Development of recombinase polymerase amplification (RPA) combined SYBR Green I dye assay for rapid detection of lipase-producing psychrotrophic bacteria in raw milk

The unseen risks of dairyborne disease: Disease burden, economic impact, and regulatory trends in raw milk and cheese in the US, 2000-2020

Thermal treatments are typically applied to milk to ensure microbial safety but can cause degradation of some bioactive proteins. Ultraviolet-C (UV-C) irradiation is an alternative treatment that can increase microbial safety and may better preserve some bioactive proteins. However, the effect of UV-C doses validated to provide ≥5-log reduction of bacteria in bovine milk on the preservation of bioactive milk proteins remains unknown. We determined the UV-C dose (fluence) required for ≥5-log inactivation of vegetative bacteria (E. coli O157: H7, Listeria monocytogenes, Salmonella Typhimurium and Staphylococcus aureus) and bacterial spores (Bacillus cereus, Bacillus subtilis) inoculated into raw bovine milk. We compared the retention of bioactive proteins (lactoferrin (Lf), immunoglobulin (Ig) G, IgM, IgA) after exposure to UV-C doses required for ≥5-log inactivation of vegetative bacteria and spores with commercially used high-temperature short-time (HTST) and ultra-high temperature (UHT) processing via ELISA. Achieving ≥5-log reductions of all four vegetative bacteria required 14kJ/L (termed UV-1), whereas 40kJ/L were required for ≥5-log reductions of all bacterial spores (termed UV-2). 14kJ/L retained IgG at higher levels than HTST (p<0.05) and preserved Lf and IgM at similar levels to HTST (p>0.05), while IgA preservation was lower than HTST (50.6% vs. 62.0%; p<0.05). 40kJ/L resulted in improved preservation of all bioactive proteins tested compared with UHT. The findings establish that UV-C may be a promising approach for processing whole bovine milk, particularly for enhancing bioactive protein preservation after spore-reduction treatment compared with the current industry standard (UHT).

Context: Attention to the potential impact of highly pathogenic avian influenza (H5N1, or “bird flu”) on human health increased following documentation of mammalian transmission in spring 2024 and the prevalence of H5N1 in the commercial dairy supply. The authors aim to quantify (a) the prevalence and (b) the sociopolitical correlates of public concern about H5N1 as well as support for policy action aimed at reducing its potential health risks. Methods: In a nationally representative survey of N = 831 US adults, the authors asked respondents to answer questions about their levels of concern about H5N1 transmission, beliefs about the safety of consuming unpasteurized milk products, and support for policy interventions. Results: Few Americans express high levels of concern about H5N1 health risks. People who express lower levels of concern about H5N1 and those who hold inaccurate or uncertain views toward raw milk safety are in turn less supportive of policies aimed at mitigating the spread of H5N1, as are people who endorse anti-intellectual attitudes and identify with the Republican Party. Conclusions: Moderate levels of concern about H5N1 risks could undermine pandemic preparedness. Lacking strong signals from the public, policy makers may be reluctant to pursue policies that reduce avian influenza's pandemic potential.

Antimicrobial resistance (AMR) in dairy production systems poses a major public health threat, particularly under the One Health framework. Raw milk can act as a reservoir for multidrug-resistant (MDR) pathogens, especially in regions like Khyber Pakhtunkhwa (KP), Pakistan, where unregulated antibiotic use and insufficient surveillance have promoted high-risk resistance hotspots. This study analyzed 172 bacterial isolates from raw milk, focusing on key pathogens, Staphylococcus aureus and Escherichia coli, to assess their prevalence, resistance profiles, and epidemiological distribution. A total of 172 bacterial isolates were analyzed using: (1) conventional microbiological isolation and identification, (2) antimicrobial susceptibility testing across twelve antibiotic classes, (3) molecular detection of resistance determinants (mecA, vanA, blaTEM, blaCTX-M-15, qnrS, aac(6')-Ib-cr) by PCR, and (4) geospatial modeling (GeoDa, R, ArcGIS) to identify AMR hotspots. E. coli was detected in 70.0% of samples, followed by S. aureus in 41.0%. Among E. coli isolates, 32.5% were MDR, with high β-lactam resistance (ampicillin 31.0%, amoxicillin 22.7%). S. aureus exhibited extensive MDR, with 55.2% resistant to three or more antibiotic classes; the Oxacillin-Penicillin-Tetracycline phenotype was most prevalent (28.6%), and 18.4% displayed complex hexa-resistant profiles. Vancomycin resistance was observed in 16.3% of S. aureus isolates, with 8.2% carrying vanA. Molecular screening confirmed mecA in 89.8% of S. aureus, blaTEM in 67.9% of β-lactam-resistant isolates, blaCTX-M-15 in 21.4% of E. coli, and plasmid-mediated quinolone/aminoglycoside determinants (qnrS 8.2%, aac(6')-Ib-cr 10.9%). Geospatial analysis identified three resistance hotspots across KP, with 68% of vancomycin-resistant S. aureus concentrated in northern districts and a strong correlation between β-lactam and tetracycline resistance ([Formula: see text], [Formula: see text]). Raw milk in KP harbors pathogens with multidrug resistance exceeding previous regional estimates by 2-3 fold, including resistance to last-resort antibiotics such as vancomycin. These findings emphasize the urgent need for enhanced veterinary antibiotic stewardship, targeted surveillance of resistance hotspots, improved dairy hygiene practices, and community education regarding raw milk consumption. Integrated One Health strategies are critical to mitigate the amplification and spread of AMR in dairy production systems.

