Fresh Bovine Milk Research Articles

Efficient secretory expression of human milk Osteopontin in Komagataella phaffii

Osteopontin (OPN), a bioactive milk protein in breast milk, has diverse applications in food and medicine. OPN is predominantly extracted from fresh bovine milk; however, the concentration of OPN in bovine milk is exceedingly low. To address these issues, in the present study, we used Komagataella phaffii for the recombinant expression of human OPN (hOPN). The results of host cell screening revealed that X33 cells were more suitable than other K. phaffii hosts for hOPN expression, yielding a titer of 340.5 μg/L. Subsequently, it was observed that the extracellular production of hOPN significantly increased to 8.02 mg/L from strain XPSA01 using the PAOX1 promoter and the hybrid signal peptide PROSCW10-α. Meanwhile, the co-expression of transcription factors, translation factors, and molecular chaperones resulted in a 2.94-fold enhancement in extracellular hOPN production compared with that in XPSA01, resulting in a titer of 23.6 mg/L. Finally, the above strategies were combined to obtain the recombinant strain XPSA01-CP, which achieved titers of 35.6 and 128.5 mg/L hOPN in shake-flask fermentation and 3-L bioreactor, respectively. To the best of our knowledge, this is the highest extracellular yield of hOPN ever reported.

Dilution Reduces Sample Matrix Effects for Rapid, Direct, and Miniaturised Phenotypic Antibiotic Susceptibility Tests for Bovine Mastitis.

The time-consuming nature of current methods for detecting antimicrobial resistance (AMR) to guide mastitis treatment and for surveillance, drives innovation towards faster, easier, and more portable technology. Rapid on-farm testing could guide antibiotic selection, reducing misuse that contributes to resistance. We identify challenges that arise when developing miniaturized antibiotic susceptibility tests (AST) for rapid on-farm use directly in milk. We experimentally studied three factors: sample matrix (specifically milk or spoiled milk); the commensal bacteria found in fresh bovine milk; and result time on the performance of miniaturised AST. Microfluidic "dip-and-test" devices made from microcapillary film (MCF) were able to monitor Gram-negative bacterial growth colourimetrically even in the presence of milk and yoghurt (used to simulate spoiled milk samples), as long as this sample matrix was diluted 1:5 or more in growth medium. Growth detection kinetics using resazurin was not changed by milk at final concentrations of 20% or lower, but a significant delay was seen with yoghurt above 10%. The minimum inhibitory concentration (MIC) for ciprofloxacin and gentamicin was increased in the presence of higher concentrations of milk and yoghurt. When diluted to 1% all observed MIC were within range, indicating dilution may be sufficient to avoid milk matrix interfering with microfluidic AST. We found a median commensal cell count of 6 × 105 CFU/mL across 40 healthy milk samples and tested if these bacteria could alter microfluidic AST. We found that false susceptibility may be observed at early endpoint times if testing some pathogen and commensal mixtures. However, such errors are only expected to occur when a susceptible commensal organism is present at higher cell density relative to the resistant pathogen, and this can be avoided by reading at later endpoints, leading to a trade-off between accuracy and time-to-result. We conclude that with further optimisation, and additional studies of Gram-positive organisms, it should be possible to obtain rapid results for microfluidic AST, but a trade-off is needed between time-to-result, sample dilution, and accuracy.

Sensitive analysis of 18 aminoglycosides in bovine milk using hydrophilic interaction chromatography combined with tandem mass spectrometry

A sensitive and selective method was developed using hydrophilic interaction chromatography combined with tandem mass spectrometry to detect 18 aminoglycosides in bovine milk. Aminoglycosides were extracted from milk using a mixture of trichloroacetic acid, ammonium acetate, ethylenediaminetetraacetic acid, and sodium chloride, and then isolated using an Oasis HLB solid-phase extraction cartridge. We evaluated the performance of chromatographic columns with different stationary phases and optimized the mobile phases to achieve satisfactory separation of the analytes. The limits of detection and quantification were 0.05–2 µg/kg and 0.1–5 µg/kg, respectively. The recoveries of the method ranged from 78.9% to 101.1%, with intra- and inter-day relative standard deviations of less than 8.1% and 9.0%, respectively. This method was applied to 30 milk samples collected from markets, and neomycin (16.8 µg/kg) was detected in one fresh bovine milk sample. The proposed method provides a powerful tool for the surveillance of aminoglycosides in bovine milk.

