Does Keeping Cows for More Lactations Affect the Composition and Technological Properties of the Milk?

This study investigated differences in the raw milk composition and technological properties between cows with different numbers of lactations. In total, 12 commercial herds were visited within a period of 12 weeks. On each farm, milk samples from five young cows (lactations 1-2) and five older cows (lactation ≥ 3) were collected. For each farm, milk samples from the young cows and the older cows, respectively, were pooled. The pooled milk samples were analyzed for gross composition and technological properties. Using principal component analysis (PCA) to assess the overall variation in milk quality attributes and the potential clustering of milk from young cows and older cows, respectively, an effect of breed, but no clear effect of lactation number, was observed. In contrast, one-way ANOVA showed higher plasmin activity (p = 0.002) in pooled milk from the older cows, whereas plasminogen-derived activity (p = 0.001) and total proteolysis (p = 0.029) were higher in milk from the young cows. Likewise, orthogonal projections to latent structure discriminant analysis (OPLS-DA) showed higher plasmin activity in milk from older cows, whereas younger cows had higher plasminogen-related activity and higher total proteolysis. To conclude, except for plasmin and plasminogen-related activities, there were no major differences in the composition and technological properties between milk from older cows and young cows.

Associations between the Bacterial Composition of Farm Bulk Milk and the Microbiota in the Resulting Swedish Long-Ripened Cheese.

The maturation of a traditional Swedish long-ripened cheese has shown increasing variation in recent years and the ripening time is now generally longer than in the past. While the cheese is reliant on non-starter lactic acid bacteria for the development of its characteristic flavour, we hypothesised that the observed changes could be due to variations in the microbiota composition and number of bacteria in the raw milk used for production of the cheese. To evaluate associations between microbiota in the raw milk and the resulting cheese, three clusters of commercial farms were created to increase variation in the microbiota of dairy silo milk used for cheese production. Cheese production was performed in three periods over one year. Within each period, milk from the three farm clusters was collected separately and transported to the cheese production facility. Following pasteurisation, the milk was processed into the granular-eyed cheese and matured at a dedicated cheese-ripening facility. For each cheese batch, farm bulk and dairy silo milk samples, a starter culture, early process samples and cheese samples from different stages of maturation (7-20 months) were collected and their microbiota characterised using 16S rRNA amplicon sequencing. The microbiota in the farm bulk milk differed significantly between periods and clusters. Differences in microbiota in dairy silo milk were observed between periods, but not between farm clusters, while the cheese microbiota differed between periods and clusters. The top 13 amplicon sequence variants were dominant in early process samples and the resulting cheese, making up at least 93.3% of the relative abundance (RA). Lactococcus was the dominant genus in the early process samples and, together with Leuconostoc, also dominated in the cheese samples. Contradicting expectations, the RA of the aroma-producing genus Lactobacillus was low in cheese during ripening and there was an unexpected dominance of starter lactic acid bacteria even at the later stages of cheese ripening. To identify factors behind the recent variations in ripening time of this cheese, future studies should address the effects of process variables and the dairy environment.

Relation between αS1-casein, genotype, and quality traits of milk from Swedish dairy goats.

Locally produced food is becoming popular among Swedish consumers. One product that has increased in popularity is artisan-manufactured goat cheese, and although the dairy goat industry in Sweden is small-scale, production is gradually increasing. In goats, the CSN1S1 gene regulates expression of the protein αS1-casein (αS1-CN), which has been found to be important for cheese yield. Over the years, breeding animals have been imported to Sweden from Norway. Historically, a high frequency of the Norwegian goat population carried a polymorphism at the CSN1S1 gene. This polymorphism, called the Norwegian null allele (D), leads to zero or significantly reduced expression of αS1-CN. Using milk samples from 75 goats, this study investigated associations between expression of αS1-CN and genotype at the CSN1S1 gene on milk quality traits from Swedish Landrace goats. Milk samples were grouped according to relative level of αS1-CN (low: 0-6.9% of total protein; medium-high: 7-25% of total protein) and genotype (DD, DG, DA/AG/AA). While the D allele leads to extremely low expression of αS1-CN, the G allele is low expressing and the A allele is highly expressing for this protein. Principal component analysis was used to explore the total variation in milk quality traits. To evaluate the effect of different allele groups on milk quality attributes, 1-way ANOVA and Tukey pairwise comparison tests were used. The majority (72%) of all goat milk samples investigated showed relative αS1-CN content of 0% to 6.82% of total protein. The frequency of individuals homozygous for the Norwegian null allele (DD) was 59% in the population of sampled goats, and only 15% carried at least one A allele. A low relative concentration of αS1-CN was associated with lower total protein, higher pH, and higher relative concentration of ß-casein and levels of free fatty acids. Milk from goats homozygous for the null allele (DD) showed a similar pattern as milk with low relative concentration of αS1-CN, but total protein was only numerically lower, and somatic cell count and αS2-CN were higher than for the other genotypes. The associations between levels of αS1-CN and the investigated genotype at the CSN1S1 gene indicate a need for a national breeding program for Swedish dairy goats.

