Bioaccessibility of D-chiro-inositol from water biscuits formulated from buckwheat flours fermented by lactic acid bacteria and fungi

LWT - Food Science and Technology 106 (2019) 37–43

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Bioaccessibility of D-chiro-inositol from water biscuits formulated from buckwheat flours fermented by lactic acid bacteria and fungi

T

Henryk Zielińskia,∗, Joanna Honkea, Natalia Bączeka, Anna Majkowskab, Małgorzata Wronkowskaa a

Department of Chemistry and Biodynamic of Food, Division of Food Sciences, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748, Olsztyn, Poland b Microbiological Laboratory, Division of Food Sciences, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748, Olsztyn, Poland

A R T I C LE I N FO

A B S T R A C T

Keywords: Fermented buckwheat flours Water biscuits Digestion D-chiro-inositol

In this study, the bioaccessibility of D-chiro-inositol (DCI) from water biscuits formulated from raw and roasted buckwheat flours originating from common buckwheat after liquid-state fermentation (LSF) by select lactic acid bacteria (LAB) and fungi Rhizopus oligosporus 2740 was studied. The LAB-dependent variation in DCI content in fermented buckwheat flours was noted. LSF by L. salivarius AWH and R. oligosporus 2740 significantly enhanced the DCI content, whereas most of the applied LAB reduced DCI in the fermented flours. Baking at 220 °C for 30 min significantly enhanced the DCI level; however, no correlation was found between the DCI content in fermented flours and the biscuits prepared from them. The potential bioaccessibility of DCI from water biscuits was low. It can be concluded that, by applying select LAB and R. oligosporus 2740 for LSF, the heat treatment and physical structure of biscuits were mainly responsible for the potential bioaccessibility of DCI.

1. Introduction The health benefits of buckwheat consumption are related to the wide range of biological and pharmacological activities: antioxidant, hypotensive, antispasmodic, hypocholesterolaemic, hypoglycaemic, anticancer, anti-inflammatory and anti-glycation activities. The D-chiroinositol (DCI), fagopyritols, well balanced amino acid profile, lysine, flavonoids and phenolic acids, phytosterols, vitamin B, thiamin-binding proteins, tocopherols, reduced glutathione, inositol phosphates and melatonin present in buckwheat have been claimed as the biologically active compounds responsible for the benefits associated with buckwheat consumption (Giménez-Bastida, Laparra, Bączek, & Zielinski, 2018; Giménez-Bastida & Zieliński, 2015; Zhang et al., 2012). DCI, also known as a biologically active isomer of vitamin B8, occurs in buckwheat seeds and buckwheat-derived food products in relatively high levels (Horbowicz & Obendorf, 2005; Przygodzka & Zieliński, 2015). DCI was able to decrease plasma glucose in obese rhesus monkeys with various degrees of spontaneous insulin resistance and streptozotocin-treated rats, and therefore it is recognized as a compound with insulin-like bioactivity (Kawa, Taylor, & Przybylski, 2003; Ortmeyer, Bodkin, Lilley, Larner, & Hansen, 1993). The use of

dietary D-chiro-inositol has been patented for treatment for type II diabetes mellitus (Larner & Kennington, 1992). Recently, it was reported that D-chiro-inositol enriched tartary buckwheat bran extract lowered the blood glucose level in mice, confirming the beneficial effect of this compound (Yao et al., 2008). Moreover, the oral DCI administration showed a positive correlation with a decreasing effect on insulin resistance in women with polycystic ovary syndrome (Cheang et al., 2008). More recently, Maurizi et al. (2017) performed a pilot study of Dchiro-inositol plus folic acid in overweight patients with type 1 diabetes. Their trial demonstrated that oral supplementation of DCI plus folic acid can improve metabolic control in overweight T1D patients. DCI naturally exists in free form and its galactosyl derivatives. The major form of these derivatives present in buckwheat is named fagopyritols which are R-D-galactopyranosyl-D-chiro-inositols with one to three galactosyl moieties (Horbowicz, Brenac, & Obendorf, 1998). It is believed that R-galactosidase can hydrolyse fagopyritols and release Dchiro-inositol (Cid, Alfonso, & Lomas, 2004). However, R-galactosidase does not occur in the human stomach; therefore, the enrichment of DCI in foods seems to be necessary. For this reason, an interest in buckwheat (Fagopyrum esculentum Moench) as a source of DCI is increasing (Yang & Ren, 2008).

