Determination of folate concentrations in diverse potato germplasm using a trienzyme extraction and a microbiological assay

Folate deficiency is a leading cause of birth defects and is implicated in several other diseases. We are interested in how much folate concentrations vary among potato germplasm. We determined total folate concentrations of potato tubers from 67 cultivars, advanced breeding lines, or wild species. Folates were extracted by a tri-enzyme treatment and analyzed by using a Lactobacillus rhamnosus microbiological assay. Folate concentrations varied from 521 +/- 96 to 1373 +/- 230 ng/g dry weight and were genotype and location dependent. The highest folate concentrations were mostly found in color-fleshed potatoes. Variations of folate concentrations within either color- or white-fleshed tubers were similar ( approximately 2-fold). Skin contained approximately 30% higher folate concentrations than flesh. Storage of tubers for 7 months generally led to an increase in folate contents. Semiquantitative RT-PCR analyses showed that higher folate contents were correlated with lower mRNA expression of some folate genes.     

Determination of folate concentrations in pulses by a microbiological method employing trienzyme extraction

Over the past two decades, the role of folate in human nutrition has been of much interest because of its relationship to diseases such as neural tube defects and heart disease. Since 1998, the U.S. Food and Drug Administration has mandated that cereal products be fortified with 140 microg of folic acid/100 g. It is important, therefore, to be able to determine accurately the folate concentrations in cereals and other grains to ensure proper dietary intake of folate. In this study, a microbiological method employing a trienzyme extraction procedure was applied to the analysis of folate in several starchy grain legumes (pulses). Differences in the folate content of dry bean were observed among some market classes but not between cultivars in the same market class. Location had a significant effect on the folate content of lentil and dry pea; cultivar did not. The significant effect of market class, cultivar, and growth environment on the levels of folate in pulses is of particular importance to pulse processors and pulse breeders.     

Computerized semiautomated microbiological assay of folacin

A semiautomated microbiological folacin assay system is described. A microcomputer controls sample dilutions, medium addition, turbidity determination, and data acquisition. Assay capacity is 600 tubes per day, approximately twice that of comparable manual assays. Using the automated equipment, more samples can be compared within one assay, eliminating many sources of between-assay variation in large studies. Additional advantages of this system are reduced human errors, flexibility of assay design, and multifunctional component equipment. Folacin results from chicken liver, spinach, and breakfast cereal samples show equivalent precision for manual and automated assays.     

Application of a tri-enzyme extraction for total folate determination in foods

A tri-enzyme digestion procedure using chicken pancreas conjugase, alpha-amylase, and Pronase was evaluated to determine its usefulness in the microbiological quantitation of total folate in foods. Folate values obtained by traditional conjugase digestion were compared to those obtained by the tri-enzyme method for 12 food products that represent diverse matrixes. The tri-enzyme treatment increased measurable folate from most foods when compared to levels found after conjugase digestion. Largest increases were noted for tuna fish (51%) and yogurt (33%) after tri-enzyme digestion. For the 12 foods, a mean increase of 19% in measurable folate was obtained with tri-enzyme treatment. The study shows that traditional conjugase treatment does not completely free folate from complex food matrixes before microbiological analysis. Further, as other investigations have suggested, current accepted methods for folate analysis may be underestimating folate levels in foods.     

A simple and rapid assay for analyzing residues of carbamate insecticides in vegetables and fruits: hot water extraction followed by liquid chromatography-mass spectrometry

A simple, specific, and rapid analytical method for determining seven largely used carbamate insecticides in tomato, spinach, lettuce, zucchini, pear, and apple is here presented. This method is based on the matrix solid-phase dispersion technique, with heated water as extractant followed by liquid chromatography (LC)-mass spectrometry (MS) equipped with a single quadrupole and an electrospray ion source. Target compounds were extracted from the vegetal matrixes by water heated at 50 degrees C. After acidification and filtration, 0.25 mL of any aqueous extract was injected in the LC column. MS data acquisition was performed in the selected ion monitoring mode, selecting three ions for each target compound. Heated water appeared to be an excellent extractant because recovery data ranged between 76 (carbaryl in spinach) and 99% (pirimicarb in spinach), with RSDs not larger than 10%. Using trimethacarb (an obsolete carbamate insecticide) as a surrogate internal standard, the accuracy of the analysis varied between 84 and 110%, with RSDs not larger than 9%. On the basis of a signal-to-noise ratio of 10, limits of quantification were estimated to range between 2 (pirimicarb) and 10 ppb (oxamyl) and were not influenced by the type of matrix. When trying to fractionate analytes by using a short chromatographic run time, marked weakening of the ion signals for oxamyl, methomyl, and aldicarb were observed. This effect was traced to polar endogenous co-extractives eluted in the first part of the chromatographic run that interfered with gas-phase ion formation for carbamates. Adopting more selective chromatographic conditions eliminated this effect.     

