Relationship between nitrate/nitrite reductase activities in meat associated staphylococci and nitrosylmyoglobin formation in a cured meat model system

Quantitative determination of catalase, nitrate reductase, nitrite reductase and nitric oxide synthase activities (NOS) was performed on 11 different bacterial strains, mainly staphylococci, isolated from fermented sausages, bacon brine or cured meat products. All except one strain possessed catalase activity in the range from 1.0 to 6.1 μmol min^− 1 ml^− 1. Ten out of 11 bacteria strains showed nitrate reductase activity in the range between 50 and 796 nmol min^− 1 ml^− 1 and nine showed nitrite reductase activity in the range between 6 and 42 nmol min^− 1 ml^− 1. No evidence of NOS activity of the selected strains was detected. In a colour formation assay containing myoglobin all strains affected nitrosylmyoglobin (MbFe^IINO) formation in assays containing nitrite, whereas only strains having nitrate reductase activity generated MbFe^IINO in assays containing nitrate as the sole nitrosylating agent. The quantitative nitrate and nitrite reductase activity did not fully explain or correlate well with the observed rate of formation of MbFe^IINO, which seemed to be more affected by the growth rate of the different strains. The mechanism of the reduction of nitrite into NO of strains not having nitrite reductase activity remains to be fully elucidated, but could be due to a dual-mode action of nitrate reductase capable of acting on nitrate.     

Determination of nitrate and nitrite in dairy samples by sequential injection using an in-line cadmium-reducing column

A sequential injection analysis (SIA) system for the spectrophotometric determination of nitrate in dairy samples was developed. A test portion was aspirated into a carrier solution containing ethylendiaminetetracetic acid (EDTA) and ammonium buffer, which flowed into a copperized cadmium reduction column installed in-line for the determination of the nitrate plus nitrite contents of samples. For the nitrite determination, another test portion of sample was aspirated and directly sent to the detector without reduction. Nitrate content was calculated from the difference between nitrate plus nitrite (expressed as nitrite) and nitrite content. The spectrophotometric determination is based on the Griess reaction. The proposed method was used to test several dairy samples (ultrapasteurized milk with 1.7% milk fat, whey, raw bovine milk and several cheese varieties). Results were statistically in good agreement with those provided by the reference procedure, with a detection limit of 0.15 mg L⁻¹. A sampling rate of 21 determinations per hour can be achieved with this procedure.     

Determination of bromate in bread additives and flours by flow injection analysis

A new, simple, rapid and sensitive spectrophotometric flow injection analysis (FIA) method was developed for the determination of bromate based on its reaction with 3,5-dibromo-PADAP and thiocyanate in a strongly acidic medium. This produced an unstable violet product with a maximum absorption at 602 nm. The calibration curve was linear in the range of 2.00×10⁻⁶–2.10×10⁻⁵ mol/l and the detection limit was 8.00×10⁻⁷ mol/l. The sampling frequency was 90 h⁻¹. The method has been successfully applied to the determination of bromate in commercial bread additives and flours.     

Intake and risk assessment of nitrate and nitrite from New Zealand foods and drinking water

Exposure to excess nitrite is a potential health risk for humans. One hundred meat and processed foods and 100 vegetable samples purchased from New Zealand retail outlets were prepared as for consumption and analysed for nitrite and nitrate concentration using a standard, validated methodology. Nitrate concentrations ranged from less than the limit of detection (LOD = 5 mg kg^-1) in cheddar cheese and cream cheese-based dips to 3420 mg kg^-1 in lettuce. Nitrite was detected in half the processed foods and meats analysed (levels up to 119 mg kg^-1), but detected in only one vegetable sample above the LOD (broccoli at 27 mg kg^-1 nitrite). Concentration data were combined with 24 h dietary recall information to generate 4398 individual adult daily exposure scenarios for exogenous nitrite and nitrate including a contribution from water assessed from 1021 drinking water samples. The mean adult daily intake of exogenous nitrate and nitrite from food and water combined was 16 and 13% of the Acceptable Daily Intake (ADI), respectively, and therefore should not pose a health risk for the average consumer. A maximally exposed New Zealand adult is estimated to have an intake of up to seven times the ADI for nitrate. When the endogenous conversion of nitrate to nitrite is taken into account, approximately 10% of people with an average rate of conversion and half of all people with a high rate of conversion are estimated to exceed the ADI. Either the ADI is inappropriate and needs to be re-evaluated, or those individuals who have a high rate of conversion of nitrate to nitrite are at risk to adverse effects of nitrite exposure.     