∗ Corresponding author. Department of Chemistry and Biodynamic of Food, Division of Food Science, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-748, Olsztyn, 10 Tuwima Str., Poland. E-mail address: [email protected] (H. Zieliński).

https://doi.org/10.1016/j.lwt.2019.02.065 Received 31 October 2018; Received in revised form 9 January 2019; Accepted 18 February 2019 Available online 19 February 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.

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dry ingredients were blended for 30 s with a planetary rotation of mixing within a 5-speed mixer (Kitchen Aid, St. Joseph, MI, USA), and then the remaining ingredients and deionized water were added and mixed again for 3 min. The dough was cut with a square cookie cutter (60 mm). Baking was carried out at 220 °C for 30 min in an electric oven DC-21 model (Sveba Dahlen AB, Fristad, Sweden). The buckwheat water biscuits obtained were lyophilized, milled and stored in a refrigerator until analysis.

Cereals and pseudocereals are good sources of nutrients for a number of species of the Lactobacillus genus (Charalampopoulos, Vazquez, & Pandiella, 2009; Muller, Wolfrum, Stolz, Ehrmann, & Vogel, 2001). The fermentation of cereal matrix leading in the degradation of anti-nutritional factors increases the nutritional value of cereal-based food (Simwaka, Chamba, Huiming, Masamba, & Luo, 2017). The benefits of lactic fermentation of buckwheat was recently described by Zieliński, Szawara-Nowak, Bączek, and Wronkowska (2019). They are include the production of organic acids (lactic and acetic), the pHlowering effect and improvement of the shelf-life and the nutritional properties (Baráth, Halász, Németh, & Zalán, 2004). In recent years, buckwheat grain has attracted interest, mainly as niche products advertised as healthier and more natural than modern wheat (Coda, Di Cagno, Gobbetti, & Rizzello, 2014). The fermentation of non-treated and thermally treated buckwheat (F. esculentum Moench) flours using select lactic acid bacteria and fungi may change the functional properties of flours and derived bakery products (Giménez-Bastida, Piskuła, & Zieliński, 2015; Zieliński et al., 2019). At present, no information is available regarding the D-chiro-inositol content in fermented buckwheat flours as well as in model water buckwheat biscuits prepared from these fermented flours. Moreover, no research has addressed the bioaccessible DCI of digested water biscuits. From a nutrition perspective, the definition of bioaccessibility is the fraction of a compound that is released from the food matrix in the gastrointestinal lumen and used for intestinal absorption (Rein, Renouf, Cruz-Hernandez, Actis-Goretta, Thakkar, & da Silva Pinto, 2013). The aim of this study was to investigate: (1) the effect of fermentation of two types of buckwheat flours originating from common buckwheat (Fagopyrum esculentum Moench) (raw and thermally treated) by select lactic acid bacteria (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, Streptococcus thermophilus Mk-10) and Rhizopus oligosporus NRRL 2740 filamentous fungus on the DCI content; (2) the effect of baking model water biscuits formulated from buckwheat fermented flours on the DCI content; and (3) the potential bioaccessibility of DCI from water biscuits after an in vitro digestion procedure that mimics the physiochemical changes occurring in gastric and small intestinal digestion.

