STORAGE STABILITY OF MILKFAT GLOBULE MEMBRANE

To investigate changes in structure and function of milk fat globule membrane (MFGM) components due to oxidation of membrane lipids, freeze-dried MFGM were stored under controlled temperature and relative humidity (RH) conditions. It was found that while membranes underwent extensive lipid oxidation when stored under air, fluorescent compounds were formed. These compounds exhibited fluorescence with excitation maxima at 350 nm and emission maxima at 440 nm; which were similar to those of the fluorescent substances derived from the reaction of oxidized fatty acids and primary amines. Formation of high molecular weight proteins was detected by SDS-PAGE in samples stored under air, but not in samples stored under nitrogen and was also affected by time and relative humidity. Activity of xanthine-oxidase, an important prooxidative agent, was greatly influenced by temperature and complete inactivation was observed when MFGM's were stored at 37°C, 50% RH for 1 day.     

Fluorescence due to interactions of oxidizing soybean oil and soy proteins

Soy proteins with soybean oil (9:1,w/w) were stored at 60°C to investigate the changes in intrinsic fluorescence during oxidation. The front-surface fluorescence of the oxidized samples showed excitation and emission maxima at 355 and 440 nm respectively. The fluorescent compounds were soluble in the organic layer of the chloroform–methanol (2:1,v/v). The solution fluorescence showed an excitation maximum at 365 nm and an emission maximum at 450 nm, and the intensity increased during storage. The interactions of oxidizing soybean oil and soy proteins also resulted in decreases of protein solubility, soluble protein hydrophobicity, and free amino groups of proteins. With an antioxidant (BHT) addition, the changes in fluorescence and in protein properties were inhibited. The intensity of the solution fluorescence showed high correlation with TBA value (r=0.968) and protein solubility (r=−0.979), which could serve as an indicator for oxidative deterioration of soy proteins and soybean oil systems.     

Influence of two lipid extraction procedures on the peroxide value in powdered infant formulas

The influence of two extraction procedures on the peroxide value (PV) of lipid extracts of powdered infant formulas (IFs) was investigated. Thirteen commercial IFs contained in their original packages were assessed after storage at room temperature for 18 months. The extraction procedures mainly differed in the extraction solvent polarity. Extraction method 1 was one of the methods more widely applied in the industry of powdered IFs. It consisted of a single extraction with a polar solvent composed of dichloromethane : methanol (2:1, v/v). Extraction method 2 consisted of disruption of the solid matrix simply by rubbing with a mortar and pestle and subsequent extraction of the lipids released with hexane, used as a non-polar solvent. The PV was contrasted with the quantitative analysis of the non-volatile lipid oxidation compounds and tocopherols. The oxidation compounds were isolated by solid phase extraction (SPE) and analyzed by high-performance size-exclusion chromatography (HPSEC) with refraction index detection. Results showed high significant differences (P < 0.0001) in the PV between the extraction methods, with lower values for method 1. However, no significant differences were found either in the levels of oxidation compounds or in those of tocopherols (P > 0.05). It was found that the lower PVs in method 1 were due to reducing substances co-extracted with the lipids by the polar solvent. Therefore, the quantitative analysis of the lipid oxidation compounds by SPE–HPSEC was more reliable for evaluation of the oxidation level in powdered IFs.     

Influence of formaldehyde in the formation of fluorescence related to fish deterioration

 In previous studies fluorescence detection at different excitation/emission maxima during common fish processing has been used. A bathochromic shift towards higher wavelength maxima was observed and measured as the ratio between absorption at two of the maxima tested. This fluorescence ratio (δF) value correlates positively with fish damage. In the present work, the influence of formaldehyde (FA) on the value δF was studied. A model system was set up in which FA reacted at 30°C for 25 days with propylamine and fish muscle. It was observed that FA was less able to produce fluorescent compounds compared with common fish oxidation products that were also tested, i. e. propanal and hexanal. However, in the presence of both lipid oxidation aldehydes, the FA-containing mixtures led to a higher δF value. Model systems consisting of FA and fatty fish (sardine) muscle produced more fluorescence than FA and lean fish (cod), because of the formation of lipid oxidation compounds under the reaction conditions of the former systems. It is thus concluded that the presence of FA in a reacting medium enhances fluorescence formation, such that δF be can used as an accurate measure of fish damage. It is thought that measurement of δF in processes such as the freezing of gadoid fish, in which both FA and lipid oxidation are produced, could be of benefit. Received: 23 June 1997     