Milking system and premilking routines have a strong effect on the microbial community in bulk tank milk.

In this study, we investigated the variation in the microbial community present in bulk tank milk samples and the potential effect of different farm management factors. Bulk tank milk samples were collected repeatedly over one year from 42 farms located in northern Sweden. Total and thermoresistant bacteria counts and 16S rRNA gene-based amplicon sequencing were used to characterize microbial community composition. The microbial community was in general heterogeneous both within and between different farms and the community composition in the bulk tank milk was commonly dominated by Pseudomonas, Acinetobacter, Streptococcus, unclassified Peptostreptococcaceae, and Staphylococcus. Principal component analysis including farm factor variables and microbial taxa data revealed that the microbial community in milk was affected by type of milking system. Milk from farms using an automatic (robot) milking system (AMS) and loose housing showed different microbial community composition compared with milk from tiestall farms. A discriminant analysis model revealed that this difference was dependent on several microbial taxa. Among farms using an automatic milking system, there were further differences in the microbial community composition depending on the brand of the milking robot used. On tiestall farms, routines for teat preparation and cleaning of the milking equipment affected the microbial community composition in milk. Total bacteria count (TBC) in milk differed between the farm types, and TBC were higher on AMS than tiestall farms (log 4.05 vs. log 3.79 TBC/mL for AMS and tiestalls, respectively). Among tiestall farms, milk from farms using a chemical agent in connection to teat preparation and a more frequent use of acid to clean the milking equipment had lower TBC in milk, than milk from farms using water for teat preparation and a less frequent use of acid to clean the milking equipment (log 3.68 vs. 4.02 TBC/mL). There were no significant differences in the number of thermoresistant bacteria between farm types. The evaluated factors explained only a small proportion of total variation in the microbiota data, however, despite this, the study highlights the effect of routines associated with teat preparation and cleaning of the milking equipment on raw milk microbiota, irrespective of type of milking system used.

Composition and properties of bovine milk: A study from dairy farms in northern Sweden; Part I. Effect of dairy farming system.

This study was part of a larger project that aimed to understand the causes for increasing variation in cheese ripening in a cheese-producing region in northern Sweden. The influence of different on-farm factors on raw milk composition and properties was investigated and is described in this paper, whereas the monthly variation in the milk quality traits during 1 yr is described in our companion paper. The dairy farming systems on a total of 42 dairy farms were characterized through a questionnaire and farm visits. Milk from farm tanks was sampled monthly over 1 yr and analyzed for quality attributes important for cheese making. On applying principal component analyses to evaluate the variation in on-farm factors, different types of farms were distinguished. Farms with loose housing and automatic milking system (AMS) or milking parlor had a higher number of lactating cows, and predominantly Swedish Holstein (SH) breed. Farms associated with tiestalls had a lower number of lactating cows and breeds other than SH. Applying principal component analyses to study the variation in composition and properties of tank milk samples from farms revealed a tendency for the formation of 2 clusters: milk from farms with AMS or a milking parlor, and milk from farms with tiestall milking. The interaction between the milking system, housing system, and breed probably contributed to this grouping. Other factors that were used in the characterization of the farming systems only showed a minor influence on raw milk quality. Despite the interaction, milk from tiestall farms with various cow breeds had higher concentrations (g/100 g of milk) of fat (4.74) and protein (3.63), and lower lactose concentrations (4.67) than milk from farms with predominantly SH cows and AMS (4.32, 3.47, and 4.74 g/100 g of milk, respectively) or a milking parlor (4.47, 3.54, and 4.79 g/100 g of milk, respectively). Higher somatic cell count (195 × 103/mL) and lower free fatty acid concentration (0.75 mmol/100 g of fat) were observed in milk from farms with AMS than in milk from tiestall systems (150 × 103/mL and 0.83 mmol/100 g of fat, respectively). Type of farm influenced milk gel strength, with milk from farms with predominantly SH cows showing the lowest gel strength (65.0 Pa), but not a longer rennet coagulation time. Effects of dairy farming system (e.g., dominant breed, milking system, housing, and herd size) on milk quality attributes indicate a need for further studies to evaluate the in-depth effects of farm-related factors on milk quality attributes.

