Heat-treated Milk Samples Research Articles

Detection of Salmonella Enteritidis in Milk Using Conductometric Immunosensor Coated on Polyaniline/Zinc Oxide Nanocomposite.

The demand for rapid and accurate detection methods for Salmonella Enteritidis necessitates the development of highly sensitive and specific biosensors to ensure proper monitoring of food safety and quality requirements in the food sector and to secure human health. This study focused on development of a polyaniline/zinc oxide (PANI/ZnO) nanocomposite film on a gold electrode conductometric immunosensor for detection of Salmonella Enteritidis. The sensor was modified with monoclonal anti-Salmonella Enteritidis antibodies as biorecognition elements. The fabricated sensor was able to detect and quantify the target pathogen within 30 min and showed a good detection range from 101 to 105 colony-forming units (CFU)/mL for Salmonella Enteritidis and a minimum detection limit of 6.44 CFU/mL in 0.1% peptone water. Additionally, the fabricated sensor showed good selectivity and detection limit toward the target bacterium and successfully determined Salmonella Enteritidis content in ultrahigh heat-treated skim milk samples without pretreatment of the food sample.

Developed rapid and simple RP-HPLC method for simultaneous separation and quantification of bovine milk protein fractions and their genetic variants

The aim was to develop a reliable rapid reversed-phase high-performance liquid chromatography (RP-HPLC) method to simultaneously determine the main bovine milk protein fractions, including their genetic variants. Compared to the previous studies, our method is able to separate the main protein fractions within 20 min of total run time. The method validation consisted of testing repeatability, reproducibility linearity, repeatability, and accuracy. The procedure was developed using raw individual, bulk, and commercially available heat-treated cow milk samples. The RSD of peak areas ranged from 1.43 to 3.16% within analytical day and from 3.29 to 6.70% across analytical days. The method can be applied to investigate both raw and heat-treated milk samples.

Heat-Treatments Affect Protease Activities and Peptide Profiles of Ruminants' Milk.

Proteases present in milk are heat-sensitive, and their activities increase or decrease depending on the intensity of the thermal treatment applied. The thermal effects on the protease activity are well-known for bovine milk but poorly understood for ovine and caprine milk. This study aimed to determine the non-specific and specific protease activities in casein and whey fractions isolated from raw bovine, ovine, and caprine milk collected in early lactation, and to determine the effects of low-temperature, long-time (63°C for 30 min) and high-temperature, short-time (85°C for 5 min) treatments on protease activities within each milk fraction. The non-specific protease activities in raw and heat-treated milk samples were determined using the substrate azocasein. Plasmin (the main protease in milk) and plasminogen-derived activities were determined using the chromogenic substrate S-2251 (D-Val-Leu-Lys-pNA dihydrochloride). Peptides were characterized using high-resolution liquid chromatography coupled with tandem mass spectrometry. The activity of all native proteases, shown as non-specific proteases, was similar between raw bovine and caprine milk samples, but lower (P < 0.05) than raw ovine milk in the whey fraction. There was no difference (P > 0.05) between the non-specific protease activity of the casein fraction of raw bovine and caprine milk samples; both had higher activity than ovine milk. After 63°C/30 min, the non-specific protease activity decreased (44%; P > 0.05) for the bovine casein fraction only. In contrast, the protease activity of the milk heated at 85°C/5 min changed depending on the species and fraction. For instance, the activity decreased by 49% for ovine whey fraction, but it increased by 68% for ovine casein fraction. Plasmin and plasminogen were in general inactivated (P > 0.05) when all milk fractions were heated at 85°C/5 min. Most of the peptides present in heat-treated milk were derived from β-casein and αS1-casein, and they matched the hydrolysis profile of cathepsin D and plasmin. Identified peptides in ruminant milk samples had purported immunomodulatory and inhibitory functions. These findings indicate that the non-specific protease activity in whey and casein fractions differed between ruminant milk species, and specific thermal treatments could be used to retain better protease activity for all ruminant milk species.

