Bioaccessibility of Phenolic Acids and Flavonoids from Buckwheat Biscuits Prepared from Flours Fermented by Lactic Acid Bacteria

The literature reports that the consumption of common buckwheat (Fagopyrum esculentum Moench), exactly the polyphenols it contains, is associated with a wide spectrum of health benefits. Therefore, the determination of the bioaccessibility of phenolic acids and flavonoids from buckwheat biscuits formulated from liquid-state fermented flours (BBF) by selected lactic acid bacteria (LAB) after gastrointestinal digestion was addressed in this study. Bioaccessibility could be defined as the fraction of a compound that is released from the food matrix in the gastrointestinal lumen and used for intestinal absorption. The bioaccessibility of eight phenolic acids (protocatechuic, vanillic, syringic ferulic, caffeic, sinapic, p-coumaric, and t-cinnamic) and six flavonoids (epicatechin, vitexin, orientin, apigenin, kaempferol, and luteolin) were provided for BBF and BBC (buckwheat biscuits prepared from fermented and unfermented flours, respectively). The bioaccessibility indexes (BI) indicated the high bioaccessibility of phenolic acids and improved bioaccessibility of flavonoids from BBF. Moreover, the data provide evidence for the suitability of selected LAB strains to be used as natural sour agents for further bakery product development rich in phenolic acids and flavonoids with LAB-dependent bioaccessibility.


Introduction
Common buckwheat is known as a gluten-free pseudocereal utilized worldwide, while other species are used as a traditional food in some regions such as south of China, Bhutan, the Himalayan hill region from northern Pakistan to eastern Tibet, and in Islek, Europe [1]. The common buckwheat is regularly consumed as raw or roasted groats, or as breakfast cereals, in various bakery products, and enriched non-bakery products (tea, honey, tarhana, sprouts) [2]. Because buckwheat does not contain gluten, it can be consumed by people with celiac disease [3]. The consumption of buckwheat-based products is related to a wide range of biological and healthy activities, such as hypocholesterolemic, hypoglycemic, anticancer, and anti-inflammatory, and buckwheat proteins and antioxidant phenolic compounds, such as phenolics, are presumed to be responsible, at least in part, for these benefits [3][4][5].
A new trend of cereal processing is natural and inoculated fermentation offering a wide range of derived fermented products. The fermentation processes, depending on the water content in the system, can be divided into solid-(SSF) and liquid-state fermentation (LSF). The positive aspects of cereal fermentation include the degradation of antinutrients but also increasing the nutritional value and availability of minerals, proteins, or carbohydrates [5][6][7]. Fermentation of cereals or pseudocereals is carried out mainly by lactic acid bacteria (LAB). An improvement in sensory and baking qualities was demonstrated as a result of the use of sourdough, which, through LAB metabolism,

Bioaccessibility of Phenolic Acids
In this study, the content of the phenolic acids identified in BB F and BB C buckwheat biscuits before and after in vitro digestion was provided. The eight phenolic acids known as derivatives of hydroxycinnamic acid (ferulic, caffeic, sinapic, p-coumaric, t-cinnamic) and derivatives of hydroxybenzoic acid (protocatechuic, vanillic, syringic) were identified. Among phenolic acids, vanillic, protocatechuic, and syringic acids were predominant. The level of phenolic acids (µg/g DM) in BB before and after digestion in vitro is presented in Tables 1 and 2, respectively.
Having the content of phenolic acids (µg/g DM) in buckwheat biscuits prepared from unfermented (BB C ) and fermented flours (BB F ) by selected lactic acid bacteria before and after in vitro digestion, the bioaccessibility indexes (BI) of phenolic acids were calculated, and they are shown in Table 3.
The content of vanillic acid in BB F prepared from fermented flours ranged from 75 to 129 µg/g DM compared with 112 µg/g DM noted in the control BBc (Table 1). Digestion of biscuits led to an increase in the content of vanillic acid, and it was almost two-threefold higher for both BB F and BB C ( Table 2). The bioaccessibility index (BI vanillic ) for both BB F and BB C was >1, indicating high bioaccessibility of vanillic acid. BI vanillic ranged from 1.77 to 3.22 compared with the 1.67 obtained for BBc. The highest BI vanillic was found for BB F baked from flour fermented by L. plantarum W42 and L. rhamnosus K (Table 3). Table 1. The content of phenolic acids (µg/g DM) in buckwheat biscuits prepared from unfermented (BB C ) and fermented flours (BB F ) by selected lactic acid bacteria. Data are expressed as mean ± standard deviation (n = 3). Means in each column followed by different letters are significantly different (p < 0.05) based on the one-way analysis of variance (ANOVA).   The protocatechuic acid was found in BB F within the range of 39-85 µg/g DM compared to 65 µg/g DM noted in BBc (Table 1). After digestion of BB F , its content increased 4-7 times, whereas a threefold higher content was noted in digested BBc ( Table 2). The bioaccessibility index (BI protocatechuic ) for BB F was >3, thus, indicating for very high bioaccessibility of this acid. BI protocatechuic ranged from 2.90 to 7.79 compared with 3.09 obtained for BB C . The highest BI protocatechuic was found for BB F formulated on fermented flours by L. rhamnosus 8/4 and L. salivarius AWH ( Table 3).

