Impact of industrial hammer mill rotor speed on extraction efficiency and quality of extra virgin olive oil

Accepted Manuscript Impact of industrial hammer mill rotor speed on extraction efficiency and quality of extra virgin olive oil Juan J. Polari, David Garcí-Aguirre, Lucía Olmo-García, Alegría CarrascoPancorbo, Selina C. Wang PII: DOI: Reference:

S0308-8146(17)31459-0 http://dx.doi.org/10.1016/j.foodchem.2017.09.003 FOCH 21672

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

8 May 2017 6 August 2017 1 September 2017

Please cite this article as: Polari, J.J., Garcí-Aguirre, D., Olmo-García, L., Carrasco-Pancorbo, A., Wang, S.C., Impact of industrial hammer mill rotor speed on extraction efficiency and quality of extra virgin olive oil, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/j.foodchem.2017.09.003

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Impact of industrial hammer mill rotor speed on extraction efficiency and quality of extra virgin olive oil Juan J. Polari1, David Garcí-Aguirre2, Lucía Olmo-García3, Alegría Carrasco-Pancorbo3, Selina C. Wang1,4*

1

Department of Food Science and Technology, University of California Davis, One Shields Avenue, Davis, CA 95616, USA. 2

Corto Olive Co., 10201 E. Live Oak Rd., Stockton, CA 95212, USA.

3

Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Ave. Fuentenueva s/n, E-18071 Granada, Spain. 4

Olive Center, University of California Davis, One Shields Avenue, Davis, CA 95616, USA.

*Corresponding author: [email protected], FAX 530-752-7080 1

Abstract Crushing is a key step during olive oil extraction. Among commercial crushers, the hammer mill is the most widely used due to its robustness and high throughput. In the present work, the impact of hammer mill rotor speed on extraction yield and overall quality of super-high-density Arbosana olive oils were assessed in an industrial facility. Our results show that increasing the rotor speed from 2400 rpm to 3600 rpm led to a rise in oil yield of 1.2%, while conserving quality parameters. Sensory analysis showed more pungency with increased rotation speed, while others attributes were unaffected. Volatile compounds showed little variation with the differences in crusher speed; however, total phenols content, two relevant secoiridoids, and triterpenoids levels increased with rotor speed. Hammer mill rotor speed is a processing variable that can be tuned to increase the extraction efficiency and modulate the chemical composition of extra virgin olive oil.

Keywords olive oil; processing; hammer mill; rotor speed; quality; extraction efficiency; phenols; volatile 2

Introduction Extra Virgin olive oil (EVOO) is the oily phase extracted from the just harvested fruit of Olea europaea L. exclusively by mechanical means. Considered one of the staples in the Mediterranean diet, it is highly appreciated for its unique nutritional and organoleptic attributes. The sensory characteristics and nutritional properties of EVOO are attributed to its composition of phenolic (Bendini et al., 2007) and volatile compounds (Kalua et al., 2007). These compounds remain in the oil after the extraction process because of the mild conditions used during processing and the lack of ulterior chemical refining. Processing variables are critical factors affecting yield, quality, and nutritional value of EVOO (Fregapane & Salvador, 2013). Modern continuous process includes crushing of the olive fruit to break the fruit’s tissues and release the oil droplets; kneading of the resulting paste to improve phase separation; and centrifugation to separate the oil from the rest of the plant constituents. Among these operations, crushing variables have been studied insufficiently compared to those of malaxation and centrifugation. The crushing step is a simple physical process used to break the fruit’s tissues and release the oil contained in the vegetal cell vacuoles. During crushing, the enzymatic reactions affecting the volatile profile and the phenolic compounds content in the final product are triggered. In addition, the physical properties of the paste going to the malaxation step are established. Therefore, this operation plays an important role in determining both yield and quality of the virgin olive oil produced (Clodoveo, Hbaieb, Kotti, Mugnozza, & Gargouri, 2014). Many types of olive crushers are currently available to processors. Studies comparing stone mills with hammer crushers (Veillet, Tomao, Bornard, Ruiz, & Chemat, 2009), hammer crushers with

3

disk crushers (Caponio, Gomes, Summo, & Pasqualone, 2003), and hammer crushers with blade crushers (Servili, Piacquadio, De Stefano, Taticchi, & Sciancalepore, 2002) show the impact of these technologies on yield, chemical composition and sensory profile. While all of these options are accessible, the hammer crusher is currently the most widely used in modern continuous facilities due to its robustness and high product throughput. The effect of hammer crusher rotor speed and screen size on olive oil chemical composition has only been studied at laboratory scale. Laboratory trials have shown a relevant impact of these variables on both phenolic and volatile compounds (Inarejos-García, Fregapane, & Salvador, 2011). Nevertheless, these results have not been validated in industrial continuous facilities. For the last decade, Arbequina, Arbosana and Koroneiki varieties have been planted in superhigh-density orchards across California, US.

