Isolation and identification of Di-D-fructose dianhydrides resulting from heat-induced degradation of inulin

Dry heating of inulin up to 200 °C for 30 min resulted in a wide range of degradation products, from which five quantitatively dominating compounds were isolated using flash chromatography and semi-preparative HPLC–RI. It was possible to identify four different DFDAs (di-d-fructose dianhydrides) by means of one- and two-dimensional NMR, namely DFA III (α-D-Fruf-1,2′:2,3′-β-D-Fruf), DFA I (α-D-Fruf-1,2′;2,1′-β-D-Fruf), DFA VII (α-D-Fruf-1,2′;2,1′-α-D-Fruf), and β-D-Fruf-1,2′;2,1′-β-D-Fruf. For the fifth compound, the structure of the difructan α-D-Fruf-1,2′-β-D-Fruf was proposed. Using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD), it was possible to quantify those isolated DFDAs in inulin caramels to a total amount of up to 25%, indicating that dry heating of inulin may be a suitable tool to enrich caramels with DFDAs.     

Combined Use of Liquid–Liquid Microextraction and Carbon Nanotube Reinforced Hollow Fiber Microporous Membrane Solid-Phase Microextraction for the Determination of Triazine Herbicides in Water and Milk Samples by High-Performance Liquid Chromatography

A simple, efficient and environmentally friendly method for the extraction and determination of five triazine herbicides in water and milk samples was developed by simultaneous liquid–liquid microextraction and carbon nanotube reinforced hollow fiber microporous membrane solid-phase microextraction coupled with high-performance liquid chromatography–diode array detection. The parameters that affect the extraction efficiencies, including the type and concentration of multi-walled carbon nanotube, type of membrane solvent and desorption solvent, the type and volume of the extraction solvent in sample solution, extraction time and temperature, the pH of sample solution, stirring rate, and ionic strength were investigated and optimized. Under the optimum conditions, the method shows a good linearity within a range of 0.5–200 ng mL⁻¹ for water samples and 1–200 ng mL⁻¹ for milk samples, with the correlation coefficients (r) varying from 0.9991 to 0.9998 and from 0.9989 to 0.9994, respectively. The limits of detection were in the range between 0.08 and 0.15 ng mL⁻¹ for water samples and 0.3 and 0.5 ng mL⁻¹ for milk samples, while the relative standard deviations varied from 4.6% to 6.9% and from 5.3% to 7.7%, respectively. The recoveries of the target analytes at spiking levels of 5.0 and 50.0 ng mL⁻¹ were in the range from 86.6% to 106.8% for water samples and from 81.3% to 97.4% for milk samples. The results demonstrated that the developed method was an efficient pretreatment and enrichment procedure for the determination of triazine pesticides in real water and milk samples.     

Application of dispersive liquid–liquid microextraction for the determination of selected organochlorine pesticides in honey by gas chromatography–mass spectrometry

Dispersive liquid–liquid microextraction (DLLME) is a rapid and easy technique that consumes minute amounts of organic solvents. In this work, we present chemometric study on optimization of DLLME parameters for the extraction of aldrin, endrin, lindane, α-endosulfan, 4,4′-DDT and its metabolites from honey matrix. Method quantification limits (MQLs) vary between 0.3 ng/g for 2,4′-DDE and 4,4′-DDE to 13.2 ng/g for α-endosulfan and enable determination at levels below EU-established Maximum Residue Limits. The developed method is linear (R ² > 0.994) in the investigated range (MQL—100 ng/g), with preconcentration factors of 13.2–30.5 and good repeatability (CV ≤ 17%). A comparison with other available methods reported in the last decade is provided. The method has been applied to 19 real samples from Poland, and the results show that organochlorine pesticides (OCPs) are present in analysed honeys at levels not posing threat to human health (below 14 ng/g for sum of 4,4′-DDT and metabolites and below 5 ng/g for aldrin, endrin and lindane). To the best of our knowledge, this is the first reported application of DLLME for the determination of OCPs in honey.     

