Influence of hormetic heat treatment on quality and phytochemical compounds of broccoli florets during storage

Postharvest Biology and Technology 128 (2017) 44–53

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Influence of hormetic heat treatment on quality and phytochemical compounds of broccoli florets during storage Arturo Duarte-Sierraa , Charles F. Forneyb,* , Dominique Michauda , Paul Angersa , Joseph Arula a b

Department of Food Science and Horticultural Research Centre, Laval University, Québec, QC, G1 V 0A6, Canada Kentville Research and Development Centre, Agriculture and Agri-Food Canada, Kentville, NS, B4N 1J5, Canada

A R T I C L E I N F O

Article history: Received 28 October 2016 Received in revised form 30 January 2017 Accepted 31 January 2017 Available online xxx Keywords: Broccoli Heat treatment Glucosinolates Hydroxycinnamic acids Respiration

A B S T R A C T

The effect of moist hot air treatment applied to broccoli florets was studied in order to maintain quality and phytochemical compounds during postharvest storage at 4
C. Exposure to hormetic heat doses of 41
C for 180 min (low temperature, LT) and 47
C for 12 min (high temperature, HT) delayed yellowing for 21 d compared with non-heated florets that yellowed after 14 d. Chlorophyll content was also higher in florets treated with both the LT and HT heat treatments. The respiration rate of heat-treated broccoli was significantly higher immediately after heat treatments, being 10-times greater in LT-treated and 15-times greater in HT-treated florets on day 0 when compared with the control florets. However, after 7 d of storage differences were not significant, even though respiration rates were lower in treated broccoli after 21 d of storage compared with non-heated florets. Off-odors were also detected in HT-treated broccoli. Titers of indole-type glucosinolates were significantly enhanced by both heat treatments, while the glucoraphanin content of florets only increased with the HT treatment. A similar pattern was observed with gene expression, where overexpression of tryptophan N-hydroxylase (CYP79B3) was greater than the expression of dihomomethionine N-hydroxylase (CYP79F1) in heat-treated broccoli florets. Titers of hydroxy-cinnamic acids of florets were increased by both heat treatments. The total antioxidant capacity was significantly enhanced by the HT treatment. Similarly, overexpression of coumarate ligase (CoL), chalcone synthase (CHS) and phenylalanine N-hydroxylase (CYP79A2) was triggered by the HT treatment. The results indicate hormetic heat treatments can enhance the content of phytochemicals in broccoli florets during storage. However, the application of heat at 41
C (LT) was superior to the HT treatment in maintaining quality, although the enhancement of phytochemicals was less. Crown Copyright © 2017 Published by Elsevier B.V. All rights reserved.

1. Introduction Broccoli (Brassica oleracea var. Italica) is a source of several vitamins (A, C, E), calcium, carotenoids, terpenes, indoles, isothiocyanates, flavonoids and glucosinolates (Nestle, 1998) and is often identified as the vegetable most frequently consumed for health reasons, including cancer prevention (Schouten et al., 2009). The most important phytochemical compounds with potential health benefits in broccoli include flavonoids and glucosinolates (Moreno et al., 2006). Flavonoids are derived from phenylalanine through the phenylpropanoid or acetate-malonate pathway catalyzed by three important enzymes, phenylalanine

* Corresponding author. E-mail address: [email protected] (C.F. Forney). http://dx.doi.org/10.1016/j.postharvbio.2017.01.017 0925-5214/Crown Copyright © 2017 Published by Elsevier B.V. All rights reserved.

ammonia-lyase (PAL), chalcone synthase (CHS) and flavonoid monooxygenase (F3H1) (Weston and Mathesius, 2013). Whereas the synthesis of glucosinolates includes the conversion of amino acids to aldoximes, which is catalyzed by cytochrome P450 from the CYP79 gene family (Mikkelsen et al., 2002). Broccoli deteriorates rapidly due to its high respiration rate that contributes to its senescence (Finger et al., 1999; Page et al., 2001). The strongest indicator of senescence in broccoli, and also an important quality factor, is yellowing of the florets (King and Morris, 1994; Tian et al., 1997; Toivonen and Forney, 2004). Nutrient and phytochemical content are also affected by senescence. Quality changes such as weight loss and chlorophyll degradation occur with a corresponding reduction of ascorbic acid and total phenolic compounds in cold-stored broccoli (Serrano et al., 2006). Similarly, the loss of glucoraphanin, the precursor of 4-methylsulfinylbutyl isothiocyanate (sulforaphane)

