Article pubs.acs.org/JAFC
Digestibility of Glyoxal-Glycated β‑Casein and β‑Lactoglobulin and Distribution of Peptide-Bound Advanced Glycation End Products in Gastrointestinal Digests Di Zhao,† Lin Li,†,‡,§ Thao T. Le,⊥ Lotte Bach Larsen,⊥ Guoying Su,† Yi Liang,∥ and Bing Li*,†,‡ †
College of Food Science and Engineering, South China University of Technology, 381 Wushan Road, Tianhe District, Guangzhou, 510640, China ‡ Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, 381 Wushan Road, Guangzhou 510640, China § School of Chemical Engineering and Energy Technology, Dongguan University of Technology, College Road 1, Dongguan 523808, China ∥ Guangdong Chongqing Font Biochemical Sci & Tech. Co., Maoming 525427, China ⊥ Department of Food Science, Aarhus University, Blichers Allé 20, Tjele 8830, Denmark ABSTRACT: This work reports the influence of glyoxal (GO)-derived glycation on the gastrointestinal enzymatic hydrolysis of β-lactoglobulin and β-casein. Reduced digestibility of glycated proteins was found in both gastric and intestinal stage. Distribution of Maillard reaction products in digests with different molecular weight ranges was investigated subsequently. The colorless and brown MRPs largely presented in the digests smaller than 20 kDa. However, the resistance of fluorescent advanced glycation end products (AGEs) to enzymatic hydrolysis gradually increased during glycation, rendering fluorescent AGEs largely present in the digests larger than 20 kDa. No free N (ε)-carboxymethyllysine (CML) was detected in digests. The relative amount of CML in digests larger than 1 kDa was higher than that of Lys, demonstrating the hindrance of CML to enzymatic hydrolysis. This study highlights the resistance of GO-derived AGEs to digestive proteases via blockage of tryptic cleavage sites or steric hindrance, which is a barrier to the absorption of dietary AGEs. KEYWORDS: glycation, advanced glycation end products, digestibility, glyoxal, β-lactoglobulin, β-casein
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INTRODUCTION The glycation of dietary proteins during food processing is an attractive area, because it leads to pleasant flavor and color, prolonged shelf life, and improved functionalities (thermal stability, emulsifying capacity, and solubility).1−3 In addition, the Maillard reaction products (MRPs) were widely found to have beneficial bioactivities, such as anticarcinogenic and prebiotic properties.4,5 However, glycation is reported to result in the formation of unwanted compounds, such as acrylamide, heterocyclic amines (HAs), and 5-hydroxymethylfurfural (HMF), which are suspected carcinogens.6−8 The term advanced glycation end products (AGEs) literally refers to nonreactive terminal products in the Maillard reaction. AGEs formed in vivo or endogenously have been demonstrated to be involved in the pathology of various diseases, such as diabetes, cardiovascular disease, and cataracts.9−11 However, understanding of AGEs present in food (dietary AGEs) remains controversial, since their bioavailability, metabolism mechanism in vivo has not been elucidated clearly.12−14 One of the main mechanisms of action of AGEs may be via AGE sensitive receptors, while interaction between AGEs and RAGE seems to depend on the molecular size of the AGE compounds.12,15 The effect of dietary AGEs on human health is first determined by their absorbility by the small intestine. Kinetics studies have estimated that only 10−30% of dietary AGEs are absorbed and enter the circulation.16,17 AGEs such as N-(ε)carboxymethyllysine (CML, Figure 1), carboxyethyllysine © 2017 American Chemical Society
(CEL), and pyrraline bound to dipeptides had been found to be transported by PEPT1 into Caco-2 cell (an in vitro model of the human small intestinal mucosa). Then, these peptidebound AGEs were further hydrolyzed into AGEs in free form and enter the circulatory system.18,19 AGEs in the unabsorbable part were found to be utilized to some extent by gut flora before being excreted with feces.20−22 Glycated proteins in foods are the most common carriers of dietary AGEs. Once ingested by humans, glycated proteins are hydrolyzed into absorbable or unabsorbable fractions during their passage through the gastrointestinal tract. Therefore, assessments of dietary AGEs are closely associated with the state of AGEs (free or peptide-bound) after digestion of glycated protein. Regarding the digestibility of glycated protein, most studies have indicated that glycation reduces the susceptibility of glycated proteins to proteolysis by blocking lysine (Lys) and arginine (Arg) residues, which are tryptic cleavage sites in intestinal digestion.23,24 Cross-linked structures in glycated protein may hinder the action of digestive enzymes. Most high-molecular-weight AGEs, which primarily contain cross-linked structure, often escape digestion in the upper Received: Revised: Accepted: Published: 5778
