Rapid determination of the various native forms of vitamin B6 and B2 in cow's milk using ultra-high performance liquid chromatography.

As the formation of pyridoxal phosphate, the active cofactor of vitamin B6, is dependent on riboflavin 5-phosphate, we propose a fast and simple ultra-high performance liquid chromatography method for the simultaneous determination of the native B6 vitamers pyridoxal, pyridoxine, pyridoxamine, their mono phosphorus esters and 4-pyridoxic acid as well as vitamin B2 as riboflavin and its phosphorus ester riboflavin 5-phosphate in milk. Separation was achieved under 6.0min by reversed-phase and pH gradient elution. Sample preparation was optimized regarding various acids and pH levels. Changes in those parameters led to significant deviations of sample matrix breakdown efficiency. The optimized method was then validated regarding specificity, accuracy, precision, linearity, range, detection and quantification limits. As the method performed satisfactory, is was used to study commercial liquid cow's milk (n=31), regarding effects of the employed preservation technique (pasteurization, extended shelf-life, ultra-high temperature) on the composition and content of B6 and B2 vitamers. In cow's milk, vitamin B6 mostly consists of pyridoxal and its phosphate ester, with pyridoxal phosphate being the bulk component. The catabolite of the vitamin B6 metabolism, 4-pyridoxic acid was present in significant amounts in all studied samples, with up to 2.69µmolL-1. Vitamin B2 was present as riboflavin and its phosphate ester up to 12.86µmolL-1.

Dietary supplementation with nonconventional feeds from the Middle East: assessing the effects on physicochemical and organoleptic properties of Awassi sheep milk and yogurt.

Increased feed costs affect the livelihoods of dairy sheep farmers in the Middle East. Farmers endure high risks with large fluctuations in the price of grain used as animal feed, which is further affected by drought and declining range productivity. Using agricultural by-products and treated straw or vetch grazing for supplementing sheep diets would provide resource-poor dairy farmers with increased options to reduce feed costs, but the effects of such feeds on the quality of yogurt (the main product) need to be better understood. Two experiments were conducted to evaluate these effects. The first trial evaluated alternative diets using locally available feedstuffs, including agricultural by-products, compared with traditional diets used by dairy sheep farmers, and was conducted on-station at the International Center for Agricultural Research in Dry Areas (ICARDA, Tel Hadya, Aleppo, Syria). Milking Awassi ewes (n=56) were used to test 6 alternative diets against a traditional control diet containing barley, wheat bran, and barley straw. The 6 alternative diets contained 4 or more of the following ingredients: barley, sugar beet pulp, molasses, cotton seed cake, wheat bran, urea-treated wheat straw, and barley straw. Ewes on one of the alternative diets grazed vetch pasture, whereas ewes on the control diet and the 5 alternative diets grazed native range pasture. The milk fat content was higher in diets containing urea-treated straw. Yogurt firmness and adhesiveness were significantly lower in energy-rich diets (e.g., the control diet) and in the diets rich in soluble sugar (molasses). The effects of diet on yogurt color and on citric and succinic acid contents were significant. A yogurt produced from the milk of the group grazing on vetch was the most yellowish in color, which is appealing to Syrian consumers. The content of citric acid tended to be higher in yogurts produced from diets containing molasses. The second trial was conducted on 3 farms in northern Syria to assess an alternative diet (1 of the 6 tested in the first trial) on 15 milking ewes compared with the farmer's traditional diet (control). The alternative diet increased yogurt firmness and adhesiveness by 7 to 9% and 10 to 16%, respectively. The use of nonconventional feeds available in the region enhances yogurt quality, may reduce requirements for expensive grains, and thus, increase farmers' livelihoods by targeting expanding markets with better quality products.

Prospects for using nonconventional feeds in diets for Awassi dairy sheep in Syria.

