Bioprocessed wheat ingredients: Characterization, bioaccessibility of phenolic compounds, and bioactivity during in vitro digestion

ORIGINAL RESEARCH published: 24 December 2021 doi: 10.3389/fpls.2021.790898 Frontiers in Plant Science | www.frontiersin.org 1December 2021 | Volume 12 | Article 790898 Edited by: Guibing Chen, North Carolina Agricultural and Technical State University, United States Reviewed by: María Janeth Rodríguez-Roque, Autonomous University of Chihuahua, Mexico Enrico Doria, University of Pavia, Italy Yingdong Zhu, North Carolina Agricultural and Technical State University, United States *Correspondence: Cristina Martínez-Villaluenga [email protected] Specialty section: This article was submitted to Crop and Product Physiology, a section of the journal Frontiers in Plant Science Received: 07 October 2021 Accepted: 29 November 2021 Published: 24 December 2021 Citation: Tomé-Sánchez I, Martín-Diana AB, Peñas E, Frias J, Rico D, Jiménez-Pulido I and Martínez-Villaluenga C (2021) Bioprocessed Wheat Ingredients: Characterization, Bioaccessibility of Phenolic Compounds, and Bioactivity During in vitro Digestion. Front. Plant Sci. 12:790898. doi: 10.3389/fpls.2021.790898 Bioprocessed Wheat Ingredients: Characterization, Bioaccessibility of Phenolic Compounds, and Bioactivity During in vitro Digestion Irene Tomé-Sánchez1, Ana Belén Martín-Diana2, Elena Peñas1, Juana Frias1, Daniel Rico2, Iván Jiménez-Pulido2and Cristina Martínez-Villaluenga1* 1Department of Characterization, Quality and Safety (DCCS), Institute of Food Science, Technology and Nutrition (ICTAN), Spanish National Research Council (CSIC), Madrid, Spain, 2Agricultural and Technical Institute of Castile and Leon (ITACyL), Sub-directorate of Research and Technology, Valladolid, Spain To enlarge the applications of whole wheat grain (WWG) and wheat bran (WB) as functional ingredients in foodstuffs that can promote human health, researchers have explored bioprocessing approaches to improve the bioaccessibility of phenolic compounds from these food matrices and, subsequently, their biological effects. The objective of this study was to compare the composition in nutrients, anti-nutrients, and bioactive compounds of WWG and WB, and their respective bioprocessed products: sprouted wheat (GERM) and WB hydrolysate (stabilized by spray-drying [SPD] and microencapsulated [MEC]). In addition, to evaluate the functional properties of these ingredients, the bioaccessibility of phenolic compounds and their potential antioxidant and anti-inflammatory activities were monitored in different digestion steps. GERM had increased amounts of insoluble dietary fiber, higher diversity of oligosaccharides, and higher concentration of monosaccharides, free phosphorous, and phenolic compounds than WWG. SPD had improved content of soluble dietary fiber, oligosaccharides, monosaccharides, free phosphorous, and phenolic compounds (vs. WB), whereas MEC was mainly composed of protein and had nearly 2-fold lower content of SPD components. All the ingredients showed lower amounts of phytic acid as compared with raw materials. In all samples, hydroxycinnamic acids were the most representative polyphenols followed by minor amounts of hydroxybenzoic acids and flavonoids. Gastrointestinal digestion of GERM, SPD, and MEC revealed high stability of total phenolic compounds in both gastric and intestinal phases. Hydroxycinnamic acids were the most bioaccessible compounds during digestion among the three bioprocessed wheat ingredients studied, although their bioaccessibility varied across ingredients. In this sense, the bioaccessibility of ferulic acid (FA) derivatives increased in GERM with progression of the digestion, while it was reduced in SPD and MEC up to the end of the intestinal phase. Microencapsulation of SPD with pea protein led to generally to lower bioaccessible amounts of phenolic acids. Comparison analysis of biological effects Tomé-Sánchez et al. Bioprocessed Wheat Ingredients: Bioaccessibility and Bioactivity highlighted SPD for its most potent antioxidant effects in the gastrointestinal tract (3 out 4 antioxidant parameters with highest values), while no clear differences were observed with regard to in vitro anti-inflammatory activity. Overall, these results support the potential application of GERM, SPD, and MEC as functional and nutraceutical ingredients. Keywords: wheat grain, bran, bioprocessing, oligosaccharides, phenolic compounds, bioactivity, bioaccessibility, digestion INTRODUCTION Wheat (Triticum spp.), one of the important staple grains in many parts of the world, is a dietary source of starch, fiber, minerals, vitamins, and phytochemicals such as phenolic compounds (phenolic acids, flavonoids, and alkylresorcinols), phytosterols, and sphingolipids, most of them concentrated in outer layers (bran) of the grain (Cheng et al., 2021). For a long time, wheat grain has been applied for white flour production used in the manufacture of a variety of foods, especially baked foods and breakfast cereals. The wheat flour milling industry produces large quantities (about 187 million tons/year) of bran, an inexpensive by-product mostly used for animal feed (Cheng et al., 2021). Based on strong scientific evidence demonstrating that consumption of dietary fiber (DF) is associated with gut health and reduction of cardiovascular diseases and colorectal cancer risk (Cãlinoiu and Vodnar, 2018), whole-wheat grain (WWG) and wheat bran (WB) are being increasingly applied in food production (Cheng et al., 2021). Several mechanisms have been proposed to explain the observed health effects such as bulking capacities of DF, production of shortchained fatty acids due to colonic microbial fermentation of DF, and antioxidant capacity of phenolic compounds (Deroover et al., 2020). Wheat bioactive compounds with recognized health benefits are naturally trapped in the food matrix and, hence, have low bioavailability (fraction that is absorbed in the gut and reaches the site of action in an active form) that limits their effectiveness to exert physiological functions linked to health effects (bioactivity). Therefore, in the last years, strategies to increase the content of dietary bioactive compounds in WWG and WB while modifying the food structure toward improved delivery of bioactive compounds in the human gastrointestinal tract have attracted the interest of scientists (Mcclements et al., 2015). Modifications of WWG and WB composition and structure through bioprocessing (germination, fermentation, enzyme treatments) improve not only their nutritional and technological properties but may also positively affect specific health effects (Coda et al., 2015; Lemmens et al., 2019; Onipe et al., 2021). For instance, sprouted grains have positive consumer perceptions due to the existing knowledge of the positive impact of germination on grain nutritional value (Lemmens et al., 2019). Activation of several biochemical events during sprouting of cereal grains results in increased soluble and total phenolic compound content, improves the digestibility of starch, proteins, and lipids, increases soluble DF (SDF), and reduces anti-nutrient content (Lemmens et al., 2019; Tome-Sanchez et al., 2020). Regarding structure, cereal grain germination has resulted in corroded starch granular surface and disrupted crystalline structure, and reduced ordered and helical structures of storage proteins in pulse grains (Xu et al., 2021). All these compositional and structural changes are often related with improved bioaccessibility of bioactive compounds, i.e., with increase in the fraction of bioactive compounds that are released from the food matrix and available for absorption, and enhanced functionality of sprouted grains (Di Stefano et al., 2019; Lemmens et al., 2019). For instance, in our recent study, wheat