Effect of co-extraction of pomegranate seed oil with green tea leaves on the extraction yield and quality of extracted oil

– Co-extraction of pomegranate seed oil (PSO) with green tea leaves (GTL) (0 [control sample], 2.5, 5, 7.5, and 10% w/w) was conducted by cold-press to evaluate the extracted oil quality during storage. The extraction yield was similar to the control sample up to 5% w/w of GTL. Total phenol and chlorophyll content were increased in the extracted oils with increasing the level of GTL. The acid and peroxide values were increased during the storage and the lowest values were achieved for PSO with 5% GTL. Rancimat analysis also con ﬁ rmed the PVresults, therefore using 5% GTL led to providing the highest induction period (11.5 h). Consequently, using 5% of GTL during the extraction of PSO by cold-press could result in an appropriate extraction yield and also present higher oxidation stability. This method does not need any antioxidant extraction from herbs and is very cost-effective, time-ef ﬁ cient, and uses no chemicals.


Introduction
Pomegranate (Punica granatum L.) has been consumed since ancient times due to its nutritional value (Khoddami et al., 2014). It has been originated in the Himalayas and Mediterranean region and has mostly grown in tropical and subtropical countries such as India, Iran, China, and the USA (Khoddami et al., 2014;Paul and Radhakrishnan, 2020).
Oil oxidative stability can be improved using antioxidants being added after extraction (Drinić et al., 2020). Antioxidants have several types and mainly they are divided into two synthetic and natural ones. There are concerns about the consumption of synthetic antioxidants due to their potential adverse health effects (Drinić et al., 2020).
Recently, attention to the use of natural antioxidants has increased, and especially consumers prefer to use natural antioxidants instead of synthetic ones (Drinić et al., 2020). The oils can be enriched with natural antioxidants and, thus, the oxidation and rancidity are retarded and oil quality is improved during storage (Osanloo et al., 2021).
Plants are rich in polyphenolic compounds, which potentially indicate antioxidant activity and radical scavenging activity. Green tea leaves (GTL) are one of the richest sources of polyphenolic compounds (Li and Jiang, 2010;Musial et al., 2020).
There are many options, such as using antioxidants, extraction of oils from mixed seedoils, and pretreatments of seedoils before oil extraction, to enhance oxidative stability of oils. Dehghan-Manshadi et al. (2020) applied infrared (IR)assisted spouted bed drying (SBD) as a potential alternative to the traditional hot air drying for heat-sensitive components of flaxseeds. Mazaheri et al. (2019a) reported that microwave treatment of black seed before the oil extraction reduced lipase activity more effectively. They reported that the treatment of black seeds with microwaves and soaking them before extracting oil in a cold-press raised the oil oxidative stability. Nain et al. (2021) reported that using green tea enhanced the oxidative stability of DHA-rich oil by reducing the formation of peroxides and secondary oxidation products. Drinić et al. (2020) used pomegranate peel extract in PSO individually or with a combination with BHT to enhance the oxidative stability of the oil. Mazaheri et al. (2019b) investigated the cold-press extraction of sunflower and black cumin seeds and reported that seeds mixing caused an increase in oxidative stability, which increased from 6.78 to 9.69 h.
All of the above-mentioned ways to enhance extracted oils' oxidative stability have advantages and disadvantages. One new way can be blending seedoils with plant materials high in bioactive compounds such as GTL and their coextraction by the press. Therefore, the aim of this work is the enhancement of PSO oxidative stability using GTL as a natural antioxidant. Co-extraction of antioxidant compounds of GTL with PSO as a new method of extraction can be highlighted in industrial applications concerning the production of oil with improved oxidative stability and high content of bioactive components.

Materials and method 2.1 Materials
Pomegranates seeds, which were separated from the Iranian pomegranate cultivar (Saveh Sweet White Peel) after juice extraction, were purchased from local market (Tabriz, Iran). Green tea leaves (Camellia sinensis L.) were obtained from local tea market (Tabriz, Iran). All chemical materials were provided from Sigma-Aldrich Co. (St. Louis, Missouri, United States).

Extraction process
PSO was coextracted with green tea leaves using a mechanical screw press apparatus (model P500R, Anton Fries, Germany) Rostami et al., 2014). First, pomegranate seeds and green tea leaves were conditioned to moisture contents of 6.4% and 3.7% based on the preliminary oil extraction yield. Then, the mixture of PSO and GTL at a level of 0 (control sample), 2.5, 5, 7.5, and 10% (w/w) simultaneously undergone the extraction process by screw press apparatus. The temperature of extracted oil was not raised over 40°C during the oil extraction by cold-press. Extracted oil samples were kept for 90 days at room condition in a dark place and their quality was analyzed on the extraction day and every 30 days of storage.

