Pork Samples Research Articles

An intelligent indicating label using mulberry pigment, polylactic acid and rutile TiO2 nanoparticle for monitoring the freshness of cold fresh pork

The purpose of this work is to develop a new type of intelligent indicating label with high sensitivity to volatile bioamine, anti-ultraviolet light and anti-pigment leakage characteristics by electrospinning for real-time visual monitoring the freshness of cold fresh pork. By investigating pH discoloration sensitivity and light stability of plant pigments with different structures, mulberry pigment was selected as the indicator of indicating label. The content of label components (mulberry pigment, polylactic acid and TiO2 nanoparticle) significantly affected label performance (light stability, responsiveness to trimethylamine, and anti-leakage of mulberry pigment), and the optimal component content was determined to be 6.01% (w/w) of polylactic acid, 0.62% (w/w) of TiO2 nanoparticles and 0.29% (w/w) of mulberry pigment by response surface test based on label performance as response value. A close correlation (the correlation coefficient R values of 0.86–0.97) between the total color difference (ΔE) of the label and the freshness indexes (total plate count, total volatile basic nitrogen content, pH, thiobarbituric acid value and sensory score) of cold fresh pork during cold storage was revealed, and the freshness level of unknown pork samples could be well quantitatively predicted by partial least squares regression models with the determination coefficient (R2) values of 0.89–0.92, facilitating producers, dealers and consumers to know pork quality promptly.

Facile construction of sandwich ELISA based on double-nanobody for specific detection of α-hemolysin in food samples

α-hemolysin (Hla), a toxin secreted by Staphylococcus aureus (S. aureus), has been proved to be involved in the occurrence and aggravation of food poisoning. Hence, it is quite essential to establish its rapid detection methods to guarantee food safety. Sandwich ELISA based on nanobody is well known to be viable for toxins, but there is absence of nanobody against Hla, let alone a pair for it. Therefore, in this paper, we screened specific nanobodies by bio-panning and obtained the optimal nanobody pair for sandwich ELISA firstly. Then, RANbody, a novel nanobody owning both recognition and catalytic capability, is generated in a single step and at low cost through molecular recombination technology. Subsequently, sandwich ELISA was developed to detect Hla based on the nanobody and RANbody, that not only eliminated the use of secondary antibodies and animal-derived antibody, but also reduced detection time and cost, compared with traditional sandwich ELISA. Lastly, the performance has been evaluated, especially for specificity which showed no response to other hemolysins and a low limit of detection of 10 ng/mL. Besides, the proposed sandwich ELISA exhibits favorable feasibility and was successfully employed for the detection of Hla in milk and pork samples.

Rapid and non-destructive microbial quality prediction of fresh pork stored under modified atmospheres by using selected-ion flow-tube mass spectrometry and machine learning

Volatile organic compounds (VOCs) indicative of pork microbial spoilage can be quantified rapidly at trace levels using selected-ion flow-tube mass spectrometry (SIFT-MS). Packaging atmosphere is one of the factors influencing VOC production patterns during storage. On this basis, machine learning would help to process complex volatolomic data and predict pork microbial quality efficiently. This study focused on (1) investigating model generalizability based on different nested cross-validation settings, and (2) comparing the predictive power and feature importance of nine algorithms, including Artificial Neural Network (ANN), k-Nearest Neighbors, Support Vector Regression, Decision Tree, Partial Least Squares Regression, and four ensemble learning models. The datasets used contain 37 VOCs' concentrations (input) and total plate counts (TPC, output) of 350 pork samples with different storage times, including 225 pork loin samples stored under three high-O2 and three low-O2 conditions, and 125 commercially packaged products. An appropriate choice of cross-validation strategies resulted in trustworthy and relevant predictions. When trained on all possible selections of two high-O2 and two low-O2 conditions, ANNs produced satisfactory TPC predictions of unseen test scenarios (one high-O2 condition, one low-O2 condition, and the commercial products). ANN-based bagging outperformed other employed models, when TPC exceeded ca. 6 log CFU/g. VOCs including benzaldehyde, 3-methyl-1-butanol, ethanol and methyl mercaptan were identified with high feature importance. This elaborated case study illustrates great prospects of real-time detection techniques and machine learning in meat quality prediction. Further investigations on handling low VOC levels would enhance the model performance and decision making in commercial meat quality control.

Assessing the Effects of Cold Atmospheric Plasma on the Natural Microbiota and Quality of Pork during Storage.

