Detection of proteolysis in raw milk stored at low temperature by an inhibition ELISA

1994 ◽  
Vol 61 (3) ◽  
pp. 395-404 ◽  
Author(s):  
Catherine Picard ◽  
Isabelle Plard ◽  
Dominique Rongdaux-Gaida ◽  
Jean-Claude Collin
Keyword(s):  
Proteolytic Activity ◽  
Limit Of Detection ◽  
Bacterial Count ◽  
Raw Milk ◽  
Enzyme Linked Immunosorbent Assay ◽  
Psychrotrophic Bacteria ◽  
Milk Samples ◽  
Inhibition Elisa ◽  
Different Strains

SummaryAn inhibition ELISA (enzyme-linked immunosorbent assay) was developed for the determination of caseinomacropeptide (CMP) in order to estimate the proteolysis of κ-casein due to the enzymes of psychrotrophic bacteria in bulk raw milk. The CMP present in milk was quantified specifically by an antibody. The limit of detection was ∽ 0·1 μg/ml and the CV was < 10%. This method was used to study the proteolytic activity of three strains of psychrotrophic Pseudomonas fluorescens in raw milk and to analyse different raw milk samples supplied by four dairy plants. The proteolytic activity for different strains of psychrotrophs and for different milk samples varied considerably, but no correlation was established between the level of microbial flora and κ-casein proteolysis. It is thus not possible to determine the extent of proteolysis from the bacterial count alone. However, by CMP determination in bulk raw milk samples after 6 d storage at 4°C, the mean κ-casein proteolysis was ∽ 4%. Among the milk samples analysed that contained < 107 cfu psychrotrophs/ml, 30% exhibited a proteolysis of κ-casein < 0·5%, i.e. < 5μg CMP/ml.

scholarly journals Determination of Aflatoxin M1 in Raw Milk Using an HPLC-FL Method in Comparison with Commercial ELISA Kits—Application in Raw Milk Samples from Various Regions of Greece

2021 ◽  
Vol 8 (3) ◽  
pp. 46
Author(s):  
Martha Maggira ◽  
Maria Ioannidou ◽  
Ioannis Sakaridis ◽  
Georgios Samouris
Keyword(s):  
Limit Of Detection ◽  
Reference Method ◽  
Raw Milk ◽  
Aflatoxin M1 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fluorescence Detector ◽  
Elisa Kit ◽  
Milk Samples ◽  
Of Greece

The highly toxic Aflatoxin M1 (AFM1) is most often detected in milk using an Enzyme-Linked-Immunosorbent Assay (ELISA) for screening purposes, while High-Performance Liquid Chromatography with Fluorescence Detector (HPLC-FL) is the reference method used for confirmation. The aim of the present study was the comparison between three commercially available ELISA kits and a newly developed HPLC-FL method for the determination of the AFM1 in milk samples. The developed HPLC-FL method was validated for the AFM1 and Aflatoxin M2 (AFM2), determining the accuracy, precision, linearity, decision limit, and detection capability with fairly good results. All three ELISA kits were also validated and showed equally good performance with high recovery rates. Moreover, the Limit Of Detection (LOD) and Limit Of Quantification (LOQ) values were found to be significantly lower than the Maximum Residue Limit (MRL) (50 ng kg−1). After the evaluation of all three commercial kits, the ELISA kit with the optimum performance along with the HPLC method was used for the determination of AFM1 in raw cow’s, goat’s, and sheep’s milk samples (396) obtained from producers in different regions of Greece. The evaluation of both methods showed that this ELISA kit could be considered as a faster and equally reliable alternative method to HPLC in routine analysis for the determination of AFM1 in milk.