Sample/Phenolic Acid
The syringic acid was present in BB C and BB F at a concentration at least threefold lower than the most abundant vanillic acid. It ranged widely from 21 to 159 µg/g DM compared with 43 µg/g DM noted in BB C (Table 1). After digestion of BB F , its content increased significantly (Table 2), resulting in high BI syringic ranging from 2.07 to 10.83 compared with 2.99 obtained for BB C .
The not predominant phenolic acids included para-coumaric, sinapic, trans-cinnamic, caffeic, and ferulic acid, and the following observations were drawn on the basis of their content in BB F and BB C before digestion (Table 1). The content of these acids in BB C was from 3.4 µg/g DM (caffeic acid) up to 21.5 µg/g DM (para-coumaric) compared with the lowest content of 1.4 µg/g DM noted for caffeic acid in BB F by L. rhamnosus 8/4, and the highest one of 28.5 µg/g DM found for para-coumaric acid in BB F by L. rhamnosus K. Generally, the content of these acids noted in BB F was decreased or not changed. There were noted some exceptions made to the selected LAB strain used for flour fermentation, where a significant increase was noted for para-coumaric in BB F by L. rhamnosus K, for sinapic acid by L. casei 2K, for trans-cinnamic by L. plantarum W42, and for caffeic and ferulic acid in BB F by L. casei 2K. Since the baking conditions were the same for BB C and BB F , it is indicated for the impact of the selected LAB on the phenolic acid contents. When the total content of phenolic acids was considered, the flour fermented by L. rhamnosus K offered the highest content in BB F , higher by almost 59% compared with their content in BB C .
In this study, it was found that the digestion of BB F and BB C led to an increase in the content of p-coumaric acid compared with undigested biscuits ( Table 2). BI p-coumaric for BB F ranged from 1.06 to 5.66 compared with 1.22 noted for BB C ( Table 3). The highest BI p-coumaric was found for BB F formulated on fermented flours by L. rhamnosus 8/4. Similar findings were found for BI sinapic , BI t-cinnamic , and BI ferulic, with the highest BI for BB F formulated on fermented flours by L. plantarum W42 (21.79), Streptococcus thermophilus MK-10 (9.15), and by L. rhamnosus GG (4.57), respectively ( Table 3). The widest range of BI was noted for caffeic acid in BB F as it ranged from 3.80 up to 31.32 for fermented flours by L. rhamnosus 8/4. In summary, the digestion in vitro released all phenolic acids in BB F, as is well seen when the average BI is compared to their BI for BB C (Table 3).

Bioaccessibility of Flavonoids
In this study, despite rutin-(quercetin-3-rutinoside) and quercetin-described bioaccessibility in our previous study [12], seven other flavonoids before and after digestion in vitro were identified in BB F and BB C, including epicatechin, vitexin, orientin, apigenin, kaempferol, and luteolin (Tables 4 and 5, respectively). Table 4. The content of flavonoids (µg/g DM) in buckwheat biscuits prepared from not fermented (BBc) and fermented flours (BB F ) by selected lactic acid bacteria. Data are expressed as mean ± standard deviation (n = 3). Means in each column followed by different letters are significantly different (p < 0.05) based on the one-way analysis of variance (ANOVA).