As a consequence of the new agronomical

practices, olive oil production in the US has been on a rapid incline, increasing from 1000 tons in 2006 to 14,000 tons in 2016 (International Olive Council, 2016). The aim of this work is to study the effect of hammer mill rotor speed on extraction efficiency, chemical composition and sensory attributes of extra virgin olive oil in an industrial mill. Arbosana cultivar grown in a super-high-density orchard has been used as a study case as it is one of the most common cultivars in the US and has not received much attention in literature. Materials and Methods Olive samples. Three batches of about 25000 kg of olives (Olea europaea L.) from super-highdensity Arbosana cultivar were used for this trial. The fruit was mechanically harvested the last week of November 2016 from the same block of trees. The fruit was immediately transported after harvest to the processing plant located in Lodi, California, US, and milled within 14 h from

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the beginning of harvest. Maturity index, moisture content and fat content were measured and considered as homogeneity indicators of the fruit (Table I). Olive oil extraction. The olives were milled with a hammer‐crusher (Manzano MT-50) operating at 2400 rpm, 3000 rpm and 3600 rpm, using a screen size of 6 mm. After crushing, the paste was transferred to the malaxer (EXNI Termobatidora WS-8000). After malaxation (60 min at 27°C), the paste was pumped into a horizontal centrifuge (GEA RCD 535) with a processing capacity of 6000 kg/h. Finally, the oil was cleaned with a vertical centrifuge (GEA OSD 50), operating at 6700 rpm. Experiments were performed in duplicate and run in random order. Temperature of the olive paste right after crushing remained in the range of 22 ºC ± 1 ºC for all the trials. Moisture content. Olive paste (60 ± 0.1 g) or olive pomace (100 ± 0.1 g) were weighed in a 600 ml beaker and placed in the oven at 105 ºC for 12 h or until constant weight. The beaker was transferred to a desiccator and the weight of the dry paste registered after 2 h. Moisture content as well as the other determinations which are going to be described were carried out in duplicate. Fat content. Previously dried sample (paste or pomace) from moisture analysis (20 ± 0.1 g) was weighed in a cellulose extraction thimble, placed in the soxhlet extractor, and extracted using nhexane for 6 h. Once the extraction finished, solvent was distilled in a rotary evaporator and residual solvent was eliminated from the oil by placing it in an oven at 105 ºC for 3 h. Fat content was expressed as wet basis and calculated according to: /   =

   1 − 100

Where  is the moisture content of paste/pomace and   the fat content of paste/pomace expressed in dry basis. 5

Efficiency. In order to calculate the extraction efficiency, samples of olive paste after crushing and pomace from the decanter were pulled at three different time points during each experiment. Each pulled sample was prepared and the moisture and fat content was determined according to the previously described methodologies. Efficiency was calculated as follows:

 !%# =

$ −   # % × 100 

where  and    are the fat content of the paste and the pomace, respectively. Quality parameters. Free fatty acids (FFA), peroxide value (PV), and UV absorbances (K232, K270) were determined according to AOCS standard methods Ca 5a-40 (09), Cd 8b-90(09) and Ch 5-91(09) (American Oil Chemist’s Society, 1998), respectively. Diacylglycerols (DAGs). The International Organization for Standardization (ISO) standard method (ISO 29822:2012) (with slight modifications) was adopted for DAGs analysis. Sample (0.1 ± 0.01 g) was dissolved in 1 mL of toluene and loaded onto a 1,000 mg/6 mL solid phase extraction (SPE) silica cartridge (Phenomenex, Torrance, CA, USA) which was previously conditioned with 4 mL of isooctane/diisopropyl ether (85:15, v/v). Two 4 mL portions of isooctane/diisopropyl ether (85:15, v/v) were added to wash off the relative hydrophobic compounds including triacylglycerols (TAGs). Two 4 mL portions of ethyl ether were then used to collect the polar fractions, including DAGs. Afterwards, the extract was evaporated to dryness and the silylation reagent (100 µL, 1-methtyl imidazole:MSHFBA, 1:20, v/v) was added to the

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reaction vial. After being sealed at room temperature for 30 min, the derivatized extract was dissolved in 900 µL of acetone solution and subsequently analyzed by GC-FID. GC analysis was conducted on a Varian 450-GC (Agilent Technologies, Santa Clara, CA, USA) equipped with a FID. Helium was used as carrier gas at a flow rate of 1.0 mL/min. DAG isomers were separated on a 30 m x 0.25 mm x 0.1 µm DB-5HT capillary column (Agilent Technologies, Santa Clara, CA, USA) with the injector held at 300 °C at a split ratio of 1:20. The GC oven program was initially held isothermally at 200 °C for 2 min, and then ramped at 15 °C/min to 330 °C and held for 8 min. The injection volume was 1 µL. Quantification was achieved by adding up the peak areas of 1,2-DAGs divided by the peak areas of both 1,2- and 1,3-DAGs (DAGs (%)). Pyropheophytins (PPP). ISO 29841:2012 standard method was adopted for PPP analysis. Three 1 mL portions of petroleum ether were used to extract about 300 mg of oil sample on a 1000 mg/6 mL SPE silica cartridge (Phenomenex, Torrance, CA, USA). The sample was then washed twice using 5 mL of petroleum ether/diethyl ether (90:10, v/v). The pheophytin fraction was later eluted using 5 mL of acetone, evaporated to dryness and reconstituted in 1 mL of acetone. An Agilent 1290 Infinity UHPLC separation system equipped with a C18 column (3.5 µm, 10 cm x 3.0 mm) was used to perform the analysis. PPP were eluted in isocratic mode using as mobile phase a mixture composed by nanopure water/methanol/acetone (4:36:60, v/v/v) at a flow rate of 0.5 mL/min. The DAD was set at 410 nm and each run lasted 15 min. Chlorophylls. Chlorophylls were determined measuring the absorbance at 670 nm, correcting the results for background absorption at 630 and 710 nm, according to AOCS method Cc 13i -96 (09) (American Oil Chemist’s Society, 1998).

