Effect of Deodorization of Camelina (Camelina sativa) Oil on Its

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Effect of deodorization of camelina (Camelina sativa) oil on its phenolic content and the radical scavenging effectiveness of its extracts Robert Hrastar, Petra Terpinc, Iztok Joze Kosir, and Helena Abramovic J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf400309j • Publication Date (Web): 08 Aug 2013 Downloaded from http://pubs.acs.org on August 13, 2013

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Journal of Agricultural and Food Chemistry

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Effect of deodorization of camelina (Camelina sativa) oil on its phenolic content and the

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radical scavenging effectiveness of its extracts1

3 Robert Hrastara, Petra Terpincb, Iztok Jože Košira, Helena Abramovičb,∗2

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a

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b

Slovenian Institute of Hop Research and Brewing, SI-3310 Žalec, Slovenia Biotechnical Faculty, University of Ljubljana, SI-1111 Ljubljana, Slovenia

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Robert Hrastar and Petra Terpinc equally contributed as first autors

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Corresponding author. Tel.: + 386 1 320 37 82; fax: + 386 1 256 62 96. E-mail adress: [email protected] (Helena Abramovič)

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ACS Paragon Plus Environment


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ABSTRACT

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The influence of deodorization parameters (temperature (T), steam flow (S), time (t)) on the

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phenolic content and radical scavenging effectiveness (RSE) of methanolic extracts of

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camelina oil was investigated and analysed by response-surface methodology (RSM). The

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phenolic content can be considered to be a linear function of all three parameters. A positive

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linear relationship between the content of phenolic compounds in deodorized oils and RSE

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was observed. Deodorization at 210 °C with a steam flow of 3 mL/h for 90 min resulted in the

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best preservation of phenolics, amounting to 29.9 mg/kg. The lowest reduction from RSE of

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12.4 µM Trolox equivalents (TE)/g oil for the crude oil was observed for oil treated at 195 °C

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and 18 mL/h for 60 min with RSE of 10.1 µM TE/g oil. The lack of correlation between RSE

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or total phenolic content with oxidative stability (OS) of the deodorized oils suggests that

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antioxidants in scavenging radicals react by different mechanisms, depending on radical type

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and reaction medium.

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Keywords: camelina oil, Camelina sativa, deodorization, polyphenols, radical scavenging

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effectiveness

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INTRODUCTION

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Camelina sativa is an important oilseed crop, belonging to Brassicaceae family. In

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recent years this crop has received great attention because of nutritional properties of the oil

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and its suitability for use in non-nutritional applications such as biofuel. Camelina meal due to

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its high content of proteins, carbohydrates and low levels of glucosinolates1, 2 can be used as

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feedstock. Camelina oil is considered to be a high value added product due to its considerable

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content of omega-3 α-linolenic acid C18:3n-3 (30 - 40 %) and low content of eruic acid C22:1n-9

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(about 3 %). The presence of tocopherols (approximately 700 mg/kg) and phenolic

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compounds (approximately 100 mg/kg as chlorogenic acid) also makes it more stable toward

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oxidation than other highly unsaturated oils such as flax oil.3-5 Camelina oil has an attractive

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yellow color, a mustard-like taste, and a characteristic pleasant odor. This type of flavor and

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odor makes it difficult to find acceptability among European and North American consumers,

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mainly because of their different expectations of the organoleptic properties of vegetable oils.6

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Namely, processing techniques such as rafination can produce oils which are flavorless,

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odorless, and with reduced acidity.7

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Phenolic compounds are among the most widely distributed secondary plant

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metabolites and are generally involved in defence against infection and injury. In foods,

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phenolic compounds may contribute to taste, aroma, color and oxidative stability.8 In

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addition, they have been associated with health benefits against the development of cancers,

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cardiovascular diseases, diabetes, osteoporosis and neurodegenerative diseases.9 The

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antioxidant activity of phenolic compounds in plants is mainly due to their redox properties

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and chemical structure, which can play important roles in scavenging free radicals, chelating

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transitional metals, and quenching singlet and triplet oxygen molecules.10 A recent

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publication11 showed that after oil pressing most of the phenolic compounds remain in the 3



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seed residues. The compounds successfully identified in the methanolic extract of camelina

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oil were: ellagic acid, p-hydroxybenzoic acid, sinapic acid, salicylic acid, catechin and

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quercetin.11 Considering its antioxidant properties revealed in the same study, especially its

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great chelating ability and significant protective effect against oxidation of the emulsion,

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camelina oil thus appears to provide a potentially useful source of antioxidants.

