Trace analysis of food by surface-enhanced Raman spectroscopy combined with molecular imprinting technology: Principle, application, challenges, and prospects.

Surface-enhanced Raman spectroscopy (SERS) is a rapid detection method with high sensitivity and simple pretreatment, but can be affected by interference from matrix components. By incorporating molecularly imprinted polymers (MIPs) that recognize specific targets, MIP-SERS sensors effectively overcome the interference of complex matrices and offer improved stability and sensitivity. This review provides a comprehensive understanding of the applications of MIP-SERS sensors for the detection of trace toxic substances in food. The underlying mechanism and development of SERS technology and the principle and classification of MIPs technology are discussed. Furthermore, the types of MIP-SERS sensors are introduced, with their advantages and disadvantages systematically illustrated. Recent advances in MIP-SERS technology for the detection of mycotoxins, additives, prohibited dyes, pesticides, veterinary drug residues, and other hazardous substances in food are highlighted. Finally, this review discusses the challenges associated with MIP-SERS technology and proposes future development prospects.

Transcriptomic analysis reveals the inhibition mechanism of pulsed light on fungal growth and ochratoxin A biosynthesis in Aspergillus carbonarius.

Pulsed light (PL) technology has a good effect on the control of fungi in postharvest fruit. In this present work, PL inhibited the growth of Aspergillus carbonarius in a dose-dependent manner, the mycelial growth decreased by 4.83 %, 13.91 % and 30.01 % at a fluence of 4.5 J·cm-2 (PL5), 9 J·cm-2 (PL10) and 13.5 J·cm2 (PL15), respectively. When inoculated with PL15 treated A. carbonarius, the scab diameter of the pears, ergosterol and OTA content was reduced by 23.2 %, 27.9 % and 80.7 % after 7 days, respectively. The third-generation sequencing technique was applied to study the transcriptome response of A. carbonarius treated with PL. Compared with the blank control, a total number of 268 and 963 differentially expressed genes (DEGs) were discovered in the group of PL10 and PL15, respectively. To be specific, a large amount of DEGs involved in DNA metabolism were up-regulated, while most of DEGs related to cell integrity, energy and glucose metabolism, ochratoxin A (OTA) biosynthesis and transport were down-regulated. In addition, the stress response of A. carbonarius was imbalanced, including up-regulation of Catalase and PEX12 and down-regulation of taurine and subtaurine metabolism, alcohol dehydrogenase and glutathione metabolism. Meanwhile, the results of transmission electron microscopy, mycelium cellular leakage and DNA electrophoresis indicated that PL15 treatment caused mitochondrial swelling, the destroyed cell membrane permeability and imbalance of DNA metabolism. The expression of P450 and Hal involved in OTA biosynthesis pathway were down-regulated in PL treated samples detected by qRT-PCR. In conclusion, this study reveals the molecular mechanism of pulsed light on inhibiting the growth, development and toxin production of A. carbonarius.

MIPs-SERS Sensor Based on Ag NPs Film for Selective Detection of Enrofloxacin in Food.

The quinolone antibiotics represented by enrofloxacin (ENRO) are harmful to the ecological environment and human health due to illegal excessive use, resulting in increasing food residues and ENRO levels in the environment. To this end, we developed a MIPs-SERS method using surface-enhanced Raman spectroscopy (SERS) and molecularly imprinted polymers (MIPs) to detect ENRO in food matrices. Firstly, a layer of silver nanoparticles (Ag NPs) with the best SERS effect was synthesized on the surface of copper rods as the enhancing material by in situ reductions, and then MIPs targeting ENRO were prepared by the native polymerization reaction, and the MIPs containing template molecules wrapped on the surface of silver nanoparticle films (Ag NPs-MIPs) were obtained. Our results showed that the Ag NPs-MIPs could specifically identify ENRO from the complex environment. The minimum detection limit for ENRO was 0.25 ng/mL, and the characteristic peak intensity of ENRO was linearly correlated to the concentration with a linear range of 0.001~0.1 µg/mL. The experimental results showed that in comparison to other detection methods, the rapid detection of ENRO in food matrices using Ag NPs-MIPs as the substrate is reliable and offers a cost-effective, time-saving, highly selective, and sensitive method for detecting ENRO residues in real food samples.

A Novel and Label-Free Chemiluminescence Detection of Zearalenone Based on a Truncated Aptamer Conjugated with a G-Quadruplex DNAzyme.

