**2. Research methods**

**1. Introduction**

of wider use of buckwheat in pastry.

240 Superfood and Functional Food - An Overview of Their Processing and Utilization

buckwheat to produce functional products.

buckwheat flour [18, 19].

Ginger cakes are traditional pastries from Central and Eastern Europe. The very important feature of ginger cakes is their ability to remain fresh and savoury for a long time. Currently, ginger cakes are baked on the basis of wheat and rye flour. However, a traditional recipe for ginger cakes was based on the use of rye flour. In Poland, rye (*Secale cereale* L.) is an important grain for bread making and cookies production; therefore, in 2012 18 % of cereal products were made of rye [1]. Rye grain is considered to be a good source of biologically active compounds like antioxidants [2]. Referring to the up-to-date literature, it is highlighted that there is a lack

The buckwheat is a rich source vitamin B1 and B2, lysine, protein with balanced amino acid composition [3], flavonoids [4], phytosterols [5], soluble carbohydrates, D-chiro-inositol and other fagopyritols [6] and thiamin-binding proteins [7]. Buckwheat is also rich in antioxidant compounds such as flavonoids, phenolic acids, tocopherols, reduced glutathione, inositol phosphates and melatonin [8]. Furthermore, buckwheat contains a high amount of rutin (quercetin-3-rutinoside) and has antioxidant, anti-inflammatory and anticarcinogenic properties [9]. According to various chemical compositions, buckwheat-based products were found to display several biological activities, including the increasing number of lactic acid bacteria in rat intestine, treatment of allergic inflammation, reducing the serum glucose level, suppressing cholesterol level, inhibiting protease and scavenging free radicals [10, 11]. These healthy and dietary benefits of buckwheat are main aspects in determining the usage of

The wide spectrum of buckwheat-based bakery and pastry products, e.g. bread, biscuits, crackers, cookies or muffins, was designed by researchers [12]. Mancebo et al. [13] observed that consumers' rating of cookies prepared from buckwheat did not reach high quality score which was mainly related to unpleasant and pungent taste of buckwheat. Therefore, Filipčev et al. [14] noted that 30 % of buckwheat flour is appropriate to create buckwheat-based product with high sensorial acceptability. Chlopicka et al. [15] also showed that 30 % addition of buckwheat flour is highly acceptable, and moreover buckwheat bread has a high antioxidant potential. Moreover, while analysing gluten-free products' sensory profiles, Loredana et al. [16] suggested that the optimum buckwheat flour addition is different for cake, cookies and muffins. The optimum amount in cake was established on the level of 30 % and 10 % for cookies, while for muffins 20 %. Not only the optimisation of the recipes but also technological process parameters for buckwheat-based product preparation have acquired an increasing interest. Lee [17] achieved high overall acceptance for steam bread (wheat with 3 % of buckwheat flour). However, the addition of buckwheat flour was not as high as in the previous studies yet, during the steaming process, not as high amount of an undesirable Maillard reaction products may be formed. Moreover, it is said that dough fermentation step can lead to nutritive and antinutritive compound formation, but some studies also suggested that fermentation process negatively influenced sensory properties of Turkish bread yufka supplemented with 10 % of

#### **2.1. The rye-buckwheat ginger cake preparation**

The ginger cakes were prepared using a mix of rye flour (70 %) and light buckwheat flour/flour from roasted buckwheat groats (30 %). Then the rye-buckwheat ginger cake recipe was modified by the addition of low (50 mg/of rutin/100 g of flour mix) and high rutin (100 mg/of rutin/100 g of flour mix) dosage. The rutin dosage added to buckwheat ginger cakes was adjusted to rutin content in one tablet of OTC drugs. The control cake was prepared from rye flour. The ingredient list used for rye-buckwheat ginger cake preparation is included in **Table 1**. All the ingredients were well mixed, and then half of dough was set aside, and the other half was cut into regular discs and baked at 180 °C for 18 min. The first half of dough was spontaneously fermented for 72 h at 21 °C in fermented chamber. Then, the fermented dough was prepared as the previous one. The cakes were freeze-dried and powdered after baking and cooling. The powdered samples were stored at −20 °C until analysis of functional properties and Maillard reaction product formation.


