Nutritional value of cold-pressed rapeseed oil during long term storage as influenced by the type of packaging material, exposure to light & oxygen and storage temperature

Amber glass and amber polyethylene terephthalate bottles specification

The dimensions of amber glass and amber PET bottles were: ∅ 6.20 cm × 19.5 cm height; ∅ 7.55 cm × 18.5 cm height, respectively. The oxygen transmission rate specified for PET bottles amounted to 7.8 cm^{/m/day (tested at 21 °C, at 0.21 atm driving force and 50 % relative humidity). The light transmittance (%) spectra of both packaging materials tested exhibited effective barriers to wavelengths shorter than 500 nm, followed by increase in light transmittance up to 31–33 % for 600 to 800 nm and up to 49 % for 680 to 710 nm wavelength for amber glass and amber PET, respectively (Fig.} ​(Fig.11).

Fig. 1

Spectra of the studied amber glass and amber polyethylene terephthalate bottles

Storage conditions

During storage oil samples were divided into two groups: (a) bottles were kept closed over entire storage period; (b) once per month (5th day of every month) bottles were opened and approximately 20 ml of oil was poured from the bottle, then bottles were re-capped and shaken to distribute the oxygen within the oil. Bottled samples were kept in three different conditions: (a) at room temperature (RT), where the average temperature during storage period was 20 °C (ranging from 18 to 22 °C), on the lab shelf exposed to daylight (day-night); (b) at room temperature (RT) in the dark; (c) and at 4 °C in a refrigerator.

Reagents

Analytical standards of γ- and α-tocopherols (purity >95 % by HPLC), a high purity standard of 5α-cholestane, and N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1 % trimethylchlorosilane (TMCS), were purchased from Sigma Aldrich (USA). HPLC-grade n-hexane, methanol (MeOH), acetonitrile (ACN), methyl tert-butyl ether (MtBE) and potassium hydroxide (KOH) were obtained from Polish Chemical Reagents (Poland). FeCl₂ > 99 % and NH₄SCN (ammonium thiocyanate) of HPLC purity were purchased from Merck Millipore (Germany), while HCl (36 %), chloroform (CHCl₃), H₂O₂ (30 %) of analysis grade were obtained from Chempur Company (Poland). All other solvents and chemicals used in this study were of analytical grade.

Fatty acid composition

Fatty acid methyl esters (1 μl), prepared by ISO 5509 (2000) standard method, were separated on a GC–FID system (Agilent 6890 N GC, Agilent Technologies, USA) equipped with a BPX 70 capillary column (60 m length, 0.22 mm i.d., 0.25 μm film thickness). Helium was used as a carrier gas at a flow rate of 1.5 ml/min. The column temperature was programmed at 2 °C/min with initial temperature 130 °C and final temperature 235 °C. The injector was set at 230 °C with split ratio of 100:1 and the detector was set at 250 °C. The fatty acids were identified by comparing their retention times with those of the authentic fatty acid methyl esters (FAME) standards under the same conditions.

Tocopherols

Samples were prepared by dissolving 0.2 g of rapeseed oil in 5 ml of ACN/MtBE mixture (4:6 by vol.), and then analyzed by VP Shimadzu HPLC system equipped with the DAD detector (SPD-M10Avp Shimadzu), the FLD detector (RF-10ALxl Shimadzu), reversed phase octadecyl silica C18 HPLC column Gemini C18 (150 mm × 2 mm × 3 μm) (Phenomenex Torrance, CA, USA), and a suitable guard column. The isocratic mobile phase was a mixture of ACN and MtBE (4:6 v/v) at a flow rate of 0.15 ml/min, and the column oven temperature was 35 °C. Tocopherols were detected by standard UV spectrum analysis (190–370 nm). Quantification of tocopherols was conducted using data from the fluorescence detector (FLD) with excitation/emission wavelengths of 290/330 nm, respectively. All samples were analysed in triplicate and the tocopherol/oil ratio was expressed in mg/100 g.

Phytosterols

Rapeseed oil (0.2 g) was dissolved in 3 ml of hexane, and then 50 μl of 5α-cholestane (0.4 mg/ml) was added as an internal standard. The mixture was saponified with 2 M methanolic KOH at room temperature for 1 h. Then, 700 μl of unsaponified fraction was transferred into 1.5 ml vial and the solvent was evaporated to dryness under nitrogen. Dry residues were dissolved in 100 μl pyridine and silylated with 100 μl BSTFA containing 1 % TMCS. Derivatives of the sterols were separated on a GC–MS system (Agilent 6890 N GC, Agilent Technologies, USA) equipped with a DB-5MS capillary column (30 m × 0,25 mm × 0,25 μm; Phenomenex Torrance, CA, USA). Column temperature was programmed in the range from 250 to 300 °C. The temperatures of detector and injector were set at 310 and 280 °C, respectively. Helium was used as carrier gas at a flow rate of 1.5 ml/min. The split ratio was 25:1. Sterols were identified based on a laboratory sterol spectra library, as well as the on-line NIST Mass Spectra Library.

