A Straightforward, Sensitive, and Reliable Strategy for Ethyl Carbamate Detection in By-Products from Baijiu Production by Enzyme-Linked Immunosorbent Assay.

Baijiu is a renowned Chinese distilled liquor, notable for its distinctive flavor profile and intricate production process, which prominently involves fermentation and distillation. Ethyl carbamate (EC), a probable human carcinogen, can be potentially formed during these procedures, thus prompting significant health concerns. Consequently, the contamination of EC during Baijiu production has become an increasingly pressing issue. In this study, we developed a rapid and easily operable immunoassay for determining EC in the fermented materials used in Baijiu production. The development of a high-quality antibody specific to EC facilitated a streamlined analytical procedure and heightened method sensitivity. Furthermore, we systematically evaluated other essential parameters. Following optimization, the method achieved an IC50 value of 11.83 µg/kg, with negligible cross-reactivity against EC analogs. The recovery study demonstrated the method's good accuracy and precision, with mean recovery rates ranging from 86.0% to 105.5% and coefficients of variation all below 10%. To validate the feasibility of the technique, we collected and analyzed 39 samples simultaneously using both the proposed immunoassay and confirmatory gas chromatography-mass spectrometry (GC-MS). A robust correlation was observed between the results obtained from the two methods (R2 > 0.99). The detected EC levels ranged from 2.36 µg/kg to 7.08 µg/kg, indicating an increase during the fermentation process.

Intelligent onsite dual-modal assay based on oxidase-like fluorescence carbon dots-driven competitive effect for ethyl carbamate detection.

Intelligent onsite accurate monitoring ethyl carbamate (EC, a group 2 A carcinogen) in environment is of great significance to safeguard environmental health and public safety. Herein, we reported an intelligent dual-modal point-of-care (POC) assay based on the bimetallic Mn and Ce co-doped oxidase-like fluorescence carbon dots (Ce&MnCDs) nanozyme-driven competitive effect. In brief, the oxidase-like activity of Ce&MnCDs was inhibited by thiocholine (TCh, originating from the hydrolysis of acetylcholinesterase (AChE) to acetylthiocholine (ATCh)), preventing the oxidation of o-phenylenediamine (OPD) to 2,3-diaminophenothiazine (DAP). However, with the aid of Br2 + NaOH, EC inactivated AChE to prevent TCh generation for re-launching the oxidase-like activity of Ce&MnCDs to trigger the oxidation of OPD into DAP, thereby outputting an EC concentration-dependent ratiometric fluorescence and colorimetric readouts by employing Ce&MnCDs and OPD as the optical signal reporters. Interestingly, these dual-modal optical signals could be transduced into the gray values that was linearly proportional to the residual levels of EC on a smartphone-based portable platform, with a detection limit down to 1.66 µg/mL, qualifying the requirements of analysis of EC residues in real samples. This opened up a new avenue for onsite assessment of the risk of residues of EC, safeguarding environmental health and public safety.

AIE fluorescent nanozyme-based dual-mode biosensor for analysis of the bioactive component hypoxanthine in meat products.

The development of facile, low-cost reliable, and precise onsite assays for the bioactive component hypoxanthine (Hx) in meat products is significant for safeguarding food safety and public health. Herein, we proposed a smartphone-assissted aggregation-induced emission (AIE) fluorogen tetraphenylethene (TPE)-incorporated amorphous Fe-doped phosphotungstates (Fe-Phos@TPE) nanozyme-based ratiometric fluorescence-colorimetric dual-mode biosensor for achieving the onsite visual detection of Hx. When the Hx existed, xanthine oxidase (XOD) catalyzed Hx into H2O2 to be further catalyzed into •OH by the prominent peroxidase activity of Fe-Phos@TPE at pH = 6.5, resulting in the oxidization of nonfluorescent o-phenylenediamine (OPD, naked-eye colorless) to be yellow fluorescent emissive 2,3-diaminophenazine (DAP, naked-eye dark yellow) at 550 nm as well as the intrinsic blue fluorescence of Fe-Phos@TPE at 440 nm to be decreased via inner-filter effect (IFE) action, thereby realizing a multi-enzyme cascade catalytic reaction at near-neutral pH to overcome the traditional acidity dependence-induced time-consuming and low sensitivity troublesome.

