Phenolic Profile and in Vitro Antioxidant Capacity of Insoluble Dietary

Article pubs.acs.org/JAFC

Phenolic Profile and in Vitro Antioxidant Capacity of Insoluble Dietary Fiber Powders from Citrus (Citrus junos Sieb. ex Tanaka) Pomace as Affected by Ultrafine Grinding Bingbing Tao,† Fayin Ye,† Hang Li,‡ Qiang Hu,§ Shan Xue,† and Guohua Zhao*,†,# †

College of Food Science, Southwest University, Chongqing 400715, People’s Republic of China Sichuan Provincial Institute for Food-Drug Control, Chengdu 610097, People’s Republic of China § Leshan Product Quality Supervision and Testing Institute, Leshan 614000, People’s Republic of China # Food Engineering and Technology Research Centre of Chongqing, Chongqing 400715, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: The effects of mechanical and jet grindings on the proximate composition, phenolics, and antioxidant capacity of insoluble antioxidant dietary fiber powder from citrus pomace (IADFP-CP) were investigated in comparison with ordinary grinding. IADFP-CP from jet grinding showed higher levels of crude fat, total sugar, and free phenolics and lower levels of crude protein and bound phenolics than that from ordinary grinding. Totally, 14 phenolics (9 free, 1 bound, and 4 free/bound) in IADFP-CP were identified by RP-HPLC-DAD/ESI-Q-TOF-MS/MS. Hesperidin accounted for >57% of total phenolics in IADFP-CP. Among IADFP-CPs, the jet-ground presented the highest free phenolics but the lowest bound phenolics. The IADFP-CP from jet grinding presented the highest antioxidant capacity of free phenolics (by DPPH and FRAP assays), followed by the ones from mechanical and then ordinary grinding. The present study suggests that jet grinding could improve the extraction of phenolic compounds from IADFP-CP and increase the antioxidant capacities of free phenolics and the resultant powder. KEYWORDS: citrus pomace, insoluble antioxidant dietary fiber, ultrafine grinding, phenolics, RP-HPLC-DAD/ESI-Q-TOF-MS/MS

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pomace is commonly used as animal feed or fertilizer.12 If not properly processed, a considerable quantity of the citrus pomace could become waste and a possible source of environmental pollution.14 DF, mainly IDF, is the main component in citrus pomace (79.10 ± 0.90 g/100 g dw) as determined in our laboratory. The phenolic content in citrus pomace is about 0.10−31.62 mg GAE/g dry peel weight.15,16 It is reported that, due to the presence of associated compounds (i.e., flavonoids), DF from citrus fruits has a comparative advantage over that from other sources.13 In this context, citrus pomace may be a good source of insoluble antioxidant dietary fiber. Micronization is an effective way to improve the functional and physical properties of food ingredients.17 Ultrafine grinding has the ability to decrease the particle size of food ingredients in the range from 1 nm to 100 μm.18 Previous investigations confirmed that ultrafine grinding could substantially affect the solubility, fluidity, and hydration properties of dietary fibers from carrot (Daucus carota) and mushroom (Lentinus edodes).17,19 However, little information is available concerning the phenolic composition and antioxidant capacity of IADFPCP and also their dependence on ultrafine grinding. Thus, the objectives of the present study were (i) to identify and quantify

INTRODUCTION Antioxidant dietary fiber (ADF), first proposed by SauraCalixto, is regarded as an abundant source of phenolic antioxidants such as phenolic acids (ferulic acid, caffeic acid, etc.), flavonoids (hesperidin, catechins, etc.), stilbenes, and tannins.1,2 In recent years, ADF has attracted increasing interest.3−5 In ADF, antioxidative compounds were associated with dietary fiber (DF) by various interactions, including hydrogen bonds, covalent bonds, and hydrophobic interactions.1 Commonly, DF has been classified into soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) on the basis of its solubility in water. Due to the digestion resistance of DF by human gastrointestinal enzymes, those phenolics associated with ADF cannot be absorbed in the small intestine but can be released by bacterial enzymes in the hindgut.1,6 The released phenolics were further metabolized by the gut flora and generated their protective health effects.7 Therefore, ADF is recently considered as a natural carrier to deliver dietary antioxidants, especially phenolics, into the hindgut.1,6 Recently, ADFs from various byproducts of plant foods, including grape pomace, apple peel, citrus peel and pulp, carrot peel, and cereal bran, were investigated.3,8−12 The chemical composition and antioxidant properties of ADF are dependent on the raw material and processing method applied in its production.6 Citrus pomace is the main byproduct of orange juice production accounting for up to 50% of fruit input.10 It is a good source of bioactive ingredients, such as essential oil, pectin, phenolics, limonene, and DF.13 Nowadays, citrus © XXXX American Chemical Society