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Sensory profile. A trained panel accredited by the American Oil Chemists’ Society performed sensory descriptive analysis. Samples were analyzed to determine the positive attributes (fruitiness, bitterness and pungency) and presence of defects (fusty, winey, musty, muddy, rancid, metallic and others), as well as ripe and green fruit character, according to COI/T.20/Doc. No 15 Rev February 2013. Volatile profile. Sample (1.0 ± 0.1 g), spiked with 4-methyl-2-pentanol as internal standard (2.5 mg/kg), was weighed into a 20 mL glass vial (Agilent Technologies, Santa Clara, CA) and sealed with a PTFE/silicon septum (Supelco, Bellefonte, PA). After 10 min at 40 °C, a solid-phase micro extraction (SPME) fiber (DVB/CAR/PDMS, Sigma-Aldrich, St. Louis, MO) was exposed to the sample headspace for 40 min for volatile extraction. The volatile compounds analysis was performed on a Varian 450-GC equipped with a Varian 220-MS ion trap (Agilent Technologies, Santa Clara, CA). A Supelcowax 10 (30 m x 0.25 mm x 0.25 µm, Sigma-Aldrich, St. Louis) was used for compounds separation. After sampling, the fiber was thermally desorbed in the GC injector for 5 min at 260 °C. Helium was used as carrier gas at a flow rate of 1 mL/min. GC oven temperature started at 40 °C and ramped at 3 °C/min after 10 min to the final temperature of 200 °C. Ionization energy of 70 eV was adopted and the ions were analyzed in the m/z range from 40 to 400. The data were recorded and analyzed using MS Workstation v6.9.3. Volatile compounds were identified by their mass spectra and using Kovatz retention index (KI). Total phenols determination. The extraction of phenolic compounds was performed according to Bajoub et al. (Bajoub et al., 2015) Briefly, sample (2.0 ± 0.1 g) was dissolved in 1 mL of hexane and extracted 3 times with 2 ml of methanol/water (60:40, v/v). After centrifugation (5000 rpm, 6 min) all the supernatants were collected together. Total phenols determination was performed 8

using Folin- Ciocalteu colorimetric method. A 0.2 mL aliquot of the phenolic extract was diluted with distilled water up to 5 mL and mixed with 0.5 mL of Folin-Ciocalteu reagent and 1 mL of sodium carbonate (35%, w/v). After bringing it to a final volume of 10 mL, the mixture was stored in the dark. After 2 hours, absorbance at 725 nm was measured and the concentration of phenolic compounds was calculated using an external calibration curve prepared with caffeic acid. Phenolic compounds profile. Extraction of phenolic compounds was performed as in the case of total phenols determination with a slight modification. Combined extracts were evaporated to dryness in a rotavap at 35 °C and reduced pressure, re-dissolved in 0.5 mL of MeOH and filtered through a syringe filter of 0.45 µm. The trimethylsilylation reaction was performed at room temperature by adding 50 µL of BSTFA+TMCS, 99:1 to the dry residue of 200 µL of the extract (after solvent evaporation with a N2 stream). An equilibration time of 30 min was required before the sample injection. The GC-MS analyses were performed on a Varian 450-GC coupled to a Varian 220-MS ion trap (Agilent Technologies, Santa Clara, CA), equipped with an HP-5-MS (30 m x 0.25 mm x 0.25 µm, Agilent Technologies, Santa Clara, CA, USA). (The same platform was used for triterpenic compounds determination). The GC-MS conditions were similar to those described by Bajoub et al. (Bajoub et al., 2016). Oven temperature was initially kept at 160 °C for 5 min, then it was increased from 160 °C to 188 °C at 3 °C/min (held for 1 min), from 188 °C to 241 °C at 15 °C/min (held for 1 min), from 241 °C to 282 °C at 2 °C/min, from 282 °C to 310 °C at 5 °C/min, keeping that value for 5 min. Identification of phenolic compounds was carried out by using commercially available standards, retention time and mass spectra of each peak together with our previous knowledge and published reports (García-Villalba et al., 2011; Ríos, Gil, & Gutiérrez-

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Rosales, 2005). Quantification was performed using an external calibration curve for each individual compound, except for oleuropein derivatives and hydroxytyrosol acetate, which were quantified in terms of hydroxytyrosol and ligstroside derivatives by using a calibration curve from tyrosol. Triterpenic compounds. Triterpenic compounds were isolated by ultrasound-assisted extraction according to a previously described procedure. Briefly, sample (0.20±0.01g) was weighed in a conical centrifuge tube and extracted two times with MeOH (5 mL). The tube was agitated in a vortex for 1 min, put in an ultrasonic bath for 30 min and centrifuged at 5000 rpm for 6 min. The combined supernatants were evaporated to dryness and redissolved in 500 µL of acetonitrile. After a further cleaning step with 1 mL of hexane, 200 µL of the filtered extract were dried under N2 flow, treated with 50 µL of the silylating reagent (BSTFA+TMCS, 99:1) and left at room temperature for 30 min before injection. For GC-MS analyses, 1 µL of the derivatized extract was injected in splitless inlet mode, with He as carrier gas (1 mL/min). Oven temperature started at 200 °C, this value was kept for 2 min and ramped at 14 °C/min to the final temperature of 300 °C, holding this temperature for 15.5 min. Injector, transfer line and source temperatures were 250 °C, 300 ºC and 210 ºC, respectively. Electron impact (EI) spectra were acquired at 70 eV (mass range from 50 to 600 m/z) with a solvent delay of 5 min at the beginning of each run. Data were recorded and analyzed using MS Workstation v6.9.3. Quantification was performed using an external calibration curve for each individual compound. Statistical analysis. ANOVA was performed for each parameter considering rotor speed as main factor. Tukey test was used to assess differences within each response factor. Significance level was set as 0.05. Minitab v16.2.4 was used for all calculations. Results and discussion 10

