Reset: 2June ma | Revised: 22 Sep 2022 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY Bioaccessibility of phenolic compounds us: ABIES WiLey ing the standardized INFOGEST protocol: A narrative review Gabriela Boscariol Rasera' © | Adriano Costa de Camargo?® | Ruann Janser Soares de Castro! "Department of Food Science and ‘Nutrition, School af Food Engineering University of Campinas, Campinas, Brazil ‘Nutrition and Food Technology Institute, University of Chile, Santiago, Chile Correspondence Gabriela Roscarol Rasera and Adriano Costa de Camargo, Department of Food Science and Nutrition, School of Food Engineering, University of Campinas, Campinas, Braz Email: gabi_boscariol@hotmail.som; ruann@unicamp.br ‘Adriano Costa de Camargo, Nutrition and Food Technology Institute, University of Chile, Santiago, Chile. Email: adrianodecamargo@inta.uchile100_bioaevessbility of main ‘with ultrasonic water bath betalains was around 15 10 ‘with ice (4 mins 5 26%, while the main mass:solvent ratio), ‘sorhamnetin glycosides were ‘centrifugation; second inthe range of 20%-26%. ‘extraction with pure methanol: ‘Losses could be related tothe supernatant collection; solvent Tink with insoluble material ‘evaporation, resuspension in such as proteins aggregates water formed during digestion Digested extract: resuspension in ‘methanol, centrifugation, and ssupematant collection Galician Starting material: Olive oi (ug phenolics in Secoirdoids represented the 2019. (Reboredo- extra-virgin olive Undigested sample extraction: dialyzed ‘most abundant phenolic Rodriguez il Extraction with aqueous fraction/ug, family (98%) of oil polar etal, 2021) methanol (60:40 viv) phenolics inil faction. After oral digestion, Digested extract: Oil phase sample) x100 phenolics acids were mainly (extraction with aqueous present in water phase and ‘methanol (60:40 vv) and Tignans and flavonoids in oll solld-phase extraction); water phase. After gastric phase, phase (solid-phase extraction) generation of tyroso, and dialysis Inydrxytyrosol, and Inydroxytyrosol acetate may be because of seciridoids extensive hyiolysis, that were ‘unstable after intestinal Aigestion, releasing simple phenols (water phase). Lignans were stable during duodenal conditions (ll phase). ‘Mexican wild Starting material: Freeze-dried © =(Undigested —Theanthocyanins contents 2019 (Stnchez- (Rubus and rehydrated (original water samples decreased after digestion (up Velazquez, liebmanii proportion) fruits extracts/final___t078%). More than 40 etal, 2021) FFocke)and _Undigested sample extraction: concentration of phenolics were identified commercial _-‘Freeze-driedfrultsextraction _phenolles) x100 during digestion, but most of (Rubus using 70% acetone—1% HCL ‘them were completely fruticosus) solution (acetone/water/HCI degraded after this process. In blackberries (70:28:, viv) or water-MeOH contrast the concentrations of (50:50 vv) and centrifugation Digested extract: Methanol addiction, centrifugation, ‘supernatant collection, filtration, lyophilization Extraction with 70% acetone—1% HCI solution {acetone/water/HC1(70:29:1 vv) or water: MeOH (50:50 yy) and centrifugation ‘many phenolic aids, such as sali, vanilic, p-coumaric, and ferulic acids, increased following digestion asa result of anthocyanins catabolism. (Continues)vo | RR TABLE 2 (Cont Starting, ‘material ‘Natural deep eutectic solvent (choline ehlo- lined) ‘Treatments beyond Bioaccessibility INFOGEST protocol, calculation Starting material: Two blueberry = (Bioaccessible ‘extracts: Natural deep eutectic frae- solvent (CE-NADES) and tion undigested rideslycerol-citric organic solvents (CE-SORG) extract) x 100, acid) and organic solvents (methanol water: formic acid) blueberry Ethanol extracts from passion fruit peel (P edulisx) Dried fruits (apricots, black cherries, sour cherries, white cherries, corinthian currants, golden yellow figs, peaches, sour prunes, sour and ‘sweet prunes) (methanol waterformie acid mixture [80:48.5:15 vey] 820 [mass:solvent ratio). Both ‘were prepared at ultrasonic bath, followed by ‘centrifugation and supernatant collection. For CE-SORG solvents, the samples were rotaevaporated and the extract ‘was resuspended in water Undigested sample extraction: Undligested CE-NADES and CE-SORG SPE purification Digested extrac: Centrifugation (supernatant = solu- bleybioaccessble digesta and sediment = soluble/non- bioaccessibe fraction). Norbioaccessible fraction ‘extraction with ‘methanol watersformic ald (65:18:05 vviy) + SPE purification. Bioaceessible fraction = SPE purification Starting material: Drying (55°); = (Fraction in the ‘grinding, extraction with dialysis bag after aqueous 70% ethanol (20 diges- masscsolvent rato; 1h) tion/undligested Sltration, rotary evaporation, sample) 100, ‘water resuspension, filtration, and lyophilization Undigested sample extraction: ‘Thesame for starting material Digested extract: Dialysis, heating to enzyme inactivation (100°C, § min), lyophilization, resuspension in water Starting material: Not mentioned Not mentioned Undigested sample extraction: Not mentioned Digested extract: Dialysis, centrifugation, ‘supernatant eollection and fitration, INFOGEST ‘Main outcomes version CE-NADES increased the 2019 stability of phenolic ‘compounds during in vivo digestion by delaying gastric cyme neutralization. Also, the increased bioavailability of anthocyanins in NADES could bea result of their better solubility wth this solvent. Phenolics were mostly retained 2019 after digestion to reach the colon than absorbed by the small intestine, maybe because of macromolecules Interactions, However, they retained good antioxidant capacity: Note that 