Rapid Detection of Psychrotrophic Bacteria In Manufacturing Grade Raw Milks

1991 ◽  
Vol 54 (11) ◽  
pp. 861-867 ◽  
Author(s):  
S. R. TATINI ◽  
P. MEKALA ◽  
A. EL-HABAZ ◽  
M. W. GRIFFITHS
Keyword(s):  
Rapid Detection ◽  
Bacterial Count ◽  
Raw Milk ◽  
Plate Count ◽  
Psychrotrophic Bacteria ◽  
Milk Samples ◽  
Microscopic Count ◽  
Direct Epifluorescent Filter Technique ◽  
Native Microflora

Methods to rapidly assess the bacteriological quality of raw milk were investigated. Whereas direct microscopic count, modified psychrotrophic plate count, and direct epifluorescent filter technique (DEFT) did not correlate well with initial psychrotrophic bacterial count of raw milk, improvements were obtained after preincubation of the milk samples. The best preincubation conditions were identified as 30°C for 6 h, 21°C for 10 h, 13°C for 15 h, 13°C for 20 h, or 7°C for 37 h. The “square root” equation was applied to the data, and a model was produced for predicting growth of the native microflora of raw milk. Using this equation, a DEFT count after preincubation of the milk at 21°C for 10 h could accurately predict the initial psychrotroph count and the count after storage of the milk at 6°C for 48 h.

scholarly journals Proteolysis in raw milk in relation to microbiological indicators

2016 ◽  
Vol 34 (No. 4) ◽  
pp. 306-312 ◽  
Author(s):  
J. Chramostová ◽  
O. Hanuš ◽  
M. Klimešová ◽  
I. Němečková ◽  
P. Roubal ◽  
...  
Keyword(s):  
Early Detection ◽  
Somatic Cell Count ◽  
Correlation Coefficients ◽  
Raw Milk ◽  
Total Count ◽  
Psychrotrophic Bacteria ◽  
Milk Samples ◽  
Crucial Parameter ◽  
Microbiological Indicators

Proteolysis in raw milk is a crucial parameter indicating both cow’s mastitis and the technological problems or spoilage risk of final products. However, a suitable analytical method for its early detection in practice is still missing. Thus, we proposed a spectrophotometric determination of milk proteolysis equivalent (MPE). We tested this method on 104 bovine raw milk samples in relation to their somatic cell count (SCC) as an indicator of native proteolysis, and the total count of mesophilic bacteria (TCMB) and the total count of psychrotrophic bacteria (TCPB) as indicators of microbial proteolysis. Correlation coefficients between log TCMB and MPE and log TCPB and MPE were 0.3651 and 0.4152, respectively (both P &lt; 0.001). SCC was not correlated with MPE (P &gt; 0.05). We estimated the MPE limit indicating an incipient risk of proteolysis in the range from 0.9366 to 1.02 mmol/l. The determination of MPE seems to be a promising method applicable in the control of raw milk.

A Novel Rapid Enzyme Immunoassay (Fluorophos BetaScreen) for Detection of β-Lactam Residues in Ex-Farm Raw Milk

1998 ◽  
Vol 61 (7) ◽  
pp. 808-811 ◽  
Author(s):  
ÅSE STERNESJÖ ◽  
GÖRAN JOHNSSON
Keyword(s):  
Enzyme Immunoassay ◽  
Limit Of Detection ◽  
Raw Milk ◽  
Penicillin G ◽  
Milk Samples ◽  
Quality Testing ◽  
Response Profile ◽  
Very High ◽  
Higher Sensitivity

A novel, qualitative enzyme immunoassay based on fluorescence detection for determination of (β-lactam antibiotics in raw, commingled milk (Fluorophos BetaScreen E. U. test) was evaluated. A dose-response profile for penicillin G was constructed by analysis of spiked milk samples. The limit of detection, defined as the concentration of penicillin G that resulted in 95% of the samples being evaluated as positive, was 1.8 μg/kg. The repeatability of the assay was very high both within and between the three participating milk quality testing laboratories. In total 5,061 randomly selected tanker milk samples were analyzed with the BetaScreen test and compared with the Delvotest SP. The agreement between the two tests was 99.7%. Probably due to a higher sensitivity to penicillin G, the BetaScreen test detected almost twice as many suspect positive tanker milk samples (0.45%) as the Delvotest SP (0.26%).

scholarly journals Quantification of psychrotrophic bacteria and molecular identification of Pseudomonas fluorescens in refrigerated raw milk

