Wide-Bore Capillary Gas Chromatographic Method for Quantification of Cholesterol Oxidation Products in Egg Yolk Powder

A wide-bore capillary gas chromatographic (GC) method was developed for quantification of cholesterol oxidation products in egg yolk powder. Baseline separations were achieved between 7α- and 7β-hydroxycholesterol, cholesterol, cholesterol-5α,6α-epoxide, cholesterol-5β,6β-epoxide and 7-ketocholesterol chromatographed as the trimethylsilylated sterols. Excellent response linearity was obtained for each cholesterol oxidation product, permitting reliable quantification. Recoveries from freeze-dried egg yolk spiked with various levels of cholesterol oxidation products ranged from 78.2 to 95.1%. The coefficients of variation compared favorably to those for packed column GC methods.     

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.     

1H-NMR Study of Cholesterol Autooxidation in Egg Powder and Cookies Exposed to Adverse Storage

The level of cholesterol oxides depends on the production and storage conditions for food. We studied the effect of prolonged storage, heat, and diffuse daylight on spray-dried egg powder and cookies containing large amounts of cholesterol. The cholesterol oxides were isolated by chromatographic procedures, and were identified by recording ¹H-NMR spectra of selected HPLC fractions. Autooxidation was quite slow when the sample was stored in a freezer at — 20°C. Diffuse daylight increased the amounts of β-epoxide of cholesterol, whereas heating caused a sharp increase in C-7 oxidized cholesterol derivatives. Extent of cholesterol oxidation depended on storage conditions.     

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.     

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.     

Cholesterol Oxides in Swedish Foods and Food Ingredients: Milk Powder Products

Cream-, whole milk-and skim milk powders had 1.6, 2.0, and 18.9 mg cholesterol/g fat, respectively. About 90% of the cholesterol was unesterified. Analyses of fresh spray-dried whole-and skim milk powders from “low and medium heat” conditions and fresh roller-dried whole milk powder from “low heat” conditions contained less than 0.1 ppm cholesterol oxides in lipids. However, spray-dried whole-and skim milk powders from “high heat” conditions had substantial levels of some important cholesterol oxides. Skim-milk powders, stored in big sacks for 11–37 months and small consumer packages for 2–23 months, contained variable amounts of 5, 6α-epoxy-5β-cholestan-3β-ol (0.3–4.0 ppm), 5,6β-epoxy-5α-cholestan-3β-ol(0.7–10.40ppm), cholest-5-ene-3β,7α-diol(0.5–16.2 ppm), cholest-5-ene-3β,7β-diol(1.3–20.8 ppm), cholest-5-ene-3β,20α-diol(0.6–2.7 ppm), cholest-5-ene-3,25-diol(0.3–0.8 ppm), 5α-cholestane-3β,5,6β-triol(1.3–2.5 ppm) and 3-hydroxy-cholest-5-en-7one(1.8-24.9 ppm).     

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.     

气质联用法测定反复冻融畜禽肉中胆固醇及其氧化物含量

本文以畜禽肉中胆固醇及胆固醇氧化物、水分、丙二醛指标变化,考察了反复冻融对畜禽肉品质的影响。采用气相色谱-串联质谱/质谱(GC-MS/MS)测定方法,对反复冻融7次(新鲜肉记为冻融0次)后鸡肉、猪肉和牛肉中胆固醇及5种胆固醇氧化物(25-羟基胆固醇、7β-羟基胆固醇、20α-羟基胆固醇、5α,6α-环氧化胆固醇、7-酮基胆固醇)的含量进行了测定。结果表明,随着冻融次数的增加,胆固醇含量逐渐降低,其中牛肉中的胆固醇含量减少尤为明显(减少量为32.47 μg/g)。在反复冻融的过程中,胆固醇氧化物的含量先逐渐增加,后趋于平稳,其中鸡腿肉中7β-羟基胆固醇含量增加最多(增加量为0.601 μg/g)。同时,通过对畜禽肉中脂质过氧化过程中的水分含量和丙二醛含量进行考察,鸡胸肉中的水分减少最多(减少量为14.472 g/100 g),牛肉中的丙二醛增加量最多(增量为1.004 μg/g)。反复冻融会导致畜禽肉胆固醇氧化增加,品质下降。     

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 Commercial Dry Egg Products: Quantitation

Cholesterol α-oxide (5α-cholestan-5,6α-epoxy-3β-ol), a suspected carcinogen and/or atherogen, and its β-isomer are found in varying amounts in commercial dried egg products. The level of total cholesterol oxides in dried eggs ranges from undetectable to 311 parts per million of total lipids. The majority of the samples (64%) had total cholesterol oxides content between 6 – 49 ppm. The level of cholesterol α-oxide ranges from undetectable to 62 ppm. The ratio of α-oxide to its β-isomer is found consistantly to be 1 to 4. The formation of cholesterol oxides is greater in samples dried by hot air heated directly with gas burner than those dried by air heated indirectly with steam. Prolonged storage increases cholesterol oxides content in dried egg products.     