Cold atmospheric plasma (CAP) is a novel non-thermal technology with significant potential for use in meat processing to prolong shelf life. The objective of the study was to evaluate the efficiency of CAP treatment on the natural microbiota and quality traits of pork stored for 8 days at 4 °C. CAP treatment was applied by employing piezoelectric direct discharge technology to treat pork samples for 0, 3, 6, and 9 min. Reductions of approximately 0.8-1.7 log CFU/g were observed in total viable counts (TVC) and Pseudomonas spp. levels for CAP treatments longer than 3 min, immediately after treatment. A storage study revealed that CAP-treated pork (>6 min) had significantly lower levels of TVC, Pseudomonas spp., and Enterobacteriaceae throughout storage. Regarding quality traits, CAP application for longer than 3 min significantly increased water retention and yellowness and decreased meat redness compared to untreated pork. However, other parameters such as pH, tenderness, and lightness exhibited no statistically significant differences between untreated and CAP-treated pork. Lipid oxidation levels were higher only for the 9-min treatment compared to untreated pork. Our results revealed that CAP is a promising technology that can extend the microbiological shelf life of pork during refrigeration storage.

Successful Primer Picking and Pooling for the Design of Multiplex PCR Primers Specific to Pork, Beef, Chicken, and Rat DNA

DNA markers and Multiplex-PCR have emerged as methods for species detection in processed meat products. The primary objective of this study is to design multiplex primer sequences for pork, rat, beef, and chicken, generating distinguishable amplicons through agarose gel electrophoresis for halal detection in processed meat products. Primer design involved utilizing mitochondrial genomic data and the NCBI-Primer BLAST site to obtain specific pork and beef primer sequences. In silico simulations, including single and multiplex-PCR, were conducted using Primer Pooler. In vitro validation encompassed Single-PCR and Multiplex-PCR annealing temperature optimization, using samples of chicken, beef, pork, and rat as well as processed meat products like meatballs, sausages, and nuggets. In vitro validation demonstrated that the halal marker gene's multiplex primer efficiently amplified the target sequence, specifically at the optimal annealing temperature of 58°C. Amplicons from beef (1,217 bp), pork (860 bp), rat (622 bp), and chicken (272 bp) primers could be distinguished on a 1.5% agarose gel. The study's results can aid in cost-effective and rapid halal testing and authentication of processed meat products, offering advantages over PCR with a single primer.

Genetic Characteristics of Extended-Spectrum Beta-Lactamase-Producing Salmonella Isolated from Retail Meats in South Korea.

Earlier studies have validated the isolation of extended-spectrum beta-lactamase-producing Salmonella (ESBL-Sal) strains from food. While poultry is recognized as a reservoir for Salmonella contamination, pertinent data regarding ESBL-Sal remains limited. Consequently, the Ministry of Food and Drug Safety has isolated Salmonella spp. from retail meat and evaluated their antibiotic susceptibility and genetic characteristics via whole-genome sequencing. To further elucidate these aspects, this study investigates the prevalence, antibiotic resistance profiles, genomic characteristics, and homology of ESBL-Sal spp. obtained from livestock-derived products in South Korean retail outlets. A total of 653 Salmonella spp. were isolated from 1,876 meat samples, including 509 beef, 503 pork, 555 chicken, and 309 duck samples. The prevalence rates of Salmonella were 0.0%, 1.4%, 17.5%, and 28.2% in the beef, pork, chicken, and duck samples, respectively. ESBL-Sal was exclusively identified in poultry meat, with a prevalence of 1.4% in the chicken samples (8/555) and 0.3% in the duck samples (1/309). All ESBL-Sal strains carried the blaCTX-M-1 gene and exhibited resistance to ampicillin, ceftiofur, ceftazidime, nalidixic acid, and tetracycline. Eight ESBL-Sal isolates were identified as S. Enteritidis with sequence type (ST) 11. The major plasmid replicons of the Enteritidis-ST11 strains were IncFIB(S) and IncFII(S), carrying antimicrobial resistance genes (β-lactam, tetracycline, and aminoglycoside) and 166 virulence factor genes. The results of this study provide valuable insights for the surveillance and monitoring of ESBL-Sal in South Korean food chain.