Received: April 7, 2014 Revised: June 22, 2014 Accepted: June 22, 2014

A

dx.doi.org/10.1021/jf501646b | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

concentrated hydrochloric acid. Hexanes were used to remove lipids in the mixture. The remaining mixture was then successively extracted with ethyl acetate (20 mL with 5 min) five times. The ethyl acetate fractions were combined and evaporated at 45 °C to dryness. The phenolics were reconstituted in 25 mL of methanol, filtered through 0.45 μm nylon membrane, and frozen at −80 °C until analysis. The determination of phenolics in the extracts was conducted on an Agilent 1260 HPLC (Agilent Technologies, USA) equipped with a diode array detector (DAD). The UV−vis spectra of phenolics were recorded in the range 190−600 nm at an acquisition rate of 2.0 s (peak width = 0.1 min). The phenolics were separated by a reverse phase Thermo BDS Hypersil C18 column (250 × 4.6 mm i.d.; 5 μm) at 30 °C with the mobile phase consisting of A (0.1% ammonium acetate) and B (acetonitrile) at a flow rate of 0.8 mL/min. A linear gradient program was used: 0−3 min, 10% B; 3−15 min, 10−30% B; 15−35 min, 30−50% B; 35−45 min, 50−90% B; 45−48 min, 90−10% B; 48− 55 min, 10% B. Each sample injection volume was 20 μL. Absorbance at 280 nm was applied to quantify the eluted phenolics. When a reference compound was not available, the calibration of a structurally related compound was employed with a molecular weight correction factor.23 The HPLC method applied in the present study was validated and given acceptable values in the correlation coefficients of calibration curves (R2 ⩾ 0.9990) and recovery (⩾80.6%) (Table S1 in the Supporting Information). Identification of Phenolics by RP-HPLC-DAD/ESI-Q-TOF-MS/ MS. To identify phenolics in the extracts, an Agilent 1290 HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with a binary pump (G4220A), a column compartment (G1316C), a DAD (G4212A), and an autosampler (CTCPAL) was coupled to an Agilent 6540 Q-TOF-MS/MS and applied in the present study. The RPHPLC-DAD was operated with parameters as described above. The effluent from the column was split using a T-type phase separator before being introduced into the mass spectrometer (split ratio = 1:3). The dual electrospray ionization (ESI) source of the MS was operated in negative mode. Mass spectra were recorded across the m/z range of 100−1100 (scan rate, 1.00 spectra/s). The operating conditions of QTOF-MS were as follows: desolvation gas (N2) temperature, 350 °C; desolvation gas flow rate, 480 L/h; nebulizer, 35 psig; capillary voltage, 3500 V; fragmentor voltage, 125 V; skimmer voltage, 65 V; Oct RFV, 750 V. The parameters of Q-TOF-MS/MS were the same as those for Q-TOF-MS except that the first quadrupole served as a mass filter and the sample injection volume was set as 10 μL. The mass spectra of all analytes were recorded at four collision energies: 10, 20, 30, and 40 V. The mass spectrum with a medium abundance for [M − H]− and high abundances for ionized fragments was selected and used to identify analytes. The whole system was controlled by MassHunter workstation software (B.05.00, Agilent Technologies). Antioxidant Capacity of IADFP-CP. DPPH Radical Scavenging Capacity (DPPH-RSC) Assay. The DPPH-RSC of IADFP-CP was determined according to the method described by Gadow et al. with some modifications.24 Briefly, 20 mg of IADFP-CP was weighed into a 10 mL centrifuge tube, and then 3.5 mL of methanol containing DPPH radicals (6 × 10−5 mol/L) was added. The tube was tightly capped and vortexed vigorously for 3 min and repeated twice with a 10 min interval. Then, it was subjected to centrifugation at 10000 rpm for 2 min. The control was prepared by replacing the phenolic extract with the same volume of methanol. The absorbance of the supernatant was recorded at 517 nm using a visible spectrophotometer (722, Shanghai Modern Science Spectral Instrument Co. Ltd., Shanghai, China) against the control. The measurement was performed in the dark and completed within 30 min. Results were expressed as micromoles of trolox equivalent (TE) per 100 g of dw of IADFP-CP. Ferric Reducing Antioxidant Power (FRAP) Assay. The FRAP of IADFP-CP was determined with the procedure described by Benzie et al. with some modifications.25 In detail, a fresh FRAP working solution was prepared by blending 25 mL of 300 mmol/L acetate buffer (pH 3.6), 2.5 mL of 10 mmol/L TPTZ in 40 mmol/L hydrochloric acid, and 2.5 mL of 20 mmol/L ferric chloride. To obtain an acceptable decoloring rate for FRAP assay, IADFP-CP was diluted with microcrystalline cellulose at a ratio of 1:4 (w/w). The diluted

phenolics in IADFP-CP and (ii) to explore the effects of ultrafine grinding on the antioxidant capacity and phenolic composition of IADFP-CP.