Composition and properties of bovine milk: A study from dairy farms in northern Sweden; Part II. Effect of monthly variation.

This study investigated the influence of monthly variation on the composition and properties of raw farm milk collected as part of a full-scale cheese-making trial in a region in northern Sweden. In our companion paper, the contribution of on-farm factors to the variation in milk quality attributes is described. In total, 42 dairy farms were recruited for the study, and farm milk samples were collected monthly over 1 yr and characterized for quality attributes of importance for cheese making. Principal component analysis suggested that milk samples collected during the outdoor period (June-September) were different from milk samples collected during the indoor period. Despite the interaction with the milking system, the results showed that fat and protein concentrations were lower in milk collected during May through August, and lactose concentration was higher in milk collected during April through July than for the other months. Concentrations of free fatty acids were generally low, with the highest value (0.86 mmol/100 g of fat) observed in February and the lowest (0.70 mmol/100 g of fat) observed in June. Plasmin and plasminogen-derived activities varied with sampling month without a clear seasonal pattern. The pH of farm tank milk ranged from 6.60 to 6.82, with the lowest and highest values in September and February, respectively. The highest somatic cell count was observed in August (201 × 103 cells/mL) and the lowest in April (143 × 103 cells/mL). The highest value of gel strength, was recorded in December (88 Pa) and the lowest in July (64 Pa). Rennet coagulation time and gel strength were inversely correlated, with the lowest rennet coagulation time value observed in December. Orthogonal projections to latent structures (OPLS) and discriminant analysis adaptation of OPLS identified casein micelle size and total proteolysis as the milk quality attributes with major responses to sampling month, with smaller casein micelle size and higher total proteolysis associated with the outdoor months. Using discriminant analysis adaptation of OPLS to further investigate causes behind the variation in milk traits revealed that there were factors in addition to feeding on pasture that differed between outdoor and indoor months. Because fresh grass was seldom the primary feed in the region during the outdoor period, grazing was not considered the sole reason for the observed difference between outdoor and indoor periods in raw milk quality attributes.

Short communication: Caseins and α-lactalbumin content of camel milk (Camelus dromedarius) determined by capillary electrophoresis.

Camel milk has unique physical, nutritional, and technological properties when compared with other milks, especially bovine. Because proteins confer many of the properties of milk and its products, this study aimed to determine the proteins of camel milk, their correlations, and relative distribution. Raw milk samples were collected from 103 dromedary camels in the morning and evening. Capillary electrophoresis results showed wide variation in the concentrations (g/L) of proteins between samples as follows: α-lactalbumin, 0.3 to 2.9; αS1-casein, 2.4 to 10.3; αS2-casein, 0.3 to 3.9; ß-casein, 5.5 to 29.0; κ-casein, 0.1 to 2.4; unknown casein protein 1, 0.0 to 3.4; and unknown casein protein 2, 0.0 to 4.6. The range in percent composition of the 4 caseins were as follows: αS1, 12.7 to 35.3; αS2, 1.8 to 20.8; ß, 42.3 to 77.4; and κ, 0.6 to 17.4. The relative proportion of αS1-, αS2-, ß-, and κ-caseins in camel milk (26:4:67:3, wt/wt) differed from that of bovine milk (38:10:36:12, wt/wt). This difference might explain the dissimilarity between the 2 milks with respect to technical and nutritional properties.

Interactive effects of casein micelle size and calcium and citrate content on rennet-induced coagulation in bovine milk.

