Conjugated Diene Hydroperoxides Research Articles

Effects of heavy metals and high temperature on Atlantic Forest species: analysis of their tolerance capacity

Changes in temperature and the deposition of potential pollutants in the soil, such as heavy metals, may damage plant communities, altering their physiological processes. High temperature may also cause a series of morpho-anatomical, physiological and biochemical changes in plants. However, tolerant plant species tend to restrict these harmful effects. The present study investigates the impact of atmospheric warming on the accumulation capacity of heavy metals (Zn, Ni, Cu) in the roots and leaves of a pioneer species (Croton floribundus) and a non-pioneer species (Esenbeckia leiocarpa) native to the Atlantic Forest of southeastern Brazil. The experimental design involved exposing the plants to two soil treatments: without excess metals (-M) and with excess metals (+M), along with varying thermoperiods of 26 ºC day/19 ºC night and 32 ºC day/20 ºC in growth chambers. Over a 28-day period, we assessed weekly metal content, translocation, growth parameters, a non-enzymatic antioxidant (glutathione) and indicators of cell damage or oxidative stress (chlorophylls a and b, total chlorophyll (a+b), carotenoids, malondialdehyde and conjugated diene hydroperoxide contents). Both species exhibited increased metal accumulation under excess metals, employing distinct translocation strategies. C. floribundus showed high translocation rates of Ni to leaves and E. leiocarpa immobilized Ni in the roots. Atmospheric warming reduced Cu and Ni translocation from roots to leaves in both species. C. floribundus displayed lower physiological damage compared to E. leiocarpa, demonstrating robust growth. We concluded that the pioneer species possessed greater tolerance to oxidative stress induced by temperature and metal-related environmental factors than the non-pioneer species, confirming our hypothesis. In addition, our finding provides valuable insights for conservation and management of ecosystems affected by climatic and pollutant changes.

Oleofoams: The impact of formulating air-in-oil systems from a lipid oxidation perspective

Air-in-oil foams, or oleofoams, have a great potential for food applications as they can at least partially replace animal or hydrogenated fats, without compromising on textural properties. Yet, there are some challenges to tackle before they can largely be implemented for real-life applications. One of those is the lack of data regarding their oxidative stability. This is an important point to consider, as although using oils rich in polyunsaturated fatty acids (PUFAs) is highly desirable from a nutritional perspective, these fatty acids are particularly prone to oxidation, which leads to major degradations of food quality. This work thus aimed to investigate the oxidative stability of oleofoams prepared with omega-3 PUFA-rich vegetable oils (rapeseed or flaxseed oil) and various types of high melting point lipid-based oleogelators (stearic acid, glyceryl monostearate and stearyl alcohol) when incubated at room temperature. The physical structure and stability of the oleofoams was monitored by various techniques (visual observations, microscopy, DSC, NMR, SAXS and WAXS). Lipid oxidation was assessed by combined measurements of primary (conjugated diene hydroperoxides) and secondary (thiobarbituric acid reactive substances – TBARS) products. We found that the oxidative stability of oleofoams was higher compared to that of the corresponding bulk oil. This protective effect was also found when the oil was simply mixed with the oleogelator without incorporation of air bubbles (i.e., forming an oleogel), and was somewhat modulated depending on the type of oleogelator. These results suggest that oleogelators and the structural changes that they induce limit the cascaded propagation of lipid oxidation in oil-continuous matrices, which is promising in the perspective of future applications.

Boosting the Antioxidant Potential of Polymeric Proanthocyanidins in Litchi (Litchi chinensis Sonn.) Pericarp via Biotransformation of Utilizing Lactobacillus Plantarum.

