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Effect of enzymatic and hydrothermal treatments of rapeseeds on quality of the pressed rapeseed oils: part II. Oil yield and oxidative stability
Process Biochemistry 45 (2010) 247–258
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Effect of enzymatic and hydrothermal treatments of rapeseeds on quality of the pressed rapeseed oils: part II. Oil yield and oxidative stability Aleksandra Szydłowska-Czerniak a,*, Gyo¨rgy Karlovits b, Gabriella Hellner c, Edward Szłyk a a
Faculty of Chemistry, Nicolaus Copernicus University, ul. Gagarina 7, 87-100 Torun´, Poland Bunge Europe Research and Development Center, ul. Niepodległos´ci 42, 88-150 Kruszwica, Poland c Bunge Europe, Research and Development Center, Kvassay J. ut. 1., Budapest H-1095, Hungary b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 June 2009 Received in revised form 31 August 2009 Accepted 14 September 2009
Response surface methodology was used to evaluate the quantitative effects of three independent variables: rapeseed moisture content (MC), enzymes dosage (ED) and conditioning temperature (T) on rapeseed oil yield (OY), efficiency of pressing (EP), and oxidative stability (OS). The highest OY (16.4%) and EP (42.8%) were obtained from pectolytic enzyme (0.1%) treated seeds (MC = 9%, T = 90 8C). The highest OS (12.6 h) was found for oil pressed from rapeseeds heated at 120 8C (MC = 11%), after the cellulolytic enzyme treatment. Results of OY, EP and OS determinations correlate with the predicted values calculated from the partial cubic models (PCMs) equations (R2 = 0.9995, 0.9994, 0.9974 for the cellulolytic enzyme-treated oils and 0.9900, 0.9900, 0.9990 for the pectolytic enzyme-treated oils). The predicted optimum MC = 9.5% and 8.6%, ED = 0.06% and 0.1%, T = 91.2 8C and 90.1 8C resulted in OY = 15.5% and 16.5%, EP = 40.4% and 43.0% for rapeseed oils from seeds treated with cellulolytic and pectolytic enzymes. OS values (12.6 h and 11.8 h) at the optimum conditions of MC = 11.0% and 10.1%, ED = 0.04% and 0.08%, T = 120.0 8C and 119.9 8C for the cellulolytic and pectolytic enzyme-treated oils were also calculated using PCM. Moreover, scanning electron microscopy revealed structural changes in the rapeseed after enzymatic treatment. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Enzyme-pressed rapeseed oils Oil yield Oxidative stability Response surface methodology Scanning electron microscopy
1. Introduction Traditional production of the crude rapeseed oil is based on seeds crushing, cleaning, flaking, conditioning, mechanical pressing and extrusion followed by solvent extraction [1]. The obtained crude oil is refined in order to remove unacceptable materials with the least possible loss of oil and without affecting desirable bioactive compounds, that are present in the crude rapeseed oil. In the mechanical pressing, the preconditioned rapeseeds are passed through a screw press where a combination of a high temperature and shear is used to crush the rapeseeds to release the oil. Rapeseed pressing involves low initial and operating costs when compared to the solvent extraction and is free of any polluting or hazardous substances [2–4]. However, this method is relatively low efficient, thus requires improvements. Moreover, after the conventional oil extraction process, still some of the oil remains in the solid residue. Several methods have been proposed to improve the oil extraction procedures, including an enzymatic pretreatment. The impact of enzymatic hydrolysis on the extraction yield and quality of the
* Corresponding author. Tel.: +48 56 611 47 86; fax: +48 56 654 24 77. E-mail address: [email protected] (A. Szydłowska-Czerniak). 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.09.014
obtained oils (sunflower, rapeseed, borage, cotton, grape seeds, soybean, olives) was studied [1,5–15]. These reports suggested, that enzymatic treatment of oilseed prior to oil extraction improves the oil yield. The oil in rapeseeds is mostly located in the vacuoles as free oil, but oil dispersed in the cytoplasm is not accessible during the extraction process and therefore is lost in the cake [1,16]. The enzymes degrade the oilseed cell wall by converting the cellulose materials into glucose, whereas the complex lipoprotein molecules into simple lipid and protein molecules [1]. This process improves release of oil and increases the yield of extraction. Enzymatic hydrolysis of sunflower [5], rapeseed [3,6], cottonseed [14] and olives [12] prior to extraction processes resulted in 4–35%, 17–29%, 14–52% and 22–152% higher yields, respectively. Moreover, the enzymatic extraction has improved functional and sensorial properties of fish by-products hydrolysates, but with lower yield of oil in comparison to chemical methods [17,18]. Although, the highest amount of oil separated from cod by-products was obtained using Alcalase and Neutrase without the water addition [19,20]. It is known that, oxidative stability (OS) determined by Rancimat method is an important parameter of the edible oil quality control and is standardized AOCS method. The method was
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Table 1 Three-level Box-Behnken design with three independent variables, experimental and predicted results for the oil yield response. Exp.
Coded level MC
1b 2 3b 4 5b 6 7b 8 9 10 11 12 13 14 15
1 1 +1 +1 0 0 0 0 1 +1 1 +1 0 0 0
Independent variables ED
1 +1 1 +1 1 +1 1 +1 0 0 0 0 0 0 0
T
0 0 0 0 1 1 +1 +1 1 1 +1 +1 0 0 0
MC [%]
7 7 11 11 9 9 9 9 7 11 7 11 9 9 9
ED [%]
0 0.1 0 0.1 0 0.1 0 0.1 0.05 0.05 0.05 0.05 0.05 0.05 0.05
Dependent variables T [8C]
105 105 105 105 90 90 120 120 90 90 120 120 105 105 105
ROHALASE1 OS-assisted
ROHAPECT1 PTE-assisted
OYa SD [%]
OYa SD [%]
Experimental
Predicted
Experimental
Predicted
5.7 0.053 11.1 0.26 11.5 0.21 12.3 0.23 14.7 0.13 15.4 0.12 9.1 0.35 9.3 0.47 7.8 0.32 13.2 0.30 8.2 0.22 9.7 0.087 13.1 0.22 12.8 0.060 12.9 0.14
5.7 11.1 11.5 12.3 14.7 15.4 9.1 9.3 7.8 13.2 8.2 9.7 12.9 12.9 12.9
5.2 0.19 14.3 0.12 11.1 0.10 12.4 0.11 14.2 0.50 16.4 0.16 8.6 0.21 12.0 0.12 12.6 0.21 14.8 0.12 10.9 0.19 11.3 0.38 12.4 0.22 11.6 0.16 12.8 0.17
5.4 14.5 10.9 12.2 14.2 16.4 8.6 12.0 12.4 15.0 10.7 11.5 12.3 12.3 12.3
SD—standard deviation; probability, p = 0.05. a n = 3. b Control sample.
also applied for evaluation of the OS of the enzyme-extracted corn [21], sunflower [22] and olive oils [9,23]. However, the correlations between antioxidant capacity, total phenolic, tocopherol and phospholipid contents and oxidative stability of the enzymetreated rapeseed oils, to the best of our knowledge, were not studied. Response surface methodology (RSM) is a collection of statistical techniques for designing experiments, building models, evaluating the effects of factors, searching optimum conditions of factors for desirable responses, which has been successfully applied in many areas of biotechnology such as: optimization of media and cultivation conditions [24,25], biotechnological parameters [26,27], lipase-catalyzed reaction [28,29], fermentation conditions [30,31]. However, the optimum conditions of hydrothermal and enzymatic treatments of rapeseeds for optimizing the extraction of rapeseed oil with high oxidative stability have not yet been reported. Therefore, RSM was used to evaluate the impact of cellulolytic and pectolytic enzymes (enzyme dosage—ED), hydrothermal parameters (moisture content—MC and temperature—T) and their interactions on response variables: oil yield (OY), efficiency of pressing (EP) and oxidative stability (OS) of the crude rapeseed oils. The main purpose of this study was to improve and optimize the oil recovery after enzymatic and hydrothermal treatments of rapeseeds using a mechanical oil expression. Moreover, the effect of cellulolytic and pectolytic enzymes concentration on physical and morphological properties of rapeseed coat and cotyledon was studied by the scanning electron microscopy (SEM). 2. Materials and methods 2.1. Sample preparation Rapeseeds (Brassica Napus cv. Oleifera) and enzymes (ROHALASE1 OS (ROS) and ROHAPECT1 PTE (RPTE)) used in the procedure of oil pressing were described in Part I [32]. 2.2. Analytical methods Oil content was determined by solvent extraction according to AOAC methods [33]. Moisture content was determined by vacuum drying at 60 8C to constant weight. Oil yield is defined as percentage of oil expelled at the pressing stage on a total oil extractable basis. Oil expelled is defined as the difference between total oil of the
original seeds and the oil in residual cake. The yield of the obtained oil (OY, %) and efficiency of pressing (EP, %) were expressed by equations: OY = (OO OC) A/B and EP = (OY/OO) 100, respectively, where OO—oil content in initial seeds (%); OC—oil content in cake (%); A—dry mater of initial material (%); B—dry mater of cake (%). Oxidative stability indexes of the obtained rapeseed oils were evaluated at 120 8C and a flow rate of air 20 L/h using the Rancimat apparatus (model 743, Metrohm). Stability was expressed as the oxidation induction time (h). Scanning electron microscopy (SEM) analysis was used to reveal the influence of enzymes treatment on the structure of the rapeseed. Scanning electron microscopic (SEM) studies of the untreated and treated seeds were carried out using a scanning electron microscope (LEO 1430 VP), at high vacuum (50 Pa), and accelerating voltage of 28 kV. 2.3. Experimental design and statistical analysis The results of OY, EP and OS obtained in this study were presented as: mean value of three replicates standard deviation (SD). Pearson correlation test was used to determine the correlations between oxidative stability results and FRAP, TPC, TTC and PL in the pressed rapeseed oils. Statistically significant differences were considered at the p < 0.05 level. The three-level Box-Behnken design with three independent variables (MC, ED and T) was applied for responses (OY, EP and OS) functions fitting. This design was selected due to the small number of experiments required to estimate complex response functions. For the three-level three-factorial Box-Behnken experimental design, a total of 15 experimental runs are necessary. The uncoded and coded independent variables and experimental design are listed in Tables 1–3. Box-Behnken design allows on the efficient estimation of the first- and secondorder coefficients. Response surface methodology (RSM) was used to study the simultaneous effects of three experimental factors (MC, ED and T) on OY, EP and OS of the pressed rapeseed oils and to find optimum conditions for a multivariable system. RSM allowed the development of a predictive mathematical model based on the experimental data, which can be employed for interpolation. In the first step of RSM the appropriate approximation for the true functional relation between response and the set of independent variables was calculated. When the response is well fitted to the linear function of the independent variables, the approximated function resulted in the first-order model. In the case when system revealed curvature, the polynomial of higher degree (second- or third-order) models must be applied for the response approximation. Therefore, all the responses observed were simultaneously fitted to the firstorder, second-order and partial cubic models. The determination coefficient (R2), the lack-of-fit of the studied models, and p-values of the parameter estimations were used to validate the models. The first-order models were used to approximate the data responses. However, the lack-of-fit p-values (0.0037–0.046) were significant (except p = 0.1305 and 0.1291 for OY and EP of the RPTE-treated oils), whereas R2 (0.6340–0.8353) were small (except R2 = 0.9531 and 0.9603, for OS of the ROS- and RPTE-treated oils, respectively). Therefore, the first-order models were not adequate approximations for the studied responses and the second-order models for data fitting were applied. Various second-order models, from interaction to partial cubic, were tested. The R2 of the second-order models were higher
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Table 2 Three-level Box-Behnken design with three independent variables, experimental and predicted results for the efficiency of pressing response. Exp.