154 phenolics decreased content after digestion, such as anthocyanins and 24 increased their amount after digestion, such as xanthohumol Despite individual polar phenols, 2014 and bioaccessibilty was different 2019 foreach dried frit digestion released the phenolis from the fruit matrix. Note that 12-82% of foal phenolics, were released in oral phase, Which further increased after gastric phase. Total polar phenols and lavones/favonols were found to enter the simutated epithelial cell wal, Reference (dasivaetal, 2021) (Caoet al, 2021) (Panagopoutou etal, 2021) (Continues)TABLE 2 (Continued) Starting. ‘Treatments beyond Bioaccessibility ‘material INFOGEST protocol, calculation ‘Opuntia stricta var Starting material: Re-hydrated = (Digested Dillenii whole lyophilized samples fractions! fruit, pee, pulp, Undigested sample extraction: undigested) x. frozen whole methanol:water (Isl vw) 100 fruits, aw juice, extraction (2 times), methanol Jam, and bagasse extraction, rotary evaporation, resuspension in water, and filtration, Digested extract: Dialysis; ‘centrifugation, supernatant collection, and filtration ‘Bee pollen and bee Starting material: Bee polen and bread bee bread Undigested sample extraction: ‘grounding and ethanol:water (80:20, xv) extraction (under stirring, 6, 240, masssolvent ratio), solvent evaporation, and lyophilization Digested extract: centrifugation, ‘supernatant collection and ‘ethanol-water (80:20, ¥¥) dilution Fermented carob Starting materia: Fermented ilk ‘carob milk Undigested sample extraction: Not mentioned Digested extract: Ie bath and lyophilization Digested undigested) 100 Digested undigested) x 100 ‘Abbreviation: SPE, solid phase extraction, Phenolic compounds, also called polyphenols, have ‘great importance as natural antioxidants, in addition to presenting anti-inflammatory, antiproliferative, antidia- betic, neuroprotective, and cardioprotective properties. ‘Therefore, they are the focus of many excellent reviews (Shahidi & Ambigaipalan, 2015; Zhang & Tsao, 016). Phe- nolics are plant secondary metabolites and participate in defense processes against fungal, insect, and bacterial inva: sion, and also include environmental stresses, such as temperature variation, providing physical and chemical barriers (Acosta-Estrada et al., 2014). Their structure con- sists of an aromatic ring containing one or more hydroxyl substituents, which is completely related to their radical- scavenging and/or metal-chelating activity. making their antioxidant action quite dependent on their structure. ‘Since hydrogens and/or electrons are donated by the feng | = INFOGEST ‘Main outcomes version Reference Fruit peel was the most 204 (Gomez-Lopez Interesting material because of its high phenolics bioaccessibilty (ranging from 30.6 to 53.7%) composed ‘mainly of pisidie acid and fsorhamnetin lucoxyl-rhamnosyl-pentoside Raw juice phenolics bioaccessibility ranged from 29.4% 10 45.3% and pulp Aavonoids bioaccessibility rom 30.6% 1058.78. Phenolics content had their bioaccessiility decreased after oral (almost 92%), gastric (eg, {sorhamnetin-O-pentosyl- hhexoside), and intestinal (57%) phases etal, 2021) 2014 (Aylancetal., 2021) Phenolic acids and flavonoids increased ater digestion: Gallic acid (481.4%), chlorogenic acid (305.19%), (#)-catechin (486-268), -myricetin (333.2%) Feri acid showed the highest loss (236.948). 1so-quercitrin was the lowest ioaccessible flavonoid (35.26%). 2014 (Chait etal, 2021) hydroxyl groups, the number of hydroxyl groups, as well as their position in relation to the carboxyl functional group, lycosylation, and the presence of substituents in the rings directly influence the antioxidant potential of phenolic compounds (Vuolo et al, 2019). When exposed to oxidative stress, nuclear factor erythroid 2-related factor 2 (NRF-2) phosphorylates and translocates into the nucleus, activating the tran: scription of antioxidant response elements, such as proteins, antioxidant enzymes (e.g., superoxide dismutase [SOD], catalase [CAT)], phospholipid hydroperoxide glutathione peroxidase [GSHpx], NAD(P)H dehydro- genase [quinone] 1 [NQO1], heme oxygenase (HO)=1 [HO-1), and non-enzymatic antioxidants (e.g., glu- tathione). 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Phenolic compounds can induce NRF-2 activation, pro- viding a prolonged antioxidant effect and preventing and/or treating several pathological and/or toxicological conditions originating from oxidative stress occurrence (Speisky et al, 2022). The longer lasting antioxidant effect via NRF-2is explained by the half-lives of de novo synthe- sized antioxidant enzymes. Rutin treatment, for example, ameliorated cardiac oxido-inflammatory dysregulation duced by plasticizers via NRF-2 activation and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-xB) suppression (Oluranti et al., 2021). NRF-2 is a nuclear factor mainly responsible for protein and enzyme modulation against oxidative stress and inflammation (Speisky et al., 2022). According to their structure and biosynthetic routes, they are categorized into several classes, but the most important phenolic compound classes found in the human diet are phenolic acids, monomeric flavonoids, and tan- nins (¢g,, proanthocyanidins and ellagitannins). They are present in nature as soluble (free, esterified, eg.,covalently bound to fatty acids, being soluble esters, and etheri fied) and insoluble-bound (covalently bound to macro- molecules, being insoluble-bound phenolics) forms (de Camargo et al., 2017; Shahidi & Yeo, 2016). Most of the insoluble-bound phenolics are linked with cell wall macromolecules, such as structural proteins, pectin, hemicellulose, and