Validation of the KangarooSci® Aerobic Count Plate for the Enumeration of Meosphilic Aerobic Bacteria in Selected Foods and on Stainless Steel Environmental Surfaces: AOAC Performance Tested MethodSM 062301.

The KangarooSci® Aerobic Count Plate is a sample-ready culture medium system, for direct counting of aerobic bacteria colonies after 48-72 h of incubation. The KangarooSci Aerobic Count Plate was evaluated for AOAC® Performance Tested Methods SM certification. The KangarooSci Aerobic Count Plate (ACP) was evaluated through matrix studies and product consistency/stability study and robustness testing. For the matrix study, nine food products (nonfat dry milk powder, fresh raw bovine milk, pasteurized liquid bovine milk, fresh raw ground beef, frozen uncooked chicken breast, cooked shredded pork, apple juice, ice cream, and fresh strawberries), and one environmental surface (stainless steel) were evaluated following the KangarooSci ACP product instructions and compared to the ISO 4833-1:2013-pat 1, Microbiology of food and animal feeding stuffs-Horizontal methods for the enumeration of microorganisms-Part 1: Colony count at 30 °C by the pour plate technique reference standard. The product consistency and stability testing evaluated three separate production lots of the KangarooSci ACP. The robustness testing examined three test parameters, volume of sample plated, incubation time, and incubation temperature using a factorial study design. Results from the matrix study demonstrated equivalent performance between the KangarooSci ACP and the ISO 4833-1:2013 reference standard. The product consistency and stability testing showed that the performance of the assay was equivalent over time up to 12 months and between production lots. Minor changes to the operational test conditions showed no significant impact on performance during the robustness testing. The KangarooSci ACP is an effective method for aerobic plate count for all matrixes evaluated. The KangarooSci Aerobic Count Plate allows for fast, reliable enumeration of aerobic bacteria. Utilizing the alternative method takes up less space in incubators, requiring no sample spreader, and requires less consumables compared to the reference method.

The mechanism and significance of the conversion of xanthine dehydrogenase to xanthine oxidase in mammalian secretory gland cells

The conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) occurs only in mammalian species. In fresh bovine milk, the enzyme exists predominantly as the oxidase form, in contrast to various normal organs where it is found primarily as the dehydrogenase: the mechanism of conversion to the oxidase in milk remains obscure. A systematic search for the essential factors for conversion from XDH to XO has been performed within fresh bovine milk using the highly purified dehydrogenase form after removal endogenous oxidase form by fractionation analysis. We find that conversion to the oxidase form requires four components under air: lactoperoxidase (LPO), XDH, SCN−, and substrate hypoxanthine or xanthine; the contribution of sulfhydryl oxidase appears to be minor. Disulfide bond formation between Cys-535 and Cys-995 is principally involved in the conversion, consistent with the result obtained from previous work with transgenic mice. In vitro reconstitution of LPO and SCN− results in synergistic conversion of the dehydrogenase to the oxidase the presence of xanthine, indicating the conversion is autocatalytic. Milk from an LPO knockout mouse contains a significantly greater proportion of the dehydrogenase form of the enzyme, although some oxidase form is also present, indicating that LPO contributes principally to the conversion, but that sulfhydryl oxidase (SO) may also be involved to a minor extent. All the components XDH/LPO/SCN− are necessary to inhibit bacterial growth in the presence of xanthine through disulfide bond formation in bacterial protein(s) required for replication, as part of an innate immunity system in mammals. Human GTEx Data suggest that mRNA of XDH and LPO are highly co-expressed in the salivary gland, mammary gland, mucosa of the airway and lung alveoli, and we have confirmed these human GTEx Data experimentally in mice. We discuss the possible roles of these components in the propagation of SARS-CoV-2 in these human cell types.