Dual-Modal Bimetallic Nanozyme-Based Sensing Platform Combining Colorimetric and Photothermal Signal Cascade Catalytic Enhancement for Detection of Hypoxanthine to Judge Meat Freshness.

Considering the enormous demand for meat in people's daily lives, the development of efficient meat freshness assays is of great significance for safeguarding food safety. Here, a novel bimetallic nanozyme Fe@CeO2 with high peroxidase-like activity was first synthesized by embedding ferrocenecarboxylic acid (Fc) into hollow CeO2 nanospheres, which combined with xanthine oxidase (XOD) to develop a self-supplying H2O2-facilitated enzymatic cascade catalytic system of XOD + Fe@CeO2, yielding a meat freshness indicator hypoxanthine (Hx)-responsive colorimetric and photothermal dual-mode analytical platform for judging meat freshness upon the assistance of 3,3',5,5'-tetramethylbenzidine (TMB). Owing to the catalytic activity of XOD to convert Hx into H2O2, Fe@CeO2 rapidly dissociated it into •OH via a peroxidase activity-triggered Fenton-like reaction, emerging a typical enzymatic cascade catalytic reaction. As a result, the colorless TMB was oxidized to be the product of dark-blue oxTMB by •OH, with a chromogenic reaction-driven absorption enhancement at 652 nm, which endowed it with a significant photothermal effect under 660 nm laser irradiation. On this basis, an Hx concentration-dependent colorimetric and photothermal dual-mode signal cascade catalytic enhancement sensing platform was proposed by integrating with a Color Picker App-installed smartphone and a 660 nm laser-equipped handheld thermal imager, achieving the onsite quantitative, reliable, and visual detection of Hx in real meat samples for judging meat freshness with acceptable results. Notably, the colorimetric and photothermal dual-mode signal cascade catalytic enhancement improved not only the reliability but also the sensitivity of the assay, which provided new insights for efficient onsite visual monitoring of meat freshness to safeguard food safety.

Levels and Health Risk Assessment of Polycyclic Aromatic Hydrocarbons in Vegetable Oils and Frying Oils by Using the Margin of Exposure (MOE) and the Incremental Lifetime Cancer Risk (ILCR) Approach in China.

A total of 139 vegetable oils and 48 frying oils produced in China were tested for the levels of 15 Environmental Protection Agency-regulated polycyclic aromatic hydrocarbons (PAHs). The analysis was completed by high-performance liquid chromatography-fluorescence detection (HPLC-FLD). The limit of detection and limit of quantitation were ranged between 0.2-0.3 and 0.6-1 µg/kg, respectively. The average recovery was 58.6-90.6%. The highest mean of total PAHs was found in peanut oil (3.31 µg/kg), while the lowest content was found in olive oil (0.39 µg/kg). In brief, 32.4% of vegetable oils exceeded the European Union maximum levels in China. The detected level of total PAHs in vegetable oils was lower than the frying oils. The mean dietary exposure to PAH15 ranged from 0.197 to 2.051 ng BaPeq/kg bw/day. The margin of exposure values was greater than 10,000, and the cumulative probabilities of the incremental lifetime cancer risk of different age groups were less than the priority risk level (10-4). Therefore, there was no potential health concern for specific populations.

An Ultrasensitive Lateral Flow Immunoassay Based on Metal-Organic Framework-Decorated Polydopamine for Multiple Sulfonylureas Adulteration in Functional Foods.

Herein, an ultrasensitive lateral flow immunoassay (LFIA), based on metal-organic framework-decorated polydopamine (PCN-224@PDA) was first established to detect multiple sulfonylureas (SUs) in functional foods. The PCN-224@PDA was synthesized using the one-pot hydrothermal method and covalently coupled with SUs antibodies, and the coupling rate was up to 91.8%. The detection limits of the developed PCN-224@PDA-LFIA for multiple SUs in functional teas and capsules were 0.22-3.72 µg/kg and 0.40-3.71 µg/kg, and quantification limits were 0.75-8.19 µg/kg and 1.03-9.08 µg/kg, respectively. The analytical sensitivity was 128-fold higher than that of similar methods reported so far. The recovery rates ranged from 83.8 to 119.0%, with coefficients of variation of 7.6-14.4%. The parallel analysis of 20 real samples by LC-MS/MS confirmed the reliability of the proposed method. Therefore, our work offers novel, ultrasensitive, and rapid technical support for on-site monitoring of SUs in functional foods.

