A unique implementation of Hantzsch reaction for determination of natamycin in Yoghurt: Hyphenated with Box-Behnken-design for optimization.

This study aims to develop a novel and selective method for the detection of natamycin (E235) in yoghurt. The suggested method adopts an application of Hantzsch reaction to turn on the fluorescence behavior of natamycin (blue fluorescence), allowing its sensitive and selective determination in yoghurt samples without any overlapping at 485 nm. The originality of the research lies in the fact that this application takes place for the first time, also the detection (LOD) and quantification (LOQ) limits were very low (0.02 and 0.06µg mL-1, respectively) with a linear concentration range of 0.1-1.0 µgmL-1. Moreover, the developed method was employed for the detection of E235 in yoghurt sample with a good recoveries (98.80 ± 1.20-99.20 ± 1.15 (%), over a concentration range of 0.5-1.0 µgmL-1, (LOD = 0.04 and LOQ = 0.12 µgmL-1). Furthermore, the specificity and convenient application of our intended method is an attempt to determine E235 in milk anddairy products with easily followable steps.

Evaluation of biological and chemical additives on microbial community, fermentation characteristics, aerobic stability, and in vitro gas production of SuMu No. 2 elephant grass.

BACKGROUND: The study was conducted to evaluate the effects of biological and chemical additives on microbial community, fermentation characteristics, aerobic stability, and in vitro gas production of SuMu No. 2 elephant grass. RESULTS: Aerobic bacteria and yeast were not affected on days 5 and 7 but were significantly (P < 0.224) reduced on days 14, 30, and 60, whereas lactic acid and lactic acid bacteria were significantly (P > 0.001) higher in all ensiling days within all treatment groups. During the ensiling days, the pH, acetic acid, butyric acid, and yeast were decreased in all treatment groups, whereas the Lactobacillus plantarum group and L. plantarum + natamycin group were highly significantly (P > 0.001) decreased. During air exposure, the water-soluble carbohydrates, ammonia nitrogen, lactic acid, and acetic acid were not affected on days 1-4, whereas pH and aerobic bacteria (were significantly (P < 0.05) increased on days 2-4. The addition of Lactobacillus plantarum and natamycin increased the gas production, in vitro dry matter digestibility, and in vitro neutral detergent fiber of SuMu No. 2 elephant grass silages. CONCLUSIONS: The addition of biological and chemical additives, such as L. plantrum alone and the combination with natamycin, affected the undesirable microbial community, fermentation characteristics, aerobic stability, and in vitro gas of SuMu No. 2 elephant grass. © 2021 Society of Chemical Industry.

Effect of chitosan and gum Arabic with natamycin on the aroma profile and bacterial community of Australian grown black Périgord truffles (Tuber melansoporum) during storage.

This study aimed to assess the effect of chitosan or gum Arabic edible coatings, with natamycin (200, 300, 400 mg/L) on the aroma profiles of Western Australian grown truffles at five storage intervals: 0, 7, 14, 21, and 28 days using solid-phase microextraction (SPME)-followed by gas chromatography-mass spectrometry (GC-MS). The population structure of the bacterial community of both untreated and chitosan-natamycin (400 mg/L) coated truffles were assessed using metagenomic sequencing analysis alongside GC-MS. The results demonstrated that all the coating treatments were able to have a positive impact in halting or delaying the changes of truffle aroma throughout the storage period, with chitosan-natamycin (400 mg/L) coating having the best preservation results compared to the other coatings. Only 9 volatile organic compounds (VOCs) were found to have significant changes in chitosan-natamycin (400 mg/L) coated truffles throughout the storage period compared to 11 VOCs in untreated controls. The result also demonstrated the gradual change of fresh truffle's bacteria communities over the storage period. Over 4 weeks of storage, the dominant bacterial classes of the truffles (α-Proteobacteria, Bacteroidia or Actinobacteria classes) were replaced by Bacteroidia, Actinobacteria, Deltaprotobacteria and γ-Proteobacteria classes. The preliminary results from this study show that edible coatings can affect the VOC and bacterial communities of the truffles which may have implications for future research into truffle preservation techniques.

