Chiral amino acid analysis of vinegars using gas chromatography – selected ion monitoring mass spectrometry

 Twenty-five vinegars were examined quantitatively for their content of free amino acid (AA) enantiomers using chiral gas chromatography/mass spectrometry. Vinegars manufactured from grape must contained l-proline (l-Pro) as the major AA. Balsamic vinegars (aceto balsamico di Modena), with one exception, contained the highest amounts of l-AAs (861–2000 mg l^–1) as well as d-AAs (46–361 mg l^–1). The amounts of d-Pro and d-alanine (d-Ala) increased in the course of maturation. Sherry vinegars had a AA pattern similar to that of balsamic vinegars but with much lower amounts: concentrations of l-AAs were 244 mg l^–1 and 456 mg l^–1 and of d-AAs were 18 mg l^–1 and 19 mg l^–1. The l-AA content of cider vinegars was very low (34 mg l^–1 and 44 mg l^–1) and only traces of d-AAs (<2 mg l^–1) were found. In spirit vinegars few d-AAs and low amounts of most l-AAs were detected, with the exception of l-glutamic acid (l-Glu) (210–847 mg l^–1), probably added as a flavour enhancer. The AA content of spirit vinegars blended with wine vinegar was influenced by the portion of wine vinegar added. Rice vinegars had concentrations of l-AAs from as low as 36 mg l^–1 to as high as 6860 mg l^–1, and the concentrations of d-AAs ranged from 6 mg l^–1 to 531 mg l^–1. All vinegars declared as "produced by microbial fermentation" contained d-Ala, d-aspartic acid, and d-Glu as typical bacterial markers. From the data it is concluded that chiral AA analysis can be used to distinguish among fermented and synthetic vinegars and to identify raw materials used for their production. In particular, the amount of d-Pro can be used as proof of the maturation process of balsamic vinegar. Received: 11 December 1997 / Revised version: 12 May 1998     

Chiral amino acid analysis of vinegars using gas chromatography – selected ion monitoring mass spectrometry

 Twenty-five vinegars were examined quantitatively for their content of free amino acid (AA) enantiomers using chiral gas chromatography/mass spectrometry. Vinegars manufactured from grape must contained l-proline (l-Pro) as the major AA. Balsamic vinegars (aceto balsamico di Modena), with one exception, contained the highest amounts of l-AAs (861–2000 mg l^–1) as well as d-AAs (46–361 mg l^–1). The amounts of d-Pro and d-alanine (d-Ala) increased in the course of maturation. Sherry vinegars had a AA pattern similar to that of balsamic vinegars but with much lower amounts: concentrations of l-AAs were 244 mg l^–1 and 456 mg l^–1 and of d-AAs were 18 mg l^–1 and 19 mg l^–1. The l-AA content of cider vinegars was very low (34 mg l^–1 and 44 mg l^–1) and only traces of d-AAs (<2 mg l^–1) were found. In spirit vinegars few d-AAs and low amounts of most l-AAs were detected, with the exception of l-glutamic acid (l-Glu) (210–847 mg l^–1), probably added as a flavour enhancer. The AA content of spirit vinegars blended with wine vinegar was influenced by the portion of wine vinegar added. Rice vinegars had concentrations of l-AAs from as low as 36 mg l^–1 to as high as 6860 mg l^–1, and the concentrations of d-AAs ranged from 6 mg l^–1 to 531 mg l^–1. All vinegars declared as "produced by microbial fermentation" contained d-Ala, d-aspartic acid, and d-Glu as typical bacterial markers. From the data it is concluded that chiral AA analysis can be used to distinguish among fermented and synthetic vinegars and to identify raw materials used for their production. In particular, the amount of d-Pro can be used as proof of the maturation process of balsamic vinegar. Received: 11 December 1997 / Revised version: 12 May 1998     

Gas chromatographic detection of d-amino acids in natural and thermally treated bee honeys and studies on the mechanism of their formation as result of the Maillard reaction