germination for 7 days at 21◦C enhanced the solubilization of phenolic compounds and improved its in vitro antioxidant and anti-inflammatory properties (Tome-Sanchez et al., 2020). Preclinical studies support these findings providing evidence on the improvement of potential health benefits of sprouted cereal grains in terms of gut microbiota composition, blood cholesterol and glucose levels, blood pressure, and mineral absorption in rats compared to a control animal group fed with un-germinated grains (Lemmens et al., 2019). Enzymatic hydrolysis using amylases, xylanases, arabinofuranosidases, cellulases, proteases, and feruloyl esterases has been applied as the main bioprocess to improve WB functional properties. This method has been adopted to enhance the solubilization of arabinoxylan and SDF, release of bound phenolic acids, production of feruloylated oligosaccharides and water-soluble antioxidants, and to increase mineral bioaccessibility (Coda et al., 2015; Bautista-Exposito et al., 2020; Martín-Diana et al., 2021). Our research group has contributed in this field by demonstrating the potential of the combination of thermomechanical (autoclave and high pressure) and enzymatic treatments (Ultraflo XL) to sequentially enhance the free to bound ratio of ferulic acid (FA) and SDF content while improving in vitro antioxidant and anti-inflammatory properties of WB (Martín-Diana et al., 2021). Encapsulating phenolic compounds inside food-grade micro-carriers is a safe and an efficient way of increasing their stability, bioactive potential, controlled release, and bioaccessibility in a specific region of the gastrointestinal tract (Huang et al., 2019). With that purpose, we applied atomization and encapsulation techniques after WB enzymatic modification [Martín-Diana, 2021 #7510]. As a result, spraydrying and its further microencapsulation with pea protein (Pisane C9) of WB hydrolysate allowed the development of two nutraceutical soluble ingredients rich in SDF and FA with higher antioxidant and anti-inflammatory properties than conventional WB. Frontiers in Plant Science | www.frontiersin.org 2December 2021 | Volume 12 | Article 790898 Tomé-Sánchez et al. Bioprocessed Wheat Ingredients: Bioaccessibility and Bioactivity Before performing preclinical and clinical trials, it is essential to advance in the knowledge about the health benefits linked to nutraceutical ingredients consumption aimed at elucidating the effect of physiological conditions on the concentration of bioactive compounds and metabolites (Ketnawa et al., 2021), helping to predict their bioaccessibility and bioactivities (antioxidant and anti-inflammatory properties) at different digestion steps. With these considerations, the aim of this study was to evaluate the effect of germination of WWG and hybrid processing of WB (thermal and enzymatic treatments) on nutritional and nutraceutical composition. Finally, changes in the bioaccessibility of phenolic compounds as well as the antioxidant and anti-inflammatory activities of wheat bioprocessed ingredients through different phases of digestion was studied. MATERIALS AND METHODS Chemicals, Standards, and Reagents Fast Blue BB (FBBB) [4-benzoylamino-2,5dimethoxybenzenediazonium chloride hemi-(zinc chloride) (Rebello et al., 2016), bile extract porcine, pancreatin from porcine pancreas, pepsin from porcine gastric mucosa, α-amylase from human saliva, 2,2′-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2′-diazobis- (2-aminodinopropane)-dihydrochloride (AAPH), fluorescein, 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich, Co. (St. Louis, MO, United States). Standards of chlorogenic acid, 2-coumaric acid, 2,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, vanillic acid, and vanillin were provided by Extrasynthese (Lyon, Genay Cedex, France). Standards of 6-hydroxy-2,5,7, 8-tetramethyl-2-carboxylic acid (Trolox), gallic acid, caffeic acid, ferulic acid, vitexin, D-(+)-glucose, D-(+)- galactose, D-(+)-xylose, D-(–)-arabinose, and stachyose were acquired from Sigma-Aldrich, Co. (St. Louis, MO, United States). Standards of 33-α-L-plus 23-α-L-arabinofuranosyl-xylotetraose (XA3XX/XA2XX), 23,33-di-α-L-arabinofuranosyl-xylotriose (A2,3XX), and xylotriose were obtained from Megazyme (Wicklow, Ireland). Raffinose and cellobiose standards were supplied by Merck (Darmstadt, Germany). Materials Whole wheat grain (WWG) (Triticum aestivum L., var. Berdún) and WB (Triticum aestivum L. var. Craklin) of <800 µm particle size were harvested in La Mudarra (2018–2019 crop year) and procured from Emilio Esteban, S.A. (Emesa S.A., Valladolid, Spain). They were stored in vacuum-sealed plastic bags at 20 ±2◦C in darkness until they were used. Pisane C9pea protein was obtained from Innovafood (Barcelona, Spain) and showed the following composition according to the product specification sheet: 0.7/100 g of carbohydrates, 1.4/100 g of dietary fiber, 9/100 g of fat of which saturated 2.1/100 g, 81.7/100 g of protein, and 5/100 g of salt. Commercial food-grade enzyme Ultraflo XL was supplied by Novozymes (Bagsværd, Copenhagen, Denmark). Enzymatic activities of Ultraflo XL include those of cellulase, endo-1,4-β-xylanase, α-L-arabinofuranosidase, and feruloyl esterase (Bautista-Exposito et al., 2020). Murine RAW 264.7 macrophages were supplied by the American Type Culture Collection (ATCC R TIB-71TM; Rockville, MD, United States). Dulbecco’s modified Eagle’s medium (DMEM) was acquired from Lonza Group (Lonza, Madrid, Spain). Penicillin, streptomycin, and fetal bovine serum (FBS) were purchased from Hyaclone (GE Healthcare, Logan, UT, United States). Lipopolysaccharide (LPS) from Escherichia coli O55:B5 was obtained from Sigma-Aldrich, Co. (St. Louis, MO, United States). The Cell Titer 96 R AQueous One Solution Proliferation Assay kit and murine interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) enzymelinked immunosorbent assay (ELISA) kits were purchased from Promega (Biotech Ibérica, Madrid, Spain) and Diaclone (Besacon Cedex, France), respectively. Production of Sprouted Wheat Grains Germination of WWG was achieved as reported earlier (TomeSanchez et al., 2020), with slight modifications. Shortly, WWG (50 g) samples were soaked with 300 ml of 0.1% sodium hypochlorite (ratio 1:6, w:v) for 30min. Subsequently, they were washed and soaked for 4 h with sterile tap water (ratio 1:6, w:v). The soaked WWG samples were spread uniformly in plastic trays over a steel grid with a moist filter paper and coated with a second moist filter paper, which were previously sterilized. Afterward, seeds were allowed to germinate in an incubator with a water circulation system (Snijders Scientific, Tilburg, The Netherlands). Frequently, the seeds were damped with sterile tap water in order to maintain relative humidity (>90%). Sprouting was performed without light at 21◦C for 7 days in duplicate. These germination parameters were determined based on our earlier optimization study that aimed to maximize soluble phenolic content in addition to anti-inflammatory and antioxidant activity in sprouted wheat flour (GERM) (Tome-Sanchez et al., 2020). The WWG samples were milled (Taurus, Oliana, Spain) and included as control. GERM was freeze-dried (Virtis Company, INC Gardiner, NY, United States), ground (Taurus, Oliana, Spain), and meshed using a 0.3-mm screen. Both powders were stored under vacuum at 4–8◦C for additional analysis. Production of Spray-Dried and Microencapsulated Wheat Bran (WB) Hydrolysate The spray-dried wheat bran hydrolysate (SPD) was produced following a multistep processing route as previously described (Martín-Diana et al., 2021). A quantity of 100g of WB soaked in 2 L deionized water (1:20, w:v) was autoclaved at 115◦C, 1.2 ×105 Pa for 15 min (Ilpra Plus 100; Ilpra Systems, Barcelona, Spain). Optimal processing conditions