In order to enhance the efficient utilization of polymeric proanthocyanidins from litchi pericarp, a process for transforming litchis' polymeric proanthocyanidins (LPPCs) by using Lactobacilli has been established for products with highly antioxidative properties. Lactobacillus plantarum was selected to enhance the transformation effect. The transformation rate of LPPCs reached 78.36%. The content of litchis' oligomeric proanthocyanidins (LOPCs) in the products achieved 302.84 μg grape seed proanthocyanidins (GPS)/mg DW, while that of total phenols was 1077.93 gallic acid equivalents (GAE) μg/mg DW. Seven kinds of substances have been identified in the products by using the HPLC-QTOF-MS/MS method, among which 4-hydroxycinnamic acid, 3,4-dihydroxy-cinnamic acid, and proanthocyanidin A2 were major components. The in vitro antioxidative activity of the products after transformation was significantly (p < 0.05) higher than those of LOPCs and LPPCs. The scavenging activity of the transformed products for DPPH free radicals was 1.71 times that of LOPCs. The rate of inhibiting conjugated diene hydroperoxides (CD-POV) was 2.0 times that of LPPCs. The scavenging activity of the products for ABTS free radicals was 11.5 times that of LPPCs. The ORAC value of the products was 4.13 times that of LPPCs. In general, this study realizes the transformation of polymeric proanthocyanidins into high-activity small-molecule substances.

Lentinula edodes, a Novel Source of Polysaccharides with Antioxidant Power.

The fruiting bodies of edible mushrooms represent an important source of biologically active polysaccharides. In this study, Lentinula edodes crude polysaccharides (LECP) were extracted in hot water, and their antioxidant and antiradical activities were investigated. The antioxidant activity of LECP was investigated against reactive species such as 1,1’-diphenyl-2-picrylhydrazyl, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, hydroxyl and superoxide anion radicals, reducing power with EC50 values of 0.51, 0.52, 2.19, 3.59 and 1.73 mg/mL, respectively. Likewise, LECP inhibited the lipid peroxidation induced in methyl linoleate through the formation of conjugated diene hydroperoxide and malondialdehyde. The main sugar composition of LECP includes mannose, galactose, glucose, fucose and glucuronic acid. Characterization by Fourier transform infrared spectroscopy and nuclear magnetic resonance determined that LECP was made up of α and β glycosidic bonds with a backbone of α-D-Glc, →6)-β-D-Glcp-(1→, →6)-α-D-Galp-(1→ and β-D-Manp-(1→ residues. The results showed that LECP can scavenge all reactive species tested in a concentration-dependent manner and with a protective effect in the initial and final stages of lipid peroxidation. The natural antioxidant activity of the LECP that was investigated strengthens the high medicinal and nutritional value of this mushroom.

Antioxidants help favorably regulate the kinetics of lipid peroxidation, polyunsaturated fatty acids degradation and acidic cannabinoids decarboxylation in hempseed oil

The seed of the hemp plant (Cannabis sativa L.) has been revered as a nutritional resource in Old World Cultures. This has been confirmed by contemporary science wherein hempseed oil (HSO) was found to exhibit a desirable ratio of omega-6 and omega-3 polyunsaturated fatty acids (PUFAs) considered optimal for human nutrition. HSO also contains gamma-linoleic acid (GLA) and non-psychoactive cannabinoids, which further contribute to its’ potential bioactive properties. Herein, we present the kinetics of the thermal stability of these nutraceutical compounds in HSO, in the presence of various antioxidants (e.g. butylated hydroxytoluene, alpha-tocopherol, and ascorbyl palmitate). We focussed on oxidative changes in fatty acid profile and acidic cannabinoid stability when HSO was heated at different temperatures (25 °C to 85 °C) for upto 24 h. The fatty acid composition was evaluated using both GC/MS and 1H-NMR, and the cannabinoids profile of HSO was obtained using both HPLC-UV and HPLC/MS methods. The predicted half-life (DT50) for omega-6 and omega-3 PUFAs in HSO at 25 °C was about 3 and 5 days, respectively; while that at 85 °C was about 7 and 5 hours respectively, with respective activation energies (Ea) being 54.78 ± 2.36 and 45.02 ± 2.87 kJ/mol. Analysis of the conjugated diene hydroperoxides (CDH) and p-Anisidine value (p-AV) revealed that the addition of antioxidants significantly (p < 0.05) limited lipid peroxidation of HSO in samples incubated at 25–85 °C for 24 h. Antioxidants reduced the degradation constant (k) of PUFAs in HSO by upto 79%. This corresponded to a significant (p < 0.05) increase in color stability and pigment retention (chlorophyll a, chlorophyll b and carotenoids) of heated HSO. Regarding the decarboxylation kinetics of cannabidiolic acid (CBDA) in HSO, at both 70 °C and 85 °C, CBDA decarboxylation led to predominantly cannabidiol (CBD) production. The half-life of CBDA decarboxylation (originally 4 days) could be increased to about 17 days using tocopherol as an antioxidant. We propose that determining acidic cannabinoids decarboxylation kinetics is a useful marker to measure the shelf-life of HSO. The results from the study will be useful for researchers looking into the thermal treatment of hempseed oil as a functional food product, and those interested in the decarboxylation kinetics of the acidic cannabinoids.

