Production and characterization of a monoclonal antibody against recombinant thermolabile hemolysin and its application to screen for Vibrio parahaemolyticus contamination in raw seafood

Thermolabile hemolysin gene (tlh) is regarded as a species-specific marker for Vibrio parahaemolyticus. To assess the utility of the tlh gene product, thermolabile hemolysin (TLH), as a marker to screen for V. parahaemolyticus-contamination in raw seafood, we generated a monoclonal antibody (MAb) against recombinant TLH and established with an enzyme-linked immunosorbent assay using the MAb (TLH-ELISA). TLH-ELISA testing of broth cultures for 78 V. parahaemolyticus strains showed positive results all around. In contrast, most broth cultures of 53 non-V. parahaemolyticus species tested yielded negative results.We devised a screening method using TLH-ELISA to check for low-level contamination of V. parahaemolyticus in raw seafood within 24 h and evaluated its ability. In testing of V. parahaemolyticus-spiked raw seafood, results suggested that our screening method can detect 100 most-probable-number (MPN) of V. parahaemolyticus/g. Further, on testing 119 commercial raw seafood samples with our screening method, 117 samples were determined to contain less than 100 MPN of V. parahaemolyticus/g. All of the 117 samples were also estimated by the MPN method to contain less than 100 MPN of V. parahaemolyticus/g. Taken together, these results suggest that our screening method using TLH-ELISA is useful to check for low-level (<100 MPN/g) of V. parahaemolyticus in raw seafood.     

Novel monoclonal antibody-based sensitive enzyme-linked immunosorbent assay and rapid immunochromatographic strip for detecting aflatoxin M1 in milk

Monoclonal antibody (mAb) that is specific to AFM1 was generated from the hybridoma cell line, 10F3C10, which was obtained by the fusion of mouse NS1 myeloma cells with the spleen cells of mouse that had been immunized with AFM1-bovine serum albumin (BSA). The 10F3C10 mAb is belong to the immunoglobulin G1 isotype. Both competitive direct and indirect enzyme-linked immunosorbent assay (ELISA) was utilized to characterize the mAb for AFM1. The concentrations of AFM1, AFB1 and AFG1 that caused 50% inhibition (IC₅₀) of the binding of AFM1-horseradish peroxidase (AFM1-HRP) to the antibody were found to be 0.022, 0.310 and 2.12 ng/mL, respectively. The immunochromatographic strip (immunostrip) assay with mAb-gold nanoparticle conjugates as a detection marker exhibited a visual limit of detection of 0.1 ng/mL for AFM1 and the analysis took a total of 10 min. Closely examining 17 milk-based samples using cdELISA revealed that four were slightly contaminated with AFM1 at concentrations from 0.002 to 0.054 ng/mL. All milk samples were negative in the immunostrip test because the levels of contaminant were below the detection limit of the strip. Notably, the presented cdELISA and immunostrip methods are highly sensitive methods for detecting AFM1 in milk.     

Development of novel monoclonal antibodies-based ultrasensitive enzyme-linked immunosorbent assay and rapid immunochromatographic strip for aflatoxin B1 detection

Monoclonal antibodies (mAbs) that are specific to aflatoxin B1 (AFB1) were produced from hybridoma cell lines 3F6G11 and 9C7C11 by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells that were isolated from a BALB/c mouse that was immunized with AFB1-bovine serum albumin (BSA). Both 3F6G11 and 9C7C11 mAbs are the immunoglobulin G1 isotypes. Competitive direct enzyme-linked immunosorbent assays (cdELISA) were established to characterize these antibodies. In the 3F6G11 mAb based cdELISA, the concentrations causing 50% inhibition of binding of AFB1-horseradish peroxidase to the antibody by AFB1, AFB2, AFG1, and AFG2 were found to be 0.051, 0.050, 1.820, and 1.270 ng/ml, respectively. Using 9C7C11 mAbs, similar IC₅₀ values for AFB1, AFB2, AFG1 and AFG2 were obtained as 0.045, 0.057, 2.530 and 2.120 ng/ml, respectively. A rapid and sensitive gold nanoparticle immunochromatographic strip (immunostrip) was also established for these antibodies. This strip has a detection limit of 1.0 ng/ml for AFB1 and the whole assay can be completed within 10 min. Extensive analysis of 20 samples by 3F6G11 mAb and 9C7C11 mAbs cdELISAs revealed that six samples were slightly contaminated by AFB1 at concentrations from 0.160 to 16.10 ng/g. Results of analyses of 20 samples with an immunostrip assay correlate well with those obtained using cdELISA. The proposed cdELISA and immunostrip methods are highly sensitive for the rapid screening of AFB1 in food samples.     