Ensuring the quality of raw milk is critical for consistent cheese manufacturing, yet traditional laboratory-based testing methods are slow, labor-intensive, and impractical for decentralized rural supply chains. This study presents a portable, fully automated robotic arm system for real-time, multi-parameter milk quality assessment. The system integrates pH, total dissolved solids (TDS), temperature, density, and color sensors into a single testing cycle of under five minutes. A four-degree-of-freedom robotic arm ensures precise and repeatable probe positioning, reducing contamination and accommodating varied container types. An AI-based Support Vector Machine (SVM) classifier, trained on multi-sensor data, achieved 97.1% classification accuracy, outperforming static threshold logic, particularly in borderline cases. Environmental control features, including an LED-based optical chamber and temperature-compensated TDS readings, improved robustness in non-climate-controlled rural conditions. Laboratory tests showed high agreement with ISO-calibrated references for pH and TDS. Field trials at rural milk collection centers in Sri Lanka demonstrated over 96.5% agreement with laboratory classifications. Although individual sensor readings (e.g., pH, temperature) are rapid, the integration of automated handling, sensor switching, and AI-driven classification reduced total testing time per sample by approximately 35% compared to manual workflows. The modular design allows for scalability, easy maintenance, and adaptability to resource-limited environments. By enabling rapid, non-destructive, and chemical-free testing, the system addresses critical challenges in rural dairy networks, improving decision-making, reducing spoilage risks, and supporting higher quality assurance standards in cheese production workflows.

This study provides a regional assessment of bacterial communities and associated metabolite profiles in raw cow's milk collected from seven commercial farms across southern and northern Xinjiang, an arid inland milk-producing region of China. By comparatively analyzing psychrophilic and thermophilic bacterial assemblages, this work aimed to clarify their community composition, explore their potential environmental origins, and identify metabolites that differ between these two microbial groups with possible implications for milk quality and safety. To achieve this, we combined 16S rRNA gene sequencing, culture-based isolation of representative strains, and untargeted liquid chromatography-mass spectrometry metabolomics. At the phylum level, three dominant bacterial phyla were identified in samples from southern farms, while four dominant phyla were observed in northern farms; at the genus level, Pseudomonas and Acinetobacter were among the most abundant taxa associated with psychrophilic or thermophilic conditions. Differential metabolomic analysis revealed 59 metabolites that were consistently upregulated in samples associated with thermophilic communities and 2 that were downregulated, whereas samples associated with psychrophilic communities showed 1consistently downregulated metabolite. One metabolite feature was annotated as 3,4-methylenedioxyamphetamine based on database matching; given the inherent uncertainty of untargeted annotations, this identification requires further targeted validation before definitive safety conclusions can be drawn. Pathway enrichment analysis implicated arginine biosynthesis and galactose metabolism, which may be related to enzymatic activities involved in milk spoilage. Overall, this study offers a regionally focused characterization of psychrophilic and thermophilic microbial and metabolite patterns in raw milk from arid inland Xinjiang and identifies microbial and metabolic features that warrant further targeted investigation for dairy quality control.IMPORTANCERaw milk from arid inland regions represents a unique ecological niche in which microbial survival, metabolic activity, and contamination dynamics differ from those in humid or temperate dairy systems. By jointly characterizing psychrophilic and thermophilic microbial communities and their associated metabolite profiles, this study provides an integrated microbe-metabolite perspective on raw milk quality under arid production conditions. The coupling of microbial source tracking with metabolomic analysis offers a framework for linking contamination sources to functional and chemical consequences, extending beyond traditional taxonomic surveys. The detection of metabolites with potential toxicological relevance highlights the importance of integrating chemical risk screening into routine microbiological assessments, while also underscoring the need for targeted confirmation in food safety surveillance. More broadly, this work demonstrates how combined microbial-metabolite approaches can support evidence-based hygiene management and risk prevention strategies in dairy production systems, with particular relevance for arid and semi-arid agricultural regions.

