Search Results (131)

Fluorescence emission-excitation matrices for cow milk samples with different fat contents in the range of 0.05–10% and a constant protein content of 3%, as well as for butter and extracted milk components such as casein and lactose, have been measured using a spectrofluorometer. The influence of the increased fat content on the shape of the fluorescence spectra of milk has been studied. In addition, fluorescence spectra measured for serial dilutions of high-fat milk with water and skim milk, along with aqueous dilutions of skim milk, have shown that the fluorescence diagnostics of fat and protein content in milk can be implemented using excitation at only two wavelengths: 280 and 320 nm. The optimal spectral ranges proposed for detecting the content of milk components via fluorescence measurements can be useful when designing UV LED-based fluorescence analyzers of milk composition. Full article

This study reports the plant-mediated synthesis of copper-oxide-decorated magnetic iron oxide composite (CuO@Fe₃O₄) nanoparticles using Dolomiaea costus extract and their application as nanocarriers for β-galactosidase immobilization. The fabricated nanocomposite exhibited favorable physicochemical properties, achieving an immobilization efficiency of 83%, with enhanced thermal and pH tolerance compared to the free enzyme. Kinetic analysis revealed a modest increase in Km and a 31% decrease in Vmax after immobilization, while maintaining 69% of the catalytic activity, confirming the system’s suitability for industrial lactose hydrolysis. Reusability and storage tests showed 79% retained activity after five cycles and 77% after 60 days at 4 °C. In milk hydrolysis, the immobilized enzyme achieved 77% conversion within 3 h, following pseudo-first-order kinetics. Biocompatibility was evaluated using HepG2 cells via the MTT assay. BDH, MDH, and ABC maintained high cell viability across the tested dilution range of 25–100% (v/v), indicating no detectable cytotoxic effect under the experimental conditions, whereas cisplatin showed marked cytotoxicity with an IC₅₀ of 14.98 µg/mL. These findings demonstrate that the green-synthesized CuO@Fe₃O₄ support provides a safe, reusable, and magnetically recoverable platform for β-galactosidase immobilization, offering a promising sustainable strategy for producing lactose-free dairy products. Full article

Persistent organic pollutants such as polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and polychlorinated biphenyls (PCBs) remain a public health concern due to their persistence, bioaccumulation, and potential health effects. Human breast milk is an important biomonitoring matrix for assessing maternal and infant exposure to lipophilic contaminants. This pilot study aimed to determine concentrations of PCDD/Fs, dioxin-like PCBs (dl-PCBs), and non-dioxin-like PCBs (ndl-PCBs) in breast milk samples collected from five lactating women residing in the Greater Poland Province and to explore potential determinants of exposure. Following participant recruitment, sample collection, and questionnaire-based assessment performed by the authors, breast milk samples were analyzed at the accredited Laboratory of Trace Analysis (Cracow University of Technology, Poland) using isotope dilution gas chromatography coupled with tandem mass spectrometry. Toxic equivalency values (TEQ) were calculated using World Health Organization 2005 toxic equivalency factors (WHO-TEFs). WHO-PCDD/F-TEQ ranged from 0.096 to 0.22 pg/g fresh weight. Median lipid-normalized WHO-PCDD/F-TEQ and total WHO-PCDD/F-PCB-TEQ concentrations were 3.5 and 4.7 pg/g lipid, respectively, remaining below the European Food Safety Authority (EFSA) reference level of 5.9 pg/g lipid; only one sample exceeded this threshold (6.2 pg/g lipid). Lipid-normalized WHO-PCB-TEQ correlated positively with maternal age (ρ = 0.949, p = 0.0389). The observed values were within the lower range reported in recent European studies. The congener patterns suggest a combination of chronic exposure to combustion by-products and long-term bioaccumulation of historical industrial pollutants. Although limited by the small sample size, this exploratory study provides preliminary regional biomonitoring data supporting future environmental exposure research. Full article