Strain/Flavonoid Epicatechin Vitexin Orientin Apigenin Kaempferol Luteolin
Control biscuits (BBc)  Table 5. The content of flavonoids (µg/g DM) in buckwheat biscuits prepared from not fermented (BB C ) and fermented flours (BB F ) by selected lactic acid bacteria after in vitro digestion. Data are expressed as mean ± standard deviation (n = 3). Means in each column followed by different letters are significantly different (p < 0.05) based on the one-way analysis of variance (ANOVA). The bioaccessibility of flavonoids provided in detail (Table 6) is based on their content in BB F and BB C before and after digestion in vitro. The epicatechin was the major flavonoid found in BB F in a wide range from 17.9 to 127.6 µg/g DM, depending on the LAB strain used for flour fermentation compared with 91.7 µg/g DM noted for BB C (Table 4). It was also found that BB F contained about threefold lower epicatechin content than BB C, with the exception of biscuits baked from fermented flours by L. rhamnosus 8/4, L. rhamnosus K, and L. salivarius AWH. The differential behavior of epicatechin was noted after digestion of BB F, as in some cases, epicatechin was released from BB F, or no changes were observed. However, the epicatechin level in BB F after digestion was increased compared with its level in BB C (Table 5). Therefore, the average BI epicatechin for BB F was 1.04, and it was almost five times higher compared with its value for BB C . The highest BI epicatechin was noted for BB F prepared from flours fermented by L. acidophilus V and L. acidophilus 145 (Table 6).

Strain/Flavonoid
A similar trend was noted for vitexin, orientin, apigenin, and luteolin; however, their content in BB F was lower than epicatechin. Their BI indexes were higher than one, indicating the high bioaccessibility in contrast to the BI value lower than one noted for BB C . The opposite data were provided for kaempferol since its content was decreased after digestion of both BB F and BB C ( Table 5), but its bioaccessibility was still very high ( Table 6).
As shown in Figure 1, all LAB strains used for buckwheat flour fermentation, despite L. casei LcY and Streptococus thermophilus MK-10, offered a buckwheat dough matrice from which phenolic acids and flavonoids were easier released into digestion fluid. It was noted that phenolic acids formed the main fraction after digestion in vitro compared with flavonoids.

Bioaccessibility of Phenolic Acids
There is an increasing interest in a healthy lifestyle and the consumption of substantial portions of secondary plant metabolites, such as polyphenols, because of their benefits for the human body. As human studies are time-consuming, costly, and restricted by ethical concerns, in vitro models for investigating the effects of digestion on these compounds have been developed to predict their release from the food matrix, as well as their bioaccessibility [16]. The most widely used procedure for screening polyphenolic compound bioaccessibility is the in vitro static GI method [17]. Contrary evidence on the bioaccessibility of phenolic compounds is available in the literature. Carbonell-Capella et al. [17] showed that gastric digestion increased polyphenolic concentration, whereas the duodenal fraction significantly diminished polyphenolic content. In contrast, Tagliazucchi et al. [18] observed an increase in the bioaccessibility of total polyphenols, flavonoids, and anthocyanins during the gastric digestion in grapes, while intestinal digestion caused a decrease in all classes of polyphenols.
It was shown that in vitro digestion released much higher levels of total phenolic compounds (TPC) from biscuits obtained from fermented buckwheat flour compared with biscuits before digestion, which indicated a much better extraction system for phenolic compounds, which was the digestion fluid, compared with the classical extraction [11]. Generally, an increase in the potential bioaccessibility of TPC was observed. As a consequence, the individual phenolic compounds responsible for this increase in the bioaccessibility of TPC should be indicated. The data on the bioaccessibility of phenolic acids from the buckwheat matrix modified by the use of fermented flour for baking are still limited. In this study, it was shown that vanillic, protocatechuic, and syringic acids were predominant in buckwheat biscuits (control and obtained from fermented flour). Previously it was shown that the baking of BBF and BBC resulted in a reduction in the average content of phenolic acids [11]. Heat treatment may enhance polyphenol bioaccessibility because of disruption of plant tissue and denaturation of polyphenols-polysaccharide complexes. However, heat treatment may also cause thermal degradation of phenolic compounds [19]. As was presented in the review by Wojtunik-Kulesza et al. [20] during consideration of in vitro bioaccessibility studies, chemical and biochemical reactions or

