Protoreactor : The innovative tool to reduce processing time, increasing phenols extraction and producing a desirable extra virgin olive oil

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1 Protoreactor : The innovative tool to reduce processing time, increasing phenols extraction and producing a desirable extra virgin olive oil Michele Balzano, Department of Agricultural, Food and Environmental Sciences, Polytechnic Marche University, m.balzano@univpm.it Deborah Pacetti, Department of Agricultural, Food and Environmental Sciences, Polytechnic Marche University, d.pacetti@univpm.it Gennaro Pieralisi, Gruppo Pieralisi, info@pieralisi.com Natale Giuseppe Frega, Department of Agricultural, Food and Environmental Sciences, Polytechnic Marche University, n.g.frega@univpm.it Abstract-The aim of a modern olive oil mill is to allow fast processing time, preserving high yield and quality. High olive quantities are hardly managed by industrial mill, thus the great dimension of the plants allows an apparently fast extraction, the oil produced is often affected by off flavor. These defects are strictly related to the prolonged processing time and extended high temperature, especially in the malaxer. In this contest fast preheating after crushing can represent an interesting tool preserving without altering extra virgin oil quality. For this purpose a new machinery, protoreactor market edition, at two different temperatures coupled with different malaxation settings was evaluated. Legal parameters, total phenols, oxidative stability, spectrophotometric constants and panel test were evaluated at time zero and during 1 year of storage. New machine was able to reduce processing time, maintaining good yield and high quality oil. As such, the protoreactor exerted an interesting extraction activity as concerns phenols compounds. In particular, protoreactor set to 36 C allowed to reduce the malaxation time till a fast passage ensuring a good oil yield, high phenols extraction and consequently excellent oxidative stability during evaluation period. For these reasons Protoreactor could represent an useful tools for the operators of the olive oil sector. I. INTRODUCTION Extra virgin olive oil (EVOO) is one of the main characteristic product in Euro-Mediterranean region countries, historically this production spreads from Portugal to Greece. Nowadays European olive farming has to deal with an enormous challenge. In fact, the growth of foreign competitors from emerging market and consequently their low cost production join to the wide economic crisis are compromising the Italian olive oil market. Furthermore EVOO importance is strictly related to remarkable health properties, which depend on many components as fatty acid, tocopherols, and hydrophilic phenols [1-5]. Actual deal for the EVOO production have been identified as high extraction yield and quality of the product. The EVOO extraction process is characterized by three principal steps: olive crusching, malaxation and oil separation using pressure or centrifugation systems. Although technological processes as crushing and malaxation play a fundamental role through the final product [6-10], malaxation phase represents one of the most critical processing point. As such, the greatest amount of olives are processed in large plants, in which due to incomplete procedures of emptying, parts of olive paste could remain in the malaxer for several cycles causing the occurrence of defect. In fact, during this prolonged malaxation, olive paste is exposed to the O 2 oxidant activity. By considering this critical issues, several works aimed to reduce the malaxation time. It is well known that high malaxation temperature, due to promoting the aggregation of oil droplets, is able to increase oil yield [11] but as discussed by Kalua [12] when the temperature increased up 45 C cause a significant yield reduction. In addition to the previous considerations, Ranalli et al. had shown how 30 C represent the ideal malaxation temperature as regards both oil yield and quality [13]. Taking into account the 1