Milk adulteration is a common form of food fraud, particularly in high-value dairy products from small ruminants. A frequent practice involves dilution with water, often combined with the addition of sugars to mask physicochemical changes and avoid detection during routine quality control. This study aimed to develop an analytical approach for detecting combined adulteration in goat and sheep milk involving both water dilution and sucrose addition. Controlled experiments were conducted by diluting milk samples with water (1–15%) followed by the addition of sucrose solutions. Changes in physicochemical parameters, including fat, protein, total solids, lactose, density, freezing point depression, mineral content, and pH, were evaluated using an automated milk analyzer. In parallel, a suspected adulterant powder was characterized using conventional chemical analysis, ICP-AES, and HPLC-RI, revealing a composition predominantly of sucrose (91.4% w/w) with elevated sodium levels. Sucrose in milk samples was subsequently quantified using an enzymatic spectrophotometric method. Water dilution reduced protein, total solids, and density, while sucrose addition partially restored these parameters, masking adulteration effects. However, sucrose was reliably detected at concentrations above 0.1%. The proposed workflow may provide a practical and cost-effective complementary tool for routine dairy authenticity surveillance and fraud prevention. Full article

Foot-and-Mouth Disease (FMD) is caused by the FMD virus. Indirect Sandwich Enzyme-Linked Immunosorbent Assay (IS-ELISA) was standardized to characterize the FMD serotype “O” virus. Total protein content in the guinea pig serum (whole serum), ammonium sulfate precipitated guinea pig serum (ASPGPS) protein and ion-exchange-based purified guinea pig serum (IEGPS) protein was measured as 52 µg/mL, 24 µg/mL and 10 µg/mL respectively. The whole serum of guinea pigs and rabbits showed the 1:32 and 1:64 anti-FMD serotype “O” virus neutralizing antibody titers, while the anti-FMD serotype “O” virus neutralizing antibody titer was 1:128 in the IEGPS proteins. IEGPS protein with 1:128 neutralizing antibody titers were used as capture/trapping antibodies in the standardization of the assay. The IEGPS protein 1:1000 diluted with 10 µg/mL of protein content was found to be optimum for capture/trapping antibodies. To cover residual blank spaces, different available blocking buffers were evaluated and Skimmed Milk Solution 5% in Phosphate-Buffered Saline (PBS5%) proved best amongst blocking buffers. Coating of 1:1000 diluted IEGPS at 37 °C for 1 h followed by storage at 4 °C for overnight was best for incubation time. FMD serotype “O” virus 1:100 diluted was optimum in IS-ELISA. Similarly rabbit anti-FMD serotype “O” virus specific immune serum 1:10,000 diluted and goat anti-rabbit IgG horseradish peroxidase conjugate 1:4000 diluted were found to be optimum during the standardization of the assay. Lastly ELISA plates proved to be best amongst the available plates for assay. In each experiment, the plateau region, test background and plate background were recorded. Lastly it became possible for the establishment of an optimized and potentially cost-effective IS-ELISA requiring further diagnostic validation in research and diagnostic laboratories in the country. Full article

This study aimed to establish an indirect ELISA for detecting the avian reovirus (ARV) epidemic strain xj-1.1 by using the purified recombinant protein pET-σC as the coating antigen. To optimize assay performance, key parameters were systematically evaluated, including antigen-coating concentration, serum dilution, blocking reagent and duration, serum incubation time, and the dilution and reaction time of the HRP-conjugated secondary antibody. The optimized conditions identified were a coating antigen dilution of 1:100, serum dilution of 1:1600, coating at 37 °C for 1 h followed by overnight incubation at 4 °C, and blocking with 5% skim milk for 2 h. The optimal serum incubation time was 1.5 h, with the secondary antibody diluted 1:1000 and incubated for 2 h, followed by a 20-min color development step. The cut-off value for distinguishing positive and negative samples was determined to be 0.121. Validation of the assay demonstrated favorable specificity, sensitivity, and repeatability, indicating that the developed indirect ELISA provides a reliable method for detecting ARV xj-1.1 infection. Full article

