High-throughput method for the determination of residues of β-lactam antibiotics in bovine milk by LC-MS/MS

This study describes the development and validation procedures for scope extension of a method for the determination of β-lactam antibiotic residues (ampicillin, amoxicillin, penicillin G, penicillin V, oxacillin, cloxacillin, dicloxacillin, nafcillin, ceftiofur, cefquinome, cefoperazone, cephapirine, cefalexin and cephalonium) in bovine milk. Sample preparation was performed by liquid-liquid extraction (LLE) followed by two clean-up steps, including low temperature purification (LTP) and a solid phase dispersion clean-up. Extracts were analysed using a liquid chromatography-electrospray-tandem mass spectrometry system (LC-ESI-MS/MS). Chromatographic separation was performed in a C18 column, using methanol and water (both with 0.1% of formic acid) as mobile phase. Method validation was performed according to the criteria of Commission Decision 2002/657/EC. Main validation parameters such as linearity, limit of detection, decision limit (CCα), detection capability (CCβ), accuracy, and repeatability were determined and were shown to be adequate. The method was applied to real samples (more than 250) and two milk samples had levels above maximum residues limits (MRLs) for cloxacillin – CLX and cefapirin – CFAP.     

Effect of heat treatments on stability of β-lactams in milk

The presence of residues of antimicrobial substances in milk may have serious toxicological and technical consequences. To date, few studies have been done to evaluate the effect of heat treatments on β-lactam residues in milk. However, the few studies that have been conducted estimate losses of antimicrobial activity under different combinations of temperature and time using microbiological methods. The aims of this study were to calculate the kinetic parameters for the degradation of β-lactam antibiotics in milk and to develop prediction models to estimate the concentration losses of these compounds in conventional dairy heat treatments. To do so, we employed a quantitative HPLC method to calculate losses in concentrations of 10 β-lactam antibiotics in milk with different combinations of temperature and time. Increasing the temperature from 60°C to 100°C decreased the half-life of amoxicillin (372 to 50 min), ampicillin (741 to 26 min), cloxacillin (367 to 46 min), and penicillin G (382 to 43 min). These increases in temperature caused further degradation in cephalosporins, which was accompanied by a decrease in half-life times to reach very low values; for instance, 4, 5, and 6 min for cefoperazone, cephurexime, and cephapirin, respectively. Kinetic equations were applied to different heat treatments used in dairy processing. Heat treatments at high temperatures and long times (e.g., 120°C for 20 min) led to a further degradation of β-lactam antibiotics with percentages close to 100% for cefoperazone and cefuroxime. In contrast, when milk was subjected to heat treatments at lower temperatures and times (e.g., 72°C for 15 s), the degradation of β-lactam in milk did not exceed 1% for the 10 antibiotics tested.     

Validation of the βeta-s.t.a.r. 1 + 1 for rapid screening of residues of β-lactam antibiotics in milk

The 2-min protocol (1 + 1) for the βeta-s.t.a.r. (manufactured by Neogen Corporation, Lansing, MI, USA) was validated at the Technology and Food Science Unit of the Institute for Agricultural and Fisheries Research according to Commission Decision 2002/657/EC. The test was very selective for the group of β-lactam compounds: the only interference found was by clavulanic acid at 2500 µg kg^?1 and above. The modified protocol (βeta-s.t.a.r. 1 + 1) detected all β-lactams with a maximum residue limit (MRL) in milk, but not all these compounds were detected at their respective MRL. The detection of cefalexin (detection capability = 6000 µg kg^?1; MRL = 100 µg kg^?1) and penethamate (detection capability = 80 µg kg^?1; MRL = 4 µg kg^?1) was especially poor, and also ceftiofur was only detected from 500 µg kg^?1 (MRL = 100 µg kg^?1). The repeatability of the reader and of the test was very good. The test was very robust: test results were not significantly influenced by small changes in the test protocol, by the milk composition or by the type of milk. The test was also suitable to test the milk of animal species other than cow. Favourable results were obtained in testing monitoring samples, in two national ring trials, and in an international proficiency test. The βeta-s.t.a.r. 1 + 1 is a very fast, simple, and reliable test that could be used at the farm level to prevent tanker milk contamination by β-lactams.     