Coded level MC
1b 2 3b 4 5b 6 7b 8 9 10 11 12 13 14 15
1 1 +1 +1 0 0 0 0 1 +1 1 +1 0 0 0
Independent variables ED
1 +1 1 +1 1 +1 1 +1 0 0 0 0 0 0 0
T
0 0 0 0 1 1 +1 +1 1 1 +1 +1 0 0 0
MC [%]
7 7 11 11 9 9 9 9 7 11 7 11 9 9 9
Dependent variables
ED [%]
T [8C]
0 0.1 0 0.1 0 0.1 0 0.1 0.05 0.05 0.05 0.05 0.05 0.05 0.05
105 105 105 105 90 90 120 120 90 90 120 120 105 105 105
ROHALASE1 OS-assisted
ROHAPECT1 PTE-assisted
EPa SD [%]
EPa SD [%]
Experimental
Predicted
Experimental
Predicted
14.8 0.14 29.0 0.69 29.9 0.54 32.1 0.59 38.4 0.33 40.1 0.30 23.8 0.92 24.2 1.22 20.4 0.85 34.4 0.77 21.4 0.56 25.3 0.23 34.2 0.61 33.3 0.16 33.7 0.36
14.8 29.0 29.9 32.1 38.4 40.1 23.8 24.2 20.4 34.4 21.4 25.3 33.7 33.7 33.7
13.6 0.48 37.3 0.32 29.0 0.22 32.4 0.29 37.1 0.21 42.8 0.42 22.4 0.55 31.3 0.16 32.9 0.54 38.6 0.32 28.4 0.51 29.5 0.99 32.4 0.58 30.3 0.42 33.4 0.44
14.1 37.8 28.5 31.9 37.1 42.8 22.4 31.3 32.4 39.1 27.9 30.0 32.0 32.0 32.0
SD—standard deviation; probability, p = 0.05. a n = 3. b Control sample.
Table 3 Three-level Box-Behnken design with three independent variables, experimental and predicted results for the oxidative stability response. Exp.
Coded level MC
1b 2 3b 4 5b 6 7b 8 9 10 11 12 13 14 15
1 1 +1 +1 0 0 0 0 1 +1 1 +1 0 0 0
Independent variables ED
1 +1 1 +1 1 +1 1 +1 0 0 0 0 0 0 0
T
0 0 0 0 1 1 +1 +1 1 1 +1 +1 0 0 0
MC [%]
7 7 11 11 9 9 9 9 7 11 7 11 9 9 9
Dependent variables
ED [%]
T [8C]
0 0.1 0 0.1 0 0.1 0 0.1 0.05 0.05 0.05 0.05 0.05 0.05 0.05
105 105 105 105 90 90 120 120 90 90 120 120 105 105 105
ROHALASE1 OS-assisted
ROHAPECT1 PTE-assisted
OSa SD [h]
OSa SD [h]
Experimental
Predicted
Experimental
Predicted
4.4 0.061 4.4 0.13 7.8 0.25 7.1 0.17 3.9 0.078 4.5 0.10 9.5 0.19 8.2 0.21 3.6 0.072 4.1 0.11 5.8 0.21 12.6 0.37 5.1 0.14 5.4 0.20 5.3 0.093
4.3 4.3 8.0 7.3 3.9 4.5 9.5 8.2 3.8 4.0 6.0 12.5 5.3 5.3 5.3
5.0 0.24 6.8 0.18 7.5 0.38 8.9 0.21 4.0 0.14 4.6 0.089 9.5 0.31 11.8 0.17 4.6 0.093 5.4 0.12 7.6 0.10 10.9 0.22 6.7 0.13 6.9 0.18 7.0 0.25
5.1 6.9 7.4 8.8 4.0 4.6 9.5 11.8 4.5 5.5 7.5 11.0 6.9 6.9 6.9
SD—standard deviation; probability, p = 0.05. a n = 3. b Control sample. (0.8761–0.9946) than the first-order (0.6340–0.8353) models, but the evaluated lack-of-fits were significant for OY and EP of ROS-treated rapeseed oils and OS of RPTE-treated oil (p = 0.0055, 0.0069, 0.0304, respectively). Based on the statistical analysis (lack-of-fit is not significant and R2 is the highest), the best-fitted model was the partial cubic model (PCM):
3. Results and discussion
Y n ¼ b0 þ b1 MC þ b2 ED þ b3 T þ b11 MC2 þ b22 ED2 þ b33 T 2
The oil yield and efficiency of pressing (oil extractability) of the enzymatic and hydro-thermally treated rapeseeds are listed in Tables 1 and 2. OY and EP results ranged between 5.2–16.4% and 13.6–42.8%, respectively. It can be noted, that the application of both procedures: enzymatic hydrolysis and hydrothermal pretreatment on rapeseeds enhanced oil yield and oil extractability in comparision to the control samples (without enzymes, at the same hydrothermal conditions). The highest OY (16.4%) and EP (42.8%) were obtained from RPTE (0.1%) treated seeds (MC = 9%, T = 90 8C), whereas, the lowest OY (5.7% and 5.2%) and EP (14.8% and 13.6%) were determined for the control samples (without enzymes) of rapeseeds contained 7% moisture and heated at 105 8C. Among two enzymes used, RPTE gave a higher OY (10.9–16.4%) and EP (28.4– 42.8%), which conforms to the fact that pectic substances are the prevalent cell wall polysaccharides in rapeseeds [34]. Moreover,
2
2
þ b12 MC ED þ b13 MC T þ b23 ED T þ b112 MC ED þ b113 MC T where Yn is one of the three responses, MC, ED and T represent the independent variables, b0 is the constant, b1, b2, b3 are the linear-term coefficients, b11, b22, b33 are the quadratic-term coefficients, and b12, b13, b23, b112, b113 are the cross-term coefficients. The fitness of the PCM was evaluated by the determination coefficient R2, the fraction of the variation explained by the model, and analysis of variance (ANOVA). The F-test was applied to confirm, whether the variance explained by the regression model was significantly larger than the variance of the residual and to evaluate the model lack-of-fit (model error). Moreover, the fitted polynomial equations were presented as surface and contour plots in order to visualize the relation between the responses and experimental levels of each factor and to deduce the optimum conditions. Regression and analysis of variance (ANOVA) were carried out using the Statistica Windows software package.