Determination of nutritional health indexes of fresh bovine milk using near infrared spectroscopy

Bovine milk is one of the most complete foods that exist. During the last decades, milk FA have shown to improve human health due to the reduction in risk of cardiovascular disease and related pathologies. The aim of this study was to evaluate the feasibility of near infrared spectroscopy (NIRS) reflectance analysis to predict the nutritional value, fatty acid (FA) composition, and health index of fresh milk from dairy cows of pastoral systems. The prediction of Atherogenicity and Thrombogenicity indexes, along with other FA ratios in fresh milk samples by NIRS were precise and accurate. In addition, the calibration model obtained by NIRS provides an opportunity for the routine quantification of milk’s healthy FA such as omega-3 and conjugated linoleic acid (CLA), with applications in the dairy industry for food labeling, and at the farm level for management of the dairy cow’s diet.

Design and synthesis of a novel nanocomposite based on magnetic dopamine nanoparticles for purification of \u03b1-amylase from the bovine milk

In this paper, a novel nanocomposite based on magnetic nanoparticles decorated by dopamine were reported. Three modified magnetic nanocomposites by dopamine were offered with different type of linkers. The mentioned magnetic nanocomposites were applied to separate α-amylase protein from fresh bovine milk. All of the magnetic nanocomposites were characterized and investigated by using Fourier-transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, field-emission scanning microscope, X-ray diffraction pattern, and vibrating-sample magnetometer analyses. To investigate the purifying application, sodium dodecyl sulfate polyacrylamide gel electrophoresis, one-dimensional isoelectric focusing gel electrophoresis, and alpha-amylase activity assay were employed. With paying attention to factors such as yield of purification and concentration of separated protein by each of magnetic nanocomposite, it could be concluded that the length of linkers played an important role in α-amylase protein separation. According to the results, the best separation and purification of α-amylase protein with 49.83% recovery and 40.11-fold purification efficiency was related to longest length linker, 1,4-butanediol diglycidyl ether, because of considerable conjugation with nanocomposite. Also, docking calculation has shown that the binding energy is − 1.697 kcal/mol and ΔG = − 6.844 kcal/mol which result that the interaction process between dopamine and α-amylase protein is spontaneous.

Enhanced Neuroprotective Effects of Epicatechin Gallate Encapsulated by Bovine Milk-Derived Exosomes against Parkinson's Disease through Antiapoptosis and Antimitophagy.

Epicatechin gallate (ECG) is a main effective catechin widely existing in natural plants and food, with well-known health benefits. The present study first designed a new exosome-based delivery system for ECG and examined its neuroprotective effects on a rotenone (Rot)-induced Parkinson's disease (PD) model in vitro. Exosomes (Exo) were isolated from fresh bovine milk, and their average size was 85.15 ± 2.00 nm. ECG was encapsulated into Exo by a sonication method, and the loading efficiency of ECG-loaded exosomes (ECG-Exo) was 25.96 ± 0.45%. The neuroprotective effects of ECG-Exo were evaluated on Rot-induced SHSY5Y cells and compared with free ECG. Cell viability, cellular reactive oxygen species, apoptosis rate, and the expressions of caspase-3, Bax, Bcl-2, parkin, PINK1, and Atg5 were determined. Our results showed that Exo delivered ECG successfully into SHSY5Y cells and exhibited enhanced neuroprotective effects. ECG-Exo might inhibit SHSY5Y cell damage induced by Rot through antiapoptosis and antimitophagy.