High feed costs are major obstacles for resource-poor dairy sheep farmers in West Asia, along with large fluctuation in grain and straw prices. Farmers need low-cost diets using locally available feeds that can provide sufficient milk of good quality. Two experimental trials were conducted on Awassi milking ewes to evaluate nonconventional and balanced low-cost diets against the traditional unbalanced diet used by farmers (control) on the total yields (milk, fat, protein, and total solids) and milk composition (fat, protein, total solids, and lactose), an important indicator of milk quality. The first trial was conducted at the research station of the International Center for Agricultural Research in the Dry Areas (ICARDA, Aleppo, Syria) to test 6 low-cost balanced diets using locally available feeds and agro byproducts against the control diet. Each diet was tested on 8 ewes that were kept on pasture as a basal diet, but received different supplements, including barley, wheat bran and nonconventional feeds (urea-treated wheat straw, molasses, sugar beet pulp, and cotton seed cake). Five balanced diets enhanced the total yields of milk, fat, protein, and total solids, in 2 cases, significantly. These diets increased total milk yield by 17.7 to 50.2% and decreased supplement feeding costs by 43% compared with the control. However, milk composition remained unaffected. The second trial was conducted on 3 different farms in northern Syria to assess in each farm a low-cost balanced diet on milking ewes (n=15) in comparison to the farmer's control (n=15). The balanced diet was a modification requested by farmers of the best performing diet in the on-station trial. Confirming the first trial's research results, the balanced diet outperformed the control in total yields; for instance, it increased total milk yield by 28 to 40% and raised net income by 30%, without affecting milk composition. Both trials showed that using locally available nonconventional feedstuffs, such as molasses, integrated into balanced dairy sheep diets can decrease feed costs of resource-poor farmers, while enhancing total yields of milk and milk constituents without compromising milk quality components. This will greatly improve the profitability of dairy sheep production in dry areas.

UPLC analysis of free amino acids in wines: profiling of on-lees aged wines.

The evolution of free amino acid (FAA) profiles intrinsic to on-lees aged white wines was determined by ultra performance liquid chromatography (UPLC™). On basis of the AccQ.Tag™ method as a commercialized amino acid analysis solution for HPLC, a new protocol for dedicated amino acid analysis using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) for pre-column derivatization was established by method transfer onto UPLC™ conditions. Since AQC derivatives enable both fluorescence (AccQ.Tag™ method) and UV detection, the performed method transfer additionally included changing to a more versatile UV detection. Emphasizing enhanced performance of UPLC™, the newly established protocol facilitated rapid and reliable separations of 24 amino acids within 23 min, hence proved to be superior compared to the original HPLC protocol due to significant improvements in resolution and reduced runtime. Applying UV detection enabled adequate quantifications of AQC amino acid derivatives at µM level (LOQs from 0.12 to 1.10 µM), thus proved sufficient sensitivity for amino acid profiling in wine samples. Moreover, this compiled methodology was successfully applied to monitor the changes of FAA concentrations in four distinct sets of on-lees aged white wines (fermented with different yeasts) at three progressing ripening periods, each (control, 3 and 6 months aging). For the control wines, the applied winery yeast significantly affected total FAA amounts (1450-1740 mg L(-1)). During maturation, the proceeding yeast autolysis implied a rather complex impact on FAAs, yielding total FAA excretions up to 360 mg L(-1). However, the magnitude for increases of specific FAAs (up to +200%) highly depended on the individual amino acids as well as on the applied fermenting yeast. Given the overall complexity of yeast autolysis in winemaking, the application of efficient LC techniques such as UPLC™ may indeed contribute as a valuable tool in wine research for product monitoring and characterization of intrinsic developments during wine maturation.

Characterization of amino acid profiles of culture media via pre-column 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization and ultra performance liquid chromatography.

The AccQ.Tag™ method, as a well-established protocol for amino acid analysis combining derivatization procedure, dedicated HPLC separation and fluorescence detection, was properly transferred and accordingly optimized for the application on ultra performance liquid chromatography (UPLC™) and UV detection. Capitalizing on sub-2 µm particles, this newly established UV-UPLC™ technique facilitated efficient chromatographic separation of 21 amino acid derivatives within 12 min. In addition, UPLC™ demonstrated significant improvements due to superior performance and reduced run times compared with the former 35 min of the original HPLC protocol. Using UV instead of fluorescence detection, amino acid quantification after pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) yielded appropriate sensitivities within the low pmol range with corresponding detection limits varying from 0.11 to 0.57 pmol per injection. Moreover, the UPLC™ method was applied to characterize changes in the free (FAA) as well as total amino acid (TAA) profiles specific to culture media at three distinctive stages of fermentation: fresh medium, fermentation broth after cell mass production prior to induction and after product expression at the end of fermentation. Amino acid profiles intrinsic to the fresh, sterilized medium indicated free, hence more bioavailable, amino acids at a FAA/TAA ratio of 40%, whereas ongoing fermentation implied a rather specific, successive decline in selective FAA concentrations. Thereby, the most distinctive variations in FAAs were highlighted by aspartic acid, serine and threonine, each exhibiting an almost complete uptake from the culture media (-96% to -99%), with remaining FAA/TAA ratios of 1%, 8%, and 1%, respectively. This indeed may indicate limitations and shortages within the nutrient broth. Thus, amino acid monitoring utilizing high-throughput chromatography, such as UPLC™, can be considered as a valuable tool to facilitate rapid adjustments of fermentation broths and to optimize culture media to specific requirements.