Comparison of real-time PCR and ELISA-based methods for the detection of beef and pork in processed meat products

Two commonly used methodologies for species detection within processed meat products are real-time polymerase chain reaction (PCR), a DNA-based method, and enzyme-linked immunosorbent assay (ELISA), a protein-based method. In this study, a real-time PCR assay was compared to a commercial ELISA kit based on sensitivity, specificity, agreement among duplicate samples, cost, time, and ease of use. Fifteen reference samples containing known percentages (0.1–99.9%, w/w) of pork and beef were analyzed in duplicate using both methods. Thirty commercial products, including sausages, pet treats, and canned meats, were also tested in duplicate with each method. Reference sample analysis showed real-time PCR was able to detect pork in duplicate samples at 0.10% and beef at 0.50% in the binary mixtures. ELISA detected pork in duplicate samples at 10.0% and beef at 1.00% in the binary mixtures. When the results of reference and commercial samples were combined, real-time PCR demonstrated the greatest agreement among duplicate samples, at 96.7%, compared to 95.6% agreement for ELISA. The real-time PCR assay used in this study was found to be less expensive, while ELISA was less time-consuming and easier to perform. Both methods were successful at identifying species in ground meats, sausage, and deli meat samples; however, pet treats and canned meats proved more challenging. Overall, it was determined that the real-time PCR assay was optimal for species identification in processed meat products when a low detection limit is required; however, the ELISA kit may be advantageous in other situations due to its ease of use.     

Ultrasonic characterization and online monitoring of pork meat dry salting process

Bearing in mind the highly variable salt content in dry-cured meat products with anatomical integrity, such as pork loin or ham, non-destructive salt content characterization and the online monitoring of dry salting are highly relevant for industrial purposes. This study explores the ability of low-intensity ultrasound to monitor the dry salting of pork Biceps femoris (BF) and Longissimus dorsi (LD) online, as well as to estimate the salt content, both in these muscles and in hams. For this purpose, meat samples were dry salted for up to 16 d at 2 °C. During the salting of the muscles, the ultrasonic velocity was continuously measured at time intervals of 5 min, while in the hams it was measured before and after salting. The ultrasonic velocity increased progressively during the salting due to salt gain and water loss, reaching a velocity variation (ΔV) of 46.8 m/s after 16 d of dry salting for hams and 59.5 and 30.6 m/s after 48 h of dry salting for LD and BF, respectively. Accurate correlations between salt gain and ΔV were obtained (R² = 0.903 in LD-BF muscles and R² = 0.758 in hams), which allowed the assessment of the salt content with an average estimation error of 0.4% w.b. for both muscles and hams. Further research should investigate the use of the time of flight obtained through the pulse-echo mode, instead of the ultrasonic velocity, in order to improve the industrial applicability.     

Development of a new broad-specific monoclonal antibody with uniform affinity for aflatoxins and magnetic beads-based enzymatic immunoassay