Black Chokeberry Fruit Polyphenols: A Valuable Addition to Reduce Lipid Oxidation of Muffins Containing Xylitol.

The study aimed at assessing effects of black chokeberry polyphenol extract (ChPE) added (0.025–0.075%) to xylitol-containing muffins to reduce lipid oxidation, especially in preventing degradation of hydroperoxides throughout the storage period. Among polyphenolic compounds (3092 mg/100 g in total) in ChPE, polymeric procyanidins were the most abundant (1564 mg/100 g). ChPE addition resulted in a significantly increased capacity of scavenging free radicals and markedly inhibited hydroperoxides decomposition, as reflected by low anisidine values (AnV: 3.25–7.52) throughout the storage. On the other hand, sucrose-containing muffins had increased amounts of primary lipid oxidation products and differed significantly from other samples in conjugated diene hydroperoxides (CD values), which was in accordance with the decrease of C18:2 9c12c in those muffins after storage. In addition, sucrose-containing muffins were found to be those with the highest level of contamination with toxic carbonyl lipid oxidation products. Throughout the storage, no yeast or moulds contamination were found in higher enriched muffins. The incorporation of polyphenols to xylitol-containing muffins resulted in preventing decomposition of polyunsaturated fatty acids (PUFAs), and in reducing the content of some toxic aldehydes. ChPE could be regarded as a possible solution to xylitol-containing muffins to extend their shelf life. The results support the use of xylitol in muffin manufacture as being favourable in terms of suitability for diabetics.

N-linoleoylamino acids as chiral probes of substrate binding by soybean lipoxygenase-1

Lipoxygenases catalyze the oxygenation of polyunsaturated fatty acids and their derivatives to produce conjugated diene hydroperoxides. Soybean lipoxygenase-1 (SBLO-1) has been the subject of intensive structural and mechanistic study, but the manner in which this enzyme binds substrates is uncertain. Previous studies suggest that the fatty acyl group of the substrate binds in an internal cavity near the catalytic iron with the polar end at the surface of the protein or perhaps external to the protein. To test this model, we have investigated two pairs of enantiomeric N-linoleoylamino acids as substrates for SBLO-1. If the amino acid moiety binds external to the protein, the kinetics and product distribution should show little or no sensitivity to the stereochemical configuration of the amino acid moiety. Consistent with this expectation, N-linoleoyl-l-valine (LLV) and N-linoleoyl-d-valine (LDV) are both good substrates with kcat/Km values that are equal within error and about 40% higher than kcat/Km for linoleic acid. Experiments with N-linoleoyl-l-tryptophan (LLT) and N-linoleoyl-d-tryptophan (LDT) were complicated by the low critical micelle concentrations (CMC = 6–8 μM) of these substances. Below the CMC, LDT is a better substrate by a factor of 2.7. The rates of oxygenation of LDT and LLT continue to rise above the CMC, with modest stereoselectivity in favor of the d enantiomer. With all of the substrates tested, the major product is the 13(S)-hydroperoxide, and the distribution of minor products is not appreciably affected by the configuration of the amino acid moiety. The absence of stereoselectivity with LLV and LDV, the modest magnitude of the stereoselectivity with LLT and LDT, and the ability micellar forms of LLT and LDT to increase the concentration of available substrate are all consistent with the hypothesis that the amino acid moiety binds largely external to SBLO-1 and interacts with it only weakly.

