Bioaccessibility of anti-AGEs activity, antioxidant capacity and phenolics from water biscuits prepared from fermented buckwheat flours

Journal Pre-proof Bioaccessibility of anti-AGEs activity, antioxidant capacity and phenolics from water biscuits prepared from fermented buckwheat flours Henryk Zieliński, Dorota Szawara-Nowak, Małgorzata Wronkowska PII:

S0023-6438(20)30039-6

DOI:

https://doi.org/10.1016/j.lwt.2020.109051

Reference:

YFSTL 109051

To appear in:

LWT - Food Science and Technology

Received Date: 31 August 2019 Revised Date:

19 December 2019

Accepted Date: 14 January 2020

Please cite this article as: Zieliński, H., Szawara-Nowak, D., Wronkowska, Mał., Bioaccessibility of anti-AGEs activity, antioxidant capacity and phenolics from water biscuits prepared from fermented buckwheat flours, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/ j.lwt.2020.109051. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

H.Z. conceived and designed the research program. D.Sz-N. performed the analysis of anti-AGEs activity, M.W. baking experiments and statistical analysis, H.Z. wrote the manuscript with input from all authors.

1 2 3 4

Bioaccessibility of anti-AGEs activity, antioxidant capacity and phenolics from water biscuits

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prepared from fermented buckwheat flours

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Henryk Zieliński*, Dorota Szawara-Nowak, Małgorzata Wronkowska

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Department of Chemistry and Biodynamic of Food, Division of Food Sciences, Institute of

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Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748

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Olsztyn, Poland

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Running title: Functional properties of processed buckwheat

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Correspondence

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Henryk Zieliński, Department of Chemistry and Biodynamic of Food, Division of Food

27

Science, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences,

28

10-748

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[email protected]

30 1

Olsztyn,

10

Tuwima

Str.,

Poland;

Fax:

+48

89

5240124;

email:

31

Abstract

32 33

The bioaccessible anti-AGEs activity of buckwheat biscuits (BB) was studied in bovine serum

34

albumin/glucose model and its relationship to the bioaccessible antioxidant/reducing capacity

35

measured by ABTS test and FRAP assay, and bioaccessible total phenolic compounds was

36

addressed. The BB were baked from common buckwheat flours after liquid-state fermentation

37

(LSF) by select lactic acid bacteria (LAB) and fungi Rhizopus oligosporus 2740. The LAB

38

and fungi-dependent variation in AGEs inhibition by BB extracts was noted. A high

39

bioaccessible anti-AGEs activity, antioxidant/reducing capacity and TPC from BB was found

40

after digestion in vitro of BB. The positive correlation noted between the anti-AGEs

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bioaccessibility indexes, antioxidant/reducing bioaccessibility indexes and total phenolic

42

compounds

43

phenolic antioxidants to the inhibitory activity of buckwheat biscuits against AGEs

44

formation.

bioaccessibility indexes indicated for the contribution of the bioaccessible

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Keywords: fermented buckwheat flours; buckwheat biscuits; digestion; bioaccessibility; anti-

48

AGEs activity; antioxidant capacity, total phenolic compounds.

49 50 51

1.

Introduction

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The term "bioaccessibility" is a key concept to ascertain nutritional efficiency of food and

54

food formula developed with the aim of improving human health. Measurement of

55

bioaccessibility provides valuable information to select the source of food matrices to ensure

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nutritional efficacy of food products (Fernández-García, Carvajal-Lérida & Pérez-Gálvez,

57

2009).

58

Advanced glycation endproducts (AGEs) are a large, heterogeneous molecules, which are

59

formed via Maillard reaction during thermal food processing or long storage at ambient

60

temperature (Rabbani & Thornalley, 2012). They share some common features such as

61

covalent cross-link formation among proteins, the effect of transforming the colour of some

62

food products into yellow-brown colours (“browning” effect) and fluorescence formation

63

(Palimeri, Palioura & Diamanti-Kandarakis, 2015). AGEs are mainly synthesized by the

64

reaction of excess reducing monosaccharides and proteins in the human body or through food 2

65

intake (Sharma, Kaur, Thind, Singh, & Raina, 2015). Intake of dietary AGEs along with the

66

in vivo formation of AGEs may result the increase of AGEs load in the human body

67

(Delgado-Andrade & Fogliano, 2018). Excessive of AGEs in the human body cause oxidative

68

stress and a series of chronic diseases in the body such as kidney disease, aging,

69

atherosclerosis and diabetic complications (Lee et al. 2010; Rabbani & Thornalley, 2018;

70

Yang, Wang, Chen, He, & Jia, 2018).