We introduce a novel integrated analytical platform for detecting antibiotic residues in raw milk. Unlike conventional approaches, this system uniquely combines polymer inclusion membrane-based extraction with an aptamer-based electrochemical sensor built on a gold-coated screen-printed graphite electrode transducer. The extraction module provides selective separation of the target analyte from the complex milk matrix, while the sensor exploits a methylene blue-tagged aptamer whose target-induced conformational change generates a measurable electrochemical signal. The integrated sensor exhibits a limit of detection of 0.267 ± 0.016 μM for oxytetracycline, below the maximum residue limit established by the U.S. Food and Drug Administration for tetracyclines. Real-time data acquisition was demonstrated in spiked raw milk samples. This approach establishes a new strategy for antibiotic monitoring in complex liquid samples, offering online detection and point-of-use capabilities.

Bovine milk has shown a positive association with child growth rates, but access in Hyderabad, India, is variable and milk-borne hazards are of concern. The aim of this study was to investigate how milk value chains (VCs) in urban Hyderabad influence microbiological and toxicological milk safety and their intersection with child stunting. A mixed-methods approach was used, encompassing a thematic reflexive analysis of qualitative interviews conducted with 12 VC key informants and risk-based sampling of 42 milk, 24 animal feed and 20 water samples that were subjected to microbiological and aflatoxin testing. Key themes identified were financial instability, trust-based processes, milk safety concerns and power imbalances in the VCs. The microbiological analysis showed contamination in unpasteurized milk at various nodes, with high levels of total colony count, faecal coliforms, Staphylococcus aureus and yeast/mould, while water used in the VCs also showed microbial contamination. A total of 67% of dairy feed samples tested positive for total aflatoxins; all of them were within Indian regulatory limits. Opportunities for safer and more nutritious milk that could help to reduce child stunting include improved food safety practices, enhanced awareness of milk-borne hazards, institutional accountability and increased agency of VC actors. This article is part of the theme issue 'Biological, biomedical and environmental drivers of stunting'.

Brazilian artisanal cheeses are widely valued for their sensory quality and cultural relevance; however, their production frequently involves raw milk and extensive handling, conditions that may favor contamination by Staphylococcus aureus. This retrospective baseline study analyzed artisanal raw-milk cheeses collected between 2014 and 2016 in Southern and Southeastern Brazil and characterized the isolates regarding antimicrobial resistance, biofilm-forming ability, and the presence of virulence and resistance genes. A total of 147 cheese samples were collected from street vendors and markets in five Brazilian states; notably, 93% of the sampling and all S. aureus-positive samples were concentrated in Paraná, São Paulo, and Minas Gerais, Brazil. S. aureus was isolated using selective media and biochemical identification, followed by antimicrobial susceptibility testing according to CLSI guidelines. Molecular analyses were performed to detect enterotoxin genes, the tst gene, the mecA gene, and SCCmec types. Biofilm production was assessed using a microtiter plate assay. Thirteen samples (8.84%) were positive for S. aureus, yielding 25 isolates. Resistance to cefoxitin (FOX) was the most frequent phenotype (20%), followed by resistance to ampicillin, ciprofloxacin, chloramphenicol, and streptomycin (8% each). Two isolates were classified as multidrug-resistant (MDR). Genotypic analysis revealed one mecA-positive isolate (methicillin-resistant S. aureus [MRSA]; SCCmec nontypable), while four FOX-resistant mecA-negative isolates (including three SCCmec type II carriers) were classified as borderline-oxacillin-resistant S. aureus. Biofilm formation was observed among resistant isolates. Among the virulence genes investigated, only one isolate carried the sea gene, and none were positive for tstH. Although the prevalence of mecA-positive MRSA and enterotoxigenic strains was low, the detection of MDR, biofilm-producing S. aureus highlights potential risks to food safety and public health. These findings reinforce the importance of continuous surveillance, molecular monitoring of resistance determinants, and strict hygienic practices in artisanal cheese production.

