargonidin 3-glucoside
• fruit quality ratings

Introduction

In North Carolina, a primary objective of the strawberry breeding program is to develop cultivars suitable for use with the annual plasticulture production system in climatic conditions encountered in the USDA hardiness zones 5 to 8 (western mountains to eastern shoreline). Strawberry production was valued at $20.3 million in 2012 (USDA, 2012) and the fruit are used primarily for direct market and local supermarket chains (www.NCstrawberry.com); therefore, the fruit are picked closer to full ripeness than those in Florida and California, which are designated for long-distance markets. In the United States east coast mid-South region, the California cultivars ‘Chandler’ and ‘Camarosa’ are used for much of the short-day acreage but tend to have one peak late in the season rather than yields distributed more evenly over the season. The industry desires strawberries with early and consistent high yields to maximize the short (4- to 6-week) harvest period, in addition to resistance to the leaf and root diseases common in this humid region. A number of advanced selections from the NC breeding program were screened from 2010 to 2014 and two promising selections were used for larger replicated trials in 2014 and 2015. In this study, postharvest storage life and fruit composition were done to provide further phenotypic data on these advanced selections.

Materials and methods

Chemicals and reagents

All chemicals were of analytical grade. Methanol (HPLC grade) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Sodium hydroxide; Folin-Ciocalteu; formic acid; gallic acid; and the standards, coumaric acid and quercetin 3-glucoside, were purchased from Sigma (St. Louis, MO, USA). Standards of cyanidin 3-glucoside, pelargonidin 3-glucoside, and ellagic acid were purchased from Chromadex (Irvine, CA, USA).

Plant material

Strawberries of ‘Chandler’, ‘Camarosa’, NCS 10-038, and NCS 10-156 were produced as plug plants and were established in plots in an annual plasticulture system at the Piedmont Research Station, Salisbury, NC in 2013 and 2014 between 15 August and 15 September. Leaf sample analysis was done weekly to monitor nutrient status and nutrients were added as needed using fertigation.

Fruit were harvested into one gallon buckets at weekly intervals between 17 April and 21 May 2014 and between 1 May and 25 May 2015. Fruit were immediately placed in a Pack N Cool trailer set at 5 °C to remove field heat. Fruit were sorted upon arrival at the lab to eliminate bruised, leaky, or small berries (<15 g) and about 230 g of intact fruit (average berry weight of 18 to 22 g) were placed into vented plastic clamshells (PRO CLM #H7357, Southern Containers, Wilson, NC, USA). Filled clamshells were weighed and placed into plastic bins with loosely fitted lids to keep relative humidity near 90% and held at 4 °C for 8 days. A total of two to three clamshells per selection/cultivar were stored per harvest date.

Ratings and quality evaluation

Following storage, clamshells were weighed and berries evaluated subjectively by three trained and experienced personnel, following rating criteria supported with written descriptions and photos. Berries were rated for overall appearance, defined as fruit shrivel, fruit darkening, calyx shrivel, calyx browning, presence of mold/decay, and firmness. A value of 5 was assigned for no mold, shrivel, browning, or darkening (i.e., close to freshly picked appearance) and a value of 1 represented the worst appearance.

For fruit composition, 20 strawberries free of injury and fully ripe were selected at each harvest for each cultivar/selection to represent day 0 of storage and divided into two sets of 10 berries. Six to 10 strawberries free of decay, bruising, or excessive shriveling were selected from each storage clamshell to determine post-storage changes. Calyxes were removed and berries cut in half lengthwise. One set of fruit halves was frozen at –20 °C for compositional analysis and the other set frozen at –80 °C for phenolic analysis by high performance liquid chromatography (HPLC).