cellulose. This accounts for a relatively large amount (20%-60% in vegetables, fruits and Jegumes/seeds) compared to the soluble phenolics in foods (Shahidi & Yeo, 2016). Therefore, ignoring the presence ‘of those linked phenolics may underestimate their poten- tial health benefits (Alshikh et al,, 2015). It is important to note that the sample analyzed, or even part of the sample (e.g, peanut skin or peanut), directly influences the pres- ‘ence and effects of insoluble bound phenolics (de Camargo et al,, 2017), However, dietary intake of free or insoluble- bound phenolic compounds depends on the desired health impact (Acosta-Estrada et al., 2014). Presently, there are ‘many processes to release insoluble-bound phenolics from the entire cell matrix, hydrolysis processes (enzymatic and non-enzymatic), biological processes (e.g., fermentation and germination), and thermal and hydrothermal process- ing (eg., boiling) can be employed (Shahidi & Yeo, 2016; ‘Vuolo etal, 2019). 4 | INFOGEST METHOD AS A STANDARDIZED PROTOCOL FOR BIOACCESSIBILITY STUDIES To start, it is important to highlight that despite often being used indistinctly, bioaccessibility and bioavailability, are different concepts and must be understood to clarify the research objectives and their results. As described by Alegria et al. (2015), “bioaccessibility is the fraction of a compound that is released from its food matrix within the gastrointestinal tract and thus becomes available for intestinal absorption (typically established from in vitro procedures). It includes the sequence of events that take place during food digestion for transformation into potentially bioaccessible material but excludes absorp- tion/assimilation through epithelial tissue and presys temic metabolism (both intestinal and hepatic)” In agree- ment, MeClements and Peng (2020) state that bioacces- sibility is “the fraction within the gastrointestinal fluids that are suitable for absorption.” On the other hand, bioavailability encompasses bioaccessibility, absorption, metabolism, excretion, and distribution, and because ofits complexity, it is usually addressed through in vivo assays (Alegria etal, 2015; MeClements & Peng, 2020). Ideally, bioaccessibility studies should be performed in vivo, which is not always technically, ethically, and financially possible. Therefore, food digestion studies are normally evaluated after in vivo simulated digestion pro- cedures. These can be divided into static, semi-dynami and dynamic models. Dynamic methods simulate the kinect aspects of digestion, such as the transport of, digested meals, continuous secretions of digestive fluids, and changes in enzyme concentrations and pH over time, In contrast, static models simplify human digestion pro- cesses, by simulating the mouth, the stomach, and the intestine conditions using static tools such as water bath shaking, air bath rotation, and a pH-stat. Various digestion models have been proposed, with different execution con ditions and, consequently, with distinet results (Li et al, 2020; Xavier & Mariutti, 2021). ‘To soften the difficulty of results comparison, a stan- dardized, static, in vivo digestion method was developed with an international consensus of more than 200 scien- tists from 32 countries in 2014 (Minekus et a, 2014) and ‘was further updated in 2019 (Brodkorb et al., 2019). This method provided the details of how to select and stan- dardize the digestive enzymes used, the execution of oral, ‘gastric, and intestinal phases, and how to analyze the data obtained. The standardized protocol can facilitate the com- parison of data from different laboratories (McClements & Peng, 2020). This protocol has good reproducibility is, robust, clear, and economically accessible, allowing easy access to each stage of digestion and, most importantly, has a good correlation with in vivo tests inthe final fractions of each stage of digestion (Brodkorb et al., 2019) By January 25, 2022, the first version of the INFOGEST protocol (Minekus et al., 2014) was cited by 2792 research articles, while the latest version (Brodkorb et al., 2019)Citations. Interestingly, some recent articles (after the 2019 update) cite the 2014 protocol. Thanks to this huge and ‘excellent initiative, data are now available for comparison. Despite the aim for this protocol creation, to our know!- ‘edge, there are no data in the current literature compar- ig the findings of phenolic compound bioaccessibility in research articles that used the INFOGEST method. Accordingly, the next topic synthesizes and discusses some ‘consensus similarities and general conclusions as a result of data comparison between the selected 79 research at cles (Figure 1) that used the INFOGEST protocol to explore phenolic compound behavior through in vivo digestion. In addition, Tables 2 and 3 (and Tables S2 and S3) show some approaches in phenolic compound bioaccessibility, ‘obtained with the in vitro digestion INFOGEST (2014 and 2019) protocol, 4.1 | To start: The metabolism of phenolic compounds ‘The metabolism of foods containing phenolic compounds ‘occurs via a common pathway and undergoes multien- zyme reactions, following alteration of physical and chem- ical properties at the oral, gastric, and intestinal phases (Manach et al, 2004; Shahidi & Yeo, 2016). Since most phenolics (except, eg, resveratrol and curcumin) are rea- sonably water soluble, their bioaccessibility depends on their release from the food matrix, their retention in the aqueous phase without complexation, and precipita- tion with proteins or minerals during digestion (Bohn et al,, 2018). In addition, anthocyanin, flavonol, and tan- nin