71

A variety of synthetic and natural products have been evaluated as inhibitors of AGE

72

formation (Thornalley, 2003). The components from natural food products have been proven

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relatively safer for human consumption when compared with synthetic compounds because

74

they are less toxic (Wu, Huang, Lin, & Yen, 2011; Jahan & Choudhary, 2015). In this regard,

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some plant extracts and food bio-active compounds have been evaluated for their effects on

76

the formation of AGEs in recent years (Ghorbani, 2017; Lu et al., 2018). In most studies,

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fluorescence spectrometry has been commonly used to determine the AGEs. Fluorescence

78

spectrometry can determine the intensity to reflect the level of AGEs however, it cannot

79

easily identify an individual AGE compound (Schmitt, Gasic-Milenkovic, & Schmitt, 2005).

80

As presented by Zhang et al. (2012) buckwheat is a good source of nutritionally

81

valuable protein, lipid, dietary fibre, and minerals, also it is know from bioactive polyphenolic

82

compounds. The mechanisms underlying beneficial effects attributed to selected buckwheat

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bioactive compounds (such as flavonoids, phenolic acids, proteins or D-chiro-inositol) were

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described in the review by Giménez-Bastida and Zieliński (2015). Numerous studies show

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that buckwheat has been widely accepted for preventing and treating diabetes, hyperlipidemia

86

and other conditions (Babu, Liu, & Gilbert, 2013; Zhang et al., 2012; Gimenez-Bastida &

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Zieliński, 2015; Giménez-Bastida, Laparra, Bączek, & Zielinski, 2018). The high anti-AGEs

88

capacity in buckwheat and buckwheat enhanced wheat bread was also reported (Lee, Lee, &

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Lai, 2015; Szawara-Nowak, Koutsidis, Wiczkowski, & Zieliński, 2014). Recently it was

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shown that the inhibitory activity of LAB fermented buckwheat flours against AGEs

91

formation was generally reduced (Zieliński, Szawara-Nowak, Bączek, &

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(2019a).

Wronkowska

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The aim of this study was to investigate: (1) the anti-AGEs activity of BB prepared

94

from non-fermented and fermented buckwheat flours in bovine serum albumin/glucose model

95

before and after digestion in vitro, (2) the bioaccessible antioxidant capacity (AC) measured

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by ABTS test and FRAP assay, and bioaccessible total phenolic compounds (TPC) after

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digestion in vitro, (3) the relationship between the bioaccessible anti-AGEs activity,

3

98

antioxidant capacity (AC) and total phenolic compounds (TPC) of buckwheat biscuits after

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digestion in vitro.

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2.

Material and methods

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2.1. Chemicals

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α-Amylase (A1031-5KU), pepsin (P7000), pancreatin (P7545), bile salts extract (B8631)

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were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and methanol

107

(HPLC-grade) were provided by Merck (Darmstad, Germany). Sodium azide, bovine serum

108

albumin

109

diammonium salt (ABTS) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid

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(Trolox) were purchased from Sigma (Sigma Chemical Company, Saint Louis, Missouri,

111

U.S.A.). All other reagents of reagent-grade quality were from POCh, Gliwice, Poland. Water

112

was purified with a Mili-Q-system (Milipore, Bedford, USA).

(BSA),

D-glucose,

2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic

acid)

113 114

2.2. Preparation of BB from fermented flours

115 116

The origin of buckwheat flour, their pre-treatment before the fermentation process, the

117

origin of lactic acid bacteria and fungi, the fermentation process and preparation of BB were

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carried out as recently described by Wronkowska, Jeliński, Majkowska and Zieliński (2018)

119

and Zielinski et al. (2019a). The water biscuit dough from fermented buckwheat flours was

120

prepared according to the AACC 10–52 method (1995) with the modification proposed by Hidalgo &

121

Brandolini (2011). The sugar, shortening and non-fat dry milk were not included in the recipe. The

122

dry ingredients were blended for 30 seconds with a planetary rotation of mixing within a 5-speed

123

mixer (Kitchen Aid, St. Joseph, MI, USA), and then the remaining ingredients and deionized water

124

were added and mixed again for 3 minutes. The dough was cut with a square cookie cutter (60 mm).

125

Baking was carried out at 220°C for 30 min in an electric oven DC-21 model (Sveba Dahlen AB,

126

Fristad, Sweden). After baking, biscuits were freeze-dried using Christ – Epsilon 2–6D LSC plus,

127

Osterode am Harz, (Germany), milled and stored in a refrigerator until analysis.

128 129

2.3. In vitro digestion of BB

130 4

131

BB prepared from non-fermented and fermented buckwheat flours were in vitro

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digested as described by Delgado-Andrade, Conde-Aguilera, Haro, De La Cueva and Rufián-

133

Henares (2010) with some modifications (Zieliński, Honke, Bączek , Majkowska, &

134

Wronkowska, 2019b). The protocol included three steps: saliva (pH 7.0), gastric (pH 2.0) and

135

intestinal digestion (pH 7.5). Briefly, 10 g of lyophilized and milled buckwheat biscuits were

136

suspended in 80 mL of deionized water. An α-amylase solution (77 U/mg solid) was added to

137

the samples at a proportion of 3.25 mg/ 10 g of sample dry matter (d.m.) in 1 mM CaCl2, pH

138

7.0. Then, samples were shaken in a water bath at 37°C for 30 minutes. For the gastric

139

digestion the pH was reduced to 2.0 with 6N HCl, and pepsin solution (738 U/mg) was added

140

in the amount of 0.5 g/10 g of sample d.m. in 0.1N HCl. The incubation was continued under

141

the same conditions for 120 minutes. In the next step the pH was adjusted to 6.0 with 6 M

142

NaOH, and a mixture of pancreatin (activity 8xUSP) and bile salts extract was added.