Characterization of β-lactamase- and efflux pump-mediated multiple antibiotic resistance in Salmonella Typhimurium

Food Science and Biotechnology - This study aimed to assess the β-lactamase- and efflux pump-mediated antibiotic resistance in Salmonella Typhimurium (WT-ST), ciprofloxacin-induced...     

Effect of Sicilian pasture feeding management on content of α-tocopherol and β-carotene in cow milk

This study was performed to evaluate α-tocopherol and β-carotene contents of pasture milk under ordinary Sicilian farming conditions. Fourteen dairy farms were allocated into 2 balanced groups on the basis of cultivated (CULT) or spontaneous (SPO) pasture type feeding. Bulk milk per farm was collected 4 times from February through April at 3-wk intervals. Pasture botanical and diet composition, diet nutritional quality, milk yield and composition were estimated each time. Pasture intake levels were calculated based on feed analyses, hay and concentrate amounts fed, and milk yield and chemical composition. According to pasture intake, the farms were split into low pasture intake (LPI; <29.5% of dry matter) and high pasture intake (HPI; >29.5% of dry matter) groups. Milk samples per farm were analyzed for α-tocopherol and β-carotene contents by HPLC. The SPO group had higher levels of α-tocopherol and β-carotene in milk (0.7 and 0.3 mg/L, respectively) and milk fat (19.0 and 7.5 mg/kg fat, respectively) compared with the CULT group in milk (0.5 and 0.2 mg/L, respectively) and milk fat (14.6 and 4.9 mg/kg, respectively). High pasture intake compared with LPI increased α-tocopherol in milk fat (18.0 and 16.0 mg/kg of fat, respectively). However, only in the SPO (not in CULT), HPI compared with LPI increased milk α-tocopherol (0.8 vs. 0.6 mg/L, respectively), milk β-carotene (0.3 vs. 0.2 mg/L, respectively), and milk fat β-carotene (8.4 vs. 6.6 mg/kg, respectively). Results may be related to the different botanical composition of the respective pasture types and pasture intake. Spontaneous pasture compared with CULT contained a higher mass proportion of Asteraceae, Fabaceae, Cruciferae, Euphorbiaceae, and Malvaceae plants. Milk and milk fat α-tocopherol levels were higher on test-days (TD)-1, TD-2, and TD-4 compared with TD-3. For HPI farms, milk fat β-carotene content was higher on the first 2 TD compared with the last 2 TD. These differences could be related to plant biological stage. On Sicilian dairy farms, the highest milk α-tocopherol and β-carotene contents may be obtained feeding high levels of SPO pasture in the spring.     

Effect of prolactin, β-lactoglobulin, and κ-casein genotype on milk yield in East Friesian sheep

The effect of prolactin (PRL), β-lactoglobulin (β-LG), and κ-casein (CSN3) on milk yield was estimated in an East Friesian dairy sheep population from Old Chatham Sheepherding Company, New York. Genotypes were determined by PCR amplification followed by digestion with HaeIII and RsaI for PRL and β-LG, respectively, and by PCR amplification for CSN3. Monthly milking records and pedigree information were used to evaluate the effect of each polymorphism on milk yield. Results indicated that PRL genotype had a significant effect on milk yield. Ewes carrying one A allele produced 110.6 g more milk per day than ewes with no A alleles. There was no statistical difference between ewes with only one A allele and ewes with 2 A alleles. No association among polymorphisms at the β-LG and CSN3 loci and milk yield was found. The results presented in this study indicate that the PRL gene is a potential marker that could be used in selection programs for improving milk yield in dairy sheep.     

