**3.4. Malaxation process**

The mixing and heating (25-35 °C) of the olive pastes during malaxation causes the breakdown of water-oil emulsion, allowing oil droplets to form larger droplets, which separate easily from the aqueous phase during the solid-liquid and liquid-liquid separation processes.

The operative conditions applied during malaxation of the olive pastes largely affect VOO quality (Servili et al., 1994; Montedoro et al., 2002; Servili et al., 2004a; Inarejos-Garcia et al., 2009). As in the case of the crushing process, malaxation can also produce significant modifications in the minor components of VOO with particular emphasis on volatile and phenolic compounds. The technological parameters of temperature and oxygen management show the highest impact on the volatile and phenolic composition of VOO.

The problems concerning temperature management during malaxation have been widely studied for over twenty years and a substantial negative relationship between the processing temperature and the quality of the VOO has been shown (Garcia et al., 2001; Di Giovacchino et al., 2002; Servili et al., 2003a; Servili et al., 2004a; Kalua et al., 2006). However some aspects of the relationship between the operative conditions of malaxation and oil quality must be better defined.

As previously reported, the most sensitive quality markers linked to the effect of processing temperature are the phenols and volatile compounds with their sensory impact. The literature on phenolic compounds clearly shows that the phenolic concentration of VOO could be more or less drastically reduced in relation to the increase in the mixing temperature. In particular, the derivatives of oleuropein, demethyloleuropein and ligstroside are highly affected by the processing temperature, whereas lignans are less affected (Servili et al., 2004a).

The optimal temperature of activity for PPO and POD may explain the loss of phenolic compounds in the oil depending on the processing temperature. These enzymes catalyze the oxidative degradation of phenolic compounds during the mixing process and show an optimal temperature of activity at approximately 50° C and 45° C respectively for PPO and POD.

As a result, the oxidation of phenols by PPO and POD within the range from 20° C to 35° C would be progressively higher, depending on the operating temperature. This explains the widely published data about the differences in phenolic concentration between oils obtained at different temperatures of between 20° and 40° C (Sánchez & Harwood, 2002; Angerosa et al., 2001).

These results are, in any case, obtained by performing malaxation with the pastes under continuous contact with air, as shown in the traditional mixer (Servili et al., 1998, 2003a).

158 Olive Germplasm – The Olive Cultivation, Table Olive and Olive Oil Industry in Italy

does not currently allow the addition of enzymatic preparations.

**3.4. Malaxation process** 

quality must be better defined.

affected (Servili et al., 2004a).

POD.

al., 2001).

processes.

It should be noted that the use of enzymatic preparations with depolymerizing activities, degrading the colloidal structure of fruit might partly solve the problem of low yields due to the extraction of pastes for stoning. So far, however, the European Union (EU regulation)

The mixing and heating (25-35 °C) of the olive pastes during malaxation causes the breakdown of water-oil emulsion, allowing oil droplets to form larger droplets, which separate easily from the aqueous phase during the solid-liquid and liquid-liquid separation

The operative conditions applied during malaxation of the olive pastes largely affect VOO quality (Servili et al., 1994; Montedoro et al., 2002; Servili et al., 2004a; Inarejos-Garcia et al., 2009). As in the case of the crushing process, malaxation can also produce significant modifications in the minor components of VOO with particular emphasis on volatile and phenolic compounds. The technological parameters of temperature and oxygen management show the highest impact on the volatile and phenolic composition of VOO.

The problems concerning temperature management during malaxation have been widely studied for over twenty years and a substantial negative relationship between the processing temperature and the quality of the VOO has been shown (Garcia et al., 2001; Di Giovacchino et al., 2002; Servili et al., 2003a; Servili et al., 2004a; Kalua et al., 2006). However some aspects of the relationship between the operative conditions of malaxation and oil

As previously reported, the most sensitive quality markers linked to the effect of processing temperature are the phenols and volatile compounds with their sensory impact. The literature on phenolic compounds clearly shows that the phenolic concentration of VOO could be more or less drastically reduced in relation to the increase in the mixing temperature. In particular, the derivatives of oleuropein, demethyloleuropein and ligstroside are highly affected by the processing temperature, whereas lignans are less

The optimal temperature of activity for PPO and POD may explain the loss of phenolic compounds in the oil depending on the processing temperature. These enzymes catalyze the oxidative degradation of phenolic compounds during the mixing process and show an optimal temperature of activity at approximately 50° C and 45° C respectively for PPO and

As a result, the oxidation of phenols by PPO and POD within the range from 20° C to 35° C would be progressively higher, depending on the operating temperature. This explains the widely published data about the differences in phenolic concentration between oils obtained at different temperatures of between 20° and 40° C (Sánchez & Harwood, 2002; Angerosa et However, when the process is performed in the new-generation malaxer, known as a "covered malaxer", which can control contact of the olive pastes with oxygen during mixing, the results obtained in terms of relationships between phenol concentrations in VOO and the processing temperature are completely different.