Interactive effects of casein micelle size and milk calcium and citrate content on rennet-induced coagulation were investigated. Milk samples containing small (SM) and large (LM) micelles, obtained from individual Holstein cows, were modified by addition of calcium and/or citrate and milk coagulation properties were evaluated in a full factorial design. The results showed that LM milk had a higher relative proportion of casein, coagulated faster, and resulted in a stronger gel than SM milk. Addition of calcium slightly decreased casein micelle size, while addition of citrate slightly increased micelle size. Calcium addition resulted in a shorter coagulation time and the strongest gels, while citrate addition increased the coagulation time and resulted in the weakest gels. Addition of calcium and citrate in combination resulted in intermediate coagulation properties. The interactive effect of micelle size and citrate was significant for gel strength. Microstructural differences between the milk gels were consistent with the rheological properties, for example, the micrographs revealed that a more homogeneous network was formed when calcium was added, resulting in a stronger gel. A more inhomogeneous network structure was formed when citrate was added, resulting in a weaker gel. Thus, variations in casein micelle size and in calcium and citrate content influence rennet-induced coagulation in bovine milk. The calcium and citrate contents in Swedish milk have changed over time, whereby calcium content has increased and citrate content has decreased. In practical cheese making, calcium is added to cheese milk, most likely altering the role of inherent citrate and possibly influencing casein micelle size. The observed interaction effect between casein micelle size and citrate in this study, suggests that larger micelles with moderate citrate level will result in firmer gels, whereas a higher citrate content reduced gel strength more in case of large than SM. Since firmer gels are likely to retain more protein and fat than less firmer gels, this interaction effect could have implications in practical cheese production.

Testing of Auxotrophic Selection Markers for Use in the Moss Physcomitrella Provides New Insights into the Mechanisms of Targeted Recombination.

The moss Physcomitrella patens is unique among plants in that homologous recombination can be used to knock out genes, just like in yeast. Furthermore, transformed plasmids can be rescued from Physcomitrella back into Escherichia coli, similar to yeast. In the present study, we have tested if a third important tool from yeast molecular genetics, auxotrophic selection markers, can be used in Physcomitrella. Two auxotrophic moss strains were made by knocking out the PpHIS3 gene encoding imidazoleglycerol-phosphate dehydratase, and the PpTRP1 gene encoding phosphoribosylanthranilate isomerase, disrupting the biosynthesis of histidine and tryptophan, respectively. The resulting PpHIS3Δ and PpTRP1Δ knockout strains were unable to grow on medium lacking histidine or tryptophan. The PpHIS3Δ strain was used to test selection of transformants by complementation of an auxotrophic marker. We found that the PpHIS3Δ strain could be complemented by transformation with a plasmid expressing the PpHIS3 gene from the CaMV 35S promoter, allowing the strain to grow on medium lacking histidine. Both linearized plasmids and circular supercoiled plasmids could complement the auxotrophic marker, and plasmids from both types of transformants could be rescued back into E. coli. Plasmids rescued from circular transformants were identical to the original plasmid, whereas plasmids rescued from linearized transformants had deletions generated by recombination between micro-homologies in the plasmids. Our results show that cloning by complementation of an auxotrophic marker works in Physcomitrella, which opens the door for using auxotrophic selection markers in moss molecular genetics. This will facilitate the adaptation of shuttle plasmid dependent methods from yeast molecular genetics for use in Physcomitrella.

Composition and enzymatic activity in bulk milk from dairy farms with conventional or robotic milking systems.

The objective of the studies reported in this research communication was to investigate differences in composition and enzymatic activities in bulk milk samples provided from Swedish dairy farms with different management systems, i.e. automated (AMS) and conventional milking systems (CMS). A bulk milk sample was collected from each of 104 dairy farms, 51 using AMS and 53 using CMS, located in the same geographical region. Sampling took place within two consecutive days during the indoor period (October). Milk samples were analysed for contents of total fat and protein, free fatty acids (FFA), caseins and whey proteins, somatic cell count (SCC), pH, plasmin and plasminogen derived activities, and total proteolysis. Our results showed a lower protein content and higher SCC in bulk milk from AMS herds compared with milk from CMS herds. Plasmin, plasminogen and total plasmin/ plasminogen derived activities were lower in milk from AMS herds but despite this, total casein and the ß-casein fraction as % of total protein were lower in milk from AMS herds than in milk from herds using CMS. Total proteolysis was higher in milk from AMS herds, suggesting that other proteases than plasmin, e.g. cellular and bacterial proteases, contributed to the degradation of casein. This was supported by a positive correlation between SCC and total proteolysis (P < 0·01), as well as a negative correlation between total proteolysis and ß-casein fraction (P < 0·05). In conclusion, comparing the quality of bulk milk from commercial dairy herds using AMS and CMS, respectively, several differences were observed, suggesting a significant effect from management system.