Bioaccessibility of Phenolic Acids
There is an increasing interest in a healthy lifestyle and the consumption of substantial portions of secondary plant metabolites, such as polyphenols, because of their benefits for the human body. As human studies are time-consuming, costly, and restricted by ethical concerns, in vitro models for investigating the effects of digestion on these compounds have been developed to predict their release from the food matrix, as well as their bioaccessibility [16]. The most widely used procedure for screening polyphenolic compound bioaccessibility is the in vitro static GI method [17]. Contrary evidence on the bioaccessibility of phenolic compounds is available in the literature. Carbonell-Capella et al. [17] showed that gastric digestion increased polyphenolic concentration, whereas the duodenal fraction significantly diminished polyphenolic content. In contrast, Tagliazucchi et al. [18] observed an increase in the bioaccessibility of total polyphenols, flavonoids, and anthocyanins during the gastric digestion in grapes, while intestinal digestion caused a decrease in all classes of polyphenols.
It was shown that in vitro digestion released much higher levels of total phenolic compounds (TPC) from biscuits obtained from fermented buckwheat flour compared with biscuits before digestion, which indicated a much better extraction system for phenolic compounds, which was the digestion fluid, compared with the classical extraction [11]. Generally, an increase in the potential bioaccessibility of TPC was observed. As a consequence, the individual phenolic compounds responsible for this increase in the bioaccessibility of TPC should be indicated. The data on the bioaccessibility of phenolic acids from the buckwheat matrix modified by the use of fermented flour for baking are still limited. In this study, it was shown that vanillic, protocatechuic, and syringic acids were predominant in buckwheat biscuits (control and obtained from fermented flour). Previously it was shown that the baking of BB F and BB C resulted in a reduction in the average content of phenolic acids [11]. Heat treatment may enhance polyphenol bioaccessibility because of disruption of plant tissue and denaturation of polyphenols-polysaccharide complexes. However, heat treatment may also cause thermal degradation of phenolic compounds [19]. As was presented in the review by Wojtunik-Kulesza et al. [20] during consideration of in vitro bioaccessibility studies, chemical and biochemical reactions or physical constraints occurring within food must be taken into account. Additionally, the release from the food matrix, particle size or pH-dependent transformations, and interactions between polyphenols and food components should be taken into account. For example, it was shown that the bioaccessibility of sinapic acid from bran-rich bread was much higher than that of ferulic acid and para-coumaric acid [21]. However, most phenolic compounds remain stable during salivary and gastric digestion [22]. Managa et al. [23] demonstrated that lactic acid bacteria used for fermentation of a smoothie composed of pineapple and chayote leaves increase the total phenol. These authors found that after in vitro digestion, fermentation improved the total phenol recovery by 66% during the intestinal phase compared with the control sample. After digestion, the TPC of mango juices decreased, while LAB-fermentation improved its bioaccessibility [24]. Bloem et al. [25] showed that Oenococcus oeni was not able to convert vanillic acid into vanillin. Micro-organisms, such as yeast, are also able to metabolize vanillin to vanillic acid or vanillyl alcohol by oxidoreductase enzymes [26]. Phelps and Young [27] demonstrated that the plant phenolic compounds ferulic and syringic acid were readily degraded by consortia of bacteria from this site under methanogenic, sulfidogenic, and denitrifying conditions.

