Analyses of Trace Crystal Violet and Leucocrystal Violet with Gold Nanospheres and Commercial Gold Nanosubstrates for Surface-Enhanced Raman Spectroscopy

Crystal violet (CV) is forbidden but still used in some aquaculture operations due to its low cost and high effectiveness against some fish diseases. Surface-enhanced Raman spectroscopy (SERS) coupled with partial least squares (PLS) regression is applied to analyze trace amounts of CV and its metabolite leucocrystal violet (LCV) in fish fillets. Two different laser sources (633 and 780 nm) and three different gold nanosubstrates (included gold nanospheres and two commercial gold substrates) were used as SERS substrates to achieve optimal analytical results. Gold nanoparticles (diameter 55.4?±?4.5 nm) as synthesized via a reduction method resulted in better sensitivity and accuracy results than the two commercial substrates. The minimum detectable concentration for CV standard solutions was 0.5 ng/mL with the use of gold nanospheres as substrate, compared to 10 and 50 ng/mL with the two commercial substrates. The R ² of actual CV concentrations versus the values predicted (cross-validation) with PLS models ranged from 0.963 to 0.989. For CV contaminated fish muscles, the minimum detectable concentration of CV was 1 ng/g, and the PLS model (n?=?64, 20 for prediction) for total CV and LCV in fish muscles was less satisfied (cross-validation R ²?=?0.889; prediction R ²?=?0.857) compared to those for standard solutions due to the interferences of nontargeted components in fish extract, but the results still indicated the possibility of applying SERS with chemometrics to determine trace amounts of CV and LCV in complex sample systems, such as fish muscles.     

Highly Broad-Specific and Sensitive Enzyme-Linked Immunosorbent Assay for Screening Sulfonamides: Assay Optimization and Application to Milk Samples

The optimum conditions of an enzyme-linked immunosorbent assay (ELISA) in regard to different monoclonal antibodies (MAbs), assay format, immunoreagents, and several physicochemical factors (pH, salt, detergent, and solvent) were investigated to develop a broad-specific and sensitive immunoassay for detection of sulfonamides in milk samples. Two previously produced broad-specific MAbs, 4D11 and 4C7, and eight structurally different haptens conjugated with bovine serum albumin (BSA) were used as coating antigens in a competitive indirect ELISA (ciELISA). In addition, six hapten-HRP conjugates and the two MAbs were evaluated in a competitive direct ELISA. After optimization, a highly broad-specific and sensitive ciELISA for screening for sulfonamides was obtained based on MAb 4D11 and the BS-BSA heterologous-coating antigen, demonstrating a 50 % specific binding (IC₅₀) for 22 sulfonamides at concentrations below 100 ng mL^?1. This is the first report of an immunoassay that is capable of detecting more than 20 sulfonamides based on MAbs. The optimized ciELISA was used to quantify the five sulfonamides, SMZ, SDM, SQX, SMM, and SMX in spiked milk samples. Recoveries of 89–104.6 % and coefficients of variation of 11.9–19.1 % demonstrated the potential of the ciELISA to simultaneously monitor multiple sulfonamides in diluted milk samples without further purification steps.     

A Direct Enzyme Immunoassay to Detect Erythrosine in Foods

As a synthetic food dye, erythrosine is associated with serious toxicity in inducing thyroid tumors, and the use of erythrosine is strictly regulated in most counties including China. In this study, a direct enzyme-linked immunosorbent assay (ELISA) has been developed for analysis of erythrosine in food products. A highly specific monoclonal antibody (MAb) for erythrosine was produced using erythrosine–bovine serum albumin (BSA) conjugate as an artificial antigen, and horseradish peroxidase (HRP)-labeled MAb for erythrosine was utilized as the detection antibody. Under the optimized condition, the UV absorbance in microplate related well with erythrosine concentration in the range of 0.1–10.0 μg/g. The proposed method could be applied to determine erythrosine in beverage and cookie, with good recoveries (80 –103 %) for the three spiked levels (30, 50, and 100 μg/g), and the relative standard deviations of detected amount were <12.3 %.     