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has been observed in broccoli during the first 3 d of storage at 20
C (Rangkadilok et al., 2002). In order to delay the senescence of broccoli, low temperature 0
C (32
F) along with high relative humidity (98–100%) and modified atmospheres (1–2 kPa O2 + 5–10 kPa CO2), are used during postharvest storage (Toivonen and Forney, 2004). Under these storage conditions, the quality of broccoli florets can be maintained for up to 21 days. In addition to the above preservation strategies, other postharvest treatments have been applied to broccoli in order to delay senescence and maintain its quality. The most effective have been heating treatment with temperatures between 41 and 52
C (Duarte-Sierra et al., 2012a; Forney, 1995; Funamoto et al., 2002); ethanol fumigation at 2500 ppm (Corcuff et al., 1996); UV-C irradiation with doses in the range of 1.2–10 kJ m2 (Costa et al., 2006; Duarte-Sierra et al., 2012b; Lemoine et al., 2007) and UV-B irradiation ranging from 4.4 to 19.0 kJ m2 (Aiamla-or et al., 2010, 2012). The potential mechanisms attributed to the beneficial effect of heat treatment in delaying ripening and/or senescence and the associated reduction of tissue softening of fresh produce involves different mechanisms. These include the induction of heat shock protein (HSP) (Paull and Chen, 2000); the inhibition of ethylene production by reducing the conversion of 1-aminocyclopropane-1carboxylic acid (ACC) to ethylene (Yu et al., 1980); and the partial inactivation of hydrolytic cell wall enzymes such as polygalacturonase, b-galactosidase, endo-1,4-b-D-glucanase (EGase) and b-xylosidase (Martínez and Civello, 2008). The cross protection of heat treatment against chilling injury has been attributed to the synthesis and accumulation of HSP (Paull and Chen, 2000). Heat treatment either by hot water or hot air was shown to delay yellowing of broccoli (Forney, 1995; Lemoine et al., 2009; Tian et al., 1996). The immersion of broccoli in 50
C water for 2 min was found to be the most effective treatment to slow yellowing and decay (Forney, 1995). The former may be in part, due to reduced chlorophyllase activity (Funamoto et al., 2002). However, negative effects of heat treatment can occur when high temperatures and longer exposure times are used. Off-odor development and increased production of ethanol were found in broccoli subjected to a hot water treatment of 52
C for 3 min (Forney and Jordan, 1998). In addition to quality maintenance, treatment of broccoli with UV-B light and the signal molecules, methyl jasmonate and salicylic acid, have altered the phytochemical composition of broccoli (Mewis et al., 2012; Pérez-Balibrea et al., 2011). Nonetheless, in broccoli, the elicitation and accumulation of phytochemical compounds induced by heat has not been completely explored, especially those with potential health benefits such as glucosinolates and flavonoids. The biological phenomenon in living organisms known as hormesis, where low doses of harmful agents or stressors elicit beneficial or adaptive responses, is generally appreciated (Chapman, 2002; Luckey, 1980). This phenomenon has been shown to occur in crops in response to UV-C, where disease resistance and delayed ripening has been demonstrated. In this work, we have examined the effect of hormetic heat doses applied at two temperatures (41
C for 180 min and 47
C for 12 min) on the quality of broccoli and its health-related phytochemical composition following storage at 4
C.

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(approximately 70 mm length) were separated from heads and 300 g of florets were randomly arranged in 0.5 L plastic punnets. The punnets were placed in ventilated 5 L plastic containers and held overnight at 4
C/90% RH. 2.2. Treatments Following storage overnight at 4
C/90% RH, heat treatment was performed on the punnets of florets in a closed 2 m3 chamber with continuous air circulation, controlled air heating and humidity control using steam injection. Broccoli florets were exposed to hormetic heat treatments of 41
C for 180 min (low-temperature dose, LT) or 47
C for 12 min (high-temperature dose, HT). These hormetic heat-dose equivalent treatments using different temperature-time conditions were determined previously for the purpose of maintaining the quality of broccoli florets based on color (Duarte-Sierra et al., 2016). The temperature of the floret stem was monitored with temperature probes (5 mm in length) placed at three locations along the length of the floret stem (bottom, middle and the top location near the buds). The time to reach the desired temperature varied between 8 min for 47
C and 10 min for 41
C. The heat treatment began once the desired temperature was reached on all 3 temperature probes on each of five florets monitored for each treatment. Heat-treated and non-exposed (control)florets were immediately cooled by immersing them into a 9 g L1 NaCl isotonic solution (to prevent cellular osmotic rupture) at room temperature (23
C) for 1 min. Florets were then surface dried and cooled to 10
C inside a disinfected 1
C chamber with constant airflow for 10 min, and subsequently stored at 4
C and 90% RH until further analysis. 2.3. Weight loss and respiration rate of broccoli florets Weight loss in broccoli florets during postharvest storage was determined in quadruplicate by subtracting sample weights from their initial recorded weights and is presented as% weight loss. Respiration rate of broccoli florets was determined by measuring the accumulation of CO2 and the depletion of O2 at room temperature (23
C). After warming to room temperature (23
C), 6 florets (ca. 30 g) were sealed in a 170 mL airtight glass container (dead volume was filled with small plastic spheres) stored at room temperature for 1 h and the concentrations of CO2 and O2 in the container atmosphere were measured using a gas analyzer (CheckMate 9900, Cambridge, ON, Canada). Respiration rate was monitored after 0, 7, 14 and 21 d of storage, and all measurements were performed in quadruplicate (four glass containers per treatment). 2.4. Color measurement of broccoli florets Color measurements of twelve florets from each treatment were performed with a Chroma meter (Minolta CR200, Osaka, Japan) equipped with an 8 mm measuring head and a D 65 illuminant. The meter was calibrated using the manufacturer’s standard white plate. Color was quantified in the L*, a* and b* color space. The hue angle (h
) was calculated as h
= tan1 (b*/a*) when a* > 0 and b* > 0, or as h
= 180 – tan1 (b*/a*) when a* < 0 and b* > 0. All measurements were performed in triplicates.

2. Materials and methods 2.5. Chemical assays 2.1. Broccoli Freshly harvested broccoli (Brassica oleraceae L. var. Italica, ‘Diplomat’) heads were obtained from a commercial farm (Ile d’Orléans, Québec, Canada). Florets of uniform size

The chemical assays were conducted on tissue powder from flower buds frozen in liquid nitrogen and lyophilized that were obtained from florets used in respiration (6) and color (12) determinations.