Phenolic compounds in frozen avocados

The ethyl acetate-soluble phenolic compounds present in frozen ripe Fuerte avocados were extracted with methanol and ethyl acetate. The compounds were separated by two-dimensional paper chromatography with n-butanol-acetic acid-water (4:1:5, v/v) and 2% acetic acid solvents. Fifteen spots were found when the chromogenic reagent was FeCl ₃-K₃Fe(CN)₆. The individual compounds were identified by their R_f values, colour reactions, fluorescent behaviour and absorption spectra. Chlorogenic and _p-coumarylquinic acid isomers, catechins, leucoanthocyanidins, isoflavone and caffeic and _p-coumaric acids were present in the extracts of frozen avocados. Ascorbic acid-treated frozen avocado chunks did not darken when kept under vacuum at —26°C, but turned dark if left exposed to air. Darkening of frozen chunks was associated with disappearance of most phenolic substances, except the simple cinnamic acid derivatives. Formation of new phenolic substances, especially leucoanthocyanidins and catechins, was observed during storage of the frozen chunks under vacuum.     

Lipid changes during long-term storage of canned tuna (Thunnus alalunga)

 In previous studies fluorescence detection at different excitation/emission maxima during common fish processing has been used. A bathochromic shift towards higher wavelength maxima was observed and measured as the ratio between absorption at two of the maxima tested. This fluorescence ratio (δF) value correlates positively with fish damage. In the present work, the influence of formaldehyde (FA) on the value δF was studied. A model system was set up in which FA reacted at 30°C for 25 days with propylamine and fish muscle. It was observed that FA was less able to produce fluorescent compounds compared with common fish oxidation products that were also tested, i. e. propanal and hexanal. However, in the presence of both lipid oxidation aldehydes, the FA-containing mixtures led to a higher δF value. Model systems consisting of FA and fatty fish (sardine) muscle produced more fluorescence than FA and lean fish (cod), because of the formation of lipid oxidation compounds under the reaction conditions of the former systems. It is thus concluded that the presence of FA in a reacting medium enhances fluorescence formation, such that δF be can used as an accurate measure of fish damage. It is thought that measurement of δF in processes such as the freezing of gadoid fish, in which both FA and lipid oxidation are produced, could be of benefit. Received: 23 June 1997     

Effect of Extraction Method on the Oxidative Stability of Camelina Seed Oil Studied by Differential Scanning Calorimetry

Camelina seed is a new alternative omega‐3 source attracting growing interest. However, it is susceptible to oxidation due to its high omega‐3 content. The objective of this study was to improve the oxidative stability of the camelina seed oil at the extraction stage in order to eliminate or minimize the use of additive antioxidants. Camelina seed oil extracts were enriched in terms of natural antioxidants using ethanol‐modified supercritical carbon dioxide (SC‐CO₂) extraction. Oxidative stability of the camelina seed oils extracted by ethanol modified SC‐CO₂ was studied by differential scanning calorimeter (DSC), and compared with cold press, hexane, and SC‐CO₂ methods. Nonisothermal oxidation kinetics of the oils obtained by different extraction methods were studied by DSC at varying heating rates (2.5, 5, 10, and 15 ° C/min). Increasing ethanol level in the ethanol‐modified SC‐CO₂ increased the oxidative stability. Based on oxidation onset temperatures (T_on), SC‐CO₂ containing 10% ethanol yielded the most stable oil. Oxidative stability depended on the type and content of the polar fractions, namely, phenolic compounds and phospholipids. Phenolic compounds acted as natural antioxidants, whereas increased phospholipid contents decreased the stability. Study has shown that the oxidative stability of the oils can be improved at the extraction stage and this may eliminate the need for additive antioxidants.     