According to the EFSA Report on Zoonoses (2024), yersiniosis was classified as the fourth most commonly reported zoonosis in humans in 2023, with a 13.5% increase in yersiniosis infections compared to 2022. In 2024, the findings were consistent with the 2020–2023 trend. Isolation and identification of enteropathogenic Yersinia is difficult and time consuming, especially when examining food and environmental samples. Among them, Y. pseudoturbeculosis poses a challenge due to the lack of a single selective medium for all bioserotypes. Therefore, faster methods for the detection of Yersinia spp. need to be implemented into the praxis. Rapid identification of pathogens in food or at the time and location of the epidemiological outbreak (point-of-care testing) enables either prevention of the outbreak or early stage diagnosis and prompt decisions. The loop-mediated isothermal amplification (LAMP) is increasingly coming to scientists’ attention as a robust and rapid methodology for pathogen detection in laboratories with limited resources and equipment. The aim of current study is to evaluate, for the first time, the sensitivity of the LAMP protocol based on colorimetric detection in the visible spectrum in comparison with that of the digital droplet PCR (ddPCR). For this aim, a series of decimal logarithmic dilutions of the pathogen Y. pseudotuberculosis in artificially contaminated raw goat milk was used. One commercial LAMP kit with two different dyes (one dsDNA-binding and one Mg²⁺-sensitive) was compared to the sensitivity of the detection to ddPCR. The results obtained revealed a high sensitivity of the kit for detection of DNA isolated from artificially contaminated milk samples in the following range: visible detection based on visible color change—3.1 × 10⁴ mL (violet dye) and 3.4 × 10³/mL (blue dye); detection with gel electrophoresis—2.0 × 10¹/mL (violet dye) and 3.4 × 10²/mL (blue dye). The enumeration of the DNA copies in the same samples was performed with ddPCR, with a detection limit of 2.0 × 10¹/mL. Our results indicate the potential and the possible applicability of the LAMP method for rapid and sensitive visual detection of Y. pseudotuberculosis in raw goat milk. The presented ddPCR protocol can be used for highly sensitive identification and enumeration of Y. pseudtuberculosis in raw goat milk. In conclusion, the conducted comparison is of importance for future implementation of LAMP protocols for on-field analysis near the sampling site and point-of-care or laboratory diagnostics of Y. pseudtuberculosis after the successful validation procedure of an appropriate LAMP protocol. Full article

A bovine milk reference material was produced containing amoxicillin with a target concentration at the maximum residue limit. For the evaluation and characterization of the reference material, an isotope dilution–liquid chromatography/tandem mass spectrometry method was developed and validated. This material was prepared by collecting milk samples from cows treated with an appropriate amoxicillin injection, followed by freeze-drying, dilution, and homogenization of the authentic samples. The amoxicillin reference material, prepared in accordance with ISO Guide 35, was homogeneous and stable. It was assigned a certified value of (4.10 ± 0.13) μg/kg with a relative standard deviation between 2.93 and 5.29%, which meets the maximum residue limit (MRL) requirement of 4.0 μg/kg established by the United States, the European Union, and China. This reference material can be used for method validation and provides internal quality control assurance for the detection of amoxicillin. Full article

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a highly contagious and fatal disease. Accurate detection in the early stages of an outbreak relies on molecular methods, but serological monitoring at the population level is also crucial for assessing the extent of exposure and past infections. This experiment developed an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against ASFV, using three ASFV RNA polymerase subunits (H359L, C147L, and D339L) as coating antigens. The recombinant proteins were successfully expressed in Escherichia coli and purified. Using a checkerboard titration method, we systematically optimized key assay parameters, determining the optimal coating conditions to be a mixture of H359L, C147L, and D339L at a volume ratio of 1:2:2, with individual concentrations of 1 μg/mL, 0.4 μg/mL, and 0.5 μg/mL, respectively. Other optimized parameters included a serum dilution of 1:200, a blocking buffer containing 5% skim milk, and specific incubation conditions for the secondary antibody and substrate. The cut-off value was established at 0.430 ($\overline{x}$ + 4SD) based on 30 negative sera. The established triple-antigen indirect ELISA exhibited high sensitivity (detecting positives at dilutions up to 1:3200) and excellent specificity (no cross-reactivity with antisera against CSFV, PRRSV, PRV, PCV2, and PEDV. Both intra and inter assay repeatability were confirmed, with coefficients of variation ranging from 1.020% to 7.600%. Validation with 123 clinical serum samples demonstrated a 96.75% concordance rate with a commercial kit. In conclusion, the three-antigen indirect ELISA established in this study exhibits high specificity and sensitivity, making it suitable for serological surveillance and exposure assessment of ASFV antibodies. It can be combined with molecular detection for epidemiological investigations and integrated prevention and control measures. Full article