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MATERIALS AND METHODS

Materials and Chemicals. Fresh citrus (Citrus junos Sieb. ex Tanak) pomace was gifted by the Citrus Research Institute of the Chinese Academy of Agricultural Sciences (Chongqing, China). Chlorogenic acid, ferulic acid, caffeic acid, sinapic acid, gallic acid, naringenin, hesperitin, naringin, hesperidin, and kaempferol (>98.0%) were purchased from Chengdu Biopurify Phytochemicals Co. Ltd. (Chengdu, China). (±)-6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) and 2,4,6-tris(2-pyridyl)-1,3,5-triazine (TPTZ) were obtained from Sigma-Aldrich Co. Ltd. (Shanghai, China). 2,2Diphenyl-1-picrylhydrazyl (DPPH) was purchased from Wako Pure Chemical Industries, Co. Ltd. (Tokyo, Japan). Methanol, acetonitrile, ammonium acetate, hexyl hydride, and ethyl acetate of HPLC grade were purchased from Merck Co. Ltd. (Darmstadt, Germany). IADFP-CP Preparation. After the removal of seeds, fresh citrus pomace was shredded and immediately immersed in a water bath at 90 °C for 30 s to inactivate enzymes; then it was filtered, air-dried (65 °C, 48 h), and ground in a high-speed pulverizer (YS-103, Yongli Pharmaceutical Machinery Co. Ltd., Wenzhou, China) to pass through a 40-mesh sieve. After the resultant powder (350 g) had been defatted in 1.0 L of anhydrous ethanol at room temperature for 1 h, the solid part was recovered by filtering through four layers of cotton gauze. Then it was air-dried at 65 °C for 30 min to remove the solvent. The dried powder was subjected to the removal of soluble components by immersing it in 11.5 L of distilled water at 60 °C for 1 h with continuous stirring. The insoluble fraction was pelleted by centrifuging the suspension at 4000 rpm for 10 min. The pellet was air-dried (60 °C for 48 h) to achieve a moisture content below 6%, and the IADFPCP was obtained. The dried IADFP-CP was subjected to ordinary grinding, which was performed with a YS-103 high-speed pulverizer. The pulverization process lasted for 3 min. Ultrafine Grinding of IADFP-CP. The powder obtained by ordinary grinding was used as the starting material for ultrafine grinding (mechanical grinding and jet grinding). The mechanically ground IADFP-CP was prepared in a YSC-701 micronizer (Yanshanzhengde Co. Ltd., Beijing, China) for 8 min by operating the lapping wheel and fan at 4500 and 3500 rpm, respectively. To perform jet grinding, a CBF-250 jet mill (Chengdu, China) was applied and operated at the following parameters: compressor power, 210 kW; nozzle speed, 1 Mach; and rotational speed of classifier, 2100 rpm. The powders obtained by ordinary grinding, mechanical grinding, and jet grinding were denoted by OGP, MGP, and JGP, respectively. All powders were hermetically packaged in plastic bags and stored at room temperature in a brown desiccator with oxygen scavenger until analysis. The particle size was measured with a Mastersizer 2000 E laser particle size analyzer (Malvern Instruments Ltd., UK). In terms of average particle size (D0.5), OGP, MGP, and JGP presented particle sizes of 347.25, 59.56, and 24.09 μm, respectively. Determination of Proximate Composition. Ash, crude fat, and crude protein were determined using AOAC methods.20 Total sugar was determined by anthrone colorimetry.21 HPLC Analysis of Phenolic Extracts. Free and bound phenolics in IADFP-CPs were extracted according to the method previously reported by Okarter et al.22 Free phenolics were extracted by blending the powder (1 g) with 50 mL of 80% chilled acetone for 10 min. After centrifugation at 4500 rpm for 10 min, the supernatant was removed. The residues were reextracted as done for the powder. The supernatants were combined and evaporated at 45 °C to dryness. The remainder was made up to 25 mL with methanol, filtered through 0.45 μm nylon membrane, and frozen at −80 °C until analysis. Bound phenolics were extracted with the residue from the free phenolic extraction as starting material. The residue was first digested with 2 mol/L sodium hydroxide at room temperature for 1 h while shaking under nitrogen. The mixture was then neutralized with B



