IJFS#1433_bozza Ital. J. Food Sci., vol. 31, 2019 - 593 PAPER INFLUENCE OF FERTILISATION TYPE ON THE QUALITY OF VIRGIN RAPESEED OIL M. KACHELa, A. MATWIJCZUKb, A. NIEMCZYNOWICZc, M. KOSZELa and A. PRZYWARAa aDepartment of Machinery Exploitation and Management of Production Processes, Głęboka 28, 20-612 Lublin, Poland bDepartment of Biophysics, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland cDepartment of Relativity Physics, University of Warmia and Mazury in Olsztyn, Słoneczna 54, 20-710 Olsztyn, Poland *Corresponding author: Tel.: +48 815317131 Email address: magdalena.kachel@up.lublin.pl ABSTRACT The aim of the study was to determine whether, and to what the type of fertilisation employed can affect the quality of the harvested spring rape and the oil extracted therefrom through virgin oil pressing. The article presents the results of research conducted with the use of ATR-FTIR absorption spectroscopy of selected cold pressed vegetable oils. The study included three types of rapeseed oil from spring plants grown with traditional NPK mixture fertilisation and digestate. The oxidative stability of the pressed oil was also determined and soil parameters such as: content of absorbable nutrients and acidity in the experimental plots. Keywords: ART-FTIR spectroscopy, cold pressed oils, fat, fatty acids, fertiliser, oxidative stability Ital. J. Food Sci., vol. 31, 2019 - 594 1. INTRODUCTION With the view of increasing rapeseed yields, numerous artificial fertilisers have been used in recent years to modify the soil content of micro-organisms and drastically alter the general environmental conditions of the soil. This situation has a negative impact on both the sustainable development of soil fertility and, above all, quality of the natural environment (RAMIREZ et al., 2012). Health and environmental concerns force societies to support a revival of traditional methods of food production that are consistent with the guidelines for sustainable agriculture (CETIN and SEVIK, 2016). The trend led to the development of the concept of the so-called eco-efficiency, which entails the use of natural fertilisers produced through natural processing of agricultural waste in agricultural biogas plants (CZEKALA et al., 2012; DENNEHY et al., 2016). As observed by MÖLLER and MÜLLER (2012), the use of digestate containing large amounts of macro- and micro- nutrients normally found in the natural environment provides a viable alternative to mineral fertilisers, capable of improving soil fertility and quality of farm produce by providing plants with easily absorbable elements (such as nitrogen, phosphorus and potassium). Depending on the quality and nutritional status, digestate application has been shown to be advantageous in terms of crop yield, plant nutrient uptake (TERHOEVEN-URSELMANS et al., 2009; ANDRUSCHKEWITSCH et al., 2013) soil health (VANEECKHAUTE et al., 2013), and enhancement observed in dehydrogenase activity (GARCÍA-SANCHEZ et al., 2015). The technological and nutritional value of rapeseeds is determined by their chemical composition, in particular the content of nutrients and anti-nutrients, which in turn is closely related to the qualities of the plant species itself and primarily defined by genetic factors. Under specific conditions, the nutrient content can, however, vary significantly depending on the cultivar, soil type, fertilisation, weather conditions, and technological processing procedures employed (heating, steaming, autoclaving, etc.) (KRASUCKI et al., 2001). Low quality raw material – containing immature seeds, contaminated, damaged, excessively moist, affected by the processes of fat hydrolysis and oxidation – will produce low quality oil characterised by short shelf life. Consequently, the most valuable oils are those extracted from the freshest, highest quality material in which the oxidative processes are still relatively unadvanced. The process of fat oxidation is the primary cause of diminished quality manifested in the loss of oil’s organoleptic and nutritional value (ZIEMLAŃSKI and BUDZYŃSKA-TOPOLOWSKA, 1991; POYATO et al., 2014). The oxidation rate is conditioned by a number of factors, including the content of fatty acids, presence of pro- and antioxidants and, particularly in the case of cold pressed oils, the storage conditions (WRONIAK and ŁUKASIK, 2007). The growing interest in natural and safe food, including oils and fats, is a testament to its value as a rich source of healthy nutrients, whose consumption facilitates prevention of numerous, particularly digestive diseases and is beneficial to the overall health of organisms (HAREL et al., 2002). The seeds of both spring and winter rape are used in the production of cooking oil and provide a key raw material in the production of (increasingly popular and sought-after) biofuels for compression-ignition engines, particularly in Europe (VAN DUREN et al., 2015, ATABANI et al., 2013) and certain countries in Africa and Asia, including China (WU and LEUNG, 2011; SHIN et al., 2012). It can be safely assumed that Europe is currently the leading global producer of rapeseed (USDA, 2016). Compared to other oils such as sunflower oil, corn oil, sesame oil or olive oil, rapeseed oil is characterised by a particularly rich chemical composition. The high content of fat and protein as well a wide range of fatty acids, including large quantities of Ital. J. Food Sci., vol. 31, 2019 - 595 n-3 fatty acids such as α-linolenic acid (C18:3), palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid, as well as its n-6/n-3 ratio, make it an attractive offering for various consumer groups. Said acids are very beneficial from the perspective of human health. However, high content of α- linolenic acid, often exceeding 10%, increases the rapeseed oil’s susceptibility to autooxidation, thus negatively affecting its oxidative stability (SHAHIDI, 2005; PARRY et al., 2005). The presence of lipids, carbohydrates, sterols, aliphatic alcohols, tocopherols and a combination of pigments ensures antioxidative activity and high quality, both in terms of nutritional and sensory value (DAMERAU, 2015; TSAKNIS et al., 1997; SZYDŁOWSKA- CZERNIAK et al., 2013). Notably, as reported in a number of studies comparing cold pressing to other methods of oil extraction, due to the presence of anti-oxidants (carotenoids, tocopherols), as well as other phenolic compounds whose small quantities are introduced into the oil in the process of pressing the seeds, cold pressed oils are characterised by one of the highest values of oxidative stability (CICHOSZ and CZECZOT, 2011). It is noteworthy that the final quality of both the seeds themselves and the oil extracted therefrom is dependent on a variety of diverse factors, including climate, type of plantation, methods of harvest, storage, preliminary processing and, above all, the process of cold pressing itself. The increased awareness of the value of oil consumption stimulated the development of better quality products and renewed interest in methods of extracting vegetable oil without the use of chemical reagents, or at least with minimum use thereof. Due to the above, in recent years, we have observed a turn back to the methods of oil extraction involving cold pressing and virgin oil pressing (SZYDŁOWSKA-CZERNIAK et al., 2013) under which, due to the low temperatures under which the process takes place, the extent of degradation of bioactive compounds can be substantially reduced (RAMADAN, 2013). The simplicity of the process of extracting the oils selected for the present study simultaneously allows them to preserve much of their original colour, high nutritional value, high bioactive compounds content, as well as the characteristic taste and smell. The aim of the study was to: 1. determine and compare selected growing conditions based on soil analysis after fertilisation with a mineral NPK mixture and digestate. 