Phenolic Composition and Antioxidant Capacity of Newly Developed Strawberry Lines from British Columbia and Quebec

Abstract

The total phenolic composition (TPC) and the total antioxidant capacity (TAC) of eleven strawberry genotypes selected from the AAFC breeding program (BC2-72-17, BC2-90-43, BC96-33-4 and BC98-49-34 from British Columbia; and APF937-1, APF939-71, LL981-24, LL9819-14, LL982-14, LL0220-10 and SJ9332-7 from Quebec) were evaluated using high-performance liquid chromatography (HPLC) and ferric reducing/antioxidant power (FRAP) assays. TPC and TAC values of these genotypes were compared to those of four commercially grown cultivars (‘Chambly,’ ‘Kent,’ ‘Veestar’ and ‘Yamaska’) and the effects on fruit quality and shelf life were studied. Several groups of phenolic compounds, including anthocyanins, flavonols, hydroxycinnamic, ellagic and benzoic acids, were identified and quantified by HPLC. Significant variation of TPC was observed between genotypes, and anthocyanins were found to be the predominant phenolic group, contributing to 79.2% of the TPC. A significant correlation (r = 0.96) was observed between TPC and anthocyanin content. BC2-72-17 had the highest anthocyanin content and differed significantly from the other genotypes. Although the highest TPC (2123.8 μg g⁻¹) was found in BC2-72-17, this genotype did not have the highest antioxidant capacity. The highest TAC (2259.0 μg g⁻¹) was found in BC2-90-43, indicating that other phenolics may have made a greater contribution than anthocyanins to the high TAC. At room temperature, LL0220-10, SJ9332-7 and ‘Yamaska’ had a three-day shelf life, while it was only two days for all other genotypes. There was a negative correlation between shelf life and ellagic acids, flavonol and anthocyanin levels but this relationship was not significant (p > 0.0.). The fruit soluble solids content (SSC) and titratable acidity (TA) varied between genotypes. ‘Veestar’ and LL981-24 had the highest SSC (7.5). These results point to the importance of identifying genotypes with high phenolic monomer activities in screening for lines with high TAC, to ensure their use in breeding programs aimed at improving the nutritional value of strawberry.

Keywords:

• Strawberry
• Total phenolic content
• Antioxidant
• FRAP
• Phenolic composition
• Breeding
• Phytochemical

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Corrigendum

INTRODUCTION

In recent years, consumers have paid much attention to the health and nutritional aspects of horticultural products. Fruits are regarded as having considerable health benefits, primarily because of their antioxidant content, which can protect the human body against cellular oxidation reactions. These benefits have stimulated research on the phenolic composition and total antioxidant capacity of fruits as well as their associated effects on shelf life. Phenolics are believed to be important components responsible for antioxidant capacity in several fruits.

Phenolics constitute one of the most numerous and widely distributed group of substances in the plant kingdom. The beneficial effects of fruit consumption include the reduction and prevention of diseases induced by oxidative stresses, such as cardiovascular diseases, cancer and inflammatory conditions. These effects may be attributed, in part, to the phenolics and the various antioxidants in fruits.[1–3] Recent studies have indicated that the antioxidant properties of strawberries are mainly due to high levels of phenolic compounds rather than to vitamin C.[4–6]

Strawberries are a good source of natural antioxidants.[7,8] Some positive correlations have been found between antioxidant capacity, shelf life and disease susceptibility, but the results are not always conclusive.[5,9,10] Although several factors influence antioxidant capacity and the content of bioactive compounds, including cultural practices, pre-harvest conditions, maturity, and post-harvest handling and processing,[11–16] the most important factor is genetic variability. Since antioxidant content is becoming an increasingly important parameter, with respect to fruit quality and shelf life, the antioxidant profile of strawberries should be considered in breeding programs in relation to environmental conditions and cultural practices.[17,18] The purpose of this study was to examine the TPC and TAC of newly developed strawberry genotypes and the possible relationships between them, and to provide theoretical data for our breeding program.

MATERIALS AND METHODS

Sample Preparation and Extraction Procedures

Fruit samples from 15 strawberry genotypes (Fragaria × ananassa Duch.) were obtained from a completely randomized design, in four replicates, established at the Agriculture and Agri-Food Canada experimental farm in L'Acadie (longitude 73.35 W; latitude 45.32 N), Quebec. Fruits (0.5 kg fresh weight/replicate) were harvested at optimum maturity, rapidly cooled to 1°C and then brought to the laboratory where they were cut in half and frozen in liquid nitrogen. Three 150 g sub-samples of each genotype were stored at −80°C until analysis.