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During refining, unfortunately, the content of valuable minor compounds, tocopherols,

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sterols and phenolic compound is reduced. Different studies on vegetable oils have shown that

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the phenol concentration decreases with increasing degree of refinement, where neutralization

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and bleaching results in major removal of these minor compounds.12-16 On the contrary, water

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degumming, as a process for removing phospholipids from oil, does not significantly affect

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the content of total polyphenols,13,16 while after mild deodorization oil samples with a

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considerable residual amount of polyphenols (50 - 75 % as compared before deodorization)

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were retained, resulting in a higher oxidative stability than fully refined oils.16 Deodorization

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is a physical process comprised holding and steam refining of oil at elevated temperatures and

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low absolute pressures to remove unwanted odoriferous compounds, namely various

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oxidation products.17 During deodorization phenolics, depending on their volatilities, are

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partly evaporated from the oil and concentrate in distillate steam as the so-called deodorizer

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distillate. When optimizing operating conditions for refining highly unsaturated camelina oil,

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it is important to find a compromise between the need to maximize the elimination of

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unwanted compounds and the desire to retain valuable compounds, thus improving the

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oxidative stability and retaining the nutritional value of the finished oil.

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Recently, we conducted a detailed study on the optimization of the deodorization

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parameters, i.e. temperature, steam flow and time.18, 19 These studies however did not indicate

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what fraction of the polyphenols are removed/retained during deodorization of camelina oil.

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Therefore, in the present work an attempt has been made to quantify the changes in phenolic 4


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content and their antioxidant activity with respect to deodorization conditions. Response-

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surface methodology was used for analyzing these experiments.

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MATERIALS AND METHODS

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Camelina oil. Camelina (Camelina sativa (L.) Crantz) seeds were purchased from a

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certified organic seed dealer and cold pressed with a screw press. The oil was centrifuged

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(4200 rpm, 30 min). The degumming step was carried out for 30 min at a temperature of

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70 °C and 100 rpm agitation, with addition of 3 % of water and 0.5 % of citric acid (30 %

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w/v). Finally, the gums were separated by precipitation and decantation. The results of

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characterization of the degummed oil, as recently published,18,

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1.6 meq/kg, p-AV = 0.41, content of total tocopherols = 780 mg/kg.

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were as follows: PV =

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Reagents and solvents. Hexane, ethanol (96 %), methanol (99.9 %) and sodium

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carbonate were obtained from Merck (Darmstadt, Germany). Folin-Ciocalteu (F.C.) reagent,

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2,2-diphenyl-1-picrylhydrazyl (DPPH·) reagent and chlorogenic acid were purchased from

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Sigma-Aldrich GmbH (Steinheim, Germany). For preparation of solutions ultrapure water

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(MiliQ, Millipore) was used.

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Deodorization process. Bench-scale deodorization of camelina oil was conducted in

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2000 g batches. The deodorization apparatus has been previously described.20 It consists of a

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5 L deodorizer flask, a steam generator with the steam tube extending to the bottom of the

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deodorizer flask, a round bottom fully-insulated condenser flask with a 50 mm cold finger

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containing ethanol and a mechanical vacuum pump. Firstly, the flow of steam was controlled

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by regulating the distance (d) between the 150 watt heating body and the glass steam

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generator. The distances of 3, 18, 30, 42 and 46 cm correspond to steam flow of 8, 6, 4, 2 and

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0 mL/h, respectively. The oil was then deaerated by setting the vacuum pump at a constant 5



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pressure of 5 mbar. Following that, the deaerated oil was heated to 85 °C and kept there for

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10 min to evaporate residual water from the degumming process. Finally, the deaerated oil

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was heated to the desired T. At the end of the desired deodorization t, the deodorized oil was

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cooled. Once the deodorized oil reached T of 60 °C, nitrogen gas was introduced into the

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apparatus to induce a normal atmosphere. The experimental was designed as follows. A

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central composite design with three-factors-five-levels was chosen. The ranges for the

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parameters, namely T (180 - 240 °C), S i.e. d (0 - 8 cm) and T (30 - 150 min), were selected to

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mimic the deodorization conditions that are usually applied in an industrial environment. This

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design generated a total of 14 experimental runs at different conditions and 6 runs at the

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central point (210 °C, 30 mL/h and 90 min) using Design-Expert 7.0.0 (Stat-Ease, Inc.;

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Minneapolis, USA) software. All experiments were conducted in randomized order as

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suggested by the software to minimize the effect of unexplained variability in the observed

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response due to extraneous factors.

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Extraction of phenolic compounds. Methanolic extracts of camelina oil were

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prepared according to the following procedure. The oil sample was first dissolved in hexane

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(1:10, w/v) and transferred to a separatory funnel. Then 80 % (v/v) methanol was added to the

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oil at a ratio of 1:4 (w/v). After 5 minutes of shaking, the lower methanol layer was removed.

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The residue was then re-extracted under the same conditions twice more. The methanol

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phases were combined and concentrated in a rotary evaporator under vacuum at 40 °C. The

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dry residues were re-dissolved in absolute methanol.

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Total phenolic content. The total content of phenolic compounds in camelina oil was

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determined spectrophotometrically.21 An appropriately diluted extract or chlorogenic acid (as

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a calibration standard) was mixed with freshly prepared F.C. reagent, sodium carbonate

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solution (20 %, w/v) and Milli-Q water. After 40 min the absorbance was measured at 765 nm

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on a model 8453 Hewlett Packard UV–Visible spectrophotometer (Hewlett Packard, 6


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Waldbronn, Germany) with a 1 cm cell. The results were expressed as mg of chlorogenic acid

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per kg of oil. The determination was conducted in triplicate and results were averaged.