Intake, digestibility and milk production and composition of dairy cows fed different levels of Yerba Mate in the diet

Eight multiparous lactating Holstein cows fitted with rumen cannulas and averaging 84±18 (mean±SD) days in milk and 598±55kg of body weight were assigned to a replicated 4×4 Latin square design to determine the effects of feeding Yerba Mate (YM) on intake, digestibility, milk production, milk composition, and milk concentration of antioxidants when cows were fed a corn silage-based diet containing ground canola seed. The treatments fed for ad libitum intake were: a total mixed ration not supplemented (0g/d), or supplemented (250, 500 or 750g/d) with dried leaves of YM. Experimental periods consisted of 14 d of diet adaptation and 7 d of data collection and sampling. Increased supplementation of the diet from 0 to 750g/d YM had no effect on milk production or yields of protein and fat, and decreased milk yields of lactose and total solids and milk urea N concentration. Digestibility of dry matter, protein and neutral detergent fiber was similar among diets and ether extract digestibility decreased with the level of YM supplementation. Concentrations of total polyphenols and production of conjugated diene hydroperoxides and thiobarbituric acid reactive substances in milk were similar among diets. The reducing power in milk increased with the inclusion level of YM in the diet, thus suggesting that antioxidant activity of milk can be enhanced with this feeding strategy although it was clearly insufficient to overcome the negative effects on milk fat synthesis probably caused by the high levels of fat added to diets as ground canola seed.

9(Z),12(Z)‐Eicosadienoic Acid as a Probe for Substrate Binding by Soybean Lipoxygenase‐1

Soybean lipoxygenase is a non‐heme iron protein that catalyzes the oxygenation of linoleic acid and other polyunsaturated fatty acids to produce conjugated diene hydroperoxides. The manner in which substrates bind to this enzyme is uncertain. One hypothesis is that the terminal methyl group of substrates binds near phenylalanine‐557. To test this hypothesis, 9(Z),12(Z)‐eicosadienoic acid (9,12‐EDA) has been synthesized and studied as a substrate of wild‐type SBLO‐1 and the F557A mutant, in which the smaller residue at position 557 might allow accommodation of a fatty acid with a longer alkyl terminus. The kinetics are complicated by pronounced substrate inhibition at high concentrations of 9,12‐EDA. At low substrate concentration, 9,12‐EDA exhibits lower activity than linoleic acid with wild type SBLO‐1, but the activities of the two fatty acids are comparable with the F557A mutant. The results are consistent with the hypothesis that phe‐557 is located at the binding site for the methyl terminus of substrates.Support or Funding InformationNSF CHE‐1213262

Using Mutagenesis and Synthetic Substrates to Explore Regiochemical Control by Soybean‐Lipoxygenase‐1

Lipoxygenases catalyze the oxygenation of polyunsaturated fatty acids to produce conjugated diene hydroperoxides. Plant lipoxygenases oxygenate linoleic acid to produce either 13‐hydroperoxy‐9,11‐octadecadienoic acid (13‐HPOD), 9‐hydroperoxy‐10,12‐octadecadienoic acid (9‐HPOD) or a mixture of both. The factors determining the position of oxygen incorporation are not well understood. Hornung et al. (Proc. Natl. Acad. Sci. USA 96 (1999) 4192–4197) identified one critical residue, corresponding to phenylalanine‐557 in soybean lipoxygenase‐1 (SBLO‐1). These workers observed that lipoxygenases such as SBLO‐1 with phenylalanine or histidine in this position generally produce primarily 13‐HPOD, whereas those with valine or threonine produce primarily 9‐HPOD. In the present work, the F557V mutant of SBLO‐1 has been prepared. Consistent with the trend noted by Hornung et al., this mutant produces 30–40% 9‐HPOD from linoleic acid at pH 7.5, compared with less than 10% for the wild‐type enzyme. Hornung et al. suggested that having a smaller residue in position 557 might enable linoleic acid to bind in a head‐first manner with the carboxylate group of the substrate interacting with arginine‐707. Consistent with head‐first binding, the 9‐HPOD formed by the F557V mutant has predominantly (86%) the S‐configuration at carbon‐9. To further test the model, we have investigated the action of SBLO‐1 on linoleoyldiethylamine (LDMA), which has been previously shown to be a good substrate for wild‐type SBLO‐1 at pH 7.0, where the dimethylamino group of LDMA is expected to be protonated and positively charged, which would disfavor interaction with arginine‐707. With this substrate, the amount of oxygenation at carbon‐9 is greater than observed with linoleic acid. This result argues against the hypothesis that oxygenation at carbon‐9 arises from binding of the polar end of the substrate near arginine‐707. Surprisingly, the F557A mutant produces only 9% 9‐HPOD from linoleic acid. This result indicates that the amount of 9‐oxygenation does not always correlate with the size of the residue at position‐557. In sum, the results confirm the importance of residue 557 in determining the regiochemistry of the products, but the structural basis for these effects remains to be elucidated.Support or Funding InformationNSF CHE‐1213262

