Identification of Aging-Associated Food Quality Changes in Citrus Products Using Untargeted Chemical Profiling.

Chemometric techniques have seen wide application in biological and medical sciences, but they are still developing in the food sciences. This study illustrated the use of untargeted LC/MS chemometric methods to identify features (retention time_m/z) associated with food quality changes as products age (freshness). Extracts of three citrus fruit varietals aged over four time points that corresponded to noted changes in sensory attributes were chemically profiled and modeled by two discriminatory multivariate statistical techniques, projection partial least-squares discrimant analysis (PLS-DA) and machine learning random forest (RF). Age-associated compounds across the citrus platform were identified. Varietal was treated as a nuisance variable to emphasize aging chemistry, and further variable selection using age-related piecewise model generation and meta filtering to emphasize features associated with general aging chemistry common to all the citrus extracts. The identified features were further replicated in a validation study to illustrate the validity and persistence of these markers for applications in citrus food platforms.

Identification and Validation of Sensory-Active Compounds from Data-Driven Research: A Flavoromics Approach.

In this study, highly predictive LC-MS features (retention time_ m/ z) derived from untargeted chemical fingerprinting-multivariate analysis (MVA) previously used to model flavor changes in citrus fruits related to aging (freshness) were further isolated and analyzed for sensory impact, followed by structural elucidation. The top 10 statistical features from two MVA approaches, partial least-squares data analysis (PLS-DA) and Random Forrest (RF), were purified to approximately 70% via multidimensional liquid chromatography-mass-directed fractionation to screen for sensory activity. When added to a 'fresh' orange flavor model system, 50-60% of the isolates were reported to cause a sensory change. From the subset of the actives identified, two compounds were selected, on the basis of statistical relevance, that were further purified to >97% for identification (MS, NMR) and for sensory descriptive analysis (DA). The compounds were identified as nomilin glucoside and a novel ionone glucoside. DA evaluation in the recombination orange model indicated both compounds statistically suppressed the perceived intensity of the "orange character" attribute, whereas the novel ionone glycoside also decreased the intensity of the floral character while increasing the green bean attribute intensity.

Identification of bitter peptides in aged cheddar cheese.

The compounds responsible for the bitter taste of aged "sharp" Cheddar cheese were characterized. Sensory-guided fractionation techniques using gel permeation chromatography and multi-dimension semi-preparative reversed-phase high-performance liquid chromatography revealed the presence of multiple bitter compounds. The compounds with the highest perceived bitterness intensity were identified by tandem mass spectrometry de novo peptide sequencing as GPVRGPFPIIV, YQEPVLGPVRGPFPI, MPFPKYPVEP, MAPKHKEMPFPKYPVEPF, and APHGKEMPFPKYPVEPF; all originated from ß-casein. Subsequent quantitative liquid chromatography-tandem mass spectrometry analysis reported that the concentrations of GPVRGPFPIIV, YQEPVLGPVRGPFPI, and MPFPKYPVEP increased during maturation by 28.7-, 3.1-, and 1.8-fold, respectively. When directly compared to young "mild" Cheddar, APHGKEMPFPKYPVEPF was reported only in the sharp Cheddar cheese, whereas the concentration of MAPKHKEMPFPKYPVEPF did not change. Further taste re-engineering sensory experiments confirmed the importance of the identified peptides to the bitterness of sharp Cheddar. The bitter intensity of the aged "sharp" Cheddar model (mild Cheddar with equivalent concentrations of the five bitter peptides in the sharp sample) was rated as not significantly different from the authentic sharp Cheddar cheese. Among the five peptides, GPVRGPFPIIV was reported to be the main contributor to the bitterness intensity of sharp Cheddar. Furthermore, a difference from control sensory test also confirmed the significance of the bitter taste to the overall perception of aged Cheddar flavor. The sharp Cheddar model was reported to be significantly more similar to aged "sharp" Cheddar in comparison to the young "mild" Cheddar cheese sample.

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.

Rapid detection of ricin in milk using immunomagnetic separation combined with surface-enhanced Raman spectroscopy.

UNLABELLED: Ricin is a potential bioterrisiom agent. There is a critical need for a method that can rapidly and simply detect ricin and other bioterrisiom agents in complex food matrices such as milk. In this study, we demonstrated a rapid method that combined immunomagnetic separation (IMS) and surface-enhanced Raman spectroscopy (SERS) to detect ricin in whole milk. IMS was used to specifically capture the ricin out of the milk. Then, SERS was applied to analyze the IMS eluate mixed with silver dendrite nanosubstrates. This approach facilitated detection and quantification down to 4 µg/mL ricin in milk within 20 min, based on the results of principal component analysis and partial least squares analysis. The feasibility of using a portable Raman instrument shows great promise for on-site detection in a processing facility. PRACTICAL APPLICATION: The method described in this manuscript that combined IMS and SERS could be used for rapid detection of ricin and other protein toxins in complex food matrices such as milk within 20 min. The use of a portable Raman could facilitate the on-site detection in a processing facility.