Characterization of isoflavone composition in soy-based nutritional supplements via ultra performance liquid chromatography.

The specific isoflavone composition of nutritional supplements is commonly not-labeled, although the stated amounts are strongly dependent on the present isoflavone conjugates. Hence, 11 soy-based dietary supplements were characterized via a newly established ultra performance liquid chromatography (UPLC) method, on both their native conjugated isoflavone spectra, as well as on quantitative amounts derived as total aglycones after enzymatic hydrolysis utilizing Helix pomatia juice. Capitalizing on sub-2 microm particles, the established RP-UPLC technique facilitated efficient chromatographic separation of all 12 soy intrinsic isoflavone forms within 10 min. Derived native isoflavone profiles implied a certain variability, comprising conjugated forms, especially glycosides, as the predominant isoflavonic constituents throughout the majority of supplements, whereas only two samples indicated the more bioavailable free aglycones as prevailing compounds. Moreover, the robust quantification as total aglycones subsequent to enzymatic hydrolysis, unexceptionally yielded negative deviations referring to the labeled specifications, thus implying that stated amounts were typically calculated on basis of the high molecular isoflavone conjugates. Thus, especially in regard to better comparability, regulations concerning an uniform labeling basis are needed.

A new ultra-pressure liquid chromatography method for the determination of biogenic amines in cheese.

A fast and reliable ultra-performance liquid chromatography (UPLC) method for the determination of biogenic amines (ethanolamine, methylamine, agmatine, histamine, dimethylamine, ethylamine, octopamine, pyrrolidine, dopamine, isopropylamine, propylamine, tyramine, putrescine, butylamine, cadaverine, tryptamine, 2-phenylethylamine, 3-methylbutylamine, spermidine, spermine) in cheese was established. After pre-column derivatization with 6-aminoquinolyl-N-hydroxy-succinimidyl carbamate (AQC), 20 primary and secondary biogenic amines were separated on an Acquity UPLC column (BEH C(18), 1.7 microm; 2.1 mm x 50 mm) within 9 min. Limits of detection (mg/100g cheese) ranged from 0.04 (ethanolamine) to 1.62 (spermine), and limits of quantification were between 0.16 (ethanolamine) and 6.09 (spermine). The UPLC method was applied to the analysis of 58 cheese samples as retailed in Austria. About 13.8% of samples had a histamine content above 10mg/100g, and 22.4% had a tyramine content above 10mg/100g. Moreover, 8.6% of samples had a putrescine or cadaverine content higher than 10mg/100g. The total concentration of biogenic amines in two cheese samples was about 194 mg/100g. Thus, obligatory monitoring of biogenic amines should be considered to ensure quality of cheese in future.

Clustering of Saccharomyces boulardii strains within the species S. cerevisiae using molecular typing techniques.

AIMS: This study was undertaken to characterize and differentiate therapeutically relevant Saccharomyces yeasts. Among the isolates were so-called Saccharomyces boulardii strains, which are considered as probiotic agents, but whose taxonomic assignment is controversial. Moreover, the discriminative power of the applied molecular typing techniques should be evaluated. METHODS AND RESULTS: Genotyping was performed using species-specific polymerase chain reaction (PCR), randomly amplified polymorphic DNA-PCR, restriction fragment length polymorphism analysis of rDNA spacer regions and pulsed-field gel electrophoresis. Species-specific PCR assigned all of the product isolates to the species S. cerevisiae. By combining the other techniques, all isolates could be discriminated. Moreover, it could be demonstrated that probiotic S. boulardii strains form a separate cluster located within the species. CONCLUSIONS: With the exception of species-specific PCR, all of the applied methodologies were suitable for subspecies typing and indicated a close relationship between the probiotic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods applied in this study are considered powerful tools for quality control of therapeutically relevant yeasts. It is of crucial importance, especially regarding S. boulardii yeasts, to verify the identity of the correct strain, since the beneficial properties are considered to be strain-specific.

Endocarditis by Lactobacillus rhamnosus due to yogurt ingestion?

A young man who ate large quantities of probiotic yogurt developed endocarditis and septic arthritis caused by Lactobacillus rhamnosus. The pathogenic isolate could not be distinguished from the yogurt microflora using methods routinely used in the clinical microbiology laboratory. Only by using more appropriate methodology, including PCR, the pathogen could be distinguished from the yogurt isolate.