This experiment was conducted to investigate the effect of using Conocarpus leaves and branches on milk production, quality, and the fatty acid profile in dairy dromedary camels. Ten Arabi camels with an average weight of 450kg, in their 4th parity, and producing approximately 2.5L of milk per day were fed either control diets or diets containing Conocarpus leaves for one month. Daily milk production, milk fat, and milk protein content increased when using Conocarpus leaves and branches (P < 0.05). The antioxidant activity of raw and fermented camel milk in the treatment containing Conocarpus was higher than that of the control (P < 0.05). According to the results, the total microbial load and mold counts in raw milk from camels fed with Conocarpus leaves and branches decreased (P < 0.05), while the population of Lactobacillus did not change compared to the control (P < 0.05). The use of Conocarpus leaves and branches in the diet increased the concentration of unsaturated fatty acids and CLA and decreased saturated fatty acids compared to the control treatment (P < 0.05). According to the results, the use of Conocarpus in the diet of dairy camels increased milk production, milk fat and protein, antioxidant activity, unsaturated fatty acids, and CLA, and reduced microbial load and molds in milk, as well as saturated fatty acids. Therefore, it may be possible to use Conocarpus at 2kg per day in dairy camel feeding.

IntroductionAntibiotic residues in milk are a growing public health concern, primarily due to their role in fostering antimicrobial resistance. Consuming contaminated milk can pose significant health risks, especially in areas with weak regulatory systems and limited public awareness.Materials and MethodsA cross‐sectional study was conducted from September 2023 to June 2024 to assess antibiotic residues in milk and stakeholder awareness. A total of 324 raw milk samples were randomly collected from dairy farms in Hawassa City (198 samples) and Wondo Genet woreda (126 samples). Samples were initially screened using the Delvotest SP sensor kit, and positives were confirmed by high‐performance liquid chromatography (HPLC). Additionally, structured questionnaires were administered to 60 dairy farmers to evaluate their knowledge and awareness of antibiotic residue.ResultsOut of 324 milk samples tested, 29 (9%) were positive for antibiotic residues using the Delvotest SP kit. (HPLC confirmed oxytetracycline (OTC) in 8 (2.5%) samples and penicillin G (PnG) in 16 (4.9%) samples. The mean OTC concentration was below the Codex Alimentarius maximum residue limit (MRL), although some samples exceeded the limit (up to 122.76 µg/L), while PnG residues (up to 142.38 µg/L) markedly exceeded the MRL, indicating potential public health risks and regulatory noncompliance. Previous treatment history, adherence to the withdrawal period, and owner awareness of drug residues were significantly associated with the prevalence of drug residues. Samples from cows with recent antibiotic treatment showed significantly higher residue levels. Among 60 dairy farmers surveyed, only 8.3% were aware of antibiotic residues in milk. All veterinarians (100%) used broad‐spectrum antibiotics for undifferentiated cases, mostly relying on clinical judgment rather than laboratory diagnosis, increasing the risk of drug residues and antimicrobial resistance.ConclusionThe presence of antibiotic residues in milk and low awareness among dairy farmers in the study areas highlight a critical public health gap. Farmers’ awareness of antibiotic residues was mainly influenced by education level, while veterinarians showed good knowledge of antibiotic risks, but gaps remain in laboratory diagnosis, record keeping, and regulatory understanding, indicating the need for targeted training and stronger residue control.

In this work, we investigated the safety characteristics and probiotic proprieties of Enterococcus faecium (E. Faecium) strains isolated from raw milk of indigenous Brown Atlas cows reared in the Tafoughalt region (Berkane, Morocco). Eleven isolates were identified as E. faecium by 16S rRNA gene sequencing and subjected to an in-vitro evaluation approach including antibiotic susceptibility, safety related enzymatic activities, tolerance to gastrointestinal stress conditions, antioxidant capacity, and antibacterial activity against selected foodborne pathogens. All isolates were susceptible to clinically relevant antibiotics, such as ampicillin, vancomycin, gentamicin, tetracycline, and chloramphenicol, and none exhibited evidence of acquired or transferable antibiotic resistance. Safety assessment revealed the absence of haemolytic, DNase, and gelatinase activities in all strains. The isolates demonstrated marked tolerance to acidic conditions (pH 2.0-3.0) and bile salt concentrations (0.15%-0.30%), indicating their ability to survive simulated gastrointestinal environments. All strains inhibited the growth of Staphylococcus aureus ATCC 29213 in a strain and concentration dependent manner, while moderate inhibitory activity was observed against Escherichia coli ATCC 25922. Neutralisation and enzymatic treatments indicated that the antibacterial activity was primarily associated with organic acid production and, to a lesser extent, with proteinaceous compounds, suggesting the involvement of bacteriocin like substances. In addition, the isolates exhibited moderate, strain dependent antioxidant activity, ranging from approximately 15% to 27%. Among the evaluated strains, E. faecium EF89 displayed the most favourable combination of safety attributes and probiotic properties. Collectively, these results show that E. faecium strains isolated from Brown Atlas cow milk in Eastern Morocco are safe and promising candidates for use in functional foods and food biopreservation, supporting the valorisation of indigenous dairy resources.