Ten grams (10 g) of fresh-frozen fruits, from each strawberry genotype, were grounded in 50 mL of 50% methanol using a Polytron® blender (Brinkmann Instruments, New York). The mixture was filtered through filter paper (Whatman no.1), followed by filtration through a 0.45-μm Acrodisc syringe filter (Gelman Lab., Michigan). The final filtrate was then stored at −20°C until needed and used in FRAP and HPLC assays to determine total phenolic content.

Chemicals

Phenolic standards and tetramethylchroman carboxylic acid were purchased from Sigma Chemical Co. (Oakville, Ontario). Ellagic, gallic, and p-coumaric acids, sodium carbonate (Na₂CO₃) and Folin-Ciocalteu reagent were obtained from Sigma Chemical Co. (St. Louis, Missouri); quercetin-3-galactoside from Fluka Chemie GmbH (Buchs, Switzerland); and cyanidin-3-galactoside from Indofine Chemical Co. (Hillsborough, New Jersey). Water used for HPLC analysis was double distilled using a NanoPure® system (Dubuque, Iowa). All other HPLC grade solvents were purchased from Caledon Laboratories Ltd. (Georgetown, Ontario).

Phytochemical Content (HPLC)

The phenolic composition of berries was analyzed by HPLC, as described by Tsao and Yang.[19] The injection volume was 20 μL for all samples. All standards except for anthocyanins were dissolved in methanol. Anthocyanins were dissolved in 1% HCl in methanol. The detector was set at 254, 280, 320, 360, and 520 nm for simultaneous monitoring of the different groups of phenolics.

Total phenolic compounds were divided into five groups and quantified as follows: anthocyanins using cyanidin-3-galactoside (520 nm); hydroxycinnamic acids using p-coumaric acid (320 nm); flavonols using quercetin-3-galactoside (360 nm); benzoic acids using gallic acid (280 nm) and the authentic standard was used for ellagic acids (254 nm), as previously described by Tsao et al.[18] and Wang et al.[19] The results were expressed as μg g⁻¹ fresh-frozen weight.

Total Phenolic Content (TPC)

TPC was determined according to the Folin-Ciocalteu (FC) method[20] with slight modifications. Standard or sample extracts (0.02 mL) were mixed with 1.58 mL of water and 0.1 mL of FC reagent, then after 3 min, 0.3 mL of Na₂CO₃ (7.5%) was added to a 10-mL vial and allowed to stand for 30 min. at 40°C. Absorption was measured at 765 nm using a Ultrospec 3100 pro UV/Visible Spectrophotometer (Fisher Scientific, Canada). Gallic acid was used as a standard and TPC was expressed as gallic acid equivalent (GAE) in μg g⁻¹ fresh-frozen weight. Concentrations exceeding the upper limit (500 μg mL⁻¹) of the linear range of the standard curve were diluted before final analysis.

Ferric Reducing/Antioxidant Power (FRAP) Assay

FRAP was performed according to the method of Benzie and Strain.[21] Briefly, 2.4 mL of freshly prepared FRAP reagent containing 10 mM TPTZ in 40 mL HCl, 20 mM FeCl₃ 6H₂O and 300 mM acetate buffer (pH 3.6), in the ratio of 1:1:10 (v:v:v), was mixed with 80 μL of appropriately diluted sample. The mixture was measured at 593 nm (Agilent 8453 Spectrophotometer, Agilent Technologies, Waldbronn, Germany). TAC of the samples was calculated on the basis of 500 μM L-ascorbic acid and expressed as μg ascorbic acid equivalent (AAE) per gram fresh-frozen weight.

Sensory and Physical Evaluation of Harvested Fruits

Five fruits of each genotype were placed in a Petri dish on a Whatman no. 12 filter paper and left at room temperature (19–22°C). Fruit weight and juice losses, glossiness and post-harvest diseases were checked daily until the fruits were no longer commercially acceptable. Fruit firmness was measured using Lloyd Instruments model LRX. The test was carried out using a constant 50 N weight with a load cell sensitivity of 100.4% and a 3.5 cm diameter probe.

SSC and TA assays were carried out on five randomly selected strawberries. They were homogenized to obtain a juice, using an ACME Supreme Juicerator (ACME JUICER MFG. CO., New Hartford, CT). SSC was determined at 20°C with a refractometer (ABBE MARK II, Reichert–Jung). TA was measured diluting each mL of strawberry juice to 10 mL with distilled water and then titrating to pH 8.1 using 0.1 N NaOH (Fisher Scientific AB 15 Accumet Basic).

RESULTS

Phenolic Composition (HPLC)

HPLC revealed that anthocyanins were the most predominant phenolic group in the strawberry extracts (79.2%) (Table 1). Total anthocyanins ranged from 191.0–1049.0 μg g⁻¹ in the different strawberry genotypes. The highest content was found in BC2-72-17, followed by LL9819-14, ‘Chambly’ and BC2-90-43, while the lowest was found in SJ9332-7. However, the total anthocyanins level in BC2-72-17 was significantly higher than in all other genotypes. Flavonols and hydroxycinnamic, benzoic, and ellagic acids accounted for the smallest proportions of the total phenolics: 6.7, 6.2, 5.4, and 2.5%, respectively.

