Identification and quantification of cholesterol oxides in grated cheese and bleached butteroil

Butteroil samples bleached with benzoyl peroxide (BP) and 17 commercial cheeses were screened for oxidized sterols by thin layer chromatography (TLC). Ungrated cheeses made from bleached milk and freshly bleached butteroil contained no detectable oxidized sterols. Oxidized sterols were detected in stored, bleached butteroils and in grated cheeses. Four major oxidation products were the isomeric 5,6-epoxycholesterols and the epimeric 7-hydroxycholesterols identified by TLC, high performance liquid chromatography (HPLC) and mass spectrometry (MS). Additional sterol oxides (tentatively identified and not quantified) present in these samples included low levels of 7-ketocholesterol and cholesta-3,5-dien-7-one. The epimeric 7-hydroxycholesterols were detected in bleached butteroils stored in air (BP-A) and nitrogen (BP-N) for 22 days at 15 C. Butteroil, after 90 days of storage at 15 C, had 30 (BP-N) and 60 (BP-A) μg total oxides/g of bleached oil and, after 1-year at −20 C, had 70 (BP-N) and 180 (BP-A) μg/g butteroil. A grated, unbleached cheese packaged in clear glass contained the most oxidized sterols (44 μg/g). Sterol oxides were not detected in bleached cream using a simulated industrial process.     

Computation of conversion factors to determine the phospholipid content in peanut oils

Peanut oil was separated into the various lipid classes by column chromatography. The polar lipid fraction which contained phospho-lipids was separated into individual major components by 2-dimen-sional thin layer chromatography. Conversion factors for calculating the concentration of total phospholipid in peanut oil from percent elemental phosphorus were determined by estimation of molecular weights for the respective components. A conversion factor of 23.6, 24.8, 26.6, 22.2, and 24.4 was found for phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidic acid, and total phospholipid, respectively. These factors also were used to convert µg of phosphorus into µg of phospholipid.     

Mutagenic potential of ammonia-related aflatoxin reaction products in cottonseed meal

In a joint research effort, the Food and Drug Administration, the National Toxicology Program and the US Department of Agriculture studied the mutagenic potential of aflatoxin reaction products following ammoniation of contaminated cottonseed meal under conditions approximating those approved for commercial ammoniation of nonaflatoxin-contaminated meal. Uniformly ring-labeled¹⁴C-aflatoxin B₁ was added to cottonseed meal that contained ca. 4000 μg naturally incurred aflatoxin B₁/kg. Distribution of the radiolabeled compound was used to trace the modification of aflatoxin B₁ after treatment with ammonia. The radioactivity-to-weight ratio of the fractions isolated by solvent extractions and chemical and enzymic treatments was used to measure the relative concentration of an aflatoxin decontamination product. All extract fractions having a radioactivity-to-weight ratio ≥1 were tested for mutagenic activity using theSalmonella/microsome mutagenicity test (Ames test). Purified aflatoxin B₁ was mutagenic at a concentration of ca. 0.005 μg/plate. The methylene chloride extract of the ammoniated meal after Pronase digestion exhibited a similar response when 180 μg of this fraction was applied to each plate. This fraction represented 0.16% of the original added radioactivity. The other fractions produced no detectable mutagenic response at the concentrations tested (10–1000 μg/plate) onSalmonella tester strain TA100. Ammonia treatment of aflatoxin-contaminated cottonseed meal significantly decreased aflatoxin levels, and the aflatoxin decontamination products formed by the treatment had little or no mutagenic potential.     

Determination of surfactant mixtures in shampoos and detergents by HPLC

A technique for separating 4 nonionic, 7 anionic and 4 amphoteric surfactants with n-dodecyl groups was studied by high performance liquid chromatography (HPLC) and applied to the determination of these surfactants in commercial shampoos and household detergents. Conditions used for the separation were: column packing and size, TSK-LS 410 (5μ) and 6 mm i.d. × 500 mm (2 connected, 250 mm columns); mobile phase, water/methanol (25/75, v/v) containing 0.25 M sodium perchlorate adjusted to pH 2.5 with phosphoric acid; column temp., 50 C; detector, RI. Surfactants in shampoos and detergents were clearly distinguished from each other and determined without column chromatographic pretreatment, e.g., ion-exchange chromatography.     

