Cholesterol oxidation in some processed fish products

Numerous foods of animal origin are reported to contain considerable levels of cholesterol oxidation products (COP); however, very few reports are available on fish products. Levels of COP were assessed in samples of fish roe, fish oil, and fish meal. Among the fish roe samples, the smoked cod roe had the highest amount of COP, 93 μg/g lipids. Refined and bleached menhaden oil had 8 μg/g, and two experimental alkali-refined, bleached, and deodorized herring fish oil samples contained similar amounts of COP. The range of total COP in the three fish meal samples ranged from 50 to 78 μg/g fish meal. Generally, processed fish roe contained high amounts of COP compared with refined fish oils, which had very low amounts of COP. Fish meal samples had very high amounts of COP.     

Effect of synthetic antioxidants on cholesterol stability during the thermal-induced oxidation of a polyunsaturated vegetable oil

As a molecule with an unsaturated bond, cholesterol is prone to oxidation. Cholesterol oxidation products (COP) are found in many common foods and have been shown to be atherogenic, cytotoxic, mutagenic, and possibly carcinogenic. Efforts to reduce the formation of oxidation products are considered important during the manufacture and processing of foods. The effect of synthetic antioxidants on cholesterol oxidation has not been extensively studied. We assayed the effect of five commonly used antioxidants—BHT, BHA, the n-propyl ester of 3,4,5-trihydroxy benzoic acid (PG), TBHQ, and 6-ethoxy-1,2-dihydro-2,4-trimethylquinoline (EQ)—on cholesterol stability when oxidation is induced in a Rancimat 679 instrument by bubbling air through the sample at 150°C. The sample consisted of 200 mg cholesterol dispersed in 100 g of a polyunsaturated vegetable oil (soybean oil). Formation of six COP was measured at the induction period, and at the 50 and 100 μS conductivity values. Under the experimental conditions, BHT and TBHQ were the most effective inhibitors of cholesterol oxidation. BHA and EQ were less effective, and PG was unable to prevent cholesterol oxidation. Synthetic antioxidants were more effective in preventing COP formation at the nucleus of the cholesterol structure than at the lateral chain.     

Enhancing Lipid Oxidative Stability of Cooked‐Chilled Lamb Meat through Dietary Rosemary Diterpenes

A dietary rosemary extract (DRE) containing carnosic acid and carnosol at 1:1 (w/w) for enhancing the lipid oxidative stability in cooked‐chilled lamb meat, is evaluated. Three diets for fattening lambs are tested: i) a cereal‐based concentrate (C‐diet); ii) the C‐diet plus 600 mg vitamin E per kg feed (E‐diet); and iii) the C‐diet plus 600 mg rosemary diterpenes per kg feed (R‐diet). Griddled‐chilled lamb patties are kept at 4 °C and lighting for 2 days, simulating catering conditions. Diterpenes have a lower deposition rate than vitamin E in lamb muscle and are completely degraded during cooking. DRE is thus less effective than dietary vitamin E in enhancing the oxidative stability of the patties. After 2‐day storage, the R‐diet shows lower (p < 0.01) peroxide values and thiobarbituric acid reactive substances than the C‐diet, while, in contrast to the E‐diet, it does not inhibit (p > 0.05) the formation of cholesterol oxidation products. The R‐diet increases (p < 0.05) the proportion of polyunsaturated fatty acids and decreases (p < 0.05) the n‐6/n‐3 ratio. These findings suggest antioxidant protection by dietary bioactive compounds beyond the direct radical scavenging activity that is able to stabilize lipids during the meat shelf‐life. Practical Applications: Cooked‐chilled meat lipids strongly oxidize in ready‐to‐eat dishes kept in retailing conditions, which may negatively affect their levels of polyunsaturated fatty acids (PUFA), cholesterol oxidation products (COP), and other lipid oxidation products. Dietary rosemary diterpenes can be used as a clean alternative to feed additives to enhance the oxidative stability of cooked‐chilled meat. Improved health and antioxidant status of the animal might be able to reduce oxidative spoilage during meat shelf‐life. Diterpenes provide lesser antioxidant protection than dietary vitamin E but may improve the PUFA content, with positive implications for the nutritional quality of lamb fat. The use of dietary antioxidants with different properties may contribute to improving the efficacy of animal feeds to improve meat quality.     