Zearalenone (ZEN), one of the most frequently occurring mycotoxin contaminants in foods and feeds, poses considerable threat to human and animal health, owing to its acute and chronic toxicities. Thus, rapid and accurate detection of ZEN has attracted broad research interest. In this work, a novel and label-free chemiluminescence aptasensor based on a ZEN aptamer and a G-quadruplex DNAzyme was constructed. It was established on a competitive assay between ZEN and an auxiliary DNA for the aptamer, leading to activation of the G-quadruplex/hemin DNAzyme and subsequent signal amplification by chemiluminescence generation after substrate addition. To maximize the detection sensitivity, numerous key parameters including truncated aptamers were optimized with molecular docking analysis. Upon optimization, our aptasensor exhibited a perfect linear relationship (R2 = 0.9996) for ZEN detection in a concentration range of 1-100 ng/mL (3.14-314.10 nM) within 40 min, achieving a detection limit of 2.85 ng/mL (8.95 nM), which was a 6.7-fold improvement over that before optimization. Most importantly, the aptasensor obtained a satisfactory recovery rate of 92.84-137.27% and 84.90-124.24% for ZEN-spiked wheat and maize samples, respectively. Overall, our label-free chemiluminescence aptasensor displayed simplicity, sensitivity, specificity and practicality in real samples, indicating high application prospects in the food supply chain for rapid detection of ZEN.

Ferulic acid improves intestinal barrier function through altering gut microbiota composition in high-fat diet-induced mice.

PURPOSE: A high-fat diet (HFD) induces gut microbiota (GM) disorders, leading to intestinal barrier dysfunction and inflammation. Ferulic acid (FA) has shown anti-obesity effects, e.g., reducing body weight and food intake. However, the mechanism linking the anti-obesity effects of FA and GM modulation remains obscure. The present study aimed to clarify the mechanism underlying the anti-obesity effects of FA and modulation of the GM. METHODS: C57BL/6 J mice were fed by a low-fat diet (LFD) and HFD with or without FA at a dose of 100 mg/kg of body weight by oral gavage for 12 weeks. Using high-throughput sequencing, gas chromatography, real-time fluorescence quantitative PCR and immunohistochemical staining, the attenuation of obesity by FA were assessed via intestinal barrier integrity, inflammation, and the GM. RESULTS: FA reduced weight gain, improved HFD-induced GM imbalance, significantly enhanced intestinal short-chain fatty acid (SCFA)-producing bacteria (e.g., Olsenella, Eisenbergiella, Dubosiella, Clostridiales_unclassified, and Faecalibaculum) along with SCFA accumulation and its receptors' expression, decreased endotoxin-producing bacteria or obesity-related bacterial genera, and serum endotoxin (lipopolysaccharides), and inhibited the colonic TLR4/NF-κB pathway. Thus, FA can mitigate colonic barrier dysfunction and intestinal inflammation, induce the production of SCFAs and inhibit endotoxins by modulating the GM. CONCLUSION: These results indicate that enhancement of intestinal barrier by altering the GM may be an anti-obesity target of FA and that FA can be used as a functional compound with great developmental values.

Rapid and Sensitive Detection of Pentachloronitrobenzene by Surface-Enhanced Raman Spectroscopy Combined with Molecularly Imprinted Polymers.

Molecularly imprinted polymers (MIPs) specifically targeting pentachloronitrobenzene (PCNB) and containing silver nanoparticles have been prepared by free radical polymerization reaction using methyl methacrylate (MMA) as a functional monomer, PCNB as a template molecule, 1,4-butanedioldimethacrylate as a cross linker, lauroyl peroxide (LPO) as an initiator, and the silver nanoparticles with the best surface-enhanced Raman scattering (SERS) effect as SERS enhancement materials. Our results indicated that MIPs specifically recognize PCNB from complex matrices. The intensity of the PCNB characteristic peak was proportional to the concentration, with a linear range of 0.005 to 0.15 µg/mL and a limit of detection of 5.0 ng/mL. The recovery rates and relative standard deviation for the detection of PCNB spiked in the rice samples were from 94.4% to 103.3% and from 4.6% to 7.4%, respectively. The experimental results are consistent with those by the GC-MS method, indicating that the rapid detection of PCNB in food matrices by SERS-MIPs is reliable. In view of the insolubility of PCNB in water, oil-soluble silver nanoparticles were synthesized which can be expanded to detect oil-soluble toxic substances. For the first time, the proposed method provides a point-of-care and cost-effective tool for rapidly detecting PCNB in food matrices with high sensitivity and selectivity by employing SERS-MIPs method.

iTRAQ proteome analysis of the antifungal mechanism of citral on mycelial growth and OTA production in Aspergillus ochraceus.