Peroxide value, acidity, specific UV extinctions (K₂₃₂ and K₂₆₈)

Peroxide value (PV) (meq O₂/kg) and acidity (expressed in %), were determined according to the ISO standard methods 3960 (1996) and 660 (2005), respectively. The conjugated dienes and trienes, expressed by absorption coefficient E1cm1%at λ_max 232 and 268 nm, were determined using Thermo Spectronic Helios β spectrophotometer, in accordance with ISO 3656 (2011) standard method.

Tocopherol contents

The total content of tocopherols in control oil sample was 57.89 mg/100 g (23.75 mg/100 g α-T, and 34.14 mg/100 g γ-T) (Table ​(Table2).2). Results presented in Table ​Table22 showed, that individual tocopherols were significantly different affected by the various storage conditions, however, in all investigated storage conditions α-T degradation proceeded more rapidly than that of γ-T. Based on the percentage of losses, the highest rate of tocopherols degradation was observed when oil was exposed to oxygen and stored at RT. Under such conditions, storage under light resulted in ca. 90–91 % loss of α-T, and ca. 80–81 % loss of γ-T. When oil samples were kept in dark, ca. 90–91 % of α-T, and ca. 77–79 % of γ-T has degraded. The substantial loss of individual tocopherols was also noted when oil samples remained closed over entire storage period. Interestingly, negligible differences in the percentage loss of tocopherols between samples kept in the dark and exposed to light, were observed. When oil samples were stored in closed containers at 20 °C under light the percentage loss of α-T was as follows: 84 % (amber glass), and 88 % (amber PET). Respective loss of γ-T, ranged from 61 % to 62 %. Storage in the dark resulted in ca. 82–84 % loss of α-T, and ca. 60–65 % loss of γ-T. The least losses in the nutritional value, and thus the highest tocopherols content was found in oil samples kept closed and stored under refrigerated temperature, where the total tocopherols content left after 12 months of storage ranged from 25.54 to 26.24 mg/100 g for oil stored in PET and glass containers, respectively. In contrast, when exposed to oxygen, the percentage loss of the total tocopherols increased to 70 % (amber glass), and 72 % (amber PET).

The results indicating losses of tocopherols and diminished nutritional value of cold-pressed rapeseed oil were consistent with numerous studies related to extra virgin olive oil and refined oils. Fadda et al. (2012) found only ca. 13 % α-T degradation in extra virgin olive oil over 18 months of storage in the dark, while Okogeri and Tasioula-Margari (2002) reported up to 50 % loss of α-T after 12 months of storage of olive oil, whereas nearly complete loss of α-T over 12 month of storage was found by Morello et al. (2004). The substantial loss of tocopherols during entire storage period may be assigned to the fact that they act as hydrogen donor or oxygen quencher to stop the chain mechanism of auto- and photo-oxidation, respectively. Tocopherols protect lipids from the oxidation by donating their hydrogen to lipid peroxy radicals (ROO·), which leads to formation of lipid hydroperoxide (ROOH) and tocopheroxy radicals (T·) (Choe and Min 2006). These radicals were more stable than lipid peroxy radicals and thereby responsible for the slowdown in the rate of oil auto-oxidation. When oil samples were exposed to light, tocopherols prevented oil from photo-oxidation by singlet oxygen (^O₂) quenching. The reaction rate of tocopherols with ^O₂ was affected by their structures. α-T exhibited the highest reaction rate, followed by β-, γ-, and δ-T, which may explain greater decline of α-T than γ-T after 12 months of storage (Choe and Min 2006). The finding that α-T decomposed faster than γ-T during long-term storage of oil has also been reported previously by Mezouari and Eichner (2007) and Samaniego-Sanchez et al. (2012).

Phytosterol content

The fresh cold-pressed rapeseed oil contained a total of 700.98 mg/100 g sterols (Table ​(Table3).3). The following phytosterols have been determined in the analysed fresh oil sample: β-sitosterol, ca. 51 %, and campesterol, ca. 32 %, followed by brassicasterol, ca. 13 %, while the content of Δ5-avenasterol and stigmasterol was approximately 3 % of total phytosterols.