Fruit composition assays of soluble solids content (SSC), pH, titratable acidity, and total anthocyanin and phenolic content were done using –20 °C fruit (about 100 g) thawed to room temperature and pureed 30 s with a homogenizer (Polytron PT10-35, Brinkmann Kinematica, Bohemia, NY, USA) equipped with a PT10-35 GT generator. Approximately 0.5 ml of puree was placed on a digital refractometer (Atago U.S.A., Bellevue, WA, USA) to determine the % SSC. A 2-g sample of puree was mixed with 60 ml of distilled water and titrated to an endpoint of 8.2 with 0.1 N NaOH using an automated titrimeter (Metrohm 862 Titrosampler, Riverview, FL, USA) and titratable acidity determined as percent citric acid. A 0.8-g puree subsample was extracted by homogenizing with 10 ml of extraction solvent (methanol, distilled deionized water, and formic acid [60:37:3 v:v:v]) and filtered through filter paper (Whatman no. 4). Filtrates were used to determine total monomeric anthocyanin and total phenolic content. Total monomeric anthocyanin was determined at 500 and 700 nm using the pH differential method (Lee et al., 2002) and a microplate reader (Biotek Powerwave35, Winooski, VT, USA). Methods for anthocyanin and phenolic content were adapted to the microplate system following the method of Heredia et al. (2006). Total phenolic content was determined according to the Folin-Ciocalteu method (Singleton et al. 1999). A standard curve of gallic acid (25 to 150 mg/kg) was used to calculate total phenolics as mg gallic acid/100 g fresh weight.

Measurement of anthocyanins and non-anthocyanin phenolics by HPLC

Strawberry samples designated for HPLC were freeze-dried using a VirTis LyoTroll (SP Scientific, Warminster, PA, USA). Freeze-dried strawberries were crushed and the powder filtered through mesh sieves. Strawberry fruit powders were extracted following the method of Bradish et al. (2015) with slight modification. Briefly, 40 mg of strawberry powder was extracted with 1.5 mL of acidified methanol (formic acid:methanol:deionized water, 1:60:39, v/v/v). Samples were vortexed and centrifuged at 10,600 g for 20 min at 4 °C. All samples were re-extracted and supernatants combined to achieve 98% of total phenolics present in samples.

Fruit extracts were filtered through 0.2 µm PTFE membranes (Fisher Scientific, Pittsburg, PA, USA). Filtered samples (20 µL) were injected into a Hitachi LaChrom (Hitachi Ltd., Tokyo, Japan), equipped with a UV-VS diode array detector (DAD), controlled temperature auto sampler (4 °C), and column compartment (30 °C). D-2000 software (Hitachi Ltd., Tokyo, Japan) was used as the system run controller and for data processing. Anthocyanin and phenolic separation were performed using a reversed phase C18 column (Synergi 4μ Hydro-RP 80Å, 6 × 250 um, Phenomenex, Torrance, CA, USA). The mobile phase consisted of 5% formic acid in water (A) and 100% methanol (B) with a flow rate of 1 ml/min using a step gradient of 0 min, 10% B; 5 min, 15% B; 15 min, 20% B; 20 min, 25% B; 25 min, 30% B; 45 min, 60% B; 47 min, 10% B; 60 min, 10% B. Compound concentrations were estimated using standard curves generated by injecting 5 µL of 0.0625–0.5 mg/mL preparations of cyanidin 3-glucoside, pelargonidin 3-glucoside, ellagic acid, coumaric acid, and quercetin 3-glucoside as external standards. Compound identification was performed based on retention time compared to authentic standards, and previously published reports (Grace et al., 2014). Each sample was run in triplicate and content reported as mg/g dry weight (DW). Sums of anthocyanins or phenolic compounds were calculated to obtain total anthocyanin and phenolic contents.

The storage and composition experiments were arranged as a completely randomized design. Analysis of variance was done using SAS, v. 9.2 (Cary, NC, USA) and mean separation by Tukey’s Honest Significance Difference t-Test (HSD).