Efficiency Extraction efficiency increased with rotor speed in the range studied (Fig. 1). While the increment was not statistically significant between 2400 rpm and 3000 rpm, and 3000 rpm and 3600 rpm, increasing the rotor speed in the hammer crusher from 2400 rpm to 3600 rpm produced a significant increment in the oil extraction efficiency of about 1.2 % (Table II). The same trend was observed using a laboratory scale extraction system with Arbequina and Cornicabra cultivars (Inarejos-García et al., 2011). Increasing the speed could produce a more efficient breakage of the cellular walls and the vacuoles where the oil is contained, releasing more oil from the vegetable matrix and therefore improving the yield. Quality parameters Quality parameters of the oil remained unaffected by the changes in rotor speed (Table II). Analogous observations were made by other authors in similar studies, where different crushing techniques (type and conditions) were employed (Di Giovacchino, Solinas, & Miccoli, 1994; Guerrini, Migliorini, Giusti, & Parenti, 2016; Inarejos-García et al., 2011; Servili et al., 2002). All the obtained values were within the limits for extra virgin olive oil according to USDA (USDA, 2010) and IOC (International Olive Council, 2016) standards. Chlorophylls are responsible for the green color of olive drupes and are principally located in the skin of the fruit, where the higher photosynthetic activity is observed (Roca & MínguezMosquera, 2000). Due to their liposolubility, chlorophylls migrate to the oil phase during the extraction process (Roca & Mínguez-Mosquera, 2001). The final concentration in the oil is affected by the initial concentration in the fruit, but also by the extraction variables (Giuliani, Cerretani, & Cichelli, 2011). In this case, a significant increase in chlorophyll oil content was

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observed when the rotor speed was increased. Furthermore, as in the case of extraction efficiency, chlorophyll concentration level increased with rotor speed. Increasing the speed during the use of a blade crusher has shown the same tendency as well (Guerrini et al., 2016). When the hammer mill and stone mills were compared (Di Giovacchino et al., 1994), oils obtained using a hammer crusher resulted in higher levels of chlorophylls. This effect was attributed to a more efficient breakage of the skin and cell walls during the use of the hammer crusher. Consistently, a higher speed in the crusher might produce a better breakage of the olive fruit cells and internal bodies (in this case, chloroplasts), releasing more chlorophylls into the oil. Sensory No sensory defects were detected by the panel members in any of the samples (Table III) and the oils obtained from all treatments were considered as extra virgin according to USDA (USDA, 2010) and IOC (International Olive Council, 2016) standards. No differences were observed regarding fruitiness, bitterness, ripe fruit and green fruit. Conversely, pungency increased with faster rotor speed, raising its value from 2.4 to 3.1. This observation is in accordance with the trend observed for the concentration of p-HPEA-EDA, which is considered to be one of the main compounds responsible for this sensory attribute (Cicerale, Breslin, Beauchamp, & Keast, 2009). Volatiles Volatile compounds are responsible of the fruity and green aroma of fresh olive oil (Aparicio & Morales, 1998). These compounds are synthetized during processing from free polyunsaturated fatty acids, through an enzymatic pathway known as lipoxygenase (LOX) pathway. The two main enzymes involved in the LOX pathway are lipoxygenase (LOX) and hydroperoxide lyase (HPL). LOX catalyzes the oxygenation of polyunsaturated fatty acids (linoleic and linolenic) to

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produce their corresponding hydroperoxides. HPL catalyzes the cleavage of fatty acid hydroperoxides at the bond situated between the carbon atom carrying the hydroperoxide group and the adjacent (E)-double bond, yielding C5 and C6 aldehydes, the main compounds identified in olive oil (Clodoveo et al., 2014). Table IV includes the C6 and C5 LOX pathway volatile compounds and total volatile content (expressed in mg/kg), including information about the different attributes generally linked with each compound. C6 volatiles were the main analytes found in the oils resulting from all treatments, representing over 90% of overall volatile fraction, (E)-2-hexenal being the one with the highest concentration, ranging from 143.6 to 150.0 mg/kg. Consistent with the results reported for a blade crusher, synthesis of volatile compounds was minimally affected by the changes in rotor speed (Guerrini et al., 2016). In addition, laboratory scale trials have shown little impact of hammer rotor speed on volatile profile, whilst different screen sizes presented a more remarkable effect on C6 volatiles (Inarejos-García et al., 2011). Total phenols and phenolic compounds profiling Oleuropein, demethyloleuropein and ligstroside are important phenolic compounds identified in olive fruit (Servili et al., 2004). These substances are hydrolyzed after crushing by the enzyme βglucosidase, leading to the formation of aglycones, which exhibit a higher lipophilicity and constitute the most abundant phenolic compounds in virgin olive oil (Romero-Segura, GarcíaRodríguez, Sánchez-Ortiz, Sanz, & Pérez, 2012). Besides β-glucosidase, polyphenol oxidase (PPO) and peroxidase (POD) oxidize the phenolic compounds during crushing and malaxation, contributing to shape the phenolic profile in the oil (García-Rodríguez, Romero-Segura, Sanz, & Pérez, 2015). As a consequence of the activity of these enzymes, and the higher water solubility

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of phenolic compounds, less than 5% of the initial phenols of the olive fruit are preserved in the oil (Jerman Klen, Golc Wondra, Vrhovšek, Sivilotti, & Vodopivec, 2015). Similar to the results observed for efficiency and chlorophylls concentration, total phenols content increased with crushing speed (Table II, Fig. 1). The total content varied from 228 to 270 mg/kg when the rotor speed changed from 2400 to 3600 rpm. The increase of total phenols with crusher speed was also observed by Inarejos-García et al. during the extraction of Arbequina and Cornicabra cultivars at laboratory scale (Inarejos-García et al., 2011) and by Guerrini et. al during industrial trials performed with Frantoio cultivar using a blade crusher (Guerrini et al., 2016). In general, when different crushing technologies are compared, those devices that cause a more violent rupture of the fruit tissues are the ones that produce a higher concentration of phenols (Gutiérrez, Arnaud, & Albi, 2002; Veillet et al., 2009). Increasing the rotor speed of the hammer crusher escalates the cutting action on the olive fruit, potentially releasing more phenolic compounds and resulting in higher levels in the oil. In addition, a higher crushing speed may decrease the oil droplets diameter, augmenting the oil/water emulsion interphase area, facilitating the mass transfer of phenols to the lipid phase after the action of βglucosidase. Ten compounds were selected for the phenolic profiling, including the aglycones from oleuropein