catabolism can increase free phenolic acids during digestion. This occurs as a result of spontaneous cleav- age, decarboxylation, carboxylation, and single hydration or dehydration processes (Sinchez-Velizquez et al., 2021). ‘The free forms can be absorbed in the small intestine and undergo various processes, such as methylation, sulfation, and glucuronidation, inside the intestinal cells. How- ever, the bond between phenolics and macromolecules ‘obstructs their absorption in the native form (Manach et al, 2004; Thakur et al., 2020). However, they can bbe released from the food matrix in the gastrointestinal tract, through enzymes and pH conditions. They are then absorbed in the small intestine, where just 5%-10% of them are introduced into the blood circulation system (Shahidi & Yeo, 2016). The unabsorbed polyphenols, also called non- extractable polyphenols, reach the colon, where they undergo fermentation by the colon microbiota. This microbial community is composed of a huge variety of microorganisms, such as different bacteria, fungi, protozoa, and archaea. These microorganisms secrete extracellular enzymes (carbohydrases, proteases, lipases, fees |= among many others) (Jia et al., 2022). Of these, the carbohydrate-active enzymes can hydrolyze the glycoside forms into aglycones and convert the non-extractable phenolics into low molecular weight phenolics; these are better absorbed and persist in circulation for more than 48 h with antioxidant activity, Additionally, the released phenolic compounds play an important rote in improving colonic environmental conditions, by decreasing pH and preventing the growth of cancer-inducing microorganisms (Gonzalez-Sarrias et al., 2017; Shahidi & Yeo, 2016) Recently, Quatrin etal, (2020) demonstrated that a large amount of ellagitannins and anthocyanins from jaboticaba peel powder was not bioaccessible after digestion. These compounds were catabolized in the intestine, yielding numerous phenolic metabolites during fecal fermenta- tion, the increase of which was parallel to the increase in short-chain fatty acids and decrease in the viability of pathogenic bacteria (Enterobacteria). In any case, free or insoluble-bound phenolic compounds, independent of phenolic bioaccessibility or bioavailability, could protect the organism from oxidative stress at the gastrointestinal level (Martini et al, 2018; Quatrin et al, 2020), due to their local effect (Silva et al., 2018). Phenolic metabolites can also be considered novel compounds, since they may be different from the parent compounds. In general, low- molecular-weight molecules can be absorbed in the small intestine, exerting other systemic effects than their precur- sor phenolic (da Fuentes et al, 2021; Quatrin et al., 2020; Silva etal, 2019; Taladrid et al., 2021). More information about phenolic metabolism is described in different reviews, such as those authored by Shahidi and Peng (2018) and Wojtunik-Kulesza et al (2020). 4.2 | Finding 1: Individual phenolic identification and/or quantification after digestion: The importance to go further Although the INFOGEST protocol is being used for a better approximation of the physiological conditions, the evalua tion of phenolics from digested samples had mostly been analyzed through indirect methods (eg., total phenolic content, scavenging of DPPH and ABTS radicals, as well as reducing power by using the FRAP assay). It is impor- tant to say that those colorimetric methods have their crucial role in samples screaming and may correspond to biological assays (de Camargo et al., 2019). However, a ‘great number of papers were excluded from this review since no phenolic identification or quantification through chromatographic methods were executed during and/or after digestion. In fact, some authors characterized the phenolics present in the samples before digestion, butthey did not track the changes in their structure and/or concentration throughout the process. Some crucial information was considered to make this decision: (1) Phenolics are not the only molecules detected by Folin methodologies, or antioxidant potential by colorimetric methods. For example, peptides can have antioxidant potential and are also known to interfere in colorimetric analyses for phenolic compound detection by Folin (Ayala-Nifto et al., 2022: Chiu et al., 2018). There- fore, the positive results observed cannot be attributed only to phenolic presence; (2) a team of well-known experts in the area of food chemistry and food bioactives have already clarified the necessity of employing state-of-the- art identification methods. Therefore, meaningful results must consider chromatographic analysis as well as bet- ter biological systems (e.g. gastric and/or colonic epithelial cells) (Fuentes et al., 2021, 2022; Granato et a, 2018). 43 | Finding 2: Bioaccessibility calculation and units: The treatment before and after simulated digestion are not in consensus ‘The INFOGEST protocol was created to facilitate compari- son between different studies. However, some variations in the design and execution of experiments and in the treat- ment of data were found during the preparation of tl review. These different approaches need to be carefully evaluated so that comparative analyses between different scientific research are reliable and valid, Important factors directly affect the bioaccessibility of phenolic compounds, and their antioxidant activity differs in several studies, such as (1) the treatment before digestion, such as process- ing impact (Section 4.4); 2) the treatment after digestion, such as extraction factors (solvent, process, time, tem- perature, pH), dialysis, filtration, and