Radiometric microbiological assay of vitamin B6: assay simplification and sensitivity study

Modification of a previously developed radiometric microbiological assay for vitamin B6 reduces assay complexity and time. Reduction of enzymatic treatment from 24 to 3 h essentially eliminates one day's time for the analysis of plasma samples. Use of lyophilized Kloeckera brevis cultures eliminates routine subculturing of the test organism, with no significant effect on test results. Modifications in test vial size and total volume in test vials have increased assay sensitivity to a level of 0.25 ng pyridoxine (PN), pyridoxal (PL), or pyridoxamine (PM) per vial level and decreased the amount of medium and labeled substrate (i.e., L-[1-14C]-valine), thus reducing assay cost.     

Improved microbiological assay for penicillin residues in tissues and stability of residues under cooking procedures

The microbiological assay for penicillin residues was improved by using centrifugation to remove physical barriers to diffusion, a small buffer/meat extraction ratio, and a more sensitive 2-layer assay system. Recoveries from muscle, kidney, and liver tissues ranged between 70.1 and 86.7% with measurable levels of 0.03--0.05 unit/g. By comparison, the Food and Drug-suggested methodology yielded recoveries of 45.9--54.0% and levels of detectability of 0.08--0.10 unit/g. Cooking of hamburger, steaks, and port chops indicated that procaine penicillin withstood cooking conditions, and significant levels of the original activity remained.     

八角茴香油提取工艺优化

采用响应面法对八角茴香油提取工艺进行优化。在选取最佳提取溶剂后,选取提取时间,液料比和温度为随机因子。在单因素试验的基础上,根据Box-Benhnken中心组合方法进行三因素三水平的试验设计。以茴香油得率为响应值,进行响应面分析(RSA),结果表明,八角茴香油提取的最佳工艺条件为：以丙酮为溶剂,提取时间2.3 h,液料比40︰5.4 （v/w）,回流温度78.6℃,在此条件下,八角茴香油的得率理论值为10.49%,验证实测值为10.56%,与理论值相对误差为0.67%。     

Determination of the total antioxidant activity of fruits and vegetables by a liposome assay

The effects of mixtures of antioxidants on the oxidation of phospholipids have been investigated in large unilamellar liposomes following initiation by 2,2'-azobis(2-aminopropane) dihydrochloride. The lag phase increased linearly with antioxidant concentration. The lag phases of mixtures containing alpha-tocopherol with ascorbic acid showed synergy between the antioxidants, but mixtures of beta-carotene with alpha-tocopherol or ascorbic acid were not synergistic. The liposome system was used to investigate the total antioxidant activity of lipid- and water-soluble extracts from 16 samples of fruits, vegetables, and related food products. The water-soluble extracts caused greater increases in lag phase than the lipid-soluble extracts. The lag phase of liposomes containing the water-soluble extracts from fruits and vegetables increased linearly with the total phenolic concentration, with the continental salad extract having the longest lag phase. The lipid-soluble extract from apples caused the largest increase in lag phase of the lipid-soluble extracts. The lag phases of the lipid-soluble and water-soluble extracts of all fruits and vegetables studied were additive, but no synergy was detected. The lag phase of the liposomes containing both the water-soluble and lipid-soluble extracts varied from 611.5 min for the continental salad extracts to 47.5 min for the cauliflower extracts.     

Study of an online molecularly imprinted solid phase extraction coupled to chemiluminescence sensor for the determination of trichlorfon in vegetables

This study reports a new online molecularly imprinted solid phase extraction coupled to chemiluminescence for the determination of trichlorfon. This molecularly imprinted polymer (MIP) was prepared through bulk polymerization, in which methacrylic acid (MAA) was used as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as the cross-linker. This novel functionalized material was characterized by FT-IR spectra and adsorption, and it exhibited good recognition and selective ability and fast adsorption-desorption dynamics toward trichlorfon. The factors affecting preconcentration of the analytes and sensitivity of the method are discussed in detail. Under the optimal condition, the linear range of the calibration graph was between 0.02 and 1.0 ng L(-1), and the detection limit was 1 × 10(3) ng L(-1). The blank cucumber samples spiked with trichlorfon at three levels were extracted and determined by the presented method with recoveries ranging from 83.5 to 94.5%, and the results were correlated well with those obtained using gas chromatography. Moreover, this developed method was successfully applied to the quantitative detection of trichlorfon residues in leek samples.     