Incidence of some spore forming bacteria in market milk

A total of one hundred raw milk samples collected from different localities of Damietta governorate (farmers, street vendors, supermarkets and, dairy shops). Storch’s test was performed for detection of heat treated milk samples that were 12, 8, 40 and, 16, respectively. Clostridium perfringens and Bacillus cereus were detected in 7 and 10% of examined raw milk samples. Alpha (cpa); enterotoxin (cpe) genes and hlbA; ces genes of isolated Clostridium perfringens and Bacillus cereus were characterized by PCR represented 85.7% (6); 0% and 60%(6); 20%(2) of each isolate, respectively. 20% of isolated B. cereus strains were not toxigenic according to the study, although all isolates were haemolytic. Growth, Lipolytic and Proteolytic activities of Bacillus cereus strains isolated from raw milk were studied at 30◦c and 7◦c in a percentage of 100, 40 and 20; 20,100 and 40, respectively. It was revealed from the previous results that 100% (10) of B. cereus isolates were mesophilic and 40% were phychrotophic. Potential health risk and economic importance of isolated strains were discussed.

Aflatoxin M1 in cow, sheep, and donkey milk produced in Sicily, Southern Italy.

Samples (n = 485) of raw (n = 394) or heat-treated (n = 91) milk of three different species (cow, n = 170; sheep, n = 133; donkey, n = 84), collected 2013-2016 in Western Sicily (Southern Italy), were analyzed for aflatoxin M1 (AFM1) by enzyme-linked immunosorbent assay (ELISA). Positive ELISA results were further analyzed by HPLC with fluorescence detection. Both methods had a detection limit for AFM1 in milk of 7ngkg-1. ELISA yielded 12.9 and 5% positives in cows and sheep milk, respectively, all samples of donkey milk were negative. Levels of AFM1 were in most cases at 0.007-< 0.05μgkg-1, only two samples (sheep milk) slightly exceeded the European Union maximum level of 0.05μgkg-1. Only 6% of the samples were positive for AFM1 in a concentration range of 0.008-0.15μgkg-1. Only milk samples collected directly from farms were positive. Overall, the levels were much lower than previously reported for Southern Italy cow and sheep milk samples purchased in retail stores. The results of this work indicate a continuous improvement of the feeding techniques on dairy farms of Southern Italy, which is essential to ensure consumers' protection.

Reducing Efficiencies of the Commonly Used Heat Treatment Methods and Fermentation Processes on Aflatoxin M1 in Naturally Contaminated Fresh Cow Milk

The reducing efficiencies of the commonly used heat treatment methods and fermentation processes on aflatoxin M1 (AFM1) in Nigeria were investigated. Seventy samples of fresh cow milk from both conventional and traditional dairy cattle herds were collected and analyzed for the determination of AFM1 using Cobra-cell incorporated high performance liquid chromatography. Of these analyzed samples, 56 (80.0%) tested positive for AFM1 out of which 3 milk samples with high AFM1 concentrations were selectively pooled and subjected to varied conditions of heat treatments and fermentation processes using both indigenized and exotic strains of lactic acid bacteria (Lactobacillus bulgaricus + Streptococcus thermophilus and L. rhamnosus and L. plantarum) as starter cultures respectively. Both processes used either singly or combined, demonstrated high degrees of reducing effects on AFM1 levels. Sterilization of the milk at 121˚C and 80˚C under the same condition of time (15 - 20) min showed significant reduction of up to 58.8% (p 0.05) in the level of AFM1 when compared with the initial mean AFM1 concentration of the untreated fresh milk. The situation was however different around the boiling temperature of 100˚C at which point the level of AFM1 reduction was found to be inconsistent. The indigenized combined strains showed some slight margins of AFM1 reduction in the proportions of (20.5, 30.8 and 43.9)% over and above that of the exotic strains (17.4, 30.0 and 41.1)% in 12 h, 48 h and 72 h of fermentation respectively. Generally, fermentation alone showed lower reduction of AFM1 in milk from 24.5% to 43.9% compared with the reducing activities of (35.4 to 58.8)% when heat-treated milk samples were subsequently subjected to varied fermentation conditions.

