4.2. Fermentation of Buckwheat Flours by LAB, Preparation of Buckwheat Biscuits from Fermented Flours (BBF), and In Vitro Digestion of BBF

Buckwheat flour originating from commercial Polish common buckwheat (Fagopyrum esculentum Moench) was purchased from local industry (Melvit S.A., Kruki, Poland). 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.

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).

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.

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 CaCl₂, 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.