3.1. Oil yield and efficiency of pressing
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RPTE increased OY and EP by 11.7–175.0%, whereas ROS improved oil recovery (OY) and extractability (EP) by only 2.2–94.7%, when compared to the control samples hydrothermal treated but without enzyme action (Tables 1 and 2, exps. 1–8). Reported by other authors, the enzyme-extracted rapeseed oils yields were higher (22.2–91.6%) [3,4]. However, OY of the discussed oils were similar to results found for olive oil (10.6–17.5%) by Aliakbarian et al. [12], sunflower (4.00–35.45%) by Sineiro et al. [5], grape seed (6.71– 19.50%) by Passos et al. [15] and cottonseed (9.70–12.89%) by Latif et al. [14]. The higher oil yield of enzyme-assisted pressing process, than the control samples (without enzymes) can be explained by the better solubilisation and hydrolysis of proteins. The network of the cotyledon cells and the protein (oleosin) based membranes were broken, thereby improving the extraction yields [1,5,8]. It can be suggested, that the increase of the enzyme concentration causes the increase the OY and EP (Tables 1 and 2). Similar results were observed by Najafian et al. [13] for the Koroneiki variety olives, when pectinex enzyme was used at the higher concentration. The opposite effect was observed when the conditioning temperature of rapeseeds was raised to 120 8C (Tables 1 and 2, exps. 5–8 and 13–15). The latter can be related to the thermal inactivation of enzymes. On the other hand, oxidative stability of the obtained crude rapeseed oils ranged over 3.6–12.6 h (Table 3). The most stable to oxidation (OS = 12.6 h) was ROS-treated oil mechanically extracted from seeds which contained 11% moisture and were heated at 120 8C. The oxidative stability of oils was elevated with an increase of the rapeseed conditioning temperature. The significant correlation (R2 = 0.6190, p < 0.000001) between induction time for all studied oils and the conditioning temperature of seeds before pressing is evident. For comparison, the oxidative stability of the olive oils obtained from a blend of Frantoio/Leccino and Coratina cultivars positively correlated with malaxation temperature (R2 = 0.818 and 0.987, respectively) [35]. The higher oil stability can be related to the inactivation of oxidative enzymes such as lipase, peroxidase and lipoxigenase during heating stage [36]. Also, the increase of MC in rapeseeds heated at 120 8C caused an elongation of the induction period of the pressed oils (Table 3). Moreover, the Rancimat stability of oils was higher as a consequence of the RPTE treatment of rapeseeds. In the case of ROS-treated seeds, the pressed oils demonstrate lower stability (except oil pressed from seeds with MC = 9% heated at 90 8C) than the control samples (without the enzyme) (Table 3, exps. 1–8). Reported enzyme processed virgin olive oils [9,11,23] and sunflower oils [22] also exhibited improved oxidative stability when compared with the one obtained without enzyme treatment. RSD values of OY: 0.47–5.05%, EP: 0.48–5.04% and OS: 1.32– 5.07% indicate reasonable repeatability of the applied analytical procedures. 3.2. Fitting the models Response surface methodology (RSM) was applied and the partial cubic response surface models were fitted to each response variable: OY, EP and OS, respectively (Tables 1–3). Regression analysis and analysis of variance (ANOVA) of the experimental data (Tables 1–3) were performed for the mathematical models fitting, determination of the regression coefficients and statistical significance examination of the model terms. The following regression equations were obtained: OYROS ¼ 425:9 þ 90:2MC þ 669:8EDROS þ 3:58T 4:74MC2 76:7ED2ROS 0:0027T 2 130:8MC EDROS 0:678MC T 0:167EDROS T þ 6:63MC2 EDROS þ 0:0358MC2 T
OYRPTE ¼ 157:1 þ 50:5MC þ 669:8EDRPTE þ 0:549T 2:64MC2 223:3ED2RPTE þ 0:0049T 2 127:5MC EDRPTE 0:375MC T þ 0:400EDRPTE T þ 6:00MC2 EDRPTE þ 0:0200MC2 T
EPROS ¼ 1110:8 þ 235:5MC þ 1794:5EDROS þ 9:32T 12:4MC2 206:7ED2ROS 0:0071T 2 351:8MC EDROS 1:76MC T 0:433EDROS T þ 17:9MC2 EDROS þ 0:0933MC2 T EPRPTE ¼ 412:7 þ 132:2MC þ 1741:5EDRPTE þ 1:48T 6:92MC2 581:7ED2RPTE þ 0:0125T 2 332:0MC EDRPTE 0:983MC T þ 1:07EDRPTE T þ 15:6MC2 EDRPTE þ 0:0525MC2 T
OSROS ¼ 30:0 þ 4:96MC þ 65:6EDROS 0:681T 0:530MC2 þ 131:7ED2ROS þ 0:0041T 2 1:75MC EDROS 0:0525MC T 0:633EDROS T 0:00MC2 EDROS þ 0:0058MC2 T
OSRPTE ¼ 163:9 34:0MC 16:3EDRPTE 1:74T þ 1:80MC2 þ 106:7ED2RPTE þ 0:0015T 2 7:75MC EDRPTE þ 0:336MC T þ 0:567EDRPTE T þ 0:375MC2 EDRPTE 0:0175MC2 T The ANOVA results exhibit the effect and the regression coefficients of each linear, quadratic and interaction terms, that were individually determined. The PCM represents responses of OY, EP and OS with the coefficients of determination, R2 = 0.9995, 0.9994, 0.9974 and 0.9900, 0.9900, 0.9990 for ROS- and RPTEtreated oils, respectively (Table 4). The calculated R2 values indicate that obtained PCMs were adequate for the description of the independent variables (MC, ED and T) impact on OY, EP and OS of the enzyme-treated rapeseed oils. The R2 and the adjusted R2 values for the studied response variables, were higher than 0.90, hence there is a close agreement between the experimental results and theoretical values predicted by the proposed models. For comparison, Aliakbarian et al. [12] used first-order model and calculated lower determination coefficient (R2 = 0.837), which demonstrates that the model can be used to explain more than 80% of the variability in the olive oil extraction yield response. The model adequacy was tested using the lack-of-fit F-test, which is not significant for p > 0.05. The ANOVA results for the studied responses revealed insignificant lack-of-fit (F ranged between 0.0536 and 7.71, p > 0.05, respectively) (Table 4). Therefore, these models were adequate for prediction, within the range of variables employed. Furthermore, the linear and quadratic effects of the rapeseed treatment variables (MC, ED and T) and their interactions on the response variables (OY, EP and OS) were analyzed by ANOVA (Table 4). The model is highly significant, when the computed F-value is greater than the tabulated F-value at low probability value (p < 0.0001). All linear (MC, ED, T), quadratic (MC2 and T2) and interactions (MC ED, MC T, MC2 ED and MC2 T) parameters of the models were statistically significant (p < 0.05) for OY and EP of the studied ROS-pressed oils. On the contrary, only
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Table 4 ANOVA results for the responses: OY, EP and OS of the enzyme-pressed rapeseed oils. Model parameters
Degree of freedom
ROHALASE1 OS-assisted Sum of squares
Oil yield (OY) Linear Quadratic Interaction Regression Residual Lack-of-fit Pure error Total R2 Adjusted R2
3 3 5 11 3 1 2 14
*
Mean square
F-value
16.34 8.78 4.38 9.56 0.016 0.00125 0.0234
700.2* 376.3* 187.8* 379.0* 0.0536
Sum of squares 61.11 8.94 22.14 98.11 0.992 0.245 0.747 99.10
0.9995 0.9979
Efficiency of pressing (EP) Linear 3 Quadratic 3 Interaction 5 Regression 11 Residual 3 Lack-of-fit 1 Pure error 2 Total 14 2 R Adjusted R2 Oxidative stability (OS) Linear Quadratic Interaction Regression Residual Lack-of-fit Pure error Total 2 R Adjusted R2
49.01 26.34 21.91 105.19 0.0480 0.00125 0.0467 105.24
ROHAPECT1 PTE-assisted
3 3 5 11 3 1 2 14
332.8 179.4 150.2 715.5 0.418 0.0112 0.407 715.9
110.9 59.80 30.04 65.05 0.139 0.0112 0.204
23.26 1.33 2.24 7.95 0.0757 0.180 0.0234
20.37 2.98 4.43 8.92 0.331 0.245 0.374
F-value 54.55* 7.98 11.86 22.45* 0.656
0.9900 0.9533 545.6* 294.1* 147.7* 296.2* 0.0553
416.7 60.34 150.2 668.1 6.72 1.71 5.01 674.8
0.9994 0.9973
69.79 3.99 11.19 87.53 0.227 0.180 0.0467 87.76
Mean square
138.9 20.11 30.04 60.74 2.24 1.71 2.51
55.48* 8.04 12.00 22.78* 0.684
0.9900 0.9535 997.0* 56.97* 95.94* 331.1* 7.71
57.88 0.719 4.54 75.54 0.0780 0.0313 0.0467 75.62
0.9974 0.9880
19.29 0.240 0.908 6.87 0.0260 0.0313 0.0234
827.0* 10.28 38.93* 246.0* 1.34
0.9990 0.9952
Significant at the p < 0.05 level.