Relative quantities of d-amino acids, (%D) calculated from the sum of d- and l-amino acids were determined in bee honeys (n=6) by GC-SIM-MS. Amino acids were isolated by treatment with Dowex 50 W X8 cation exchanger and converted into N(O)-perfluoroacyl amino acid propyl esters. In all honeys d-Ala, ranging from 2.2–6.2% d-Ala, was detected. Other d-amino acids were also found, albeit not in all honeys and approached 5.9% d-Glx, 5.4% d-Lys, 3.0% d-Phe, 2.1% d-Orn, 1.7% d-Asx, 1.5% d-Ser, 0.1% d-Pro, and 0.4% d-Val in certain honeys. Quantities of d-amino acids increased very much on experimental heating of honeys in an oven and on a microwave treatment. Conventional heating of a forest honey (no. 1) at 65 °C for 450 h leads to an increase of d-Ala (2.2–12.5%), d-Pro (0.0–5.0%), d-Ser (1.5–9.1%), d-Asx (1.7–9.8%), d-Phe (0.4–5.0%) and d-Glx (1.5–5.8%); first numbers in parentheses refer to unheated honeys. Relative quantities of other d-amino acids also increased. Experimental heating of another forest honey (no. 2) in a microwave oven for 3 min at 180 W leads to an increase of d-Ala (3.7–11.0%), d-Glx (1.5–13.7%), d-Asx (0.7–10.2%), d-Phe (0.3–4.8%), d-Val (0–4.2%), and d-Pro (0.1–2.3%). Microwave treatment at 700 W for 1 min of a blossom honey (no. 3) leads to an increase of d-Ala (6.2–26.7%) and of d-Phe (3.0–10.9%). Microwave treatments were accompanied by intensive destruction of amino acids. Heating of a model mixture mimicking the major components of honey (d-glucose, d-fructose, and l-amino acids at 20% water content) at pH 2.6–9.0 and at 180 W for 1–3 min leads to the generation of d-amino acids and was also accompanied by intensive decay of amino acids. From the data it is concluded that d-amino acids are formed in honeys in the course of the Maillard reaction. A mechanism is presented based on amino acid racemization of reversibly formed Heyns and Amadori compounds (fructose-amino acids).Parts of the results have been presented at 9th International Congress on Amino Acids and Proteins, August 8–12, 2005, Vienna, Austria, and Euro Food Chem XIII, September 21–23, 2005, Hamburg, Germany.     

Microwave treatment of dietary gelatin does not generate cis-4-hydroxy-l-proline, an inhibitor of collagen biosynthesis

 Aqueous solutions (5 g/100 ml) of commercial preparations of (a) an enzymatic partial hydrolysate of gelatin and (b) type A gelatin were subjected to threefold heating to boiling in a domestic microwave oven at 750 W and to conventional heating. Then samples were totally hydrolyzed (6 M hydrochloric acid, 110  °C, 24 h) and investigated for the presence of eight possible stereoisomers of 3- and 4-hydroxyproline (Hyp) using capillary gas chromatography. Amino acids were analyzed as N(O)-trifluoroacetyl 2-propyl esters on Chirasil-l-Val and detected by selected ion monitoring mass spectrometry. Blanks of (a) and (b) were analyzed in parallel. Relative amounts of 5.0±0.2% cis-4-d-Hyp were generated from native trans-4-l-Hyp as a result of total hydrolysis in all samples and independent of previous treatment. Notably, neither cis-3-l-Hyp nor cis-4-l-Hyp could be detected in either of the gelatin samples. Thus a report on the generation of antifibrotic and therefore potentially hazardous cis-3-l-Hyp and cis-4-l-Hyp from protein-bonded native trans-3-l-Hyp and trans-4-l-Hyp on microwave heating of infant formulae could not be confirmed.     

Production of Cell Membrane-Bound α- and β-Glucosidase by Lactobacillus acidophilus

Hydrolytic enzymes, viz. α- and β-glucosidase, were produced from indigenous isolate, Lactobacillus acidophilus, isolated from fermented Eleusine coracana. Production of these enzymes was enhanced by optimizing media using one factor at a time followed by response surface methodology. The optimized media resulted in a 2.5- and 2.1-fold increase in α- and β-glucosidase production compared with their production in basal MRS medium. Localization studies indicated 80% of the total activity to be present in the cell membrane-bound fraction. Lack of sufficient release of these enzymes using various physical, chemical, and enzymatic methods confirmed their unique characteristic of being tightly cell membrane bound. Enzyme characterization revealed that both α- and β-glucosidase exhibited optimum catalytic activity at 50 °C and pH 6.0 and 5.0, respectively. K _m and V _max of α-glucosidase were 4.31 mM and 149 μmol min⁻¹ mL⁻¹ for p-nitrophenyl-α-d-glucopyranoside as substrate and 3.8 mM and 120 μmol min⁻¹ mL⁻¹ for β-glucosidase using p-nitrophenyl-β-d-glucopyranoside as the substrate.     