April 27, 2017 June 23, 2017 June 27, 2017 June 27, 2017 DOI: 10.1021/acs.jafc.7b01951 J. Agric. Food Chem. 2017, 65, 5778−5788
Article
Journal of Agricultural and Food Chemistry
Figure 1. Chemical structures of several glyoxal (GO)-derived AGEs. These mixtures (8 mL) were heated in a water bath at 98 ± 2 °C for 2 h in 10 mL sealed glass vials. β-CN and β-LG were also heated independently as control samples. After incubation and before simulated digestion, the protein mixtures were dialyzed at 4 °C for 3 days using dialysis tubes with a 3 kDa MW cutoff to ensure the removal of excessive amounts of carbonyls and other small-molecule compounds. The dialyzed solution was lyophilized and stored at −20 °C until the digestion assay. All samples were prepared in triplicate. Simulated Gastrointestinal Digestion. The simulated gastrointestinal digestion of the control and glycated samples was adapted from Martinez and Pinto.23,32 Freeze-dried powder (24 mg) of each sample was dissolved in 8 mL of simulated gastric fluid (SGF) containing 0.6 M NaCl, and the pH was adjusted to 2.5 by 0.1 M HCl. A 0.5% (w/v) solution of porcine pepsin was added to each sample to obtain a final concentration of 165 units per mg of protein. Digestion was performed at 37 °C for 120 min. At the end of the reaction, pepsin was inactivated by the addition of 0.1 mL of 1 M NaHCO3. For the following intestinal digestion, the pH was adjusted to 6.5 with 26.1 mM Bis-Tris buffer (pH 6.5, containing 4 mM sodium taurocholate and 4 mM sodium glycodeoxycholate). Chymotrypsin (5 mg/mL) and trypsin (5 mg/mL) were prepared before use and added to a final concentration of 0.4 units and 34.5 units per mg of protein, respectively. Digestion in simulated intestinal fluid (SIF) was performed at 37 °C for 240 min and was stopped by heating for 3 min at 100 °C. A 0.2 mL aliquot of each digested solution was withdrawn at 1 min, 3 min, 5 min, 10 min, 30 min, 120 min, and 240 min during the gastric or intestinal digestion stage. These digests were used for ophthaldialdehyde (OPA) and SDS-PAGE analyses. A 10 μL aliquot of 1 M NaHCO3 was mixed with each gastric digest to immediately inactivate the pepsin, and each gastrointestinal digest was heated in boiling water for 3 min to stop the action of trypsin and chymotrypsin. OPA Assay. The level of primary amino groups was measured using a modified OPA method to investigate the extent of glycation during incubation and the dynamic hydrolytic process during simulated gastrointestinal digestion.33 The OPA reagent was prepared as follows: 40 mg of OPA in 25 mL of 10 mM sodium tetraborate was mixed with 2.5 mL of 20% (w/w) SDS and 100 μL of βmercaptoethanol. Then, the mixed solution was diluted with distilled water to a final volume of 50 mL as working solution. During the assay, 100 μL of glycated protein or digests was mixed with 2 mL of working solution and incubated at 40 °C for 3 min. The absorbance at 340 nm was measured with a Varian Cary 50 (Varian, USA) which is controlled by Cary WinUV software version 2.0. The loss of amino groups during glycation was expressed as a relative amount, assuming that 100% was equal to the content of amino group in the control proteins before heat treatment. Increased absorbance during digestion was calculated as a difference between the absorbance values before and after gastric or intestinal digestion. SDS-PAGE. Digested samples were electrophoresed by reducing (by dithiothreitol (DTT)) SDS-PAGE on a Mini Protean II system (Bio-Rad Laboratories, Richmond, CA, USA) according to the method
gastrointestinal tract and are eventually excreted in feces.20 Contrarily, it has been reported that the conformational changes in glycated proteins may induce more accessible cleavage sites for digestive proteases.25 Although the digestibility of glycated proteins has been extensively studied, the status of AGEs in digests (free or peptide-bound), which quantitatively determines their absorptivity by the intestinal epithelium, has rarely been assessed. Therefore, this study was designed to emphasize the detection of AGEs in protein digests with varying molecular weight (MW) ranges. Subsequently, β-casein (β-CN) and β-lactoglobulin (β-LG), which are commonly used components in food production and research, were selected as the glycation substrates. Glyoxal (GO), one of the major precursors of AGEs, such as CML, carboxymethylarginine (CMA, Figure 1), GO-derived hydroimidazolone (G-H1, Figure 1), and glyoxal lysine dimer (GOLD, Figure 1), was used to prepare protein-bound AGEs.26,27 GO extensively exists in both food production and storage, and has been detected in common foods, such as honey, jams, bakery products, and sweets.28 The GO content of milk has been reported to increase from less than 5 μM to approximately 40 μM, that is 5−300-fold higher than the levels of methylglyoxal (MGO), 3-deoxyglucosone (3-DG), and diacetyl (DA), which are also precursors of relevant AGEs, after ultra heat treatment (UHT) or other processes.29 Furthermore, GO was also indicated to be the major αdicarbonyl compound being generated during oil oxidation.30,31 After in vitro gastrointestinal digestion, digests of GOglycated protein were separated into fractions with different MW ranges, followed by the detection of MRPs with UV absorption, fluorescent AGEs, and CML in fractions with different MWs ranges. This work should help to increase our understanding of the health significance of dietary AGEs.
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MATERIALS AND METHODS
Materials. Bovine β-LG (≥90% identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)), β-CN (≥98% identified by SDS-PAGE), GO (40% aqueous solution, w/ w), the SEC analysis calibration standards, including BSA, cytochrome C, bacitracin, lysozyme, Gly-Gly-Tyr-Arg, Gly-Gly-Gly, Tyr, and Trp, and all the enzymes used for simulated gastrointestinal digestion, including pepsin (3800 units/mg protein), trypsin (10000 units/mg protein), and chymotrypsin (40 units/mg protein), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Standard CML was obtained from Toronto Research Chemicals (Toronto, Canada). Glycation Model. GO was used in a model system to prepare protein rich in AGEs. Glycation was performed in phosphate buffer (100 mM, pH 7) containing 5 mg/mL of protein and 1 or 10 mM GO. 5779
DOI: 10.1021/acs.jafc.7b01951 J. Agric. Food Chem. 2017, 65, 5778−5788
Article
Journal of Agricultural and Food Chemistry
Figure 2. Increase in free amino groups in both control and glycated β-CN/β-LG during in vitro gastric and subsequent intestinal digestion. (A): simulated gastric digestion of β-CN; (B): simulated gastric digestion of β-LG; (C): simulated intestinal digestion of β-CN; (D): simulated intestinal digestion of β-LG. Each data point is an average of triplicate data from two independent assays, and the error bars indicate the standard deviation. of Schagger and von Jagow.34 Precast gels (Novex, 10−20%, Thermo Fisher) were used for the analysis of dynamic proteolysis during digestion. Digested samples were diluted 5-fold with sample buffer (containing 0.2 M Tris, 0.2% SDS, 4% glycerol, 0.05% Coomassie G250, and 0.03 mM DTE) and heated to 90 °C for 5 min. Then, 20 μL of each diluted sample was loaded onto a precast gel and electrophoresed at 125 V before staining with Coomassie G250. Chromatography Coupled to an Electrospray Ionization− Tandem Mass Spectrometry (LC-ESI-MS/MS). Gastrointestinaldigested peptide sequences and their modifications were identified using LC-ESI-MS/MS according to the method of Le et al.35 Gastrointestinal digested samples were mixed with 0.2% formic acid at a ratio of 1:1 before filtration through a 10 kDa cutoff filter (14000 g, 4 °C for 15 min). The filtered solution (10 μL; MW < 10 kDa) was loaded onto an Aeris Peptide C18 column (250 mm × 2.1 mm, 3.6 μm, Phenomenex, Torrance, CA, USA) in an Agilent LC 1200 series directly connected to an HCT Ultra Ion Trap (Bruker Daltonics, Bremen, Germany). Solvent A was 0.1% formic acid, and solvent B was 90% acetonitrile with 0.1% formic acid. The following gradient run was performed: 2% B for 5 min, 40% B for 80 min, 80% B for 20 min, and 2% B for 5 min. The MS mode was set to MS and MS/MS mass scans from 400 to 1800 m/z and 150 to 1800 m/z, respectively. The MS/MS spectra were recorded and analyzed with Data Analysis and Biotools software (Bruker Daltonics, Bremen, Germany). The obtained tandem mass spectra were analyzed with Mascot v2.4 (Matrix Science, London, UK) according to Rauh et al., with some adjustment in the choice of modification.36 The tandem mass spectra were searched against a custom database containing known genetic and post-translational modification (PTM) variants of major milk proteins (β-CN, αs1-CN, αs2-CN, κ-CN, β-Lg, and α-lactalbumin). The following search parameters were applied: protease: unspecific; mass tolerance for precursor ions 15 ppm and 0.6 Da for product ions; variable modifications: phosphorylated Ser (for β-CN), carboxymethylated Lys, and G-H1 modified Arg. Only peptides identified as significant (Mascot score >26, P < 0.05) were considered.