(3,4-DHPEA-EA)

and

ligstroside

(p-HPEA-EA),

along

with

their

decarboxymethylated forms (3,4-DHPEA-EDA and p-HPEA-EDA, respectively), the most abundant phenolic compounds found in the oil, as shown in Table V. Simple phenols (tyrosol, hydroxytyrosol and hydroxytyrosol acetate) were not affected by changes in the hammer mill rotor speed. Tyrosol concentrations were found within the range 3.0-3.2 mg/kg; hydroxytyrosol concentrations minimally fluctuated from 2.4 to 2.8 mg/kg; and the levels of hydroxytyrosol 14

acetate were determined within the interval defined from 10.8 to 12.6 mg/kg. p-HPEA-EDA and 3,4-DHPEA-EDA, belonging to complex phenols, showed an increase when faster rotor speeds were used, from 45.2 to 54.0 mg/kg in the case of p-HPEA-EDA, and from 50.7 to 86.5 mg/kg for 3,4-DHPEA-EDA, respectively. Guerrini et al. showed a very similar behavior for these two compounds when increasing blade crusher speeds were tested (2200, 2700 and 3200 rpm). The effect of the rotor speed on 3,4-DHPEA-EDA levels was not as clear; it went up from 21.2 to 27.2 mg/kg after changing the rotor speed from 2400 to 3000 rpm and then declined when the acceleration of the rotor was set at 3600 rpm (25.4 mg/kg). p-HPEA-EA concentrations remained unaffected by rotor speed modifications. The findings published by Guerrini et al. were also comparable regarding these two compounds. Flavonoids did not experience drastic changes; apigenin remained stable regardless of rotor speed; and luteolin showed a smooth and slight rise. As found in previous reports (Inarejor-García et al., 2011; Guerrini et al., 2016), lignans showed a limited fluctuation with the rotation speed changes. Pinoresinol decreased its concentration (from 9.1 to 7.6 mg/kg) when the rotor speed increased from 2400 to 3000 rpm, however, its concentration increased from 7.6 mg/kg to 8.3 mg/kg when the speed increased from 3000 to 3600 rpm. Triterpenic compounds Some triterpenic compounds, including oleanolic acid, maslinic acid, uvaol and erythrodiol, can be found in the olive fruit epicarp. Several investigations have shown their potential as antioxidant (Assimopoulou, Kaliora, Assimopoulou. & Papageorgiou, 2002), anti-inflammatory (Marquez-Martin, Puerta, Fernandez-Arche, Ruiz-Gutierrez, & Yaqoob, 2006) and antitumoral (Juan, Planas, Ruiz-Gutierrez, Daniel, & Wenzel, 2008) agents in biological systems. A study performed at laboratory scale has shown the impact of the screen size of the hammer mill on 15

triterpenic acids and dialcohols concentration (Allouche et al., 2010). The authors considered as variable factors three level combinations of sieve size of the hammer mill and two level factorial combinations of malaxing temperature and time, however, to the best of knowledge, no references to the effect of the crusher speed on the concentration of these compounds have been reported so far. In our experiment, the concentration of the four studied triterpenoids increased with the rotor speed (Table V). The enhancement observed in their concentrations was more remarkable for triterpenic acids than for dialcohols; indeed, levels of oleanolic and maslinic acids increased about 40% and 70%, respectively, when the speed changed from 2400 to 3600 rpm. To the best of our knowledge, this is the first report on how this processing parameter affects the triterpenic compounds content in the oils. Further research will be necessary to confirm the cause of this observation.

Conclusions The effect of hammer mill rotor speed on extraction yield and overall quality of super-highdensity Arbosana olive oils was evaluated. Quality parameters such as FFA, PV, UV absorbances, DAGs and PPP were unaltered by crushing speed, but extraction efficiency and chlorophylls content increased linearly with faster hammer mill rotor speed in a continuous industrial facility. As far as descriptive sensory analysis is concerned, results showed no noticeable differences in fruitiness and bitterness, though higher pungency scores were assigned to the oils obtained using faster rotor speeds. Volatile compounds remained unaffected by the changes in rotor speed. Quantitative values of total phenols and some individual phenolic compounds, such as 3,4-DHPEA-EDA and p-HPEA-EDA, increased with rotor speed. Similarly, the level of triterpenic compounds, such as oleanolic acid and maslinic, increased significantly when higher crushing speeds were applied. Based on our findings, hammer mill rotor speed is a 16

processing variable that can easily be controlled to increase the extraction efficiency and modulate the chemical composition of the olive oil. Processors may use this information to tailor the phenolic content and pungency of olive oil by adjusting the rotor speed. Future industrialscale experiments with a wider range of speeds will be studied in order to understand the potential impact of this variable in optimizing extra virgin olive oil extraction process.

Acknowledgements The authors would like to thank Xueqi Li for the analysis on DAGs and PPP and Ava Moin for the assistance with moisture and fat content measurements. We also thank Sue Langstaff for the sensory evaluation of the oil. Juan Polari is deeply grateful to Fulbright Foundation for supporting his graduate studies at UC Davis. We also want to express our gratitude to the University of Granada for the Mobility Program for young researchers CEI BioTic 2015-2016, which made this collaboration possible and supported a pre-doctoral stay for Lucía Olmo-García at UC Davis.

