Characterization of the products
The two products used in this study (red grape skin extract (RG) and AH) were manufactured by ILSA (Arzignano-Vicenza, Italy). AH was produced by a fully controlled enzymatic hydrolysis using vegetal material from alfalfa (Medicago sativa L.) plants. The elemental composition, organic matter content, and physical properties of AH were previously reported by Schiavon et al. . Specifically, the water percentage in AH was 43.5% (v/v), and the amount of organic matter was low 37.6% (w/v). The content of ash and the electrical conductivity (ECw) were both high, with values of 18.9% (w/v) and 16 dS m-1, respectively. The percentage of inorganic nitrogen in the form of ammonia and nitrates was low (0.38% and 0.03 w/w, respectively), whereas the total amount of free amino acids was high up to 1.916% (w/w) and correlated with the free α-amino nitrogen (α-NH2-N). The hydrolytic process employed for EM production was effective in the weight-average molecular weight (MW) reduction, as confirmed by data on EM hydrolysis degree (DH) and MW.
RG was obtained from the skin of red grape wine via cool extraction according to the method of Machado . RG aqueous extract was prepared by extracting 10 g of dried ground plant material with 50 mL of deionized water in a shaker for 2 h at room temperature. The extract was then filtered with cellulosic membrane filters at 0.8 μm (Membra-Fil® Whatman Brand, Whatman, Milan, Italy). The pH was determined in water (3:50 w/v). Total organic carbon (TOC) was measured using an element analyzer (varioMACRO CNS, Hanau, Germany).
Total phenols in RG extracts were determined according to Arnaldos et al. . Specifically, RG (1 mL) was maintained in ice with pure methanol (1:1, v/v) for 30 min and centrifuged at 5,000g for 30 min at 4°C. One mL of 2% Na2CO3 and 75 μL of Folin-Ciocalteau reagent (Sigma-Aldrich, St. Louis, MO, USA) were added to 100 μL of phenolic extract. After 15 min of incubation at 25°C in the dark, the absorbance at 725 nm was measured. Gallic acid was used as standard according to Meenakshi et al. .
For reducing sugars determination, a sample of RG product was dried for 48 h at 80°C, ground in liquid nitrogen, and then 100 mg were extracted with 2.5 mL 0.1 N H2SO4. Samples were incubated in a heating block for 40 min at 60°C and then centrifuged at 6,000g for 10 min at 4°C. After filtration (0.2 μm, Membra-Fil® Whatman Brand, Whatman, Milan, Italy), the supernatants were analyzed by HPLC (Perkin Elmer 410, Perkin Elmer, Waltham, MA, USA). The soluble sugars were separated through a Biorad Aminex 87 C column (300 × 7.8 mm; Bio-Rad Laboratories, Inc., Hercules, CA, USA) using H2O as eluent at a flow rate of 0.6 mL min−1.
Indole-3-acetic acid (IAA) in the RG was determined using an enzyme-linked immuno-sorbent assay (ELISA) standardized with methylated IAA (Phytodetek-IAA, Sigma, St. Louis, MO, USA). The ELISA test utilized a monoclonal antibody to IAA and was sensitive in the 0.05-to-100 pmol range. The tracer and standard solutions were prepared following the manufacturer’s instructions, and absorbances were read at 405 nm with a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Isopentenyladenosine (IPA) in RG and AH was determined by ELISA, a Phytodetek-IPA with an anti-IPA monoclonal antibody was used (Sigma-Aldrich, St. Louis, MO, USA). The competitive antibody-binding method was adopted to measure the IPA concentration. IPA labeled with alkaline phosphatase (tracer) was added with the sample to antibody coated microwells. A competitive binding reaction was set up between a constant amount of the IPA tracer, a limited amount of the antibody and the unknown sample containing IPA (Sigma-Aldrich, St. Louis, MO, USA). One hundred μL of standard IPA concentration or serial dilutions of RG and AH and 100 μL diluted tracer were added to each well. For the standard curve, progressions of 100, 50, 20, 5, 1, 0.1, and 0.02 pmol IPA 100 μL−1 were used, whereas for RG and AH, the progressions were 20, 10, 7.5, 5.0, 3.5, and 2.5 μg C 100 μL−1. After incubation at 4°C for 3 h, the wells were decanted and any unbound tracer was washed out by adding 200 μL of washing solution before adding 200 μL of substrate solution for colorimetric detection. After 60 min at 37°C, the optical density (OD) was read at 405 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Experimental design and growth conditions
The experimental trial was derived from the factorial combination of three types of treatment: no biostimulant, a cool extract from RG, and an AH. Two doses (50 and 100 mL L−1 for RG, and 25 and 50 mL L−1 for AH) of biostimulants were applied to two randomized blocks of pots, each consisting of 50 pots (ten pots per treatment to test biostimulants at flowering stage and ten pots per treatment to test biostimulants at maturity stage). The two doses for each biostimulant were in accordance with previous tests showing that doses in the 1-to-200 mL L−1 range were those that more interfered with pepper metabolism. Each pot was filled with 2 L perlite/vermiculite mixture. At the beginning of the trial, 7-day-old seedlings of chili pepper (Capsicum chinense L. cv. Fuoco della Prateria) were transplanted at a density of one plant per pot. Plants were grown until maturity in a tunnel maintained at 25°C/15°C day/night, receiving natural light. Plants were treated twice, i.e., at the second and fourth week after transplanting, by spraying each one with 4.5 mL of RG or AH on the leaves. All treatments were irrigated with a half-strength Hoagland’s nutrient solution. Plants from 50 pots were sampled at flowering (4 weeks after transplanting), and plants from the remaining 50 pots at maturity (6 weeks after transplanting), harvesting leaves and fruits separately.