2 polyphenols, they can be considered as EVOO quality markers, for this reason their behavior to the temperature was widely investigated. There was a well known inverse relationship between phenols content and temperature, widely accepted in traditional literature [1, 5] but recent studies have shown that phenols amount increase as response to the temperature increase [12, 14]. As such, it is notable to underline how traditional malaxation process is characterized by low thermal transfer efficiency, for this reason, the thermal conditioning of olive paste is relatively long compared to the optimal processing temperature. In order to obtain a more efficient heat exchange between the small malaxer surface area and the volume of olive paste, the use of flash thermal conditioning treatment of the olive paste before malaxation process was supposed. Recently Esposto et al. [15], Leone et al. [16], Veneziani et al. [17] and Fiori et al. [18] provided an analytical support to flash thermal conditioning before malaxation process. Esposto et al. shown how virgin oil flash thermal conditioning did not affect the concentration of volatile compounds; Leone et al. observed how a tubular heat exchanger is able to improve phenolic and volatile compounds, while Fiori et al. set up different preliminary experiments in order to evaluate the effects of fast preheating of olive paste after crushing. As result, Fiori et al. supposed that a fast preheating not longer than 72 s at 38 C followed by 10 malaxation lead to an EVOO characterized by a mild sensory profile and a shelf life of at least 12 months [18]. The present work had the aim to point out the role of a rapid fast preheating in a olive oil processing. First of all, it is necessary to underline how the machinery was not a prototype as in the previous study [18], but the fast preheating machine was the protoreactor as the final commercial edition. Taking into account the temperature, it can be assumed that there are not prescriptions on the temperature requirements during olive processing (temperature requirements are only specified in the EU Reg. 29/2012, as an optional indication for cold pressing/extraction). Although the PDO (protected designation of origin) and PGI (protected geographical indication) disciplinary tend to limit the processing temperature. The study was aimed to observe how protoreactor, in a final commercial edition at two different heat levels coupled with variable malaxer settings, influenced yield and quality parameters as at the production than during storage period. The impact of two protoreactor temperatures combined with four different malaxation settings was evaluated. II. MATERIALS AND METHODS A. Olive oil extraction process A blend of defoliated and washed olives of the cultivar Frantoio and Leccino in the same proportion (600 kg) were processed with a modified two-phase continuous plant (Pieralisi Group, Jesi, Italy). The system consisted of a fast preheater (protoreactor by Pieralisi Group, Jesi, Italy), a mobile hammer crusher, and a malaxer (Genius P4 model, Pieralisi group Jesi, Italy). Successively, the oil was extracted using a horizontal centrifuge (decanter) operating at 2410 rpm (Maior special model, Pieralisi Group, Jesi, Italy). Using protoreactor at temperature T1 the paste heated until 36 C while during preheating at temperature T2 the past reach 43 C. Operating temperatures in the protoreactor hardware were reported in Table I. 2

3 TABLE I TEMPERATURES DETECTED IN THE DIFFERENT DEVICE COMPONENTS OF PROTOREACTOR. Temperatures T1 T2 C water inlet first hardware (without olive paste) C water outlet first hardware (without olive paste) C water outlet second hardware (without olive paste) C water inlet first hardware (with olive paste) C water outlet first hardware (with olive paste) C water outlet second hardware (with olive paste) Olive paste temperatures C Eight different experiments (Fig. 1) were carried out in order to evaluate the two different protoreactor temperatures (T1 versus T2) in relationship to malaxation time and temperature. The eight experiments were performed on different days of the 2013 oil campaign, from November 2 until November 30. Two control samples were produced, they were obtained by-passing protoreactor and malaxer was at room temperature (17 C) respectively for 15 and 30 minutes. The control sample using malaxer for zero minutes was omitted, due to the lower oil yied. The fast preheater tool called by Pieralisi group protoreactor was a cylindrical segment with an inner cavity for the passage of the olive paste by means of a screw feeder. It was 6 m long and was surrounded by a hollow space (16 cm internal diameter) in which hot water flowed in counter current to the passage of the olive paste. For each trial have been made three replicates. To investigate the shelf-life of the olive oils so obtained, two bottles for each analysis time (0, 6, and 12 months) were filled with the oil samples prepared during each experimental procedure. The bottles (750 ml) were sealed with a screw cap and were kept in the dark and at room temperature for the entire period of experimentation (12 months). 3

4 Figure 1. Extra Virgin Olive Oil processing protocols and trials plan. B. Determination of legal quality parameters and oxidative stability Determination of free acidity (g oleic acid per 100 g olive oil), peroxide index (meq O 2 kg -1 oil) and panel test were performed according to the official European Commission methods, K232 and K270 extinction coefficients were calculated from absorption at 232 and 270 nm, respectively, with a UV spectrophotometer CARY 5000 UV-Vis-NIR spectrophotomer (Varian, Walnut Creek, CA, USA) [19-23]. The oxidative stability was determined with a Rancimat apparatus (Metrohm model 679, Herisau, Switzerland). The oil samples (5 g each) were heated to 120 C under an air stream of 20 L h -1. The induction period was determined by drawing the two tangents of the time conductivity curve and projecting the intersection onto the time axis. A sensory profile for each sample was obtained by each of the eight judges; the medians were calculated and reported as radar charts. The determinations were performed at each analysis time (t0, t1, t2, respectively at time 0, 6, and 12 months). C. Analysis of the phenolic fraction The phenolic compounds were extracted and determined according to the procedure described by Boselli et al. [14]. Briefly, the methanolic extract of minor polar compounds was used for the spectrophotometric determination of total phenols with the Folin Ciocalteu reagent by using a CARY 5000 UV-Vis-NIR spectrophotomer (Varian, Walnut Creek, CA, USA) at 765 nm. The results were expressed as gallic acid equivalents (mg kg -1 oil) based on a calibration curve (r 2 = 0.999). 4