During processing, the olive pastes release CO2 and the dissolved O2 is simultaneously consumed by the oxidoreductase activities. As a result, the reduction of the O2 content obtained in the covered malaxer inhibits the PPO and POD activities, improving the concentration of hydrophilic phenols in the olive pastes and in the corresponding VOO (Figure 3) (Servili et al., 2008).

**Figure 3.** Phenolic composition (mg/Kg) of VOOs obtained after malaxation in different initial atmospheric compositions (Servili et al., 2008). 0 kpa saturated with N2; 30 kpa corresponding to the air composition. The vertical lines are the mean

values of three independent experiments, standard deviation is reported in brackets.

As a result, the oxidative reactions occurring in the pastes during malaxation can explain the relationships between VOO phenolic concentration and malaxation temperatures (Servili et al., 2004a; 2009a; 2009b). The O2 dissolved in the pastes during malaxation, activate POD and PPO, which oxidize phenolic compounds according to the temperature and consequently reduce their concentration in VOOs obtained by pastes malaxed at high temperatures. The traditional malaxer, which contains a high amount of O2 dissolved in the paste during the process due to contact with the air, represents a classical example of the aforementioned relationship between high temperatures and VOO phenolic loss. Low

amounts of O2, on the contrary, inhibit the oxidative reactions of phenols during malaxation and, in this case, their concentration in the VOO increases according to the temperatures, because of a major release of phenols into the oil (Servili et al., 2008; 2009a; 2009b).

Technological Aspects of Olive Oil Production 161

From the aforementioned considerations, it is, therefore, clear that the mixing temperature control at levels lower than the 28-30 °C represents an advisable stage in the extraction process in order to get high quality VOO (Angerosa et al., 2001; Angerosa et al., 2004; Servili et al., 2009a). We must also take into account that even temperatures of paste below 22 °C in the new generation of confined malaxer lead to a decrease in the solubilisation of phenolic

Considering the relationships between the volatile composition and O2 concentration of VOO during malaxation, the results reported in literature indicate that the O2 concentration in the pastes seems to have no effect on LPO activity during the malaxation (Table 2) (Servili

2-Pentenal (*E*) *z* 291.5 (31.8)ab 343.0 (31.1)a 247.5 (11.7)b 269.5 (13.5)b Hexanal 939.5 (9.2)a 1546.0 (200.8)b 1011.5 (27.6)a 1499.5 (16.3)b 2-Hexenal (*E*) 43645.0 (912.2)a 39130.0 (1054.7)b 37315.0 (233.3)b 38170.0 (1258.7)b

1-Pentanol 28.5 (2.1)a 128.0 (6.8)b 122.5 (3.5)b 158.0 (1.4)c 2-Penten-1-ol (*E*) 55.5 (3.5)a 63.0 (4.6)a 50.5 (9.2)ab 38.5 (6.4)b 1-Penten-3-ol 567.0 (17)a 871.0 (4.7)b 690.0 (1.4)c 809.5 (3.5)d 1-Hexanol 8357.0 (102.6)a 9699.0 (106.1)b 11660.0 (99)c 13675.0 (63.6)d 3-Hexen-1-ol (*E*) 35.0 (1.2)a 41.0 (3.5)a 47.5 (2.1)b 61.5 (2.1)c 3-Hexen-1-ol (*Z*) 286.5 (4.9)a 434.0 (20.6)b 341.0 (11.3)c 400.5 (7.8)d 2-Hexen-1-ol (*E*) 7662.5 (75.7)a 8616.0 (87.9)b 9355.0 (353.6)c 9780.0 (60.8)c

2-Pentenal (*E*) 548.5 (16.3)ab 509.7 (5.8)b 636.7 (17.9)c 613.0 (51.2)ac Hexanal 1187.0 (9.9)a 1624.3 (30)bc 1532.1 (27.3)b 1744.0 (121.2)c 2-Hexenal (*E*) 51565.0 (827.3)a 52900.0 (565.7)ab 54340.5 (355.7)b 53920.0 (332.1)b