Occurrence of Furfural and Its Derivatives in Coffee Products in China and Estimation of Dietary Intake.

This is the first report on the content of furfural and its derivatives in coffee products in China. The concentrations of furfural and its derivatives in 449 sampled, commercially available coffee products in China were analyzed through a GC-MS technique, and the associated health risks were estimated. As a result, 5-hydroxymethyl furfural (5-HMF) was identified as the predominant derivative compound, with the highest concentration of 6035.0 mg/kg and detection frequency of 98.7%. The mean dietary exposures of 5-HMF, 5-MF(5-methylfurfural), and 2-F(2-furfural) in coffee products among Chinese consumers were 55.65, 3.00, and 3.23 µg/kg bw/day, respectively. The ranges of mean dietary intake of furfural and its derivatives based on age groups were all lower than the acceptable daily intake (ADI) and the toxicological concern threshold (TTC). Risk evaluation results indicate that coffee product intake did not pose potential risks to consumers. Notably, the analysis revealed that children aged 3-6 years had the highest mean exposure due to their low body weight.

Temperature-dependent mycotoxins production investigation in Alternaria infected cherry by ultra-high performance liquid chromatography and Orbitrap high resolution mass spectrometry.

For temperature-dependent Alternaria mycotoxins production analysis, cherry samples were inoculated with Alternaria sp. and incubated at two different temperatures (4 °C and 25 °C). Six Alternaria mycotoxins, including altenuene (ALT), alternariol monomethyl ether (AME), alternariol (AOH), altertoxin-I (ATX-I), tenuazonic acid (TeA), and tentoxin (TEN), in cherries were detected with integrated visible data-processing tools. Maximum concentration of these mycotoxins reached 71,862.2 µg/kg at 25 °C. Notably, considerable amount of TeA (290.4 µg/kg) was detected at 4 °C, which indicated that low temperature is not a safe storage condition for fruits. A total of 102 compounds were detected with a neutral loss of 162.0528 Da, and TeA-glucose was identified in this work. Based on MS/MS cosine similarity, products were verified and annotated with feature based molecular networking (FBMN) in global natural products social networking (GNPS). The results showed Alternaria mycotoxins in cherry samples were mainly demethylation, hydrogenation, and dehydration. This work revealed the production of Alternaria mycotoxins in cherries under different storage temperature, which will provide theoretical basis for the control of mycotoxin contamination in food commodities.

Metabolism Profile of Mequindox in Sea Cucumbers In Vivo Using LC-HRMS.

In this work, the metabolism behavior of mequindox (MEQ) in sea cucumber in vivo was investigated using LC-HRMS. In total, nine metabolites were detected and identified as well as the precursor in sea cucumber tissues. The metabolic pathways of MEQ in sea cucumber mainly include hydrogenation reduction, deoxidation, carboxylation, deacetylation, and combinations thereof. The most predominant metabolites of MEQ in sea cucumber are 2-iso-BDMEQ and 2-iso-1-DMEQ, with deoxidation and carbonyl reduction as major metabolic pathways. In particular, this work first reported 3-methyl-2-quinoxalinecarboxylic acid (MQCA) as a metabolite of MEQ, and carboxylation is a major metabolic pathway of MEQ in sea cucumber. This work revealed that the metabolism of MEQ in marine animals is different from that in land animals. The metabolism results in this work could facilitate the accurate risk assessment of MEQ in sea cucumber and related marine foods.

Rainbow latex microspheres lateral flow immunoassay with smartphone-based device for simultaneous detection of three mycotoxins in cereals.