during enzymatic hydrolysis of WB were identified in our previous study (Bautista-Exposito et al., 2020). Malic acid was used to reach a pH of 5. Quickly, Ultraflo XL was incorporated at 1% (ratio enzyme:WB, w:w) and then placed in a temperature-controlled water bath (47◦C, 20 h) with a magnetic stirrer (Unitronic Vaivén C; Selecta, S.A., Spain) at 1,000 rpm. Enzyme activity was stopped by heat inactivation using a water bath at 95◦C for 10 min. WB soluble fraction was Frontiers in Plant Science | www.frontiersin.org 3December 2021 | Volume 12 | Article 790898 Tomé-Sánchez et al. Bioprocessed Wheat Ingredients: Bioaccessibility and Bioactivity collected after removing insoluble particles by filtration using nylon membranes of 0.2 mm pore size. Spray-drying of the WB hydrolysate (SPD) was carried out in a MM basic rotary spray-dryer (GEA Mobile MinorTM, Düsseldorf, Germany) composed of a chamber coupled to a peristaltic pump (Watson Marlow 520S, Wilmington, MA, United States). Spray-drying was performed at 0.6 MPa (compressed air pressure) with a flow rate of 0.78 L/h and an air inlet/outlet temperature of 130/85◦C, according to Martín-Diana et al. (2021). Microencapsulated WB hydrolysate (MEC) was obtained under the abovementioned conditions after a mixing step of SPD with pea protein (ratio 1:1, SPD:Pisane C9). Afterward, the spraydried powders were collected in a cyclone placed outside the dryer and stored at 4–8◦C until further use. Simulated Gastrointestinal Digestion The INFOGEST 2.0 method (Brodkorb et al., 2019) was performed to simulate the gastric and intestinal phase digestion of GERM, SPD, MEC, and Pisane C9. Before the in vitro digestion, enzyme activities, and bile concentration were determined. Briefly, 3 g of the sample were mixed (ratio 1:1, w:v) with a simulated salivary fluid containing salivary amylase (75 U/ml) and calcium chloride (0.3 M) for 2 min at 37◦C in a Büchi B-491 (Marshall Scientific, Hampton, NH, United States) heating bath. Subsequently, pH value was quickly adjusted to 3 by adding HCl (1M). Oral bolus was diluted (ratio 1:1, v:v) with simulated gastric containing a pepsin solution (2,000 U/ml) and calcium chloride (0.3 M). In order to finish the gastric phase, pH was adjusted to 7 with sodium hydroxide (1 M) and then a simulated intestinal fluid containing calcium chloride (0.3 M), pancreatin (800 U/ml) and bile (10 mmol/L) were added to gastric chyme (ratio 1:1, v:v). Both gastric and intestinal phases were incubated for 2 h at 37◦C and 150 rpm in a G25-controlled environment incubator shaker (New Brunswick Scientific Co, Inc. Edison, NJ, United States). Finally, enzymes were inactivated by heating in a water bath (95◦C for 10 min). Digestion phases were freeze-dried (Virtis Company, INC Gardiner, NY, United States) and stored at 4–8◦C until additional analysis. All the phases were performed in duplicate. Chemical Composition of Wheat Nutraceutical Ingredients Determination of total starch, dietary fiber, β-glucan, and phytic acid/free phosphorus content was carried out using K-TSTA100A, K-RINTDF, K-BGLU, and K-PHYT assay kits (Megazyme, Wicklow, Ireland), respectively. Total nitrogen, determined using the Dumas method with a nitrogen analyzer (LECO Corp., St. Joseph, MI, United States), was converted to total protein by conversion factor 6.25. All analyses were performed in duplicate and expressed as g per 100g of dry weight (d.w.). Monosaccharide Analysis A quantity of 50–100 mg of the sample powder was mixed with 1 ml of deionized water and homogenized. After incubation for 1 h at 40◦C and 2,000 rpm in a thermomixer (ThermoMixer Compact; Eppendorf, AG, Hamburg, Germany), the mixture was centrifuged for 10 min at 21◦C and 13,000 rpm (Centrifuge 5424 R; Eppendorf AG, Hamburg, Germany). Deionized water was added to the collected supernatant until a final volume of 1 ml was reached. Monosaccharide extracts were stored at −20◦C until they were used. Monosaccharide composition was determined by highperformance anion-exchange chromatography (HPAEC) in water-soluble extracts of WWG flour, WB, and derived wheat ingredients (GERM, SPD, and MEC). The samples were injected into an ion chromatographic system, which comprises an 800 Dosino dispenser with a Bioscan module coupled to a degasser, and a pulse amperometric detector (Metrohm, Heriau, Switzerland). Monosaccharide separation was performed in a Hamilton RCX-30 column (4.1 mm ×250 mm, 7 µm) with sodium hydroxide (120 mM) as mobile phase at a flow rate of 1 ml/min. Column temperature and injection volume were maintained at 30◦C and 20 µl, respectively. The Metrodata IC Net 2.3. software was used for data acquisition and processing. Monosaccharide identification was carried out by comparison of retention time (RT) and sample spike with authentic standards. Calibration curves of a multi-standard with good linearity were used for the quantification of monosaccharides (R2>0.99). Results were expressed as mean ±standard deviation of two independent replicates (g per 100 g d.w.). Extraction and Quantification of Oligosaccharides A sample of 50 mg was accurately weighed in a 1.5-ml polypropylene microtube, and then 1 ml of 50% ethanol was added and vigorously mixed by vortexing. Then, the sample was incubated at 70◦C for 30 min and 2,000 rpm in ThermoMixer Compact Shaker (Eppendorf, AG, Hamburg, Germany). The mixture was centrifuged for 7 min at 4◦C and 10,000 g (Centrifuge 5424 R; Eppendorf AG, Hamburg, Germany). The supernatant was carefully collected, and extraction was performed once again. Afterward, both supernatants were mixed and adjusted to a final volume of 5 mL with 50% ethanol (v:v). The mixture was filtered through a 0.45-µm nylon syringe filter and stored at −20◦C until further analysis. Determination of oligosaccharides was performed with HPLC-ESI-QTOF-MS operating in negative mode with the same equipment described in section Determination of Phenolic Profile and Bioaccessibility by HPLC-ESI-QTOF-MS. Soluble oligosaccharide extracts were injected into a Hypercarb ThermoTeknokroma (100 mm ×2.1 µm×5µm) (Teknokroma, Barcelona, Spain) column coupled to a guard column at 30◦C. Injection volume was set at 10 µl, and the flow rate was 0.4 mL/min. The mobile phase consisted of ammonium formate (5 mM) (solvent A) andacetonitrile (solvent B), using a gradient elution from 5 to 10% B (5 min); from 10 to 50% B (15 min); from 50 to 5% B (10 min). A time of 10 min was used before the following injection. Mass range was 100–1,000 (m/z). The electrospray ionization source conditions were: drying gas (N2) flow rate, temperature, and nebulizer pressure, 12 L/min, 350◦C, and 45 psi, respectively, and sheath gas temperature and flow rate, 300◦C and 6.5 L/min, respectively. Capillary voltage was set at 4 kV, and nozzle and fragmentation, at 0 and 200 V, respectively. Collision energy was fixed at 20 V for targeted MS/MS experiments. MassHunter Data Acquisition Frontiers in Plant Science | www.frontiersin.org 4December 2021 | Volume 12 | Article 790898 Tomé-Sánchez et al. Bioprocessed Wheat Ingredients: Bioaccessibility and Bioactivity (version B.05.00) and Qualitative Analysis (version B.07.00) Workstation (Agilent Technologies, Waldbroon, Germany) were used for data acquisition and processing. Calibration curves of authentic standards were used (0–250 µg/ml, R2>0.96). Data were expressed as mean ±standard deviation of three independent replicates (mg per 100 g d.w.). The relative amount of each degree of polymerization of soluble oligosaccharides tentatively identified (%) was calculated as a percentage of the sum of the areas of extracted precursor ions (m/z) with the same degree of polymerization relative to the total area of extracted oligosaccharides. Extraction and Quantification of Total Soluble Phenolic Compounds (TSPCs) In order to avoid