2.4. In vitro digestion of buckwheat water biscuits The buckwheat water biscuits baked with fermented unroasted and roasted buckwheat flours were in vitro digested as described by Delgado-Andrade, Conde-Aguilera, Haro, De La Cueva and RufiánHenares (2010) with some modifications. The protocol included three steps: saliva (pH 7.0), gastric (pH 2.0) and intestinal digestion (pH 7.5). Briefly, 10 g of lyophilized and milled buckwheat water biscuits were 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 the gastric digestion the pH was reduced to 2.0 with 6N HCl, and pepsin solution (738 U/mg) was added in the amount of 0.5 g/10 g of sample d.m. in 0.1N 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 buffered 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 D-chiro-inositol from water biscuits. 2.5. Extraction and quantification of D-chiro-inositol The lyophilized fermented flours and water buckwheat biscuits (0.25 g) were extracted with 2 mL of ethanol/water (1:1, v/v) solution using a thermomixer (1400 rpm, 1 h, 25 °C). Then, the samples were centrifuged (16100×g, 20 min, 4 °C). Then, 1 mL of the supernatant was removed and concentrated to approximately half of the initial volume using a concentrator (1400 rpm, 1 h, 30 °C). The supernatants obtained after digestion in vitro of buckwheat water biscuits (1 mL) were also concentrated to approximately half of the initial volume using a concentrator (1400 rpm, 1 h, 30 °C). One millilitre of a 2 M solution of TFA (2,2,2-trifluoroacetic acid) was added to the resulting concentrated samples, which were then subjected to hydrolysis (4 h, 70 °C). After hydrolysis, the sample were evaporated to dryness under a nitrogen atmosphere. The samples prepared in this way were stored at −20 °C until analysis. The methods developed to determine the DCI content are mainly based on the application of gas chromatography or HPLC. Yang and Ren (2008) published method based on gas chromatography, in which a silylation process with trimethylsilylation imidazole (TMSI) was needed, making the detection complicated and time-consuming. In this work, detection of DCI by HPLC was conducted according to the modified method of Yang and Ren (2008), however we chose the refractive index detector (RID) and Unison UK-Amino (3 μm) column. The samples were analysed by HPLC using a Shimadzu chromatograph (LC10 AD pump, refractometric detector RID-6A, CTO 6A column oven) and a 250 × 4.6 mm Unison UK-Amino (3 μm) column at 35 °C. Directly before the HPLC analysis, the samples were dissolved in 0.2 mL of methanol. The chromatogram was developed isocratically using 80% acetonitrile as the mobile phase at a flow rate of 0.8 mL/min. The identification of D-chiro-inositol in the test material was made on the

2. Material and methods 2.1. Chemicals α-Amylase (A1031-5KU), pepsin (P7000), pancreatin (P7545), bile salts extract (B8631), trifluoroacetic acid (TFA, 99%, bp 70 °C) and Dchiro-inositol standard (99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and ethanol (HPLC-grade) were provided by Merck (Darmstad, Germany), All other reagents were from POCh, (Gliwice, Poland). Water was purified with a Milli-Q-system (Millipore, Bedford, USA). 2.2. Buckwheat fermented flours The origin of buckwheat flours, their pre-treatment before the fermentation process, the origin of lactic acid bacteria and fungi, and the fermentation process were recently described by Wronkowska, Jeliński, Majkowska, and Zieliński (2018) and Zielinski et al. (2019). After fermentation the samples were freeze-dried (Christ – Epsilon 2-6D LSC plus, Germany). 2.3. Preparation of buckwheat water biscuits The water biscuit dough from fermented buckwheat flours was prepared according to the AACC 10–52 method (1995) with the modification proposed by Hidalgo and Brandolini (2011). The sugar, shortening and non-fat dry milk were not included in the recipe. The 38

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Fig. 1. HPLC-RID chromatograph of D-chiro-inositol standard. The chemical structures of D-chiro-inositol are inserted.

