Buckwheat hull, a valuable bakery product ingredient: assessment of bioaccessible phenolics and antioxidant capacity

In this study, the bioaccessible total phenolic compounds (TPC) and antioxidant capacity (AC) of commonly consumed bakery products enriched with raw (BH) or roasted (RBH) buckwheat hull were evaluated. The soluble and insoluble fractions obtained after in vitro enzymatic digestion of mixed rye/wheat bread with 4% of RBH (BRBH), wheat bread with 3% of BH (BBH), and their control counterparts were separated (C-BRBH and C-BBH, respectively). The addition of buckwheat hull, raw and roasted, significantly increased the values of analyzed parameters compared to control samples. Before the digestion, the highest values of TPC and AC were found for bread with 4% of RBH. After in vitro digestion of the bakery products, the content of TPC and AC in the soluble fraction was 75–90% higher compared to the values found in the undigested fraction. Generally, a decrease in the bioaccessibility index of enriched bakery products compared to control samples was observed. The obtained results indicate that buckwheat by-products may be used as a valuable ingredient for commonly used bakery products. Also, it was shown that the in vitro digestion model may be of relevance in assessing the bioaccessibility of bioactive compounds in commonly used bakery products.


Introduction
Buckwheat contains many nutrients and bioactive components that positively affect the human body as was presented by Kreft [1]. This pseudocereal is a good food source of nutritionally valuable components: protein with well-balanced amino acid composition, fiber, minerals, and in combination with other health-promoting components. Buckwheat is widely used in the world in various food products due to its proven hypotensive, hypocholesterolemic, hypoglycemic, neuroprotective, and antioxidant properties [2]. The main by-product during the obtaining of kasha or flour from buckwheat is the hull. From the literature data, it is known that buckwheat hull could be used as an additive in solid biofuel production [3,4]. Also, there is some information on the application of these by-products to yogurt [5], noodles [6], or tea [7]. There are also studies on the use of an extract obtained from buckwheat hull in frozenstored meat products [8] or mayonnaise [9], in both cases for the prolonged shelf life of products. As was indicated by Liu et al. [6], buckwheat hull has many already proven nutritional properties. It has more bioactive compounds than buckwheat flour, as presented by Zhang et al. [10]. Dziadek et al. [11] found in six selected cultivars and strains of common buckwheat (whole seeds, dehulled seeds, and hulls) the highest content of dietary fiber and total polyphenols as well as the highest antioxidant activity in the hulls compared to other examined samples. As presented in our previous investigations [12], commonly consumed bakery products can be enriched by the addition of buckwheat flour or hull and has a positive influence on the antioxidative potential as well as on the sensory and storage properties of the obtained products.
The antioxidant capacity of cereals or pseudocereals is difficult to measure because the compound responsible for this capacity is bound to the cell wall [13]. The main problem during the analysis of antioxidant capacity 1 3 is encountered during the extraction of bioactive compounds from the food matrix, because their health benefits depend on bioaccessibility. Wojtunik-Kulesza et al. [14] in the review presented how the in vitro digestion conditions could influence the composition, bioaccessibility, and antioxidant activity of food polyphenols. The constituents which are released from the solid food matrix into a soluble phase may be expected to be more likely absorbable by the organism [15]. The bioavailability of bioactive compounds, which have high antioxidant activity, administered orally in pure form is much greater than the bioavailability of the same compounds from the food product matrix as presented by Dima et al. [16]. The method called QUENCHER, elaborated by Gökmen et al. [17], can be used to assess in vitro the total antioxidant capacity of solid foods or solid fractions after digestion without extraction. It enables evaluating the antioxidant activity in the residue portion obtained when food is exposed to in vitro intestinal digestion [18].
The review of Angelino et al. [19] showed potential strategies to improve phenolic bioaccessibility and bioavailability in bread. These strategies include the use of raw materials with high phenolic content or different processing technologies, such as fermentation or enzymatic treatment of ingredients. During in vitro hydrolysis of buckwheat products, Choi et al. [20] found that the baking process positively affects flavonoid digestibility and bioaccessibility. Whereas Świeca [21] showed a positive effect of elicitation, supported by the phenylpropanoids precursors, there was an increase in the phenolics content and antioxidant capacity of the potentially bioaccessible fraction of buckwheat sprouts.
This study aimed to investigate the effect of in vitro digestion of enriched commonly produced and consumed bakery products on the bioaccessible and non-bioaccessible total phenolic compounds (TPC) and antioxidant capacity (AC) content.