Production, composition, and oxidative stability of milk highly enriched in polyunsaturated fatty acids from dairy cows fed alfalfa protein concentrate or supplemental vitamin E

Given its elevated content of carotenoids, alfalfa protein concentrates (APC) have the potential to prevent oxidation of milk enriched in polyunsaturated fatty acids. The effects of feeding APC or supplemental vitamin E on production, composition, and oxidative stability of milk enriched in polyunsaturated fatty acids were evaluated using 6 lactating Holstein cows (224±18d in milk) in a replicated 3×3 Latin square (21-d periods, 14d for adaptation). Treatment diets contained (dry matter basis) (1) 9% soybean meal (control, CTL); (2) 9% soybean meal + 300 IU of vitamin E/kg (VitE treatment); or (3) 9% APC (APC treatment). Cows received a continuous abomasal infusion of 450g/d of linseed oil. As a result, milk fat content of cis-9,cis-12 18:2 increased from 1.08±0.13 to 3.9±0.40% (mean ± SD), whereas cis-9,cis-12,cis-15 18:3 increased from 0.40±0.04 to 14.27±1.81% during the experimental period compared with the pretrial period. Milk yield tended to be higher for APC (14.7kg/d) compared with CTL (13.4kg/d), and was greater than that for VitE (13.0kg/d). Protein yield was higher in cows fed APC (518g/d) compared with VitE (445g/d) but was not different from that in cows fed CTL (483g/d). These effects resulted in improved milk N efficiency in cows fed APC (26.1% of N intake secreted in milk) compared with CTL (23.0%) and VitE (22.9%). Feeding APC increased milk fat content of lutein (252μg/g) compared with CTL (204μg/g) and VitE (190μg/g). Milk fat content of vitamin E was higher for APC (34.5μg/g) compared with CTL (19.0μg/g) and tended to be lower than that with VitE (44.9μg/g). Redox potential of fresh milk from cows fed APC (152mV) was similar to that of VitE (144mV), but lower than that of CTL (189mV). Treatments had no effect on fresh milk contents of dissolved oxygen (8.1±1.5mg/L), and conjugated diene hydroperoxides (2.7±0.5mmol/L). The concentrations of volatile lipid oxidation products (propanal, hexanal, hept-cis-4-enal, 1-octen-3-one) tended to be decreased by APC relative to CTL, whereas similar values were observed for VitE, except for hexanal, which was reduced by 40% in VitE. In conclusion, feeding APC to lactating dairy cows could serve as a source of dietary protein that improves dietary N utilization efficiency, and also as a preharvest technology to increase natural antioxidant levels in milk to limit oxidation.