2. analyse the quality of seeds in terms of fat and protein content in spring rape seeds after using fertilisers containing different amounts of nitrogen. 3. analyse selected quality parameters of seeds intended for oil extraction and the quality of the thus obtained virgin rapeseed oil. The scope of the study included: determination of the content of moisture, fats, fatty acids, and protein in the respective seeds, an analysis of the oil extracted therefrom and determination of its oxidative stability. A comparative analysis of ATR-FTIR spectra was employed for the purposes of identifying/highlighting differences in the chemical makeup of the respective oils. 2. MATERIALS AND METHODS The research material comprised three spring rape cultivars: ‘Markus’ – ‘M’, ‘Bios’ – ‘B’ and ‘Feliks’ – ‘F’ obtained from Hodowla Roślin Strzelce Sp. z o.o. The aforementioned rapeseed cultivars were grown in two combinations on experimental plots of 27 m2 located in the Lublin Voivodship (51o16’19.8”N 22o24’34.0”E), Poland (Fig. 1). In the first version of the experiment, natural fertiliser (digestate) obtained from the agricultural biogas farm Wikana Bioenergia Sp. z o.o. in Piaski, Lublin Voivodship, was used, while in the second Ital. J. Food Sci., vol. 31, 2019 - 596 version, the traditional multi-compound fertilizer Yara NPK 5-14-28 was applied (ammonium nitrogen - 5 %, P- 14% K - 28 %, SO3 – 12.5%, CaO - 3%). The digestate in the amount of 97 L was distributed in a plot of 27 m2 (36,000 l/ha). The pH of the digestate used in the cultivation of spring rape was 8.73. The cultivars grown in the control plot – C did not receive any of the aforementioned fertilisers. The products included in the study were obtained from a two-year experimental plot cultivation (2015 and 2016). After harvest, the seeds were cleaned and stored in laboratory conditions for a period of one month in 20oC ambient temperature and 70% ambient humidity to equalise the moisture content. Subsequently, the seeds were cold pressed to extract oil. The oil was then refrigerated for a period of one week in 4oC, in dark bottles, to separate the oil from impurities. Calculations were conducted for triplicate mean values. All the results obtained in the course of the two-year experiment were averaged and tabularised (see tables below). Figure 1. Distribution of experimental spring rape plots. Weather information relating to the period from March to September 2015 and 2016 was obtained from the Institute of Meteorology and Water Management National Research Institute IMGW-PIB as recorded at the weather station Lublin-RADAWIEC. The measurements of soil microelement content prior to the application of the selected fertilisers and after the harvest were conducted at the Regional Chemical-Agricultural Station in Lublin. Soil samples were tested for the available nutrient content, in particular phosphorus and potassium, in accordance with the applicable norms with respect to those nutrients, i.e. PN-R-04024:1997, as well as magnesium in mg per 100g od soil, in accordance with PN-ISO 10390:1997. Soil acidity, pH (KCl) and potential liming requirements were also considered. The digestate content of major nutrients and heavy metals was determined. The laboratory tests were conducted at the Regional Chemical-Agricultural Station in Lublin in accordance with KQ/PB-17-76-77 rev. 04 of 02.07.12. The biogas plant feedstock used in the production of biogas and digestate consisted of: corn silage, whey and plant waste. Rape seeds were analysed in terms of fat content. The fat content was determined using Soxhlet method in accordance with PN- EN 1163:1999, i.e. by multiple, continuous extraction from pulverised and pre-dried product using an organic extraction solvent, followed by removal of the solvent and determination of the fat content by weight. The Ital. J. Food Sci., vol. 31, 2019 - 597 tests were conducted at the Central Agro-Energy Laboratory of the University of Life Sciences in Lublin. Rapeseeds were tested for fatty acids content in accordance with PM-EN ISO 5509:2001. “Vegetable and animal fats and oils” – The analysis of fatty acid methyl esters was performed by way of gas chromatography at the Central Agro-Energy Laboratory of the University of Life Sciences in Lublin. Rape seeds were analysed in terms of protein content. The protein content was determined using the Kjeldahl method, in accordance with PN – A – 04018/A z 3:2002, i.e. by way of sulphation of organic nitrogen compounds using concentrated sulphuric acid in the presence of a catalyst, and subsequent alkalization of the solution, distillation and titration with sulphuric acid of ammonia bound by boronic acid. The oil was extracted using a continuous action screw press with exchangeable nozzles, 8 mm in diameter with a set of microscopic sieves, manufactured by Farmet DUO. Before activation, the press was preheated to 60oC ±10oC. Once the press was active and the pressing temperature equalised, the pressing process itself was initiated. Stabilisation was achieved after pressing oil from approximately 1kg of seeds, with the temperature stabilised at approximately 70oC. The pressing temperature was measured using an ama- digit thermometer. After the extraction, the oil was stored in dark-glass bottles at 5oC to achieve natural decantation over a period of 6 days. Afterwards, the oil was ready for laboratory analyses. The oxidative stability was measured using a Rancimat 670 apparatus (Metrohm AG, Herisau, Switzerland). Oil samples (2.5 g) were weighed into reaction vessels and heated to 1200C under a dry air flow of 20 l h-1. The volatile compounds released during oxidation were collected into a cell containing distilled water, and the increasing water conductivity was continually measured. The time taken to reach the conductivity inflection point was recorded as the induction period (IT) expressed in hours. All determinations were carried out in triplicate. The normal time was calculated using StabNet software, which controlled the Rancimat apparatus. Infrared absorption spectra were recorded with a Vertex 70 FTIR (Bruker) spectrometer. The attenuated total reflection (ATR) configuration was used with 20 internal reflections of the ZnSe crystal plate (45° cut). Typically, 16 scans were collected, Fourier-transformed, and averaged for each measurement. Absorption spectra at a resolution of one data point per 1 cm-1 were obtained in the region between 4000 and 600 cm-1. The instrument was continuously purged with N2 for 40 min. before and during measurements. The ZnSe crystal plate was cleaned with ultra-pure organic solvents from Sigma-Aldrich. The spectral analysis was performed with Grams/AI software from ThermoGalactic Industries (USA). All experiments were carried out at 25°C. In the recent years, chemometric methods have played an important role in studies on food quality as well as the identification and quantification of major constituents of foods. The use of chemometric methods combined with spectroscopic techniques has been analysed in previous research. The literature is extensive and details the theoretical aspects of applying such methods