significantly decreased the DCI content in fermented raw flour from 10% (L. casei Lcy) up to 59% (L. rhamnosus 8/4) compared with the non-fermented raw flour (Table 1). No changes in DCI content were noted after fermentation by L. rhamnosus GG. In contrast, fermentation using L. salivarius AWH yielded a DCI content more than twofold higher in fermented flour, whereas R. oligosporus increased the DCI concentration by 30%. The similar decreases in DCI within the range of 6% (L. rhamnosus GG) to 47% (L. rhamnosus 8/4 and L. rhamnosus K) were noted after LSF of the roasted buckwheat flour (Table 2). It was also confirmed that LSF by L. salivarius AWH and R. oligosporus significantly enhanced the DCI content in fermented flours as a nearly twofold higher concentration was noted compared with non-fermented roasted buckwheat flour. The influence of fermentation on DCI has scarcely been investigated in buckwheat flours, and currently no reports are available to confirm our findings. However, our findings clearly indicate the possibility of selecting LAB and fungi to enhance the content of DCI in LSF buckwheat flours because different microbes possess the capacity of decomposing or synthesizing bioactive compounds (Bremus, Herrmann, Bringer-Meyer, & Sahm, 2006). In our study, LSF of raw or roasted buckwheat flours by L. salivarius AWH and R. oligosporus proved to be the most beneficial for enhancing the DCI concentrations in flour. Recently, Zieliński et al. (2017) showed that fermented buckwheat flour by L. plantarum could offer an innovative functional products. Moreover, R. oligosporus is considered the most suitable for improving functional properties and reducing the levels of allergenic proteins in cereals, pseudocereals and legumes. Handoyo, Maeda, Urisu, Adachi, and Morita (2006) found that buckwheat grain fermented by R. oligosporus had a higher content of amino acids and minerals but a lower content of allergic proteins than the non-fermented grain. It was suggested that R. oligosporus could possibly be used to produce hypoallergenic buckwheat with higher functional and good rheological properties, and thus the fermented buckwheat flours produced are considered to become a big prospect for the development of new foods in the future. This statement is in accordance with recent work done by Wronkowska, Honke, and Piskula (2015) in that solid-state fermentation with R. oligosporus could be recommended for the production of tempeh-like functional buckwheat-based foods with valuable nutritive substances.

basis of the retention time, and the content was calculated on the basis of a standard curve made for the D-chiro-inositol standard in the range of 0.1–1.0 mg/mL. The analysis was performed in triplicate for each trial. The results were expressed as mg DCI per gram of sample d.m. As shown in Fig. 1, the applied HPLC coupled with RID was suitable for detecting DCI. 2.6. Statistical analysis The analyses were performed in triplicate, and the results are displayed as the mean ± standard deviation (SD). The differences in DCI content in fermented flours in relations to control sample, also in water biscuits in relations to control sample and in digested biscuits in relations to control sample were evaluated using a Student's t-test for less numerous groups (P < 0.05). The differences in DCI content between the all analysed samples: fermented flours, water biscuits and sample after digestion were determined by a one-way analysis of variance (ANOVA) with Fisher's Least Significant Difference test (P < 0.05). The correlation analysis (P < 0.05) was performed and the Pearson correlation coefficient was calculated. All analyses were made using STATISTICA for Windows (StatSoft Inc., Tulsa, USA, 2001). 3. Results and discussion 3.1. Effect of fermentation on D-chiro-inositol content of raw and roasted buckwheat flours In this study, the average DCI content in raw and roasted buckwheat flours before fermentation was 1.06 and 0.75 mg/g d.m., respectively. These results were consistent with the DCI values determined by Horbowicz et al. (1998) which were up 1.05 mg/g in mature seed (embryo and endosperm). However, Steadman, Burgoon, and Lewis (2001) reported a lower level of DCI in buckwheat groats ranging from 0.21 to 0.42 mg/g d.m. The DCI content of fermented raw and roasted buckwheat flours is shown in Table 1 and Table 2, respectively. The fermented buckwheat raw flours contained a higher concentration of DCI than the respective fermented roasted buckwheat flours. Most of the applied LAB 39