1-Pentanol 40.0 (5.7)a 54.3 (5)b 39.4 (5)a 48.0 (3.2)ab 2-Penten-1-ol (*E*) 87.5 (0.7)a 67.0 (0.2)b 105.8 (5.7)c 105.0 (8.3)c 1-Penten-3-ol 890.0 (2.8)a 820.0 (1.2)b 1093.5 (33.7)c 1185.0 (91.2)c 1-Hexanol 2326.0 (49.5)a 3694.2 (2)b 1788.0 (57.2)c 2170.0 (123.1)a 3-Hexen-1-ol (*E*) 25.5 (0.7)ab 31.6 (3.8)a 20.0 (1.9)b 21.0 (1.9)b 3-Hexen-1-ol (*Z*) 561.0 (4.2)a 513.6 (9.6)b 486.3 (11.1)b 498.0 (31.2)b 2-Hexen-1-ol (*E*) 3654.5 (30.4)a 5905.0 (321)b 3350.1 (80.5)a 4185.0 (35.6)c

**Table 2.** Volatile composition (μg/kg) of EVOOs obtained after malaxation in different initial

a saturated with N2; b corresponding to the air composition. z Data are the mean values of three independent experiments, standard deviation is reported in brackets. Values in each row with different

0*<sup>a</sup>* 30*<sup>b</sup>*

Initial O2 partial pressure in the malaxer chamber headspace (kPa)

OGLIAROLA cv.

CORATINA cv.

50 100

compounds and chlorophylls.

et al., 2008).

ALDEHYDES

ALCOHOLS

ALDEHYDES

ALCOHOLS

atmospheric compositions (Servili et al. 2008).

letters (a-d) are significantly different from one another at p < 0.01.

Thus, O2 control during malaxation can be considered a new technological parameter which, in combination with the traditional ones (time and temperature of the process), can be used to optimize the VOO phenolic and volatile concentrations (Servili et al., 2004a, 2008, 2009a). In this regard, the time of exposure of the olive pastes to air contact (TEOPAC) was studied as a process parameter to regulate the O2 availability in the paste and, as a result, the amount of phenols in the VOO (Servili et al., 2003a, 2003b).

Furthermore, the natural increase of an inert gas of this respiration catabolite, such as CO2, released during malaxation after the destruction of the olive cell, may be combined with the use of nitrogen or argon to reduce the O2 contact with the olive pastes during malaxation (Parenti et al. 2006a, 2006b; Servili et al., 2008).

The latter have to be carefully controlled according to the time of malaxation. Moreover, special attention must be paid to the traditional malaxers which work continuously in an air saturated atmosphere. In fact, in this case, the longer the time at the same temperature conditions, the greater the loss of phenolic compounds in the oil (Servili et al., 1994; Di Giovacchino et al., 2002; Servili et al., 2004a). On the other hand, there is not a direct link in the confined malaxer between the time of malaxation and the loss of phenolic compounds. However, malaxation times of over 35-40 minutes do not involve an extraction yield increase and, therefore, even though a loss in the quality of the oil is not observed, longer periods of malaxation are negative for a correct plant management.

Time and temperature of malaxation also affect the volatile profile and, therefore, the sensory characteristics of the resulting EVOOs (Angerosa et al. 2004; Servili et al. 2009a, 2009b). A large part of the volatile compounds, which explain the flavour of VOO, is due to the activity of the enzymes involved in the lipoxygenase pathway (Figure 1).

This group of enzymes promotes the formation of aldehydes, alcohols at C5 and C6 saturated and unsaturated and esters. The main effect of malaxation time is the increase of C6 and C5 carbonyl compounds, especially of trans-2-hexenal, which represent an important contribution to the flavour of olive oil due to their low odour threshold, whereas high temperatures of malaxation promote a fall of esters and cis-3-hexen-1-ol and an accumulation of hexan-1-ol and trans-2-hexen-1-ol, both considered by some authors as producing a not entirely agreeable odour (Angerosa et al., 2004; Servili et al., 2009a).

The enzymes involved in the LOX pathway such as lipoxygenase, hydroperoxidelyase, alcoholdehydrogenase and alcohol acyltransferase show an optimal temperature between 15 and 25 °C, whereas their activity decreases after 30 °C. Therefore, the malaxing process carried out at temperatures of over 35 °C can produce a reduction in the volatile compounds generation during malaxation.