as e.g. Principal Component Analysis (PCA), Hierarchical Cluster Analysis (HCA) or Partial Last Squares Regression (PLS). Examples are the analysis pork fat or lard as a whole of matter extracted from pork (ROHMAN et al., 2011), the determination of fat in milk samples (INON et al., 2004), analysis of extra virgin olive oil adulterated with palm oil (ROHMAN and CHE MANA, 2010). Principal Component Analysis (PCA) is the most popular mathematical method of reducing data dimensionality with a minimum loss of information. PCA allows us to visualize and interpret data. Another multivariate method with which we can hierarchize the data is Ital. J. Food Sci., vol. 31, 2019 - 598 Hierarchical Cluster Analysis (HCA), which searches for objects, which are close together in the variable space. HCA and PCA methods combined with spectroscopic techniques have been successfully used for the purposes of determining food quality as well as identifying and quantifying major constituents of foods. The objective of the present study was to overview the similarities and differences observed between oil samples extracted through cold pressing and also involved the content of fatty acid composition in rapeseeds. The results obtained for the selected oil products were analysed statistically by way of mean value and ± standard deviation determination, as wells as post–hoc Tukey's test, with the level of significance of p < 0.05, using Statistica 10 software. Furthermore, correlations between the respective variables were evaluated using PCA and HCA. PCA transforms the original, measured variables into new uncorrelated variables. HCA was applied to the data in order to identify similarities between different oils samples. HCA calculates the distances (or correlations) between all oils samples based on Euclidean distance. 3. RESULTS AND DISCUSSION Table 1 presents the relevant weather conditions including atmospheric pressure, air temperature and rainfall in the years of cultivation, 2015 and 2016, for the period from March (sowing) to August when the crop was collected by way of two-stage harvest. As shown in the relevant data, 2015 was characterised by lower rainfall (10.08 mm H2O) relative to 2016, when the total rainfall in the analysed period was (12.59 mm H2O). The mean daily air temperature in 2015 was higher than in 2016, with the exception of April and May, respectively 7.75 and 12.37°C. Table 1. Weather conditions in 2015 and 2016. Month Year 2015 Year 2016 Pressure (hPa) Temperature ( oC) Rainfall (mm H2O) Pressure (hPa) Temperature (oC) Rainfall (mm H2O) 3 991.41 4.70 1.31 985.26 3.46 2.04 4 986.86 7.75 1.20 983.69 8.91 1.20 5 986.75 12.37 2.20 985.82 14.44 1.09 6 989.60 26.63 0.63 986.30 18.34 1.78 7 986.81 21.60 1.42 987.12 18.91 4.49 8 990.38 25.15 0.30 990.73 18.07 1.52 9 989.19 14.63 3.02 990.73 15.15 0.47 An important gap given that a significant popularisation of inorganic N fertilizers in the coming decades is expected to occur in developing countries as a consequence of population growth and increasing food demands (FARNWORTH et al., 2017). Oil and nitrogen (N) content in soil and rapeseed is an important quality indicator affecting oil yield and protein content of the meal (GRANT et al., 2011). Nitrogen content has a significant impact on protein synthesis and must be supplied in adequate amounts to Ital. J. Food Sci., vol. 31, 2019 - 599 ensure optimum crop yield. Too much N in the soil can lead to a nutrient imbalance that can restrict protein synthesis and reduce rapeseed growth and seed yield (GRANT et al., 2012; LEMKE et al., 2009). According to RATHKE et al. (2005), the yield of oilseed rape involves balancing the synthesis of oil and crude protein in the seeds, as well as the energy and carbon dioxide (CO2) budget of the photosynthetic pool. Table 2 presents the results of the analysis of major nutrient and heavy metal content in the digestate used for the spring rape cultivation. The same confirm the presence of numerous major nutrients. However, one should not that with respect to primary major nutrients such as: nitrogen, phosphorus and potassium, the levels observed in the applied digestate (respectively: 0.119; 0.12; 5.37 g/L) were significantly lower than those present in industrial fertilisers. ODLARE et al. (2008) reported in his research high content of mineral nutrients (nitrogen, phosphorus, potassium) in fermentation wastewater, while Rehl (2011) observed that in terms of response time digestate was in fact comparable to mineral fertilizers due to that fact that the N, P, and K nutrients were more readily accessible and available for the plants. Based on the subsequent analyses of the obtained results it can be reported that the tested digestate samples contained virtually no heavy metals, while the overall high major nutrient content supported the usability of digestate as a potential alternative fertiliser. Furthermore, COMPARETTI (2013) and KOUŘIMSKÁ et al. (2012) observed that digestate contains organic matter that has a significant positive influence on the physicochemical quality of the fertilised soil. Table 2. Content of major nutrients and selected heavy metals in digestate used for cultivation of spring rape. Element Unit Content Nitrogen (g/l) 0.119 Phosphorus (g/l) 0.12 Potassium (g/l) 5.37 Calcium (g/l) 0.28 Magnesium (g/l) 0.07 Cadmium (mg/l) <0.43 Lead (mg/l) <0.43 Nickel (mg/l) <0.43 Chromium (mg/l) <0.43 Copper (mg/l) 0.43 Zinc (mg/l) 2.00 Manganese (mg/l) 2.26 Iron (mg/l) 70.82 Table 3 presents the averaged results obtained in the course of the two-year study in terms of major nutrient content in the soil prior to an after the application of selected fertilisers for the purposes of spring rape cultivation. Based on the obtained, data it was observed that the pH of the soil in both cases remained relatively stable and did not change significantly after the sowing. With respect to nutrient content in plants relative to the levels or phosphorus, potassium and magnesium, the values varied significantly in the samples selected for the study. In all cases, the content of said nutrients was significantly Ital. J. Food Sci., vol. 31, 2019 - 600 higher in the fertilised plots when compared to the control plots, both prior to sowing and after the harvest. The analysis of soil content after the harvest revealed similar values for both experimental variants, which in both cases remained higher than those observed for the control, and were e.g.: 64.5 mg /100g of phosphorus in the plot fertilised with digestate compared to 25.1 mg /100g in the control plot. In the case of potassium, its concentration in the control plot was 20.04 mg/100g, while in the digestate plot it was 60.05 mg/100g. Data related to magnesium content analyses returned very similar results, i.e. 9.6 mg/100g in control plots and 21.8 mg/100g in lots fertilised with digestate. Given the above, the primary agricultural use of digestate, given its physical and chemical properties, is biofertilisation. Such usability of fermentation wastewater was also suggested, based on obtained research results, by KOUŘIMSKÁ et al., (2012), EICKENSCHEIDT et al., (2014), VÁZQUEZ-ROWE et al., (2015). KOUŘIMSKÁ et al., (2012) reported that digestate used in fertilisation improved soil fertility, as well as quality of the crop and the plants’ resistance to biotic and abiotic factors. The statistical analysis using the post-hoc Tukey test at the significance level of p<0.05 also revealed statistically significant differences in the content of the respective