BACKGROUND: Aspergillus ochraceus causes food spoilage and produces mycotoxin ochratoxin A (OTA) during storage of agricultural commodities. In this study, citral was used to inhibit A. ochraceus growth and OTA accumulation, proteomic analysis was employed to verify the mechanism of citral. RESULTS: Citral was found to significantly inhibit fungal growth and mycotoxin production in A. ochraceus. Specifically, 75, 125, 150 and 200 µL L-1 citral suppressed mycelial growth by 33%, 46%, 50% and 100%, respectively. Additionally, 75 µL L-1 citral inhibited OTA accumulation by 25%. Proteomic analysis was performed to elucidate the inhibitory mechanism of citral on mycelial growth and OTA production at subinhibitory concentrations (75 µL L-1 ). Proteomics analysis identified 2646 proteins in A. ochraceus fc-1, of which 218 were differentially expressed between control and 75 µL L-1 citral treatment samples. Differentially expressed proteins were identified by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of biological process, cellular component and molecular function terms. Potential factors affecting mycelial growth and OTA production were analysed, and OTA production was revealed to be a complex process involving many associated factors related to various processes including nutrient intake, sterol biosynthesis, ribosome biogenesis, energy metabolism, oxidative stress and amino acid metabolism. In addition, citral at 75 µL L-1 down-regulated OTA biosynthetic genes including pks and nrps, but slightly up-regulated the global regulatory factors veA, velB and laeA. CONCLUSION: The findings further demonstrate the potential of citral for the preservation of grains and other agricultural products, and provide new insight into its antifungal mechanisms at subinhibitory concentrations. © 2021 Society of Chemical Industry.

Quantitative Proteomic Profiling of Fungal Growth, Development, and Ochratoxin A Production in Aspergillus ochraceus on High- and Low-NaCl Cultures.

Dry-cured meat products are worldwide food with high-salt content, and filamentous fungi are beneficial to the maturation process. However, some salt-tolerant strains of Aspergillus and Penicillium produce ochratoxin A (OTA) on these products and thus threaten food safety. In our study, proteomic analysis was performed to reveal the mechanism of adaptability to high-salt environment by Aspergillus ochraceus. Twenty g/L and 70 g/L NaCl substrates were used to provide medium- and high-NaCl content environments, respectively. The NaCl addition could induce fungal growth, but only 20 g/L NaCl addition could induce spore production while 70 g/L repressed it. Proteomics analysis identified 2646 proteins in A. ochraceus fc-1, of which 237 and 251 were differentially expressed with 20 g/L and 70 g/L NaCl addition, respectively. Potential factors affecting fungal growth and development were identified by GO and KEGG analyses of biological process, cellular component, and molecular function terms. The results revealed that ergosterol synthesis pathway was significantly upregulated with 20 g/L and 70 g/L NaCl addition. However, fungal growth and development including OTA production were complex processes associated with many factors including nutrient uptake, cell membrane integrity, cell cycle, energy metabolism, intracellular redox homeostasis, protein synthesis and processing, autophagy, and secondary metabolism. Reactive oxygen species may be an important window to understand the mechanism that medium-salt content was conducive to intracellular signal transduction while high-salt content caused oxidative stress. The findings would help to improve the processes and storage conditions of dry-cured meat products.

Citrus Physiological and Molecular Response to Boron Stresses.

Since the essentiality of boron (B) to plant growth was reported nearly one century ago, the implication of B in physiological performance, productivity and quality of agricultural products, and the morphogenesis of apical meristem in plants has widely been studied. B stresses (B deficiency and toxicity), which lead to atrophy of canopy and deterioration of Citrus fruits, have long been discovered in citrus orchards. This paper reviews the research progress of B stresses on Citrus growth, photosynthesis, light use efficiency, nutrient absorption, organic acid metabolism, sugar metabolism and relocation, and antioxidant system. Moreover, the beneficial effects of B on plant stress tolerance and further research in this area were also discussed.