Thermal degradation of long‐chain polyunsaturated fatty acids during deodorization of fish oil

Long‐chain polyunsaturated fatty acids (LC‐PUFA) of the n‐3 series, particularly eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid, have specific activities especially in the functionality of the central nervous system. Due to the occurrence of numerous methylene‐interrupted ethylenic double bonds, these fatty acids are very sensitive to air (oxygen) and temperature. Non‐volatile degradation products, which include polymers, cyclic fatty acid monomers (CFAM) and geometrical isomers of EPA and DHA, were evaluated in fish oil samples obtained by deodorization under vacuum of semi‐refined fish oil at 180, 220 and 250 °C. Polymers are the major degradation products generated at high deodorization temperatures, with 19.5% oligomers being formed in oil deodorized at 250 °C. A significant amount of CFAM was produced during deodorization at temperatures above or equal to 220 °C. In fact, 23.9 and 66.3 mg/g of C20 and C22 CFAM were found in samples deodorized at 220 and 250 °C, respectively. Only minor changes were observed in the EPA and DHA trans isomer content and composition after deodorization at 180 °C. At this temperature, the formation of polar compounds and CFAM was also low. However, the oil deodorized at 220 and 250 °C contained 4.2% and 7.6% geometrical isomers, respectively. Even after a deodorization at 250 °C, the majority of geometrical isomers were mono‐ and di‐trans. These results indicate that deodorization of fish oils should be conducted at a maximal temperature of 180 °C. This temperature seems to be lower than the activation energy required for polymerization (intra and inter) and geometrical isomerization.     

Supercritical fluid chromatographic analysis of fish oils

Various natural and processed fish oil triglyceride mixtures have been analyzed by capillary supercritical fluid chromatography (SFC). The analyses were performed on nonpolar columns to separate the components by lipid class and by the number of carbon atoms. The compounds separated included free fatty acids, squalene, α-tocopherol, cholesterol, wax esters, cholesteryl esters, di- and triglycerides. This kind of analysis is not possible by gas chromatography or high-performance liquid chromatography methods without prior treatment of the fish oil, making SFC superior for this application. Applications of SFC to fish oils are given, including a control analysis of the various process steps in the refining of a fish oil, analysis of a lipase-catalyzed transesterification of a fish oil and the detection of polymeric artifacts.     

Separation and quantitation of cholesterol oxides by HPLC with an evaporative light scattering detector in a model system

A method to analyze cholesterol and 10 of its oxidation products, ranging from the weakly polar cholest-4-ene-3,6-dione to moderately polar cholest-5-ene-3β,7α-diol, in a single run is described. The separation was achieved by normal-phase gradient high-performance liquid chromatography with an evaporative light-scattering detector. This universal mass detector does not detect changes in solvent composition; this makes it possible to employ gradients, an essential technique whenever a wide range of compounds with diverse characteristics is to be separated. Standards at concentrations from 0.1–1.0 μg were separated within 37 min on an alumina/silica column with a gradient elution system that contained dichloromethane, acetonitrile, and water.     

Oxidative interactions of cholesterol with triacylglycerols

Triacylglycerols (TGs) accelerated the decomposition of cholesterol at 130°C. Addition of stearic and linoleic acids also accelerated cholesterol decomposition and produced characteristic cholesterol oxide profiles, qualitatively different from those produced in the presence of TGs. Milk fat accelerated cholesterol decomposition at 130°C and produced a cholesterol oxide pattern similar to that arising from the addition of pure TG. Not only did TGs affect cholesterol oxidation, but cholesterol influenced the decomposition of TGs. Addition of cholesterol accelerated the destruction of TGs at the beginning of heating while protecting them later. A similar pattern,i.e., acceleration followed by protection, could also be seen when triacylglycerols were heated in the presence of other triacylglycerols. The results of this work demonstrate that the stability of lipid components in complex mixtures is influenced by interactions among these components and/or their decomposition products. Such interactions do not merely shift,i.e., accelerate or delay, the oxidation rate, they may also modify the shape of the oxidation curve itself.     

Oxidative effects of animal-originated porphyrins and riboflavin on cholesterol oxidation in an aqueous model system

This study was executed to investigate effects of animal-originated porphyrins and riboflavin on cholesterol oxidation in aqueous model systems. Changes of headspace oxygen contents, cholesterol, and cholesterol oxide products (COP) in the model systems were measured by gas chromatography during storage under light. As concentration of protoporphyrin increased, contents of headspace oxygen decreased and COP increased. The same trend as that of protoporphyrin occurred with riboflavin in terms of contents of headspace oxygen, but production of COP was the highest at 5 ppm riboflavin. As concentrations of hemoglobin and myoglobin increased, headspace oxygen content and COP production were not changed significantly. Consequently, protoporphyrin could be the most active catalyst on the cholesterol oxidation in the aqueous system, but myoglobin and hemoglobin did not accelerate cholesterol oxidation.     