Development of a closed-tube, calcein-based loop-mediated isothermal amplification assay to detect Salmonella spp. in raw meat samples

Foodborne pathogens compromise food safety and public health, and Salmonella spp. are among the major pathogenic bacteria that cause outbreaks worldwide. Proper surveillance through timely and cost-effective detection methods across the food animal production chain is crucial to prevent Salmonella outbreaks and agricultural losses. Traditional culture methods are labor- and resource-intensive, with lengthy turnaround times. Meanwhile, conventional molecular tools, such as PCR and qPCR, are expensive and require technical skills and equipment. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, inexpensive, highly sensitive, and specific molecular assay that does not require expensive equipment. Hence, this study developed and optimized a closed-tube, calcein-based LAMP assay to detect Salmonella using the invA gene and performed evaluation and validation against conventional PCR. The LAMP assay showed high specificity and sensitivity. It showed 10-fold higher sensitivity than conventional PCR, at <1 ng/μL DNA concentrations. Meanwhile, for CFU/mL, LAMP assay showed 1000-fold higher sensitivity than conventional PCR at 4.8 × 103 cells/mL than 4.8 × 107 cells/mL, respectively. For parallel testing of 341 raw meat samples, after conventional culture enrichment (until Rappaport-Vassiliadis broth), the optimized LAMP assay showed 100% detection on all samples while conventional PCR showed 100%, 99.04%, and 96.64% for raw chicken, beef, and pork samples, respectively. Meanwhile, a shortened enrichment protocol involving 3-h incubation in buffered peptone water only, showed lower accuracy in tandem with the optimized LAMP assay ranging from 55 to 75% positivity rates among samples. These suggest that the optimized LAMP assay possesses higher sensitivity over conventional PCR for invA gene detection when coupled with conventional enrichment culture methods. Hence, this assay has potential as a powerful complementary or alternative Salmonella detection method to increase surveillance capacity and protect consumer food safety and public health worldwide.

Development of an Open Droplet Microchannel-Based Magnetosensor for Immunofluorometric Assay of Trimethoprim in Chicken and Pork Samples with a Wide Linear Range.

Trimethoprim (TMP), functioning as a synergistic antibacterial agent, is utilized in diagnosing and treating diseases affecting livestock and poultry. Human consumption of the medication indirectly may lead to its drug accumulation in the body and increase drug resistance due to its prolonged metabolic duration in livestock and poultry, presenting significant health hazards. Most reported immunoassay techniques, such as ELISA and immunochromatographic assay (ICA), find it challenging to achieve the dual advantages of high sensitivity, simplicity of operation, and a wide detection range. Consequently, an open droplet microchannel-based magnetosensor for immunofluorometric assay (OMM-IFA) of trimethoprim was created, featuring a gel imager to provide a signal output derived from the highly specific antibody (Ab) targeting trimethoprim. The method exhibited high sensitivity in chicken and pork samples, with LODs of 0.300 and 0.017 ng/mL, respectively, and a wide linear range, covering trimethoprim's total maximum residue limits (MRLs). Additionally, the spiked recoveries in chicken and pork specimens varied between 81.6% and 107.9%, maintaining an acceptable variation coefficient below 15%, aligning well with the findings from the ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technique. The developed method achieved a much wider linear range of about 5 orders of magnitude of 10-2-103 levels with grayscale signals as the output signal, which exhibited high sensitivity, excellent applicability and simple operability based on magnetic automation.

Determination of erythromycin, clarithromycin and N-desmethyl-erythromycin A residues in pork, beef and lamb based on a simple and fast extraction procedure followed by ultrahigh-performance liquid chromatography with triple quadrupole/linear ion trap mass spectrometry

A modified quick, easy, cheap, effective, rugged and safe (QuEChERS) method was developed for the simultaneous determination of erythromycin (ERY), clarithromycin (CLA) and N-desmethyl-erythromycin A (N-D-ERY) in pork, beef and lamb via ultrahigh-performance liquid chromatography with triple quadrupole/linear ion trap mass spectrometry (UHPLC-QTRAP-MS/MS). The sample preprocessing and detection conditions were optimized, and the obtained parameters of all target compounds met the EU and FDA requirements. In the sample preprocessing procedure, acetonitrile-water (80:20, v/v) was used as an extractant, and a Cleanert MAS-Q cartridge was utilized and achieved good extraction recovery. The matrix-matched calibration curves showed good linearities, with determination coefficients of 0.9983–0.9999. The recoveries ranged from 88.96% to 101.76% with relative standard deviations (RSDs) ≤5.95%, and the intraday and interday RSDs ranged from 2.64% to 6.65% and 3.08–6.38%, respectively. The limits of detection and quantification for the pork, beef and lamb samples were 0.4 μg/kg and 2.0 μg/kg, respectively. The detection limits and detection capabilities were in the range of 2.15–215.16 μg/kg and 2.26–230.32 μg/kg, respectively. This method was successfully applied to the analysis of the pork, beef and lamb samples, demonstrating its applicability and suitability for the routine analysis of ERY, CLA and N-D-ERY residues in meat samples.

