High-Resolution NMR Detection of Cholesterol Oxides in Spray-Dried Egg Yolk

A new approach, alternative to currently available methods, for studying cholesterol oxidation in egg powder is reported. The quantitative analysis of cholesterol oxides was carried out by high-resolution proton nuclear magnetic resonance (¹H-NMR). The following oxides were isolated from spray-dried egg powder by rapid chromatographic procedures and detected by selecting “marker”¹H-NMR signals: 7-ke-tocholesterol, 7α-hydroxy-cholesterol, 7β-hydroxy-cholesterol, cholesterol-α-epoxide and cholesterol-β-epoxide. The quantified cholesterol derivatives ranged from 4.9 to 9.1 ppm with a detection limit of 0.3 ppm (5 μ.g from 16g of matrix). The main usefulness of the method should be in investigating intermediates and products due to chemical transformation of cholesterol during storage and heating of foodstuffs.     

Enzymatic Determination of Cholesterol Oxides

For measurement of the concentration of oxidised cholesterols in foodstuffs, a method was developed by adapting the well-known enzymatic determination of cholesterol. A linear correlation was found between the absorbance measured after enzymatic reaction and the concentrations of 7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, cholesterol-5α, 6α-epoxide in the range 1–12 μg. The usefulness of this method was demonstrated by determination of the four cholesterol oxidation products separated by TLC from the non-saponifiable lipid fraction of 5-month-old whole-egg powder (stored under different conditions) and of biscuit enriched with egg powder. The main advantages of the combination of TLC and the enzymatic method, beyond its rapidity and sensitivity, are that the determination of cholesterol oxidation products can be carried out in parallel with many samples containing small amounts of oxidised cholesterols even in the presence of a large quantity of cholesterol.     

Antioxidant Inhibition of Cholesterol Oxidation in a Spray-Dried Food System during Accelerated Storage

Spray-dried egg yolk was used to evaluate antioxidant inhibition of cholesterol oxidation during accelerated (CU⁺² and heat) storage. Liquid yolk was treated with equimolar amounts of BHA (0.01%, w/w of lipid), ascorbyl palmitate (0.023%), or a tocopherol blend (0.023%). Yolk batches were spray-dried, stored at 60 ± 2°C for up to 28 days, and analyzed for cholesterol oxidation products (COPS) using gas chroma-tography. COP levels generally increased during storage with 7-ketocholesterol predominant. Significant antioxidant effects were manifest in decreased levels of 7-ketocholesterol, 7α- and 7β-hydroxycholesterol; while cholestane-triol and cholesterol-5,6-epoxide levels were not affected. All antioxidants showed significant inhibitive effects relative to the control, and tocopherols were more effective than ascorbyl palmitate.     

Effect of cooking and storage on lipid oxidation and development of cholesterol oxidation products in water buffalo meat

Buffalo meat was subjected to two cooking methods viz. broiling and pressure cooking and two storage procedures viz. refrigerated (4 °C) storage for six days and frozen (-10 °C) storage for 90 days. Changes in lipid oxidation and development of cholesterol oxidation products were studied in raw as well as cooked meat samples. Total lipid, phospholipid, cholesterol, free fatty acid, glycolipid and glyceride contents increased significantly on cooking of meat but did not show any significant changes during either refrigerated or frozen storage except for free fatty acid content which showed an increase. The TBA values also increased during storage but not to the extent of indicating rancidity. Cholesterol oxidation products separated by thin layer chromatography were: cholestanetriol, 7-α-hydroxycholesterol, 19-hydroxycholesterol, 7-ketocholesterol, cholesterol-α-epoxide, cholesterol-β-epoxide and an unidentified fraction. All these fractions, except for the unidentified fraction, increased on cooking and storage. The cholesterol-β-epoxide fraction was resistant to changes. Changes in broiled meat were more pronounced compared to pressure cooked meat. Frozen storage did not prevent the development of cholesterol oxidation products in buffalo meat.     