Bioaccessibility of Flavonoids
Since the beneficial health effects of flavonoids depend on their absorption in the gut [28,29], their bioaccessibility is important to indicate their possible influence on the human organism. Rutin is the main buckwheat flavonoid, whereas quercetin is present in significantly lower concentrations [30], and our previous investigation showed that fermentation, baking, and in vitro digestion significantly affect their content [12]. It was found that the expanded bioaccessibility of rutin from BB F was low, and the BI of quercetin was greater than 1. Payne et al. [31] found that epicatechin, compared with catechin, is as much as 30 times greater in fresh and dried cocoa beans, but as conventional processing occurs, there is a loss in epicatechin and, at times, an increase in catechin.
Choi et al. [32] showed that the total flavonoid contents of the various buckwheat food matrices were higher after digestion compared with the predigested form, which indicated that flavonoids are easily released by in vitro digestion. These authors found that processed buckwheat samples had improved flavonoid bioaccessibility upon baking, which indicated that they are easily released from the food matrix by both digestion and baking. A significant increase of 7 out of 11 flavonoid compounds after in vitro gastrointestinal digestion of quinoa products was presented by Balakrishnan and Schneider [33]. Thilakarathna and Rupasinghe [34], in the review, showed that flavonoids had shown promising health-promoting effects in human cell culture, experimental animal, and human clinical studies. Still, an investigation is required to enhance the bioavailability and subsequent efficacy of certain flavonoids using consumer-friendly technologies.

Chemicals
Reagents in MS grade, including acetonitrile, methanol, water, and formic acid, were purchased from Sigma Chemical Co.  According to the produced declaration, the carbohydrate, dietary fiber, proteins, and fat content of buckwheat flour and roasted buckwheat groats were 62%, 2.3%, 7.2%, and 0.7% on a dry basis, respectively. Before fermentation, the buckwheat flour was pretreated as follows: about 50 g of flour was suspended with 950 mL of distilled water, heated at 90 • C for 45 min, then autoclaved at 121 • C for 15 min, and finally cooled to 37 • C. The pretreatment was carried out to reduce microbial populations in buckwheat flour before fermentation since they would compete with and inhibit the growth of inoculated microbes during the fermentation process.

Fermentation of Buckwheat Flours
The following selected lactic acid bacteria were used: L. acidophilus (145, La5, V); L. casei (LcY, 2K); L. delbruecki subsp. bulgaricus (151, K); L. plantarum (W42, IB); L. rhamnosus (GG, 8/4, K); L. salivarius AWH and Strepcococcus thermophilus Mk-10, all strains originated from the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences' collections. The Lactobacillus rhamnosus GG was purchased from ATCC ® . Fermentation of buckwheat flours was carried out as follows: the 5% suspension of pretreated buckwheat flour in distilled water was inoculated with selected lactic acid bacteria with an amount of 8.00 log CFU/mL, and fermentation was performed at 37 • C for 24 h. The pretreated buckwheat flour not subjected to a fermentation process was used as a control sample. After fermentation, the samples were freeze-dried (Christ-Epsilon 2-6D LSC plus, Germany).

Preparation of BB F from Fermented Flour
The biscuit dough was prepared according to the AACC 10-52 method [35], with the modification proposed by Hidalgo and Brandolini [36]. The dough was cut with a square cookie cutter (60 mm). BBs were baked at 220 • C for 30 min (electric oven DC-21 model, Sveba Dahlen AB, Fristad, Sweden). The control biscuits (BBc) were formulated on unfermented buckwheat flour. The buckwheat biscuits were lyophilized, milled, and stored in a refrigerator until analysis.

In Vitro Digestion of Buckwheat Biscuits
The BB F and BB C were in vitro digested as described by Delgado-Andrade et al. [37] with some modifications [38]. Briefly, the three steps of digestion were saliva (pH 7.0), gastric (pH 2.0), and intestinal digestion (pH 7.5). Briefly, 10 g of lyophilized and milled buckwheat biscuits was suspended in 80 mL of deionized water. An α-amylase solution (77 U/mg solid) was added to the samples at a proportion of 3.25 mg/10 g of sample dry matter (d.m.) in 1 mM CaCl 2 , pH 7.0. Then, samples were shaken in a water bath at 37 • C for 30 min. For gastric digestion, the pH was reduced to 2.0 with 6 N HCl, and pepsin solution (738 U/mg) was added in the amount of 0.5 g/10 g of sample d.m. in 0.1 N HCl. The incubation was continued under the same conditions for 120 min. In the next step, the pH was adjusted to 6.0 with 6 M NaOH, and a mixture of pancreatin (activity 8xUSP) and bile salts extract was added. Subsequently, the pH was increased to 7.5 with 6 M NaOH, and water buffering to a pH of 7.5 was introduced to obtain a final volume of 150 mL. Then, the samples were incubated at 37 • C for 120 min. After incubation, the digestive enzymes were inactivated by heating at 100 • C for 4 min and cooled for centrifugation at 5000 rpm for 60 min at 4 • C in an MPV-350R centrifuge (MPW Med. Instruments, Warsaw, Poland). The supernatants obtained were stored at −18 • C for the evaluation of the bioaccessibility of phenolic acids and flavonoids from water biscuits.