In Situ Fabrication of Photoluminescent Hydrogen-Bonded Organic Framework-Functionalized Ca (II) Hydrogel Film for the Tetracyclines Visual Sensor and Information Security.

A facilely in situ fabricated hydrogen-bonded organic framework (HOF) hydrogel film with perfect photoluminescent performance was designed for visual sensing of tetracycline antibiotics (TCs) and information security. Luminescent HOF (MA-IPA) was combined with sodium alginate (SA) through hydrogen bonding actions and electrostatic interactions, then cross-linked with Ca2+ ions to form HOF hydrogel film (Ca@MA-IPA@SA). The HOF hydrogel film exhibited exceptional mechanical robustness along with stable blue fluorescence and ultralong green phosphorescence. After exposure to TCs, Ca2+ was combined with TCs to generate a new green fluorescence exciplex (TC-Ca2+) in hydrogel films. Due to fluorescence resonance energy transfer, the fluorescence of MA-IPA was quenched, and the fluorescent color of the HOF hydrogel film was changed from blue to green. This dichromatic fluorescent response is convenient for the visual and rapid detection of TCs. The detection limits of tetracycline (TC), oxytetracycline (OTC), and chlortetracycline (CTC) were 5.1, 7.7, and 32.7 ng mL-1, respectively. Importantly, this hydrogel sensing platform was free of tedious operation and enabled the ultrasensitive and selective detection of TCs within 6 min. It has been successfully applied to TC detection in pork and milk samples. Based on the stable photoluminescence performance of HOF hydrogel films and fluorescent-responsive properties to TCs, two types of anticounterfeiting arrays were fabricated for information encryption and decryption. This work provides a novel approach for on-site detection of TCs and offers valuable insights into information security.

SERS detection of volatile gas in spoiled pork with the Ag/MoS2 nano-flower cavity/PVDF micron-bowl cavity (FIB) substrate.

Putrescine and cadaverine are significant volatile indicators used to assess the degree of food spoilage. Herein, we propose a micro-nano multi cavity structure for surface-enhanced Raman spectroscopy (SERS) to analyze the volatile gas putrescine and cadaverine in decomposing food. The MoS2 nano-flowers are inserted into a PVDF micro-cavity through in-situ growth, followed by vacuum evaporation technology of Ag nanoparticles to form an Ag/MoS2 nano-flower cavity/PVDF micron-bowl cavity (FIB) substrate. The micro-nano multi cavity structure can improve the capture capacity of both light and gas, thereby exhibiting high sensitivity (EF = 7.71 × 107) and excellent capability for gas detection of 2-naphthalenethiol. The SERS detections of the putrescine and cadaverine are achieved in the spoiled pork samples with the FIB substrate. Therefore, this substrate can provide an efficient, accurate, and feasible method for the specific and quantitative detection in the food safety field.

Development of triplex assay for simultaneous detection of Escherichia coli, methicillin resistant and sensitive Staphylococcus aureus in raw pork samples of retail markets.

Escherichia coli and Staphylococcus aureus are the most important food borne pathogen transmitting from animal meat and meat products. Therefore, it is vital to design an accurate and specific diagnostic tool for identifying those food-borne pathogens in animal meat and meat products. In the current study, E. coli, methicillin-resistant and sensitive S. aureus (MRSA and MSSA) were simultaneously detected using a developed triplex PCR-based technique. To obtain an optimal reaction parameter, the multiplex assay was optimised by changing just one parameter while holding the others constant. Specificity of the assay was assessed using several porcine bacterial template DNA. The plasmid DNA was used to test the multiplex PCR assay's sensitivity and interference in spiked pork samples. E. coli, MRSA, and MSSA each have PCR amplified products with sizes of 335, 533, and 209bp, respectively. The assay detects a minimum microbial load of 102CFU/μl for all the three pathogens and can identify bacterial DNA as low as 10-2ng/µl. The assay was validated employing 210 pork samples obtained from retail meat shops and slaughter houses, with MRSA, E. coli, and MSSA with the occurrence rate of 1.9%, 42.38%, and 18.1%, respectively. The rate of mixed bacterial contamination in pork meat samples examined with the developed method was 6.19%, 1.43%, 1.90%, and 1.43% for MSSA & E. coli, MRSA & E. coli, MSSA & MRSA, and E. coli, MSSA & MRSA, respectively. The developed multiplex PCR assay is quick and efficient, and it can distinguish between different bacterial pathogens in a single reaction tube.

