Palestinian ecosystem is rich in biodiversity, where olive trees are considered among the most abundant trees (Olea Europaea L.) . Olive oil is the vegetable oil obtained from olive tree fruit (Olea europea sative) by mechanical extraction .
Filtration means removal of humidity and suspended solids from VOO before bottling . Tri-glycerides hydrolyzed because of lipolytic enzymes (lipases) that cause the release of free fatty acids (FFA) and mono-glycerides or di-glycerides. These lipases are present naturally in olive, and they are hydrophilic. Thus, oil filtration is crucial to remove partial amounts of water and suspended solids and cells residues [4-7].
Fregapane et al.  and Lozano-Sánchez et al.  reported findings with no significant changes in FFA for filtered VOO samples. On the other hand, Valli et al. (2019) found that % FA values were significantly higher in unfiltered EVOO than in clarified and filtered samples .
Free fatty acidity increase in unfiltered samples could be attributed to the presence of molds and microorganisms in precipitate which produce large amounts of very active lipases . Moreover, Valli et al., 2019; Bendini et al., 2013; Sanchez et al., 2012, and Yun et al., 2012 observed that both clarification and filtration treatments were very effective in reducing the water content, and consequently, the rate of hydrolysis .
Both clarification and filtration treatments were very effective in reducing the water content and in removing the suspended solids enriched with polyphenols .
Commercial filtration (CF) is a common filtration, achieved using filter aids such as diatomaceous earth (diatomite and extracted from the fossil of microscopic algae) of different particle sizes and different permeabilities. It is normally mixed in various steps with oil to get a filter membrane, which results from the aggregation of the filter aid with the suspended solids during the filtration process. Filtration is considered complete when the maximum partial pressure (insert a number of this pressure in psi or bar) is reached .
Inert gas clarification is another method that was developed recently to extract moisture and suspended solids from virgin olive oils. It is based on the insufflation of flow of inert gases (nitrogen or argon) directly into the middle of the virgin olive oil mass from the bottom of filtration tank containing the cloudy virgin olive oil. As far as carbon dioxide clarification is concerned, to our knowledge, there are no studies describing this clarification technique.
The existence of microorganisms in oil could be attributed to prolonged storage of the olive fruits . It should also be noted that olive oil is not free from moisture, which plays an important role in microbial growth .
The presence of microorganisms in olive oil could lower its quality and could make it harmful to human health .
In the current study, different clarification and filtration techniques were applied to virgin olive oil samples such as (1) commercial filtration (CF) using diatomaceous earth as a filter aid, (2) clarification by insufflating of carbon dioxide, and (3) argon gas.
Materials and Methods
VOO was extracted from olives of ‘nabali balasi’ cultivar from Ramallah region (Palestine) in November 2019, and then divided into four parts. An aliquot of this extract was subjected to a commercial filtration system using diatomaceous earth as a filter aid to produce filtered VOO sample. A second and a third aliquots of cloudy VOO were clarified by directly injecting inert gases; namely, carbon dioxide or argon (at a flow rate of 12 L/min for 2 hours), into the center of VOO mass using a pilot clarification system, that was developed and patented by the University of Bologna and Sapio . In this system, CO2 gas was directly injected into the veiled VOO bulk mass (pressure = 1 bar for 2 hours) to produce CO2-clarified VOO. Another part of the veiled VOO was injected. The remaining VOO was kept unfiltered. Clarification and filtration were conducted at room temperature.
All VOO samples were filled in 150 mL dark and clear glass bottles (with approximately 4% v/v of head-space) immediately after production and filtration/clarification treatments. The hermetically sealed bottles were stored inside a storage room in the dark or under artificial and diffused day light. The samples were stored for 6 months (December 8th, 2019 to June 6th, 2020) at room temperature.
The samples were analyzed for microbial and chemical quality parameters at time zero and after 45, 90, 135, and 180 days of storage. Analysis included basic quality parameters (acidity percentage, peroxide value, and extinction coefficients), and total phenols to evaluate the oxidation stability of such treated VOO during storage under different conditions. Two bottles of each stored sample were removed from the storage room at each respective time of analysis, and aliquots of the oil (drawn from the geometrical center of each bottle) analyzed in triplicates, two replicates were obtained from the same bottle, while the third one was obtained from a separate bottle of the olive oil.
Quality chemical parameters and microbial analysis
Stored samples were analyzed for, peroxides value, free acidity (expressed as mill-equivalents of oxygen per kg of oil meq O2/kg), and percentage of oleic acid (%), respectively. The UV absorption coefficients (K232 and K270) were also measured according to the official methods described in European Union council regulations EEC. Reg 2568/91 of the European Commission.
Yeasts and molds were enumerated in malt extract agar medium, clostridium perfringens were enumerated in Sulfite Polymyxin Sulfadiazine (SPS) agar, Bacillus cereus were enumerated in Bacara agar, and total plate count, all of which were determined for each sample at each respective time of analysis according to the methods described in FDA Bacteriological analytical manual FDA- BAM. The results were expressed as colony forming unit of microorganism per ml of sample (CFU/ ml).
Results and Discussion
Changes in quality parameters
Basic quality indicators (Free fatty acid, peroxide value, K232, and K270) of the unfiltered, commercial filtered, carbon dioxide, and argon gas clarified VOO samples were evaluated before storage and at different time intervals after storage. Two storage conditions were studied; namely, dark and diffused day light. As listed in Tables 1 and 2, the acidity of filtered and clarified VOO were lower than unfiltered. In filtered VOO samples stored exposed to diffusive light, % FA followed the following order Ar <CF<CO22 clarification, the second highest was measured in the UF samples whereas the lowest % acidity was found in the oil samples subjected to Ar clarification.