Size Exclusion Chromatograph (SEC) Separation. The separation of gastrointestinal-digested mixtures was performed using an FPLC system (AKTA, GE Life Sciences, USA). All mixtures (approximately 5 mL) after the entire simulated digestion were concentrated by freeze-drying and were subsequently redissolved in 1.5 mL of Milli-Q water. These concentrated samples were membrane filtered (0.22 μm) prior to analysis. The separation procedure followed the method of Hellwig et al.37 Superdex peptide HR 10/30 (Pharmacia Biotech, Uppsala, Sweden) was used for analytical runs. Each run was performed for 70 min with phosphate-buffered saline (300 mM PBS and 150 mM NaCl, pH 7.0) at a flow rate of 0.5 mL/min. The injection volume was 500 μL. Absorbance at 280 nm was measured for detection, and fractions were collected every 5 min from 10 to 65 min. BSA (66.446 kDa), cytochrome C (12.384 kDa), bacitracin (6.511 kDa), lysozyme (1.450 kDa), Gly-Gly-Tyr-Arg (0.451 Da), and Gly-Gly-Gly (0.189 kDa) were used for mass calibration. Standard Trp and Tyr were also used to determine their retention time in digests. These collected fractions were stored at −20 °C prior to detection of ultraviolet−visible (UV) MRPs, fluorescent AGEs, and CML. Liquid Chromatography with Mass Spectrometry (LC-ESIMS). CML content in the glycated proteins and digested fractions collected from SEC was measured by LC-ESI-MS. It should be noted that pretreatments for glycated protein and their digested fractions were different. Glycated proteins were prepared according to the protocol of Lima et al.38 Glycated protein (0.5 mL containing 2.5 mg) was reduced with sodium borohydride (0.1 M as the final concentration) overnight at 4 °C, precipitated with trichloroacetic acid (20% as the final concentration), and concentrated at 5000g for 10 min. Then, the supernatant was removed and the precipitated protein was hydrolyzed in 6 M HCl (1 mL) at 110 °C for 24 h in 2 mL sealed polypropylene vials. Vacuum drying was used for 5 h at 50 °C to remove HCl and water. Then, hydrolyzed samples were reconstituted in 1 mL of 10% methanol and filtered through a 0.22 μm membrane before LC-MS analysis. 5780
DOI: 10.1021/acs.jafc.7b01951 J. Agric. Food Chem. 2017, 65, 5778−5788
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Journal of Agricultural and Food Chemistry
Figure 3. Electropherogram of digests from both control and glycated β-CN/β-LG. (A and D): control β-CN and β-LG; (B and E): β-CN and β-LG glycated with 1 mM GO; (C and F): β-CN and β-LG glycated with 10 mM GO. Lane 0: samples before digestion; lanes 1, 2, 3, 4, and 5: samples digested for 1, 5, 15, 60, and 120 min in simulated gastric digestion, respectively; lanes 6, 7, 8, 9, and 10: samples digested for 120 min in gastric digestion followed by 1, 5, 15, 60, and 120 min of simulated intestinal digestion, respectively. volume before detection by UV or fluorescence spectra. The UV absorbance from 250 to 600 nm of the glycated samples after dialysis and each digested group was measured with a Varian Cary 50 which is controlled by Cary WinUV software. Fluorescence Spectra (FS). Fluorescent AGEs present in the glycated proteins and each digested group (>20 kDa, 4.5−20 kDa, 1− 4.5 kDa, and 20 kDa, 4.5−20 kDa, 1−4.5 kDa and 20 kDa, respectively (Figure 7D). A comparison of the fluorescent spectra of the glycated proteins (Figures 7A and 7B) and their digests (Figures 7C and 7D) reveals that the resistance of fluorescent AGEs to enzymatic hydrolysis gradually increased during 2 h of glycation. Prolonged glycation could result in fluorescent AGEs with complicated structures in the final stage of glycation through carbonyl-amine condensation, aldolization, and cyclization.57 These compounds usually exhibit intermolecularly cross-linked structures with high MWs, which are speculated to largely resist the access of pepsin, trypsin, and chymotrypsin due to their strong steric hindrance or other sources of repulsion. 5784