Determination of benzo[α]pyrene in edible oil using tetraoxocalix[2]arene[2]triazine bonded silica SPE sorbent

Benzo[α]pyrene (BaP) is a well-known carcinogen in edible oil. In this study, a method combined solid-phase extraction (SPE) with fluorescent detection was developed using tetraoxocalix[2]arene[2]triazine sorbent (SiO₂-OCA) for the clean-up and enrichment of BaP. The interaction between SiO₂-OCA and BaP involves a donor–acceptor complex mechanism. The experimental procedure was as follows: BaP was extracted from edible oil with DMF/H₂O (9:1, v/v). Then, the ratio of DMF/H₂O was adjusted to 1:2 prior to SPE. The final concentrate was analysed using a fluorescence detector at excitation and emission wavelengths of 255 and 420 nm. The method was fully validated. The linearity was in the range of 0.1–100 μg kg^?1 with a coefficient of 0.999. The limits of detection and quantification were 0.03 and 0.1 μg kg^?1, respectively. The average recoveries were in the range of 88.0 ? 122.3%. The intraday and interday precisions were 6.8% and 9.2%, respectively. Compared with other methods, the method reported in this article shows a good detection limit, high reproducibility and recovery and linearity over a broad concentration range. This established method was also applied to evaluate real samples. The concentration of six tested samples was below 5 μg kg^?1.     

Interaction between the peptide glutathione and linoleic acid hydroperoxide

Incubation experiments in order to study the influence of some parameters in the fluorescence products formation have been carried out. It has been observed a greater efficiency with the relation linoleic acid hydroperoxide:glutathione, 2:1, and a temperature of 37 °C. The complex formed working with labelled linoleic acid has been separated in two main fractions. The first one (without radioactivity) is fluorescent with excitation and emission maxima at 350 nm and 440 nm, respectively. It has been confirmed that this fraction consists of a complex of glutathione and short chain aldehydes. The fluorescence of the complex did not decrease by treatment with NaBH₄ or with pH. The NH₂ and SH groups take part in the lipid-peptide complex formation. The second, highly radioactive fraction includes free hydroperoxides and short chain hydroxy fatty acids.     

Determination of oxytetracycline in tomatoes by HPLC using fluorescence detection

An analytical method for the determination of oxytetracycline (OTC) in tomatoes was developed and validated. Liquid–liquid extraction (LLE) and a solid-phase extraction (SPE) were used for sample preparation. Reversed-phase high performance liquid chromatography (RP-HPLC) using a C₁₈ column and a mobile phase containing MeOH: calcium chloride, disodium ethylenediaminetetraacetate (EDTA) and sodium acetate, pH 7.3 (30:70, v/v), with fluorescence detection at 390 nm excitation and 512 nm emission, was used for separation and quantitation of OTC. The method was validated through the following performance criteria: linearity and linear range, sensitivity, selectivity, intra-day and inter-day precision, detection and quantitation limits and accuracy. Limit of quantitation show that the method developed is suitable for the determination of OTC at a level below the maximum residue limits established by the Brazilian legislations (250 μg kg⁻¹). Of 40 samples analyzed, none contained OTC above the limit of quantitation.     

A novel approach to monitor the oxidation process of different types of heated oils by using chemometric tools

The oxidative stability of seven oils with different fatty acid profiles was assessed. Oxidation at 0, 2 and 4 h at 180 °C was monitored by measuring the absorbance of thiobarbituric acid reactive substances (TBARS) along the absorption spectrum (300–600 nm), the volatile aldehydes (HS-SPME–GC–MS) and the fatty acid profile (FID-GC).TBARS absorption spectrum behavior depended on the lipid composition of heated oils. Higher absorbance increments during heating were noticed at 390 nm compared to 532 nm (from 2 to 21 fold higher depending on the oil), pointing to its better sensitivity to detect oxidation. Furthermore, a close relationship between ABS₃₉₀, the loss of polyunsaturated fatty acids and their corresponding oxidation compounds (volatile aldehydes) was revealed by Principal Component Analysis.Multiparametric equations allowed predicting the formation of volatile aldehydes of heated oils by measuring only two parameters: TBARS₃₉₀ during their heating, and the lipid profile in unheated oils (MUFA, ω-3 and ω-6). Results pointed out the interest of choosing ABS₃₉₀ when the oxidative evolution of vegetable oils under heating is assessed by the TBARS test.     

