ELISA to detect proteolysis of ultrahigh-temperature milk upon storage

Casein proteolysis can occur in milk during storage leading to its gelation. The two main proteolytic systems suspected to be involved are the plasmin and the proteases produced by psychrotrophic bacteria. The latter have been shown to cleave kappa-casein at the Phe105-Met106 bond. Although several techniques allow the determination of plasmin in milk, few rapid and easy-to-perform analytical techniques are available to check for bacterial proteolytic activity. This study presents the development of an inhibition ELISA allowing for the quantification of the kappa-casein intact at the Phe105-Met106 bond. It uses a monoclonal antibody specifically directed against this peptide bond that binds to the protein as long as the molecule's cleavage site is intact but not when it is cleaved. This simple technique allows for the rapid analysis of more than 20 samples within 3 h. Applied to commercial milks, this assay allowed for the detection of unstable milk.     

Effect of high-pressure processing on activity and structure of alkaline phosphatase and lactate dehydrogenase in buffer and milk

Changes in the activity and structure of alkaline phosphatase (ALP) and L-lactate dehydrogenase (LDH) were investigated after high pressure processing (HPP). HPP treatments (206-620 MPa for 6 and 12 min) were applied to ALP and LDH prepared in buffer, fat-free milk, and 2% fat milk. Enzyme activities were measured using enzymatic assays, and changes in structure were investigated using far-ultraviolet circular dichroism (CD) spectroscopy and dynamic light scattetering (DLS). Kinetic data indicated that the activity of ALP was not affected after 6 min of pressure treatments (206-620 MPa), regardless of the medium in which the enzyme was prepared. Increasing the processing time to 12 min did significantly reduce the activity of ALP at 620 MPa (P < 0.001). However, even the lowest HPP treatment of 206 MPa induced a reduction in LDH activity, and the course of reduction increased with HPP treatment until complete inactivation at 482, 515, and 620 MPa. CD data demonstrated a partial change in the secondary structure of ALP at 620 MPa, whereas the structure of LDH showed gradual denaturation after exposure at 206 MPa for 6 min, leading to a random coil structure at both 515 and 620 MPa. DLS results indicated aggregation of ALP only at HPP treatment of 206 MPa and not above and enzyme precipitation as well as aggregation at 345, 415, 482, and 515 MPa. The loss of LDH activity with increasing pressure and time treatment was due to the combined effects of denaturation and aggregation.     

Determination of alkaline phosphatase in casein: collaborative study

A collaborative study was made to determine alkaline phosphatase in casein samples by the rapid colorimetric test. Six to eight collaborators tested 10 unknown casein samples containing various amounts of residual phosphatase with and without the addition of magnesium acetate. Results indicated that magnesium acetate significantly increased phosphatase activity. The collaborators correctly analyzed 95% of the samples with the added magnesium acetate. The method has been adopted official first action.     

Analysis of fenbendazole residues in bovine milk by ELISA

Fenbendazole residues in bovine milk were analyzed by ELISAs using two monoclonal antibodies. One monoclonal antibody (MAb 587) bound the major benzimidazole anthelmintic drugs, including fenbendazole, oxfendazole, and fenbendazole sulfone. The other (MAb 591) was more specific for fenbendazole, with 13% cross-reactivity with the sulfone and no significant binding to the sulfoxide metabolite. The limit of detection of the ELISA method in the milk matrix was 7 ppb for MAb 587 and 3 ppb for MAb 591. Fenbendazole was administered in feed, drench, and paste form to three groups of dairy cattle. Milk was collected immediately before dosing and then every 12 h for 5 days. The ELISA indicated that residue levels varied widely among individual cows in each group. Fenbendazole levels peaked at approximately 12-24 h and declined rapidly thereafter. Metabolites were detected at much higher levels than the parent compound, peaked at approximately 24-36 h, and declined gradually. Residue levels were undetectable by 72 h. The ELISA data correlated well with the total residues determined by chromatographic analysis, but the use of the two separate ELISAs did not afford an advantage over ELISA with the single, broadly reactive MAb 587. The ELISA method could be used to flag high-residue samples in on-site monitoring of fenbendazole in milk and is a potential tool for studying drug pharmacokinetics.     

