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The Advanced Approaches to Nutritional and Breadmaking Quality of Wheat, Barley and Rye Flour

This work is focused on the characterization and rapid analytical determination of cereal flour quality with regard to nutritional and breadmaking quality. Starch, protein and non-starch polysaccharides are the main components of cereals. The content and quality of proteins and content of damaged starch is important because of the technological quality of flours. The high content of high molecular weight proteins is substantial for bread technology especially, while soluble protein fractions and non-starch polysaccharides are important for nutrition. The set of wheat, barley and rye flours and their blends were analyzed and their properties and their qualitative parameters were determined. Principal component analysis (PCA) was used on Fourier transform-infrared (FT-IR) spectra in the 1,200-800 cm^(-1) wavenumber region and significant correlations of various nutritional and breadmaking parameters were observed. Results showed that the FT-IR spectroscopy and PCA can serve for rapid screening and classification of cereal flour quality.


Introduction 
Cereals play the main role in the agricultural production of the majority of countries. This fact is connected first of all with the importance of cereals in nutrition. The greatest part of wheat and rye is used for bread and bakery products in Northern, Central and Eastern Europe and they contribute to an important source of protein and dietary fiber in the diet as well as in the Czech Republic. Wheat is the most important crop for breadmaking because of its supreme baking performance in comparison with all other cereals. Only wheat proteins can make the three-dimensional network (gluten structure) of wheat dough during mixing and kneading of dough. However, wheat is lower in dietary fiber content and the nutritive value of bread could be increased by a supplementation of rye and barley or an addition of isolated dietary fiber into the bread formula [1][2][3]. However, Hung [4] presented a new kind of whole waxy wheat with higher dietary fiber content than regular whole wheat flour. Among commonly grown cereals, whole grain rye has the highest dietary fiber content [5]. Rye is considered to be a healthy cereal with regard to a number of bioactive compounds such as phenolic acids, lignans and also alkylresorcinols [6]. Recently we have observed a renewed interest in barley for food production [7]. As the latest investigations were accomplished in barley breeding, the new beta-glucan-rich barley for baking was developed [8]. Hull-less cultivars of barley have better nutritional value than hulled ones with regards to higher protein and soluble dietary fiber content. A hull-less barley flour was incorporated into white and wholegrain wheat bread and the bread showed acceptable and desirable nutritional and sensory properties [9].
The main components of cereals are carbohydrates (unavailable: non-starch polysaccharides, dietary fiber and available: starch) and proteins. Starch is a major food reserve providing a bulk nutrient and energy source with high glycemic response in the human diet [10]. The content of starch in a cereal grain varies but generally it is between 60% and 75% of the weight of the grain. During cereal processing and grain milling in particular, the content of damaged starch is raised in the flour and can influence the technological quality of flour and handling of dough. Starch granule size distribution is an important factor that affects the quality of final cereal products. The effects of wheat starch granules size distribution were observed on mixing and breadmaking quality [11]. The determination of total or damaged starch can be carried out by chemical end group analysis or modern enzymatic methods. Determination of starch content by hydrochloric acid dissolution consists of break-down of starch in acid and measuring of the optical rotation of formed products in solution. The determination is more exacting and Carrez solutions (one of them contains cyanide) are necessary to protein precipitate. Measurement of total or damaged starch by enzymatic kit is rapid, easy but expensive.
The content of proteins in the fully-grown grains varies from 8% to 15% in dry matter. Dominant part of proteins is stored in endosperm and aleuronic layer of cereal grain. Cereal proteins have a high content of glutamine and proline and a very low content of essential amino acids as lysine and threonine. The earliest classification of cereal storage proteins is based on their solubility properties and it is often called Osborne fractionation [12]. Albumins and globulins belong to water-soluble fractions with low molecular weight and especially have the nutritive importance. Gliadins (prolamins) and glutelins have higher molecular weight (from 30 kDa to 20 MDa) and they are extracted in alcohol-water mixture and alkaline-water mixture, respectively. The ratio between high and low molecular weight gluten fractions is predominant for baking performance. Therefore, it seems that the greater the content of large molecular weight glutenin subunits, the larger the glutenin polymers and the stronger the flour [13]. Tatham [14] published the up-to-date review of wheat, barley and rye prolamins structure, physical chemistry and their functional properties. Protein in flour can be determined chemically by the Kjeldahl method, but the procedure is very complex. The indirect test by near-infrared spectroscopy (NIR) is very widely used in the milling industry but calibration should be carrying out carefully. The particular protein fractions can be separated by liquid chromatography with reverse phase and UV detection. The baking quality of gluten can be expressed by degree of sedimentation (according to Zeleny) of flour suspended in a lactic acid solution during standard time interval.
Dietary fiber is the edible part of the plants or analogous carbohydrates that are resistant to digestion and absorption in the human small intestine with complete or partial fermentation in the large intestine [15]. Detailed medical experiments indicated [16,17] that dietary fiber can stimulate weakening of hunger, stimulating peristaltic movements, reducing the level of blood glucose, regulating the activity of microbial flora, lowering of cholesterol blood serum level and decreasing of the risk of civilization diseases [18].
Dietary fiber can be classified into water-soluble (SDF) and water-insoluble dietary fiber (IDF) according to their water solubility. The significant components of cereal water-soluble fiber are -glucans, arabinoxylans, galactomannans, fructans and arabinomannans which can form viscous solutions. Water-insoluble dietary fiber consists of cellulose, hemicelluloses and lignin primarily. Lignin is a lipophilic phenolic polymer that can absorb bile acid [3]. The content of -glucans and fiber is determined mostly enzymatically. Determination of fibre content is time consuming and expensive (enzyme assay kit, reagents).
Original gravimetric, enzymatic and chemical methods of analysis were not capable to determine reproducibly and quickly the properties and constituents of grain mixtures. On the other hand, vibrational spectra have recently found their importance in qualitative food analysis [19]. Mid infrared spectroscopy in the wavenumber range 4,000-400 cm -1 is a rapid, sensitive and versatile tool for elucidating the structure and physical properties of starch [20] and other carbohydrates [21]. The most important strong absorptions for starch are at 1,150, 1,075 and 1,015 cm -1 assigning the bending vibration of (CO) groups, and medium absorptions at 845 cm -1 assigning the bending vibration (C 1 -H) of -anomer [20,22].  [24]. It was also proved to be useful for a quick evaluation of polysaccharides used as additives in foodstuffs [27].
The aim of this work was to determine the quality of selected flours using several analytical methods to find the relationship among flour quality parameters. The set of Czech and Moravian wheat, barley and rye flours and their blends were analyzed and their qualitative parameters, such as content of moisture, ash, protein and starch, protein fractions, value of sedimentation Zeleny test, solvent retention capacity profile, gluten index, content of soluble, insoluble and total dietary fiber and -glucans, were determined.
Fourier transform-infrared spectroscopy and principal component analysis were applied to correlate various nutritional and breadmaking parameters.