Short communication: Influence of shortening the dry period of Swedish dairy cows on plasmin activity in milk.

The aim of this study was to evaluate the influence of shortening the dry period of Swedish dairy cows on plasmin activity and casein composition in milk. Swedish Holstein and Swedish Red cows, 45 in total, were assigned to a dry period of either 4 or 8wk. Milk samples were taken 10 and 5wk prepartum, and 6 and 12wk postpartum. Plasmin activity and plasminogen activity were measured with a spectrophotometric assay. Casein composition was measured by capillary zone electrophoresis. Prepartum plasminogen activity increased by 22% between 10 and 5wk prepartum, whereas no change in plasmin activity was observed during the same period. Cows with a 4-wk dry period had 61% higher plasmin activity in postpartum milk than cows with an 8-wk dry period. Cows of third or greater parity tended to have a stronger increase in plasmin activity as a result of applying a short dry period than cows of second parity. Although the αS1- and ß-casein fractions declined with increasing plasmin activity, no dry period effects were found. Based on postpartum differences in plasmin activity, it was concluded that particularly multiparous cows require more than 4wk between lactations for recovery of the mammary epithelium. Changes in casein composition as an effect of plasmin activity are not expected to have a great effect on processing quality of milk, although future work is needed to verify this.

Effect of shortening or omitting the dry period of Holstein-Friesian cows on casein composition of milk.

The aim of this study was to evaluate the effect of shortening or omitting the dry period of dairy cows on milk casein composition. For this study, we analyzed milk samples of 90 cows with a dry period of 0, 30, or 60d and either a glucogenic or a lipogenic ration in early lactation. Milk was sampled at 6 and 2wk prepartum and at 2, 6, and 12wk postpartum. Milk was analyzed for casein (CN) composition by capillary zone electrophoresis, and isoforms of κ-CN were measured by reversed phase-HPLC. Shortening the dry period from 60 to 30d reduced the αS1-CN fraction by 3.8% and increased the αS2-CN fraction by 5.5%. In milk from cows with a 0-d dry period, the glycosylated κ-CN fraction in late lactation increased from 8 to 12% between 6 and 2wk prepartum. After calving, the glycosylated κ-CN fraction in milk was higher for cows with a 0-d dry period (6.7%) compared with cows with a 60-d dry period (5.2%). The glycosylated κ-CN fraction at 2wk postpartum was negatively correlated with milk yield, suggesting that glycosylation was related to reduced productivity of mammary epithelial cells. In early lactation, the ß-CN fraction was reduced in milk of cows with a 0-d dry period. A lowered ß-CN fraction was associated with high somatic cell count and greater parity, indicating that it was the result of proteolytic activity. In conclusion, casein composition changes that result from shortening the dry period from 60 to 30d are not expected to affect processing characteristics of milk. Applying a 0-d dry period may affect processability of milk because of a higher glycosylated κ-CN fraction, and possibly because of higher proteolytic activity compared with a 60-d dry period.

Short communication: Lipolytic activity on milk fat by Staphylococcus aureus and Streptococcus agalactiae strains commonly isolated in Swedish dairy herds.