Amyloglucosidase-catalysed synthesis of curcumin-bis-α-d-glucoside—a response surface methodological study

Response surface methodology (RSM) was employed to optimize amyloglucosidase-catalyzed synthesis of curcumin-bis-α-d-glucoside. A central composite rotatable design (CCRD) was employed involving five variables (enzyme concentration, curcumin concentration, incubation period, buffer concentration and pH) at five levels. A second-order polynomial equation with a R ² value 0.9 showed good correspondence between experimental and predicted yields. Three-dimensional surface and contour plots generated described the catalytic efficiency of amyloglucosidase under the reaction conditions employed. A maximum conversion of 35% was predicted for curcumin concentrations above 0.55 mmol at 35–60% (w/w d-glucose) amyloglucosidase concentrations. While lower (<35%) amyloglucosidase concentrations converted less, concentrations >60% could be inhibitory to curcumin. Extent of glucosylation was governed by a critical buffer (0.95–1.0 ml of 0.01 M pH 6.0) to enzyme ratio (15–45% w/w d-glucose). Experiments under optimum predicted conditions of 16.9% (w/w d-glucose) amyloglucosidase, 0.33 mmol curcumin, 120 h incubation period, 0.1 mM (1.0 ml of 0.01 M) buffer concentration at pH 7.5 gave a conversion yield of 56.3%. Validation experiments carried out under selected random conditions also showed good correspondence between experimental and predicted yields.     

Determination of Free Arginine, Glutamine, and β-alanine in Nutritional Products and Dietary Supplements

A method for the determination of free arginine, glutamine, and β-alanine in nutritional products and dietary supplements is described. The amino acids are derivatized with the fluorescent tag 9-fluorenylmethoxycarbonyl (FMOC), and the derivatives are then determined by reversed phase HPLC. Method suitability was defined by experimental assessments of linearity (R ² > 0.999), precision (day-to-day RSD ≤ 1.0%), accuracy (spike recoveries=98.7%–101.8%, n = 18), and selectivity (baseline resolution from the other common amino acids). The method provides for an accurate and precise quantification of the three amino acids, when present at concentrations >0.2% (w/w) in nutritional products and dietary supplements. Assessments of free l-glutamine stability in three different reconstituted powder products, as performed by the method, found recoveries >97% through 24 h at room temperature.     

Effects of nitrates and nitrites on Lactobacillus helveticus and Lactobacillus casei dairy cultures

 The aim of this work was to observe the effects of nitrites and nitrates on the titratable acidity of milk after addition of Lactobacillus helveticus (TX 121) and L. casei (CAD 154) dairy cultures and on the levels of nitrate and nitrite. After adding L. helveticus and nitrites the increasing concentrations of the latter brought about a marked decrease in titratable acidity. In milk containing 100 mg·kg^–1 nitrite the resulting concentration was 39.33 mg·kg^–1 NaNO₂. Nitrates caused a less obvious decrease in titratable acidity, giving 13.27 g·l^–1 lactic acid. In milk containing 100 mg·kg^–1 NaNO₃ the resulting concentration was 24.99 mg·kg^–1 NaNO₃. Experiments with L. casei and a nitrite additive revealed a decrease in titratable acidity to 8.89 g·l^–1 lactic acid. After incubation, nitrite levels were reduced from 100 mg·kg^–1 NaNO₂ to 37.81 mg·kg^–1 NaNO₂. Nitrates were also stated to inhibit the titratable acidity of the sample, which decreased to 11.42 g·l^–1 lactic acid. Nitrates were reduced to 46.99 mg·kg^–1 NaNO₃. The present study shows that nitrites, more than nitrates decrease the titratable acidity of milk after addition of L. helveticus and L. casei dairy cultures. A reduction of nitrates and nitrites in milk samples by Lactobacillus was also found. The results of this study can be used in the dairy industry in the production of several types of hard cheese as well as fermented milk products that use L. helveticus and L. casei dairy cultures. Received: 31 March 1999 / Revised version: 6 September 1999     