The Influence of WPC 80 Additive on the Stability of Water-In-Oil Emulsions

Abstract The effect of 0, 1.5, 5, 10 and 15 (g 100 g-1 of emulsion) WPC 80 additive (80% whey protein concentrate) on the pH, physical, oxidative and microbiological stability of the water-in-oil emulsion was studied during 16-week storage at ~20°C at 4-week intervals. All determined features were significantly affected by the supplementation. The most beneficial as regards storage stability was the emulsion with 5% of WPC 80. This treatment was the most resistant to oxidative changes showing low increase of the concentration of conjugated diene hydroperoxides (from 0.92 to 1.04 mg g-1) and of the thiobarbituric acid reactive substances (from 0.83 to 1.37 mg malondialdehyde g-1) as well as only slight increment (by 0.47 log CFU g-1) of the microorganisms number during storage. Thus, the results of the present study revealed that whey proteins can be applied in the proper amount to produce cosmetic emulsions composed of natural ingredients and with reasonable storage stability.

Assessment of antioxidant potential of &lt;i&gt;Moringa stenopetala&lt;/i&gt; leaf extract

This study was conducted to assess the antioxidant potential of Moringa stenopetala leaf obtained from a private garden in Bahir Dar City and powdered Moringa leaf purchased from a supermarket in Bahir Dar City by using ferric reducing antioxidant power, 2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity, peroxide value and conjugated diene hydroperoxide assays. The powdered Moringa stenopetala leaf extract was invariably found to have a higher antioxidant capacity than the purchased Moringa powder. In the conjugated diene hydroperoxide and peroxide value assays, sunflower oil was used as an oxidation substrate. Both peroxide value and conjugated diene concentration for sunflower oil containing extracts of Moringa leaf and purchased Moringa powder were found to be lower than the corresponding values observed for the control showing the effectiveness of the extracts in delaying oxidation of the oil. The total phenolic content, in terms of mg gallic acid equivalent per 100 g of dry weight of sample was found to be 92.8 ±1.01 and 75.5 ± 2.28 for, respectively, powdered leaves of M. stenopetala and purchased powder of Moringa leaf. The antioxidant capacity of the powdered Moringa stenopetala leaf and purchased Moringa powder were found to be, respectively, 442.0±10.58 and 291.3±15.52 mg of ascorbic acid equivalent per 100 g of dry weight of plant sample in the FRAP assay. The corresponding values for the powdered Moringa stenopetala leaf and purchased Moringa powder in the DPPH assay were found to be, respectively, 144.0±0.53, 138.8±1.05 mg of ascorbic acid equivalent per 100 g of dry weight of the sample. This difference in antioxidant capacity of these two samples can be attributed to differences in their total phenolic content. It is suggested that this antioxidant potential of the leaves of Moringa stenopetala may underlie the widespread use of the plant in traditional medicine.Key words: Moringa stenopetala, Antioxidant potential, Ferric reducing antioxidant power (FRAP), 2,2-diphenyl- 1-picrylhydrazyl (DPPH) radical, Folin-Ciocalteu’s phenol reagent (FCR), Peroxide value (PV).

Functional properties of ultrasonically generated flaxseed oil-dairy emulsions

This study reports on the functional properties of 7% flaxseed oil/milk emulsion obtained by sonication (OM) using 20kHz ultrasound (US) at 176W for 1–8min in two different delivery formulae, viz., ready-to-drink (RTD) and lactic acid gel. The RTD emulsions showed no change in viscosity after sonication for up to 8min followed by storage up to a minimum of 9days at 4±2°C. Similarly, the oxidative stability of the RTD emulsion was studied by measuring the conjugated diene hydroperoxides (CD). The CD was unaffected after 8min of ultrasonic processing. The safety aspect of US processing was evaluated by measuring the formation of CD at different power levels. The functional properties of OM gels were evaluated by small and large scale deformation studies. The sonication process improved the gelation characteristics, viz., decreased gelation time, increased elastic nature, decreased syneresis and increased gel strength. The presence of finer sono-emulsified oil globules, stabilized by partially denatured whey proteins, contributed to the improvements in the gel structure in comparison to sonicated and unsonicated pasteurized homogenized skim milk (PHSM) gels. A sono-emulsification process of 5min followed by gelation for about 11min can produce gels of highest textural attibutes.