Optimisation and validation of a quantitative and confirmatory LC-MS method for multi-residue analyses of β-lactam and tetracycline antibiotics in bovine muscle

A multi-residue method for the determination of the β-lactam antibiotics ampicillin, cefazolin, cloxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin V and the tetracyclines chlotetracycline, tetracycline and oxytetracycline was optimised and validated in bovine muscle. The method is based on the extraction of the residues from muscle using water/acetonitrile (2/8, v/v) with subsequent use of dispersive solid-phase C18 and hexane for purification. Extracts were analysed using ultra-performance liquid chromatography (UPLC-MS/MS) coupled with the mass spectrometer in positive electrospray ionisation mode (ESI+) for all analytes. The method was validated according to the requirements of European Commission Decision 2002/657/EC. The validation results were obtained within the MRL range of 0-1.5 of the MRL, with recoveries varying from 90% to 110% and CV < 20% (n = 54), except for cloxacillin, dicloxacillin and nafcillin. However, matrix interference was observed. The decision limit (CCα) ranged from 10% to 15% of the MRL. The uncertainty measurement was estimated based on both bottom-up and top-down strategies and the uncertainty values were found to be lower than 20% of the MRL. The method has a simple extraction procedure whereby analytes are separated with reasonable resolutions in a single 11-min chromatographic run. According to the validation results, this method is suitable for monitoring β-lactams and tetracyclines according to National Program for Residue and Contaminant Control - Brazil (NPRC-Brazil) in bovine muscle.     

Effect of heating on the distribution of transforming growth factor-��2 in bovine milk

The objective of this study was to characterize the impact of heat treatments on the distribution of transforming growth factor-beta (TGF-??2) between cream and skim milk and between the casein and whey fractions of skim milk. Skimming removed 45% and 62% of the TGF-??2 from raw and pasteurized milks and only 8% of the total TGF-??2 in skimmed pasteurized milk was found in whey, compared to 37% in whey from raw skimmed milk. The TGF-??2 content of whey decreased as the heat treatment of the milk increased in intensity (thermization > pasteurization > UHT sterilization). Using milk held for 1 or 2 min at temperatures ranging from 57 to 84 °C, it was shown that TGF-??2 in the whey portion decreases at temperatures above 66 °C and becomes undetectable at temperatures higher than 76 °C. Altogether, these data on the heat-induced changes in TGF-??2 content of cream, skim milk, casein and whey reveal a potentially negative impact of certain heat treatments in developing TGF-??2-enriched fractions from milk.     

Effect of pulsed-light processing on the surface and foaming properties of β-lactoglobulin

Pulsed-light processing was used to treat β-lactoglobulin (BLG) solutions. The impact of pulsed light (PL) on the structural properties of this protein was explored through far-UV, CD spectral analysis, size exclusion chromatography, surface hydrophobicity and NMR spectroscopy. Changes on these physicochemical properties were related to surface rheology, surface tension, foam stability and foam capacity of the non-treated and treated BLG to elucidate adsorption mechanism and consequences on foaming capacity. Conformational modification of BLG was related with PL total fluence as important conformational changes increased when total fluence was higher. Consequently, adsorption rate of treated BLG at the air/water interface was faster than native BLG. Additionally, treated BLG formed highly elastic interfaces. This was found to have an impact on the foam stability. Pulsed-light treatment seemed to enhance the overall strength of the interface, resulting in more stable foams.     

Effect of glycation on the gastrointestinal digestibility and immunoreactivity of bovine β-lactoglobulin

Immunoreactivity of bovine β-lactoglobulin (β-Lg) hydrolysates obtained after a simulated gastrointestinal digestion and previously glycated via Maillard reaction with galactose, tagatose, and dextran of 10 or 20 kDa has been determined, with a view to study the effect of glycation and aggregation degree of β-Lg on its residual immunoreactivity. High levels of glycation impaired β-Lg proteolysis and, consequently, increased the IgG- and IgE-reactivities of hydrolysates, regardless of the carbohydrate used. Protein aggregation during the advanced stages of Maillard reaction had a masking effect on β-Lg epitopes, counteracting the negative effect of the lower digestibility of glycated protein on its allergenicity. Finally, the use of polysaccharides as glycation agents did not contribute to enhancement of the masking effect of the attached carbohydrate on β-Lg epitopes. These findings stress the importance of evaluating the impact of glycation on protein gastrointestinal digestibility prior to investigation of the immunoreactivity of protein Maillard complexes.     

