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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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 %.     

The probiotic characteristics and GABA production of Lactobacillus plantarum K154 isolated from kimchi

In order to identify strains with a high GABA production ability and glutamate decarboxylase activity, 273 bacteria were isolated from kimchi. K154 produced 154.86 μg/mL of GABA in an MRS broth containing 1% MSG, 170.42 μg/mL of GABA in an MRS broth containing 2% MSG, and 201.78 μg/mL of GABA in an MRS broth containing 3% MSG. K154 was identified as Lactobacillus plantarum based on API carbohydrate fermentation pattern testing. The 16s rDNA sequence was investigated in order to determine physiological characteristics. The optimum growth temperature of K154 was 37°C. K154 was more sensitive to novobiocin and bacitracin than to other antibiotics, and exhibited greater resistance to polymyxin B and vancomycin. K154 was comparatively tolerant to bile juice and acid, and displayed resistance to Escherichia coli, Salmonella Typhimurium, and Staphylococcus aureus at rates of 19.0, 18.9, and 13.6% respectively.     

Carpesium abrotanoides extract inhibits inducible nitric oxide synthase expression induced by toll-like receptor agonists

Inflammation is a pathological and physiological process which is known to be involved in numerous diseases, while it is notable that a considerable proportion of chronic inflammatory diseases overlap with the development of cancer. One of the most important proteins for inflammatory responses is inducible nitric oxide synthase (iNOS). The present study investigated the effect of the extract of Carpesium abrotanoides L. (ECA) on inflammation by modulating iNOS expression induced by toll-like receptors (TLRs) agonists in murine macrophages. ECA suppressed iNOS expression induced by lipopolysaccharide (TLR4 agonist), macrophage-activating lipopeptide 2-kDa (TLR2 and TLR6 agonist), and polyriboinosinic polyribocytidylic acid (TLR3 agonist). All the results suggest that ECA can modulate TLR signaling pathways and subsequent chronic inflammatory responses.     

Novel bioconversion of sodium glutamate to γ-poly-glutamic acid and γ-amino butyric acid in a mixed fermentation using Bacillus subtilis HA and Lactobacillus plantarum K154

Both γ-poly-glutamic acid (γ-PGA) and γ-amino butyric acid (GABA) were produced from sodium glutamate in a mixed fermentation. A culture broth was obtained using a defined medium including 3% glutamate and Bacillus subtilis HA for 3 days at 42°C, with 1.20×10⁸ CFU/mL viable cells, a 0.46 Pa·sⁿ consistency index, and a 1.94% mucilage content. In a second fermentation using Lactobacillus plantarum K154 at 37°C for 3 days, 1.29% of the remaining glutamic acid in the viscous culture broth was converted to 0.86% GABA. Glutamic acid in the first culture broth fortified with 5% skim milk was completely converted to GABA. The final co-cultured broth had 0.48% GABA, a 0.294% tyrosine content, and viable cells of 1.06×10⁹ CFU/mL (L. plantarum K154) and 5.42×10⁵ CFU/mL (B. subtilis HA). Serial co-culturing of these two bacteria can provide novel ingredients fortified with γ-PGA, GABA, peptides, and probiotics.     

Quality Changes and Establishment of Predictive Models for Bighead Carp (Aristichthys nobilis) Fillets During Frozen Storage

The objectives of this study were to investigate quality changes of bighead carp fillets during frozen storage by measuring free fatty acids (FFA), salt extractable protein (SEP) content, Ca²⁺-ATPase activity, and total sulfhydryl (SH) content and to develop predictive models based on them. FFA increased with the extension of storage time. Meanwhile, SEP content, SH content, and Ca²⁺-ATPase activity all decreased during storage. A marked inhibition (p?2+-ATPase activity and SH content decrease was observed during storage of bighead carp fillets at ?30 °C, compared to those stored at ?10 and ?20 °C. Additionally, the relative errors of models based on Ca²⁺-ATPase and SH were all below 10 %. Thus, the lower frozen temperature can inhibit quality damage effectively, and a reliable estimation of quality changes could be performed by models based on Ca²⁺-ATPase and SH.     

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 %.