Comparison of the Phenolic Compounds, Carotenoids and Tocochromanols Content in Wheat Grain under Organic and Mineral Fertilization Regimes

A field study was performed to evaluate the effect of mineral (NPK) and organic-based fertilizers such as compost (C), manure (FYM) and meat and bone meal (MBM) on the appearance (dimensions and color) of spring wheat kernels and on the total content in grain of main its phytochemicals (polyphenols, carotenoids and tocochromanols) and phenolic acids composition. Total phenolic compounds were determined using the Folin-Ciocalteu assay after alkaline hydrolysis of grain and carotenoids were analyzed spectrophotometrically. Composition of tocochromanols and phenolic acids was determined using RP-HPLC techniques. Only insignificant differences in the appearance of kernels and small changes in the content and composition of grain phytochemicals were noted between the studied fertilization systems. Among the analyzed phytochemicals the greatest variation was observed in the group of polyphenol compounds, with a stated increase of their total content of 6.7 and 11.2% in grain fertilized with MBM and compost, respectively. Simultaneously the grain from organic fertilization contained significantly less phenolic acids, and the decrease in their content ranged from 10.0% for FYM to 24.8% for MBM+EM-1. Organically and conventionally fertilized grain had similar amounts of tocochromanols and carotenoids. Comparison of MBM and MBM+EM-1 variants showed that application of effective microorganisms decreased carotenoids and tocochromanols content by 8.5 and 9.7%, respectively.

by applying effective microorganisms (EM) which are able to carry out targeted processing of nutrients present in soil and to make it more fertile [21,22].
There have been many reports lately on the effect of organic fertilization on the content of the main plant storage polymers (carbohydrates and proteins) as well as micro-and macroelements [16][17][18]. However, there have been few reports comparing the effect of different forms of fertilization on the content of nutritionally valuable phytochemicals. Zuchowski et al. [11], Taie et al. [23], and Omar et al. [24] observed an increase in concentration and change of the composition of phenolic compounds in wheat grain, soy bean and cassava tubers, respectively. On the other hand, Hussain et al. [25] found that in organically grown wheat the content of tocochromanols was in a similar range as reported for conventionally grown wheat. Stracke et al. [26] concluded that climate factors have a greater impact on the carotenoids and phenolic acids concentrations in wheat grain than production methods (organic vs. conventional).
The present study aimed at evaluating whether wheat grain produced conventionally and organically (with the use of compost, manure and meat and bone meal) differs in the content of total polyphenols and phenolic acids, carotenoids and tocochromanols.

Kernel Characteristics
Thousand kernels weight (TKW) ranged from 33.79 to 35.29 g (Table 1). These are typical values for spring wheat kernels, which are usually smaller than those of winter wheat [26][27][28][29]. It has been shown that only compost and MBM+EM−1 changed TKW of wheat grains in comparison to the NPK system. The kernels from plots fertilized with compost were 2.2% heavier, and kernels from plots fertilized with MBM+EM−1 of 2.1% lighter than traditionally fertilized ones. The average length and width of the kernels ranged from 7.15 to 7.30 mm and from 3.15 to 3.24 mm, respectively (Table 1). Although the differences relative to the NPK fertilization variant did not exceed 2%, compost fertilization resulted in slight, but significant shortening of kernels. The kernels were also wider than those obtained with the other variants of organic fertilization ( Figure 1). This resulted in the lowest value of the length/width ratio (2.21). On the contrary the highest value of that index was determined for the sample fertilized with MBM+EM−1. The fertilization variants caused only slight differences in the color of the kernel surface ( Table 1). The most varied values of the color components were observed for kernels fertilized with NPK (H = 27.23, S = 28.01, I = 60.13) and those treated with FYM (H = 28.02, S = 30.76, I = 57.70). This suggests that the kernels from conventional cultivation were slightly lighter and more red than others.