As can be seen from Table ​Table3,3, the highest degradation rate of total sterols was found in rapeseed oil samples exposed to oxygen and stored at RT with the access of light. Under such conditions, the percentage loss of total sterols ranged from 15.1 % (amber glass) to 16.3 % (amber PET). Respective loss of total sterols in oil samples kept closed over entire storage period was 1.7 % and 2.9 %, for glass and PET containers, respectively. Storage in the dark (at RT) resulted in similar degradation rate of total sterols, regardless of the access of oxygen. Of the total sterol content, ca. 4.7–5.5 % were lost in oil samples kept closed over entire storage period, while respective loss of sterols in samples exposed to oxygen ranged from 4.4 to 5.5 %. The type of packaging material was found to have statistically significant (p < 0.05) impact on the degradation rate of sterols in oil samples stored under refrigerated temperature. When samples were stored in amber glass containers, the percentage loss of total sterols ranged from 0.5 to 2.6 %, for containers kept closed throughout entire storage period and opened once per month, respectively. Respective loss of sterols in samples stored in amber PET containers amounted to 2.6 % and 5.0 %.

In contrary to tocopherols, sterols showed high storage stability over the entire storage period. This may be explained by the fact, that tocopherols were consumed first when protecting PUFAs from oxidation (Pekkarinen et al. 1998). Similar dependence between tocopherol and sterol stability during prolonged crude rice oil storage was observed by Mezouari and Eichner (2007), where 4.9 % loss of the total sterols content, and about 72 % decrease of the total tocopherol content was reported in oil samples kept in the dark. However, when oil samples were exposed to light, the percentage loss of the total sterols increased to 49.2 %, with practically complete loss of tocopherols after 240 days of storage.

Acidity, peroxide value, K₂₃₂ and K₂₆₈

After the entire storage period, the degree of hydrolysis, assessed in terms of changes in acidity, remained below the threshold limit of 0.8 %, specified for extra virgin olive oil, regardless of the type of packaging and storage conditions (Table ​(Table4).4). This may be linked to the fact that the FA composition showed slight alterations over the 12 months of storage, and hence, a small rise in the formation of FFA occurred.

Initial value of PV of cold-pressed rapeseed oil was 2.9 meq O₂/kg (Table ​(Table4).4). Contrary to acidity, PV showed statistically significant (p < 0.05) variations highly correlated with the storage conditions. The PVs for samples stored at 4 °C in closed containers were as follows: 4.8 meq O₂/kg, for amber glass, and 6.0 meq O₂/kg for amber PET. Respective PVs in the presence of oxygen at the same temperature were 11.21 meq O₂/kg, for amber glass, and 12.7 meq O₂/kg, for amber PET. When oil samples were stored in closed containers at 20 °C in the dark the PVs were as follows: 6.0 meq O₂/kg (amber glass), and 7.6 meq O₂/kg (amber PET). Respective PVs in the presence of oxygen, for samples kept in the dark, at the same temperature were 29.6 meq O₂/kg, for amber glass, and 30.3 meq O₂/kg, for amber PET. When samples were kept in closed containers and stored under the same temperature in the presence of light the PVs were 5.7 meq O₂/kg (amber glass), and 7.7 meq O₂/kg (amber PET), and the respective PVs in the presence of oxygen were as follows: 28.2 meq O₂/kg and 28.3 meq O₂/kg, for amber glass and amber PET, respectively. A strong correlation has been found between peroxides formation and oil exposure to oxygen, leading to conclusion that the most critical factor affecting the rate of oxidation in the cold-pressed rapeseed oil is the oxygen concentration. Similar findings were stated by Pristouri et al. (2010) who reported higher losses in extra virgin olive oil quality stored with high oxygen headspace concentrations as compared to those stored without the access of oxygen (no headspace).

As a result of auto-oxidation and by oxygen initially dissolved in the oil, K₂₃₂ increased from 1.88 to 2.10 (amber glass), and 2.26 (amber PET) after 12 months of storage in the dark at 20 °C. In contrast, when exposed to oxygen, K₂₃₂ increased to 5.50 (amber glass), and 4.73 (amber PET). In the presence of light, for samples stored at 20 °C, K₂₃₂ increased to 2.10 (amber glass), and 2.05 (amber PET), when exposed to oxygen, K₂₃₂ increased to 3.11 (amber glass), and 3.47 (amber PET). On the other hand, storage at 4 °C resulted in the slowest dienes formation, K₂₃₂ increased to 1.89 (amber glass), and 2.06 (amber PET), and to 2.19 (amber glass), and 2.10 (amber PET) in the presence of oxygen in the headspace. Similarly to Pristouri et al. (2010), the most pronounced changes in K₂₃₂ were caused by the access of oxygen, followed by storage temperature and the presence of light.

The initial value for K₂₆₈ was 0.11. Respective K₂₆₈ for oil samples stored at 4 °C, regardless of the access of oxygen, remained practically unchanged over entire storage period (Table ​(Table44).

Similar changes in K₂₆₈ were caused by the presence of light, K₂₆₈ increased to 0.13–0.14, regardless of the presence of light, and to 0.19–0.23, in the presence of the oxygen in the headspace. The above results were in general in agreement with the results obtained by Guillaume and Ravetti (2012) and Méndez and Falqué (2007), who reported that amber PET bottles are able to provide similar protection of olive oil against light as amber glass bottles.