interferences, TSPCs were analyzed by FBBB reaction according to Pico et al. (2020), with slight modifications. A quantity (50–100 mg) of the milled sample was extracted with 1 ml of 80% methanol in 0.1% formic acid. The sample was vortexed and then incubated for 15 min at 30◦C and 2,000 rpm (ThermoMixer Compact; Eppendorf AG, Hamburg, Germany). Subsequently, the sample was centrifuged for 5 min at 5◦C and 10,000 rpm (Centrifuge 5424 R; Eppendorf AG, Hamburg, Germany). The sample solution was collected, and a second extraction cycle was performed with 1 ml of 70% acetone in 0.1% formic acid. Both methanolic and acetone extracts were combined and increased to 2 ml with deionized water. A volume of 1 mL of TPSC extract was mixed with 100 µL of a freshly prepared FBBB reagent (0.1% in distilled water) and vortexed for 1 min. Immediately, the extract solution was shaken after adding 100 µL of 5% NaOH and allowed to incubate for 120 min in darkness at room temperature (20 ±2◦C). Finally, 200 µl of the incubated mixture was placed in a 96-well-plate, and absorbance was measured in triplicate at 420 nm using a Synergy HT (BioTek Instruments, Winooski, VT, United States) microplate reader. Quantification of TSPCs was performed with linear calibration curves of gallic acid (0–225 µg/ml) and showed good linearity (R2>0.99). All analyses were performed in duplicate. Data were expressed as g of gallic acid equivalents (GAEs) per 100 g of sample d.w. Determination of Phenolic Profile and Bioaccessibility by HPLC-ESI-QTOF-MS Determination of individual phenolic compounds was performed using an Agilent (Agilent Technologies, Santa Clara, CA, United States) system, which includes an Agilent 1200 series liquid chromatograph (LC) coupled with a G1315B diode array detector (DAD) and an Agilent G6530A accuratemass quadrupole-time of flight mass spectrometer with an electrospray-ionization source with JetStream technology (HPLC-ESI-QTOF-MS). Before being analyzed, phenolic extracts, as prepared in section Extraction and Quantification of Total Soluble Phenolic Compounds (TSPC), were centrifuged at 4◦C and 10,000 rpm. Chromatographic separation of the phenolic extracts (20 µl) was performed at 40◦C using an Agilent ZORBAX Eclipse XDB-C18 (4.6 mm ×150 mm ×5µm) (Agilent Technologies, Santa Clara, CA, United States) analytical column, and DAD chromatograms were acquired at 280, 320, 360, and 520 nm. The mobile phases and gradient program, as well as MS and MS/MS runs, were set as previously described (Martín-Diana et al., 2021). Data acquisition and processing software used were MassHunter Data Acquisition (version B.05.00) and Qualitative Analysis (version B.07.00) Workstation (Agilent Technologies, Waldbroon, Germany). Calibration curves of standards were used for quantification (0–25 µg/ml, R2 >0.98). Data were expressed as mean ±standard deviation of two independent replicates (mg per 100 g sample d.w.). Bioaccessibility (%) is defined as the ratio between the concentration of phenolic compounds released in the simulated digestion compared to the concentration of phenolic compounds in the non-digested functional ingredient. Bioaccessibility (%)=      Concentration of compound released during digestion Concentration of compound in the food matrix before digestion      ×100 Total Antioxidant Capacity (TAC) DPPH Radical Scavenging Activity 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging potential was determined according to the modification of the method previously described by Brand-Williams et al. (1995). Briefly, a DPPH solution was dissolved with absolute methanol (120 µM). A final volume of 250 µl containing wheat extract, milliQ water, and DPPH solution (ratio 1:4:5, v:v:v) was added on a 96-well-microplate. The absorbance was recorded at 525 nm for 30 min using a microplate reader (FLUOstar Omega; BMG, Ortenberg, Germany). The calibration curve of Trolox solution (7.5–240 µM) was determined. Results were expressed as µmol Trolox equivalents (TE) per 100 g d.w. Oxygen Radical Absorbance Capacity (ORAC) The ORAC of samples was determined based on a modified procedure described by Ou et al. (2001). The samples, standards, and blanks were diluted in a phosphate buffer (10 mM, pH 7.4). The intensity of the fluorescence was recorded for 2.5 h using a FLUOstar Omega (BMG, Ortenberg, Germany) microplate reader. The wavelengths of excitation and emission were 485 nm and 520 nm, respectively. A Trolox standard curve (7.5–240 mM) was prepared. Results were calculated using the net area under the curves, between the blank and the sample. The ORAC value was expressed as µmol TE per 100 g d.w. ABTS Radical Cation Scavenging Activity The antioxidant activity of the extracts was also determined by the ABTS›+procedure (Miller and Rice-Evans, 1997). Briefly, a 7-mM ABTS aqueous solution was mixed with 2.45 mM potassium persulfate at 2:1 ratio (v:v) and kept in the dark at room temperature overnight before use. The ABTS›+solution was diluted with a 100-mM phosphate buffer solution (pH 7.4) until absorbance at 734 nm reached a value of 0.7 ±0.02 and equilibrated at 30◦C. Ten picoliters of the samples or Trolox standards (at 20–800 µM concentrations) were dissolved in 190 Frontiers in Plant Science | www.frontiersin.org 5December 2021 | Volume 12 | Article 790898 Tomé-Sánchez et al. Bioprocessed Wheat Ingredients: Bioaccessibility and Bioactivity µl of the ABTS›+solution, and absorbance (at 734 nm) kinetic was measured after initial shaking every 1 min up to 10 min at 30◦C using a microplate reader (FLUOstar Omega; BMG, Ortenberg, Germany). Antioxidant activity was expressed as µmol TE per 100 g d.w. Ferric-Reducing Antioxidant Power (FRAP) The FRAP activity of the samples was determined using a protocol according to [(Benzie and Strain, 1996) #27]. The FRAP reagent was prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mM HCl, and 20 mM FeCl3·6H2O in a relative proportion of 10:1:1 (v:v:v). The FRAP reagent was prepared fresh daily and was warmed to 37◦C prior to use. A volume of 20 µl of H2O (blank), FeSO4(400 µM – 3 mM) standard solution or sample extracts was mixed with 1.9 ml of the FRAP reagent. The mixture was incubated at 37◦C for 2 h, and the absorbance was read at 593 nm using a FLUOstar Omega (BMG, Ortenberg, Germany) microplate reader. The solution without extract was used as a blank. Data were expressed as mmol Fe+2equivalents per 100 g d.w. Determination of Anti-inflammatory Activity Cell viability and anti-inflammatory activity of undigested and digested wheat ingredients and Pisane C9were determined in murine RAW 264.7 macrophages as previously reported (MartínDiana et al., 2021), with minor modifications. The inflammatory response of treated cells was induced by the addition of 40 ng/ml of LPS. Positive control was LPS-treated cells without sample extracts. In both assays, the cells were treated with increasing concentration of wheat and Pisane C9extracts (0.05, 0.1, and 0.5 mg/ml and 0.025, 0.05, and 0.25 mg/ml of DMEM supplemented with 100 U/ml of penicillin, 100 mg/L of streptomycin, and 0.1% of FBS, respectively). Absorbance was determined on a Synergy HT (Biotek Instruments, VT, United States) microplate reader at 490 and 450 nm. One hundred percent viability of cell was determined with regard to the untreated cells. Anti-inflammatory activity was calculated as percentage inhibition relative to positive control. Data represent the mean ±standard deviation of five biological replicates. All analyses were carried out in duplicate. Statistical Analysis The data were expressed as mean ±standard deviation. Statgraphics Centurion XVIII (Statgraphics Technologies, The Plains, VA, United States) was used to perform all the statistical analyses. Analysis of variance (ANOVA) and post-hoc Duncan’s test were carried out to determine