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2.5.1. Total phenolic content Determination of the total phenolic content of broccoli samples was carried out using the Folin-Ciocalteau method (Ainsworth and Gillespie, 2007). Flower buds, the tissue that contains the greatest concentration of glucosinolates (RybarczykPlonska et al., 2016), were cut from florets using a sharp blade and buds were immediately dropped in liquid nitrogen to avoid tissue degradation and glucosinolate conversion. Samples were ground using liquid nitrogen and immediately lyophilized. A 10 mg sample of the lyophilized broccoli samples was extracted for 5 min with 1 mL of deionized water at 70
C in a sonication bath followed by 1 min of vortexing. The suspension was centrifuged at 11,270g at 4
C for 1 min. The pellets were re-extracted for 5 min using 70
C deionized water, and the supernatants of each extraction were pooled. The solution (0.1 mL) was transferred to an assay tube and mixed with 0.5 mL of deionized water, 20 mL of Folin–Ciocalteau reagent, and 400 mL of 700 mM sodium carbonate (Na2CO3). After incubation at room temperature for 30 min, 200 mL of sample, standard (Gallic acid) or blank (water) from the assay tube were transferred to a clear 96-well microplate, and the absorbance was read at 765 nm on a spectrophotometer (Benchmark Plus, Bio-Rad, Philadelphia, USA). Using a 5 data point standard curve (0–250 mg L1) the total phenolic content in broccoli was determined in triplicate, and the phenolic content was expressed as mg equivalents of gallic acid on a dry mass basis. All measurements were conducted in triplicates on three broccoli samples 2.5.2. Total flavonoid content Total flavonoid content was determined by the aluminum chloride colorimetric method (Lin and Tang, 2007). The extract used was the same used for the determination of total phenolics. Samples of the extracts (0.1 mL) were mixed with 300 mL of ethanol, 20 mL of 100 g L1 aluminum chloride (AlCl3), 20 mL of 1 M potassium acetate and 600 mL of water. After incubation at room temperature for 30 min, 200 mL of sample, standard (quercetin) or blank (water) were transferred to a clear 96-well microplate, and the absorbance was read at 415 nm on a spectrophotometer (Benchmark Plus, Bio-Rad, Philadelphia, USA). Using a 5 data point quercetin standard curve (0–100 mg L1), the total flavonoid content was determined in triplicate and expressed as mg equivalents of quercetin on a dry mass basis. All measurements were conducted in triplicates on three broccoli samples. 2.5.3. Assay of reduced and total ascorbic acid Reduced and total ascorbic acid content were determined using the method of Gillespie and Ainsworth (2007). Extracts from lyophilized broccoli samples (ca. 10 mg) were conducted with 1 mL of 60 g L1 trichloroacetic acid (TCA). The suspension was centrifuged at 11,270g at 4
C for 5 min. The pellets were reextracted using 1 mL of TCA, and the supernatants of each extraction were pooled. To determine total ascorbic acid (AA) content, 200 mL of the sample extract was incubated at room temperature with 100 mL of 75 mM phosphate buffer (pH 7) and 100 mL of 10 mM dithiothreitol (DTT) for 10 min. The excess DTT was removed by the addition of 100 mL of 5 g L1 N-ethylmaleimide (NEM) and incubating at room temperature for at least 30 s. Reduced ascorbic acid content was determined on 200 mL of the extract mixed with 100 mL of 75 mM phosphate buffer (pH 7.0) and 200 mL of deionized water. To the solution, 500 mL of 100 g L1 TCA, 400 mL of 430 mL L1 phosphoric acid (H3PO4), 400 mL of 40 g L1 a-a’- bipyridine and 200 mL of 30 g L1 AlCl3 were added. The assay tubes containing this mixture were incubated at 37
C for 1 h, and 200 mL of the samples, standards (AA) or blank (60 g L1 TCA) were transferred

to a clear 96-well microplate, and the absorbance was read at 525 nm and quantified using a 5 data point standard curve (0–5 mM). The levels of reduced and total ascorbic acid were determined in triplicate, and the values were expressed as mg equivalents of ascorbic acid on a dry mass basis. All measurements were conducted in triplicates on three broccoli samples. 2.5.4. Total antioxidant capacity ORAC (Oxygen Radical Absorbance Capacity) assay was performed following the method of Gillespie et al. (2007). Extractions were carried out from lyophilized broccoli samples (ca. 0.02 g) with 1.5 mL of 500 mL L1 acetone. The suspension was centrifuged at 1585g at 4
C for 30 min. The pellets were re-extracted using 1.5 mL of 50% acetone and supernatants of each extraction were pooled, and the extract was diluted 80 times. An aliquot of 25 mL of the diluted extract, 25 mL of 75 mM phosphate buffer (pH 7.0) as a blank, or 25 mL of 20 mM Trolox as a standard was added to each well of a clear microplate containing 150 mL of 0.08 mM fluorescein. In addition, 25 mL of 150 mM 2,20 azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) was added to the samples, blanks and standards and ORAC measurements were performed at 37
C on a microplate reader (FluoStar Galaxy DMG, Vienna, VA, USA) with excitation wavelength of 485 nm and emission wavelength of 530 nm. The ORAC values were calculated using the regression equation relating Trolox concentration and the net area under the curve (AUC) [Net AUC = AUC (extract) – AUC (blank)]. Using a five-point standard curve (0–50 mM Trolox) the total antioxidant capacity was determined in triplicate, and the values were expressed as mg Trolox equivalents on a dry mass basis. All measurements were conducted in triplicates on three broccoli samples. 2.5.5. Chlorophyll content Chlorophyll was extracted from lyophilized broccoli samples (0.01 g) by vortexing in 1 mL of methanol for 30 s (Genie 2, Vernon Hills, IL, USA) following the method of Warren (2008). Samples were centrifuged 2 min at 11,270g (Microcentrifuge 5418, Hamburg, Germany), and the supernatant was transferred to another microtube. The pellet was re-extracted by adding 1 mL of ethanol to the pellet under agitation and centrifuged as previously described. The two supernatants were pooled and assayed by spectrophotometry. Measurements were conducted by adding 200 mL of samples or blanks into a 96-well flat bottom polystyrene plate. The absorbance of samples was measured on a spectrophotometer (Benchmark Plus, Bio-Rad, Philadelphia, USA). The absorbance at 652 and 665 nm was converted into 1 cm path length using the measured path length of 0.58 cm by: A652;1cm ¼

ðA652;microplate  blankÞ 0:58cm

ð1Þ

A665;1cm ¼

ðA665;microplate  blankÞ 0:58cm

ð2Þ

Chlorophyll concentration was calculated from 1-cm corrected pathlength absorbance value with the following formulae: Chlaðmg=mLÞ ¼ 8:09A652;1cm þ 16:51A665;1cm

ð3Þ

Chlbðmg=mLÞ ¼ 27:44A652;1cm  12:16A665;1cm

ð4Þ

Total chlorophyll was the sum of Chl a and Chl b. Each measurement was performed in triplicate for each of the four storage intervals of 0, 7, 14 and 21 d at 4
C.