Detection of an angiotensin converting enzyme inhibitory peptide from peanut protein isolate and peanut polypeptides by western blot and dot blot hybridization

The peptide P7, with an amino acid sequence of Cys-Val-Thr-Pro-Ala-Leu-Arg, inhibits angiotensin I-converting enzyme (ACE). In this paper, P7 was identified in peanut protein isolate (PPI) and peanut polypeptides (PPs) with a new method. The P7 peptide was synthesized and used in the preparation of an antiserum. Using the antiserum, P7 was specifically identified in PPI by western blot, and its level in PPI and PPs was assayed by dot blot hybridization. The results showed that two bands of P7 expression with molecular weight 18–25 and 25–35 kDa were seen in PPI by glucan gel chromatography. The positive reaction rate of P7 in PPs was higher than in PPI, consistent with the measured ACE inhibitory activity. The rate of P7 in sample no. 3 reached 39.29% of the positive control, using a dose of 20 μg/mL. This sample had an ACE inhibitory activity of 89.73%. Therefore, western blot and dot blot hybridization with prepared antibody against synthetic peptide was a very sensitive detection method for peptide.     

Integrated structure and event-specific real-time detection of transgenic <Emphasis Type="Italic">cry1Ac/SCK</Emphasis> rice Kefeng 6

Transgenic rice Kefeng 6 is a transformation event containing two insect-resistant genes, cry1Ac and SCK (modified CpTI gene) in China. In order to monitor the probable release of Kefeng 6 in the future and execute the labeling requirements, it is necessary to develop a rapid and reliable detection method. In this study, both the 5′ and 3′-junction sequences spanning the plant DNA and the integrated gene construct of the rice event Kefeng 6 were isolated by genome walking and long-distance PCR (LD-PCR), successively. Multiple copies of truncated SCK gene and cry1Ac gene were found to integrate into the host rice genome. The event-specific real-time detection method for Kefeng 6 event based on its 5′-junction sequence was established using one plasmid molecule pMD-KF6 containing both 5′-junction sequence and rice endogenous gene gos9 sequence as the reference material (RM) with an absolute limit of quantification (LOQa) around 10 template copies. Thereafter, three different transgenic amounts of w/w mixed samples (5, 1, and 0.5%, respectively) were quantified to assess the performance characteristics of the established real-time PCR method. The accuracy expressed as bias deviated from the 4.00–26.00%, the precision expressed as standard deviation (SD) and relative standard deviation (RSD) deviated from 0.03–0.19 and 3.42–4.76%, respectively. Based on the earlier results, we concluded that the qualitative and quantitative PCR assays were reliable and accurate for Kefeng 6 measurement, and the reference plasmid pMD-KF6 could be a good substitute for the reference material for Kefeng 6 quantification.     

Determination of Vanadium in Food Samples by Cloud Point Extraction and Graphite Furnace Atomic Absorption Spectroscopy

A preconcentration methodology utilizing the cloud point phenomenon is described for the determination of total vanadium by graphite furnace atomic absorption spectrometry. Cloud point extraction method was based on the formation of a ternary complex between vanadium, 2-(2′-thiazolylazo)-p-cresol, and ascorbic acid, with subsequent extraction–preconcentration of the formed complexes using Triton X-100. Optimization of different parameters was evaluated. Under optimal conditions, a calibration curve was constructed, showing a linear range of 1.0–60 ng mL⁻¹ and the limit of detection and relative standard deviation for preconcentration of a 10-mL sample were found to be 0.05 ng mL⁻¹ and 3.9%, respectively. The preconcentration factor was found to be tenfold for 10 mL of water sample. The technique has been applied succesfully to the determination of vanadium traces in wine, tea, and tomato samples and the recoveries of added vanadium were in the range 96–102%.     