Extraction, Isolation, and HPLC Analysis of Phenolic Compounds from BB F before and after In Vitro Digestion
The analysis of polyphenols (phenolic acids and flavonoids) was conducted according to the modified method of Wiczkowski et al. [39]. In the first step, about 0.05 g of freezedried samples was extracted 5 times with 80% MeOH. Next, polyphenolic compounds (forms released from soluble esters and soluble glycosides as well as free forms) were separated from the methanolic extracts in several stages. In the case of free forms of polyphenols, after adjusting the primary extract to pH 2 with 6 M HCl, the isolation by diethyl ether was carried out. However, in the case of conjugated forms (esters and glycosides), before adjusting the extract to pH 2 and the extraction of released forms of polyphenols by diethyl ether, the hydrolysis under a nitrogen atmosphere was executed for 4 h at room temperature with 4 M NaOH and subsequently in the condition of 6 M HCl for 1 h at 100 • C. After each hydrolysis, the extraction process was conducted in triplicates by utilizing sonication and centrifugation, and the collected ether extracts were evaporated to dryness under a nitrogen atmosphere at 35 • C. For the analysis of the profile and content of phenolic acids and flavonoids, the HPLC system (LC-200, Eksigent, Vaughan, ON, Canada) coupled with a mass spectrometer (QTRAP 5500, AB Sciex, Vaughan, ON, Canada) consisting of a triple quadrupole, ion trap, and ion source of electrospray ionization (ESI) was used. The chromatographic separation was conducted with a HALO C 18 column (50 mm × 0.5 mm × 2.7 µm, Eksigent, Vaughan, ON, Canada) at 45 • C, at the flow rate of 15 µL/min. Identification and quantitation of the phenolic acids and flavonoids were based on the comparison of their retention times and the presence of the respective parent and daughter ion pairs (Multiple Reaction Monitoring method, MRM) with data obtained after analysis of the authentic standards.

Calculation of the Bioaccessibility Index of Phenolic Compounds
In this study, we determined the bioaccessibility index (BI) [38] of individual phenolic acids and flavonoids, which was calculated according to the following formulas: BI PA = PA GD /FA BB and BI F = F GD /F BB where PA GD is the indicated phenolic acid content after simulated gastrointestinal digestion (GD), F GD is indicated flavonoid content after simulated gastrointestinal digestion (GD), PA BB is the indicated phenolic acid content in BB, and F BB is indicated flavonoid content in BB. BI PA and BI F values > 1 indicate high bioaccessibility of phenolic acids and flavonoids from BB (BB F and BB C) ; BI FA and BI F values < 1 indicate low bioaccessibility.

Statistical Analysis
Results are given as the average ± standard deviation (SD) of n = 3 independent experiments. They were determined by one-way analysis of variance (ANOVA) with Fisher's least significant difference test (p < 0.05). All analyses were made using STATISTICA for Windows (StatSoft Inc., Tulsa, OK, USA, 2001).

Conclusions
The bioaccessibility indexes of phenolic acids and flavonoids from buckwheat biscuits formulated from flours fermented by selected LAB are important factors in understanding the bioavailability of these compounds. The eight phenolic acids (protocatechuic, vanillic, syringic, ferulic, caffeic, sinapic, p-coumaric, and t-cinnamic) and seven other flavonoids than rutin and quercetin, including epicatechin, vitexin, orientin, apigenin, kaempferol, and luteolin were identified in buckwheat biscuits before and after digestion in vitro. The obtained data indicated the high bioaccessibility of phenolic acids and improved bioaccessibility of flavonoids under the influence of the fermentation and baking processes used. The study provides evidence for the suitability of selected LAB strains to be used as natural selected sour agents for further bakery product development rich in indicated phenolic acids and flavonoids with high bioaccessibility.