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2.6. Glucosinolates and hydroxycinnamic acid analysis Separation of glucosinolates (GLS) and hydroxycinnamic acids (HCA) was done using the method of Nadeau et al. (2012). Broccoli inflorescence samples from florets used for respiration (6) and color determination (12) experiments stored for 0, 3, 7 and 14 d after treatment were sampled in triplicate, frozen in liquid nitrogen, and lyophilized. The dried samples were pulverized, packed in plastic laminate pouches under vacuum, and stored at 30
C until extraction. GLS and HCA were extracted from the dried sample (0.5 g) with 10 mL of 700 mL L1 aqueous methanol containing 800 mg of sinigrin (internal standard) at 70
C in a sonication bath for 10 min. The suspension was centrifuged at 4528g at 4
C. The pellets were re-extracted twice using 10 mL of 700 mL L1 methanol and sonication for 10 min, and the supernatants of each extraction were pooled together. The extract was concentrated to dryness by evaporation under vacuum using a Rotavapor (Büchi R215, Flawil, Switzerland), and was dissolved in 10 mM ammonium acetate/formic acid at pH 4.4, which was the mobile phase used for LC analysis. Compounds were separated on a LC equipped with MS detector (HP series 1100 LC/MSD) using a Phenomenex Synergi Hydro-RP column (250 mm  2 mm, 80 Å) at 30
C. The analytes were eluted using a mobile phase A: (10 mM ammonium acetate/formic acid pH 4.4) and B (Acetonitrile). The gradient was 0–7 min 1–3% B; 7–13 min 3–15% B and 13–26 min 15–30% B. Identification was achieved by electrospray ionization MS on negative ion mode. Separation of GLS and HCA was achieved by the following retention times and molecular weights: 1 = sinigrin (8.31 min, 358.85 M); 2 = glucoraphanin (9.93 min, 436.91 M); 3 = 4-hydroxyglucobrassicin (23.02 min, 463.91 M); 4 = chlorogenic acid (31.25 min, 447.93 M); 5 = glucobrassicin (36.91 min, 477.99 M); 6 = 4-methoxyglucobrassicin (41.81 min, 477.95 M); 7 = neoglucobrassicin (50.52 min, 478.49 M); 8 = 1,2 disinapoyl gentiobiose (51.26 min; 724.2 M); 9 = 1-sinapoyl-2feruloyl gentiobiose (51.85 min, 694.21 M); 10 = 1,2-diferuloyl gentiobiose (53.63 min, 960.23 M); 11 = 1,2,20 –trisinapoyl gentiobiose (54.36 min, 930.22 M); 12 = 1,2-disinapoyl-2-feruloyl gentiobiose (55.19 min, 900.27 M). Quantification of the GLS was carried out with UV detection at 224 nm and HCA at 330 nm using a diode array detector. 2.7. Gene expression Genes responsible for quality (chlorophyllase), phenylpropanoids biosynthesis (PAL, CoL:, CHS and F3H1) and GLS biosynthesis (CYP79A2, CYP79B3 and CYP79F1) were assessed following heat treatment to gain insights on their potential impact on quality and enhancement of phytochemical compounds. RNA was extracted from lyophilized broccoli inflorescence samples (0.02 g) at 0, 48 and 96 h after the heat treatments were applied to florets using a RNeasy plant mini kit (Qiagen, Germany). Residual DNA was removed from RNA extracts with deoxyribonuclease (DNase) (TURBO DNA-free Kit, Ambion, USA). The integrity of RNA was verified by running 1 mg of RNA samples on a 1% denaturing agarose gel (formaldehyde 37%; formamide; and 10X MOPS (pH 7)). Subsequently, cDNA synthesis was carried out using 3 mg of the DNase-treated RNA; oligo dT; and SuperScript Reverse Transcriptase II (Invitrogen, Carlsbad, CA, USA) following the kit instructions. Polymerase chain reaction (PCR) was conducted using a commercial kit (Taq PCR) (New England Biolabs, Ipswich, MA, USA) with 0.25 mM forward and reverse primers, 1 unit of polymerase and 3 mg of cDNA in a total volume of 20 mL. The PCR program was set in a thermocycler (TPersonal, Biometra, Goettingen, Germany) to perform an initial denaturation for 3 min at 95
C and n cycles (depending on primer used) of [30 s–95
C; 45 s (temperature depending on primers) and 1 min–68
C] and a final extension step