**Table 1.** The list of ingredients used for rye-buckwheat ginger cake formulation.

*Sample description*: RGC (control), rye ginger cake; BERGC-1, buckwheat-enhanced rye ginger cake formulated on (1) light buckwheat flour; BERGC-1L, buckwheat-enhanced rye ginger cake with low rutin dose; BERGC-1H, buckwheat-enhanced rye ginger cake with high rutin dose; BERGC-2, buckwheat-enhanced rye ginger cake formulated on (2) flour from roasted buckwheat groats; BERGC-2L, buckwheat-enhanced rye ginger cake with low rutin dose; and BERGC-2H, buckwheat-enhanced rye ginger cake with high rutin dose.

#### **2.2. The determination of total phenolic, rutin contents and antioxidant capacity in ginger cakes**

The initial step included preparation of ginger cake extracts. Therefore, 100 mg of powdered samples was extracted with 1 ml of 80 % (v/v) methanol solution. Then, the mixture was treated by ultrasounds (30 s) and vortexed (30 s) three times. After centrifugation (6860 rpm at controlled temperature 4 °C, 5 min) the supernatant was collected into 5-ml flask. That step was repeated five times to achieve the final extract concentration 20 mg/ml.

The total phenolic content (TPC) was measured using Folin-Ciocalteu reagent according to Przygodzka et al. [30], whereas the rutin content was determined with HPLC with UV detector (330 nm), reported by Zielińska [31]. The antioxidant properties were determined by measurement of scavenging ability against ABTS radical cation, DPPH radical and superoxide anion radical (O2 •−) [30]. The measurements were carried out using spectrophotometer UV-160 1PC (Shimadzu, Japan) and Photochem® apparatus (Analytical Jena, Germany). The Trolox was used as a standard.

#### **2.3. The Maillard reaction product determination: furosine, fluorescent intermediary compounds (FIC), carboxymethyllysine (CML) and melanoidins**

baking and cooling. The powdered samples were stored at −20 °C until analysis of functional

**Ingredients RGC BERGC-1 BERGC -1L BERGC -1H BERGC -2 BERGC -2L BERGC -2H**

– – – – 30 30 30

2 2 2 2 2 2 2

Rye flour [g] 100 70 70 70 70 70 70 Light buckwheat flour [g] – 30 30 30 – – –

Buckwheat honey [g] 50 50 50 50 50 50 50 Sugar [g] 20 20 20 20 20 20 20 Baking soda [g] 3 3 3 3 3 3 3 Butter [g] 25 25 25 25 25 25 25

Rutin [mg] 0 0 50 100 0 50 100

*Sample description*: RGC (control), rye ginger cake; BERGC-1, buckwheat-enhanced rye ginger cake formulated on (1) light buckwheat flour; BERGC-1L, buckwheat-enhanced rye ginger cake with low rutin dose; BERGC-1H, buckwheat-enhanced rye ginger cake with high rutin dose; BERGC-2, buckwheat-enhanced rye ginger cake formulated on (2) flour from roasted buckwheat groats; BERGC-2L, buckwheat-enhanced rye ginger cake with low rutin dose; and

The initial step included preparation of ginger cake extracts. Therefore, 100 mg of powdered samples was extracted with 1 ml of 80 % (v/v) methanol solution. Then, the mixture was treated by ultrasounds (30 s) and vortexed (30 s) three times. After centrifugation (6860 rpm at controlled temperature 4 °C, 5 min) the supernatant was collected into 5-ml flask. That step

The total phenolic content (TPC) was measured using Folin-Ciocalteu reagent according to Przygodzka et al. [30], whereas the rutin content was determined with HPLC with UV detector (330 nm), reported by Zielińska [31]. The antioxidant properties were determined by measurement of scavenging ability against ABTS radical cation, DPPH radical and superoxide anion

(Shimadzu, Japan) and Photochem® apparatus (Analytical Jena, Germany). The Trolox was

•−) [30]. The measurements were carried out using spectrophotometer UV-160 1PC

**Table 1.** The list of ingredients used for rye-buckwheat ginger cake formulation.

BERGC-2H, buckwheat-enhanced rye ginger cake with high rutin dose.

was repeated five times to achieve the final extract concentration 20 mg/ml.