Chemometrics-Assisted GC-MS Analysis of Volatile and Semi-Volatile Constituents of Elettaria cardamomum

Multivariate curve resolutions (MCR) along with other chemometric techniques are proposed to improve the analysis of Iranian Elettaria cardamomum (E. cardamom) essential oil with gas chromatography-mass spectrometry (GC-MS). In addition, multivariate curve resolution-alternating least squares (MCR-ALS) is used to obtain pure elution and mass spectral profiles for the components present in each chromatographic segment as well as their relative concentrations. This strategy was also used to overcome the problems of baseline offset, asymmetric peaks, retention time shifts, and overlapped and embedded peaks occurring during GC-MS analysis. The analysis of GC-MS data revealed that 42 components exist in the E. cardamom essential oil. However, with the help of MCR and different chemometric methods, this number was extended to 73 components. The most important components of the Iranian E. cardamom are α-terpineol acetate (11.78 %), linalool (10.15 %), nerolidol (8.82 %), α-pinene (8.11 %), geranyl acetate (3.47 %),1,8-cineole (4.25 %), and γ-terpinene (3.88 %).     

Optimization of multiplex PCR for the identification of animal species using mitochondrial genes in sausages

Detection of species fraud in meat products is very important in order to protect consumers from undesirable adulteration, as well as for the economic, religious and health aspects. The most important reason for verification of the labeling statements is to detect fraudulent substitution of expensive meat components with other cheaper animals or mislabeling. The aim of this study was to develop a multiplex PCR that could be used in the simultaneous identification of multiple meat species. In this study, ten sausages with a minimum beef content of 55 %, from ten different manufacturing companies, and five samples of cow, chicken, goat, camel and donkey raw meats, for the purpose of positive control, were collected from food markets in Tehran, Iran. Total DNA was extracted from each sausage and the raw meats. Primers were selected in different regions of mitochondrial DNA (12S rRNA, cytochrome b and NADH dehydrogenase subunits 2) for identification of meat species. 12S rRNA and NADH dehydrogenase subunits 2 primers generated specific fragments of 183 and 145 bp length, for chicken and donkey, respectively. Three different specific primers were used for amplification of cytochrome b gene in goat, camel and cattle species and amplified species-specific DNA fragments of 157, 200 and 274 bp, respectively. The results proved that half of the specimens were contaminated with chicken meat, and this was greater than the proportion of beef stated on the label, while the other half only had chicken residuals, and no beef content. No contamination was found with goat, donkey or camel meats. These findings showed that molecular methods, such as multiplex PCR, is a potentially reliable, sensitive and accurate assay for the detection of adulterated meat species in mixed meat products.     

Quantitation of Selected Polyphenols in Plant-Based Food Supplements by Liquid Chromatography–Ion Trap Mass Spectrometry

A high-performance liquid chromatography method with electrospray ionization mass spectrometric detection (HPLC-ESI-MS/MS) for the determination of some of the most important polyphenols present in botanical supplements has been developed. The target analytes were five flavonoids (diosmin, hesperidin, quercetin, rutin and troxerutin) and the flavolignan silybin. The extraction of the analytes from the supplements was carried out by ultrasound-assisted extraction using 100 % dimethyl sulfoxide (or methanol) for 15 min. After centrifugation, 1 μL of the diluted supernatant was injected in the HPLC system and the quantitation was performed by ESI-MS using the negative ionization mode, with methylparaben as internal standard. The validation of the method was performed with recovery experiments, observing recoveries in the range of 85–112 %, and relative standard deviations lower than 10 % for the complete analytical procedure, including the extraction. The limits of detection were in the 2.5–120 μg L^?1 range.     

Analysis of Essential Oils from Cassia Bark and Cassia Twig Samples by GC-MS Combined with Multivariate Data Analysis

Cinnamomum cassia is one of several species of Cinnamomum which has been widely used as a spice. In this study, the chemical profiles of its bark and twig were characterized by gas chromatography-mass spectrometry (GC-MS) and multivariate data analysis. Principle component analysis (PCA) and orthogonal projection to latent structures discriminate analysis (OPLS-DA) of GC-MS data provided a clear separation between those samples. The result obviously showed that the metabolome of cinnamon bark (CB) and cinnamon twig (CT) is different, and the corresponding loading S-plots revealed that the differential metabolites between the essential oils of CB and CT were trans-cinnamaldehyde, trans-anethole, α-cubebene, γ-muurolene, γ-amorphene, δ-cadinene, (?)-calamenene, and o-methoxycinnamaldehyde. Our study demonstrates that the combination of GC-MS spectrum and multivariate data analysis can be applied to identify essential oils from different parts of C. cassia.     