Trifunctional Nanocomposites with Colorimetric Magnetic Catalytic Activities Labels in Sandwich Immunochromatographic Detection of Escherichia coli O157:H7.

The emergence of nanoenzymes has catalyzed the robust advancement of the lateral flow immunoassay (LFIA) in recent years. Among them, multifunctional nanocomposite enzymes with core-shell architectures are considered preferable for promoting the sensing ability due to their good biocompatibility, precise control over size, and surface properties etc. Herein, we developed a dual-channel ensured lateral flow immunoassay (DFLIA) platform utilizing a magnetic, colorimetric, and catalytic multifunctional nanocomposite enzyme (Fe3O4@TCPP@Pd) [TCPP, Tetrakis (4-carboxyphenyl) porphyrin] for the ultrasensitive and highly accurate rapid detection of Escherichia coli O157:H7 (E. coli O157:H7). Fe3O4@TCPP@Pd-mAb exhibits superior performance compared to traditional AuNPs, including enhanced sensitivity and an extended linear detection range, benefiting from its high brightness signal, strong magnetic separation ability, and high peroxidase activity (Vmax = 2.32 μM S1-). Moreover, the Fe3O4@TCPP@Pd-labeled mAb probe exhibited exceptional stability and high affinity toward E. coli O157:H7 (with an affinity constant of approximately 1.723 × 109 M-1), indicating its potential for the efficient capture of the pathogen. Impressively, the developed Fe3O4@TCPP@Pd-DFLIA achieved ultrasensitive detection for E. coli O157:H7 with pre- and postcatalytic naked-eye detection sensitivities of 255 cfu/mL and 77 cfu/mL, respectively, representing an approximately 41-fold improvement over the conventional AuNP-based LFIA and also possessed good specificity and reproducibility [relative standard deviation (RSD) < 10%]. Additionally, the established DFLIA exhibited satisfactory recoveries in detecting pork and milk samples, further validating the reliability of this platform for immunoassays and demonstrating its potential for utilization in bioassays and clinical diagnostics.

A cheaper substitute for HRP: ultra-small Cu-Au bimetallic enzyme mimics with infinitesimal steric hindrance to promote catalytic lateral flow immunodetection of clenbuterol.

A highly sensitive lateral flow immunoassay (LFIA) is developed for the enzyme-catalyzed and double-reading determination of clenbuterol (CLE), in which a new type of probe was adopted through the direct electrostatic adsorption of ultra-small copper-gold bimetallic enzyme mimics (USCGs) and monoclonal antibodies. In the assay, based on the peroxidase activity of USCG, the chromogenic substrate TMB-H2O2 was introduced to trigger its color development, and the results were compared with those before catalysis. The detection sensitivity after catalysis is 0.03 ng mL-1 under optimal circumstances, which is 6-fold better than that of the traditional Au NPs-based LFIA and 2-fold greater than that before catalysis. This approach was successfully applied to the detection of CLE in milk, pork and mutton samples with an optimum assay time of 7 min and best catalytic time of 80 s, after which satisfactory recoveries of 98.53-117.79% were obtained. Cu-Au nanoparticles as a signal tag and the use of their nanozyme properties are the first applications in the field of LFIA. This work can be a promising exhibition for the application of a cheaper substitute for HRP, ultra-small bimetallic enzyme mimics, in LFIAs.

Distribution and spread of tigecycline resistance gene tet(X4) in Escherichia coli from different sources.