**2.2. The determination of total phenolic, rutin contents and antioxidant capacity**

properties and Maillard reaction product formation.

242 Superfood and Functional Food - An Overview of Their Processing and Utilization

Flour from roasted buckwheat groats [g]

Spice mix for ginger

**in ginger cakes**

radical (O2

used as a standard.

cakes [g]

In these buckwheat ginger cakes, markers of early (furosine), advanced (carboxymethyllysine (CML), total fluorescent intermediate compounds) and final (melanoidins) Maillard reaction compounds were identified and quantified.

The furosine content was determined as described by Delgado-Andrade et al. [32], using HPLC (Shimadzu, Japan) with PDA detector setup at 280 nm. To determine the furosine quantity, the external standard of furosine at concentration range 0.2–9 μg/ml was applied. In the next step, the fluorescent intermediary compounds (FIC) was measured. The total sum of intermediatory compounds in buckwheat ginger cake extracts was determined according to procedure described by Delgado-Andrade et al. [21]. The analysis was followed by enzymatic hydrolysis step using pronase E to break the bindings between intermediatory compounds and proteins. The fluorescent readings were registered at extinction wavelength 347 nm and emission, 415 nm, using a luminescent spectrofluorometer (LS-50B, PerkinElmer, USA). The results are expressed as fluorescence intensity (FI) per milligram of dry matter.

The degradation of proteins (nutritional value) was expressed as FAST index according to Damjanovic Desic and Birlouez-Aragon's procedure [24]. The FAST index was calculated as a ratio of the fluorescence of intermediatory compounds, measured at extinction wavelength 347 nm and emission, 415 nm, using a luminescent spectrofluorometer (LS-50B, PerkinElmer, USA), to fluorescence of tryptophan (extinction ,290 nm, and emission, 340 nm) and described as a percentage.

The carboxymethyllysine (CML), one of the intermediatory compounds, was quantified and determined by HPLC method. The CML extraction was followed by a detailed description of Peng et al. [9]. The OPA reagent solution, which is a mixture of 10 mg of *o*-phthaldialdehyde (OPA) in 2 ml of methanol and the CML determination, was evaluated by HPLC (Dionex, USA) with fluorescent detector (SFLD-3400RS, Dionex, USA). The detector settings were established as the excitation wavelength 455 nm and emission, 340 nm, whereas the oven temperature was adjusted at 35 °C and flow rate 0.2 ml/min. The CML was separated on Luna® 3 μm C18 column (Phenomenex, USA) and eluted in isocratic gradient by water with 0.05 % of *o*-phosphoric acid and acetonitrile with 0.05 % of *o*-phosphoric acid. For quantitative analysis, calibration curve of CML standard was prepared in the range from 2.5 to 20 μM. The results were expressed in μg per gram of dry matter.

The formation of final Maillard reaction products was estimated as reported previously by Zieliński et al. [33]. The absorbance of buckwheat ginger cake methanolic extracts was measured at 410 nm using UV-Vis spectrophotometer (Shimadzu, Japan). Final results were expressed as the absorbance units (AU).

#### **2.4. The evaluation of buckwheat ginger cake inhibitory activity against advanced glycation end-product formation**

The inhibitory effect on formation of advanced glycation end products (AGEs) in ginger cakes was studied according to the procedure described by Szawara-Nowak et al. [29] in two in vitro model systems: bovine serum albumin-glucose (BSA-glu) and bovine serum albuminmethylglyoxal (BSA-MGO). The fluorescence intensity was measured at the excitation wavelength 330 nm and emission 410 nm using a luminescent spectrofluorometer (LS-50B, PerkinElmer, USA). The results are expressed in percentage inhibition of AGE formation. Aminoguanidine solution (1 mmol/l) was used as a positive control in this experiment.