5 D. Statistical Analysis The statistical analysis of data was performed by one way ANOVA carried out with software R Project for Statistical Computing (R Foundation for Statistical Computing, Wien, Austria). To explain significant means differences, post-hoc analysis have been performed through Tukey test, suitably fitted to the repeated measure design (TukeyC R package). Significance was accepted at probability of 0.05 (P < 0.05). Finally, Principal Component Analysis (PCA) have been performed using FactoMineR R package. III. RESULT AND DISCUSSION E. Effects of the protoreactor on the Extra Virgin Olive Oil yield Oil yield were reported in Fig. 2, the lowest value was obtained from Ctrl1, in this case the protoreactor was by-passed and the malaxation was performed only for 15 minutes. On the contrary the best yields were obtained in samples E4, E5, E6, E7, E8. All these samples were carried out using protoreactor respectively at temperature T1 for E4 and T2 for the other samples. Taking into account these results, it was possible to confirm that protoreactor at higher temperature was able to influence positively the oil yield extraction. At the same time a high yield performance was reached using T1, but in this case the malaxation played a fundamental role, in fact, only coupling protoreactor T1 with 30 minutes of heated malaxation (45 C) was possible to reach a yield comparable to T2 experimental samples. Oil yield % a 12.7 b bc b bc d cd cd cd 14.6 cd 15.2 Ctrl1 Ctrl2 E1 E2 E3 E4 E5 E6 E7 E8 Figure 2. Oil yields kg/100kg. Oil percentage extracted mechanically from 8 experimental trials and two controls. Means in the oil yield histogram (experimental data) bearing different letters differ significantly (P < 0.05). F. Effects of the protoreactor on the quality of Extra Virgin Olive Oil All EVOO analysis were reported in Table II. At the production the protoreactor T1 coupled with a room temperature malaxation for 30 minutes (E2) ensured both the lowest free acidity and peroxide index. All experimental samples had higher amount of total phenols than controls, in particular protoreactor T1 coupled with malaxation heat for 0 minutes (E3) allowed to reach the best extraction. 5

6 Taking into account the closed relationship between phenols content and oxidative stability, identical situation was repeated for the oxidative stability. Likewise, protoreactor T2 coupled with heat malaxation for 0 and 30 minutes (E7, E8) reached the best performance. As regards spectrophotometric analysis the K232 and K270 extinction coefficients were investigated and their values were far below the maximus level in every sample (data not shown). The multivariate analysis was a powerful tool in order to get an overview of the experimental samples, in fact, it was possible plotting the data grouped by oil mill settings utilized. Principal component analysis was able to explain at least the 92% of variance. Taking into account the score plot, it was possible to underline the great homogeneity between the two control groups, Ctrl1 and Ctrl2, which were located in the upper left quadrant. E1 and E2 were in the lower left quadrant, while E3, E6, E7, and E8 were in the right quadrants. Finally E4 and E5 were in the middle between left and right quadrants (Fig. 3a). This overview allowed to distingue clearly three cluster, the first represented by controls groups (protoreactor by-passed), the second containing samples obtained by protoreactor T1 coupled with malaxation at room temperature (low processing temperature groups), and finally the third cluster containing samples obtained by protoreactor T1 coupled with heated malaxation and protoreactor T2 (high processing temperature group). While the score plot was able to grouped samples, using loading plot (Fig. 3b) it was possible underline how the variables investigated had influenced the final result. As such, the control groups sited in the upper left quadrant were in the opposite position of total phenols and oxidative stability. At the same way the groups E1 and E2 (characterized by lower processing temperature) were in the opposite positon as regards free acidity and peroxide index, due to their lower value. In conclusion, PCA was able to ensure an overall of the collected results but also it represented an interesting tool in order to carry on a post hoc test approach able to identify the processing temperature. 6