Table 1 Phenolic composition (μg g-¹ fresh-frozen weight) of 15 advanced strawberry lines and cultivars

Genotype	Total anthocyanins (520 nm)	Total hydroxycin-namic acids (320 nm)	Total flavonols (360 nm)	Ellagic Acids (254 nm)	Total benzoic acids (280 nm)	Total phenolic content - sum of 5 groups
APF937-1	420.1	122.9	42.0	17.8	94.8	697.6
APF939-71	473.5	47.2	68.2	12.8	20.6	622.2
BC2-72-17	1049.0	31.0	52.3	22.7	8.2	1163.3
BC2-90-43	713.8	26.2	40.7	17.0	29.0	826.7
BC96-33-4	564.7	9.8	25.1	15.0	14.6	629.2
BC98-49-34	389.0	65.1	49.8	15.0	82.8	601.6
Chambly	772.0	44.7	76.1	16.0	29.4	938.1
Kent	506.5	22.5	56.2	30.0	58.2	673.4
LL981-24	448.8	14.3	60.4	18.7	12.7	555.0
LL9819-14	796.9	43.8	30.8	19.0	40.3	930.9
LL982-14	615.8	32.9	43.0	12.8	13.7	718.2
LL0220-10	431.8	8.1	37.4	16.1	9.7	503.2
SJ9332-7	191.0	62.7	30.2	21.8	59.6	365.4
Veestar	559.7	20.7	49.3	19.8	28.4	677.8
Yamaska	648.3	119.9	64.1	21.1	83.2	936.6
% total	79.2%	6.2%	6.7%	2.5%	5.4%	100%
LSD0.05	161.9	19.4	21.8	8.2	22.6	199.8
Data represent the average of triplicates. Phenolic groups were quantified as follows: ellagic acids as ellagic acid, anthocyanins as cyanidin-3-galactoside, hydroxycinnamic acids as p-coumaric acid, flavonols as quercetin-3-galactoside and benzoic acids as gallic acid. LSD0.05: Least significant difference at the 0.05 level.

TPC, based on the sum of the five groups analyzed by HPLC, ranged from 365.4 to 1163.3 μg g⁻¹. The highest TPC was found in BC2-72-17 (1163.3 μg g⁻¹), followed by ‘Chambly,’ ‘Yamaska,’ LL9819-14 and BC2-90-43, while SJ9332-7 had the lowest (365.4 μg g⁻¹). There was a high correlation between anthocyanins, flavonols and TPC (sum of five groups) (r = 0.96, r = 0.47, respectively).

Ferric Reducing/Antioxidant Power (FRAP)

The highest TAC was observed in BC2-90-43, which differed significantly from the other genotypes but was similar to ‘Yamaska’ (Table 2). The lowest was detected in ‘Kent’ and LL9819-14 while the rest of the genotypes were intermediate.

Table 2 Total phenolic content (TPC) and antioxidant capacity (TAC) of 15 advanced strawberry lines and cultivars

Genotype	TPC ^a (μg GAE/g)	TAC ^b (μg AAE/g)	Firmness	Shelf life at room temperature (day)	SSC (Soluble solids content)	TA (Titratable acidity)
APF937-1	1016.8	1301.0	12.1	2	6.7	0.9
APF939-71	1026.5	1412.0	–	–	–	–
BC2-72-17	2123.8	1400.7	–	–	–	–
BC2-90-43	1696.8	2259.0	–	–	–	–
BC96-33-4	1035.6	1401.0	–	–	–	–
BC98-49-34	1214.5	1690.7	–	–	–	–
Chambly	920.4	1512.6	7.5	2	6.7	0.8
Kent	1706.8	1183.4	11.6	2	7.1	0.7
LL981-24	1127.4	1435.7	6.7	2	7.5	0.6
LL9819-14	1396.4	1224.0	10.1	2	6.5	0.5
LL982-14	750.7	1356.7	–	–	–	–
LL0220-10	649.3	1318.7	9.6	3	7.0	0.6
SJ9332-7	1351.7	1583.5	14.2	3	6.0	0.6
Veestar	1311.5	1526.3	6.0	2	7.5	0.7
Yamaska	1950.7	1795.3	12.4	3	6.6	0.9
MEAN	1285.26	1493.4	10.0	–	6.8	0.7
LSD0.05	358.9	515.5	1.5	–	0.9	0.1
FRAP and FC analyses were performed in triplicate. ^aTPC expressed as μg gallic acid equivalent (GAE) per gram fresh-frozen weight. ^bTAC expressed as μg ascorbic acid equivalent (AAE) per gram fresh-frozen weight. LSD0.05: Least significant difference at the 0.05 level. – means missing data due to the missing plots in 2^nd year.