Multiresidue method for the analysis of pesticide residues in fruits and vegetables by accelerated solvent extraction and capillary gas chromatography

An analytical procedure using accelerated solvent extraction and capillary gas chromatography with electron capture and flame photometric detections was developed to simultaneously determine residues of different pesticides in fruits and vegetables. Single laboratory validation of the method was carried out for 28 compounds selected from eight pesticide classes, in blank and fortified samples of fresh pear, cantaloupe, white potato, and cabbage. The method had to meet specific established validation criteria for regulatory purposes applicable to our laboratory. At each of the two fortification levels studied, 24 of the 28 pesticides gave recoveries of more than 70% with a coefficient of variation of less than 10%. With respect to existing procedures, the method showed acceptable limits of detection (from 0.0019 to 0.14 microg/g depending on the pesticide and matrix) while minimizing environmental concerns, time, and labor.     

Simultaneous extraction and quantitation of carotenoids, chlorophylls, and tocopherols in Brassica vegetables

Brassica oleracea vegetables, such as broccoli (B. oleracea L. var. italica) and cauliflower (B. oleracea L. var. botrytis), are known to contain bioactive compounds associated with health, including three classes of photosynthetic lipid-soluble compounds: carotenoids, chlorophylls, and tocopherols. Carotenoids and chlorophylls are photosynthetic pigments. Tocopherols have vitamin E activity. Due to genetic and environmental variables, the amounts present in vegetables are not constant. To aid breeders in the development of Brassica cultivars with higher provitamin A and vitamin E contents and antioxidant activity, a more efficient method was developed to quantitate carotenoids, chlorophylls, and tocopherols in the edible portions of broccoli and cauliflower. The novel UPLC method separated five carotenoids, two chlorophylls, and two tocopherols in a single 30 min run, reducing the run time by half compared to previously published protocols. The objective of the study was to develop a faster, more effective extraction and quantitation methodology to screen large populations of Brassica germplasm, thus aiding breeders in producing superior vegetables with enhanced phytonutrient profiles.     

水相酶解法提取菜籽油与菜籽蛋白工艺的优化

为提高菜籽蛋白的利用率，采用水相酶解法提取菜籽油与菜籽蛋白，以超滤法分离菜籽蛋白，重点对酶处理的工艺参数进行了研究。试验对各种酶的作用效果进行了比较，确定采用纤维素酶、果胶酶按活力比3∶1配方的复合酶处理效果较好；由优化试验得出酶处理的工艺参数为：固液比1∶5，酶用量30 U/g，酶处理时间100 min；在最佳工艺条件下获得高质量的菜籽油与菜籽蛋白，油与蛋白质得率分别为92.6%、82.3%。     

Improved folate extraction and tracing deconjugation efficiency by dual label isotope dilution assays in foods

A dual label stable isotope dilution assay was developed to trace the deconjugation efficiency of polyglutamic folate vitamers converted to their monoglutamic analogues. For this purpose, [(13)C(5)]-pteroylheptaglutamate was synthesized and added during extraction of foods as a tracer isotopologue along with [(2)H(4)]-5-methyltetrahydrofolate, [(2)H(4)]-5-formyltetrahydrofolate, [(2)H(4)]-tetrahydrofolate, [(2)H(4)]-10-formylfolate, and [(2)H(4)]-folic acid. The [(2)H(4)]-labeled folates were used as internal standards for the monoglutamates. Deconjugation converted the addition tracer [(13)C(5)]-pteroylheptaglutamate to the detection tracer [(13)C(5)]-folic acid, which was quantified along with unlabeled folic acid using [(2)H(4)]-folic acid as the internal standard. LC-MS/MS enabled the unequivocal differentiation of the three isotopologues. This tracing was used to optimize deconjugation efficiency, which was achieved by using 4-morpholineethanesulfonic acid buffer for extraction at pH 5.0 . The optimized assay revealed limits of detection for the folate vitamers ranging between 2.0 and 5.6 pmol per assay (equivalent to 2.2-6.6 μg/100 g dry mass), recoveries ranging between 98 and 105% and relative standard deviations in inter-assay precision ranging between 2 and 6%. The assay was applied to quantitate folates in spinach, beans, cheeses, bread, wheat germs, and yeast .     

超临界CO2流体萃取杏仁油工艺研究

该研究以单因素试验和正交试验相结合的方法对苦杏仁脂肪油的超临界CO2萃取工艺进行了研究.确定了超临界CO2萃取杏仁油的最佳工艺为:萃取压力35 MPa、萃取温度50 ℃、CO2流量24 L/h、粒径60目、萃取时间2 h.各因素影响杏仁的得率的顺序为:粒径＞时间＞萃取压力＞CO2流量＞萃取温度.最佳工艺验证试验的杏仁油的得率为52.98%.本研究的结果为下一步综合、无毒、高效地开发利用苦杏仁奠定了基础.