Tigecycline serves as a last-resort antimicrobial agent against severe infections caused by multidrug-resistant bacteria. Tet(X) and its numerous variants encoding flavin-dependent monooxygenase can confer resistance to tigecycline, with tet(X4) being the most prevalent variant. This study aims to investigate the prevalence and characterize tigecycline resistance gene tet(X) in E. coli isolates from various origins in Yangzhou, China, to provide insights into tet(X) dissemination in this region. In 2022, we tested the presence of tet(X) in 618 E. coli isolates collected from diverse sources, including patients, pig-related samples, chicken-related samples, and vegetables in Yangzhou, China. The antimicrobial susceptibility of tet(X)-positive E. coli isolates was conducted using the agar dilution method or the broth microdilution method. Whole genome sequencing was performed on tet(X)-positive strains using Illumina and Oxford Nanopore platforms. Four isolates from pig or pork samples carried tet(X4) and exhibited resistance to multiple antimicrobial agents, including tigecycline. They were classified as ST542, ST10, ST761, and ST48, respectively. The tet(X4) gene was located on IncFIA8-IncHI1/ST17 (n=2), IncFIA18-IncFIB(K)-IncX1 (n=1), and IncX1 (n=1) plasmids, respectively. These tet(X4)-carrying plasmids exhibited high similarity to other tet(X4)-bearing plasmids with the same incompatible types found in diverse sources in China. They shared related genetic environments of tet(X4) associated with ISCR2, as observed in the first identified tet(X4)-bearing plasmid p47EC. In conclusion, although a low prevalence (0.65%) of tet(X) in E. coli strains was observed in this study, the horizontal transfer of tet(X4) among E. coli isolates mediated by pandemic plasmids and the mobile element ISCR2 raises great concerns. Thus, heightened surveillance and immediate action are imperative to curb this clinically significant resistance gene and preserve the efficacy of tigecycline.

Investigating the mechanistic impact of pork soft tissue preparation techniques on the classification precision of laser-induced breakdown spectroscopy.

The investigation of the mechanism underlying the impact of biological soft tissue sample preparation methods on laser-induced breakdown spectroscopy (LIBS) signals can enhance the stability of LIBS signals. Our study focused on four specific preparation methods applied to pork samples: rapid freezing, fresh slicing, drying, and pressing. The influence of various preparation techniques on the signal-to-noise ratio and fluctuation of Ca, Na, Mg, and CN bands within the sample spectra was assessed. The signal-to-noise ratios for samples that were dried and pressed notably improved. And the pressing method effectively mitigated the uneven distribution of pork tissue components, displaying superior spectral line stability. To explain this phenomenon, we used the Saha-Boltzmann diagram to estimate the plasma temperature. Remarkably, there was a significant reduction in plasma temperature fluctuations across four pressed samples, with a standard deviation of 108.53. Furthermore, we undertook a classification analysis employing support vector machine models to corroborate the generalization efficacy of the sample preparation technique. Dried and pressed samples demonstrated notably higher classification accuracy, precision, and recall (all >93%) compared to frozen and fresh samples, where these metrics remained below 86%. The performance of the SVM model was ultimately evaluated using Receiver Operating Characteristic (ROC) curves and the Area Under the Curve (AUC). The AUC for the frozen, fresh, dried, and pressed samples was 0.854, 0.907, 0.989, and 0.996, respectively. The findings revealed that the pressing method exhibited superior performance, followed by drying, fresh slicing, and freezing, in descending order of effectiveness.

Changes in the Microbiota from Fresh to Spoiled Meat, Determined by Culture and 16S rRNA Analysis

Growth of meat microbiota usually results in spoilage of meat that can be perceived by consumers due to sensory changes. However, a high bacterial load does not necessarily result in sensory deviation of meat; nevertheless, this meat is considered unfit for human consumption. Therefore, the aims of this study were to investigate changes in the microbiota from fresh to spoiled meat and whether the proportions of certain bacteria can probably be used to indicate the hygiene status of meat. For this purpose, 12 fresh pork samples were divided into two groups, and simultaneously aerobically stored at 4°C and 22°C. At each time-temperature point (fresh meat, days 6, 13, and 20 at 4°C, and days 1, 2, 3, and 6 at 22°C), 12 meat subsamples were investigated. Sequences obtained from next-generation sequencing (NGS) were further analyzed down to species level. Plate counting of six bacterial groups and NGS results showed that Pseudomonas spp. and lactic acid bacteria (LAB) were found in a high proportion in all stored meat samples and can therefore be considered as important “spoilage indicator bacteria”. On the contrary, sequences belonging to Staphylococcus epidermidis were found in a relatively high proportion in almost all fresh meat samples but were less common in stored meat. In this context, they can be considered as “hygiene indicator bacteria” of meat. Based on these findings, the proportion of the “hygiene indicator bacteria” in relation to the “spoilage indicator bacteria” was calculated to determine a “hygiene index” of meat. This index has a moderate to strong correlation to bacterial loads obtained from culture (p < 0.05), specifically to Pseudomonas spp., LAB and total viable counts (TVCs). Knowledge of the proportions of hygiene and spoilage indicator bacteria obtained by NGS could help to determine the hygiene status even of (heat-) processed composite meat products for the first time, thus enhancing food quality assurance and consumer protection.