Semi-industrial production of a minimally processed infant formula powder using membrane filtration

Infant formula (IF) is submitted to several heat treatments during production, which can lead to denaturation or aggregation of proteins and promote Maillard reaction. The objective of this study was to investigate innovative minimal processing routes for the production of first-age IF powder, thus ensuring microbial safety with minimal level of protein denaturation. Three nutritionally complete IF powders were produced at a semi-industrial scale based on ingredients obtained by fresh bovine milk microfiltration (0.8 and 0.1-µm pore size membranes). Low-temperature vacuum evaporation (50°C) and spray-drying (inlet and outlet temperatures of 160 and 70°C, respectively) were conducted to produce the T- formula with no additional heat treatment. The T+ formula was produced with a moderate heat treatment (75°C for 2 min) applied before spray-drying, whereas the T+++ formula received successive heat treatments (72°C for 30 s on the milk; 90°C for 2-3 s before evaporation; 85°C for 2 min before spray-drying), thus mimicking commercial powdered IF. Protein denaturation and Maillard reaction products were followed throughout the production steps and the physicochemical properties of the powders were characterized. The 3 IF powders presented satisfactory physical properties in terms of aw, free fat content, glass transition temperature, and solubility index, as well as satisfactory bacteriological quality with a total flora <103 cfu/g and an absence of pathogens when a high level of bacteriological quality of the ingredients was ensured. Protein denaturation occurred mostly during the heat treatments of T+ and T+++ and was limited during the spray-drying process. The IF powder produced without heat treatment (T-) presented a protein denaturation extent (6 ± 4%) significantly lower than that in T+++ (58 ± 0%), but not significantly different from that in T+ (10 ± 4%). Although T- tended to contain less Maillard reaction products than T+ and T+++, the Maillard reaction products did not significantly discriminate the infant formulas in the frame of this work. The present study demonstrated the feasibility of producing at a semi-industrial scale an infant formula being bacteriologically safe and containing a high content of native proteins. Application of a moderate heat treatment before spray-drying could further guarantee the microbiological quality of the IF powders while maintaining a low protein denaturation extent. This study opens up new avenues for the production of minimally processed IF powders.

Casein micelles from bovine Milk exerts Neuroprotection by stalling metabolic complications linked to oxidative brain injury.

Caseins are the most abundant milk proteins in mammalian species and are assembled in supra-macromolecular structures called micelles. In this study, the microstructural properties, particle size, and elemental composition of isolated casein from bovine milk and its therapeutic effect on oxidative and cholinergic activities linked to dementia in oxidative brain injury were investigated. SEM analysis of the isolated casein micelles from skimmed fresh bovine milk revealed spherical colloid aggregates, while TEM analysis revealed dispersed spherical particles with a mean size of 63.15 ± 4.77nm. SEM-EDX analysis revealed clusters of carbon, oxygen, sulfur, copper, sodium, magnesium, potassium, iron, and selenium. Treatment of oxidative brain injury with the isolated casein micelles led to elevated levels of GSH, SOD, catalase, ENTPDase, 5'NTPase activities, while concomitantly suppressing MDA, cholesterol, HDL-c levels, acetylcholinesterase and lipase activities. Treatment with the isolated casein micelles led to complete depletion of oxidative generated lipid metabolites, while deactivating oxidative-activated lipid metabolic pathways. These results indicate the microstructural properties, particle size, elemental composition, and antioxidant neuroprotective effect of casein micelles from bovine milk. Thus, demonstrating the nutraceutical properties of milk in the management of oxidative induced cognitive impairment.

Monolayers of bovine milk‐derived stem cells present with distinctive multinucleated syncytial cells following self‐induced differentiation.