centrifugation; and (3) the units and formula used for bioaccessibility calcula- tion, Some data compare the dialysable fraction with the non-dialysable fraction, and others to crude material or extracts obtained with conventional chemical extraction methods. However, most researchers use bioaccessibility calculations as follows: Digested sample (crated same) * 1° Even if the formula is often the same, it is important to note that the phenolic extraction (mainly the solvent type and methodology employed) significantly affects the results, since phenolic polarity changes depending on their structure, In addition, when different extraction meth- ‘ds are applied for each phase (undigested or digested), it can result in underestimated values for phenolies after digestion. This assessment may indicate low bioaccessi- bility of phenolic compounds, since higher denominators rather than numerator values decrease bioaccessibility val- tues (Ozdal et al, 2019). For example, when digesta from propolis were compared to ethanolic extracts, much lower bioaccessibility values were found than when compared to the oral phase, Finally, the lack of standardization in bioaccessibility expression units results in different interpretations and, consequently, different conclusions (Gayoso et al, 2016). Figure 2 summarizes this information, Because of all these differences, the results are difficult to compare, ‘which creates a huge challenge even when the INFOGEST. protocol is used (Eran Nagar et al., 2021; Gayoso et al., 2016). 4.4 | Finding 3: Phenolic compounds are unstable during simulated digestion, especially at the intestinal phase Phenolic instability and peculiar behavior through the digestion process are a consensus finding in most research data obtained using the INFOGEST protocol. In agree- ment, Shahidi and Peng (2018) and Thakur et al. (2020) made excellent reviews reporting phenolic compound bioaccessibility and bioavailability - The polemic oral phase ral phase or salivary a-amylase addition is often not considered in research involving the bioaccessibility of phenolic compounds (Carrasco-Sandoval et a, 2021; Eran Nagar et al, 2021; Lopez-Gamez et al., 2021a, 2021b; Lyu et al., 2021; Mashitoa et al, 2021). They argue that the oral phase does not interfere with phenolic bioaccessibil- ity, mostly due to the short time of amylase action, or due to the nature of the systems studied and short resi- dence times of purees and liquid food. Indeed, the original protocol (Minekus et al., 2014) described the oral phase as optional. However, when updated in 2019 (Brodkorb et al., 2019), the authors claimed that, “The oral phase must be included in all simulated digestion procedures, regardless ofthe state of the food (liquid or solid) in order to provide consistency of dilution” and “the formation of a swallowable bolus.” In accordance, Dou et al. (2019) observed an important role of the oral phase in phenolic release, especially for caffeoylquinic acid, 3-caffeoylquinic acid, and catechin, In addition, mastication can contribute to food structure disruption, collaborating with phenolic release and increasing the surface area for amylase action (Panagopoulou et al., 2021). Furthermore, it is already known that phenolic compounds can inhibit c-amylase action, a well-studied mechanism for diabetes treatmentINFOGEST protocol ‘Treatment after Extraction Dyalisis Fitration Centrifugation FIGURE 2 ii es ‘Treatment before High Hydrostatic Pressure _| Sermination Freezing Fermentation Heat Drying Extraction Pulsed Electric Fields, Pasteurization Enzymatic Hydrolysis, Units used/bioaccessibility formu ‘Beyond digestion factors, the different treatments before (eg, high hydrostatic pressure, germination, freeing, ‘fermentation, hea, drying, extraction, pulsed electric felds, pasteurization, and enzymatic hydrolysis) and after simulated digestion (extraction, dialysis, tration, and centrifugation) make ect comparisons between different studies dificult. The units used to report the results andthe formula to caleulate bioaccessibility of phenolic compounds are also challenging and must be standardized. (Casarin etal, 2021; Gomes et al., 2021). Therefore, during the oral phase, phenolic bioaccessibility can indeed change (Aylanc et al, 2021). It is important to consider the phytochemical and hostile environment of moisture—pH, electrolytes, tem- perature, darkness, decreasing O) content, and enzymes. Asaconsequence, phenolics can be released through enzy- matic hydrolysis or degraded, especially anthocyanins, which can be oxidized by O>, deprotonated at pH 7 and spontaneously cleaved into phenolic derivatives (Sanchez- Velzquez et al., 2021). - The gastric phase favors phenolic stability ‘At the gastric phase, pepsin can hydrolyze the peptide chains, releasing phenolic acids, flavonoid-diglycosidic compounds, and monoglycosides (Guo et al., 2017). In general, pH 2 maintains phenolic stability, mainly anthocyanins (Gomes et al., 2019; Mihailovié et al., 2019; Pineda-Vadillo et al., 2016; Quatrin et al., 2020; Stiibler et al,, 2020), but it may also decrease rutin levels, due to its cleavage from quercetin, which consequently increases (Ma et al, 2019). However, Tomé-Sanchez etal, (2021) reported flavonoid glucoside conversion to aglycones, which decreased flavonoid glucoside bioaccessibility at the end of the gastric phase. Intestinal phase and its controversies In contrast, phenolics can be degraded, precipitated, and become unstable during the intestinal phase (Mihailovié et al., 2016; Pineda-Vadillo et al., 2016) as a result of enzy- matic action and pH on hydroxyl groups. These changes are mostly