143

Subsequently, the pH was increased to 7.5 with 6 M NaOH, and water buffered to a pH of 7.5

144

was introduced to obtain a final volume of 150 mL. Then, the samples were incubated at 37°C

145

for 120 minutes. After incubation, the digestive enzymes were inactivated by heating at

146

100°C for 4 minutes and cooled for centrifugation at 5000 rpm for 60 minutes at 4°C in an

147

MPV-350R centrifuge (MPW Med. Instruments, Warsaw, Poland). The fresh supernatants

148

obtained after digestion was directly used for the measurement the inhibitory activity against

149

AGEs formation, the antioxidant capacity and the content of total phenolic compounds.

150

2.4. Evaluation of the in vitro inhibitory activity of non-digested and digested BB

151

formulated on fermented buckwheat flours on AGEs formation (anti-AGEs)

152 153

Determination of the inhibitory effects of extracts obtained from non-digested (200

154 155

mg/mL)

156

buckwheat flour on the formation of AGEs was performed in vitro in bovine serum

157

albumin/glucose (BSA/Glu) according to Szawara-Nowak et al. (2014). Aminoguanidine

158

(AG) 1 mM was used as positive control and its concentration vs. anti-AGEs activity was

159

measured. Triplicate samples were run for each set and the percent inhibition of AGEs

160

formation by each non-digested and digested BB extracts was calculated using the following

161

equation:

162

5

and digested BB (55-60 mg/mL) formulated on fermented raw and roasted

fluorescence of the solution with inhibitors (Ex 330 nm; Em 410 nm)

163 164

% inhibition = {1 – ( —————————————————

)} x 100%

fluorescence of the solution without inhibitors (Ex 330 nm; Em 410 nm)

165 166 167 168

2.5. Determination of total phenolic compounds (TPC) before and after digestion in vitro

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BB samples (300 mg) were extracted with 80% aqueous methanol (5 mL) for 40 min of

171

shaking at room temperature. Samples were then centrifuged at 3000 x g for 15 min in a

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Beckman GS-15 R centrifuge (Beckman Instruments, Fullerton CA, USA). All extractions

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were performed in triplicate. The crude extracts and fresh supernatants obtained after

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digestion were used for TPC as described by Zielinski et al. (2019a). TPC content was

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standardized against gallic acid and linearity range for this assay was determined at 0.025 –

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0.5 mg/mL (y= 2.243x + 0.044; R2=0.99). Analyses were carried out in triplicate and results

177

are reported as mean values (n=3) expressed as mg gallic acid equivalents (GAE)/g DM.

178 179

2.6. Determination of the antioxidant/reducing capacity (AC) of BB before and after digestion

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in vitro

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Extraction. About 100 mg of freeze-dried BB was extracted by 30 s sonication with 1 mL of

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solution containing 80% MeOH. Next, the mixture was vortexed for 30 s, again sonicated and

184

vortexed, and centrifuged for 5 min (5 000 x g at 4°C). This procedure was repeated 5 times

185

and finally, the supernatants obtained was collected in 5 mL flask. The final extract

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concentration was 20 mg/mL.

187

Determination of the antioxidant capacity.

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The antioxidant capacity against ABTS•+ radical cation was measured using a

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temperature-controlled spectrophotometer UV‑160 1PC with CPS-Controller (Shimadzu,

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Japan). For measurement the ABTS•+ solution was diluted with 80% (v/v) methanol to the

191

absorbance of 0.70±0.02 at 734 nm. Solution of the ABTS•+ (1.48 mL) and BB extracts before

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and after digestion (20 µL) were mixed for the spectrophotometric assay, then absorbance was 6

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measured immediately after 6 min at 734 nm at 30°C. The obtained results were expressed as

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µmol Trolox per gram of dry matter (DM) sample (Zieliński et al., 2019a).

195

The ferric reducing ability of BB extracts before and after digestion was analysed by

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FRAP assay according to Zieliński et al. (2017). The method utilized the antioxidant power of

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flour extracts to cause Fe+3 to Fe+2 reduction after that is formed a coloured complex with

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2,4,6-tri(2-pirydyl)-s-triazine (TPTZ). The increase in absorbance of the TPTZ- Fe+2 complex

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is proportional to antioxidant amount in the test tube. The results were expressed as µmol

200

Trolox per per gram of dry matter (DM) sample.