Production, composition and antioxidants in milk of dairy cows fed diets containing soybean oil and grape residue silage

Four primiparous Holstein cows averaging 504kg of body weight and 136 days in milk were used in a 4×4 Latin square design with four 21 days experimental periods to determine the effects of feeding four concentrations (0, 50, 75 and 100g/kg of dry matter (DM)) of grape residue silage on DM intake, total tract apparent digestibility (TTAD), milk production, milk composition, milk fatty acid profile and milk concentration of antioxidants when cows were fed a diet containing soybean oil. Intake of DM and nutrients was similar among diets, except for intake of ether extract (EE) which increased with the proportion of grape residue silage in the diet. The TTAD of DM, crude protein (CP), EE, acid detergent fiber and neutral detergent fiber decreased linearly with higher proportions of grape residue silage in the diet. Milk production and concentrations of CP, fat and lactose were similar among treatments, but increased levels of grape residue silage reduced milk urea N content. There was a little effect of diet on milk fatty acid composition, although there was a trend to increased proportion of polyunsaturated fatty acids in milk fat when feeding increased levels of grape residue silage. Concentrations of total polyphenols and flavonoids, and production of conjugated diene hydroperoxides in milk, were not altered by diets. Reducing power in milk was higher with increased dietary levels of grape residue silage. Results suggest that it is possible to improve milk fat quality by feeding cows with grape residue silage as shown by enhanced antioxidant activity, although it was clearly insufficient to overcome the negative effects on milk fat synthesis probably caused by the high levels of fat added to diets as soybean oil.

Introducing Pistacia khinjuk (Kolkhoung) Fruit Hull Oil as a Vegetable Oil with Special Chemical Composition and Unique Oxidative Stability

The chemical and qualitative characteristics of P. khinjuk hull oil (PKHO), P. atlantica hull oil (PAHO), and Sesamum indicume L. cv. Dezful seed oil (SISO) were compared. The high values of wax and tocopherol compounds and low values of the saponification number of PKHO were interesting. PKHO was also unique due to its high content of tocotrienols (92.6% of total tocol compounds). Oxidation stability data showed that PKHO was the most stable oil against lipid oxidation, followed by PAHO and SISO. The conjugated diene hydroperoxides increase during heat treatment (8 h) at 170°C for PKHO, PAHO, and SISO and were 18.41, 25.28, and 300.66%, respectively. Moreover, the carbonyl value was significantly increased at the lowest speed for PKHO, followed by PAHO and SISO. During heat treatment, there was no difference between PKHO and PAHO concerning total polar compounds, whereas the parameter showed a linear increase for SISO. The antioxidative activity of PKHO at low concentration (0.0099%) in olive oil was also almost equal to that of TBHQ (100 ppm).

An improved spectrophotometric phospholipase A2 assay using 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine as substrate and lipoxygenase as coupled enzyme

An improved spectrophotometric assay of phospholipase A2 (PLA2) activity based on the coupled PLA2/lipoxygenase (LOX) reactions using 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PCLIN) as substrate is reported. The PLA2-mediated release of free linoleate is continuously monitored by following the absorbance increase at 234 nm caused by its conversion into the conjugated diene hydroperoxide catalyzed by the coupled soybean LOX-1 reaction. The new protocol includes the use of Tween 20 (3 μL/μmol phospholipid) as surfactant and of ethanol (15 μL/mL reaction mixture), that ensure clearness of reaction mixture and linear increase of absorbance in the course of reaction. This method was tested on a purified secretory PLA2 from honey bee venom (HBV-PLA2). The enzyme did not discriminate among PCLIN, phosphatidylcholine, and phosphatidylethanolamine, but showed the highest rate using 1,2-dilinoleoyl-sn-glycero-3-phosphatidylcholine (PCDILIN). Nevertheless, the use of PCDILIN is not recommended, as it may induce an overestimation of enzyme activity, because not only the free linoleate, but also the reaction product 1-linoleoyl-lysophosphatidylcholine, are known to be oxidized by LOX. HBV-PLA2 showed maximal activity at pH 9.0, hyperbolic kinetics (Km, 74.2±2.9 μM; Vmax, 827±7 μmol/min/mg protein) and competitive inhibition (Ki about 5 μM) by palmityl trifluoromethyl ketone, a classical PLA2 inhibitor. Interestingly, the HBV-PLA2/soybean LOX-1 coupled reactions also allow an accurate assay of PCLIN concentration. In the whole, these results demonstrate that this improved PLA2/LOX assay allows a very accurate, simple, and rapid measurement of enzyme activity and substrate concentration.