related to oxidation and/or polymerization reactions, and the formation of high molecular weight phe- nolic derivates with low solubility and different chemical properties (Ucar & Karadag, 2019), such as tion, hydrolyzation, protein binding, low water solubility, and complexation with pancreatin proteins (Thumann et al., 2020), The effects are dependent on the chemical class of the phenolic compounds, and the most affected polyphenols from the intestinal environment are antho- cyanins, resulting in lower bioaccessibility (Eran Nagar et al,, 2021). However, pancreatin can release flavonoid- monoglycosides coupled with lipase and bile extracts, increasing their bioaccessibility (Guo etal, 2017). Knowing that phenolic structures are related to their activity, itis obvious that these changes can modify antiox- dant potential and phenolic bioaccessibility (da Silva cet al, 2019; Gomez-Maqueo et al., 2020; Melo et a., 2020; Pineda-Vadillo etal, 2016; Stiibler et al., 2020). - It is not just a matter of increase or decrease, the transformation of phenolics is also important!= LR Increases and decreases in phenolic bioaccessibility are both observed in the reviewed studies. When diges- tion increases phenolic compound bioaccessibility, it is mainly related to the release of phenolic compounds linked to other components in the vegetal matrix and is ‘a consequence of enzymatic action during the digestion process (Pineda-Vadillo et a., 2016), or to the breakdown of polyphenols with a high degree of polymerization or conjugation in their respective derivatives (Eran Nagar etal., 2021). Additionally, the interactions of phenolic com: pounds with sugars or other dietary compounds (e.g, lipids) released during digestion or present in the food matrix could play a protective role against degradation, affecting their solubility and augmenting their bioaccess bility. This has been observed for catechin and epicatechin from cacao after the digestion process (Martinez-Las Heras ct al, 2017). In agreement, an increase in antioxidant potential during the intestinal phase could be a con- sequence of the formation of new oxidation products, metabolites with a higher antioxidant activity than that of their precursors (Pineda-Vadillo et al, 2016); or the presence or liberation of other antioxidant molecules that are not phenolic compounds (Ng & See, 2019). On the other hand, association with intestinal enzymes, carbohy- rates, or dietary fiber from the food matrix can reduce polyphenol bioaccessibility (Quatrin et al. 2020). Furthermore, digestive enzymes can induce changes in the chemical structures of phenolic compounds, espe- cially hydroxyl groups, which is mainly related to their antioxidant potential and solubility (Neto et al., 2017) Ellagitannins from jaboticaba peel powder have an ele- vated number of hydroxyl groups, and consequently, low. bioaccessibility. In the same food matrix, ellagic acid had lower bioaccessibility than gallic acid (Quatrin etal.,2020). Additionally, phenolics such as procyanidins and epicate- chin gallate may be converted into epicatechin, wl more stable in acidic conditions, while gallic acid can be released from galloylated catechins (de Morais et al., 2020). 45 | Finding 4: Processing extensively impacts phenolic bioaccessibility Phenolic bioaccessibility in vivo is significantly influ- ‘enced by many factors, including (1) the physicochemical characteristics of phenolic compounds, molecular size, sol- ubility, polymerization or glycosylation and conjugation with other compounds (Silva et al., 2018); (2) individual physiological parameters; (3) composition and possible interactions between the different constituents of the food matrix and phenolics (Paz-Yépez et al, 2019; Pesié et a, 2019), as well as its physical structure, which influences the diffusion rate of digestion fluids (Ucar & Karadag, 2019); (4) time of ingestion (empty or full stomach) (Sun et al., 2020); (5) Assay conditions (Martini et al., 2018) and methods applied; and (6) processes applied—as exten sively reviewed by Cilla et a. (2018)—and the focus ofthis topic. ‘Sample structure (Bochnak & Swieca, 2020) or particle size (Loper-Gamez et al, 202), drying temperature and conditions (Bochnak & Swieca, 2020; Takécs et al., 2018), pasteurization, dehydration, frying, freezing, extraction methods—as conventional chemical extraction—addit of oil, fat, and certain enzymes (Thakur et al., 2020), time of drinking—before or after a meal (Sun et al., 2020) or even the parts of a fruit analyzed affects phenolic bioaccessibility Table 3 and Table S3 indicate several studies that reported the impact of processing on phenolic bioaccessi bility after INFOGEST protocol application. Processing especially impacts the chemical stability of phenolics, phenolic interactions, and associations in solutions. Despite causing changes at the beginning of digestion, processing also affects the bioaccessibility pro- file along the digestive tract (Eran Nagar et al., 2021). Alongi et al. (2019) gives an example that shows that ther- mal and ultrasound pasteurization treatments increased the phenolic compound content and antioxidant activ- ity of apple juice. This observation was confirmed by the increased bioaccessiblity of the compounds of interest after simulated digestion using the INFOGEST protocol. Additionally, Beres et al. (2019) demonstrated that the aqueous extract from the white wine Pinot noir grape “Pomace” provided @ higher bioaccessiblity of macro- molecular complexed phenolics after simulated digestion by INFOGEST than the flour from the same material Those results corroborate with the importance of the extraction