nutrients between the control plots and the experimental fertilised plots. Statistically significant differences were also observed between the content of the analysed nutrients in the soil prior to sowing and after the harvest, with the exception of control plots relate to the use of traditional NPK fertilisation. Table 3. Content of available nutrients and soil acidity. Experimental variant Plot type Acidity Liming needed Content of available nutrients (mg 100/g) pH in KCL reaction Phosphorus P2O3 Potassium K2O Magnesium Mg Prior to sowing Digestate Control 6.97 neutral no 23.8a 16.3a 10.3a Exp. plot 7.22 alkaline no 42.17b 53.17b 13.75b NPK fertilizer Control 7.06 alkaline no 21.1c 15.7a 9.5a Exp. plot 5.51 acidic yes 23.57a 20.55c 14.05b After harvest Digestate Control 6,99 neutral no 25.1d 20.04c 9.6a Exp. plot 7.52 alkaline no 64.5e 60.5e 21.8c NPK fertilizer Control 7.06 alkaline no 21.6c 16.7a 9.55a Exp. plot 5.37 acidic yes 26.5d 23.3d 17.2b a, b, c, d, e – statistically significant cultivar differences relative to the control; mean values marked with the same letter are not statistically significantly different (p > 0.05). The yield obtained from spring rape plants in 2015/2016 was below expectations and the unfavourable weather conditions observed in the summer in both years resulted in yield discrepancies. It is assumed that the correct moisture content of rapeseed suitable for further storage ought to be between 5 and 9%, with 10% being within the admissible range in some countries, e.g. Canada (GAWRYSIAK-WITULSKA et al., 2012). Table 4 presents Ital. J. Food Sci., vol. 31, 2019 - 601 the values of initial moisture during harvest, moisture reported during analyses, as well as fat and protein content of the rapeseeds. The initial moisture content during harvest was between 8.3 and 10% dry mass, respectively for all the samples. During storage, the value dropped on average by 1% relative to the initial moisture content, which rendered it acceptable by oil and fat industry standards, i.e. the harvested seeds would be accepted for further potential storage. The tests conducted with the aim of determining the fat content in seeds revealed that this particular characteristic was primarily dependent on genetic conditions. Seeds of spring rape are characterised by slightly lower fat and protein content (respectively 48.6% and 24%) compared to winter rapeseed (WARMIŃSKI et al., 2001). In our study, the protein concentration wasn’t strongly affected by the application of N, regardless of the fertilizer used. Since N is a major structural component of protein, increasing N supply frequently leads to an increase in protein concentration (MALHI and GILL, 2007). In both experimental cases, the fat content turned out to be fairly similar, varying between 40.23 and 42.57% for digestate cultivations, with the lowest value recorded for ‘Feliks’ control, and between 41.33 and 42.53% for ‘Feliks’ control cultivar on traditional NPK fertiliser. The protein content is undoubtedly an important parameter influencing the usefulness of various oils, which is why its measurement is necessary when determining the potential usability of the fat-free leftover material found in rapeseeds as e.g. raw material in the production of dietary protein supplements (GAWRYSIAK-WITULSKA et al., 2012). The analysis of protein content revealed that the same was slightly higher in seeds cultivated using natural fertiliser (digestate) and was 23.30% (for ‘Bios’ cultivar) and between 23.23% and 22.47% (for ‘Feliks’ cultivar). We concluded that the application of digestate had a subtle but noticeable influence on yield and quality compared to mineral fertilization. The obtained results in terms of fat and protein content were consistent with values reported by other authors (MURAWA and WARMIŃSKI, 2005). In the research of RATHKE et al. (2005) analyzing the effect of applying cattle manure, it was observed that seed yield of winter oilseed rape tended to increase with increasing N fertilization rate, while the oil content in the seeds declined. CHEEMA et al., (2001) and MASON and BRENNAN, (1998) reported the negative influence of N fertilization on the oil (fat) content in seeds. The statistical post-hoc Tukey test conducted at the significance level of p<0.05 revealed no statistically significant differences in terms of the protein content between the respective rapeseed cultivation variants. At the same time, such significant differences were observed for the fat content. MENSINK and KATAN (1990) say that high fatty acids content in oils is desirable because of their health benefits. The fatty acid content varies significantly between vegetable oils obtained from different sources, particularly depending on the actual plant cultivar used and plant maturity. Other factors with significant influence include: the region in which the plants are grown and specific climatic conditions (MURKOVIC et al., 1996). Given the above, Table 4 presents the primary fatty acids content in spring rapeseeds during the first stage of storage treated as the point of reference for the subsequent extracted oil measurements. The fatty acid content in oils was determined for specific rape cultivars (as reflected by the obtained measurement results) and returned the approximate percentages of 60% oleic acid, 21% linoleic acid, and 10% linolenic acid (KRYGIER, 1997). Comparable results were obtained in the cultivation and harvest of winter rape, where the highest reported content of MUFAs and PUFAs was respectively 68.33% and 34.55% (TYS et al., 2006). In terms of the analysed seeds, the fatty acid content was within the range approved by the CODEX ALIMENTARIUS (2011). Ital. J. Food Sci., vol. 31, 2019 - 602 Table 4. Moisture, fat and protein content in seeds. Experiment Cultivar Initial moisture (%) Moisture content (%) Fat content (% dry mass) Protein content (% dry mass) Control ‘Bios’ 10.00±0.06aA 9.93±0.06aA 42.57±0.15aA 23.35±0.35aA ‘Feliks’ 9.50±0.06aA 8.80±0.10bB 40.23±0.85cB 22.47±0.21aA ‘Markus’ 9.90±0.08aA 8.93±0.21bA 40.73±0.64cB 23.37±0.15aB Digestate ‘Bios’ 9.20±0.05bAa 8,17±0.06aAa 41.37±0.12bAa 23.30±0.10aAa ‘Feliks’ 8.30±0.06bAa 7.27±0.06aAa 41.67±0.38bAa 23.23±0.06aAa ‘Markus’ 9.03±0.07bAa 7.97±0.12aAa 41.97±0.06bAa 22.83±0.21aAa NPK fertilizer ‘Bios’ 8.75±0.05bAa 7.97±0.06aAa 41.80±0.10aAa 22.73±0.06aAa ‘Feliks’ 8.60±0.05bAa 7.57±0.06aAa 42.00±0.44bAa 23.57±0.31aAa ‘Markus’ 8.95±0.09bAa 7.67±0.06aAa 41.33±0.06bAa 22.37±0.40aAa a, b, c - statistically significant cultivar differences relative to the control; A, B - statistically significant differences between cultivars under the same field experiment; a, b, - statistically significant differences between the respective fertilizer type experiments relative to the same parameters; mean values marked with the same letter are not statistically significantly different (p > 0.05). Based on the obtained results (Table 5) it can be concluded that in the case of seeds grown with the use of digestate, the content of oleic acid (C18:1) and linoleic acid (α-C18:3) was lower in the control than in the “ecologically” grown samples of ‘Bios’, ‘Markus’ and ‘Feliks’ cultivars. An analogous situation was also observed in seeds cultivated using traditional NPK fertilisation. The statistical Tuckey’s post-hoc analysis revealed statistically significant differences in terms of acid content between the cultivars and their respective control, in terms of C18:1n9c for ‘Bios’ and ‘Feliks’, in terms of C18:2n6c (only for ‘Feliks’); and in terms