2019 ◽  
Vol 86 ◽  
Author(s):  
Camila Lampugnani ◽  
Mykaella Zanatta Was ◽  
Maike Taís Maziero Montanhini ◽  
Luis Augusto Nero ◽  
Luciano dos Santos Bersot
Keyword(s):  
Pseudomonas Fluorescens ◽  
Proteolytic Activity ◽  
Molecular Identification ◽  
Raw Milk ◽  
Chain Reaction ◽  
Psychrotrophic Bacteria ◽  
Milk Samples ◽  
16S Gene ◽  
Polymerase Chain ◽  
Proteolytic Capacity

ABSTRACT In this study, we investigated the contamination of refrigerated raw milk produced in the western region of Paraná, southern Brazil, with psychrotrophic microorganisms, aiming to assay the proteolytic activity of the isolates and to identify Pseudomonas fluorescens, the main proteolytic species associated with the spoilage of milk products. Raw milk samples from 50 dairy farms were submitted to the counting of psychrotrophic microorganisms, being the microbiota characterized by its mesophilic behavior and proteolytic capacity, besides molecular identification of P. fluorescens. Of the samples evaluated, 94% had psychrotrophic counts ranging from 3 to 7.1 log CFU mL-1, and 48.5% of these showed mesophilic behavior. Of the isolates, 48.0% had proteolytic activity in at least one evaluated temperature (21 and 30°C), and 39.3% had proteolytic activity in both temperatures. Among the 61 isolates submitted to molecular identification by polymerase chain reaction (PCR), 86.8% contained the expression of the 16S gene characteristic for P. fluorescens. In this study, we demonstrated that P. fluorescens is the most prevalent psychrotrophic bacteria species in raw refrigerated milk and their proteolytic ability poses high risks to the dairy industry.

scholarly journals Reduction of Aflatoxin M1 Levels during Ethiopian Traditional Fermented Milk (Ergo) Production

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Tsige Shigute ◽  
Alemayehu P. Washe
Keyword(s):  
Incubation Time ◽  
Raw Milk ◽  
Fermented Milk ◽  
Aflatoxin M1 ◽  
Chemical Components ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bacterial Cells ◽  
Milk Samples ◽  
Significant Difference

In this study, the reduction of aflatoxin M1 (AFM1) levels during lab-scale ergo production was investigated through determination of the residual levels of AFM1 using Enzyme Linked Immunosorbent Assay. The results showed gradual and incubation time dependent reduction of AFM1 level in the raw milk samples being fermented to ergo. The maximum reductions of 57.33 and 54.04% were recorded in AFM1 in natural and LAB inoculums initiated fermentations, respectively, in 5 days of incubation. Although a significant difference (P=0.05) in the AFM1 decrease in the two types of fermentations was recorded, such findings could vary with milk samples depending on initial load of the microorganisms as determined by hygienic conditions. However, the level of AFM1 in control (sterilized) samples showed only a 5.5% decrease during the entire period of incubation. Microbiological investigation showed increasing LAB counts with incubation time. A gradual decrease in pH of the milk samples was observed during fermentation. Considering the fact that both viable and dead bacterial cells could remove AFM1 during ergo production, the mechanism is proposed as predominantly involving noncovalent binding of the toxin with the chemical components of the bacterial cell wall.

scholarly journals Pitfalls in the Immunochemical Determination of β‑Lactam Antibiotics in Water

Antibiotics ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 298
Author(s):  
Alexander Ecke ◽  
Rudolf J. Schneider
Keyword(s):  
Limit Of Detection ◽  
Cost Effective ◽  
Enzyme Linked Immunosorbent Assay ◽  
Degree Of Hydrolysis ◽  
Site Analysis ◽  
Antibody Recognition ◽  
Immunochemical Determination ◽  
Key Factor ◽  
Lactam Ring

Contamination of waters with pharmaceuticals is an alarming problem as it may support the evolution of antimicrobial resistance. Therefore, fast and cost-effective analytical methods for potential on-site analysis are desired in order to control the water quality and assure the safety of its use as a source of drinking water. Antibody-based methods, such as the enzyme-linked immunosorbent assay (ELISA), can be helpful in this regard but can also have certain pitfalls in store, depending on the analyte. As shown here for the class of β-lactam antibiotics, hydrolysis of the β‑lactam ring is a key factor in the immunochemical analysis as it influences antibody recognition. With the antibody used in this study, the limit of detection (LOD) in the immunoassay could be significantly reduced by hydrolysis for the five tested penicillins, with the lowest LOD for carbenicillin (0.2 nmol/L) and the greatest impact on penicillins G and V (reduction by 85%). In addition to enhanced quantification, our strategy also provides access to information about the degree of hydrolysis in water samples as shown for the most abundant penicillin amoxicillin.