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2.6.Statistical analysis

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Results of the analyses are illustrated as mean values and the standard deviation of three

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independent measurements. The differences in the anti-AGEs activity, AC and TPC content

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of BB in relations to control sample and in the digested biscuits in relations to control sample

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were evaluated using a Student’s t-test for less numerous groups (P<0.05). The differences in

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the anti-AGEs, AC and TPC in BB before and after digestion were determined by a one-way

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analysis of variance (ANOVA) with Fisher’s Least Significant Difference test (P<0.05). The

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correlation analysis was performed and the Pearson correlation coefficient was calculated. All

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analyses were made using STATISTICA for Windows (StatSoft Inc., Tulsa, USA, 2001).

212 213

3. Results and Discussion

214 215

3.1. The anti-AGEs activity of non-digested and digested BB formulated on fermented

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buckwheat flours

217 218

The bovine serum albumin (BSA)-glucose model adopted in this study provides a

219

useful tool for the evaluation of the inhibitory activity of BB against AGEs formation whereas

220

aminoquanidine (AG) has served as a reference compound (Lee et al., 2015; Szawara-Nowak

221

et al., 2014). AG inhibited in a dose dependent manner (from 0.01 to 1.0 Mm) the formation

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of the AGEs reaching an inhibition above 68% at 1 mmol/L (Figure 1a). Moreover, the linear

223

dose-dependent relationship was found for BB extracts ranged from 50 mg/mL to 200 mg/mL

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(y=0.24 x + 1.96) as it is shown on Figure 1b.

7

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Recently we showed reduction of the inhibitory activity of buckwheat flours against

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AGEs formation after liquid-state fermentation (LSF) by selected lactic acid bacteria (LAB)

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and Rhizopus oligosporus (Zielinski et al., 2019a). In this study, based on the linear dose-

228

dependent relationship between concentration of BB extracts and anti-AGEs activity, we used

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the concentration of 200 mg/mL of BB extracts to study their anti-AGEs activity and the data

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were extrapolated to the concentration of soluble fraction obtained after digestion in vitro..

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The anti-AGEs activity of BB biscuits prepared from non-fermented flour (control

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biscuits) was 12.05% as compared to 68.3 % noted for 1 mM aminoguanidine. The anti-AGEs

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activity of non-digested BB formulated on fermented buckwheat flours ranged from 8.06 to

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13.65% as it is shown in Table 1. The LAB and fungi-dependent variation in anti-AGEs

235

activity of BB extracts formulated on fermented buckwheat flours was noted. Compared to

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control biscuits prepared from non-fermented flour the higher inhibitory activity up to 13%

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was found for BB obtained from flour fermented by the following lactic acid bacteria: L.

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plantarum W42, L. casei Lcy, L. acidophilus La5, L. casei 2K, L. rhamnosus GG as well as

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for sample fermented by fungi R. oligosporus 2740 (Table 1). In contrast, a reduction of the

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anti-AGEs activity up to 33 % was noted for BB obtained from flour fermented by the nine

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remaining lactic acid bacteria. This finding indicates that not only baking at 220°C for 30

242

min had an impact on the inhibitory activity of BB on the AGEs formation but also the

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fermented buckwheat flours by specific LAB and fungi.

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The bioaccessible anti-AGEs activity of BB formulated on fermented buckwheat

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flours flours is shown in Table 1. From a nutrition perspective, the classic definition of

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bioaccessibility is the fraction of a compound that is released from the food matrix in the

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gastrointestinal lumen and used for intestinal absorption (Rein, Renouf, Cruz‐Hernandez,

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Actis‐Goretta, Thakkar, & da Silva Pinto, 2013). However, this definition may be extended

249

also for the functional properties of food, including anti-AGEs activity.

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The anti-AGEs activity of the digested BB formulated on fermented buckwheat flours

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ranged from 51% to 65% as compared to 59% noted for digested BB from non-fermented

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flour. Some supernatants obtained after digestion of BB from fermented flours showed

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slightly higher inhibitory activity as compared to the digested control BB prepared from non-

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fermented flour while the remaining ones showed the same level of the inhibitory activity

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(Table 1). It was worthy to note that those BB with high anti-AGEs activity showed also

256

higher activity after digestion. The anti-AGEs activity of BB before and after digestion was

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positively correlated (r= 0.57).

8

258

For better evaluation of the bioaccessibility in vitro we determined the anti-AGEs

259

bioaccessibility index (BIAnti-AGEs) of BB which was calculated according to the following

260

formula:

261

BIAnti-AGEs = Anti-AGEsGD/Anti-AGEsBB

262 263 264

where

265

Anti-AGEsGD is the inhibitory activity of BB after simulated gastrointestinal digestion (GD),

266

Anti-AGEsBB is the inhibitory activity of BB before digestion. The BIAnti-AGEs value ˃ 1

267

indicates high bioaccessibility; BI value < 1 indicates low bioaccessibility.