The Influence of NaCl and Glucose Content on Growth and Ochratoxin A Production by Aspergillus ochraceus, Aspergillus carbonarius and Penicillium nordicum.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin, which deserves particular attention for its widespread contamination of a variety of food and feed. Aspergillus ochraceus, Aspergillus carbonarius, and Penicillium nordicum are an important source of OTA in three different kinds of food commodities, including cereals, grape and dried fruit products, and dry-cured meat products. Deeper knowledge of OTA production and mycelium growth related to the high-sugar or NaCl-rich environments was gained in this manuscript. A. ochraceus and P. nordicum were likely to have greater growth rates in medium supplied with certain concentrations of NaCl (0-80 g/L), and the colony diameter was the largest at the salt content of 40 g/L. P. nordicum was more suitable to grow in NaCl-riched medium, the OTA production was increased to 316 ppb from 77 ppb when 20 g/L NaCl was added. The capability of OTA production was inhibited when salt content was 40 g/L and 60 g/L in A. ochraceus and P. nordicum, respectively. As the glucose content increased to 250 g/L, the capacity of mycelium growth and sporulation was increased significantly in A. ochraceus and A. carbonarius. A. carbonarius was more suitable to grow in high-sugar grape products. OTA production was significantly promoted with an added 100 g/L glucose in A. carbonarius. OTA production was inhibited when glucose content was 150 g/L and in 200 g/L in A. ochraceus and A. carbonarius, respectively. NaCl and glucose have an effect on fungal growth and OTA production, and the activation of biosynthetic genes of OtaA. These results would allow designing new strategies to prevent OTA accumulation on sugar or NaCl-riched foodstuffs and achieve the objective to manufacture cereals, dried vine fruits and dry-cured ham, free of OTA.

Application of surface-enhanced Raman spectroscopy in fast detection of toxic and harmful substances in food.

Surface-enhanced Raman spectroscopy (SERS) is being considered as a powerful technique in the area of food safety due to its rapidity, sensitivity, portability, and non-destructive features. This review aims to provide a comprehensive understanding of SERS applications in fast detection of toxic and harmful substances in food matrix. The enhancement mechanism of SERS, classification of active substrates, detection methods, and their advantages and disadvantages are briefly discussed in the review. The latest research progress of fast SERS detection of food-borne pathogens, mycotoxins, shellfish toxins, illegal food additives, and drug residues are highlighted in sections of the review. According to the current status of SERS detection of food-derived toxic and harmful substances, the review comes up with certain problems to be urgently resolved in SERS and brings up the perspectives on the future directions of SERS based biosensors.

In vitro prebiotic activities of oligosaccharides from the by-products in Ganoderma lucidum spore polysaccharide extraction.

Until recently, a variety of oligosaccharides from fruits, vegetables and mushrooms have demonstrated positive prebiotic effects. Ganoderma lucidum, a well-known traditional medicine and tonic in East Asia, has been utilized in the prevention and treatment of a broad range of illnesses. In this study, each of three oligosaccharides was obtained from the polysaccharide extraction by-products of sporoderm: the unbroken and broken spores of Ganoderma lucidum (UB-GLS, B-GLS). Their molecular weight distribution, monosaccharide composition and preliminary structures were analyzed using gel permeation chromatography (GPC), GC-MS, UV and FTIR, respectively. All of the oligosaccharides were found to exhibit prebiotic activities, evaluated by detecting growth stimulation on Lactobacillus in vitro. Among these, UB-O80 and B-O80 displayed the most significant effects (p < 0.05) in these groups, and UB-O80 showed higher resistance to hydrolysis by artificial human gastric juice compared with inulin, giving a maximum hydrolysis rate of 1.65%. Compared with inulin media, Lactobacillus also revealed high tolerance to lower pH levels and simulated gastric juices in UB-O80 and B-O80 media. Compared with a control in gut microbiota fermentation, the abundance of some beneficial bacteria increased and some harmful bacteria declined in the groups of UB-O80 and B-O80. In conclusion, the results suggest that GLS oligosaccharides could be exploited as promising prebiotics for the enhancement of human health.

Detection of Multiple Pathogens in Serum Using Silica-Encapsulated Nanotags in a Surface-Enhanced Raman Scattering-Based Immunoassay.

A robust immunoassay based on surface-enhanced Raman scattering (SERS) has been developed to simultaneously detect trace quantities of multiple pathogenic antigens from West Nile virus, Rift Valley fever virus, and Yersinia pestis in fetal bovine serum. Antigens were detected by capture with silica-encapsulated nanotags and magnetic nanoparticles conjugated with polyclonal antibodies. The magnetic pull-down resulted in aggregation of the immune complexes, and the silica-encapsulated nanotags provided distinct spectra corresponding to each antigen captured. The limit of detection was ∼10 pg/mL in 20% fetal bovine serum, a significant improvement over previous studies in terms of sensitivity, level of multiplexing, and medium complexity. This highly sensitive multiplex immunoassay platform provides a promising method to detect various antigens directly in crude serum samples without the tedious process of sample preparation, which is desirable for on-site diagnostic testing and real-time disease monitoring.