Total Phenolic Content (TPC)

TPC, determined by the FC method, of the 15 selected strawberry genotypes (expressed as μg GAE/g of fresh-frozen weight) is shown in Table 2. Significant differences in TPC were found among the tested genotypes. The TPC ranged from 649.3 to 2123.8 μg g⁻¹. BC2-72-17 was found to have the highest followed by ‘Kent,’ BC2-90-43 and LL9819-14 (1706.8, 1696.8, and 1396.4 μg g⁻¹, respectively), whereas LL0220-10 (649.3 μg g⁻¹) had the lowest. The remaining genotypes were intermediate. There was a high correlation between TPC, anthocyanins and ellagic acids (r = 0.38, r = 0.66, respectively).

Firmness and Shelf Life

SJ9332-7 had the firmest fruits, followed by ‘Yamaska,’ APF937-1 and LL9819-14, while ‘Veestar’ was the least firm (Table 2). LL0220-10, SJ9332-7, and ‘Yamaska’ had a three-day shelf life at room temperature, while it was of only two days for the other genotypes. There was a negative correlation between shelf life and ellagic acids, flavonols and anthocyanins (data not shown), which is similar to the results reported by Tao et al.[5] However, there were no significant differences between them.

Soluble Solids Content (SSC) and Titratable Acidity (TA)

Strawberry flavour derives from the interactive tastes and aromas of many chemical constituents.[22] High sugars and relatively high acid contents are required for good flavour. The SSC and TA contents varied among genotypes (Table 2). ‘Veestar’ and LL981-24 had the highest SSC (7.5), followed by ‘Kent,’ LL0220-10 and ‘Chambly,’ while SJ9332-7 showed the lowest. TA content was highest in ‘Yamaska’ and APF937-1 but lowest in LL9819-14.

DISCUSSION

The high variation in phenolic composition of strawberry genotypes has been confirmed by several researchers.[23–25] HPLC was used to identify and quantify several groups of phenolic compounds in the 15 strawberry genotypes. The results in this study showed a very wide variation in the composition and distribution of these phenolic compounds among the different genotypes. In all cultivars studied, anthocyanins were the predominant group of phenolics, which is consistent with previous reports on selected genotypes.[18,25,27,28] They accounted for 79.2% of the total phenolics, and the BC2-72-17 strawberry cultivar contained the highest concentration. Some studies have shown a correlation between antioxidant capacity, TAC and anthocyanins rather than between antioxidant capacity and the concentration of any given compound in strawberries, blueberries and grapes, regardless of the assays used.[14,29] Also, the correlation found between anthocyanin content and antioxidant capacity, based on oxygen radical absorbance capacity from the pink stage to the ripe stage,[14] was observed in 10 tested cranberry cultivars.[15] However, in our study, no correlation was observed between anthocyanins and total antioxidant activity, which is similar to the report by Meyers et al.[24] This result is probably due to other un-quantified phenolics, major phenolics affecting correlations between them and TAC, or synergism among these compounds, a possibility which deserves further investigation.

It has been shown that phenolic compounds play an important role in extending shelf life and enhancing the quality of fresh fruits by delaying senescence induced by oxidative degradation.[30] Some positive correlations have been found between antioxidant capacity, shelf life and disease susceptibility,[5,9,10] but we were unable to detect a significant relationship in our study, probably due to the sample size.

Tao et al.[5] reported that ellagic acid has a negative effect on germ tube and mycelial growth at a low concentration (36 ppm), whereas a higher level promotes germ tube elongation and mycelial growth. Hébert et al.[9] reported a higher level of resistance to grey mould growth in fruit extracts from six strawberry cultivars, but the authors did not indicate which phenolic compositions or combinations were responsible for inhibiting fungal growth.

CONCLUSION

In summary, a significant variation has been observed between newly developed June bearing strawberry lines and established cultivars and clearly shows the potential value of advanced lines and their possible use in breeding programs to develop lines with improved antioxidant compounds.[33] The genotypes with the highest TPC did not have the highest antioxidant capacity, suggesting that the activities of other compounds were responsible for the antioxidant capacity. A more in-depth investigation should therefore be undertaken, because minor components like epicatechin and hydroxycinnamic acids may have stronger antioxidant and biological activities, and these may also have a synergistic role. Therefore, we should not only consider the TPC but also phenolic monomers like epicatechin and hydroxycinnamic acids, along with the presence of newly synthesized isozymes[31–33] with high antioxidant activity, in breeding programs, in order to select new lines with high antioxidant capacity.