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Table 1 The content of D-chiro-inositol in fermented raw buckwheat flour and model water buckwheat biscuits before and after digestion in vitro (mg/g d.m.). Strain/sample Control – non fermented L. plantarum IB L. plantarum W42 L. delbrucki subsp. bulgaricus 151 L. casei Lcy Streptococcus thermophilus MK-10 L. acidophilus La5 L. acidophilus V L. acidophilus 145 L. casei 2K L. delbrucki subsp. bulgaricus K L. rhamnosus GG L. rhamnosus 8/4 L. rhamnosus K L. salivarius AWH Rhizopus oligosporus 2740

Fermented raw buckwheat flour 1.06 0.70 0.79 0.64 0.95 0.70 0.74 0.78 0.76 0.54 0.65 0.94 0.44 0.73 2.29 1.37

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

b

0.015 0.02∗b 0.03∗b 0.01∗b 0.02∗b 0.01∗b 0.01∗b 0.02∗c 0.04∗b 0.02∗c 0.02∗b 0.02∗ 0.02∗c 0.01∗b 0.09∗a 0.01∗b

Buckwheat biscuits from fermented raw flour 1.90 1.54 1.46 1.23 1.36 1.24 1.62 1.74 1.71 1.16 1.88 1.10 1.42 2.03 1.72 2.14

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

∗a

0.05 0.18∗a 0.25∗a 0.08∗a 0.12∗a 0.13∗a 0.27∗a 0.07∗a 0.30∗a 0.19∗a 0.15∗a 0.17∗ 0.07∗a 0.06∗a 0.07∗b 0.31a

(79%↑) (122%↑) (85%↑) (91%↑) (43%↑) (78%↑) (117%↑) (123%↑) (125%↑) (115%↑) (190%↑) (18%↑) (225%↑) (177%↑) (25%↓) (56%↑)

Digested buckwheat biscuits from fermented raw flour 0.97 0.64 1.24 0.35 0.51 1.01 0.78 1.03 0.81 0.86 0.46 0.81 0.73 0.75 1.08 1.70

± 0.18b ± 0.08∗b ± 0.24ab ± 0.04∗c ± 0.05∗c ± 0.19ab ± 0.13b ± 0.06b ± 0.14b ± 0.08b ± 0.09∗b ± 0.13 ± 0.13b ± 0.13b ± 0.19c ± 0.32*ab

Data are expressed as mean ± standard deviation (n = 3). Means in each column followed by upper star are significantly different (P < 0.05) based on the Student's t-test for less numerous groups (compared to control sample). Means in each row followed by different letters for DCI content in fermented raw flours, biscuits and digested biscuits are significantly different (P < 0.05) based on the one-way analysis of variance (ANOVA). In the brackets the percentage changes in DCI content after baking is shown in comparison to the respective flours.

by 7% and 13% was found in biscuits prepared from fermented raw flour by L. rhamnosus K and R. oligosporus, respectively, compared with the biscuits based on non-fermented raw flour. It is known that DCI exists naturally in two major forms, the free form and its galactosyl derivatives named fagopyritols (Horbowicz et al., 1998). Fagopyritols, chemically named as R-DgalactopyranosylD-chiro-inositols with one to three galactosyl moieties, are the major form of these derivatives present in buckwheat (Horbowicz et al., 1998). Therefore, it can be suggested that L. salivarius AWH was able to disrupt the galactosidic bonds and release the free form of DCI, and then no available galactosidic bonds were present in the buckwheat biscuits to further release the free form of DCI during baking as the heat treatment. This hypothesis can also be applied for explaining the significantly enhanced DCI content in fermented raw flour by R. oligosporus; however, in this case some galactosidic bonds were still available to release the free form of DCI during baking. The buckwheat biscuits prepared from non-fermented roasted buckwheat flour also contained a nearly twofold higher concentration of DCI than the non-fermented roasted buckwheat flour (Table 2). This increase was also confirmed in the buckwheat biscuits prepared from