Optimization of extraction process for phenolic acids from black cohosh (Cimicifuga racemosa) by pressurized liquid extraction

An investigation to optimize the extraction of phenolic acids from black cohosh using a pressurized liquid extractor system was studied with the aim of developing a generalized approach for sample preparation of phenolic compounds from plant matrices. Operating parameters such as solvent composition, solid‐to‐solvent ratio, temperature, particle size distribution, and number of extraction cycles were identified as main variables that influence extraction efficiency. A mixture of methanol and water (60:40 v/v) was found to be the best solvent for total phenolics (TP) and individual phenolic acids. The four phenolic acids extracted from black cohosh were identified by HPLC and LC‐MS as caffeic acid, ferulic acid, sinapic acid and isoferulic acid. Over 96% of the measured phenolics were extracted in first two cycles. The extraction efficiency for black cohosh with MeOH:H₂O (60:40 v/v) was found to be maximum at a solid‐to‐solvent ratio of 80 mg ml^?1. TP content of the extract was found to increase with temperature up to 90 °C. Particle size was found to have a large impact on extraction efficiency of TP. Samples with particle size between 0.25 mm and 0.425 mm provided optimum extraction of phenolics from black cohosh. Published in 2005 for SCI by John Wiley & Sons, Ltd.     

Lipid rafts in the bovine milk fat globule membrane revealed by the lateral segregation of phospholipids and heterogeneous distribution of glycoproteins

This study reveals the lateral organisation of the milk fat globule membrane (MFGM). Using confocal laser scanning microscopy (CLSM) and a lipid soluble molecule, an exogenous phospholipid and two lectins as fluorescent probes we located triacylglycerols in the core of fat globules and investigated the organisation of the polar lipids and glycoproteins of the MFGM, in situ in milk. Lipid rafts corresponding to the lateral segregation of sphingolipids in liquid-ordered phases surrounded by liquid-disordered domains composed by the glycerophospholipids were observed in the MFGM. These lipid rafts which correspond to rigid sphingolipid-rich domains have a circular shape at room temperature. CLSM experiments revealed that glycoproteins and glycolipids are heterogeneously distributed around fat globules and that they are not located in the lipid rafts. The characterisations performed by in depth thin sectioning of fat globules and in dynamic as a function of time revealed chemical and structural heterogeneities in the MFGM. Schematic 3D and 2D representations of the MFGM are proposed and discussed. The physiological and nutritional consequences of the lateral organisation of polar lipids and glycoproteins in the MFGM are discussed but remain to be elucidated.     

Phenolic compounds from winemaking waste and its antioxidant activity towards oxidation of rapeseed oil

The effects of extraction parameters on bioactive compound contents of winemaking waste extracts (WWE) and its effect on rapeseed oil oxidative stability were evaluated. Research showed that the total phenols and anthocyanin contents and antioxidant activity (AA) of WWE significantly depended on the extraction parameters. Increasing the temperature (60 °C) and time (5 h) of extraction and an addition of water to ethanol statistically improved the rate of active component extraction. HPLC analysis showed that procyanidin B_2, catechin, gallic acid, γ‐resorcylic acid and p‐coumaric acid were the major phenolic compounds of WWE. Important correlations between total phenolic compounds quantified by HPLC and both DPPH and (ferric reducing antioxidant power) (FRAP) values were found. WWE added to the oil at three different levels clearly slowed down the process of fatty acid oxidation, inhibiting hydroperoxide formation by about 86%, comparable with BHT, while it was more effective than that of α‐tocopherol. When using volatile compound formation as a marker of lipid oxidation, WWE at the level of 2000 ppm were the most effective inhibitors of the decomposition of hydroperoxides. The research showed that the WWE are a rich source of phenolic compounds with powerful antioxidant activities and are suitable for preventing rapeseed oil oxidation.     