Results and discussion

Fruit quality after 8 days of refrigerated storage was used as a way to assess storage life from time of harvest to the consumer. Under these conditions, the advanced selection NCS 10-038 was comparable to both ‘Camarosa’ and ‘Chandler’ in postharvest ratings of quality for overall appearance, fruit shrivel, calyx browning, and % moldy berries (Table 1). NCS 10-156 had more mold and was softer compared to the other selection and cultivars. Fruit darkness was notably less for NCS 10-038, while NCS 10-156 had the lowest fruit darkness rating (darkest appearing) compared to ‘Camarosa’ and ‘Chandler’. A subjective ranking below 3 indicates that fruit were no longer marketable. The 2.2 firmness ranking for NCS 10-156 indicates that these fruit were too soft for market after 8 days of storage. Weight loss of all genotypes was similar (1.9% to 2.2%).

Table 1. Subjective ratings of strawberry fruit held at 4 °C for 8 days averaged for 2014 and 2015 seasons.^z

Selection	Overall appearance^y	Fruit shrivel	Fruit darkness	Calyx brown	Calyx shrivel	Fruit firmness	Berries with mold (%)
Camarosa	3.8a	3.9ab	3.7bc	3.8a	3.6a	4.3a	3.1a
Chandler	4.0a	4.3a	3.8b	3.9a	3.8a	3.2b	4.6ab
NCS 10-038	4.0a	4.4a	4.2a	3.4a	3.6a	3.8b	6.1ab
NCS 10-156	3.3b	3.3b	3.2c	3.0b	3.0a	2.2c	12.3b

^zAll fruit quality attributes were given subjective ratings of 1 to 5 where a higher number indicates better fruit quality. Berries with mold was determined by 100% × (no. berries with mold/total no. berries).

^yMeans within column with same letter indicate no significant difference using Tukey’s HSD, p < 0.05.

The selection, NCS 10-156, was highest in SSC at both storage days 0 and 8 compared to the other genotypes (Table 2). Fruit pH ranged from 3.7 to 4.1 and titratable acidity (TA) from 0.4% to 0.7% across genotypes and storage days. The SSC/TA ratio was lowest for NCS 10-038 at both 0 and 8 days storage and reflects the lower SSC for this genotype. These values are slightly higher than those reported for California, Oregon, and Florida fruit (Diamanti et al., 2014; Finn et al., 2013) and most likely reflect the higher night temperatures in North Carolina during the strawberry season. Total anthocyanin content was lowest for NCS 10-038 fruit, reflecting the light color of this selection after storage. ‘Camarosa’, a noted deep red strawberry, was highest in total monomeric pigment at both 0 and 8 days storage. In spite of its dark red appearance after storage, NCS 10-156 was not as high in total anthocyanin content as ‘Camarosa’. Fruit shrivel may have made NCS 10-156 appear darker than would be expected from the anthocyanin content. The total phenolic content was lowest for NCS 10-038 compared to the other genotypes and remained lowest after storage.

Table 2. Fruit composition of fully ripe freshly harvested strawberry selections grown at Piedmont, NC in 2014 and 2015.^z

Selection	SSC (%)^y	pH	Titratable acidity (TA) (% as citric acid)	SSC/TA	Total anthocyanin (mg P3G/ 100 g fwt)^x	Total phenolic content (mg GA/ 100 g)
Day 0
Camarosa	7.1b	3.82a	0.69b	10.6ab	41.27a	155.62a
Chandler	6.8b	3.71b	0.69b	10.0ab	48.55a	157.57a
NCS 10-038	7.1b	3.67b	0.75a	9.8b	28.87b	142.05b
NCS 10-156	7.8a	3.78a	0.72ab	11.0a	33.95ab	152.23a
Day 8
Camarosa	7.6b	3.97a	0.64b	12.1a	51.52a	152.16bc
Chandler	7.6b	3.87b	0.66b	11.6a	45.39ab	158.50a
NCS 10-038	6.9c	3.81b	0.68b	10.2b	29.86c	147.59c
NCS 10-156	8.7a	3.87b	0.77a	11.6a	37.04b	153.21b

^zEach selection consists of a mean of three to seven samples, representing three harvest dates per year.