Chemical Composition
The results of determination of free and total phenolic compounds are presented in Table 2. The total content of polyphenols ranged from 2,710 to 3,016 µg/g of grain. The grains from NPK fertilization contained the lowest concentration of these compounds, and the highest value was seen in grain which was fertilized with compost. The ether-soluble fractions accounted for 42% to 49% of them. Only a small part of the total polyphenols occurred as soluble forms extracted by 80% methanol. The lowest concentration was found in the sample of grain fertilized with NPK (16.6%) and the highest was in the sample fertilized with MBM+EM−1 (19.1%).
An analysis of the phenolic acids composition has shown that the wheat grain contained the following acids: ferulic, sinapic, p-coumaric, vanillic and p-OH benzoic ( Table 2). The dominant ferulic acid accounted for 75% to 81% of the total content, and the proportions of the other acids were as follows: sinapic acid-from 12% to 15%, p-coumaric acid-from 4% to 7% and both of vanillic and p-OH benzoic acids-approx. 2% of the total content. All the samples from organic farming contained significantly less phenolic acids as compared with the grain treated with NPK. It was the most visible in grain from MBM−EM−1 cultivation system that contained about 1/3 less total phenolic acids than the grain from NPK fertilization.
The content of free and total carotenoids is presented in Table 3. Depending on the fertilization systems, the wheat grain contained from 1.38 to 1.54 μg/g of petroleum ether soluble pigments and from 3.54 to 3.87 μg/g of total yellow pigments. Generally, the differences between the samples were small, but the grain treated with MBM was found to contain the largest amounts of carotenoids. Supporting the fertilization with effective microorganisms reduced the accumulation of carotenoids in wheat grain. The ether-soluble fractions accounted for 36.4% to 42.1% of total carotenoids, and was the highest in NPK fertilized grain. This suggests that more polar than carotene xantophylls predominated in the tested grain. Table 3. Content of carotenoids (CAR), tocopherols (T) and tocotrienols (T3) in wheat grain from different systems of fertilization (μg/g of seed dry mass). The wheat samples contained both forms of tocochromanols (Table 3). Approximately 90%-94% of them were extractable by petroleum ether. Analysis of (total) water-butanol soluble tocochromanols showed that α-and β-tocopherol ranged from 13.56 to 15.48 μg/g and from 5.15 to 6.10 μg/g, respectively. Their total content was the highest in the grain fertilized with MBM (21.34 µg/g), but use of EM−1 removed that positive effect. The tocotrienol fraction was found to contain two main forms: β-T3 (from 17.55 to 19.14 μg/g), α-T3 (from 5.38 to 6.29 g/g) and traces of γ-T3 (below 0.4 μg/g-data not presented).

Compound * System of fertilization
The total tocotrienol content ranged from 22.93 to 25.15 g/g. The combined content of both forms of tocochromanols was the highest in the C (compost) and MBM samples, but they were not statistically different from the NPK variant. Using effective microorganisms reduced the total content of tocochromanols by as much as 8%, to a lower level than that determined for the NPK variant. The ratio of both forms in water-butanol extracts ranged from 1.15 to 1.24, with tocotrienols dominating.