268

In our study, the anti-AGEs bioaccessibility index of digested BB made of fermented

269

raw flours ranged from 4.39 to 6.68. The highest index was noted for digested BB prepared

270

from fermented flour by L. rhamnosus 8/4 (6.68), Streptococcus thermophilus MK-10 (6.37),

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L. acidophilus 145 (5.89), L. delbrucki subsp. bulgaricus K (5.50). L. rhamnosus 8/4 (5.92),

272

L. rhamnosus K (5.87), Streptococcus thermophilus MK-10 (5.75) and L. salivarius AWH

273

(5.17) as compared to the anti-AGEs bioaccessibility index for digested BB made of non-

274

fermented flour (4.95) (Table 1).

275

Currently research aimed to study of anti-AGEs activity has become a new interesting

276

direction (Szawara-Nowak et al., 2014; Zielinska, Szawara-Nowak, & Zielinski, 2009;

277

Przygodzka, & Zieliński, 2015) and the high anti-AGEs activity of buckwheat was also

278

reported (Lee et al., 2015). The BIAnti-AGEs values provided clearly indicate the very high

279

bioaccessible anti-AGEs activity of BB prepared from fermented buckwheat. Different

280

contributors may affect the potential bioaccessible inhibitory activity of BB against AGEs

281

formation. It can be affected by the composition of the digested food matrix, the synergisms

282

and antagonisms of the different components, and the pH, temperature, and texture of the

283

matrix (Fernàndez‐Garcìa et al., 2009). The provided data indicates that selected LAB for

284

LSF, baking process and enzymes used for digestion in vitro are an important factors

285

affecting the potential anti-AGEs bioaccessibility index. The physical structure of BB seems

286

to be also important as recently we demonstrated the impact of selected LAB on some

287

physical properties of BB prepared from fermented buckwheat flour (Wronkowska et al.,

288

2018). The use of selected LAB such as Streptococcus thermophilus MK-10 and L. delbrucki

289

subsp. bulgaricus K for LSF for obtaining fermented flours appears to be the most beneficial

290

for enhancing the bioaccessible anti-AGEs activity of BB. The anti-AGEs activity of BB 9

291

before and after digestion in vitro is regarded as important effect of buckwheat phenolic

292

compounds with antioxidant activity which may an impact on the anti-AGEs activity.

293 294

3.3. Bioaccessibility of total phenolic compounds from BB after digestion in vitro

295 296

Total phenolic compounds (TPC) content in BB prepared from fermented buckwheat

297

flours before and after in vitro digestion is presented in Table 2. As it was presented in our

298

previous investigation, fermentation caused a slight, specific LAB-dependent increase in TPC

299

in fermented flours (Zieliński et al. 2019a). In this study, an increase of TPC in BB prepared

300

from fermented flours up to 113% was noticed as compared to control BB prepared from non-

301

fermented flour with exception made to BB from flours fermented by L. delbrucki subsp.

302

bulgaricus K and L. rhamnosus K.

303

Samples obtained after digestion of BB formulated on fermented flours showed

304

increased TPC up to 42% as compared to control BB prepared from unfermented flours. The

305

ANOVA analysis of the differences in TPC content in BB before and after digestion showed

306

that TPC content was significantly higher in all samples after digestion compared to other. In

307

this study a positive weak correlations were found between TPC content and anti-AGEs

308

activity of BB formulated on the fermented flours before (r= 0.47) and after in vitro

309

digestion (r= 0.47). In this study we determined the bioaccessibility index of total phenolic compounds

310 311

(BITPC), which was calculated according to the following formula:

312

BITPC = TPCGD/ TPCBB

313 314 315

where TPCGD is the phenolics content after simulated gastrointestinal digestion (GD) and

316

TPCBB is the phenolic content in BB. BITPC value ˃ 1 indicates high bioaccessibility; BITPC

317

value < 1 indicates low bioaccessibility.

318

In our study, the BITPC values ranged from 2.72 to 7.47 for digested BB made of

319

fermented raw flours (Table 2). The highest BITPC were noted for digested BB prepared from

320

fermented flour by L. delbrucki subsp. bulgaricus K (7.47), L. rhamnosus GG (6.06) and L.

321

delbrucki subsp. bulgaricus 151 (5.62) as compared to the digested BB formulated on non-

322

fermented flour (4.58). The provided BITPC values clearly indicated for the high 10

323

bioaccessibility of TPC from BB and varied effect of LSF on the TPC content in fermented

324

flours used for biscuits preparation. These findings clearly indicate for the contribution of

325

bioaccessible BB phenolic compounds to their inhibitory activity against AGEs formation.

326

However, as there were more than one effective ingredient of BB, it’s hard to distinguish the

327

AGEs inhibitory effect of each compound itself (Jing & Weibiao, 2018).

