Stable carbon isotopic characterization of rice vinegar protein as an intrinsic reference for discriminating the authenticity of brewed rice vinegar.

Rice vinegar plays an important role in daily life. However, some unscrupulous manufacturers may deliberately add synthetic acetic acid in vinegar products to reduce fermentation time and save production costs. To protect the rights and health of consumers, vinegar authenticity must be controlled. The rice vinegar protein was used as an intrinsic reference and its stable carbon isotope ratio (δ13Cprotein) was analyzed by elemental analyzer-isotope ratio mass spectrometry. The stable carbon isotope ratio difference between the acetic acid and the rice vinegar protein (Δδ13Cacetic acid-protein) was calculated to evaluate vinegar authenticity. Sixteen rice vinegar samples were analyzed and a stable carbon isotopic pattern of rice vinegar was established by the 95% confidence interval for Δδ13Cacetic acid-protein (0.27‰-2.10‰). An acetic acid adulteration curve of Δδ13Cacetic acid-protein was also assumed according to the data from rice vinegar samples, and its validity was confirmed by rice vinegar deliberately blended with acetic acid at different ratios (25, 50, and 75%). The Δδ13Cacetic acid-protein values of the adulterated vinegars decreased with increasing amounts blended acetic acid, but the δ13Cprotein values did not, showing that rice vinegar protein could be used as an intrinsic reference for identifying the adulterated rice vinegar. The rice vinegar adulterated with acetic acid at higher than approximately 10% could be detected.

Determination of glycidyl esters and 3-MCPD esters in edible oils by sample pretreatment with the combination of lipase hydrolysis and modified QuEChERS for GC-MS analysis.

Glycidyl esters (GEs) and 3-chloroprapane-1,2-diol esters (3-MCPDEs) are processing contaminants in refined edible oils that have raised concerns globally owing to their potentially carcinogenic properties. Official analytical methods for GEs and 3-MCPDEs, such as AOCS Cd 29a-13 and AOCS Cd 29b-13, require up to 16 h for chemical hydrolysis. Also, parallel experiments should be conducted to correct for the conversion of analytes during hydrolysis in AOCS Cd 29b-13. For AOCS Cd 29c-13 with the shortest operating time, the reaction time (3.5-5.5 min) and temperature of alkaline hydrolysis should be carefully controlled, implying the accuracy may be influenced by human errors. Here, we propose a novel method based on Candida rugosa lipase hydrolysis and direct detection of free form GEs, glycidol, which was achieved by sample preparation with modified QuEChERS, to prevent side reactions in previous approaches, and also to shorten the overall sample preparation time. Glycidol was directly analyzed without halogenation and derivatization, whereas 3-MCPD required derivatization for analysis by GC-MS. Our method showed good accuracy and precision in terms of repeatability, intermediate precision, and reproducibility (inter-laboratory precision). The limit of detection (LOD) and limit of quantification (LOQ) for glycidol were 0.02 and 0.1 mg/kg, which is sufficient for practical applications. The proposed method was further compared with AOCS Cd 29c-13 by determination of GEs content in commercial oil samples and spiked samples. Our method with a streamlined procedure seems to possess potential advantage of reduced errors from operational factors. This proposed method based on direct detection of glycidol may serve as a simplified alternative for routine analysis of GEs and 3-MCPDEs in edible oils.

Simultaneous Analysis of the Stable Carbon Isotope Ratios of Acetoin and Acetic Acid by GC-C-IRMS for Adulteration Detection in Brewed Rice Vinegar Products.

In this study, we developed a method to simultaneously measure the stable carbon isotope ratio for acetic acid (δ 13Cacetic acid) and acetoin (δ13Cacetoin) in rice vinegar by gas chromatography-combustion-isotope ratio mass spectrometry. The method showed good precision and accuracy. With this method, data from 16 brewed rice vinegars and 10 acetic acid samples were used to evaluate the feasibility of adulteration detection. On the basis that all δ13Cacetoin values of brewed rice vinegars are nearly constant, a characteristic pattern of the stable carbon isotope in rice vinegar was built with the 95% confidence intervals for δ13Cacetic acid (-26.97 to -25.38‰), δ13Cacetoin (-28.14 to -27.09‰), and Δδ13C (0.61 to 2.27‰). An adulteration detection curve of Δδ13C was proposed based on the results of vinegar and acetic acid samples and confirmed by vinegar spiked with different amounts of acetic acid. This method could be useful in estimating the blending ratio of adulterated rice vinegar products. Products containing more than 10% of synthetic acetic acid could be possibly identified.

Effect of charged amino acid side chain length on lateral cross-strand interactions between carboxylate- and guanidinium-containing residues in a ß-hairpin.