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Table 1. Proximate Composition and Phenolics in Insoluble Antioxidant Dietary Fiber Powders from Citrus Pomacea component ash crude fat crude protein total sugar free phenolics bound phenolics total phenolics

OGP (g/100 g dw) 3.07 2.31 5.28 2.71 1250.41 589.69 1845.10

± ± ± ± ± ± ±

MGP (g/100 g dw)

0.08b 0.10b 0.18c 0.12b 16.26c 9.46c 25.72c

3.15 6.22 4.99 2.78 1022.01 680.01 1712.01

± ± ± ± ± ± ±

0.15b 0.27c 0.24bc 0.12b 7.01b 9.59d 16.76b

JGP (g/100 g dw) 3.10 10.33 4.67 3.19 1412.74 483.46 1894.2

± ± ± ± ± ± ±

0.09b 0.97d 0.18b 0.16c 10.95d 3.26b 13.21c

a Data in the same row bearing different lower case letters are significantly different (p < 0.05). OGP, ordinary-ground powder; MGP, mechanicalground powder; JGP, jet-ground powder.

Figure 1. Base peak chromatograms of free (A, jet grinding) and bound (B, mechanical grinding) phenolics in insoluble antioxidant dietary fiber powders from citrus pomace.

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IADFP-CP (20 mg) was weighed into a 10 mL centrifuge tube and then 3.5 mL of fresh FRAP working solution was added. The suspension was shaken and kept in a water bath at 37 °C for 5 min, followed by centrifugation at 10000 rpm for 5 min. The absorbance of the resultant supernatant was monitored at 593 nm using a 722 visible spectrophotometer against FRAP working solution. All solutions were used on the day of preparation. Results were expressed as micromoles of FeSO4·7H2O equivalent (FE) per 100 g of dw of IADFP-CP. Antioxidant Capacity of Phenolic Extracts. The DPPH and FRAP assays of free and bound phenolic extracts were performed as done with IADFP-CP by omitting vortex and centrifugation treatments. Statistical Analysis. Results were presented as the mean value ± standard deviation (SD) of triplicate determinations. ANOVA and LSD comparison tests were performed to identify differences between values using SPSS (version 19.0, SPSS Inc., Chicago, IL, USA). Significant differences were declared at p < 0.05.

RESULTS AND DISCUSSION Proximate Composition of IADFP-CP. The proximate composition of IADFP-CP is presented in Table 1. In contrast to ordinary grinding, mechanical grinding generated significant effects only on the contents of crude fat and free, bound, and total phenolics (p < 0.05), but jet grinding presented significant changes in the contents of crude fat, crude protein, total sugar, and free and bound phenolics (p < 0.05). The contents of crude fat, total sugar, free, and bound and total phenolics in MGP and JGP were significantly different (p < 0.05). With regard to the increases in crude fat induced by MGP and JGP and in total sugar induced by JGP, the increases in their extraction efficiencies from micronized powders were assumed to be factual. Ultrafine grindings effectively disrupted the cell structure of the citrus pomace and substantially increased the specific surface of IADFP-CP.18 The images of IADFP-CP recorded by scanning electron microscope confirmed this fact (Figure S1 in the Supporting Information). Furthermore, the C



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Table 2. Retention Time, Identification, UV Spectra, Mass Spectrometric Data, and Source of Phenolic Compounds in Insoluble Antioxidant Dietary Fiber Powders from Citrus Pomace peak

tRa (min)

identity e

MWb

[M − H]−c (m/z)

325 322

354.1 194.0

353.1 193.0

283

594.2

593.2

284

650.4

649.4

224, 267, 334 223, 272

449.2 762.8

448.2 761.8

DAD λmax (nm)

fragment ion (m/z)

sourced

ref

191.1; 85.1; 93.1; 161.1; 127.1 134.0; 178.1; 117.1; 106.1; 149.1 285.0; 309.0; 338.0; 351.0

FO, FM, FJ FO, FM, FJ FO, FM, FJ

26

283.3; 255.3;

FO, FM, FJ

26

385.2; 445.2 290.9; 487.0;

FO, FM, FJ FO, FM, FJ

27 28

579.0

605.7; 347.2; 236.9 162.0; 223.1; 726.0; 696.0; 264.8 460.0; 271.1;