References Allouche, Y., Jiménez, A., Uceda, M., Paz Aguilera, M., Gaforio, J. J., & Beltrán, G. (2010). Influence of olive paste preparation conditions on virgin olive oil triterpenic compounds at laboratory-scale. Food Chemistry, 119(2), 765–769. American Oil Chemist’s Society. (1998). Official methods and recommended practises of the American Oil Chemists’ Society (5th ed.). Champaign: American Oil Chemists’ Society. Aparicio, R., & Morales, M. T. (1998). Characterisation of olive ripeness by green aroma compounds of virgin olive oil. Journal of Agricultural and Food Chemistry, 46(3), 1116–

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1122. Andrikopoulos, N. K., A. N., Kaliora. A. C., Assimopoulou, A. N., & Papageorgiou, V.P., (2002). Inhibitory Activity of Minor Polyphenolic and Nonpolyphenolic Constituents of Olive Oil Against In Vitro Low-Density Lipoprotein Oxidation. Journal of Medicinal Food, 5(1). Bajoub, A., Hurtado-Fernández, E., Ajal, E. A., Ouazzani, N., Fernández-Gutiérrez, A., & Carrasco-Pancorbo, A. (2015). Comprehensive 3-year study of the phenolic profile of Moroccan monovarietal virgin olive oils from the meknes region. Journal of Agricultural and Food Chemistry, 63(17), 4376–4385. Bajoub, A., Pacchiarotta, T., Hurtado-Fernández, E., Olmo-García, L., García-Villalba, R., Fernández-Gutiérrez, A., … Carrasco-Pancorbo, A. (2016). Comparing two metabolic profiling approaches (liquid chromatography and gas chromatography coupled to mass spectrometry) for extra-virgin olive oil phenolic compounds analysis: A botanical classification perspective. Journal of Chromatography A, 1428, 267–279. Bendini, A., Cerretani, L., Carrasco-Pancorbo, A., Gómez-Caravaca, A. M., Segura-Carretero, A., Fernández-Gutiérrez, A., & Lercker, G. (2007). Phenolic molecules in virgin olive oils: A survey of their sensory properties, health effects, antioxidant activity and analytical methods. An overview of the last decade. Molecules, 12(8), 1679–1719. Caponio, F., Gomes, T., Summo, C., & Pasqualone, A., (2003). Influence of the type of olivecrusher used on the quality of extra virgin olive oils Research Paper. European Journal of Lipid Science and Technology, 105, 201–206. Cicerale, S., Breslin, P. A. S., Beauchamp, G. K., & Keast, R. S. J. (2009). Sensory 18

characterization of the irritant properties of oleocanthal, a natural anti-inflammatory agent in extra virgin olive oils. Chemical Senses, 34(4), 333–339. Clodoveo, M. L., Hbaieb, R. H., Kotti, F., Mugnozza, G. S., & Gargouri, M. (2014). Mechanical Strategies to Increase Nutritional and Sensory Quality of Virgin Olive Oil by Modulating the Endogenous Enzyme Activities. Comprehensive Reviews in Food Science and Food Safety, 13(2), 135–154. Di Giovacchino, L., Solinas, M., & Miccoli, M. (1994). Effect of extraction systems on the quality of virgin olive oil. Journal of the American Oil Chemists’ Society, 71(11), 1189– 1194. Fregapane, G., & Salvador, M. D. (2013). Production of superior quality extra virgin olive oil modulating the content and profile of its minor components. Food Research International, 54(2), 1907–1914. García-Rodríguez, R., Romero-Segura, C., Sanz, C., & Pérez, A. G. (2015). Modulating oxidoreductase activity modifies the phenolic content of virgin olive oil. Food Chemistry, 171, 364–369. García-Villalba, R., Pacchiarotta, T., Carrasco-Pancorbo, A., Segura-Carretero, A., FernándezGutiérrez, A., Deelder, A. M., & Mayboroda, O. A. (2011). Gas chromatographyatmospheric pressure chemical ionization-time of flight mass spectrometry for profiling of phenolic compounds in extra virgin olive oil. Journal of Chromatography A, 1218(7), 959– 971. Giuliani, A., Cerretani, L., & Cichelli, A. (2011). Chlorophylls in olive and in olive oil: chemistry and occurrences. Critical Reviews in Food Science and Nutrition, 51(7), 678– 19

690. Guerrini, L., Migliorini, M., Giusti, M., & Parenti, A. (2016). The influence of crusher speed on extra virgin olive oil characteristics. European Journal of Lipid Science and Technology, 1600156, 1–7. Gutiérrez, F., Arnaud, T., & Albi, M. a. (2002). Influence of olive processing on virgin olive oil quality. Journal of the American Oil Chemists’ Society, 104(5), 587–601. Inarejos-García, A. M., Fregapane, G., & Salvador, M. D. (2011). Effect of crushing on olive paste and virgin olive oil minor components. European Food Research and Technology, 232(3), 441–451. International Olive Council. (2015). World olive oil figures. Retrieved from http://www.internationaloliveoil.org/estaticos/view/131-world-olive-oil-figures International Olive Council. (2016). International Trade Standard Applying to Olive Oils and Olive-Residue Oils (COI/T.15/No.1), (3), 1–17. Retrieved from file:///C:/Users/Simon/Downloads/TRADE STANDARD-REV 11_ENGLISH.pdf Jerman Klen, T., Golc Wondra, A., Vrhovšek, U., Sivilotti, P., & Vodopivec, B. M. (2015). Olive Fruit Phenols Transfer, Transformation, and Partition Trail during Laboratory-Scale Olive Oil Processing. Journal of Agricultural and Food Chemistry, 63(18), 4570–4579. Juan, M. E., Planas, J. M., Ruiz-Gutierrez, V., Daniel, H., & Wenzel, U. (2008). Antiproliferative and apoptosis-inducing effects of maslinic and oleanolic acids, two pentacyclic triterpenes from olives, on HT-29 colon cancer cells. The British Journal of Nutrition, 100(1), 36–43.