3.2. Effect of baking on D-chiro-inositol content in water biscuits formulated from fermented buckwheat flours The D-chiro-inositol content in model water buckwheat biscuits prepared from fermented raw buckwheat flours ranged from 1.10 to 2.14 mg/g d.m., whereas in those prepared from fermented roasted flours it ranged from 0.65 to 1.54 mg/g d.m. as shown in Tables 1 and 2, respectively. In this study, a nearly twofold higher concentration of DCI was noted in the water buckwheat biscuits prepared from non-fermented raw buckwheat flour than in the non-fermented raw buckwheat flour (Table 1). The D-chiro-inositol content in the buckwheat biscuits prepared from LAB and R. oligosporus fermented raw buckwheat flours was also nearly or more than twofold higher than that noted in the respective fermented flours. However, one exception was noted with respect to the biscuits prepared from fermented raw buckwheat flour by L. salivarius AWH, for which a nearly twofold higher concentration in flour was previously noted after LSF than in non-fermented buckwheat flour. In this case, the DCI content in biscuits was decreased by 25% compared with the content in fermented flour. The highest DCI content

Table 2 The content of D-chiro-inositol in fermented roasted buckwheat flour and model water buckwheat biscuits before and after digestion in vitro (mg/g d.m.). Strain/sample

Fermented roasted buckwheat flour

Control – non fermented L. plantarum IB L. plantarum W42 L. delbrucki subsp. bulgaricus 151 L. casei Lcy Streptococcus thermophilus MK-10 L. acidophilus La5 L. acidophilus V L. acidophilus 145 L. casei 2K L. delbrucki subsp. bulgaricus K L. rhamnosus GG L. rhamnosus 8/4 L. rhamnosus K L. salivarius AWH Rhizopus oligosporus 2740

0.53 0.59 0.58 0.55 0.59 0.51 0.55 0.45 0.43 0.43 0.49 0.71 0.40 0.40 1.30 1.21

± 0.02b ± 0.01∗b ± 0.01*b ± 0.02∗b ± 0.01∗b ± 0.01∗b ± 0.01∗b ± 0.02∗b ± 0.02∗b ± 0.01∗b ± 0.02∗b ± 0.016a ± 0.01∗b ± 0.01∗b ± 0.05∗a ± 0.03∗b

Buckwheat biscuits from fermented roasted flour

1.16 1.01 0.99 0.92 1.11 0.83 0.88 0.90 1.08 0.67 1.48 0.66 0.65 1.54 1.39 1.43

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.92a 0.031a 0.07∗ 0.08∗a 0.01a 0.09∗a 0.11∗a 0.10∗a 0.12a 0.04∗a 0.16∗a 0.09∗a 0.06∗a 0.26a 0.14a 0.15a

(54%↑) (71%↑) (72%↑) (67%↑) (86%↑) (82%↑) (60%↑) (60%↑) (153%↑) (51%↑) (202%↑) (7%↓) (63%↑) (284%↑) (7%↑) (18%↑)

Digested buckwheat biscuits from fermented roasted flour 0.34 0.42 0.66 0.20 0.31 0.61 0.31 0.73 0.25 0.15 0.18 0.12 0.21 0.20 0.13 0.90

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.06c 0.06c 0.12∗b 0.03∗c 0.05c 0.08∗b 0.05c 0.13∗a 0.05c 0.02∗c 0.02∗c 0.03∗b 0.03∗c 0.02∗b 0.02∗b 0.04∗c

Data are expressed as mean ± standard deviation (n = 3). Means in each column followed by upper star are significantly different (P < 0.05) based on the Student's t-test for less numerous groups (compared to control sample). Means in each row followed by different letters for DCI content in fermented roasted flours, biscuits and digested biscuits are significantly different (P < 0.05) based on the one-way analysis of variance (ANOVA). In the brackets the percentage changes in DCI content after baking is shown in comparison to the respective flours. 40