^yMeans within column with same letter indicate no significant difference using Tukey’s HSD, p < 0.05.

^xP3G and GA are pelargonidin 3-glucoside and gallic acid equivalents, respectively.

HPLC analysis of phenolics showed that NCS 10-156 was strikingly low in total phenolic acids, compared to the other genotypes (Table 3). P-coumaroyl-glucoside was determined from the literature (Urrutia et al., 2016) to be the dominant peak in this group, and was much less in NCS 10-156 than in the other genotypes. NCS 10-156 was also lower than other genotypes in flavonols, while relatively high in total anthocyanin. This is in contrast to Hernanz et al. (2007), who noted that strawberry cultivars low in flavonols were also low in anthocyanin. Yoshida and Tamura (2005) found that flavanols and P3M were much higher in strawberry achene tissue compared to epidermal tissue. Differences found among genotypes in our study in these compounds may be affected by type and amount of achenes in the fruit powders, as achenes were not excluded from samples prepared for HPLC analysis.

Table 3. Phenolic and anthocyanin composition of North Carolina strawberry selections at harvest determined by high performance liquid chromatography.

Selection	Total flavanols and tannins^z	Total phenolic acids	Total flavonols	Total antho-cyanins	C3G^y	P3G	P3R	P3M	%C3G	%P3G	%P3R	%P3M
	mg/100 g dry wt fruit tissue				%
Camarosa	624.86a	228.22a	62.02a	621.47a	44.51a	499.43a	82.65a	10.03b	7.4b	79.9a	13.4a	1.7b
Chandler	570.01b	123.42b	60.61a	617.24a	20.52b	522.77a	48.23b	17.30b	3.3c	83.6a	7.6c	3.8b
NCS 10-038	529.18c	254.16a	61.63a	441.36b	50.29a	305.47b	43.46b	62.72a	11.6a	69.4c	9.9b	14.1a
NCS 10-156	599.24ab	48.71c	50.65b	590.46a	15.69b	452.76a	46.24b	80.80a	2.8c	74.6b	7.8c	13.7a

^zMeans within columns with same letters indicate no significant difference, Tukey’s HSD, p < 0.05.

^yC3G (Cyanidin 3-glucoside); P3G (pelargonidin 3-glucoside); P3R (pelargonidin 3-rutinoside); P3M (pelargonidin 3-malonylglucoside). Percent of individual pigments was calculated as pigment/total anthocyanins.

Pelargonidin 3-glucoside was the major pigment in the cultivars/selections used in this study, representing 69% to 84% of the total anthocyanins identified (Table 3). Cyanidin 3-glucoside, pelargonidin 3-rutinoside, and pelargonidin 3-malonylglucoside were also present in much smaller amounts. Pelargonidin 3-glucoside imparts a red-orange color to strawberries (Fossen et al., 2004); as this pigment dominated the anthocyanin profile of the genotypes in this study, it may explain why NCS 10-38 appeared lighter but not different in red color compared to the other genotypes. Pelargonidin 3-rutionoside made up 8% to 13% of identified pigments in this study and was highest in ‘Camarosa’.

In conclusion, NCS 10-38 was similar to ‘Camarosa’ and ‘Chandler’ in postharvest storage, and was lighter in fruit color. NCS 10-156 was softer and sweeter than ‘Camarosa’ and ‘Chandler’. Both selections had acceptable postharvest performance after 8 days storage at 4 °C and NCS 10-038 may be acceptable for shipping to regional markets.

Acknowledgment

We wish to thank Joyce Edwards, Absalom Shank, Rocco Schiavone, and the staff at the Piedmont Research Farm for their assistance.

Funding

This work was supported by University of Arkansas National Strawberry Sustainability Initiative (UA28932-13).

Additional information

Funding

This work was supported by University of Arkansas National Strawberry Sustainability Initiative (UA28932-13).