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Kalua, C. M., Allen, M. S., Bedgood, D. R., Bishop, A. G., Prenzler, P. D., & Robards, K. (2007). Olive oil volatile compounds, flavour development and quality: A critical review. Food Chemistry, 100(1), 273–286. Marquez-Martin, A., Puerta, R. D. La, Fernandez-Arche, A., Ruiz-Gutierrez, V., & Yaqoob, P. (2006). Modulation of cytokine secretion by pentacyclic triterpenes from olive pomace oil in human mononuclear cells. Cytokine, 36(5–6), 211–217. Ríos, J. J., Gil, M. J., & Gutiérrez-Rosales, F. (2005). Solid-phase extraction gas chromatography-ion trap-mass spectrometry qualitative method for evaluation of phenolic compounds in virgin olive oil and structural confirmation of oleuropein and ligstroside aglycons and their oxidation products. Journal of Chromatography A, 1093(1–2), 167–176. Roca, M., & Mínguez-Mosquera, M. I. (2000). Changes in Chloroplast Pigments of Olive Varieties during Fruit Ripening, 832–839. Roca, M., & Mínguez-Mosquera, M. I. (2001). Change in the natural ratio between chlorophylls and carotenoids in olive fruit during processing for virgin olive oil. Journal of the American Oil Chemists’ Society, 78(2), 133–138. Romero-Segura, C., García-Rodríguez, R., Sánchez-Ortiz, A., Sanz, C., & Pérez, A. G. (2012). The role of olive β-glucosidase in shaping the phenolic profile of virgin olive oil. Food Research International, 45(1), 191–196. Servili, M., Piacquadio, P., De Stefano, G., Taticchi, A., & Sciancalepore, V. (2002). Influence of a new crushing technique on the composition of the volatile compounds and related sensory quality of virgin olive oil. European Journal of Lipid Science and Technology, 104(8), 483–489. 21

Servili, M., Selvaggini, R., Esposto, S., Taticchi, A., Montedoro, G., & Morozzi, G. (2004). Health and sensory properties of virgin olive oil hydrophilic phenols : agronomic and technological aspects of production that affect their occurrence in the oil, 1054, 113–127. USDA. (2010). United States Standards for Grades of Olive Oil and Olive-Pomace Oil Effective, 21. Retrieved from https://www.ams.usda.gov/sites/default/files/media/Olive_Oil_and_OlivePomace_Oil_Standard%5B1%5D.pdf Veillet, S., Tomao, V., Bornard, I., Ruiz, K., & Chemat, F. (2009). Chemical changes in virgin olive oils as a function of crushing systems: Stone mill and hammer crusher. Comptes Rendus Chimie, 12(8), 895–904.

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b

Efficiency (%)

86.7 86.4

a,b

86.1 85.8 85.5

a

85.2 84.9 84.6

290 Total phenols (mg/Kg)

87.0

c

280 270 b

260 250 240

a

230 220 210

2400 RPM

3000 RPM

3600 RPM

2400 RPM

3000 RPM

3600 RPM

Fig. 1. Extraction efficiency (%) and total phenols content (mg/kg) as a function of hammer crusher rotor speed. (a,b,c) indicates significant differences according to Tukey test.

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Table I. Homogeneity parameters for the different batches of olive fruit used for the trialsa. Analytical determination Maturity index Moisture content (g/100g) Fat Content (WB, g/100g) a

Batch #1 1.1±0.1 60.5±0.5 23.2±0.2

Batch #2 1.1±0.1 59.8±0.5 23.5±0.2

Batch #3 1.1±0.1 60.5±0.5 23.3±0.3

Values are expressed as mean±SD.

Table II. Efficiency and quality parameters for oils obtained at different rotor speeda. Analytical determination Efficiency (%) Free Fatty Acids (%) Peroxide Value (mEq O2/Kg) K232 K270 DAGs (%) PPP (%) Chlorophylls (mg/kg) Total phenols (mg/kg)

2400 rpm 85.3±0.1 (a) 0.39±0.06 4.1±0.8 1.45±0.03 0.11±0.01 91.7±1.3 ND 12.9±0.5 (a) 228±3 (a)

3000 rpm 86.0±0.2 (a,b) 0.37±0.06 3.2±0.2 1.40±0.03 0.10±0.01 93.1±1.3 ND 14.8±0.6(b) 255±1 (b)

3600 rpm 86.5±0.2 (b) 0.38±0.06 3.7±0.2 1.40±0.01 0.11±0.01 91.8±1.3 ND 17.7±0.2(c) 273±3 (c)

a

ND, not detected; values are expressed as mean±SD; (a,b,c) indicates significant differences according to Tukey test.

Table III. Sensory evaluation medium scores (1-10 scale) for oils obtained at different rotor speeda. Sensory attribute Defects Fruitiness Bitterness Pungency Ripe fruit Green fruit

2400 rpm ND 3.6±0.1 2.4±0.1 2.4±0.1 (a) 1.8±0.1 2.2±0.1

3000 rpm ND 3.6±0.1 2.5±0.1 2.6±0.1 (a) 1.8±0.1 2.2±0.1

3600 rpm ND 3.5±0.1 2.6±0.1 3.1±0.1 (b) 1.7±0.1 2.2±0.1

a

ND, not detected; values are expressed as mean±SD; (a,b) indicates significant differences according to Tukey test.