Analysis of sugars in plant material
Leaf and fruit samples (2 g) were homogenized in water (20 mL) with an Ultra Turrax T25 (IKA, Staufen, Germany) at 13,500 rpm until uniform consistency. Samples were filtered with filter paper (589 Schleicher) and further filtered through cellulose acetate syringe filters (0.45 μm). The analysis of the extracts was performed using an HPLC apparatus (Jasco X.LC system, Jasco Co., Tokyo, Japan) consisting of a model PU-2080 pump, a model RI-2031 refractive index detector, a model AS-2055 autosampler and a model CO-2060 column oven. ChromNAV Chromatography Data System was used as software. Sugars were separated on a HyperRez XP Carbohydrate Ca++ analytical column (7.7 × 300 mm, ThermoScientific, Waltham, MA, USA) operating at 80°C. Isocratic elution was effected using water at a flow rate of 0.6 mL/min. The peaks were identified by comparing the retention time with those of standard compounds. To calculate the concentrations in the extract, a calibration curve was drawn for four solutions of known concentration in water.
Determination of total phenols in plant material
The concentration of total phenols was determined by the Folin-Ciocalteau (FC) assay with gallic acid as calibration standard, using a Shimadzu UV-1800 spectrophotometer (Shimadzu Corp., Columbia, MD, USA). The FC assay was performed by pipetting 200 μL of plant extract (obtained as described above for sugars analysis) into a 10-mL PP tube. This operation was followed by addition of 1 mL of Folin-Ciocalteau’s reagent. The mixture was vortexed for 20 to 30 s. Eight hundred microliters of sodium carbonate solution (20% w/v) was added to the mixture 5 min after the addition of the FC reagent. This was recorded as time zero; the mixture was then vortexed for 20 to 30 s after addition of sodium carbonate. After 2 h at room temperature, the absorbance of the colored reaction product was measured at 765 nm. The total phenols concentration in the extracts was calculated from a standard calibration curve obtained with different concentrations of gallic acid, ranging from 0 to 600 μg mL−1. Results were expressed as milligrams of gallic acid equivalent per kilogram of fresh weight .
Determination of total antioxidant activity by ferric reducing antioxidant power
The assay was based on the methodology of Benzie and Strain . Ten grams of plant material was homogenized in 20 mL of HPLC grade methanol using an Ultra-Turrax tissue homogenizer (Takmar, Cincinnati, OH, USA) at moderate speed (setting of 60) for 30 s. The ferric-reducing antioxidant power (FRAP) reagent was freshly prepared, containing 1 mM 2,4,6-tripyridyl-2-triazine (TPTZ) and 2 mM ferric chloride in 0.25 M sodium acetate buffer at pH 3.6. One hundred microliters of the methanol extract was added to 1,900 μL of FRAP reagent and accurately mixed. After leaving the mixture at 20°C for 4 min, the absorbance was determined at 593 nm. Calibration was against a standard curve (0 to 1,200 μg mL−1 ferrous ion) obtained by the addition of freshly prepared ammonium ferrous sulfate. FRAP values were calculated as microgram per milliliter ferrous ion (ferric-reducing power) and are presented as milligram per kilogram of Fe2+E (ferrous ion equivalent).
Extraction and determination of ascorbic acid
After harvest, the samples were immediately stored at −80°C before analysis. Five-gram samples were homogenized until uniform consistency in a meta-phosphoric acid and acetic acid solution. This solution was used for the ascorbic acid extraction after quantitative reduction of 2,6-dichlorophenolindophenol dyestuff by ascorbic acid and extraction of the excess dyestuff using xylene. The excess of ascorbic acid was measured at 500 nm in a Shimadzu UV 160A spectrophotometer (Shimadzu Corp., Columbia, MD, USA) and compared with a vitamin C reference standard (ISO/6557-2-1984 method).
Capsaicin and dihydrocapsaicin determination
Fresh leaves and green and red peppers (2 g) were extracted with 20 mL of acetone, followed by homogenization with an Ultra-Turrax T 25 (IKA, Staufen, Germany) for 30 s at 17,500 rpm. The extract was filtered with filter paper and then through regenerated cellulose syringe filters (0.45 μm). The analysis of the extracts was performed using an HPLC system (X-LC Jasco Co., Tokyo, Japan) equipped with a DAD detector (MD-2015, Jasco Co., Tokyo, Japan) and autosampler (AS-2055, Jasco Co., Tokyo, Japan). Samples (20 μL injection volume) were separated on a Tracer Extrasil ODS-2 (250 × 45 mm, 5 μm, Teknokroma, Barcelona, Spain) HPLC column. The mobile phase consisted of two solvents: water (A) and methanol (B) (50:50, v/v). Isocratic elution for 10 min was used, followed by gradient elution 50% to 90% B for 10 min. The flow rate was 1 mL/min and column temperature was 25°C. Detection was set at 278 nm. To calculate the concentrations in the extract, a calibration curve was drawn for four solutions of known concentration in acetone.
The data represent the means of measurements from five plants per treatment, each representing one biological replicate. Analysis of variance (ANOVA) was performed using the SPSS for Windows software, version 18.0 (SPSS, Chicago, IL, USA) and was followed by pair-wise post hoc analyses (Student-Newman-Keuls test) to determine which means differed significantly at p < 0.05.