Effect of α‐, γ‐, and δ‐tocopherol on the distribution of volatile secondary oxidation products in fish oil

The relative ability of α‐, γ‐ and δ‐tocopherol (TOH) to influence the distribution of volatile secondary oxidation products in fish oil was studied, with particular emphasis on oxidation products expected to be important for adverse flavour formation. Purified fish oil samples with 100 ppm or 1000 ppm of the different tocopherols were analysed by dynamic headspace analysis of the volatiles formed after 2, 5 and 8 d of storage at 30 �C. The tocopherol type and concentration affected not only the overall formation of volatile secondary oxidation products, but also the composition of this group of oxidation products. Principal component analysis of the data obtained suggested that high tocopherol hydrogen‐donating power, i.e. a high tocopherol concentration or the use of αTOH as opposed to γTOH or δTOH, directs the formation of hydrocarbons, unsaturated carbonyl compounds of relatively high molecular weight, as well as the formation of cis, trans isomers of unsaturated aldehydes. Although an active inhibitor of overall volatile formation, αTOH at a high concentration thus appears to direct the formation of the more flavour‐potent aldehydes, such as those linking the carbonyl group with ethylenic conjugated unsaturation.     

Oxidative flavour deterioration of fish oil enriched milk

The oxidative deterioration of milk emulsions supplemented with 1.5 wt‐% fish oil was investigated by sensory evaluation and by determining the peroxide value and volatile oxidation products after cold storage. Two types of milk emulsions were produced, one with a highly unsaturated tuna oil (38 wt‐% of n‐3 fatty acids) and one with cod liver oil (26 wt‐% of n‐3 fatty acids). The effect of added calcium disodium ethylenediaminetetraacetate (EDTA) on oxidation was also investigated. Emulsions based on cod liver oil with a slightly elevated peroxide value (1.5 meq/kg) oxidised significantly faster than the tuna oil emulsions, having a lower initial peroxide value (0.1 meq/kg). In the tuna oil emulsions the fishy off‐flavour could not be detected throughout the storage period. Addition of 5—50 ppm EDTA significantly reduced the development of volatile oxidation products in the cod liver oil emulsions, indicating that metal chelation with EDTA could inhibit the decomposition of lipid hydroperoxides in these emulsions. This study showed that an oxidatively stable milk emulsion containing highly polyunsaturated tuna fish oil could be prepared without significant fishy off‐flavour development upon storage, provided that the initial peroxide value was sufficiently low.     

Gamma-irradiation of individual cholesterol oxidation products

Cholesterol and seven of its oxidation products in aqueous suspensions of multilamellar vesicles or sonicated aqueous suspensions were subjected individually to γ-radiation (10 KGy) at 0–4°C in air, N₂ or N₂O. All compounds underwent some changes under the influence of radiation. β-Epoxide (cholesterol 5β,6β-epoxide) and, to a much lesser extent, α-epoxide (cholesterol 5α,6α-epoxide) were converted in low yield to 6-ketocholestanol (5α-cholestan-3β-ol-6-one). 7β-Hydroxycholesterol (cholest-5-ene-3β,7β-diol) and, to a lesser extent, 7α-hydroxycholesterol (cholest-5-ene-3β,7α-diol) gave low yields of 7-ketocholestanol (5α-cholestan-3β-ol-7-one). The latter compound also was obtained by irradiation of 7-ketocholesterol (cholest-5-ene-3β-ol-7-one). 6-Ketocholestanol and 7-ketocholestanol are potential biomarkers for irradiated meat and poultry.     

Oxidative stability and acceptability of camelina oil blended with selected fish oils

The effects of blending camelina oil with a number of fish oils on oxidative stability and fishy odour were evaluated. Camelina oil was found to be more stable than tuna oil, ‘omega‐3’ fish oil and salmon oil as indicated by predominantly lower ρ‐anisidine (AV), thiobarbituric acid reactive substances (TBARS) and conjugated triene levels (CT) during storage at 60 °C for 20 days (p < 0.05). Peroxide values (PV) were similar for all oils until Day 13 when values for camelina oil were higher. Values for blends of the fish oils (50, 25, 15, 5%) with camelina oil were generally between those of their respective bulk oils indicating a dilution effect. Camelina oil had a similar odour score (p < 0.05) to sunflower oil (9.2 and 9.6, respectively) indicating, as expected, an absence of fishy odours. In comparison, the fish oils had lower scores of 6.1 to 6.6 (p < 0.05) indicating mild to moderate fishy odours. Odour scores were improved at the 25% fish oil levels (p < 0.05) and were not different to camelina oil at the 15 or 5% levels (p < 0.05). Practical applications: Camelina oil is a potentially important functional food ingredient providing beneficial n‐3 PUFA. Oil extracted from Camelina sativa seeds contains greater than 50% polyunsaturated fatty acids of which 35‐40% is α‐linolenic acid (C18:3ω3, ALA), an essential omega‐3 fatty acid 1 . While EPA and DHA from fish oils are more potent nutritionally, they are less stable than ALA. This work evaluated innovative blends of fish oil with camelina oil for stability and acceptability. The results demonstrate that there is potential for use of blends of camelina oil with fish oils in food products, as the results show some benefits in terms of reduction of fishy odours. Such information could be valuable in relation to formulation of food products containing high levels of n‐3 PUFA from both plant and fish sources.     