Microbiological quality of fresh and heat treated cow's milk during storage

The aim of the present study was to evaluate the microbiological quality of raw milk from milk vending machine and heat treated milk during storage. There were analyzed 120 samples of milk (30 samples of fresh milk, 30 samples of raw milk stored 4 day at 4 °C, 30 samples of heat treated milk - 70 °C stored 4 day at 4 °C and 30 samples of heat treated milk - 100 °C stored 4 day at 4 °C). Total viable counts (TVC), coliform bacteria (CB) and microscopic filamentous fungi (MFF) were determined by microbiological analysis. Plate dilution method were used for microbiological analysis. The number of total viable counts (TVC) in fresh milk ranged from 4.08 log KTJ.mL-1 to 4.89 CFU.mL-1. TVC in raw milk after storage ranged from 5.31 log CFU.mL-1 to 6.81 log CFU.mL-1. TVC in heat treated milk with temperature 70 °C after storage ranged from 3.89 log CFU.mL-1 to 4.45 log CFU.mL-1 and TVC in heat treated milk with temperature 100 °C after storage ranged from 2.96 log KTJ.mL-1 to 3.91 log KTJ.mL-1 in heat treated milk with temperature 100 °C after storage. The number of CB were in range from 1.49 log CFU.mL-1 to 1.89 log CFU.mL-1 in fresh milk, from 1.99 log CFU.mL-1 to 2.61 log CFU.mL-1 in raw stored milk. Coliform bacteria were not present in heat-treated milk samples. The values of MFF ranged from 0 log CFU.mL-1 to 2.01 log CFU.mL-1 in fresh milk, from 1.43 log CFU.mL-1 to 3.98 log CFU.mL-1 in raw milk after storage, from 1.33 log CFU.mL-1 to 3.41 log CFU.mL-1 in heat treated milk with temperature 70 °C after storage and from 1.30 log CFU.mL-1 to 3.32 log CFU.mL-1 in heat treated milk with temperature 100 °C after storage.

Isıl İşlem Görmüş Sütlerde Salmonella Typhimurium Canlı Hücrelerinin PMA/Real-Time PCR ile Belirlenmesi

Applying different technological processes during the production of food has a lethal effect on the bacteria but DNA of these bacterial strains may cause false positive results when detected by real time PCR technique because they preserve their existence for a certain period of time. To overcome this shortcoming of the real time PCR technique, a new method has been developed in recent years, based on the removal of dead cell DNA from the medium by treatment with Propodium Monoazide (PMA) before DNA extraction. In this study, real-time PCR method was combined with PMA application for the detection of live cells of Salmonella Typhimurium in heat treated milk samples. For this purpose, milk samples inoculated with S. Tyhimurium were heat treated at different temperatures (60, 65, 70 and 75°C) and times (15, 60, 300, 900 sec) and number of live bacteria was determined comparatively by direct real-time PCR, PMA/real-time PCR and conventional cultural method. As a result, unlike the direct real time PCR technique, PMA/real-time PCR method prevents to a certain extent of false positive results from dead cells at all tested temperatures and times but higher results were obtained from PMA/real-time PCR method when compared to conventional cultural results. Therefore, further studies should be carried out to optimize the conditions of the PMA application in order to eliminate the high positive results detected by the PMA / real-time PCR method