scholarly journals Simultaneous Determination of C18 Fatty Acids in Milk by GC-MS

Separations ◽  
2021 ◽  
Vol 8 (8) ◽  
pp. 118
Author(s):  
Meiqing Chen ◽  
Yangdong Zhang ◽  
Fengen Wang ◽  
Nan Zheng ◽  
Jiaqi Wang
Keyword(s):  
Fatty Acids ◽  
High Sensitivity ◽  
Raw Milk ◽  
Uht Milk ◽  
Milk Samples ◽  
Accuracy And Precision ◽  
Different Temperatures ◽  
Chromatographic Resolution ◽  
Commercial Milk

The determination of C18 fatty acids (FAs) is a key and difficult aspect in FA profiling, and a qualified method with good chromatographic separation and high sensitivity, as well as easy methylation, is required. A GC-MS method was established to simultaneously determine C18 FAs in milk. To simplify the methylation protocol for milk samples, besides a base-catalyzation methylation (50 °C for 20 min), the necessity of an additional acid-catalyzation was also studied using different temperatures (60 °C, 70 °C, 80 °C, and 90 °C) and durations (90 min and 150 min). The results showed that the chromatographic resolution was improved, although three co-eluted peaks existed. The base-catalyzation was sufficient, and an additional acid-catalyzation was not necessary. The proposed method was validated with good sensitivity, linearity, accuracy, and precision, and then applied in determining C18 FAs in 20 raw milk and 30 commercial milk samples. UHT milk presented a different profile of C18 FAs from raw milk and PAS milk samples, which indicated that excessive heating could change the profile. Overall, the proposed method is a high-throughput and competent approach for the determination of C18 FAs in milk, and which presents an improvement in chromatographic resolution and sensitivity, as well as a simplification of methylation.

scholarly journals Characterization and validation of fragment antigen-binding (Fab) antibody-based immunoassay for deoxymiroestrol, a potent phytoestrogen from Pueraria candollei

2021 ◽  
Author(s):  
Worapol Sae-foo ◽  
Supaluk Krittanai ◽  
Wipawee Juengsanguanpornsuk ◽  
Gorawit Yusakul ◽  
Tharita Kitisripanya ◽  
...  
Keyword(s):  
Quantitative Method ◽  
Estrogenic Activity ◽  
Limit Of Detection ◽  
Hormone Replacement ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigen Binding ◽  
Pueraria Candollei ◽  
Specific Determination ◽  
Fragment Antigen Binding

Deoxymiroestrol is the most potent phytoestrogen in chromenes group that has been found in Pueraria candollei, Thai name known as Kwao Krua Khao. Several studies reported estrogenic activity of P. candollei in order to using as hormone replacement therapy for postmenopausal women. Previously, specific determination of deoxymiroestrol content by enzyme-linked immunosorbent assay (ELISA) using polyclonal antibody (pAb) have been reported. However, production of pAb has limitation and variability from different batches. Therefore, in this study, we established quantitative method for determination of deoxymiroestrol using fragment antigen-binding (Fab) antibody-based immunoassay. The developed immunoassay has specificity to deoxymiroestrol with a calibration range of 15.6-1000 ng mL-1. Precision including intra-assay and inter-assay are 1.48-7.11 and 0.58-9.31%, respectively. Accuracy of the assay showed in recovery between 99.77-101.61% when spike deoxymiroestrol standard into the samples. The limit of detection (LOD) is 30.80 ng mL-1. Comparation antibody-based immunoassay for determination of deoxymiroestrol using Fab with pAb was represented consistency (R2 = 0.9807) when analysis roots bark of Pueraria candollei from difference areas. Therefore, this development assay can apply to determine deoxymiroestrol content in the plant samples.

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