Buckwheat-enriched bakery product
All ingredients and procedures used for preparing bakery products were presented by Wronkowska et al. [12]. Briefly, commonly produced and consumed bakery products (in the northern part of Poland) were enriched with buckwheat hull: raw (BH) or roasted (RBH). In this study, wheat bread with 3% BH and rye/wheat bread with 4% RBH were used. The control samples were products made without the hull addition. Both types of buckwheat hulls were obtained from the common buckwheat (Fagopyrum esculentum Moench) during the dehulling process used in the preparation of unroasted and roasted buckwheat groats. Both hulls were milled into powder to particles < 400 μm before addition to bakery products (mill WZ-1, ZBPP, Bydgoszcz, Poland).

In vitro digestion of bakery products
The in vitro digestion was performed according to Delgado-Andrade et al. [22] using three steps to mimic digestion in the digestive tract: the mouth, stomach, and intestine. Fractions obtained after in vitro digestion, soluble and insoluble ones, were used for further analysis. The insoluble fraction was freeze-dried, and the soluble fraction was used in liquid form.

Determination of TPC and AC in bakery products before in vitro digestion
The extraction procedure and analysis of TPC content and AC (measured by ABTS) and photochemiluminescence (PCL) assay in bakery products before digestion were done according to Zieliński et al. [23].

Determination of TPC and AC in bakery products after in vitro digestion
The bioaccessible TPC and AC of the soluble fraction after digestion were evaluated according to Szawara-Nowak et al. [24]. The determination of non-bioaccessible TPC and nonbioaccessible AC of the insoluble fraction after sample digestion was conducted by the QUENCHER method as described by Szawara-Nowak et al. [22].

Statistical analysis
All types of analyzed samples were baked in two replications (3 loaves in each). Results are given as the means and the standard deviation of six independent measurements. To compare TPC and AC data obtained for buckwheatenhanced bakery products and control samples, the analysis of variance (one-way ANOVA) was used. Also, the same analysis was done to compare data obtained before and after in vitro digestion of the examined bakery products. The Tukey test at a significance level of p < 0.05 was performed for post hoc comparison (Statistica 7.1, StatSoft, Kraków, Poland). Bioaccessible total phenolic compounds (TPC) and antioxidant capacity (AC) were assessed.

Results and discussion
In this study, commonly produced and consumed bakery products (wheat and mixed rye/wheat) before and after enrichment with buckwheat hull, raw (BH) or roasted (RBH) (by-products obtained during the buckwheat dehulling process), were analyzed for the content and bioaccessibility of total phenolic compounds and antioxidant capacity pre-and post-digestion in the in vitro model.

Total phenolic compound content before and after in vitro digestion
Before digestion, bakery products enriched by both used BH showed significantly higher TPC content compared to control samples ( Table 1). The addition to the bread of roasted BH (BRBH) had a significantly greater influence on TPC content compared to the addition of raw BH (BBH). Li et al. [25] showed that extract from buckwheat hull exhibited higher TPC and AC compared to extract from buckwheat flour. Zhang et al. [10] found higher total phenolic content of buckwheat hull and bran compared to flour. Baking was reported to lead to a TPC decrease in wheat cookies enriched with buckwheat flour, but the TPC value of buckwheat cookies was higher compared to the wheat ones, as presented by Jan et al. [26].
In all the soluble fractions obtained after in vitro digestion, a significant increase of bioaccessible TPC compared to the samples before digestion was observed (Table 1). It was found that the insoluble solid fraction, resistant to enzymatic digestion, was still the source of non-bioaccessible TPC, which was still present in a sample but in a significantly lower amount compared to samples before digestion. Chandrasekara and Shahidi [27] found TPC increase after in vitro digestion of millet grains. The same observation was made by Gawlik-Dziki et al. [28] for the TPC of wheat bread under simulated digestion. Szawara-Nowak et al. [24] demonstrated that the TPC content was higher in the soluble fraction obtained after digestion of wheat bread enriched with buckwheat flour compared to the insoluble fraction. During the gastric phase of grain digestion, proteins are digested and some phenolic bound to them may be released as was shown by Chandrasekara and Shahidi [27]. But some parts of nonbioaccessible TPC may reach the colon unchanged, and in this part of the gastrointestinal tract may be transformed by the gut microbiota enzymes into a wide range of phenolic acids [29].