Novel electrochemical sensing strategy for ultrasensitive detection of tetracycline based on porphyrin/metal phthalocyanine-covalent organic framework

In this work, a novel two-dimensional semiconducting metal covalent organic framework (CuTAPc-TFPP-COF) was synthesized and used as biosensing platform to construct aptasensor for trace detection of tetracycline (TC). The CuTAPc-TFPP-COF integrates the highly conjugated structure, large specific surface area, high porosity, abundant nitrogen functional groups, excellent electrochemical activity, and strong bioaffinity for aptamers, providing abundant active sites to effectively anchor aptamer strands. As a result, the CuTAPc-TFPP-COF-based aptasensor shows high sensitivity for detecting TC via specific recognition between aptamer and TC to form Apt-TC complex. An ultralow detection limit of 59.6 fM is deduced from the electrochemical impedance spectroscopy within a wide linear range of 0.1–100000 pM for TC. The CuTAPc-TFPP-COF-based aptasensor also exhibits good selectivity, reproducibility, stability, regenerability, and excellent applicability for real river water, milk, and pork samples. Therefore, the CuTAPc-TFPP-COF-based aptasensor will be promising for detecting trace harmful antibiotics residues in environmental water and food samples.

Development of Filtering Electrospray Ionization-Ion Trap Mass Spectrometry Technique for the Rapid Detection of Antibiotic Residues in Food

This article reports on the development of an atmospheric pressure filtering electrospray ionization (FESI) source – composed of a syringe, a filter, and the ESI source – for the determination of furazolidone, amoxicillin, nitrofurantoin, and nitrofural, common antibiotics. Spiked food samples were also analyzed; the analytes were placed in the filter head, and the flow rate of the extraction solution from the syringe pump was adjusted. Using a mixture of oleic acid and antibiotics, hydrophilic and hydrophobic filters were compared, demonstrating the advantage of FESI in reducing the background and noise for the mixture. The device simplifies not only the experimental procedure but also the sample pretreatment process. Additionally, the limits of detection of the antibiotics were determined to be 1 mg L−1 for furazolidone, amoxicillin, and nitrofurantoin and 2 mg L−1 for nitrofural. The linear regression coefficients (R 2) of the antibiotic standard solutions were all ≥0.99. Furthermore, the spiked recovery rates of pork, chicken, and egg samples ranged from 73.3% to 107.5%, with relative standard deviations (RSDs) from 1.1% to 15.6%. This study uncovers the potential capabilities of ion trap mass spectrometry (ITMS) for the on-site detection of antibiotics and offers a new method for ensuring food safety.

PH-responsive color-indicating film of pea protein isolate cross-linked with dialdehyde carboxylated cellulose nanofibers for pork freshness monitoring

The limited mechanical performance and responsiveness of protein-based smart packaging materials have hindered their development. To address these issues, this study prepared a pH-responsive smart film by introducing dialdehyde carboxylated cellulose nanofibers (DCCNFs) as the cross-linking agent capable of covalently reacting with proteins, and bilberry extract (BE) as a pH-responsive indicator into pea protein isolate (PPI) matrix. The results demonstrated that adding DCCNF and BE enhanced the PPI film's thermal stability, density, and UV barrier properties. Tensile tests revealed significant improvements in both tensile strength and elongation at the break for the resulting film. Furthermore, films containing DCCNF and BE exhibited lower moisture content, swelling ratio, water vapor permeability, and relative oxygen transmission compared to PPI films. Notably, the anthocyanins in BE endowed the film with visual color changes corresponding to different pH values. This feature enabled the film to monitor pork freshness; a transition from acidic to alkaline in pork samples was accompanied by a color change from brown to brownish green in the film as storage time increased. Overall, these findings highlight that this developed film possesses excellent physicochemical properties and sensitive pH response capabilities, making it a promising candidate for future smart packaging applications.