**2. Materials and methods**

#### **2.1. Chemicals and reagents**

**1. Introduction**

Development of products which positively affect the consumer health is an important aspect followed by the food industry. This assignment may be realized when process of the industry is concentrated on the natural antioxidants. Antioxidants present in food can protect against lipid and protein autoxidation. It is very important to quantify the antioxidant properties of different types of food. In addition, a special attention is devoted to the processing methods to

Presently, wheat flour is widely used in bread making; however, other types of flour are also used. Rye and spelt types of flour are preferred due to the content of micro- and macronutrients and fibre [2, 3]. Recently, the potential usage of buckwheat flour as a functional component in food has been demonstrated [4]. Buckwheat (*Fagopyrum esculentum* Moench), commonly, cultivated in Russia and China, is added to the other cereal grains because of likenesses in usage [5]. Buckwheat is a rich source of nutrients (lysine, vitamins B, carbohydrates) [6] and antioxidants such as vitamin E, glutathione, phytic acid [7], phenolic acids and flavonoids mainly rutin, with anti-inflammatory, anti-carcinogenic and anti-glycation properties [4, 8, 9]. Buckwheat polyphenols can function as antioxidants in one or more possible ways: as reducing agents, as compounds that scavenge free radicals, as chelating agents of metals that catalyse oxidation reactions and thus limiting their ability to initiate free radical chain reactions or by inhibiting oxidative enzymes such lipoxygenases [10–13]. However, processing conditions may considerably affect biological activity of polyphenols. There are many studies regarding negative effects of thermal processing on the phenolic compounds including flavonoids. The type of heat transfer and processing conditions are the major factors responsible for the observed decrease in the flavonoid content in food [7, 14–16]. Having all these evidences, buckwheat mill products seem to be an attractive ingredient in the bakery industry [17–20]. The recent evidences have shown that the intake of bread with addition of buckwheat flour in the recipe resulted in a positive increase of antioxidant potential in humans [17]. This finding, due to the quality properties of buckwheat bread as described by Lin et al. [21], can make it favourable for developing a healthy diet. Recently, a number of food products containing buckwheat has been investigated such as buckwheat-enhanced ginger nutty cakes [18], buckwheat enriched wheat bread [17, 20, 21] and buckwheat cakes [18, 19, 22]. Therefore, due to the nutritional value and beneficial effects on human health, buckwheat and partially

buckwheat-based products form a pool of potential functional food [4, 10].

reagents and are quick [13].

For the overall characterization of a new food product, more often the antioxidative capacity is used. There is a variety of analytical methods to assess the antioxidant capacity of food. However, there is no single standard method that would be used to determine the antioxidant capacity of the complex matrix and give consistent, unquestionable results in confrontation with other analytical methods. Therefore, it is advisable to use more than one method. A part of analytical methods is based on scavenging of non-natural free radicals, other deal with lipid peroxidation chemical markers. These methods need a little preparation, small amounts of

maintain the beneficial antioxidant properties of food [1].

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

n-Hexane and methanol (HPLC-grade) were provided by Merck (Darmstadt, Germany). 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), L-ascorbic acid (AA) and sodium dodecyl sulfate (SDS) were from Sigma Chemical Co. (St. Louis, MO, USA). PCL ACW (antioxidant capacity of water-soluble substances) and PCL ACL (antioxidant capacity of lipid-soluble substances) kits were from Analytik Jena AG (Jena, Germany). Monobasic potassium phosphate, dibasic potassium phosphate, ethylenediaminetetraacetic acid (EDTA), ferric chloride and ferrous chloride were purchased from POCh (Gliwice, Poland). Water was purified with a Mili-Q-system (Millipore, Bedford, USA).

#### **2.2. Preparation of buckwheat-enhanced dark wheat breads**

Dark wheat flour (DWF) and buckwheat (variety Kora) flour were purchased from a healthy food store in Olsztyn, Poland. The flour from roasted buckwheat groats (BFR) was procured from a local company in Poland. The dry matter in BF, BFR and DWF was 87.6, 89.7 and 87.0%, whereas protein content was 10.6, 14.3 and 8.1%, respectively. BF or BFR was used to replace DWF at levels of 10, 20, 30 and 50% (w/w). Buckwheat-enhanced dark wheat breads (BEDWBs) and reference dark wheat bread (DWB) were baked in a laboratory bakery. **Table 1** shows the buckwheat-enhanced dark wheat breads formulation and baking conditions. Three pieces of each type of bread was baked. Samples were freeze-dried, milled and sieved through of 0.6 mm, and then were stored at –20°C before using for analysis.


**Table 1.** Buckwheat and reference dark wheat breads formulation and baking conditions.
