Application of the relative molar sensitivity method using GC-FID to quantify safranal in saffron (Crocus sativus L.).

Safranal is one flavor component of saffron, which is used as a spice, food additive, and crude drug. In ISO3632, safranal is defined as the compound that contributes to the quality of saffron, and many quantitative determination methods for safranal have been reported. However, safranal is volatile and degrades easily during storage, and an analytical standard with an exact known purity is not commercially available, making it difficult to quantify accurately the content of safranal in saffron. Here, we developed a method for quantifying safranal using relative molar sensitivity (RMS), called the RMS method, using a GC-flame ionization detector (GC-FID). We determined the RMS of safranal to 1,4-bis(trimethylsilyl)benzene-d4, a certified reference material commercially available, by a combination of quantitative NMR and chromatography. Using two GC-FID instruments made by different manufacturers to evaluate inter-instrument effect, the resultant RMS was 0.770, and the inter-instrument difference was 0.6%. The test solution, with a known safranal concentration, was measured by the RMS method, with an accuracy of 99.4-101%, repeatability of 0.81%, and reproducibility of 0.81-1.3%. Given the ease of degradation, high volatility, and uncertain purity of safranal reagents, the RMS method is a more accurate quantification approach compared to the calibration curve method and methods based on absorption spectrophotometry. Moreover, our findings revealed that the GC-FID makeup gas affected the RMS and quantitative values.

Ester synthesis reaction with CALB displaying yeast whole cell biocatalyst: effect of organic solvent and initial water content.

The effect of the reaction conditions on the ester synthesis reaction with CALB displaying yeast whole cells was determined. Utilization of hydrophobic organic solvent improved the efficiency of the ester synthesis reaction. Also the initial water content was important for the expression of the ester synthesis activity of CALB displaying yeast whole cells.

Development of yeast cells displaying Candida antarctica lipase B and their application to ester synthesis reaction.

We isolated the lipase B from Candida antarctica CBS 6678 (CALB CBS6678) and successfully constructed CALB-displaying yeast whole-cell biocatalysts using the Flo1p short (FS) anchor system. For the display of CALB on a yeast cell surface, the newly isolated CALB CBS6678 exhibited higher hydrolytic and ester synthesis activities than the well-known CALB, which is registered in GenBank (Z30645). A protease accessibility assay using papain as a protease showed that a large part of CALB, approximately 75%, was localized on an easily accessible part of the yeast cell surface. A comparison of the lipase hydrolytic activities of yeast whole cells displaying only mature CALB (CALB) and those displaying mature CALB with a Pro region (ProCALB) revealed that mature CALB is preferable for yeast cell surface display using the Flo1p anchor system. Lyophilized yeast whole cells displaying CALB were applied to an ester synthesis reaction at 60 degrees C using adipic acid and n-butanol as substrates. The amount of dibutyl adipate (DBA) produced increased with the reaction time until 144 h. This indicated that CALB displayed on the yeast cell surface retained activity under the reaction conditions.