151.1; 119.1

610.0

609.0

301.1; 286.3

217, 280 203, 283

610.0 594.2

609.0 593.2

301.1; 286.3 285.0; 309.0; 338.0; 351.0

225, 282

294.2

293.2

236.1; 221.1; 71.0

naringenine

288

272.0

271.0

hesperitine feruloylquinic acid derivative

285 244, 328

302.1 530.2

301.1 529.2

151.0; 119.1; 107.1; 177.1; 93.1 164.1; 286.1 154.9; 162.0; 204.9; 248.9; 366.6

FO, FM, BJ FO, FM, BJ BO, BM FO, FM, BJ FO, FM, BJ FO, FM,

1 2

4.2 4.6

chlorogenic acid ferulic acide

3

9.5

4

10.5

5 6

11.2 11.5

kaempferol rutinoside isomer I galloyl HHDPf glucoside isomer sinapoyl hexoside derivative sinapic acid derivative

7

15.5

naringine

282

580.0

8

16.4

hesperidine

217, 280

9 10

17.7 20.5

11

21.7

hesperidin isomere kaempferol rutinoside isomer II unknown

12

23.2

13 14

24.2 31.6

FJ, BO, BM, FJ, BO, BM,

FJ, BO, BM,

26

FJ, BO, BM, FJ

FO, FM, FJ FO, FM, FJ

29−31

tR, retention time. bMW, molecular weight. c[M − H]−, quasi-molecular ion. dFO, free phenolics of ordinary-grinded IADFP-CP; FM, free phenolics of mechanical-grinded IADFP-CP; FJ, free phenolics of jet-grinded IADFP-CP; BO, bound phenolics of ordinary-grinded IADFP-CP; BM, bound phenolics of mechanical-grinded IADFP-CP; BJ, bound phenolics of jet-grinded IADFP-CP. eConfirmed with standards. fHHDP, hexahydroxydiphenoyl. a

For the identification of peak 4, the deprotonated molecule at m/z 649.4 was in agreement with the data of laggerstannin C (galloyl HHDP glucoside) reported in the literature.26 Moreover, the MS/MS fragment ions at m/z 605.7 and 283.3 of the peak were in agreement with MS3 fragment ion data of trisgalloyl HHDP glucoside.26 Regrettably, the characteristic fragment ions of galloyl HHDP glucoside at m/z 301 and 497 have been not detected in the mass spectrum of peak 4. Thus, we tentatively characterized peak 4 as galloyl HHDP glucoside isomer. Peak 5 at 11.2 min showed an [M − H]− ion at m/z 448.2 and MS/MS fragment ions at m/z 223.1, 162.0, and 385.2. According to the literature, the MS/MS fragment ion at m/z 223.1 resulted from the loss of a hexosyl unit (162.0 amu) from the sinapoyl hexoside moiety (385.2 amu).27 Therefore, peak 5 was tentatively assigned as sinapoyl hexoside derivative. Peak 6 at 11.5 min showed an [M − H]− ion at m/z 761.8. The MS/MS fragment ions at m/z 726, 487, and 290.9 were sinapic acid derivatives according to ref28. Moreover, the DAD profile of peak 6 was similar to that of sinapic acid. On the basis of their molecular ions, UV−vis spectra, and literature data, a sinapoyl unit was probably present in the compound, so peak 6 was assigned as a sinapic acid derivative. Peak 9 at 17.7 min showed identical UV−vis spectra and identical [M − H]− ion (at m/z 609.0) and MS/MS fragment (at m/z 301.1, hesperitin) patterns to hesperidin. Because the retention time of peak 9 did not match the retention time of hesperidin (16.4 min), peak 9 was tentatively assigned as a hesperidin isomer. Peak 11 (at 21.7 min), with an [M − H]− ion at m/z 293.2, was assigned as unknown. Although MS/MS fragments at m/z 236.1 (−57 amu, loss of −C(CH3)3) and m/z 221.1 (loss of a