Antioxidant capacity before and after in vitro digestion
In our study, the AC of bakery products before and after in vitro digestion was analyzed using two assays: against ABTS +• radicals and against superoxide anion radicals (O 2 −• ). Obtained data were shown in Table 1. Before digestion, the AC measured against ABTS +• radicals of the examined samples were as follows: C-RBH < RBH < C-BRBH < BRBH. Products BRBH and RBH showed significantly higher AC compared to control samples before digestion. After the in vitro digestion, the bioaccessible and non-bioaccessible AC significantly increased in all soluble and insoluble fractions obtained from the experimental bakery products. The ability of soluble fractions to scavenge ABTS +• radicals was more than 96% higher compared to samples before digestion. The highest value was found for the soluble fraction obtained from bread with 3% of raw BH (BBH) and its control (C-BBH), 66.81 and 67.28 μmol Trolox g −1 DM, respectively. After digestion, the insoluble fractions obtained from the buckwheat-enriched bakery products were still a source of compounds capable of scavenging ABTS +• radicals. In our study, the differences in the AC of the soluble and insoluble fraction were as large as those presented by Szawara-Nowak et al. [24] for wheat bread enriched with buckwheat flour. Baublis et al. [30] showed that simulated gastrointestinal pH treatments and enzymatic hydrolysis could increase the AC of wheat and wheat-based breakfast cereal extracts. Rufián-Henares and Delgado-Andrade [18] found that all the digestive processes occurring in the gastrointestinal tract could enhance the AC of grains, flour, or bread. As presented by Marques et al. [31], polyphenols may affect the activity of digestive enzymes through binding within the active site and changing their secondary structure. Phenolic compounds may also reduce the bile salts' emulsification capacity. The results presented in Table 1 showed that compared to the ABTS assay, lower values of the scavenging effect obtained by the PCL method were obtained. Before digestion, the highest AC measured by PCL ACW assays was observed in extracts from BRBH (1.23 μmol Trolox g −1 DM).
For all analyzed samples (control and buckwheat enriched) the increase of PCL ACW values in the soluble fraction, obtained after digestion, compared to samples before digestion was noticed. The ability to scavenge superoxide anion radicals was not determined in the insoluble fraction due to the limitations of the methodology. Chandrasekara and Shahidi [27] found that millet grain phenolics bound to the insoluble fiber could be released during in vitro gastrointestinal digestion. They also showed that different millet types could demonstrate different AC under the digestion conditions. The contents of polyphenols and thus the AC of extracts obtained during digestion of cereal samples were significantly higher compared to aqueous-organic extracts, as presented by Perez-Jimenez and Saura-Calixto [32]. This phenomenon suggested that the number of antioxidants released during digestion in the human intestines may be higher than could be expected from the data based on chemical extracts.

Potential bioaccessibility of TPC and AC of the examined bakery products
For better evaluation of the bioaccessibility of TPC and AC in vitro, the bioaccessibility index (BI TPC , BI ABTS, and BI PCL ) was determined and defined as follows: Similarly, the BI was analyzed for AC determined by ABTS and PCL ACW assays. BI value higher than 1 indicates high bioaccessibility, and lower than 1 indicates low bioaccessibility. Gawlik-Dziki et al. [33] introduced BI as a useful parameter to study the bioaccessibility of phenolics from coffee and coconut. As it was shown in Fig. 1, the values of BI TPC , BI ABTS , and BI PCL were higher than 1 for all bakery products studied, which is indicated by their high bioaccessibility. Generally, the BI values for all investigated enriched bakery products were lower compared to control samples. The only exception is the values obtained for the BI TPC for bread BBH. For bread BRBH and its control, no statistically significant difference was found for the indicator BI TPC and BI PCL . Hemery et al. [34] found that the amounts of bioaccessible phenolic acids were higher in whole-grain bread and bran-rich bread than in white bread, and the finer the bran particles in bran-rich bread, the more bioaccessible were the phenolic acids. However, Irakli et al. 35 showed that the amounts of bioaccessible phenolic acids released in vitro digestion of wheat bread with the addition of hop sourdough combined with rice bran were lower compared to the control sample.

Conclusions
Commonly consumed bakery products enriched with buckwheat by-products were subjected to in vitro digestion and the bioaccessible TPC and AC from soluble fraction obtained after digestion were determined, whereas the non-bioaccessible TPC and AC were investigated in the insoluble fraction. Also, for the examined products, the bioaccessibility index (BI) was calculated. Before digestion, the highest TPC among analyzed bakery products had the bread with 4% of roasted buckwheat hull (1.80 mg GAE g −1 DM). After in vitro digestion of the examined products, the content of TPC and AC in the soluble fraction was 75-90% higher as compared to the insoluble ones. The values of BI showed that enrichment with raw or roasted buckwheat hull (BH, BRH) does not affect the bioaccessibility of TPC and AC from bakery products. BI TPC = (TPC of soluble fraction after digestion)∕ (TPC before digestion).
However, it was shown that the in vitro digestion model may be of relevance in assessing the bioaccessibility of bioactive compounds in bakery products.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The bioaccessibility index (BI) indicates the potential bioaccessibility of TPC and AC of the examined bakery products. BRBH bread with 4% of roasted buckwheat hull, C-BRBH control for BRBH, BBH bread with 3% of raw buckwheat hull, C-BBH control for BBH. Values with different letters (a,b) are significantly different; (p < 0.05), differences between the control and sample with buckwheat hull