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of 5 min at 68
C. Primers and Tm’s used in this experiment were: Actin (GenBank accession number: AF044573) sense: 50 -GGCATCACACTTTCTACA-30 antisense: 50 -CCTTAATCTTCATGCTGC-30 and Tm = 49
C/28cycles. Dihomomethionine N-hydroxylase CYP79F1 (GenBank accession number: GU385846) sense: 50 TCTCGAGGGTTTATGGTT-30 antisense: 50 -CCATGTTATTTGCCGGATT-30 and Tm = 62
C/36cycles. Tryptophan N-hydroxylase CYP79B3 (GenBank accession number: FJ376047) sense: 50 GGCTGCTCCAGACAATCCATCG-30 antisense: 50 -GTTCCTCCGA0 CAACCTCAATTCTCC-3 and Tm = 61
C/45 cycles. Phenylalanine N-hydroxylase CYP79A2 (GenBank accession number: EU877074) sense: 50 -AGAACCGAAGAGGCTGAT-30 antisense: 50 CCTTCTCATGGCCTTCCA-30 Tm = 60
C/36 cycles. Phenylalanine ammonia lyase PAL (GenBank accession number: HM623311) sense: 50 -TCAACACTCTCCTCCAAG-30 antisense: 50 -GAAGTTACCACCGTGAAT-30 and Tm = 49
C/36 cycles. Chalcone synthase CS (GenBank accession number: AY228486) sense: 50 -ACTTCCGCATCACCAACA-30 antisense: 50 -CACTCCAACCCTTCTCCT-30 and Tm = 62
C/36 cycles. Flavanone 3-hydroxylase F3H1 (GenBank accession number: EU402420) sense: 50 -GACAGGAAGAGGTTGGAA-30 antisense: 50 -TGAAGGTAAGGAAGCTGA-30 and Tm = 57
C/36 cycles. PCR products were run on 1% agarose gel at 160 V/60 min using 1 x Borax buffer (pH 8.0), normalized with actin and analyzed by densitometry with ImageJ (Wayne Rasband, NIH, USA). 2.8. Statistical analysis The experiment was conducted as a complete randomized design and data analyzed by one-way analysis of variance (oneway ANOVA) using a significance level of 0.05. Least significant difference test was used to report significant differences. The statistical analysis was executed using the Statistical Analysis System version 9.3 (SAS Institute Inc. 2011. Base SAS1 9.3 Procedures Guide. Cary, NC, USA). No interaction between treatment and storage time was observed for total phenols and flavonoids, ascorbic acid and ORAC values. Therefore the means over storage times (0, 7, 14 and 21 d) were determined. 3. Results and discussion Based on membrane integrity of broccoli florets from previous results (Duarte-Sierra et al., 2012a), hormetic heat doses were applied at two temperatures: 1) 41
C applied for 180 min (LT) and 2) 47
C for 12 min (HT). Both doses were equivalent based on electrolyte leakage of the tissue, 41
C being within the sub-critical zone (<42
C), and 47
C in the upper-critical zone (>45
C) as described by Duarte-Sierra et al. (2016). These two hormetic heat doses were used to characterize the qualitative, physiological and nutraceutical properties of broccoli florets in response to hormetic heat treatments. 3.1. Physiological and biochemical characteristics 3.1.1. Respiration rate and weight loss The respiration rate of florets increased substantially 6 h following exposure to heat in both the LT and HT treatments on day 0 (Fig. 1A). On this day, significant differences were observed among the treatments. The respiration rate of the control florets measured as CO2 production was lowest at 2.56 nmol kg1 s1, followed by the LT treatment at 19.5 and the HT treatment at 32.6 nmol kg1 s1. On day seven, respiration rate of the florets reached steady levels, with florets receiving the LT treatment decreasing to rates lower than that of the control and the HT treatment. At this point, respiration rates of florets were 2.0, 1.25 and 1.84 nmol kg1 s1 for the control, LT and HT treated florets, respectively. Respiration rates of heat-treated florets remained

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Fig. 1. Respiration rate (A) and weight loss (B) of heat-treated broccoli florets exposed to three different heat doses: (*), control; (), 41
C/180 min (LT); and (!), 47
C/12 min (HT). Vertical bars represent standard deviation, n = 4.

lower than that of the control from day 14 until the end of storage (21 d), when respiration rates of the florets were 2.40, 0.95 and 1.09 nmol kg1 s1 for the control, LT and HT treatments, respectively. The rapid increase of floret respiration in response to heat treatment may be a consequence of a general adaptation process, which requires large amounts of energy in the form of ATP for the synthesis of protective/defense compounds. In plants exposed to heat, a general response is the production of HSP (Paull and Chen, 2000). The rise in CO2 production in response to heat treatments may also be a result of induced fermentation. Hypoxic conditions in the heat-treated tissue resulting from a decrease in oxygen solubility with the increased temperature could result in anaerobic respiration that would substantially increase CO2 production. This would be associated with the generation and accumulation of ethanol, which was previously described in heat-treated broccoli (Duarte-Sierra et al., 2012a, 2016; Forney and Jordan, 1998). Heat treatments may also cause membrane damage, resulting in the disruption of oxidative phosphorylation, which would induce fermentation (Forney and Jordan, 1998). Hot water treatments of broccoli florets that inhibited yellowing were reported to inhibit chlorophyll fluorescence (Fv) (Tian et al., 1996), which supports the hypothesis that the heat treatment disrupted normal membrane function in broccoli. The elevated production of CO2 in heat-treated florets immediately after treatment was transient and respiration rate declined to rates lower than that of the control florets from day 7 until the end of storage. Similar observations were previously reported in apples (Lurie and Klein, 1990) and mangoes (Mitcham and McDonald, 1993), where respiration rates of heat-treated tissue were lower compared with non-treated plant tissue. These authors attributed lower rates of respiration as a consequence of