of C18:3n3 again for ‘Bios’ and ‘Feliks’, where the experimental plots were cultivated on digestate. Statistical differences were observed in terms of the C18:1n9c content between particular seed cultivars from the cultivation fertilized traditionally with NPK, specifically between ‘Bios’ seeds on the one hand, and ‘Feliks’ and ‘Markus’ seeds on the other. Differences were also observed in terms of C18:3n3 acid between seed cultivars grown on digestate, specifically between Bios’ seeds on the one hand, and ‘Feliks’ and ‘Markus’ seeds on the other. In terms of SFAs in the ‘Bios’ cultivar, the highest values were observed in the control, where it reached 7.85%, compared to 7.53% for traditionally fertilised crops. In terms of the other two cultivars, the highest Saturated Fatty Acids content was observed in the digestate cultivations 7.99% followed by the control 7.76% (‘Feliks’) and 7.76% for traditional fertilisation (‘Markus’). The lowest content of those fatty acids was observed in ‘Markus’ seeds grown on digestate: 7.55%. A very similar situation could be observed with respect to Monounsaturated Fatty Acids. After analysing the results obtained for Omega 3 acids, it can be concluded that the same fluctuated between 6.22 and 7.64%, with the lowest values observed for Feliks seeds both in the control (6.90%) and the experimental cultivations, respectively 6.22% for digestate and 6.31% for NPK. The highest content of the same was observed for ‘bios’ seeds cultivated using the biofertilizer (7.64%). The content of Omega 6 acids in the seeds of analysed cultivars varied between 17.90 and 20.98%. The highest values were observed for the control ‘Feliks’ cultivar (20.48%) and the lowest for ‘Markus’ at 17.90% (on traditional fertiliser). Ital. J. Food Sci., vol. 31, 2019 - 603 The statistical analysis using Tukey’s post-hoc test at the level of significance of p<0.05 revealed clear statistical difference in the content of MUFAs between the control plots and plots fertilised with digestate for the ‘Bios’ cultivar, as well as between the control plots and experimental plots fertilised with either digestate of NPK mixture for the ‘Feliks’ cultivar. The statistical analysis also revealed significant differences in terms of the content of MUFA acids in the analysed seeds, specifically for ‘Bios’ vs. ‘Feliks’ and ‘Markus’ grown on digestate, and between ‘Bios’ and ‘Markus’ in the NPK cultivation. In terms of PUFAs, statistical differences were also observed for the ‘Bios’ and ‘Feliks’ cultivars relative to seeds obtained from their respective control plots. The statistical analysis revealed the existence of certain differences between the respective analysed cultivar types. Significant differences in the content of PUFAs were observed in the control cultivation between ‘Bios’ and ‘Markus’ seeds on the one hand and ‘Feliks’ seeds on the other. Moreover, Discrepancies were observed between the three cultivars grown on digestate as well as between ‘Bios’ on the one hand and ‘Markus’ and ‘Feliks’ on the other in the NPK cultivation. Statistical differences were also observed in the content of Omega 6 acids for the ‘Feliks’ cultivar when comparing the control plots to the experimental plots, regardless of the fertilisation method. Furthermore, significant differences in terms of their levels were noted in the control between ‘Bios’ and ‘Markus’ seeds on the one hand and ‘Feliks’ seeds on the other. Simultaneously, the statistical analysis revealed no statistical differences in terms of the content of Omega 3 acids between the experimental cultivations and the control. Differences of borderline statistical significance were observed between particular cultivars fertilized with digestate, specifically between ‘Bios’ seeds on the one hand and ‘Feliks’ and ‘Markus’ on the other. According to JELEŃ et al. (2000) major components of vegetable oils are fatty acids, both saturated and unsaturated, mainly bound to glycerol as triacylglycerols. The allyl groups in unsaturated fatty acids are highly susceptible to free-radical reactions. DE LEONARDIS and MACCIOLA (2012) reported that the influence of fatty acid composition on oil stability has been varied between respective cases. In general, oils that are more unsaturated oxidise more readily than their less unsaturated counterparts. In the presence of oxygen, unsaturated fatty acids undergo decomposition even at low temperatures. The process of lipid oxidation results in deterioration of food quality, which can manifest itself through unpleasant odour leading consumers to reject the product (SHAHIDI, 2005). It is the decomposition of hydroperoxides that constitute the main non-volatile compounds directly involved in the decomposition of volatile compounds, such as alkenes, aldehydes, alcohols, esters, acids and carbohydrates, that deteriorates the taste of food and is responsible for the rancid smell of soap. Moreover, the oxidative degradation of lipids can damage biological membranes and protein, and as such can pose a direct threat to human life (MALHEIRO et al., 2013). Ital. J. Food Sci., vol. 31, 2019 - 604 Table 5. Fatty acid content of the rapeseeds. a, b - statistically significant cultivar differences relative to the control; A, B - statistically significant differences between cultivars under the same field experiment; a, b, - statistically significant differences between the respective fertilizer type experiments relative to the same parameters; mean values marked with the same letter are not statistically significantly different (p > 0.05). Specification (%) Rapeseeds Control ,B’ ‘B’- digestate ‘B’ – NPK fertilizer Control ‘F’ ,F’ - digestate ‘F’ – NPK fertilizer Control ‘M’ ‘M’ - digestate ‘M’ – NPK fertilizer C14:0 0.07±0.02 aAa 0.08±0.02aAa 0.07±0.02aAa 0.09±0.02aAa 0.08±0.02aAa 0.09±0.02aAa 0.09±0.02aAa 0.08±0.02aAa 0.07±0.02aAa C16:0 4.47±0.71aAa 4.24±0.68aAa 4.26±0.68aAa 4.74±0.76aAa 4.66±0.74aAa 4.52±0.72aAa 4.39±0.70aAa 4.46±0.71aAa 4.39±0.70aAa C16:1 0.27±0.01aAa 0.25aAa 0.28aAa 0.31±0.01aAa 0.30±0.01aAa 0.28aAa 0.27±0.01aAa 0.27aAa 0.26aAa C17:0 0.05±0.01aAa - - 0.05±0.01aAa - - 0.05±0.01aAa - - C17:1 0.07±0,01aAa 0.10±0.01aAa 0.08aAa 0.08±0.01aAa 0.08±0.01aAa 0.08aAa 0.09±0.01aAa 0.07aAa 0.07±0.01aAa C18:0 2.05±0.21aAa 1.99±0.21aAa 2.01±0.21aAa 1.71±0.18aAa 2.04±0.21aAa 2.08±0.22aAa 1.91±0.20aAa 1.87±0.19aAa 2.11±0.22aAa C18:1n9c +C18:1n9t 65.01±0.60aAa 63.81±0.48bBa 64.24±0.52aAa 62.41±0.33cB b 65.76±0.67aCa 65.56±0.65aCa 64.60±0.56aAa 64.69±0.57aBa 65.24±0.62aCa C18:2n6c+C18:2n6t 18.19±0.57aAa 18.74±0.74aAa 18.52±0.67aAa 20.48±0.27aB b 17.99±0.51aAa 18.04±0.53aAa 18.49±0.66aAa 18.78±0.75aAa 17.90±0.48aAa C18:3n6 (gamma) - - - - - - - - - C18:3n3 (alpha) 6.72±0.19aAa 7.64±0.22aAa 7.14±0.20aAa 6.90±0.20aAa 6.22±0.18aBa 6.31±0.18aAa 7.17±0.20aAa 6.38±0.18aBa 6.50±0.18aAa C20:0 0.69±0.06aAa 0.68±0.06aAa 0.67±0.06aAa 0.63±0.06aAa 0.69±0.06aAa 0.72±0.06aAa 0.64±0.06aAa 0.65±0.06aAa 0.72±0.06aAa C20:5 - - - - - - - - - C20:1 1.50±0.23aAa 1.54±0.23aAa 1.65±0.25aAa 1.66±0.25aAa 1.38±0.21aAa 1.44±0.22aAa 1.45±0.22aAa 1.65±0.25aAa 1.77±0.27aAa C22:0 0.35±0.03aAa 0.34±0.03aAa 0.35±0.03aAa 0.37±0.03aAa 0.36±0.03aAa 0.37±0.03aAa 0.33±0.03aAa 0.35±0.03aAa 0.33±0.03aAa C22:2 - - - - - - - - - C24:1 0.17±0.03aAa 0.14aAa 0.15aAa 0.19±0.01aAa 0.14aAa 0.14aAa 0.14±0.03aAa 0.16±0.03aAa 0.10aAa SFA 7.85aAa 7.49aAa 7.52aAa 7.76aAa 7.99aAa 7.95aAa 7.56aAa 7.55aAa 7.76aAa MUFA 67.13aAa 65.98bAa 66.68aAa 64.71bBb 67.66aBa 67.49aAa 66.67aAa 67.15aBa 67.74aBa PUFA 25.10aAa 26.60bAa 26.03bAa 27.53aBb 