Hypnotic effect of GABA from rice germ and/or tryptophan in a mouse model of pentothal-induced sleep

γ-Aminobutyric acid (GABA), a primary inhibitory neurotransmitter, and tryptophan (Trp), a substrate for melatonin, are found in functional foods and exert hypnotic effects. The hypnotic effects of 3 doses of GABA and a combined-preparation of GABA and Trp (GABA+Trp) were investigated in mice. Hypnotic activity was evaluated using pentothal-induced sleep time testing. Treatments included low, middle, and high doses of GABA and GABA+Trp. Low doses of GABA (low-GABA) and low-GABA+Trp reduced sleep latency and significantly (p<0.05) prolonged the sleep time induced by pentothal, compared with controls, although the melatonin concentration in the serum was not affected. On the other hand, the adenosine A1 receptor (AA1R) immunoreactivity in the suprachiasmatic nucleus of the hypothalamus was significantly (p<0.05) increased after administration of low-GABA and/or low-GABA+Trp, compared to controls. Low doses of GABA and/or Trp cause hypnotic effects that may be related to AA1R activation.     

High-resolution melting analysis: a promising molecular method for meat traceability

To find a promising molecular method for meat traceability, three methods of single nucleotide polymorphism (SNP) detection: RFLP-PCR analysis, high-resolution melting (HRM) analysis, and TaqMan probe analysis, have been compared in terms of accuracy, ease of use, throughput capability, and cost. We genotyped ten pork DNA samples across three SNPs. The results showed that the HRM genotyping method was the most accurate and easiest to use with the lowest cost, while TaqMan probe analysis provided similar results, but its cost was much higher.     

Physicochemical properties and cell permeation efficiency of l-ascorbic acid loaded nanoparticles prepared with N-trimethyl chitosan and N-triethyl chitosan

The physicochemical properties and permeation efficiency of l-ascorbic acid (AA)-loaded nanoparticles (NPs) prepared using N-trimethyl chitosan (TMC) and Ntriethyl chitosan (TEC) were investigated. The sizes of AA-TMC-NPs and AA-TEC-NPs were 568±50 and 150±15 nm, respectively and both zeta potential values were slightly positively charged. The encapsulation efficiency (EE) of AA-TMC-NPs was consistently between 40 and 45%, but the EE of AA-TEC-NPs varied between 18 and 56%. The release rate increased with increased temperatures for both NPs. AA-TMC-NPs exhibited an initial burst release, while AA-TEC-NPs exhibited a controlled release, increasing rapidly after 2 h. The pH-related release pattern was similar to the temperature-related release pattern. The permeation efficiency into Caco-2 cells, in order from lowest to highest, was AA, an AA mixture with TMC, an AA mixture with TEC, AA-TMC-NPs, and AA-TEC-NPs. Both NPs showed potential to enhance the permeability of AA.     

Antimicrobial Properties of Ethylene Vinyl Alcohol/Epsilon-Polylysine Films and Their Application in Surimi Preservation

Polymer films based on ethylene vinyl copolymers (EVOH) containing a 29 % (EVOH 29) and a 44 % molar percentage of ethylene (EVOH 44), and incorporating ε-polylysine (EPL) at 0 %, 1 %, 5 % and 10 % were successfully made by casting. The optical properties and the amount of EPL released from the films to phosphate buffer at pH 7.5 were evaluated, films showing great transparency and those of EVOH 29 copolymer releasing a greater amount of EPL. The antimicrobial properties of the resulting films were tested in vitro against different foodborne microorganisms and in vivo in surimi sticks. With regard to the antimicrobial capacity tested in vitro in liquid medium at 37 °C and 4 °C against Listeria monocytogenes and Escherichia coli over a period of 72 h, films showed a considerable growth inhibitory effect against both pathogens, more notably against L. monocytogenes, and being EVOH 29 more effective than EVOH 44 films. At 37 °C, total growth inhibition was observed for EVOH 29 films incorporating 10 % EPL against both microorganisms whereas the copolymer EVOH 44 did show total inhibition against L. monocytogenes and the growth of E. coli was reduced by 6.64 log units. At 4 °C, no film was able to inhibit completely bacterial growth. Scanning electron microscopy micrographs showed corrugated cell surfaces with blisters and bubbles, and collapse of the cells appearing shorter and more compact after treatment with EPL. Finally, the films were successfully used to increase the shelf life of surimi sticks. The results show the films developed have a great potential for active food packaging applications.     