DOI: 10.1021/acs.jafc.7b01951 J. Agric. Food Chem. 2017, 65, 5778−5788
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Figure 7. Fluorescence emission spectra of 10 mM GO-glycated proteins (A and B) during 2 h of incubation and their gastrointestinal digests with different MW ranges after SEC separation (C and D). (A and C): glycated β-CN and its digested fractions; (B and D): indicate β-LG and its digested fractions.
Table 2. Possible Peptide-Bound AGEs in the Gastrointestinal-Digests of β-CN or β-LG Incubated with 1 or 10 mM GO for 2 h, as Identified by LC-ESI-MS/MS Identified mass (Da)
Theoretical mass (Da)
1087.452 1344.565 1601.811 827.429 979.537 1499.870
1087.570 1343.637 1600.774 827.403 979.529 1499.886
731.533 800.551 871.446 1100.674 1329.682 1646.414 873.472 727.297 1108.540 1386.552
731.407 800.447 871.502 1100.681 1329.786 1646.888 873.435 727.369 1108.588 1386.679
Sequence
Modification
β-CN Carboxymethyl (+58.015) Carboxymethyl (+58.015) Carboxymethyl (+58.015) G-H1 (+40.017) G-H1 (+40.017) GH-1 (+40.017 Da) β-LG Carboxymethyl (+58.015) Carboxymethyl (+58.015) Carboxymethyl (+58.015) Carboxymethyl (+58.015) Carboxymethyl (+ 58.015) Carboxymethyl (+58.015) GH-1 (+40.017) GH-1 (+40.017) GH-1 (+40.017) GH-1 (+40.017)
Distribution of Peptide-bound CML. CML was analyzed using LC-ESI-MS as a marker to further elucidate the status of AGEs after digestion. Since free amino acids and di- and tripeptides are commonly objects for carriers during intestinal absorption, smaller peptides after gastrointestinal digestion are more likely to be absorbed after passing through the epithelial mucus layer and being hydrolyzed to absorbable products by peptidases.58 Consequently, CML bound to small peptides is
Origin
K(99)EAMAPKQK AMAPK(105)HKEMPF KEAMAPKHK(107)EMPF R(1)ELEEL GPVR(202)GPFPI PVLGPVR(202)GPFPIIV
β-CN β-CN β-CN β-CN β-CN β-CN
+ + + + + +
GO GO GO GO GO GO
(10 mM) (1 mM) (1 mM) (10 mM) (10 mM) (1 and 10 mM)
IIAEK(75)T ALK(141)ALPM ENK(91)VLVL K(70)IIAEKTKI EKTKIPAVFK(83)I KPTPEGDLEILLHK(60) R(148)LSFNPT CLVR(124)TP LDAQSAPLR(40)V DAQSAPLR(40)VYVE
β-LG β-LG β-LG β-LG β-LG β-LG β-LG β-LG β-LG β-LG
+ + + + + + + + + +
GO GO GO GO GO GO GO GO GO GO
(1 mM) (1 and 10 mM) (10 mM) (10 mM) (1 mM) (1 mM) (10 mM) (1 mM) (1and 10 mM) (10 mM)
apt to enter the human circulatory system after being absorbed in the small intestine. In the in vitro digestion of 3-deoxyglucosone-glycated casein by Hellwig et al., less than 4% of pyrraline was digested into the free form.59 In this study, gastrointestinal digests of GO-derived glycated samples without HCl hydrolysis were also directly identified by LC-ESI-MS, and no free CML was detected. Several possible peptide-bound AGEs were identified in digests using LC-ESI-MS/MS, as shown in Table 2; AGEs (CML or G5785
DOI: 10.1021/acs.jafc.7b01951 J. Agric. Food Chem. 2017, 65, 5778−5788
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Journal of Agricultural and Food Chemistry
Figure 8. Relative amounts of CML and Lys in gastrointestinal digests of glycated β-CN (A) and β-LG (B) with different MW ranges. The sum of Lys or CML in the all fractions is equal to 100%. C1 and C2 indicate digests from β-CN incubated with 1 and 10 mM GO, respectively; L1 and L2 indicate digests from β-CN incubated with 1 and 10 mM GO, respectively. Each data point is an average of triplicates from two independent assays, and the error bars indicate the standard deviation.