process, Heat treatment may be an allied process to i phenolic bioaccessibility Heat treatment is extensively discussed in the litera- ture, and its effects on phenolic bioaccessibility through the INFOGEST protocol were highlighted as a positive pro- cessing result (Cattivelli et al., 2021; de Santiago et al., 2018; Lafarga et al., 2019). Some hypotheses are in con- sensus, such as the additive or synergistic effects of phytochemicals released from the matrix during process- ing; polymerization due to heating or concentration during processing (Tomas et al., 2017); Maillard reactions and con sequently the formation of typical high molecular weight end-products, such as melanoidins, could include or retain phenolic compounds in their structures, providing protec- tion Guaniz etal, 2017); and cell wall rupture during heat treatment and the breakdown of covalent bonds resultedin phenolic release, higher extractability and can also inac- tive oxidase enzymes (polyphenol oxidase and peroxidase, for example) in the food matrix, which plays an important role in phenolic oxidation during digestion (Martini etal. 2021). It is important to highlight that even different heat treatments exert different phenolic bioaccessibility behav- iors. As an example, 31 phenolic compounds in tomato changed specifically during heat processing in relation to non-treated samples—rutin for example, increased 36% in industrially processed sauce and decreased 26% in home processed sauce when compared to tomato fruit (Tomas et al., 2017). - Food enrichment and phenolic-macromolecule interactions during digestion, a complex point Another positive application process addressed by the articles that used the INFOGEST protocol to investigate phenolic bioaccessibility or antioxidant properties was food enrichment. Lucas-Gonzilez, Angel Pérez-Alvarez, ct al. (2021) did not see any improvement in spaghetti enrichment with persimmon flour concentrates. On the other hand, phenolics released from bread enriched with artichoke stem flour during digestion correlated with the ‘amount of artichoke used in the formulation (Colantuono etal., 2018), Protein binding may protect phenolic compounds from degradation during digestion. In fact, dairy matrices forti- fied with aqueous extracts, such as those from cinnamon, green coffee, or warrow, exhibited improved phenolic bioaccessibility (Acevedo-Fani et al., 2021; Chait et al., 2021; Helal & Tagliazucchi, 2018; Sgczyk et al., 2017; Villalva et al., 2020). However, these results may be inter- preted as not so promising because, when connected to other molecules, they may not be available for absorption and/or show a lower biological activity (Montiel-Snchez cet al, 2021; Paz-Yépez. et al. 2019). Czubinski et al. (2019) showed that phenolic complexation with proteins decreased protein digestibility. ‘Thus, beyond proteins, dietary fiber and lipids inter: fere with the behavior of phenolics during digestion (Gong cet al., 2019), Rocehetti etal (2021) speculated that a higher dietary fiber content in fortified breads with grape extracts ‘would determine a major protection effect on antocyanins, due to their interactions. A hypothesisis that the fibers can trap the compounds within their matrix during digestion, protecting them from the hostile digestion environment (Bas-Bellver et al., 2020). Also, the bioaccessibility of free phenolic acids, especially ellagitannins, can be reduced with dietary fiber associations (Quatrin et al, 2020). In fact, the results depend on fiber content and type. Sugars and soluble fibers were already associated with a fevEWS | => protective role against pH changes and enzymatic action on phenolic compounds during digestion, while insoluble fibers were reported to be responsible for lower pheno: lic bioaccessibility due to their strong physical interaction through covalent bonds (Bas-Bellver et al., 2020; Giusti et al,,2019).In accordance, Beres etal. (2019) observed that macromolecular complexed phenolics were more bioac- cessible in aqueous grape extracts rich in soluble fiber than in a grape flour rich in insoluble fiber. - Encapsulation and its protective role Encapsulation studies were evaluated as favorable for phenolic recovery, when digestion was followed using the INFOGEST protocol (Ferreira-Santos et al., 2021; Lachowiczetal.,2021). Encapsulation techniques may play a role in phenolic protection, probably by the formation of inclusion complexes and prevention of phenolic adhesion to bile salts or other food matrix compounds tested dur- ing simulated digestion. Consequently, their application promotes an increase in phenolic bioaccessibility (Paulo & Santos, 2020). Interestingly, Carrasco-Sandoval et al. (2021) observed that the composition ofthe encapsulating matrices was not as relevant as the chemical nature of phenolic compounds, and their respective polarity for phenolic bioaccessibility The results indicated that the most polar phenolics were the most bioaccessible with encapsulation Fermentation requires further investigation In general, fermentation impacts are not consistent, since the results are strain dependent (Mashitoa et al. 2021). The environmental conditions (pH during fermen- tation and digestion, fermentation and digestive enzymes, and strain survival during and after digestion) facilitate the release of phenolics and improves their bioaccessibility, such as quercetin, as observed by Mashitoa eal (2021) in pasteurized papaya puree, and by de Assis et al. (2021) in Brazilian fruits. The latter explains such improvement by phenolic transformation into metabolites during fermen- tation, which are then better absorbed during