The coexistent pollution of multiple mycotoxins displays a synergistic toxicity effect that significantly threatens human health. Therefore, it is essential to establish a rapid detection method for multi-mycotoxins in food. In this study, red, green, and blue latex microspheres (LMs) were applied as the aflatoxin B1 (AFB1), T-2 toxins (T-2), and zearalenone (ZEN) antibodies labeled tracer, respectively. Based on the principle of spatial resolution, a rainbow "traffic light" pattern latex microspheres lateral flow immunoassay (LMs-LFIA) integrated with a portable and user-friendly smartphone-based device was first developed to detect three kinds of mycotoxins simultaneously. The cut-off values of the method for AFB1/T-2/ZEN in cereals were 1/15/40 µg kg-1, the limits of detection were 0.04/0.40/1.21 µg kg-1, respectively. The recoveries ranged from 82.1% to 107.5%, with the coefficient of variation from 3.0% to 8.1%. A parallel analysis in 26 naturally contaminated cereal samples was confirmed by commercial ELISA kits; the results showed a good correlation (R2＞0.99), indicating the practical reliability of the rainbow LMs-LFIA. This method provided a visually enjoyable, portable, and sensitive detection mode for multi-target detection of mycotoxins or other small molecule hazard factors in food.

A highly sensitive and quantitative time resolved fluorescent microspheres lateral flow immunoassay for streptomycin and dihydrostreptomycin in milk, honey, muscle, liver, and kidney.

Streptomycin (STR) and dihydrostreptomycin (DHSTR) are clinically widely used in the prevention and treatment of animal diseases. Unreasonable use or abuse can easily cause adverse effects on human health. Thus, it is very necessary to establish a rapid detection method for STR and DHSTR in animal-derived foods. In this study, a time-resolved fluorescent microspheres lateral flow immunoassay (TRFM-LFIA) integrated with a portable fluorescence reader was developed for STR and DHSTR detection. The cut-off values of the TRFM-LFIA for STR and DHSTR in milk/honey/muscle/liver/kidney were both 30/15/80/25/60 µg kg-1, respectively, the limits of detection were 1.10/0.28/2.82/1.52/3.02, 0.75/0.23/1.76/1.21/2.35 µg kg-1, respectively, and the limits of quantitation were 3.62/0.93/9.31/5.02/9.96, 2.48/0.76/5.81/3.99/7.76 µg kg-1, respectively. The recoveries ranged from 80.0% to 120.1%, with coefficient of variation from 2.8% to 14.3%, respectively. The parallel experiment of 25 samples showed excellent correlation (R2 > 0.99) between ELISA kit and TRFM-LFIA. The sample pretreatment is very simple, and the results can be achieved in 8 min. This sensitive, simple, rapid and portable analysis strategy can provide universal technical support for the residue detection of veterinary drugs and even small molecular hazard factors.

Data mining of natural hazard biomarkers and metabolites with integrated metabolomic tools.

Data mining was one of the most important challenges in natural product analysis and biomarker discovery. In this work, we proposed an integrated data analysis protocol for natural products annotation and identification in data-dependent acquisition. Firstly, natural products and structure-related compounds could be identified by comparing mass spectrum behavior with commercial standard. Secondly, diagnostic fragmentation filtering (DFF) function in MZmine (http://mzmine.github.io/) was investigated for screening specific conjugation compounds with the same neutral loss. Thirdly, we present feature-based molecular networking (FBMN) in GNPS (https://gnps.ucsd.edu/) as a chromatographic feature detection and alignment tool. In addition, FBMN could enable natural products analysis based on molecular networks. This proposed integrated protocol should facilitate metabolomic data mining and biomarker discovery.

A Rapid and Sensitive Fluorescent Microsphere-Based Lateral Flow Immunoassay for Determination of Aflatoxin B1 in Distillers' Grains.

Aflatoxin B1 (AFB1) is a toxic compound naturally produced by the genera Aspergillus. Distillers' grains can be used as animal feed since they have high content of crude protein and other nutrients. However, they are easily contaminated by mycotoxins, and currently there are no rapid detection methods for AFB1 in distillers' grains. In this study, a lateral flow immunoassay (LFIA) based on red fluorescent microsphere (FM), is developed for quantitative detection of AFB1 in distillers' grains. The whole test can be completed within 15 min, with the cut-off value being 25.0 µg/kg, and the quantitative limit of detection (qLOD) being 3.4 µg/kg. This method represents satisfactory recoveries of 95.2-113.0%, and the coefficients of variation (CVs) are less than 7.0%. Furthermore, this technique is successfully used to analyze AFB1 in real samples, and the results indicates good consistency with that of high-performance liquid chromatography (HPLC). The correlation coefficient is found to be greater than 0.99. The proposed test strip facilitates on-site, cost-effective, and sensitive monitoring of AFB1 in distillers' grains.