THE OCCURRENCE OF BACILLUS CEREUS IN HEAT TREATED MILK

Fifty random heat treated milk samples (25 each, pasteurized and UHT milk samples) were collected from different supermarkets in Mansoura City, Egypt. The collected samples were examined for quantitative detection of psychrotrophicBacillus cereus strains.The obtained results revealed that the incidence of psychrotrophicBacillus species was 19(76%) in pasteurized milk samples and 3(12%) in UHT milk samples and the total psychrotrophic Bacillus counts in pasteurized and UHT milk samples, were ranged from 1X 102 –5 X 102and zero– 1cells/ml with an average counts of 2.37 X102 and 1cells/ml, respectively. While the isolated strains were purified and identified as Bacillus cereus, B. circulans, B. coagulans, B. licheniformis, B. mycoides, B. pumilus, B. subtilis, B. sphaericus and B. thuringiensis. ,

Ribonucleosides in raw milk and heat-treated milk samples from cows, sheep, goats and camels and their potential biotechnological application

The aim of the present study was to investigate the content and pattern of unmodified and modified ribonucleosides in raw and heat-treated milk from sheep, goats, and camels compared to raw and heat-treated milk from cows. The data were compared with published results from other studies on ribonucleosides in non-bovine milk. In the raw milk samples, different ribonucleoside concentrations and patterns were determined. The resulting concentration ratios and the statistical analysis indicated the species-specificity of milk ribonucleosides. In pasteurized milk samples, only slight changes in ribonucleoside concentrations were observed. The investigations have shown that sheep and goat milk are suitable sources for ribonucleosides. In particular goat milk is a valuable source of nucleo monomers and, due to the legal conditions, it is very suitable for the preparation of infant formula.

Formation of Kokumi-Enhancing γ-Glutamyl Dipeptides in Parmesan Cheese by Means of γ-Glutamyltransferase Activity and Stable Isotope Double-Labeling Studies.

Recently, γ-glutamyl dipeptides (γ-GPs) were found to be responsible for the attractive kokumi flavor of Parmesan cheese (PC). Quantitation of γ-GPs and their parent amino acids in 13-, 24-, and 30-month ripened PC samples by LC-MS/MS and stable isotope dilution analysis (SIDA), in-cheese (13)C-labeling studies, followed by analysis of the γ-glutamyl transferase (GGT) activity revealed γ-GPs to be generated most efficiently after 24 months of ripening by a GGT-catalyzed transfer of the γ-glutamyl moiety of L-glutamine onto various acceptor amino acids released upon casein proteolysis. Following the identification of milk as a potential GGT source in PC, the functionality of the milk's GGT to generate the target γ-GPs was validated by stable isotope double-labeling (SIDL) experiments. Therefore, raw and heat-treated milk samples were incubated with L-glutamine-[U-(13)C] and acceptor amino acids (X) and the hetero- (γ-Glu-[(13)C5]-X) and homotranspeptidation products (γ-Glu-Gln-[(13)C10]) were quantitated by LC-MS/MS-SIDA using γ-Glu-Ala-[(13)C3] as the internal standard. High GGT activity to generate the γ-GPs and preference for L-phenylalanine and L-methionine as acceptor amino acids were found in raw milk and milk samples heat-treated for 10 min up to a maximum of 65 °C. In comparison, GGT activity and SIDL studies performed with inoculated Lactobacillus strains, including Lactobacillus harbinensis and Lactobacillus casei identified in PC by means of 16S rRNA gene sequencing, did not show any significant GGT activity and unequivocally demonstrated unpasteurized cow's milk, rather than microorganisms, as a key factor in γ-glutamyl dipeptide generation in Parmesan cheese.