328

The in vitro digestion of BB showed that most of phenolic compounds which exhibit

329

the antioxidant activity were soluble in the medium used for digestion. The increased content

330

of phenolic compounds was due to the fact that phenolics entrapped in the structures of the

331

buckwheat biscuits matrix could be released during the gastrointestinal digestion. Gawlik-

332

Dziki, Dziki, Baraniak, and Lin (2009) observed the gradually release of phenolic compound

333

during the in vitro hydrolysis of wheat bread enriched in an extract from the green parts of

334

buckwheat plant. Also, in the fractions obtained after in vitro digestion of wheat breads

335

enhanced by buckwheat an increase of TPC content was showed by Szawara-Nowak et al.

336

(2016). Liyana-Pathirana and Shahidi (2005) demonstrated significantly increased of the TPC

337

content of extracts obtained from wheat whole grains and their flour, germ and bran fractions

338

after in vitro digestion . Keeping in mind the limitations of the Folin-Ciocalteu (FC) assay, the

339

obtained data should be always interpreted with great caution, especially in situations where

340

the system contains a complex food matrix. It has been determined previously thatthe FC

341

reagent can be non-specifically reduced by reducing sugars, aromatic amines, organic acids,

342

fatty acids and Fe2+ ions, as well as by proteins and small peptides that are formed during

343

digestion of food proteins (Prior, Wu, & Schaich, 2005).

344 345

3.4. Bioaccessible antioxidant/reducing capacity measured by ABTS test and FRAP assay

346

The antioxidant/reducing capacity of BB prepared from fermented buckwheat flours

347

before and after in vitro digestion is shown in Table 3. As it was presented in our previous

348

investigation, fermentation caused a LAB-dependent variation in antioxidant/reducing

349

capacity of buckwheat flours (Zieliński et al. 2019a). In our study, the antioxidant and

350

reducing capacity of BB prepared from fermented flours by L. plantarum (W42, IB), (L.

351

acidophilus (145, La5, V), L. delbruecki subsp. bulgaricus (151) was increased up to 36% and

352

70% as compared to control BB prepared from non-fermented flour however these findings

353

were

354

antioxidant/reducing capacity was found for BB baked from flour fermented by Rhizopus 11

not

observed

after

digestion.

The

highest,

almost

two-fold

increase

of

355

oligosporus 2740 and this effect was also noted after digestion. The antioxidant capacity of

356

BB after digestion was almost five-fold higher whereas reducing capacity was almost three-

357

fold increased as compared to non-digested samples. The data provided for BB by ABTS and

358

FRAP were highly correlated before (r= 0.97) and after digestion (r= 0.95).

359

In this study, similarly to BITPC, the bioaccessibility index of antioxidant capacity

360

(BIABTS) and reducing capacity (BIFRAP) was calculated. The BIABTS values ranged from 3.22

361

to 5.73 for digested BB made of fermented flours as compared to value of 5.20 provided for

362

BB baked from non-fermented flour (Table 3). The BIFRAP values ranged from 1.65 to 3.42

363

for digested BB made of fermented flours as compared to value of 3.14 provided for BB

364

baked from non-fermented flour (Table 3). These findings indicate for general lower

365

bioaccessible antioxidant/reducing capacity from BB prepared from fermented flours as

366

compared to that one non-fermented buckwheat flour.

367

The anti-AGEs bioaccessibility indexes (BIAnti-AGEs) were positively correlated with

368

BITPC and BIABTS values and the correlations coefficient had value r= 0.66 and r= 0.42

369

whereas no correlation was found for and BIFRAP (r= 0.08). It may be suggested that anti-

370

AGEs activity is rather related to the phenolic compounds with free radical scavenging

371

activity that to those with reducing properties. These findings clearly indicate for the

372

contribution of bioaccessible BB phenolic compounds to their anti-AGEs activity. So far as

373

an oxidation reaction occurs during the nonenzymatic glycosylation reaction then substance

374

with antioxidant activity has potential anti-AGEs activity. Research shows that the natural

375

phenolics have good antioxidant activity and the high anti-AGEs capacity in buckwheat was

376

reported by Lee et al. (2015). AGEs contribute to the development of diabetes has been

377

widely recognized as important factors. Substances with antioxidant capacity also had

378

substantial anti-AGEs activity (Ahmed, 2005; Byun et al. 2017). Previous studies have shown

379

that in terms of free radical scavenging capacity, antioxidant activity is the major action for

380

phenolic compounds to suppress AGEs generation (Navarro, Fiore, Fogliano, & Morales,

381

2015; Zhang, Hu, Chen, & Wang, 2014).

382 383

4. Conclusions

384 385

This study provided report on the bioaccessible anti-AGEs activity of BB formulated on

386

fermented flours against AGEs formation and its relationship to the bioaccessible

12

387

antioxidant/reducing capacity and total phenolic compounds. A high bioaccessible anti-AGEs

388

activity, antioxidant/reducing capacity and TPC from BB was found after digestion in vitro

389

of BB. The positive correlation noted between the anti-AGEs bioaccessibility indexes,

390

antioxidant/reducing bioaccessibility indexes and total phenolic compounds bioaccessibility

391

indexes indicated for the contribution of the bioaccessible phenolic antioxidants to the

392

inhibitory activity of buckwheat biscuits against AGEs formation. These findings provided

393

extensive utilization prospect in the development of buckwheat fermented flours as a

394

functional food. The use of select LAB for LSF for LSF for LSF, for example such as

395

Streptococcus thermophilus MK-10 and L. rhamnosus 8/4 for obtaining fermented flours

396

appears to be the most beneficial for enhancing the potential anti-AGEs activity of BB

397

prepared from fermented flours. The future research are ongoing on the phytochemical profile

398

of buckwheat biscuits before and after digestion and anti-AGEs activity of each identified

399

compound.