Discussion
The size, shape, weight and color of kernels determine the commercial value of wheat grain. These features are associated with its technological and milling quality and the baking value of flour. In general, big grains containing large amounts of nutrients, with uniform color and dimensions, are more desirable for food processing. In hexaploid wheat, these traits are controlled by a range of quantitative traits loci (QTL) on chromosomes of all the three genomes A, B and D [27,28]. However, it has been well-documented that the environmental conditions can also change the cultivar characteristics. For example, high temperatures and a water deficit significantly reduce kernel weight and dimensions (thickness and width) [29,30].
Of the phytochemicals under investigation, polyphenols are a more diverse group, both in the qualitative and quantitative aspect. Their content lies within a wide range from about 800 to 2,400 µg/g of dry matter of grain [3,31,32]. They include phenolic acids at up to 700 µg/g [11], flavonoids at up to 500 µg/g [32,33], condensed tannins at up to 700 µg/g [34], alkylresorcinols at up to 800 µg/g [35][36][37] and lignans at up to 4 µg/g [33,38]. The results of our study indicate that the total content of phenolic compounds is equal to 2,700-3,000 µg/g of dry matter, which is slightly higher than in the papers cited above. This may be explained by the effect of alkaline hydrolysis of grain, that releases phenol aglycons from ester and glycoside compounds with cell-wall polysaccharides [39,40] and decomposes of other grain components, which can subsequently react with the Folin-Ciocalteu (F-C) reagent [41]. Everette et al. [41] even suggest that F-C assay should be rather seen as a method to measure total antioxidant capacity than phenolic content. From this point of view the values of approximately 3,000 µg/g indicate high antioxidative potential of the grain samples under investigation, which was the highest in the sample fertilized with compost and the lowest in the sample treated with NPK. Phenolic acids were responsible only for approx. 20%-25% of the potential. The main phenolic acid was ferulic acid, that may account for as much as 90% of total phenolic acids [3,11].
Although there have been many reports on polyphenols in cereal grain, data on the effect of cultivation/fertilization on their content are scarce. Most of them indicate that phenolic compounds content is higher in grain from organic cultivation. According to Zuchowski et al. [11] the observed higher concentration of phenolic acids in organic wheat is caused mainly by the smaller kernel size. Taie et al. [23] explained this phenomenon by the action of bioorganic fertilizers that help plants to fix nitrogen from the air and utilize it to production phytohormones and other growth-promoting compounds [42], such as phenolics. Our results confirmed the higher accumulation of total polyphenols in grain from organic cultivation, although those kernels contained significantly less phenolic acids (on average of 18%).
One of the factors which affects the biological activity of phenolic compounds is their bioavailability from the diet. First of all, the form in which they enter alimentary tract (free or bound) is important. Monomers and dimers which are part of the free fraction are more easily available and they are imbibe in the higher sections of the alimentary tract [43,44]. From this point of view, grain produced by organic cultivation is more valuable because it contains more free polyphenols, with the largest increase observed for MBM+EM-1 fertilization. This may have been affected by enzymatic activity of grain microflora. Suproniene et al. [45] have shown that increasing the amount of mineral fertilizers favors infection of the surface of spring wheat grain by Fusarium and Penicillium species.
These fungi produce esterases, which are part of the enzymatic spectrum employed to degrade plant polysaccharides and to release phenolic compounds [46][47][48].
Moreover, the amount and proportions of tocochromanols in wheat grain samples vary and lie within a wide range from 10.2 to 74.3 μg/g for the content, and from 1.2 to 5.3 for the tocotrienol/tocopherol ratio [3,25,31,[49][50][51]. The results of this study lie in the middle of the range for the content and at the lower boundary of the range for the ratio. The reports on the effect of fertilization for this group of compounds have also been scarce. According to Hussain et al. [25] organically grown wheat contains similar amounts of tocochromanols to that found in conventionally grown wheat (a study conducted in Sweden). These conclusions have been confirmed by our results, because the differences relative to the NPK sample did not exceed 5%. Synthesis of tocochromanols in plastids and chloroplasts is affected by stress and/or senescence of grain [52][53][54]. The main function of tocochromanols is to protect PUFA in cellular membranes against oxidative stress; they also regulate the process of adaptation to cold and the germination process [53,54]. Studies conducted for several crop species have shown that synthesis of α-tocopherol is favored by such factors as drought, heat, salinity and UV/light [55,56]. This is probably one of the reasons for the large variability in tocochromanols content in wheat grain which have been reported in the literature. Another reason is the different extraction techniques, which was mentioned by Okarter et al. [3].
Wheat grain usually contains 1.3 to 5.0 µg/g of carotenoids [3,34,57]. They are deposited throughout the whole kernel in different wheat species. The main compound of these pigments is lutein, whose total content ranges from about 50% to 95% [3,58,59]. The present results found the content of carotenoids to be within the cited range. We can state that organic fertilizers only slightly affected accumulation of these compounds (differences not exceed 7%). Similar results were previously obtained by Stracke et al. [26] who stated that climate has a greater impact on the carotenoids concentration than the production method. Organic fertilization have also slightly changed the ratio of ether-soluble and water-butanol-soluble fractions of carotenoids. Generally, the lowest content of less polar forms (ether-soluble) has been found in grain fertilized with MBM (alone and with addition of effective microorganisms). This may possibly affect antioxidative function of carotenoids in grain-based diet.