which cannot totally represent a real food system. In addition, the subsequent fate of AGEs bound to these putatively unabsorbable peptides in the intestinal microflora remains unknown and requires further enquiry.
H1) were found to bind to peptides ranging from 6 to 14 amino acids in length. Recent studies have reported being transported by PEPT 1 as a potential means for AGEs to enter intestinal epithelial cells; CML, CEL, and pyrraline bound to dipeptides were shown to be transported more efficiently than free AGEs.18,19 However, transmembrane transportation of AGEs bound to longer peptides requires further work. Figure 8 shows the relative amounts of CML in different fractions. When the GO concentration was increased from 1 mM to 10 mM in the glycation of β-CN, the CML content in the fraction with MWs < 1 kDa decreased from 70.6% to 31.4%; and CML in the fraction with MWs > 20 kDa increased from 0.3% to 21.7%. Lys content also migrated to digests fractions with higher MW when GO concentration was increased to 10 mM. These findings correspond with the SEC profiles shown in Figure 5. When comparing the distribution of CML and Lys, lower relative content of CML was found in the fraction with MWs < 1 kDa than that of Lys. For example, approximately 20% of CML was present in smaller digests (MWs < 1 kDa) of 10 mM GO-glycated β-LG, which is compared with approximately 30% of Lys in the same fractions. This result suggested that a carboxymethylation on the ε-NH2 of Lys directly impaired the digestibility of selected proteins by blocking the tryptic cleavage site (Lys) and increasing the steric hindrance. Reduced digestibility induced by GO-derived glycation in simulated gastric digestion was found in this study, which was not found in previous studies. In addition to blocking Lys and Arg residues, elevated steric hindrance and other types of repulsion caused by glycation were speculated to hinder hydrolysis progress and alter the cleavage pattern. Promising results regarding the distribution of fluorescent AGEs and CML were found: GO-derived AGEs were shown to hinder the enzymatic hydrolysis process and largely remained in larger peptides (>1 kDa). This antidigestion behavior of AGEs could reduce their absorption in the small intestine and serve as a natural barrier to the absorption of protein-bound AGEs. This study could also indicate that protein-bound AGEs have lower bioavailability than free or peptide-bound AGEs generated in food production due to their lower likelihood of being absorbed in the small intestine. The model we established in this study may represent foods, such as cooked meat and dairy products, which contain a high level of CML. But, the GO-derived glycation is only a simplified system which is abstracted from complex food production,
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AUTHOR INFORMATION
Corresponding Author
*Tel: +86 87113252; Fax: +86 87113252; E-mail:
[email protected]. cn. ORCID
Bing Li: 0000-0002-4407-2132 Funding
This work was funded by the National Key R&D Program of China (grant no. 2016YFD0400203), the National Natural Science Foundation of China (nos. 31371833 and 31671961), and the Fundamental Research Funds for the Central University SCUT (no. 2015zp040). Notes
The authors declare no competing financial interest.
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REFERENCES
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DOI: 10.1021/acs.jafc.7b01951 J. Agric. Food Chem. 2017, 65, 5778−5788
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Journal of Agricultural and Food Chemistry
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DOI: 10.1021/acs.jafc.7b01951 J. Agric. Food Chem. 2017, 65, 5778−5788
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DOI: 10.1021/acs.jafc.7b01951 J. Agric. Food Chem. 2017, 65, 5778−5788