In an ongoing series of studies, our laboratory has been examining the biology of a putative stem cell population harvested from fresh bovine milk samples. Among the observations previously reported have been the long‐term survival of ductal‐like monolayer colonies which clearly undergo differentiation and generate several cell types consistent with the normal mammary gland. These include cells which produce large fat globules as well as supporting cells. In addition, after several month of maintenance, these colonies eventually generate localized 3D tissue features representative of mammary gland ducts and acinar structures. Now we are reporting on the development within these cultures of a subset of multinucleated syncytial cells. These cells appear to form in specific regional distributions and in smaller colonies are often spaced out relative to each other suggestive of a regulatory function in tissue feature development. In these studies, we have examined the relative development of milk ductal syncytial cells and sought evidence of a regulatory role using morphologic analysis and labeling studies. In addition, we are also examining the potential to artificially induce such syncytial cell formation using the cAMP activator forskolin. Preliminary experiments in this regard have focused on the effects of forskolin on the ductal cell line MCF10A. To date, results suggest that forskolin will in fact increase the incidence of syncytial cells in these monolayers. It remains to be examined whether this will also occur in our milk samples and then whether these syncytia are functionally equivalent to those already spontaneously occurring. While formation of syncytial cells is a common observation in breast ductal cancers and precancerous tissues, there is no indication in our bovine milk samples of any neoplastic transformation and the position, developmental time‐frame and relatively ubiquitous observation of these in every sample suggest a potentially functional regulatory role for these cells.

Characterization of difference in structure and function of fresh and mastitic bovine milk fat globules.

Characterization of milk fat globule (MFG) was performed to investigate the difference in MFG membrane (MFGM) between fresh and mastitis Holstein Friesian cow milk. Lipid distribution investigated by exogenous phospholipids using microscopy showed higher phospholipid content in fresh compared to mastitic MFGM. Xanthine oxidase assay indicative of membrane impairment revealed lower activity in mastitic samples compared to fresh globules. Of note, significantly higher roughness of globule surface and zeta potential was observed in mastitis compared to fresh globules. Influence of globule membrane on the interaction with L. fermentum demonstrated preferential adhesion of bacteria to fresh compared to mastitic globules including enhanced extent of binding. Results of the present study provides an insight of the interfacial changes occurring at the globule surface as well as highlighting the importance of selective bacterial interaction with milk components for the potential development of functional food with relevance to human health.

Enhanced Antibacterial Activity of Lactoperoxidase-Thiocyanate-Hydrogen Peroxide System in Reduced-Lactose Milk Whey.

The product of the lactoperoxidase system (LPOS) has been developed as a preservative agent to inhibit foodborne bacteria, but its action was, heretofore, limited to several original compounds in milk. This research was conducted to analyze the application of the lactoperoxidase system against Escherichia coli in fresh bovine milk and its derivative products to determine the strength of antibacterial activity. Lactoperoxidase was purified from bovine whey using the SP Sepharose Big Beads Column. The enzymatic reaction involving lactoperoxidase, thiocyanate, and hydrogen peroxide was used to generate the antibacterial agent from LPOS. This solution was then added to milk, skimmed milk, untreated whey, reduced-LPO whey, reduced-lactose whey, and high-lactose solution containing E. coli at an initial count of 6.0 log CFU/mL. LPOS showed the greatest reduction of bacteria (1.68 ± 0.1 log CFU/mL) in the reduced-lactose whey among the products tested. This result may lead to a method for enhancement of the antimicrobial activity of LPOS in milk and derived products.

Prevalence of Staphylococcus aureus contamination in bovine milk from local dairy herd in Oyo State, Nigeria