400 401

Acknowledgment

402

This work was supported by grant No 2014/15/B/NZ9/04461 from the National

403 404

Science Centre, Poland.

405 406

Authors’ contribution

407 408

H.Z. conceived and designed the research program. D.Sz-N. performed the analysis of

409

anti-AGEs activity, M.W. baking experiments and statistical analysis, H.Z. wrote the

410

manuscript with input from all authors.

411 412

Conflicts of interest

413 414

The authors declare no competing financial or other interests.

415 416

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FIGURE legends

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Figure 1. The dose-dependent relationship between anti-AGEs activity and (a)

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aminoguanidine (AG) and (b) BB extracts before digestion in vitro.

535

17

Table 1. The anti-AGEs activity of extracts from buckwheat biscuits formulated on fermented flours before and after digestion in vitro measured in BSA/Glucose system (%).

Strain/sample Aminoguanidine Biscuits from non-fermented raw flour Biscuits from raw flour fermented by: 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 1)

Buckwheat biscuits before digestion 1

Buckwheat biscuits after digestion2

68.26 ± 0.05 12.05 ± 0.19b

68.92 ± 0.05 59.41 ± 1.46a

Anti-AGEs bioaccessibility index BIAnti-AGEs 1.01 4.95 ± 0.10

11.76 ± 0.66b 12.96 ± 0.18b* 10.60 ± 0.22b* 13.65 ± 0.25b* 8.06 ± 0.28b* 12.99 ± 0.23b* 11.83 ± 0.10b 10.48 ± 0.36b* 12.64 ± 0.26b* 10.65 ± 0.35b* 13.05 ± 0.18b* 9.09 ± 0.24b* 9.97 ± 0.48b* 11.64 ± 0.44b 12.95 ± 0.34b*

55.93 ± 3.98a 65.49 ± 1.12a* 53.64 ± 0.72a* 61.82 ± 0.62a 51.37 ± 1.01a* 62.85 ± 1.10a* 62.20 ± 0.98a 61.72 ± 0.68a 64.47 ± 2.24a* 58.56 ± 0.47a 64.98 ± 1.61a* 60.72 ± 1.55a 58.49 ± 0.56a 60.17 ± 3.26a 56.91 ± 1.82a

4.75 ± 0.40 5.05 ± 0.14 5.06 ± 0.05 4.53 ± 0.12* 6.37 ± 0.18* 4.84 ± 0.08 5.25 ± 0.08* 5.89 ± 0.20* 5.10 ± 0.13 5.50± 0.32* 4.98 ± 0.09 6.68 ± 0.26* 5.87 ± 0.18* 5.17 ± 0.08* 4.39 ± 0.25

Final concentration of the extract used for the anti-AGEs test was 200 mg/mL. Data were recalculated to the extract concentration equal to the concentration obtained after digestion. 2) Final concentration of the supernatant obtained after digestion used for the anti-AGEs test was within the range of 55-60 mg/mL. 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. Means in each row followed by different letters for the inhibitory activity against AGEs formation of buckwheat biscuits before and after digestion in vitro are significantly different (P < 0.05) based on the one-way analysis of variance (ANOVA).

Table 2. The content of total phenolic compounds (TPC) in buckwheat biscuits (BB) formulated on fermented flours before and after digestion in vitro (mg GAE/g d.m.) and TPC bioaccessibility index. Strain/sample Biscuits from non-fermented raw flour (control) Biscuits from raw flour fermented by: 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

Buckwheat biscuits before digestion

Buckwheat biscuits after digestion

1.30 ± 0.04

6.01 ± 0.33

1.83 ± 0.04*b 1.48 ± 0.02*b 1.42 ± 0.05*b 1.95 ± 0.02*b 1.42 ± 0.04*b 1.94 ± 0.04*b 1.90 ± 0.04*b 1.70 ± 0.03*b 1.27 ± 0.02b 1.09 ± 0.03*b 1.25 ± 0.05b 1.42 ± 0.09b 1.21 ± 0.01*b 1.34 ± 0.03b 2.54 ± 0.05*b

8.33 ± 0.25*a 7.32 ±0.25*a 7.99 ± 0.34*a 7.41 ± 0.20*a 7.57 ± 0.18*a 7.69 ± 0.08*a 8.49 ± 0.09*a 7.24 ± 0.23*a 6.78 ± 0.24*a 8.15 ± 0.14*a 7.54 ± 0.15*a 7.28 ± 0.26*a 6.72 ± 0.10*a 7.03 ± 0.47*a 6.92 ± 0.31*a