Two quantitative multiplex real-time PCR systems for the efficient GMO screening of food products

According to the EU and Swiss food legislation, only deregulated traits of transgenic plants are allowed to be imported and sold to the consumer. In order to control imports of soya and maize products from retailers, efficient and reliable methods for the detection and quantification are a prerequisite. The screening for specific DNA elements characteristic of transgenic plants is crucial for further analysis and has a major impact on the efficiency of the whole analysis workflow. To allow laboratories to efficiently and reliably screen food products for transgenic plant products, two novel multiplex real-time polymerase chain reaction (PCR) systems were developed and validated. One system determines DNA contents from maize, soya, cauliflower mosaic virus (CaMV) 35S promoter (P35S), NOS terminator from Agrobacterium tumefaciens and CaMV, and the second PCR system simultaneously detects DNA sequences from figwort mosaic virus promoter (PFMV), from bar gene of Streptomyces hygroscopicus, from gene coding for phosphinothricin acetyltransferase (PAT) and from a DNA construct of enolpyruvyl shikimate phosphate synthase gene (CP4-EPSPS) and Arabidopsis thaliana (CPT2). The tests exhibit good specificity and a limit of detection of at least 0.1 % for all analytes.     

Development of a Broad Specific Monoclonal Antibody for Fluoroquinolone Analysis

Fast and sensitive screening of antibiotics is a great need in food quality assurance. A monoclonal antibody specific to fluoroquinolones (FQ) was obtained using ciprofloxacin (CPF) derivant as hapten. The antibody was characterized with broad recognition to CPF (100 %), enrofloxacin (ENF, 73.89 %), norfloxacin (73.57 %), nadifloxacin (67.28 %), danofloxacin (53.09 %), pefloxacin (50.26 %), lomefloxacin (35.66 %), enoxacin (12.40 %), and sarafloxacin (3.23 %). Then, an enzyme linked immunosorbent assay (ELISA) was established. The optimized concentrations of coating antigen and antibody were 0.1 and 0.5 μg/ml, respectively. Various parameters including pH values, ionic strength and the concentration of antibody and antigen were optimized for this assay. The ELISA was found to have the best sensitivity in assay buffer of pH 6 and sodium chloride content 1.6 % (m/v) using CPF as tested compound. Fortified milk samples with CPF and ENF (50 and 250 μg/l) and fortified chicken samples (10 and 50 μg/kg) were analyzed with the proposed measure. The ELISA was examined with recovery range of 94–104 % for milk detection and 93–108 % for chicken detection. The coefficient variation data for intra-assay and inter-assay ranged from 4.24 % to 12.16 %. All results indicate the potential extensive application prospects of this ELISA in FQ monitoring for food safety.     

Effect of gamma-aminobutyric acid produced by Lactobacillus sakei B2-16 on diet and exercise in high fat diet-induced Obese rats

Effect of gamma-aminobutyric acid (GABA) produced by Lactobacillus sakei B2-16 on diet and acute swimming exercise was investigated in rats with high fat diet (HFD)-induced obesity. The body weight gain in the GABA+Exercise group was significantly (p<0.05) lower than in the HFD group. Combined treatment with GABA and exercise decreased the body weight gain by 25.6%, compared to the HFD group. On the other hand, neither GABA supplementation nor exercise alone significantly (p>0.05) influenced reduction in body weight gain, compared to the HFD group. The weights of abdominal and epididymal fat tissues and the liver in the GABA+Exercise group were significantly (p<0.05) lower than in the HFD group. The activity of citrate synthase was significantly (p<0.05) elevated in the soleus muscle by GABA supplementation. GABA contributes to reduction in body weight gain and fat tissue weight by increasing physical activity during exercise.     

Use of highly variable gene (yycH) as DNA marker to resolve interspecific relationships within the Lactobacillus casei group and a target for developing novel species-specific PCR primers

Identifying Lactobacillus species group using only phenotypic and genotypic (16S rDNA sequence homology analysis) techniques yields inaccurate results. In this study, the partial yycH gene sequence was used for species discrimination by direct DNA sequencing and as target in a species-specific PCR assay. All examined Lactobacillus strains were clearly distinguished from the closely related species by comparative sequence analysis of the yycH gene. The average sequence similarity for the yycH gene (78.5 %) among type strains was significantly lower than that of the 16S rRNA sequence (99.0 %). Therefore, the yycH gene can be proposed as an additional molecular marker for Lactobacillus casei group that provides higher resolution than 16S rRNA. In addition, the species-specific primers were also developed based on the yycH gene sequences, which were then employed for PCR using the template DNA of Lactobacter strains. The PCR primer pairs were shown to be specific for Lactobacillus paracasei and Lactobacillus rhamnosus. Our data indicate that the phylogenetic relationships in the L. casei group are easily resolved by direct sequencing of the yycH gene and combined with species-specific PCR assays.     