Effects of the degree of unsaturation of coexisting triacylglycerols on cholesterol oxidation

In order to evaluate the effects of the degree of unsaturation of triacylglycerols on cholesterol oxidation, mixtures of purified sardine oil triacylglycerols (iodine value, IV=182.6) and cholesterol; of partially hydrogenated sardine oil triacylglycerols (IV=174.5) and cholesterol; and of fully hydrogenated sardine oil triacylglycerols (IV=92.0) and cholesterol were incubated at 25°C in the dark. The oxidative stability of the samples decreased with increasing degree of unsaturation of the triacylglycerols in the sample mixtures; the induction period for peroxide values (PV) of the sardine oil triacylglycerols and cholesterol was shorter than that of the partially hydrogenated sardine oil triacylglycerols and cholesterol. Certain polyunsaturated fatty acids (PUFAs) in the constituent fatty acids of sardine oil triacylglycerols started to decrease after a shorter induction period compared with that of the partially hydrogenated triacylglycerols. The prominent cholesterol oxides accumulated in the samples were 7β-hydroxycholesterol, 7-ketocholesterol, β-epoxide and cholestane triol. The tendency for accumulation of cholesterol oxides in the time course coincided with the changes in PV as well as the decrease in PUFAs. Cholesterol was oxidized in conjunction with autoxidation of coexisting fish oil triacylglycerols. Although lowering the degree of unsaturation of fish oil triacylglycerols was effective in prolonging the induction period of cholesterol oxidation, the rate of cholesterol oxidation in the cholesterol oxides' formation phase after the induction period was not affected by the difference in the proportion of highly unsaturated fatty acids in the natural and partially hydrogenated triacylglycerols of fish oils.     

Dietary fish oil inhibits Δ6-desaturase activity in vivo

Liver Δ6-desaturase activity was determined in mice which were made deficient in (i) n-6 and n-3 polyunsaturated fatty acids (PUFA), (ii) n-6 PUFA, or (iii) arachidonic acid (AA). Initially, the mice were subjected to two cycles of a fasting (1 d)/refeeding (2–3 d) protocol in which they were refed an essential fatty acid-deficient (EFAD) diet during the refeeding period. This 1-wk fasting/refeeding protocol, referred to as F/R EFAD, produced a rapid and substantial decline in tissue n-3 and n-6 PUFA and a corresponding increase in n-9 fatty acids, notably oleic acid and Mead acid (20:3n-9). Combined liver Δ6-desaturase/elongase/Δ5-desaturase activities in vivo were quantified by measuring the conversion of ¹⁴C-linoleic acid (LA) to ¹⁴C-AA in mouse liver. Although F/R EFAD caused, as expected, a substantial decline in liver AA and LA content, the conversion of ¹⁴C-LA to ¹⁴C-AA was the same in these mice as in chow-fed controls (approximately 33–34%). Subsequent refeeding of F/R EFAD mice with an EFAD diet, supplemented with corn oil, restored tissue n-6 PUFA levels without altering the conversion of ¹⁴C-LA to ¹⁴C-AA. In contrast, refeeding with an EFAD diet, supplemented with fish oil, inhibited ¹⁴C-LA to ¹⁴C-AA conversion by 78%. Significantly, inhibition of conversion of ¹⁴C-LA to ¹⁴C-AA was maintained in F/R EFAD mice that were subsequently fed an EFAD diet supplemented with a 1:1 mixture of fish oil/corn oil. This latter protocol yielded a unique liver fatty acid composition in which AA was selectively depleted, whereas LA and the n-3 PUFA were increased. The data suggest that dietary n-3 C_20–22 PUFA negatively regulate the in vivo synthesis of n-6 PUFA at the level of the Δ6-desaturase.     