Effect of reaction conditions on lactulose-derived trisaccharides obtained by transgalactosylation with β-galactosidase of <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

We have studied the effect of pH (6.5, 7.5), temperature (40, 50 °C), time (2–24 h) as well as lactulose (250, 450 g/L) and enzyme (3, 6, 9 U/mL) concentration on the formation of 6′-galactosyl-lactulose (β-D-Galp(1 → 6)-β-D-Galp(1 → 4)-D-Fru) and 1-galactosyl-lactulose (β-D-Galp(1 → 4)-β-D-Frup(1 → 1)-D-Gal) formed during the transgalactosylation of lactulose with β-galactosidase from Kluyveromyces lactis. The most striking feature was the different susceptibility of both trisaccharides to the reaction conditions, being 1-galactosyl-lactulose more susceptible to degradation than 6′-galactosyl-lactulose. With the exception of pH, all the parameters studied presented an important effect on the formation of both trisaccharides. Among the reaction conditions assayed, the most favourable to obtain the highest yields of 6′-galactosyl-lactulose (10.4 g/100 g of total carbohydrates) and 1-galactosyl-lactulose (11.5 g/100 g of total carbohydrates) were 50 °C, pH 6.5, 250 g/L of lactulose, 6 U/mL of enzyme and 2 h. In addition, the reaction products galactose and fructose presented an inhibitory effect on the reaction, particularly noticeable in the case of fructose.     

Rheological properties of whey protein isolate stabilized emulsions with pectin and guar gum

Dynamic oscillatory and steady-shear rheological tests were carried out to evaluate the rheological properties of whey protein isolate (WPI) stabilized emulsions with and without hydrocolloids (pectin and guar gum) at pH 7.0. Viscosity and also consistency index of emulsions increased with hydrocolloid concentration. At γ = 20 s⁻¹, the value of viscosity of the emulsion with 0.5% (w/v) pectin was about fivefold higher than that of the emulsion without pectin. Flow curves were analyzed using power law model through a fitting procedure. Flow behaviour index of all emulsions except for containing 0.5% (w/v) guar gum was approximately in the range of 0.9–1.0, which corresponds to near-Newtonian behaviour. The shear thinning behaviour of emulsions containing 0.5% (w/w) guar gum was confirmed by flow behaviour index, n, of 0.396. Both storage (G′) and loss modulus (G″) increased with an increase in frequency. Emulsions behaved like a liquid with G″ > G′ at lower frequencies; and like an elastic solid with G′ > G″ at higher frequencies. Effect of guar gum was more pronounced on dynamic properties. Phase angle values decreased from 89 to <10° with increasing frequency and indicated the viscoelasticity of WPI-stabilized emulsions with and without pectin/guar gum.     

Improved Sample Preparation for Real-Time PCR Detection of Listeria monocytogenes in Hot-Smoked Salmon using Filtering and Immunomagnetic Separation Techniques

This work evaluated the application of filtration and immunomagnetic separation (IMS) as sample pretreatments for use in combination with real-time polymerase chain reaction (PCR) to detect and quantify Listeria monocytogenes in hot-smoked salmon. Salmon was artificially inoculated with L. monocytogenes at levels ranging from 8 × 10⁰ to 8 × 10⁵ cfu/g of sample, and homogenates obtained from these samples were filtered to recover bacterial cells without a pre-enrichment step. High recovery of bacterial cells was achieved using standard coffee filters. IMS significantly reduced the co-extraction of PCR inhibitors present in the samples to increase the assay sensitivity with regression line parameters applicable for quantification. The limit of detection and quantification were equal to 2 × 10¹–4 × 10¹ and 2 × 10² cfu/g of sample, respectively. The entire detection procedure could be completed within 3.5 h. This study demonstrated that coupling filtration and IMS with real-time PCR has contributed to improve the sensitivity of L. monocytogenes detection from hot-smoked salmon.     