7 TABLE II. CHEMICAL PROFILE (MEAN, N=3) OF THE VIRGIN OLIVE OILS OF EXPERIMENT 1 AT DIFFERENT STORAGE TIME (FROM TIME 0 TO 12 MONTHS). Oxidative stability 12 months Oxidative stability 6 months Oxidative stability T0 Total phenols 12 months Total phenols 6 months Total phenols T0 Peroxide value 12 months Peroxide value 6 months Peroxide value T0 Free acidity 12 months Free acidity 6 months Free acidity T0 Malaxation time (min.) Malaxatio ID Protoreactor n Ctrl1 Room ±0.01b 0.45±0.02a 0.70±0.04ab 2.5±0.2cd 5.3±0.6b 6.5±0.5ab 259±5a 158±10a 109±6a 25±1a 16±1a 15±1a Bypassed Ctrl2 temp ±0.01b 0.46±0.03a 0.71±0.02ab 2.0±0.2b 5.7±0.6b 7.0±1.0bc 253±13a 131±9a 98±2a 25±1a 16±1a 15±0a E1 Room ±0.02a 0.48±0.01ab 0.75±0.02b 2.0±0.3b 6.3±0.6bc 8.3±0.6cd 377±15b 352±8e 302±7e 32±1c 22±1b 16±1a E2 temp ±0.01a 0.45±0.01a 0.75±0.01b 1.5±0.1a 3.7±0.3a 5.2±0.4a 454±21c 272±8c 207±7d 32±1c 23±0bc 22±1c T1 E ±0.03de 0.60±0.04cd 0.75±0.04b 3.1±0.1f 4.0±0.2a 6.2±0.6ab 592±20e 424±16f 124±10ab 36±1d 32±0f 28±1d Heat E ±0.3bc 0.54±0.04bc 0.70±0.02ab 2.2±0.2bc 3.6±0.2a 3.1±0.5a 425±25c 227±13b 149±11bc 32±1c 25±1d 23±1c E5 Room ±0.02bc 0.51±0.03ab 0.67±0.02a 3.0±0.1de 7.3±0.3c 9.3±0.6d 414±13bc 314±14d 156±12c 29±1b 23±1bc 20±1b E6 temp ±0.02e 0.65±0.02d 0.77±0.01bc 3.4±0.2ef 4.0±0.3a 6.1±0.3ab 438±13c 435±12f 230±15d 32±1c 25±2cd 23±1c T2 E ±0.04bd 0.50±0.03ab 0.86±0.03d 2.0±0.1b 4.0±0.3a 6.0±0.3ab 518±10d 309±12d 164±16c 35±1d 28±1e 22±1c Heat E ±0.03cd 0.55±0.04bc 0.84±0.02cd 3.0±0.2e 3.6±0.2a 5.0±0.5a 541±25d 548±19g 100±10a 35±1d 31±1f 22±1c 7 MAYFEB Journal of Agricultural Science Free acidity as g free oleic acid kg -1 oil; peroxide index as meq O2 kg -1 oil; Phenolic compounds are reported in mg kg -1 of oil oxidative stability (induction time). Different letters in each column for each storage time represent significant differences (p < 0.05).

8 Figure 3. Principal component analysis (PCA) of EVOO across the different system combination at the production time (time 0). (a) Score plot of the samples; (b)) loading plot of the t variables. 3a) 3b) Dim 2 (32.47%) Free.Acidity Peroxide.value e Total.phe enols Oxidative.stability Dim 1 (60.11%) G. Effects of the protoreactor on the shelf life of Extra Virgin Olive Oil Extra virgin olive oils were evaluated at time 0 (just produced), after 6 and 12 months of storage in order to investigate the evolution of oil quality during a year of storage. These data were reported in Table II. The free acidity did not show relevant differencess at 6 months storage, while it was possible underlinee that after 12 month the samples producted by protoreactor at T22 temperature, malaxation heat h for both 0 and 30 minutes (E7, E8) losted the EVOO denomination only due to values slightly exceeding the limit. 8