linear terms of RPTE dosage and conditioning temperature, and interaction MC ED produced a significant effect on OY and EP of RPTE-assisted oils. Also, all linear parameters (MC, ED, T) and interactions (MC T, ED T, MC2 T) significantly influenced OS of rapeseed oils obtained from seeds after RPTE treatment (Table 4). Besides, only two interactions (MC T, ED T) and hydrothermal conditioning (MC, T and T2) of seeds revealed significant effects on OS of ROS-pressed oils. The ANOVA results indicate, that linear terms of the enzymes dosage and the conditioning temperature and interaction MC ED caused significant impact on OY and EP of all crude rapeseed oils. It can be noted, that impact of the RPTE dosage on OY and EP of the studied oils was greater, than the processing temperature. On the contrary, MC and T were more evident independent variables on OY, EP of ROS-assisted oils and the oxidative stability of all oils. For comparison, only linear terms (enzyme concentration and malaxation time) had statistically significant effect on the olive oil extraction yield [12]. The highest yield (17.5 goil/100 gpaste) was determined after 150 min malaxation time and upon enzymes addition (25 mL/kgpaste) [12]. In the case of sunflower oil, the maximum extractability (35.65%) was obtained from sunflower grinded seeds, adjusted to 5 g of water/1 g of seeds and with 2 g of enzyme/100 g of seeds, after 2 h of enzymatic treatment extraction [5]. To enhance oil availability and extractability, Kashyap et al. [37] used a mixed-activity crude enzyme. Authors applied second-order response surface methodology to calculate optimal process conditions for soyflakes and soybrokens with 24.6% and 24.7% moisture content subjected to hydrolysis, along with 14.2% and 7.1% of
enzyme concentration, and 13.3 h and 13.9 h hydrolysis time, respectively. 3.3. Analysis of response surfaces The effects of the three independent variables (MC, ED and T) on OY, EP and OS of the obtained oils were illustrated by the surface response and the contour plots of the PCM for different interactions of any two independent variables, while holding the value of the third variable as constant (0 level) (Figs. 1–3). The relations between MC, ED and T of seed conditioning and the OY and EP of ROS-treated rapeseed oils as well as their interactions are presented in Figs. 1a–c and 2a–c. The elliptical shape of the contour plots indicates the significant interactions between the corresponding variables MC ED, MC T, MC2 ED and MC2 T. The significant positive linear effect of MC in rapeseeds and ROS dosage on the OY and EP of ROS-treated rapeseed oils resulted in the enhance of OY and EP along with the increase of MC and ROS concentration (Figs. 1a and 2a). Besides, the OY and EP of ROS-pressed rapeseed oils were improved at conditioning temperature below 105 8C and higher levels of MC (>7%) and ROS (>0.05%) dosage (Figs. 1b, c and 2b, c). Figs. 1d and 2d exhibit that at the lowest level of MC, the OY and EP of the RPTE-treated oils rise rapidly with the increasing RPTE concentration, while at the highest level of MC, the OY and EP enhanced to a certain level due to the contribution by the interaction term of RPTE concentration and MC in rapeseeds. The RPTE dosage and conditioning temperature displayed a significant linear (positive and negative, respectively) influence on OY and EP of RPTE-assisted oils. In Figs. 1f and 2f, RSM revealed the maximum
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Fig. 1. Response surfaces and contour plots for OY of the enzyme-pressed rapeseed oils expressed as a function of (a and d) MC and ED (at T = 105 8C), (b and e) MC and T (at ED = 0.05%), (c and f) T and ED (at MC = 9%).
of OY and EP at the highest RPTE (0.1%), while the lowest T (90 8C) and an intermediate MC = 9% were observed. Moreover, as the T of seed conditioning after addition of RPTE has decreased, the OY and EP responses were higher for all studied MC (Figs. 1e and 2e).
The effects of the ROS dosage and one of variables (MC or T) demonstrate similar response surfaces of OS (Fig. 3a and c). It is evident that, higher MC and T resulted in the progressive OS increase, whereas the effect of ED was insignificant. Moreover, the
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Fig. 2. Response surfaces and contour plots for EP of the enzyme-pressed rapeseed oils expressed as a function of (a and d) MC and ED (at T = 105 8C), (b and e) MC and T (at ED = 0.05%), (c and f) T and ED (at MC = 9%).
oxidative stability of the enzyme-assisted oils rapidly increased, along with growth of the conditioning temperature and the moisture content in rapeseeds (Fig. 3b and e). These two parameters (MC and T) affected positively on OS of all studied oils. On the contrary, RPTE concentration displayed the positive
and significant linear effect on OS values (Fig. 3d and f). Therefore, more stable oils were pressed from rapeseeds with higher moisture (MC > 9%), conditioning at higher temperature (T > 105 8C), after treatment with more concentrated RPTE (c > 0.05%).
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Fig. 3. Response surfaces and contour plots for OS of the enzyme-pressed rapeseed oils expressed as a function of (a and d) MC and ED (at T = 105 8C), (b and e) MC and T (at ED = 0.05%), (c and f) T and ED (at MC = 9%).
By the analysis of regression equations and response surface contour plots, the optimum MC, ED and T were found to be 9.5%, 0.06% and 91.2 8C, where the OY and EP of ROS-pressed oils were predicted to be 15.5% and 40.4%, respectively. However, the optimum conditions for pressing oils from seeds with RPTE were:
MC = 8.6%, ED = 0.1% and T = 90.1 8C, under which the predicted OY = 16.5% and EP = 43.0% were found. Besides, the models predicted the OS values for ROS-assisted (12.6 h) and RPTE-assisted (11.8 h) oils at the optimum conditions of MC = 11.0% and 10.1%, ED = 0.04% and 0.08% and T = 120.0 8C and 119.9 8C, respectively.
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Fig. 4. Scanning electron micrographs of rapeseed untreated and treated with cellulolytic enzyme ROHALASE1 OS (c = 0.05% and c = 0.1%).
3.4. Effect of enzymatic treatment on the physical structure of rapeseed Scanning electron micrographs (Figs. 4 and 5) present differences between untreated rapeseed and after enzymatic treatment at both concentrations (ED = 0.05% and 0.1%) of ROS and RPTE. Images of the untreated rapeseed did not exhibit distortion or rupture of the cells and only lipid can be observed in the spaces between the closely packed, intact cells (Figs. 4 and 5, column 1). In
the control sample (without enzymes), the spherical oil bodies were surrounded by an intact biological membrane. Oil-rich droplets located inside the rapeseed cotyledon cell can be seen. The applied enzymes distorted the original structure of rapeseed and the oil droplets (white points) appeared (Figs. 4 and 5, columns 2 and 3). The similar morphologic changes of rapeseed after cellulolytic (ROS) and pectolytic (RPTE) enzymes adding were observed (Figs. 4 and 5). Enzymatic treatment caused destruction of the oil bodies membranes and oil located in the lipid bodies coalesced. The SEM images confirmed, that the enzymes are able to
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Fig. 5. Scanning electron micrographs of rapeseed untreated and treated with pectolytic enzyme ROHAPECT1 PTE (c = 0.05% and c = 0.1%).
breakdown the cell structure of rapeseed and induce the release of oil from cells. However, the seed coat retains its integrity, in spite of enzymatic treatment, hence antioxidants were retained inside the layer. Therefore, the highest antioxidant capacity (1220.0, 964.8 mmolTE/100 g) and total phenolic content (83.3, 74.0 mgSA/100 g) were found for two rapeseed oils extruded from seeds heated at 120 8C (MC = 11%) and treated with less concentrated cellulolytic and pectolytic enzymes (ED = 0.05%) (see Part I) [32].
3.5. Correlations between antioxidant capacity (AC), total phenolic (TPC), tocopherol (TTC) and phospholipid (PL) contents and oxidative stability of enzyme-treated oils The oxidative stability of oil is affected by some minor compounds such as phenols, tocopherols and other antioxidants present in oil [38]. Hence, regression analysis was performed to calculate the correlations between AC, TPC, TTC, PL (results were presented in Part I) [32] and OS of rapeseed oils pressed from seeds
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after hydrothermal and enzymatic treatments. The oxidative stability data for all studied rapeseed oils correlate significantly positively with AC, TPC and PL (y = 61.97x + 198.6, R2 = 0.6090, y = 6.05x + 2.16, R2 = 0.7939, y = 1177.8x 1165.9, R2 = 0.7026, respectively; p < 0.0000001). Similarly, significant linear correlation between free radical antioxidant capacity measured by DPPH and oxidative stability determined by Rancimat method (R2 = 0.83, p = 0.032) for nut oils was found by Arranz et al. [39]. Furthermore, significant positive correlations (R2 = 0.8986, 0.786, 0.948 p < 0.001) between oxidative stability and total phenols in virgin olive oils were observed by others [40,41]. Likewise, phospholipids contribute to the stability of vegetable oils [39,42]. Therefore, positive linear correlation (r = 0.9849) between the induction time of olive oils and lecithin concentration was demonstrated by Koprivnjak et al. [43]. However, the lack of correlation between oxidative stability and total tocopherol content in all studied oils (y = 0.675x + 646.9, R2 = 0.0041, p = 0.7362) was observed. Also, Bozan and Temelli [44] did not find correlation between oxidative stability and tocol (tocopherols and tocotrienols) levels of flax, safflower and poppy oils. Response surface methodology (RSM) appeared to be useful way of studies the influence of three experimental factors (rapeseed moisture content, enzyme dosage and conditioning temperature) on OY, EP and OS of the pressed rapeseed oils. The highest OY (16.4%) and EP (42.8%) were obtained from RPTE (0.1%) treated rapeseeds (MC = 9%, T = 90 8C), whereas the most stable to oxidation (OS = 12.6 h) was noted for ROS-treated oil mechanically extracted from seeds with 11% of water heated at 120 8C. The effect of a pectolytic enzyme concentration on OY and EP of the RPTEassisted rapeseed oils was greater, than the processing temperature, whereas conditioning temperature was more effective independent variable on OY, EP of ROS-assisted oils and oxidative stability of all oils. Besides, the significant positive correlations between AC, TPC, PL values and OS of rapeseed oils pressed from seeds after hydrothermal and enzymatic treatments were observed. Moreover, SEM analysis demonstrated, that the studied enzymes promote splitting and destructive effects in rapeseed cells, resulting in enhanced oil yield and the efficiency of pressing. Therefore, biotechnological processes by using the proposed enzymes could improve the quality and quantity of the pressed rapeseed oils.