24.27bBa 24.41bBa 25.87aAa 25.53aCa 24.70aBa OMEGA 3 6.72aAa 7.64aAa 7.14aAa 6.90aAa 6.22aBa 6.31aBa 7.17aAa 6.38aBa 6.50aAa OMEGA 6 18.19aAa 18.74aAa 18.52aAa 20.48bBb 17.99aAa 18.04aAa 18.49aAa 18.78aAa 17.90aAa Ital. J. Food Sci., vol. 31, 2019 - 605 The oxidative stability of the analysed oil samples was determined using the Rancimat method. Said stability is affected by a number of factors, including above all: the content of fatty acids, antioxidants and a whole range of other minor compounds. Table 6 presents the results of analyses performed in relation to the oxidative stability of the studied oils. In the case of oils extracted from plants cultivated on digestate, the shortest induction time was observed for the ‘Bios’ cultivar and was 5.21 h, while in the case of the respective control the same was three hours longer (8.31 h). The longest period of oxidative stability was recorded for the ‘Feliks’ oil, specifically 8.17 h, the same result as that observed in the respective control – 8.17 h. The results of measurements conducted for oil extracted from seeds cultivated using NPK and digestate fertilisation revealed relatively high oxidative stability compared to that reported by other authors. In a study by CICHOSZ and CZECZOT (2011), the induction time of oxidative processes (Rancimat test) in pressed and refined rapeseed oil was respectively: 4.5 and 4.7 h. Results obtained by WRONIAK and ŁUKASIK (2007) with respect to the oxidative stability of cold-pressed rapeseed oils with various antioxidative additives varied between 3.76 and 4.38 h. In the case of oils obtained from traditionally fertilised cultivations, the longest induction time was observed for control ‘Markus’ oil – 9.30 h, and the shortest for ‘Bios’ oil – 8.41. In terms of oxidative stability of oils analysed relative to normal time, a discrepancy was observed for ‘Bios’ oil obtained from seeds grown on digestate, NPK mixture and the respective control, where it was 24.4 h (control), 20.96 h (digestate) and 22.02 h (NPK). A similar situation was recorded for ‘Markus’ oil (Tale 6). The conducted statistical analysis using Tukey’s post-hoc test for the significance level of p<0.05 also revealed slight statistical induction time differences between ‘Bios’ grown on digestate and the respective control plants. In the case of the ‘Markus’ cultivar, slight statistical differences were observed between the control and oil from the NPK plots. The results of the statistical analysis also indicated the presence of significant differences between the respective cultivars from particular field experiments. Seeds grown on digestate showed differences in terms of the time of oil induction, specifically between ‘Bios’ seeds on the one hand and ‘Markus’ and ‘Feliks’ seeds on the other. Significant differences were also observed between the fertilizer types for the respective rape cultivars. Specifically, for ‘Bios’ seeds in the case of the control and ‘Bios’ and ‘Markus’ seeds grown on digestate and NPK. In the discussed case it should be noted that the seeds were subjected to no heating processes prior to extraction. The obtained oxidative stability values were considerably higher than those reported in other studies (4.35 or 4.39h) (KONDRATOWICZ- PIETRUSZKA, 2014; CIEMNIEWSKA-ŻYTKIEWICZ et al., 2014). Fig. 2 presents the ATR-FTIR spectra for selected oil samples. For easier interpretation of the results, Table 7 provides a breakdown of all characteristic bands (maximums) for selected samples together with the corresponding vibration of the respective functional groups. Samples were placed on a ZnSe crystal under N2 atmosphere (as described above in Materials and Methods). Spectra in the infrared region (ATR-FTIR) have well resolved bands that can be assigned to functional groups of particular components of food and biodiesel. Edible fats and oils as well as some biodiesel substances consist basically of triglycerides groups with different substitution patterns, which are mainly differentiated by the degree and form of unsaturation of the acyl groups and their length (GUILLEN and COBO, 1997; HONG et al., 2005; SCHOLZE and MEIER, 2005; ROHMAN and CHE MAN, 2010; Van de VOORT, 1992). Fig. 2 shows the infrared spectrum of selected oil samples chosen for the spectroscopic research. Ital. J. Food Sci., vol. 31, 2019 - 606 Table 6. Analysis of the induction time for oils. Parameter Experiment Cultivar Measurements Induction time Control ‘Bios’ 8.31±0.02aAa Normal time 24.40±0.53aAa Oxidative Stability (h) Induction time ‘Markus’ 8.58±0.55aAa Normal time 21.39±0.46aBa Induction time ‘Feliks’ 8.17±0.04aAa Normal time 21.11±0.93aBa Induction time Digestate ‘Bios’ 5.21±0.13bAb Normal time 20.96±0.02bAb Oxidative Stability (h) Induction time ‘Markus’ 7.71±0.21aBa Normal time 21.42±0.11aAa Induction time ‘Feliks’ 8.17±0.05aBa Normal time 21.66±0.38 aAa Induction time NPK fertilizer ‘Bios’ 8.41±0.07aAa Normal time 22.02±0.23bAc Oxidative Stability (h) Induction time ‘Markus’ 9.30±0.08cAb Normal time 23.89±0.65aBa Induction time ‘Feliks’ 8.51±0.18aAa Normal time 21.77±0.77aBa a, b, c - statistically significant cultivar differences relative to the control; A, B - statistically significant differences between cultivars under the same field experiment; a, b, c, - statistically significant differences between the respective fertilizer type experiments relative to the same parameters; mean values marked with the same letter are not statistically significantly different (p > 0.05). Figure 2. ATR-FTIR spectra for selected rapeseed oil samples for the following cultivars: ‘Bios’, ‘Feliks’ and ‘Markus’, respectively, ‘Bios’ NPK– (7) solid black line, ‘Bios’ digestate – (6) solid grey line, ‘Feliks’ NPK – (5) solid blue line, Feliks digestate – (4) solid light-blue line and ‘Markus’ NPK – (3) solid red line, ‘Markus’ digestate – (2) solid brown line. The spectra are presented in the spectral range of 600 – 3800/cm. Ital. J. Food Sci., vol. 31, 2019 - 607 Although some authors have suggested specific assignments of spectral bands in oils and fats (GUILLEN and COBO, 1997; LI et al., 2013; QIAN et al., 2007), there are some, which are notoriously difficult to decidedly assign to a specific group. Table 7 shows the frequencies of the characteristic bands or shoulders in the spectra of 7 oil samples (GUILLEN and COBO, 1997), as well as their assignment to respective functional groups (GUILLEN and COBO, 1997; YANG et al., 2005; HONG et al., 2005; SCHOLZE and MEIER, 2005; ROHMAN and CHE MAN, 2010), their vibration mode and intensity in spectra typical for the IR region. It should be noted that the assignment of bands corresponding to the stretching vibration in FTIR modes is usually easier than the assignment of bands corresponding to the bending vibration modes due to overlap or vibration of this group. Thus, in the absorption spectra of our oil samples, we can observe that some bending vibrations of the methylene group are produced between 1350 and 1150/ cm. This stretching vibration originates from –C-H vibrations in –CH3 (~1350/cm) groups and deformation vibrations in this group (~1150 cm-1). On the other hand, stretching vibrations of the C-O bond of esters are composed of two coupled asymmetric vibrations, C-C(=O)-O and O-C-C, the first being more important (GUILLEN and CABO, 1997; HONG et al., 2005; ROHMAN and CHE MAN, 2010); these bands occur in the region between 1300 (some C-C(=O)-O) and 1000/cm (of this combination’s group). The C-C(=O)-O band of saturated esters appears between 1240 and 