Determination of Histamine in Some Foods by Isotachophoretic Method with Simple Sample Preparation

A one-dimensional capillary isotachophoretic method in cationic system of the separation has been applied for histamine determination in food samples. The proposed electrolyte system consisted of 0.01 M potassium hydroxide with l-valine to pH = 9.9 as the leading electrolyte and 0.02 M 2-amino-2-hydroxymethyl-propane-1,3-diol adjusted to pH = 8.3 with 0.1 M hydrochloric acid as terminating electrolyte. Proposed method was characterized by linearity range 5–50 mg L⁻¹ and R ² = 0.9982, accuracy (recoveries ranged from 95% to 102%), detection (2.10 mg L⁻¹), and quantification (7.01 mg L⁻¹) limits. The sample preparation for proposed electrophoretic method included only simple extraction with trichloroacetic acid with filtration and derivatisation stage are avoided. The histamine concentration was determined in meat (turkey, chicken, beef and pork) and meat products (ripened sausage and dry-cured ham), fish (smoked salmon and mackerel), and different kind of mildew and mold ripened cheeses samples. The histamine content ranged from not detected level for fresh meat to 29.63 mg 100 g⁻¹ for cheese samples. The reversed phase HPLC was applied as reference method and the F-Snedecor test and the t test were employed to compare the precision and accuracy of the both methods. Positive correlations were found between the two analytical methods for histamine determination in food products. The obtained results indicate that the proposed electrophoretic method is simple, precise, accurate, and convenient.     

Electrochemical Determination of Ascorbigen in Sauerkrauts

Ascorbigen (ABG), 2-C-(indol-3-yl)methyl-α-l-xylo-hex-3-ulofuranosono-1,4-lactone, was synthesised and its structure and purity was confirmed by means of ¹H NMR and ¹³ C NMR spectra and HPLC-PDA, respectively. The electrochemical behaviour of ABG was studied by cyclic voltammetry (CV) method for pH within the range of 3.0–7.0 and further characterisation was performed by differential pulse voltammetry (DPV) at pH 5.0. Voltammetric studies of ABG at glassy carbon electrode showed one irreversible oxidation peak (centred at E _p = 0.950 V for pH 5.0). The anodic peak current potential related to the irreversible oxidation of ABG was shifted towards more positive potentials with decreasing pH values. The linear response between concentration of ABG within the range of 0.08–0.75 mM and recorded current was observed. The DPV method was shown to be more sensitive when compared to the CV method. ABG showed a significant reducing activity provided by CV, whilst the antioxidant activity of ABG against O₂^−• was negligible. Electrochemical behaviour of ABG standard was applied successfully for the quantification of natural ABG in sauerkrauts by DPV and HPLC-CoulArray methods. Based on the developed analytical methods, ABG content increased during natural fermentation of cabbage at 0.5% and 1.5% NaCl levels, and results were comparable with those found in the literature.     

Spectrophotometric determination of glucose in foods by flow injection analysis with an immobilized glucose oxidase reactor

 A stopped-flow injection analysis system for the determination of glucose is described, based on the iodometric measurement of hydrogen peroxide generated in a glucose oxidase reactor. The detection of the iodine–starch complex was carried out spectrophotometrically. The calibration curve was linear up to 2 mmol l^-1 glucose, with a correlation coefficient, r, of 0.9996. The relative standard deviation was 1.6% (n=5) for 1.0 mmol l^-1 glucose. A sample frequency of 25 samples h^-1 was achieved. This method was applied to the determination of glucose in fruit products and the results obtained using this method were in good agreement with those of a routine enzymatic method. A high dilution of the sample avoided interference from ascorbic acid. The enzyme reactor was stable for 1 month after 200 measurements with no loss of activity. Received: 22 March 1996     

Biosensing Applications of Modified Core–Shell Magnetic Nanoparticles

Fe₃O₄ magnetic nanoparticles (MNPs) were synthesized and silanized to form a core–shell (Fe₃O₄–SiO₂) structure. Afterwards, surface modification with amino silane was carried out to produce amino groups on the MNPs for the biomolecule immobilization. In order to test the performance of amino functional MNPs as immobilization platform in biosensing applications, glucose oxidase was immobilized on the surface via glutaraldehyde. Obtained Bio-MNPs were then fixed onto the carbon paste electrode by the aid of magnetic force and used as the working electrode during the amperometric measurements at −0.7 V versus Ag/AgCl. After optimization of some parameters affecting the biosensor performance, analytical characterization was carried out. Linearity was found in the range of 0.25–2.0 mM glucose and defined by the equation of y = 8.366x + 1.819, (R ² = 0.996). Proposed biosensor was then applied for the glucose analysis in various beverages. Finally, data were compared with a commercial enzyme assay kit based on spectrophotometric Trinder reaction as a reference method.     

