Compositional Analyses and Shelf-Life Modeling of Njangsa (Ricinodendron heudelotii) Seed Oil Using the Weibull Hazard Analysis.

This study investigated the compositional characteristics and shelf-life of Njangsa seed oil (NSO). Oil from Njangsa had a high polyunsaturated fatty acid (PUFA) content of which alpha eleostearic acid (α-ESA), an unusual conjugated linoleic acid was the most prevalent (about 52%). Linoleic acid was also present in appreciable amounts (approximately 34%). Our investigations also indicated that the acid-catalyzed transesterification of NSO resulted in lower yields of α-ESA methyl esters, due to isomerization, a phenomenon which was not observed under basic conditions. The triacylglycerol (TAG) profile analysis showed the presence of at least 1 α-ESA fatty acid chain in more than 95% of the oil's TAGs. Shelf-life was determined by the Weibull Hazard Sensory Method, where the end of shelf-life was defined as the time at which 50% of panelists found the flavor of NSO to be unacceptable. This was determined as 21 wk. Our findings therefore support the potential commercial viability of NSO as an important source of physiologically beneficial PUFAs.

Assessment of the inhibition of ricin toxicity by lactose in milk.

The effect of lactose at the concentration typically found in milk (134 mM) on the ability of ricin to inhibit protein synthesis in HeLa cells was studied. Ricin (0.001 to 300 µg/ml) that was either not treated or treated with 134 mM lactose was added to test tubes containing 1 ml of HeLa cells (approximately 3 × 10(5) cells in a low-leucine medium). After 2 h of incubation at 37°C, 0.5 µCi of L-[U-(14)C]-leucine was added to each tube and incubated for another 60 min. The cells were harvested by centrifugation and lysed, and cellular proteins were separated. The amount of radioactivity incorporated into the proteins was determined by liquid scintillation. The biological activity of ricin, i. e., the amount of radioactivity in a sample relative to that of the control (cells not treated with ricin), was calculated for each treatment. The inhibitory effect of 134 mM lactose on the biological activity of ricin was only significant at concentrations of ricin below 1 µg/ml. At higher ricin concentrations, the effect of 134 mM lactose decreased as the concentration of ricin increased, resulting in an increase in the inhibition of proteins synthesis. Our results also indicated that bovine milk, when used in place of 134 mM lactose, was more effective for reducing the activity of ricin at concentrations below 1 µg/ml but was ineffective against ricin concentrations greater than 1 µg/ml. These results suggest that milk may not protect against ricin intoxication at the concentration (0.89 µg/ml) equivalent to the lowest limit of its 50 % lethal dose for a 20-kg child consuming 225 ml (8 oz) of milk.

Comparison of the presence of Shiga toxin 1 in food matrices as determined by an enzyme-linked immunosorbent assay and a biological activity assay.

This study was conducted to compare the identification of Shiga toxin 1 (Stx1) based on its specific biological activity and based on results of a commercial enzyme-linked immunosorbent assay (ELISA) kit. Stx1 was thermally treated for various periods in phosphate-buffered saline, milk, and orange juice. The residual Stx1 concentration was determined with the commercial ELISA kit, and its residual enzymatic activity (amount of adenine released from a 2,551-bp DNA substrate) was determined with a biological activity assay (BAA). Regression analysis indicated that the inactivation of Stx1 as a function of time followed first-order kinetics. The half-lives determined at 60, 65, 70, 75, 80, and 85°C were 9.96, 3.19, 2.67, 0.72, 0.47, and 0.29 min, respectively, using the BAA. The half-lives determined by the ELISA with thermal treatments at 70, 75, 80, and 85°C were 40.47, 11.03, 3.64, and 1.40 min, respectively. The Z, Q(10), and Arrhenius activation energy values derived by both assays were dissimilar, indicating that the rate of inactivation of the active site of Stx1 was less sensitive to temperature change than was denaturation of the epitope(s) used in the ELISA. These values were 10.28°C and 9.40 and 54.70 kcal/mol, respectively, with the ELISA and 16°C and 4.11 and 34 kcal/mol, respectively, with the BAA. Orange juice enhanced Stx1 inactivation as a function of increasing temperature, whereas inactivation in 2% milk was not very much different from that in phosphate-buffered saline. Our investigation indicates that the ELISA would be a reliable method for detecting the residual toxicity of heat-treated Stx1 because the half-lives determined with the ELISA were greater than those determined with the BAA (faster degradation) at all temperatures and were highly correlated (R(2) = 0.994) with those determined with the BAA.

Detection and inactivation of saxitoxin in skim milk.