results implied that jet grinding was more effective in promoting extraction efficiencies than mechanical grinding, which can be explained by the smaller particle size of JGP (24.09 ± 0.67 μm) than of MGP (59.56 ± 0.66 μm). Identification of Phenolics. The chromatographic profiles of free (jet grinding) and bound (mechanical grinding) phenolics in IADFP-CP are shown in Figure 1 (for the other four profiles see the Supporting Information, Figure S2). The RP-HPLC-Q-TOF-MS/MS in negative ionization mode allowed the separation and tentative identification of 14 phenolics (9 free, peaks 1−6 and 10−14; 1 bound, peak 9; and 4 free/bound, peaks 7, 8, 10, and 11) in IADFP-CP (Figure S3 in the Supporting Information). The retention time (tR), molecular weight (MW), quasi-molecular ion [M − H]−, and MS/MS fragment ions for each phenolic compound are summarized in Table 2. Among them, six phenolics including chlorogenic acid (peak 1), ferulic acid (peak 2), naringin (peak 7), hesperidin (peak 8), naringenin (peak 12), and hesperitin (peak 13) were identified using authentic reference compounds. Another eight detected phenolics were tentatively identified by comparing their UV−vis and mass spectra data with the literature data. Peak 3 and 10, showing an [M − H]− ion at m/z 593.2 and MS/MS fragment ions at m/z 285.0, 309.0, 338.0, and 351.0, were observed at 9.5 and 20.5 min, respectively. The MS/MS fragment ion at m/z 309 could be assigned as a rutinosyl group.26 However, fragment ions of glucosyl (−162 amu) or rhamnosyl (−146 amu) groups were absent in the MS/MS chart. In addition, the DAD profiles of peaks 3 and 10 were more similar to that of kaempferol than to that of luteolin. According to the evidence above, peaks 3 and 10 were assigned as kaempferol rutinoside isomers I and II, respectively. D



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Table 3. Free and Bound of Phenolics in Insoluble Antioxidant Dietary Fiber Powders from Citrus Pomace As Affected by Grinding Methodsa free phenolics (mg/100 g dw) compound

standards to quantify

phenolic acids and their derivatives chlorogenic chlorogenic acid acid ferulic acid ferulic acid galloyl HHDPc gallic acid glucoside isomer sinapoyl sinapic acid hexoside derivative sinapic acid sinapic acid derivative unknown sinapic acid feruloylquinic caffeic acid acid derivative total (calculation) flavanones and their derivatives naringin naringin hesperidin hesperidin hesperidin hesperidin isomer naringenin naringenin hesperitin hesperitin total (calculation) flavonols and their derivatives kaempferol kaempferol rutinoside isomer I kaempferol kaempferol rutinoside isomer II total (calculation)

OGP

MGP

bound phenolics (mg/100 g dw) JGP

OGP

MGP

JGP

1.88 ± 0.03d

1.88 ± 0.05d

2.15 ± 0.25e

ndb

nd

nd

0.70 ± 0.14d 15.00 ± 0.19d

1.13 ± 0.13e 15.09 ± 0.13d

1.13 ± 0.23e 19.58 ± 0.90e

nd nd

nd nd

nd nd

25.56 ± 1.06d

27.67 ± 0.84d

32.06 ± 0.57e

nd

nd

nd

6.49 ± 0.26d

6.04 ± 0.11d

7.10 ± 0.49d

nd

nd

nd

100.18 ± 3.11e 18.02 ± 1.05d

127.04 ± 1.26f 19.85 ± 0.37d

132.19 ± 1.76g 20.04 ± 0.69d

139.76 ± 1.80h nd

130.25 ± 4.33fg nd

76.59 ± 3.25d nd

167.83 ± 5.84g

198.7 ± 2.89f

214.25 ± 4.89f

139.76 ± 1.80f

130.25 ± 4.33e

76.59 ± 3.25d

113.20 ± 2.53g 891.33 ± 10.50g nd

144.18 ± 1.05h 583.68 ± 3.58f nd

184.30 ± 1.51i 927.93 ± 6.38h nd

41.75 ± 0.70e 355.35 ± 5.70d 26.13 ± 0.52e

53.00 ± 0.83f 464.43 ± 6.85e 1.16 ± 0.08d

36.07 ± 0.43d 338.73 ± 2.15d nd

0.83 ± 0.10d 0.85 ± 0.07d 1006.21 ± 13.21h

0.98 ± 0.28d 0.97 ± 0.04d 729.81 ± 4.88g

1.25 ± 0.28d 1.73 ± 0.12e 1115.21 ± 8.22i

nd nd 423.23 ± 6.99e

nd nd 518.59 ± 8.49f

nd nd 374.81 ± 2.58d

15.39 ± 0.32d

16.05 ± 0.18d

21.04 ± 0.58e

51.16 ± 0.75f

63.99 ± 2.02h

59.66 ± 1.5g7

25.09 ± 1.40d

31.15 ± 0.48e

29.71 ± 0.42e

66.55 ± 1.17f

80.04 ± 2.22g

81.72 ± 2.25g

25.09 ± 1.40d

31.15 ± 0.48e

29.71 ± 0.42e

nd

nd

nd

a

Data in the same row bearing different lower case letters are significantly different (p < 0.05). OGP, ordinary-grinded powder; MGP, mechanicalgrinded powder; JGP, jet grinded-powder. bnd, not detected. cHHDP, hexahydroxydiphenoyl.