Plot Experiments
Samples of spring wheat grain of the Tybalt cultivar, cultivated in 2009 in the experimental field in Bałcyny in the north-eastern part of Poland (53°36'N, 19°51'E) were used as the study material. A single-factorial field study was carried out. The experiment was set up in a random block design, with four replicates. The experimental design covered five systems of fertilization (Table 4).
Effective microorganisms were used only on the plot fertilized with meat and bone meal at 5 dm 3 ha −1 , in two doses: (a) 3 dm 3 ha −1 -immediately before sowing, (b) 2 dm 3 ha −1 -before the first weeding procedure. The EM-1 preparation made by the Greenland Technologia EM Company (Trzcianki, Poland) was used. Microorganisms were revived by adding 1 dm 3 of EM−1 with 4 dm 3 of water sweetened with 40 g of molasses and leaving it for 12 h at the temperature of 20 ± 2 °C. The preparation was water-diluted and used as a spray in the dose of 300 dm 3 ha −1 . Spraying was done on humid and cloudy days, immediately before mechanical agricultural procedures. The composition of the microflora in used preparation was not determined, but Szymański and Patterson [60] and Valarini et al. [61] have previously reported that it contains bacteria-Lactobacillus plantarum, L. casei, Streptococus lactis, Rhodopseudomonas palustrus, Rhodobacter spae; yeast-Saccharomyces albus, Candida utilis; Actinobacteria-Streptomyces albus, S. griseus and fungi-Aspergillus oryzae, Mucor hiemalis.

Grain Features Measurement
Harvested grain was dried to a moisture content below 15%. It was then cleaned of dust and broken kernels on a laboratory air-sieve separator (fragments with width/thickness below 2.2 mm were rejected) and stored at a temperature of 6 ± 2 °C. The weight of 1,000 kernels (TKW) was analyzed using an LN-S-50 seed counter in five replicates. The kernel dimensions: length, width and length/width ratio (elongation) and color of surface were determined for 60 kernels using the digital image analysis according to Konopka et al. [57]. The images were acquired by a high resolution, low-noise CCD Nikon DXM-1200 color camera and analyzed by LUCIA G ver. 4.8 software. The results are presented in HSI (H-hue, S-saturation, I-intensity) color space, where H is expressed in degrees, and S and I in percentages.

Extraction of Grain Phytochemicals
Before the chemical analyses, the grain was ground to obtain particles smaller than 300 µm. The carotenoids, tocochromanols and polyphenols extraction scheme is presented on Figure 2. Free lipophilic compounds (carotenoids, tocochromanols) were extracted without a saponification step by petroleum ether and free polyphenols by 80% methanol. Total carotenoids and tocochromanols were extracted by water-saturated butanol. Extraction of total polyphenols was preceded by alkaline hydrolysis of wheat samples with 2 N NaOH × 4 h at room temperature. After hydrolysis, the mixture was neutralized (2 N HCl) and evaporated to dryness. Released polyphenols were extracted in two steps (1) by the use of diethyl ether and (2) by the use of 80% methanol. Additionally, the composition of phenolic acids was determined in the extract of total polyphenols according to method of Robbins [62]. To prevent phytochemicals oxidation the extraction procedures were done in the presence of a mixture of butylated hydroxytoluene (BHT). Extractions with petroleum ether were conducted in a FoodALYT RT 60-type Soxhlet apparatus (Omnilab). Extractions with water-saturated butanol were conducted by the method proposed by Kaneko [63], and that of polyphenols using the modified method proposed by Irmak et al. [64]. All the extracts were concentrated to dryness at temperatures below 50 °C.