Microbiological and physicochemical quality of raw materials and Cabacinha artisanal cheese: a traditional stretched-curd cheese from Vale do Jequitinhonha, Minas Gerais, Brazil.

Since 2021, avian influenza A(H5N1) clade 2.3.4.4b viruses have spread widely among wild birds and domesticated poultry in the United States, with sporadic spillover into mammals. During November 2024-January 2025, 19 domestic cats in Los Angeles County, California, became ill after consumption of commercially purchased raw milk, raw meat, or raw pet food; nine cats tested positive for influenza A(H5N1) virus (clade 2.3.4.4b genotype B3.13). Overall, 139 persons were exposed to the 19 infected cats, and all were monitored for symptoms. Although 30 persons reported influenza-like illness symptoms, none received a positive influenza A(H5) reverse transcription-polymerase chain reaction (RT-PCR) test result. In April 2025, the Los Angeles County Department of Public Health and CDC invited all exposed persons to participate in an influenza A(H5N1) serosurvey to determine whether transmission of influenza A(H5N1) virus occurred, including in those without symptoms. Sera from 25 (18%) of the 139 exposed persons were tested. Among these, antibodies specific to A(H5N1) clade 2.3.4.4.b (antigenically similar to the clade 2.3.4.4.b influenza A[H5N1] virus isolated from the infected cats) were detected in serum from one veterinary professional, who was asymptomatic. This person did not use respiratory or eye protection during the exposure, did not report influenza-like illness after the exposure, and reported no other known risk factors for A(H5N1) infection. These findings represent serologic evidence of possible transmission of influenza A(H5N1) clade 2.3.4.4.b virus from a domestic cat to a human, highlighting concerns about potential cat-to-human transmission of influenza A(H5N1) virus and the importance of infection control practices in veterinary settings.

Evaluation of PCR-Based H5N1 Influenza Detection Methods in Milk from an Interlaboratory Comparison Study Demonstrating Method-Dependent Sensitivity Variability.

Raw goat milk was explored as a source of lactic acid bacteria (LAB) with potential probiotic properties. Following 16S rRNA gene sequencing, Lactiplantibacillus plantarum PP101-STR and Lactococcus lactis PP104-STR were selected for further probiotic assessment. L. plantarum PP101-STR exhibited broad antagonistic activity against both Gram-positive and Gram-negative pathogens, especially Salmonella enterica ATCC 13312 and maintained high viability (> 83%) under simulated gastric conditions (pH 2.0-3.0) and bile salt exposure. This strain also demonstrated high cell surface hydrophobicity, along with strong auto-aggregation and co-aggregation capacities, which were associated with significantly enhanced adhesion to Caco-2 and HT-29 cell lines and effective competitive exclusion of pathogenic bacteria (p≤0.05). In addition, L. plantarum PP101-STR displayed strong antioxidant activity, as determined by total phenolic content, FRAP, DPPH, and ABTS assays, exceeding that of L. lactis PP104-STR. Its cell-free supernatant (CFS) exhibited notable α-glucosidase and α-amylase inhibitory activities, indicating potential for glucose regulation. Antiproliferative evaluation revealed that L. plantarum PP101-STR markedly suppressed colorectal cancer cell growth in a dose-dependent manner. Importantly, low lactate dehydrogenase release (8.11-8.86%) and maintained viability in MRC-5 cells indicated minimal membrane damage and low cytotoxicity toward normal cells, supporting a non-lytic mechanism potentially involving apoptosis-related pathways. On the basis of the present finding, L. plantarum PP101-STR demonstrates strong probiotic potential and multiple biologically relevant activities in vitro, supporting its further investigation in applications related to intestinal health.

Molecularly imprinted metal-organic framework-based potentiometric sensor for sensitive detection of torsemide in dietary supplements and milk.