Chemical Analysis
Moisture content was determined by drying at 105 °C to constant weight [28]. Ash content was determined by burning the samples at 925 °C to constant weight [28]. The soluble, insoluble and total dietary fiber content was determined according to the method AOAC 985.29 [29] combined with total dietary fiber assay kit K-TDFR (Megazyme International Ireland Ltd., Wicklow, Ireland). The content of mixed linkage (1→3)(1→4)--D-glucans was determined according to the AACC Method 32-23 [30] combined with McCleary method assay procedure K-BGLU (Megazyme International Ireland  [30] was based on the reaction of individual components (gluten, damaged starch, arabinoxylans-pentosans) with water and aqueous solvents of sucrose, sodium carbonate or lactic acid. All the determinations were carried out in duplicate and were expressed on dry weight basis. Fourier transform infrared spectra (FT-IR) of flours and their blends were measured and processed by principal component analysis (PCA).

FT-IR Spectroscopy and Statistical Analyses
Fourier transform infrared spectra (FT-IR) of flours and their blends were measured in the wavenumber region of 4,000-400 cm -1 (mid infrared region) in KBr tablet, transmittance mode and at the resolution of 2 cm -1 . Collected spectra were processed by PCA within the 1,200-800 cm -1 region (software package Statistica, version 7.1, StatSoft CR, Czech Republic). The relationships among the different analytical parameters of analyzed flours were tested by a simple correlation (Pearson correlation). Significant differences were declared at P < 0.05.