Development of a capillary zone electrophoresis method to determine natamycin in food and a comparison with a liquid chromatography method.

BACKGROUND: Natamycin is often added to pastries, cheeses, and beverages. The residual amount of natamycin should be less than 10 mg kg-1 . The current method for its determination in various foodstuffs is high-performance liquid chromatography (HPLC). Capillary electrophoresis (CE) is a simple, fast, and environmentally friendly method with low reagent consumption and comparable separation performance. However, no reports were found on the determination of natamycin in foods by CE. A CE method to determine natamycin is therefore sought. RESULTS: Natamycin in foods was determined by the capillary zone electrophoresis (CZE) method with ultraviolet-visible (UV) detection. Separation conditions were optimized as 20 mM Na2 HPO4 , pH 9.2, with 25 kV applied voltage, and UV detection at 306 nm. Under optimal conditions, electrophoretic analysis was completed in less than 4 min, with a limit of detection (LOD) of 0.065 µg mL-1 and limit of quantitation (LOQ) of 0.22 µg mL-1 . A good linear relationship (r2 = 0.999) was obtained at the range of 0.1-25 µg mL-1 . A comparison with the HPLC-UV method was also carried out according to the National Standards of the People's Republic of China. CONCLUSION: The results obtained by the CZE and HPLC methods are comparable but the proposed CZE method can help us obtain a shorter detection time at low cost. © 2019 Society of Chemical Industry.

Comparison of the Ocular Penetration and Pharmacokinetics Between Natamycin and Voriconazole After Topical Instillation in Rabbits.

PURPOSE: To investigate the ocular penetration of natamycin (NAT) and voriconazole (VRC) after topical instillation in New Zealand white rabbits using simplified liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high-performance liquid chromatography. METHODS: Seventy-eight healthy rabbits were randomly divided into 3 groups. In the first 2 groups, 72 rabbits were used for single-dose testing (36 for NAT, 36 for VRC), in which 50 µL of 5.0% NAT or 1.0% VRC was instilled into the rabbits' left eyes. In the 3rd group, 6 rabbits were used for repeated-dose testing in which 50 µL of 5.0% NAT was instilled into their left eyes 12 times (once per hour) during the daytime. These animals were sacrificed immediately to collect their aqueous humors and corneas. RESULTS: After a single topical instillation, the highest concentrations in the cornea and aqueous humor for VRC were 34.1 µg/g and 14.7 µg/mL, respectively. The permeability ratios of aqueous/cornea were from 0.1 to 1.26. The highest concentrations in cornea and aqueous humor for NAT were 299.3 ng/g and 27.1 ng/mL, respectively. The permeability ratios of aqueous/cornea were from 0.02 to 0.23. In the repeated-dose group, the NAT concentrations in the cornea and aqueous humor were 10,569 ng/g and 54.4 ng/mL, respectively. The permeability ratio was as low as 0.0051. CONCLUSION: The better corneal penetration of VRC suggests that it is more suitable for deep corneal fungal infections than NAT via topical ocular administration.

Development of a LC-MS/MS method for the simultaneous determination of sorbic acid, natamycin and tylosin in Dulce de leche.

A simple extraction, rapid routine method for the simultaneous determination of sorbic acid, natamycin and tylosin in Dulce de leche, a traditional South American product, by liquid chromatography-tandem mass spectrometry has been developed and fully validated. The limits of detection were set to 24.41mgkg(-1) (sorbic acid), 0.10mgkg(-1) (natamycin) and 2µgkg(-1) (tylosin). Recoveries ranged from 95% to 110%. Proportionally, internal standardization was more efficient than external standard, resulting in a smaller measurement of uncertainty. In total, 35 commercial samples from Brazil, Argentina and Uruguay have been assessed. The proposed method was tested on other dairy desserts, demonstrating to be versatile. Although tylosin was not detected in any sample, a high rate of non-compliance was found, with 67.39% of samples above the maximum allowed for sorbic acid and a maximum concentration of 2105.36±178.60mgkg(-1). In two samples, natamycin was irregularly found.