Analytical Methods for the Evaluation of α-<Emphasis Type="SmallCaps">l</Emphasis>-Fucosidase Activity in Sheep Milk

The lack of analytical methods for measuring the activity of highly thermolable endogenous enzymes in sheep milk is a factor that hampers the protection of typical Protected Designation of Origin (PDO) dairy products made from raw milk. In order to provide a solution, this study assesses, tests, and fully validates analytical procedures for the determination of α-l-fucosidase activity in sheep milk. While the UV–VIS method has been optimized for this matrix in order to solve clarification problems before the spectrophotometric reading, a reversed phase high-performance liquid chromatography literature method proposed for bovine milk has been successfully applied also to sheep milk. Both methods have been fully validated in terms of sensitivity, linearity, precision, and accuracy, displaying low detection and quantification limits, excellent linearity over a wide enzymatic activity interval, very good repeatability and reproducibility, and the lack of any bias. The analysis of a number of real samples of whole sheep milk has allowed the evaluation of an average value (46.78 ± 5.49 U mL⁻¹) and range (from 29.27 ± 2.60 to 72.64 ± 1.17 U mL⁻¹) of α-l-fucosidase activity. Such activity does not seem to differ substantially from those measured for bovine milk. Finally, marked seasonal variability has been observed in this preliminary dataset.     

Chemical characterization and antioxidant activity of an exopolysaccharide fraction isolated from <Emphasis Type="Italic">Bifidobacterium animalis</Emphasis> RH

The major exopolysaccharide fraction (EPSa) of Bifidobacterium animalis RH was purified to illustrate the chemical characterization and the antioxidant activity in vitro. The molecular weight of EPSa was 2.31 × 10⁴ Da determined by the gel permeation chromatography (GPC). Fourier-transform infrared spectra (FT-IR) and the monosaccharide analysis of high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) showed that it was a heteropolysaccharide mainly composed of d-glucose, d-mannose, d-galactose, l-arabinose, d-fructose, and l-rhamnose in molar ratios of 43:34:18:4:1:1. In antioxidant assays, the EPSa had a higher activity for antilipid peroxidation and a stronger scavenging activity of hydroxyl radical and superoxide radical than ascorbic acid. It also had a similar α,α-diphenyl-β-picrylhydrazyl (DPPH) radical-scavenging activity with ascorbic acid at low concentration and had concentration-dependent inhibitory ability on erythrocyte hemolysis. These results indicated that the EPSa fraction had strong antioxidant activities and could be explored as novel potential antioxidants.     

Enzymatische Bestimmung von S?uren in Wein I. Mitteilung ?pfels?ure, Milchs?ure, Citronens?ure

Zusammenfassung Nach Ausschaltung von Störungsmöglichkeiten durch Farbstoffe und Gerbstoffe kann die enzymatische Bestimmung vonl-Malat,l-Lactat und Citrat in Wein einwandfrei durchgeführt werden. Äpfelsäure kommt in Wein nur in derl-Form vor, Lactat dagegen sowohl in derd- als auch in derl-Form. Die beiden Antipoden stehen in keinem bestimmten Verhältnis zueinander, jedoch überwiegt bei hohen Milchsäuregehalten der Anteil derl-Form.

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Enzymatic determination of acids in wine I. Malic-, lactic- and citric-acid

Summary The enzymatic determination ofl-malate with MDH,l-lactate with LDH and citrate with citrate — lyase was carried out in more than 100 samples of white and red wine, after elimination of interfering substances, such as tans and colouring matters. The contents ofl-malate and citrate agreed very well with the results, found by a fractionation method on silica gel. Comparing the content of total lactate, found by the latter method, with the results of enzymatic determination ofl-lactate, it was obvious, thatd- andl-compounds occur in a different ratio, but at high lactic acid concentrations, however, the level of thel-form is higher.