Optimisation of calcium-dependent protease and cathepsin D assays in ostrich muscle and the effect of chemical and physical dry-curing parameters

The best conditions for the assay of cathepsin D and Ca²⁺-dependent pro tease (CDP) activity in ostrich muscle was established in order to have a simple, rapid and reliable method for its determination. Measurements of A_280nm of TCA-soluble peptides and amino acid digests of casein and haemoglobin were used for measuring proteolytic activity in muscle extracts. The best conditions for the reliable determination of cathepsin D activity were found to be the incubation of an enzyme extract for 1 hr at 55 °C in a reaction mixture containing 0.9% (w/v) haemoglobin in 50 mM sodium formate buffer, pH 3.7. Characterization of the assay system for CDPs, obtained after phenyl-Sepharose chromatography, indicated that proteolytic degradation of casein by CDPs was linear with time up to 30 min at 30 °C and up to 0.1 units of activity. The effect of NaCl, KCl, nitrate, ascorbic acid, phosphate, glucose and sucrose on ostrich muscle CDP and cathepsin D activities has been studied. Salt (NaCl and KCl) acts as a strong inhibitor of proteolytic activity. Sodium and potassium nitrates (in the range 0–1000 mg l⁻¹) affected activity to varying degrees. CDP activity was enhanced by sodium nitrate concentrations below 700 mg l⁻¹ and unchanged by potassium nitrate. Cathepsin D activity was inhibited to some extent by sodium nitrate above 200 mg l⁻¹ and completely by potassium nitrate. Results showed that phosphate is an inhibitor of both activities. High concentrations of ascorbic acid (above 6 g l⁻¹) inhibited cathepsin D activity. Glucose (up to 2g l⁻¹) activated cathepsin D activity and inhibited CDP activity (up to 1 g l⁻¹). Sucrose activated enzyme activities at very low concentrations (1 × 10⁻³ M) and inhibited activities above 1 × 10⁻³ M.     

Changes of alanyl aminopeptidase activity and free amino acid contents in biceps femoris during processing of Jinhua ham

Sixty experimental Jinhua hams were processed by a traditional method. The potential alanyl aminopeptidase (AAP) activity in biceps femoris was determined. The effects of temperature, salt content, sodium nitrate content and pH on muscle AAP were evaluated using response surface methodology. Porcine muscle was found to possess very strong potential AAP activity that decreased gradually during processing from 201,635 U g⁻¹ before salting to 6147 U g⁻¹ after aging. Temperature, pH and salt content had significant exponential effects on AAP activity (P < 0.001). Both temperature and salt content interacted with pH in their effects on AAP activity (P < 0.01). However, 0–50 mg L⁻¹ sodium nitrate had no detectable effect on AAP activity (P > 0.05). The regression model showed muscle AAP maintaining its activity all through Jinhua ham processing, indicating that muscle AAP may generate free amino acids during the processing and storage of Jinhua ham. The concentrations of free amino acids increased significantly (P < 0.05) during Jinhua ham processing, except for arginine and cystine. The concentrations of most free amino acids were 5–20 times higher in the final product than in hams before salting. Final concentrations exceeded thresholds for sensory detection, thus implicating an important role of free amino acids in the determination of Jinhua ham flavor.     