Carotenoids and tocols of corn grain determined by HPLC

A high performance liquid chromatographic (HPLC) procedure has been developed that permits determinationof carotenoids and tocols in the same sample preparation of corn grain. For 15 inbreds, the total carotenoids ranged from 16 to 77 μg/g dry wt and the total tocols from 30 to 128 μg/g dry wt. For four inbreds, total carotenoids were concentrated in the horny endosperm (83±2%) and total tocols in the germ (77±6%). After six months storage at room temperature, the mean loss of total carotenoids for four inbreds was 42±4%, while the tocols had a mean loss of 5%. Presented at the AOCS meeting in Honolulu, HI in May 1986.     

Physicochemical characterization of galactosyldiglycerides and their quantitation in wheat flour lipids by high performance liquid chromatography

A high performance liquid chromatographic (HPLC) method was developed for analyzing digalactosyldiglycerides (DGDG) and monogalactosyldiglyceride (MGDG) in polar lipids fractionated from lipid extracts of wheat or flour. Wheat lipid samples were prepared by solvent extraction, then fractionated on a silica gel packed open column. A Spherisorb ODS (octadecyl silane) column with methanol/water elution system was used for separation of glycolipids in the polar lipid fractions. The detection limit of the refractive index detector with interferometric optics was 0.25μg for both DGDG and MGDG. Separating on nonpolar bonded phase columns permitted us to differentiate, based on fatty acid composition and position, among components within the specific glycolipid classes. Semipreparative HPLC on analytical columns was used to subfractionate the polar lipids. The glycolipids were collected for functional group characterization. Approximately 35% of each DGDG subfraction was accounted for as carbohydrate. The absence of phosphorus precluded phospholipids. Fatty acid analysis by gas chromatography showed the first DGDG to be linoleic acid, whereas the second DGDG peak was composed of linoleic, oleic and palmitic acids. Mass spectrometric analysis of the first DGDG peak showed linoleic acid in both the SN-1 and 2 positions. Mass spectrometric analysis revealed that palmitic or oleic acid in the second peak was preferentially located on the SN-1 position; linoleic acid was on the SN-2 position. Contribution no. 80-207J, Department of Grain Science and Industry, Kansas Agricultural Experiment Station, Kansas State University, Manhattan, KS. Part of a dissertation submitted by T.N. Tweeten in partial fulfillment of the PhD degree. Honored Student Award Presentation at AOCS annual meeting, San Francisco, April 1979.     

Analysis of milkfat by HPLC

A routine method for HPLC analysis of milkfat has been developed, which takes into account the specific phenomena of this complex and far-ranging system of triglycerides. The usual amount of injection of 1 mg has been reduced to 30 or 10 μg of fat. By this the composition of the mobile phase (acetone:acetonitrile, 35/65) and the temperature of the column (Nucleosil C18-5 μ, 15 cm + Microspher C18-3μ, 10 cm, in series, T=30–35 C) could be adjusted to a relatively high selectivity without inducing the high-melting fat components to crystallize on the column. A further advantage resulting from this consisted in permitting the eluent to be recycled over long periods of time. The extremely low amounts of samples necessitated a highly sensitive detection (Δn=5 × 10⁻⁷ RI units full scale deflection) which could be realized by an interferential refractometer in connection with a thermostat to keep up a stable temperature of the whole HPLC system (ΔT<0,005 K). By this it was possible to separate milkfat into 45 to 50 different types of triglycerides. By comparing soft and hard milkfats and fractions of milkfats, every fourth peak, starting with C 42, could be attributed to saturated triglycerides; apart from this, easily recognizable qualitative features appearing in the chromatograms permitted conclusions about the feeding regimen and energy supply of the cattle. Furthermore, due to the high stability of separating conditions achieved and due to computer software developed for this purpose it was possible to obtain and compare a large number of chromatograms under the same conditions.     

Method of analysis for deoxynivalenol and zearalenone from cereal grains

A method was developed to determine deoxynivalenol and zearalenone in corn, wheat, oats, rice and barley. The toxins are extracted with methanol/water (50:50, v/v) (2×) and partially purified by partitioning into ethyl acetate and then defatting with acetonitrile-petroleum ether. Toxins are isolated by silica gel column chromatography. Interfering materials are removed from the column with benzene; zearalenone is eluted with benzene/acetone (95:5, v/v), and after a column wash of chloroform/methanol (95:2, v/v), deoxynivalenol is eluted with chloroform/methanol (95:5, v/v). Zearalenone is quantitated by thin-layer chromatography and deoxynivalenol by gas-liquid chromatography of the trimethylsilyl derivative. The detection limit is about 0.02 μg/g for each toxin. Recoveries of added toxins varied with substrate and level of toxins. Recovery of deoxynivalenol ranged from 58% for 1 ppm in rice to 108% for 1 ppm in corn. Average recoveries for all levels (1, 2 and 5 ppm) ranged from 69% for barley to 89% for oats. Recovered zearalenone ranged from 40% for 5 ppm in wheat to 100% for 1 ppm in barley. Average recoveries for zearalenone at 1, 2 and 5 ppm varied from 53% for wheat to 87% for rice.     