From the aforementioned considerations, it is, therefore, clear that the mixing temperature control at levels lower than the 28-30 °C represents an advisable stage in the extraction process in order to get high quality VOO (Angerosa et al., 2001; Angerosa et al., 2004; Servili et al., 2009a). We must also take into account that even temperatures of paste below 22 °C in the new generation of confined malaxer lead to a decrease in the solubilisation of phenolic compounds and chlorophylls.

160 Olive Germplasm – The Olive Cultivation, Table Olive and Olive Oil Industry in Italy

amount of phenols in the VOO (Servili et al., 2003a, 2003b).

periods of malaxation are negative for a correct plant management.

the activity of the enzymes involved in the lipoxygenase pathway (Figure 1).

(Parenti et al. 2006a, 2006b; Servili et al., 2008).

2009a).

generation during malaxation.

amounts of O2, on the contrary, inhibit the oxidative reactions of phenols during malaxation and, in this case, their concentration in the VOO increases according to the temperatures,

Thus, O2 control during malaxation can be considered a new technological parameter which, in combination with the traditional ones (time and temperature of the process), can be used to optimize the VOO phenolic and volatile concentrations (Servili et al., 2004a, 2008, 2009a). In this regard, the time of exposure of the olive pastes to air contact (TEOPAC) was studied as a process parameter to regulate the O2 availability in the paste and, as a result, the

Furthermore, the natural increase of an inert gas of this respiration catabolite, such as CO2, released during malaxation after the destruction of the olive cell, may be combined with the use of nitrogen or argon to reduce the O2 contact with the olive pastes during malaxation

The latter have to be carefully controlled according to the time of malaxation. Moreover, special attention must be paid to the traditional malaxers which work continuously in an air saturated atmosphere. In fact, in this case, the longer the time at the same temperature conditions, the greater the loss of phenolic compounds in the oil (Servili et al., 1994; Di Giovacchino et al., 2002; Servili et al., 2004a). On the other hand, there is not a direct link in the confined malaxer between the time of malaxation and the loss of phenolic compounds. However, malaxation times of over 35-40 minutes do not involve an extraction yield increase and, therefore, even though a loss in the quality of the oil is not observed, longer

Time and temperature of malaxation also affect the volatile profile and, therefore, the sensory characteristics of the resulting EVOOs (Angerosa et al. 2004; Servili et al. 2009a, 2009b). A large part of the volatile compounds, which explain the flavour of VOO, is due to

This group of enzymes promotes the formation of aldehydes, alcohols at C5 and C6 saturated and unsaturated and esters. The main effect of malaxation time is the increase of C6 and C5 carbonyl compounds, especially of trans-2-hexenal, which represent an important contribution to the flavour of olive oil due to their low odour threshold, whereas high temperatures of malaxation promote a fall of esters and cis-3-hexen-1-ol and an accumulation of hexan-1-ol and trans-2-hexen-1-ol, both considered by some authors as producing a not entirely agreeable odour (Angerosa et al., 2004; Servili et al.,

The enzymes involved in the LOX pathway such as lipoxygenase, hydroperoxidelyase, alcoholdehydrogenase and alcohol acyltransferase show an optimal temperature between 15 and 25 °C, whereas their activity decreases after 30 °C. Therefore, the malaxing process carried out at temperatures of over 35 °C can produce a reduction in the volatile compounds

because of a major release of phenols into the oil (Servili et al., 2008; 2009a; 2009b).

Considering the relationships between the volatile composition and O2 concentration of VOO during malaxation, the results reported in literature indicate that the O2 concentration in the pastes seems to have no effect on LPO activity during the malaxation (Table 2) (Servili et al., 2008).


**Table 2.** Volatile composition (μg/kg) of EVOOs obtained after malaxation in different initial atmospheric compositions (Servili et al. 2008).

a saturated with N2; b corresponding to the air composition. z Data are the mean values of three independent experiments, standard deviation is reported in brackets. Values in each row with different letters (a-d) are significantly different from one another at p < 0.01.