statistical differences between mean values. RESULTS Chemical Characterization The chemical composition of WWG, WB, and derived products is shown in Table 1. Although starch was the predominant component in both WWG and GERM, a significant reduction (p≤0.05) was observed after germination (from 60.96 ±3.9 to 26.11 ±1.39 g/100 g). However, germination led to a significant increase in total dietary fiber (TDF) and insoluble dietary fiber (IDF) contents (1.24and 1.54-fold increase, respectively), with no significant differences in soluble dietary fiber (SDF) and a significant reduction in β-glucan content (22.08%). Although GERM showed similar SDF content compared to WWG, higher diversity of oligosaccharides was detected in the former (Supplementary Material 1). Compared to WWG, raffinose and sucrose were reduced up to undetectable values in GERM, whereas cellobiose was detected in quantifiable amounts (2.28 ±0.21 g/100 g). A significant increase was observed for total monoand disaccharides after germination (7.94-fold increase), having an order of abundance of glucose >cellobiose >fructose >arabinose >galactose. The Protein content of WWG (12 g/100 g) remained unaltered after sprouting. While phytic acid content decreased (32.89% reduction), free phosphorous values were higher (8-fold increase) in GERM than in WWG. Regarding phenolic compounds, a significantly higher amount of TSPC and soluble FA isomers was observed in GERM (2.69and 1.25-fold increase, respectively) compared to WWG. A significant reduction in starch values was observed in SPD and MEC when compared to WB (1.86and 13.76-fold decrease, respectively; Table 1). This effect was also observed in TDF where the values decreased at least twice in SPD and MEC compared to WB (1.75and 2.61-fold decrease, respectively). Unlike WB where IDF was the most abundant compound, SPD mainly constituted of SDF (31.65 ±1.70 g/100 g). Total soluble polysaccharides in SPD and MEC were 1.65and 2.04-fold higher, respectively, than in WB, whereas amounts of total soluble oligosaccharides and β-glucan were 88.39 and 28.21% higher in SPD than in WB, respectively. Nevertheless, no significant differences were observed in total soluble oligosaccharides, and β-glucan content was significantly reduced from 1.95 ±0.10 g/100 g in WB to 1.35 g/100 g in MEC. With regard to oligosaccharide content, stachyose was significantly reduced in both powders, and no significant differences were observed in raffinose when compared to WB. Conversely, the applied processing technologies favored the release of xylotriose, 33-α-L-arabinofuranosylxylotetraose/33-α-L-plus-23-α-L-arabinofuranosyl-xylotetraose, 23,33-di-α-L-arabinofuranosyl-xylotriose, and O-(5-O-feruloylα-L-arabinofuranosyl)-(1-3)-O-β-D-xylopyranosyl-(1-4)- D-xylopyranose, and other unidentified oligosaccharides (Supplementary Material 1). Regarding monosaccharide content, a higher amount was found in SPD and MEC than in WB (7.85and 4.78fold increase, respectively). Unlike WB, both SPD and MEC contained significant amounts of maltose and cellobiose, where maltose was the major sugar, followed by smaller amounts of glucose, fructose, sucrose, arabinose, cellobiose, xylose, and galactose (decreasing order). Because Pisane C9(81.7% of protein) was used as encapsulating agent, the main compound of MEC was protein showing a 23.56% higher concentration than WB. In contrast, protein content was significantly reduced in SPD compared to that in WB (from 15.62 ±0.09 g/100 g to 10.36 ±0.18 g/100 g). SPD and MEC showed a significant increase in free phosphorous values (19.17and 12-fold increase, respectively) and a significant reduction in phytic acid content (3.31and 1.89-fold decrease, respectively). Frontiers in Plant Science | www.frontiersin.org 6December 2021 | Volume 12 | Article 790898 Tomé-Sánchez et al. Bioprocessed Wheat Ingredients: Bioaccessibility and Bioactivity TABLE 1 | Nutritional and nutraceutical composition of whole wheat grain (WWG)- and wheat bran (WB)-derived ingredients. Components (g 100/g d.w.) WWG GERM WB SPD MEC Starch 60.96 ±3.90b26.11 ±1.39a19.13 ±0.56c10.28 ±0.72b1.39 ±0.04a Total insoluble dietary fiber 10.62 ±1.48a16.35 ±0.78b37.83 ±0.40and nd Total soluble polysaccharides 3.74 ±0.53a4.52 ±0.55a5.90 ±0.40a9.74 ±1.34b12.03 ±0.97b β-glucan 0.77 ±0.04b0.60 ±0.09a1.95 ±0.10b2.50 ±0.23c1.35 ±0.15a Total soluble oligosaccharides 4.85 ±1.08a3.05 ±0.17a11.63 ±0.03a21.91 ±3.05b9.13 ±2.32a Raffinose 0.88 ±0.00 nd 1.42 ±0.17a1.38 ±0.23a0.92 ±0.14a Stachyose <LOQ <LOQ 0.14 ±0.03c0.11 ±0.02b0.04 ±0.01a Xylotriose nd nd nd 4.78 ±0.59b2.98 ±0.29a XA3XX/XA2XX nd nd nd 0.09 ±0.02b0.01 ±0.01a A2,3XX nd nd nd 0.97 ±0.12b0.65 ±0.03a FAXX nd nd nd 0.10 ±0.08a0.07 ±0.00a Total dietary fiber 19.22 ±0.14a23.92 ±1.50b55.36 ±0.03c31.65 ±1.70b21.17 ±1.35a Glucose 0.62 ±0.02a8.03 ±0.27b1.19 ±0.01a4.40 ±0.21c2.35 ±0.06b Galactose 0.05 ±0.01a0.10 ±0.01b0.11 ±0.02a0.23 ±0.02b0.11 ±0.05a Arabinose 0.03 ±0.00a0.21 ±0.01b0.04 ±0.00a1.09 ±0.05c0.51 ±0.01b Xylose <LOQ* <LOQ* <LOQ* 0.70 ±0.05b0.33 ±0.04a Fructose 0.43 ±0.03a2.05 ±0.04b1.05 ±0.07a2.18 ±0.09c1.18 ±0.06b Sucrose 0.18 ±0.00 <LOQ* 0.17 ±0.01a1.12 ±0.06c0.75 ±0.04b Maltose nd nd nd 9.34 ±0.31b6.49 ±0.78a Cellobiose nd 2.28 ±0.21 nd 1.03 ±0.09b0.51 ±0.06a Total diand monosaccharides 1.31 ±0.04a10.40 ±0.27b2.56 ±0.05a20.10 ±0.39c12.23 ±0.80b Protein 12.17 ±1.21a12.07 ±1.14a15.62 ±0.09b10.36 ±0.18a39.18 ±0.87c Phytic acid 0.76 ±0.02b0.51 ±0.01a2.82 ±0.07c0.85 ±0.10a1.49 ±0.08b Free phosphorous 0.01 ±0.00a0.08 ±0.00b0.06 ±0.00a1.15 ±0.06c0.72 ±0.03b Phenolic compounds TSPC (g GAE/100 g d.w.) 0.16 ±0.01a0.43 ±0.01b0.49 ±0.01a0.93 ±0.02c0.60 ±0.01b Soluble FA isomers (mg/100 g d.w.) 1.25 ±0.04a1.56 ±0.20b4.03 ±0.29a177.81 ±1.21c104.98 ±1.39b Data are mean values ±standard deviation of at least three replicates. Different letters in the same row indicate significant differences among the mean values of different treatments (WWG vs. GERM; WB vs. SPD vs. MEC) (one-way analysis of variance, ANOVA, post-hoc Duncan’s test, p ≤0.05). A2,3XX, 23,33-di-α-L-arabinofuranosyl-xylotriose; XA3XX/XA2XX, 33-α-L-arabinofuranosyl-xylotetraose/33-α-L-plus 23-α-L-arabinofuranosyl-xylotetraose; d.w., dry weight; FA, ferulic acid; FAXX, O-(5-O-feruloyl-α-L-arabinofuranosyl)-(1-3)-O-β-Dxylopyranosyl-(1-4)-D-xylopyranose; GAE, gallic acid equivalents; GERM, germinated whole-wheat grain; SPD, hydrolyzed WB spray-dried at 130◦C; MEC, hydrolyzed WB spray-dried at 130◦C and microencapsulated with Pisane C9; LOQ, limit of quantification; nd, not detected; TSPC, total soluble phenolic compounds; WB, wheat bran; WWG, whole wheat grain. *LOQ =0.1 µg/mL. Finally, TSPC and soluble FA isomers significantly increased in SPD (1.89and 43.97-fold, respectively) and MEC (1.22and 26.05-fold, respectively) with respect to WB. Characterization of Phenolic Compounds The phenolic profile of GERM, SPD, and MEC was determined by high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry in tandem (HPLC-ESI-QTOF-MS/MS) in negative ionization mode ([M - H]−) and classified according to peak elution, identification/tentative identification, molecular formula, retention time (RT), fragmentation score, mass error (in ppm), precursor, and product ions (m/z). Phenolic compounds were identified by comparison of reference standards, or tentatively identified by comparison of RT, fragmentation pattern, error between observed and theoretical mass, and molecular formulas with those reported in the literature for wheat. In GERM, 20 phenolic compounds were tentatively identified (Table 2). Using reference standards, three of the 11 phenolic acids were identified, as previously described by Gawlik-Dziki et al. (Gawlik-Dziki et al., 2016) in wheat extracts germinated at 20◦C for 4 days: 4-hydroxybenzoic acid (i1) (1), caffeic acid (3) and trans-FA (12), respectively. Similar to compound 1, peak 17 with observed [M—H]−at m/z 137 and RT at 22.8 min was tentatively characterized as isomer 2 of 4-hydroxybenzoic acid. Compounds 2 and 7 with theoretical [M—H]−at m/z 371 and compound 4 with theoretical [M—H]− at 367 and a daughter ion at m/z 193 (corresponding to a deprotonated FA), were identified as FA derivatives (i1 and i2) and 3-feruloylquinic acid, respectively (Barros Santos et al., 2019). Compounds 6, 10, and 13 with detected precursor ion [M—H]−at m/z 385 and main fragment ions at m/z 205, 267, and 175, respectively, were tentatively characterized as 1-O-sinapoyl-beta-D-glucose (i1, i2, and i3, respectively) (Barros Santos et al., 2019). Regarding the flavonoid class, compound 9 with molecular formula C26H28O15 and [M—H]−at m/z 579 and lacking fragment ions was tentatively identified as lucenin1/3 (luteolin-6/8-C-xyloside-8/6-C-glucoside), in agreement with data previously reported by other authors in un-germinated wheat (Dinelli et al., 2009; Leoncini et al., 2012). Similarly, Frontiers in Plant Science | www.frontiersin.org 7December 2021 | Volume 12 | Article 790898 Tomé-Sánchez et al. Bioprocessed Wheat Ingredients: Bioaccessibility and Bioactivity TABLE 2 | Tentative identification of GERM phenolic compounds by HPLC-ESI-QTOF-MS/MS in negative ion mode. No Proposed compounds Molecular formula RT (min) Score Error (ppm) Precursor ion (m/z) MS/MS (relative intensity, %) 1 4-hydroxybenzoic acid (i1) C7H6O37.6 70.12 −5.42 137.0242 nd 2 Ferulic acid derivative (i1) C16H20O10 9.6 73.94 −5.46 371.0999 75.0027 (9.90); 134.0363 (22.99); 193.0529 (100); 259.0270 (7.51); 371.1049 (9.58) 3 Caffeic acid C9H8O410.8 65.52 −7.13 179.0369 nd 4 3–Feruloylquinic acid C17H20O911.2 79.64 −7.58 367.1065 134.0364 (30.30); 193.0491 (100); 278.0699 (0.36); 367.1019 (1.70) 5 Unidentified C16H20O912.9 75.67 −7.24 355.1029 101.0134 (6.55); 175.0446 (100); 268.9671 (0.99) 6 1–O–Sinapoyl-beta-D-glucose (i1) C17H22O10 14 48.93 −7.34 385.1146 89.0247 (20.72); 149.019 (8.54); 205.0531 (100); 327.0984 (5.30) 7 Ferulic acid derivative (i2) C16H20O10 16.4 81.86 −4.89 371.1002 121.0287 (74.52); 192.0509 (5.36); 249.0575 (100); 371.0996 (55.15) 8 Syringic acid C9H10O516.5 85.32 −2.14 197.0455 nd 9 Lucenin-1/3 (luteolin-6/8-Cxyloside-8/6-C-glucoside) C26H28O15 17.8 90.54 −2.9 579.1370 nd 10 1-O-Sinapoyl-beta-D-glucose (i2) C17H22O10 18 85.98 −4.55 385.1146 113.0225 (41.91); 180.9091 (27.15); 267.0778 (100); 307.1036 (8.86); 385.1130 (21.92) 11 Apigenin-6-C-arabinoside-8-Chexoside isomer (i1) C26H28O14 18.8 92.4 −3.28 563.1406 119.0276 (1.22); 163.2720 (0.33); 265.0513 (1.39); 353.0729 (8.61); 443.1027 (7.72); 519.9548 (2.22); 563.1417 (100) 12 Trans-ferulic acid C10H10O420 64.01 9.33 193.0484 nd 13 1-O-Sinapoyl-beta-D-glucose (i3) C17H22O10 20.1 46.96 −6.73 385.1146 129.0205 (24.84); 175.0386 (100); 265.0693 (33.25); 353.0627 (7.5) 14 Apigenin-6-C-arabinoside-8-C– hexoside isomer (i2) C26H28O14 20.6 83.84 −5.75 563.1406 117.6116 (0.17); 178.0311 (1.00); 233.0466 (0.48); 311.0466 (0.48); 383.0793 (5.21); 443.0973 (9.30); 563.1445 (100) 15 Unidentified C11H12O520.7 94.28 −4.04 223.0622 59.0163 (29.91); 91.0523 (100); 117.0739 (26.17); 176.9861 (31.09); 223.0803 (3.50) 16 Apigenin-6-C-arabinoside-8-Chexoside isomer (i3) C26H28O14 22.5 61.45 −5.11 563.1440 nd 17 4-hydroxybenzoic acid (i2) C7H6O322.8 47.11 −2.08 137.0247 nd 18 5,7,4’-trihydroxy-3’,5’- dimethoxy-flavone (tricin) C17H14O734.5 76.24 −7.79 329.0694 nd 19 Formononetin (Glycosylated and methylated) (i1) C23H24O934.9 52.19 −9.38 443.1390 nd 20 Formononetin (Glycosylated and methylated) (i2) C23H24O935.5 92.9 −3.15 443.1362 nd RT, retention time; i, isomer; nd, not detected. compound 18 with [M—H]−at m/z 329 and compounds 19 and 20 with [M—H]−at m/z 443 were assigned as 5,7,4′- trihydroxy-3′,5′-dimethoxy-flavone (tricin) and isomers 1 and 2 of formononetin (glycosylated and methylated), respectively (Dinelli et al., 2011). Peaks 11, 14, and 16, which showed the same precursor ion at m/z 563 [M—H]−and gave main daughter ions at m/z 353, 443, and 383, were identified as isomers 1, 2, and 3 of apigenin-6-C-arabinoside-8-C-hexoside isomers (Dinelli Frontiers in Plant Science | www.frontiersin.org 8December 2021 | Volume 12 | Article 790898 Tomé-Sánchez et al. Bioprocessed Wheat Ingredients: Bioaccessibility and Bioactivity TABLE 3 | Tentative identification of SPD and MEC phenolic compounds by HPLC-ESI-QTOF-MS/MS in negative ion mode. No Proposed compounds Molecular formula Ingredient RT (min) Score Error (ppm) Precursor ion (m/z) MS/MS (relative intensity, %) 1 2,4-dihydroxybenzoic acid (i1) C7H6O4SPD 4.3 79.72 −1.56 153.0193 82.9958 (100); 153.0128 (72.61) MEC 4.2 70.71 −6.02 153.0198 nd 2 2,4-dihydroxybenzoic acid (i2) C7H6O4SPD 5.2 80.71 −6.71 153.0203 53.0384 (19.17); 91.0125 (10.41); 109.0284 (100) MEC 5.2 65.7 −12.56 153.0208 nd 3 4-hydroxybenzoic acid (i1) C7H6O3SPD 7.7 81.68 −12.88 137.0263 65.0072 (13.63); 108.0191 (33.55); 137.0221 (100) MEC 7.7 84.01 −4.53 137.025 137.0221 (100) 4 Vanillic acid C8H8O4SPD 9.9 73.17 −7.33 167.0364 nd MEC 10.0 62.52 −5.11 167.0365 nd 5 2,4-dihydroxybenzoic acid (i3) C7H6O4SPD 10.7 99.14 −0.2 153.0194 65.0371 (34.28); 109.0259 (100); 153.0091 (23.74) MEC 10.7 72.47 −6.58 153.0199 nd 6 Caffeic acid C9H8O4SPD 11.3 56.04 −12.96 179.0367 93.0352 (22.58); 135.0432 (100) C9H8O4MEC 11.3 64.89 −5.45 179.0353 nd 7 Vanillin C8H8O3SPD 15.1 74.7 −3.39 151.0408 nd MEC 15.1 65.53 −10.59 151.0414 nd 8 Apigenin-6-C-arabinoside-8-Chexoside (i1) C26H28O14 SPD 16.7 60.69 3.81 563.1431 210.8969 (4.49); 277.1295 (2.48); 325.0840 (3.25); 383.0773 (17.20); 415.2465 (10.63); 528.2741 (7.92); 563.1344 (100) MEC 16.6 67.22 −0.77 563.1397 nd 9 Apigenin-6-C-arabinoside-8-Chexoside (i2) C26H28O14 SPD 18.9 80.57 −5.2 563.1431 202.0162 (2.19); 260.5279 (0.36); 311.1749 (1.53); 353.0562 (8.48); 413.0764 (1.31); 443.1070 (11.44); 563.1475 (100) MEC 18.8 88.05 0.11 563.1407 nd 10 Trans-ferulic acid C10H10O4SPD 20.0 96.39 −3.95 193.0513 51.2765 (0.20); 106.0406 (10.73); 134.0377 (100); 178.0224 (14.22) MEC 20.0 93.27 −4.59 193.0517 90.1390 (4.93); 134.0316 (100); 167.0645 (19.17) 11 Apigenin−6–C–arabinoside-8-Chexoside (i3) C26H28O14 SPD 20.7 71.64 −7.11 563.1431 205.0360 (2.22); 259.1010 (0.84); 325.0530 (2.24); 383.0832 (13.22); 443.1033 (8.06); 527.2743 (2.66); 563.1449 (100) MEC 20.6 82.52 −5.81 563.1442 123.0069 (2.14); 197.8871 (2.20); 353.0616 (15.28); 383.0952 (8.47); 426.9918 (4.60); 503.1427 (4.89); 563.1469 (100) 12 Cis-ferulic acid C10H10O4SPD 21.7 80.41 −10.5 193.0528 89.0308 (23.47); 137.0334 (100); 179.0332 (6.09) MEC 21.7 80.41 −10.4 193.0528 63.0035 (47.37); 133.0224 (43.97) 13 4–hydroxybenzoic acid (i2) C7H6O3SPD 23.0 81.68 −12.88 137.0263 nd MEC 22.8 84.01 −4.53 137.0250 nd 14 Apigenin-6-C-arabinoside-8-Chexoside (i4) C26H28O14 SPD 23.5 59.91 −5.18 563.1431 263.0646 (6.96); 383.0582 (12.52); 443.1119 (7.22); 503.1066 (2.86); 563.1420 (100) MEC 23.4 44.05 −8.29 563.1444 nd SPD, hydrolyzed WB spray-dried at 130◦C; MEC, hydrolyzed WB spray-dried at 130◦C and microencapsulated with Pisane C9; i, isomer; nd, not detected; RT, retention time. Frontiers in Plant Science | www.frontiersin.org 9December 2021 | Volume 12 | Article 790898 Tomé-Sánchez et al. Bioprocessed Wheat Ingredients: Bioaccessibility and Bioactivity Encapsulation of SPD with a commercial pea protein isolate (Pisane C9) at ratio 1:1 (w:w) explained most differences observed between the chemical composition of SPD and MEC. On the one hand, the amounts of β-glucan, TDF,SDF, total monoand disaccharides, free P and TSPC (Table 1), and individual phenolic compounds (Table 5) in MEC were lower than the amounts observed in SPD because of the dilution effect caused by the use of the encapsulation agent in the atomization process (Table 1). On the other hand, the higher amounts