Chemicals and raw material
CA and Folin − Ciocalteu phenol reagent were purchased from Sigma-Aldrich Chemical Co, Ltd, (St. Louis, MO, USA). EC, ECG, procyanidin B1, procyanidin B2 were purchased from Chengdu Manset Biotechnology Co, Ltd, (Chengdu, Sichuan, China). Procyanidin B3 and procyanidin C1 were purchased from Shanghai yuanye Bio-Technology Co, Ltd, (Shanghai, China). Cinnamtannin A2 was purchased from Shanghai ZZBIO Co, Ltd, (Shanghai, China). Gallic acid (GA), 2–20-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) were purchased from TCI (Shanghai) Development Co, Ltd, (Shanghai, China). Vanillin, benzyl mercaptane, 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Shanghai Macklin Biochemical Co, Ltd, (Shanghai, China). T-AOC Assay Kit was provided by Beyotime Biotechnology Co, Ltd, (Shanghai, China). Unless stated otherwise, the solvents used for chromatography were of high-performance liquid chromatography grade (HPLC-grade) and the other chemicals were of analytical reagent grade (AR-grade). Water was purified using a Milli-Q water purification system (Milford, MA, USA).
Grape seeds of “Chardonnay” were purchased from Xi'an Haoxuan Biotechnology Co, Ltd. The grape seeds were washed with deionized water for 3 times, then they were ground into powder by a high-speed blender and freeze-dried at − 40 °C in a vacuum freeze drier (SCIENTZ-18 N, Sunway Hanguang Electric Manufacturing Limited, Ningbo, China). The freeze-dried powder of grape seeds was sieved with a 20-mesh sieve, then sealed with N2 and kept in -20 ºC for further experiments.
Steam explosion pretreatment, structural observation and procyanidins extraction
SE was carried out according to Zhang et al (2014). 100 g of freeze-dried grape seeds powder was mixed with 200 mL deionized water and the mixture was kept for 60 min at room temperature. The mixture was put into the SE equipment and pretreated at the combination of different pressures (0.3, 0.6, 0.9, 1.2 or 1.5 MPa) and different time-courses (30 s or 60 s), and then the pressure was reduced abruptly to atmospheric pressure. After SE, the pretreated grape seeds were collected and freeze-dried.
The unexploded and exploded samples were treated by dehydration, drying and coating with gold. Then, these samples were analyzed by scanning electron microscopy (SEM), (Japan Electronics Co, Ltd, Japan), which was operated at an accelerating voltage of 15 kV.
The extracting process of procyanidins was modified according to previous reports (Gu et al. 2002; Hellström and Mattila 2008). Two grams of freeze-dried unexploded and exploded samples were weighed. Then, they were defatted for 12 h with 20 mL hexane in 50 mL centrifuge tubes. After centrifugation at 9000 rpm for 10 min, the supernatants were discarded. The centrifuge tubes were placed into fume hood for 12 h to remove residual hexane. One gram of defatted samples was mixed with 10 mL of acetone/water/acetic acid (70/29.5/0.5, v/v/v) mixture in a 50 mL centrifuge tubes. The tubes were vortexed for 30 s, and then kept at 37 ℃ for 10 min with ultrasound treatment. Procyanidins were extracted for 50 min at ambient temperature under 300 rpm shaking. After centrifugation at 9000 rpm for 15 min, 7.5 mL supernatants were pipetted out and filtered using a polypropylene filter membrane (0.45 µm) for further analysis.
Procyanidins content assessment
Procyanidins content assay was modified according to previous reports (Çam and Hışıl 2010), using an ultraviolet–visible V-5100B spectrophotometer (Shanghai Metash Instruments Co, Ltd, China). One milliliter of 60-fold diluted extract was first mixed with 2.5 mL of 1% vanillin in methanol solution, and then mixed with 2.5 mL of 25% H2SO4 in methanol solution. After keeping in water bath at 30 ℃ for 15 min, the absorbance of mixtures was read at 500 nm. Pure methanol was used as a blank. CA was used as a standard control, and the calibration curve has a good linear relationship at the range: 10–100 µg/mL (R2 = 0.9998). According to a calibration curve, the results were described as catechin equivalents (CE) (µg CA /g dry matter (DM)).