To reduce the incidence of false-positive and false-negative results caused by high or low cross-reactivity (CR%) values of the antibodies for total aflatoxins (AFs, AFB₁+AFB₂+AFG₁+AFG₂) detection, a new broad-specific monoclonal antibody (MAb) with uniform affinity, named 5H3, was developed. Moreover, magnetic beads (MBs) replaced microplates as immobile phase to improve the sensitivity of the enzymatic immunoassay. Then, a direct competitive enzyme-linked immunosorbent assay (ELISA) based on MBs (MBs-dcELISA) that could simultaneously detect the total AFs with similar sensitivity was developed. Following optimization of conditions, the half maximal inhibitory concentrations (IC₅₀) of the MBs-dcELISA in buffer were 0.05 ng/mL for AFB₁, 0.04 ng/mL for AFB₂, 0.05 ng/mL for AFG₁, 0.06 ng/mL for AFG₂. The corresponding CR% values were 100%, 125%, 100% and 83.3%, respectively. The limit of detection (LOD) of the MBs-dcELISA for the total AFs was 0.21 ng/g with a working range from 0.22 ng/g to 19.8 ng/g, and the recoveries for the total AFs ranged from 74.5% to 96.5% with coefficients of variation (CV) under 12.1% in spiked maize samples. In addition, the MBs-dcELISA was more sensitive than the conventional dcELISA. Finally, the MBs-dcELISA was applied to screen 9 naturally contaminated maize samples and 6 spiked samples and the results indicated a good agreement with that obtain by HPLC-MS/MS method.     

Effect of pH,temperature and storage time on the stability of bovine myelin basic protein

Bovine central nervous system tissue (CNT) harboring highest levels of the infectious agent for prion diseases, has been banned from food and feed supplies. Effective detection of CNT in excessively processed meat and feedstuffs requires the assay based on a stable marker in CNT as the analyte. Myelin basic protein (MBP), the major central nervous system (CNS) myelin protein, was reported to be detectable up to 115 °C in the literature. This study further investigated the effects of pH, temperature and storage time on the stability of purified bovine MBP (18.5 kDa) in solutions. Purified MBP dissolved in 10 mM PBS was adjusted to pH 3.0, 7.2 and 10.5, respectively. Sample at each pH was subjected to three heat treatments, 100 °C, 121 °C, and 133 °C for 30 min. The unheated and 100 °C heated samples at pH 7.2 were stored at 4 °C for 19 days to study the storage stability of bovine MBP. The immunoreactivity of all samples was examined using indirect non-competitive enzyme-linked immunosorbent assay and immunoblotting with an anti-MBP monoclonal antibody. The protein degradation was observed by analyzing samples using SDS-PAGE. Results show that bovine MBP was most stable at pH 7.2 while the protein was least stable at pH 10.5. MBP started to degrade after heating at 121 °C for 30 min at pH 7.2. Storage up to 19 days at 4 °C did not significantly affect the immunoreactivity of unheated and heated samples. However, unheated MBP samples started to show degradation from day 8 of storage; while no degradation was observed in 100 °C heated samples over the storage time. This study demonstrated that bovine MBP can be a suitable marker for the detection of bovine CNT in highly processed food and feed supplies.     

Mass spectrometry quantification of beef and pork meat in highly processed food: Application on Bolognese sauce

Food frauds have become a very important issue in the field of food quality and safety. The risk of food adulteration is higher in highly processed food and mainly affects high added value foodstuff. The methods currently available to face this issue, PCR and ELISA, are very sensitive and specific, but they have some limitations. In the present work, tandem mass spectrometry is presented as an emerging approach to detect beef and pork meat in very complex and highly processed food matrices, such as Bolognese sauce, both in qualitative than in quantitative way. The detection is achieved using two different marker peptides, specific for beef and pork meat, both deriving from α2-collagen chain. Then, a calibration curve is set up using real sauces made by different percentages of pork and beef meat in a working range from 0 to 100%. The method here developed allows to quantify beef and pork meat in a complex product such as Bolognese sauce.     