Acknowledgement The authors thank Dirk Brouwer from AB Enzymes, an ABF Ingredients Company (Germany) for kindly providing ROHALASE1 OS and ROHAPECT1 PTE enzymes. References [1] Rosenthal A, Pyle DL, Niranjan K. Aqueous and enzymatic processes for edible oil extraction. Enzyme Microb Technol 1996;19:402–20. [2] Willems P, Kuipers NJM, De Haan AB. Hydraulic pressing of oilseeds: experimental determination and modeling of yield and pressing rates. J Food Eng 2008;89:8–16. [3] Latif S, Diosady LL, Anwar F. Enzyme-assisted aqueous extraction of oil and protein from canola (Brassica napus L.) seeds. Eur J Lipid Sci Technol 2008;110:887–92. [4] Zhang SB, Wang Z, Xu SY. Optimization of the aqueous enzymatic extraction of rapeseed oil and protein hydrolysates. J Am Oil Chem Soc 2007;84:97–105. ˜ ez MJ, Lema JM. Optimization of the enzymatic [5] Sineiro J, Domı´nguez H, Nu´n treatment during aqueous oil extraction from sunflower seeds. Food Chem 1998;61:467–74. [6] Sosulski K, Sosulski FW. Enzyme-aided vs two stage processing of Canola: technology, product quality and cost evaluation. J Am Oil Chem Soc 1993;70:825–9.
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Effect of enzymatic and hydrothermal treatments of rapeseeds on quality of the pressed rapeseed oils: part II. Oil yield and oxidative stability Aleksandra Szydłowska-Czerniak a,*, Gyo¨rgy Karlovits b, Gabriella Hellner c, Edward Szłyk a a
Faculty of Chemistry, Nicolaus Copernicus University, ul. Gagarina 7, 87-100 Torun´, Poland Bunge Europe Research and Development Center, ul. Niepodległos´ci 42, 88-150 Kruszwica, Poland c Bunge Europe, Research and Development Center, Kvassay J. ut. 1., Budapest H-1095, Hungary b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 June 2009 Received in revised form 31 August 2009 Accepted 14 September 2009
Response surface methodology was used to evaluate the quantitative effects of three independent variables: rapeseed moisture content (MC), enzymes dosage (ED) and conditioning temperature (T) on rapeseed oil yield (OY), efficiency of pressing (EP), and oxidative stability (OS). The highest OY (16.4%) and EP (42.8%) were obtained from pectolytic enzyme (0.1%) treated seeds (MC = 9%, T = 90 8C). The highest OS (12.6 h) was found for oil pressed from rapeseeds heated at 120 8C (MC = 11%), after the cellulolytic enzyme treatment. Results of OY, EP and OS determinations correlate with the predicted values calculated from the partial cubic models (PCMs) equations (R2 = 0.9995, 0.9994, 0.9974 for the cellulolytic enzyme-treated oils and 0.9900, 0.9900, 0.9990 for the pectolytic enzyme-treated oils). The predicted optimum MC = 9.5% and 8.6%, ED = 0.06% and 0.1%, T = 91.2 8C and 90.1 8C resulted in OY = 15.5% and 16.5%, EP = 40.4% and 43.0% for rapeseed oils from seeds treated with cellulolytic and pectolytic enzymes. OS values (12.6 h and 11.8 h) at the optimum conditions of MC = 11.0% and 10.1%, ED = 0.04% and 0.08%, T = 120.0 8C and 119.9 8C for the cellulolytic and pectolytic enzyme-treated oils were also calculated using PCM. Moreover, scanning electron microscopy revealed structural changes in the rapeseed after enzymatic treatment. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Enzyme-pressed rapeseed oils Oil yield Oxidative stability Response surface methodology Scanning electron microscopy
1. Introduction Traditional production of the crude rapeseed oil is based on seeds crushing, cleaning, flaking, conditioning, mechanical pressing and extrusion followed by solvent extraction [1]. The obtained crude oil is refined in order to remove unacceptable materials with the least possible loss of oil and without affecting desirable bioactive compounds, that are present in the crude rapeseed oil. In the mechanical pressing, the preconditioned rapeseeds are passed through a screw press where a combination of a high temperature and shear is used to crush the rapeseeds to release the oil. Rapeseed pressing involves low initial and operating costs when compared to the solvent extraction and is free of any polluting or hazardous substances [2–4]. However, this method is relatively low efficient, thus requires improvements. Moreover, after the conventional oil extraction process, still some of the oil remains in the solid residue. Several methods have been proposed to improve the oil extraction procedures, including an enzymatic pretreatment. The impact of enzymatic hydrolysis on the extraction yield and quality of the
* Corresponding author. Tel.: +48 56 611 47 86; fax: +48 56 654 24 77. E-mail address: [email protected] (A. Szydłowska-Czerniak). 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.09.014
obtained oils (sunflower, rapeseed, borage, cotton, grape seeds, soybean, olives) was studied [1,5–15]. These reports suggested, that enzymatic treatment of oilseed prior to oil extraction improves the oil yield. The oil in rapeseeds is mostly located in the vacuoles as free oil, but oil dispersed in the cytoplasm is not accessible during the extraction process and therefore is lost in the cake [1,16]. The enzymes degrade the oilseed cell wall by converting the cellulose materials into glucose, whereas the complex lipoprotein molecules into simple lipid and protein molecules [1]. This process improves release of oil and increases the yield of extraction. Enzymatic hydrolysis of sunflower [5], rapeseed [3,6], cottonseed [14] and olives [12] prior to extraction processes resulted in 4–35%, 17–29%, 14–52% and 22–152% higher yields, respectively. Moreover, the enzymatic extraction has improved functional and sensorial properties of fish by-products hydrolysates, but with lower yield of oil in comparison to chemical methods [17,18]. Although, the highest amount of oil separated from cod by-products was obtained using Alcalase and Neutrase without the water addition [19,20]. It is known that, oxidative stability (OS) determined by Rancimat method is an important parameter of the edible oil quality control and is standardized AOCS method. The method was
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Table 1 Three-level Box-Behnken design with three independent variables, experimental and predicted results for the oil yield response. Exp.
Coded level MC
1b 2 3b 4 5b 6 7b 8 9 10 11 12 13 14 15
1 1 +1 +1 0 0 0 0 1 +1 1 +1 0 0 0
Independent variables ED
1 +1 1 +1 1 +1 1 +1 0 0 0 0 0 0 0
T
0 0 0 0 1 1 +1 +1 1 1 +1 +1 0 0 0
MC [%]
7 7 11 11 9 9 9 9 7 11 7 11 9 9 9
ED [%]
0 0.1 0 0.1 0 0.1 0 0.1 0.05 0.05 0.05 0.05 0.05 0.05 0.05
Dependent variables T [8C]
105 105 105 105 90 90 120 120 90 90 120 120 105 105 105
ROHALASE1 OS-assisted
ROHAPECT1 PTE-assisted
OYa SD [%]
OYa SD [%]
Experimental
Predicted
Experimental
Predicted
5.7 0.053 11.1 0.26 11.5 0.21 12.3 0.23 14.7 0.13 15.4 0.12 9.1 0.35 9.3 0.47 7.8 0.32 13.2 0.30 8.2 0.22 9.7 0.087 13.1 0.22 12.8 0.060 12.9 0.14
5.7 11.1 11.5 12.3 14.7 15.4 9.1 9.3 7.8 13.2 8.2 9.7 12.9 12.9 12.9
5.2 0.19 14.3 0.12 11.1 0.10 12.4 0.11 14.2 0.50 16.4 0.16 8.6 0.21 12.0 0.12 12.6 0.21 14.8 0.12 10.9 0.19 11.3 0.38 12.4 0.22 11.6 0.16 12.8 0.17
5.4 14.5 10.9 12.2 14.2 16.4 8.6 12.0 12.4 15.0 10.7 11.5 12.3 12.3 12.3
SD—standard deviation; probability, p = 0.05. a n = 3. b Control sample.