1163/cm, and in unsaturated esters the vibration is produced at lower frequencies. On the other hand, the O-C-O band of esters derived from primary alcohols appears in the zone between 1064 and 1031/cm, while for those derived from secondary alcohols the band appears approximately at 1100/cm (ROHMAN and CHE MAN, 2010; LI et al., 2013). Both kinds of esters are present in triglyceride molecules. However, some authors assign the band at about 1238/7 /cm exclusively to out-of-plane bending vibrations of the methylene group (GUILLEN and CABO, 1997; LI et al., 2013; QIAN et al., 2007). Two bands in Table 7 have been assigned in a different way: the bands at approximately 1400/cm and 1319/cm. The first band, at about 1400/cm, has been assigned by some authors to terminal methyl groups on the aliphatic chains of oil components (GURDENIZ and OZEN, 2009). The second band, at about 1319/cm, was observed in all samples where absorption reached between 967 and 914/cm. In this context, it must be pointed out that the band at about 914/cm, which appears in all oil samples, has been related to the bending vibration of cis- disubstituted olefinic groups (GUILLEN and CABO, 1997; QIAN et al., 2007) and to vinyl groups. Although the oil spectra examined in this articles seem to be similar, they show differences in the intensity of their bands as well as in the exact frequency at which the maximum absorbance is produced in each case, due to the different nature and composition of the respective oil samples under study. Further characteristic areas of vibration are found in the bands with the maximum of approximately 1740/cm, typically associated with vibrations of the C=O carbonyl group in esters (HONG et al., 2005; SCHOLZE and MEIER, 2005). At the same time, a very weak band, or more specifically extension of the band with the maximum at approximately 1705/cm, reveals very weak vibrations of the carbonyl group in acids. In turn, bands with the maximum at approximately 1650/cm correspond to vibrations originating from stretching vibrations of the –C=C- group (due to cis- transformation). A characteristic area is also found in bands with the maximum of approximately 1460/cm originating from deformation vibrations in the -C-H groups of CH2 and CH3 (bending (scissoring)). The subsequent area of bands from 890 to 660/cm represents the characteristic deformation vibrations of -HC=CH- groups (cis- conformation, extra plain) and ring vibrations of the groups (δ(-(CH2)n- and – Ital. J. Food Sci., vol. 31, 2019 - 608 HC=CH- (cis-)) (GUILLEN and CABO, 1997; SCHOLZE and MEIER, 2005; LI et al., 2013; QIAN et al., 2007). As we progress to vibrations within higher wavenumber ranges, one should mention the significant stretching vibrations =C-H (trans-) with the maximum at approximately 3060/cm which originate from the vibrations of triglyceride fractions. A very characteristic feature of stretching =C-H vibrations in the cis- configuration are the relatively intensive vibrations at approximately 3006/7 /cm. Next, a number of vibrations with the maxima at approximately 2955, 2920 and 2855/cm originate from the stretching vibrations of–C-H in the respective -CH3, CH2 groups belonging to the aliphatic groups in triglycerides (HONG et al., 2005; SCHOLZE and MEIER, 2005; QIAN et al., 2007). Characteristic vibrations associated with specific functional groups were described in detail for all studied samples in Table 7. Furthermore, it is noteworthy that in the spectra of the studied samples of oil extracted from rape grown on digestate and traditional fertilizer mixture (Fig. 2), there are clearly visible differences in terms of the shape of spectra in the 1770 – 1670/cm area. In most of the studied samples there is apparent, slight amplification of the band at 1742/3/cm (responsible for vibrations of the C=O group) on the side of lower wave numbers, with the clearly defined extreme at approximately 1710/cm (with the relatively lowest intensity in the control spectrum), which can be associated with the formation of a hydrogen bond between the C=O…H-O-H groups in oil samples selected for the study. Simultaneously to the appearance of the band at 1710/cm (under both fertilisation regimens), there is a clear increase in the intensity of bands at approximately 1360, 700/cm, which is associated to stretching vibrations in C-O and C-C groups (as described above). The area between 1100 – 1300/cm is also responsible for the stretching vibrations of the C- O group, but the same varied only slightly between the studied oil samples, regardless of their origin (i.e. cultivation with the use of digestate or traditional fertiliser) (LI et al., 2013; QIAN et al., 2007). With the reducing affinity of the constituent molecules for the formation of the hydrogen bond between C=O…H-O-H, the bands show a slight increase in intensity. A similar correlation can be observed both in rapeseed oil samples originating from traditionally fertilised plots and from plots fertilised with digestate. Moreover, it would be prudent to once again mention bands from the range of 2960 to 2840/cm, which are responsible for the symmetrical and asymmetrical vibrations of CH2 and CH3 groups in the aliphatic sections of triglycerides. Their rates vary significantly depending on the origin of the respective samples, i.e. from rapeseed grown in plots fertilised traditionally or with the use of digestate. The variations observed in this area are very well correlated with variations in the fatty acids profile presented in Table 5. The aforementioned spectral changes also confirm the slight discrepancies in the overall fat content in the studied oil samples, as presented in Table 4 above. One should also point out the clear discrepancies in terms of band intensity between the particular rapeseed cultivations, relative to the method of fertilisation. In all spectrum-wide band intensity measurements the same was clearly lower in in the case of plots fertilised with digestate as compared to traditional fertilisation. With regard to a comparison between specific cultivars, the most intensive bands were observed for ‘Bios’, followed by ‘Markus’, with the lowest values observed for ‘Feliks’. The described differences correspond to the respective overall nutrient content in the studied samples of rapeseed oil. The main band differences for all samples, relative to the method of fertilisation, can be observed in bands with the respective maxima at 1745, 1709, 1368, 1220, 1154 and 1146/cm, which confirms the discrepancies in the overall fatty acids profile of the analysed samples. Ital. J. Food Sci., vol. 31, 2019 - 609 Table 7. Position of the maxima of absorption spectra in relation to the respective vibrations for selected spring rapeseeds grown with the use of traditional and digestate fertilisation, within the spectral range of 3800-550/cm. FTIR Types and origin of vibration Position of the maximum (/cm) NPK fertilizer Digestate Control ’B’ ’F’ ’M’ ’B’ ’F’ ’M’ ’B’ ’F’ ’M’ – 3463 3476 3476 3474 3476 3474 3476 3474 -C=Ow (overtone) and ν(=C-Hvw, trans-) 3005 3066 3064 3063 3061 3066 3067 3181 – – 3006 3007 3007 3007 3007 3006 3059/3007 3062/ 3007 ν(=C-Hm, cis-) – 2969 2954 2954 2953 2955 2954 – – νas(-C-Hm, -CH3) – 2955 2923 2922 2921 2922 2922 2953 2956 νas(-C-Hvst, -CHa) and νs(-C-Hvst, -CHa) (aliphatic groups in triglycerides) 2922 2923 – – – – – 2922 2923 2854 2871/2853 2853 2853/ 2872 2853/ 2870 2853/ 2872 2852/ 2872 2854 2852 2731 2731 – 2731 2730 – 2729 2730 2729 -C=Ovw Fermi Resonance 2676 2677 2676 2635/2680 