Identification of Host Blends that Attract the African Invasive Fruit Fly,Bactrocera invadens

Bactrocera invadens, an invasive fruit fly species in the Afro-tropical region belonging to the Bactrocera dorsalis complex, causes considerable damage to fruit production and productivity. We sought to find attractants from hosts of B. invadens that could serve as baits in traps for monitoring and management of this pest. The attractiveness of volatiles from four different fruit species (mango, guava, banana and orange) at two stages of ripeness (ripe or unripe) was tested in an olfactometer assay. All fruits were attractive against a clean air control. Using hexane extracts of volatile collections of fruits, we demonstrated that male flies preferred the volatiles of ripe guava and orange over unripe fruit extracts. There was a slight difference in preference between females and males; females preferred orange to guava and mango, whereas males preferred mango and guava to orange. Gas chromatography/electroantennographic detection (GC/EAD) and GC/mass spectrometry (GC/MS) were used to identify compounds to which B. invadens antennae were sensitive. GC/EAD recordings from distal and medio-central parts of the fly antenna showed responses to a number of compounds from each fruit species, with esters dominating the responses. Synthetic blends were made for each fruit species using the shared antennally active compounds in ratios found in the extracts. In the olfactometer, B. invadens was most attracted to the banana and orange blends, followed by the mango and guava blends. The synthetic banana blend was as attractive as the volatile collection of banana, although both were less attractive than the fruit. The results demonstrate that composing attractive blends from GC/EAD-active constituents shared by host fruits can be effective for formulating attractive synthetic host mimics for generalist fruit fly species, such as B. invadens.     

Confectionery fat analysis with high performance liquid chromatography

Natural triglyceride mixtures have been analyzed by a variety of techniques. Earlier methods, such as crystallization, were unsuitable because they were very time-consuming, required large amounts of material and were not reproducible. Later advances in chromatography gave successful separations of triglycerides but required a combination of techniques to give suitable separations. High performance liquid chromatography (HPLC) with very efficient columns has been successfully applied to triglyceride separations on a routine basis. In the present work, triglycerides of cocoa butter as well as several replacement fats were separated by HPLC. The system consisted of a highly efficient column packed with octadecylbonded spherical silica (5 μ) and a mobile phase composed of acetone and acetonitrile. Peaks corresponding to eluted components were collected and their composition determined by gas chromatography (GC) of the corresponding methyl esters to allow unambiguous assignment of triglyceride structure to the components. A profile of the triglycerides present in such fats can be obtained within 30 min.     

Peanut oil

Conventional expeller and expeller/solvent extraction processes for peanuts are compared to the nonconventional processes of direct solvent extraction, cold pressing and nonhexane solvent processes. Peanut composition, cleaning and specific extraction procedures have a major impact on finished crude oil composition, refining characteristics, final oil and meal quality and utility. Special care in raw material and process selection must be taken when using crude peanut oil for direct edible or biofuel applications. Changes in world oil prices and protein market, US peanut quota and price support program and plant breeding will have a major impact on peanut oil availability and prices in the future.     

Densitometric thin-layer chromatographic analyses of cholesterol inSchistosoma mansoni (Trematoda) adults and their excretory-secretory products

Densitometric thin-layer Chromatographic analysis was used to quantitate cholesterol in 6-week-old male, female, and worm-pairs ofSchistosoma mansoni, and their excretory-secretory (E-S) products. Males extracted immediately after removal from mice had 1.2–1.5 μg cholesterol/worm, whereas those incubated for 0.5 hr in Earle's balanced solution at 37 ° C contained 0.8–1.5 μg cholesterol/ worm. Females extracted immediately after removal from hosts contained 130 ng cholesterol/worm. Females accumulated considerable cholesterol during incubation and had 420 ng cholesterol/ worm at 0.5 hr. Worm-pairs extracted at 0 hr had 1.9–2.8 μg cholesterol/pair and 1.2–1.5 μg cholesterol/ pair when extracted at 0.5 hr postincubation. Following incubation for 0.5 hr, males released 12–28 ng cholesterol/ worm (average 21), females released 8–13 ng cholesterol/ worm (average 11), and worm-pairs released 3–13 ng cholesterol/worm-pair (average 8).     