the heating process resulting in damage to the respiratory apparatus of the tissue. The higher respiration rate of non-treated florets after 21 d of storage could also reflect the development of fungal infection during storage or increased metabolism associated with senescence, which appeared to be delayed in heat-treated florets. Heat treatments also may affect ethylene production. An increase in CO2 production is generally followed by a decrease of ethylene synthesis (Klein and Lurie, 1992). Hot air was attributed to inhibit the ripening of climacteric fruits by inhibiting the production of the ethylene precursor 1-aminocyclopropane-1carboxylic acid (ACC) (Lurie, 1998). While broccoli is not climacteric, it is sensitive to ethylene action, which stimulates senescence (Toivonen and Forney, 2004). Hot water dips delayed yellowing of broccoli florets during postharvest storage (Forney, 1995; Tian et al., 1996) and this delay in yellowing was associated with an inhibition of ethylene production (Tian et al., 1997). Although ethylene measurements were not carried out in this study, evidence of high initial CO2 production and delay of yellowing by the LT and HT treatments were observed, giving credence to the hypothesis that heat may inhibit ethylene production or its action. In addition to the increase of respiration rate, heat treatments altered weight loss of broccoli florets (Fig. 1B). Florets exposed to the HT treatment had higher rates of weight loss during storage than control florets, while those exposed to the LT treatment had a lower rate. An analogous observation made by Williams et al. (1994) in oranges showed that fruit immersed in 53
C water had greater weight loss than fruit immersed in 45
C water, but fruit treated at 45
C had less weight loss than non-treated fruit. It was suggested that this reduction in weight loss was a result of the spreading of softened or molten cuticular waxes resulting in a greater barrier to water loss. Waxes are also present on the surface of broccoli, and their melting may have protected the tissue from desiccation, as observed with oranges. However, florets treated at 47
C for 12 min (HT), which had greater weight loss, may have resulted in excess melting and a reduced moisture barrier. In addition, the release of volatile plant acids and other volatiles generated at this temperature could also affect weight loss since emissions of biogenic volatile organic compounds (BVOCs), especially terpenes, is a defense mechanism of plants and they increase exponentially with increasing temperature up to a maximum rate (Grote et al., 2013). 3.1.2. Color and chlorophyll content in florets Delay of yellowing of florets, measured as hue angle, by heat treatments was evident after 21 d of storage (Fig. 2A). No significant differences were observed between the control and heat-treated broccoli florets during the first 14 d of storage at 4
C. Nevertheless, it was clear after 21 d of storage that florets treated with both the LT and HT treatments had higher h
values compared with the control. At this time, h
values were 115 for control florets, compared to 128 and 131 for florets exposed to the LT and HT treatments, respectively. The higher h
values indicate darker green color. Similarly, after 21 d of storage, the chlorophyll content of heat-treated broccoli was significantly higher compared with control florets (Fig. 2B). Chlorophyll content on a dry mass basis was 1.8, 3.0 and 3.1 g kg1 for control, LT and HT treatments, respectively. A closer inspection of color revealed differences between the LT and HT treated florets. Visual examination revealed that broccoli florets treated at 47
C appeared greener than those treated at 41
C. Forney (1995) reported that hot water treatments of 52
C for 3 min made broccoli flower buds appear dark green and suggested this change in color was a result of alteration of the surface wax,

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hot water at >50
C resulting in off-odor generation and ethanol production (Forney, 1995; Forney and Jordan, 1998). These authors suggested that ethanol production was due to fermentation induced by the heat-induced stress. They also suggested that even if the visual quality of florets was adequate when exposed to the high dose of heat, cellular damage may have occurred as a result of the heat treatment. Induced fermentation can accelerate senescence due to the low amount of energy as a consequence of reduced ATP production and protein turnover, as well as induce the production of off-odors in broccoli (De Vries, 1975; Forney et al., 1991) 3.1.3. Phenylpropanoid compounds, ascorbic acid and ORAC The concentration of total phenols, 16 g kg1 and flavonoids, 5.4 g kg1 was very similar to values reported by Costa et al. (2006), 12.62 g kg1 and 9.5 g kg1, respectively. Nevertheless, the concentration of 16.9 g kg1 of ascorbic acid was higher compared to 6 g kg1 reported by Koh et al. (2009). Florets exposed to heat showed no significant differences during storage at 4
C in the content of total phenols, total flavonoids or total ascorbic acid (Table 1). However, ORAC (oxygen radical absorbance capacity) of HT-treated florets was 37% greater than the control and 19% greater than the LT-treated florets (Table 1). The ORAC value and level of oxidized ascorbic acid was greatest in the HT-treated florets (Table 1) presumably as a result of increased activity of ascorbate oxidases and reactive oxygen species (ROS) generation in response to heat stress (Mittler et al., 2012) Fig. 2. Color (hue angle h
) (A) and chlorophyll content (B) of heat-treated florets exposed to three different heat doses: (*), control; (), 41
C/180 min (LT); and (!), 47
C/12 min (HT). Bars represent the LSD (0.05), where h
LSD = 4.28, n = 9 and chlorophyll LSD = 0.25, n = 3.

which was not observed at lower temperatures or shorter treatment times. Improved green color due to heat treatment has been reported for other vegetables. For instance, the green color of asparagus increased with heat treatment between 70 and 98
C (Lau et al., 2000). The ratio of a*/b*, which expresses green color, was consistently superior immediately after heat treatments in the range of 40–90
C in green beans and increased as temperature increased (Tijskens et al., 2001b). This ratio was also superior on heat-treated florets compared to non-treated florets at the end of storage, but differences were more evident expressed as h
. The effect of heat treatments on broccoli florets also improved the green-blue color of florets. Although excessively heated green vegetables turn olive green and eventually brown by pheophytin formation caused by the replacement of Mg2+ by H+ in the chlorophyll due to the decrease in pH by the liberation of plant acids during heating (Tijskens et al., 2001a); if the process is performed in an adiabatic system, as was in this study, plant acids would escape, favoring alkalization of cells and reducing the formation of pheophytins. In addition, the enhancement of color in florets by heat treatments may be a result of the alteration of surface reflecting properties of vegetables due to air removal between cells (Tijskens et al., 2001b). In spite of the enhancement of greenness by the exposure of broccoli to heat at 47
for 12 min, a deleterious effect was evident by the perception of off-odors after treatment, which was not present in LT treated florets. Ethanol and off-odor were perceived on florets treated at 47 and 52
C in a previous experiment conducted by the authors (Duarte-Sierra et al., 2016). This can be related with protein degradation increasing with temperature (Ferguson et al., 1994), which eventually produces free sulfur containing amino acids that are the substrate for off-odor volatiles. A comparable observation was made when broccoli was dipped in