Total phenolic content (TPC) assessment
TPC was determined according to the early-reported method (Xu et al. 2014) using an ultraviolet–visible V-5100B spectrophotometer (Shanghai Metash Instruments Co, Ltd, China). One hundred microliters of 40-fold diluted extracts were placed into 50 mL centrifuge tubes. Subsequently, 3900 µL of distilled water, 250 µL of 2 mol/L Folin–Ciocalteau reagent and 750 µL of 20% Na2CO3 were added. The mixtures were kept in water bath for 15 min at 30 ℃. Then, the absorbance of mixtures was read at 760 nm. Pure methanol was used as a blank control, gallic acid was used as a standard control and the calibration curve has a good linear relationship at the range: 100–800 µg/mL (R2 = 0.9999). According to a calibration curve, the final results were described as gallic acid equivalents (GAE) (µg GA/g DM).
Mean degree of polymerization (mDP) assessment
The thiolysis of procyanidins was modified according to previous paper (Gu et al. 2002). Fifty microliters of extracts were mixed first with 50 µL 3.3% HCl in methanol solution in a 250-µL polypropylene insert, and then 100 µL 5% benzylmercaptan in methanol were added. The 250-µL polypropylene insert was placed into a 1.5-mL vial and the vial was quickly sealed with a cap. The mixtures were kept in water bath at 40 ℃ for 30 min, and then reacted at room temperature for 10 h for complete degradation. The final mixtures were kept at − 20 ℃ and measured by reversed phase high performance liquid chromatography (RP-HPLC). Thiolysis products of procyanidin B1, procyanidin B3, and the CA, CA benzylthioether, the EC, EC benzylthioether were used as standards to identify their counterparts (Furuuchi et al. 2011). Standard solutions for CA, EC, ECG, procyanidin dimers B1 and procyanidin dimers B3 were prepared at 1000 µg/mL.
Chromatographic analysis was modified according to previous papers (Gu et al. 2002). HPLC analysis was performed using a Shimadzu LC-20AT (Shimadzu, Japan) equipped with a UV-detector, a binary pump, an auto-sampler and a column compartment. Separation was carried out using a Diamonsil C18 column (5 μm, 250 × 4.6 mm i.d.) from Dikma Technologies (Beijing, China). The detection wavelength and column temperature were set at 280 nm and 35 ℃. The mobile phases were the mixture of solvent A (water/acetic acid = 98/2) and solvent B (methanol, 100%), which was developed as 0 min (A = 85%, B = 15%) → 45 min (A = 20%, B = 80%) → 50 min (A = 20%, B = 80%) → 55 min (A = 85%, B = 15%) → 70 min (A = 85%, B = 15%). The flow rate was set at 1.0 mL/min and the injection volume was set as 20 μL. Then, standard solutions were used to identify and quantify the samples. Although the presence of EGC-thiol and ECG-thiol was ignored in the early reports for mDP calculation (Furuuchi et al. 2011), their peaks were included in the present paper to increase the accuracy of the mDP calculation. The mDP of extracts can be calculated with the Eq. (1):
$$\mathrm{mDP}=\frac{Total\, area\, ratio\, of\, extension\, units\, benzlthioether}{Total\, area\, ratio\, of\, terminal\, units}+1$$
(1)
Measurements of antioxidant activity
DPPH• radical-scavenging capacity, ferric reducing power and ABTS•+ radical-scavenging capacity were used to analyze the antioxidant activity of extracts. Diluted extract was prepared at same concentration (µg CA/g DM).
The DPPH assay was modified according to the reported papers (Xu et al. 2014). One hundred microliters of diluted extracts were placed into the tube. Then, 3900 µL of 25 µg/mL DPPH in methanol solution was added. The mixtures were kept in water bath for 60 min at 30 ℃ in darkness. Finally, the absorbance of mixtures was read at 515 nm. The scavenging capacity of diluted extracts on DPPH can be calculated with the following equation:
$$\mathrm{Scavenging\,rate}=\frac{({A}_{0}-\mathrm{A})}{{A}_{0}}\times 100 (\%)$$
(2)
where A0 was the absorbance of blank control (methanol solution); A was the absorbance of the diluted extracts. Trolox was used as a standard control, and the calibration curve has a good linear relationship between scavenging rate and concentration of Trolox solutions at the range: 100–1000 µmol/L (R2 = 0.9996). According to a calibration curve, the final radical scavenging activities were described as Trolox equivalent (TE) (µmol T/g DM).