digestion as a consequence of f-plycoside hydrolysis by B-glucosidase, produced by intestinal microbiota (de Assis et al. 2021). ~ Pulsed electric fields (PEFs) improved phenolic ioaccessibility due to their release In general, PEF treatment improved the phenolic bioac- cessibility of different samples—fresh strawberries and kale juice (Stibler et a., 2020), apple (Ribas-Agusti et a., 2019) and whole carrot, juice or purees (Lépez-Gimez= ABIES et al,, 2021a), 2021; Lépez-Gimez, Elez-Martinez, et al. 2021). For other treatments, pulsed electric field (PEF) causes chemical modifications to phenotics—hydroxylation, methylation, formation of phenolic derivatives—and food matrix integrity. Cell wall degradation as a result of PEF application facilitates the release of phenolics during digestion, leading them to be more readily absorbed. On the other hand, despite the positive consequences of PEF processing on phenolic bioaccessibility, some ‘exceptions observed could be a result of released phenolic ‘compounds, which are as follows: entrapment with other molecules, which avoids their crossing through the dial- ysis membrane, and degradation, since modifications in phenolic structure may affect their bioaccessibility (Lopez- Gamez etal, 2021a) , 2021; Lépez- Gamez, Elez-Martinez, et al., 2021), - Not less important Curiously, freezing also showed a positive impact on phenolic extractability, due to its damage tothe food matrix as a consequence of ice crystal formation (Kamiloglu, 2019). Microwave extraction was also largely and posi tively used asan allied process for phenolic bioaccessibility (de Santiago et al., 2018; Ng & Rosman, 2019). The oil matrix nanoemulsion also influenced phenolic bioaccessi- bility (Zhow et al, 2021). Interestingly, germination released phenolics after digestion, as observed by researchers who used the INFO- GEST protocol. The opposite behavior was perceived by the same research group, corroborating the objective of the INFOGEST group and the present review (Tomé-Sanchez cet al, 2021), Gu et al. (2021) explained that such improve- ment reduces the damage to cell structure provided by germination, and consequently, reduces polyphenol oxidase release and phenolic degradation. ‘Additionally, enzymatic hydrolysis improved isoflavone bioaccessibility (de Queirés et al, 2021), and active coat- ings on apple cubes were reported to play an impor- tant protective role in improving phenolic bioaccessibility (Bambace et al, 2021). Furthermore, atmospheric cold plasma when applied to acai samples (Dantas et al, 2021) and extrusion cooking when applied to lupin seed coat (Zhong etal, 2021) also improved bioaccessibility. Finally, high hydrostatic pressure treatment was regarded as an allied process to improve phenolic bioac- cessibility (Eran Nagar et al., 2021; Stiibler et al., 2020; Gémez-Maqueo et al, 2021). Its application increases phe- nolic extractability and improves digestive stability, due to the microstructural changes caused in the food matrix, proteins and pectin modifications (protective effect on phenolics), and active/inactive endogenous enzymes of the food matrix (Gémez-Maqueo et al., 2021) 5 | FUTURE TRENDS As clearly seen, a huge amount of quality data are available. Some remarks already explored in this review emphasize the same trends and need for future research: a standardized pretreatment and post-treatment protocol, utilization of optimized extraction conditions for each food matrix as a way to quantify phenolic amounts correctly, and units and formula standardization. ‘To date, the importance and impact of processing before simulated digestion on phenolic bioaccessibility are clear. Each one contributes differently and causes changes in several proportions. Interestingly, germination and chem- ical and enzymatic hydrolysis as transformation processes were scarcely addressed in studies that employed the INFOGEST protocol. This is intriguing due to their impor- tance and potential application in the procurement of functional foods and ingredients, as well as nutraceuticals and dietary supplements. ‘Acid hydrolysis and its effects on phenolic bioacces- sibility with the INFOGEST protocol were also reported (Mihailovié et al,, 2016) (Table $3), as well as enzymatic hydrolysis, which showed great potential for improved isoflavone bioaccessibility from soy (de Queirés et al., 2021), These processes have already been studied in com- bination with lentils (Casarin et al., 2021). Additionally, de Souza Rocha et al. 2014) indicated the impact of germi- nation and enzymatic hydrolysis of cowpea bean (Vigna unguiculata) on the generation of peptides after simu- lated digestion; however, they did not use the INFOGEST protocol. Tomé-Sénchez et al. (2021) applied two difer- ent bioprocess approaches: germination for wheat and hydrolysis for wheat bran, with the latter stabilized by spray drying and microencapsulation. The positive results emphasize the potential of such bioprocess applications in obtaining functional and nutraceutical ingredients. ‘To our knowledge, some premises could be formulated if the INFOGEST protocol is applied to germinated and/or hydrolyzed grains: (1) germination and hydrolysis as a sin- gle process could improve phenolic bioaccessibility due to phenolic release into free or soluble conjugate forms from their insoluble-bound counterparts before digestion, (2) both processes used together could enhance free or sol- uuble conjugate release, and (3) digestion could play the same role as those processes but render a different phenolic profile. Within this context, both processes are reviewed and detailed in the following sections.