also applied for evaluation of the OS of the enzyme-extracted corn [21], sunflower [22] and olive oils [9,23]. However, the correlations between antioxidant capacity, total phenolic, tocopherol and phospholipid contents and oxidative stability of the enzymetreated rapeseed oils, to the best of our knowledge, were not studied. Response surface methodology (RSM) is a collection of statistical techniques for designing experiments, building models, evaluating the effects of factors, searching optimum conditions of factors for desirable responses, which has been successfully applied in many areas of biotechnology such as: optimization of media and cultivation conditions [24,25], biotechnological parameters [26,27], lipase-catalyzed reaction [28,29], fermentation conditions [30,31]. However, the optimum conditions of hydrothermal and enzymatic treatments of rapeseeds for optimizing the extraction of rapeseed oil with high oxidative stability have not yet been reported. Therefore, RSM was used to evaluate the impact of cellulolytic and pectolytic enzymes (enzyme dosage—ED), hydrothermal parameters (moisture content—MC and temperature—T) and their interactions on response variables: oil yield (OY), efficiency of pressing (EP) and oxidative stability (OS) of the crude rapeseed oils. The main purpose of this study was to improve and optimize the oil recovery after enzymatic and hydrothermal treatments of rapeseeds using a mechanical oil expression. Moreover, the effect of cellulolytic and pectolytic enzymes concentration on physical and morphological properties of rapeseed coat and cotyledon was studied by the scanning electron microscopy (SEM). 2. Materials and methods 2.1. Sample preparation Rapeseeds (Brassica Napus cv. Oleifera) and enzymes (ROHALASE1 OS (ROS) and ROHAPECT1 PTE (RPTE)) used in the procedure of oil pressing were described in Part I [32]. 2.2. Analytical methods Oil content was determined by solvent extraction according to AOAC methods [33]. Moisture content was determined by vacuum drying at 60 8C to constant weight. Oil yield is defined as percentage of oil expelled at the pressing stage on a total oil extractable basis. Oil expelled is defined as the difference between total oil of the
original seeds and the oil in residual cake. The yield of the obtained oil (OY, %) and efficiency of pressing (EP, %) were expressed by equations: OY = (OO OC) A/B and EP = (OY/OO) 100, respectively, where OO—oil content in initial seeds (%); OC—oil content in cake (%); A—dry mater of initial material (%); B—dry mater of cake (%). Oxidative stability indexes of the obtained rapeseed oils were evaluated at 120 8C and a flow rate of air 20 L/h using the Rancimat apparatus (model 743, Metrohm). Stability was expressed as the oxidation induction time (h). Scanning electron microscopy (SEM) analysis was used to reveal the influence of enzymes treatment on the structure of the rapeseed. Scanning electron microscopic (SEM) studies of the untreated and treated seeds were carried out using a scanning electron microscope (LEO 1430 VP), at high vacuum (50 Pa), and accelerating voltage of 28 kV. 2.3. Experimental design and statistical analysis The results of OY, EP and OS obtained in this study were presented as: mean value of three replicates standard deviation (SD). Pearson correlation test was used to determine the correlations between oxidative stability results and FRAP, TPC, TTC and PL in the pressed rapeseed oils. Statistically significant differences were considered at the p < 0.05 level. The three-level Box-Behnken design with three independent variables (MC, ED and T) was applied for responses (OY, EP and OS) functions fitting. This design was selected due to the small number of experiments required to estimate complex response functions. For the three-level three-factorial Box-Behnken experimental design, a total of 15 experimental runs are necessary. The uncoded and coded independent variables and experimental design are listed in Tables 1–3. Box-Behnken design allows on the efficient estimation of the first- and secondorder coefficients. Response surface methodology (RSM) was used to study the simultaneous effects of three experimental factors (MC, ED and T) on OY, EP and OS of the pressed rapeseed oils and to find optimum conditions for a multivariable system. RSM allowed the development of a predictive mathematical model based on the experimental data, which can be employed for interpolation. In the first step of RSM the appropriate approximation for the true functional relation between response and the set of independent variables was calculated. When the response is well fitted to the linear function of the independent variables, the approximated function resulted in the first-order model. In the case when system revealed curvature, the polynomial of higher degree (second- or third-order) models must be applied for the response approximation. Therefore, all the responses observed were simultaneously fitted to the firstorder, second-order and partial cubic models. The determination coefficient (R2), the lack-of-fit of the studied models, and p-values of the parameter estimations were used to validate the models. The first-order models were used to approximate the data responses. However, the lack-of-fit p-values (0.0037–0.046) were significant (except p = 0.1305 and 0.1291 for OY and EP of the RPTE-treated oils), whereas R2 (0.6340–0.8353) were small (except R2 = 0.9531 and 0.9603, for OS of the ROS- and RPTE-treated oils, respectively). Therefore, the first-order models were not adequate approximations for the studied responses and the second-order models for data fitting were applied. Various second-order models, from interaction to partial cubic, were tested. The R2 of the second-order models were higher
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Table 2 Three-level Box-Behnken design with three independent variables, experimental and predicted results for the efficiency of pressing response. Exp.
Coded level MC
1b 2 3b 4 5b 6 7b 8 9 10 11 12 13 14 15
1 1 +1 +1 0 0 0 0 1 +1 1 +1 0 0 0
Independent variables ED
1 +1 1 +1 1 +1 1 +1 0 0 0 0 0 0 0
T
0 0 0 0 1 1 +1 +1 1 1 +1 +1 0 0 0
MC [%]
7 7 11 11 9 9 9 9 7 11 7 11 9 9 9
Dependent variables
ED [%]
T [8C]
0 0.1 0 0.1 0 0.1 0 0.1 0.05 0.05 0.05 0.05 0.05 0.05 0.05
105 105 105 105 90 90 120 120 90 90 120 120 105 105 105
ROHALASE1 OS-assisted
ROHAPECT1 PTE-assisted
EPa SD [%]
EPa SD [%]
Experimental
Predicted
Experimental
Predicted
14.8 0.14 29.0 0.69 29.9 0.54 32.1 0.59 38.4 0.33 40.1 0.30 23.8 0.92 24.2 1.22 20.4 0.85 34.4 0.77 21.4 0.56 25.3 0.23 34.2 0.61 33.3 0.16 33.7 0.36
14.8 29.0 29.9 32.1 38.4 40.1 23.8 24.2 20.4 34.4 21.4 25.3 33.7 33.7 33.7
13.6 0.48 37.3 0.32 29.0 0.22 32.4 0.29 37.1 0.21 42.8 0.42 22.4 0.55 31.3 0.16 32.9 0.54 38.6 0.32 28.4 0.51 29.5 0.99 32.4 0.58 30.3 0.42 33.4 0.44
14.1 37.8 28.5 31.9 37.1 42.8 22.4 31.3 32.4 39.1 27.9 30.0 32.0 32.0 32.0
SD—standard deviation; probability, p = 0.05. a n = 3. b Control sample.
Table 3 Three-level Box-Behnken design with three independent variables, experimental and predicted results for the oxidative stability response. Exp.
Coded level MC
1b 2 3b 4 5b 6 7b 8 9 10 11 12 13 14 15
1 1 +1 +1 0 0 0 0 1 +1 1 +1 0 0 0
Independent variables ED
1 +1 1 +1 1 +1 1 +1 0 0 0 0 0 0 0
T
0 0 0 0 1 1 +1 +1 1 1 +1 +1 0 0 0
MC [%]
7 7 11 11 9 9 9 9 7 11 7 11 9 9 9
Dependent variables
ED [%]
T [8C]
0 0.1 0 0.1 0 0.1 0 0.1 0.05 0.05 0.05 0.05 0.05 0.05 0.05
105 105 105 105 90 90 120 120 90 90 120 120 105 105 105
ROHALASE1 OS-assisted
ROHAPECT1 PTE-assisted
OSa SD [h]
OSa SD [h]
Experimental
Predicted
Experimental
Predicted
4.4 0.061 4.4 0.13 7.8 0.25 7.1 0.17 3.9 0.078 4.5 0.10 9.5 0.19 8.2 0.21 3.6 0.072 4.1 0.11 5.8 0.21 12.6 0.37 5.1 0.14 5.4 0.20 5.3 0.093
4.3 4.3 8.0 7.3 3.9 4.5 9.5 8.2 3.8 4.0 6.0 12.5 5.3 5.3 5.3
5.0 0.24 6.8 0.18 7.5 0.38 8.9 0.21 4.0 0.14 4.6 0.089 9.5 0.31 11.8 0.17 4.6 0.093 5.4 0.12 7.6 0.10 10.9 0.22 6.7 0.13 6.9 0.18 7.0 0.25
5.1 6.9 7.4 8.8 4.0 4.6 9.5 11.8 4.5 5.5 7.5 11.0 6.9 6.9 6.9
SD—standard deviation; probability, p = 0.05. a n = 3. b Control sample. (0.8761–0.9946) than the first-order (0.6340–0.8353) models, but the evaluated lack-of-fits were significant for OY and EP of ROS-treated rapeseed oils and OS of RPTE-treated oil (p = 0.0055, 0.0069, 0.0304, respectively). Based on the statistical analysis (lack-of-fit is not significant and R2 is the highest), the best-fitted model was the partial cubic model (PCM):
3. Results and discussion
Y n ¼ b0 þ b1 MC þ b2 ED þ b3 T þ b11 MC2 þ b22 ED2 þ b33 T 2
The oil yield and efficiency of pressing (oil extractability) of the enzymatic and hydro-thermally treated rapeseeds are listed in Tables 1 and 2. OY and EP results ranged between 5.2–16.4% and 13.6–42.8%, respectively. It can be noted, that the application of both procedures: enzymatic hydrolysis and hydrothermal pretreatment on rapeseeds enhanced oil yield and oil extractability in comparision to the control samples (without enzymes, at the same hydrothermal conditions). The highest OY (16.4%) and EP (42.8%) were obtained from RPTE (0.1%) treated seeds (MC = 9%, T = 90 8C), whereas, the lowest OY (5.7% and 5.2%) and EP (14.8% and 13.6%) were determined for the control samples (without enzymes) of rapeseeds contained 7% moisture and heated at 105 8C. Among two enzymes used, RPTE gave a higher OY (10.9–16.4%) and EP (28.4– 42.8%), which conforms to the fact that pectic substances are the prevalent cell wall polysaccharides in rapeseeds [34]. Moreover,
2
2
þ b12 MC ED þ b13 MC T þ b23 ED T þ b112 MC ED þ b113 MC T where Yn is one of the three responses, MC, ED and T represent the independent variables, b0 is the constant, b1, b2, b3 are the linear-term coefficients, b11, b22, b33 are the quadratic-term coefficients, and b12, b13, b23, b112, b113 are the cross-term coefficients. The fitness of the PCM was evaluated by the determination coefficient R2, the fraction of the variation explained by the model, and analysis of variance (ANOVA). The F-test was applied to confirm, whether the variance explained by the regression model was significantly larger than the variance of the residual and to evaluate the model lack-of-fit (model error). Moreover, the fitted polynomial equations were presented as surface and contour plots in order to visualize the relation between the responses and experimental levels of each factor and to deduce the optimum conditions. Regression and analysis of variance (ANOVA) were carried out using the Statistica Windows software package.