Background: Exposure to environmental pollutants, especially endocrine-disrupting chemicals, disproportionately affects vulnerable populations like pregnant women, lactating mothers, and preterm infants. This study aimed to assess the detection patterns of DiNP-, DEP-, and DEHP-related metabolites in maternal urine and breast milk, examine agreement between matrices, and explore maternal factors associated with phthalate exposure. Methods: Fifty-five mothers who delivered at ≤32 gestational weeks and whose infants were hospitalized in the Neonatal Intensive Care Unit (NICU) were enrolled. Breast milk and urine samples were analyzed using a validated isotope-dilution LC–MS/MS method. Urinary phthalate metabolite concentrations were adjusted for specific gravity. Linear mixed-effects models with a random intercept for mother were used to examine associations between urinary and breast milk phthalate metabolite concentrations, assess temporal changes, and evaluate the influence of breast milk lipid content. Results: DEHP and DiNP metabolites were detected in nearly all maternal urine samples. Breast milk contained predominantly primary metabolites (MEHP and MiNP), while secondary oxidative metabolites were rarely detected. Urine concentrations consistently exceeded breast milk concentrations. Urinary and breast milk phthalate concentrations were not correlated across sampling periods, indicating limited matrix concordance. Conclusions: Mothers of very preterm infants experience sustained phthalate exposure in the postpartum period; however, limited metabolite transfer to breast milk indicates that maternal urine remains the preferred biomonitoring matrix for assessing systemic phthalate exposure. Breast milk phthalate profiles exhibit compound-specific temporal changes and appear largely independent of concurrent urinary exposure biomarkers. Full article

Chymosin is an aspartyl protease widely used in the food industry for milk coagulation during cheesemaking. Although recombinant production has replaced natural extraction from rennet, current heterologous expression systems still face significant challenges, including low solubility, costly purification steps, and enzyme instability after activation. To address these limitations, we sought to develop a more efficient and economical production strategy for bovine chymosin by cloning its codon-optimized prochymosin A gene into Escherichia coli using the pBAD/His vector under the control of the L-arabinose-inducible PBAD promoter. Overexpression of the recombinant gene resulted in the formation of inclusion bodies, which were solubilized with NaOH and refolded by dilution and pH adjustment with glycine. The folded prochymosin was then activated by acidification. To simplify the downstream process and improve enzyme recovery, different immobilization strategies were explored to combine activation, purification, and immobilization in a single step. While polymeric agarose-based supports showed low immobilization efficiency (<20%) due to pore clogging, magnetic nanoparticles completely overcame these limitations, achieving nearly 100% immobilization yield and retaining about 85% of enzymatic activity. This integrated magnetic-based approach provides a cost-effective and scalable alternative for the production and stabilization of active chymosin. Full article

Reticuloendotheliosis virus (REV) is an immunosuppressive virus in poultry that can cause acute reticular neoplasms, chronic lymphoid tumors, stunting syndrome, and secondary infections. In many countries, the lack of effective vaccines has resulted in a high prevalence of REV infections and substantial economic losses. Enzyme-linked immunosorbent assay (ELISA)-based antibody detection is an important tool for monitoring the REV prevalence in poultry farms. ELISA coating antigens generally consist of either whole virus or viral protein; however, most commercially available REV antibody ELISA detection kits use whole virus as the coating antigen, which limits their applicability in certain diagnostic and research settings. In this study, the gp90 protein from a dominant REV strain was expressed and purified using 293F suspension cell eukaryotic expression system. Using recombinant gp90 protein as the coating antigen, an indirect ELISA for detecting gp90 antibodies (gp90-ELISA) was developed. After optimization, the optimal conditions were as follows: coating antigen concentration of 4 µg/mL with overnight incubation at 4 °C; blocking with 5% skim milk at 37 °C for 1.5 h; serum dilution of 1:200 with incubation at 37 °C for 45 min; secondary antibody dilution of 1:1000 with incubation at 37 °C for 30 min; and color development using TMB substrate at room temperature in the dark for 10 min. The cut-off value was defined as an OD₄₅₀ ≥ 0.22 for positive samples and <0.22 for negative samples. The developed gp90-ELISA specifically detected REV-positive sera at a maximum serum dilution ratio of 1:3200. Intra- and inter-assay variation coefficients were ≤10%, indicating that the gp90-ELISA had good specificity, sensitivity, and reproducibility. Laboratory serum testing showed that the gp90-ELISA successfully detected sera from chickens immunized with the gp90 protein or infected with REV. Furthermore, analysis of clinical serum samples demonstrated 100% concordance between the gp90-ELISA results and a commercial whole-virus-coated ELISA kit. These results indicate that the gp90-ELISA is a reliable supplementary method to whole-virus-coated ELISA and has potential utility in disease surveillance and evaluation of immune responses. Full article