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Auszug aus der Dissertation vonSiegfried Looser: Beitrag zur enzymatischen Analyse von organischen Säuren und Kohlenhydraten in Lebensmitteln. Technische Hochschule München 1968.     

Physicochemical and Enzymatic Properties of Five Kiwifruit Cultivars during Cold Storage

Samples of Abbot, Alison, Bruno, Monty, and Hayward cultivars of kiwifruit (Actinidia deliciosa) were obtained from the Iran Research Center of Citrus (Tonekabon, located in north of Iran) and their physicochemical properties were studied during cold storage (at T = 1 ± 1 °C, RH = 80 ± 5%) at 0-, 9-, and 18-week intervals. The mean chemical composition of the fruits were as follows: ash = 0.66–0.96%, moisture = 75.2–84.7%, starch = 0.3–7.0%, and ascorbic acid = 54.8–261.0; K = 125.0–372.0 mg 100 g⁻¹ fresh weight, Mg = 18.0–32.0 mg 100 g⁻¹ fresh weight, Na = 1.4–3.1 mg 100 g⁻¹ fresh weight, Fe = 0.17–0.52 mg 100 g⁻¹ fresh weight, Cu = 0.04–0.24 mg 100 g⁻¹ fresh weight, Zn = 0.16–0.49 mg 100 g⁻¹ fresh weight, Mn = 0.04–0.10 mg 100 g⁻¹ fresh weight, and P = 25.2–49.3 mg 100 g⁻¹ fresh weight; glucose = 0.7–2.39%, fructose = 1.20–3.13%, and sucrose = 0.0–5.8%. At the same time, the values of the parameters °Brix = 6.5–14.8% and acidity = 1.8–2.5% of the studied cultivars (mutual effects of cultivar and storage time) were investigated. The increase in peroxidase (POX = 0.0–6.65 U ml⁻¹) and the decrease in pectinesterase (PE; poor activity to 0) activities were also determined. The statistical analysis showed that the Bruno cultivar had the highest content of ascorbic acid (115.0–261.0 mg 100 g⁻¹ fresh weight), which is an important compound in fruits during storage, while Hayward had the best overall quality particularly with regards to its resistance to softening. This study confirms that long-term cold storage at 1 ± 1 °C and 80 ± 5% RH is suitable for maintaining the highest quality of Iranian grown cultivars of kiwifruit.     

Storage Properties of Low Fat Fish and Rice Flour Coextrudates

In the present study, response surface method (RSM) and genetic algorithm (GA) were used to study the effects of process variables like screw speed, rpm (x ₁), L/D ratio (x ₂), barrel temperature (°C; x ₃), and feed mix moisture content (%; x ₄), on flow rate of biomass during single-screw extrusion cooking. A second-order regression equation was developed for flow rate in terms of the process variables. The significance of the process variables based on Pareto chart indicated that screw speed and feed mix moisture content had the most influence followed by L/D ratio and barrel temperature on the flow rate. RSM analysis indicated that a screw speed > 80 rpm, L/D ratio > 12, barrel temperature > 80 °C, and feed mix moisture content > 20% resulted in maximum flow rate. Increase in screw speed and L/D ratio increased the drag flow and also the path of traverse of the feed mix inside the extruder resulting in more shear. The presence of lipids of about 35% in the biomass feed mix might have induced a lubrication effect and has significantly influenced the flow rate. The second-order regression equations were further used as the objective function for optimization using genetic algorithm. A population of 100 and iterations of 100 have successfully led to convergence the optimum. The maximum and minimum flow rates obtained using GA were 13.19 × 10⁻⁷ m³/s (x ₁ = 139.08 rpm, x ₂ = 15.90, x ₃ = 99.56 °C, and x ₄ = 59.72%) and 0.53 × 10⁻⁷ m³/s (x ₁ = 59.65 rpm, x ₂ = 11.93, x ₃ = 68.98 °C, and x ₄ = 20.04%).     