TPC bioaccessibility index BITPC 4.58 ± 0.15

4.55 ± 0.11 4.97 ± 0.23* 5.62 ± 0.12* 3.80 ± 0.13* 5.35 ± 0.12* 3.96 ± 0.04* 4.46 ± 0.11 4.27 ± 0.19 5.35 ± 0.10* 7.47 ± 0.27* 6.06 ± 0.43* 5.15 ± 0.50 5.53 ± 0.06* 5.25 ± 0.28* 2.72 ± 0.13*

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. Means in each row followed by different letters for TPC of buckwheat biscuits before and after digestion in vitro are significantly different (P < 0.05) based on the one-way analysis of variance (ANOVA).

Table 3. The antioxidant and reducing capacity of buckwheat biscuits (BB) formulated on fermented flours before and after digestion as measured by ABTS and FRAP assays (μmol TE/g d.m.) and bioaccessibility indexes (BIABTS, BIFRAP).

Strain/sample Biscuits from non-fermented flour Biscuits from raw flour fermented by: 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 1)

Antioxidant capacity by ABTS assay Buckwheat Buckwheat Bioaccessibili biscuits after ty index biscuits before 1 2 digestion digestion BIABTS 5.20 13.81 ± 0.51 b 71.87 ± 1.49 a 16.66 ± 0.48*b 15.84 ± 0.81*b 15.59 ± 0.24*b 19.01 ± 0.52*b 14.05 ± 0.76 b 18.85 ± 0.66*b 17.69 ± 0.24*b 16.93 ± 0.14*b 13.02 ± 0.27 b 11.78 ± 0.58*b 12.92 ± 0.58 b 14.30 ± 0.51 b 12.01 ± 0.13*b 12.45 ± 0.52*b 27.95 ± 1.29*b

74.24 ± 4.33 a 66.86 ± 3.85 a 71.48 ± 1.78 a 69.98 ± 3.27 a 69.02 ± 0.61 a 67.27 ± 3.51 a 56.95 ± 2.56* a 70.18 ± 0.95 a 63.01 ± 3.82* a 62.84 ± 1.87* a 69.38 ± 2.09 a 68.76 ± 1.33 a 68.88 ± 0.79 a 71.07 ± 0.38 a 108.67 ± 2.68*a

4.46 4.22 4.59 3.68 4.91 3.57 3.22 4.15 4.84 5.33 5.37 4.81 5.73 5.71 3.89

Reducing capacity by FRAP assay Buckwheat Bioaccessibility Buckwheat biscuits after index biscuits before 1 2 digestion digestion BIFRAP 3.14 4.49 ± 0.26 b 14.10 ± 0.27 a 7.01 ± 0.28* b 6.30 ± 0.19* b 5.04 ± 0.23 b 7.62 ± 0.29*b 5.87 ± 0.32* b 7.29 ± 0.22* b 7.21 ± 0.08*b 6.79 ± 0.07* b 4.75 ± 0.27 b 4.26 ± 0.11 b 4.68 ± 0.24 b 5.52 ± 0.31* b 4.40 ± 0.20 b 5.23 ± 0.23* b 10.75 ± 0.41*b

15.07 ± 0.08*a 13.13 ± 0.22*a 12.20 ± 0.27*a 12.64 ± 0.03*a 14.45 ± 0.09*a 12.77 ± 0.15*a 11.88 ± 0.13*a 13.18 ± 0.15*a 11.38 ± 0.14*a 11.66 ± 0.01*a 12.42 ± 0.23*a 12.44 ± 0.18*a 11.65 ± 0.18*a 11.79 ± 0.15*a 36.81 ± 0.26*a

2.15 2.08 2.42 1.66 2.46 1.75 1.65 1.94 2.40 2.74 2.65 2.26 2.65 2.25 3.42

Final concentration of the extract used for the anti-AGEs test was 200 mg/mL. Data were recalculated to the extract concentration equal to the concentration obtained after digestion. 2) Final concentration of the supernatant obtained after digestion used for the anti-AGEs test was within the range of 55-60 mg/mL.

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. Means in each row followed by different letters for antioxidant and reducing capacity measured by ABTS and FRAP of BB before and after digestion in vitro are significantly different (P < 0.05) based on the one-way analysis of variance (ANOVA).

Highlights: • The extracts of buckwheat biscuits from fermented flours showed anti-AGEs effects. • A high bioaccessible anti-AGEs activity of buckwheat biscuits was found. • A high bioaccessibility of antioxidant capacity and total phenolic compounds from buckwheat biscuits was noted. • The anti-AGEs activity correlated with antioxidant capacity and total phenolic compounds. • Phenolic compounds contributed to the anti-AGEs activity.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

On behalf of the all authors Prof. Henryk Zieliński