Surface-enhanced Raman scattering (SERS) detection of multiple viral antigens using magnetic capture of SERS-active nanoparticles.

A highly sensitive immunoassay based on surface-enhanced Raman scattering (SERS) spectroscopy has been developed for multiplex detection of surface envelope and capsid antigens of the viral zoonotic pathogens West Nile virus (WNV) and Rift Valley fever virus (RVFV). Detection was mediated by antibody recognition using Raman reporter-coated Au nanoparticles (GNPs) and paramagnetic nanoparticles (PMPs) conjugated with polyclonal antibodies specific for each antigen target, followed by 785nm laser excitation of magnetically concentrated GNP/antigen/PMP complexes. The discrimination of WNV and RVFV antigen detection in mixed Raman spectra was achieved by SERS enhancement of Raman spectra specific for the Raman reporter dyes Infrared 792 (IR-792) and Nile Blue (NB), respectively. Assay reactions containing dilutions of both target antigens yielded a reduction in the intensification of IR-792 and NB signature spectrum peaks and provided a conservative limit of detection of ∼5fg/ml for assays conducted in phosphate buffered saline buffer (PBS) and ∼25pg/ml for assays containing PBS spiked with fetal bovine serum. Based on the inherent simplicity of the assay, magnetic capture-based SERS assays afford promise as a biosensor platform that provides high-level multiplex detection sensitivity and can be adapted for portable diagnostic applications in limited resource settings.

A versatile SERS-based immunoassay for immunoglobulin detection using antigen-coated gold nanoparticles and malachite green-conjugated protein A/G.

A surface enhanced Raman scattering (SERS) immunoassay for antibody detection in serum is described in the present work. The developed assay is conducted in solution and utilizes Au nanoparticles coated with the envelope (E) protein of West Nile Virus (WNV) as the SERS-active substrate and malachite green (MG)-conjugated protein A/G (MG-pA/G) as a bi-functional Raman tag/antibody binding reporter. Upon incubation of these reagents with serum collected from rabbits inoculated with E antigen, laser interrogation of the sandwiched immunocomplex revealed a SERS signaling response diagnostic for MG. The intensification of signature spectral peaks is shown to be proportionate to the concentration of added serum and the limit of antibody detection is 2 ng/ml of serum. To assess assay performance relative to more a traditional immunoassay, indirect enzyme-linked immunosorbent assays conducted using the same concentrations of reagents were found to be >400-fold less sensitive. Quartz crystal microbalance with dissipation (QCM-D) monitoring of immunocomplex film deposition on solid Au surfaces also confirmed the formation of antigen-antibody-protein A/G trilayers and provided quantitative measurements of film thickness which likely position MG within the sensing distance of laser-elicited, enhanced electromagnetic fields. The sensitivity and inherent versatility of the assay, which is provided by the binding of pA/G to a broad spectrum of immunoglobulins in different mammalian species, suggest that it could be developed as an alternative immunoassay format to the ELISA.

Low dose streptozotocin (STZ) combined with high energy intake can effectively induce type 2 diabetes through altering the related gene expression.

High energy-intake is a major factor revolved in type 2 diabetes. A number of animal models have been adopted for studying the type 2 diabetes, but they differ greatly from human type 2 diabetes. The objectives of the present study are to set up a suitable animal model, which is similar to the human type 2 diabetes, and then to understand possible molecular mechanisms underlying type 2 diabetes. Twenty five-week-old Wistar male rats were randomized into four groups. One group was fed with basal diet (BD) whereas the others consumed high-energy diet (HD) of 20% sucrose and 10% lard. Four weeks later, BD and one of HD were sampled. Other groups continued to consume HD, but one of them was treated by one injection of streptozotocin (STZ) (30 mg/kg body weight). After another four weeks, all were sacrificed. Changes in body weight were recorded, and levels of glucose, TG, TC, LDL in serum were analyzed by standard methods. Moreover, expressions of genes related to energy metabolism in liver, muscle and fat were measured by real-time RT-PCR. HD had no notable differentiation with BD on bodyweight and serum indices, but it altered gene expressions in a tissue-specific manner. Two receptors of adiponectin, leptin, PPARgamma, UCP2 mRNA levels in fat were up regulated, whereas most of them were down regulated in liver. STZ treatment induced symptoms of diabetes, and the gene expression mentioned above exhibited changes in both tissue- and gene-specific manners. The results suggest that a combination of low dose STZ and high-energy intake can effectively induce type 2 diabetes by altering the related gene expressions in major metabolic tissues.