Quantification of 5α-Cholestane-3β,5,6β-triol and Other Cholesterol Oxidation Products in Fast Food French Fried Potatoes

Concerns about toxicity of cholesterol oxidation products (COPS) prompted this study. Two restaurants were selected which use animal-vegetable (A/V) shortening for deep-frying. The survey of COPS for 30 days indicated values for total COPS in French fried potatoes were 20 ± 9 ppm to 24 ± 6 ppm. 5α-Cholestane-3β,5,6β-triol (triol) was identified in French fried potatoes from one restaurant. The mean for triol was 9 ± 3 ppm. Triol, 7α-hydroxycholesterol, 7β-hydroxy-cholesterol and 7-ketocholesterol were confirmed by co-chromatography and mass spectrometry. Triol is one of the most potent of angiotoxic COPS. This and other studies suggested French fried potatoes and other deep fried foods cooked in A/V shortening are a major source of COPS.     

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.     

Oxidation of cholesterol in mayonnaise during storage

The oxidative stability of cholesterol in commercial mayonnaise under different storage conditions was evaluated by measuring cholesterol oxides (COs), 7-ketocholesterol (7-Keto), 25-hydroxycholesterol (25-OH), 7α-hydroxycholesterol (7α-OH) and 7β-hydroxycholesterol (7β-OH) using HPLC. Oxidation of cholesterol was indicated within about 15 days after manufacture by the presence of 7-Keto. Oxidation increased during storage at 4 and 25 °C (being greater at 25 °C) for 165 days in darkness, as indicated by the presence of 7-Keto, 25-OH, 7α-OH and 7β-OH. There was a strong correlation between COs and PV (peroxide value) [r²=0.95 (4 °C) and r²=0.96 (25 °C)] during the process of oxidation. The pattern of fatty acids was not affected during the period of the experiment. Temperature and time were important factors in the oxidative stability of cholesterol. Total formation of COs during 165 days was 20.3 μg/g at 4 °C and 30.2 μg/g at 25 °C.     

Cholesterol Oxidation in Egg Yolk Powder During Storage and Heating as Affected by Dietary Oils and Tocopherol

The effects of feeding flax, sunflower, palm and fish oils, with and without tocopherols, to laying hens was investigated on oxidative stability of cholesterol in egg yolk powders. Storage of spray-dried egg yolk powders at room temperature resulted in loss (p <0.05) of tocopherols. The initial levels of spray-dried yolk cholesterol oxides were 7–10 ppm and increased (p <0.05) with storage. The cholesterol oxide contents in the egg yolk powders were: fish > flax > sunflower > palm. Heating egg yolk powder increased (p <0.05) the amounts of total cholesterol oxides. Diets with tocopherol supplementation resulted in a decrease (p <0.05) of cholesterol oxides during storage and heating.     

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.     

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.     

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.     

Effect of garlic powder on the performance,egg traits and blood parameters of laying hens

This study was conducted to investigate the effects of garlic powder on the performance, egg traits and blood parameters of laying hens. One hundred and sixty‐two SHSY‐type brown layers aged 21 weeks were chosen at random from a large flock. They were allocated to three dietary treatments. Each treatment comprised six replicates of nine layers in groups of three. The diets were supplemented with 0, 5 and 10 g kg^?1 garlic powder. The experimental period lasted 22 weeks. Garlic powder addition did not significantly affect body weight, egg production, feed consumption, feed efficiency, egg shell index, egg breaking strength, egg shell thickness, egg albumen index, egg yolk index, egg Haugh unit, egg yolk weight and serum protein concentration. Egg weight increased (P < 0.01) with garlic powder supplementation. There was a significant (P < 0.01) reduction in egg cholesterol concentration as mg g^?1 yolk when the dietary level of garlic powder was increased from 0 to 10 g kg^?1. Hen serum triglyceride (P < 0.05) and total cholesterol (P < 0.01) concentrations decreased with garlic powder supplementation. This study demonstrated that garlic powder addition increased egg weight and decreased egg yolk cholesterol concentration (mg g^?1 yolk) and serum triglyceride and cholesterol concentrations without adverse effects on performance and egg traits. Copyright © 2006 Society of Chemical Industry     

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.     

Influence of Surface Structure on Cholesterol Oxidation in Model Food Powders

Three milk-resembling powders having different oil phases were prepared and stored for six months at room temperature. Samples were taken monthly for estimation of the surface composition by ‘electron spectroscopy for chemical analysis’ (ESCA) and analyses of the level of cholesterol oxides by gas chromatography. The highest fat coverage was obtained with technical tristearin as the oil phase. This preparation had around 75% of the powder surface covered by fat; during storage this coverage decreased. This powder also had the largest increase in cholesterol oxides during storage, the final level being 159 μg g⁻¹ cholesterol. The powder containing high-melting pure tristearin had a low surface coverage of fat, originally about 25%, which decreased during the storage period. Little increase in cholesterol oxides was observed, the final level being 52 μg g⁻¹ cholesterol. The third powder containing liquid triolein as oil phase, had a surface coverage of about 50% throughout the storage period. The cholesterol oxidation rate was in between that of the two tristearin powders, the final level of oxides being 75 μg g⁻¹ cholesterol. The results for the investigated powders indicate that the surface composition is of major importance for the oxidation of cholesterol. No correlation between cholesterol oxidation and solvent extractable fat (free fat) was found.