Highly photoluminescent carbon dots-based immunosensors for ultrasensitive detection of aflatoxin M1 residues in milk.

Here, a facile hydrothermal method was used to synthesize highly photoluminescent N-doped carbon dots, and the quantum yields reached 97.1%. Then, a label-free immunosensor based on the inner filter effect of carbon dots was developed for ultrasensitive detection of aflatoxin M1 residues in milk. The detection limit was 0.0186 ng/mL (equivalents to 18.10 ng/kg), which satisfied the most stringent maximum tolerable limit value of 25 ng/kg. Besides, the immunosensor showed a good linear relationship from 0.003 ng/mL to 0.81 ng/mL, and the average recoveries ranged from 79.6% to 112.5% for spiked milk samples, with relative standard deviations ranging from 6.7% to 13.3%. Compared with other immunoassays, the inner filter effect-based immunosensor incorporating fluorescent detection into conventional enzymatic cascade amplification systems and could be a reliable on-site screening method for aflatoxin M1 residue analysis.

In vitro study of the antioxidative and antiproliferative capabilities of Lactobacillus casei 16-fermented soymilk.

In this study, soymilk was fermented with Lactobacillus casei 16. The contents of aglycone isoflavones, saponins, total phenolic, and free amino acid in the fermented soymilk, and the protection for the HepG2 cells against 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP) damage and the antiproliferative effects of the fermented soymilk on the HT-29 cells and Caco-2 cells were studied. The results showed that the levels of total phenolic, aglycone isoflavones, and free amino acids in the L. casei 16-fermented soymilk were enhanced. The ethanol extract and the water extract of the L. casei 16-fermented soymilk showed protection for HepG2 cells against ABAP damage and inhibited the proliferation of the HT-29 cells and Caco-2 cells, which may be attributed to the enhanced level of total phenolic, aglycone isoflavones, and free amino acids in the L. casei 16-fermented soymilk.

Hypoglycemic and hypolipidemic activities of Grifola frondosa polysaccharides and their relationships with the modulation of intestinal microflora in diabetic mice induced by high-fat diet and streptozotocin.

This study aimed to investigate the hypoglycemic and hypolipidemic activities of polysaccharides from Grifola frondosa (GFP) in diabetic mice induced by high-fat diet (HFD) and streptozotocin (STZ). Results showed that oral administration of GFP markedly reduced the serum levels of fasting blood glucose (FBG), oral glucose tolerance (OGT), cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C), and significantly decreased the hepatic levels of TC, TG and free fatty acids (FFA). Meanwhile, high-dose of GFP supplementation (900 mg/kg day) also showed powerful effects on moderating the composition of intestinal microflora in diabetic mice, especially altering the functionally relevant intestinal microbial phylotypes. Spearman's correlation network analysis revealed that key microbial phylotypes responding to GFP intervention were strongly correlated with the glucose and lipid metabolic disorders associated parameters. Moreover, GFP treatment regulated mRNA expression levels of the genes responsible for hepatic glucose and lipid metabolism. It is noteworthy that GFP treatment markedly increased mRNA expression of cholesterol 7α-hydroxylase (CYP7A1) and bile salt export pump (BSEP), suggesting an enhancement of bile acids (BAs) synthesis and excretion in liver. These findings demonstrated that GFP could prevent hyperglycemia and hyperlipidemia in diabetic mice by altering gut microbiota and regulating hepatic glycolipid metabolism related genes, and therefore could be used as potential functional food ingredients for the prevention or treatment of hyperglycemia and hyperlipidemia.

Highly Regioselective and Stereoselective Hydroxylation of Free Amino Acids by a 2-Oxoglutarate-Dependent Dioxygenase from Kutzneria albida.