In this study, saxitoxin dihydrochloride in skim milk was reacted with sodium hydroxide and hydrogen peroxide to yield nontoxic 8-amino-6-hydroxymethyl-iminopurine-3(2H)-propionic acid (AHIPA), which was quantified by fluorescence spectroscopy using excitation and emission wavelengths of 330 and 425 nm, respectively. Samples of saxitoxin dihydrochloride (in 20% ethanol, vol/vol) were used as controls. The limits of detection of AHIPA, based on the concentration of saxitoxin prior to inactivation, were 5 and 10 µg/ml for the control and skim milk, respectively. These values are considerably below the concentration of saxitoxin that corresponds to the lethal dosage of 1 mg for an adult of average weight (70 kg). The inactivation of saxitoxin proceeded at a lower rate in skim milk than in the control, as its reaction rate constant was only 0.004 min(-1) compared with 0.011 min(-1) for the control. We were unable to detect AHIPA in 2% milk contaminated with saxitoxin because of possible interference from what we believed were products of secondary reactions involving milk fat and sodium hydroxide. Our results also indicated that the conversion of saxitoxin to AHIPA increased initially with temperature up to 40°C but decreased thereafter. We observed a decrease in the formation of AHIPA when the concentration of hydrogen peroxide was increased except at 22°C, where there was an initial increase in AHIPA formation between 1.2 and 2.4 mg/ml hydrogen peroxide but its formation decreased thereafter.

Validation of immunodetection (ELISA) of ricin using a biological activity assay.

The suitability of enzyme-linked immunosorbent assay (ELISA) for residual ricin toxicity determination was investigated in this study. Ricin was thermally treated at 80 to 90 °C for up to 9 min, and its residual concentration was determined by means of a commercial ELISA kit, and its bioactivity (amount of adenine released from DNA) was determined by means of a biological activity assay (BAA). Results showed that inactivation of ricin followed 1st-order kinetics. The half-life values for loss of bioactivity at 80, 85, and 90 °C were 1.93, 0.65, and 0.41 min, respectively. Similarly, the half-life values for reduction in ricin concentration determined by ELISA were 3.06, 0.79, and 0.43 min, respectively. The half-lives determined by both assays were only significantly different at 80 °C. The Z, Q(10), and Arrhenius activation energy values determined by both assays were dissimilar: 11.74 ˚C, 7.12 and 50.1 kcal/mol, respectively, by ELISA; and 14.87 °C, 4.71 and 39.5 kcal/mol, respectively, by BAA. Nevertheless, our findings indicate that the 2 assays were highly correlated (R(2) = 1), and it can be concluded that ELISA would be a reliable method for detecting residual toxicity of heat-treated ricin based on fraction lost. Practical Application: The results of this study indicate that immunodetection, even though not a direct measurement of the biological activity of ricin, is suitable for determining the residual bioactivity of ricin since immunodetection and the biological activity assay used in this investigation were highly correlated. Therefore, ELISA can be used for routine assessment of residual activity or toxicity of ricin in thermally treated foods.

Detection of warfare agents in liquid foods using the brine shrimp lethality assay.

The brine shrimp lethality assay (BSLA) was used for rapid and non-specific detection of biological and chemical warfare agents at concentrations considerably below that which will cause harm to humans. Warfare agents detected include T-2 toxin, trimethylsilyl cyanide, and commercially available pesticides such as dichlorvos, diazinon, dursban, malathion, and parathion. The assay was performed by introducing 50 µL of milk or orange juice contaminated with each analyte into vials containing 10 freshly hatched brine shrimp nauplii in seawater. This was incubated at 28 °C for 24 h, after which mortality was determined. Mortality was converted to probits and the LC(50) was determined for each analyte by plotting probits of mortality against analyte concentration (log(10)). Our findings were the following: (1) the lethal effects of toxins dissolved in milk were observed, with T-2 toxin being the most lethal and malathion being the least, (2) except for parathion, the dosage (based on LC(50)) of analyte in a cup of milk (200 mL) consumed by a 6-y-old (20 kg) was less than the respective published rat LD(50) values, and (3) the BSLA was only suitable for detecting toxins dissolved in orange juice if incubation time was reduced to 6 h. Our results support the application of the BSLA for routine, rapid, and non-specific prescreening of liquid foods for possible sabotage by an employee or an intentional bioterrorist act. Practical Application: The findings of this study strongly indicate that the brine shrimp lethality assay can be adapted for nonspecific detection of warfare agents or toxins in food at any point during food production and distribution.

Esterification and hydrolytic activities of Candida rugosa lipase isoform 1 (LIP1) immobilized on celite 545, duolite A7, and sephadex G-25.