Kinetic behaviour of alkaline phosphatase desorbed from different clay minerals

The kinetic (K_m, V_max) of alkaline phosphatase (AP) desorbed from different Ca-homoionic clay minerals (montmorillonite, illite, and kaolinite) by extraction with Tris-Malate-Citrate buffer solution (pH 9.6) was studied in model experiments. After extraction (shaking for 15 min.) the K_m and V_max were measured in the extract, the remaining sediment and in the whole set-up. With kaolinite and illite, V_max of the desorbed AP was lower than that of the sediment. However, with montmorillonite, V_max of AP in the extract and whole system increased if compared to the control, but decreased in the sediment. The K_m of desorbed AP increased from 4.3 × 10^?3 (control) to 5.0 × 10^?3 M (illite), 5.4 × 10^?3M (kaolinite), and 5.5 × 10^?3M (montmorillonite). These values were lower than those obtained with the various sediments and whole experimental systems. An aberrant behaviour was recorded with the illite sorbed AP which showed an increase in affinity towards the substrate. Generally speaking, AP desorbed from clays may be reduced in its affinity towards the substrate p-nitrophenylphosphate by residual inhibitor and/or conformational change of the enzyme.     

Development of a cell culture/ELISA assay to detect anticoagulant rodenticides and its application to analysis of rodenticide treated grain

This study describes a generic biological screening assay designed to detect anticoagulant rodenticides based on their inhibitory action on the vitamin K epoxide reductase protein complex, resulting in an accumulation of under-carboxylated prothrombin or proteins induced by vitamin K antagonism (PIVKA-II). A combined cell culture/ELISA assay was optimized to measure PIVKA-II production by the human hepatoma HepG2 cell line cultured in the presence of anticoagulant rodenticides. The specificity and sensitivity of the assay was validated using 41 grain extracts containing representative concentrations of rodenticide or appropriate nonrodenticide control compounds. In all cases, PIVKA-II produced by HepG2 cells in response to grain extracts spiked with rodenticides was detected by ELISA, while PIVKA-II was not detected in supernatants collected from cells exposed to nonrodenticide controls. This represents a novel, class-specific biological assay for the detection of anticoagulant rodenticides present in contaminated grain.     

Reaction rate modeling in cryoconcentrated solutions: alkaline phosphatase catalyzed DNPP hydrolysis

The hydrolysis of disodium p-nitrophenyl phosphate catalyzed by alkaline phosphatase was chosen as a model to study the kinetics of changes in frozen food products. The initial reaction rate was determined in concentrated sucrose solutions down to -24 degrees C, and the enzymatic characteristics K(M) and V(max) were calculated. The experimental data were compared to the kinetics predicted by assuming that the reaction was viscosity dependent. Indeed, an analysis of the enzymatic reaction demonstrated that both the diffusion of the substrate and the flexibility of the enzyme segments were controlled by the high viscosity of the media. When the temperature was too low for the viscosity to be measured simply, the Williams-Landel-Ferry equation was used to predict the viscosity, taking, as reference temperature, the glass transition temperature (T(g)) corresponding to the concentration of the freeze-concentrated phase at the test temperature. Predicted values of the reaction rate were very close to the experimental ones in the studied temperature range.     

Rapid screening method for alkaline phosphatase activity in cheese: collaborative study

The method developed for developed for determining alkaline phosphatase activity in cheese, in which phenolphthalein monophosphate is used as the substrate, was collaboratively studied. A 7.5% butanol extract of cheese is reacted with phenolphthalein monophosphate; phenolphthalein is released and yields a red solution that is compared visually with a standard (s) prepared from the same extract. Seven collaborators analyzed 8 samples of cheese, in duplicate, by the screening method and Scharer I method. Of the 208 observations returned, only 4 were incorrect. The alkaline phosphatase method has been adopted as official first action.     

Fertilization management affects the alkaline phosphatase bacterial community in barley rhizosphere soil

There is increasing interest in good agriculture practices that address the issues of sustainability, reduction in inputs such as fertilizers and pesticides while maintaining crop yield and soil fertility. It is important that soil microbial diversity and function are not impaired by altered agricultural practice. In this study, as indicators of soil quality, the bacterial community structure was evaluated from a long-term field trial managed with conventional and low-input fertilization/pesticide regimes. The low-input plots under study received approximately one fifth less N fertilizer than the conventional-input plots, a maximum of half the recommended application rates of fungicides and pesticides and no externally added P source. A non-culturable approach was taken using polymerase chain reaction–denaturing gradient gel electrophoresis analysis of 16S rRNA and alkaline phosphomonoesterase [phosphatase] (ALP) genes in an attempt to relate bacterial community structure to respective field management regimes. To identify the ALP bacteria in these plots, randomly selected ALP clones were sequenced. The results based on Shannon diversity indices and community structure analysis of ALP genes suggest differences in community diversity and structure under conventional and low-input barley sites in most sampling seasons. We conclude that soil fertilization management affects the ALP bacteria in the barley rhizosphere, while the overall changes in bacterial community in these sites are prominently due to seasonal variation compared to crop or input regimes. The randomly selected ALP sequences identified from these sites were mostly from the Alpha and Gamma classes of Proteobacteria.     