Effect of lyoprotectants on β-glucosidase activity and viability of Bifidobacterium infantis after freeze-drying and storage in milk and low pH juices

The benefit of disaccharide protectants for maintaining viability and β-glucosidase activity of Bifidobacterium infantis UV16PR during freeze-drying and storage in different food matrices was investigated. Protectants used were cellobiose, lactose, sucrose and trehalose.     

Effect of different β-glucans on the gelatinisation and retrogradation of rice starch

Four β-glucan preparations, i.e., curdlan (CL), oat (OG), barley (BG) and yeast (YG) β-glucans, were compared for their effects on the gelatinisation and retrogradation of rice starch (RS). Rapid visco-analysis (RVA) showed that addition of any of these β-glucans significantly increased the peak, breakdown, final, and setback viscosities of RS, whereas the pasting temperatures were significantly decreased by OG or CL addition, but were unaffected by BG or YG addition. Differential scanning calorimetry (DSC) demonstrated that all the β-glucans had a negligible effect on the onset (T_o), peak (T_p), and conclusion (T_c) temperatures but slightly decreased the gelatinisation enthalpy (△H₁) of RS. Storage of all the gels at 4 °C resulted in a marked decrease in the T_o, T_p, T_c, and melting enthalpy (△H₂) values. The retrogradation ratio (△H₂/△H₁) and the phase transition temperature range (T_c–T_o) of all the gels increased with storage time. Dynamic viscoelastic measurements revealed weak gel-like behaviour of all the gels, in which their storage modulus (G′) increased and their loss tangent (tan δ) decreased during storage. Steady flow tests illustrated time-dependent shear-thinning (thixotropic) behaviour of all the gels. The hysteresis loop area and the gel hardness increased with storage time. However, the rate and extent of retrogradation and the rheological and textural changes of the RS gels were reduced by addition of any of these β-glucans. The extent of the aforementioned effects differed among the different β-glucan preparations, generally in the order OG ≈ BG > CL ≈ YG.     

Effect of milk protein composition and amount of β-casein on growth performance,gut hormones,and inflammatory cytokines in an in vivo piglet model

The objective of this work was to better understand the effect of differences in milk protein composition, and specifically, a change in β-casein to total casein in a milk-based matrix, on growth performance and metabolic and inflammatory responses using a piglet model. Three formulas were optimized for piglets, with similar metabolizable energy, total protein content, and other essential nutrients. Only the protein type and ratio varied between the treatments: the protein fraction of the control diet contained only whey proteins, whereas 2 other matrices contained a whey protein to casein ratio of 60:40, and differed in the amount of β-casein (12.5 and 17.1% of total protein). Piglets fed formula containing whey proteins and caseins, regardless of the concentration of β-casein, showed a significantly higher average daily gain, average daily feed intake, and feed efficiency compared with piglets consuming the formula with only whey protein. Consumption of the formula containing only whey protein showed higher levels of plasma glucagon-like peptide-1 and ghrelin compared with the consumption of formula containing casein and whey protein. A positive correlation was observed between postprandial time and glucagon-like peptide-1 response. The intestinal pro-inflammatory cytokine tumor necrosis factor α increased significantly in piglets fed the whey protein/casein diet compared with those fed whey protein formula. All formula-fed piglets showed a lower level of IL-6 cytokine compared with the ad libitum sow-fed piglets, regardless of composition. No significant differences in the anti-inflammatory IL-10 concentration were observed between treatment groups. Milk protein composition contributed to the regulation of piglets‘ metabolic and physiological responses, with whey protein/casein formula promoting growth performance and a different immune regulatory balance compared with a formula containing only whey protein. Results indicated no differences between treatments containing different levels of β-casein.     