基于三维温度场的热采井水泥环完整性破坏的研究及对策

稠油热采井固井问题一直是制约稠油资源开发的关键技术难题之一,注蒸汽高温热载荷作用下的水泥环完整性破坏导致的井口抬升现象普遍存在于海上稠油热采井开发过程中,危害极大,严重影响油井生产寿命和井控风险。以渤海热采井固井水泥环完整性为研究背景,基于热传导分析,建立了热采井注热井筒三维温度场分析模型和热载荷、注氮载荷和非均匀地应力耦合条件下的水泥环完整性分析模型,并从水泥浆体系、工艺技术、隔热油管性能等方面提出了增强热采井水泥环完整性的应对措施。应用表明,该套理论可有效模拟热采井注蒸汽三维温度场剖面,判断热载荷条件下的水泥环完整性破坏机理及失效形式,为新开发热采井提供借鉴与参考,应用前景广阔。      

Enumeration and characterization of Listeria monocytogenes in novelty ice cream samples manufactured on a specific production line linked to a listeriosis outbreak

Listeriosis is an invasive illness typically caused by the ingestion of foods contaminated with Listeria monocytogenes. In 2015, an outbreak of listeriosis was linked to ice cream products produced on a specific production line at Facility X. The United States Food and Drug Administration (FDA) obtained samples representing several lots of three products manufactured on that line from May 2014 through January 2015. Two of these products, A and B, while not linked to any reported illnesses, were analyzed to determine the frequency of contamination and the contamination level for risk assessment and dose-response analyses. These enumerations were performed utilizing a Most Probable Number (MPN) method, with a lower detection limit of 0.03 MPN/g, on 344 samples of Product A and 95 samples of Product B. Ten lots of Product A were analyzed and 77% of the samples tested were found to be positive for L. monocytogenes. Five lots of Product B were analyzed and 46% of the tested samples were found to be positive. Additionally, the level of contamination of positive Product B samples was always less than 1 MPN/g. The contamination levels of both products, overall, were low with median values of 0.1 MPN/g and 0.02 MPN/g for Products A and B, respectively. A majority of Product A samples (52%) were contaminated at levels of less than 1 MPN/g and only one sample was above 100 MPN/g. Whole genome sequencing (WGS) of L. monocytogenes isolated from ice cream samples produced in the line suggested minor strain differences related to product type, possibly due to differences in the food matrices and/or differences in the manufacturing equipment. Overall, the data showed a consistent low level of contamination in products produced from a single production line over a nine month period.     

Pretreatment with citric acid or a mixture of nitric acid and citric acid to suppress egg white protein deposit formation on stainless steel surfaces and to ease its removal during cleaning

Fouling, adhesion of protein onto a food contact surface, is an important difficulty hindering the pasteurization processing of egg products. To explore a strategy for efficient cleaning of a food contact surface fouled by adherent egg protein, this study investigated the effects of stainless steel surface pretreatment with citric acid or a mixture of nitric acid and citric acid on the adhesion and removability of egg white protein. The 1.05% citric acid pretreatment for 120 min was effective to suppress egg white protein adhesion to a stainless steel surface at 30–80 °C. Pretreatment with nitric acid (1.05% or 4.55%) containing 1.05% citric acid was also effective at 60 °C, which is relevant as a practical pasteurization temperature of egg products. Reducing the pretreatment time from 120 to 15 min was still effective to suppress egg white protein adhesion significantly. Pretreatment with 1.05% nitric acid containing 1.05% citric acid caused higher removability of adhered protein during the cleaning process, especially at higher temperatures. These results demonstrate that pretreatment with nitric acid containing citric acid might be an excellent choice for promoting the efficient cleaning of food manufacturing equipment that has been fouled with egg products.     

Thermal markers arising from changes in the protein component of milk

Classification of heat load applied to milk requires the detection of parameters appropriately related to the intensity of the heat treatment. Current analytical methods based on heat-induced changes in the protein component of milk have been directed either to determine the amount of protein-derived products arised from heat treatments or to evaluate the extent of thermal denaturation of milk proteins. Lately, a new analytical strategy has been developed according to the occurrence of three major whey proteins, namely bovine serum albumin (BSA), beta-lactoglobulin (βlg) and alfa-lactalbumin (αla), normally soluble at pH 4.6 in raw milk, in the pH 4.6 insoluble protein fraction recovered from heat-treated milk. The results have shown that pH 4.6 insoluble BSA, βlg and αla, as detected by ELISA in milk, can be regarded as thermal markers suited for either dairy process control or regulation purposes.