Cloud point extraction and spectrofluorimetric determination of aluminium and zinc in foodstuffs and water samples

Cloud point methodology was successfully used for the extraction of trace amounts of aluminium and zinc as a prior step to their determination by spectrofluorimetry. Aluminium and zinc react with 8-hydroxyquinoline in a surfactant solution yielding a hydrophobic complex, which then is entrapped in surfactant micelles. Optimization was performed of the variables affecting complexation and phase separation. Under the experimental conditions used, preconcentration of 25 ml of sample in the presence of 0.12% (v/v) Triton X-114 permitted the detection of 0.79 μg l⁻¹ of aluminium and 1.2 μg l⁻¹ of zinc, respectively. The relative standard deviation for five replicate determinations of aluminium and zinc at 40 and 100 μg l⁻¹ concentration level, were 2.72 and 2.1%, respectively. Good recoveries in the range of 95–104% were obtained for spiked samples. The proposed method was applied to the determination of aluminium and zinc in different samples.     

Analysis of vinylidene chloride and 1-chlorobutane in foods packaged with polyvinylidene chloride casing films by headspace gas chromatography/mass spectrometry (GC/MS)

A headspace gas chromatography/mass spectrometry method was developed for the simultaneous determination of vinylidene chloride and 1-chlorobutane in foods packaged with polyvinylidene chloride casing films. The solid foodstuff was homogenized with an equal mass of distilled water. The homogenate was incubated for 1 h at 90°C in a sealed headspace vial, and the headspace gas was then analysed by gas chromatography/mass spectrometry in selected ion-monitoring mode using a bonded porous polymer-coated capillary column. The recovery rates of vinylidene chloride and 1-chlorobutane in foodstuffs were 94.5-103.9 and 85.8-120.3%, respectively. Among 13 samples tested, vinylidene chloride was detected at 0.001-0.020 µg g^-1 in 11 foodstuffs, and 1-chlorobutane was detected at 0.004-0.040 µg g^-1 in all 13 foodstuffs. Furthermore, vinylidene chloride was detected at 0.04 µg g^-1 in one casing film, and 1-chlorobutane was detected in all casing films. The results indicate that these compounds migrated from the casing films into the foodstuffs.     

Selenium content in selected Slovenian foodstuffs and estimated daily intakes of selenium

Food is the primary source of selenium for man and, since selenium is an essential trace element, the nutritional selenium status is of importance. Data on this topic are currently lacking in Slovenia. In the present study, selenium contents of some selected foods purchased on the Slovenian market were determined and estimation of the daily dietary intake by analysing 20 diet samples collected in four Slovenian Army barracks was made. In determination of the selenium content in selected food, the highest values were found, as expected, in protein-rich food such as fish, meat and eggs (33–686 ng g⁻¹), but lower values in milk and dairy products (12–30 ng g⁻¹) and vegetables and fruits (0.3–77 ng g⁻¹). Analysis of 20 military total daily diet samples, gave an average selenium daily intake of 87 μg.     

Corn leaf nitrate reductase--a nontoxic alternative to cadmium for photometric nitrate determinations in water samples by air-segmented continuous-flow analysis

Development, characterization, and operational details of an enzymatic, air-segmented continuous-flow analytical method for colorimetric determination of nitrate + nitrite in natural-water samples is described. This method is similar to U.S. Environmental Protection Agency method 353.2 and U.S. Geological Survey method 1-2545-90 except that nitrate is reduced to nitrite by soluble nitrate reductase (NaR, EC 1.6.6.1) purified from corn leaves rather than a packed-bed cadmium reactor. A three-channel, air-segmented continuous-flow analyzer-configured for simultaneous determination of nitrite (0.020-1.000 mg-N/L) and nitrate + nitrite (0.05-5.00 mg-N/L) by the nitrate reductase and cadmium reduction methods-was used to characterize analytical performance of the enzymatic reduction method. At a sampling rate of 90 h(-1), sample interaction was less than 1% for all three methods. Method detection limits were 0.001 mg of NO2- -N/L for nitrite, 0.003 mg of NO3-+ NO2- -N/L for nitrate + nitrite by the cadmium-reduction method, and 0.006 mg of NO3- + NO2- -N/L for nitrate + nitrite bythe enzymatic-reduction method. Reduction of nitrate to nitrite by both methods was greater than 95% complete overthe entire calibration range. The difference between the means of nitrate + nitrite concentrations in 124 natural-water samples determined simultaneously bythe two methods was not significantly different from zero at the p = 0.05 level.     