ß-Sheet is one of the major protein secondary structures. Oppositely charged residues are frequently observed across neighboring strands in antiparallel sheets, suggesting the importance of cross-strand ion pairing interactions. The charged amino acids Asp, Glu, Arg, and Lys have different numbers of hydrophobic methylenes linking the charged functionality to the backbone. To investigate the effect of side chain length of guanidinium- and carboxylate-containing residues on lateral cross-strand ion pairing interactions at non-hydrogen-bonded positions, ß-hairpin peptides containing Zbb-Agx (Zbb = Asp, Glu, Aad in increasing length; Agx = Agh, Arg, Agb, Agp in decreasing length) sequence patterns were studied by NMR methods. The fraction folded population and folding energy were derived from the chemical shift deviation data. Peptides with high fraction folded populations involved charged residue side chain lengths that supported high strand propensity. Double mutant cycle analysis was used to determine the interaction energy for the potential lateral ion pairs. Minimal interaction was observed between residues with short side chains, most likely due to the diffused positive charge on the guanidinium group, which weakened cross-strand electrostatic interactions with the carboxylate side chain. Only the Aad-Arg/Agh interactions with long side chains clearly exhibited stabilizing energetics, possibly relying on hydrophobics. A survey of a non-redundant protein structure database revealed that the statistical sheet pair propensity followed the trend Asp-Arg < Glu-Arg, implying the need for matching long side chains. This suggested the need for long side chains on both guanidinium-bearing and carboxylate-bearing residues to stabilize the ß-hairpin motif.

Effect of charged amino acid side chain length on lateral cross-strand interactions between carboxylate-containing residues and lysine analogues in a ß-hairpin.

ß-Sheets are one of the fundamental three-dimensional building blocks for protein structures. Oppositely charged amino acids are frequently observed directly across one another in antiparallel sheet structures, suggesting the importance of cross-strand ion pairing interactions. Despite the apparent electrostatic nature of ion pairing interactions, the charged amino acids Asp, Glu, Arg, Lys have different numbers of hydrophobic methylenes linking the charged functionality to the backbone. Accordingly, the effect of charged amino acid side chain length on cross-strand ion pairing interactions at lateral non-hydrogen bonded positions was investigated in a ß-hairpin motif. The negatively charged residues with a carboxylate (Asp, Glu, Aad in increasing length) were incorporated at position 4, and the positively charged residues with an ammonium (Dap, Dab, Orn, Lys in increasing length) were incorporated at position 9. The fraction folded population and folding free energy were derived from the chemical shift deviation data. Double mutant cycle analysis was used to determine the interaction energy for the potential lateral ion pairs. Only the Asp/Glu-Dap interactions with shorter side chains and the Aad-Orn/Lys interactions with longer side chains exhibited stabilizing energetics, mostly relying on electrostatics and hydrophobics, respectively. This suggested the need for length matching of the interacting residues to stabilize the ß-hairpin motif. A survey of a nonredundant protein structure database revealed that the statistical sheet pair propensity followed the trend Asp-Lys < Glu-Lys, also implying the need for length matching of the oppositely charged residues.

Helix formation and capping energetics of arginine analogs with varying side chain length.

Arginine (Arg) has been used for recognizing negatively charged biological molecules, cell penetration, and oligosaccharide mass signal enhancement. The versatility of Arg has inspired the need to develop Arg analogs and to research the structural effects of incorporating Arg analogs. Accordingly, we investigated the effect of Arg side chain length on helix formation by studying 12 Ala-based peptides containing the Arg analogs (S)-2-amino-6-guanidino-hexanoic acid (Agh), (S)-2-amino-4-guanidinobutyric acid (Agb), and (S)-2-amino-3-guanidinopropionic acid (Agp). Solid phase guanidinylation with orthogonal protection strategies was necessary to synthesize Agb- and Agp-containing peptides using Fmoc-based chemistry. The fraction helix for the peptides was determined by circular dichroism spectroscopy, and used to derive the statistical mechanical parameters and energetics for N-capping, C-capping, and helix propagation (propensity). All four Arg analogs were unfavorable for N-capping. The C-cap parameter followed the trend AgpAgh, highlighting the uniqueness of the Arg side chain length in helix formation. Molecular mechanics calculations and a survey on protein structures were consistent with the experimental results. Furthermore, calculations and survey both showed that the g- conformation for the χ1 dihedral was present for the first two residues at the N-terminus of helices, but not favored in the center or C-terminus of helices due to sterics. These results should serve as the foundation for developing Arg-related bioactive compounds and technologies.

Influence of nutritive factors on C50 carotenoids production by Haloferax mediterranei ATCC 33500 with two-stage cultivation.

The production of pigments by Haloferax mediterranei ATCC 33500 with two-stage cultivation in response to nutritive factors in culture media was studied. Sodium chloride and magnesium sulfate in the second-stage media showed a marked effect upon the production of pigments, and sodium acetate could enhance the production. As the cells were harvested at mid-log phase of growth in first-stage cultivation and transferred to the defined media containing 5% sodium chloride, 0.1% sodium acetate and 8% magnesium sulfate at 37 degrees C, 120 rpm for further 24 h of cultivation, H. mediterranei exhibited to be an efficient producer of pigments. The yield of pigments could reach up to 0.604 A(494 nm) mL(-1) broth. TLC analysis and the UV-Vis spectra of individual spots thereof revealed that H. mediterranei produced three red pigments of C(50) carotenoid, namely bisanhydrobacterioruberin, monoanhydrobacterioruberin and bacterioruberin, as well as a C(45) carotenoid, 2-isopentenyl-3,4-dehydrorhodopin.