3.2. Phytochemical compound analysis Glucosinolates and hydroxycinnamic acids are phytochemical compounds characteristic of Brassicas that participate in defense responses to pathogens, insects and wounding. Heat, as well as other biotic and abiotic stresses, affects the stability of various

Table 1 Concentration of oxygen radical absorbance capacity (ORAC, trolox equivalents), ascorbic acid, total phenols (gallic acid equivalents), total flavonoids (quercetin equivalents) and rutin (sinigrin equivalents) of treated broccoli florets. Florets were exposed to heat: control; 41
C/180 min (LT); and 47
C/12 min (HT) and stored 21 days at 4
C in darkness. The values  SD (n = 3) are on a dry weight basis and are the averages of 0, 7, 14 and 21 d. ORAC (g kg1) Control 41
C/12 min 47
C/180 min

152.0  14.6 174.1  17.0 208.9  16.8*

Ascorbic acid (g kg1) Control 41
C/12 min 47
C/180 min

Oxidized 3.8  0.2 3.8  0.4 4.2  0.2

Reduced 13.1  6.0 12.8  5.0 11.9  5.6

Total phenols (g kg1) Control 41
C/12 min 47
C/180 min

16.2  3.6 17.4  2.8 18.2  2.7

Total flavonoids (g kg1) Control 41
C/12 min 47
C/180 min

5.4  0.7 5.6  0.9 5.8  0.7

Rutin (g kg1) Control 41
C/12 min 47
C/180 min

0.6  0.1 0.6  0.0 0.7  0.01

Total 16.9  5.7 16.6  5.0 16.1  5.6

The asterisk indicates that the value is significantly different from the corresponding control at p < 0.05.

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cellular structures including membranes and proteins causing a state of imbalance. This imbalance leads to the generation and accumulation of undesirable products such as ROS, a response consequent of heat and other stresses (Mittler, 2002). One of the most important protective responses of plants is the enhancement of phytochemical compounds. Changes of these compounds were followed by gene expression during the first two days after the imbalance of homeostasis in florets was produced due to heat. Eventually, the concentration profile was quantified previous to yellowing, when commercial quality ends. The concentration of glucosinolates in broccoli florets in this study were slightly superior to those examined by RybarczykPlonska et al. (2016). Briefly, it was found, on average 8 g kg1 of glucobrassicin, 4 g kg1 of neoglucobrassicin, 2 g kg1 of 4-methoxy-glucobrassicin, and 2 g kg1 of 4-hydroxy-glucobrassicin compared to 3.41 g kg1; 1.94 g kg1, 0.4 g kg1 and 0.1 g kg1 of the same compounds extracted from buds. Total indole glucosinolates (sum of glucobrassicin, neo glucobrassicin, 4-methoxyglucobrassicin and 4-hydroxyglucobrassicin) were significantly (p < 0.05) enhanced by both heat treatments. After 14 d of storage, the concentration of total glucobrassicins in HT-treated florets was 21% greater than that of untreated florets and 11% greater than that of LT-treated florets (Fig. 3A). The glucoraphanin content of the HT-treated folorets was enhanced only during the first four days of storage; and thereafter no difference was observed among the other treatments (Fig. 3B). In general, tryptophan-derived indole glucosinolates are more enhanced by biotic stresses than aliphatic glucosinolates (Brader

et al., 2001). The difference in gene expression between tryptophan N-hydroxylase (CYP79B3) and dihomomethionine N-hydroxylase (CYP79F1) also supports this observation of heat treatment effects. Titer of glucobrassicin in heat-treated florets was relatively constant during storage compared with the titer of the control florets, which decreased after 4 d (Fig. 3C). A similar pattern was observed with neo glucobrassicin. Its concentration in heat-treated florets was significantly higher (p < 0.05) compared with the untreated florets throughout the storage period (Fig. 3D). However, other derivatives of glucobrassicin including 4-methoxyglucobrassicin (Fig. 3E) and 4-hydroxyglucobrassicin (Fig. 3F) showed an accumulation pattern different from the other glucobrassicins, although these two were relatively minor glucobrassicins. The concentration of these two glucobrassicin derivatives increased in the untreated florets during storage and was enhanced following heat treatments. The accumulation of 4-hydroxyglucobrassicin was seen in B. rapa when infected with F. oxysporum (Abdel-Farid et al., 2010). Likewise, 4-methoxyglucobrassicin appears to be necessary for resistance to pathogens and callose formation in Arabidopsis (Clay et al., 2009). Therefore, the gradual induction of these compounds during storage in untreated florets suggests there may have been development of fungal disease in untreated florets, although infection was not visually apparent. The steady increase of both 4-methoxyglucobrassicin and 4-hydroxyglucobrassicin in the untreated florets during storage, as opposed to the evolution in heat-treated florets, may support the hypothesis that untreated florets are more susceptible to

Fig. 3. Glucosinolate content, expressed as sinigrin equivalents, of heat-treated broccoli florets during storage at 4
C. Glucosinolate content was measured in florets exposed to three different heat doses: (*), control; (), 41
C/180 min (LT); and (!), 47
C/12 min (HT). Values are g equivalents of sinigrin on a dry weight basis. Total Glucobrassicins (A) LSD (0.05) = 0.69; Glucorapharin (B) LSD (0.05) = 0.35; Glucobrassicin (C), LSD (0.05) = 0.40; Neoglucobrassicin (D), LSD (0.05) = 0.51; 4-Methoxyglucobrassicin (E), LSD (0.05) = 0.27; 4-Hydroxyglucobrassicin (F), LSD = 0.21; n = 3. The bar in each graph represents the LSD value.