Extraction yield
The extraction yield (EY) was calculated based on the weight of obtained oil after extraction and the weight of pomegranate seed powder as the following equation (Rezvankhah et al., 2018):

Determination of total phenolic content
The total phenolic content of PSO enriched with GTL was determined using the Folin-Ciocalteu method according to the method of Womeni et al. (2016) with brief modification. Firstly, 1 mL of oil samples (1 mg/mL) that dissolved in ethanol was mixed with 1 mL of diluted Ciocalteu reagent (1:10, v/v) and 3 mL of sodium carbonate 7%. The prepared mixtures were kept in a dark place at an ambient temperature for 30 min. The absorbance of oil mixtures was determined at 760 nm using a UV-Vis spectrophotometer. Also, pure ethanol was used as the control, and gallic acid at the concentration of 0-0.06 mg/mL was used as a standard. Eventually, the TPC of oil mixtures was expressed as milligram gallic acid equivalents (GAE)/gram samples.

Determination of chlorophyll
Chlorophyll content of PSO enriched with GTL was determined at 670 nm in cyclohexane using the method of Sahin et al. (2017). The chlorophyll concentration was expressed as mg of pheophytin per kg of oil.

Determination of acid value (AV)
AV was determined by potentiometric titration in an automatic titrator model G20 (Mettler Toledo, Urdrof, Switzerland) according to Costa et al. (2019). Briefly, oil acidity was analyzed by titrating PSO enriched with GTL dissolved in 45 mL acetone: ethanol (1:1, v/v) with 0.4 M NaOH until pH 11. AV was expressed as % punicic acid.

Determination of peroxide value (PV)
The PV was measured according to the method described by Rezvankhah et al. (2019). Determination was conducted by titration of 0.1 N KI saturated solutions of the oil with 0.1 N Na 2 S 2 O 3 and starch as an indicator. PV was expressed in mg oxygen equivalent per kg of oil (meqO 2 /kg-oil).

Rancimat analysis
The oxidative stability of PSO before and after enrichment was evaluated by the Rancimat method at 110°C in a Rancimat apparatus (Metrohm 743; Metrohm Co., Herisau, Switzerland) according to Costa et al. (2019). The oxidation process was conducted upon 2.5 g oil samples at the mentioned temperature at an air velocity of 20 L/h while monitoring mixtures until a sharp increase in the water conductivity corresponded to the oil stability index. The stability of oil mixtures was expressed as the induction period (IP).

Statistical analysis
All data were conducted in three replications and provided in mean and standard deviation. The statistical analysis was conducted by one-way ANOVA and the mean difference between the data was implemented by the Duncan test at the probable level of 5% (P < 0.05) using SPSS software (IBM SPSS Statistics 23).

Extraction yield
The extraction yield in the cold-press extraction method is affected mainly by the temperature and also moisture content of the seed (Naebi et al., 2022). The effects of the incorporation of green tea leaves on the extraction yield of PSO were evaluated during the mechanical press. Incorporation of GTL at a concentration ranging between 2.5 to 5% did not have a significant effect (P < 0.05) on the extraction yield (6.3 to 6.1%) (Fig. 1). However, incorporation of higher than 7.5% significantly reduced the extraction yield. It was thought that GTL at higher concentrations made a mechanical obstacle against the elicitation of oil from the ground seed texture. Eikani et al. (2012) reported that the cold-pressing method indicated 4.29% extraction efficiency. Our extraction yield values were higher which can be attributed to the variety and also process condition.
It has been reported that a heating process by temperature 75°C and moisture content of output seeds in the confine of 6.3-6.5% have yielded the highest extraction efficiency that resulted in high-quality oil production and by-products with the minimum remaining oil content according to the previous studies (Rostami et al., 2014). Also, Naebi et al. (2022) declared that the optimum moisture content which gave the highest oil yield (33.5%) was 7.5%. The extracted PSO main fatty acids were punicic acid (C18:3) up to 76% as the main fatty acid followed by linoleic acid (C18:2) about 8.2%, oleic acid (C18:1) about 7.6%, palmitic acid (C16:0) about 4.6% and stearic acid (C18:0) about 2.6% (Khoddami et al., 2014).
TPC ranged between 2.7 to 12.1 mg GAE/g for control and PSO with 10% incorporated GTL, respectively, which shows . Extraction yield of pomegranate seed oil mixed with green tea leaves at the conditioned moisture content of 6.4% and 3.75%. T0, T2.5, T5, T7.5, and T10 indicate pomegranate seed (control sample), pomegranate seed with 2.5% green tea leaves, pomegranate seed with 5% green tea leaves, pomegranate seed with 7.5% green tea leaves, pomegranate seed with 10% green tea leaves, respectively. the positive effect of GTL incorporation on the TPC on PSO. High TPC (10.44 mg GAE/g sample) content was reported for the PSO extracted by cold-pressing from the variety Torshe Malas Iran (Khoddami et al., 2014).
It has been reported that GTL has a high content of polyphenolic compounds, which can donate antioxidant activity (Hu et al., 2021;Musial et al., 2020;Palit et al., 2008). Catechins are the prominent polyphenols of GTL that can render antioxidant and preservation effects (Palit et al., 2008;Unno et al., 2018). The addition of tea polyphenols to oil has shown other beneficial impacts (Palit et al., 2008;Samadi and Raouf Fard, 2020). For instance, it has been reported that rice bran oil cold performance quality was improved after the addition of tea polyphenols. Indeed, rice bran oil retained clear at 0°C for 5.5 h when tea polyphenols concentration was 0.08% w/w, which could recrystallize in rice bran oil before rice bran crystallization and further promote the nucleation of rice bran oil (Wang et al., 2021).