Effect of β-lactoglobulin A and B whey protein variants on cheese yield potential of a model milk system

Cheese yield mainly depends on the amount and proportion of milk constituents; however, genetic variants of the proteins present in milk may also have an important effect. The objective of this research was to study the effect of the variants A and B of β-lactoglobulin (LG) on cheese yield using a model system consisting of skim milk powder fortified with different levels of a mixture containing α-lactalbumin and β-LG genetic variants (A, B, or A-B) in a 1:2 ratio. Fortified milk samples were subjected to pasteurization at 65°C for 30 min. Miniature cheeses were made by acidifying (pH = 5.9) fortified milk and incubating with rennet for 1 h at 32°C. The clot formed was cut, centrifuged at 2,600 × g for 30 min at 20°C and drained for determining cheese yield. Cheese-yielding capacity was expressed as actual yield (grams of cheese curd per 100 g of milk) and dry weight yield (grams of dried cheese curd per 100 g of milk). Free-zone capillary electrophoresis was used for determining β-LG A or B recovery in the curd during rennet-induced coagulation. The presence of β-LG variant B resulted in a significantly higher actual and dried weight cheese yield than when A or A-B were present at levels ≤0.675% of whey protein (WP) addition. Results of free-zone capillary electrophoresis allowed us to infer that β-LG B associates with the casein micelles during renneting, as shown by an increase in the recovery of this variant in the curd when β-LG B was added up to a maximum at 0.45% (equivalent to 0.675% WP). In general, actual or dried weight cheese yield increased as WP addition was increased from 0.225 to 0.675%. However, when WP addition ranged from 0.675 to 0.90%, a drastic drop in cheese yield was observed. This behavior may be because an increase in the aggregation of casein micelles with a concomitant inclusion of whey protein in the gel occurs at low levels of WP addition, whereas once the association of WP with the casein micelles reach a saturation point at addition levels higher than 0.675%, rearrangements of the gel network result in larger whey expulsion and syneresis. This knowledge is expected to be useful to maximize cheese yield and optimize processing conditions during cheese and cheese analogs manufacturing.     

Effect of β-cyclodextrin on the long-term retrogradation of rice starch

In this study, the impact of the addition of β-cyclodextrin (β-CD) on long-term retrogradation of rice starch was investigated by texture profile analysis (TPA), differential scanning calorimeter (DSC), and X-ray diffraction (XRD). Results showed that the addition of β-CD presented a crucial role in retarding the long-term retrogradation of starch. The present β-CD also significantly decreased crystallization rate (k), and increased avrami exponent (n). The increase in exponent value indicated that the addition of β-CD transformed the nucleation type due to the formation of amylose–lipid-β-CD complex. Further, the presence of β-CD produced an additional peak 5.2 Å of the gelatinized gel, and retarded crystalline type of the retrograded gel from V- to B-pattern, probably corresponding for the nucleation type transformation.     

Effect of storage conditions on the solubility and viscosity of β-glucan extracted from bread under in vitro conditions

The viscosity and solubility of β-glucan in muffins have been shown to be reduced by certain storage conditions, though the effect of storage on bread fortified with barley β-glucan concentrate has not been investigated. Therefore, this study investigated the effect of storage temperature and time (23 °C for 1, 4, and 7 d, 4 °C for 4, 7, and 14 d, and -20 °C for 1, 2, 4, and 8 wk) on the solubility and viscosity of β-glucan upon incorporation into bread at levels corresponding to 0 or 1.5 g β-glucan/serving, with or without vital gluten addition. The firmness and moisture content of bread following each storage treatment were also evaluated. The highest moisture and lowest firmness values were found in fresh bread, though these parameters were still maintained at appreciable levels upon room temperature storage of the 1.5 g β-glucan/serving bread with added gluten and at either room temperature or frozen storage for the 1.5 g β-glucan/serving bread for 4 d. If it is desirable to store bread for 7 d or more, frozen storage should be utilized in order to best maintain bread moisture and firmness levels. It is recommended that β-glucan-fortified bread be consumed fresh for greatest β-glucan solubility and viscosity, though β-glucan solubility of approximately 40% is still achievable upon frozen storage of the bread for up to 2 wk. It is still unclear, however, as to what extent of reductions in the solubility and viscosity of β-glucan would lower its physiological effectiveness. PRACTICAL APPLICATION: Previous research has demonstrated that solubility and thus viscosity of β-glucan, which is an important property associated with its health benefits can be impacted by different storage conditions applied to some bakery products, like muffins. This study demonstrates the extent of changes in the solubility and viscosity of β-glucan incorporated into bread. Therefore, storage time and temperature should be optimized to minimize changes in β-glucan for maintaining its efficacy for its health benefits.