This study examines the critical role of whole milk or milk replacer as a liquid diet (LD) with 15% solids in combination with different physical forms of starter as a solid diet (SD), on performance, health, and behavior of pre-weaned calves. Sixty male Holstein calves were used in a 2 × 2 factorial design, and randomly distributed into the following treatments: Whole milk powder diluted to 12.5% of solids and enriched with 25 g/L of milk replacer to achieve 15% solids, associated with either micropelleted stater (WM+micro) or texturized stater (WM+text); milk replacer diluted to 15% solids associated with either micropelleted stater (MRmicro) or texturized stater (MRtext). Starter intake and, consequently, total DMI were higher in the MRtext treatment compared to WM+micro. Calves fed texturized starter showed higher DMI, starter intake time, and rumination time. Calves in the WM+Text group showed greater ADG compared with MR treatments, regardless of starter type. Calves fed WM+ presented a lower number of days with fecal score ≥2, and the first day of diarrhea occurred at older ages. Calves fed MR showed more health challenges but similar feed efficiency with WM+, while texturized starter increased intake, eating duration, and rumination compared with micropelleted starter. Full article

Donkey milk is increasingly recognized as a functional food due to its unique nutritional profile and richness in bioactive compounds. This longitudinal observational study investigated changes in both chemical composition (total solids, protein, fat, lactose, and ash) and immune-active proteins (lactoferrin, α-lactalbumin, β-lactoglobulin, and lysozyme) across lactation. A total of 153 donkey milk samples were collected from five farms from very early (1–3 days in milk) to late lactation (30–210 days in milk). Chemical composition was determined using mid-infrared spectroscopy, while the concentrations of the immune-active proteins were determined by ELISA Quantitative Sandwich. Chemical analysis showed high values in colostrum, including total solids (10.13%), protein (3.1%), and ash (0.73%), which declined progressively during lactation to 8.45%, 1.14%, and 0.64%, respectively. Fat varied modestly, between 0.55 and 0.25%, while lactose remained stable at 5.75–6.41%. In parallel, bioactive proteins measured between 31 and 210 days exhibited distinct trajectories. Lactoferrin increased from 0.07 to 0.14 mg/mL, α-lactalbumin peaked mid-lactation at 2.58 mg/mL (compared with 1.91 mg/mL early and 2.25 mg/mL late), β-lactoglobulin declined from 0.84 to 0.55 mg/mL, and lysozyme decreased from 0.95 mg/mL early to 0.64 mg/mL late. Across lactation, we observed dilution of total solids and protein, relatively stable lactose and fat, and distinct trajectories of lactoferrin, α-lactalbumin, β-lactoglobulin, and lysozyme, indicating that donkey milk modulates rather than loses its protective protein profile. These results refine reference values for donkey milk and support its nutraceutical relevance for human nutrition and health. Full article

This study aimed to evaluate the supplementation of aqueous extract of Moringa oleifera (AEMO) as a natural ruminal modulator to improve the lactation performance of ewes. The AEMO was prepared by chopping Moringa oleifera leaves and diluting them in distilled water (163.3 g DM/L). Twelve ewes were used in a replicated 3 × 3 Latin square, with periods of 14 days (assessments on the last five days of each period). Treatments were as follows: 20 mL of water as Control, 20 mL of AEMO (20-AEMO), and 40 mL of AEMO (40-AEMO). Ewes were milked twice a day (7:30 a.m. and 2:30 p.m.). Diet corresponds to grain mix (at 3% of BW) and hay ad libitum. We determined the intake, digestibility, fermentative measurements, metabolic measurements, and milk production and composition. Intake and digestibility were not affected by AEMO. Milk yield and the concentrations of fat, protein, and lactose were numerically lower in ewes supplemented with 20-AEMO. A linear decrease in milk protein yield was observed when the highest extract level (40-AEMO) was used. Ruminal pH did not differ among treatments; however, there was a tendency for reduced acetate and increased propionate concentrations, which corresponded with a non-significant numerical decrease in methane estimates in 40-AEMO group. Blood and urinary parameters were not affected by AEMO supplementation. Inclusion of Moringa extracts as an additive in lactating ewes diet does not affect intake and nutrient digestibility, but tends to affect ruminal fermentation and microbial synthesis, with possible changes in methane emission estimation, and impair milk protein production. Therefore, we recommend studies with different extract concentrations to investigate possible effects on rumen fermentation and the synthesis of milk compounds. Full article