Cholesterol Oxides in Swedish Foods and Food Ingredients: Fresh Eggs and Dehydrated Egg Products

Cholesterol oxides were enriched from lipid extracts of fresh and dehydrated egg yolk products by chromatography on Lipidex and TEAP-Lipidex. Appropriate fractions from TEAP-Lipidex were analyzed by capillary GLC as their TMS derivatives. At the detection limit level, ca 0.2 ppm in total lipids, no cholesterol oxides could be detected in fresh egg yolk. Similarly, spray dried egg yolk powder contained only traces of cholesterol oxides when fresh or stored for 2 months at 4°C. Prolonged storage gave lipid extracts which contained variable levels (0.2–12 ppm) of known oxidation products, viz., cholest-5-ene-3β, 7α-diol, cholest-5-ene-3β, 7β-diol, 5,6α-epoxy-5α-cholestan-3β-ol, 5,6 β-epoxy-5 β-cholestan-3β-01, cholest-5-ene-3β, 20α-diol, 3 β-hydroxycholest-5-en-7-one. Traces or small quantifiable amounts of cholest ?5-ene-3β, 25-diol and 5α-cholestane-3β, 5, 6β-triol were observed in some samples at longer storage periods.     

Influence of carcass weight on instrumental and sensory lamb meat quality in intensive production systems

The effects of cooking viz. pressure-cooking and broiling and storage at 4 °C for six days and -10 °C for 90 days on lipid oxidation and development of cholesterol oxidation products in mutton were studied. Results revealed that cooking of meat significantly increased the total lipids, phospholipids, cholesterol, glycolipids, free fatty acids and glycerides, but they did not change during refrigerated and frozen storage. The TBA values increased on cooking and during storage. However, the values were below the threshold level for rancidity development. The following cholesterol oxidation products were separated by thin layer chromatography cholestanetriol, 7-α-hydroxy cholesterol, 19-hydroxycholesterol, 7-ketocholesterol, cholesterol-α-epoxide, cholesterol-β-epoxide and an unidentified fraction. All these fractions except the unidentified fraction increased on cooking. On refrigerated and even on frozen storage all these fractions increased except the unidentified fraction, which showed a concomitant reduction. The changes in broiled meat were more pronounced compared to pressure-cooked meat. Results clearly indicated that even frozen storage of cooked meat did not prevent the development of cholesterol oxidation products.     

Quantification of Three Cholesterol Oxidation Products in Raw Meat and Chicken

Raw veal, beef, pork, and chicken muscle tissues were extracted by a modified dry column procedure in which silicic acid was incorporated in the trap of the column. This method separated cholesterol derivatives from the bulk of neutral lipids, phospholipids and cholesterol. Isolation of sterols by preparative thin layer chromatography followed by quantification by direct on-column capillary gas chromatography permitted measurement of 7-ketocholesterol, cholesterol 5α, 6α-epoxide, and cholesterol 5β 6β-epoxide at concentrations less than 1 ppm. All muscle tissues contained the three cholesterol products in measurable quantities. 7-Ketocholesterol constituted more than 50% of the oxidation products in all samples.     

Separation and Identification of Cholesterol Oxidation Products in Dried Egg Preparations

A technique is described for the simultaneous determination of a wide range of angiotoxic sterols in foods produced by the oxidation of cholesterol. Gas chromatography using on-column injection onto a bonded phase fused silica capillary column gave excellent separation of the oxides examined. Combined gas chromatography- mass spectrometry was used to confirm the identities of the trimethylsilyl ether derivatives of the various sterols and 6-ketocholestanol was found to be an acceptable internal standard for determination. Analysis of powdered scrambled egg mix showed the presence of several cholesterol oxides, with cholesterol-5α,6α-epoxide, cholesterol-5β,6β-epoxide and 7-ketocholesterol predominating. Other atherogenic sterols identified included 25-hydroxycholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol and 5α-cholestane-3β,5,6β-triol.     

7α and 7β-Hydroxycholesterols Formed in a Dry Egg Nog Mix Exposed to Fluorescent Light

Light-induced generation of two cholesterol oxidation products, 7α- and 7β-hydroxycholesterol, was measured in an egg nog mix using an improved isolation procedure and high performance liquid chromatography (HPLC). Dry egg nog mix was exposed continuously to fluorescent light at room temperature for 90 days. The 7α-hydroxycholesterol was formed in consistently greater amounts than the 7β-form. Amounts of both forms increased for up to 80 days (23 ± 2°C), then declined. Mix stored in the dark did not contain detectable amounts of 7α- or 7β-hydroxycholesterol. Purification techniques enabled HPLC quantification of cholesterol oxidation products in a food.     