A new measure of uptake: desorption of unreacted phosphine from susceptible and resistant strains of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

Previous studies of phosphine (PH₃) uptake by insects have concentrated on the process as a whole (“gross uptake”), without distinguishing between absorption of the gas and oxidation to non-volatile products. The lower gross uptake by phosphine-resistant (R) strains of stored product pests has given some insights into resistance mechanism(s). In this study, a recently described method of fumigant residue analysis in grains (microwave irradiation followed by headspace gas chromatography) was adapted to measure absorbed unreacted PH₃ (“reversible uptake”) in a susceptible (S) and an R strain of the rust red flour beetle Tribolium castaneum. At a concentration of 0.9 mg l⁻¹, S insects contained 20 ng g⁻¹ after 15 min exposure, rising slowly to 50 ng g⁻¹ after 5 h. The R strain yielded 190 ng g⁻¹ after 15 min, falling to 50 ng g⁻¹ over 5 h. Falling PH₃ content corresponded with increasing mortality in the R strain, while all except the shortest exposure killed 97% or more of the S strain. Insects of either strain, killed prior to PH₃ exposure by freezing in liquid nitrogen, contained 130–140 ng g⁻¹ after 30 min, rising to 190–200 ng g⁻¹ after 5 h. Gross uptake under the same conditions was 50 μg g⁻¹ (S) and 8 μg g⁻¹ (R) after 5 h, which accords with the literature. Reversible uptake by living insects of either strain under anoxia was 40–50 ng g⁻¹ over 30 min to 2 h. By examining the time-course of reversible PH₃ uptake, a new hypothesis of phosphine action and uptake, in which PH₃ oxidation in vivo is a consequence of reactive oxygen species generation, rather than a direct cause of toxicity, is discussed.     

Total and soluble contents of calcium, magnesium, phosphorus and zinc in yoghurts

This paper examines the content of major minerals (calcium, magnesium, phosphorus and zinc) and its distribution between soluble (non-sedimentable) and micellar fractions of 16 commercial yoghurts from five different branches. These elements were determined by atomic absorption spectrometry after mineralisation by dry ashing. Total mineral contents showed wide intervals of variation (calcium 1090–2050 mg l⁻¹, magnesium 101–177 mg l⁻¹, phosphorus 878–1560 mg l⁻¹ and zinc 4.0–7.3 mg l⁻¹) due to the addition during manufacturing of different dairy products or fractions. Most of the minerals were found in the non-sedimentable fraction. Practically all calcium was measured in the supernatants of ultracentrifugation. Lower proportions of magnesium and zinc were determined in the non-sedimentable fraction (between 87 and 96%) whereas the lowest ones (63–77%) were found for phosphorus.     

Effect of nitric oxide on ethylene production in strawberry fruit during storage

Strawberry is a non-climacteric fruit, with low ethylene production rate after harvest. Its response to nitric oxide (NO), which can be released from sodium nitroprusside (SNP), was studied. We have examined the effect of 1.0, 5.0 and 10.0 μmol l⁻¹ SNP aqueous solution on ethylene production, respiration rate, 1-aminocyclopropane-1-carboxylic acid (ACC) content and the activities of ACC synthase and ACC oxidase in post-harvest strawberry (“Fengxiang”). The most remarkable effect was obtained with 5 μmol l⁻¹ SNP aqueous solution, which significantly inhibited ethylene production, respiration rate, the activity of ACC synthase and reduced the content of ACC, but did not significantly affect the activity of ACC oxidase. SNP at 10 μmol l⁻¹ harmed the fruits; 1 μmol l⁻¹ SNP was too low to significantly extend strawberry storage life. It was suggested that NO could decrease ethylene output, through inhibiting ACC synthase activity reducing ACC content.     