Inhibition of Soybean Lipoxygenase‐1 by N‐oleoyl‐Dtryptophan

Soybean lipoxygenase‐1 (SBLO‐1) is a nonheme iron protein that catalyzes the oxygenation of polyunsaturated fatty acids using molecular oxygen, resulting in conjugated diene hydroperoxides. Although the three‐dimensional structure of SBLO‐1 is known, it is not certain how substrates bind. Following the discovery that N‐linoleoyl‐ D‐tryptophan is an excellent substrate for SBLO‐1, N‐oleoyl‐ D‐tryptophan (ODT) was synthesized as a potential competitive inhibitor. Potent inhibition was observed at micromolar concentrations of ODT. Kinetic analysis demonstrated that ODT had only small effects on Vmax, indicating that the inhibition is nearly competitive; however, the inhibition is strikingly non‐linear, which implies that OTD and substrate can bind simultaneously. Similar behavior was observed with N‐oleoyl‐ L‐tryptophan and N‐oleoyl‐D‐phenylalanine. Slow oxygenation of ODT was detected by HPLC, which indicates that ODT can access the active site. However, the inhibition kinetics imply that ODT binds more effectively somewhere else. Funded by NSF grant CHE‐1213262.

Crosslinking Studies to Probe Substrate Binding by Soybean Lipoxygenase‐1

Soybean lipoxygenase‐1 (SBLO‐1) catalyzes the oxygenation of polyunsaturated fatty acids into conjugated diene hydroperoxides. The three dimensional structure of SBLO‐1 is known, but it is not certain how substrates bind. One hypothesis involves the transient separation of helix‐2 and helix‐11 located on the exterior of the molecule in front of the active site iron. To test this hypothesis, site‐directed mutagenesis was used to create a cysteine mutation on each helix, which could allow for the formation of a disulfide linkage. Disulfide formation between the two cysteines in the T259C, S545C mutant was found to be unfavorable. Treatment of this mutant with the crosslinker 2,3‐dibromomaleimide (DBM) resulted in a 50% reduction in catalytic activity. No loss of activity was observed when the single mutant S545C or the wild type was treated with DBM. Additionally, no loss in activity occurred when the double mutant was treated with N‐ethylmaleimide. The results suggest that restricting the mobilities of helices 2 and 11 reduces but does not eliminate catalytic activity. Funded by NSF grant CHE‐1213262.

Oxidative stability and chemical safety of mayonnaise enriched with grape seed extract

Grape seed extract (GSE), a by-product from the wine industry, was explored for its use as enrichment to mayonnaise, due to its potential health effects. Mayonnaises were enriched with 0 mg GSE per mL, 0.5 mg GSE per mL (~0.050%), 0.9 mg GSE per mL (~0.10%) and 1.4 mg GSE per mL (~0.15%) during preparation and stored in the dark at room temperature for 8 weeks. The antioxidative capacity of the mayonnaises was evaluated by their ability to scavenge the stable radical TEMPO by electron spin resonance (ESR) spectroscopy. The oxidative stability of the mayonnaises was determined by the content of lipid hydroperoxides (peroxide value, POV), the content of conjugated diene hydroperoxides and the content of thiobarbituric acid reactive substances (TBARS). The highest antioxidative capacity and the lowest content of lipid hydroperoxides and TBARS were found in the mayonnaise with the highest percentage of GSE (0.15%). Therefore, the oxidative stability of the mayonnaises enriched with GSE was slightly improved through storage. However, mayonnaise without GSE had the highest sensorial acceptability compared to mayonnaise enriched with GSE. In the Artemia salina assay, a fast screening method for overall toxicity, the death rate of brine shrimps larvae was found to increase for increasing percentage of GSE. A level of 0.05% GSE in mayonnaise is concluded not to constitute any toxicological risks, but to provide significant protection against oxidation during storage.