Influence of packaging atmosphere on the formation of cholesterol oxides in γ-irradiated egg powder

In a previous work the effect of ionising radiation on formation of cholesterol oxidation products (COPs) under aerobic conditions was studied. In the present work the influence of aerobic and anoxic conditions have been comparatively investigated to study the chemical changes of cholesterol in γ-irradiated egg powder. The irradiation treatment was carried out with powdered egg packed under air and also under vacuum in polyethylene (PE) bags and in laminated, oxygen impermeable three-layer (polyester-aluminium-polyethylene) foil to dosage levels 2 and 4 kGy at room temperature. The cholesterol oxidation is demonstrated by thin-layer chromatograms. In the egg powder wrapped in PE bags the predominant cholesterol derivatives?7-hydroxycholesterol isomers (α and β)—accumulated in significant amounts (increasing with dose) while vacuum-packaging in laminated foil considerably suppressed the quantity of these products and prevented the formation of cholesterol 5α, 6α-epoxide as well as 7-ketocholesterol. Little or no change was observed in non-irradiated (control) vacuum-packed egg powder stored at approximately 22°C for up to 5 months. Peroxide values showed changes parallel to the formation of COPs.     

Effect of Ionizing Radiation on Cholesterol in Aqueous Dispersion

Aqueous sodium stearate dispersions of cholesterol were irradiated at 0-2°C with absorbed doses ranging from 2.5 to 50 kGy. The resulting mixture of cholesterol derivatives was isolated and examined for 7-ketocholesterol and cholesterol 5α,6α-epoxide and 5β,6β-epoxide content. Concentrations of all three compounds increased with dose, while the ratio of 7-ketocholesterol to total epoxides decreased with increasing dose. The ratio of 7-ketocholesterol to the epoxides was approximately 1 or below at all dose levels while the same ratio in autoxidations of cholesterol in dispersions was normally 6 or greater. The change in the keto/epoxide ratio may be a means for determining whether meat or other foods containing cholesterol have been subjected to ionizing radiation.     

Cholesterol Oxidation Products in Some Muscle Foods

Cholesterol oxidation products were estimated in some meat products by capillary gas chromatography after trimethylsilylation. Peak identities were confirmed by mass spectrometry. Freeze-dried pork, stored in contact with air at 22°C for ca. 3 yr, revealed 7α- and 7β-hydroxycholesterol, 7-ketocholesterol, α- and β-epoxide and cholestane-triol. The total concentration of oxidation products reached almost half of the remaining cholesterol content with 7-ketocholesterol as the predominant species. Some oxidation products were noted at a few ppm levels in broiled beef steaks, but not in precooked beef products. As rancidity development advanced in comminuted and cooked meats during storage, the oxidation of cholesterol became apparent.     

Cholesterol Oxides in Commercial Dry Egg Products: Isolation and Identification

5α-Cholestan-5,6α epoxy-3β-ol (cholesterol α-oxide) and 5β-cholestan-5,6β-epoxy-3β-ol (cholesterol β-oxide) were isolated from freshly dehydrated commercial whole egg and yolk powders. The isolates were shown to have identical HPLC retention volumes to those of synthesized cholesterol α- and β-oxides, respectively. Their mass (MS), nuclear magnetic resonance (NMR) and infrared (IR) spectra were also proven to be identical. Cholesterol oxides found in dehydrated egg products may have been formed during the commercial drying process since no oxides were found in fresh shell eggs nor in the egg samples lyophilized in the laboratory.     

Photooxidation of Cholesterol in Butter

The effect of exposure to fluorescent light on the oxidative stability of cholesterol in unpackaged butter was investigated. Oxidation of cholesterol in butter occurred, although the oxidation products were detectable only after prolonged exposure to light. Cholesterol oxides were more concentrated at the surface than throughout the entire butter block and were detectable after 8 days of exposure to light. 5-Cholesten-313, 7α-diol and its 7β-epimer, products of free radical attack on cholesterol, were detected among the oxidation products, along with a compound suspected to be 6-cholesten-3β, 5α-diol. This latter compound is only known to form following singlet oxygen attack on cholesterol.     

Cholesterol oxides and atherosclerosis: A review

Cholesterol is an important constituent of animal food products and has often been implicated in the etiology of atherosclerosis and coronary heart diseases. Recent reports have, however, shown the possible role of cholesterol oxidation products (COP), rather than cholesterol, in the initiation of atherosclerotic plaque formation. Cholestan-3β,5α,6β-triol and 25-hydroxycholesterol have been reported as the most potent atherogenic agents. Inhibition of HMG-CoA reductase (EC 1.1.1.34) enzyme activity and cholesterol biosynthesis by cholesterol oxidation products has also been thoroughly investigated in cultured cells. Various animal food products, viz meat products, egg products and dairy products (especially butter, butter oil, ghee, cheese etc) have been reported to contain various COP developed during certain processing treatments. The literature on the presence of COP in food products and their cytotoxic and atherogenic effects is reviewed.     