Inactivation of Enterobacter sakazakii by pulsed electric field in buffered peptone water and infant formula milk

The effect of high-intensity pulsed electric field (PEF) treatment on the survival of Enterobacter sakazakii suspended in buffered peptone water (BPW) and powdered infant formula milk (IFM) was evaluated. Reference medium and IFM samples were treated with PEF. Electric field intensity and treatment time were varied from 10 to 40 kV cm⁻¹ and from 60 to 3895 μs, respectively. Samples of buffered peptone water (3 g L⁻¹) and IFM were inoculated with E. sakazakii (CECT 858) (10⁹ cfu mL⁻¹) and then treated with PEF. The inactivation data were adjusted to the Weibull frequency distribution function and Bigelow model, and constants were calculated for both substrates. A maximum 2.7 log (cfu mL⁻¹) reduction was achieved in BPW after exposure of E. sakazakii to PEF for 360 μs (2.5 μs pulse width) at 40 kV cm⁻¹. In IFM, exposure of E. sakazakii to PEF, with the same conditions, led to a 1.2 log (cfu mL⁻¹) reduction. The greater the field strength and treatment time, the greater the inactivation achieved in both substrates. Even though further research will be necessary, according to the results, there are good prospects for the use of PEF in hospitals to achieve safe reconstituted infant formula before storage at refrigerated temperatures.     

Diffusion of thiocyanate and hypothiocyanite in whey protein films incorporating the lactoperoxidase system

The diffusion of the thiocyanate (SCN⁻) and hypothiocyanite (OSCN⁻) components of a lactoperoxidase system (LPOS) in whey protein isolate (WPI) films was investigated. Diffusion coefficients for these molecules were measured for the LPOS-incorporated WPI films prepared with different WPI:glycerol ratios (1:1, 3:1, and 5:1). WPI film disks were coated on the surfaces of smoked salmon samples, and the samples were stored at 4, 10 and 22 °C. The diffusion coefficients were determined by fitting a mathematical model to the amounts of SCN⁻ and OSCN⁻ released from the disks during a period of time. The diffusion coefficients for SCN⁻ (D₁) and OSCN⁻ (D₂) in the films were 0.19–5.2 × 10⁻¹² m² s⁻¹ and 0.13–6.5 × 10⁻¹³ m² s⁻¹, respectively. The D₁ and D₂ decreased as the WPI:glycerol ratio increased and the storage temperature decreased. The E_a values for diffusion in 1:1, 3:1, and 5:1 WPI:glycerol films were 13.3, 29.5, and 35.6 kJmol⁻¹, respectively, for SCN⁻ and 15.8, 30.1, and 39.9 kJmol⁻¹, respectively, for OSCN⁻.     

Effects of ultrafiltration of whole milk on some properties of spray-dried milk powders

The objective was to produce spray-dried milk powders for assessment subsequently in chocolate. Milks were ultrafiltered to increase their protein content (3.08–5.33 g 100 g⁻¹), concentrated to different solids levels (42.8–52.3 g 100 g⁻¹) and spray-dried to produce powders (26–59 g 100 g⁻¹ fat). The relationships between the milk protein content, concentrate viscosity and some powder properties were quantified. The free-fat content of the powders increased linearly to 74 g 100 g⁻¹ fat with milk protein content for 26 and 40 g 100 g⁻¹ fat powders. The particle size and moisture content of the powders increased linearly with concentrate viscosity for 26 g 100 g⁻¹ fat powders. Differences between the control and ultrafiltration-treated milk powders were explained. The free-fat content and the particle size increased with the fat content of the control powders. The vacuole volume of the powders was inversely more related to the free-fat content than to the fat content of the control powders.     

Spectrophotometric Determination of Nitrite and Nitrate in Cured Meat by Sequential Injection Analysis

ABSTRACT: A robust, automated, labor-saving, accurate, and economical sequential injection system was developed for simultaneous determination of nitrite and nitrate in cured meat samples, based on the Shinn reaction. Nitrite is coupled and diazotized with sulfanilamide and N-(1-naphtyl)-ethylenediamine dihydrochloride, to form a colored compound that absorbs at 538 nm. Nitrate is previously in-line reduced to nitrite in a copperized cadmium column and measured as nitrite. The solutions' aspiration sequence, the influence of reagent and buffer concentrations, the manifold parameters, and the characteristics of the reducing column were studied. Nitrite and nitrate can be determined within the 0.030 to 1.22 of N-NO₂− and 0.034 to 3.95 mg/L of N-NO₃− ranges, respectively, at a sampling rate of 9/h. Detection limits of 9 μg/L of N for nitrite and 9 μg/L of N for nitrate were obtained, and the conversion rate of nitrate to nitrite was 100.6%± 1.8%. The results were in good agreement with those obtained by the reference methods, with relative standard deviations (r.s.d.) better than 3.70% for nitrites and 2.42% for nitrates.     