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Radical scavenging effectiveness. The radical scavenging effectiveness of phenolic

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extracts was evaluated as their capability to scavenge free radicals by DPPH· assay.22 Briefly,

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2.9 mL of a solution of DPPH· (0.1 mM) in ethanol (96 % (v/v)) was added to 0.1 mL of

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phenolic extract and 30 min later, the absorbance was measured at 517 nm against ethanol

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(96 %) as a blank (As 517). The absorbance of the control (Ac 517) was obtained by replacing

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the phenolic extract with 96 % ethanol. A decrease in absorbance from Ac 517 to As 517 occurs

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when an antioxidant reacts with the DPPH· radical. The RSE was expressed in µM TE

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(calculated as Trolox concentration needed to reach the same effect as phenolic extract) per g

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of oil. Triplicate analyses were run for each extract.

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Statistical analysis. The data were statistically analyzed and interpreted with RSM by

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statistical software as mentioned above. First or second order coefficients were generated by

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least-squares multiple regression analysis with hierarchical backward elimination at the 95 %

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significance level (p < 0.05). Insignificant (p > 0.05) factors and interactions between factors

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were removed. Responses (total phenolic content, RSE) were fitted to the parameters (T, S, t)

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by multiple regression to produce a mathematical model: 3

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3

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Y = β 0 + ∑ β i X i + ∑ β ii X i2 + ∑ i =1

i =1

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∑β

ij

Xi X j

(1)

i =1 j =i +1

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The fit of the model was evaluated according to the coefficient of determination (R2)

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and analysis of variance (ANOVA). The main effect plot displays the predicted change in the

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response when parameters vary from their lowest to their highest level, while the third

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parameter in the design is set to its average value.

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RESULTS

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Main Effects on total phenolic content. Table 1 shows the total phenolic content for

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the 20 experimental runs carried on at different deodorization conditions. All three

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deodorization parameters significantly affected the content of total phenolic compounds in

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deodorized oil. Only first order parameters were significant, while those of second order and

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interactions between them did not influence the content of total phenolics, so the total

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phenolic content could be considered as a simple linear function of all three parameters (R2 =

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0.68) (Equation 2, Figure 1). The actual coded values for the S were the following: 46 mL/h =

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0 cm, 42 mL/h = 2 cm, 30 mL/h = 4 cm, 18 mL/h = 6 cm, 3 mL/h = 8 cm.

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TPC = – 0.21*T + 1.19*d – 0.07*t + 66.5

(2)

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In this study, the lowest reduction of total phenolic content was achieved in oil treated

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at 210 °C with S of 3 mL/h for 90 min (Experimental number (Eno) 12) (Figure 2). At the

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same T and t of deodorization, when S increased to 30 mL/h (Eno 15 - 20) a more profound

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decrease of phenolic compounds was observed. A subsequent increase in deodorization T

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(240 °C) at unchanged t resulted in still greater losses of phenolic compounds (Eno 10). It was

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found that shortening t to 30 min in comparison to oils deodorized for 90 min at the same

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conditions (210 °C, 30 mL/h) resulted in better preservation of phenolic compounds (Eno 13).

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Lower reduction of total phenolics was observed in oil treated at relatively mild

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conditions (180 °C, 30 mL/h, 90 min) (Eno 9) and (195 °C, 18 mL/h, 60 min) (Eno 3). An

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increase in T to 225 °C at unchanged S and t (18 mL/h and 60 min, respectively) (Eno 4)

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resulted in a profound lowering of phenolic content. On the other hand, an appreciably lower

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decrease was observed when T remained at 195 °C but S increased to 42 mL/h (Eno 1).

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The highest reduction of total phenolic content was obtained when the oil was

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subjected to the most severe deodorization conditions (225 °C, 42 mL/h, 120 min) (Eno 6). 8


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Lowering S to 18 mL/h resulted in better preservation of phenolic compounds in the

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deodorized oil (Eno 8). Lowering T from 225 °C to 195 °C with S remaining unchanged (Eno

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5) resulted in an even better preservation of phenolic compounds in the deodorized oil. A

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subsequent lowering of S to 18 mL/h resulted in a still greater preservation of phenolic

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compounds (Eno 7). This is similar to that for 60 min deodorization t (as described in the

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previous paragraph) where increased S had a lower impact on the loss of phenolic compounds

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than increased T.

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As evident, around 50 % of the original total phenolic content was retained when the

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deodorization parameters were adjusted to the central point (210 °C, 30 mL/h, 90 min) (Eno

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15 - 20). According to published results,18, 19 the same camelina oil deodorized under these

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conditions showed good organoleptic results and chemical properties (free fatty acids

Effect of Deodorization of Camelina (Camelina sativa) Oil on Its

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