Determination of Total Carotenoids Content
Carotenoids content was determined spectrophotometrically by the method described by Craft [65]. To this end, 2.5% extract solutions in hexane were prepared and their absorbance was measured at the wavelength of 454 nm (maximum of lutein absorption). The measurements were carried out with a UNICAM UV/Vis UV2 spectrophotometer. Carotenoid content was calculated based on molar absorptivity coefficient (for lutein dissolved in hexane is equal to 147,300 L/molcm) and molar mass of lutein (equal to 568.87 g/mol) [65]. The results are presented as μg/g of a sample dry mass.

Determination of Tocopherols and Tocotrienols Content
The tocopherols and tocotrienols content was determined by the RP-HPLC technique using the method described by Gimeno et al. [66]. One % solutions of ether extract and butanol-water extract in  hexane were prepared. After being stirred and centrifuged in a 5417R type Eppendorf centrifuge (10 min, 25,000 × g), they were transferred to chromatography vials. The analysis was carried out in a series 1200 Agilent Technologies apparatus, fitted out with a fluorescence detector of the same manufacturer. Separation was done on a LiChrospher Si 60 column (5 μm, 250 mm × 4 mm) manufactured by Merck, with 0.7% isopropanol solution in hexane as the mobile phase. Identification of the isomers was based on retention times, determined for reference standards of tocopherols and tocotrienols (Sigma-Aldrich). The content of each isomer was determined from calibration curves of the reference standards and expressed as g/g of a sample dry mass.

Determination of Total and Free Polyphenols Content
The content of phenolic compounds (free and total) was determined spectrophotometrically with Folin-Ciocalteau reagent by the method described by Ribereau-Gayon [67]. The color reaction was carried out by adding Folin-Ciocalteau reagent (0.5 mL), 14% sodium carbonate (3 mL) and distilled water (6.5 mL) to the polyphenols extract. After mixing, the solutions were left for 60 min and their absorbance was then measured against the reagent sample (without the phenolics extract) at the wavelength of 720 nm, with a UNICAM UV/Vis UV2 spectrophotometer. The content of phenolic compounds was expressed as g of D-catechin equivalent in 1 g of a sample dry mass.

Determination of Phenolic Acids Content
The phenolic acid content was determined by the RP-HPLC technique using the method described by Ogrodowska et al. [68]. Phenolic acids were extracted with ethyl ether from flour sample hydrolysates (hydrolysis as for total phenols), acidified to pH 2. Then the ether was evaporated, and the dry residue was dissolved in methanol, centrifuged in a 5417R-type Eppendorf centrifuge (10 min, 25,000 × g) and transferred to chromatography vials. Liquid chromatography (RP-HPLC) was performed on an Agilent Technologies 1200 series system fitted with a photodiode detector and with a Phenomenex Synergi Fusion RP18 column (4 m, 2 mm, 150 mm) at the temperature of 30 °C. The mobile phase consisted of two solvents: A-0.15% formic acid (FA) in acetonitrile and B-0.15% FA in water. The gradient applied was: 0-7 min 10% of eluant A, followed by linear increase up to 100% of eluant A over 43 min. The flow rate was equal to 0.2 mL/min. Detection was performed at the wavelength of 280 and 320 nm. Phenolic acids were identified by comparing with absorption spectra of the reference phenolic acids. The content of phenolic acids was determined from calibration curves of phenolic acid reference standards and expressed as g of D-catechin equivalent in 1 g of a sample dry mass.

Statistical Analysis
All the chemical determinations were performed in triplicate. The experimental results were analyzed using Statistica 8.0 software. ANOVA analysis with Duncan tests was performed at the significance level of p < 0.05.

Conclusions
The data suggest that despite the use of diverse fertilization systems, wheat grain did not vary much, either in terms of the content of the phytochemicals under investigation or the morphological features of the grain. Organically and conventionally fertilized grain had similar amounts of tocochromanols and carotenoids. However, organic wheat grain was more abundant in total polyphenols, while less abundant in phenolic acids. Only minor variations were found in the content of phytochemicals among the tested organic fertilizers. Applied effective microorganisms had a negative influence because they contributed to a decrease of tocochromanols, carotenoids and phenolic acids content in the grain, along with a decrease in kernel weight.