Hydroxyl amino acids have tremendous potential applications in food and pharmaceutical industries. However, available dioxygenases are limited for selective and efficient hydroxylation of free amino acids. Here, we identified a 2-oxoglutarate-dependent dioxygenase from Kutzneria albida by gene mining and characterized the encoded protein (KaPH1). KaPH1 was estimated to have a molecular weight of 29 kDa. The optimal pH and temperature for its l-proline hydroxylation activity were 6.5 and 30 °C, respectively. The K m and k cat values of KaPH1 were 1.07 mM and 0.54 s-1, respectively, for this reaction by which 120 mM l-proline was converted to trans-4-hydroxy-l-proline with 92.8% yield (3.93 g·L-1·h-1). EDTA, [1,10-phenanthroline], Cu2+, Zn2+, Co2+, and Ni2+ inhibited this reaction. KaPH1 was also active toward l-isoleucine for 4-hydroxyisoleucine synthesis. Additionally, the unique biophysical features of KaPH1 were predicted by molecular modeling whereby this study also contributes to our understanding of the catalytic mechanisms of 2-oxoglutarate-dependent dioxygenases.

Comparison of soybean peroxidase with horseradish peroxidase and alkaline phosphatase used in immunoassays.

Enzyme labeling of an antigen or an antibody helps to visualize and amplify the signal and is an important reagent used in immunoassays for the detection of a target of interest. In this research, soybean peroxidase (SBP), a less commonly used enzyme reporter, was compared in immunoassays with the two most commonly used reagents, horseradish peroxidase (HRP) and alkaline phosphatase (ALP). The enzyme-antibody conjugates were evaluated by their performance in an indirect competitive enzyme-linked immunosorbent assay (icELISA) and in an indirect competitive chemiluminescent enzyme immunoassay (icCLEIA) for ractopamine (RAC). The results revealed that the more affordable SBP offers a long-lasting chemiluminescent signal, which outperformed ALP and HRP. SBP-antibody conjugate (SBP-Ab) based immunoassays produced lower limits of detection (LODs) and better accuracy in the detection of RAC in animal urine samples. Additionally, SBP-Ab has advantages in being more resistant to heat, acid and organic solvents. These results suggest that SBP could be a potentially excellent alternative to HRP and ALP for the development of immunoassay in food safety field.

A Dual-Color Quantum Dots Encoded Frit-Based Immunoassay for Visual Detection of Aflatoxin M1 and Pirlimycin Residues in Milk.

Mycotoxins and antibacterial agents are the main chemical hazards that lead to several health problems. Nowadays, multiplex immunoassay is a primary goal throughout the world. Here, aflatoxin M1 and pirlimycin were selected as models, and a novel dual colorimetric encoded frit-based immunoassay was developed for simultaneously screening of aflatoxin M1 and pirlimycin residues in milk. This multiplex frit-based immunoassay combined two monoclonal antibodies to extend the spectrum of analytes and to enable detection of two classes of analytes in a single test. The cutoff values were 0.02 µg/kg for aflatoxin M1 and 0.5 µg/kg for pirlimycin, which satisfied the requirement to measure the maximum residue levels. The novel colorimetric frit-based immunoassay has the advantage of high throughput, short analysis time, reduced overall cost per assay, and can be used as a rapid screening technique for simultaneously detecting aflatoxin M1 and pirlimycin residues in milk.

Development of a heterologous enzyme-linked immunosorbent assay for the detection of clindamycin and lincomycin residues in edible animal tissues.

In this study, new clindamycin (CLIN) artificial antigens were prepared and used to produce broad-specificity monoclonal antibodies. Based on the as-produced mAbs, a heterologous ELISA was developed to detect CLIN and lincomycin (LIN) residues in edible animal tissues. The IC50 values of the developed assay were 0.3ng/mL (CLIN) and 1.2ng/mL (LIN) in buffer, respectively. The detection limits were estimated to be 1.8µg/kg (CLIN) and 6.8µg/kg (LIN) in bovine, chicken, porcine and fish muscles. In the spike and recovery tests, the mean recovery rate ranged from 76% to 112% at different spiked levels, and the intra-/inter-assay coefficients of variation were in the range of 7.1% to 13.2%. This method was verified using LC-MS/MS with a correlation coefficient >0.97. The developed ELISA is therefore well suited for simultaneous determination of CLIN and LIN residues in bovine, chicken, porcine and fish muscles.