Distribution of ammonia-related aflatoxin reaction products in cottonseed meal

The fate of aflatoxin during ammoniation of contaminated cottonseed meal was studied under conditions approximating those approved for commercial ammoniation of nonaflatoxin-contaminated meal. Uniformly ring-labeled¹⁴C-aflatoxin B₁ was added to 27.7 kg meal (14% moisture) that contained ca. 4000 μg naturally incurred aflatoxin B₁/kg. Distribution of the radiolabeled compound was used to trace the modification of aflatoxin B₁ after treatment with 4% ammonia at 40 psi, 100 C for 30 min. This treatment reduced the chemically detected aflatoxin B₁ to less than 4 μg/kg. In control nonammoniated meals, 90% of the radiolabeled material was accounted for in the methylene chloride extract. Duplicate 2-kg samples of the ammoniated meal were fractionated and the distribution of radioactivity was determined. Ca. 86% of the radioactivity was detected in the meal after initial air-drying. Ca. 25% of the added radioactivity was extracted from the air-dried meal with methylene chloride and another ca. 5% was extracted from this residue with methanol. Weak acid released 3% of the added radioactivity from the residue after methanol extraction, bicarbonate released 1% and Pronase digestion, including methylene chloride extraction of the residue, accounted for nearly 19% of the total added radioactivity. Only 37% of the added radioactivity remained in the meal matrix following solvent extractions and chemical and enzymic treatments.     

Chemical studies on corn embryos infected by various fungi

The occurrence of various fungi in corn kernels obtained from eight localities in Egypt in two successive years was studied. Values for refractive index, color, acid value, saponification value, iodine value, peroxide value and unsaponifiable matter content of oils extracted from corn embryos that were deliberately infected by various fungi were compared to those for oil extracted from healthy embryos. Spectrometric analyses (UV, visible and IR) were done to deduce differences in the functional groups of the oils. Corn oil extracted from embryos infected with various fungi contained the same lipid classes as the oil extracted from healthy embryos. Contents of mono- and diglycerides and free fatty acids were much smaller for the oil extracted from healthy embryos. The fatty acid and unsaponifiable compositions of oils were studied by gas liquid chromatography. The fatty acid composition of corn oil extracted from infected embryos showed that some new and short-chain fatty acids had appeared and that some of the 18:2 was converted to 18:0. Analysis of the hydrocarbon fraction of the unsaponifiables showed also that some new compounds had appeared and others disappeared. The sterols were greatly influenced by the fungi and the ratio between different sterols might be used to characterize the effect of fungi. Aflatoxin B₁ content of oil extracted from corn embryos infected byA. flavus was 300 μg/kg.     

Triacylglycerol composition of Pinus koraiensis seed oil

The triacylglycerol (TG) composition of Pinus koraiensis seed oil, which contains Δ5 nonmethylene-interrupted (NMI) fatty acids (FA) (the main acid is pinolenic, 18:3 Δ5, 9, 12), was determined. TG were preliminarily separated by argentation thin-layer chromatography (TLC), and the obtained fractions were analyzed by high-temperature gas chromatography (GC) on a capillary column with methyl phenyl silicone phase. Additionally, high-performance liquid chromatography (HPLC) of TG was applied. The FA composition of all TG fractions was identified. The identification of TG was carried out by combining TLC, GC, HPLC, and calculated equivalent carbon numbers of TG standards. The TG species identification was confirmed by comparison of the theoretical recalculated and directly analyzed FA compositions of all TLC fractions of TG. Species of TG with unsaturation degrees of 1 to 7 and trace amounts of saturated and octaenoic TG species were found. Except for minor compounds, 26 TG molecular species of 32 main components were quantitatively determined. The main species were oleoyl dilinoleoylglycerol (14.7%), dilinoleoyl pinolenoylglycerol (10.7%), palmitoyl oleoyl linoleoylglycerol (8.3%), triolein (7.6%), and dioleoyl, linoleoylglycerol (7.4%). Seven TG species contained Δ5 NMI acyl groups. Of these, the major were dilinoleoyl pinolenoyglycerol (10.7%), stearoyl linoleoyl pinolenoylglycerol (6.5%) dioleoyl, pinolenoylglycerol (5.4%), and palmitoyl linoleoyl pinolenoyl-glycerol (5.5%). TG species with two or three NMI acyl groups were not detected.     