methyl group from fragment at m/z 236.1) were observed, MS/ MS fragments at m/z 236.1 and 221.1 were unclear. Therefore, peak 11 cannot be identified. Peak 14 (at 31.6 min) showed an [M − H]− ion at m/z 529.2 and MS/MS fragments at m/z 154.9, 162, 204.9, 248.9, and 367, respectively. On the basis of the literature data, the fragment ion at m/z 367 resulted from the compound of peak 14 losing a caffeoyl moiety (−162 amu).29,30 In addition, the MS/MS fragment ion at m/z 154.9 suggested the presence of a feruloyl moiety.31 Therefore, the compound of peak 14 was assigned to feruloylquinic acid derivative. Accordingly, phenolics in IADFP-CP mainly consisted of phenolic acids, flavonoids, and their derivatives. All phenolic acids were identified as hydroxycinnamic compounds, including chlorogenic acid (peak 1), ferulic acid (peak 2), sinapic acid derivative (peak 6), peak 11, and feruloylquinic acid derivative (peak 14). Two classes of flavonoids, including five flavanones (peaks 7−9, 12, and 13) and two flavonols (peak 3 and 10), have been characterized. Among the 14 phenolic compounds characterized in the present study, 7 (peaks 1, 2, 7−9, 12, and 13) identified phenolics were reported in citrus peel in the literature.32−34 Quantification of Phenolics. The quantitative results of free and bound phenolics in IADFP-CP are presented in Table 3. The free phenolic content (1022.01−1412.74 mg/100 g dw)

was much higher than the bound phenolic content (483.46− 680.01 mg/100 g dw) in IADFP-CP regardless of the grinding method. The study done by Oboh et al. revealed that the free phenolic content of citrus peels (6.5−13.1 mg/g) was significantly higher than the bound phenolic content (0.7−6.8 mg/g).35 The total content of phenolic acids and their derivatives (328.95−290.84 mg/100 g) was much lower than that of flavonoids and their derivatives (1359.6−1600.42 mg/ 100 g). Among phenolic acids and their derivatives, peak 11 presented the highest abundance (257.29−208.78 mg/100 g) and was the unique bound phenolic acid derivative detected in all IADFP-CP samples. Meanwhile, in flavonoids and their derivatives, hesperidin (1048.11−1266.66 mg/100 g) was the most abundant monomer, which accounted for >70% (w/w) in both free and bound phenolics in all samples. It is well documented that flavonoids (mainly naringenin, hesperidin, naringin, and neohesperidin) were the predominant phenolics in citrus peels,32 and their total content highly exceeded the content of phenolic acids by up to 10 times or more.14,34 With regard to the effects of ultrafine grinding on free and bound phenolics in IADFP-CP, MGP and JGP presented a bound phenolic content comparable to that of OGP. Contrary to our expectations that the free phenolic content in IADFP-CP would be increased by ultrafine grinding, the free phenolic content in MGP was significantly lower than that in OGP. The possible E



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Figure 2. Antioxidant capacities (A, DPPH radical scavenging capacity; B, ferric reducing antioxidant power) of insoluble antioxidant dietary fiber powders from citrus pomace and their phenolic extracts as determined by the DPPH and FRAP assays. Columns bearing different lowercase letters (a−c) with the same sample form (powder, free and bound phenolic) were of significant differences (p < 0.05). Columns bearing different capital letters (A−C) with the same grinding method (ordinary (OG), mechanical (MG), and jet grindings (JG)) were of significant differences (p < 0.05).

our study) compared with mechanical grinding.18 Interestingly, the bound phenolic content in JGP was dramatically lower than that in OGP. Although the reasons for the result were not entirely understood, it is worthwhile noting that a redistribution of bound phenolics to free phenolics possibly happened during jet grinding.33,36 Antioxidant Capacity. Numerous studies demonstrated that the DPPH and FRAP assays are simple and cost-effective

explanation for the result would be the thermal damage of phenolics during mechanical grinding. This was evidenced by the fact that the free phenolic content in JGP was significantly greater than that in OGP. As reported, jet grinding is a good choice in producing thermally sensitive biomaterials. Jet grinding could produce impacting, colliding, and friction among materials by the energy of high-speed or steam and bring about less thermal energy (cavity temperature 32 °C in F



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research, the in vivo antioxidant activity of ultrafine ground IADFP-CP and their potential applications in food systems should be intensively addressed.