Natamycin content and quality evaluation of yoghurt from small- and large-scale brands in Turkey.

In this study, the presence of natamycin and quality parameters of yoghurt samples manufactured by small- and large-scale dairy firms in Turkey were investigated. Physicochemical and microbiological results revealed that, except Lactobacillus bulgaricus and Streptococcus thermophilus counts, the majority of the yoghurts manufactured by small-scale dairy firms were found to be out of the limits. Natamycin was detected in 31 and 2 yoghurt samples from small- and large-scale brands, respectively. The levels of natamycin in small-scale brand yoghurts were higher than those in large-scale brand yoghurts. Of the analysed samples, 42.3% did not comply with the Turkish Food Codex.

Determination of natamycin in rabbit cornea by high-performance liquid chromatography-tandem mass spectrometry with protective soaking extraction technology.

A new selective and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the quantification of natamycin (NAT) in rabbit corneas with amphotericin B as the internal standard (IS). The cornea samples were processed by a simple and protective methanol soaking extraction technology. The NAT could be extracted completely from rabbit cornea after 24h of soaking with methanol under a mild condition. Chromatographic separation was performed on a C18 column (2.1mm×50mm, 3.5µm) using mobile phase with ammonium acetate buffer (pH 4.5; 4.0mM):acetonitrile (40:60, v/v) at a flow rate of 0.25ml/min. Quantification was performed using the transitions 666.2→503.2 m/z for NAT and 924.5→906.6 m/z for IS by positive ion electrospray ionization in multiple reaction monitoring mode. The assay was validated over a concentration range of 8.64ng/ml to 843ng/ml with lower limit of detection of 4.32ng/ml. The method was validated with respect to linearity, accuracy, precision, recovery, stability and extracting efficiency. The extraction recovery of NAT from cornea samples was approximately 100% with the new methanol soaking extraction procedure. The method has been successfully applied to the ocular pharmacokinetic studies of NAT eye drops in the cornea of Japanese white rabbit.

A novel and rapid method for determination of natamycin in wines based on ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry: validation according to the 2002/657/EC European decision.

A novel, simple, and rapid reversed-phase liquid chromatography-tandem mass spectrometric methodology was developed for the analysis of natamycin in wine samples. Natamycin was protonated to form singly charged ions in an electrospray positive ion mode. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring (MRM) of three fragment ion transitions (666.3 → 648.2, 666.3 → 503.3, and 666.3 → 485.2) to provide a high degree of sensitivity and specificity. Chromatographic separation was performed on a rapid resolution column using a mobile phase consisting of an acetonitrile/water mixture with a total run time of 5.0 min. After only filtration as pretreatment, the sample was injected into the chromatographic system. The proposed method was validated in terms of selectivity, trueness, precision, decision limit (CCα), and detection capability (CCß) according to 2002/657/EC Commission decision. The values for trueness, reported as bias (%), agreed with those established by the aforementioned document. Repeatability (intraday variability) values were 12.37% at a concentration of 1.0 µg L(-1) and 8.99-4.19% at concentrations between 2.5 and 10 µg L(-1). The overall within-laboratory (interday variability) reproducibility was 15.47% at a concentration of 1.0 µg L(-1), which was significantly lower than the indicative value reported in the EU decision. The results indicated that the proposed approach is a sensitive, fast, reproducible, and robust methodology suitable for the analysis of natamycin levels in wine samples.

Bioanalytical method development and validation of natamycin in rabbit tears and its application to ocular pharmacokinetic studies.