2675 2674 2678 2678 2679 – 2584 – – – – 2335/2361 – 2339/23 60 2360/ 2336 2362/ 2337 2361 – – 2362 – – – 1743 1743 1744 1743 1743 1743 1743 1743 1744 ν(-C=Ovst) in ester 1704 1704 1704 1705 1705 1705 1704 1702 1705 ν(-C=Ovw) in acid – 1654 1654/ 1559 1654 1654 1654 1653/ 1558 1653 1653 νvw(-C=C-, cis-) 1462 1462 1462 1462 1462 1462 1463 1462 1462 δvw(-C-H) in CH2 and CH3 group, bending (scissoring) 1417 1441/1418 1440/ 1417 1416 – 1418 1440/ 1418 1439/ 1418 1440/ 1418 νvw(-C-H, cis-) bending (rocking) – 1401 1400 1400 – 1400 1400 1400 1401 νw, m, vw(-C-H, -CH3), bending 1377 1376 1377 1377 1377 1351/ 1377 1377/ 1350 1376/ 1349 1377 1367 – – – – – – 1365 1349 1353 1350 1349 1350 1318 1316 1317 1351 1317 1318 1318 1317 1318/ 1302 1301 1300 1300 1300 1300 1301 1299 1279 1277 1278 1278 1277 1279 νm(-C-O) or δm(-CH2-) 1278 1278 1277 – – – – – – – 1265 1262 1264 1261 1262 – 1237 1229 1237 1237 1237 1236 1237 1237 1234 1161 1160 1160/ 1141 1160 1143/ 1159 1141/ 1160 1161 1142/ 1160 1160/ 1143 νst(-C-O) or δst(-CH2-) 1120 1119 1119 1119 1119 1119 1120 1120 1119 νm(-C-O) 1097 1097 1096 1097 1096 1096 1097 1097 1097 νm,vw(-C-O) 1066 1060 1061 1062 1062 1059 1060 1063 1060 1030 1030 1030 1030 1031 1030 1030 1030 1031 966 965 967 966 914/965 913/966 913/ 967 912/ 965 913/969 /949 δw(-HC=CH-, trans-) bending out of plane Ital. J. Food Sci., vol. 31, 2019 - 610 913/ 867/ 851 911 912/ 867/ 849 848/ 868/ 913 – 870 689/ 867 694/ 869 791 δvw(-HC=CH-, cis-) bending out of plane 766 767 765 765 – 766 770 765 765 δ(-(CH2)n- and –HC=CH- (cis-) bending (rocking) 723 722 722 721 – 722 722 722 722 – 689 691 694 – 687 662 664 690 ν – stretching, δ – deformation, s – symmetrical, as – asymmetrical, st – strong, vst – very strong, w – weak PCA is a mathematical tool, which performs a reduction in data dimensionality and allows the visualization of underlying structure in experimental data and relationships between data and samples. FTIR spectra data of 7 oil samples were used in Principal Component Analysis (PCA). PCA of FTIR spectra using absorbencies at 16 wavenumber regions was carried out using Statistica 13 advanced statistics software. The PCA score plot (Fig. 3) was obtained from the correlation matrix of peak absorbencies at 16 frequency regions, namely 3007, 2921, 2853, 1744, 1654, 1461, 1418, 1373, 1235, 1161, 1119, 1096, 1031, 966, 913, 724/cm. In PCA, the first principal component (PC1) and the second principal component (PC2) account the largest and the next largest of variable variation. PC 1 explain 99,32% variance, meanwhile PC 2 accounted 0,62%; therefore the variance can be described by the first two PCs. Figure 3. Projection of cases on the PC 1 and PC 2 plane. Ital. J. Food Sci., vol. 31, 2019 - 611 Hierarchical cluster analysis (HCA) was performed in order to observe similarities or dissimilarities between the ‘Bios’, ‘Markus’ and ‘Feliks’ oil samples from spring plants grown with traditional NPK mixture fertilisation and digestate (D). The dissimilarity of different clusters was defined by Euclidean distance and calculated by single linkeage method. The results obtained are presented in the dendrogram structure, showing the different groups. On the Fig. 4 we present the plot obtained from the 7 oil samples of ‘Bios’, ‘Markus’ and ‘Feliks’. Considering the cut-off of 0.1 dissimilarity units we can distinguish three clusters (in red, green and blue colour, respectively). The first cluster, green coloured, is a small cluster, aggregated on the far left arm of the dendrogram. This cluster is formed by F-NPK oil of ‘Feliks’ sample. The second cluster, red coloured, is composed of all ‘eco-digestate’ oil samples including ‘Bios’, ‘Markus’ and ‘Feliks’ samples and samples of Control and of M-NPK. This result suggests that these oils have a physical- chemical properties more similar than the others. The sample of B-NPK oil is aggregated in third cluster (blue coloured) on the right arm of dendrogram. Figure 4. Dendrogram analysis (HCA) of all samples in the 3800-600/cm spectral region. The PCA analysis was applied to fatty acid data. The obtained results are presented in Figs. 5-6. The greatest effect on PC 1 in PCA is positively correlated with the content of C18:1N9c +C18:1n9t (0.017), C16:0 (0.0127) and is negatively correlated with the content of PUFA (-0.0471). In turn, PC2 is negatively correlated with C18:2n6c+C18:2n6t and OMEGA 6 (-0,0091) and positively correlated with the content of C18:3n3 (alpha) and OMEGA 3 (0,0096). Ital. J. Food Sci., vol. 31, 2019 - 612 Figure 5. The plot of two first principal components after PCA analysis of fatty acid compositions. Projection of the cases on the PC 1 and PC 2 plane. Figure 6. The plot of two first principal components after PCA analysis of fatty acid compositions. Projection of the variables on the PC 1 and PC 2 plane. Ital. J. Food Sci., vol. 31, 2019 - 613 4. CONCLUSIONS The obtained results reveal a correlation between the employed method of fertilisation, using digestate or a traditional NPK mixture, and the soil condition as well as the quality of the products, i.e. spring rapeseed and the oil cold-pressed therefrom. Based on said results it can be concluded that statistically significant differences can be observed in terms of the content of nutrients available to plants between the control and fertilised plots. Statistically significant differences were also observed between the content of the analysed nutrients in the soil prior to sowing and after the harvest, with the exception of control plots related to the use of NPK fertiliser. Analyses of the oil in terms of its oxidative stability revealed no direct correlation between the qualities of particular oils and the method of cultivating the seeds from which they were extracted, although the statistical analysis did reveal significant differences in terms of oil obtained from the ‘Bios’ cultivar, which was characterised by the lowest stability relative to digestate fertilisation. It is noteworthy that clear, although not particularly big, differences in terms of band intensity in the general cross-section of the FTIR spectrum were observed for samples fertilised with the mineral mixture, as compared to digestate. The same signify the possibility of a very efficient use of this method of field fertilisation. It is also noteworthy that FTIR spectroscopy techniques can be used for the purposes of rapid classification of oil samples without the need for sample preparation. FTIR spectroscopy techniques provide exquisite structural insights into the functional groups of oils, as required for discriminant analyses. FTIR spectroscopy has been found to be the most efficient method of oil discrimination and classification. To recapitulate, FTIR spectroscopy constitutes a simple, efficient and low-cost quantitative quality control tool available to the fat and oil industry. ACKNOWLEDGEMENTS Research supported by the Polish Ministry of Science and Higher Education as part of the statutory activities of the Department of Machinery Exploitation and Production Process Management, University of Life Sciences. REFERENCES Andruschkewitsch M., Wachendorf C. and Wachendorf M. 2013. Effects of digestates from different biogas production systems on above and belowground grass growth and the nitrogen status of the plant-soil-system. Jpn. Grassl. Sci. 183. Atabani A., Silitonga A., Ong H., Mahlia T., Masjuki H., Badruddin I.A. and Fayaz H. 2013. 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Paper Received November 4, 2018 Accepted May 6, 2019