Apoptosis accompanies a change in the phenotypic distribution and functional capacity of murine bone marrow B-cells chronically exposed to prednisolone.

Prednisolone (PD) is commonly used for the treatment of inflammation accompanying diseases such as arthritis, allergy, asthma, and autoimmunity. While it is well documented that PD induces apoptosis in immature T-cells of the mouse, the effects of PD on development of immature B-cells in normal bone marrow (BM) was not known. An implantation system was developed which chronically delivered PD at a rate of a few nanograms per milliliter of plasma to mice. Ten days of exposure to such levels of PD caused splenic and thymic atrophy, which was accompanied by a 50% decrease in the numbers of circulating lymphocytes. Flow cytometric analysis (FACS) of the effects of PD on the BM revealed a threefold decrease in the proportion of B220+IgM- pre-B-cells and immature IgM+IgD- B-cells. However, the mature IgM+IgD+ cells were reasonably resistant to the effects of PD. A 25% decrease in small nucleated cells presumed to be part of the lymphocyte compartment was also noted from the scatter profiles of the marrow of PD-treated mice. These marked changes in BM composition were also accompanied by significant reductions in capacity of the BM to respond to trinitrophenylated-lipopolysaccharide (TNP-LPS) after exposure to PD either in vivo or in vitro. Studies to ascertain whether apoptosis played a role in the decline in the number of developing B-cells of marrow exposed to PD were performed in vitro in order to reduce the possibility of phagocytosis of apoptotic cells. A recent modification of FACS cell cycle analysis, which is highly quantitative and allows rapid analysis of heterogeneous tissues such as the marrow, was used to detect the apoptotic cells. After 16 hr of culture in 10(-7) M PD, approximately 40% of IgM+ and B220+ cells of BM resided to the left of G0/G1 in a region associated with apoptotic cells previously termed the A0 or "hypodiploid" region. Thus, these data indicate that chronic exposure to low levels of PD significantly altered the B-cell compartment of the murine bone marrow both in vivo and in vitro, potentially inducing apoptosis in these cells.

In vitro depression of bovine lymphocyte function by treatment of cultured bovine lymphocytes with physiologic concentrations of hydrocortisone.

The effect of hydrocortisone (hydrocortisone sodium succinate) on bovine lymphocyte blastogenesis in response to Staphylococcus aureus antigens and phytohemagglutinin was measured in vitro. Lymphocytes isolated from the blood of cows were treated for 6 to 8 days with physiologic hydrocortisone concentrations known to be inducible by environmental stress (10 ng/ml), acute clinical mastitis (25 ng/ml), or adrenocorticotropin treatment (45 ng/ml). All 3 concentrations of hydrocortisone caused a depression (P less than 0.01) in lymphocyte blastogenesis in response to phytohemagglutinin and S aureus antigen extract. Hydrocortisone concentrations as low as 10 pg/ml caused a depression in the lymphocyte blastogenic response to phytohemagglutinin. Marked variation existed among cows in the normal response of their nontreated lymphocytes and in the degree of depression of lymphocyte function after the in vitro treatment with hydrocortisone. Macrophage depletion experiments showed that the suppressive effect of hydrocortisone was not mediated by induction of suppressor macrophages. The data suggest that T-cell function was impaired directly by hydrocortisone treatment.

Failure of cortisol injected prior to milking to inhibit milk ejection in dairy cattle.

To test the potential for cortisol to inhibit milk ejection directly, 18 Holstein cows were divided equally into control and treatment groups based on milk yields. For treated animals, a single injection of cortisol was made into the saphenous vein 15 min before milkings. Increasing amounts of cortisol (0, 25, 50, and 100 mg) were injected for one morning and one evening milking, with the exception that the treated cows received only one 100 mg injection. Control animals received injections of 0.9% (w/v) NaCl. Cortisol injections had no effect on milk yields. However, a potential inhibitory mechanism might involve a delay, perhaps due to the necessity of synthesizing a regulatory protein. Therefore, to test the potential for increased cortisol over a period of hours to inhibit milk ejection, six of the nine cows in the treatment group were injected with 100 mg of cortisol at 3.25, 2.25, 1.25 and 0.25 h before sequential morning and evening milkings. In blood samples taken 1 min before and after injections, base-line cortisol concentrations averaged 10.2 mg/ml; after injection they were 984.1 ng/ml, and before subsequent injections they were 37.6 ng/ml. Again cortisol injections had no effect on milk yields.