Chlorophyll content
The plants are rich in natural pigments such as carotenoids and chlorophylls. GTL is also rich in chlorophylls and incorporation of them enhances the chlorophyll content. The addition of GTL significantly (P < 0.05) increased the chlorophyll content of PSO on all days of storage (Tab. 2). It has been reported that extracted PSO by the supercritical CO 2 method did not have detectable chlorophyll (de O Silva et al., 2019). Storing had also a significant effect and chlorophyll content was decreased during 90 days of storage. This reduction was associated with chlorophyll degradation (Li et al., 2019;Sahin et al., 2017). It should be also pointed out that the addition of a high amount of chlorophyll to the oils with higher PUFAa has sensitizing effects and can increase oxidation (Machado et al., 2022;Rezvankhah et al., 2018Rezvankhah et al., , 2019Roshanak et al., 2016). Chlorophylls can promote the initial autooxidation by their ability to generate alkyl hydroperoxides (Machado et al., 2022;. Rezvankhah et al., 2018). Based on current reports, the presence of high content of chlorophylls in the extracted oil can reduce the oil oxidative stability (Rezvankhah et al., 2018(Rezvankhah et al., , 2019. So, the incorporation of a high amount of GTL into PSO can have adverse effects on oxidative stability.

Acid value
AV is associated with the free fatty acids and for PSO, punicic acid is principal (Goula and Adamopoulos, 2012;Keskin Çavdar et al., 2017;Tian et al., 2013). The higher the AV determination, the lower the quality of the obtained oil (Drinić et al., 2020). Degradation of triacylglycerol molecules especially occurs during storage due to the presence of oxidation parameters such as inducing moisture, metals, oxygen, etc. (Rezvankhah et al., 2018(Rezvankhah et al., , 2019(Rezvankhah et al., , 2020. It was observed that incorporation of GTL especially at higher concentrations increased the AV (Tab. 3). Also, the incorporation of the GTL enhanced the AV during the storage time. The lowest amount of AV (0.1%) was obtained for the 12.1 Aa 10 Ba 12.2 Aa 11.9 Aa * For samples see Figure 1. ** Different small superscripts indicate the significant difference between the samples while the different capital superscripts indicate the significant difference between the days (P < 0.05). sample with 5% GTL on day 1 and the highest amount (2.4%) was obtained for PSO with 10% GTL on day 90. Drinić et al. (2020) reported that the AV was 0.84 mL/g, Dadashi et al. (2013) reported 3.78 and 8.36 mL/g for n-hexane extraction while 0.63 mL/g for cold-press technology (de Melo et al., 2016).