Methods of sample preparation for detecting alkaline phosphatase in casein: collaborative study

A collaborative study was conducted in which 2 different sample preparation techniques were used to determine alkaline phosphatase in casein by the rapid colorimetric test. Seven collaborators tested 10 unknown casein products containing different amounts of residual phosphatase. Results indicated that the phosphatase contents of casein prepared by the 2 methods were not significantly different. The collaborators correctly analyzed 100% of the test samples that were ground and 98% of the test samples that were unground. The alternative rapid sample preparation method has been adopted official first action.     

酶联免疫法快速筛查牛奶中的二噁英

二噁英是最受人们关注的持久性有机污染物之一。本研究建立了ELISA快速筛查牛奶基质中二噁英的检测方法。牛奶样品经真空冷冻干燥,加速溶剂萃取法萃取、用酸性硅胶柱、活性炭柱净化后,浓缩氮吹后复溶,进行ELISA检测。结果表明,方法回收率为72.0%~125.2%,相对偏差<30%。本法对牛奶中二噁英毒性当量(TEQ)测定值与高分辨气相色谱-高分辨质谱法(HRGC-HRMS)测定值吻合度高,检测结果误差<20%。本方法具有操作简便、快速、检测结果可靠等特点,适用于基层检测机构对牛奶中二噁英的快速筛查。     

Application of a real time polymerase chain reaction method to detect castor toxin contamination in fluid milk and eggs

The castor seed contains ricin, which is one of the most potent biological toxins and is widely considered to be a threat agent for bioterrorism. In this study, a rapid and sensitive PCR method was applied to the detection of castor contamination in milk and liquid egg samples. The targeting gene sequence of the primer set, Ricin-F4/R4, was not found in either the bovine or chicken genome. Primers against a highly conserved sequence from the 18S ribosomal RNA gene were used as a positive control for DNA extraction and PCR reaction efficiency. The quantity and quality of DNA prepared from castor spiked or nonspiked milk and egg samples obtained from three different DNA extraction methods were compared. The cetyl trimethylammonium bromide (CTAB) method yielded the highest quality of DNA and is most suitable for the sensitive detection of castor DNA by real-time PCR in both milk and liquid egg matrixes. However, taking time and cost into consideration, a commercial kit designed for extraction of DNA from stool samples could be used as an alternative method for the routine extraction of DNA from milk for real-time PCR assays. The egg matrix was found to inhibit PCR amplification and interfere with two of the three methods tested for DNA extraction. Egg yolk had a greater negative effect on PCR amplification than the egg white matrix. Our results affirm the necessity of performing individual validations for each food matrix. Both real-time PCR systems used in this study, TaqMan and SYBR Green I dye, were capable of detecting 100 ng of castor acetone powder, corresponding to 5 ng of ricin, in 1 mL of milk or liquid egg, well below the toxic dose for humans. On the basis of these results, the real-time PCR method for detection of intentional castor contamination is applicable to milk and egg matrixes.     

Analysis of bacterial communities on alkaline phosphatase genes in soil supplied with organic matter

Abstract

We studied the effects of the application of organic matter (OM) and chemical fertilizer (CF) on soil alkaline phosphatase (ALP) activity and ALP-harboring bacterial communities in the rhizosphere and bulk soil in an experimental lettuce field in Hokkaido, Japan. The ALP activity was higher in soils with OM than in soils with CF, and activity was higher in the rhizosphere for OM than in the bulk soil. Biomass P and available P in the soil were positively related to the ALP activity of the soil. As a result, the P concentration of lettuce was higher in OM soil than in CF soil. We analyzed the ALP-harboring bacterial communities using polymerase chain reaction based denaturing gradient gel electrophoresis (DGGE) on the ALP genes. Numerous ALP genes were detected in the DGGE profile, regardless of sampling time, fertilizer treatment or sampled soil area, which indicated a large diversity in ALP-harboring bacteria in the soil. Several ALP gene fragments were closely related to the ALP genes of Mesorhizobium loti and Pseudomonas fluorescens. The community structures of the ALP-harboring bacteria were assessed using principal component analysis of the DGGE profiles. Fertilizer treatment and sampled soil area significantly affected the community structures of ALP-harboring bacteria. As the DGGE bands contributing to the principal component were different from sampling time, it is suggested that the major bacteria harboring the ALP gene shifted. Furthermore, there was, in part, a significant correlation between ALP activity and the community structure of the ALP-harboring bacteria. These results raise the possibility that different ALP-harboring bacteria release different amounts and/or activity of ALP, and that the structure of ALP-harboring bacterial communities may play a major role in determining overall soil ALP activity.     