Increasing n-3 polyunsaturated fatty acid content of fish oil by temperature control of lipase-catalyzed acidolysis

An attempt was made to further increase the content of n-3 polyunsaturated fatty acid (n-3 PUFA) of fish oil by lipase-catalyzed acidolysis (reaction between fish oil and n-3 PUFA-enriched free fatty acid) without solvent. A bioreactor system was constructed composed of a water-jacketed packed-bed column and a substrate reservoir with a circulation pipeline between the packed-bed column and the reservoir. By keeping the temperature of the reservoir at −10°C (for the first 20 h), followed by −20°C (for the subsequent 40 h) during the batch acidolysis, crystals of free fatty acid appeared, which were removed intermittently by a cotton plug packed in the tip of the outlet pipe in the reservoir. The n-3 PUFA content of the triacylglycerol fraction increased a further 10% by the reduced temperature of the reservoir. Bioreactors for Enzymatic Reaction of Fats and Fatty Acid derivatives, Part XV.     

The antioxidant properties of ethoxyquin and of some of its oxidation products in fish oil and meal

Ethoxyquin is an effective antioxidant for both fish oil and fish meal but its antioxidant activity often follows an initial burst of pro-oxidant activity. Some oxidation products of ethoxyquin are shown to be powerful antioxidants as determined from measurements by the Warburg manometric technique. Of the compounds examined, 2,6-dihydro-2,2,4-trimethyl-6-quinone imine and ethoxyquin nitroxide showed marked antioxidant activity.     

Capabilities of different cooking oils in prevention of cholesterol oxidation during heating

The potential of various cooking oils to prevent cholesterol degradation and/or oxidation, as measured by the production of 7-ketocholesterol during heating at different temperatures, was studied using a cholesterol model system. In the control group (without cooking oil), cholesterol was relatively stable, and 73% of its initial concentration was present after 30 min of heating at 125°C. Less than 30 and 10% of cholesterol remained at 150 and 175°C after 30 min, respectively, and 10% at 200°C after 10 min. In the treatment group, cholesterol mixed with corn, canola, soybean, or olive oil had significantly improved thermal stability. More than 60 and 40% of cholesterol remained at 150 and 175°C after 30 min, respectively. In the control group, 7-ketocholesterol was produced when samples were heated above 150°C, and levels increased consistently during 30 min of heating. At 175 or 200°C, the level of 7-ketocholesterol did not increase further after reaching the highest level after 10 min of heating. 7-Ketocholesterol is not stable above 175°C, and its degradation rate could be much faster than its production at 200°C. 7-Ketocholesterol was not found in samples of cholesterol mixed with corn oil or laboratory-prepared soybean and rice bran oils until the heating temperature was raised to 175°C for 20 min. The levels of 7-ketocholesterol in those treatment groups were greater than that in the control group at 175°C for 30 min. These oils may increase the thermal stability of 7-ketocholesterol and retard its degradation rate.     

Application of Saponin for Cholesterol Removal from Pacific White Shrimp (Litopenaeus vannamei) Lipid

The removal of cholesterol from lipid obtained from Pacific white shrimp (Litopenaeus vannamei) cephalothorax using saponin in conjunction with celite under various conditions is investigated. Various lipid:saponin ratios (1:2, 1:4, and 1:6, W/W) with different lipid:solvent ratios (1:20, 1:40, and 1:80, W/V) are employed with a treatment time of 4 h. The highest cholesterol removal (88.77 ± 0.18%) is obtained with a lipid:saponin ratio of 1:6 and 20 volumes of 50% ethanol. As the treatment time is increased from 1 to 4 h, higher cholesterol removal is achieved. Lipid peroxides and conjugated dienes are higher in the lipid after cholesterol removal compared to untreated lipid, while thiobarbituric acid reactive substances, the ρ‐anisidine value and free fatty acids are reduced. However, the polyunsaturated fatty acid (PUFA) content in the saponin‐treated lipid (18.06 ± 0.37 g/100 g^?1) is higher than that of untreated lipid (15.80 ± 0.17 g/100 g^?1). FTIR spectra confirmed the formation of hydroperoxides, with lower aldehyde, phospholipid and free fatty acid contents in the cholesterol‐removed lipid. The process developed employing saponin is able to produce crude shrimp lipid with a pronounced decrease in cholesterol, but augmented PUFAs, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Practical Applications: Shrimp lipid is a vital byproduct capable of extraction from cephalothorax. Due to the presence of astaxanthin, EPA and DHA, shrimp lipid is considered to be a highly useful nutraceutical product. However, the cholesterol content of shrimp lipid is a major constraint on its human consumption. The negative impact of cholesterol content can be overcome by lowering the cholesterol content. Saponin treatment of shrimp lipid is able to reduce the cholesterol level and increase the PUFA, thus solving the high cholesterol problem and also gaining augmented health benefits.