Two year survey on the occurrence and seasonal variation of aflatoxin M1 in milk and milk products in Serbia

The incidence of contamination of aflatoxin M1 (AFM1) in milk and milk products samples collected in Serbia was investigated by using the competitive enzyme linked immunosorbent assay (ELISA) technique. A total of 1438 samples composed of 678 raw milk, 438 heat treated milk and 322 milk product samples that were analyzed during the period of 2013–2014, including all seasons. The AFM1 levels exceeded the European Union maximum residue permitted amount (EU MRL) in 56.3% raw milk, 32.6% heat treated milk and 37.8% of milk product samples. Milk powders had the highest mean AFM1 concentration (0.847 μg/kg) of all types of milk products examined. Mean concentration of AFM1 in raw milk samples during the period of winter in Serbia was 0.358 μg/kg and did not significantly differ from the mean concentrations of 0.375 μg/kg during the spring. However, the AFM1 raw milk concentration in the summer (0.039 μg/kg) and autumn season (0.103 μg/kg) was significantly lower. Seasonal variation of AFM1 concentrations in heat treated milk samples followed the trend observed in raw milk. Mean raw milk AFM1 concentration has dropped down by 10 fold from 0.314 μg/kg in 2013 to 0.035 μg/kg in 2014. The fraction of raw milk samples exceeding the EU MRL has decreased from 62.3% to 11.5% by the end of 2014.

Microbiological Quality of Milk in Tanzania: From Maasai Stable to African Consumer Table

In Tanzania, pastoralists such as the Maasai and small urban farmers are responsible for the country's milk production, and 95% of the national milk supply is sold without regulation. This study was conducted using hygiene checklists and milk sampling to investigate milk quality and safety at various steps throughout the milk production chain. In regions of Dar es Salaam and Lake Victoria, 196 milk samples were collected: 109 samples of raw milk, 41 samples of packed or open served heat-treated products, and 46 samples of fermented products. Samples were taken from (i) the production level (pastoralists and urban farmers), (ii) the collection level (middlemen and depots), (iii) processors (dairies), and (iv) retailers (kiosks). Samples were analyzed for hygiene criteria (total bacteria, total coliforms, Escherichia coli, and coagulase-positive staphylococci) and foodborne pathogens such as Salmonella, enterohemorrhagic E. coli O157:H7, and Listeria monocytogenes. Adequate heating of milk for drinking was determined via heat labile alkaline phosphatase and lactoperoxidase analysis. Total bacterial counts indicated that only 67% (73 of 109) of raw milk samples and 46% (19 of 41) of heat-treated samples met national Tanzanian standards. Bulk milk samples taken from the traditional milking vessels of Maasai pastoralists had the lowest total bacterial counts: ≥ 1 × 10(2) CFU/ml. Foodborne pathogens such as E. coli O157:H7 and Salmonella were isolated from 10.1% (11 of 109) of raw milk samples but were not detected in heat-treated or fermented products, and 83% of heat-treated milk samples were lactoperoxidase negative, indicating overpasteurization. Coliforms were detected in 41% (17 of 41) of processed milk samples, thus indicating a high rate of recontamination. A progressive decrease in microbial quality along the milk production chain was attributed to departures from traditional methods, inadequate milk containers, long transport distances, lack of cooling, and lack of a basic understanding of hygiene.

Microwave milk pasteurization without food safety risk

According to nutrition science, milk and milk products are essential food for humans. The primary processing of milk includes its storage, separation, homogenization and the pasteurization process as well. The latter is a kind of heat treatment, which has been used to extend the storage life of food since the late 18th century. Although heat treatment of milk can be achieved through the use of microwave technology, the inhomogeneity of electromagnetic fields leads to an uneven distribution of temperature in the food products, therefore precluding their use in industry. The pasteurization operation is very often Critical Controll Point (CCP) according of food safety systems. In recent years our research team has developed continuously operating heat treatment pilot-plant equipment, capable of measuring and contrasting the effects of different heat treatment methods, such as thermostat-controlled water baths and microwave energy, on liquid food products. We examined and compared protein, fat and bacterial content in samples of fresh cow milk with heat-treated cow milk samples. In addition, storage experiments were carried out under a microscope and recordings made of fat globules. Our results so far show that the microwave heat treatment is equivalent to the convection manner pasteurization technology, as we found no difference between the heat-treated products.