The impact of cream churning conditions on xanthine oxidase activity and oxidation–reduction potential in model emulsion systems

Six types of milk fat globule membrane (MFGM) fractions were isolated from fresh raw cream using various churning conditions at different temperature (10, 15 or 20 °C) and pH values (5.5 or 6.6) and characterised for protein composition, xanthine oxidase (XO) content and activity, and oxidation–reduction potential (E_h). A greater proportion of non-membrane proteins was found at higher temperatures and higher cream pH, and the XO content decreased significantly under these conditions. XO activity was greater at lower temperatures and pH, yielding a more positive E_h value for the emulsions. Storage at 4 °C for 14 d significantly reduced the Eh of the emulsions to between −320 mV and −580 mV for low pH/low temperature, and high pH/high temperature emulsions, respectively. This study suggests that it is possible to obtain MFGM fractions with specific Eh reducing ability by altering churning conditions.     

Milk fat globule membrane proteins are involved in controlling the size of milk fat globules during conjugated linoleic acid–induced milk fat depression

Milk fat globule membrane (MFGM) proteins surround the triacylglycerol core comprising milk fat globules (MFG). We previously detected a decrease in the size of fat globules during conjugated linoleic acid (CLA)-induced milk fat depression (MFD), and other studies have reported that some MFGM proteins play a central role in regulating mammary cellular lipid droplet size. However, little is known about the relationship between MFD, MFG size, and MFGM proteins in bovine milk. The aim of this study was to investigate the profile of MFGM proteins during MFD induced by CLA. Sixteen mid-lactating Holstein cows (145 ± 24 d in milk) with similar body condition and parity were divided into control and CLA groups over a 10-d period. Cows were fed a basal diet (control, n = 8) or control plus 15 g/kg of dry matter (DM) CLA (n = 8) to induce MFD. Cow performance, milk composition, and MFG size were measured daily. On d 10, MFGM proteins were extracted and identified by quantitative proteomic analysis, and western blotting was used to verify a subset of the identified MFGM proteins. Compared with controls, supplemental CLA did not affect milk production, DM intake, or milk protein and lactose contents. However, CLA reduced milk fat content (3.73 g/100 mL vs. 2.47 g/100 mL) and the size parameters volume-related diameter D_[4,3] (3.72 μm vs. 3.35 μm) and surface area-related diameter D_[3,2] (3.13 μm vs. 2.80 μm), but increased specific surface area of MFG (1,905 m²/kg vs. 2,188 m²/kg). In total, 177 differentially expressed proteins were detected in milk from cows with CLA-induced MFD, 60 of which were upregulated and 117 downregulated. Correlation analysis showed that MFG size was negatively correlated with various proteins, including XDH and FABP3, and positively correlated with MFG-E8, RAB19, and APOA1. The results provide evidence for an important role of MFGM proteins in regulating MFG diameter, and they facilitate a mechanistic understanding of diet-induced MFD.     

Isolation of the sweet components from Siraitia grosvenorii

Powdered concentrate from dried Luo Han Guo fruit was subjected to liquid extraction by Soxhlet (hexane:ethanol, 1/4 v/v) or with subcritical water (scH₂O) or with supercritical ethanol carbon dioxide (scCO₂). Whereas exhaustive Soxhlet extraction of the crude dried fruit powder (CDFP) was inefficient, pressurized water extraction in the presence of chromatographic support (Alumina, Celite or Silica gel) was beneficial to the scH₂O recovery of mogrosides as determined by colourimetry. With a flow rate of 0.7 mL min⁻¹ scH₂O and a back pressure of 11.7 MPa, a maximum recovery was obtained at 150 °C; yet increases in recovery for extractions beyond 10 min were marginal. The recovery of target compounds were very inefficient for scCO₂ alone but was improved with the addition of 0.3 mL min⁻¹ ethanol as co-solvent to the mobile phase and by adding chromatographic support to the substrate. Increased pressure during the scCO₂ extractions were beneficial to the recoveries that were maximized at 60 °C. However, increases in the recoveries of mogrosides for extractions beyond 90 min for the dried fruit powder or beyond 30 min for the partially purified concentrate were very modest. Of the three extraction techniques, Soxhlet, scCO₂ or scH₂O, the latter technique, in tandem with ultra-sonication of the dried fruit powder proved to be very efficient so that there was little value to partially purifying this substrate prior to pressurized fluid extraction.     