Also, for peroxide value (PV), the results indicated that there was no significant difference among all samples stored in dark. A higher PV values were found in samples stored under the light compared to those measured in samples stored under dark conditions. The data showed higher peroxide values in samples subjected to Ar clarification and stored under the light than other samples.
Nevertheless, as indicated in Tables 1 and 2, all stored VOO samples yielded PV values of ≤ 20%, and were within the limits of regulations (for example, EU 1348/2013).
These results agreed with those reported by Valli et al. (2019) who found that PV of EVOO during storage was relatively unchanged .
As shown in Tables 1 and 2, there was no significant difference in K232 among VOO samples stored in dark, as well as for VOO samples stored in daylight except, for Ar clarified VOO samples, where K232 values increased significantly after the end of storage (6 months).
This increase in K232 values might be attributed to the progress in oxidation reaction during storage. As a comparison, Brenes et al. (2001) reported higher K232 values of for UF oil due to more rapid oxidation during storage .
These changes in PV values might be explained by the presence of oxidative enzymes in the suspended solids, as suggested by Georgalaki et al. (1998) and Valgimigli, et al. (2001) [17, 18].
However, upon evaluating K270 results for all treated samples as shown in Tables 1 and 2, higher K270 values were observed for VOO samples stored under diffused daylight compared to that found in VOO samples stored in dark conditions. However, no significant difference was found between the results of the filtered and clarified samples. In contrast, a significant increase in the K270 values measured in the unfiltered samples at the end of storage was observed.
Change in phenolic compounds
As shown in Tables 1 and 2, there was no significant difference in the measured phenolic compounds among clarified/filtered and unfiltered VOO samples in both storage conditions, in dark and under diffused daylight after the end of storage time (6 months). However, the UF VOO samples stored in Dark conditions had higher phenolic compounds values and less reduction than what was found in samples stored under diffused daylight.
Phenolic compounds react as an antioxidant, as they prevent lipids autoxidation via trapping intermediate peroxyl radical, as a result the total phenol decrease . Moreover, as a consequence of light, the action of photo oxidation is higher compared to auto-oxidation process .
As a comparison, Ciafardini et al. (2002a) reported values of polyphenols lower in the filtered olive oil than what was observed in the unfiltered VOO samples . Okogeri et al. (2002) reported that the decrease of total phenol measured in samples stored light was less than that measured in samples stored in dark .
On the other hand, Bendini et al. (2013) found that there was no clear difference in the observed total phenols for inert gas clarified VOO and non-clarified samples . Valli et al. (2019) reported that phenolic compounds decreased significantly after the end of storage of virgin olive oil, a trend similar to what was observed in the study .
Quantity of microorganism
As presented in Table 3, Y&M microbes were found in all VOO stored samples (filtered, clarified, and unfiltered) while total plate count, clostridium perfringens, and bacillus cereus were not found in any stored VOO samples. As illustrated in Table 3, Y & M in CF and clarified VOO samples decreased and reached to undetectable level at the end of storage time (i.e. 6 months) whereas Y & M in UF VOO samples remained detectable (present) even at storage at the end of 6 months.
The presence of droplet water in oil (oil-water emulsions) played an important role in microorganism's growth . Likewise, as Ciafardini et al. (2002a) reported that fewer types of yeast in the filtered olive oil were found; this might be due to the mechanical removal of the suspended solids . Microorganisms that might be present in water droplets and sediments particles of the fruit will be removed upon subjecting the oil to filtration treatment. Hence, filtration and clarification are considered as effective methods for lowering microorganism levels. Many studies reported the effectiveness of filtration and clarification in reducing the levels of bacteria in VOO (Valli et al., 2019; Bendini, et al., 2013; Lozano-Sa ́ nchez et al., 2012; Ciafardini, et al., 2002a; Peri, 2014) [3, 9, 10, 21, 24].
In addition, the phenolic compounds could have antimicrobial activity against microorganism, as suggested by Karaosmanoglu et al. .
This study showed that clarification and filtration treatments might have be beneficial effects in filtered VOO compared to UF VOO in terms of reducing the precipitate, decreasing microbial count, preserving the chemical quality of VOO. Hydrolytic degradation and evaluated by FA levels were more obvious in UF VOO than those in filtered and clarified VOO at the end of the storage (6 months). For total polar phenol measured at the end of the 6-month storage, no significant difference was observed between all VOO samples. Similar trend was observed for the peroxide values, where no significant difference was also observed for all stored VOO samples after 6-month storage. It should be noted that the lowest peroxide value was found in the VOO clarified with argon gas. As far as K232 and K270 results are concerned, the lowest values measured were those for VOO samples clarified with CO2. Nevertheless, no significant difference was observed in the K270 values measure in clarified and filtered samples after 6-month storage. It was evident that filtration is mostly necessary as a final step to eliminate moisture or suspended solids and to increase the brilliance of olive oil for consumer admission. Moreover, the VOO clarification resulted in eliminating most of Yeasts and Molds. Moreover, clarification and filtration might enhance the VOO quality during storage as well. In addition, clarification seemed to be a good treatment to avoid potential interaction between VOO and filter aid. Generally speaking, and upon inspecting all quality indices, storage under dark conditions was better and yielded better quality of VOO compared to what was observed in the oil samples stored in day light.
No potential conflict of interest was reported by the authors.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
No potential conflict of interest was reported by the authors.
HOW TO CITE THIS ARTICLE
Ziad Ayyad, Wafaa Alsioury, Zaher Barghouthi, Diab Qadah, Mohannad Qurie*. Effects of Different Filtration and Clarification Techniques on Palestinian Virgin Olive Oil Chemical and Microbial quality. J. Med. Chem. Sci., 2023, 6(11) 2699-2706.