Comparison of phosphorus fractions and alkaline phosphatase activity in sludge,soils, and sediments

Purpose  

The land application or disposal of sewage sludge generally leads to phosphorus (P) loss in aquatic environments and often results in eutrophication. The nature of P fractions plays a key role in the transport of P within the environment, and alkaline phosphatase is important for the transformation of the P fractions. The objective of this study was to assess the P fractions and alkaline phosphatase activity in sewage sludge, soils, and sediments, in order to effectively evaluate their P bioavailability and mobility.     

Preparation of anti-tetracycline antibodies and development of an indirect heterologous competitive enzyme-linked immunosorbent assay to detect residues of tetracycline in milk

Tetracycline (TC) is a broad-spectrum antibiotic used increasingly in animal husbandry to treat diseases or to promote growth as feed additives. To avoid using labor-intensive instrumental methods to detect residues of TC in food and food products, a simple and convenient indirect heterologous competitive enzyme-linked immunosorbent assay (ELISA) method for TC was developed using polyclonal antibody prepared in this study. Three new immunogens, TC-o-tolidine-bovine serum albumin (BSA), TC- 4-aminobenzoic acid-cationized BSA (cBSA), and TC-1,1'-carbonyldiimidazole-cBSA, were synthesized in this research to develop anti-TC antibodies. All antibodies raised in rabbits and coating antigens synthesized were screened and characterized using homologous and heterologous ELISA formats to select the best combination. An optimized ELISA gave an IC50 value of 3.92 mug/mL toward TC in PBS buffer. The specificity of the assay was studied by measuring cross-reactivity of the antibody with the structurally closely related compounds of chlortetracycline (112%) and oxytetracycline (<2%). The recovery rates from the TC-fortified raw milk samples were in the range of 74-116%, while the intra- and interassay coefficients of variation were <14.5 and <25.0, respectively.     

Colorimetric determination of alkaline phosphatase as indicator of mammalian feces in corn meal: collaborative study

In the official method for rodent filth in corn meal, filth and corn meal are separated in organic solvents, and particles are identified by the presence of hair and a mucous coating. The solvents are toxic, poor separation yields low recoveries, and fecal characteristics are rarely present on all fragments, especially on small particles. The official AOAC alkaline phosphatase test for mammalian feces, 44.181-44.184, has therefore been adapted to determine the presence of mammalian feces in corn meal. The enzyme cleaves phosphate radicals from a test indicator/substrate, phenolphthalein diphosphate. As free phenolphthalein accumulates, a pink-to-red color develops in the gelled test agar medium. In a collaborative study conducted to compare the proposed method with the official method for corn meal, 44.049, the proposed method yielded 45.5% higher recoveries than the official method. Repeatability and reproducibility for the official method were roughly 1.8 times more variable than for the proposed method. The method has been adopted official first action.     

Effect of humic amendments on inorganic N, dehydrogenase and alkaline phosphatase activities of a Mediterranean soil

Dehydrogenase activity, alkaline phosphatase activity and NH₄ ⁺, NO₂ ⁻ and NO₃ ⁻ concentrations were monitored in an aridisol treated with three commercially available humic amendments. The materials were of plant residue, lignite and peat origins. The humus plant residues, fulvic acids, with a high content of Kjeldahl-N, sustained high enzyme activities and highest levels of NH₄ ⁺, NO₂ ⁻ and NO₃ ⁻. Humus lignite (mainly humic acids) produced the highest dehydrogenase activity, whereas the alkaline phosphatase activity was not as high as that amendment with humus plant residues. The lower activity of alkaline phosphatase could not be attributed to the higher P content of humus lignite. Nitrification was also low, probably due to the low N content of this fertilizer. The amendment of humus peat origin (only humic acids) did not increase enzyme activity or inorganic N concentrations of soil. Our results show that although these materials are widely utilized and recommended as microbial and plant activators, they all behave very differently, and the effects on soil microbiological activity cannot be predicted solely on the basis of their humic and/or fulvic acid contents.