Ferulic acid dehydrodimers as structural elements in cereal dietary fibre

 Investigations on insoluble dietary fibre (IDF) of wheat, rye, barley, oat, maize, rice and millet led to the identification of several new dehydrodimers of ferulic acid (DFA). These compounds arise from 8–8′, 8–5′, 8–O–4′ and 5–5′ coupling. Esterified phenolics were set free by mild alkali hydrolysis, total amounts of phenolics (ester- plus etherified) were determined by alkali hydrolysis under pressure. Phenolic acids were analysed by gas chromatography – mass spectrometry (GC-MS) as their trimethylsilyl (TMS) derivatives and by high performance liquid chromatography – diode array detection (HPLC-DAD). In esterified form 8–8′aryl DFA and 5–5′ DFA dominate in most cereal IDF with, together, 45–60% of the DFA sum. More than 60% of total bound DFA are involved in ether linkages. Highest amounts of esterified as well as etherified DFA are estimated in millet, followed by maize. DFA contents of wheat, rye and barley are about two- to threefold lower than in millet but about twofold higher than in oat or rice. Received: 27 January 2000     

Quality of mashed potatoes: effect of adding blends of kappa-carrageenan and xanthan gum

Blends of kappa-carrageenan (K) and xanthan gum (X) were added to mashed potatoes. Product was tested by instrumental texture profile analysis and cone penetration tests, oscillatory and steady rheology, colour, drip loss, total soluble solids and sensory analyses. A central composite rotatable design was used to study the effects of variation in levels of K (1.5–4.5 g kg⁻¹) and X (0.5–2.5 g kg⁻¹) concentrations. Addition of K had a major impact on gel strength, viscoelastic behaviour, sensory attributes and overall acceptability, whereas addition of X influenced textural and steady properties and colour. Mainly, elastic modulus (G′) was strongly dependent on the K concentration in mashed potatoes containing amylose. As compared to mashed potatoes with 1.5 g kg⁻¹ added X, additional incorporation of 5.12 g kg⁻¹ K increased G′ approximately twofold, possibly due to exclusion effect of the swollen starch granules and synergistic effect of K and denatured protein. The function of X in the mashed potatoes may in fact be confined to that of a filler rather than a dynamic constituent, although it does affect yield stress behaviour. When K/X blends (each biopolymer at 1.5 g kg⁻¹) were included in the formulation, the product exhibited very acceptable sensory quality. K provided the appropriate texture, while X imparted creaminess and mouthfeel to the product.     

Detection and Quantification of Enterobacter sakazakii by Real-time 5′-Nuclease Polymerase Chain Reaction Targeting the palE Gene

Enterobacter sakazakii is an emerging food-borne pathogen causing invasive infection with high mortality rates in neonates and infants. The aim of this study was to develop, optimize, and evaluate real-time 5′-nuclease polymerase chain reaction (PCR) for the specific detection and quantification of E. sakazakii. Original primers and TaqMan probe targeting a sequence of E. sakazakii palE gene were designed. The developed real-time PCR system was highly specific for E. sakazakii with 100% inclusivity determined using 54 E. sakazakii strains and 100% exclusivity determined using 99 other strains. Detection limits of 4 × 10² and 4 × 10¹ CFU ml⁻¹ were determined with 100% and 90% probability, respectively. The response of the 5′-nuclease PCR system was linear (correlation coefficient ≥ 0.997) in the range of 10¹ to 10⁸ CFU ml⁻¹. Five methods of DNA sample preparation were compared. The methods of DNA preparation using the InstaGene Matrix and the simple lysis by boiling with the Triton X-100 were the most sensitive with calibration lines applicable for quantification. The developed real-time PCR targeted to the palE gene provides an alternative possibility for the detection and quantification of E. sakazakii after the suitable sample preparation.     