spectrophotometric methods to evaluate the potential antioxidant properties of biomaterials.37,38 Therefore, they were applied in the present study to evaluate the antioxidant capacities of IADFP-CP and their phenolic extracts. The obtained results are presented in Figure 2. With regard to DPPH-RSC (Figure 2A), in powder form, OGP, MGP, and JGP presented antioxidant values of 131.93, 172.44, and 179.12 μmol TE/100 g dw, respectively. By comparison, the DPPH-RSCs of MGP and JGP were significantly higher than the value of OGP, but no significant difference was observed between the values of MGP and JGP. Te effects of ultrafine grinding on the DPPH-RSC of free phenolics in IADFP-CP revealed that jet grinding significantly improved the antioxidant capacity, whereas mechanical grinding did not significantly change it. Commonly, the decrease in particle size of a powder could facilitate the extraction of antioxidants and increase their activity. This has been confirmed in numerous researche studies.39,40 However, this law was not effective in comparing the DPPH-RSCs of OGP and MGP. MGP presented a significantly smaller particle size than OGP, but generated an antioxidant value equivalent to that of OGP. This discrepancy was possibly due to the thermal effects of mechanical grinding, which damaged the free phenolic components and thus deteriorated their antioxidant capacities.18 Without exception, ultrafine grindings significantly decreased the antioxidant capacity of bound phenolics (p < 0.05). This should be related to the differences in total bound phenolics induced by the various grindings as mentioned above. With regard to FRAP (Figure 2B), similar results were obtained as with DPPH-RSC. Moreover, significant differences were observed between the antioxidant values of MGP and JGP powder and those from free phenolics in OGP and MGP. The antioxidant capacity of OGP was lower than that of MGP and JGP, which may be due to the enhanced solubilization of antioxidants, including phenolic and nonphenolic substances, by the decrease in particle size.19,41 The antioxidant capacities of both ultrafine grinding powders and their phenolic extracts followed the order of powder > free phenolic extract > bound phenolic extract. This sequence reflected that the antioxidant capacity of the powder came not only from its phenolic components but also from other nonphenolic components, such as carotenoids and Maillard compounds.1 The differences in the antioxidant capacities of free and bound phenolic extracts could be explained by the higher phenolic content in free extracts than in bound extracts, which is reflected by the high correlation coefficients of the relationships between the antioxidant values and phenolic content (r ⩾ 0.8418) (Figure 2). Likewise, the DPPH-RSC (r = 0.939) and FRAP (r = 0.906) of 30 plant extracts were found highly dependent on their total phenolic contents.42 The free and bound phenolics in IADFP-CP and their changes induced by two kinds of ultrafine grindings (mechanical grinding and jet grinding) were first investigated in the present study. Fourteen phenolic compounds, mainly including phenolic acids, flavonoids, and their derivatives, were identified and quantified. The free phenolic content was much higher than the bound phenolic content in IADFP-CP. Hesperidin was identified as the component with the highest abundance, which accounted for >57% of total phenolics. As far as the protective effects on phenolics, the redistribution of bound phenolics to free phenolics, and the antioxidant capacity of powders were concerned, jet grinding was superior to mechanical grinding in producing IADFP-CP. For further

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S3 and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(G.Z.) Phone: +86 23 68 25 03 74. Fax: +86 68 25 19 47. Email: [email protected]. Funding

This work was supported by the National High-tech R&D Program (863 Program) of China (2011AA100805-2) and the National Natural Science Foundation of China (31371737). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The citrus pomace was kindly supplied by the Citrus Research Institute of the Chinese Academy of Agricultural Sciences. We are grateful for the helpful assistance from Dr. Yong Tang (College of Food Science, Sichuan Tourism University) in jet grinding.

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ABBREVIATIONS USED ADF, antioxidant dietary fiber; DPPH, 2,2-diphenyl-1-picrylhydrazyl; DPPH-RSC, DPPH radical scavenging capacity; DF, dietary fiber; [M − H]−, deprotonated molecule; dw, dry weight; FEAC, FeSO4·7H2O equivalent antioxidant capacity; FRAP, ferric reducing antioxidant power; GAE, gallic acid equivalent; HHDP, hexahydroxydiphenoyl; IADFP-CP, insoluble antioxidant dietary fiber powder from citrus pomace; IDF, insoluble dietary fiber; JGP, jet ground powder; MGP, mechanical grinded powder; MW, molecular weight; OGP, ordinary ground powder; tR, retention time; TEAC, trolox equivalent antioxidant capacity

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Phenolic Profile and in Vitro Antioxidant Capacity of Insoluble Dietary

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