A new selective and sensitive high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of natamycin in rabbit tears using amphotericin B as internal standard (IS). Chromatographic separation was achieved on a Luna Cyano column (100 mm × 2 mm, 3 µm) using ammonium acetate buffer (pH 4; 3.5mM): methanol (10:90, v/v) as the mobile phase. The run time was 5 min. Detection was performed by negative ion electrospray ionization in multiple reaction monitoring (MRM) mode. The calibration curve was linear over the concentration range from 25 to 800 ng/ml, and lower limit of detection of 12.5 ng/ml. The accuracy and precision of the method were within the acceptable limit of ± 20% at the lower limit of quantitation and ± 15% at other concentrations. Natamycin was stable during the battery of stability studies viz., bench-top, auto-sampler, freeze/thaw cycles and 30 days storage in a freezer at -70 ± 10 °C. The method was successfully applied to the ocular pharmacokinetic studies of natamycin eye drops in New Zealand rabbit tears.

[Analytical method for natamycin in wine using high-performance liquid chromatography].

An analytical method was developed for determining amount of natamycin in wine using a C18 minicartridge column and high-performance liquid chromatography (HPLC) with photodiode array (PDA) detection. Natamycin purified from wine was identified in accordance with the retention time and UV spectrum obtained from PDA detection. The limit of quantification of natamycin in wine was estimated as 0.05 microg/ml. Recovery of natamycin in wine was acceptable at 91.0% with low relative standard deviation (2.3%).

Rapid ultraviolet spectrophotometric and liquid chromatographic methods for the determination of natamycin in lactoserum matrix.

Two rapid and simple methods were developed for the determination of natamycin in lactoserum matrix by ultraviolet (UV) spectrophotometry and liquid chromatography (LC) with diode-array detection. The methods involve protein precipitation with methanol, followed by centrifugation. No cleanup is necessary. The applicable concentrations of natamycin in lactoserum range from 2 to 500 mg/L for samples analyzed by both methods. The detection and quantitation limits are 0.07 and 0.23 mg/L, respectively, for the UV spectrophotometric method and 0.1 and 0.32 mg/L, respectively, for the LC method. The methods were applied satisfactorily to the determination of natamycin in various commercial lactosera. Both methods were validated independently by standard additions and Youden methodologies, which verified their accuracy. Once the 2 proposed methods were validated independently, the validation of one method was carried out with the other.

[Determination of natamycin residues in ripening cheeses by high-performance liquid chromatography]. / Oznaczanie pozostalosci natamycyny w serach dojrzewajacych metoda wysokosprawnej chromatografii cieczowej.

The purpose of the study was to determine the usefulness of high performance liquid chromatography (HPLC) for natamycin determination in routine control of ripening cheeses. In the method the antibiotic is extracted from the studied sample with a 2:1 methanol/water solution, freezing of contaminants at -18 degrees C and determination of HPLC using a RP C8 column and UV detection. In case of low concentrations of the antibiotic the extract was condensed by extraction to solid phase (SPE). In the study of the fortified samples the basic analytical parameters of the method were tested (determinability, repeatability, recovery) and its usefulness in the 0.05-0.4 mg/kg concentration range (by SPE) and above 0.5 mg/kg of cheese was checked. Recovery was from 87 to 98%, repeatability was 1.3-7.3% and determinability was 0.5 mg/kg (using SPE 0.05 mg/kg). The obtained results demonstrated a good usefulness of HPLC for routine control of natamycin content in ripening cheeses. It seems recommendable to apply HPLC for natamycin determination in ripening cheese by sanitary-epidemiological stations.

Rapid spectrophotometric method for analyzing natamycin in cheese and cheese rind.