9 Unlike free acidity, the peroxide index in all samples from the entire investigation period was much lower than the legal limit (20 meq O 2 kg -1 oil) for EVOO [23]. Taking into account the phenols content, a relevant decrease affected the oils that were resulted the best at time zero, as E7 and E8. As such, the best resuls at 12 month storage had been showed by protoreactor working with lower temperature and room temperature malaxation for 0 minutes (E1). As regard oxidative stability the sample E3 rapresented the best system combination. Finally the K232 and K270 extinction coefficients were far below the maximus level in every samples, during each storage time (data not shown). H. Sensory profile Sensory profiles were evaluated as radar chart, particularly in Fig. 4a sensory profiles of extra virgin olive oils were evaluated at zero time, whereas in Fig. 4b the evolution of characteristic parameters during one year of storage were reported. The sensory profile of the oils collected at time 0 were reported in Fig. 4a in the form of radar chart. All the samples featured as EVOO. The EVOOs were characterized by a middle/high value of fruity, olfactory intensity and well balanced, whereas there were a wide range as regards intensity of herbaceous, pungency and bitter. Within the first six months there were not relevant differences and all the samples belonged to EVOO product category, moreover until this time each samples were characterized by a high homogeneity (results not shown). However, after twelve months of storage, the samples Ctrl1 and Ctrl2 had a defect score higher than 0, thus they lost their attributes of EVOO and classified as VOO, probably due to their low phenols content which led to a low oxidative resistance. The different defects, due to their presence only as rancidity, were unified in the chart. The minor phenolic content in the Ctrl samples led an oil deterioration after twelve months while all the preheatered sample maintained the EVOO quality. 9

10 Figure 4. a) Sensory profiles of extra virgin olive oils evaluatedd in samples from the eight experiments plus two controls at time zero. b) Sensory profiles of extra virgin olive oils evaluated in samples from the eight experiments pluss two controls after one year storage. 4a) 4b) 10

11 IV. CONCLUSION The work was aimed to collect a deeper knowledge and potentiality of a new plant called protoreactor. As such, final market edition by Gruppo Pieralisi (Jesi, Italy) at two different preheater temperatures was evaluated. Protoreactor was able to guarantee a good yield. As a matter of fact using preheater at higher temperature T2 it was even possible to reduce malaxation time till 0 minutes. Moreover it was demonstrated how protoreactor exerts an high extraction capacity as regards polyphenol compounds. It is well known that the antioxidant activity of phenol compounds, due to their high redox properties and chemical structure, are responsible for neutralizing free radicals, chelating transitional metals and quenching singlet and triplet oxygen by delocalization or decomposing peroxides [24-25]. In particular the best EVOO under this point of view was obtained using respectively protoreactor T1, malaxer heated for zero minutes or protoreactor T2, malaxer heat for thirty minutes. Due to the close relationship between polyphenol content and oxidative stability, high polyphenols contents allowed a good storage performance. Taking into account the sensorial point of view, high phenols amount involved a strong intensity of positive olfactory and taste sensations. Taking into account the EVOO shelf live, the data showed that in order to produce an high preservable olive oil it is suggested to set protoreactor at T1, in fact, higher temperature processing (E7 and E8) led a downgraded product due to exceeded free acidity limit. The system combination which promoted the best results after twelve months storage supporting higher total phenols content were obtained using preheather T1 coupled with room temperature malaxation (E1), while preheather T1 coupled with heat malaxation for 0 minutes (E3) allowed to reach a best oxidative stability. Finally, comparing two different preheating temperatures it was possible to show how protoreactor at T2 was able to promote a better quality olive oil at time zero while carry on limit value as regards free acidity after one year storage. For this reason the treatment at T1 resulted the best in order to produce a long oil shelf life. As such, it was possible to underline that protoreactor at T1 coupled to room temperature malaxer for 0 minutes led a better sensory evaluation due to the higher total phenols amount extracted after one year storage while protoreactor T2 coupled with heath malaxer led a best product at time zero as regard sensory test and phenols but these optimal parameters notably decreased during storage. As such, protoreactor can be considered an interesting tool able to reduce processing time and consequently the risk of occurrence of defects. Depending on the presumed storage period or the favorite organoleptic oil characteristic, it can be possible to choose the most appropriate protoreactor settings. Using protoreactor at two different temperature in combination with malaxer settings allows to guarantee different extra virgin olive oil, focus on yield, sensorial characteristics or oil shelf life. In conclusion, protoreactor is able to produce good quality EVOO, as you want, saving time and energy. 11

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