3.1. Oil yield and efficiency of pressing
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RPTE increased OY and EP by 11.7–175.0%, whereas ROS improved oil recovery (OY) and extractability (EP) by only 2.2–94.7%, when compared to the control samples hydrothermal treated but without enzyme action (Tables 1 and 2, exps. 1–8). Reported by other authors, the enzyme-extracted rapeseed oils yields were higher (22.2–91.6%) [3,4]. However, OY of the discussed oils were similar to results found for olive oil (10.6–17.5%) by Aliakbarian et al. [12], sunflower (4.00–35.45%) by Sineiro et al. [5], grape seed (6.71– 19.50%) by Passos et al. [15] and cottonseed (9.70–12.89%) by Latif et al. [14]. The higher oil yield of enzyme-assisted pressing process, than the control samples (without enzymes) can be explained by the better solubilisation and hydrolysis of proteins. The network of the cotyledon cells and the protein (oleosin) based membranes were broken, thereby improving the extraction yields [1,5,8]. It can be suggested, that the increase of the enzyme concentration causes the increase the OY and EP (Tables 1 and 2). Similar results were observed by Najafian et al. [13] for the Koroneiki variety olives, when pectinex enzyme was used at the higher concentration. The opposite effect was observed when the conditioning temperature of rapeseeds was raised to 120 8C (Tables 1 and 2, exps. 5–8 and 13–15). The latter can be related to the thermal inactivation of enzymes. On the other hand, oxidative stability of the obtained crude rapeseed oils ranged over 3.6–12.6 h (Table 3). The most stable to oxidation (OS = 12.6 h) was ROS-treated oil mechanically extracted from seeds which contained 11% moisture and were heated at 120 8C. The oxidative stability of oils was elevated with an increase of the rapeseed conditioning temperature. The significant correlation (R2 = 0.6190, p < 0.000001) between induction time for all studied oils and the conditioning temperature of seeds before pressing is evident. For comparison, the oxidative stability of the olive oils obtained from a blend of Frantoio/Leccino and Coratina cultivars positively correlated with malaxation temperature (R2 = 0.818 and 0.987, respectively) [35]. The higher oil stability can be related to the inactivation of oxidative enzymes such as lipase, peroxidase and lipoxigenase during heating stage [36]. Also, the increase of MC in rapeseeds heated at 120 8C caused an elongation of the induction period of the pressed oils (Table 3). Moreover, the Rancimat stability of oils was higher as a consequence of the RPTE treatment of rapeseeds. In the case of ROS-treated seeds, the pressed oils demonstrate lower stability (except oil pressed from seeds with MC = 9% heated at 90 8C) than the control samples (without the enzyme) (Table 3, exps. 1–8). Reported enzyme processed virgin olive oils [9,11,23] and sunflower oils [22] also exhibited improved oxidative stability when compared with the one obtained without enzyme treatment. RSD values of OY: 0.47–5.05%, EP: 0.48–5.04% and OS: 1.32– 5.07% indicate reasonable repeatability of the applied analytical procedures. 3.2. Fitting the models Response surface methodology (RSM) was applied and the partial cubic response surface models were fitted to each response variable: OY, EP and OS, respectively (Tables 1–3). Regression analysis and analysis of variance (ANOVA) of the experimental data (Tables 1–3) were performed for the mathematical models fitting, determination of the regression coefficients and statistical significance examination of the model terms. The following regression equations were obtained: OYROS ¼ 425:9 þ 90:2MC þ 669:8EDROS þ 3:58T 4:74MC2 76:7ED2ROS 0:0027T 2 130:8MC EDROS 0:678MC T 0:167EDROS T þ 6:63MC2 EDROS þ 0:0358MC2 T
OYRPTE ¼ 157:1 þ 50:5MC þ 669:8EDRPTE þ 0:549T 2:64MC2 223:3ED2RPTE þ 0:0049T 2 127:5MC EDRPTE 0:375MC T þ 0:400EDRPTE T þ 6:00MC2 EDRPTE þ 0:0200MC2 T
EPROS ¼ 1110:8 þ 235:5MC þ 1794:5EDROS þ 9:32T 12:4MC2 206:7ED2ROS 0:0071T 2 351:8MC EDROS 1:76MC T 0:433EDROS T þ 17:9MC2 EDROS þ 0:0933MC2 T EPRPTE ¼ 412:7 þ 132:2MC þ 1741:5EDRPTE þ 1:48T 6:92MC2 581:7ED2RPTE þ 0:0125T 2 332:0MC EDRPTE 0:983MC T þ 1:07EDRPTE T þ 15:6MC2 EDRPTE þ 0:0525MC2 T
OSROS ¼ 30:0 þ 4:96MC þ 65:6EDROS 0:681T 0:530MC2 þ 131:7ED2ROS þ 0:0041T 2 1:75MC EDROS 0:0525MC T 0:633EDROS T 0:00MC2 EDROS þ 0:0058MC2 T
OSRPTE ¼ 163:9 34:0MC 16:3EDRPTE 1:74T þ 1:80MC2 þ 106:7ED2RPTE þ 0:0015T 2 7:75MC EDRPTE þ 0:336MC T þ 0:567EDRPTE T þ 0:375MC2 EDRPTE 0:0175MC2 T The ANOVA results exhibit the effect and the regression coefficients of each linear, quadratic and interaction terms, that were individually determined. The PCM represents responses of OY, EP and OS with the coefficients of determination, R2 = 0.9995, 0.9994, 0.9974 and 0.9900, 0.9900, 0.9990 for ROS- and RPTEtreated oils, respectively (Table 4). The calculated R2 values indicate that obtained PCMs were adequate for the description of the independent variables (MC, ED and T) impact on OY, EP and OS of the enzyme-treated rapeseed oils. The R2 and the adjusted R2 values for the studied response variables, were higher than 0.90, hence there is a close agreement between the experimental results and theoretical values predicted by the proposed models. For comparison, Aliakbarian et al. [12] used first-order model and calculated lower determination coefficient (R2 = 0.837), which demonstrates that the model can be used to explain more than 80% of the variability in the olive oil extraction yield response. The model adequacy was tested using the lack-of-fit F-test, which is not significant for p > 0.05. The ANOVA results for the studied responses revealed insignificant lack-of-fit (F ranged between 0.0536 and 7.71, p > 0.05, respectively) (Table 4). Therefore, these models were adequate for prediction, within the range of variables employed. Furthermore, the linear and quadratic effects of the rapeseed treatment variables (MC, ED and T) and their interactions on the response variables (OY, EP and OS) were analyzed by ANOVA (Table 4). The model is highly significant, when the computed F-value is greater than the tabulated F-value at low probability value (p < 0.0001). All linear (MC, ED, T), quadratic (MC2 and T2) and interactions (MC ED, MC T, MC2 ED and MC2 T) parameters of the models were statistically significant (p < 0.05) for OY and EP of the studied ROS-pressed oils. On the contrary, only
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Table 4 ANOVA results for the responses: OY, EP and OS of the enzyme-pressed rapeseed oils. Model parameters
Degree of freedom
ROHALASE1 OS-assisted Sum of squares
Oil yield (OY) Linear Quadratic Interaction Regression Residual Lack-of-fit Pure error Total R2 Adjusted R2
3 3 5 11 3 1 2 14
*
Mean square
F-value
16.34 8.78 4.38 9.56 0.016 0.00125 0.0234
700.2* 376.3* 187.8* 379.0* 0.0536
Sum of squares 61.11 8.94 22.14 98.11 0.992 0.245 0.747 99.10
0.9995 0.9979
Efficiency of pressing (EP) Linear 3 Quadratic 3 Interaction 5 Regression 11 Residual 3 Lack-of-fit 1 Pure error 2 Total 14 2 R Adjusted R2 Oxidative stability (OS) Linear Quadratic Interaction Regression Residual Lack-of-fit Pure error Total 2 R Adjusted R2
49.01 26.34 21.91 105.19 0.0480 0.00125 0.0467 105.24
ROHAPECT1 PTE-assisted
3 3 5 11 3 1 2 14
332.8 179.4 150.2 715.5 0.418 0.0112 0.407 715.9
110.9 59.80 30.04 65.05 0.139 0.0112 0.204
23.26 1.33 2.24 7.95 0.0757 0.180 0.0234
20.37 2.98 4.43 8.92 0.331 0.245 0.374
F-value 54.55* 7.98 11.86 22.45* 0.656
0.9900 0.9533 545.6* 294.1* 147.7* 296.2* 0.0553
416.7 60.34 150.2 668.1 6.72 1.71 5.01 674.8
0.9994 0.9973
69.79 3.99 11.19 87.53 0.227 0.180 0.0467 87.76
Mean square
138.9 20.11 30.04 60.74 2.24 1.71 2.51
55.48* 8.04 12.00 22.78* 0.684
0.9900 0.9535 997.0* 56.97* 95.94* 331.1* 7.71
57.88 0.719 4.54 75.54 0.0780 0.0313 0.0467 75.62
0.9974 0.9880
19.29 0.240 0.908 6.87 0.0260 0.0313 0.0234
827.0* 10.28 38.93* 246.0* 1.34
0.9990 0.9952
Significant at the p < 0.05 level.