The FRAP assay was carried out with a T-AOC Assay Kit (Xia et al. 2017). The experiments of the FRAP assay was performed in 96-well polystyrene microplates. Pure methanol solution was used as a blank control. Trolox was used as a standard control, and the calibration curve indicated a significant linear relationship between ferric reducing power and concentration of Trolox solutions at the range: 150–1500 µmol/L (R2 = 0.9983). According to a calibration curve, the final ferric reducing power was described as TE (µmol T/g DM).
The ABTS assay was carried out according to early reports (Oldoni et al. 2016). The 7 mmol/L ABTS and 140 mmol/L potassium persulfate were reacted for forming ABTS•+ radical at 25 ℃ in darkness for 12 h. To get absorbance value of 0.70 at 734 nm, the prepared ABTS•+ radical solution was diluted with ethanol. Thirty microliters of diluted extracts were mixed with 3.0 mL of the diluted ABTS•+ radical solution and kept at 30 ℃ in darkness for 6 min. At the end, absorbance of mixture was measured at 734 nm. Pure ethanol was as a blank. The radical scavenging capacity of the diluted extracts on ABTS•+ can be calculated with the following equation:
$$\mathrm{Scavenging\, rate}=\frac{({A}_{0}-\mathrm{A})}{{A}_{0}}\times 100 (\%)$$
(3)
where A0 was the absorbance of blank; A was the absorbance of diluted extracts. Trolox was used as a standard control, and the calibration curve has a good linear relationship between scavenging rate and concentration of Trolox solutions at the range: 100–2000 µmol/L (R2 = 0.9959). According to calibration curve, the final radical scavenging activities was described as TE (µmol T/g DM).
NP-HPLC analysis of procyanidins
Chromatographic analysis was modified according to the early paper (Choy et al. 2013). Normal phase high performance liquid chromatography (NP-HPLC) analysis was carried out by a Shimadzu LC-20AT (Shimadzu, Japan). Separation was carried out on a Develosildiol 100 column (5 μm, 250 × 4.6 mm i.d.) from Nomura Chemical Co, Ltd, (Japan). The mobile phase was the mixture of solvent A (acetonitrile/water = 98/2, v/v) and solvent B (methanol/water/acetic acid = 95/3/2, v/v/v). The gradient elution program was developed as: 0 min (A = 93%, B = 7%) → 3 min (A = 93%, B = 7%) → 53 min (A = 62.4%, B = 37.6%) → 56 min (A = 0%, B = 100%) → 69 min (A = 0%, B = 100%) → 75 min (A = 93%, B = 7%) → 85 min (A = 93%, B = 7%). The flow rate was 1.0 mL/min and the injection volume was 20 μL. The detection wavelength and column temperature were set at 280 nm and 30 ℃. The peaks of procyanidins with different DP were assigned by comparing retention time with the standards (Additional file 1: Table S1) (Choy et al. 2013). Standard solutions of CA, EC, ECG, procyanidin B1, procyanidin B2, procyanidin B3, procyanidin C1 and Cinnamtannin A2 were prepared with concentration of 1000 µg/mL. The solutions of the standards were filtered using a polypropylene filter membrane (0.45 µm). One milliliter of 1 mg/mL CA, EC, ECG standards were mixed in screwed glass tubes to get procyanidin standards 1. One milliliter of 1 mg/mL procyanidin B1, procyanidin B2, procyanidin B3 standards were mixed to get procyanidin standards 2. One milliliter of 1 mg/mL CA, EC, ECG, procyanidin B1, procyanidin B2, procyanidin B3, procyanidin C1 and innamtannin A2 standards were mixed to get procyanidin standards 3. Standard solutions were used to quantify chemicals. Peak area growth ratio of procyanidins (RAGR) with different DP can calculated with the following equation:
$$\mathrm{RAGR}=\frac{{A}_{s-n}}{{A}_{u-n}}\left(\mathrm\,{n}=1,\mathrm{ECG}, 2, 3, 4, \ge 5\right)$$
(4)
where As-n was peak area of steam-exploded procyanidins with DP = n; Au–n was peak area of unexploded procyanidins with DP = n.
Statistical analysis
Statistical analysis was carried out with Origin 2017. Means were calculated using one sample t-Test. Significance level was set at P ≤ 0.05.