of protein and phytic acid found for MEC (vs. SPD) were contributions of Pisane C9 (composed of 81.7% protein and 4.05% phytic acid) (MartínDiana et al., 2021). Hydroxycinnamic Acids Are the Major Bioaccessible Phenolic Compounds of Wheat Bioprocessed Ingredients Food matrix composition, food processing, and chemical structure influence the bioaccessibility of dietary phytochemicals (Shahidi and Pan, 2021). In line with this statement, the bioaccessibility of phenolic compounds varied across wheat derived ingredients (GERM, SPD, and MEC) and among different individual compounds. Simulated digestion of GERM increased the total amounts of bioaccessible TSPC (41.69% relative increase vs. undigested sample) more extensively in the gastric phase (32% relative increase) than in the intestinal phase (9.5% relative increase) (Table 4). FA derivatives were the major bioaccessible phenolic compounds in GERM because of their high stability to digestion conditions as reported previously for barley and wheat sprouts (Aborus et al., 2018; Drawbridge et al., 2021). Maximum bioaccessible amounts of phenolic compounds were observed at the end of the intestinal phase for GERM, consistent with previous studies on in vitro digestion of conventional and bioprocessed (fermentation and enzymatic treatments) whole wheat bread (De Almeida et al., 2020). The main difference between our results and those obtained by previous studies regarding the in vitro digestion of sprouted cereal grains (oat, barley, and wheat) was higher release of phenolic compounds during the intestinal phase (Aborus et al., 2017, 2018). This difference may be attributed to the use of different digestion models. In fact, an obstacle in the interpretation of results from existing scientific literature is the large number of in vitro digestion models that differ on stages of digestion, conditions (enzymes, pH, electrolytes, and bile acids concentration, etc.), and modes of operation (static or dynamic) (Alminger et al., 2014). For this reason, in this article, we used a harmonized digestion protocol developed by the international INFOGEST network that sets parameters and conditions for static in vitro simulation of adult digestion suitable for food (Brodkorb et al., 2019). There was a variation in the digestion behavior of individual phenolic compounds in GERM (Table 4). For instance, FA bioaccessibility increased gradually during all the stages of digestion in GERM probably because of physical, chemical, and enzymatic breakdown of the food matrix by the acidic environment of the stomach and pepsin action (Li et al., 2022). Moreover, in the intestinal phase, the esterase activity of lipases could contribute to the breakdown of covalent bonds and release of FA from the GERM food matrix (Shahidi and Pan, 2021). Gastric and intestinal digests contained too low amounts of 1-O-synapoyl-β-D-glucose to be detected possibly because of its transformation to other compounds either by hydrolysis or binding to other compounds such as proteins and starch. The bioaccessibility of apigenin and formonetin glycosides in GERM showed an opposite trend compared to that of FA. The conversion of flavonoid glucosides to aglycones induced by acidic conditions in the stomach might be a plausible mechanism that explains the lower content of these flavonoids at the end of gastric digestion (Ketnawa et al., 2021). Unlike GERM, all phenolic compounds in SPD were in soluble forms as a result of the WB processing applied in this study. TSPC showed high stability during simulated digestion (<10% loss, Table 5) in SPD. Trans-ferulic acid was the major bioaccessible phenolic compound in SPD, with greater amounts during the gastric phase of digestion as compared with the end of intestinal digestion. A similar behavior was mostly observed for other hydroxycinnamic acids, hydroxybenzoic acids, apigenin glycosides, and vanillin during the digestion of SPD. Protein hydrolysis by pepsin and acidic pH during the gastric phase of digestion could have resulted in the disruption of hydrophobic interactions between free phenolic compounds and soluble proteins/peptides formed during WB processing. Previous studies have shown that caffeic and FAs are chemically unstable (oxidation or degradation) at the alkaline pH of pancreatic fluid (Chen et al., 2019; Ketnawa et al., 2021), explaining the decrease in phenolic compound bioaccessibility found for SPD after intestinal digestion. As observed for MEC, 80% of TSPC were bioaccessible in the gastric phase, whereas greater bioaccessibility was observed at the end of the intestinal phase (120.14% of bioaccessibility, Table 5) that could be attributed to Pisane C9contribution (0.1 g/100g of bioaccessible TSPC in the intestinal phase). Similar to SPD, FA isomers had good stability to digestion; therefore, hydroxycinnamic acids (with trans-FA as the most representative compound) were the major bioaccessible compounds in MEC. During digestion, the bioaccessibility of all the identified compounds showed a decreasing trend with the lowest values at the end of the intestinal phase. Interactions between phenolic compounds and food proteins is reported to be weakened during digestion (Chen et al., 2019). For example, the binding rate of oat aventhramides and caseins decreased from 70.8 to 38% after intestinal digestion, although new interactions may also appear between phenolic compounds and peptides (Chen et al., 2019). These peptides could be the carriers of phenolic compounds to small intestine where they could be metabolized by gut microbiota (Shahidi and Pan, 2021). All these effects may explain the lower bioaccessibility of individual phenolic compounds observed for MEC at either gastric or intestinal phase of digestion as compared to SPD. Consistent with our results, differences in food matrix composition have been associated to differential phenolic compound bioaccessibility in different cereal flours (whole meal wheat, rice, and corn). Seczyk et al. (2021) suggested that food matrices with high content of dietary Frontiers in Plant Science | www.frontiersin.org 16 December 2021 | Volume 12 | Article 790898 Tomé-Sánchez et al. Bioprocessed Wheat Ingredients: Bioaccessibility and Bioactivity fiber and proteins showed stronger interactions with polyphenols and lower digestibility (Seczyk et al., 2021). These findings also support the differences found between SPD and MEC, in which the higher protein content of the latter can be reflected by the higher affinity of phenolic acids with protein and their hydrolysis products (peptides). Hydroxycinnamic acids, in particular ferulic acid or its derivatives, were the most abundant bioaccessible compounds during the digestion of the three bioprocessed wheat ingredients studied. Based on existing evidence, free FA is absorbed almost completely before reaching the colon (Zhao et al., 2003). In contrast, FA moieties in feruloyl arabinooligosaccharides or arabinoxylans could be released and then metabolized predominantly by colonic microbiota (Zhao et al., 2003). The main ferulic acid microbial colonic metabolites were reported to include 3-(3-hydroxyphenyl)propionic acid, dihydrocaffeic acid [3-(3,4-dihydroxyphenyl)propionic acid], 3-phenylpropionic acid, 3-(2-hydroxyphenyl)propionic acid (melilotic acid), and 3-(4-hydroxyphenyl)propionic acid (phloretic acid) (Anson et al., 2009; Koistinen et al., 2017). Interactions between cereal phenolic acids and gut bacteria may eventually exert functions linked to the observed health benefits of its consumption. For instance, ferulic acid metabolism has been reported to induce marked structural changes in the gut microbiota reducing the relative abundance of Fimicutes,Erysipelotrichaceae, and Ileibacterium, which were positively correlated with serum lipid levels in mice with atherosclerosis (Gu et