Automated AOM test for fat stability

A home-built version of the automated AOM test was used with Canola, corn, sunflower, olive and Crisco? oils, shortening and lard. The endpoint was found by measuring the conductivity of a solution of the exit gas from the reaction tube. Coefficients of variability of the samples ranged from 1.1% to 8.3%. The endpoint of the test was ca. 100 PV for Canola oil, ca. 200 PV for corn oil and 35 PV for lard. The aqueous solutions of the volatiles of three oils were used to determine the TBA value. Canola, sunflower and olive oil had TBA values ranging from 6–60 μg malonaldehyde/g at the end point. No apparent relationship was found between the TBA values of the volatiles’ solutions and the PV’s of the oils. Presented at the 73rd AOCS Annual Meeting, Toronto, 1982.     

Allelopathic effects ofCitrus aurantium L.

Field observations on undisturbed stands of sour orange revealed thatCynodon dactylon, Chenopodium album, Avena sativa, andAmaranthus retroflexus were not able to grow normally and complete their life cycles under its canopies, although the same species grow well under adjacent trees of date palm. Investigations revealed that the failure of the test species to grow normally under sour orange was not due to competition for light, moisture and minerals or to differences in soil texture or pH. Soil under sour orange trees drastically reduced seed germination and/or seedling growth of test species. Aqueous extracts, decaying materials, and volatile compounds of senescent and nonsenescent sour orange leaves were found to inhibit seed germination and/or seedling growth of test species. Therefore, allelopathy appeared to be the basic factor responsible for the reduction in plant growth with competition propably accentuating its effects.     

Membrane phospholipids and sterols in microfiltered milk fat globules

Native milk fat globules of various mean diameters, ranging from d₄₃ = 2.3 µm to 8.0 µm, were obtained using microfiltration of raw whole milk. After milk fat globule washing, the milk fat globule membrane (MFGM) was separated by manual churning. After total lipid extraction and separation of polar lipids, their phospholipid (PL) and sterol composition was measured using thin‐layer chromatography, methyl ester analyses by gas chromatography, and gas chromatography coupled to mass spectrometry. The main PL species were phosphatidylethanolamine, phosphatidylcholine and sphingomyelin. The respective fatty acid composition of each PL species was measured. Many different minor bioactive sterols were detected in the MFGM, e.g. lanosterol, lathosterol, desmosterol, stigmasterol and β‐sitosterol. No significant differences in the PL and sterol profile were found between MFGM extracted from small and large milk fat globule fractions.     

Fractional crystallization and gas chromatographie analysis of fatty acids as a means of detecting butterfat adulteration

A method has been devised which gives the distribution of saturated and unsaturated fatty acids of pure and adulterated cow and buffalo ghee with lard or margarine. It involves fractionation of pure and adulterated butterfat into fractions by fractional crystallization. The composition of the fatty acids liberated by the hydrolysis of each of the fractions was determined by gas chromatography. Adulteration of cow and buffalo ghee with various levels of lard or margarine caused significant changes in certain fatty acids, i.e., 22:0, 18:1, 18:0 and 16:0. It is possible to determine the extent of admixture of lard or margarine to either cow or buffalo ghee by applying a simple regression equation for certain fatty acids. This technique provides a basis for the detection of lipid adulteration.     

Comparison of Free Fatty Acids Composition of Cuticular Lipids of <Emphasis Type="Italic">Calliphora vicina</Emphasis> Larvae and Pupae

The chemical characterization of the free fatty acid (FFA) fractions of the cuticular lipids of Calliphora vicina larvae and pupae was performed by separating the FFA fraction using high-performance liquid chromatography with laser light scattering detection (HPLC–LLSD) and quantitatively analyzing the FFA using gas chromatography–electron impact mass spectrometry (GC–MS). Thirty-two saturated and unsaturated FFA were identified and quantified in the insect lipids. Cuticular FFA profiles of C. vicina larvae and pupae were compared. Cuticular FFA of larvae and pupae accounted for 70.8 and 77.8 % of the total lipids, respectively. The cuticular lipids of C. vicina larvae contained 24 FFA ranging from 8:0 to 24:0, whereas the cuticular lipids of pupae contained 32 FFA ranging from 6:0 to 26:0. The cuticular lipids of the larvae contained 16 saturated, five monounsaturated, one diunsaturated, and two polyunsaturated FFA. The cuticular lipids of the pupae contained 18 saturated, nine monounsaturated, two diunsaturated, and three polyunsaturated FFA. The major cuticular FFA in C. vicina larvae and pupae was 18:1 (47.6 and 41.7 %, respectively). The highest amounts of total cuticular FFA were detected in larvae of C. vicina (1.7 mg/g of the insect body). The quantities of total cuticular FFA in pupae were smaller (1.4 mg/g of the insect body).