Real-Time PCR Detection of Paenibacillus spp. in Raw Milk To Predict Shelf Life Performance of Pasteurized Fluid Milk Products

Psychrotolerant sporeformers, specifically Paenibacillus spp., are important spoilage bacteria for pasteurized, refrigerated foods such as fluid milk. While Paenibacillus spp. have been isolated from farm environments, raw milk, processing plant environments, and pasteurized fluid milk, no information on the number of Paenibacillus spp. that need to be present in raw milk to cause pasteurized milk spoilage was available. A real-time PCR assay targeting the 16S rRNA gene was designed to detect Paenibacillus spp. in fluid milk and to discriminate between Paenibacillus and other closely related spore-forming bacteria. Specificity was confirmed using 16 Paenibacillus and 17 Bacillus isolates. All 16 Paenibacillus isolates were detected with a mean cycle threshold (C(T)) of 19.14 ± 0.54. While 14/17 Bacillus isolates showed no signal (C(T) > 40), 3 Bacillus isolates showed very weak positive signals (C(T) = 38.66 ± 0.65). The assay provided a detection limit of approximately 3.25 × 10(1) CFU/ml using total genomic DNA extracted from raw milk samples inoculated with Paenibacillus. Application of the TaqMan PCR to colony lysates obtained from heat-treated and enriched raw milk provided fast and accurate detection of Paenibacillus. Heat-treated milk samples where Paenibacillus (≥1 CFU/ml) was detected by this colony TaqMan PCR showed high bacterial counts (>4.30 log CFU/ml) after refrigerated storage (6°C) for 21 days. We thus developed a tool for rapid detection of Paenibacillus that has the potential to identify raw milk with microbial spoilage potential as a pasteurized product.

Short communication: Appropriate and alternative methods to determine viable bacterial counts in cow milk samples

Farm milk consumption is reported to be inversely related to the development of asthma and atopy in children and it has been hypothesized that microorganisms in milk might contribute to this protective effect. The GABRIEL study was designed to investigate this hypothesis in a large population of European children, calling for a rapid alternative to classical culture techniques to determine bacteriological properties of milk samples. One objective was to evaluate 2 different rapid methods to determine bacteriological properties in a large number of cow milk samples collected under field conditions. BactoScan (Foss Analytical, Hillerød, Denmark), an automated standard flow cytometric method utilized for routine testing of milk quality, and TEMPO (bioMérieux, Marcy l’Etoile, France), an automated most-probable-number method, were used to assess the total viable bacterial count in farm and commercial milk samples. Both methods were compared with standard plate count method and each other. Measurements based on the TEMPO method were in good agreement with the standard plate count method and showed reliable results, whereas BactoScan results did not correlate with standard plate count measurements and yielded higher bacteria counts in heat-treated milk samples compared with raw milk samples. Most likely, these discrepant results were due to inferences with staining reactions and detection of bacteria in heat-treated milk samples. We conclude that, in contrast to the routinely used BactoScan method, the TEMPO method is an inexpensive and rapid alternative to standard culture methods suitable to assess total bacterial counts in processed and raw milk samples.

PAHs concentration in heat-treated milk samples

In this study the presence of residual levels of Polycyclic Aromatic Hydrocarbons (PAHs) in milk samples from Calabria was evaluated. A comparative analysis of PAHs concentrations was conducted on raw, pasteurized, UHT semi-skimmed and whole milk. Quantitative determination of PAHs was performed by HPLC using a fluorescence detector and analysis in HPLC–MS was conducted to confirm the presence of these compounds. Residual levels of PAHs were found in all milk samples analyzed, showing higher concentrations in pasteurized and UHT milk than in raw milk samples. The results obtained demonstrate that PAHs presence also in raw milk is dependent from environmental pollution but pasteurization and UHT treatments of milk can influence PAHs formation; the differences found between whole and semi-skimmed samples can be due to different fat content of milk.