Extended-spectrum β-lactamase-producing K. pneumoniae (ESBL-PKP) and carbapenemase-producing K. pneumoniae (CPKP) are major causes of life-threatening infections in humans and animals. In 2019, the bacterium was responsible for 100,000 to 200,000 deaths globally attributable to antimicrobial resistance (AMR). Ethiopia, home to the largest livestock population in Africa, is characterized by close human-animal interaction and common practices of raw milk and meat consumption that favor the transmission of resistant bacteria. Despite these risks, the epidemiology of ESBL-PKP and CPKP has not been investigated using a one-health approach. This study aimed to determine the prevalence of ESBL-PKP and CPKP among humans, animals, and the environment in northwest Amhara, Ethiopia. A cross-sectional study was conducted from January 1 to August 30, 2025. A total of 972 samples (508 humans, 158 animals, and 306 environmental samples) were analyzed. Sociodemographic and related data were collected from human participants using a structured questionnaire. Urine, blood, environmental swab, wastewater, and rectoanal mucosal swab samples were collected and processed for the isolation of K. pneumoniae using standard microbiological methods. ESBL-PKP and CPKP were identified phenotypically using a combination disk diffusion test and a modified carbapenem inactivation method, respectively, in accordance with the Clinical Laboratory Standard Institute guidelines. Data were analyzed using SPSS version 25 and R software. The prevalence of ESBL-PKP and CPKP among human isolates (n = 71) was 49.3% (35/71, 95% CI: 38-60.7) and 40.8% (29/71, 95% CI: 30.2-52.5), respectively. Meanwhile, the prevalence of ESBL-PKP and CPKP among environmental isolates (n = 56) was 39.3% (22/56, 95% CI: 27.6-52.4) and 25% (14/56, 95% CI: 15.5-37.7), respectively. No ESBL-PKP or CPKP were detected in animal samples. All ESBL-PKP and CPKP isolates were multidrug resistant (MDR) with more than 70% co-resistance to ciprofloxacin, co-trimoxazole, gentamicin, and tetracycline. Among K. pneumoniae isolates, 93% (56/61) of humans, and 73.2% (41/56) of environmental isolates were MDR. Though the number of animal isolates was small, all K. pneumoniae isolates from animals (n = 5) were MDR. Nearly all (40/43, 93%) CPKP isolates were resistant to the last-resort antibiotics, ceftazidime-avibactam and meropenem-vaborbactam. Neonates had higher odds of culture-positive K. pneumoniae compared with adults aged 18-49 years (adjusted odds ratio [AOR]: 27.79, 95% CI: 3.47-222.42, p = 0.002). The high prevalence of ESBL-PKP and CPKP in humans and hospital environments, coupled with widespread multidrug resistance, underscores the presence of a serious public health threat. The nearly universal resistance of CPKP isolates to the last-resort antibiotics is particularly alarming. However, molecular characterization was not performed, limiting the inference about specific carbapenemase genes. Cleaning of the targeted hospital environment and implementing integrated AMR surveillance under a one health framework are essential to prevent cross-sectoral spillover.

Abstract Milk is one of the most widely consumed staple foods worldwide. Its increasing commercial importance both increases the need for quality classification and creates a breeding ground for fraudulent practices, such as adding water to dilute. In this work, some milk varieties, including raw (unheated), skimmed, semi-skimmed, and fat (whole) milk, as well as boiling raw milk, were analyzed using the Time Domain Nuclear Magnetic Resonance (TD-NMR) methodology in parallel with the Microwave (MW) dielectric method. The study also examined the effects of adding water to certain kinds of milk. Spin lattice (T 1 ) and spin–spin (T 2 ) relaxation times and dielectric permittivity constants Ɛ 1 and Ɛ 2 were used to classify milk, and the Inverse Laplace Transform (ILT) method was used to examine these relaxation times further. Furthermore, the effects of temperature categorization on milk were examined using temperature-dependent T 1 and T 2 measurements. As a result, the relaxation times of different types of milk are listed from longest to shortest: skimmed milk, raw milk, semi-skimmed milk, fat (whole) milk, and raw milk (boiled). It was found that T 1 and T 2 values increased with the increase in temperature. And also, skimmed milk has a greater Ɛ 1 value compared to fat (whole) milk. Furthermore, it has been observed that adding water to milk increases the dielectric constant (Ɛ 1- Ɛ 2 ) as the water concentration increases. Overall, our results demonstrate that the TD- NMR and MW approach is a sensitive and efficient method for identifying various milk kinds and detecting the presence of water adulteration.