Development and Optimization of a SERS Method for On-site Determination of Nitrite in Foods and Water

A rapid, sensitive, and selective detection method of nitrite in foods and water was established by surface-enhanced Raman spectroscopy (SERS) based on the diazo reaction of nitrite with p-nitroaniline and 1-naphthylamine in acidic solution. The azo dye (4-(4-nitrophenyldiazenyl)naphthalene-1-aminium, NNA), which was derived from the diazo reaction, was determined by SERS using citrate-coated silver nanoparticles (AgNPs) as SERS substrates. The concentrations of nitrite in samples were finally calculated from the intensities of SERS signals generated by NNA in the testing solutions. By using the present method combined with a portable miniature Raman spectrometer, on-site determination of nitrite could be performed easily and efficiently. The effects of several experimental parameters on the intensity of SERS signals, such as the volume of AgNP solution and the mixing time of AgNPs and NNA, were investigated. The linear range of the method was 0.1–10.0 mg L^?1. The limits of detection (LODs) were 0.01, 0.03, and 0.05 mg L^?1 at 720, 1,459, and 1,609 cm^?1, respectively. The method was applied to the determination of nitrite in real food and water samples, with recoveries in the range of 86.9–103.4 % and relative standard deviations (RSDs) less than 9.64 %.     

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 %.     

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.     

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.     

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.     

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.     

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.     

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.     

Alleviating effects of Opuntia ficus indica extracts on psychomotor alterations induced by ethanol in rats

Effects of Opuntia ficus indica (OFI) extracts on ethanol-induced psychomotor alterations were studied using Sprague-Dawley rats orally administered 4 g/kg of ethanol (EtOH group) or distilled water (control group). An OFI-group received OFI extracts (50, 100, and 200 mg/kg) 30 min prior to EtOH administration. Behavioral and hematological tests were evaluated 1, 2, 4, and 8 h after EtOH administration. Electroencephalogram (EEG) assessment was also performed. EtOH significantly (p<0.001) induced psychomotor alterations (reduced locomotion, balance, coordination, muscle strength, and tolerance to cold), compared with control group rats. Pre-treatment with OFI extracts alleviated alterations and also inhibited elevation of blood ethanol levels. EEG (percent of total power for delta, theta, alpha, and beta) results for OFI group rats were similar to control rats, indicating a countering of EtOHinduced EEG changes. Extracts of OFI can be effective for alleviation of psychomotor alterations induced by EtOH administration.     

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.     

Ethylacetate extracts of the muscles of Anguilla japonica suppress glucose levels in db/db mice via activation of AMP-activated protein kinase

Hypoglycemic effects of ethylacetate extracts of Anguilla japonica (EMA) muscles in db/db mice were investigated. To understand the mechanism responsible for the hypoglycemic effects of EMA, the effects of EMA on AMP-activated protein kinase (AMPK) activation in L6 myotubes and in vivo using type II diabetic db/db mice were analyzed. In L6 myotubes, the phosphorylation degrees of AMPK and acetyl-CoA carboxylase (ACC) were markedly increased and glucose uptake was significantly (p<0.001) increased by EMA, compared with untretaed L6 myotubes. However, in L6 myotubes, these effects were abolished by compound C, an AMPK inhibitor. Moreover, EMA significantly reduced non-fasting blood glucose and serum insulin levels, and strongly induced AMPK phosphorylation in skeletal muscle tissues of db/db mice. EMA regulates glucose levels in L6 myotubes and in diabetic mice by activation of AMPK. Beneficial effects for diabetes treatment are indicated.     

Trans-caryophyllene suppresses tumor necrosis factor (TNFα)-induced inflammation in human chondrocytes

Proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) have been shown to be associated with the pathogenesis of cartilage damage in arthritis. Trans-caryophyllene (TC) is an important constituent of the essential oils of several species of plants. In this study, we found that TC treatment could inhibit TNF-α-induced matrix metallopeptidase 13, cyclooxygenase-2, and prostaglandin E2 (PGE2) production in human chondrocytes. Moreover, the increase in inducible nitric oxide synthase expression and NO production induced by TNF-α was able to be suppressed by TC treatment. Importantly, treatment with TC was also found to inhibit activation of the main proinflammatory regulator, interferon regulatory factor-1, which is increased in the presence of TNF-α. Interestingly, we also proved that TC’s effects on these mechanisms are dependent on the type 2 cannabinoid receptor. Taken together, the results of this study show that TC exerts anti-inflammatory effects in TNF-α-stimulated chondrocyte models.     

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.