al., 2021). Antioxidant Activity of Bioprocessed Wheat Ingredients Increased During Digestion Dietary antioxidants may have a crucial function in the gastrointestinal tract by reducing the oxidative stress produced by respiratory bursts of immune cells activated by diet-related toxins and bacteria (Halliwell et al., 2005). Our results have demonstrated that undigested bioprocessed wheat ingredients studied herein were able to reduce free radicals through hydrogen (DPPH, ABTS, and ORAC) or electron donation (FRAP test) (Table 6). There was a variation in the overall antioxidant activity across bioprocessed ingredients (SPD >MEC >GERM) that was consistently associated to their TSPC, which is consistent with previous studies (Bautista-Exposito et al., 2020; Tome-Sanchez et al., 2020). As digestion progressed, a common increasing trend in most of the antioxidant parameters measured was observed for all the samples. The comparison among ingredients highlighted SPD (highest DPPH and FRAP values) as the most promising sample with the ability to prevent oxidative stress in the gastrointestinal tract. In agreement with our results, Li et al. (2022) highlighted the increased radical scavenging activity of WB with different particle sizes during digestion (Li et al., 2022). Most studies have attributed this behavior to the higher bioaccessibility of total phenolic compounds during digestion (Sánchez-Velázquez et al., 2021; Li et al., 2022), as could be observed for GERM (in the gastric and intestinal phases, Table 4) and MEC (intestinal phase, Table 5). In contrast, this association could not be established for SPD in which bioaccessible TSPC were slightly reduced (< 10%, Table 4). It has been demonstrated that in addition to the concentration of bioaccessible phenolic compounds, the pH of oral, gastric, and intestinal fluids play a role in the antioxidant activity of phenolics (Bouayed et al., 2011). The transition from acidic to alkaline environments enhances the antioxidant power of phenolics by causing deprotonation of hydroxyl moieties of aromatic rings, which could have contributed to the increased trend in the antioxidant activity observed during digestion of all the ingredients. Moreover, enzymatic hydrolysis in the stomach and intestine may contribute to the release of other compounds with antioxidant activity. For instance, the hydrolytic action of digestive enzymes in proteins during digestion releases peptides and free amino acids with radical scavenging activity (Vanvi and Tsopmo, 2016; Phongthai et al., 2018). Potential Anti-inflammatory Activity of Bioprocessed Wheat Ingredients Increased During in vitro Digestion The bioprocessed wheat ingredients were able to inhibit the release of TNF-αand IL-6 by LPS-stimulated macrophages at the doses tested before and during simulated digestion (Table 7). This result indicated that the three ingredients studied have the potential to attenuate inflammation in the gut. Our study is in accordance with Gabriele et al. (2018), which demonstrated the anti-inflammatory effects of fermented whole wheat on TNFαinflamed human intestinal epithelial cells (HT-29) (Gabriele et al., 2018). Particularly, the authors pointed out that free phenolic compounds released by fermentation reduced the expression of IL-8 and COX-2 inflammatory mediators through inhibition of the NF-κB signaling pathway. The results indicated that there was a greater release of anti-inflammatory compounds during the intestinal phase of digestion than during the gastric phase (Table 7). Among the different factors involved in this effect, phenolic compounds might have played a role, since wheat hydroxycinnamic acids are able to inhibit the expression of IL-6 and TNF-αin LPSactivated RAW 264.7 macrophages (Luyen et al., 2014; Lampiasi and Montana, 2016). Nevertheless, other compounds might have contributed to the anti-inflammatory activity of bioprocessed ingredients. The presence of XOs in WB functional ingredients could promote their anti-inflammatory activity. In un-stimulated macrophages, xylooligosaccharides induced TNF-α, IL-1β, IL-6 and NO release, while inhibiting their production in a dosedependent manner in LPS-stimulated RAW264.7 macrophages (Chen et al., 2012). CONCLUSIONS This study demonstrate that WWG germination and WB bioprocessing result in promising outcomes for the creation of functional ingredients with improved nutritional profile. In particular, wheat germination was a useful method to enrich wheat flour in TDF, IDF, oligoand monosaccharides, free phosphorous, and phenolic compounds, mainly FA derivatives, sinapoyl glucose, apigenin diglucosides, and formononetin. Similarly, the sequential combination of transformation (thermal and enzymatic treatments), separation (filtration), and drying (atomization) methods applied on WB served to obtain a soluble powder rich in SDF, oligoand monosaccharides, and Frontiers in Plant Science | www.frontiersin.org 17 December 2021 | Volume 12 | Article 790898 Tomé-Sánchez et al. Bioprocessed Wheat Ingredients: Bioaccessibility and Bioactivity phenolic compounds that include FA as the most abundant compound followed by lower amounts of hydroxybenzoic acids, apigenin diglucosides, and vanillin. Another advantage of both bioprocessing methods was their effectiveness in dephytinization and increasing free P concentrations. Microencapsulation of SPD with Pisane C9contributed to the increase in protein and phytic acid contents while at the same time diluting other SPD components. Gastrointestinal digestion of GERM, SPD, and MEC revealed high stability of TSPC in both the gastric and intestinal phases. Hydroxycinnamic acids were the most bioaccessible compounds during digestion in the three bioprocessed wheat ingredients studied, although their bioaccessibility varied across ingredients. In this sense, the bioaccessibility of FA derivatives increased in GERM with progression of digestion while it was reduced in SPD and MEC up to the end of the intestinal phase. Microencapsulation of SPD with pea protein generally led to lower bioaccessible amounts of phenolic acids. Regarding biological properties, it was confirmed that digestion generally increased the ability of the ingredients to scavenge radicals and inhibit the production of proinflammatory cytokines. These findings highlight the potential of bioprocessing as a technological approach to valorize WWG and WB into addedvalue, and potential functional and nutraceutical properties of ingredients for innovative food applications. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s. AUTHOR CONTRIBUTIONS CM-V: conceptualization and project administration. DR, EP, IJ-P, and IT-S: methodology. EP, IT-S, and IJ-P: software. CM-V, AM-D, DR, IT-S, EP, and IJ-P: validation. IT-S and IJ-P: formal analysis and data curation. IT-S, CM-V, EP, AM-D, IJ-P, DR, and JF: investigation. AM-D, CM-V, and DR: resources and supervision. AM-D, IT-S, and CM-V: writing-original draft preparation. AM-D, DR, IT-S, CM-V, EP, and JF: writingreview and editing. CM-V, AM-D, DR, IT-S, EP, IJ-P, and JF: visualization. JF, CM-V, and AM-D: funding acquisition. All authors have read and agreed to the published version of the manuscript. FUNDING This research was funded by FEDER/Ministry of Science, Innovation and Universities-State Agency of Research (AEI/Spain and FEDER/UE) grant number AGL2017-83718-R. IT-S thanks to AEI/Spain and ESF/UE for her FPI fellowship (PRE2018-086464) and IJ-P to AEI/Spain and ESF/UE for his FPI fellowship (PRE2019-087824). ACKNOWLEDGMENTS The authors acknowledge Emesa, S.A., for providing wheat bran and for the scientific and technical advice provided during the study. Novozymes Ltd. is also acknowledged for kindly donating one of the food-grade enzymes used in this study. 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