Breadmaking Characteristics of Flours
The higher content of ash had barley bran (3.8%) and blends (1.1%) (all values are related to dry matter) ( Table 2). The content of total starch was determined from 62% to 73%. The average content of protein in flours was 12.7%, whereas the highest content of protein (15.2%) was found in sample no. 25   addition of barley and rye to wheat flour caused a decrease of protein content in blends (decrease of value of Zeleny test and gluten index) and pronounced deterioration of breadmaking quality of flours. Wheat flours for bread production showed the higher content of gluten (Zeleny test, SRC of lactic acid) in comparison with blends. Wheat flours and blends did not show different value of gluten index.
Solvent retention profile of selected flours and their blends is showed in Fig. 1. The value of SRC of lactic acid characterized the high content of glutenins (high molecular fraction of gluten) in wheat flours and the low content of hordenins in barley flours ( Table 2). The highest content of protein, glutenin (determined by

Nutritional Characteristics of Flours
Generally, wheat flours had the lowest content of dietary fiber and -glucans (Table 3). An addition of barley or rye increased the content of fiber and -glucans in blends. Brennan [5] and Rakha [18] described that the content of -glucans is about 1% in wheat grains, 1%-2% in rye and 5%-11% in barley. In our case, barley flour contained from 2.5% of -glucan (sample no. 10) to 4.2% (sample no. 13).
The portion of protein fractions of Czech flours and blends were not significantly different (Table 3). Only, rye flour (sample no. 14) had evidently higher content of albumins and globulins in comparison with other cereals. The most often published wheat and barley protein fraction proportions are 20%-25%, 40%-50%, and 35%-40% for albumins and globulins, gliadins and glutenins, respectively [12]. Different fraction proportion is showed in rye: albumins 44%, globulins 10%, gliadins 21% and glutelins 25%. Some Moravian flours came from same place of origin (e.g. samples no. 23-25, or samples no. 29-31) and they had comparable contents of protein fractions. The higher content of glutenins and the lowest content of gliadins were found in samples no. 29, 30 and 31. The content of glutelins decreased in blends in accordance with a decrease of total protein content determined by Kjeldahl.
Statistically significant positive correlations were observed between -glucan and protein (r = 0.85, P < 0.05), -glucan and SRC of sucrose (r = 0.93, P < 0.05), between -glucan and soluble dietary fiber (r = 0.88, P < 0.05), and -glucan and gluten value of Zeleny test (r = 0.81, P < 0.05). A strong positive correlation between the content of -glucan and protein and between -glucan and soluble fiber was confirmed with the results obtained by Holtekjølen [33]. Positive correlations were found between soluble dietary fiber and SRC of water (r = 0.95, P < 0.05), between proteins and total dietary fiber (r = 0.88, P < 0.05), and SRC of lactic acid and gluten value of Zeleny test (r = 0.70, P < 0.05). Significant negative correlations were observed between content of -glucan and starch (r = -0.69, P < 0.05) and between proteins and starch (r = -0.83, P < 0.05).

FT-IR Spectroscopy and Principal Component Analysis
The transmission FT-IR spectra of flours and blends were recorded. The characteristic region of starch, non-starch polysaccharides and protein absorption was observed. PCA was used on IR data and PCA score allowed to separate the flours and their blends according to the content of starch and non-starch polysaccharides, especially -glucan and fiber content. The flours were separated to three clusters (wheat, blends, barley and rye) (Fig. 2).
The application of a PCA to the FT-IR spectra in the mid-infrared region was effective for fast distinction of various different types of flours on the basis of nutritional and breadmaking flour parameters.

Conclusions
An addition of barley or rye to wheat flour influenced the quality of flour blends significantly. The content of nutritive and healthy components (soluble proteins, fiber, -glucans and pentosans) was increased in blends, but the breadmaking quality decreased (higher content of damaged starch, higher water capacity and sticky dough).
FT-IR spectroscopy and chemometric analysis (PCA analysis) can be used to a fast screening of the cereal flour quality and to classification of flours on the basis the specific purpose of their uses.