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and 28% was found, respectively (Table 1). This finding was also confirmed by a nearly threefold higher DCI level in the digested biscuits prepared from roasted buckwheat flour fermented by R. oligosporus, and a twofold higher DCI after application of the flours fermented by select LAB such as L. acidophilus V, L. plantarum W42 and Streptococcus thermophilus MK-10 (Table 2). In this study, the DCI contents in the digested biscuits prepared from fermented raw flours were significantly higher than those noted in the digested biscuits obtained from fermented roasted flours. Therefore, for better evaluation of the bioaccessibility in vitro we determined the DCI bioaccessibility index (PAC), which is an indication of the potential bioaccessibility of DCI:

fermented roasted buckwheat flours (Table 2), thus indicating the importance of the heat treatment on the D-chiro-inositol content. The higher DCI content by 20–28% was found in biscuits prepared from fermented roasted flour by L. delbrucki subsp. bulgaricus K, L. rhamnosus K, L. salivarius AWH and R. oligosporus compared with the biscuits based on non-fermented roasted flour. A higher DCI concentration by 19–54% was noted in biscuits prepared from fermented raw buckwheat flour compared with the biscuits from fermented roasted buckwheat flours. In this study, no correlation was found between the DCI content in fermented flours and the biscuits prepared from them. The correlation coefficient was r = 0.34 (biscuits from fermented raw flours) and r = 0.54 (biscuits from fermented roasted flours), thus indicating the heat treatment as the factor enhancing the DCI concentration in biscuits. This finding is in agreement with the study performed by Yao et al. (2008), who showed that steaming tartary buckwheat bran in an autoclave at 1.6 MPa and 120 °C for 60 min significantly enriched the DCI level in tartary buckwheat bran extract from 0.03 to 0.22%, thus suggesting that pressure and heat can disrupt galactosidic bonds and release the free form of DCI. Therefore, we can suggest that baking at 220 °C for 30 min significantly enhanced the DCI level in buckwheat biscuits as was shown in our study.

PAC= CGD/ CWBB Where CGD is the D-chiro-inositol content after simulated gastrointestinal digestion (GD) and CWBB is the D-chiro-inositol content in water buckwheat biscuits (WBB). PAC value ˃ 1 indicates high bioaccessibility; PAC value < 1 indicates low bioaccessibility. The PAC factor was introduced by Gawlik-Dziki, Durak, Jamioł, and Świeca (2016) as a useful parameter to study the bioaccessibility of phenolics from coffee and coconut. In our study, the PAC values ranged from 0.25 to 0.85 for digested buckwheat biscuits made of fermented raw flours, and amongst them the highest PACs were noted for digested biscuits prepared from fermented flour by R. oligosporus (0.79) and L. plantarum W42 (0.85) (Fig. 2). PAC values from 0.10 to 0.81 were noted for the digested buckwheat biscuits prepared from fermented roasted flours, and the highest values were found for L. plantarum W42 (0.67), Streptococcus thermophilus MK-10 (0.74), L. acidophilus V (0.81) and R. oligosporus (0.63). The PAC values provided clearly indicate the low potential bioaccessibility of DCI from both types of biscuits. Moreover, when the same LAB strain or R. oligosporus was applied, the PAC value of the digested biscuit prepared from fermented raw flour was higher than those for the digested biscuits prepared from roasted buckwheat. Different contributors may affect DCI bioaccessibility. The provided data indicates that the physical structure of biscuits is an important factor affecting the potential bioaccessibility of DCI. This is in accordance with our recent work demonstrating the impact of select LAB on some physical properties of the water biscuits prepared from fermented buckwheat flour (Wronkowska et al., 2018). In summary, it can be stated that the bioaccessibility of D-chiro-inositol from water biscuits formulated from fermented buckwheat flours is rather low. The use of select LAB such as L. plantarum W42, Streptococcus thermophilus MK-10, L. acidophilus V and fungi R. oligosporus for LSF appears to be beneficial for enhancing the potential bioaccessibility of DCI from water buckwheat biscuits.