Unhygienic practices, handling and transportation; pooling of milk from unscreened mastitic cows and indiscriminate prophylactic, therapeutic and growth promotion use of antibiotics in Nigerian cattle production could result in dissemination of resistant bacteria including Staphylococcus aureus and leading to food poisoning. This work aims to determine the prevalence of Methicllin Resistant Staphylococcus aureus (MRSA) in bovine milk from local herds. A total of 165 fresh bulk milk samples were collected from local herds at Ibarapa, Oyo and Oke-Ogun in Oyo State for isolation of Staphylococcus aureus. Standard bacteriological methods were used to determine the bacterial load, isolate and characterize Staphylococcus aureus in fresh bovine bulk milk from dairy herds in Oyo State. Antibiotics sensitivity of the isolates was determined using disc diffusion method. Bacteriological assay revealed high total mean bacterial counts ranging from 1.5+0.069 to 6.1+23.19 log cfu/ml, with the highest contamination in 13.9% of milk from Oke-Ogun, followed by 4.2% from Oyo and Ibarapa respectively. There was a prevalence of 31.5% coagulase positive Staphylococcus aureus with 7.9% isolates indentified as MSRA. The isolates showed very high resistance to several antibiotics with the highest resistance to augmentin (88.5%) and the highest sensitivity to ofloxacin (96.1%). This study established the presence of Staphylococcus aureus in milk from local herds with a prevalence of 33.1%. This signified the high risk of mastitis in the dairy cows from the study area. The high prevalence of MRSA is of food safety concern and could have resulted from indiscriminate use of antibiotics. Enforcement of milk hygiene practices, constant screening and treatment of mastitic cow as well as effective milk pasteurization are hereby suggested to ensure food safety.Keywords: Animal, Antimicrobial agents, Bulk milk, Staphylococcus aureus

Extending Shelf Life of Indonesian Soft Milk Cheese (Dangke) by Lactoperoxidase System and Lysozyme.

Dangke, a type of fresh soft cheese made of bovine and buffalo milk, is a traditional dairy product used in South Sulawesi, Indonesia. It is prepared from fresh milk using the conventional method, which easily destroys the quality. This study was conducted to assess whether using lactoperoxidase system and lysozyme as preservative agents could suppress the growth of bacteria in dangke. The pH value, total microbial count, and hardness of dangke were determined to measure the quality. Lactoperoxidase and lysozyme were purified from fresh bovine milk, and their purity was confirmed using SDS-PAGE. The combination of lactoperoxidase system and lysozyme was able to remarkably suppress the total microbial count in dangke from 7.78 ± 0.67 to 5.30 ± 0.42 log CFU/ml during 8 h of storage at room temperature. Preserving dangke in this enzyme combination affected its hardness, but there was no remarkable change in the pH value. Results of this study may provide knowledge to utilize a new method to preserve the quality of dangke.

Protective effect of bovine milk against HCl and ethanol–induced gastric ulcer in mice

The purpose of this study was to investigate the gastroprotective effects of bovine milk on an acidified ethanol (HCl-ethanol) mixture that induced gastric ulcers in a mouse model. Mice received different doses of commercial fresh bovine milk (5, 10, and 20 mL/kg of body weight) by oral gavage once a day for 14 d. One hour after the last oral administration of bovine milk, the HCl-ethanol mixture was orally intubated to provoke severe gastric damage. Our results showed that pretreatment with bovine milk significantly suppressed the formation of gastric mucosa lesions. Pretreatment lowered gastric myeloperoxidase and increased gastric mucus contents and antioxidant enzymes catalase and superoxide dismutase. Administration of bovine milk increased nitrate/nitrite levels and decreased the malondialdehyde levels and the expression of proinflammatory genes, including transcription factor nuclear factor-κB, cyclooxygenase-2, and inducible nitric oxide synthase in the stomach of mice. These results suggest that bovine milk can prevent the development of gastric ulcer caused by acid and alcohol in mice.

Cheese Manufacturing and Bioactive Substance Separation: Separation and Preliminary Purification of cAMP from Whey.

Cheese consumption has been gradually increased in China. However, both the manufacturing process of cheese and the utilization of its main by-product were not well developed. Based on the sensory evaluation, Box-Behnken Design (BBD) was performed in the present study to optimize the cheese processing, which was proved more suitable for Chinese. The optimal parameters were: rennet 0.052 g/L, start culture 0.025 g/L and CaCl2 0.1 g/L. The composition analysis of fresh bovine milk and whey showed that whey contained most of the soluble nutrients of milk, which indicated that whey was a potential resource of cyclic adenosine-3’, 5’-monophosphate (cAMP). Thus, the cAMP was isolated from whey, the results of high-performance liquid chromatography (HPLC) analysis showed that the macroporous adsorption resins (MAR) D290 could increase the concentration of cAMP from 0.058 µmol/mL to 0.095 µmol/mL. We firstly purified the cAMP from the whey, which could become a new source of cAMP.