The objective of this study was to determine the lipolytic activity on milk fat of 2 bovine mastitis pathogens, that is, Staphylococcus aureus and Streptococcus agalactiae. The lipolytic activity was determined by 2 different techniques, that is, thin-layer chromatography and an extraction-titration method, in an experimental model using the most commonly occurring field strains of the 2 mastitic bacteria isolated from Swedish dairy farms. The microorganisms were inoculated into bacteria-free control milk and incubated at 37°C to reflect physiological temperatures in the mammary gland. Levels of free fatty acids (FFA) were analyzed at time of inoculation (t=0) and after 2 and 6h of incubation, showing significant increase in FFA levels. After 2h the FFA content had increased by approximately 40% in milk samples inoculated with Staph. aureus and Strep. agalactiae, and at 6h the pathogens had increased FFA levels by 47% compared with the bacteria-free control milk. Changes in lipid composition compared with the bacteria-free control were investigated at 2 and 6h of incubation. Diacylglycerols, triacylglycerols, and phospholipids increased significantly after 6h incubation with the mastitis bacteria, whereas cholesterol and sterol esters decreased. Our results suggest that during mammary infections with Staph. aureus and Strep. agalactiae, the action of lipases originating from the mastitis pathogens will contribute significantly to milk fat lipolysis and thus to raw milk deterioration.

A general analytical platform and strategy in search for illegal drugs.

An effective screening procedure to identify and quantify active pharmaceutical substances in suspected illegal medicinal products is described. The analytical platform, consisting of accurate mass determination with liquid chromatography time-of-flight mass spectrometry (LC-QTOF-MS) in combination with nuclear magnetic resonance (NMR) spectroscopy provides an excellent analytical tool to screen for unknowns in medicinal products, food supplements and herbal formulations. This analytical approach has been successfully applied to analyze thousands of samples. The general screening method usually starts with a methanol extraction of tablets/capsules followed by liquid chromatographic separation on a Halo Phenyl-Hexyl column (2.7µm; 100mm×2.1mm) using an acetonitrile/0.1% formic acid gradient as eluent. The accurate mass of peaks of interest was recorded and a search made against an in-house database containing approximately 4200 substances, mostly pharmaceutical compounds. The search could be general or tailored against different classes of compounds. Hits were confirmed by analyzing a reference substance and/or by NMR. Quantification was normally performed with quantitative NMR (qNMR) spectroscopy. Applications for weight-loss substances like sibutramine and orlistat, sexual potency enhancement (PDE-5 inhibitors), and analgesic drugs are presented in this study. We have also identified prostaglandin analogues in eyelash growth serum, exemplified by isopropyl cloprostenate and bimatoprost. For creams and ointments, matrix solid-phase dispersion (MSPD) was found to give a clean extracts with high recovery prior to LC-MS analyses. The structural elucidation of cetilistat, a new weight-loss substance recently found in illegal medicines purchased over the Internet, is also presented.

Use and qualification of primary and secondary standards employed in quantitative ¹H NMR spectroscopy of pharmaceuticals.

Standards are required in quantitative NMR (qNMR) to obtain accurate and precise results. In this study acetanilide was established and used as a primary standard. Six other chemicals were selected as secondary standards: 3,4,5-trichloropyridine, dimethylterephthalate, maleic acid, 3-sulfolene, 1,4-bis(trimethylsilyl)benzene, and 1,3,5-trimethoxybenzene. The secondary standards were quantified using the primary standard acetanilide. A protocol for qualification and periodic checks of these secondary standards was developed, and used for evaluation of the stability of the compounds. Periodic monitoring of purity was performed for several years. The purity was higher than 99% for all secondary standards. All standards maintained the initial purity during the time period of monitoring, with very small variations in purity (0.3-0.4%). The selected secondary standards were shown to be suitable qNMR standards and that periodic requalification of the standards by qNMR ensures reliable analytical results. These standards have been used in our laboratory for compliance testing of pharmaceutical active substances and approved medicinal products as well as for analysis of suspected illegal medicines. In total more than 1000 samples have been tested using both internal and external standardization and examples are given.

Casein breakdown in bovine milk by a field strain of Staphylococcus aureus.

The objectives of this study were to establish the proteolytic effects of Staphylococcus aureus during mastitis on economically important milk proteins. Concentrations of milk proteins were determined by capillary electrophoresis in an experimental model using a field strain of S. aureus. The pathogen was inoculated into bacteria-free control milk to imitate proteolysis caused by the pathogen in the mammary gland between milkings. Milk content of caseins (CN) α(S1), α(S2), κ, ß(A1), and ß(A2) and whey proteins α-lactalbumin and ß-lactoglobulin were analyzed initially and after 6 h of incubation. After 6 h, the overall CN content was significantly reduced (21%) in milk inoculated with S. aureus compared with the bacteria-free control milk. S. aureus significantly lowered concentration of α(S1)-CN (2.5%), ß(A1)-CN (3%), and ß(A2)-CN (5%). S. aureus also hydrolyzed κ-CN into para-κ-CN, with significant reduction of κ-CN (7.4%) as a consequence.