Evaluation of the Nanostructure of Pectin,Hemicellulose and Cellulose in the Cell Walls of Pears of Different Texture and Firmness

The nanostructure of polysaccharides is supposed to determine properties such as stiffness or diffusivity of cell walls and their functionality for various tailored properties of food. However, at present, a relation of these nano-properties with sensory texture and firmness remains to some degree unknown. In this work, water (WSP), calcium chelator (CSP) and sodium carbonate (DASP) soluble pectins, hemicellulose and cellulose, extracted from cell walls of two pear cultivars ‘Xenia’ and ‘Conference’ at their harvest times, were studied. An atomic force microscope and image analysis were used to evaluate diameter and branching of the molecules. Sensory texture of ‘Xenia’ was considered as better and its firmness (87 N) was higher than ‘Conference’ (76 N). WSP molecules were present as short molecules with a height of about 0.5 nm for both cultivars. A chain-like and branched CSP fraction had diameter of about 0.3–0.4 nm for both cultivars with a pronounced contribution of molecules with diameter of about 1 nm for ‘Xenia’, which had also higher branching index. DASP revealed similar regular structures for both cultivars however the network was much denser for ‘Xenia’. A rod-like hemicellulose molecules had length of about 20–400 nm and diameter of 1 nm for ‘Xenia’ and 1–4 nm for ‘Conference’. Cellulose diameter for both cultivars was about 23 nm. This study showed that less degraded, thicker and more branched pectin molecules were associated with higher firmness and more favourable texture. Hemicellulose provided a positive contribution to texture when they were thinner and more flexible.     

Punicalagin exhibits negative regulatory effects on LPS-induced acute lung injury

Punicalagin, mainly isolated from the fruit of Pomegranate (Punica granatum L.), is a natural polyphenolic compound. In the present study, we investigated the negative regulatory effect of punicalagin on acute lung injury (ALI) induced by Lipopolysaccharide (LPS). In the murine model of ALI, the data showed that punicalagin inhibited the production of TNF-α, IL-1β, and IL-6 and decreased protein concentration and myeloperoxidase activity with a single 4 mg/kg dose of punicalagin prior to the administration of intratracheal LPS in the bronchoalveolar lavage fluid. Furthermore, we investigated the effects of punicalagin how to modulate signal transduction. MAPK and NF-κB activation were measured by Western blot and immunocytochemical analysis. The data showed that punicalagin significantly inhibited phosphorylated p38 MAPK protein expression and shocked p65-NF-κB translocation into the nucleus. These results indicated punicalagin may exert negative regulatory effects on ALI partly through suppressing p38 MAPKs or/and NF-κB pathways. This study offered a novel therapeutic strategy for improving clinical effects of acute lung injury (ALI)/acute respiratory distress syndrome and provided more evidence for the health benefits of pomegranate fruits.     

Determination of Phenolic Compounds in Artichoke,Garlic and Spinach by Ultra-High-Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry

Phenolic compounds were determined in artichoke (Cynara scolymus), garlic (Allium sativium) and spinach (Spinacia oleracea) using a single method based on simple extraction and ultra-high-performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). Several compounds belonging to different families, such as phenolic acids, isoflavones, flavones and flavonols, were determined. The analytical procedure was validated in all the matrices, obtaining recoveries ranging from 60 to 120 % with reproducibility values (expressed as relative standard deviations (RSDs)) lower than 26 %. Limits of quantification (LOQs) were always equal to or lower than 50 μg/kg, except to kaempferol and its glucosides in spinach (LOQs?=?75 μg/kg). Artichoke showed higher concentration of phenolic compounds (837.2 mg/kg dry weight (DW)) than garlic (26.5 mg/kg DW) or spinach (64.5 mg/kg DW). Apigenin 7-O-glucoside (from 147.0 to 722.7 mg/kg DW) was found to be the major flavonoid in all samples of artichoke investigated, while chlorogenic acid, whose concentration ranged from 37.8 to 734.7 mg/kg DW, is the major phenolic acid in this matrix. Regarding garlic, caffeic acid (from 1.7 to 28.3 mg/kg DW) and quercetin (from 9.0 to 18.9 mg/kg DW) were the compounds detected at higher concentrations, although in general the total content was very low in relation to other matrices. In relation to spinach, ferulic acid was the major phenolic compound, and its concentration ranged from 18.0 to 41.4 mg/kg DW.