Simultaneous Determination of 69 Pesticide Residues in Coffee by Gas Chromatography?CMass Spectrometry

A method using gel permeation chromatography (GPC) combined with solid-phase extraction (SPE) cleanup followed by gas chromatography–mass spectrometry (GC-MS) has been established for quantitative determination of 69 pesticide residues in coffee. Based on an appraisal of the characteristics of GC-MS, validation experiments were conducted for 69 pesticides. In the method, 2.0 g samples were mixed with 5 ml water and 1 g sodium chloride and extracted with 5 ml of ethyl acetate by blender homogenization, centrifugation, and filtration. Evaporation was conducted and the sample was injected into a 250 mm × 10 mm S-X3 GPC column, with ethyl acetate–n-hexane (1:2 v/v) as the mobile phase at a flow rate of 3 ml/min. The 4–15 min fraction was collected for the SPE cleanup, which was Envi-Carb SPE cartridge coupled with NH₂-LC SPE cartridge with acetone–ethyl acetate (2:5 v/v) as the eluted solvent. The eluents were collected and then evaporated to dryness, which was redissolved in 0.5 ml ethyl acetate for GC-MS analysis. For the 69 pesticides determined by GC-MS, the portions collected from GPC were concentrated to 0.5 ml and exchanged with 5 ml n-hexane. In the linear range of each pesticide, the correlation coefficient was R ² ≥ 0.99. At the low, medium, and high fortification levels of 0.05–1.0 mg/kg, recoveries fell within 60–120%. The relative standard deviation was between 1.3% and 22.3% for all 69 pesticides. The limits of detection for the method were 10 μg/kg to 150 μg/kg, depending on each pesticide.     

Chemiluminescence Detection of 2,4,5-Trichlorophenoxy Acetic Acid in Apple Juice by Digital Image Analysis

An image-based detection of an enhanced chemiluminescence enzyme-linked immunosorbent assay was developed for 2,4,5-trichlorophenoxy acetic acid with an anti-rabbit secondary antibody conjugated to peroxidase (goat anti-rabbit IgG–HRP). Data acquisition on microtiter wells is performed using a low-cost charge-coupled device camera for capturing images. The standard curve was produced for 0.01–5,000 ng mL⁻¹ of 2,4,5-trichlorophenoxy acetic acid. The minimum detectable concentration was 24 pg mL⁻¹, and the relative standard deviation was 5.5% (n = 10) for a 5 ng mL⁻¹ sample concentration. Similar sensitivity was obtained with enzyme-linked immunosorbent assay using the same polyclonal antibody and data acquisition by a spectrofluorometer. This method was applied to apple juice with recoveries between 95% and 111% and relative standard deviation between 6.1% and 11.3%.     

Determination of Anthraquinone Derivatives in Chang-Qing Tea by Using Cloud-Point Extraction and High-Performance Liquid Chromatography

In this paper, a cloud-point extraction method was developed for the determination of five anthraquinone derivatives in Chang-Qing tea by high-performance liquid chromatography. The optimum conditions for micelle extraction were obtained as follows—15% (w/v) Genapol X-080 as extractant, pH 3.5, liquid/solid ratio 80, and extraction time, 40 min. For cloud point preconcentration, 20% (w/v) NaCl was added, and the solution was incubated at 55 °C for 30 min. The detection limits for the five anthraquinone derivatives were in the range of 0.55–3.30 ng ml⁻¹. Average recoveries for the anthraquinone derivatives at three spiked levels were in the range of 84.3–104.1%. Relative standard deviations for six replicate determinations of Chang-Qing tea sample were below 2.39. The established method has been successfully applied to the determination of anthraquinone derivatives in Chang-Qing tea products from three different manufacturers.     