Peroxide value
Although the incorporation of GTL increased the oil AV, the presence of phenolic compounds can reduce the oxidation rate associated with their antioxidant activity (Maqsood et al., 2014). Also, PSO inherently has antioxidant activity mainly due to the presence of tocopherols and phytosterols (Costa et al., 2019;de Melo et al., 2016). According to Table 4, PV was determined after the incorporation of various concentrations of GTL and during storage. PV was decreased after the addition of 2.5% to PSO while higher concentration had even inducing effects on the oxidation of PUFAs (Bahmaei et al., 2005;Palit et al., 2008). The highest PV (4.7 meq-O 2 /kg-oil) was obtained for PSO enriched with 10% GTL and the lowest amount (2.1 meq-O 2 /kg-oil) was obtained for PSO with 2.5% GTL. The addition of a sufficient not excessive amount of polyphenol-rich source to the oils with high susceptibility to oxidation can retard the oxidation and inhibit the propagation (Mazaheri et al., 2019a(Mazaheri et al., , 2019bPalit et al., 2008). However, the higher the polyphenol content, and also the higher the chlorophyll content the higher peroxyl radical production, and thus, oxidation was increased (Li et al., 2019;Rezvankhah et al., 2019). Li et al. (2019) declared that residues of chlorophyll induced oxidative rancidity and deterioration through photooxidation in rapeseed oil. They reported that PV of rapeseed oil exposed to light increased significantly, especially for the oil with a high content of chlorophyll. It was also consistent with reports by Bahmaei et al. (2005) that pigments present in oil impart an undesirable color and also promote oxidation in the presence of light. Regarding the storage, it was observed that PV was increased in all samples. However, the sample with 2.5% GTL indicated lower PV than control and other samples with higher GTL concentrations (Maqsood et al., 2014). Incorporation of the high amount of GTL into the PSO-rich PUFAs leads to an increase in sensitivity of oil to oxidation since chlorophylls act as light adsorbents and, therefore, oil oxidation be propagated (Rezvankhah et al., 2018(Rezvankhah et al., , 2019. In addition, based on the current studies, the addition of herbs event without extraction can raise the oil oxidative stability (Ebrahimi Monfared et al., 2022;Nain et al., 2021). As reported by Ebrahimi Monfared et al. (2022), the addition of matcha GTL increased the oil oxidative stability led to a higher shelf-life of muesli. Also, Kırmızıkaya et al. (2021) reported that black, green, and white tea infusions and powder improved the oxidative stability of minced beef throughout refrigerated storage.

Rancimat
Plants due to their phenolic compounds can enhance the oxidative stability of oils. In the present study, the incorporation of GTL increased the oxidative stability of PSO (Fig. 2). Concerning this, IP values were determined and the lowest  Figure 1. ** Different small superscripts indicate the significant difference between the samples while the different capital superscripts indicate the significant difference between the days (P < 0.05). amount was obtained for control and the highest amount was obtained for PSO with 5% GTL. Based on the PV and AV results, a higher concentration of GTL induces oxidation due to the high content of chlorophyll and degradation of phenol functional hydroxyl groups, which propagate the oxidation (Li et al., 2019;Womeni et al., 2016;Ye et al., 2020). Thus, a moderate amount of GTL can be more effective in retarding the oxidation. Womeni et al. (2016) used old Cameroonian GTL and BHT to evaluate the oxidative stability of RBD palm olein under forced storage conditions. Based on the Rancimat results, the oil with GTL had a higher induction time (24.8-28.9 h) compared with BHT (20.1-22.7 h). Due to the presence of gallic acid, epicatechin gallate, gallocatechin, and epigallocatechin gallate as the main phenolic antioxidants in GTL, the oils with these natural antioxidants would have higher oxidation stability (Womeni et al., 2016). Mazaheri et al. (2019b) reported that co-extraction of sunflower oil with black seed oil increased the oxidative stability related to the presence of high phenolic compounds in black seed oil.

Conclusion
PSO lacking high oxidation stability was enriched with GTL at various concentrations (2.5 to 10%). Using up to 5% GTL did not affect the oil extraction yield. TPC as health-promoting components was increased in the PSO after the inclusion of GTL. In conclusion and general view and based on the factors related to the oxidative stability (AV, PV, and Rancimat analysis), it can be suggested that that PSO coextracted with 5% GTL has good quality and high oxidative stability. Therefore, the coextraction of oil from a mixture of plant materials and seed oils can be suggested as a safe, cost-effective, and time-efficient process for the industry. The induction period of pomegranate seed oil with and without green tea leaves at various concentrations (2.5 to 10%). T0, T2.5, T5, T7.5, and T10 indicate pomegranate seed (control sample), pomegranate seed with 2.5% green tea leaves, pomegranate seed with 5% green tea leaves, pomegranate seed with 7.5% green tea leaves, pomegranate seed with 10% green tea leaves, respectively.