Levels of heavy metals in candy packages and candies likely to be consumed by small children

Ninety two samples of child-consumed candies and candy packages were analyzed for seven heavy metals. Lead (Pb) was detected at concentrations of 110.3–6394.1 mg kg⁻¹ in ten of 92 candy packages. The directive factor of Pb contamination had originated in the lead-based ink of the outer cover. Particularly, Pb was detected at high concentrations in case of green- or yellow-colored packages. Chromium (Cr) was detected at high concentrations in cases where Pb was also detected at high concentrations, and the Cr levels ranged from 136.9 mg kg⁻¹ to 1429.3 mg kg⁻¹ in seven of the 92 candy packages. Hexavalent chromium [Cr(VI)] was detected at 87–105.0% of the total Cr in polypropylene-coated wrappers with printed outer covers. The migration of Cr(VI) increased with elution time up to 0.20 μg (cm²)⁻¹ for 30 days in basic (pH 10.0) solution; however, there were no migrations in acidic (pH 4.0) and neutral (pH 7.0) solutions. The migration of Pb increased with elution time up to 0.65 μg (cm²)⁻¹ and 0.28 μg (cm²)⁻¹ in basic (pH 10.0) and acidic (pH 4.0) solutions, respectively. However, any migration was hardly observed in neutral (pH 7.0) solution.     

A survey of nitrate and nitrite content of fruit and vegetables grown in Slovenia during 1996-2002

Monitoring of nitrate (NO₃^-) and nitrite (NO₂^-) content in agricultural products in Slovenia has been carried out since 1996. The results of monitoring over the period 1996-2002 are presented. During this time 924 samples of 14 different agricultural products (potato, lettuce, apples, carrot, silage maize, cabbage, grapes, peaches, string beans, cereals, pears, cucumbers, strawberries and tomato) were analysed. The samples were taken at the time of maturity directly from growing sites and they were analysed using segmented flow analysis. The average nitrate contents were the highest in lettuce (1074 mg kg^-1), cabbage (881 mg kg^-1), string beans (298 mg kg^-1) and carrot (264 mg kg^-1), and they were moderately high in potato (158 mg kg^-1), silage maize (122 mg kg^-1), strawberries (94 mg kg^-1), cucumbers (93 mg kg^-1) and cereals (49 mg kg^-1). Low nitrate contents (below 6 mg kg^-1) were found in fruit (grapes, peaches, apples and pears) and tomato. With the exception of cereals (8.9 mg kg^-1), apples (1.5 mg kg^-1), potato (1.2 mg kg^-1) and pears (1.0 mg kg^-1) the content of nitrites did not exceed 0.5 mg kg^-1. It may be concluded that the results of the monitoring were in most cases similar to the results of investigations obtained in other countries.     

Modelling the mass transfer during convective, microwave and combined microwave-convective drying of solid slabs and cylinders

In the present work, the mass transfer characteristics, namely moisture diffusivity and moisture transfer coefficient, of slab and cylindrical potato samples were evaluated by adopting the analytical model proposed by Dincer and Dost [Drying Technology, 13,1/2 (1995), 425]. As part of the experimental work, the moisture contents of slab and cylindrical potato samples dried under convective, microwave and combined convective–microwave conditions (air temperature, 30–60 °C; air velocity, 1–2 ms⁻¹; microwave output power, 30–650 W) were measured. From the data collected the drying coefficients and lag factors were calculated and incorporated into the model. The results showed a reasonably good agreement between the values predicted from the correlation and the experimental observations; average error between experimental and predicted results less that 10%. The experimental system exhibited mass transfer Bi numbers in the range 0.038–11.4, thus indicating the presence of finite internal and external resistances. Moisture diffusivity values in the range 0.13×10⁻⁸ to 24.22×10⁻⁸ m²s⁻¹ were calculated.