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Table IV. C6 and C5 LOX pathway volatile compounds and total volatiles content (mg/kg) in oils obtained at different rotor speedb. Volatile compound

2400 rpm

3000 rpm

3600 rpm

OT

Hexanal

0.2±0.0 (a)

0.2±0.0 (a)

0.1±0.0 (b)

0.08

(E)-2-Hexenal

143.6±9.4

142.1±8.4

150.0±10.2

1.13

(Z)-3-Hexenyl Acetate

50.1±8.5

43.9±5.1

46.0±4.1

0.75

(Z)-3-Hexen-1-ol

7.6±0.5(a)

6.6±0.9(a)

5.8±0.6(b)

6.0

Hexyl acetate

5.2±0.8(a)

4.6±0.5(a)

3.8±0.4(b)

1.0

1-Hexanol

2.1±0.3

1.9±0.3

1.9±0.2

0.4

(Z)-2-Hexen-1-ol

1.0±0.0

0.8±0.1

1.0±0.1

1.0

(Z)-2-Hexenal

0.7±0.1

0.6±0.2

0.3±0.1

NA

(Z)-3-Hexenal

0.1±0.0

ND

ND

0.003

(E)-3-Hexen-1-ol

0.1±0.0

ND

ND

NA

Sum C6

210±22 (a)

201±7.0 (a)

209±23 (a)

-

1-Penten-3-one

1.6±0.1(a)

2.0±0.2(a,b)

2.7±0.3(b)

0.05

1-Penten-3-ol

0.7±0.0

0.8±0.1

0.9±0.1

0.4

(Z)-2-Pentenol

0.5±0.0

0.4±0.1

0.4±0.1

0.25

0.3±0.0 (a) 0.4±0.0 0.2±0.1(a) 0.3±0.0 (a) 0.1±0.1 0.2±0.0 3.1±0.1 (a) 222±22

0.4±0.0(a,b) 0.4±0.0 0.5±0.1(a,b) 0.4±0.0 (a,b) ND ND 3.6±0.4 (a,b) 212±9

0.5±0.1(b) 0.7±0.1 0.7±0.1(b) 0.5±0.0(b) 0.1±0.0 0.3±0. 4.6±0.6 (b) 223±22

NA NA NA NA NA NA -

3-Pentanone 3-Ethyl-1,5-Octadiene (I) 3-Ethyl-1,5-Octadiene (II) 3,7-Decadiene (I) 3,7-Decadiene (II) 3,7-Decadiene (III) Sum C5 Total Volatiles

Attributes Green, sweet (Ramón Aparicio & Luna, 2002) Apple, green (Ramón Aparicio & Luna, 2002) Green (Ramón Aparicio & Luna, 2002) Green (Ramón Aparicio & Luna, 2002) Green, fruity, sweet (Ramón Aparicio & Luna, 2002) Fruity, banana (Ramón Aparicio & Luna, 2002) Green grass, leaves (Luna, Morales, & Aparicio, 2006) Fruity, almonds (Kiritsakis, 1998) Leaf, green (Ramón Aparicio & Luna, 2002) Green leaf, nuts (Kiritsakis, 1998) Green, pungent (Ramón Aparicio & Luna, 2002) Fruity, butter, green (Luna et al., 2006) Banana, grass, green (Luna et al., 2006) Fruity, green, sweet NA NA NA NA NA -

b

ND, not detected; NA, not available; OT, odor threshold; values are expressed as mean±SD; (a,b) indicates significant differences according to Tukey test.

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Table V. Main phenolic and triterpenic compounds concentration (mg/kg) in oils obtained at different rotor speeda. Family

Phenolic compounds

Triterpenic compounds

Compound Tyrosol p-HPEA-EDA p-HPEA-EA Hydroxytyrosol Hydroxytyrosol acetate 3,4-DHPEA-EDA 3,4-DHPEA-EA Apigenin Luteolin Pinoresinol Erythrodiol Uvaol Oleanolic Acid aslinic Acid

2400 rpm 3.1±0.5 45.2±1.1 (a) 12.1±2.3 2.4±0.3 10.8±1.8 50.7±0.8 (a) 21.2±2.6 (a) 2.9±0.1 2.8±0.1 (a) 9.1±0.3 (a) 6.4±0.7 (a) 1.5±0.1 (a) 51.3±1.0 (a) 63.5±5.4 (a)

3000 rpm 3.0±0. 51.3±1.0 (b) 13.8±0.5 2.5±0.2 12.2±0.4 62.1±1.7 (b) 27.2±0.4 (c) 3.0±0.1 (a) 3.1±0.1 (b) 7.6±0.1 (b) 7.2±0.2 (a) 1.5±0.1 (a) 56.0±0.8 (b) 76.7±6.2 (b)

3600 rpm 3.2±0.1 54.0±0.3 (c) 12.1±0.5 2.8±0.1 12.6±0.5 86.5±0.8 (c) 25.4±0.1(b) 3.1±0.2 (a) 3.5±0.5 (b) 8.3±0.5 (a) 9.1±0.6 (b) 1.9±0.1 (b) 72.6±1.2 (c) 108.4±6.0 (c)

a

Values are expressed as mean±SD; (a,b,c) indicates significant differences according to Tukey test.

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• • • • •

Effect of hammer crusher rotor speed was studied during continuous olive oil extraction. Hammer mill rotor speed increased extraction efficiency, phenolic and triterpenic compounds content in extra virgin olive oil. FFA, PV, UV absorbances, DAGs and PPP were not affected by changes in hammer mill rotor speed. Pungency increased with hammer mill rotor speed. Hammer mill rotor speed may be used to tailor minor components content during industrial olive oil extraction.

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