A rapid spectrophotometric method for determining natamycin in cheese and cheese rind has been developed. Samples are homogenized with acidified aqueous acetonitrile and homogenates are filtered. Natamycin is directly quantitated in filtered extracts on the basis of the characteristic third-derivative trough at 322.6 nm. Additional cleanup of extracts is not required because derivative transformation of the conventional analytical band at around 319 nm eliminates spectral interferences from other compounds. The analysis is simple and can be completed in 6 min. The equipment is easily accessible because most modern spectrophotometers allow instant generation of derivative spectra. The method needs small amounts of solvents and has good analytical characteristics. Overall recovery was 98.4 +/- 0.7%, and linearity was excellent (r = 0.9998) in the range examined (0.5-20 mg/kg). Quantitation and detection-limits were estimated at 0.5 and 0.25 mg/kg, respectively. Precision statistics based on within-day and between-days variations suggest an overall relative standard deviation of 1.4%.

Determination of natamycin in cheese and cheese rind: interlaboratory collaborative study.

A collaborative test on the determination of natamycin in cheese and cheese rind was conducted. Participants were from 37 laboratories in 13 countries. Eight samples, consisting of 4 duplicates, were investigated by a spectrometric method and a liquid chromatographic (LC) method. The spectrometric method gave good results (coefficient of variation [CV] = 12%) and the LC method with ultraviolet detection gave reasonable results (CV = 25%) for levels down to 15 mg/kg (0.9 mg/dm2). For very low levels, a preconcentration step is necessary, but even then quantitation is poor (CV = 35-37%) for both methods at 1.7 mg/kg, although the presence of natamycin can be detected qualitatively. For a level of 0.3 mg/kg, quantitation is poor (CV = 39%) for the LC method and impossible (CV = 60%) for the spectrometric method.

Spectrometric and liquid chromatographic determination of natamycin in cheese and cheese rind.

Methods for determining natamycin content of cheese rind and cheese are presented. Cheese and rind samples are extracted with methanol and the fat precipitated by cooling the sample solution in methanol-water at -15 to -20 degrees C for ca 1 h. Natamycin levels are measured by UV spectrometric detection at absorbance minimum 311 nm, maximum 317 nm, and at exactly 329 nm, or by LC separation over Lichrosorb RP-8 column with detection at 303 nm. For measuring low levels, a concentration step is provided. The method is applicable to natamycin in cheese rind and in the interior of the cheese. Detection limit is 0.5 mg/kg. The method is suitable for controlling a maximum tolerance of natamycin on the cheese rind, at a level of 1 mg/dm2, and for detecting migration of natamycin into the cheese.

[The spectrophotometric determination of natamycin in cheese]. / Zur Spektralphotometrischen Bestimmung von Natamycin auf Käse.

Derivative spectroscopy was used for quantitative determination of natamycin in cheese. When measuring a methanolic cheese extract against methanol, the second derivative of the UV-spectrum is measured between 340 and 290 nm. The natamycin concentration can be determined by measuring the vertical distance between the minimum at 318 nm and the maximum at 311 nm. Under these conditions the detection limit of natamycin in a pure methanolic solution lies at 20 ng/ml, in cheese extracts at 150 ng/ml. The latter corresponds to a natamycin concentration of 2.5 ppm in the case of a 3 g test sample or 0.03 mg/dm2 in the case of a 25 cm2 cheese surface. The introduction of derivative spectroscopy makes it possible to reduce the interference of cheese substances in the photometric measurements and to increase the sensitivity and selectivity of the detection process. Besides the advantage that work and expenses are reduced - as no pimaricin-free sample has to be extracted from the interior of the cheese - it is also possible to determine natamycin photometrically in cheese, in which it is distributed homogeneously.

Liquid chromatographic determination of natamycin in cheese at residue levels.

A high performance liquid chromatographic method, with special reference to the cleanup procedure, is described for the measurement of natamycin in cheese. Co-extracted fats and proteins are removed by cooling the methanol extract of the cheese. Amounts as low as 1 ng natamycin can be detected; the limit of determination is better than 0.05 mg/kg. Recovery at the 1 mg/kg level is better than 90%. Reproducibility at the 0.4 mg/kg level shows a coefficient of variation of 10%. Some results obtained with Dutch cheese samples from the local market are reported.