Liquid organic fertilizer sprayed on shoots of Phaseolus vulgaris stimulates arbuscular mycorrhiza colonization and phosphate solubilizing microorganisms in soil

ABSTRACTProcessed organic agricultural byproducts such as liquid organic fertilizer (LOF) are promising alternatives that can improve crop productivity while reducing mineral fertilizer use and improving sustainability. The effects on beneficial soil organisms, such as arbuscular mycorrhizal (AM) fungi and phosphate solubilizing microorganisms (PSM), caused by LOF spraying on shoots is poorly understood. Therefore, we evaluated how AM colonization and soil PSM are affected by LOF spraying on common beans (Phaseolus vulgaris L.) grown in a greenhouse. The LOF was obtained by anaerobically fermenting a mixture of fresh water, cattle manure, bovine milk, sugarcane molasses and mineral salts. LOF was applied twice on the plant shoots. Fresh and dry mass of root and shoot, P content, P accumulation in the shoots and AMF spore counts in the soil were not LOF dose dependent. However, we found that a 5% LOF application stimulated AM colonization and led to dose-dependence for calcium and aluminium phosphate solubilizing bacteria and fungi. These results show that LOF sprayed on shoots causes responses on soil communities. We therefore endorse the use of LOF in low input agriculture.

Development and application of a high-throughput sample cleanup process based on96-well plate for simultaneous determination of 16 steroids in biological matrices using liquid chromatography-triple quadrupole mass spectrometry.

A novel high-throughput sample pretreatment system was developed by the integration of protein precipitation (PP), phospholipid removal (PPR), and hollow fiber liquid-phase microextraction (HF-LPME) into two simple 96-well plates and a matching 96-grid lid. With this system, 16 steroids were separated from biological matrices of plasma, milk, and urine and analyzed by liquid chromatography-triple quadrupole mass spectrometry. In the tandem sample cleanup process, the prepositive PP and PPR step preliminarily removed some of the interferences from the biological matrices. The following HF-LPME step kept the residual interference out of the hollow fiber and enriched the steroids in the hollow fiber to achieve high sensitivity. By a series of method optimizations, acetonitrile was chosen as the crash solvent for PP and PPR. A mixture of octanol and toluene (1:1 v/v) was used as the acceptor phase for HF-LPME. The extraction was conducted at 80 rpm for 50 min in a donor phase containing 1 mL 20% sodium chloride at 25 °C. Under these conditions, the limits of detection for the 16 steroids were 3.6-300.0 pg(.)mL(-1) in plasma, 3.0-270.0 pg·mL(-1) in milk, and 2.2-210.0 pg(.)mL(-1) in urine. The recoveries of the 16 steroids were 81.9-97.9% in plasma (relative standard deviation 1.0-8.0%), 80.6-97.7% in milk (relative standard deviation 0.8-5.4%), and 87.3-98.7% in urine (relative standard deviation 1.0-4.9%). Further, the integrated 96-well platform of PP, PPR, and HF-LPME enabled us to run this assay in an automatic and high-throughput fashion. The reliability of the method was further corroborated by evaluation of its applicability in plasma and urine samples from volunteers and fresh bovine milk from local dairy enterprises.

Concentration characteristics of bovine β-defensin 1 and 2 in fresh bovine milk and infant formula

The aim of this study was to determine the concentration of defensins BNBD-1 and BNBD-2 in fresh bovine milk and infant formula, and to compare the defensin concentrations of bovine milk and human milk. Abundant BNBD-1 and BNBD-2 was found in fresh bovine colostrum. However, compared to fresh bovine milk, BNBD-1 and BNBD-2 concentrations in infant formula were very low. The BNBDs concentration ranges were also less than β-defensins in human milk. The BNBD-2 concentration in fresh bovine milk was higher than that of BNBD-1.