FTNIR Spectroscopic Method for Determination of Moisture Content in Green Tea Granules

The feasibility of measuring moisture content in green tea by Fourier transform near infrared (FTNIR) spectroscopic technique was investigated. Green tea granules samples with different moisture contents were scanned using FTNIR spectroscopy. The spectra were measured in diffused reflectance mode by keeping 4–5 g samples in small sample bottle. A partial least-square regression model was developed with vector normalization method in the near-infrared region (4,000–12,000 cm⁻¹ or 800–2,500 nm). The developed model was validated using cross-validation technique. Maximum coefficient of determination (r ²) value of 0.997 was obtained for the calibration model developed. The developed method was used further for quantification of moisture content in fresh green tea samples, and the results were compared with other methods like gravimetric method and moisture analyzer. Results indicated that FTNIR spectroscopy could be used for rapid detection of moisture content in green tea granules without destruction of samples. The measurement will take only 5–10 s.     

Effect of lactobacillus fermented beetroot juice on composition and activity of cecal microflora of rats

This study aimed at determination of the effect caused by ingestion of beetroot juice fermented by Lactobacillus casei 0920 and Lactobacillus brevis 0944 strains on the state of cecal ecosystem of experimental rats. The intake of fermented beetroot juice containing 3.5–4.0 × 10⁹ CFU/mL live Lactobacillus sp. cells positively modulated the cecal microflora of the rats and its metabolic activity. The counts of Lactobacillus sp., Bifidobacterium sp., Bacteriodes sp., and Enterococcus sp. were maintained at the level of 8.2–8.6, 6.2–7.5, 8.0–8.3, and 7.3–7.7 log units, respectively, while the number of Clostridium sp. cells was increased by 1.1–1.6 log units and Enterobacteriaceae bacteria were reduced by 0.8–2.1 log units. In this study, the selected cecal enzymes such as β-glucosidase, β-glucuronidase, and β-galactosidase as well as the profile and concentration of short chain fatty acids (SCFA) were the biochemical markers of metabolic activity of the intestinal ecosystem. The considerable decrease in activities of β-glucosidase and β-glucuronidase was observed in all three experimental groups fed with the fermented beetroot juice. Total concentration of SCFA was the highest (78.1 μmol/100 g BW vs. 59.2 μmol/100 g BW in control group) in intestines of rats fed with 6 mL of fermented beetroot juice daily. These results prove that the fermented beetroot juice benefits cecal microbial activity.     

Quantitation of the homocysteine content in wine

Alcohol consumption has been previously shown to correlate with elevated plasma homocysteine levels, but investigations have not been carried out on the possible availability of this compound in alcoholic beverages such as wine or spirits. Therefore, in this study we investigated the levels of homocysteine in various Bulgarian wines. A total of 36 different Bulgarian wines with known origins were studied. The measured values were in the range of 0.09–0.64 mg l⁻¹ for the tested white wines and in the range of 0.10–1.37 mg l⁻¹ for the red wines. The method used for homocysteine determination was based on RP-HPLC with fluorescent detection after derivatization with N-(2-acridonyl)maleimide. The method was linear in the range of 0.0070–1.35 mg l⁻¹ homocysteine and showed low limits of detection and quantification (LOD = 6 fmol, LOQ = 68 fmol). The within-run precision expressed as relative standard deviation (RSD, %) was 2.2–2.4% and the between-run precision was 2.6–3.9%. Enzyme immunoassay and LC-MS ⁿ analyses were used for confirmation of presence of homocysteine in wine.     

A Method for the Detection of Cryptosporidium parvum Oocysts in Milk Based on Microfiltration and Real-Time Polymerase Chain Reaction

A method for the detection of Cryptosporidium parvum oocysts in milk was developed on the basis of optimizing microfiltration and elution of the material from the filter, and using a previously developed highly sensitive downstream detection by real-time polymerase chain reaction. The method involves heating of milk to 40°C, microfiltration through a membrane microfilter made of a mixture of cellulose acetate and cellulose nitrate (pore size, 3.0 μm), elution of the material from the filter by a solution containing sodium pyrophosphate and Tween 80 in a shaker, rapid DNA extraction using a Chelex-based agent, and single-tube nested real-time PCR. The detection limit of the method is 10 C. parvum oocysts per 100 ml of milk. The developed method may be useful for specific and sensitive control of contamination of milk by C. parvum oocysts.