A. Duarte-Sierra et al. / Postharvest Biology and Technology 128 (2017) 44–53

infection. It also suggests that these two derivatives of glucobrassicin are more effective compounds against infection than the glucosinolates, glucobrassicin, neo glucobrassicin and glucoraphanin, which tend to decrease towards the end of storage in all treatment groups. Similar to glucosinolates, it was observed that the HT treatment significantly enhanced the level of HCAs in broccoli. Total HCAs increased during storage in the untreated florets, but greater increases were observed with the HT dose followed by the LT dose (Fig. 4), although the increase in the untreated florets was greater after 7 d of storage. Sharp increases of 1,2-disynalpoyl-2-feruloyl gentiobiose (Fig. 4B), 1,2,20 -trisinapoyl gentiobiose (Fig. 4E) and 1,2-disynapoyl gentiobiose (Fig. 4F), were also observed in the untreated florets after 7 d of storage similar to that of total HCA and their levels reached levels close to those observed in florets treated with the HT dose. The other HCAs, 1-sinapoyl-2-feruloyl gentiobiose (Fig. 4C, the major HCA), 1,2-diferuloyl gentiobiose (Fig. 4D)

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and sinapoyl-diferuloyl gentiobiose (Fig. 4G), showed small increases during storage, presumably as senescence of the florets progressed. However, the sharp increase in the former HCA may not be attributable solely to senescence but may be in response to other events that occur simultaneously, such as the infection of the untreated florets. As mentioned before; these compounds may accumulate in response to infection and may provide antimicrobial activity against diseases. The induction of HCA was concomitant with the increased expression of the phenylpropanoid-related genes coumarate ligase (CoL); chalcone synthase (CHS) and phenylalanine N-hydroxylase (CYP79A2) (Fig. 5). Usually, the induction of chalcone synthase along with phenylalanine ammonia-lyase precedes increases in the levels of constitutive phenols and provide adequate substrates for the synthesis of fungitoxic compounds such as quinones (Lattanzio et al., 2006). The initial content of glucosinolates and HCA in heat-treated florets either with LT or HT doses were consistently higher to those

Fig. 4. Hydroxy-cinnamic acid (HCA) content, expressed as sinigrin equivalents, in heat-treated broccoli florets exposed to three different heat doses: (*), control; (), 41
C/180 min (LT); and (!), 47
C/12 min (HT). Total HCA (A); LSD (0.05) = 0.63; 1,2-Disynalpoyl-2-feruloyl gentiobiose (B), LSD (0.05) = 0.17; 1-Sinapoyl-2-feruloyl gentiobiose (C), LSD (0.05) = 0.37; 1,2-Diferuloyl gentiobiose (D), LSD (0.05) = 0.09; 1,2,20 –Trisinapoyl gentiobiose (E), LSD (0.05) = 0.08; 1,2-Disinapoyl gentiobiose (F), LSD (0.05) = 0.11; Sinapolyl-diferuloyl gentiobiose (G), LSD(0.05) = 0.04; n = 3. The bar in each graph represents the LSD value.

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Fig. 5. Gene expression analysis of heat-treated broccoli florets on day 0. Florets were exposed to three different heat doses: control; 41
C/180 min (LT); and 47
C/12 min (HT). Gene expression was measured immediately after treatments on chlorophyllase (Chlase); coumarate ligase (CoL); chalcone synthase (CHS); phenylalanine Nhydroxylase (CYP79A2); tryptophan N-hydroxylase 2 (CYP79B3); dihomomethionine N-hydroxylase (CYP79F1); flavanone 3-hydroxylase (F3H1) and phenylalanine ammonia-lyase (PAL). Bars represent the standard deviation (n = 3).

in the untreated florets. It is not clear as to the contributing factors for this observation. The enhanced synthetic enzyme activities and mobility of the substrates at higher ambient temperatures during heat treatment may play a role in higher initial values of the compounds. In addition, the improved extractability of the compounds from heat-treated tissue could contribute in part. This possibility has been observed on water-blanched vegetables (Kaiser et al., 2013). 4. Conclusions Hormetic heat doses delivered at 41
C and 47
C showed different effects on quality and phytochemical content of broccoli florets during postharvest storage at 4
C. Enhanced levels of glucosinolates and HCA by heat treatment is likely a response to ROS accumulation in the tissue. The accumulation of 4-methoxyglucobrassicin, 4-hydroxyglucobrassicin, 1,2-disynalpoyl-2-feruloyl gentiobiose and 1,2,20 –trisinapoyl gentiobiose in the untreated florets may be in response to infection, and these compounds may likely be effective antimicrobials. The HT treatment showed higher levels of glucosinolates and HCA during storage than the LT treatment, however, the storability factors such as weight loss and generation of off-odors can be limiting, although the greenness of the florets was quite acceptable. The LT treatment maintained the quality of the florets, although elevation of phytochemicals was moderate, and at the least maintained at their initial levels throughout the storage period. Thus, the application of the hormetic heat dose of 41
C may be beneficial in terms of both maintaining quality attributes as well as enhancing phytochemical content. Acknowledgements This study was supported by the Natural Science and Engineering Research Council (NSERC) and the Quebec Ministry of Agriculture, Fisheries and Food (MAPAQ). Arturo Duarte-Sierra acknowledges the award of scholarship from the National Council of Science and Technology of Mexico (CONACyT). References Abdel-Farid, I.B., Jahangir, M., Mustafa, N.R., van Dam, N.M., van den Hondel, C.A.M.J. J., Kim, H.K., Choi, Y.H., Verpoorte, R., 2010. Glucosinolate profiling of Brassica

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