RP-HPLC analysis of furosine and acid-soluble β-lactoglobulin to assess the heat load of extended shelf life milk samples in Austria

Recent trends to extend the shelf life of pasteurized milk, without the negative flavour normally associated with ultra-high-temperature (UHT) milk, have resulted in the development of extended shelf life (ESL) milk. Acid-soluble β-lactoglobulin (β-Lg) and furosine contents were chosen as relevant indicators for heat load of ESL milk products. RP-HPLC methods were developed using the same column (Symmetry 300™, Waters), which enabled the separation of whey proteins within 22 min; furosine was analysed using ion-pair RP-HPLC within 8 min. Electrophoresis was used as a high-throughput and cost-effective screening method to assess the impact of thermal processes on milk and to distinguish different categories of heat-treated milk samples. Liquid milk samples (n = 128; including 7 raw, 33 pasteurized, 71 ESL, and 17 UHT milk samples) were obtained from retail outlets in Austria and analysed. Only 45% of the analysed samples designated as ESL milk showed furosine contents 1800 mg·L−1 milk, which had been discussed as threshold levels for ESL milk. A further 55% of the analysed ESL milk samples had low acid-soluble β-Lg ( 40 mg·100 g−1 protein), levels comparable to the excessive heat load of UHT milk. Thus, there is an urgent need for an EU regulation to define legal limits for the tolerable heat load of ESL milk as soon as possible.

Antigenicity of heat-treated and trypsin-digested milk samples studied by an optical immunochip biosensor

Individuals with known hypersensitivity or food allergy need to avoid ingestion of provoking food. Correct labelling of allergenic content in manufactured food products and the reliable determination of its residual immunoreactivity after several processing steps are therefore a major concern for the food industry. We evaluated the applicability of a new immunochip biosensor system to reveal the allergenic profile of the whey protein β-lactoglobulin (β-LG) in its natural biological cow’s milk matrix upon processing by tryptic digestion and extensive heat treatment. Colorimetric immunochemical signals generated by gold nanoparticles (Au NPs), in particular their functional optical property based on resonance-enhanced absorption of mirror-reflected light, were directly visible to the ‘naked’ eye of the analyst without the need of any instrumentation or enzyme-substrate for read-out. By using affinity-purified polyclonal rabbit IgG against the native protein, no antigenicity was detected for tryptic fragments. Both heat-denatured whey proteins and cow’s whole milk, however, did not lose their antibody-binding capacity even after a processing time of 20 min at 95°C for the whey proteins, and 60 min at 90°C for the milk, though the immunochemical response was considerably low compared to the unprocessed β-LG. Additionally, cross-reactivity and the false positive as well as false negative predictive value of the chip system were highlighted critically.

Detection of pH 4.6 Insoluble β-Lactoglobulin in Heat-Treated Milk and Mozzarella Cheese

Different protein aggregates including beta-lactoglobulin (beta lg) were detected in the pH 4.6 insoluble fraction recovered from actual heat-treated milk samples by gel electrophoresis and immunoblotting. A competitive enzyme-linked immunosorbent assay (ELISA) using anti-beta lg polyclonal antibodies was developed to analyze the beta lg partition in the protein fractions obtained upon acidification of both milk and Mozzarella cheese at pH 4.6. According to ELISA determinations, nearly 90% of the pH 4.6 soluble beta lg included in raw milk was found in the pH 4.6 insoluble fraction of ultrahigh temperature (UHT)-treated milk. As concerns Mozzarella cheese analysis, ELISA results indicated that about 36% of the total beta lg milk content was transferred from pasteurized milk to Mozzarella cheese, whereas less than 0.5% was transferred from raw milk. The pH 4.6 insoluble beta lg proved to be a suitable indicator of the intensity of the heat treatment applied to milk. The ELISA-based detection of this parameter was suggested for quality control of both drinking milk and raw milk cheese.