DIETARY OILS AND TOCOPHEROL SUPPLEMENTATION ON CHOLESTEROL OXIDE FORMATION IN FREEZE-DRIED CHICKEN MEAT DURING STORAGE

The influence of dietary oils [fish (FO), flax (LO), palm (PO) and sunflower (SO) oil] and tocohperol supplementation on the oxidation of cholesterol and lipids of chicken meat was investigated. Lipid oxidation in chicken meat powders was determined by thiobarbituric acid reactive substances (TBARS) and cholesterol oxidation products were estimated by capillary gas chromatography (GC) after trimethylsilylation. Peak identities from GC were confirmed by mass spectrometry. Freeze-dried chicken meat powder, stored in contact with air at room temperature for four months showed the presence of 7α- and 7β-hydroxycholesterol, 7-ketocholesterol, α and β-epoxide, and cholestane-triol. Total cholesterol oxides contents significantly (P < 0.05) increased with storage. The amount of cholesterol oxides in the meat powder was the highest in FO and SO group, and lowest in PO group. Lipid and cholesterol oxidation were accelerated by the presence of long-chain PUFA. Lipid and cholesterol stability of chicken meat were improved by the dietary supplementation of tocopherol.     

β-环糊精脱除蛋黄中胆固醇的研究

研究了β-环状糊精(β-CD)包合法脱除鸡蛋蛋黄中胆固醇,应用DX7Trial对试验数据进行分析并建立了数学模型,得出了胆固醇脱除的最佳工艺为:pH10.5,加热温度50.18℃,搅拌时间10.94min,n(β-CD):n (蛋黄液中胆固醇)=4.11:1,在此条件下,胆固醇脱除率为92.23%,产品中胆固醇含量为0.7536mg/g。     

AN IMPROVED METHOD OF CHOLESTEROL DETERMINATION IN EGG YOLK BY HPLC

An improved method for cholesterol determination in egg yolk is reported in this paper. Egg yolk was first diluted. Cholesterol was then extracted with ether and petroleum ether, and quantified by reverse phase chromatography on a Zorbax ODS column (0.46 × 15cm, 5–6 μm) using a mobile phase of acetonitrile and 2-propanol (4:1) with a flow rate of 0.6 mL/min. A linear correlation was observed between cholesterol concentration at 0.05–0.40 mg/mL and its peak heights with a correlation coefficient of 0.9993 (n = 5). The average recovery was 98.9%, and detection limit was 0.02 mg/mL. No differences in cholesterol concentration were observed between egg yolk samples with and without saponification. Rapid and reproducible quantification of cholesterol in egg yolk can be completed with this simplified method.     

Effect of processing conditions on the oxidation of cholesterol in ghee

The cholesterol oxidation products (COP) in fat-rich dairy products were identified and quantified. Fresh cream contained no COP whereas fresh butter contained trace levels of 7β-hydroxy- and 5,6α-epoxycholesterol. Low levels of various major COP were present in ghee (clarified butter fat). Intermittent heating and frying of ghee induced severe oxidation of cholesterol and a number of COP were detected by GC and TLC. Most atherogenic COP. viz 25-hydroxycholesterol and cholestantriol, were formed more in intermittently heated ghee (8.1-9.2% of the total COP) than in fried ghee samples (7.1 %).     

Ultrasonic-assisted enzymatic degradation of cholesterol in egg yolk

An ultrasonic-assisted enzymatic process was developed for effective degradation of cholesterol in natural egg yolk. The objective of this study was to follow the catalytic activity of cholesterol oxidase against egg yolk cholesterol in an effort to obtain a cholesterol reduced egg yolk without affecting the major nutrients composition of egg yolk. Cholesterol oxidase was used to catalyze the degradation of cholesterol in egg yolk. Firstly, a 30 g portion of the egg yolk was pretreated by ultrasonic for 15 min at 200 W and then incubated for 10 h with cholesterol oxidase concentration of 0.6 U/g egg yolk at 37 °C. Finally, the cholesterol level in egg yolk was reduced to 8.32% of its original concentration without affecting the quality attributes of the yolk.

Industrial relevance

With the increase of public awareness to the dangers of cholesterol in recent years, a large number of popular food products have drawn criticism for containing high levels of cholesterol. Many reduced cholesterol egg products have been developed for market requirement. The results indicate that the ultrasonic-assistant extraction is an effective method for effective degradation of cholesterol in natural egg yolk. The knowledge gained from this study should be useful for further exploitation and application of the resource. Cholesterol-reduced egg yolk promises to be a value-added functional food with all the health enhancing attributes associated with egg yolk.