In conclusion, it can be considered that, as regards the process variables adopted during malaxation, the temperature should be set within the range of 24-27 °C, both for the traditional and for the new malaxer (i.e., the confined malaxer), while process times greater than 35-40 minutes may result in a large loss in terms of product quality (in particular in the traditional malaxer) without producing significant positive effects on the oil extraction yield. Technological Aspects of Olive Oil Production 163

malaxed paste + enzyme preparations

439 ± 16d

mature olives, on which the aforementioned enzymes should act, have already been degraded by the endogenous enzymatic activity during the ripening of the fruit (Heredia et al., 1993). Therefore, the addition of enzymatic preparations would be useless in this case.

However, the EU regulations 2568/1991 and 1989/2003 do not allow the addition of enzymatic preparations, whereas they authorise the use of micronized talc as co-adjuvant. The use of talc as a co-adjuvant has been proved important to increase oil extraction yield without any interference to the quality of VOO. The amount of talc used ranges from 0.7 to 1.5% of the weight of the olives being milled after the first 10 minutes of the process. In fact, its addition to difficult pastes improves the paste structure and reduces emulsions. This product acts on the olive pastes by increasing the drainage effect and, therefore, improving the efficiency of solid-liquid separation during centrifugation of the crushed pastes (Servili et al., 2004b). The micronized talc can be added to the pastes during malaxation (0.7-1.5% of the paste weight) after the first 10 minutes of the trial. Several studies carried out on Italian cultivars show that the use of the micronized talc does not involve any negative effects on oil quality and, in some cases, its use leads to a meaningful increase in the extraction yield

> malaxed paste

2.7 ± 0.3a 0.7 ± 0.1b 1.09 ± 0.1c

*p*-HPEA 2.3 ± 0.4a 1.2 ± 0.1b 1.02 ± 0.1b

*p*-HPEA-EDA 24.8 ± 1.9a 25.8 ± 1.4ab 29.4 ± 0.8c Lignans 32.5 ± 1.4a 24.2 ± 0.8b 28.5 ± 0.9c 3,4-DHPEA-EA 357.0 ± 13a 177.0 ± 8.0b 218 ± 8.0c **Table 3.** Phenolic composition of virgin olive oil (mg/kg) with and without enzymatic treatment during

a The phenolic content is the mean value of three independent experiments (standard deviation). Values in each row bearing the same superscripts are not significantly different from one another (P < 0.05).

Different extraction technologies, such as pressure and centrifugation and selective filtration (i.e. "surface tension" or "percolation") enabling the separation of oily must from the olive

Pressing is one of the oldest methods of oil extraction and has evolved considerably over the centuries. In olive oil mills equipped with this system the press separation of the oil from the paste is currently carried out using open hydraulic presses, whereas close cage presses have almost disappeared not only due to high purchase prices, but also to their maintenance costs. The previously malaxed paste is subsequently stratified on stacked filter mats, each

crushed paste

3,4-DHPEA-EDA 515.0 ± 23ab 317.0 ± 16c

paste can be used (Boskou, 1996; Di Giovacchino et al., 1994, 1995).

(Servili et al., 2004b).

malaxation (Vierhuis et al., 2001).

**3.5. Olive oil extraction systems** 

*3.5.1. Pressure extraction system* 

3,4-DHPEA*<sup>a</sup>*

Special attention must be paid to the control and regulation of oxygen percentage in contact with the olive paste during malaxation to optimize the phenolic content as well as the flavour of VOO. This new operating parameter can be used to act on the phenolic concentration of the pastes and, therefore, of the oils, excluding negative collateral effects on the volatile compound content of the product (Servili et al., 2003a; Migliorini et al., 2006; Servili et al., 2008). In fact, the traditional Italian *cvs* differ with respect to their content of phenolic substances and, because of this difference, the phenolic concentration in oil must be optimized; this optimization can be obtained by regulating their oxidative degradation level during malaxation. Thus, malaxation should be carried out without oxygen for the *cvs* with low phenolic concentration, whereas malaxation should be carried out with controlled supplementation of oxygen for those *cvs* characterized by higher phenolic concentrations (Figure 3, Table 2).

Moreover, it must be pointed out that no additional gases, such as nitrogen or argon, are required inside the head space in the confined malaxer to avoid the presence of oxygen. In fact, if the malaxer is filled with crushed paste during the process, the olive tissues of pastes naturally release carbon dioxide (CO2) (Weichmann, 1987), whereas the limited amount of oxygen they adsorb during the crushing process will be consumed rapidly by endogenous enzyme activities. As a consequence, the malaxer head space will be naturally saturated by an inert gas, such as carbon dioxide (Servili et al., 2003a; Parenti et al., 2006a, 2006b; Servili et al., 2008).