Occurrence of aflatoxin M1 in raw, pasteurized and UHT milk commercialized in Esfahan and Shahr-e Kord, Iran

Between September 2006 and September 2007, 236 samples of raw (n = 140), pasteurized (n = 48) and UHT (n = 48) milk were collected from supermarkets and from bulk milk tanks of eight dairy plants in the cities of Esfahan and Shahr-e Kord, Iran. All samples were analyzed for aflatoxin M₁ (AFM₁) contamination by ELISA and 213 (90.3%) were positive with mean concentrations 65 ng.l⁻¹. These concentrations are lower than the standards of Codex Alimentarius and FDA (500 ng.l⁻¹), but 119 samples (55.9%) had higher concentrations than the maximum tolerance accepted by some European countries (50 ng.l⁻¹). Mean concentrations of AFM₁ in raw, pasteurized and UHT milk were 68, 56, and 65 ng.l⁻¹, respectively. Mean concentrations of AFM₁ in autumn and winter samples were significantly higher (P < 0.05) than those of spring and summer but differences between AFM₁ concentrations of spring and summer samples were not significantly different. Concentrations of AFM₁ in milk from Shahr-e Kord were significantly lower (P ≤ 0.05) than those from Esfahan.     

Sorption Isotherms of Barnyard Millet Grain and Kernel

The moisture sorption isotherms of grain and kernel of barnyard millet (Echinochloa frumentacea) were determined at 20, 30, 40, and 50 °C. A gravimetric static method was used under 0.112–0.964 water activity (a _w) range for the determination of sorption isotherms. The models were compared using the coefficient of determination (r ²), reduced chi-square (χ ²) values, and on the basis of residual plots. In grain, modified Chung–Pfost (r ² > 0.99; χ ² < 0.7) and modified Oswin (r ² > 0.99; χ ² < 0.55) models were found suitable for predicting the M _e –a _w relationship for adsorption and desorption, respectively. Modified Henderson model was found to give the best fit (r ² > 0.99 and χ ² < 0.55) for describing the adsorption and desorption of the kernel. The isosteric heat, calculated using Clausius–Clapeyron equation, was varied between 46.76 and 61.71 kJ g⁻¹ mol⁻¹ at moisture levels 7–21% (d.b.) for grain and 47.11–63.52 kJ g⁻¹ mol⁻¹ at moisture level between 4% and 20% (d.b.) for kernel. The monolayer moisture content values ranged from 4.3% to 6% d.b. in the case of adsorption of barnyard millet grain and 5.2–6.6% d.b. in the case of desorption at the temperature ranges of 50–20 °C. The monolayer moisture values of barnyard millet kernel ranged from 4.4% to 6.67% d.b. in adsorption and 4.6% to 7.3% d.b. in desorption in the temperature ranges of 50–20 °C.     

Synthesis of oligosaccharides with lactose and N-acetylglucosamine as substrates by using β-d-galactosidase from Bacillus circulans

In the present study, β-d-galactosidase from Bacillus circulans was proved to be a suitable biocatalyst for the production of N-acetyl-oligosaccharides with lactose and N-acetylglucosamine (GlcNAc) as biocatalyst. During the hydrolysis of lactose, apart from no ultraviolet absorption oligosaccharides such as β-d-Galp-(1 → 6)-d-Glcp (6′-allolactose) and β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-d-Glcp (4′-galactosyl-lactose), the formation of four N-acetyl-oligosaccharides was followed by high-performance liquid chromatography with a diode-array detector. The four N-acetyl-oligosaccharides were isolated from the reaction mixture and identified to be as β-d-Galp-(1 → 4)-d-GlcpNAc (LacNAc, I), β-d-Galp-(1 → 6)-d-GlcpNAc (allo-LacNAc, II), β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-d-GlcpNAc (III), β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-d-GlcpNAc (IV) by authentic standards and the spike technique or high-resolution mass spectrometry with an electrospray ionization source and nuclear magnetic resonance spectroscopy. Furthermore, the effects of synthetic conditions including reaction temperature, concentration of substrate, molar ratio of donor/acceptor and enzyme concentration on the formation of N-acetyl-oligosaccharides were examined. We found that the optimal synthetic conditions were different for production of oligosaccharides with β-(1 → 4) linkages and β-(1 → 6) linkage. The optimal reaction conditions for I, III and IV were 40 °C, 0.50 M lactose and 0.50 M GlcNAc and 1.0 U/mL of enzyme. Under such conditions, the N-acetyl-oligosaccharides formed were composed of 28.75% of I, 2.29% of II, 9.47% of III and 5.67% of IV. On the other hand, suitable reaction conditions found for II were 40 °C, 0.50 M lactose and 0.50 M GlcNAc and 2.0 U/mL of enzyme.