The esterification and hydrolytic activities of free and immobilized Candida rugosa lipase isoform 1 (LIP1) were investigated. Esterification activity was determined by reacting caprylic acid with glycerol in the presence of molecular sieves (30%, w/w), and the volume of 1.0 M NaOH consumed by the reaction products upon titration was used to calculate esterification activity. Caprylic acid was also reacted with cottonseed oil, and the amount of caprylic acid incorporated after 12 h of reaction was determined. Results indicated that LIP1 had little esterification activity, which was not significantly improved upon immobilization. Hydrolytic activity was determined by incubating tricaprylin emulsion (15%, w/w) with the respective lipases for 60 min, and the reaction products were titrated against 0.5 M NaOH. LIP1 showed hydrolytic activity comparable to Lipozyme RM IM. The hydrolytic activity improved significantly upon immobilization. Immobilization on Celite 545 produced the highest increase in hydrolytic activity.

Optimization of solid fat content and crystal properties of a trans-free structured lipid by blending with palm midfraction.

The optimization of solid fat content (SFC) and crystal properties of trans-free structured lipids (SL) synthesized by incorporating stearic acid into canola oil was investigated. The SLs were blended with varying amounts of palm midfraction (PMF). The SFC and crystal polymorphism were improved. The addition of sucrose stearate (S-170), sorbitan tristearate (STS), and distilled monoglycerides (DMG) to one of the blends, SL40:PMF (70:30, w/w), did not improve crystal polymorphism but had significant effects on crystal morphology. The emulsifiers significantly delayed crystal growth, resulting in smaller crystal sizes as compared to the control. They were unable to inhibit the formation of granular crystals (30-140 microm), which are undesirable in margarine, after 4 weeks of storage at 0 degrees C. Blends treated with S-170 and STS showed many small evenly distributed crystals interspersed with large crystal aggregates (after 4 weeks of storage), whereas the blend treated with DMG and the control showed irregularly shaped globular crystals, also interspersed with large crystal aggregates. However, these crystal aggregates were not observed upon visual and physical examination and may therefore not impart the sensory properties of the finished products negatively.

trans-Free margarines prepared with canola oil/palm stearin/palm kernel oil-based structured lipids.

Structured lipids (SLs) for formulating trans-free margarines were synthesized by lipase-catalyzed interesterification of the blends of canola oil (CO), palm stearin (PS), and palm kernel oil (PKO) in weight ratios (CO/PS/PKO) of 40:60:0, 40:50:10, 40:40:20, 40:30:30, 50:30:20, and 60:25:15. The atherogenicity was determined using fatty acid profiles. We also determined the physical properties (melting/crystallization profiles, solid fat content, polymorphism, and microstructure) of SLs and the textural properties of margarines made with the SLs. The SLs from the 50:30:20 and 60:25:15 blends had atherogenic indices similar to or lower than those of the commercial trans (CTMF) and similar to the trans-free margarine fats (CTFMF). SLs from the blends with PKO contained a wide range of fatty acids (C6-C20) and had more beta' than beta polymorphs. Margarines made with SLs from 50:30:20 and 60:25:15 blends possessed similar hardness, adhesiveness, or cohesiveness to margarines made with CTMF and CTFMF, respectively. Therefore, CO/PS/PKO-based SLs were suitable for formulating trans-free margarines with low atherogenicity and desirable textural properties.

Synthesis and characterization of canola oil-stearic acid-based trans-free structured lipids for possible margarine application.

Incorporation of stearic acid into canola oil to produce trans-free structured lipid (SL) as a healthy alternative to partially hydrogenated fats for margarine formulation was investigated. Response surface methodology was used to study the effects of lipozyme RM IM from Rhizomucor miehei and Candida rugosa lipase isoform 1 (LIP1) and two acyl donors, stearic acid and ethyl stearate, on the incorporation. Lipozyme RM IM and ethyl stearate gave the best result. Gram quantities of SLs were synthesized using lipozyme RM IM, and the products were compared to SL made by chemical catalysis and fat from commercial margarines. After short-path distillation, the products were characterized by GC and RPHPLC-MS to obtain fatty acid and triacylglycerol profiles, 13C NMR spectrometry for regiospecific analysis, X-ray diffraction for crystal forms, and DSC for melting profile. Stearic acid was incorporated into canola oil, mainly at the sn-1,3 positions, for the lipase reaction, and no new trans fatty acids formed. Most SL products did not have adequate solid fat content or beta' crystal forms for tub margarine, although these may be suitable for light margarine formulation.