Extraction,distribution and characterisation of phenolic compounds and oil in grapeseeds

Five factors, extraction means, extraction repeats, extraction solvents, sample granularities, and grinding manners, were investigated and compared to optimise the extraction methods of phenolic compounds from grapeseed. Twenty minutes sequential sonication for three repeats was a preferred extraction means for assessment of phenolic content in grapeseed over three repeats 100-min sequential mechanical agitation. Methanol/water/HCl (70:29:1, v/v/v) was the best among the solvent compositions tested. Grinding granularity showed a significant (p = 0.05) effect on the yield of phenolic compounds when the sample was ground with a grinder and separated to different granularities by sieves. Grinding sample with mortar in liquid nitrogen could provide samples with uniform size and thus give a more consistent evaluation of phenolic compounds in grapeseed. Significant correlation coefficients (p < 0.01) among total phenols, total flavan-3-ols, and total proanthocyanidins contents in grapeseed extracts were observed. In addition, phenolic compounds and oil were very unevenly distributed in grapeseed as about 90% of phenolic compounds were observed in seed coat while about 60% of oil was in the endosperm and embryo.     

Antioxidant Activity of Pea (Pisum sativum L.) Seed Coat Acetone Extract

The contents of phenolic compounds in seed coat of pea and their antioxidative properties were examined. The pea seed coat was extracted with acetone-water (7:3 v/v) mixture and the extract was separated into five (?V) fractions using a Sephadex LH-20 column chromatography. Antioxidative activity of extract and fractions was measured by the oxidation of phosphatidylcholine to hydroxyperoxidephosphatidylcholine in liposome model and by scavenging effects of superoxide radical anion in xanthine-xanthine oxidase system. Phenolic compounds of extract and fractions were determined by spectrophotometric methods and characterized by HPLC analysis. Strong antioxidative properties were noted for extract and its five fractions measured by liposome method. The extract and fractions I, IV and V also showed scavenging effects of superoxide radical anion. A statistically significant correlation (P≤0.05) was found between the inhibition of PC oxidation in the system tested and contents of either total phenols or tannins. However no statistically significant correlation was found between O^•−₂ scavenging effect and contents of either total phenols or tannins. The HPLC analysis of phenolic compounds of extract and active fractions showed the presence of some phenolic acids (benzoic and cinnamic acids, and cinnamic acid derivatives), flavone and flavonol glycoside.     

HPLC determination of ethoxyquin and its major oxidation products in fresh and stored fish meals and fish feeds

A new method was developed to determine 1,2‐dihydro‐6‐ethoxy‐2,2,4‐trimethylquinoline (EQ, ethoxyquin), 2,6‐dihydro‐2,2,4‐trimethyl‐6‐quinolone (QI) and 1,8′‐di(1,2‐dihydro‐6‐ethoxy‐2,2,4‐trimethylquinoline) (DM) in fish meals or fish feeds, QI and DM being the oxidation products of EQ. The sample was first extracted with hexane. After the removal of hexane the three analytes were extracted from the resulting oil with acetonitrile and determined by C₁₈ reverse phase high‐performance liquid chromatography with a UV detector set at λ = 280 nm. The mobile phase was acetonitrile–0.01 M ammonium acetate (80:20 v/v). The recoveries for EQ, QI and DM from the samples spiked at different levels varied in the ranges 90–100 per cent, 75–85 per cent and 90–100 per cent respectively. At room temperature, QI and DM were the major oxidation products of EQ in stored fish meals and fish feeds. Loss of EQ from fish meals is faster than that from feeds, resulting in relatively higher accumulations of QI and DM in the fish meals. Both QI and DM, especially the former, were not stable during storage of either and could be further oxidised to unidentified compounds. The residue levels of these two compounds were thus unpredictable during storage intervals. When the storage temperature was increased to 50 °C, EQ disappeared more rapidly, but neither QI nor DM accumulated. © 2000 Society of Chemical Industry