Screening of counterfeit corticosteroid in creams and ointments by NMR spectroscopy.

It has been shown that NMR spectroscopy is an effective analytical method to rapidly screen creams and ointments for counterfeit corticosteroids. Extraction and NMR procedures have been developed. Ten over the counter creams and ointments sold in health care shops were screened and two creams were found to contain counterfeited corticosteroids.

Protein degradation in bovine milk caused by Streptococcus agalactiae.

Streptococcus (Str.) agalactiae is a contagious mastitis bacterium, often associated with cases of subclinical mastitis. Different mastitis bacteria have been evaluated previously from a diagnostic point of view, but there is a lack of knowledge concerning their effect on milk composition. Protein composition is important in achieving optimal yield and texture when milk is processed to fermented products, such as cheese and yoghurt, and is thus of great economic value. The aim of this in vitro study was to evaluate protein degradation mainly caused by exogenous proteases originating from naturally occurring Str. agalactiae. The samples were incubated at 37°C to imitate degradation caused by the bacteria in the udder. Protein degradation caused by different strains of Str. agalactiae was also investigated. Protein degradation was observed to occur when Str. agalactiae was added to milk, but there were variations between strains of the bacteria. Caseins, the most economically important proteins in milk, were degraded up to 75% in milk inoculated with Str. agalactiae in relation to sterile ultra-high temperature (UHT) milk, used as control milk. The major whey proteins, α-lactalbumin and ß-lactoglobulin, were degraded up to 21% in relation to the sterile control milk. These results suggest that different mastitis bacteria but also different strains of mastitis bacteria should be evaluated from a milk quality perspective to gain knowledge about their ability to degrade the economically important proteins in milk.

Simultaneous determination of cytochrome P450 1A, 2A and 3A activities in porcine liver microsomes.

The aim of this study was to develop a robust method for the simultaneous determination of the activities of three porcine CYP450 enzymes in hepatic microsomes. A cocktail consisting of three selective CYP450 probe substrates, 7-ethoxyresorufin (CYP1A), coumarin (CYP2A) and 7-benzyloxy-4-trifluoromethylcoumarin (BFC; CYP3A), was incubated with porcine liver microsomes. The presence of 7-ethoxyresorufin appears to significantly influence the kinetics of coumarin hydroxylation and BFC O-debenzylation. These results indicate that the use of 7-ethoxyresorufin in substrate cocktails together with coumarin and BFC should be avoided.

Homologues of the Arabidopsis thaliana SHI/STY/LRP1 genes control auxin biosynthesis and affect growth and development in the moss Physcomitrella patens.

The plant hormone auxin plays fundamental roles in vascular plants. Although exogenous auxin also stimulates developmental transitions and growth in non-vascular plants, the effects of manipulating endogenous auxin levels have thus far not been reported. Here, we have altered the levels and sites of auxin production and accumulation in the moss Physcomitrella patens by changing the expression level of homologues of the Arabidopsis SHI/STY family proteins, which are positive regulators of auxin biosynthesis genes. Constitutive expression of PpSHI1 resulted in elevated auxin levels, increased and ectopic expression of the auxin response reporter GmGH3pro:GUS, and in an increased caulonema/chloronema ratio, an effect also induced by exogenous auxin application. In addition, we observed premature ageing and necrosis in cells ectopically expressing PpSHI1. Knockout of either of the two PpSHI genes resulted in reduced auxin levels and auxin biosynthesis rates in leafy shoots, reduced internode elongation, delayed ageing, a decreased caulonema/chloronema ratio and an increased number of axillary hairs, which constitute potential auxin biosynthesis sites. Some of the identified auxin functions appear to be analogous in vascular and non-vascular plants. Furthermore, the spatiotemporal expression of the PpSHI genes and GmGH3pro:GUS strongly overlap, suggesting that local auxin biosynthesis is important for the regulation of auxin peak formation in non-vascular plants.