linear terms of RPTE dosage and conditioning temperature, and interaction MC ED produced a significant effect on OY and EP of RPTE-assisted oils. Also, all linear parameters (MC, ED, T) and interactions (MC T, ED T, MC2 T) significantly influenced OS of rapeseed oils obtained from seeds after RPTE treatment (Table 4). Besides, only two interactions (MC T, ED T) and hydrothermal conditioning (MC, T and T2) of seeds revealed significant effects on OS of ROS-pressed oils. The ANOVA results indicate, that linear terms of the enzymes dosage and the conditioning temperature and interaction MC ED caused significant impact on OY and EP of all crude rapeseed oils. It can be noted, that impact of the RPTE dosage on OY and EP of the studied oils was greater, than the processing temperature. On the contrary, MC and T were more evident independent variables on OY, EP of ROS-assisted oils and the oxidative stability of all oils. For comparison, only linear terms (enzyme concentration and malaxation time) had statistically significant effect on the olive oil extraction yield [12]. The highest yield (17.5 goil/100 gpaste) was determined after 150 min malaxation time and upon enzymes addition (25 mL/kgpaste) [12]. In the case of sunflower oil, the maximum extractability (35.65%) was obtained from sunflower grinded seeds, adjusted to 5 g of water/1 g of seeds and with 2 g of enzyme/100 g of seeds, after 2 h of enzymatic treatment extraction [5]. To enhance oil availability and extractability, Kashyap et al. [37] used a mixed-activity crude enzyme. Authors applied second-order response surface methodology to calculate optimal process conditions for soyflakes and soybrokens with 24.6% and 24.7% moisture content subjected to hydrolysis, along with 14.2% and 7.1% of
enzyme concentration, and 13.3 h and 13.9 h hydrolysis time, respectively. 3.3. Analysis of response surfaces The effects of the three independent variables (MC, ED and T) on OY, EP and OS of the obtained oils were illustrated by the surface response and the contour plots of the PCM for different interactions of any two independent variables, while holding the value of the third variable as constant (0 level) (Figs. 1–3). The relations between MC, ED and T of seed conditioning and the OY and EP of ROS-treated rapeseed oils as well as their interactions are presented in Figs. 1a–c and 2a–c. The elliptical shape of the contour plots indicates the significant interactions between the corresponding variables MC ED, MC T, MC2 ED and MC2 T. The significant positive linear effect of MC in rapeseeds and ROS dosage on the OY and EP of ROS-treated rapeseed oils resulted in the enhance of OY and EP along with the increase of MC and ROS concentration (Figs. 1a and 2a). Besides, the OY and EP of ROS-pressed rapeseed oils were improved at conditioning temperature below 105 8C and higher levels of MC (>7%) and ROS (>0.05%) dosage (Figs. 1b, c and 2b, c). Figs. 1d and 2d exhibit that at the lowest level of MC, the OY and EP of the RPTE-treated oils rise rapidly with the increasing RPTE concentration, while at the highest level of MC, the OY and EP enhanced to a certain level due to the contribution by the interaction term of RPTE concentration and MC in rapeseeds. The RPTE dosage and conditioning temperature displayed a significant linear (positive and negative, respectively) influence on OY and EP of RPTE-assisted oils. In Figs. 1f and 2f, RSM revealed the maximum
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Fig. 1. Response surfaces and contour plots for OY of the enzyme-pressed rapeseed oils expressed as a function of (a and d) MC and ED (at T = 105 8C), (b and e) MC and T (at ED = 0.05%), (c and f) T and ED (at MC = 9%).
of OY and EP at the highest RPTE (0.1%), while the lowest T (90 8C) and an intermediate MC = 9% were observed. Moreover, as the T of seed conditioning after addition of RPTE has decreased, the OY and EP responses were higher for all studied MC (Figs. 1e and 2e).
The effects of the ROS dosage and one of variables (MC or T) demonstrate similar response surfaces of OS (Fig. 3a and c). It is evident that, higher MC and T resulted in the progressive OS increase, whereas the effect of ED was insignificant. Moreover, the
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Fig. 2. Response surfaces and contour plots for EP of the enzyme-pressed rapeseed oils expressed as a function of (a and d) MC and ED (at T = 105 8C), (b and e) MC and T (at ED = 0.05%), (c and f) T and ED (at MC = 9%).
oxidative stability of the enzyme-assisted oils rapidly increased, along with growth of the conditioning temperature and the moisture content in rapeseeds (Fig. 3b and e). These two parameters (MC and T) affected positively on OS of all studied oils. On the contrary, RPTE concentration displayed the positive
and significant linear effect on OS values (Fig. 3d and f). Therefore, more stable oils were pressed from rapeseeds with higher moisture (MC > 9%), conditioning at higher temperature (T > 105 8C), after treatment with more concentrated RPTE (c > 0.05%).
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Fig. 3. Response surfaces and contour plots for OS of the enzyme-pressed rapeseed oils expressed as a function of (a and d) MC and ED (at T = 105 8C), (b and e) MC and T (at ED = 0.05%), (c and f) T and ED (at MC = 9%).
By the analysis of regression equations and response surface contour plots, the optimum MC, ED and T were found to be 9.5%, 0.06% and 91.2 8C, where the OY and EP of ROS-pressed oils were predicted to be 15.5% and 40.4%, respectively. However, the optimum conditions for pressing oils from seeds with RPTE were:
MC = 8.6%, ED = 0.1% and T = 90.1 8C, under which the predicted OY = 16.5% and EP = 43.0% were found. Besides, the models predicted the OS values for ROS-assisted (12.6 h) and RPTE-assisted (11.8 h) oils at the optimum conditions of MC = 11.0% and 10.1%, ED = 0.04% and 0.08% and T = 120.0 8C and 119.9 8C, respectively.
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Fig. 4. Scanning electron micrographs of rapeseed untreated and treated with cellulolytic enzyme ROHALASE1 OS (c = 0.05% and c = 0.1%).
3.4. Effect of enzymatic treatment on the physical structure of rapeseed Scanning electron micrographs (Figs. 4 and 5) present differences between untreated rapeseed and after enzymatic treatment at both concentrations (ED = 0.05% and 0.1%) of ROS and RPTE. Images of the untreated rapeseed did not exhibit distortion or rupture of the cells and only lipid can be observed in the spaces between the closely packed, intact cells (Figs. 4 and 5, column 1). In
the control sample (without enzymes), the spherical oil bodies were surrounded by an intact biological membrane. Oil-rich droplets located inside the rapeseed cotyledon cell can be seen. The applied enzymes distorted the original structure of rapeseed and the oil droplets (white points) appeared (Figs. 4 and 5, columns 2 and 3). The similar morphologic changes of rapeseed after cellulolytic (ROS) and pectolytic (RPTE) enzymes adding were observed (Figs. 4 and 5). Enzymatic treatment caused destruction of the oil bodies membranes and oil located in the lipid bodies coalesced. The SEM images confirmed, that the enzymes are able to
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Fig. 5. Scanning electron micrographs of rapeseed untreated and treated with pectolytic enzyme ROHAPECT1 PTE (c = 0.05% and c = 0.1%).
breakdown the cell structure of rapeseed and induce the release of oil from cells. However, the seed coat retains its integrity, in spite of enzymatic treatment, hence antioxidants were retained inside the layer. Therefore, the highest antioxidant capacity (1220.0, 964.8 mmolTE/100 g) and total phenolic content (83.3, 74.0 mgSA/100 g) were found for two rapeseed oils extruded from seeds heated at 120 8C (MC = 11%) and treated with less concentrated cellulolytic and pectolytic enzymes (ED = 0.05%) (see Part I) [32].
3.5. Correlations between antioxidant capacity (AC), total phenolic (TPC), tocopherol (TTC) and phospholipid (PL) contents and oxidative stability of enzyme-treated oils The oxidative stability of oil is affected by some minor compounds such as phenols, tocopherols and other antioxidants present in oil [38]. Hence, regression analysis was performed to calculate the correlations between AC, TPC, TTC, PL (results were presented in Part I) [32] and OS of rapeseed oils pressed from seeds
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after hydrothermal and enzymatic treatments. The oxidative stability data for all studied rapeseed oils correlate significantly positively with AC, TPC and PL (y = 61.97x + 198.6, R2 = 0.6090, y = 6.05x + 2.16, R2 = 0.7939, y = 1177.8x 1165.9, R2 = 0.7026, respectively; p < 0.0000001). Similarly, significant linear correlation between free radical antioxidant capacity measured by DPPH and oxidative stability determined by Rancimat method (R2 = 0.83, p = 0.032) for nut oils was found by Arranz et al. [39]. Furthermore, significant positive correlations (R2 = 0.8986, 0.786, 0.948 p < 0.001) between oxidative stability and total phenols in virgin olive oils were observed by others [40,41]. Likewise, phospholipids contribute to the stability of vegetable oils [39,42]. Therefore, positive linear correlation (r = 0.9849) between the induction time of olive oils and lecithin concentration was demonstrated by Koprivnjak et al. [43]. However, the lack of correlation between oxidative stability and total tocopherol content in all studied oils (y = 0.675x + 646.9, R2 = 0.0041, p = 0.7362) was observed. Also, Bozan and Temelli [44] did not find correlation between oxidative stability and tocol (tocopherols and tocotrienols) levels of flax, safflower and poppy oils. Response surface methodology (RSM) appeared to be useful way of studies the influence of three experimental factors (rapeseed moisture content, enzyme dosage and conditioning temperature) on OY, EP and OS of the pressed rapeseed oils. The highest OY (16.4%) and EP (42.8%) were obtained from RPTE (0.1%) treated rapeseeds (MC = 9%, T = 90 8C), whereas the most stable to oxidation (OS = 12.6 h) was noted for ROS-treated oil mechanically extracted from seeds with 11% of water heated at 120 8C. The effect of a pectolytic enzyme concentration on OY and EP of the RPTEassisted rapeseed oils was greater, than the processing temperature, whereas conditioning temperature was more effective independent variable on OY, EP of ROS-assisted oils and oxidative stability of all oils. Besides, the significant positive correlations between AC, TPC, PL values and OS of rapeseed oils pressed from seeds after hydrothermal and enzymatic treatments were observed. Moreover, SEM analysis demonstrated, that the studied enzymes promote splitting and destructive effects in rapeseed cells, resulting in enhanced oil yield and the efficiency of pressing. Therefore, biotechnological processes by using the proposed enzymes could improve the quality and quantity of the pressed rapeseed oils.
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