3.3. Bioaccessibility of D-chiro-inositol from water biscuits formulated from fermented buckwheat flours after digestion in vitro procedure From a nutrition perspective, the definition of bioaccessibility is the fraction of a compound that is released from the food matrix in the gastrointestinal lumen and used for intestinal absorption (Rein et al., 2013). The in vitro digestion model has been widely used to study the complex multistage process of human digestion (Li, Deng, Liu, Loewen, & Tsao, 2014). Bioaccessibility is a major factor that should be taken into account when assessing the health-benefits potential of functional foods. Different contributors affect bioaccessibility. It can be affected by the composition of the digested food matrix, the synergisms and antagonisms of the different components, and the pH, temperature, and texture of the matrix (Fernàndez-Garcìa, Carvajal-Lérida, & PérezGàlvez, 2009). Bioactive compounds are susceptible to degradation during digestion due to the effects of pH and enzymes, and in the present study the bioaccessibility of D-chiro-inositol from buckwheat water biscuits was determined for the first time after an in vitro digestion. The DCI content after in vitro digestion of the buckwheat biscuits prepared from fermented raw and roasted flours is shown in Tables 1 and 2, respectively. Data were recalculated from the DCI concentration in the supernatants obtained after digestion to the digestible matter of buckwheat biscuits. The insoluble matter left after digestion was 27–31% of the initial dry matter of biscuits (data not shown). The DCI content in the digested buckwheat biscuits was statistically significant lower than its content before digestion. In relation to the control biscuits, a twofold lower content was found in the digested biscuits prepared from non-fermented buckwheat flour (Table 1), whereas it was at least threefold lower in those prepared from non-fermented roasted buckwheat flour (Table 2). The use of fermented raw buckwheat flours for water biscuit preparation and then for its digestion had no effect on the DCI content as the correlation coefficient between the DCI content in the fermented flours and digested biscuits was r = 0.46 and that between the DCI content in the buckwheat biscuits before and after fermentation was r = 0.39. A similar finding was noted in relation to the use of fermented roasted buckwheat flours for biscuit preparation and then its digestion as the correlation coefficients were r = 0.21 and r = 0.71, respectively. The DCI content in the digested biscuits prepared from raw fermented buckwheat flours was lower than in the control biscuits except for the digested biscuits prepared from flour fermented by R. oligosporus and L. plantarum W42. In this case, an increase in DCI content by 76%

4. Conclusions The study showed that LSF, baking and digestion significantly affected the DCI content in fermented flours, biscuits and the digestible matter of buckwheat biscuits. LSF by L. salivarius AWH and R. oligosporus 2740 significantly enhanced the DCI content, whereas most of the applied LAB reduced DCI in the fermented flours. In contrast, baking significantly enhanced the DCI level in buckwheat biscuits; however, no correlation was found between the DCI content in the fermented flours and the water biscuits prepared from them. The potential bioaccessibility of DCI from water biscuits was lower than 1, thus indicating its low bioaccessibility. Moreover, when the same LAB strain or R. oligosporus 2740 was applied, the PAC value of the digested biscuit prepared from fermented raw flour was higher than those for the digested biscuits prepared from roasted buckwheat. It can be concluded that in applying select LAB and R. oligosporus 2740 for LSF, the heat treatment and physical structure of biscuits were mainly responsible for the potential bioaccessibility of DCI. These factors should be taken into account when DCI-enhanced water buckwheat biscuits are prepared. 41

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Fig. 2. The bioaccessibility index (PAC) indicating the potential bioaccessibility of DCI from water biscuits formulated from buckwheat flours fermented by lactic acid bacteria and fungi; PAC value gt; 1 indicates high bioaccessibility; PAC value < 1 indicates low bioaccessibility.

Authors’ contribution

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